Hexose Monophosphate Shunt Activity Research Articles

Dietary fat- and phenobarbital-induced alterations in hepatic antioxidant functions of mice.

Inbred strains of mice with differential response to known tumor promoters were compared with respect to their susceptibility to modulation of hepatic antioxidant enzymes by long-term treatment with high fat diet (HF) and phenobarbital (PB). Mice of the C57BL/6J (C57), C3H/HeOuJ (C3H) and DBA/2J (DBA) strains were fed diets containing low (5%) or high (15%) amounts of fat (sunflower oil) for 26 weeks from the age of 6 weeks onwards. Groups of mice on the 5% fat diet received 0.05% PB in their drinking water from 12 to 22 weeks of age. Mice of the C57 strain are known to be refractory to promotion of hepatocarcinogenesis, the C3H strain has a high incidence of spontaneous tumors and is sensitive to promotion by HF and PB, and the DBA strain is especially sensitive to promotion by PB. Within all strains of mice, and in both dietary groups, the degree of oxidative stress in the liver was found to increase with age, as was indicated by the increased amounts of TBA reactive material (lipid peroxidation) and decreased glutathione (GSH) and phospholipid contents of the tissue. HF elevated the amount of TBA reactive material in the liver of C57 and C3H mice, induced GSH-peroxidase and Mn-superoxide dismutase activities in the C3H strain, and depressed the hexose monophosphate shunt activity within all mouse strains. PB drastically decreased the amount of TBA reactive material in the liver in all mouse strains, increased catalase activity in all strains and the activity of GSH-peroxidase in the C3H and DBA strains. The above strain differences in responses of hepatic antioxidant functions to HF and PB parallel the differential responsiveness of these mouse strains to promotion of hepatocarcinogenesis by these agents, and the increased antioxidant capacity was proportional to susceptibility to tumor promotion.

Suppression of monocyte and neutrophil function by recombinant IL‐2

Little IS known about the influence of IL-2 on phagocytes. We now describe the effects of human recombinant IL-2 on human neutrophil and monocyte functions related to mobility, phagocytosis, glucose uptake, respiration and degranulation. Neutrophil adherence and hexose monophosphate shunt activities were both suppressed after incubation with IL-2. IL-2 had no effect on neutrophil migration, phagocytosis, deoxyglucose uptake or degranulation, ionocytes demonstrated a greater sensitivity to IL-2 with suppression of monocyte adherence, random and stimulated migration, glucose uptake and hexose monophosphate shunt activity, even after addition of phorbol myristate acetate. Monocyte phagocytosis and degranulation were not affected. All of the effects observed were dose-dependent within a biologically active range for IL-2. These studies suggest that IL-2 may have an important down-regulatory role across a broad range of monocyte functions including movement, deoxyglucose uptake and respiration. However, its role in regulation of neutrophil function is limited to adherence and respiration. IL-2 may be a more versatile cytokine than has previously been appreciated.

TNF‐specific deactivation of granulocytes in vivo: A possible mechanism of self‐protection

Granulocytes (PMN) have recently been shown to be specifically deactivated towards tumor necrosis factor (TNF) by preexposure to TNF itself and to other stimuli such as endotoxin and complement C5a in vitro. When TNF (200 micrograms/m2) was infused to a male volunteer, we found ex vivo an almost complete TNF-specific deactivation of PMN adhesion (57 +/- 3* vs 6 +/- 2%**), of exocytosis of secondary granules (37 +/- 1* vs 7 +/- 1%**) and of the respiratory burst as measured by hexose monophosphate shunt activation (74 +/- 1* vs 12 +/- 3** nM/10(7) PMN/45 min). Furthermore, up- and down-regulation, respectively, of the PMN surface adhesion molecules Mac-1 and LAM-1 became TNF-resistant. Similar to the results obtained in vitro, deactivation correlated with a decrease in the number of cellular TNF receptors. In PMN of 4 patients exposed to activated complement during hemodialysis, a state of TNF-specific cross-deactivation could also be demonstrated.

