Maureen E. Garnett

Antioxidant enzyme alterations in experimental and clinical diabetes

Previous studies from our laboratory have demonstrated the presence of complex alterations in the activities of antioxidant enzymes in various tissues of rats with streptozotocin (STZ)-induced diabetes. In the present investigation, it is shown that rats made diabetic with alloxan (ALX), an agent differing from STZ both chemically and in its mechanism of diabetogenesis, show virtually identical tissue antioxidant enzyme changes which, as is the case with STZ, are preventable by insulin treatment. The finding that the patterns of antioxidant enzyme alterations in chemically-induced diabetes are independent of the diabetogenic agent used and the presence of similar abnormalities in tissues of spontaneously diabetic (BB) Wistar rats (particularly when diabetic control is less than optimal) suggest that the changes observed are a characteristic feature of the uncontrolled diabetic state and that these may be responsible for (or predispose to) the development of secondary complications in clinical diabetes. Comparative studies involving red cells of diabetic rats and human diabetics revealed a number of common changes, namely an increase in glutathione reductase activity, a decreased susceptibility to oxidative glutathione depletion (which was related to the presence of hyperglycemia) and an increased production of malondialdehyde (an indirect index of lipid peroxidation) in response to in vitro challenge with hydrogen peroxide. In the diabetic patients, the extent of this increase in susceptibility of red cell lipids to oxidation paralleled the severity of diabetic complications. Our results suggest that increased (or uncontrolled) oxidative activity may play an important role in the pathogenesis of complications associated with the chronic diabetic state.

Propofol enhances red cell antioxidant capacity in swine and humans

PurposeTo determine the effect of an anaesthetic with antioxidant potential, propofol, on red blood cell (RBC) antioxidant enzyme activities and RBC susceptibility to peroxidative challenge.MethodsPropofol was administered by intravenous bolus (2.5 mg·kg−1) and continuous infusion (36 and 72 ml·hr−1 in nine swine; 216 ml·hr−1 in two swine), to achieve serum concentrations between 5 and 30μg·ml−1 for two hours at each rate. Arterial blood sampling was at 0,10, 30, 60, and 120 min for each rate of infusion, for measurement of plasma propofol concentration, activities of plasma and RBC Superoxide dismutase, glutathione peroxidase, gluthathione reductase, RBC catalase, and RBC malondialdehyde (MDA) formation in response to exvivo oxidative challenge with t-butyl hydrogen peroxide (tBHP; 1.5mM). Antioxidant mechanisms were determined byin vitro study of MDA formation, GSH depletion, and oxidation of haemoglobin to methaemoglobin in human erythrocytes exposed to propofol 0–75 μM. The antioxidant potential of propofol was compared with that of alpha-tocopherol utilising the reaction with 2,4,6-tripyridyl-s-triazine (TPTZ).ResultsPropofol had no effect on plasma or RBC antioxidant enzyme activities. It inhibited RBC MDA production over the range of 0–20 μg·ml−1 (y = −18.683x + 85.431 ; R2 = 0.8174). Effective propofol concentrations for 25% and 50% reductions in MDA levels were 7–12 and 12–20 μg·ml−1, respectively. Propofol has a similar effect on human erythrocytesin vitro (R2 = 0.98).ConclusionPropofol antagonises the effects of forced peroxidation of red cells at anaesthetic and sub-anaesthetic concentrations in swine. Its actions include scavenging of oxygen derived free radicals in a tocopherol-like manner.RésuméObjectifDéterminer l’effet d’un agent anesthésique possédant un potentiel antioxydant, le propofol, sur l’activité d’un enzyme antioxydant des globules rouges (GR) et sur la susceptibilité des GR à une provocation peroxydative.MéthodesLe propofol a été administré en bolus intraveineux (2,5 mg·kg−1) et en infusions continues (36 et 72 ml·h−1 chez 9 porcs; 216 ml·h−1 chez 2 porcs) pour obtenir des concentrations sériques entre 5 et 30 μg·ml−1 durant deux heures à chaque vitesse d’infusion. Des prélèvements sanguins par voie artérielle ont été réalisés à 0, 10, 30, 60 et 120 min. pour chaque vitesse d’infusion; on a mesuré la concentration de propofol, l’activité de la superoxyde dismutase du plasma et des GR, de la peroxydase du glutathion, de la réductase du glutathion, de la catalase du GR, ainsi que de la formation dans le GR de la malondialdehyde (MDA) en réponse à une provocation oxydative exvivo avec le peroxyde d’hydrogène t-butylique (tBHP, 1,5 mM). Les mécanismes antioxydants ont été déterminés par l’étudein vitro de la formation de MDA, de la déplétion de GSH ainsi que de l’oxydation de l’hémoglobine en methémoglobine dans des GR humains exposés au propofol 0–75 μM. Le potentiel antioxydant du propofol a été comparé à celui de l’alpha-tocophérol en utilisant la réaction avec le 2,4,6-tripyridyl-s-triazine (TPTZ).RésultatsLe propofol n’a pas eu d’effet sur l’activité de l’enzyme antioxydant du plasma ou des GR. Il a inhibé la production de MDA par les GR pour tout le spectre de 0–20 μg·ml−1 (y = −18.683x + 85.431 ; R2 = 0,8174). Les concentrations de propofol efficaces pour obtenir une réduction des taux de MDA de 25 et de 50% étaient respectivement de 7–12 et de 12–20 μg·ml−1. Le propofol a un effet analogue sur les globules rouges humainsin vitro (R2 = 0,98).ConclusionLe propofol, à des concentrations anesthésiques et subanesthésiques chez le porc, antagonise les effets d’une peroxydation forcée des globules rouges. Son mode d’action comporte l’épuration des radicaux libres provoqués par l’oxygène comme le fait le tocophérol.

