Microdetermination of Individual Neutral and Amino Sugars and N-Acetylneuraminic Acid in Complex Saccharides*

J. Biochem., 76, 783-789 (1974)

Seiichi TAKASAKI and Akira KOBATA Department of Biochemistry, Kobe University School of Medicine, Kusunoki-cho, Ikuta-ku, Kobe 650

Received for publication, April 4, 1974

A very sensitive method for the quantitative determination of individual neutral and amino sugars and N-acetylneuraminic acid in complex saccharides has been developed. The usefulness of this method was confirmed by applying it to the analysis of several milk oligosaccharides of known structure.

Heterosaccharide complexes on the cell surface commonly found in glycoproteins and glyco are important as immunodeterminants, func lipids. tional structures of viruses and hormone re This method is based on quantitative radio ceptors, and may also play a key role in cell isotope labelling of sugars with [3H]-sodium to-cell interactions. However, in the charac borohydride, as first applied by Howell et al. terization of heterosaccharides, the determina for the analysis of total hexose and hexosamine tion of the sugar components has always been contents of IgG (4), and separation of sugar a problem. alcohols by borate paper electrophoresis. The sugar composition in heterosaccharide complexes is usually determined by an appro MATERIALS AND METHODS priate method after hydrolysis in acidic con ditions. Sugars and Reagents-Sorbitol and man After the development of the gas chromato nitol were purchased from Seikagaku Kogyo graphic (1, 2) and ion exchange chromato Co., Ltd. N-Acetylneuraminic acid and galac graphic methods (3), the required sample size titol were purchased from Sigma Chemical Co., was reduced down to scores of micrograms, Ltd. Fucitol and N-acetylneuraminic acid al but this is still too large to be applicable for cohol** were synthesized from fucose and N- the study of cell surface components. acetylneuraminic acid by sodium borohydride This paper deals with a sensitive ana reduction (5). N-Acetyglucosaminitol and N- simple method for the determination of sugars acetylgalactosaminitol were synthesized by the method of Crimmin (6). Glucosaminitol hydro chloride and galactosaminitol hydrochloride * This work has been supported in part by Research Grants from the Jane Coffin Childs Memorial Fund ** The reduction product of N-acetylneuraminic acid for Medical Research, the Naito Foundation, and the Scientific Research Fund (1973-1974) of the Ministry in which the ketone group is converted to a secondary of Education of Japan. alcohol group.

Vol. 76, No. 4, 1974 783 784 S. TAKASAKI and A. KOBATA

were obtained from their N-acetyl derivatives again evaporated to dryness. The evaporation by hydrolysis in 2 N HC1 for 2 hr at 100•Ž. with 1 M acetic acid was repeated five times After decolorization with a small amount of to ensure the removal of any exchangable active charcoal, the mixtures were filtered. tritium as [3H]-H2O. The filtrates were lyophilized and crystallized For the simple measurement of radioac from aqueous ethanol. Milk oligosaccharides tivity incorporation, the residue was dissolved were isolated from human milk as reported in 1.0 ml of water and a 0.1-0.5 ml aliquot

previously (7). [3H]-Sodium borohydride, spe was counted with 7 ml of Bray's solution (10) cific activity 346 mCi/mMole, was purchased using a Packard liquid scintillation counter from Amersham Radiochemical Centre. It was model 3320. Since comMercial [3H]-sodium dissolved in freshly distilled anhydrous di borohydride contains several percent of non methylformamide at 32 ƒÊmoles/ml and stored volatile radioactive material, a blank experi in a tube with an airtight screw cap at -10•Ž. ment without sugar was always done with this Under these conditions no detectable change simple determination method. The blank was of the reductant was observed even after 5 unnecessary in the case of paper electrophore months. Sialidase [EC 3.2.1.18] purified from tic determination of radioactive sugar alcohol Clostridium perfringens was purchased from mixtures (vide infra), as the nonvolatile con Worthington Biochemical Co., Ltd. taminant remains at the origin as a single Buffers-Borate buffer (8), 0.2 M in borate, peak in the electrophoretogram and does not was prepared by dissolving 19.1 gm of borax interfere with the determination of any of the in 500 ml of water, adjusting the pH to 9.50 sugar alcohols. with 1.0 M NaOH, and adding water to make Determination of the Monosaccharide Com a final volume of one liter. Borate buffers of position of Complex Sugars-i) Measurements 0.06 and 0.04 M were prepared by simply dilut of reducing values: For the correct determina ing the 0.2 M solution with distilled water. tion of the monosaccharide composition of Paper Electrophoresis-High voltage paper complex sugars, the total reducing value of electrophoresis of sugar alcohols was performed the acid hydrolysate must be known. For this in various concentrations of borate buffer, pH purpose, complex sugars containing approxi 9.50 on Whatman No. 1 paper. mately 1-2 nanomoles of monosaccharides

