Impaired endothelium-dependent vasodilation in type 2 diabetes mellitus and the lack of effect of...

DIABETES CARE, VOLUME 22, NUMBER 6, JUNE 1999 973

Im p a i red endothelium-dependent vaso-dilation in fore a rm resistance vessels char-acterizes individuals who are at risk of

developing athero s c l e rosis (1). In hyperc h o-l e s t e rolemic patients, total and LDL choles-t e rol concentrations are inversely corre l a t e dwith the in vivo vasodilatory response toendothelium-dependent vasodilators, suchas acetylcholine (ACh), in both fore a rmresistance (2–4) and coro n a ry vessels (5).This is not true for patients with type 1 dia-betes, in whom the degree of chronic hyper-glycemia appears to be the most import a n td e t e rminant of the endothelial dysfunction(6). Regarding data on endothelium-depen-dent vasodilation in patients with type 2diabetes, endothelium-dependent bloodflow responses have been impaired in most(7–12) although not all (13) studies.Responses to sodium nitro p russide (SNP)have been subnormal in many (7,10–12)but not all (8,13) studies. In none of thestudies has the defect in endothelium-dependent vasodilation in type 2 diabeticpatients been explained by total or LDL cho-l e s t e rol concentrations (7–12). The lattero b s e rvation is consistent with epidemiolog-ical data demonstrating a two- to fourf o l di n c rease in cardiovascular disease in patientswith type 2 diabetes that cannot beexplained by classic cardiovascular risk fac-tors such as age, sex, hypertension, ands e rum cholesterol concentration (14).

Several new potential card i o v a s c u l a rrisk factors have recently been identifie d .Use of moderate doses of vitamin E hasbeen shown to decrease nonfatal myocard i a li n f a rctions in nondiabetic subjects (15). Inpatients with type 2 diabetes, intra-art e r i a ladministration of large doses of vitamin Cacutely improves endothelium-dependentvasodilation (9). Such data raise the possi-bility that levels of plasma antioxidantsmight be related to endothelium-depen-dent vasodilation in type 2 diabetes. Levelsof circulating antioxidants have not, how-e v e r, been measured in previous studiesexamining endothelial function in type 2diabetes. Another relatively recent card i o-vascular risk predictor is the size of LDLp a rticles (16). LDL size, although oftenclosely correlated with serum triglycerides

F rom the Departments of Medicine (S.M., M.L., J.V., A.S., S.L., M.S., R.B., M.-R.T., H.Y.-J.) and Ophthal-mology (P.S.), Helsinki University Central Hospital; and the Research Institute of Military Medicine (M.M.),Central Military Hospital, Helsinki, Finland.

A d d ress correspondence and reprint requests to Hannele Yki-Järvinen, MD, Professor of Medicine, Uni-versity of Helsinki, Department of Medicine, Division of Endocrinology and Diabetology, Haartmaninkatu 4,FIN-00290 Helsinki, Finland. E-mail: ykijarv i @ h e l s i n k i . fi.

Received for publication 11 December 1998 and accepted in revised form 25 Febru a ry 1999.A b b reviations: ACh, acetylcholine; AER, albumin excretion rate; apo, apolipoprotein; ECG, electro c a r-

diogram; HPLC, high-pre s s u re liquid chromatography; IDL, intermediate-density lipoprotein; L-NMMA, NG-m o n o m e t h y l -L- a rginine; NO, nitric oxide; SH, sulfhydryl; SNP, sodium nitro p russide; TRAP, total pero x y lradical-trapping capacity; TRAPC A L C, calculated total peroxyl radical-trapping capacity; TRAPO B S, observ e dtotal peroxyl radical-trapping capacity.

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

I m p a i red Endothelium-DependentVasodilation in Type 2 DiabetesRelation to LDL size, oxidized LDL, and antioxidants

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 search for determinants of endothelial dysfunction in type 2 diabetes.

RESEARCH DESIGN AND METHODS — We perf o rmed a comprehensive analysis ofc a rdiovascular risk markers and measured blood flow responses to endothelium-dependent(acetylcholine [ACh] and NG- m o n o m e t h y l -L- a rginine) and -independent (sodium nitro p ru s-side [SNP]) vasoactive agents in 30 nonsmoking men with type 2 diabetes (age 51 ± 1 years,BMI 27.8 ± 0.4 kg/m2, HbA1 c 7.4 ± 0.3%) and 12 matched normal control men.

R E S U LT S — ACh-induced vasodilation was 37% lower in type 2 diabetic (6.1 ± 0.5) than inn o rmal subjects (9.7 ± 1.5 ml ? d l21 ? m i n21, P , 0.01), while flows during SNP were similar(9.1 ± 0.6 vs. 9.9 ± 1.3 ml ? d l21 ? m i n21, NS). The ratio of endothelium-dependent vs. -inde-pendent flow (ACh:SNP ratio) was 31% lower in type 2 diabetic (0.70 ± 0.05) than in norm a lsubjects (1.10 ± 0.18, P , 0.01). Total (2.2 ± 0.4 vs. 1.3 ± 0.2 mmol/l, P , 0.05), VLDL, andi n t e rmediate-density lipoprotein triglycerides were significantly higher, and the mean LDL par-ticle diameter was significantly smaller in type 2 diabetic than in normal subjects. The lag timesfor LDL oxidation by Cu21 in vitro were similar in patients with type 2 diabetes (183 ± 7) andin normal subjects (183 ± 9 min, NS). Measured and calculated (sum of concentration of indi-vidual antioxidants in serum) total peroxyl radical-trapping capacities (TRAPs) were compa-rable between the groups. In the patients with type 2 diabetes, LDL size was signific a n t l yc o rrelated with endothelium-dependent vasodilation (r = 0.43, P , 0.05), serum triglycerides(r = 20.75, P , 0.001), and the lag time for LDL oxidation in vitro (r = 0.38, P , 0.05). HbA1 c

was inversely correlated with the lag time for LDL oxidation in vitro (r = 20.41, P , 0.05) andT R A P.

