Adaptation of the phosphotungstate method to determine reduced and oxidized vitamin C in blood plasma.

The phosphotungstate reagent (PTR) was used for quantitative spectrophotometric determination of physiological forms of vitamin C in blood plasma. An immediate action of PTR on the first half of the tested samples allowed to determine reduced vitamin C concentrations (I) at 700 nm. 10 mM dithiothreitol added to the second half of the samples reduced oxidized vitamin C in it--hence the total amount of this vitamin was reduced with a concentration (II) determined as above (remains of dithiothreitol were removed with N-ethylmaleimide). The difference of results (II) and (I) gave the concentration of oxidized vitamin C. The method is characterised by fault-less analytical parameters: correlation coefficients of analytical curves > 0.99, recovery factor 100.5%, variation coefficients intra- and inter-serial < 3% and < 5%, respectively, detection limit 0.05 microM. The simplicity of the method enables an easy control of the ratio of oxidized and reduced vitamin C concentrations in blood plasma--the biomarker of the level of oxidative damage to cells.

Concomitant S-, N-, and heme-nitros(yl)ation in biological tissues and fluids: implications for the fate of NO in vivo.

There is growing evidence for the involvement of nitric oxide (NO) -mediated nitrosation in cell signaling and pathology. Although S-nitrosothiols (RSNOs) have been frequently implicated in these processes, it is unclear whether NO forms nitrosyl adducts with moieties other than thiols. A major obstacle in assessing the significance of formation of nitrosated species is the limited reliability of available analytical techniques for measurements in complex biological matrices. Here we report on the presence of nitrosated compounds in plasma and erythrocytes of rats, mice, guinea pigs, and monkeys under basal conditions, in immunologically challenged murine macrophages in vitro and laboratory animals in vivo. Besides RSNOs, all biological samples also contained mercury-stable nitroso species, indicating the additional involvement of amine and heme nitros(yl)ation reactions. Significant differences in the amounts and ratios of RSNOs over N- and heme-nitros(yl)ated compounds were found between species and organs. These observations were made possible by the development of a novel gas-phase chemiluminescence-based technique that allows detection of nitroso species in tissues and biological fluids without prior extraction or deproteinization. The method can quantify as little as 100 fmol bound NO and has been validated extensively for use in different biological matrices. Discrimination between nitrite, RSNOs, and N-nitroso or nitrosylheme compounds is accomplished by use of group-specific reagents. Our findings suggest that NO generation in vivo leads to concomitant formation of RSNOs, nitrosamines, and nitrosylhemes with considerable variation between rodents and primates, highlighting the difficulty in comparing data between different animal models and extrapolating results from experimental animals to human physiology.

Separation of o-phthalaldehyde-mercaptoethanol derivatives of amino acids from blood plasma on reversed-phase Nova-Pak C18 cartridges.

A separation of 25 o-phthalaldehyde-mercaptoethanol derivatives of primary amino acids in plasma prepared from human blood has been developed for Waters 10 cm x 0.8 cm I.D., 4-microns Nova-Pak C18 Radial-Pak cartridges. A binary gradient system with solvent-switching capability for the A pump is required. Computer methodologies have been utilized to develop mobile phase mixtures of phosphate buffer (pH 6.9), water, methanol and tetrahydrofuran. Advantages of the method include simple sample preparation, fast turnover time (67 min including the pre-column Autotag derivatization procedure) and exceptional column durability (several hundred analyses).

Mechanism of augmentation of the antibody response in vitro by 2-mercaptoethanol in murine lymphocytes. III. Serum-bound and oxidized 2-mercaptoethanol are available for the augmentation.

The fetal calf serum (FCS) that was incubated with 2-mercaptoethanol (2-ME) followed by the removal of free 2-ME could support the antibody response to sheep erythrocytes in vitro as effectively as native FCS plus 2-ME. The supporting activity of 2-ME-pulsed FCS was reversibly abrogated by the treatment with dithiothreitol followed by dialysis. In addition, iodoacetamide-treated FCS did not acquire the supportiveness by 2-ME pulsing. These observations suggest that the activity of 2-ME-pulsed FCS would be due to the mixed disulfide between 2-ME and FCS components. On the other hand, the disulfide form of 2-ME (2-MEox) could also augment the antibody response as effectively as fresh 2-ME (the reduced form). These derivatized forms of 2-ME as well as fresh 2-ME was found to stimulate the transport of [35S]cystine into murine lymphocytes when the uptake was examined by the long-term experiments (24 hr). These stimulations were thought to be mediated by the formation of the mixed disulfide between 2-ME and cysteine because the lymphocytes promoted the reaction of [35S]cystine with 2-MEox- or 2-ME-pulsed FCS to produce the mixed disulfide that had been shown to be taken up by the lymphocytes four to five times more rapidly than cystine. Therefore, it was suggested that 2-MEox, and 2-ME-pulsed FCS could augment the antibody response in a similar fashion to 2-ME by stimulating the uptake of cystine, an essential amino acid.