The pathophysiological significance of red blood cell nitric oxide concentrations in inflammatory Behçet's disease.

Abstract

Nitric oxide (NO) is a free oxygen radical with powerful vasodilator properties studied in many tissues. It is produced by NO synthases in endothelial cells upon stimulation by various agents like cytokines and tumor necrosis factor. The end-products of NO are nitrite and nitrate. Their levels are used biochemically to determine NO synthase activity. Evidence is accumulating for the role of NO in the pathophysiology of inflammatory Behcet’s disease (BD). Indeed, we first demonstrated that serum NO production is increased in active BD, suggesting a possible new activity marker. More recently, we have reported that pro-inflammatory cytokines, inducers of NO, and lipid peroxidation are increased and associated with decreased antioxidant enzyme activities in BD. Our findings were then supported by four independent investigations, one of which was from the present group. 7 Moreover, aqueous humor NO levels were found to be increased in uveitic BD patients. Furthermore, Salvarani et al . have recently shown that the GluAsp298 polymorphism of endothelial NO synthase gene is associated with BD susceptibility. Therefore, to support our previous studies, this report further investigated for the first time NO levels in red blood cells of BD patients compared with age-matched and sex-matched healthy control volunteers. A total of 20 patients with BD (16 men, four women), with a mean age of 33.4 years, and 15 healthy control subjects (12 men, three women), with a mean age of 32.1 years, were enrolled in this study. Patients with BD had to fulfil the International Study Group criteria for the diagnosis of BD. Fasting whole-blood samples were taken from BD patients and control subjects by venipuncture from an antecubital peripheral vein into heparinised plain tubes to prepare the erythrocyte sediment. The buffy coat on the erythrocyte sediment was carefully separated. The erythrocyte sediment was subsequently washed three times with 10-fold volumes of 0.9% sodium chloride solution to remove the plasma remnants. Following this, the erythrocyte sediment was treated with four-fold volumes of ice-cold deionised water to obtain hemolysate. NO is a labile compound and has a brief half-life, and therefore its detection as the native NO molecule is difficult. It is rapidly converted to the stable end-products nitrate (NO3 ) and nitrite (NO2 ) in typical oxygenated aqueous solutions and tissues. Thus, erythrocyte total nitrite levels were measured as an index of NO production. For total nitrite detection, lysate was treated with copperised cadmium in glycine buffer at pH 9.7 (2.5 /3.0 g of cadmium granules for a 4 ml reaction mixture) to reduce NO3 to NO2 . Measurement of total nitrite was based on the Griess reaction, in which a chromofore with a strong absorbance at 540 nm is formed by the reaction of nitrite with a mixture of sulphanilamide and N -(1Letter