2-4-dinitrophenylhydrazine and 1-2-diaminobenzene

Allantoinase hydrolyzes allantoin, a purine metabolite and a nitrogen transport molecule in plants, to form allantoic acid. The standard enzyme assay involves acid-catalyzed product decomposition to form urea and glyoxylate, reaction of glyoxylate with phenylhydrazine, and oxidative conversion of phenylhydrazone to 1, 5-diphenylformazan that is measured colorimetrically. When used with crude cell extracts this assay is problematic and its complexity is a hindrance to detailed enzyme characterization; thus, three alternative assays were developed. In the first assay, 2, 4-dinitrophenylhydrazine was reacted with allantoate-derived glyoxylate and the concentration of hydrazone was measured directly by its absorbance at 450 nm. This assay exhibited enhanced reproducibility compared to the standard method and entailed fewer steps, but was 3-fold less sensitive. The second assay combined allantoate decomposition and glyoxylate reaction with o-phenylenediamine to yield a quinoxalone that was detected by its absorbance at 340 nm. This one-step method was the least error prone of those examined, but was more than 10-fold less sensitive than the standard assay. The third assay involved urease-catalyzed hydrolysis of allantoate-derived urea, followed by reaction of the released ammonia to form indophenol. This was the most laborious of the assays, but was more sensitive than the standard method.

Topics: Amidohydrolases; Phenylenediamines; Phenylhydrazines; Substrate Specificity; Urease

We demonstrate that total ascorbic acid (TAA, the sum of ascorbic acid and dehydroascorbic acid) in properly prepared human plasma is stable at -70 degrees C for at least 6 years when preserved with dithiothreitol. TAA in human plasma or serum preserved with metaphosphoric acid degrades slowly, at the rate of no more than 1% per year. As assessed from our stability data and from data obtained from 23 laboratories over a period of > 2 years, the intralaboratory repeatability of TAA measurement is approximately 2 mumol/L, irrespective of TAA concentration. Nonchromatographic analytical methods involving dinitrophenylhydrazine and 0-phenylenediamine yield biased results relative to chromatographic methods. Within groups of laboratories that use roughly similar analytical methods, the interlaboratory measurement reproducibility CV for TAA is 15%.

Topics: Ascorbic Acid; Chromatography; Dehydroascorbic Acid; Dithiothreitol; Drug Stability; Freezing; Humans; Kinetics; Laboratories; Phenylenediamines; Phenylhydrazines; Phosphorous Acids; Plasma; Reproducibility of Results; Specimen Handling; Stereoisomerism; Time Factors

A high-performance liquid chromatographic method was developed for the determination of glyoxylate in the liver. Alpha-keto acids in charcoal-treated acid-extract of the liver were converted to the corresponding 2,4-dinitrophenylhydrazones and purified as the derivatives by successive extractions with ethyl acetate and sodium bicarbonate solution. The dinitrophenylhydrazones were then quantitatively converted to the corresponding substituted 2-hydroxyquinoxalines by reaction with o-phenylenediamine, followed by analysis by high-performance liquid chromatography with fluorescence detection. As a control to correct the recovery of tissue glyoxylate, an acid-extract of the liver prepared with the addition of standard glyoxylate (25-50 nmol/g wet weight of tissue) was simultaneously subjected to the analytical procedure. The maximum sensitivity of the glyoxylate measurement as 2-hydroxyquinoxaline (the quinoxaline derivative corresponding to glyoxylate) was defined as the peak area reading five times as high as the blank value obtained without sample and was approximately 10 pmol per injection. Glyoxylate in the addition compound with tris(hydroxymethyl)aminomethane was quantitatively recovered as 2-hydroxyquinoxaline. The addition compounds of glyoxylate with bisulfite and cysteine did not react with 2,4-dinitrophenylhydrazine under the conditions employed and were not detectable as glyoxylate by this method, while the adduct-forming substances added to the acid-extract of the liver did not interfere with the glyoxylate determination. No glyoxylate was detected when the liver extract had been incubated at neutral pH with a large excess of cysteine, indicating that little artificial production of glyoxylate occurred during the analytical procedure. Among 64 compounds tested for possible artificial production of glyoxylate or possible interference with the chromatographic determination of 2-hydroxyquinoxaline, p-hydroxyphenylpyruvate was the only compound which was converted to glyoxylate during the procedure. However, p-hydroxyphenylpyruvate was easily removed from the acid-extract of the tissue by charcoal treatment. The amount of glyoxylate in the liver of fasted rat was measured by the present method to be approximately 5 nmol per g of wet weight.

Topics: Animals; Chromatography, High Pressure Liquid; Glyoxylates; Liver; Male; Phenylenediamines; Phenylhydrazines; Rats; Rats, Inbred Strains