Vitamin A supplementation inhibits chemiluminescence and lipid peroxidation in isolated rat liver microsomes and mitochondria

In the present study we investigated if administration of vitamin A could protect rat liver microsomes and mitochondria from in vitro peroxidation. Appreciable decrease of chemiluminescence and lipid peroxidation was measured in microsomal membranes from rats receiving vitamin A, with respect to control animals. In membranes derived from control animals, the fatty acid composition was profoundly modified when subjected to in vitro peroxidation mediated by ascorbate-Fe⁺⁺, with a considerable decrease of 20:4 n6 and 22:6 n3 in mitochondria and 18:2 n6 and 20:4 n6 in microsomes. As a consequence the peroxidizability index, a parameter based on the maximal rate of oxidation of specific fatty acids was higher in supplemented animals than in control group when both kind of membranes were analyzed. These changes were less pronounced in membranes derived from rats receiving vitamin A. These results are in agreement with previous results that indicated that vitamin A may act as an antioxidant protecting membranes from deleterious effects.Abbreviations BHT butylated hydroxytoluene - BSA bovine serum albumin - CL chemiluminescence - PI peroxidizability index Member of Carrera del Investigador Científico, Consejo Nacional de Investigaciones Cientificas y Técnicas de la Republica Argentina     

Non-enzymatic lipid peroxidation of microsomes and mitochondria from liver, heart and brain of the bird Lonchura striata: relationship with fatty acid composition

The aim of this study was to examine the fatty acid composition and non-enzymatic lipid peroxidation (LP) of mitochondria and microsomes obtained from liver, heart and brain of Lonchura striata. The percentage of total unsaturated fatty acid was approximately 30-60% in the organelles from all tissues studied. Brain mitochondria and both organelles of liver exhibited the highest percentage of polyunsaturated fatty acid (PUFA) (30 and 18%, respectively). The arachidonic acid (AA) content was 7% in mitochondria of liver and brain and 3% in heart mitochondria. The percentage of docosahexanoic acid (DHA) was 8% in brain mitochondria and approximately 2-3% in heart and liver mitochondria. The peroxidizability index (PI) of brain mitochondria and both organelles from liver was higher than that of organelles from heart and brain microsomes. Liver organelles and brain mitochondria were affected by LP, as indicated by the increase in chemiluminescence and a decrease of AA and DHA. These changes were not observed during LP of brain microsomes and both organelles from heart. These results indicate: 1) PI positively correlates with PUFA percentage and LP; 2) The resistance to LP detected in heart organelles would contribute to the cardiac protection against oxidative damage.     

Dehydroepiandrosterone-induced lipid peroxidation in rat liver mitochondria

Administration of dehydroepiandrosterone (DHEA), a steroid hormone of the adrenal cortex which acts as a peroxisome proliferator and hepatocarcinogen in the rat, caused an increase in NADPH-dependent lipid peroxidation in mitochondria isolated from the liver, kidney and heart, but not from the brain. The effect of DHEA on rat liver mitochondrial lipid peroxidation became discernible after feeding steroid-containing diet (0.6% w/w) for 3 days, and reached maximal levels between 1 and 2 weeks. DHEA in the concentration range 0.001–0.02% did not significantly increase lipid peroxidation compared to the control. Lipid peroxidation was significantly enhanced in animals given a diet containing ≥ 0.05% DHEA. The addition of DHEA in the concentration range 0.1–100 μM to mitochondria isolated from control rats had no effect on lipid peroxidation. It seems, therefore, that the steroid effect is mediated by an intracellular process. Our data indicate that induction of mitochondrial membrane lipid peroxidation is an early effect of DHEA administration at pharmacological doses.     

Fatty acid profiles and lipid peroxidation of microsomes and mitochondria from liver, heart and brain of Cairina moschata

