Showing papers in "Lipids in 1994"

Garlic reduces plasma lipids by inhibiting hepatic cholesterol and triacylglycerol synthesis.

Abstract: Prompted by the reported hypolipidemic activity of garlic, the present study was undertaken to elucidate the mechanism(s) underlying the cholesterol-lowering effects of garlic. Rat hepatocytes in primary culture were used to determine the short-term effects of garlic preparations on [1-14C]acetate and [2-3H]glycerol incorporation into cholesterol, fatty acids and glycerol lipids. When compared with the control group, cells treated with a high concentration of garlic extracts [i.e., petroleum ether- (PEF), methanol- (MEF) and water-extractable (WEF) fractions from fresh garlic] showed decreased rates of [1-14C]acetate incorporation into cholesterol (by 37–64%) and into fatty acids (by 28–64%). Kyolic containingS-allyl cysteine and organosulfur compounds inhibited cholesterogenesis in a concentration dependent manner with a maximum inhibition of 87% at 0.4 mM. At this concentration, Kyolic decreased [1-14C]acetate incorporation into fatty acids by 67%.S-allyl cysteine at 2.0 and 4.0 mM inhibited cholesterogenesis by 20–25%. PEF, MEF and WEF depressed the rates of [2-3H]glycerol incorporation into triacylglycerol, diacylglycerol and phospholipids in the presence of acetate, but not in the presence of oleate. The results suggest that the hypocholesterolemic effect of garlic stems, in part, from decreased hepatic cholesterogenesis, whereas the triacylglycerol-lowering effect appears to be due to inhibition of fatty acid synthesis. Primary hepatocyte cultures as used in the present study have been proven useful as tools for screening the anticholesterogenic properties of garlic principles.

Singlet oxygen quenching ability of naturally occurring carotenoids

Abstract: The singlet oxygen quenching ability of various naturally occurring carotenoids was examined by measuring toluidine blue-sensitized photooxidation of linoleic acid. To assess quenching, the oxidation of linoleic acid was followed by measuring oxygen consumption and ultraviolet absorbance at 235 nm. We found that oxygen quenching increased as the number of conjugated double bonds in the carotenoids increased, but quenching varied with chain structure and functional groups. Acyclic carotenoids enhanced quenching more than did cyclic carotenoids. Conjugated keto groups and the presence of a cyclopentane ring stimulated quenching, while hydroxy, epoxy and methoxy groups showed lesser effects. The photosynthetic bacterial carotenoids, spirilloxanthin and rhodopin, were found to be most effective as quenchers, followed by the cayenne carotenoid, capsorbin.

Evidence that palmitic acid is absorbed as sn-2 monoacylglycerol from human milk by breast-fed infants.

Abstract: Milk fatty acids consist of about 20-25% palmitic acid (16:0), with about 70% of 16:0 esterified to the sn-2 position of the milk triacylglycerols. Hydrolysis of dietary triacylglycerols by endogeneous lipases produces sn-2 monoacylglycerols and free fatty acids, which are absorbed, reesterified, and then secreted into plasma. Unesterified 16:0 is not well absorbed and readily forms soaps with calcium in the intestine. The positioning of 16:0 at the sn-2 position of milk triacylglycerols could explain the high coefficient of absorption of milk fat. However, the milk lipase, bile salt-stimulated lipase, has been suggested to complete the hydrolysis of milk fat to free fatty acids and glycerol. These studies determined whether 16:0 is absorbed from human milk as sn-2 monopalmitin by comparison of the plasma triacylglycerol total and sn-2 position fatty acid composition between breast-fed and formula-fed term gestation infants. The human milk and formula had 21.0 and 22.3% of 16:0, respectively, with 54.2 and 4.8% 16:0 in the fatty acids esterified to the 2 position. The plasma triacylglycerol total fatty acids had 26.0 +/- 0.6 and 26.2 +/- 0.6% of 16:0, and the sn-2 position fatty acids had 23.3 +/- 3.3 and 7.4 +/- 0.7% of 16:0 in the three-month-old exclusively breast-fed (n = 17) and formula-fed (n = 18) infants, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of n−3 and n−6 fatty acids on the activities and expression of hepatic antioxidant enzymes in autoimmune‐prone NZB×NZW F1 mice

Abstract: Menhaden fish oil (FO) containing n−3 fatty acids dramatically extends the life span and delays the onset and progression of autoimmune disease in (NZB×NZW)F1 (B/W) female mice as compared to those fed corn oil (CO) rich in n−6 lipids. As an inefficient antioxidant defense system has been linked to autoimmune diseases, the present study was undertaken to determine whether the protective action of n−3 lipids is mediated through their antioxidant defense system. Weanling B/W mice were fed a nutritionally adequate, semipurified diet containing CO or krill oil (KO) or FO at 10% level (w/w)ad libitum until the mice were 6.5 months old. All diets contained the same level of vitamin E (21.5 mg/100 g diet). We compared the effects of feeding n−6 and n−3 lipids on survival, kidney disease, hepatic microsomal lipid composition, peroxidation, and on the activity and mRNA expression of the antioxidant enzymes catalase, glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) in 6.5-month-old B/W mice. The results showed that when compared to livers from CO-fed mice, livers from KO- and FO-fed mice showed: (i) significantly higher (P<0.001) activities and expression of CAT, GSH-Px and SOD; (ii) significantly lower (P<0.001) arachidonic acid (20∶4n−6) and linoleic acid (18∶2n−6) and higher (P<0.001) eicosapentaenoic acid (20∶5n−3) and docosahexaenoic acid (22∶6n−3) levels in hepatic microsomes; and (iii) significantly lower (P<0.001) estimated peroxidation indices and thiobarbituric acid reactive substances generation. The data indicate that one of the mechanisms through which the n−3 lipids delay the onset of autoimmune diseases in B/W mice may be through maintenance of higher activities and expression of hepatic antioxidant enzymes.

