Soy Isoflavones in Epidemiological Serum Samples: what are the Optimal Time Window and Concentration Cutoffs for Assignment of Equol Producer Status?

Research Article

Austin J Nutri Food Sci. 2014;2(6): 1034.

Soy Isoflavones in Epidemiological Serum Samples: what are the Optimal Time Window and Concentration Cutoffs for Assignment of Equol Producer Status?

Melissa K Melby1* and Shaw Watanabe2

1Departments of Anthropology and Behavioral Health and Nutrition, University of Delaware, USA

2Department of Anthropology, Graduate Academy of Integrative Medicine, Japan

*Corresponding author: :Melissa K Melby, Departments of Anthropology and Behavioral Health and Nutrition, University of Delaware, 46 W. Delaware Ave., Newark, DE, USA

Received: April 29, 2014; Accepted: June 09, 2014; Published: June 13, 2014


Background: The ability to produce equol from daidzein may explain observed inconsistencies in isoflavones' health effects. Reliable identification of Equol Producers (EqPs) in non-clinical studies is limited by poor understanding of pharmacokinetics in producers and background concentrations in Equol Nonproducers (EqNPs).

Objective: To characterize equol pharmacokinetics and identify optimal time windows and concentration cutoffs for assignment of EqP phenotype.

Design: An optimal time window was identified using a pharmacokinetic study (N=10) with sampling every 2-3hrs for 24hrs following ingestion of 4mgdaidzein. An optimal cutoff was conducted through analysis of samples collected at baseline, 3 stages following 1 month intake of: (1) 38.5mg/day, (2) 77.0mg/day; (3) 115.4mg/day daidzein aglycone equivalents; and post onemonth washout period (N=7).

Result: Serum equol peaked between 12-22 hrs following isoflavone intake. A 22nM threshold clearly distinguished between samples collected from EqPs and EqNPs 12-22hours post-ingestion, while ratios of Equol/Daidzein did not. Following high isoflavone intake, no dose response in equol concentrations was observed in EqNPs.

Conclusion: Serum equol concentrations greater than 22nM reliably indicate intestinal microflora ability to metabolize daidzein to Equol (EqP phenotype). Sampling 12-24hr post-ingestion maximizes equol producer status assignment accuracy.

Keywords: Futsal; Nutritional status; Anthropometry


D: Daidzein; G: Genistein; Eq: Equol; EqP: Equol Producer; EqNP: Equol Non-producer.


Research suggests that many health benefits of isoflavones may depend on an individual's ability to metabolize the isoflavone Daidzein (D) into the isoflavan Equol (Eq), because of Eq's greater estrogenic activity and affinity for both estrogen receptors [1,2]. The ability to metabolize Eq from D depends on the presence of certain intestinal microflora [3,4]. People who harbor the necessary microflora to metabolize daidzein into equol are called Equol Producers (EqP) and show measurable concentrations of equol in blood and urine following ingestion of soy or other daidzein-containing foods or supplements, and those who do not are considered Equol Non-Producers (EqNP). The prevalence of equol producers varies from 30%to 59% in human populations, and is higher in Asian and vegetarian populations [5-9].

No published data exist on the average or maximum background serum concentrations of equol in non-producers, or on the pharmacokinetics of equol that would enable optimal sampling in the post-ingestion window when equol peaks above background concentrations in producers. Thus, reliable guidelines for assigning equol producer status based on serum samples, the most routinely collected samples in epidemiological studies, are nonexistent.

Methods for assessing EqP ability generally involve analysis of fecal microflora [3] or asoy or daidzein challenge test followed by 24-hour urine analysis [8]. Although some researchers have found strong correlations between urinary Eq excretion and plasma Eq concentrations [10], others argue that while urinary isoflavone concentrations may reflect average exposure, they correlate poorly with maximal serum concentrations [5], and thus may be of limited utility for assessing systemic bioavailability and cellular and organ exposure. Furthermore, collection of fecal or 24-hour urine samples is rarely feasible in large-scale epidemiological studies examining the effects of soy on cholesterol, heart disease, menopausal symptoms, and osteoporosis. Thus it is important to develop algorithms for reliable assignment of EqP status from serum samples.

Pharmacokinetic studies have observed peaks in blood Genistein (G) and daidzein concentrations in the first 8 hours post-ingestion and reported maximal Eq concentrations at 24 hours post-ingestion [11-13]. However, the report of an Eq maximum at 24 hrs may be an artifact of sampling frequency and schedule. Most studies sampled frequently during the first 12 hours, but subsequently only every 12 hours, presumably because of researchers' and participants' need for sleep. Thus the observed Eq peak at 24 hours could mean that Eq reached maximum concentration anywhere between 12-36 hours. The use of our validated finger-prick dried blood spot method [14] represents a significant advance for pharmacokinetic studies of isoflavones and metabolites, as self-sampling by participants at home enables more frequent collection throughout a 24-hour period.

To identify the optimal time window for assigning EqP status from serum samples collected in epidemiological studies, we carried out pharmacokinetic studies with sampling every 2-3 hours for the 24 hours following isoflavone ingestion. Since background levels of equol may be observed in EqNP due to the presence of equol in cow's milk (metabolized by cows from daidzein in forage legumes), it is important to establish a cutoff for equol in serum above which a person can be reliably considered to be an equol producer. To identify the optimal Eq serum concentration cutoff to reliably assign EqP status (i.e., to distinguish between EqNPs and EqPs), we conducted a study of repeated high dose isoflavone intake. This simulates a longterm daidzein challenge test and permits identification of maximal Eq concentration in EqNPs.

Materials and Methods

Two formulations of purified isoflavones derived from soy hypocotyls were provided by Fuji Oil Company Ltd. (Osaka, Japan), containing a total of 2.08mg and 27.75mg isoflavones per 300mg tablet in a base of lactose (78.5%) and glycerin fatty acid esters (3.0%) in Formulations 1 and 2 respectively (Table 1). The study tablets used here had higher ratios of D:G (approximately 4.8:1 and 2.8:1) than the 1:1.5 ratio typically observed in foods [15] in order to administer maximum D substrate for assessing equol production ability. Study protocols were approved by the Ethical Committee of the Life Science Foundation, and Tokyo University of Agriculture. Informed consent was obtained from all study participants and the procedures followed were in accordance with the Helsinki Declaration of 1975 as revised in 1983.