Erythrocyte catalase. A somatic oxidant defense?

Mammalian erythrocytes have large amounts of catalase, an enzyme which catabolizes hydrogen peroxide (H2O2). Because catalase has a low affinity for H2O2, others have suggested that glutathione peroxidase clears most H2O2 within the erythrocyte and that catalase is of little import. We hypothesized that erythrocyte catalase might function to protect heterologous somatic cells against challenge by high levels of exogenous H2O2 (e.g., in areas of inflammation). We find that, whereas nucleated cells (L1210 murine leukemia) are readily killed by an enzymatically generated flux of superoxide (and, therefore, H2O2), the addition of human and murine erythrocytes blocks lethal damage to the target cells. Inhibition of erythrocyte superoxide dismutase, depletion of glutathione, and lysis of the erythrocytes do not diminish this protection. However, inhibition of erythrocyte catalase abrogates the protective effect and the addition of purified catalase (but not superoxide dismutase) restores it. Furthermore, erythrocytes derived from congenitally hypocatalasemic mice (in which other antioxidant systems are intact) do not protect L1210 cells. Our results raise the possibility that the erythrocyte may serve as protection against by-products of its own cargo, oxygen.

Hemolytic and microbicidal actions of diethyldithiocarbamic acid.

Micromolar concentrations of diethyldithiocarbamic acid (DDC) kill fungi, bacteria and malaria. DDC forms chelates with copper and the microbicidal effectiveness of this drug is enhanced greatly by small amounts of copper. DDC, in the presence of at least 1 molar equivalent of copper, also causes lysis of human erythrocytes. To explore the cytocidal actions of DDC and copper, we have used human erythrocytes and Escherichia coli as models. We found that: (1) the combination of DDC and copper also lysed E. coli spheroplasts, suggesting a possible common mechanism of hemolytic and microbicidal action; (2) higher ratios of drug: metal (greater than 4:1) diminished hemolytic and, as observed earlier, microbicidal effects; (3) cobalt, known to suppress the microbicidal effects of DDC:Cu, also prevented red cell lysis; (4) despite the necessary involvement of copper in DDC-mediated hemolysis, there was no evidence of oxidative damage to erythrocytes, and both lysis of erythrocytes and killing of E. coli were undiminished in the absence of oxygen; (5) the DDC:Cu chelate preferentially located in organic solvents and in membranes of erythrocytes. The chelate was quite soluble in chloroform but much less so in a C-16 hydrocarbon (hexadecane) which resembled erythrocyte membrane lipid. In hexadecane and at greater than 10(-4) M DDC and 5 x 10(-5) copper, an amphipathic drug:metal complex accumulated at the organic:aqueous interface; and (6) this amphipathic complex may permeabilize the lipid bilayer, causing leakage of ions and cell water and eventuating in colloid osmotic lysis. Red cells and E. coli exposed to the chelate showed early loss of intracellular rubidium (86Rb+). Furthermore, lysis of erythrocytes and E. coli spheroplasts was suppressed by the inclusion of either dextran or sucrose. Thus, it appears that DDC:Cu chelates are cytocidal by virtue of concentrating in the lipid bilayer and, perhaps, forming amphipathic complexes which disrupt membrane integrity. Drugs with similar behavior hold promise for therapy of malaria because metals capable of forming such complexes may accumulate within parasitized red cells.

Hematology and erythrocyte biochemistry of spectacled hare-wallaby, Lagorchestes conspicillatus (Macropodidae Marsupialia).

Hematology and the red cell enzymes, metabolites, and rates of lactate production were measured in a small marsupial, the spectacled hare-wallaby Lagorchestes conspicillatus. In common with other marsupials of similar body weights, the red cells of this wallaby were found to have low levels of ATP and higher levels of 2,3-diphosphoglycerate (DPG) and produced no lactate when incubated with inosine. One of the five animals examined was suffering from anemia (hematocrit of 22.0% and hemoglobin concentration of 7.2 g/dl compared with the normal values of 46.6% and 15.8 g/dl, respectively); its red cells had almost twice the concentrations of ATP and DPG and had significantly higher activities of several enzymes of the glycolytic pathway.

