Gait kinematics and kinetics in patients with chronic ankle instability and healthy controls: A statistical parametric mapping analysis.

BACKGROUND: Chronic ankle instability (CAI) is associated with changes in gait biomechanics which may be related to chronic dysfunction. Traditional statistical models may be limited in their ability to assess the complex 3D movement of the lower extremity during gait. Multivariate analysis of the lower extremity kinematics may reveal unique biomechanical differences associated with CAI. RESEARCH QUESTION: Do patients with CAI differ from healthy controls in their lower extremity biomechanics and GRF when comparing 3D biomechanics? METHODS: Thirty-nine young, active adults participated in this study. Data was collected using a 3D motion analysis system while patients walked and jogged. Statistical parametric mapping (SPM) was used to explore 3D GRF, kinematics and kinetics of the of the lower extremity of CAI and healthy patients. RESULTS: During walking, patients with CAI had greater inversion from 68-100% of gait cycle (p < 0.001, mean difference=3.2°). During jogging, patients with CAI had greater inversion from 20-92% (p < 0.001, mean difference=4.6°). Greater plantar flexion moments were found from 65-71% (p = 0.05, mean difference=347.4Nm/kg) and greater eversion moments were found from 95-100% (p = 0.03, mean difference=74.6Nm/kg) in the CAI group. No differences in GRF were found. SIGNIFICANCE: Greater inversion may present a potentially injurious position. A faulty position of the rearfoot may require greater muscle function in order to correct the position of the joint resulting in greater eversion moments at the ankle. However, this kinetic change does not appear to correct the ankle position.

Analysis of historical negative control group data from the rat in vivo micronucleus assay.

A database of micronuclei counts for historical negative control data from rat in vivo micronuclei tests performed in 10 different laboratories was established. Data were available from over 4000 negative control rats from 10 laboratories. The mean frequency of micronucleated cells (MN)/1000 cells ranged from 0.44 to 2.22, a 5-fold range. Overall there were no major sex or strain differences in frequency, although there were some small but statistically significant differences within laboratories. There was appreciable variability between experiments compared with variability within experiments in some laboratories. No specific factor was identified which could explain this variability although it was noted that many different vehicles were used in the experiments. It is hoped that these data will help laboratories beginning studies with the rat micronucleus assay and those involved in the assessment of micronucleus assay results.

Potential impurities in drug substances: Compound-specific toxicology limits for 20 synthetic reagents and by-products, and a class-specific toxicology limit for alkyl bromides.

This paper provides compound-specific toxicology limits for 20 widely used synthetic reagents and common by-products that are potential impurities in drug substances. In addition, a 15 µg/day class-specific limit was developed for monofunctional alkyl bromides, aligning this with the class-specific limit previously defined for monofunctional alkyl chlorides. Both the compound- and class-specific toxicology limits assume a lifetime chronic exposure for the general population (including sensitive subpopulations) by all routes of exposure for pharmaceuticals. Inhalation-specific toxicology limits were also derived for acrolein, formaldehyde, and methyl bromide because of their localized toxicity via that route. Mode of action was an important consideration for a compound-specific toxicology limit. Acceptable intake (AI) calculations for certain mutagenic carcinogens assumed a linear dose-response for tumor induction, and permissible daily exposure (PDE) determination assumed a non-linear dose-response. Several compounds evaluated have been previously incorrectly assumed to be mutagenic, or to be mutagenic carcinogens, but the evidence reported here for such compounds indicates a lack of mutagenicity, and a non-mutagenic mode of action for tumor induction. For non-mutagens with insufficient data to develop a toxicology limit, the ICH Q3A qualification thresholds are recommended. The compound- and class-specific toxicology limits described here may be adjusted for an individual drug substance based on treatment duration, dosing schedule, severity of the disease and therapeutic indication.

Novel statistical approach for evaluating flow cytometric in vitro micronucleus data.

