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A critical analysis of total sialic acid and sialoglycoconjugate contents of bovine milk-based infant formulas

BACKGROUND Several infant formulas were bovine milk-based products. Mature bovine milk has a very low sialoglycoconjugate content compared with human milk from the first phases of lactation. METHODS The present study was undertaken to determine total sialic acid and oligosaccharide, glycoprotein, and ganglioside sialic acid contents of bovine milk-based formulas. RESULTS Starter formulas, designed for the first days/weeks after birth, have very similar sialic acid contents (233-266 mg/L fresh milk). We found more oligosaccharide-bound sialic acids (167-174 mg/L fresh milk) than those bound to proteins (53-84 mg/L fresh milk) in these formulas. The ganglioside sialic acid contents of starter formulas (952-1135 micrograms/L fresh milk) vary slightly from formula to formula. However, all the above-mentioned contents are lower than in human colostrum or transitional milk. CONCLUSIONS Infants fed starter formulas have total sialic acid and oligosaccharide, glycoprotein, and ganglioside sialic acid intakes of 36, 28, 50, and 20%, respectively, of those fed human colostrum or transitional milk. By contrast, follow-on formulas, used from 4 to 5 months of age, provide total sialic acid and oligosaccharide, glycoprotein, and ganglioside sialic acid contents similar to those furnished by mature human milk. Since the reference standard for optimal nutrition in the early months of infancy is human milk, a supplementation with sialic acid-containing glycoconjugates of infant formulas recommended for the first days after delivery could be advisable when breast-feeding is not possible.

Glycosphingolipids from bovine milk and milk fat globule membranes: a comparative study. Adhesion to enterotoxigenic Escherichia coli strains

Abstract Several components of milk fat globule membranes (MFGMs) have been reported to display beneficial health properties and some of them have been implicated in the defense of newborns against pathogens. These observations prompted us to determine the glycosphingolipid content of MFGMs and their interaction with pathogens. A comparative study with whole milk components was also carried out. Milk fat globules and MFGMs were isolated from milk. Gangliosides and neutral glycosphingolipids were obtained from MFGMs and whole milk and their fatty acid contents were determined by gas chromatography-mass spectrometry (GC-MS). MFGMs and whole milk showed similar ganglioside and neutral glycosphingolipid contents, with whole milk having more GM3 and glucosylceramide and less GD3, O-acetyl GD3, O-acetyl GT3, and lactosylceramide. The fatty acid content of gangliosides from both sources showed a similar composition. However, the neutral glycosphingolipid fatty acid content seemed to be quite different. Whole milk had fewer very-long-chain fatty acids (18.1% vs. 46.4% in MFGMs) and more medium-chain and unsaturated C18:1 and C18:2 fatty acids. Milk fat globules, MFGMs, lactosylceramide, and gangliosides GM3 and GD3 were observed to bind enterotoxigenic Escherichia coli strains. Furthermore, bacterial hemagglutination was inhibited by MFGMs and glycosphingolipids.

Bovine milk gangliosides: Changes in ceramide moiety with stage of lactation

The stage of lactation is one of the most important factors that influence milk composition. Changes in fatty acids from triacylglycerols and phospholipids have already been reported. In this study, we looked for a lactational change in the ganglioside lipid moeity since ganglioside contents and patterns vary strongly with stage of lactation. Individual gangliosides from four stages were isolated, methanolyzed to cleave the bonds between individual constituents, and derivatized for gas-liquid chromatography and gas chromatography/mass spectrometry analyses. Ceramide components, both fatty acids (as methyl esters derivatives) and long-chain bases, were identified and quantified. The results pointed to a marked change in ceramide from colostrum to milk that was characterized by a dramatic decrease in saturated and the longest-chain fatty acids as well as an increase in 18∶1 and 18∶2. The major long-chain base along lactation was a recently described structure, 3-ethoxy-15∶0 sphinganine. Other new long-chain base structures appeared in these gangliosides. All these changes suggest differences in the fluidity of the fat globule membrane, reflecting physiological variations in cows with respect to milk production.