C O N C L U S I O N S — In summary, patients with type 2 diabetes exhibited impaired endothe-lium-dependent vasodilation in vivo, elevated serum triglycerides, decreased LDL size, and nor-mal antioxidant capacity. Of these parameters, LDL size was significantly correlated withendothelial function.

Diabetes Care 22:973–981, 1999

SARI MÄKIMATTILA, MD, MSC

MING-LIN LIU, MD

JUHA VAKKILAINEN, MD

ANNA SCHLENZKA, MD

SANNI LAHDENPERÄ, MD

MIKKO SYVÄNNE, MD

MATTI MÄNTYSAARI, MD

PAULA SUMMANEN, MD

ROBERT BERGHOLM, MD

MARJA-RIITTA TASKINEN, MD

HANNELE YKI-JÄRVINEN, MD

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

974 DIABETES CARE, VOLUME 22, NUMBER 6, JUNE 1999

Endothelial function and lipids in type 2 diabetes

(17), was recently shown to predict theincidence of ischemic heart disease, evenindependent of serum triglyceride concen-trations in nondiabetic men (16). O’Brien etal. (12) compared LDL size and endothelialfunction between obese patients with type 2diabetes and normal-weight nondiabeticsubjects. Type 2 diabetic patients also hads i g n i ficantly higher blood pre s s u re than thenonobese subjects. In the study by O’Brienet al., endothelial function and LDL sizew e re significantly correlated, but itremained unclear whether endothelial dys-function was indeed due to type 2 diabetesor to obesity or hypert e n s i o n .

The present study was undertaken tod e t e rmine whether endothelium-depen-dent or -independent vasodilation is altere din carefully characterized groups of type 2diabetic patients compared with matchednondiabetic subjects and, if so, whetherthe levels of circulating antioxidants, lipids,or lipoproteins, LDL size, or the suscepti-bility of LDL to oxidation in vitro explaininterindividual variation in endothelialfunction in these patients.

RESEARCH DESIGN ANDM E T H O D S

SubjectsA total of 30 men with type 2 diabetes and12 normal men were studied. The twog roups were matched with respect to age,weight, and body composition (Table 1). Allstudy participants were nonsmokers.

Patients with type 2 diabetes were re c ru i t e df rom diabetes outpatient clinics in theHelsinki area based on the following crite-ria: 1) age 35–65 years, 2) age at diagnosisof diabetes $35 years. Exclusion criteriaincluded 1) a history of cere b rovascular orischemic heart or peripheral vascular dis-eases, 2) symptoms of neuro p a t h y, 3) sig-n i ficant renal impairment (abnorm a lc reatinine or macroalbuminuria), 4) hyper-tension (blood pre s s u re .160/90 mmHg),and 5) use of cardiovascular drugs. Patientsw e re treated with diet alone (n = 18) or withdiet plus sulfonylurea or biguanide pre p a-rations (n = 12). Drugs were discontinuedfor 2 days before the studies to avoid possi-ble confounding effects of the drugs on vas-cular function (18). Subjects underwent acomplete history and physical examinationand laboratory tests, including an electro-c a rdiogram (ECG), to exclude diseasesother than type 2 diabetes and clinicallys i g n i ficant macrovascular disease. ECGsw e re evaluated in a blinded fashion by ac a rdiologist (M.S.). Abnormalities suggest-ing coro n a ry heart disease (Minnesota Codeitems A3.1.2.1, A3.1.2.4, A3.1.2.5, andA3.1.2.7) were not found in any of thestudy subjects. The screening examinationalso included careful assessment of the pre s-ence of autonomic neuro p a t h y, since thiscomplication modulates vascular re s p o n s e sto nitrovasodilators (19). To exclude pro l i f-erative or moderate to severe nonpro l i f e r a-tive diabetic retinopathy or maculopathy,two 45° fundus color slides of the macular

and disc/nasal fields of each eye were takent h rough dilated pupils and scored by anophthalmologist (P.S.) in a blinded fashionusing the modified Early Treatment of Dia-betic Retinopathy Study scoring system(20). Three timed overnight urine collec-tions were perf o rmed to determine the uri-n a ry albumin excretion rate (AER) of thediabetic patients (Table 1). Urine albuminwas measured by an immunoturbidimetric(Hitachi, Tokyo) method using an anti-s e rum against human albumin (Orion Diag-nostica, Espoo, Finland). Micro a l b u m i n u r i awas defined as an AER of 20–200 µg/min.I n f o rmed written consent was obtainedafter the purpose, nature, and potential risksw e re explained to the subjects. The experi-mental protocol was designed and per-f o rmed according to the principles of theDeclaration of Helsinki and was appro v e dby the ethical committee of the HelsinkiUniversity Central Hospital.

