Phenylalanine kinetics differ between formula-fed and human milk-fed preterm infants

Infants fed casein-dominant formulas have higher plasma phenylalanine and tyrosine concentrations than those fed mother's milk. Conversely, elevated plasma threonine concentrations are observed in infants fed whey-dominant formulas. We recently showed that formula-fed preterm infants have a lower capacity to degrade threonine than do preterm infants fed mother's milk. We hypothesized that these same infants (n = 18) would differ in their catabolism of phenylalanine in response to phenylalanine loads provided by formulas with increasing casein content of formulas (whey:casein 60:40, 40:60, and 20:80) compared with preterm infants fed mother's milk. Plasma phenylalanine concentrations significantly rose (49, 46, 79 micromol . L(-1) for whey:casein 60:40, 40:60, and 20:80, respectively, pooled SD 8, P < 0.05); and plasma phenylalanine concentrations in infants fed mother's milk were low (40 +/- 4 micromol . L(-1)). Using [1-(13)C]phenylalanine tracer and (13)CO(2) production in breath we found that although there was a significant positive relation between phenylalanine oxidation and phenylalanine intake in formula-fed infants (r(2) = 0.43, P = 0.03), these infants were not able to increase their oxidation of phenylalanine enough to prevent a significant rise in plasma phenylalanine when fed the 20:80 formula. Compared to infants fed mother's milk, formula-fed infants had significantly lower phenylalanine oxidation (39.1 vs. 30.7% of phenylalanine intake, respectively, P < 0.05). We conclude that one of the mechanisms for the differences in plasma amino acid concentration between formula-fed and mother's milk-fed preterm infants may be in vivo down-regulated catabolism of 2 important essential amino acids (phenylalanine in addition to threonine) in formula-fed preterm infants.     

human and bovine milk gangliosides differ in their fatty acid composition

Gangliosides are considered bioactive components in human infant nutrition, and their fatty acid composition alters their biological effects. We used matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) MS and GLC to analyze the fatty acid composition of the predominant gangliosides, the monosialoganglioside GM(3) [sialic acid (Sia) alpha2-3 galactose (Gal) beta1-4 glucose (Glc) beta1-1 ceramide] and the disialoganglioside GD(3) (Sia alpha2-8 Sia alpha2-3 Gal beta1-4 Glc beta1-1 ceramide), in pooled human and bovine milk, the latter being a source for gangliosides in infant formula. Compared with whole milk lipids, both human and bovine milk gangliosides were selectively enriched with certain fatty acids, and the fatty acid composition of milk gangliosides in the 2 species was significantly different. The amount of long-chain fatty acids (> or =20 C atoms) was higher in bovine milk gangliosides (GM(3): 73.71 +/- 3.39%; GD(3): 79.19 +/- 2.79%) than in human milk gangliosides (GM(3): 51.25 +/- 0.65%; GD(3): 34.04 +/- 1.80%). Tricosanoic acid (23:0) dominated in bovine milk gangliosides (GM(3): 24.05 +/- 1.37%; GD(3): 26.66 +/- 1.24%), whereas it only played a minor role in human milk gangliosides (GM(3): 2.88 +/- 0.10%; GD(3): 1.84 +/- 0.29%). We hypothesized that the differences in the fatty acid composition of milk gangliosides result in physiological distinctions between breast-fed and formula-fed infants and therefore are of importance for human infant nutrition.     

Profiles of human milk oligosaccharides and production of some human milk oligosaccharides in transgenic animals

During the decade of the 1990s and the first years of the current century, our group embarked on a project to study and synthesize human milk oligosaccharides. This report describes 2 unexpected collateral observations from that endeavor. The first observation was the detection and confirmation of 2 rare neutral human milk oligosaccharides profiles that were uncovered while assessing oligosaccharide content in hundreds of samples of human milk. One of these lacked fucosylated structures altogether, and the other lacked the oligosaccharide 3-fucosyllactose [Galβ1-4(Fucα1-3)Glc]. We used glycoconjugate probes to determine whether the unusual profiles were mirrored by fucosylation of milk glycoproteins. The results show that the lack of fucosylated oligosaccharides in these samples corresponds to the absence of equivalent fucosylated motifs in milk glycoproteins. The second finding was a shortened and distinct lactation process in transgenic rabbits expressing the human fucosyltransferase 1. During the first day of lactation, these animals expressed milk that contained both lactose and 2'-fucosylactose, but on the second day, the production of milk was severely diminished, and by the fourth day, no lactose was detected in their milk. Meanwhile, the concentration of fucosylated glycoproteins increased from the onset of lactation through its premature termination. These 2 findings may shed light on the glycobiology of milk and perhaps on mammary gland differentiation.     

