Extracellular Glutathione Peroxidase - Purification, Immunoassay, Nutritional Regulation and Clinical Aspects

Detta är en avhandling från Department of Applied Nutrition and Food Chemistry, Lund University, P.O.Box 124, S-221 00 Sweden

Sammanfattning: Extracellular glutathione peroxidase (eGSHPx) is a selenoprotein catalysing the reduction of hydrogen peroxide, organic peroxides and lipid peroxides in the presence of glutathione. A scaled-up purification procedure for obtaining eGSHPx from human plasma was established based on ammonium sulphate fractionation, hydrophobic interaction chromatography, ion-exchange chromatography and gel chromatography. The purified eGSHPx was used for immunoassay development and for studies of its crystallization and its reducing substrates. Thioredoxin reductase, and the thioredoxin and glutaredoxin systems seem to be more efficient reducing substrates than glutathione. Anti-eGSHPx antisera were generated by immunizing rabbits with eGSHPx. A radioimmunoassay of eGSHPx was developed, which had the advantages of increased specificity, easier handling of samples and requiring smaller sample volumes. Sera from eight animal species did not interfere with the assay. The amount of serum eGSHPx measured by radioimmunoassay was closely correlated to its activity in serum samples with widely varying selenium concentration. Among 50-69-year-old Swedish subjects the concentration of eGSHPx tended to be higher in men than in women, whereas serum selenium tended to be higher in women. Serum selenoprotein P or urinary selenium excretion did not differ between men and women. Serum selenium was positively correlated to eGSHPx in men, but not in women. The intakes of fish and milk products were significantly associated with selenium status in women. In another study of middle-aged men, fish intake was found to be positively associated to plasma selenium, but apparently not to the levels of functional selenoproteins in plasma. In patients treated with home parenteral nutrition without selenium supplementation the mean concentrations in serum of both eGSHPx and selenium were only half of the reference levels. Also, eGSHPx was correlated to selenoprotein P in serum, suggesting that both selenoproteins could be used as functional indices of selenium status in patients on home parenteral nutrition. eGSHPx was also demonstrated in aqueous humour. The level of aqueous humour eGSHPx was 18% of its level in serum, and it had a positive association with serum selenium. There was no relation between eGSHPx in aqueous humour and that in serum. Serum creatinine was inversely associated with serum eGSHPx, but not with aqueous humour eGSHPx, in agreement with the fact that the kidney is the main source of serum eGSHPx. These findings suggest that the maintenance of eGSHPx levels in the two fluids is controlled by different mechanisms in addition to selenium status.

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