Preparation of fully deuterated fatty acids by simple method

Lipids - An economical, simple, efficient system of deuterating saturated fatty acids at atmospheric pressure has been developed. Exchange is carried out between fatty acids and deuterium gas over...     

Dual-labeled technique for human lipid metabolism studies using deuterated fatty acid isomers

Two deuterated fatty acids, elaidate-d ₂ and oleate-d ₄, were fed simultaneously to a human subject as a mixture of trielaidin-d ₆ and triolein-d ₁₂. Periodically, blood samples were drawn, and red blood cells were separated from the plasma. Red blood cells and plasma lipids were fractionated and analyzed by combined gas chromatography—multiple ion mass spectroscopy. Dual deuterium-labeling allows rate and extent of fatty acid incorporation to be followed in various plasma and red cell neutral and phospholipid fractions. Maximum amount of deuterated fat varied from 4% in cholesterol ester to 64% in phosphatidyl ethanolamine. The highest levels of deuterated fat occurred in either 6-, 8-, or 12-hr samples; generally, <1% labeled fatty acids could be detected in 72-hr samples. Because the method is based on dual-labeling, differences in the relative incorporation of both fatty acid isomers can be compared directly. Differences in rates of incorporation, rates of removal, and extent of incorporation of labeled fatty acids into blood plasma can also be determined reliably. Our experimental labeling of fats with deuterium permits for the first time the metabolism of two fatty acid isomers to be compared simultaneously in human subjects. This new method should be applicable to a variety of other lipid metabolic studies. Presented at the AOCS meeting Dallas, Texas, April 27–30, 1975.     

Dietary fatty acids: Their metabolic fate and influence on fatty acid biosynthesis

Adult rats fed a low-fat diet or diets containing 15% of either tripalmitin, triolein or trilinolein were injected intraperitoneally with H³-labeled acetate. Those which received fat were also given by mouth, simultaneously with acetate, the 1-C¹⁴-labeled sodium salt of the respective dietary fatty acid. The fate of the tagged material was followed by time-spaced biopsies of subcutaneous adipose tissue and by collection of the expired C¹⁴O₂. After 72 hr, 51, 64, and 52% of the dietary palmitate, oleate, and linoleate, respectively, were catabolized, as indicated by the corresponding percentages of the label having been excreted as C¹⁴O₂. Dietary linoleate was relatively less incorporated into body triglycerides than palmitate and oleate. Animals ingesting diets of 15% triolein had only about one-half the amount of phospholipids in their tissues as had the other groups. The distribution of both the C¹⁴ and H³ labels in the tissue triglycerides showed that all diets containing fats decreased fatty acid synthesis but did not inhibit conversion of palmitate to oleate. Conversions of oleate or linoleate appeared to be through acetate. As a result of these factors, the fatty acid composition of the tissue triglycerides after 3 months’ ingestion of tripalmitin was essentially the same as that of the low-fat group, whereas the ingestion of triolein produced triglycerides with a very high content of oleic acid. Trilinolein ingestion produced effects similar to triolein but to a less pronounced degree. Both the respiratory C¹⁴O₂ and the C¹⁴- and H³-labeled fatty acids in subcutaneous adipose tissue exhibited a second rise in specific activity 12 to 24 hours after the administration of the label. Presented at the AOCS meeting, Chicago, 1964.     

