Gut microbiota in nonalcoholic fatty liver disease: a PREDIMED-Plus trial sub analysis.

To evaluate the changes in the gut microbiota associated with changes in the biochemical markers of nonalcoholic fatty liver disease (NAFLD) after a lifestyle intervention with the Mediterranean diet. Participants (n = 297) from two centers of PREDIMED-Plus trial (Prevención con Dieta Mediterránea) were divided into three different groups based on the change tertile in the Hepatic Steatosis Index (HSI) or the Fibrosis-4 score (FIB-4) between baseline and one year of intervention. One-year changes in HSI were: tertile 1 (T1) (-24.9 to -7.51), T2 (-7.5 to -1.86), T3 (-1.85 to 13.64). The most significant differences in gut microbiota within the year of intervention were observed in the T1 and T3. According to the FIB-4, participants were categorized in non-suspected fibrosis (NSF) and with indeterminate or suspected fibrosis (SF). NSF participants showed higher abundances of Alcaligenaceae, Bacteroidaceae, Bifidobacteriaceae, Clostridiaceae, Enterobacteriaceae, Peptostreptococcaceae, Verrucomicrobiaceae compared to those with SF. Then, participants were divided depending on the FIB-4 tertile of change: T1 (-89.60 to -5.57), T2 (-5.56 to 11.4), and T3 (11.41 to 206.24). FIB-4 T1 showed a decrease in Akkermansia and an increase in Desulfovibrio. T2 had an increase in Victivallaceae, Clostridiaceae, and Desulfovibrio. T3 showed a decrease in Enterobacteriaceae, and an increase in Sutterella, Faecalibacterium, and Blautia. A relation between biochemical index changes of NAFLD/NASH (HSI and FIB-4) and gut microbiota changes were found. These observations highlight the importance of lifestyle intervention in the modulation of gut microbiota and the management of metabolic syndrome and its hepatic manifestations.

What You Need to KnowWhat is the context:Obesity and metabolic syndrome have been associated with nonalcoholic fatty liver disease (NAFLD). Gut microbiota and its interaction with the environment may play a key role in NAFLD.What is new:Mediterranean diet and physical activity can modify the scores for liver steatosis (HSI) and liver fibrosis (FIB−4) in only one year. A relation between the changes in these scores and gut microbiota changes was found.What is the impact:The discovery of microbiota-based biomarkers for NAFLD and the development of strategies to modulate gut microbiota in the treatment of NAFLD.

Water intake, hydration status and 2-year changes in cognitive performance: a prospective cohort study.

BACKGROUND: Water intake and hydration status have been suggested to impact cognition; however, longitudinal evidence is limited and often inconsistent. This study aimed to longitudinally assess the association between hydration status and water intake based on current recommendations, with changes in cognition in an older Spanish population at high cardiovascular disease risk. METHODS: A prospective analysis was conducted of a cohort of 1957 adults (aged 55-75) with overweight/obesity (BMI between ≥ 27 and < 40 kg/m2) and metabolic syndrome from the PREDIMED-Plus study. Participants had completed bloodwork and validated, semiquantitative beverage and food frequency questionnaires at baseline, as well as an extensive neuropsychological battery of 8 validated tests at baseline and 2 years of follow-up. Hydration status was determined by serum osmolarity calculation and categorized as < 295 mmol/L (hydrated), 295-299.9 mmol/L (impending dehydration), and ≥ 300 mmol/L (dehydrated). Water intake was assessed as total drinking water intake and total water intake from food and beverages and according to EFSA recommendations. Global cognitive function was determined as a composite z-score summarizing individual participant results from all neuropsychological tests. Multivariable linear regression models were fitted to assess the associations between baseline hydration status and fluid intake, continuously and categorically, with 2-year changes in cognitive performance. RESULTS: The mean baseline daily total water intake was 2871 ± 676 mL/day (2889 ± 677 mL/day in men; 2854 ± 674 mL/day in women), and 80.2% of participants met the ESFA reference values for an adequate intake. Serum osmolarity (mean 298 ± 24 mmol/L, range 263 to 347 mmol/L) indicated that 56% of participants were physiologically dehydrated. Lower physiological hydration status (i.e., greater serum osmolarity) was associated with a greater decline in global cognitive function z-score over a 2-year period (ß: - 0.010; 95% CI - 0.017 to - 0.004, p-value = 0.002). No significant associations were observed between water intake from beverages and/or foods with 2-year changes in global cognitive function. CONCLUSIONS: Reduced physiological hydration status was associated with greater reductions in global cognitive function over a 2-year period in older adults with metabolic syndrome and overweight or obesity. Future research assessing the impact of hydration on cognitive performance over a longer duration is needed. TRIAL REGISTRATION: International Standard Randomized Controlled Trial Registry, ISRCTN89898870. Retrospectively registered on 24 July 2014.

