The effect of extracellular NADH on the rate of reduction of nitrite-induced methaemoglobin in erythrocytes from man, cattle, dog, horse, grey kangaroo, pig and sheep was investigated. Extracellular NADH was found to enhance the rate of methaemoglobin reduction in man, dog, pig and kangaroo erythrocytes, but had essentially no effect on the rate of methaemoglobin reduction in erythrocytes from cattle, horse and sheep. In erythrocytes of those animals affected by extracellular NADH the rate of reduction of metHb in the presence of NADH was the same or greater than that observed in the presence of nutrients such as glucose and inosine. The combination of nutrient and NADH produced a more profound increase in the rate of methaemoglobin reduction. The rate of methaemoglobin reduction in all cases was significantly less than that observed with methylene blue, the standard treatment of methaemoglobinaemia. Extracellular NADH was found to indirectly increase the intracellular NADH concentration through displacement of the pseudo-equilibrium of the intracellular LDH reaction and relied upon the presence of sufficient LDH activity released into the extracellular medium through haemolysis. The lack of response of cattle, horse and sheep RBCs to extracellular NADH was found to derive mainly from their low extracellular LDH activity, but also correlated with their lower NADH-methaemoglobin reductase activity compared to the other species.

In the current work, we investigated the effect of ascorbic acid on GSH-mediated haemin degradation. GSH-mediated haemin degradation in the presence of ascorbic acid in phosphate-buffered saline and in erythrocyte ghosts was determined by recording absorbance at 365 and 399nm, respectively. Generation of intracellular H(2)O(2) was measured indirectly in terms of the inactivation of endogenous catalase in erythrocytes in the presence of 3-amino-1,2,4-triazole. Although ascorbic acid itself did not induce haemin degradation, it enhanced GSH-mediated haemin degradation. Experiments with catalase showed that H(2)O(2) was essential in this process. The oxidation of ascorbic acid in the presence of haemin was stimulated by GSH, suggesting that ascorbic acid can alter the mechanism of H(2)O(2) generation observed with GSH and haemin alone. These results suggest that enhancement of GSH-mediated haemin degradation by ascorbic acid may be due to an increase in the production of H(2)O(2) generated by GSH and haemin in the absence of ascorbic acid.

1-chloro-2,4-dinitrobenzene (CDNB), an intracellular glutathione-depleting agent, has been shown to have an adverse effect on erythrocyte membrane integrity. In the current study, we have demonstrated that CDNB caused haemolysis of human red blood cells (RBC) at higher concentrations (>or= 5 mM). The haemolysis induced by CDNB was preceded by the leakage of K(+) from the cells suggesting the colloid-osmotic nature of this lysis. The inclusion of molecules of increasing size in the extracellular media inhibited both the rate and extent of haemolysis thus supporting the proposal of CDNB-induced pore formation. The size of membrane lesions increased with an increase in the concentration of CDNB. SDS-PAGE demonstrated that CDNB causes the polymerisation and/or fragmentation of membrane proteins. Although CDNB has been shown to cause a drastic reduction in membrane thiols, our data suggest that the CDNB-induced formation of membrane disulfide bonds as a prima facie cause of permeability enhancement is unlikely.

The red blood cells (RBC) of bats (Order Chiroptera) are morphologically similar to those of other mammals, but the suborder Megachiroptera are unique in responding to very high demands for oxygen supply, more than 30 times that of resting conditions. Although greater efficiency of these cells to transport oxygen is advantageous to the animal, it could potentially expose the RBC to higher than average risk of oxidant damage. The aim of the present study was to investigate the capacity of RBCs of Pteropus poliocephalus, the greyheaded flying fox, to defend itself against oxidant stress. RBC of flying fox and human were challenged with 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH). The RBC from the two species were found to have similar reduced glutathione (GSH) reduction kinetics and succumbed to haemolysis at similar rates. However, haemoglobin oxidation was much faster in the flying fox suggesting that the RBC of flying fox may not enjoy as good protection as those of humans in the event of an oxidant stress.

Oxidant sensitivity was investigated in erythrocytes from Barrow Island euro (Macropus robustus isabellinus), mainland euro (Macropus robustus erubescens) and brushtailed-bettong (Bettongia pericillata). Oxidant sensitivity was tested by measuring methaemoglobin formation in haemolysates treated with H2O2 or NaNO2. Barrow Island euro produced the highest methaemoglobin formation with H2O2. There was a significant negative correlation between catalase activity and H2O2-induced methaemoglobin formation. Our results provide further evidence supporting previous findings on eight species of marsupials.