GC-MS外标法分析核桃仁中的脂肪酸含量

利用气相色谱-质谱联用(GC-MS)的外标法对核桃仁中的脂肪酸进行定量分析。核桃仁脂肪酸种类丰富,常见的4种脂肪酸在核桃仁中的含量:亚油酸油酸棕榈酸硬脂酸。相对标准偏差RSD(n=5)在2.27%~11.98%之间,样品回收率测定在81.0%~109.0%之间。     

Effect of dietary fatty acid composition on the biosynthesis of unsaturated fatty acids in rat liver microsomes

A study was made of the influence of semisynthetic diets of low and high unsaturation on the fatty acid composition and desaturation-chain elongation enzymatic activity of the liver microsomal fractions of male Sprague-Dawley rats of different ages. Groups of rats were fed 5 or 20% coconut oil (CO), or a 5 or 20% mixture of corn and menhaden oils (3∶7) (CME) from weaning to 100 wk of age. Growth rate and food consumption were measured during this period in which animals were sacrificed at 36, 57, 77 and 100 wk of age. Both the level and composition of the dietary fat supplements produced marked effects on the fatty acid composition of the liver microsomal lipids. In general, the fatty acid composition of the microsomal fractions reflected that of the dietary fat and was more unsaturated with the higher level of fat fed. The rate of conversion of linoleic to arachidonic acid in assays performed in vitro with liver microsomal preparations from animals of the different groups also showed marked differences. The 6-desaturase-chain elongation activity was higher in the 5% than 20% group and corresponded to the essential fatty acid (EFA) status of the animals in these groups as represented by the triene-tetraene ratio of the microsomal lipid. The relationship of the 6-desaturase activity to fatty acid composition of the microsomal lipid indicated that if varied directly with the level of 20∶3ω9, 18∶1 and 16∶1 and was inhibited by arachidonic acid. The activity of the 6-desaturase enzyme system was lowest in the liver microsomal fraction obtained from the animals fed the CME diets and appeared to be suppressed by the high levels of 20∶5 and 22∶6 that accumulated in the microsomal lipid. Accordingly, the levels of arachidonic acid were lower in the microsomal lipid of these groups than those of the corresponding CO groups in spite of a greater abundance of linoleic acid in the diet. The data suggest that the activity of the 6-desaturase-chain elongation system is regulated by the fatty acid composition of the microsomal lipid as influenced by the composition of the dietary fat.     

单体酸混合物成分的GC-MS分析

研究了生产二聚酸时得到的副产品单体酸的化学组成的GC MS分析方法,样品首先用质量浓度为140g/L KOH M eOH进行甲酯化,然后用GC MS进行分析和鉴定,共鉴定出棕榈酸、肉豆蔻酸、10甲基月桂酸、12甲基十四烷酸、十八烯酸等13种物质,总脂肪酸的质量分数占76.71%,其中饱和酸占66.36%,不饱和酸占10.35%,其中直链酸占饱和脂肪酸的41.02%,支链酸即异构酸占饱和脂肪酸的58.98%。     

The biosynthesis of polyunsaturated fatty acids in plants

This paper is a review of some of the work being done at the author's laboratory. The phospholipids and glycolipids of the alga,Chlorella vulgaris, have been implicated in fatty acid transformations such as chain elongation and desaturation. Labeling studies with [¹⁴C] acetate have shown that newly synthesized galactosyl glycerides have mainly saturated fatty acids. Subsequent to de novo synthesis, a series of alterations of fatty acid structure takes place within the same glycolipid molecules. The specific incorporation of [¹⁴C] oleic acid intoChlorella phosphatidyl choline provides a convenient model system for studying the lipid dependent desaturation of oleic to linoleic acid. The inhibitor of fatty acid desaturation, sterculic acid, only inhibits the conversion of oleate into linoleate if added before the precursor fatty acid has been incorporated into a complex lipid. Studies with isomeric monoenoic fatty acids have suggested that there are two enzymes which catalyze the formation of linoleic from oleic acid. One measures the position of the second double bond from the carboxyl group, the other, from the methyl end of the chain. The latter enzyme probably requires the complex lipid substrate.     

