Promotion of iron-induced rat liver microsomal lipid peroxidation by copper

Although copper has been demonstrated to promote lipid peroxidation in a number of systems, the mechanisms involved have not been fully defined. In this study, the role of copper in modifying lipid peroxidation has been explored in rat hepatic microsomes. In an in vitro system containing reduced glutathione (GSH, 200 μM) and Tris buffer, pH 7,4, cupric sulfate (1–50 μM) potentiated lipid peroxidation induced by ferrous sulfate (10 μM) but was unable to elicit peroxidation in the absence of iron. Higher levels of cupric sulfate (100 μM or greater) were inhibitory. The nature as well as the extent of the peroxidative response of microsomes to cupric sulfate were dependent on glutathione levels in addition to those of iron. Cupric sulfate (100 μM) strongly potentiated ferrous ion-induced lipid peroxidation in the presence of 400–800 μM GSH, while it inhibited peroxidation at lower levels of GSH (0–200 μM) and did not affect ferrous ion-induced peroxidation with glutathione levels of 3–10 mM. The potentiating effect of copper on ferrous ion-induced lipid peroxidation was further explored by investigating: (1) potential GSH-mediated reduction of cupric ions; (2) potential copper/GSH-mediated reduction of ferric ions (formed by oxidation during incubation); and (3) possible promotion of propagation reactions by copper/GSH. Our results indicate that cupric ions are reduced by GSH and thus are converted from an inhibitor to an enhancer of iron-induced lipid peroxidation. Cuprous ions appear to potentiate lipid peroxidation by reduction of ferric ions, rather than by promoting propagation reactions. Iron (in a specific Fe⁺²/Fe⁺³ ratio) is then an effective promoter of initiation reactions.     

Involvement of vitamin E and protein thiols in the inhibition of microsomal lipid peroxidation by glutathione

Iron-ascorbate stimulated lipid peroxidation in rat liver microsomes can be inhibited by glutathione (GSH). The role of protein thiols and vitamin E in this process was studied in liver microsomes isolated from rats fed diets either sufficient or deficient in vitamin E and incubated at 37°C unde 100% O₂. Lipid peroxidation was induced by adding 400 μM adenosine 5′-triphosphate, 2.5 to 20 μM FeCl₃, and 450 μM ascorbic acid. One mL of the incubation mixture was removed at defined intervals for the measurement of thiobarbituric acid reactive substances (TBARS), protein thiols and vitamin E. In vitamin E sufficient microsomes, the addition of GSH enhanced the lag time prior to the onset of maximal TBARS accumulation and inhibited the loss of vitamin E. Treatment of these microsomes with the protein thiol oxidant diamide resulted in a 56% loss of protein thiols, but did not significantly change vitamin E levels. However, diamide treatment abolished the GSH-mediated protection against TBARS formation and loss of vitamin E during ascorbate-induced peroxidation. Liver microsomes isolated from rats fed a vitamin E deficient diet contained 40-fold less vitamin E and generated levels of TBARS similar to vitamin E sufficient microsomes at a 4-fold lower concentration of iron. GSH did not affect the lag time prior to the onset of maximal TBARS formation in vitamin E deficient microsomes although total TBARS accumulation was inhibited. Similar to what was previously found in vitamin E sufficient microsomes [Palamanda and Kehrer, (1992)Arch. Biochem. Biophys. 293, 103–109], GSH prevented the loss of protein thiols in vitamin E deficient microsomes. However, GSH did not protect efficiently against the loss of residual vitamin E in deficient microsomes. These data provide support for the concept that GSH protects against microsomal lipid peroxidation by maintaining protein thiols, and consequently vitamin E, in the reduced state. The lack of protection in vitamin E deficient microsomes may be related to the inability of such low levels of vitamin E to inhibit peroxidation.     