Factores predictores de resultado en tratamiento de tabaquismo en población de adultos mayores de la Comunidad Valenciana / Predictors of smoking treatment outcome in a population of older adults in the Valencian Community

El consumo de tabaco es uno de los más importantes factores de riesgo de enfermedad y muerte en España. Por ello, el objetivo de este estudio fue investigar las características epidemiológicas de 239pacientes fumadores mayores de 50 años en una unidad hospitalaria de deshabituación de tabaco, así como analizar los principales factores predictores que pueden influir en sus intentos de cese a losdoce meses. Es un estudio analítico transversal con tratamiento multicomponente combinando terapia psicológica e intervención farmacológica. Para comparar el éxito de abandono de los pacientes yconocer los posibles factores predictores, se llevó a cabo un análisis multivariante y de regresión logística binaria. De todos los pacientes, el 49,7% estableció el día D y la tasa de abandono final fue de 41,4%. Los predictores de intentos de abandono significativos fueron: índice paquetes-año entre 30 y 60, valor de cooximetría ≤ 10ppm, con una o más veces de intentos previos, tiempo máximo de cese superior a tres meses y grado alto en el test de Richmond. La escala de Minnesota con un valor inferior a 5 puntos fue el único predictor de abstinencia puntual a los doce meses. Los pacientes que consumieron menos tabaco y manifestaron intentos previos y alto grado de motivación tuvieron mayor posibilidad de tomar la decisión con éxito para dejar de fumar. Además, controlar el síndrome de abstinencia fue el aspecto más importante para tratar y reducir la tasa de recaída. (AU)

Tobacco consumption is one of the most important risk factors for disease and death in Spain. Therefore, the aim of this study was to investigate the epidemiological characteristics of 239 smoking patients over 50 years of age in a hospital smoking cessation unit, as well as to analyse the main predictors that may influence their cessation attempts at 12 months. It is a cross-sectional analytical study with multicomponent treatment combining psychological therapy and pharmacological intervention. A multivariate and binary logistic regression analysis was carried out to compare patients’ cessation success and to identify possible predictors. Of all patients, 49.7% established D-day and the final quit rate was 41.4%. Significant predictors of quit attempts were: pack-year index between 30 and 60, cooximetry value ≤ 10ppm, with one or more times of previous attempts, maximum cessation time greater than three months and high grade on the Richmond test. The Minnesota scale with a value of less than 5 points was the only predictor of timely abstinence at 12 months. Patients who used less tobacco and reported previous attempts and high motivation were more likely to make a successful decision to quit smoking. In addition, controlling the withdrawal syndrome was the most important aspect to treat and reduce the relapse rate. (AU)

Timing of Meals and Sleep in the Mediterranean Population: The Effect of Taste, Genetics, Environmental Determinants, and Interactions on Obesity Phenotypes.

Circadian rhythms regulate the sleep-wake and feeding-fasting cycles. Sleep and feeding constitute a complex cycle that is determined by several factors. Despite the importance of sleep duration and mealtimes for many obesity phenotypes, most studies on dietary patterns have not investigated the contribution of these variables to the phenotypes analyzed. Likewise, they have not investigated the factors related to sleep or mealtimes. Thus, our aims were to investigate the link between taste perception and eating/sleep patterns and to analyze the effect of the interactions between sleep/meal patterns and genetic factors on obesity phenotypes. We conducted a cross-sectional analysis on 412 adults from the Mediterranean population. We measured taste perception (bitter, sweet, salty, sour, and umami) and assessed sleep duration and waketime. The midpoint of sleep and social jetlag was computed. From the self-reported timing of meals, we estimated the eating window, eating midpoint, and eating jetlag. Adherence to the Mediterranean diet was measured with a validated score. Selected polymorphisms in the TAS2R38, CLOCK, and FTO genes were determined, and their associations and interactions with relevant phenotypes were analyzed. We found various associations between temporal eating, sleep patterns, and taste perception. A higher bitter taste perception was associated with an earlier eating midpoint (p = 0.001), breakfast time (p = 0.043), dinner time (p = 0.009), waketime (p < 0.001), and midpoint of sleep (p = 0.009). Similar results were observed for the bitter taste polymorphism TAS2R38-rs713598, a genetic instrumental variable for bitter perception, increasing the causality of the associations. Moreover, significant gene-sleep interactions were detected between the midpoint of sleep and the TAS2R38-rs713598 (p = 0.032), FTO-rs9939609 (p = 0.037), and CLOCK-rs4580704 (p = 0.004) polymorphisms which played a role in determining obesity phenotypes. In conclusion, our study provided more information on the sleep and mealtime patterns of the general Spanish Mediterranean population than on their main relationships. Moreover, we were able to show significant associations between taste perception, specifically bitter taste; sleep time; and mealtimes as well as an interaction between sleep time and several genetic variants linked to obesity phenotypes. However, additional research is needed to better characterize the causality and mechanisms behind these associations.

