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PubMed2002

High-performance liquid chromatographic determination of biogenic amines in poultry carcasses.

Biogenic amines, produced by bacterial decarboxylation of amino acids, have been associated with toxicological symptoms in broilers fed various poultry byproducts. A reversed-phase high-performance liquid chromatographic method is described for the quantitation of eight biogenic amines (tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine) in chicken carcasses. Amines were extracted with perchloric acid, derivatized with dansyl chloride, separated using gradient elution (methanol and water), and detected by fluorescence. Benzylamine was used as the internal standard. Linearity, repeatability, and recovery of the method were evaluated. The method was linear for all of the amines studied at concentrations ranging from 0.05 to 25 microg/mL. Average recoveries ranged from 92.6% to 96.8% for all amines except for histamine, which was 74.6%.

Animals↗

PubMed1982

Sulphydryl oxidation in the mechanism of molecular-weight conversion of renin in dog kidney.

1. A high-molecular-weight renin (M(r) 60 000) was formed by the reaction of a low-molecular-weight renin (M(r) 40 000) with a renin-binding substance in canine renal cortical extract in the presence of the sulphydryl (SH) group oxidizing agent potassium tetrathionate; thus the reaction required SH oxidation. 2. Renin extracted from isolate renin granules was adsorbed on to thiopropyl Sepharose 6B, and then liberated with dithiothreitol (50 mmol/1), indicating that it possessed on SH moiety(s). 3. However, the renin was capable of reaction with the renin-binding substance even after its SH moiety (or moieties) was protected with 5,5'- dithiobis-(2-nitrobenzoic acid). 4. The high-molecular-weight renin was converted into the low-molecular-weight renin by incubation (37 degrees C, 15 min) with cytosol (soluble fraction) of renal cortex and liver. Such converting ability was diminished after the cytosol was treated with perchloric acid or potassium tetrathionate. 5. These results suggest that the reaction of renin with the renin-binding substances does not require disulphide bond(s) and that an enzyme-like substance which is sensitive to SH oxidation is involved in the conversion from the high molecular-weight renin into the low-molecular weight renin.

Animals↗

PubMed1982

Isolation and characterization of histones and other acid-soluble chromosomal proteins from Physarum polycephalum.

Chromosomal basic proteins were isolated from amoebal and plasmodial stages of the acellular slime mold Physarum polycephalum. Polyacrylamide electrophoresis on high resolution acid-urea gels separated the five histone fractions in the sequence H1, H2A, H2B, H3, and H4. Under these electrophoretic conditions Physarum histones migrated more like plant (rye) than animal (calf) histones. Furthermore, Physarum histones H1, H2A, and H2B have higher molecular weights on sodium dodecyl sulfate (SDS) gels than the corresponding calf fractions. No differences were detected between amoebal and plasmodial histones on either acid-urea or SDS-polyacrylamide gel electrophoresis. Amoebal basic proteins were fractionated by exclusion chromatography. The five histone fractions plus another major acid-soluble chromosomal protein (AS) were isolated. The Physarum core histones had amino acid compositions more closely resembling those of the calf core histones than of rye, yeast, or Dictyostelium. Although generally similar in composition to the plant and animal H1 histones, the Physarum H1 had a lower lysine content. The AS protein was extracted with 5% perchloric acid or 0.5 M NaCl, migrated between histones H3 and H4 on acid-urea polyacrylamide gels, and had an apparent molecular weight of 15 900 on SDS gels. It may be related to a protein migrating near H1. Both somewhat resembled the high mobility group proteins in amino acid composition.

Amino Acids↗

PubMed2006

Pyruvate improves recovery after PARP-1-associated energy failure induced by oxidative stress in neonatal rat cerebrocortical slices.