Advances in analysis of human milk oligosaccharides

Oligosaccharides in human milk strongly influence the composition of the gut microflora of neonates. Because it is now clear that the microflora play important roles in the development of the infant immune system, human milk oligosaccharides (HMO) are studied frequently. Milk samples contain complex mixtures of HMO, usually comprising several isomeric structures that can be either linear or branched. Traditionally, HMO profiling was performed using HPLC with fluorescence or UV detection. By using porous graphitic carbon liquid chromatography MS, it is now possible to separate and identify most of the isomers, facilitating linkage-specific analysis. Matrix-assisted laser desorption ionization time-of-flight analysis allows fast profiling, but does not allow isomer separation. Novel MS fragmentation techniques have facilitated structural characterization of HMO that are present at lower concentrations. These techniques now facilitate more accurate studies of HMO consumption as well as Lewis blood group determinations.     

Glucose-based oligosaccharides exhibit different in vitro fermentation patterns and affect in vivo apparent nutrient digestibility and microbial populations in dogs

To evaluate the potential of indigestible oligosaccharides (OS) to serve as "dietary fiber-like" ingredients, it is necessary to determine their extent of indigestibility. In vitro fermentation characteristics of two novel OS, alpha-glucooligosaccharides (GOS) and a maltodextrin-like OS (MD), were compared to those of fructooligosaccharides (FOS), gum arabic (GA), guar gum (GG) and guar hydrolysate (GH). Total short-chain fatty acid (SCFA) production (micromol/g dry matter) as a result of MD fermentation was higher initially compared with GA (P<0.01), but GA was more extensively fermented at 24 h (P<0.01). Total SCFA production for GOS was similar to that for FOS, GG, GH and GA. In the second experiment, GOS and MD were added at 6% to an enteral formula control diet (Control) and fed to ileal-cannulated dogs in a 3x3 replicated Latin-square design. Ileal digestibility of glucose was lower (P<0.05) and carbohydrate (CHO) numerically lower (P = 0.08) for both GOS and MD compared with the Control. Total tract digestibility of CHO and glucose was lower only for MD (P<0.01) compared with the Control. Total fecal weights were higher (P<0.01) for both GOS and MD treatments. Fecal concentration of bifidobacteria was numerically increased by GOS and MD supplementation (P = 0.13 and 0.23, respectively). Thus, GOS and MD are indigestible yet fermentable OS, and may act as "dietary fiber-like" ingredients.     

Lactose-derived oligosaccharides in the milk of elephants: comparison with human milk

Human milk is commonly considered to be unique when compared with the milk of other species with regard to its high content of complex fucosylated and sialylated lactose-derived oligosaccharides. We describe the application of high-pH anion-exchange chromatography with pulsed amperometric detection and TLC to characterize and quantitate neutral and sialylated lactose-derived oligosaccharides in milk from three Asian elephants and human milk. The lactose contents of elephant and human milks were 25-30 g/l and about 66 g/l respectively, whereas total oligosaccharide concentration was about three times higher in elephant milk and comprised up to 40% (10% in human milk) of the carbohydrate content. The ratio neutral: acidic components was different in the milk of the two species; in elephant milk, the N-acetylneuraminic acid-containing oligosaccharides made up almost half of the total amount v. 30% in human milk. Most oligosaccharides in elephant milk were more fucosylated and/or sialylated compared with human milk components. By mild acid hydrolysis, fucose and N-acetylneuraminic acid were cleaved off from complex components, and this resulted in increased amounts of fucose, galactose, N-acetylneuraminic acid, lactose and lacto-N-neo-tetraose. Unique to elephant milk are the high levels of 3'-galactosyllactose (up to 4 g/l) and lacto-N-neo-tetraose which are present in human milk only in trace amounts. Elephant and human milks have high levels and unique patterns of oligosaccharides which may reflect the relative importance of these components in neonatal host defence, in endothelial leucocyte interactions or in brain development.     