DNA-Methylation Signatures of Tobacco Smoking in a High Cardiovascular Risk Population: Modulation by the Mediterranean Diet.

Biomarkers based on DNA methylation are relevant in the field of environmental health for precision health. Although tobacco smoking is one of the factors with a strong and consistent impact on DNA methylation, there are very few studies analyzing its methylation signature in southern European populations and none examining its modulation by the Mediterranean diet at the epigenome-wide level. We examined blood methylation smoking signatures on the EPIC 850 K array in this population (n = 414 high cardiovascular risk subjects). Epigenome-wide methylation studies (EWASs) were performed analyzing differential methylation CpG sites by smoking status (never, former, and current smokers) and the modulation by adherence to a Mediterranean diet score was explored. Gene-set enrichment analysis was performed for biological and functional interpretation. The predictive value of the top differentially methylated CpGs was analyzed using receiver operative curves. We characterized the DNA methylation signature of smoking in this Mediterranean population by identifying 46 differentially methylated CpGs at the EWAS level in the whole population. The strongest association was observed at the cg21566642 (p = 2.2 × 10-32) in the 2q37.1 region. We also detected other CpGs that have been consistently reported in prior research and discovered some novel differentially methylated CpG sites in subgroup analyses. In addition, we found distinct methylation profiles based on the adherence to the Mediterranean diet. Particularly, we obtained a significant interaction between smoking and diet modulating the cg5575921 methylation in the AHRR gene. In conclusion, we have characterized biomarkers of the methylation signature of tobacco smoking in this population, and suggest that the Mediterranean diet can increase methylation of certain hypomethylated sites.

Association between ultra-processed food consumption and gut microbiota in senior subjects with overweight/obesity and metabolic syndrome.

The production and consumption of ultra-processed foods (UPF) has increased considerably during the last years worldwide. Collective evidence shows the association between UPF consumption and adverse health outcomes, including inflammatory gastro-intestinal disorders and obesity. The gut microbiota has been suggested as potential mediator of the effects of UPF consumption on metabolism and health. However, few studies have been conducted in order to elucidate these aspects. Therefore, the aim of the present study was to assess the cross-sectional associations between UPF consumption and gut microbiota in a population of senior subjects (n = 645) within the frame of the PREDIMED-Plus trial. Eligible participants were men and women (aged 55-75 years), without documented history of cardiovascular disease at enrollment, with overweight/obesity (body mass index ≤ 27 and <40 kg/m2) and metabolic syndrome. Using the information of food frequency questionnaires, the consumption of UPF, expressed as a percentage of total dietary energy intake in kcal/day, was calculated considering those food items classified in group 4 of NOVA system. Population was categorized according to tertiles of UPF consumption. Taxonomic fecal microbiota information, along with blood biochemical parameters, anthropometric measurements and clinical data were obtained. Bioinformatics analysis was performed to study the association between fecal microbiota composition and UPF consumption. We observed that subjects allocated in the highest tertile of UPF consumption (21.4 ± 5.0 % kcal/day) presented lower adherence to MedDiet (p < 0.001) and higher total energy intake (p < 0.001). The taxonomic analysis of the fecal microbiota revealed a significant (Benjamini-Hochberg adjusted p < 0.2) positive association between specific taxa and tertiles (T) of UPF consumption: Alloprevotella (p = 0.041 vs. T2; p = 0.065 vs. T3), Negativibacillus (p = 0.096 vs. T3), Prevotella (p = 0.116 vs. T3), and Sutterella (p = 0.116 vs. T2). UPF consumption was positively associated with lower adherence to MedDiet and higher total energy intake in senior subjects with overweight obesity and metabolic syndrome. In addition, positive association with specific fecal microbiota taxa related to inflammatory gastro-intestinal diseases and low consumption of fruits and vegetables, was observed.