Alteration of chain length selectivity of a rhizopus delemar lipase through site-directed mutagenesis

Abstract: The coding sequences of theRhizopus delemar lipase and prolipase were altered by oligonucleotide-directed mutagenesis to introduce amino acid substitutions. The resulting mutant enzymes, synthesized by the bacterial hostEscherichia coli BL21 (DE3), were tested for their ability to hydrolyze the triglycerides triolein (TO), tricaprylin (TC) and tributyrin (TB). Mutagenesis and lipase gene expression were carried out using plasmid vectors derived from previously described recombinant plasmids [Joerger and Haas (1993)Lipids 28, 81–88] by introduction of the origin of replication of bacteriophage fl. Substitution of threonine 83 (thr83), a residue thought to be involved in oxyanion binding, by alanine essentially eliminated lipolytic activity toward all substrates examined (TB, TO and TC). Replacement of thr83 with serine caused from two- to sevenfold reductions in the activity toward these substrates. Introduction of tryptophan (trp) at position 89, where such a residue is found in closely related fungal lipases, reduced the specific activity toward the three triglyceride substrates. For the mutagenesis of residues in the predicted acyl chain binding groove, mutagenic primers were designed to cause the replacement of a specific codon within the prolipase gene with codons for all other amino acids. Phenylalanine 95 (phe95), phe112, valine 206 (val206) and val209, were targeted. A phenotypic screen was successfully employed to identify cells producing prolipase with altered preference for olive oil, TC or TB. In assays involving equimolar mixtures of the three triglycerides, a prolipase with a phe95→asparatate mutation showed an almost twofold increase in the relative activity toward TC. Substitution of trp for phe112 caused an almost threefold decrease in the relative preference for TC, but elevated relative TB hydrolysis. Replacement of val209 with trp resulted in an enzyme with a two- and fourfold enhanced perference for TC and TB, respectively.

Differential effects of eicosapentaenoic acid and docosahexaenoic acid on human skin fibroblasts

Abstract: To better understand the mode of action of ω3 fatty acids in cell membranes, human foreskin fibroblasts were grown in serum-free medium supplemented with 50 μM oleic acid linoleic acid, eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), and the effects on membrane composition, fluorescence polarization and enzyme activities were followed. The cells were enriched with EPA and DHA up to 7 and 13% of total lipids, respectively, of which >95% was associated with phospholipids. In addition, the concentration of 22∶5n−3 increased with both EPA and DHA to 7.5, and 2.1% of the total fatty acids, respectively. When compared to controls (oleic acid), cells treated with DHA showed a decrease in cholesterol, phospholipids, arachidonic acid (AA) and free cholesterol/phospholipid ratio (P<0.05). In the presence of EPA, only decreases in AA and cholesterol were significant (P<0.05). Membrane fluidity, assessed by fluorescence anisotropy, was increased 16% in cells enriched with DHA (P<0.05), but showed no change with EPA or linoleic acid. There was an increase in membrane-associated 5′-nucleotidase (+27%) and adenylate cyclase (+19%) activities (P<0.05), in DHA-enriched, but not in EPA-enriched cells, when compared with oleate controls. The studies show that incorporation of DHA, but not EPA, into cell membranes of fibroblasts alters membrane biophysical characteristics and function. We suggest that these two major n−3 fatty acids of fish oils have differential effects on cell membranes, and this may be related to the known differences in their physiological effects.

Lymphatic absorption of oxidized cholesterol in rats.

Abstract: The absorption of cholesterol and of cholesterol oxidation products (oxidized cholesterols) was compared in lymph-cannulated rats. We found that the lymphatic absorption of an intragastrically administered, emulsified lipid meal containing 25 mg of cholesterol or 25 mg of oxidized cholesterols, within 24 h, was approximately 67 and 30%, respectively. The absorption rate of individual oxidized cholesterols differed considerably and was approximately 30% for 7α-hydroxycholesterol, 42% for 7β-hydroxycholesterol, 32% for 5β-epoxycholesterol, 28% for 5α-epoxycholesterol, 15% for cholestanetriol and 12% for 7-ketocholesterol. Moreover, cholesterol oxidation products delayed the absorption of oleic acid as triolein. Approximately 35 and 48% of cholesterol was recovered in chylomicrons (CM) and very low density lipoprotein (VLDL), respectively. In contrast, 54 and 40% of the oxidized cholesterols was recovered in CM and VLDL, respectively, although there was a significant difference in the distribution of individual oxidized cholesterols. The results of the present study indicate that oxidized cholesterols are absorbed to a lesser extent than is cholesterol, that they disturb fat absorption and that they distribute differently between lymphatic lipoproteins.

Lipid peroxidation and antioxidant status is affected by different vitamin E levels when feeding fish oil.

