Diabetes has been considered to be associated with oxidative stress. It has been suggested that increased free radicals and decline of antioxidant defense mechanisms induce diabetic micro- and macrovascular complications (1–3). Vitamin C is one of the major antioxidants and is detected in various blood components (4). However, measurements of vitamin C levels have shown inconsistent results, and the interpretation of vitamin C levels in diabetes as an antioxidant biomarker has not been clarified (5–8). In this study, we investigated the lymphocyte and plasma vitamin C levels in type 2 diabetic patients with and without diabetes complications.
RESEARCH DESIGN AND METHODS
Forty-one patients with type 2 diabetes (63 ± 8.9 years [mean ± SD]; 25 men and 16 women) attending the Department of Endocrinology and Metabolism at Shizuoka City Hospital were recruited. Type 2 diabetes was diagnosed according to the American Diabetes Association criteria. The duration of illness was 11 ± 8.3 years, fasting plasma glucose was 137 ± 43 mg/dl, and HbA1c levels were 7.1 ± 1.0%. Twenty-six patients had diabetes complications with neuropathy, retinopathy, or nephropathy, and 15 patients had no complications. Both diabetic groups were matched by age, sex, fasting plasma glucose, and HbA1c level (63 ± 9.7 years, 18 men and 8 women, 137 ± 45 mg/dl, and 7.2 ± 1.0% for diabetic patients with complications compared with 64 ± 7.5 years, 7 men and 8 women, 137 ± 42 mg/dl, and 6.8 ± 0.8% for diabetic patients without complications, respectively). The duration of illness was longer in the diabetic patients with complications than in diabetic patients without complications (13 ± 9.1 vs. 7.7 ± 5.2 years, respectively, P = 0.051). For the normal control subjects, 50 age- and sex-matched healthy volunteers (63 ± 5.7 years, 31 men and 19 women) were recruited. The participants taking vitamin supplements were excluded from the study. All participants gave informed consent before entering the study. The study was conducted in accordance with the Declaration of Helsinki and was approved by the ethics committee at the hospital.
Blood samples were obtained by vein puncture in the morning while the patients were in the fasting state. Lymphocytes and plasma were prepared by centrifugation and the Ficoll gradients method, then immediately treated with metaphosphoric acid (final 5% wt/wt) to stabilize vitamin C (9,10). These processes were performed within 2 h under cooled conditions on ice to obtain reliable data. The vitamin C samples were stored at −80°C until analyzed, and the vitamin C (ascorbic acid, reduced form) levels were measured by high-performance liquid chromatography with the electrochemical detector method (11). All samples were handled and stored similarly in both diabetic patients and control subjects.
The lymphocyte and plasma vitamin C levels in type 2 diabetic patients were compared with those of the control subjects. The differences between the vitamin C levels in type 2 diabetic patients with and without diabetes complications were also studied. Statistical analysis was performed with the unpaired Student’s t test to compare the data between diabetic patients and control subjects and between type 2 diabetic patients with and without diabetes complications. A P value <0.05 was considered significant.
The lymphocyte vitamin C level in diabetic patients was significantly lower than in control subjects (18 ± 4.5 vs. 28 ± 7.9 nmol/mg protein, P < 0.0001), whereas the plasma vitamin C level was not different (59 ± 19 vs. 53 ± 18 μmol/l, P = 0.17) (Fig. 1A and B). There were no significant linear correlations between the lymphocyte and plasma vitamin C levels in diabetic patients (r = 0.011, P = 0.95) as well as in control subjects (r = 0.14, P = 0.35). The lymphocyte vitamin C level in diabetic patients with complications was significantly lower than in those without complications (17 ± 3.3 vs. 21 ± 5.4 nmol/mg protein, P = 0.011) (Fig. 1C), whereas the plasma vitamin C level was not different (59 ± 18 vs. 59 ± 21 μmol/l, P = 0.97).
Increased oxidative stress in diabetes could contribute to depletion of antioxidants such as vitamin C (2,3). In this report, we demonstrated that the lymphocyte vitamin C level is significantly lower in type 2 diabetic patients, but we could not observe such an association in plasma vitamin C levels. The plasma concentration of vitamin C is considered to be strongly correlated with transient consumption of foods such as fruit, supplements, and vegetables (4). Compared with plasma, lymphocyte has been reported to maintain a vitamin C concentration as large as 80- to 100-fold across the plasma membrane (12,13) and to have cell-membrane transporting mechanisms between vitamin C and glucose (14,15). In diabetes, therefore, the measurement of lymphocyte vitamin C might be expected to be a more reliable antioxidant biomarker than plasma vitamin C level.
It is unclear whether leukocyte vitamin C correlates with diabetes complications. VanderJagt et al. (5) reported that vitamin C levels in mononuclear leukocytes were decreased in the whole group of type 1 diabetic patients compared with control subjects but were not different between patients with and without long-term complications. We showed the significant lower lymphocyte vitamin C levels in patients with type 2 diabetes with complications compared with those without complications. However, the results should be interpreted carefully because of the small sample size and because the differences of lymphocyte vitamin C level among different diabetes complications are not fully clarified. Further studies are required to investigate the precise correlations of lymphocyte vitamin C with duration or severity of diabetes and to establish the clinical usefulness of lymphocyte vitamin C level as a biomarker in developing diabetes complications.
This study was supported by a grant from Japanese Ministry of Health, Labour and Welfare.
We thank Reiko Akiyama for her skillful assistance.
A table elsewhere in this issue shows conventional and Système International (SI) units and conversion factors for many substances.