gy, Part A 145 (2006) 468–478www.elsevier.com/locate/cbpa
Comparative Biochemistry and Physiolo
Structural determination of the oligosaccharides in the milk of an Asianelephant (Elephas maximus)
Yusuke Uemura a,b, Sadaki Asakuma c, Lisa Yon d, Tadao Saito e, Kenji Fukuda c,Ikichi Arai a, Tadasu Urashima c,⁎
a Department of Bioresource Chemistry, Obihiro University of Agriculture and Veterinary Medicine, Inada cho, Obihiro, Hokkaido 080-8555, Japanb Course of the Science of Bioresources, The United Graduate School of Agricultural Science, Iwate University, Morioka, Iwate 020-8550, Japanc Graduate School of Food Hygiene, Obihiro University of Agriculture and Veterinary Medicine, Inada cho, Obihiro, Hokkaido 080-8555, Japan
d Department of Population Health and Reproduction, University of California, Davis, One Shields Avenue, Davis, CA 95616, USAe Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan
Received 25 April 2006; received in revised form 28 July 2006; accepted 1 August 2006Available online 4 August 2006
Keywords: Milk oligosaccharides; Asian elephant; Elephas maximus; Type II chain; NMR spectroscopy; Sialyl Lewis x; Isoglobotriose; Lacto-N-neotetraose;Para lacto-N-neohexaose
1. Introduction
Mammalian milk usually contains lactose as the dominantcarbohydrate (Jenness et al., 1964). Exceptions include the milkof monotremes, marsupials and of some eutherians (Ezo brownbear, Japanese black bear, polar bear, giant panda and mink)(Messer and Urashima, 2002; Urashima et al., 1997a,b, 1999a,b,2000, 2003, 2004, 2005; Nakamura et al., 2003); these milksgenerally contain greater amounts of free oligosaccharides thanof lactose. On the other hand, it has been reported that the milksof some Pinnipedia species (California sea lion, Northern furseal, crabeater seal, hooded seal and Australian fur seal)
unusually contained no or quite little carbohydrate (Pilson andKelly, 1962; Dosako et al., 1983; Messer et al., 1988; Urashimaet al., 2001a). The compositions of the free saccharides (milkoligosaccharides) other than lactose vary between differentmammalian species (Urashima et al., 2001b).
Human milk contains 12–13 g/L of more than 100 differentoligosaccharides that can be divided into twelve groups based ontheir core structures (Haeuw-Fievre et al., 1993; Newburg andNeubauer, 1995). Almost all of these oligosaccharides have alactose unit at their reducing end. It is generally considered thatfree lactose serves mainly as an available energy source forhuman infants while the milk oligosaccharides act as prebioticsand soluble receptor analogues which protect against infectionby pathogenic bacteria and viruses (Messer and Urashima,2002).
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It has been reported that African elephant (Loxodontaafricana) and Asian elephant (Elephas maximus) milks contain3.7% and 4.0–8.4% of carbohydrate, respectively (McCullaghand Widdowson, 1970; Peters et al., 1972). Osthoff et al. (2005)reported that the lactose content decreased from 52.5 to 11.8 g/kgin African elephant milk at 4 and 47 days post partum, while theoligosaccharide content increased from 11.8 to 15.2 g/kg. Thismeans that the oligosaccharide content gained ascendancy overthat of lactose at 47 day post partum in themilk. Kunz et al. (1999)found that Asian elephant milk carbohydrate consists of 60%lactose and 40% of a variety of milk oligosaccharides. It is ofinterest whether these oligosaccharides have the same biologicalsignificance for the elephant calves as it does for human infants.The elephant milk oligosaccharide fraction contained a high ratioof sialyl oligosaccharides; this may be significant with respect tothe formation of brain components, such as gangliosides, of thesuckling calves (Messer and Urashima, 2002).
Kunz et al. (1999) characterized their elephant milkoligosaccharides by comparison of the retention time of eachpeak in HPAEC-PAD with that of human milk oligosaccharide.They reported that the oligosaccharides comprising the type IIchain (Gal(β1-4)GlcNAc) were dominant in the Asian elephantmilk, however, the type I chain (Gal(β1-3)GlcNAc) had alsobeen observed.
In this study, we purified each of the major oligosaccharidesfrom Asian elephant milk and characterized their chemicalstructures using 1H-NMR techniques. The oligosaccharidesobserved by us differed in some respects from those reported byKunz et al.; most notably, we found only the type II chainamong the oligosaccharides of the elephant milk.
2. Materials and methods
2.1. Materials
Asian elephant milk (100 mL) was collected from an animalbred at the Ayutthaya Elephant Palace and Royal Kraal inAyutthaya, Thailand. The milk was obtained manually on 4October 2003 at 11 days postpartum and was stored at −20 °C.