In vivo endothelial function testIn vivo endothelial function was determ i n e dby measuring fore a rm blood flow re s p o n s e sto intra-arterial infusions of endothelium-dependent and -independent vasodilators.The study was begun after a 10- to 12-h fastat 7:30 A.M. Venous blood samples werewithdrawn for measurement of plasma glu-cose and serum insulin concentrations andfor the other laboratory analyses. Plasmaglucose averaged 9.3 ± 0.6 and 8.7 ± 0.5mmol/l before and at the end of theendothelial function test in the patients withtype 2 diabetes. A 27-G unmounted steelcannula (Coopers Needle Works, Birm i n g-ham, U.K.) connected to an epiduralcatheter (Portex; Hythe, Kent, U.K.) wasi n s e rted into the left brachial art e ry. Dru g sw e re infused at a constant rate of 1 ml/minwith infusion pumps (Braun, Mesungen,G e rmany). Subjects rested supine in a quiete n v i ronment for 30 min after needle place-ment before blood flow measure m e n t s .N o rmal saline was first infused for 18 min.D rugs were then infused in the followingsequence: SNP (Roche, Basel, Switzerland)at 3 (low dose) and 10 (high dose) µg/min;ACh (Iolab, Claremont, CA) at 7.5 (lowdose) and 15 (high dose) µg/min; and coinfusion of NG- m o n o m e t h y l -L- a rginine (L-NMMA) (Clinalfa, Läufelfingen, Switzer-land) at a rate of 4 µmol/min with ACh atrates of 7.5 (low dose) and 15 (high dose)µg/min. Each dose was infused for 6 min,and the infusion of each drug was separatedby infusion of normal saline for 18 min,during which blood flow re t u rned to basal

Table 1—Characteristics of the study gro u p s

Type 2 diabetic patients Normal subjects

n 3 0 1 2Age (years) 51 ± 1 51 ± 1Duration of diabetes (years) 3.5 ± 0.7 —BMI (kg/m2) 27.8 ± 0.4 27.1 ± 0.6Waist-to-hip ratio 0.98 ± 0.01 0.97 ± 0.01Fasting plasma glucose (mmol/l) 9.3 ± 0.6† 5.6 ± 0.1Fasting seru m - f ree insulin (pmol/l) 55 ± 6 42 ± 9H b A1 c ( % ) 7.4 ± 0.3* 5.4 ± 0.1Systolic blood pre s s u re (mmHg) 138 ± 3‡ 127 ± 4Diastolic blood pre s s u re (mmHg) 86 ± 2‡ 81 ± 2Resting heart rate (beats/min) 63 ± 1 60 ± 3M i c ro a l b u m i n u r i a 6 / 3 0 0 / 1 2Evidence for cardiovascular autonomic neuro p a t h y 2 / 3 0 0 / 1 2Sympathetic vasomotor function 257 ± 3 250 ± 4

(% change in fore a rm blood flo w )

Data are means ± SEM or n. Cardiovascular autonomic neuropathy is defined by controlled and deep-bre a t h-ing test, spectral analysis of heart rate variability, Valsalva test, isometric handgrip test, and orthostatic test (19).Sympathetic vasomotor function is measured by re flex blood flow response to cold immersion (30). *P , 0 . 0 0 1 ,†P , 0.01, ‡P , 0.05 for type 2 diabetic patients versus normal subjects.


Mäkimattila and Associates

values. Fore a rm blood flow was re c o rd e dfor 10 s at 15-s intervals during the last 3min of each drug and saline infusion periodwith merc u ry - i n - rubber strain-gaugevenous occlusion plethysmography (EC 4Strain Gauge Plethysmograph; Hokanson,Bellevue, WA) combined with a rapid cuffi n flator (E 20, Hokanson), an analog-to-digital converter (McLab/4e; AD Instru-ments, Castle Hill, Australia), and a personalc o m p u t e r, as previously described (21). Them e a s u rement was perf o rmed simultane-ously in the infused (experimental) and con-t rol arm. The mean of the final fiv em e a s u rements of each re c o rding period wasused for analysis. Reactive hyperemic bloodflow was determined in each subject beforethe endothelial function test as a measure ofv a s o d i l a t o ry capacity (21).

Plasma oxidized LDLIsolation of LDL. LDL was isolated bys h o rt - run density ultracentrifugation.Plasma (up to 5 ml) was adjusted withsolid NaBr to a density of 1.5 g/ml and lay-e red on the bottom of a centrifuge tube.This layer was then successively overlaidwith 2.5 ml each of 1.21 g/ml and 1.063g/ml NaCl solutions and 2.0 ml of distilledw a t e r. All solutions contained 1 mg/ml ofE D TA. Tubes were centrifuged in a Beck-man SW 40-ti rotor (Fullerton, CA) in aBeckman L8-70 centrifuge (Palo Alto, CA)at 40,000 rpm at 4°C for 2.5 h. After cen-trifugation, the LDL fraction was well sep-arated, and EDTA was removed using smalldextran-sulfate affinity columns (Liposor-ber LA-15; Kaneka, Osaka, Japan).Susceptibility of LDL to oxidation inv i t ro. In vitro LDL oxidation was perf o rm e dusing a modification of the pro c e d u redescribed by Esterbauer et al. (22). LDL oxi-dation was initiated by adding freshly pre-p a red CuSO4 ( final concentration of 10.64µmol/l) to the LDL subfraction, the pro t e i nconcentration of which was similar in allsubjects. The formation of conjugateddienes was followed by monitoring thechange in absorbance at 234 nm in a motorized Schimadzu spectrophotometer (UV 1201; Kyoto, Japan) equipped with a 6-cuvette cell connected to a micro c o m-puter at 24°C. Absorbance was re c o rd e de v e ry 1.5 min. The change in absorbance at234 nm over time can be divided into thre econsecutive phases: lag phase, pro p a g a t i o nphase, and decomposition phase (22). Theresistance of LDL to oxidation was judgedf rom the length of the lag time before pro p-agation of the re a c t i o n .

