Distribution of newly formed fatty acids among glycerolipids of isolated perfused rat liver.

Livers of chow fed rats were perfused 1-3 h with buffer, glucose, albumin, and red blood cells, made up in 100 percent D(2)O. Glycerolipids were isolated and the deuterated fatty acids determined by gas chromatography with mass spectrometry on Silar 5 CP. Percentage of replacement by deuterated acids ranged from 1 to 14, of which palmitate was 87 percent. Differences were found in total lipid class and in subcellular distribution of the newly synthesized acids. Microsomes had 37 percent more deuterated acids than the total or floating fat. At 3 h the highest replacement was found in diacylglycerols (17 percent) and free fatty acids (11 percent). Of the palmitate in hepatic choline and ethanolamine phosphatides, 6.9 percent and 4.7 percent, respectively, contained dueterium. The serine and inositol phosphatides had a higher proportion of deuterated palmitate (7.7 percent) than other phosphatides. The data support the hypothesis that palmitate is incorporated into glycerolipids largely via de novo synthesis while stearate enters them by deacylation-acyl transfer replacement.     

Incorporation of chlorinated alkanes into fatty acids of hydrocarbon-utilizing mycobacteria

The cellular fatty acid composition of Mycobacterium vaccae JOB5 and Mycobacterium convolutum R22 was examined after growth on n-alkanes and compared with the fatty acids of the organisms after growth on 1-chlorohexadecane and 1-chlorooctadecane. Growth on n-alkanes resulted in normal fatty acid profiles. Mass spectral analyses indicated that, after growth on the terminally chlorinated n-alkanes, 75 to 86% of the fatty acids in M. convolutum and ca. 55% of the fatty acids in M. vaccae contained chlorine. Neither organism could utilize chloroacetate or 3-chloropropionate as sole source of carbon and energy. When these compounds were added to a growth medium with n-hexadecane as substrate, there was no evidence that chlorinated fatty acids were produced. Terminally chlorinated n-alkanes can be added to the list of n-alkanes, alkenes, and cyclohexylalkane derivatives that can be directly incorporated into cellular fatty acids of hydrocarbon-utilizing organisms.     

Subcellular distribution of newly synthesized glycerolipids in intact and homogenized rat adipocytes.

1. Homogenization of rat adipocytes was found to inhibit the transfer of newly synthesized phospholipids and glycerides from the membranes to the storage lipids. 2. In intact adipocytes, only 23 +/- 3.4% of the phospholipids and 0.44 +/- 0.12% of the glycerides remained bound to the membranes, while for the homogenate, the comparable values obtained were 74 +/- 5.2% and 90 +/- 0.3% respectively. 3. Various factors that might be involved in the liberation of the glycerolipids from the adipocyte esterification sites are discussed.     

Incorporation of spin-labeled fatty acids into bovine brain clathrin coated vesicles

Stearic acids with a nitroxide radical at selected positions have been incorporated in the phospholipid bilayers of clathrin coated vesicles, uncoated vesicles and sonicated liposomes made from the lipids extracted from the uncoated vesicles. The extent of incorporation was found minimum for stearic acids labeled on C-12 and for bilayers of uncoated vesicles. The ESR spectra of the spin-labeled fatty acids incorporated in the bilayers showed a pronounced temperature dependence (without discontinuity) and a decrease in the hyperfine splitting as the nitroxide group was inserted deeper in the hydrophobic core of the membranes. An abrupt phospholipid phase transition or a phase separation could be excluded. The presence of the external proteins (the clathrin coat) on the membranes was not found to noticeably influence the gradient of flexibility of the fatty acid chains of the phospholipids. The influence of the internal proteins embedded in the bilayers was evidenced by a detailed analysis of the ESR spectra of (7,8)SA in terms of two components: one component arising from the labels surrounded exclusively by phospholipids, the other component arising from labels of reduced mobility perturbed by the vicinity of the proteins. These results support the persistence of lipidic domains in the endocytic vesicles despite the accumulation of receptors which follows their formation.     

alpha-Hydroxylation of newly synthesised fatty acids by a soluble fraction from germinating pea.

A soluble fraction from germinating pea (Pisum sativum) seeds alpha-hydroxylated newly-synthesised fatty acids to form alpha-hydroxypalmitic and alpha-hydroxystearic acids. In contrast to fatty acid synthesis from [14C] malonyl CoA, alpha-hydroxylation was inhibited by exogenous phospholipids. alpha-Hydroxylation was optimal at pH 8, required reduced pyridine nucleotides and was inhibited by EDTA and imidazole.     

