Turnover of plasma free arachidonic and oleic acids in men and women.

The turnover of plasma free arachidonic and oleic acids was determined in healthy men and women. The plasma pool of arachidonic acid in the men was 75 per cent higher than in the women. The fractional turnover of arachidonate was 0.80 +/- 0.04 min(-1) in the women and 0.47 +/- 0.04 min(-1) in the men. The turnover rate of arachidonic acid was similar in both sexes; calculated per kg body weight it was significantly higher in the women. No sex differences were observed in the concentration or turnover of plasma free oleic acid when corrected for differences in body size. The composition of the free fatty acid fraction differed between the sexes, the female subjects having a lower proportion of saturated fatty acids and higher proportions of oleic and eicosenoic acids. The results indicate that the metabolism of polyunsaturated fatty acids in man is influenced by gonadal steroid hormones.     

Use of radio-gas chromatography in the study of plant metabolism

The parameters controlling the counting rate and the peak resolution in radio-gas chromatography (rgc) from a typical “effluent combustion” system have been examined, with special emphasis placed on the precautions necessary for the operation of rgc with temperature programming. The potential of rgc for study of the dynamics of carbohydrate transport and metabolism in plants has also been demonstrated.     

Determination of plasma oxalic acid by high-pressure liquid chromatography

A new specific and sensitive method for determination of oxalic acid in plasma by High Performance Liquid Chromatography (HPLC) is described. The plasma sample is deproteinized by ultrafiltration. The oxalic acid in the ultrafiltrate is purified by precipitation with CaCl2, new dilution of calcium oxalate precipitate, oxalic acid extraction with diethyl-ether and total dryness of the sample. The losses of oxalic acid during this process are evaluated by the addition of oxalic acid (U-14C) before the precipitation step. The dried samples are redissolved in mobile phase (o-H3PO4, 0.05 M) and injected into a HPLC chromatograph, with reversed phase column (Lichrosorb RP-8, Merck). Oxalate peak is detected spectrophotometrically at 220 nm with a retention time of 3.20 minutes. The method shows a mean recovery value of 82.11, with an intra-run and between-run CV values of 2.54 and 6.95 respectively. The oxalic acid measured in plasma by this method is 291 +/- 89 micrograms/100 ml plasma ultrafiltrate, in 16 normal subjects.     

Microbial oxidation of oleic acid.

Resting cells of Saccharomyces cerevisiae (baker's yeast, type II; Sigma) were used to convert oleic acid into 10-hydroxyoctadecanoic acid with a 45% yield. Nocardia aurantia (ATCC 12674), Nocardia sp. (NRRL 5646), and Mycobacterium fortuitum (UI 53378) all converted oleic acid into 10-oxo-octadecanoic acid with 65, 55, and 80% yields, respectively. Structures of all metabolites were suggested by 1H and 13C nuclear magnetic resonance and by infrared and mass spectrometry. Structures of isomeric hydroxystearate and oxostearate derivatives and the stereochemical purity of hydroxystearates are difficult to prove unambiguously unless authentic standard compounds are available for spectral comparison. We describe the use of the chemical Baeyer-Villiger oxidation technique with 10-oxo-octadecanoic acid followed by mass spectral analysis of neutral extracts as a simple method to confirm the position of oxo-functional groups in the structures of fatty acid ketones. We further introduce a simple method based on 1H nuclear magnetic resonance analysis of diastereomeric S-(+)-O-acetylmandelate esters of hydroxystearates as a means of ascertaining stereochemical purities of hydroxy fatty acids.     

Microbial oxidation of oleic acid.

Resting cells of Saccharomyces cerevisiae (baker's yeast, type II; Sigma) were used to convert oleic acid into 10-hydroxyoctadecanoic acid with a 45% yield. Nocardia aurantia (ATCC 12674), Nocardia sp. (NRRL 5646), and Mycobacterium fortuitum (UI 53378) all converted oleic acid into 10-oxo-octadecanoic acid with 65, 55, and 80% yields, respectively. Structures of all metabolites were suggested by 1H and 13C nuclear magnetic resonance and by infrared and mass spectrometry. Structures of isomeric hydroxystearate and oxostearate derivatives and the stereochemical purity of hydroxystearates are difficult to prove unambiguously unless authentic standard compounds are available for spectral comparison. We describe the use of the chemical Baeyer-Villiger oxidation technique with 10-oxo-octadecanoic acid followed by mass spectral analysis of neutral extracts as a simple method to confirm the position of oxo-functional groups in the structures of fatty acid ketones. We further introduce a simple method based on 1H nuclear magnetic resonance analysis of diastereomeric S-(+)-O-acetylmandelate esters of hydroxystearates as a means of ascertaining stereochemical purities of hydroxy fatty acids.     

Turnover rates of different collagen types measured by isotope ratio mass spectrometry.

