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Patients with type 2 diabetes have atwo- to fourfold increased risk of car-diovascular morbidity and mort a l i t y

c o m p a red with age-matched nondiabeticsubjects (1–3). Known risk factors, such assmoking, dyslipidemia, and hypert e n s i o n ,can explain only part of this increase, and

recent epidemiological studies have sug-gested that hyperglycemia contributes toi n c reased cardiovascular risk (4–6).

One of the potential mechanisms bywhich hyperglycemia may induce card i o-vascular disease is through the formation of advanced glycation end products ( A G E s ) .

AGEs have been suggested to participate inthe development of both micro- and macro-vascular complications in diabetes.

AGE deposits have been demonstratedin athero s c l e rotic plaques and myocard i u mby immunohistochemistry in patients withdiabetes and athero s c l e rosis (7), and thec ross-linking abilities of AGEs may con-tribute to the increased stiffening of collagenand possibly to vascular hypert rophy (8,9).Because calcification of the media of art e r i e sand reduced arterial compliance have beenshown to be significant predictors of futurec o ro n a ry heart disease (CHD) in patientswith type 2 diabetes (10), formation of AGEsmay be one important mechanism.

AGEs are also, through interaction withreceptors, able to increase the level of NF-kB, a transcription factor suggested to beinvolved in the development of athero s c l e-rosis and in apoptosis (11). In addition,AGEs can quench nitric oxide and mayimpair endothelial function (12,13). Modi-fication of LDL as a result of glycation maycontribute to foam cell formation andi n c reased athero s c l e rosis (14,15).

Given these potential contributions ofAGEs to the development of athero s c l e ro-sis, and the fact that increased serum levelsof AGEs have been demonstrated to bei n c reased and to predict development ofm i c rovascular complications in patientswith type 1 diabetes (16,17), we investi-gated whether serum levels of AGEs wereelevated in patients with type 2 diabetesand whether the levels were diff e rent inpatients with type 2 diabetes with andwithout CHD. In addition, we examinedwhether there were any diff e rences ins e rum levels of the glycoxidation epitope,Nε-(carboxymethyl)lysine (CML) by usinga newly developed immunoassay (18).

RESEARCH DESIGN AND M E T H O D S

SubjectsThe study involved 53 patients with type 2diabetes (32 men and 21 women) with a

F rom the Aker Diabetes Research Centre, Department of Endocrinology (B.K.K., T.J.B., K.F.H.), Horm o n eL a b o r a t o ry (K.I.B., P. T.), Aker University Hospital, Oslo, Norw a y.

A d d ress correspondence and reprint requests to Bente Kilhovd, MD, Aker Diabetes Research Centre, AkerUniversity Hospital, 0514 Oslo, Norw a y. E-mail: b.k.kilhovd@ioks.uio.no.

Received for publication 30 December 1998 and accepted in revised form 19 May 1999.A b b re v i a t i o n s : AGE, advanced glycation end product; BSA, bovine serum albumin; CHD, coro n a ry heart dis-

ease; CML, Nε-(carboxymethyl)lysine; CV, coefficient of variation; ECG, electro c a rdiogram; GDR, glucose disposalrate; GDRI, glucose disposal rate index; ICD-9, I n t e rnational Classification of Diseases, Ninth Revision; OCTOPUS,Oslo Comparative Trial of Peroral Versus Insulin Treatment in Type 2 Diabetes.

A table elsewhere in this issue shows conventional and Système International (SI) units and conversionfactors for many substances.

S e rum Levels of Advanced Glycation End Products Are Increased in PatientsWi thType 2 Diabetes and Coro n a ryH e a rt D i s e a s e

O R I G I N A L A R T I C L E

O B J E C T I V E — To investigate whether serum levels of advanced glycation end pro d u c t s(AGEs) and the glycoxidation product Nε-(carboxymethyl)lysine (CML) are increased inpatients with type 2 diabetes compared with nondiabetic control subjects and whether levelsof AGEs and/or CML differ in patients with type 2 diabetes with or without coro n a ry heartdisease (CHD).

