Chemical characterisation of the oligosaccharides in hooded seal (Cystophora cristata) and Australian fur seal (Arctocephalus pusillus doriferus) milk

Comparative Biochemistry and Physiology Part B 128 Ž2001. 307᎐323

Chemical characterisation of the oligosaccharides in hooded seal ž Cystophora cristata/ and Australian fur seal ž Arctocephalus pusillus doriferus/ milk Tadasu Urashimaa,U , Megumi Aritaa , Maho Yoshidaa , Tadashi Nakamuraa , Ikichi Arai a , Tadao Saito b, John P.Y. Arnould c , Kit M. Kovacs d, Christian Lydersen d a

Department of Bioresource Science, Obihiro Uni¨ ersity of Agriculture and Veterinary Medicine, Inada cho, Obihiro, Hokkaido 080-8555, Japan b Department of Bio Production, Graduate School of Agriculture, Tohoku Uni¨ ersity, Tsutsumidori-Amamiya machi 1-1, Aoba-Ku, Sendai 981-8555, Japan c Marine Mammal Research Group, Graduate School of the En¨ ironment, Macquarie Uni¨ ersity, NSW 2109, Australia d Norwegian Polar Institute, N-9296 TroⲐ mso, Norway Received 1 June 2000; received in revised form 18 October 2000; accepted 23 October 2000

Abstract Carbohydrates were extracted from hooded seal milk, Crystophora cristata Žfamily Phocidae.. Free oligosaccharides were separated by gel filtration and then purified by ion exchange chromatography, gel filtration and preparative thin layer or paper chromatography and their structures determined by 1 H-NMR. The hooded seal milk was found to contain inositol and at least nine oligosaccharides, most of which had lacto-N-neotetraose or lacto-N-neohexaose as core units, similar to those in milk of other species of Carnivora such as bears ŽUrsidae.. Their structures were as follows: GalŽ␤1-4.Glc Žlactose.; FucŽ ␣1-2.GalŽ␤1-4.Glc Ž2⬘-fucosyllactose.; GalŽ␤1-4.GlcNAcŽ␤1-3.GalŽ␤1-4.Glc Žlacto-Nneotetraose.; FucŽ ␣1-2.GalŽ␤1-4.GlcNAcŽ␤1-3.GalŽ␤1-4.Glc Žlacto-N-fucopentaose IV.; GalŽ␤1-4.GlcNAcŽ␤13.wGalŽ␤1-4.GlcNAcŽ␤1-6.xGalŽ1-4.Glc Žlacto-N-neohexaose.; FucŽ ␣1-2.GalŽ␤1-4.GlcNAcŽ␤1-3.wGalŽ␤1-4.GlcNAcŽ␤16.xGalŽ␤1-4.Glc Žmonofucosyl lacto-N-neohexaose a.; GalŽ␤1-4.GlcNAcŽ␤1-3.wFucŽ ␣1-2.GalŽ␤1-4.GlcNAcŽ␤16 .xGalŽ␤1-4 .Glc Žmonofucosyl lacto-N-neohexaose b .; Fuc Ž ␣ 1-2 .GalŽ␤1-4 .GlcNAc Ž␤1-3 .wFuc Ž ␣ 1-2 .GalŽ␤14.GlcNAcŽ␤1-6.xGalŽ␤1-4.Glc Ždifucosyl lacto-N-neohexaose.; GalŽ␤1-4.GlcNAcŽ␤1-3.GalŽ␤1-4.GlcNAcŽ␤1-3.GalŽ␤14.Glc Ž para lacto-N-neohexaose.; FucŽ ␣1-2.GalŽ␤1-4.GlcNAcŽ␤1-3.GalŽ␤1-4.GlcNAcŽ␤1-3.GalŽ␤1-4.Glc Žmonofucosyl para lacto-N-neohexaose.. Milk of the Australian fur seal, Arctophalus pusillus doriferus Žfamily Otariidae. contained inositol but no lactose or free oligosaccharides. These results, therefore, support the hypothesis that the milk of otariids, unlike that of phocids, contains no free reducing saccharides. 䊚 2001 Elsevier Science Inc. All rights reserved. Keywords: Hooded seal; Milk; Oligosaccharides; Chemical structure; Cystophora cristata; Phocidae; Australian fur seal; Otariidae

U

Corresponding author. Tel.: q81-155-49-5566; fax: q81-155-49-5577. E-mail address: [email protected] ŽT. Urashima.. 1096-4959r01r$ - see front matter 䊚 2001 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 6 - 4 9 5 9 Ž 0 0 . 0 0 3 2 7 - 4

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T. Urashima et al. r Comparati¨ e Biochemistry and Physiology Part B 128 (2001) 307᎐323

1. Introduction

2. Materials and methods

The milk of pinnipeds has attracted considerable attention because of its high lipid content, particularly among the phocid seals. The relative composition of the milk of a broad range of pinniped species has been documented ŽOftedal et al., 1988; Iverson et al., 1993; Kretzmann et al., 1993. and the pattern of changes in the composition throughout lactation has been studied and integrated into various lactation energetic analyses ŽLydersen and Kovacs, 1999.. In addition, the fatty acid composition of the milk of various seals was recently studied to explore changes in the nature of the maternal diet ŽIverson et al., 1995.. Hooded seals have the shortest lactation period among mammals ŽBowen et al., 1985; Lydersen et al., 1997.; concomitantly their milk has the highest fat concentration measured to date on any species while the daily output of their milk and growth rate of their pups is exceptional ŽLydersen and Kovacs, 1999.. In contrast to milk fat, the carbohydrate of the milk of pinnipeds has focused little attention, chiefly because studies on milk of the California sea lion, and other species of the family Otariidae, have shown that lactose, the major milk sugar in most eutherians, is either entirely absent or present in only very low concentrations ŽPilson and Kelly, 1962; Kerry and Messer, 1968.. Subsequent studies on the crabeater seal, Lobodon carcinophagus, showed, however, that milk of this species of the family Phocidae contains several oligosaccharides of unknown structure, albeit at low concentration, in addition to a trace of free lactose ŽMesser et al., 1988.. Urashima et al. Ž1997b. subsequently identified 2⬘-fucosyllactose as one of the oligosaccharides in the milk of this species. These results suggested that the milk of phocids contains free oligosaccharides whereas that of otariids does not ŽMesser et al., 1988.. In this study, we isolated a number of free oligosaccharides from milk of the hooded seal, Cystophora cristata, and compared their structures with those of milk oligosaccharides of other species of Carnivora, including bears ŽUrashima et al., 1997a, 1999a. and the coati ŽUrashima et al., 1999b.. In addition we established that milk of the Australian fur seal, Arctocephalus pusillus doriferus ŽOtariidae. contains neither lactose nor any other free reducing saccharides.

