Optimized steps in fluorometric determination of thiobarbituric acid-reactive substances in serum: importance of extraction pH and influence of sample preservation and storage.

A simple, reliable, and reproducible fluorometric method for measuring thiobarbituric acid-reactive substances (TBARS) in serum is proposed, based on the reaction between malondialdehyde (MDA) and thiobarbituric acid. Formation of TBARS was complete at pH 2.4-2.6, but extraction with n-butanol proved complete only at lower pH, i.e., 1.6-1.7. Analytical recoveries of MDA added to serum were 94%-101%; within- and between-run CVs were 2.4-3.6% and 4.6-5.5%; and the detection limit for TBARS in serum was 0.10 mumol/L. Optimized conditions included: (a) collection of either serum or heparinized plasma, (b) preservation from in vitro autoxidation by glutathione and EDTA, and (c) storage at -20 degrees C up to 35 days. The mean (+/- SD) TBARS concentration in 47 healthy adults was 1.01 (0.21) mumol/L; no sex-related difference was observed. Higher concentrations were measured in patients with renal insufficiency undergoing hemodialysis and in patients with insulin-dependent diabetes, chronic pancreatitis, or liver cirrhosis.

[1]  E. Bartnikowska,et al.  Lipid peroxides in blood from patients with atherosclerosis of coronary and peripheral arteries. , 1992, Clinica chimica acta; international journal of clinical chemistry.

[2]  F. Muskiet,et al.  Optimized determination of malondialdehyde in plasma lipid extracts using 1,3-diethyl-2-thiobarbituric acid: influence of detection method and relations with lipids and fatty acids in plasma from healthy adults. , 1992, Clinica chimica acta; international journal of clinical chemistry.

[3]  A. Favier,et al.  High-performance liquid chromatographic separation of malondialdehyde-thiobarbituric acid adduct in biological materials (plasma and human cells) using a commercially available reagent. , 1992, Journal of chromatography.

[4]  A. Favier,et al.  Malondialdehyde kit evaluated for determining plasma and lipoprotein fractions that react with thiobarbituric acid. , 1992, Clinical chemistry.

[5]  R. Cohen,et al.  Plasma lipoprotein peroxidation potential: a test to evaluate individual susceptibility to peroxidation. , 1991, Clinical chemistry.

[6]  G. Lepage,et al.  Preparative steps necessary for the accurate measurement of malondialdehyde by high-performance liquid chromatography. , 1991, Analytical biochemistry.

[7]  P. Canioni,et al.  Free and bound malondialdehyde measured as thiobarbituric acid adduct by HPLC in serum and plasma. , 1991, Clinical chemistry.

[8]  P. Morand,et al.  Improved fluorometric determination of malonaldehyde. , 1991, Clinical chemistry.

[9]  B. Halliwell,et al.  The measurement and mechanism of lipid peroxidation in biological systems. , 1990, Trends in biochemical sciences.

[10]  A. Legrand,et al.  Distribution of thiobarbituric acid-reactive substances in lipoproteins and proteins in serum. , 1989, Clinical chemistry.

[11]  J. Knight,et al.  Specificity of the thiobarbituric acid reaction: its use in studies of lipid peroxidation. , 1988, Clinical chemistry.

[12]  T. Hendriks,et al.  On the fluorometric assay of circulating lipoperoxides. , 1988, Clinica chimica acta; international journal of clinical chemistry.

[13]  J. Knight,et al.  Reference intervals for plasma lipoperoxides: age-, sex-, and specimen-related variations. , 1987, Clinical chemistry.

[14]  S. Wong,et al.  Lipoperoxides in plasma as measured by liquid-chromatographic separation of malondialdehyde-thiobarbituric acid adduct. , 1987, Clinical chemistry.

[15]  J. Michalak,et al.  The relationship between plasma triglycerides, cholesterol, total lipids and lipid peroxidation products during human atherosclerosis. , 1986, Clinica chimica acta; international journal of clinical chemistry.

[16]  A. V. Van rij,et al.  Quantitation of malondialdehyde (MDA) in plasma, by ion-pairing reverse phase high performance liquid chromatography. , 1985, Biochemical medicine.

[17]  M. Foglietti,et al.  Plasma malonaldehyde levels during myocardial infarction. , 1983, Clinica chimica acta; international journal of clinical chemistry.

[18]  D. M. Lee.  Malondialdehyde formation in stored plasma. , 1980, Biochemical and biophysical research communications.

[19]  T. Kamada,et al.  Serum lipoperoxide level in patients suffering from liver diseases. , 1977, Clinica chimica acta; international journal of clinical chemistry.

[20]  K. Yagi,et al.  A simple fluorometric assay for lipoperoxide in blood plasma. , 1976, Biochemical medicine.

[21]  P. Odetti,et al.  Malondialdehyde (MDA) level in diabetic subjects. Relationship with blood glucose and glycosylated hemoglobin. , 1991, Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie.

[22]  T. Hendriks,et al.  Spectrophotometric correction for bile pigments in the thiobarbituric acid test for malondialdehyde-like substances in plasma. , 1990, Medical laboratory sciences.

[23]  D. Janero,et al.  Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. , 1990, Free radical biology & medicine.

[24]  T. Bigwood,et al.  Pseudo malonaldehyde activity in the thiobarbituric acid test. , 1989, Free radical research communications.

[25]  S. Jain.  Evidence for membrane lipid peroxidation during the in vivo aging of human erythrocytes. , 1988, Biochimica et biophysica acta.

[26]  V. Walker,et al.  Plasma malondialdehyde: a poor measure of in vivo lipid peroxidation. , 1988, Clinical chemistry.

[27]  B. Goldstein,et al.  The reaction of 2-thiobarbituric acid with biologically active alpha,beta-unsaturated aldehydes. , 1986, Journal of free radicals in biology & medicine.

[28]  J. Gutteridge,et al.  Free-radical damage to lipids, amino acids, carbohydrates and nucleic acids determined by thiobarbituric acid reactivity. , 1982, The International journal of biochemistry.