Long chain polyenoic acid levels in viably sorted,highly enriched mouse testis cells

Twenty-and 22-carbon fatty acids of the linoleic (n-6) and linolenic (n-3) acid families were measured in murine spermatogonia and preleptotene spermatocytes (early), pachytene primary spermatocytes (1⁰) round spermatids (RS), condensing spermatids (CS) and Leydig cells enriched by staput velocity sedimentation at 1 G, followed by viable microflow sorting on the basis of light scatter and DNA content. 22: 5(n-6) increased progressively from 2 to 20% of total fatty acid in the progression of germinal cell differentiation, early »1⁰»RS»CS, but decreased in mature sperm. The precursor 20:4(n-6) showed a roughly reciprocal relationship. 22:6(n-3) showed no significant correlation with cell type. 22:5(n-6) was found highest in triglycerides of later differentiation stages whereas 20:4(n-6) and 22:6(n-3) were found primarily in phospholipid in all cell fractions.     

Biosynthesis of long-chain polyenoic acids from arachidonic acid in cultures of enriched spermatocytes and spermatids from mouse testis

Primary spermatocytes (PS), round spermatids (RS) and condensing spermatids (CS) from mouse testes were enriched on Sta-Put 1 X g density gradients and cultured for 22 or 44 hr in the presence of [1-14C]arachidonate. Mass and radioactivity were measured by gas radiochromatography of constituent fatty acids of the various complex lipid classes fractionated by thin layer chromatography. Patterns and levels of incorporation were compared with those of whole testis, both in vitro and in vivo. The 20:4, 22:4, 22:5, 24:4 and 24:5 of the germinal cells contained levels of radioactivity in each lipid class which were consistent with an important role for the germinal cells in long-chain polyenoic acid (LCPA) metabolism. Cells which represented later stages of spermatogenesis (RS, CS) incorporated much higher percentages and absolute amounts of radioactivity into the fatty acids derived from 20:4 by elongation-desaturation pathways than did PS or whole testis in vitro. These differences were most pronounced in triacylglycerol of CS. Distributions of mass and radioactivity among lipid classes suggest synthesis of triacylglycerol by CS with a high degree of specificity for 22 or 24 carbon LCPA at the sn-3 position.     

Occurrence of long and very long polyenoic fatty acids of the n-9 series in rat spermatozoa

Dietary deficiency of essential fatty acids of the n−3 and n−6 series is known to promote a compensatory increase in polyenoic fatty acids of the n−9 series in the lipids of mammalian tissues. In the present study long-chain n−9 polyenes were found to be normal components of the epididymis and especially of sperm isolated from that tissue, in healthy, well-fed, fertile rats maintained on essential fatty acid-sufficient diets. The n−9 polyenes occurred in large concentrations in the choline glycerophospholipids (CGP), the major phospholipid class of spermatozoa in epididymalcauda, and were highly concentrated in plasmenylcholine, the major subclass of CGP. The uncommon polyene 22∶4n−9 was found in the highest proportion, followed in order of relative abundance by 22∶3n−9, 20∶3n−9 and 24∶4n−9. These polyenes were probably derived from oleate (18∶1n−9) in much the same way as long-chain polyenes of the n−6 and n−3 series are derived from linoleate (18∶2n−6) and linolenate (18∶3n−3), respectively.     

Metabolism of arachidonate in rat testis: Characterization of 26–30 carbon polyenoic acids

Fatty acid methyl esters of long-chain polyenoic fatty acids (LCPA) from rat testis injected with [1-¹⁴C] arachidonate were analyzed and separated by reversed-phase high performance liquid chromatography (RP-HPLC). Earlier, all previously identified LCPA were prepared in high purity along with 4 previously unidentified fatty acids, which were further characterized by capillary gas chromatography (GC), catalytic hydrogenation and alkaline isomerization. Unidentified fatty acids proved to be 26∶4, 26∶5, 28∶5 and 30∶5. All of these LCPA incorporated¹⁴C from arachidonate (20∶4) to specific activities that were comparable to that of 20∶4 and previously identified metabolites of 20∶4 and much greater than specific activities of 18∶1n−9 or 22∶6n−3. LCPA were analyzed on a capillary GC system capable of resolving knowncis-trans and positional isomers of the n−3, n−6, n−7 and n−9 families of unsaturated fatty acids. Log plots of isothermal retention times and normal plots of temperature programmed retention times were linear (r=0.999) in carbon number when values for known and previously unidentified LCPA of 4 or 5 double bonds, respectively, were coplotted. Thus, the newly identified fatty acids belong to the n−6 family of fatty acids synthesized from arachidonic acid.     

