Chemical characterization of oligosaccharides in the milk of six species of New and Old World monkeys

Abstract

Human and great ape milks contain a diverse array of milk oligosaccharides, but little is known about the milk oligosaccharides of other primates, and how they differ among taxa. Neutral and acidic oligosaccharides were isolated from the milk of three species of Old World or catarrhine monkeys (Cercopithecidae: rhesus macaque (Macaca mulatta), toque macaque (Macaca sinica) and Hamadryas baboon (Papio hamadryas)) and three of New World or platyrrhine monkeys (Cebidae: tufted capuchin (Cebus apella) and Bolivian squirrel monkey (Saimiri boliviensis); Atelidae: mantled howler (Alouatta palliata)). The milks of these species contained 6-8% total sugar, most of which was lactose: the estimated ratio of oligosaccharides to lactose in Old World monkeys (1:4 to 1:6) was greater than in New World monkeys (1:12 to 1:23). The chemical structures of the oligosaccharides were determined mainly by (1)H-NMR spectroscopy. Oligosaccharides containing the type II unit (Gal(β1-4)GlcNAc) were found in the milk of the rhesus macaque, toque macaque, Hamadryas baboon and tufted capuchin, but oligosaccharides containing the type I unit (Gal(β1-3)GlcNAc), which have been found in human and many great ape milks, were absent from the milk of all species studied. Oligosaccharides containing Lewis x (Gal(β1-4)[Fuc(α1-3)]GlcNAc) and 3-fucosyl lactose (3-FL, Gal(β1-4)[Fuc(α1-3)]Glc) were found in the milk of the three cercopithecid monkey species, while 2-fucosyl lactose (5'-FL, Fuc(α1-2)Gal(β1-4)Glc) was absent from all species studied. All of these milks contained acidic oligosaccharides that had N-acetylneuraminic acid as part of their structures, but did not contain oligosaccharides that had N-glycolylneuraminic acid, in contrast to the milk or colostrum of great apes which contain both types of acidic oligosaccharides. Two GalNAc-containing oligosaccharides, lactose 3'-O-sulfate and lacto-N-novopentaose I (Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc) were found only in the milk of rhesus macaque, hamadryas baboon and tufted capuchin, respectively. Further research is needed to determine the extent to which the milk oligosaccharide patterns observed among these taxa represent wider phylogenetic trends among primates and how much variation occurs among individuals or species.

Fig. 1

Gel chromatograms of the carbohydrate fraction from milk of a rhesus macaque, b toque macaque and c Hamadryas baboon. Elution from a BioGel P-2 column (2.6 × 100 cm) was done with distilled water at a flow rate of 15 mL/h, and of 5.0 mL fractions were collected. Each fraction was monitored by the phenol-H₂SO₄ method at 490 nm (as shown in solid line) and the periodate-resorcinol method at 630 nm (as shown in dot line)

Fig. 2

Gel chromatograms of the carbohydrate fraction from milk of a tufted capuchin, b mantled howler and c Bolivian squirrel monkey. The gel chromatographies were done as in Fig. 1

Fig. 3

Anion exchange chromatograms of a R-1, b T-1, c B-1 and d M-1 separated from rhesus macaque, toque macaque, Hamadryas baboon and mantled howler milk, respectively, by gel chromatography on BioGel P-2. A DEAE-Sephadex A-50 column (1.5 × 20 cm) equilibrated with 50 mM tris-HCl buffer solution (pH 8.7) was used. Elution was done first with 250 mL of the same solution, and then with a linear gradient of the same containing NaCl from 0 to 0.5 M. The flow rate was 15 mL/h and 5 mL fractions were collected. Each fraction was monitored by the phenol-H₂SO₄ method

Fig. 4

HPLC of a R-1-2, b T-1-2 and c B-1-1 separated from rhesus macaque, toque macaque and Hamadryas baboon milk, respectively. HPLC was done using a Shimadzu LC-10 AT VP pump on a TSK-gel Amido-80 column (4.6 × 250 mm, pore size 80 Å, particle size 5 μm). The mobile phase was 80% and 50% acetonitrile in 15 mM potassium phosphate buffer solution, denoted buffer A and buffer B. Elution was done using a linear gradient of 0% to 50% of B buffer for 15 min, followed by 50% to 100% of B buffer for 65 min at 60°C at a flow rate of 1 mL/min. Detection of peaks was done by UV absorption at 195 nm

Fig. 5

HPLC of a C-1, b M-1-2 and c S-1 separated from tufted capuchin, mantled howler and Bolivian squirrel monkey milk, respectively. The chromatographies were performed as described in Fig. 4