Different effects of Triton X-100, deoxycholate, and fatty acids on the kinetics of glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase.

The effects of Triton X-100, deoxycholate, and fatty acids were studied on the two steps of the ping-pong reaction catalyzed by Se-dependent glutathione peroxidases. The study was carried out by analyzing the single progression curves where the specific glutathione oxidation was monitored using glutathione reductase and NADPH. While the "classic" glutathione peroxidase was inhibited only by Triton, the newly discovered "phospholipid hydroperoxide glutathione peroxidase" was inhibited by deoxycholate and by unsaturated fatty acids. The kinetic analysis showed that in the case of glutathione peroxidase only the interaction of the lipophilic peroxidic substrate was hampered by Triton, indicating that the enzyme is not active at the interface. Phospholipid hydroperoxide glutathione peroxidase activity measured with linoleic acid hydroperoxide as substrate, on the other hand, was not stimulated by the Triton concentrations which have been shown to stimulate the activity on phospholipid hydroperoxides. Furthermore a slight inhibition was apparent at high Triton concentrations and the effect could be attributed to a surface dilution of the substrate. Deoxycholate and unsaturated fatty acids were not inhibitory on glutathione peroxidase but inhibited both steps of the peroxidic reaction of phospholipid hydroperoxide glutathione peroxidase, in the presence of either amphiphilic or hydrophilic substrates. This inhibition pattern suggests an interaction of anionic detergents with the active site of this enzyme. These results are in agreement with the different roles played by these peroxidases in the control of lipid peroxide concentrations in the cells. While glutathione peroxidase reduces the peroxides in the water phase (mainly hydrogen peroxide), the new peroxidase reduces the amphyphilic peroxides, possibly at the water-lipid interface.

Phospholipid hydroperoxide glutathione peroxidase.

In acute inflammation the activated leukocytes generate cytotoxic oxygen free radicals. The role of these radical species in the cellular damage following an acute inflammatory reaction is well known. On the other hand the extent of the cellular damage must be dependent on both the rate of the free-radical generation and the scavenging capacity of the tissues. Among the enzymes acting in the inhibition of this damage, a key role seems to be played by the new selenoenzyme phospholipid hydroperoxide glutathione peroxidase. Indeed the reduction of membrane hydroperoxides constitutes a secondary line of defence against lipid peroxidation, preventing the decomposition of hydroperoxides leading to the formation of new radicals. This enzyme inhibits lipid peroxidation and is as active as glutathione peroxidase on phospholipid hydroperoxides, on which no previously known peroxidase is active. Its protective activity for biomembranes, and the kinetic analysis in the presence of detergents, suggest its interfacial character. The inhibition of lipid peroxidation in the membranes apparently requires this enzyme, along with glutathione and vitamin E, in order to reduce the rate of the initiation reactions. This synergism bears out the role of this enzyme in the multilevel defence system against free-radical damage in tissues.

The selenoenzyme phospholipid hydroperoxide glutathione peroxidase.

The reduction of membrane-bound hydroperoxides is a major factor acting against lipid peroxidation in living systems. This paper presents the characterization of the previously described 'peroxidation-inhibiting protein' as a 'phospholipid hydroperoxide glutathione peroxidase'. The enzyme is a monomer of 23 kDa (SDS-polyacrylamide gel electrophoresis). It contains one gatom Se/22 000 g protein. Se is in the selenol form, as indicated by the inactivation experiments in the presence of iodoacetate under reducing conditions. The glutathione peroxidase activity is essentially the same on different phospholipids enzymatically hydroperoxidized by the use of soybean lipoxidase (EC 1.13.11.12) in the presence of deoxycholate. The kinetic data are compatible with a tert-uni ping-pong mechanism, as in the case of the 'classical' glutathione peroxidase (EC 1.11.1.9). The second-order rate constants (K1) for the reaction of the enzyme with the hydroperoxide substrates indicate that, while H2O2 is reduced faster by the glutathione peroxidase, linoleic acid hydroperoxide is reduced faster by the present enzyme. Moreover, the phospholipid hydroperoxides are reduced only by the latter. The dramatic stimulation exerted by Triton X-100 on the reduction of the phospholipid hydroperoxides suggests that this enzyme has an 'interfacial' character. The similarity of amino acid composition, Se content and kinetic mechanism, relative to the difference in substrate specificity, indicates that the two enzymes 'classical' glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase are in some way related. The latter is apparently specialized for lipophylic, interfacial substrates.

Enzymatic determination of membrane lipid peroxidation.

