Faculty Bibliography

Glucose metabolism was studied in human red blood cells incubated in the presence of physiologic concentrations of ascorbate (0.1 mM) and/or lactate (2 mM) plus pyruvate (0.1 mM). The total flux through glycolysis, as measured by 14C-labeling of glycolytic intermediates, was increased about 15% by ascorbate, 30% by lactate plus pyruvate, and 40% by ascorbate plus lactate plus pyruvate. We found, however, that physiologic concentrations of ascorbate and/or lactate plus pyruvate had no effect on flux of glucose or recycling of pentoses through the hexose monophosphate shunt. Increased formation of lactate accounted for most of the observed increase in glycolysis with little change in pyruvate formation, indicating that the increased flux of reducing equivalents from glucose was stored as lactate rather than being consumed by red cell metabolism. In all experiments, there was a net increase with time in the absolute amount of both lactate and pyruvate in red cell suspensions, indicating that lactate or pyruvate present at zero time did not function as a stoichiometric source or sink for reducing equivalents. There was little effect on steady-state levels of ATP or 2,3-diphosphoglycerate. Equilibration of ascorbate between red cells and the medium was complete before the addition of 14C-labeled glucose to the medium. Glucose metabolism prevented net oxidation of ascorbate in the incubation medium. Physiologic concentrations of ascorbate, lactate and pyruvate appear to increase flux through glycolysis by increasing the turnover of ATP and/or 2,3-diphosphoglycerate. Red cells were exposed to mild oxidative stress by incubation with 0.27 mM 6-hydroxydopamine, 0.27 mM 6-aminodopamine, 0.13 mM 1,4-naphthoquinone-2-sulfonic acid or 0.27 mM phenylhydrazine. The metabolic response to oxidative stress was determined by measuring the formation of methemoglobin, pyruvate, lactate and CO2 in the presence and absence of physiologic concentrations of lactate, pyruvate and ascorbate. Lactate, pyruvate and ascorbate had no effect on the net methemoglobin accumulation but rather on the distribution of the metabolic sources of reducing equivalents and on the flux of reducing equivalents to oxygen. Physiologic lactate and pyruvate allowed increased flow of reducing equivalents from glycolysis to methemoglobin and ultimately oxygen without the necessity of increased flux through glycolysis. This was accomplished by a decrease in the ratio of newly formed lactate to newly formed pyruvate with no increase in total lactate plus pyruvate.(ABSTRACT TRUNCATED AT 400 WORDS)

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

Red cells exposed to t-butyl hydroperoxide undergo lipid peroxidation, haemoglobin degradation and hexose monophosphate-shunt stimulation. By using the lipid-soluble antioxidant 2,6-di-t-butyl-p-cresol, the relative contributions of t-butyl hydroperoxide and membrane lipid hydroperoxides to oxidative haemoglobin changes and hexose monophosphate-shunt stimulation were determined. About 90% of the haemoglobin changes and all of the hexose monophosphate-shunt stimulation were caused by t-butyl hydroperoxide. The remainder of the haemoglobin changes appeared to be due to reactions between haemoglobin and lipid hydroperoxides generated during membrane peroxidation. After exposure of red cells to t-butyl hydroperoxide, no lipid hydroperoxides were detected iodimetrically, whether or not glucose was present in the incubation. Concentrations of 2,6-di-t-butyl-p-cresol, which almost totally suppressed lipid peroxidation, significantly inhibited haemoglobin binding to the membrane but had no significant effect on hexose monophosphate shunt stimulation, suggesting that lipid hydroperoxides had been decomposed by a reaction with haem or haem-protein and not enzymically via glutathione peroxidase. The mechanisms of lipid peroxidation and haemoglobin oxidation and the protective role of glucose were also investigated. In time-course studies of red cells containing oxyhaemoglobin, methaemoglobin or carbonmono-oxyhaemoglobin incubated without glucose and exposed to t-butyl hydroperoxide, haemoglobin oxidation paralleled both lipid peroxidation and t-butyl hydroperoxide consumption. Lipid peroxidation ceased when all t-butyl hydroperoxide was consumed, indicating that it was not autocatalytic and was driven by initiation events followed by rapid propagation and termination of chain reactions and rapid non-enzymic decomposition of lipid hydroperoxides. Carbonmono-oxyhaemoglobin and oxyhaemoglobin were good promoters of peroxidation, whereas methaemoglobin relatively spared the membrane from peroxidation. The protective influence of glucose metabolism on the time course of t-butyl hydroperoxide-induced changes was greatest in carbonmono-oxyhaemoglobin-containing red cells followed in order by oxyhaemoglobin- and methaemoglobin-containing red cells. This is the reverse order of the reactivity of the hydroperoxide with haemoglobin, which is greatest with methaemoglobin. In studies exposing red cells to a wide range of t-butyl hydroperoxide concentrations, haemoglobin oxidation and lipid peroxidation did not occur until the cellular glutathione had been oxidized. The amount of lipid peroxidation per increment in added t-butyl hydroperoxide was greatest in red cells containing carbonmono-oxyhaemoglobin, followed in order by oxyhaemoglobin and methaemoglobin. Red cells containing oxyhaemoglobin and carbonmono-oxyhaemoglobin and exposed to increasing concentrations of t-butyl hydroperoxide became increasingly resistant to lipid peroxidation as methaemoglobin accumulated, supporting a relatively protective role for methaemoglobin. In the presence of glucose, higher levels of t-butyl hydroperoxide were required to induce lipid peroxidation and haemoglobin oxidation compared with incubations without glucose. Carbonmono-oxyhaemoglobin-containing red cells exposed to the highest levels of t-butyl hydroperoxide underwent haemolysis after a critical level of lipid peroxidation was reached. Inhibition of lipid peroxidation by 2,6-di-t-butyl-p-cresol below this critical level prevented haemolysis. Oxidative membrane damage appeared to be a more important determinant of haemolysis in vitro than haemoglobin degradation. The effects of various antioxidants and free-radical scavengers on lipid peroxidation in red cells or in ghosts plus methaemoglobin exposed to t-butyl hydroperoxide suggested that red-cell haemoglobin decomposed the hydroperoxide by a homolytic scission mechanism to t-butoxyl radicals