High dose propofol enhances red cell antioxidant capacity during CPB in humans.

PurposeTo compare low vs high dose propofol and isoflurane on red cell RBC antioxidant capacity in patients during aortocoronary bypass surgery (ACBP).MethodsTwenty-one patients, for ACBR were anesthetized with sufentanil 0.5–10 μg·kg−1 and isoflurane 0–2%; ISO = control; n=7), or sufentanil 0.3 μg·kg−1, propofol 1–2.5 μg·kg−1 bolus then 100μg·kg−1 before, and 50 μg·kg−1·min−1 during CPB (LO; n=7), or sufentanil 0.3 μg·kg−1, propofol 2–2.5 μg·kg−1 bolus then 200 μg·kg−1·min−1 (HI; n=7). Venous blood was drawn pre-and post-induction, after 30 min CPB, 5, 10, and 30 min of reperfusion, and 120 min post-CPB to measure red cell antioxidant capacity (malondialdehyde (MDA) production in response to oxidative challenge with t-butyl hydrogen peroxide) and plasma propofol concentration. Preinduction blood samples were analyzed for antioxidant effects of nitrates on red cells. The tBHP concentration response curves for RBC MDA in ISO, LO and HI were determined.ResultsPreoperative nitrate therapy did not effect RBC MDA production. Perioperative RBC MDA production was similar in ISO and LO groups. Sustained intraoperative decrease in RBC MDA was seen with propofol 8.0 ± 2.4 − 11.8 ± 4.5 μg·ml−1 in HI (P < 0.05- 0.0001). MDA production vs log plasma propofol concentration was linear in HI dose.ConclusionsDuring CPB, RBC antioxidant capacity is enhanced and maintained with HI dose propofol. Propofol, at this dose, may prove useful in protecting against cardiopulmonary ischemia-reperfusion injury associated with ACBRRésuméObjectifComparer une faible dose (LO) vs une forte dose (HI) de propofol et d’isoflurane sur la capacité antioxydante des globules rouges (GR) lors d’un pontage aortocoronarien (PAC).MéthodeLors d’un PAC, 21 patients ont reçu une anesthésie avec du sufentanil 0,5–10 μg·ml−1 et de l’isoflurane 0–2 %; (ISO = témoin, n = 7) ou du sufentanil 0,3 μg·kg−1 un bolus de propofol 1–2,5 μg·kg−1 suivi d’une perfusion de 100 μg·kg−1·min−1 avant le PAC et de 50 μg·kg−1·min−1 pendant le PAC (LO, n = 7), ou du sufentanil 0,3 μg·kg−1, un bolus de propofol 2–2,5 μg·kg−1 et une perfusion de 200 μg·kg−1·min−1 (HI, n = 7). Le sang veineux a été prélevé avant et après l’induction, 30 min après le PAC, à 5, 10 et 30 min pendant la reperfusion et 120 min après la CEC afin de mesurer la capacité antioxydante des GR (production de dialdéhyde malonique DAM en réponse à la provocation oxydante avec du peroxyde d’hydrogène t-butyl PHtB) et la concentration plasmatique de propofol. Les échantillons de sang prélevés avant l’induction ont été analysés pour vérifier les effets antioxydants des nitrates sur les GR. Les courbes illustrant la réaction des GR au DAM chez les patients des groupes ISO, LO et HI ont été déterminées.RésultatsLa thérapie préopératoire aux nitrates n’a pas changé la capacité antioxydante des GR, donc la production de DAM a été semblable dans les groupes ISO et LO. Une baisse peropératoire de production de DAM a toutefois été observée avec 8,0 ± 2,4 – 11,8 ± 4.5 μg·ml−1 de propofol dans le groupe HI (P < 0,05 - 0,0001). La production de DAM vs le logarithme de la concentration plasmatique de propofol était linéaire dans le groupe HI.ConclusionPendant la CEC, la capacité antioxydante des GR a été améliorée et maintenue par une forte dose de propofol. Administré selon cette dose, le propofol peut se révéler utile pour protéger des lésions cardio-pulmonaires liées à l’ischémie de reperfusion associée au PAC.