Breast-fed and formula-fed infants do not differ in immunocompetent cell cytokine production despite differences in cell membrane fatty acid composition

BACKGROUND: Breast-fed and formula-fed infants differ in the amount and type of polyunsaturated fatty acids consumed. The fatty acid composition of cell membranes is related to dietary fatty acids and, in adults, changes in membrane fatty acid composition are accompanied by changes in monocyte cytokine production and hence a modification of the immunologic response. OBJECTIVE: Our objective was to determine whether production by immunocompetent cells of the proinflammatory cytokines interleukin 1 (IL-1) and tumor necrosis factor (TNF) differs between breast-fed and formula-fed infants. DESIGN: Twenty-six healthy infants (13 breast-fed and 13 fed modified cow-milk formula) aged 2-4 mo were studied. The fatty acid composition of red blood cell (RBC) membrane phospholipids was measured by gas-liquid chromatography and IL-1 and TNF release were measured in whole blood culture in bacterial-endotoxin-stimulated and unstimulated cells. RESULTS: The infants' ages, weights, hemoglobin concentrations, and white blood cell counts did not differ significantly between groups. The percentage of n-3 fatty acids of total RBC phospholipid fatty acids was significantly higher in breast-fed than in formula-fed infants (6.31 +/- 2.5% compared with 2.98 +/- 0.97%); docosahexaenoic acid (22:6n-3) concentrations were also markedly higher in breast-fed infants (5.1 +/- 1.2% compared with 2.2 +/- 0.9%, P: < 0.001), but eicosapentaenoic acid (20:5n-3) and docosapentaenoic acid (22:5n-3) concentrations did not differ significantly between groups. The percentage of n-6 fatty acids was not significantly different between groups. The percentage of oleic acid (18:1) was higher in formula-fed than in breast-fed infants (16.2 +/- 0.7% compared with 20.6 +/- 1.1%; P: < 0.001). IL-1 and TNF release in whole blood culture did not differ significantly between groups. CONCLUSION: The release of proinflammatory cytokines by immunocompetent cells does not differ significantly in breast-fed and formula-fed infants despite differences in cell membrane fatty acid composition.     

低敏配方喂养的牛奶过敏儿童生长及体成分发展的纵向随访性研究

目的 纵向观察生后早期低敏配方(HF)喂养的牛奶过敏(CMA)婴儿的体格生长特别是体成分(BC)的发展情况,以便对该群体进行营养与生长的评估和监测。方法 于2014年1月-2015年9月将60名HF喂养且不满4月龄的CMA婴儿纳入本次研究。给婴儿进行常规健康体检直至其满1周岁,并在6月龄及此之前采用PEA POD^®婴儿体成分测量仪、12月龄采取皮褶厚度测量的方法评估其BC发展。结果 婴儿的平均入组年龄为(2.9±1.0)个月。与年龄配对的健康对照组儿童相比,HF喂养的CMA婴儿在6～12月龄的年龄别体重、9～12月龄的身长别体重Z评分均更低;体脂百分比在6月龄(26.2% vs 30.3%,P=0.015)和12月龄(23.8% vs 27.1%,P=0.028)时均明显落后;平均脂肪组织指数(FMI)较对照组落后至少0.2个单位,但差异无统计学意义(P>0.05)。结论 生后早期HF喂养的CMA婴儿有可能出现体重增长和体脂储存的受损。     

Mass spectrometric methods for analysis of oligosaccharides in human milk

Establishing the analytical platforms for characterizing human milk oligosaccharides is important to fully assess their specific functionalities. The characterization of these biomolecules, however, is still considered challenging, owing to their overall complexity and diversity. Addressed here are the technical difficulties with an emphasis on the application of mass spectrometry to rapidly profile and quantify human milk oligosaccharides. Fundamental concepts and improvements in instrumentation and an overview of the biological functions and structures of these compounds are also discussed. Results reveal that small-chain oligosaccharides, evident in abundance in the early stage of lactation, are selectively consumed by specific stains of Bifidobacterium longum biovar, infantis.     

