Hemoglobin-free Perfused Rat Liver Research Articles

Increase in hepatic mixed disulphide and glutathione disulphide levels elicited by paraquat

Paraquat (1 mM), when added to isolated haemoglobin-free perfused rat liver, leads to an increase of intracellular mixed disulphides from 1.3 μmole GSH equivalents per g wet weight in the controls to 2.5 μmole/g. This raises the proportion of mixed disulphides to total glutathione equivalents from about 0.2 at the beginning of the perfusion to about 0.4. The mixed disulphides are predominantly protein-bound, with low molecular weight compounds being quantitatively negligible. The content of intracellular glutathione disulphide (GSSG) is increased from 17 nmole/g in the controls to 38 nmole/g in the presence of paraquat. In addition, there is an increased rate of release of GSSG into the extracellular (biliary) space, reported previously. It is suggested that, in a reaction catalysed by thioltransferase(s), the rise in GSSG is correlated with the rise in mited disulphides (reaction 1). Occupancy of potential cellular mixed disulphide sites is about 1 2 in the controls, and rises to about 2 3 in the presence of paraquat. The ratio of cellular contents, NADPH/NADP +, is decreased from 5.1 in the controls to 2.3 in the presence of paraquat, while the sum of NADPH plus NADP + remains unaltered. The perturbation in the glutathione status may be related to metabolic effects such as the stimulation of the pentose-phosphate pathway activity, and possibly also to the expression of toxic effects.

Peroxisomal fatty acid oxidation as detected by H2O2 production in intact perfused rat liver.

1. H2O2 formation associated with the metabolism of added fatty acids was quantitatively determined in isolated haemoglobin-free perfused rat liver (non-recirculating system) by two different methods. 2. Organ spectrophotometry of catalase Compound I [Sies & Chance (1970) FEBS Lett. 11, 172-176] was used to detect H2O2 formation (a) by steady-state titration with added hydrogen donor, methanol or (b) by comparison of fatty-acid responses with those of the calibration compound, urate. 3. In the use of the peroxidatic reaction of catalase, [14C]methanol was added as hydrogen donor at an optimal concentration of 1 mM in the presence of 0.2 mM-L-methionine, and 14CO2 production rates were determined. 4. Results obtained by the different methods were similar. 5. The yield of H2O2 formation, expressed as the rate of H2O2 formation in relation to the rate of fatty-acid supply, was less than 1.0 in all cases, indicating that, regardless of chain length, less than one acetyl unit was formed per mol of added fatty acid by the peroxisomal system. In particular, the standard substrate used with isolated peroxisomal preparations (C16:0 fatty acid) gave low yield (close to zero). Long-chain monounsaturated fatty acids exhibit a relatively high yield of H2O2 formation. 6. The hypolipidaemic agent bezafibrate led to slightly increased yields for most of the acids tested, but the yield with oleate was decreased to one-half the original yield. 7. It is concluded that in the intact isolated perfused rat liver the assayable capacity for peroxisomal beta-oxidation is used to only a minor degree. However, the observed rates of H2O2 production with fatty acids can account for a considerable share of the endogenous H2O2 production found in the intact animal.

Transport of inorganic anions in perfused rat liver.

