Determination of individual long-chain fatty acyl-CoA esters in heart and skeletal muscle

A method has been developed for determination of individual long-chain fatty acyl-CoA esters from heart and skeletal muscle using high performance liquid chromatography (HPLC). The esters were extracted from freezeclamped tissue of pig and rat hearts and rat skeletal muscle for analysis on a radially compressed C₁₈ 5μ reversephase column. Nine peaks in the extract with carbon chain lengths from C₁₂ to C₂₀ that subsequently disappeared on alkaline hydrolysis were identified. The major acyl-CoA peaks were 14∶1, 18∶2, 16∶0 and 18∶1 and additionally in rat heart 18∶0. Total long-chain acyl-CoA esters obtained by summation of the individual molecular species was 11.34±1.48 nmol/g wet wt. pig heart; 14.51±2.11 nmol/g wet wt. in rat heart, and 4.35±0.71 nmol/g wet wt. in rat skeletal muscle. These values were approximately 132% of those obtained using a separate procedure that measured total CoA by HPLC after alkaline hydrolysis of the esters. The described method demonstrates the quantitation of individual acyl-CoA species in muscle tissue. Therefore, it has a number of advantages in that it permits information to be obtained on the individual molecular species under various nutritional and metabolic conditions.     

Effect of dietary fat on individual long-chain fatty acyl-CoA esters in rat liver and skeletal muscle

The effect of dietary fat on the long-chain acyl-CoA ester profile of liver and skeletal muscle was investigated by feeding weanling rats 12%-fat diets composed of high-linoleic safflower oil (73% 18∶2n−6), high-oleic safflower oil (70% 18∶1n−9) or olive oil (70% 18∶1n−9) for six and ten weeks. Approximately 50% of both hepatic and skeletal muscle acyl-CoA esters comprised linoleoyl-CoA or oleoyl-CoA with high-linoleic or oleic feeding, respectively. Total hepatic acyl-CoA ester concentration was 40% higher (p<0.05) in rats fed 12% fat compared with controls fed a 4%-fat diet. These data demonstrate that the long-chain acyl-CoA ester profile of liver and skeletal muscle reflects the dietary fatty acid profile.     

β-oxidation of the coenzyme A esters of vaccenic,elaidic, and petroselaidic acids by rat heart mitochondria

Rates of β-oxidation of the coenzyme A esters of vaccenic, elaidic, and petroselaidic acids as well as their respectivecis isomers by rat heart mitochondria were measured and compared. At all concentrations studied, vaccenoyl-CoA was oxidized more rapidly than elaidoyl-CoA, but more slowly than oleoyl-CoA except at high substrate concentrations. Alltrans octadecenoyl-CoA esters were oxidized at a slower rate than their respectivecis or saturated isomers. Oxidation rates decreased as the double bond approached the carboxyl-end.     

Immediate no-flow ischemia decreases rat heart nonesterified fatty acid and increases acyl-CoA species concentrations

Tissues changes in FA metabolism can occur quite rapidly in response to ischemia and may require immediate microwave fixation to determine basal concentrations. The present study aimed to quantify the effects of immediate no-flow ischemia on concentrations of individual nonesterified FA (NEFA) and acyl-CoA species in the rat heart. Male CDF 344 rats were anesthetized and decapitated either 5 min prior to being microwaved (5.5 kW, 3.4 s, twice) to produce ischemia or microwaved prior to decapitation (nonischemic). Hearts were then removed and used to measure the concentrations of acyl-CoA species and FA in several lipid classes. The ischemic heart total NEFA concentration was significantly lower than that in the nonischemic heart (11.9 vs. 19.0 nmol/g). Several individual NEFA concentrations were decreased by 31–85%. Ischemic heart total long-chain acyl-CoA concentrations (21.0 nmol/g) were significantly higher than those in nonischemic hearts (11.4 nmol/g). Increased concentrations of individual acyl-CoA species occurred in palmitoyl-CoA, stearoyl-CoA, oleoyl-CoA, and linoleoyl-CoA. Concentrations of short-chain acetyl-CoA and β-hydroxy-β-methylglutaryl-CoA were also two- to three-fold higher in ischemic hearts than in nonischemic hearts. The FA concentration in TG and phospholipids generally did not differ between the groups. Decreases in concentrations of individual FA and increases in acyl-CoA species during no-flow ischemia occur very rapidly within the heart. Although it is not clear how these alterations contribute to the pathogenesis of ischemia, it is evident that future studies attempting to quantify basal levels of these metabolites could use microwave fixation.     