The metabolism of polyenoic fatty acids

Feeding experiments with C¹⁴-labeled and unlabeled unsaturated fatty acids have been used to study the possible routes of formation of the C₂₀- and C₂₂-polyenoic fatty acids of rat liver phosphatides. The acids of the palmitoleate, oleate, linoleate, and linolenate types (considered on the basis of the position of the double bond closest to the methyl end) are apparently formed from the C₁₆ and C₁₈ unsaturated acids of the corresponding types. The results rule out possible transformations of the C₂₀- and C₂₂-polyenoic acids from one type to another, and demonstrate the exclusive introduction of new double bonds toward the carboxyl group. Isomers of linoleate or linolenate in which the double bonds were shifted by one carbon atom toward the carboxyl or methyl groups were incorporated into the phosphatides only to a negligible extent in the form of polyenoic acids.     

Autoxidative rates of nonmethylene-interrupted polyenoic fatty acids

Autoxidative rates of free fatty acids and methyl esters increased in the following order: oleic (c9–18:1), c5, c9–18:2, linoleic (c9,c12–18:2), c5,c9,c12–18:3, t5,c9,c12–18:3, and linolenic (c9,c12,c15–18:3). The increase of the rates due to the Δ5-olefinic bond was much lower than that due to an olefinic bond extension in a methylene-interrupted sequence. Agreement of the oxidative rates obtained from conjugated diene content with those obtained from POV showed that oxygen attack did not occur at the isolated olefinic bond, but at the methylene-interrupted diene in the oxidation of 5,9,12–18:3. The Δ5-olefinic bond may promote the oxidation of the methylene-interrupted diene intramolecularly.     

Reaction of long chain triol acids with hydrogen bromide

The reaction of hydrogen bromide (HBr) with long chain triol acids has been investigated in some detail. The 9, 10, 12-trihydroxy stearic acid (I) on treatment with HBr yielded three products, namely 9(10)-bromo-10(9)-hydroxy-12-bromo (III), 9(10)-bromo-10(9)acetoxy-12-bromo (IV) and 9,10-dibromo-12-hydroxy (V) stearic acids. When 9,12,13-trihydroxy stearic acid (II) was subjected to the same reaction under similar conditions, 12,13-dibromo-9-hydroxy (VI), 12(13)-bromo-13(12) acetoxy-9-bromo (VII) stearic acids were obtained. The structure of these products were established on the basis of their elemental values, spectral data and chemical evidence.     

Derivatives of long chain fatty acids as lubricant components

Soaps ofcis-9,10-epiminostearic acid have been synthesized and tested as components of lubricating greases. The lithium, sodium and potassium soaps were obtained by the reaction ofβ-iodocarbamates with the proper alkali metal hydroxides in the ring closure step of the iodine isocyanate (INCO) procedure for the synthesis of internal aziridines. The soaps of other metals, namely barium, lead, aluminum and indium, were prepared from the alkali metal epiminostearates by metathetical reactions. As a result of this method of preparation, the aluminum and the indium soaps were isolated as the dicarboxylates rather than as the tricarboxylates. For the purpose of comparison a sample of lithium 9,10-epiminostearate was also prepared from theβ-chlorocarbamate obtained by the addition of dichlorourethane (DCU) to methyl oleate. Since DCU addition is nonstereospecific, the soap obtained in this preparation was a mixture ofcis- andtrans-aziridines. Evaluation of the metal soaps as grease thickeners was made. The lithium derivative showed considerable promise as thickener having additional desirable properties. Presented at the AOCS-ISF World Congress, Chicago, September 1970. E. Market. Nutr. Res. Div. ARS, USDA.     