The rates of collagen turnover in different tissues have been estimated in growing rats previously exposed to gaseous 18O2. The abundance of the stable isotope was measured using isotope ratio mass spectrometry following combustion of isolated collagen-derived hydroxyproline. Using this method, problems of label reutilization associated with radiolabelling methods are avoided. In general the results confirm the slow turnover rates with half-lives of total collagen in skin, muscle and gut of 74, 45 and 244 d, respectively. The use of cyanogen bromide digests of whole tissues followed by isolation of collagen type-specific peptides has allowed the comparison of turnover rates of collagen types I and III, indicating that collagen type III is turned over more rapidly than type I.     

Rapid flip-flop of oleic acid across the plasma membrane of adipocytes

Nonesterified long-chain fatty acids may enter cells by free diffusion or by membrane protein transporters. A requirement for proteins to transport fatty acids across the plasma membrane would imply low partitioning of fatty acids into the membrane lipids, and/or a slower rate of diffusion (flip-flop) through the lipid domains compared to the rates of intracellular metabolism of fatty acids. We used both vesicles of the plasma membrane of adipocytes and intact adipocytes to study transmembrane fluxes of externally added oleic acid at concentrations below its solubility limit at pH 7.4. Binding of oleic acid to the plasma membrane was determined by measuring the fluorescent fatty acid-binding protein ADIFAB added to the external medium. Changes in internal pH caused by flip-flop and metabolism were measured by trapping a fluorescent pH indicator in the cells. The metabolic end products of oleic acid were evaluated over the time interval required for the return of intracellular pH to its initial value. The primary findings were that (i) oleic acid rapidly binds with high avidity in the lipid domains of the plasma membrane with an apparent partition coefficient similar to that of protein-free phospholipid bilayers; (ii) oleic acid rapidly crosses the plasma membrane by the flip-flop mechanism (both events occur within 5 s); and (iii) the kinetics of esterification of oleic acid closely follow the time dependence of the recovery of intracellular pH. Any postulated transport mechanism for facilitating translocation of fatty acid across the plasma membrane of adipocytes, including a protein transporter, would have to compete with the highly effective flip-flop mechanism.     

A novel phospholipase D of Arabidopsis that is activated by oleic acid and associated with the plasma membrane.

Oleate-dependent phospholipase D (PLD; EC 3.1.4.4) has been reported in animal systems, but its molecular nature is unkown. Multiple PLDs have been characterized in plants, but none of the previously cloned PLDs exhibits the oleate-activated activity. Here, we describe the biochemical and molecular identification and characterization of an oleate-activated PLD in Arabidopsis. This PLD, designated PLDdelta, was associated tightly with the plasma membrane, and its level of expression was higher in old leaves, stems, flowers, and roots than in young leaves and siliques. A cDNA encoding the oleate-activated PLD was identified, and catalytically active PLDdelta was expressed from its cDNA in Escherichia coli. PLDdelta was activated by free oleic acid in a dose-dependent manner, with the optimal concentration being 0.5 mM. Other unsaturated fatty acids, linoleic and linolenic acids, were less effective than oleic acid, whereas the saturated fatty acids, stearic and palmitic acids, were totally ineffective. Phosphatidylinositol 4,5-bisphosphate stimulated PLDdelta to a lesser extent than oleate. Mutation at arginine (Arg)-611 led to a differential loss of the phosphatidylinositol 4,5-bisphosphate-stimulated activity of PLDdelta, indicating that separate sites mediate the oleate regulation of PLDdelta. Oleate stimulated PLDdelta's binding to phosphatidylcholine. Mutation at Arg-399 resulted in a decrease in oleate binding by PLDdelta and a loss of PLDdelta activity. However, this mutation bound similar levels of phosphatidylcholine as wild type, suggesting that Arg-399 is not required for PC binding. These results provide the molecular information on oleate-activated PLD and also suggest a mechanism for the oleate stimulation of this enzyme.     

Determination of plasma fatty acid composition in neonates by gas chromatography

Total fatty acids in plasma of neonates have been analysed as their methyl esters by gas chromatography. They were separated on a capillary column coated with a SP-2380 stationary phase. As little as 100 μl of plasma is used for the analysis. The extraction procedure was performed with dichloromethane—methanol (2:1) and fatty acids were methylated with boron trifluoride—methanol. The quantification of fatty acids is based on an internal standard method. Absolute values (μg fatty acid per 100 μl plasma) are given together with relative values (%). At a signal-to-noise ratio of 3, the detection limits for flame ionisation detection are between 0.08 to 0.51 ng. The high sensitivity and precision permits the effective determination of the fatty acids in neonate plasma.     