Glutathione and antioxidants protect microsomes against lipid peroxidation and enzyme inactivation

The study investigated the relationship between lipid peroxidation and enzyme inactivation in rat hepatic microsomes and whether prior inactivation of aldehyde dehydrogenase (ALDH) exacerbated inactivation of other enzymes. In microsomes incubated with 2.5 μM iron as ferric sulfate and 50 μM ascorbate, ALDH, glucose-6-phosphate (G6Pase) and cytochrome P450 (Cyt-P450) levels decreased rapidly and concurrently with increased levels of thiobarbituric acid-reactive substances. Microsomal glutathioneS-transferase and nicotinamide adenine dinucleotide phosphate-cytochromec reductase were little affected during 1 hr of incubation. Addition of reduced glutathione partially protected, andN,N′-diphenyl-p-phenylenediamine and butylated hydroxytoluene completely protected microsomes against inactivation of ALDH, G6Pase and Cyt-P450, as well as lipid peroxidation induced by iron and ascorbate. ALDH was more susceptible than G6Pase to inactivation by iron and ascorbate, and was thus an excellent marker for oxidative stress. Inhibition of ALDH by cyanamide injection of rats exacerbated the inactivation of G6Pase in microsomes incubated with 0.1 mM, but not 25 μM 4-hydroxynonenal (4-HN). 4-HN did not stimulate lipid peroxidation. Thus, 4-HN may play a minor role in microsomal enzyme inactivation. In contrast, lipid, peroxyl radicals play an important role in microsomal enzyme inactivation, as evidenced by the prevention of both lipid peroxidation and enzyme inactivation by chain-breaking antioxidants.     

Reduced glutathione effects on α-tocopherol concentration of rat liver microsomes undergoing NADPH-dependent lipid peroxidation

Factors involved in reduced glutathione (GSH) and vitamin E-mediated inhibition of NADPH-dependent rat liver microsomal lipid peroxidation were examined. Lipid peroxidation was monitored over a time-course of 180 min by thiobarbituric acid reactive product formation. The addition of 5 mM GSH to the reaction system containing microsomes from rats fed a diet supplemented with 150 IU/kg of α-tocopherol acetate for eight weeks produced a lag in peroxidation of >30 min. This effect was not observed for microsomes prepared from rats fed a diet deficient in vitamin E. Indeed, a prooxidant effect of 5 mM GSH was observed in assays containing microsomes from rats fed a diet deficient in vitamin E. The inhibition by GSH of lipid peroxidation in microsomes prepared from livers of vitamin E supplemented rats was not restricted by its availability, for it was found that approximately 92% of the GSH remained in the reduced form after 60 min. Additional experiments revealed that the α-tocopherol content of peroxidizing microsomes decreased rapidly in the absence of GSH. The addition of 5 mM GSH to the assay system markedly depressed the loss of microsomal α-tocopherol. The results ofin vivo labeling of liver microsomes with [¹⁴C] α-tocopherol demonstrated that i) GSH addition to thein vitro peroxidizing medium reduced the disappearance of α-tocopherol, and ii) a compound that interfered with the determination of α-tocopherol was separated by HPLC and was not an oxidation product of α-tocopherol. A portion of the microsomal¹⁴C-labeled α-tocopherol was converted to an unidentified product with HPLC retention characteristics that was similar, but not identical, to α-tocopherol quinone.     

Microsomal lipid peroxidation: Effect of vitamin E and its functional interaction with phospholipid hydroperoxide glutathione peroxidase

The role of vitamin E in the protection against iron dependent lipid peroxidation was studied in rat liver microsomes and Triton-dispersed microsomal lipid micelles. In these systems, an antioxidant effect of vitamin E at a physiological ratio to phospholipids could be observed only in the presence of phospholipid hydroperoxide glutathione peroxidase (PHGPX) and glutathione. The rationale of this cooperation is discussed on the basis of the hydroperoxyl radical scavenging capacity of vitamin E and the reduction of membrane hydroperoxides by PHGPX. The scavenging of lipid hydroperoxyl radicals by vitamin E, although inhibiting propagation of the peroxidative chain, produces lipid hydroperoxides from which ferrous iron generates alkoxyl radicals that react with vitamin E almost as fast as with fatty acids. Therefore, only if membrane hydroperoxides are continuously reduced by this specific peroxidase does the scavenging of hydroperoxyl radicals by vitamin E lead to an effective inhibition of lipid peroxidation.     

