Comparison of the Inactivation of Microsomal Glucose-6-Phosphatase by in Situ Lipid Peroxidation-Derived 4-Hydroxynonenal and Exogenous 4-Hydroxynonenal

1) The effect of 4-hydroxynonenal and lipid peroxidation on the activities of glucose-6-phosphatase and palmitoyl CoA hydrolase were studied.

2) 4-Hydroxynonenal inactivates glucose-6-phosphatase but has no effect on palmitoyl-CoA hydrolase. These effects are similar with those observed during lipid peroxidation of microsomes.

3) The inhibition of glucose-6-phosphatase by 4-hydroxynonenal can be prevented by glutathione but not by vitamin E. The inactivation of glucose-6-phosphatase during lipid peroxidation is prevented by glutathione and delayed by vitamin E.

4) The formation of 4-hydroxynonenal during lipid peroxidation was followed in relation to the inactivation of glucose-6-phosphatase. At 50% inactivation of glucose-6-phosphatase the 4-hydroxynonenal concentration was 1.5μM. To obtain 50% inactivation of glucose-6-phosphatase by added 4-hydroxynonenal a concentration of 150μM or 300μM was needed with a preincubation time of 30 and 60 min, respectively.

5) It is concluded that the glucose-6-phosphatase inactivation during lipid peroxidation can be due to the formation of 4-hydroxynbnenal. The formed 4-hydroxynonenal which inactivates glucose-6-phosphatase is located in the membrane. If this mechanism is valid it implies that a functional SH group of glucose-6-phosphatase is layered in the membrane. However, an inactivation of glucose-6-phosphatase by desintegration of the membrane by lipid peroxidation cannot be ruled out.     

Alterations of the microsomal glucose-6-phosphatase system evoked by ferrous iron- and haloalkane free-radical-mediated lipid peroxidation

Alterations of catalytic activities of the microsomal glucose-6-phosphatase system were examined following either ferrous iron- or halothane (CF3CHBrCl) and carbon tetrachloride (CCl4) free-radical-mediated peroxidation of the microsomal membrane. Enzyme assays were performed in native and solubilized microsomes using either glucose 6-phosphate or mannose 6-phosphate as substrate. Lipid peroxidation was assessed by the amounts of malondialdehyde equivalents formed. Regardless of whether the experiments were performed in the presence of NADPH/Fe3+, NADPH/CF3CHBrCl, or NADPH/CCl4, with the onset of lipid peroxidation, mannose-6-phosphatase activity of the native microsomes increased immediately, while further alterations in catalytic activities were only detectable when lipid peroxidation had passed characteristic threshold values: above 2 nmol malondialdehyde/mg microsomal protein, glucose-6-phosphatase activity of the native microsomes was lost, and at 10 nmol malondialdehyde/mg microsomal protein, glucose-6-phosphatase and mannose-6-phosphatase activity of the solubilized microsomes started to decline. It is concluded that the latter alterations are due to an irreversible damage of the phosphohydrolase active site of the glucose-6-phosphatase system, while the changes observed at earlier stages of microsomal lipid peroxidation may also reflect alterations of the transporter components of the glucose-6-phosphatase system. Virtually no changes in the catalytic activities of the glucose-6-phosphatase system occurred under anaerobic conditions, indicating that CF3CHCl and CCl3 radicals are without direct damaging effect on the glucose-6-phosphatase system. Further, maximum effects of carbon tetrachloride and halothane on lipid peroxidation and enzyme activities were observed at an oxygen partial pressure (PO2) of 2 mmHg, providing additional evidence for the crucial role of low PO2 in the hepatotoxicity of both haloalkanes.     