Association between Microbiome-Related Human Genetic Variants and Fasting Plasma Glucose in a High-Cardiovascular-Risk Mediterranean Population.

Background and Objectives: The gut microbiota has been increasingly recognized as a relevant factor associated with metabolic diseases. However, directly measuring the microbiota composition is a limiting factor for several studies. Therefore, using genetic variables as proxies for the microbiota composition is an important issue. Landmark microbiome-host genome-wide association studies (mbGWAS) have identified many SNPs associated with gut microbiota. Our aim was to analyze the association between relevant microbiome-related genetic variants (Mi-RSNPs) and fasting glucose and type 2 diabetes in a Mediterranean population, exploring the interaction with Mediterranean diet adherence. Materials and Methods: We performed a cross-sectional study in a high-cardiovascular-risk Mediterranean population (n = 1020), analyzing the association of Mi-RSNPs (from four published mbGWAS) with fasting glucose and type 2 diabetes. A single-variant approach was used for fitting fasting glucose and type 2 diabetes to a multivariable regression model. In addition, a Mendelian randomization analysis with multiple variants was performed as a sub-study. Results: We obtained several associations between Mi-RSNPs and fasting plasma glucose involving gut Gammaproteobacteria_HB, the order Rhizobiales, the genus Rumminococcus torques group, and the genus Tyzzerella as the top ranked. For type 2 diabetes, we also detected significant associations with Mi-RSNPs related to the order Rhizobiales, the family Desulfovibrionaceae, and the genus Romboutsia. In addition, some Mi-RSNPs and adherence to Mediterranean diet interactions were detected. Lastly, the formal Mendelian randomization analysis suggested combined effects. Conclusions: Although the use of Mi-RSNPs as proxies of the microbiome is still in its infancy, and although this is the first study analyzing such associations with fasting plasma glucose and type 2 diabetes in a Mediterranean population, some interesting associations, as well as modulations, with adherence to the Mediterranean diet were detected in these high-cardiovascular-risk subjects, eliciting new hypotheses.

Glycemic Dysregulations Are Associated With Worsening Cognitive Function in Older Participants at High Risk of Cardiovascular Disease: Two-Year Follow-up in the PREDIMED-Plus Study.

Introduction: Type 2 diabetes has been linked to greater cognitive decline, but other glycemic parameters such as prediabetes, diabetes control and treatment, and HOMA-IR and HbA1c diabetes-related biomarkers have shown inconsistent results. Furthermore, there is limited research assessing these relationships in short-term studies. Thus, we aimed to examine 2-year associations between baseline diabetes/glycemic status and changes in cognitive function in older participants at high risk of cardiovascular disease. Methods: We conducted a 2-year prospective cohort study (n=6,874) within the framework of the PREDIMED-Plus study. The participants (with overweight/obesity and metabolic syndrome; mean age 64.9 years; 48.5% women) completed a battery of 8 cognitive tests, and a global cognitive function Z-score (GCF) was estimated. At baseline, participants were categorized by diabetes status (no-diabetes, prediabetes, and <5 or ≥5-year diabetes duration), and also by diabetes control. Furthermore, insulin resistance (HOMA-IR) and glycated hemoglobin (HbA1c) levels were measured, and antidiabetic medications were recorded. Linear and logistic regression models, adjusted by potential confounders, were fitted to assess associations between glycemic status and changes in cognitive function. Results: Prediabetes status was unrelated to cognitive decline. However, compared to participants without diabetes, those with ≥5-year diabetes duration had greater reductions in GCF (ß=-0.11 (95%CI -0.16;-0.06)], as well as in processing speed and executive function measurements. Inverse associations were observed between baseline HOMA-IR and changes in GCF [ß=-0.0094 (95%CI -0.0164;-0.0023)], but also between HbA1c levels and changes in GCF [ß=-0.0085 (95%CI -0.0115, -0.0055)], the Mini-Mental State Examination, and other executive function tests. Poor diabetes control was inversely associated with phonologic fluency. The use of insulin treatment was inversely related to cognitive function as measured by the GCF [ß=-0.31 (95%CI -0.44, -0.18)], and other cognitive tests. Conclusions: Insulin resistance, diabetes status, longer diabetes duration, poor glycemic control, and insulin treatment were associated with worsening cognitive function changes in the short term in a population at high cardiovascular risk. Clinical Trial Registration: http://www.isrctn.com/ISRCTN89898870, identifier ISRCTN: 89898870.

