Neutral glycosphingolipid content of ovine milk.

Milk glycosphingolipids (GSL) have been reported to participate in the newborn's defense against pathogens. Taking this into account, in this study we determined the neutral GSL content of ovine milk, including its fatty acid profile. Its role in bacterial adhesion was also addressed by immunodetection of separate GSL in a high-performance thin-layer chromatography overlay assay. Ovine milk has a neutral GSL pattern similar to human milk and includes lactosylceramide (LacCer; 45.7%), monohexosylceramide (glucosylceramide and galactosylceramide, 31.2%), globotriaosylceramide (Gb3; 19.1%), and globotetraosylceramide (Gb4; 3.5%). Globotriaosylceramide and Gb4 are present in human but not bovine milk. Neutral GSL contained C23:0 and C24:0 as the most abundant fatty acids, a finding consistent with its high content of very long chain fatty acids (longer than C20). Most fatty acids were saturated and had a low content of polyunsaturated fatty acids. Bovine enterotoxigenic Escherichia coli strains bound strongly to LacCer and showed a weak binding to monohexosylceramide. The K99 strain also bound strongly to Gb3, and F41 to Gb4. Lactosylceramide, monohexosylceramide, and Gb3 were also observed to bind to human uropathogenic E. coli strains. The results reported here show the ability of neutral GSL in ovine milk to bind to E. coli strains. These compounds could be used as an alternative and available source to supplement infant or bovine formulas with a view to preventing bacterial infections.

Sialyloligosaccharides in human and bovine milk and in infant formulas: variations with the progression of lactation.

Several lines of research support a role for human milk oligosaccharides in the defense of breast-fed infants against pathogens. Some ofthese oligosaccharides contain at least one moiety of sialic acid and are, thus, termed sialyloligosaccharides. These constitute a significant component (>1 g/L) of human milk. It is well established that milk composition varies among species, and previous reports have indicated that one ofthe differences between human and bovine milk is precisely their contents of sialyloligosaccharides. Because most infant formulas are manufactured with bovine milk components, it follows that formula-fed and breast-fed infants ingest dissimilar quantities of these carbohydrate structures. To ascertain these differences and their impact along lactation, the contents of oligosaccharide-bound sialic acids and major sialyloligosaccharides in samples of human and bovine milk (obtained at different lactation stages) were determined. In addition, infant formulas were assayed for their sialyloligosaccharide contents. Seven sialyloligosaccharides were identified in human milk; namely, 3'-sialyl-3-fucosyllactose and sialyllacto-N-tetraoses (a and b+c), the predominant structures at all lactation stages. Five sialyloligosaccharides were identified in bovine milk, of which 6'-sialyllactosamine and 3'-sialyllactose were the most abundant. In addition, sialyloligosaccharides in human and bovine milk decreased along lactation, and infant formulas did not contain significant amounts of sialyloligosaccharides. The results point to the general conclusion that regarding both qualitative and quantitative aspects, milk from humans and cows and infant formulas have different oligosaccharide contents. In this sense, bottle-fed infants are subject to reduced sialyloligosaccharide intake as compared to breast-fed infants.

Distribution of bovine milk sialoglycoconjugates during lactation.

The sialoglycoconjugate content of human milk has been extensively studied. However, little attention has been paid to the changes occurring in these compounds in bovine milk during lactation. Since sialoglycoconjugates are very abundant in milk from the early stages of lactation, they have been suggested to be important for the nutrition of the newborn during the first months of life. The distribution of sialoglycoconjugates (expressed as glycoconjugate-bound sialic acid) from four different stages of lactation (colostrum, transitional, mature, and late-lactation milks) was investigated in four Spanish-Brown cows. All the fractions studied (total sialic acids, glycoproteins, oligosaccharides, casein, and gangliosides) showed a similar trend. We found the highest values in the colostrum, these decreasing in transitional and mature milks and increasing again in late-lactation milk. We also found a selective change in the relative contents of glycoprotein- and oligosaccharide-bound sialic acids. In mature milk, the latter increased up to 80% (59% in colostrum) and the former decreased to 3.9% (35.3% in colostrum). It would appear that the decrease in oligosaccharide-bound sialic acid is compensated by the increase in glycoprotein-bound sialic acid. From these results, it is deduced that newborn infants or calves fed with infant formulas or milk replacers, respectively, should be supplemented with sialoglycoconjugates to approximate the composition of human and cow milk as far as is practicable.

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 moiety since ganglioside contents and patterns vary strongly with stage of lactation. Individual gangliosides from four stages were isolated, methanolized 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.

Single-step gas chromatography-mass spectrometry analysis of glycolipid constituents as heptafluorobutyrate derivatives with a special reference to the lipid portion.