Statistical methods currently recommended for analysis of in vitro micronucleus data are based on small sample sizes. The tests are designed to evaluate linear trends and differences between treated and control samples. When using flow cytometric analysis, >5 times the number of cells are easily evaluated, and the variance estimates from these large samples are small. Application of these recommended tests to large samples resulted in statistically significant outcomes which were not considered to be biologically meaningful. Alternative statistical methods for testing trends and differences among treatments that were either widely used, or sample-size independent, were investigated. Using data from 95 experiments (from 2011-2013) where 19% of the experiments were considered positive, results for the various statistical methods were compared. When using either the recommended or alternate methods, 42-68% of the experiments resulted in statistically significant results (p < 0.05). A new concept was then tested using the same data sets: the "z' factor", designed to identify 'hits' during high throughput screening. Using this simple-to-compute statistic the number of significant calls was reduced to 27%. Then, when combined with a biological criterion based on historical vehicle control data, there was restoration of the original positive frequency (19%). Given the larger sample sizes evaluated using flow cytometry, we have demonstrated that traditional statistical tests may be overly sensitive to small changes in micronucleus induction, and that a simple-to-compute index of separation (z') may be a better tool for analysis, provided that the response is first determined to be biologically meaningful. Environ. Mol. Mutagen. 57:589-604, 2016. © 2016 Wiley Periodicals, Inc.

Coenzyme Q10, carotenoid, tocopherol, and retinol levels in cord plasma from multiethnic subjects in Hawaii.

Coenzyme Q10 (Q10), carotenoids, tocopherols, and retinol are the major circulating lipid-phase micronutrients (LPM) known to help mitigate oxidative damage and prevent chronic diseases. However, the functions of these compounds in newborns are little understood. This is due, in part, to the paucity of studies reporting their concentrations in this population. We measured Q10, carotenoids, tocopherols, and retinol in cord plasma from 100 multiethnic subjects living in Hawaii using HPLC with diode array and electrochemical detection. Appropriate internal standards were used including, for the first time, custom designed oxidized (UN10) and reduced (UL10) Q10 analogues. These compounds reflected the oxidation of UL10 to UN10 that occurred during sample processing and analysis and thus permitted accurate adjustments of natively circulating Q10 levels. All LPM measured were much lower in cord than in peripheral plasma. Cord plasma levels of total carotenoids, tocopherols, and retinol were approximately 10-fold, 3- to 5-fold and 1.5- to 3-fold lower than those in children or women. Cord plasma levels of total Q10 (TQ10; median, 113 ng/mL) were approximately 2-fold or 7- to 9-fold lower than peripheral plasma levels of neonates or children and adults, respectively. In contrast, the UN10/TQ10 ratio was substantially higher in cord (24%) than in peripheral plasma of children (3-4%) or adults (9%). Among the 5 ethnic groups in our cohort, no differences were observed in the levels of UN10, UL10, or TQ10. However, significant differences in many of the LPM were observed between ethnicities. More research is needed to explain these phenomena.

Bacterial mutagenicity screening in the pharmaceutical industry.

Genetic toxicity testing is used as an early surrogate for carcinogenicity testing. Genetic toxicity testing is also required by regulatory agencies to be conducted prior to initiation of first in human clinical trials and subsequent marketing for most small molecule pharmaceutical compounds. To reduce the chances of advancing mutagenic pharmaceutical candidates through the drug discovery and development processes, companies have focused on developing testing strategies to maximize hazard identification while minimizing resource expenditure due to late stage attrition. With a large number of testing options, consensus has not been reached on the best mutagenicity platform to use or on the best time to use a specific test to aid in the selection of drug candidates for development. Most companies use a process in which compounds are initially screened for mutagenicity early in drug development using tests that require only a few milligrams of compound and then follow those studies up with a more robust mutagenicity test prior to selecting a compound for full development. This review summarizes the current applications of bacterial mutagenicity assays utilized by pharmaceutical companies in early and late discovery programs. The initial impetus for this review was derived from a workshop on bacterial mutagenicity screening in the pharmaceutical industry presented at the 40th Annual Environmental Mutagen Society Meeting held in St. Louis, MO in October, 2009. However, included in this review are succinct summaries of use and interpretation of genetic toxicity assays, several mutagenicity assays that were not presented at the meeting, and updates to testing strategies resulting in current state-of the art description of best practices. In addition, here we discuss the advantages and liabilities of many broadly used mutagenicity screening platforms and strategies used by pharmaceutical companies. The sensitivity and specificity of these early mutagenicity screening assays using proprietary compounds and their concordance (predictivity) with the regulatory bacterial mutation test are discussed.

Urinary estrogen metabolites in two soy trials with premenopausal women.

