Monitoring the effect of triamterene and hydrochlorothiazide on dihydrofolate reductase activity using a new spectrophotometric method

Abstract

A new spectrophotometric method is developed and applied for the study of the inhibitory effect of triamterene, hydrochlorothiazide and their combinations on the in vitro activity of dihydrofolate reductase enzyme. The method is based on incubating the drug (0.1–1.0 μM) or a buffer control with a solution containing reduced nicotinamide adenine dinucleotide phosphate (0.5 mM), magnesium chloride (1.29 mM), and folic acid as a substrate (0.01–0.1 mM) with the dihydrofolate reductase (0.25 unit). The resulting tetrahydrofolic acid is determined by first hydrolysing it by a methanol—hydrochloric acid mixture to produce p-aminobenzoyl glutamic acid, then adding p-dimethylaminocinnamic aldehyde reagent to form a stable pink coloured product. The colour is found to develop within 5 min and is stable over 12 h, with a maximum absorption at 545 nm. A linear calibration curve is formed by using standard solutions of tetrahydrofolic acid. The presence of the studied drugs did not interfere with the determination. Lineweaver—Burk plots of the reaction kinetics, in the presence of triamterene and/or hydrochlorothiazide showed a competitive inhibition of the dihydrofolate reductase in the presence of triamterene with or without hydrochlorothiazide. A 100% inhibition is obtained by 1 μM solution of triamterene at a folic acid concentration of 0.01 mM. No measurable effect of hydrochlorothiazide at the studied concentration range is demonstrated.