Vitamin D and Diabetic Neuropathy

Vitamin D and Diabetic Neuropathy

A thesis submitted to the University of Manchester for the degree of

Doctor of Philosophy in Medicine

in the Faculty of Medical and Human Sciences

2013

Uazman Alam

SCHOOL OF MEDICINE

Institute of Human Development, Centre for Endocrinology and Diabetes

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Table of Contents

1 Chapter I - INTRODUCTION ......................................................................... 35 1.1 Aetiopathology of Diabetes ........................................................................... 36 1.2 Latent Autoimmune Diabetes in Adults (LADA) ........................................... 40

1.2.1 Screening and Treatment in LADA ............................................................... 40 1.2.2 Auto Antibodies in LADA .............................................................................. 43 1.2.3 Microvascular complications in LADA ........................................................... 44

1.3 Complications of Diabetes............................................................................. 45 1.3.1 Diabetic Neuropathy ..................................................................................... 46 1.3.2 Painful Diabetic Neuropathy ......................................................................... 51 1.3.3 Pathogenesis of Diabetic Neuropathy ........................................................... 53

1.4 Vitamin D deficiency ...................................................................................... 62 1.4.1 Vitamin D deficiency, Hyperlipidaemia and Hypertension ............................. 63 1.4.2 Vitamin D deficiency and Macrovascular Complications ............................... 64 1.4.3 Vitamin D and Diabetic Neuropathy .............................................................. 65 1.4.4 Vitamin D and Diabetic Retinopathy ............................................................. 67 1.4.5 Vitamin D and Diabetic Nephropathy ............................................................ 68

1.5 Clinical Evaluation and Investigations of Neuropathy ................................. 69 1.5.1 Diagnosis of Diabetic Distal Symmetrical Polyneuropathy ............................ 70

1.6 REFERENCES ................................................................................................. 79

2 CHAPTER II - METHODS ........................................................................... 113 2.1 Synopsis ....................................................................................................... 114 2.2 Hypothesis and aims.................................................................................... 114 2.3 Study Design ................................................................................................ 116 2.4 METHODS: Neuropathy Studies - Chapters 3, 4, 5 &8 ............................... 116

2.4.1 Patient Enrolment ....................................................................................... 116 2.4.2 Inclusion Criteria for Chapters 3, 4, 5 & 8 ................................................... 117 2.4.3 Exclusion Criteria for Chapters 3, 4, 5 & 8 .................................................. 118 2.4.4 Questionnaire Administration ...................................................................... 118 2.4.5 Neuropathy Disability Score (NDS) ............................................................. 119 2.4.6 Neuropad ................................................................................................... 121 2.4.7 Heart Rate Variability to Deep Breathing .................................................... 122 2.4.8 Quantitative Sensory Testing ...................................................................... 122 2.4.9 Electrophysiology ....................................................................................... 123 2.4.10 Skin Biopsies and Immuno-histology ...................................................... 125 2.4.11 Corneal sensitivity .................................................................................. 126 2.4.12 Corneal Confocal microscopy ................................................................. 127 2.4.13 Manual Image Analysis: Chapter 4,5 & 8 ................................................ 130 2.4.14 Automated Image Analysis: Chapter 3 only ............................................ 131

2.5 METHODS: Vitamin D Studies – Chapters 6, 7 & 9 .................................... 132 2.5.1 Participant Exclusion Criteria for Chapters 6, 7 & 9 .................................... 132 2.5.2 Blood pressure and Anthropometric measurements ................................... 132 2.5.3 Assessment of Demographics, Cardiovascular disease and Medications ... 133 2.5.4 Laboratory Measurements .......................................................................... 133 2.5.5 Diabetic Retinopathy Assessment .............................................................. 134 2.5.6 25(OH) Vitamin D Assay ............................................................................. 135

2.6 REFERENCES ............................................................................................... 136

3 Chapter III - Rapid Nerve Fibre Decline in patients with Type 1 diabetes can be readily detected using Corneal Confocal Microscopy. ..................... 138

3.1 ABSTRACT ................................................................................................... 139 3.2 INTRODUCTION ............................................................................................ 141 3.3 RESEARCH DESIGN AND METHODS ......................................................... 143

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3.3.1 Selection of patients ................................................................................... 143 3.3.2 Assessment of neuropathy ......................................................................... 143 3.3.3 Corneal Confocal Microscopy ..................................................................... 144 3.3.4 Statistical analysis and Power calculation ................................................... 145

3.4 RESULTS ...................................................................................................... 147 3.4.1 Demographics ............................................................................................ 147 3.4.2 Metabolic and Anthropometric measurements ............................................ 149 3.4.3 Neuropathy evaluation ................................................................................ 151

3.5 DISCUSSION ................................................................................................. 159 3.6 REFERENCES ............................................................................................... 162

4 Chapter IV - Diagnostic Utility of Corneal Confocal Microscopy and Intra-Epidermal Nerve Fibre Density in Diabetic Neuropathy ................................ 167


4.3.1 Selection of patients ................................................................................... 172 4.3.2 Definition of Neuropathy ............................................................................. 172 4.3.3 Assessment of neuropathy ......................................................................... 172 4.3.4 Corneal Confocal Microscopy ..................................................................... 173 4.3.5 Skin biopsy and immunohistochemistry ...................................................... 174 4.3.6 Statistical analysis ...................................................................................... 174

4.4 RESULTS ...................................................................................................... 176 4.4.1 Demographics, Metabolic and Anthropometric Assessment ....................... 176 4.4.2 Symptoms and deficits ............................................................................... 178 4.4.3 Neuropathy evaluation ................................................................................ 180


5 Chapter V - Enhanced Small Fibre Neuropathy in Patients with Latent Autoimmune Diabetes in Adults ...................................................................... 201

5.1 ABSTRACT ................................................................................................... 202 5.2 INTRODUCTION ............................................................................................ 203 5.3 RSEARCH DESIGN AND METHODS ............................................................ 205

5.3.1 Selection of patients ................................................................................... 205 5.3.2 LADA definition ........................................................................................... 205 5.3.3 Assessment of neuropathy ......................................................................... 205 5.3.4 Corneal Confocal Microscopy ..................................................................... 206 5.3.5 Skin biopsy and immunohistochemistry ...................................................... 207 5.3.6 Statistical analysis ...................................................................................... 208

5.4 RESULTS ...................................................................................................... 209 5.4.1 Demographics and Clinical Neuropathy ...................................................... 209 5.4.2 Metabolic and Anthropometric measurements ............................................ 211 5.4.3 Neuropathy evaluation ................................................................................ 213


6 Chapter VI - Marked vitamin D deficiency in patients with diabetes from secondary care: Ethnic and seasonal differences and an association with dyslipidaemia .................................................................................................... 226


6.3.1 Subjects ..................................................................................................... 231 6.3.2 Blood pressure and Anthropometric measurements ................................... 231 6.3.3 Assessment of Demographics, Cardiovascular disease and Medications ... 232

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6.3.4 Laboratory Measurements .......................................................................... 232 6.3.5 Statistical analysis ...................................................................................... 233

6.4 RESULTS ...................................................................................................... 235 6.5 DISCUSSION ................................................................................................. 241 6.6 REFERENCES ............................................................................................... 245

7 Chapter VII - Differential effects of different vitamin D replacement strategies in patients with diabetes ................................................................ 249


7.3.1 Blood pressure and Anthropometric measurements ................................... 253 7.3.2 Assessment of Demographics, Cardiovascular disease and Medications ... 254 7.3.3 Laboratory Measurements .......................................................................... 254 7.3.4 Statistical analysis ...................................................................................... 255


8 Chapter VIII - Vitamin D Deficiency Contributes to Painful Diabetic Neuropathy ........................................................................................................ 272


8.3.1 Selection of patients ................................................................................... 276 8.3.2 Assessment of neuropathy ......................................................................... 276 8.3.3 Corneal Confocal Microscopy ..................................................................... 277 8.3.4 Skin biopsy and immunohistochemistry ...................................................... 278 8.3.5 25(OH) Vitamin D Assay ............................................................................. 279 8.3.6 Statistical analysis ...................................................................................... 280

8.4 RESULTS ...................................................................................................... 281 8.4.1 Demographics, Metabolic and Anthropometric Assessment ....................... 281 8.4.2 Symptoms and deficits ............................................................................... 284 8.4.3 Quantitative sensory tests .......................................................................... 286 8.4.4 Electrophysiology ....................................................................................... 286 8.4.5 Autonomic function, IENFD and CCM ......................................................... 286 8.4.6 25(OH)D status .......................................................................................... 289


9 Chapter IX - Vitamin D Deficiency is not associated with Diabetic Retinopathy or Maculopathy: A Cross Sectional study ................................ 300


9.3.1 Subjects ..................................................................................................... 305 9.3.2 Blood pressure and Anthropometric measurements ................................... 305 9.3.3 Assessment of Demographics, Cardiovascular disease and Medications ... 306 9.3.4 25(OH) vitamin D Assay ............................................................................. 307 9.3.5 Statistical analysis ...................................................................................... 308


10 Chapter X - Discussion .......................................................................... 327

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10.1 Rapid Nerve Fibre Decline in patients with Type 1 diabetes can be readily detected using Corneal Confocal Microscopy ........................................... 330 10.2 Diagnostic Utility of Corneal Confocal Microscopy and Intra-Epidermal Nerve Fibre Density in Diabetic Neuropathy .............................................. 330 10.3 Enhanced Small Fibre Neuropathy in Patients with Latent Autoimmune Diabetes in Adults ........................................................................................ 331 10.4 Marked vitamin D deficiency in patients with diabetes from secondary care: Ethnic and seasonal differences and an association with dyslipidaemia ............................................................................................... 332 10.5 Differential effects of different vitamin D replacement strategies in patients with diabetes. ............................................................................................... 333 10.6 Vitamin D Deficiency Contributes to Painful Diabetic Neuropathy ........... 334 10.7 Vitamin D Deficiency is not associated with Diabetic Retinopathy or Maculopathy: A Cross Sectional study ...................................................... 335 10.8 Study limitations .......................................................................................... 336 10.9 Future Work .................................................................................................. 338 10.10 Conclusion ................................................................................................ 339 10.11 REFERENCES ........................................................................................... 340

11 REFERENCES ......................................................................................... 350

12 APPENDIX 1– Study Related Documents ............................................. 403

13 APPENDIX 2 – Additional Analyses ...................................................... 413

Word count: 81807

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List of Tables

Table 1-1 Diagnosis of DM ................................................................................... 37

Table 1-2 Details of major epidemiological studies of DSPN ................................ 49

Table 1-3 Abnormal sensations in DSPN .............................................................. 52

Table 1-4 Treatment of DSPN based on the putative pathogenetic mechanisms

and outcome of clinical trials. ................................................................................ 61

Table 1-5 Serum 25(OH) vitamin D concentrations and status ............................. 63

Table 1-6 Type of nerve fibres in the peripheral nervous system.......................... 70

Table 3-1 Participant demographics and clinical neuropathy parameters in control

subjects and patients with T1DM at baseline and 2 year follow up ..................... 148

Table 3-2 Metabolic parameters in control subjects and T1DM patients at baseline

and 2 year follow up ............................................................................................ 150

Table 3-3 Small and large fibre tests of nerve structure and function in control

subjects and diabetic patients at baseline and 2 year follow up .......................... 153

Table 3-4 T1DM subjects with Rapid Nerve Fibre Decline at baseline and 2 year

follow up .............................................................................................................. 156

Table 4-1 Participant demographics and metabolic parameters in control subjects

and diabetic patients without (T1DM) and with neuropathy (DSPN) ................... 177

Table 4-2 Neuropathy symptoms and deficits in control subjects and diabetic

patients without (T1DM) and with neuropathy (DSPN) ....................................... 179


subjects and diabetic patients without (T1DM) and with neuropathy (DSPN) ..... 183

Table 4-4 Spearman’s rank correlation of CNFD, CNBD, CNFL and IENFD versus

NDS, McGill VAS, NSP, IENFD, thermal thresholds, VPT and nerve conduction

studies ................................................................................................................ 186

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Table 4-5 ROC analysis with area under the curve, optimal cut off and respective

sensitivity and specificity with 95% confidence interval in T1DM versus DSPN for

CNFD, CNBD, CNFL, IENFD, VPT, CST and WST ............................................ 188

Table 5-1 Participant demographics in Controls, T2DM and LADA .................... 210

Table 5-2 Clinical and metabolic parameters in control subjects and patients with

T2DM and LADA ................................................................................................. 212

Table 5-3 Small and large fibre neuropathy tests in control subjects, T2DM and

LADA .................................................................................................................. 216

Table 6-1 Baseline characteristics of subjects with and without diabetes ........... 235

Table 6-2 Characteristics by categories of 25(OH)D in 563 unselected subjects

with diabetes. ...................................................................................................... 238

Table 6-3 Regression analyses of 25(OH)D and T-CHL, HDL and triglycerides in

subjects with diabetes ......................................................................................... 239

Table 6-4 Characteristics of subjects with diabetes based on ethnicity .............. 240

Table 7-1 Clinical and demographic data and Characteristics of Anti-Diabetic

therapy in all three cohorts of all three intervention cohorts ................................ 257

Table 7-2 Change in 25(OH)D status, metabolic data and anthropometric

measurements following intervention in all three cohorts .................................... 259

Table 8-1 Participant demographics and metabolic parameters in control subjects

and diabetic patients with DPN and PDN ............................................................ 282

Table 8-2 Clinical neuropathy symptoms and deficits in control subjects and

diabetic patients with DPN and PDN ................................................................... 285


subjects and diabetic patients with DPN and PDN .............................................. 287

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Table 9-1 Demographic and metabolic parameters in subgroups based on severity

of retinopathy ...................................................................................................... 312

Table 9-2 Logistic regression analyses for the relationship between retinopathy,

25(OH)D status and other confounding variables ............................................... 314

Table 9-3 Demographic and metabolic parameters in subgroups based on

maculopathy........................................................................................................ 316

Table 9-4 Logistic regression analyses for the relationship between maculopathy,

25(OH)D status and other confounding variables ............................................... 317

Table 9-5 Frequencies of retinopathy, maculopathy and photocoagulation scarring

categorised by 25(OH)D status ........................................................................... 319

Table 13-1 Baseline characteristics of the complete Control group (n=16) and the

Control group with only white European participants (n=12) ............................... 414


Control group with only white European participants (n=21) ............................... 415


Control group with participants of an older age matched to the DSPN group (n=13)

............................................................................................................................ 416

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List of Figures

Figure 1-1 The pathogenesis of DSPN ................................................................. 56

Figure 1-2 Polyol Pathway in DSPN ..................................................................... 57

Figure 1-3 Putative effect of C-peptide on peripheral nerve structure and function

.............................................................................................................................. 60

Figure 1-4 Montage of the whole human subbasal nerve plexus .......................... 77

Figure 2-1 The Neuropathy Disability Score ....................................................... 120

Figure 2-2 Testing vibration sensation with a 128 Hz tuning fork as part of the NDS

............................................................................................................................ 120

Figure 2-3 Testing temperature sensation on the dorsum of foot as part of the NDS

............................................................................................................................ 121

Figure 2-4 The Neuropad .................................................................................... 121

Figure 2-5 The user interface for thermal threshold and pain measurement ...... 123

Figure 2-6 Electrodiagnostic studies of the peroneal and sural nerves ............... 124

Figure 2-7 Skin biopsies stained for PGP9.5 in a healthy control and subjects with

DM ...................................................................................................................... 126

Figure 2-8 Estimation of corneal sensation using NCCA .................................... 127

Figure 2-9 Corneal confocal microscopy ............................................................. 128

Figure 2-10 Positioning of the TomoCap® on the cornea ................................... 129

Figure 2-11 Image of the corneal subbasal nerves using corneal confocal

microscopy .......................................................................................................... 129

Figure 2-12 A preview of an image from the corneal subbasal nerves on the

Heidelberg eye explorer. ..................................................................................... 130

Figure 2-13 An CCM image of a control subject analysed to quantify corneal

subbasal nerve morphology in DSPN.. ............................................................... 131

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Figure 3-1a, b & c. CNFD, CNBD and CNFL and baseline and 2 year follow up.

............................................................................................................................ 157

Figure 3-2 a & b. CCM images of a subject with RNFD at baseline and 2 year

follow up .............................................................................................................. 158

Figure 4-1a, b & c. Skin biopsy images of IENF in Controls, T1DM and DSPN ........

............................................................................................................................ 182

Figure 4-2a, b & c. CCM images in Controls, T1DM and DSPN ......................... 182

Figure 4-3 Receiver-operated characteristic (ROC) curves, based on the analysis

of CNFD and IENFD in T1DM versus DSPN ...................................................... 189

Figure 5-1 Skin biopsies stained for PGP9.5. in a healthy control, LADA patient

and T2DM patient with neuropathy ..................................................................... 214

Figure 5-2 Corneal confocal microscopy in a healthy control, LADA patient and

T2DM patient with neuropathy ............................................................................ 215

Figure 6-1 Histogram of 25(OH)D Status in subjects with diabetes .................... 237

Figure 6-2 Relationship between serum 25(OH)D and high-density lipoprotein

cholesterol (HDL) in subjects with diabetes ........................................................ 239

Figure 7-1a, b, c Box and Whisker plots for 25(OH)D pre- and post-intervention in

ECC, CCC and CC subdividing into <15ng/ml and >15ng/ml .................... 260 - 262

Figure 8-1 Graph showing 25(OH)D levels in ng/ml in C, DPN and PDN ........... 289

Figure 9-1 Frequencies of retinopathy, maculopathy and photocoagulation scarring

categorised by 25(OH)D status ........................................................................... 318

Figure 12-1Patient information sheet .................................................................. 404

Figure 12-2 Patient consent form ........................................................................ 407

Figure 12-3 Physical measurements and peripheral neuropathy assessment forms

............................................................................................................................ 408

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Figure 12-4 Ophthalmic examination sheet ........................................................ 410

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The University of Manchester

ABSTRACT OF THESIS submitted by Uazman Alam for the degree of Doctor of

Philosophy and entitled Vitamin D and Diabetic Neuropathy

September 2013

ABSTRACT

The accurate assessment of human diabetic somatic polyneuropathy (DSPN) is

important to define at risk patients, predict deterioration, and assess the efficacy of

pathogenetic treatments. Corneal confocal microscopy (CCM) has been proposed

as a surrogate endpoint for DSPN. Approximately 50% of patients with DSPN

experience neuropathic pain or symptoms and the underlying reasons are not

clearly elucidated. Vitamin D deficiency has been associated with diabetic

complications including DSPN and diabetic retinopathy (DR). However there is a

paucity of data regarding the interaction of vitamin D status with diabetic

complications.

This thesis shows that CCM can readily detect small fibre neuropathy prior to large

fibre involvement and assess rapidly progressive nerve fibre loss prior to

conventional thermal threshold testing. CCM has a superior diagnostic capabilities

compared to intra-epidermal nerve fibres and correlates better with nerve

conduction studies. Patients with LADA have a greater prevalence of small fibre

neuropathy compared to matched patients with type 2 diabetes. Vitamin D

deficiency is highly prevalent in patients with diabetes and despite relatively

aggressive replacement regimens are inadequate in raising vitamin D levels in a

significant proportion of patients. Vitamin D deficiency is not associated with DR

but there is a strong association between painful DSPN and vitamin D insufficiency

and more so with overt deficiency.

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DECLARATION

No portion of the work referred to in this thesis has been submitted in support of an

application for another degree or qualification of this or any other university or other

institute of learning.

COPYRIGHT STATEMENT

i. The author of this thesis (including any appendices and / or schedules to

this thesis) owns certain copyright or related rights in it (the “Copyright”) and he

has given The University of Manchester certain rights to use such Copyright,

including for administrative purposes.

ii. Copies of this thesis, either in full or in extracts and whether in hard or

electronic copy, may be made only in accordance with the Copyright, Designs and

Patents Act 1988 (as amended) and regulations written under it or, where

appropriate, in accordance with the licensing agreements which the University has

from time to time. This page must form part of any such copies made.

iii. The ownership of certain copyright, patents, designs, trademarks and other

intellectual property (the “Intellectual Property”) and any reproductions of copyright

works in this thesis, which may be described in this thesis, may not be owned by

the author and may be owned by third parties. Such Intellectual Property and

Reproductions cannot and must not be made available for use without the prior

written permission of the owner(s) of the relevant Intellectual Property and / or

Reproductions.

iv. Further information on the conditions under which disclosure, publication

and commercialisation of this thesis, the Copyright and any Intellectual Property

and / or Reproductions described in it may take place is available in the University

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IP Policy (see

http://www.campus.manchester.ac.uk/medialibrary/policies/intellectual-

property.pdf), in any relevant Thesis restriction declarations deposited in the

University Library, The University Library’s regulations (see

http://www.manchester.ac.uk/library/aboutus/regulations) and in the University’s

policy on presentation of Theses.

http://www.campus.manchester.ac.uk/medialibrary/policies/intellectual-property.pdf

http://www.campus.manchester.ac.uk/medialibrary/policies/intellectual-property.pdf

http://www.manchester.ac.uk/library/aboutus/regulations

15

CONTRIBUTION

This section is to confirm that Uazman Alam, the author of this thesis, was actively

involved and had a significant contribution in all chapters/studies presented and

discussed in this thesis. Briefly, he recruited the vast majority of subjects with type

1 diabetes and latent autoimmune diabetes, a large portion of control subjects and

a smaller portion of subjects with type 2 diabetes. He consented subjects,

performed peripheral neuropathy assessments and undertook skin biopsies in the

majority of subjects in the included studies. He also partook in a portion of

laboratory skin biopsy processing and undertook intra-epidermal nerve fibre

analysis for all controls, subject with type 1 diabetes and latent autoimmune

diabetes as the dual examiner (along with Dr Maria Jeziorska). For chapters 4, 5

and 7, which are analyses of vitamin D data, he designed the studies and

undertook data extraction with co-researchers. He performed all of the statistical

analyses in this thesis with knowledge gained through an MSc in Population

Health, postgraduate certificate in statistics for clinical trials and masters level

professional development modules in advanced epidemiology. Finally, he has

written all the chapters of this thesis which have been reviewed by his supervisor,

Professor Rayaz Malik. The following tasks were performed by other members of

the research team:

Electrodiagnostic studies by Dr Andrew Marshall, consultant

neurophysiologist.

Ophthalmic examinations were performed by Dr Ioannis Petropulos, Dr.

Mitra Tavakoli and Maryam Ferdousi.

Peripheral neuropathy assessments and skin biopsies were also performed

by Dr Omar Asghar and Dr Hassan Fadavi.

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Skin biopsy processing was undertaken by Dr Maria Jeziorska, Simon

Forman, Louisa Nelson, Aisha Meskiri, and Wendy Jones.

Skin biopsy analyses were also Dr Maria Jeziorska and Professor Rayaz

Malik.

Patient recruitment was also conducted by Georgios Ponirakis, Professor

Rayaz Malik and Dr Hassan Fadavi.

Software engineering to develop image analysis was undertaken by Dr

Mohammad A Dabbah, Dr Xin Chen and Dr James Graham.

Blood and Urine sample collections and anthropometric measurements by

the nursing staff in the Wellcome Trust Clinical Research Facility.

Haematology, immunology and clinical biochemistry analysis was performed

and reported by the relevant departments under the directorate of Laboratory

Medicine, Central Manchester University Hospitals, NHS foundation trust, UK.

Vitamin D laboratory analyses were undertaken by the vitamin D laboratory

at the Central Manchester Foundation Trust under the guidance of Dr Jacqueline

Berry.

Co-researchers assisting with trial design and data extraction on chapters 4,

5 and 7 were Professor J Kennedy Cruickshank, Dr Sara Al-Himdani, Dr Sophie

Benoliel, Dr Yasar Amjad, Dr Agnes Chan, Dr Ravinder Jugdey and Dr Osman

Najam.

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ALTERNATIVE THESIS FORMAT

The author has been granted permission to submit this Ph.D. thesis in an

alternative format by his supervisor Professor Rayaz A. Malik approved under the

University of Manchester, Faculty of Medical and Human Sciences regulations,

including sections which are in a format suitable for submission for publication or

dissemination. The following chapters in this thesis have been published or will be

submitted for publication:

- Chapter 3: To be submitted for publication.



- Chapter 6: Published in the journal Diabetic Medicine, 2012.

- Chapter 7: Accepted for publication in the Journal of Diabetes and its

Complications, 2013.

- Chapter 8: Submitted for publication in the journal Diabetes and is currently

being revised for resubmission, 2013.


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LIST OF ABBREVIATIONS

1,25(OH)2D: 1,25 vitamin D: 1,25 hydroxyvitamin D

25(OH)D: 25(OH) vitamin D: 25 hydroxyvitamin D

Ab: antibody

ACCORD: Action to Control Cardiovascular Risk in Diabetes

ACR: albumin creatinine ratio

AE: adverse event

AGE: advanced glycation end products

ALADIN: Alpha Lipoic in Diabetic Neuropathy

Alb: Albumin

ALP: Alkaline phosphatise

BDR: background diabetic retinopathy

BMI: body mass index

BP: blood pressure

CCa2+: corrected calcium

CCM: corneal confocal microscopy

CI: confidence interval

CNBD: corneal nerve branch density

CNFD: corneal nerve fibre density

CNFL: corneal nerve fibre length

CNFT: corneal nerve tortuosity coefficient

CNS: central nervous system

CBC: complete blood count

CKD: chronic kidney disease

CRGP: calcitonin gene-related peptide

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CST: cold sensation threshold

CVA: Cerebrovascular accident

CVD: cardiovascular disease

DCCT: diabetes control and complications trial

DD: disc diameter

DM: diabetes mellitus

DNS: Diabetic Neuropathy Symptom Score

DPN: diabetic peripheral neuropathy

DR: diabetic retinopathy

DSPN: diabetic somatic polyneuropathy

EDIC: Epidemiology of Diabetes Interventions and Complications

eGFR: estimated Glomerular Filtration Rate

eNOS: endothelial nitric oxide synthase

fMRI: functional magnetic resonance imaging

GAD: anti-glutamic acid decarboxylase

HbA1c: glycated haemoglobin A1c

HDL: high density lipoprotein cholesterol

ICA: islet cell antibodies

IDF: International Diabetes Federation

IENF: intra-epidermal nerve fibre

IENFD: intra-epidermal nerve fibre density

IFN: interferon

IHD: ishaemic heart disease

IQR: interquartile range

IU: international units

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LADA: latent autoimmune diabetes in adults

LANDMARK: Longitudinal Assessment of Opthalmic Diabetic Neuropathy

LDL: low density lipoprotein cholesterol

LFT: liver function tests

LURIC study: ludwigshafen risk and cardiovascular health study

MI: myocardial infarction

McGill VAS: McGill visual analogue score

NBF: nerve blood flow

NCCA: non-contact corneal aesthiometer

NCS: nerve conduction studies

NCV: nerve conduction velocity

NDR: no diabetic retinopathy

NDS: neuropathy disability score

NGF: nerve growth factor

NGSP: National Glycohaemoglobin Standardization Program

NHANES: National Health and Nutrition Examination study

NSP: neuropathy symptom profile

NCS: nerve conduction study

OGTT: oral glucose tolerance test

OR: odds ratio

PDR: proliferative diabetic retinopathy

PPDR: pre-proliferative diabetic retinopathy

PKC: protein kinase C

PMNCV: peroneal motor nerve conduction velocity

PMNamp: peroneal motor nerve amplitude

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PVD: peripheral vascular disease

ROC: receiver operating characteristic

QST: quantitative sensory testing

RAGE: receptor for AGE

RAS: renin angiotensin aldosterone system

RCT: randomised clinical trial

RNFD: rapid nerve fibre decline

SD: standard deviation

SSNCV: sural sensory nerve conduction velocity

SSNAmp: sural sensory nerve amplitude

TGF-β1: transforming growth factor β1

TRIG: triglycerides

TRPV1: transient receptor potential channel V1

T-CHL: total cholesterol

UE: urea and electrolytes

VAS: visual analogue score

RDA: recommended daily allowance

UKPDS: United Kingdom prospective diabetes study

VDR: vitamin D receptor

VEGF: vascular endothelial growth factor

VITAL: Vitamin D and Omega-3 Trial

VPT: vibration perception threshold

WST: warm sensation threshold

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In the name of Allah, the beneficent, the merciful.

La-ilaha-iLLaLLah- Muhammadur-Rasulullah.

“There is no wealth like knowledge, no poverty like ignorance”.

“There is no knowledge and science like pondering and thought; and there is

no prosperity and advancement like knowledge and science”.

Hazraat Ali bin Abu-Talib (RA), Fourth Caliph of Islam

DEDICATION

This Thesis is dedicated to my beloved mother,

my first and most important teacher.

She continues to be the most inspirational person in my life.

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ACKNOWLEDGEMENTS

Even before I decided to practice medicine and become a physician, I always had

a clear aspiration to undertake research. The completion of this PhD has brought

together a number of years of work which started well before this project

commenced. I have thoroughly enjoyed my time undertaking this novel work. It

would not have been possible without the support of many individuals.

First and foremost, I would like to thank Professor Rayaz Malik who has been my

friend and mentor for over ten years. He has guided me from a fourth year medical

student working on peripheral nerve histology to my current role as a SpR in

endocrinology and on the cusp of completing a PhD. He has been a continual

source of personal encouragement and has a enthusiastic passion for research

and clinical excellence which he projects on all his co-workers and students. A

special mention must be given to Dr Kashif Khawaja who took an interest in me as

a medical student on a paediatric placement when I said ‘I am interested in

learning research’ and introduced Professor Malik and I in 2002.

My co-supervisor Dr Maria Jeziorska has spent much time and effort in providing

me with skills of skin biopsy processing and analysis. Her help with reviewing

chapters of the thesis and overall encouragement were invaluable. I would also like

to thank my advisor Professor Andrew J M Boulton for allowing me to gain early

research skills in the diabetic foot at the Diabetes Research Institute, University of

Miami whilst an undergraduate medical student and for all his advice and support

during the PhD.

Drs Ioannis Petropoulos, Omar Asghar, Hassan Fadavi, Maryam Ferdousi, Mitra

Tavakoli, Andrew Marshall, Ravinder Jugdey, Georgios Ponirakis, Osman Najam,

Yasar Amjad, Agnes Chan, Sara Al-Himdani, Sophie Benoliel, Cristiano Van Zeller,

24

and April Buazon have provided significant support to the series of studies

presented and I am indebted for their efforts in this work. Dr Ioannis Petropoulos

and Dr Salik Kakar require further mention, they have both provided me with

detailed technical support of Microsoft office© Word.

Both my family and close friends were very important in bringing all this together. I

must show an immense amount of gratitude to my wife, Iram who is expectant with

our first child at the time of writing this thesis. She has encouraged me throughout

the writing process and has been very patient and understanding of my work

commitments, particularly as I have combined the thesis writing with working as an

SpR. I would also like to thank my parents, my brother Kashif and two sisters,

Afshaan and Saima, eldest niece Marihah and my nephew, Danyaal for all their

support during this PhD and the many previous years.

I would like to acknowledge the significant contribution of all of the subjects who

participated in this research and without their efforts none of this work would be

possible. The support of the nursing staff at the Wellcome Trust Clinical Research

Facility was of course invaluable. The Juvenile Diabetes Research Foundation and

the National Institute of Health must be generously acknowledged for their financial

and honourable commitment to these projects. Finally, I want to thank my close

friends Ravinder Jugdey and Mohammed Qadir Hussain for their continual

personal support and guidance.

Addendum to the Acknowledgements

My daughter, Inara, was born on Sunday 20th October 2013. I hope her life is full of

happiness (with Allah’s will); she has brought immense happiness to my wife and I

and my extended family.

25

PREFACE

Uazman Alam graduated with a BSc in Medical Science from the University of St.

Andrews, Scotland, UK in 2001, before he went on to the University of Manchester,

UK in 2001 to undertake Medicine (MBChB) and graduated from this in 2005. Part

way through studying Medicine he took a year out to study for an MSc in

Population Health (MPHe) in 2003 until 2004 and graduated from this in 2005. He

has also completed other academic courses in Advanced Epidemiology and he has

advanced his knowledge of statistics by completing a Post-Graduate Certificate in

Statistics for Clinical Trials. Uazman has a plethora of work based clinical and

research experience as well as experience of academic teaching. He has been

actively involved in research from a fourth year medical student. The genesis of

Uazman’s research education began working on peripheral nerve histology in

experimental models of diabetes under the auspices of Professor Rayaz Malik.

Uazman worked as a ‘houseman’ at the Pennine Acute Hospital NHS Trust in 2005

and rotated in A&E, chest medicine and general surgery. He successfully applied

for a prestigious academic FY2 post at the North West Lung Centre, Wythenshawe

Hospital and was involved in a randomised controlled trial on cough and

inflammation which involved clinical assessment of subjects, undertaking flow

volume loops, body plethysmography and laboratory analysis of sputum. From

there, Uazman went on to complete his senior house officer training in South

Yorkshire at Rotherham General Hospital and the Northern General Hospital

(2007-2009). He is currently an SpR (ST5) in Endocrinology and Diabetes at

Pennine Acute Hospital Trust.

Uazman’s interest in Diabetes and Endocrinology has its foundations as a 4th year

medical student in 2003 working with Professor Rayaz Malik on animal models of

26

peripheral nerve histology and during his elective period at the Diabetes Research

Institute, University of Miami in 2005. Uazman became involved in diabetes during

this period of time and then advanced his interest in this speciality in 2009 when he

was awarded a National Training Number in Endocrinology and Diabetes and the

same year he took time out of programme as a Clinical Research Fellow to

undertake a PhD. He undertook both clinical work and research at the Manchester

Diabetes Centre, Manchester Royal Infirmary and Centre for Endocrinology and

Diabetes, University of Manchester. The three years he spent working and

researching at these latter two locations have allowed him to conduct research and

extrapolate data for his PhD focusing on Longitudinal Assessment of Ophthalmic

Diabetic Neuropathy Markers (LANDMARK) study and the role of vitamin D in

diabetes under the supervision of Professor Rayaz A Malik.

During his PhD, he presented his work at regional, national and international

conferences between 2010 and 2013 including the American Diabetes Association,

NeuroDiab - Diabetic Neuropathy Study Group of the EASD, Diabetes UK and at

the International Diabetes Federation’s World Diabetes Congress.

Uazman is a member of the Royal College of Physicians and a council member for

Lipids, Metabolism and Vascular risk section of the Royal Society of Medicine. He

is also an examiner for the University of Manchester Medical School and has been

the assistant lead examiner for the final MB exams from 2010-2012 at the

Manchester Royal Infirmary.

27

LIST OF PUBLICATIONS

Research Publications

1. Alam U, Chan AWS, Buazon A, Van Zeller C, Berry JL, Jugdey RS, Asghar

O, Cruickshank JK, Petropoulos IN, Malik RA. Differential effects of different

vitamin D replacement strategies in patients with diabetes. J Diabetes

Complications 2013; [Epub ahead of print]

2. Petropoulos IN, Alam U, Fadavi H, Asghar O, Green P, Ponirakis G,

Marshall A, Boulton AJ, Tavakoli M, Malik RA: Corneal Nerve Loss Detected With

Corneal Confocal Microscopy Is Symmetrical and Related to the Severity of

Diabetic Polyneuropathy. Diabetes Care 2013; [Epub ahead of print]

3. Tavakoli M, Mitu-Pretorian M, Petropoulos IN, Fadavi H, Asghar O, Alam U,

Ponirakis G, Jeziorska M, Marshall A, Efron N, Boulton AJ, Augustine T, Malik RA:

Corneal confocal microscopy detects early nerve regeneration in diabetic

neuropathy after simultaneous pancreas and kidney transplantation. Diabetes

2013;62:254-260

4. Petropoulos IN, Manzoor T, Morgan P, Fadavi H, Asghar O, Alam U,

Ponirakis G, Dabbah MA, Chen X, Graham J, Tavakoli M, Malik RA: Repeatability

of in vivo corneal confocal microscopy to quantify corneal nerve morphology.

Cornea 2013;32:e83-89

5. Alam U, Najam O, Al-Himdani S, Benoliel S, Jinadev P, Berry JL, Kew M,

Asghar O, Petropoulos IN, Malik RA: Marked vitamin D deficiency in patients with

diabetes in the UK: ethnic and seasonal differences and an association with

dyslipidaemia. Diabetic Medicine 2012;29:1343-1345

28

6. Marsden PA, Smith JA, Kelsall AA, Owen E, Naylor JR, Webster D, Sumner

H, Alam U, McGuinness K, Woodcock AA: A comparison of objective and

subjective measures of cough in asthma. J Allergy Clin Immunol 2008;122:903-907

Review Publications

1. Asghar O, Alam U, Hayat SA, Aghamohammadzadeh R, Heagerty AM,

Malik RA: Obesity, diabetes and atrial fibrillation; epidemiology, mechanisms and

interventions. Curr Cardiol Rev 2012;8:253-264

2. Alam U, Asghar O, Malik RA: Diabetic gastroparesis: Therapeutic options.

Diabetes Ther 2010;1:32-43

3. Tavakoli M, Asghar O, Alam U, Petropoulos IN, Fadavi H, Malik RA: Novel

insights on diagnosis, cause and treatment of diabetic neuropathy: focus on painful

diabetic neuropathy. Ther Adv Endocrinol Metab 2010;1:69-88

4. Rogers LC, Alam U, Tesfaye S, Malik RA. Treatment of painful diabetic

neuropathy: A review of the most efficacious pharmacological treatments. Practical

Diabetes International 2004;7(21):301-306

Editorials

1. Alam U, Asghar O, Malik RA: Are vitamin D and B deficiency relevant to the

pathogenesis and treatment of diabetic neuropathy? Future Neurology 2012;7:235-

238

2. Alam U, Asghar O, Malik RA: Vitamin D deficiency and cardiovascular

disease: the missing link. Diabetes Management 2011;1:151-155

3. Alam U, Malik RA. New NICE guidance for DPNP: Evidence based and cost

effective. The Diab Foot J 2010;13(2):56.

29

LIST OF ABSTRACTS

1. Alam U, Petropoulos IN, Fadavi H, Asghar O, Marshall A, Ponirakis G, Al-

Ahmar A, Kheyami A, Ferdousi M, Azmi S, Tavakoli M, Boulton AJM, Malik RA.

Corneal Confocal Microscopy is as Proficient as Electrophysiology and Skin Biopsy

in Detecting Neuropathy in Subjects with Type 1 Diabetes Mellitus. American

Diabetes Association conference 21th-25th June 2013. Chicago, USA (Oral)

2. Azmi S, Alam U, Fadavi H, Asghar O, Petropoulos IN, Ponirakis G, Marshall

A, Ferdousi M, Kheyami A, Al-Ahmar A, Tavakoli M, Boulton AJM, Malik RA.Early

Non-Progressive Small Fibre Neuropathy in Type 2 Diabetes. American Diabetes

Association conference 21th-25th June 2013. Chicago, USA (Poster)

3. Pritchard N, Edwards K, Vagenas D, Sampson G, Russell A, Petropoulos

IN, Alam U, Fadavi H, Asghar O, Ponirakis G, Marshall A, Tavakoli M, Malik RA,

Efron N. Corneal sensitivity and corneal confocal microscopy in a large cohort with

type 1 diabetes: the LANDmark study. Annual meeting of the Diabetic Neuropathy

Study Group of the EASD, 27th – 30th September 2012. Dresden, Germany (Oral

presentation)

4. Green P, Tavakoli M, Petropoulos IN, Marshall A, Alam U, Fadavi H,

Asghar O, Chan A, Malik RA. Neuropathy precedes retinopathy and nephropathy

in type 1 diabetes. Annual meeting of the Diabetic Neuropathy Study Group of the

EASD, 27th – 30th September 2012. Dresden, Germany (Poster presentation)

http://www.abstractsonline.com/Plan/ViewAbstract.aspx?sKey=e65210ee-64a7-4098-9d29-ef6907504715&cKey=d11d1cf2-7602-410d-872f-e49f5370e83d&mKey=%7b89918D6D-3018-4EA9-9D4F-711F98A7AE5D%7d

http://www.abstractsonline.com/Plan/ViewAbstract.aspx?sKey=e65210ee-64a7-4098-9d29-ef6907504715&cKey=d11d1cf2-7602-410d-872f-e49f5370e83d&mKey=%7b89918D6D-3018-4EA9-9D4F-711F98A7AE5D%7d

30

4. Alam U, Petropoulos IN, Asghar O, Fadavi H, Marshall A, Ponirakis G,

Boulton AJM, Tavakoli M, Malik RA. Small fibre dysfunction and vitamin D

deficiency may underlie the symptoms of painful neuropathy in subjects with IGT

and diabetes. Annual meeting of the Diabetic Neuropathy Study Group of the

EASD, 27th – 30th September 2012. Dresden, Germany (Oral presentation)

5. Ponirakis G, Petropoulos IN, Fadavi H, Alam U, Asghar O, Tavakoli M,

Malik RA. Diagnostic accuracy of the Neuropad response to assess small and

large fibre in diabetic peripheral neuropathy. Annual meeting of the Diabetic

Neuropathy Study Group of the EASD, 27th – 30th September 2012. Dresden,

Germany (Oral presentation)

6. Petropoulos IN, Ponirakis G, Green P, Chen X, Dabbah MA, Graham J,

Fadavi H, Asghar O, Alam U, Tavakoli M, Malik RA. Quantification of corneal

nerve fibre morphology using a novel automated algorithm. Annual meeting of the

Diabetic Neuropathy Study Group of the EASD, 27th – 30th September 2012.

Dresden, Germany (Poster presentation)

7. Petropoulos IN, Asghar O, Alam U, Fadavi H, Ponirakis G, Marshall A,

Tavakoli M, Boulton AJM, Malik RA. Corneal confocal microscopy detects and

tracks progression of neuropathy in subjects with impaired glucose tolerance.

Annual meeting of the Diabetic Neuropathy Study Group of the EASD, 27th – 30th

September 2012. Dresden, Germany (Oral presentation)

31

8. Buazon A, Alam U, Chan AWS, Petropoulos IN, Asghar O, Malik RA.

Endemic Vitamin D Deficiency: Inadequate Replacement Strategies. American

Diabetes Association conference 12th-18th June 2013. Philedelphia, USA (Poster)

9. Petropoulos IN, Chan AWS, Asghar O, Alam U, Malik RA. Vitamin D: a

novel risk factor for diabetic retinopathy. International Diabetes Federation World

Diabetes Congress, 4th – 8th December 2011. Dubai, United Aram Emirates (Poster

presentation)

10. Petropoulos IN, Asghar O, Fadavi H, Alam U, Ponirakis G, Marshall A,

Nelson L, Forman S, Jeziorska M, Dabbah MA, Tavakoli M, Malik RA. Corneal

confocal microscopy detects significant neuropathy in subjects with impaired

glucose tolerance. Annual meeting of the Diabetic Neuropathy Study Group of the

EASD, 8th – 11th September 2011. Porto, Portugal (Oral presentation)

11. Alam U, Fadavi H, Petropoulos IN, Ponirakis G, Asghar O, Marshall A,

Tavakoli M, Boulton AJM, Malik RA. Protection from Neuropathy: lessons from

Patients with Long Duration Type 1 Diabetes. American Diabetes Association 24th-

28th June 2011. San Diego, USA. (Poster presentation)

12. Alam U, Petropoulos IN, Fadavi H, Jinadev P, Asghar O, Ponirakis G,

Boulton AJM, Tavakoli M, Malik RA. Corneal confocal microscopy detects

neuropathy in subjects with latent autoimmune diabetes in adults. Diabetes UK,

30th March-1st April 2011. London, UK. (Poster presentation)

32

13. Asghar O, Petropoulos IN, Alam U, Fadavi H, Ponirakis G, Dabbah MA,

Marshall A, Tavakoli M, Boulton AJM, Malik RA. Corneal confocal microscopy

detects significant neuropathy in subjects with impaired glucose tolerance.

Diabetes UK, 30th-1st April 2011. London, UK. (Oral & Poster presentation)

14. Petropoulos IN, Fadavi H, Asghar O, Alam U, Ponirakis G, Dabbah M,

Marshall A, Tavakoli M, Boulton AJM, Malik RA. Corneal Confocal Microscopy

Detects minimal small nerve fibre regeneration following combined pancreas and

kidney transplantation in patients with Type 1 diabetes. Diabetes UK, 30th March-

1st April 2011. London, UK. (Poster presentation)

15. Najam O, Alam U, Petropoulos IN, Asghar O, Malik RA. Lack of placebo

worsening in trials of human diabetic neuropathy. 20th Annual meeting of the

Diabetic Neuropathy Study Group of the EASD, 17th – 19th September 2010.

