Emerging role of thiamine therapy for prevention and treatment of early-stage diabetic nephropathy.

Thiamine supplementation may prevent and reverse early-stage diabetic nephropathy. This probably occurs by correcting diabetes-linked increased clearance of thiamine, maintaining activity and expression of thiamine pyrophosphate-dependent enzymes that help counter the adverse effects of high glucose concentrations-particularly transketolase. Evidence from experimental and clinical studies suggests that metabolism and clearance of thiamine is disturbed in diabetes leading to tissue-specific thiamine deficiency in the kidney and other sites of development of vascular complications. Thiamine supplementation prevented the development of early-stage nephropathy in diabetic rats and reversed increased urinary albumin excretion in patients with type 2 diabetes and microalbuminuria in two recent clinical trials. The thiamine monophosphate prodrug, Benfotiamine, whilst preventing early-stage development of diabetic nephropathy experimentally, has failed to produce similar clinical effect. The probable explanations for this are discussed. Further definitive trials for prevention of progression of early-stage diabetic nephropathy by thiamine are now required.

Increased protein damage in renal glomeruli, retina, nerve, plasma and urine and its prevention by thiamine and benfotiamine therapy in a rat model of diabetes.

AIMS/HYPOTHESIS: The aim of this study was to quantify protein damage by glycation, oxidation and nitration in a rat model of diabetes at the sites of development of microvascular complications, including the effects of thiamine and benfotiamine therapy. METHODS: Diabetes was induced in male Sprague-Dawley rats by 55 mg/kg streptozotocin and moderated by insulin (2 U twice daily). Diabetic and control rats were given thiamine or benfotiamine (7 or 70 mg kg(-1) day(-1)) over 24 weeks. Plasma, urine and tissues were collected and analysed for protein damage by stable isotopic dilution analysis MS. RESULTS: There were two- to fourfold increases in fructosyl-lysine and AGE content of glomerular, retinal, sciatic nerve and plasma protein in diabetes. Increases in AGEs were reversed by thiamine and benfotiamine therapy but increases in fructosyl-lysine were not. Methionine sulfoxide content of plasma protein and 3-nitrotyrosine content of sciatic nerve protein were increased in diabetes. Plasma glycation free adducts were increased up to twofold in diabetes; the increases were reversed by thiamine. Urinary excretion of glycation, oxidation and nitration free adducts was increased by seven- to 27-fold in diabetes. These increases were reversed by thiamine and benfotiamine therapy. CONCLUSIONS/INTERPRETATION: AGEs, particularly arginine-derived hydroimidazolones, accumulate at sites of microvascular complication development and have markedly increased urinary excretion rates in experimental diabetes. Thiamine and benfotiamine supplementation prevented tissue accumulation and increased urinary excretion of protein glycation, oxidation and nitration adducts. Similar effects may contribute to the reversal of early-stage clinical diabetic nephropathy by thiamine.

High-dose thiamine therapy for patients with type 2 diabetes and microalbuminuria: a randomised, double-blind placebo-controlled pilot study.

AIMS/HYPOTHESIS: High-dose supplements of thiamine prevent the development of microalbuminuria in experimental diabetes. The aim of this pilot study was to assess whether oral supplements of thiamine could reverse microalbuminuria in patients with type 2 diabetes. METHODS: Type 2 diabetic patients (21 male, 19 female) with microalbuminuria were recruited at the Diabetes Clinic, Sheikh Zayed Hospital, Lahore, Pakistan, and randomised to placebo and treatment arms. Randomisation was by central office in sequentially numbered opaque, sealed envelopes. Participants, caregivers and those assessing the outcomes were blinded to group assignment. Patients were given 3 x 100 mg capsules of thiamine or placebo per day for 3 months with a 2 month follow-up washout period. The primary endpoint was change in urinary albumin excretion (UAE). Other markers of renal and vascular dysfunction and plasma concentrations of thiamine were determined. RESULTS: UAE was decreased in patients receiving thiamine therapy for 3 months with respect to baseline (median -17.7 mg/24 h; p < 0.001, n = 20). There was no significant decrease in UAE in patients receiving placebo after 3 months of therapy (n = 20). UAE was significantly lower in patients who had received thiamine therapy compared with those who had received placebo (30.1 vs 35.5 mg/24 h, p < 0.01) but not at baseline. UAE continued to decrease in the 2 month washout period in both groups, but not significantly. There was no effect of thiamine treatment on glycaemic control, dyslipidaemia or BP. There were no adverse effects of therapy. CONCLUSIONS/INTERPRETATION: In this pilot study, high-dose thiamine therapy produced a regression of UAE in type 2 diabetic patients with microalbuminuria. Thiamine supplements at high dose may provide improved therapy for early-stage diabetic nephropathy. TRIAL REGISTRATION: CTRI (India) CTRI/2008/091/000112. FUNDING: Pakistan Higher Education Commission.

