European Journal of Nutrition (v.51, #4)

Does equol production determine soy endocrine effects? by Dana Shor; Thozhukat Sathyapalan; Stephen L. Atkin; Natalie J. Thatcher (389-398).
Isoflavones, a group of phytoestrogens, are selective oestrogen receptor (ER) modulators. They may positively impact endocrine-related conditions but the current evidence is sparse. Equol, a non-steroidal oestrogen, is produced by the metabolism of the isoflavone daidzein by intestinal bacteria. In Western countries, 30–50% of individuals metabolize daidzein into equol and are known as equol producers. Equol production may be the source of benefit from isoflavones in endocrine disease.
Keywords: Equol; Phytoestrogen; Isoflavone; Soy; Cardiovascular disease; Osteoporosis; Breast cancer

High-fat diets affect energy and bone metabolism in growing rats by Elisa V. Macri; Macarena M. Gonzales Chaves; Patricia N. Rodriguez; Patricia Mandalunis; Susana Zeni; Fima Lifshitz; Silvia M. Friedman (399-406).
High-fat diets are usually associated with greater weight (W) gain and body fat (BF). However, it is still unclear whether the type and amount of fat consumed influence BF. Additionally, dietary fat intake may also have consequences on skeletal health.To evaluate in healthy growing rats the effects of high-fat diets and type of dietary fat intake (saturated or vegetable oils) on energy and bone metabolism.At weaning, male Wistar rats (n = 50) were fed either a control diet (C; fat = 7% w/w) or a high-fat diet (20% w/w) containing either: soybean oil, corn oil (CO), linseed oil (LO), or beef tallow (BT) for 8 weeks. Zoometric parameters, BF, food intake and digestibility, and total and bone alkaline phosphatase (b-AP) were assessed. Total skeleton bone mineral density (BMD) and content (BMC), BMC/W, spine BMD, and bone volume (static-histomorphometry) were measured.Animals fed BT diet achieved lower W versus C. Rats fed high-fat vegetable oil diets showed similar effects on the zoometric parameters but differed in BF. BT showed the lowest lipid digestibility and BMC. In contrast, high vegetable oil diets produced no significant differences in BMC, BMC/W, BMD, spine BMD, and bone volume. Marked differences were observed for LO and BT groups in b-AP and CO and BT groups in bone volume.BT diet rich in saturated fatty acids had decreased digestibility and adversely affected energy and bone metabolisms, in growing healthy male rats. There were no changes in zoometric and bone parameters among rats fed high vegetable oil diets.
Keywords: Healthy growing rats; High-fat diets; Saturated and vegetable oil diets; Bone densitometry and histomorphometry

To prospectively evaluate the association of vitamin/mineral supplementation with cancer, cardiovascular, and all-cause mortality.In the Heidelberg cohort of the European Prospective Investigation into Cancer and Nutrition (EPIC-Heidelberg), which was recruited in 1994–1998, 23,943 participants without pre-existing cancer and myocardial infarction/stroke at baseline were included in the analyses. Vitamin/mineral supplementation was assessed at baseline and during follow-up. Cox regression models were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs).After an average follow-up time of 11 years, 1,101 deaths were documented (cancer deaths = 513 and cardiovascular deaths = 264). After adjustment for potential confounders, neither any vitamin/mineral supplementation nor multivitamin supplementation at baseline was statistically significantly associated with cancer, cardiovascular, or all-cause mortality. However, baseline users of antioxidant vitamin supplements had a significantly reduced risk of cancer mortality (HR: 0.52; 95% CI: 0.28, 0.97) and all-cause mortality (HR: 0.58; 95% CI: 0.38, 0.88). In comparison with never users, baseline non-users who started taking vitamin/mineral supplements during follow-up had significantly increased risks of cancer mortality (HR: 1.74; 95% CI: 1.09, 2.77) and all-cause mortality (HR: 1.58; 95% CI: 1.17, 2.14).Based on limited numbers of users and cases, this cohort study suggests that supplementation of antioxidant vitamins might possibly reduce cancer and all-cause mortality. The significantly increased risks of cancer and all-cause mortality among baseline non-users who started taking supplements during follow-up may suggest a “sick-user effect,” which researchers should be cautious of in future observational studies.
Keywords: Supplements; Cancer; Cardiovascular disease; Mortality; Cohort study

