Autoantibodies against oxidized LDL and endothelium-dependent vasodilation in insulin-dependent diabetes mellitus

Atherosclerosis 147 (1999) 115 – 122 www.elsevier.com/locate/atherosclerosis

Autoantibodies against oxidized LDL and endothelium-dependent vasodilation in insulin-dependent diabetes mellitus Sari Ma¨kimattila a, Jukka S. Luoma b, Seppo Yla¨-Herttuala b, Robert Bergholm a, Tapio Utriainen a, Antti Virkama¨ki a, Matti Ma¨ntysaari c, Paula Summanen d, Hannele Yki-Ja¨rvinen a,* a

Di6ision of Endocrinology and Diabetology, Department of Medicine, Helsinki Uni6ersity Central Hospital, Helsinki, Finland b A.I. Virtanen Institute and Department of Medicine, Uni6ersity of Kuopio, Kuopio, Finland c Research Institute of Military Medicine, Central Military Hospital, Helsinki, Finland d Di6ision of Ophthalmology, Department of Medicine, Helsinki Uni6ersity Central Hospital, Helsinki, Finland Received 13 October 1998; received in revised form 5 March 1999; accepted 21 April 1999

Abstract We determined whether autoantibodies against oxidized LDL are increased in patients with IDDM, and if so, whether they are associated with endothelial dysfunction in vivo. Autoantibodies against oxidized LDL (ratio of antibodies against oxidized vs. native LDL, oxLDLab) were determined in 38 patients with IDDM (HbA1c 8.490.2%), who were clinically free of macrovascular disease, and 33 healthy normolipidemic subjects (HbA1c 5.190.1%, PB 0.001 vs. IDDM). The groups had comparable serum total-, LDL- (2.9 9 0.1 vs. 2.89 0.1 mmol/l, IDDM vs. controls), and HDL-cholesterol concentrations. OxLDLab were 1.5-fold higher in the IDDM patients (1.890.1) than in the normal subjects (1.29 0.1, P B0.001). OxLDLab were correlated with age in normal subjects, but not with age, duration of disease, LDL-cholesterol, HbA1c or degree of microvascular complications in patients with IDDM. To determine whether oxLDLab are associated with endothelial dysfunction in vivo, blood flow responses to intrabrachial infusions of acetylcholine, sodium nitroprusside and L-NMMA were determined in 23 of the patients with IDDM (age 3391 years, body mass index 24.3 90.6 kg/m2, HbA1c 8.590.3%) and in the 33 matched normal males. OxLDLab were 41% increased in IDDM (1.7 90.2 vs. 1.2 90.1, PB 0.01). Within the group of IDDM patients, HbA1c but not oxLDLab or LDL-cholesterol, was inversely correlated with the forearm blood flow response to acetylcholine (r= − 0.51, PB 0.02), an endothelium-dependent vasodilator, but not to sodium nitroprusside (r= 0.06, NS). These data demonstrate that oxLDLab concentrations are increased in patients with IDDM, but show that chronic hyperglycemia rather than oxLDLab, is associated with impaired endothelium-dependent vasodilation in these patients. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Nitric oxide; Atherosclerosis; Glucose; Blood vessels

1. Introduction Impaired endothelium-dependent vasodilation characterizes individuals who are at risk of developing atherosclerosis [1]. In hypercholesterolemic patients, the in vivo vasodilatory response to endothelium-dependent vasodilators, such as acetylcholine, is inversely correlated with total- and LDL-cholesterol concentrations in * Corresponding author. Present address: Academy of Finland, University of Helsinki, Department of Medicine, Division of Endocrinology and Diabetology, Haartmaninkatu 4, FIN-00290 Helsinki, Finland. Tel.: +358-9-4712350; fax: +358-9-4712250. E-mail address: [email protected] (H. Yki-Ja¨rvinen)

both forearm resistance [1–4], and the coronary vessels [5]. Endothelial function was also recently shown to be inversely related to the concentration of plasma oxidized LDL antibodies in hypercholesterolemic patients [6]. The latter are thought to reflect the amount of extensively oxidized LDL (oxLDL) that is present in the circulation [7–10]. Patients with IDDM have an increased risk of atherosclerotic vascular disease, which is inadequately explained by classic risk factors such as increases in the concentrations of total- or LDL-cholesterol, hypertension or smoking [11]. Consistent with the predisposition to atherosclerosis, endothelium-dependent vasodilation