Stockholm, Sweden (Oral presentation)

16. Petropoulos IN, Fadavi H, Asghar O, Alam U, Ponirakis G, Dabbah MA,

Tavakoli M, Boulton AJM, Malik RA. Corneal confocal microscopy detects minimal

neuropathy in patients with impaired glucose tolerance which progresses with

diabetes. Annual meeting of the Diabetic Neuropathy Study Group of the EASD,

17th – 19th September 2010. Stockholm, Sweden (Oral presentation)

17. Petropoulos IN, Asghar O, Alam U, Fadavi H, Dabbah MA, Marshall A,

Tavakoli M, Boulton AJM, Malik RA. Corneal Confocal Microscopy Detects

33

Neuropathy in Subjects with Impaired Glucose Tolerance. American Diabetes

Association conference 25th-29th June 2010. Orlando, USA. (Poster presentation)

18. Alam U, Jinadev P, Asghar O, Petropoulos IN, Davies R, Rutter M, Boulton

AJM, Malik RA. Marked Vitamin D Deficiency in a General Diabetic Population.

Diabetes UK, 3rd-5th March 2010. Liverpool, UK. (Oral & Poster presentation)

19. Petropoulos IN, Manzoor T, Alam U, Fadavi H, Asghar O, Dabbah MA,

Tavakoli M, Boulton AJM, Malik RA. Corneal confocal microscopy demonstrates

significant neuropathy in south Asian patients with diabetes. Diabetes UK, 3rd- 5th

March 2010. Liverpool, UK. (Poster presentation)

20. Alam U, Kelsall A, Sumner H, Donnoley I, Thompson NC, Smith JA,

Woodcock A. Voluntary Coughing and Sputum Inflammatory Markers in Healthy

Volunteers. American Thoracic Society conference, 16th – 21st May 2008.

Toronto, Canada. (Poster)

21. Alam U, Siddique I, Patel R, Chung SK, Malik RA. Sensory nerve

morphology in nitric oxide synthase-knockout and diabetic mice: relevance to

diabetic neuropathy. International Diabetes Federation Congress, 24th – 29th Aug

2003. Paris, France. (Poster)

22. Alam U, Siddique I, Patel R, Wong AKC, Boulton AJM, Chung SK, Malik

RA. Sensory nerve morphology in nitric oxide synthase-knockout and diabetic

34

mice: relevance to diabetic neuropathy. Annual meeting of the Diabetic Neuropathy

Study Group of the EASD 30th Aug – 2nd Sept 2003. St Malo, France. (Oral)

23. Patel R, Alam U, Boulton AJM, Kador P, Malik RA. Endoneurial

microangiopathy without neuropathy or apoptosis in the long duration galactosemic

dog. Annual meeting of the Diabetic Neuropathy Study Group of the EASD 30th

Aug – 2nd Sept 2003. St Malo, France. (Oral)

35

1 Chapter I - INTRODUCTION

36

Diabetes Mellitus (DM) has been recognised for millennia and an Egyptian

manuscript described a condition as “too great emptying of the urine” as early as

1550 BC. Around the same time, Indian physicians identified the disease and

classified it as ‘madhumeha’ or honey urine noting that the urine would attract ants.

However, it was not until the 18th century that Western physicians began to study

diabetes and its complications (1). The islets of Langerhans were discovered in

1869 by the anatomist Paul Langerhan. The endocrine role of the pancreas and

the existence of insulin were not fully clarified until the epochal discovery in 1921

when Frederick Banting and George Best demonstrated they could reverse

induced diabetes in dogs, by extirpating the pancreas and then administrating an

extract from the pancreatic islets of Langerhans of healthy dogs (2-3).

DM is group of disorders which are characterised by hyperglycaemia due to an

absolute or relative deficit in insulin production or action (4). The chronic

hyperglycaemia of DM is associated with end-organ damage, dysfunction and

failure, including the retina, kidney, nerves, heart and blood vessels (4). The

International Diabetes Federation (IDF) estimates an overall prevalence of DM to

be 366 million in 2011, and this is expected to rise to 552 million by 2030 (5).

1.1 Aetiopathology of Diabetes

DM is a heterogeneous group of metabolic disorders fall into two broad categories

characterised by raised plasma glucose concentrations due to absolute (type 1

DM) or relative (type 2 DM) lack of insulin secretion, action or both and thus

resulting in aberrations in fat, carbohydrate and protein metabolism. Diagnosis may

be made through glucose tolerance test, fasting and/or random plasma glucose or

glycated haemoglobin A1c (HbA1c) (Table 1-1).

37

Table 1-1 Diagnosis of DM

Diagnostic Parameter Comment

Fasting Plasma Glucose

≥7.0 mmol/l

Fasting is regarded as no caloric intake for

8 hours

2 hour plasma glucose ≥11.1 mmol/l

during an OGTT

The test should be performed according to

the World Health Organisation guidelines,

using a glucose load containing the

equivalent of 75 g anhydrous glucose

dissolved in water.

In a patient with classic symptoms of

hyperglycaemia, a random plasma glucose

≥11.1 mmol/l

HbA1C ≥6.5% / 48mmol/mol

Its role in diagnosis has been cemented by

the American Diabetes Association,

although some consider there is a further

case for discussion in its precise role (6).

The test should be performed in a

laboratory using a method that is NGSP

Certified and Standardized to the DCCT

assay.

Adapted from Diagnosis and Classification of Diabetes Mellitus (7).

Table key

OGTT – Oral Glucose Tolerance Test.

DCCT – Diabetes Control and Complication Trial.

NGSP - National Glycohaemoglobin Standardization Program.

In type 1 DM, individuals are often metabolically normal before the disease is

clinically manifest, but the process of autoimmune β-cell destruction can be

detected before overt diabetes develops. The presence of this autoimmune

disorder is characterised by the presence of auto-antibodies such as anti-glutamic

acid decarboxylase (GAD), islet (ICA) or insulin antibodies (8). Type 1 DM is

38

associated with HLA DR3 and DR4 (9-11) and it has been proposed that an

interplay of a genetic predisposition with environmental factors may lead to an

autoimmune response (12). Environmental factors implicated include viral

infections, early childhood cow’s milk administration, malnutrition, and

vaccinations, which trigger an immune cascade leading to cross reactivity with

pancreatic β-cells, however to date no definitive causal link has been identified (12-

14). Furthermore, there is a putative link of vitamin D deficiency and the

occurrence of type 1 DM (15) with a positive impact of supplementation with

cholecalciferol (vitamin D3) during the first year of life on the risk of developing type

1 DM (16) and a meta-analysis of five observational studies has confirmed this risk

reduction (17). Importantly in the Finnish Birth cohort study (16), children with

suspected rickets had a threefold increase in their risk of type 1 DM. There is a

striking geographical variation in the incidence of type 1 diabetes and seasonal

variation with the highest incidence of onset during the winter months and at

greater latitudes; thus suggesting that variations in vitamin D synthesis in the skin

and subsequent status may play a significant role in the pathogenesis (18-20). The

destruction of pancreatic β-cells is an autoimmune T-helper cell mediated disorder

and activated vitamin D (1,25(OH)2D) has immunomodulatory actions in T-cells

and leads to a reduction in the induction of Th1 cytokines such as interferon (IFN) γ

(15; 21-23).

Type 2 DM has become an epidemic in Western society and virtually no health

care professional is without patients who have the disease. The majority of patients

with DM have type 2 DM (~90%) with only a small proportion having the

monogenic forms such as maturity onset diabetes of the young (24-25). Type 2 DM

results from defects in insulin secretion, but with a major contribution from insulin

39

resistance (7). There is subsequently an inadequate compensatory insulin

secretary response resulting in hyperglycaemia. Numerous mechanisms have

been implicated, including changes in fatty acid fractions, interplay of inflammatory

cytokines and adipokines, mitochondrial dysfunction with insulin resistance, and

glucotoxicity, lipotoxicity, and amyloid formation and deposition leading to β-cell

dysfunction (26). Type 2 DM has a strong genetic component, however, only a

small number of genes have been identified to date (26). A family history confers a

2-4 fold increase for type 2 DM. The lifetime risk of type 2 DM is approximately

38% and 60% if one or both parents have the disease respectively (27-28). This

genetic predisposition is highlighted by the fact that monozygotic twins have higher

concordance rates of type 2 DM than dizygotic twins (29-30). Vitamin D may play

an important role in the pathogenesis of type 2 DM. Insulin sensitivity is improved

significantly in adults with impaired fasting glucose who were randomised to

calcium and vitamin D supplementation (31). Furthermore, baseline 25(OH) vitamin

D levels in non-diabetic subjects were shown to predict future glycaemia and

insulin resistance (32). In the meta-analysis by Parker et all (33), the risk of type 2

diabetes may be reduced by 55% in individuals with the highest levels of vitamin D.

Despite the majority of cases of diabetes falling into the two broad

aetiopathogenetic categories of type 1 and type 2 DM often classified on their

clinical presentation. In some individuals this classification is not so well defined. It

is common practice to classify individuals based on the following variables:

1. Age at onset of diabetes

2. The abruptness of hyperglycaemia

3. Presence of ketosis

40

4. Degree of obesity

5. Need for insulin at diagnosis

This however, does not always accurately classify the aetiology of diabetes.

1.2 Latent Autoimmune Diabetes in Adults (LADA)

Latent Autoimmune Diabetes in Adults (LADA) is an under diagnosed form of

diabetes. Particularly, as the age at onset is later than in classical type 1 diabetes.

The finding of auto antibodies is characteristic of type 1 DM but can be found in 10-

30% of those who have phenotypically type 2 DM (34-35). It is this subset that is

termed as LADA. Furthermore, the U.K. prospective diabetes study (UKPDS)

demonstrated that approximately 10% of adults with type 2 diabetes have LADA

(36). Although the prevalence may be as low as 3.6% of all newly diagnosed

apparent type 2 diabetic patients (37). Despite the initial lack of requirement of

exogenous insulin the underlying process is that of β-cell destruction as opposed

to the insulin resistance of type 2 DM, it does however occur slower than the

classical young onset type 1 DM (38).

1.2.1 Screening and Treatment in LADA

Insulin dependency can occur after a relatively short period of around 6 months or

up to 10 years or more (39-40). The time to insulin therapy use can vary depending

on local clinical judgement and use of laboratory GAD antibody testing (41).

However, despite the variances, the median time to failure of oral medication and

exogenous insulin use is less than 5 years (41). In fact, a clinical screening tool

has been developed which can help to identify individuals with LADA (42). These

criteria are based upon the clinical presentation of this subgroup of individuals. The

five clinical features that are more common in LADA compared to type 2 DM are:

41

a. Age of onset less than 50 years

b. Acute symptoms (polyuria, polydipsia, osmotic visual changes etc)

c. BMI ≤25kg/m2

d. Personal history of autoimmune disorders

e. Family history of autoimmune disease

In one particular study (42) the presence of at least two of these distinguishing

features (clinical risk score ≥2) had a 90% sensitivity and 71% specificity for

identifying LADA. The negative predictive value for a LADA clinical risk score ≤1

was 99% (42). The most common diagnostic test for LADA is a GAD antibody

assay (43). GAD antibodies may also be present in individuals with type 1 DM and

thus generally an initial exogenous insulin free period is often considered a

requirement for a definitive diagnosis of LADA. GAD antibodies are not found in

patients with pure type 2 DM (37).

Individuals with LADA may require insulin at an earlier in the disease compared to

those with type 2 DM. The slow process of β-cell destruction occurs to the point

that oral agents poorly control hyperglycaemia. However, a number of interventions

have been trialled to evaluate the possible beneficial effects of different classes of

diabetes medications (44). There are a paucity of data on the effects of oral agents

alone in LADA. Sulphonylurea treatments work by stimulating the β-cells to

produce endogenous insulin. This class of medication has been associated with a

more rapid progression to insulin treatment in those who are well controlled at

baseline compared to other treatment groups (44). It is possible that β-cell failure is

accelerated through the action of sulphonylureas. Furthermore, in the systematic

42

review by Brophy et al (44) individuals who were poorly controlled at baseline

progressed to insulin treatment rapidly (60% in 2 years). There may be some

beneficial effect on β-cell preservation in those with LADA who are treated with the

now withdrawn rosiglitazone (when compared to sulphonylureas) and this may

highlight the possibility that there is a degree of insulin resistance despite the

autoimmune nature of the disorder (45-46). Other interventions have also been

shown to have a protective effect on β-cell function. Li et al (47) undertook a

randomised trial of insulin versus 1-α-hydroxyvitamin D3 plus insulin and in a

subgroup of those with a duration of diabetes less than 1 year they showed a

significantly elevated fasting c-peptide and postprandial c-peptide in those who

were treated with insulin and 1-α-hydroxyvitamin D3, suggesting that β-cell function

was better preserved. Indeed there is some evidence that vitamin D deficiency may

be related to type 1 DM (48). Furthermore, vitamin D deficiency and receptor gene

polymorphisms may be a risk factor for the development of type 1 DM and other

forms of diabetes that have an autoimmune element to their pathogenesis (49-51).

There is evidence to suggest that autoimmune diabetes presents a continuum of

genetic susceptibility, which extends from a strong effect in childhood-onset type 1

DM to a more limited effect in LADA (52). The predominant imbalanced production

of T helper 1 cells in part through the production of interleukin (IL) 12 in

autoimmune diabetes is destructive to the pancreatic β-cell (52). Activated vitamin

D may inhibit the production of IFN-γ, IL-2 and IL-12 (53) and thus possibly

disrupting the T helper 1 mediated pathogenesis.

This highlights a possible opportunity to preserve β-cell function as there is a

slower rate of β-cell decline than in the classical type 1 DM. Maximising β-cell

43

function through interventions may lead to better glycaemic control and hence

reduce the rate of microvascular complications (54).

1.2.2 Auto Antibodies in LADA

Generally a diagnosis of LADA is based upon a phenotypically type 2 individual

testing positive for GAD antibodies, as this is the most common antibody present

in this cohort (55). Even in the absence of GAD antibodies some individuals may

test positive for other β-cell auto-antibodies (56) conferring a diagnosis of LADA.

Furthermore, other autoimmune markers such as ICA, and tyrosine phosphatase

auto-antibodies (anti-IA2 or anti-ICA512) have been found in LADA. A combination

of ICA and GAD antibodies and the levels of their titres have been shown by

Lohmann et al 2001 (57) to help differentiate two clinical and phenotypical variants

of LADA. Those who are ICA positive and have high titres of GAD antibodies are

clinically more similar to individuals with type 1 diabetes whereas those who are

negative for ICA and have low titres of GAD antibodies are clinically similar to

individuals with type 2 diabetes (57). The levels of GAD antibody titres are not

thought to predict disease progression (58) but a combination of thyroid peroxidase

antibodies (which one study occurred at a possible prevalence of 29% (59)), anti-

IA2 and C-peptide levels may predict β-cell failure (60).

Previous studies have shown co-existing coeliac disease in those with type1 DM

with a rate of between 1-7% (61-62) and anti-gliadin antibodies may have a high

prevalence in those with type 1 DM even without symptoms of coeliac disease

(63). It seems that there is an increased frequency of humoral markers of

autoimmunity and this is also mirrored in LADA (64). A number of non β-cell

antibodies are associated with LADA and include anti-gliadin and thyroid

44

peroxidase antibodies (64). It has been suggested that a susceptibility to anti-

gliadin antibodies may reflect changes in the mucosal barrier function and thus

cause changes in antigen exposure (64).

Defining LADA through testing for auto-antibodies itself can pose a significant

diagnostic challenge as these markers can change over time including absence of

ICA and GAD antibodies after the presenting illness. Defining cut off levels of these

antibodies for diagnosis is troublesome as there is a continuous spectrum of

distribution in both diabetic and control populations. This also highlights the

possibility of false positives, as these markers may be present in non-diabetic

populations.

1.2.3 Microvascular complications in LADA

Historically, complications in LADA have been poorly studied. Baum et al (65),

compared patients with LADA (n=14) versus type 1 DM (n=9) and type 2 DM

(n=14) through clinical assessment, quantitative sensory testing (QST) and

electrophysiology. Individuals with LADA had fewer features of diabetic neuropathy

in particular DSPN in the early stages of the disease and were similar to the

classical type 1 DM individuals who develop neuropathy at a later stage after

diagnosis (65). In an epidemiological study (Freemantle study), increased

prevalence of retinopathy compared with GAD negative controls with type 2

diabetes which was primarily attributable to poorer glycaemic control (66). All the

studies relating to prevalence of LADA have a common limitation of small study

size samples particularly for epidemiological studies. The LADA cohort requires

further investigation with regard to the microvascular complications so that future

care can be tailored to this group.

45

1.3 Complications of Diabetes

Diabetes is a major risk factor for macro and micro-vascular complications, such as

strokes and amputations, nephropathy, retinopathy and neuropathy. These

complications are a major cause of illness and an enormous economic burden

especially with the increasing prevalence of diabetes in western society (67).

Diabetes is a leading cause of end-stage renal disease in many developed

countries accounting for up to 50% of patients receiving renal replacement therapy

(67). Kidney disease is common at the time of diagnosis of DM with a prevalence

of ~20% (68) but also accounts for 21% of deaths in type 1 DM and 11% of deaths

in type 2 DM (69). Within 20 years after disease onset, nearly all adults with type 1

DM and more than 60% of adults with type 2 DM develop the complication of DR

(70-71). In an analysis in the UK, it is believed that there is a prevalence of

blindness of 4200 and an annual incidence of blindness of 1280 people with

diabetes (72) and in a retrospective analysis (n=20,686) from 1990-2006 of

national diabetic retinopathy screening program, the first retinal examination

(baseline), 79% did not have retinopathy, 18% had non-proliferative retinopathy,

and 2.9% had pre-proliferative retinopathy (73). In this same study after 10 years,

patients without retinopathy at baseline had an increased incidence of pre-

proliferative retinopathy (cumulative incidence 16.4%), sight-threatening

maculopathy (1.2%), or proliferative retinopathy (1.5%) (73). In Wisconsin

Epidemiologic Study, the 14-year rate of progression of retinopathy was 86%,

regression of retinopathy was 17%, progression to proliferative retinopathy was

37% and incidence of macular oedema was 26% (74). Diabetic neuropathy is

discussed in more detail below.

46

Diabetic patients from ethnic minorities have higher mortality rates and higher risk

of diabetes complications (75). In the study by Lanting et al (75), additional

adjustment for risk factors in most instances ethnic differences disappear.

However, blacks and Hispanics in the U.S. and Asians in the U.K. have an

increased risk of end-stage renal disease, and blacks and Hispanics in the U.S.

have an increased risk of retinopathy (75). In terms of neuropathy, there appears to

be a tendency for white individuals to have a higher prevalence of sensorimotor

neuropathy than the groups they were compared (76). In a community based study

in the U.K., painful DSPN was more prevalent in patients with type 2 diabetes,

women, and people of South Asian origin despite a lower prevalence of neuropathy

in the latter group (77).

1.3.1 Diabetic Neuropathy

The complication of neuropathy has been well documented since the end of the

19th century, when a physician by the name of Purdy concluded: ‘It is rare to meet

with a case of diabetes in which there is not more or less nervous disturbance’

(78). Since those early days of investigation there has been extensive research of

diabetic neuropathies. The most common presentation of neuropathy in diabetes is

a DSPN and is considered to be the presence of symptoms and/or signs of

peripheral nerve dysfunction in people with diabetes after the exclusion of other

causes. DSPN is a symmetrical, length-dependent sensorimotor polyneuropathy

attributable to metabolic and microvessel alterations as a result of chronic

hyperglycaemia exposure and cardiovascular risk covariates (79). The occurrences

of diabetic retinopathy and nephropathy in a given patient strengthen the case that

the polyneuropathy is attributable to diabetes (79). The prevalence of DSPN in

47

diabetes can be as high as 50% (80) and symptomatic diabetic neuropathy can

affect up to 30-40% of diabetic patients with neuropathy (81). Traditionally

symptoms have been denoted as either positive (aching, burning, sharp or

pressure pains) or negative (numbness or dead feeling). Both ‘negative and

positive symptoms’ may coexist.

1.3.1.1 Prevalence of Distal Symmetrical Polyneuropathy

The prevalence of DSPN is thought to be around 30% (80; 82) and increases with

the duration of the disease. When diabetes has been present for greater than 25

years, the prevalence rises to approximately 50% (82). The Rochester Diabetic

Neuropathy Study reported a prevalence of 54% and 45% for patients with type 1

and type 2 DM respectively, amongst 380 patients in total, with the majority

however not exhibiting neuropathic symptoms (83). The 1989 National Health

Interview Survey consisted of a representative sample of 84,572 persons in the

U.S.A. aged 18 years or over (84). Those with DM (n=2405) were identified and

administered a questionnaire to define the prevalence of symptoms of sensory

neuropathy which was found to be 30.2% among subjects with type 1 DM. This

prevalence was 36.0% for men and 39.8% for women with type 2 DM, compared

with 9.8 and 11.8% for non-diabetic men and women, respectively (84). Symptoms

of sensory neuropathy affect 30-40% of diabetic patients and the prevalence of

these symptoms increase with longer duration of diabetes; hypertension and

hyperglycaemia (84). However, importantly there was neither a clinical or

quantitative assessment of neuropathy in this study. In a survey of 10 English

practices based in the community (85), 1077 subjects with diabetes were screened

for neuropathy by eliciting two of the following five:

48

1. Neuropathic foot symptoms

2. Loss of light touch sensation

3. Impaired pin prick sensation

4. Absent ankle jerk reflexes

5. Vibration perception threshold greater than 97.5% of an age-standardised

value.

There was an age and sex matched non-diabetic control group (n=480) (85). The

prevalence of neuropathy was 16.3% in diabetic patients and 2.9 % in non-diabetic

subjects, yielding a prevalence odds of 6.75 (95% CI 3.87-11.79), p<0.001 (85).

49

Table 1-2 Details of major epidemiological studies of DSPN

Table 1-2 Prevalence and risk factors for DSPN

Study Prevalence

(type of DM) Risk factors

Rochester Diabetic Neuropathy Study

(n=380) (83)

54% / 45%

(Type 1 (n=102) / Type 2 DM

(n=278))

Not reported

San Luis Valley Study (n=277)

(86-87) 26% (Type 2 DM)

Age, male sex, HbA1c,

duration of disease,

insulin use, lower

fasting C-peptide, DR,

diabetic nephropathy

Pittsburgh Epidemiology of Diabetes

Complications Study (n=363) (88-89)

34% (Type 1 DM)

Duration of diabetes,

HbA1c reduced HDL,

current smoking,

macrovascular disease

Dutch Population study (n=137) (90)

53-63% Dependent on

method of assessment (Type

2 DM)

Macrovascular disease

EURODIAB IDDM Complications

Study (n=3,250) (91) 28% (Type 1 DM)

Age, diabetes duration,

weight, HbA1c,

smoking status, severe

ketoacidosis,

microalbuminuria,

retinopathy

United Kingdom Prospective Diabetes

Study (cross sectional analysis, n=2,337)

(68; 92)

5-7% Dependent on method

of assessment (Type 2 DM) HbA1c, hypertension

National Health and Nutrition

Examination Survey (n=419) (93)

28.5% (Type 1 DM/Type 2

DM)

13.3% in Controls

Age, ethnicity

50

1.3.1.2 Diabetic Neuropathy, Glycaemia Control and other risk factors

The role of tight glycaemic control in improving microvascular outcomes is

irrefutable. The United Kingdom Prospective Diabetes Study (UKPDS)

demonstrated a significant risk reduction for cardiovascular disease (CVD) and

amputation for every 1% reduction in mean HbA1c (94). The Diabetes Control and

Complications Trial (DCCT) is another landmark study conducted in type 1 DM

(95). This prospective study evaluated 1441 subjects aged 13 to 39 years old who

were followed up for 5 years (96). 726 had type 1 DM for 1 to 5 years and had no

retinopathy at baseline (primary prevention cohort); 715 had type 1 DM for 1 to 15

years and had minimal to moderate non-proliferative retinopathy at baseline

(secondary intervention cohort) (96). Intensive therapy reduced the development of

confirmed clinical neuropathy by 64% in the combined cohorts after 5 years of

follow-up (96). This was defined as a history or physical examination consistent

with clinical neuropathy confirmed by either abnormal nerve conduction or

autonomic nervous system testing. The prevalence of abnormal nerve conduction

and abnormal autonomic nervous system function were reduced by 44% and 53%

respectively (96). The Epidemiology of Diabetes Interventions and Complications

(EDIC) Study (this assessed the 14-year cumulative benefits of prior intensive

insulin treatment in the DCCT) found a lower incidence of DSPN (22.0% vs.

28.0%) amongst patients assigned to the intensive arm compared to the

conventional arm (97). Therefore, the benefit of former intensive insulin treatment

persisted for 13-14 years after DCCT closeout (97). These data introduced the

concept of ‘metabolic memory’ in which provides significant evidence of a durable

effect of prior intensive treatment on neuropathy (98).

51

The Eurodiab study (91), assessed the risk factors for the development of distal

symmetric neuropathy in 1172 patients with type 1 DM. Neuropathy was assessed

at baseline (1989 to 1991) and at follow-up (1997 to 1999), with a follow-up of

7.3±0.6 years. A standardised protocol for the evaluation of DSPN included clinical

evaluation, quantitative sensory testing and autonomic-function tests. Serum lipids

and lipoproteins, HbA1c, and the urinary albumin excretion rate were also

measured. Duration of diabetes, HbA1c status, change in glycosylated

haemoglobin value during the follow-up period, body-mass index and smoking

were all independently associated with the incidence of neuropathy (91). Other

than glycaemic control, the incidence of neuropathy is associated with potentially

modifiable cardiovascular risk factors, including a raised triglyceride level, body-

mass index, smoking and hypertension (91).

1.3.2 Painful Diabetic Neuropathy

DSPN has an insidious onset and is often indicated by sensory symptoms that start

in the toes and then progress proximally to involve the feet and legs in a stocking

distribution. DSPN results in the loss of sensation, which may lead to foot

ulceration and eventual lower limb amputation, representing the commonest cause

of hospitalisation amongst patients with DM (99). Approximately 50% of patients

with DSPN experience neuropathic symptoms (100) and these symptoms include;

burning pain, electrical or stabbing sensations, paraesthesiae, hyperaesthesia and

a deep aching pain (Table 1.3).

52

Table 1-3 Abnormal sensations in DSPN

Sensation Description

Dysaesthesia Spontaneous pain

Allodynia Stimulus which does not usually evoke pain

Hyperalgesia Exaggerated pain

Paraesthesia Abnormal touch sensation

In a study conducted in the U.S.A., painful DSPN was associated with decrements

in many aspects of patients' lives: physical and emotional functioning and

significant sleep disturbance (101) with a greater negative impact in patients with

greater pain severity which has also been noted in other studies (100-101).

The precise reasons for the development of neuropathic pain despite loss of

sensation is not clearly elucidated but suggests that aberrations in pain signalling

in the peripheral and central nervous system occur (100). Capsaicin found in chilli

peppers activates transient receptor potential channel (TRP) V1 on the peripheral

nerve and topical application of capsaicin produces an analgesia effect at higher

doses (102-103) and polymorphisms in TRPV1 can explain some of the variation in

pain seen after neuropathy in patients (104). Alterations in ion channels have been

noted in damaged peripheral nerve (105). Changes in sodium and calcium channel

distribution and expression impact on the pattern of neuronal excitability (100) and

inherited pain disorders are known to occur due to mutations in sodium channel

NAV 1.7 (106). Voltage gated calcium channels induces release of

neurotransmitter and subsequent to peripheral nerve injury the α2δ subunit is

unregulated thus resulting in increased released of neurotransmitter such as

glutamate and substance P (100; 107). The α2δ subunit is the target of both

gabapentin and pregabalin and these drugs result a reduction in aberrant

neurotransmitter release and subsequent pain relief (106; 108). Aβ nerve fibre

53

sprouting into dorsal horn, and reduced inhibition via descending pathways (109)

together with axonal atrophy in peripheral nerves have been demonstrated in

patients with painful diabetic neuropathy (110). Painful diabetic neuropathy has

also been associated with significantly greater autonomic dysfunction than painless

DPN (111). Central nervous system (CNS) involvement also seems to be a

hallmark of DSPN but may actually be related to nocturnal exacerbations typically

noted by suffers which impairs sleep (100). A combination of central sensitisation,

changes in inhibition of descending pathways and increased thalamic vascularity

may all play a significant role in painful DSPN and the latter has been noted in

studies of functional magnetic resonance imaging (fMRI) (112-113).

Currently there are two licenced treatments for the treatment of painful DSPN

within the UK, namely pregabalin and duloxetine with amtriptylline also commonly

used particularly within primary care. There is no definitive accepted cut off for a

reduction in pain which is deemed clinically significant. However, an end-point of

50% improvement in pain relief is generally considered an adequate response to

an intervention considering a placebo response may be as great as 35% (114).

The treatment of painful DSPN remains inadequate and in recent studies in DSPN

have shown a placebo effect which has approached and in the study by Selvarajah

et al (115), a medicinal cannabis-based product even surpassed that of the active

therapy, obscuring the precise treatment effect of the active treatment (116). There

is an urgent need for novel therapeutic agents in the treatment of painful DSPN.

1.3.3 Pathogenesis of Diabetic Neuropathy

The pathogenesis of diabetic neuropathy is yet to be fully elucidated but is known

to involve metabolic effects mediated directly and indirectly by hyperglycemia,

54

resulting in oxidative stress, accelerated polyol pathway metabolism, and

generation of advanced glycation end-products (117-118). Both metabolic and

microvascular factors are considered to play important roles (119-120). Chronic

hyperglycaemia is a pivotal mediator. Even pre-diabetic or intermittent

hyperglycaemia may be related to painful neuropathy (121). In one study, 25% of

apparent idiopathic painful neuropathy and electrodiagnostic evidence of axonal

injury with loss of epidermal nerve fibres had IGT (122). Diabetic neuropathy is

initially a predominantly small fibre neuropathy which begins at the distal

extremities moving proximally with time (123). In fact, around 56% of those

presenting with the burning feet of idiopathic small fibre neuropathy have impaired

glucose tolerance (124). Many individuals with small fibre neuropathy will only

have mild, subclinical large fibre disease (125) and thus there is a cohort of those

with small fibre neuropathy not detectable with traditional investigations.

The Diabetes Control and Complications Trial (96) and the United Kingdom

Prospective Diabetes Study (126) have demonstrated that poor glycaemic control

is related to increased prevalence of neuropathy and other microvascular

complications. Improvement in glycaemic control may even prevent or slow

elements of diabetic neuropathy (96). The results of the DCCT (96) were

particularly important; this randomised, prospective study showed a significant

reduction in the development and progression of clinical neuropathy (64%), motor

nerve conduction velocity (44%) and autonomic dysfunction in type 1 diabetic

patients with optimal glycaemic control.

After many years of research the precise mechanism of hyperglycaemia leading to

diabetic neuropathy is still to be fully explained. The numerous pathological

changes are mediated through competing or parallel pathological pathways;

55

glucose-induced activation of protein kinase C isoforms; increased formation of

advanced-glycation end-products (AGE); and increased glucose flux through the

aldose reductase pathway (127). Brief accounts of the postulated pathways which

are the focus of therapeutic interventions are described below.

Over 80% of amputations follow a foot ulcer or injury (82), early recognition of at

risk individuals and appropriate foot care may result in a reduced incidence of

ulceration and consequently amputation. However, the study by Lincon et al (128)

has challenged this notion with an observer-blind, randomised controlled trial to

assess the effect of a foot care education programme in the secondary prevention

of foot ulcers. Intervention was associated with improved foot care behaviour,

however there was no evidence that a programme of targeted education was

associated with clinical benefit over and above usual care (128). The ever

increasing prevalence of DM and consequently lower limb amputation in the

western world (80) highlights the need to understand and accurately assess DSPN.

Figure 1.1 highlights the main mechanisms contributing to DSPN. Cellular factors

derived from the bone marrow may produce chimeric cells in peripheral nerves of

diabetic animals thus elicit nerve injury and cellular components from the bone

marrow have an influence on the nerve pathology in diabetes (129).

56

Figure 1-1 The pathogenesis of DSPN

Schematic adapted from Yagihashi et al (129)

1.3.3.1 Polyol Pathway

Polyol pathway (Figure 1.2) hyperactivity has been postulated as a mechanism

linking hyperglycaemia to neuropathy (130). Animal models have consistently

demonstrated an association between increased polyol pathway flux and a

reduction in nerve conduction velocity, which may be ameliorated with aldose

reductase inhibitors (131). It is thought that hyperglycaemia leads to sorbitol

accumulation in the peripheral nerve due to increased conversion from glucose, via

the enzyme aldose reductase. This is evidenced by elevated sorbitol in diabetic

nerves (132-133) however, it is now thought that this accumulation may actually

not be as important as previously considered (134). The high rate of flux through

the pathway may contribute to oxidative stress (134). This pathway has been the

focus of much of the therapeutic interventions that have been trialled. Aldose

57

reductase inhibitors have shown an improvement in nerve conduction velocity and

some improvement in nerve fibre count (135) but have consistently failed in phase

III clinical trials (136). It is postulated that the role of the polyol pathway in humans

perhaps is not as crucial as described in animal models. Furthermore, there may

be a considerable heterogeneity in the level of the polyol pathway metabolites as

shown by numerous studies (137-140).

Figure 1-2 Polyol Pathway in DSPN

NADPH NADP+ NAD+ NADH

Glucose Sorbitol Fructose

Aldose reductase Sorbitol dehydrogenase

1.3.3.2 Non-enzymatic glycation

Glucose and other sugars can form covalent bonds with proteins (non-enzymatic

glycation) which has been documented in diabetes (141). Advanced Glycation End

products have also been identified that may contribute to oxidative stress and

interplay with other pathways (134). AGEs can quench nitric oxide and thus lead to

impaired tissue blood flow (142).

1.3.3.3 Oxidative stress

In diabetes, there is increased free radical production due to hyperglycaemia and a

reduced ability to neutralize free radicals. This is partly the result of non-enzymatic

glycation and polyol pathway hyperactivity (134). There is acceleration in free-

radical formation that may be associated with a deficiency of antioxidant and

detoxification pathways (134). With polyol pathway hyperactivity, the ability to

neutralize free radicals is reduced, as there is NADH depletion (143). Oxidative

58

stress impairs nerve function by either a direct toxic effects or by reducing nitric

oxide. This in turn may impinge on blood flow in endoneurial capillaries.

A powerful antioxidant (alpha lipoic acid) which scavenges hydroxyl radicals,

superoxide and peroxyl radicals and generates glutathione has shown some

benefit (144) and the Alpha Lipoic in Diabetic Neuropathy (ALADIN) study

demonstrated an improvement of neuropathic symptoms with 3 weeks of

intravenous treatment with alpha lipoic acid (145) although with such a short

duration of intervention a considerable placebo effect is likely. The ALADIN II study

which comprised of 5 days of intravenous treatment with alpha lipoic acid followed

by 2 years of oral treatment showed a significant improvement in sural and tibial

nerve conduction parameters (146). However, the more recent study by Nathan I

study (147), which was a 4 year randomized double-blind parallel-group trial

enrolling 460 diabetic patients with mild to moderate DSPN who were randomly

assigned to oral treatment with 600 mg alpha lipoic acid once daily (n = 233) or

placebo (n = 227) did not achieve the primary end point. However, importantly the

primary end point did not deteriorate significantly in placebo treated subjects and

brings into question the reoccurring dilemma of the lack of placebo decline in trials

of human DSPN (148).

1.3.3.4 Protein Kinase C (PKC) activation

Hyperactivity of Protein Kinase C (PKC) by 1,2-diacylglycerol inducement occurs in

diabetes and is associated with abnormalities in vascular function. It can improve

Na+-K+- ATPase activity, which is normally suppressed (134). PKC inhibitors have

been shown to correct nerve conduction velocity and perfusion deficits and to

protect endothelial dependent relaxation in animal models (149-150). A PKC-β

59

inhibitor, Ruboxistaurin, has been studied in randomised placebo controlled trials

and failed in its primary outcome measure (151-152).

1.3.3.5 Renin-Angiotensin-Aldosterone System (RAS)

A small open label study of lisinopril in hypertensive subjects (n=13) showed

improvements in nerve conduction measures over 12 weeks (153). One double

blind randomised controlled study has found benefit of trandolopril in normotensive

patients with DM and mild neuropathy over a one year period (154). The

mechanism of action is thought to be mediated through protection from nerve

hypoxia.

1.3.3.6 C-Peptide, Complications and DSPN

C-peptide deficiency is an important contributing factor to the characteristic

functional and structural abnormalities of the peripheral nerves (155). C-peptide

binds to cell membranes, resulting in stimulation of endothelial nitric oxide

synthase (eNOS) and Na+, K+ -ATPase (Figure 1.3) (156). In the Joslin 50-Year

Medallist Study (157), protection from complications (retinopathy, nephropathy and

neuropathy) was thought to be due to the presence of enriched protective factors

against microvascular complications. It was also noted that there was residual C-

peptide production (6% of subjects) which may itself play an important role (157).

In two double-blind, placebo-controlled studies in type 1 DM patients, C-peptide

replacement or placebo was given with the patients’ regular insulin therapy (158-

159). Sensory NCV assessed in the sural nerve and showed a significant

improvement. The two studies had different populations: In the first study, patients

were at an early stage of nerve dysfunction with an average age of 29 years and

approximately 10 years diabetes duration. There was an increase after 3 months

60

which amounted to 2.7 m/s, corresponding to an 80% correction of the initial

conduction velocity deficit in these patients (158). In the second study, patients

presented with sensory NCV in the sural nerve that was at baseline on average 2.6

SD below normal after correction for body height thus suggesting that some

patients had quite marked nerve conduction deficits prior to the 6 months of

intervention (159). Further detailed large randomised controlled trials of sufficient

quality and length are required to truly assess the effectiveness of C-peptide as a

therapeutic intervention. Figure 1.3 shows the postulated mechanism by which C-

peptide may exert an effect on nerve structure and function.

Figure 1-3 Putative effect of C-peptide on peripheral nerve structure and function

Adapted from Ekberg et al (156).

1.3.3.7 Treatments for DSPN based on pathogenetic mechanisms

Treatments based on the pathogenetic mechanisms of DSPN have dramatically

failed at the clinical trial stage (82; 136; 160-162). In part, the failure of these

C-Peptide

eNOS ↑ Improved

Endothelial Function

Improved Nerve function

Na+, K+ -ATPase ↑

Na+ - Retention ↓

Energy status ↑

Improved Nerve function

Amelioration of Nerve Structural

Abnormalities

Neurotrophic Factors ↑

NGF, IGF-1, NT3 ↑ Amelioration of Nerve Structural

Abnormalities

61

treatments may be due to inadequate trial design, inappropriate surrogate end

points and a lack of decline in the placebo arms of clinical trials (148). Currently,

there is a single treatment which is licensed only in Japan (163), although its

clinical effectiveness is debatable. There are no licensed treatments in the UK.

Treatments based on their putative mechanism and effects in RCTs are detailed in

Table 1.4.

1-4 Treatment of DSPN based on the putative pathogenetic mechanisms and outcome of

clinical trials.

Abnormality Compound Aim of treatment Status of RCTs

Polyol pathway↑ Aldose reductase

inhibitors Nerve sorbitol↓

Sorbinil/Tolrestat/

Zenarestat/Lidorestat Withdrawn (AE)

Zopolrestat

Withdrawn

No published clinical

trials

Ponalrestat

Ineffective

Epalrestat

Marketed in Japan

Ranirestat

In phase III trials (157)

Oxidative stress↑ α-Lipoic acid Oxygen free radicals↓

Effective in RCTs,

Nathan I (147) –

ineffective at primary

endpoint

Nerve hypoxia↑ ACE inhibitors NBF↑ Effective in one RCT

Protein kinase C↑ Protein kinase C-β

inhibitor NBF↑ RCTs ongoing,

C-peptide↓ C-peptide NBF↑ Some data available,

Studies ongoing

Neurotrophism↓ Nerve growth factor Nerve regeneration,

growth↑ Ineffective

Adapted from Boulton et al: Diabetes Neuropathies (162).

AE adverse event, NBF nerve blood flow, RCT randomised clinical trial.

62

1.4 Vitamin D deficiency

It estimated that vitamin D deficiency affects one billion people worldwide (164).

Previous estimates of vitamin D deficiency in the UK suggest it may affect up to

87% of adults, depending on the season (165). Considerable attention has recently

been focused on the role of vitamin D deficiency on cardiovascular health (161;

166-167). The vitamin D receptor (VDR) is known not only to be expressed in bone

but ubiquitously in other tissues and cells including lymphocytes, cardiomyocytes,

endothelium, pancreatic β-cells, and lipid laden macrophages (foam cells) (168).

25(OH) vitamin D is used to determine vitamin D status as it accurately represents

body stores and has a half-life of approximately 15 days (169-170), whereas the

active form (1,25(OH)2 vitamin D) has a short half life 15 hours and can alter over a

24 hour period (169).

Current ‘healthy’ or sufficient levels of 25(OH) vitamin D are suggested to be

>30ng/dl although this advice is based on data derived from bone metabolic health

(171) (Table 1.5) and there is no definitive consensus on optimal levels (164).

Vitamin D deficiency is defined by some experts as a 25(OH) vitamin D of less than

20 ng/ml (172-174), however, 25(OH) vitamin D levels are inversely associated

with parathyroid hormone levels until 25(OH) vitamin D reaches 30 to 40 ng/ml at

which point parathyroid hormone levels reach their nadir (175-177).

63

1-5 Serum 25(OH) vitamin D concentrations and status

Serum 25(OH) vitamin D concentrations and status (164)

25(OH) vitamin D Concentration 25(OH) vitamin D status

<10ng/ml Severely Deficient

10-<20ng/ml Deficient

20-<30ng/ml Insufficient

≥30ng/ml Adequate

≥150ng/ml Possible toxicity

1.4.1 Vitamin D deficiency, Hyperlipidaemia and Hypertension

1.4.1.1 Hyperlipidaemia

Vitamin D and cholesterol share a common metabolic pathway through 7-

dehydrocholesterol and this metabolite is a precursor for both. Lower levels of

25(OH) vitamin D have been associated with lower high density lipoprotein

cholesterol (HDL) and hyper triglyceridaemia (178). Statin therapy has shown to

raise vitamin D levels (179) and a study in macrophages from obese hypertensive

patients with DM established that culturing with 1,25(OH)2 vitamin D suppressed

foam cell formation by reducing oxidized low-density lipoprotein cholesterol (LDL)

uptake (180). The deletion of the vitamin D receptor in macrophages in the same

study accelerated foam cell formation induced by modified LDL (180). 25(OH)

64

vitamin D is sequestered in adipose tissue and this may partly explain the low

levels associated with obesity by reducing the release of this inactive form into the

circulation for transformation to active vitamin D (181) as well as being

independently associated with HDL and metabolic syndrome (178).

1.4.1.2 Hypertension

Vitamin D is implicated in hypertension by its role as a negative regulator of the

RAS and in experimental studies, VDR knockout mice have elevated renin,

angiotensin and aldosterone, suggesting that vitamin D may be a potent inhibitor of

the RAS axis (182). The NHANES III study (183) described a lower blood pressure

in those in the highest deciles of 25(OH) vitamin D and vitamin D3 with calcium

supplementation has been shown to reduce blood pressure compared to calcium

supplementation alone (184). However, the largest interventional study was the

Women's Health Initiative (n = 36 282), which was designed to evaluate the effects

of 400 IU of daily vitamin D3 with calcium supplementation on fracture and cancer

risk when compared to placebo showed no change in blood pressure or incident

hypertension over 7 years (185).

1.4.2 Vitamin D deficiency and Macrovascular Complications

The Framingham offspring study longitudinally followed up individuals (n=1739)

with no prior history of cardiovascular disease in this cohort (167). During the follow

up period (mean 5.4 years) a composite of cardiovascular events was assessed

and after multivariate adjustment those with 25(OH) vitamin D of <15ng/ml had a

hazard ratio of 1.62 (95% confidence interval (CI) 1.11-2.36, p=0.01) for incident

cardiovascular events compared to 25(OH) vitamin D ≥15ng/ml (167). This

increased risk was even more evident in those with hypertension (167). Vitamin D

65

deficiency has been related to cardiac disease in the form of coronary artery

calcification, myocardial infarction, stroke and congestive cardiac failure (170). The

Ludwigshafen risk and cardiovascular health study (LURIC study) assessed a

consecutive cohort of 3258 individuals scheduled for coronary angiography (186)

for their vitamin D status. Sudden cardiac death and death due to heart failure

were independently and inversely associated with 25(OH) vitamin D, and CVA was

related to both 25(OH) vitamin D and 1,25(OH)2 vitamin D levels (186).

Furthermore, the NHANES III study subgroup (n=3408) analysis supported these

findings as 25(OH) vitamin D was inversely associated with all-cause mortality over

a mean period of 7.3 year (187). Compared to individuals with 25(OH) vitamin D of

≥40ng/ml, those with 25(OH) vitamin D of <10ng/ml the adjusted risk was

approximately 83% higher (187). A study vitamin D deficiency was associated with

an increased amputation risk in veterans with peripheral arterial disease (188).

