Quantitative and qualitative analyses of isolated lipid droplets from interstitial cells in renal papillae from various species

The lipid droplets of renal papillae homogenates from four different species were obtained by ultracentrifugation. Ca. 80–98% of the lipids (triglycerides, phospholipids, free fatty acids, and cholesterol esters) consist of triglycerides. The triglycerides were fractionated by argentation thin layer chromatography and each fraction characterized by gas liquid chromatography. No fraction contained any unique triglyceride. The fatty acid composition of the total triglycerides, as analyzed by gas liquid chromatography and ozonolysis, differed markedly from the fatty acid composition of the corresponding plasma triglycerides. The papillary triglycerides were characterized by higher concentrations of stearic acid, arachidic acid, and polyunsaturated acids with 20 or more carbon atoms. Particularly interesting was the presence in the lipid droplets of docosa-7,10,13,16-tetraenoic acid. This acid has been shown to be a major component in the cholesterol ester fraction of rat and canine adrenal lipids. In the papillary triglycerides, this acid accounted for 7%, 15%, and more than 20% of the total fatty acids in the dog, rat, and rabbit, respectively. The pig differs from these three species in having only ca. 1% of this acid. These observations suggest that the interstitial cells produce these triglycerides. This production could occur either by a transacylation from phospholipids and cholesterol esters and by a de novo synthesis from locally produced fatty acids. The possibility that the triglyceride production may be involved in a control of the prostaglandin production of the renal medulla is discussed.     

Hydrocarbon gases produced during in vitro peroxidation of polyunsaturated fatty acids and decomposition of preformed hydroperoxides

Hydrocarbon gases have been used previously as an index of lipid peroxidation in vivo and in vitro. In vitro experiments are reported on the formation of hydrocarbon gases from peroxidizing ω-3 and ω-6 fatty acids. Hydrocarbon gases were not related during a 20-hr peroxidation phase but were released following the decomposition of hydroperoxides by addition of excess ascorbic acid. The major hydrocarbon gas products in iron, copper, or hematin catalyzed peroxidation systems were ethane or ethylene from linolenic acid, and pentane from linoleic acid and arachidonic acid. Calculations of the ratios of hydrocarbon gases formed were based on fatty acid decrease and/or change in diene conjugation and peroxide values. Depending on the fatty acid, catalyst, and calculation basis used, pentane formation was a high as 1.3 mol %, ethanol 4.3 mol %, and ethylene 10.6 mol %.     

Relative susceptibility of microsomes from lung,heart, liver,kidney, brain and testes to lipid peroxidation: Correlation with vitamin E content

Rates of in vitro lipid peroxidation of microsomes and homogenates were found to vary widely among different tissues and species. In rats and rabbits, lung microsomes peroxidized at a 25- to 50-fold lower rate than liver, kidney, testes and brain microsomes. Heart microsomes peroxidized at a rate slightly greater than, but most similar to, lung microsomes. Comparison of tissue homogenates also revealed the unique resistance of lung and heart to lipid peroxidation. The ratio of vitamin E to peroxidizable polyunsaturated fatty acids in lung and heart microsomes was several-fold higher than in microsomes from the other tissues studied, which accounted for the relative resistance of lung and heart to lipid peroxidation. Liposomes of extracted rat lung microsomal lipid were also resistant to peroxidation and the amount of vitamin E contained in the lung lipid extract was sufficient to confer the same degree of resistance when incorported into an equivalent amount of rat liver lipid. Higher rates of peroxidation in mouse lung microsomes relative to rabbit, rat and human lung microsomes were similarly correlated with a lower ratio of vitamin E to peroxidizable fatty acids in mouse lung microsomes. These data provide strong support for the role of vitamin E as the major cellular antioxidant, especially in the highly oxygenated tissues of heart and lung, and demonstrate the utility of the microsomal system in characterizing tissue differences in susceptibility to peroxidative membrane decomposition.     