We compared the effects of consumption of n-3 polyunsaturated fatty acids (PUFA)-enriched tomato juice versus plain tomato juice on the serum lipid profile and levels of biomarkers related to antioxidant status and cardiovascular disease (CVD) risk in women.Eighteen healthy women participated in a 2-week intervention trial involving the daily intake of 500 mL of n-3 PUFA-enriched juice (n = 11) or plain tomato juice (n = 7). Each serving of enriched juice provided 250 mg of eicosapentaenoic acid (EPA) plus docosahexanoic acid (DHA). Both juices provided natural antioxidant compounds such as phenolics (181 mg) and lycopene (26.5 mg).Intervention with the enriched juice had no effect on the lipid profile, and serum levels of triglycerides and cholesterol (total, LDL, and HDL) remained unchanged. The serum antioxidant status improved following juice intake, as revealed by an increase in total antioxidant capacity and a slight decrease in lipid peroxidation. The serum levels of homocysteine, a cardiovascular risk factor, decreased following n-3 PUFA-enriched juice consumption. A decrease in vascular adhesion molecule 1 (VCAM-1) levels was also noted after intake of either plain or enriched tomato juice, whereas intercellular adhesion molecule 1 (ICAM-1) levels only decreased following intake of the enriched juice.Overall, stronger positive amelioration of CVD risk factors was observed following the intake of n-3 PUFA-enriched juice than after plain tomato juice consumption, which suggested a possible synergistic action between n-3 PUFAs and tomato antioxidants.
Keywords: Cardiovascular diseases (CVD); PUFAs; Tomato juice; Antioxidants; Homocysteine; Lipid oxidation; VCAM-1; ICAM-1

Micronutrient status in lactating mothers before and after introduction of fortified flour: cross-sectional surveys in Maela refugee camp by Wolfgang Stuetz; Verena Ilona Carrara; Rose McGready; Sue Jean Lee; Juergen Georg Erhardt; Joern Breuer; Hans Konrad Biesalski; François Henry Nosten (425-434).
Deficiency of micronutrients is common in refugee populations.Identify deficiencies and whether provided supplements and wheat flour fortified with 10 micronutrients impacts upon status among breast-feeding women from Maela refugee camp.Two sequential cross-sectional studies were conducted in different groups of lactating mothers at 12 weeks postpartum. The first survey was before and the second 4–5 months after micronutrient fortified flour (MFF) had been provided to the camp (in addition to the regular food basket). Iron status and micronutrients were measured in serum, whole blood, and in breast milk samples.Iron and zinc deficiency and anemia were highly prevalent while low serum retinol and thiamine deficiency were rarely detected. Iron and zinc deficiency were associated with anemia, and their proportions were significantly lower after the introduction of MFF (21 vs. 35% with soluble transferrin receptor (sTfR) >8.5 mg/L, P = 0.042, and 50 vs. 73% with serum zinc <0.66 mg/L, P = 0.001). Serum sTfR, whole-blood thiamine diphosphate (TDP) and serum β-carotene were significant predictors (P < 0.001) of milk iron, thiamine and β-carotene, respectively. Lower prevalence of iron deficiency in the MFF group was associated with significantly higher iron and thiamine in breast milk.High whole-blood TDP and breast milk thiamine reflected good compliance to provided thiamine; high prevalence of iron deficiency suggested insufficient dietary iron and low acceptance to ferrous sulfate supplements. MFF as an additional food ration in Maela refugee camp seemed to have an effect in reducing both iron and zinc deficiency postpartum.
Keywords: Micronutrients; Lactation; Refugee; Iron; Zinc; Anemia

Four polyphenols: ferulic acid and p-coumaric acid (hydroxycinnamic acids), quercetin (flavonol) and cyanidin 3-glucoside (anthocyanin) were selected, and their antioxidant properties and their influence on cholesterol concentration in hypercholesterolemic and normal erythrocytes were investigated.To determine the effect of phenolic compounds, we prospectively studied cholesterol concentration, lipid peroxidation and membranes fluidity. Whole-blood and isolated erythrocytes (2% hematocrit) were incubated for 24 h with selected compounds at concentration 1, 10 and 100 μmol/L. All investigated compounds decreased lipid peroxidation in whole blood. Cyanidin 3-glucoside and quercetin showed higher antioxidant properties than hydroxycinnamic acids (ferulic acid and p-coumaric acid).Incubation of whole blood of hypercholesterolemic patients with quercetin and cyanidin 3-glucoside resulted in statistically significant reduction of cholesterol concentration in erythrocytes down to 75% (at 10 μmol/L of polyphenols) and 69% (at 100 μmol/L of polyphenols) of initial values. The effect of both compounds on isolated erythrocytes was even more pronounced, reduction down to 70% (at 10 μmol/L of polyphenols) and 58% (at 100 μmol/L of polyphenols) of initial values. After incubation of isolated erythrocytes of hypercholesterolemic patients with quercetin and cyanidin 3-glucoside, increase of membrane fluidity was noticed. After incubation of isolated erythrocytes of healthy donors with investigated compounds, no changes in membrane fluidity were observed.Our results indicate that flavonols and anthocyanins have higher antioxidant properties and higher influence on cholesterol concentration in erythrocytes membranes than simple hydroxycinnamic acids.
Keywords: Hypercholesterolemia; Lipid peroxidation; Cholesterol; Quercetin; Cyanidin 3-glucoside; Ferulic acid; Coumaric acid; Membrane fluidity

