FIG. 2.

MQ-induced changes in NADH/NAD⁺ and NADPH/NADP⁺ contents in HT29 cells under normal glucose conditions. (A) Time course of pyridine nucleotide changes. Cells (5 × 10⁶) were treated with 50 or 200 μM MQ for 30 and 60 min in 3 g/l glucose. At designated times, cell aliquots were removed, total cell lysates were prepared, and cellular contents of NADH plus NAD⁺ and NADPH plus NADP⁺ were determined by enzyme cycling assays. Nucleotide contents were calculated based on reaction velocities of known standards. Results are mean ± SEM for 5 cell preparations. *p < 0.05 versus untreated controls (zero time); ^#p < 0.05 versus 50 μM MQ. (B) Changes in cytosolic (a, b) and mitochondrial (c, d) NADH/NAD⁺ and NADPH/NADP⁺. Cells (5 × 10⁶) were incubated with 50 or 200 μM MQ in 3 g/l (18.3 mM) glucose for 30 min. At the end of the incubation, cytosolic and mitochondrial fractions were prepared by digitonin fractionation as described in the Methods section. KCN (0.2 M) was added to stabilize the pyridine nucleotides as nicotinamide-cyanide derivatives, which were separated by high-performance liquid chromatography. Nucleotide contents were quantified by comparison to authentic standards derivatized in the same manner. Results are expressed as nmol per 5 × 10⁶ cells and are mean ± SEM for four cell preparations. *p < 0.05 versus untreated controls. SEM, standard error of the mean.