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has been impaired in some [12 – 14], although not in all [15 – 17] studies in patients with IDDM. In contrast to the studies performed in non-diabetic individuals, however, the degree of endothelial dysfunction does not correlate with either total- or LDL-cholesterol concentrations [13]. Multiple mechanisms link chronic hyperglycemia to endothelium-dependent vascular dysfunction. Hyperglycemia increases auto-oxidation of glucose [18–21], formation of advanced glycosylation end (AGE)-products [18,21] and vasoconstrictor prostanoids [22,23], all of which generate free radicals, especially superoxide. Superoxide may then quench nitric oxide (NO) directly [24,25] or it may oxidate LDL [26,27]. These data imply that hyperglycemia may impair endothelial function independently of oxidized LDL. There are currently no studies in which in vivo endothelial function would have been related to circulating levels of modified LDL or its markers in patients with IDDM. The present study was undertaken to determine whether the concentration of oxidized LDL antibodies is increased in patients with IDDM, and to determine, in a subgroup of IDDM patients, whether the titer of oxidized LDL antibodies is correlated with endothelial dysfunction as in non-diabetic patients [6], or whether other parameters, such as the glycosylated hemoglobin concentration, are more closely related to vascular function.

2. Methods

2.1. Subjects and study design 2.1.1. Comparison of oxLDLab le6els between IDDM patients and normal subjects OxLDLab concentrations were determined in 38 men with IDDM and 33 normal men. The patients with IDDM were recruited from the outpatient clinic on the basis of the following criteria: (1) age 18–60 years, (2) age at diagnosis of diabetes B30 years, and (3) undetectable fasting C-peptide concentration (B 0.1 nmol/l). The subjects underwent history and physical examination, and laboratory tests were performed to exclude diseases other than IDDM. The screening examination also included careful assessment of the presence of autonomic neuropathy [28], retinopathy [29] and renal function (vide infra). Patient characteristics are shown in Table 1. All patients and normal subjects had normal blood counts, electrolyte concentrations, liver enzymes and thyroid function tests. None of the IDDM patients had signs or symptoms of ischemic heart disease, and had normal electrocardiograms according to Minnesota criteria [30]. The diabetic patients were treated with two (n= 3), three (n=5) or four (n=26) injections of a combination of intermediate- and short-acting insulins. A total of four patients were using continuous subcutaneous insulin infusion therapy. None of the patients with IDDM or normal subjects was taking antioxidants

Table 1 Characteristics of the study groupsa IDDM patients, all patients (n= 38) Age (years) Body mass index (kg/m2) % Body fat HbA1c (%) Serum triglycerides (mmol/l) Serum cholesterol (mmol/l) LDL-cholesterol (mmol/l) HDL-cholesterol (mmol/l) Mean arterial pressure (mmHg) Urinary albumin excretion (mg/min) ETDRS scoreb Valsalva ratio E/I ratio HFc LFd

389 1*** 24.6 9 0.5 189 1 8.4 90.2*** 1.19 0.1* 4.79 0.1 2.99 0.1 1.39 0.1 96 9 1** 3599 140*** (median 70) 469 3 1.59 0.1*** 1.29 0.03** 52 9 23***,+ 88923***,++

IDDM patients, endothelial (n =23) 33 91 24.3 90.6 18 91 8.5 9 0.3*** 1.190.1 4.790.2 2.8 90.1 1.3 9 0.1 929 1 150 9 71*** (median 27) 40 94 1.6 90.1* 1.3 90.05 83 9 44* 146 953***

