Daily consumption of milk enriched with fish oil, oleic acid, minerals and vitamins reduces cell adhesion molecules in healthy children.

BACKGROUND AND AIMS: Several studies have suggested that polyunsaturated fatty acids, vitamins and minerals have beneficial effects on lipid profile and systemic inflammation in adults. METHODS AND RESULTS: We examined the effects of a daily intake of milk enriched with longchain polyunsaturated fatty acids, oleic acid, carbohydrates, vitamins, minerals and low in saturated fatty acids (SFAs) for 5 months, on several cardiovascular (CVD) risk biomarkers in healthy children aged 8-14 years. In a randomized double-blind placebo-controlled trial, a total of 107 children of both genders were assigned to two study groups: 1) a supplemented group (SG, n=53) who consumed 0.6 L/day of an enriched dairy product, and 2) a control group (CG, n=54) who consumed 0.6 L/day of standard whole milk. Both groups consumed the dairy drinks for 5 months, in addition to their usual diet. Serum levels of adhesion molecules as indices of vascular endothelial cell activation were assessed in both groups at 0 and 5 months as well as white blood cell counts, lipid profile, serum proteins, total serum calcium, 25-OH vitamin D, glucose, insulin and adiponectin. In the enriched dairy drink supplemented group, adhesion molecules E-selectin and ICAM-1 as well as lymphocyte levels decreased while plasma docosahexaenoic acid (DHA) and serum calcium concentrations increased. In the control group, serum total protein, transferrin, total cholesterol, HDL-cholesterol and adiponectin concentrations decreased. CONCLUSION: The consumption of a milk enriched with fish oil, oleic acid, minerals and vitamins reduced indices of endothelial cell activation in the studied group of healthy children.

[Intake of an iron-supplemented milk formula as a preventive measure to avoid low iron status in 1-3 year-olds]. / Ingesta de una fórmula láctea suplementada con hierro como medida preventiva del déficit de hierro en niños de 1 a 3 años de edad.

OBJECTIVE: Low iron status is a well known risk factor for iron deficiency anemia in infants and young children. The aim of the present study was to evaluate the influence of an iron-fortified toddler formula on iron status in 1-3 year-olds. PATIENTS AND METHODS: Thirty-three healthy infants and young children were assigned to two groups that received 500 mL/day of and iron-fortified toddler formula or 500 mL/day of unmodified cow's milk for 4 months. Allocation was random and double-blind. Daily dietary intake was calculated by dietary evaluation, and iron nutritional status was assessed (hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, serum iron, ferritin, and transferrin). RESULTS: At enrollment, no anemia was found in either group, although hemoglobin concentration and hematocrit were significantly lower in the toddler formula group than in the unmodified cow's milk group. However, these differences disappeared at the end of the intervention period. After 4 months, the toddler formula group showed significantly higher serum ferritin and lower serum transferrin concentrations than the cow's milk group. CONCLUSION: Intake of iron-supplemented toddler formula for 4 months in 1-3 year-olds is more effective in maintaining iron nutritional status than cow's milk.

[Cardiovascular effects of omega-3-fatty acids and alternatives to increase their intake]. / Efectos cardiovasculares de los ácidos grasos omega-3 y alternativas para incrementar su ingesta.

Cardiovascular diseases are the main mortality cause in Europe, the USA and a great extent of Asia. There are several risk factors associated with cardiovascular diseases, such as increased total cholesterol, homocysteine and triglycerides, hypertension, diabetes, and reduced levels of HDL-cholesterol. Many of these risk factors are diet influenced. In spite of the great amount of foods enriched with n-3 fatty acids available at the market, the knowledge about the effects produced by regular intake of these foods still is a challenge in the majority of cases. It appears that intake of foods enriches with n-3 polyunsatured fatty acids is an option that may be effective in reducing risk factors for diseases, by substituting supplements without modifying consumer's alimentary habits. Also shown are the outcomes from a nutritional study undergone with a functional milk-bases food that contains n-3 fatty acids, oleic acid and vitamins.

A rapid gas-liquid chromatography method for the determination of lactulose and mannitol in urine: clinical application in studies of intestinal permeability.