Metformin Use and Cognitive Function in Older Adults With Type 2 Diabetes Following a Mediterranean Diet Intervention.

Background and Purpose: Both adherence to the Mediterranean diet (MedDiet) and the use of metformin could benefit the cognitive performance of individuals with type 2 diabetes, but evidence is still controversial. We examined the association between metformin use and cognition in older adults with type 2 diabetes following a MedDiet intervention. Methods: Prospective cohort study framed in the PREDIMED-Plus-Cognition sub-study. The PREDIMED-Plus clinical trial aims to compare the cardiovascular effect of two MedDiet interventions, with and without energy restriction, in individuals with overweight/obesity and metabolic syndrome. The present sub-study included 487 cognitively normal subjects (50.5% women, mean ± SD age of 65.2 ± 4.7 years), 30.4% of them (N = 148) with type 2 diabetes. A comprehensive battery of neurocognitive tests was administered at baseline and after 1 and 3 years. Individuals with type 2 diabetes that exhibited a good glycemic control trajectory, either using or not using metformin, were compared to one another and to individuals without diabetes using mixed-effects models with inverse probability of treatment weights. Results: Most subjects with type 2 diabetes (83.1%) presented a good and stable glycemic control trajectory. Before engaging in the MedDiet intervention, subjects using metformin scored higher in executive functions (Cohen's d = 0.51), memory (Cohen's d = 0.38) and global cognition (Cohen's d = 0.48) than those not using metformin. However, these differences were not sustained during the 3 years of follow-up, as individuals not using metformin experienced greater improvements in memory (ß = 0.38 vs. ß = 0.10, P = 0.036), executive functions (ß = 0.36 vs. ß = 0.02, P = 0.005) and global cognition (ß = 0.29 vs. ß = -0.02, P = 0.001) that combined with a higher MedDiet adherence (12.6 vs. 11.5 points, P = 0.031). Finally, subjects without diabetes presented greater improvements in memory than subjects with diabetes irrespective of their exposure to metformin (ß = 0.55 vs. ß = 0.10, P < 0.001). However, subjects with diabetes not using metformin, compared to subjects without diabetes, presented greater improvements in executive functions (ß = 0.33 vs. ß = 0.08, P = 0.032) and displayed a higher MedDiet adherence (12.6 points vs. 11.6 points, P = 0.046). Conclusions: Although both metformin and MedDiet interventions are good candidates for future cognitive decline preventive studies, a higher adherence to the MedDiet could even outweigh the potential neuroprotective effects of metformin in subjects with diabetes.

Sweet Taste Preference: Relationships with Other Tastes, Liking for Sugary Foods and Exploratory Genome-Wide Association Analysis in Subjects with Metabolic Syndrome.

Taste perception and its association with nutrition and related diseases (type 2 diabetes, obesity, metabolic syndrome, cardiovascular, etc.) are emerging fields of biomedicine. There is currently great interest in investigating the environmental and genetic factors that influence sweet taste and sugary food preferences for personalized nutrition. Our aims were: (1) to carry out an integrated analysis of the influence of sweet taste preference (both in isolation and in the context of other tastes) on the preference for sugary foods and its modulation by type 2 diabetes status; (2) as well as to explore new genetic factors associated with sweet taste preference. We studied 425 elderly white European subjects with metabolic syndrome and analyzed taste preference, taste perception, sugary-foods liking, biochemical and genetic markers. We found that type 2 diabetic subjects (38%) have a small, but statistically higher preference for sweet taste (p = 0.021) than non-diabetic subjects. No statistically significant differences (p > 0.05) in preferences for the other tastes (bitter, salty, sour or umami) were detected. For taste perception, type 2 diabetic subjects have a slightly lower perception of all tastes (p = 0.026 for the combined "total taste score"), bitter taste being statistically lower (p = 0.023). We also carried out a principal component analysis (PCA), to identify latent variables related to preferences for the five tastes. We identified two factors with eigenvalues >1. Factor 2 was the one with the highest correlation with sweet taste preference. Sweet taste preference was strongly associated with a liking for sugary foods. In the exploratory SNP-based genome-wide association study (GWAS), we identified some SNPs associated with sweet taste preference, both at the suggestive and at the genome-wide level, especially a lead SNP in the PTPRN2 (Protein Tyrosine Phosphatase Receptor Type N2) gene, whose minor allele was associated with a lower sweet taste preference. The PTPRN2 gene was also a top-ranked gene obtained in the gene-based exploratory GWAS analysis. In conclusion, sweet taste preference was strongly associated with sugary food liking in this population. Our exploratory GWAS identified an interesting candidate gene related with sweet taste preference, but more studies in other populations are required for personalized nutrition.