The transport of inorganic anions (sulfate, chloride, phosphate and bicarbonate) across the plasma membrane was investigated in hemoglobin-free perfused rat livers, applying the multiple-indicator dilution technique (pulse labelling of inorganic anions and indicator substances). The following results were obtained: 1. Chloride and phosphate exchanged very slowly between extracellular and intracellular spaces, whereas the exchange of sulfate was rapid. 2. The exchange of sulfate exhibited saturation kinetics with half-maximal rates at approximately 8mM sulfate. The maximal steady-state exchange rate was near 60 micromol x min-1 x (g liver wet wt)-1. 3. The exchange of sulfate was inhibited completely by cyanocinnamate; the inhibition was fully reversible. It was also completely, but irreversibly inhibited by diisothiocyanostilbenedisulfonic acid; the amount of the inhibitor bound by the liver that was required for half-maximal effect was considerably less than that required for the inhibition of D-lactate transport. 4. The exchange of sulfate was also inhibited by pyruvate and L-lactate at high concentrations; the inhibition was not competitive. 5. The exchange of D-lactate was not inhibited by sulfate or chloride. 6. Bicarbonate exchanges very rapidly; this process cannot be inhibited by cyanocinnamate. From these observations the following conclusions were drawn. A carrier system for the transport of inorganic anions, at least for sulfate, exists in the plasma membrane of liver. This carrier is specific for inorganic anions and is independent of the carrier system for the transport of monocarboxylates, in contrast to the findings with erythrocytes. Moreover, this carrier does not transport bicarbonate at a significant rate. Most likely, the rapid exchange of bicarbonate reflects the free diffusion of carbon dioxide.

Intracellular redox state and stimulation of gluconeogenesis by glucagon and norepinephrine in the perfused rat liver.

The role of the cellular redox state in the hormonal stimulation of gluconeogenesis was studied in hemoglobin-free perfused rat liver, by fluorimetric measurement of the redox states of intracellular pyridine nucleotides. The maximum rate of glucose production from lactate/pyruvate mixture was observed with a lactate/pyruvate ratio of 10/1, which corresponds to the ratio observed in vivo. Increased reduction of pyridine nucleotides on infusion of ethanol or octanoate was associated with an increased production of glucose from pyruvate, whereas glucose production from lactate decreased. Stimulation of gluconeogenesis from lactate by glucagon was affected by the lactate/pyruvate ratio; a decrease of the lactate/pyruvate ratio resulted in a decrease of the efficacy of glucagon. Stimulation by glucagon of glucose production from pyruvate was abolished during octanoate infusion, although it was still observable during ethanol infusion. In contrast to glucagon, the stimulatory effect of norepinephrine on gluconeogenesis was unaffected by the ratio of lactate to pyruvate. Norepinephrine in the presence of octanoate and ethanol still induced stimulation of glucose production from lactate and pyruvate, which was always accompanied by a transient reduction of pyridine nucleotides. The results demonstrate that the regeneration of NADH in the cytosol is one of the regulatory factors in gluconeogenesis, and that the effects of glucagon and norepinephrine on gluconeogenesis and on the redox state of pyridine nucleotides are not identical.

Ethanol-induced changes in the intralobular oxygen gradient of perfused rat liver

A new tissue fluorometric method was developed to estimate the intralobular oxygen gradient in hemoglobin-free perfused rat liver. The method employs a two-branch micro-light guide with a tip diameter of 170 mu. With this light guide, it was possible to measure pyridine nucleotide fluorescence (366 nm leads to 450 nm) from periportal and pericentral regions of the liver lobule. By measuring inflow PO2 values at which pyridine nucleotide fluorescence increased in the pericentral regions of the liver lobule, the mean intralobular oxygen gradient was estimated. The measured gradient was approximately 180 torr in livers from sucrose-treated control rats. Chronic treatment with ethanol increased both the mean intralobular oxygen gradient and the rate of hepatic oxygen uptake by 30%. The antithyroid drug, 6-propyl-2-thiouracil, completely reversed the effects of ethanol on both the intralobular oxygen gradient and the rate of oxygen uptake. These data present direct physical evidence that the increased tissue respiration induced by chronic ethanol treatment indeed accentuates the intralobular oxygen gradient and thus support the hypothesis that selective depletion of oxygen in the pericentral region of the liver lobule may underlie ethanol-induced cellular injury confined to this site.

Transport of D-lactate in perfused rat liver.

