Francene M. Steinberg

Chronic consumption of flavanol-rich cocoa improves endothelial function and decreases vascular cell adhesion molecule in hypercholesterolemic postmenopausal women

Endothelial dysfunction characterizes many disease states including subclinical atherosclerosis. The consumption of flavanol-rich cocoa and cocoa-based products has been shown to improve endothelial function in both compromised and otherwise normal, healthy individuals when administered either acutely or over a period of several days, or weeks. Women experience increased risk for cardiovascular disease after menopause, which can be associated with endothelial dysfunction. Whether a flavanol-rich cocoa-based product can improve endothelial function in hypercholesterolemic postmenopausal women is not known. The purpose of the present study was to determine whether chronic dietary administration of flavanol-rich cocoa improves endothelial function and markers of cardiovascular health in hypercholesterolemic postmenopausal women. Thirty-two postmenopausal hypercholesterolemic women were randomly assigned to consume a high-flavanol cocoa beverage (high cocoa flavanols (CF)—446 mg of total flavanols), or a low-flavanol cocoa beverage (low CF—43 mg of total flavanols) for 6 weeks in a double-blind study (n=16 per group). Endothelial function was determined by brachial artery-reactive hyperemia. Plasma was analyzed for lipids (total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol), hormones (follicle-stimulating hormone), total nitrate/nitrite, activation of cellular adhesion markers (vascular cell adhesion molecule 1, intercellular adhesion molecule 1, E-Selectin, P-Selectin), and platelet function and reactivity. Changes in these plasma markers were then correlated to brachial reactivity. Brachial artery hyperemic blood flow increased significantly by 76% (P<0.05 vs. baseline) after the 6-week cocoa intervention in the high CF group, compared with 32% in the low CF cocoa group (P=ns vs. baseline). The 2.4-fold increase in hyperemic blood flow with high CF cocoa closely correlated (r2=0.8) with a significant decrease (11%) in plasma levels of soluble vascular cell adhesion molecule-1. Similar responses were not observed after chronic use of low CF. There were no significant differences between high and low CF in other biochemical markers and parameters measured. This study is the first to identify beneficial vascular effects of flavanol-rich cocoa consumption in hypercholesterolemic postmenopausal women. In addition, our results suggest that reductions in plasma soluble vascular cell adhesion molecule-1 after chronic consumption of a flavanol-rich cocoa may be mechanistically linked to improved vascular reactivity.

Soy isoflavone supplementation and bone mineral density in menopausal women: a 2-y multicenter clinical trial

BACKGROUND Isoflavones are naturally occurring plant estrogens that are abundant in soy. Although purported to protect against bone loss, the efficacy of soy isoflavone supplementation in the prevention of osteoporosis in postmenopausal women remains controversial. OBJECTIVE Our aim was to test the effect of soy isoflavone supplementation on bone health. DESIGN A multicenter, randomized, double-blind, placebo-controlled 24-mo trial was conducted to assess the effects of daily supplementation with 80 or 120 mg of soy hypocotyl aglycone isoflavones plus calcium and vitamin D on bone changes in 403 postmenopausal women. Study subjects were tested annually and changes in whole-body and regional bone mineral density (BMD), bone mineral content (BMC), and T scores were assessed. Changes in serum biochemical markers of bone metabolism were also assessed. RESULTS After study site, soy intake, and pretreatment values were controlled for, subjects receiving a daily supplement with 120 mg soy isoflavones had a statistically significant smaller reduction in whole-body BMD than did the placebo group both at 1 y (P < 0.03) and at 2 y (P < 0.05) of treatment. Smaller decreases in whole-body BMD T score were observed among this group of women at 1 y (P < 0.03) but not at 2 y of treatment. When compared with the placebo, soy isoflavone supplementation had no effect on changes in regional BMD, BMC, T scores, or biochemical markers of bone metabolism. CONCLUSION Daily supplementation with 120 mg soy hypocotyl isoflavones reduces whole-body bone loss but does not slow bone loss at common fracture sites in healthy postmenopausal women. This trial was registered at clinicaltrials.gov as NCT00665860.