Species-related variations in tissue antioxidant status—II. Differences in susceptibility to oxidative challenge

1. In order to investigate possible species-related variations in antioxidant capacity, the susceptibility of red cells from various species (e.g. rat, rabbit, pig and quail) to depletion of glutathione (GSH) and formation of malondialdehyde (MDA), an indirect measure of lipid peroxidation, following in vitro oxidative challenge with t-butylhydroperoxide (tBHP) has been examined. 2. Marked differences in sensitivity were found, although the relative order of susceptibilities varied depending on the index of oxidation used. 3. For example, pig erythrocytes showed the highest sensitivity to depletion of GSH but the greatest resistance to tBHP-induced MDA formation. 4. Red cell susceptibility to oxidative stress under the experimental conditions used was not predictable from basal levels of GSH or from the activities of antioxidant enzymes, suggesting a prominent role of non-enzymatic antioxidants. 5. Species-dependent differences in antioxidant capacity were also found to extend to myocardial tissue homogenates and some degree of parallelism was noted with tBHP-induced MDA formation in red cells of the same species. 6. Thus, the relative resistance of both tissues from pig contrasted with the high susceptibility of red cells and myocardium from rat and quail. 7. This parallelism allows the suggestion that the functional consequences of antioxidant interventions might be discernible from measurements involving red cells. 8. Our findings may have potentially important implications in the interpretation and comparison of data obtained with experimental models of disease states in which oxidative processes are implicated when differences in species are involved.

Dietary cholesterol-induced xanthomatosis in atherosclerosis-susceptible Japanese quail (Cotunix japonica)

