Abstract
3'-Phosphoadenosine 5'-phosphosulfate (PAPS) and UDP-glucuronic acid (UDPGA) in cultured rat hepatocytes were directly assayed by isocratic reverse- phase HPLC. The sample preparation involves the extraction of nucleotide cofactors with cold alkaline solvent and clearing treatment by ultra-filtration. Nucleotides were separated on HPLC equipped with a RPAQUEOUS C-30 column adjusted to 20°C using 100 mM sodium potassium buffer (pH 5.5) as elution solvent and detected by UV absorption at 260 nm. Lin- ear calibration curves were obtained over the ranges from 0.1 to 1.0 M for PAPS and 1.0 to 10 M for UDPGA in both distilled water and hepatic cell ex- tracts. Peaks in the cell extracts were identified as PAPS and UDPGA by comparison of the retention times and co-elution with each of their corresponding authentic standards. Assignment of peak identity was addition- ally supported by the preferential decrease in PAPS and UDPGA in cultured hepatocytes which were in- cubated with quercetin or D-galactosamine. This HPLC method was found to be sensitive enough to accurately quantify the cellular contents of PAPS and UDPGA far below that normally found in cultured rat hepatocytes.
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