RESEARCH DESIGN AND METHODS — S e rum levels of AGEs and CML were meas-u red with an immunoassay in 32 men and 21 women aged 59.3 ± 6.2 years (means ± SD) withtype 2 diabetes for 7.3 1 3.1 years and in 17 men and 17 women aged 56.2 ± 4.2 years with-out diabetes. Of the patients with diabetes, 18 had CHD.

R E S U LT S — The serum levels of AGEs and CML were significantly increased in patients withtype 2 diabetes compared with nondiabetic control subjects (median [5th–95th perc e n t i l e ] :AGEs 7.4 [4.4–10.9] vs. 4.2 [1.6–6.4] U/ml, P , 0.0001; CML 15.6 [5.6–29.9] vs. 8.6[4.4–25.9] U/ml, P , 0.0001). The median level of AGEs but not CML was signific a n t l yi n c reased in patients with type 2 diabetes and CHD compared with patients without CHD (8.1[6.4–10.9] vs. 7.1 [3.5–9.8] U/ml, P = 0.03). There were significant positive corre l a t i o n sbetween serum levels of AGEs and CML in both patients and control subjects.

C O N C L U S I O N S — Levels of AGEs and CML were significantly increased in patients withtype 2 diabetes compared with nondiabetic control subjects, and levels of AGEs but not CMLw e re significantly higher in patients with type 2 diabetes and CHD than in patients without dia-betes. These results may indicate a role for non-CML AGEs in the development of macro v a s-cular disease in patients with type 2 diabetes.

Diabetes Care 22:1543–1548, 1999

BENTE K. KILHOVD, MD

TORE JULSRUD BERG, MD

KÅRE I. BIRKELAND, MD, PHD

PER THORSBY, MD

KRISTIAN F. HANSSEN, MD, PHD

P a t h o p h y s i o l o g y / C o m p l i c a t i o n s

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mean age of 59.3 ± 6.2 years (means ± SD)and a known duration of diabetes of 7.3 ±3.1 years. Patients with established type 2diabetes of .2 years duration with an initialH b A1 c of 7–10% who had stable glycemicc o n t rol during a run-in period of 3 monthsconsisting of diet and glibenclamide wererandomized to either continue gliben-clamide or start insulin treatment in theOslo Comparative Trial of Peroral Ve r s u sInsulin Treatment in Type 2 Diabetes(OCTOPUS) Study (19). The patients wereexamined every 3 months for a median of 5 years. The OCTOPUS study was designedto investigate the relationship between long-t e rm metabolic control and the develop-ment of micro- and macrovascular compli-cations in type 2 diabetes.

I n i t i a l l y, half of the patients were ran-domized to start insulin treatment and theother half was to continue sulfonylurea tre a t-ment, but most of the patients (n = 51) tookinsulin 5 years from randomization to main-tain acceptable blood glucose levels. At startof this study, mean BMI was 26.6 ± 3 . 7k g / m2, HbA1 c was 8.6 ± 1.2%, and fastingblood glucose was 11.0 ± 2.6 mmol/l. Therew e re no significant diff e rences between menand women re g a rding baseline values.

The control group for the AGE andCML measurements consisted of 34 ran-domly selected healthy blood donors (17men and 17 women with a mean age of56.2 1 4.2 years). The mean age in thec o n t rol group was slightly but signific a n t l ylower than in the patient gro u p .

CHD was diagnosed on the basis of at h o rough clinical examination and a med-ical history of angina pectoris or myocar-dial infarction. Medical re c o rds wereexamined, and the diagnosis of myocard i a li n f a rction (I n t e rnational Classification of Dis -e a s e s, Ninth Revision [ICD-9] codes 410and 412) and angina pectoris (code 413)or the use of coro n a ry intervention pro c e-d u res (e.g., percutaneous transluminalc o ro n a ry angioplasty or coro n a ry art e rybypass grafting) was verified. Angina pec-toris was defined on the basis of a medicalh i s t o ry of exercise-induced central chestpain relieved by stopping the activity or by

sublingual nitroglycerin. Resting electro-c a rdiogram (ECG) confirmed CHD whenpathological Q-waves (i.e., Q-wave dura-tion of .0.04 s or Q . 1/4 of the QRScomplex in more than one lead) were pre s-ent. Exercise ECG was defined as confirm-ing CHD if horizontal ST- s e g m e n td e p ressions of at least 1 mm combinedwith characteristic chest pain appeare dduring or immediately after exerc i s e .