OBJECTIVE: The purpose of this study was to develop a gas-liquid chromatography method for the determination of the urinary excretion of two nonmetabolizable sugars (lactulose and mannitol) to estimate the intestinal permeability. METHODS: Two internal standards (alpha-methyl-D-glucopyranosyde and sucrose) were added to the urine samples prior to derivatization with the aid of a solution of pyridine, [N, O-bis (trimethylsilyl)]-acetamide and chlorotrimethylsilan. Sample preparation was simpler and faster than in other previous methods and the four sugars were resolved within 27 min. RESULTS: The inclusion of internal standards reduced the coefficient of variation from 9.44% to 4.78%. The method provided better sensitivity, range of analysis, and linearity than a previously published HPLC method reducing variances in the recovery of lactulose and mannitol added to urine. The clinical application of this method was tested in preterm infants fed human milk or cow's milk based formula and in breast fed term infants. CONCLUSION: The method described here for the determination of urinary lactulose and mannitol is more rapid, simpler, and more accurate than other previously published methods.

Neither glutamine nor arginine supplementation of diets increase glutamine body stores in healthy growing rats.

The aim of the work was to resolve whether glutamine and arginine supplemented diets affect plasma and tissue (muscle, liver and intestinal mucosa) glutamine concentrations, as well as glutaminase and glutamine synthetase specific activities. The trial was performed in growing rats fed 10% protein diets for 3 weeks. Protein sources were: whey proteins (W); whey proteins+free glutamine (WG); whey proteins+arginine (WA); and casein+wheat protein hydrolysate+acid whey (39:39:22), as source containing protein-bound glutamine (CGW). Rats fed the control diet (6.4% glutamine) (W) showed comparable glutamine body stores to those of rats fed the WG diet. In fact, glutamine sup- plementation down-regulated the hepatic glutamine synthetic capacity of growing rats (W/WG: 6.8+/-0.3 vs 6.0+/-0.2 nmol/min/mg protein). Arginine supplementation of the diet (up to 9% of the protein content) resulted in a decrease in plasma and tissue glutamine concentrations (W/WA: plasma, 1218+/-51 vs 1031+/-48 micromol/L; liver 7.5+/-0.4 vs 6.5+/-0.2 micromol/g; muscle: 5.7+/-0.2 vs 4.0+/-0.2 micromol/g). These data suggest that glutamine supplementation of the diet does not increase plasma and tissue glutamine concentrations in healthy growing rats, while the addition of arginine to the diet decreases glutamine body stores.

n-3 Fatty acids plus oleic acid and vitamin supplemented milk consumption reduces total and LDL cholesterol, homocysteine and levels of endothelial adhesion molecules in healthy humans.

BACKGROUND AND AIMS: Numerous studies suggest n -3 polyunsaturated fatty acids (n -3 PUFA) and oleic acid intake have beneficial effects on health including risk reduction of coronary heart disease. The purpose of this study was to evaluate the effect of a commercially available skimmed milk supplemented with n -3 PUFA, oleic acid, and vitamins E, B(6), and folic acid (Puleva Omega3) on risk factors for cardiovascular disease. (CVD). METHODS: Thirty volunteers were given 500 ml/day of semi-skimmed milk for 4 weeks and then 500 ml/day of the n -3 enriched milk for 8 further weeks. Plasma and LDL lipoproteins were obtained from volunteers at the beginning of the study (T(pre)), and at 4, 8 and 12 weeks. RESULTS: The consumption of n -3 enriched milk produced a significant decrease in plasma concentration of total and LDL cholesterol accompanied by a reduction in plasma levels of homocysteine. Plasma and LDL oxidability and vitamin E concentration remained unchanged throughout the study. A significant reduction in plasma levels of vascular cell adhesion molecule 1, and an increase in plasma concentration of folic acid were also observed. CONCLUSION: Daily intake of n -3 PUFA and oleic acid supplemented skimmed milk plus folic acid and B-type vitamins has favourable effects on risk factors for CVD.

Plasma glutamine response to enteral administration of glutamine in human volunteers (free glutamine versus protein-bound glutamine).