The effect of glutathione on the vitamin E requirement for inhibition of liver microsomal lipid peroxidation

Vitamin E dependent inhibition of rat liver microsomal lipid peroxidation in an NADPH and ADP-Fe⁺³ containing system occurred at lower vitamin E concentrations in the presence of glutathione (GSH). Using microsomes from rats fed a vitamin E deficient diet, vitamin E was shown to be rquired for inhibition. Inhibition also required the presence of a storage labile microsomal component, since no inhibition was observed when using microsomes that had been stored for one moth. This observation provides evidence that direct reduction of reversibly oxidized vitamin E by GSH does not appear to contribute significantly to inhibition of peroxidation. During GSH and vitamin E dependent inhibition of lipid peroxidation, vitamin E (reduced form) concentrations remained constant, indicating that GSH maintained vitamin E concentrations. Without GSH, vitamin E concentrations dropped rapidly. By adding vitamin E to microsomes, it was found that inhibition of lipid peroxidation in the presence of GSH occurred at about five-fold less vitamin E than in the absence of GSH. Inhibition at these lower levels of vitamin E was 85–90% complete. Results indicate that GSH can be used to maintain vitamin E (reduced form) concentrations, thereby lowering the concentration of vitamin E necessary to inhibit microsomal lipid peroxidation.     

The effect of vitamin C on in vivo lipid peroxidation in guinea pigs as measured by pentane and ethane production

Measurements of pentane and ethane as indices of in vivo lipid peroxidation were made on samples of breath from vitamin C-sufficient and vitamin C-deficient guinea pigs injected with 23 μl carbon tetrachloride (CCl₄)/100 g body wt. Vitamin C-deficient animals produced significantly more pentane and ethane after CCl₄ treatment than did vitamin C-sufficient guinea pigs. Pretreatment of vitamin C-deficient animals with 75 mg ascorbic acid/100 g body wt significantly lowered both pentane and ethane evolution. Protection against in vivo lipid peroxidation similar to that provided by ascorbic acid was also found when vitamin C-deficient guinea pigs were pretreated with isoascorbic acid, reduced glutathione, α-tocopherol or β-carotene. When animals were pretreated with the radical scavenger mannitol, a protective effect was also observed as measured by pentane evolution.     

An enzymatic protective mechanism against lipid peroxidation damage to lungs of ozone-exposed rats

The effects of whole animal exposure to ozone and of dietary α-tocopherol on the occurrence in rat lung of lipid peroxidation and alteration of the activity of enzymes important in detoxification of lipid peroxides were studied. Exposure to 0.7 and 0.8 ppm ozone continuously for 5 and 7 days, respectively, significantly elevated the concentration of TBA reactants, primarily malonaldehyde, produced by lipid peroxidation, as well as the activities of glutathione (GSH) peroxidase, GSH reductase and glucose-6-phosphate (G-6-P) dehydrogenase. As a logarithmic function of dietary α-tocopherol (0, 10.5, 45, 150 and 1500 mg/kg), the increase in formation of malonaldehyde and the increase in activities of GSH peroxidase and G-6-P dehydrogenase were partially inhibited. The activity of GSH reductase was not affected by dietary α-tocopherol. The concentration of malonaldehyde and the activity of GSH peroxidase in lung were linearly correlated (p<0.001). This study confirmed the occurrence of lipid peroxidation in the lung during ozone exposure and revealed an enzymatic mechanism against damage. An apparent compensation mechanism is that with increased lipid peroxides there is increased activity of GSH peroxidase, which in turn increases lipid peroxide catabolism. The increased activities of GSH reductase and G-6-P dehydrogenase also function in the protective chain by providing increased levels of GSH and NADPH, respectively. Postdoctoral fellow of the American Society for Clinical Nutrition sponsored by the National Vitamin Foundation.     

