Effect of ß-cryptoxanthin plus phytosterols on cardiovascular risk and bone turnover markers in post-menopausal women: a randomized crossover trial.

BACKGROUND AND AIM: Post-menopausal women are at higher risk of cardiovascular disease and bone demineralization. Phytosterols (PS) may be used for hypercholesterolemia in some groups and ß-cryptoxanthin (ß-Cx) displays a unique anabolic effect on bone. Our aim was to assess the changes in cardiovascular and bone turnover markers from the oral intake of ß-Cx and PS in post-menopausal women. METHODS AND RESULTS: A randomized, double-blind, crossover study with ß-Cx (0.75 mg/day) and PS (1.5 g/day), single and combined, was performed in 38 postmenopausal women. Diet was supplemented with 1 × 250 mL milk-based fruit drink/day for 4 weeks with a wash-out period of 4-weeks in between. Serum ß-Cx and PS were determined by UPLC and CG-FID respectively. Outcome variables included markers of bone turnover and cardiovascular risk. Biological effect was assessed by paired t test and generalized estimating equations analysis that included the previous treatment, the order of intervention and the interactions. The intake of beverages containing ß-Cx and PS brought about a significant increase in serum levels of ß-Cx, ß-sitosterol and campesterol. Intervention caused changes in almost all the markers while the order, previous treatment and the interaction did not reach statistical significance. Only the intake of the beverage containing ß-Cx plus PS brought about significant decreases in total cholesterol, c-HDL, c-LDL and bone turnover markers. CONCLUSIONS: ß-Cx improves the cholesterol-lowering effect of PS when supplied simultaneously and this combination may also be beneficial in reducing risk of osteoporosis. CLINICAL TRIAL REGISTRY: ClinicalTrials.gov number NCT01074723.

Gangliosides and sialic acid effects upon newborn pathogenic bacteria adhesion: an in vitro study.

The effect of the main gangliosides (GM(1), GM(3), GD(3)) and free sialic acid (Neu5Ac) upon the adhesion of pathogenic bacteria implicated in infant diarrhoea is assessed in vitro using the Caco-2 cell line. Concentrations of the bioactive compounds found in the bioaccessible (soluble) fraction of infant formula and human milk are employed. Bacterial adhesion behaviour included enterotoxigenic Escherichia coli (ETEC), enteropathogenic E.coli (EPEC), Listeria monocytogenes, Salmonella entericaserovartyphi, Shigella sonnei, Campylobacter jejuni and Helicobacter pylori. Three different approaches were assayed: pre-incubation of bacteria and compounds before addition to cells (competition); pre-incubation of the cells with compounds (exclusion); and pre-incubation of cells with bacteria (displacement). Furthermore, the spatial localization of the most abundant gangliosides, GM(3) and GD(3), in Caco-2 cells has been determined using confocal microscopy. Results show that GM(3), GD(3), GM(1) and Neu5Ac at the assayed concentrations are able to interfere with the adhesion of several pathogenic bacteria involved in neonatal diseases-the greatest effect corresponding to Neu5Ac, followed by GD(3), GM(1) and GM(3). Gangliosides GM(3) and GD(3) are located in the apical and basolateral membranes of the Caco-2 cells.

Polyphenolic profile and antiproliferative activity of bioaccessible fractions of zinc-fortified fruit beverages in human colon cancer cell lines.

INTRODUCTION: Colorectal cancer risks could be reduced by polyphenol-rich diets that inhibit tumour cell growth. AIMS: To determine the polyphenolic profile of four fruit beverages (FbZn, FbZnFe, FbZnM and FbZnFeM) as affected by the presence of Zn with/without Fe and with/without skimmed milk, and the digestion conditions. To evaluate the antiproliferative activity of bioaccessible fractions against Caco-2 and HT-29 cells. To clarify whether cell cycle arrest and/or apoptosis is involved in their possible antiproliferative activity. METHODS: The polyphenolic profiles were analyzed by RP-HPLC-DAD before and after in vitro gastrointestinal digestion. Cell proliferation and viability were measured using Trypan blue test, mitochondrial enzyme activity by means MTT test, cell cycle distribution using flow cytometry and apoptosis by means Hoechst dye. RESULTS AND DISCUSSION: The presence of zinc, iron and/or milk decreased the soluble extractable phenolic content before digestion probably by chelate formation, FbZn and FbZnFe being the samples with the highest soluble extractable phenolics. After digestion, a decrease in phenolics was observed in all zinc-fortified samples (up to 32% with respect to the original fruit beverages) - the FbZnFeM sample showing the lowest soluble extractable phenolic content, though with the lowest percentage decrease in phenolics (14%). FbZnM digest (approximately 50 microM total soluble extractable phenolics) was the sample that most inhibited Caco-2 and HT-29 cell proliferation after 24 h of incubation, without cytotoxicity. The specific combination of phytochemicals in FbZnM digest proved cytostatic and significantly suppressed proliferation through cell cycle arrest in the S-phase in both cell lines, without apoptosis.