Metabolic and behavioural effects of sucrose and fructose/glucose drinks in the rat by Anastasia Sheludiakova; Kieron Rooney; Robert A. Boakes (445-454).
Overconsumption of sugar-sweetened beverages, in particular carbonated soft drinks, promotes the development of overweight and obesity and is associated with metabolic disturbances, including intrahepatic fat accumulation and metabolic syndrome. One theory proposes that drinks sweetened with high-fructose corn syrup are particularly detrimental to health, as they contain fructose in its ‘free’ monosaccharide form. This experiment tested whether consuming ‘free’ fructose had a greater impact on body weight and metabolic abnormalities than when consumed ‘bound’ within the disaccharide sucrose.Male Hooded Wistar rats were given free access for 56 days to 10% sucrose (Group Suc), 10%, 50/50 fructose/glucose (Group FrucGluc) or water control drinks (Group Control), plus chow. Caloric intake and body weights were measured throughout the protocol, and the following metabolic indices were determined between days 54 and 56: serum triglycerides, liver triglycerides, retroperitoneal fat and oral glucose tolerance.Animals with access to sugar beverages consumed 20% more calories, but did not show greater weight gain than controls. Nevertheless, they developed larger abdominal fat pads, higher triglyceride levels and exhibited impaired insulin/glucose homeostasis. Comparison of the two sugars revealed increased fasting glycaemia in the FrucGluc group, but not in Suc group, whereas the Suc group was more active in an open field.A metabolic profile indicating increased risk of diabetes mellitus and cardiovascular disease was observed in animals given access to sugar-sweetened beverages. Notably, ‘free’ fructose disrupted glucose homeostasis more than did ‘bound’ fructose, thus posing a greater risk of progression to type 2 diabetes.
Keywords: Metabolic syndrome; Sucrose; Fructose; Glucose; High-fructose corn syrup; Insulin resistance; Glucose tolerance; Triglycerides; Behaviour

Anti-proliferative and pro-apoptotic activities of hydroxytyrosol on different tumour cells: the role of extracellular production of hydrogen peroxide by Roberto Fabiani; Maria Vittoria Sepporta; Patrizia Rosignoli; Angelo De Bartolomeo; Marilena Crescimanno; Guido Morozzi (455-464).
Several recently published data suggest that the anti-proliferative and pro-apoptotic properties of hydroxytyrosol [3,4-dihydroxyphenyl ethanol (3,4-DHPEA)] on HL60 cells may be mediated by the accumulation of hydrogen peroxide (H2O2) in the culture medium. The aim of this study was to clarify the role played by H2O2 in the chemopreventive activities of 3,4-DHPEA on breast (MDA and MCF-7), prostate (LNCap and PC3) and colon (SW480 and HCT116) cancer cell lines and to investigate the effects of cell culture medium components and the possible mechanisms at the basis of the H2O2-producing properties of 3,4-DHPEA.The proliferation was measured by the MTT assay and the apoptosis by both fluorescence microscopy and flow cytometry. The concentration of H2O2 in the culture medium was measured by the ferrous ion oxidation–xylenol orange method.It was found that the H2O2-inducing ability of 3,4-DHPEA is completely prevented by pyruvate and that the exposure of cells to conditions not supporting the H2O2 accumulation (addition of either catalase or pyruvate to the culture medium) inhibited the anti-proliferative effect of 3,4-DHPEA. Accordingly, the sensitivity of the different cell lines to the anti-proliferative effect of 3,4-DHPEA was inversely correlated with their ability to remove H2O2 from the culture medium. With regard to the mechanism by which 3,4-DHPEA causes the H2O2 accumulation, it was found that superoxide dismutase increased the H2O2 production while tyrosinase, slightly acidic pH (6,8) and absence of oxygen (O2) completely prevented this activity. In addition, different transition metal-chelating compounds did not modify the H2O2-producing activity of 3,4-DHPEA.The pro-oxidant activity of 3,4-DHPEA deeply influences its ‘in vitro’ chemopreventive activities. The main initiation step in the H2O2-producing activity is the auto-oxidation of 3,4-DHPEA by O2 with the formation of the semiquinone, superoxide ions (O2 ) and 2H+.
Keywords: Hydroxytyrosol; Apoptosis; Hydrogen peroxide; Olive oil; Tumour cells