Previous neuron and glial cell culture studies of excessive poly (ADP-ribose) polymerase (PARP-1) activation found NAD(+) depletion, glycolytic arrest, and cell death that could be avoided by exogenous tricarboxylic acid cycle (TCA) metabolites, especially pyruvate (pyr). Pyruvate neuroprotection has been attributed to cytosolic NAD(+) replenishment, TCA metabolism, and antioxidant activity. We investigated the first two mechanisms in respiring cerebrocortical slices after a 1-h H(2)O(2) exposure to activate PARP-1. H(2)O(2) was followed by a 4-h recovery with oxy-artificial cerebrospinal fluid superfusion having either: (1) no glucose (glc) or pyruvate; (2) 10 mmol/L glc only; (3) 10 mmol/L pyruvate only; (4) both 10 mmol/L glc and 10 mmol/L pyruvate. Poly-ADP-ribosylation was quantified from Western blots and immunohistochemistry. Perchloric acid extracts were quantified with 14.1 T (31)P nuclear magnetic resonance spectroscopy. Just after H(2)O(2) exposure, ATP and NAD(+) decreased by approximately 50%, PCr decreased by 75%, and the ADP/ATP ratio approximately doubled. ATP and NAD(+) changes, but not PCr changes, were nearly eliminated if PARP inhibitors accompanied the H(2)O(2). Recovery with both pyruvate and glc was better than with glc alone, having higher ATP (0.161 versus 0.075, P<0.01) and PCr levels (0.144 versus 0.078, P<0.01), and higher viable cell counts in TUNEL and Fluoro-Jade B staining. Two-dimensional [(1)H-(13)C] HSQC spectra showed metabolism during recovery of (13)C glc or pyr. Pyruvate metabolism was primarily via pyruvate dehydrogenase, with some via pyruvate carboxylation. Pyruvate superfusion of PARP-injured brain slices helps replenish NAD(+) while providing metabolic fuel. Although this augments recovery, a strong antioxidant role for pyruvate has not been ruled out.

Animals↗

PubMed1977

Metabolism of dog gastric mucosa. Levels of glycolytic, citric acid cycle and other intermediates.

Several metabolites, including those of glycolysis, the citric acid cycle, the hexose monophosphate shunt, glutamate, aspartate, and Coenzyme A were measured in defined parietal cell-enriched freeze-dried sections of dog gastric biopsies derived from nonsecreting and secreting tissue. In addition, NH3, ribulose 5-phosphate, glycerol, and succinate were measured in perchloric acid extracts of biopsies. The onset of secretion increased the level of glycolytic intermediates including pyruvate and lactate with the most marked increase being in fructose 1,6-diphosphate levels. The level of 6-phosphogluconate and ribulose 5-phosphate also increased, in spite of a constant NADP+/NADPH ratio. The levels of all the citric acid cycle intermediates measured also rose, the most marked rise being in malate and fumarate. The levels of glycerol, acetyl-CoA, and CoA increased, but the ratio of the latter intermediates remained constant. Calculation of the ratio of the oxidized to reduced form of diphosphopyridine nucleotide indicated a fall of the ratio in the cytoplasm and a rise in the mitochondria. From these data, it is concluded that the major energy source for acid secretion is due to an increase in citric acid cycle activity and that glycolysis, and probably also fatty acid oxidation, is stimulated to provide mitochondrial substrate.

Ammonia↗

PubMed2005

Determination of phenolic preservatives in gelatin and vacant capsules for medicine use by ion-suppression reversed-phase high performance liquid chromatography.

A reliable method for the simultaneous determination of phenolic preservatives parahydroxybenzoic acid, methyl, ethyl, propyl, butyl and pentyl parahydroxybenzoates in gelatin and vacant capsules for medical use has been developed by ion-suppression reversed-phase high performance liquid chromatography. Separation was carried out on a Kromasil C(18) column by isocratic elution using methanol-perchloric acid (pH 2.0; 10 mM) (60:40, v/v) at a flow-rate of 1.0 ml min(-1), and detection by UV absorbance at a wavelength of 254 nm. This method has been successfully applied to the routine analyses of these preservatives in the real samples.

Calibration↗

PubMed1979

Determination of apomorphine in plasma and brain tissue by ion-pair extraction and liquid chromatography.