Sugar alcohols (less than 10 ƒÊg) were were heated in 1 N HCl for 2 hr at 100•Ž. spotted and were located after electrophoresis The hydrolysate was freed from acid by re with periodate-benzidine reagent (9). peated evaporation with water (five times). Paper electrophoresis in pyridine-acetate The residue was dissolved in 5 pl of 0.05 N buffer, pH 5.4 (13 ), was used to separate the NaOH and mixed with 5, 10, and 20 ƒÊl of reduction product of the acid hydrolysate of [3H]-sodium borohydride solution, as in the 3•Œ-sialyllactose. standard reduction procedure. The radioac Standard Reduction Procedure for Mono tivity incorporated was measured by the simple saccharides with [3H]-Sodium Borohydride method described in the standard reduction Five nanomoles or less of monosaccharides in procedure. This preliminary experiment gives 5 ƒÊ1 of water was mixed with 20 pl of alkaline an approximate reducing value by simple cal [3H]-sodium borohydride solution. The solu culation from the specific activity of the re tion was prepared by mixing 40 pl of dimethyl agent. The specific activity can be obtained formamide solution of [3H]-sodium borohydride by determining the radioactivity incorporated (vide supra) with 1 ml of 0.05 N NaOH. The into 5 nmoles of glucose by the standard re pH of the mixture was 12.0. The reaction duction procedure. mixture was incubated at 30•Ž for 4 hr, and The optimal conditions for the hydrolysis the reaction was stopped by adding 100 ƒÊ1 of of various complex heterosaccharides into glacial acetic acid. The acidic mixture was monosaccharides could vary according to the then evaporated to dryness and the residue sample, and a time course study for each sam was dissolved in 0.5 ml of 1 M acetic acid and ple may be necessary for the accurate deter-

J. Biochem. MICRODETERMINATION OF SUGAR COMPOSITION 785

mination of monosaccharide composition by this neutral oligosaccharide fraction, the number method. The following procedures were con of N-acetylneuraminic acid moieties in the firmed to be useful for the analysis of sugar original sample could be obtained. A blank composition in milk oligosaccharides, experiment without sugar was necessary for

úA) Procedure I: The complex sugar sam estimation of the radioactivity incorporated into ple, 0.5-1.0 jug, was heated in 0.4 ml of 1.0 N the neutral oligosaccharide fraction, since the HCI for 2 hr at 100•Ž. The hydrolysate was nonvolatile radioactive contaminant in [3H]- evaporated to dryness and the residue was sodium borohydride was included in this frac freed from acid by evaporation with 0.5 ml of tion. The neutral oligosaccharide fraction can water five times. The residue was dissolved further be analyzed for its monosaccharide in 20 pl of 0.05 N NaOH and subjected to the composition by procedure I. standard reduction procedure. The labelled sample was spotted on Whatman No. 1 paper, RESULTS AND DISCUSSION and subjected to paper electrophoresis in borate Separation of Sugar Alcohols by Paper buffer. The electrophoretogram was monitored Electrophoresis-Nine sugar alcohols derived with a Packard radiochromatoscanner model from monosaccharides usually found in glyco 7201 and the radioactive region was cut out, proteins and glycolipids were subjected to paper extracted with 1 ml of water in a counting electrophoresis in various concentrations of vial and the radioactivity determined with a borate buffer, pH 9.5. As shown in Fig. 1-B, Packard liquid scintillation spectrometer model N-acetylneuraminic acid alcohol, galactitol, 3320 with 7 ml of Bray's solution (10 ). fucitol, mannitol, sorbitol, N-acetylgalacto- As will be discussed later, N-acetyl- saminitol, and N-acetylglucosaminitol were suc neuraminic acid was completely destroyed un- cessfully separated from each other when elec der these conditions and did not give any false trophoresis was carried out with 0.06 M borate radioactive peaks. buffer, 40 V/cm for 2.5 hr, Glucosaminitol and úB) Procedure II: This procedure is ap galactosaminitol could not be separated, but plicable for the determination of N-acetyl- were separated from seven other sugar alco neuraminic acid in oligosaccharides. O1igo hols under these conditions. saccharides, 0.5-1.0 ƒÊg, were dissolved in 0.2 These two amino sugar alcohols, however, ml of 0.01 N HC1 and heated for 10 min at were clearly separated when the electrophore- 100•Ž. The reaction mixture was lyophilized and subjected to the standard reduction pro cedure. The labelled sample was freed from boric acid by repeated evaporation with 2% acetic acid in methanol, and then separated into N-acetylneuraminic acid alcohol and a neutral oligosaccharide either by paper electro