During follow-up, yearly clinical exam-inations with resting ECGs were perf o rm e d ,and ECGs were examined for newly devel-oped Q-waves. Patients with new symp-toms suggesting angina pectoris duringfollow-up were exercise tested and re f e rre dfor coro n a ry angiography if the exerc i s eECG indicated ischemia. Medical re c o rd sf rom hospital admissions during the studyw e re collected. Medical re c o rds with ICD-9codes 410–414 confirmed CHD.

The number of patients with CHD wasthe sum of those who had CHD at the startof the study and those who developed itduring follow-up.

MethodsS e rum levels of AGEs were measured witha competitive immunoassay (16) by using

Table 1—S e rum levels of AGEs and CML in patients with type 2 diabetes and control subjects

P a t i e n t s C o n t rol subjects

n 5 3 3 4S e rum AGEs (U/ml) 7.4 (4.4–10.9) 4.2 (1.6–6.4)*S e rum CML (U/ml) 15.6 (5.9–29.9) 8.6 (4.4–25.9)*

Data are medians (5th–95th percentiles). *P , 0 . 0 0 0 1 .

Figure 1—A: Distribution of serum levels of AGEs (U/ml) in patients with type 2 diabetes (h) andnondiabetic control subjects ( j). Percentages indicate patients with a given level of serum value ofAGEs. B: Serum levels of CML in patients and control subjects.

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polyclonal anti-AGE antibodies made fro mrabbit immunized with AGE-RNase (20)and europium-labeled anti-rabbit antibod-ies as detecting antibodies. AGE-bovines e rum albumin (BSA) was used as a stan-d a rd. Both AGE-BSA and polyclonal anti-AGE antibodies were provided by Pro f .R i c h a rd Bucala of the Picower Institute forMedical Research (Manhasset, NY). Resultsa re expressed as AGE units (1 U = 1 µg/mlAGE-BSA standard) and are adjusted fortotal serum protein concentration in eachsample. All analyses were perf o rmed dur-ing the same run, and the intra-assay coef-ficient of variation (CV) was ,1 2 % .

S e rum levels of CML were measure din a newly developed immunoassay (18)by using monoclonal anti-CML antibodiesas detecting antibodies and CML-BSA asthe standard. Both anti-CML antibodiesand CML-BSA were supplied by JesClausen of Novo Nordisk AS (Bagsværd ,Denmark). Europium-labeled anti-mouseantibodies were used as indicators. Resultsa re expressed as CML units (1 U = 1 µg/mlCML-BSA standard) and are adjusted fortotal serum protein concentration in eachsample. All analyses were perf o rmed dur-ing the same run, and the intra-assay CVwas ,1 2 % .

H b A1 c was analyzed with a high-per-f o rmance liquid chromatography method(Diamat analyzer; Bio-Rad, Richmond, CA)that has a normal range of 4.3–6.1% withan interassay CV of ,3 % .

F ructosamine was measured as an i t robleu-tetrazolium reaction product in a

photometric analyzer (Cobas BIO/UnimatRoche, Basel, Switzerland) with a norm a lrange of 230–300 µmol/l for nondiabeticsubjects and an interassay CV of ,4 % .

S e rum total cholesterol, HDL choles-t e rol, and triglycerides were measured enzy-matically (Boehringer Ingelheim/BoehringerMannheim, Mannheim, Germany), andLDL cholesterol was calculated with theFriedewald formula (21). Total protein ins e rum samples was measured by using the

B i u ret method (Boehringer Mannheim,Mannheim, Germany) with a CV of ,2 % .M i c roalbuminuria was defined as an albu-min excretion rate of .20 and ,2 0 0µg/min in an overnight timed urine sample.Blood pre s s u re was measured with a stan-d a rd sphygmomanometer with the patientsitting after a 5- to 10-min rest. Diastolicblood pre s s u re was re g i s t e red at the disap-pearance of Koro t k o ff’s V sound.

