A synbiotic mixture of scGOS/lcFOS and Bifidobacterium breve M-16V increases faecal Bifidobacterium in healthy young children.

Little is known about the impact of nutrition on toddler gut microbiota. The plasticity of the toddler gut microbiota indicates that nutritional modulation beyond infancy could potentially impact its maturation. The objective of this study was to determine the effect of consuming Young Child Formula (YCF) supplemented with short chain galactooligosaccharides and long chain fructooligosaccharides (scGOS/lcFOS, ratio 9:1) and Bifidobacterium breve M-16V on the development of the faecal microbiota in healthy toddlers. A cohort of 129 Thai children aged 1-3 years were included in a randomised controlled clinical study. The children were assigned to receive either YCF with 0.95 g/100 ml of scGOS/lcFOS and 1.8×107 cfu/g of B. breve M-16V (Active-YCF) or Control-YCF for 12 weeks. The composition and metabolic activity of the faecal microbiota, and the level of secretory immunoglobulin A were determined in the stool samples. The consumption of Active-YCF increased the proportion of Bifidobacterium (mean 27.3% at baseline to 33.3%, at week 12, P=0.012) with a difference in change from baseline at week 12 between the Active and Control of 7.48% (P=0.030). The consumption of Active-YCF was accompanied with a more acidic intestinal milieu compared to the Control-YCF. The pH value decreased statistically significantly in the Active-YCF group from a median of 7.05 at baseline to 6.79 at week 12 (P<0.001). The consumption of Active-YCF was associated with a softer pudding-like stool consistency compared to the Control-YCF. At week 6 and week 12, the between-group difference in stool consistency was statistically significant (P=0.004 and P<0.001, respectively). A Young Child Formula supplemented with scGOS/lcFOS and B. breve M-16V positively influences the development of the faecal microbiota in healthy toddlers by supporting higher levels of Bifidobacterium. The synbiotic supplementation is also accompanied with a more acidic intestinal milieu and softer stools.

Stimulating program implementation via a Community of Practice: a responsive evaluation of care programs for frail older people in the Netherlands.

Evaluation is often used as a vehicle to improve program implementation. To evaluate the implementation of programs that provide care for frail older people a Community of Practice (CoP) was developed in the Netherlands. The purpose of this paper is to describe and reflect on the role of a CoP in the implementation of these programs. Using a responsive evaluation approach this study was based on interviews with participating stakeholders and transcripts of the CoP meetings with 13 professionals, project managers and patient representatives. Findings showed that CoP members had unanticipated concerns regarding the pro-active approach of the programs and older people not being open to receiving care. The heterogenic composition was appreciated and fostered learning. A social infrastructure was created for active learning inside and outside the CoP. We conclude that a CoP is a useful strategy as part of an evaluation aimed at improving program implementation. Lessons learned include the importance of creating ownership among CoP members by sharing responsibilities and paying attention to the heterogenic group composition and professional language spoken to involve all members.

Effect of a nutrient-enriched drink on dietary intake and nutritional status in institutionalised elderly.

OBJECTIVE: (1) To determine whether nutritional supplementation (energy and micronutrients) in institutionalised elderly has a positive effect on dietary intake and nutritional status. (2) To investigate whether individuals tend to compensate for the energy content of the intervention product by decreasing their habitual food consumption. METHODS: A 24-week, randomised, double-blind, placebo-controlled, intervention trial in homes for the elderly (n=3), in nursing homes (n=3) and 'mixed' homes (n=3) in The Netherlands. Institutionalised elderly people (n=176) older than 60 years of age, with a body mass index < or =30 kg/m(2) and a Mini-Mental State Examination score of 10 points or higher, randomly received a nutrient-enriched drink or a placebo drink twice a day during 24 weeks in addition to their usual diet. Allocation to treatment took into account of sex, the Mini-Mental State Examination score and the plasma homocysteine level. Body weight and several nutrition-related analyses in fasting blood samples were measured in all participants. Data on dietary intake were collected in a subsample (n=66). RESULTS: A significantly favourable effect (P<0.001) of the intervention drink was observed on vitamin intake, mineral intake and vitamin status in blood (for example, homocysteine decreased from 14.7 to 9.5 micromol/l in the intervention group as compared with that in the placebo group (17.2-15.9)). The difference in change in total energy intake between the two treatment groups was 0.8 MJ/day (P=0.166). Energy intake from food decreased in both groups to the same extent (-0.5 MJ/day). Therefore, this decrease cannot be considered as compensation for the energy content of the product. CONCLUSIONS: This group of institutionalised elderly people does not compensate for the energy content of a concentrated nutritional supplement. Therefore, this supplement is effective for counteracting the development of malnutrition in this population.