BACKGROUND: Soy consumption may protect against breast cancer through modification of estrogen metabolism. OBJECTIVES: We examined the effect of soy foods on urinary estrogens and the 2-hydroxy (OH)/16α-OH estrone (E(1)) ratio in two dietary interventions with premenopausal women. SUBJECTS/METHODS: The Breast, Estrogens, And Nutrition (BEAN1) study was a 2-year randomized trial and BEAN2 a 13-month randomized crossover study. In both interventions, study participants consumed a high-soy diet with 2 soy food servings/day and a low-soy diet with <3 servings of soy/week. Urine samples were collected at baseline and at the end of the diet periods, analyzed for nine estrogen metabolites by liquid chromatography mass spectrometry, and adjusted for creatinine levels. For BEAN1, two samples for 188 participants and for BEAN2, three samples for 79 women were analyzed. We applied mixed-effects regression models with log-transformed values of estrogen metabolites and soy intake as the exposure variable. RESULTS: In BEAN1, no effect of the high-soy diet on individual estrogen metabolites or hydroxylation pathways was observed. The median 2-OH/16α-OHE(1) ratio decreased non-significantly in the intervention group from 6.2 to 5.2 as compared with 6.8 and 7.2 in the control group (P=0.63). In BEAN2, only 4-OHE(1) was significantly lower after the high-soy diet. Interaction terms of the high-soy diet with equol producer status, ethnicity and weight status revealed no significant effect modification. CONCLUSIONS: Contrary to our hypothesis and some previous reports, the results from two well-controlled dietary interventions do not support an effect of a high-soy diet on a panel of urinary estrogen metabolites and the 2-OH/16α-OHE(1) ratio.

The role of genetic toxicology in drug discovery and optimization.

Genetic toxicology data is used as a surrogate for long-term carcinogenicity data during early drug development. The aim of genotoxicity testing is to identify potentially hazardous drug candidates. Results from genetic toxicology tests in combination with acute and subchronic animal data are used as the basis to approve clinical trials of drug candidates. With few exceptions, mutagenic compounds are dropped from development and clastogenic compounds result in unfavorable labeling, require disclosure in clinical trial consent forms, and can impact the marketability of a new drug. Therefore, genetic toxicology testing in drug discovery and optimization serves to quickly identify mutagens and remove them from development. Additionally, clastogenicity can delay drug development by requiring additional testing to determine in vivo relevance of in vitro clastogenic responses. Clastogenicity screening is conducted so any additional testing can be planned and perhaps integrated into other toxicity studies to expedite progression of drugs into the clinic. Commercially available genotoxicity and carcinogenicity predictive software systems used for decision support by ICSAS, FDA/CDER is described along with the strengths and weakness of each system. The FDA has concentrated on using a consensus approach to maximize certainty for positive predictions at the expense of sensitivity. The consensus approach consists of requiring 2 complementary software packages, such as MC4PC and MDL QSAR models, to agree that a compound has a genotoxic or carcinogenic liability. Mutagenicity and clastogenicity screening tests are described along with advantages and disadvantages of each test. Several testing strategies are presented for consideration.

Combined effects of well-done red meat, smoking, and rapid N-acetyltransferase 2 and CYP1A2 phenotypes in increasing colorectal cancer risk.

Heterocyclic amines (HAAs) are suspected carcinogens that are formed in meat when it is cooked at high temperature for long durations. These compounds require metabolic activation by CYP1A2 and N-acetyltransferase (NAT) 2 or NAT1 before they can bind to DNA. It has been hypothesized that well-done meat increases the risk of colorectal cancer (CRC), especially in individuals with the rapid phenotype for CYP1A2 and NAT2. This association may be particularly strong in smokers because smoking is known to induce CYP1A2. We conducted a population-based case-control study on Oahu, Hawaii to specifically test this hypothesis. An in-person interview assessed the diet and preference for well-done red meat of 349 patients with CRC and 467 population controls. A urine collection after caffeine challenge and a blood collection were used to assess phenotype for CYP1A2 and NAT2 and genotype for NAT2 and NAT1, respectively. No statistically significant main effect association with CRC was found for red meat intake, preference for well-done red meat, the NAT2 rapid genotype, the CYP1A2 rapid phenotype or the NAT1*10 allele. However, in ever-smokers, preference for well-done red meat was associated with an 8.8-fold increased risk of CRC (95% confidence interval, 1.7-44.9) among subjects with the NAT2 and CYP1A2 rapid phenotypes, compared with smokers with low NAT2 and CYP1A2 activities who preferred their red meat rare or medium. No similar association was found in never-smokers, and there was no increased risk for well-done meat among smokers with a rapid phenotype for only one of these enzymes or for smokers with both rapid phenotypes who did not prefer their red meat well-done. These data provide additional support to the hypothesis that exposure to carcinogens (presumably HAAs) through consumption of well-done meat increases the risk of CRC, particularly in individuals who are genetically susceptible (as determined by a rapid phenotype for both NAT2 and CYP1A2) and suggest that smoking, by inducing CYP1A2, facilitates this effect.