Oxidative insult in sheep red blood cells induced by T-butyl hydroperoxide: the roles of glutathione and glutathione peroxidase.

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.

Oxidative insult to human red blood cells induced by free radical initiator AAPH and its inhibition by a commercial antioxidant mixture.

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.

Phospholipid composition of erythrocyte membranes and plasma of mammalian blood including Australian marsupials; quantitative 31P NMR analysis using detergent.

The phospholipid classes of erythrocyte membranes and plasma from several domestic animals and marsupials were quantified by 31P NMR using detergents. Washed erythrocyte samples were thoroughly haemolysed by tip-sonication and dissolved in sodium cholate; plasma samples were dissolved in Triton X-100. The species studied were: common wombat (Vombatus ursinus), black-striped wallaby (Macropus dorsalis), bandicoot (Isoodon macrocarpus), Eastern grey kangaroo (Macropus giganteus), Tammar wallaby (Macropus eugenii), sheep (Ovis aries), goat (Capra hircus), cattle (Bos taurus), horse (Equus caballus), dog (Canus familiaris) and rabbit (Orytolagus caniculus). There were considerable species variations in the relative abundance of erythrocyte and plasma phospholipid classes. The variations may be attributed to the habitats and diets of the animals as well as to their phylogenetic differences.

Comparative erythrocyte metabolism in marsupials and monotremes.

Concentrations of ATP and DPG, activities of 10 enzymes and the glycolytic rates were measured in the erythrocytes of 11 species of marsupials and two species of monotremes. Mean DPG concentrations were greater in the erythrocytes of marsupials than those of eutherian mammals. The opposite is true of ATP. Significant findings from the results of enzyme activities were: high activity of hexokinase (7.39 +/- 0.82 EU/g Hb) in the short-beaked echidna, pyruvate kinase (37.49 +/- 1.0 EU/g) Hb in bridled nailtail wallaby and glucose-6-phosphate dehydrogenase (G6PD; 41.66 +/- 1.24 EU/g Hb) in black-striped wallaby. About 6- to 7-fold difference in the activity of G6PD levels between the two species of wombats was confirmed. Glucose phosphate isomerase activity was also shown to be twice as high in the red cells of the common wombat compared with those of the southern hairy nosed wombat. There were wide variations in the glycolytic rate among the species examined.

Comparative study of the antioxidant defence systems in the erythrocytes of Australian marsupials and monotremes.

A comparison of the erythrocyte (RBC) antioxidant metabolites and enzymes in nine marsupial and two monotreme species was carried out. Reduced glutathione (GSH) concentrations were comparable with those reported for other marsupial and eutherian species. An important finding was that the erythrocytes of the southern hairy nosed wombat regenerated GSH faster than the erythrocytes from its close relative, the common wombat. The activities of glutathione-S-transferase, NADH-methaemoglobin reductase, superoxide dismutase, and glutathione peroxidase (GSH-Px), showed similar levels and extents of variation as those observed in other marsupial and eutherian species. Catalase activities in the marsupials were lower than those measured in the two monotreme species and much lower than those reported in eutherian species. A negative correlation, significant at P < 0.05, was observed between GSH-Px and catalase activities in the RBC of the marsupials. Since both these enzymes "detoxify" H2O2, there appears to be a reciprocal relationship between the activities of these enzymes in marsupial RBC.

Glutathione reductase activity and flavin concentration in guinea-pig tissues.

Glutathione reductase (GR) activity and flavin concentration were studied in systemic tissues (brain, heart, lung, liver, spleen, stomach, pancreas, muscle, kidney, testis) and blood components (erythrocytes and plasma) from male guinea-pigs. GR activity and the flavin concentration were high in kidney and liver, and low in muscle. GR activity in erythrocytes was found in a range of tissues, but flavin concentration in erythrocytes was lower than in any tissues. GR was saturated with flavin adenine dinucleotide (FAD) in almost all tissues, but not in muscle or erythrocytes.