Neu5Ac(α2-3)Gal(β1-4)Glc (3′-sialyllactose) and Neu5Ac(α2-6)Gal(β1-4)Glc (6′-sialyllactose) were purchased fromSigma (St. Louis, USA). Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc (Sialyl lacto-N-neotetraose c, LSTc) waspurchased from Seikagaku (Tokyo, Japan). Neu5Ac(α2-3)Gal(β1-4)[Fuc(α1-3)]Glc (monofucosyl monosialyllactose),Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-3)[Gal(α1-3)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (monogalactosyl monosialyllacto-N-neohexaose) and Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-3){Gal(α1-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-6)}Gal(β1-4)Glc (monogalactosyl monofucosyl monosialyl lacto-N-neohexaose) were purified from the milks of giant panda(Nakamura et al., 2003), mink (Urashima et al., 2005) andJapanese black bear (Urashima et al., 2004), respectively.Isoglobotriose (Gal(α1-3)Gal(β1-4)Glc) was purified from themilks of giant panda (Nakamura et al., 2003) and mink(Urashima et al., 2005). All other reagents were of analyticalgrade.
2.2. Colorimetric assays
The carbohydrate content of the Asian elephant milk wasassayed for total hexose using the phenol-H2SO4 method(Dubois et al., 1956), with lactose as the standard. The sialicacid content was determined by the periodate-resorcinol methodusing N-acetylneuraminic acid as the standard (Jourdian et al.,1971). Assays were made in triplicate.
2.3. Preparation of the oligosaccharides from Asian elephantmilk
The milk was thawed and 5 mL were extracted with fourvolumes of chloroform/methanol (2:1, v/v). After agitation, theemulsion was centrifuged at 5000×g, 4 °C for 30 min, and thelower chloroform layer and the denatured protein werediscarded. The methanol was evaporated from the upper layer,and the lyophilized residue was designated as the carbohydratefraction.
The carbohydrate fraction was dissolved in 2mL of water andthe solution passed through a Bio-Gel P-2 (b45 μm, BioRad,USA) column (2.6×100 cm, void volume=135 mL) that hadbeen calibrated with 2 mg each of galactose (monosaccharide),lactose (disaccharide) and raffinose (trisaccharide). Elution wasdone with distilled water at a flow rate of 15 mL/h, and 5 mLfractions were collected. Aliquots (40 μL) of each fraction wereanalyzed for hexose with the phenol-H2SO4 method and forsialic acid with the periodate-resorcinol method (chromatogramin Fig. 1). The peak fractions, Em 1 (see Fig. 1), which reactedpositively in the periodate-resorcinol assay were pooled andlyophilized. The components in peak Em 1-2 and Em 1-3 wereanalyzed by thin layer chromatography (TLC) on Silica Gel 60(20×20;Merck, Darmstadt, Germany) with acetone/2-propanol/0.1M lactic acid (2:2:1, v/v/v) as a developing solvent. The spotswere detected by spraying with 5% sulfuric acid in 99.5%ethanol and heating above a flame. The components in Em 2 andEm 3 were characterized by 1H-NMR.
The fractions Em 1 (see Fig. 1) were dissolved in 2 mL of50 mM Tris-hydroxyaminomethane-HCl buffer (pH 8.7) andsubjected to anion exchange chromatography using a DEAE-Sephadex A-50 (Pharmacia Biotech, USA) column (1.5×35 cm,void volume=15 mL) equilibrated with the same buffer(chromatogram in Fig. 2a). The unadsorbed components wereeluted with 250 mL of the buffer, after which elution wascontinued using a linear gradient of 0–0.5 M NaCl in the buffer.Elution was done at a flow rate of 15 mL/h and fractions of 5 mLwere collected. Aliquots (50 μL) of each fraction were analyzedfor hexose. The adsorbed components were barely found in thefractions eluted with the linear gradient of the buffer. The peakfractions Em 1-1, 1-2 and 1-3 containing unadsorbed materialswere pooled and lyophilized. The products from these fractionswere each dissolved in 2 mL of water and passed through a Bio-Gel P-2 column under the same conditions as described above toremove salt. All the obtained fractions were pooled andlyophilized (Fig. 2b).
Each compound in Em 1-2 (Fig. 2b) was further separated byhigh-performance liquid chromatography (HPLC) on a TSK gel
Fig. 1. Gel chromatogram of the carbohydrate fraction from Asian elephant milk. Elution from a Bio-Gel P-2 column (2.6×100 cm) was done with distilled water at aflow rate of 15 mL/h, and fractions of 5.0 mL were collected. Each fraction was monitored by the phenol-H2SO4 method (at 490 nm,—) and the periodate-resorcinolmethod (at 630 nm, ♦). The void volume (V0) is indicated by an arrowhead.
