Impaired Synthesis Is Not the Reason for Decreased Activity of Extracellular Superoxide Dismutase in Patients with Diabetes

Background

The aim of the study was to find the cause of decreased activity of extracellular superoxide dismutase (EC SOD) in patients with diabetes—is it the decreased synthesis or increased glycation?

Methods

Total EC SOD activity, the activity of its fractions (A, B, and C) and its glycated form were determined in basal state and 30 min after intravenous (i.v.) administration of 50 mg of heparin. Patients were given i.v. heparin at a dose of 10,000 IU (100 mg) each 6 h for at least 3 days, and the activity of EC SOD was determined before the first heparin administration, just before each subsequent administration, and 30 min after heparin administration.

Results

Pre- and postheparinic activities of EC SOD and its fraction C in the group of patients with diabetes were significantly lower (p <0.001) than in control group. Preheparinic activities of EC SOD did not differ between the examined groups of patients. The postheparinic activities were different during the first 18 h of treatment. They were significantly lower in the group of patients with diabetes. During the following hours, after subsequently administered doses, there were no differences in the activity of EC SOD between the examined groups. Decline of EC SOD activity was observed after administration of repeated doses of heparin both in the examined and in the control groups.

Conclusions

The decrease of extracellular superoxide dismutase activity in diabetes develops due to excessive glycation but not due to impaired synthesis. Therefore, appropriate glycemic control can lead to normalization of EC SOD activity.

Introduction

It is assumed that non-enzymatic protein glycation can lead to the development of diabetic angiopathy by the following mechanisms: 1) disturbances in microcirculation hemodynamics due to increased plasma viscosity and decreased erythrocyte compliance to deformation 1, 2; 2) activation of coagulation system due to decreased activity of glycated proteins: antithrombin III, protein C, and decreased fibrinolytic activity due to plasminogen glycation 1, 3; 3) increase of vascular permeability caused by vasoactive factors released by macrophages stimulated with glycated proteins 3, 4; 4) thickening of the vascular basement membrane as a result of crossing bond between the molecules of glycated collagen and between the collagen and glycated plasma proteins, and as a result of elongated half-life of such modified proteins and accumulation of immunological deposits in basement membrane 1, 2, 3, 5, 6.

Morphological changes analogical to the first phase of diabetic microangiopathy (i.e., increased vascular permeability in the venous part of microcirculation) appear as a result of the insertion of the system (enzyme−substrate) generating superoxide radical into the bloodstream 7, 8 or after histamine administration (9). Free oxygen species are also released in the course of non-enzymatic protein glycation 10, 11, 12, 13. In addition, glycated proteins can themselves generate free radicals (14). The activity of cytoplasmic superoxide dismutase (CuZnSOD) that “scavenges” superoxide radical in diabetes decreases as a result of its glycation (15).

Superoxide dismutase, the specific scavenger of superoxide radical, protects against the pathological increase of microvenule permeability 8, 16, 17. Similar action can be exhibited by intravenously administered heparin (18). Repeated doses of heparin are less and less effective and produce the expected protective result only after a few days interval in heparin administration (18). Extracellular superoxide dismutase (EC SOD–E.C.1.15.1.1) is the only enzyme that breaks down the superoxide radical in extracellular space 19, 20, 21, 22. It is released by fibroblasts into the bloodstream and appears in three fractions: A, B and C, which differ in their in vivo affinity to heparan sulfate and in vitro affinity to heparin-Sepharose 13, 24, 25, 26, 27.

EC SOD C appears to have the strongest affinity, EC SOD B affinity is weaker, and fraction A does not show any affinity to heparin-Sepharose 13, 24, 27.

EC SOD C, which shows the strongest activity in plasma, is the only enzyme that protects the surface of the vessels from the action of superoxide radical 13, 19, 22, 24, 28, 29. The doses of heparin releasing EC SOD C from the vascular wall must be 10-fold higher than those that can release diamine oxidase (DAO) or lipoprotein lipase 24, 25. The studies of Adachi et al. (30) showed that non-enzymatic EC SOD C glycation led to its decreased affinity to heparin-Sepharose without any changes in its activity. Patients with diabetes appear to have a greater percentage of glycated extracellular superoxide dismutase (30). Superoxide dismutase protects from the increase in vascular permeability caused by superoxide radical (8) and by ischemia (31). The EC SOD C “contents” are, due to its glycation, in such a state that can lead to increased concentration of superoxide radical released on the vascular surface, e.g., by DAO.

Therefore, the reactions leading to the release of superoxide radical can start the pathological events leading in consequence to the increased vascular permeability which initiates the development of diabetic angiopathy.

The aim of the study was to find the cause of decreased activity of superoxide dismutase in patients with diabetes—is it the decreased synthesis or increased glycation?