Abstract
An isocratic reverse-phase high-performance liquid chromatography method for the separation and quantitation of total pyridine dinucleotides in hepatocyte cultures is described. Cells are extracted with cold 3 m perchloric acid or 0.5 n sodium hydroxide containing 50% ( v v) ethanol and 35% cesium chloride for the determination of the oxidized or reduced pyridine dinucleotides, respectively. Pyridine dinucleotides in the neutralized extracts were separated on an Excellopak ODS C18 (4.6 × 150 mm) column with 0.1 m potassium phosphate, pH 6.0, containing 3.75% methanol as the mobile phase. NAD+ and NADP+ were detected spectrophotometrically at 254 nm. The response was linear from 5 to 4000 pmol with recoveries of NAD+ and NADP+ of 98 and 101.1%, respectively. NADH and NADPH were monitored fluorometrically by activation at 370 nm and emission in the 400-700 nm range. The reduced pyridine dinucleotides had a linear response from 7.5 to 60 pmol with recoveries of NADH and NADPH of 99.4 and 101.3%, respectively. The coefficients of variation for all of the pyridine dinucleotide standards were less than 3.5%.
Original language | English (US) |
---|---|
Pages (from-to) | 34-36 |
Number of pages | 3 |
Journal | Analytical biochemistry |
Volume | 179 |
Issue number | 1 |
DOIs | |
State | Published - May 15 1989 |
ASJC Scopus subject areas
- Biophysics
- Biochemistry
- Molecular Biology
- Cell Biology