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Determination of picomole amounts of dopamine, noradrenaline, 3,4-dihydroxyphenylalanine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindolacetic acid in nervous tissue after one-step purification on Sephadex G-10, using high-performance liquid chromatography with a novel type of electrochemical detection.

A determination of dopamine (DA), noradrenaline (NA), 3,4-dihydroxyphenylalanine (DOPA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindolacetic acid (5-HIAA) in nervous tissue is described. The method is based on a rapidly performed isolation of DA, NA, DOPA, DOPAC, HVA, and 5-HIAA from one single nervous tissue sample on small columns of Sephadex G-10, followed by reverse-phase high-performance liquid chromatography with electrochemical detection. A new type of electrochemical detector based on a rotating disk electrode (RDE) was used. The rotating disc electrode was found to be a reliable and sensitive amperometric detector with several advantages over the currently used thin-layer cells. The detector appeared very useful for routine analysis. Practical details are given for the routine use of the RDE. Brain samples containing no more than 75-150 pg (DA, DOPA, DOPAC, HVA, and 5-HIAA) or 500 pg (NA) could be reproducibly assayed with high recovery (approx. 85%) and precision (approx. 5%), without the use of internal standards. Endogenous concentrations of DA, NA, DOPA, DOPAC, HVA, and 5-HIAA were determined in eight brain structures.

3,4-Dihydroxyphenylacetic Acid↗

An amino acid substitution in the human intestinal fatty acid binding protein is associated with increased fatty acid binding, increased fat oxidation, and insulin resistance.

The intestinal fatty acid binding protein locus (FABP2) was investigated as a possible genetic factor in determining insulin action in the Pima Indian population. A polymorphism at codon 54 of FABP2 was identified that results in an alanine-encoding allele (frequency 0.71) and a threonine-encoding allele (frequency 0.29). Pimas who were homozygous or heterozygous for the threonine-encoding allele were found to have a higher mean fasting plasma insulin concentration, a lower mean insulin-stimulated glucose uptake rate, a higher mean insulin response to oral glucose and a mixed meal, and a higher mean fat oxidation rate compared with Pimas who were homozygous for the alanine-encoding allele. Since the FABP2 threonine-encoding allele was found to be associated with insulin resistance and increased fat oxidation in vivo, we further analyzed the FABP2 gene products for potential functional differences. Titration microcalorimetry studies with purified recombinant protein showed that the threonine-containing protein had a twofold greater affinity for long-chain fatty acids than the alanine-containing protein. We conclude that the threonine-containing protein may increase absorption and/or processing of dietary fatty acids by the intestine and thereby increase fat oxidation, which has been shown to reduce insulin action.

Adult↗

Protein binding of quinolonecarboxylic acids. I. Cinoxacin, nalidixic acid and pipemidic acid.

Binding of cinoxain (CINX), nalidixic acid (NA) and pipemidic acid (PPA) to serum proteins was investigated by equilibrium dialysis, ultrafiltration and circular dichroism (CD) spectroscopy. CINX and NA were found to bind mainly to albumin in human serum, the latter interacting with the protein about ten times as strongly as CINX at pH 7.4 and 37 degrees C. PPA showed little or no significant binding to human serum albumin (HSA), alpha 1-acid glycoprotein, and globulins, but showed 20-30% binding to protein in human serum. The CD results were suggestive of some weak interaction of PPA with human apotransferrin. Binding of the three drugs to HSA was found to depend on the lipophilicity of their substituents at the 7-position. The degree of protein binding for human, dog and rat sera at 37 degrees C was in the order of NA (92-97%) greater than CINX (68-90%) greater than PPA (20-30%) at drug concentrations of 10-30 micrograms/ml. CINX showed relatively large species dependence in serum protein binding, which seemed to be due to different affinities of this drug to the respective albumins. CINX was found to bind to rat serum albumin as strongly as NA.

Animals↗

Lipid content and fatty acid composition in foods commonly consumed by nursing Congolese women: incidences on their essential fatty acid intakes and breast milk fatty acids.

