Comparative determination of the potential for activating lipid peroxidation of the adrenal cortex, liver and heart in various animals

Studies have been made on the rate of spontaneous, ascorbate- and NADP.H-dependent lipid peroxidation in the adrenal cortex, liver heart of rats, rabbits and cattle. It was shown that in spite of some species differences in predisposition of tissues to lipid peroxidation. The highest rate of this process is characteristic of the liver of all the animals investigated.     

Synthesis and release of low density lipoproteins by the isolated perfused pig liver.

In previous studies, we have shown that a relatively large amount of low density lipoproteins is released into the perfusate during isolated pig liver perfusion. The present studies were done to determine the source of these lipoproteins. Breakdown of the very low density lipoproteins to low density lipoproteins by the perfusion apparatus or by hepatic catabolism was excluded by adding 125I very low density lipoproteins to the perfusate in the presence and absence of a liver and then measuring the radioactivity in the low density lipoprotein fraction after rate-zonal ultracentrifugation. Release of preformed low density, lipoproteins from the liver was investigated by injecting iodine-labeled low density lipoproteins in vivo several hours prior to perfusion of the liver and then measuring the release of labeled low density lipoproteins into the perfusate. It was shown that intact labeled low density lipoproteins were released by the perfused liver. De novo synthesis of the low density lipoproteins was established by measuring the incorporation of [1-14C]leucine into this lipoprotein fraction. The radioactivity in the low density lipoprotein fraction increased with time and accounted for 20 to 25% of the total radioactivity incorporated into all the lipoprotein fractions. The incorporation of [1-14C]leucine into the low density lipoproteins was confirmed by rate-zonal analysis. We conclude that the low density lipoproteins in the perfusate from pig liver perfusions were derived mainly from a preformed liver pool, but also partly from de novo synthesis by the liver.     

Cholesterol-rich diets have different effects on lipid peroxidation, cholesterol oxides, and antioxidant enzymes in rats and rabbits

The objective of this study was to compare the effect of cholesterol feeding of rats and rabbits. The levels of lipid peroxidation products and oxysterols in the plasma of the two species plus the antioxidant enzyme activities in the liver and erythrocytes were measured to explain their different susceptibilities to atherosclerosis. Our study showed that rats are less susceptible than are rabbits to the atherogenic effect of a cholesterol-rich diet because of differences in lipid peroxidation products as well as antioxidant enzymes activities in their livers. In rabbits, cholesterol feeding produced severe hypercholesterolemia (43-fold increase) and increased plasma and liver lipid peroxidation. Total as well as the individual oxysterol contents of 7alpha-, 7beta-hydroxycholesterol, alpha-epoxy, beta-epoxycholesterol, cholestanetriol, 7-keto, and 27-hydroxycholesterol significantly increased in the plasma of hypercholesterolemic (HC) rabbits. Erythrocyte glutathione peroxidase (GSH-Px) activity significantly decreased whereas catalase activity significantly increased in HC rabbits. In rats cholesterol feeding increased the plasma cholesterol only twofold and had no effect on plasma or liver lipid peroxidation. Only 7alpha- and 7beta-hydroxycholesterol increased and no change was observed in any of the antioxidant enzymes activity in the erythrocytes. Although cholesterol feeding caused a 10-fold increase of liver cholesterol as ester in both rats and rabbits, the antioxidant enzyme GSH-Px and catalase activities in the liver significantly increased in rats but significantly decreased in rabbits. The increase of GSH-Px and catalase activities in the liver of cholesterol fed rats could have a protective role against oxidation, thus preventing the formation of lipid peroxidation and oxysterols.     

Interrelation of oxygen anion-radical generation and lipid peroxidation in liver microsomes]

Consumption of O2-radicals and malondialdehyde accumulation was studied in the liver microsomes (LM) of rat after phenobarbital and 3-methylcholantrene induction, and in the LM of rabbits; there was revealed a suppression of lipid peroxidation, but a sufficiently high level of O2-radical generation. It is supposed that the absence of any direct association between the lipid peroxidation activity and the O2-radical generation in the LM of rats induced with the mentioned agents and in the LM of rabbits depended on increase in the antioxidant level.     

Lipid peroxidation and free Fe2+ during ischemia and reoxygenation of the liver

Ischemia and reoxygenation were experimentally induced in thin liver sections. It has been shown that free iron decompartmentalization takes place 30 min after the induction of ischemia, with no lipid peroxidation activation observed. In reoxygenation, activation of lipid peroxidation and decrease in free iron concentration take place in the liver cells. It is suggested that free iron accumulation in the tissues during ischemia causes lipid peroxidation activation during reoxygenation.     