Purification of cyclic fatty acid esters: a GC-MS study

Gas chromatographic analysis of cyclic monomeric concentrates and fractions from argentation chromatography on packed columns containing SE-30, OV-25 and Apiezon L stationary phases yielded incompletely separated peaks representing the various isomers present in the mixture. Somewhat better separation was achieved using a 6 ft×1/8 in. column packed with 15% EGS on Chromosorb W. This column, when coupled to a mass spectrometer, yielded information concerning the composition of each of the isomeric components. Comparable results were obtained using a 50 ft×0.02 in. S.C.O.T. column with DEGS stationary phase and a 150 ft×0.01 in. capillary column coated with Apiezon L. While argentation thin layer chromatography proved useful, an argentation column method using silicic acid coated with 10% AgNO₃ proved more efficient for larger scale preparations. Elution of the column with 2% diethyl ether in petroleum ether yielded material essentially free of conjugated linolenate. A comparison of the behavior upon argentation thin layer chromatography of conjugated methyl linolenate, methyl linoleate and cyclic monomer esters indicated that these esters migrated to the same relative position as methyl oleate. Presented at the AOCS Meeting, Los Angeles, April 1972. Abstracted in part from the dissertation to be submitted to the University of Illinois Graduate College in partial fulfillment of the requirements for the Ph.D. degree.     

In vitro biosynthesis of fatty acids inDrosophila melanogaster

A new in vitro technique, utilizing ruptured larvae ofDrosophila melanogaster, was employed to study the incorporation of³H-acetate into long-chain fatty acids. Preparative gas-liquid chromatography and scintillation spectroscopy were used to determine the relative activity of each fatty acid from total lipid extracts. Quantitative changes were observed in the distribution of label during the course of the incubation times, which ranged from five minutes to nine hours. All fatty acids which were observed to incorporate acetate in previous in vivo studies also showed incorporation of label under these in vitro conditions. It is concluded that this system may be useful for studying aspects on insect metabolism for short intervals of time.     

A pathway for biosynthesis of divinyl ether fatty acids in green leaves

[1-¹⁴C]α-Linolenic acid was incubated with a particulate fraction of homogenate of leaves of the meadow buttercup (Ranunculus acris L.). The main product was a divinyl ether fatty acid, which was identified as 12-[1′(Z),3′(Z)-hexadienyloxy]-9(Z), 11(E)-dodecadienoic acid. Addition of glutathione peroxidase and reduced glutathione to incubations of α-linolenic acid almost completely suppressed formation of the divinyl ether acid and resulted in the appearance of 13(S)-hydroxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid as the main product. This result, together with the finding that 13(S)-hydroperoxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid served as an efficient precursor of the divinyl ether fatty acid, indicated that divinyl ether biosynthesis in leaves of R. acris occurred by a two-step pathway involving an ω6-lipoxygenase and a divinyl ether synthase. Incubations of isomeric hydroperoxides derived from α-linolenic and linoleic acids with the enzyme preparation from R. acris showed that 13(S)-hydroperoxy-9(Z), 11(E)-octadecadienoic acid was transformed into the divinyl ether 12-[1′(Z)-hexenyloxy]-9(Z), 11(E)-dodecadienoic acid. In contrast, neither the 9(S)-hydroperoxides of linoleic or α-linolenic acids nor the 13(R)-hydroperoxide of α-linolenic acid served as precursors of divinyl ethers.     

Composition and biosynthesis of fatty acids inPyramimonas grossii

The green algaPyramimonas grossii orginating in the coastal waters of the Atlantic Ocean Argentina was subcultured until a monoalgal culture was obtained. The fatty acid composition of the alga grown in a mineral medium at 12 C was determined by gas liquid chromatography (GLC) on 2 columns. The major fatty acids were oleic, linoleic, palmitic and α-linolenic acids, but the 20-carbon polyunsaturated acids, 20∶4ω6 and 20∶5ω3, respectively, belonging to the linoleic and α-linolenic series, were also found. Incubation with [¹⁴C] oleate, [¹⁴C] acetate, [¹⁴C] linoleate and [¹⁴C] α-linolenate suggests that linoleate is not directly converted to α-linolenate. [¹⁴C] Acetate was easily converted to palmitic, palmitoleic and oleic acids. However, after 48 hr of incubation, only traces of radioactivity were detected in linoleic acid and no label was found in α-linolenic acid.     