The fat content and fatty acid (FA) composition of nearly 40 foods, currently consumed by 102 nursing Congolese mothers living in Brazzaville, were determined to assess their impact on mothers' essential fatty acid (EFA) intakes and breast milk FA. Data on mothers' milk FA and dietary habits which allowed food selection were recently published (Rocquelin et al., 1998). Most foods were locally produced. Food samples were collected at local markets, bleached if necessary to avoid microbial degradation, and stored at +4 degrees C or -20 degrees C. They were lyophilized upon their arrival in the laboratory before lipid analyses. FA composition of food lipids was determined by capillary gas chromatography. Staple diets included low-fat, high-carbohydrate foods (processed cassava roots, wheat bread) and high-polyunsaturated fatty acid (PUFA) foods: soybean oil (high in 18 : 2 n-6 and alpha-18 : 3 n-3), bushbutter (dacryodes edulis), peanuts, avocado (high in fat and 18 : 2 n-6), freshwater and salt-water fish (high in LC n-3 and/or n-6 PUFA), and leafy green vegetables (low in fat but very high in alpha-18 : 3 n-3). Their frequent consumption by nursing mothers provided enough EFA to meet requirements due to lactation. It also explains why mothers' breast milk was rich in C8-C14 saturated FA (26% of total FA) and in n-6, n-3 PUFA (respectively 15.0% and 2.4% of total FA) highly profitable for breastfed infants' development. From this point of view, dietary habits of Congolese mothers have to be sustained for they are more adequate than most Western-type diets.

Animals↗

The effects of plant phenolics, caffeic acid, chlorogenic acid and ferulic acid on arylamine N-acetyltransferase activities in human gastrointestinal microflora.

The possible effects of naturally occurring plant phenolics, caffeic acid (CA), chlorogenic acid (CGA) and ferulic acid (FA) on arylamine N-acetyltransferase (NAT) activities on human gastrointestinal microflora, Escherichia coli, Klebsiella pneumoniae, Enterobacter aerogenes, Citrobacter koseri and Pseudomonas aeruginosa, were examined. The bacterial NAT activities were determined by HPLC measuring the acetylation of 2-aminofluorene (2-AF). Among all examined bacteria, P. aeruginosa exerted the highest NAT activity while C. koseri possessed the lowest NAT activity. CA, CGA and FA could suppress the bacterial NAT activities dose-dependently both in the intact cell and cytosolic fraction analysis. According to the analysis of kinetic parameters in E. coli and P. aeruginosa, CA, CGA and FA were shown to be potent noncompetitive inhibitors of bacterial NAT activities. For the time course experiment, 4 mM of CA and FA could inhibit bacterial NAT activities for at least 4 hour but 4 mM of CGA could only significantly suppress NAT activity in E. coli for the same reaction time. These results strongly demonstrated that CA, CGA and FA inhibited NAT activities in human gastrointestinal bacteria.

Arylamine N-Acetyltransferase↗

Effects of caffeic acid, chlorogenic acid and ferulic acid on growth and arylamine N-acetyltransferase activity in Shigella sonnei (group D).

Arylamine N-acetyltransferase (NAT) activities with 2-aminofluorene (2-AF) as substrates were determined in Shigella sonnei (group D) collected from patients with diarrhoeal disease. The NAT activity was determined using an acetyl CoA recycling assay and high pressure liquid chromatography. Inhibition of growth studies from S. sonnei (group D) demonstrated that caffeic acid (CA), chlorogenic acid (CGA) and ferulic acid (FA) elicited a dose-dependent bactericidal effect in S. sonnei (group D) cultures, i.e. the greater the concentration of CA, CGA and FA, the greater the inhibition of growth of S. sonnei (group D). Cytosols or suspensions of S. sonnei (group D) with and without selected concentrations of CA, CGA and FA co-treatment showed different percentages of 2-AF acetylation. The data indicated that there was reduced NAT activity associated with increased CA, CGA and FA in Shigella dysenteriae (group D) cytosols and intact cells. For the cytosol and intact bacteria examinations, the apparent values of K(m) and Vmax decreased after being co-treated with 400 microM CA, CGA and FA. This report is the first demonstration of plant phenolic inhibition (CA, CGA and FA) of arylamine NAT activity and growth in the bacterium S. sonnei (group D).

Arylamine N-Acetyltransferase↗

[Effects of arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid on tension of rabbit aortic strips].

