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

The role of oxidative stress resulting from production of reactive oxygen species and/or from suppression of the cellular antioxidant capacity 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 mechanisms. For studying this system in parasitic diseases such as malaria new or modified methods are required. Total glutathione comprising GSH and glutathione disulphide (GSSG) in blood samples was assayed as follows. One volume of blood (greater than or equal to 10 mu l) is mixed with two volumes of 5% sulphosalicylic acid; after centrifugation (5 min, 10000 g), 10 mu l of supernatant is taken for spectrophotometric analysis using the 5,5'-dithiobis(2-nitrobenzoate) (DTNB)-glutathione recycling assay. When compared with the original method, the procedure reported here is more sensitive, less time-consuming, avoids unfavourable pH-values and leads to a sample which when frozen is stable for months. 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 controls (2.11+/-0.45 mM. P<0.011. The findings are contrasted with P. falciparum cultures in vitro where glutathione levels are known to be elevated. 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 potential since the dithiol-disulphide interconversion at the enzyme's active site is associated with a change in colour. Here we describe a simple overlay technique for crystallizing recombinant human glutathione reductase. This procedure may also be applicable to the crystallization of other proteins.