Autoxidation Products Of Linoleic Acid Research Articles

Separation of linoleic acid oxidation products by micellar electrokinetic capillary chromatography and nonaqueous capillary electrophoresis.

In this work the suitability of micellar electrokinetic capillary chromatography (MEKC) and nonaqueous capillary electrophoresis (CE) to the analysis of the primary oxidation products of linoleic acid was studied with uncoated fused-silica capillaries. The primary autoxidation products of linoleic acid are the four hydroperoxide isomers 13-hydroperoxy-cis-9, trans-11-octadecadienoic acid, 13-hydroperoxy-trans-9, trans-11-octadecadienoic acid, 9-hydroperoxy-trans-10,cis-12-octadecadienoic acid, 9-hydroperoxy-trans-10, trans-12-octadecadienoic acid. Addition of a surfactant such as sodium dodecyl sulfate (SDS) or sodium cholate (SC) into the running buffer (20-30 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) or ammonium acetate, pH 9.5-11) was required to enhance the water solubility of the sample and selectivity of the separation. MEKC proved to be a promising new technique for the separation of the primary oxidation products of lipids giving results comparable to high performance liquid chromatography (HPLC). Partial separation of hydroperoxide isomers was also achieved using nonaqueous CE with methanol-acetonitrile-sodium cholate as running buffer.

Generation of α-hydroxyaldehydic compounds in the course of lipid peroxidation

Based on 18O-labeling experiments a general scheme for the generation of hydroxy aldehydic compounds in the course of lipid peroxidation of linoleic acid is developed. Key intermediates are obviously dioxygenated fatty acids, since after reduction with either NaBH 4 or Rh/H 2 1,2 and 1,6 dihydroxy fatty acids can be identified. The postulated mechanism not only explains the formation of 2-hydroxyalkanals but also supports earlier hypothesis concerning the generation of 4-hydroxyalkenals. In addition it predicted the occurrence of ( n − 1)-hydroxy-n-oxo fatty acids as additional oxidation products. A search for these previously unknown autoxidation products of linoleic acid was indeed successful.

Effects of Peroxidation Products of Linoleic Acid on Tryptophan–nicotinamide Metabolism in Rats

The flavin and pyridine nucleotide coenzymes are involved in the detoxication of autoxidation products of lipids. In tryptophan-nicotinamide metabolism, kynurenine 3-hydroxylase and N1-methylnicotinamide (MNA) oxidase contain FAD as a coenzyme. So, the effects of dietary autoxidation products of linoleic acid on the metabolism of tryptophan-nicotinamide were investigated using rats. The administration of linoleic acid hydroperoxides or secondary products reduced the urinary excretion of xanthurenic acid, nicotinamide and its metabolites such as MNA, N1-methyl-2-pyridone-5-carboxamide (2-Py), and N1-methyl-4-pyridone-3-carboxamide (4-Py) as compared with the group administered saline or linoleic acid. Among the enzyme activities involved in the tryptophan-nicotinamide metabolism, the activity of NAD+ synthetase was decreased by the administration of linoleic acid hydroperoxides or secondary products. The activities of tryptophan oxygenase and 4-Py-forming MNA oxidase were also decreased by the administrati...

リノール酸自動酸化産物の肝毒性の解明

リノール酸自動酸化産物の肝毒性の解明

Linoleic acid hydroperoxide concentration in relation to mutagenicity of repeatedly used deep-frying fats.

A high-performance liquid chromatography procedure was developed to measure linoleic acid hydroperoxides (LAHPO) in 19 used, deep-frying fat samples that were screened for mutagenicity. The detection limit of the method was found to be 10 nmol of LAHPO/g of fat. In eight samples, LAHPO were not detected; levels in the other samples ranged from 17 to 267 nmol/g of fat. LAHPO were not detectable in unused hydrogenated frying fat samples. Concentrations of LAHPO correlated positively with mutagenicity to Salmonella tester strains TA97 and TA100, in presence of S9 mix only; coefficients of correlation were, respectively, r=0.48 (p less than 0.05) and r=0.24 (n.s.). Without metabolic activation no significant associations or secondary autoxidation products of linoleic acid generated in presence of liver S9 mix may contribute to mutagenicity of some of the used deep-frying fat samples.