Data are expressed as mean 9S.E.M. HbA1c reference range 4–6%. ETDRS score for grading of severity of diabetic retinopathy (score 10 = normal) [29]. c HF, high frequency. d LF, low frequency component of heart rate variability in spectral analysis performed in head-up position [28]. * PB0.05, ** PB0.01, *** PB0.001 for IDDM patients versus normal subjects. + PB0.05, ++ PB0.01 for all IDDM patients versus IDDM patients in endothelial test.

a

b

Normal subjects (n = 33) 30 9 1 23.69 0.5 16 91 5.19 0.1 0.99 0.1 4.49 0.2 2.890.2 1.3 90.1 899 2 7 9 1 (median 8) – 1.990.1 1.39 0.04 153951 308 954

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or drugs known to affect glucose or lipid metabolism, or drugs known to affect sympathetic or parasympathetic activity. Informed written consent was obtained after the purpose, nature and potential risks were explained to the subjects. The experimental protocol was designed and performed according to the principles of the Declaration of Helsinki, and was approved by the Ethical Committee of the Helsinki University Central Hospital.

2.1.2. OxLDLab and HbA1c as determinants of endothelial function in patients with IDDM In vivo endothelial function was determined in 23 of the diabetic patients and in all 33 normal subjects by measuring the effects of intra-arterial infusions of endothelium-dependent and -independent vasodilators on forearm blood flow. The 23 IDDM patients were selected from the 38 screened patients by excluding those with the most severe signs of autonomic neuropathy or macroalbuminuria as these complications may alter lipids, lipoproteins [31] and vascular function [13] independent of glycemia (Table 1). 2.2. Measurements 2.2.1. Autoantibodies against oxidized and nati6e LDL Blood samples were obtained after an overnight 12-h fast. Plasma was separated and aliquots were stored at − 20°C. Autoantibodies against oxidized and native LDL were measured according to a modification of a published method [32]. For each set of samples, three identical 96-well microtitre plates (NUNC MaxiSorp, Nunc A/S, Roskilde, Denmark) were used: one plate was coated with native LDL, a second plate with LDL oxidized with copper for 24 h. Plates were coated with 50 ml antigen (5 mg/ml) per well in phosphate buffered saline (PBS) overnight at 4°C. To prevent oxidation of native LDL, 0.27 mmol/l EDTA and 20 mmol/l butylated hydroxytoluene (BHT) were added to PBS. Each well was washed three times with PBS containing 0.05% Tween 20 and once with water. Plates were blocked with 2% bovine serum albumin (BSA; Sigma, St. Louis, MO) in PBS containing 0.27 mmol/l EDTA and 20 mmol/l BHT for 2 h at 4°C. Samples (50 ml per well) were pipetted on the plates at 1:50 dilution; plates were incubated overnight and washed as above. Horseradish peroxidase-conjugated anti-human IgG (Cappel, Organon Teknika, Durham, NC) diluted 1:5000 was added to the wells in PBS containing 1% BSA, 0.05% Tween 20, 0.27 mmol/l EDTA, and 20 mmol/l BHT, and the plates were incubated at 4°C for 4 h. Wells were then washed as above and incubated with Ophenylene-diamine (Fluka Chemie, Neu-Ulm, Germany) for 5 min. Absorbances were measured at 492 nm with a microplate reader (Multiscan RC, Labsystems Oy, Helsinki, Finland). All measurements were

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done in duplicate without knowledge of the forearm blood flow measurements (J.L. and S.Y.-H.). Results were expressed as a ratio of epitopes characteristic of oxidized LDL to native LDL. In the present article ‘oxLDLab’ refers to this ratio.

