Hexose Monophosphate Pathway Research Articles

Hypoxic toxicity of misonidazole in a glucose-6-phosphate dehydrogenase deficient mutant chinese hamster ovary cell Line

The metabolic activation of misonidazole (MISO) and its effects on the hexose monophosphate pathway (HMP) and on cell viability were studied in hypoxic mutant Chinese hamster ovary (CHO) cells deficient in glucose-6-phosphate dehydrogenase and their parent wildtype cells. The metabolic activation of MISO was similar in both cell lines as indicated by the binding of 14C-MISO to the acid-insoluble fraction of these cells; it was decreased by the absence of glucose. In the wildtype CHO cells, MISO caused a significant stimulation of the activity of the HMP while in the mutant CHO cells no HMP activity was measurable, even in the presence of MISO. In both cell lines clonogenicity began to decline after 2 hr and trypan blue exclusion after 4 hr of hypoxic incubation. The effect of MISO on both parameters of cell viability was somewhat more pronounced in the wildtype CHO cells. This difference became especially significant at the longer incubation times. The results indicate that reducing equivalents for the metabolic activation of MISO are provided not only by the HMP but that pathways other than the HMP, such as glycolysis or pathways starting from mitochondrial tricarboxylates, are of similar or even greater importance in this respect.

Role of hexokinase in the regulation of erythrocyte hexose monophosphate pathway under oxidative stress

Human erythrocytes overloaded with homogeneous human hexokinase (up to 15-times the activity of normal RBC) show almost unmodified rates of glucose metabolized in the HMP, however hexokinase-loaded RBC are able to metabolize 1.5 fold more glucose than controls through the HMP when an oxidizing agent like methylene blue (5 to 100 microM) is present. Similarly, RBC loaded with inactivating anti-hexokinase IgG (12 +/- 3% residual hexokinase activity) show HMP rates unchanged under resting conditions, but only 12% of the HMP rate found in normal controls under oxidative stress. These data provide clear evidence that the HMP rate under conditions of oxidative stress is controlled by hexokinase activity and suggest that RBC from patients with hexokinase deficiency are not able to increase the HMP rate under oxidative stress like erythrocytes from individuals with G6PD deficiency.

Hexose monophosphate pathway activity in normal and hypoxic rat brain

Experiments were undertaken to define the role of the alternative route of glucose metabolism in the hexose monophosphate pathway (HMP) during energy balanced, mild brain hypoxia. In similar hypoxic model in spite of the lack of the deficit of high energy compounds, the significant acceleration of glycolysis and inhibition of macromolecular syntheses (lipid, proteins, and nucleic acids) were previously observed. The HMP activity, although directly coupled to intracellular synthetic processes, has not been defined and little is known about the mechanisms of its regulation under brain hypoxia. HMP activity was examined in the rat brain in vivo by estimation of the increment of 6-phosphogluconate concentration after inhibition of its oxidation as achieved by injection of 6-aminonicotinamide. The activity of this alternative route of glucose metabolism was estimated to be 0.4 mmol/h/kg w.w. in the brain cortex and 0.7 mmol/h/kg w.w. in the brain stem. During 2 h of mild hypoxia (7% O2 in N2) the HMP activity dropped to 30% of control level, whereas during first hour of reoxygenation increased to 200% of control. Increased activity of HMP in posthypoxic brain during reoxygenation also was observed in vitro by measuring the rate of [1-14C]- and [6-14C]glucose conversion to 14CO2 in cerebral cortical slices. The possible mechanism of the rapid changes in the activity of HMP induced by hypoxia is discussed. The results suggest that the brain glucose metabolism under mild hypoxia is reoriented toward energy producing pathway (glycolysis) partially at the expense of HMP. The mechanism of this regulation seems not to be directly triggered by energy deficit. Activity of HMP in the brain is in accord with the intracellular synthetic processes and their demands on the metabolites produced by this pathway. Relying upon that, the posthypoxic stimulation of HMP would indicate the metabolic recovery during reoxygenation.

A study of the role of the hexose monophosphate pathway with respect to fatty acid biosynthesis in sporulation of Saccharomyces cerevisiae.