Depletion of hepatic coenzyme A derivatives is one of the markers of the toxicity of orally administered secondary autoxidation products of linoleic acid in rat.

When 400 mg/rat/day of secondary autoxidation products of linoleic acid was orally administered 3 times to rats, they died at 30-40 h after the third dose. To search the markers of the toxicity of secondary products in vivo, the rats were killed at 24h after the third dose, and conditions of their digestive tracts and liver were analyzed. In the stomach, macroscopically, inflation, retention of undigested food, and edema were seen. Slight congestions were detected in the small intestines. It was considered that these injuries led to reduction in food consumption and then depression of the growth, but did not lead to the death of the animals. The lipid peroxide levels in the liver and the activities of its detoxifying enzymes were increased as compared to those in the control groups. The hepatic lipid contents and unsaturated fatty acid compositions were also not changed. The endogenous lipid peroxidation, therefore, did not give the rats a severe stress. The activities of hepatic acetyl-CoA carboxylase and carnitine palmitoyltransferase were 20 and 35% lower than those of control, respectively. The levels of CoASH, acetyl-CoA, and long-chain acyl-CoA were 1/9, 1/2, and 1/4 of those in control, respectively. Thus, one of the markers of the toxicity of secondary products was the depletion of hepatic CoA derivatives. In rat, bio-energy was reduced by the decrease in the intestinal absorption of nutrients, and the depletion of hepatic CoA derivatives also failed to supply energy with beta-oxidation.

Succinate Dehydrogenase and Synthetic Pathways of Glucose 6-Phosphate Are also the Markers of the Toxicity of Orally Administered Secondary Autoxidation Products of Linoleic Acid in Rat Liver

In order to find the markers of the toxicity of the autoxidized lipids in the liver, rats were given a lethal amount of secondary autoxidation products of linoleic acid (400 mg/rat/day for 3 days) and then changes in the hepatic metabolic functions were analyzed. A decrease in acetyl-CoA level to half caused by the depletion of CoASH was reported in an associated paper (J. Nutr. Sci. Vitaminol., 35, 11-23, 1989). Citrate, isocitrate, and 2-oxoglutarate also decreased to half the level of those of the control group. Reduction in isocitrate dehydrogenase activity was only 25%, while NADH2 and ATP levels remained unchanged. Thus, the reduction in the citrate cycle activity was due to the decrease in acetyl-CoA. The activity of mitochondrial succinate dehydrogenase was decreased to 1/5. Other appreciable changes were depletion of glucose 6-phosphate and fructose 6-phosphate, accumulation of glucose 1-phosphate, reductions in hexokinase, phosphofructokinase, glucose-6-phosphatase, phosphoglucomutase, and phosphogluconate dehydrogenase activities, and decrease in the NADPH2 level. It was considered that these changes were caused by the depletion of glucose 6-phosphate whose synthetic pathways were abnormal. Therefore, the markers of the hepatotoxicity of secondary products were the changes in the CoASH level and the activities of succinate dehydrogenase and synthetic pathways for glucose 6-phosphate.

Effect of orally administered secondary autoxidation products of linoleic acid on carbohydrate metabolism in rat liver