Temperature-mediated hyperinduction of fatty acid desaturation in pre-existing and newly formed fatty acids synthesized endogenously in Bacillus megaterium

3H-labedeled fatty acids synthesized endogenously by Bacillus megaterium ATCC 14581 growing at 35 degrees C in the presence of L-[G-3H]valine exhibited the same time-course of hyperinduced desaturation following a temperature decrease to 20 degrees C as was observed previously with exogenously supplied 14C-labeled fatty acids. Radioactive fatty acids synthesized in the presence of [U-14C]glucose during hyperinduction at 20 degrees C following a shift-down from 35 degrees C were desaturated at the same relative rate as 14C-labeled fatty acids synthesized previously at 35 degrees C, suggesting that the newly synthesized fatty acids equilibrate with a large portion of the preexisting moieties before becoming susceptible to desaturation.     

Positional distribution of isomers of monoenoic fatty acids in animal glycerolipids

The distributions of the following monoenoic acids were determined [notation: (position of double bond)-(chain length): (no. of double bonds)]: 7-, 9-, and 11-16:1; 7-, 9-, 11-, and 13-18:1; 9-, 11-, and 13-20:1; 9 + 11-22:1 and 13-22:1. As a rule, all isomers of a group show different distribution patterns. In the phospholipids of fish and mammals, the 7- and 13-isomers of 18:1 accumulate in position 1. In triglycerides of mammals fed on fish they accumulate in positions 1 plus 3, and this distribution is shared by 7-16:1 and 11-16:1 and by the groups 20:1 and 22:1. The positional distribution of the acids seems to depend on their structure, the 9-isomers in general accumulating in position 2; but in triglycerides, at least, the origin of the acid also seems to play a directing role, the exogenous acids being incorporated into positions 1 and 3. The variability of the distribution patterns of 9-16:1, 9-18:1, and 11:18:1, which contrasts with the regularity of the patterns for saturated and polyenoic acids, may be connected with the ability of the endogenous monoenoic acids to balance fluctuations in the supply of the exogenous polyenoic acids, and with the role of the fatty acid 9,10-dehydrogenation mechanism in the maintenance of structural and physical properties of phospholipids and triglycerides.     

Activity of the phosphatidylcholine biosynthetic pathway modulates the distribution of fatty acids into glycerolipids in proliferating cells

PtdCho accumulation is a periodic, S phase-specific event that is modulated in part by cell cycle-dependent fluctuations in CTP:phosphocholine cytidylyltransferase (CCT) activity. A supply of fatty acids is essential to generate the diacylglycerol (DG) precursors for phosphatidylcholine (PtdCho) biosynthesis but it is not known whether the DG supply is also coupled to the cell cycle. Although the rate of fatty acid synthesis in a macrophage cell line was dramatically stimulated in response to the growth factor, CSF-1, it was not regulated by the cell cycle. Increased fatty acid synthesis correlated with elevated acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) steady-state mRNA levels. Cellular fatty acid synthesis was essential for membrane PL synthesis. Cerulenin inhibition of endogenous fatty acid synthesis also inhibited PtdCho synthesis, which was not relieved by exogenous fatty acids. Inhibition of CCT activity by the addition of lysophosphatidylcholine (lysoPtdCho) or temperature-shift of a conditionally defective CCT diverted newly synthesized DG to the TG pool where it accumulated. Enforced expression of CCT stimulated PtdCho biosynthesis and reduced TG synthesis. Thus, the cellular DG supply did not regulate PtdCho biosynthesis and CCT activity governs the partitioning of DG into either the PL or TG pools, thereby controlling both PtdCho and TG biosynthesis.     

Incorporation of radioiodinated fatty acids into cardiac phospholipids of normoxic canine myocardium

The aim of this study was to assess the phospholipid distribution of radioiodinated 17-iodoheptadecanoic acid (IHDA), 15-(p-iodophenyl)pentadecanoic acid (p-IPPA) and 15-(p-iodophenyl)-3,3-dimethylpentadecanoic acid (DMIPPA) under normoxic conditions and to compare these data with the fatty acid composition of the phospholipid classes. After simultaneous i.v. injection of the radioiodinated fatty acids (1-123-IHDA; 1-131-p-IPPA; 1-125 DMIPPA) in open-chest dogs seven myocardial biopsies were taken over 40 min (n = 26). After lipid extraction of the biopsies the organic phase was analyzed for both neutral and polar lipids by two different TLC systems. The following polar lipid fractions were analyzed: lysophopshatidylcholine (LPC), sphingomyelin (SPH), phosphatidy1choline (PC; lecithin), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG; cardiolipin) and neutral lipids. Fractions were counted in a gamma well counter and corrected for cross-over and recovery. Results of the polar phospholipids analysis showed that IHDA has the highest incorporation into the phospholipids. The IHDA was mainly incorporated into PI (45.6%) followed by PC (30.9%), PE (14.0%) and PS (5.6%). The p-IPPA was predominantly incorporated incorporated into PC (37.2%), followed by PS (20.1%) and PE (13.7%). In contrast to IHDA, incorporation of p-IPPA into PI was small (6.4%). The DMIPPA analogue was incorporated into phopsholipids to only a very small degree, compared to IHDA and p-IPPA. PS (27.4%) was the only considerable phospholipid fraction into which DMIPPA was incorporated.The results clearly demonstrated that these radioiodinated fatty acid analogues have entirely different patterns of phospholipid incorporation. Major resemblances have been found between the incorporation into phospholipids of IHDA and the phospholipid distribution of the natural counterpart: stearic acid. The p—IPPA phospholipid incorporation only partly resembles the phospholipid distribution of palmitic acid. DMIPPA is because of its modified structure, incorporated into phospholipids to a low extent, mainly into PS. (Mol Cell Biochem116: 79–87, 1992)     