Quantitation of LDL sizeNondenaturing PAGE was perf o rmed ons e rum samples and stored at 28 0°C usinggels casted in our laboratory, as pre v i o u s l ydescribed in detail (23). Gels were stainedwith Sudan Black B lipid stain and scannedwith a computer-assisted laser scanningdensitometer (Personal Densitometer;Molecular Dynamics, Sunnyvale, CA) usinga 50-nm pixel size and 12-bit signal re s o-lution. Particle diameters of the re f e re n c eLDL preparations were determined by elec-t ron microscopy as previously described(23). Coefficients of variation for interg e land intragel precisions for the control sam-ple were 2.0 and 1.2%.

Composition of lipoproteinsubfractions and apolipoproteinconcentrationsIsolation of lipoprotein subfractions.S e rum lipoproteins were isolated as pre v i-ously described (24) by sequential ultra-centrifugation using the following densities(d): VLDL d ,1.006 g/ml, interm e d i a t e -density lipoprotein (IDL) d = 1.006–1.019g/ml, LDL d = 1.019–1.063 g/ml, and HDLd = 1.063–1.210 g/ml.C h o l e s t e rol, triglyceride, phospholipid,f ree cholesterol, and protein concentra-tions in lipoprotein subfractions. C o n-centrations of cholesterol and triglyceridesin serum and lipoprotein subfractions wered e t e rmined by enzymatic colorimetricassays (Hoffman-La Roche, Basel, Switzer-land) in an autoanalyzer (Cobas Mira F;H o ffman-La Roche). Commercial kits werealso used to measure the concentration ofphospholipids (Wako Chemicals, Neuss,G e rmany) and free cholesterol (BoehringerMannheim, Mannheim, Germany) inl i p o p rotein subfractions. Protein concentra-tions in the lipoprotein fractions were meas-u red by the method of Kashyap et al. (25).S e rum apolipoprotein concentrations.Concentrations of serum apolipopro t e i n(apo) AI, AII, and B were determined usingimmunoturbidimetric kits from BoehringerMannheim for apo AI and apo AII and animmunochemical assay from Orion Diag-nostica (Espoo, Finland) for apo B.

Total peroxyl radical-trappingcapacity (TRAP)The combined capacity of all antioxidantsto neutralize free radicals in seru m ,o b s e rved TRAP (TRAPO B S), was determ i n e ds p e c t rophotometrically using a recently val-idated technique (26,27). This methoduses a free radical probe, dichloro flu o re s-

cein-diacetate, the oxidation of which byradicals from the azo-compound 2,29- d i a-zobis (2-amidinopropane) dihydro c h l o r i d egenerates highly flu o rescent dichloro flu o-rescein diacetate. The latter compound alsohas absorbance at 504 nm, enabling spec-t rophotometric quantitation. TRAPO B S

m e a s u red by this method increases withvitamin E supplementation in normal sub-jects and with the addition of variousantioxidant substances to serum samples inv i t ro (27). To estimate to what extento b s e rved changes in circulating antioxi-dants (urate, ascorbate, sulfhydrul gro u p s ,a- t o c o p h e rol) explained changes inT R A PO B S, the contribution of each antioxi-dant to TRAP was calculated by multiply-ing its concentration by its stoichiometricvalue (molar amount of free radicalstrapped by mole of each antioxidant) (cal-culated TRAP [TRAPC A L C]) (27).

Circulating antioxidantsPlasma concentrations of a- t o c o p h e rol, b- c a rotene, and retinol were measured byreverse-phase high-pre s s u re liquid chro-matography (HPLC), as described byS c h ä f e r-Elinder and Walldius (26). Weused a Hewlett-Packard re v e r s e - p h a s eHPLC column (ODS Hypersil 5 µm, 200mm 3 2.1 mm) connected to a Wa t e r sHPLC system consisting of an M600 con-t ro l l e r, M486 tunable UV- a b s o r b a n c ed e t e c t o r, M7171 a u t o s a m p l e r, and a Mille-nium 2010 single-system chro m a t o g r a p h ymanager (Waters, Milford, MA). The UVdetector was set at 326 nm for retinol, 292nm for a- t o c o p h e rol, and 450 nm for b- c a rotene. Lipid-standardized a- t o c o-p h e rol was calculated by dividing the a-t o c o p h e rol concentration by the sum ofs e rum total cholesterol and triglycerideconcentrations. Serum total ascorbic acid(ascorbate and dehydroascorbate) wasm e a s u red using the spectro p h o t o m e t r i cmethod of Denson and Bowers (28). Seru ms u l f h y d ryl (SH) groups were determined asdescribed by Ellman (29). Plasma uric acidconcentrations were measured by an enzy-matic colorimetric assay (Roche Unimate 5UA; Roche, Neuilly-sur-Seine, France).

Autonomic function testsBecause altered autonomic control of bloodvessel tone influences blood flow re s p o n s e sto nitrovasodilators (19), detailed character-ization of autonomic function was per-f o rmed in each subject by measuring re fle xf o re a rm blood flow response to cold immer-sion, which is a test of re flex sympat h e t i c

vasomotor function (30), and by per-f o rming a set of cardiovascular autonomicfunction tests (controlled and deep-b reathing test, spectral analysis of heartrate variability, Valsalva test, isometrichandgrip test, and orthostatic test). Detailsof these pro c e d u res and their re p ro-ducibility in our laboratory have re c e n t l ybeen described in detail (19).

