Reversed-phase Performance Liquid Chromatography Research Articles

Pancreatic secretory trypsin inhibitor from human amniotic fluid and fetal and neonatal urine: Concentrations and physicochemical characterization

The levels and physicochemical properties of the pancreatic secretory trypsin inhibitor, also known as Kazal type trypsin inhibitor, were studied in human amniotic fluid. In the second trimester, the median concentration was 160 ng/ml, which exceeds the maternal serum levels 20-fold. Towards term, the amniotic fluid levels declined about 5-fold, whereas the maternal serum values remained constant. In fetal urine, the concentration of the trypsin inhibitor was similar to that in amniotic fluid in early gestation, whereas in newborn urine, the median level was 4-to 5-fold higher than in term amniotic fluid. The physicochemical characteristics of the trypsin inhibitor in amniotic fluid, neonatal urine and cancer urine from an ovarian cancer patient were similar, as studied by gel filtration, high performance reverse phase liquid chromatography, and complete immunological identity in immunodiffusion. The physicochemical similarity and levels in various compartments suggest fetal contribution to amniotic fluid levels of the trypsin inhibitor.

Interaction of cotton tissue culture cells andVerticillium dahliae

Elicitation of sesquiterpenoid aldehyde phytoalexins inGossypium arboreum cell suspension cultures was confirmed by thin layer chromatography, high performance reverse phase liquid chromatography, and an aniline-reaction assay after inoculation with heat-treated conidia ofVerticillium dahliae A 2.3X mean increase in total terpenoids was observed. Component phytoalexins varied, with either hemigossypol and gossypol being detected or the O-methylated terpenoids hemigossypol-6-methyl ether and related compounds. Long-termGossypium suspension cultures were mixoploid with an increase in chromosome number and mean DNA content. Addition ofV. dahliae elicitor(s) to the medium for embryo-proliferating callus ofG. hirsutum inhibited growth and embryo production with a linear correlation (r=−0.87;P<0.01) between the elicitor concentration and the number of embryos. Addition of14C-labeled NaOAc to suspension cells gave 30% incorporation, and from13C-NaOAc addition, labeled sesquiterpenoid aldehydes were recovered. The cotton-Verticillium system is another case of secondary metabolite elicitation in plant tissue culture and might be used for basic studies of hostpathogen interaction as well as for a selection tool to obtain resistance to an important disease.

Is cytochrome aa3 from thermus thermophilus a single subunit oxidase?

Abstract A reliable procedure has been developed for the purification of the cytochrome c 1 aa 3 complex from the plasma membrane of T. thermophilus . The ratios heme C:heme A:Fe:C were found to be 1:2:3:2 confirming previous results, however, the molecular weight was found to be ∼92,000 rather than the ∼200,000 reported earlier [1]. Polyacrylamide gel electrophoresis under strongly denaturing conditions and high performance reverse phase liquid chromatography showed that cytochrome c 1 aa 3 is composed of only two subunits in 1:1 ratio. Both polypeptides have blocked N-termini. The smaller subunit (∼33,000) binds heme c and presumably no other metals. The larger subunit (∼55,000) is thus thought to contain the elements of cytochrome aa 3 and therefore be considered a single subunit cytochrome oxidase. The bacterial cytochrome c 1 aa 3 has been compared with beef heart cytochrome oxidase with a number of techniques including optical, EPR [1], Raman, MCD, and Mossbauer [2] spectroscopies. These experiments establish that the fundamental chemical properties of the redox centers are substantially similar in these two proteins. Cytochrome c 552 (from Thermus ), horse heart cytochrome c , and tetramethylphenylenediamine greatly stimulate the ascorbate oxidase activity of cytochrome c 1 aa 3 . This enhancement is characterized by a ‘high affinity’ component which results in only a small velocity increase and a ‘low affinity’ component which gives a large velocity increase. Very similar behavior has been previously observed with mammalian cytochrome oxidase [3]. Preliminary experiments show that vesicularized c 1 aa 3 is capable of proton pumping.

Characterization of mutagenic fractions in beef extract and in cooked ground beef. Use of blue-cotton for efficient extraction.