GSH-dependent inhibition of lipid peroxidation: Properties of a potent cytosolic system which protects cell membranes

Properties of a heat labile, nondialyzable cytosolic factor which prevents lipid peroxidation in membranous organelles are described. The factor is present in liver and other animal tissues, and its capacity to inhibit lipid peroxidation in membranes subjected to oxidative stress is greatly potentiated by glutathione (GSH), although GSH by itself has no inhibitory effect on lipid peroxidation. The data obtained thus far indicate that one or more sulfhydryl groups associated with the factor is required for the inhibition. The mechanism by which lipid peroxidation is inhibited must involve prevention of initiation of peroxidation in the membranes, presumably by a process requiring one or more sulfhydryl groups associated with the heat labile factor. The latter appears to be protected by GSH while the factor is exerting its inhibitory effect on lipid peroxidation. The factor is not one of the known GSH-dependent enzymes, and appears to be a potent and ubiquitous system for stabilizing cell membranes against oxidative damage.     

Influence of vitamin E and nitrogen dioxide on lipid peroxidation in rat lung and liver microsomes

Rat lung and liver microsomes were used to examine the effects of dietary vitamin E deficiency on membrane lipid peroxidation. Microsomes from vitamin-E-deficient rats displayed increased lipid peroxidation in comparison to microsomes from vitamin-E-supplemented controls. The extent of lipid peroxidation, as determined by measurement of thiobarbituric acid reacting materials, was enhanced by addition of reduced iron and ascorbate (or NADPH). Rats fed a vitamin-E-supplemented diet and exposed to 3 ppm NO₂ for 7 days did not exhibit increases in microsomal lipid peroxidation compared to air-breathing controls. However, increases were found in microsomes prepared from rats fed a vitamin-E-deficient diet and exposed to NO₂. Lung microsomes from vitamin-E-fed rats contained almost 10 times as much vitamin E as liver microsomes when expressed in terms of polyunsaturated fatty acid content. The extent of lipid peroxidation was, in turn, considerably less in lung than in liver microsomes. Lipid peroxidation in lung microsomes from vitamin-E-deficient rats was comparable to liver microsomes from vitamin-E-supplemented rats as was the content of vitamin E in these respective microsomal samples. A combination of vitamin E deficiency and NO₂ exposure resulted in the greatest increases in lung and liver microsomal lipid peroxidation with the largest relative increases occurring in lung microsomes. An inverse relationship was found between the extent of lipid peroxidation and vitamin E content. Most of the peroxidation in lung microsomes appeared to proceed nonenzymatically whereas peroxidation in liver was largely enzymatic. Vitamin E appears to be assimilated by the lung during oxidant inhalation, but with dietary vitamin E deprivation, the margin for protection in lung may be less than in liver.     

Lipids and lipid antioxidant systems in developing eggs of salmon (Salmo salar)

Lipid class and fatty acid analyses were carried out on developing salmon eggs at four clearly defined pre-feeding stages, namely, fertilization, eyed egg stage (50 days), hatching (yolk sac fry, 98 days) and swim up fry (138 days). Measurements of components of the system considered to be involved in defense of cells against lipid peroxidation (glutathione peroxidase, EC 1.11.1.9, glutathione S-transferase, EC 2.5.1.18, reduced glutathione [GSH], α-tocopherol and ascorbic acid) were made at the same time. Levels of triacylglycerol decreased markedly during development, but there were few changes in fatty acid composition, indicating a non-selective utilization of fatty acids. Phosphatidylcholine was the dominant polar lipid (>94% by weight) in fertilized eggs. It was used preferentially during development so that in swim up fry the ratio phosphatidylcholine: phosphatidylethanolamine approached that found in fish muscle. Amounts of docosahexaenoic acid and arachidonic acid in polar lipids were significantly greater (p<0.01) in swim up fry than in fertilized eggs. Activities of the two enzymes were very low in the fertilized egg and remained low until hatching, when there was a concerted increase in their activity and in the concentration of GSH. Egg tocopherol concentrations decreased significantly during development, but whole body concentrations in swim up fry were not dissimilar from those in normal juvenile fish. Ascorbic acid, on the other hand, declined to very low levels in swim up fry; the restoration of this vitamin during first feeding seems vital to the well being of the fish.     