The euglycemic glucose clamp tech-nique was perf o rmed in the patients after ano v e rnight fast. Insulin (7.2 pmol ? k g– 1 ?m i n– 1) was infused at fixed rate until eugly-cemia was reached, and a variable glucoseinfusion (concentration 240 mg/l) was thens t a rted. The infusion rate of glucose wasadjusted to maintain a stable blood glucoselevel at 5 mmol/l for 120 min, and theamount of glucose infused during the last20 min was defined as the glucose disposalrate (GDR). The insulin concentration wasm e a s u red in arterialized blood samplese v e ry 10–20 min. The GDR index (GDRI)was expressed as the GDR relative to thep revailing serum insulin levels.

Blood pre s s u re and levels of HbA1 c,lipids, AGEs, and CML were measured aftera 10-h fast before randomization into theOCTOPUS Study.

Statistical analyses were perf o rmed withthe Number Cruncher Statistical System(NCSS, Kaysville, UT) and SAS statisticals o f t w a re (Cary, NC). Nonparametric tests

Figure 2—Serum levels of AGEs correlate to serum levels of CML (r = 0.61, P , 0.0001) in patientswith type 2 diabetes.

Table 2—Characteristics of patients with type 2 diabetes with or without CHD

With CHD Without CHD P

n 1 8 3 5 —Sex (M/F) 1 0 / 8 2 2 / 1 3 N SAge (years) 61.5 (50–69) 60 (47.2–67.2) N SDiabetes duration (years) 6.5 (3–13) 7 (2.4–14.2) N SH b A1c ( % ) 8.3 (6.4–10.9) 8.5 (6.9–11.1) N SFasting blood glucose (mmol/l) 10.5 (6.8–15.3) 11.2 (7.5–16.8) N SS e rum AGEs (U/ml) 8.1 (4.4–11.3) 7.1 (3.5–9.8) 0 . 0 3S e rum CML (U/ml) 16.2 (8.3–27) 13.7 (2.9–31.9) N STotal cholesterol (mmol/l) 6.8 (5.1–9.6) 6.0 (4.9–8.0) N STriglycerides (mmol/l) 1.89 (0.60–6.65) 1.60 (0.47–3.61) N SHDL cholesterol (mmol/l) 1.07 (0.68–1.76) 1.12 (0.76–2.12) N SLDL cholesterol (mmol/l) 4.46 (3.37–7.18) 4.0 (2.90–6.19) N SSystolic blood pre s s u re (mmHg) 160 (120–180) 140 (114–204) 0 . 0 3Diastolic blood pre s s u re (mmHg) 90 (80–120) 90 (74–106) N SBMI (kg/m2) 26.0 (22.0–33.1) 26.1 (20.4–33.2) N SG D R I 4.31 (0.07–12.7) 4.43 (1.14–12.0) N SSmokers (current or form e r / n e v e r ) 1 3 / 5 1 7 / 1 8 N SM i c roalbuminuria (with/without) 4 / 1 4 6 / 2 9 N S

Data are medians (5th–95th percentiles). Microalbuminuria is defined as two out of three or one out of two con-secutive measurements of albumin excretion rate .20 and ,200 mg/min the last year before inclusion/neverm i c ro a l b u m i n u r i a .

w e re used because of the nonequal distri-bution of data, and diff e rences betweeng roups were tested with a two-tailed Mann-Whitney U test. Univariate corre l a t i o n sw e re tested with the use of Spearman rank-o rder correlations, and a stepwise logisticalre g ression analysis was perf o rmed withCHD as the dependent variable. The two-sided significance level was 5%.

