Normal Fatty Acid Research Articles

Micro-scale separation of normal and hydroxy fatty acid methyl esters on a Florisil column

Micro-scale separation of normal and hydroxy fatty acid methyl esters on a Florisil column

Poly(Crown Ether) Catalyzed Derivatization of Lower Fatty Acids for Gas Chromatography

Abstract Poly (crown ether) was employed as a catalyst of derivatization of lower fatty acids to the p-bromophenacyl esters for gas chromatographic determination of them. The esterification reaction proceeds quantitatively under mild conditions, i.e. at room temperature and within 30 min. The poly (crown ether) did not interfere with the gas chromatogram of the esters unlike monomeric crown ethers. Normal fatty acid (C1-C6) and the isomers could be determined simultaneously near to the detection limit of FID.

Synthesis of long chain acyl-enzyme thioesters by modified fatty acid synthetases and their hydrolysis by a mammary gland thioesterase

The fatty acid synthetase multienzyme from lactating rat mammary gland was modified either by removal of the two thioesterase I domains with trypsin or by inhibiting the thioesterase I activity with phenylmethanesulfonyl fluoride. The modified multienzymes are able to convert acetyl-CoA, malonyl-CoA, and NADPH to long chain acyl moieties (C 16C 22), which are covalently bound to the enzyme through thioester linkage, but they are unable to release the acyl groups as free fatty acids. A single enzyme-bound, long chain acyl thioester is formed by each molecule of modified multienzyme. Kinetic studies showed that the modified multienzymes rapidly elongate the acetyl primer moiety to a C 16 thioester and that further elongation to C 18, C 20, and C 22 is progressively slower. Thioesterase II, a mammary gland enzyme which is not part of the fatty acid synthetase multienzyme, can release the acyl moiety from its thioester linkage to either modified multienzyme. Kinetic data are consistent with the formation of an enzyme—substrate complex between thioesterase II and the acylated modified multienzymes. The present study demonstrates that the ability of thioesterase II to modify the product specificity of normal fatty acid synthetase is most likely attributable to the capacity of thioesterase II for hydrolysis of acyl moieties from thioester linkage to the multienzyme.

Biosynthesis of β-diketones and hydrocarbons in barley spike epicuticular wax

Aided by the analysis of induced, single gene mutants in barley, independent elongation systems were inferred for the synthesis of β-diketones (98% hentriacontan-14,16-dione) and hydrocarbons (primarily hentriacontane). This proposal has been substantiated by comparing the effects of preincubations of inhibitors on the ability of whole spikes to incorporate [2- 14C]acetate into the various epicuticular wax lipids. Dithiothreitol and mercaptoethanol inhibited the incorporation of label into hydrocarbons, but not into β-diketones. Cyanide blocked the synthesis of β-diketones, while stimulating hydrocarbon formation more than twofold. β-diketone synthesis was far more sensitive to arsenite than was synthesis of hydrocarbons. Degradation of the asymmetric β-diketone molecules by base hydrolysis and determination of the amount of label in the resulting fragments revealed a specific inhibition by arsenite of label incorporation into the C-31 end, i.e., the end from which previous studies have shown elongation to proceed. Tissue slices prepared from spikes minus awns were able to incorporate into β-diketones 1abel from [1- 14C]palmitate and shorter evenchained fatty acids, but not from [1- 14C]stearate. However, all fatty acids tested served equally well as hydrocarbon precursors. The elongation systems leading to the β-diketones and hydrocarbons are thought to diverge when the chain has 16 carbons. Thus, when a C 16 chain is elongated by the addition of a C 2 unit to form a C 18 β-keto acyl chain, the β-keto group is not reduced as in normal fatty acid synthesis, but both carbonyl groups are protected and retained during further elongation. After reaching a C 32 chain length, decarboxylation and release of the protected carbonyl groups yield the β-diketone.

Metabolism of a n-paraffin, heptadecane, in rats.