Isolation of phenotypically and functionally distinct macrophage subpopulations from human bronchoalveolar lavage

Bronchoalveolar lavage was used to obtain alveolar macrophages (AM) from the lower respiratory tract of healthy normal volunteers. Monoclonal antibody (MoAb) probes specific against macrophage determinants were then applied, in conjunction with density separation techniques, to identify and isolate three relatively homogeneous subpopulations from the AM pool. The MoAbs used, RFD1 and RFD7, have previously been shown to differentiate between "dendritic" cells and mature macrophages, respectively, in normal tissue. In addition to these two phenotypically distinct AM subsets (RFD1+D7- and RFD1-D7+ AM), a third AM subpopulation was isolated, which appeared to express both markers (RFD1+D7+). All three separated macrophage subsets were morphologically similar but exhibited distinct differences in surface receptor expression, enzyme content and physiology. Isolated RFD1+D7- AM (the phenotype of "dendritic" cells) did not adhere to the glass, had weak expression of C3b and FcR1 receptors, low fibronectin content and lysosomal activity; only a small proportion of these cells exhibited phagocytosis. The other two isolated AM subsets adhered to glass, expressed C3b and FcR1 receptors, had high fibronectin and acid phosphatase content, and a large majority exhibited phagocytic capacity; qualitative and quantitative differences in these features existed between the two AM subtypes. Furthermore, a diverse spectrum of hexose monophosphate shunt activity was observed throughout all three AM subpopulations, with the highest activity being recorded in the non-adherent AM. These data support the concept of a dynamic heterogeneity within the AM population. The variation in surface antigen expression and physiological capabilities observed amongst the three isolated AM subsets implies the presence of functionally distinct AM within the human lung, which, during steady-state conditions, may be critically balanced under the influence of stimuli in their local microenvironment. In support, proportional and functional shifts have been witnessed amongst these three AM subpopulations with the advent of disease.

Energy metabolism in developing brain cells.

During development different energy substrates are available to cells in brain in plentiful supply. The metabolic environment, which is dictated by the milk diet rich in fat, ensures that substrates in addition to glucose are available as fuels. Some substrates serve readily as primary fuels for respiration, whereas other substrates can serve other functions in addition to serving as primary fuels. Primary fuels for respiration serve to supply acetyl CoA directly and as a result always have first priority. With this criteria in mind, a consideration of substrate priority for respiration by developing brain is presented. Many studies in the decade, 1970-1980, in human infants and in the rat pup model show that both glucose and the ketone bodies, acetoacetate and D-(-)-3-hydroxybutyrate, are taken up by brain and used for energy production and as carbon sources for lipogenesis. Products of fat metabolism, free fatty acids, ketone bodies, and glycerol dominate metabolic pools in early development as a consequence of the milk diet. This recognition of a distinctive metabolic environment from the well-fed adult was taken into consideration within the last decade when methods became available to obtain and study each of the major cell populations, neurons, astrocytes, and oligodendrocytes in near homogeneous state in primary cultures. Studies on these cells made it possible to examine the distinctive metabolic properties and capabilities of each cell population to oxidize the metabolites that are available in development. Studies by many investigators on these cell populations show that all three can use glucose and the ketone bodies in respiration and for lipogenesis. Only one cell type, the astrocytes, can beta-oxidize fatty acids such as octanoate. By comparing the production of labeled carbon dioxide from glucose labeled on carbon-1 compared with carbon-6, it is clear that all three cell populations are capable of active hexose monophosphate shunt activity. Neurons and oligodendrocytes are capable of making good use of acetoacetate and D-(-)-3-hydroxybutyrate, whereas the best substrate for astrocytes is fatty acid. Under comparable conditions of incubation with astrocytes, fatty acids serve better than ketones, which in turn serve better than glucose in respiration. Some of the major factors that can explain the differing observations by different investigators on the capacity for substrate oxidation are presented. Over the last decade, astrocytes have captured the attention of neurobiologists because they have special attributes as metabolic support cells for the management of intermediary metabolism in brain.(ABSTRACT TRUNCATED AT 400 WORDS)

Quinone induced stimulation of hexose monophosphate shunt activity in the guinea pig lens: role of zeta-crystallin