1.4.3 Vitamin D and Diabetic Neuropathy

The effective treatment of DSPN remains a major challenge and anticonvulsants

and anti-depressants remain the mainstay of symptom relief, but provide no benefit

for underlying nerve damage. However, increasing data suggest that vitamin D

may have not only comparable analgesic but also play a pivotal role in the

peripheral nervous system and in particular diabetic neuropathy (189-192).

Vitamin D deficiency may occur in a significant proportion of UK patients; with

diabetes ~90% classified as being insufficient (<30ng/ml) with severe deficiency in

over 30% (<10ng/ml) (193). Nerve Growth Factor (NGF) and neuronal Ca2+

homeostasis, both may play a neuroprotective role in the peripheral nerve have

been linked to vitamin D experimental studies through the regulation of

66

neurotrophins (194). NGF is depleted in experimental diabetes (195). Sciatic nerve

NGF was preserved in animals exposed to a vitamin D analogue (CB1093) (194)

and another vitamin D analogue (MC903) is known to increase NGF synthesis

(196). Also in the peripheral nervous system, VDR are found in predominantly

nociceptive neurons of dorsal root ganglia and their expression is influenced by

ovarian hormones and a significant increase in VDR expression has been

observed in DRG neurons of diabetic rats (197).

Observational studies have demonstrated a significant link between vitamin D

deficiency and DSPN (190-191). In the National Health and Nutrition Examination

Survey (NHANES) 2001-2004, an unweighted sample of 591 subjects with

diabetes demonstrated a significant association between vitamin D deficiency and

both paraesthesiae (odds ratio 2.12; 95% CI 1.17–3.85) and numbness (odds ratio

2.04; 95% CI 1.18–3.52) after adjusting for confounders (191). Similarly in a cross

sectional study by Shebab et al (190), vitamin D deficiency was an independent

risk factor for DSPN assessed using the neuropathy symptom score, as well as

clinical and electrophysiological measures of DSPN.

Vitamin D deficiency has been implicated in numerous pain syndromes (198-199).

Vitamin D supplementation may improve musculoskeletal pain and non-traumatic

back pain as shown in some studies (200), although the data on vitamin D

deficiency prevalence and non-specific musculoskeletal pain is not as compelling

(201). The data supporting a benefit of vitamin D treatment in painful DSPN is as

yet limited. However, in a non-randomised non-placebo controlled but prospective

study with cholecalciferol (vitamin D3) at a mean dose of 2059 IU daily for 3

months in painful DSPN an improvement of ~50% on the Visual Analogue (pain)

Scores (VAS) was observed (202). Furthermore Valensi et al (203), showed use of

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a compound (QR-333) containing a vitamin D analogue in a double blind

randomised controlled trial reduced the severity of numbness, jolting pain, and

irritation in subjects with painful DSPN although QR-333 also contained an aldose

reductase inhibitor compound, which may have had an impact on the pathogenetic

mechanisms causing neuropathy. The largest observational study of patients with

in the community in northwest England (n = 15,692) has shown that the prevalence

of painful symptoms and painful DSPN was 34 and 21% respectively (77).

Additionally, despite a lower prevalence of neuropathy in South Asians compared

to Europeans and Afro-Caribbeans, painful symptoms were greater in South

Asians (77). These differences may partly be explained in relation to vitamin D

deficiency as these groups have been shown to have excess vitamin D deficiency

(204). Thus the role of vitamin D supplementation in painful DSPN requires further

study in terms of mechanisms but also demands good quality randomised

controlled trials.

1.4.4 Vitamin D and Diabetic Retinopathy

There is a possible direct link between vitamin D deficiency and the development

and progression of diabetic retinopathy. Not only have immunostaining in animal

models shown that vitamin D receptor (VDR) is expressed in the ganglion cells, the

inner and outer plexiform layer and the photoreceptor layer (205) but VDR

dependent calcium binding proteins have been isolated in the photoreceptor layer

of the human retina (206). In experimental models of ischaemic retinopathy,

calcitriol is a potent inhibitor of retinal neovascularisation (207). Other experimental

studies show similar findings with administration of active vitamin D having a

partially protective effect on diabetic retinopathy of diabetic rats, through a

68

mechanism which may involve inhibition of vascular endothelial growth factor

(VEGF) and transforming growth factor (TGF)-β1 expressions in the retinal tissues

(208).

Furthermore, retinoblastoma tissue expressing VDR has shown reduction of

growth with subsequent apoptosis of the retinoblastoma cells after

supplementation with vitamin D (209). VDR genotypes have been linked with the

cumulative prevalence of diabetic retinopathy (210). The VDR gene in the French

population, FokI and TaqI single nucleotide polymorphisms have been associated

with DR (211-212). In a Turkish study, there was a demonstrated inverse

correlation between worsening diabetic retinopathy and lower activated vitamin D

in a population of 66 subjects (213). Cross-sectional studies in children and adults

with DM, show positive associations with vitamin D deficiency and DR (214-216).

The NHANES III study showed an association between severity of diabetic

retinopathy and prevalence of vitamin D deficiency, but the findings were

inconclusive about the existence of a significant relationship (216). These findings

were also mirrored in a smaller cross sectional study of type 1 DM and controls

and those with proliferative diabetic retinopathy having the lowest vitamin D status

(215). Further evaluation of this possible important relationship is required.

1.4.5 Vitamin D and Diabetic Nephropathy

Vitamin D therapies for renal disease have been used for over a half century and

are likely to be utilised for many more years (217). Vitamin D has paracrine

functions through local activation by 1-alpha-hydroxylase and thus maintain

vascular function, cardiomyocyte health, and abrogate inflammation and insulin

resistance (218). Low serum 25(OH) vitamin D status is a common complication in

69

patients with chronic kidney disease (CKD) and the severity of deficiency increases

with the progression of CKD (219). The RAS plays a critical role in the

development of diabetic nephropathy, and blockade of the RAS is currently used

for treatment of diabetic nephropathy (220). Vitamin D plays a significant role as a

RAS inhibitor (220). In an experimental study, combination of vitamin D analogues

and RAS inhibitors prevented renal injury in diabetic nephropathy (220) and a

vitamin D analogue (paricalcitriol) has shown an improvement in microalbuminuria

in a human study through a mechanism related to inhibition of RAS (221).

1.5 Clinical Evaluation and Investigations of Neuropathy

The assessment of DSPN is a common clinical entity facing the general physician.

A simple approach to the assessment of neurological deficits is essential and can

be undertaken using the neuropathy disability score (NDS). Further evaluation

includes symptom questionnaires, quantitative sensory testing, electrophysiology,

autonomic tests and/or skin biopsy. Sural sensory nerve biopsy is a well

established method of accurately assessing neuropathy. However, due to the

invasive nature of the investigation it is not commonly used and is reserved for

diagnoses that are inconclusive, often when a vasculitic phenomena is a

differential (222). Sensory nerve biopsy will not be considered further in this thesis

for this reason. A thorough examination should include evaluation of large

myelinated nerve fibres (Aβ) by evaluating vibration perception, small myelinated

(Aδ) fibres by evaluating sharp pain and cold sensation and small unmyelinated (C)

nerve fibres by evaluating warm and pain perception. Types of nerve fibres in

peripheral nervous system are shown in table 1.6.

70

1-6 Type of nerve fibres in the peripheral nervous system

Motor Sensory Autonomic

Myelination Myelinated Myelinated Thinly

myelinated Unmyelinated Unmyelinated

Fibre type

Aα

Aα/Aβ

Aδ

C C

Function Motor control

Coarse Touch Proprioception

Vibration perception

Cold perception

Pain

Warm perception

Pain

Cardiovascular control (heart

rate, blood pressure),

genitourinary and sexual

function diaphoresis and gastrointestinal

control.

1.5.1 Diagnosis of Diabetic Distal Symmetrical Polyneuropathy

A number of approaches are employed to diagnose and evaluate the severity of

neuropathic deficits and painful symptoms in diabetic neuropathy. In a clinical

environment, bedside screening instruments are usually sufficient in screening for

neuropathy (136). The diagnosis of DSPN can only be made after a careful history

and clinical examination. The consensus is all patients with diabetes should be

screened annually for DSPN by examining:

1. Pinprick

2. Temperature

3. Vibration perception (using a 128-Hz tuning fork)

4. Ankle reflexes

5. 10g monofilament pressure sensation at the distal halluces.

71

A combination of more than one test has >87% sensitivity in detecting DSPN (4).

The modified neuropathy disability score (NDS) is a screening instrument for signs

of neuropathy. It comprises three sensory modalities and one assessment of

reflexes: 1) pain sensation (pin prick), 2) temperature sensation (hot and cold

rods), 3) vibration perception (128Hz tuning fork), and 4) absence/presence of

Achilles tendon reflexes. Thus the total maximum abnormal score possible is 10. A

score of 3-5 is regarded as evidence of mild neuropathic signs, 6-8 as moderate

and a score of 9 or 10 as severe signs of neuropathy. The original NDS in the

Rochester Diabetic Neuropathy study (223) and more recently a study by Abbott et

al, (224) using the modified NDS showed this evaluation to be reliable and

reproducible as a screening instrument of peripheral neuropathy. An increasing

NDS and Vibration Perception Thresholds (VPT) along with abnormal plantar foot

pressures can be used in identifying those at risk of foot ulceration (225). An NDS

>6 was an independent risk factor for diabetic foot ulceration (224). Also the loss of

vibration perception and 10g monofilament alone can accurately predict the risk for

future foot ulceration (82) which is an important factor in the morbidity that can be

associated with diabetes. A NDS of >6 has however been shown to be a better

predictor than the 10g monofilament and is only second to past or present history

of ulceration (224). The NDS does not diagnose the cohort with small fibre

neuropathy or with subclinical large fibre disease and should be used as a guide to

the risk of neuropathic ulceration.

Two major end-points are used particularly when assessing neuropathy for either

research purposes or quantifying the benefits of an intervention (81). These are:

a. Assessment of symptoms in defining efficacy of pain relief agents.

b. Assessment of neurological deficits in defining efficacy of nerve repair agents.

72

An alteration of symptoms does not necessarily constitute an improvement in nerve

function itself and changes in nerve ultrastructure may not equate to improved

nerve function. The criteria which is used to determine the effectiveness of an

intervention on symptoms is thus varied and lacks any specialist consensus.

1.5.1.1 Neuropathy Symptom Questionnaires

Diabetic patients usually find it difficult to describe the neuropathic pain they

experience and thus numerous questionnaires and scoring systems have been

developed to quantify the subjective feeling of neuropathic pain. Some of the more

commonly used questionnaires are discussed below.

Neuropathy Symptom Profile (NSP) assesses neuropathy in general but has been

validated in diabetes (226). It comprises of 38 questions with further subsections

which are subdivided on anatomy. It is useful in detecting neuropathy and staging

severity (226). The Rochester Diabetic Neuropathy Study (227) concluded that

NSP, combined with other neurological examinations is a valid means to assess

neuropathy.

The McGill Pain Questionnaire is the most frequently used questionnaire, however

is not specific for diabetic neuropathy. This questionnaire is a valid measure of

neuropathic pain symptoms (228). A more recently developed questionnaire, the

Diabetic Neuropathy Symptom Score (DNS) is a simple and easily reproducible

test in clinical practice. It is a 4-item questionnaire and the DNS score has been

shown to differentiate between individuals with and without diabetic neuropathy

(229) but is used more as a screening questionnaire. These questionnaires,

however valid at assessing symptoms are unable to differentiate accurately

between large and small nerve fibre deficit.

73

1.5.1.2 Quantitative Sensory Testing (QST)

QST involves the delivery of a stimulus which is reproducible and controlled, has

an established normal range and allows for serial evaluation of a standardised

stimuli (81). The biothesiometer is a QST of vibration and the other widely used

QST is that of thermal and pain thresholds (136). Both can track progression (230)

and predict those at risk of ulceration (231). The advantages and limitations of QST

are established. The most important advantage is that it requires little expertise to

perform and has a reasonable sensitivity, accuracy and reproducibility when

evaluating different stages of neuropathy at multiple anatomical sites (136). The

main limitations are variability and subjectivity of the test, patient motivation,

attention and expectation bias (232). Thermal testing consists of graded and

standardised warm and cold stimuli delivered through a thermode attached to the

foot. Thermal testing is able to quantify warm and cold sensation thresholds,

temperature induced pain and temporal summation. Room temperature, inter-

stimulus intervals, gender, age and lifestyle may also affect the outcomes (233).

However, QST including thermal threshold assessment for cold sensation (A

fibres) and warm sensation (C fibres) can assess small fibre dysfunction but are

subjective, lack accuracy and reproducibility (136; 232).

The San Antonio consensus (234-235) and now the Toronto consensus (79) states

that nerve conduction studies and symptoms and/or signs should be used as a

definitive diagnosis of DSPN. However, a significant limitation is that this approach

primarily assesses large myelinated nerve fibres (235) and only then the neural

activity of a specific small subset of large, myelinated fibres (236). Despite this

obvious draw back nerve electrophysiology is used as a surrogate marker in

clinical trials (237).

74

1.5.1.3 Electrophysiology

Electrophysiological procedures in the form of nerve conduction studies (NCS) are

one of the fundamental tests in the diagnosis of diabetic neuropathy and are

particularly used in clinical trials as a measure of effectiveness. Indeed the San

Antonio and now the Toronto consensus on diabetic neuropathy recommends the

use of electrophysiological testing along with other tests such as QST and

autonomic function to assess clinical deficits of neuropathy (79, 235). The

procedure is not invasive, but objective and is highly sensitive and therefore

accurately measures and classifies DSPN. The drawback is an inability to target

the fibre types most commonly affected by diabetes (small fibres) as it assesses

large myelinated fibres (238) and also has limited ability to demonstrate

regeneration and repair. Electrophysiological tests measure conduction of both

sensory and motor nerves, the amplitude of the propagating neural signal, the

density and synchrony of muscles activated by maximal nerve simulation and the

integrity of neuromuscular transmission (236). There are several factors

contributing to nerve conduction velocity (NCV) slowing: 1) stage of nerve

demyelination, 2) mean diameter of the conducting axons, 3) internodal distance in

the segment under study and 4) the nodal microenvironment (136).

In a study by Dyck et al, (239) when compared to nerve conduction studies,

individual physicians clinical diagnosis was highly variable with a propensity to over

diagnose DSPN. Although considered a ‘Gold Standard’ test, confounding factors

can lead to decreased sensitivity and specificity (240). Gender, weight, age and

height may all affect the results of NCS with the latter two variables having the

most significant effect (240). In a 10-year follow-up study of newly diagnosed

patients with type 2 DM, NCV slowing and decrease in sensory and motor

75

amplitudes were reported in 16.7% of the patients at 5 years and 41.9% at 10

years, indicating an increase in the severity of DPN with time (241). Although more

recent experimental studies have shown significant variability in measures of nerve

conduction with up to 56.4% variability in motor amplitude in serial measures over

a 4.5 month period (242).

Abnormal NCV is known to correlate with morphologic features in small fibre

neuropathy and QST (243) and is still used as an end point in

interventional/observational trials of DSPN.

1.5.1.4 Skin Biopsy

The only techniques which allow a direct examination of thinly myelinated and

unmyelinated nerve fibre damage and repair, are those of sural nerve biopsy with

electronmicroscopy (119; 244) and the newly refined skin-punch biopsy (124; 245).

This technique allows a reliable and reproducible means of assessing the small (C)

fibres which previously were limited to nerve biopsies, in particular that of the sural

nerve (246). Intraepidermal nerve fibres (IENF) are stained using

immunohistochemistry with the antibody to protein gene products 9.5 (120). The

densities are calculated in at least three sections as the number of IENF per length

of the section (IENF/mm) (247). According to the European Federation of

Neurological Societies/Peripheral Nerve Society Guideline on the use of skin

biopsy in the diagnosis of small fibre neuropathy, single IENF crossing the dermal–

epidermal junction should be counted, whereas secondary branching is excluded

from quantification (247). Intra- and inter observer variability, and inter laboratory

agreement on IENF counts has been assessed with good reliability scores

obtained (kappa values > or = 0.90) (248).

76

Previous studies have associated reduced IENF density with small fibre

neuropathy and more so with painful DSPN (123; 125) with excellent sensitivity

(123). There is increased small fibre loss at >5 years duration of diabetes with

shortening of IENF at all durations (123). Also reduced rates of nerve regeneration

have been seen in individuals with diabetes but without signs or symptoms of

disease or evidence of neuropathy (249). Unsurprisingly skin biopsy analysis may

be more sensitive than sural nerve conduction studies for diagnosing small fibre

neuropathy (250). The advantages of this method of assessing small fibres are:

1. Quantitative

2. Reproducible

3. Can be used for longitudinal assessment

4. Can be used at multiple sites

However, there are also major disadvantages as the technique is limited

significantly by its availability and is certainly not commonly used in clinical

practice, it is invasive and requires potentially intensive complicated laboratory

analysis.

1.5.1.5 Corneal Confocal Microscopy (CCM)

The cornea is one the most densely innervated organs in the body (251). Most

corneal nerves are derived from the ophthalmic branch of the trigeminal nerve and

constitute both Aδ and C fibres. Approximately 70 – 80 trunks of large diameter (~

6μm), myelinated nerves (Aδ) containing 900 – 1200 axons enter the cornea at the

stroma and run anteriorly towards the midpoint of the cornea. The nerves penetrate

the Bowman’s membrane and form the subbasal nerve plexus (Figure 1.3) in the

77

boundary between the Bowman’s layer and the epithelium and finally terminate in

the superficial layers of epithelium.

Figure 1-4 Montage of the whole human subbasal nerve plexus [Image adapted from: Patel

and McGhee (252).

78

Recently developed, CCM is a rapid, reiterative, non-invasive procedure which

allows microscopic examination of the cellular and nerve structures of the cornea.

Conventional techniques of electrophysiology and quantitative sensory testing

along with an assessment of neurological disability offer a relatively robust means

of defining neuropathic severity (136). However, they have major shortcomings

when they are employed to define therapeutic efficacy in clinical intervention trials

(253). New therapies in the treatment of diabetic neuropathy have been hampered

by the lack of clinically relevant end points (136) as historically quantification of

large nerve fibres were used to diagnose neuropathy and not in assessing nerve

regeneration or a response to an intervention (254). CCM may provide an ideal

surrogate end-point and has been shown to demonstrate corneal nerve fibre

pathology which is correlated with DSPN (255-257). Numerous studies have

looked at the relationship of corneal nerve pathology and diabetic neuropathy

suggesting it may be used as a surrogate marker of small fibre neuropathy (258-

263). Significant changes can be detected in corneal nerve fibre density,

branching, and tortuosity which relate to the severity of DSPN (256; 264). CCM has

also been shown to detect the benefits of therapeutic intervention with pancreas

transplantation after 6 months of the intervention (265) and more recently with

simultaneous pancreas and kidney transplantation after 12 months (266). This

highlights the ability of this modality to be potentially used in assessing therapeutic

interventions. It has been argued that however significant the changes to nerve

structure this may not necessarily equate to alterations in function. However,

corneal sensitivity is reduced and is directly related to the severity of the

neuropathy in diabetes (267). This modality has shown significant potential as a

surrogate test of nerve function.

79

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2 CHAPTER II - METHODS

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2.1 Synopsis

The ability to define the ‘at risk’ patient, anticipate deterioration, and assess new

therapies is of paramount importance in DSPN and there is also an urgent need for

‘new’ surrogate endpoints for this condition. Current techniques lack the sensitivity

(QST), require expert assessment or target inappropriate nerve fibre types (NCS),

are invasive (skin biopsy) and are not routinely performed across health systems.

A surrogate endpoint needs to reliably reflect the activity of the underlying disease

process, which ultimately causes significant morbidity such as foot ulceration and

lower limb amputation. Vitamin D has long been linked with bone metabolic

disease however, more recently there have been postulated links to DM and it

complications. There is a subsequent need to assess the role of vitamin D in

diabetic microvascular complications, the effectiveness of therapeutic interventions

and the prevalence of vitamin D deficiency in patients with DM.

2.2 Hypothesis and aims

The primary hypothesis of this study is corneal nerve morphology using CCM is a

valid surrogate marker for human DSPN and the vitamin D status has a definitive

role in the diabetic microvascular complications of DR and DSPN. This research

aims:

i.To delineate subjects who have rapidly progressive DSPN and assess whether this

may be discerned through the novel modality of CCM. It will also aim to assess

corneal nerve fibre damage visualised with CCM and evaluate if it is progressive in

nature and in agreement with the natural history of the disease relating to clinical

(NDS, QST, NCS) and laboratory (intra-epidermal nerve fibre density) markers of

DSPN.

115

ii.To establish whether subjects with LADA have a different neuropathic phenotype

than those with type 2 DM. We aim to establish the ability of CCM to discernibly

validate small fibre changes compared to conventional small and large fibre

neurological tests.

iii.To estimate the prevalence of neuropathy measured with CCM and Non Contact

Corneal Aesthesiometry (NCCA) in a cohort of patients with type 1 DM and

compare this with the results of neurological deficits (NDS); nerve

electrophysiology, quantitative sensory testing (thermal thresholds and VPT), IENF

quantitative analysis and establish the relationship with risk factors.

iv.To establish the local prevalence of vitamin D deficiency and its association with

the constituents of metabolic syndrome and cardiovascular disease risk which may

also drive DSPN.

v.To establish the improvement in vitamin D status through the effects of 3 different

vitamin D replacement regimens (using combinations of ergocalciferol,

cholecalciferol and calcium carbonate/cholecalciferol) for the treatment of vitamin D

deficiency.

vi.To establish any phenotypic and metabolic differences between groups with mild

neuropathy with and without painful DSPN and to compare the results of

neurological deficits (NDS); nerve electrophysiology, quantitative sensory testing

(thermal thresholds and VPT), IENF quantitative analysis and vitamin D status.

vii.To establish the relationship of vitamin D status to DR alongside conventional risk

factors such as duration of diabetes, glycaemic control, lipids and blood pressure.

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2.3 Study Design

1. Chapter III – Prospective longitudinal, investigator-masked observational

study.

2. Chapter IV, V – Prospective cross-sectional, investigator-masked

observational study.

3. Chapter VI – Retrospective cross-sectional, case-control study

4. Chapter VII – Retrospective analysis of clinical therapeutic intervention

5. Chapter VIII – Prospective cross-sectional, investigator-masked

observational study.

6. Chapter IX – Retrospective cross-sectional study

2.4 METHODS: Neuropathy Studies - Chapters 3, 4, 5 &8

Ethical approval was obtained prior to the initiation of the study by the North

Manchester and Salford and Trafford Research Ethics Committee, and written

informed consent was obtained according to the Declaration of Helsinki. This study

is conducted in accordance with the stipulations stated in the Declaration of

Helsinki and along with Good Clinical Practice guidance. All participants were

supplied with study literature at least 24 hours prior to informed consent being

obtained.

2.4.1 Patient Enrolment

Informed written consent was obtained from all subjects prior to their participation

and they were provided with the opportunity to discuss any concerns about the

117

experimental protocol and their safety with a trained member of the research team

(a copy of the patient consent and study forms can be found in Appendix 2). Upon

enrolment to the study, all control and diabetes participants underwent detailed

screening by means of personal and medical history and a set of blood and urine

tests to determine their metabolic status and ensure eligibility for this study. The

specific inclusion / exclusion criteria used are presented below. Appointments were

offered to participants during normal working hours and if a participant could not

complete the full visit assessment in a single visit, a second visit was scheduled

within a month.

Patients with type 1 and 2 DM and LADA were recruited from the Manchester

Diabetes Centre and general medical clinics at the Manchester Royal Infirmary,

Manchester, UK. Controls are recruited from the University of Manchester and via

social networks of the study investigators.

2.4.2 Inclusion Criteria for Chapters 3, 4, 5 & 8

Participants were expected to satisfy the following criteria prior to inclusion in the

study:

a) Aged 14 to 80 years

b) Signed written informed consent

c) Type 1 or 2 DM or LADA

d) Absence of diabetes for the control group

e) Be willing to participate and comply with the experimental protocol.

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2.4.3 Exclusion Criteria for Chapters 3, 4, 5 & 8

Any of the following criteria rendered the participant ineligible for inclusion:

a) History of corneal trauma or surgery (cataract surgery does not preclude

enrolment unless surgery occurred in the 12 months prior to enrolment date)

b) History of ocular disease or systemic disease which may affect the cornea

c) Concurrent ocular disease, infection or inflammation

d) History of systemic disease (e.g. malignant disease, congestive heart failure

NYHA Grade III or IV, major psychosis (i.e. schizophrenia or bipolar), certain

autoimmune diseases – hypothyroidism, Addison’s disease, vitiligo)

e) History of neuropathy due to non-diabetic cause e.g. alcoholism,

amyloidosis, autoimmune disorders, chronic kidney failure, connective tissue

disease, infectious disease (e.g. Lyme disease, HIV/AIDS, hepatitis B, leprosy),

liver failure, radiculopathy, vitamin deficiencies (e.g. pernicious anaemia, B12

deficiency)

f) Current or active diabetic foot ulcer or infection

g) Participating in any other interventional (e.g. drug) research trial.

2.4.4 Questionnaire Administration

The NSP, DNS and McGill Pain Questionnaire were either administered by a

researcher or they were self-completed. When questionnaires were self completed,

careful instructions were provided to the patient on how to score the questionnaire

in order to avoid any misinterpretation.

119

2.4.5 Neuropathy Disability Score (NDS)

The NDS is derived from a basic neurological examination testing vibration, pin-

prick and temperature perception in addition to deep tendon reflexes. A score

ranging between 0 and 10 is calculated and the degree of neuropathy is

categorised as no neuropathy [0-2], mild neuropathy [3-5], moderate neuropathy

[6-8] and severe [9-10] (Figure 2.1).

Pain sensation (C fibres) was tested using a neurotip. Sharp and blunt ends were

applied and the patient then distinguished the sharp sensation. Vibration sensation

was tested using a 128 Hz tuning fork. The vibrating fork was first applied on the

subject’s forearm/sternum and was then applied three times to the bony

prominence of the first hallux of each foot. Two correct responses were deemed as

being normal (Figure 2.2). Temperature sensation was tested using two metal rods

separately placed in hot and cold water (Figure 2.3). The patient was then asked to

ascertain the correct sensation and two or more correct responses were deemed

as being normal. Deep tendon reflexes were assessed by the achilles tendon

reflex. If absent, subjects were asked to ‘reinforce’ the reflex. The score was then

recorded (0: normal, 1: present with reinforcement and 2: abnormal). All

procedures were repeated for the opposite foot.

120

Figure 2-1 The Neuropathy Disability Score

From Boulton, A.J.M (1).

Figure 2-2 Testing vibration sensation with a 128 Hz tuning fork as part of the NDS

121

Figure 2-3 Testing temperature sensation on the dorsum of foot as part of the NDS

2.4.6 Neuropad

The Neuropad is an early detection test to ascertain the hydrosis status (moisture

content) of the sole of the foot (Figure 2.4). Those with chrome, nickel and/or

cobalt allergy were excluded from the test. Shoes and socks were removed at least

5 minutes prior to application of the neuropad on the ball of the small toe. After 10

minutes the colour of the pad was assessed and a gross % assessment was

made. 100% pink is considered normal whilst the pad remaining blue or patchy is

considered abnormal (Figure 2.4).

Figure 2-4 Neuropad: a) 100% pink- normal response, b) no change- abnormal and c) patchy-

abnormal

122

2.4.7 Heart Rate Variability to Deep Breathing

Computer-aided sensory evaluator IV (CASE IV) was used to measure the heart

rate response to deep breathing. In this test the patient was asked to inhale and

exhale deeply six to eight times in a row in the supine position, whilst following the

rhythm of a “breathing cue” in the form on an LED light. The changes in heart rate

are displayed on an ECG monitor. One eight-cycle breathing series was completed

and then a subsequent six-cycle breathing series interspersed by two minutes of

normal breathing. The acquired data was analysed by calculating the mean

difference between the highest and lowest heart rate for five consecutive, artefact-

free cycles in each series.

2.4.8 Quantitative Sensory Testing

The patient was made comfortable in a semi-recumbent position, with heels resting

on the bed. The patient was familiarised with the sensation of vibration from the

neurothesiometer at the wrist/forearm. The tip of the neurothesiometer was rested

on the great toe (right and left). The amplitude of vibration for the probe was

increased from zero, by turning the dial slowly, whilst the patient has their eyes

closed. The patient was instructed to say “Yes” when he/she first detects the

slightest “buzzing” or vibration sensation and this is recorded as the vibration

perception threshold which was repeated three times on each test site. VPT is

measured in volts and has a range between 0-50 volts.

Thermal tests were performed with a Medoc TSA 2001 device (Medoc Ltd,

Minneapolis, MN). To avoid tactile or pressure stimulation the probe was kept in

contact with the skin for the entire duration of the test. The starting temperature

(adaptation temperature) was 32°C. The thermode is controlled by a computer

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software program (MEDOC TSA II, Medoc Ltd, Ramat Yishai 30095, Israel) (Figure

2.5). The room temperature ranged between 18-22°C as per standard protocol.

The thermode contacts the skin and a subject is asked to report a sensation of

temperature change or heat pain. Cold sensation threshold (CST) is tested initially

by a gradual decrease in the thermode temperature and the subject is asked to

press the computer mouse button when they first become aware of a cold

sensation. This was repeated a further three times and a similar procedure was

carried out to test warm sensation threshold (WST). The mean value is recorded.

Figure 2-5 The user interface for thermal threshold and pain measurement

2.4.9 Electrophysiology

All nerve conduction studies were performed by a consultant neurophysiologist (Dr

Andrew Marshall) using an MS92a EMG machine (Medelec Limited, Old Woking

Surrey UK) using surface stimulation and recording electrodes), maintaining skin

temperature at 31oC (Figure 2.6). Any deviations from the standard temperature

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were recorded. Lower limb nerves were assessed, namely, motor peroneal and

tibial nerves and the sensory sural nerve. Nerve conduction velocity (m/s),

maximum amplitude (mV), and baseline to peak and minimum F-wave latency (ms)

were recorded for the motor nerves. For the sensory nerve, baseline to peak

amplitude of the sensory action potential (μV), latency to onset (ms) and

conduction velocity were recorded. Graded stimulation of nerves was undertaken

by increasing the strength of the stimulus until a maximal response was achieved

and then the stimulus was increased by 10-15% above maximal response to

ensure a supra-maximal response.

Figure 2-6 Electrodiagnostic studies of the peroneal and sural nerves

Motor amplitude was measured from the baseline to the negative peak. The

amplitude was reported to the nearest 0.1 mV. Sensory amplitude of the sensory

potential was measured from the baseline to the negative peak. If there was a

positive preceding the negative, the amplitude was measured from the base of the

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positive peak to the negative peak. The sensory nerve action was reported to the

nearest whole number.

2.4.10 Skin Biopsies and Immuno-histology

A sub-cohort of participants underwent a 3-mm punch skin biopsy from the dorsum

of the foot; 2 cm above the second metatarsal head after local anaesthesia (1%

lidocaine). The biopsy site was closed using Steristrips, and the specimen was

immediately fixed in PBS-buffered 4% paraformaldehyde for 18-24 h, washed in

Tris-buffered saline, cryoprotected in sucrose, frozen in liquid nitrogen and stored

at -80°C and subsequently cut into 50-μm sections on a cryostat microtome. Five

floating sections per subject were immunostained for PGP 9.5 neuronal marker.

Briefly, the non-specific protein binding and endogenous peroxidase activity were

blocked by incubation in 5% goat serum and 0.3% hydrogen peroxide,

respectively. The anti-PGP9.5 antibody (Milipore 1:1000; Billerica,MA) was

followed first by goat anti-rabbit IgG and then by HRP-Streptavidin (both diluted

1:1000, both from Vector Laboratories, Peterborough, UK). Nerve fibres were

visualised by SG chromogen (Vector Laboratories). Intraepidermal nerve fibre

density (IENFD) was calculated as the number of nerve fibres crossing the

basement membrane of the epidermis and expressed per millimetre length of

epidermis (Figure 2.7).

IENFD were assessed by two independent assessors as per European Federation

of Neurological Societies/Peripheral Nerve Society Guideline on the use of skin

biopsy in the diagnosis of small fibre neuropathy (2).

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Figure 2-7 Skin biopsies stained for PGP9.5. Healthy control (A) shows numerous long

intraepidermal nerve fibres and a well-developed subepidermal nerve plexus, compared with

short intraepidermal nerves and less pronounced subepidermal plexus in subjects with DM

(B & C) (Bar = 100 µm).

2.4.11 Corneal sensitivity

Corneal sensitivity was measured using an NCCA (constructed for the IHBI,

Anterior Eye Lab by Kimble Dunster and Lincoln Hudson). It uses a puff of air

lasting 0.9s expressed through a bore of 0.5-mm diameter with an electronic

pressure sensor, which displays the force exerted in millibars (3). The patient

places their head in the head and chin-rests and the NCCA table and equipment

are adjusted for comfort. The patient was instructed to look straight ahead and

gaze at the fixation target. The stimulus jet was mounted on a slit lamp positioned

1 cm from the eye and aligned to the centre of the cornea. Corneal sensitivity was

assessed by the patient being administered a high test stimulus (Figure 2.8). Thus

the patient is familiarised with ‘normal’ stimulus. The stimulus was then decreased

until it is just detectable by the subject (absolute threshold). The stimulus was

subsequently increased and repeated on 3 occasions with an average

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measurement derived. This was recorded and the procedure repeated for the

alternate eye.

Figure 2-8 Estimation of corneal sensation using NCCA

2.4.12 Corneal Confocal microscopy

Previously established indicators of corneal nerve pathology which have been

shown to be valid markers of peripheral neuropathy include corneal nerve fibre

(CNFD) and branch density (CNBD), length (CNFL) and the corneal nerve

tortuosity coefficient (CNFT):

i. CNFD: the number of main nerve fibres / mm2.

ii. CNBD: the number of main nerve branches / mm2.

iii. CNFL: the sum of the length (mm./mm2) of all nerve structures.

iv. CNFT: the tortuosity or non-linearity of the main nerve fibres.

Patients underwent examination with a corneal confocal microscope. Images are

obtained using the HRT III-RCM (Heidelberg Engineering GmBH, Heidelberg,

Germany) (Figure 2.9). The laser is a 670 nm red wavelength Helium Neon diode

laser and does not pose any danger to ocular structures. The corneal sub-basal

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nerve plexus is determined by continuous imaging of the cornea through use of an

OCD camera.

Figure 2-9 Corneal confocal microscopy

Patient demographics were entered in the Heidelberg Eye Explorer software

(Heidelberg Engineering GmBH, Heidelberg, Germany) and the confocal

microscope is prepared for examination. One eye was selected and anesthetized

with one drop of 0.4% benoxinate hydrochloride (oxybuprocaine hydrochloride)

(Chauvin Pharmaceuticals Ltd., Essex, UK). The objective lens of the confocal

microscope was disinfected (isopropyl alcohol 70% vol/vol, swabs), and 1 drop of

Viscotears liquid gel (Carbomer 980, 0.2%; Novartis, UK) was applied onto the tip

of the lens and advanced forward until the gel touched the cornea thus allowing

visual but not physical contact between the lens and corneal epithelium (Figure

2.10). The cornea was scanned in 2 min and en face two-dimensional images were

acquired. Approximately three to five high-quality images of Bowman’s layer were

examined as it contains the main nerve plexus (Figure 2.11 and 2.12). When

enough images had been captured the procedure was repeated for the other eye.

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Morphometric measurements of the images were undertaken and the assessors of

the images were masked with respect to the identity of the patient.

Figure 2-10 Positioning of the TomoCap® on the cornea

Figure 2-11 Image of the corneal subbasal nerves using corneal confocal microscopy

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Figure 2-12 A preview of an image from the corneal subbasal nerves on the Heidelberg eye

explorer with a simultaneous view of the location of the image through the CCD camera.

2.4.13 Manual Image Analysis: Chapter 4,5 & 8

Images are analysed manually by the assessor using purpose built software called

“CCM image analysis tools v0p6” (CCMIATv0p6) (M.A. Dabbah, Imaging Science,

The University of Manchester). The following parameters were quantified to define

corneal nerve fibre damage and repair: 1) nerve fibre density (CNFD), the total

number of major nerves per square millimetre of corneal tissue; 2) nerve fibre

length (CNFL), the total length of all nerve fibres and branches per square

millimetre of corneal tissue; 3) nerve branch density (CNBD), the number of

branches emanating from each nerve trunk per square millimetre of corneal tissue;

and 4) nerve fibre tortuosity (CNFT), a parameter that is mathematically derived

from the images (4).

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Figure 2-13 An CCM image of a control subject analysed to quantify corneal subbasal nerve

morphology in DSPN. The red colour corresponds to CNFD and the CNFT, the green dots

highlight CNBD at the points of junction with main nerve fibres and CNFL, the length of

nerve structures in the entire CCM image is highlighted under the red and blue colour.

2.4.14 Automated Image Analysis: Chapter 3 only

Automated corneal nerve fibre quantification consists of two steps: (1) CCM image

enhancement and nerve fibre detection and (2) quantification of CNFD, CNBD and

CNFL. The detection of nerve fibre is a challenging task, as the nerve fibres often

show poor contrast in the relatively noisy images. As described in our earlier work

(5-6), a dual-model feature descriptor combined with a neural network classifier

was used to train the computer to distinguish nerve fibres from the background

(noise and underlying connective tissue). In the nerve fibre quantification process,

all the end points and branch points of the detected nerve fibres are extracted and

used to construct a connectivity map. Each segment in the connectivity map can

then be connected and classified as main nerve fibres or branches according to the

nerve intensity, orientation and length.

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2.5 METHODS: Vitamin D Studies – Chapters 6, 7 & 9

Studies were undertaken from a population-based cohort at the Central

Manchester University Hospitals NHS Foundation Trust, Manchester, UK of those

with Type 1 and 2 DM attending a tertiary diabetes centre. Ethical approval was not

required for this specific analysis as the data were extracted retrospectively from

clinical databases and did not extend beyond standard clinical practice.

Retrospective vitamin D studies undertaken are as follows:

a. Chapter VI: Marked vitamin D deficiency in patients with diabetes from

secondary care: Ethnic and seasonal differences and an association with

dyslipidaemia.

b. Chapter VII: Differential effects of different vitamin D replacement strategies

in patients with Diabetes

c. Chapter IX: Vitamin D Deficiency is not associated with Diabetic

Retinopathy or Maculopathy in Diabetes Mellitus

2.5.1 Participant Exclusion Criteria for Chapters 6, 7 & 9

Those with renal impairment (eGFR <30 mL/min/1.73m2 (CKD stage 4 and below),

granulomatous diseases (tuberculosis, sarcoidosis etc), malabsorption syndromes

(Coeliac disease, bacterial overgrowth, concomitant orlistat treatment), pregnant

and lactating women.

2.5.2 Blood pressure and Anthropometric measurements

Body mass index (BMI) was measured as per the standard equation (mass

(kg)/(height(m))2. Weight was measured with a digital scale (Seca 701, Seca,

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Hamburg, Germany) to the nearest 0.1kg and height to the nearest 0.1cm. Blood

pressure (BP) measurements were obtained with the use of an automated BP

device (Dinamap pro 100v2, GE Medical Systems, Freiburg, Germany) with an

appropriate cuff size. A minimum of two measurements of systolic and diastolic BP

were made five minutes apart with the lowest reading recorded.

2.5.3 Assessment of Demographics, Cardiovascular disease and

Medications

An assessment of patient demographics, previous cardiovascular events and

medications were made through analysis of medical records and an in-hospital

medical record database (Diamond database, Hicom, Surrey, UK). The subject

demographics extracted were age, sex, ethnicity (Caucasian, South Asian, Far

East Asian and Afro-Caribbean descent), smoking status (never, previous and

current) and type (Type 1 and 2 diabetes) and duration of diabetes. Previous

cardiovascular events extracted were ischaemic heart disease (IHD), myocardial

infarction (MI), cerebrovascular accidents (CVA) and peripheral vascular disease

(PVD).

2.5.4 Laboratory Measurements

Standard assessment of 25(OH)D was instituted in the Manchester Diabetes

Centre, Manchester, UK in August 2009 as part of routine haematological and

biochemical laboratory measurements which included HbA1c, (Complete Blood

count (CBC)), Urea and Electrolytes (UE), Liver function tests (LFT), bone profile

(Corrected Calcium (CCa2+), Alkaline Phosphatase (ALP), Albumin (Alb), and

Lipid profile (Total Cholesterol (T-CHL), High Density lipoprotein Cholesterol

(HDL), Triglycerides (TRIG)). Low Density lipoprotein (LDL) analyses were not

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undertaken and patients were routinely advised that bloods were to be collected

under fasting conditions.

2.5.5 Diabetic Retinopathy Assessment

The retinopathy data were collected according to the grading criteria set out to a

national standard by the National Screening Committee (NSC)(7-8). Previous

studies have shown acceptable levels of quality and accuracy of grading compared

to expert graders within the English NSC (9). The national guidelines do not

contain R1.5 or M0.5 grades and are categorised as Pre-Proliferative Diabetic and

Diabetic Maculopathy being present respectively. These sub-gradings are used

locally in screening centres have thus been included. The grading of retinopathy

are as follows:

R0 - No Diabetic retinopathy (NDR)

R1 - Background Diabetic Retinopathy (NDR): microaneurysms, retinal

haemorrhages, exudates

R1.5 - Moderate numbers of intra-retinal haemorrhages, hard exudates >1 disc

diameter (DD) from fovea, 3-6 cotton wool spots visible

R2 - Pre-Proliferative Diabetic Retinopathy (PPDR): Venous beading or looping,

deep haemorrhages visible, other microvascular anomaly visible.

R3 - Proliferative Diabetic Retinopathy (PDR): New vessel formation, vitreous

haemorrhage, pre- retinal haemorrhage or fibrosis and/or retinal detachment.

The outlines for maculopathy grading are as follows:

M0 - No maculopathy.

M0.5 - Hard exudates within the arcades >1DD from the centre of the fovea.

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M1- Exudates <1DD within the centre of the fovea, retinal thickening <1DD of the

centre of the fovea.

The outlines for photocoagulation grading are as follows:

P0 – No photocoagulation scarring

P1 – Photocoagulation scarring present

2.5.6 25(OH) Vitamin D Assay

The specific laboratory used for the biochemical assay measurements (Vitamin D

Research Group Manchester Royal Infirmary, UK) was accredited to ISO

9001:2008 and ISO 13485:2003 by Lloyd’s Register Quality Assurance, certificate

number LRQ 4001542 and participated successfully in the Vitamin D quality

assurance scheme (DEQAS). Serum was separated from whole blood and stored

at -20 C until assay. The assay used was an automated platform assay

(ImmunoDiagnostic Systems Ltd, Bolden, Tyne and Wear, UK) and is based on

chemiluminescence technology. Briefly, samples were subjected to a pre-treatment

step to denature the vitamin D binding protein. The treated samples were then

neutralised in assay buffer and a specific anti-25(OH)D antibody labelled with

acridinium was added. Following an incubation step, magnetic particles linked to

25(OH)D were added. Following a further incubation step, the magnetic particles

were “captured” using a magnet. After a washing step and addition of trigger

reagents, the light emitted by the acridinium label was inversely proportional to the

concentration of 25(OH)D in the original sample. The concentration of 25(OH)D

was calculated automatically using a 4-point logistic curve. The cross reactivity for

vitamin D2 (of the assay) as per manufacturers assertion was 100% (relative to

vitamin D3) and the assay has excellent correlation to existing globally recognised

136

assays, in combination with good sensitivity and precision (10). The reportable

range of the assay was 5-140 ng/mL. Inter- and intra- assay variation of the in-

house control was 5.6% and 9.7% respectively. Vitamin D deficiency (<20ng/ml)

and insufficiency (<30ng/ml) were defined according to the Institute of Medicine

(IOM) of the National Academies (11).