2.1. Materials The hooded seal milk was collected from animals on the drifting pack-ice in the southern part of the Gulf of St. Lawrence, Canada in March 1995. Adult females with pups were captured using an A-frame sling net that provided the ability to restrain the animals without the use of drugs. The sample was taken approximately 10 min after an intra muscular injection of 20 IU oxytocin. Milk was collected via gentle manual suction into a 10-ml collection tube, repeatedly as necessary. The milk was then stored at y20⬚C until analysis. The Australian fur seal milk was collected from animals in a breeding colony in Bass Strait, southeastern Australia, 1997᎐1998. Randomly selected adult females nursing pups were captured during lactation using a hoop-net and a small milk sample Ž5᎐50 ml. was collected by manual expression following an intra-muscular injection Ž0.5᎐1.0 ml, 10 UIr7 ml. of oxytocin. The samples were stored in plastic vials at y20⬚C until analysis. 2.2. Chemicals Lactose monohydrate was purchased from Kanto Co., Tokyo, Japan. Lacto-N-neotetraose wGalŽ␤1-4.GlcNAcŽ␤1-3.GalŽ␤1-4.Glcx and lactoN-neohexaose wGalŽ␤1-4.GlcNAcŽ␤1-3.wGalŽ␤14.GlcNAcŽ␤1-6.xGalŽ␤1-4.Glcx were obtained from Seikagaku Co., Tokyo, Japan. FucŽ ␣1-2. G al Ž ␤1-4 . G lc Ž 2⬘-fucosyllactose . and Nacetylneuraminic acid were purchased from Sigma Co., St. Louis, MO. Fuc Ž ␣ 1-2 . Gal Ž ␤1-4 . GlcNAcŽ␤1-3.GalŽ␤1-4.Glc was isolated from coati milk ŽUrashima et al., 1999b.. 2.3. Colorimetric assays The carbohydrate content of hooded seal milk was assayed as total hexose using the phenolH 2 SO4 method ŽMesser and Green, 1979.. Lactose was used as the standard. The sialic acid content was determined by the periodateresorcinol method ŽJourdian et al., 1971. using N-acetylneuraminic acid as the standard.

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309

2.4. Preparation of oligosaccharides from hooded seal milk The milk Ž20 ml. was thawed, diluted with four volumes of distilled water and extracted with 400 ml of chloroformrmethanol 2:1 Žvrv.. The emulsion was centrifuged at 4⬚C and 4000 = g for 30 min. The lower chloroform layer and the denatured protein were discarded. The methanol was removed from the upper layer by rotary evaporation, and the residue was freeze-dried. The resulting white powder was called the ‘carbohydrate fraction’. The carbohydrate fraction was dissolved in 2-ml of water and the solution was passed through a Bio Gel P-2 Ž- 45 ␮m. column Ž2.5= 100 cm., which had been calibrated with 2 mg of each of galactose Žmonosaccharide., lactose Ždisaccharide. and raffinose Žtrisaccharide ., at 30⬚C using a water jacket. Elution was done with water at a flow rate of 15 mlrh and 5-ml fractions were collected. Aliquots Ž0.5 ml. of each fraction were analysed for hexose and for sialic acid. Peak fractions were pooled and freeze-dried. The above procedures were performed twice with 40 ml of milk. The pooled fraction referred to as HSM-2 ŽFig. 1a., was dissolved in 2 ml of 50 mM Tris-hydroxyaminomethane-HCl buffer ŽpH 8.7. and passed through a DEAE-Sephadex A-50 column Ž1.5= 35 cm. equilibrated with the same buffer to remove peptides. Elution was done with the same buffer at a flow rate of 15 mlrh and fractions of 5 ml were collected. Aliquots Ž0.5 ml. of each fraction were analysed for hexose. Peak fractions were pooled and freeze-dried. The product was dissolved in 2 ml of water and passed through a Bio Gel P-4 Ž- 45 ␮m. column Ž2.5= 100 cm. at 30⬚C. Elution was performed with distilled water at a flow rate of 15 mlrh and fractions of 5 ml were collected. Aliquots Ž0.5 ml. of each fraction were analysed for hexose ŽMesser and Green, 1979.. Peak fractions were pooled and freezedried. Two peak fractions, named HSM-2-1 and HSM-2-2 Žsee Fig. 1b, elution volumes; 240 and 255 ml, respectively. were subjected to preparative thin-layer chromatography ŽTLC. with acetoner2-propanolr0.1 M lactic acid Ž2r2r1, vrv. as developing solvent, and the component in HSM-2-1 with R Lac s 0.85 and in HSM-2-2 with R Lac s 0.94 were purified by passage through a

Fig. 1. Gel chromatograms of the saccharides of hooded seal milk on Bio Gel P Ž2.5= 100 cm.. Elution was done with water at a flow rate of 15 mlrh and fractions of 5 ml were collected. An aliquot Ž0.5 ml. of each fraction was analysed for hexose with phenol-H 2 SO4 at 490 nm Ž䉫. and for sialic acid with periodate-resorcinol at 630 nm ŽB.. Ža. Gel chromatogram of the carbohydrate fraction from hooded seal milk on Bio Gel P-2. Žb. Gel chromatogram of fraction HSM-2 from the total carbohydrate fraction of hooded seal milk on Bio Gel P-4. Žc. Gel chromatogram of fraction HSM-1 from the total carbohydrate fraction of hooded seal milk on Bio Gel P-4.

Bio Gel P-2 column under the same conditions as described above.

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T. Urashima et al. r Comparati¨ e Biochemistry and Physiology Part B 128 (2001) 307᎐323

Fractions HSM-3 and HSM-4 ŽFig. 1a. were dissolved in 2 ml of water and passed though Sep-pak cartridge columns ŽWaters, MA, USA. to remove peptides, followed by freeze-drying. They were then dissolved in 1 ml of water and subjected to preparative paper chromatography using butanolrpyridinerwater Ž6:4:3, vrv. as a developing solvent with eight developments. Saccharides were detected with alkaline AgNO3 reagent ŽTrevelyan et al., 1950.. The components with R Lac s 0.97 from HSM-3 and R Lac s 1.0 and 0.43 from HSM-4 were isolated and then purified by passage through the Bio Gel P-2 column, as above. Fraction, HSM-1 was dissolved in 100 ml of 50 mM Tris-hydroxyaminomethane HCl buffer ŽpH 8.7.. DEAE-Sephadex A-50 resin Ž100 ml. was added, the solution was mixed slowly for 2 h, and then filtered to remove the resin. The filtrate was freeze-dried, dissolved in 5 ml of water and passed through a Bio Gel P-4 column as above. Peak fractions detected by the phenol-H 2 SO4 method were pooled and freeze-dried. The two fractions, HSM-1-1 and HSM-1-2, ŽFig. 1c, elution volumes 205 and 230 ml, respectively. were subjected to preparative TLC as above, and the components in HSM-1-1 with R Lac s 0.24 and 0.19 and in HSM1-2 with R Lac s 0.58, 0.50, 0.42 0.36 and 0.29 were purified by passage through Bio Gel P-2 as above. 2.5. Attempted separation of oligosaccharides from Australian fur seal milk The milk Ž100 ml. was thawed, diluted with four volumes of water and extracted with 2000 ml of chloroformrmethanol 2:1 Žvrv.. The ‘carbohydrate fraction’ was separated as described above for the hooded seal milk. It was then resolved into several fractions on Bio Gel P-2. These were passed through Sep-pak cartridge columns to remove peptides.

