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PubMed1976

The mass spectrometric identification of dipeptides in the urine of a patient suffering from chronic skin ulceration and oedema.

The identification of a complex ninhydrin positive mixture present in the urine of a child suffering from chronic skin ulceration and oedema by direct chemical ionisation mass spectrometric analysis is described. The compounds were shown to be dipeptides, of which glycylproline was the major constituent. At least 15 dipeptides were identified in the urine, most of which contained proline or hydroxyproline in the carboxy terminal position. The results suggested that the patient suffered from a defect in collagen metabolism. This hypothesis was subsequently confirmed by a grossly diminished level of prolidase in cultured fibroblasts and erythrocytes.

Amino Acids↗

PubMed1977

A fluorometric assay for beta-phenylethylamine in human urine.

A sensitive and specific method for the fluorometric determination of beta-phenylethylamine (PEA) in human urine is described. PEA in urine was separated from phenylalanine and 5-hydroxytryptamine by n-heptane extraction, and determined by the ninhydrin reaction in the presence of L-leucyl-L-alanine. Using this method, the amounts of free PEA in urine samples of normal subjects and schizophrenics were measured. Normal subjects excreted 15.9 +/- 6.4 microgarm/day of free PEA, and chronic schizophrenics 9.3 +/- 2.7 microgram/day of free PEA. In normal subjects, the highest excretion of free PEA was observed in the period from 4:00 p.m. to 12:00 p.m. in a day.

Adult↗

PubMed1978

Fluorimetric determination of free hydroxyproline and proline in blood plasma.

A selective and sensitive assay of hydroxyproline in blood plasma is presented. The technique uses ion exchange chromatography followed by reaction with 7-chloro-4-nitrobenzofurazan (NBD choloride) and continuous recording of the resulting fluorescence (gamma ex=465 nm, gamma fl=535 nm). Proline and prolyl-and hydroxyprolyl-peptides can also be measured by this system. For hydroxy proline, the method is more sensitive than procedures using ninhydrin or p-dimethylaminobenzaldehyde.

Fluorometry↗

PubMed1978

3-Amino-2-piperidone in the urine of patients with hyperornithinemia.

3-Amino-2-piperidone, a delta-lactam of ornithine, has been detected in the urine of a number of patients with hyperornithinemia. The distinctive yellow colour formed with a ninhydrin-cadmium reagent should help in the detection of hyperornithinemia by urinary chromatography.

Adolescent↗

PubMed1978

A routine method for determination of glycine and taurine distribution in bile acids.

A procedure is described in which the content of glycine and taurine in bile acids is determined. Following an alkaline hydrolysis, the amino acids are separated on a column of Dowex 50-X8 and quantitatively estimated by a ninhydrin colorimetric method. The taurine/taurine + glycine ratios in a series of samples were determined by this method and by an enzymatic bile acid analysis and a good accordance between the two methods was obtained.

Bile Acids and Salts↗

PubMed1978

Isolation and identification of a new potent inhibitor of creatine kinase from human serum.

Human serum contains a very potent inhibitor of creatine kinase in addition to urate which was characterized as an inhibitor of this enzyme by Warren (Warren, W.A. (1975) Clin. Biochem. 8, 247). This new inhibitor was isolated from human serum by dialysis, negative DEAE-cellulose adsorption and a series of gel filtration chromatographies. The inhibitor so isolated was an organic compound. Kinetic investigation showed noncompetitive inhibition versus creatine, as well as MgATP. Infrared spectroscopy revealed amino and carboxyl groups. The isolated material was ninhydrin-positive. Thin-layer chromatography of the dansyl derivative showed several fluorescent spots. Quantitative amino acid analysis revealed the presence of several amino acids. Of these only cystine and cysteine were inhibitory in the standard inhibitor assay, using rabbit MM creatine kinase. Testing with human MM creatine kinase only cystine was found inhibitory. Cystine has a specific inhibitor activity of 600 anti-Units per mg, using rabbit or human MM creatine kinase, respectively. Urate by comparison has a specific inhibitor activity of 45 anti-Units per mg using human MM creatine kinase.

Creatine Kinase↗

PubMed1979

Determination of D-penicillamine in serum and urine of patients with rheumatoid arthritis.