Characterization of nascent lipoproteins produced by perfused rat liver: evidence of hepatic secretion and post-secretory modification of nascent lipoproteins

We characterized the lipoproteins produced by perfused rat liver in recirculating and non-recirculating systems. The apolipoprotein (apo) B of the perfusate very low density lipoprotein (VLDL) and low density lipoprotein (LDL) were labeled with a radioactive precursor amino acid in both systems, suggesting that newly synthesized apo B was secreted in association with VLDL and LDL. When the lipoproteins obtained from the non-recirculating perfusate were injected into rats in vivo, the half life of the VLDL was 13 min and most of it was converted to LDL, while that of the LDL was 5.2 h, indicating that the perfusate LDL was different from the VLDL with respect to its metabolic fate. These observations suggest that both VLDL and LDL are produced as independent primary products in the liver, although the majority of LDL is derived from VLDL in vivo. The nascent lipoproteins in the non-recirculating perfusate were richer in apo E than those in the recirculating perfusate, and a part of the apo E disappeared when the VLDL was added to the recirculating perfusate. The particle sizes of the VLDL and LDL were examined by electron microscopy, which revealed that those in the non-recirculating perfusate were more homogeneous and smaller than the plasma counterparts, while those in the recirculating perfusate were more heterogeneous and their mean diameter was closer to that of the plasma lipoproteins, than in the case of non-recirculating perfusate. These observations suggest that apo E secreted with the nascent lipoproteins may be picked up by the liver just after secretion, causing the heterogeneity in size, as observed in the case of plasma lipoproteins.     

Nascent lipoproteins from recirculating and nonrecirculating liver perfusions and from the hepatic Golgi apparatus of African green monkeys

Perfusate apoB-100-containing lipoproteins from the isolated, perfused livers of African green monkeys consist of significant amounts of d greater than 1.006 g/ml particles in addition to very low density lipoproteins (VLDL). Distinguishing characteristics of these perfusate lipoproteins are the relative abundance of surface lipids and deficiency of core lipids. The present studies were performed to determine the likelihood that the d greater than 1.006 g/ml perfusate lipoproteins are secretion products instead of products of post-secretory modification (e.g., lipolysis) of secreted VLDL. [14C]Leucine from the perfusate became incorporated into the apoB of each of the perfusate lipoprotein classes to a similar extent in both recirculating and nonrecirculating perfusions. When endogenously radiolabeled perfusate VLDL from one liver was recirculated through a second liver, only about 15% of the radiolabeled protein appeared in the d greater than 1.006 g/ml fraction. The particle morphology and the cholesterol and apoB distribution between VLDL and d greater than 1.006 g/ml fractions were similar in recirculating and nonrecirculating perfusions. A Golgi apparatus-rich fraction was isolated from the homogenates of fresh liver samples and the isolated Golgi VLDL and d greater than 1.006 g/ml lipoproteins exhibited morphologic evidence of extra surface material analogous to that seen in perfusate. Taken together, these data support the possibility that significant amounts of d greater than 1.006 g/ml lipoproteins, many with surface-rich properties, are nascent, secretory products of the primate liver. The low level of lecithin:cholesterol acyltransferase (LCAT) in this perfusion system appears to permit detection of these secretion products and it is significant to note that the perfusate lipoprotein profile, which is unlike that of normal plasma, is similar to that of LCAT-deficient patients.     

Effect of chronic ethanol treatment on the t-butyl hydroperoxide-dependent lipid peroxidation in rat liver

1. The effect of chronic ethanol consumption on the level of the t-butyl hydroperoxide (Bu'OOH)-induced lipid peroxidation in rat liver homogenate and subcellular fractions was measured using chemiluminescence technique and malondialdehyde formation. 2. It was shown that under the action of ethanol the rate of lipid peroxidation was decreased in the whole and "postnuclear" liver homogenates. 3. Ethanol significantly decreased the intensity of lipid peroxidation in microsomes, but did not affect the Bu'OOH-dependent process in mitochondria. 4. The level of lipid peroxidation was reduced after incubation of the total particulate fraction (mitochondria plus microsomes) with the undialysed cytosol from ethanol-treated rat liver. Dialysis of the cytosol prevented depressive effect of ethanol treatment on lipid peroxidation. 5. Reduced glutathione (0.1-1.0 mM) was shown to decrease the rate of lipid peroxidation in rat liver microsomes, but did not affect its level in mitochondria. 6. Pyrazole injections to rats reduced and phenobarbital treatment increased the level of the Bu'OOH-dependent lipid peroxidation in liver microsomes. 7. The data obtained indicate that the Bu'OOH-dependent lipid peroxidation is not an appropriate marker of the ethanol-induced oxidative stress in rat liver cells.     

