Oxidative stress indices in IDDM subjects with and without long-term diabetic complications.

BACKGROUND: Numerous animal and population studies of diabetes have identified markers of oxidative stress. However, for most markers that have been measured the results are not consistent. In addition, it is less clear whether oxidative stress is related to the development of diabetic complications. The objective of this study was to evaluate a series of plasma markers and leukocyte markers to test the hypothesis that type 1 Insulin Dependent Diabetes Mellitus (IDDM) subjects experience oxidative stress. A related question was whether markers of oxidative stress are higher in IDDM subjects who have developed long-term complications. METHODS: The study population consisted of 22 IDDM subjects with diabetic complications and 22 IDDM subjects without complications, both groups matched by age and gender and with similar HbA1c levels, and 16 nondiabetic control subjects. Plasma levels of organoperoxides were determined by the ferrous oxidation/xylenol orange (FOX) assay, malondialdehyde by the thiobarbituric acid (TBARS) assay, and vitamin E by HPLC. Mononuclear cells and polymorphonuclear cells were analyzed for ascorbic acid by HPLC and for glutathione (GSH) by enzymatic recycling. In addition, GSH peroxidase, GSH transferase and glucose-6-phosphate dehydrogenase levels were determined in both cell fractions. RESULTS: Plasma organoperoxides were significantly elevated in the IDDM subjects compared to controls (p = 0.02) while TBARS and vitamin E levels were not significantly different. In the IDDM subjects, mononuclear cell levels of ascorbic acid were significantly lower (p < 0.02) and levels of GSH were lower, approaching significance (p = 0.07), compared to controls. Ascorbic acid and GSH levels in polymorphonuclear cells were not significantly different between IDDM subjects and controls, nor were enzyme levels different. In addition, the plasma and intracellular indices of oxidative status in IDDM subjects were not different when IDDM subjects with complications were compared to IDDM subjects without complications. CONCLUSION: Demonstration of oxidative stress in IDDM subjects depends upon which markers are measured. This is in agreement with previous studies of oxidative stress in various disease states including diabetes. Plasma levels of organoperoxides may be the most reliable indicators of oxidative stress. However, it is unclear whether elevated plasma organoperoxides indicate a generalized systemic stress or are produced in localized areas. By comparison, oxidative stress indices determined with isolated blood cells may provide a clearer picture. Depressed levels of ascorbic acid and GSH were observed only in mononuclear cells, which are mainly long-lived T lymphocytes. Mononuclear cells antioxidant status may reflect systemic oxidative stress. In this study, neither plasma markers nor intracellular markers of oxidative stress were different in IDDM subjects with long-term diabetic complications compared to subjects without complications.

Increased levels of methylglyoxal-metabolizing enzymes in mononuclear and polymorphonuclear cells from insulin-dependent diabetic patients with diabetic complications: aldose reductase, glyoxalase I, and glyoxalase II--a clinical research center study.

Levels of aldose reductase, glyoxalase I, and glyoxalase II in mononuclear and polymorphonuclear cells from insulin-dependent diabetes mellitus (IDDM) patients with long term diabetic complications were compared to levels in IDDM patients without complications and to those in nondiabetic controls. Cells were isolated from 22 asymptomatic long term IDDM patients, 22 symptomatic IDDM patients, and 16 controls, using a double gradient centrifugation procedure. Aldose reductase was determined by Western blots using polyclonal antiserum to human aldose reductase purified from skeletal muscle. Glyoxalase I and glyoxalase II were determined spectrophotometrically. Aldose reductase in mononuclear cells from symptomatic IDDM patients is significantly elevated compared to that in asymptomatic IDDM patients (mean +/- SEM, 0.96 +/- 0.20 vs. 0.46 +/- 0.08 microgram/mg protein; P < 0.02). Aldose reductase was not detected in polymorphonuclear cells. Glyoxalase I in mononuclear and polymorphonuclear cells from symptomatic IDDM patients is significantly elevated compared to that in controls [mean for mononuclear cells, 0.46 +/- 0.03 vs. 0.37 +/- 0.03 mumol/min.mg (P < 0.05); mean for polymorphonuclear cells, 0.16 +/- 0.01 vs. 0.10 +/- 0.01 mumol/min.mg (P < 0.002)]. Glyoxalase II is significantly elevated only in polymorphonuclear cells from symptomatic IDDM patients compared to controls (mean, 0.13 +/- 0.01 vs. 0.063 +/- 0.016 mumol/min.mg; P < 0.005). Glutathione peroxidase and glutathione S-transferase were not significantly different in these populations. Aldose reductase, glyoxalase I, and glyoxalase II are involved in the metabolism of methylglyoxal, suggesting that methylglyoxal may play a role in the etiology of diabetic complications.