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At least 703 records · Page 39Linked to original sources
PubMed1997

Generation and remodeling of highly polyunsaturated molecular species of rat hepatocyte phospholipids.

Freshly isolated rat hepatocytes were incubated for 20 min with [U-14C]glycerol in the presence or absence of unlabeled linoleic (18:2n-6), arachidonic (20:4n-6), or docosahexaenoic (22:6n-3) acid, added as albumin complex in 10% ethanol. Most of the radioactivity (approximately 95%) recovered in hepatocyte lipids was present in phosphatidylcholine (PC), phosphatidylethanolamine (PE), and triacylglycerol (TAG). The presence of exogenous fatty acids resulted in (i) higher incorporation of [U-14C]glycerol, (ii) higher percentage of label in TAG, and (iii) enhanced formation of PC and PE molecular species bearing the exogenous fatty acid at both the sn-1 and sn-2 positions of glycerol. In each case, these molecular species contained 60 to 70% of the label in that lipid class. Further incubation of the cells for 40 and 80 min in the absence of labeled substrate and exogenous fatty acids resulted in a redistribution of label among PC and PE molecular species due to deacylation-reacylation at the sn-1 position of glycerol.

Animals↗
PubMed1988

Separation of formyl-methionyl transfer RNA, methionyl transfer RNA, and transfer RNAfmet using mixed-mode high-performance liquid chromatography on C6-modified aminopropylsilyl-hypersil.

Preparative amounts of formyl-methionyl-tRNAfmet, methionyl-tRNAfmet, and tRNAfmet were separated from each other with baseline resolution in 30 min using mixed-mode HPLC on hexanoic anhydride-modified aminopropylsilyl-Hypersil 2. Pure tRNAfmet was aminoacylated with [35S]methionine in the presence or absence of a formyl donor and was immediately fractionated on the column. Two isoacceptors, tRNA1fmet and tRNA2fmet, as well as aminoacyl-tRNA synthetases were clearly separated from each other. The purified f[35S]-methionyl-tRNA was biologically active in that as much as 98% could be bound to ribosomes in response to AUGUAA in vitro. Formyl-methionine was released from this complex by the action of termination factor and greater than 92% of bound formyl-methionine was released by puromycin.

Caproates↗
PubMed1989

Protein modification by diazotized arsanilic acid: synthesis and characterization of the phenylthiohydantoin derivatives of azobenzene arsonate-coupled tyrosine, histidine, and lysine residues and their sequential allotment in labeled peptides.

Analytical procedures are elaborated for the sequential allotment of azobenzene arsonate binding sites in proteins and peptides. The reaction of diazotized arsanilic acid with proteins leads to covalent modification of tyrosine, histidine and, in part, lysine residues. Synthetic peptides containing these amino acids were modified with diazotized arsanilic acid and subjected to N-terminal sequence analysis. The amino acid derivatives phenylthiohydantoin(Pth)-azobenzene-arsonate-tyrosine, Pth-azobenzene-arsonate-histidine, and alpha-Pth-epsilon-hydroxycaproic acid are recovered upon Edman degradation of selected peptides. Phenylthiohydantoins of modified and nonmodified amino acids are fully separated by reverse-phase HPLC on a Zorbax-PTH column. For identification purposes, phenylthiohydantoins of azobenzene arsonate-labeled amino acids have been synthetized. They are characterized with respect to spectral absorption characteristics and retention times on reverse-phase supports.

Amino Acid Sequence↗
PubMed1990

Determination of alpha-keto acids including phenylpyruvic acid in human plasma by high-performance liquid chromatography with chemiluminescence detection.