The response of the hexose monophosphate shunt (HMS) in organ-cultured guinea pig lens to 1,2-naphthoquinone and 5-hydroxy-1,4-naphthoquinone (juglone)_has been investigated. Both these compounds, which are substrates of guinea pig lens zeta-crystallin (NADPH:quinone oxidoreductase), were found to cause increases in the rate of 14CO 2 production from 1- 14C-labelled glucose. Exposure of lenses to 15 μM 1,2-naphthoquinone or 20 μM juglone yielded 5.9- and 7-fold stimulation of HMS activity, respectively. Unlike hydrogen peroxide-induced sitmulation of HMS activity, these effects were not abolished by preincubation with the glutathione reductase inhibitor, 1,3-bis(2-chloroethyl)-1 nitrosourea (BCNU). While hydrogen peroxide produced substantial decrements in lens glutathione (GSH) levels, incubation with quinones was not associated with a similar reduction in GSH concentration. Protein-bound NADPH content in quinone-exposed guinea pig lenses was decreased, with a concomitant increase in the amounts of free NADP +. This finding supported the involvement of zeta-crystallin bound NADPH in the in vivo enzymic reduction of quinones. Hydrogen peroxide, on the other hand, caused decreases in the level of free NADPH alone, serving to confirm our earlier inference that quinone stimulated increases in the guinea pig lens HMS could be mediated through zeta-crystallin NADPH:quinone oxidoreductase activity.

Oxidative metabolic status of blood monocytes and alveolar macrophages in the spectrum of human pulmonary tuberculosis

The oxidative metabolic status of blood monocytes (BM) and alveolar macrophages (AM) in patients with active pulmonary tuberculosis (TB) (n = 40) and in successfully treated patients (n = 40) was assessed and compared with that of healthy control subjects (n = 40). Oxygen free radical (OFR) generation, measured by chemiluminescence (CL) and cytochrome c reduction assay and confirmed by using scavengers of different OFR, was suppressed in AM of the pulmonary TB group compared with healthy controls, whereas it was enhanced in BM. Successfully treated patients showed partial recovery of CL and cytochrome c reduction in AM. There was no significant change in BM of patients after having been treated. The overall capacity to generate OFR was markedly suppressed upon in vitro stimulation with latex in both BM and AM of TB patients. The observed suppressed oxidative metabolic activity in BM and AM was further elucidated by studying the molecular mechanism of respiratory burst. The activities of NADPH oxidase and enzymes of the hexose monophosphate (HMP) shunt were significantly (p less than 0.05) decreased in BM and AM of pulmonary TB patients compared with healthy controls. Patients who had been treated showed marked recovery of NADPH oxidase and HMP shunt activity. The present study suggests that tubercle bacilli escape the microbicidal action of macrophages as a result of suppressed OFR generation caused by decreased activity of HMP shunt, leading to decreased levels of NADPH, thereby preventing NADPH oxidase from working at its full capacity.

Vascular cells under peroxide induced oxidative stress: a balance study on in vitro peroxide handling by vascular endothelial and smooth muscle cells.

Enzymes such as glutathione peroxidase and catalase play an important role in the cellular defence against (per)oxidative stress. Balance- and inhibitor-studies were undertaken with in vitro cultured human vascular endothelial cells (EC) and smooth muscle cells (SMC) to assay the relative importance of these enzymes in the handling of cumene hydroperoxide (Chp) and hydrogen peroxide (H2O2). Low concentrations of Chp (up to 80 microM) could be removed to near completion within the first hour of incubation by stimulation of the hexose monophosphate shunt (HMS) of both cell types. The HMS activity reached a plateau upon incubation with higher concentrations of Chp (> 80 microM). The non-converted Chp in the higher concentrations could be detected quantitatively in the incubation solution. After inhibition of the glutathione reductase by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), incubation with Chp (40 microM) did not result in a stimulation of the HMS activity. Moreover the added Chp could be recovered from the medium. So Chp is exclusively handled by the GSH-redox cycle. When low concentrations of H2O2 (up to 80 microM) were added to EC or SMC approximately 50% of the peroxide loss could not be accounted for. Inhibitor studies with aminotriazole proved that catalase was responsible for the handling of this unaccounted H2O2. In both ECs and SMCs at lower concentrations of H2O2 the GSH-redox cycle was as effective as catalase and at higher H2O2 concentrations the catalase pathway plays the major role.