Faecal SIgA secretion in infants fed on pre- or probiotic infant formula.

Secretory immunoglobulin A (SIgA) plays an important role in the defence of the gastrointestinal tract. The level of faecal SIgA antibody is associated with increased neutralization and clearance of viruses. Formula-fed infants who lack the transfer of protective maternal SIgA from breast milk may benefit from strategies to support maturation of humoral immunity and endogenous production of SIgA. We aimed at studying the effects of standard, prebiotic and probiotic infant formulas on the faecal SIgA levels. At birth, infants of whom the mother had decided not to breastfeed were allocated to one of three formula groups in a randomized, double-blind fashion. Nineteen infants received standard infant formula; 19 received prebiotic formula containing a specific mixture of 0.6 g galacto-oligosaccharides (GOS)/fructo-oligosaccharides (FOS)/100 ml formula and 19 received probiotic formula containing 6.0 x 10(9) cfu Bifidobacterium animalis/100 ml formula. Faecal samples were taken on postnatal day 5, day 10, wk 4 and every 4 wk thereafter until wk 32. SIgA in faeces was determined by an enzyme-linked immunosorbent assay. During the intervention, infants fed on prebiotic formula showed a trend towards higher faecal SIgA levels compared with the standard formula-fed infants reaching statistical significance at the age of 16 wk. In contrast, infants fed on the probiotic formula showed a highly variable faecal SIgA concentration with no statistically significant differences compared with the standard formula group. Formula-fed infants may benefit from infant formulas containing a prebiotic mixture of GOS and FOS because of the observed clear tendency to increase faecal SIgA secretion. Adding viable B. animalis strain Bb-12 to infant formula did not reveal any sign for such a trend.

Homocysteine and cognitive function in institutionalised elderly A cross-sectional analysis.

BACKGROUND: Several cross-sectional, case-control and prospective studies revealed a relation between homocysteine and cognitive function or dementia. These studies included either patient populations or healthy, community- dwelling elderly people. AIM OF THE STUDY: In this study we tested the hypothesis that homocysteine was inversely associated with cognitive function in a population of institutionalised elderly (aged >/= 60 y; n = 157). METHODS: For testing this hypothesis baseline data of a recently conducted intervention study in institutionalised elderly (median age 83 years) were used. Cognitive function was evaluated by the cognitive subscale of the Alzheimer's disease Assessment Scale (ADAS-cog). The association between fasting plasma homocysteine level and cognitive function was investigated by multiple linear regression analysis. RESULTS: In the crude model homocysteine concentration was not significantly related to ADAS-cog score (beta = 0.061; p = 0.45). Age was found to be related to ADAS-cog score (beta = 0.161; p < 0.05). Adjusting for age did however not result in a relation between homocysteine and cognitive function. CONCLUSIONS: In our study no association was found between homocysteine and cognitive function in a population of very old institutionalised subjects.

The effect of supplementation with an enriched drink on indices of immune function in frail elderly.

Ageing induces a change in immune responses. Besides this, impaired nutritional status is considered to have a critical influence on immune function, which may be reversed by nutritional supplementation. We evaluated the effect of an enriched drink on immune function in the elderly. 33 frail elderly subjects (aged > or = 65 years and body mass index < or = 25) received two 125 ml packages of either an enriched drink (n=20) or placebo (n=13) daily for 6 months. The enriched drink contained macro- and micronutrients. At baseline and after 6 months blood samples were drawn and PBMC's were isolated. ConA stimulated proliferation and IL-2 production of PBMC's were measured. There was a significant difference between groups in proliferation over the study period. The supplement group remained stable whereas the placebo group showed a reduction in proliferation over the 6-month period. There was no significant difference in IL-2 production between groups. Our study adds to the evidence that nutritional supplementation can affect immune function in the elderly.

The effect of a liquid nutrition supplement on body composition and physical functioning in elderly people.