Apomorphine is extracted from plasma or tissue homogenate with ethyl acetate. After back-extraction into hydrochloric acid, the apomorphine is extracted as an ion pair with 3,5-di-tert.-butyl-2-hydroxybenzene sulphonate into a small volume of methylene chloride and the solution is injected into the chromatographic column. Apomorphine is separated on microporous silica with a mixture of aqueous perchloric acid, methanol and methylene chloride as the mobile phase. With absorbance measurement of the eluent at 254 nm the method permits the determination of 15 pmol of apomorphine in 1 ml of plasma or in a rat brain. The coefficient of variation was 4% at the 100 pmol level.

Animals↗

PubMed2000

Urinary phenylmercapturic acid as a marker of occupational exposure to benzene.

A hand-saving HPLC method to measure urinary phenylmercapturic acid (PMA) was developed which allows about 35 PMA determinations per day. The method involves conversion of pre-PMA to PMA by the addition of sulfuric acid to a urine sample, extraction into an ether-methanol mixture followed by condensation under a nitrogen stream. The condensate was introduced to a ODS-3 column in a HPLC system, and PMA in the column was eluted into a mobile phase of acetonitrile: methanol: perchloric acid: water. The elution of PMA was monitored at 205 nm. One determination will be completed in 40 min. The method was applied to analysis of end-of-shift urine samples from 152 workers exposed up to 210 ppm benzene, 66 workers exposed to a mixture of benzene (up to 116 ppm) and toluene + xylenes (up to 118 ppm), and 131 non-exposed controls of both sexes. A linear regression was established between time-weighted average intensity of exposure to benzene and urinary PMA. From the regression, it was calculated that urinary PMA level will be about 6.4 mg/l after 8-hour exposure to benzene at 100 ppm, and that PMA in urine accounted for about 0.1% of benzene absorbed. No effects of sex, age, and smoking habit of individuals were detected, and the effect of co-exposure to toluene + xylenes at the levels comparable to that of benzene was essentially nil, which indicates an advantage of PMA as a benzene exposure marker over monoto tri-phenolic metabolites or t,t-muconic acid.

Acetylcysteine↗

PubMed1987

Effect of fasting and of methionine deficiency on L-methionine, DL-methionine and DL-2-hydroxy-4-methylthiobutanoic acid metabolism in broiler chicks.

Metabolism of L-[1-14C]methionine, DL-[1-14C]methionine and DL-[1-14C]2-hydroxy-4-methylthiobutanoic acid (DL-HMB) by broiler chicks which had been fasted overnight or given a methionine-deficient diet was compared with fed (control) birds. The excretion of 14C-labelled material, total 14CO2 exhaled, 14C incorporation into tissue proteins and the 14C-labelled material in perchloric-acid-soluble tissue fractions were measured 6 h after injection of the 14C-labelled materials. The incorporation of 14C into tissue proteins and the relative rates of conversion of D-methionine and DL-HMB to L-methionine in tissues under different nutritional regimens were compared using protein-bound 14C:protein-free 14C values. Fasted birds exhaled more 14CO2 than control birds but excreted less 14C, while methionine-deficient birds behaved very similarly to the control animals in these respects. Fasted birds incorporated much less 14C into proteins of tissues other than liver and kidney from all three labelled tracers. The values for protein-bound 14C:protein-free 14C were lower in all tissues. Methionine-deficient birds had similar levels of 14C in tissue proteins but lower values for protein bound 14C:protein-free 14C. Examination of the values for protein-bound 14C:protein-free 14C suggest that brain and probably liver tissues from fasted and methionine-deficient birds showed improved rates of conversion of D-methionine and DL-HMB to L-methionine compared with control animals.

Animals↗

PubMed1998

Procedure for the sample preparation and handling for the determination of amino acids, monoamines and metabolites from microdissected brain regions of the rat.

