© 1996 American Heart Association, Inc.
Articles |
Autoantibodies Against Oxidized LDL Do Not Predict Atherosclerotic Vascular Disease in Non–Insulin-Dependent Diabetes Mellitus
the Departments of Clinical Nutrition (M.I.J.U., L.N.) and Medicine (L.N., S.Y.-H.), the Kuopio Research Institute of Exercise Medicine (R.R.), and the A.I. Virtanen Institute (J.L., S.Y-H.), University of Kuopio, Kuopio, Finland; the Medical School (P.V.), University of Tampere, Tampere, Finland; and the Division of Vascular Ultrasound Research (M.M.), Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC.
Correspondence to Matti Uusitupa, MD, Department of Clinical Nutrition, University of Kuopio, PO Box 1627, FIN-70211 Kuopio, Finland.
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Abstract |
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Accelerated atherosclerosis in diabetes has been suggested as being due to an enhanced oxidative modification of LDL. We hypothesized that the titers of autoantibodies against oxidized LDL (oxLDL) may be increased in patients with non–insulin-dependent diabetes mellitus (NIDDM) and that they may contribute to various manifestations of atherosclerosis among such patients. In a 10-year follow-up study of 91 newly diagnosed NIDDM patients and 82 nondiabetic control subjects, autoantibodies against oxLDL (expressed as the ratio of autoantibodies against oxLDL and native LDL) were measured at baseline and after 10 years. Quantitative ultrasonography to examine the intimal-medial thickness of the common carotid artery (a morphological index of arterial wall injury) and carotid bifurcation was performed at the 10-year examination. The relationship of autoantibodies against oxLDL to the occurrence of cardiovascular death, fatal and nonfatal myocardial infarction, stroke, and any cardiovascular event as well as to the intimal-medial thickness of the common carotid artery and carotid bifurcation was evaluated. Associations between these autoantibodies and metabolic variables (fasting glucose, glycosylated hemoglobin, insulin, and serum lipids) in NIDDM patients were also examined. Autoantibodies against oxLDL did not differ between NIDDM and control subjects (NIDDM: baseline, 1.63 and 0.61 to 23.6; 10-year examination, 1.64 and 0.06 to 59.0; control group: baseline, 1.84 and 0.13 to 36.0; 10-year examination, 1.50 and 0.25 to 8.29; median and range, P=.62, baseline; P=.45, 10 year). In both groups, the titers of these autoantibodies measured at baseline and after 10 years significantly correlated with each other (r=.63 for the diabetic and r=.51 for the control group, respectively, P<.001 for each). The frequency of all cardiovascular events was markedly higher in the NIDDM group than in the control group, but autoantibodies against oxLDL had no significant association with any of these events, including cardiovascular mortality. At the 10-year examination the intimal-medial thicknesses of the common carotid artery (1.24±0.36 versus 1.06±0.30 mm, P=.002) and carotid bifurcation (2.11±0.73 versus 1.77±0.82 mm, P=.01) were greater in NIDDM patients than in control subjects, but autoantibodies did not show any association with the intimal-medial thicknesses in either the diabetic or control groups. Autoantibodies against oxLDL indicate the presence of oxidatively modified LDL in vivo, but their titers in the serum do not seem to associate with the excess cardiovascular mortality, morbidity, or intimal-medial thickness of the carotid artery.
Key Words: oxidized LDL • non–insulin-dependent diabetes mellitus • atherosclerosis • cardiovascular mortality • morbidity
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Introduction |
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Atherosclerotic vascular diseases are more common in both insulin-dependent diabetes and NIDDM than among nondiabetic populations. Furthermore, these diseases constitute the principal cause of mortality among diabetic patients.1 2 3 4 5 6 7 8 Cardiovascular risk factors, such as elevated serum levels of TC and LDL-C, high BP, and smoking, increase the risk of atherosclerotic vascular diseases approximately to the same extent in diabetic subjects as they do in the nondiabetic population. However, some of the excess occurrence of atherosclerotic vascular diseases in diabetic subjects remains unexplained after considering these risk factors alone.7
OxLDL may play a fundamental role in the pathogenesis of atherosclerosis.9 10 As chronic hyperglycemia and its consequences11 12 may increase oxidative stress in diabetes,13 14 15 16 it is conceivable to hypothesize that oxidatively modified lipoproteins could explain the increased susceptibility of diabetic patients for atherosclerosis. In addition to their role in the formation and accumulation of foam cells in the arterial intima during the development of early atherosclerosis,9 10 oxLDL particles may accelerate the progression of more advanced atherosclerotic lesions.17 18 19 The measurement of autoantibodies against oxLDL offers an elegant model for examining in vivo the role of this potential risk factor in the pathogenesis of atherosclerotic vascular diseases.17 The aims of the present study were to investigate (1) whether patients with NIDDM have an elevated level of autoantibodies against oxLDL, (2) the main correlates of these autoantibodies in NIDDM patients and control subjects, (3) the predictive role of the autoantibodies against oxLDL with regard to clinically significant atherosclerotic vascular diseases in NIDDM, and (4) the relationship between carotid atherosclerosis and oxLDL in NIDDM and control subjects.
