ANALYTICAL BIOCHEMISTRY 115, 410-418 (1981) 4889

Differential Gas-Liquid Chromatography Method for Determination of Uronic Acids in Mixtures

JACOB LEHRFELD Northern Regional Research Center. Agricultural Research. Science and Education Administration. U. S. Department of Agriculture.' Peoria. Jllinois 61604 Received March 17, 1981

An integrated gas-liquid chromatography method is described for the quantitation of mix­ tures containing simple monosaccharides in addition to mannuronic, glucuronic, and/or ga­ lacturonic acids. A hydrolyzed sample is divided into two portions. One portion is analyzed by the standard aldononitrile method. Glucuronic, galacturonic, and mannuronic acids are converted into compounds that do not chromatograph in the region of the standard aldononitrile acetates. Thus, this analysis gives an accurate estimation of the neutral monosaccharide con­ tent. The second portion is analyzed by a modified alditol acetate procedure. The reduction step is repeated three times to convert mannuronic, galacturonic, and glucuronic acids to their corresponding alditols via their intermediate lactones. The results of this gas-liquid chro­ matography analysis reflect the sum of the monosaccharides present plus their corresponding uronic acids. The difference between the values obtained by the aldononitrile acetate method and the modified alditol acetate method, therefore, is a measure of the uronic acid(s) present. Heteropolysaccharides containing only heating time must be carefully controlled, neutral sugars are readily analyzed by the as well as the temperature of the reaction alditol acetate (1-4) or the aldononitrile ac­ when the sulfuric acid is added. The presence etate (5-9) procedures. However, the pres­ of other neutral sugars requires the addition ence of uronic acids complicates the analysis. of a correction factor that will vary with the Out of necessity, a supplemental spectro­ amount of sugar present. Additionally, the photometric (4) reaction generally is run on ability to differentiate between the various the mixture to determine how much uronic uronic acids in mixtures is limited (13). acid is present. For example, uronic acids, Other more specific methods have been when treated with sulfuric acid and either reported. For example, Spiro (14) reported carbazole (10,11) or harmine (12), develop that an Aminex A-25 column can separate a color quantitatively. However, if the so­ a number of uronic acids. Quantitation is lution is colored to start with, the analysis accomplished by coupling the column to a is difficult or impossible to perform. Technicon sugar analyzer using the orcinol Furthermore, the carbazole analysis is reagent. Enzymic methods are also avail­ very sensitive to slight procedural changes able. For example, D-galacturonic acid (IS) (13). For example, small amounts of im­ can readily be analyzed. These supplemen­ purities in the sulfuric acid, carbazole, or tary methods require additional equipment absolute ethanol can markedly change the and/or specialty reagents. results. The temperature of the bath and In contrast, the differential method re­ ported here uses the same basic methodol­ , The mention of firm names or trade products does ogy, reagents, and equipment used for not imply that they are endorsed by the U. S. Depart­ standard gas-liquid chromatography (glc)2 ment of Agriculture over other firms or similar products not mentioned. 2 Abbreviation used: glc, gas-liquid chromatography.