The goal of the present work was to compare the plasma glutamine response to exogenous glutamine administration in human volunteers; glutamine was provided as a free amino acid, bound to proteins, or in the form of peptides. Plasma glutamine concentrations were measured in eight human volunteers at 30, 60, 90, 120, and 240 min after receiving a drink containing 30 g of protein from one of the five different proteins tested (sodium caseinate, sodium caseinate + free glutamine, carob germ flour, carob protein concentrate, and carob protein hydrolysate). Peak plasma glutamine concentrations were 42% higher than postabsorptive basal values when exogenous glutamine was administered in the form of free glutamine added to caseinate (925.9 +/- 67.7 versus 651.3 +/- 44.0 micromol/L, respectively). In contrast, when glutamine was offered 100% bound to proteins (carob proteins), peak plasma glutamine concentration increased only between 18% and 23% from basal values, possibly because of the lower digestibility of carob proteins versus that of caseinate + free glutamine, to a different glutamine utilization at the gut level, or to a different response in endogenous glutamine kinetics to enteral administration of glutamine, depending on the molecular form of the glutamine source (free or protein bound).

Effect of glutamine supplementation of the diet on tissue protein synthesis rate of glucocorticoid-treated rats.

Although glutamine status in the critically ill patient can be improved by nutritional means, the most effective way of effecting such supplementation has received little attention. We evaluated two different ways of supplementing clinical nutrition products with glutamine, either with free glutamine or by providing a glutamine-rich protein source, in acute glucocorticoid-treated (intraperitoneal dexamethasone, 120 mg/kg) rats. During the recovery period, the animals received isonitrogenous and isoenergetic diets containing either casein, mixed whey proteins with or without glutamine, or carob protein plus essential amino acids. Plasma and tissue amino acids and glutathione as well as tissue protein synthesis were measured. Dexamethasone treatment lowered weight gain, muscle glutamine, and muscle and jejunal protein synthetic rate. Muscle protein synthesis was increased (from 15.9% to 24.2%/d) only when glutamine was included in the diet as a free amino acid. This increase paralleled a rise in plasma glutamine. We speculate that glutamine provided in dietary protein is extensively metabolized by the splanchnic tissues and does not influence peripheral glutamine status to the same extent as glutamine provided in a free amino acid form. However, both forms of glutamine supplementation were equally effective in increasing protein synthesis in the jejunum (by 25%). This is likely the main benefit of glutamine supplementation of enteral nutrition formulas.

Methods for measuring tissue RNA turnover.

Measuring RNA turnover is important because of the significance of rRNA, tRNA and mRNA in tissue protein synthesis. Changes in turnover of each of these species precede important cellular events such as hormone or cytokine action or cell-division itself. Isotopic methods have relied on decay of pulse-labelled RNA or on incorporation of isotopically-labelled precursors. However, recycling of labels may lead to under or overestimation of synthesis rates respectively. The labelling of the intracellular precursor pool must be known if accurate RNA synthesis rates are to be calculated from the degree of incorporation. However, the intracellular nucleotide pools may be anatomically or metabolically compartmented (i.e. via de novo or salvage synthesis routes) and this complicates many study designs. The use of[methyl-14C]- or [methyl-3H]methionine as a means of labelling methylated nucleosides in RNA and protein simultaneously is described in addition to new stable isotopic techniques based on 13C-glycine as a de novo label. Urinary excretion of the numerous modified nucleosides in cellular RNA can be used to calculate whole-body turnover rates of each of the major RNA species. Examples of the effects of critical-illness and glutamine supplementation on RNA turnover are given. We conclude by suggesting that whole-body RNA turnover rates have been significantly underestimated and that this has implications for nutritional therapy, especially with regard to nucleotide supplementation.

Ribonucleic acid nucleotides in maternal and fetal tissues derive almost exclusively from synthesis de novo in pregnant mice.