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Amido-80 column (4.6×250 mm, pore size 80 Å, particle size5 μm; Tosoh, Tokyo, Japan) using a LC-10ATVP pump(Shimadzu, Tokyo, Japan) (see chromatogram in Fig. 3a). Themobile phase was 50% and 80% (v/v) acetonitrile (CH3CN) in15 mM potassium phosphate buffer (pH 5.2). Elution was doneusing a linear gradient of acetonitrile from 80% to 50% at 40 °Cat a flow rate of 1 mL/min. Eluate was detected by measuringthe absorbance at 195 nm. The peak fractions of oligosacchar-
Fig. 2. (a) Anion exchange chromatogram of Em 1 separated by gel chromatograequilibrated with 50 mM Tris–HCl buffer (pH 8.7) was used. Elution was done first wcontaining NaCl from 0 to 0.5 M. The flow rate was 15 mL/h and fractions of 5 mL wvoid volume (V0) is indicated by an arrowhead. (b) Gel chromatography on Bio Gel P-(a). The gel chromatography was done as in Fig. 1.
ides were pooled, concentrated by rotary evaporation andpassed through the Bio-Gel P-2 column to remove salt,followed by lyophilization.
2.4. 1H-NMR spectroscopy
The NMR spectra were recorded in D2O (100.00 atom D%,Aldrich, Milwaukee, USA) at 500 or 600 MHz for 1H-NMR
phy on Bio-Gel P-2 (Fig. 1). A DEAE-Sephadex A-50 column (1.5×35 cm)ith 250 mL of the same buffer and then with a linear gradient of the same bufferere collected. The fractions were monitored by the phenol-H2SO4 method. The2 of the peak fraction, Em 1-2, from the anion exchange chromatogram shown in
Fig. 3. HPLC of the carbohydrate fraction Em 1-2 separated from Asian elephant milk. HPLC was done using a Shimadzu LC-10ATVP pump on a TSK-gel Amido-80column (4.6×250 cm, pore size 80 Å, particle size 5 mm). The mobile phase was 50% and 80% acetonitrile (CH3CN) in 50 mM potassium phosphate buffer. Elutionwas done using a linear gradient of CH3CN from 80% to 50% at 40 °C at a flow rate of 1 mL/min. The detection of peaks was done by UV absorption.
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with a JEOL ECP-500 FT-NMR or Varian INOVA 600spectrometer operated at 293.1 K. Chemical shifts are expressedin ppm from down-field from internal 3-(trimethylsilyl)-1-propane sulfonic acid, sodium salt (TPS), but actually measuredby reference to an internal acetone (δ=2.225).
3. Results
The sample of Asian elephant milk contained 91 and 3 g/L oftotal hexose and total sialic acid, respectively, by thecolorimetric assays as described above with the standard curveof lactose and N-acetylneuraminic acid. As shown in the sizeexclusion chromatogram in Fig. 1, the carbohydrate fractionresolved into several peaks. Only the components in fraction Em1 contained sialic acid, reacting positively with the periodate-resorcinol assay. It was assumed from the eluted positions inFig. 1 that the fractions Em 2, Em 3 and Em 4 containedtrisaccharide, disaccharide and monosaccharide, respectively.As the component in Em 3 had the same 1H-NMR spectrum(Table 1) and the same Rf value as lactose in TLC, using acetone/2-propanol/0.1 M lactic acid (2:2:1, v/v/v) as a developingsolvent, it was characterized to be lactose. The components inEm 2 were characterized by 1H-NMR as described below.
3.1. Neutral oligosaccharide: Em 2
As the 1H-NMR spectrum of the oligosaccharide in Em 2(chemical shifts in Table 1) was completely identical with that
Table 11H-NMR chemical shifts of saccharides in Em 2 and Em 3
Reportergroup
Residue Chemical shifts, δ (Coupling constants, Hz)
of isoglobotriose and with the published data for thattrisaccharide (Urashima et al., 2005), it was characterized tobe Gal(α1-3)Gal(β1-4)Glc. The spectrum had the anomericshifts of α-Glc, β-Glc, α(1-3)-linked Gal and β(1-4)-linked Galat δ 5.225, 4.668, 5.147 and 4.524, respectively, and thecharacteristic H-5 shift of α(1-3)-linked Gal at δ 4.197 and theH-4 shift of β(1-4)-linked Gal, which was substituted by α(1-3)-linked Gal, at δ 4.183.