2.7. 1H-NMR 1

H-NMR spectra were recorded in D 2 O Ž100.00 atom %D, Aldrich, Milwakee, WI. at 600 MHz with a Varian INOVA 600 spectrometer operated at 293.1 K. Chemical shifts are expressed in ppm down-field from internal 3-Žtrimethylsilyl .-1-propane sulfonic acid, sodium salt ŽTPS., but were actually measured by reference to an internal acetone Ž ␦ s 2.225..

3. Results The hooded seal milk sample contained 2.0% hexose carbohydrate and 0.60% sialic acid. The carbohydrate fraction separated into at least five peaks on Bio Gel P-2 ŽFig. 1a.. Oligosaccharides were present in fractions HSM-1 to HSM-4. 3.1. HSM-4 The 1 H-NMR spectrum of HSM-4 showed that it contained saccharides along with other components. The saccharides were purified using paper chromatography Žsee Section 2. and two components ŽR Lac s 1.0 and 0.43. were isolated. The 1 H-NMR spectrum of the component with R Lac s 0.43 did not have any ␣- and ␤-anomeric signals of a reducing residue and was not characterised further in this study. The saccharide with R Lac s 1.0 was re-designated as HSM-4 and characterised by 1 H-NMR. The 1 H-NMR Žchemical shifts in Table 1. of HSM-4 had the anomeric resonances of reducing ␣-Glc and ␤-Glc, and ␤Ž1-4. linked Gal at ␦ 5.224, 4.668 and 4.453, respectively. This pattern was essentially similar to that of lactose; HSM-4 was therefore characterised to be GalŽ␤1-4.Glc. 3.2. HSM-3

2.6. Isolation of inositols The inositols in the hooded seal milk were separated from fraction HSM-5 ŽFig. 1a., using Bio Gel P-2 chromatography. The inositols in the Australian fur seal milk were separated from the fraction, which eluted at 360 ml on Bio Gel P-2. The resulting fractions were then subjected to preparative paper chromatography as described above and the components with R Lac s 0.79 were isolated.

The 1 H-NMR of fraction HSM-3 showed that it contained a free oligosaccharide along with other components. The oligosaccharide was purified by paper chromatography Žsee Section 2.. The 1 H-NMR spectrum Žchemical shifts in Table 1. of the purified oligosaccharide had the anomeric signals of reducing ␣-Glc and ␤-Glc, ␣ Ž1᎐2. linked Fuc and ␤Ž1᎐4. linked Gal at ␦ 5.228, 4.640, 5.311 and 4.528, respectively. The NMR had the characteristic shifts of H-5 and H-6

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Table 1 H-NMR chemical shifts of oligosaccharides in HSM-2 to HSM-4 separated from hooded seal milk

1

Reporter group

Residue

Chemical shifts, ␦ Žcoupling constants, Hz. HSM-2-1a

HSM-2-2b

HSM-3c

HSM-4d

5.220 Ž3.8. 4.663 Ž8.0. 4.439 Ž8.0. 4.480 Ž7.7. ᎐ 4.706 Ž8.2. 4.703 Ž8.3. 4.158 Ž3.3. ᎐ ᎐ 2.033

5.228 Ž3.6. 4.640 Ž8.0. 4.528 Ž7.7. ᎐ 5.311 Ž2.5. ᎐ ᎐ ᎐ 4.257 4.228 1.224 Ž6.9. ᎐

5.224 Ž3.8. 4.668 Ž8.0. 4.453 Ž7.7. ᎐ ᎐ ᎐ ᎐ ᎐ ᎐ ᎐ ᎐ ᎐

H-1

Glc ␣ Glc␤ Gal⬘Ž␤1-4. Gal⬘⬘⬘Ž␤1-4. Fuc⬘⬘⬘⬘Ž ␣1-2. GlcNAc⬘⬘Ž␤1-3.

H-4 H-5

Gal⬘Ž␤1-4. Fuc⬘⬘⬘⬘Ž ␣1-2.

5.219 Ž3.8. 4.662 Ž8.0. 4.441 Ž8.0. 4.549 Ž7.7. 5.308 Ž3.0. 4.701 Ž8.2. 4.697 Ž8.5. 4.147 Ž3.3. 4.219

H-6 NAc

Fuc⬘⬘⬘⬘Ž ␣1-2. GlcNAc⬘⬘Ž␤1-3.

1.227 Ž6.6. 2.038

a

HSM-2-1: FucŽ ␣1-2.GalŽ␤1-4.GlcNAcŽ␤1-3.GalŽ␤1-4.Glc. HSM-2-2: GalŽ␤1-4.GlcNAcŽ␤1-3.GalŽ␤1-4.Glc. c HSM-3: FucŽ ␣1-2.GalŽ␤1-4.Glc. d HSM-4: GalŽ␤1-4.Glc. b

of ␣ Ž1᎐2. linked Fuc at ␦ 4.257 Ž ␣ ., 4.228 Ž␤ . and 1.224, respectively. The pattern was essentially similar to that of 2⬘-fucosyllactose ŽUrashima et al., 1994, 1997b. and this oligosaccharide was characterised to be FucŽ ␣1-2.GalŽ␤1᎐4.Glc. 3.3. HSM-2 Fraction HSM-2 resolved into two peaks, designated HSM-2-1 and HSM-2-2, during chromatography on Bio Gel P-4 ŽFig. 1b.. Both fractions were still found to be mixtures of free oligosaccharides and other components. Hence, their oligosaccharides were further purified Žsee Section 2. and then investigated by 1 H-NMR ŽFig. 2, Table 1.. 3.3.1. HSM-2-2 The characteristic NAc shift at ␦ 2.033 in the 1 H-NMR showed that this oligosaccharide contained N-acetylhexosamine. The spectrum had the characteristic anomeric signals of ␣-Glc, ␤Glc, ␤Ž1-3. linked GlcNAc and two of ␤Ž1-4. linked Gal at ␦ 5.220, 4.663, 4.706 Ž ␣ . and 4.703 Ž␤ ., 4.480 and 4.439. The shift at ␦ 4.158 was assigned to H-4 of a ␤Ž1-4. linked Gal which was substituted at OH-3. Since its 1 H-NMR pattern was essentially similar to that of lacto-Nneotetraose ŽUrashima et al., 1997b., HSM-2-2 was characterised to be GalŽ␤1-4.GlcNAcŽ␤13.GalŽ␤1-4.Glc.