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Methods |
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Subjects
The original study population consisted of 133 newly diagnosed patients with NIDDM who were 45 to 64 years old at the time of diagnosis and 144 randomly selected nondiabetic subjects in the same age group, all of whom were investigated from 1979 through 1981.8 20 Both groups were recruited from a defined area of 180 000 inhabitants in the county of Kuopio in eastern Finland.
The 10-year examination of the same study population was performed during 1991 and 1992.8 21 During the 10-year follow-up, 36 diabetic and 8 nondiabetic subjects died. Five diabetic and 8 nondiabetic subjects declined to participate, but data concerning cardiovascular mortality and morbidity were obtained from all subjects originally examined.8 Autoantibodies against oxLDL could not be obtained from all subjects examined. Therefore, the mortality and morbidity data of the present group are different from those presented previously.8
Both the baseline and 10-year studies were approved by the ethics committee of the University of Kuopio. Informed consent was obtained from all the subjects participating in the study.
Diabetic Patients
General practitioners working in community health centers and private practitioners in the survey area were asked to refer newly diagnosed diabetic patients with a fasting venous whole blood glucose level 
7.0 mmol/L and who were 45 to 64 years old to the outpatient department of Kuopio University Hospital. All patients were examined within 4 weeks after the detection of diabetes. The diagnosis was confirmed by an oral glucose–tolerance test using the diagnostic criteria of the World Health Organization.22 Patients with secondary diabetes and those in whom fasting blood glucose exceeded 7.0 mmol/L for more than 6 months were excluded.20 All diabetic subjects were nonketotic at the time of diagnosis, and none needed insulin treatment during the 3 months after diagnosis.
Control Subjects
A random control sample of subjects who were 45 to 64 years old was selected from the population registers of the study area, taking into consideration the distribution of the population living in rural and urban communities. Of 183 subjects originally contacted, 9 had diabetes, 1 had hypothyrosis, and 29 refused to participate. The final study population consisted of 144 nondiabetic control subjects.
Baseline Examination
The same methodological protocol was used to examine diabetic and nondiabetic control subjects. Medical history included a history of cardiovascular, renal, and other significant long-term disease and the use of drugs and vitamin preparations. Height, weight, and BMI were measured in the morning. BP was measured in the sitting position after a 5-minute rest. Hypertension was diagnosed as systolic BP >160 mm Hg and/or diastolic BP >95 mm Hg and a history of antihypertensive drug treatment.8 20
A conventional 12-lead resting ECG was recorded from each subject and interpreted according to the Minnesota code. Ischemic ECG abnormalities (MC 1.1-3, 4.1-3, 5.1-3, and 7.1) included Q-QS abnormalities, various degrees of ST segment depression, T wave changes, and left bundle branch block. A diagnosis of MI was based on the presence of major Q-QS abnormalities and/or previously documented MI.8 20
An oral glucose–tolerance test was performed after a 12-hour overnight fast. Samples for blood glucose and serum insulin determinations were taken before the test (fasting) and at 1 and 2 hours after glucose intake. Samples for serum insulin were placed in prechilled tubes, centrifuged, and stored without delay at -20C° until analyzed. Blood glucose levels were measured by using a glucose-oxidase method (Glox; Kabi Ab) and serum insulin by a double-antibody radioimmunoassay (antiserum M 8309; Novo Industries). The variation coefficient for the insulin assay was 5.4%; its sensitivity was 2 mU/L.