Determination of the surface charge of lipoproteins and its changes during lipid peroxidation

Surface potential of human plasma lipoproteins was studied by the use of positively charged spin probe. The calculated values of surface potential of high and low density lipoproteins appeared to be -29 +/- 1 and -16 +/- 1 respectively. It was shown that lipid peroxidation process induces an increase of surface potential of both high and low density lipoproteins. Probably, it is connected with the increase of the negative charge density on their surface. This fact can play an important role in pathogenesis of diseases with lipid metabolism and lipid peroxidation level disorders in plasma (atherosclerosis, ischemic heart disease etc.).     

Radiation induced lipid peroxidation: factors which determine the oxidizability of lipids

Lipids are the essential components of cell membranes and lipoproteins. Their peroxidation plays an important role in numerous pathologies in which oxidative stress is involved. Lipid peroxidation occurs through a chain reaction that contributes to membrane damage in cells. It results in the conversion of fatty acids to polar hydroperoxides and leads to the breakdown or malfunction of the membrane. Lipids are amphiphilic molecules that aggregate in aqueous solutions into micelles and liposomes. The effect of this structural organization is significant in studies of radiation-induced peroxidation damage in highly ordered biological systems such as biological membranes. In this paper, a synthesis of the data concerning radioinduced lipid peroxidation is completed by an original review of the different parameters that determine lipid oxidizability. In addition, the influence of lipid aggregation and the effect of molecular packing are discussed.     

Role of lipid peroxidation in the inhibition of mononuclear cell proliferation by normal lipoproteins

Stimulated peripheral blood mononuclear cells (PBMC) can oxidize normal lipoproteins, and sufficiently oxidized lipoproteins are cytotoxic. However, the role of lipid peroxidation in the inhibition of mitogen-stimulated PBMC proliferation by physiologic concentrations of normal lipoproteins is unclear. In the present investigation, normal low density lipoprotein (LDL) and very low density lipoprotein (VLDL) suppressed [3H]thymidine incorporation and gamma interferon production in concanavalin A-stimulated PBMC without causing cell death. This suppression was accompanied by parallel increases in lipid peroxidation products measured as thiobarbituric acid reactive substances (TBARS). In contrast, high density lipoprotein (HDL) failed to inhibit PBMC and TBARS remains low. Differences between the PBMC suppression from LDL, VLDL, and HDL were best accounted for by normalizing the lipoprotein concentrations by their total lipid content. Moreover, the antioxidants superoxide dismutase and butylated hydroxytoluene each substantially ameliorated the inhibition of PBMC caused by LDL, and reduced the levels of lipid peroxidation products that were generated. Altogether, these results suggest that reactive oxygen species generated by stimulated PMBC may cause oxidative alterations of normal lipoproteins that may, in turn, account for much of the previously reported inhibition of PBMC by normal lipoproteins.     

Apoproteins of the lipoproteins in a nonrecirculating perfusate of rat liver.

The apoproteins of serum lipoproteins and of lipoproteins present in a nonrecirculating perfusate of rat liver were compared by immunochemical, gel electrophoretic, and solubility techniques. Serum and perfusate very low density lipoprotein apoprotein composition were not different. No evidence for the presence of a lipoprotein resembling serum low density lipoprotein was obtained. However, the apoprotein composition of circulatory high density lipoprotein was quantitatively different from the secretory product in the density 1.06-1.21 range. As measured by stained sodium dodecyl sulfate gel electrophoretic patterns, the arginine-rich protein was the major secretory apoprotein while the A-I protein was the major apoprotein in circulating high density lipoprotein. A very similar pattern was seen in perfusates of orotic acid-fatty livers. It was concluded that although the liver secrets lipoproteins in the high density class, circulatory high density lipoprotein is largely a product of catabolic processes.     

Haemoglobin denaturation, lipid peroxidation and haemolysis in phenylhydrazine-induced anaemia

Temporal changes in the levels of denatured haemoglobin (Heinz bodies) and fluorescent lipid peroxidation products in the red cells of rabbits administered phenylhydrazine have been followed. Heinz bodies were maximal just before the period when most of the cell destruction occurred, whereas lipid peroxidation products were maximum when reticulocyte levels were highest. This implies that lipid peroxidation occurs mainly in immature cells and that haemoglobin denaturation is more likely than lipid peroxidation to be a major contributor to haemolysis.     