The effects of orally administered secondary autoxidation products of linoleic acid in rat liver were investigated. Their administration led to two toxic effects on hepatic carbohydrate metabolism, as compared to the administration of saline or linoleic acid used as controls. One effect was depletion of glucose 6-phosphate and fructose 6-phosphate caused by the reduction of glycolysis and glycogenolysis, accompanied by decreases in glycogen synthesis and pentose phosphate cyclic activity. The reduction in these metabolic systems seems unlikely to occur because phosphofructokinase was regulated by ATP or citrate enzymatically, because their accumulation in the liver was not detected in the secondary products. Another toxic effect was the depletion of oxaloacetate and isocitrate caused by the reduction in enzyme activity of the mitochondrial citrate cycle. On the basis of these results, the hepatotoxic effects of secondary products are discussed as follows: the incorporated secondary products impaired the activities of hexokinase and phosphoglucomutase in the liver. The reduction in these enzyme activities resulted in the depletion of glucose 6-phosphate and fructose 6-phosphate, which led ultimately to decreases in the activities of phosphofructokinase, the pentose phosphate cycle, and glycogen synthesis. Moreover, the secondary products disturbed the mitochondrial membrane, resulting in a decrease in the activity of the citrate cycle, which was accompanied by depletion of its metabolites.

Decrease of the NADPH level in rat liver on oral administration of secondary autoxidation products of linoleic acid.

The hepatotoxicity of orally administered secondary autoxidation products of linoleic acid was investigated, as compared with the administration of a saline solution and linoleic acid as controls. The de novo synthesis of fatty acids was strongly reduced in the secondary products group. The level of NADPH in the liver significantly decreased while that of NADH did not. The activities of glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase apparently decreased. The activities of NAD+ kinase and NAD+ synthetase decreased and that of NAD+ nucleosidase increased in the secondary products group. Therefore, the depletion of NADPH can be attributed to the inhibition of two metabolic systems (an NADPH-supplemental system, and a synthetic system of NADP and NAD), and resulted in the reduction of lipogenesis in the liver.

The effect of orally administered secondary autoxidation products of linoleic acid on the activity of detoxifying enzymes in the rat liver

Radioactive secondary autoxidation products of linoleic acid were administered orally to rats and the incorporation of radioactive substances into lipids was investigated in the liver. The radioactive substances were significantly incorporated into hepatic mitochondrial and microsomal lipids 12 h after the administration. 80% of the radioactivity in mitochondria was detected in neutral lipids. The radioactivity in microsomal neutral lipids significantly decreased and the activity in phospholipids increased 12 h after the administration. On the other hand, contents of lipid peroxide and thiobarbituric acid reactive substances in liver were significantly increased by 40% at 15 h after the administration of the secondary autoxidation products. Activity of marker enzymes used for an indication of the hepatic injury was also elevated. Glutathione peroxidase activity increased 3-fold and catalase activity increased 1.5-fold. Activity of mitochondrial NAD-dependent aldehyde dehydrogenase, however, was decreased by 50%. It seems likely that the secondary autoxidation products orally administered are detoxified in the hepatic mitochondria, metabolized to neutral lipids, and further metabolized to phospholipids in microsomes, while as the incorporated secondary autoxidation products induces hepatic injury by lipid peroxidation.

Identifications of 9-oxononanoic acid and hexanal in liver of rat orally administered with secondary autoxidation products of linoleic acid.

Secondary autoxidation products (SP) of [14C-U]linoleic acid were administered orally to rats and the incorporation of SP into the body was investigated. The radioactivity of SP was incorporated into the liver after 12 hr to the extent of 1-2.5%. An attempt was then made to detect the major low molecular-weight components of SP, 9-oxononanoic acid (9-ONA) and hexanal, in the hepatic mitochondrial and microsomal lipids. The fraction containing SP could be separated from the neutral and phospholipids of the organelles and their peroxides by silica gel column chromatography. The specific activity of the thiobarbituric acid-reactive substances of the fraction approached the value of SP per se, and with gas chromatographic-mass spectrometric analysis, 9-ONA and hexanal were identified in the fraction. These detected compounds could not be artifacts and, therefore, were originated from the SP orally administered.

Calcium-translocating and cardiotonic properties of oxidation products of linoleic acid.