0003-2697/81/120410-09502.00/0 410 Purchased by U.S. Dept. of Agriculture for Official Use. Copyright 1981 by Academic Press. Inc. All rights of reproduction in any form reserved. URONIC ACID DETERMINATION IN CARBOHYDRATE MIXTURES 411 analysis of neutral sugars by alditol acetate periods of time.) The solution was heated at and aldononitrile acetate procedures. In ad­ 100°C for I h and then cooled. One-half dition, the uronic acids can readily be dif­ milliliter of acetic anhydride (0.5 ml) was ferentiated be they glucuronic, galacturonic, added and the solution was reheated at or mannuronic. 100°C for I h. The resultant solution was satisfactory for glc analysis. A word of cau­ MATERIALS AND METHODS tion is needed: Hydroxylamine has been shown to be a mutagen and, as such, should Materials. Extracellular polysaccharides, be handled with caution (16). NRRL B-1973 and NRRL B-1459, were a Preparation ofalditol acetate. A standard gift from C. A. Knutson of the Northern containing up to 30 mg of carbohydrate (in Regional Research Center. Carbohydrate a 16 X 125-mm Teflon-lined screw-capped standards D-galactose, D-, D-man­ tube) was dissolved in a solution of 25 mg nose, D-ribose, D-arabinose, D-xylose, ga­ sodium borohydride in 1 ml water. The so­ lactitol, D-glucitol, D-mannitol, ribitol, D-ar­ lution was kept at room temperature for 1.5 abinitol, xylitol, D-glucono-I,5-lactone, D­ h. Sufficient 10% acetic acid in water was glucurono-6,3-lactone, D-galactopyranuronic added dropwise (10-20 drops) until bub­ acid monohydrate, and sodium D-gluconate bling stopped. The solution was percolated were obtained from Pfanstiehl Laboratories through a column made from a Pasteur pipet Inc. Potassium D-gluconate, D-mannuronic and I to 1.5 ml of a cation-exchange resin. acid-6,3-lactone, L-mannonic acid-I A-lac­ The resin was washed with 4-6 ml of deion­ tone, and D-galactonic acid-I A-lactone were ized water and the combined eluate and obtained from Sigma Chemical Company. washings were evaporated to dryness. Meth­ Sodium borohydrate was obtained from anol (4 ml) containing HCI (0.1%) was Ventron Company. Barium carbonate, silver added to the residue and the solution was carbonate, hydroxylamine hydrochloride, shaken while being heated at 50-55°C for pyridine, and acetic anhydride were obtained about 5 min and then was flash evaporated. from Fisher Scientific. The cation-exchange This procedure was repeated twice. A Buch­ resin was AG 50W-X8, 200-400 mesh, H+ ler Vortex evaporator can rapidly and ex­ from Bio-Rad Laboratories. The X8 is a peditiously handle up to 72 samples. The better choice than the X4 because it has a reduction and deionization were repeated higher capacity, i.e., 1.7 mEq/ml vs 1.2 twice. mEq/ml. The final residue was dissolved in 0.5 ml HI-EFF 3BP (neopentyl glycol succinate) pyridine and 0.5 ml acetic anhydride and on Gas-Chrom Q, 100-120 mesh, and JXR heated at 100°C for I h. This reaction mix­ (methyl silicone) on Gas Chrom Q, 100-120 ture is suitable for glc analysis without fur­ mesh, were obtained from Applied Science ther treatment. Inc., and SP 2340 (Cyano-silicon) on Su­ glc analysis. A Packard Instrument Model pelcoport, 100-120 mesh, was obtained from 428 gas chromatographic unit equipped with Supelco Inc. dual F.LD. and dual electrometers was out­ Preparation of aldononilrile acetates. A fitted with two glass columns 2 mm X 2 m. standard containing up to 30 mg of carbo­ Column A was used to separate the aldon­ hydrate (in a 16 X 125-mm Teflon-lined onitrile acetate and contained 3% HI-EFF screw-capped tube) was dissolved in 0.5 ml 3BP on Gas-Chrom Q, 100-120 mesh. Col­ pyridine containing 30 mg hydroxylamine umn B was used to separate the alditol ac­ hydrochloride. (Solutions containing 60 mg etates and contained a I: I mixture of 3% hydroxylamine hydrochloride/ml pyridine HI-EFF 3BP on Gas-Chrom Q, 100-120 can be prepared readily and stored for long mesh, and 3% SP 2340 on Supelcoport, 100- 412 JACOB LEHRFELD