Serum and lipoprotein lipids were measured from 12-hour fasting samples. Lipoproteins were separated by ultracentrifugation at d=1.006 g/mL to remove VLDL, after which the infranatant fraction was precipitated by using dextran sulfate and magnesium chloride.23 Enzymatic methods were used for the determination of cholesterol24 and TGs25 from the whole serum, the top layer after ultracentrifugation of VLDL, and the supernatant after precipitation of LDL. LDL was calculated as the difference between the whole serum and the sum of VLDL and HDL. The intraassay variations for TC, HDL-C, and TGs were 1.3%, 0.95%, and 3.1%, and the interassay variations were 3.3%, 1.9%, and 5.2%, respectively.
Ten-Year Examination
The data on medical history and resting ECG were obtained similarly to the baseline determinations. The incidences of fatal cardiovascular events, nonfatal MI, and stroke were derived by reviewing patient records, medical histories, clinical examination, and death certificates. MI was verified by using the baseline criteria.8 20 The category "any cardiovascular event" included cardiovascular death, fatal and nonfatal MI, or stroke.
Laboratory tests for glucose and insulin determinations were performed by using slightly different procedures from baseline. Glucose was analyzed by using a glucose-oxidase method (Daiichi Co) from plasma samples. Insulin was analyzed by a double-antibody assay using a different kit (Phasedeph, Pharmacia). However, these tests have similar detection limits and coefficients of variation. Glycosylated hemoglobin was measured by liquid cation-exchange chromatography (normal range, 4.0% to 6.0%).
Carotid Ultrasonography Examination
The scanning and reading protocols26 and the measurement reproducibility for this laboratory are available.27 Two certified sonographers performed the B-mode carotid ultrasonography examination. The imaging protocol was designed to ensure valid and reliable identification of arterial carotid references and the definition of near and far wall interfaces. The carotid artery was divided into two segments based on arterial anatomy and geometry, ie, the distal CCA and the carotid bifurcation (bulb). The key anatomic features defining these segments were the proximal origin of the bulb and the tip of the flow divider, which separates the internal from the external carotid artery. In longitudinal arterial images the adventitial-medial and intimal-lumenal interfaces on the near wall and the lumenal-intimal and medial-adventitial interfaces on the far wall were the specific anatomic boundaries that defined intimal-medial thickness.28 Quantitative carotid ultrasonography was performed by using a B-mode ultrasound imaging device equipped with a 10-MHz annular array (Biosound Inc). A computerized reading station was used to analyze videorecorded examinations according to a described protocol.26 The mean of the maximum far wall intimal-medial thickness on each right and left CCA and carotid bifurcation was chosen as the ultrasonographic end point for this analysis. These end points are used in epidemiological studies and interventional clinical trials as an index of arterial injury of an atherosclerotic nature.
Autoantibodies Against OxLDL
Autoantibodies were determined for 91 NIDDM patients and 82 control subjects from the samples obtained in 1979 through 1981. At the 10-year examination these antibodies were measured for all diabetic patients participating in the follow-up study (n=92) and for 80 of the control subjects for whom the baseline serum samples were also available. All serum samples were stored at -70°C until analyzed. Autoantibodies against oxLDL were measured according to a modification of a published method.17 The enzyme-linked immunosorbent assay used for the determinations does not measure acetylated LDL or other types of nonoxidized LDL modifications. The three determinations made for each sample were identical except for antigen coating of microtiter plates (maxiSorb, Nunc): one plate was coated with native LDL, a second with 24-hour copper-oxidized LDL, and a third with postcoat only (see below). Plates were coated with antigen (5 µg/mL) in PBS overnight at 4°C. To prevent oxidation of native LDL, PBS contained 0.27 mmol/L EDTA and 20 µmol/L butylated hydroxytoluene. Plates were washed three times with PBS containing 0.5% Tween 20 and twice with water. Plates were blocked with 2% bovine serum albumin (Sigma Chemical Co) and washed as above. Serum samples in PBS containing 1% bovine serum albumin, 0.27 mmol/L EDTA, 20 µmol/L butylated hydroxytoluene, and 0.05% Tween 20 were placed by pipette on plates at 1:20, 1:50, and 1:100 dilutions. Plates were incubated overnight at 4°C and washed. Horseradish peroxidase–conjugated anti-human IgG (Cappel, Organon Teknika Corp) diluted 1:5000 in the previous buffer was added, and plates were incubated at 4°C for 4 hours. After washing, the plates were incubated with peroxidase substrate (o-phenylenediamine) for 5 minutes. Color development was stopped with 2 mol/L H2SO4, and absorbances were measured at 492 nm by using a Multiskan microplate reader (Eflab). The reaction volume was 50 µL for each step except 150 µL for postcoating. All measurements were done in duplicate. Baseline and 10-year follow-up samples were always measured simultaneously on the same plate. Postcoat values for each dilution were subtracted from the analyzed samples. Results were calculated as the ratio of oxLDL to native LDL at each dilution. In the present article "anti-oxLDL antibodies" refers to this ratio.