In a previous work (Zanetta et al. Glycobiology 9, 255-266 (1999)), it was reported that all constituents of gangliosides could be obtained as heptafluorobutyrate derivatives after methanolysis in a single gas chromatography analysis. This report demonstrates that gas chromatography coupled with mass spectrometry in the electron impact mode allows identification and quantification of long-chain bases and fatty acids without interference from monosaccharides. On the basis of ions specific for families and for individual compounds, sphingosines, sphinganines, and phytosphingosines (including ramified, unsaturated, hydroxylated, and etherified compounds) can be identified. Fatty acid methyl esters, including linear, ramified, unsaturated, and hydroxylated species, are identified and quantified in the same way. Possible extensions of this method to the fatty moiety of other lipids (alkylacylglycerol and dimethyl acetal) are discussed.

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 degrees 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 degrees C (control or sucrose groups) or 34-35 degrees 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 consequently the behaviour of acetylcholinesterase.

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.

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.

New data on content and distribution of gangliosides in human milk.

The content and distribution of gangliosides, and total lipid content, were studied in human milk samples from different periods of lactation. We found a significant correlation (r = 0.5564; p = 0.0165) between ganglioside and total lipid contents. There was a selective change in the relative concentrations of GD3 and GM3 during lactation. The most abundant ganglioside in samples from the first three weeks of lactation was GD3, whereas after the first month, GM3 was the major ganglioside. In addition to GD3 and GM3, previously known to be present in human milk, we detected several previously unreported highly polar gangliosides.

Cocaine exposure induces changes in the ganglioside content of rat liver.

During cocaine exposure, the liver undergoes significant morphological and biochemical changes. We report here changes in the ganglioside pattern of rat liver after repeated administration (over 5 hours, one injection per hour) of a moderate dose of cocaine (10 mg/kg body weight). Cocaine exposure results in an accumulation of more complex gangliosides (GM1, GD1a, GD1b, GT1b and GQ1b) and a reduction of precursors (GM3, GM2, GD3 and GD2). Our results suggest that ganglioside biosynthesis could be affected by an alteration of vesicular transport from cis- to trans-Golgi cisternae produced either by cocaine itself or by some product of cocaine metabolism.

Ganglioside content of rat liver after administration of ethanol and pentazocine or sucrose supplemented diets.

In a previous paper, we determined the effect of either ethanol or pentazocine administered separately on the ganglioside content of rat liver. In the present paper we have investigated the effect of pentazocine injection on the liver ganglioside contents of chronic alcoholic rats. The effect of high carbohydrate ingestion was also studied. Thirty male Wistar rats were divided into three experimental groups that received ethanol and pentazocine, a carbohydrate supplemented diet or a laboratory diet and water. Liver ganglioside contents were slightly increased in the ethanol plus pentazocine group as compared to the control and high carbohydrate diet groups. No differences were found between the two latter groups. The percentage distribution of individual gangliosides (ganglioside pattern) was also modified. A decrease in gangliosides belonging to the b-series (GD3, GD1b, GT1b and GQ1b) in parallel with an increase in that of the a-series (GM2, GM1 and GD1a) were found for both the ethanol plus pentazocine and the high-carbohydrate fed rats. The results suggest that ethanol or high carbohydrate ingestion diminishes the activity of GD3 synthase, a key enzyme in the metabolism of gangliosides, which determines the proportion of gangliosides, belonging to the a- and b-series.

Changes in ganglioside and sialic acid contents of goat milk during lactation.

The ganglioside content of goat milk has been determined from d 1 after parturition to d 60 of lactation. Marked changes occurred in milk over the course of lactation; the highest ganglioside content occurred in d-1 colostrum and then decreased to the end of the period studied. At least seven different ganglioside species were detected; three gangliosides containing sialyllactosylceramide accounted for 66 to 92% of the total lipid-bound sialic acid; this result reflected a very simple core structure of goat milk gangliosides. The most abundant ganglioside, II3(N-acetylneuraminic acid)2-lactosylceramide, was about 35 to 56%. The sialic acid content exhibited a trend similar to that of gangliosides; during early lactation sialic acid content was higher than in mature milk. Fat, protein, and total solids were high at initiation of lactation and decreased thereafter. However, lactose content remained almost unchanged during the period studied.

Lactational changes in the N-glycoloylneuraminic acid content of bovine milk gangliosides.

The N-glycoloylneuraminic acid (Neu5Gc) contents of milk and milk gangliosides from bovines were investigated during the different stages of lactation. The Neu5Gc content of milk is high in the colostrum (32% of the total sialic acid content of milk) and decreases thereafter until the end of the period considered (6% on day 30). When the Neu5Gc content of gangliosides was evaluated a similar profile to that of Neu5Gc in total sialic acids was found. Gangliosides from colostrum showed the highest Neu5Gc content (21-22% of the total sialic acid content of milk gangliosides). This content dropped towards the end of the period studied (8% on day 90). Our results indicate that a significant supply of Neu5Gc by the milk could be important for the newborn during the first days after parturition.

Gangliosides in bovine milk. Changes in content and distribution of individual ganglioside levels during lactation.