Decrease in platelet reduced glutathione increases lipoxygenase activity and decreases vitamin E

Unstimulated normal human blood platelets were treated with azodicarboxylic acidbis(dimethylamide) (diamide), a thiol-oxidizing agent. Oxygenated arachidonic acid (AA) metabolites, malondialdehyde (MDA), and tocopherols were then quantified by high-performance liquid chromatography (HPLC). Diamide treatment partially decreased the amount of reduced glutathione (GSH) content and induced a subsequent decrease in peroxidase activity. However, formation of 12-hydroxy-eicosatetraenoic acid (12-HETE), the end-product of lipoxygenation of AA, increased. Formation of MDA, a marker of overall lipid peroxidation, was also enhanced. Furthermore, platelet α-tocopherol, but not γ-tocopherol, significantly decreased. These results indicate that enhanced “basal” lipoxygenase activity, as a marker of specific AA oxygenation, may be linked to decreased platelet antioxidant status.     

Effect of dietary vitamin E and selenium on susceptibility of brain regions to lipid peroxidation

The effect of dietary vitamin E and/or selenium (Se) supplementation (200 IU and/or 0.2 ppm, respectively) or deficiency for two months on lipid peroxidation in cerebrum, cerebellum, mid-brain, and brain stem of one-month-old male F344 rats was investigated. Dietary treatment had a minimal effect on weight gain of rats for the period tested. Plasma α-tocopherol (α-T) concentration and glutathione peroxidase (GSH-Px) activity were reflective of dietary treatments. Supplementation of diets with vitamin E and/or Se increased plasma α-T and/or GSH-Px activity, while diets devoid of these nutrients reduced them significantly. Increased GSH-Px activity in Sesupplemented rats was further enhanced by vitamin E supplementation. Differential concentrations of α-T among brain regions were affected by dietary vitamin E but not by Se. In vitro lipid peroxidation of brain homogenates was inhibited by dietary vitamin E supplementation and increased by deficiency. Addition of 0.25 mM ascorbic acid or 0.1 mM of Fe²⁺ to brain homogenates markedly increased in vitro lipid peroxidation. Ascorbic acid-induced lipid peroxidation was inversely correlated with dietary vitamin E and Se in cerebrum. In vitro Fe²⁺-addition induced the greatest stimulation of lipid peroxidation, with cerebellum and brain stem of vitamin E-deficient rats showing the highest response to Fe²⁺ challenge. These findings indicate that concentrations of α-T among the brain regions are different and can be altered by dietary vitamin E treatments, cerebellum and brain stem are more susceptible to in vitro challenge by peroxidative agents than other regions, and the degree of lipid peroxidation of brain regions is partially affected by dietary vitamin E but not by Se in the levels tested.     

Inhibition of soybean lipoxygenase-1 by chain-breaking antioxidants

The aim of this investigation was to determine whether chain-breaking antioxidants able to prevent lipid peroxidation can inhibit lipoxygenase-1 (EC 1.13.11.12). Therefore, the effects of ascorbic acid, 6-palmitoylascorbic acid and trolox on the enzyme activity were analyzed by means of Lineweaver-Burk double reciprocal plots and Yoshino's graphical method. The effect of these compounds on the formation of free radicals during lipoxygenase-1 reaction was investigated as well, by monitoring the enzymic formation of oxodienes. We present evidence that the chain-breaking antioxidants ascorbic acid, 6-palmitoylascorbic acid and trolox inhibit soybean lipoxygenase-1 in the micromolar concentration range (Ki 27, 3 and 18 μM, respectively). The inhibition is competitive, complete and reversible. All three compounds trap the free radicals formed during the lipoxygenase-catalyzed reaction, which might substantially contribute to their inhibitory ability. These findings can have physiological significance in the light of the lipoxygenase involvement in biomembrane remodelling.     