2.6 REFERENCES

1. Boulton AJM: Management of Diabetic Peripheral Neuropathy. Clinical Diabetes

2005;23:9-15

2. Lauria G, Hsieh ST, Johansson O, Kennedy WR, Leger JM, Mellgren SI, Nolano

M, Merkies IS, Polydefkis M, Smith AG, Sommer C, Valls-Sole J: European


use of skin biopsy in the diagnosis of small fibre neuropathy. Report of a joint task



3. Murphy PJ, Lawrenson JG, Patel S, Marshall J: Reliability of the Non-Contact

Corneal Aesthesiometer and its comparison with the Cochet–Bonnet

aesthesiometer. Ophthalmic and Physiological Optics 1998;18:532-539

4. Kallinikos P, Berhanu M, O'Donnell C, Boulton AJM, Efron N, Malik RA: Corneal

Nerve Tortuosity in Diabetic Patients with Neuropathy. Invest Ophthalmol Vis Sci

2004;45:418-422

5. Dabbah MA, Graham J, Petropoulos IN, Tavakoli M, Malik RA: Automatic

analysis of diabetic peripheral neuropathy using multi-scale quantitative

morphology of nerve fibres in corneal confocal microscopy imaging. Medical Image

Analysis 2011;15:738-747

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6. Dabbah M, Graham J, Petropoulos I, Tavakoli M, Malik R: Dual-Model Automatic

Detection of Nerve-Fibres in Corneal Confocal Microscopy Images

Medical Image Computing and Computer-Assisted Intervention – MICCAI 2010.

Jiang T, Navab N, Pluim J, Viergever M, Eds., Springer Berlin / Heidelberg, 2010,

p. 300-307


diabetic retinopathy. J Med Screen 2008;15:1-4

8. Jyothi S, Elahi B, Srivastava A, Poole M, Nagi D, Sivaprasad S: Compliance with

the quality standards of National Diabetic Retinopathy Screening Committee. Prim

Care Diabetes 2009;3:67-72

9. Patra S, Gomm EMW, Macipe M, Bailey C: Interobserver agreement between

primary graders and an expert grader in the Bristol and Weston diabetic

retinopathy screening programme: a quality assurance audit. Diabetic Medicine

2009;26:820-823

10. ASBMR 31st Annual Meeting MO0001–MO0445. Journal of Bone and Mineral

Research 2009;24:S370-S496

11. Rosen CJ, Gallagher JC: The 2011 IOM report on vitamin D and calcium

requirements for north america: clinical implications for providers treating patients

with low bone mineral density. J Clin Densitom 2011;14:79-84

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3 Chapter III - Rapid Nerve Fibre Decline in patients with

Type 1 diabetes can be readily detected using Corneal

Confocal Microscopy.

Contribution: Uazman Alam contributed to the conception and design of the study

and made a major contribution to the recruitment of subjects, clinical neuropathy

assessments, all statistical analyses and writing of this chapter.

To be submitted for publication

Uazman Alam, Omar Asghar, Ioannis N Petropoulos, Hassan Fadavi, Georgios

Ponirakis, Maria Jeziorska, Andrew Marshall, Mitra Tavakoli, Andrew JM Boulton,

Nathan Efron, Rayaz A Malik.

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3.1 ABSTRACT

Diabetic neuropathy is associated with increased morbidity and is an independent

risk factor for mortality. Defining a population which has progressive rather than

stable neuropathy would allow risk stratification and also perhaps identify those

who may respond optimally to therapeutic intervention. We have assessed whether

corneal confocal microscopy (CCM) can detect progression of neuropathy by

establishing a decline in corneal nerve morphology.

Fifty subjects with Type 1 diabetes (T1DM) and sixteen non-diabetic healthy

control subjects (C) underwent assessment of neurologic deficits; quantitative

sensory testing (QST), electrophysiology and CCM at baseline and two year follow

up.

Neuropathic symptom profile (NSP) (P<0.0006), McGill pain index (P=0.008),

neuropathy disability score (NDS) (P=0.008), HbA1c (P<0.0001), and vibration

perception threshold (VPT) (P<0.0001) were significantly increased and sural

nerve conduction velocity and amplitude (P<0.0001 and P=0.0003 respectively),

peroneal nerve conduction velocity and amplitude (P<0.0001 and P=0.0003

respectively) were significantly reduced in T1DM compared to controls. Corneal

nerve fibre density (CNFD) (P<0.0001), corneal nerve branch density (CNBD)

(P=0.007) and corneal nerve fibre length (CNFL) (P<0.0001) were significantly

reduced at baseline in T1DM compared with control subjects. There was no

significant change for: NDS, vibration perception threshold (VPT), warm sensation

threshold (WST), electrophysiology and CCM measures in both T1DM and controls

at 2 year follow up. However, we identified 11/50 T1DM patients who had a greater

than 14.4% decline in CNFL over 2 years based on a 2 standard deviation cut off

for intra-individual variation in the control group. In this cohort: sural sensory

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(P=0.04) and peroneal motor (P=0.05) nerve conduction velocities, CNFD

(18.8±10.2 vs 11.5±9.4 no./mm2, P=0.0006), CNBD (24.1±15.6 vs 10.7±12.8

no./mm2, P=0.0002) and CNFL (12.1±5.0 vs 8.5±4.5 mm/mm2, P=0.0002) were

significantly reduced from baseline to follow up after 2 years, which was not related

to any change in HbA1c, lipids or BP.

A rapid small fibre decline is readily detectable in a subgroup of subjects with Type

1 diabetes using CCM and this novel modality provides an ideal surrogate endpoint

in defining those who at risk of progressive DPN.

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3.2 INTRODUCTION

Diabetic peripheral neuropathy (DPN) is a distressing condition which affects

around one half of patients during the natural history of the disease (1) although

prevalence data varies (2-3). DPN is a confirmed risk factor for debilitating lower

limb amputation (4) and large nerve fibre dysfunction in diabetes is itself predicted

by cardiovascular disease (5) and is one of only three independent and significant

risk factors for mortality as confirmed through the Action to Control Cardiovascular

Risk in Diabetes (ACCORD) study (6). Risk factors for DPN include duration of

diabetes, glycaemic control and elevated triglycerides (7) and improvement of

glycaemic control as shown through the Diabetes Control and Complications Trial

(DCCT) and the Epidemiology of Diabetes Interventions and Complications (EDIC)

to improve neuropathy outcomes both in the short and long term respectively (8-9).

An analysis of a placebo control arm in a multi-centre, double-blind study showed

that short term metabolic improvements in glycaemic control and serum triglyceride

levels have an independent, additive and durable effect on restoration of nerve

function as evaluated by neurophysiology (10). We have previously shown that

improvements in corneal nerve fibre density (CNFD) occur with an improvement in

HbA1c (11) and corneal nerve fibre density (CNFD) and branch density (CNBD)

improves with simultaneous pancreas and kidney transplantation (12). Smith et al

(13) also showed that intra-epidermal nerve fibre density (IENFD) improved with

diet and exercise in a pre-diabetic group. Deterioration in small nerve fibres may

progress overtime and indeed a postulated longitudinal decline in IENFD (14) and

small fibre function with diminished axon reflex-mediated vasodilator response is

thought to occur (15). Longitudinal studies of neuropathy are limited; however, they

do show deterioration of neuropathy overtime in both Type 1 diabetes (16) and

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Type 2 diabetes using electrophysiology (17). The progression of DPN is thought

to occur much slower than previously thought and is likely in part due to the

positive impact on the peripheral nerve from commonly prescribed agents such as

ACE inhibitors (18) and statins (19). This lack of placebo decline is of course one

of the postulated reasons for the dramatic failure of pathogenetic treatments for

DPN and Dyck et al concluded that future clinical trials should include subjects who

are developing neuropathy rather than those with established neuropathy (20).

In the current study, we evaluated a cohort of subjects with Type 1 Diabetes at

baseline and 2 year follow up in relation to their neuropathy status with the aim of

delineating subjects who have rapidly progressive DPN through the novel modality

of CCM.

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3.3 RESEARCH DESIGN AND METHODS

3.3.1 Selection of patients

Fifty subjects with Type 1 diabetes (T1DM) (n=50) and sixteen age and sex

matched non-diabetic healthy control subjects (C) (n=16) were assessed at

baseline and two year follow up. Control subjects were selected through the

University of Manchester staff network or friends and family of diabetic participants.

Both diabetic patients and control subjects underwent full neurologic assessment

and were included in final analyses on completing the protocol of baseline and 2

year follow up visit. Retinopathy status evaluation through retinal photography was

not a part of the experimental protocol. All causes of peripheral neuropathy (except

diabetes in T1DM) and those with a history of ocular trauma or previous ocular

surgery were excluded. This study was of an observational nature only with no

active study intervention although alterations to anti-glycaemic, anti-hypertensive

and lipid modifying medication may have been instituted either in primary or

secondary care. The study was approved by the North West Research Ethics

committee, and written informed consent was obtained according to the

Declaration of Helsinki.

3.3.2 Assessment of neuropathy

All patients and control subjects underwent a detailed evaluation of neurologic

symptoms according to the neuropathy symptom profile (NSP), and the McGill

visual analogue score (VAS) was used to assess the severity of painful

neuropathy. Clinical neurologic deficits were assessed using the modified

neuropathy disability score (NDS), which includes evaluation of vibration, pin prick,

and temperature perception as well as the presence or absence of ankle reflexes

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to establish the severity of neuropathy (21). Quantitative sensory testing included

an assessment of vibration perception threshold (VPT), measured using a

Neurothesiometer (Horwell, Scientific Laboratory Supplies, Wilford, Nottingham,

U.K.), cold sensation threshold (CST) (Aδ fibres) and warm sensation threshold

(WST) (22) (c fibres) using the method of limits with the MEDOC TSA II (Medoc,

Ramat Yishai, Israel) on the dorsum of the left foot.

Electro-diagnostic studies were undertaken using a Dantec “Keypoint” system

(Dantec Dynamics, Bristol, U.K.) equipped with a DISA temperature regulator to

keep limb temperature constantly between 32°C and 35°C. Peroneal motor and

sural sensory nerves were assessed in the right lower limb by a consultant

neurophysiologist. The motor study was performed using silver-silver chloride

surface electrodes at standardized sites defined by anatomical landmarks, and

recordings for the sural nerve were taken using antidromic stimulation over a

distance of 100 mm.

3.3.3 Corneal Confocal Microscopy

Patients underwent examination with the Heidelberg retina tomography III in vivo

corneal confocal microscope employing our established methodology for image

acquisition (23). Several scans of the entire depth of the cornea were recorded by

turning the fine focus of the objective lens backward and forward for ~2 min using

the section mode, which enables manual acquisition and storage of single images

of all corneal layers. This provides en face two-dimensional images with a lateral

resolution of ~2 mm/pixel and final image size of 400 x 400 pixels of the subbasal

nerve plexus of the cornea from each patient and control subject. Each sub-basal

nerve fibre bundle contains unmyelinated fibres, which run parallel to Bowman’s

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layer before dividing and terminating as individual axons underneath the surface

epithelium. Five images per patient from the centre of the cornea were selected

and examined in a masked and randomised fashion (24). Three corneal nerve

parameters were quantified: 1) CNFD, the total number of major nerves per square

millimetre of corneal tissue (no.mm2); 2) corneal nerve branch density (CNBD), the

number of branches emanating from all major nerve trunks per square millimetre of

corneal tissue (no.mm2); and 3) corneal nerve fibre length (CNFL), the total length

of all nerve fibres and branches (mm/mm2) within the area of corneal tissue.

Automated corneal nerve fibre quantification (ACCMetrics software, University of

Manchester, Manchester, UK) was undertaken and consists of two steps: (1) CCM

image enhancement and nerve fibre detection and (2) quantification of three

morphometric parameters i.e. CNFD, CNBD and CNFL. In the nerve fibre

quantification process, all the end points and branch points of the detected nerve

fibres are extracted and used to construct a connectivity map. Each segment in the

connectivity map can then be connected and classified as main nerve fibres or

branches according to the nerve properties such as intensity, orientation and

length.

3.3.4 Statistical analysis and Power calculation

Previous natural history studies are lacking in assessing CCM parameters in the

progression of diabetic neuropathy and thus power calculations are based on

unpublished data. For the diabetic cohort, an assumption of paired groups was

used to calculate the sample size considering a change in CNFD over 2 years. The

standard deviation between groups is likely to be 9 nerves/mm2. Therefore,

recruiting a minimum of 28 patients for the diabetic group will provide 80% chance

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to detect a clinically meaningful change in CNFD of 5 nerves/mm2 and an

assumption of a type 1 error (α-level) of 0.05. Considering a greater change of 7.5

nerves/mm2 at 80% power to detect a clinically meaningful change, 14 patients

would be required with an assumption of type 1 error of 0.05. Statistical analyses

were undertaken on Statsdirect (Statsdirect, Cheshire, UK). The data are

expressed as Mean ± standard deviation (SD). Paired t-test or a non-parametric

counterpart, Mann-Whitney-U were used to assess differences between baseline

and follow up data on normality of the data. Unpaired t-test or Mann-Whitney-U test

were used to assess differences between baseline control and T1DM data,

depending on normality of the data. Chi squared analyses were used to assess

frequencies of gender and ethnicity. A significant P value was considered to be

≤0.05 corrected for multiple comparison tests. A post hoc analysis was undertaken

of those showing a worsening neuropathy through assessment of decline in CNFL

defined as rapid nerve fibre decline based on a two standard deviation cut off of

CNFL variation over 2 years in the control group.

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3.4 RESULTS

3.4.1 Demographics (Table 3.1)

The participant demographics and metabolic and anthropometric measurements in

diabetic subjects and age-matched control subjects are summarized in Table 3.1.

There were no significant differences in age (Control baseline: 41.4±11.4 vs T1DM

baseline: 48.2±15.5 years, P=NS) and duration of diabetes was 33.1±16.7 years at

baseline in the T1DM group. There were a no differences in gender between

controls and T1DM but there were a higher proportion of white Europeans in T1DM

(Chi2 P<0.0001).

148

Table 3-1 Participant demographics and clinical neuropathy parameters in control subjects

and patients with T1DM at baseline and 2 year follow up, with statistically significant

differences between groups

Control

BL (n=16)

Control FU

(n=16) P

T1DM

BL (n=50)

T1DM FU

(n=50) P

Age (years) 41.4±11.4 48.2±15.5

Gender

(Male) (%) 63 54

Ethnicity

(White

European)

(%)

75 96

Duration of

Diabetes

(years)

Median(IQR)

N/a 33.1±16.7

NDS (-/10)

Median(IQR)

0.5±1.1*

0(0-0)

0.4±1.1

0(0-0) NS

3.4±3.5*

2(0-6)

3.3±3.6

2(0-6) NS

NSP (-/38)

Median(IQR)

0.1±0.25**

0(0-0)

0

0(0-0) NS

3.8±5.3**

1(0-5)

4.1±6.7

1(0-5.5) NS

McGill VAS

(-/10cm)

Median(IQR)

0.3±1.25†

0(0-0)

0

0(0-0) NS

2.3±3.3†

0(0-4)

1.5±3.0

0(0-0) NS

Post hoc analyses (Control Baseline vs T1DM Baseline)

* P=0.0006; **P= <0.0001; † P=0.008.

Table key

BL – Baseline; FU – Follow Up; McGill VAS – McGill Visual Analogue Score; NDS – Neuropathy

Disability Score; NSP – Neuropathy Symptom Profile.

149

3.4.2 Metabolic and Anthropometric measurements (Table 3.2)

BMI did not differ between patients with T1DM and control subjects at baseline.

HbA1c was significantly higher in T1DM (P<0.0001) compared to controls with no

significant change at 2 year follow up. Although, there was a significant reduction in

HbA1c in controls at follow up, both baseline and follow up values were in the

normal non-diabetic range. The total cholesterol was significantly lower (P<0.0001)

in diabetic patients due to greater statin use, whilst triglycerides and HDL were

comparable between T1DM and control subjects with no changes at follow up.

Systolic BP was no different in controls compared to T1DM at baseline. T1DM

(P=0.04) exhibited a mild drop in systolic BP at 2 year follow up with no change in

controls at baseline and follow up. There were no differences in the estimated

glomerular filtration rate between T1DM and controls at baseline, however, there

was a decline in eGFR in T1DM at 2 year follow up (P=0.02).

150

Table 3-2 Metabolic parameters in control subjects and T1DM patients at baseline and 2 year

follow up, with statistically significant differences between groups

Control

BL (n=16)

Control FU

(n=16) P

T1DM

BL (n=50)

T1DM FU

(n=50)

P

HbA1c (%) 5.6±0.3 5.3±0.3

0.003

8.2±1.3* 8.1±1.6

NS HbA1c

(mmol/mol) 38.0±3.3 34.8±3.4 66.2±14.3* 65.2±17.1

BMI (kg/m2) 25.7±4.2 24.7±3.8 NS 26.9±4.4 27.1±4.7 NS

T-CHL

(mmol/l) 5.1±0.9 4.8±0.7 NS 4.3±0.9** 4.3±0.9 NS

HDL (mmol/l) 1.5±0.3 1.5±0.3 NS 1.7±0.5 1.7±0.5 NS

Triglycerides 1.4±0.7 1.2±0.5 NS 1.1±0.7 1.1±0.5 NS

Systolic BP

(mmHg) 125±21 120±18 NS 132±18 127±21 0.04

Diastolic BP

(mmHg) 75±12 74±11 NS 73±8 66±9 0.004

eGFR

(ml/min/1.73) 86±7 83±7 NS 81±19 75±17 0.02


*P<0.0001; ** P=0.003.

Table key

BL – Baseline; BMI – Body Mass Index; BP – Blood Pressure; eGFR – estimated Glomerular

Filtration Rate; FU – Follow Up; HbA1c – Glycated Haemoglobin A1c; HDL – High Density

Lipoprotein Cholesterol; T-CHL – Total Cholesterol.

151

3.4.3 Neuropathy evaluation (Table 3.3)

3.4.3.1 Symptoms and Deficits

Neuropathic symptoms as assessed with the NSP were significantly greater in

diabetic patients than in control subjects at baseline (P<0.0006), but there was no

significant change at 2 year follow up for either controls or T1DM. The McGill pain

index was significantly (P=0.008) greater at baseline compared with control

subjects and did not show a significant change at 2 years follow up in T1DM. The

modified NDS was significantly (P=0.008) greater at baseline compared with

control subjects, indicating a mild to moderate neuropathy, and did not change

significantly at 2 year follow up in T1DM.

3.4.3.2 Vibration Perception and Thermal Thresholds

VPT was significantly greater in T1DM (P<0.0001) compared with control subjects

at baseline with no significant change from baseline to 2 year follow up in either

controls or T1DM. CST was significantly greater in T1DM (P=0.002) compared with

control subjects at baseline and further increased at 2 year follow up in T1DM

(P=0.02) although the latter isolated change may be due to chance. WST did not

differ between T1DM and control subjects at baseline and did not change at follow

up.


Sural nerve conduction velocity and amplitude were significantly lower (P<0.0001

and P=0.0003 respectively) at baseline in T1DM compared with control subjects.

Peroneal nerve conduction velocity and amplitude were significantly lower

(P<0.0001 and P=0.0003 respectively) at baseline in T1DM compared with control

152

subjects. There were no significant changes in electrophysiology from baseline to 2

year follow up in either controls or T1DM.

3.4.3.4 Corneal sensation and structure

NCCA was significantly greater in T1DM compared to control subjects and did not

progress over 2 years of follow up in either T1DM or controls. CNFD (P<0.0001),

CNBD (P=0.007) and CNFL (P<0.0001) were reduced in T1DM compared to

controls at baseline with no significant changes at 2 year follow up in either

controls or T1DM.

153

Table 3-3 Small and large fibre tests of nerve structure and function in control subjects and

diabetic patients at baseline and 2 year follow up, with statistically significant differences

between groups.

Control

BL (n=16)

Control FU

(n=16) P

T1DM

BL

(n=50)

T1DM FU

(n=50)

P

NCCA

(mBar) 0.6±0.3 0.6±0.4 NS 1.4±2.3† 1.5±2.3 NS

CNFD

(no/mm2) 30.1±4.9 28.3±5.5 NS 19.5±9.1* 18.7±9.9 NS

CNBD

(no/mm2) 36.6±15.5 39.3±18.0 NS 23.9±15.0** 22.6±15.8 NS

CNFL

(mm/mm2) 16.9±2.8 16.6±3.0 NS 12.0±4.6*** 12.1±5.0 NS

CST (˚C) 28.6±2.1 26.8±4.8 NS 24.8±6.9˚ 22.5±8.4 0.02

WST(˚C) 37.6±3.5 38.9±3.8 NS 39.6±4.6 40.2±5.7 NS

VPT (volts) 5.3±4.8 6.0±5.2 NS 15.5±13.6ª 15.9±12.6 NS

SSNCV

(m/s) 49.4±3.9 48.1±4.7 NS 43.6±6.3

ƒ 41.3±6.4 NS

SSNAmp

(µV) 21.0±10.8 18.6±8.7 NS 10.1±6.7

ƒƒ 8.7±6.8 NS

PMNCV

(m/s) 47.8±3.6 47.3±4.2 NS 40.6±7.0^ 40.0±6.6 NS

PMNAmp

(mV) 6.0±1.6 5.8±1.2 NS 3.4±2.4^^ 3.2±2.0 NS


†P=0.02; *P<0.0001; **P=0.007; ***P<0.0001; ˚P=0.002; ªP<0.0001; fP<0.0001;

ƒƒP=0.0003;

^P<0.0001; ^^P=0.0003.

Table key

BL – Baseline; CNBD – Corneal Nerve Branch Density; CNFD – Corneal Nerve Fibre Density;

CNFL – Corneal Nerve Fibre Length; CST – Cold Sensation Threshold; FU – Follow Up, PMNAmp

– Peroneal Motor Nerve Amplitude; PMNCV – Peroneal Motor Nerve Conduction Velocity;

SSNAmp – Sural Nerve Sensory Nerve Amplitude; SMNCV – Sural Nerve Conduction Velocity;

VPT – Vibration Perception Threshold; WST – Warm Sensation Threshold.

154

3.4.3.5 Rapid Nerve Fibre Decline (RNFD) in T1DM

(Table 3.4, Figures 3.1 & 3.2)

The mean intra-individual change in CNFL over the 2 year follow up in the control

group was 7.9±7.2%. A 2 standard deviation intra-individual change is therefore

14.4%. We thus used this parameter as a cut off for RNFD in the T1DM group. We

identified eleven subjects (n=11) out of fifty in the T1DM group who had a greater

than 14.4% decline in CNFL over 2 years. This analysis is based on the

unpublished data presented by Professor Bruce Perkins (at the American Diabetes

Association conference 2013) in which rapid nerve fibre decline was defined as a

change in CNFL of -14% (25). The mean intra-individual change in CNFL over 2

year follow up in the T1DM group was 27.8±32.4%.

There were no significant differences for age HbA1c, BMI, T-CHL and systolic BP

in those with RNFD. There were no significant changes in VPT (Baseline:

17.5±13.1 vs 2 yr FU: 19.7±14.2 volts, P=NS), CST (Baseline: 25.5±4.0 vs 2 year

FU: 23.3±4.4 (˚C), P=NS) and WST (Baseline: 39.2±4.2 vs 2 year FU: 40.1±4.3

(˚C), P=NS).

There were concomitant reductions at 2 year follow up in sural sensory and

peroneal motor nerve conduction velocities (P=0.04 and P=0.05 respectively) with

no change in sural and peroneal amplitudes over the same period.

All measures of CCM were significantly reduced at follow up: CNFD (Baseline:

18.8±10.2 vs 2 yr FU: 11.5±9.4 no./mm2, P=0.0006), CNBD (Baseline: 24.1±15.6

vs 2 yr FU: 10.7±12.8 no./mm2, P=0.0002) and CNFL (Baseline: 12.1±5.0 vs 2 yr

FU: 8.5±4.5 mm/mm2, P=0.0002) (Figure 3.1a, b & c). Figure 3.2a & b shows

images from a subject with RNFD at baseline and 2 year follow up. In assessing

155

subjects with reductions in peroneal motor and sural sensory nerve conduction

velocity, concomitant reductions in CNFD, CNBD and CNFL were also noted,

however data are analysed based on those with a reduction of 14.4% in CNFL or

greater.

156

Table 3-4 T1DM subjects with Rapid Nerve Fibre Decline at baseline and 2 year follow up

with statistically significant differences

T1DM BL

(n=11)

T1DM FU

(n=11) P

Age (years) 54.8±9.2 N/a -

HbA1c (%) 7.9±0.6 7.7±1.0 NS

BMI (kg/m2) 27.9±3.3 28.8±4.3 NS

T-CHL (mmol/l) 4.6±1.2 4.6±1.3 NS

Systolic BP (mmHg) 141±20 132±24 NS

SSNCV (m/s) 42.7±6.1 38.1±7.0 0.04

SSNAmp (µV) 9.3±8.7 6.3±5.8 NS

PMNCV (m/s) 42.4±3.3 41.1±4.2 0.05

PMNAmp (mV) 2.8±2.1 3.0±1.5 NS

Table key

BMI – Body Mass Index; BP – Blood Pressure; PMNAmp – Peroneal Motor Nerve Amplitude;

PMNCV – Peroneal Motor Nerve Conduction Velocity; SSNAmp – Sural Sensory Nerve Amplitude;

SMNCV – Sural Nerve Conduction Velocity; T-CHL – Total Cholesterol; VPT – Vibration Perception

Threshold.

157

Figure 3-1a, b & c. CNFD, CNBD and CNFL and baseline and 2 year follow up.

C

B

A

158

Figure 3-2 a & b. CCM images of a subject with RNFD at baseline and 2 year follow up

CCM progressionBaseline (A) vs. 2 Years (B)

A B

Nerve Branches

Nerve Fibres

159

3.5 DISCUSSION

The major findings of this study are: 1) Type 1 diabetes patients show no

significant progression of neuropathy over 2 years; 2) A subgroup of patients with

Type 1 diabetes shows rapidly progressive and readily detectable decline in

corneal nerve fibre structure using CCM 2) and 3) CCM provides an ideal

surrogate endpoint for the assessment and progression of diabetic neuropathy.

These findings are both of importance in identifying those who have progression of

DPN with subsequent risk of ensuing complications such as foot ulceration and

lower limb amputation but also mortality. Furthermore, it may also be important in

identifying patients who should be recruited into clinical trials of DPN as Dyck et al

(20) concluded that future trials of DPN should include those with developing

neuropathy and also select endpoints which show a discernible worsening. CCM

provides a seemingly suitable surrogate endpoint for the assessment of DPN and

has also shown an excellent correlation with the current gold standard IENFD (26).

At 2 years there were no significant changes in the majority of our diabetic subjects

and this is in keeping with the notion that DPN progresses much slower than

previously thought (27). This may provide a partial explanation for the failure of

pathogenetic treatments in clinical trials of DPN. Nerve conduction velocity is only

gradually diminished by DPN, with estimates of a loss of ~0.5m/s/ year (28). In the

10 year natural history study by Partanen et al (17), nerve conduction velocity

deteriorated in all nerve segments evaluated with the largest deficit of 3.9 m/s for

the sural nerve. The DCCT showed a similar slow progression of change in nerve

conduction velocity over a 5 year period (29). Previously, a placebo controlled

study has shown improvements in signs and VPT in the placebo-treated arm with a

small reduction in electrophysiological measures (30) and this has also been

160

demonstrated in another placebo controlled study (31). Both the study by Partanen

et al (17) and the DCCT (16) were undertaken prior to the widespread use of ACE

inhibitors and statins, both of which are likely to impact positively on the peripheral

nerve (18-19). Electrophysiology, of course focuses on large fibre function wherein

the primary pathology lies in small fibres and predates abnormalities in nerve

conduction studies (32). Therefore a measure of small fibre damage may be the

most sensitive to detect an early abnormality and progression. Small fibre

neuropathy in particular that of sudomotor dysfunction may result in dryness

of foot skin and has been associated with foot ulceration (33) and CCM provides

an ideal surrogate marker for small fibre neuropathy.

The Toronto criteria for DPN (32) advocates electrophysiology for epidemiologic

surveys or controlled clinical trials of DPN as an early and reliable indicator of the

occurrence of neuropathy. However, our study clearly shows a lack of concomitant

reductions in nerve amplitudes particularly sural amplitude which is a considered a

particularly valid and sensitive marker of nerve damage (34). Furthermore,

variability in measurements (intra-observer variability) can be a significant concern

when interpreting electrophysiological results, particularly in multi centre trials (35)

and short term improvements in metabolic control can cause significant increases

in nerve conduction parameters (10) although whether this correlates to actual

repair or enhancement of large nerve fibre structure is unknown. Our data shows

globally reduced measures of corneal nerve structure at baseline confirming

previous studies which have shown that corneal small nerve fibre damage can be

detected prior to abnormalities in electrophysiology and quantitative sensory

testing (26). CCM also has the ability to detect nerve fibre repair and regeneration

161

as evidenced by our recently published data on subjects undergoing simultaneous

pancreas and kidney transplantation with amelioration of Type 1 diabetes (12).

We have no obvious metabolic explanation for the basis of rapid nerve fibre decline

observed in the subset of patients with T1DM as there were no significant changes

in HbA1c, lipids and blood pressure in the overall T1DM cohort. This is clearly

different from the results of the Eurodiab study which showed that the development

of neuropathy was related to a range of cardiovascular risk factors (7). However,

the control group was relatively small and the cut offs used for RNFD may be

different if derived from a larger cohort and the duration of follow up was relatively

short.

In conclusion, we have shown that CCM can identify a subcohort of patients with

T1DM with a rapid degree of small fibre decline. Furthermore, given that we have

recently shown that CCM can detect early nerve fibre repair after SPK (12) this

adds to the evidence that CCM may be an ideal surrogate endpoint for clinical trials

of new therapies in diabetic neuropathy.

162

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threshold testing for the assessment of small fibre dysfunction: normal values and

reproducibility. Diabet Med 1992;9:546-549

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G, Dabbah MA, Chen X, Graham J, Tavakoli M, Malik RA: Repeatability of in vivo

corneal confocal microscopy to quantify corneal nerve morphology. Cornea

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technique to quantify small fibre pathology in peripheral neuropathies. J Vis Exp

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25. Halpern EM, Lovblom LE, Orlov S, Bril V, Perkins BA: The Existence of Rapid

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29. Effect of intensive diabetes treatment on nerve conduction in the Diabetes

Control and Complications Trial. Ann Neurol 1995;38:869-880

30. Tesfaye S, Tandan R, Bastyr EJ, 3rd, Kles KA, Skljarevski V, Price KL: Factors

that impact symptomatic diabetic peripheral neuropathy in placebo-administered

patients from two 1-year clinical trials. Diabetes Care 2007;30:2626-2632

31. Brown MJ, Bird SJ, Watling S, Kaleta H, Hayes L, Eckert S, Foyt HL: Natural

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33. Stewart JD, Low PA, Fealey RD: Distal small fiber neuropathy: results of tests

of sweating and autonomic cardiovascular reflexes. Muscle Nerve 1992;15:661-

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34. Freeman TL, Johnson E, Freeman ED, et al.: Nerve Conduction Studies

(NCS). S C, Ed. New York, Demos Medical Publishing, 2004

35. Keith M, Goldstein R, Nesathurai S, Graham W: Serial nerve conduction

studies of the tail of rhesus monkey Macaca mulatta and potential implications for

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167

4 Chapter IV - Diagnostic Utility of Corneal Confocal

Microscopy and Intra-Epidermal Nerve Fibre Density in

Diabetic Neuropathy


and made a major contribution to the recruitment of subjects, clinical tests including

neuropathy assessments and skin biopsies, skin biopsy analyses, all statistical

analyses and writing of this chapter.


Uazman Alam, Maria Jeziorska, Ioannis N Petropoulos, Omar Asghar, Hassan

Fadavi, Georgios Ponirakis, Andrew Marshall, Mitra Tavakoli, Andrew JM Boulton,

Nathan Efron, Rayaz A Malik

168

4.1 ABSTRACT

Corneal confocal microscopy (CCM) is a rapid, non-invasive, reproducible

technique that quantifies small nerve fibres. We have compared the diagnostic

capability of CCM against a range of established measures of nerve damage in

patients with diabetic neuropathy.

Thirty subjects with Type 1 diabetes without neuropathy (T1DM), thirty one

subjects with Type 1 diabetes and neuropathy (DSPN) and twenty seven non-

diabetic healthy control subjects underwent detailed assessment of neuropathic

symptoms and neurologic deficits, quantitative sensory testing (QST),

electrophysiology, skin biopsy and CCM.

Comparing subjects with DSPN and T1DM, those with DSPN they were older

(P=0.0002), had a longer duration of diabetes (P<0.0001), lower eGFR (P=0.006)

and higher albumin-creatinine ratio (ACR) (P=0.03) with no significant difference

for HbA1c, BMI, lipids and blood pressure. Patients with DSPN had more

symptoms (neuropathy symptom profile (NSP) (P<0.0001), McGill visual analogue

score (McGill VAS) (P=0.01) and McGill pain score (P=0.02), higher neuropathy

disability score (NDS) (P<0.0001), CST (P=0.007), WST (P=0.0004), vibration

perception threshold (VPT) (P<0.0001) and lower peroneal motor nerve conduction

velocity (PMNCV) and amplitude (PMNAmp) (P<0.0001 and P<0.0001), sural

sensory nerve conduction velocity (SSNCV) and amplitude (SSNAmp) (P<0.0001),

intra-epidermal nerve fibre density (IENFD) (P=0.001), corneal nerve fibre density

(CNFD) (P<0.0001), corneal nerve branch density (CNBD) (P=0.02) and corneal

nerve fibre length (CNFL) (P=0.001). CNFD correlated better with PMNCV

(Spearman’s Rho= 0.60 P<0.0001) than IENFD with PMNCV (Spearman’s Rho=

0.56 P<0.0001). For the diagnosis of diabetic neuropathy the sensitivity for CNFD

169

was 0.77 and specificity was 0.79 with Wilcoxon estimate of area under the ROC

curve of 0.81. IENFD had a diagnostic sensitivity of 0.61, specificity of 0.80 and

area under the ROC curve of 0.73.

CCM is a valid accurate non-invasive method which is superior to skin biopsy in

diagnosing DPN.

170

4.2 INTRODUCTION

Diabetic peripheral neuropathy is a debilitating condition which may lead to pain,

foot ulceration and eventual amputation (1). Therefore it is important to accurately

diagnose diabetic neuropathy at the earliest stage of damage. Clinical neurological

assessment is poorly reproducible (2), and whilst QST can measure both large and

small nerve fibre function it is subjective and requires an alert and motivated

subject. Nerve conduction studies remain the mainstay for the diagnosis and

progression of diabetic neuropathy (3). However, the earliest nerve fibres to

degenerate (4-5) and regenerate (6) are the small unmyelinated fibres and indeed

they are central to the genesis of pain and the development of foot ulceration (7).

In 2005 the European Federation of Neurological Societies published guidelines on

the use of skin biopsy in the diagnosis of peripheral neuropathies (8) and more

recently the value of the technique has been further emphasized (9). Currently,

skin biopsy with an assessment of intra-epidermal nerve fibres (IENF) is

considered the gold standard for the evaluation of small fibre neuropathy and has

been advocated as measure of treatment response in clinical trials (10). Previous

studies have demonstrated good diagnostic ability of skin biopsy for small fibre

neuropathy (11-14), however, these studies have assessed mixed populations of

small fibre neuropathy and the diagnostic utility of CCM versus IENF have not

been evaluated. Hence reproducible and reliable processing and accurate

quantification methods have been established for assessing IENF pathology

against normative ranges (15). However, despite being advocated as an endpoint

in clinical trials of diabetic neuropathy there is surprisingly scarce data which have

established the diagnostic ability of skin biopsy for diabetic neuropathy (7).

171

Furthermore skin biopsy is invasive with a small but significant risk for bleeding and

infection and requires expertise in laboratory assessment (16).

CCM is a non-invasive ophthalmic application, which is rapid, non-invasive and

readily reproducible for quantifying small nerve fibres and thereby has been shown

to diagnose and track the progression of diabetic neuropathy (17-19). CCM has

been shown to have reasonable diagnostic utility in detecting DPN diagnosed

using NDS (20) with good reproducibility (21). More recently CCM has been shown

to correlate with functional measures of small nerve fibre injury (22). However, the

differential ability of CCM against currently accepted gold standard FDA approved

methods such as skin biopsy, QST and nerve conduction studies for the diagnosis

of diabetic neuropathy has not been evaluated to date. We have therefore

compared the ability of CCM, QST and skin biopsy in the diagnosis of diabetic

neuropathy using the Toronto criteria (3).

172



Sixty one consecutive subjects with Type 1 diabetes were assessed and

subsequently divided into two groups based their neuropathy status. Patients were

recruited from general diabetes clinics in the Manchester Diabetes Centre, Central

Manchester Foundation Trust. Thirty subjects with Type 1 diabetes without

neuropathy (T1DM) (n=30), thirty one T1DM subjects with neuropathy (DSPN)

(n=31) and twenty seven non-diabetic healthy control subjects were studied.

Subjects with a history of neurologic conditions, ocular trauma or previous ocular

surgery were excluded. The study was approved by the North Manchester

Research Ethics committee, and written informed consent was obtained according

to the Declaration of Helsinki.

4.3.2 Definition of Neuropathy

Diabetic neuropathy was defined according to the Toronto criteria by the presence

of an abnormality of electrophysiology and a symptom or symptoms or a sign or

signs of neuropathy (3).



symptoms according to the NSP, and the McGill VAS was used to assess the

severity of painful neuropathy. Clinical neurologic deficits were assessed using the

modified neuropathy disability score, which includes an evaluation of vibration, pin

prick, and temperature perception as well as the presence or absence of ankle

reflexes (23). Quantitative sensory testing included an assessment of the VPT,

173

measured using a neurothesiometer (Horwell, Scientific Laboratory Supplies,

Wilford, Nottingham, U.K.), CST (Aδ fibres) and WST (24) (c fibres) thresholds

using the method of limits with the MEDOC TSA II (Medoc, Ramat Vishay, Israel)

on the dorsum of the left foot. Electro-diagnostic studies were undertaken using a

Dante “Key point” system (Dante Dynamics, Bristol, U.K.) equipped with a DISA

temperature regulator to keep limb temperature constantly between 32°C and

35°C. Peroneal motor and sural sensory nerves were assessed in the right lower

limb by a consultant neurophysiologist. The motor study was performed using

silver-silver chloride surface electrodes at standardized sites defined by anatomical

landmarks, and recordings for the sural nerve were taken using antidromic

stimulation over a distance of 100 mm.









nerve plexus of the cornea from each patient and control subject. Each sub-basal




and examined in a masked and randomized fashion (26). Three corneal nerve

174


millimetre of corneal tissue (no.mm2); 2) CNBD, the number of branches

emanating from all major nerve trunks per square millimetre of corneal tissue

(no.mm2); and 3) CNFL, the total length of all nerve fibres and branches (mm/mm2)

within the area of corneal tissue (27).

4.3.5 Skin biopsy and immunohistochemistry

Participants underwent a 3-mm punch skin biopsy from the dorsum of the foot; 2

cm above the second metatarsal head after local anaesthesia (1% lidocaine). The

biopsy site was closed using Steristrips, and the specimen was immediately fixed

in PBS-buffered 4% paraformaldehyde for 18-24 hours, washed - in Tris-buffered

saline, cryoprotected in sucrose, frozen in liquid nitrogen and stored at -80°C- and

subsequently cut into 50-μm sections on a cryostat microtome. Five floating

sections per subject were immunostained for PGP9.5 neuronal marker. Non-

specific protein binding and endogenous peroxidase activity were blocked by

incubation in 5% goat serum and 0.3% hydrogen peroxide, respectively. The anti-

PGP9.5 antibody (Milipore 1:1000; Billerica,MA) was followed first by goat anti-

rabbit IgG and then by HRP-Streptavidin (both diluted 1:1000, both from Vector

Laboratories, Peterborough, UK). Nerve fibres were visualised by SG chromogen

(Vector Laboratories). IENFD was calculated as the number of nerve fibres

crossing the basement membrane of the epidermis and expressed per millimetre

length of epidermis (28).

4.3.6 Statistical analysis

Statistical analyses were undertaken on Statsdirect (Statsdirect, Cheshire, UK).

The data are expressed as Mean ± standard deviation (SD). ANOVA method or a

175

non-parametric counterpart, Kruskal-Wallis were used to assess differences

between groups depending on normality of the data. The Mann-Whitney U test was

used to compare T1DM with DSPN for the duration of diabetes. Chi squared

analyses were used to assess frequencies of gender, and ethnicity. Overall the P

value was maintained at 0.05 for multiple comparison tests (Bonferoni adjustment

or Conover-Inmann pairwise comparison). Spearman’s rank correlation was

undertaken for CNFD, CNBD, CNFL and IENFD versus NDS, McGill VAS, NSP,

IENFD, thermal thresholds, VPT and nerve conduction studies. ROC curve

analyses were used to define the Wilcoxon estimate of area under ROC curve,

optimal cut offs with associated sensitivity and specificity for CCM parameters,

IENFD, VPT and thermal thresholds. Positive predictive value (PPV) and negative

predictive value (NPV) were calculated for the three diagnostic measures, which

had the greatest Wilcoxon estimate of area under the ROC curve.

176

4.4 RESULTS

4.4.1 Demographics, Metabolic and Anthropometric Assessment (Table 4.1)


diabetic patients and control subjects are summarized in Table 4.1. Those with

DSPN (53.3±11.9) were significantly older compared to controls (41.0±14.9 years,

P=0.0008) and T1DM (38.8±12.5, P<0.0001) and the duration of diabetes was

greater in DSPN compared to T1DM (P<0.0001). The non white European

ethnicities in table 4.1 were Asian with one participant of African origin in the

control and T1DM group. HbA1c (P<0.0001) was significantly higher in diabetic

patients compared with control subjects with no difference between patients with

T1DM and DSPN. Total cholesterol was significantly lower in diabetic patients with

T1DM (P=0.006) and DSPN (P=0.002) compared to control subjects. BMI, HDL,

triglycerides, systolic and diastolic blood pressure were comparable between

diabetic patients and control subjects. The estimated glomerular filtration rate

(eGFR) was lower in DSPN compared to T1DM (P=0.006), the Albumin-Creatinine

Ratio (ACR) was higher in DSPN compared to T1DM (P=0.03) and controls

(P=0.004), although median values for controls, T1DM and DSPN were all within

the normal range for ACR.

177

Table 4-1 Participant demographics and metabolic parameters in control subjects and

diabetic patients without (T1DM) and with neuropathy (DSPN), with statistically significant


C (n=27) T1DM (n=30) DSPN (n=31) T1DM v DSPN

Age (years) 41.0±14.9 38.8±12.5 53.3±11.9 0.0002

Gender (Male) (%) 59 43 61 -

Ethnicity (White

European) (%) 77 90 97 -

Aetiology of

Diabetes (Type 1

DM) (%)

- 100 100 NS

Duration of

Diabetes (years) - 17.2±12.0 37.2±13.1 <0.0001

HbA1c (%) 5.5±0.3 8.0±1.3 8.5±1.5 NS

HbA1c (mmol/mol) 36.9±3.4 61.0±21.0 70.0±17.0 NS

BMI (kg/m2) 26.9±4.0 26.3±4.4 27.2±4.2 NS

T-CHL (mmol/l) 5.0±0.8 4.4±0.9 4.3±0.9 NS

HDL (mmol/l) 1.6±0.4 1.6±0.4 1.6±0.5 NS

Triglycerides 1.3±0.6 1.2±0.8 1.3±0.7 NS

Systolic BP (mmHg) 128±18 126±17 132±22 NS

Diastolic BP

(mmHg) 70±10 71±10 72±9 NS

ACR (mg/mmol)

((Median)IQR)

0.4±0.4

(0.3(0.2-0.4))

0.7±0.9

(0.3(0.2-0.5)

2.8±4.8

(0.7(0.2-2.7) 0.03

eGFR (ml/min/1.73) 85±7 90±3 80±18ƒ 0.006

178

Post Hoc Analyses

Age: C vs T1DM (NS); C vs DSPN (P=0.0008); T1DM vs DSPN (P<0.0001).

Gender: Chi2 – P=0.02.

Ethnicity: Chi2 – P<0.0001.

HbA1c: C vs T1DM (P<0.0001); C vs DSPN (P<0.0001); T1DM vs DSPN (NS).

T-CHL: C vs T1DM (P=0.006); C vs DSPN (P=0.002); T1DM vs DSPN (NS).

ACR: C vs T1DM (NS); C vs DSPN (P=0.004); T1DM vs DSPN (P=0.03).

eGFR: C vs T1DM (NS); C vs DSPN (NS); T1DM vs DSPN (P=0.006).

Table key

ACR – Albumin Creatinine Ratio; BMI – Body Mass Index; BP – Blood Pressure; C – Controls,

estimated Glomerular Filtration Rate; HbA1c – Glycated Haemoglobin A1c; T-CHL – Total

Cholesterol; HDL – High Density Lipoprotein Cholesterol.

4.4.2 Symptoms and deficits (Table 4.2)

The NDS was significantly increased in patients with DSPN compared with control

subjects (P<0.0001) and T1DM (P<0.0001), with no significant difference between

controls and T1DM. The NSP was significantly higher in patients with DSPN

(P<0.0001) compared to control subjects and T1DM (P<0.0001). The McGill pain

score and McGill VAS were significantly increased in diabetic patients with DSPN

compared with control subjects (P=0.001 and P=0.0007 respectively) and patients

with T1DM (P=0.01 and P=0.02 respectively). There were no differences between

in NDS, NSP, McGill pain score and VAS between controls and T1DM.