Activation of polyunsaturated fatty acids by rat tissues in vitro

The conversion of labeled palmitic, linoleic, arachidonic and docosahexaenoic acids to their respective acyl CoA’s was studied in homogenates and microsomes of rat tissues. The highest activity, both in homogenates and microsomes, was seen in liver and heart. There was moderate activity in retina, brain, lung, kidney and testes and the lowest activity was found in spleen. Docosahexaenoic acid was activated much less actively in heart tissue than the other fatty acids. In all tissues examined, the highest activation was observed with arachidonic acid and the lowest with docosahexaenoic acid. Except for liver, those tissues that contained high levels of docosahexaenoic acid also had the highest activation capacity for this fatty acid.     

Metabolic effects of selenium as related to vitamin E

Trace amounts of dietary selenium, which prevent certain vitamin E deficiency symptoms, have been found to affect the composition of chick tissues. Both selenium and vitamin E increased liver coenzyme A levels to normal when cystine was omitted from the diet. Liver and plasma total fatty acids were unaltered by selenium but vitamin E increased the arachidonic acid content slightly. There was no effect of either selenium or vitamin E on total lipid, cholesterol, phopholipid or coenzyme Q in liver. Low dietary levels of either selenium or vitamin E, which were insufficient in preventing deficiency symptoms, completely eliminated mortality from toxic amounts of a dietary antioxidant. The ability of dietary selenium to reducein vitro lipid peroxidation in chick liver homogenates was not demonstrable with rat liver. Presented at the AOCS meeting in Toronto, Canada, 1962.     

Acetyl carnitine formation in rat heart

The comparative incorporation of acetate into long chain fatty acids and acetyl carnitine by cell-free preparations of rat heart has been investigated. Whereas the addition of 1 mM carnitine stimulated (45%) fatty acid synthesis by liver preparations in citrate-containing media, fatty acid synthesis from acetate in rat heart homogenates under the same incubation conditions was markedly depressed. This depression by carnitine of acetate incorporation into long chain fatty acids in 105,000 × g soluble fractions of heart was associated with increased acetyl carnitine formation. Thus in heart tissue acetyl CoA is effectively shuttled into acetyl carnitine and is unavailable for synthesis of fatty acids. These data are in agreement with results obtained earlier in studies with perfused rat heart. A similar conversion of added acetyl CoA to the carntine derivative occurred when labeled malonyl CoA was used as fatty acid precursor, again resulting in reduced fatty acid synthesis. It was shown by direct measurement that acetyl carnitine formation in the absence of carnitine was greatest in heart mitochondria and least in microsomes. In the presence of carnitine, acetyl carnitine formation was increased in all subcellular fractions, with the greatest change again occurring with mitochondria.     

Lipid peroxidation in rat tissue homogenates: Interaction of iron and ascorbic acid as the normal catalytic mechanism

Iron and ascorbic acid appear to be the normal catalytic components responsible for the lipid peroxidation reaction in aerobically incubated rat tissue homogenates. The amounts of each present in the catalytically-active fractions of rat liver, brain, testis, and kidney are appropriate to explain the lipid peroxidation reaction measured. Utilization of ascorbic acid as part of the normal catalytic mechanism is indicated by the following: The catalytic activity of the tissue soluble phase occurs only in the small molecule fraction eluted from Sephadex, and ascorbic acid occurs only in this fraction; the extent of catalysis by the small molecule fractions of the soluble phases from several tissues is proportional to their ascorbic acid content; and pH effect on lipid peroxidation is the same for both soluble-phase and ascorbic acid catalysis. Utilization of iron as part of the normal catalytic mechanism is indicated by EDTA inhibition studies and by measurements of pH effects. Previous studies have demonstrated the lack of catalytic activity by cations other than iron for the lipid peroxidation reaction in homogenates. Lipid peroxidation is inhibited at high tissue concentration and the inhibition is due to components occurring in the large molecule fraction of the soluble phase.     