3.2. Acidic oligosaccharides
The components in Em 1, which contained sialic acid, weresubjected to anion exchange chromatography. The un-adsorbedfractions were designated as Em 1-1, 1-2 and 1-3 as shown inFig. 2a. The crude sialyl oligosaccharide fraction, Em 1-2, waspassed through the Bio Gel P-2 column to remove salt (Fig. 2b),and attempts were made to purify each of the oligosaccharidesby HPLC (Fig. 3) prior to characterization by 1H-NMR.However, as the compounds in Em 1-1 and Em 1-3 were notfully separated by HPLC, they were not characterized in thisstudy.
3.2.1. Em 1-2-2As the 1H-NMR spectrum of the oligosaccharide in Em 1-2-
2 (chemical shift in Table 2) was completely identical with thatof 3′-SL and its published data (Urashima et al., 1999b), it wascharacterized to be Neu5Ac(α2-3)Gal(β1-4)Glc. The spectrumhad the anomeric shifts of α-Glc, β-Glc and β(1-4)-linked Galat δ 5.220, 4.663 and 4.531, respectively, and the characteristicdown field shift of H-3 of β(1-4)-linked at δ 4.115. Thespectrum also had H-3 axial and H-3 equatorial shifts of α(2-3)linked Neu5Ac at δ 1.801 and 2.757, respectively, and its NAcshift at δ 2.030.
3.2.2. Em 1-2-3As the 1H-NMR spectrum of the oligosaccharide in Em 1-2-3
(chemical shift in Table 2) was completely identical with that of6′-SL, it was characterized to be Neu5Ac(α2-6)Gal(β1-4)Glc.The spectrum had the anomeric shifts of α-Glc, β-Glc and β(1-4)-linked Gal at δ 5.223, 4.668 and 4.427, respectively, and H-3axial, H-3 equatorial and NAc shifts of α(2-6) linked Neu5Ac atδ 1.746, 2.713 and 2.028, respectively.
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3.2.3. Em 1-2-4As the chemical shifts (Table 2) of Em 1-2-4 were similar to
those of the published data of 3′-sialyl-3-fucosyllactose (Naka-mura et al., 2003), it was characterized to be Neu5Ac(α2-3)Gal(β1-4)[Fuc(α1-3)]Glc. The spectrum had the anomeric shifts ofα-Glc, β-Glc, β(1-4)-linked Gal at δ 5.176, 4.652, 4.502, re-spectively, and α and β of α(1-3)-linked Fuc linked to reducingGlc at δ 5.377 and 5.435, respectively. The shifts at δ 1.798, 2.763and 2.029 were assigned to H-3 axial, H-3equatorial and NAc ofα(2-3) linked Neu5Ac, respectively. The two signals at δ 1.177and 1.182 arose from α and β of H-6 of the Fuc residue.
3.2.4. Em 1-2-7Because the 1H-NMR spectrum (chemical shifts in Table 1)
of Em 1-2-7 was essentially the same as that of LSTc and its
published data (Urashima et al., 2002), it was characterized tobe Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc. Thespectrum had the characteristic H-3 axial, H-3 equatorial andNAc resonances of Neu5Ac residue at δ 1.726, 2.670 and 2.028,respectively, showing the presence of a Neu5Ac(α2-6)Gal unit.The spectrum had the anomeric shifts of α-Glc, β-Glc, β(1-3)-linked GlcNAc and two β(1-4)-linked Gal at δ 5.220, 4.665,4.728, 4.442 and 4.457, respectively, and H-4 shift of β(1-4)-linked Gal which was substituted at OH-3 by β linked GlcNAc,at 4.160.
3.2.5. Em 1-2-8The 1H-NMR spectrum (Fig. 4 and chemical shifts in Table 2)
of Em 1-2-8 had the anomeric shifts of α-Glc, β-Glc, β(1-3)-linked GlcNAc and two β(1-4)-linked Gal at δ 5.219, 4.662,4.702, 4.532 and 4.434, respectively, showing that the oligosac-charide in this fraction contained lacto-N-neotetraose unit. Thespectrum had the H-1 and H-6 shifts of Fuc of δ 5.121 and 1.167,respectively. From the chemical shift at δ 5.121, it is assumed thatthe Fuc is linked to theGlcNAc byα(1-3) linkage (Gronberg et al.,1992), showing that the saccharide contained a Lex (Gal(β1-4)[Fuc(α1-3)]GlcNAc) unit. The NAc shift of β(1-3)-linkedGlcNAc at δ 2.020 moved upfield due to the substitution of thisresidue byα(1-3)-linked Fuc. The spectrum had theH-3 axial, H-3equatorial and NAc shifts of Neu5Ac at δ 1.794, 2.764 and 2.030,respectively. In addition, the spectrum had the doublet-doubletsignal ofβ(1-4)-linkedGal at δ 4.087. From these observation, thisoligosaccharide contained Neu5Ac(α2-3)Gal unit. The downfieldshift of H-1 of β(1-4)-linked Gal at δ 4.532 also indicated that thisresidue was substituted by α(2-3)-linked Neu5Ac.