A chromatographic auto-analyser method is described for the determination of total D-penicillamine in biological fluids. After oxidation with performic acid, D-penicillaminic acid is separated from other ninhydrin-positive acidic amino acids by anion-exchange chromatography. Total cysteine/cystine is simultaneously determined as cysteic acid, which makes the method equally suitable for patients with cystinuria. The detection limit in serum and urine is 2 microM D-penicillamine, which is more than sufficient for clinical application. The metabolite S-methyl-D-penicillamine is oxidized by performic acid to a sulphone, which can be determined after separation by cation-exchange chromatography.

Amino Acids↗

PubMed1983

The determination of pipecolic acid: method and results of hospital survey.

A method is described for the quantitative assay of pipecolic acid in biological fluids using column chromatography, with acid ninhydrin for color development. The technique permits measurement of plasma pipecolic acid in normal individuals with blood samples of 1 ml. The mean value in 29 pediatric patients, aged 4 months to 17 years, hospitalized with a variety of diagnoses was 2.1 mumol/1 +/- 1.6. Newborn infants had slightly elevated plasma levels with a mean of 12 mumol/1 +/- 5.6. Among the diagnostic categories, patients with liver disease were distinctive in commonly having levels at the upper limits of normal. In some cases, the levels were exceedingly high, reaching 78 mumol/1 in one fatal case.

Amino Acids↗

PubMed1986

HPLC assay of phenylalanine and tyrosine in blood spots on filter paper.

Blood spots on filter paper are used to screen for phenylketonuria. We have developed a high pressure liquid chromatographic assay of phenylalanine and tyrosine after elution of these amino acids from filter paper. The analysis time is 20 min. The amino acids are separated using an ion-exchange resin with post-column ortho-phthalaldehyde derivatization and subsequent fluorescence detection. Other common amino acids do not interfere. The assay is linear from 20 pmol to at least 2,000 pmol with a coefficient of variation of 3%. The assay is as accurate as the determination of phenylalanine by ion-exchange ninhydrin-reactive methods currently in use, but is 100 times more sensitive. The method has the advantages of avoiding venipuncture, is cost effective, has a high sensitivity, and a rapid turnaround.

Adult↗

PubMed1990

O-phosphohydroxylysinuria: a new inborn error of metabolism?

An abnormal ninhydrin positive compound was observed in the urine of two unrelated patients with neurological abnormalities. The compound was isolated by cation exchange followed by preparative paper chromatography and finally purified via cation exchange column chromatography. Its identification as O-phosphohydroxylysine resulted from FAB mass spectrometry and NMR spectroscopy. Chemical synthesis confirmed the structure. It was tentatively postulated that these patients had a defect of the metabolism of hydroxylysine, viz., a deficiency of the enzyme O-phosphohydroxylysine phospholyase.

Adolescent↗

PubMed1995

A scheme for the interpretation of primary and secondary disturbances of plasma and urinary amino acid profiles. A possible way to an expert system.

A general scheme for the interpretation of primary and secondary abnormalities of plasma and urine amino acid concentrations is described. The key steps of this scheme are: analytical assessment of the measurements, comparison of results obtained with the reference values expressed in absolute and/or relative concentrations and identification of abnormally increased ninhydrin-positive compounds. The interpretation of results takes account of the various abnormalities induced by drugs or diet. The origins of these abnormalities are ordered by their frequency. A part of the proposed scheme is now computerized as the first step in the development of an expert system.

Amino Acid Metabolism, Inborn Errors↗

PubMed1986

Quantitation of lens protein mixed disulfides by ion-exchange chromatography.

A sensitive and high resolution ion-exchange chromatographic method was employed to analyse lens protein mixed disulfides. The mixed disulfides were analysed and quantified as glutathione sulfonic acid and cysteic acid after the lens proteins were subjected to performic acid oxidation. The oxidized product was applied on a 0.9 X 15 cm column packed with BioRad Aminex A-28 resin (9 microns beads). The column was maintained at 55 degrees C and eluted with 0.3 M sodium acetate buffer, pH 5.1 at 60 ml hr-1 for 40 min before switching to 0.5 M of the same buffer. The glutathione subsequently reacted with ninhydrin and monitored for absorbance at 570 nm. The glutathione sulfonic acid emerged at 69 min, cysteic acid at 35 min, and both were clearly resolved from neighboring peaks and could be detected at less than nmol level. In the lens, both glutathione and cysteine protein mixed disulfides were only found in the TCA-insoluble protein fraction. Normal rat lens contained 0.35 nmol per lens of protein-bound glutathione but 10-fold this amount of protein-bound cysteine. The hyperglycemic condition had little effect on the protein thiol mixed disulfide level except that at a more advanced diabetic condition, equal depletion of cysteine and glutathione mixed disulfides were found. On the other hand, oxidative conditions induced a marked elevation of glutathione mixed disulfide but not of the cysteine mixed disulfide.