14C-heptadecane incorporated in rat diet was largely absorbed, and a balance study showed extensive 14CO2 excretion (65%). There was no elimination of the hydrocarbon in the urine, and only minute quantities of labeled metabolites. Radioactivity in the feces was entirely in heptadecane. About 7% of the heptadecane absorbed was stored in the carcass, whereas the rest was omega-oxidized to heptadecanoic acid. This fatty acid was incorporated into neutral lipids and phospholipids, underwent the normal fatty acid degradation pathway, and contributed to the synthesis of lipids, including fatty acids, squalene and cholesterol, and nonlipids (7-10%). Heptadecanoic acid was desaturated to heptadecenoic acid. The even distribution of radioactivity in the fatty acids of the various phospholipid classes indicated that heptadecane did not interfere with the biochemical mechanisms of these functional lipids.

Fatty acid composition of myelin isolated from the brain of a patient with cellular deficiency of co-enzyme forms of vitamin B12

Myelin fractionation and subsequent lipid isolation have been carried out on a brain from a patient who suffered from a cellular deficiency of the adenosylcobalamin and methylcobalamin co-enzyme forms of vitamin B12. Examination of the fatty acid composition of choline and ethanolamine glycerophospholipids indicated a relative enrichment of odd-chain fatty acids which were identified by gas-liquid chromatography-mass spectroscopy as C15, C15:1, C17 and C17:1. A mixture of methyl branched C17 fatty acids was also identified. Odd-chain fatty acids accounted for 9.8% of the total fatty acid in the myelin choline phospholipid compared to control values of 1.2%. The affected brain myelin phospholipids had a lower unsaturated fatty acid content. Examination of the myelin sphingolipids, sphingomyelin, cerebroside and sulfatide, yielded abnormal fatty acid profiles. The sphingomyelin contained only small amounts of C24:1 fatty acid. Both normal and hydroxy fatty acid containing cerebroside and sulfatide had reduced levels of C24 fatty acid. Determination of the relative hydroxy and normal fatty acid content of the galactolipids indicated an abnormally high hydroxy fatty acid level. Abnormal fatty acid profiles of brain cerebral sphingolipids have not been previously described in cases of vitamin B12 deficiency. Whether or not these alteration are characteristic will only be established by estimating sphingolipids in other such cases.

The Prader-Willi syndrome. Regulation of fat transport.

Studies of fat mobilization and transport are reported in six patients with the Prader-Willi syndrome. Two patients had carbohydrate intolerance. One of these had a low and the other an augmented insulin response to glucose challenge. Following challenge with glucose, three of the four nondiabetics had normal insulin responses or increased responses consistent with their obesity; the other nondiabetic had insulinopenia. Measurements of the effects of norepinephrine, insulin, glucose, and 5-methylpyrazole-3-carboxylic acid on plasma levels of FFA, glycerol, and ketones provide no evidence for abnormal regulation of mobilization of fat from adipose tissue. Measurements of plasma lipids and postheparin lipolytic activity are consistent with normal uptake of fat into adipose tissue, and normal fatty acid composition of adipose tissue gives no evidence for abnormal lipogenesis.

Effects of temperature and host cell genetic characteristics on the replication of the lipid-containing bacteriophage PR4 in Escherichia coli

The lipid-containing bacteriophage PR4 is of special intest because it can replicate in various gram-negative bacteria, including Escherichia coli, that carry one of a group of drug resistance plasmids. PR4 grown in E. coli strain PS2R contains about 10% lipid by weight, with the negatively charged phospholipid phosphatidylglycerol being the most abundant lipid in the virion. We now report the following. (i) PR4 attaches to E. coli with an attachment rate constant of Ka approximately 6.2 X 10(-10) ml/min, which is about twice that of the enveloped phage phi6 (to Pseudomonas phaseolicola), but a factor of 5 less than that of phage PM2 (to Pseudomonas BAL-31). (ii) Use of an E. coli glycerol auxotroph indicated that a normal amount of PR4 replication occurs only if glycerol starvation (inhibition of all phospholipid synthesis) begins no earlier than about halfway through the lytic cycle. (iii) Use of an E. coli fatty acid synthesis temperature-sensitive mutant and an E. coli phosphatidylethanolamine synthesis temperature-sensitive mutant indicate that PR4 replication can occur in the absence of either normal fatty acid synthesis or normal phospholipid synthesis if the infection takes place prior to the termination of overall cell growth and the onset of cell death, (iv) Whereas PR4 burst size in nutrient media at 30 degrees C to 42%C is about 40, the burst size at 20 degrees C is less than 3, Temperature-shift experiments show that the temperature late in infection determines the burst size.