Stimulation of human neutrophil oxidative metabolism by nonopsonized Neisseria gonorrhoeae.

Nonopsonized gonococci possessing opacity-associated (Opa; previously PII) outer membrane proteins stimulate neutrophils to undergo a vigorous oxidative response when measured by luminol-dependent chemiluminescence (LDCL). In these studies, we characterized the mechanism of this stimulation. No gonococci that we tested induced measurable release of neutrophil superoxide anion (O2-) or hydrogen peroxide (H2O2) as measured by reduction of cytochrome c or the oxidation of scopoletin, respectively. Neutrophils pretreated with gonococci and then exposed to phorbol myristate acetate, the chemotactic peptide formylmethionylleucylphenylalanine, or opsonized zymosan released levels of neutrophil O2- and H2O2 comparable to controls, indicating that gonococci were not preventing or inhibiting neutrophil O2- or H2O2 release. To ascertain a possible explanation for these seemingly contradictory observations (i.e., induction of LDCL, but no release of O2- or H2O2), we further characterized the ability of Opa+ gonococci to stimulate LDCL. By using 1 mM azide and 4 U of horseradish peroxidase to monitor extracellular LDCL selectively and 2,000 U of catalase to monitor intracellular LDCL selectively, we determined that greater than 80% of total gonococcus-induced neutrophil LDCL occurred intracellularly. In addition, neutrophils stimulated with Opa+ gonococci showed a marked increase in O2 uptake and hexose monophosphate shunt activity. We conclude that Neisseria gonorrhoeae induces neutrophil oxidative metabolism without causing release of detectable amounts of reactive oxygen intermediates into the surrounding milieu. The gonococcus apparently directs oxidase assembly and activity to the phagolysosomal membrane. This could be a mechanism by which extracellular gonococci persist for extended periods in vivo in the presence of high concentrations of neutrophils.

Interleukin-2 Therapy Enhances Salicylate Oxidation by Blood Granulocytes

AbstractThese studies determined the effect of interleukin-2 (IL-2) immunotherapy on the oxidative metabolism of the blood granulocytes of eight patients with metastatic renal cancer. We quantitated the rate of the hexose monophosphate shunt activity (HMPS), hydrogen peroxide (H2O2) production, and salicylate oxidation of the unstimulated and phorbol myristate acetate (PMA)-stimulated granulocyte cultures before, during, and after a 5-day continuous infusion of IL-2. There was no change in the rate of HMPS activity. However, the rate of salicylate oxidation of the unstimulated and PMA-stimulated cultures of these patients was significantly increased after the therapy was complete. Overall, there was no increase in the rate of H2O2 production, although the PMA-stimulated cultures of three of eight patients had a twofold higher production of H2O2 after treatment compared with the pretreatment values. The enhanced rate of salicylate oxidation by the granulocytes after treatment indicates that these cells were “stimulated” in vivo to produce a potent oxidant, which is most likely hydroxyl radical or an oxidant of comparable activity. Further, the granulocytes were primed (“activated”), since they had an augmented response to PMA. IL-2 did not stimulate the oxidative metabolism of granulocyte cultures in vitro, suggesting that the IL-2 effect in vivo is not a direct one. Our results indicate that IL-2 immunotherapy is associated with the activation of blood granulocyte oxidative metabolism and that these activated granulocytes may be related to some of the toxic side effects of IL-2 therapy such as the capillary leak syndrome. Further oxidant injury to the granulocytes may explain the reported defect in chemotaxis.