This study was carried out to investigate sequel of oxidative insult to human erythrocytes induced by a water-soluble radical initiator, 2,2'-azobis-(amidinopropane) dihydrochloride (AAPH) and the effect of a commercially available mixed antioxidant (Blackmores, BioAce Excel), containing alpha-tocopherol, ascorbic acid, beta-carotene and some herbal extracts (containing grape seed catechins and milk thistle derived silybin), on lipid peroxidation, degradation of membrane proteins and haemolysis. We performed this study in order firstly to clarify aspects of the mechanism of AAPH induced free radical damage in human erythrocytes and secondly to establish in vitro conditions by which the efficacy of mixed antioxidant preparations may fairly and objectively be compared. In the process of oxidation initiated by peroxyl radical, a rapid loss of reduced glutathione occurred in the first 60 min. Formation of thiobarbitric acid-reactive substances indicative of lipid peroxidation increased subsequently and almost reached maximal levels at 180 min before significant apparent degradation of membrane proteins was detected. At this point, a significant haemolysis occurred. This sequence of events is consistent with the idea that haemolysis is a consequence of lipid peroxidation and the degradation of membrane proteins. The mixed commercial antioxidant, which suppressed lipid peroxidation and protected membrane proteins against degradation induced by peroxyl radicals, also effectively delayed AAPH induced haemolysis. The system we describe provides a sound objective basis for the in vitro comparison of the potential efficacy of the hundreds of antioxidant nutritional supplements currently available in the market place.

Three different types of red blood cells (RBC) were used: (i) RBC from sheep having genetically high GSH (ii) RBC from sheep with genetically low GSH and (iii) RBC from high-GSH sheep treated with CDNB to deplete GSH. Incubation of these RBC with t-butyl hydroperoxide (tBHP, 3 mM) for 10 min caused the formation of TBARS, oxidation of haemoglobin and degradation and aggregation of membrane proteins in RBC from low-GSH sheep and GSH-depleted RBC. By contrast, RBC from high-GSH sheep (normal RBC) did not show the degradation and aggregation of membrane proteins within the first 10 min. Dithiothreitol (DTT) was highly effective in preventing the tBHP-mediated oxidation of haemoglobin, the formation of TBARS and the degradation and aggregation of membrane proteins in both normal RBC and low-GSH RBC. However, DTT did not provide protection in GSH-depleted RBC or normal RBCs in the presence of 1.5 mM mercaptosuccinate (MCS), a potent inhibitor of GSH peroxidase (GSHPx). The ability of GSH to prevent the oxidation of haemoglobin and the degradation and aggregation of membrane proteins was abolished in the presence of MCS. These results indicate that the protective function of DTT involves a GSH-dependent mechanism. Both GSH and GSHPx play key roles in this enzymatic system. In the light of the complete protection of RBC against oxidation induced by tBHP in the presence of DTT or GSH, the GSH/GSHPx system appears to act directly as a tBHP scavenger. The activities of four well-known antioxidants, Butylated hydroxytoluene, ascorbate, alpha-tocopherol and desferrioxamine were also tested in this study to cast further light on the role of free radical scavenging in protection from tBHP mediated free radical insult.

Although extensive studies have been performed on human erythrocytes, there is a shortage of information on marsupial erythrocytes. Studies on haematology and biochemistry are useful in the ecomanagement of these animals especially those in wildlife parks and zoos. The present review summarises our findings from ∼30 species of marsupials. As marsupials show great diversity in physical and behavioural characteristics, it is not surprising that examination of their red blood cells reveals variation in the biochemical features. Many variations in red cell biochemistry appear to be species specific and, although interesting, are too numerous to list here and have been discussed in the relevant sections. Red blood cells of several species of marsupial differ from those in placental mammals by having higher haemoglobin concentration and haematocrit, lower ATP and higher 2,3-diphosphoglycerate concentrations. These features are consistent across the majority of marsupial species, but the relevance of these variations from red cell metabolism in the placental mammals is unknown.

Haemolysis of red blood cells (RBC) in glycerol media may be measured spectrophotometrically. The haemolytic process in a rapid phase obeys a first order rate law. The rate constant expresses the rate of haemolysis. To gain a better understanding of the mechanism of haemolysis in glycerol media, the effects of pH and band 3 inhibitors on the rate of haemolysis in human and sheep RBC were observed. Over the pH range used (pH 5.8-10.0), the rate of haemolysis decreased with increase in pH in sheep RBC. By contrast, the rate of haemolysis increased from pH 5.8 to 6.4 and decreased above pH 6.4 in human RBC. The different effects of pH on the rate of haemolysis are due to inhibition of glycerol permeability by H(+) in human RBC but not in sheep RBC. This is supported by the different effects of temperature and Cu(2+) on the rate of haemolysis in human and sheep RBC. We did not observe complete inhibition of haemolysis by the classical band 3 inhibitor, 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). Another band 3 inhibitor 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) showed only weak inhibition. Phenylgloxal (PG), another band 3 inhibitor, had no effect whatsoever on the rate of haemolysis. These results indicate that the anion pathway of band 3 is not the preferred route of transport of glycerol in mammalian RBC.