The effect of non-steroid anti-inflammatory drugs, dibutyryl cyclic 3′,5′-adenosine monophosphate and phosphodiesterase inhibitors on platelet aggregation and the platelet release reaction in normal and essential fatty acid deficient rats

A comparison was made of the action of three classes of substances with platelet aggregation inhibiting effect in normal and essential fatty acid deficient rats. In the latter group, the effect of indomethacin was considerably reduced, whereas the difference was smaller with aspirin. Dibutyryl cyclic AMP had the same inhibiting effect in both groups. Of the phosphodiesterase inhibitors tested, dipyridamole was inactive, whereas the inhibiting effects of caffeine and papaverine are slightly reduced in the deficient animals. The same differences between the two groups were seen in the magnitude of the release reaction after 14C-serotonin labelling. These data support the idea that the non-steroid anti-inflammatory drugs act by inhibiting the formation of an aggregation-inducing substance from arachidonic acid.

Characterization of docosa-4,15-sphingadienine and 4-hydroxy-docosa-15-sphingenine in sphingophosphonolipids from Turbo cornutus by gas chromatographymass spectrometry

In previous papers [1–2], it was reported that sphingophosphonolipids obtained from the viscera of Turbo cornutus consisted of two types of ceramide 2-N-methyl-aminoethylphosphonates, CMAEP-I and CMAEP-II. ∗ ∗ Ceramide 2-N-methylaminoethylphosphonates are abbreviated as CMAEP. 1 means the combination of dihydroxy long chain base and normal fatty acid in ceramide moiety, II trihydroxy long chain base and hydroxy fatty acid. These sphingophosphonolipids were found to contain uncommon long chain bases, which were identified as C 22-sphingadienine in CMAEP-I and C 22-dehydrophytosphingosine in CMAEP-II by gas chromatography-mass spectrometric analysis of their corresponding trimethylsilyl derivatives. However, the double bond positions of these two long chain bases remained to be clarified. In this communication, we wish to report that the structures of these two long chain bases are docosa-4,15-sphingadienine and 4-hydroxy-docosa-15-sphingenine

Mutants of Escherichia coli with Temperature-sensitive Malonyl Coenzyme A-Acyl Carrier Protein Transacylase

Abstract We have characterized two mutants of Escherichia coli in which fatty acid biosynthesis is adversely affected by elevated temperature. Under conditions which do not alter the activity of wild type cell-free extracts, virtually all of the malonyl coenzyme A-acyl carrier protein (ACP) transacylase and fatty acid synthetase activities present in mutant extracts are thermolabile. Malonyl transacylase from wild type cells restores normal fatty acid synthetase activity to inactivated mutant extracts. Furthermore, fatty acid synthesis in mutant extracts is corrected by malonyl-ACP. Malonyl transacylase and fatty acid synthetase activities are also restored in revertants which were selected for their ability to grow at elevated temperatures. These experiments furnish evidence that the mutants are altered in the structural gene for malonyl transacylase and that this is their only genetic defect. When these strains, defective in the synthesis of a substrate vital for fatty acid elongation, are grown at elevated temperatures, their phospholipids become deficient in long chain acyl groups. Supplementation of these cultures with long chain fatty acids permits sustained growth and the maintenance of a normal fatty acid composition.

