Helmut Sies

The role of H2O2 generation in perfused rat liver and the reaction of catalase compound I and hydrogen donors

Abstract 1. 1. Hydrogen peroxide production in hemoglobin-free perfused rat liver was measured quantitatively by analyzing the steady state level of catalase-H2O2 intermediate ( p m e ). The method is based on a relationship between the ratio of H2O2 generaztion rate ( dx n dt ) to catalase-heme concentration (e) and the concentration of methanol ( a 1 2 ) which causes a decrease in the p m e value to half of its saturation value ( p m e ). Endogenous hydrogen donor in the perfused liver was estimated to be equivalent to about 30 μ m methanol. 2. 2. The rate of H2O2 production in the perfused liver with 2 m ml -lactate and 0.3 m m pyruvate was 49 nmol/min/g wet wt liver at 30 °C. This rate corresponds to about 1.7% of the total liver respiration rate. 3. 3. Mitochondrial production of H2O2 was proved by the fact that fatty acids enhance the H2O2 production and that this enhancement was inhibited by either rotenone or antimycin A. Perfusion of antimycin A caused stimulation of H2O2 production by a factor of 1.7. In the presence of 0.3 m m octanoate, H2O2 generation increased to 170 nmol/min/g wet wt liver. The results strongly suggest that one of the major sources for H2O2 production is the mitochondrial system participating in fatty acid oxidation. 4. 4. Changes in the redox state of cytosolic pyridine nucleotides by xylitol or by lactate did not alter the H2O2 production signficantly. 5. 5. Peroxisomal H2O2 production was observed when the liver was perfused with either urate or glycolate. The maximum rate of H2O2 production in the presence of urate and glycolate were 750 and 490 nmol/min/g wet wt liver, respectively. 6. 6. The total role of H2O2 production and utilization is considered and H2O2 metabolism in the peroxisomes is compared with that of other systems. The general conclusion is that, the body regulates the intracellular H2O2 level below about 10−7 m , and with this level biological oxidations of considerable significance are possible through the catalase system.

Oxidation in the NADP system and release of GSSG from hemoglobin-free perfused rat liver during peroxidatic oxidation of glutathione by hydroperoxides

Both of these enzyme activities have been studied extensively in recent years in rat liver homogenates and subcellular fractions [l-6]. In addition to Hz 02, several organic hydroperoxides were reported as active substrates for rat liver glutathione peroxidase [2]; some of these, e.g. t-butyl hydroperoxide or cumene hydroperoxide, do not react with catalase, and the corresponding alcohols do not react with alcohol dehydrogenase. Therefore, a selective transition in the 2GSH/GSSG redox system is effected in intact liver cells by these substrates.

Increased biliary glutathione disulfide release in chronically ethanol-treated rats.

tion was: carbohydrate, 54%; lipid, 193; and protein 27%. Glutathione &sulfide (GSSG) release frc3 liver [I ] has been evaluated as an indicator of oxidative stress, including lipid peroxidation (cf. hydroperoxide metabolism review in [2] )_ At present, the occurrence of enhanced rates of lipid peroxidation or of Ns02 production in the liver of rats c!lronically treated with ethanol is controversial (cf. review in ]3] and references therein) but appears to be a central concept in experimental pathology_ Therefore, we have applied a non-invasive approach and have examined the release of both oxidized and reduced ghrtathione from the liver of rats chronically treated, i.e., for 6 weeks with ethanol, essentially as in [4]. According to the finding that GSSG release occurs into bile [5], measurements were carried out in bile samples obtained from anesthetized rats. The calory percentages of the ingredients in th: final regimen, i.e., basal diet plus ethanol solution, consumed by the animals of the alcohol group were as follows:-ethanol, 41.5%; carbohydrate> 42.6%; lipid, 6.6% and protein, 9.3%. The composition of the diet of the control group was the same except that the ethanol-derived calories were replaced by sucrose. The animals in the ethanol group lere kept witbhout ethanol for 18 h prior to the experiment but were allowed access to the basal diet and to drinking water.

Hepatic mitochondrial and cytosolic glutathione content and the subcellular distribution of GSH-S-transferases.

1. Introduction Glutathlone participates m a number of redox and alkylatlon reactions m the hepatocyte which have been studied either m mtact cells or m isolated sub- cellular fractions (cf. recent symposmm [ 11). How- ever, mformatlon on the relevant subcellular concen- trations of glutathione m the mtact cell 1s lacking. Therefore, we have studied here the subcellular dlstn- butron of glutathione between the mitochondrial matrur (M) and cytosolic (C) spaces. In addition, the activity of the glutattione(GSH)-S-transferases was also measured, since it 1s known that the protective action of glutathlone against certain types of liver damage 1s due to this S-conJugatmg activity [2]. 2. Materials and methods 2 1.