From these observations, the oligosaccharide was character-ized to be Neu5Ac(α2-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3)Gal(β1-4)Glc.
3.2.6. Em 1-2-11The oligosaccharide in fraction Em 1-2-11 was characterized
by comparison of its 1H-NMR spectrum (Fig. 5, chemical shiftsin Table 3) with that of Em 1-2-7 (LSTc). The spectrum had theanomeric signals of α-Glc, β-Glc, β(1-3)-linked GlcNAc andtwo of β(1-4)-linked Gal at δ 5.219, 4.663, 4.726, 4.455 and4.438, respectively, and the H-4 shift of β(1-4)-linked Gal,which was substituted by β(1-3)-inked GlcNAc, at δ 4.159;these were similar to those of Em 1-2-7 that had lacto-N-neo-tetraose unit. However, the spectrum had the additionalanomeric shifts at δ 4.697 and 4.469, which were assigned toH-1 signals of β(1-3)-linked GlcNAc and β(1-4)-linked Gal,respectively. In addition, it is concluded from its signalintensities that the signal at δ 4.159 corresponded to tworesonances. These data showed that the oligosaccharidecontained an additional Gal(β1-4)GlcNAc(β1-3) unit; namelyit had a para-lacto-N-neohexaose unit (Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc). The spectrum had theH-3 axial, H-3 equatorial and NAc shifts at δ 1.725, 2.669 and2.028 of α(2-6)-linked Neu5Ac.
From these observations, the oligosaccharide was character-ized to be Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc.
Fig. 4. 500 MHz 1H-NMR spectrum of Em 1-2-8 isolated from Asian elephant milk. The spectrum was obtained in D2O at 500 MHz with a JEOL ECP-500FTspectrometer operated at a probe temperature of 293.1 K.
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3.2.7. Em 1-2-17The oligosaccharide in fraction Em 1-2-17 was characterized
by comparison of its 1H-NMR spectrum (Fig. 6, chemical shifts inTable 3) with that of Em 1-2-11. The spectrum had the anomericshifts of α-Glc, β-Glc, two of β(1-3)-linked GlcNAc and three ofβ(1-4)-linked Gal at δ 5.219, 4.663, 4.721, 4.708, 4.456, 4.451and 4.434, respectively, and two H-4 shifts of β(1-4)-linked Gal,which were substituted by β(1-3)-linked GlcNAc, at δ 4.158 and4.104; these showed that it contained a para-lacto-N-neohexaoseunit. The spectrum had the additional H-1 and H-6 shifts of Fuc atδ 5.116 and 1.153. From the chemical shifts, it was thought that thefucose is linked to β(1-3)-GlcNAc via α(1-3)-linkage. The H-1signal at δ 4.708 of β(1-3)-linked GlcNAc moved more downfield than the shift at δ 4.697 of Em 1-2-11,while theNAc signal atδ 2.021 ofβ(1-3)-linked GlcNAcmoved upfield than the shift at δ2.028 of Em 1-2-11; the difference would be the result of the
Fig. 5. 600 MHz 1H-NMR spectrum of Em 1-2-11 isolated from Asian elephant mispectrometer operated at a probe temperature of 293.1 K.
attachment of an α(1-3)-linked Fuc to this residue. The spectrumhad the H-3 axial, H-3 equatorial and NAc shifts of α(2-6)-linkedNeu5Ac at δ 1.725, 2.667 and 2.028, respectively.
From these observation, the oligosaccharide in Em 1-2-17was characterized to be Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc with one α(1-3)-linkedFuc residue that attached to either GlcNAc residue.
3.2.8. Em 1-2-18As the chemical shifts (Table 2) of Em 1-2-18 were quite
similar to those of the published data for monosialyl mono-galactosyl lacto-N-neohexaose separated from mink milk (Ura-shima et al., 2005), it was characterized to be Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-3)[Gal(α1-3)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc. The spectrum (chemical shifts in Table 3) had theanomeric shifts at δ 5.219, 4.668, 4.433, 4.455, 4.545, 4.730 and
lk. The spectrum was obtained in D2O at 600 MHz with a Varian INOVA 600
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4.648 of α-Glc, β-Glc, three of β(1-4)-linked Gal, β(1-3)-linkedGlcNAc and β(1-6)-linked GlcNAc, respectively. The shifts at δ5.145 and 4.193 arose from H-1 and H-5 of α(1-3)-linked Gal,respectively, and the shift at δ 4.184 of H-4 of β(1-4)-linked Gal,which was substituted by an α(1-3)-linked Gal. The shifts at δ1.723, 2.669 and 2.028 were assigned to H-3 axial, H-3 equatorialand NAc of α(2-6) linked Neu5Ac, respectively. The respectiveNAc signals at δ 2.052 and 2.062 were assigned to a β(1-3)- andβ(1-6)-linked GlcNAc.