2.2.2. Forearm blood flow responses to endothelium-independent (SNP) and -dependent (ACh and L -NMMA) 6asoacti6e agents The study was begun at 07:30 h after a 10–12-h fast. To avoid possible confounding effects of acute hyperglycemia on endothelial function [33], an intravenous infusion of insulin (0.1 mU/kg per min) was started at 08:00 h to normalize the plasma glucose concentration. Insulin was infused and blood samples were drawn through a 18-G (Venflon, Viggo-Spectramed, Helsingborg, Sweden) catheter inserted into the right antecubital vein. Normoglycemia was reached after 175917 min. Glucose was infused if necessary to maintain normoglycemia on the basis of plasma glucose measurements performed at 20-min intervals. The plasma glucose concentration averaged 5.990.3 mmol/l in the IDDM patients, and 5.290.1 mmol/l in the normal subjects (NS), immediately before the endothelial function test. Serum free insulin concentrations averaged 4196 and 419 4 pmol/l, respectively (NS). A 27-G unmounted steel cannula (Coopers Needle Works, Birmingham, UK), connected to an epidural catheter (Portex, Hythe, Kent, UK), was inserted into the left brachial artery. Drugs were infused at a constant rate of 1 ml/min with infusion pumps (Braun AG, Mesungen, Germany and Harvard Apparatus model 22, South Natic, MA). Subjects rested supine in a quiet environment for 30 min after needle placement before any blood flow measurements. Normal saline was first infused for 12 min. Drugs were then infused in the following sequence: SNP (Roche, Basel, Switzerland), 3 and 10 mg/min, ACh (Iolab Corporation, Claremont, CA) 7.5 and 15 mg/min, and L-NMMA (Clinalfa AG, Switzerland) at a rate of 4 mmol/min. Each dose was infused for 6 min, and the infusion of each drug was separated by infusion of normal saline for 18 min, during which blood flow returned to basal values. Forearm blood flow was recorded for 10 s at 15-s intervals during the last 3 min of each drug and saline infusion period. The measurement was performed simultaneously in the infused (experimental) and control arm. Forearm blood flow was recorded with mercury-in-rubber strain-gauge venous occlusion plethysmography (EC 4 Strain Gauge Plethysmograph, Hokanson, Bellevue, WA) combined with a rapid cuff inflator (E 20, Hokanson), an analog-to-digital converter (McLab/ 4e, AD Instruments, Castle Hill, Australia) and a personal computer, as previously described [28]. The mean of the final five measurements of each recording period was used for analysis.

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Fig. 1. Individual concentrations of circulating autoantibodies against native LDL, oxidized LDL and the ratio of autoantibodies against oxidized versus native LDL (oxLDLab) in normal subjects (CONT, n= 33) and patients with IDDM (n =38).

2.3. Other measurements

2.4. Statistical analyses

A total of three timed overnight urine collections were performed to determine the urinary albumin excretion rate (UAER) of the diabetic patients (Table 1). Urine albumin was measured by immunoturbidimetric (Hitachi, Tokyo, Japan) method with an antiserum against human albumin (Orion Diagnostica, Espoo, Finland). Plasma glucose concentrations were measured in duplicate with the glucose oxidase method, using the Beckman Glucose Analyzer II (Beckman Instruments, Fullerton, CA). HbA1c was measured by high-pressure liquid chromatography using the fully automated Glycosylated Hemoglobin Analyzer System (BioRad, Richmond, CA). Serum free insulin was determined by double antibody radioimmunoassay (Pharmacia Insulin RIA kit; Pharmacia, Uppsala, Sweden) after precipitation with polyethylene glycol [34]. The serum concentrations of cholesterol, triglycerides and high-density lipoprotein (HDL) cholesterol were determined by enzymatic colorimetric assays with an automated analyzer (Cobas Mira; Hoffmann La Roche; Diagnostica, Basel, Switzerland). The concentration of low-density lipoprotein (LDL) cholesterol was calculated using the formula of Friedewald. Blood pressure was measured using a mercury sphygmomanometer. Pulse pressure (PP) was calculated from the difference between systolic and diastolic blood pressures, and mean arterial pressure (MAP) by adding 1/3 of the pulse pressure to the diastolic blood pressure. Fat free mass and the percentage of body fat were determined using a single frequency bioelectrical impedance device (Bio-Electrical Impedance Analyzer System, Model cBIA-101A, RJL Systems, Detroit, MI).