Effect of seminal plasma antioxidant on lipid peroxidation in spermatozoa, mitochondria and microsomes

Seminal plasma antioxidant inhibited ascorbate/iron-induced lipid peroxidation in spermatozoa, brain and liver mitochondria. The concentration required to produce inhibition in brain and liver mitochondria was high. Denaturation of spermatozoa resulted in complete loss of antioxidant action. Maintenance of native structure was essential for action of seminal plasma antioxidant in spermatozoal lipid peroxidation. The antioxidant inhibited NADPH, Fe3+-ADP induced lipid peroxidation in microsomes and consequences of lipid peroxidation such as glucose-6-phosphatase inactivation were prevented by presence of antioxidant. It did not inhibit microsomal lipid peroxidation induced by ascorbate and iron and xanthine-xanthine oxidase.     

The role of lipid peroxidation in CCl4-induced damage to liver microsomal enzymes: comparative studies in vitro using microsomes and isolated liver cells

The question as to whether CCl4 decreases the activities of glucose-6-phosphatase and cytochrome P-450 in liver endoplasmic reticulum mainly through its action in stimulating lipid peroxidation has been investigated using Promethazine to block lipid peroxidation. The investigation, moreover, has compared the effects of CCl4, with and without Promethazine, on isolated rat hepatocytes with corresponding effects on rat liver microsomal suspensions. Our data give no support for the view that products of lipid peroxidation are the main cause of the decrease in cytochrome P-450 observed in CCl4-intoxication. However, our present results are consistent with lipid peroxidation being a major contributory factor to the decrease in glucose-6-phosphatase activity observed in CCl4-induced liver injury.     

Further studies on lipid-peroxide formation in isolated hepatocytes.

Lipid peroxide formation was initiated by the addition of either ADP-complexed Fe3+ or cumene hydroperoxide to a suspension of isolated hepatocytes. The reaction was monitored by malonaldehyde measurements. Upon the addition of iron, malonaldehyde production in the cells started immediately but ceased within 30-60 min, and the response was dose-related with iron concentrations ranging from 19 to 187 muM. Malonaldehyde formation was associated with increased oxygen uptake and conjugated diene production. The addition in vitro of N,N,N',N'-tetramethyl-p-phenylenediamine, menadione or p-benzoquinone inhibited the iron-induced malonaldehyde production. It was also possible to demonstrate an apparent disappearance of malonaldehyde from fresh cells by addition of adequate amounts of N,N,N',N'-tetramethyl-p-phenylenediamine (100 muM). The attenuation of the iron-induced malonaldehyde production was found to be correlated with an increased binding of iron to an intracellular ferritin fraction. Further, malonaldehyde formation was also associated with a conversion of reduced glutathione to the oxidized form which, in turn, revealed a faster permeation out of the cells into the surrounding medium of the oxidized than of the reduced thiol. So, concomitant with the redox alterations, there was also an overall loss of glutathione from the cells. Cumene hydroperoxide-induced malonaldehyde production could be initiated by the addition of this peroxide in concentrations ranging from 150 muM to the liver cell incubate. With concentrations below 150 muM, a lag phase was present which seemed to be glutathione-dependent. It is concluded that iron enters the cell, then is probably reduced inside the cell by NADPH via the NADPH-cytochrome P-450 reductase, and in the reduced state initiates lipid peroxidation. The reaction is inhibited by intracellular mechanisms, the glutathione redox system being of principal importance, and possibly terminated by the iron-apoferritin complex formation.     

Biochemical evidence for chemical and/or topographic differences in the lipoperoxidative processes induced by CCl4 and iron

Isolated rat hepatocytes, treated with CCl4 or ADP-Fe3+ complex show an enhanced lipid peroxidation and a decreased glucose 6-phosphatase activity. Lipid peroxidation is much more stimulated by ADP-Fe3+ or Fe3+ than by CCl4, when the metal and the haloalkane are used at a similar concentration. Increasing rates of lipid peroxidation in the different experimental conditions do not correlate with the degree of glucose 6-phosphatase inactivation, which is produced by CCl4 and not by a similar amount of ferric iron. In the case of iron, its intracellular concentration must be higher to give the enzyme inactivation exerted by CCl4. Higher intracellular levels of iron are reached when the metal is added to the cell suspension together with ADP. Under these conditions there is inactivation of glucose 6-phosphatase. Possible mechanisms accounting for a different enzyme sensitivity to iron and CCl4 are discussed.     