Preparation of esters and anhydrides from long chain fatty acid chlorides

Conclusions The use of reduced pressure to remove the HCl formed in the reaction of acid chlorides with alcohols and acids appears to be generally applicable to reactants of low volatility. The lowest yield of recrystallized product obtained was 85.0%, and in most cases tested the yield was more than 90%. Any side reactions occurring are apparently minor as a single crystallization gave products of high purity. This paper was awarded an honorable mention in the 1957 Fatty Acid Producers' Award Competition administered by the American Oil Chemists' Society.     

Association of phospholipid-cholesterol micelles with rat heart mitochondria: Stimulators and inhibitors

The uptake of emulsified labeled lipids by rat heart mitochondria was studied. Divalent cations greatly stimulated uptake of cholesterol in emulsions containing phospholipids; the effect increased with increasing atomic number. Epinephrine and norepinephrine were also stimulatory. Lipid-depleted mitochondria were less effective in taking up cholesterol unless lecithin or serum lipids were included in the emulsion. Addition of iodoacetamide did not inhibit uptake, while use of heated mitochondria or extremes of pH augmented lipid uptake. Thus the process appears to be non-enzymatic. While lecithin emulsions showed no visible change on addition of calcium ions, phosphatidylethanolamine emulsions became turbid and the turbidity was largely removed by addition of heparin. Heparin as well as chondroitin sulfate B and nonionic detergent did inhibit lipid uptake by mitochondria. The possible role of such nonenzymatic lipid uptake in membrane formation and cholesterol accumulation is discussed.     

Ketonization of long chain esters from transesterification of technical waste fats

The methyl ester mixture obtained from rape oil, and the mixture of butyl esters produced from animal fatty acids, were converted into mixtures of ketones with a total yield above 65%. The experiments were performed at atmospheric pressure in a temperature range of 300–400 °C using a standard flow system, with an iron catalyst modified with silicon, chromium, and potassium oxides, in the molar ratio of 100 : 2 : 1 : 0.1. The described method may extend the prospects of using waste technical fats. © 2001 Society of Chemical Industry     

Influence of elaidate and erucate on heart mitochondria

Male, weanling rats were fed, for up to six weeks, corn oil (CO), rapeseed oil (RSO), partially hydrogenated fat (HF), or a mixture of partially hydrogenated fat and corn oil (HF+CO). The respiratory activity of their isolated heart mitochondria, their hormone-sensitive lipase activity, and the fatty acid compositions of the phospholipids of the mitochondria were determined. The results indicated that heart mitochondria isolated from rats which had been fed corn oil (CO) had a higher rate of oxygen uptake, showed higher respiratory control ratios, higher ADP/0 ratios and a higher rate of ATP synthesis than the heart mitochondria isolated from those fed rapeseed oil or hydrogenated fats. The oxygen uptake rates of the rat heart mitochondria isolated from each dietary group of rats was in order: oleyl carnitine ≫ erucyl carnitine > elaidyl carnitine. The decreased capacity to oxidize substrate by heart mitochondria which had been isolated from the hearts of rats fed rapeseed or hydrogenated soybean oil as compared with those fed corn oil as a sole source of dietary fat seemed related to the mitochondria lipid composition. The type of dietary fat fed had a pronounced influence on the mitochondrial fatty acid compositions of phosphatidylcholine, phosphatidylethanolamine, and cardiolipin. The lipase activity of the RSO-fed group did not show any increment with either epinephrine or supplemental ATP treatment. The substrate preference for lipase activity in myocardium was corn oil-triglycerides > trierucin > trielaidin > tripalmitin. However, cardiac lipid accumulation did not seem related to lipase activity in the myocardium. Taken from a thesis submitted by Chi Ming Lee Hsu in partial fulfillment of the Ph.D. degree in Food Science, University of Illinois at Urbana-Champaign.     