The syrian hamster embryo (SHE) cell transformation assay: review of the methods and results.

The Syrian hamster embryo (SHE) cell-transformation assay represents a short-term in vitro assay capable of predicting rodent carcinogenicity of chemicals with a high degree of concordance (LeBoeuf et al [1996]. Mutat Res 356: 85-127). The SHE assay models the earliest identifiable stage in carcinogenicity, morphological cell transformation. In contrast to other short-term in vitro assays, both genotoxic and epigenetic carcinogens are detected. The SHE assay, originally developed by Berwald and Sachs (J Natl Cancer Inst 35: 641-661) and modified as described by LeBoeuf and Kerckaert (Carcinogenesis 7: 1431-1440), was included in the International Life Sciences Institute, Health and Environmental Sciences Institute (ILSI/HESI). Alternative Carcinogenicity Testing (ACT) collaboration to provide additional information on the use of short-term in vitro tests in predicting carcinogenic potential. A total of 19 ILSI compounds have been tested in the SHE assay: 15 were tested for this project, whereas clofibrate, methapyrilene, reserpine, and Di(2-ethylhexyl)phalate (DEHP) were tested previously. Of the 3 noncarcinogenic compounds tested, 2 were negative in the SHE assay, whereas ampicillin was tested positive. The remaining 16 compounds tested were either known rodent carcinogens and/or human carcinogens. From this group, 15 tested positive in the SHE assay whereas phenacetin, a genotoxic carcinogen, was tested negative. Therefore, overall concordance between the SHE assay and rodent bioassay was 89% (17/19), whereas concordance with known or predicted human carcinogens was 37% (7/19). Based on these data, it is concluded that the SHE cell-transformation assay has utility for predicting the results of the rodent carcinogenesis bioassay but lacks the selectivity to distinguish between rodent and human carcinogens.

Effects of dietary sesame seeds on plasma tocopherol levels.

The tocopherols, the major vitamers of vitamin E, are believed to play a role in the prevention of human aging-related diseases such as cancer and heart disease, yet little is known concerning determinants of their plasma concentrations. Evidence from animal studies suggests that the dietary source of gamma-tocopherol can significantly affect plasma levels of this tocopherol as well as its functional vitamin E activity. To determine whether plasma levels of tocopherols in humans are similarly altered, a study was undertaken in which subjects (n = 9) were fed muffins containing equivalent amounts of gamma-tocopherol from sesame seeds, walnuts, or soy oil. We observed that consumption of as little as 5 mg of gamma-tocopherol per day over a three-day period from sesame seeds, but not from walnuts or soy oil, significantly elevated serum gamma-tocopherol levels (19.1% increase, p = 0.03) and depressed plasma beta-tocopherol (34% decrease, p = 0.01). No significant changes in baseline or postintervention plasma levels of cholesterol, triglycerides, or carotenoids were seen for any of the intervention groups. All subjects consuming sesame seed-containing muffins had detectable levels of the sesame lignan sesamolin in their plasma. Consumption of moderate amounts of sesame seeds appears to significantly increase plasma gamma-tocopherol and alter plasma tocopherol ratios in humans and is consistent with the effects of dietary sesame seeds observed in rats leading to elevated plasma gamma-tocopherol and enhanced vitamin E bioactivity.

Evaluation of objects and food for environmental enrichment of NZW rabbits.

