Reaction conditions affecting the relationship between thiobarbituric acid reactivity and lipid peroxides in human plasma.

The thiobarbituric acid (TBA) reactivity of human plasma was studied to evaluate its adequacy in quantifying lipid peroxidation as an index of systemic oxidative stress. Two spectrophotometric TBA tests based on the use of either phosphoric acid (pH 2.0, method A) or trichloroacetic plus hydrochloric acid (pH 0.9, method B) were employed with and without sodium sulfate (SS) to inhibit sialic acid (SA) reactivity with TBA. To correct for background absorption, the absorbance values at 572 nm were subtracted from those at 532 nm, which represent the absorption maximum of the TBA:MDA adduct. Method B gave values of TBA-reactive substances (TBARS) 2-fold higher than those detected with method A. SS lowered TBARS by about 50% with both methods, indicating a significant involvement of SA in plasma TBA reactivity. Standard SA, at a physiologically relevant concentration of 1.5 mM, reacted with TBA, creating interference problems, which were substantially eliminated by SS plus correction for background absorbance. When method B was carried out in the lipid and protein fraction of plasma, SS inhibited by 65% TBARS formation only in the latter. Protein TBARS may be largely ascribed to SA-containing glycoproteins and, to a minor extent, protein-bound MDA. Indeed, EDTA did not affect protein TBARS assessed in the presence of SS. TBA reactivity of whole plasma and of its lipid fraction was instead inhibited by EDTA, suggesting that lipoperoxides (and possibly monofunctional lipoperoxidation aldehydes) are involved as MDA precursors in the TBA test. Pretreatment of plasma with KI, a specific reductant of hydroperoxides, decreased TBARS by about 27%. Moreover, aspirin administration to humans to inhibit prostaglandin endoperoxide generation reduced plasma TBARS by 40%. In conclusion, reaction conditions affect the relationship between TBA reactivity and lipid peroxidation in human plasma. After correction for the interfering effects of SA in the TBA test, 40% of plasma TBARS appears related to in vivo generated prostaglandin endoperoxides and only about 60% to lipoperoxidation products. Thus, the TBA test is not totally specific to oxidant-driven lipid peroxidation in human plasma.     

High-performance liquid chromatographic determination of iothalamic acid in human plasma and urine

A simple, rapid and sensitive method for the determination of iothalamic acid (IA) in both plasma and urine is reported. After extraction with ethyl acetate, IA was determined by strong anion-exchange high-performance liquid chromatography with ultraviolet detection at 254 nm. The lower limit of detection was 0.5 μg/ml. The average recovery was 73 and 57% from plasma and urine, respectively. Linearity was found over the investigated concentration range (up to 500 μg/ml for plasma and up to 10.0 mg/ml for urine). The reproducibility of the technique was good (coefficient of variation less than 6%) as was the precision and accuracy (coefficient of variation less than 2.5%). No interference from endogenous substances or any of the common drugs tested was found.     

The determination of allopurinol and oxipurinol in human plasma and urine

A method is described for allopurinol and oxipurinol assay within human plasma and urine in the range expected during therapy. The method is based on high-performance ion-exchange chromatography following an efficient sample purification step using Chelex-100 resin in the Cu²⁺ form. Linear calibration curves are produced for allopurinol over the range 0.05–10 μmole/1 (0.068–1.36 μg/ml) in plasma and 0.005–1 mmole/1 (0.68–136 μg/ml) in urine and for oxipurinol 0.5–100 μmole/1 (0.076–15.2μg/ml) in plasma and 0.1–2 mmole/1 (15.2–304 μg/ml) in urine.     

Gas chromatographic determination of low concentrations of benzoic acid in human plasma and urine

A method for the determination of benzoic acid down to concentrations of 10 ng/ml in plasma or urine is described. After addition of an internal standard, benzoic acid is extracted at acid pH into diethyl ether. Both compounds are derivatized with pentafluorobenzyl bromide. The derivatives are determined by gas chromatography using a ⁴³Ni electron-capture detector. Hippuric acid is hydrolysed in plasma and urine and total benzoic acid is determined by the same technique.     

Interpretation of the thiobarbituric acid reactivity of rat liver and brain homogenates in the presence of ferric ion and ethylenediaminetetraacetic acid.

The thiobarbituric acid (TBA) reactivity of rat liver and brain homogenates was characterized to elucidate what kinds of aldehyde species contributed to the reactivity. Characteristic pH dependence of the reactivity with a maximum at around pH 3 and marked enhancement of the reactivity by t-butyl hydroperoxide (t-BuOOH) and ferric ion were similar to those of alkadienals. The amounts of aldehyde species, including alkadienals determined as 2,4-dinitrophenylhydrazones, were high enough to account for the enhanced reactivity. The reactivity was inhibited by ethylenediaminetetraacetic acid (EDTA) but not completely, suggesting the presence of malonaldehyde whose reactivity was not affected by EDTA. The amounts of malonaldehyde determined as 1-(2,4-dinitrophenyl)pyrazole could account for a part of the reactivity in the presence of EDTA. Hence, the TBA reactivity of liver and brain homogenates at around pH 3 in the presence of t-BuOOH and ferric ion may be accounted for by alkadienals and malonaldehyde and that in the presence of EDTA by malonaldehyde.     

