Plasma Homocysteine Is not Increased in Microalbuminuric Patients With Type 2 Diabetes Without Clinical Cardiovascular Disease

Although mild hyperhomocysteinemia (MHH) has been considered as an independent risk factor for cardiovascular disease (CVD) (1,2), some investigators have cast doubts about this statement (2–4). Subjects with type 2 diabetes and microalbuminuria (MA) are particularly at risk of developing CVD. In these subjects, there is some controversy as to whether total plasma homocysteine (tHcy) levels are increased (5–7) or not (8–11). Usually, these studies, which include subjects with and without CVD, do not take into account this variable when analyzing their results. It is well known that there is a positive relationship between the presence of CVD and MA (12). In addition, CVD itself is associated with MHH (1,2). We hypothezised that studies that found an increase in tHcy levels in subjects with MA did not take into account the presence of a preexisting CVD as a confounding variable. Therefore, as subjects with type 2 diabetes and MA have a higher prevalence of preexisisting CVD, higher values of tHcy could be expected as a result of CVD and not because of MA.

To test this hypothesis, we studied 93 subjects with type 2 diabetes (55 with normoalbuminuria and 38 with MA) and 86 nondiabetic control subjects matched for age and sex, all of whom were recruited at a primary care center. The exclusion criteria were: age <35 or >85 years, serum creatinine >1.4 mg/dl, uncontrolled hypertension (systolic >160 mmHg and/or diastolic >95 mmHg), congestive heart failure, major invalidating disease, pregnancy, hypothyroidism, preexisting clinical CVD (including coronary heart disease, stroke, or peripheral vascular disease), use of oral drugs that could have elevated tHcy in the previous 3 months (notably drugs for dyslipemia and metformin), or macroalbuminuria (urinary albumin excretion rate [UAER] ≥200 ugr/min). The following data were collected for each subject: age, sex, duration of type 2 diabetes, BMI, and blood pressure. Blood pressure was measured twice via the right arm after 10 min rest in the supine position by a trained staff member using a standard manometer. Hypertension was defined as blood pressure ≥140/90 mmHg or being under antihypertensive treatment. After an overnight fast, blood was drawn and analyzed for fasting plasma glucose, HbA_1c, serum creatinine, total cholesterol, triglycerides, HDL cholesterol, thyroid-stimulating hormone, serum vitamin B₁₂, serum folate, and tHcy. Information about alcohol intake and smoking habits was obtained through a questionnaire. During 3 months, subjects collected triplicate 24-h urine samples, which were analyzed for UAER and urinary creatinine excretion rate. MA was diagnosed when the geometric mean of the three values of UAER was between 20 and 200 μg/min. Otherwise, subjects were classified as normoalbuminuric (<20 μg/min) or macroalbuminuric (≥200 μg/min). Estimated creatinine clearance was calculated by the Cockroft-Gault formula (13). Retinopathy was assessed by a consultant ophthalmologist. In the statistical analysis, skewed variables were logarithmically transformed to reduce kurtosis.

