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At least 649 records · Page 36Linked to original sources
PubMed2006

Determination of homocitrulline in urine of patients with HHH syndrome by liquid chromatography tandem mass spectrometry.

A liquid chromatography tandem mass spectrometric method is described for the analysis of homocitrulline in human urine, a key metabolite in the differential diagnosis of hyperammonemia, hyperornithinemia, homocitrullinuria (HHH) syndrome. Urine samples were prepared by mere five-fold dilution with a mixture of internal standards (2H2-citrulline and 2H3-creatinine) used for the simultaneous quantification of creatinine. Analytes were separated on a cyano column and eluted isocratically within seven min. Detection was achieved by monitoring transitions of 190 > 84 and 190 > 127 for homocitrulline, 178 > 115 for 2H2-citrulline, 114 > 44 for creatinine and 117 > 47 for 2H3-creatinine. Calibration curves were linear up to 100 micromol/L. Intraday (n = 7) and interday (n = 6) variations were less than 10%. In urine samples from three siblings confirmed to have HHH syndrome, homocitrulline levels were at 13.3 (74), 21.1 (50) and 108.2 (103) mmol/mol creatinine (micromol/L). Control values were 0-9 mmol/mol creatinine (n = 120). The current method solves specificity issues in homocitrulline determination often encountered with some ninhydrin-based systems (coelution with methionine) and some o-phthalaldehyde-based ones (coelution with taurine), and presents an attractive alternative with a relatively high throughput.

Ammonia↗
PubMed1996

Immunological determination of triazine pesticides bound to soil humic acids (bound residues).

An immunological method for the determination of triazine herbicides covalently bound to soil humic acids has been developed. A sandwich-immunoassay has been performed, based on both polyclonal humic acid-antibodies and monoclonal triazineantibodies. A peroxidase-labelled third antibody has been used for the photometric detection. A triazine-humic acid conjugate served as calibration standard. The coupling density for this conjugate has been determined by measuring the difference of free amino groups both with ninhydrin and with the trinitrobenzene sulfonic acid method. In addition, the coupling density has been confirmed by scintillation counting using a (14)C-atrazine derivative. Due to nonspecific interactions between antibody proteins and humic acids, different blocking steps had to be performed. Finally, the assay has been applied to a triazine contaminated soil sample. Humic acids (including bound residues) have been extracted by diluted sodium carbonate solution. Concentrations of bound atrazine residues have been found in the range of 2 mg/kg soil on fields where triazine herbicides has been applied over a period of 21 years. These results are comparable to both the applied amount and the nonextractable fraction.

Journal Article↗
PubMed2005

A novel crude oil emulsifier excreted in the culture supernatant of a marine bacterium, Myroides sp. strain SM1.

A marine bacterium, Myroides sp. SM1, can grow on weathered crude oil and show emulsification of it. The biosurfactant able to emulsify crude oil was excreted in culture supernatant of Myroides sp. SM1 grown on marine broth, which was extracted with chloroform/methanol (1:1) at pH 7 and purified by normal and reverse phase silica gel column chromatographies. The compound was ninhydrin-positive, and the chemical structure was elucidated by nuclear magnetic resonance (NMR), infrared spectroscopy (IR), fast atom bombardment mass spectrometry, and gas chromatography-mass spectrometry (GC-MS) to be a mixture of L: -ornithine lipids, which were composed of L: -ornithine and a different couple of iso-3-hydroxyfatty acid (C(15)-C(17)) and iso-fatty acid (C(15) or C(16)) in a ratio of 1:1:1. The critical micelle concentration for a mixture of ornithine lipids was measured to be approximately 40 mg/l. A mixture of ornithine lipids exhibited emulsifying activity for crude oil in a broad range of pH, temperature, and salinity and showed higher surface activity for oil displacement test than other several artificial surfactants and a biosurfactant, surfactin.