179

Table 4-2 Neuropathy symptoms and deficits in control subjects and diabetic patients

without (T1DM) and with neuropathy (DSPN), with statistically significant differences

between groups


NDS (-/10)

(Median (IQR)

0.4±0.8

0(0-1)

1.2±2.0

0(0-2)

4.6±3.3

5(2-7)

<0.0001

NSP (-/38)

(Median (IQR)

0.1±0.4

0(0-0)

1.3±2.0

0(0-2)

5.0±6.2

2.5(0-6)

<0.0001

McGill VAS

(-/10cm)

(Median (IQR)

0.2±1.0

(0(0-0)

1.0±2.3

(0(0-0)

3.3±3.8

(0(0-0)

0.01

McGill Pain score

(Median (IQR)

0.1±0.4

(0(0-0)

1.9±6.5

(0(0-0)

4.2±6.5

(2(0-5) 0.02

Post Hoc Analyses

NDS: C vs T1DM (NS); C vs DSPN (P<0.0001); T1DM vs DSPN (P<0.0001).

NSP: C vs T1DM (NS); C vs DSPN (P<0.0001); T1DM vs DSPN (P<0.0001).

McGill VAS: C vs T1DM (NS), C vs DSPN (P=0.001); T1DM vs DSPN (P=0.01).

McGill Pain score: C vs T1DM (NS); C vs DSPN (P=0.0007); T1DM vs DSPN (P=0.02).

Table key

C – Controls; McGill VAS – McGill Visual Analogue Score; NDS – Neuropathy Disability Score,

NSP – Neuropathy Symptom Profile.

180



Peroneal nerve conduction velocity was significantly lower in DSPN compared to

controls (P<0.0001) and T1DM (P<0.0001) and between T1DM compared to

controls (P<0.0001). Peroneal nerve amplitude was significantly lower in DSPN

compared with T1DM (P<0.0001) and controls (P<0.0001). Sural nerve conduction

velocity and amplitude were significantly lower in DSPN (P<0.0001 and P<0.0001

respectively) compared with control subjects and T1DM (P<0.0001 and P<0.0001

respectively). Sural nerve conduction velocity was lower in T1DM compared to

controls (P=0.0008). However, values for sural and peroneal nerve conduction

velocities and amplitudes were within the normal reference range in T1DM

suggesting that there was minimal large fibre deficit in this group.

4.4.3.2 Vibration Perception and Thermal Thresholds

VPT was significantly greater in DSPN compared to T1DM (P<0.0001) and control

subjects (P<0.0001). CST was significantly greater in DSPN compared to T1DM

(P=0.0007) and control subjects (P<0.0001). WST was significantly greater in

DSPN compared to T1DM (P=0.0004) and control (P<0.0001). There were no

differences in VPT, CST and WST between controls and T1DM.

4.4.3.3 IENFD and CCM

Figure 4.1 shows skin biopsy specimens with highlighted IENF in controls, T1DM

and DSPN. IENFD was significantly reduced in subjects with DSPN compared to

T1DM (P=0.001) and control subjects (P<0.0001) and in T1DM compared to

controls (P=0.02).

181

Figure 4.2 shows CCM images with highlighted corneal nerves in controls, T1DM

and DSPN from the same subjects as in Figure 4.1. CNFD, CNBD and CNFL were

significantly lower in DSPN compared with both T1DM (CNFD: P<0.0001, CNBD:

P=0.02 and CNFL: P=0.001) and controls (CNFD: P<0.0001, CNBD: P<0.0001

and CNFL: P<0.0001). These parameters were also significantly reduced in

patients with T1DM compared to controls (CNFD: P<0.0001, CNBD: P=0.0008 and

CNFL: P<0.0001) suggesting early small fibre damage.

Excluding non-white European subjects from the control group (6 participants)

(n=21) made no significant difference to any of the demographic, metabolic or

neuropathy parameters. Therefore the control group in this study had no significant

confounding effect through ethnicity (Appendix 2, Table 13.2). There were no

significant differences when analysing an older control subgroup (Age: 54.4±9.9

years, n=13) which was more closely matched in age to the DSPN group (Age:

53.3±11.9) compared to the overall complete control group. There were no

significant differences in demographic, metabolic and neuropathy parameters

between the older control subgroup (n=13) and complete control group (n=27). The

control group in this study is therefore representative sample which may be

appropriately used for comparison to both T1DM and DSPN (see appendix 2,

Table 13.3).

182

Figure 4-1a, b & c. Skin biopsy images of IENF in Controls (A), T1DM (B) and DSPN (C)

Red arrows show intra-epidermal nerve fibres

Figure 4-2a, b & c. CCM images in Controls (A), T1DM (B) and DSPN (C)

183


diabetic patients without (T1DM) and with neuropathy (DSPN), with statistically significant



NCCA (mBar) 0.5±0.3 0.8±0.7 1.3±2.6 NS

CNFD (no/mm2) 37.2±5.1 30.1±6.7 19.8±9.2

<0.0001

CNBD (no/mm2) 92.0±36.2 60.7±27.9 45.4±32.0

0.02

CNFL (mm/mm2) 26.6±3.8 21.5±4.8 15.8±7.0

0.001

IENFD (no/mm) 10.2±3.3 8.3±5.5 4.7±4.3

0.001

CST (˚C) 28.6±2.0 27.5±2.0 21.4±9.1 0.0007

WST(˚C) 36.4±2.0 38.1±3.4 41.8±4.5 0.0004

VPT (volts) 5.3±4.1 5.6±2.5 18.4±12.2 <0.0001

SSNCV (m/s) 50.6±4.2 47.1±4.1 39.4±6.1 <0.0001

SSNAmp (µV) 20.2±8.8 15.1±6.1 5.5±4.2 <0.0001

PMNCV (m/s) 49.2±3.7 45.5±2.2 35.4±8.6 <0.0001

PMNAmp (mV) 6.1±2.4 7.3±9.7 2.4±2.1 <0.0001

184

Post hoc analyses

CNFD: C vs T1DM (P<0.0001); C vs DSPN (P<0.0001); T1DM vs DSPN (P<0.0001).

CNBD: C vs T1DM (P=0.0008); C vs DSPN (P<0.0001); T1DM vs DSPN (P=0.02).

CNFL: C vs T1DM (P<0.0001); C vs DSPN (<0.0001); T1DM vs DSPN (P=0.001).

IENFD: C vs T1DM (P=0.02); C vs DSPN (P<0.0001); T1DM vs DSPN (P=0.001).

CST: C vs T1DM (NS); C vs DSPN (P<0.0001); T1DM vs DSPN (P=0.0007).

WST: C vs T1DM (NS); C vs DSPN (P<0.0001); T1DM vs DSPN (P=0.0004).

VPT: C vs T1DM (NS); C vs DSPN (P<0.0001); T1DM vs DSPN (P<0.0001).

SSNCV: C vs T1DM (P=0.0008); C vs DSPN (P<0.0001); T1DM vs DSPN (P<0.0001).

SNAmp: C vs T1DM (NS); C vs DSPN (P<0.0001); T1DM vs DSPN (P<0.0001).

PMNCV: C vs T1DM (P<0.0001); C vs DSPN (P<0.0001); T1DM vs DSPN (P<0.0001).

PMNAmp: C vs T1DM (NS); C vs DSPN (P<0.0001); T1DM vs DSPN (P<0.0001).

Table key

C – Controls; CNFD – Corneal Nerve Fibre Density; CNBD – Corneal Nerve Branch Density; CNFL

– Corneal Nerve Fibre Length; CST – Cold Sensation Threshold; IENFD – Intra Epidermal Nerve

Fibre Density; PMNAmp – Peroneal Motor Nerve Amplitude; PMNCV – Peroneal Motor Nerve

Conduction Velocity; SSNAmp – Sural Nerve Sensory Nerve Amplitude; SSNCV – Sural Motor

Nerve Conduction Velocity; VPT – Vibration Perception Threshold; WST – Warm Sensation

Threshold.

185

4.4.3.4 Correlates of CCM & IENFD (Table 4)

To explore the relationship of CCM parameters and IENFD with other diagnostic

modalities, Spearman’s rank correlations of these measures were undertaken and

are highlighted in table 4.4. The strongest correlation was between CNFD and

peroneal motor nerve conduction velocity (Rho= 0.60, P<0.0001). Other

neurophysiology measures correlated well with CNFD (peroneal motor nerve

amplitude: Rho= 0.52 P<0.0001, sural sensory nerve conduction velocity: Rho=

0.52 P<0.0001, sural sensory nerve amplitude: Rho= 0.48 P<0.0001). IENFD

correlated less well (peroneal motor nerve conduction velocity Rho= 0.56

P<0.0001, peroneal motor nerve amplitude: Rho= 0.45 P<0.0001, sural sensory

nerve conduction velocity: Rho= 0.45 P<0.0001, sural sensory nerve amplitude:

Rho= 0.50 P<0.0001). Only a low to moderate correlation (Rho=0.3-0.49) was

found between CCM parameters and IENFD (CNFD: Rho= 0.33 P=0.001, CNBD:

Rho= 0.33 P=0.003, CNFD: Rho= 0.32 P=0.002). However, both signs (NDS) and

in particular symptoms (NSP and McGill VAS) correlated better with CNFD and

IENFD.

186

Table 4-4 Spearman’s rank correlation of CNFD, CNBD, CNFL and IENFD versus NDS, McGill

VAS, NSP, IENFD, thermal thresholds, VPT and nerve conduction studies

CNFD CNBD CNFL IENFD

NDS (-/10) Rho= -0.45

P<0.0001

Rho= -0.27

P=0.01

Rho= -0.34

P=0.001

Rho= -0.47

P<0.0001

McGill VAS

(-/10cm)

Rho= -0.43

P<0.0001

Rho= -0.34

P=0.002

Rho= -0.43

P<0.0001

Rho= -0.45

P<0.0001

NSP (-/38) Rho= -0.51

P<0.0001

Rho= -0.28

P=0.009

Rho= -0.39

P=0.0002

Rho= -0.51

P<0.0001

IENFD (no/mm) Rho= 0.33

P=0.001

Rho= 0.31

P=0.003

Rho= 0.32

P=0.002

CST (˚C) Rho= 0.37

P=0.0005

Rho= 0.23

P=0.04

Rho= 0.26

P=0.02

Rho= 0.33

P=0.002

WST(˚C) Rho= -0.39

P=0.0003

Rho= -0.35

P=0.0009

Rho= -0.33

P=0.002

Rho= -0.52

P<0.0001

VPT (volts) Rho= -0.49

P<0.0001

Rho= -0.31

P=0.004

Rho= -0.37

P=0.0004

Rho= -0.47

P<0.0001

SSNCV (m/s) Rho= 0.52

P<0.0001

Rho= 0.40

P=0.0002

Rho= 0.43

P<0.0001

Rho= 0.45

P<0.0001

SSNAmp (µV) Rho= 0.48

P<0.0001

Rho= 0.28

P=0.01

Rho= 0.34

P=0.002

Rho= 0.50

P<0.0001

PMNCV (m/s)

Rho= 0.60

P<0.0001

Rho= 0.46

P<0.0001

Rho= 0.54

P<0.0001

Rho= 0.46

P<0.0001

PMNAmp (mV) Rho= 0.52

P<0.0001

Rho= 0.40

P=0.0002

Rho= 0.52

P<0.0001

Rho= 0.45

P<0.0001

The strongest correlations are for CCM parameters and IENFD are highlighted in bold.

187

Table key

CNFD – Corneal Nerve Fibre Density; CNBD – Corneal Nerve Branch Density; CNFL – Corneal

Nerve Fibre Length; CST – Cold Sensation Threshold; IENFD – Intra Epidermal Nerve Fibre

Density; PMNAmp – Peroneal Motor Nerve Amplitude; PMNCV – Peroneal Motor Nerve

Conduction Velocity; SSNAmp – Sural Nerve Sensory Nerve Amplitude; SMNCV – Sural Motor

Nerve Conduction Velocity; VPT – Vibration Perception Threshold; WST – Warm Sensation

Threshold.

4.4.3.5 Receiver-Operating Characteristic (ROC) analysis (Table 4.5, Figure

4.3)

To assess the diagnostic ability of small and large fibre tests including optimal cut

offs, sensitivity and specificity ROC analysis was undertaken for all measures of

neuropathy. As the delineation of T1DM from DSPN was based on the nerve

conduction studies (Toronto criteria for definite diabetic neuropathy (3)) we have

used the ROC analysis for peroneal motor nerve conduction velocity as the

referent value for Wilcoxon estimate of area under ROC curve = 0.98 (95% CI:

0.49 – 1), sensitivity = 0.94 (95% CI: 0.79 - 0.99) and specificity = 1 (95% CI: 0.88

– 1). The Wilcoxon estimate of area under the ROC curve was greatest for VPT at

0.85 with an optimal cut off of 13 volts, sensitivity of 0.67, specificity of 1, PPV of

1.0 and NPV 0.75. The small fibre test with the greatest Wilcoxon estimate of area

under the ROC curve was CNFD at 0.81 (Figure 4.3), which was superior to IENFD

(Figure 4.3). The PPV and NPV for CNFD were 0.8 and 0.77 respectively for a cut

off of 25 fibres/mm. IENFD had a lower Wilcoxon estimate of area under the ROC

curve at 0.73, which was similar to CNFL (0.74), CST (0.76) and WST (0.74). The

PPV and NPV were also lower for IENFD at 0.76 and 0.67 respectively for a cut off

of 4.5 fibres/mm.

188

Table 4-5 ROC analysis with area under the curve, optimal cut off and respective sensitivity

and specificity with 95% confidence interval in T1DM versus DSPN for CNFD, CNBD, CNFL,

IENFD, VPT, CST and WST

Optimal Cut off AUC

(95% CI)

Sensitivity

(95% CI)

Specificity

(95% CI)

CNFD (no/mm2)

25.0

0.81

(0.47 – 1.0)

0.77

(0.59 - 0.90)

0.79

(0.60 - 0.92)

CNBD (no/mm2) 36.5

0.67

(0.45 - 0.90)

0.58

(0.39 - 0.75)

0.79

(0.60 - 0.92)

CNFL (mm/mm2) 16.8

0.74

(0.46 – 1.0)

0.61

(0.42 - 0.78)

0.86

(0.68 -0.96)

IENFD (no/mm) 4.5 0.73

(0.46 - 1.0)

0.61

(0.42 - 0.78)

0.80

(0.61 - 0.92)

VPT (Volts) 13 0.85

(0.74 - 0.95)

0.67

(0.47 - 0.83)

1

(0.88 – 1)

CST (˚C) 24.7 0.76

(0.46 – 1.0)

0.57

(0.37 - 0.76)

0.89

(0.72 - 0.98)

WST (˚C) 38

0.74

(0.61 - 0.88)

0.86

(0.67 - 0.96)

0.64

(0.44 - 0.81)

Table key


Nerve Fibre Length; CST – Cold Sensation Threshold; IENFD – Intra Epidermal Nerve Fibre

Density; VPT – Vibration Perception Threshold; WST – Warm Sensation Threshold.

189

Figure 4-3 Receiver-operated characteristic (ROC) curves, based on the analysis of CNFD

and IENFD in T1DM versus DSPN

Black line – CNFD

Red line – IENFD

ROC plot for CNFD & IENFD in T1DM vs DSPN

0.00 0.25 0.50 0.75 1.000.00

0.25

0.50

0.75

1.00

Sensitivity

1-Speci ficity

190

4.5 DISCUSSION

There is a need for surrogate end points of diabetic neuropathy, which accurately

detect early disease, quantify disease progression and measure therapeutic

response (29). The current ‘gold’ standard for the diagnosis of neuropathy is

neurophysiology, a robust measure that also predicts foot ulceration and mortality

in diabetes (30). Other measures of neuropathy such as neurological assessment

are poorly reproducible (2), QST is subjective and more accurate measures such

as skin and nerve biopsy are invasive and require specialist analysis (3) . Small

fibre neuropathy has direct pathophysiological relevance to the main outcomes of

pain and foot ulceration (7) and therefore skin biopsy assessment of IENF has

been proposed as a valid measure of diabetic neuropathy (8). Whilst skin biopsy

detects early small nerve fibre damage even when electrophysiology and QST are

still normal (4-5), the use of this test in clinical trials is limited by its invasive nature.

CCM is a novel, rapid and readily reiterative technique, which quantifies small

nerve fibres non-invasively and shows promise (17-18; 31-34). The major findings

of this study which address major questions in relation to the diagnostic ability of

different small fibre tests in diabetic neuropathy are: 1) early subclinical small nerve

fibre decline can be detected by CCM and IENFD; 2) CNFD has higher diagnostic

utility than IENFD in the diagnosis of DPN; 3) CNFD correlates better with nerve

conduction studies than IENFD.

Early intervention with improved glycaemic control in type 1 DM can lead to a

durable reduction in DPN (35-36). Furthermore, the need for early evaluation of

subclinical small fibre neuropathy has been demonstrated by Smith et al (6) where

lifestyle intervention with diet and exercise in a pre-diabetic neuropathy group lead

to cutaneous re-innervation and improved pain. Previous studies have employed

191

ROC curve analysis of IENFD at the distal leg and shown a specificity of 95%-97%

and sensitivity of 45%- 80% (15; 37) for small fibre neuropathy but these studies

were not specifically in patients with DPN. Recently Nebuchennykh et al (13) in a

study of patients with polyneuropathy from varying causes showed a sensitivity of

35% and specificity of 95% using a cut off point of 6.7 fibres/mm for IENFD. In

another study by Vlckova-Moravcova et al (12), the diagnostic sensitivity for

detecting neuropathy was 80% and the specificity was 82% with an optimal IENFD

cut off point of ≤8.8 fibres/mm. Although, ROC curve analysis is a standard and

appropriate method for establishing diagnostic validity these studies were flawed

as they assessed a disease group against a healthy control population and thus

sensitivities and specificities will be inappropriately high and the delineation of the

optimal cut off points inaccurate. The need to identify DPN in diabetic subjects

should mean that optimal cut off points for neuropathy should be based on data

from a population with diabetes with and without neuropathy rather than a healthy

control population versus DPN. Therefore we have performed the present study in

a population of diabetic patients with and without neuropathy using the robust

Toronto criteria and utilised a best fit ROC curve analysis (38-39) to derive optimal

cut off points, sensitivities, and specificities to assess the diagnostic validity of

CCM measures and IENFD. ROC curve analysis in this study showed that IENFD

had a sensitivity of 61% and specificity of 80% at an optimal cut off point of 4.5

fibres/mm. These diagnostic validity measures are clearly lower than in the current

published literature (12-14), but we believe are more representative. Using exactly

the same population and methods we show that CNFD has a better sensitivity of

77% and an almost identical specificity of 79% at an optimal cut off point of 25.0

fibres/mm.

192

Previous studies of IENFD have found either absent or weak correlations with

nerve conduction studies (12) and sural nerve action potentials (15; 40-41). In the

present study CNFD showed a superior correlation than IENFD with peroneal

motor nerve conduction velocity and amplitude and sural sensory nerve conduction

velocity. These data support the study of Shun et al (14) which showed a

significant correlation between IENFD with sural nerve action potential and warm

sensation threshold. Thermal thresholds continue to an important psychophysical

test in evaluating small nerve fibres and our data show that the strongest

correlation was indeed between IENFD and WST. For DPN, thermal thresholds

have previously shown a sensitivity which ranges from 36%-85% (6; 42-43).

Sensitivities reported in our study are 57% and 86% respectively for CST and

WST. Furthermore, we have shown negative correlations of WST with CCM

measures and IENFD confirming a previous study (42). Interestingly CNBD, CNFD

and CNFL correlated with IENFD but the association was a low one (Spearman’s

Rho 0.33, 0.31 and 0.32 respectively). Although both CCM and skin biopsy

measure small nerve fibres, the sites of assessment are anatomically distinct.

There are no reported significant variations of IENFD calculated in adjacent

sections from the same biopsy or in adjacent biopsies from the same site (15).

However, even with IENFD differences in mean values exist between differing sites

on the lower limb (15; 44) although no direct correlations were assessed between

sites. Although DPN is considered a length dependent neuropathy, recent studies

suggest that lesions may occur in a proximal (45) multifocal fascicular pattern (46).

IENFD correlates somewhat better with NDS, McGill VAS and NSP than CNFD.

Clinical measures of neuropathy in part directly assess distal symptoms which

would be in keeping with distal measures such as IENFD. We suggest that CCM

193

measures are similar to IENFD despite assessing a distinct clinical site than

typically assessed through clinical measures of diabetic neuropathy. VPT had the

greatest AUC and specificity but this reflects the method of delineation of

neuropathy through the Toronto criteria which uses electrophysiology with signs

and/or symptoms. Both VPT and electrophysiology are large fibre measures and

therefore the ability of VPT as a diagnostic measure for diabetic neuropathy to be

high particularly as the groups were divided based on electrophysiology. However,

CNFD was still superior to IENFD despite the delineation of the groups with a large

fibre test.

The current study provides the first direct comparison between IENFD and CCM in

the assessment DPN. Both CNFD and IENFD correlated with clinical signs (NDS),

and symptoms (McGill VAS and NSP). Furthermore, CNFD had better diagnostic

utility for DPN through ROC curve analyses and correlated better with

electrophysiology than the current ‘gold’ standard of IENFD.

194

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201

5 Chapter V - Enhanced Small Fibre Neuropathy in

Patients with Latent Autoimmune Diabetes in Adults




analyses and writing of this chapter.


Uazman Alam, Maria Jeziorska, Ioannis N Petropoulos, Omar Asghar, Hassan

Fadavi, Georgios Ponirakis, Andrew Marshall, Mitra Tavakoli, Andrew JM Boulton,


202

5.1 ABSTRACT

Latent Autoimmune Diabetes in Adults (LADA) is often misdiagnosed and patients

have relatively poorer glycaemic control with a potential for an increased risk of

neuropathy. Surprisingly, few studies have been undertaken in these patients in

relation to the prevalence of neuropathy.

Subjects with LADA (LADA) (n=19), type 2 DM (T2DM) (n=22) and non-diabetic

healthy control subjects (C) (n=33) underwent detailed assessment of neurologic

deficits, quantitative sensory testing (QST), electrophysiology, skin biopsy and

corneal confocal microscopy (CCM).

There was no significant difference for age, duration of diabetes, lipids and blood

pressure, but HbA1c was higher (P<0.0001) and the BMI was lower (P=0.008) in

patients with LADA compared with T2DM. There was no significant difference in

the neuropathy disability score (NDS), vibration perception threshold (VPT) or

electrophysiology between LADA and T2DM. However, cold sensation threshold

(CST) (P=0.008), and warm sensation threshold (WST) (P=0.008), were

significantly higher and intra-epidermal nerve fibre density (IENFD) (P=0.02),

corneal nerve fibre density (CNFD) (P=0.03), corneal nerve fibre length (CNFL),

(P=0.02), and corneal nerve branch density (CNBD) (P=0.003) were significantly

lower in LADA compared to T2DM.

Despite, comparable age, duration of diabetes and cardiovascular risk factors,

subjects with LADA have a significant small fibre neuropathy compared to matched

patients with T2DM.

203

5.2 INTRODUCTION

LADA is often misdiagnosed as type 2 diabetes mellitus (DM), but

pathophysiologically is more closely related to type 1 DM. (1). Hence, these

patients present at a younger age due to a slow autoimmune destruction of

pancreatic β-cells (2-5). The prevalence of LADA in patients with type 2 DM

(T2DM) ranges from a minimum of 4% (6-7) to 10% as shown in the UKPDS (3).

Thus patients with LADA represent a significant proportion of patients with a faster

decline in C-peptide (6) and delayed treatment with insulin resulting in a significant

period of poor glycaemic control (8). Given, that early and optimal treatment of

glycaemia reduces the incidence of neuropathy in Type 1 DM as shown by the

DCCT and EDIC studies (9-10) then patients with LADA with poorer glycaemic

control may well have an increased risk of developing the long term complications

(11-12).

However, the long term complications in patients with LADA, in particular diabetic

somatic polyneuropathy (DSPN) have been poorly studied. Hence, surprisingly

individuals with LADA had a lower prevalence of diabetic neuropathy assessed

using electrophysiology, quantitative sensory testing and autonomic function,

despite having a HbA1c which was 1.1% (~12mmol/mol) higher when compared to

patients with T2DM (13). Yet in another study the prevalence of neuropathy

defined using the Michigan Neuropathy screening instrument was higher in the

LADA group (~38%) when compared to T2DM (~30%) (14). Whilst there was no

difference for neuropathy in LADA compared to Type 2 DM when assessed using

the Neuropathy Disability score (NDS) and Vibration Perception Threshold (VPT)

(15). These data reflect no clear consensus on the prevalence of DSPN in patients

with LADA and raise the important issue of the sensitivity of different tests to

204

diagnose diabetic neuropathy. Intra-epidermal nerve fibre density (IENFD) is

significantly reduced in patients with normal electrophysiology suggesting early

damage to small nerve fibres (16). Similarly, corneal confocal microscopy (CCM) is

a non-invasive technique that can detect early small sensory nerve fibre loss (17)

and repair on normalisation of glycaemia after pancreas transplantation in Type 1

diabetes (18). Therefore the assessment of small fibres may be the most sensitive

means to detect early neuropathy in diabetes (19).

We have undertaken a comprehensive study phenotyping diabetic neuropathy in

subjects with LADA to identify large and small fibre neuropathy compared to

matched patients with Type 2 DM.

205

5.3 RSEARCH DESIGN AND METHODS


19 subjects with LADA, 22 subjects with T2DM and 33 age and sex matched non-

diabetic control subjects were studied. Subjects with a history of neurologic

conditions, ocular trauma or previous ocular surgery were excluded. The study was

approved by the North West and Salford and Trafford Research Ethics committee,

and written informed consent was obtained according to the Declaration of

Helsinki.

5.3.2 LADA definition

LADA was defined as follows: age of onset less than 50 years, anti-glutamic

decarboxylase (GAD) antibodies (GAD-Ab) positivity, exclusion of patients with

ketonuria/diabetic ketoacidosis at diagnosis and a lack of insulin treatment within

the first six months after diagnosis of diabetes mellitus, suggesting type 1 DM (1).

All subjects with LADA had elevated positive GAD-Ab titres which were consistent

with autoimmune diabetes. All subjects with T2DM had a clinical LADA screening

score of 0 or 1 and thus low risk of autoimmune diabetes as defined in the study by

Fourlanos et al (20).



symptoms according to the neuropathy symptom profile (NSP), and the McGill

visual analogue score (McGill VAS) VAS was used to assess the severity of painful

neuropathy. Clinical neurologic deficits were assessed using the modified

neuropathy disability score, which includes evaluation of vibration, pin prick, and

206

temperature perception as well as the presence or absence of ankle reflexes to

establish the severity of neuropathy (21). Quantitative sensory testing (QST)

included an assessment of vibration perception threshold (VPT), measured using a



(WST) (22) (c fibres) thresholds using the method of limits with the MEDOC TSA II

(Medoc, Ramat Yishai, Israel) on the dorsum of the left foot. Electro-diagnostic

studies were undertaken using a Dantec “Keypoint” system (Dantec Dynamics,

Bristol, U.K.) equipped with a DISA temperature regulator to keep limb temperature

constantly between 32°C and 35°C. Peroneal motor and sural sensory nerves

were assessed in the right lower limb by a consultant neurophysiologist. The motor

study was performed using silver-silver chloride surface electrodes at standardized

sites defined by anatomical landmarks, and recordings for the sural nerve were

taken using antidromic stimulation over a distance of 100 mm.









nerve plexus of the cornea from each patient and control subject. Each subbasal


207



and examined in a masked and randomized fashion (24). Four corneal nerve


millimetre of corneal tissue (no.mm2); 2) CNBD, the number of branches

emanating from all major nerve trunks per square millimetre of corneal tissue

(no.mm2); 3) CNFL, the total length of all nerve fibres and branches (mm/mm2)

within the area of corneal tissue and 4) corneal nerve fibre tortuosity (CNFT), a

parameter mathematically derived from the images (25).


A sub-cohort of participants (Controls n=19, T2DM n=12, and LADA n=9)

underwent a 3-mm punch skin biopsy from the dorsum of the foot; 2 cm above the

second metatarsal head after local anaesthesia (1% lidocaine). The biopsy site

was closed using Steristrips, and the specimen was immediately fixed in PBS-

buffered 4% paraformaldehyde. After 18–24 hours, it was rinsed in Tris-buffered

saline and soaked in 33% sucrose (2–4 hours) for cryoprotection. It was then

embedded in optimal cutting temperature–embedding compound, rapidly frozen in

liquid nitrogen, and cut into 50-μm sections using a cryostat (model OTF; Bright

Instruments, Huntington, U.K.). Four floating sections per subject underwent

melanin bleaching (0.25% KMnO4 for 15 min followed by 5% oxalic acid for 3 min),

a 4-h protein block with a Tris-buffered saline solution of 5% normal swine serum,

0.5% powdered milk, and 1% Triton X-100, and overnight incubation with 1:1,200

Biogenesis polyclonal rabbit anti-human PGP9.5 antibody (Serotec, Oxford, U.K.).

Biotinylated swine anti-rabbit secondary antibody (1:300; DakoCytomation, Ely,

208

U.K.) was then applied for 1 hour; sections were quenched with 1% H2O2 in 30%

MeOH-PBS (30 min) prior to a 1 hour incubation with 1:500 horseradish

peroxidase–streptavidin (Vector Laboratories, Peterborough, U.K.). Nerve fibres

were demonstrated using 3, 3-diaminobenzidine chromogen (Sigma-Aldrich,

Manchester, U.K.). Sections were mildly counterstained with eosin to better

localize the basement membrane to identify nerve fibres passing through it.

Negative control subjects consisted of replacing the anti- PGP9.5 antibody with

rabbit immunoglobulin (DakoCytomation) at a concentration matching that of the

primary antibody, which showed no immunostaining. IENFD, i.e., the number of

fibres passing through the dermo-epidermal junction per millimetre of basement

membrane was quantified in accord with established criteria and techniques and

expressed as number per millimetre (26).



The data are expressed as Mean ± standard deviation (SD). ANOVA method or a

non-parametric counterpart, Kruskal-Wallis were used to assess differences

between groups depending on normality of the data. The Mann-Whitney U test was

used to compare T2DM with LADA for the duration of diabetes. Chi squared

analyses were used to assess frequencies of gender, ethnicity and aetiology of

diabetes. Overall the P value was maintained at 0.05 for multiple comparison tests

(Bonferoni adjustment or Conover-Inmann pairwise comparison).

209

5.4 RESULTS

5.4.1 Demographics and Clinical Neuropathy (Table 5.1)

The participant demographics and clinical neuropathy measurements in subjects

with diabetes and age-matched control subjects are summarized in Table 5.1.

There were no significant differences in age (C: 53.3±9.8 vs T2DM: 54.9±6.8 vs

LADA: 50.6±11.9 years, P=NS) and duration of diabetes (T2DM: 9.8±5.4 vs LADA:

11.4±10.2 years, P=NS). There were no differences between the groups in their

ethnic mix. The NDS was significantly greater in patients with T2DM (P=0.02) and

LADA (P=0.0003) compared to control subjects, consistent with a mild neuropathy,

but did not differ between T2DM and LADA. The NSP was significantly higher in

T2DM and LADA (P<0.0001 and P=0.0002 respectively) with no differences in the

McGill VAS compared to controls. There were no significant difference in the

degree of neuropathic pain in T2DM (Median (IQR) (0.5(0-4)) compared to LADA

(0(0-0) (P=NS).

210

Table 5-1 Participant demographics in Controls, T2DM and LADA

C

(n=20)

T2DM

(n=17)

LADA

(n=19)

T2DM v LADA

Age (years) 53.3±9.8 54.9±6.8 50.6±11.9 NS

Gender (Male)

(%) 65 76 58 NS

Ethnicity (White

European) (%) 60

59

58 NS

Duration of

Diabetes (years)

Median (IQR)

- 9.8±5.4 11.4±10.2 NS

NDS (-/10)

Median(IQR)

0.5±1.0

0(0-1)

2.1±1.9*

1(0-4)

3.9±3.6**

3(1-6) NS

NSP (-/38)

Median (IQR)

0.1±0.3

0(0-0)

4.5±5.6^

2(1-4)

4.4±6.0^^

2.5(0-6) NS

McGill VAS

(-/10cm)

Median (IQR)

0.6±1.8

0(0-0)

1.9±2.5

0.5(0-4)

0.5±1.2

0(0-0)

NS

Post hoc analyses

NDS *C vs T2DM – P=0.02; **C vs LADA– P=0.0003.

NSP ^C vs T2DM – P<0.0001; ^^C vs LADA – P=0.0002.

McGill VAS C vs T2DM – P=NS; C vs LADA – P=NS.

Table Key

IQR – Inter quartile Range; McGill VAS – McGill Visual Analogue Score; NDS – Neuropathy

Disability Score; NSP – Neuropathy Symptom Profile.

211

5.4.2 Metabolic and Anthropometric measurements (Table 5.2)

Metabolic and anthropometric measurements are detailed in Table 5.2. HbA1c was

significantly higher in subjects with LADA compared to T2DM and controls

(P<0.0001). BMI was significantly higher in T2DM subjects (P=0.001 and P=0.0005

respectively) compared to subjects with LADA and control subjects. The total

cholesterol was significantly lower (P<0.0001) in diabetic patients due to greater

statin use with no difference between the patients with T2DM and LADA. HDL was

significantly lower in T2DM compared to controls (P=0.002) and subjects with

LADA (P=0.03). There were no differences between blood pressure either systolic

or diastolic in any group. There were no differences in the estimated glomerular

filtration rate (eGFR) between diabetic subjects and controls.

212

Table 5-2 Clinical and metabolic parameters in control subjects and patients with T2DM and

LADA

C

(n=20)

T2DM

(n=17)

LADA

(n=19)

T2DM v LADA

P Value

HbA1c (%) 5.7±0.4 7.0±0.7* 9.8±2.3** <0.0001

HbA1c

(mmol/mol) 38.4±4.2 52.8±7.4* 83.4±24.7** <0.0001

BMI (kg/m2) 27.5±4.5 32.8±4.6ƒ

27.1±4.0 0.001

T-CHL (mmol/l) 5.5±0.7 4.1±1.3† 4.5±1.3†† NS

HDL (mmol/l) 1.6±0.4 1.1±0.4˚ 1.4±0.4 0.03


Systolic BP

(mmHg) 133±16 137±25 137±22 NS

Diastolic BP

(mmHg) 76±10 79±11 76±11 NS

eGFR

(ml/min/1.73) 80±9 79±16 82±13 NS

Post hoc analyses

HbA1c: *C vs T2DM – P<0.0001; **C vs LADA – P<0.0001.

BMI: ƒC vs T2DM – P=0.0005; C vs LADA – P=NS.

T-CHL: †C vs T2DM – P<0.0001; ††C vs LADA – P=0.003.

HDL: ˚C vs T2DM – P=0.002; C vs LADA – P=NS.

Table key

ACR – Albumin Creatinine Ratio; BMI – Body Mass Index; BP – Blood Pressure; estimated

Glomerular Filtration Rate; HbA1c – Glycated Haemoglobin A1c; HDL – High Density Lipoprotein

Cholesterol; T-CHL – Total Cholesterol.

213



Peroneal motor nerve conduction velocity was significantly lower in T2DM

(P=0.004) and LADA (P=0.0001) compared with control subjects with no difference

between the T2DM and LADA groups. Peroneal motor nerve amplitude was

significantly lower inT2DM (P=0.01) and LADA (P=0.0o2) compared with control

subjects. Sural sensory nerve conduction velocity and amplitude were significantly

lower in LADA (P=0.01 and P=0.03 respectively) with no difference in T2DM

compared with control subjects with no difference between T2DM and LADA

groups. However, values for sural sensory and peroneal motor nerve conduction

velocities and amplitudes were within the normal reference range for T2DM and

LADA groups suggesting that there was minimal large fibre deficit.

5.4.3.2 Vibration Perception, Thermal Thresholds & Neuropad

VPT was significantly greater in T2DM (P=0.009) and LADA (P=0.004) compared

with control subjects with no difference between T2DM and LADA. CST was

significantly lower in T2DM (P=0.002) and LADA (P<0.0001) compared with control

subjects and was significantly lower in LADA (P=0.03) compared to T2DM. WST

was significantly greater in T2DM (P<0.0001) and LADA (P<0.0001) compared

with control subjects and was significantly greater in LADA (P=0.02) compared to

T2DM. There were no differences between controls, T2DM and LADA in the

neuropad assessment.

214

5.4.3.3 IENFD and CCM

Figure 5.1 shows skin biopsy specimens with highlighted IENF in controls, LADA

and T2DM (A, B and C respectively). IENFD was significantly reduced in subjects

with LADA compared to control subjects and T2DM (P=0.001 and P=0.03

respectively) with no difference between controls and T2DM. Those undergoing

skin biopsy were representative of the overall cohorts in terms of age, ethnicity,

gender and duration of diabetes. There were no significant differences in age and

duration of diabetes between those subjects who had skin biopsy (controls vs

T2DM vs LADA).

Figure 5-1 Skin biopsies stained for PGP9.5. Healthy control (A) shows numerous long intra-

epidermal nerve fibres and a well-developed sub-epidermal nerve plexus, compared with

short intra-epidermal nerves and less pronounced sub-epidermal plexus in a LADA patient

(B) and comparable sub-epidermal plexus but more numerous intra-epidermal nerves in a

T2DM patient with neuropathy (C). (Bar = 100 µm).

215

Figure 5.2 shows CCM images with highlighted corneal nerves in controls, LADA

and T2DM (A, B and C respectively). CNFD was significantly reduced in both

T2DM (P=0.003) and LADA (P=0.001) compared with control subjects. CNBD

(P=0.0008) and CNFL (P=0.0005) were significantly lower in LADA with no

significant difference in T2DM compared to controls. CCM parameters were further

significantly reduced in patients with LADA compared to T2DM (CNFD (P=0.03),

and CNFL (P=0.01)).

Figure 5-2 Healthy control (A) shows numerous corneal nerve fibres and branches compared

with much fewer and less pronounced subbasal nerve plexus in a LADA patient (B) and

comparable subepidermal plexus but greater numbers of corneal nerves and braches in a

T2DM patient with neuropathy (C).

Control (A) vs LADA (B) vs Mild Neuropathy (C)

BA C

Nerve Branches

Nerve Fibres

216

Table 5-3 Small and large fibre neuropathy tests in control subjects, T2DM and LADA

C

(n=20)

T2DM

(n=17)

LADA

(n=19)

T2DM v LADA

P Value

NCCA (mBar) 0.7±0.8 1.0±0.8 1.0±1.2 NS

CNFD (no/mm2) 36.7±5.1 30.5±11.25* 24.2±5.3** P=0.03

CNBD (no/mm2) 93.4±37.1 70.1±29.4 60.4±29.3†† NS

CNFL

(mm/mm2)

26.1±5.3 24.7±6.9 19.9±4.8¥¥ P=0.01

CNFT 16.0±3.7 19.7±4.9¤ 19.5±5.3¤¤ NS

IENFD (no/mm) 9.9±3.2

(n=9)

7.4±4.4

(n=8)

3.6±3.0ƒƒ

(n=8) P=0.03

CST (˚C) 28.6±2.0 26.0±3.1æ

22.6±6.9ææ

P=0.03

WST(˚C) 36.8±2.6 41.3±3.4£ 43.9±3.6

££ P=0.02

Neuropad (%) 87±20 79±33 68±35 NS

VPT (volts) 6.0±3.5 10.5±6.9β 13.5±11.8

ββ NS

SSNCV (m/s) 49.6±4.0 46.6±5.7 45.2±6.4˚ NS

SSNAmp (µV) 17.8±8.2 12.6±8.2 10.8±6.0€ NS

PMNCV (m/s) 47.8±3.6 42.5±7.4ª 40.9±7.2ªª NS

PMNAmp (mV) 6.1±1.9 4.2±2.1§ 3.8±2.8§§ NS

Post hoc analyses

CNFD: *C vs T2DM – P=0.003; **C vs LADA – P=0.001.

CNBD: C vs T2DM – P=NS; ††C vs LADA – P=0.0008.

CNFL: C vs T2DM – P=NS; ¥¥C vs LADA – P=0.0005.

CNFT: ¤C vs T2DM – P=NS; ¤¤C vs LADA – P=NS.

IENFD: C vs T2DM – P=NS; ƒƒ

C vs LADA – P=0.001.

CST: æ

C vs T2DM – P=0.002; ææ

C vs LADA – P<0.0001.

WST: £C vs T2DM – P<0.0001;

££C vs LADA – P<0.0001.

VPT: βC vs T2DM – P=0.009;

ββC vs LADA – P=0.004.

SSNCV: C vs T2DM – P=NS; ˚C vs LADA– P=0.01.

SSNAmp: C vs T2DM – P=NS; €C vs LADA – P=0.03.

PMNCV: ªC vs T2DM – P=0.004; ªªC vs LADA – P=0.0001.

217

PMNAmp: §C vs T2DM – P=0.01, §§C vs LADA – P=0.002.

Table Key

CNBD – Corneal Nerve Branch Density; CNFD – Corneal Nerve Fibre Density; CNFL – Corneal

Nerve Fibre Length; CNFT – Corneal Nerve Fibre Tortuosity; CST – Cold sensation Threshold;

IENFD – Intra Epidermal Nerve Fibre Density; NCCA- Non-Contact Corneal Aesthesiometry;

PMNAmp – Peroneal Motor Nerve Amplitude; PMNCV – Peroneal Motor Nerve Conduction

Velocity; SSNAmp – Sural Nerve Sensory Nerve Amplitude; SSNCV – Sural Sensory Nerve

Conduction Velocity; VPT – Vibration Perception Threshold; WST – Warm Sensation Threshold.

218

5.5 DISCUSSION

LADA is a slowly progressive form of autoimmune diabetes which is often

misclassified as type 2 diabetes and therefore results in a protracted period of

relatively poor glycaemic control due to a delay in the introduction of insulin (1).

Longitudinal studies in patients with LADA have found a relatively rapid decline in

insulin secretion in the first few years after diagnosis of diabetes (27-28). In a

recent study, individuals with LADA had worse glycaemic control than patients with

type 2 diabetes despite a longer time on insulin therapy (29). Two small studies

have shown beneficial effects on β-cell reserve of early insulin therapy in patients

with LADA (27; 30). Our LADA cohort also had a greater degree of hyperglycaemia

reflecting this late recognition and late introduction of insulin.

Given that poorer glycaemic control is associated with an increased risk for the

development of neuropathy in both type 1 (31-32) and type 2 diabetes (19; 33),

patients with LADA may therefore have a greater propensity for the development of

neuropathy (29). However, in subjects with LADA there are limited studies (13-15)

which have shown a varying severity of neuropathy (6; 13; 34). In a recent study

current perception thresholds and electrophysiology were found to be similar in

patients with LADA and type 1 and 2 diabetes (6). In the few other studies which

have been undertaken, the assessment of neuropathy was crude as symptoms

and clinical neurological deficits or vibration perception were assessed (14-15),

groups were not age matched. Given that small nerve fibre damage precedes large

fibre deficits (17; 35-37), and no direct assessment of small fibre function or

structure was undertaken (13; 15), neuropathy may not have been detected.

Thus in the present study we show that subjects with both T2DM and LADA have

an abnormal NDS, VPT and electrophysiology. However, patients with LADA have

219

comparable measures of large fibre neuropathy to subjects with T2DM. Cold and

warm thermal thresholds were increased, and both IENFD and corneal nerve

parameters were reduced in patients with T2DM and LADA. However, these small

fibre parameters were further significantly reduced in patients with LADA compared

to patients with T2DM. This relative more advanced pathology of small fibres may

explain the discrepant findings in previous studies where patients with LADA have

been shown to have either no abnormality or indeed mild comparable neuropathy

to patients with T2DM. However, despite less small fibre neuropathy in T2DM

compared to LADA there was no difference in levels of neuropathic pain as

demonstrated by the McGill VAS. In a previous community based study an excess

of pain was South Asian subjects with diabetes (38) although in our study, groups

were accurately matched for ethnicity.

Of potential pathogenetic relevance, GAD-Ab, which are used to diagnose LADA

have been linked to other neurological conditions such as stiff man syndrome (39),

however there has been no direct correlation of GAD-Ab status with

peripheral/autonomic neuropathy in diabetes mellitus (40-41).