The Guide for the Care and Use of Laboratory Animals states that both structural and social environments should be considered when addressing the husbandry needs of laboratory animals. The purpose of this study was to investigate environmental enrichment strategies that could potentially enhance the well-being of rabbits. Male and female 6-week old New Zealand White rabbits were divided into three groups: food-enriched (Bunny Stix, Bunny Blocks, or celery), non-food enriched (Jingle Ball, Kong toy, or Nylabone), and not enriched. Animals were given a particular enrichment for 1 h daily for 15 days. Home cages were fitted with specially designed plexiglass doors, which allowed the animals' interactions with the objects to be videotaped. The amount of time the animal interacted with each object and the total activity during the 1-h taped session were recorded for each rabbit. Rabbits were weighed weekly. Rabbits spent significantly more time interacting with the Bunny Stix than any other food item or non-food object. In addition, total activity time was significantly greater for all rabbits enriched with food versus any of the non-food items. Weight gains after 15 days did not differ significantly, but there was a trend towards increased weight gains in food-enriched rabbits. In this study, food was a stronger, more sustained enrichment device than were non-food objects.

A refined protocol for conducting the low pH 6.7 Syrian hamster embryo (SHE) cell transformation assay.

The Syrian hamster embryo (SHE) cell transformation assay evaluates the potential of chemicals to induce morphological transformation in karyotypically normal primary cells. Induction of transformation has been shown to correlate well with the carcinogenicity of many compounds in the rodent bioassay. Historically the assay has not received wide-spread use due to technical difficulty. An improved protocol for a low pH 6.7 assay was developed by LeBoeuf et al. [R.A. LeBoeuf, G.A. Kerckaert, M.J. Aardema, D.P. Gibson, R. Brauninger, R.J. Isfort, Mutat. Res., 356 (1996) 85-127], that greatly reduced many of the technical difficulties associated with the SHE assay. The purpose of this paper is to describe the most current execution of the pH 6.70 protocol including protocol refinements made since the publication of a comprehensive protocol for this assay in Kerckaert et al. [G.A. Kerckaert, R.J. Isfort, G.J. Carr, M.J. Aardema, Mutat. Res., 356 (1996) 65-84].

Comparison of analytical methods for quantitation of isolated porcine hepatocyte yields.

As cell-based therapies receive approval for clinical evaluation and use, the development of reliable methods to quantify cell number and control the dose of therapy delivered is becoming increasingly important. An example is the determination of the number and volume of primary porcine hepatocytes used in an extracorporeal treatment for patients with liver disease. Conventional cell counting using optical microscopy was compared against two alternate methods to quantify isolated porcine hepatocytes: (1) automated cell counting using a commercially available particle characterization instrument, and (2) quantitation by cell mass. Methods were compared based on accuracy, precision, specificity, linear range, and ruggedness. The automated method delivered substantially improved accuracy, precision, and ruggedness when compared to the conventional optical method. It also provided valuable information about the size distribution of cell preparations, which often contained clumps of cells, and showed that processing steps such as cryopreservation can alter the size characteristics of a cell population. The automated method was also faster, and was well suited to use in a commercial manufacturing process. The mass-based method was simple and inexpensive, but suffered from nonlinearity at low cell concentrations. Automated cell quantitation using a commercially available particle characterization instrument proved to be the preferred method for obtaining accurate and consistent porcine hepatocyte counts in a timely manner.

Isoflavone levels in soy foods consumed by multiethnic populations in Singapore and Hawaii.

Concentrations and glucosidic conjugation patterns of isoflavones were determined in soy foods consumed by multiethnic populations in Singapore and Hawaii. Six raw and 11 cooked food groups traditionally consumed in Singapore and 8 food groups consumed in Hawaii were analyzed by reversed-phase high-pressure liquid chromatography with diode array detection. Mean total isoflavone levels varied between 35 and 7500 ppm, with the lowest values found in soy milk and burgers and the highest levels observed in soybean and its seeds and in supplements. Total isoflavone levels and conjugation patterns varied as a function of soybean variety, storage conditions, and food processing. A large contribution to the differences in total isoflavone content between food groups was due to the water content in foods and to leaching of polar analytes into the water phase during boiling. Soy protein drinks and traditional soy foods were found to possess very similar isoflavone amounts considering usual serving sizes.

Overview of extracorporeal liver support systems and clinical results.