Effect of Dietary Constituents With Chemopreventive Potential on Adduct Formation of a Low Dose of the Heterocyclic Amines PhIP and IQ and Phase II Hepatic Enzymes

We conducted a study to evaluate dietary chemopreventive strategies to reduce genotoxic effects of the carcinogens 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). PhIP and IQ are heterocyclic amines (HCAs) that are found in cooked meat and may be risk factors for cancer. Typical chemoprevention studies have used carcinogen doses many thousand-fold higher than usual human daily intake. Therefore, we administered a low dose of [14C] PhIPand [3H] IQand utilized accelerator mass spectrometry to quantify PhIP adducts in the liver, colon, prostate, and blood plasma and IQadducts in the liver and blood plasma with high sensitivity. Diets supplemented with phenethylisothiocyanate (PEITC), genistein, chlorophyllin, or lycopene were evaluated for their ability to decrease adduct formation of [14C] PhIPand [3H] IQin rats. We also examined the effect of treatments on the activity of the phase II detoxification enzymes glutathione S-transferase (GST), UDP-glucuronyltransferase (UGT), phenol sulfotransferase (SULT) and quinone reductase (QR). PEITC and chlorophyllin significantly decreased PhIP-DNA adduct levels in all tissues examined, which was reflected by similar changes in PhIP binding to albumin in the blood. In contrast, genistein and lycopene tended to increase PhIP adduct levels. The treatments did not significantly alter the level of IQ-DNA or -protein adducts in the liver.With the exception of lycopene, the treatments had some effect on the activity of one or more hepatic phase II detoxification enzymes. We conclude that PEITC and chlorophyllin are protective of PhIP-induced genotoxicity after a low exposure dose of carcinogen, possibly through modification of HCA metabolism.

Effect of grape seed extract on blood pressure in subjects with the metabolic syndrome

This study was undertaken to determine whether grape seed extracts (GSE) that contain powerful vasodilator phenolic compounds lower blood pressure in subjects with the metabolic syndrome. The subjects were randomized into 3 groups-(a) placebo, (b) 150 mg GSE per day, and (c) 300 mg GSE per day-and treated for 4 weeks. Serum lipids and blood glucose were measured at the beginning of the study and at the end. Blood pressure was recorded using an ambulatory monitoring device at the start of the treatment period and at the end. Both the systolic and diastolic blood pressures were lowered after treatment with GSE as compared with placebo. There were no significant changes in serum lipids or blood glucose values. These findings suggest that GSE could be used as a nutraceutical in a lifestyle modification program for patients with the metabolic syndrome.

Cocoa procyanidin chain length does not determine ability to protect LDL from oxidation when monomer units are controlled.

Cocoa flavan-3-ols (catechin, epicatechin and oligomeric procyanidins) were tested for their ability to decrease LDL oxidative susceptibility and spare alpha-tocopherol (alpha-toc) in vitro. Physiologic concentration (0.10-0.50 &mgr;M) of flavanols were used. The flavanols increased LDL conjugated diene lag times dose-dependently from 23-207% and 15-143% in response to copper and AAPH oxidation, respectively, and delayed alpha-toc consumption. Sparing of LDL alpha-toc represents a possible mechanism for flavanols to enhance the resistance of plasma and LDL to oxidative stress. Procyanidins decreased LDL oxidative susceptibility with increasing chain length. However, when based on equivalent amounts of monomeric units, they inhibited LDL oxidation to a similar extent. This suggests that antioxidant activity of procyanidins with biologic substrates is not attributable to chain length or charge delocalization through polymeric linkages, but primarily to ring structures and catechol groups. Additionally, human plasma was analyzed for the presence of oligomeric procyanidins following consumption of a flavanol-rich cocoa product. Procyanidin dimers were detected in plasma concordant with the appearance of monomeric flavanols, with a peak of 0.08 +/- 0.01 &mgr;mol/L (n = 6) at two hours after consumption. Thus, this paper confirms the occurrence of procyanidins in human plasma, and extends previous structure-function observations regarding flavanoid protection of LDL.