Statistical Methods
Differences between groups were analyzed by using Student's t test or the Mann-Whitney U test. Serum TGs, VLDL TGs, LDL TGs, insulin, and anti-oxLDL antibodies were analyzed after logarithmic transformation. 
2 tests were used for the analyses of categorical variables. Intergroup associations were tested by using Spearman correlation coefficients. Multiple logistic regression analyses (forward method) were performed to determine the independent association of anti-oxLDL antibodies with atherosclerotic vascular diseases. All the data are given as mean (SD) or as percentages.
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Results |
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Table 1
summarizes the clinical data of the study subjects for whom anti-oxLDL antibody measurements were available at the baseline and 10-year follow-up examinations. Patients with newly diagnosed NIDDM were more obese than control subjects, and fasting serum insulin levels were higher in diabetic subjects. Serum TC, LDL-C, and HDL-C values were lower but VLDL-C values higher in the diabetic than the control group. Serum total TGs as well as lipoprotein TGs were increased in the diabetic subjects. At baseline, hypertension, history of MI, and the presence of ischemic ECG abnormalities were more prevalent among diabetic subjects. Consequently, the use of both ß-blockers and diuretics was more common in the diabetic cohort.
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Of the present study group, 18 diabetic subjects died, 15 of them from cardiovascular causes. The incidences of MI, stroke, and any major cardiovascular event were higher in the diabetic than the control group. Intimal-medial thicknesses of the CCA and carotid bifurcation were significantly greater in diabetic than nondiabetic subjects (Table 2).
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No significant difference was observed in the mean BMI between the groups after 10 years, but diabetic subjects still had higher fasting serum insulin values. Serum TC and LDL-C values were similar in both groups, but VLDL-C and serum total and VLDL TGs were higher and HDL-C was lower in diabetic patients. The prevalence of hypertension no longer differed significantly between the groups.
Ten years after diagnosis, 20.3% of the diabetic subjects were treated with diet only, 26.1% had sulfonylurea treatment, 2.9% were treated with metformin, 27.5% with a combination of sulfonylurea and metformin, 8.7% with insulin, and 14.5% received both insulin and sulfonylurea for treatment of hyperglycemia. The use of vitamin preparations was uncommon and inconsistent in both groups.
Table 3 provides data on anti-oxLDL antibodies for both groups. There were no significant differences in anti-oxLDL antibodies between the two groups at baseline or 10 years later, and no significant change was observed in this variable with time in either group. In each group, measurements of anti-oxLDL antibodies taken at baseline and after 10 years were significantly correlated (r=.63 for diabetic and r=.51 for control subjects, P<.001 for both).
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At baseline, LDL-C correlated with anti-oxLDL antibodies in the control but not the diabetic group. In diabetic subjects serum total TGs inversely correlated with anti-oxLDL antibodies (Table 4). No significant correlations between anti-oxLDL antibodies and serum lipids, insulin, glucose, glycosylated hemoglobin, BP, and urinary albumin at baseline or 10-year follow-up were found. Smoking was not significantly associated with anti-oxLDL antibodies.
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Table 5 shows the relationship between anti-oxLDL antibodies and the prevalence of MI and ischemic ECG at baseline and the association of anti-oxLDL antibodies with the incidence of cardiovascular events during the 10-year follow-up. No statistically significant relationships were observed at baseline. Baseline anti-oxLDL antibody titers were not associated with cardiovascular death, incidence of MI, incidence of stroke, or any cardiovascular event in either NIDDM patients or control subjects.