Distribution of sialic acids in the milk of spanish mothers of full term infants during lactation.

Objectives: The protective effect of human milk against infection is well known. Several non-immunologic components, including complex carbohydrates, have been described. The present study was undertaken to determine the sialic acid distribution in different milk fractions (complex carbohydrates). Methods: Milk samples from 12 Spanish women at three different lactational stages (colostrum, transitional milk and mature milk) were analyzed. Total and glycoprotein-bound, oligosaccharide-bound, casein-bound, and lipid-bound sialic acids were determined. Results: Sialic acids from human milk are mainly bound to oligosaccharides and only a small amount is present bound to glycoproteins or in the free form. All the fractions analyzed showed a similar trend: sialic acids decrease rapidly along lactation. Casein-bound sialic acid does not follow this trend. We detected the presence of an O-acetylated species of N-acetylneuraminic acid. Conclusions: In human milk from Spanish women we observed slightly different values than those previously reported. This could be a result of population differences but nutritional or methodological aspects can not be discarded.

Binding of milk oligosaccharides by several enterotoxigenic Escherichia coli strains isolated from calves

Milk oligosaccharides have been proposed to play an important role in newborn defense, blocking bacterial adhesion to the intestinal mucosa and preventing infections. Some studies have been performed on human milk oligosaccharides. Here we checked whether bovine milk oligosaccharides would achieve the same protective action against the most common calf enteric pathogens. Seven enterotoxigenic Escherichia coli strains, isolated from diarrheic calves, were selected. All strains managed to agglutinate horse erythrocytes, and we therefore used the inhibition of hemagglutination in the presence of oligosaccharides as an indicator of the union between oligosaccharide and bacterial adhesins. Oligosaccharides from different stages of bovine lactation and standard oligosaccharides were assayed. Midlactation milk, in particular that corresponding to the transition period, proved to be the most efficient at inhibiting hemagglutination. The standard oligosaccharides used pointed to the preference of several strains (K99-, F41-, and F17-fimbriated) for α2,6-linked sialic acid. By contrast, B23 fimbriae exhibited higher affinity for α2,3-sialylated isomers and B64 seemed to require N-acetylglucosamine for binding.Our results suggest a general trend for milk oligosaccharides. Probably they participate in the protection of newborn mammals from pathogens.

Enterotoxigenic Escherichia coli strains bind bovine milk gangliosides in a ceramide-dependent process

Diarrhea caused by enterotoxigenic Escherichia coli (ETEC) is the main infectious disease of newborn calves. The first step of infection involves bacterial attachment to the intestinal mucosa. This adhesion is mediated by fimbriae that recognize some glycoconjugates on the host cell surface, in particular, several gangliosides. Because milk also contains gangliosides, these have been suggested to serve as ligands for bacterial fimbriae and thus prevent the bacterial attachment to mucosa. The most relevant ETEC strains in calves, including those with K99 and F41 fimbriae, were assayed to determine whether they are able to bind gangliosides isolated from several stages of bovine lactation. Both GM3 and GD3, the main gangliosides of milk, were recognized by ETEC strains, although the different fimbriae showed diverse levels of affinity. Unexpectedly, the adhesion to colostral gangliosides was considerably weaker than that to gangliosides from the other stages of lactation. Because the carbohydrate moiety did not change and because differences in the percentages of unsaturated FA and sphingosine between colostrum and other stages were observed, we conclude that the differences in adhesion could be due to a different composition of the ganglioside caramide.

Developmental Changes in UDP-N-Acetylglucosamine 2-Epimerase Activity of Rat and Guinea-Pig Liver

The activity of UDP-N-acetylglucosamine 2-epimerase was determined in the liver of rats and guinea-pigs of different ages. The activity of this enzyme in rats was low at birth, increased to a maximum value on day 15, and fell gradually until day 30. Thereafter, it increased up to the 60th day. The activity profile of the enzyme from guinea-pig liver was very similar. However, guinea-pig activity was 2-5 times lower than in rats. Both rats and guinea-pigs displayed similar liver sialic acid contents which increased from birth to 2 months of age. Rats also showed a N-glycolylneuraminic acid content that decreased from birth to 2 months. From these results we can inferred that postnatal UDP-N-acetylglucosamine 2-epimerase activity seems to be correlated with age and the developmental states of rats and guinea-pigs.

