ABSTRACT. Milk gangliosides inhibit Vibrio cholerae enterotoxin and Escherichia coli heat-labile enterotoxin. Human milk gangliosides showed considerably higher enterotoxin-inhibitory activity compared to bovine and formula milk gangliosides as measured in vitro by enzymelinked immunosorbent assay and in vivo in rabbit small bowel loops. While gangliosides from less than 1 ml human milk inhibited 0.1 pg choleratoxin in vitro and in vivo, five to 10 times higher amounts of bovine milk gangliosides were necessary to achieve similar results. Analysis of the ganglioside composition in human, bovine, and bovine milkbased formula milk showed that the ganglioside patterns in human and bovine milk differed markedly. The ganglioside patterns of bovine milk and formula milk appeared identical. In human or bovine milk, the total amount of gangliosides was 11 mgjliter compared to 6 mglliter in formula milk. The predominating ganglioside in human milk, monosialoganglioside 3 (74% of total gangliosides), was only a minor component (3%) of bovine milk gangliosides. Disialoganglioside 3 represented 80% of bovine milk gangliosides compared to 25% of the human milk gangliosides. Trace amounts of monosialoganglioside 1 were detected in human, as well as in bovine, milk by a sensitive high performance thin-layer chromatography immunoassay. The monosialoganglioside 1 content in human milk was 10 times higher than in bovine milk. We conclude that the higher nonimmunoglobulin enterotoxin-inhibitory activity in human milk compared to bovine milk is associated with the differences in the ganglioside fraction. (Pediatr Res 20: 416-421,1986)

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1. The amino acid composition of purified Bacillus cereus phospholipase C is reported. The enzyme contains one methionine residue and two fragments are obtained after cyanogen bromide cleavage. The sequence of the amino-terminal fragment (25 residues) is reported.2. Antisera were raised against the enzyme and purified by affinity chromatography. The antisera were monospecific and gave one precipitation line with purified as well as with crude phospholipase C, showing that no antigenic contaminants were present in the purified preparations used as antigen. The antibodies were purified to the extent that about two molecules neutralized one enzyme molecule.3. The enzyme is quite resistant to denaturation by urea, sodium dodecyl sulphate or heat (in the presence of 1 mM Zn").4. Phospholipase C hydrolyses phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine. Under the conditions used phosphatidylglycerol, cardiolipin, phosphatidylinositol, sphingomyelin, lysophosphatidylcholine and lysophosphatidyl ethanolamine were not substrates. Replacement of Zn2+ by Co2+ or Ni2+ or variation of pH (7.2-8.3), did not change the range of substrates. Phosphatidylcholine was the best substrate among the isolated phospholipids and dicaproylphosphatidylcholine was clearly a better substrate than dipalmitoylphosphatidylcholine.

Inhibitory activity of enterotoxin from Escherichia coli and Vibrio cholerae was associated with the ganglioside fraction of human milk. Both the milk fat and skim milk contained gangliosides that inhibited the toxins. The most purified milk fraction contained three glycolipid components, of which two migrated close to ganglioside GM1 on thin-layer chromatography plates. A component with a slightly different mobility from GM1 appeared to be associated with the inhibitory activity. Milk ganglioside fraction, derived from 2 ml of human milk, contained 1 to 4 micrograms of lipid-bound sialic acid and completely inhibited 0.1 micrograms of cholera toxin in rabbit intestinal loop experiments. It is suggested that human milk gangliosides, although present only in trace amounts, may be important in protecting infants against enterotoxin-induced diarrhea.