Abstract: The protective role of vitamin E and changes in the status of several physiological antioxidants after feeding rats a fish oil diet were investigated. Six-week-old male Sprague-Dawley rats were divided into four groups and fed experimental diets for 8 wk. Three fish oil (FO) groups were fed a menhaden fish oil and soybean oil (SO) (9∶1) mixture as 10% (w/w) of the diet. These groups were provided with ≤3, 45 or 209 IU of vitamin E/kg diet. One SO group was used as control and was fed ≤45 IU of vitamin E/kg diet. Plasma vitamin E levels, when expressed as vitamin E per mL plasma, were extremely low in the group fed FO and ≤3 IU of vitamin E, and were lower in the groups fed FO than in the group fed SO. However, plasma vitamin E levels when expressed per mg plasma lipid were higher in the FO groups provided with ≤45 and 209 IU of vitamin E than in the SO group. Compared with the SO group, plasma levels of thiobarbituric acid reactive substances (TBARS), when expressed per mg lipid, were higher in the three FO groups, plasma retinol levels were lower in the FO groups provided with ≤3 and 45 IU of vitamin E, and ascorbic acid levels were lower only in the FO group provided with ≤3 IU of vitamin E. Blood glutathione (GSH) levels were lower in all three FO groups than in the SO group. Liver vitamin E levels increased as the dietary level of vitamin E increased, but all FO groups had higher liver levels of TBARS than the SO group. The dietary vitamin E levels were correlated positively with plasma vitamin E (r=0.71) and negatively with TBARS in both the plasma and liver of rats fed FO. Among the antioxidants measured, correlations were found between plasma retinol and vitamin C (r=0.64), and plasma vitamin C, uric acid (r=0.72) and blood GSH (r=0.60). Weaker correlations were found between plasma retinol, uric acid and blood GSH. It is concluded that vitamin E requirements are higher when feeding fish oil. Vitamin E seems necessary to prevent enhanced lipid peroxidation and to maintain appropriate levels of other physiological antioxidants.

An improved method for the measurement of malondialdehyde in biological samples

Abstract: An improved method was developed for measuring malondialdehyde (MDA) as its thiobarbituric acid (TBA) complex. Samples were initially incubated with 1% potassium iodide and 0.1% butylated hydroxytoluene at 50°C for 20 min, and then with 0.4% TBA at 60°C for 60 min. The MDA-TBA complex formed was extracted with isobutyl alcohol and measured by high-performance liquid chromatography with fluorescence detection. The improved method allows for a more specific determination of MDA present in biological samples.

Effect of dietary carnosine on plasma and tissue antioxidant concentrations and on lipid oxidation in rat skeletal muscle.

Abstract: The effect of dietary carnosine supplementation on plasma and tissue carnosine and α-tocopherol concentrations and on the formation of thiobarbituric acid reactive substances (TBARS) in rat skeletal muscle homo-genates was evaluated. Plasma, heart, liver and hind leg muscle was obtained from rats fed basal semipurified diets or basal diets containing carnosine (0.0875%), α-tocopheryl acetate (50 ppm), or carnosine (0.0875%) plusα-tocopheryl acetate (50 ppm). Dietary carnosine supplementation did not increase carnosine concentrations in heart, liver and skeletal muscle. Dietary supplementation with both carnosine and α-tocopherol increased carnosine concentrations in liver 1.56-, 1.51- and 1.51-fold as compared with diets lacking carnosine, α-tocopherol or both carnosine and α-tocopherol, respectively. Dietary supplementation with both carnosine and α-tocopherol also increased α-tocopherol concentrations in heart and liver 1.38-fold and 1.68-fold, respectively, as compared to supplementation with α-tocopherol alone. Dietary supplementation with carnosine, α-tocopherol or both car-nosine and α-tocopherol was effective in decreasing the formation of TBARS in rat skeletal muscle homogenate, with dietary α-tocopherol and α-tocopherol plus carnosine being more effective than dietary carnosine alone. The data suggest that dietary supplementation with carnosine and α-tocopherol modulates some tissue carnosine and α-tocopherol concentrations and the formation of TBARS in rat skeletal muscle homogenates.

Diets rich in lean beef increase arachidonic acid and long-chain ω3 polyunsaturated fatty acid levels in plasma phospholipids

Abstract: Diets rich in meat are claimed to contribute to the high tissue arachidonic acid (20:4 omega 6) content in people in Westernized societies, but there are very few direct data to substantiate this assertion. Because meat contains a variety of long-chain polyunsaturated fatty acids (PUFA) that are susceptible to oxidation, we initially examined the effect of cooking on the long-chain PUFA content of beef, and then determined the effect of ingestion of lean beef on the concentration of long-chain PUFA in plasma phospholipids (PL). First, we examined the effect of grilling (5-15 min) and frying (10 min) different cuts of fat-trimmed lean beef on the long-chain PUFA content. Second, we investigated the effect of including 500 g lean beef daily (raw weight) for 4 wk on the fatty acid content and composition of plasma PL in 33 healthy volunteers. This study was part of a larger trial investigating the effect of lean beef on plasma cholesterol levels. In the first two weeks, the subjects ate a very low-fat diet (10% energy) followed by an increase in the dietary fat by 10% each week for the next 2 wk. The added fat consisted of beef fat, or olive oil (as the oil or a margarine) or safflower oil (as the oil or a margarine). This quantity of beef provided 60, 230, 125, 140 and 20 mg/d, respectively, of eicosatrienoic acid (20:3 omega 6), 20:4 omega 6, eicosapentaenoic acid (20:5 omega 3), docosapentaenoic acid (22:5 omega 3) and docosahexaenoic acid (22:6 omega 3).(ABSTRACT TRUNCATED AT 250 WORDS)