3.3.2. HSM-2-1 This oligosaccharide was shown to contain ␣ Ž12. linked Fuc because of chemical shifts at ␦ 5.308, 4.219 and 1.227, which were assigned to H-1, H-5 and H-6 of a fucose residue. The anomeric shift at ␦ 4.549 of ␤Ž1-4. linked Gal indicated that the residue was substituted, because it had shifted down-field compared with the H-1 of the unsubstituted ␤Ž1-4. linked Gal at ␦ 4.480 in lacto-N-neotetraose. The ␤Ž1-4. linked Gal residue was assumed to be substituted by a non-reducing ␣ Ž1-2. linked Fuc. The other anomeric shifts at ␦ 5.219, 4.662, 4.701 Ž ␣ . and 4.697 Ž␤ . and 4.441, which arose from H-1 of ␣-Glc, ␤-Glc, ␤Ž1-3. linked GlcNAc and ␤Ž1-4. linked Gal, respectively, were similar to those of lacto-N-neotetraose. Therefore, a lacto-Nneotetraose unit was present. From the above assignments, and the fact that the characteristic resonances were essentially similar to those of ␣ Ž 1-2 . linked fucosyllacto-N -neotetraose ŽUrashima et al., 1999b., HSM-2-1 was characterised to be FucŽ ␣1-2.GalŽ␤1-4.GlcNAcŽ␤13.GalŽ␤1-4.Glc Žlacto-N-fucopentaose IV.. 3.4. HSM-1 During Bio Gel P-2 chromatography of the ‘carbohydrate fraction’ of hooded seal milk ŽFig. 1a., most of the components detected by the phenol-H 2 SO4 method eluted in the first Žvoid

312

T. Urashima et al. r Comparati¨ e Biochemistry and Physiology Part B 128 (2001) 307᎐323

Fig. 2. 600 MHz 1 H-NMR spectra of the oligosaccharides in Ž1. HSM-2-2 and Ž2. HSM-2-1. The spectrum obtained at 293.1 K and recorded in D 2 O Ž100.00% D.. Chemical shifts Žppm. are expressed down-field from internal TPS, but were actually measured by reference to acetone Ž ␦ s 2.225..

T. Urashima et al. r Comparati¨ e Biochemistry and Physiology Part B 128 (2001) 307᎐323

313

Table 2 H-NMR chemical shifts of the oligosaccharides in HSM-1-2-1and HSM-1-2-2 separated from hooded seal milk

1

Reporter group

Chemical shifts ␦ Žcoupling constants, Hz .

Residue

HSM-1-2-1a

HSM-1-2-2b

H-1

Glc ␣ Glc␤ Gal⬘Ž␤1-4. Gal⬘⬘⬘Ž␤1-4. Gal⬘⬘⬘⬘⬘Ž␤1-4. GlcNAc⬘⬘Ž␤1-3. GlcNAc⬘⬘⬘⬘Ž␤1-3. Fuc⬘⬘⬘⬘⬘Ž ␣1-2.

5.219 Ž3.6. 4.663 Ž8.0. 4.436 Ž7.4. 4.466 Ž8.0. 4.479 Ž7.7. 4.698 Ž8.5. 4.698 Ž8.5. ᎐

5.219 Ž3.7. 4.662 Ž8.1. 4.436 Ž7.3. 4.467 Ž8.0. 4.550 Ž7.6. 4.695 Ž8.1. 4.695 Ž8.1. 5.307 Ž2.7.

H-4

Gal⬘Ž␤1-4. Gal⬘⬘⬘Ž␤1-4.

4.159 Ž2.7. 4.155 Ž2.7.

4.154 Ž2.7. 4.154 Ž2.7.

H-5 H-6 NAc

Fuc⬘⬘⬘⬘⬘Ž ␣1-2. Fuc⬘⬘⬘⬘Ž ␣1-2. GlcNAc⬘⬘Ž␤1-3. GlcNAc⬘⬘⬘⬘Ž␤1-3.

᎐ ᎐ 2.032 2.032

4.222 1.227 Ž6.6. 2.031 2.037

a b

HSM-1-2-1: GalŽ␤1-4.GlcNAcŽ␤1-3.GalŽ␤1-4.GlcNAcŽ␤1-3.GalŽ␤1-4.Glc. HSM-1-2-2: FucŽ ␣1-2.GalŽ␤1-4.GlcNAcŽ␤1-3.GalŽ␤1-4.GlcNAcŽ␤1-3.GalŽ␤1-4.Glc.

volume. peak, HSM-1; the components in this peak also reacted positively with the resorcinolperiodate method. As the components in HSM-1 were assumed to be primarily a mixture of glycoproteinsrglycopeptides and small amounts of free oligosaccharides, the components in the fraction were further separated by passage through DEAE-Sephadex A-50 and Bio Gel P-4 Žsee Sec-

tion 2.; this yielded two fractions, HSM-1-1 and HSM-1-2 ŽFig. 1c.. 3.4.1. HSM-1-2 During TLC, five spots were observed in this fraction; these corresponded to five oligosaccharides designated HSM-1-2-1, HSM-1-2-2, HSM-12-3, HSM-1-2-4, and HSM-1-2-5. Each of these

Table 3 1 H-NMR chemical shifts of the oligosaccharides in HSM-1-2-3; HSM-1-2-5 separated from hooded seal milk a Reporter group

Residue

Chemical shifts Žcoupling constants, Hz . HSM-1-2-3

HSM-1-2-4-1

HSM-1-2-4-2

HSM-1-2-5

5.218 Ž3.9. 4.665 Ž8.1. 4.430 Ž7.3. 4.471 Ž7.8. 4.552 Ž7.8. 4.696 Ž8.3. 4.640 Ž8.3. 4.635 Ž8.3. 5.311 Ž2.4.

5.218 Ž3.9. 4.665 Ž8.1. 4.430 Ž7.3. 4.481 Ž7.5. 4.542 Ž7.8. 4.696 Ž8.3. 4.595 Ž7.1.

5.217 Ž3.9. 4.664 Ž8.1. 4.431 Ž7.3. 4.551 Ž7.6. 4.541 Ž7.6. 4.695 Ž8.3. 4.594 Ž7.8.

Fuc⬘⬘⬘⬘Ž ␣1-2.

5.218 Ž3.7. 4.666 Ž7.8. 4.427 Ž7.8. 4.481 Ž7.8. 4.471 Ž7.8. 4.697 Ž8.3. 4.644 Ž8.2. 4.637 Ž8.2. ᎐

5.311 Ž2.4.

5.309 Ž2.7.

H-4

Gal⬘Ž␤1-4.

4.148 Ž3.1.

4.140 Ž3.4.

4.140 Ž3.4.

4.140 Ž3.6.

H-5 H-6 NAc

Fuc⬘⬘⬘⬘Ž ␣1-2. Fuc⬘⬘⬘⬘Ž ␣1-2. GlcNAc⬘⬘Ž␤1-3. GlcNAc⬘⬘Ž␤1-6.

᎐ ᎐ 2.031 2.060

4.224 1.229 Ž6.6. 2.031 2.059

4.224 1.229 Ž6.6. 2.036 2.059

4.224 1.229 Ž6.6. 2.036 2.063

H-1

Glc ␣ Glc␤ Gal⬘Ž␤1-4. Gal⬘⬘⬘Ž␤1-4. GlcNAc⬘⬘Ž␤1-3. GlcNAc⬘⬘Ž␤1-6.