Data between patients with IDDM and normal subjects were compared using Student’s t-test. Data between the three study groups (see Section 3) were analyzed using analysis of variance followed by pairwise comparison using Fisher’s least-significant-difference test. Simple correlations between selected study variables were calculated using Spearman’s rank correlation coefficient. Multiple linear regression analysis within patients with IDDM was used to analyze the causes of variation in parameters of endothelial function, glycemic control and oxLDLab. All calculations were made using the SYSTAT statistical package (SYSTAT, Evanston, IL). All data are expressed as means9 S.E.M.

3. Results

3.1. Plasma oxLDLab concentrations in patients with IDDM and in normal subjects The plasma titer of autoantibodies against native LDL were comparable in patients with IDDM and in normal subjects (0.0990.01 vs. 0.089 0.01, NS, Fig. 1), while the antibodies against oxidatively modified LDL were significantly higher in patients with IDDM (0.1390.01) than in the normal subjects (0.0990.01, PB 0.001, Fig. 1). OxLDLab (ratio of oxidized to native LDL) were 1.5-fold higher in patients with IDDM (1.89 0.1, range 0.6–4.6) than in the normal subjects (1.290.1, range 0.6–2.1, PB 0.001) (Fig. 1). This increase could not be attributed to differences in the lipid or lipoprotein pattern or body mass index

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Table 2 Simple correlations between plasma oxLDLab and selected study variables r IDDM patients (n = 38) Age (years) Body weight (kg) Body mass index (kg/m2) Waist to hip ratio Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Mean arterial pressure (mmHg) Serum triglycerides (mmol/l) Serum cholesterol (mmol/l) LDL-cholesterol (mmol/l) HDL-cholesterol (mmol/l) HbA1c (%) Serum creatinine (mmol/l) Urinary albumin excretion (mg/min) Valsalva ratioa ETDRS scoreb

Normal subjects (n = 33)

0.05 −0.13 −0.12 −0.02 0.29

0.34* 0.35* 0.31 0.37* −0.20

0.16

−0.27

0.21

−0.28

0.08 −0.12 −0.08 −0.22 0.07 −0.10 0.16

0.16 0.16 0.15 −0.19 −0.22 −0.06 –

−0.24 0.18

−0.26 –

a

Other tests of autonomic function were not significantly associated with levels of plasma oxLDLab concentrations. b ETDRS score for grading of severity of diabetic retinopathy (score 10 = normal). * PB0.05.

Fig. 2. Mean 9S.E.M. forearm blood flows in the experimental ( – ) and control (- -) forearm in normal subjects (CONT, n = 33), and IDDM patients in good (GOOD, n= 11) and poor (POOR, n = 12) glycemic control. SNP was infused at rates of 3 (12–18 min) and 10 mg/min (18 – 24 min), ACh at rates of 7.5 (42–48 min) and 15 mg/min (48–54 min), and L-NMMA at rate of 4 mmol/min for 72–78 min. ** P B0.01 for IDDM patients in POOR versus CONT. + P B0.05 for POOR versus GOOD.

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between the two groups (Table 1). In analysis of covariance using plasma oxLDLab as the dependent variable, the difference between groups remained significant (P=0.008), also after adjusting for age (P= 0.63, NS) and mean arterial pressure (P=0.67, NS). To determine the possible causes of the higher plasma oxLDLab concentrations in the patients with IDDM, we calculated simple correlation coefficients between various clinical and biochemical features and plasma oxLDLab levels. In normal subjects, plasma oxLDLab was significantly correlated with age, body weight and waist to hip ratio but not with parameters such as blood pressure, serum total- or LDL-cholesterol or serum triglyceride concentrations (Table 2). In patients with IDDM, no significant correlations were observed between plasma oxLDLab concentrations and any of the clinical or biochemical parameters (Table 2).

