Fatty acid methyl esters, Serum

Normal human fibroblasts grown in media with 12% fetal calf serum were treated with or without galactose oxidase and then subjected to reduction with potassium borotritiide. GSLg were isolated and subjected to acid-catalyzed methanolysis (62) (see Materials and Methods). Methyl glycosides, methyl sphingosine, and fatty acid methyl esters were isolated by extraction of the total hydrolysate with solvents as described previously (62). Radioactivity was measured in triplicate in aliquots of these extracts. 

Radiolabeled [l- C]18 3n-3 was purchased from Perkin-Elmer Life Sciences (Boston, MA). The free acid form of 24 5n-3 and 24 6n-3 were generous gifts from A. Spector and H. Sprecher. Human skin fibroblasts from normal controls and patients with peroxisomal or mitochondrial disorders were received from the Mental Retardation Research Center of the Kennedy Krieger Institute. Cells at 90% confluence were incubated with 0.05 pCi albumin-bound [1- C] 18 3n-3 in Dul-becco s modified Eagle s minimum essential medium supplemented with 10% fetal bovine serum for 3 d. At harvest, cells were removed and washed with Hanks solution. An aliquot of cells was removed for protein determination, and the remainder was used for analysis of fatty acids after conversion to their methyl esters (17). Radiolabeled fatty acid methyl esters were separated by reversed-phase high performance liquid chromatography (18), and collected by a fraction collector. The radioactivity was counted by liquid scintillation counting. 

As stationary phase for the resolution of fatty acid mixtures polar (polyesters of short chain dicarbonic acids and low molecular diols) and unpolar (hydrocarbons, silicones) substances can be used. Gas-chromatography under standardized conditions permits the tentative identification of separated fatty acids from the time elapsing between application of the sample and the emergence of the acid in question. Figure 6 shows the separation of serum fatty acid methyl esters using a polar stationary phase. 

An aliquot of the corresponding fraction of the samples was counted in a Packard Tricarb Scintillation counter. Other aliquots were esterified. The distribution of the radioactivity between the fatty acids was determined by gas liquid radiochromatography (Alaniz et al, 1976). The labeled methyl esters were identified by equivalent chain length determination and comparison with authentic standards. The fatty acid composition of serum, HTC cells and lipid fractions of culture cells was analyzed by gas liquid chromatography. The specific radioactivities for linoleic and arachidonic acids were calculated with those data after measuring the radioactivity in an aliquot in which the mass distribution of the fatty acids had previously been determined by gas liquid chromatography in the presence of an internal standard of eicosaenoic acid. 

Cholesterol is formed in the liver (85%) and intestine (12%) - this constitutes 97% of the body s cholesterol synthesis of 3.2 mmol/day (= 1.25 g/day). Serum cholesterol is esterized to an extent of 70-80% with fatty acids (ca. 53% linolic acid, ca 23% oleic acid, ca 12% palmitic acid). The cholesterol pool (distributed in the liver, plasma and erythrocytes) is 5.16 mmol/day (= 2.0 g/day). Homocysteine stimulates the production of cholesterol in the liver cells as well as its subsequent secretion. Cholesterol may be removed from the pool by being channelled into the bile or, as VLDL and HDL particles, into the plasma. The key enzyme in the synthesis of cholesterol is hydroxy-methyl-glutaryl-CoA reductase (HGM-CoA reductase), which has a half-life of only 3 hours. Cholesterol is produced via the intermediate stages of mevalonate, squalene and lanosterol. Cholesterol esters are formed in the plasma by the linking of a lecithin fatty acid to free cholesterol (by means of LCAT) with the simultaneous release of lysolecithin. (s. figs. 3.8, 3.9) (s. tab. 3.8)... 

Preparation from [i- C] Linoleic Acid. (9Z,llf )-[l-i" C]-Octadecadienoic acid was prepared by incubating [l-i" C]-linoleic acid with an enzyme, Unoleate isomerase, obtained from the rumen bacterium Butyrivibrio fibrisolvens (11). This isomerization was carried out with a mixture of 0.2 mL of the enzyme preparation, [l-i" C]-linoleic acid (0.2 pCi 0.12 mg) and 4 mg of bovine serum albumin in 1 mL of 0.1 moI/L potassium phosphate buffer (pH 7). After separation of fatty acids from the reaction mixture and formation of methyl esters with diazomethane, methyl (9Z,ll )-[l-i C]-octadecadienoate was purified by preparative silver nitrate thin-layer chromatography. 

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