BACKGROUND & AIMS: The elderly are at an increased risk of poor nutritional status which is mutually interacting with functional status. We evaluated the effects of a liquid nutrition supplement on anthropometric and functional indices in elderly people. METHODS: Subjects (n=68; mean age=82+/-7 years) with body mass index

Study of the effect of a liquid nutrition supplement on the nutritional status of psycho-geriatric nursing home patients.

OBJECTIVES: To evaluate the acceptance of a multinutrient liquid nutrition supplement in psycho-geriatric nursing home patients and the effect on weight, plasma nutrients and activities of daily life. DESIGN: Double-blind, placebo-controlled 12-week intervention study. SETTING: Two nursing homes in Boxtel, The Netherlands. SUBJECTS: Forty-two (body mass index (BMI) <23 kg/m2 for men or <25 kg/m2 for women) psycho-geriatric nursing home patients aged 60 y or over. INTERVENTIONS: Provision with a complete micronutrient-enriched liquid nutrition supplement of 125 ml and 0.6 MJ (135 kcal) or placebo twice daily during daytime between main meals. Study parameters were assessed at 0, 6 and 12 weeks. MAIN OUTCOME FOR MEASURES: Weight, Barthel index of daily activities, several plasma values (albumin, C-reactive protein (CRP), homocysteine, thiamine, thiamine diphosphate (TDF), vitamin B6, vitamin B12, folic acid, vitamin D), bowel function. RESULTS: The supplement was well accepted. Thirty-five patients completed the intervention period (16 control group; 19 supplement group). Baseline daily nutrient intake was low. A statistically significant improvement was observed for body weight (difference between groups 2.2 kg, P=0.03), and homocysteine, vitamin B1, TDF, vitamin B6, vitamin B12, folate and vitamin D in the supplement group compared to the placebo group. No significant difference was observed in the Barthel index (mean difference -0.3+/-1.1 for both groups). No difference in occurrence of diarrhoea was observed. CONCLUSIONS: The study shows that nutritional supplementation is well accepted and can improve the nutritional status of psycho-geriatric nursing home patients. SPONSORSHIP: Numico Research BV.

Analysis of intestinal flora development in breast-fed and formula-fed infants by using molecular identification and detection methods.

BACKGROUND: An obvious difference between breast-fed and formula-fed newborn infants is the development of the intestinal flora, considered to be of importance for protection against harmful micro-organisms and for the maturation of the intestinal immune system. In this study, novel molecular identification methods were used to verify the data obtained by traditional culture methods and to validate the culture independent fluorescent in situ hybridization (FISH) technique. METHODS: From each of six breast-fed and six formula-fed newborn infants, six fecal samples were obtained during the first 20 days of life. The microbial compositions of the samples were analyzed by culturing on specific media and by FISH, by using specific 16S rRNA-targeted oligonucleotide probes. The colonies growing on the media were identified by random amplified polymorphic DNA pattern analysis and by polymerase chain reaction amplification and subsequent analysis of the 16S rRNA gene. RESULTS: Molecular identification of the colonies showed that the selective media are insufficiently selective and unsuitable for quantitative analyses. Qualitative information from the culturing results combined with the data obtained by the FISH technique revealed initial colonization in all infants of a complex (adult-like) flora. After this initial colonization, a selection of bacterial strains began in all infants, in which Bifidobacterium strains played an important role. In all breast-fed infants, bifidobacteria become dominant, whereas in most formula-fed infants similar amounts of Bacteroides and bifidobacteria (approximately 40%) were found. The minor components of the fecal samples from breast-fed infants were mainly lactobacilli and streptococci; samples from formula-fed infants often contained staphylococci, Escherichia coli, and clostridia. CONCLUSIONS: This study confirms the differences in development of intestinal flora between breast-fed and formula-fed infants. The results obtained from the FISH technique were consistent. Although the repertoire of probes for this study was not yet complete, the FISH technique will probably become the method of reference for future studies designed to develop breast-fed-like intestinal flora in formula-fed infants.

Double-blind, randomized trial of a synthetic triacylglycerol in formula-fed term infants: effects on stool biochemistry, stool characteristics, and bone mineralization.