Other measurementsPlasma glucose concentrations weremeasured in duplicate with the glucoseoxidase method, using the Beckman Glu-cose Analyzer II (Fullerton, CA). HbA1c

was measured by HPLC using the fullyautomated Glycosylated HemoglobinAnalyzer System (Bio-Rad, Richmond,CA). Seru m -f ree insulin concentrationsw e re determined by double antibodyradioimmunoassay (Pharmacia InsulinRIA kit; Uppsala, Sweden) after pre c i p i t a-tion with polyethylene glycol (31).

Statistical analysesData between the patients with type 2 dia-betes and the normal subjects were com-p a red using Student’s t test. Simplec o rrelations between selected study vari-ables were calculated using Spearm a n ’srank correlation coefficient, becauseparameters such as the urinary AER, somelipid concentrations (Table 2), and LDLsize were not normally distributed. All cal-

culations were made using the SYSTAT sta-tistical package (Evanston, IL). All data aree x p ressed as means ± SEM.

R E S U LT S

In vivo endothelial functionF i g u re 1 depicts fore a rm blood flow duringthe endothelial function test. Basal bloodflows were slightly, but not signific a n t l y,lower in the patients with type 2 diabetesthan in normal subjects throughout theendothelial function test (Fig. 1). During thelow-dose infusion of ACh, blood flow wass l i g h t l y, but not signific a n t l y, lower in thepatients with type 2 diabetes (5.4 ± 0.5)than in the normal subjects (6.7 ± 1.1 ml ?d l– 1 ? m i n– 1, NS). Blood flow during thehigh-dose ACh infusion was signific a n t l ylower in the patients with type 2 diabetes(6.1 ± 0.5) than in the normal subjects (9.7 ±1.5 ml ? d l– 1 ? m i n– 1, P , 0.01). During thelow-dose SNP infusion, blood flows werecomparable between the groups and aver-aged 6.9 ± 0.5 ml ?d l– 1 ? m i n– 1 in the patientswith type 2 diabetes and 7.3 ± 0.9 ml ? d l– 1 ?m i n– 1 in the normal subjects. Blood flo w sw e re also comparable during the high-doseSNP infusion and averaged 9.1 ± 0.6 and 9.9 ±1.3 ml ? d l– 1 ? m i n– 1, re s p e c t i v e l y.

The ratio of blood flows during infusionof the high dose of ACh to that during infu-sion of the high dose of SNP was signifi-



Table 2—S e rum lipid, lipoprotein, and apolipoprotein concentrations

Type 2 diabetic patients Normal subjects

n 3 0 1 2Triglycerides (mmol/l)

To t a l 2.22 ± 0.36† 1.27 ± 0.18V L D L 1.61 ± 0.28† 0.82 ± 0.17I D L 0.14 ± 0.01* 0.09 ± 0.01L D L 0.23 ± 0.01 0.22 ± 0.01H D L 0.17 ± 0.01 0.14 ± 0.01

C h o l e s t e rol (mmol/l)To t a l 5.38 ± 0.20 5.69 ± 0.23V L D L 0.73 ± 0.13† 0.40 ± 0.08I D L 0.25 ± 0.02 0.18 ± 0.03L D L 3.21 ± 0.15† 3.89 ± 0.20H D L 1.17 ± 0.05 1.23 ± 0.10

A p o l i p o p roteins (mg/dl)A I 136 ± 4 127 ± 6A I I 39 ± 1 38 ± 2B 120 ± 8 112 ± 6LDL particle diameter (Å) 262 ± 2† 268 ± 2

Data are means ± SEM. VLDL, HDL, and total triglycerides are log-transformed for statistical analyses. *P ,0.001, †P , 0.05 for type 2 diabetic patients versus normal subjects.

Figure 1—Means ± SEM blood flows in the experimental and control forearm and the ratio (insert)of blood flows during infusion of a high dose of ACh to that during infusion of a high dose of SNP in nor -mal subjects and in patients with type 2 diabetes. SNP was infused at rates of 3 (18–24 min, low dose)and 10 (24–30 min, high dose) µg/min. ACh was infused at rates of 7.5 (48–54 min, low dose) and 15(54–60 min, high dose) µg/min. L-NMMA (4 µmol/min) was coinfused with ACh at rates of 7.5 (84–90min) and 15 (90–96 min) µg/min. **P 0.01 for patients with type 2 diabetes versus normal subjects.



cantly lower in the patients with type 2 dia-betes (0.70 ± 0.05) than in the normal sub-jects (1.10 ± 0.18; P , 0.01). Duringcoinfusion of L-NMMA with the low dose ofACh, blood flow averaged 4.1 ± 0.3 in thepatients with type 2 diabetes and 4.5 ± 0.8ml ? d l– 1 ? m i n– 1 in the normal subjects(NS). The corresponding blood flows duringcoinfusion of L-NMMA with the high doseof ACh were 4.7 ± 0.5 and 4.9 ± 0.8 ml ? d l– 1 ?m i n– 1, respectively (NS). Thus, when thenitric oxide (NO)-dependent component ofACh-stimulated blood flow was abolished,blood flows were similar between thepatients with type 2 diabetes and the norm a lsubjects. The percent inhibition of ACh-stimulated blood flow by L-NMMA wass i g n i ficantly lower in the patients with type 2diabetes (21 ± 5%) than in the normal sub-jects (45 ± 7%, P , 0 . 0 0 5 ) .

