2003 Volume 49 Issue 5 Pages 395-400
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. Linear calibration curves were obtained over the ranges from 0.1 to 1.0 μM for PAPS and 1.0 to 10 μM for UDPGA in both distilled water and hepatic cell extracts. 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 additionally supported by the preferential decrease in PAPS and UDPGA in cultured hepatocytes which were incubated 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.