A method is described for the analysis of amino acids, monoamines and metabolites by high-performance liquid chromatography with electrochemical detection (HPLC-ED) from individual brain areas. The chromatographic separations were achieved using microbore columns. For amino acids we used a 100x1 mm I.D. C8, 5 microm column. A binary mobile phases was used: mobile phase A consisted of 0.1 M sodium acetate buffer (pH 6.8)-methanol-dimethylacetamide (69:24:7, v/v) and mobile phase B consisted of sodium acetate buffer (pH 6.8)-methanol-dimethylacetamide (15:45:40, v/v). The flow-rate was maintained at 150 microl/min. For monoamines and metabolites we used a 150X1 mm I.D. C18 5 microm reversed-phase column. The mobile phase consisted of 25 mM monobasic sodium phosphate, 50 mM sodium citrate, 27 microM disodium EDTA, 10 mM diethylamine, 2.2 mM octane sulfonic acid and 10 mM sodium chloride with 3% methanol and 2.2% dimethylacetamide. The potential was +700 mV versus Ag/AgCl reference electrode for both the amino acids and the biogenic amines and metabolites. Ten rat brain regions, including various cortical areas, the cerebellum, hippocampus, substantia nigra, red nucleus and locus coeruleus were microdissected or micropunched from frozen 300-microm tissue slices. Tissue samples were homogenized in 50 or 100 microl of 0.05 M perchloric acid. The precise handling and processing of the tissue samples and tissue homogenates are described in detail, since care must be exercised in processing such small volumes while preventing sample degradation. An aliquot of the sample was derivatized to form the tert.-butylthiol derivatives of the amino acids and gamma-aminobutyric acid. A second aliquot of the same sample was used for monamine and metabolite analyses. The results indicate that the procedure is ideal for processing and analyzing small tissue samples.

Amino Acids↗

PubMed1981

Analysis of laboratory animal feed for toxic and essential elements by atomic absorption and inductively coupled argon plasma emission spectrometry.

Analytical procedures are described for the determination of arsenic, cadmium, calcium, copper, lead, mercury, selenium, and zinc in animal feed. Mercury is determined by digesting the feed sample in a mixture of concentrated nitric and sulfuric acids with vanadium pentoxide added as an oxidation catalyst, reducing with stannous chloride, and sweeping the elemental mercury into an absorption tube for measurement by atomic absorption (AA) spectrophotometry. Arsenic and selenium are determined simultaneously by digesting the sample with a mixture of concentrated nitric, sulfuric, and perchloric acids; the hydrides of arsenic and selenium, which are formed with the addition of sodium borohydride, are swept into an argon-hydrogen flame for analysis by AA. A low temperature ash is prepared and dissolved in IN HNO3 for the analysis of calcium, copper, and zinc by emission spectroscopy using the inductively coupled argon plasma source; the same solution is used for the determination of cadmium and lead by flameless AA. Animal feed spiked with 3 levels of each of the 8 elements gave recoveries that ranged from 80 to 107%.

Animal Feed↗

PubMed1998

Spectral editing technique for the in vitro and in vivo detection of taurine.

In vivo 1H NMR spectroscopy has proven to be a useful noninvasive tool for the investigation of numerous metabolic and physiological states. Taurine is potentially a useful indicator in neonate development and is involved in a number of physiological processes. However, it could not previously be observed in the in vivo 1H spectrum because of overlap with adjacent resonances. We have developed a spectral editing technique based upon double quantum filtration which allows the taurine resonances to be resolved from adjacent peaks. The experiment is demonstrated both on perchloric acid rodent brain extract and on rodent brain homogenate.

Algorithms↗

PubMed1989

Measurement of adrenolutin as an oxidation product of catecholamines in plasma.