Studies of biosynthesis of waxes by developing jojoba seed: III. Biosynthesis of wax esters from Acyl-CoA and long chain alcohols

Acyl-CoA: alcohol transacylase activity was demonstrated in cell-free homogenates of developing jojoba seeds. The optimal pH was 8.0–8.1. Under optimal conditions, wax formation had a nearly linear relationship with extract concentration; the time course of wax formation was also linear up to 30 min.cis-11-Eicosenol was the most effective alcohol substrate whereas tetradecanol, octadecanol, dodecanol,cis-9-octadecanol, andcis-13-eicosenol gave progressively lower activities. Either saturated or unsaturated acyl-CoA with 18 or 20 C-atoms had similar activity. The enzyme was fairly stable at 0 C, less stable at RT and labile above 30 C. Differential centrifugation showed that the 12,000 × g fat pad was the most active in wax formation; to maximize the activity, a 12,000 × g supernatant appeared to be necessary. This factor in the supermatant was thermolabile and nondialyzable.     

Studies on the influence of long chain acrylic esters polymers with polar monomers as crude oil flow improver additives

This study investigated the efficiency of 22 polymer additives of defined and controlled structural characteristics in improving the flow properties of crude oil. The additives were synthesized by the solution polymerization of alkyl acrylates of various alkyl chain lengths with styrene, acrylic acid and 1-vinyl-2-pyrrolidone as copolymers or terpolymers. Polymerization was performed by tert-butylperoxy-2-ethylhexanoate as initiator in xylene at 92 °C, up to high conversion. The influence of polymerization conditions on additive composition and properties, and their effect on the pour point and rheological properties of crude oil samples from the fields of ?tevkovica, Obod and ?eletovci located in the northern Croatia were studied. Additive efficiency was dominantly influenced by the alkyl acrylate chain length. The influence of component with carboxyl functional group or heterocyclic-nitrogen compound on additive quality was less obvious. Additive efficiency significantly depended on the properties of crude oil, and n-paraffin content and distribution. The main advantage of the process is that additives can be directly used without purification.     

Oxidation of cholesterol in synaptosomes and mitochondria isolated from rat brains

Cholesterol and α-tocopherol oxidations were studied in brain subcellular fractions isolated from cerebral hemispheres of 4-month-old, male Fischer 344 rats. The fractions were suspended in buffered media (pH 7.4, 37°C) and oxidized by adding (i) ferrous iron (Fe²⁺) with or without ascorbate or (ii) peroxynitrite (an endogenous oxidant produced by the reaction of superoxide and nitric oxide). Treatment of subcellular fractions with Fe²⁺ in the presence or absence of ascorbate produced primarily 7-keto- and 7-hydroxy-cholesterols and small amounts of 5α,6α-epoxycholesterol. Since brain contains high levels of ascorbate, any release of iron could result in oxysterol formation. Peroxynitrite oxidized α-tocopherol but not cholesterol. Hence, the toxicity of peroxynitrite or nitric oxide could not be due to cytotoxic oxysterols. When synaptosomes were incubated for 5 min in the presence of 0.5 to 2 μM Fe²⁺ and ascorbate, α-tocopherol was oxidized while cholesterol remained unchanged. Thus, α-tocopherol is functioning as an antioxidant, protecting cholesterol. Diethylenetriaminepentaacetic acid blocked production of oxysterols, whereas citrate, ADP and EDTA dit not. A significant percentage of mitochondrial cholesterol was oxidized by treatment with Fe²⁺ and ascorbate. Hence, mitochondrial membrane properties dependent on cholesterol could be particularly susceptible to oxidation. The oxysterols formed were retained within the membranes of synaptosomes and mitochondria. The 7-oxysterols produced are known to be inhibitors of membrane enzymes and also can modify membrane permeability. Hence, oxysterols may play an important role in brain tissue damage during oxidative stress.     

Modification of alkenyl chain profile in plasmalogens of rat heart mitochondria by dietary trielaidin

Effects of dietary trielaidin upon the alkenyl chain profile of plasmalogens were studied using heart mitochondria of rats fed a semi-purified diet containing 10% of fat supplement in which elaidic acid accounted for 69% of total fatty acids. Alkyl substituted dioxane (ASD) derivatives of the alkenyl groups of plasmalogens were prepared and analyzed by silver nitrate TLC and by GLC on different phases (BDS and OV-275). After two months of feeding the experimental diet, 40% of the ASD contained atrans-octadecenyl chain, suggesting that dietary elaidic acid was reduced in vivo to the corresponding alcohol and incorporated into plasmalogens. There was a simultaneous decrease in the percentage of ASD containing saturated chains, but the percentage of ASD substituted withcis-octadecenyl chains was not significantly affected. These observations suggested that elaidic acid may compete with saturated fatty acids, but not withcis-octadecenoic acids during the plasmalogen biosynthesis. Feeding trielaidin did not seem to have any significant influence on the relative proportions of plasmalogens, which accounted for 11–12%, on a phosphorus basis, of total heart mitochondria phospholipids.     