S u l f o n y l u rea preparations may blockthe vasodilation to ACh by inhibiting thee ffects of an endothelium-derived hyperpo-larizing factor of the vascular smooth muscle (18). Although drugs had been dis-continued for 2 days before the endothelialfunction test, and are there f o re unlikely tohave influenced the results, we compare dblood flow responses in type 2 diabeticpatients subdivided according to the modeof antidiabetic therapy. Blood flo wresponses to both ACh/L-NMMA and SNPw e re comparable in patients on sulfonyl-u rea drugs (n = 12) and in those treated bydiet alone (n = 18, data not shown). Therew e re also no significant diff e rences in theblood flow responses to ACh or SNP amongthe six patients with micro a l b u m i n u r i ac o m p a red with those with norm o a l b u m i n-

uria (data not shown). Reactive hypere m i cblood flow was comparable in the patientswith type 2 diabetes (36 ± 2) and in the nor-mal subjects (42 ± 3 ml ? d l– 1 ? m i n– 1, NS).

Serum lipids, lipoproteins, andapolipoproteinsS e rum triglycerides were 74% higher in thepatients with type 2 diabetes than in then o rmal subjects. This increase was due to as i g n i ficant increase in the triglyceride con-tent of VLDL and IDL particles. Mean LDLp a rticle size was significantly smaller in thepatients with type 2 diabetes than in then o rmal subjects, and it was inversely corre-lated with serum triglyceride concentrationsin both the normal subjects (r = 20.61, P ,0.05) and in the patients with type 2 dia-betes (r = 20.75, P , 0.001). Total choles-t e rol concentrations were comparablebetween the two groups. This apparent nor-mality was explained by a signific a n t l ylower cholesterol concentration in the LDLsubfraction and a higher cholesterol con-centration in the VLDL subfraction in thepatients with type 2 diabetes than in then o rmal subjects (Table 2). The lag times forLDL oxidation in vitro were similar for bothg roups (183 ± 7 vs. 183 ± 9 min, type 2 dia-betic versus normal subjects).

The phospholipid (9.7 ± 0.6 mg%)and free cholesterol (0.10 ± 0.01 mmol/l)content of IDL particles was increased inthe patients with type 2 diabetes compare dwith the normal subjects (6.8 ± 0.9 mg%,P , 0.005 and 0.07 ± 0.01 mmol/l, P ,0.05, respectively). In the LDL particles, thephospholipid content was lower in thepatients with type 2 diabetes (81.6 ± 3.7)

than in the normal subjects (99.6 ± 4.4mg%, P , 0.005). The phospholipid andf ree cholesterol content of other lipopro t e i np a rticles was comparable between theg roups (data not shown). Serum concen-trations of apo AI, AII, and B were compa-rable between the two gro u p s .

Circulating antioxidants and TRAPBoth TRAPO B S and TRAPC A L C w e re compa-rable between the patients with type 2 diabetes and the normal subjects. Regard-ing the individual antioxidants, plasma b- c a rotene concentrations were signifi-cantly (P , 0.01) lower and serum SHg roups significantly higher (P , 0.01) inthe patients with type 2 diabetes than in then o rmal subjects. Plasma concentrations ofa- t o c o p h e rol, retinol, ascorbic acid, anduric acid were comparable between thestudy groups (Fig. 2).

Interrelationships betweencardiovascular risk markers andendothelial functionIn re g ression analysis using Spearm a n ’snonparametric correlation coefficient, LDLsize, but not parameters such as seru mtriglycerides, LDL or HDL cholesterol con-centrations, the lag time for LDL oxidation,phospholipid or free cholesterol content ofl i p o p rotein fractions, AER, blood pre s s u re ,indexes of glycemia (plasma glucose,H b A1 c), or BMI, was significantly corre l a t e dwith endothelium-dependent vasodilationin the patients with type 2 diabetes (Fig. 3).LDL size was correlated with the lag time forLDL oxidation in vitro and inversely withs e rum triglycerides (Fig. 3). Glycemic con-t rol, as measured with HbA1 c, was inverselyc o rrelated with the lag time for LDL oxida-tion in vitro and with TRAPO B S (Fig. 3).

C O N C L U S I O N S — We found im-p a i red endothelium-dependent vasoactiveresponses in fore a rm resistance vessels ofpatients with type 2 diabetes with a re l a-tively short duration of disease with noclinical signs of macrovascular complica-tions. Patients with type 2 diabetes alsoexhibited hypertriglyceridemia, decre a s e dLDL size, subnormal LDL cholesterol con-centrations, and normal circulating antiox-idant capacity. Of these parameters, LDLsize was significantly correlated withendothelial function.

