An in vitro ESR study of uncatalyzed rat liver protein-catalyzed spin-labeled phosphatidylcholine exchange.

ESR spectrometry has been used to study fatty acid spin-labeled phosphatidylcholine exchange from single bilayer donor vesicles to various acceptor systems, such as intact or differently treated mitochondria, phospholipid multilamellar vesicles or single bilayer vesicles. This exchange is catalyzed by soluble non-specific rat liver protein, first investigated by Bloj and Zilversmit in 1977 (J. Biol. Chem. 252, 1613--1619). Non-catalyzed phosphatidylcholine exchange has also been studied. Full inhibition of both mechanisms occurs with lipid-depleted acceptor mitochondria, while N-ethylmaleimide-treated mitochondria behave as good acceptors during catalyzed exchange but are in no way effective during spontaneous exchange. Non-catalyzed exchange does not take place with phospholipase D-treated mitochondria as acceptors, while the pure catalyzed mechanism is inhibited by 28%. Neither multilamellar nor single bilayer phospholipid vesicles exchange spin-labeled phosphatidylcholine in the absence of protein, the former being a poorer acceptor system than the latter during catalyzed exchange, when this activity is 31 and 80%, respectively, of that of intact mitochondria. The hypothesis is made that the spontaneous mechanism is active among intact natural membranes and could be of some importance in vivo. Furthermore, the biomembrane protein moiety is assumed to be involved in the catalyzed exchange more as a phospholipid spacer than as a binder between the exchange protein and the membrane involved. Phospholipids, on the contrary, appear to be important for both functions.

Effect of hyperthyroidism on lipogenesis in brown adipose tissue of young rats.

Fatty acid synthetic capacity, investigated both in subcellular fractions and in vivo, is very active in brown adipose tissue of room temperature-acclimated rats. In hyperthyroid animals this tissue, analogously to the liver, exhibits an increased activity of acetyl-CoA carboxylase, fatty acid synthetase and microsomal fatty acid chain elongation, this last mechanism remaining unaffected in mitochondria. An enhancement of reducing capacities of a group of cytoplasmic NADP-dependent enzymes has also been observed in brown adipose tissue of hyperthyroid rats, probably due to a greater use of NADPH in lipogenesis under these conditions. An increase in palmitate oxidation and in polyenoic fatty acids was observed in mitochondria of brown adipose tissue from hyperthyroid animals. The latter increase is related to the importance of these compounds in the regulation of membrane fluidity and probably to an increased resistance to cold in the hyperthyroid state.

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.

A possible mechanism for initiation of lipid peroxidation by ascorbate in rat liver microsomes.

The mechanism by which lipid peroxidation progresses has been known for years, but there is disagreement regarding the mode of its initiation. The aim of this study was to examine: (a) the role of endogenous iron in the initiation of ascorbate-induced lipid peroxidation in microsomal and liposomal membranes; (b) the role of oxygen-free radicals in this process; and (c) the redox state of ascorbate during the course of lipid peroxidation. Ascorbate-induced lipid peroxidation was assessed by measuring hydroperoxide and thiobarbituric acid reactive substances (TBARS) formation in membranes after incubation in Tris-HCl buffer (pH 7.4) for 15 min. To confirm the role of endogenous iron and oxygen-free radicals, the effect of iron chelating agents (EDTA and thiourea) and radical scavengers (benzoate, mannitol, catalase and SOD) on lipid peroxidation was examined. Spectrophotometric measurements and ESR spectra have made it possible to determine ascorbate concentration and its redox state. Ascorbate promoted lipid peroxidation in both rat liver microsomes and liposomes without addition of exogenous iron. Iron chelating agents such as EDTA and thiourea inhibited lipid peroxidation, while SOD, catalase, mannitol and benzoate had no effect. The addition of 5 microM Fe2+ (or Fe3+) to the incubation mixture did not significantly alter hydroperoxide production, but that of TBARS was increased. Lipid peroxidation significantly altered the fatty acid profile in microsomes and liposomes, the most affected being the C20:4 and C22:6 species. Ascorbate in Tris-HCl buffer (pH 7.4) autoxidized very slowly. Its oxidation was catalyzed by Fe3+ ions at a rate determined by incubation time and iron concentration. In contrast, no ascorbate oxidation occurred in the presence of microsomes when lipid peroxidation was proceeding at a maximal rate. Under these conditions a typical ascorbyl radical ESR spectrum signal greater than that arising from ascorbate alone was obtained and the magnitude of this signal was unchanged by variations of microsome or ascorbate concentrations. A ferrous ion ascorbyl radical complex was responsible for this signal. These results suggest that an ascorbate-microsomal iron complex is responsible for the initiation of lipid peroxidation, and that during this process ascorbate remains in its reduced form.