120 mesh. A helium flow rate of 25 milmin been used for over a year under the condi­ was maintained. Temperature programming tions described under Materials and Meth­ was employed for maximum resolution in ods with little loss in resolution. minimum analysis time. For simple mixtures The aldononitrile acetate method for neu­ of , an initial temperature of 210°C tral sugars is simple. The sugars are dis­ was maintained for 3 min and then the tem­ solved in pyridine containing hydroxylamine perature was programmed upward at 2°C hydrochloride. Solutions more than a year per minute to 234°C. A complete analysis old have been used with no problem. The took 10-12 min. Complex mixtures were in­ solution is heated (IOO°C) for a short time jected into the column kept at an initial tem­ (1 h) to convert the sugars to their corre­ perature of 184°C for 3 min and pro­ sponding oximes. Then, acetic anhydride is grammed upward at 2°C/min to 234°C. added and the oxime is dehydrated to the Trimethylsilylated lactones were analyzed corresponding nitrile. Actually, an initial on a column of 3% JXR on 100- to 120-mesh heating period of 1-2 min is all that is re­ Gas-Chrom Q 2 mm X 2.5 M. This column quired. For example, glucose is completely was kept at 184°C for 5 min and then pro­ converted into its oxime within 1 min. Pyr­ grammed up to 204°C at 2° Imin. idine solution containing xylitol, glucose, and hydroxylamine hydrochloride was heated RESULTS AND DISCUSSION at 100°C (Fig. 1). Samples removed after lOs and after 1 min were treated with an Accurate and meaningful component excess of acetic anhydride. Any D-glucose analysis of a polysaccharide presupposes ho­ that was not converted into its oxime ap­ mogeneity of the sample and a hydrolysis peared as glucose pentaacetate; any oxime technique (4,17,18) that results in complete present at the time acetic anhydride was breakdown of the polymer and, at the same added was converted into the corresponding time, minimal degradation of the compo­ D-glucononitrile. After lOs, 40% of the glu- nents. Once this is accomplished, the sample then can be treated as described to obtain useful component composition data. Simple compositional data on neutral sug­ ars are readily obtained by the alditol ace­ tate or the aldononitrile acetate methods. Aldononitrile acetate method. Per-O-ace­ tyl aldononitriles are well-characterized, sta­ ble, sugar derivatives that have been known for over 70 years (19). Lance and Jones (20), who were the first to suggest that these de­ rivatives be used for the characterization of methylated , readily distin­ guished 2-0-methyl xylose from 4-0-methyl FIG. I. A pair of glc chromatograms that show glucose xylose. Easterwood and Huff (5) suggested oxime formation is essentially complete after I min. that the method be used to characterize the Samples were removed after 1a s and I min. The re­ sugars in polysaccharides. 1n 1971, Dimi­ action with hydroxylamine at I aaoc was stopped by triev et al. (6) demonstrated the suitability adding a large excess of acetic anhydride. The large of the neopentyl glycol succinate (NGS) col­ initial peak is xylitol acetate (I) an internal standard. the doublet is a.,S-glucose pentaacetate (2.3) and the umn for separation of the aldononitrile ac­ final peak is the aldononitrile acetate of glucose (4). At etate derivatives. This is still one of the best I min the only peaks showing are the xylitol acetate and glc columns. It is remarkably stable and has the aldononitrile of glucose. URONIC ACID DETERMINATION IN CARBOHYDRATE MIXTURES 413 cose had already been converted into its ox­ 123456 ime. After 1 min, all the glucose had been 3% HI·EFF 3BP 6 It converted into the oxime and no glucose pen­ 210° 1 min 2° /min to 230°C taacetate could be detected. Peak identifi­ cation was confirmed by spiking with known standard. Even so, a l-h heating period is recommended to accommodate any unusual sugars and to completely eliminate uronic and aldonic acids that might be present. The solutions are usable without further treatment. If the sample size is small, or if it contains short-chain sugars, it is advisable ~~ to remove the pyridine by a water-methy­ : G,I"to"""","",, ,," lene chloride partition or by evaporation to C Y·Mannonolactone ------A..... 0 D·Glucuronolaclone minimize interference by the tailing solvent peak. The samples are remarkably stable. A B 12 16 20 variation of ± 3% occurred in a sample re­ Time, min peatedly run during a 5-month period. How­ FIG. 2. A series of chromatographic runs demonstrate ever, if the sample was partitioned between insignificant peaks from uronic acids following their treatment with hydroxylamine and acetic anhydride. water and methylene chloride, it degraded Chromatograph (A) a standard carbohydrate mixture within a few days due to residual water in containing the aldononitrile acetates of arabinose (I). the methylene chloride solution. The sample xylose (2). mannose (3), glucose (4), galactose (5), and generally can be returned to its former value inositol (6); (B) galactopyranuronic acid; (C) mannon­ by treatment with acetic anhydride. A major olactone; (D) glucuronolaclone. advantage over the alditol acetate method is that the basic symmetry of the sugar is check the precision and accuracy, eight stan­ maintained. dard samples were prepared and run as de­ Uronic and aldonic acids do not interfere scribed under Materials and Methods. There with the assay. They either are destroyed or is an average overall margin of error of are converted into other compounds that do ± 2.7%. Xylitol also can be used as an in­ not cochromatograph on this column with ternal standard. It elutes right after the al­ the simple neutral sugars. Some typical ex­ dononitrile acetate of xylose. Its response amples are depicted in Fig. 2. Equal weights factor is 1.00 relative to inositol. Sugars of sugars and acid derivatives were used. The which form the nitrile functional group, largest pen excursion on the acid chromato­ however, have a response factor of about grams represented less than 0.3% the size of 0.57-0.69. a comparable neutral sugar peak and oc­ Alditol acetate method. The alditol ace­ curred near the xylose peak. In similar runs tate method for neutral sugars, even though made with mannuronolactone, glucononlac­ it requires a number of separate operations, tone, and galactonolactone the chromato­ is straightforward and simple. The sample gram showed no significant peaks. is reduced by dissolving it in an aqueous so­ A typical chromatogram on column A is lution containing sodium borohydride (21). shown in Fig. 3. Xylitol and inositol acetates The reduction is complete after 1-2 h at serve as internal standards. The chromato­ room temperature (22). If the borate is not graphic run can be reduced by one-third to removed, the subsequent acetylation pro­ about 10 min, by starting at 210°C with lit­ ceeds with difficulty. The residue then is tle loss in resolution. The column is efficient treated with pyridine-acetic anhydride to and has about 6000 theoretical plates. To form the acetate and is suitable for analysis 414 JACOB LEHRFELO by glc. A mixture of arabitol, xylitol, man­ nitol, galactitol, glucitol, inositol, and the 1 2 3 4 5 6 7 internal standard, methyl a-o-glucopyrano­ 3% HI·EFF 3BP 3% SP 2340 side tetraacetate, were dissolved in pyridine 190 0 3 min and treated with acetic anhydride. These 2' Imin to 230 c C common alditols are acetylated completely after 20 min. A typical chromatographic run is depicted in (Fig. 4). A sample run is com­ pleted in about 14 min. A comparable sep­ aration can be obtained with 3% SP2340 and 3% Silar 10C. The mixed SP2340jHI-EFF 3BP column gives a shorter total run time and an improved separation between ribitol and arabitol. Some internal standards that have been used in the alditol acetate pro­ ~ cedure are arabinose (23), 2-deoxy-o-glu­ '---- cose (1), rhamnose (24), and myo-inosi­ 4 6 10 12 Time, min. tol (25). To check the precision and accuracy of FIG. 4. The glc separation of some alditol acetates. this method, eight standard samples were The order of elution is arabitol (I), xylitol (2). methyl ,B-D-g]ucopyranoside (3). mannitol (4). galactitol (5). prepared and run as described under Ma­ glucitol (6), and inositol (7). terials and Methods. The standard deviation