R E S U LT S — Levels of serum AGEs were7.4 (4.4–10.9) U/ml (median [5th–95th per-centiles]) in the patients with type 2 diabetesand 4.2 (1.6–6.4) U/ml in the control gro u p(P , 0.0001). Levels of serum CML were15.6 (5.6–29.9) U/ml in the patients withtype 2 diabetes, and 8.6 (4.4–25.9) U/ml inthe control group (P , 0.0001) (Table 1).The frequency distribution of serum levels ofAGEs and CML are shown in Fig. 1.

The serum levels of AGEs corre l a t e ds i g n i ficantly to levels of CML in bothpatients (r = 0.61, P , 0.0001) (Fig. 2) andc o n t rol subjects (r = 0.57, P , 0 . 0 0 1 ) .T h e re were no significant corre l a t i o n sbetween age and serum levels of AGEs orCML in our material, neither in patients norc o n t rol subjects (Fig. 3).

The median serum levels of AGEs were8.1 (6.4–10.9) U/ml in patients with type 2diabetes and CHD compared with 7.1(3.5–9.8) U/ml in patients without CHD (P = 0.03). Systolic blood pre s s u re wass i g n i ficantly higher in patients with CHDthan in those without CHD (Table 2).

S e rum triglycerides showed a tenden-cy toward higher values in patients withCHD compared with patients withoutCHD, but the diff e rence did not reach sig-n i ficance (P = 0.07).

In a stepwise logistical re g ression anal-ysis with CHD as the dependent variable(including blood pre s s u re, diabetes dura-tion, HbA1 c, GDRI, micro a l b u m i n u r i a ,s e rum AGEs, serum CML, total cholestero l ,triglycerides, HDL and LDL cholestero l ,age, BMI, and smoking status as indepen-dent variables), serum triglyceride, seru m

AGEs, and patient age were signific a n t l yassociated with CHD (Table 3).

T h e re were no significant diff e rences ins e rum levels of CML, HbA1 c, fasting bloodglucose, or total, LDL, or HDL cholestero lbetween patients with and without CHD( Table 2), nor was there a diff e rence ininsulin resistance measured with the eugly-cemic hyperinsulinemic glucose clampt e c h n i q u e .

T h e re were no significant corre l a t i o n sbetween serum levels of AGEs and CML andfasting blood glucose, HbA1 c, or cholestero llevels in patients with type 2 diabetes. Therew e re no diff e rences in serum levels of AGEsor CML between men and women.

A total of 10 patients had micro a l b u-minuria, 4 in the group with CHD and 6 inthe group without CHD.

C O N C L U S I O N S — This study showedhighly significantly increased serum levelsof both AGEs and CML in patients withtype 2 diabetes. The diff e rences are compa-rable with those in patients with type 1 dia-betes (16,18).

We also found significantly incre a s e ds e rum levels of AGEs but not CML inpatients with CHD (n = 18). This is the fir s tstudy to show such a diff e rence, and thisraises the possibility that AGEs may part i c-ipate in the development of CHD inpatients with type 2 diabetes.

T h e re may be several potential mecha-nisms for AGE involvement in macro v a s-cular complications in diabetes. Whenexamining the in vitro pro p e rties of AGEssuch as activation of cytokines (22) and

transcription factors (10), chemotaxis andactivation of monocytes through the acti-vation of adhesion molecules (23,24), ande x p ression of platelet-derived growth factor(25), one finds possible mechanisms ofaction for AGEs in the development of ath-e ro s c l e rosis. There is also indirect evidenceof the involvement of AGEs in the athero-s c l e rotic process via the effect of amino-guanidine, an inhibitor of AGE form a t i o n ,on the development of athero s c l e rosis innondiabetic rats (26) and in cholestero l - f e drabbits (27). Aminoguanidine can pre v e n td e c reased myocardial compliance in dia-betic rats (28) and age-related arterial stiff-ening and cardiac hypert rophy in nondia-betic rats (29). Recently, a soluble re c e p t o rfor AGEs (RAGE) has been shown toinhibit athero s c l e rosis in mice (30).

The polyclonal anti-AGE antibody re c-ognizes several epitopes, one of which isCML (18). However, anti-AGE antibodiesrecognize other epitopes as well, and someof them are not known. An increasing inter-est in non-CML immunoreactivity in dia-betic complications is now evident (31–34).