The contributions of dietary nucleotides and nucleotides synthesized de novo to ribonucleic acid synthesis in vivo were estimated by feeding, from d 13 to 18 of gestation, two groups of five pregnant mice a defined diet that contained either uniformly [U13C]-labeled nucleotides or [U13C]-algal amino acids isolated from algal biomass. Ribonucleic acid and protein were isolated from mucosa, liver and fetus. Nucleosides and amino acids were isolated and converted to their trimethylsilyl and n-propyl ester, heptaflurobutyramide derivatives, respectively. The isotopic enrichments of all isotopomers were determined by gas chromatography-mass spectrometry. In the mice that ingested [U13C]-nucleotides, the isotopic enrichment of [Ul3C]-purines (0.03-0.2 mol/100 mol) was significantly (P < 0.001) less than that of [U13C]-uridine (1.5-4.2 mol/100 mol). [13C5]-Purines (0.1-0.8 mol/100 mol) and [13C4]-uridine (0.2-0.5 mol/100 mol) were detected, showing that some dietary bases and ribose were incorporated via the salvage pathway. In mice that Ingested U13C-amino acids, the isotopic enrichment (2-4.6 mol/100 mol) of the [13C2]-purines, which derive from [Ul3C]-glycine, was between 73 (liver) and 113% (fetus) of protein-bound 13C2-glycine. The isotopic enrichment (0.8-1.6 mol/100 mol) of [13C3]-uridine, an isotopomer that derives from [U13C]-aspartate, was 50 (liver) to 126% (mucosa) of [13C4]-protein-bound aspartate. The results suggest that a large majority of the bases incorporated into maternal and fetal ribonucleic acids derive from synthesis de novo.

Nutritional value and antigenicity of two milk protein hydrolysates in rats and guinea pigs.

Two milk protein (whey protein and casein) hydrolysates were obtained by enzymatic hydrolysis. In the casein hydrolysate, an ultrafiltration stage was performed to remove all the peptides with molecular weights > 2500. A nutritional value study of both hydrolysates was undertaken. No differences were found between the native proteins and their enzymatic hydrolysates. Neither the enzymatic hydrolysis nor the heat treatments used affected the nutritional value of the protein sources. The two hydrolysates were studied for antigenic properties. The enzymatic hydrolysis of the whey protein concentrate followed by a thermal treatment at 90 degrees C for 10 min reduced significantly its antigenicity. However, the in vivo allergenicity tests showed some positive reactions in both systemic anaphylaxis and passive cutaneous anaphylaxis. The casein hydrolysate, after being ultrafiltered, was devoid of sensitizing capacity by the oral route and was also ineffective in producing local or systemic anaphylaxis in previously sensitized animals, as shown by in vivo assays.

Serum amino acid concentrations in growing rats fed intact protein versus enzymatic protein hydrolysate-based diets.

The aim of this study was to evaluate the effect of the molecular form of dietary protein (native or enzymatically hydrolyzed) on the total serum protein concentrations and the serum amino acid profile of growing rats at weaning. Wistar male rats at weaning were randomly assigned to one of the four isocaloric and isonitrogenous (12% protein equivalent content) diets and fed for 7 days. The protein sources of the diets were: whey protein, casein and their respective hydrolysates. Differences in the serum amino acid profiles exclusively related to the amino acid composition of the protein (casein or whey proteins) were observed, but differences due to their molecular form were not observed. It is concluded that the use of enzymatic hydrolysates of whey proteins and casein has the same effects as their native proteins on nitrogen intake, body weight gain and serum amino acid profile of growing rats at weaning.

Protein v. enzymic protein hydrolysates. Nitrogen utilization in starved rats.

The present study was carried out to compare the effects of four isoenergetic and isonitrogenous diets on the N utilization, total serum protein concentration and serum amino acid profile in starved rats at weaning. These diets differed only in the molecular form of two milk proteins (whey protein and casein), which were either native or partly hydrolysed. Male Wistar rats at weaning were fasted for 3 d and then refed with one of the four diets for 48 h. No differences were observed in the body weight gain, protein digestibility and total serum protein concentration between groups after the refeeding period and all the N balances were positive. N retention was higher in the two groups of rats given the protein-hydrolysate-based diets compared with those given the intact-protein-based diets. This was associated with a lower urinary N excretion in rats, given the whey-protein-hydrolysate and the casein-hydrolysate diets. Despite this fact, the serum amino acid pattern of rats given the hydrolysed protein diet was very similar to that of those given the corresponding native protein diet. In conclusion, we have proved that enzymic hydrolysates from milk proteins have equivalent effects to native proteins in recovery after starvation in rats at weaning, on N absorption, total serum protein concentration and serum amino acid profile, and even give a higher N retention. We did not observe any harmful effect in using protein hydrolysates instead of native proteins.