Turnover of fatty acids in rat liver cardiolipin: comparison with other mitochondrial phospholipids.

Following intraperitoneal administration of 1-14C-linoleic acid or 2-3H-acetate to rats, the specific radioactivities of both liver cardiolipin and other mitochondrial phospholipids after different time intervals were measured. Comparison of the data obtained with those from another stock of rats treated with 32P-phosphate or 2-3H-glycerol showed that the fatty acids of cardiolipin, like those of other phospholipids, exhibit an independent turnover with respect to the remaining parts of the molecule. The half-life of acyl moieties of cardiolipin is ca. 20% higher than that of the same components of other mitochondrial phospholipids. Moreover, it appears that, in both cardiolipin and other phospholipids, linoleyl residues turn over faster than nonessential fatty acids. Discussion is made as to whether this characteristic can be related to the role of phospholipids in the functioning of some enzymes bound to the inner mitochondrial membrane.

Lipid composition of liver mitochondria and microsomes in hyperthyroid rats.

Triiodothyronine-induced alteration of the lipid pattern in rat-liver mitochondria and microsomes has been investigated. In mitochondria, a 25% total cholesterol decrease and a 14% phospholipid increase have been detected. In these hyperthyroid rat liver organelles, a strong decrease in the total cholesterol/phospholipid molar ratio occurs. On the contrary, in microsomes from the same animals, a decrease of about 23% has been measured for both total cholesterol and phospholipids; hence, in this fraction, the total cholesterol/phospholipid molar ratio is unaffected by hyperthyroidism. The liver mitochondrial phospholipid composition, unlike the microsomal composition, is altered significantly in hyperthyroid rats; a 7.4% phosphatidylcholine decrease is accompanied by a similar additive percentage increase of both phosphatidylethanolamine and cardiolipin. In regard to total phospholipid fatty acid composition in liver microsomes from hyperthyroid rats, no variation has been observed compared with the control rats, whereas in mitochondria from the same animals, a meaningful linoleic acid decrease with a similar arachidonic acid increase has been found. In addition to fatty acid alteration, the separated mitochondrial phospholipid classes also exhibit some increase in stearic acid. Among phospholipids, cardiolipin changes the most of the esterified fatty acids in hyperthyroid rat liver. In this compound, a strong increase in the percentage of both palmitic and stearic acid and a 32.4% decrease of linoleic acid have been found.

Effect of N-ethylmaleimide on rat liver phosphatidylcholine-specific and non-specific transfer protein activities: its dependence on donor liposome composition.

Phosphatidylcholine exchange between liposomes and mitochondria catalyzed by rat liver phosphatidylcholine transfer protein is strongly stimulated by N-ethylmaleimide (NEM) when PC/PI (molar ratio, 4:1) donor liposomes are used. In the presence of PC/PE or PC liposomes the exchange activity by this protein is unaffected. In the same experimental conditions, the activity of rat liver non-specific transfer protein is always stimulated by N-ethylmaleimide with all the types of liposomes tested in the order PC/PI greater than PC/PE greater than PC. Since the effect of NEM depends on the type of liposomes used and appears to be similar for both phospholipid transfer proteins, the possibility that their mode of action implies the formation of a ternary complex should be considered. As far as non-specific transfer protein is concerned, its interaction could vary depending on the nature of the exchanging membranes. Data are also presented indicating that when the two transfer proteins are together their activity is additive, therefore suggesting a specific role in phospholipid biomembrane assembly for each of them.

Changes in liver enzyme activity in the teleost Sparus aurata in response to cadmium intoxication.

