Reducing Agent2 | Control rats1 | DCA-pretreated rats1 | ||
---|---|---|---|---|
(nmol glyoxylate formed/min/mg protein) | ||||
Cytosol Incubated with NADPH3 | Cytosol Incubated with Water3 | Cytosol Incubated with NADPH3 | Cytosol Incubated with Water3 | |
GSH (1 mM) | 1.43 ± 0.134,a | 0.58 ± 0.16b | 0.45 ± 0.10c | 0.09 ± 0.06d |
NADPH (2 mM) | 0.02 ± 0.02e | < 0.015 | < 0.01 | <0.01 |
NADH (2 mM) | 0.14 ± 0.17d | < 0.01 | < 0.01 | <0.01 |
1 Rats were treated by oral gavage with water (controls) or NaDCA, 50 mg/kg, for 2 days before preparation of hepatic cytosol fractions.
2 Assay tubes contained 0.2 mM DCA (0.18 mM 1,2-13C-DCA with 0.02 mM 1-14C-DCA, 55.5 μCi/mmole), 0.5 mg cytosolic protein and GSH, NADPH or NADH at the indicated concentrations in 0.25 ml 0.1 M HEPES-NaOH buffer pH 7.4. After incubation at 37°C for 15 min, the reaction was stopped by addition of methanol, and product formation was measured by HPLC as described in the Methods section.
3 Dialysis conditions: Hepatic cytosol samples from four individual rats for each pretreatment (water or DCA) were dialyzed at 4°C against two changes of 1.15% KCl, 0.05 M potassium phosphate buffer pH 7.4, then incubated at 37°C for 15 min with one-tenth volume of NADPH, final concentration 1 mM, or an equal volume of water. Incubated cytosol fractions were redialyzed at 4°C against two changes of 1.15% KCl/0.05M potassium phosphate pH 7.4 and used in assays.
4 Values shown are mean ± SD,N = 4. Mean values with differing superscripts indicate significant differences between groups, p < 0.001.
5 Not detectable.