The metabolism of trans,trans-muconaldehyde (MUC), a hematotoxic agent which is a presumed in vivo metabolite of benzene, was studied in mouse liver cytosol. MUC was incubated for 30 min at 37 degrees C with mouse liver cytosol (from CD-1 mice) supplemented with NAD+ and the products were analyzed by reverse phase HPLC. Two products were detected in addition to the previously identified acid-aldehyde 6-oxo-trans,trans-2,4-hexadienoic acid (COOH-M-CHO) and the diacid trans,trans-muconic acid (COOH-M-COOH). Based on the molecular weight (112) obtained by thermo-spray LC-mass spectrometry and the absorbance maximum (269 nm), one of the products was identified as the aldehyde-alcohol 6-hydroxy-trans,trans-2,4-hexadienal (CHO-M-OH). The second product was identified as 6-hydroxy-trans,trans-2,4-hexadienoic acid (COOH-M-OH) by coelution with authentic standard, the fragmentation pattern obtained by electron impact mass spectrometry and the absorbance maximum (258 nm). Time course and concentration dependency studies indicate that COOH-M-OH and COOH-M-COOH are end products of MUC metabolism while CHO-M-OH, and COOH-M-CHO, the initially formed mono-reduction and mono-oxidation products, respectively, are the intermediates leading to these end products. The metabolite COOH-M-OH is formed mainly by oxidation of CHO-M-OH and to a much lesser extent by reduction of CHO-M-COOH, whereas COOH-M-COOH is formed solely by oxidation of COOH-M-CHO. The reduction of MUC to CHO-M-OH is reversible, whereas oxidation to COOH-M-CHO is not. The compound CHO-M-OH is not only oxidized to COOH-M-OH by oxidation of the aldehyde functional group, but is also converted back to MUC by oxidation of the alcohol functional group.
Muconic acid (MA) is a urinary metabolite of benzene and has been used as a biomarker of exposure to benzene in humans exposed to levels as low as 1 ppm. We have modified a high-pressure liquid chromatography (HPLC) based assay for urinary MA (Ducos et al., 1990) by the use of a diode array detector. This modification increases the specificity of the HPLC-based assay by identifying false positives. In addition, we have developed a gas chromatography (GC) based assay that uses a flame ionization detector (GC-FID). Both assays identified and quantified MA in human urine at concentrations greater than 40-50 ng/ml. Assay precision was within 10% relative standard deviation for MA concentrations above 90 ng/ml using the HPLC assay and above 40 ng/ml using the GC-FID assay. Quantitative accuracy of the assays was evaluated by determining MA in human urine samples using both methods and also a gas chromatography-mass spectrometry (GC-MS) procedure. Numerical correlation among the three assays was good at MA concentrations above 100 ng/ml.
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