Peroxidation reactions in plant membranes: effects of free fatty acids

Abstract: Free fatty acids accumulate in plant membranes after exposure of plants to environmental stress, such as freezing and desiccation. Fatty acid accumulation has been linked to various biophysical changes and to the occurrence of lipid peroxidation, but the relationships appear complex and inconsistent. The interactions between oxygen free radicals, free fatty acids and lipid peroxidation in plant membranes were examined further by studying peroxidation reactions in a model membrane system composed of a complex mixture of plant phospholipids, including various free fatty acids. Multilamellar liposomes were treated with oxygen free radicals generated from iron ascorbate. Increased concentrations of free palmitic acid up to 10 mol% (fatty acid/phospholipid) reduced the production of aldehydes detected by the thiobarbituric acid assay, but enhanced the production of fluorescent products. By contrast, increased concentrations of free linolenic acid increased aldehyde production and reduced the formation of fluorescent products. The two free fatty acids both enhanced the susceptibility of phospholipids to degradation as shown by the reduced recovery of esterified polyunsaturated fatty acids (linoleic and linolenic). The free radical reactions with or without free fatty acid additions catalyzed the selective degradation of phospholipids in the order phosphatidylethanolamine > phosphatidylcholine > phosphatidylinositol > phosphatidylglycerol. Selective degradation of phospholipids is often observed after periods of environmental stress or during senescence of plants, and has been cited as evidence for the involvement of phospholipases in these degenerative processes. The results indicate that selectivity is not a criterion for eliminating the involvement of oxygen free radicals in these degenerative processes. Furthermore, the results suggest that modifications of lipid composition during a plant's acclimation to adverse environments may determine the types of free radical reactions that occur due to stress.

Effect of n−3 fatty acid-rich fish oil supplementation on the oxidation of low density lipoproteins

Abstract: This study was aimed at determining the effect of fish oil supplementation on copper-catalyzed oxidation of low density lipoproteins (LDL) from nine hypertriglyceridemic human subjects. A rapid headspace gas chromatographic method was used to measure the volatile oxidation products from LDL. Propanal and hexanal were the major volatile products formed in the oxidation of n−3 and n−6 polyunsaturated fatty acids (PUFA), respectively. Fish oil supplementation resulted in a significant increase in propanal formation from 3.7 to 13.4 nmol/mL LDL (P<0.01); it also resulted in small decreases in pentanal formation from 14.7 to 11.4 nmol/mL LDL and in hexanal formation from 138 to 108 nmol/mL LDL (P<0.05). The changes in peroxidation products paralleled the changes in LDL composition, which showed a significant increase in n−3 PUFA from 3.2 to 14.6% (P<0.01) and a decrease in n−6 PUFA from 43.7 to 35.0% (P<0.05). Propanal formation was highly and significantly correlated with n−3 PUFA content (r=0.950,P<0.001). Since total volatiles remained unchanged, this indicated that the two groups of LDL samples did not differ in overall oxidative susceptibility. Although fish oil intake did not alter the oxidative susceptibility of LDL, the chemically modified LDL particles generated a distinct pattern of volatile oxidation products that reflected changes in their fatty acid composition.

Effect of dietary fish oil on blood levels of free fatty acids, ketone bodies and triacylglycerol in humans

Abstract: Although the reduction of serum triacylglycerol concentrations by dietary fish oil is a well-known effect, the exact mechanism of this effect has not been previously studied in human subjects. Therefore, the aim of this study was (i) to examine the effect of short-term fish oil supplementation on blood concentrations of ketone bodies, free fatty acids and triacylglycerol in healthy humans and (ii) to verify whether the observed relationships between these variables would be consistent with reduced lipolysis and/or enhanced hepatic fatty acid oxidation after fish oil supplementation. Twenty subjects (21–23 years, normal liver function tests) were randomly divided into two groups to supplement their usual diet with either 30 g/d of fish oil (n=11) or olive oil (n=9). Venous blood samples were drawn after an overnight fast, before and after 1, 3 and 7 d of fish oil/olive oil supplementation. Blood concentrations of triacylglycerol and free fatty acids decreased consistently after fish oil supplementation; the reduction was already significant after one day of fish oil (P 0.10). No significant changes in glucose, insulin or ketone body levels were observed in either group after supplementation. After fish oil, but not after olive oil supplementation, the ratio of blood ketone body levels to free fatty acid levels increased significantly (P<0.05). Furthermore, after fish oil supplementation only, free fatty acid levels were significantly correlated with levels of ketone bodies (day 7 of supplementation: r=0.90,P<0.001) and triacylglycerol (maximum value on day 3: r=0.77,P<0.01). These findings suggest that reduced lipolysis and increased hepatic β-oxidation/ketogenesis may contribute to reduced triacylglycerol levels after ω3 fatty acid supplementation in humans. Turnover studies are needed in order to further quantitate these processes.