Culture Media, Conditioned↗
PubMedDate not supplied

Cathepsin A activity in primary and metastatic human melanocytic tumors.

Several lysosomal proteases including cathepsins B, D, H and L have been found to play a role in the metastasis of tumor cells. However, up to now no information on the role of cathepsin A, a lysosomal multifunctional peptidase, in the proliferative, invasive, and metastatic potential of malignant tumors has been available. In the present study we compared the activity of cathepsin A in lysates of 34 human melanocytic tumors: primary (n = 12) and metastatic (n = 5) malignant melanoma, dysplastic pigmented nevi (n = 6) and pigmented nevi without evidence of dysplastic melanocytes (n = 11). The carboxypeptidase activity of cathepsin A was assayed at pH 5.0 with its specific substrate Cbz-Phe-Ala. The amount of released C-terminal alanine was measured by the ninhydrin method. We found that lysates of primary malignant melanoma lesions exhibited significantly higher cathepsin A activity than lysates of dysplastic and normal pigmented nevi. The cathepsin A activity in lysates of metastatic lesions of malignant melanoma was significantly higher than in primary focus lysates. It seems that cathepsin A may play a role in malignant transformation and metastatic dissemination of malignant melanoma.

Carboxypeptidases↗
PubMed1999

Long-term follow up of a new case of hawkinsinuria.

UNLABELLED: Hawkinsinuria is a rarely diagnosed autosomal dominantly transmitted inborn error of tyrosine metabolism with impaired conversion of 4-hydroxyphenylpyruvate to homogentisate. As a consequence of the defective 4-hydroxyphenylpyruvate dioxigenase activity, large amounts of the unusual, ninhydrin-positive amino acid hawkinsin and later on in life 4-hydroxycyclohexylacetic acid are formed and excreted. Clinically the disease is characterised mainly by chronic metabolic acidosis and severe growth retardation as a result of protein overload. As the ability to form 4-hydroxycyclohexylacetic acid and thereby to cope with the still not very well defined reactive and toxic intermediates increases, clinical symptoms vanish. We report here a new patient with hawkinsinuria having experienced a series of admissions because of unclear hepatopathy, growth retardation, and renal tubular acidosis. CONCLUSION: Prolonged tyrosyluria in the newborn and young baby should cause the clinical chemist not only to exclude tyrosinaemia, galactosaemia, and fructose intolerance but also to look carefully for hawkinsin in the aminoacid chromatogram.

4-Hydroxyphenylpyruvate Dioxygenase↗
PubMed2003

Taurine concentration in the brain and in the plasma following intraperitoneal injections.

Summary. The effect of different taurine doses (0.050, 0.125, 0.250, 0.500 and 1.000 g/kg) administered intraperitoneally to Wistar rats was studied in both the plasma and the hippocampal microdialysate content. The samples were analyzed by reverse phased HPLC for the microdialysate samples and by HPLC with ion-exchange post-column derivatization (ninhydrin) for the plasma samples. In both plasma and microdialysate, we observed a dose dependent increase of taurine concentration. The AUC curves obtained from both microdialysate and plasma samples showed that the increase of taurine concentrations were linear. The mean ratio between AUC's microdialysate and plasma was 1.63+/-0.21 showing thus an unbalance between plasma and brain taurine content; a mechanism which enhance taurine transfer from the plasma to the brain was assumed.

Animals↗
PubMed2006

Rapid analysis of taurine in energy drinks using amino acid analyzer and Fourier transform infrared (FTIR) spectroscopy as basis for toxicological evaluation.