Functional impairment of intact rat liver cells due to biological aldehydes

The addition of lipid peroxidation end-products, 4-hydroxynonenal (HNE) or hexanal (HEX) to the incubation medium of rat hepatocytes caused significant decrease of cell cytochrome P-450 content and inactivation of total cell glucose-6-phosphatase. Both the tested aldehydes exerted a marked inhibition of triglyceride secretion by liver cells. The reported results on intact cells furtherly support a possible damaging effect of aldehydes in pathological conditions in which a stimulation of lipid peroxidation occurs.     

Lipid peroxidation in plumbagin administered rats

Plumbagin was administered to rats at a concentration of 1,2,4,8 and 16 mg per kg body weight. After 24 h lipid peroxide levels were found to decrease in subcellular fractions of liver. Plumbagin inhibited ascorbate and nicotinafde adenine dinucleotide phosphate (reduced) dependent lipid peroxidation but was without any effect on cumene hydroperoxide dependent lipid peroxidation. Injection of 16 mg of plumbagin per kg body weight was found to decrease liver total reduced glutathione and also fcrosomal glucose-6-phosphatase. The results are discussed with reference to the anti- and prooxidant properties of plumbagin.     

Effect of alpha-tocopherol on doxorubicin-induced changes in rat liver and heart microsomes.

Rats were treated with doxorubicin (2.5 mg/kg body wt, iv) once a week for 8 weeks. Alpha-Tocopherol (400 mg/kg body wt/day) was co-administered orally for 2 months. Cytochrome-P450 (Cyt-P450) and Cytochrome-b5 (Cyt-b5) levels decreased significantly in doxorubicin treated rats. Significant decreases were observed in glucose-6-phosphatase, Cyt-P450 and Cyt-b5 reductase activities. In vitro lipid peroxidation study showed that alpha-tocopherol significantly minimises the lipid peroxide formation by doxorubicin. There was a significant change in microsomal cholesterol and phospholipid levels. Alpha-Tocopherol co-administration reduced the alterations in xenobiotic metabolising system and microsomal lipid levels. The results were discussed with reference to drug metabolising enzymes, lipid peroxidation and antioxidant nature of alpha-tocopherol.     

Lipid peroxidation in rat liver microsomes. II. Response of hydroperoxide formation to iron concentration

When rat liver microsomes were incubated with NADPH, the major products were hydroperoxides which increased with time indicating that endogenous iron content is able to promote lipid peroxidation. The addition of either 5 microM Fe2+ or Fe3+ ions strongly enhanced the hydroperoxide formation rate. However, due to the hydroperoxide breakdown, hydroperoxide concentration decreased with time in this case. Higher ferrous or ferric iron concentration did not change the situation much, in that both hydroperoxide breakdown and formation were similar to those when NADPH only was present in the incubation medium. After lipid peroxidation, analysis of fatty acids indicated that the highest amount of peroxidized PUFA occurred in the presence of 5 microM of either Fe2+ or Fe3+. This analysis also showed that after 8 min incubation with low iron concentration, PUFA depletion was about 77% of that observed after 20 min, whereas without any iron addition or in the presence of 30 microM of either Fe3+, PUFA decrease was only about 37% of that observed after 20 min. As far as the optimum Fe2+/Fe3+ ratio required to promote the initiation of microsomal lipid peroxidation in rat liver is concerned, the highest hydroperoxide formation was observed with a ratio ranging from 0.5 to 2. These results indicate that microsomal lipid peroxidation induced by endogenous iron is speeded up by the addition of low concentrations of either Fe2+ or Fe3+ ions, probably because free radicals generated by hydroperoxide breakdown catalyze the propagation process. In experimental conditions unfavourable to hydroperoxide breakdown the principal process is that of the initiation of lipid peroxidation.     