The reaction of low levels of nitrogen dioxide with methyl linoleate in the presence and absence of oxygen

Abstract: The reaction of methyl linoleate with low levels of nitrogen dioxide in a carrier gas, such as helium or air, at nitrogen dioxide concentrations ranging from 2 to 228 ppm was studied and the products formed were monitored. In both aerobic and anaerobic conditions, low concentrations of nitrogen dioxide reacted with methyl linoleate predominately to form allylic products. When a 1∶1 mixture of methyl palmitate/methyl linoleate was layered over an aqueous buffer and a nitrogen dioxide stream was passed from underneath, so that the stream passed through the aqueous layer before contacting the organic layer, allylic products again predominated. In the absence of air, the allylic products consisted of allylic nitro and nitrite derivatives of linoleate, whereas in the presence of air, allylic hydroperoxides were the principal products. The findings suggest that fatty acids with doubly allylic hydrogen atoms react preferentially by a hydrogen atom abstraction reaction rather than by the addition of nitrogen dioxide to a double bond.

Production of docosahexaenoic acid by marine bacteria isolated from deep sea fish.

Abstract: Five bacterial strains isolated from the intestine of deep sea fish were shown to produce docosahexaenoic acid (22∶6n−3; DHA) at a level of 6.4 to 11.6% of total fatty acids when incubated in DHA-free medium. In all of the strains examined, other polyunsaturated fatty acids were barely detectable, except for eicosapentaenoic acid (20∶5n−3). A typical strain, such as T3615, produced DHA at a concentration of about 0.8 mg/L within six days of aerobic incubation at 5°C and under atmospheric pressure. The T3615 strain, belonging to the genusVibrio, is rod-shaped, Gram-negative, motile and facultatively anaerobic.

Trp89 in the lid of Humicola lanuginosa lipase is important for efficient hydrolysis of tributyrin.

Abstract: To determine whether Trp89 located in the lid of the lipase (EC 3.1.1.3) fromHumicola lanuginosa is important for the catalytic property of the enzyme, site-directed mutagenesis at Trp89 was carried out. The kinetic properties of wild type and mutated enzymes were studied with tributyrin as substrate. Lipase variants in which Trp89 was changed to Phe, Leu, Gly or Glu all showed less than 14% of the activity compared to that of the wild type lipase. The Trp89Glu mutant was the least active with only 1% of the activity seen with the wild type enzyme. All Trp mutants had the same binding affinity to the tributyrin substrate interface as did the wild type enzyme. Wild type lipase showed saturation kinetics against tributyrin when activities were measured with mixed emulsions containing different proportions of tributyrin and the nonionic alkyl polyoxyethylene ether surfactant, Triton DF-16. Wild type enzyme showed a Vmax=6000±300 mmol·min−1·g−1 and an apparent Km=16±2% (vol/vol) for tributyrin in Triton DF-16, while the mutants did not show saturation kinetics in an identical assay. The apparent Km for tributyrin in Triton DF-16 was increased as the result of replacing Trp89 with other residues (Phe, Leu, Gly or Glu). The activities of all mutants were more sensitive to the presence of Triton DF-16 in the tributyrin substrate than was wild type lipase. The activity of the Trp89Glu mutant was decreased to 50% in the presence of 2 vol% Triton DF-16 compared to the activity seen with pure tributyrin as substrate. Wild type lipase and all mutants except Trp89Glu had the same affinity for the substrate interface formed by 15.6 vol% tributyrin in Triton DF-16. The Trp89Glu mutant showed a lower affinity than all the other lipase variants for the interface of 15.6 vol% tributyrin in Triton DF-16. The study showed that Trp89 located in the lid ofH. lanuginosa lipase is important for the efficient hydrolysis of tributyrin and that this residue plays a role in the catalytic steps after adsorption of the lipase to the substrate interface.

Lipid composition of glucose-stimulated pancreatic islets and insulin-secreting tumor cells

Abstract: The effect of glucose stimulation (25 mM for 5 min) on the phospholipid and neutral lipid composition of isolated pancreatic islets was studied to find out whether there is a change in the mass of potential lipid mediators or modulators of insulin secretion. For comparison, the lipid compositions of homogenates and subcellular fractions from RINm5F insulin-secreting tumor cells and of glucose-stimulated streptozotocin/nicotinamide-induced islet cell tumors were analyzed. After separation of the lipid extract into a neutral and an acidic fraction by anion-exchange chromatography, lipids were separated by high-performance thin-layer chromatography and quantitated by in situ densitometry of the cupric sulfate-charred bands. In glucose-stimulated islets, the molar percentages of phosphatidic acid (PA) and of phosphatidylinositol were significantly increased (3.1 vs. 4.7 mol% and 8.6 vs. 11.8 mol%), while those of all other phospholipids and neutral lipids, including 1,2-diacylglycerol, were not significantly changed. In stimulated islet cell tumors, an increase of PA was visible in the microsomal fraction, and there was an increase of lysophosphatidylcholine in the mitochondrial fraction. However, in both tumoral tissues, particularly in RINm5F cells, the lipid distribution pattern showed abnormalities which can be regarded as a loss of differentiation and which limit the usefulness of these tissues for the study of the physiological regulation of lipid metabolism during glucose stimulation. In conclusion, the data are in accordance with a role of PA early in stimulus-secretion coupling. The well-known stimulation of phospholipid synthesis in pancreatic islets during glucose-induced insulin secretion does not result in an increase in the total phospholipid mass.