Antioxidant capacity of infant fruit beverages: influence of storage and in vitro gastrointestinal digestion.

AIM: The total antioxidant capacity of three beverages based on fruit juice, milk and cereals, intended for infants and young children up to 3 years of age was evaluated by two methods Trolox Equivalent Antioxidant Capacity and Oxygen Radical Absorption Capacity. RESULTS: According to the total antioxidant values obtained by both methods, the beverages can be ranked as follows: grape-orange-banana > peach-apple > pineapple-banana. Ascorbic acid was the main contributor (60%) to the total antioxidant capacity, while the contribution of skimmed milk was less than 1.2%. After one month of storage at -20 degrees C, significant losses (p < 0.05) in total antioxidant capacity were found, though these were lower than 3% and therefore lacked nutritional significance. The bioaccessible fractions (maximum soluble fraction in simulated gastrointestinal media) of the beverages, obtained by in vitro gastrointestinal digestion, had antioxidant activities significantly lower (p < 0.05) than the original beverages, though the loss of antioxidant activity was always lower than 19%--thus indicating the stability of the total antioxidant capacity under the applied conditions. CONCLUSIONS: The total antioxidant capacity values of the bioaccessible fraction show that most antioxidants are available for absorption after digestion, and might contribute to the beneficial effects attributed to antioxidants.

Methylmercury determination in fish and seafood products and estimated daily intake for the Spanish population.

The mercury content of 25 samples of fish and seafood products most frequently consumed in Spain was determined. A simple method comprising cold vapour and atomic absorption spectrometry was used to determine separately inorganic and organic mercury. In all samples inorganic mercury content was below 50 microg kg(-1). There was wide variability, among not only the mercury levels of different fish species, but also for different samples of the same species - with the methylmercury content ranging from below 54 to 662 microg kg(-1). The highest mean methylmercury content was found in fresh tuna. Based on an average total fish consumption of 363 g/person week(-1), the methylmercury intake was estimated to be 46.2 microg/person week(-1). Therefore, the mercury intake of Spanish people with a body weight < or = 60 kg is lower than the Joint FAO/WHO Expert Committee on Food Additives (JECFA) provisional tolerable weekly intake (PTWI) of 1.6 microg kg(-1) body weight, but exceeds the US National Research Council (NRC) limit of 0.7 microg kg(-1) body weight week(-1) based on a benchmark dose.

Analysis of phytosterols in foods.

Phytosterols are bioactive compounds, one of their most studied and outstanding properties being their cholesterol-lowering activity. This explains the growing interest in the phytosterol contents of foods as either intrinsic or added components. The different steps (extraction, saponification, clean up, chromatographic determination) of plant sterol determination are reviewed, and emphasis is placed on the methods used to assay different phytosterols in food.

Optimización de la determinación porpolarografía diferencial de impulsos de ácidoascórbico en fórmulas para lactantes / Optimization of a differential pulse polarography determination of ascorbic acid in infant formulas

El objeto del estudio es la selección de las condiciones adecuadas para la preparación de la muestra y las instrumentales para la determinación de ácido ascórbico en fórmulas de base láctea para lactantes por polarografía diferencial de impulsos. La bondad del método se estima determinando los parámetros analíticos. Se obtiene un límite de detección equivalente a 15 mg de ácido ascórbico por 100 g de fórmula, una respuesta lineal en el intervalo de 15 a 550 mg de ácido ascórbico por 100g de muestra, una precisión interdía del 4,33 por ciento y una recuperación del 95 por ciento, valores que indican la bondad del método para el fin propuesto (AU)

Copper, iron, and zinc contents in human milk during the first three months of lactation: a longitudinal study.

The aim of the study has been to analyze the evolution of copper, iron, and zinc contents in human milk, from colostrum to the third postpartum month, following a longitudinal design, under specific conditions of sample collection and to apply an analytical procedure previously optimized to reduce any variation outside physiological lactation. The copper, iron, and zinc concentrations in 144 milk samples from 39 healthy puerpera women, were analyzed in five stages by flame atomic absorption spectrometry, following a standardized protocol. Copper presented a gradual decrease from 0.38 mg/L to 0.19 mg/L by the 90th day; the particular analysis from colostrum to transitional milk manifested the following two tendencies. Whereas an increase from 0.19 to 0.42 mg/L was observed in some women, a decrease from 0.53 to 0.45 mg/L was detected in others; therefore, copper presented two significant behaviors in the evolution from colostrum to transitional milk. In both cases, the evaluated changes were significant. The iron content varied from 0.56 to 0.40 mg/L by the 30th day, remaining constant until the first trimester concluded. The average zinc concentration decreased sharply from 7.99 to 3.3 mg/L on d 15; the rate of decrease slowed down gradually until 1.05 mg/L.