Interleukin-2 therapy enhances salicylate oxidation by blood granulocytes

These studies determined the effect of interleukin-2 (IL-2) immunotherapy on the oxidative metabolism of the blood granulocytes of eight patients with metastatic renal cancer. We quantitated the rate of the hexose monophosphate shunt activity (HMPS), hydrogen peroxide (H2O2) production, and salicylate oxidation of the unstimulated and phorbol myristate acetate (PMA)-stimulated granulocyte cultures before, during, and after a 5-day continuous infusion of IL-2. There was no change in the rate of HMPS activity. However, the rate of salicylate oxidation of the unstimulated and PMA-stimulated cultures of these patients was significantly increased after the therapy was complete. Overall, there was no increase in the rate of H2O2 production, although the PMA-stimulated cultures of three of eight patients had a twofold higher production of H2O2 after treatment compared with the pretreatment values. The enhanced rate of salicylate oxidation by the granulocytes after treatment indicates that these cells were “stimulated” in vivo to produce a potent oxidant, which is most likely hydroxyl radical or an oxidant of comparable activity. Further, the granulocytes were primed (“activated”), since they had an augmented response to PMA. IL-2 did not stimulate the oxidative metabolism of granulocyte cultures in vitro, suggesting that the IL-2 effect in vivo is not a direct one. Our results indicate that IL-2 immunotherapy is associated with the activation of blood granulocyte oxidative metabolism and that these activated granulocytes may be related to some of the toxic side effects of IL-2 therapy such as the capillary leak syndrome. Further oxidant injury to the granulocytes may explain the reported defect in chemotaxis.

Analysis of concurrent glucose consumption by the hexose monophosphate shunt, glycolysis, and the polyol pathway in the crystalline lens

A method based on 13C-NMR spectroscopy is described, in which the metabolism of [ 13C 2]glucose was assessed in intact rat lenses. By analysing the ratio of 13 C 3 ( 13C 2 + 13 C 3) lactate, a quantitative measurement of the hexose monophosphate shunt (HMPS) activity could be derived. Similarly, measurements of the time-dependent increase of lactate and sorbitol permitted the determination of the activity of the Embden-Meyerhof pathway and the polyol pathway, respectively. This method offers significant advantages over other biochemical and spectroscopic methods and allows for the first time a direct correlation between the activities of the polyol pathway and the HMPS.

Up-regulation of glucose metabolism in Kupffer cells following infusion of tumour necrosis factor

Alterations of glucose metabolism and the oxidation of glutamine and palmitate were studied, by using specifically labelled substrates, in freshly isolated Kupffer cells and hepatic endothelial cells after infusion in vivo of human recombinant tumour necrosis factor-alpha (TNF; 7.5 x 10(5) IU/30 min per kg body wt., intravenously). Cells were incubated in a medium containing 5 mM-glucose, 0.4 mM-palmitate, 1 mM-lactate and 0.5 mM-glutamine. Administration of TNF in vivo increased glucose use in Kupffer cells by 70%. Glucose oxidation in the tricarboxylic acid cycle and flux in the Embden-Meyerhof (EM) pathway were elevated by 40 and 80% respectively. Treatment in vitro with 1 microM-phorbol 12-myristate 13-acetate (PMA) resulted in a similar percentage increase in glucose use by Kupffer cells prepared from either saline- or TNF-treated rats. However, PMA increased the activity of the hexose monophosphate shunt (HMS) by 3- and 10-fold in cells isolated from saline- or TNF-infused animals respectively. A phagocyte stimulus in vitro, opsonized zymosan, increased glucose use by 30% and doubled the flux through the HMS in Kupffer cells from saline-infused animals. The activity of the HMS in response to zymosan was increased by 400% after TNF treatment. In endothelial cells, basal glucose utilization was not altered by TNF treatment. PMA increased HMS activity in endothelial cells to a similar degree after saline or TNF infusion. Zymosan, however, increased HMS activity only in endothelial cells from TNF-treated rats. Oxidation of palmitate or glutamine was not affected by TNF treatment either under basal conditions or after challenge in vitro. Our data indicate that, after phagocytosis in vitro or protein kinase C activation, glucose use and flux through the HMS increase in Kupffer cells. This is accompanied by increased glycolytic flux, with no changes in glucose oxidation in the tricarboxylic acid cycle. After TNF exposure, followed by a secondary stimulus, the enhanced glucose use by Kupffer cells is primarily channelled through the HMS pathway. These data suggest that the increased glucose use in vivo by Kupffer cells found after immune-stimulated conditions may subserve primarily the increased need for NADPH and HMS intermediates.

