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S-nitrosothiols (RSNO) are involved in post-translational modifications of many proteins analogous to protein phosphorylation. In addition, RSNO have many physiological roles similar to nitric oxide ((•)NO), which are presumably involving the release of (•)NO from the RSNO. However, the much longer life span in biological systems for RSNO than (•)NO suggests a dominant role for RSNO in mediating (•)NO bioactivity. RSNO are detected in plasma in low nanomolar levels in healthy human subjects. These RSNO are believed to be redirecting the (•)NO to the vasculature. However, the mechanism for the formation of RSNO in vivo has not been established. We have reviewed the reactions of (•)NO with oxygen, metalloproteins, and free radicals that can lead to the formation of RSNO and have evaluated the potential for each mechanism to provide a source for RSNO in vivo.

S-nitrosothiols present in nanomolar concentrations in cells and body fluids play an important role in vasodilation, in preventing platelet aggregation, leukocyte adhesion, and for cellular signaling. However, because of the low levels of s-nitrosothiols and interference with other nitric oxide species, reliable assays that measure both high molecular weight and low molecular weight s-nitrosothiols in plasma and red blood cells red blood cells have been difficult to develop. We have previously developed a sensitive method using Cu(II)-ascorbic acid Cu(II)-ascorbic acid at a neutral pH, which was specific for s-nitrosothiols without interference of nitrite or other NOx species. However, due to neutral pH foaming, this method was not suitable for determinations in plasma or red blood cells with high protein content. This method has now been modified by using copper (II) chloride (CuCl(2)) and ascorbic acid in glacial acetic acid. The low pH solves the foaming problem. However, protonation of nitrite under acidic conditions facilitates the formation of s-nitrosothiols. For this method to specifically measure s-nitrosothiols in the sample, the unreacted thiols are blocked by reacting with N-ethylmaleimide and nitrite is blocked by reacting with acidified sulfanilamide before being analyzed by chemiluminescence. Using this method, s-nitrosothiols have been determined in the range of 2 nM to 26 nM (mean ± SE = 10.18±2.1) in plasma and up to 88.1 nM (mean ± SE = 51.27 ± 10.5) in red blood cells.

Altered copper homeostasis and oxidative stress have been observed in patients with hepatocellular carcinoma. Non-ceruloplasmin copper, the free form, is a potent pro-oxidant than the protein bound copper. The aim of the present study was to evaluate which form of copper can be correlated with the oxidative stress in the circulation and in the malignant liver tissues of hepatocellular carcinoma patients. Hepatocellular carcinoma patients (grades II and III, n = 18) were enrolled in this study. Serum levels of total, free and bound copper, ceruloplasmin, iron, iron-binding capacity, lipid peroxidation products, and enzymatic and non-enzymatic antioxidants were quantified in serum and in malignant liver tissues and compared with those of normal samples (n = 20). A significant positive correlation between the serum non-ceruloplasmin copper and lipid peroxidation products and negative correlation with antioxidants were observed in hepatocellular carcinoma patients. In liver tissue, glutathione peroxidase, superoxide dismutase, and catalase activity were significantly decreased with concomitant elevation in oxidative stress markers. Our experiment revealed that the elevation in non-ceruloplasmin copper has high relevance with the oxidative stress than the bound copper.