A study of factors that may influence the determination of copper, iron, and zinc in human milk during sampling and in sample individuals.

The aim of this study was to establish the possible effects of the sampling protocol (between-breast, within-feed, and diurnal differences) and the mother's personal factors (age, parity, iron supplementation, smoking habits, and lactation period) on the copper, iron, and zinc contents in human milk. One hundred thirty-six human milk samples identified by their origin and sampling conditions were analyzed. The samples were obtained from the 2nd to 15th d postpartum from 62 women. The data on the individuals required for the study were available. Mineral determinations were analyzed by flame atomic absorption spectrometry following a standardized protocol. The results showed that iron contents were higher in hind-milk samples and at the nighttime feeding and depended on the breast from which the sample was taken. The copper and zinc concentrations showed no significant variations. There was no significant relationship among the mothers' age, parity, smoking habits, iron supplementation, and copper content. Milk from older women had lower zinc contents than that of younger women. Increased amounts of iron were found in multiparous women. Between colostrum and transitional milk, a sharp decrease in zinc content was observed, whereas copper and iron contents remained constant. All of these results make it clear that standardized sampling protocols are needed in order to obtain comparable values.

In vitro dialyzability of zinc from different salts used in the supplementation of infant formulas.

Seven zinc salts--acetate, chloride, lactate, sulfate, citrate, gluconate, and oxide--were added to milk--and soy-based infant formulas to estimate possible differences in zinc availability depending on the type of salt used. For this purpose, an in vitro method that estimates the dialyzability of the element (i.e., the fraction available for absorption) was applied. Zinc dialyzability is always higher in milk-based products than in soy products, even when the total zinc contents are higher in the latter. The salts can be classified according to the zinc dialyzability in the two types of formulas as follows: oxide > gluconate = chloride = lactate > citrate = acetate > sulfate. Therefore, according to the dialysis percentage, oxide and gluconate are the compounds of choice for zinc supplementation of infant formulas.

Selenium, copper, and zinc indices of nutritional status: influence of sex and season on reference values.

The objective was to estimate the possible influence of season or season and sex on the selenium, glutathione peroxidase (GSH-Px), copper, zinc, and superoxide dismutase (SOD) values in plasma with a view to establishing reference values. For this purpose, 55 healthy volunteers (36 women and 19 men) were selected and their whole-blood samples were collected four times a year at the beginning of each season. In the population as a whole, no statistically significant seasonal differences were detected in copper, selenium, and GSH-Px values in plasma, whereas zinc and SOD values in plasma depended on the season. In general, the variability was higher among the women. In view of the differences detected according to sex and/or season in the parameters studied, we recommend taking samples from men and women and from different seasons to establish reference values.

Effect of proteins, phytates, ascorbic acid and citric acid on dialysability of calcium, iron, zinc and copper in soy-based infant formulas.

The possible effect of ascorbic acid, citric acid, proteins and phytate on dialysability of Ca, Fe, Zn and Cu in soy-based infant formulas is studied, taking dialysability as a measure of the amount of element available for absorption. Different dialysis percentages for similar element contents in different formulas are found. A regression analysis was applied between Ca, Zn, Cu and Fe dialysis percentages and soy-based formula components to estimate the possible influence of the latter on the dialysability of the elements. Significant correlations were found between citric acid contents and dialysability of Zn and Fe. No correlations were found between protein, ascorbic acid and phytic acid contents and the dialysis percentages of the four minerals. However, we must to point out that the range of protein contents was narrow and the ascorbic acid: iron ratio was high in our formulas.

Lipid peroxidation and antioxidant enzyme activities in patients with type 1 diabetes mellitus.