: Glutathione (GSH) regeneration is a process in which cells reduce oxidised glutathione (GSSG) to GSH after exposure of cells to oxidants in the presence of suitable energy source such as glucose. This reaction consists of (1) membrane glucose transport, (2) phosphorylation of glucose by hexokinase (HK) utilising adenosine 5′-triphosphate (ATP), (3) reduction of nicotinamide–adenine dinucleotide phosphate (NADP) to its reduced form (NADPH) by glucose-6-phosphate dehydrogenase (G-6-PD) and 6-phosphogluconate dehydrogenase (6-PGD) and (4) reduction of GSSG to GSH by glutathione reductase (GR) using NADPH. The rate of GSH regeneration is thus dependent on the enzymatic activity and concentration of substrate. G-6-PD deficiency and enzyme inhibitory chemicals reduce GSH regeneration and concentration of substrate greatly influences the rate of GSH regeneration. Not only glucose but also mannose, fructose and galactose may also be used as energy source. Interspecies and intraspecies differences occur in the rate of GSH regeneration. These differences cannot be explained by variations in enzymatic activities. Although physiological relevance of GSH regeneration is still unclear, it is known that erythrocytes from G-6-PD-deficient patients have lowered erythrocyte deformability and shortened erythrocyte life-span. In-vitro experiments show lowered GSH regeneration enforces loss of deformability. These findings suggest that GSH regeneration plays an important role in erythrocyte biology.

Although extensive studies have been performed on human erythrocytes, there is a shortage of information on marsupial erythrocytes. Studies on haematology and biochemistry are useful in the ecomanagement of these animals especially those in wildlife parks and zoos. The present review summarises our findings from ∼30 species of marsupials. As marsupials show great diversity in physical and behavioural characteristics, it is not surprising that examination of their red blood cells reveals variation in the biochemical features. Many variations in red cell biochemistry appear to be species specific and, although interesting, are too numerous to list here and have been discussed in the relevant sections. Red blood cells of several species of marsupial differ from those in placental mammals by having higher haemoglobin concentration and haematocrit, lower ATP and higher 2,3-diphosphoglycerate concentrations. These features are consistent across the majority of marsupial species, but the relevance of these variations from red cell metabolism in the placental mammals is unknown.

Effects of potassium concentrations on glycolytic rate and pyruvate kinase (PK) activity were investigated in high potassium (HK) and low potassium (LK) erythrocytes from the common brushtail possum (Trichosurus velpecula). Increasing concentrations of potassium resulted in an increase in glycolytic rate and PK activity in both HK and LK possum erythrocytes. Our results are similar to those from HK/LK sheep. We conclude that there is a close link between Na-pump and glycolysis in erythrocytes from sheep and possum.

This study was undertaken with a view to examine certain aspects of erythrocyte metabolism in the developing sheep foetus. The parameters measured were: the concentrations of adenosine triphosphate (ATP); 2,3-diphosphoglycerate (DPG); and the activities of three enzymes associated with antioxidant defense, namely glucose-6-phosphate dehydrogenase (G6PD), glutathione S-transferase (GST) and reduced nicotinamide adenine dinucleotide-dependent methaemoglobin reductase (NADH-MR). The level of both, ATP and DPG, and all three enzyme activities were considerably greater in foetal erythrocytes compared to those of the mothers. These results suggest that foetal cells are better equipped to defend from oxidative insult.

The effect of potassium concentration on glycolysis was studied in the erythrocytes of Japanese Shiba dogs with high potassium (HK) and low potassium (LK) concentrations. When intracellular Na and K concentrations were changed in intact erythrocytes, lactate formation increased with increasing K concentration in both HK and LK dogs. In the reconstituted haemolysates, lactate formation, phosphofructokinase (PFK) and pyruvate kinase (PK) activities increased with increasing K concentration in both HK and LK dogs. These results suggest that glycolysis in erythrocytes of HK and LK dogs is dependent on K concentration because PFK and PK require K for their activities. In the reticulocytes of a LK dog possessing high K and low Na concentrations, PK and PFK activities were markedly elevated from the levels in mature erythrocytes and also dependent on K concentration. As in HK dog erythrocytes, high K concentration is required for glucose metabolism in LK dog reticulocytes. It is suggested that the glycolytic system of LK dog erythrocytes retains the potential to be stimulated with high K concentration even after cell maturation.

