Urinary estrogen metabolites during a randomized soy trial.

One of the hypothesized protective mechanisms of soy against breast cancer involves changes in estrogen metabolism to 2-hydroxy (OH) and 16α-OH estrogens. The current analysis examined the effect of soy foods on the 2:16α-OH E(1) ratio among premenopausal women during a randomized, crossover intervention study; women were stratified by equol producer status, a characteristic thought to enhance the protective effects of soy isoflavones. The study consisted of a high-soy diet with 2 soy food servings/day and a low-soy diet with <3 servings of soy/wk for 6 mo each; estrogen metabolites were measured in 3 overnight urines (baseline and at the end of the low- and high-soy diet) using gas chromatography mass spectrometry for the 82 women who completed the study. Urinary isoflavonoids were assessed by liquid chromatography mass spectrometry. When applying mixed models, the 2:16α-OH E(1) ratio increased (P = 0.05) because of a nonsignificant decrease in 16α-OH E(1) (P = 0.21) at the end of the high-soy diet. Similar nonsignificant increases in the 2:16α-OH E(1) ratio were observed in equol producers (P = 0.13) and nonproducers (P = 0.23). These findings suggest a beneficial influence of soy foods on estrogen metabolism regardless of equol producer status.

Analysis of urinary estrogens, their oxidized metabolites, and other endogenous steroids by benchtop orbitrap LCMS versus traditional quadrupole GCMS.

Estrogens and other endogenous steroids are known risk markers for cancer. Gas chromatography (GC) with mass spectrometry (MS) has traditionally predominated the analysis of estrogens and other endogenous steroids, but liquid chromatography (LC) MS is increasingly favored. Direct comparisons of the two technologies have hitherto not been performed. Steroids were analyzed from 232 urine samples of 78 premenopausal women in a blinded fashion by benchtop orbitrap LCMS and single quadrupole GCMS. Sixteen steroidal estrogens including oxidized metabolites could be analyzed by LCMS. LCMS-GCMS Spearman rank correlations of the major estrogens E(1), E(2), E(3), 16α-OHE(1), and 2-OHE(1) were very high (r = 0.72-0.91), and absolute concentrations also agreed (<5% difference for E(1), E(2), E(3), 16α-OHE(1)). LCMS allowed reinterrogation of the acquired data due to orbitrap technology, which permitted post-analysis quantitation of progesterone, cortisol, and cortisone (LCMS-GCMS Spearman rank correlations = 0.80-0.84; absolute difference, <7%; n = 137). GCMS allows the measurement of a wide range of steroids including non-polar analytes that escape the presented LCMS assay. In contrast, orbitrap-based LCMS can detect more estrogens, is faster, less costly, allows post-data acquisition reinterrogation of certain analytes that had not been targeted a priori, and requires much less urine.

Urinary sex steroid excretion levels during a soy intervention among young girls: a pilot study.

Soy intake early in life may protect against breast cancer later in life, possibly by altering sex hormone metabolism. We evaluated the feasibility of assessing urinary sex steroid excretion among 20 young girls aged 8-14 yr in an 8-wk trial. The girls consumed one daily soy serving, collected weekly overnight urine samples, and reported Tanner stages for breast and pubic hair development. Sex steroid excretion was measured in duplicate by gas chromatography-mass spectrometry and adjusted for urinary creatinine. The respective coefficients of variation for estrone, estradiol, estriol, testosterone, pregnanediol were 11.4%, 10.4%, 8.4%, 12.8%, and 4.6%. The statistical analysis included t-tests, Spearman's correlations, and analysis of variance. Seventeen girls completed the study and showed good compliance with the intervention strategy. We observed nonsignificant increases in total androgens (0.11 microg/mg creatinine) and total estrogens (0.001 microg/mg creatinine) and a nonsignificant decrease in pregnanediol (-0.03 microg/mg creatinine) during the study period. Higher Tanner stages for pubic hair development were associated with ninefold higher estrogen, fourfold higher androgen, and twofold higher pregnanediol excretions (P=0.01, P<0.001, and P=0.047, respectively). Similar differences were observed after stratification by breast development and menarcheal status. The association of sex steroid levels with pubertal development supports the validity of the sex steroid measurements.

Medroxyprogesterone acetate and dihydrotestosterone induce coronary hyperreactivity in intact male rhesus monkeys.

Coronary hyperreactivity (CH), characterized by persistent severe vasoconstrictions in response to vasoconstrictor challenge, is oppositely influenced by progesterone (P) and medroxyprogesterone acetate (MPA) treatment in surgically menopausal primates. In this study we tested whether multiweek MPA or dihydrotestosterone (DHT) exposure induced CH in intact male rhesus monkeys. Coronary angiographic experiments with intracoronary serotonin and the thromboxane A(2) analog U46619 stimulated brief vasoconstriction (for 1-3 min) in large epicardial coronaries in untreated male monkeys. In contrast, MPA- and DHT-treated monkeys displayed long-duration constrictions (>5 min), with significantly greater reductions in the minimal diameters of epicardial coronaries. Immunocytochemistry demonstrated androgen receptors (AR) and P receptors in aorta and coronary arteries, and immunocytochemistry and Western blotting showed AR and P receptors in rhesus coronary vascular muscle cells. In vivo, MPA or DHT increased thromboxane prostanoid (TP) receptor expression in the aorta. In vitro, MPA or DHT increased, whereas P did not change, TP receptor expression in primary coronary vascular muscle cell. This MPA- or DHT-mediated increase in TP receptor expression was attenuated by the AR antagonist flutamide. MPA or DHT induction of CH in intact adult male primates, hypothesized to occur via androgenic up-regulation of vascular muscle TP receptor expression, could predispose to CH-mediated myocardial ischemia.

Prevention of coronary hyperreactivity in preatherogenic menopausal rhesus monkeys by transdermal progesterone.

OBJECTIVE: To test if transdermal progesterone (P) confers coronary vascular protection in surgically menopausal preatherosclerotic rhesus monkeys. METHODS AND RESULTS: Ovariectomized rhesus monkeys fed an atherogenic diet (AD) for 19 months were treated with an investigational transdermal P cream (n=7) or identical placebo cream (n=5) for 4 weeks. Aorta and carotids showed fatty streaks and Oil Red O staining demonstrated lipid deposition. Serum P levels in P-treated rhesus monkeys (0.6 ng/mL) were significantly greater than placebo (0.2 ng/mL). Significant elevation of cholesterol, LDL cholesterol, and HDL cholesterol, was noted in all animals. Lp(a) was significantly attenuated in the AD-fed P-treated monkeys. Coronary angiographic experiments stimulating vasoconstriction by intracoronary injections of serotonin plus U46619 showed exaggerated prolonged actions amplified by AD, but significant protection against severe prolonged vasoconstriction in P-treated monkeys. Immunocytochemistry confirmed co-expression of P and thromboxane prostanoid (TP) receptors in coronaries and aorta. Western blotting demonstrated TP receptor attenuation in vascular muscle after P treatment. CONCLUSIONS: Coronary hyperreactivity, a putative component of coronary artery disease mediated via increased vascular muscle thromboxane prostanoid receptors, can be prevented by subphysiological levels of P, not only in nonatherosclerotic (previously shown) but also in preatherosclerotic primates.