Enzyme activity modulation by cadmium in the liver of the teleost fish Sparus aurata was investigated in vivo following 3 and 6 days of CdCl2 administration (2.5 mg/kg body wt). The specific activities of the mitochondrial enzymes NAD-isocitrate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase were stimulated by approximately 20% after 3 days administration and were further increased (by about 40%) after 6 days treatment. In comparison with these enzymes, the activities of glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) in mitochondria were less stimulated after the two indicated intervals of treatment. Cadmium significantly reduced the activities of liver cytoplasmic GOT and GPT while a simultaneous increase occurred in the serum activities of these same enzymes. The activity of liver NADPH-cytochrome P450 reductase was stimulated by 25 and 40% after 3 and 6 days cadmium intoxication, respectively. Lastly, the antioxidant enzymes glutathione peroxidase and glutathione reductase in liver and catalase in both liver and blood were strongly reduced after 3 and 6 days cadmium administration. These data suggest that cadmium in fish hepatocytes alters cell membrane structure and concomitantly induces some perturbation in the integrity of the mitochondrial membrane.

Ketone-body metabolism in hyperthyroid rats: reduced activity of D-3-hydroxybutyrate dehydrogenase in both liver and heart and of succinyl-coenzyme A: 3-oxoacid coenzyme A-transferase in heart.

The specific activity of D-3-hydroxybutyrate dehydrogenase is reduced by about a third in liver and heart mitochondria of hyperthyroid rats. State 3 respiration is also reduced in isolated mitochondria from the same animals when DL-3-hydroxybutyrate is the substrate. Determination of the kinetic parameters of the membrane-bound D-3-hydroxybutyrate dehydrogenase in liver of hyperthyroid rats reveals a decreased in maximal velocity (Vmax). The Michaelis and dissociation constants of NAD+ and D-3-hydroxybutyrate are also significantly influenced, thus indicating that both the affinity and the binding of this enzyme toward its substrates are affected. In hyperthyroid rats a significant ketone-body increase is found in both liver and heart: in blood, an almost doubled concentration can be measured. At the same time, in heart mitochondria of these animals the activity of succinyl-coenzyme A: 3-oxoacid coenzyme A-transferase is significantly reduced. The decrease in both D-3-hydroxybutyrate dehydrogenase and 3-oxoacid coenzyme A-transferase associated with the increase in ketone bodies supports the suggestion that there is a lower utilization of these compounds by peripheral tissues. In the blood of hyperthyroid rats a higher D-3-hydroxybutyrate/acteoacetate ratio is also found, probably resulting from a selective utilization of the two compounds in this pathological state.

Enzyme activity alteration by cadmium administration to rats: the possibility of iron involvement in lipid peroxidation.

The specific activities of D-3-hydroxybutyrate dehydrogenase (BDH) and glutamate dehydrogenase (GDH) are reduced in the liver and kidney of rats intoxicated with 2.5 mg Cd/kg body wt and sacrificed after 24 h; conversely ketone-body concentration is strongly increased in both of these organs and blood. In the same animals a great stimulation of antioxidant enzymes glutathione reductase and glutathione peroxidase occurs. The prooxidant state induced by cadmium in liver mitochondria and microsomes is unaffected by superoxide dismutase, catalase, or mannitol, whereas it is completely blocked by vitamin E thus excluding the involvement of reactive oxygen species in this process. The mechanism by which cadmium induces lipid peroxidation has been investigated by measuring the effect of this metal on liposomes. Ninety-minute treatment of liposomes with CdCl2 does not induce any lipid peroxidation. In contrast, Fe2+ ions under the same conditions cause strong liposome peroxidation. It has also been observed that cadmium promotes a time-dependent iron release from biological membranes. When lipid peroxidation is induced by a low concentration (5 microM) of FeCl2, in place of CdCl2, the characteristics of this process and the sensitivity to the various antioxidants used are similar to those observed with Cd. From these results we conclude that the prooxidative effect of cadmium is an indirect one since it is mediated by iron. With regard to the inhibitory effect on BDH and GDH following cadmium intoxication, it does not appear to be imputable to lipid peroxidation since in vitro investigations indicate that the presence of vitamin E does not remove the inhibition at all.

Effect of thyroid hormones on microsomal fatty acid chain elongation synthesis in rat liver.