To maximize the likelihood of detectingan association between a cardiovascular riskfactor and endothelial function, we tried toavoid confounding factors such as those

Figure 2—T R A PO B S, TRAPC A L C, and circulating antioxidants of the study groups. **P , 0.01 for type 2diabetic patients versus normal subjects.



arising from endogenous or exogenouse s t rogen (32), conduit vessel length (33),m a c roalbuminuria (19), and insulin therapy(34) when selecting the study subjects.C o m p a red with previous studies, thepatients re c ruited for the present study hada shorter duration of type 2 diabetes andw e re extensively characterized for the pre s-ence of complications, especially micro v a s-cular complications. This was considere di m p o rtant because both autonomic neu-ropathy (19) per se and nephropathy (35)have been associated with altered endothe-lial function in patients with type 2 diabetes.Lack of quantitation of these parametersmay have confounded results in pre v i o u sstudies addressing endothelial function inpatients with type 2 diabetes (7–13). Ourpatients had a marginally elevated AER thatwas not correlated with endothelial func-tion. Results of the autonomic function testsrevealed slight alterations in measures ofparasympathetic function in 2 of 30 patients( Table 1), but these changes were trivial inmagnitude compared with changes associ-ated with altered responses to nitro v a-sodilators (19). Also, we chose to studytype 2 diabetic patients who were tre a t e dwith diet alone or who were off oral agentsand who were carefully matched to norm a lsubjects with respect to body weight. Thiscontrasts the patient selection of Ting et al.(9) and Williams et al. (11), who includedi n s u l i n - t reated type 2 diabetic patients intheir studies, and the recent studies of Wa t t set al. (10) and O’Brien et al. (12), in whichtype 2 diabetic patients with a BMI of 29.9w e re compared with normal subjects with aBMI of 25.8 kg/m2.

In the present study, reactive hyper-emic blood flow responses were similar

between the study groups, suggesting thats t ructural changes in fore a rm re s i s t a n c evessels did not attenuate vasoactiveresponses. Va s o d i l a t o ry responses to theextrinsic NO donor SNP were also compa-rable between the patients with type 2 dia-betes and the normal subjects, suggestingp re s e rved function of vascular smoothmuscle. Blood flow responses to SNP org l y c e ryl trinitrate have been impaired insome (7,10–12), but not in all (8,13), pre-vious studies in patients with type 2 dia-betes. The reason for the discrepant re s u l t sis unclear but could be explained by diff e r-ences in patient characteristics, such asduration of diabetes and degree of obesityand hypertension. Diabetic autonomic neu-ropathy is also known to influence vesseltone and response to nitrovasodilators (19).In the present study, this was considered inthe patient characterization. Responses tocold immersion, which is a test of sympa-thetic vasomotor function, were similarbetween the study groups, suggesting thatthe results of endothelial function testinga re not explained by altered sympatheticvascular re g u l a t i o n .

Our results confirm the presence of as i g n i ficant defect in fore a rm blood flo wresponses to ACh (7,10,12) and metha-choline (9,11). Only ,40% of the ACh-induced increase in blood flow can beabolished by inhibiting NO synthesis withL-NMMA (36). The other componentappears to be due to release of othervasodilators, such as prostacyclin (37), andthe endothelium-derived hyperpolarizingfactor (38). To establish whether the defectin ACh-induced vasodilation in type 2 dia-betes was caused by the NO-dependentcomponent of ACh-stimulated blood flo w,

we coinfused L-NMMA with ACh. In keep-ing with a previous re p o rt (10), blood flo w sw e re similar between the type 2 diabeticpatients and the normal subjects duringcoinfusion of these drugs. These data implythat endothelial dysfunction in our patientswas due to a vascular defect that was spe-c i fic to the NO pathway, in keeping with thedata of Watts et al. (10), who found a defectin NO synthesis in obese type 2 diabeticpatients compared with norm a l - w e i g h tnondiabetic individuals. These data,together with the normal response of vas-cular smooth muscle to SNP in the pre s e n tand in previous (8,13) studies and lack ofan increased production of vasoconstrictorp rostanoids in type 2 diabetes (11), implythat neither accelerated NO destruction nori m p a i red NO action explains in vivoendothelial dysfunction in type 2 diabetes.Endothelial dysfunction in type 2 diabetes ist h e re f o re most likely a consequence of adefect in NO production. The present datado not allow distinction between the causesof the latter defect, which could involvesignaling defects between muscarinic re c e p-tors and stimulation of NO synthase, lack ofNO synthase or its substrates, or acceleratedd e s t ruction of NO in endothelial cells.

As in all previous studies in patientswith type 2 diabetes (7–12) and in contrastto data on endothelial function in nondia-betic individuals (2,4,39), serum LDL cho-l e s t e rol did not correlate with endothelialfunction. Indeed, in the present study,s e rum LDL cholesterol concentrations wereeven subnormal in type 2 diabetes, in keep-ing with some (17,40,41) but not all (42)p revious studies. Considering the impor-tance of oxidized LDL in the pathogenesisof athero s c l e rosis, at least in nondiabeticindividuals, one might have expected oxi-dized LDL rather than LDL size to corre l a t ewith endothelial function. Several factorscould contribute to the lack of such anassociation. First, multiple methods existfor measurement of various stages of LDLoxidation (43), but it is currently unknownwhich methods, if any, re flect alterations invascular function. Second, in type 2 dia-betes, oxidized LDL may also be an inferiormarker of atherogenic LDL, since part ofLDL may be modified by glucose, glucoseoxidation products, and advanced glyca-tion end product peptides (44). Althoughin vivo glycation of LDL increases its oxid-a b i l i t y, as also suggested by the corre l a t i o nbetween HbA1 c and the susceptibility ofLDL to oxidation in vitro in the pre s e n ts t u d y, glycated lipoproteins, both LDL and

Figure 3—Simple correlations between endothelium-dependent vasodilation (ratio of endothelium-dependent [ACh 15 µg/min] to -independent [SNP 10 µg/min] forearm blood flow), HbA1c, serum totaltriglycerides (S-Tg), TRAP, lag time of LDL to oxidation, and LDL size in patients with type 2 diabetes.

others, may also impair endothelial func-tion by mechanisms that are independentof LDL oxidability (44).