CYP1A1 is overexpressed upon incubation of breast cancer cells with a polyphenolic cocoa extract by Carlota Oleaga; Miriam García; Anna Solé; Carlos J. Ciudad; Maria Izquierdo-Pulido; Véronique Noé (465-476).
To evaluate the effect of cocoa flavonoids in breast cancer cells at the molecular level, a functional genomic analysis was performed using a polyphenolic cocoa extract (PCE) in MCF-7 and SKBR3 cell lines.The expression profile of 84 genes included in the Stress & Toxicity PathwayFinder™ PCR Array was analyzed after PCE incubation for 24 h. mRNA and protein levels were analyzed by RT-PCR and western blot, respectively. Gel shift assays were used to evaluate DNA–protein complexes. Protein complexes were identified by co-immunoprecipitation. Cell viability was evaluated by MTT assays.Upon PCE incubation, 7 genes were overexpressed and 1 underexpressed in MCF-7 cells, whereas 9 genes were overexpressed in SKBR3 cells. Among the differentially expressed genes in both cell lines, cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1) was chosen for further study. CYP1A1 mRNA and protein levels and enzymatic activity increased upon PCE incubation. CYP1A1 transcriptional activation by PCE was mediated through AhR binding to XRE elements within the CYP1A1 promoter in MCF-7 cells. A protein complex including AhR and ERα was detected. The combination of PCE with tamoxifen caused a synergistic cytotoxicity in both cell lines and was due to an increase in apoptosis in MCF-7 cells.The interaction between ERα and AhR upon incubation with PCE leads to CYP1A1 induction in breast cancer cells. The synergy between PCE and non-cytotoxic tamoxifen concentrations opens the possibility for a combination therapy based on polyphenols from cocoa that increased tamoxifen efficacy.
Keywords: AhR; Cocoa; CYP1A1; ERα; Tamoxifen; Breast cancer

To explore whether there are regional differences in iodine status and in prevalence of thyroid diseases in the two main regions of Belgium.A national survey of iodine status among children was performed in 1998. The raw data of this survey were reanalyzed to explore regional differences. The total number of thyroidectomies, carried out for multinodular goiter or solitary nodules, was obtained from the Minimal Clinical Summary hospital discharge database. Percentage of people with thyroid diseases going to the general practitioner or the specialist was assessed by means of data about the number of adults using anti-thyroid medications. Food consumption patterns were explored using national food consumption data.In Flanders, median urinary iodine concentration (UIC) was higher than in Wallonia, 84 μg/L (n = 1,316) and 78 μg/L (n = 1,268), respectively (p < 0.001). There were no differences in goiter prevalence and thyroid volume between the regions among children. Data from the food consumption survey showed a significant higher consumption of seafood in Flanders compared to Wallonia. Further, it was observed that the number of thyroidectomies, carried out for MNG or solitary nodules, and the use of anti-thyroid medication were significantly higher in Wallonia than in Flanders.Iodine status in children was found slightly different in both regions of the country. This finding is in agreement with a higher incidence of thyroidectomies and more extensive use of anti-thyroid medications in the adult population in the region with the lowest iodine excretion.
Keywords: Iodine status children; Belgium; Thyroid diseases adults

The purpose of the study was to evaluate hypolipidemic and hypocholesterolemic activities of conjugated linolenic acid (CLnA) isomers, present in bitter gourd and snake gourd seed, in terms of amelioration of plasma lipid profile, lipoprotein oxidation and erythrocyte membrane fluidity after oral administration.Male albino rats were divided into six groups. Group 1 was control, and others were induced with oxidative stress by oral gavage of sodium arsenite (Sa). Group 2 was kept as treated control, and groups 3–6 were further treated with different oral doses of seed oils to maintaining definite concentration of CLnA isomers (0.5 and 1.0% of total lipid for each CLnA isomer).CLnA isomers normalized cholesterol, LDL-cholesterol, HDL-cholesterol and triglyceride contents in plasma and body weight of experimental rats and decreased cholesterol synthesis by reducing hepatic HMG-CoA reductase activity. Administration of Sa caused alteration in erythrocyte membrane fluidity due to increase in cholesterol and decrease in phospholipid content. Tissue cholesterol and lipid contents were also increased by Sa administration. These altered parameters were reversed by experimental oil administration. Protective effect of CLnA isomers on erythrocyte morphology was observed by atomic force microscopy (AFM). Fatty acid composition of erythrocyte membrane showed decrease in polyunsaturated fatty acid (PUFA) and increase in arachidonic acid content after Sa administration, which was normalized with the treatment of these oils. Supplementation of CLnA isomers restored erythrocyte membrane (EM) lipid peroxidation and lipoprotein oxidation.CLnA isomers, present in vegetable oils, showed potent hypolipidemic and hypocholesterolemic activities against biochemical perturbations.
Keywords: Conjugated linolenic acid; α-eleostearic acid; Punicic acid; Hypocholesterolemic; Membrane fluidity; Hypolipidemic