Animals↗

PubMed1975

[Isolation of a new keto-acid, produced from epsilon-N-trimethyllysine by Neurospora crassa].

A new epsilon-N-trimethyllysine metabolite has been isolated from the mycelium of Neurospora crassa. The labelled compound produced from incubations in vivo and in vitro from epsilon-N-trimethyl ([14-C]H3)L-lysine has been identified as 2-keto-epsilon-N-trimethyl-hexanoic acid by reducing its 2,4-dinitrophenyl hydrazone back to epsilon-N-trimethyllysine by hydrogenolysis in a Parr bomb. Analyses on TLC and in four different ion exchange chromatographic systems show the appearance of a ninhydrin positive product having the same Rf and the same retention time as epsilon-N-trimethyllysine; it contains more than 85% of the radioactivity of the 2,4-dinitrophenylhydrazone of the keto acid.

Chromatography, Ion Exchange↗

PubMed1986

Decrease in plasma amino acid level after secretin and pancreozymin as an indicator of exocrine pancreatic function.

Total plasma amino acids were determined by the ninhydrin method in 37 controls and 30 patients with chronic pancreatitis and normal (n = 7) pancreatic enzyme output or mildly (n = 6), moderately (n = 8), and severely (n = 9) reduced pancreatic enzyme output. Intravenous injection of synthetic secretin did not change plasma amino acid levels. During a combined intravenous infusion of secretin (1 CU/kg X h) and pancreozymin (1 Ivy dog unit/kg X h), amino acid concentrations decreased maximally by 31% +/- 19% (mean +/- SD) in controls, but only by 6.3% +/- 4.7% in patients with exocrine pancreatic insufficiency (p less than 0.001 vs. controls). At a cutoff limit of less than or equal to 12% for the decrease in total amino acids, mild exocrine insufficiency (20%-40% of mean normal chymotrypsin output) was identified with a sensitivity of 67%, whereas moderately to severely impaired function was detected in every case (overall sensitivity 91%). Pancreatic function, as assessed by duodenal intubation and the tubeless amino acid test, was significantly correlated (e.g., rs = 0.73 for chymotrypsin output, p much less than 0.001). In 15 controls and 13 patients with mildly (n = 5) to severely impaired pancreatic function, individual amino acids were estimated. Plasma serine kinetics completely distinguished both groups. Kinetics of serine, valine, isoleucine, and histidine correlated even better with pancreatic function than those of total amino acids.

Adult↗

PubMed1983

Estradiol-induced hepatic protein synthesis and transaminase activity in the male flounder, Platichthys flesus (L.).

A continued accumulation of vitellogenin was observed in the serum through 28 days after administration of spaced large doses of estradiol. The level of estradiol in the serum reached a maximum on Day 4, followed by a continued decrease until a basal concentration was reached at Day 15. The estradiol treatment increased the in vitro hepatic protein synthesis activity, measured as polyphenylalanine synthesis per unit cytoplasmic RNA, within 2-4 days reaching a maximum after 7 days. The effect was transitory and reduced to less than 50% within 15 days. After restimulation a second rise in protein synthesis activity was observed. The cellular content of bulk RNA i the liver showed a slow but steady accumulation through the 28 days. The specific activity of glutamate-oxaloacetate transaminase and glutamate-pyruvate transaminase in the liver decreased 35 and 20% relative to control, respectively, within 7 days after the first stimulation and again 14 days after restimulation. The concentration of serum protein showed a steady increase throughout the experimental period. The concentration of serum ninhydrine positive substances was lowered to 50% relative to the controls within the first 7 days after start of treatment with hormone.