In conclusion the present study shows a clear adverse consequence of LADA with

a greater degree of small fibre neuropathy compared to matched patients with

T2DM. This likely consequence is due to the poorer glycaemic control, as the other

major risk factors for neuropathy such as lipid profile were comparable and BMI

was in fact lower (32). Thus patients with LADA require assessment for small fibre

neuropathy, perhaps to enable risk stratification and earlier commencement of

insulin, limiting the progression of neuropathy. Our study also highlights the need

to undertake detailed assessment of small fibres using either skin biopsy or corneal

confocal microscopy to identify small fibre neuropathy.

220

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226

6 Chapter VI - Marked vitamin D deficiency in patients with

diabetes from secondary care: Ethnic and seasonal

differences and an association with dyslipidaemia

Contribution: Uazman Alam conceived the study and subsequently designed it,

extracted data, performed statistical analyses and wrote the manuscript which

constitutes the basis for this chapter and was submitted and accepted for

publication in the journal Diabetic Medicine 2012;29:1343-1345

Uazman Alam, Osman Najam, Sarah Al-Himdani, Sophie Benoliel, Pushpa

Jinadev, Jacqueline Berry, Michelle Kew, Omar Asghar, Ioannis Petropolous,

Rayaz A Malik

227

6.1 ABSTRACT

Vitamin D deficiency has been implicated in cardiovascular disease in subjects with

diabetes. We aimed to assess the 25-hydroxyvitamin D (25(OH)D) status in

patients from a secondary care hospital.

This cross-sectional population-based cohort study assessed 25(OH)D levels in

563 unselected subjects with diabetes and 44 age and ethnicity matched subjects

without diabetes, in relation to metabolic, anthropometric and demographic factors.

The mean 25(OH)D was lower in subjects with diabetes (15.7 ± 9.1 ng/ml)

compared to age and ethnicity subjects without diabetes (21.3 ± 14.6 ng/ml)

(p=0.009) with vitamin D deficiency (<30 ng/ml) in 91% compared to 78% in the

respective groups. The severest (<10ng/ml) vitamin D deficiency occurred in South

Asian subjects (54%) compared to white Europeans (22%). There were no

significant differences in BMI, systolic and diastolic blood pressure, ALP, corrected

calcium or HbA1c between the different quartiles of 25(OH)D (<10ng/ml, 10-

19.9ng/ml, 20-29.9ng/ml, >30ng/ml). However, HDL (1.3±0.7, 1.3±0.4, 1.4±0.5,

1.6±0.6mmol/l, p=0.003) was higher and triglycerides (2.2±2.8, 1.7±1.5, 1.5±0.9,

1.6±1.3mmol/l, p=0.04) were significantly lower in subjects with diabetes with

25(OH)D >30ng/ml. There was a statistically but clinically non-significant seasonal

variation in the 25(OH)D status in the white European cohort in summer

(20.1±8.8ng/ml) versus winter (16.0±6.1ng/ml) (p=0.007), with no change in the

South Asian/Middle Eastern cohort.

There is profound vitamin D deficiency in the North West of England, particularly in

South Asians which is associated with lower HDL levels, possibly mediating

elevated cardiovascular risk. Further research is warranted to define whether

replacement of vitamin D may alter cardiometabolic risk.

228

6.2 INTRODUCTION

Over one billion people worldwide are vitamin D deficient (1). Vitamin D deficiency

is highly prevalent in the UK (2) and in a nationwide cohort study, 87.1% and

60.9% were deficient in vitamin D in the winter/spring and summer/autumn months

respectively (2) using ≥30ng/ml as a cut off for adequate levels (1; 3). At risk

groups for deficiency include those with pigmented skin (4) and severe deficiency

is highly prevalent in South Asians living in northern latitudes (5).

The role of Vitamin D is well established in the regulation of bone metabolism and

calcium homeostasis. Hence concentrations of ≥30ng/ml of vitamin D have been

identified as necessary for optimal bone health (3) by maximal suppression of

parathyroid hormone, greatest calcium absorption, and highest bone mineral

density (6). Concentrations ≥10ng/ml are sufficient to prevent the severe skeletal

manifestations leading to rickets in children and osteomalacia in adults (7). Thus a

serum concentration of ≥30 ng/ml is considered sufficient, 20 to <30ng/ml

insufficient, 10 to <20ng/ml deficient and <10ng/ml as severe deficiency (1).

Vitamin D may play a pivotal role in several chronic diseases such as diabetes and

cardiovascular disease (1; 4). The vitamin D receptor (VDR) is expressed

ubiquitously including in tissues such as cardiomyocytes, vascular endothelium

and lymphopoeitic cells (1). With regard to diabetes, the binding of the activated

hormonal form of vitamin D (1,25-hydroxyvitamin D [1,25(OH)2D]) to VDR in

pancreatic β-cells leads to the production of insulin (8). The development of both

type 1 and 2 diabetes has been causally associated with vitamin D deficiency and

has been validated in both experimental and human interventional studies (9-11).

In the Finnish birth cohort study, more than 10,000 individuals were followed over

30 years and children who were supplemented with 2000IU daily of vitamin D for

229

the first year of life had a relative risk of 0.22 (95% CI 0.05-0.89) for the

development of type 1 diabetes when compared to those who were not

supplemented (9). There is also a propensity for vitamin D deficiency, particularly

in Type 2 diabetes (12). VDR activation may be beneficial in terms of proliferation

of vascular smooth muscle and cardiomyocytes (1). Indeed two large prospective

studies have demonstrated increased cardiovascular mortality in subjects with

severe deficiency (≤10ng/ml) (13-14). The Framingham cohort study has also

shown similar results with a cut off of 15ng/ml (15) and a recent meta-analysis

showed a significant association between high levels of vitamin D and a reduction

of cardiovascular disease risk (33% reduction compared to low levels of vitamin D),

and metabolic syndrome (51% reduction) (16). The relationship of vitamin D to

cardiovascular disease is further supported considering the shared metabolic

pathway with cholesterol production, via 7-dehydrocholesterol (17). In a recent

study an approximate increase of 4.2mg/dl in HDL was associated with every

10ng/ml increase of 25(OH)D (18). Indeed, activated vitamin D inhibits foam cell

formation and an impairment of VDR signalling results in acceleration of this

process in patients with diabetes (19). This postulated link to cholesterol

metabolism and cardiovascular outcomes is further strengthened by the

demonstration that statins increase vitamin D levels (20). However, large

prospective randomised trials of vitamin D intervention assessing cardiovascular

outcomes as the primary endpoint are absent, thus the relationship may be

regarded by some as post hoc ergo propter hoc (correlation not causation).

This cross-sectional study was undertaken in a group of unselected subjects with

diabetes attending a hospital based diabetes clinic and a control group without

diabetes from a general medical clinic. We defined the prevalence of vitamin D

230

deficiency and its association with the constituents of metabolic syndrome and

cardiovascular disease risk.

231


Data from a population-based cohort of 563 subjects with diabetes and 44 age and

ethnicity matched subjects without diabetes from Central Manchester Foundation

Trust, Manchester, UK were analyzed. All patients attending the clinics were

assessed irrespective of a history suggestive of vitamin D deficiency. Ethical

approval was not required as the data were extracted retrospectively and did not

extend beyond standard clinical practice. All those found to be deficient in

25(OH)D, were provided with appropriate replacement therapy in accordance with

standard clinical practice.

6.3.1 Subjects

This cross sectional study was conducted in an unselected group of subjects with

and without diabetes in a general medical population. All participants were aged ≥

18 years old attending clinics at the Central Manchester Foundation Trust,

Manchester from August 2009 to July 2010. Those with renal impairment (eGFR

<30 mL/min/1.73m2 (CKD stage 4 and below), granulomatous diseases

(tuberculosis, sarcoidosis etc), malabsorption syndromes (Coeliac disease,

bacterial overgrowth, concomitant orlistat treatment), pregnant and lactating

women, and those currently on vitamin D supplementation were excluded from the

analysis. From previous prevalence studies in the UK we anticipated the proportion

of individuals with vitamin D deficiency to be greater than 50% (4).




232

Hamburg, Germany) to the nearest 0.1kg and height to the nearest 0.1cm. Blood

pressure (BP) measurements were obtained with the use of an automated BP


appropriate cuff size. A minimum of two measurements of systolic and diastolic BP

were made five minutes apart with the lowest reading recorded.


Medications



medical record database (Diamond database, Hicom, Surrey, UK). Subject

demographics extracted were age, sex, ethnicity (White European, South Asian,

Far East Asian and Afro-Caribbean descent), smoking status (never, previous and

current) and type (Type 1 and 2 diabetes) and duration of diabetes. Previous

cardiovascular events extracted were ischaemic heart disease (IHD), myocardial

infarction (MI), cerebrovascular accidents (CVA) and peripheral vascular disease

(PVD).


Standard assessment of 25(OH)D was instituted in August 2009 as part of routine

haematological and biochemical laboratory measurements which included HbA1c,

(Complete Blood count (CBC)), Urea and Electrolytes (UE), Liver function tests

(LFT), bone profile (Corrected Calcium (CCa2+), Alkaline Phosphatase (ALP),

Albumin (Alb), and Lipid profile (Total Cholesterol (T-CHL), High Density

lipoprotein Cholesterol (HDL), Triglycerides (TRIG)). Low Density lipoprotein (LDL)

233

analyses were not undertaken and patients were routinely advised that bloods

were to be collected under fasting conditions.

For 25(OH)D measurement, serum was separated from whole blood and stored at

-20 C until assay. The laboratory used for the biochemical assay measurements

(Vitamin D Research Group Manchester Royal Infirmary, UK) was accredited to

ISO 9001:2008 and ISO 13485:2003 by Lloyd’s Register Quality Assurance

certificate number LRQ 4001542 and participated successfully in the Vitamin D

quality assurance scheme (DEQAS). Serum 25(OH)D was measured using the

IDS-iSYS multi-discipline automated analyzer (Immunodiagnostic Systems Ltd,

Boldon, Tyne and Wear, UK). The assay is based on chemiluminescent technology

and was performed exactly as per the manufacturer’s instructions. Briefly, samples

were subjected to a pre-treatment step to denature the vitamin D binding protein.

The treated samples were then neutralised in assay buffer and a specific anti-

25(OH)D antibody labelled with acridinium was added. Following an incubation

step, magnetic particles linked to 25(OH)D were added. Following a further

incubation step, the magnetic particles were “captured” using a magnet. After a

washing step and addition of trigger reagents, the light emitted by the acridinium

label was inversely proportional to the concentration of 25(OH)D in the original

sample. Concentration of 25(OH)D was calculated automatically using a 4-point

logistic curve. The reportable range of the assay was 5-140 ng/mL. Inter- and

intra- assay variation of the in-house control was 5.6% and 9.7% respectively.



Mean 25(OH)D ± standard deviation (SD) are presented as a whole and per

234

season and subdivided for ethnic background. ANOVA method or a non-parametric

counterpart, Kruskal-Wallis were used to assess differences between groups

depending on normality of the data. Overall the P value was maintained at 0.05 for

multiple comparison tests (Bonferoni adjustment or Dwass-Steel-Chritchlow-Fligner

pairwise comparison). Further descriptive statistics are presented for

characteristics of 25(OH)D (≥30ng/ml, 20 to <30ng/ml, 10 to <20ng/ml and

<10ng/ml). Multiple linear regression models, after assessment of normality, were

used to examine relationships between vitamin D level and lipids, adjusting for

BMI/type of diabetes and ethnicity. A P value of <0.05 was considered to be

statistically significant. All values are presented as mean ± SD.

235

6.4 RESULTS

The mean serum 25(OH)D concentration was 15.7 ± 9.1ng/ml in an unselected

consecutive sample of 563 subjects with diabetes. The smaller cohort of 44

subjects without diabetes from a general medical clinic had a significantly higher

25(OH) vitamin D of 21.3 ± 14.6ng/ml (Table 6.1). The two cohorts did not differ in

either age or ethnicity.

Table 6-1 Baseline characteristics of subjects with and without diabetes

Subjects with

Diabetes

Control subjects

(without Diabetes) P

N 563 44

Age (years) 59.7±13.3 58.1±15.6 NS

White European, n= (%) 334 (59%) 28 (63%) NS

25(OH)D (ng/ml) 15.7±9.1 21.3±14.6 0.009

Subjects with diabetes were divided into categories based on their 25(OH)D status

and their respective characteristics are detailed in table 3.2. Using the clinically

recognised cut off of 30ng/ml, 511 (91%) subjects had vitamin D deficiency and

179 (32%) had severe (<10ng/ml) vitamin D deficiency (Figure 6.1). The severest

vitamin D deficiency occurred in those of South Asian/Middle Eastern descent with

54% of subjects falling into this group, although 22% of white Europeans were also

severely deficient. Despite marked deficiency in vitamin D there were no

significant differences in either ALP or CCa and these were well within the normal

range in each category. There was no difference in the percentage free of

cardiovascular events in the categories although this was marginally higher in

category 4. Although chi2 analyses did not reach significance (P=0.06), there

seemed to be a greater medication load in the form of multidrug therapy for

236

hypertension in the lower echelons (category 1 and 2) of vitamin D status

compared to category 3 and 4. A significant confounding factor is possibly the

longer duration of diabetes in category 4 and the presence of asymptomatic

undeclared CVD, thus obscuring the true cardiovascular event profile.

Nevertheless, we demonstrate a significant difference in HDL (P=0.003) and

triglycerides (P=0.04) between the categories. On post hoc analysis, HDL in

category 3 and 4 were significantly higher than category 1 (1.4±0.5mmol/l vs

1.3±0.7mmol/l, P=0.02 and 1.6±0.6mmol/l vs 1.3±0.7mmol/l, P=0.002 respectively)

and a similar relationship was found with triglycerides, with triglycerides being

significantly lower in categories 2, 3 and 4 compared to category 1 (1.7±1.5mmol/l

vs 2.2±2.8mmol/l, p=0.002, 1.5±0.9mmol/l vs 2.2±2.8mmol/l, P=0.02 and

1.6±1.3mmol/l vs 2.2±2.8mmol/l, P=0.05 respectively). However, after adjusting for

BMI, season, ethnicity and lipid medication, regression analyses between 25(OH)D

and triglycerides was no longer significant (Table 6.3 (r=-0.06, P=NS), particularly

as the lowest 25(OH)D (category 1) had a higher proportion of Asian subjects with

type 2 diabetes and features of metabolic syndrome. Table 6.3 shows a regression

analyses of lipid fractions with 25(OH)D levels and demonstrates a significant

relationship with HDL (P=0.006) after adjusting for BMI, ethnicity, season and lipid

medication. Figure 6.2 represents a scatter plot showing a positive linear trend of

25(OH)D versus HDL.

237

Figure 6-1 Histogram of 25(OH)D Status in subjects with diabetes*

*Minimal reported 25(OH)D was 5ng/ml

238

Table 6-2 Characteristics by categories of 25(OH)D in 563 unselected subjects with diabetes.

Serum 25(OH)D

Category 1 Category 2 Category 3 Category 4

<10ng/ml 10-<20ng/ml 20-<30ng/ml ≥30ng/ml

N 179 233 99 52

Men (%) 53 57 48 48

Type 2 DM (%) 85 75 69 52

White European, n (%)

71 (40) 146 (63) 73 (74) 44 (84)

South Asian/ Middle East, n

(%) 96 (54) 61 (26) 16 (16) 6 (12)

Afro-Caribbean, n (%)

8 (4) 14 (6) 8 (8) 2 (4)

Far-East Asian, n (%)

4 (2) 12 (5) 2 (2) 0 (0)

% free of Cardiovascular

events 79 80 77 84

% on CHL meds 88 85 82 81

% on BP meds (No

therapy/single therapy/multidrug

therapy)

76 (25/31/45)

76 (24/23/53)

69 (31/30/39)

76 (23/39/38)

Mean±SD P

Age (years) 58.8±13.7 59.6±13.5 61.0±12.4 60.2±13.0 NS

Duration of Diabetes (years)

15.4±9.4 17.9±11.9 19.1±11.1 22.3±15.1 0.03

BMI (kg/m2) 31.2±6.5 30.5±6.4 31.5±7.0 29.1±6.4 NS

Systolic BP (mmHg)

129±17 129±16 133±18 131±17 NS

Diastolic BP (mmHg)

69±10 70±9 69±9 71±11 NS

HbA1c (%) (IFCC (mmol/mol))

8.6±1.9, 69±24

8.4±1.7, 68±20

8.2±1.5, 66±17

8.8±1.9, 73±21

NS

T-CHL (mmol/l) 4.2±1.3 4.1±1.2 4.2±0.9 4.5±1.6 NS

HDL (mmol/l) 1.3±0.7 1.3±0.4 1.4±0.5 1.6±0.6 0.003

TRIG (mmol/l) 2.2±2.8 1.7±1.5 1.5±0.9 1.6±1.3 0.04

ALP (U/L) 94±43 86±33 85±48 85±29 NS

CCa (mmol/l) 2.3±0.1 2.3±0.1 2.4±0.1 2.4±0.1 NS

eGFR (ml/min/l) 73±20 73±19 75±16 73±21 NS

Table key

ALP – Alkaline Phosphatase; BMI – Body Mass Index; BP – Blood Pressure; CCa – Corrected

Calcium; eGFR – estimated Glomerular Filtration Rate; HDL – High-density Lipoprotein Cholesterol;

T-CHL – Total Cholesterol; TRIG – Triglycerides;

239

Table 6-3 Regression analyses of 25(OH)D and T-CHL, HDL and triglycerides in subjects with

diabetes

r

(unadjusted)

P

(unadjusted)

r *

(adjusted)

P*

(adjusted)

T-CHL 0.014 NS -0.007 NS

HDL 0.158 0.0002 0.12 0.006

TRIG -0.112 0.009 -0.06 NS

*adjusted for BMI, ethnicity, season and lipid medication.

Table key

HDL – High-density Lipoprotein Cholesterol; T-CHL – Total Cholesterol; TRIG – Triglycerides.

Figure 6-2 Relationship between serum 25(OH)D and high-density lipoprotein cholesterol

(HDL) in subjects with diabetes

*adjusted for BMI, ethnicity, season and lipid medication.

0

0.5

1

1.5

2

2.5

3

3.5

0 10 20 30 40 50

HDL-C (mmol/l)

25(OH) vit D status (ng/ml)

25(OH)D vs HDL-C

r=0.12, p=0.006

240

Table 6.4 shows ethnic differences in 25(OH)D with a further sub-categorisation for

South Asian/Middle Eastern and white European subjects. There were significant

differences between the South Asian/Middle Eastern group and white Europeans

(11.3±9.6ng/ml vs 18.0±7.0ng/ml, P<0.0001), African-Caribbean (11.3±9.6ng/ml vs

16.4±7.8ng/ml, P=0.001) and Far-East Asian groups (11.3±9.6ng/ml vs

14.5±4.5ng/ml, P=0.02). There were no differences in BMI between the ethnic

subcategories. There was a statistically significant but clinically non-significant

seasonal variation in the 25(OH)D status in the white European group in summer

(20.1±8.8ng/ml) versus winter (16.0±6.1ng/ml) (P=0.007) but there was no

seasonal variation in the South Asian/Middle Eastern group. There were

insufficient data to assess seasonal variation in either the Afro-Caribbean or the

Far-East Asian group.

Table 6-4 Characteristics of subjects with diabetes based on ethnicity

White

European

South

Asian/Middle

East

Afro-

Caribbean*

Far East

Asian*

P

for

comparison

of means

Mean 25(OH)D

(ng/ml)

18.0±7.0 11.3±9.6 16.4±7.8 14.5±4.5 <0.0001

Season Seasonal variation

Spring

(ng/ml)

17.2±9.9 10.6±6.1

Summer

(ng/ml)

20.1±8.8 11.0±5.5

Autumn (ng/ml) 17.8±9.9 10.9±6.8

Winter

(ng/ml)

16.0±6.1 12.5±8.9

P

for comparison

of means

0.009 NS

*Insufficient number to undertake seasonal analysis.

241

6.5 DISCUSSION

Vitamin D deficiency is endemic in the United Kingdom and our data confirm this

showing a pronounced severe (<10ng/ml) vitamin D deficiency in 32% of

individuals, with more than half of South Asian and Middle Eastern subjects

demonstrating severe deficiency. Whilst this highlights the need for routine testing

in patients with diabetes, the general patient population are also deficient as shown

in this study and others (2; 21). However, we have clearly shown a higher 25(OH)D

in the age and ethnicity matched subjects without diabetes in this study. Vitamin D

deficiency has not only been implicated in the causation of both type 1 and 2

diabetes, but those with type 2 diabetes are particularly prone to vitamin D

deficiency, in part due to obesity with the proposed sequestration of 25(OH)D in

adipose tissue (22). Although, a previous study has shown a propensity for vitamin

D deficiency even when adjusting for BMI (23). The reasons for this may be

multifactorial including genetic polymorphisms and variants of vitamin D binding

protein (24). The VDR is expressed on the pancreatic β-cell and activated vitamin

D affects insulin production (8). The predisposition for the development of vitamin

D deficiency may contribute to worsening glycaemic control, leading to the

development of impaired glucose tolerance and eventually diabetes. Unfortunately

most interventional studies of vitamin D have either been secondary analyses of

osteoporosis interventions or have generally had small numbers with low doses of

vitamin D replacement. This has significant implications on the guidelines for

supplementation of vitamin D particularly as the current recommended daily

allowance (RDA) of 400IU, which is less than the RDAs elsewhere, appears

woefully inadequate in the absence of adequate sunlight exposure (25).

242

Seasonal variation in levels of vitamin D are often cited as plausible reasons for

overestimation of vitamin D deficiency, particularly in South Asians, but this is a

controversial issue and previous studies have had conflicting results (26-27). In the

present study in white European subjects, the difference of 4.1 ng/ml from winter to

summer is not considered to be clinically significant. Furthermore, a lack of

seasonal variation in South Asian and Middle Eastern subjects, which may in-part

be related to lack of sunlight exposure through body attire, particularly in women of

South Asian origin, further blunts this minimal seasonal effect. Current data on the

association of vitamin D deficiency with glucose intolerance seems to be

population specific (24). Reduction in serum 25(OH)D concentration has been

noted in a UK South Asian population at risk for Type 2 diabetes compared with

subjects not at risk (28). In the same South Asian population, short-term vitamin D

supplementation increased insulin secretion (28). The data from the Third National

Health and Nutrition Examination study (NHANES) also showed an inverse

association between vitamin D status and diabetes in non-Hispanic white and

Mexican American people but not in non-Hispanic black people (29). However,

serum 25(OH)D levels have not been related to glucose status in a white European

population from the UK (30). Assessing vitamin D status in population specific

targets may an important facet in addressing poorer glycaemic control particularly

in ethnic minorities at risk of vitamin D deficiency living in the UK.

Cholesterol and vitamin D share a common metabolic pathway through 7-

dehydrocholesterol which is a precursor for both. Our study has shown a positive

but albeit weak correlation between 25(OH)D and HDL, confirming the results of

other studies (18). Our subjects are largely (approximately 80%) on statin therapy

which, of course, impacts on HDL and probably also on 25(OH)D. Atorvastatin and

243

rosuvastatin have been shown to increase levels of 25(OH)D, and statins may

require adequate levels of vitamin D to function optimally (20). However, statins

may worsen diabetes and predispose to dysglycaemia which would go against the

notion of statins increasing vitamin D levels may protect against diabetes mellitus.

However, the mechanism by which statins may cause dysglycaemia is distinct from

the vitamin D pathway and may be via inhibition of β-cell glucose transporters,

delayed ATP production, pro-inflammatory and oxidative β-cell effects of plasma-

derived cholesterol, inhibition of calcium channel-dependent insulin secretion and

β-cell apoptosis (31). In a recent study culturing macrophages from obese,

diabetic, hypertensive patients with 1,25(OH)2D suppressed foam cell formation by

reducing LDL uptake, whilst deletion of the VDR in these macrophages accelerated

foam cell formation through increased LDL uptake (19). Thus vitamin D may play

an important, perhaps even permissive role in atherosclerosis.

Whilst this cross-sectional study has limitations as it provides association and not

cause and effect a major strength is that all participants were from a single centre

and from a small geographical locality. The north west of England is known to have

lower levels of sunlight when compared to other areas of the UK (2) and this may

potentially impact on transferability of results. However, despite these

shortcomings, we are mindful that the results of this study are not dissimilar to

those of other studies in the UK (2).

In conclusion, we provide further confirmation of the epidemic of vitamin D

deficiency in diabetic patients (85% in Type 1 diabetes and 93% in Type 2

diabetes) in England. Furthermore, we provide an association with lipid

abnormalities providing a potential mechanistic link observed in the landmark

longitudinal studies such as the Framingham cohort study (15) which showed

244

approximately twice the cardiovascular risk in those with low levels of 25(OH)D

compared to higher levels. We also confirm the lower levels in those from South

Asian, Far Eastern and Afro-Caribbean ethnicity. Moreover we show that seasonal

variation contributes minimally to differences in vitamin D status, particularly in

South Asians. These data therefore highlight the need to assess 25(OH)D levels in

all patients with diabetes, not only those with symptoms of osteomalacia and also

confirms the inability of corrected calcium or alkaline phosphatase levels to target

individuals for assessment. Whether supplementation will lower cardiovascular risk

is not known and the results of the VITAL study (a randomised placebo controlled

trial of 25(OH)D and omega-3) are awaited (clinicaltrials.gov - NCT01169259).

245

6.6 REFERENCES


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Pinacho F, Duenas A: Effect of atorvastatin on bone mineral density in patients

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25-hydroxyvitamin D concentrations fluctuate seasonally in young adults of diverse

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and impairment of insulin secretion in relation to vitamin D deficiency in east

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ethnicity in the Third National Health and Nutrition Examination Survey. Diabetes

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249

7 Chapter VII - Differential effects of different vitamin D

replacement strategies in patients with diabetes


acquired data, performed statistical analysis and wrote the manuscript which

constitutes the basis for this chapter and was submitted and accepted for

publication in the Journal of Diabetes and its Complications 2013 (Epub ahead of

print).

Uazman Alam, Agnes WS Chan, April Buazon, Cristiano Van Zeller, Jacqueline L

Berry, Ravinder S Jugdey, Omar Asghar, John Kennedy Cruickshank, Ioannis N

Petropoulos, Rayaz A Malik

250

7.1 ABSTRACT

The optimal treatment regimen for correcting vitamin D insufficiency in diabetic

patients has not been established.

Two hundred and forty four adult diabetic patients with vitamin D insufficiency were

enrolled to receive: Ergocalciferol (D2) 50,000 IU daily over 10 days (500,000 IU)

followed by Calcichew D3 (calcium carbonate/Cholecalciferol) BID (~24,000IU

cholecalciferol/month) (ECC) (n=53); Cholecalciferol (D3) 40,000 IU daily over 10

days (400,000 IU) followed by Calcichew D3 BID (~24,000IU cholecalciferol/month)

(CCC) (n=94) or Cholecalciferol 40,000 IU daily over 10 days (400,000 IU) followed

by Cholecalciferol 40,000 IU monthly (CC) (n=97). The 25(OH)D, HbA1c, lipids,

blood pressure and eGFR were assessed at baseline and after a mean follow up of

8.0±4.0 months.

Treatment increased 25(OH)D concentrations significantly in ECC (17.4±13.8 vs

29.9±9.6ng/ml, P<0.0001), CCC (14.2±6.6 vs 30.9±13.1ng/ml, p<0.0001) and CC

(13.5±8.4 vs 33.9±14.4ng/ml, P<0.0001). The relative increase in 25(OH)D was

significantly lower with ECC compared to CC (+14.6±12.2 vs +20.6±15.0, P=0.01)

and the majority of subjects in the ECC group (63%) remained vitamin D deficient

(25(OH)D<30ng/ml) compared to CCC (46%) and CC (36%)(P=0.0005).

This study demonstrates that relatively aggressive treatment regimens of both

vitamin D2 and D3 increase 25(OH)D concentrations in diabetic patients, but the

ability to raise 25(OH)D status to ‘sufficient’ levels is inadequate in a large

proportion of individuals.

251

7.2 INTRODUCTION

There is an epidemic of vitamin D deficiency worldwide (1). Vitamin D has been

implicated not only in bone health but also a host of cardiovascular diseases

including diabetes, hypertension and metabolic syndrome (2). Vitamin D deficiency

poses a significant health problem in many countries irrespective of economic,

cultural or geographic diversity (3).

There is currently consensus that levels below 10ng/ml (25nmol/L) qualify as

‘deficient’ and require treatment, but beyond this there is currently no standard

definition of ‘optimal’ 25(OH)D with some sources suggesting that levels above

20ng/ml (50nmol/L) are ‘sufficient’, while 28-32ng/ml (70–80nmol/L) are ‘optimal’

(4). The Department of Health in the UK has set dietary recommendations for

children aged 6 months to 5 years, those aged 65 years and over (5), and pregnant

or lactating women and also recommends that people at risk of low sun exposure

should get 10 µg of vitamin D a day, through supplementation (6). However, the

recommended daily allowance (RDA) has not changed despite its inability to

prevent severe vitamin D deficiency and ensuing bone or metabolic complications

in the absence of adequate sunlight exposure (7-9).

Recent data confirm the high prevalence of vitamin D deficiency and no impact of

dietary vitamin D intake in the UK due to low amounts of fortification (10).

Furthermore, proposed treatment strategies recommending ‘over the counter’

vitamin D preparations have been found to be largely ineffective in correcting

vitamin D deficiency (2; 7; 11). Currently there are limited data to guide policy on

replacement strategies (2; 7). Previous studies have suggested that vitamin D2 is

2-3 times less effective at increasing serum 25-hydroxyvitamin D (25(OH)D) than

252

vitamin D3, but that vitamin D2 taken on a daily basis in high enough doses may be

as effective in maintaining 25(OH)D as vitamin D3 (12-14).

We assessed the effects of 3 different vitamin D replacement regimens (using

combinations of ergocalciferol, cholecalciferol and calcium

carbonate/cholecalciferol) dependent on the clinician’s preference for treatment of

vitamin D deficiency.

253


The study was undertaken at the Central Manchester University Hospitals NHS

Foundation Trust, Manchester, UK. Data from a population-based cohort of 244

patients with Diabetes (Type 1 and 2 Diabetes Mellitus) attending a tertiary

diabetes centre were analyzed. Ethical approval was not required for this analysis

as all the data were extracted retrospectively. This therefore did not extend beyond

standard clinical practice. All patients with a 25(OH)D <30ng/ml were allocated to 3

different treatment regimens based on the prescribing choice of three different

clinicians: Ergocalciferol (D2) 50,000 IU daily over 10 days (500,000 IU) followed by

Calcichew D3 (calcium carbonate/Cholecalciferol) BID (~24,000IU

cholecalciferol/month) (ECC) (n=53); Cholecalciferol (D3) 40,000 IU daily over 10

days (400,000 IU) followed by Calcichew D3 BID (~24,000IU cholecalciferol/month)

(CCC) (n=94) or Cholecalciferol 40,000 IU daily over 10 days (400,000 IU) followed

by Cholecalciferol 40,000 IU monthly (1st day of each month (CC (n=97). All

subjects were provided with vitamin D regimens as soon as vitamin D results were

made available, however, data on medication compliance were unavailable.




Hamburg, Germany) to the nearest 0.1kg and height to the nearest 0.1cm with a

calibrated stadiometer. Both height and weight were taken at comparable times at

baseline and follow up. Blood pressure (BP) was assessed using an automated BP


254

appropriate cuff size. Two measurements of systolic and diastolic BP were made

five minutes apart with the lowest reading recorded.


Medications

An assessment of patient demographics was made through analysis of medical

records and an in-hospital medical record database (Diamond database, Hicom,

Surrey, UK). Subject demographics extracted were age, sex, ethnicity (White

European, South Asian, Far East Asian and Afro-Caribbean descent), type

(T1DM/T2DM), duration of diabetes and current anti-diabetic therapy with any

alterations to treatment during the intervention period.


Assessment of 25(OH)D was instituted in August 2009 as part of routine


Complete Blood count (CBC), Urea and Electrolytes (UE), Liver function tests


Albumin (Alb), and Lipid profile (Total Cholesterol (T-CHL), High Density lipoprotein

Cholesterol (HDL), Triglycerides (TRIG)). For 25(OH)D measurement, serum was

separated from whole blood and stored at -20 C until assay. The laboratory used

for the biochemical assay measurements (Vitamin D Research Group Manchester

Royal Infirmary, UK) was accredited to ISO 9001:2008 and ISO 13485:2003 by

Lloyd’s Register Quality Assurance certificate number LRQ 4001542 and

participated successfully in the Vitamin D quality assurance scheme (DEQAS).

Serum 25(OH)D was measured using the IDS-iSYS multi-discipline automated

255

analyzer (Immunodiagnostic Systems Ltd, Boldon, Tyne and Wear, UK). The assay

is based on chemiluminescent technology and was performed exactly as per the

manufacturer’s instructions. The cross reactivity for vitamin D2 (of the assay) as per

manufacturers assertion was 100% (relative to vitamin D3) and the assay has

excellent correlation to existing globally recognised assays, in combination with

good sensitivity and precision (15). The reportable range of the assay is 5-140

ng/ml Inter- and intra- assay variation of the in-house control was 5.6% and 9.7%

respectively.


Statistical analyses were undertaken using Statsdirect (Statsdirect, Cheshire, UK).

ANOVA method or a non-parametric counterpart, Kruskal-Wallis were used to

assess differences between groups depending on normality of the data. Overall the

P value was maintained at 0.05 for multiple comparison tests (after Bonferoni

adjustment or Conover-Inman pairwise comparison). Paired t-tests were used for

intra-individual observations. A P value of ≤0.05 was considered to be statistically

significant. All values are presented as mean ± standard deviation (SD).

256

7.4 RESULTS

Demographics of the three cohorts are shown in Table 7.1. They did not differ in for

age, type of diabetes, sex and ethnicity and there were no differences in the period

of follow up. A significant proportion of subjects still remained deficient on ECC

regimen of supplementation (P=0.0005) compared to the other regimens (CCC and

CC). Anti-diabetic therapies used in each cohort are detailed in Table 7.1. There

were significant differences in the types of anti-diabetic regimens used (chi2

P=0.01) across the 3 groups, however, there were no significant differences in the

numbers of individuals who had intensification of their diabetes treatment in each

cohort from baseline to follow up. In those individuals requiring insulin

administration, mean doses of insulin units required per day did not differ.

257

Table 7-1 Clinical and demographic data and Characteristics of Anti-Diabetic therapy in all

three cohorts of all three intervention cohorts

ECC (n=53) CCC (n=94) CC (n=97)

Age (years) 60.7±12.1 58.2±13.3 57.0±12.2

Sex (% male) 55 57 52

Type 2 DM (%) 81 68 82

Duration of diabetes (years) 16.7±11.2 21.1±12.8 17.1±10.3

Ethnicity (% White European)

55 57 52

% on lipid lowering therapy 80 90 87

Period of follow up (months)

8.0±3.7 7.8±3.9 8.1±4.3

Insufficient after supplementation

with 25(OH)D (<30ng/ml) n (%)

32 (63)* 43 (46)* 35 (36)*

Treatment regimens^

Diet controlled n (%) 0 (0) 3 (3) 1 (1)

Single oral therapy n (%) 8 (15) 7 (7) 4 (4)

Oral combination therapy n (%)

20 (38) 22 (23) 31 (32)

Oral and/or Insulin + GLP-1 mimetic n (%)

1 (2) 8 (9) 10 (10)

Oral + Insulin therapy n (%) 15 (28) 21 (22) 30 (31)

Insulin alone n (%) 12 (23) 31 (33) 20 (21)

CSII therapy n (%) 2 (4) 2 (2) 1 (1)

Mean Insulin dose/day (mean±SD) Median(IQR)

86±81** 64(44-76)

92±149** 68.5(48-88)

84±63** 65(52-104)

Escalation in Anti diabetic Therapy n (%)

11 (19)*** 20 (21)*** 17 (18)***

No. of subjects with increase in insulin n (%)

1 (2) 10 (11) 2 (2)

Average dose increase in insulin/day (units)

(mean±SD) 6±0 4±1.3 2±0

Post hoc analyses

*P=0.0005 (Chi2 test); ^Chi

2 for treatment regimens – P=0.01; **KrusKal-Wallis – P=NS.

***Chi 2 for intensification of anti-diabetic therapy – P=NS.

Type 2 DM – Type 2 Diabetes Mellitus.

Table key

CSII – Continuous Subcutaneous Insulin Infusion, DM – Diabetes Mellitus, IQR – Interquartile

range, SD – Standard deviation.

258

25(OH)D status and metabolic data are presented in Table 7.2. The 25(OH)D

concentrations at baseline were higher in the ECC cohort when compared to CCC

and CC (17.4±13.8 vs 14.2±6.6 vs 13.5±8.4ng/ml), respectively, although this did

not reach statistical significance. After treatment 25(OH)D concentrations

increased significantly in the ECC (17.4±13.8 vs 29.9±9.6, P<0.0001), CCC

(14.2±6.6 vs 30.9±13.1, P<0.0001) and CC (13.5±8.4 vs 33.9±14.4ng/ml,

P<0.0001) cohorts, respectively. There was no difference in the increase in

25(OH)D comparing ECC to CCC (+14.6±12.2 vs +16.7±13.4ng/ml). However,

there was a significantly greater increase in CC compared to ECC (+20.6±15.0 v

+14.6±12.2, P=0.01) with no difference compared to CCC. However, 63% of

patients remained deficient (25(OH)D <30ng/ml) after treatment in the ECC cohort

compared to 46% and 36% in the CCC and CC cohorts, respectively (chi2

P=0.0005). When further subdividing the three cohorts into those with an initial

25(OH)D <15ng/ml and >15ng/ml (based on the study by Wang et al (16)) there

were significant differences between pre- and post-intervention, with a more

pronounced increase in 25(OH)D in those with a lower baseline 25(OH)D (ECC:

+21.2±8.4 vs +6.4±11.4ng/ml, p<0.0001, CCC: +20.1±13.8 vs +12.3±11.6ng/ml,

P=0.005, CC: +24.4±14.5 vs +12.1±12.9ng/ml, P=0.01)(figures 7.1a, b and c).

There were no significant changes in BMI, systolic and diastolic blood pressure,

lipid profile (T-CHL, HDL and TRIG) and eGFR after treatment in any of the

cohorts. However, there was a significant reduction in HbA1c after treatment in

ECC (-0.5% or 5.2mmol/mol, P=0.02), with no change in the CCC (8.3±1.7 vs

8.3±1.7%) or CC (8.2±1.6 vs 8.1±1.6%) cohorts.

259

Table 7-2 Change in 25(OH)D status, metabolic data and anthropometric measurements

following intervention in all three cohorts

ECC (n=53) CCC (n=94) CC (n=97)

Baseline Follow up Baseline Follow up Baseline Follow up

25(OH)D

(ng/ml) 17.4±13.8* 29.9±9.6^/* 14.2±6.6^^ 30.9±13.1^^ 13.5±8.4** 33.9±13.6^/**

BMI (kg/m2) 29.6±8.6 30.0±7.3 31.4±7.3 31.0±6.9 31.7±6.7 31.2±6.3

Systolic BP

(mmHg) 126±16 132±16 128±15 128±18 130±18 132±18

Diastolic BP

(mmHg) 68±10 67±13 68±10 70±16 70±12 70±19

HbA1c (%)

(IFCC

(mmol/mol))

8.5±1.7***

(69.8±18.6)

8.0±1.5***

(64.6±16.8)

8.3±1.7

(66.8±18.7)

8.3±1.7

(66.7±18.8)

8.2±1.6

(66.3±17.3)

8.1±1.6

(64.9±17.2)

T-CHL

(mmol/l) 4.1±1.3 4.3±1.5 4.0±1.0 4.1±1.4 4.1±1.2 4.0±0.9

HDL (mmol/l) 1.4±0.5 1.3±0.5 1.4±0.5 1.4±0.5 1.2±0.3 1.3±0.4

TRIG(mmol/l) 1.7±1.3 2.0±2.4 1.6±0.9 1.4±1.2 2.0±2.2 1.7±1.0

eGFR

(ml/min/l) 75±18 77±16 74±17 74±17 79±17 77±17

Post hoc analyses

25(OH)D - *p<0.0001; ^ ECC vs CC (p=0.01); **P<0.0001, ^^P<0.0001.

HbA1c - ***P=0.02.

Table key

BMI – Body Mass Index; BP – Blood Pressure; eGFR – estimated Glomerular Filtration Rate; HDL

– High-density Lipoprotein Cholesterol; T-CHL – Total Cholesterol; TRIG – Triglycerides.

260

Figure 7-1a Box and Whisker plots for 25(OH)D pre- and post-intervention in ECC

subdividing into <15ng/ml and >15ng/ml

1st VIT D ECC <15ng/ml 2nd Vit D ECC <15ng/ml 1st VIT D ECC >15ng/ml 2nd Vit D ECC >15ng/ml

0

10

20

30

40

50

6025 (OH)D pre- and post-intervention dependent on baseline Vitamin D status

25

(O

H)D

ng

/ml

P<0.0001 +6.4 ± 11.4

ng/ml

+21.2 ± 8.4

ng/ml

261

Figure 7-2b Box and Whisker plots for 25(OH)D pre- and post-intervention in CCC

subdividing into <15ng/ml and >15ng/ml

1st VIT D CCC <15 ng/ml 2nd Vit D CCC <15 ng/ml 1st VIT D CCC >15 ng/ml 2nd Vit D CCC >15 ng/ml

0

10

20

30

40

50

60

70

25

(O

H)D

ng

/ml


+12.3 ± 11.6

ng/ml

+20.1 ± 13.8

ng/ml

P=0.005

0.)00.00

5

262

Figure 7-1c Box and Whisker plots for 25(OH)D pre- and post-intervention in CC subdividing

into <15ng/ml and >15ng/ml

1st VIT D CC <15ng/ml 2nd Vit D CC <15ng/ml 1st VIT D CC >15ng/ml 2nd Vit D CC >15ng/ml

0

10

20

30

40

50

60

70


25

(O

H)D

ng

/ml

P = 0.01 +12.1 ± 12.9

ng/ml

+24.4 ± 14.5

ng/ml

263

7.5 DISCUSSION

Vitamin D deficiency is highly prevalent and current guidelines recommending

replacement regimes appear inadequate (17). In 2011, the US Institute of Medicine

(IOM) Recommended Dietary Allowance (RDA) of vitamin D was 800 IU per day

for older adults. A serum 25(OH)D of 20 ng/ml is considered to be sufficient by the

IOM. However, recently the US Endocrine Society's Clinical Practice Guideline

suggest 1500-2000 IU per day for adults aged 19 years or more to maintain

25(OH)D above the optimal level of 30 ng/ml (18). Earlier data suggested that at

least 55 µg (2200IU)/day is necessary to increase 25(OH)D concentrations from

20- 40 nmol/L (8-16 ng/ml) to 80 nmol/L (32 ng/ml) (19). Yet both the UK

Department of Health and NICE continue to endorse dietary supplementation of 10

µg (400IU)/day and UK recommendations, particularly for primary care advocate

‘over the counter’ supplements or dual calcium carbonate and cholecalciferol

supplementation which only contain 100-400IU of cholecalciferol/per tablet (7; 20).

Recent guidelines by Holick et al (21) have suggested in adults that a dose of

400,000IU vitamin D2 over 8 weeks followed by a maintenance therapy of 1500-

2000IU daily is adequate in the treatment of deficiency. In a retrospective cohort

study from the US, a dosing schedule of D2 (50,000IU on alternate weeks) has

recently been shown to be effective, with a baseline 25(OH)D of 31.0 ± 10.6 ng/mL

increasing to 48.3 ± 13.4 ng/mL (22). Our treatment protocols at the Manchester

Diabetes centre consisted of a loading dose of vitamin D2 (ergocalciferol) or D3

(cholecalciferol) at non-bioequivalent doses of 500,000IU and 400,000IU

respectively, followed by a maintenance dose of cholecalciferol with or without

calcium carbonate. In our study the baseline 25(OH)D concentration was lower,

264

than the previous study, and therefore despite a higher dose replacement the

increase in 25(OH)D was not as marked (D2 ~12-13ng/ml, D3 ~20ng/ml). Most

treatment protocols do not differentiate between the therapeutic effect of vitamin D2

and D3 (7; 23), although, clearly this has implications on therapeutic effects and

monitoring of treatment. The difference in increasing the concentrations of

25(OH)D is both physiologically and pharmacologically meaningful with studies

showing that increasing 25(OH)D improves calcium absorption (2), reduces fall

frequency (24) and osteoporotic fracture risk (25). Previous studies have

suggested that 50,000IU of vitamin D2 may be biologically equivalent to ~15,000IU

of vitamin D3 (13). Of course, vitamin D2 is effective in elevating 25(OH)D

concentrations, but has been considered not to be bio-equivalent with a

suggestion for dose adjustment in treatment protocols (26). However, conclusions

regarding the biological consequence and functional effects of the D2 and D3

cannot clearly be drawn. Additional studies are needed to elucidate the

pharmacokinetic parameters and the varying vitamin D2 and vitamin D3 25(OH)D

metabolites produced which may have differing biological affinity and thus differing

physiological responses (19; 27).