Reduced and oxidized glutathione efflux from liver.

Recently, evidence for interorgan relationships in the turnover of glutathione has accumulated [l-4] , and a steady state plasma glutathione concentration of 5 PM measured in rat plasma [5] . Thus, the concept is emerging of the kidney as a major site of glutathione degradation to the constituent amino acids, whereas other organs, e.g., the liver, synthesize their own glutathione pool and, furthermore, contribute to the plasma pool in an as yet ill-defined mapner. Several studies have shown that cells release glutathione disulfide (GSSG) when exposed to ‘oxidative stress’, most probably as a consequence of an increased intracellular concentration of GSSG due to the action of glutathione peroxidase, e.g., in lens cells [6], erythrocytes [7-91 and liver cells [lo-131 . However, the release of glutathione from cells in the absence of experimentally-imposed oxidative conditions has been less extensively studied. Isolated perfused rat liver releases GSSG at a small but significant rate of l-2 nmol GSSG/min/g liver (wet wt), as was found in our earlier studies [ 10,141, and total glutathione, GSSG t GSH, was found to be released at rates between 6-l 2 nmol GSH equivalents/ min/g perfused liver at 30°C [ 12,131, or about 6 nmol/min/g (10s cells) isolated hepatocytes [ 151. Results of separate measurement of GSH and GSSG released from the perfused liver are presented here, indicating that glutathione efflux occurs largely as GSH, whereas the extra release of glutathione elicited by an acceleration of the glutathione peroxidase reaction occurs as GSSG. Further, it is estimated that the observed rates are compatible with the in vivo turnover of glutathione.

Glutamine metabolism in mammalian tissues

Enzymology and Transport.- Enzymology of Glutamine.- Enzymes of Renal Glutamine Metabolism.- Enzymes of Cerebral Glutamine Metabolism.- Glutamine Transport Across Biological Membranes.- Intestine and Liver.- Metabolism of Vascular and Luminal Glutamine by Intestinal Mucosa in Vivo.- Hepatic Glutamine and Ammonia Metabolism Nitrogen and Redox Balance and the Intercellular Glutamine Cycle.- Cellular Distribution and Regulation of Glutamine Synthetase in Liver.- Liver Glutaminase.- Mechanism and Control of Deprivation-Induced Protein Degradation in Liver: Role of Glucogenic Amino Acids.- Kidney.- Renal Glutamine Metabolism and Hydrogen Ion Homeostasis.- Effects of 2-Oxoglutarate and Glutamate on Glutamine Metabolism by Rat Kidney Mitochondria.- Role of Fatty Acids in Simultaneous Regulation of Flux Through Glutaminase and Glutamine Synthetase in Rat Kidney Cortex.- Other Tissues.- Cyclic Nucleotide Regulation of Glutamine Metabolism in Skeletal Muscle.- Cerebral Glutamine/Glutamate Interrelationships and Metabolic Compartmentation.- Glutamine Metabolism in Lymphoid Tissues.- Glutamine Metabolism by Cultured Mammalian Cells.- Clinical Aspects.- Ammonia Detoxication and Glutamine Metabolism in Severe Liver Disease and its Role in the Pathogenesis of Hepatic Encephalopathy.- Molecular Targets of Anti-Glutamine Therapy with Acivicin in Cancer Cells.

The steady state level of catalase compound I in isolated hemoglobin-free perfused rat liver☆