Calcium-translocating activity of linoleic acid and its lipoxygenase (linoleate: oxygen oxidoreductase; EC 1.13.11.12) metabolites or autoxidation products was determined in vitro by estimation of 45Ca transport from a bulk aqueous to a bulk organic phase. Fresh commercial linoleic acid, tested immediately after removal from a sealed vial, stimulated calcium translocation only at concentrations greater than 1 mM. In contrast, 45Ca translocation by linoleic acid exposed to air was detectable at 10 microM. Oxidation products of linoleic acid obtained either by incubation with lipoxygenase or by autoxidation were much less potent than the calcium ionophore A23187. The products obtained by enzymic oxidation of linoleic acid enhanced contractility in the Langendorff-perfused guinea pig heart up to 45% over control (at 3 X 10(-8) M). The inotropic response was transient with rapid onset and not affected by the beta-adrenergic antagonist, propranolol. The autoxidation products of linoleic acid increased cardiac contractility up to 43% at 10(-6) M. In contrast, fresh linoleic acid caused only a negative inotropic effect at 10(-8) to 3 X 10(-7) M, progressing to contracture at 10(-6) M. These findings suggest that conflicting reports on the cardiostimulant effect of linoleic acid may be due to varying levels of the autoxidation products. Linoleic acid metabolites in vivo may have a physiological role in myocardial function related to their Ca2+-ionophoric activity.

The induction of lipid peroxidation in rat liver by oral intake of 9-oxononanoic acid contained in autoxidized linoleic acid.

A component in the autoxidation products of linoleic acid (LA) which induced the endogenous lipid peroxidation was searched for. Secondary autoxidation products (SP) of LA induced the production of thiobarbituric acid reactive substances (TEARS) in liver as well as the LA hydroperoxides stimulated. SP were separated chromatographically into some fractions which were administered orally to rats. One fraction composed mainly of 9-oxononanoic acid (9-ONA) markedly stimulated the TEARS production in liver. Authentic 9-ONA also increased the lipid peroxide level as determined by a new test, as well as the activities of glutathione peroxidase and reductase in liver. Thus, hepatic lipid peroxidation was induced by orally given 9-ONA which was present in the autoxidation products of LA.

Some analytical observations of autoxidation products of linoleic acid and their thiobarbituric acid reactive substances.

Autoxidation products of linoleic acid (LA) were analyzed, when the weight became 1.14-fold under the autoxidation conditions of satisfactory atmospheric oxygen, at 37°C, in the dark, for 7 days. The LA absorbed 2.8 mol of oxygen to form secondary degradation products. This autoxidized LA consisted of 45% intact substance, 22% a mixture of polymers and endoperoxides, 18% LA hydroperoxides, 3% polar products, 1.7% azelaldehydeic acid, 1.3% hexahal, 0.9% azelaic acid, 0.6% octanoic acid, 0.3% suberaldehydeic acid, and so on. Thus, unstable 2,4-dienoic carbonyls were the main intermediate products of autoxidation of LA. Therefore, malonaldehyde was not a main product nor a major thiobarbituric acid reactive substance.

Immunophoretic evaluation of blood serum proteins in chickens. II. Patterns in vitamin E deficiency encephalomalacia.

Summary and Conclusions1. Twenty-five newly hatched or very young chickens were subjected to a purified diet, deficient in vit E or any synthetic antioxidant and supplemented with 4.8% linoleic acid. If permitted to survive, these chicks developed encephalo-malacia between the 10th and 26th day. Forty-two chicks, fed on a conventional stock laboratory diet, developed normally and were well at the time blood samples were examined. 2. Immunophoresis patterns of blood sera from experimental and normal chickens were obtained. 3. In the experimental sera, an antigen-antibody precipitation curve with the mobility of beta lipoprotein persisted whereas this curve did not persist after the 10th day in normal chickens. This curve was interpreted as due to the presence of an abnormal protein, possibly a denatured beta lipoprotein. 4. The role of possible denaturation of beta lipoproteins in the blood serum in the presence of autoxidation products of linoleic acid in the serum as a possible mechanism for production o...