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.     

Effects of lipid peroxidation on membrane-bound enzymes of the endoplasmic reticulum

1. Induction of the formation of lipid peroxide in suspensions of liver microsomal preparations by incubation with ascorbate or NADPH, or by treatment with ionizing radiation, leads to a marked decrease of the activity of glucose 6-phosphatase. 2. The effect of peroxidation can be imitated by treating microsomal suspensions with detergents such as deoxycholate or with phospholipases. 3. The substrate, glucose 6-phosphate, protects the glucose 6-phosphatase activity of microsomal preparations against peroxidation or detergents. 4. The loss of glucose 6-phosphatase activity is not due to the formation of hydroperoxide or formation of malonaldehyde or other breakdown products of peroxidation, all of which are not toxic to the enzyme. 5. All experiments lead to the conclusion that the loss of activity of glucose 6-phosphatase resulting from peroxidation is a consequence of loss of membrane structure essential for the activity of the enzyme. 6. In addition to glucose 6-phosphatase, oxidative demethylation of aminopyrine or p-chloro-N-methylaniline, hydroxylation of aniline, NADPH oxidation and menadione-dependent NADPH oxidation are also strongly inhibited by peroxidation. However, another group of enzymes separated with the microsomal fraction, including NAD⁺/NADP⁺ glycohydrolase, adenosine triphosphatase, esterase and NADH–cytochrome c reductase are not inactivated by peroxidation. This group is not readily inactivated by treatment with detergents. 7. Lipid peroxidation, by controlling membrane integrity, may exert a regulating effect on the oxidative metabolism and carbohydrate metabolism of the endoplasmic reticulum in vivo.     

Lipid peroxidation inactivates rat liver microsomal glycerol-3-phosphate acyl transferase. Effect of iron and copper salts and carbon tetrachloride

Lipid peroxidation is known to affect the activity of several enzymes including microsomal enzymes such as glucose-6-phosphatase; but its effect on the enzymes of lipid biosynthesis has not been investigated. Glycerol-3-phosphate acyltransferase (GPAT) represents the first committed step and probably the rate limiting step in glycerolipid synthesis and thus may be a good candidate for study. Rat liver microsomal GPAT was assayed after preincubating the microsomes under conditions known to induce peroxidation. In 30 min, 10 microM Fe2+ can diminish the activity by as much as 80%. The inactivating effect can be blocked to different extents by several antioxidants, while ascorbic acid enhances it. These effects, along with the concomitant measurement of lipid peroxidation, indicate that microsomal GPAT activity is inactivated by lipid peroxidation in a sensitive and rapid fashion. This is further confirmed by the inactivating effect of carbon tetrachloride, which is known to induce lipid peroxidation in microsomes. Fe3+ also inactivates the enzyme, but at a higher concentration. Copper salts inactivate GPAT by a mechanism apparently different from that of iron. The mechanism might involve a direct sulfhydryl modification by copper and lipid peroxidation apparently different from that induced by iron. It is suggested that the inactivation of GPAT by lipid peroxidation could accelerate the process of membrane disintegration caused by lipid peroxidation in pathological conditions involving free radical-mediated tissue injury.     

Stimulatory effect of glucose 6-phosphate on the non-mitochondrial Ca2+ uptake in permeabilized hepatocytes and Ca2+ release by inositol trisphosphate

The relationships between Ca2+ transport and glucose-6-phosphatase activity, previously studied in isolated liver microsomes, were investigated in permeabilized hepatocytes in the presence of mitochondrial inhibitors. It was found that the addition of glucose 6-phosphate to the cells markedly stimulates the MgATP-dependent Ca2+ uptake. A progressive increase in the stimulation of Ca2+ uptake was seen with increasing amounts of glucose 6-phosphate up to 5 mM concentrations. Vanadate, when added in adequate concentrations (20-40 microM) to the hepatocytes inhibits both the glucose-6-phosphatase activity and the stimulation of Ca2+ uptake by glucose 6-phosphate, while not affecting the MgATP-dependent Ca2+ uptake. The addition of inositol 1,4,5-trisphosphate to permeabilized hepatocytes in which Ca2+ had been accumulated in the presence of MgATP and glucose 6-phosphate, results in a rapid release of Ca2+.     