Primaquine, an 8-aminoquinoline, and chloroquine, a 4-aminoquinoline, both stimulate ATP hydrolysis in human red blood cells incubated in the absence of glucose. In the presence of glucose, ATP levels are partially maintained by increased flux of glucose through glycolysis. Glucose dependence of chloroquine uptake and the activity of primaquine as a redox reagent explain quantitative differences in ATP hydrolysis and accumulation of specific glycolytic products

Stimulation of the hexose monophosphate shunt by primaquine results from the oxidation of NADPH by primaquine. This conclusion was based on the observations that primaquine lowered cellular NADPH but not GSH and that, in red cells in which the GSH was unavailable for reaction, primaquine still stimulated the rate of the hexose monophosphate shunt. In a non-cellular system, primaquine interacted with NADPH, but not GSH, to produce H2O2. Stimulation of the hexose monophosphate shunt by primaquine does not primarily involve H2O2 accumulation since stimulation of the pathway by primaquine was also observed in red cells containing methemoglobin, a red cell preparation in which no H2O2 accumulates. Methemoglobin prevented the formation and/or accumulation of H2O2 in intact red cells incubated with primaquine as well as in a non-cellular system containing primaquine plus Fe2+-EDTA as an H2O2 source. Methemoglobin probably acts by scavenging reactive intermediates since oxyhemoglobin was formed from methemoglobin in the non-cellular experiments. In the red cell, primaquine stimulated glucose-dependent conversion of methemoglobin to oxyhemoglobin

Lipid peroxidation and haemoglobin degradation were the two extremes of a spectrum of oxidative damage in red cells exposed to t-butyl hydroperoxide. The exact position in this spectrum depended on the availability of glucose and the ligand state of haemoglobin. In red cells containing oxy- or carbonmono-oxy-haemoglobin, hexose monophosphate-shunt activity was mainly responsible for metabolism of t-butyl hydroperoxide; haem groups were the main scavengers in red cells containing methaemoglobin. Glutathione, via glutathione peroxidase, accounted for nearly all of the hydroperoxide metabolizing activity of the hexose monophosphate shunt. Glucose protection against lipid peroxidation was almost entirely mediated by glutathione, whereas glucose protection of haemoglobin was only partly mediated by glutathione. Physiological concentrations of intracellular or extracellular ascorbate had no effect on consumption of t-butyl hydroperoxide or oxidation of haemoglobin. Ascorbate was mainly involved in scavenging chain-propagating species involved in lipid peroxidation. The protective effect of intracellular ascorbate against lipid peroxidation was about 100% glucose-dependent and about 50% glutathione-dependent. Extracellular ascorbate functioned largely without a requirement for glucose metabolism, although some synergistic effects between extracellular ascorbate and glutathione were observed. Lipid peroxidation was not dependent on the rate or completion of t-butyl hydroperoxide consumption but rather on the route of consumption. Lipid peroxidation appears to depend on the balance between the presence of initiators of lipid peroxidation (oxyhaemoglobin and low concentrations of methaemoglobin) and terminators of lipid peroxidation (glutathione, ascorbate, high concentrations of methaemoglobin)

Changes in hemoglobin status and lipid peroxidation were followed in red cells containing either oxy-met-, or carbonmonoxyhemoglobin, incubated with t-butyl hydroperoxide in a medium with or without glucose. Loss of intact hemoglobin (the sum of oxyhemoglobin and methemoglobin) was inversely proportional to the degree of lipid peroxidation in red cells containing either oxy- or methemoglobin. When glucose was added to the medium, lipid peroxidation increased while there was a decreased loss of intact hemoglobin in red cells containing either oxy- or methemoglobin, while both lipid peroxidation and changes in hemoglobin decreased in red cells containing carbonmonoxyhemoglobin. Methemoglobin formation and loss of intact hemoglobin were directly proportional to the degree of lipid peroxidation in red cells containing carbonmonoxyhemoglobin. The greatest amount of lipid peroxidation occurred in red cells containing carbonmonoxyhemoglobin, incubated without glucose. These results indicate that methemoglobin and non-intact hemoglobin may protect the membrane against lipid peroxidation. We propose that, depending on the availability of glucose and the liganded state of hemoglobin, lipid peroxidation and hemoglobin alterations represent extremes of a spectrum of oxidative damage