Vitamin E dependent reduced glutathione inhibition of rat liver microsomal lipid peroxidation

Effects of reduced glutathione (GSH) were investigated on $\text{in}$$\text{vitro}$ lipid peroxidation of hepatic microsomes obtained from Long-Evans Hooded rats fed chemically defined, purified diets containing adequate or documented deficiencies of vitamin E (E), selenium (Se) or both. Glutathione inhibited lipid peroxidation mediated by both NADPH-dependent enzymatic and ascorbate-dependent non-enzymatic systems. The inhibitory effect of GSH was observed in microsomes obtained from E supplemented groups whereas it had no effect on microsomes from E deficient animals. Selenium status had no effect on GSH inhibition. Glutathione was found to be specific for the E dependent inhibition of lipid peroxidation and could not be substituted by other sulfhydryl compounds tested. Also, GSH did not inhibit non-enzymatic lipid peroxidation of heat-denatured microsomes from either E-supplemented groups or any of the other dietary regimens.     

Enhanced hepatic chemiluminescence following carbon tetrachloride or hydrazine administration

Lipid peroxidation has been implicated as a mechanism of cellular injury due to a variety of hepatotoxic agents. In this study a scintillation spectrophotometer was employed to measure photon emission as an index of lipid peroxidation. Chemiluminescence (CL) from rat liver homogenates was significantly enhanced when animals were pretreated with hepatotoxic agents such as hydrazine or CCl₄. The enhanced CL was markedly reduced by the addition of antioxidants $\text{in}$$\text{vitro}$ or incubation with 100% nitrogen, events which have been demonstrated to decrease lipid peroxidation. Compared to results obtained employing the thiobarbituric acid assay, the CL procedure appears to be a more sensitive index of lipid peroxidation. The chemiluminescence procedure appears to merit further evaluation in the assessment of the role of lipid peroxidation in the pathogenesis of cell injury.     

Effect of dietary antioxidants and phenobarbital pretreatment on microsomal lipid peroxidation and activation by carbon tetrachloride.

The effect of the dietary antioxidants, vitamin E and selenium, and the effect of phenobarbital pretreatment on $\text{in}$$\text{vitro}$ NADPH-dependent microsomal lipid peroxidation and the activation of microsomal lipid peroxidation by CCl₄ were studied. The rate of microsomal lipid peroxidation decreased as a function of dietary anti-oxidant, while the degree of CCl₄ activation increased. Phenobarbital pretreatment diminished the antioxidant inhibition of microsomal lipid peroxidation found with microsomes from rats fed high levels of antioxidant. Phenobarbital pretreatment lowered the extent of lipid peroxidation as measured by malonaldehyde production but had little effect on the rate of lipid peroxidation as measured by oxygen uptake. The kinetics of lipid peroxidation and the stoichiometry of the reaction were assessed as a function of dietary antioxidant.The findings suggest that at low microsomal antioxidant concentrations, the lipid peroxidation reaction occurs at a maximal rate dependent upon some rate-limiting step, such as the reduction of Fe⁺³, which is unaffected by CCl₄ addition. Conversely, at high microsomal antioxidant concentrations, the antioxidant termination reactions appear to determine the overall reaction rate.     

NADPH-dependent reductase solubilized from microsomes by peroxidation and its activity

Isolated rat liver microsomes were subjected to enzymatic or non-enzymatic lipid peroxidation $\text{in vitro}$. NADPH-dependent cytochrome $\text{c}$ reductase activity was released from the microsomes into the media during peroxidation. This activity could be recovered from the media by DEAE-cellulose chromatography. The recovered enzyme retained high activity for the reduction of cytochrome $\text{c}$ and a lower level of activity for the reduction of cytochrome P-450. The active fractions were capable of enzymatically supporting the peroxidation of isolated mitochondria in the presence of organically complexed Fe⁺³ and NADPH, and in this respect the specific activity was found to be about ten times higher than in microsomes.     