CHEMICAL PATHOLOGY OF KRABBE'S DISEASE II. Futty Acid Composition of Cerebrosides, Sulfatides and Sphingomyrlins in Brain

ABSTRACT: Vanier, M.‐Th. and Svennerholm, L. (Department of Neurochemistry, Psychiatric Research Centre, University of Göeborg, Göteborg, Sweden). Chemical Pathology of Krabbe's Disease. II. Fatty acid composition of cerebrosides, sulfatides and sphingomyelins in brain. Acta Paediatr Scand, 63: 501, 1974.—The fatty acid composition of sphingomyelins, cerebrosides and sulfatides was determined in cerebral tissue from 18 Swedish children aged 7–32 months, with Krabbe's disease. Sphingomyelins of the white matter had a fatty acid pattern which indicated a serious deficiency of myelin. The fatty acid pattern also suggested that a substantial amount of the sphingomyelins was derived from cells of mesenchymal origin. The cerebrosides, and to a certain extent the sulfatides of cerebral cortex and white matter, had a higher concentration of saturated very‐long‐chain C23‐C26 acids than normal brains. The difference from the normal fatty acid pattern of cerebrosides was smaller in myelin. This suggests that the cerebrosides stored in the globoid cells have an abnormal fatty acid composition.

Alterations in normal fatty acid composition in a temperature-sensitive mutant of a thermophilic bacillus

A temperature-sensitive mutant of a thermophilic bacillus was isolated which was unable to maintain membrane integrity at high temperature. The mutant appeared to lose cytoplasmic contents, as indicated by a decrease in turbidity and cell refractivity, when shifted from a permissive (52° C) to a restrictive (65° C) temperature. Cell number remained fairly constant, however. At the approximate onset of the decline in turbidity, viability decreased and net synthesis of ribonucleic acid, deoxyribonucleic acid, and protein ceased. Both chloramphenicol and sucrose were effective in retarding the decline in turbidity at 65° C. An abnormal fatty acid composition at high temperature suggested that the lesion in the mutant involved lipid synthesis. The proportion of fatty acids with a high melting point (> 55° C) increased in the parent from 42% at 42° C to 69% at 65° C. Similar changes were not made by the mutant. An abnormal phospholipid composition was also observed in the mutant at 42° C and 52° C. However, at 58° C, the maximum growth temperature of the mutant, the proportion of major phospholipids (phosphatidylglycerol, phosphatidylethanolamine, and cardiolipin) was similar to the parent strain. The mutant's apparent loss of membrane stability at high temperature and its inability to regulate fatty acid and phospholipid composition in a normal manner suggested that (i) the temperature-sensitivity of the mutant may be a result of a defect in normal lipid metabolism at high temperature and (ii) the normal changes in fatty acid composition observed at increased growth temperatures may be an essential feature of thermophily.

Biological studies with cyclopropenoids inactivated with fatty acids.

AbstractCyclopropenoids inactivated by reactingSterculia foetida oil with cottonseed oil fatty acids were fed at three dietary levels to growing rats and laying hens for 4 weeks. At the termination of the experiments, all animals were autopsied and examined microscopically for pathological lesions, but no pathology that could be related to dietary treatment was observed. Hemoglobin, packed cell volume and plasma cholesterol were similar in animals fed all of the diets. Growth rate of rats and egg production of hens fed the experimental diets were similar to those of animals fed the control diet. After 3 and 6 months of storage, eggs from hens fed the inactivated cyclopropenoids were normal and showed no evidence of the unusual characteristics of cyclopropenoid feeding. Lipids of heart, liver and adipose tissues of all the rats and hens varied little from the normal fatty acid composition. Small amounts of three unidentified fatty acids were found in the adipose tissues of rats fed the higher levels of inactivated cyclopropenoids. The results of these feeding studies suggest that inactivation of cyclopropenoids with fatty acids eliminates the unusual biological effects attributable to cyclopropenoids.

Mass spectra of acetyl derivatives of homogeneous cerebrosides (monoglyco-sylceramides)

Mass spectra of acetyl derivatives of a series of homogeneous cerebrosides have been recorded. The compounds studied had a D-galactopyranose in β-glycosidic linkage to different ceramides (containing dihydroxy or trihydroxy long-chain base and normal or hydroxy fatty acid). The composition of the major fragments was established by high resolution analysis. Molecular ions were found for all compounds ( m/ e 713–1055). Dominating fragments were obtained from the acetylated glycose residue, with a similar pattern as for acetylated simple carbohydrates. Fragments containing base plus fatty acid were of medium intensity and may be used for the analysis of natural compounds containing a mixture of fatty acids and long-chain bases. Specific fragments of the fatty acid or longchain base were not found, except for some fragments of low intensity containing part of or the complete normal fatty acid.