The incorporation of oleic and linoleic acids and their desaturation products into the glycerolipids of maize leaves

[1-¹⁴C]Oleic and [1-¹⁴C]linoleic acids were rapidly desaturated when incubated with maize leaves from 8-day-old plants and the labeled fatty acids, and their desaturation products, were rapidly incorporated into glycerolipids. Oleic acid was desaturated to linoleate at the rate of 0.7 nmol/100 mg tissue/h and further desaturated to linolenate at about one-third this rate. The rates of linolenate formation were similar when either oleic acid or linoleic acid was the substrate although there was a 2-h lag period when oleic acid was substrate. When radioactive oleic, linoleic, and linolenic acids were substrates, phosphatidylcholine was the most extensively labeled glycerolipid followed by monogalactosyldiacylglycerol. The relative rates of incorporation of label into individual glycerolipids are consistent with a movement of labeled fatty acids from phosphatidylcholine to monogalactosyldiacylglycerol and then to diagalactosyldiacylglycerol. The rates of labeling of phosphatidylcholine oleate and of phosphatidylcholine linoleate are consistent with a precursor-product relationship in that there was a delayed accumulation of phosphatidylcholine linoleate relative to that of phosphatidylcholine oleate and phosphatidylcholine linoleate continued to accumulate while phosphatidylcholine oleate declined. Linoleate formed from oleate was widely distributed in glycerolipids but neither phosphatidylcholine linolenate nor linolenate-containing diacylglycerol was detected at short and intermediate incubation times when either oleic or linoleic acid was substrate. The kinetics of incorporation of linoleate and linolenate into monogalactosyldiacylglycerol suggest a transfer of linoleate from phosphatidylcholine. The initial rate of accumulation of labeled linolenate in monogalactosyldiacylglycerol was very similar to the rate of desaturation of linoleate and it is suggested that desaturation of linoleate occurs while associated with monogalactosyl-diacylglycerol.     

Influence of dexamethasone on the lipid distribution of newly synthesized fatty acids in fetal rat lung

There is a developmental increase in fatty acid biosynthesis and surfactant production in late-gestation fetal lung and both are accelerated by glucocorticoids. We have examined the distribution of the newly synthesized fatty acids to determine whether they are preferentially incorporated into surfactant. Explants of 18 day fetal rat lung were cultured with and without dexamethasone for 48 h and then with [3H]acetate for 4 h after which labeled fatty acids were measured. Incorporation of radioactivity from acetate was considered a measure of newly synthesized fatty acids. Phospholipids contained 86% of the newly synthesized fatty acids of which approx. 80% were in phosphatidylcholine. Phosphatidylcholine and disaturated phosphatidylcholine contained a much greater percentage of the labeled fatty acids than of the phospholipid mass determined by phosphorus assay while phosphatidylethanolamine, phosphatidylserine and sphingomyelin contained less. Dexamethasone increased the rate of acetate incorporation into total lipid fatty acids but it had little effect on fatty acid distribution, except that it increased the percentages in phosphatidylglycerol and disaturated phosphatidylcholine. The hormone also increased the mass of these two phospholipids to a greater extent than that of the total. These data suggested that the newly synthesized fatty acids are preferentially incorporated into surfactant phospholipids and that this process is accelerated by dexamethasone. However, since phosphatidylcholine and phosphatidylglycerol are not exclusive to surfactant, we compared isolated lamellar bodies with a residual fraction not enriched in surfactant. The rate of acetate incorporation into fatty acids in lamellar body phosphatidylcholine as well as its specific activity (radioactivity per unit phosphorus) were both increased by dexamethasone. Specific activity, however, was no greater in the lamellar bodies than in the residual fraction in both control and dexamethasone-treated cultures. Therefore, there is no preferential incorporation of newly synthesized fatty acids into phospholipids in surfactant as opposed to those in other components of the lung.