BACKGROUND: The low sn-2 palmitate content of infant formulas results in formation of fatty acid calcium soaps in the stools and reduced calcium absorption. OBJECTIVE: Our objective was to test the hypotheses that increasing the proportion of sn-2 palmitate in formula for term infants would result in greater skeletal mineral deposition and reduced stool hardness. DESIGN: Healthy term neonates were randomly assigned to receive standard formula (n = 103) or formula containing 50% sn-2 palmitate (high-sn-2 formula; n = 100) for 12 wk. One hundred twenty breast-fed infants were also studied. The main outcome measures were 1) radial (single-photon absorptiometry) and whole-body (dual-energy X-ray absorptiometry) bone mineral content (WBBMC) at 12 wk and 2) stool frequency, volume, and consistency at 6 and 12 wk. Secondary outcome measures included stool fatty acid content. RESULTS: Infants receiving high-sn-2 formula had higher WBBMC (128.1 +/- 9.7 compared with 122.7 +/- 10.1 g, adjusted for size and sex), softer stools at 6 and 12 wk, and a lower proportion of stool soap fatty acids than did infants receiving the control formula. Breast-fed infants had adjusted WBBMC values (128.3 +/- 9.1 g) similar to those of infants fed high-sn-2 formula and significantly higher than those of infants fed the control formula. CONCLUSIONS: Changing the stereoisomeric structure of palmitate in infant formula resulted in higher WBBMC, reduced stool soap fatty acids, and softer stools more like those of breast-fed infants. The greater bone mass measured could be important if it persists beyond the trial period; this merits further investigation.

Growth, development and differentiation: a functional food science approach.

Few other aspects of food supply and metabolism are of greater biological importance than the feeding of mothers during pregnancy and lactation, and of their infants and young children. Nutritional factors during early development not only have short-term effects on growth, body composition and body functions but also exert long-term effects on health, disease and mortality risks in adulthood, as well as development of neural functions and behaviour, a phenomenon called 'metabolic programming'. The interaction of nutrients and gene expression may form the basis of many of these programming effects and needs to be investigated in more detail. The relation between availability of food ingredients and cell and tissue differentiation and its possible uses for promoting health and development requires further exploration. The course of pregnancy, childbirth and lactation as well as human milk composition and the short- and long-term outcome of the child are influenced by the intake of foods and particularly micronutrients, e.g. polyunsaturated fatty acids, Fe, Zn and I. Folic acid supplementation from before conception through the first weeks of pregnancy can markedly reduce the occurrence of severe embryonic malformations; other potential benefits of modulating nutrient supply on maternal and child health should be further evaluated. The evaluation of dietary effects on child growth requires epidemiological and field studies as well as evaluation of specific cell and tissue growth. Novel substrates, growth factors and conditionally essential nutrients (e.g. growth factors, amino acids, polyunsaturated fatty acids) may be potentially useful as ingredients in functional foods and need to be assessed carefully. Intestinal growth, maturation, and adaptation as well as long-term function may be influenced by food ingredients such as oligosaccharides, gangliosides, high-molecular-mass glycoproteins, bile salt-activated lipase, pre- and probiotics. There are indications for some beneficial effects of functional foods on the developing immune response, for example induced by antioxidant vitamins, trace elements, fatty acids, arginine, nucleotides, and altered antigen contents in infant foods. Peak bone mass at the end of adolescence can be increased by dietary means, which is expected to be of long-term importance for the prevention of osteoporosis at older ages. Future studies should be directed to the combined effects of Ca and other constituents of growing bone, such as P, Mg and Zn, as well as vitamins D and K, and the trace elements F and B. Pregnancy and the first postnatal months are critical time periods for the growth and development of the human nervous system, processes for which adequate substrate supplies are essential. Early diet seems to have long-term effects on sensory and cognitive abilities as well as behaviour. The potential beneficial effects of a balanced supply of nutrients such as I, Fe, Zn and polyunsaturated fatty acids should be further evaluated. Possible long-term effects of early exposure to tastes and flavours on later food choice preferences may have a major impact on public health and need to be further elucidated. The use of biotechnology and recombinant techniques may offer the opportunity to include various bioactive substances in special dietary products, such as human milk proteins, peptides, growth factors, which may have beneficial physiological effects, particularly in infancy and early childhood.

Influence of long chain unsaturated fatty acids in formula feeds on lipid peroxidation and antioxidants in preterm infants.