To evaluate oxidative stress in type I diabetes mellitus, two antioxidant enzymes in erythrocytes, copper-zinc superoxide dismutase (SOD EC 1.15.1.1.) and seleno-dependent glutathione peroxidase (GSH-Px; EC 1.11.19), and two indexes of peroxidation in plasma, thiobarbituric acid reactive substances (TBARS) and organic hydroperoxides (OHP), were measured in 118 patients with insulin-dependent diabetes mellitus (IDDM), classified in accordance with the presence or absence of vascular complications and the degree of metabolic control established by the HbA1c level. Ninety healthy subjects made up the control group. According to our results, plasmatic TBARS and OHP concentrations are significantly higher in diabetics than in controls, and these differences are accentuated in diabetic people with vascular disorders. The GSH-Px activity was significantly reduced in diabetic patients with poor and medium metabolic control in relation to the control group, regardless of the existence or absence of vascular disorders. No differences in SOD activity between diabetic and control groups were found. A significant positive correlation between TBARS and HPO (r=0.683, p<0.001) was found in both the control and diabetic groups. Among the lipid parameters studied, there were only significantly positive correlations between TBARS and total cholesterol; TBARS and triglycerides; OHP and total cholesterol and OHP and triglycerides. Positive correlations between TBARS and HbA1c and between OHP and and HbA1c, and negative correlations between GSH-Px and HbA1c and between SOD and HbA1c were also found. The multiple regression analysis shows that TBARS and HPO correlate negatively with GSH-Px. There was no significant correlation with SOD.

Selenium contents of human milk and infant formulas in Spain.

The selenium content of Spanish human milk samples and different milk-based and soy-based infant formulas has been estimated by using a flow injection hydride atomic absorption spectrometric method after microwave digestion of the organic matter. Mean values of 11.4 +/- 3.7 and 10.7 +/- 4.6 ng/ml for colostrum and transitional milk, 8.4 +/- 3.4 and 5.3 +/- 1.9 ng/ml for mature milk at 1 month and up to 2 months respectively, was obtained. These values are close to those reported by others authors in Europe, and lower than the ones from the US, Japan and Korea. Selenium contents of the analyzed infants' formulas ranged from 2.7 to 9.6 ng/ml and from 1.8 to 7.5 ng/ml for soy and milk-based infant formulas, respectively. The variability in selenium contents is large, although mean values are close to the ones given in other European countries. Selenium contents are not usually given on the product. The selenium intakes were estimated assuming that infants fed only human milk. The intakes ranged from 2.0 to 8.4 micrograms/day and from 3.4 to 12.9 micrograms/day for colostrum and transitional milk, respectively, and from 2.6 to 10.3 micrograms/day for mature milk at 1 month, and from 1.2 to 8.3 micrograms/day for milk up to 2 months. The analyzed infant formulas provide significantly less selenium than the 10 micrograms/day corresponding to the recommended daily allowance for infants from 0 to 6 months.

Whole blood selenium content in pregnant women.

The selenium content in the blood of pregnant women in the Valencian Community, Spain was determined until the end of gestation in order to assess its evolution and detect possible differences in relation to the values corresponding to non-pregnant women of fertile age. A total of 158 blood samples were obtained from 137 pregnant women. Samples were classified as: (a) first; (b) second; and (c) third trimester. Selenium was determined by a flow injection hydride atomic absorption spectrometric method (Seronorm whole blood was used as a quality control check). The selenium values obtained for the three trimesters followed a Gaussian distribution. The intervals for mean found were: (a) 75.7-85.5 micrograms/l; (b) 72.6-81.4 micrograms/l; and (c) 69.9-77.5 micrograms/l. Although a tendency to decrease was observed, no statistical significant differences between the three trimesters of the gestational period were observed. When we compare the values obtained with the blood selenium content of women of fertile age in the Valencian community (87.8-98.8 micrograms/l) by using an ANOVA test, statistically significant differences are found between the selenium values of women of fertile age and those corresponding to pregnant women. The differences, however, are very small and can be partially explained by the level of hemodilution during pregnancy, however, reference values of blood selenium contents in pregnant women should be taken into account.

Optimization of selenium determination in human milk and whole blood by flow injection hydride atomic absorption spectrometry.

A flow injection hydride atomic absorption spectrometric (FI-HAAS) method was developed for determining selenium in human milk and whole blood after microwave digestion of the sample. The sample (2 mL human milk or 0.25 mL blood) was introduced into the microwave vessel with 1.5 mL HNO3 and 0.25 mL H2O2 and 300 W (4 min) and 600 W (4 min) were applied. The digestion was completed by heating to 140 degrees C (2-3 h). Se (VI) was reduced to Se (IV) with hydrochloric acid. The instrumental conditions for FI-HAAS (concentrations of reducing agent and carrier acid, flow rate of argon carrier gas, and sample volume injected) were optimized. The detection limit of the proposed method was 0.23 ng/mL (assay) or 115 pg Se (absolute) in biological samples (1.15 ng/mL milk, 10.4 ng/mL blood). The precision values were 5.0% for milk and 4.0% for blood. The accuracy was evaluated with 2 reference materials, National Institute of Standards and Technology Non-Fat Milk Powder (found: 104.3 +/- 7.2 ng/g, certified: 110 +/- 10 ng/g) and Whole Blood Seronorm (found: 81 +/- 7.3 ng/mL, reference: 83 +/- 4 ng/mL). The results show the suitability of the method for selenium determination in human milk and whole blood. The method was applied to whole blood samples obtained from pregnant women and to human milk.