Pyrroloquinoline Quinone Improves Growth and Reproductive Performance in Mice Fed Chemically Defined Diets

Growth, reproductive performance, and indices of collagen maturation and expression were investigated in Balb/c mice fed chemically defined, amino acid-based diets with or without the addition 6 <M pyrroloquinoline quinone (PQQ)/kg diet. The diets were fed to virgin mice for 8 weeks before breeding. At weaning, the pups from successful pregnancies were fed the same diet as their respective dams. Reproductive performance was compromised in mice fed diets devoid of PQQ, and their offspring grew at slower rates than offspring from mice fed diets supplemented with PQQ. Successful mating (confirmed vaginal plugs) was not affected by the presence or absence of PQQ; however, pup viability (number of pups at parturition/number of pups at Day 4 of lactation) was decreased in PQQ-deprived mice. Conception (percentage of females giving live births) and fertility (percentage of births) were also decreased in PQQ-deprived mice. The slower rates of growth in offspring from PQQ-deprived mice were associated with decreased steady-state mRNA levels for Type I procollagen α1-chains in skin and lungs from neonatal mice. Values for lysyl oxidase accumulation as protein in PQQ-deficient mice also tended to be lower than corresponding values from PQQ-supplemented or -replete mice. Skin collagen solubility was increased in PQQ-deprived mice. These results indicate that PQQ supplementation can improve reproductive performance, growth, and may modulate Indices of neonatal extracellular matrix production and maturation in mice fed chemically defined, but otherwise nutritionally complete diets.

Various Doses of Soy Isoflavones Do Not Modify Mammographic Density in Postmenopausal Women

Soy isoflavones have functional similarity to human estrogens and may protect against breast cancer as a result of their antiestrogenic activity or increase risk as a result of their estrogen-like properties. We examined the relation between isoflavone supplementation and mammographic density, a strong marker for breast cancer risk, among postmenopausal women. The Osteoporosis Prevention Using Soy (OPUS) study, a multi-site, randomized, double-blinded, and placebo-controlled trial assigned 406 postmenopausal women to 80 or 120 mg/d of isoflavones each or a placebo for 2 y. Percent densities were assessed in digitized mammograms using a computer-assisted method. The mammogram reader did not know the treatment status and the time of mammograms. We applied mixed models to compare breast density by treatment while considering the repeated measures. The mammographic density analysis included 358 women, 88.2% of the OPUS participants; 303 had a complete set of 3 mammograms, 49 had 2, and 6 had only 1 mammogram. At baseline, the groups were similar in age, BMI, and percent density, but mean breast density differed by study site (P = 0.02). A model with all mammograms did not show a treatment effect on any mammographic measure, but the change over time was significant; breast density decreased by 1.6%/y across groups (P < 0.001). Stratification by age and BMI did not reveal any effects in subgroups. In this randomized 2-y trial, isoflavone supplements did not modify breast density in postmenopausal women. These findings offer reassurance that isoflavones do not act like hormone replacement medication on breast density.

Clinical outcomes of a 2-y soy isoflavone supplementation in menopausal women

BACKGROUND Soy isoflavones are naturally occurring phytochemicals with weak estrogenic cellular effects. Despite numerous clinical trials of short-term isoflavone supplementation, there is a paucity of data regarding longer-term outcomes and safety. OBJECTIVE Our aim was to evaluate the clinical outcomes of soy hypocotyl isoflavone supplementation in healthy menopausal women as a secondary outcome of a trial on bone health. DESIGN A multicenter, randomized, double-blind, placebo-controlled 24-mo trial was conducted to assess the effects of daily supplementation with 80 or 120 mg aglycone equivalent soy hypocotyl isoflavones plus calcium and vitamin D on the health of 403 postmenopausal women. At baseline and after 1 and 2 y, clinical blood chemistry values were measured and a well-woman examination was conducted, which included a mammogram and a Papanicolaou test. A cohort also underwent transvaginal ultrasound measurements to assess endometrial thickness and fibroids. RESULTS The baseline characteristics of the groups were similar. After 2 y of daily isoflavone exposure, all clinical chemistry values remained within the normal range. The only variable that changed significantly was blood urea nitrogen, which increased significantly after 2 y (P = 0.048) but not after 1 y (P = 0.343) in the supplementation groups. Isoflavone supplementation did not affect blood lymphocyte or serum free thyroxine concentrations. No significant differences in endometrial thickness or fibroids were observed between the groups. Two serious adverse events were detected (one case of breast cancer and one case of estrogen receptor-negative endometrial cancer), which was less than the expected population rate for these cancers. CONCLUSION Daily supplementation for 2 y with 80-120 mg soy hypocotyl isoflavones has minimal risk in healthy menopausal women. This trial was registered at clinicaltrials.gov as NCT00665860.