3.2.9. Em 1-2-19The oligosaccharide in fraction Em 1-2-19 was characterized
by comparison of its 1H-NMR (Fig. 7, chemical shifts in Table 3)with that of Em 1-2-8. The spectrum had the H-1 shifts of α-Glc,β-Glc,α(1-3)-linked Fuc,β(1-3)-linkedGlcNAc and twoβ(1-4)-linked Gal at δ 5.218, 4.662, 5.126, 4.707, 4.535 and 4.432,respectively. TheH-6 shift ofα(1-3)-linked Fuc at δ 1.167, andH-4 shift of β(1-4)-linked Gal, which was substituted by β(1-3)-linked GlcNAc, at δ 4.156, resemble those of Em 1-2-8, showingthat it contained Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3)Gal(β1-4)unit. However, the spectrumhad the additional shift of H-1 andH-6 of α(1-3)-linked Fuc at δ 5.114 and 1.146, respectively, H-1 ofβ(1-3)-linked GlcNAc at δ 4.692, H-1 of β(1-4)-linked Gal at δ4.443, and H-4 of β(1-4)-linked Gal, which was substituted byβ(1-3)-linked GlcNAc, at δ 4.097, indicating that the oligosac-charide had an additional Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3)unit. The spectrum had the H-3 axial, H-3 equatorial and NAcshifts of α(2-3)-linked Neu5Ac at δ 1.793, 2.764 and 2.030,respectively.
From these observations, the oligosaccharide in Em 1-2-19was characterized to be Neu5Ac(α2-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3)Gal(β1-4)Glc. The two NAc shifts of β(1-3)-linked GlcNAc at δ 2.014and 2.019 had moved upfield compared with the usual signalsbecause these residues were substituted by α(1-3)-linked Fucresidues. The H-3 shift of β(1-4)-linked Gal, which wassubstituted by α(2-3)-linked Neu5Ac, at δ 4.084, partiallyoverlapped with the shift of H-4 of β(1-4)-linked Gal.
3.2.10. Em 1-2-21As the chemical shifts (Table 3) of Em 1-2-21 were similar as
those of the published data (Urashima et al., 2004) for monosialylmonogalactosyl monofucosyl lacto-N-neohexaose separated fromJapanese black bearmilk, it was characterized to beNeu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-3){Gal(α1-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-6)}Gal(β1-4)Glc. The spectrum had the anomericshifts of α-Glc, β-Glc, β(1-3)-linked GlcNAc, three of β(1-4)-linked Gal and β(1-6)-linked GlcNAc at δ 5.219, 4.668, 4.725,4.433, 4.455, 4.525 and 4.642, respectively, and the shift at δ 4.147was assigned toH-4 ofβ(1-4)-linkedGal,whichwas substituted atOH-3. The anomeric shifts at δ 5.143 and 5.115 were assigned toH-1 ofα(1-3)-linkedGal andα(1-3)-linked Fuc consisting of aGal(α1-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc unit. The shifts at δ 4.196and 4.157 were assigned to H-5 of α(1-3)-linked Gal and H-4 ofβ(1-4)-linked Gal consisting of Gal(α1-3)Gal(β1-4) unit, respec-tively, and the shift at δ 1.179 to H-6 of α(1-3)-linked Fuc. Theshifts at δ 1.725 and 2.668 assigned to H-3 axial and H-3
Fig. 6. 600 MHz 1H-NMR spectrum of Em 1-2-17 isolated from Asian elephant milk. The spectrum was obtained in D2O at 600 MHz with a Varian INOVA 600spectrometer operated at a probe temperature of 293.1 K.
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equatorial of α(2-6) linked Neu5Ac, and the NAc shifts at δ 2.027,2.052 and 2.052 were assigned to α(2-6) linked Neu5Ac, β(1-4)-linked GlcNAc and β(1-6)-linked GlcNAc, respectively.