Catecholamine-induced changes in transmembrane potential and temperature in normal and dystrophic hamster brown adipose tissue

Previous studies have shown that norepinephrine (NE) elicits trans-membrane potential changes in skeletal muscle cells from normal and dystrophic (BIO 14.6) hamsters, with the magnitude of these changes being significantly less in dystrophic cells. To determine if the decreased response of the dystrophic muscle cells reflects a more generalized phenomenon, the present study was designed to evaluate the effects of NE on membrane properties of brown adipocytes. $\text{In vivo}$ techniques using glass microelectrodes were similar to those used in the muscle studies. NE injection (2 to 5 μg/kg body wt, i.v.) into anesthetized hamsters was followed by membrane depolarization, the magnitude of which did not significantly differ in the dystrophic and normal adipocytes. For example, upon administration of 5 μg NE/kg body wt, the average depolarization was $\text{14.5 ± 1.3}\text{mV}\text{(}\text{X}\text{±}\text{S.E.}\text{)}$ for 20 dystrophic cells and 14.1 ± 1.8 mV for 18 normal cells. The depolarizations following i.v. infusion of isoproterenol and phenylephrine also had similar amplitudes in both normal and dystrophic cells. Despite this lack of difference in plasma membrane responses, NE induced a significantly smaller rise in interscapular brown fat temperature in the dystrophic (0.09°C) than in the normal hamsters (0.26°C) following administration of 5 μg NE/kg body wt. Thus, the decreased responsiveness to NE of dystrophic sarcolemma did not occur with the plasma membrane of brown adipocytes, although brown fat temperature changes in the dystrophic hamsters were decreased in amplitude.     

Studies on the effects of alloxan and streptozotocin induced diabetes on lipid metabolism in the isolated perfused rat lung.

The effects of experimentally induced diabetes on the conversion of glucose to lipid in the isolated perfused rat lung were examined. Alloxan diabetes and streptozotocin diabetes reduced the incorporation of glucose into the neutral lipid and phospholipid fractions of the lung to a rate less than 40% of that observed in normal animals. This phenomenon appears to be related to insulin deficiency as lungs from diabetic rats treated for one week with insulin were capable of incorporating glucose at a rate comparable to that observed in normal animals. While insulin $\text{in}$$\text{vivo}$ altered lipid metabolism in perfused lung, $\text{in}$$\text{vitro}$ insulin had no demonstrable effect on lipid metabolism in the perfused lung, an indication that the effects of the hormone may be long term rather than short term. These data indicate that pulmonary lipid metabolism may be regulated by the action of insulin.     

The requirement for ferric in the initiation of lipid peroxidation by chelated ferrous iron

When certain ferrous chelates are added to lipid, peroxidation of the lipid occurs following a short lag. This suggests that a product of ferrous autoxidation is required to initiate lipid peroxidation. This autoxidation product is apparently ferric iron, rather than the oxygen radicals which also result from ferrous autoxidation. Studies with oxy-radical scavengers and catalase suggest that $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ H₂O₂, or the ·OH are not involved in the initiation reactions, therefore, we propose that a ferrous-dioxygen-ferric chelate complex may be the initiating species.     

Rat liver cytosolic glutathione peroxidase: reactivity with linoleic acid hydroperoxide and cumene hydroperoxide.

The reactivity of rat liver glutathione (GSH) peroxidase with two hydroperoxides was determined using integrated rate equations. The bimolecular rate constant for the reaction of GSH peroxidase with linoleic acid hydroperoxide is approximately four times the rate constant with cumene hydroperoxide. The reactivity toward reduced glutathione is not altered by different hydroperoxides. The $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for lipid hydroperoxide in rat liver is approximated at 9.5 × 10^?5 min.     

A defect of purine nucleotide cycle in the skeletal muscle of hereditary dystrophic mice

The purine nucleotide cycle in the hind leg skeletal muscle of hereditary dystrophic mice ($\text{C57BL}\text{6J}\text{-}\text{dy}\text{dy}$) was investigated. The amount of adenine nucleotide produced from adenylosuccinate by the muscle extract in the dystrophic group was less than 3 % of that in the control group, while adenine nucleotide plus adenylosuccinate converted from IMP in the dystrophic group was about 70 % of that of the control group. Moreover, the activity of AMP deaminase of the dystrophic group was about 50 % of that of the control group. These results indicate that the purine nucleotide cycle is defective in the dystrophic muscle. This abnormality was suggested to be caused by the considerably low activity of adenylosuccinase.     