13C NMR was used to study the pattern of label incorporation from [2-13C]acetate into trehalose during sporulation in Saccharomyces cerevisiae. A wild-type strain and a strain homozygous for the zwf1 mutation (which affects glucose-6-phosphate dehydrogenase) were used. In the wild-type it was possible to deduce the cycling of glucose 6-phosphate around the hexose monophosphate pathway whilst in the mutant strain this did not occur. The requirement of the hexose monophosphate pathway for providing NADPH for fatty acid biosynthesis was examined using 13C NMR and GC/MS. The wild-type strain produced a typical profile of fatty acids with palmitoleic acid being the most abundant whereas the mutant contained only one-quarter the amount of total fatty acid. As zwf1 homozygous diploids are able to sporulate this indicates that the large amount of fatty acid biosynthesis observed in sporulation of wild-type strains is not essential to the process.

Characterization of oxygen-tolerant Chinese hamster ovary cells: II. Energy metabolism and antioxidant status

Further characteristics of an oxygen-tolerant variant of Chinese hamster ovary cells (CHO-99) capable of stable proliferation at 99% O 2/1% CO 2, an O 2 level that is lethal to the parental line (CHO-20), are described. Previous work has revealed that CHO-99 cells have 2- to 4-fold increased activities of superoxide dismutases, catalase and glutathione peroxidase, and substantially increased relative volumes of mitochondria and peroxisomes. To document possible additional mechanisms of O 2 tolerance we compared CHO-20 cells growing at 20% O 2 (normoxia) and CHO-99 cells at 99% O 2 (normobaric hyperoxia). We show the following: (1) the estimated total (oxidative and glycolytic) ATP production in CHO-99 cells was 36% decreased. ATP production through oxidative phosphorylation was 52% lower in CHO-99 cells, while the relative contribution from glycolysis was increased from 6% to 30%. The ATP content was 29% lower in CHO-99 cells, the adenylate energy charge being also significantly decreased, indicating that energy production through oxidative phosphorylation is compromised in CHO-99 cells. Cyanide-resistant respiration was 4-fold higher in CHO-99 cells, probably reflecting, at least partly, the increased peroxisomal activity in these cells. (2) The level of reduced glutathione was several fold increased in CHO-99 cells, oxidized glutathione being unaltered; (NADPH + NADP +) levels were elevated 2.7-fold, while the ratio of NADPH to NADP + was increased almost two-fold. These changes were associated with a 50% increased metabolism of glucose through the hexose monophosphate pathway. (3) No evidence was obtained for an increased steady-state level of endogenous lipid peroxidation in CHO-99 cells, in spite of a 50% increased content of polyunsaturated fatty acids in the phospholipid fraction.

Metabolic studies on dysmyelinating mutant "pt" rabbit brain in vitro.

"Paralytic tremor" (pt) rabbit mutant is characterized by a severe hypomyelination of the CNS, however, it is not defined if the defect in myelinogenesis is an "assembly" or "synthesis" type. In this study, we have compared the general metabolic and biosynthetic properties of the myelinating mutant brain with unaffected controls of the same age. In the brain slices of 4 wk old "pt" rabbits the incorporation of U-[14C]glucose, 6-[3H] galactose, and U-[14C] leucine into macromolecules (total lipids and proteins, galactolipids, and myelin basic protein) was substantially elevated. In isolated myelin fraction, the total reduction of the radioactivity was followed by the increased specific activity of all examined macromolecules. The myelin to homogenate specific activity ratio was similar in control and "pt" rabbits. Distribution of the label and myelin marker, cyclic nucleotide 3'-phosphodiesterase (CNP-ase) among the membranous fractions suggests the partial inhibition of myelin formation in "pt" rabbits on the step of premyelin, unilamellar membranes. 14CO2 yields derived from differently labeled glucose were used for the evaluation of the basal oxidative metabolism in "pt" brain slices. 14CO2 production from U-[14C] glucose was normal. The depolarization of the slices by 50 mM K+ stimulated glucose oxidation to a higher extent in "pt" than in control. Hexose monophosphate pathway (HMP), the route providing much of NADPH required for lipid biosynthesis, did not change significantly by mutation. The activity of glucose 6-phosphate dehydrogenase (Glc-6-P DH), an oligodendroglia enriched, HMP connected enzyme, was slightly lower in "pt" homogenates by 13-17%, whereas CNP-ase was lowered more than 30% in the same samples. All this data suggest that the capacity for the synthesis of myelin constituents is well preserved in the mutant brain and the impairment of myelogenesis is probably caused by increased elimination of already synthesized, myelin-related components.

