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.

Activation of Proestrogens from Hops (Humulus lupulus L.) by Intestinal Microbiota; Conversion of Isoxanthohumol into 8-Prenylnaringenin

“…However, as only one-third of the samples converted IX, interindividual variability in microbial community activity could strongly influence this in vivo conversion. Finally, Schaefer et al (35) noted that urinary 8-PN excretion after beer consumption was slower than expected (up to several days), which could very well be due to delayed conversion of IX into 8-PN in the intestine, followed by absorption and renal excretion. Enterohepatic circulation is another important factor determining the retention of IX and 8-PN.…”

In vivo estrogenic comparisons of Trifolium pratense (red clover) Humulus lupulus (hops), and the pure compounds isoxanthohumol and 8-prenylnaringenin

“…Phase II metabolites of 8-PN, including glucuronides and sulfate conjugates, have been identified in incubations with human Caco-2 cells and human hepatocytes [24] (see structures in Figure 1). Conjugates of 8-PN were also reported in urine of men after consuming beer or beer spiked with 8-PN [25], but the radioimmunoassay used in this study did not allow for the identification of specific conjugates. However, little information is available concerning the formation of metabolites of 8-PN in vivo.…”

Analytical Methods for Quantitation of Prenylated Flavonoids from Hops

“…Defatting the serum with hexane prior to extraction as carried out by Bolca et al may help alleviate this problem [49]. Excellent recoveries (> 90%) from serum, urine and fecal cultures were observed for all prenylflavonoids [22,23,50,51], except in the report by Wyns et al [39], who reported recoveries of 78.7% and 74.1% for 8-PN and XN, respectively. SPE was also used for extraction of IX and 8-PN from urine [21] and serum [39].…”

“…Schaefer et al reported a radioimmunoassay (RIA) that used tritiated 8-PN [22]. The monoclonal antibody against 8-PN employed in the final method showed very little crossreactivity against other prenylated flavonoids.…”

“…Isoxanthohumol (IX), the 5-O-methyl analog of 8-PN, has much weaker estrogenic activity [17]. However, several in vitro and in vivo studies have shown that IX can be metabolically converted into either by the action of cytochrome P450 or by intestinal microflora [20][21][22][23]. Thus, IX can be considered a pro-estrogen, which provides an important rationale for inclusion of this compound in the standardization of various hop extracts.…”