A comparative study was carried out to investigate various aspects of erythrocyte metabolism in three species of marsupials located in the western part of Australia. The burrowing bettong ( Bettongia lesueur ) and the Barrow Island euro ( Macropus robustus isabellinus ) were captured on Barrow Island whereas the mainland euro ( Macropus robustus erubescens ) was captured from the Pilbara region of northwest Western Australia. Aspects of erythrocyte metabolism studied included: levels of glycolytic intermediates, enzyme activities, sensitivity of erythrocytes to oxidants and methaemoglobin reducing capacity. Some important findings were: (1) no relationship was observed between haemoglobin and diphosphoglycerate levels; (2) both species from Barrow Island had lower levels of catalase than the mainland species; and (3) methaemoglobin reductive capacity was greater in the erythrocytes of the burrowing bettong compared to the two species of euro.

The oxidant effects of two fractions of eucalyptus oil; monoterpenes (C10) and the larger sesquiterpenes (C15), were investigated in the erythrocytes of the koala (Phascolarctos cinereus). The effects studied included the degree of haemolysis and changes in the intracellular levels of glutathione (GSH) and adenosine triphosphate (ATP) and in the membrane phospholipids. The results indicate that koala erythrocytes are susceptible to eucalyptus oil-induced oxidative damage. The two types of eucalyptus oils have different effects on the erythrocytes; monoterpenes appear to induce haemolysis through oxidative damage to the intracellular constituents, whereas sesquiterpenes may attack the red cell membrane.

Measurements were made of glutathione (GSH) levels, catalase activity and the oxidant sensitivity of the erythrocytes from the koala ( Phascolarctos cinereus ) and the common brushtail possum ( Trichosurus vulpecula ). The oxidant sensitivity was tested by treating the haemolysates with either 0.55 him H 2 O 2 or 1.4 mm NaNO 2 . The erythrocytes of the koala had greater levels of GSH and catalase and yet were found to be more susceptible to oxidation induced by both these oxidants.

The effect of ATP levels on GSH regeneration was examined in canine erythrocytes. The main findings were: (1) The GSH regeneration was dependent on glucose and ATP; (2) cytochalasin B and polymyxin B, both glucose transport inhibitors, reduced ATP synthesis and GSH regeneration; (3) inosine, a substrate of the salvage pathway, was not effective for ATP synthesis and GSH regeneration. These results indicate that glucose transport and its metabolism play an important role in oxidant defence systems in general and GSH regeneration in particular in canine erythrocytes.

The efficacy of hexoses, their intermediates and analogues, as substrates for glutathione (GSH) regeneration, were tested in rabbit and guinea-pig erythrocytes. Glucose was found to be the most effective substrate in both species. Compared to glucose, the efficacy of other substrates were: fructose (85%), mannose (68%), galactose (47%), 2-deoxyglucose (43%), glucose-1-phosphate (29%), 2-deoxyglucose-6-phosphate (21%), fructose-6-phosphate (12%) and glucose-6-phosphate (11%) in rabbit erythrocytes, and mannose (93%), fructose (52%) and 2-deoxyglucose-6-phosphate (11%) in guinea-pig erythrocytes. Our results provide evidence that in addition to enzyme activities, the availability of the substrate is also an important factor in determining GSH regeneration in mammalian erythrocytes.

The effect of heavy metals (copper (Cu2+), iron (Fe2+), lead (Pb2+) and cobalt (Co2+)) on glutathione (GSH) regeneration were examined in erythrocytes from rabbits and guinea pigs. Cu2+ and Fe2+ oxidised GSH, and decreased GSH regeneration rate. Pb2+ did not oxidise GSH, but decreased GSH regeneration rate. Co2+ did not show any effects. In an in vitro study using haemolysate, Cu2+ inhibited the activities of hexokinase and 6-phosphogluconate dehydrogenase (6-PGD); Pb2+ also inhibited 6-PGD; Fe2+ inhibited 6-PGD, glucose-6-phosphate dehydrogenase (G-6-PD) and glutathione reductase (GR). There were no striking differences between the two species used. The present results suggest that the decline of GSH generation by heavy metals involves oxidation of GSH and inhibition of its associated enzymes, and that the inhibitory mechanism is different for different metals.