Vitamin C Increases Hexose Monophosphate Pathway Activity but Not Chemiluminescence in Neutrophils from Chronic Hemodialysis Patientsa

Annals of the New York Academy of SciencesVolume 498, Issue 1 p. 494-499 Vitamin C Increases Hexose Monophosphate Pathway Activity but Not Chemiluminescence in Neutrophils from Chronic Hemodialysis Patientsa JOSEPH NICOTRA, JOSEPH NICOTRA The Renal Laboratory and Service Department of Medicine St. Joseph's Hospital and Medical Center University of Medicine and Dentistry of New Jersey Paterson, New Jersey 07503Search for more papers by this authorPAULETTE NOVEMBRE, PAULETTE NOVEMBRE The Renal Laboratory and Service Department of Medicine St. Joseph's Hospital and Medical Center University of Medicine and Dentistry of New Jersey Paterson, New Jersey 07503Search for more papers by this authorMARK A. NEEDLE, MARK A. NEEDLE The Renal Laboratory and Service Department of Medicine St. Joseph's Hospital and Medical Center University of Medicine and Dentistry of New Jersey Paterson, New Jersey 07503Search for more papers by this authorVINCENT A. DeBARI, Corresponding Author VINCENT A. DeBARI The Renal Laboratory and Service Department of Medicine St. Joseph's Hospital and Medical Center University of Medicine and Dentistry of New Jersey Paterson, New Jersey 07503To whom all correspondence should be addressed at the Renal Lab., St. Joseph's Hospital and Medical Center, 703 Main St., Paterson, N.J.Search for more papers by this author JOSEPH NICOTRA, JOSEPH NICOTRA The Renal Laboratory and Service Department of Medicine St. Joseph's Hospital and Medical Center University of Medicine and Dentistry of New Jersey Paterson, New Jersey 07503Search for more papers by this authorPAULETTE NOVEMBRE, PAULETTE NOVEMBRE The Renal Laboratory and Service Department of Medicine St. Joseph's Hospital and Medical Center University of Medicine and Dentistry of New Jersey Paterson, New Jersey 07503Search for more papers by this authorMARK A. NEEDLE, MARK A. NEEDLE The Renal Laboratory and Service Department of Medicine St. Joseph's Hospital and Medical Center University of Medicine and Dentistry of New Jersey Paterson, New Jersey 07503Search for more papers by this authorVINCENT A. DeBARI, Corresponding Author VINCENT A. DeBARI The Renal Laboratory and Service Department of Medicine St. Joseph's Hospital and Medical Center University of Medicine and Dentistry of New Jersey Paterson, New Jersey 07503To whom all correspondence should be addressed at the Renal Lab., St. Joseph's Hospital and Medical Center, 703 Main St., Paterson, N.J.Search for more papers by this author First published: July 1987 https://doi.org/10.1111/j.1749-6632.1987.tb23792.x a This work was supported, in part, by a grant-in-aid from the New Jersey Lions District 16A Charitable Foundation. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Volume498, Issue1Third Conference on Vitamin CJuly 1987Pages 494-499 RelatedInformation

Early metabolic effects of platelet-derived growth factor and transforming growth factor-beta in rat liver in vivo.