Binding of long chain fatty acids to β-lactoglobulin

β-lactoglobulin (BLG), a bovine milk protein that is available commercially in crystalline form, binds long chain free fatty acids (FFA). The binding data were analyzed with a model containing one primary FFA binding site and a large number of weak secondary binding sites. At 37C and pH 7.4, the apparent association constant for binding of FFA to the primary site was of the order of 10⁵ M⁻¹ and that for binding to the secondary sites was approximately 10³ M⁻¹. The strength of binding was: palmitate > stearate > oleate > laurate. The affinity of BLG for palmitate increased as the pH of the incubation medium was raised from 6.5 to 8.7 and decreased as the ionic strength of the medium was raised. Palmitate binding was decreased in the presence of 6 M urea and when the protein either was exposed to elevated temperature or was acetylated prior to incubation. BLG took up methyl palmitate, cetyl alcohol, hexadecane and cholesterol to a lesser extent than FFA. Binding of FFA to BLG was associated with a small increase in the intensity of the fluorescent emission of the protein at 333 mμ. BLG can serve as an FFA acceptor or carrier in biological experiments. FFA released from adipose tissue during in vitro incubation was taken up by BLG. Net transfer of fatty acid to the incubation medium ceased when the molar ratio of FFA to BLG exceeded 1.1.¹⁴C-1-Palmitate bound to BLG was taken up by Ehrlich ascites tumor cells in vitro. At a given palmitate-protein molar ratio, much more labeled fatty acid was taken up by these cells from media containing BLG than from those containing bovine albumin, apparently because FFA is bound less firmly to BLG than to albumin. Special abbreviations used in this text: ν, average molar ratio of bound FFA to total protein; c, molar concentration of FFA in free solution and in equilibrium with that bound to protein; n, number of binding sites in a given class; k’, apparent association constant for binding to a given class of sites.     

Reaction of lipoxidase with polyenoic acids in marine oils

Eight samples of raw oils from fresh water and marine fish, and two from marine mammals, were examined for polyenoic fatty acids susceptible to the action of lipoxidase. The results agreed well in most instances with gas chromatographic data and indicated that only one peroxide group was formed in polyenoic fatty acids with more than one ostensibly suitable 1,4-pentadiene system. Four commercially available preparations of polyenoic fatty acids showed varying degrees of susceptibility to lipoxidase, indicating the probable presence of artifacts formed in purification steps. The marine polyenoic acids were suitable substrates for lipoxidase. Presented at the AOCS Meeting, New York October, 1968.     

Fecal long chain fatty acids and colon cancer risk

To determine whether excretion of high concentrations of long chain fatty acids might be associated with high colon cancer risk, we compared concentrations of major long chain fatty acids in the feces of four populations at different risk for colon cancer. Concentrations of C18∶1 were found to be significantly higher (P<0.05) in the feces of the two high risk populations than in the feces of the two low risk populations.     

Chromatographic analysis of sucrose esters of long chain fatty acids

The procedure of the quantitative analysis of the components of sucrose ester products is described. By thin-layer chromatography, sucrose ester products have been separated into their components: mono-,di-,tri-, and higher esters. The individual components have been extracted from the plate and determined colorimetrically by using anthrone reagent. Standard substances of monoesters, diesters and triesters, which have been synthesized from gas-chromatographically pure methyl esters of fatty acids, have been isolated by column chromatography and identified by elemental analysis, colorimetric determination and N.M.R. determination.     

Study of binary systems of long chain alcohols and acids

Several binary phase diagrams of primary alcohol-secondary alcohol, secondary alcohol-secondary alcohol, odd-even fatty acids, and fatty acid-alcohol systems have been worked out. In many of these systems, freezing point curves give rise to eutectics. In some cases, molecular compound formation was detected; and in one system, dodecanoic acid-tridecanoic acid, the freezing points show solid solution type of curve with a minimum temperature. The solid state transition points of the pure compounds are found, invariably, to become lower when the second component is added. The freezing and transition point behaviors have been explained in terms of miscibility of the components in the solid state.     

