Guanosine triphosphate cyclohydrolase I assay in human and rat liver using high-performance liquid chromatography of neopterin phosphates and guanine nucleotides

Abstract

D-erythro-7,8-Dihydroneopterin triphosphate (NH2TP) formed from guanosine triphosphate (GTP) by GTP cyclohydrolase I (EC 3.5.4.16) in the presence of EDTA was oxidized to neopterin triphosphate (NTP) by iodine, separated from substrate and other compounds by ion-paired reverse-phase HPLC, and quantitated by fluorometric detection at 365/446 nm. Excess GTP at the end of reaction was controlled by simultaneous detection of guanine nucleotides at 254 nm. The method required only 15 mg of liver tissue for the measurement of GTP cyclohydrolase I and is suitable for activity measurement in liver biopsies. The detection limit was 4 pmol of NTP at a signal to noise ratio of 10:1. The activity of GTP-cyclohydrolase I in homogenates of human liver (n = 10) was 45 pmol NH2TP (range 32-60) formed per milligram protein per hour at 37 degrees C. Liver homogenates from Wistar rats (n = 8) formed 47 pmol NH2TP (range 35-61) per milligram protein per hour.