3.2. Endothelial function in patients with IDDM and normal subjects When the entire group of diabetic patients was compared with the normal subjects, no significant differences were observed between blood flow basally or during the ACh, SNP, and L-NMMA infusions (data not shown). Forearm blood flow during the vasoactive drug and saline infusions in the control arm was stable and comparable between the groups (data not shown). In simple linear regression analysis within the group of IDDM patients, HbA1c, but not the concentration of plasma oxLDLab, serum total-, LDL-, or HDL-cholesterol or triglycerides, was inversely correlated with forearm blood flow during the submaximal dose of ACh (r= − 0.51, PB 0.02). Neither HbA1c (r=0.06 and r= 0.14) nor oxLDLab (r= − 0.22 and r= − 0.15, NS for submaximal and maximal doses) was significantly correlated with blood flow during infusion of SNP. HbA1c was also inversely correlated (r= − 0.46, PB 0.02) with the ratio of blood flow during the submaximal ACh dose (7.5 mg/min) divided by blood flow during the submaximal SNP dose (3 mg/min). The independence of the association between HbA1c and endothelium-dependent vasodilation was confirmed in multiple linear regression analysis (PB0.01 for HbA1c, NS for oxLDLab). HbA1c tended to be correlated with serum triglycerides (r=0.36, P= 0.097) but serum triglycerides were not related to any measure of vascular function (data not shown). To illustrate the impact of glycemia on endothelial function, Fig. 2 depicts forearm blood flow during the entire endothelial function test in patients with IDDM subdivided into those with an HbA1c below (7.59 0.2%, n= 11) and above (9.49 0.3%, n= 12) the median HbA1c, and in normal subjects. The ratio of blood flow during infusion of the submaximal ACh dose (7.5 mg/min) to that during infusion of the submaximal SNP dose (3 mg/min) was

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significantly lower in the IDDM patients in poor control (0.79 0.1) than in those with good glycemic control (1.19 0.1; PB0.01) or in normal subjects (1.0 90.04; PB 0.005). During infusion of L-NMMA, blood flow decreased by 219 4 and 25 9 6% in the IDDM patients with poor and good glycemic control (NS), respectively, and by 2892% in the normal subjects (NS vs. both IDDM groups).

4. Discussion In the present study we found that despite comparable and on average normal concentrations of LDLcholesterol between relatively young IDDM patients and normal subjects, the ratio of autoantibodies against oxidized versus native LDL was 50% increased in IDDM. In a subgroup of IDDM patients with no clinically significant macrovascular complications and with no severe microvascular complications, we determined whether oxLDLab is associated with defects in endothelium-dependent vasodilation. We found HbA1c, but not oxLDLab, to be inversely related with endothelium-dependent but not -independent vasodilation in forearm resistance vessels. Advances in understanding of the atherogenic process have provided many new tools for clinical characterization of lipoprotein abnormalities. In non-diabetic subjects, the exact factors which oxidize LDL in vivo are still unclear, but several lines of evidence suggest that formation of oxidized LDL is of critical importance for the atherogenic process (see [35] for review). Consequently, measurement of the state of oxidation of circulating LDL lipids or its protein moiety is of obvious interest. Assessment of the latter is most commonly done by following conjugated diene formation after copper catalyzed oxidation of LDL in vitro [36], by directly quantitating lipid peroxides [37], or by determining the amount of soluble bifunctional aldehydes derived from lipid peroxidation (TBARS) [38]. Oxidation of LDL apoB makes LDL antigenic and induces the production of autoantibodies against oxidized LDL [7]. The titer of these autoantibodies may reflect oxidation of LDL apoB in vivo since autoantibodies against oxidized LDL have predicted the appearance of myocardial infarction in case-control studies [39], and the progression of carotid atherosclerosis [8] in non-diabetic subjects. On the other hand, it is unknown to what extent, if any, oxidized LDL antibodies reflect the quantity of oxidized LDL in the arterial wall. The assay for autoantibodies against oxidized LDL might lack specificity in vivo as the epitopes on oxidized LDL are shared with other oxidized proteins and lipids. Most of the studies examining the clinical significance of oxidized LDL autoantibodies are cross-sectional [40]. However, the immunological technique used in the