Lack of effects oftrans fatty acids on eicosanoid biosynthesis with adequate intakes of linoleic acid

The minimum requirement of linoleic acid to prevent effects of dietary C18trans fatty acids on eicosanoid biosynthesis in rats was assessed. In a first experiment, six groups of animals were fed diets with a high content oftrans fatty acids [20% of energy (en%)], and increasing amounts of linoleic acid (0.4 to 7.1 en%). In a second experiment, four groups of rats were fed diets designed to comparetrans fatty acids with saturated andcis-monounsaturated fatty acids of the same chain length at the 2 en% linoleic acid level. After 9–14 weeks the biosynthesis of prostacyclin by pieces of aorta and the biosynthesis of hydroxy-heptadecatrienoic acid and 12-hydroxy-eicosatetraenoic acid by platelets were measured. The fatty acid compositions of aorta phospholipid and platelet lipid were also determined. Both the prostacyclin-production by aorta pieces and the production of hydroxy-heptadecatrienoic acid and 12-hydroxy-eicosatetraenoic acid by platelets appeared to be a linear function of the arachidonic acid level in aorta phospholipid and platelet lipid, irrespective of thetrans fatty acid content in the diet. This indicates thattrans fatty acids do not directly influence enzymes involved in eicosanoid biosynthesis. In a direct comparison withcis-monounsaturated or saturated fatty acids with 2 en% linoleic acid in the diet, only a moderate reduction in arachidonic acid level in aorta phospholipids in the group fedtrans fatty acids was observed. The geometry of the double bond did not influence the arachidonic acid level in platelet lipid, although the diet rich in saturated fatty acids increased arachidonic acid levels significantly compared with all other diets. Neither prostacyclin-production nor hydroxy-heptadecatrienoic acid or 12-hydroxy-eicosatetraenoic acid-production were significantly affected bytrans fatty acids when 2 en% linoleic acid was present in the diet. Our study indicates that in rats 2 en% linoleic acid is sufficient to prevent effects of dietarytrans fatty acids on eicosanoid synthesis.     

Octadecatrienoic acids as the substrates for the key enzymes in glycerolipid biosynthesis and fatty acid oxidation in rat liver

The activities of key enzymes in glycerolipid biosynthesis and fatty acid oxidation were compared using CoA esters of naturally occurring positional isomers of octadecatrienoic acids (18∶3) as the substrates. The trienoic acids employed were 9,12,15–18∶3 (α-18∶3), 6,9,12–18∶3 (γ-18∶3), and 5,9,12–18∶3 (pinolenic acid which is a fatty acid contained in pine seed oil, po-18∶3). The activities of microsomal glycerol 3-phosphate acyltransferase obtained with various 18∶3 were only slightly lower than or comparable with those obtained with palmitic (16∶0), oleic (18∶1), and linoleic (18∶2) acids. Mitochondrial glycerol 3-phosphate acyltransferase was exclusively specific for saturated fatty acyl-CoA. The activities of microsomal diacylglycerol acyltransferase measured with various polyunsaturated fatty acyl-CoAs were significantly lower than those obtained with 16∶0- and 18∶1-CoAs. Among the polyunsaturated fatty acids, γ-18∶3 gave the distinctly low activity. The V_max values of the mitochondrial carnitine palmitoyltransferase I were significantly higher with α-18∶3 and po-18∶3 but not γ-18∶3, than with 16∶0 and 18∶2, while the apparent K_m values were the same irrespective of the types of acyl-CoA used except for the distinctly low value obtained with γ-18∶3. The response to an inhibitor of the acyltransferase reaction, malonyl-CoA, was appreciably exaggerated with 18∶2, α-18∶3, and po-18∶3 more than with 16∶0 and 18∶1. However, the response with γ-18∶3 was the same as with 16∶0. Thus, some of glycerolipid biosynthesis and fatty acid oxidation enzymes could discriminate not only the differences in the degree of unsaturation of fatty acids but also the positional distribution of double bond among the naturally occurring 18∶3 acids.     