In vivo lipogenesis and enzyme levels in adipose and liver tissues from pair-fed genetically obese and lean rats

Genetically obese Zucker rats pair-fed to lean controls were similar in body weight and food intake, however, epididymal fat pads were considerably larger than lean controls. $\text{In}$$\text{vivo}$ incorporation of acetate-¹⁴C into adipose tissue lipid was not significantly different, however, $\text{in}$$\text{vivo}$ liver lipogenesis was elevated in the obese rat. Characterization of enzyme profiles in both liver and adipose tissues revealed that enzymes normally associated with lipogenesis were elevated in liver tissue from obese rats. Malic enzyme and citrate cleavage enzyme were both depressed in adipose tissue of obese animals. From these data, it appears that the liver may be prominently involved in the development of excessive blood lipid and enlarged fat cells in the Zucker obese rat.     

Ferrous ion-mediated cytochrome P-450 degradation and lipid peroxidation in adrenal cortex mitochondria

The relationship between the degradation reaction of cytochrome $\text{P-450}$ and lipid peroxidation was studied utilizing bovine adrenal cortex mitochondria. The two reactions were found to be closely correlated in terms of their response to storage of the mitochondrial preparation, stimulation by Fe²⁺, inhibition by EDTA and their initiation by cumene hydroperoxide. Both reactions were also found not to be inhibited by catalase, superoxide dismutase, 1,4-diazabicyclo-(2,2,2)-octane and alcohols, indicating that H₂O₂, superoxide, singlet oxygen and hydroxyl radicals do not participate in these reactions. Yet, diphenylamine proved to be a powerful inhibitor for both reactions, suggesting the involvement of a radical species. Cumene hydroperoxide could induce these two reactions at below 0.1 mM concentrations in the presence of molecular oxygen. The chemiluminescence observed during the Fe²⁺-mediated lipid peroxidation reaction which was not inhibited by either superoxide dismutase or 1,4-diazabicyclo-(2,2,2)-octane, was biphasic: one was a rapid burst; and the other was a slowly increasing emission. The latter portion of the emission of light coincided with the formation of malondialdehyde. These results indicate that in adrenal cortex mitochondria the degradation of cytochrome $\text{P-450}$ is closely related to lipid peroxidation.     

Lipid metabolism and in vitro production of progesterone and prostaglandin F during induced regression of porcine corpora lutea

The effects of Cloprostenol administration on porcine luteal lipid and arachidonic acid accumulation were examined in relation to luteal $\text{in vitro}$ progesterone and prostaglandin F synthesis in 18 mature gilts at day 12 of the estrous cycle. Basal and net $\text{in vitro}$ release of progesterone from luteal tissue was depressed at 8 hr after treatment whereas net $\text{in vitro}$ release of prostaglandin F was elevated at 8 hr. Inclusion of copper dithiothreitol or reduced glutathione in the incubation media resulted in minor alterations of $\text{in vitro}$ release of progesterone and prostaglandin F and no changes in composition of luteal lipids or fatty acids. Luteal contents of triglyceride had increased by 8 hr after treatment whereas contents of free and esterified cholesterols had increased by 32 hr after Cloprostenol administration. Luteal contents of phospholipid and free fatty acids were not affected by Cloprostenol administration. At 32 hr after treatment, percentages and content of arachidonic acid had increased in luteal cholesterol esters and triglycerides. Although arachidonic acid percentages increased in luteal free fatty acids and phospholipids, calculated arachidonic acid contents did not change following Cloprostenol administration. Induced luteal regression was associated with decreased $\text{in vitro}$ progesterone release, increased $\text{in vitro}$ prostaglandin F release, and accelerated lipid and arachidonic acid accumulation within the corpus luteum. The effects of altered lipid metabolism on release of prostaglandin F could not be defined. However, availability of arachidonic acid did not appear to be rate-limiting in relation to luteal $\text{in vitro}$ prostaglandin F synthesis.     