Iodothyronines: Oxidative deiodination by hemoglobin and inhibition of lipid peroxidation

Purified rat hemoglobin catalyzes the oxidative degradation of iodothyronines to form iodide and an iodine-containing intermediate that reacts with protein. Hemoglobin also catalyzes peroxidation of linoleic acid. These observations are consistent with the reported intrinsic peroxidase activity of hemoglobin and other heme-proteins. However, incubations containing both linoleic acid and an iodothyronine produced a surprising result: deiodination was stimulated rather than competitively inhibited. In contrast, linoleic-acid peroxidation was inhibited by iodothyronines. Thus, low levels of iodothyronines (2.6×10⁻⁷M) are effective inhibitors of linoleic-acid peroxidation. Thyroxine and reverse T₃ were found to be more effective in this antioxidant activity than vitamin E, glutathione, ascorbic acid and DTT. Since linoleic-acid peroxidation proceeds by a propagating free-radical mechanism, we have concluded that iodothyronines can effectively terminate the free-radical chain reaction to become oxidatively deiodinated. Consistent with this antioxidant mechanism, reverse T₃ is effective in preserving red cell membranes as measured by the inhibition of erythrocyte hemolysis. The opinions or assertions contained here are the authors' and are not to be construed as official or as reflecting the views of the Department of Defense or the Uniformed Services University of the Health Sciences. The experiments reported here were conducted according to the principles in the “Guide for the Care and Use of Laboratory Animals,” Institute of Laboratory Animal Resources, National Research Council DHEW Pub. No. (NIH) 74-23. Supported by NIH Postdoctoral fellowship No. 5F32-AM-0610502.     

A microsomal membrane component associated with iron reduction in NADPH-supported lipid peroxidation

This study was conducted to determine whether a factor responsible for reduced nicotinamide adenine dinucleotide phosphate (NADPH)-supported lipid peroxidation in rat liver microsomes is involved in iron reduction by cooperation with NADPH-cytochrome P450 reductase. Under anaerobic conditions, NADPH-dependent reduction of ferric pyrophosphate in microsomes was not dependent on cytochrome P450 levels and was not inhibited by carbon monoxide (CO). All of the iron complexes with chelators such as adenosine 5′-diphosphate, pyrophosphate, nitrilotriacetate, oxalate or citrate were reduced in microsomes, although in the reconstituted system containing purified NADPH-cytochrome P450 reductase little or no iron reduction was found. A cytochrome P450-free fraction from a cholate-solubilized preparation of microsomes after passage through a laurate sepharose column was required for reduction of iron pyrophosphate in the reconstituted system leading to lipid peroxidation. The iron reduction was not inhibited by CO and was destroyed by heat treatment or trypsin digestion of the fraction. All iron complexes were reduced in the presence of the fraction, using a reducing equivalent of NADPHvia NADPH-cytochrome P450 reductase. The results indicate that a heat-labile component, which is probably a protein distinct from cytochrome P450, is associated with iron reduction responsible for lipid peroxidation in microsomes.     

Specific susceptibility of docosahexaenoic acid and eicosapentaenoic acid to peroxidation in aqueous solution

The peroxidation of different polyunsaturated fatty acids (PUFA) after photoirradiation in aqueous solution was evaluated by measuring fatty acid loss and malonaldehyde production in medium. The oxidation rates of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), two highly unsaturated fatty acids of the n−3 series, were surprisingly lower (14 and 22%, respectively) than the oxidation rates of linoleic, α-linolenic, γ-linolenic, dihomo γ-linolenic, and arachidonic acids (62–90%). The quantities of malonaldehyde (MA) produced were assayed simultaneously by gas chromatography (GC) and high performance liquid chromatography (HPLC). MA production was found to be related to both the degree of unsaturation and the metabolic series of the fatty acid. The maximum value was observed with arachidonic acid (MA production from 2 mM arachidonic acid in aqueous solution was estimated at 44.9±6.0 μM by GC and 46.8 ±4.0 μM by HPLC). Eicosapentaenoic acid and docosahexaenoic acid produced lower MA quantities compared to arachidonic acid (MA production from 2 mM EPA and 2 mM DHA was estimated at 17.9±1.5 μM and 37.9±0.7 μM, respectively, by GC, and 26.3±4.9 μM and 37.3±4.2 μM, respectively, by HPLC). The MA yield, defined as the amount of MA (nmols) produced per 100 nanomoles of oxidized fatty acid, was used to express the susceptibility of individual PUFA to peroxidation. The MA yield correlated well with the degree of unsaturation, but was independent of carbon chain length and metabolic series. The study suggests that adequate assessment of lipid peroxidation cannot be achieved by measuring MA formation alone, but it also requires knowledge of the fatty acid composition of the system studied.     