The transport of D-lactate across the plasma membrane was investigated in hemoglobin-free perfused rat livers, applying the multiple-indicator dilution technique (pulse labelling of D-lactate and indicator substances). The following results were obtained: 1. The steady state exchange rate at 1 mM D-lactate was 2.5 mumol x min-1 x g wet wt-1. It was proportional to the extracellular concentration in the range between 0.1 and 70 mM. 2. The transport of D-lactate was inhibited by L-lactate and pyruvate; 50% inhibition was observed at 40 mM L-lactate or 5 mM pyruvate. 3. The transport was also inhibited by alpha-cyanocinnamate and 4,4'-diisocyanostilbene-2,2'-disulfonic acid. The inhibition by cyanocinnamate was complete (with 25 mM) and fully reversible, whereas the inhibition by diisothiocyanostilbenedisulfonic acid was incomplete and irreversible; it was dependent upon the amount of diisothiocyanostilbenedisulfonic acid bound by the liver. Maximal inhibition (80%) was observed with 2 mumol diisothiocyanostilbenedisulfonic acid bound per g wet weight. 4. The intracellular concentration (ci) of D-lactate was proportional to the extracellular concentration (ce); the ratio ci/ce was 0.5 throughout the concentration range studied. It decreased in the presence of L-lactate or pyruvate. It is concluded that the transport of D-lactate is carrier-mediated, and, at least partially, electroneutral.

Glutathione S-conjugate formation from 1-chloro-2,4-dinitrobenzene and biliary S-conjugate excretion in the perfused rat liver.

The isolated hemoglobin‐free perfused rat liver was used as a model to study the formation of a glutathione S‐conjugate and its disposition into the biliary as well as other extracellular (caval perfusate) compartments. 1‐Chloro‐2,4‐dinitrobenzene was chosen as substrate because it reacts with the glutathione‐S‐transferases at high activity to give rise to an S‐conjugate, S‐(2,4‐di‐nitrophenyl)‐glutathione, a product readily discernible from the parent compound. S‐(2,4‐Dinitrophenyl)‐glutathione formation rates were equal to the infusion rates of 1‐chloro‐2,4‐dinitrobenzene up to 0.3 μmol × min−1× g liver−1. Steady‐state rates of S‐conjugate release were maintained until the glutathione content of the liver had decreased to about 40% of the control value. Biliary excretion of the S‐conjugate was practically quantitative at low rates of S‐conjugate formation, up to about 30 nmol × min−1× g liver−1. and release of S‐conjugate into the caval perfusate gradually increased with the rate of formation. Maximal biliary S‐conjugate concentration was 36 mM. Choleresis associated with biliary S‐conjugate excretion amounted to 18.6 pi of extra bile per μmol of net S‐conjugate excreted. This would correspond to a limiting concentration of 54 mM S‐conjugate in the water compartment associated with biliary transport, e.g. exocytotic vesicle in the context of an exocytosis‐like mechanism of bile formation. The system described here is suited for further investigation of properties of bile formation, as is illustrated by the sensitive detection of changes in the concentration of S‐conjugate in the perfusate upon Ca2+‐depletion.

Biochemical and ultrastructural evaluation of isolated rat liver systems perfused with a hemoglobin-free medium.

The viability of hemoglobin-free perfused rat liver was examined with respect to several liver functions and to the intactness of subcellular structures under electron microscopic observation. Provided that rat livers were perfused with the oxygenated buffer solution at a flow rate between 3 and 3.5 ml/min per g of liver, all the biochemical parameters measured in the perfused liver system, i.e. the rates of glucose, pyruvate, and lactate production, the rate of oxygen consumption and the tissue contents of adenine nucleotides, were similar to those observed with perfusion systems containing erythrocytes or albumin. The perfused liver showed a sensitive response to norepinephrine, involving a reduction of pyridine nucleotides and enhancements of glucose production and oxygen consumption. On electron microscopic examination, changes in hepatic-structure indicative of hypoxic injury particularly vacuolar degeneration and mitochondrial swelling, were not detected in the liver after 70 min of perfusion; the fact that the fine structure of the hepatocyte was preserved in all parts of the organ confirmed that the supply of oxygen to the perfused liver was sufficient under the conditions employed. From viewpoint of the generally accepted criteria for the viability of perfused liver, therefore, the results confirmed that the perfusion of liver with a hemoglobin- and albumin-free medium is a convenient and reliable tool for biochemical investigation of the reactions occurring in whole liver.