Unsaturated fatty acids in the postnatally developing rat lung

Fatty acid desaturase activity specific for the C-9 position is present in lung microsomes prepared from rats of all ages. This activity is significantly lower in neonatal rat lung compared with adult lung. A rapid increase in C-9 fatty acid desaturase activity seen at the approximate time of weaning may be related to a decrease in the polyunsaturated fatty acid (PUFA) content of the diet as the rat begins to consume laboratory chow instead of mother's milk. The 900×g supernatant fraction of rat lung parenchymal cell homogenates is capable of incorporating linoleate, linolenate, and arachidonate into both triacylglycerols and phospholipids. Lung tissue from rats less than 20 days old incorporates these PUFA into phospholipids at a greater rate than lung, tissue from adult rats. The incorporation of these PUFA into phospholipids in neonatal lung tissue occured at a greater rate than their incorporation into triacylglycerols. In contrast, lung tissue from adult rats incorporated PUFA into triacylglycerols at a greater rate than into phospholipids. These data show that PUFA, known to be elevated in neonatal rat lungs, are used primarily for phospholipid biosynthesis in neonatal lung tissue whereas in adult lung tissue they are preferentially esterified to glycerol.     

Lipid composition of morris hepatoma 5123c,and of livers and blood plasma from host and normal rats

The lipid composition of Morris hepatoma 5123c was analyzed together with that of liver and blood plasma from both normal and tumor-bearing rats. The results showed that the liver of tumor-bearing rats contained higher amounts of glycerides, cholesteryl esters, free fatty acids and phospholipids than the liver of normal rats. In the blood plasma of tumor-bearing rats, there was an increase of free cholesterol and triglycerides; this latter difference, however, was not statistically significant. Acyl chain changes in the liver of tumor-bearing rats consisted of an increase of palmitic and oleic acids and a decrease of stearic and arachidonic acids in phosphatidylinositol. Morris hepatoma 5123c contained a lower amount of triglycerides than the livers (both host and normal) and showed a significant decrease of total phospholipids when compared to the host liver. The major acyl chain changes found in Morris hepatoma 5123c compared with both normal and host rat livers were: a) a higher percentage of arachidonic acid together with a lower proportion of palmitic acid in cholesteryl esters; b) an increase of stearic and arachidonic acids and a decrease of palmitic acid in triglycerides; and c) a higher level of palmitic and oleic acids associated with a lower percentage of stearic and C₂₂ polyunsaturated acids in phosphatidylcholine.     

Lipid metabolism in plasma,liver, and adipose tissue of rats with experimental chronic nephrotic syndrome

Plasma, liver, and adipose tissue lipid composition and synthesis from [1-¹⁴C] acetate were studied three months following induction of nephrotic syndrome in rats by injection of antiglomerular basement membrane protein. Plasma triglyceride concentrations and specific radioactivities were elevated, and the triglycerides contained increased proportions of oleic acid. Plasma cholesterol and phospholipid concentrations were also increased, but free fatty acid levels were not. Liver triglyceride concentrations were decreased and incorporation of [1-¹⁴] acetate into liver triglycerides was also depressed below that of normal controls. Nephrotic rat liver triglycerides contained a higher proportion of oleic acid and lower arachidonic acid than did controls. Incorporation of [1-¹⁴C] acetate into adipose tissue lipids of the nephrotic rats was increased, and the proportion of palmitic acid was decreased. In the chronic nephrotic rat, the major source of the increased plasma triglycerides may be fatty acids mobilized from adipose tissue stores.     

Serum fatty acid profiles after intravenous medium chain triglyceride administration

The serum fatty acid profiles of patients receiving either intravenous medium or long chain triglycerides were studied. Seventeen hospitalized patients, dependent on total parenteral nutrition, were randomly enrolled into a prospective study. The total parenteral nutrition (TPN) delivered amino acids and glucose and either a 75% medium chain triglyceride and 25% long chain triglyceride (MCT group) physical mixture or all long chain triglyceride (LCT group), as the respective fat sources. The amino acids and glucose were given continously, and the lipid was given for 10 hours each day over five days. Fatty acid profiles on serum triglycerides and free fatty acids were done in the morning before any lipid was given and also later in the afternoon, near the end of the lipid administration, on days 1, 3 and 5. Medium chain fatty acids rose quickly in the triglyceride fraction in patients given MCT. Rapid MCT hydrolysis occurred as evidenced by the appearance of medium chain fatty acids in the free fatty acid fraction in the afternoon sampling. Clearance of the hydrolyzed medium chain free fatty acids (MCFFA) occurred so that little, if any, were present in the morning sampling one day later. Long chain fatty acids, as either triglycerides or free fatty acids, showed expected increases during the daily infusion, but not of such relative magnitude as the medium chain fatty acids. Medium chain fatty acid incorporation into the phospholipid or cholesterol ester fractions by the end of the five-day feeding period was present but minimal. As opposed to conventional long chain triglycerides, intravenously administered medium chain triglycerides are hydrolyzed and cleared rapidly and do not accumulate in other lipid fractions, and are therefore a more readily available lipid fuel.     