Mutagenic components in commercial beef extract and in cooked ground beef were adsorbed from their aqueous solutions on cotton bearing covalently linked trisulfo-copper-phthalocyanine residues (blue-cotton). By repeating the adsorption and elution, efficient concentration of the mutagenic components with a satisfactory overall recovery was achieved. Carboxymethyl cellulose column chromatography was found to be an excellent means to separate 2-amino-3,8-dimethylimidazo-[4,5-f]quinoxaline (MeIQx) from 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), two strong mutagens that have previously been found in heated beef samples. Chromatography of the mutagenic components of beef extract on this column gave two mutagenic fractions which corresponded to MeIQx and IQ in elution profile. In reversed-phase high performance liquid chromatography, the major active component of the MeIQx fraction and that of the IQ fraction behaved identically with standard samples of MeIQx and IQ, respectively. The contents of these mutagens in a sample of Difco beef extract were estimated at 200-300 ng of MeIQx and 20-40 ng of IQ per gram. By the same fractionation procedures, mutagenic substances in the cooked beef were fractionated into MeIQx-type and IQ-type components. The activity distribution among these two fractions was similar to that found for beef extract.

Molecular species of marine animal lipid-I. Molecular species of fish muscle lecithin.

A satisfactory separation of the molecular species of fish muscle lecithin was made by modifying the lecithin into diglceride acetate for high performance reversed phase liquid chromatography (HPLC), The molecular species of each chromatographic peak was determined by fatty acid composition and by total acyl carbon number analysis subsequent to separation, using a thin layer chronatographic technique with silver nitrate impregnated silica ged plated (Ag+-TLC). After plotting the relative retention time (RRT) of each molecular species from soybean, egg yolk, chum salmon, big-eyed tuna, Alaska pollack, and carp muscle lecithins semilogarithmically against the total cayl carbon number of the number of total double bonds of each molecular species, a set of parallel straight oblique lines was obtained. Thus we can express the molecular species in matrix relation by giving a variable integer x for the acyl carbon number and a variable integery for the number of double bonds of each fatty acid in the molecular species. By using the correlations between the RRTs and the corresponding molecular species on semilogarithmic plots, it was conclude that an identification of molecular species from fish lipids could be done by RRTs from HPLC.

High performance reversed-phase liquid chromatography of trace amounts of inorganic and organic mercury as diethyldithiocarbamate chelates.

The separation of diethyldithiocarbamate (DDTC) chelates of Hg(II), CH3Hg(I), C2H5Hg(I) and C6H5Hg(I) was done by reversed phase partition chromatography. The separation was accomplished within 20 min. The column was a 3.9 mm × 300 mm μ-Bondapak C18 column, and the eluent was 85 % (V/V) methanol-water mixture containing 10-4mol dm-3 of ethylenediamine tetraacetic acid (EDTA).

Catecholamine biosynthesis in specific brain areas of the rat as determined by liquid chromatography and amperometric detection

Catecholamine turnover is examined using both steady and non-steady state methods. The steady state technique involved the injection of 5 μCi of 3H-tyrosine into the lateral cerebral ventricle in chronically cannulated rats. Catecholamines were separated by high performance reverse phase liquid chromatography and content analyzed with amperometric detection. Catecholamine synthesis was examined in the anterior hypothalamus, medial basal hypothalamus, brain stem and caudate-putamen. Dopamine synthesis was greater than norepinephrine in all areas but the brain stem. The decline in catecholamine content was demonstrated with a synthesis inhibitor, αMpT, and comparisons were made between the two methods. This study indicated that tyrosine hydroxylase inhibition alters catecholaminergic neuronal activity in the brain areas that we examined.

Determination of polycyclic aromatic hydrocarbons in aqueous samples by reversed-phase performance liquid chromatography

Determination of polycyclic aromatic hydrocarbons in aqueous samples by reversed-phase performance liquid chromatography

Bioanalysis of Naproxen by High Performance Reversed Phase Liquid Chromatography with Photometric and Fluorimetric Detection

Abstract Methods for the quantitative determination of NAPROXEN and its main metabolite in plasma and urine are described. The separation is based on reversed phase liquid chromatography with LiChrosorb RP 8 (5 μm) as the support and methanol/phosphate buffer pH 7 as mobile phase, in some cases with addition of tetrabutyl ammonium ion as ion-pairing agent to improve the chromatographic selectivity. With UV-detector and a simple filter fluorometer an extraction-evaporation procedure is used for both plasma and urine determinations, while the high selectivity and sensitivity of a sophisticated fluorescence detector permits the direct injection of diluted samples on to the column. Use of an internal standard improves the within-run precision (srel%), which for plasma determinations of NAPROXEN are - with UV-detection, 0.2 – 1.7% (range 10 – 40 μg/ml), with filter fluorometer, 2.4 – 5.9% (range 12 – 58 μg/ml), and with fluorescence detector, 0.8 – 4.1% (range 5 – 20 μg/ml).