Formation of bile acids in hemoglobin-free perfused rat livers.

Formation of bile acids in hemoglobin-free perfused rat livers.

Peroxide removal by selenium-dependent and selenium-independent glutathione peroxidases in hemoglobin-free perfused rat liver.

Peroxide removal by selenium-dependent and selenium-independent glutathione peroxidases in hemoglobin-free perfused rat liver.

Effect of oxygen shortage on the metabolism of oestrone in the hemoglobin-free perfused rat liver.

The isolated rat liver was perfused with a haemoglobin-free, albumin containing salt solution at 28 degrees and 37 degrees C, respectively, with [4-14C]oestrone as substrate. During perfusion, the functional state of the liver was checked by continuously measuring the oxygen pressure and hydrogen ion concentration in the perfusion medium, flow rate, oxygen tension at various areas of the liver surface, and oxygen consumption; in addition, the following biochemical parameters were determined: ATP, ADP, lactate and pyruvate in liver tissue, and lactate and pyruvate in the perfusion medium. After 30 min of perfusion, free steroids, steroid glucuronides, steroid sulphates and the remaining water-soluble metabolites, present in liver tissue, perfusion medium and bile, were separated from each other and characterised by thin-layer and paper chromatography. It was found that, during perfusion at 37 degrees C, less hydroxylated metabolites were formed than at 28 degrees C. In contrast, metabolites whose formation is not directly oxygen-dependent, such as glucuronides and sulphates, arose in higher amounts at 37 degrees C than at 28 degrees C. It may be concluded that the relative shortage of oxygen at 37 degrees C leads to a selective impairment of metabolic pathways requiring a sufficient supply of molecular oxygen. Since oxidative processes play an important role in the biogenesis and metabolism of steroid hormones, it is evident that results, obtained in perfusion experiments with haemoglobin-free media at 37 degrees C, must be treated with reserve.

Hydroperoxide-metabolizing systems in rat liver.

1. Metabolism of added hydroperoxides was studied in hemoglobin-free perfused rat liver and in isolated rat hepatocytes as well as microsomal and mitochondrial fractions. 2. Perfused liver is capable of removing organic hydroperoxides [cumene and tert-butyl hydroperoxide] at rates up to 3--4 mumol X min-1 X gram liver-1. Concomitantly, there is a release of glutathione disulfide (GSSG) into the extracellular space in a relationship approx. linear with hydroperoxide infusion rates. About 30 nmol GSSG are released per mumol hydroperoxide added per min per gram liver. GSSG release is interpreted to indicate GSH peroxidase activity. 3. GSSG release is observed also with added H2O2. At rates of H2O2 infusion of about 1.5 mumol X min-1 X gram liver-1 a maximum of GSSG release is attained which, however, can be increased by inhibition of catalase with 3-amino-1,2,4-aminotriazole. 4. A contribution of the endoplasmic reticulum in addition to glutathione peroxidase in organic hydroperoxide removal is demonstrated (a) by comparison of perfused livers from untreated and phenobarbital-pretreated rats and (b) in isolated microsomal fractions, and a possible involvement of reactive iron species (e.g. cytochrome P-450-linked peroxidase activity) is discussed. 5. Hydroperoxide addition to microsomes leads to rapid and substantial lipid peroxidation as evidenced by formation of thiobarbituric-acid-reactive material (presumably malondialdehyde) and by O2 uptake. Like in other types of induction of lipid peroxidation, malondialdehyde/O2 ratios of 1/20 are observed. Cumene hydroperoxide (0.6 mM) gives rise to 4-fold higher rates of malondialdehyde formation than tert-butyl hydroperoxide (1 mM). Ethylenediamine tetraacetate does not inhibit this type of lipid peroxidation. 6. Lipid peroxidation in isolated hepatocytes upon hydroperoxide addition is much lower than in isolated microsomes or mitochondria, consistent with the presence of effective hydroperoxide-reducing systems. However, when NADPH is oxidized to the maximal extent as evidenced by dual-wavelength spectrophotometry, lipid peroxidation occurs at large amounts. 7. A dependence of hydroperoxide removal rates upon flux through the pentose phosphate pathway is suggested by a stimulatory effect of glucose in hepatocytes from fasted rats and by an increased rate of 14CO2 release from [1-14C]glucose during hydroperoxide metabolism in perfused liver.