Plasma thiobarbituric acid reactivity: reaction conditions and the role of iron, antioxidants and lipid peroxy radicals on the quantitation of plasma lipid peroxides

The effects of Fe3+, lipid peroxy radicals and the antioxidant butylated hydroxytoluene on the 2-thiobarbituric (TBA) acid quantitation of plasma lipid peroxides were investigated. Whole plasma and plasma fractions prepared by trichloroacetic acid (TCA) protein precipitation and lipid extraction, demonstrated markedly differing TBA reactivities in the presence or absence of added Fe3+. Examination of the spectral profiles of the TBA reacted whole plasma and TCA precipitated fractions demonstrated the presence of interfering compounds which gave rise to an artifactual increase in lipid peroxide concentrations. In contrast the TBA reacted lipid extracts had low levels of interfering compounds that could be removed by our previously described high pressure liquid chromatographic method (Wade, Jackson and van Rij (1985) Biochem. Med. 33, 291-296). Further characterization of the TBA reactivity of the lipid extract showed that Fe3+ at an optimal concentration of 0.5 mM was necessary for the quantitative decomposition of the lipid peroxides to the TBA reactive product malondialdehyde (MDA). However the presence of Fe3+ resulted in further peroxidation of any unsaturated lipids present. Butylated hydroxytoluene (BHT) at an optimal concentration of 1.4 mM inhibited Fe3+ stimulated peroxidation without affecting the formation of the MDA-TBA chromogen. Using a standardized TBA test with plasma lipid extracts and the addition of optimal concentrations of Fe3+ and BHT, we have determined the mean concentration of lipid peroxides in 30 healthy human subjects to be 102.7 +/- 20.0 ngm/ml.     

Measurement of epsilon-N-trimethyllysine in human blood plasma and urine

A method for measurement of epsilon-N-trimethyllysine in human blood plasma and urine is described. An internal standard, delta-N-trimethylornithine, was added to plasma and urine specimens and the mixtures were deproteinized and/or hydrolyzed. Preliminary purification of epsilon-N-trimethyllysine and delta-N-trimethylornithine was achieved by sequential cation-exchange--anion-exchange chromatography. Amino acids in the column eluates were derivatized with o-phthalaldehyde and mercaptoethanol, and were separated by isocratic reversed-phase high-performance liquid chromatography in the presence of an ion-pairing reagent. Quantitation was achieved by post-column fluorometry. The limit of detection was 5 pmol of epsilon-N-trimethyllysine injected into the chromatograph. The procedure was suitable for determination of epsilon-N-trimethyllysine in 1 ml of plasma or 0.2-0.4 ml of urine. The method was applied to measurements of epsilon-N-trimethyllysine in plasma and urine of four systemic carnitine deficiency patients and six normal subjects. Plasma epsilon-N-trimethyllysine concentration was significantly lower in systemic carnitine deficiency patients compared to normal individuals, but no significant difference in urinary epsilon-N-trimethyllysine excretion was observed between the two groups.     

Radioimmunoassay of 6beta-hydroxycortisol in human plasma and urine

A sensitive and reliable radioimmunoassay for urine and plasma 6β-hydroxycortisol has been developed. Antiserum showing high specificity against 6β-hydroxycortisol was produced in rabbits immunized with 6β-hydroxycortisol 21-hemisuccinate-bovine serum albumin. The sensitivity of the assay was 25 pg on a diluted sample equivalent to 1 μl of urine, and on 50 μl of plasma after separation by celite chromatography. The intra- and inter-assay coefficients of variation for urine were 4.8 and 6.7% and those for plasma were 4.2 and 12.1%. Concentrations were determined in patients with bronchogenic carcinoma, in patients treated with dilantin, in neonates, and in infants aged 5–12 months.     

alpha-Hydroxy-beta-keto-gamma-aminobutyric acid in human urine.

A new amino acid has been isolated from the normal human urine. The chemical structure of the amino acid was determined to be alpha-hydroxy-beta-keto-gamma-aminobutyric acid based on its physical properties involving NMR, infrared and mass spectra, as well as chemical degradation and synthesis. In six healthy adults the urinary contents of the new amino acid were 3.2--4.5 mumol/24 h.     

Identification of delta-guanidinovaleric acid in human urine

delta-Guanidinovaleric acid (DGVA) was identified in human urine using thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS). In the TLC, all Rfs of sample from urine developed by 6 solvent systems were identical to that of authentic DGVA. In the GC/MS, the mass spectrum of the sample was identical to the trifluoroacetylated dimethylpyrimidyl derivative of DGVA butylester (M+ = 375). In the HPLC analysis, the DGVA peak was observed just before 15 min in either chromatogram obtained by analysis of human urine or authentic DGVA, and the content of DGVA in pooled human urine was calculated at 2.4 nmol/ml.     

Isolation and characterisation of three fucosyloligosaccharide-1-phosphates from normal human urine

Three phosphate-containing fucosyloligosaccharides were isolated from normal human urine using charcoal adsorption, gel-filtration, ion-exchange chromatography and paper chromatography. Chemical investigations and 400 MHz¹H-NMR spectroscopy analyses led to the following structures: Their oligosaccharide chains are identical with, or similar to the fucosyloligosaccharides and urinary compounds synthesized by the stepwise transfer ofN-acetylglucosamine, galactose, fucose andN-acetylgalactosamine to free galactose or glucose residues. A transfer reaction of monosaccharides to free hexose-1-phosphates or glycosylnucleotides is proposed for explaining the origin of these sugar-phosphates.