生体試料中の4-Nitroquinoline 1-Oxide及びその代謝産物の定量法

Abstract

4-Nitroquinoline 1-oxide (4-NQO), which is a potent carcinogen, is metabolized to 4-hydroxyaminoquinoline 1-oxide (4-HAQO), 4-aminoquinoline 1-oxide (4-AQO), and 4-hydroxyquinoline 1-oxide (4-(OH) QO) by some microbes and mammalian tissues, but the separation and sensitivity in the determination of 4-NQO and its metabolites are not yet adequate. Therefore, an attempt was made to determine 4-NQO and its metabolites in biological materials more sensitively. 4-NQO was extracted from the homogenized tissue with benzene, and absorption of the extract was measured at 392 mμ by a spectrophotometer. After removal of 4-NQO by benzene, the aqueous layer was washed with chloroform. After addition of ascorbic acid and HCl-ethanol, the aqueous layer was evaporated to dryness under a reduced pressure at room temperature. The metabolites in the residue were extracted with warm ethanol-water (1 : 1) solution. An aliquot of the extract was spotted on a silica gel thin-layer plate, and metabolites were separated by using the upper layer of a mixture of sec-butanolethyl acetate-water (2 : 1 : 1) as the developing solvent. Two main bands having blue fluorescence were detected under UV light. One band having Rf 0.60 was identified as 4-HAQO, the other band of Rf 0.45 contained 4-AQO and 4-(OH) QO. The band corresponding to 4-HAQO was extracted with isobutanol-water, while from the other band 4-AQO and 4-(OH) QO were extracted with isoamyl alcohol and Na2CO3 solution, respectively. The fluorescence intensities of 4-HAQO in isobutanol, of 4-AQO in isoamyl alcohol, and of 4-(OH) QO in Na2CO3 solution were measured at 475 mμ, excited by 375 mμ, at 480 mμ excited by 370 mμ, and at 480 mμ excited by 360 mμ, respectively. Each range of determination for 4-NQO, 4-HAQO, 4-AQO, and 4-(OH) QO was 1-25, 0.05-0.2, 0.02-0.5, and 0.02-0.5μg/ml, respectively.