Studies were done to analyze the fatty acid composition and sensitivity to lipid peroxidation (LP) of mitochondria and microsomes from duck liver, heart and brain. The fatty acid composition of mitochondria and microsomes was tissue-dependent. In particular, arachidonic acid comprised 17.39+/-2.32, 11.75+/-3.25 and 9.70+/-0.40% of the total fatty acids in heart, liver and brain mitochondria respectively but only 13.39+/-1.31, 8.22+/-2.43 and 6.44+/-0.22% of the total fatty acids in heart, liver and brain microsomes, respectively. Docosahexahenoic acid comprised 17.02+/-0.78, 4.47+/-1.02 and 0.89+/-0.07% of the total fatty acids in brain, liver and heart mitochondria respectively but only 7.76+/-0.53, 3.27+/-0.73 and 1.97+/-0.38% of the total fatty acids in brain, liver and heart microsomes. Incubation of organelles with ascorbate-Fe(2+) at 37 degrees C caused a stimulation of LP as indicated by the increase in light emission: chemiluminescence (CL) and the decrease of arachidonic acid to: 5.17+/-1.34, 8.86+/-0.71 and 5.86+/-0.68% of the total fatty acids in heart, liver and brain mitochondria, respectively, and to 4.10+/-0.61 in liver microsomes. After LP docosahexahenoic acid decrease to 7.29+/-1.47, 1.36+/-0.18 and 0.30+/-0.11% of the total fatty acids in brain, liver and heart mitochondria. Statistically significant differences in the percent of both peroxidable fatty acids (arachidonic and docosahexaenoic acid) were not observed in heart and brain microsomes and this was coincident with absence of stimulation of LP. The results indicate a close relationship between tissue sensitivity to LP in vitro and long chain polyunsaturated fatty acid concentration. Nevertheless, any oxidative stress in vitro caused by ascorbate-Fe(2+) at 37 degrees C seems to avoid degradation of arachidonic and docosahexaenoic acids in duck liver and brain microsomes. It is possible that because of the important physiological functions of arachidonic and docosahexaenoic acids in these tissues, they are protected to maintain membrane content during oxidative stress.     

The effect of alpha-tocopherol on lipid peroxidation of microsomes and mitochondria from rat testis

The testis is a remarkably active metabolic organ; hence it is suitable not only for studies of lipid metabolism in the organ itself but also for the study of lipid peroxidation processes in general. The content of fatty acids in testis is high with a prevalence of polyunsaturated fatty acids (PUFA) which renders this tissue very susceptible to lipid peroxidation. Studies were carried out to evaluate the effect of alpha-tocopherol in vitro on ascorbate-Fe(++) lipid peroxidation of rat testis microsomes and mitochondria. Chemiluminescence and fatty acid composition were used as an index of the oxidative destruction of lipids. Special attention was paid to the changes produced on the highly PUFA [C20:4 n6] and [C22:5 n6]. Lipid peroxidation of testis microsomes or mitochondria induced a significant decrease of both fatty acids. Total chemiluminescence was similar in both kinds of organelles when the peroxidized without (control) and with ascorbate-Fe(++) (peroxidized) groups were compared. Arachidonic acid was protected more efficiently than docosapentaenoic acid at all alpha-tocopherol concentrations tested when rat testis microsomes or mitochondria were incubated with ascorbate-Fe(++). The maximal percentage of inhibition in both organelles was approximately 70%; corresponding to an alpha-tocopherol concentration between 1 and 0.25 mM. IC50 values from the inhibition of alpha-tocopherol on the chemiluminescence were higher in microsomes (0.144 mM) than mitochondria (0.078 mM). The protective effect observed by alpha-tocopherol in rat testis mitochondria was higher compared with microsomes, associated with the higher amount of [C20:4 n6]+[C22:5 n6] in microsomes that in mitochondria. It is proposed that the vulnerability to lipid peroxidation of rat testis microsomes and mitochondria is different because of the different proportion of PUFA in these organelles The peroxidizability index (PI) was positively correlated with the level of long chain fatty acids. The results demonstrated the protective effect of alpha-tocopherol on lipid peroxidation in microsomes and mitochondria from rat testis.     

The effect of alpha-tocopherol on the lipid peroxidation of mitochondria and microsomes obtained from rat liver and testis.