Fatty acids of long chain length in baltic herring lipids

The component fatty acids with carbon numbers exceeding 22 in flesh lipids of Baltic herring caught in May and September 1967 in the Turku archipelago have been studied. The total lipid content of the flesh of the herring was 3.5% on average in May and 7.2% on average in September. The fatty acids in the lipids were converted to methyl esters which were resolved and analyzed by urea adduct fractionation, thin layer chromatography and programmed temperature gas liquid chromatography (GLC). The lipids of the herring caught in May were found to contain 15 fatty acids with 24–32 carbon atoms, whereas the lipids of the herring caught in September were found to contain only nine fatty acids with 24–28 carbon atoms. The differences are probably due to nutritional factors. The long chain fatty acids in the lipids of the herring caught in September were isolated by preparative GLC and their structures were studied by UV spectroscopy before and after alkali isomerization, by IR spectroscopy and by GLC of their ozonization products. The identified acids were tetracosanoic, 15-tetracosenoic, 12,15,18,21-tetracosatetraenoic, 9,12,15,18,21-tetracosapentaenoic, 6,9,12,15,18,21-tetracosahexaenoic, 17-hexacosenoic, 11,14,17,20,23-hexacosapentaenoic, 8,11,14,17,20,23-hexacosahexaenoic and 4,7,10,13,16,19,22-octacosaheptaenoic acids. The proportion of the fatty acids containing over 22 carbon atoms in the lipids of fall herring is much higher than has been found earlier in the lipids of marine teleost fish; the reason may be due at least partly to differences in analytical methods.     

N,N-dialkylamides of long chain fatty acids as plasticizers

A number of N,N-dialkylamides have been prepared, characterized and evaluated as plasticizers for poly (vinyl chloride-vinyl acetate) copolymer. Among these are the N-oleoyl derivatives of diisopropyl, dibutyl, diisobutyl, diamyl, dihexyl, diheptyl, dioctyl, di-2-ethylhexyl and didecylamines. Also included are the N,N-dibutylamides of 2-ethylhexanoic, neodecanoic, neotridecanoic, palmitic, stearic, linoleic, erucic, ricinoleic, naphthenic, dimer, pinic, epoxystearic, animal, cottonseed, hydrogenated cottonseed, rapeseed,Limnanthes douglasii seed and parsley seed acids. Optimum low-temperature plasticizing properties are achieved for the N-oleoyl derivatives of dibutyl, diamyl and dihexyl amines. These low-temperature properties are comparable to those of the adipate and sebacate plasticizers without the adverse volatility characteristics of the adipates. Compatibility of the N,N-dialkyloleamides extends through the dihexyl derivative. A brief heat stability study of some selected plasticized polylvinyl chloride copolymer compositions indicates that the thermal stabilization of amide plasticizers is not an insurmountable problem. Presented at the AOCS Meeting at Houston, Texas, April 1965. Laboratory of the So. Utiliz. Res. Devel. Div., ARS, USDA.     

Marine phospholipids as dietary carriers of long‐chain polyunsaturated fatty acids

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are polyunsaturated fatty acids (PUFA) of the n‐3 series. Fish oil is a classical source of n‐3 PUFA, where they occur in the form of triacylglycerols (TAG). However, new sources of n‐3 PUFA esterified in phospholipids (PL) are emerging. We prepared liposomes from a natural marine lipid extract and examined their behaviour under conditions mimicking that of the gastrointestinal tract. This physicochemical approach proved that liposomes could be used as an effective oral PUFA delivery system. In vivo studies in rats were performed to examine the metabolic fate of EPA (20:5 n‐3) and DHA (22:6 n‐3) delivered either in PL from liposomes or in TAG from oil. Liposome ingestion increased PUFA bioavailability in lymph compared with fish oil. The proportion of n‐3 PUFA esterified in the sn‐2 position of chylomicron TAG depended on the dietary lipid source. Complex time‐course profiles were observed for plasma lipids with liposome supplementation over a 2‐week period, suggesting time‐dependent regulations. Taken together, the type of PUFA, EPA or DHA, as well as its intramolecular distribution in chylomicron TAG seemed to influence the metabolic fate of the fatty acids and their physiological activities.     

Identification of some polyenoic acids isolated from human testicular tissue

Five polyenoic acids present in human testicular tissue have been isolated in pure form by gas chromatography and chemically identified using procedures of hydrogenation and UV absorption spectrometry following alkaline isomerization and ozonolysis. Three of these (20∶3, 22∶4 and 22∶5) belong to the linoleic acid and the other two (22∶6 and 22∶5), to the α-linolenic acid family. The latter (22∶5ω3) had not been reported previously in human testes; the others had been tentatively identified by retention time using gas chromatography.     

长链二元酸及其延伸产品的生产工艺和应用

介绍了轻蜡油分离、长链二元酸及其延伸产品尼龙1212联合装置的生产流程及其产品的应用前景,并对投资效益进行了分析。