So-called energy drinks with very high amounts of taurine (up to 4000 mg/l are usually granted by certificates of exemption) are increasingly offered on the market. To control the currently valid maximum limits of taurine in energy drinks, a simple and rapid analytical method is required to use it routinely in food monitoring. In this article, we describe a fast and efficient analytical method (FTIR-spectroscopy) that is able to reliably characterize and quantify taurine in energy drinks. The determination of taurine in energy drinks by FTIR was compared with amino acid analyzer (ion chromatography with ninhydrin-postcolumn derivatization). During analysis of 80 energy drinks, a median concentration of 3180 mg/l was found in alcohol-free products, 314 mg/l in energy drinks with spirits, 151 mg/l in beer-containing drinks and 305 mg/l in beverages with wine. Risk analysis of these products is difficult due to the lack of valid toxicological information about taurine and its interferences with other ingredients of energy drinks (for example caffeine and alcohol). So far, the high taurine concentrations of energy drinks in comparison to the rest of the diet are scientifically doubtful, as the advertised physiological effects and the value of supplemented taurine are unproven.

Adult↗
PubMed2001

A fast and sensitive method for measuring picomole levels of total free amino acids in very small amounts of biological tissues.

In the present study we describe a simple and fast method to measure the concentration of total free amino acids in very small amounts of biological tissues. The procedure described here is based on the reaction of free amino acids with o-phthaldialdehyde (OPA) in the presence of a reducing agent, beta-mercaptoethanol (MET), to give a complex which can be measured by fluorescence. It is a very rapid process and has the same reliability as the conventional ninhydrin method of Moore and Stein but is about 500 times more sensitive. The sensitivity of the new protocol is such to permit the determination with high reliability of very small amounts of free amino acids at picomole levels, either in a standard amino acid mixture or in biological tissues, without chromatographic separation of the amino acids. It is particularly useful when the amount of the sample is very low, e.g. on a single pituitary or pineal gland of small animals or on single cells, such as oocytes or eggs, as well as single ganglions or axons of marine invertebrates.

Amino Acids↗
PubMed2005

Determination of the GABA analogue succinic semialdehyde in urine and cerebrospinal fluid by dinitrophenylhydrazine derivatization and liquid chromatography-tandem mass spectrometry: application to SSADH deficiency.

Succinic semialdehyde (SSA) accumulates in the inborn error of meta- bolism succinic semialdehyde dehydrogenase deficiency owing to impaired enzymatic conversion to succinic acid. We developed a stable-isotope dilution liquid chromato- graphy-tandem mass spectrometry method for the determination of SSA in urine and cerebrospinal fluid samples. Stable-isotope-labelled [13C4]SSA, serving as internal standard, was prepared by reaction of ninhydrin with L-[13C5]glutamic acid. SSA in body fluids was converted to its dinitrophenylhydrazine (DNPH) derivative, without sample purification prior to the derivatization procedure. The DNPH derivative of SSA was injected onto a C18 analytical column and chromatography was performed by isocratic elution. Detection was accomplished by tandem mass spectrometry operating in the negative multiple-reaction monitoring mode. The limit of detection was 10 nmol/L and the calibration curves over the range 0-500 pmol of SSA showed good linearity (r2 > 0.99). The intra-day coefficient of variation (n = 10) for urine was 2.7% and inter-day coefficient of variation (n = 5) for urine was 8.5%. The average recoveries performed on two levels by enriching urine and cerebrospinal fluid samples ranged between 85 and 115%, with coefficients of variation < 8%. The method enabled the first determination of normal values for SSA in urine and pathological values of SSA in urine and cerebrospinal fluid samples derived from patients with succinic semialdehyde dehydrogenase deficiency.

Blood Chemical Analysis↗
PubMed1978

Chromatographic determination of angiotensin-converting enzyme and angiotensinase activity.