The serum levels of CML were nots i g n i ficantly increased in patients withCHD versus the levels of AGEs. This mayindicate that there are AGE fractions otherthan CML that are of importance in thedevelopment of athero s c l e rosis in patientswith diabetes and CHD (7).

T h e re were no correlations betweenage and serum levels of AGEs or CML inour study. So far, there has been a tendencyto believe that serum levels of AGEs andCML increase with age because tissue lev-

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Table 3—Risk factors for CHD in a multi-variate model using stepwise forw a rd logisticalre g re s s i o n a n a l y s i s

Odds ratioVa r i a b l e (95% CI) P

Triglycerides (mmol/l) 2.4 (1.2–4.8) 0 . 0 1 8S e rum AGEs (U/l) 1.9 (1.2–3.1) 0 . 0 0 8Age (years) 1.1 (1.0–1.3) 0 . 0 4 9

F i g u re 3—S e rum levels of AGEs in patients with diabetes (d) and in nondiabetic control subjects (j), ands e rum levels of CML in patients with diabetes (s) and in nondiabetic control subjects (h) a c c o rding to age.

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els of AGEs and CML increase with age(20,35–38), possibly because a slowert u rnover in the tissues that allows AGEsand CML to accumulate with age. No stud-ies so far have shown any increase in seru mlevels with age.

Some of the increase in serum AGEsmay re p resent increased AGE-LDL, and gly-cation of LDL has been shown to increase itsuptake by the macrophage scavenger re c e p-tors and contribute to foam cell form a t i o n(14,15). It is also possible that modific a t i o nof serum proteins through glycation maycontribute to increased athero s c l e ro s i st h rough activation of cytokines and gro w t hfactors as shown in vitro .

Our results alone cannot be considere devidence for the involvement of AGEs ina t h e ro s c l e rosis in humans, but they raise itas a hypothesis and may give rise to furt h e ri n v e s t i g a t i o n s .

The lack of a significant corre l a t i o nbetween serum AGEs and CML and HbA1 c

in our study compared with the pre v i o u s l yshown associations between skin collagenAGEs and CML and previous mean HbA1 c

(39) may re flect the diff e rent turnover ofthe components in serum and skin (40)because these are diff e rent enviro n m e n t s .

Only four patients in the study hadboth microalbuminuria and CHD, but thereis no indication that patients with micro a l-buminuria and CHD in our study had anyhigher levels of serum AGEs or CML thanpatients with CHD alone (data not shown).This contrasts with previous publicationsthat showed increased levels of AGEs(41,42) and CML (40,43) in patients withm a c roalbuminuria and on dialysis and mayindicate that kidney damage in micro a l b u-minuria is not severe enough to causedefects in handling AGEs in the kidneys.

We did not find any significant corre-lation between either previous or curre n tsmokers and levels of AGEs or CML, andthus our data do not support the hypothe-sis that persons with diabetes who are orhave been smokers have increased ACElevels (44).

In summary, our results show signifi-cantly increased serum levels of both AGEsand CML in patients with type 2 diabetesc o m p a red with nondiabetic control subjects.We also found that patients with type 2 d i a-betes and CHD have a significantly highers e rum level of AGEs (but not CML) thanpatients with diabetes but without CHD.These findings may suggest that AGEs con-tribute to the development of macro v a s c u-lar complications in diabetes.

A c k n o w l e d g m e n t s — B.K. received a grantf rom the Norwegian Foundation for Health andRehabilitation. Anti-AGE antibodies and AGE-BSA were gifts from Prof. Richard Bucala (ThePicower Institute for Medical Research, NewYork), and anti-CML antibodies and CML-BSAw e re provided by Jes Clausen (Novo Nord i s kAS, Bagsværd, Denmark). The OCTOPUS studyhas received grants from Novo Nordisk Pharm aAS, Hoechst Marion Roussel, The Norw e g i a nDiabetes Association, and Aker DiabetesR e s e a rch Fund.

We thank Turi A. Siegwarth for excellenttechnical assistance.

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