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Since anti-oxLDL antibodies did not show a normal distribution, an additional analysis on mortality and morbidity data was performed by comparing the frequencies of diabetic subjects in the two lower tertiles of the baseline anti-oxLDL antibodies to those in the highest tertile (see Table 6 for tertile limits). Total mortality (the two lower tertiles versus the highest one) and cardiovascular mortality did not differ significantly in this analysis: 24.2% versus 10.3%, P=.12, age-adjusted OR 0.32, 95% CI 0.08-1.25; 19.4% versus 10.3%, P=.28, OR 0.42, CI 0.10-1.73. Note that the fatal events were even somewhat lower in the highest tertile. The incidence of MI was 22.4% versus 39.1% (P=.14, OR 1.99, CI 0.68-5.80), and the respective figures for stroke and any cardiovascular event were 14.8% versus 20.7% (P=.48, OR 1.40, CI 0.44-4.46) and 35.4% versus 43.5% (P=.51, OR 1.25, CI 0.45-3.5).
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In the forward stepwise multiple logistic analyses on diabetic patients, of the risk factors included in the model (age, BMI, baseline glucose, fasting insulin, LDL-C, HDL-C, TGs, anti-oxLDL antibodies, systolic BP, and smoking), only baseline blood glucose was a significant predictor of stroke (P=.0075, OR 1.26, CI 1.06-1.49), while baseline glucose (P=.013, OR 1.24, CI 1.05-1.53) and fasting insulin (P=.054, OR 5.79, CI 0.99-34.7) showed a significant contribution to the incidence of any major cardiovascular event. Detailed data on the predictive values of different baseline variables for cardiovascular mortality and morbidity in the original study population are available.8
Finally, the relationship of anti-oxLDL antibodies measured at baseline and after 10 years to the intimal-medial thickness of the CCA and carotid bifurcation was examined according to the tertiles of anti-oxLDL antibodies. Neither the baseline nor the 10-year anti-oxLDL antibodies showed any association with the intimal-medial thickness in either group (Table 6
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Discussion |
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The main goal of this study was to investigate the role of anti-oxLDL antibodies in the occurrence of clinical and morphological signs of cardiovascular disease in NIDDM patients. Oxidative modification of LDL may be a prerequisite for macrophage uptake and cellular accumulation of cholesterol.9 10 In this process, many components of LDL are damaged, including apoB. The oxidative modification of the apolipoprotein moiety of LDL makes it antigenic and causes it to elicit the production of autoantibodies against oxLDL. These autoantibodies may reflect LDL oxidation in vivo,17 18 29 and oxidatively modified LDL particles do occur in atherosclerotic lesions.30 31 32 33 34 35 Furthermore, some studies suggest that the presence of a high titer of autoantibodies against oxLDL is associated with the severity of carotid atherosclerosis.18 In case-control studies of nondiabetic subjects autoantibodies against oxLDL predicted the appearance of MI19 and the progression of carotid atherosclerosis.17 The clinical significance of autoantibodies against oxLDL remains unresolved,36 however, and needs further investigation.
Recent studies of NIDDM suggest that the characteristic lipoprotein abnormalities of NIDDM contribute to the increased risk of atherosclerosis.8 37 38 Furthermore, there is evidence that chronic hyperglycemia could also account for the high cardiovascular mortality and morbidity rates among diabetic patients.8 39 Since diabetes per se is said to increase oxidative stress,13 14 and glycosylation of LDL12 could lead to an enhanced production of oxidized or glycoxidized LDL,15 and since atherosclerosis is accelerated in diabetes, NIDDM seems to be a natural in vivo experimental model for confirming the role of oxidation in the pathogenesis of atherosclerosis.