Patients with acute liver failure (ALF) continue to have an almost 50% mortality rate despite improvements associated with the use of orthotopic liver transplantation (OLT). Numerous ex vivo methods have been developed in attempts to improve patient survival. These methods can be divided into three groups: detoxification (e.g., dialysis, charcoal adsorption, plasma exchange), which only provides excretory function; ex vivo liver perfusion (e.g., whole organ or tissue perfusion), which provides some metabolic function; and bioartificial or cell-based systems, which combine elements of the first two methods. Clinical trials have shown minimal efficacy of the various detoxification methods in terms of ALF patient survival, while the relative success of OLT has shown the importance of providing metabolic as well as excretory functions. Attempts to provide those additional functions with ex vivo tissue perfusion have been fraught with complications such as clotting and acute tissue rejection, leading to the conceptual development of cell-based bioreactor systems. A number of these bioartificial systems have been clinically evaluated, and the preliminary patient survival rates have encouraged further work in this area.

Usual dietary consumption of soy foods and its correlation with the excretion rate of isoflavonoids in overnight urine samples among Chinese women in Shanghai.

Soy foods and certain soy constituents, particularly isoflavones, have been suggested to have potential cancer-inhibitory effects in laboratory and epidemiological studies. Chinese women in Shanghai consume high levels of soy foods and have low incidence rates of breast and other hormone-related cancers. To assess the usual dietary consumption of soy foods and evaluate the correlation of soy food consumption with the urinary excretion of isoflavonoids in overnight urine samples in this population, we analyzed data from 60 healthy women included in an ongoing population-based case-control study of breast cancer in Shanghai. Usual consumption of soy foods in the previous five-year period was assessed using a food-frequency questionnaire, and urinary excretion of daidzein, genistein, glycitein, equol, and O-desmethylangolensin was measured from overnight urine samples collected at the time of dietary assessment. Virtually all women (96.7%) in Shanghai consumed soy foods at least once a week. The median intake of soy food was 100.6 g/day, with 25th and 75th percentiles of 36.8 and 238.2 g, respectively. The median intake of isoflavones was 39.26 mg/day, and there was a nearly fourfold difference between the 25th and 75th percentiles of this measurement. With the increasing intake of soy foods, urinary excretion rates of total isoflavonoids and all individual major isoflavonoids were increased in a dose-response manner (trend test p < or = 0.05). At individual levels the urinary excretion rate of total isoflavonoids was correlated closely with dietary soy food intake, with a correlation coefficient of around 0.5 (p < 0.001). These results indicate that the urinary excretion rate of total isoflavonoids measured from overnight urine samples may reflect reasonably well the usual intake of soy foods in a population with a high level of soy food consumption.

Serum levels and metabolic clearance of the isoflavones genistein and daidzein in hemodialysis patients.

Genistein and daidzein are biologically active isoflavones that are especially abundant in soybeans. After intestinal absorption, circulating genistein and daidzein are eliminated primarily by the kidneys. This study was undertaken to assess the metabolism of genistein and daidzein in patients with end-stage renal disease (ESRD) on hemodialysis therapy, and to test whether this treatment modality can replace the lack of kidney function, with respect to the elimination of the isoflavones. Twenty-three hemodialysis patients and 10 healthy subjects were studied. While consuming a self-selected low isoflavone diet, baseline blood levels were undetectable in eight of 10 healthy subjects and in 14 of 23 dialysis patients. The remaining participants had detectable levels, with the nine dialysis patients displaying much higher blood concentrations than the two healthy control subjects. After the evening intake of one dose of an isoflavone-rich soy protein isolate drink, the early morning blood levels of genistein and daidzein were higher in seven dialysis patients than in eight healthy subjects (genistein 1271+/-321 versus 425+/-104, P<0.05; daidzein 1304+/-352 versus 292+/-78, P<0.05). The blood clearance of the isoflavones was studied in two healthy subjects and in three dialysis patients. Genistein and daidzein were eliminated within 2 d in the healthy subjects, but had not returned to baseline in two of three ESRD patients, 7 d after intake. The half-life of both compounds was estimated to be 10-fold longer in the ESRD patients than in the healthy subjects. Finally, genistein and daidzein levels were measured before and after dialysis in five patients, both while on their regular diet and after one dose of a soy protein isolate drink. In both instances, the dialysis treatment did not affect the blood isoflavone levels. In conclusion, approximately one-third of hemodialysis patients eating the standard American renal diet experience high blood levels of the isoflavones genistein and daidzein, while the remaining two-thirds have undetectable levels. After ingestion of isoflavone-rich food such as soy products, all patients have detectable levels that remain very high for several days due to lack of renal excretion.