Synthesis of deuterated cyclopropene fatty esters structurally related to palmitic and myristic acids

To develop a synthesis of tritiated cyclopropene fatty acids (CPFA), compounds that should prove useful for affinity labeling of desaturases in insect pheromone biosynthetic studies, a series of novel, selectively deuterated CPFA analogues was prepared and characterized. In methyl [16-²H]12,13-methylene-12-hexadecenoate, the incorporation of deuterium was achieved by treatment of the corresponding ω-chloro derivative with sodium borodeuteride in dimethylsulfoxide at 70°C for 24 h (67% yield) following conventional procedures. Alkylation of the tetrahydropyranyl derivative of 13-tridecynol in the presence of lithium diisopropylamide in tetrahydrofuran at −20°C with 1-chloro-3-iodopropane in hexamethylphosphoramide, followed by Jones oxidation of the crude product, yielded 16-chloro-12-hexadecynoic acid (54%), which was esterified to the corresponding methyl ester by treatment with potassium carbonate and methyl iodide in dimethylformamide. Treatment of this acetylenic ester with ethyldiazoacetate in the presence of activated copper-bronze as catalyst followed by hydrolysis in KOH solution at room temperature yielded 16-chloro-12,13-(carboxymethylene)-12-hexadecenoic acid. This diacid was treated with excess oxalyl chloride to give the corresponding diacyl chloride, which was decarbonylated in a diethyl ether solution with zinc chloride, and the cyclopropenium ions thus formed were added at −40°C to a methanolic sodium hydroxide solution of sodium borohydride to give methyl 16-chloro-12,13-methylene-12-hexadecenoate. Analogous procedures were followed to prepare methyl [17-²H]10,11-methylene-10-hexadecenoate, methyl [17-²H]11,12-methylene-11-hexadecenoate and methyl [17-²H]12,13-methylene-12-hexadecenoate from the corresponding diacids using sodium borodeuteride in the reduction of the cyclopropenium ions. Alternatively, methyl [2,2,3,3-²H₄]hexadecynoate, prepared by reaction of methyl 2,11-hexadecadiynoate with magnesium in deuterated methanol at room temperature, was submitted to the above cyclopropenylation and reductive decarbonylation sequence to give methyl [2,2,3,3,17-²H₅]-11,12-methylene-11-hexadecenoate. In summary, complementary methods for the selective incorporation of one to five deuterium atoms into cyclopropene fatty acids, at different sites, in moderate to high yields have been developed. The methods should easily be applicable to the preparation of the corresponding tritiated analogues.     

Occurrence of n−5 monounsaturated fatty acids in jujube pulp lipids

The pulp lipids of jujube (Zizyphus jujuba var.inermis) fruit have been shown by chromatographic, spectrometric and chemical analyses to contain a series ofcis-monoenoic fatty acids with n−5 unsaturation as major acyl moieties. The total concentration of these n−5 fatty acids, such as 14∶1n−5, 16∶1n−5 and 18∶1n−5, ranged from 22 to 54% of total fatty acids in the pulp lipids of 11 different sources. The main component of the n−5 homologues was 16∶1n−5 in all cases. Other monoenoic acids with n−7 unsaturation, namely palmitoleic (cis-9-hexadecenoic) acid andcis-vaccenic (cis-11-octadecenoic) acid, as well as with n−9 unsaturation, namely oleic acid, were also identified. In the seed lipids of jujube fruit, none of the n−5 monoenoic acids could be detected. Thus the jujube pulp lipids are characterized by the predominance of n−5 monoenoic acid isomers.     