Soy isoflavones increase quinone reductase in hepa-1c1c7 cells via estrogen receptor beta and nuclear factor erythroid 2-related factor 2 binding to the antioxidant response element

Soy protein and isoflavones (genistein and daidzein) have been demonstrated to increase quinone reductase (QR) activity, protein, and mRNA in animal and cell culture models. However, their mechanism of action has not been completely characterized. Additionally, it has not been determined if equol, a daidzein metabolite, can modulate QR activity and expression. Estrogen receptor beta (ERβ) is thought to be involved in stimulating QR gene transcription by anti-estrogens and phytoestrogens, along with nuclear factor erythroid 2-related factor 2 (Nrf2). This study tested the hypothesis that genistein, daidzein and equol increase quinone reductase activity, protein and mRNA via ERβ and Nrf2 binding to the QR antioxidant response element (ARE). QR expression and activity were determined using TaqMan polymerase chain reaction, protein immunoblots and activity assays. Molecular events were investigated using luciferase reporter gene assays and chromatin immunoprecipitation (ChIP). Hepa-1c1c7 cells were treated with control [0.1% (v:v) dimethyl sulfoxide (DMSO)]; 1 μmol/L β-naphthoflavone (positive control); 5 μmol/L resveratrol (ChIP positive control for ERβ binding) and 1, 5 and 25 μmol/L genistein, daidzein or equol. Treatment durations were 1 h (ChIP), 24 h (mRNA and luciferase assays) and 24 and 48 h (protein and activity). Genistein, daidzein and equol increased QR activity, protein and mRNA, with daidzein and equol having more of an impact at physiologic concentrations (1 and 5 μmol/L) compared to genistein. Furthermore, the study results demonstrate that genistein, daidzein and equol interact with the QR ARE and that daidzein and equol act via both ERβ and Nrf2 binding strongly to the QR ARE.

Soy provides modest benefits on endothelial function without affecting inflammatory biomarkers in adults at cardiometabolic risk

SCOPE Systemic inflammation, endothelial dysfunction, and oxidative stress are involved in the pathogenesis of the metabolic syndrome (MetS). Epidemiological evidence supports an association between whole soy food consumption and reduced risk of cardiovascular disease (CVD). The objective of this randomized, controlled, cross-over study was to evaluate the effects of soy nut consumption on inflammatory biomarkers and endothelial function and to assess whether isoflavone metabolism to secondary products, equol, and/or O-desmethylangolensin (ODMA), modifies these responses. METHODS AND RESULTS n = 17 adults at cardiometabolic risk were randomly assigned to the order of two snack interventions, soy nuts, and macronutrient-matched control snack, for four weeks each, separated by a two week washout period. Outcome measures included biomarkers of inflammation, oxidative stress, and glycemic control (ELISA and clinical analyzers), endothelial function, and arterial stiffness (peripheral arterial tonometry (PAT)), and isoflavone metabolites (LC-MS/MS). Results revealed that consuming soy nuts improved arterial stiffness as assessed by the augmentation index using PAT (p = 0.03), despite lack of improvement in inflammatory biomarkers. Addition of equol and/or ODMA production status as covariates did not significantly change these results. CONCLUSION Soy nuts when added to a usual diet for one month provide some benefit on arterial stiffness in adults at cardiometabolic risk.