Influence of extra virgin olive oil diet enriched with hydroxytyrosol in a chronic DSS colitis model by Susana Sánchez-Fidalgo; L. Sánchez de Ibargüen; A. Cárdeno; C. Alarcón de la Lastra (497-506).
Recent epidemiological studies have shown that habitual consumption of extra virgin olive oil (EVOO), the characteristic culinary fat of the Mediterranean area, is effective in the prevention of diverse types of digestive disorders such as inflammatory bowel disease. Many of these benefits are, in addition to its high proportion of oleic acid, due to the high content of phenolic compounds.Six-week-old mice were randomized into three dietary groups: standard, EVOO and hydroxytyrosol-enriched EVOO. After 30 days, mice that were exposed to 3% DSS for 5 days developed acute colitis that progressed to severe chronic inflammation during a regime of 21 days of water.Diets enriched with EVOO significantly attenuated the clinical and histological signs of damage, improving results from disease activity index and reducing about 50% the mortality caused by DSS. Moreover, hydroxytyrosol supplement showed better results. Cytokines study showed that TNF-α was maintained near to sham control and IL-10 levels were significantly improved in EVOO and EVOO plus hydroxytyrosol diet-DSS groups. In the same way, COX-2 and iNOS were downregulated, and the activation of p38 MAPK was reduced. We also observed a higher significant reduction in iNOS in hydroxytyrosol-enriched EVOO compared with EVOO alone.EVOO diets exerted a noteworthy beneficial effect in chronic DSS-induced colitis by cytokine modulation and COX-2 and iNOS reduction via downregulation of p38 MAPK. In addition to the beneficial effect by EVOO, supplementation of the diet with hydroxytyrosol may improve chronic colitis through iNOS downregulation plus its antioxidant capacity.
Keywords: Extra virgin olive oil; Hydroxytyrosol; Chronic colitis; Cytokines; COX-2; iNOS

The sourdough fermentation may enhance the recovery from intestinal inflammation of coeliac patients at the early stage of the gluten-free diet by Maria Calasso; Olimpia Vincentini; Francesco Valitutti; Cristina Felli; Marco Gobbetti; Raffaella Di Cagno (507-512).
This study aimed at investigating the effect of corn, rice and amaranth gluten-free (GF) sourdoughs on the release of nitric oxide (NO) and synthesis of pro-inflammatory cytokines by duodenal mucosa biopsies of eight coeliac disease (CD) patients.Selected lactic acid bacteria were used as starters for the manufacture of corn, rice or amaranth sourdoughs. Chemically acidified doughs, without bacterial starters, and doughs started with baker’s yeast alone were also manufactured from the same GF matrices. Pepsin-trypsin (PT) digests were produced from all sourdoughs and doughs, and used to assay the rate of recovery of biopsy specimens from eight CD patients at diagnosis. The release of NO and the synthesis of pro-inflammatory cytokines interferon-γ (IFN-γ) were assayed.During fermentation, lactic acid bacteria acidified and grew well (ca. log 9.0 CFU/g) on all GF matrices, showing intense proteolysis. Duodenal biopsy specimens still released NO and IFN-γ when subjected to treatments with basal medium (control), PT-digest from chemically acidified doughs and PT-digest from doughs fermented with baker’s yeast alone. On the contrary, the treatment of all the biopsy specimens with PT-digests from all GF matrices subjected to sourdough fermentation significantly decreased the release of NO and the synthesis of IFN-γ.During manufacture of GF baked goods, the use of sourdough fermentation could be considered as an adjuvant to enhance the recovery from intestinal inflammation of coeliac patients at the early stage of the gluten-free diet.
Keywords: Coeliac disease; Sourdough; Nitric oxide; Interferon-γ; Biopsies of duodenal mucosa