HSM-1-2-3: GalŽ␤1-4. GlcNAcŽ␤1-3. wGalŽ␤1-4. GlcNAc Ž␤1-6.x GalŽ␤1-4. Glc, HSM-1-2-4-1: Fuc Ž ␣1-2. GalŽ␤1-4. GlcNAcŽ␤1-3. wGalŽ␤1-4. GlcNAcŽ␤1-6.x GalŽ␤-4.Glc, HSM-1-2-4-2: GalŽ␤1-4. GlcNAcŽ␤1-3. wFucŽ ␣-2. GalŽ␤1-4. GlcNAcŽ␤1-6.x GalŽ␤-4.Gsc, HSM-1-2-5: FucŽ ␣1-2. GalŽ␤1-4.GlcNAcŽ␤1-6.x wFuc Ž ␣-2. GalŽ␤1-4. GlcNAcŽ␤1-6.x GalŽ␤1-4.Glc.

314

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Fig. 3. 600 MHz 1 H-NMR spectra of the oligosaccharides in Ž1. HSM-1-2-1 and Ž2. HSM-1-2-2.

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315

oligosaccharides was further purified Žsee Section 2. and characterised by 1 H-NMR ŽFigs. 3 and 4, Tables 2 and 3.. 3.4.2. HSM-1-2-1 (R L ac s 0.58) The chemical structure of HSM-1-2-1 was characterised by comparison of its 1 H-NMR spectrum with the spectra of HSM-2-2 and authentic lactoN-neotetraose. The spectrum had the anomeric shifts of ␣-Glc, ␤-Glc, ␤Ž1-3. linked GlcNAc and two shifts due to ␤Ž1-4. linked Gal at ␦ 5.219, 4.663, 4.698, 4.479 and 4.436, respectively, similar to those of lacto-N-neotetraose. However, the spectrum had an additional H-1 of ␤Ž1-4. linked Gal at ␦ 4.466. In addition, the intensity of ␤Ž1-3. linked GlcNAc at ␦ 4.698 showed that the signal arose from two protons of that anomeric shift. This indicated that the saccharide had an additional ␤Ž1-3. linked GlcNAc. The spectrum had two resonances of H-4 of ␤Ž1-4. linked Gal, which were substituted by GlcNAc at OH-3, at ␦ 4.155 and 4.159. These observations indicated that the saccharide had an un-branched unit of GalŽ␤14 . G lcNAc Ž ␤1-3 . G al Ž ␤1-4 . G lcNAc Ž ␤1-3 . G al. From the assignments, the oligosaccharide of HSM-1-2-1 was characterised to be GalŽ␤1-4. GlcNAcŽ␤1-3.GalŽ␤1-4.GlcNAcŽ␤1-3.GalŽ␤1-4. Glc Žpara lacto-N-neohexaose.. 3.4.3. HSM-1-2-2 (R L ac s 0.50) The chemical structure of HSM-1-2-2 was characterised by comparison of its 1 H-NMR spectrum with that of HSM-1-2-1. The spectrum had the anomeric shifts of ␣-Glc, ␤-Glc, ␤Ž1-3. linked GlcNAc and two shifts due to ␤Ž1-4. linked Gal at ␦ 5.219, 4.662, 4.695 and 4.467 and 4.436, respectively, similar to those of HSM-1-2-1. The anomeric shift of ␤Ž1-3. linked GlcNAc and H-4 shift of ␤Ž1-4. linked Gal, which were substituted at OH-3, at ␦ 4.154 arose from these two protons as concluded by their intensities. These observations showed that the structure contained the sequence G al Ž ␤ 1-4 . G lcN A c Ž ␤ 1-3 . G al Ž ␤ 14.GlcNAcŽ␤1-3.GalŽ␤1-4.Glc. The spectrum had the additional resonances of H-1, H-5 and H-6 of ␣ Ž1-2. linked Fuc at ␦ 5.307, 4.222 and 1.227. The H-1 shift of ␤Ž1-4. linked Gal at ␦ 4.550 was thought to have shifted down-field compared with the H-1 shift at ␦ 4.479 in HSM-1-2-1, showing that the residue in HSM-1-2-2 was substituted by

Fig. 4. 600 MHz 1 H-NMR spectra of the oligosaccharides in Ž1. HSM-1-2-3, Ž2. HSM-1-2-4 and Ž3. HSM-1-2-5.

a non-reducing residue. It was concluded that HSM-1-2-2 was FucŽ ␣1-2.GalŽ␤1-4.GlcNAcŽ␤13.GalŽ␤1-4.GlcNAcŽ␤1-3.GalŽ␤1-4.Glc Žmonofucosyl para lacto-N-neohexaose..

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3.4.4. HSM-1-2-3 (R L ac s 0.42) The chemical structure of HSM-1-2-3 was characterised by comparison of its 1 H-NMR spectrum with that of authentic lacto-N-neohexaose. The spectrum had the anomeric shifts of ␣-Glc, ␤-Glc, ␤Ž1-3. linked GlcNAc, ␤Ž1-6. linked GlcNAc and three shifts of ␤Ž1-4. linked Gal at ␦ 5.218, 4.666, 4.697, 4.644 and 4.637 and 4.481, 4.471 and 4.427, respectively. The spectrum had two NAc shifts of ␤Ž1-3. linked GlcNAc and ␤Ž1-6. linked GlcNAc at ␦ 2.031 and 2.060, respectively. It also had the characteristic H-4 shift of ␤Ž1-4. linked Gal, which was substituted at OH-3, at ␦ 4.148. The spectrum was essentially similar to that of authentic lacto-N-neohexaose; HSM-1-2-3 was therefore characterised to be GalŽ␤1᎐4 .GlcNAc Ž␤13.wGalŽ␤1᎐4.GlcNAcŽ␤1᎐6.xGalŽ␤1᎐4.Glc. 3.4.5. HSM-1-2-5 (R L ac s 0.29) The chemical structure of HSM-1-2-5 was characterised by comparison of its 1 H-NMR with the spectrum of HSM-1-2-3 and authentic lacto-Nneohexaose. The spectrum had the characteristic anomeric shifts of ␣-Glc, ␤-Glc, ␤Ž1-3. linked GlcNAc, ␤Ž1-6. linked GlcNAc and three shifts of ␤Ž1-4. linked Gal at ␦ 5.217, 4.664, 4.695, 4.594 and 4.551, 4.541 and 4.431, respectively, the H-4 shift of ␤Ž1-4. linked Gal, which was substituted at OH-3, at ␦ 4.140, and the NAc shifts of ␤Ž1-3. linked GlcNAc and ␤Ž1-6. linked GlcNAc at ␦ 2.036 and 2.063, respectively. These observations showed that the structure contained a lacto-Nneohexaose unit. However, the shifts of ␤Ž1-4. linked Gal at ␦ 4.551 and 4.541 were shifted down-field compared to those of lacto-Nneohexaose, indicating that the residues were substituted. The spectrum had the characteristic H-1, H-5 and H-6 shifts of ␣ Ž1-2. linked Fuc at ␦ 5.309, 4.224 and 1.229, respectively. Based upon their intensities, these resonances were concluded to correspond to two protons. From these observations, the two ␤Ž1-4. linked Gal residues of this saccharide were thought to be substituted by two non-reducing ␣ Ž1-2. linked Fuc. It was concluded that the saccharide was FucŽ ␣ 1-2.GalŽ␤ 14.GlcNAcŽ␤ 1-3.wFucŽ ␣ 1-2.GalŽ␤ 1-4.GlcNAcŽ␤ 1-6.xGalŽ␤ 1-4.Glc Ždifucosyl lacto-N-neohexaose.. The shift of the ␤Ž1-6. linked GlcNAc at ␦ 4.594 was slightly shifted up-field compared with that of lacto-N-neohexaose, as a result of substitution of