4. Discussion
The Asian elephant milk obtained at 11 days postpartumanalyzed in this study contained 91 g/L of carbohydrates. Thisvalue is significantly higher than those for human (70 g/L) andbovine (45 g/L) milk. Lactose was dominant in the carbohy-drate, as in most mammalian milks, but the milk carbohydrate ofAsian elephant also contained a variety of oligosaccharides atrelatively high concentration (see Fig. 1); these results weresimilar to those of Kunz et al. (1999). The concentrations oftotal milk oligosaccharides, lactose and monosaccharide in thismilk were calculated from the ratios of the peak areas in Fig. 1,
Fig. 7. 600 MHz 1H-NMR spectrum of Em 1-2-19 isolated from Asian elephant mispectrometer operated at a probe temperature of 293.1 K.
to be 53.7, 36.4 and 0.9 g/L, respectively. Peters et al. (1972)reported that the concentrations of lactose in Asian elephantmilk were 40 and 83.6 g/L at 5 and 141 days postpartum,respectively. These values represented the sum of lactose andmilk oligosaccharides. Kunz et al. (1999) reported that Asianelephant milk obtained at 45, 72 and 234 days post partumcontained 21.0, 21.8 and 19.3 g/L of milk oligosaccharides,respectively, and 25.8, 29.6 and 30.7 g/L of lactose, respec-tively. On the other hand, McCullagh and Widdowson (1970)reported that milk of the African elephant (L. africana)contained 80 g/L lactose at 6 months postpartum, as determinedby the anthrone method (Slater, 1961); this value would haveincluded oligosaccharides. Osthoff et al. (2005) reported thatAfrican elephant milk obtained at 4 and 47 days postpartumcontained 11.8 and 15.2 g/L of milk oligosaccharides, res-pectively, and 52.5 and 11.8 g/L of lactose, respectively. From
lk. The spectrum was obtained in D2O at 600 MHz with a Varian INOVA 600
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the above values and our data it can be concluded that theconcentrations of milk oligosaccharides and lactose of elephantmilk are rather close to those of human milk, insofar as maturehuman milk contains about 12-14 g/L of oligosaccharides and60 g/L of lactose (Newburg and Neubauer, 1995). As lactose isconsidered to be a significant energy source for human neo-nates, it can be assumed that elephant calves similarly utilize thedisaccharide for the provision of energy.
The milk oligosaccharides characterized in this study con-sisted of one neutral oligosaccharide and ten sialyl oligosacchar-ides (Fig. 8). Isoglobotriose, which was dominant among theAsian elephant milk oligosaccharides, has not been reported inhuman milk. However, it has been found in the milks of cow(Urashima et al., 1991), sheep (Urashima et al., 1989), goat(Nakamura et al., 2000), bears (Urashima et al., 1997a, 1999a,2000), giant panda (Nakamura et al., 2003), white nosed coati(Urashima et al., 1999b) andmink (Urashima et al., 2005). 3′-SLand 6′-SL have been found in the milk or colostrum of manymammals including domestic cow (Kuhn and Gauhe, 1965;Schneir and Rafaelson, 1966; Veh et al., 1981), goat (Urashimaet al., 1997b), human (Newburg and Neubauer, 1995) etc, andalso in the previous study of Asian elephant milk (Kunz et al.,1999). 3′-Sialyl-3-fucosyllactose (Neu5Ac(α2-3)Gal(β1-4)[Fuc(α1-3)]Glc), which has been also found in human milk(Gronberg et al., 1989) and giant panda milk (Nakamura et al.,2003), is the shortest unit of sialyl Lex. The present paper reportstwo oligosaccharides, Neu5Ac(α2-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3)Gal(β1-4)Glc (Em 1-2-8, Fig. 8) and Neu5Ac(α2-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3)Gal(β1-4)Glc (Em 1-2-19, Fig. 8), withterminal sialyl Lex structure, occur in the Asian elephant milk.
Fig. 8. Structures of the oligosaccharides
This is the first time these saccharides have been observed as freesugars in milk or colostrum. As saccharides containing sialylLex have been regarded as analogues of the selectin ligand(Vestweber and Blanks, 1999), it is possible that these oligo-saccharides function as colonic anti-inflammation factors in theelephant calves.
LST c has been found in human (Kuhn and Gauhe, 1965) andminke whale milks (Urashima et al., 2002), while Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc(Em 1-2-11, Fig. 8) has not as yet been reported in any othermammalianmilk or colostrum.Monosialylmonogalactosyl lacto-N-neohexaose (Em 1-2-18, Fig. 8) and monosialyl monogalacto-syl monofucosyl lacto-N-neohexaose (Em 1-2-21, Fig. 8) havebeen found in the milk of mink (Urashima et al., 2005) andJapanese black bear (Urashima et al., 2004), respectively.