The recently purified "phospholipid hydroperoxide glutathione peroxidase" has been used to measure the membrane hydroperoxides formed during lipid peroxidation that are not substrates for the "classical" glutathione peroxidase. A spectrophotometric test in the presence of glutathione, glutathione reductase and NADPH has been used. The peroxidized membranes were added directly to the reaction mixture and the reaction was started by the addition of the enzyme. Triton X-100 exerted a stimulatory effect. Phospholipid hydroperoxide glutathione peroxidase allows a rapid, sensitive, accurate and specific determination of membrane hydroperoxides, the most quantitative index of lipid peroxidation. Glutathione peroxidase can be used in the same test to measure other hydroperoxides such as the cumene hydroperoxide used to induce the peroxidation.

Formation of alpha-tocopherol radical and recycling of alpha-tocopherol by ascorbate during peroxidation of phosphatidylcholine liposomes. An electron paramagnetic resonance study.

The events accompanying the inhibitory effect of alpha-tocopherol and/or ascorbate on the peroxidation of soybean L-alpha-phosphatidylcholine liposomes, which are an accepted model of biological membranes, were investigated by electron paramagnetic resonance, optical and polarographic methods. The presence of alpha-tocopherol radical in the concentration range 10(-8)-10(-7) M was detected from its EPR spectrum during the peroxidation of liposomes, catalysed by the Fe3+-triethylenetatramine complex. The alpha-tocopherol radical, generated in the phosphatidylcholine bilayer, is accessible to ascorbic acid, present in the aqueous phase at physiological concentrations. Ascorbic acid regenerates from it the alpha-tocopherol itself. A kinetic rate constant of about 2 X 10(5) M-1 X s-1 was estimated from the reaction as it occurs under the adopted experimental conditions. The scavenging effect of alpha-tocopherol on lipid peroxidation is maintained as long a ascorbic acid is present.

Purification from pig liver of a protein which protects liposomes and biomembranes from peroxidative degradation and exhibits glutathione peroxidase activity on phosphatidylcholine hydroperoxides.

The cell sap from pig liver contains a protein which protects phosphatidylcholine liposomes and biomembranes from peroxidative degradation in the presence of glutathione. The activity of this protein has been assayed by measuring the inhibition of aged phosphatidylcholine liposome peroxidation induced by the Fe3+-triethylenetetramine complex. The peroxidation-inhibiting protein from pig liver has been purified 585-fold to homogeneity with overall recovery of activity of 12%. (NH4)2SO4 precipitation, ion-exchange chromatography on DEAE-Sepharose CL-6B and CM23-cellulose, affinity chromatography on glutathione-bromosulfophthalein-Sepharose and gel filtration on Sephadex G-50 were used. Gel filtration and SDS- polyacrylamide gel electrophoresis indicated a molecular weight of approximately 20 000. The protein inhibited peroxidation by Fe3+-triethylenetetramine following a 15 min preincubation of phosphatidylcholine liposomes in the presence of 5mM glutathione or 2-mercapthoethanol. The pure protein exhibited glutathione peroxidase activity on hydroperoxide groups of phosphatidylcholine and on cumene and t-butyl hydroperoxides, with specific activities of 2.2, 3.8 and 0.9 mumol/min per mg protein, respectively. The protein appears to be distinct from the selenoenzyme glutathione peroxidase and from any known glutathione S-transferase. The peroxidation was studied also with fresh phosphatidylcholine liposomes and was induced in this case by Fe-ascorbate. To obtain protection by the peroxidation-inhibiting protein and glutathione, preincubation was not necessary, but alpha-tocopherol, incorporated in the liposomes in the molar ratio 1:250 to phosphatidylcholine, was required. Lipid peroxidation of rat liver mitoplasts and microsomes was blocked when these preparations were incubated in the peroxidizing mixture in the presence of peroxidation-inhibiting protein and glutathione. The protection from Fe3+-triethylenetetramine-induced peroxidation is related apparently to reduction of hydroperoxide groups in polyunsaturated fatty acid residues of phospholipids and to inhibition of free radicals formation by chain branching. Protection from the Fe-ascorbate-induced peroxidation is apparently attributable to the same mechanism. However, the requirement of alpha-tocopherol for protection in the Fe-ascorbate-induced peroxidation suggests that the cooperation of a free-radical scavenger is necessary. It is probable that the glutathione peroxidase activity is involved also in the glutathione-dependent protection exhibited by the protein on lipid peroxidation of biomembranes.

Hydrogen peroxide and hematin in microsomal lipid peroxidation.