Activation of long chain fatty acids by subcellular fractions of rat liver: II. Effect of ethylenic bond position on acyl-CoA formation oftrans-octadecenoates

The rates of acyl-CoA biosynthesis of thetrans Δ⁴ to Δ¹⁵ octadecenoates have been studied using rat liver microsomes and mitochondria as the enzyme(s) source. Acyl-CoA formation was lowest when the Δ⁹ isomer was the substrate and increased as the double bond was moved to either end of the fatty acid chain. Maximal activation was observed when the Δ⁶ and Δ¹⁴ trans-octadecenoates were substrates. All-trans retinoate caused differential inhibition in the activation rates of the varioustrans-octadecenoate isomers. In contrast, the activation profile relative to double bond position was not altered by changing the incubation temperature, pH, or buffer or by adding fatty acid free albumin to the incubation medium or by aging the enzyme. These results do not support the suggestion that more than one enzyme activates the varioustrans-octadecenoate positional isomers. Triton X-100 differentially activated acyl-CoA biosynthesis of the various isomers resulting in a uniform rate of activation. Release of the isomers from the detergent micelle is suggested to be rate-limiting under these conditions.     

Activation of long chain fatty acids by subcellular fractions of rat liver: III. Effect of ethylenic bond position on acyl-CoA formation ofcis-octadecenoates

The rates of rat liver microsomal and mitochondrial activation of the Δ⁴ to Δ¹⁷ cis-octadecenoate positional isomers have been investigated. The fatty acid to protein ratios required for maximum activation of the Δ⁸, Δ⁹ and Δ¹⁰ isomers were much lower than the corresponding ratios required for maximum activation of thecis-octadecenoates with double bonds at either end of the fatty acid molecule. Also, as the incubation temperature was raised from 22–38 C the Δ⁸ and Δ⁹ isomers exhibited little change in their rates of activation, while large increases in activation rates of the isomers with the double bond at either end of the fatty acid chain were observed. Differential inhibition of the activation of the various positional isomers was observed when anionic, cationic, or nonionic detergents were included in the incubation medium. The different responses to fatty acid concentration, temperature and detergents are attributed to enzyme specificity and to differences in solution properties of thecis-octadecenoates, rather than to the presence of separate rat liver enzymes that catalyze acyl-CoA ester formation of the various positional isomers.     

Mass spectrometric analysis of long chain alk-1-enyl ether esters and alkyl ether esters of diols

The mass spectra of homologous series of long chain alk-1-enyl ether esters and alkyl ether esters of short chain diols were determined, and general patterns of fragmentation were established. Both types of diol lipids yielded ions characteristic of the alkoxy or alk-1-enyloxy moiety and the acyl moiety, as well as ions indicative of the constituent short chain diol. Prominent ions were formed from both types of ether esters due to the loss of the alkoxy or alk-1-enyloxy moieties giving rise to ions for which cyclic structures are proposed. High resolution mass spectrometry and deuterium labeling techniques were used to verify the composition of ions and to substantiate fragmentation mechanisms. This is part VII in the series “Naturally Occurring Diol Lipids” (part VI is Reference 1) and part VIII in the series “Mass Spectrometry of Lipids” (part VII is Reference 23).     

Studies on photo-crosslinkable polyphosphoramide esters containing divanillylidene cycloalkanone units

Two series of photo-crosslinkable polyphosphoramide esters were synthesized from 2,5-bis(4-hydroxy-3-methoxy benzylidene)cyclopentanone and 2,6-bis(4-hydroxy-3-methoxy benzylidene)cyclohexanone with various N-arylphosphoramide dichlorides by interfacial polycondensation using hexadecyl trimethylammonium bromide as phase transfer catalyst at ambient temperature. The resulting polymers were characterized by inherent viscosity, Fourier transform infra-red, ¹H and ¹³C nuclear magnetic resonance spectroscopy. The thermal stability of the polymers synthesized was evaluated by thermogravimetric analysis. These polymers were studied for their photo-chemical reactions. The divanillylidene cycloakanone groups in the polymer chain function as photo-active centres. Crosslinking proceeds via 2π + 2π cycloaddition reaction of the divanillylidene cycloalkanone moieties. The rate of crosslinking decreases with increase in the size of the cycloalkanone ring, while the thermal stability increases with increase in the size of the cycloalkanone ring.     