Dexamethasone inhibits the hexose monophosphate shunt in activated rat peritoneal macrophages by reducing hexokinase-dependent sugar uptake

Dexamethasone decreases 2-D-deoxyglucose (2-dGlc) uptake and accumulation into rat peritoneal macrophages in vitro in a concentration- and time-dependent manner (Ki for 1 microM-dexamethasone after a 2 h exposure = 0.71 +/- 0.21 microM; Ki for 0.1 microM-dexamethasone after exposure for 4 h = 0.10 +/- 0.06 microM). The inhibition of 2-dGlc uptake is consistent with a decrease in the coupling between endofacial hexokinase activity and the sugar transporter. The evidence for this is: (1) the Km for zero-trans 2-dGlc uptake in quiescent macrophages was increased by dexamethasone, but there was no significant effect on the Vmax.; (2) dexamethasone increased the rate of exit of sugar from cells preloaded with 2-dGlc; (3). the free sugar accumulation within the cytosol of the cells above the external solution concentration was significantly decreased by dexamethasone. These effects of dexamethasone on 2-dGlc transport were antagonized by simultaneous exposure to the steroid RU 38486 (Ki = 0.04 +/- 0.01 microM; 4 h incubation). Although dexamethasone inhibited zero-trans uptake, the maximum rate of infinite-trans exchange uptake of 2-dGlc into cells preloaded with 3-O-methyl-D-glucose (40 mM) was unaltered by dexamethasone or RU 38486, indicating that the dexamethasone-dependent decrease in zero-trans uptake was not due to a change in the number of transporters in the plasma membrane. Dexamethasone also inhibited the phorbol myristate acetate-induced stimulation of hexose monophosphate shunt (HMPS) activity, and this was reversed by RU 38486. Cytochalasin B, the potent sugar-transport inhibitor, inhibited HMPS activity and 2-d[2,6-3H]Glc uptake equally, indicating a single site of action. By contrast, dexamethasone showed differential inhibition of HMPS activity and 2-d[2,6-3H]Glc uptake, suggesting that it not only acts by decreasing the coupling between hexokinase and sugar transport, but also at one or more additional points.

Effects of macrophage colony-stimulating factor and phorbol myristate acetate on 2-D-deoxyglucose transport and superoxide production in rat peritoneal macrophages.

2-D-Deoxyglucose (2-dGlc) uptake and accumulation into rat peritoneal macrophages was increased by colony-stimulating factor (mCSF) by stimulating the coupling between endofacial hexokinase activity and the sugar transporter. The evidence for this is as follows: (1) mCSF significantly decreased the Km for zero-trans uptake (P less than 0.05), without altering Vmax.; (2) the accumulation of free 2-dGlc was increased by mCSF (P less than 0.05); (3) mCSF retarded the rate of exit of accumulated free 2-dGlc. The mCSF-dependent increase in 2-dGlc uptake by macrophages was enhanced by preincubation of the cells in mCSF-free solution. The activity of the hexose monophosphate shunt (HMPS) measured by the differential uptake of 2-d[1-3H]Glc and 2-d[2,6-3H]Glc was not stimulated by mCSF. Also, in quiescent cells, superoxide production, as determined by cytochrome c reduction, was unaffected by mCSF. Phorbol myristate acetate (PMA; 40 nM) stimulated both the HMPS activity and superoxide production. Both these effects were dependent on the uptake of external sugar (2-dGlc). Incubation of the macrophages with mCSF enhanced the sugar transport and PMA-dependent stimulation of HMPS activity and superoxide production, indicating a role for mCSF in the 'priming' of macrophage functions. Both HMPS activity and superoxide production are entirely dependent on uptake of exogenous sugar, since the potent sugar-transport inhibitor cytochalasin B competitively inhibited 2-dGlc uptake, HMPS activity and superoxide generation in PMA-activated cells (Ki approximately 0.3 microM for all three processes). Over a wide range of 2-dGlc concentrations, 4 mol of superoxide were generated/mol of 2-dGlc metabolized in the HMPS pathway, indicating coupling between these processes. The Km of 2-d[2,6-3H]Glc uptake in PMA-treated cells was 0.45 +/- 0.07 mM, and Vmax. was 1.32 +/- 0.05 mumol.min-1.ml of cell water-1. It is evident that there is a large degree of slippage between HMPS activity and membrane-associated hexokinase activity, since the Km for HMPS activity was 0.06 +/- 0.02 mM and the Vmax. was 0.10 +/- 0.03 mumol.min-1.ml of cell water-1.