The purpose of the investigation was to determine the effects of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (AA) on the isolated rabbit aortic strips. The results showed that (1) the rabbit aortic strips with intact endothelium contracted in a dose-dependent manner to exogenous AA between the concentrations of 2.5 x 10(-6) mol/L and 4 x 10(-5) mol/L. EPA did not affect the tension of the relaxing strips, but inhibited the contractile response to AA (2 x 10(-5) mol/L), IC50 = 9.94 x 10(-6) mol/L. DHA showed no significant effect on the contractile response to AA. (2) In the endothelium-damaged rabbit aorta, the contraction of strips to AA was drastically diminished. The inhibitory effect of EPA on the contraction to AA was almost vanished. (3) The contraction of rabbit aortic strips to AA was shown to be abolished by indomethacin (1.5 x 10(-5) mol/L), a cyclooxygenase inhibitor. (4) Radioimmunoassay showed that exogenous AA increased the tissue levels of 6-keto-PGF1 alpha, TXB2 and their ratio. Lower dose of EPA (1.5 x 10(-6) or 1.5 x 10(-5) mol/L) did not affect the tissue levels of 6-keto-PGF1 alpha and TXB2. However, EPA at higher concentrations decreased the metabolites of AA significantly. These findings suggest that exogenous AA induces the contraction of rabbit aortic strips in vitro, which is related to the endothelial cells and mediated by the metabolites(s) of cyclooxygenase, likely endothelium-derived contracting factor (EDCF).(ABSTRACT TRUNCATED AT 250 WORDS)

6-Ketoprostaglandin F1 alpha↗

[Comparison of central effects produced by intracerebral injection of glutamic acid, quisqualic acid, and kainic acid].

Icv glutamic acid (Glu), quisqualic acid (QA) and kainic acid (KA) significantly increased spontaneous activity of mice in photecell box, and induced dose-dependent rise of blood pressure in anesthetized rats. Their intensities were arranged in the order of KA greater than Glu greater than QA. In mice step-through test Glu 0.1 micrograms icv improved learning and memory; KA 1 ng had no evident effect; and QA 0.1 microgram impaired learning and memory, which were also confirmed by step-down test in normal mice. Therefore, the non-NMDA (N-methyl-D-aspartate) receptor subtype might be different from the NMDA receptor subtype in the action of learning and memory.

Animals↗

[Preparation and analysis of hydroxycarboxylic acid, sugar acid and amino acid complexes of gallium(III)].

Neutral, stable in water and not hydrolysing gallium(III) complexes were prepared and analysed by instrumental analytical methods. Nine hydroxy-carboxylic acids, four sugar acids and four amino acids were used as ligands. The chemical and physical characteristics of complexes (composition, stability, water solubility, pH in aqueous solution) significantly depend on the quality of gallium salt and ligand, the ratio of gallium and ligand, the medium used during the preparation (pH and temperature).

Amino Acids↗

Simultaneous liquid-chromatographic determination of urinary vanillylmandelic acid, homovanillic acid, and 5-hydroxyindoleacetic acid.

We describe a liquid-chromatographic method for quantifying, simultaneously by a single procedure, vanillylmandelic acid (VMA), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) in urine. After solvent extraction of acidified urine, the analytes were chromatographed on a C8 column, with use of a mobile phase of phosphate buffer (20 mmol/L, pH 4.0) and methanol with a variable gradient elution, and detected fluorometrically. We report the analytical recovery, sensitivity, precision, working linear range, and potential for interference from similar molecules or drugs. The results of such tests demonstrate that the proposed method is sensitive and reproducible. It is, furthermore, easy to perform, and thus is suitable for use in the clinical laboratory.

Chromatography, High Pressure Liquid↗

Studies of the fatty acid-binding site of rat liver fatty acid-binding protein using fluorescent fatty acids.

Rat liver fatty acid-binding protein (FABP) is a 14.3-kDa cytosolic protein which binds long chain free fatty acids (ffa) and is believed to participate in intracellular movement and/or distribution of ffa. In the studies described here fluorescently labeled ffa were used to examine the physical nature of the ffa-binding site on FABP. The fluorescent analogues were 16- and 18-carbon ffa with an anthracene moiety covalently attached at eight different points along the length of the hydrocarbon chain (AOffa). Emission maxima of all FABP-bound AOffa were found to be considerably blue-shifted with respect to emission of phospholipid membrane-bound AOffa, suggesting a high degree of motional constraint for protein-bound ffa. Large fluorescence quantum yields and long excited state life-times indicate that the FABP-binding site for ffa is highly hydrophobic. Analysis of rotational correlation times for the FABP-bound AOffa suggest that the ffa are tightly bound to the protein. Variation of the quantum yield with attachment site suggests that the carboxylic acid group of the fatty acyl chain is located near the aqueous surface of the FABP. The rest of the ffa hydrocarbon chain is buried within the protein in a hydrophobic pocket and is particularly constrained at the midportion of the acyl chain.