Purification of the agent inducing lipid peroxidation with dihydroxyfumaric acid in rat liver mitochondria

Dihydroxyfumaric acid induces lipid peroxidation in rat liver mitochondria as reported previously. When the mitochondria were solubilized with 0.35% ($\text{W}\text{V}$) sodium cholate, the supernatant itself could not catalyze lipid peroxidation with dihydroxyfumaric acid, but the precipitate slightly induced the reaction. The supernatant produced lipid peroxide in the presence of the precipitate and dihydroxyfumaric acid. The supernatant was heat sensitive contrary to the stability of the precipitate. An attempt was made to isolate active entity through a sephadex G-200 column and a DEAE-cellulose column, resulting in about 10-fold purification. At 408–410 nm the partially purified agent showed a maximum absorption, which disappeared rapidly after reduction with sodium dithionite and was slowly diminished with dihydroxyfumaric acid. The molecular weight was much larger than that of oxidized cytochrome c.     

Serum glutathione S-transferases: Perinatal development,sex difference,and effect of carbon tetrachloride administration on enzyme activity in the rat

Glutathione $\text{S}$-transferase activity was determined in rat, rabbit, and guinea pig serum using styrene 7,8-oxide (SO) and benzo (a) pyrene 4,5-oxide (4,5-BPO) as substrates. Of the species tested, rat had the highest transferase activity (62.5 and 3.2 nmol/min/ml serum for SO and 4,5-BPO, respectively) and rabbit had the lowest activity. Glutathione $\text{S}$-transferase activity was 60% higher in serum from male rats than in female rats. In rats, serum enzyme specific activities (nmol/min/mg protein) were less than 1% of hepatic enzyme activities with SO, 4,5-BPO, 1,2-dichloro-4-nitrobenzene (DCNB), and 1-chloro-2,4-dinitrobenzene (DNCB). Glutathione $\text{S}$-transferase activity was also determined in rat serum during perinatal development. Serum from rats at 18 days of gestation or from 1- and 4-day-old animals had barely detectable transferase activity. Activity increased with age and reached a maximum in 140-day-old animals. The intraperitoneal administration of diethyl maleate (DEM) (0.8 ml/kg) or L-methionine-DL-sulfoximine (MS) (200 mg/kg) to male rats had no effect on serum or hepatic glutathione $\text{S}$-transferase activities 2 or 26 hr after dosing. Treatment with carbon tetrachloride (CCl₄) (1 m1/kg) caused an 11-fold increase in serum transferase activity and a 40% decrease in liver specific activities 24 hr after administration.     

Drug-induced changes in lipid composition of E. coli and of mammalian cells in culture: ethanol, pentobarbital, and chlorpromazine.

Both Chinese hamster ovary cells in culture and $\text{E.}$$\text{coli}$ cells change their lipid composition when grown in the presence of ethanol, pentobarbital, and chlorpromazine. The effects of ethanol and the cross-tolerant drug, pentobarbital, are similar. Both cause a shift from 18:0 fatty acid to 16:0 fatty acids in CHO cells and a decrease in the proportion of saturated fatty acids in $\text{E.}$$\text{coli}$. Chlorpromazine, a non-cross-tolerant drug, causes the opposite effect in $\text{E.}$$\text{coli}$, a decrease in the proportion of unsaturated fatty acids. Chlorpromazine has little effect on the fatty acid composition of CHO cells. These changes in lipid composition are proposed as an adaptive response and a part of the mechanism for the development of drug tolerance.     

Studies of a possible relationship of brain proteins to changes in behavioral sensitivity to DFP

Whether behavioral differences in selectively bred lines of rats could be attributed to changes in the enzymes acetylcholinesterase and glutathione S-transferase in different brain regions have been examined. Electrophoresis of the brain proteins has been studied and compared to one another and to that of control animals. The results presented suggest that differences in behavior are associated with changes in the level of brain enzymes, although not with changes in the properties of the enzymes $\text{per}$$\text{se}$.     