Effect of simulated gastrointestinal digestion on sialic acid and gangliosides present in human milk and infant formulas.

The effects of simulated gastrointestinal digestion upon sialic acid and gangliosides in infant and follow-on formulas and human milk, as well as their bioaccessibility, have been evaluated. The gastric stage is the step that causes a greater decrease in sialic acid and ganglioside contents. The intestinal stage only decreases the total and individual contents of gangliosides. After gastrointestinal digestion, neither sialic acid nor gangliosides were found in the nonbioaccessible fraction. The highest bioaccessibility (100 × content in soluble fraction after gastrointestinal digestion/total content) of sialic acid is found in human milk (87%), followed by infant formula (77%) and follow-on formula (16%). In the case of gangliosides, the highest bioaccessibility is present in the follow-on formula (51%), followed by human milk (29%) and infant formula (5%).

Ganglioside alterations in various brain areas, liver and kidney from chronic alcoholic rats

Ethanol was administered to Wistar male rats for 4 or 10 months and to female rats for 21 2 months (including gestation), using a 20% ethanol-water solution as the only fluid. Gangliosides (expressed as NeuAc) from forebrain, cerebellum, brain stem, liver and kidney of the alcoholic rats and their newborns were determined by densitometry. Forebrain and liver from males showed a statistically significant increase in their ganglioside-NeuAc content after 4 months of alcohol ingestion. In addition, when the treatment lasted up to 10 months the increase was larger. In contrast, a significant decrease of cerebellar and kidney ganglioside-NeuAc content was found after 10 months. The ganglioside pattern of the different sources displayed a variable profile. Moreover, while alcohol fed mothers showed a significant increase in the ganglioside-NeuAc content of cerebellum and liver, and a decrease in the brain stem, newborns of mothers given alcohol in their drinking water exhibited an increase of ganglioside-NeuAc content in cerebellum, liver and kidney and a decrease in forebrain.

Changes of acetylcholinesterase activity in brain areas and liver of sucrose- and ethanol-fed rats.

The effects of chronic ethanol or sucrose administration to rats on acetylcholinesterase from brain and liver were investigated. Membrane-bound and soluble acetylcholinesterase activities were determined in fractions prepared by centrifugation. The thermal stability and the effects of temperature and different types of alcohols on acetylcholinesterase activity were also studied. Membrane-bound acetylcholinesterase activity increased (p < 0.01) in the liver after chronic ethanol administration, whereas no differences among groups in the encephalic areas, except in the brain stem soluble form, were found. Membrane-bound acetylcholinesterase from the ethanol- and sucrose-treated groups was more stable at the different temperatures assayed between 10 and 50°C than that corresponding to the control group. Non-linear Arrhenius plots were obtained with preparations of membrane-bound acetylcholinesterase from rat liver, with discontinuities at 30°C (control or sucrose groups) or 34–35°C (alcohol group). Assays made with membrane-bound or soluble enzyme from brain showed linear Arrhenius plots in all groups studied. The inhibitory effects of increasing concentrations of ethanol, n-propanol and n-butanol on acetylcholinesterase preparations from forebrain, cerebellum, brain stem and liver of the three experimental groups (control, sucrose-fed and ethanol-fed) were very similar. However, n-butanol displayed a biphasic action on particulate or soluble preparations of rat forebrain. n-butanol inhibited (competitive inhibition) at higher concentrations (250–500 mM), while at lower concentrations (10–25 mM), the alcohol inhibited at low substrate concentrations but activated at high substrate concentration. These results suggest that the liver is more affected by ethanol than the brain. Moreover, the lipid composition of membranes is probably modified by ethanol or sucrose ingestion and this would affect membrane fluidity and consecuently the behaviour of acetylcholinesterase.