Using the reverse phase high-performance liquid chromatography (HPLC) with mobile phases composed of simple acids, we have developed an assay technique for the measurement of adrenolutin, one of the oxidation products of catecholamines, in rat plasma. Ion-pairing chromatography permits the separation and quantitation of plasma adrenolutin (microM) in a linear manner. Sample preparation involved the precipitation of plasma proteins with perchloric acid and it is easier to handle a large number of samples at a time. However, we were unable to demonstrate the presence of adrenochrome, another oxidation product of catecholamines, in plasma since adrenochrome was rapidly destroyed in acid as well as in blood and was quickly changed into adrenolutin. Adrenolutin peak in HPLC was confirmed by 1) the retention time; 2) co-injection of adrenolutin and; 3) the appearance of 3H-adrenolutin after injection of 3H-norepinephrine. Administration of different catecholamines as well as adrenochrome and adrenolutin in rats also increased the level of adrenolutin in plasma. Adrenolutin was found to be present in plasma in other species including dog, rabbit and pig. High level of adrenolutin, which may represent total concentration of aminolutin in plasma, suggests the presence of an efficient mechanism for the oxidation of catecholamines under in vivo conditions.

Adrenochrome↗

PubMed2006

Determination of green tea catechins in human plasma using liquid chromatography-electrospray ionization mass spectrometry.

A method for the sensitive and specific determination of eight green tea catechins, consisting of catechin (C), epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), catechin-3-gallate (CG), epicatechin-3-gallate (ECG), gallocatechin-3-gallate (GCG) and epigallocatechin-3-gallate (EGCG), in human plasma was established. For optimization of conditions for LC-ESIMS, the separation of the eight catechins was achieved chromatographically using Inertsil ODS-2 column combined with a gradient elution system of 0.1M aqueous acetic acid and 0.1M acetic acid in acetonitrile. Detection using a mass spectrometer was performed with selected ion monitoring at m/z=289 for E and EC, 305 for GC and EGC, 441 for CG and ECG, and 457 for GCG and EGCG under negative ESI. A preparative procedure, consisting of the addition of perchloric acid and acetonitrile to the plasma for deproteinizing and the subsequent addition of potassium carbonate solution to remove excess acid, was developed. In six different plasma with the eight catechins spiked at two different concentrations, the average recoveries were in the range between 72.7 and 84.1%, which resulted from the matrix effect and preparative loss, with coefficients of variance being 8.2-19.8% among individuals. The levels of the catechins in prepared plasma solutions that were kept at 5 degrees C within 24h were stable, which allows us to simply analyze many prepared plasma solutions using an autosampler overnight. When using this method to analyze the eight catechins in human plasma after oral ingestion of a commercial green tea beverage, we detected all the catechins absorbed into human blood for the first time. This also suggested that extremely small amounts of the eight catechins orally ingested may be absorbed based on each absorptive property for the catechins. The method should enable pharmacokinetic studies of green tea catechins in humans.

Catechin↗

PubMed1992

Simultaneous high-performance liquid chromatographic determination of salicylates in whole blood, plasma and isolated erythrocytes.

A method using liquid-liquid extraction has been developed for the isolation of acetylsalicylic acid and its metabolites, salicylic, gentisic or possibly salicyluric acids, from whole blood, isolated erythrocytes and plasma. Methylene chloride proved to be the best of the organic solvents tested. For whole blood and isolated erythrocytes it was necessary to carry out haemolysis prior to their extraction. The high-performance liquid chromatographic conditions for the quantitation of acetylsalicylic acid and its metabolites from samples of whole blood, erythrocytes and whole plasma were optimized. Separation was performed using reversed-phase chromatography on Separon SGX C18 and ultraviolet detection at 236 nm. A mixture of methanol-water (80:100, v/v) was the mobile phase, acidified with perchloric acid to pH 2.5.

Animals↗

PubMedDate not supplied

[Hydroxyapatite thin-layer chromatography of nucleic acid].