Rat liver chromatin phospholipids

Abstract: To shed light on the question whether the phospholipids present in chromatin are native or are due to contamination from nuclear membranes, we labeled the phospholipids of isolated nuclei and determined the amount of phospholipids (PL) and PL fatty acid composition in nuclei and chromatin. The hepatocyte nuclei were isolated and radioiodinated by the lactoperoxidase method under saturating and nonsaturating conditions, and the radioactivity associated with chromatin extracted from these nuclei was monitored. Whereas 97% the label was recovered in the nuclear membranes, only 0.08–0.6% was found in chromatin. The PL present in chromatin were relative to the amounts present in the entire nuclei and calculated as percentage of total, phosphatidylethanolamine (10%), phosphatidylserine (22%), phosphatidylinositol (19%) phosphatidylcholine (14%), and sphingomyelin (35%). In sphingomyelin of chromatin-associated PL an enrichment in polyunsaturated fatty acids was seen. The data indicated that the PL found in isolated chromatin do not seem to be due to contamination from the nuclear membrane.

Dietary lipids modify the fatty acid composition of cartilage, isolated chondrocytes and matrix vesicles.

Abstract: The effects of dietary lipids on the fatty acid composition of hyaline cartilage, epiphyseal chondrocytes (EC) and matrix vesicles (MV) were evaluated in chicks. A basal semipurified diet was fed to chicks containing one of the following lipid sources at 70 g/kg: soybean oil, butter+corn oil, margarine+corn oil or menhaden oil+corn oil (MEC). Articular and epiphyseal growth cartilage were isolated from the proximal tibiotarsus; EC and MV were subsequently released by trypsin (EC 3.4.21.4) and collagenase (EC 3.4.24.3) digestion followed by ultracentrifugation. The fatty acid composition of polar lipids in chick epiphyseal cartilage at three and six weeks, as well as articular cartilage, EC and MV at eight weeks of age revealed the presence of high levels of saturated and monounsaturated fatty acids (up to 85.5%) but low levels of n−6 polyunsaturated fatty acids (PUFA) (2.6–10.2%). Mead acid (20∶3n−9,>3%) was also present in cartilage, EC and MV lipids, and was unaffected by the dietary lipid treatments. Total n−3 PUFA concentrations were the highest in cartilage, EC and MV of chicks consuming MEC. Feeding MEC lowered the levels of 20∶4n−6 in cartilage, but increased 20∶5n−3 levels. The data are consistent with those reported previously which showed that cartilage tissues are low in n−6 PUFA and that they contain 20∶3n−9. We furthermore demonstrated that the PUFA composition of cartilage can be modified by dietary lipids.

Incorporation of trans long-chain n-3 polyunsaturated fatty acids in rat brain structures and retina.

Abstract: During heat treatment, polyunsaturated fatty acids and specifically 18∶3n−3 can undergo geometrical isomerization. In rat tissues, 18∶3 Δ9c, 12c, 15t, one of thetrans isomers of linolenic acid, can be desaturated and elongated to givetrans isomers of eicosapentaenoic and docosahexaenoic acids. The present study was undertaken to determine whether such compounds are incorporated into brain structures that are rich in n−3 long-chain polyunsaturated fatty acids. Two fractions enriched intrans isomers of α-linolenic acid were prepared and fed to female adult rats during gestation and lactation. The pups were killed at weaning. Synaptosomes, brain microvessees and retina were shown to contain the highest levels (about 0.5% of total fatty acids) of thetrans isomer of docosahexaenoic acid (22∶6 Δ4c, 7c, 10c, 13c, 16c, 19t). This compound was also observed in myelin and sciatic nerve, but to a lesser extent (0.1% of total fatty acids). However, the ratios of 22∶6trans to 22∶6cis were similar in all the tissues studied. When the diet was deficient in α-linolenic acid, the incorporation oftrans isomers was apparently doubled. However, comparison of the ratios oftrans 18∶3n−3 tocis 18∶3n−3 in the diet revealed that thecis n−3 fatty acids were more easily desaturated and elongated to 22∶6n−3 than the correspondingtrans n−3 fatty acids. An increase in 22∶5n−6 was thus observed, as has previously been described in n−3 fatty acid deficiency. These results encourage further studies to determine whether or not incorporations of suchtrans isomers into tissues may have physiological implications.

Lipoprotein lipase and hepatic lipase in Wistar and Sprague-Dawley rat tissues. Differences in the effects of gender and fasting

Abstract: To evaluate the effects of strain, gender and fasting in the regulation of lipoprotein lipase (LPL) and hepatic lipase (HL) activities were measured in tissues of male and female Wistar and Sprague-Dawley rats after feeding or a 24-h starvation period. It is noteworthy that an effect of gender on LPL activity was observed in Wistar, but not in Sprague-Dawley rats, not only in the basal (fed) activity in several tissues, such as white and brown adipose tissues, heart, and brain, but also in response to fasting which affected LPL activity in brown adipose tissue, heat and lung of female but not of male Wistar rats. By contrast, HL activity in liver, plasma and adrenals of Sprague-Dawley rats was higher in females than in males. No effect of gender on HL activity was observed in Wistar rats. Our results indicate that differences exist between Wistar and Sprague-Dawley rats in the regulation of both LPL and HL. Some of the contradictory results found in the literature may be explained by the differences between rat strains and gender, as well as differences in the nutritional status of the animals.