Lipid peroxidation during n−3 fatty acid and vitamin E supplementation in humans

The purpose of this study was to investigate in healthy humans the effect of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) intake, alone or in combination with dL-α-tocopherol acetate (vitamin E) supplements on lipid peroxidation. Eightly men were randomly assigned in a double-blind fashion to take daily for 6 wk either menhaden oil (6.26 g, n−3 fatty acids) or olive oil supplements with either vitamin E (900 IU) or its placebo. Antioxidant vitamins, phospholipid composition, malondialdehyde (MDA), and lipid peroxides were measured in the plasma at baseline and week 6. At the same time, breath alkane output was measured. Plasma α-tocopherol concentration increased in those receiving vitamin E (P<0.0001). In those supplemented with n−3 fatty acids, EPA and DHA increased in plasma phospholipids (P<0.0001) and plasma MDA and lipid peroxides increased (P<0.001 and P<0.05, respectively). Breath alkane output did not change significantly and vitamin E intake did not prevent the increase in lipid peroxidation during menhaden oil supplementation. The results demonstrate that supplementing the diet with n−3 fatty acids resulted in an increase in lipid peroxidation, as measured by plasma MDA release and lipid peroxide products, which was not suppressed by vitamin E supplementation.     

Rapid headspace gas chromatography of hexanal as a measure of lipid peroxidation in biological samples

A rapid, sensitive and convenient capillary gas chromatographic-headspace method was developed to determine hexanal as an important volatile decomposition product of hydroperoxides formed from n−6 polyunsaturated fatty acids in rat liver samples. Total volatiles were also determined as a measure of overall lipid peroxidation. Samples of headspace taken from sealed serum bottles incubated at 37°C were injected into a gas chromatograph. It was possible to make 15 determinations per hour. This method is convenient because no special sample manipulations are necessary. The addition of 0.5 mM ascorbic acid prior to gas chromatographic, analysis significantly increased hexanal production. The applicability of the method was demonstrated in studies of the effect of iron in the presence or absence of hydroperoxides of methyl linoleate and methyl linolenate andtert-butyl hydroperoxide on rat liver homogenates, slices and microsomes. A rapid silica cartridge chromatographic procedure was used to purify hydroperoxides from autoxidized methyl linoleate and methyl linolenate, and hydroperoxy epidioxides (cyclic peroxides) from autoxidized methyl linolenate in 20–40 mg quantities. The hydroperoxides and hydroperoxy epidioxides of methyl linolenate were, effective inducers n−6 polyunsaturated fatty acid peroxidation in liver homogenates. Hexanal and thiobarbituric acid-reacting substances were signficantly correlated in liver homogenates and microsomes but not in slices. This specific method for hexanal, a known product of peroxidation of n−6 polyunsaturated fatty acids, can be used as a good measure of lipid peroxidation. Presented at the joint meeting of the Amerian Society for Cell Biology and the American Society for Biochemistry and Molecular Biology, San Francisco, CA January 29–February 2, 1989.     

Altered microsomal phospholipid composition in the streptozotocin diabetic rat

Streptozotocin diabetes in the rat alters liver microsomal membrane fatty acid composition. The present study was undertaken to determine if such changes in fatty acid composition were due to changes in the amount of individual phosphoglycerides or to disproportionate changes in fatty acid composition in any of the individual phosphoglycerides. The diabetic animals showed a small increase in total microsomal phospholipid, which is due to a selective increase in the phosphatidylethanolamine fraction. The changes in fatty acid composition in the total lipid extract (decreased palmitoleic, oleic and arachidonic acids and increased linoleic and docosahexaenoic acids) from the diabetic animals were present in both the major phosphoglycerides, phosphatidylcholine and phosphatidylethanolamine, with very little change in fatty acid composition in the phosphatidylserine and inositol fraction. Further studies are necessary to delineate the cause of the abnormal membrane phospholipid composition in the diabetic animal. Abbreviations: The abbreviated fatty acid nomenclature refers to the number of carbon atoms in the chain, the number of unsaturated bonds, and the position of the first unsaturated bond counting from the terminal methyl group; thus arachidonic acid, 5,8,11,14-eicosatetraenoic acid, is 20∶4ω6.     