The effect of intraperitoneal administration of alpha-tocopherol (100 mg/kg wt/24 h) on ascorbate (0.4 mM) induced lipid peroxidation of mitochondria and microsomes isolated from rat liver and testis was studied. Special attention was paid to the changes produced on the highly polyunsaturated fatty acids C20:4 n6 and C22:6 n3 in liver and C20:4 n6 and C22:5 n6 in testis. The lipid peroxidation of liver mitochondria or microsomes produced a significant decrease of C20:4 n6 and C22:6 n3 in the control group, whereas changes in the fatty acid composition of the alpha-tocopherol treated group were not observed. The light emission was significantly higher in the control than in the alpha-tocopherol treated group. The lipid peroxidation of testis microsomes isolated from the alpha-tocopherol group produced a significant decrease of C20:4 n6 , C22:5 n6 and C22:6 n3, these changes were not observed in testis mitochondria. The light emission of both groups was similar. The treatment with alpha-tocopherol at the dose and times indicated showed a protector effect on the polyunsaturated fatty acids of liver mitochondria, microsomes and testis mitochondria, whereas those fatty acids situated in testis microsomes were not protected during non enzymatic ascorbate-Fe2+ lipid peroxidation. The protector effect observed by alpha-tocopherol treatment in the fatty acid composition of rat testis mitochondria but not in microsomes could be explained if we consider that the sum of C20:4 n6 + C22:5 n6 in testis microsomes is 2-fold than that present in mitochondria.     

Mechanism of citrinin-induced dysfunction of mitochondria. VI. Effect on iron-induced lipid peroxidation of rat liver mitochondria and microsomes

The inhibition by citrinin (CTN) of lipid peroxidation of mitochondria, sub-mitochondrial particles (SMP) and microsomes was studied. This effect was reversed by the presence of high concentrations of Fe³⁺ (0·4 and 0·5 mM ), suggesting chelation of the mycotoxin with iron or interference in the reduction of Fe³⁺. © 1998 John Wiley & Sons, Ltd.     

Effect of thiols on lipid peroxidation in rat liver microsomes

The stimulatory or inhibitory effects of various thiol compounds on in vitro lipid peroxidation by iron-ascorbate in rat liver microsomes were determined. Glutathione had no measurable pro-oxidant capacity, in contrast, it protected against lipid peroxidation. N-Acetyl l-cysteine and S-methyl-glutathione had no effect on in vitro lipid peroxidation. l-Cysteine stimulated lipid peroxidation and also of d-penicillamine and dl-dithiothreitol the pre-oxidant capacity predominated the anti-oxidant capacity. Cysteamine afforded a pronounced protection against in vitro lipid peroxidation. In contrast to the labile character of the glutathione dependent protection, the protection by cysteamine was not affected by heat-pretreatment of the liver microsomes or alkylating protein sulfhydryl groups by N-ethyl maleimide. Again in contrast to glutathione, the protection against in vitro microsomal lipid peroxidation by cysteamine was not reduced after in vivo lipid peroxidation induced by CC14. This suggests that even after the process of lipid peroxidation has been started, administration of cysteamine might still be beneficial.     

NADPH-dependent inhibition of lipid peroxidation in rat liver microsomes.

Microsomal NADPH-driven electron transport is known to initiate lipid peroxidation by activating oxygen in the presence of iron. This pro-oxidant effect can mask an antioxidant function of NADPH-driven electron transport in microsomes via vitamin E recycling from its phenoxyl radicals formed in the course of peroxidation. To test this hypothesis we studied the effects of NADPH on the endogenous vitamin E content and lipid peroxidation induced in liver microsomes by an oxidation system independent of iron: an azo-initiator of peroxyl radicals, 2,2'-azobis (2,4-dimethylvaleronitrile), (AMVN), in the presence of an iron chelator deferoxamine. We found that under conditions NADPH: (i) inhibited lipid peroxidation; (ii) this inhibitory effect was less pronounced in microsomes from vitamin E-deficient rats than in microsomes from normal rats; (iii) protected vitamin E from oxidative destruction; (iv) reduced chromanoxyl radicals of vitamin E homologue with a 6-carbon side-chain, chromanol-alpha-C-6. Thus NADPH-driven electron transport may function both to initiate and/or inhibit lipid peroxidation in microsomes depending on the availability of transition metal catalysts.     

Senescence-associated decrease of NADPH-induced lipid peroxidation in rat liver microsomes

Senescence is associated with decrease in the NADPH-induced lipid peroxidation in liver homogenate as well as rough and smooth microsomes of female rats. In the microsomal fractions, sensitivity to NADPH-induced lipid peroxidation is high in young adults (3-month-old), decreases in middle aged (12-month-old) and reaches lowest levels in senescent (30-month-old) rats. Increasing the concentration of co-factors or time of incubation does not alter this resistance observed in the senescent rats. Major factors responsible for this resistance in senescent rats seem to be low levels of substrate in the c reductase, cytochrome P-450 and high cholesterol:phospholipid ratios besides enhanced levels of superoxide dismutase, alpha-tocopherol and reduced glutathione.     