Absorption of isomeric, palmitic acid-containing triacylglycerols resembling human milk fat in the adult rat

Abstract: The effect of the positional distribution of palmitic acid (16∶0) in triacylglycerols (TAG) on 16∶0 apparent absorption in adult rats was investigated. The rats were fed two diets which contained 30 energy % as fat with identical total fatty acid compositions, both containing 30% 16∶0. The Betapol diet contained TAG with 73% of total 16∶0 in thesn-2 position, the control diet contained TAG with 6% of total 16∶0 in thesn-2 position. After six weeks on these diets, the rats were killed two or six hours after the last meal, and the small intestine was removed, cut into 10-cm segments, and the fatty acid composition of the segment's contents was determined. At both time points the amount of 16∶0 in the intestinal segments starting at 40 cm from the stomach was much lower in the animals fed Betapol than in the animals fed the control diet. Overall absorption of 16∶0 and stearic acid was significantly greater in the Betapol group. Absorption of oleic and linoleic acid from the small intestine was similar in both groups, although the overall absorption was significantly greater in the animals fed Betapol. Total fat absorption was significantly higher in the Betapol-fed rats than in the control-fed rats. No effect on calcium and nitrogen absorption, on plasma total cholesterol and TAG levels, and on bodyweights (growth) was seen. The data demonstrate that the positional distribution of the fatty acids in the TAG molecule affects the site of absorption in the small intestine and particularly the net absorption of saturated fatty acids.

Blood polyunsaturated fatty acids in patients with peroxisomal disorders. A multicenter study.

Abstract: The purpose of the study was to compare the polyunsaturated fatty acid (PUFA) status in patients with X-linked adrenoleukodystrophy or adrenomyeloneuropathy (X-ALD/AMN) with that in disorders of peroxisome biogenesis (PB). Total fatty acids and plasmalogens were quantified in plasma and red cells from 28 patients with X-ALD/AMN, 26 patients with generalized peroxisomal disorders, and 37 controls. Total fatty acid methyl esters and plasmalogen dimethyl acetals were obtained by direct transmethylation and separated by capillary column gas chromatography. The results confirm previous findings in that docosahexaenoic acid (DHA, 22∶6n−3) was greatly decreased in both plasma and erythrocytes from patients with PB disorders. When nutritional conditions were adequate, patients with X-ALD/AMN had normal levels of DHA. A highly significant positive correlation was found between the levels of DHA and those of plasmalogens in peroxisomal patients. As in other tissues, the parent n−6 fatty acid, linoleic acid (LA, 18∶2n−6) was significantly increased in red cells from PB patients, whereas arachidonic acid (20∶4n−6) was virtually within normal limits. In clear contrast to red cells and other tissues, arachidonate was significantly lower in plasma from PB patients. The decrease in plasma arachidonate and the high tissue levels of LA suggest a defect of Δ6 desaturase and/or Δ5 desaturase in PB patients. The n−6 fatty acids were normal in X-ALD/AMN patients. The present data show that X-ALD/AMN patients do not have the profound PUFA alterations that PB patients have, at least in blood.

Vitamin E isomers in grain amaranths (Amaranthus spp.)

Abstract: Vitamin E isomers are important antioxidants, but their variation is poorly documented in pseduocereal grains such as amaranths. Using normal-phase, high-performance liquid chromatography with fluorescence detection, seeds of thirteen amaranth (Amaranthus cruentus L.,A. hypochondriacus L.) accessions were surveyed for the composition of tocols. The most common tocols found were α-tocopherol (2.97 to 15.65 mg/kg seed) and β-tocotrienol (5.92 to 11.47 mg/kg seed) and γ-tocotrienol (0.95 to 8.69 mg/kg seed), while someA. cruentus accessions contained δ-tocotrienol (0.01 to 0.42 mg/kg seed). This is the first report of tocotrienols in amaranths.Amaranthus cruentus grain-types of Mesoamerican origin had significantly (P≤0.01) greater levels of four tocols than didA. cruentus African vegetable-types. Unlike many cereal grains, amaranths have significant amounts of both β- and γ-tocotrienols; however, β-tocopherol was not detected in any of the amaranths. Using multiple linear regressions, α-tocopherol variation of both species and types was consistently explained by variation in tocols other than α-tocopherol. On the whole, fresh amaranth samples of both species tended to have higher levels of tocotrienols than samples stored for two years. Storage effects on amaranth tocol composition are suspected.

Gangliosides and atherosclerosis

Abstract: The ganglioside levels in atherosclerotic lesions of human aorta are considerably higher than those in unaffected areas of aorta, and atherosclerotic patients frequently have increased concentrations of serum gangliosides. The present review summarizes recent findings that suggest the possible involvement of aortic gangliosides in platelet activation and adhesion of platelets to the vessel wall. The effect of gangliosides on the structure of low density lipoproteins (LDL), on the interaction of LDL with macrophages and hepatic cells and on the LDL-regulated biosynthesis of cholesterol is also discussed.In vitro experiments have demonstrated that a major ganglioside of the intima of atherosclerotic aorta induces rapid adhesion, aggregation and spreading of platelets. Moreover, gangliosides present in elevated amounts in the intercellular space of atherosclerotic aortic tissue modify the surface structure and stimulate aggregation of LDL. Ganglioside-modified LDL are readily recognized and taken up by macrophages, while preincubation of LDL with low concentrations of gangliosides inhibits LDL binding to hepatic cells. Thus, ganglioside enrichment of LDL is likely to interfere with LDL clearancevia the hepatic cells. Thus, ganglioside enrichment of LDL is likely to interfere with LDL clearancevia the hepatic LDL receptor, and to stimulate binding of LDL to the scavenger receptor of macrophages. It is postulated that high ganglioside levels in the aorta and serum may be an additional risk factor in atherosclerosis.