Functional efficiency of mitochondrial membrane of rats with hepatic chronic iron overload

The effect of $\text{in}\text{vivo}$ hepatic iron overload, induced by two different amounts of iron, on the energy-transducing efficiency of the mitochondrial membrane has been examined. It has been found that when the epatic iron concentration is up to a threshold value mitochondria present an anomalous membrane potential. Addition of oligomycin fully restitutes it. A low content of intramitochondrial K⁺ is connected with this pathological condition. A relative lack of antioxidant capability is parallely exhibited by these mitochondria. A possible involvement of lipid peroxidation process $\text{in}\text{vivo}$ in causing the membrane potential drop and the net efflux of intramitochondrial K⁺ is suggested.     

Specificity in the interaction of phospholipids and fatty acids with vesicle reconstituted cytochrome P-450. A spin label study

The association of fatty acids, androstane, phosphatidylcholine, phosphatidylethanolamine, and phosphatidic acid with purified and phospholipid-vesicle reconstituted cytochrome $\text{P-450}$ was studied by spin labeling. Spin-labeled fatty acids were found to be motionally restricted by cytochrome $\text{P-450}$ in both phospholipid vesicles and in microsomes to a much greater extent than spin-labeled phospholipids. The equilibrium of spin-labeled fatty acid between the bulk membrane lipid and the protein interface could be shifted towards an increased amount in the bulk phospholipid phase by the addition of oleic acid or lysophosphatidylcholine, but not by sodium cholate. Microsomes from different animals showed a variable extent of motional restriction of fatty acids, independent of pretreatment of the animals with phenobarbital or β-naphthoflavone, of cytochrome $\text{P-450}$ content, of the presence of type I and type II substrates for cytochrome $\text{P-450}$. These differences are attributed to the presence of varying amounts of lipid breakdown products in the microsomal membrane such as lysolipids or fatty acids which compete with the externally added spin-labeled fatty acids, or with spin-labeled androstane for the binding to cytochrome $\text{P-450}$. The negative charge of the fatty acid was found to be involved in its association with the protein. Cytochrome $\text{P-450}$ was shown to interact only with a few spin-labeled phospholipid molecules in such a way that the motional restriction of the spin acyl chains can be detected by electron paramagnetic resonance ($\text{τ}{}_{\mathrm{<mtext>R</mtext>}}\text{> 10}{}^{\mathrm{?8}}\text{s}$). The number of associated lipid molecules per protein probably is too small to form a complete shell around the protein. This lipid-protein interaction could be destroyed by the addition of sodium cholate, in contrast to the fatty acid-protein interaction.     

Myogenic defect in muscular dystrophy of the chicken.

Inherited muscular dystrophy of the chicken is thought to arise from abnormal development of trophic regulation of skeletal muscles by their innervating nerves. To determine whether expression of muscular dystrophy in the chicken is a property of the nerves or of the muscles, wing limb buds were transplanted between normal and dystrophic chick embryos at $\text{3}\text{1}\text{2}$ days of incubation (stage 19–20). Muscles of donor limbs innervated by nerves of the hosts were compared to contralateral unoperated host limb muscles in chicks from 6 to 25 weeks after hatching. Expression of normal or dystrophic phenotype was determined by examination of five different properties which are altered in dystrophic chick muscle: electromyographic evidence of myotonia; fiber diameter; acetylcholinesterase activity, localization, and isozymes; lactic dehydrogenase activity; and succinic dehydrogenase activity. Genetically normal muscle innervated by nerves of normal or dystrophic hosts was phenotypically normal while genetically dystrophic muscle innervated by normal nerves was phenotypically dystrophic. The results suggest that inherited muscular dystrophy of the chicken arises from a defect of muscle rather than from a lesion in the nerves themselves.     

Inhibition of glycolytic enzymes by endogenous aldehydes: a possible relation to diabetic neuropathies

Endogenous saturated and unsaturated aldehydes were found in significant elevations in serum of diabetic humans and rats. These compounds, originating from the lipid peroxidation processes, are shown here to be potent inhibitors of the glycolytic enzymes, phosphofructokinase and glyceraldehyde-3-phosphate dehydrogenase. The inhibition process is non-competitive and progressive. The aldehyde mixture, when supplemented to the standard rat diet at $\text{1}\text{100}$ ratio, caused nerve damage that is reminiscent of diabetic polyneuropathies.