Apoptotic effect of a phytosterol-ingredient and its main phytosterol (ß-sitosterol) in human cancer cell lines.

Dietary interventions may effectively control cancer development, with phytosterols (PS) being a class of cancer chemopreventive dietary phytochemicals. The present study, for the first time, evaluates the antiproliferative effects of a PS-ingredient used for the enrichment of several foods and its main PS, ß-sitosterol, at physiological serum levels, in the most prevalent cancer cells in women (breast (MCF-7), colon (HCT116) and cervical (HeLa)). In all three cell lines, these compounds induced significant cell viability reduction without a clear time- and dose-dependent response. Moreover, all treatments produced apoptotic cell death with the induction of DNA fragmentation through the appearance of a sub-G1 cell population. Thus, the use of PS as functional ingredients in the development of PS-enriched foods could exert a potential preventive effect against human breast, colon and cervical cancer, although further in vivo studies are required to confirm our preclinical findings.

A positive impact on the serum lipid profile and cytokines after the consumption of a plant sterol-enriched beverage with a milk fat globule membrane: a clinical study.

The hypocholesterolemic effect and the modification of serum biomarkers of a dietary plant sterol (PS) intake, cholesterol precursors and cytokines after the consumption of milk-based fruit beverages with a milk fat globule membrane were evaluated by a randomized, double-blind, crossover, multiple dose bioavailability study. Postmenopausal women (n = 38) consumed daily 250 mL of a beverage with or without 2 g of PS added during 6 weeks in each of the study periods. With the intake of the PS-added beverage, significant decreases (mg dL-1) in serum total cholesterol (pre-treatment: 220.0 ± 27.8 vs. post-treatment: 212.9 ± 25.8; p < 0.05) and LDL-cholesterol (129.4 ± 28.5 vs. 121.7 ± 24.4; p < 0.05) were detected. The cholesterol precursor lathosterol (11.2%), markers of the dietary PS intake (campesterol 43.1% and ß-sitosterol 32.5%), and anti-inflammatory IL-10 cytokine (22.5%) increased significantly, with a concomitant significant reduction in pro-inflammatory IL-1ß (6.7%). No variations in HDL-cholesterol, other sterols (desmosterol and stigmasterol) or cytokines (IL-6, IL-8, IL-12p70 and TNF-α) were detected. These results indicated that this kind of PS-enriched milk-based fruit beverage is suitable during the period of clinical intervention, and its consumption may be an adequate way to improve PS functionality since a significant reduction in cholesterol levels has been observed. Therefore, the intake of this beverage could contribute to reduce the risk of cardiovascular disease also obtaining a beneficial effect on the serum inflammatory status in postmenopausal women.

Effects of Plant Sterols or ß-Cryptoxanthin at Physiological Serum Concentrations on Suicidal Erythrocyte Death.

The eryptotic and hemolytic effects of a phytosterol (PS) mixture (ß-sitosterol, campesterol, stigmasterol) or ß-cryptoxanthin (ß-Cx) at physiological serum concentration and their effect against oxidative stress induced by tert-butylhydroperoxide (tBOOH) (75 and 300 µM) were evaluated. ß-Cryptoxanthin produced an increase in eryptotic cells, cell volume, hemolysis, and glutathione depletion (GSH) without ROS overproduction and intracellular Ca2+ influx. Co-incubation of both bioactive compounds protected against ß-Cx-induced eryptosis. Under tBOOH stress, PS prevented eryptosis, reducing Ca2+ influx, ROS overproduction and GSH depletion at 75 µM, and hemolysis at both tBOOH concentrations. ß-Cryptoxanthin showed no cytoprotective effect. Co-incubation with both bioactive compounds completely prevented hemolysis and partially prevented eryptosis as well as GSH depletion induced by ß-Cx plus tBOOH. Phytosterols at physiological serum concentrations help to prevent pro-eryptotic and hemolytic effects and are promising candidate compounds for ameliorating eryptosis-associated diseases.