An analytical method utilizing an automatic amino acid analyzer is described for the separation, identification, and measurement of 5 to 50 nmol of angiotensin I, angiotensin II, [Des-Phe8]angiotensin II, Phe-His-Leu, His-Leu, isoleucine, leucine, tyrosine, and phenylalanine. Aminex A-5 cation-exchange resin (0.9 x 15 cm) is sequentially eluted with three sodium citrate buffers: pH 3.25, 0.2 N; pH 4.85, 0.54 N, and pH 6.5, 0.39 N at 60 and 80 degrees C. Reaction with ninhydrin is used for detection. This chromatographic system was used to determine angiotensin-converting enzyme activity and the angiotensinase activity of rabbit brain endopeptidase B. In each assay, the unhydrolyzed substrate and both products were measured simultaneously in one step without pretreatment of the hydrolysate. Products were recovered in 1:1 molar ratios and the overall recovery of an hydrolyzed substrate of products was quantitative.

Amino Acids↗
PubMed1978

The effect of thiodiglycol and dithiothreitol on the alkaline hydrolysis products of certain amino acid phenylthiohydantoins.

The thiazolinone and phenylthiohydantoin derivatives of most amino acids can be hydrolyzed with alkaline dithionite to generate the free amino acid. The acidification of this hydrolysate with 3 N HCl containing thiodiglycol leads in the case of glutamic acid, glutamine, aspartic acid, asparagine, and S-carboxy-methylcysteine to the generation of ninhydrin-reacting components having the chromatographic properties of other amino acids. The use of dithiothreitol instead of thiodiglycol appears to be more satisfactory in most instances.

Amino Acid Sequence↗
PubMed1983

Measurement of ferredoxin-dependent sulfite reductase activity in crude extracts from leaves using O-acetyl-L-serine sulfhydrylase in a coupled assay system to measure the sulfide formed.

Ferredoxin-dependent sulfite reductase (EC 1.8.7.1) catalyses the reduction of sulfite to sulfide, using reduced ferredoxin as an electron donor. An assay system was developed for measuring this enzyme activity in crude extracts and broken chloroplast preparations from leaves. The assay consists of a coupled system in which the sulfide formed is used for cysteine synthesis by added O-acetyl-L-serine sulfhydrylase (EC 4.2.99.8). Cysteine thus formed is determined with ninhydrin under conditions where O-acetylserine does not react and serves as a measure for ferredoxin-dependent sulfite reductase activity. Cysteine synthesized in the assay can be determined from 10 to 200 nmol. One assay per minute can be performed.

Cysteine↗
PubMed1983

Chromophoric peptide substrates for activity determination of animal aspartic proteinases in the presence of their zymogens: a novel assay.

Solid-phase synthesis was used for the preparation of pyroglutamyl-histidyl-p-nitrophenylalanyl-phenylalanyl-alanyl-leucine amide (I) and glycyl-glycyl-histidyl-p-nitrophenylalanyl-phenylalanyl-alanyl-leucine amide (II), two water-soluble and sensitive chromophoric substrates of chicken pepsin, hog pepsin A, and bovine spleen cathepsin D. The kinetic constants of hydrolysis of the p-nitrophenylalanyl-phenylalanyl bond of the substrates were measured by difference spectrophotometry at 308 nm (delta epsilon = 860 M-1 cm-1) and by ninhydrin colorimetry (substrate I, epsilon 570 = 2.31 X 10(4) M-1 cm-1). The pH optimum of cleavage is 5 for the pepsins and 3.7 for cathepsin D. Since all three proteinases still have a significant activity at pH 5.5-6 a new, simple assay was designed for submicrogram quantities of pepsins in the presence of pepsinogens without interference of the latter. The method is particularly suitable for the analyses of the zymogen activation mixtures.

Animals↗
PubMed1984

Cysteine: a potential source of error in amino acid analysis of mercaptoethane sulfonic or hydrochloric acid hydrolysates of proteins and peptides.

Hydrolysis of proteins and peptides with mercaptoethane sulfonic acid is liable to produce overestimation of the proline content owing to the production of ninhydrin-positive material (probably cysteine) which coelutes with proline on many ion-exchange analytical systems. A similar error occurs with HCl hydrolysis (especially in the presence of mercaptoethanol or thioglycollic acid) if care is not taken to oxidize cysteine during reconstitution of the hydrolysate before amino acid analysis.

