2-4-dinitrophenylhydrazine and thiobarbituric-acid

A selective and sensitive method based on derivatisation with 2,4-dinitrophenylhydrazine (DNPH) and consecutive HPLC gradient separation is described for the determination of malondialdehyde (MDA) in urine. Preparation of urine samples involved a one-step derivatisation/extraction procedure. Separation was achieved using a Waters SymmetryC(18) column (3.9 x 150 mm) and linear gradient of acetonitrile in water (from 30% to 70% in 30 min). The overall detection limit of the method was 56 nM of MDA in urine. The recovery of MDA was 94.3+/-8.6%. MDA in urine of healthy volunteers, measured using the method of standard additions, was 0.019+/-0.012 microM/mmol creatinine. MDA in the same samples measured using the 2-thiobarbituric acid (TBA) assay was 0.181+/-0.063 microM/mmol creatinine. We demonstrate that the commonly used TBA assay in conjunction with HPLC may overestimate the MDA concentration in human urine by almost 10-fold.

Topics: Chromatography, High Pressure Liquid; Humans; Malondialdehyde; Phenylhydrazines; Reproducibility of Results; Thiobarbiturates

A 532 nm red pigment formed in the thiobarbituric acid (TBA) assay of human urine was characterized after separation of the pigment by high-performance liquid chromatography. The yield of the red pigment was somewhat higher at pH 2 than at pH 5; its development was not inhibited by ethylenediaminetetraacetic acid. The characteristics of the pigment were similar to those of the pigment derived from standard malonaldehyde. The amount of the pigment formed was roughly equal to the content of malonaldehyde derivatives estimated as 1-(2,4-dinitrophenyl)pyrazole. Pigment formation was significantly enhanced by t-butyl hydroperoxide (t-BuOOH) and ferric ions, which may be due to pigment formed from aldehydes other than malonaldehyde; the presence of these aldehydes was confirmed by the formation of the corresponding 2,4-dinitrophenylhydrazones. The amount of pigment produced from 24-h urine samples of 12 healthy subjects was estimated to be 26-95 nmol/kg, and 65-182 nmol/kg in the presence of t-BuOOH. These values are lower than those for urine of rabbit or rat. The TBA reactivity in the absence and presence of t-BuOOH of human urine was not related to age or sex. The TBA reactivity of human urine collected in the afternoon and in the evening was higher than that of urine collected in the morning.

Topics: Animals; Edetic Acid; Female; Ferric Compounds; Humans; Hydrazones; Lipid Peroxidation; Male; Malondialdehyde; Peroxides; Phenylhydrazines; Pigments, Biological; Rabbits; Rats; Rats, Wistar; Reference Standards; tert-Butylhydroperoxide; Thiobarbiturates; Thiobarbituric Acid Reactive Substances; Time Factors; Urine