Activation of pyruvate dehydrogenase during metabolism of ammonium ions in hemoglobin-free perfused rat liver.

1 Ammonium chloride addition to perfused rat liver results in a shift of the steady state of the pyruvate dehydrogenase system towards the active (dephospho) form. This is evidenced by direct measurement of enzyme activity in freeze-stopped liver tissue, by net uptake of pyruvate from the perfusate, and also by an increased release of labeled CO2 from [1-14C]pyruvate. Half-maximal concentration of NH4Cl for extra pyruvate uptake is 0.5–0.7 mM. The effect is readily reversible. 2 Extra urea formation upon ammonia addition is matched by an extra O2 uptake as that required theoretically for the two ATP-consuming steps of urea synthesis. At 1.5 mM NH4Cl, there is no significant difference of ATP levels compared to the controls. 3 When extra urea formation and the concomitant O2 uptake are suppressed due to limitation at carbamoylphosphate synthesis in carbon-dioxide-free media, the activation of pyruvate dehydrogenase by NH4Cl is still observed. 4 When ammonia is generated intracellularly from glutamine, an extra uptake of pyruvate is not observed. 5 The NH4Cl-induced activation of pyruvate dehydrogenase is increased in presence of acetoacetate, and decreased in presence of 3-hydroxybutyrate. 6 Possible mechanisms are discussed. While some of the observations could also be explained by a direct interaction of ammonium ions with the pyruvate dehydrogenase interconversion system, the effect of ammonium ions on pyruvate dehydrogenase is interpreted to be mediated in an indirect manner by changes of the mitochondrial concentration of 2-oxoglutarate. As 2-oxoglutarate decreases during reductive amination to glutamate, a pool of high-energy compounds (eg. GTP or related substances) dependent upon 2-oxoglutarate oxidation is depleted, resulting in a de-inhibition of pyruvate dehydrogenase by the interconversion system. The detailed mechanism of the linkage between 2-oxoglutarate oxidation and pyruvate oxidation remains to be elucidated.

Mitochondrial nicotinamide nucleotide systems: ammonium chloride responses and associated metabolic transitions in hemoglobin-free perfused rat liver.

Mitochondrial nicotinamide nucleotide systems: ammonium chloride responses and associated metabolic transitions in hemoglobin-free perfused rat liver.

The properties of the secondary catalase—peroxide complex (Compound II) in the hemoglobin-free perfused rat liver

The possibility of the occurrence of the secondary catalase-peroxide complex (Compound II) in the isolated peroxisomal-mitochondrial fraction of rat liver and in the perfused rat liver has been examined under various conditions. The steady state of Compound I is maintained by either an endogeneous or a urate and glycolate-supplemented H 2O 2 generation in both systems, but Compound II is not detectable. Significant accumulation of Compound II, which is identified by the measurement of its difference spectrum and by its response to hydrogen donors, is observed only when Compound I is converted to Compound II by an appropriate concentration of p-cresol. The properties of Compound II observed in the perfused liver are similar to those observed with isolated catalase.

Carbon-dioxide concentration and the distribution of monocarboxylate and H+ ions between intracellular and extracellular spaces of hemoglobin-free perfused rat liver.