phoresis in pyridine-acetate buffer, or on an ion exchange column as follows. The residue was dissolved in 0.5 ml of H20 and passed through a column (0.5 cm di

ameter) containing 3 cm of Amberlite AG-50 Fig. 1. Paper electrophoresis of sugar alcohols in (H+) in a lower layer and 3 cm of Dowex-1 various concentrations of borate buffer. Electro (acetate form) in an upper layer. The column phoresis was performed using A, 0.04 M ; B and C, was washed with water to get a neutral oligo 0.06 M ; and D, 0.2 M borate buffer. Spots repre saccharide fraction. N-Acetylneuraminic acid sent sugar alcohols visualized with periodate-benzi dine alcohol was eluted from the column with 4 ml reagent (9). 1, glucosaminitol ; 2, galactosaminitol ; r,f 0 1 M sodium acetate. 3, N-acetylgalactosaminitol ; 4, N-acetylglucosami- By dividing the radioactivity of the N- nitol ; 5, sorbitol ; 6, mannitol ; 7, fucitol ; 8, galac titol ; and 9, N-acetylneuraminic acid alcohol. acetvlneuraminic acid alcohol by that of the

Vol. 76, No. 4, 1974 786 S. TAKASAKI and A. KOBATA sis conditions were changed to 60 V/cm for 2 radation of sodium borohydride increased hr and 45 min (Fig. 1-C). sufficiently to compete with the reducing reac The concentration of borate buffer seems tion at this limited amount of reductant. to be critical for the separation of these sugar To find out if the concentration of the alcohols, since with 0.04M buffer, the sugar reactants had some effect on incorporation, the alcohols started tailing (Fig. 1-A) and with 0.2 standard reaction mixture was diluted one M buffer, N-acetylglucosaminitol and N-acetyl- hundred times by adding 2.5 ml of 0.05 M galactosaminitol or glucosaminitol and galacto- NaOH solution, and the radioactivity incor saminitol gave a single spot (Fig. 1-D). porated was measured after 2, 4, or 8 hr in Consequently, the following experiments cubation. As shown in Fig. 3 (black circles), were performed using 0.06 M borate buffer. radioactivity incorporation occurred in exactly Quantitative Reduction of Monosaccharides the same way as in the case of the standard with [3H]-Sodium Borohydride-i) Time course reduction procedure. study: To determine the time required for úB) Dose response curve by the labelling the quantitative conversion of monosaccharides method: To determine the linearity of molar into their alcohols, each monosaccharide was response by reduction, various amounts of subjected to the standard reduction procedure (see "MATERIALS AND METHODS") for 2, 4, and 8 hr and the radioactivity incorporated was measured. As shown in Fig. 2, reduction was completed within 4 hr for all nine sugars. This experiment also confirmed that the radio activities incorporated into these nine sugars were the same on a molar basis. úA) Optimal pH and effect of concentration of reactants: When the pH of the reaction mixture was reduced from that of the standard reduction procedure (pH 12.0) to 9.5, the re duction again reached a maximum at 4 hr, Fig. 3. Effect of pH and concentration of reactants but the counts incorporated in this case were on the rate and extent of radioactivity incorporation. only 70% of those obtained at pH 12.0 (Fig. 3, 0, pH 12.0 ; A , pH 9.5; and O, reaction carried out at one hundred times dilution over the standard open triangles). This may mean that the deg- reduction procedure.

Fig. 2. Reaction time and incorporation of radioactivity into each monosaccharide. •ü, glucose ; •œ, galactosamine hydrochloride ; ƒ¢, fucose ; and •£, Fig. 4. The linearity of molar response . Glucose N-acetylneuraminic acid. Other sugars (glucosamine (•ü), galactosamine hydrochloride (•œ), fucose (ƒ¢), hydrochloride, N-acetylglucosamine, N-acetylgalac- and N-acetylneuraminic acid (•£) are shown in this tosamine, mannose, and galactose) were labelled in figure. Other sugars also behaved in a similar the same manner. manner.