We found no significant difference in the mean level of autoantibodies against oxLDL at baseline or after the 10-year follow-up between the diabetic patients and control subjects. These autoantibodies did not significantly contribute to the prevalence of coronary heart disease at baseline, nor did they significantly predict the incidence of total cardiovascular mortality or any other atherosclerotic vascular disease events. Furthermore, our 10-year examination data did not show any association between the titer of autoantibodies against oxLDL and morphological signs of carotid artery injury as determined by quantitative ultrasonography. Although baseline samples for autoantibody analysis were not available for all the diabetic and nondiabetic subjects originally examined, we believe that the data presented here is representative of the population that was originally selected for the study.8 20 21 40 The repeated measurements of autoantibodies strengthen our findings on the group differences and the associations of the outcome variables of atherosclerotic vascular disease. In the NIDDM group the incidence of various cardiovascular manifestations, including mortality from a cardiovascular cause, was high enough to evaluate the effect of other risk factors on these events.8 If the autoantibodies against oxLDL were a major contributor to diabetic vascular disease then they would have shown a clear association with cardiovascular events as well. Diabetic patients had a markedly increased intimal-medial thickness of the CCA, and various risk factors, eg, apoB and LDL TGs as well as fasting insulin, were related to intimal-medial thickness in NIDDM patients.41 However, the autoantibodies against oxLDL showed no such association. One may argue that the intimal-medial thickness of the carotid arteries as measured by ultrasonography does not reflect the severity of atherosclerosis. However, this technique has been shown to be valid and reliable in estimating the extent and severity of atherosclerosis,26 27 and the intimal-medial thickness of the carotid arteries predicts different clinical end points of atherosclerotic vascular diseases.42 43
In the current autoantibody assay, oxLDL was used as the antigen. MDA-modified LDL (MDA-LDL) has also been used as the antigen in a similar assay. MDA-LDL contains MDA-lysine adducts, which represent one class of oxidation-derived antigen epitopes present in vivo. Other oxidation-derived epitopes include HNE-modified LDL (HNE-LDL) and presumably some other peroxidation-derived products. OxLDL was chosen as the antigen for the present study because it contains a collection of various epitopes typically required for the oxidation process and may thus be a more physiologically relevant antigen than MDA-LDL or HNE-LDL alone. However, in oxLDL the density of various oxidation-derived epitopes is likely to be much lower than in MDA-LDL or HNE-LDL, which rely only on one or a few epitopes generated during the reaction with aldehydes. Consequently, assays that use oxLDL as the antigen may be less sensitive than assays using MDA-LDL or HNE-LDL, but they should reflect a more generalized immune response against oxLDL. Thus, this study does not entirely rule out the possibility that autoantibodies against MDA-LDL or HNE-LDL may better predict the appearance of clinical end points or intimal-medial thickness of carotid arteries.
While a number of studies show that the LDL particles of diabetic subjects are more susceptible to oxidative modification in vitro than those of control subjects,14 16 studies of autoantibodies against oxLDL in NIDDM are almost lacking, except one that reports a higher ratio of autoantibodies against modified versus native LDL in NIDDM than control subjects.15 Differences in the study population, methods, and antigens (see above) may explain these discrepancies. Of note, autoantibodies against oxLDL were not associated with either microvascular or macrovascular complications in NIDDM subjects in the cross-sectional study.15
The finding of a positive correlation between the autoantibodies against oxLDL and LDL-C, particularly in control subjects, fits well with the hypothesis that the amount of LDL available for oxidation affects the magnitude of the oxidative modification of LDL. This relationship was not significant in NIDDM patients, but an inverse association between serum TGs and these autoantibodies was found. It is important to note that glycemia, HDL-C, VLDL-C, and LDL TGs, which are all related to atherosclerotic vascular disease in diabetes,8 did not show significant correlations with autoantibodies against oxLDL. This also suggests that the accelerated atherosclerosis in NIDDM that is associated with poor glycemic control and characteristic lipoprotein abnormalities8 may not be mediated through oxLDL.
On the other hand, these autoantibodies could indicate the presence of oxidatively modified LDL in vivo,44 45 but they may rather appear as innocent bystanders that reflect the immunologic phenomena occurring in the arterial wall than as active agents that directly predict the atherosclerotic process. Accepting this concept implies that autoantibodies against oxLDL can not be used to estimate the progression of atherosclerosis.
There are no studies on the stability of autoantibodies against oxLDL after storage for a long period of time. In this respect our findings on the quite stable titers from baseline and 10-year examinations as well as the reasonably high correlations observed between the determinations of autoantibodies from the 10-year-old samples and more recent ones are of interest.
In conclusion, autoantibodies against oxLDL were not significantly increased in patients with NIDDM either at the time of diagnosis or after a 10-year follow-up. These antibodies did not predict cardiovascular mortality or morbidity, nor were they associated with the intimal-medial thickness of the CCA and carotid bifurcation in either NIDDM or nondiabetic control subjects.
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Acknowledgments |
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This study was supported by grants from the Finnish Foundation of Diabetes Research and the Medical Council of the Academy of Finland (Dr Uusitupa).
Received December 11, 1995;
revision received April 1, 1996;
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