Amino Acids↗
PubMed1985

Periodate oxidation products of hydroxylysine in the synthesis of 5-substituted prolines.

The amino acid hydroxylysine was subjected to oxidation by sodium metaperiodate under various conditions. It was found that in acid and high temperature, the initial oxidation product alpha-aminoglutaric gamma-semialdehyde was converted to glutamic acid with a yield of 60%. The use of alkaline conditions of oxidation favored the cyclization of alpha-aminoglutaric gamma-semialdehyde to form delta 1-pyrroline 5-carboxylic acid. Addition of NaCN to this intermediate generated new proline analogs, likely a mixture of cis- and trans-5-cyanoprolines, with a yield of 30%. Upon hydrolysis, the 5-cyanoprolines were converted to a probable mixture of cis- and trans-5-carboxyprolines. Infrared and high-resolution mass spectral data of the analogs and visual absorption spectra of the ninhydrin products were obtained to confirm the structures.

Hydrogen-Ion Concentration↗
PubMed1985

An amino-terminal tryptophan derivative which is refractory to Edman degradation.

A new derivative of tryptophan is proposed to account for the observation that some peptides having amino-terminal tryptophan residues become refractory to Edman degradation. An acid-catalyzed oxidation of the indole nucleus and subsequent cyclization to a unique 3-anilinopyrrolidin-2-one derivative with cleavage of the peptide chain is the most likely chemical explanation. This amino acid is reactive with ninhydrin and contains an aryl amine. However, the amine does not bond to the alpha carbon so while reactive to phenyl isothiocyanate, distances are too great for the residue to be cleaved from the peptide during Edman degradation.

Amino Acid Sequence↗
PubMed1985

An o-phthalaldehyde spectrophotometric assay for proteinases.

A rapid and convenient spectrophotometric assay has been devised to measure proteolysis. The assay is based on the reaction of o-phthalaldehyde (OPA) and 2-mercaptoethanol with amino groups released during proteolysis of a protein substrate. The reaction is specific for primary amines in amino acids, peptides, and proteins, approaches completion within 1 to 2 min at 25 degrees C (half-times of approx 10-15 s), and requires no preliminary heating or separation of the hydrolyzed products from the undegraded protein substrate prior to performing the assay. The OPA assay was relatively as successful as a 2,4,6-trinitrobenzenesulfonic acid (TNBS) procedure in predicting the extent of hydrolysis of a protein substrate. The utility of the OPA method was demonstrated by measuring the degree of proteolytic degradation caused by trypsin, subtilisin, Pronase, and chymotrypsin of various soluble protein substrates. Ethanethiol (instead of 2-mercaptoethanol) or 50% of dimethyl sulfoxide can be included in the assay solution to stabilize certain OPA-amine products. The present method approaches the sensitivity of ninhydrin and TNBS procedures, is more convenient and rapid, and could substitute for these reagents in most assay systems.

Aldehydes↗
PubMed1986

Quantification of chymosin action on nonlabeled kappa-casein-related peptide substrates by ultraviolet spectrophotometry: description of kinetics by the analysis of progress curves.

A method is described for quantifying the proteolytic action of the milk-clotting enzyme chymosin on small and medium-sized peptide substrates by monitoring the decrease of absorbance at 230 nm during cleavage. The method is illustrated by the determination of the kinetic parameters of the specific splitting of a kappa-casein-related hexa- and pentadecapeptide by chymosin. The results are in good agreement with those found earlier with the same enzyme/substrate system by using an automated ninhydrin method. Erroneous results were obtained when the kinetic data were derived from one single progress curve. The significance of initial rate measurements for calculating correct kinetic parameters is briefly discussed. The usefulness of single progress curves measured at different initial substrate concentrations for obtaining information about the mechanism of the enzymic reaction is demonstrated.

Animals↗