Overexpression of glyoxalase-I in bovine endothelial cells inhibits intracellular advanced glycation endproduct formation and prevents hyperglycemia-induced increases in macromolecular endocytosis.

Methylglyoxal (MG), a dicarbonyl compound produced by the fragmentation of triose phosphates, forms advanced glycation endproducts (AGEs) in vitro. Glyoxalase-I catalyzes the conversion of MG to S-D-lactoylglutathione, which in turn is converted to D-lactate by glyoxalase-II. To evaluate directly the effect of glyoxalase-I activity on intracellular AGE formation, GM7373 endothelial cells that stably express human glyoxalase-I were generated. Glyoxalase-I activity in these cells was increased 28-fold compared to neo-transfected control cells (21.80+/-0.1 vs. 0. 76+/-0.02 micromol/min/mg protein, n = 3, P < 0.001). In neo-transfected cells, 30 mM glucose incubation increased MG and D-lactate concentration approximately twofold above 5 MM (35.5+/-5.8 vs. 19.6+/-1.6, P < 0.02, n = 3, and 21.0+/-1.3 vs. 10.0+/-1.2 pmol/ 10(6) cells, n = 3, P < 0.001, respectively). In contrast, in glyoxalase-I-transfected cells, 30 mM glucose incubation did not increase MG concentration at all, while increasing the enzymatic product D-lactate by > 10-fold (18.9+/-3.2 vs. 18.4+/- 5.8, n = 3, P = NS, and 107.1+/-9.0 vs. 9.4+/-0 pmol/10(6) cells, n = 3, P < 0.001, respectively). After exposure to 30 mM glucose, intracellular AGE formation in neo cells was increased 13.6-fold (2.58+/-0.15 vs. 0.19+/-0.03 total absorbance units, n = 3, P < 0.001). Concomitant with increased intracellular AGEs, macromolecular endocytosis by these cells was increased 2.2-fold. Overexpression of glyoxalase-I completely prevented both hyperglycemia-induced AGE formation and increased macromolecular endocytosis.

Modification of the glyoxalase system during the functional activation of human neutrophils.

The glyoxalase system catalyses the metabolism of methylglyoxal to D-lactic acid, via the intermediate S-D-lactoylglutathione. It is present in human neutrophils and undergoes a significant modification during functional activation--induction of chemotaxis, phagocytosis and degranulation. During the activation of neutrophils with serum-opsonised zymosan and the tumour-promoting phorbol diester 12-O-tetradecanoylphorbol 13-acetate, the activity of glyoxalase I increases and the activity of glyoxalase II decreases by 20-40% of their activities in resting cells, in the initial 10 min of the activation period. Determination of the Michaelis constant, Km, and the apparent maximum velocity, Vmax, for these enzymatic reactions indicates that the change in activity is due to a non-competitive activation and inhibition of glyoxalase I and glyoxalase II, respectively. This is consistent with a modification of the glyoxalase enzyme protein during the activation response. This modification occurs under aerobic and anaerobic incubation conditions. The concentration of S-D-lactoylglutathione increases approx. 100% of the resting cell concentration during the initial 10 min of the activation period. The presence of S-D-lactoylglutathione in neutrophils may be related to its ability to stimulate microtubule assembly.

Possible role for metallothionein in protection against radiation-induced oxidative stress. Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals.

Rabbit liver metallothionein-1 (Mr 6500), which contains zinc and/or cadmium ions, appears to scavenge free hydroxyl (.OH) and superoxide (O-.2) radicals produced by the xanthine/xanthine oxidase reaction much more effectively than bovine serum albumin (Mr 65 000) which was used as a control. Kinetic competition studies between metallothionein and either a spin trap for .OH or ferricytochrome c for O-.2 radicals, gave bimolecular rate constants of the order of kOH/MT approximately equal to 10(12) M-1 X s-1 and kO-2/MT approximately equal to 5 X 10(5) M-1 X s-1, respectively. The former value suggests that all 20 cysteine sulfur atoms are involved in this quenching process and that they all act in the diffusion control limit. The aerobic radiolysis of an aqueous solution of metallothionein, generating O-.2 and .OH radicals, induced metal ion loss and thiolate oxidation. These effects could be reversed by incubation of the irradiated protein with reduced glutathione and the appropriate bivalent metal ion. Metallothionein appears to be an extraordinarily efficient .OH radical scavenger even when compared to proteins 10-50-times its molecular weight. Moreover, hydroxyl radical damage to metallothionein appears to occur at the metal-thiolate clusters, which may be repaired in the cell by reduced glutathione. Metallothionein has the characteristics of a sacrificial but renewable cellular target for .OH-mediated cellular damage.

