Oxidative Stress in Families of Type 1 Diabetic Patients: Further evidence

We have recently reported evidence of increased oxidative stress in nondiabetic first-degree relatives of patients with type 1 diabetes(1). Elevated circulating markers of lipid peroxidation and increased cellular fragility seemed to be associated with supposed markers of inflammation. Transition metals may promote free radical reactions. Indeed, among radical-scavenging defenses of extracellular fluids, there are proteins involved in the sequestration of transition metals. Further-more it has been reported that cellular-redox modification influences the activity of ion-transport systems(2,3,4,5,6),including sodium/hydrogen exchange (NHE), and we have found increased erythrocyte NHE activity in families of patients with type 1 diabetes(7). Because we had previously examined the plasma thiol groups and the erythrocyte antioxidant reserves in type 1 families (8), we completed our investigation of possible first-chain initiating or stimulating factors.

In these families, we searched for the contribution of extracellular antioxidants to the increased levels of oxidative stress. We also investigated the eventual relationship between oxidative stress and abnormal NHE activity. The study groups were the same type 1 diabetic patients, first-degree relatives, and control subjects previously examined(1). We selected 30 type 1 diabetic patients (mean duration 20 ± 8 years; 10 without diabetic complications, 10 with retinopathy, and 10 with nephropathy and retinopathy),36 nondiabetic normotensive siblings, 37 parents, and three groups of healthy subjects without family history of diabetes. During the previous investigation, these patients underwent the following analyses: erythrocyte sedimentation rate analysis; blood and platelet counts (Technicon System H^*1; Bayer Diagnostics, Milan, Italy); serum and urine urea,creatinine, uric acid, electrolytes, and L-gamma-glutamyl transferase measurements; and serum bilirubin, glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, alkaline phosphatase; iron assays using BM/HITACHI SYSTEM 717 model) and reagents from Boehringer Mannheim (Mannheim, Germany). Serum transferrin, ceruloplasmin, and serum and urine albumin were quantified by the kinetic immunonephelometric method (Behring Institute nephelometer and reagents; Scoppitto, L'Aquila, Italy). Serum ferritin was measured by the IMX System (Abbott SpA, Divisione Roma, Italy). Serum copper was determined with a Varian SpectrAA (Varian Techtron, Victoria, Australia). Erythrocyte NHE activity was measured as previously described(7).

In comparison with the control subjects, the type 1 diabetic patients had lower levels of plasma uric acid (0.2 ± 0.1 vs. 0.3 ± 0.1 mmol/l, P < 0.05) and sodium (136 ± 2 vs. 140 ± 2 mEq/l, P < 0.001), higher levels of plasma potassium (4.2 ±0.3 vs. 3.9 ± 0.3 mEq/l, P < 0.001), and an over-active erythrocyte NHE (7.06 ± 1.89 vs. 5.16 ± 1.78 mmol/l red blood cell (RBC) per h, P < 0.01). Urinary sodium excretion in type 1 diabetic patients nearly reached statistical significance (median 154 vs. 175 mEq/24 h, P = 0.05). Siblings of type 1 diabetic patients showed a lower amount of circulating sodium (139 ± 2 vs. 140 ± 2 mEq/l, P < 0.05) and an enhanced erythrocyte NHE activity count (8.25± 2.78 vs. 5.34 ± 1.75 mEq/l, P < 0.001). Parents differed from control subjects only in erythrocyte NHE activity (7.88 ±2.74 vs. 6.22 ± 2.33, P < 0.01).

The multiple regression analysis included all of the biochemical measurements performed in these families and the assays previously reported in our study on oxidative stress. NHE activity was significantly correlated with erythrocyte glutathione (GSH) content, plasma advanced oxidation protein product (AOPP) concentration, basal plasma metal deactivator additive (MDA),basal RBC osmotic fragility, and the amount of MDA accumulated in the RBCs during a 3-h incubation under oxidative stress (R = 0.4, P< 0.001). Among the diabetic patients, plasma sodium concentration was strictly associated with plasma levels of glucose (R 0.6, P< 0.001), whereas among the siblings, plasma concentrations of lipoprotein(a) [Lp(a)] and fibrinogen (R = 0.5, P <0.001) were associated.

These data, with further biochemical measurements that were performed in the same experimental session and in the same study group, complete and integrate the previous study on oxidative stress in type 1 diabetic families(1). To our knowledge, this study provides the first in vivo demonstration of a significant association between oxidative stress and NHE upregulation. We were unable to reveal any abnormalities in circulating metal ions or extracellular antioxidant defenses that could favor oxidative stress in nondiabetic relatives of type 1 patients. On the other hand, we confirmed our previous finding of a generalized increase in NHE activity, which was significantly associated with both RBC and GSH content and some markers of radical-induced damage, such as plasma AOPP, MDA,RBC osmotic fragility, and RBC MDA accumulation under oxidative stress. First,intracellular GSH content is essential in maintaining the functional integrity of NHE (2, 5, 6, 9). Second, many ion transport pathways have been reported to be under redox control(2,3,4,5,6). A direct stimulatory effect of oxidative stress on NHE can be hypothesized on the basis of recent observations in hepatoma cells(6). Low concentrations of H₂O₂ activated mitogen-activated protein (MAP) kinases that stimulated NHE activity during reperfusion injury(10). Svegliati-Baroni et al.(11) gave the first demonstration in vitro of a direct stimulating effect of oxidative stress on NHE in hepatic stellate cells.

In our opinion, there are two possible mechanisms of NHE upregulation by oxidative stress: 1) MAP kinase activation of the transport system that has a protective role for restoration of intracellular pH, such as during ischemia-reperfusion (10); and 2) oxidative modifications of a cellular membrane cytoskeleton network that is considered a “solid-state,” signaling and facilitating cross-talk among multiple signaling pathways(12). Both markers of oxidative stress and erythrocyte NHE activity were increased in all members of type 1 diabetic families (probands and nondiabetic relatives included),whereas abnormalities in circulating electrolytes were observed only in type 1 diabetic patients and their siblings, thus seeming to exclude NHE as a contributing factor. Abnormalities in electrolyte handling have been reported in type 1 diabetic patients and ascribed to the metabolic control of the disease (13). Indeed, our type 1 diabetic patients showed a striking association between plasma values of sodium and glucose (translocational hyponatremia). On the contrary, we found that plasma sodium is significantly reduced also in nondiabetic siblings of type 1 diabetic patients and therefore correlated with plasma concentrations of Lp(a) and fibrinogen. Cases of hyponatremia have been correlated with inflammation (14) and increased plasma interleukin -6 concentration(15). These findings are consistent with our hypothesis of an association among inflammatory markers,oxidative stress, and susceptibility to type 1 diabetes. Moreover, the familiarly overactive NHE could be viewed independently as further evidence for the presence in these families of a redox disequilibrium in which oxidation seems to be dominant.