Inhibition of hexose monophosphate shunt in young erythrocytes by pyrimidine nucleotides in hereditary pyrimidine 5‘ nucleotidase deficiency

Recent reports have suggested that haemolytic anaemia in pyrimidine 5' nucleotidase (P5'N) deficiency might be due to impaired erythrocyte hexose monophosphate shunt (HMS). To investigate the relationship between pyrimidine accumulation, HMS impairment and shortened red-cell survival, we tested glucose 6-phosphate dehydrogenase (G-6PD), HMS, P5'N activities and the UV spectrum in whole red cells and in red cells of different age from 2 P5'N-deficient patients with different degrees of haemolytic anaemia. In whole red cells we found a reduction of both G-6PD and stimulated HMS activity in the presence of a variable amount of pyrimidine nucleotides (37.79 and 17.88 mumol/gHb respectively). A drastic inhibition of stimulated HMS activity was already present in the lightest red-cell fractions from patient 1, who presented a more severe haemolytic anaemia. The variable degree of pyrimidines found among red cell fractions, with a minor accumulation in the older red cells, supports the hypothesis that pyrimidine accumulation and HMS impairment occur in the younger erythrocytes of P5'N-deficient patients.

Auranofin inhibits the activation pathways of polymorphonuclear leukocytes at multiple sites

In order to characterize the mechanism by which the anti-rheumatic gold complex auranofin (AF) affects the functions of resting and activated polymorphonuclear leukocytes (PMN) the following studies were performed: (1) The effect of AF on the major processes involved in the respiratory burst of PMN: glucose transport and phosphorylation; hexose monophosphate (HMP) shunt activity in intact cells and in a cell-free system; Superoxide production by paniculate fractions and intact PMN measured as lucigenin-dependent chemiluminescence. (2) A comparison of the effects of AF added to the PMN before, at the time of, or subsequent to the stimulants [ N-formyl-methionyl-leucyl phenylalanine (FMLP), concanavalin A (ConA), calcium ionophore (A23187) and phorbol myristate acetate (PMA)]. (3) The effect of AF on PMN activated by two stimulates (PMA, ConA) added sequentially. AF (0.1–10 μM) caused a dose-dependent inhibition of lucigenin-dependent chemiluminescence regardless of the activator (FMLP, ConA, A23187, PMA) when AF was added before the activator. In contrast, when AF was added to PMN after stimulation, it inhibited only the chemiluminescence of PMN stimulated by PMA. Furthermore, the chemiluminescence was largely unaffected by AF in sequentially activated PMN. The relative sensitivity to AF of the various processes studied indicates that blockade of the activation signal appears to be responsible for inhibition of the respiratory burst of PMN.

The role of endotoxin in the pathogenesis of bacterial peritonitis with special reference to superoxide in polymorphonuclear leukocytes stimulated by endotoxin

It is well known that endotoxin (Et) plays an important role in severe surgical infectious diseases such as peritonitis. Recently, it has been reported that increased superoxide (O2-) formation and accelerated lipid-peroxidation cause the progress of Et shock. The present study was designed to estimate the changes in the amount of lipid-peroxides in the liver and the relationship between Et and lipid-peroxidation in bacterial peritonitis. Plasma Et levels, lipid-peroxides in the liver, the number of leukocytes in the blood and the number of bacteria in the blood and peritoneal cavity were determined using an experimental peritonitis model that was induced by intraperitoneal (i.p.) injection of E. coli, E. faecalis and B. fragilis, as well as experimental endotoxemia model induced i.p. injection of Et. The influence of ET on the function of polymorphonuclear leukocytes (PMN), that was considered to be one of the origins O2- production, was studied using PMN from the peritoneal cavity of rats. The plasma Et level was increased in an E. coli group and mixed injection group, and the lipid-peroxide levels in the liver were increased in these two groups as well as in a B. fragilis group. Plasma Et and lipid-peroxide levels in the liver were also increased in Et injected mice. In the study of the influence of Et on PMN function, O2- formation of PMN was increased when PMN was stimulated by Et with a high concentration and hexose monophosphate shunt activity was increased in all PMN stimulated by Et. These results suggest that O2- from PMN stimulated by Et is related to lipid-peroxidation in the liver, which is considered an index of injury in bacterial peritonitis.