The effect of glyceraldehyde on red cells. Haemoglobin status, oxidative metabolism and glycolysis.

Glyceraldehyde induces changes in the flux of glucose oxidised through the hexose monophosphate pathway, the concentrations of intermediates in the Embden-Meyerhoff pathway, the oxidative status of haemoglobin and levels of reduced and oxidised pyridine nucleotides and glutathione in red cells. Glyceraldehyde autoxidises in the cellular incubations, consuming oxygen and producing glyoxalase I- and II-reactive materials. Major fates of glyceraldehyde in red cells appear to be: (i) adduct formation with reduced glutathione and cellular protein; (ii) autoxidation and reaction with oxyhaemoglobin and pyridine nucleotides, and (iii) phosphorylation of D-glyceraldehyde and entry into the glycolytic pathway as glyceraldehyde 3-phosphate. The production of glycerol from glyceraldehyde by red cell L-hexonate dehydrogenase appears not to be a major reaction of glyceraldehyde in red cells. These results indicate that high concentrations of glyceraldehyde (1-50 mM) may induce oxidative stress in red cells by virtue of the spontaneous autoxidation of glyceraldehyde, forming hydrogen peroxide and alpha-ketoaldehydes (glyoxalase substrates). The implications of glyceraldehyde-induced oxidative stress for the in vitro anti-sickling effect of DL-glyceraldehyde and for the polyol pathway metabolism of glyceraldehyde are discussed.

Free radical production from the aerobic oxidation of reduced pyridine nucleotides catalysed by phenazine derivatives.

Free radical production from the reaction of reduced pyridine nucleotides with phenazine derivatives in aerobic media at pH 7.5 has been studied by ESR spectroscopy and the ESR technique of spin trapping. With the spin trapping agent, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), the oxidation of NADH and NADPH catalysed by phenazine methosulphate, phenazine ethosulphate and 1-methoxyphenazine methosulphate gave exclusively the hydroxyl radical spin adduct of DMPO, 2-hydroxy-5,5-dimethylpyrrolidino-1-oxyl (DMPO-OH). DMPO-OH production was inhibited from these systems by catalase and sodium benzoate whereas superoxide dismutase gave a small increase in the rate of DMPO-OH production. NADH gives a higher rate of DMPO-OH production than NADPH with initial rates of DMPO-OH production in the order 1-methoxyphenazine methosulphate greater than phenazine ethosulphate greater than phenazine methosulphate. However, for an oxygen-limited system, the maximum DMPO-OH concentration attained varied in the order 1-methoxyphenazine methosulphate greater than phenazine methosulphate greater than phenazine ethosulphate. DMPO-OH production occurred in both the aerobic and anaerobic phases of the reaction with these phenazine derivatives. A similar system with pyocyanine gave DMPO-OH and the superoxide spin adduct of DMPO, 2-hydroperoxy-5,5-dimethylpyrrolidino-l-oxyl (DMPO-OOH). Addition of superoxide dismutase to this system stimulated the rate of DMPO-OH production and inhibited DMPO-OOH production. Addition of catalase and sodium benzoate decreased the production of DMPO-OH only. No DMPO-OH production was observed in the anaerobic phase of the reaction. The auto-oxidation of fully reduced phenazine methosulphate, 5,10-methylhydrophenazine methosulphate, produced the phenazine methosulphate radical cation PMSH+ and DMPO-OH in the presence of DMPO. A mechanism for the auto-oxidation of reduced phenazine derivatives is proposed where superoxide production occurs in discrete steps in the auto-oxidation of fully reduced pyocyanine whereas for the auto-oxidation of fully reduced phenazine methosulphate, phenazine ethosulphate and 1-methyoxyphenazine methosulphate, the production of superoxide appears masked by the rapid further reduction to hydrogen peroxide.

The effect of spin traps on phenylhydrazine-induced haemolysis.