Effect of liposomal antitumor preparation 5,6-benzocoumarin-5-uracil on intensity of free-radical processes in the liver microsomes and tumor cells of rats with transplanted Guerin's carcinoma

The contents of primary and secondary (TBA-active) products of lipid peroxidation were investigated in microsomal fraction of the liver and tumor cells of rats with transplanted Guerin's carcinoma and under the condition of antitumor liposomal preparation 5,6-benzcumarine-5-uracil (BCU) action. High level of lipid peroxidation process in the microsomal fraction is shown in the rat liver and tumor cells under the condition of BCU action in the period of intensive carcinoma growth. It remains till the period of tumor growth braking. This fact testifies to the prooxidation action of the preparation. Liposomal antitumor preparation BCU raises the process of lipid peroxidation in microsomal fraction of tumor cells and its action increases according to the malignant growth. The processes of lipid peroxidation in microsomal rat liver fraction approach the control data under the condition of the mentioned preparation. The investigated liposomal form of BCU possesses directed prooxidation action on the malignant tissue.     

Lipid peroxidation and glutathione system in hyperlipemic rabbits: influence of olive oil administration

We studied the effect of supplementation (10% w/w) of a hyperlipemic diet (1% cholesterol) with olive oil (OLIV) for 6 weeks in four groups of 10 rabbits each. At the end of this period, we determined lipid peroxidation, glutathione content, and glutathione peroxidase, reductase and transferase activities in liver, brain, heart, aorta and platelets. The atherogenic diet increased tissue lipid peroxidation and decreased the protective antioxidant effect of glutathione. Dietary supplementation with olive oil reduced tissue lipid peroxidation by 71.6% in liver, 20.3% in brain, 84.5% in heart, 63.6% in aorta, 72% in platelets. The ratios total/oxidized glutathione were increased in all tissues (49% in liver, 48% in brain, 45% in heart, 83% in aorta, 70% in platelets). Olive oil increased glutathione peroxidase and transferase activities in all tissues. We conclude that in rabbits made hyperlipemic with a diet rich in saturated fatty acids, olive oil decreased tissue oxidative stress.     

Secretion of cholesteryl-ester-rich lipoproteins by the perfused livers of rabbits fed a wheat-starch-casein diet

Rabbits fed a cholesterol-free semi-synthetic wheat-starch-casein diet had a high plasma cholesterol concentration; most of the cholesterol was associated with low-density lipoproteins (LDL). Chemical analyses of plasma lipoproteins revealed that very-low-density lipoproteins (VLDL), intermediate lipoproteins and LDL from casein-fed rabbits contained more cholesteryl ester than that of lipoproteins isolated from chow-fed animals. The fatty acid composition of cholesteryl esters of plasma lipoproteins showed that there were higher contents of oleic acid than linoleic acids in lipoproteins from casein-fed rabbits. Lipoproteins isolated from liver perfusates of casein-fed rabbits had higher cholesteryl oleate content than lipoproteins from chow-fed rabbit liver perfusates. There was a marked increase in secretion of apolipoproteins from perfused livers of casein-fed rabbits. We conclude that the high levels of plasma cholesterol in casein-fed rabbits are of hepatic origin and that one of the hypercholesterolemic actions of dietary casein in rabbits is the induction of hepatic synthesis and secretion of cholesteryl-ester-rich lipoproteins.     

应用冷Schiff组织化学法检测人体组织脂质过氧化反应的研究

自由基引发的生物膜不饱和脂肪酸脂质过氧化反应涉及多种疾病过程,多年来检测脂质过氧化反应一直沿用生物化学（如硫代巴比妥酸法测定丙二醛）或生物物理技术（如分光光度法测定共轭双烯）。自从冷Schiff组织化学染色技术用于自由基研究以来,使形态学方法研究脂质过氧化反应成为可能,当前,应用冷Schiff组织化学法进行组织细胞的脂质过氧化反应检测大多限于动物实验研究,本研究对多种人体离体新鲜组织的冰冻切片应用冷Schiff组织化学法进行检测,未发现被组织存在组织化学水平上的脂质过氧化反应;胆对被测组织人为施加氧化攻击（用Fe-NADPH促氧化剂孵育）后,肝、肾及胃的泌酸细胞与其它组织相比呈现较明显的脂质过氧化反应;皮肤、脂肪组织几科不出现脂质过氧化反应;甲状腺C细胞、肌肉、骨骼等与钙代谢、贮存及利用相关的组织也出现较明显的脂质过氧化反应。结论：冷Schiff组织化学方法检测人体组织脂持过氧化反应具有简便易行、同时可以形态学定位的优点,在医学生物学、肿瘤学及老年医学研究中具有应用前景。     