Animals↗

[Behavior of blood amino acids and fatty acids during complete parenteral hyperalimentation with carbohydrates, amino acids and fats].

Over a period of 12 hours a total amount of 190 g fructose, 190 g glucose, 145 g xylitol, 96 g fat and 99 g amino acids were infused. The utilization of carbohydrate was not diminished despite the high fat load. In addition, the antiketogenic action of carbohydrate was still provable. The nitrogen balance was positive as an indicator of good utilization of amino acids. Therefore, simultaneous administration of a carbohydrate mixture and a fat emulsion as energy sources during application of amino acids is recommended.

Acid-Base Equilibrium↗

Cholesterol-ascorbic acid interactions in guinea pig liver homogenates, and in the in situ perfused liver: relationship between ascorbic acid status and cholesterol and bile acid synthesis from mevalonate.

Cell free homogenates, and in situ perfused livers were used to study cholesterol synthesis and catabolism in control and latently scorbutic guinea pigs. In the homogenate studies, cholesterol synthesis from (2-14C) mevalonate was significantly depressed in latently scorbutic guinea pigs when compared to controls (0.02 less than p less than 0.05). Synthesis of cholesterol from (1-14C) acetate and n (1-14C) octanoate was minimal in guinea pigs. In the in situ liver perfusions, the synthesis of cholesterol from (2-14C) mevalonate was 40% lower in latently scorbutic guinea pigs than in control animals (p less than 0.005). The synthesis of cholesterol from (1-41C) acetate did not show the same clear cut effect. Bile acid production in the in situ perfused liver was unexpectedly higher in the latently scorbutic animals than in control animals (p = 0.005), although the incorporation of the label from (2-14C) mevalonate into the bile acids from the two groups was not significantly different. This finding led to the latently scorbutic group having a far lower specific activity in the bile acid fraction than the replete group.

Animals↗

Simultaneous analysis of homovanillic acid, 5-hydroxyindoleacetic acid, 3-methoxy-4-hydroxyphenylethylene glycol and vanilmandelic acid in plasma from alcoholics by high-performance liquid chromatography with electrochemical detection. Critical comparison of solid-phase and liquid-liquid extraction methods.

A method is described for the simultaneous determination of vanilmandelic acid, 3-methoxy-4-hydroxyphenylethylene glycol, 5-hydroxyindoleacetic acid, and homovanillic acid in a human plasma sample using reversed-phase high-performance liquid chromatography with column switching and amperometric detection. Two methods of sample preparation were tested. Liquid-liquid extraction yields better recoveries, is more selective and precise than solid-phase extraction and allows a shorter time of chromatographic analysis. Estimated plasma values of the metabolites from healthy controls are in good agreement with previously reported levels. Studies of alcoholics at the beginning of the delirium tremens provided different plasma levels of the metabolites, dependent on the different duration--and hence the severity--of the delirium.

Alcohol Withdrawal Delirium↗

Liquid chromatographic determination of flumequine, nalidixic acid, oxolinic acid, and piromidic acid residues in catfish (Ictalurus punctatus).