Spin-trapping agents have been used to study the involvement of free radicals in phenylhydrazine-induced haemolysis. Spin traps were found to decrease the rate of oxygen uptake and the rate of haemoglobin oxidation in the reaction of phenylhydrazine with oxyhaemoglobin. Spin traps were also found to inhibit haemolysis and lipid peroxidation in phenylhydrazine-treated fresh human erythrocytes, with a concomitant production of phenyl radical spin adducts. Lipophilic spin traps were found to be more effective inhibitors of haemolysis than their hydrophilic analogues.

Free radical involvement in the oxidative phenomena induced by tert-butyl hydroperoxide in erythrocytes.

Free radical involvement in the oxidative events induced by tert-butyl hydroperoxide in erythrocytes has been demonstrated by the use of the electron spin resonance technique of spin trapping with the spin trap 5.5-dimethyl-1-pyrroline-N-oxide (DMPO). The reactions of tert-butyl hydroperoxide with haemoglobins and intact cell systems were studied. Oxyhaemoglobin-containing system showed exclusive production of the t-butyloxy radical spin adduct of DMPO (DMPO-OBut), indicating t-butyloxy radical production. Methaemoglobin-containing systems showed the production of an oxidised derivative of DMPO, 5,5-dimethyl-2-ketopyrrolidino-1-oxyl (DMPOX)-previously associated with the generation of highly oxidised haem-iron. Carbon monoxyhaemoglobin-containing systems show the production of both DMPO-OBut and DMPOX but markedly slower than in either of the other haemoglobin systems. Generally, free radical production in haemoglobin systems was faster than in intact cell systems, indicating a membrane transport rate-limiting step for the tert-butyl hydroperoxide-mediated effects. Data from the use of free radical scavengers to inhibit DMPO-OBut production was consistent with the known reactivities of the scavengers toward t-butyloxy radicals. These and previously reported results (Trotta, R. J., Sullivan, S. G. and Stern, A. (1981) Biochim. Biophys. Acta 679, 230-237 and (1982) Biochem. J. 204, 405-415) implicate important roles for t-butyloxy radicals and haem intermediates in tert-butyl hydroperoxide-induced lipid peroxidation and haemoglobin oxidation in erythrocytes, respectively.

Modification of the glyoxalase system in human HL60 promyelocytic leukaemia cells during differentiation to neutrophils in vitro.

The glyoxalase system of human promyelocytic leukaemia HL60 cells was substantially modified during differentiation to neutrophils. The activity of glyoxalase I was decreased and the activity of glyoxalase II was markedly increased relative to the level in control HL60 promyelocytes. There was a decrease in the apparent maximum velocity, Vmax, of glyoxalase I, and an increase in the Vmax of glyoxalase II. The apparent Michaelis constants for both enzymes remained unchanged. The flux of intermediates metabolised via the glyoxalase system increased during differentiation, as judged by the formation of D-lactic acid, whereas the percentage of glucotriose metabolised via the glyoxalase system remained unchanged. The cellular concentrations of the glyoxalase substrates, methylglyoxal and S-D-lactoylglutathione, were markedly decreased during differentiation. The maturation of HL60 promyelocytes is associated with an increased ability to metabolise S-D-lactoylglutathione by glyoxalase II and a concomitant decrease in the mean intracellular concentrations of S-D-lactoylglutathione and methylglyoxal. The maintenance of a high concentration of S-D-lactoylglutathione in HL60 promyelocytes may be related to the status of the microtubular cytoskeleton, since S-D-lactoylglutathione potentiates the GTP-promoted assembly of microtubules.

Methylglyoxal-modified arginine residues--a signal for receptor-mediated endocytosis and degradation of proteins by monocytic THP-1 cells.

Non-enzymatic glycosylation or glycation of proteins to form advanced glycation endproducts (AGE) has been proposed as a process which provides a signal for the degradation of proteins. Despite this, the AGE which act a recognition factor for receptor-mediated endocytosis and degradation of glycated proteins by monocytes and macrophages has not been identified. Methylglyoxal, a reactive alpha-oxoaldehyde and physiological metabolite, reacted irreversibly with arginine residues in proteins to form Ndelta-(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine and Ndelta-(5-methyl-4-imidazolon-2-yl)ornithine residues. Human serum albumin minimally-modified with methylglyoxal (MG(min)-HSA) was bound by cell surface receptors of human monocytic THP-1 cells in vitro at 4 degrees C: the binding constant K(d) value was 377 +/- 35 nM and the number of receptors per cell was 5.9 +/- 0.2 X 10(5) (n = 12). N alpha-Acetyl-Ndelta-(5-hydro-5-methyl-4-imidazolon-2-yl)orni thine displaced MG(min)-HSA from THP-1 cells, suggesting that the Ndelta-(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine residue was the receptor recognition factor. At 37 degrees C, MG(min)-HSA was internalised by THP-1 cells and degraded. Similar binding and degradation of human serum albumin modified by glucose-derived AGE was found but only when highly modified. MG(min)-HSA, therefore, is the first example of a protein minimally-modified by AGE-like compounds that binds specifically to monocyte receptors. The irreversible modification of proteins by methylglyoxal is a potent signal for the degradation of proteins by monocytic cells in which the arginine derivative, Ndelta-(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine, is the receptor recognition factor. This factor is not present in glucose-modified proteins.