Bovine milk undergoes changes in its ganglioside contents during the different stages of lactation. These contents are higher in colostrum (7.5 mg of lipid-bound NeuAc/kg) than in transitional (2.3 mg) or mature (1.4 mg) milk. The sialic acid content of milk follows a similar profile to that of gangliosides with the highest content during the first few days post partum followed by a gradual decrease towards the end of the period studied. When the individual distribution of gangliosides was examined throughout the course of lactation, several changes were also found. GD3 is the major ganglioside (about 60-70%) found; its content decreases from the first to the fifth day, increasing towards the end of the period considered. GM3, GD3 and GT3, sialyllactosylceramide-containing gangliosides account for 80-90% of the total lipid-bound NeuAc content. The most striking change in the ganglioside pattern was the gradual increase in G3.

Ganglioside and phospholipid composition of forebrain, cerebellum, and brain stem from adult and newborn rats.

The aim of this study was to elucidate whether sex or pregnancy state might affect the content and/or pattern of gangliosides from the forebrain, cerebellum and brain stem of rats. Adult male, mother (1-day after offspring) and nonpregnant rats of similar age were analyzed. Non-significant differences in ganglioside concentrations and patterns were found for the respective neural area of adult male and female rats except for a decrease in cerebellum and brain stem content from mothers and 12.0 months-old males, respectively. Thus, it seems that neither sex nor pregnancy hormones affect these parameters. By contrast, significant differences were found for pattern and ganglioside contents between adult (male and female) rats and newborns (1 day-old). Newborns showed a significant decrease in their forebrain (2.5-fold), cerebellum (2.0-fold) and brain stem (2.0-fold) ganglioside content when compared with adult (male and female) rats. Significant increases (p less than 0.001) were found in the phospholipid and cholesterol contents in the different brain areas in mothers versus their newborns. The phospholipid pattern also showed significant changes in all brain areas, with an increase (p less than 0.001) in phosphatidylethanolamine percentage in adult animals, among the main variations. An explanation for these facts is suggested.

Lectin-resistant variants of mouse Lewis lung carcinoma cells. II. Altered glycosylation of membrane glycoproteins.

Lectin-resistant variants of mouse Lewis lung carcinoma LL2 cell line, selected with wheat germ agglutinin (WGAR), Ricinus communis agglutinin II (RCA IIR) and Aleuria aurantia agglutinin (AAAR) were studied. Total cellular glycopeptides of the parent LL2 line and of the five lectin-resistant variants were analyzed by gel filtration and affinity chromatography on immobilized concanavalin A and Lens culinaris agglutinin. The results revealed that low-metastatic WGAR and RCA IIR variants possessed less highly branched tri- and tetra-antennary N-acetyllactosaminic type glycans with a simultaneous increase in biantennary N-acetyllactosaminic type, oligomannosidic type or hybrid type glycans, as compared to the parent metastasizing LL2 cell line. These findings imply that cell surface carbohydrate changes may possibly be relevant for metastasis. However, the AAAR variant, which possessed reduced spontaneous metastatic ability after s.c. administration, but increased experimental metastatic ability after i.v. inoculation, exhibited apparently the same glycan pattern than the parent LL2 line. This particular variant is under investigation in order to find specific modification(s) of glycan(s) which could play a specific role in the metastatic process.

O-acetylated sialic acids in gangliosides from pig spleen lymphocytes.

The sialic acid content of gangliosides from pig spleen lymphocytes was studied by thin-layer chromatography. N-glycolylneuraminic acid and N-acetylneuraminic acid were detected for the first time in this material as the major sialic acids. In addition, two other sialic acids, tentatively designated O-acetylated sialic acids, according to their RF values on cellulose plates, were also found. We have detected several gangliosides showing a retarded migration pattern in two dimensional thin-layer chromatography with an intermediate ammonia treatment. One of these gangliosides could be an O-acetylated derivative of the disialoganglioside GD3, since after de-O-acetyation it co-migrates with GD3. Another ganglioside co-migrated with GM2 before the alkaline treatment; however, after the treatment it was also retarded and co-migrates with GD3.

Ganglioside composition of liver and kidney from rat after pentazocine treatment.

1. Female non-pregnant rats were intramuscularly injected with pentazocine for 3 months. Liver showed a statistically significant (P less than 0.05) increase in its ganglioside content after the pentazocine treatment; in addition, no changes were found in the kidney ganglioside content. 2. We have also found changes in the ganglioside pattern of these rats after the pentazocine injection. The GM1 and GD1b liver content was decreased (P less than 0.05) in parallel with an increase (P less than 0.05) in GD3 and GT1b content; kidney showed a decrease (P less than 0.05) in GM1, GD1a and GD1b content and an increase (P less than 0.05) in GM4, GD2, GT1b and GQ content. 3. Female pregnant rats were also injected with pentazocine from the first to the nineteenth day of the gestation period. The total ganglioside content of liver and kidneys from mothers and their newborns did not show statistically significant differences after the treatment. 4. Mothers showed a decrease (P less than 0.05) in the GM1 content of liver and an increase (P less than 0.05) in the GT1b content of liver and GM1, GD3 and GD1a content of kidney. Only the GM3 content from kidney was increased (P less than 0.001). 5. Newborns showed minor changes in their ganglioside pattern. GT1b content from liver and GD2 and GQ content from kidneys were decreased (P less than 0.05).

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.