椰子油单乙醇酰胺丙氧基醚的合成研究

以椰子油单乙醇酰胺、环氧丙烷为原料,进行开环聚合反应合成椰子油单乙醇酰胺丙氧基醚,考察了椰子油单乙醇酰胺丙氧基醚合成的条件,实验表明,在催化剂氢氧化钾用量为反应物总物质的量的0.5%,反应物椰子油单乙醇酰胺与环氧丙烷物质的量比为1：2,反应温度145℃,反应时间3h的条件下,环氧丙烷的转化率可达99.9%。经测试,椰子油单乙醇酰胺丙氧基醚与椰子油单乙醇酰胺的稳泡性能相近,具有黏度低,水溶性好的特点。     

Very long chain PUFA in murine testicular triglycerides and cholesterol esters

Very long chain (VLC) PUFA of the n−6 and n−3 series are known to occur in mammalian testis. The aim of this work was to characterize further two testicular lipid classes with VLCPUFA, cholesterol esters (CE) and total triglycerides (TG) in rat and mouse testis. The VLCPUFA predominating in these lipids were a series of n−6 pentaenes and tetraenes with 24 to 32 carbons, including small amounts of odd-chain PUFA, 28∶5n−6 and 24∶5n−6 prevailing in CE and TG, respectively. Most of the VLCPUFA of TG were concentrated in a small fraction of TG, made up by 1-O-alkyl-2,3-DAG. This TG subclass was absent altogether from the TG of sexually immature testis. The TG and the CE with VLCPUFA only occurred in testis of adult fertile animals. The proportion of VLCPUFA in total TG and CE was higher in rodents than in other mammals. In the n−6 PUFA-rich adult mouse testis, the amounts of testicular triacylglycerols decreased significantly after consumption of fish oil for 2 wk. Whereas 18∶2n−6 was significantly reduced, the amounts of 22∶5n−6 and longer n−6 PUFA were less affected in all mamor testicular lipids including PC and PE, where they were unchanged. The 1-O-alkyl-2,3-DAG and their n−6 VLCPUFA were virtually unaffected by the diet. The VLCPUFA-containing molecular species of CE and TG may represent a form of storage of cholesterol and polyenoic FA required to sustain spermatogenesis. Via chain-shortening, VLCPUFA stored in the neutral lipids may serve as precursors of the major C₂₂ PUFA typical of cell membrane glycerophospholipids, protecting testicular cells against shifts in FA composition induced by dietary changes.     

Studies on kinetics and preparation of C36 dimer acid esters

C₃₆ Dimer acids were esterified with various short-chain alcohols, namely 2-propanol, n-butanol, n-hexanol, n-octanol, 2-octanol and 2-ethyl-1-hexanol by using sulfuric acid as catalyst and benzene as an azeotropic solvent. Various reaction parameters were standardized. In case of isopropyl esters, acid-to-alcohol mole ratio of 1:5 and sulfuric acid concentration of 2% based on the weight of dimer acids were found to be optimum. In case of straight-chain primary alcohols, namely n-butanol, n-hexanol and n-octanol, 1:2.5 mole ratio of acid to alcohol and 1% by weight of sulfuric acid were found satisfactory. Esterification reaction rates were determined from the fall in acid value of the product. The reaction followed pseudo first order kinetics. The reaction rates increased with the increase in chainlength of straight-chain primary alcohols from n-butanol to n-octanol. The rate of reaction decreased from n-octanol to 2-ethyl-1-hexanol to 2-octanol due to the branching of the chain in 2-ethyl-1-hexanol and secondary nature of the −OH group in 2-octanol.     

Allyl esters and allyl epoxy esters from crambe oil

Room temperature transesterification of crambe oil with allyl alcohol gave allyl esters previously prepared by hydrolysis of the oil and reesterification of the mixed acids at elevated temperatures. Treating the esters with m-chloroperbenzoic acid in the presence of sodium bicarbonate resulted in the selective epoxidation of ethylenic bonds and suppression of side reactions. Bifunctional allyl epoxy esters produced in 88% overall yield by this method contain 5.08% oxirane oxygen and an unsaturation equivalent to 91% allyl group; they are prospective monomers for various types of polymerization.