Japanese quail of a strain (SUS) susceptible to dietary cholesterol-induced atherosclerosis were fed a diet supplemented with cholesterol (0.5% w/w) for 4, 8 or 12 weeks. Plasma cholesterol increased significantly from 240-1550 mg/dl at 4 weeks and remained at that concentration for 8 and 12 weeks on the same diet. Plasma triglycerides (TGs) increased from 112-384 mg/dl after 4 weeks but showed no significant increases thereafter. Striking eruptive xanthomatous lesions were noticed on the feet of 50% of these birds at 4 weeks, and the percentage of birds affected increased to 85 after 12 weeks on the cholesterol-supplemented diet. This is the first report of xanthomatosis in birds. These birds had also developed atherosclerotic plaques in the aorta and brachiocephalic arteries by 4 weeks. There was no significant correlation between xanthoma scores and plasma cholesterol and TG concentrations at any of the three sampling periods (4, 8 and 12 weeks of cholesterol feeding). There was, however, a significant negative correlation (r = -0.61) between xanthoma scores and atherosclerotic plaque scores at 4 weeks. The correlation became non-significant at later stages of cholesterol exposure. Similarities between mammalian and SUS Japanese quail xanthomatosis may make the SUS quail a useful model for the study of this disorder.

Alterations in aortic antioxidant components in an experimental model of atherosclerosis: a time-course study.

Antioxidant component alterations in the aorta during atherogenesis were examined in atherosclerosis-susceptible (SUS) Japanese quail fed a cholesterol-supplemented (0.5% w/w) diet. Birds fed a non-supplemented diet provided information on the effects of aging on endogenous antioxidants. One hundred adult SUS males were used. Birds were sacrificed after 0, 4, 8 and 12 weeks on the diets and were examined for plaque development and corresponding antioxidant component alterations in aorta and myocardium. With aging, superoxide dismutase (SOD) activity was increased in both tissues, whereas aortic glutathione peroxidase (GPx) activity and myocardial glutathione reductase (GRd) activity decreased. Myocardial ascorbate levels increased with aging, with a reciprocal decrease in myocardial tocopherol levels. Following 4 weeks of cholesterol supplementation, aortic GRd decreased, SOD activity increased, but activities of GPx and catalase were unchanged. This same qualitative pattern of antioxidant enzyme changes was also found in myocardium. Thus, although aortic antioxidant enzyme changes produced by cholesterol feeding and aging showed some similarities, the early phase of atherogenesis does not simply reflect accelerated aging. In the late stages of atherogenesis, SOD activity returned to baseline, but other antioxidant enzymes remained unaltered from levels characterizing the early phase of lesion development. There was no detectable functional coupling between changes in GPx and GRd, nor between SOD (which produces hydrogen peroxide) and GPx or catalase (which utilize hydrogen peroxide as substrate). Previously reported alterations in erythrocyte antioxidant enzyme components during atherogenesis in quail were not predictive of changes in the corresponding enzymes in the aorta and myocardium.

Membrane perturbational effects of antiarrhythmic drugs.

Abstract The structural and functional consequences of the interaction of various antiarrhythmic agents with human erythrocyte membranes were analyzed using drug concentrations influencing the stability of intact erythrocytes to hypotonic lysis, with the assumption that such stabilization may bear some molecular analogy to the stabilizing properties of these molecules in excitable tissues. Most of the agents tested, including quinidine, lidocaine, the verapamil analogue D-600 and the two quaternary analogues QX-572 and pranolium, produced concentration-dependent perturbations of membrane structural components as reflected by increases in the incorporation of 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) and trinitrobenzenesulfonic acid (TNBS) into membrane sulfhydryl and amino groups respectively. Practolol, a β-adrenergic antagonist which, unlike the foregoing agents, lacks significant cardiodepressant actions, did not appreciably modify the membrane incorporation of TNBS or DTNB, despite the fact that this molecule possessed marked antihemolytic properties. It, therefore, appears that the membrane perturbational actions of antiarrhythmics as analyzed here by means of group-specific chemical probes are a better index of the direct myocardial membrane actions than erythrocyte stabilization. The diverse perturbational characteristics of the various antiarrhythmics studied, as revealed by their different effects on the incorporation of TNBS into membrane protein and phospholipid components suggested the possibility that the molecular mechanisms by which these drugs alter cardiac automaticity may not be identical. This, in turn, might underlie the different spectra of clinical effectiveness exhibited by these agents.