J. Biochem. MICRODETERMINATION OF SUGAR COMPOSITION 787 monosaccharides in 5 tel of water were sub in addition to N-acetylneuraminic acid alcohol, jected to the standard reduction procedure. As was obtained (Fig. 6-D). shown in Fig. 4, all nine monosaccharides in When 3•Œ-sialyllactose was hydrolyzed with corporated radioactivity linearly with amounts sialidase, it gave the same radioelectrophoreto up to at least 6 nmoles. gram in pyridine acetate buffer as the acid The counts incorporated for the nine mono hydrolysate (Fig. 6-B). However, in this case, saccharides were approximately 1.4•~101 dpm/ the acidic peak was composed solely of N- nmole. acetylneuraminic acid alcohol, and the minor Behaviour of N-Acetylneuraminic Acid peak was not detected in borate buffer (Fig. after Treatment under Various Acidic Condi 6-E). tions-Sialic acids are known to be modified To investigate the discrepancy between and finally degraded by heating in acidic so the results obtained under the two different lutions. hydrolysis conditions, authentic N-acetyl- Therefore, the behaviour of this sugar neuraminic acid was heated at 100•Ž in 0.01 N after acid hydrolysis of complex sugars should HCl for 50 min and lyophilized. When the be clarified before application of the analytical residue was labelled with [3H]-sodium boro method to sialic acid-containing heterosac hydride and examined by paper electrophoresis charides. in borate buffer, a radioactive peak with the N-Acetylneuraminic acid could be com same mobility as the minor peak was obtain- pletely released from 3•Œ-sialyllactose by heated (Fig. 6-F). ing in 0.01 N HCl at 100•Ž for 10 min (Fig. These observations showed that the IN- 5). acetylneuraminic acid residues in sugar chains The hydrolysate after 10 min was lyo could be completely released by acid treat philized and labelled by the standard reduction ment, but were partially modified during the procedure with [3H]-sodium borohydride. acidic treatment ; they were probably converted Examination of the reaction product by paper electrophoresis in pyridine-acetate buffer revealed two radioactive peaks ; a neutral peak and an acidic peak corresponding to N-acetylneuraminic acid alcohol (Fig. 6-A). When the acidic radioactive peak was eluted with water and examined by paper electrophoresis in borate buffer, a minor peak,

Fig. 6. Paper electrophoretic analysis of the hydrol

ysis products of 3•Œ-sialyllactose. A, products obtained by heating 3•Œ-sialyllactose in 0.01 N HCI at 100•Ž for 10 min; B, products obtained by digesting 3•Œ-sialyllactose with sialidase ; C, sialidase without 3•Œ-sialyllactose was labelled by the standard reduction pro

cedure ; D, the acidic peak in A ; E, the acidic peak in B ; and F, N-acetylneuraminic acid heated in 0.01 N HCl at 100•Ž for 50 min. Spots are standard

sugars as follows : 1, N-acetylneuraminic acid alcohol ; Fig. 5. Release of N-acetylneuraminic acid from 2, 3•Œ-sialyllactitol ; and 3, lactitol. Conditions of elec 3•Œ-sialyllactose by acid hydrolysis (in 0.01 N HCl at trophoresis were : in pyridine-acetate buffer, 74 V/cm, 100•Ž). The liberated N-acetylneuraminic acid was 1 hr for A, B, C, and in 0.06 M borate buffer, 40 determined by thiobarbituric acid assay (11) using V/cm, 2 hr for D, E, F. N-acetylneuraminic acid as a standard.