Because LDL size and serum triglyc-erides, and triglycerides and HDL choles-t e rol (data not shown), were closelyc o rrelated, it would be somewhat art i fic i a lto try to distinguish, using, e.g., multiplelinear re g ression analysis, which of thet h re e —1) serum, total, or VLDL triglyc-erides, 2) HDL cholesterol, or 3) LDLsize—would be most closely corre l a t e dwith endothelial function. Regardless, LDLsize was the only one that significantly cor-related with endothelial function. Themechanisms responsible for the athero-genicity of small dense LDL particles arepoorly understood, but it has been sug-gested that small LDL particles filtrate morereadily into the arterial wall than large ones,and these particles also appear to be morep rone to modification once they enter thea rterial wall (45).

In a previous study perf o rmed inpatients with type 1 diabetes (6), hyperg l y-cemia was the most important independentd e t e rminant of impaired endothelium-dependent vasodilation. However, in thep resent study in patients with type 2 dia-betes, neither fasting plasma glucose norH b A1 c was significantly associated withendothelium-dependent vasodilation. Thisfinding is consistent with previous studiesin patients with type 2 diabetes (7,10,11).H b A1 c might still be an important determ i-nant of endothelial function in patientswith type 2 diabetes, but the coexistence ofseveral pathophysiological and metabolica b n o rmalities in type 2 diabetes may maskits independent impact.

The TRAP of plasma can be eitherm e a s u red directly (TRAPO B S) or calculatedby multiplying the concentrations of indi-vidual antioxidants by their stoichiometricvalue (TRAPC A L C) (27). The diff e re n c ebetween TRAPO B S and TRAPC A L C re fle c t seither the contribution of hitherto uniden-t i fied plasma antioxidants or synerg i s mbetween known antioxidants (27). BothT R A PC A L C and TRAPO B S w e re comparablebetween the patients with type 2 diabetesand the normal subjects, implying thatdepletion of circulating antioxidants doesnot explain endothelial dysfunction inpatients with type 2 diabetes. On the otherhand, lack of a decrease in TRAP does notexclude the possibility that antioxidantsmay regulate endothelial function in type 2diabetes. For example, large doses of vita-min C have been shown to acutely re s t o re

endothelial function in these patients (9).R e g a rding the individual antioxidants, wefound unaltered levels of a- t o c o p h e ro l ,ascorbic acid, uric acid, and re t i n o l ,w h e reas SH groups were slightly incre a s e dand b- c a rotene decreased in the patientswith type 2 diabetes compared with then o rmal subjects. Serum bilirubin was notm e a s u red in the present study, since biliru-bin concentrations are unlikely to bea b n o rmal (over 20 µmol/l) in patients withtype 2 diabetes with normal transaminasesand because its contribution to TRAP isnegligible in such individuals (2–20 31.964 µmol/l, i.e., the stoichiometric valuefor bilirubin in our assay [27], which is,0.4–4% of a TRAP of 1,000 µmol/l). Pre-vious data on individual antioxidants intype 2 diabetes are highly variable, since a- t o c o p h e rol levels have been re p o rted tobe unchanged in the U.K. (46), decre a s e din India (47), and increased in Italy (48).Ascorbic acid levels have been found to bed e c reased in India (47) and in one study inCaucasian type 2 diabetic patients (49) andunchanged in one study (48) and incre a s e din another (50). TRAPO B S, TRAPC A L C, andSH groups have been measured only oncep reviously in type 2 diabetes, and theyw e re decreased (48). b- c a rotene was foundto be unaltered in type 2 diabetes in onep revious study (51). Retinol concentrationshave been either unchanged (47) ori n c reased (52) in patients with type 2 dia-betes. Uric acid concentrations in individ-uals with normal renal function ared e t e rmined by the combined effects ofh y p e rglycemia (53) and hyperinsulinemia(54), which increases plasma uric acid con-centration. The apparent normality of uricacid concentrations could re flect the nete ffects of all these factors in our patientswith type 2 diabetes. Taken together, thesedata do not provide convincing evidencefor altered circulating antioxidant concen-trations in type 2 diabetes. The inverse cor-relation between HbA1 c and TRAP (Fig. 3)is, however, consistent with recent re p o rt s(55,56) and with the idea that chro n i ch y p e rglycemia increases oxidative stress viamechanisms such as glucose auto-oxida-tion or nonenzymatic glycosylation (57).

In conclusion, patients with type 2 dia-betes exhibit a defect in endothelium-depen-dent vasodilation that cannot be explainedby traditional cardiovascular risk factors,such as hypertension or LDL cholestero lconcentrations, and that also does not seemto directly relate to chronic hyperg l y c e m i a ,as it does in patients with type 1 diabetes. In

this study, we searched for new determ i n a n t sof endothelial dysfunction in these patientsand found LDL size but not the lag time forLDL oxidation or levels of antioxidants inplasma to be significantly related to endothe-lium-dependent vasodilation.

A c k n o w l e d g m e n t s — This study was sup-p o rted by grants from the Duodecim (S.M.), theLehikoinen (S.M.), the Academy of Finland( H . Y.-J.), the Sigrid Juselius (H.Y.-J.), and theAhokas (H.Y.-J.) foundations.

We thank Hannele Hilden and Kati Tu o-mola for excellent technical assistance, SoileA a rnio for drawing the fig u res, and the volun-teers for help.

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