There were no differences between the subjects with type 2 diabetes and control subjects with respect to age (66.0 ±11.5 vs. 65.4 ±12.8 years, P=0.74), sex (49.5 vs. 50.0% of women, P=0.94), and the other variables evaluated, except for fasting plasma glucose (151.9 ±61.7 vs. 100.8 ±14.0 mg/dl, P=0.00), HbA_1c (6.3 ±1.4 vs. 5.0 + 0.6%, P=0.00), and UAER (39.9 ±33.6 vs. 4.3 ±3.6 μg/min, P=0.00). All subjects had serum vitamin B₁₂ and folate concentrations within the reference range. No significant differences were found in relation to tHcy levels between type 2 diabetic and control subjects (7.4 ±2.6 vs. 7.3 ±2.8 μmol/l, P=0.81). There were no differences between type 2 diabetic subjects with and without MA in relation to sex (47.4 vs. 50.9% of women, respectively), age (66.4 ±10.5 vs. 65.8 ±10.5 years), BMI (27.4 ±4.7 vs. 28.4 ±4.8 kg/m²), alcohol intake >20 g/day (10.5 vs. 10%), percentage of current smokers (15.8 vs. 27.3%), prevalence of diabetic retinopathy (21.1 vs. 10.9%), fasting plasma glucose (155.2 ±66.0 vs. 149.7 ±53.1 mg/dl), total cholesterol (223.0 ±41.1 vs. 217.1 ±38.0 mg/dl), triglycerides (134.1 ±71.7 vs. 141.1 ±91.9 mg/dl), HDL cholesterol (46.6 ±13.3 vs. 47.7 ±15.3 mg/dl), LDL cholesterol (149.6.2 ±42.4 vs. 140.4 ±34.2 mg/dl), creatinine (0.95 ±0.19 vs. 0.95 ±0.17 mg/dl), creatinine clearance (79.0 ±25.3 vs. 88.2 ±26.2 ml/min), UAER (57.9 ±30.5 vs. 10.6 ±5.3 μgr/min), serum folate (9.2 ±3.4 vs. 9.7 ±7.3 ng/ml), serum vitamin B₁₂ (431 ±228 vs. 427 ±210 pg/ml), tHcy (7.3 ±2.8 vs. 7.4 ±2.4 μmol/l), and percentage of subjects with MHH (31.6 vs. 25.5%). However, subjects with MA had a longer duration of the disease (12.2 ±8.7 vs. 8.2 ±9.5 years, P=0.04), a higher prevalence of hypertension (65.8 vs. 41.8%, P=0.01), and higher HbA_1c levels (6.7 ±1.5 vs. 6.0 ±1.2 mg/dl, P=0.00). In the control group, the 75th percentile of the tHcy values distribution was ≥8.2 umol/l. This cutoff value was used to classify subjects with type 2 diabetes as having MHH. A total of 14 subjects with normoalbuminuria (25.5%) and 12 with MA (31.6%) had MHH, but the differences were not statistically significant. In the whole group of subjects with type 2 diabetes, we found higher tHcy values in male as compared with female subjects (8.0 ±2.9 vs. 6.4 ±2.0 μmol/l, P=0.02). We also found a significant positive correlation (Pearson’s correlation coefficient) between Log tHcy and age (r=0.25, P=0.02) and a significant negative correlation between Log tHcy and both serum folate (r=−0.23, P=0.03) and serum B₁₂ vitamin (r=−0.24, P=0.03). No significant correlation was found between Log UAER and Log tHcy (r=0.15, P=0.17). By stepwise logistic regression analysis, serum folate (negative relationship, P=0.01) and serum creatinine (positive relationship, P=0.01), but not UAER, emerged as the independent variables related to MHH in subjects with type 2 diabetes. Additional preliminary data in a reduced group of 22 subjects with type 2 diabetes and CVD (aged 67.6 ±9.2 years, sex 50.0% of women, 10.7 ±6.9 years of duration of type 2 diabetes, 45.5% of them with MA, 68.1% hypertensive, and 40.9% with MHH) supported our hypothesis. This subgroup of patients had higher values of tHcy than subjects with MA but without CVD (8.5 ±2.5 vs. 7.3 ±2.8 μmol/l, P=0.05).

Some previous studies have evaluated the relationship between MA and tHcy levels in subjects with type 2 diabetes. Some of these studies have found a positive relationship between MA and tHcy values (5–7), but this finding has not been confirmed by others (8–11). The majority of these studies are cross-sectional and have not adjusted their results according to the presence of CVD. Uncontrolled confounding variables must always be considered as a possible noncausal explanation for any observed association between tHcy levels and MA. Therefore, if an association between tHcy and MA is found, it is mandatory to adjust for the presence of CVD before concluding that an association between tHcy and MA exists. This adjustment is very important, especially if we take into account that in some of the above-mentioned studies (5), ∼30% of subjects with type 2 diabetes have CVD as compared with an absence of CVD in the control group. In conclusion, the present findings suggest that in type 2 diabetic subjects with MA but without clinical CVD, there is no association between MA and tHcy levels.