Bifidobacterial enzymes involved in the metabolism of human milk oligosaccharides

Intestinal colonization of bifidobacteria is important for the health of infants. Human milk oligosaccharides (HMO) have been identified as growth factors for bifidobacteria. Recently, a bifidobacterial enzymatic system to metabolize HMO was identified. 1,3-β-Galactosyl-N-acetylhexosamine phosphorylase (GLNBP, EC 2.4.1.211), which catalyzes the reversible phosphorolysis of galacto-N-biose (GNB) (Galβ1→3GalNAc)] and lacto-N-biose I (LNB) (Galβ1→3GlcNAc), is a key enzyme to explain the metabolism of HMO. Infant-type bifidobacteria possess the intracellular pathway to specifically metabolize GNB and LNB (GNB/LNB pathway). Bifidobacterium bifidum possesses extracellular enzymes to liberate LNB from HMO. However, Bifidobacterium longum subsp. infantis imports intact HMO to be hydrolyzed by intracellular enzymes. Bifidobacterial enzymes related to the metabolism of HMO are useful tools for preparing compounds related to HMO. For instance, LNB and GNB were produced from sucrose and GlcNAc/GalNAc in 1 pot using 4 bifidobacterial enzymes, including GLNBP. LNB is expected to be a selective bifidus factor for infant-type strains.     

Human and bovine milk oligosaccharides inhibit Neisseria meningitidis pili attachment in vitro

Milk oligosaccharides have been shown to interfere with adhesion of many pathogens to host mucosal surfaces. Characterization of the adhesion mechanisms of the bacteria to host cell surface is needed to develop novel functional food, infant formulas, and anti-infective drugs. Adhesion of Neisseria meningitidis, a human specific pathogen causing meningitis and septicemia, is not completely understood but is mediated by type IV pili. Here, we developed a microtiter well pili binding assay to investigate the binding activities of N. meningitidis isolated type IV pili to different glycoproteins. Pili binding activities to bovine thyroglobulin and human salivary agglutinin but not to chicken ovalbumin were present. Inhibition of these binding activities was demonstrated by fractionated human or bovine milk oligosaccharides. The binding of neisserial pili to bovine thyroglobulin was most effective and was clearly inhibited by human milk neutral or bovine milk acidic oligosaccharides.     

Human milk oligosaccharides affect P-selectin binding capacities: in vitro investigation

OBJECTIVE: In the initial phase of cellular immune response, selectins mediate the emigration of leukocytes from the blood stream into inflammatory regions. Human milk oligosaccharides (HMOs) possess binding epitopes of selectin ligands such as sialyl Lewis(x) and sialyl Lewis(a) and therefore might impair the interaction of selectins with cellular ligands. Neutral, acidic, sialylated, or fucosylated HMO fractions with polymerization degrees of 3 to 50 were investigated regarding this interaction in a dynamic flow chamber model that considers physiologic shear stress conditions. METHODS: Human milk oligosaccharides were compared with kappa-carrageenans and pectin oligosaccharides to deduce structure-activity relations. Fucoidan and sialyl Lewis(x) served as positive controls. RESULTS: All HMO fractions affected P-selectin ligand binding capacity but were not comparable to fucoidan. The activity of the acidic HMO fraction resembled sialyl Lewis(x) in decreasing the binding of the ligand to P-selectin. CONCLUSION: Human milk oligosaccharides modulate rather than block the function of P-selectin.     

N-acetylneuraminic acid concentrations in human milk oligosaccharides and glycoproteins during lactation

Human milk contains a variety of N-acetylneuraminic acid (NANA)-containing oligosaccharides, but the expected range of intake of sialic acid in this form by infants fed human milk is unknown. Two quite different amounts have been reported: 120 mg/liter in pooled, mature human milk (1) and 1400 mg/liter in the milk of a single woman on the 1st day of lactation (2). The normal range of NANA intake in human milk glycoproteins likewise does not appear to have been analyzed previously. Data presented here indicate that both human milk oligosaccharide and glycoprotein NANA decline exponentially over the first 2 months of lactation, decreasing little thereafter. During the first 2 months of lactation, milk from women delivering at term cannot be distinguished from that of women delivering significantly before term (less than 32 wks gestation) with regard to oligosaccharide and glycoprotein NANA. The parallel decrease of sialic acid in these fractions suggests a relationship between sialydation of human milk oligosaccharides and glycoproteins. Human milk NANA concentrations are discussed with regard to reports that exogenous administration of NANA can increase cerebral and cerebellar concentrations of NANA in glycoproteins and gangliosides, and produce long term changes in behavior in rats.     

Secretory patterns of biotin in human milk.