Stimulation of hepatic cholesterol biosynthesis by oleic acid

Livers from normal fed male rats were perfused $\text{in vitro}$ with an erythrocyte-free, bloodless medium containing serum albumin (3%), and glucose (100 mg %). Oleic acid (663 μmoles) bound to albumin, or albumin alone, was infused at a constant rate. Biosynthesis of cholesterol was evaluated by incorporation of radioactivity from ³H₂O. Oleic acid stimulated output of cholesterol (1.60 ± 0.08 SEM vs 1.18 ± 0.04 μmoles/g) but did not change the concentration of cholesterol in the liver or hepatic microsomes. Incorporation of ³H into cholesterol was stimulated by oleate; dpm per μmole cholesterol/dpm per μg atom H was 3.94 ± 0.33, 3.46 ± 0.32, and 4.46 ± 0.37 in the total cholesterol of liver, perfusate, and microsomes, respectively, when oleate was infused. Corresponding values when oleate was not infused were 1.71 ± 0.23, 1.62 ± 0.20, and 2.09 ± 0.26, respectively (P<0.001 in all cases). It is suggested that the stimulation of biosynthesis of cholesterol by oleate results from the obligatory requirement of cholesterol, as a moiety of the very low density lipoprotein, for the secretion of triglyceride by the liver.     

Rapid determination of mycophenolic acid in plasma by reversed-phase high-performance liquid chromatography

A simple, accurate and sensitive high-performance liquid chromatographic method with UV detection was carried out to measure plasma concentrations of mycophenolic acid. Following a simplified acid hydrolysis of the sample, the separation was carried out in 4 min using a Zorbax Eclipse C(8) reversed-phase column with a flow-rate of 1.5 ml/min, and monitoring the absorbance at 250 nm. Throughput was up to 100 samples in 24 h. Within the investigated concentration ranges of mycophenolic acid (0-100 mg/l), good linearity (r>0.99) was obtained. The method is sensitive (the limit of detection was about 20 microg/l) and precise (for 0.49 mg/l added to plasma, within-run C.V. was 2% and between-run was 4.2%; for 2.88 mg/l, within-run C.V. was 0.35% and between-run C.V. was 0.69%; for 24.38 mg/l, within-run C.V. was 0.77% and between-run C.V. was 3.1%). Analytical recoveries were 96% for 0.5 mg/l mycophenolic acid added to plasma, 100% for 12 mg/l and 102.5% for 24 mg/l.     

Determination of indomethacin and mefenamic acid in plasma by high-performance liquid chromatography

Indomethacin and mefenamic acid are widely used clinically as non-steroidal anti-inflammatory agents. Both drugs have also been found effective to produce closure of patent ductus arteriosus in premature neonates. A simple, rapid, sensitive and reliable HPLC method is described for the determination of indomethacin and mefenamic acid in human plasma. As these drugs are not applied together, the compounds are alternately used as analyte and internal standard. Plasma was deproteinized with acetonitrile, the supernatant fraction was evaporated to dryness and the resulting residue was reconstituted in the mobile phase and injected into the HPLC system. The chromatographic separation was performed on a C₁₈ column (250 × 4.6 mm I.D.) using 10 mM phosphoric acid—acetonitrile (40:60, v/v) as the mobile phase and both drugs were detected at 280 nm. The calibration graphs were linear with a correlation coefficient (r) of 0.999 or better from 0.1 to 10 μg/ml and the detection limits were 0.06 μg/ml for indomethacin and 0.08 μg/ml for mefenamic acid, for 50μl plasma samples. The method was not interfered with by other plasma components and has been found particularly useful for paediatric use. The within-day precision and accuracy of the method were evaluated for three concentrations in spiked plasma samples. The coefficients of variation were less than 5% and the accuracy was nearly 100% for both drugs.     

5-Methyltetrahydrofolic acid and folic acid measured in plasma with liquid chromatography tandem mass spectrometry: applications to folate absorption and metabolism

We describe a liquid chromatography (LC) tandem mass spectrometry (MS-MS) method for the determination of 5-methyltetrahydrofolic acid (5-methylTHF) and folic acid concentrations and enrichments in human plasma. It was used to study absorption and initial metabolism in five volunteers with two simultaneously administered oral test doses ([(13)C(6)]folic acid in capsules and [(2)H(2)]folic acid in a drink). [(13)C(5)]5-methylTHF and [(2)H(4)]folic acid were used as internal standards. Plasma samples (2 ml) were purified using folate binding protein affinity columns, followed by a concentration step. After LC separation, folates were detected using positive electrospray ionization MS-MS under multiple reaction monitoring conditions. Calibrations were linear for 5-methylTHF over the range 1.2 x 10(-11) (=limit of detection) to 3.2 x 10(-7)mol/L and for folic acid over the range 5 x 10(-10) (=limit of detection) to 4.5 x 10(-8)mol/L. For 5-methylTHF concentration in plasma, intraassay coefficient of variation was within 8.6% (and for unlabeled 5-methylTHF it was within 2.8%) and interassay coefficient of variation was within 9.0%. For folic acid concentrations these coefficient of variations were within 7.5% and within 6.5%, respectively. The [(13)C(6)] and [(2)H(2)] isotopomers of folic acid and 5-methylTHF were measured in the plasma of each volunteer for 8h. After accounting for the time delay due to capsule opening, the modeling results showed no significant differences in absorption time, first pass effect, and elimination rate in the folic acid test doses in capsule or drink. We conclude that LC-MS-MS offers increased sensitivity for quantification of plasma concentrations and enrichments of 5-methylTHF and folic acid and is applicable to stable-isotope studies in humans.