Quantitative analysis of the principle soy isoflavones genistein, daidzein and glycitein, and their primary conjugated metabolites in human plasma and urine using reversed-phase high-performance liquid chromatography with ultraviolet detection

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Abstract

Soy isoflavones are becoming of increasing interest as nutritional agents which can be used to combat osteoporosis and hyperlipidemia, and are also being considered as potential cancer chemopreventive compounds. However, prior to their formulation and distribution as therapeutic agents, thorough pharmacokinetic and toxicological assessment needs to be completed in men and women in a variety of health conditions in order to ensure their therapeutic efficacy and safety. At this time, studies of purified soy isoflavones are possible, and are being designed to fully evaluate the pharmacological utility of these preparations. In support of these studies, quantitative analysis of soy isoflavones in biological fluids can be accomplished with a wide variety of methods and analytical instrumentation. However, the relatively ubiquitous presence of high-performance liquid chromatography with ultraviolet detection (HPLC–UV) in most analytical laboratories, the relative ease of its operation, and the lesser expense of this instrumentation as compared to more sophisticated techniques such as liquid chromatography–mass spectrometry, offers some distinct advantages for its use in pharmacokinetic studies. In this manuscript, the development and validation of an HPLC–UV method for the quantitation of the principal soy isoflavones, genistein, daidzein, and glycitein, and their primary metabolites, in human plasma and urine is described. This analytical approach allows for pharmacologically relevant concentrations of the analytes and their principle metabolites to be detected, and has been validated in close agreement with the US Food and Drug Administration’s guidelines for the validation of methods to be used in support of pharmacokinetic studies.

Introduction

Genistein (GEN), daidzein (DDZ), and glycitein (GCT), the principal soy isoflavones (Fig. 1), are being considered as potential chemopreventive agents for breast and prostate cancers [1]. In addition to their putative anticarcinogenic effects, soy isoflavones have also been investigated as an antihyperlipidemic agent (reduces the level of low-density lipoprotein [LDL] cholesterol) and as a therapeutic substance to combat osteoporosis. Because of these putative therapeutic and preventative pharmacological properties, consumption of soy or soy isoflavone preparations is increasing, particularly in Western countries where dietary intake of soy-based food is typically 20–50 times less per capita than that of Asians [2], [3].

In general, isoflavones are efficiently absorbed from the gut. However, genistein can be glucuronidated in human colon microsomes (UGT 1A10 isoform), suggesting a role for glucuronidation of genistein in the intestine concomitant with absorption [4]. Once absorbed, genistein, daidzein, and glycitein are subject to hepatic glucuronidation at the 7 or 4′-positions, and are also substrates for sulfotransferases. Kidney microsomes are also capable of glucuronidating isoflavones once in circulation [4]. The glucuronides are excreted in the bile where they can be reabsorbed, either before [5] or after cleavage by bacterial gluronidases. The biological activity of the conjugated forms is not well known, but they have been reported to have some therapeutic effects at nutritionally or pharmacologically relevant concentrations, including weak estrogenic effects and activation of natural killer cells [6], [7]. Because of their extensive glucuronidation and sulfate conjugation, it is difficult to determine concentrations of free isoflavones; instead, concentrations of total isoflavones or free plus sulfate conjugates in blood and biological matrices are often reported. The low level of free isoflavones, and the formation of biologically active conjugates, has led to experimental difficulty in establishing a relationship between dose and pharmacological effect, identifying the concentration-dependent range of the effect, and identifying doses that produce toxic responses in humans.

A wide variety of analytical techniques have been applied to the quantitation of soy isoflavones in foods and biological fluids, including high-performance liquid chromatography (HPLC) [4], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], gas chromatography (GC) [7], [26], [27], [28], [29], [30], [31], [32], [33], [34], capillary electrophoresis (CE) [35], [36], time-resolved fluoroimmunoassay (TR-FIA) [37], [38], enzyme-based immunoassays, e.g., enzyme-linked immunosorbent assay (ELISA) [39], [40], radioimmunoassay (RIA) [41], [42], [43], and other analytical methodologies [44], [45]. However, methodological improvements for the quantitative analyses of the free plus conjugated forms of genistein, daidzein, and glycitein in human urine and plasma continue to be needed for clinical trials aimed at defining single and multiple-dose safety, pharmacokinetic, and efficacy profiles. This is particularly important because many of the existing analytical techniques have limited validation data, and therefore their ability to meet the US Food and Drug Administration’s (FDA) draft guidelines on the validation of analytical procedures for pharmaceuticals is (in most instances) unknown. In addition, few relatively simple and robust methods have been described for the quantitation of all three of the principle isoflavones in their free as well as conjugated forms, in both plasma and urine. This manuscript reports the development of validated analytical methods using HPLC with UV detection which enable the measurement of (1) the free, non-conjugated molecules, (2) the combined “free plus the sulfate-conjugated molecules”, or “free plus sulfate fraction”, and (3) the total conjugated and free molecules in plasma and urine.

Section snippets

Materials

Genistein and daidzein were obtained from Indofine (Somerville, NJ, USA). Glycitein was obtained from Ralston Analytical Labs. (St. Louis, MO, USA) and from Indofine. Formic acid (96%, ACS reagent grade), 4-hydroxybenzophenone (98%), ammonium formate (97%), ammonium acetate, and dimethylformamide (99.8%, ACS reagent grade) were obtained from Aldrich (Milwaukee, WI, USA). Acetic acid (glacial, ACS certified) was obtained from Fisher Scientific (Pittsburgh, PA, USA). Ascorbic acid (crystalline

Recovery studies

The extraction recovery (Table 1) of isoflavones in urine was quite high, with genistein tending to have the lowest recovery (ranging from 90 to 94%). Daidzein, glycitein, and the internal standard appeared to be completely extracted using this procedure. In contrast to the extraction procedure for urine, plasma recovery of all three analytes and the internal standard were considerably lower, ranging between ca. 40 and 60%. The variability of the extraction recovery was greater in the plasma

Discussion

The relatively high extraction recovery of the analytes in urine is consistent with the results obtained by other investigators using a similar method [22] and is comparable to that obtained with alternative procedures including column chromatography [17], [46], [47]. However, our extraction recovery from plasma was considerably lower than that reported by Supko and Phillips [22]. Our average plasma extraction recovery was approximately 50%, whereas Supko and Phillips [22] reported over 90%

Acknowledgements

This work was funded by a contract from The National Cancer Institute (N65117 to S.Z.), with additional assistance from the UNC Clinical Nutrition Research Center (DK56350).

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