Impact of Lipid Components and Emulsifiers on Plant Sterols Bioaccessibility from Milk-Based Fruit Beverages.

Sterol bioaccessibility (BA) of three plant sterol (PS)-enriched milk-based fruit beverages (MFb) with different fat contents (1.1-2.4%), lipid sources (animal or vegetable), and without or with emulsifiers (whey proteins enriched with milk fat globule membrane (MFGM) or soy lecithin) was evaluated after simulated gastrointestinal digestion. The BA of total PS followed the order 31.4% (MFbM containing milk fat and whey proteins enriched with MFGM) = 28.2% (MFbO containing extra virgin olive oil and soy lecithin) > 8.7% (MFb without fat addition). Total and individual PS content in the bioaccessible fractions followed the order MFbM > MFbO > MFb. Consequently, formulation with MFGM is proposed in beverages of this kind to ensure optimum bioavailability of PS. Our results suggest that the BA of PS is influenced by the type and quantity of fat and the emulsifier type involved.

DETERMINATION OF CHOLESTEROL IN HUMAN MILK: AN ALTERNATIVE TO CHROMATOGRAPHIC METHODS.

INTRODUCTION: human milk (HM) is considered the best option for feeding healthy infants. Cholesterol (CHOL) is important for proper development of the nervous system, and for hormone and vitamin synthesis in growing infants. Breastfeeding and dietary CHOL intake during infancy have been suggested to affect blood lipid levels and the risk of cardiovascular disease in adulthood. Gas chromatography is the technique most widely used to determine CHOL in HM. Chromatographic methods are specific for the determination of CHOL and other sterols present in HM, but are extremely time consuming, and the costs and equipment requirements mean that they are not accessible to all laboratories. AIM: the present study describes the optimization and validation of an enzymatic-spectrophotometric method for CHOL determination in mature HM. METHOD: determination of CHOL involves fat extraction with chloroform:methanol, hot saponification and extraction of the unsaponifiable fraction with diethyl ether. CHOL was determined by an enzymatic method in which the concentration of the lutidine dye formed is stoichiometric to the amount of CHOL, and is measured by the increase in light absorbance at 405 nm. RESULTS: human milk fat (mg/mL) (27.5 ± 1.3) and CHOL (0.113 ± 0.004) in analyzed HM were within the ranges reported by others authors. Analytical parameters of the proposed method were assessed: The precision values (%) (expressed as the relative standard deviation (RSD)) were: 3.5 and 6.7 for intra- and inter-day, respectively. Accuracy, estimated by recovery assays, was 110 ± 1.6%. CONCLUSION: the validated enzymatic-spectrophotometric method for determining CHOL in HM constitutes an alternative for fast and simple analysis of CHOL with equipment requirements accessible to any laboratory.

Introducción: la leche humana (HM) se considera el modo óptimo de alimentación en lactantes sanos. El colesterol (CHOL) es importante para el correcto desarrollo del sistema nervioso y la síntesis de hormonas y vitaminas en el crecimiento del lactante. Se ha constatado que la lactancia materna y la ingesta dietética de CHOL durante la infancia influye en los niveles de lípidos en sangre, así como en el riesgo de enfermedad cardiovascular en la edad adulta. La técnica más utilizada para determinar el CHOL en HM es la cromatografía de gases. Los métodos cromatográficos son específicos para la determinación del CHOL y otros esteroles presentes en la HM, pero el elevado tiempo consumido, los costes y la necesidad de una instrumentación específica hacen que no sea accesible para cualquier laboratorio. Objetivo: el presente estudio describe la optimización y validación de un método enzimático-espectrofotométrico para la determinación del CHOL en HM madura. Métodos: la determinación del CHOL requiere una extracción lipídica con cloroformo:metanol, saponificación en caliente y extracción del insaponificable con dietil éter. El CHOL fue determinado por un método enzimático en el que la concentración de lutidina formada es estequiométrica a la cantidad de CHOL, y se mide a 405 nm. Resultados: la cantidad de grasa (mg/mL) (27,5 ± 1,3) y de CHOL (0,113 ± 0,004) en la HM analizada se halla en el intervalo indicado por otros autores. Se evalúan parámetros analíticos del método propuesto: la precisión (expresada como desviación estándar relativa en %) fue de 3,5 y 6,7 para intra- e interdía, respectivamente. La exactitud, estimada mediante ensayos de recuperación, fue de 110 ± 1,6%. Conclusión: el método enzimático-espectrofotométrico validado para determinar el CHOL en HM constituye una alternativa para el análisis rápido y sencillo de CHOL empleando equipos accesibles para cualquier laboratorio.