Detergent induced changes in serum lipid composition in rats

The influence ofin vivo administration of detergents on serum lipid composition was studied in rats. Male Wistar rats received 50 mg Emulgen 913 (polyoxyethylene nonylphenylether, a nonionic detergent) or SDS (sodium dodecylsulfate, an anionic detergent) per kg of body weight intraperitoneally for 3 consecutive days. Emulgen 913 and SDS administration increased the level of cholesterol esters and phospholipids, respectively. But Emulgen 913 administration reduced the level of triglycerides in the Serum, and SDS administration reduced also the levels of triglycerides and cholesterol esters. In spite of the changes in serum lipid composition, the administration of these detergents did not affect the amount of total lipids in rat serum. The proportion of palmitic, oleic, and docosahexaenoic acids in phospholipids was decreased by the administration of Emulgen 913 while the level of arachidonic acid was raised. However, the level SDS administration had no effect on the fatty acid composition of the serum phospholipids. On the other hand, both Emulgen 913 and SDS administration showed an effect on the fatty acid composition of triglycerides. It is postulated that liver damage due to administration of detergents is responsible for the changes in serum lipid and fatty acid composition in detergent-treated rats.     

Thiobarbituric acid-reactive malondialdehyde formation during superoxide-dependent,iron-catalyzed lipid peroxidation: Influence of peroxidation conditions

A systematic study of the influence of biological lipid peroxidation conditions on lipid hydroperoxide decomposition to thiobarbituric acid-reactive malondialdehyde is presented. A superoxide-dependent, iron-catalyzed peroxidation system was employed with xanthine oxidase plus hypoxanthine plus ferric iron-adenosine diphosphate complex as free radical generator. Purified cardiac membrane phospholipid (as liposomes) was the peroxidative target, and 15-hydroperoxy-eicosatetraenoic acid was used as a standard lipid hydroperoxide. Exposure of myocardial phospholipid to free radical generator at physiological pH (7.4) and temperature (37°C) was found to support not only phospholipid peroxidation, but also rapid lipid hydroperoxide breakdown and consequent malondialdehyde formation during peroxidation. Under lipid peroxidation conditions, oxidative injury to the phospholipid polyunsaturated fatty acids required superoxide radical and ferric iron-adenosine diphosphate complex, whereas 37°C temperature and trace iron were sufficient for lipid hydroperoxide decomposition to malondialdehyde. Harsh thiobarbituric acid-test conditions following peroxidation were not mandatory for either lipid hydroperoxide breakdown or thiobarbituric acid-reactive malondialdehyde formation. However, hydroperoxide decomposition that had begun in the peroxidation reaction could be completed during a subsequent thiobarbituric acid test in which no lipid autoxidation took place. Iron was more critical than heat in promoting the observed hydroperoxide decomposition to malondialdehyde during the lipid peroxidation reaction at 37°C and pH 7.4. These data demonstrate that the radical generator, at physiological pH and temperature, serves a dual role as both initiator of membrane phospholipid peroxidation and promotor of lipid peroxide breakdown and thiobarbituric acid-reactive malondialdehyde formation. Consequently, peroxidation reaction conditions can directly influence lipid hydroperoxide decomposition, malondialdehyde production and system thiobarbituric acid-reactivity. In vivo, decomposition of lipid peroxides to malondialdehyde during radical-mediated, metal-catalyzed membrane peroxidation may represent an integral component of oxidative tissue injury rather than a mere consequence of hydrolyzing the peroxidized biological sample in a thiobarbituric acid test.     