The current recommendation for safe and adequate daily dietary intake of biotin for infants is based on measurements of biotin concentration in human milk and calculations of biotin intake that tacitly assume that biotin content of human milk is reasonably uniform for a given subject. This assumption of uniformity was tested by examining the effects of several factors on the biotin concentration. The degree of breast emptying had little effect on biotin concentration. However, in three of five individuals studied, the biotin concentration varied significantly over 24 h. In two of five subjects, there was a consistent difference between breasts of approximately 16%. In the first 18 d postpartum, the milk concentration of biotin increased steadily in four of the eight individuals studied, remained low in two and increased erratically in two. Rather than reaching a stable plateau in mature milk, biotin concentration varied substantially in most of the subjects. These observations provide evidence that an adequate scheme for estimating total biotin intake of the breastfed infant will require sampling from both breasts frequently over the 24-h cycle and frequently as a function of time postpartum.     

Effects of fermentation products of pro- and prebiotics on trans-epithelial electrical resistance in an in vitro model of the colon

Evidence from in vivo and in vitro studies suggests that the consumption of pro- and prebiotics may inhibit colon carcinogenesis; however, the mechanisms involved have, thus far, proved elusive. There are some indications from animal studies that the effects are being exerted during the promotion stage of carcinogenesis. One feature of the promotion stage of colorectal cancer is the disruption of tight junctions, leading to a loss of integrity across the intestinal barrier. We have used the Caco-2 human adenocarcinoma cell line as a model for the intestinal epithelia. Trans-epithelial electrical resistance measurements indicate Caco-2 monolayer integrity, and we recorded changes to this integrity following exposure to the fermentation products of selected probiotics and prebiotics, in the form of nondigestible oligosaccharides (NDOs). Our results indicate that NDOs themselves exert varying, but generally minor, effects upon the strength of the tight junctions, whereas the fermentation products of probiotics and NDOs tend to raise tight junction integrity above that of the controls. This effect was bacterial species and oligosaccharide specific. Bifidobacterium Bb 12 was particularly effective, as were the fermentation products of Raftiline and Raftilose. We further investigated the ability of Raftilose fermentations to protect against the negative effects of deoxycholic acid (DCA) upon tight junction integrity. We found protection to be species dependent and dependent upon the presence of the fermentation products in the media at the same time as or after exposure to the DCA. Results suggest that the Raftilose fermentation products may prevent disruption of the intestinal epithelial barrier function during damage by tumor promoters.     

Recent advances on structure, metabolism, and function of human milk oligosaccharides

Human milk is often the sole dietary source for the first few months in life. It contains all the nutrients necessary for the infant to thrive, but also ingredients that may provide health benefits beyond those of traditional nutrients. Human milk oligosaccharides (HMO) comprise part of these functional ingredients; 1 L of mature human milk contains approximately 5-10 g unbound oligosaccharides, and >130 different HMO have been identified. Both their high amount and structural diversity are unique to humans. Only trace amounts of these oligosaccharides are present in mature bovine milk and, as a consequence, in bovine milk-based infant formula. The potential health benefits of HMO that were uncovered over the years may affect breast-fed infants both locally and systemically. Recent advances in glycobiology and nutrition, including the use of stable isotopes, frontal-affinity chromatography, glycan microarrays, MS, and automated solid-phase carbohydrate synthesis, will help verify hypotheses and unravel the mysteries behind HMO.     

Length of gestation and nutritional composition of human milk

A comparative analysis of the composition of milk produced during the first 14 days of lactation by mothers who deliver prematurely and those who deliver at term is described and these values are contrasted with the composition of donor milk specimens. Twenty-four-hour milk collections (days 3, 7, and 14 postpartum) were obtained from nine mothers delivered between 37 to 42 wk gestation (term) and from 14 mothers who delivered between 28 to 36 wk gestation (preterm). A single spot milk collection was obtained from nine mothers who were 6 to 10 months postpartum (donor). Term and preterm milk was compared on specific postpartum days using an analysis of covariance controlling for 24-h milk volume. The protein, carbohydrate, fat, and energy content varied in a similar fashion in term and preterm milk over the 14 postpartum days studied. Furthermore, there was no significant difference between the two milk groups on any single postpartum day evaluated in terms of protein, carbohydrate, fat, or energy concentration. The milk volumes were significantly greater from the mothers delivered at term on days 7 and 14 (p less than 0.01) and the protein content of both term and preterm milk was negatively correlated with milk volume (r = -0.6 or more on each day studied). The nutrient and energy composition of spot donor milk was highly variable and frequently quite different from either term or preterm 24-h milk collections. These data indicate that milk from mothers who deliver prematurely does not contain significantly different concentrations of nutrients or energy than milk from mothers delivered at term and suggest that the differences previously noted between the two groups may be related to 24-h milk volume.