Dihydroxyfumaric acid induced lipid peroxidation in rat liver microsomes.

Dihydroxyfumaric acid induced lipid peroxidation in rat liver microsomes. This reaction was heat-insensitive contrary to the mitochondrial peroxidation reported in the previous paper, and was enhanced by p-chloromercuribenzoate. Additions of Fe²⁺ and Fe³⁺ stimulated both the lipid peroxidation and the disappearance of dihydroxyfumaric acid. On the other hand, addition of Mn²⁺ or Cu²⁺, which stimulated the disappearance of dihydroxyfumaric acid, inhibited the lipid peroxidation. Hydroxyl radical scavengers, superoxide dismutase and catalase had no effect on this lipid peroxidation and dihydroxyfumaric acid disappearance. The cytochrome p-450 content decreased about 70 % in parallel with the lipid peroxidation.     

Iron content and degree of lipid peroxidation in liver mitochondria isolated from iron-loaded rats

1.The content of non-heme iron and the degree of lipid peroxidation were measured in liver mitochondria isolated from rats injected with either Jectofer (an iron-sorbitol-citric acid complex) or iron-nitrilotriacetate. 2. The sedimentation profiles of the mitochondria from controls and iron-treated rats as revealed by analytical differential centrifugation, indicated single population of mitochondria with $\text{s}{}_{\mathrm{4,B}}$ values of 13200± 560 S and 14200±590 S for controls and iron-loaded animals, respectively. In contrast, the sedimentation profiles of the acid phosphatase activity and the non-heme iron revealed marked polydispersities with at least three populations of particles for both controls and iron-loaded animals. 3. The mitochondria and iron-rich lysosomes were separated by density-gradient centrifugation in an isotonic medium of Percoll and sucrose. With this technique, the amount of non-heme iron in a mitochondrial fraction by differential centrifugation decreased from 69±28 nmol/mg protein to 5.6±1.1 nmol/mg protein and from 19.3±5.6 nmol/mg protein to 3.3±0.6 nmol/mg protein for Jectofer and iron-nitrilotriacetate injected rats, respectively. For control rats the amount of mitochondrial non-heme iron was about 2.7 nmol/mg protein both before and following density gradient centrifugation. The extra amount of non-heme iron still present in the purified mitochondrial fraction from iron-loaded rats, as compared to controls, was further characterized by the reactivity towards bathophenanthroline sulfonate. The results suggest that the extra iron was due to a small amount of either ferritin or hemosiderin still contaminaning the mitochondrial fraction. The amount of mitochondrial heme iron was the same in iron-loaded rats and controls. 4. The degree of lipid peroxidation in the mitochondria was estimated from the amount of malondialdehyde. The thiobarbituric acid method used for the quantitation of malondialdehyde was modified so that it was insensitive to variable amounts of iron present in the samples. No difference in the degree of lipid peroxidation was observed between the mitochondria from iron-loaded rats and controls. 5. In contrast to recent proposals (Hanstein, E.G. et al. (1981) Biochim. Biophys. Acta 678, 293–299), the present study showed that the amounts of non-heme iron and the degrees of lipid peroxidation are the same in mitochondria isolated from iron-loaded and control animals.     

NADPH-dependen lipid peroxidation catalyzed by purified NADPH-cytochrome C reductase from rat liver microsomes

A purified preparation of rat liver microsomal NADPH-cytochrome c reductase has been shown to catalyze the NADPH-dependent peroxidation of isolated microsomal lipid. In addition to ADP and ferric ion required for NADPH-dependent lipid peroxidation in whole microsomes, this system requires high ionic strength and a critical concentration of EDTA. The peroxidation activity can be inhibited by superoxide dismutase suggesting that the superoxide anion, produced by this flavoprotein, is involved in the lipid peroxidation reaction.     