Determination of cholesterol in human milk: an alternative to chromatographic methods / Determinación del colesterol en leche humana: una alternativa a los métodos cromatográficos

Introduction: human milk (HM) is considered the best option for feeding healthy infants. Cholesterol (CHOL) is important for proper development of the nervous system, and for hormone and vitamin synthesis in growing infants. Breastfeeding and dietary CHOL intake during infancy have been suggested to affect blood lipid levels and the risk of cardiovascular disease in adulthood. Gas chromatography is the technique most widely used to determine CHOL in HM. Chromatographic methods are specific for the determination of CHOL and other sterols present in HM, but are extremely time consuming, and the costs and equipment requirements mean that they are not accessible to all laboratories. Aim: the present study describes the optimization and validation of an enzymatic-spectrophotometric method for CHOL determination in mature HM. Method: determination of CHOL involves fat extraction with chloroform:methanol, hot saponification and extraction of the unsaponifiable fraction with diethyl ether. CHOL was determined by an enzymatic method in which the concentration of the lutidine dye formed is stoichiometric to the amount of CHOL, and is measured by the increase in light absorbance at 405 nm. Results: human milk fat (mg/mL) (27.5 ± 1.3) and CHOL (0.113 ± 0.004) in analyzed HM were within the ranges reported by others authors. Analytical parameters of the proposed method were assessed: The precision values (%) (expressed as the relative standard deviation (RSD)) were: 3.5 and 6.7 for intra- and inter-day, respectively. Accuracy, estimated by recovery assays, was 110 ± 1.6%. Conclusion: the validated enzymatic-spectrophotometric method for determining CHOL in HM constitutes an alternative for fast and simple analysis of CHOL with equipment requirements accessible to any laboratory (AU)

Introducción: la leche humana (HM) se considera el modo óptimo de alimentación en lactantes sanos. El colesterol (CHOL) es importante para el correcto desarrollo del sistema nervioso y la síntesis de hormonas y vitaminas en el crecimiento del lactante. Se ha constatado que la lactancia materna y la ingesta dietética de CHOL durante la infancia influye en los niveles de lípidos en sangre, así como en el riesgo de enfermedad cardiovascular en la edad adulta. La técnica más utilizada para determinar el CHOL en HM es la cromatografía de gases. Los métodos cromatográficos son específicos para la determinación del CHOL y otros esteroles presentes en la HM, pero el elevado tiempo consumido, los costes y la necesidad de una instrumentación específica hacen que no sea accesible para cualquier laboratorio. Objetivo: el presente estudio describe la optimización y validación de un método enzimático-espectrofotométrico para la determinación del CHOL en HM madura. Métodos: la determinación del CHOL requiere una extracción lipídica con cloroformo:metanol, saponificación en caliente y extracción del insaponificable con dietil éter. El CHOL fue determinado por un método enzimático en el que la concentración de lutidina formada es estequiométrica a la cantidad de CHOL, y se mide a 405 nm. Resultados: la cantidad de grasa (mg/mL) (27,5 ± 1,3) y de CHOL (0,113 ± 0,004) en la HM analizada se halla en el intervalo indicado por otros autores. Se evalúan parámetros analíticos del método propuesto: la precisión (expresada como desviación estándar relativa en %) fue de 3,5 y 6,7 para intra- e interdía, respectivamente. La exactitud, estimada mediante ensayos de recuperación, fue de 110 ± 1,6%. Conclusión: el método enzimático-espectrofotométrico validado para determinar el CHOL en HM constituye una alternativa para el análisis rápido y sencillo de CHOL empleando equipos accesibles para cualquier laboratorio (AU)