Liver glucose-6-phosphatase activity is modulated by physiological intracellular Ca2+ concentrations

The effect of varying concentrations of free Ca2+ on the formation of Pi from mannose-6-P or of Pi and [U-14C]glucose from [U-14C]glucose-6-P was investigated in isolated fasted rat hepatocytes made permeable by freezing and in liver microsomes. Free Ca2+ concentration was adjusted by the use of Ca-EGTA buffers. In permeabilized cells, glucose-6-phosphatase (EC 3.1.3.9) activity was inhibited up to 50% and in intact microsomes up to 70% by increasing free Ca2+ concentrations from 0.01 to 10 microM. The inhibition was reversible and competitive with respect to glucose-6-P. Treatment of microsomes with 0.4% deoxycholate exposed 90% of latent mannose-6-phosphatase activity which was insensitive to Ca2+. The results indicate that Ca2+ affects the glucose-6-P translocase rather than the phosphohydrolase component. It is concluded that the glucose-6-phosphatase system is modulated by changes in Ca2+ concentrations in the range of those occurring in the liver cell upon hormonal stimulation.     

Effects of hormones on the activity of glucose-6-phosphatase in primary cultures of rat hepatocytes

Although the activity of glucose-6-phosphatase in rat liver is altered markedly following the administration of a variety of hormones in vivo, it is not certain whether the hormones act directly on the hepatocyte. To study this problem hepatocytes were isolated by a collagenase-perfusion technique and cultured on collagen gel/nylon mesh membranes. The activity of glucose 6-phosphatase in cells cultured with fetal calf serum and with Dulbecco's modified Eagle's medium or Leibovitz L-15 medium decreased to less than 10-30% of the activity in freshly isolated cells by 96 h. However, when L-15 plus newborn or fetal calf serum was supplemented with glucagon (10(-6)M), epinephrine (10(-6)M), triiodothyronine (10(-6)M), and dexamethasone (10(-5)M) (L-15-GETD), the activity of glucose-6-phosphatase was maintained so that, after 144 h, the activity was at least 80% of that detected in freshly isolated cells. In cells cultured in L-15 plus serum for 72 or 96 h and then in L-15-GETD, glucose-6-phosphatase increased 30-50% over that in control cultures after 24 h. Insulin, which decreases glucose-6-phosphatase activity when administered to intact animals, also decreased the glucose-6-phosphatase activity in cultured hepatocytes to 20-50% of that in controls.     

Inhibition of lipid peroxidation by disulfiram and diethyldithiocarbamate does not prevent hepatotoxin-induced cell death in isolated rat hepatocytes. A study with allyl alcohol, tert-butyl hydroperoxide, diethyl maleate, bromoisovalerylurea and carbon tetrachloride

The relationship between lipid peroxidation and cell death, induced by a number of hepatotoxins, was studied in isolated rat hepatocytes. Disulfiram (DSF) and diethyldithiocarbamate (DDC) completely prevented lipid peroxidation, induced by allyl alcohol, tert-butyl hydroperoxide (t-BHP), diethyl maleate (DEM), bromoisovalerylurea (BIU) and carbon tetrachloride (CCl4). Lipid peroxidation was measured by the formation of both thiobarbituric acid positive material and conjugated dienes. However, DSF and DDC did not protect against cell death, induced by these hepatotoxins. In the presence of DSF or DDC, cell death occurred even earlier in time. We conclude that cell death can occur in the absence of lipid peroxidation. Therefore, lipid peroxidation is not a requisite for the induction of cell death.     