一种聚酯污水中挥发性脂肪酸气相色谱法的建立

聚酯装置产生的废水含有大量的有机物,通过厌氧处理产生大量的挥发性脂肪酸,挥发性脂肪酸是污水系统运行的重要控制指标.公司采用传统蒸馏滴定法测定挥发性脂肪酸总量,检测时间长,存在安全隐患,且污水系统运行异常时,数据准确度不高,经改进,采用分析气相色谱法,检测时间缩短至0.5 h,且数据度高,提高分析效率,完全满足污水正常及异常情况的调整需要.     

Methods for the determination of cyclopropenoid fatty acids. I. Aqueous hydrochloric acid method

An analytical method is described for the estimation of long-chain cyclopropenoid fatty acid derivatives. It is based upon the quantitative addition of a molecule of hydrogen chloride at the cyclopropene ring when the sample is shaken with concentrated hydrochloric acid. The cyclopropenoid content can be calculated, as sterculic acid, from the increase in chlorine content. Epoxy compounds and hydroperoxides interfere and must be removed by one of the accepted pretreatment methods. A laboratory of the So. Utiliz. Res. & Dev. Div., ARS, U.S.D.A.     

Identification and characterization of fully deuterated fatty acids isolated fromScenedesmus obliquus cultured in deuterium oxide

The algaScenedesmus obliquus cultured in deuterated water, synthesized fully deuterated saturated and unsaturated long chain fatty acids. Their methyl esters were purified and their equivalent chain lengths were determined by gas liquid chromatography (GLC). They were characterized by mass spectroscopy, IR and near-IR spectroscopy, and NMR spectroscopy. Hexadeca-3,6-dienoic acid was identified. The fatty acid compositions of the total lipid and of individual lipid classes were measured. The melting point of methyl perdeuteriohexadecanoate was lower than that of its hydrogen counterpart. Methyl esters of perdeuterio fatty acids had shorter retention times in GLC chromatography on polar and nonpolar phases. Presented before ISF-AOCS Congress, Chicago, September 1970.     

The biosynthesis of cyclopropane and cyclopropene fatty acids in plant tissues

The biosynthesis of cyclopropane and cyclopropene fatty acids was investigated in seeds of several species of the order Malvales, including species with a high content of sterculic acid, a high content of malvalic acid, and a low content of these cyclopropene fatty acids. The fatty acid composition of the lipids in young and developing seeds is compared with particular attention to variations in cyclopropane and cyclopropene fatty acid contents. Incubation studies employing several¹⁴C compounds indicate that the methyl group of methionine is the most likely precursor of the ring-methylene carbon. A pathway for the synthesis of the cyclopropane and cyclopropene fatty acids is postulated. The origin of malvalic acid is also considered.     

The desaturation step in the animal biosynthesis of polyunsaturated fatty acids

The aerobic desaturation of unsaturated fatty acids in the microsomes has been systematically explored and some of the relevant experimental works have been assembled. The contribution of the microsomal electron transport chain and lipoproteic structure of the microsomes is analyzed. Evidences of linoleyl-CoA and α-linolenyl-CoA being desaturated by the same enzyme are presented. The linoleic acid desaturation is shown to be different in different tissues and to decrease with aging. The effect of competitive reactions with acids of the same or different series, the competition of desaturation and transesterification, and dietary and hormonal contributions to unsaturated fatty acids desaturation are summarized. Alltrans linoleic acid and elaidic acid were not desaturated in our experimental conditions by rat liver microsomes. From the bulk of data collected, a hypothetical model of 6-olefinase is drawn. The main features of the model are: The existence of both binding and desaturating sites; the binding of acyl-CoA and enzyme through hydrophobic forces of the Van der Waals type and weak polar attractions due to double bonds; the orienting binding characteristics of double bond proximate to the place where olefination will take place; and the importance of enzyme conformation that requires that the orienting double bond must have acis structure.