Vol. 76, No. 4, 1974 788 S. TAKASAKI and A. KOBATA

to the 42-pyrroline derivatives as proposed by small residual activity at the origin was found Gottschalk (12). after paper electrophoresis in borate buffer. As is evident from Fig. 5, the release of This result shows that sialic acid does not N-acetylneuraminic acid from 3•Œ-sialyllactose interfere with the determination of other mono on acid hydrolysis reached its maximum value saccharides by the labelling technique reported at 10 min, and then the color yield with thio here. barbituric acid decreased linearly with time of Determination of the Monosaccharide Com hydrolysis. The value at 10 min was about positions of Milk Oligosaccharides-The reso 90%0 of the amount actually present in the lution possibilities of the sugars by this meth sample. If the line after 10 min is extrapolated od are shown by the radiochromatogram in to zero time (shown by the dotted line in Fig. Fig. 7-A. 5) the value obtained corresponds to almost Four neutral milk oligosaccharides with 100% recovery. various sugar compositions were analyzed by The ƒ¢1-pyrroline derivative structurally procedure I, described in "MATERIALS AND cannot produce ƒÀ-formylpyruvic acid , which METHODS." The radiochromatogram obtained is the chromogen of the thiobarbituric acid re action, and the loss of color yield can probably be ascribed to the formation of this deriva tive. Since the radioactivity incorporated into N-acetylneuraminic acid and the ƒ¢1-pyrroline derivative was the same of an equimolar basis , and their mobilities were the same in pyridine acetate buffer, the assay method reported here may give a more accurate value than the thio barbituric acid assay. In order to determine the behaviour of N- acetylneuraminic acid after strong acid treat ment, the reaction product of N-acetylneurami Fig. 7. Radio electrophoretograms of the products obtained by the standard reduction procedure nic acid in 1 N HC1 at 100•Ž for 2 hr (general . A : mixture of glucose, galactose, mannose hydrolysis conditions for investigating the , fucose, gluco samine, galactosamine, N-acetylglucosamine, and N- monosaccharide composition of milk oligosac acetylgalactosamine ; B : acid hydrolysis products of charides) was examined by the labelling tech lacto-N-fucopentaose I; C: standard sugar alcohols; nique. No radioactive peak other than the for numbers see legend to Fig. 1.

TABLE I. Monosaccharide content of milk oligosaccharides determined by procedure I.

J. Biochem. MICRODETERMINATION OF SUGAR COMPOSITION 789 from lacto-N-fucopentaose I is shown in Fig. but sugars were isomerized at the C-2 position 7-B as a representative case. The mean values and hexosamines were partially decomposed obtained for the four oligosaccharides in five under these conditions. analyses are shown in Table I with their standard deviations. The authors gratefully acknowledge the skillful The data show that the new method re secretarial assistance of Miss Miyoko Inohara. ported here is applicable to the analysis of complex sugars. The radioactivities obtained REFERENCES in this experiment were approximately 1.2 x 105 1. C.C. Sweeley, R. Bentley, M. Makita, and W.W. dpm/nmoles. Wells, J. Am. Chem. Soc., 85, 2497 (1963). Determination can be carried out with one 2. J.H. Kim, B. Shome, T.H. Liao, and J.G. Pierce, tenth of the amount reported here. This Anal. Biochem., 20, 258 (1967). means that the sample size necessary for deter 3. Y.C. Lee, J.F. McKelvy, and D. Lang, Anal. mination can be decreased down to several Biochem., 27, 567 (1969). hundred nanograms. The experiments report 4. H.M. Howell, H.E. Conrad, and E.W. Voss, Jr., ed here were all done using [3H]-sodium boro Federation Proc., 30, 594 (1971). hydride with moderate specific activity. By 5. M.L. Wolfrom and A. Thompson, " Methods in using reductant of higher specific activity, the Carbohydrate Chemistry," ed. by R.L. Wistler, M.L. Wolfrom, J.N. Be Miller, Academic Press amount of sample necessary for determination Inc., New York, Vol. II, p. 65 (1962). of the monosaccharide composition could be 6. W.R.C. Crimmin, J. Chem. Soc., 2838 (1957). reduced even more. 7. A. Kobata, " Methods in Enzymology," ed. by Mclean et al. (14) recently reported the V. Ginsburg, Academic Press Inc., New York, use of [3H]-potassium borohydride for the Vol. XXVIII, p. 262 (1972). quantitative determination of reducing sugars 8. G.E. Delory and E.J. King, Biochem. J., 39, 245 without isolating each sugar alcohol. We also (1945). attemted to use [3H]-potassium borohydride be 9. H.T. Gordon, W. Thornburg, and L.N. Werum, cause it is easier to handle as an aqueous so Anal. Chem., 28, 849 (1956). 10. G. Bray, Anal. Biochem., 1, 279 (1960). lution than sodium borohydride. As they re 11. L. Warren, J. Biol. Chem., 234, 1971 (1959). ported, the amount of the potassium boro 12. A. Gottschalk, "The Chemistry and Biology of hydride necessary for quantitative reduction Sialic Acids and Related Substances," University of the reducing sugar was more than ten times Press, Cambridge (1960). the amount of [3H]-sodium borohydride because 13. A. Kobata and V. Ginsburg, J. Biol. Chem., 245, of the lower reactivity of the former reagent. 1484 (1970). Sugars could be quantitatively reduced 14. C. Mclean, D.A. Werner, and D. Aminoff, Anal. with 5 molar excess of [3H]-potassium boro Biochem., 55, 72 (1973). hydride in 0.05 N NaOH at 60•Ž within 4 hr,

Vol. 76, No. 4, 1974