H+ movement and monocarboxylate‐monocarboxylate exchange accompanying monocarboxylate gradient equilibration in hemoglobin‐free perfused rat liver have been studied in an isopH system using different buffer systems.1. At 5–10 mM concentrations of added monocarboxylate, there is an approximately linear dependence of net H+ uptake by the liver on added monocarboxylate. Net H+ uptake at 10 mM additions is 1.5–1.6 μmol per gram liver from aromatic weak acids like benzoate or salicylate, and 0.6–0.8 μmol per gram liver for aliphatic weak acids like acetate, lactate, pyruvate, and also 5,5‐dimethyloxazolidine‐2,4‐dione.2. Practically no net H+ movement is observed with dicarboxylates like phthalate or succinate and also not with choline chloride.3. Monocarboxylate‐monocarboxylate exchange is demonstrated by preloading the hepatocytes with benzoate, lactate and pyruvate, and observation of their release upon addition of other monocarboxylates.4. Net H+ uptake increases as total CO2 in perfusate is decreased at constant extracellular pH, in accordance with an intracellular acidification by the CO2 system. This is supported by measurement of distribution of 5,5‐dimethyloxazolidine‐2,4‐dione, and by monocarboxylate release from the cells during increases of CO2 concentration.5. Intracellular pH poise is low when bicarbonate buffer is replaced by zwitterionic buffers like N‐2‐hydroxyethylpiperazine‐N′‐ethane sulfonic acid or morpholinopropane sulfonic acid, resulting in calculated intracellular alkalinization of up to 0.5 pH unit. 5,5‐Dimethyloxazolidine‐2,4‐dione as a permeant buffer system is suggested as a more suitable replacement of bicarbonate buffer than the zwitterionic buffers with respect to intracellular pH poise.

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, k 4′ k 1 , is equal in vitro and in vivo. Catalase 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. Meeting 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.

The role of H 2O 2 generation in perfused rat liver and the reaction of catalase compound I and hydrogen donors

1. 1. Hydrogen peroxide production in hemoglobin-free perfused rat liver was measured quantitatively by analyzing the steady state level of catalase-H 2O 2 intermediate ( p m e ). The method is based on a relationship between the ratio of H 2O 2 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 H 2O 2 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 H 2O 2 was proved by the fact that fatty acids enhance the H 2O 2 production and that this enhancement was inhibited by either rotenone or antimycin A. Perfusion of antimycin A caused stimulation of H 2O 2 production by a factor of 1.7. In the presence of 0.3 m m octanoate, H 2O 2 generation increased to 170 nmol/min/g wet wt liver. The results strongly suggest that one of the major sources for H 2O 2 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 H 2O 2 production signficantly. 5. 5. Peroxisomal H 2O 2 production was observed when the liver was perfused with either urate or glycolate. The maximum rate of H 2O 2 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 H 2O 2 production and utilization is considered and H 2O 2 metabolism in the peroxisomes is compared with that of other systems. The general conclusion is that, the body regulates the intracellular H 2O 2 level below about 10 −7 m, and with this level biological oxidations of considerable significance are possible through the catalase system.

Problems involved in the measurement of microcirculation by means of microelectrodes.

Until now, the interpretation of local wash-out curves, measured by means of microelectrodes and using hydrogen as diffusible indicator, is an unsettled problem. It has been shown, that even the simultaneous measurement by means of a surface-multiwire-Pt-microelectrode at the isolated and hemoglobin-free perfused rat liver yields different wash-out curves (4). These differences occur with regard to the height of the curves as well as to the moment, when the maximum of the indicator’s concentration is seen by the microelectrode. Fig.1 represents four simultaneously recorded curves. The difference in sensitivity of each micro-electrode has been compensated by a computer. The fast curve corresponds the real input function to the liver and is directly measured in the inflow by means of a non-compensated electrode. Usually the evaluation of local hydrogen wash-out curves is performed in the same way as the evaluation of those clearance curves, which are measured by a large-scale detector, using 85-Krypton or 133-Xenon as indicator. The most commonly used methods are the slope-method (2) and the heightover-area-method (5). Application of these methods to local hydrogen wash-out curves yields different values for the local perfusion rate. Fig.2 shows the comparison between the mean perfusion rates, determined by means of the total inflow, and the local perfusion rates. In fig.2A the local perfusion rates were calculated using the height-over-area-method, whereas in fig.2B the slope-method was applied to the same curves.

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.