Development of a radioimmunoassay for the quantitative determination of 8-prenylnaringenin in biological matrices

Abstract

Seven carboxylic acid haptens of 8-prenylnaringenin (8-PN) were synthesized, coupled to cationized bovine serum albumin, and employed to raise specific antisera in rabbits. Two linkers of different lengths (C3H6COOH and C6H12COOH) were coupled to the C7-OH group and separated into their respective enantiomers yielding the first four haptens. Racemic derivatives with C4'-OH coupled linkers C5H10COOH and C9H18COOH were synthesized carrying a methylated C7-OH. Another racemic C4'-OH hapten (CH2COOH) was prepared starting from naringenin. The haptens elicited variable antibody titers dependent on linker lengths, with short linkers giving the best results. Three antisera were characterized in detail: anti-C7-carboxy-propyloxy-2S-(-)-8-PN (anti-H-11), anti-C7-carboxy-propyloxy-2R-(+)-8-PN (anti-H-10), and anti-C4'-carboxy-methoxy-rac-8-PN (anti-H-25). anti-H-10 and anti-H-11 showed about 9% enantiomeric cross-reactivity, and anti-H-11 did not discriminate between isoxanthohumol (IX) and 8-PN (84% cross-reactivity). For anti-H-10, cross-reactivities in the range of 2-5% were found for xanthohumol, IX, and 6-prenylnaringenin. Respective numbers for anti-H-25 were 0.02, 0.1, and 0.2%. Tritiated 8-PN was synthesized yielding a 3H-tracer of high specific radioactivity (2.22 GBq/mg). A radioimmunoassay using anti-H-25 and 3H-8-PN was established and used for the quantitative determination of 8-PN in various beer brands and in the urine of six men after the consumption of three different brands of beer. Furthermore, the dose-dependent excretion of 8-PN was tested after the consumption of a higher volume of a single beer brand with and without spiking with 8-PN and a small oral dose of authentic 8-PN, respectively. Conflicting results led to a pilot test on the in vivo conversion (demethylation) of IX into 8-PN in two men. Conversion rates of 1.9 and 4.4% were estimated. Thus, the total 8-PN dose in beer brands spiced with natural hop or hop products seems to be the sum of the 8-PN amount in a consumed volume and the amount arising from the conversion of IX.