The influence of long chain polyunsaturated fatty acids (LCP) in formula feeds on lipid peroxidation and antioxidants was studied in 35 healthy preterm infants (gestational age 30-35 wk) during the first 6 postnatal weeks. Infants received a preterm formula supplemented with n-3 LCP (LCP group, n = 13), or standard preterm formula (NO-LCP group, n = 15); 7 infants fed human milk served as a reference group. With LCP supplementation, erythrocyte C22:6n-3 levels were stable; without supplementation, the levels declined (difference p < 0.001). LCP supplementation did not decrease vitamin E or C levels, or increase lipid peroxidation products (thiobarbituric acid-reactive substances) in plasma. In erythrocytes, LCP supplementation did not markedly influence the reduced/oxidized glutathione ratio; however, the susceptibility to H2O2-induced oxidative stress was reduced. Our results suggest that healthy preterm infants are able to cope with any extra peroxidative stress produced by n-3 LCP supplementation. However, these findings might not be generally applicable to other formulas containing LCP supplements.

Symptomatology and growth in infants with cow's milk protein intolerance using two different whey-protein hydrolysate based formulas in a Primary Health Care setting.

Both growth and the course of allergic symptoms were evaluated in 79 infants with cow's milk protein intolerance, aged three months or younger, diagnosed by standard elimination/provocation and treated with a whey-hydrolysate based infant formula: Nutrilon Pept or Pepti Junior. The efficacy of both products, in terms of symptomatology and growth, was compared with each other. The products differ in fat source (Pepti Junior 50% of its fat as MCT, Nutrilon Pepti normal LCT fat blend) and the presence of lactose (Pepti Junior: lactose free; Nutrilon Pepti: 40% of its carbohydrate as lactose). The study was part of a large project that aimed at standardising the approach towards cow's milk protein intolerance in Baby Health Clinics. Nearly 50 Baby Health Clinics participated in this project. In this study, growth and symptomatology (skin, respiratory tract, gastrointestinal tract) were monitored during an intervention period of at least 10 weeks. Infants in both feeding groups showed normal growth, and in at least 80% of the infants an improvement of the overall symptomatology could be seen during the intervention period. Most profound were the decreases in prevalence and severity of eczema and infantile colic. No differences in efficacy were found in this study between the two infant formulas. It was concluded that the exclusive use of either whey hydrolysate based infant formula resulted in an improvement of allergic symptoms and in normal growth in infants diagnosed by elimination/provocation for cow's milk protein intolerance in a Primary Health Care setting.

Modification of an in-vitro method for estimating the bioavailability of zinc and calcium from foods.

A continuous flow in-vitro method for estimating the bioavailability of minerals and trace elements was modified. This modified method includes a simulated gastric digestion with pepsin, gradual pH change during the first 30 min of dialysis in an Amicon stirred cell, and a further 2 h of continuous dialysis accompanied by intestinal digestion with pancreatin-bile extract. The percentage of continuously dialysed minerals or trace elements (dialysability) is used to express the bioavailability. Comparison of different in-vitro methods by using the dialysability of zinc and calcium from milk- and soy-based formula samples revealed that with the modified method the results are closer to the in-vivo situation and could be used as a relative index for predicting the bioavailability of some minerals and trace elements.

Biochemistry of human milk in early lactation.

With the analysis of more than 550 human milk samples we showed that triglycerides (accounting for 96-99% of the total lipids) increased from 2 to 3.5 g/100 ml mainly during the first week postpartum and remained constant thereafter. In contrast, both cholesterol and vitamin E concentrations decreased from 35 to 20 mg/100 ml and from 1.7 to 0.30 mg/100 ml, respectively. The phospholipids remained constant (40 mg/100 ml). Also the fatty acid composition of total lipids changed. Mid chain fatty acids (C10, C12, C14) increased, whereas the long chain polyunsaturated fatty acids decreased. The P/S-ratio of 0.32 remained constant throughout lactation. The mean protein content of human milk decreased from approximately 2 g/100 ml at day 2 to approximately 1 g/100 ml at day 36 of lactation. The content of each individual amino acid decreased likewise. However, when we expressed the amino acid values in relation to the protein value--that is as g amino acid per g protein--some decreased, some remained constant and some increased indicating a changing protein pattern. From these data we computer-calculated a sharp decrease in IgA content, a moderate decrease for lactoferrin and constant values for casein and a-Lactalbumin. Throughout the whole period of investigation, non protein nitrogen accounted for about 20% of total nitrogen. Although the absolute values decreased according to total nitrogen, the sum of free amino acids, as well as the amount of taurine, remained constant. Lactose increased from about 4 g/100 ml to 6 g/100 ml during the first days of lactation. The calcium content increased from a mean value of 25 mg/100 ml at day 1 to 32 mg/100 ml at day 5 and remained constant at 30 mg/100 ml up to day 36. Phosphorus content increased from 10 mg/100 ml at day 1 to 17 mg/100 ml at day 8 and then continuously decreased to 13 mg/100 ml at day 36. The protein-bound part of the calcium remained constant during the period investigation, the fat-associated part increased from 11% to 26%. Protein-bound phosphorus decreased from 45% in colostral milk to 29% in transitional and 23% in mature milk.