Occurrence of 5c,8c,11c,15t‐eicosatetraenoic acid and other unusual polyunsaturated fatty acids in rats fed partially hydrogenated calola oil

Abstract: Uncommoncis andtrans fatty acids can be desaturated and elongated to produce unusual C18 and C20 polyunsaturated fatty acids in animal tissues. In the present study we examined the formation of such metabolites derived fromcis andtrans isomers of oleic and linoleic acids of partially hydrogenated vegetable oil origin in rats. For two months, aduut male rats were fed a partially hydrogenated canola oil diet containing moderately high levels oftrans fatty acids (9.6 energy%) and an adequate level of linoleic acid (1.46 energy%). Analysis of the phospholipid (PL) fatty acids of liver, heart, serum and brain showed no new C18 polyunsaturated fatty acids, except for those uncommon 18∶2 isomers originating from the diet. However, minor levels (each <0.3% PL fatty acids) of six unusual C20 polyunsaturated fatty acids were detected in the tissues examined, except in brain PL. Identification of their structures indicated that the dietary 9c,13t−18∶2 isomer, which is the majortrans polyunsaturated fatty acid in partially hydrogenated vegetable oils, was desaturated and elongated to 5c,8c,11c,15t−20∶4, possibly by the same pathway that is operative for linoleic acid. Furthermore, dietary 12c−18∶1 was converted to 8c,14c−20∶2 and 5c,8c,14c−20∶3; dietary 9c,12t−18∶2 metabolized to 11c,14t−20∶2 and 5c,8c,11c14t−20∶4, and dietary 9t,12c to 11t,14c−20∶2. These results suggested that of all the possible isomers of oleic and linoleic acids in partially hydrogenated vegetable oils, 12c−18∶1, 9c,13t−18∶2, 9c,12t−18∶2 and 9t,12c−18∶2 are the preferred substrates for desaturation and elongation in rats. However, their conversions to C20 metabolites were not as efficient as that of oleic or linoleic acids.

Thermal adaptation affects the fatty acid composition of plasma phospholipids in trout

Abstract: The adaptive changes in the fatty acid (FA) composition of plasma phospholipids (PL) in response to alterations in environmental water temperature were investigated in juvenile rainbow trout (Oncorhynchus mykiss). The changes observed during thermal adaptation from 22°C in summer to 8°C in winter were reproduced by laboratory cold acclimation (CA) at 6°C of 22°C-summer-acclimated animals. In cold-acclimated and winter-acclimated trout, the increase in the unsaturation of PL fatty acids was mainly due to an enrichment of approximately 7% in the total weight percentage of 22∶6n−3, while a concomitant significant decrease in the levels of 18∶0 and of the monoenoic n−9 FA was observed. A time course study revealed that the changes in PL fatty acids became significant after 10 d of CA and were complete after one month. These changes in the composition of the fatty acyl chains of plasma total PL indicate that the FA composition of plasma lipoprotein PL does not remain constant during thermal adaptation. This would suggest that plasma lipoproteins provide a rapid systemic supply of lipids containing more or less unsaturated FA during thermal adaptation of poikilothermic animals.

Serum fatty acid profiles in cystic fibrosis patients and their parents

Abstract: Fatty acid compositions of the major serum lipid classes from 43 cystic fibrosis (CF) homozygotes (CF patients), 36 obligate heterozygotes (parents of CF patients) and 34 controls were determined by capillary gas chromatography. Fatty acid compositions of the homozygote CF patients were skewed in the direction of relative essential fatty acid deficiency in comparison with the controls. Less pronounced, but similar deviations from normal, were observed in the heterozygotes. Homozygotes with normal fatty acid compositions and heterozygotes with considerably disturbed fatty acid profiles were found.

β-Carotene transport in human lipoproteins. Comparisons with a-tocopherol

Abstract: The purpose of this study was to investigate the temporal relationships of the transport of β-carotene in human lipoproteins. We administered 60 mg β-carotene with breakfast to nine fasting subjects, then blood samples were collected at intervals of up to 75 h, lipoproteins were isolated, and β-carotene was quantitated. β-Carotene concentrations in chylomicrons and very low density lipoproteins (VLDL) peaked at 6 and 9 h, respectively. Nonetheless, at all time points the majority of plasma β-carotene was contained in low density lipoproteins (LDL), while high density lipoproteins (HDL) carried a smaller portion (at 24 h, 73±8% in LDL as compared with 23±5% in HDL). In three subjects, transport of β-carotene was compred with the results of earlier studies on the transport of stereoisomers of α-tocopherol. Unlike plasmaRRR-α-tocopherol concentrations, which are maintained by the preferential incorporation ofRRR-α-tocopherol into VLDL by the liver, β-carotene increased and decreased in VLDL similarly toSRR-α-tocopherol, a stereoisomer whose concentrations are not maintained in plasma. In conclusion, β-carotene is primarily transported in the plasma in LDL, but its incorporation by the liver into lipoproteins does not appear to be enhanced.