Dialyzability of iron, zinc, and copper of different types of infant formulas marketed in Spain.

The bioavailability of trace elements in infant formulas is affected by different physiological and dietetic factors. In vitro methods based on element dialyzability have been proposed to estimate the bioavailability. Infant formulas of the same type but from different manufacturers can differ in the salt used for supplementation and in the contents of other components that can affect mineral bioavailability. The aim of our study is to estimate the dialyzability of iron, zinc, and copper of formulas marketed in Spain, in order to detect possible differences in formulas of the same type coming from different manufacturers. At the same time, the effects of the type of formula, the composition of the protein fraction, and the mineral content on the element dialyzability are also studied. Differences are found in the dialysis percentages of the elements studied in formulas of the same type but from different manufacturers. The formulas giving the highest dialysis percentages for the three considered elements are the hypoallergenic ones based on protein hydrolysates. No differences are observed in formulas having whey or casein as the main protein fraction. Significant correlations are obtained between the element contents and the dialyzability of the elements.

Determination of plasma lipid hydroperoxides by an NADPH/NADP+ coupled enzyme reaction system. Evaluation of a method.

Several techniques based on different principles have been proposed to measure lipid hydroperoxides. Enzymatic methods are sensitive and can be quite specific but they are subject to interference by inhibitors and not all are stoichiometric. The present work proposes some modifications of the Heath & Tappel (Anal Biochem 1976; 7:184-91) enzymatic method of determination of lipid hydroperoxides in order to standardize and automate it and to meet the analytical criteria required for a biological assay. The proposed new protocol and the automated assay give acceptable within-run and between-run precisions, with coefficients of variation of 3.34% and 5.80%, respectively, at the usual plasma lipid hydroperoxides content. The recovery is 98.7 +/- 4.89%, and the method is linear for a wide range of contents and sensitive (10 mumol/l) enough to measure the plasma lipid hydroperoxides content.

Selenium and glutathione peroxidase reference values in whole blood and plasma of a reference population living in Valencia, Spain.

In order to assess the reference values for selenium nutritional status, adequate indicators (selenium concentration and glutathione peroxidase activity) were determined in whole blood and blood derivates of a healthy population (n = 287) from the province of Valencia, Spain. The reference population was selected by applying preestablished criteria. Selenium in whole blood and plasma was measured by graphite furnace atomic absorption spectrometry (GFAAS), with a deuterium correction, after addition of Pd/Mg(NO3)2 as the matrix modifier and appropriate dilution. Accuracy was checked by means of a reference material (Seronorm Trace Metals serum and whole blood). The population's reference intervals for selenium content at a 95% confidence level were: 53.03-108.96 and 66.71-119.4 mg/L for plasma and whole blood selenium concentration respectively. GPX activity was measured using a modification of the Paglia and Valentine method, and the reference intervals obtained ranged from 196 to 477 U/L in plasma, from 49 to 93 U/gHb in erythrocytes and from 52 to 96 U/gHb in whole blood. The only statistically significant differences detected between men and women are for to the GPX activity in whole blood. The results obtained are in the range of values found by others authors in healthy populations residing in different European countries.

Calcium, magnesium, sodium, potassium and iron content of infant formulas and estimated daily intakes.

The calcium, magnesium, sodium, potassium and iron content of a total of 22 different infant formulas marketed in Spain were measured by atomic spectrometry, and the mineral intake of infants fed exclusively with these formulas was estimated. The contents (mg/100kJ) are in the following ranges: Ca, 14-24; Mg, 1.1-2.8; Na, 5.6-9.8; K, 19-35; Fe, 0.02-0.50. These values coincide with those recommended by the Codex and European Society for Paediatric Gastroenterology and Nutrition (ESPGAN), and do not exceed the limits established by the European Union (EU). The mean values and ranges of estimated intakes for each formula type and period of infancy (0-1, 1-2, 2-3, 3-4 and 4-5 months) expressed in mg element/kg body weight are tabulated. The mean Ca, Mg, Na, K and Fe daily intakes of infants (0-5 months) fed with infant formulas meet the recommended values (RDA), except for the iron intake when non-iron supplemented formulas were used.