A peer-verified, liquid chromatographic (LC) method for simultaneous determination of residues of flumequine (FLU), nalidixic acid (NAL), oxolinic acid (OXO), and piromidic acid (PIR) in catfish muscle is presented. Sample workup involves homogenizing tissue with acetone, defatting with hexane, and extracting quinolones into chloroform. Sample is purified further by partitioning into base and then subsequently back-extracting into chloroform after acidifying the aqueous phase. After solvent is evaporated, the residue is diluted with mobile phase, and analytes are introduced into an LC system where separations are made with a 5 microns, reversed-phase polymer column and an isocratic, buffered acetonitrile-tetrahydrofuran mobile phase. Determinations are made by UV detection at 280 nm for PIR and by fluorescence detection (excitation at 325 excitation and emission at 365 nm) for the other 3 analytes. Each quinolone was used to fortify catfish muscle at 5, 10, 20, 40, and 80 ng/g. The following recoveries and relative standard deviation (RSD) values represent an average of the 5 levels for each analyte: FLU, 79.7% (RSD = 5.7%); OXO, 80.8% (RSD = 6.3%); PIR, 75.0% (RSD = 5.9%); and NAL, 87.1% (RSD = 10%). Assay of 5 levels (base incurred catfish, plus 4 dilutions with control catfish) of catfish muscle incurred with the 4 quinolones gave the following averages: FLU: base, 198 ng/g (RSD = 2.3%); dilutions, 98.0 ng/g (RSD = 4.2%), 61.6 ng/g (RSD = 4.4%), 21.6 ng/g (RSD = 2.8%), 9.24 ng/g (RSD = 8.7%); OXO, base, 257 ng/g (RSD = 6.9%); dilutions, 146 ng/g (RSD = 5.5%), 95.0 ng/g (RSD = 4.1%), 30.7 ng/g (RSD = 3.8%), 13.7 ng/g (RSD = 4.6%); PIR, base, 22.1 ng/g (RSD = 4.2%); dilutions, 13.7% ng/g (RSD = 6.7%), 6.49 ng/g (RSD = 15%), 2.65 ng/g (RSD = 15%); and NAL, base, 75.1 ng/g (RSD = 3.8%); dilutions, 42.3 ng/g (RSD = 5.1%), 24.1 ng/g (RSD = 6.3%), 8.59 ng/g (RSD = 4.8%). A second multiresidue analysis of the 4 quinolones was performed by an outside analyst. Average recoveries from catfish fortified at 5, 10, 20, and 40 ng/g were FLU, 75.9% (RSD = 4.0%); OXO, 84.0% (RSD = 5.5%); NAL, 85.6% (RSD = 8.9%); and PIR, 66.2% (RSD = 8.7%).

Animals↗

High-pressure liquid chromatography analysis of salicylic acid, salicyluric acid, and gentisic acid in biological matrixes.

An assay is reported for salicylic acid and two of its metabolites, salicyluric acid and gentisic acid, in mouse and rat blood, urine, and feces and rat embryos. The procedure involves aqueous dilution of the biological sample, addition of methanol-1% acetic acid (in water) (40:60), centrifugation, and injection of the supernate onto a commerical high-pressure liquid chromatographic column. Detection is via a variable-wavelength UV detector set at 296 nm. Quick and easy sample preparation, good accuracy and precision, and sufficient sensitivity make analysis of large numbers of samples feasible.

Animals↗

Models of interaction between nucleic acids and proteins. Hydrogen bonding of arginine with nucleic acid bases, phosphate groups and carboxylic acids.

Complex formation between the side chain of arginine and nucleic acid bases has been investigated by proton magnetic resonance in dimethylsulfoxide. Simultaneous formation of two hydrogen bonds leads to a selectivity of arginine interaction towards cytosine and guanine. A comparison is made of the interaction of arginine side chain with nucleic acid bases, phosphate and carboxylate anions. It is shown that interaction between carboxylate and arginine is stronger than between phosphate and arginine. These results are discussed with respect to the selective recognition of nucleic acid bases by arginine side chains and by the arginyl-glutamyl ion pair which could form in proteins interacting with nucleic acids.

Arginine↗

The contribution of alkali soluble (humic acid-like) and unhydrolyzed-alkali soluble (core-humic acid-like) fractions extracted from maize plant to the formation of soil humic acid.

Alkali soluble (humic acid-like material) (HA-like) (yield of 132 gkgdm(-1)) and the unhydrolized-alkali soluble (core-humic acid-like material) (core-HA-like) (yield of 33.4 gkgdm(-1)) fractions were extracted from maize plants and characterized by C and N determinations, DRIFT, and 1H and 13C-NMR spectroscopy. Fresh plants were subsequently incubated for 6 months in an artificial mineral soil, and the HA-like and core-HA-like trends were monitored quantitatively (C fraction content) and qualitatively (spectroscopic approach) in order to study their contribution to the formation of soil humic acid. During incubation the HAC-like partially degraded (loss of 320 gkgHAC(-1)) and partially formed new fulvic-like acids (160 gkgHAC(-1)). On the contrary, the stable fraction of HAC, the core-HAC-like, was maintained (loss of 153 kgcore-HAC(-1)), representing, after incubation, 846 gkg(-1) of the initial core-HAC-like content. The core-HA-like fraction is composed of lignin residues, polysaccharides, lipids and proteins, probably structured into a well-defined network, i.e. the plant cell wall.

Alkalies↗