The short term metabolic effects of the in vivo administration of platelet-derived growth factor have been examined in the liver of the rat. Meal-fed male Wistar rats weighing between 150-180 g received an intraperitoneal injection of platelet-derived growth factor (17 units/100 g weight), transforming growth factor-beta (185 ng/100 g weight), or saline. At 5 min after injection, the livers were freeze-clamped. Samples of the tissue were subsequently assayed for metabolite content and enzyme activities. Platelet-derived growth factor injection caused an elevation in the liver content of pyruvate from 0.14 +/- 0.012 to 0.19 +/- 0.009 mumol/g wet weight liver (p less than or equal to 0.01) and an increase in the cytosolic phosphorylation potential [sigma ATP]/[sigma ADP][sigma Pi] from 6670 +/- 540 to 8970 +/- 750 (p less than or equal to 0.01). In addition an increase in the hepatic content of the hexose monophosphate pathway metabolites, 6-phosphogluconate (0.027 +/- 0.004 to 0.037 +/- 0.005 mumol/g wet weight) (p less than or equal to 0.05), ribulose 5-phosphate (0.013 +/- 0.001 to 0.017 +/- 0.001 mumol/g wet weight) (p less than or equal to 0.05) and combined sedoheptulose 7-phosphate and ribose 5-phosphate (0.052 +/- 0.007 to 0.062 +/- 0.004 mumol/g wet weight) (p less than or equal to 0.05) was observed. The elevation in the hexose monophosphate pathway metabolites resulted from a 1.3-fold elevation in the activity of glucose-6-phosphate dehydrogenase [EC 1.1.1.49] when measured in a crude homogenate. Kinetic analysis performed on partially purified glucose-6-phosphate dehydrogenase demonstrated no significant change in the Km of the enzyme for either NADP+ or glucose 6-phosphate, while a 2.4-fold increase in the Vmax was observed. In view of the rapidity of the change in total measured enzyme activity and increase in the Vmax of glucose-6-phosphate dehydrogenase, it is postulated that platelet-derived growth factor causes a covalent modification of the existing enzyme. Transforming growth factor-beta caused no change in the hepatic metabolite content in the treated animals when compared to saline treated controls.

The role of glycolysis and hexose monophosphate pathway in the hypoxic toxicity of misonidazole.

The metabolic activation of misonidazole (MISO) and its effects on the hexose monophosphate pathway (HMP) and clonogenicity were studied in hypoxic EMT6/Ro, wildtype Chinese hamster ovary (CHO) and mutant CHO cells deficient in glucose-6-phosphate dehydrogenase. In all three cell lines metabolic activation of MISO, as indicated by the binding of 14C-MISO to the acid-insoluble fraction of these cells, was increased by the presence of glucose. In EMT6/Ro cells and wildtype CHO cells, MISO caused a significant stimulation of the activity of the HMP while in the mutant CHO cells no HMP activity was measurable, even in the presence of MISO. Loss of clonogenicity induced by MISO occurred markedly earlier in EMT6/Ro cells than in the CHO cells. In the latter cells, however, only a small difference was observed between the wildtype and mutant cell line. From these results it is concluded that not only the HMP but also glycolysis and other, glucose-independent, metabolic pathways are able to provide electrons for the reductive activation of MISO and hence contribute to the hypoxic toxicity of this compound.

Changes of hexose monophosphate pathway and methemoglobin reductase enzyme activity after radiation in guinea pigs

1. 1. HMP pathway activity changes occurring after exposure to ionizing radiation (LD 50 dose) have been investigated. The study was carried out on 18 experimental guinea pigs subjected to 5 successive exposures of 150 rads 3 or 4 days apart. The control animals were sham radiated but were otherwise treated identically as those of the experimental groups. 2. 2. Blood samples were taken by cardiac puncture before radiation and 30 min after each exposure of 150 rads. The red cells were re-suspended in their own plasma and HMP pathway activity was measured in the suspension. 3. 3. The pathway activity showed a consistent but minor reduction in the experimental group, which became statistically significant after the total dose of 750 rads ( P < 0.020). 4. 4. In a separate study the changes induced by ionizing radiation in the erythrocyte enzyme NADH-methemoglobin reductase were measured using the same experimental protocol. 5. 5. The enzyme activity in the red cells of the experimental group varied between 34.90 ± 2.17 to 161.95 ± 5.34 I.U./ml erythrocyte pack. 6. 6. Its activity declined toward the initial value after reaching the peak by the 12th day of ionizing radiation with 600 rads ( P < 0.001).