Phagocytosis of Leishmania enhances macrophage activation by IFN-gamma and lipopolysaccharide.

This investigation describes the ability of Leishmania promastigotes to enhance activation of bone marrow-derived murine macrophages in vitro if added together with rIFN-gamma in the presence or absence of LPS. Activation was defined as the capacity for arginine-derived NO2- production and the killing of intracellular Leishmania. Enhanced NO2- production was observed for either CBA or C3H/HeJ macrophages undergoing phagocytosis at the time of activation. Other phagocytic stimuli including inert polystyrene latex beads were as effective as Leishmania. No correlation could be demonstrated between the enhanced NO2- release and secretion of products of the respiratory burst or PGE2. However, TNF-alpha secretion was elevated in cultures undergoing phagocytosis and a relationship between hexosemonophosphate shunt activity and NO2- levels was evident. These studies confirm and extend previous reports that phagocytosis plays an important role in the regulation of macrophage physiology.

Mechanisms of fat-related modulation of N-nitrosodiethylamine-induced tumors in rats: organ distribution, blood lipids, enzymes and pro-oxidant state.

Groups of rats, either dosed with N-nitrosodiethylamine (NDEA) for 10 weeks (from the age of 7 to 17 weeks) or untreated, were fed diets containing either 2% (low fat, LF) or 30% polyunsaturated fat (high fat, HF) on an equicaloric basis from 5 weeks until rats were 43 weeks old. Biochemical parameters were measured during and at the end of the experiment in various organs, blood, urine and exhaled air, for correlation with the presence or absence of tumors. The HF diet tended to increase the number of hepatic tumors induced by NDEA, while the number of extrahepatic tumors was higher in rats fed on the LF diet; also the overall tumor incidence was higher in the LF group. In the HF/NDEA group, only two benign extrahepatic tumors were found. Plasma total and free cholesterol and triglyceride concentrations were lower in the HF than the LF group without NDEA treatment. In animals bearing liver and/or extrahepatic tumors all plasma lipid concentrations were lower than in tumor-free animals. Only minor or no changes were detected in blood catalase activity, malondialdehyde level, reduced glutathione (GSH) level or GSH-related enzymes and excretion of thioethers in the urine due to dietary modulation or NDEA. Changes in the liver that were associated with the HF diet were: (i) increased amounts of some polyunsaturated fatty acids and of total phospholipids in liver microsomes; (ii) an enhanced level of lipid peroxidation in liver; (iii) a decrease in liver glutathione levels during NDEA treatment, with a simultaneous adaptive increase in superoxide dismutase levels, and a decrease in renal glutathione levels in both treated and untreated groups; (iv) enhanced microsomal induction of aminopyrine N-demethylase and epoxide hydrolase activities by NDEA, and (v) decreased hexose monophosphate shunt (HMS) activity. All mono-oxygenase activities were lower, and the activities of epoxide hydrolase, UDP-glucuronosyltransferase and HMS were higher, in liver tumors than in non-tumorous liver of similarly-treated rats. Neither diet nor NDEA had a major effect on drug-metabolizing enzyme activities in lung and kidney. HF diet significantly increased ethane exhalation (an indicator of the whole-body pro-oxidant state) over those on the LF diet: in rats on either diet, it was further increased when NDEA was given. Ethane exhalation was still elevated 30 weeks after the cessation of NDEA treatment. Our results suggest an association between the observed changes in biochemical parameters, notably oxidative stress, due to dietary modulation and the altered tumor incidence and organ distribution of tumors induced by NDEA.