Administration of the tris salt of alpha-tocopheryl hemisuccinate inactivates CYP2E1, enhances microsomal alpha-tocopherol levels and protects against carbon tetrachloride-induced hepatotoxicity

A series of tocopherol compounds were examined for their capacity to protect against carbon tetrachloride (CCl4)-induced hepatotoxicity in rats. Of the tocopherol compounds tested in our study, only the tris salt of d-alpha-tocopheryl hemisuccinate (TS-tris) protected against CCl4-induced hepatotoxicity. The administration of d-alpha-tocopherol (alpha-T) and the nonhydrolyzable tocopherol ether, d-alpha-tocopheryloxybutyrate tris salt (TSE-tris), failed to protect against CCl4-induced hepatotoxicity. TS-tris was the only tocopherol which significantly decreased CYP2E1 activity after 18 h. This decrease in CYP2E1 activity is likely to limit the activation of CCl4 and protect against CCl4-induced hepatotoxicity. Our results also suggest that TS-tris protection against CCl4-induced hepatotoxicity correlates with the enhanced capacity of TS-tris to deliver alpha-T and increase the antioxidant status of hepatocytes. TSE-tris did not increase cellular alpha-T levels, while administration of TS-tris produced large increases in alpha-T levels in liver homogenates as well as in liver nuclei, microsomes, mitochondria and plasma membranes. This enhanced ability to deliver tocopherol equivalents to parenchymal liver cells may be related in part to the ability of TS-tris to form liposomes in aqueous solutions. TS-tris administration protected against CCl4-induced microsomal lipid peroxide formation and inactivation of the microsomal enzyme glucose-6-phosphatase (G6Pase). Supplementation of animals with alpha-T protected against microsomal lipid peroxide formation but not against the inactivation of G6Pase. Based on our findings, we propose that high cellular levels of alpha-T protect against CCl4-induced hepatotoxicity by scavenging CCl4 radicals as well as protecting against lipid peroxidation. Our results do not support the importance of microsomal lipid peroxidation as an early event in acute CCl4-induced hepatic necrosis.     

Loss of latent activity of liver microsomal membrane enzymes evoked by lipid peroxidation. Studies of nucleoside diphosphatase, glucose-6-phosphatase, and UDP glucuronyltransferase

The effects of lipid peroxidation on latent microsomal enzyme activities were examined in NADPH-reduced microsomes from phenobarbital-pretreated male rats. Lipid peroxidation, stimulated by iron or carbon tetrachloride, was assayed as malondialdehyde formation. Independent of the stimulating agent of lipid peroxidation, latency of microsomal nucleoside diphosphatase activity remained unaffected up to microsomal peroxidation equivalent to the formation of about 12 nmol malondialdehyde/mg microsomal protein. However, above this threshold a close correlation was found between lipid peroxidation and loss of latent enzyme activity. The loss of latency evoked by lipid peroxidation was comparable to the loss of latency attainable by disrupting the microsomal membrane by detergent. Loss of latent enzyme activity produced by lipid peroxidation was also observed for microsomal glucose-6-phosphatase and UDPglucuronyltransferase. In contrast to nucleoside diphosphatase, however, both enzymes were inactivated by lipid peroxidation, as indicated by pronounced decreases of their activities in detergent-treated microsomes. According to the respective optimal oxygen partial pressure (po2) for lipid peroxidation, the iron-mediated effects on enzyme activities were maximal at a po2 of 80 mmHg and the one mediated by carbon tetrachloride at a po2 of 5 mmHg. Under anaerobic conditions no alterations of enzyme activities were detected. These results demonstrate that loss of microsomal latency only occurs when peroxidation of the microsomal membrane has reached a certain extent, and that beyond this threshold lipid peroxidation leads to severe disintegration of the microsomal membrane resulting in a loss of its selective permeability, a damage which should be of pathological consequences for the liver cell. Because of its resistance against lipid peroxidation nucleoside diphosphatase is a well-suited intrinsic microsomal parameter to estimate this effect of lipid peroxidation on the microsomal membrane.