Biochemistry of maternal milk in early lactation.

The analysis of more than 550 human milk samples showed that triglycerides (accounting for 96-99 per cent of the total lipids) increased from 2 to 3.5 g/100 ml mainly during the first week postpartum and remained constant thereafter. In contrast, both cholesterol and vitamin E concentrations decreased from 35 to 20 mg/100 ml and from 1.7 to 0.30 mg/100 ml, respectively. The phospholipids remained constant (40 mg/100 ml). The fatty acid composition of total lipids showed remarkable changes. Mid-chain fatty acids (C10, C12, C14) increased, whereas the long-chain polyunsaturated fatty acids decreased. The P/S ratio of 0.32 remained constant throughout lactation. The mean protein content of human milk decreased from approximately 2 g/100 ml at day 2 to approximately 1 g/100 ml at day 36 of lactation. The content of each individual amino acid decreased likewise. However, when we expressed the amino acid values in relation to the protein value--that is as gram of amino acid per gram of protein--some decreased, some remained constant and some increased indicating a changing protein pattern. Throughout the whole period of investigation, non-protein nitrogen accounted for about 20 per cent of total nitrogen, taurine ranging at about 6 mg/100 ml. The calcium content increased from a mean value of 25 mg/100 ml at day 1 to 32 mg/100 ml at day 5 and remained constant at 30 mg/100 ml up to day 36. The mean phosphorus content increased from 10 mg/100 ml at day 1 to 17 mg/100 ml at day 8 and then continuously decreased to 13 mg/100 ml at day 36.

The reaction of citraconic anhydride with bovine alpha-crystallin lysine residues. Surface probing and dissociation-reassociation studies.

Citraconic anhydride reacts readily with alpha-crystallin's lysine residues at pH 7.4. Upon addition of 2 equivalents of citraconic anhydride per equivalent lysine, 24% of the lysine residues were modified without disrupting the native quaternary structure. Further citraconylation led to dissociation into 10 S aggregates. Complete dissociation into subunits (1.4 S) occurred after adding 100 equivalents of citraconic anhydride, resulting in 98% modification. Decitraconylation did not lead to reaggregates identical with the native ones. The unmodified and the once and twice citraconylated alpha-crystallin subunits were discerned by isoelectric focusing according to their theoretical isoelectric points. In the native alpha-crystallin aggregates, nearly all B chains and approx. 60% of the A chains were found to possess at least one surface-exposed lysine residue. No differences between the susceptibilities to citraconylation of the in vivo deamidated (A1 and B1) and the de novo synthesized (A2 and B2) subunits were found. These results support the three-layer spherical assembly model for the alpha-crystallin quaternary structure.

Human milk nonprotein nitrogen components: changing patterns of free amino acids and urea in the course of early lactation.

Free amino acids and urea were analyzed in 78 human milk samples obtained during the first 5 wk of lactation from 10 mothers delivering at term. Significant differences (p less than 0.05) in the concentrations between colostral and mature milk were found for glutamic acid, glutamine, alanine, glycine, cystine, and phosphoethanolamine which increased, and with serine, phosphoserine, aspartic acid + asparagine, arginine, lysine, isoleucine, phenylalanine, proline, methionine, tryptophan, and beta-alanine which decreased. Some of these changes occurred within the first 5 days of lactation, so that differences between transitional and mature milk became negligible (glutamic acid, alanine, and serine, aspartic acid + asparagine, lysine, isoleucine, methionine, tryptophan, respectively). No significant differences between any of the three stages of lactation were found regarding the concentrations of total free amino acids, urea, taurine, threonine, valine, leucine, histidine, and tyrosine. Possible relevances for free amino acids, including nonprotein ones, in human milk are discussed.