Postnatal changes in the levels of 2,3-diaphosphoglycerate, reduced glutathione and some enzyme activities in the erythrocytes of lambs.

The concentration of 2,3-diphosphoglycerate, and the activities of the enzymes hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glutathione reductase and NADH-dependent methaemoglobin reductase in the erythrocytes of newborn and adult sheep were investigated. All the enzyme activities and the concentration of 2,3-diaphosphoglycerate were found to be significantly greater in the erythrocytes of newborn lambs than in those of adult sheep.

Comparative red cell metabolism in the domestic dog and the dingo.

A comparative study of the red cell characters, glycolytic enzymes and intermediates was made in the domestic dog and the dingo. No significant differences were found in any of the parameters studied except the enzyme level of NADH-MR which was significantly lower in dingoes (P less than 0.05).

The effect of phlebotomy on canine erythrocyte metabolism.

Enzyme activity declines with erythrocyte age in most mammals. To test this concept in the dog, we decreased the PCV to less than 20 by phlebotomy. The erythrocytes were restored rapidly (1.57 per cent per day). The resulting decline in the mean erythrocyte age was accompanied by increased activity by most of the erythrocyte enzymes studied. Enzymes with lower initial enzymatic activity (hexokinase, pyruvate kinase, 6-phosphogluconate dehydrogenase and glutathione reductase) increased proportionally more than those with higher initial activity (lactate dehydrogenase, 3-phosphoglycerate kinase, glyceraldehyde-3-dehydrogenase and glucose-6-dehydrogenase). Among species, increases in enzyme activity after phlebotomy appear to be related to each species' life span. Most of the metabolites increased concomitantly with the highest reticulocyte period. Diphosphoglycerate concentrations did not change significantly during the experiment.

Glutathione and 2,3-diphosphoglycerate in the blood of hypoxic ruminants.

Two merino sheep and two angora goats were subjected to simulated altitudes of up to 5500 m for about 10 h per day for 38 days. Packed cell volume (PCV), haemoglobin (Hb) concentration and red blood cell (RBC) levels of reduced glutathione (GSH) and 2,3-diphosphoglycerate (2,3-DPG) were measured at regular intervals. Mean PCV increased from 33 per cent to 60 per cent in the sheep and from 33 per cent to 45 per cent in the goats. Mean Hb concentration rose from 11.7 g/dl to 23.1 g/dl in the sheep, and from 11.4 g/dl to 16.0 g/dl in the goats. The level of GSH began to rise in the sheep only after the animals were no longer being subjected to the hypoxic stress, increasing by about 20 mg/dl RBC. In the goats, the levels of GSH rose during, as well as after, the period of hypoxic stress, also by about 20 mg/dl RBC. No change in 2,3-DPG levels was found in either species.

Studies on erythrocyte metabolism following acute blood loss in the horse.

Anaemic horses do not provide reticulocytes during an erythropietic response. Acute blood loss was studied in 4 ponies as a model to monitor changes in erythrocyte metabolism during active erythropoiesis. Following phlebotomy, erythrocyte adenosine-5-triphosphate, adenosine-5-diphosphate, glucose-6-phosphate, fructose-6-phosphate, and phosphoenolpyruvate increased signifcantly. The remaining glycolytic intermediates did not change. It is suggested that the determination of erythrocyte adenosine-5-triphosphate may be used to evaluate erythrocyte response following acute blood loss in the horse.

Erythrocyte metabolism in normal and glutathione-deficient sheep.

Glucose utilization and lactate formation in erythrocytes from normal and glutathione (GSH)-deficient sheep were similar. Significant differences were observed, however, between the 2 groups of sheep in the production of 14-CO2 from erythrocytes incubated with ascorbic acid or methylene blue, or both. The greater response of normal erythrocytes compared to erythrocytes deficient in GSH suggests that there are some metabolic differences in the pentose phosphate pathway (ppp) activity of the erythrocytes. The nature and site of these differences are, however, not known. When sheep were kept in a decompression chamber for 2 weeks and subjected to a simulated altitude of 7,000 m for 12 hours/day, the erythrocytes showed a four- to six-fold increase in the activity of the ppp.