Alanine Transaminase↗

PubMed1995

Determination of the relative amounts of the B and C components of neomycin by thin-layer chromatography using fluorescence detection.

The determination of the relative amounts of the B and C components of neomycin sulphate by thin-layer chromatography using silica gel plates from Whatman as the stationary phase is described. The mobile phase consisted of methanol-20% (m/v) sodium chloride solution (15:85). Fluorescence detection was performed after derivatization with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole. The influence of different parameters on the separation was investigated. A number of commercial samples was analysed using this method and the results were compared with results obtained with ion-exchange chromatography and ninhydrin colorimetric detection, which is the official method prescribed by the European Pharmacopoeia. The described method is much easier to perform than the official method.

4-Chloro-7-nitrobenzofurazan↗

PubMed1978

A micro-procedure for quantitative precipitin tests.

A new method for the quantitative analysis of antigens and antibodies has been based on (1) ultrafiltration of the antigen-antibody precipitates through silver membranes of 0.2 micrometer pore size in a specially designed multisample apparatus, and (2) spectrophotometric determination at 210 nm of the amount of proteins in the antigen-antibody precipitates dissolved in 0.01 N HCl. At this wavelength, the = C = O group of the polypeptide chains constitutes the main chromophoric group. In comparisons with the ninhydrin color reaction, protein determination by low UV spectrophotometry, e.g., at 210 nm, was shown to be about 6 times more sensitive, permitting analysis of samples containing from 1.0 to 35.0 microgram of antigen. The concentration range of protein solutions in 0.01 N HCl which is measured at low UV can be regulated by a factor of 8--10 by changing absorption between 200 and 230 nm. Comparison of the ultrafiltration microtechnique with the standard quantitative precipitin microtechnique involving centrifugation of precipitates was made in 4 different antigen-antibody systems. The new technique was found to be as accurate as the standard technique. It allows completion of analysis within only 5--6 h. The standard precipitin technique, by contrast, requires a 5--7-day reaction period for completion.

Animals↗

PubMed1992

Convulsive action and toxicity of uremic guanidino compounds: behavioral assessment and relation to brain concentration in adult mice.

Four guanidino compounds that are known to accumulate in uremia, namely creatinine, guanidine, guanidinosuccinic acid and methylguanidine, were administered intraperitoneally and intracerebroventricularly to adult albino mice and the compounds epileptogenic and toxic properties were behaviorally assessed. After intraperitoneal injection, brain concentration of the compounds as a function of injected dose was monitored additionally. Guanidino compound brain concentration was determined by cation exchange chromatography with fluorescence ninhydrin detection. After systemic administration, especially guanidinosuccinic acid and methylguanidine induced long-lasting generalized convulsions which gradually increased in severity. Increasing the dose injected intraperitoneally resulted in linear increase in brain concentration of the injected compounds, in parallel with increase in proportion of animals presenting with convulsions and/or severity of convulsions. Guanidinosuccinic acid brain concentration increased more slowly than that of the other 3 compounds and guanidinosuccinic acid also exerted its effect later than the others. Since none of the other metabolically related guanidino compounds determined was significantly increased in the brains of the injected animals, the observed behavior was most certainly induced by the compounds injected and not by some secondary metabolite. Epileptogenic properties of the four compounds were markedly and qualitatively different in systemic administration, but rather similar in intracerebral administration. A tentative epileptogenic potency order was inferred from the combined behavioral and biochemical results. All 4 of the compounds tested displayed the ability to induce full-blown clonic-tonic convulsions and they did so in a dose-related manner. Guanidinosuccinic acid appeared to be slightly more potent than methylguanidine, but both guanidinosuccinic acid and methylguanidine were considerably more potent than guanidine. Creatinine was many times less potent than the other 3 guanidino compounds. Revised epileptogenic potency order on the basis of guanidino compound brain concentration after systemic administration as well as potency order after intracerebral administration paralleled the potency order of these compounds in their GABA antagonism reported earlier. It was therefore postulated that the GABA antagonism of uremic guanidino compounds could underlie their epileptogenic character. Moreover, these compounds could very likely be at the basis of the neurological complications including epilepsy of uremic patients in whom they accumulate in physiological fluids and brain.

Animals↗