A highly sensitive method for the determination of alpha-keto acids including phenylpyruvic acid in human plasma is investigated. The method employs high-performance liquid chromatography with chemiluminescence detection. The acids and alpha-ketocaproic acid (internal standard) in human plasma are isolated by anion-exchange chromatography on a Toyopak DEAE cartridge, and then converted into the corresponding chemiluminescent derivatives with 4,5-diaminophthalhydrazide dihydrochloride, a chemiluminescence derivatization reagent for alpha-keto acids. The derivatives are separated within 50 min on a reversed-phase column, TSKgel ODS-120T, with isocratic elution, followed by chemiluminescence detection; the chemiluminescence is produced by the reaction of the derivatives with hydrogen peroxide in the presence of potassium hexacyanoferrate(III). The detection limits for the acids are in the range 9-92 pmol/ml in plasma (signal-to-noise ratio = 3). This sensitivity permits precise determination of several alpha-keto acids including phenylpyruvic acid, which cannot be determined by other HPLC methods, in 10 microliters of normal human plasma. The chemiluminescent product from phenylpyruvic acid was characterized as 3-benzyl-7,8-dihydropyridazino[4,5-g]quinoxaline-2,6,9(1H)-trione.

Adult↗
PubMed1990

Versatile stable isotope technique for the measurement of amino acids and keto acids: comparison with radioactive isotope and its use in measuring in vivo disposal rates.

Tracer methods using both carbon-13 and -14 have been utilized for determination of ovine fetal amino acid disposal and the results compared in seven animals. We infused [1-13C]leucine simultaneously with [1-14C]leucine into the fetal circulation of pregnant sheep chronically catheterized during late gestation. Radioactive and stable isotope enrichments of leucine (Leu) and stable isotope enrichments of ketoisocaproic acid (KIC) in the umbilical artery and vein and the maternal artery and uterine vein were measured. Stable isotope enrichments and concentrations of both Leu and KIC were determined from a single 0.2-ml sample by the use of internal standards and electron ionization GC/MS analysis after a simple isolation and derivatization procedure. The KIC/Leu enrichment ratio was measured for the first time in fetal arterial plasma and was 0.66 +/- 0.05 (SE). Fetal leucine disposal rate was 9.0 +/- 0.5 (SE) micron/min/kg. Disposal rates determined by stable isotopes were not different from those determined by radioactive isotopes. The GC/MS stable isotope method provided higher precision in both leucine concentration and enrichment measurements and has been shown to be a general method for the determination of concentration and isotopic enrichment of other amino acids and their corresponding keto acids. Furthermore, this method is ideally suited to clinical studies where large numbers of samples of rather small volume can easily be studied with a short turnaround time.

Amino Acids↗
PubMed1990

Analysis of beta-hydroxy-beta-methyl butyrate in plasma by gas chromatography and mass spectrometry.

A method for measuring the branched chain hydroxy acid beta-hydroxy-beta-methyl butyrate (HMB, a product of leucine catabolism) has been described. A [2H6]HMB internal standard was added to plasma and standards, and samples were extracted with diethyl ether, backextracted into neutral phosphate, dried, and derivatized for gas chromatography and mass spectrometry. The natural HMB was monitored at 175 amu and the deuterated HMB was monitored at 181 amu. Standard curves were linear to at least 25 microns and were quantitatively recovered from plasma. Basal concentrations of plasma HMB were from 1 to 2 microM in sheep and increased three- to fourfold when leucine's alpha-ketoacid (alpha-ketoisocaproate, KIC) was fed to lambs. This method can also be adapted to quantitate KIC and other branched chain ketoacids in plasma during the same run.

Animals↗
PubMed1992

Gas-liquid chromatographic analysis of volatile short chain fatty acids in fecal samples as pentafluorobenzyl esters.

A protocol was developed for the analysis of volatile short chain fatty acids in microsamples of feces, short chain fatty acid (SCFA) extraction was from fecal samples using ethanol incorporating n-hexanoic acid as an internal standard. The SCFAs were converted to pentafluorobenzyl esters with alpha-2,3,4,5,6-pentafluorotoluene and analyzed on a gas-liquid chromatograph equipped with an electron capture detector. One hundred milligrams of sample was routinely used but analysis could be carried out on 20 mg of sample.

Animals↗