Evidence is presented that rat liver microsomal fatty acid chain elongation synthesis and desaturation, as well as acetyl-CoA carboxylase and fatty acid synthetase, are strongly influenced by thyroid hormone level. Conversely, the fatty acid chain elongation system in mitochondria, unlike the oxidative capacity of palmitate, NADH, succinate and malate, does not seem significantly affected by the thyrotoxic state. In triiodothyronine-induced or thyroxine-induced hyperthyroidism, rat liver acetyl-CoA carboxylase, fatty acid synthetase and microsomal chain elongation and desaturation reactions are not greatly affected after the first 10 days of treatment, while after longer intervals a respective increase in these activities is shown of up to 87, 116 and 65% after 22 days. In propylthiouracil-induced hypothyroidism, all the above synthetic activities are strongly reduced immediately after three days of drug administration and diminished no further following longer periods. Although the pattern of synthesized fatty acids in the thyrotoxic state is similar to that obtained from normal subcellular rat fractions, the esterification process of fatty acids in microsomal lipids appears to be slightly inhibited in hypothyroid rats and increased following triiodothyronine or thyroxine administration. Finally, a reduction in the hepatic cyclic AMP level of about 41% is reported after 19 days of triiodothyronine-administration to rats. On the basis of the observed insensitivity of the mitochondrial fatty acid chain elongation system to the thyrotoxic state, a tentative interpretation of its role in the hepatic cell is postulated.

Cadmium-dependent enzyme activity alteration is not imputable to lipid peroxidation.

The effect of cadmium on the liver-specific activities of NADPH-cytochrome P450 reductase (CPR), malic dehydrogenase (MDH), glyceraldehyde-3-phosphate dehydrogenase (GADPH), and sorbitol dehydrogenase (SDH) was assessed 6, 24, and 48 h after administration of the metal to rats (2.5 mg/kg of body weight, as CdCl2, single ip injection). CPR specific activity increased after 6 h and afterward decreased significantly, while MDH specific activity increased up to 24 h and then remained unchanged. Both SDH and GADPH specific activities reduced after 6 h, the former only a little but the latter much more, and after 24 and 48 h were strongly inhibited. In vitro experiments, by incubating rat liver microsomes, mitochondria, or cytosol with CdCl2 in the pH range 6.0-8.0, excluded cadmium-induced lipid peroxidation as the cause of the reduction in enzyme activity. In addition, from these experiments, we obtained indications on the type of interactions between cadmium and the enzymes studied. In the case of CPR, the inhibitory effect is probably due to Cd2+ binding to the histidine residue of the apoenzyme, which, at physiological pH, acts as a nucleophilic group. In vitro, mitochondrial MDH was not significantly affected by cadmium at any pH, indicating that this enzyme is probably not involved in the decrease in mitochondrial respiration caused by this metal. As for GADPH specific activity, its inhibition at pH 7.4 and above is imputable to the binding of cadmium to the SH groups present in the enzyme active site, since in the presence of dithiothreitol this inhibition was removed. SDH was subjected to a dual effect when cytosol was exposed to cadmium. At pH 6.0 and 6.5, its activity was strongly stimulated up to 75 microM CdCl2 while at higher metal concentrations it was reduced. At pH 7.4 and 8.0, a stimulation up to 50 microM CdCl2 occurred but above this concentration, a reduction was found. These data seem to indicate that cadmium can bind to different enzyme sites. One, at low cadmium concentration, stimulates the SDH activity while the other, at higher metal concentrations, substitutes for zinc, thus causing inhibition. This last possibility seems to occur in vivo essentially at least 24 h after intoxication. The cadmium-induced alterations of the investigated enzymes are discussed in terms of the metabolic disorders produced which are responsible for several pathological conditions.

The response of rat liver lipid peroxidation, antioxidant enzyme activities and glutathione concentration to the thyroid hormone.

1. In liver microsomes from hyperthyroid rats NADPH-dependent lipid peroxidation induces a hydroperoxide formation 56% higher than that in euthyroid ones. 2. The addition of 5 microM Fe2+ (or Fe3+) strongly decreases the hydroperoxide level in favour of that of TBA-reactive substances. Higher iron concentrations (30 microM) have no significant effect. 3. In hepatocytes from hyperthyroid rats CCl4-induced lipid peroxidation produces an amount of TBA-reactive substances four times higher than that in those from euthyroid rats. 4. In the liver of hyperthyroid rats a GSH concentration decrease (by about 35%) is found while the opposite occurs in the blood of the same animals where GSH increases 2.5 times. 5. It is shown that in the liver of hyperthyroid rats, besides higher lipid peroxidation, a more active defense mechanism is operating since both glutathione peroxidase and glutathione reductase specific activities are higher than in euthyroid rats.