A new kinetic model for the mode of action of soluble and membrane-immobilized glutathione peroxidase from bovine erythrocytes--effects of selenium.

Kinetic studies on the oxidative reaction of glutathione by hydrogen peroxide were performed using soluble and membrane-bound ox erythrocyte glutathione peroxidase of various types. The effects of organic and inorganic selenium on the glutathione peroxidase activity were also examined. The kinetic behaviour of the enzyme was investigated using a coupled reaction within a relatively large range of hydrogen peroxide and glutathione concentrations. Non-parallel double-reciprocal plots were obtained which suggested that a sequential ordered rather than a ping-pong mechanism was involved. Similar results were obtained with soluble and membrane-bound enzyme, whatever the type of crosslinking used. Crosslinking was performed on a nylon support using various alkylating agents and bifunctional molecules. With all three types of immobilized enzyme thus obtained, a slight but significant increase in the Km was observed. The effects of selenium were then studied. Using soluble enzyme, a slight increase in the activity was observed in the presence of inorganic selenium (sodium selenite) but not with organic selenium (seleno-L-methionine). Inorganic selenium alone was also found to have a slight effect on the membrane-bound enzyme. An increase in the catalytic efficiency was observed when glutathione peroxidase was bound using lysine as the bifunctional agent and either glutaraldehyde or triethyloxonium tetrafluoroborate as the reticulation agent, after a three-month period of incubation.

Gas chromatographic analysis of resveratrol in plasma, lipoproteins and cells after in vitro incubations.

Resveratrol is a trihydroxystilbene present in certain red wines. It may play a role in the inhibition of lipoprotein oxidation and platelet activity. We have developed the first method to measure resveratrol in animal and human samples and to study its incorporation in vitro. After adding epicoprostanol as an internal standard, samples are subjected to lipid extraction in the presence of antioxidant and under dim light to minimize both denaturation and isomerization of the trans-resveratrol to the cis-form. Extracts were purified by cold acetone precipitation and the resveratrol-containing acetone phase was evaporated under nitrogen. The resveratrol was analyzed as a trimethylsilyl derivative by capillary gas chromatography which resolved the cis- and trans-resveratrol (6.6 and 12.9 min, respectively). Analyses of samples spiked with pure trans-resveratrol (0.1 to 10 microg) indicated that the method was specific and gave excellent linearity and recovery (96.8%) with a high reproducibility (coefficient of variation: 3.3%). The detection limit was about 50 ng/ml. Applications show that resveratrol was incorporated into blood cells and lipoproteins after in vitro incubations with plasma, lipoproteins and cells.

Comparative study of methods for measuring cholesterol in biological fluids.

A method combining enzymatic and electrochemical detection of cholesterol in biological fluids was compared with conventional detection methods: two chromatographic methods--gas chromatography or high performance liquid chromatography--and two enzymatic methods with colorimetric detection either by kinetic measurement or at the end of the reaction. For serum, enzymatic determination is accurate with both colorimetric detection methods; but for bile, colorimetric detection is difficult to perform due to interference from bile pigments. Enzymatic cholesterol determination, combined with electrochemical detection, is simpler and gives results in good agreement with those of chromatographic methods.

ESR analysis of irradiated frogs' legs and fishes.

Electron spin resonance (ESR) spectral analysis of different parts (bones, scales, jaw, etc.) from ionized (irradiated) frozen frogs' legs and fishes (brown trout and sardine) were recorded. There is always present, after treatment, a signal due to the irradiation. ESR and ENDOR experiments lead us to assign it to h1 centers from hydroxyapatite, as in the case of other irradiated meat bones. The use of ESR to prove whether one of these foods has been irradiated or not is discussed.