Acetylcholinesterase: a probe for the study of antiarrhythmic drug-membrane interactions.

Structural consequences of antiarrhythmic drug interaction with erythrocyte membranes were analyzed in terms of resulting changes in the activity of membrane-associated acetylcholinesterase. When enzyme inhibitory effects of drugs were compared at concentrations producing an equivalent degree of erythrocyte antihemolysis, a number of distinct groupings emerged, indicating that the molecular consequences of drug-membrane interaction are not identical for all agents examined. Differences in drug-induced acetylcholinesterase inhibition in intact erythrocytes, erythrocyte membranes and a brain synaptic membrane preparation emphaized the role of membrane structural organization in determining the functional consequences of antiarrhythmic interaction in any given system. While the inhibitory actions of lidocaine, D-600 and bretylium in intact red cells were not altered by an increased transmembrane chloride gradient, enhanced enzyme inhibition by quinidine and propranolol was observed under these conditions. The diverse perturbational actions of these membrane-stabilizing antiarrhythmics observed here may be indicative of a corresponding degree of complexity in the mechanisms whereby substances modify the potential-dependent properties of excitable tissues.

Perturbational effects of inorganic cations on human erythrocyte membranes

SummaryThe perturbational effects of monovalent and divalent cations on human erythrocyte membranes were analyzed by examining their influence on kinetic and structural characteristics of trinitrobenzenesulfonic acid (TNBS) incorporation into the amino groups of protein and phospholipid structural components. The stimulatory effects of monovalent cations on TNBS incorporation, which were size-independent and attributed to nonspecific membrane alterations resulting from ionic strength factors, contrasted with the more pronounced stimulatory properties of divalent cations which were markedly size-dependent. These stimulatory effects of cations on TNBS incorporation were associated with alterations not only in rate but also in activation energy of incorporation. Changes in activation energy produced by divalent cations paralleled their ability to perturb membrane protein components and probably reflected changes in probe permeation. The rate of TNBS incorporation exhibited a dependence on divalent cation ionic radius which paralleled ion-induced perturbations in the labelling of the membrane amino phospholipid phosphatidylethanolamine. Divalent cations differed both in the relative extent and in the characteristics of protein and phospholipid perturbation. Alkaline earth cations behaved as a rather homogeneous group while Ni++, Co++ and Mn++ constituted a second heterogeneous group. The influence of monovalent and divalent cations on the hemolytic behavior of intact erythrocytes paralleled their effects on TNBS incorporation into isolated membranes rather closely. It is suggested that TNBS incorporation may provide a valuable means of analyzing functionally relevant cation-induced alterations in biological membranes in general.

In vivo and in vitro studies of bacterial endotoxin-membrane interactions and the effects of membrane-active agents

1 The characteristics of 51Cr‐labelled E. coli endotoxin binding to human erythrocyte membranes in vitro have been investigated. A saturable component of binding was apparent at low endotoxin concentrations (<50 μg ml−1) relevant to its in vivo actions, while at higher concentrations binding was non‐saturable and increased in linear fashion. Experiments examining the ability of unlabelled endotoxin to antagonize the binding of labelled toxin provided further evidence for these specific and non‐specific modes of endotoxin‐membrane interaction. 2 Membrane‐active agents previously shown to reduce endotoxin toxicity in vivo decreased endotoxin binding to erythrocyte membranes in vitro, with propranolol and pranolium being the most effective in this regard. 3 Tissue distribution studies following administration of radiolabeled endotoxin to guinea‐pigs showed a positive correlation between the accumulation of 51Cr‐endotoxin in lung and elevations in plasma acid phosphatase activity, a measure of in vivo endotoxin toxicity. 4 The in vivo accumulation of 51Cr‐endotoxin in guinea‐pig lung was reduced by prior treatment with (+)‐propranolol or pranolium, paralleling the results of the in vitro binding studies. 5 Our results suggest that membrane‐active agents such as (+)‐propranolol may be useful adjuncts to antimicrobial drugs in the therapy of gram‐negative endotoxaemia.