The production of oxygen-centered radicals by bacillus-Calmette-Guerin-activated macrophages. An electron paramagnetic resonance study of the response to phorbol myristate acetate.

The spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide of free radicals formed from Bacillus-Calmette-Guerin elicited peritoneal macrophages stimulated with phorbol myristate acetate resulted in the formation of a superoxide and hydroxyl spin adducts. The formation of both spin adducts was inhibited by copper/zinc superoxide dismutase. Only 70% of the hydroxyl spin adduct could be inhibited by catalase or the scavenger dimethyl sulfoxide. This suggests that the production of hydroxyl radicals involves prior formation of both superoxide radicals and hydrogen peroxide, implicating a Fenton catalysed Haber-Weiss reaction. The metal scavenger desferrioxamine also reduced the hydroxyl radical signal by 70%. The unaccounted 30% hydroxyl radical-like signals are probably due to carbon-centered free radicals formed by the lipoxygenase reaction. Spin trapping in the presence of the lipid-soluble spin trap, 5-octadecyl-5,3,3-trimethyl-1-pyrroline-N-oxide, resulted in a spectrum consistent with the presence of an oxaziridine nitroxide. This results from the free radical-induced cyclisation of a nitrone with an unsaturated fatty acid.

Enhanced production of hydroxyl radicals by the xanthine-xanthine oxidase reaction in the presence of lactoferrin.

The generation of hydroxyl radicals by the xanthine-xanthine oxidase reaction (C. Beauchamp and I. Fridovich (1970) J. Biol. Chem. 245, 4641-1616) has been shown to be increased by iron-saturated lactoferrin isolated from pig neutrophils. Hydroxyl radical production, measured by EPR spin trapping and by ethylene production from alpha-keto-gamma-methiol butyric acid, has been demonstrated to be produced by a Fenton-type Haber-Weiss reaction catalysed by lactoferrin. The possibility that lactoferrin catalyses such a reaction in vivo is considered.

The production of free radicals during the autoxidation of cysteine and their effect on isolated rat hepatocytes.

Autoxidizing cysteine has been shown to produce thiyl and hydroxyl radicals. Hydrogen peroxide increased the yield of both radicals which was inhibited by catalase but stimulated by copper/zinc superoxide dismutase. This effect is due to increased hydrogen peroxide production by copper/zinc superoxide dismutase as a result of superoxide dismutation. The production of superoxide radicals could not be detected probably because of its low reactivity, however, measurement of oxygen uptake and reduction of ferricytochrome c by autoxidizing cysteine clearly implicate the involvement of super oxide radicals. The production of hydroxyl radicals is postulated to proceed through a fenton reaction, however, this may not necessarily be metal ion controlled. Autoxidizing cysteine disrupts the integrity of hepatocytes causing release of glutathione, adenosine triphosphate and lactate dehydrogenase indicating that it is of little use as a therapeutic agent.

Teratogenicity of 3-deoxyglucosone and diabetic embryopathy.

The increased rate of embryonic dysmorphogenesis in diabetic pregnancy is correlated with the severity and duration of the concurrent hyperglycemia during early gestation. Whole embryo culture was used to investigate a possible association of hyperglycemia-induced disturbances of embryo development with tissue levels of the three alpha-oxoaldehydes: glyoxal, methylglyoxal, and 3-deoxyglucosone (3-DG). Rat embryos exposed to high glucose levels in vitro showed severe dysmorphogenesis and a 17-fold increased concentration of 3-DG compared with control embryos cultured in a low glucose concentration. Exogenous 3-DG (100 micromol/l) added to the medium of control cultures yielded an increased embryonic malformation rate and a 3-DG concentration similar to that of embryos cultured in high glucose. Addition of superoxide dismutase (SOD) to the culture medium decreased the malformation rates of embryos exposed to either high glucose or high 3-DG levels, but it did not decrease the high embryonic 3-DG concentrations caused by either agent. Our results implicate the potent glycating agent 3-DG as a teratogenic factor in diabetic embryopathy. In addition, the anti-teratogenic effect of SOD administration appears to occur downstream of 3-DG formation, suggesting that 3-DG accumulation leads to superoxide-mediated embryopathy.