Analyses of renal medullary lipid droplets from normal,hydronephrotic, and indomethacin treated rabbits

Lipid droplets isolated from rabbit renal medullary tissue were analyzed and found to be composed of triglyceride and free fatty acids in a ratio of 2.9:1. These triglycerides were unique when compared to triglycerides of other rabbit tissues examined, in that they contained high percentages of octadecanoic acid (stearic acid, 9.8%), 5,8,11,14-eicosatetraenoic acid (arachidonic acid, 6.8%), and 7,10,13,16-docosatetraenoic acid (adrenic acid, 10%). Lipid droplet triglycerides were found to increase during experimental hydronephrosis and after administration of indomethacin, a prostaglandin synthetase and phosphodiesterase inhibitor. From gas liquid chromatography of fatty acid methyl esters of these triglycerides, it was determined that they were enriched further in their percent composition of 9,12-octadecadienoic acid (linoleic acid) and arachidonic acid, a prostaglandin precursor. The inverse relationship between lipid droplets and prostaglandin content in the inner medulla suggested a significant role of lipid droplet triglycerides as storage pools for prostaglandin precursors.     

Fatty acids and phospholipids of adult and newborn rat hearts and of cultured,beating neonatal rat myocardial cells

Fatty acids and phospholipids of adult and newborn rat hearts and of cultured, neonatal rat heart cells were determined by gas liquid and thin layer chromatographies. In adult heart, the proportion of linoleic acid was higher and that of palmitic acid lower than in newborn hearts or in cultured cells. The relative amounts of linoleic and arachidonic acids in adult heart were affected by the source and amount of dietary fat. In heart cells, after 3 days in culture, the proportion of arachidonic acid resembled that in the newborn and adult rat hearts but showed a gradual and significant decline with age. The gradual shift in fatty acid composition as the cells aged in culture was attributed to outgrowth of mesenchymal cells (fibroblasts and endothelioid cells) characterized by a low relative proportion of arachidonic acid. The amounts of phospholipids in heart cells after 3 days in culture differed from those in the newborn or adult rat hearts. Phosphatidylethanolamine was highest in adult heart (34% of lipid phosphorus) and lowest in cells (26%); lecithin was higher in newborn heart (43%) than in adult heart (37%) or in cells (39%), while sphingomyelin was higher in cells (8%) than in newborn (5%) or adult heart (3%). Phospholipid levels in cultured heart cells were unrelated to those of serum in the growth medium. The absence of a significant change in phospholipid composition after continued incubation of the heart cell cultures for periods up to 3 weeks reflected the major structural role of these lipid components in cell membranes.     

Fatty acid composition of rat hearts as influenced by age and dietary fatty acids

Fatty acid composition of neutral and polar lipid fractions from rat hearts was determined in rats of different ages as their diet source changed. Piebald rats were weaned at 21 days and were fed standard lab chow. Lipids from rat hearts, mothers milk and lab chow were purified on a Sephadex G-25 fine column and separated into neutral and polar lipid fractions by silicic acid column chromatography. These lipid fractions were then hydrolyzed and methylated with BF₃ in methanol, prior to gas liquid chromatographic separation on a 1/8 in. × 10 ft aluminum column of 15% EGS on 80–100 mesh acid-washed Chromosorb W. Three major fatty acids in the neutral lipid fraction comprised 72% of total neutral lipid fatty acids from young hearts. At sexual maturity (at least 74 days old) C_18∶1 was the major fatty acid, followed by C_16∶0 and C_18∶0. The same three fatty acids comprised 83% of total polar lipid fatty acids, but C_18∶0 was the major fatty acid, followed by C_16∶0 and C_18∶1. The fatty acid composition of dietary lipids influenced the total neutral lipid fatty acid composition of the rat heart, but had little influence on the fatty acid composition of the polar lipid fraction. Presented in part at the AOCS Meeting, New Orleans, April 1970.     

Dipyridamole inhibits lipid peroxidation and scavenges oxygen radicals

Dipyridamole incubatedin vitro in systems which peroxidize fatty acids inhibited arachidonic acid, gammalinolenic acid and linoleic acid peroxidation. In the xanthine-xanthine-oxidase system, which produces superoxide anion, dipyridamole inhibited the reduction of cytochrome-C in a dose-dependent fashion. In systems generating hydroxyl radicals, dipyridamole reduced deoxyribose degradation in a dose-dependent manner. The study suggests that dipyridamole inhibits lipid peroxidation, probably by scavening oxygen free radicals.