␤Ž1-4. linked Gal by ␣ Ž1-2. linked Fuc in the branched unit. 3.4.6. HSM-1-2-4 (R L ac s 0.36) The chemical structure of HSM-1-2-4 was characterised by comparison of its 1 H-NMR spectrum with the spectra of lacto-N-neohexaose and HSM1-2-5. The spectrum had the characteristic anomeric shifts of ␣ Ž1-2. linked Fuc, ␣-Glc, ␤-Glc, ␤Ž1-3. linked GlcNAc, and ␤Ž1-4. linked Gal at ␦ 5.311, 5.218 and 4.430, respectively. Although it had the anomeric shifts of ␤Ž1-6. linked GlcNAc, there appeared to be two sets of the signals at ␦ 4.640 Ž ␣ . plus 4.635 Ž␤ . and another at ␦ 4.595, showing that this fraction was a mixture of two oligosaccharides. In addition, the H-1 shifts of two kinds of ␤Ž1-4. linked Gal had two sets of the resonances at ␦ 4.552, 4.542, and ␦ 4.471, 4.481, as well. This, too, supported the conclusion that the fraction contained two oligosaccharides. The shifts at ␦ 4.552 and 4.542 of ␤Ž1-4. linked Gal were shifted down-field compared to that of lactoN-neohexaose, due to substitution of this residue by an ␣ Ž1-2. linked Fuc. From these observations, it was concluded that each oligosaccharide in this fraction had a lacto-N-neohexaose unit, in which a ␤Ž1-4. linked Gal residue was substituted by an ␣ Ž1-2. linked Fuc residue. Each of these oligosaccharides was thought to contain this one residue from the intensities of the H-1, H-5 and H-6 shifts of the ␣ Ž1-2. linked Fuc at ␦5.311, 4.224 ans 1.229, respectively. The ␣ Ž1-2. linked Fuc could be attached to two possible positions in the structure, namely either the non-reducing end of GalŽ␤1-4.GlcNAcŽ␤1-6. or that of a GalŽ␤14.GlcNAcŽ␤1-3. unit. The minor H-1 shift of ␤Ž16. linked GlcNAc at ␦ 4.595 most likely arose from the FucŽ ␣1-2.GalŽ␤1-4.GlcNAcŽ␤1-6. unit, as shown by the similar shift at ␦ 4.594 of HSM-12-5, whereas the major H-1 shift of this residue at ␦ 4.640 plus 4.635 arose from the non-substituted GalŽ␤1-4.GlcNAcŽ␤1-6. unit. In the major saccharide in this fraction, ␣ Ž1-2. linked Fuc was thought to attach to the non-reducing end of the GalŽ␤1-4.GlcNAcŽ␤1-3. unit. In conclusion, these two oligosaccharides were characterised to be FucŽ ␣1-2.GalŽ␤1-4.GlcNAcŽ␤1-3.wGalŽ␤1-4. GlcNAcŽ␤1-6.xGalŽ␤1-4.Glc ŽHSM-1-2-4-1. and GalŽ␤1-4.GlcNAcŽ␤1-3.wFucŽ ␣1-2.GalŽ␤1-4.GlcNAcŽ␤1-6.xGalŽ␤1-4.Glc ŽHSM-1-2-4-2., i.e. two isomers of monofucosyl lacto-N-neohexaose. The spectrum had two NAc shifts of ␤Ž1-3. linked

T. Urashima et al. r Comparati¨ e Biochemistry and Physiology Part B 128 (2001) 307᎐323

GlcNAc at ␦ 2.031 and 2.036, with the NAc shift of ␤Ž1-6. linked GlcNAc at ␦ 2.059.

317

3.4.7. HSM-1-1 During TLC, two spots were observed in the fraction; these corresponded to two oligosaccharides designated HSM-1-1-1 and HSM-1-1-2. Both oligosaccharides were further purified Žsee Section 2. and characterised by 1 H-NMR ŽFig. 5, Table 4..

sion that it contained the above unit. The presence of one residue of non-reducing ␣ Ž1-2. linked Fuc was concluded by the shifts of H-1, H-5 and H-6 at ␦ 5.308, 4.220 and 1.227 and their signal intensities. The shift at ␦ 4.549 corresponded to H-1 of ␤Ž1-4. linked Gal which was substituted by a non-reducing ␣ Ž1-2. linked Fuc. However, it was still unclear whether a GalŽ␤1-4.GlcNAc Ž␤1-3.GalŽ␤1-4.GlcNAc unit was attached to the galactose by a ␤Ž1-3. or a ␤Ž1-6. linkage.

3.4.8. HSM-1-1-1 (R L ac s 0.24) The chemical structure of HSM-1-1-1 was explored via comparison of its 1 H-NMR spectrum with those of HSM-1-2-2 and lacto-N- neohexaose. The spectrum had the characteristic anomeric shifts of ␣-Glc, ␤-Glc, ␤Ž1-3. linked GlcNAc, ␤Ž1-6. linked GlcNAc, ␤Ž1-4. linked Gal at ␦ 5.218, 4.664, 4.698 Ž ␣ . and 4.693 Ž␤ ., 4.639 and 4.426, respectively, showing that it contained a lacto᎐N-neohexaose unit. The shifts of H-1 of ␤Ž1-3. linked GlcNAc and H-4 of ␤Ž1-4. linked Gal, which was substituted at OH-3, arose from two protons as shown by their intensities. This indicated that the saccharide also contained a GalŽ␤1-4.GlcNAcŽ␤1-3.GalŽ␤1-4.GlcNAc unit. The H-1 shifts of ␤Ž1-4. linked Gal at ␦ 4.479 plus 4.472 arose from three protons as indicated by their signal intensity, supporting the conclu-

3.4.9. HSM-1-1-2 (R L ac s 0.19) The chemical structure of HSM-1-1-2 was explored via comparison of its 1 H-NMR spectrum compared with that of HSM-1-1-1. The structure had the characteristic anomeric shift of ␣-Glc, ␤-Glc, ␤Ž1-3. linked GlcNAc, ␤Ž1-6. linked GlcNAc and ␤Ž1-4. linked Gal at ␦ 5.220, 4.665, 4.694, 4.639 and 4.427, respectively. In addition, the spectrum had other shifts of ␤Ž1-4. linked Gal at ␦ 4.457᎐4.486; the signals were well resolved from each other. These observations showed that this saccharide contained a lacto-N-neohexaose unit. Similar to HSM-1-1-1, the shifts of H-1 of ␤Ž1-3. linked GlcNAc and H-4 shift of ␤Ž1-4. linked Gal, which was substituted at OH-3, arose from two protons as shown by their intensities. This indicated that it contained a GalŽ␤14.GlcNAcŽ␤1-3.GalŽ␤1-4.GlcNAc unit. The pres-

Table 4 ⬘H-NMR chemical shifts of the oligosaccharides in HSM-1-1 seperated from hooded seal milk Reporter group

H-1

Residue

Glc ␣ Glc ␤ Gal⬘Ž␤1-4. GalŽ␤1-4.