Advanced glycation end product free adducts are cleared by dialysis.

Plasma advanced glycation end product (AGE) free adducts are increased up to 50-fold among patients on dialysis. We examined the ability of hemodialysis (HD) and continuous ambulatory peritoneal dialysis (CAPD) to clear these compounds. The AGE free adducts Nepsilon-carboxymethyl-lysine (CML) and Nepsilon-(1-carboxyethyl)lysine (CEL) and the hydroimidazolones derived from glyoxal (G-H1), methylglyoxal (MG-H1), and 3-deoxyglucosone (3DG-H) were determined by LC-MS/MS and pentosidine by HPLC with fluorimetric detection in ultrafiltrates of plasma, urine, or PD effluent as appropriate from patients on HD (n = 8) or PD (n = 8), and from healthy controls (n = 8). Among patients on HD, all free AGEs predialysis were significantly higher than in controls and were decreased with dialysis. The removal of MG-H1 and 3DG-H was comparable to that of urea, whereas that of CML and pentosidine was some 20% higher; in contrast, the removal of CEL and G-H1 was 25% lower. Among patients on CAPD, free AGEs in PD effluent increased with increasing dwell time. The combined renal and peritoneal 24-h excretion rates of CML (4.7 micromol), CEL (6.5 micromol), 3DG-H (16.6 micromol), and pentosidine (0.08 micromol) were twofold higher than the amount excreted in healthy controls, whereas MG-H1 was ninefold higher (59 micromol); the combined clearances of all free AGEs except pentosidine were lower than in healthy controls. Impaired renal clearance contributes to increased plasma free AGEs in uremia, but the increased excretion rate among patients on PD demonstrates that there was also an increased synthesis of free AGEs. Both HD and PD are able to remove free AGEs.

The production of hydroxyl radicals by adriamycin in red blood cells.

Spin trapping of the free radicals formed from the interaction between adriamycin and red blood cells resulted in the formation of a hydroxyl spin adduct. The formation of hydroxyl radicals was found to be inhibited by mannitol. Hemoglobin was found not to be obligatory for the formation of hydroxyl radicals which probably result from the reduction of hydrogen peroxide by adriamycin semiquinone.

Glyoxalase activity in human red blood cells fractioned by age.

Human red blood cells were fractionated by density, which correlates with cell age, and the activities of glyoxalase I and glyoxalase II were determined for each fraction. The activity of glyoxalase I and glyoxalase II both significantly increased during maturation of the red blood cells (P less than 0.001), except in the most dense, old cell fraction where both glyoxalase activities decreased. The increase in glyoxalase activity from the reticulocyte-rich fraction to mature erythrocytes was substantial and markedly different from other glycolytic enzymes which typically decrease. This suggests that glyoxalase activity changes markedly during and probably after the maturation of reticulocytes to erythrocytes. The decrease in glyoxalase activity from the mature to old red blood cell fractions may be caused by oxidative inactivation of glyoxalases. The decreased capacity to metabolise methylglyoxal may be an important factor in red blood cell senesence. This is expected to be particularly important in diabetes mellitus where the rate of methylglyoxal formation is increased during hyperglycaemia.

Antitumor activity of S-(p-bromobenzyl)glutathione diesters in vitro: a structure-activity study.

S-(p-Bromobenzyl)glutathione is a competitive inhibitor of human glyoxalase I which is part of the cytosolic glyoxalase system. It may be delivered into the cystosol of cells by diesterification wherein it is deesterified by cytosolic nonspecific esterases. S-(p-Bromobenzyl)glutathione diesters had antitumor activity in vitro and in vivo. The inhibition of human leukemia 60 cell growth in vitro by a series of alkyl and cycloalkyl diesters of S-(p-bromobenzyl)glutathione was investigated. For n-alkyl diesters, the n-propyl diester was the most potent derivative with a median growth inhibitory concentration GC50 value of 7.77 +/- 0.01 microM (N = 18). The most potent derivative was S-(p-bromobenzyl)glutathione cyclopentyl diester which had a GC50 value of 4.23 +/- 0.01 microM (N = 21) and also had antitumor activity in vivo.