Helmut SIES lnstitut ff~r Physiologische Chemie und Physikalische Biochemie, der Universittit Mftnchen, Germany and Britton CHANCE Johnson Research Foundation, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA Received 25 September 1970 1. Introduction The long study of the catalytic and peroxidatic ac- tivities of catalase has led to little information on its function in vivo, particularly in mammalian systems [1, 2]. In bacteria, e.g. Micrococcus lysodeikticus, spectroscopic evidence indicated that catalase is large- ly saturated with H202 in the form of the primary intermediate, compound I [3]. In mammalian tissue, the correspondence of the order of reaction and the overall flux rates for methanol metabolism in the rab- bit were suggestive of catalases role in methanol oxi- dation [ 1 ]. Similar conclusions were reached by Tephly, Parks and Mannering [4] for methanol metab- olism in the rat. Heppel and Porterfield [5] found coupled nitrite oxidation in rat liver homogenates, and Portwich and Aebi [6] demonstrated the coupled oxidation of formate in rat liver slices. In the present investigation, spectrophotometric measurements of absorbance in the 660 nm band re- gion of catalase compound I from isolated perfused rat liver have been performed. Experimental evidence which supports an interpretation of changes of the ob- served signal to be due to the catalase system will be * Postal address: Dr. H. Sies, Institut fllr Physiologische Chemie und Physikalische Biochemie, Goethestrasse 33, 8-Miinchen- 15, Germany. presented; it includes the effects of the above-men- tioned hydrogen donors, of hydrogen peroxide gener- ating substance, of oxygen tension, and of inhibitors of the catalase system. 2. Experimental Spectrophotometry of light transmitted through a lobe of perfused liver was performed with the Rapid- spektroskop of Howaldtswerke Deutsche Werf, Kiel, as adapted by Brauser [7]. Dual wavelength absor- bance photometry by sinusoidal wavelength modula- tion [7] was used for more sensitive study of absor- bance changes as a function of time (see also [8]). Bandwidth of monochromatic light was 4 nm. Com- pensation for spectral characteristics of the apparatus was afforded by a reference beam which was reflected from a magnesia surface. In preliminary experiments, the rotating filter ap- paratus of Chance [9] as adapted for absorbancy measurements [ 10] was used with a 660 nm mea- suring filter and a 720 nm reference filter, having bandwidths of + 30 and + 7 nm, respectively. Light transmitted through a liver lobe was collected by a light pipe and was detected by an infrared-guarded silicone diode. Oxygen uptake was followed with Ag-Pt-micro- electrodes inserted into the perfusion circuit before 172 North.Holland Publishing Company - Amsterdam

Effect of oxygen concentration on the reaction of halothane with cytochrome P450 in liver microsomes and isolated perfused rat liver

Abstract The influence of oxygen on the complex formation of reduced cytochrome P450 with halothane has been investigated with liver microsomes and perfused livers from phenobarbital-pretreated rats. The reductive formation of the trifluoro carbene complex from halothane in liver microsomes was inhibited at high oxygen concentrations but started to appear below 50 μM oxygen and was maximal under anaerobic conditions. Metyrapone was an efficient inhibitor of the carbene complex formation. Organ spectrophotometry of isolated perfused livers established that the complex appeared already under slightly hypoxic conditions and that metyrapone addition to the perfusion medium abolished its formation. The results indicate the possibility of a reductive in vivo -metabolism of halothane to reactive intermediates when the oxygen concentration of the cell becomes lower than about 50 μM.

Assessment of the kidney function in maintenance of plasma glutathione concentration and redox state in anaesthetized rats

Substantial evidence has been provided for a role of the kidney in the metabolic turnover of extracellular glutathione [l-12]. Glutathione has been found in the extracellular space at 3-5 PM in the arterial plasma of the rat [7,13] and in human venous plasma [ 141. In studies of the fate of extracellular glutathione the degradation has been proposed to occur mainly on the luminal surface of the renal brush-border membrane and that the y-glutamyl transpeptidase is a gluta~ionase acting on extracellular glutathione. However, a quantitative assessment of this function of the kidney enzyme in the intact animal was not performed. In order to provide more information on this hypothesis, we measured glutathione uptake by the kidney in the anaesthetized rat 171. Interestingly, renal glutatl~ione extraction was -90%. In view of the renal Rltration fraction of -30% this indicated that, in addition to the intratubular degradation, there must be a further uptake mechanism for glutathione by the kidney, in accor-

Heme occupancy of catalase in hemoglobin-free perfused rat liver and of isolated rat liver catalase.

Maximal heme occupancy, the maximal proportion of total catalase heme present in the form of Compound I, is found to be 0.4 both in the enzyme isolated from rat liver and in the peroxisomal enzyme as present in the intact cells of perfused rat liver. This indicates that the ratio of second order rate constants for catalatic decomposition and for formation of Compound I, k4′k1, is equal in vitro and in vivo. n nCatalase was isolated from rat liver, and the extinction coefficients for Compound I and for cyanide-catalase at 640 minus 660 nm were determined. The measurement of heme occupancy of catalase in hemoglobin-free perfused rat liver was made possible by wavelength scanning as well as by dual wavelength absorbance photometry. Thus, Compound I and cyanide-catalase were demonstrated in the red region and in the Soret band region. n nMeeting the particular needs of organ photometry, specific metabolic transitions were used to visualize specific transitions of absorbing pigments. Compound I is specifically demonstrated by its decomposition by the hydrogen donor, methanol. A measure for total catalase heme is provided by formation of cyanide-catalase. The cyanide concentrations required are well below appearance of possible interference by other cyanide-binding hemoproteins at 640–660 nm.