As described above, themilk oligosaccharides characterized inthis study had a lactose, lacto-N-neotetraose or para-lacto-N-neohexaose as well as a sialyl Lex unit, with non-reducingα(2-6)-linked Neu5Ac or non-reducing α(1-3)-linked Gal. Interestingly,all of the higher oligosaccharides had only the type II chain (Gal(β1-4)GlcNAc) but not the type I chain (Gal(β1-3)GlcNAc). Thefucosyl oligosaccharides contained only the Fuc(α1-3) residuebut not the α(1-2)-linkage. The components in several minorpeaks remained unidentified, but any signals of their 1H-NMRspectrum did not show the presence of the type I chain in thestructures (data not shown). On the other hand, Kunz et al. (1999)identified Neu5Ac(α2-3)Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc(LSTa), which contains the type I chain (Gal(β1-3)GlcNAc), intheir Asian elephant milk. They characterized the oligosacchar-ides by comparing the band mobility or the retention time of eachcarbohydrate in HPTLC or HPAEC analysis with that in human
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milk carbohydrates. Their identification of LSTa may be due tothe differences in analytical techniques; alternatively, theinconsistency in this respect between our results and those ofKunz et al. (1999) may simply be due to differences between themilk samples. It may be worth mentioning the higher oligosac-charides with sialyl Lex or α-Gal residue have not as yet beenfound in human milk; this is a noticeable difference between theoligosaccharide compositions of human and Asian elephant milk.
As mentioned above, lactose and milk oligosaccharidesprobably act as an energy source and anti-infection factors forneonates (Dai et al., 2000; Sharon and Ofek, 2000; Morrow et al.,2005), respectively. It is also reported that human milkoligosaccharides promote the growth of the bifidus flora in thenewborn's intestine (Gyorgy and Rose, 1955; Kuhn, 1958). Theneonates of humans and elephants suckle their mother's milk forabout 12 and 22 months, respectively; these periods are relativelylonger than most of other mammalian species. During thesesuckling periods, the neonates apparently require relatively largeamounts of lactose as well as of milk oligosaccharides. Bycomparison of these species, the heterogeneity of milk oligo-saccharides between these two species, human and Asianelephant, may reflect differences in pathogenic microorganismspresent in the colons of the neonates. Additionally, as observed inhuman milk oligosaccharide (Wang et al., 2004; Gnoth et al.,2001), sialylated oligosaccharides in the milk of Asian elephantmay have a significant function in the development of the neonatebrain, subsequent to absorption of the sialic acidwithin their smallintestine.
As mentioned in Introduction, the milk of monotremes,marsupials and of some eutherians of Carnivora (Ezo brownbear, Japanese black bear, polar bear, giant panda and mink)contain greater amounts of free oligosaccharides than of lactose(Messer and Urashima, 2002). It has been also reported that themilk of some Pinniped species (California sea lion, Northern furseal, crabeater seal, hooded seal and Australian fur seal)contained no or quite little carbohydrate (Pilson and Kelly,1962; Dosako et al., 1983; Messer et al., 1988; Urashima et al.,2001a). Lactose is synthesized within the mammary glands fromUDP-galactose (donor) and glucose (acceptor) by a transgalac-tosylation catalyzed by lactose syntase that is a complex of a β4-galactosyltranferase I and α-lactalbumin. On the other hand,milk oligosaccharides are synthesized by the action of severalglycosyltransferase using lactose as an acceptor. It is thought thatthe ratio of milk oligosaccharides to lactose in milk is due to therelative ratio of the expression of the glycosyltransferases to α-lactalbumin in lactating mammary glands (Messer and Ura-shima, 2002; Urashima et al., 2005). It is hypothesized that thisrelative ratio is higher in the mammary glands in monotremes,marsupials and the above eutherian species than that in othereutherians. It is also speculated that the relative ratio in themammary glands of humans and elephants is rather higher thanthat in other species such as cows etc. On the other hand, theabsence of lactose and milk oligosaccharides in fur seal and sealion milk is almost certainly due to non-expression of α-lactalbumin within their mammary glands. Indeed, this proteinhas been show to be entirely absent from lactating mammaryglands of the California sea lion (Johnson et al., 1972).
Acknowledgements
This study was partially supported by a Grant from the 21stCentury COE Program (A-1), Ministry of Education, Culture,Sports, Science and Technology, Japan, and a Grant-in-Aidfrom the Bio-oriented Technology Research AdvancementInstitution of the National Agriculture and Food ResearchOrganization.
We would like to thank Dr. Narongsak Chaiyabutr, Dean ofthe Faculty of Veterinary Science, at Chulalongkorn Universityin Thailand for his support and assistance, and Mr. SampastMeepan of the Ayutthaya Elephant Palace and Royal Kraal forsupplying the elephant milk. We would also like to thank Dr.Michael Messer of the University of Sydney for helpful advice.
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