Lipids of rat liver microsomes underwent peroxidation with production of malondialdehyde in the presence of H2O2 and hematin. Rates of peroxidation of 27-33 nmol of MDA formed/mg of microsomal protein/30 min were measured with 5 mM H2O2 and 10 microM hematin at 22 degrees C. Histidine (0.01 M) caused a 55% inhibition. Hematin could be added to the reaction mixtures either simultaneously with H2O2 or afterwards, when all H2O2 had been destroyed by catalase present in the microsomal preparation. Catalase was necessary for formation of MDA. Indeed, when heat-denatured microsomes were employed, incubation with H2O2 and the iron complex led to formation of lipid hydroperoxides; however, no production of MDA was observed, unless exogenous catalase was added together with H2O2 and hematin to the reaction mixture. The role of H2O2 in microsomal lipid peroxidation is that of promoting the formation of fatty acid hydroperoxides. These are decomposed in the presence of hematin, with formation of free radicals, bicyclic endoperoxides and MDA. Catalase is necessary to remove H2O2, which, after starting the peroxidation process, blocks the decomposition of lipid hydroperoxides, apparently by binding to the iron complex.

Renal handling of sodium and water in early chronic liver disease. Evidence for a reduced natriuretic activity of the cirrhotic urinary extracts in rats.

Acute expansion of extracellular fluid volume during maximal water diuresis was induced in 8 chronic liver disease patients without clinical evidence of fluid retention, and in 8 controls. Fractional reabsorption of sodium was inferred in the proximal tubule, in the ascending limb of Henle's loop, and in the more distal site of the tubule. The results indicate that the significantly reduced increment of sodium excretion in cirrhotic patients was due to its augmented reabsorption in the proximal tubule. To establish whether there was a reduced activity of a natriuretic factor, a biologic assay was performed in 16 albino Wistar rats by using urine samples collected immediately after completion of a saline load and processed with gel filtration. The infusion of this fraction resulted in a significant lowering of the increment of urine output, and absolute and fractional sodium excretion only in the rats infused with urine extracts from cirrhotic patients. The results of this study raise the possibility that a reduced production of a natriuretic factor may play some role in the pathogenesis of sodium retention, which is observed in patients with cirrhosis of the liver.

Perfused liver carnitine palmitoyl-transferase activity and ketogenesis in streptozotocin treated and genetic hyperinsulinemic rats. Effect of glucagon.

Perfused liver carnitine palmitoyl transferase (CPT) activity and ketone body output were determined in streptozotocin -- treated and untreated Sprague-Dawley and Zucker rats. Streptozotocin enhanced liver ketogenic capacity and CPT activity in both these strains. No difference was observed in CPT activity or in ketone body production between the fatty and lean Zucker strains. Glucagon, added directly to the perfusate, had no influence on ketone body output and only in the livers of obese Zücker rats increased CPT activity.

Lecithin-cholesterol acyltransferase (LCAT) activity in chronic uremia.

High plasma concentrations of triglycerides and low plasma concentrations of esterified cholesterol and lysolecithin, with an impaired rate of VLDL and LDL catabolism, have been reported in chronic uremic patients. An important contribution to these abnormalitites might be an impaired activity of the (LCAT). Serum LCAT activity and cholesteryl ester clearance were determined in 11 patients with chronic renal failure and in 10 controls. LCAT activity was determined by using the serum of each patient both as a source of enzyme and as a substrate ("intrinsic" activity) and was compared with the activity determined on a standard substrate ("extrinsic activity), so as to ascertain the presence of inhibitory factors in the patients' sera. Both activityes have been found to be significantly (P less than 0.01) lower in chronic uremic patients than in controls. The cholesteryl ester clearance apparently did not respond to the stimulatory effect of hypertriglyceridemia, as observed in other cases of dislipoproteinemias. The parallel decrease of both enzyme activities makes it unlikely that it is due to the presence of "uremic toxins" inhibiting the enzyme activity. LCAT synthesis in the liver is probably reduced in chronic uremia. These results suggest that in chronic uremia the VLDL fail to cooperate in their own catabolism.

Sodium retention in chronic liver disease: lack of a natriuretic factor?

An acute volume expansion in water diuresis has been induced in 8 patients with liver cirrhosis and in 8 normal subjects, taken as controls. A two hour postinfusion urine sample has been utilized to obtain the urine fraction with natriuretic activity. This activity was assayed in 16 rats. Our results confirm a significantly lower natriuretic activity of cirrhotic urine fractions than those of controls.

Formation of acetoacetate from 3-hydroxy-3-methylglutarate by rat liver and isolation of a mitochondrial coenzyme A-transferase activity involved.

1. Formation of acetoacetate from 3-hydroxy-3-methylglutarate was observed in the perfused rat liver. Production of 3.5mumol of acetoacetate/h per g of tissue was obtained. 2. Formation of acetoacetate was catalysed mainly by the mitochondrial fraction of the homogenized liver, at a rate of 62nmol/h per mg of protein. 3. Experiments with hydroxy-[3-(14)C]methylglutarate demonstrated that the acetoacetate formed was derived mainly from this compound. 4. A mitochondrial transferase activity catalysing the transfer of a CoA molecule from succinyl-CoA (3-carboxypropionyl-CoA) to hydroxymethylglutarate was shown. The K(m) value for hydroxymethylglutarate was 5x10(-3)m.