Protein hydrolysate vs free amino acid-based diets on the nutritional recovery of the starved rat.

BACKGROUND AND AIMS: To test the hypothesis that a peptide-based enteral product was equivalent to a low-fat, free amino acid-based formula in the nutritional and functional recovery of the starved rat. METHODS: Sixteen male Wistar rats were starved for 3 days. Then, rats were randomised to a whey protein hydrolysate-based diet or a free amino acid-based diet and refed for 3 days. The experiment was designed to provide the same energy intake in both groups. The parameters studied included body weight gain, nitrogen retention, plasma free amino acid concentrations, muscle glutamine concentrations and glutathione levels in gut mucosa and liver. RESULTS: Weight gain was statistically higher on the peptide-based diet than on the elemental diet after the refeeding period. This difference in weight gain was associated with a statistically higher nitrogen retention. Plasma and muscle free glutamine concentrations were higher in rats fed the whey protein hydrolysate-based diet than those in rats refed the free amino acid-based diet, even though the glutamine intake was higher in the latter group. Glutathione concentrations in liver and gut mucosa were similar in the groups. CONCLUSION: We conclude that enteral diets containing peptides were more effective than a diet containing free amino acids in the nutritional recovery of the starved rat.

Role of glutamine on the de novo purine nucleotide synthesis in Caco-2 cells.

BACKGROUND: The body's nucleotide pool derives from three potential sources: de novo synthesis, salvage of preformed-nucleosides/bases or the diet. The relative contributions of these pathways of assimilation are poorly understood in vivo. Dietary nucleotides have been suggested to have beneficial effects an the development and repair of the gastrointestinal tract. Tissues with a rapid turnover, such as the gut and the immune system cells, may utilise preformed nucleotides (coming from the diet), in situations in which there is a high demand of nucleotides for nucleic acid synthesis. Therefore, nucleotides could be considered as conditionally essential nutrients. AIM OF THE WORK AND METHODS: Development of a method to measure synthesis de novo of RNA-purine nucleotides in Caco-2 cells, relying an the incorporation of 14C-glycine into the purine ring of the nucleotide. To establish the fractional synthesis rate of RNA purine nucleotides in Caco-2 cells, grown in culture medium containing different concentrations of glutamine, in the presence or absence of added nucleotides. To investigate the degree to which tissue ribonucleosides are derived from the culture medium or from de novo synthesis in the presence of different concentrations of glutamine, using undifferentiated Caco-2 cells, stressed or not by the addition of IL-1 beta to the medium. RESULTS AND CONCLUSIONS: The presence of high levels of glutamine in the culture medium is essential for cell proliferation (estimated by measurement of the fractional synthesis rate of purine nucleotides) and the presence of nucleotides cannot replace the glutamine dependence of Caco-2 cell proliferation. The incorporation of exogenous purine nucleotides into RNA of Caco-2 cells is rather limited, and it becomes important when cells are stressed by glutamine deprivation. Stress by addition of interleukin-1 beta resulted in the maintenance or the increase in de novo synthesised RNA-purine nucleotides, even in the presence of exogenous nucleotides. However, the addition of interleukin-1 beta to the culture medium led to an enhanced salvage of preformed pyrimidine nucleotides for nucleic acid synthesis when glutamine was present in the medium at a concentration of 0.5 mmol/L.

Dietary nucleotides might influence the humoral immune response against cow's milk proteins in preterm neonates.

The objective of this study was to evaluate the influence of dietary nucleotide supplementation in preterm infants during the first month of life on the intestinal permeability to lactulose, mannitol and to beta-lactoglobulin and on the development of circulating antibodies to beta-lactoglobulin and alpha-casein. Twenty-seven preterm infants were enrolled in the study; 11 of them were fed a standard low-birth weight milk formula and 16 infants were fed the same formula supplemented with nucleotides at similar levels to those found in human milk. Blood and urine samples were obtained at 1, 7 and 30 days of age. Serum beta-lactoglobulin, serum IgG antibody to alpha-casein and serum IgG antibody to beta-lactoglobulin were measured by ELISA. The lactulose/mannitol urinary excretion rate was measured by gas liquid chromatography. Neither the intestinal permeability to saccharides nor the intestinal absorption of beta-lactoglobulin were affected by the nucleotide supplementation. However, serum concentrations of IgG antibody to beta-lactoglobulin were higher in preterm neonates fed the supplemented formula than in those fed the standard formula. According to these results, dietary nucleotides might influence the maturation of the humoral immune response in preterm newborn infants.