GlcNAcŽ␤1-3.

Chemical shifts ␦ Žcoupling constants, Hz . HSM᎐1-1-1

HSM-1-1-2

5.218 Ž4.1. 4.664 Ž8.0. 4.426 Ž7.7. 4.472 Ž7.7. 4.479 Ž7.7. 4.549 Ž7.4.

5.220 Ž3.6. 4.665 Ž7.3. 4.427 Ž7.1. 4.457᎐4.486 4.537 Ž8.2. 4.564 Ž8.2. 4.694 Ž8.3.

GlcNAc⬘⬘Ž␤1-6. FucŽ ␣1-2.

4.693 Ž7.7. 4.698 Ž7.7. 4.639 Ž8.3. 5.308 Ž2.3.

H-4 H-5 H-6

GalŽ␤1-4. FucŽ ␣1-2. FucŽ ␣1-2.

4.147 4.220 1.227 Ž6.9.

4.145 4.221 1.228 Ž6.6.

NAc

GlcNAcŽ␤1-3.

2.029 2.034 2.039 2.057

2.036

GlcNAc⬘⬘Ž␤1-6.

4.639 Ž7.9. 5.311

2.058

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T. Urashima et al. r Comparati¨ e Biochemistry and Physiology Part B 128 (2001) 307᎐323

Fig. 5. 600 MHz 1 H-NMR spectra of the oligosaccharides in Ž1. HSM-1-1-1 and Ž2. HSM-1-1-2.

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ence of two residues of non-reducing ␣ Ž1-2. linked Fuc was illustrated by the shifts of H-1, H-5 and H-6 at ␦ 5.311, 4.221 and 1.228, and their signal intensities. The shifts at ␦ 4.564 and 4.537 corresponded to H-1 of two ␤Ž1-4. linked Gal which were substituted by two non-reducing ␣ Ž1-2. linked Fuc residues. It is still unknown whether the GalŽ␤1-4.GlcNAc Ž␤1-3.GalŽ␤1-4.GlcNAc unit was attached to the galactose by a ␤Ž1-3. or a ␤Ž1-6. linkage. 3.5. Carbohydrate in Australian fur seal milk The carbohydrate fraction from the Australian fur seal milk was subjected to Bio Gel P-2 chromatography. The fraction resolved into several peaks. The 1 H-NMR of the peak fractions did not indicate the presence of any free oligosaccharides or lactose. 3.6. Inositols The inositols isolated from hooded seal and Australian fur seal milk by paper chromatography were identified by 1 H-NMR. The 1 H-NMR spectrum of the inositol from the hooded seal milk had the following characteristic resonances: ␦ 4.049 Žtriplet, coupling constant: 3.0, 2.7.; 3.622 Ždoublet, 9.9.; 3.606 Ždoublet, 9.6.; 3.532 Ždoublet, 2.7.; 3.516 Ždoublet, 3.0.; 3.337 Žsinglet.; and 3.266 Žtriplet, 9.6, 9.3.. The resonances at ␦ 4.049, 3.622, 3.606, 3.532, 3.516, 3.266 arose from myo-inositol, whereas the resonance at ␦ 3.337 arose from scyllo-inositol. Thus, the inositols were identified as myo- and scyllo-inositol. From the intensities of the above resonances the ratio of myo-inositol to scyllo-inositol was determined to be 1:0.85. The 1 H-NMR spectrum of the inositol from the Australian fur seal milk had characteristic resonances, as follows: ␦ 4.05 Žtriplet, coupling constant: 3.0, 2.7.; 3.621 Ždoublet, 9.6.; 3.605 Ždoublet, 9.6.; 3.533 Ždoublet, 3.0.; 3.517 Ždoublet, 2.7.; 3.337 Žsinglet.; and 3.267 Žtriplet, 9.3, 9.3.. From these resonances, the inositols were again identified to be myo- and scyllo-inositol. The ratio of myo-inositol to scyllo-inositol was determined to be 1:0.04.

4. Discussion The dominant sugar in milk is generally the

319

disaccharide lactose, but the milk of many species contains, in addition, a variety of oligosaccharides whose total concentration can exceed that of lactose ŽJenness et al., 1964.. Among eutherian mammals, examples include some of the Carnivora, such as the Ezo brown bear ŽUrashima et al., 1997a. and the Japanese black bear ŽUrashima et al., 1999a., in whose milk free lactose was found to be only a minor component relative to ␣1-3galactosyllactose and higher oligosaccharides. In this study, we isolated and identified several oligosaccharides in hooded seal milk. Most of the carbohydrate of the milk sample eluted in the void volume during Bio Gel P-2 chromatography, suggesting that it was protein-bound. The remaining carbohydrate consisted of lactose and the following nine oligosaccharides: 2⬘-fucosyllactose, lacto-N-neotetraose, lacto-N-fucopentaose IV, lacto-N-neohexaose, two monofucosyl lacto-Nneohexaoses, difucosyl lacto-N-neohexaose, para lacto-N-neohexaose, monofucosyl para lacto-Nneohexaose. The concentration of the trisaccharide 2⬘fucosyllactose was similar to that of lactose ŽFig. 1a.; in this character the hooded seal milk resembled that of the coati ŽUrashima et al., 1999b.. The other oligosaccharides comprised free lactoN-neotetraose and saccharides which contained lacto-N-neotetraose or lacto-N-neohexaose as core units. Lacto-N-neotetraose and lacto-Nneohexaose were first isolated from human milk ŽKuhn and Gauhe, 1962; Kobata and Ginsburg, 1972. and have since been found in horse colostrum ŽUrashima et al., 1991., and also as core units of coati ŽUrashima et al., 1999b. and bear milk oligosaccharides ŽUrashima et al., 1997a, 1999a.; they have not been previously detected in milk of any pinniped. Thus, in this respect, hooded seal milk resembles coati and bear milk Žsee Fig. 6.. Oligosaccharides containing ␣-Gal at their non-reducing ends, which are present in bear ŽUrashima et al., 1997a, 1999a. and coati ŽUrashima et al., 1999b. milk, were not, however, detected in the hooded seal milk Žsee Fig. 6 . , suggesting that ␣ Ž 1-3 . galactosyltransferase activity is absent from the mammary glands of lactating hooded seals. The GlcNAc residues of lacto-N-tetraose and neohexaose units in hooded seal milk oligosaccharides, like those of the coati ŽUrashima et al., 1999b., were not fucosylated, in contrast to most

