REDOX PROCESSES IN MALARIA AND OTHER PARASITIC DISEASES - DETERMINATION OF INTRACELLULAR GLUTATHIONE

The role of oxidative stress resulting from production of reactive oxy gen species and/or from suppression of the cellular antioxidant capaci ty in parasitic infections is shortly reviewed. The experimental part of the paper deals with the glutathione (GSH)- glutathione reductase ( GR) system, a cornerstone of intracellular antioxidant defence mechani sms. For studying this system in parasitic diseases such as malaria ne w or modified methods are required. Total glutathione comprising GSH a nd glutathione disulphide (GSSG) in blood samples was assayed as follo ws. One volume of blood (greater than or equal to 10 mu l) is mixed wi th two volumes of 5% sulphosalicylic acid; after centrifugation (5 min , 10000 g), 10 mu l of supernatant is taken for spectrophotometric ana lysis using the 5,5'-dithiobis(2-nitrobenzoate) (DTNB)-glutathione rec ycling assay. When compared with the original method, the procedure re ported here is more sensitive, less time-consuming, avoids unfavourabl e pH-values and leads to a sample which when frozen is stable for mont hs. In a pilot study, the method was applied to 14 patients suffering from malaria caused by Plasmodium 2 falciparum. The concentrations of erythrocyte glutathione were significantly decreased in the patients ( 1.42+/-0.37 mM, mean+/-SD) when compared to age- and sex-matched contr ols (2.11+/-0.45 mM. P<0.011. The findings are contrasted with P. falc iparum cultures in vitro where glutathione levels are known to be elev ated. Based on the characteristics of GR a concept of determining the redox state of single cells is introduced. Microcrystals of the enzyme are expected to be suitable indicators for the intracellular redox po tential since the dithiol-disulphide interconversion at the enzyme's a ctive site is associated with a change in colour. Here we describe a s imple overlay technique for crystallizing recombinant human glutathion e reductase. This procedure may also be applicable to the crystallizat ion of other proteins.