Mass spectral discrimination between monoenoic and cyclopropanoid, and between normal, iso, and anteiso fatty acid methyl esters

Measurement of the ratio of the intensity of the parent molecular ion to that of the parent molecular ion minus 32 (the mass of methanol) can distinguish between monoenoic and cyclopropanoid fatty acid methyl esters with the same carbon number. A similar technique can be used to distinguish between isomeric normal, iso, and anteiso fatty acid methyl esters.

A physicochemical study of fat absorption in rats limitation of methods in vitro

1. 1. The hypothesis that the first stage of normal fatty acid absorption represents a partition between a luminal bile salt mixed micellar phase and mucosal membrane lipid phase has been investigated. 2. 2. Everted segments of rat jejunum incubated with [ 14C]oleic acid in 10 mM sodium taurocholate accumulated a greater amount of 14C-labelled free fatty acid when incubated at pH 5.8 then at pH 7.3 but this was unrelated to incorporation into jejunal [ 14C]triglyceride. 3. 3. Uptake of [ 14C]oleic acid into mucosal free fatty acid from 15 mM taurocholate (20 μmoles/ml) in vitro was less than from 6 mM taurocholate, but the triglyceride accumulation was greater. 4. 4. pH had no effect on the uptake in vitro of the non-ionized oleyl alcohol from taurocholate mixed micellar solution but with increasing taurocholate concentration there was decreased uptake of [ 14C] oleyl alcohol. 5. 5. Increasing concentrations of [ 14C]oleic acid in bile salt mixed micelles caused an increase both of 14C-labelled free fatty acid and [ 14C]triglyceride in vitro. 6. 6. Absorption of [ 14C]oleic acid from perfused or closed loops of jejunum in vivo was increased with increased oleic acid concentration but was not influenced by pH or bile salt concentration. 7. 7. These findings suggest that although uptake of free fatty acid by the mucosal cell in vitro may be predicted on the basis of a partition between micelle and lipid membrane via a molecular phase, this is not an important mechanism during normal fatty acid absorption.

Separation of lipids containing phytanic acid by thin‐layer chromatography

Cholesteryl phytanate and triglycerides containing phytanic acid were separated from their normal fatty acid analogs by thin-layer chromatography. The presence of the branched-chain fatty acid makes the lipid less polar, and this effect becomes more pronounced as the number of phytanic acid residues in the triglycerides is increased. Phytanic acid was prepared from commercial phytol by catalytic hydrogenation, followed by catalytic oxidation. It contained 5% pristanic acid.

Studies of the metabolism of isolated livers of normal and alloxan-diabetic rats perfused with insulin.

Livers of fed normal and diabetic rats were perfused in the presence of glucose-l-C-14 and acetate-H-3 in order to study their metabolism with and without addition of insulin to the medium. Glucose was added to bring the initial medium level to 436 ± 6 mg./100 ml. Net glucose removal occurred only with normal rat livers. Livers of diabetic rats showed progressive impairment of glucose uptake and increase in glucose production as the interval from the last maintenance insulin injection was prolonged. Whereas these chronically diabetic animals had livers with low glycogen content, decreased lipogenesis, and increased uro genesis, rats which had received 1.5 U. protamine insulin on the previous day were fully hyperglycemic but yielded livers showing normal fatty acid and urea synthesis along with excessively high glycogen. Insulin addition lowered perfusate amino nitrogen levels with normal and diabetic livers but did not significantly affect glycemia in any series and had only small stimulatory effects on lipogenesis and glucose oxidation in livers of rats three to five days off insulin. The data suggest that although insulin has important direct effects on liver metabolism, its hepatic action does not explain its early in vivo hypoglycemic effect in diabetes.

The Normal Fatty Acid Constituents of Depot Fat of Cats and Guinea Pigs

AbstractThe normal fatty acid contents of omental fat were investigated in cats and guinea pigs. Sex appears to have no effect on the distribution of saturated and unsaturated fatty acids; the latter are always in higher proportion than the former, although guinea pigs show greater variations in this proportion. Palmitic acid was the principal constituent of the saturated portion and linoleic acid, that of the unsaturated portion in both animals.