Modification of the glyoxalase system in clinical diabetes mellitus.

The glyoxalase system cata lyses t h e conversion o f methy lg lyoxal t o 0 l a c t i c acid, v i a the in termediate S0-1 a c t o y l g l u t a t h i one. It comprises two enzymes , glyoxalase I and glyoxalase 11, and a c a t a l y t i c amount o f reduced g lu tath ione. Glyoxalase I cata lyses t h e format ion o f S-Dlactoy lg lu tath ione from methy lg lyoxal and reduced g lu tath ione. Glyoxalase I 1 cata lyses t h e hyd ro l ys i s o f S-D-1 ac toy l g l u ta th ione t o D-1 ac ta te and reduced g lu tath ione. The glyoxalase system i s present i n the cytosol o f a l l c e l l s [l]. Recent research has shown t h a t t he g lyoxalase system i s modi f ied du r ing hyperglycaemia i n human red b lood c e l l s i n v i t r o [2], and i n red b lood c e l l s from (ob/ob) obese mice and streptozotocininduced d i a b e t i c mice i n vivo [3]. The mod i f i ca t i on o f t he glyoxalase system has been l i n k e d t o t h e development o f c l i n i c a l d i a b e t i c compl icat ions [l]. We descr ibe here c h a r a c t e r i s t i c s o f t h e red blood c e l l g lyoxalase system i n c l i n i c a l d iabetes m e l l i t u s . Blood samples were taken from 43 insul in-dependent d i a b e t i c p a t i e n t s (IDDM) , 107 noninsul in-dependent d i a b e t i c p a t i e n t s (NIDDM) and 21 normal con t ro l s . The blood samples were s tored a t -196OC p r i o r t o ana lys i s as p e r c h l o r i c ac id e x t r a c t s (analyzed f o r methy lg lyoxal and Dlactate content) o r n a t i v e s t a t e ( f o r assay o f glyoxalase a c t i v i t i e s and measurement o f t h e concentrat ion o f GSH) f o r upto 6 weeks and showed no s i g n i f i c a n t change i n glyoxalase a c t i v i t i e s and metabol i te concentrat ions dur ing t h i s per iod. The concentrat ion o f methy lg lyoxal i n blood samples was determined by d e r i v a t i s a t i o n w i t h 1,2-diamino-4,5dimethoxy-benzene and a n a l y t i c a l reverse hase h igh performance 1 i q u i d chromatography (HPLCf o f t h e u inoxa l i ne adduct w i t h spectrophotometr ic de tec t i on 1141 . The concentrat ion o f S-olactoy lg lu tath ione i n blood samples was determined, a f t e r s t rong anion exchange so l i d phase ex t rac t i on , by reverse phase HPLC w i t h spectrophotometr ic de tec t i on a t 233 nm [5]. The concentrat ion o f o l ac ta te i n b lood samples was determined by endpoint enzymatic assay w i t h o l a c t i c dehydrogenase and f l uo romet r i c de tec t i on [6]. The concentrat ion o f reduced g lu ta th ione (GSH) i n b lood samples was determined as non-protein su lphydry l by reac t i on w i t h Ellman's reagent [7]. The a c t i v i t i e s o f g lyoxalase I and glyoxalase I 1 i n red blood c e l l s were determined by the spectrophotometr ic methods descr ibed