Modulation of the hypoxic toxicity and binding of misonidazole by glucose.

The hypoxic toxicity and binding of misonidazole (MISO) requires metabolic reduction. The influence of glucose on the toxicity and binding of MISO was studied because glucose is a major substrate for the supply of NADPH through the hexose monophosphate pathway (HMP). Hypoxic EMT6/Ro cells (10(6) cells ml-1) were incubated with varying concentrations of glucose (0.015 mM to 5 mM). The initial rate of glucose transport was found to increase linearly with the extracellular glucose concentration up to 5 mM (0.038 nmol glucose 10(-6) cells sec-1). About 1.5 percent of the total glucose consumed went through the HMP for hypoxic cells in 5 mM glucose. The rate of HMP progressively decreased as the glucose concentration was lowered. When exposed to 5 mM MISO, the HMP was stimulated. This stimulation declined from 3.2 times in 5 mM glucose to barely detectable below 1 mM glucose. Both the hypoxic toxicity and binding of 5 mM MISO to the acid-insoluble fraction were decreased as the concentration of glucose was lowered. Below 0.5 mM glucose, no significant toxicity due to MISO was observed. There was an initial burden of 2.5 nmol MISO 10(-6) cells bound with little toxicity. After this initial burden, the terminal slope was 1.8 mol MISO bound 10(-6) cells (63 percent decrease in the surviving fraction). These results indicate that glucose concentrations lower than 5 mM can decrease the HMP rate and the toxicity and binding of MISO to hypoxic cells, and imply that calibration curves with normal and low glucose concentrations should be used to estimate the possible hypoxic fraction when MISO is used as a hypoxic probe in vivo.

Redox balances in the metabolism of sugars by yeasts

The central role of the redox couples NAD+/NADH and NADP+/NADPH in the metabolism of sugars by yeasts is discussed in relation to energy metabolism and product formation. Besides their physical compartmentation in cytosol and mitochondria, the two coenzyme systems are separated by chemical compartmentation as a consequence of the absence of transhydrogenase activity. This has considerable consequences for the redox balances of both coenzyme systems and hence for sugar metabolism in yeasts. As examples, the competition between respiration and fermentation of glucose, the Crabtree effect, the Custers effect, adaptation to anaerobiosis, the activities of the hexose monophosphate pathway, and the fermentation of xylose in yeast are discussed.

Increased cerebral glucose-6-phosphate dehydrogenase activity in Alzheimer's disease may reflect oxidative stress.

The activities of the hexose monophosphate pathway enzymes glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were measured at autopsy in control and Alzheimer's disease brains. Enzyme activities did not vary between different areas of brain and were unaltered by age. In Alzheimer's disease, the activities of both enzymes were increased, the glucose-6-phosphate dehydrogenase activity being almost double the activity of normal controls. We propose that this increased enzyme activity is a response to elevated brain peroxide metabolism.

Binding of C-reactive protein to human polymorphonuclear leukocytes: evidence for association of binding sites with Fc receptors.

The functional similarities between C-reactive protein (CRP) and IgG raised the question as to whether human phagocytes are stimulated by CRP in the same way as by binding of antigen-complexed or aggregated IgG to their Fc receptors. Studies with the use of highly purified 125I-labeled CRP showed specific and saturable binding to human polymorphonuclear leukocytes (PMN) with a KD of 10.5 +/- 5.7 X 10(-8) M only when carried out in heat-inactivated plasma. The number of specific binding sites per cell was estimated at 1 to 3 X 10(6). Competitive inhibition of CRP binding by antigen-complexed or aggregated IgG suggests CRP binding sites to be associated with PMN Fc receptors. Only when assayed in heat-inactivated plasma did CRP binding induce adherence of cells to tissue culture dishes. However, no metabolic and potentially cytotoxic stimulation of PMN was detected during CRP plasma-dependent attachment to surfaces: induction of aggregation, release of secondary granule constituents, and activation of the hexose monophosphate pathway were not observed. These results imply that CRP-PMN interactions is dependent on an additional factor present in heat-inactivated plasma and is followed only by a complement-independent increase in PMN attachment to surfaces. Because CRP was found to be deposited at sites of tissue injury, the CRP-mediated adherence of PMN may be an important step in localizing an inflammatory focus.