Carbon tetrachloride-induced lipid peroxidation dependent on an ethanol-inducible form of rabbit liver microsomal cytochrome P-450

Treatment of rats with ethanol or rabbits with either imidazole or pyrazole, agents known to induce the ethanol-inducible form of liver microsomal cytochrome P-450 (P-450 LMeb), caused, compared to controls, 3-25-fold enhanced rates of CCl4-dependent lipid peroxidation or chloroform production in isolated liver microsomes. No significant differences were seen when the rate of CCl4-dependent lipid peroxidation was expressed relative to the amount of P-450 LMeb in the various types of microsomal preparations. In reconstituted membranous systems, this type of P-450 was a 100-fold more effective catalyst of CCl4 metabolism than either of the cytochromes P-450 LM2 or P-450 LM4. It is proposed that the induction of this isozyme provides the explanation on a molecular level for the synergism seen of ethanol on CCl4-dependent hepatotoxicity.     

Quantification of the hepatic contribution to the catabolism of high density lipoproteins in rats.

Isolated rat livers were perfused for four hours in a recirculating system containing washed rat erythrocytes. Biologically screened radioiodinated rat high density lipoproteins (1.090 < d < 1.21 g/ml) were added to the perfusate with different amounts of whole serum to supply unlabeled rat high density lipoproteins. The protein moiety of the lipoprotein contained more than 95% of the radioiodine. The fraction of apolipoprotein mass degraded during the perfusion was quantified by the linear increment of non-protein-bound radioiodine in the perfusate, corrected for the increment observed during recirculation of the perfusate in the absence of a liver. The small amount of (131)I secreted into bile was added to calculate the fractional catabolic rate. The fractional catabolic rate ranged from 0.22 to 0.63% per hour in 12 experiments and was inversely related to the size of the perfusate pool of high density apolipoprotein. The absolute catabolic rate of high density apolipoprotein (fractional catabolic rate x pool size) in three livers in which the concentration of rat HDL in the perfusate approximated that in intact rats was 69.5 +/- 10.4 micro g hr(-1) (mean +/- SD). The rate of disappearance of cholesteryl esters of rat high density lipoproteins (labeled biologically by injecting donor rats with [5-(3)H]mevalonic acid) from the liver perfusate did not exceed that of the apoprotein component. These rates were compared with catabolic rates for rat high density lipoproteins in intact rats. Fractional catabolic rate in vivo, obtained by multicompartmental analysis of the disappearance curve of (131)I-high density apolipoprotein from blood plasma, was 11.9 +/- 1.3% hr(-1) (mean +/- SD). Total catabolic rate in vivo (fractional catabolic rate x intravascular pool of high density apolipoprotein) was 986 +/- 145 micro g hr(-1) (mean +/- SD). The results suggest that only a small fraction of high density lipoproteins in blood plasma of rats is degraded directly by the liver.-Sigurdsson, G., S-P. Noel, and R. J. Havel. Quantification of the hepatic contribution to the catabolism of high density lipoproteins in rats.     

Mutation induction in Escherichia coli incubated in the reaction mixture of NADPH-dependent lipid peroxidation of rat-liver microsomes

Experiments were carried out to examine mutation induction in E. coli cells incubated in the reaction mixture of NADPH-dependent lipid peroxidation of microsomes isolated from rat liver. The results obtained were as follows: (1) Lipid peroxidation of microsomes occurred extensively on incubation with NADPH and Fe2+. In the E. coli WP2uvrA(pKM101) system, the mutation frequency to streptomycin resistance increased markedly when the cells were incubated in the reaction mixture of microsomal lipid peroxidation. The induced mutation frequencies were dependent on the extent of the lipid peroxidation. (2) It was also found that the mutations were induced at the same rate as in the case of (1) when the cells were added to the microsomal suspensions after the reactions due to the short-lived free radicals had terminated. (3) The cytotoxicity of the lipid peroxidation products was larger in the DNA repair-defective mutant, E. coli SR18 (uvrArecA) than the wild-type strain, SR749. From these results it is concluded that some DNA-damaging and mutagenic substances are indeed produced in the degradation process of peroxidized polyunsaturated fatty acids in liver microsomal lipids.