The validity of extrinsic stable isotopic labeling for mineral absorption studies in rats.

The use of extrinsic stable and radioisotopic labels (Fe, Zn, Cu and Se) was compared with the use of intrinsic labels by measuring label retention in rats. Saccharomyces cerevisiae (Hansen strain CBS 1171) was prepared intrinsically enriched with a stable isotope of iron, zinc, copper or selenium, and unenriched freeze-dried yeast was extrinsically labeled with the appropriate stable and/or radioisotope. Male Wistar rats, weighing 80-100 g and fed a purified diet, were given a test meal of one of the above labeled yeasts. Isotopic retention was determined by fecal monitoring. Retention of the stable isotopes was determined by thermal ionization quadruple mass spectrometry (TIQMS) and retention of the radioisotopes by counting feces in a whole-body counter. The results indicated that the behavior of the labels differed among the minerals, with copper as the only one in which the intrinsic and extrinsic stable isotopes were comparably retained. With zinc, retention of the extrinsic radiolabel and intrinsic label was similar, but retention of the extrinsic stable isotope label was higher. With iron, the intrinsic label had a significantly lower retention than the two extrinsic labels; with selenium, retention of all three labels was different, but these differences were not of a sufficient magnitude to conclude that extrinsic stable isotopic labelling is not valid. These results demonstrate that an extrinsic stable isotope label can be used for copper, selenium and inorganic iron, but that such a label is not valid for studies on zinc.

Use of different dietary protein sources for lactating goats: milk production and composition as functions of protein degradability and amino acid composition.

To establish the effect of the nature of four different protein sources [fababeans, 27.8% crude protein (CP); sunflower meal, 41.7% CP; corn gluten feed, 18.8% CP; and cottonseed, 18.3% CP] on milk protein production by goats, the ruminal degradation of these feeds was studied as was the amino acid (AA) composition of the original material and that of the undegradable fractions of the protein sources. Four diets were designed; 20% of their protein was supplied by each of the different sources. Four groups of 5 Granadina goats were used to study the utilization of these diets for milk production. No significant differences were observed in dry matter intake or milk production. The milk produced by goats fed the diet containing sunflower meal had the lowest protein concentration; the highest milk protein concentration was observed for goats fed the diet containing corn gluten feed. From a multivariate analysis, it was deduced that the quickly degradable protein fraction in the rumen and the ruminally undegradable protein fraction were the components of the protein sources most directly related to the milk protein produced. Given the similar AA profiles of the undegradable fractions of the different protein sources, the possible supplementation achieved from these ruminally undegradable fractions must be established by the amount of protein supplied regardless of AA composition.

Food deprivation and refeeding influence growth, nutrient retention and functional recovery of rats.

The objective of this work was to determine the effects of starvation and refeeding on growth, nutritional recovery and intestinal repair in starved rats. Male Wistar rats, weighing 200 g, were starved for 3 d, then refed a soy-based diet for another 3 d. Normally fed rats were given the same diet and used as controls. The variables assessed were as follows: body weight gain and nitrogen retention during recovery after starvation; muscle glutamine concentration; tissue protein content; gut mucosa and liver glutathione levels; intestinal permeability to ovalbumin, lactulose and mannitol; and intestinal tissue apoptosis. Starvation was associated with lower muscle glutamine levels and intestinal mucosa impairment, including a lower content of mucosal protein, a higher level of oxidized glutathione, enhanced permeability to macromolecules and greater numbers of apoptotic cells. Refeeding for 3 d resulted in rapid repair of gut atrophy and normalization of not only intestinal permeability but also of the majority of metabolic markers assessed in other tissues. In conclusion, with the use of severely starved rats, we have established a reversible experimental animal model of malnutrition that might prove useful in comparing the effectiveness of different enteral diets.