The present work is concerned with a sensitive and fast micromethod for separation of single- and double-stranded molecules of nucleic acid by hydroxyapatite (HAP) thin-layer chromatography. The thin layers were obtained by precipitation of ground HAP particules into the surface of the plates in water. Chromatography in sodium phosphate buffer makes it possible to separate from 1 to 50 micrograms of nucleic acids for 30--50 sec. Thereby double-stranded molecules remain at the starting line, whereas single-stranded DNA or RNA follow up the solvent. For quantitative assay of nucleic acids by HAP thin-layer chromatography, the plates were scanned in UV light, radioactivity was measured without extracting substances from HAP and DNA and RNA were eluted with the help of phosphate buffer. A simple and accurate determination method has been suggested consisting in dissolving HAP in perchloric acid followed by hydrolysis of nucleic acids and spectrophotometry of solutions. The retrieval of the material after chromatography in 99 +/- 2%, the mean determinations error is 2--3%. The conditions are described for extraction, after thin-layer chromatography, of desalted and concentrated DNA, ready for use in later experiments. The paper describes a method: for determination of the degree of DNA nativity; quantitative determination of DNA in solutions, containing admixtures; separation of synthesized RNA from its precursors and from the DNA template; assay of DNA thermostability; investigation of the kinetics of DNA reassociation and DNA-DNA hybridization. Some results obtained from hydroxyapatite thin-layer chromatography are discussed.

Chromatography, Thin Layer↗

PubMed1984

A facile route to semi-synthesis of acetyl glycerylether phosphoethanolamine and its choline analogue.

A facile route to the semi-synthesis of acetyl glycerylether phosphoethanolamine and, subsequently, its choline analogue (platelet-activating factor) has been developed. In essence, this technique takes advantage of the fact that the phosphatidylethanolamine fraction of bovine erythrocytes contains 75-80% of a 1-O-alkyl-2 fatty acyl derivative. Isolation of the latter by silicic acid chromatography followed by base-catalyzed methanolysis allowed good recovery (60-70%) of the 1-O-alkyl-(lyso)-sn-glyceryl-3-phosphoethanolamine, which contained a mixture of long chain alkyl ethers. This compound was treated with acetic anhydride in the presence of trace amounts of perchloric acid for 45 sec to give, in excellent yield, 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphoethanolamine (AGEPE). This procedure gave a 70% yield of purified AGEPE, based on the starting component, 1-O-alkyl-(lyso)-sn-glyceryl-3-phosphoethanolamine. Separation of AGEPE into fractions individually enriched in the 16:0, 18:0, and 18:1 alkylether substituents was accomplished by silica gel G combined with silver nitrate-impregnated silica gel H thin-layer chromatography. The AGEPE or its individual molecular species can be converted in high yields to the corresponding 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphocholine (AGEPC) analogues by reaction with methyl iodide in the presence of a crown ether. Characterization of the derivatives was achieved through thin-layer chromatography, infrared spectroscopy, gas-liquid chromatography, and combined gas-liquid chromatography-mass spectrometry. The ability of these analogues to induce irreversible aggregation and secretion of serotonin from washed rabbit platelets was evaluated.

Acetylation↗

PubMed1982

Simultaneous determination of 3,4-dihydroxyphenylalanine, 5-hydroxytryptophan, dopamine, 4-hydroxy-3-methoxyphenylalanine, norepinephrine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, serotonin, and 5-hydroxyindoleacetic acid in rat cerebrospinal fluid and brain by high-performance liquid chromatography with electrochemical detection.

A method using reversed-phase ion-pair high-performance liquid chromatography with electrochemical detection for the simultaneous determination of tryptophan (TRP), 3,4-dihydroxyphenylalanine (DOPA), and their metabolites in whole brain, small-brain parts, and cerebrospinal fluid of rats has been developed. The sample preparation requires only homogenization in perchloric acid and centrifugation before injection onto the column. With a LiChrosorb RP-18 (10 micrometer) column and a mobile phase consisting of a phosphate (NaH2PO4, 0.1 M)-methanol mixture with octylsulfonate (2.6 x 10(-3) M) at pH 3.35 and 26 degrees C, the separation of DOPA, dopamine, norepinephrine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, 4-hydroxy-3-methoxyphenylalanine, TRP, 5-hydroxytryptophan (5-HTP), serotonin, and 5-hydroxyindoleacetic acid was achieved. The method has been applied to study the effect of alpha-monofluoromethyldopa alone and in combination with L-DOPA or L-5-HTP, on the catechol and 5-OH indole levels in brain and CSF of the rat.

3,4-Dihydroxyphenylacetic Acid↗