Antioxidant effect of conjugated linoleic acid and vitamin A during non enzymatic lipid peroxidation of rat liver microsomes and mitochondria

In the study reported here the effect of conjugated linoleic acid (CLA) and vitamin A on the polyunsaturated fatty acid composition, chemiluminescence and peroxidizability index of microsomes and mitochondria isolated from rat liver was analyzed. The effect of CLA on the polyunsaturated fatty acid composition of native microsomes was evidenced by an statistically significant p < 0.007 decrease of linoleic acid C18:2 n6, whereas in mitochondria it was observed a decrease p < 0.0001 of arachidonic acid C20:4 n6 when compared with vitamin A and control groups. Docosahexaenoic acid C22:6 n3 in mitochondria was reduced p < 0.04 in CLA and vitamin A groups when compared with control. After incubation of microsomes or mitochondria in an ascorbate (0.4 mM)-Fe⁺⁺ (2.15 M) system (120 min at 37°C) it was observed that the total cpm/mg protein originated from light emission: chemiluminescence was lower in liver microsomes or mitochondria obtained from CLA group (received orally: 12.5 mg/daily during 10 days) than in the vitamin A group (received intraperitoneal injection: daily 0.195 g/kg during 10 days). CLA reduced significantly maximal induced chemiluminescence in microsomes relative to vitamin A and control groups, whereas in mitochondria the effect was observed relative to control group The polyunsaturated fatty acid composition of liver microsomes or mitochondria changed by CLA and vitamin A treatment. The polyunsaturated fatty acids mainly affected when microsomes native and peroxidized from control group were compared were linoleic, linolenic and arachidonic acids, while in vitamin A group linoleic and arachidonic acid were mainly peroxidized, whereas in CLA group only arachidonic acid was altered. In mitochondria obtained from the three groups arachidonic acid and docosahexaenoic acid showed a significant decrease when native and peroxidized groups were compared. As a consequence the peroxidizability index, a parameter based on the maximal rate of oxidation of fatty acids, show significant changes in the CLA group compare vitamin A and control groups. The simultaneous analysis of peroxidizability index, chemiluminescence and fatty acid composition demonstrated that CLA is more effective than vitamin A protecting microsomes or mitochondria from peroxidative damage.     

Enhancement of hydroperoxide-dependent lipid peroxidation in rat liver microsomes by ascorbic acid

Simultaneous addition of ascorbic acid and organic hydroperoxides to rat liver microsomes resulted in enhanced lipid peroxidation (approximately threefold) relative to incubation of organic hydroperoxides with microsomes alone. No lipid peroxidation was evident in incubations of ascorbate alone with microsomes. The stimulatory effect of ascorbate on linoleic acid hydroperoxide (LAHP)-dependent peroxidation was evident at all times whereas stimulation of cumene hydroperoxide (CHP)-dependent peroxidation occurred after a lag phase of up to 20 min. EDTA did not inhibit CHP-dependent lipid peroxidation but completely abolished ascorbate enhancement of lipid peroxidation. Likewise, EDTA did not significantly inhibit peroxidation by LAHP but dramatically reduced ascorbate enhancement of lipid peroxidation. The results reveal a synergistic prooxidant effect of ascorbic acid on hydroperoxide-dependent lipid peroxidation. The inhibitory effect of EDTA on enhanced peroxidation suggests a possible role for endogenous metals mobilized by hydroperoxide-dependent oxidations of microsomal components.     

Lipid peroxidation in mitochondria and microsomes from adult and fetal rat tissues

Pregnant female Wistar rats that received a control (100 ppm Zn) or a Zn-deficient diet (1.5 ppm Zn) from d 0 to 21, or nonpregnant normally fed female rats without or with five daily oral doses of 300 mg/kg salicylic acid were used for the experiments. In isolated mitochondria or microsomes from various maternal and fetal tissues, lipid peroxidation was determined as malondialdehyde formation measured by means of the thiobarbiturate method. Zn deficiency increased lipid peroxidation in mitochondria and microsomes from maternal and fetal liver, maternal kidney, maternal lung microsomes, and fetal lung mitochondria. Lipid peroxidation in fetal microsomes was very low. Zn deficiency produced a further reduction of lipid peroxidation in fetal liver microsomes. Salicylate increased lipid peroxidation in liver mitochondria and microsomes after addition in vitro and after application in vivo. The increase of lipid peroxidation by salicylate may be caused by two mechanisms: an increased cellular Fe uptake that, in turn, can increase lipid peroxidation and chelating Fe, in analogy to the effect of ADP in lipid peroxidation. The latter effect of salicylate is particularly expressed at increased Fe content.     

Stimulation of lipid peroxidation in rat liver microsomes by tetraphenylporphine and its metallocomplexes

It is shown that tetraphenylporphyrin (TPP) and its complexes with metals decrease the rate of the diene conjugate formation. The above compounds increase the malonic dialdehyde accumulation. The effect of TPP and its complexes with metals is connected with stimulation of lipid peroxidation in biomembranes.