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Fig. 6. Structures of hooded seal, white nosed coati and Ezo brown bear milk oligosaccharides.

of these residues in bear milk sugars, which are fucosylated at OH-3 ŽUrashima et al., 1997a, 1999a. Žsee Fig. 6.. It would appear, therefore, that ␣ Ž1-3.fucosyltransferase activity, too, is absent from the seal mammary glands, as it is in the coati ŽUrashima et al., 1999b.. Although trifucosyl derivatives of para lacto-N-neohexaose have been found in human milk ŽBruntz et al., 1988., para lacto-N-neohexaose itself and its monofucosyl derivative, as found in the hooded seal milk, are novel oligosaccharides. The presence of these and other oligosaccharides containing two GlcNAc residues suggests that hooded seal mammary glands contain ␤ Ž1-3 . N-acetylglucosaminyltransferase which is thought to be a key enzyme for the elongation of glycoconjugates containing such structures ŽVan den Eijnden et al., 1988.. Many of the hooded seal milk oligosaccharides were found to have an ␣ Ž1-2. linked fucose at the non-reducing end. This suggests that ␣ Ž1-2.fucosyltransferase activity is present in the seal mammary glands. Some brown bear and white-nosed coati oligosaccharides also have FucŽ ␣1-2.Gal-R unit ŽH antigen. in the non-reducing ends as is the case for most of the hooded seal milk oligosaccharides Žsee Fig. 6.. In this respect, hooded seal milk is similar to both coati and brown bear milk. It seems likely that the ancestral lineage of the

bear, coati and seal had lacto-N-neotetraose, lacto-N-neohexaose and non-reducing FucŽ ␣12.Gal units in some milk oligosaccharides. It is assumed that non-reducing ␣-Gal in milk oligosaccharides has been lost in the seal during the evolution process from the common ancestor, or alternatively that this unit has been obtained in the bear and coati during the evolution. The oligosaccharide content of bear milk appears to be much higher than that in hooded seal milk. We used 1 ml of bear milk to separate each oligosaccharide ŽUrashima et al., 1997a, 1999a., whereas each hooded seal oligosaccharide was separated from 40-ml of milk in this study. The difference of the content of milk oligosaccharides between bears and the hooded seal is thought to be due to differences in the biosynthetic activity of lactose in their respective mammary glands during lactation. The lactose unit is thought to be a template for biosynthesis of other oligosaccharides, because the milk oligosaccharides always have a lactose unit at the reducing end. The biosynthesis of lactose, and of saccharide containing lactose, is dependent on the presence of ␣lactalbumin within the mammary glands ŽBrodbeck et al., 1967.. The strength of the biosynthetic activity of lactose in the mammary glands is assumed to dependent on the amount of ␣lactalbumin in the glands. The amount of ␣-

T. Urashima et al. r Comparati¨ e Biochemistry and Physiology Part B 128 (2001) 307᎐323

lactalbumin in bear milk, or in the mammary glands, is assumed to be higher than that in the hooded seal milk or in the mammary glands. The amount of ␣-lactalbumin in the seal milk might have become lower during the evolution from the common ancestor of bears and seals. Milk of the crabeater seal ŽMesser et al., 1988. and the hooded seal have been shown to contain both lactose and free saccharides. However, a number of otariid species including the California sea lion, Zalophus californianus ŽPilson and Kelly, 1962., the Northern fur seal, Callorhinus ursinus, ŽDosako et al., 1983. and the Australian fur seal Žthis study. contain neither. ␣-Lactalbumin has been shown to be entirely absent from mammary glands of the California sea lion ŽJohnson et al., 1972., consistent with the lack of lactose in the milk of this species. It is therefore very likely that ␣-lactalbumin is absent also from the mammary glands of the Australian and the Northern fur seals and perhaps all otariids. Phocids more closely resemble the terrestrial Carnivora Že.g. bears. with respect to these traits. It is assumed that Otariids lost ␣-lactalbumin completely during evolution, whereas Phocids still have small amount of this protein. This possible difference between the two pinniped families may have interesting implications with respect to their evolutionary history and warrants further investigation through studies of a broader range of species within the two families. Examination of lactose and other milk sugars in the walrus Ž Odobenus rosmarus. might also provide valuable insight. The presence of lactose and other oligosaccharides in the hooded seal milk leads to the question if the Phocids pups digest them in the tract. Lactase activity has been investigated in the digestive tract of pinniped pups. Kretchmer and Sunshine Ž1967. found a small amount of lactase activity, which was approximately 1r60 to 1r100 of that encountered in the 2᎐5-day-old-rat, in the intestine of a Harbor seal pup. They were unable to detect either lactase or O-nitrophenyl-␤galactosidase activity in California or Steller sea lion pups ŽSunshine and Kretchmer, 1964; Kretchmer and Sunshine, 1967.. Kerry and Messer Ž1968. did detect a small amount of lactase activity, 0.8% of that encountered in man, in the intestine of a suckling Australian fur seal pup. These data show that lactase activity in the digestive tract of pinniped pups are minimal, even

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though these activities are detectable. Phocid pups do not use lactose as a major source for energy. It is unclear if eutherian mammals’ neonates digest milk oligosaccharides other than lactose, as energy sources. In humans, it is suggested that milk oligosaccharides resist digestion in the small intestine of most breast-fed infants ŽBrand-Miller et al., 1998; Engfer et al., 2000. and that the undigestable oligosaccharides, which can inhibit enteropathogen binding to host cell receptors, could serve a protective function ŽDai et al., 2000.. This possible biological function of milk oligosaccharides may be applicable for bear, coati and even for Phocids. It is clearly not in the case for fur seals, because their milk does not contain free-reducing saccharides. The milk of both seal species we studied, like that of the Northern fur seal ŽDosako et al., 1983., contained significant amounts of inositol. The ratio of myo-inositol to scyllo-inositol, which was 1.00:0.85 in the hooded seal milk, was much greater at 1.00:0.04 in Australian fur seal milk. The biological significance of this difference, if there is one, is unknown.

Acknowledgements We thank Prof. T. Itoh of Tohoku University for recording the 1 H-NMR spectra. We also thank Dr M.O. Hammill of the Department of Fisheries and Oceans, Canada, for his help with collecting the hooded seal milk. We are indebted to Dr M. Messer, of the University of Sydney, for valuable advice. References Bowen, W.D., Oftedal, O.T., Boness, D.J., 1985. Birth to weaning in four days: remarkable growth in the hooded seal, Cystophora cristata. Can. J. Zool. 63, 2841᎐2846. Brand-Miller, J.C., McVeagh, P., McNeil, Y., Messer, M., 1998. Digestion of human milk oligosaccharides by healthy infants evaluated by the lactulose hydrogen breast test. J. Pediatr. 133, 95᎐98. Brodbeck, U., Denton, W.L., Tanashi, N., Ebner, K.E., 1967. The isolation and identification of the B protein of lactose synthase as ␣-lactalbumin. J. Biol. Chem. 242, 1391᎐1397.

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