Relation between reduced glutathione content and Heinz body formation in sheep erythrocytes.

To clarify the oxidant defense functions of reduced glutathione (GSH) in erythrocytes, the effect of GSH deficiency on in vitro oxidant defense was studied, using GSH-deficient sheep erythrocytes (low-GSH cells). The formation of Heinz bodies in low-GSH cells was higher than that in high-GSH cells when the cells were incubated with an oxidant drug, acetylphenylhydrazine (APH). Artificial depletion of GSH by 1-chloro-2,4-dinitrobenzene in high-GSH cells resulted in increased Heinz body formation in these cells incubated with APH. Furthermore, high negative correlation was observed between Heinz body formation and GSH content in sheep erythrocytes exposed to APH. These results clearly indicate that erythrocyte GSH is indispensable for erythrocyte defense against oxidative damage induced by APH, and support the previous observations that sheep with low-GSH erythrocytes were more susceptible to oxidative agents than were sheep with high-GSH erythrocytes.

Certain features of erythrocytes of normal and glutathione-deficient sheep.

Studies were made of certain characters of erythrocytes of normal and glutathione (GSH)-deficient sheep. The results indicate that erythrocytes of GSH-deficient sheep are no more sensitive to oxidative damage than those of normal sheep.

Oxygen-hemoglobin equilibrium of normal and glutathione-deficient sheep.

The affinity of hemoglobin for oxygen did not differ significantly between normal and glutathione-deficient sheep. Although phlebotomy in sheep of both types did not significantly alter the partial pressure values at which hemoglobin is 50% saturated with oxygen, it did increase significantly the erythrocyte glutathione and 2,3-diphosphoglycerate (GSH) and (DPG) concentrations. Because DPG does not bind to sheep hemoglobin, its increase in hypoxic sheep must mean DPG is metabolically controlled in the absence of physiologic effect.

The relationship between reduced glutathione level and glutathione S-transferase activity in sheep erythrocytes.

The relationship between reduced glutathione (GSH) level and glutathione S-transferase (GST) activity in erythrocytes was examined, using sheep erythrocytes, which have varying GSH concentrations, and dog erythrocytes with an inherited high concentration of GSH. There was a positive correlation (r = 0.529, p < 0.001) between the GSH level and GST activity in sheep erythrocytes. In dog erythrocytes, the GST activity in high-GSH cells was significantly (p < 0.001) higher than that in normal-GSH cells. These results indicate that the activity of GST in erythrocytes is directly correlated with the intracellular GSH level.

A cutaneous lymphoma international prognostic index (CLIPi) for mycosis fungoides and Sezary syndrome.

BACKGROUND: There is no prognostic index for primary cutaneous T-cell lymphomas such as mycosis fungoides (MF) and Sezary syndrome (SS). METHOD: Two prognostic indices were developed for early (IA-IIA) and late stage (IIB-IVB) disease based on multivariate data from 1502 patients. End-points included overall survival (OS) and progression free survival (PFS). External validation included 1221 patients. FINDINGS: Significant adverse prognostic factors at diagnosis consisted of male gender, age >60, plaques, folliculotropic disease and stage N1/Nx for early stage, and male gender, age >60, stages B1/B2, N2/3 and visceral involvement for late stage disease. Using these variables we constructed two separate models each defined using 3 distinct groups for early and late stage patients: 0-1 (low risk), 2 (intermediate risk), and 3-5 factors (high risk). 10 year OS in the early stage model was 90.3% (low), 76.2% (intermediate) and 48.9% (high) and for the late stage model 53.2% (low), 19.8% (intermediate) and 15.0% (high). For the validation set significant differences in OS and PFS in early stage patients (both p<0.001) were also noted. In late stage patients, only OS differed between the groups (p=0.002). INTERPRETATION: This proposed cutaneous lymphoma prognostic index provides a model for prediction of OS in early and late stage MF/SS enabling rational therapeutic choices and patient stratification in clinical trials.