An esterification protocol for cis-parinaric acid-determined lipid peroxidation in immune cells1,2

Loss of fluorescence from cis-parinaric acid (cPnA) is a sensitive indicator of lipid peroxidation. The purpose of this study was to utilize cPnA to determine, at the level of the intact immune cell, whether enrichment of membranes with polyunsaturated fatty acids (PUFA) increased lipid peroxidation. P388D1 macrophages were labeled by addition of cPnA as an ethanolic solution. Within two minutes of addition, in the absence of serum, cPnA rapidly intercalated into the plasma membrane. Lipid peroxidation was initiated by addition of Fe²⁺-EDTA resulting in a dose-dependent decrease in fluorescence with increased oxidant concentration. Cells previously enriched with PUFA and labeled by intercalation showed no differences in spontaneous or Fe²⁺-induced lipid peroxidation. In separate experiments, 20 μM cPnA in ethanolic solution was injected into cell culture media containing 0.1% essentially fatty acid free bovine serum albumin (BSA). Cells were resuspended and incubated for 90 min at 37°C. After washing with BSA to remove cPnA which had not incorporated, 0.5% (0.1 μM) of the added cPnA was found esterified within cellular lipids. This level of cPnA provided a 100-fold increase over basal autofluorescence levels. Cells labeled in this manner also lost fluorescence in a dose-dependent manner as levels of oxidant stress increased. Cells enriched with PUFA and labeled by esterification had significantly increased rates and total amounts of lipid peroxidation. Co-incubation with α-tocopherol and PUFA resulted in a decrease in lipid peroxidation which was not significantly different from control cells. In conclusion, esterification of cPnA into membrane phospholipids can sensitively detect changes in lipid peroxidation induced by alteration of membrane PUFA and/or vitamin E content. Presented in part at the Experimental Biology Meetings, Anaheim, California, April 1994. Contribution from the Missouri Agriculture Extention Station, Journal #12,495.     

Influence of vitamin C on hydroxylation and side chain oxidation of cholesterol in vitro

The addition of ascorbic acid (20–160 μg) to mitochondrial preparations of rat or guinea pig liver has no effect upon the oxidation of [26-¹⁴C]-cholesterol to¹⁴CO₂. The 7α-hydroxylation of cholesterol by rat liver microsomes is also unaffected by addition of ascorbic acid. Hydroxylation by guinea pig liver microsomes is increased in the presence of ascorbic acid, but the results are not statistically significant.     

Oxidative catabolism of α-tocopherol in rat liver microsomes

The goal of this study was to clarify the mechanism responsible for the catabolism of α-tocopherol. The vitamin, bound to albumin, was incubated with rat liver microsomes and appeared to be broken down. Optimal production of the metabolite was obtained when 1 mg of microsomal protein was incubated with 36 μM of α-tocopherol in the presence of 1.5 mM of NADPH. Chromatographic and mass spectrometric analyses of the metabolite led to the conclusion that it consists of an ω-acid with an opened chroman ring, although we could not perform nuclear magnetic resonance analysis to confirm this. Our data show that α-tocopherol is ω-oxidized to a carboxylic acid and that this process can occur in rat liver microsomes in the presence of NADPH and O₂. The oxidation to the quinone structure appears to be a subsequent event that may be artifactual and/or catalyzed by a microsomal enzyme(s).