Blood cell phosphogluconolactonase: assay and properties.

6-Phosphogluconolactonase (6-PGL) catalyses the second reaction of the hexosemonophosphate pathway. Although the delta-lactone of 6-phosphogluconic acid is the natural substrate for this enzyme, the more stable gamma-lactone may also be used. We prepared the gamma-lactone of 6-phosphogluconic acid from 6-phosphogluconate. When stored in dimethylsulfoxide, this material was found to be stable in liquid nitrogen for several months. A method for measuring 6-phosphogluconolactonase (6-PGL) using the gamma-lactone as substrate has been developed, after defining conditions under which spontaneous hydrolysis of the lactone is relatively slow and the enzymatic velocity is relatively rapid. The enzyme had no divalent cation requirement and was not significantly inhibited by a 50-fold excess of gluconolactone. It was distinct, therefore, from gluconolactonase. At 25 degrees C normal human red cells were found to contain approximately 50 IU of 6-phosphogluconolactonase/g Hb. The activity of the enzyme was independent of red cell age. Based on protein content, human lymphocytes, monocytes, granulocytes and platelets, contained approximately 10 times the activity of red blood cells. The activity of 6-PGL was stable for at least 6 d in red cells stored at 22 degrees C and for at least 20 d in red cells stored at 4 degrees C.

A Radiorespirometric Study on the Contribution of the Hexose Monophosphate Pathway to Glucose Metabolism in Candida utilis CBS 621 Grown in Glucose-limited Chemostat Cultures

Summary: A radiorespirometric study of glucose metabolism in Candida utilis CBS 621 was done using glucose-limited chemostat cultures growing at a dilution rate of 0.1 h−1 with ammonium or nitrate as the nitrogen source. From a steady-state analysis of 14CO2 yields from [1-14C]- and [6-14C]glucose supplied in the medium feed it appeared that during growth with nitrate the flow of glucose through the hexose monophosphate (HMP) pathway was much higher than during growth with ammonium as the nitrogen source. The same phenomenon was apparent from an analysis of the rate of 14CO2 production after administration of small amounts of labelled glucose to samples withdrawn from steady-state cultures. Additionally, these experiments revealed not only that the initial fraction of glucose 6-phosphate routed into the HMP pathway increases, but also that recycling of hexose phosphates via this pathway increases when nitrate is used as the nitrogen source. From a quantitative analysis of the results it is concluded that the contribution of the HMP pathway to glucose metabolism is close to the theoretical minimum required to cover the NADPH requirement for biosynthesis.

13C NMR studies of histidine fermentation with a Corynebacterium glutamicum mutant.

13C NMR spectroscopy was applied to studies on histidine biosynthesis in Corynebacterium glutamicum N-730, a histidine producing mutant. When it was cultured in a medium containing [1-13C]glucose as the carbon source, the 13C isotopomer was abundantly incorporated into C-6 (28 atom%) and C-l (21%), and to a lesser extent into C-5 (7%) of the histidine accumulated. The high incorporation into C-6 was explained as being due to that most of the one-carbon unit for histidine biosynthesis is derived from serine. On the bases of this view and the 13C population value at C-6, the contributions of the Embden-Meyerhof and hexose monophosphate pathways to the histidine production were calculated to be 56 and 44%, respectively. The significance of the label incorporation into C-l and C-5 was discussed.

The effect of short chain fatty acid administration on hepatic glucose, phosphate, magnesium and calcium metabolism.