present study has been shown to reflect epitopes characteristic of oxidized LDL, which predict the progression of atherosclerosis [8]. In vitro, hyperglycemia, at physiologically relevant glucose concentrations, oxidizes reactive lysine groups in LDL particles [41]. Our finding of increased levels of autoantibodies against oxidized LDL is consistent with these in vitro data as well as with reports of increased concentrations of oxidized LDL, measured by conjugated diene formation during in vitro oxidation of LDL, in poorly controlled patients with IDDM [42]. Evidence for an increased susceptibility of circulating LDL particles to oxidative modification in vivo under hyperglycemic conditions is also supported by studies demonstrating increased serum malondialdehyde concentrations [43], and conjugated dienes in LDL [42] in these patients. Titers of autoantibodies against oxidized LDL have been reported to be either elevated [9,10] or normal [32] in patients with NIDDM. In patients with IDDM, previous data on endothelium-dependent vasodilation are heterogeneous. When determined using intra-arterial infusions of muscarinic agonists to increase endothelium-dependent blood flow, or using LNMMA to decrease endothelium-dependent blood flow, blunted responses have been found in four [12– 14,44] out of seven studies and normal responses in three [15–17] studies. The exact cause of these varying results is unclear but could involve factors such as gender and differences in glycemic control [12–17,44]. Study of both men and women increases the possibility of a type 2 error because women have shorter forearms than men, and forearm length [45] affects metabolism of acetylcholine but not sodium nitroprusside [45]. Regarding the effect of glycemia, we found no defect in acetylcholine induced vasodilation in our moderately controlled patients (Fig. 2). Whether this reflects the existence of a threshold for hyperglycemia induced endothelial dysfunction, or can be attributed to other factors, is unclear. In the present study we found an inverse correlation between the degree of chronic hyperglycemia and endothelium-dependent vasodilation in vivo. In contrast, the concentration of autoantibodies against oxidized LDL and endothelial function were unrelated to each other. To explain why hyperglycemia per se might be better correlated to endothelial function than the amount of oxidized LDL, one has to consider current data linking hyperglycemia and oxidized LDL to nitric oxide (NO) production. Production of superoxide, which is functionally an NO-antagonist, seems to be a key factor linking hyperglycemia to defects in endothelium-dependent vasodilation [41]. Both glucose autooxidation [19,21,46] and non-enzymatic glycosylation of proteins [18] generate free radicals such as superoxide. Superoxide reacts with NO to form peroxynitrite [47], which nitrates protein tyrosines in human atheroscle-

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rotic lesions [48] and via its reaction with NO reduces the amount of NO available for stimulation of vasodilation. These data imply that oxidative modification of LDL is not a prerequisite for hyperglycemia induced production of superoxide. Our finding of an inverse correlation between HbA1c but not oxLDLab, and endothelium dependent vasodilation could thus favor the conclusion that generation of superoxide via pathways not involving oxidation of LDL is a more important determinant of endothelium-dependent vasodilation than superoxide generation via oxidation of LDL, or direct quenching of NO by oxidized LDL. Obviously, the cross-sectional nature of the present study does not allow causal conclusions to be made. In conclusion, the present data demonstrate that the degree of chronic hyperglycemia in patients with IDDM, who were clinically free of macrovascular disease, is inversely correlated with endothelium-dependent vasodilation in vivo. The latter may reflect either reduced production or increased destruction of NO, perhaps via hyperglycemia induced generation of superoxide [41,49], and is thought to precede the development of macrovascular disease [50]. In contrast to non-diabetic subjects [6], however, we found no correlation between the concentration of autoantibodies against oxidized LDL and endothelial function. Our data are consistent with epidemiological evidence linking chronic hyperglycemia to macrovascular disease in diabetes [51], and suggest that at least early defects in vascular function are more dependent on hyperglycemia than the concentration of oxidized or modified LDL.

Acknowledgements This study was supported by grants from the Academy of Finland (H.Y.), and the Sigrid Juselius (H.Y.) and Ahokas (H.Y.) foundations. We thank Sari Ha¨ma¨la¨inen and Kati Tuomola for excellent technical assistance, Soile Aarnio for drawing figures and the volunteers for their help.

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