Despite these differences a considerable proportion of individuals still remain

vitamin D deficient on these regimens. Thus even with doses of cholecalciferol

which were approximately equivalent to the current recommended maintenance

therapy in addition to an ‘adequate’ loading dose still resulted in around 46% of

individuals being below the target 25(OH)D of 30ng/ml. This cannot be due to

inadequate time of exposure to treatment as the time to equilibrium of vitamin D3 is

approximately 5 months (28). Our data confirm a significant disparity of effect

between those supplemented at lower compared to higher baseline levels (29). It

265

has been previously suggested that there is essentially a linear relationship up to

10000IU (4), however there are minimal data directly assessing baseline 25(OH)D

status and response to a standardised vitamin D intervention. Variations in

circulating concentrations can also be attributed to differences in the transport of

vitamin D in the circulation following exposure to the sun and oral routes (30) with

an initial rapid hepatic delivery of vitamin D via the oral route but the increase in

concentrations is not as well sustained. However, in the present study all three

interventions were oral and of sufficiently long duration to ensure a steady state

concentration. Therefore the differences can be primarily attributed to

pharmacological differences in the replacement regimens, although genetic

polymorphisms may affect the vitamin D metabolic pathway (31). Furthermore, we

show that patients with a lower baseline show the most marked rise in

concentrations of 25 (OH)D, despite equivalent doses. This may be important for

dosing considerations when establishing replacement protocols and the need for

less conservative treatment (32). In fact vitamin D shares a similar intake-response

curve as other nutrients and in persons whose baseline values differ, an identical

nutrient intake may or may not produce a measurable response (33).

Unfortunately, most of the randomized, controlled trials of vitamin D that have been

published to date have paid little attention to baseline status (33).

A range of potentially beneficial metabolic effects of vitamin D have been proposed

(34-36). Vitamin D receptors are of course expressed ubiquitously along with

vitamin D activating enzymes (2) and are also present on pancreatic β-cells (2).

Circulating 25(OH)D is inversely associated with insulin resistance and possibly

insulin secretion (37), and there is a inverse relationship between 25(OH)D

concentrations and the risk of Type 2 DM (38). Moreover, a recent study has

266

shown an improvement in HbA1c when diabetic patients were given vitamin D (39).

A recent meta-analysis, stated ‘there was a insufficient evidence of beneficial effect

to recommend vitamin D supplementation as a means of improving glycaemia or

insulin resistance in patients with diabetes, normal fasting glucose or impaired

glucose tolerance’, although the doses used for replacement were not adequate

(40). In the present study, whilst there was an overall intensification of anti-diabetic

treatment in all groups there was no significant difference between the cohorts.

Importantly the administration of D2 did not achieve adequate plasma

concentrations of 25(OH)D which was lower than CCC and CC cohorts which did

not produce significant drops in glycaemia. Therefore the improvement in blood

glucose control cannot be entirely accredited to ergocalciferol administration. This

study provides hypothesis generating data to assess any differential effect of

vitamin D2 and D3 on glycaemic control.

A major limitation of the present retrospective analysis was that it was not

randomised (to treatment groups) and there was no placebo arm and was

therefore subject to a greater degree of confounding. However, it provides

interesting hypotheses generating ‘real world’ data on the ‘correct’ dosing regimens

in vitamin D deficiency and the need for more intensive dosing regimens for

patients with vitamin D deficiency. This highlights the significant need for high

quality randomised controlled trials assessing the effectiveness of cholecalciferol

and ergocalciferol in the treatment of vitamin D deficiency and metabolic outcomes

in diabetes.

267

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markers during fall and winter in schoolchildren. Eur J Clin Nutr 2010;64:1415-

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Lanham-New SA: Vitamin D deficiency in UK South Asian Women of childbearing

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Arvizu RA, Gallagher JC, Gallo RL, Jones G, Kovacs CS, Mayne ST, Rosen CJ,

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25-hydroxycholecalciferol response to extended oral dosing with cholecalciferol.

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vitamin D3 increases serum 25-hydroxyvitamin D more efficiently than does

vitamin D2. The American Journal of Clinical Nutrition 1998;68:854-858

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transport of vitamin D after its endogenous synthesis. J Clin Invest 1993;91:2552-

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Vitamin D. Annals of Epidemiology 2009;19:441-445

33. Heaney RP: Vitamin D — Baseline Status and Effective Dose. New England


34. Holick MF: Evidence-based D-bate on health benefits of vitamin D revisited.

Dermatoendocrinol 2012;4:183-190

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Endocrinol 2012;8:388-390

36. Holick MF: Vitamin D: extraskeletal health. Rheum Dis Clin North Am

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37. Peechakara SV, Pittas AG: Vitamin D as a potential modifier of diabetes risk.

Nat Clin Pract End Met 2008;4:182-183

38. Mitri J, Muraru MD, Pittas AG: Vitamin D and type 2 diabetes: a systematic

review. Eur J Clin Nutr 2011;65:1005-1015

39. Ali AM, Awad, T.G., Ali, O.M.: Effect of Short-Term Vitamin D Supplementation

On Glycemic Control In Newly-Diagnosed Type 2 Diabetics. Australian Journal of

Basic and Applied Sciences 2011;5:1235-1239

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8 Chapter VIII - Vitamin D Deficiency Contributes to

Painful Diabetic Neuropathy




analyses and the writing of this chapter.

Submitted to Diabetes awaiting revision and resubmission

Uazman Alam, Ioannis N Petropoulos, Georgios Ponirakis, Omar Asghar, Maria

Jeziorska, Hassan Fadavi, Andrew Marshall, Mitra Tavakoli, Andrew JM Boulton,


273

8.1 ABSTRACT

The aetiology of painful diabetic neuropathy (PDN) is unclear. Low vitamin D levels

have been associated with a number of painful conditions. We have evaluated

vitamin D levels in diabetic patients with and without painful neuropathy.

Forty three type 1 diabetic patients subdivided into painless neuropathy (DPN)

(n=20) and painful neuropathy (PDN) (n=23) and 14 non-diabetic healthy control

subjects (C) underwent detailed assessment of neurologic deficits, quantitative

sensory testing (QST), electrophysiology, skin biopsy and corneal confocal

microscopy (CCM) together with measurement of serum 25(OH)D.

There were no significant differences for age, BMI, HbA1c, lipids, neurological

evaluation, QST, electrophysiology, intra-epidermal nerve fibre density (IENFD)

and corneal nerve morphology when comparing DPN with PDN. Both positive

(hyperalgesia and allodynia) and negative symptoms (paraesthesia and

numbness) of diabetic neuropathy were greater in PDN compared to DPN

(P=0.009 and P=0.02 respectively). Serum 25(OH)D level were significantly lower

in PDN (24.0±14.1ng/ml) compared to DPN (34.6±15.0ng/ml, P=0.01) and controls

(34.1±8.6ng/ml, P=0.03). The odds ratio in favour of painful diabetic neuropathy

was 9.8 (P=0.003 (95% CI 2.2-76.4) for vitamin D deficiency (<20ng/ml) and 4.4

(P=0.03 (95% CI 1.1-19.8)) for vitamin D insufficiency (<30ng/ml).

This study provides a novel association between vitamin D deficiency and painful

diabetic neuropathy.

274

8.2 INTRODUCTION

A large population based study has recently shown that the prevalence of painful

diabetic neuropathy is ~21% and painful symptoms are more prevalent in patients

with type 2 diabetes, females and South Asians (1). It is characterised by

symmetrical lower limb paraesthesiae, dysaesthesiae, lancinating pains and

allodynia with nocturnal exacerbation (2) which are associated with significant

sleep disturbance and reduced quality of life (3). National and international

guidelines advocate a range of therapies providing symptomatic relief (4-5).

However, the therapeutic efficacy for all recommended medications is at best

~50% pain relief and is limited due to unwanted side effects (2; 6). Apart from

peripheral and central alterations (7), metabolic alterations such as increased

glycaemic flux (8) and elevated plasma methylglyoxal levels have been implicated

in patients with painful diabetic neuropathy (9).

Two recent observational studies in diabetic patients have demonstrated a

significant association between vitamin D deficiency and both paraesthesiae and

numbness (10) and diabetic neuropathy per se, defined using the neuropathy

symptom score, neurological deficits and electrophysiology (11). However, neither

of these studies delineated negative from positive symptoms and there was no

definitive assessment of pain. There is of course an association between vitamin D

deficiency and painful symptoms in the general population (12). In relation to a

mechanistic link between vitamin D and pain, a recent study has shown that

nociceptive calcitonin gene-related peptide (CGRP)-positive neurones have a

distinct vitamin D phenotype with hormonally-regulated ligand and receptor levels

(13). Vitamin D deficiency results in increased numbers of axons containing CGRP,

and in culture vitamin D receptor (VDR) expression is increased in growth cones

275

and sprouting appears to be regulated by VDR-mediated rapid response signalling

pathways (14). Nerve Growth Factor (NGF) is known to be depleted in

experimental diabetes (15) and in a study of patients with diabetic neuropathy NGF

immunostaining on skin keratinocyte correlated with skin axon-reflex vasodilation

(16). In an experimental study a preservation of NGF expression was shown in

sciatic nerves of diabetic animals treated with a vitamin D analogue (CB1093).

Similarly Tacalcitol, an active vitamin D3 induces NGF production in human

epidermal keratinocytes (17). Given that demyelination and axonal degeneration

are the hallmarks of diabetic neuropathy (18-20), of potential relevance, treatment

with vitamin D3 has been shown to reduce demyelination in a cuprizone

experimental model of demyelination (21) and in a separate spinal cord

compression model it has been shown to induce axonal regeneration (22).

In relation to the potential therapeutic benefits of vitamin D therapy, a prospective

study by Lee et al (23) of 51 patients with Type 2 diabetes and painful neuropathy

given ~2000 IU of cholecalciferol daily for 3 months showed a ~50% decrease in

pain scores as measured by Visual Analogue Score (VAS). Similarly a dramatic

improvement in neuropathic symptoms has been described recently after treatment

with 50000U of D2 weekly in a type 1 diabetic patient who had been refractory to

treatment with tricyclics, gabapentin, pregabalin and oxycodone (24).

In the present study we have evaluated the association between the levels of

25(OH)D and painful versus painless diabetic neuropathy matched for all other

measures of neuropathy.

276



Forty three type 1 diabetic patients were categorized into two groups: painless

neuropathy (DPN) (n=20) and painful neuropathy (PDN) (n=23) using the McGill

Visual Analogue Score (McGill VAS) and McGill pain score, and compared with 14

age, sex and ethnicity matched non-diabetic healthy control subjects (C). Subjects

with any history of neurologic conditions, ocular trauma or ocular surgery were

excluded. The study was approved by the North West and Salford and Trafford

Research Ethics committee, and written informed consent was obtained according

to the Declaration of Helsinki.



symptoms according to the neuropathy symptom profile (NSP), and the McGill VAS

was used to assess the severity of painful neuropathy. Neurologic deficits were

assessed using the modified neuropathy disability score (NDS), which includes

evaluation of vibration, pin prick, and temperature perception as well as the

presence or absence of ankle reflexes (25). Quantitative sensory testing included

an assessment of vibration perception threshold (VPT), measured using a



(WST) (26) (C fibres) thresholds using the method of limits with the MEDOC TSA II

(Medoc, Ramat Yishai, Israel) on the dorsum of the left foot. Computer-Aided

Sensory Evaluator (CASE IV) (WR Medical Electronic Ltd, Maplewood, Maine,

USA) was used to measure the heart rate response to deep breathing (HRV-DB)

277

over two 8-cycle breathing series interspersed by a 5-min period of normal

breathing. Electro-diagnostic studies were undertaken using a Dantec “Keypoint”

system (Dantec Dynamics, Bristol, U.K.) equipped with a DISA temperature

regulator to keep limb temperature constantly between 32°C and 35°C. Peroneal

motor and sural sensory nerves were assessed in the right lower limb by a

consultant neurophysiologist. The motor study was performed using silver-silver

chloride surface electrodes at standardized sites defined by anatomical landmarks,

and recordings for the sural nerve were taken using antidromic stimulation over a

distance of 100 mm.




acquisition (27). Several scans of the entire depth of the central cornea were

recorded by turning the fine focus of the objective lens backward and forward for

~2 min using the section mode, which enables manual acquisition and storage of

single images of all corneal layers. This provides en face two-dimensional images

with a lateral resolution of ~2 mm/pixel and final image size of 400 x 400 pixels of

the subbasal nerve plexus of the cornea. Each nerve fibre bundle contains

unmyelinated fibres, which run parallel to Bowman’s layer before dividing and

terminating as individual axons underneath the surface epithelium. Five images per

patient from the centre of the cornea were selected and examined in a masked and

randomized fashion (28). Three corneal nerve parameters were quantified: 1)

corneal nerve fibre density (CNFD), the total number of major nerves per square

millimetre of corneal tissue (no.mm2); 2) corneal nerve branch density (CNBD), the

278

number of branches emanating from all major nerve trunks per square millimetre of

corneal tissue (no.mm2); and 3) corneal nerve fibre length (CNFL), the total length

of all nerve fibres and branches (mm/mm2) within the area of corneal tissue.


Intra-epidermal nerve fibre density (IENFD) was assessed in a sub-cohort of

participants (Controls n=10, DPN n=11, and PDN n=9) who agreed to undergo a 3-

mm punch skin biopsy from the dorsum of the foot; 2 cm proximal to the second

metatarsal head, after local anaesthesia (1% lidocaine). The biopsy specimen was

immediately fixed in PBS-buffered 4% paraformaldehyde and after 18–24 hours

rinsed in Tris-buffered saline and soaked in 33% sucrose (2–4 hours) for

cryoprotection. It was then embedded in optimal cutting temperature-embedding

compound, rapidly frozen in liquid nitrogen, and cut into 50- μm sections using a

cryostat (model OTF; Bright Instruments, Huntington, U.K.). Four floating sections

per subject were subjected to melanin bleaching (0.25% KMnO4 for 15 min

followed by 5% oxalic acid for 3 min), a 4-h protein block with a Tris-buffered saline

solution of 5% normal swine serum, 0.5% powdered milk, and 1% Triton X-100,

and overnight incubation with 1:1,200 Biogenesis polyclonal rabbit anti-human

PGP9.5 antibody (Serotec, Oxford, U.K.). Biotinylated swine anti-rabbit secondary

antibody (1:300; DakoCytomation, Ely, U.K.) was then applied for 1 hour; sections

were quenched with 1% H2O2 in 30% MeOH-PBS (30 min) before a 1 hour

incubation with 1:500 horseradish peroxidase–streptavidin (Vector Laboratories,

Peterborough, U.K.). Nerve fibres were demonstrated using 3, 3-diaminobenzidine

chromogen (Sigma-Aldrich, Manchester, U.K.). Sections were mildly

counterstained with eosin to better localize the basement membrane to identify

279

nerve fibres passing through it. Negative control subjects consisted of replacing the

anti-PGP9.5 antibody with rabbit immunoglobulin (DakoCytomation) at a

concentration matching that of the primary antibody and showed no

immunostaining. IENFD, i.e., the number of fibres per millimetre of basement

membrane, was quantified in accord with established criteria and techniques and

expressed as number per millimetre (29).

8.3.5 25(OH) Vitamin D Assay

The laboratory used for the biochemical assay measurements (Vitamin D

Research Group Manchester Royal Infirmary, UK) was accredited to ISO

9001:2008 and ISO 13485:2003 by Lloyd’s Register Quality Assurance certificate

number LRQ 4001542 and participated successfully in the Vitamin D quality

assurance scheme (DEQAS). Serum was separated from whole blood and stored

at -20 C until assay. The assay used was an automated platform assay

(ImmunoDiagnostic Systems Ltd, Bolden, Tyne and Wear, UK) and is based on

chemiluminescence technology. Briefly, samples were subjected to a pre-treatment

step to denature the vitamin D binding protein. The treated samples were then

neutralised in assay buffer and a specific anti-25(OH)D antibody labelled with

acridinium was added. Following an incubation step, magnetic particles linked to

25(OH)D were added. Following a further incubation step, the magnetic particles

were “captured” using a magnet. After a washing step and addition of trigger

reagents, the light emitted by the acridinium label was inversely proportional to the

concentration of 25(OH)D in the original sample. The concentration of 25(OH)D

was calculated automatically using a 4-point logistic curve. The cross reactivity for

vitamin D2 (of the assay) as per manufacturers assertion was 100% (relative to

280

vitamin D3) and the assay has excellent correlation to existing globally recognised

assays, in combination with good sensitivity and precision (30). The reportable

range of the assay was 5-140 ng/mL. Inter- and intra- assay variation of the in-

house control was 5.6% and 9.7% respectively. Vitamin D deficiency (<20ng/ml)

and insufficiency (<30ng/ml) were defined according to the Institute of Medicine

(IOM) of the National Academies (31).


Statistical analyses were undertaken on Statsdirect (Statsdirect, Cheshire, UK). All

values are presented as mean ± SD. ANOVA method or a non-parametric

counterpart, Kruskal-Wallis were used to assess differences between groups

depending on normality of the data. Overall the p value was maintained at 0.05 for

multiple comparison tests (Bonferoni adjustment or Dwass-Steel-Chritchlow-Fligner

pairwise comparison). Unpaired t-test or Mann-Whitney U test were used for

analysis for DPN versus PDN for the duration of diabetes and IENFD. Chi2

analyses were used to assess frequencies of gender, ethnicity and aetiology of

diabetes. Odds Ratios for painful symptoms were calculated by further delineating

DPN and PDN groups based on the cut offs for vitamin D deficiency (<20ng/ml)

and insufficiency (<30ng/ml).

281

8.4 RESULTS

8.4.1 Demographics, Metabolic and Anthropometric Assessment (Table 8.1)


diabetic patients and control subjects are summarized in Table 8.1. There were no

significant differences in age (C: 59.3±7.8 vs DPN: 57.1±13.5 vs PDN: 59.8±11.7

years, P=NS), duration of diabetes (DPN: 36.0±17.5 vs PDN: 35.5±14.9 years,

P=NS) gender, type of diabetes and all subjects were of white European origin.

BMI was lower in diabetic patients but this was not significant. HbA1c (P<0.0001)

was significantly higher in diabetic patients compared with control subjects with no

difference between patients with PDN and DPN. The total cholesterol was

significantly lower in diabetic patients with DPN (P=0.003) and PDN (P=0.02)

compared to control subjects, due to greater statin use. HDL, triglycerides, systolic

and diastolic blood pressure were comparable between diabetic patients and

control subjects. The estimated glomerular filtration rate was comparable between

diabetic patients and control subjects, but the Albumin-Creatinine Ratio (ACR) was

higher in the DPN (P=0.009) and PDN (P=0.002) groups versus control subjects

with no difference between DPN and PDN. Serum vitamin B12 levels were

comparable between groups.

282

Table 8-1 Participant demographics and metabolic parameters in control subjects and

diabetic patients with DPN and PDN, with statistically significant differences between groups

C (n=14) DPN (n=20) PDN (n=23) DPN v PDN

Age (years) 59.3±7.8 57.1±13.5 59.8±11.7 NS

Gender (Male) (%) 57 60 53 NS

Ethnicity (White

European) (%) 100 100 100 NS

Aetiology of

Diabetes (Type 1

DM) (%)

- 85 91 NS

Duration of

Diabetes (years) - 36.0±17.5 35.5±14.9 NS

HbA1c (%) 5.7±0.2 8.1±1.1* 8.0±1.5** NS

HbA1c (mmol/mol) 38.3±2.5 65.2±12.0* 63.7±16.3** NS

BMI (kg/m2) 31.0±4.2 28.1±4.1 26.6±4.9 NS

T-CHL (mmol/l) 5.1±1.2 4.2±1.0† 4.4±0.8†† NS

HDL (mmol/l) 1.7±0.5 1.7±0.4 1.7±0.6 NS


Systolic BP

(mmHg) 138±15 141±26 141±23 NS

Diastolic BP

(mmHg) 77±8 71±7 71±11 NS

ACR (mg/mmol)

((Median)IQR)

0.4±0.3

(0.2(0.2-0.7))

7.1±16.7¥

(0.2(0.2-0.7)

5.3±6.7¥¥

(2.2(0.5-10.2) NS

eGRF

(ml/min/1.73) 85±7 77±16 78±15 NS

Serum Vitamin

B12 (ng/l) 247±69 342±121 317±111 NS

283

Post hoc analyses

HbA1c: *C vs DPN (P<0.0001); **C vs PDN (P<0.0001); DPN vs PDN (NS).

T-CHL: †C vs DPN (P=0.003); ††C vs PDN (P=0.02); DPN vs PDN (NS).

ACR: ¥C vs DPN (P=0.009); ¥¥C vs PDN (P=0.002); DPN vs PDN (NS).

Table key

ACR – Albumin Creatinine Ratio; BMI – Body Mass Index; C – Controls; DPN – Diabetic Peripheral

Neuropathy; estimated Glomerular Filtration Rate; HDL – High Density Lipoprotein Cholesterol;

PDN – Painful Diabetic Neuropathy; T-CHL – Total Cholesterol.

284

8.4.2 Symptoms and deficits (Table 8.2)

The NSP was significantly higher in patients with PDN (P<0.0001) compared to

control subjects and between patients with PDN compared to DPN (P<0.0005).

Positive symptoms (out of 6) were significantly greater in PDN compared to control

subjects (P=0.05) and DPN (P=0.009). Negative symptoms (out of 4) were

significantly greater in PDN compared to control subjects (P=0.009) and DPN

(P=0.02). The McGill pain score and McGill VAS were significantly increased in

diabetic patients with PDN compared with control subjects (P<0.0001) and patients

with DPN (P<0.0001). The NDS was significantly increased in patients with DPN

(P=0.01) and PDN (P=0.002) compared with control subjects, but there were no

significant difference between DPN and PDN.

285

Table 8-2 Clinical neuropathy symptoms and deficits in control subjects and diabetic

patients with DPN and PDN, with statistically significant differences between groups

Post hoc analyses

NDS: *C vs DPN (P=0.01); **C vs PDN (P=0.002); DPN vs PDN (NS).

NSP: C vs DPN (NS); ^C vs PDN (P<0.0001); DPN vs PDN (P=0.0005).

+ve Symptoms: C vs DPN (NS); σC vs PDN (P=0.05); DPN vs PDN (P=0.009).

-ve Symptoms: C vs DPN (NS); ¥C vs PDN (P=0.009); DPN vs PDN (P=0.02).

McGill VAS: C vs DPN (NS); †C vs PDN (P<0.0001); DPN vs PDN (P<0.0001).

McGill Pain score: C vs DPN (NS); ††C vs PDN (P<0.0001); DPN vs PDN (P<0.0001).

Table key

C – Controls; DPN – Diabetic Peripheral Neuropathy; IQR – Inter Quartile Range; McGill VAS –

McGill Visual Analogue Score; NDS – Neuropathy Disability Score; NSP – Neuropathy Symptom

Profile; PDN – Painful Diabetic Peripheral Neuropathy.

C

(n=14)

DPN

(n=20)

PDN

(n=23) DPN v PDN

NDS (-/10)

(Median(IQR)

1.4±1.5

1(0-2)

3.9±3.2*

3.5(1.5-5.5)*

4.5±3.2**

4(2-6)** NS

NSP (-/38)

(Median(IQR)

1±1.5

0(0-1)

2.2±2.6

1(0.5-2.5)

6.3±5.5^

5(2-11)^ <0.0005

+ve Symptoms

on NSP (-/6)

(Median(IQR)

0.5±0.9

(0(0-1)

0.25±0.6

(0(0-0)

1.6±1.8σ

(1(0-3)σ 0.009

-ve symptoms on

NSP (-/4)

(Median(IQR)

0±0

(0(0-0)

0.3±0.4

(0.5(0-1)

1.1±1.2¥

(1(0-2)¥ 0.02

McGill VAS

(-/10cm) 0.5±1.4 0±0 5.7±2.3† <0.0001

McGill Pain score

(Median (IQR)

0.4±0.9

(0(0-0)

0±0

(0(0-0)

6.1±6.5††

(3(2-10) <0.0001

286

8.4.3 Quantitative sensory tests (Table 8.3)

VPT, CST and WST did not differ between diabetic patients and control subjects or

between patients with DPN and PDN.

8.4.4 Electrophysiology (Table 8.3)

Peroneal motor nerve conduction velocity and amplitude were significantly lower in

DPN (P=0.004 and P=0.003, respectively) and PDN (P=0.0008 and P=0.001,

respectively) compared with control subjects, but there was no difference between

patients with DPN and PDN. Sural sensory nerve conduction velocity and

amplitude were significantly lower in DPN (P=0.02 and P=0.007, respectively) and

PDN (P=0.005 and P=0.04, respectively) compared with control subjects, but there

was no significant difference between patients with DPN and PDN.

8.4.5 Autonomic function, IENFD and CCM (Table 8.3)

HRV-DB did not differ between diabetic patients and control subjects or between

patients with DPN and PDN. IENFD did not differ between patients with DPN and

control subjects but was significantly reduced in PDN compared to control subjects

(P=0.05) with no difference between DPN and PDN. CNFD was significantly

reduced in patients with DPN (P=0.0008) and PDN (P<0.0001) compared with

control subjects with no difference between DPN and PDN. CNBD was significantly

reduced only in patients with PDN (P<0.03) compared with control subjects with no

difference between DPN and PDN. CNFL was significantly reduced in patients with

DPN (P=0.03) and PDN (P<0.0009) compared with control subjects with no

difference between DPN and PDN.

287


diabetic patients with DPN and PDN, with statistically significant differences between groups

C (n=14) DPN (n=20) PDN (n=23) DPN v PDN

CNFD (no/mm2) 34.6±5.4 24.5±8.4* 20.4±10.0**

NS

CNBD (no/mm2) 75.9±24.2 56.1±31.8 45.5±29.3

†

NS

CNFL (mm/mm2) 24.3±3.6 19.8±5.7

¥ 15.8±7.2

¥¥

NS

IENFD (no/mm) 7.6±3.4

(n=10)

5.2±3.7

(n=11)

3.9±2.9a

(n=9)

NS

DB-HRV (beats/min) 23±10 18±9 18±14 NS

CST (˚C) 27.2±1.9 24.0±6.6 23.5±6.8 NS

WST(˚C) 39.8±3.8 40.7±4.7 42.0±4.9 NS

VPT (volts) 10.7±6.6 17.0±14.0 19.5±13.1 NS

SSNCV (m/s) 47.7±5.1 41.9±6.2˚ 42.2±4.6˚˚ NS

SSNAmp (µV) 9.6±2.7 6.9±4.8€ 5.3±5.3

€€

NS

PMNCV (m/s) 45.9±3.3 38.3±9.3ª 38.4±8.7ªª NS

PMNAmp (mV) 4.9±1.0 2.6±1.9§ 2.6±2.5

§§

NS

Post hoc analyses

CNFD: *C vs DPN (P=0.0008); **C vs PDN (P<0.0001); DPN vs PDN (NS).

CNBD: C vs DPN (NS); †C vs PDN (P=0.03); DPN vs PDN (NS).

CNFL: ¥C vs DPN (P=0.03); ¥¥C vs PDN (P=0.0009); DPN vs PDN (NS).

IENFD: C vs DPN (NS); ªC vs PDN (P=0.05); DPN vs PDN (NS).

SSNCV: ˚ ˚C vs DPN (P=0.02); ˚˚C vs PDN (P=0.005); DPN vs PDN (P=NS).

SSNAmp: €C vs DPN (P=0.007);

€€C vs PDN (P=0.04); DPN vs PDN (NS).

PMNCV: ªC vs DPN (P=0.004); ªªC vs PDN (P=0.0008); DPN vs PDN (P=NS).

PMNAmp: §C vs DPN (P=0.003); §§C vs PDN (P=0.001); DPN vs PDN (P=NS).

288

Table Key

C – Controls; CNBD – CNFD – Corneal Nerve Fibre Density; Corneal Nerve Branch Density; CNFL

– Corneal Nerve Fibre Length; CST – Cold Sensation Threshold; DB-HRV – Deep Breathing –

Heart Rate Variability; DPN – Diabetic Peripheral Neuropathy; IENFD – Intra Epidermal Nerve Fibre

Density; PDN – Painful Diabetic Neuropathy; PMNAmp – Peroneal Motor Nerve Amplitude; PMNCV

– Peroneal Motor Nerve Conduction Velocity; SSNAmp – Sural Nerve Sensory Nerve Amplitude;

SSNCV – Sural Sensory Nerve Conduction Velocity; VPT – Vibration Perception Threshold; WST –

Warm Sensation Threshold.

289

8.4.6 25(OH)D status (Figure 8.1)

The serum 25(OH)D level was significantly lower in PDN (24.0±14.1 ng/ml)

compared to DPN (34.6±15.0 ng/ml, P=0.01) and control subjects (34.1±8.6 ng/ml,

P=0.03). The odds ratio in favour of painful diabetic neuropathy was 9.8 (P=0.003

(95% CI 2.2-76.4) for vitamin D deficiency (<20ng/ml) and 4.4, (P=0.03 (95% CI

1.1-19.8)) for vitamin D insufficiency (<30ng/ml).

Figure 8-1 Graph showing 25(OH)D levels in ng/ml in C, DPN and PDN

Post hoc analyses

C vs DPN – P=NS; *C vs DPN – P=0.03; **DPN vs PDN – P=0.01.

(Overall P for Kruskal Wallis = 0.02).

Controls DPN PDN 0

10

20

30

40

50

60

25

(OH

)D

in n

g/m

l

25(OH)D levels in C, DPN & PDN

*/** ** *

290

8.5 DISCUSSION

Painful neuropathy is an extremely disabling condition which may be present in at

least one fifth of diabetic patients (1). The treatment of this disabling condition has

remained unsatisfactory with a ‘good’ response to conventional medication rated at

between 30-50% pain relief (5). Available drugs are often moderately effective and

their use is limited by side effects.

The aetiology of painful diabetic neuropathy is not clear. Central changes

comprising of increased thalamic vascularity (32), Aβ fibre sprouting into lamina II

of the dorsal horn, and reduced inhibition via descending pathways (2) together

with axonal atrophy in peripheral nerves have been demonstrated in patients with

painful diabetic neuropathy (33). In the present study we carefully phenotyped

diabetic patients into those with painful and painless diabetic neuropathy. Painful

diabetic neuropathy has also been associated with significantly greater autonomic

dysfunction than painless DPN (34). Previously we have shown that the LDIflare, a

measure of small fibre function, is abnormal in patients with painful diabetic

neuropathy, whilst conventional quantitative sensory testing and dermal nerve fibre

density did not differ from those with painless neuropathy (35). Given the results of

these studies suggesting greater small fibre, particularly autonomic dysfunction we

undertook detailed assessment of large and small fibre neuropathy. There was no

difference for electrophysiology, quantitative sensory testing and autonomic

function between diabetic patients with painful and painless neuropathy. We have

also shown a greater reduction in both intraepidermal nerve and corneal nerve

fibre length in diabetic patients with painful neuropathy (36). A recent detailed

immunophenotyping study has shown increased axonal growth through growth

associated proteins (higher GAP43/PGP) and axonal swellings, positive for

291

tropomyosin-receptor-kinase A and substance P in patients with painful compared

to painless neuropathy (37). In the present study, although patients with painful

diabetic neuropathy had greater deficits for IENFD and corneal nerve morphology

compared to diabetic patients with painless neuropathy, this was not significantly

different between diabetic patients with painful and painless neuropathy. Although

IENFD was lower in PDN, however due to a fewer number of subjects consenting

for skin biopsy compared to the overall group this did not reach significance.

Painful neuropathic symptoms tend to vary in their severity over time and therefore

it is difficult to reconcile these ‘hard wired’ changes with the fluctuating symptoms.

Changes in sodium channel distribution and expression, altered peripheral blood

flow and glycaemic flux have also been implicated (2). Given the potential link

between vitamin D and pain, together with the high prevalence of vitamin D

deficiency in diabetic populations (38) we have explored the link with painful

diabetic neuropathy. Two recent studies have shown a relationship between

vitamin D deficiency and diabetic neuropathy (10-11), but did not assess the link

between positive and negative neuropathic symptoms in these cohorts. In the

present study we have shown a markedly increased risk of diabetic painful

neuropathy in diabetic patients with vitamin D deficiency and insufficiency. We

observed no relationship for painful diabetic neuropathy with any other risk factor

for neuropathy such as glycaemic control or severity of neuropathy, which

challenges the two recent studies showing a link between vitamin D deficiency and

diabetic neuropathy (10-11). Previous epidemiological studies have shown a

higher prevalence of painful diabetic neuropathy in South Asians despite a lower

prevalence of neuropathy (1) compared to other ethnic groups and of course South

Asian are at the highest risk of vitamin D deficiency (38). In our previous study

292

(38), 55% of our South Asian patients were severely deficient with a 25(OH)D

<10ng/ml. The cohort of subjects evaluated in this study was exclusively of white

European origin thus minimising ethnicity as a confounding factor.

Whilst vitamin D supplementation has been used to treat pain particularly in

rheumatological conditions (39) a Cochrane review (40) concluded that there was a

poor evidence base for the use of vitamin D in chronic pain. Intervention studies in

diabetic neuropathy are limited. Whilst Valensi et al (41) showed benefit for painful

symptoms with a topical compound (QR-333) containing quercetin (aldose

reductase inhibitor effects), ascorbyl palpitate and vitamin D3, with three different

compounds that may benefit painful neuropathy it is difficult to define the relative

effectiveness of vitamin D3. Lee et al (23) showed benefit with oral cholecalciferol

in painful diabetic neuropathy, however, this study had neither a placebo group nor

was it randomised. Most recently a dramatic improvement was observed in a single

type 1 patient with painful neuropathy, refractory to a range of standard therapies

(24). However given that recent studies of novel drugs in the treatment of painful

diabetic neuropathy have dramatically failed (42-43) with active treatment being

barely superior to placebo there is an urgent need to explore new mechanisms and

hence treatments for diabetic painful neuropathy.

Whilst this is a small study a major strength is the detailed phenotyping undertaken

to ensure that diabetic patients with and without painful neuropathy were absolutely

matched for all other measures of neuropathy and demographic characteristics,

except painful symptoms. The results appear unequivocally in favour of a

relationship between vitamin D deficiency and painful neuropathic symptoms. A

well constructed controlled trial of vitamin D supplementation in painful diabetic

293

neuropathy is required in order to assess the effectiveness of a potentially simple

treatment with no obvious side effects.

294

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Journal of Pain 2013;14:941-947





39. Catalano A, Morabito N, Atteritano M, Basile G, Cucinotta D, Lasco A: Vitamin

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40. Straube S, Andrew Moore R, Derry S, McQuay HJ: Vitamin D and chronic pain.

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41. Valensi P, Le Devehat C, Richard JL, Farez C, Khodabandehlou T,

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300

9 Chapter IX - Vitamin D Deficiency is not associated with

Diabetic Retinopathy or Maculopathy: A Cross Sectional

study


extracted data, performed statistical analyses and wrote the manuscript which

constitutes the basis for this chapter.


Uazman Alam, Yasar Amjad, Omar Asghar, Ioannis N Petropoulos, Rayaz A Malik

301

9.1 ABSTRACT

Experimental data and several small clinical studies suggest a possible association

between vitamin D deficiency and both diabetic retinopathy and maculopathy.

We have performed a cross-sectional study in adults aged 18-80 years with Type 1

and 2 Diabetes Mellitus. The relationship between the presence and severity of

diabetic retinopathy and maculopathy with serum 25-hydroxyvitamin D

concentration was evaluated using logistic regression analyses in the presence of

demographic and clinical covariates, such as age, race, gender, duration of

diabetes, anthropometric measures (Body Mass Index (BMI) and Blood Pressure

(BP)) and metabolic parameters (HbA1c and lipid fractions).

657 adults with diabetes were stratified based on retinopathy grading: No Diabetic

retinopathy (NDR) (39%), Background Diabetic Retinopathy (BDR) (37%), Pre-

Proliferative Diabetic Retinopathy (PPDR) (21%) and Proliferative Diabetic

Retinopathy (PDR) (3%), respectively. There were no differences in serum 25-

hydroxyvitamin D concentrations (25(OH)D) between the groups (15.3±9.0 vs

16.4±10.5 vs 15.9±10.4 vs 15.7±8.5ng/ml, P=NS). Logistic regression analysis

demonstrated no statistically significant relationship between the severity of

retinopathy and serum 25(OH)D. Furthermore, there was no difference in serum

25(OH)D between those with (n=94, 14%) and without (n=563, 86%) diabetic

maculopathy (16.2±10.0 vs 15.8±9.8, p=NS) and no relationship was demonstrated

by logistic regression analyses between the two variables.

This study has found no association between serum 25(OH)D concentrations and

the presence and severity of diabetic retinopathy or maculopathy.

302

9.2 INTRODUCTION

The prevalence of diabetic retinopathy (DR) approaches 93 million people

worldwide (1) and is one the leading causes of premature visual loss in the UK and

worldwide (2-3). Indeed, the World Health Organization estimates that whilst DR

accounts for approximately 5% of the global prevalence of blindness, the

prevalence rises sharply to 15-17% in developed countries (4). Several risk factors

are implicated in the aetiology of DR with hyperglycemia and hypertension showing

the strongest association (5), yet interventions aimed at correcting these risk

factors have demonstrated moderate success (6-7). Therefore the interactions

between neural and retinal vascular dysfunction and the mechanisms resulting in

retinal pathology including neovascularisation have been questioned recently (8).

Furthermore, micronutrients including Vitamin C, Vitamin E and magnesium have

been postulated to play a role in DR (9).

Vitamin D deficiency has been linked to a host of cardiovascular diseases including

diabetes and hypertension (10-11). Vitamin D receptor (VDR) genotypes have

been associated with the cumulative prevalence of diabetic retinopathy (12). In two

separate studies of the VDR gene in the French population, FokI and TaqI single

nucleotide polymorphisms have been associated with DR (13-14). In a study of

Caucasians with C-peptide-negative type 1 diabetes, there was a novel association

between the functional FokI VDR polymorphism and severe DR (13). VDR

dependent calcium binding proteins have been isolated in the human retina,

particularly in the photoreceptor layer of the cones (15) and immunostaining in

animal models has shown that VDR is expressed in the ganglion cells, the inner

and outer plexiform layer and the photoreceptor layer (16). In an in-vitro study of

303

retinoblastoma tissue expressing VDR, supplementation with vitamin D resulted in

a reduction of growth and apoptosis of the retinoblastoma cells (17). 1,25-

dihydroxyvitamin D3 (1,25(OH)2D3) closely regulates Vascular Endothelial Growth

Factor in experimental models (18) and there is an inverse correlation of 25(OH)D

with Vascular Endothelial Growth Factor, postulated to be related to tissue hypoxia

(19). In a mouse model of ischaemic retinopathy, 1,25(OH)2D3 was shown to inhibit

neovascularisation in retinal tissue (20). Vitamin D may also have a direct effect on

the renin-angiotensin-aldosterone-system (RAS) and the RAS is known to be

overexpressed in type 1 diabetic patients with retinopathy (21) and blockade of this

system reduces DR progression (22). A vitamin D analogue (paricalcitriol) has

shown an improvement in microalbuminuria through a mechanism related to

inhibition of RAS (23).

Aksoy H et al, demonstrated an inverse correlation in a Turkish cohort between

worsening diabetic retinopathy and lower 1,25-hydroxyvitamin D3 (active vitamin D)

in a population of 66 subjects (24). Furthermore, severe vitamin D deficiency has

been shown to predict not only mortality but the development of nephropathy and

retinopathy in type 1 diabetes (5). In a recent cross-sectional study of children and

adolescents with type 1 diabetes, retinopathy prevalence was higher in children

and adolescents with lower levels of vitamin D (25). Other cross-sectional studies

which have assessed vitamin D status in relation to DR in adults have either had

small numbers (26) or have been based on retrospective analysis of data collected

from the National Health and Nutrition Examination Survey between 1988 and

1994 (27). However, since then the targets for glycaemia, blood pressure and

lipids have changed and also this study made no assessment of diabetic

maculopathy (27). Therefore we have undertaken a study to establish the

304

relationship between vitamin D status and the severity of DR and maculopathy in a

large adult population with type 1 and type 2 diabetes.

305


All patients attending clinics were assessed for the level of 25(OH)D, irrespective

of a history suggestive of vitamin D deficiency. Ethical approval was not required

as the data were extracted retrospectively and did not extend beyond standard

clinical practice. 25(OH)D was added as a standard routine test from June 2009

due to the high levels of deficiency noted.

9.3.1 Subjects

All participants were aged ≥ 18 years old attending clinics at the Central

Manchester Foundation Trust, Manchester from August 2009 to May 2011. Those

with renal impairment (eGFR <30 mL/min/1.73m2 (CKD stage 4 and below),

granulomatous diseases (tuberculosis, sarcoidosis etc), malabsorption syndromes

(Coeliac disease, bacterial overgrowth, concomitant Orlistat treatment), pregnant

and lactating women, and those currently on vitamin D supplementation were

excluded from the analysis.


BMI was measured as per the standard equation (mass (kg)/(height(m))2. Weight

was measured with a digital scale (Seca 701, Seca, Hamburg, Germany) to the

nearest 0.1kg and height to the nearest 0.1cm. Blood pressure measurements

were obtained with the use of an automated device (Dinamap pro 100v2, GE

Medical Systems, Freiburg, Germany) with an appropriate cuff size. A minimum of

two measurements of systolic and diastolic blood pressures were made five

minutes apart with the lowest reading recorded and the mean of the preceding 2

years blood pressure results were used. Metabolic variables were also recorded

306

with a mean of 2 year retrospective readings for glycosylated haemoglobin A1c

(HbA1c) and components of the lipid profile (total cholesterol (T-CHL), high density

lipoprotein cholesterol (HDL) and triglycerides). The following measurements were

taken as ‘spot readings’ at the same date as baseline 25(OH)D measurements:

BMI; bone profile markers such as corrected calcium (CCa), alkaline phosphatase

(ALP) and estimated glomerular filtration rate (eGFR).


Medications



medical record database (Diamond database, Hicom, Surrey, UK). Subject

demographics extracted were age, sex, ethnicity (Caucasian, South Asian, Far

East Asian and Afro-Caribbean descent), smoking status (never, previous and

current) and type (type 1 and 2 diabetes) and duration of diabetes. Dates of

baseline 25(OH)D were used to obtain respective retinopathy screening data. Only

retinopathy screening data within 1 year of the baseline 25(OH)D and prior to

vitamin D supplementation were included.

Baseline 25(OH)D status and retinopathy data were collected for 657 patients who

had attended their retinopathy screening appointments. The retinopathy data were

collected according to the grading criteria of the National Screening Committee

(28-29). Previous studies have shown acceptable level of quality and accuracy of

grading compared to expert graders within the English National Screening

Committee (30). The national guidelines do not contain R1.5 or M0.5 grades and

are categorised as Pre-Proliferative Diabetic Retinopathy and Diabetic

307

Maculopathy, respectively. These sub-gradings were used locally in screening

centres and have been included. Retinopathy was graded as follows:

R0 - No Diabetic retinopathy (NDR).

R1 - Background Diabetic Retinopathy (BDR): microaneurysms, retinal

haemorrhages, exudates.

R1.5 - Moderate numbers of intra-retinal haemorrhages, hard exudates >1 disc

diameter (DD) from fovea, 3-6 cotton wool spots visible.

R2 - Pre-Proliferative Diabetic Retinopathy (PPDR): Venous beading or looping,

deep haemorrhages visible, other microvascular anomaly visible.

R3 - Proliferative Diabetic Retinopathy (PDR): New vessel formation, vitreous

haemorrhage, pre- retinal haemorrhage or fibrosis and/or retinal detachment.

M0 - No maculopathy.

M0.5 - Hard exudates within the arcades >1 disc diameter from the centre of the

fovea.

M1- Exudates <1 DD from the centre of the fovea, retinal thickening <1 disc

diameter from the centre of the fovea.

P0 – No photocoagulation scarring.

P1 – Photocoagulation scarring.

9.3.4 25(OH) vitamin D Assay

Assessment of 25(OH)D was instituted in August 2009 as part of routine


(Complete Blood count (CBC)), Urea and Electrolytes (UE), Liver function tests


Albumin (Alb), Phosphate (phos)), and Lipid profile (Total Cholesterol (T-CHL),

308

High Density lipoprotein Cholesterol (HDL), Triglycerides (TRIG)). Serum was

separated from whole blood and stored at -20 C until assay. The laboratory used

for the biochemical assay measurements (Vitamin D Research Group Manchester

Royal Infirmary, UK) was accredited to ISO 9001:2008 and ISO 13485:2003 by

Lloyd’s Register Quality Assurance certificate number LRQ 4001542 and

participated successfully in the Vitamin D quality assurance scheme (DEQAS).