Intra peritoneal administration of the short chain fatty acids, acetate, propionate and butyrate, in amounts calculated to reach 20 mM in total body water were given to fed and 48 hour starved male Wistar rats. One half hour after administration, the livers were freeze-clamped and the hepatic contents of various intermediary metabolites were measured. The liver content of total glycolytic intermediates was elevated by short chain fatty acids. In fed animals, the portion of glycolysis from fructose 1,6-bisphosphate (FBP) to PEP was elevated 2 to 4 fold. In 48 hour starved animals, where gluconeogenesis is active, the portion of the gluconeogenetic pathway from FBP to glucose was elevated 1.5 to 3.5 fold with the exception of the butyrate treated animals where blood glucose was not elevated. The metabolites of the hexose-monophosphate pathway that were measured, namely 6-phosphogluconate, ribulose 5-phosphate and xylose 5-phosphate were increased in both fed and starved animals. The free cytoplasmic [NAD+]/[NADH], [NADP+]/[NADPH], and [epsilon ATP]/[epsilon ADP] X [epsilon Pi] ratios were all decreased in both fed and starved animals after short chain fatty acid administration. The liver content of calcium increased 1.2 to 2 fold in fed animals and 2 to 3 fold in starved animals while total liver magnesium was either unchanged or increased only 1.2 times. The liver pyrophosphate (PPi) content increased a minimum of 10 fold in fed animals and over 100 fold in starved animals. In all cases no PPi could be detected in vivo by 31P NMR even though in the starved rats the PPi levels approached those of ATP. The liver content of inorganic Pi increased 1.3 to 1.5 fold in fed animals and 1.5 to 2 fold in starved animals. The total "rapidly metabolizing" Pi pool, that includes adenine and guanine nucleotides, glycolytic and shunt intermediates, Pi and PPi increased 1.3 times in fed animals (from 13.8 mumole/g fresh weight) and 1.5 to 1.7 fold in starved animals (from 15.7 mumol/g fresh weight). The total phosphate taken up from blood and entering the rapidly turning over pool of liver phosphate ranged between 4 and 12 mumols/g of liver.(ABSTRACT TRUNCATED AT 400 WORDS)

Production of oxygen-centered radicals by neutrophils and macrophages as studied by electron spin resonance (ESR).

Neutrophils and macrophages undergo a respiratory burst and an increase in the activity of the hexose monophosphate pathway in response to particulate or soluble agents. The increase in oxygen consumption was found to be associated with the production of oxygen-centered radicals. The ESR technique of spin trapping showed that besides a superoxide spin adduct, a hydroxyl spin adduct is also produced. ESR is considered to be the least ambiguous technique for the detection of free radicals. The spin-trapping agents used for oxygen-centered radical detection are usually nitrones. The most commonly used nitrone is 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), which reacts with O2-. to form 5,5-dimethyl-2-hydroperoxypyrroline-N-oxide (DMPO-OOH) and with OH. to form 5,5-dimethyl-2-hydroxypyrroline-N-oxide (DMPO-OH). Although spin-adduct formation is considered to be the most direct technique for the detection of free radicals, some disadvantages are encountered. There has been considerable interest in the isolation of the O2-. generating activity from phagocytic cells. The enzyme can be extracted with deoxycholate and gel filtration indicates that it is a high molecular weight complex. Maximum activity was between pH 7.0 and pH 7.5. The Km value was 15.8 microM for NADPH and 434 micron for NADH, indicating that NADPH is the preferred substrate.

Stimulation of the hexose monophosphate pathway by pyrroline-5-carboxylate reductase in the lens

Addition of pyrroline-5-carboxylate (P5C) or its precursors to rat lenses cultured for 24 hr in TC-199 medium containing 14C-glucose results in an apparent concentration-dependent increase in hexose monophosphate-pentose (HMP) pathway activity. Addition of proline, the reduction product of P5C, did not result in an increase, suggesting that stimulation of the HMP pathway is related to the reduction of P5C to proline by the enzyme P5C reductase. No apparent feedback inhibition on P5C reductase was observed. Stimulation of HMP pathway activity by P5C was also observed in the lenses of the Philly and Nakano mouse, two models of congenital osmotic cataracts. Compared with its genetic control, the Swiss-Webster mouse, generally no difference in the lenticular levels of HMP pathway activity was observed in the Philly mouse — even after the onset of cataract. Stimulation of the HMP pathway in the Philly lens by P5C, however, was consistently lower than its control. In the lenses from the Nakano mouse and its genetic control, the Balb c mouse, no difference in the percentage stimulation of the HMP pathway resulting from the addition of P5C was observed, but HMP pathway activity in the Nakano lens was consistently lower than that of the control.