Serum 25(OH)D was measured using the IDS-iSYS multi-discipline automated

analyzer (Immunodiagnostic Systems Ltd, Boldon, Tyne and Wear, UK). The assay

is based on chemiluminescent technology and was performed exactly as per the

manufacturer’s instructions. The cross reactivity for vitamin D2 (of the assay) as per

manufacturers assertion was 100% (relative to vitamin D3) and the assay has

excellent correlation to existing globally recognised assays, in combination with

good sensitivity and precision (31). The reportable range of the assay is 5-140

ng/ml Inter- and intra- assay variation of the in-house control was 5.6% and 9.7%

respectively.


Data were analysed using StatsDirect (StatsDirect, Altringham, Cheshire, UK). The

data were stratified according to retinopathy (NDR, BDR, PPDR and PDR) and

maculopathy (No maculopathy and maculopathy) status and a comparison of

means was undertaken using either ANOVA or Krus-Kal Wallis for DR data and

Unpaired t-test or Mann-Whitney U for maculopathy data. Chi Squared test were

used for aetiology of diabetes, ethnicity, gender and smoking status. Logistic

regression analyses were undertaken to assess the association between serum

25(OH)D levels and retinopathy and maculopathy status (either present (1) or not

309

present (0)), adjusting for mean values of duration of diabetes, smoking status,

HbA1c, total cholesterol, HDL, triglycerides, systolic and diastolic blood pressure.

Further assessment of the results comparing vitamin D categories (Severely

deficient (<10ng/ml), Deficient (10-<20ng/ml), Insufficient (20-<30ng/ml) and

Sufficient (>30ng/ml)) and retinopathy (NDR, BDR, PPDR and PDR), maculopathy

(No maculopathy and maculopathy) and photocoagulation status (No

photocoagulation and photocoagulation) were performed using chi2 testing.

Appropriate statistical analyses were employed depending on the normality of the

data. Overall the P value was maintained at 0.05 for multiple comparison tests

(Bonferoni adjustment or Dwass-Steel-Chritchlow-Fligner pairwise comparison).

310

9.4 RESULTS

657 patients were stratified according to their retinopathy status: NDR (n=257,

39%), BDR (n= 243, 37%), PPDR (n=135, 21%) and PDR (n=22, 3%); No Diabetic

Maculopathy (n=563, 86%) and Diabetic Maculopathy (n=94, 14%). 206 (31%) of

the patients had severe vitamin D deficiency with 25(OH)D levels below 10ng/ml,

284 (43%) were deficient with 25(OH)D of 10-<20ng/ml, 101(14%) were insufficient

with 25(OH)D of 20-<30ng/ml. Only 65 (10%) individuals had ‘adequate’ levels of

25 (OH)D at >30ng/ml. The mean 25(OH)D for the population was 15.8 ± 9.4

ng/ml.

Table 9.1 shows demographic and metabolic data based on retinopathy grading;

NDR, BDR, PPDR and PDR respectively. There were no differences in 25(OH)D

status between the groups (15.3±9.0 vs 16.4±10.5 vs 15.9±10.4 vs 15.7±8.5,

P=NS). Subjects were matched for age (59.8±13.8 vs 58.8±13.3 vs 60.8±10.9 vs

55.1±13.6 years), however, the duration of diabetes was significantly lower in NDR

(11.3±8.7 vs 18.7±11.7 vs 21.0±9.8 vs 19.7±10.0 years, P<0.0001). The median

number of metabolic and anthropometric measurements over the preceding two

year period from the baseline 25(OH)D result was 4 (Interquartile range 3-5). Two

year mean HbA1c (%) (8.2±1.6 vs 8.6±1.7 vs 8.9±1.6 vs 8.9±1.5, P<0.0006)

showed a significantly lower HbA1c, lower systolic Blood pressure (129±13 vs

131±15 vs 134±15 vs 134±11mmHg, P=0.007) and higher eGFR (76.3±16.9 vs

75.9±17.5 vs 70.9±16.9 vs 69.0±21.6, P=0.02) in NDR (Table 9.1). There was no

difference for aetiology of diabetes, ethnicity, sex, smoking status, BMI, lipid and

bone parameters and diastolic blood pressure between the grades of DR. Table

9.2 shows logistic regression analyses for DR status with Odds Ratios (OR) and

95% CI. There was no correlation of DR with 25(OH)D (OR 1.00 (95% CI 0.98-

311

1.02), P=NS), gender or ethnicity. However, lower age (OR 0.97 (95% CI 0.96-

0.99), P=0.01), longer duration of diabetes (OR 1.09 (95% CI 1.06-1.13),

P<0.0001), higher HbA1c (OR 1.22 (95% CI 1.07-1.39), P=0.003) and systolic

blood pressure (OR 1.02 (95% CI 1.00-1.04), P=0.02) were all associated with DR.

312

Table 9-1 Demographic and metabolic parameters in subgroups based on severity of

retinopathy

No Diabetic

Retinopathy

(NDR)

(n=257)

Background

Diabetic

Retinopathy

(BDR)

(n=243)

Pre-

Proliferative

Diabetic

Retinopathy

(PPDR)

(n=135)

Proliferative

Diabetic

Retinopathy

(PDR)

(n=22)

P value

Age (years) 59.8±13.8 58.8±13.3 60.8±10.9 55.1±13.6 NS

Duration of

Diabetes

(years)

11.3±8.7† 18.7±11.7† 21.0±9.8† 19.7±10.0† <0.0001

Type 2 DM (%) 88 75 80 77 NS

Ethnicity (White

European/South

Asian (%))

48/45 55/38 50/45 52/48 NS

Sex (male (%)) 51 50 48 53 NS

Current

Smokers/Past

smokers/Never

smokers (%)

14/25/61 14/33/53 14/26/60 4/23/71 NS

BMI (kg/m2) 31.6±10.6 31.1±7.1 31.9±6.4 31.1±6.9 NS

25(OH)D (ng/ml) 15.3±9.0 16.4±10.5 15.9±10.4 15.7±8.5 NS

HbA1c (%) 8.2±1.6* 8.6±1.7* 8.9±1.6* 8.9±1.5 <0.0001

T-CHL (mmol/l) 4.1±1.0 4.2±1.1 4.2±1.3 4.3±1.1 NS

HDL (mmol/l) 1.3±0.5 1.4±0.5 1.4±0.7 1.3±0.4 NS

TRIG (mmol/l) 1.8±0.9 1.8±1.5 1.8±0.9 2.5±2.9 NS

eGFR (ml/min/l) 76.3±16.9** 75.9±17.5 70.9±16.9** 69±21.6 0.02

Systolic BP

(mmHg) 129±13*** 131±14.6 134±15*** 134±11 0.007

Diastolic BP

(mmHg) 70±7 69±7 70±8 71±8 NS

ALP (u/L) 87±39 83±34 84±32 93±43 NS

CCa (mmol/l) 2.4±0.1 2.4±0.1 2.3±0.1 2.4±0.2 NS

313

Post hoc analyses

Duration of Diabetes – †NDR vs BDR (P<0.0001); NDR vs PPDR (P<0.0001);

NDR vs PDR (P=0.001).

HbA1c – *NDR vs BDR (P=0.01); NDR vs PPDR (P<0.0001).

eGFR – **NDR vs PPDR (P=0.04).

Systolic BP – ***NDR vs PPDR (P=0.008).

Table key



T-CHL – Total Cholesterol; TRIG – Triglycerides.

314

Table 9-2 Logistic regression analyses for the relationship between retinopathy, 25(OH)D

status and other confounding variables

Odds Ratio 95% CI p

25(OH)D (ng/ml) 1.00 0.98-1.02 NS

Age (years) 0.97 0.96-0.99 0.01

Duration of Diabetes (years) 1.09 1.06-1.13 <0.0001

Never Smoker 0.48 0.21-1.09 NS

HbA1c (%) 1.22 1.07-1.39 0.003

T-CHL (mmol/l) 1.09 0.88-1.36 NS

HDL (mmol/l) 0.88 0.55-1.41 NS

Triglycerides (mmol/l) 0.98 0.77-1.25 NS

Systolic BP (mmHg) 1.02 1.00-1.04 0.02

Diastolic BP (mmHg) 0.98 0.94-1.01 NS

eGFR (ml/min/l) 0.99 0.98-1.00 NS

Table key

BMI – Body Mass Index, BP – Blood Pressure, eGFR – estimated Glomerular Filtration Rate, HDL

– High-density Lipoprotein Cholesterol, T-CHL – Total Cholesterol.

315

Table 9.3 shows demographic and metabolic data in diabetic patients with (n=94,

14%) and without (n=563, 86%) maculopathy. There were no differences in

25(OH)D status between patients with and without maculopathy (16.2±10.0 vs

15.8±9.8 ng/ml, P=NS). Subjects were matched for age (59.1±11.5 vs 59.5±13.3

years), however, the duration of diabetes was significantly longer in patients with

maculopathy (15.9±11.1 v 19.2±9.7 years, P=0.0003). Two year mean HbA1c (%)

(8.4±1.6 vs 9.1±1.5, P<0.0001) and systolic blood pressure (130±14 vs

134±14mmHg, P=0.01) were significantly higher in diabetic patients with

maculopathy. There were no differences for type of diabetes, ethnicity, sex,

smoking status, BMI, lipid and bone parameters and diastolic blood pressure

between patients with and without maculopathy. Table 9.4 shows logistic

regression analyses for diabetic maculopathy status with Odds Ratios and 95% CI.

There was no relationship of maculopathy status with 25(OH)D (OR 1.00 (95% CI

0.98-1.03), P=NS), age, gender, ethnicity, systolic blood pressure or lipid fractions.

However, a longer duration of diabetes (OR 1.03 (95% CI 1.00-1.05), P=0.01) and

higher HbA1c (OR 1.22 (95% CI 1.05-1.43), P=0.009) were associated with

maculopathy status.

316

Table 9-3 Demographic and metabolic parameters in subgroups based on maculopathy

No Diabetic

Maculopathy

(n=563)

Diabetic

Maculopathy

(n=94 )

P

Age (years) 59.5±13.3 59.1±11.5 NS

Duration of

Diabetes (years) 15.9±11.1 19.2±9.7 0.0003

Type 2 DM (%) 82 80 NS

Ethnicity (White

European/South

Asian (%))

40/50 49/48 NS

Sex (male (%)) 51 47 NS

Current/Past/Never

smokers (%) 15/28/57 16/27/57 NS

BMI 31.4±8.8 31.3±6.2 NS

25(OH)D (ng/ml) 15.8±9.8 16.2±10.0 NS

HbA1c (%) 8.4±1.6 9.1±1.5 <0.0001

T-CHL (mmol/l) 4.1±1.1 4.4±1.4 NS

HDL (mmol/l) 1.3±0.5 1.4±0.8 NS

TRIG (mmol/l) 1.8±1.2 1.9±1.6 NS

eGFR (ml/min/l) 75±18 73±20 NS

Systolic BP

(mmHg) 130±14 134±14 0.01

Diastolic BP

(mmHg) 71±7 70±7 NS

ALP (u/L) 87±53 100±129 NS

CCa (mmol/l) 2.3±0.1 2.4±0.1 NS

Table key



T-CHL – Total Cholesterol; TRIG – Triglycerides.

317

Table 9-4 Logistic regression analyses for the relationship between maculopathy, 25(OH)D

status and other confounding variables

OR 95% CI p

25(OH)D 1.00 0.98-1.03 NS

Age (years) 0.99 0.97-1.02 NS

Duration of Diabetes (years) 1.03 1.00-1.05 0.01

Never Smoker 1.24 0.73-2.12 NS

HbA1c (%) 1.22 1.05-1.43 0.009

T-CHL (mmol/l) 1.05 0.84-1.33 NS

HDL (mmol/l) 1.12 0.73-1.73 NS

Triglycerides (mmol/l) 1.19 0.77-1.25 NS

Systolic BP (mmHg) 1.01 0.99-1.04 NS

Diastolic BP (mmHg) 1.03 0.98-1.07 NS

eGFR (ml/min/l) 0.99 0.97-1.00 NS

Table key


Calcium; eGFR – estimated Glomerular Filtration Rate; HDL– High-density Lipoprotein Cholesterol;

T-CHL – Total Cholesterol.

318

The frequencies for both severity of retinopathy, maculopathy and

photocoagulation were similar between the four vitamin D categories (severely

deficient (<10ng/ml), deficient (10-<20ng/ml), insufficient (20-<30ng/ml) and

sufficient (>30ng/ml) (Figure 9.1, Table 9.5).

Figure 9-1 Frequencies of retinopathy, maculopathy and photocoagulation scarring

categorised by 25(OH)D status

0

10

20

30

40

50

60

70

80

90

100

%

Retinopathy, Maculopathy and Photocoagulation status subdivided by 25(OH)D categories

Severely Deficient (<10ng/ml)

Deficient (10-<20ng/ml)

Insufficient (20-<30ng/ml)

Sufficient (=/>30ng/ml)

319

Table 9-5 Frequencies of retinopathy, maculopathy and photocoagulation scarring

categorised by 25(OH)D status

<10ng/ml 10-<20ng/ml 20-<30ng/ml >30ng/ml p

No Diabetic

Retinopathy 37 43 39 30

NS

Background

Diabetic

Retinopathy

36 35 35 45

Pre-Proliferative

Diabetic

Retinopathy

25 19 21 23

Proliferative

Diabetic

Retinopathy

2 3 5 2

No Maculopathy 85 84 87 83 NS

Maculopathy 15 16 13 17

No

Photocoagulation 90 87 80 83

NS

Photocoagulation 10 13 20 17

320

9.5 DISCUSSION

Vitamin D deficiency has wide ranging implications for insulin resistance, beta cell

dysfunction and hypertension and therefore provides a potential link with diabetic

complications (32). Experimental studies have postulated an important link

between vitamin D deficiency and retinopathy (33) and an increased risk of diabetic

retinopathy has been demonstrated in the presence of VDR polymorphisms (13).

However, our study has shown no relationship between the vitamin D status and

the severity of diabetic retinopathy or maculopathy in a large cohort of patients with

predominantly type 2 diabetes, after correcting for glycaemic control, blood

pressure and lipids. We confirm that the ‘usual culprits’ of longer duration of

diabetes, higher HbA1c and systolic blood pressure are directly related to

retinopathy and maculopathy (1), thereby providing confidence in the validity of our

data. Furthermore, the metabolic and anthropometric measurements used in the

regression analysis were taken over an extended period of time as opposed to

‘spot’ readings taken in other studies (24; 26). A possible explanation for the lack of

relationship between vitamin D deficiency and retinopathy could be the striking

extent of vitamin D deficiency in this population, although this is consistent with our

previous data (34). Thus the majority of patients demonstrated deficiency (~90%)

and indeed severe deficiency (~31%). Therefore any relationship between

retinopathy and adequacy of vitamin D could not be explored adequately. Only a

limited number of clinical studies have investigated the role of vitamin D deficiency

in DR. In one of the earliest studies Aksoy et al showed an inverse relationship

between 1,25(OH)2D3 and worsening retinopathy, although the short half life of

1,25(OH)2D3 may limit the interpretation of any such relationship (24). Another

smaller North American study has shown that subjects with DR, in particular PDR,

321

have lower levels of 25(OH)D (26). Whilst in a recent study the percentage of

individuals with vitamin D deficiency increased with the severity of retinopathy,

regression analysis did not demonstrate a statistically significant relationship

between retinopathy severity and serum 25(OH)D concentration (27). In a

prospective observational follow-up study of a cohort of type 1 diabetic patients,

although severe vitamin D deficiency independently predicted all-cause mortality, it

was not related to the development of either retinopathy or nephropathy (5).

In conclusion, this large cross-sectional study found no association of vitamin D

status with diabetic retinopathy or maculopathy. A population with a larger spread

of vitamin D levels may provide further insight into a possible association, but this

may not be possible due to the high prevalence of vitamin D deficiency.

322

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Combined intensive blood pressure and glycemic control does not produce an

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8. Hammes HP, Feng Y, Pfister F, Brownlee M: Diabetic retinopathy: targeting

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in the start codon of the vitamin D receptor gene and severe diabetic retinopathy in

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327

10 Chapter X - Discussion

328

The chronic hyperglycaemia of DM is associated with end-organ damage,

dysfunction and failure, including the retina, kidney, nerves, heart and blood

vessels (1). The IDF estimates an overall prevalence of DM to be 366 million in

2011, and this is expected to rise to 552 million by 2030 (2). The prevalence of

DSPN is thought to be around 30% (3-4) and increases with a longer duration of

the disease. When diabetes has been present for greater than 25 years the

prevalence rises to approximately 50% (4).

In the same manner that identifying and treating microalbuminuria allows the

prevention of diabetic nephropathy, identifying and treating abnormalities in small

nerve fibre function may help to prevent diabetic neuropathy and its sequelae.

Furthermore, earlier diagnosis of neuropathy would allow for the optimisation of

conventional risk factors and consequently reduce the morbidity associated with

neuropathy. The accurate detection and quantification of DSPN are important to

identify at risk patients, anticipate deterioration of nerve function, and assess

endpoints in clinical trials. Current methods lack sensitivity (QST), primarily focus

on large fibres (NCS) or are invasive and require detailed laboratory processing

(skin/nerve biopsy) and thus are not routinely performed within the health care

system. CCM has been proposed as a surrogate endpoint of DSPN and allows for

rapid, reiterative and non-invasive assessment of the corneal subbasal nerve

plexus. The evaluation of corneal subbasal nerve morphology has shown

significant diagnostic potential for a number of peripheral neuropathies and in

particular DSPN (5-8). Corneal nerve pathology correlates well with IENF loss (9)

and currently prospective natural history studies are underway will investigate the

utility of CCM to assess the status and progression of DSPN in subjects with type 1

329

and type 2 DM (9). Furthermore, recent studies using CCM have also shown nerve

fibre regeneration after simultaneous pancreatic and kidney transplant (10).

The UKPDS demonstrated a significant risk reduction for cardiovascular disease

and amputation for every 1% reduction in mean HbA1c (11). Intensive therapy

reduced the development of confirmed clinical neuropathy by 64% in the DCCT

after 5 years of follow-up (12). However, approximately 50% of patients with DSPN

experience neuropathic symptoms (13) and these symptoms include; burning pain,

electrical or stabbing sensations, paraesthesia, hyperaesthesia and a deep aching

pain. The precise reasons for the development of neuropathic pain despite loss of

sensation is not clearly elucidated but suggests that aberrations in pain signalling

in the peripheral and central nervous system occur (13). Furthermore, there is an

epidemic of vitamin D deficiency worldwide with over one billion people thought to

be affected (14). Until recently, studies of vitamin D have largely focussed on bone

health and calcium metabolism (14), however, there is an increasing body of

evidence implicating the role of vitamin D in the nervous system (15-17). In

experimental studies, vitamin D has been linked to the regulation of neurotrophins

such as Nerve Growth Factor (NGF) and neuronal Ca2+ homeostasis, both may

play a neuroprotective role in the peripheral nerve (18). Previous studies have

shown improvements in diabetic and non-diabetic nephropathy with activated

vitamin D administration through a mechanism linked to inhibition of the RAS

system (19-21). Vitamin D is a potent RAS inhibitor (22). RAS over activity impacts

on the prevalence of diabetes related complications and inhibition of this system

may have a dramatic effect on the improvement of diabetic complications including

retinopathy and neuropathy (23-24).

330

10.1 Rapid Nerve Fibre Decline in patients with Type 1 diabetes can be

readily detected using Corneal Confocal Microscopy (To be submitted)

DSPN is a confirmed risk factor for debilitating lower limb amputation (25) and is

one of only three independent and significant risk factors for mortality as confirmed

through the ACCORD study (26). Longitudinal studies show deterioration in

neuropathy overtime in both type 1 diabetes (12) and type 2 diabetes by means of

electrophysiology (27). However, the dramatic failure of pathogenetic treatments in

DSPN (28) has been considered to be related to inappropriate surrogate endpoints

and selection of patients for studies. The progression of DSPN has been shown to

occur much slower than previously thought, likely due to overall improved diabetes

care with better glycaemic control and the more prevalent use of ACE inhibitors

(24) and statins (29), which are likely to have an impact on neuropathy. Therefore it

has been proposed that subjects with deteriorating as opposed to stable nerve

function should be recruited into clinical trials of DSPN (28). Delineating this cohort

is essential in undertaking a well conducted clinical trial and also identifying the at

risk patient for timely intervention. In this study, we have shown that CCM can be

used to identify a subgroup of subjects with type 1 diabetes undergoing a more

accelerated degree of small fibre decline. Therefore, in-vivo CCM provides an ideal

surrogate endpoint in identifying those at risk of worsening DSPN and also for

recruitment into future clinical trials of pathogenetic treatments for DSPN.

10.2 Diagnostic Utility of Corneal Confocal Microscopy and Intra-Epidermal

Nerve Fibre Density in Diabetic Neuropathy (To be submitted)

DSPN is a debilitating condition which may lead to pain, foot ulceration and

eventual amputation (30). Therefore it is important to accurately diagnose diabetic

331

neuropathy at the earliest stage of damage. However, the earliest nerve fibres to

degenerate (31-32) and regenerate (33) are the small unmyelinated fibres and

indeed they are central to the genesis of pain and the development of foot

ulceration (34). CCM is a non-invasive ophthalmic application, which is rapid, non-

invasive and readily reproducible for quantifying small nerve fibres and thereby has

been shown to diagnose and track the progression of diabetic neuropathy (5; 7;

35). CNFD has a superior sensitivity and an almost identical specificity compared

to IENFD. Subsequently, CNFD had better diagnostic utility for DSPN and

correlated better with electrophysiology than the current ‘gold’ standard of IENFD.

CCM is a valid accurate non-invasive method which is superior to skin biopsy in

diagnosing DSPN.

10.3 Enhanced Small Fibre Neuropathy in Patients with Latent Autoimmune

Diabetes in Adults (To be submitted)

Latent Autoimmune Diabetes in Adults is an under recognized (~10%) form of

diabetes which is often misdiagnosed as type 2 diabetes mellitus (36). The

pathophysiology is more closely related to autoimmune destruction of β-cells rather

than the insulin resistance found in type 2 diabetes. Patients with LADA are

autoantibody positive at a younger age of presentation, with a relatively low BMI

and have an earlier loss of glycaemic control which may influence the development

of complications (37-41). The complications of LADA have been poorly phenotyped

with only a handful of studies assessing neuropathy in this group (41-44). We

clearly demonstrate a greater degree of small fibre neuropathy in subjects with

LADA who have poorer glycaemic control. This was detected using conventional

small fibre tests such as thermal thresholds and IENFD but in particular the novel

332

non-invasive test of CCM. Hyperglycaemia predisposes to microvascular

complications including DSPN (11; 45-46) and therefore poorer glycaemic control

due to continued conventional oral hypoglycaemic treatment as opposed to earlier

treatment with insulin in LADA (47) may result in the more prevalent small fibre

damage. Furthermore, given the potential link between C-peptide and neuropathy,

the faster decline in C-peptide levels may also predispose to more prevalent

neuropathy (43). Thus patients with LADA require early and accurate assessment

for neuropathy to identify those individuals who may be at greater risk for

progression to early β-cell failure and loss of glycaemic control.

10.4 Marked vitamin D deficiency in patients with diabetes from secondary

care: Ethnic and seasonal differences and an association with dyslipidaemia

(48) (Alam U, et al. Diabet Med 2012;29:1343-1345)

The purpose of this cross sectional study was to assess the prevalence of vitamin

D deficiency in a diabetic population in relation to metabolic parameters. Using

≤30ng/ml as a cut off for deficiency (49) we found that 91% of our subjects with

diabetes, compared to 78% age and ethnicity matched subjects without diabetes,

were deficient (48). Despite particularly low vitamin D concentrations there was no

association with an increase in ALP or low corrected calcium status, both of which

are considered to be good metabolic indicators of vitamin D deficiency (50-51).

This is an important observation as current local, national and international

guidelines endorse a view that vitamin D should only be assessed if there are overt

symptoms of osteomalacia or if the calcium is reduced and or ALP is elevated, in

predefined high risk groups. An association was found, albeit weak with HDL

cholesterol, suggesting an important relationship with lipids and a possible

333

explanation for the excess cardiovascular disease described in vitamin D

deficiency states. Significant ethnic differences were seen with the lowest levels in

South Asian/middle Eastern patients but the differences were not marked, again

challenging the sterotype that vitamin D deficiency occurs predominantly in South

Asians. Furthermore, there was a minimal seasonal variation in white European

patients and there were no significant variation of note in South Asian/middle

Eastern subjects. Seasonality is often cited as a reason for vitamin D insufficiency;

however with a lack of sunlight exposure in most patients in all seasons this not

likely to be a valid contributing factor. This study demonstrates an important

deficiency of vitamin D which is not routinely assessed in diabetic patients and may

play a role in cardiovascular disease states.

10.5 Differential effects of different vitamin D replacement strategies in

patients with diabetes. (Alam U et al. J Diabetes Complications 2013.

Accepted, [Epub ahead of print])

Sunlight exposure in the U.K. is insufficient in providing an adequate 25(OH)D

status (52-53) and effective replacement strategies are required particularly in high

risk groups. This study is an evaluation of vitamin D status at baseline and after

intervention with vitamin D in over two hundred and forty participants assigned to

three treatment strategies of Vitamin D2 or D3. Despite relatively aggressive

regimens of vitamin D replacement there was significant vitamin D deficiency in a

large subgroup, particularly in those treated with vitamin D2. Furthermore, there

was a significant disparity in regimens of vitamin D2 and vitamin D3 in relation to

the relative effectiveness of the improvement in 25(OH)D concentrations which has

also been suggested in previous literature (54). Current regimens do not adjust for

334

these differences (55) and the relative disparity of treatments may lead to

inadequate replacement with consequent complications. Vitamin D shares a similar

intake-response curve to other nutrients and in persons whose baseline values

differ then an identical nutrient intake may or may not produce a measurable

response (56). We have found reduced efficacy of vitamin D replacement in those

with higher baseline vitamin D status. Differences in baseline concentrations and

type of vitamin D (D2/D3) replacement need to be considered for future policies and

guidelines. We believe our data provides considerable insights to enable more

evidence based guidance to be developed.

10.6 Vitamin D Deficiency Contributes to Painful Diabetic Neuropathy (Alam

U et al. Submitted Diabetes awaiting revision)

We have undertaken a detailed assessment in forty three subjects with mild

diabetic neuropathy based on the neuropathy disability score with and without

painful neuropathy compared to a healthy age matched control group. A previous

study has shown a potential therapeutic benefit of modest intervention with vitamin

D therapy (~2000 IU cholecalciferol daily) in patients with type 2 diabetes and

painful neuropathy (57). Other than potential analgesic effects, active vitamin D3

induces NGF (58) and NGF is known to be depleted in experimental diabetes (59).

This suggests vitamin D may have a direct effect on the pathogenesis of diabetic

neuropathy, particularly when considering that vitamin D3 has been shown to

reduce demyelination in a cuprizone experimental model of demyelination (60).

The data provides a novel postulated link between vitamin D deficiency and painful

diabetic neuropathy. Despite identical severity of neuropathy and metabolic control,

the only abnormality noted was a lower level of vitamin D in a cohort of patients

335

with painful diabetic neuropathy. Furthermore, there was a significantly greater

odds ratio for painful neuropathy in those with vitamin D insufficiency and in

particular deficiency. This study provides a novel association between vitamin D

deficiency and painful diabetic neuropathy. Recent studies of novel drugs in the

treatment of painful diabetic neuropathy have dramatically failed (61-62) with active

treatment being barely superior to placebo. There is an urgent need to explore new

mechanisms and hence clinical trials assessing the efficacy of vitamin D

replacement in painful diabetic neuropathy are urgently required.

10.7 Vitamin D Deficiency is not associated with Diabetic Retinopathy or

Maculopathy: A Cross Sectional study (To be submitted).

A cross sectional evaluation of retinopathy and maculopathy in subjects with type 1

and type 2 diabetes in relation to vitamin D status, demographic, anthropometric

measurement, blood pressure and blood metabolic chemistry was undertaken in

six hundred and fifty seven participants. VDR genotypes have been associated

with the cumulative prevalence of diabetic retinopathy (63) and the VDR is

expressed in the ganglion cells, the inner and outer plexiform layer and the

photoreceptor layer of the retina as demonstrated by immunostaining in animal

models (64). Several risk factors are implicated in the aetiology of DR with

hyperglycaemia and hypertension showing the strongest association (65).

However, interventions aimed at correcting these risk factors have demonstrated

moderate success (66-67). Recent studies have suggested a link with vitamin D

status and DR in adolescents (68) and adults (69). Whilst we have shown there is

a marked vitamin D deficiency in this population, we found no association between

vitamin D status and the presence and severity of diabetic retinopathy or

336

maculopathy. However, well established risk factors such as HbA1c, age, duration

of diabetes and systolic blood pressure were significantly associated with the

severity of retinopathy. We conclude that further studies, in particular longitudinal

evaluation is required to better understand the potential interplay between vitamin

D and diabetic retinopathy and maculopathy.

10.8 Study limitations

There are several limitations to the current studies and should be acknowledged. A

major limitation in all but the first study (chapter 3) is the cross-sectional design.

Furthermore, the studies of vitamin D deficiency prevalence, differential vitamin D

intervention and vitamin D status in relation to retinopathy were retrospective

analyses of clinically derived data. Nevertheless, this work provides a real world

analysis of the extent of this significant public health problem and detailed

effectiveness of commonly used replacement regimens. This is particularly

important as there is no consensus agreement on the optimal replacement

strategies in vitamin D deficiency and many of the current proposed strategies lack

an evidence base. The study of vitamin D status in relation to painful diabetic

neuropathy provides a novel clinical association which demands further

mechanistic studies and human clinical trials. A lack of association between

vitamin D status and diabetic retinopathy and maculopathy in our study does not

exclude causation, particularly as most of the subjects had a low level of vitamin D.

Of course, epidemiological studies are liable to ecological fallacy.

We also present a prospective study evaluating the ability to predict the

development and progression of neuropathy through CCM. This builds on our

previously published data showing that CCM can accurately stratify the severity of

337

neuropathy (70) and track an improvement in nerve structure (5). A large

longitudinal study of CCM as a surrogate marker of DSPN is currently ongoing to

establish the predictive value of this novel modality (9). There was an absence of

skin biopsy data in the longitudinal study over 2 years. This therefore limits

interpretation as IENFD is the current gold standard technique in assessing small

nerve fibres and would allow direct comparison versus corneal nerve structure and

peripheral nerve function thus allowing a more accurate prediction of the ability of

CCM in the diagnosis and assessment of progression of DSPN. The

restoration/regeneration of the peripheral nerve is of paramount importance and

future studies of pathogenetic treatments that may show regression of neuropathy

detected by CCM are of key significance. For recruitment into these studies the

correct patient population and surrogate endpoints are required. CCM may indeed

be a game changer if this current data are mirrored in longitudinal studies with

accurate comparisons to IENFD and electrophysiology. It has been argued that the

human cornea is avascular and immune privileged and therefore assessment of

corneal nerves which are of course also much shorter has limited relevance to

peripheral nerves and hence a dying back sensorimotor neuropathy which affects

the most distal nerves first. However, the corneal subbasal nerve plexus is the

most distal portion of the ophthalmic branch of the trigeminal nerve with its cell

body in the trigeminal ganglion and therefore is directly comparable to the anatomy

of any peripheral nerve. Furthermore, the observed alterations in corneal nerves

appear to be the result of vascular-mediated effects which occur in the trigeminal

ganglion and accompanying neurovascular bundle (71-72).

338

10.9 Future Work

In terms of CCM, the prognostic value of the corneal nerve morphometric

parameters is currently being established through a number of longitudinal studies.

A community based study, investigating the ability of CCM as a screening tool for

DSPN would be invaluable and the rates of subsequent ‘hard’ endpoints such as

foot ulceration would provide valuable evidence on the utility of the technique.

Clearly subjects with LADA may have a significant predisposition to poorer

glycaemic control and subsequent small fibre neuropathy and this population may

require more intensive screening for diabetic complications, and CCM may provide

a unique opportunity for non-invasive, reiterative screening of small nerve fibres.

Future epidemiological studies of vitamin D status relating to diabetic complications

should be prospective and data derived, from multi-national sources consisting of a

spread of vitamin D status including a significant proportion that have sufficient

vitamin D concentrations. Of course, the definitive scientific evaluation should be

through double blinded randomised placebo controlled trials of vitamin D

supplementation for metabolic outcomes, diabetic complications and painful

diabetic neuropathy. However, a number of obstacles must be considered; the

likely effective dose as under dosing has been an issue in other studies, the type of

vitamin D preparation and metabolites and the length of treatment required with the

relevant vitamin D formulation. Vitamin D deficiency in relation to painful diabetic

neuropathy is an important avenue which clearly needs urgent scientific and

clinical trial evaluation as it represents an almost perfect treatment option with once

weekly or even monthly dosing, minimal side effects and of course the potential for

multiple other benefits.

339

10.10 Conclusion

In conclusion, this thesis provides evidence of marked vitamin D deficiency in a

population with diabetes and a novel association with DSPN but not retinopathy.

We also show that corneal nerve loss is readily detected with CCM and allows the

identification of both progressive DSPN and early small fibre neuropathy in patients

with LADA and type 1 diabetes.

340

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12 APPENDIX 1-STUDY RELATED DOCUMENTS

404

12-1Patient information sheet - Ophthalmic markers of diabetic neuropathy

407

12-2 Patient consent form

Participant Study Number:

Participant identification Number:

CONSENT FORM

Title of Project: Ophthalmic markers of diabetic neuropathy

Investigators: Prof. Rayaz. A Malik, Consultant Physician, MB ChB, FRCP, PhD.

Prof. Andrew Boulton, Consultant Physician, MBBS, MD, FRCP, DSc.

Dr. Andrew Marshall, Consultant Clinical Neurophysiologist BSc, BM CHB, MRCP

Dr. Mitra Tavakoli, Post-Doctoral Research Fellow BSc (Hons), MSc, PhD

Please initial box:

I confirm that I have read and I understand the information sheet dated…26/04/2011

(Version 5...) for the above study and have had the opportunity to ask questions.

I understand that my participation is voluntary and that I am free to withdraw at any time,

without giving any reason, without my medical care or legal rights being affected

I understand that sections of any of my medical notes may be looked at by responsible

individuals from University of Manchester and NHS Trust where it is relevant to my

taking part in research. I give permission for these individuals to have access to my records.

I agree to take part in the above study.

I agree that you may contact my GP regarding my participation in this study

I also agree that you can contact me in the future to see how my circumstances have

changed.

I understand that this study requires two small samples of skin to be removed from the

top of the foot. I agree to have this procedure undertaken.

……………………………. ……….………………. …………….... Name of Patient Signature Date

…………………………… ……….………………. …………..... Name of Person Signature Date

taking consent

1 for participant; 1 for researcher

408

12-3 Physical measurements and peripheral neuropathy assessment forms

Check List: Surrogate markers of diabetic neuropathy ( IGT- Diabetes- Transplant- JDRF) Name of Patient:

Date Notes

Travel costs

Information sheet

Consent forms

NCS

NDS

VPT

Neuropad

Medoc

Blood pressures

Blood samples

NCCA

CCM

Fundus camera

Skin biopsy

Medical history

Hypertension Stroke High cholesterol Heart problems Breathing problems

Other health issues: Medication

Beta blockers Warfarin Synthrome Aspirin Clopidogrel ACE inhibitor

A2RB Statin Fibrates

Other anti-hypertensive medication Neuropathy Exclusion criteria

409

Severe systemic diseases (e.g. congestive cardiac failure,

rheumatoid disease, SLE)

Chronic renal

impairment (serum creatinine >250 umol/l)

Known peripheral vascular

disease (e.g. previous bypass surgery, angioplasty or

claudication)

Alcohol intake >21 units

per week (males) or >14

units/week (females)

Non-diabetic peripheral

neuropathy Aspirin and Clopidrogrel

410

12-4 Ophthalmic examination sheet

Ophthalmic markers Ophthalmic record sheet

Participant’s Full Name: Date of Birth:

Date of Visit: Investigator(s): Study & Visit ID:

IF NOT PART OF A TRIAL

Patient referred from: Hospital No.:

Address details:

Medical History :

Type of Diabetes:

Duration of Diabetes:

Family History of Diabetes (quote parental/maternal side):

Other systemic disease (e.g. Heart Failure, Liver Failure, Hep B, HIV+, Vit. Deficiencies, Alcohol abuse, MS, Connective Tissue

Disease, SLE, psoriasis):

Medication (quote reason e.g. hypertension, cholesterol, diabetes, other CVD-related etc.):

Ocular History:

History of previous ocular disease (e.g. systemic, infections) / trauma:

History of operations (quote year, eye, type of operation):

History of contact lens use (quote type and frequency):

History of retinopathy (official grading):

For Transplant Study:

Date of Transplant:

Type of Transplant:

Duration on Renal Dialysis:

Smoking: per day

Drinking: units per week

Ophthalmic Examinations:

Slit Lamp Biomicroscopy (draw findings):

OD OS

Comments: Cornments:

411

Corneal Aesthesiometry: NCCA (mbar) CB-A

OD OS

Puppilometry (ensure 10 min. dark adaptation before examination):

Tear Tests:

BUT:

Schirmer Test:

Tear Sample collection:

- Schirmer strips:

- Microcappillary tubes:

Corneal Confocal Microscopy (HRT III-RCM)

Epithelium Bowman’s Layer/Nerve Plexus Stroma Endothelium

OD

OS

Corneal nerve parameters (values):

NFD (no./mm2) NBD (no./mm

2) NFL (mm/mm

2) NFT

OD

OS

Fundoscopy (Mydriatic/Non-Mydriatic) and Ophthalmoscopy (draw findings):

OD OS

Comments: Comments:

http://www.google.co.uk/search?hl=en&rlz=1T4GGLL_en-GBGB324GB324&ei=2S-mSqLPHJ_ajQflw8CYDg&sa=X&oi=spell&resnum=0&ct=result&cd=1&q=Schirmer+Test&spell=1

412

Additional Notes

413

13 APPENDIX 2 – Additional Analyses

414


Control group with only white European participants (n=12) with significant

differences.

Complete

Control BL

(n=16)

White European

Control BL

(n=12)

P value

Age (years) 41.4±11.4 42.6±12.5 NS

Gender (Male) (%) 63 58 NS

Ethnicity (White

European) (%) 75 100 NS

NDS (-/10)

Median(IQR)

0.5±1.1

0(0-0)

0.6±1.3

0(0-1)

NS

NSP (-/38)

Median(IQR)

0.1±0.25

0(0-0)

0.1±0.29

0(0-0)

NS

McGill VAS (-/10cm)

Median (IQR)

0.3±1.25

0(0-0)

0.4±1.40

(0-0)

NS

HbA1c (%) 5.6±0.3 5.6±0.3 NS

BMI (kg/m2) 25.7±4.2 24.9±4.2 NS

T-CHL (mmol/l) 5.1±0.9 5.1±1.0 NS

HDL (mmol/l) 1.5±0.3 1.5±0.3 NS

Triglycerides 1.4±0.7 1.4±0.8 NS


Diastolic BP (mmHg) 75±12 75±13 NS

eGFR (ml/min/1.73) 86±7 87±5 NS

NCCA (mBar) 0.6±0.3 0.7±0.3 NS

CNFD (no/mm2) 30.1±4.9 30.1±4.7 NS

CNBD (no/mm2) 36.6±15.5 34.1±16.1 NS

CNFL (mm/mm2) 16.9±2.8 16.6±2.9 NS

CST (˚C) 28.6±2.1 28.5±2.2 NS

WST(˚C) 37.6±3.5 38.2±3.9 NS

VPT (volts) 5.3±4.8 6.0±5.5 NS

SSNCV (m/s) 49.4±3.9 49.2±3.9 NS

SSNAmp (µV) 21.0±10.8 21.6±12.1 NS

PMNCV (m/s) 47.8±3.6 47.2±4.1 NS

PMNAmp (mV) 6.0±1.6 5.4±1.0 NS

415


Control group with only white European participants (n=21) with significant

differences.

Controls (n=27)

Controls White

European only

(n=21)

P value

Age (years) 41.0±14.9 42.9±16.0 NS


HbA1c (%) 5.5±0.3 5.5±0.3 NS

HbA1c (mmol/mol) 36.9±3.4 36.9±3.4 NS

BMI (kg/m2) 26.9±4.0 26.9±4.3 NS

T-CHL (mmol/l) 5.0±0.8 5.0±0.9 NS

HDL (mmol/l) 1.6±0.4 1.6±0.4 NS




eGFR (ml/min/1.73) 85±7 85±7 NS

NDS (-/10)

Median(IQR)

0.4±0.8

0(0-1)

0.5±0.9

0(0-1) NS

NSP (-/38)

Median(IQR)

0.1±0.4

0(0-0)

0.1±0.5

0(0-0) NS

McGill VAS (-/10cm)

Median(IQR)

0.2±1.0

(0(0-0)

0.2±1.1

(0(0-0) NS

NCCA (mBar) 0.5±0.3 0.6±0.3 NS

CNFD (no/mm2) 37.2±5.1 37.7±4.8 NS

CNBD (no/mm2) 92.0±36.2 92.2±34.1 NS

CNFL (mm/mm2) 26.6±3.8 27.0±2.8 NS

IENFD (no/mm) 10.2±3.3 10.2±3.3 NS

CST (˚C) 28.6±2.0 28.5±2.1 NS

WST(˚C) 36.4±2.0 36.6±2.2 NS

VPT (volts) 5.3±4.1 5.9±4.5 NS

SSNCV (m/s) 50.6±4.2 50.4±4.6 NS

SSNAmp (µV) 20.2±8.8 19.2±9.3 NS

PMNCV (m/s) 49.2±3.7 48.8±4.0 NS

PMNAmp (mV) 6.1±2.4 5.5±2.5 NS

416


Control group with participants of an older age matched to the DSPN group (n=13)

with significant differences.

Controls (n=27) Older Controls

(n=13) P value

Age (years) 41.0±14.9 54.4±9.9 0.007


Ethnicity (White

European) (%) 77 84

NS

HbA1c (%) 5.5±0.3 5.7±0.3 NS

BMI (kg/m2) 26.9±4.0 28.5±3.2 NS

T-CHL (mmol/l) 5.0±0.8 5.0±0.8 NS

HDL (mmol/l) 1.6±0.4 1.6±0.4 NS




eGFR (ml/min/1.73) 85±7 83±9 NS

NDS (-/10)

Median(IQR)

0.4±0.8

0(0-1)

0.4±0.8

0(0-1) NS

NSP (-/38)

Median(IQR)

0.1±0.4

0(0-0)

0.2±0.6

0(0-0) NS

McGill VAS (-/10cm)

Median(IQR)

0.2±1.0

(0(0-0)

0.4±1.4

(0(0-0) NS

NCCA (mBar) 0.5±0.3 0.6±0.3 NS

CNFD (no/mm2) 37.2±5.1 38.2±5.1 NS

CNBD (no/mm2) 92.0±36.2 99.7±40.5 NS

CNFL (mm/mm2) 26.6±3.8 27.8±3.8 NS

IENFD (no/mm) 10.2±3.3 9.1±2.3 NS

CST (˚C) 28.6±2.0 28.0±2.3 NS

WST(˚C) 36.4±2.0 37.2±2.0 NS

VPT (volts) 5.3±4.1 6.9±5.4 NS

SSNCV (m/s) 50.6±4.2 48.4±4.1 NS

SSNAmp (µV) 20.2±8.8 16.0±8.9 NS

PMNCV (m/s) 49.2±3.7 47.7±3.7 NS

PMNAmp (mV) 6.1±2.4 5.6±2.3 NS

417

Key for Table 13.1, 13.2 & 13.3

ACR – Albumin Creatinine Ratio; BMI – Body Mass Index; BP – Blood Pressure; C – Controls;


Nerve Fibre Length; CST – Cold Sensation Threshold; estimated eGFR – Glomerular Filtration

Rate; HbA1c – Glycated Haemoglobin A1c; HDL – High Density Lipoprotein Cholesterol; IENFD –

Intra Epidermal Nerve Fibre Density; McGill VAS – McGill Visual Analogue Score; NDS –

Neuropathy Disability Score, NSP – Neuropathy Symptom Profile; PMNAmp – Peroneal Motor

Nerve Amplitude; PMNCV – Peroneal Motor Nerve Conduction Velocity; SSNAmp – Sural Nerve

Sensory Nerve Amplitude; SSNCV – Sural Motor Nerve Conduction Velocity; T-CHL – Total

Cholesterol; VPT – Vibration Perception Threshold; WST – Warm Sensation Threshold.

Vitamin D and Diabetic Neuropathy

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