is ± 1.5%. Variations as large as ± 3%, how­ 3% HI-EFF 3BP 6 It. ever, have been encountered. The response 190° 3 min factor for all the samples varied from 0.97 2° /min to 230°C to 1.0. Uronic acids in the a/dito! acetate method. 2 4 Uronic acids interfere with the alditol method 3 because the lactones that can form are readily reducible by sodium borohydride. The problem is especially acute with gluc­ 6 7 uronic acid and mannuronic acids, which readily form their corresponding lactones. Galacturonic acid, on the other hand, does not readily form a lactone. Consequently, values obtained by the alditol acetate method are inflated when glucuronic or mannuronic acids are present and are not appreciably inflated in the presence of galacturonic acid. ~, The extent of lactonization and the degree '---...... J v v "--- II of reduction are not reproducible (26,27). 0 2 4 6 8 10 12 14 Therefore, Sloneker (I) recommended that Time, min the sample be treated with 0.01 M sodium carbonate to convert the lactone into its cor­ FIG. 3, The glc separation of some sugar aldononitrile acetates at 190°C 230°C order of their elution time: responding sodium carboxylate salt, which ribose (]). arabinose (2). xylose (3). xylitol (4), mannose is not reducible by sodium borohydride; how­ (5). glucose (6). galactose (7). and inositol (8). ever, this does not completely resolve the URONIC ACID DETERMINATION IN CARBOHYDRATE MIXTURES 415

problem. If appreciable amounts of some ductions with sodium borohydride. Under uronic acids are present during the sample the experimental conditions cited under the preparation, the excess acids are converted Materials and Methods section, i.e., three ultimately into aldonolactones. Aldonolac­ reductions with borohydride essentially all tones, when treated with acetic anhydride, of the aldonic and uronic acids lactones and can form dehydrated products (28) that will salts are completely reduced to their corre­ chromatograph in the region of the alditol sponding alditols. The following experiment acetates (Fig. 5). was performed to confirm the need for three Jones and Albersheim (3), using different reductions. experimental conditions, reported the quan­ Samples containing potassium glucuron­ titative conversion of some uronic acids to ate, glucurono-6,3-lactone, mannurono-6,3­ alditols. This result is in keeping with the lactone, galacturonic acid, galactono- I,4­ observation of Sjorstrom et al. (29) that lactone, glucono-l,5-lactone, mannono- 1,4­ some lactones can be quanti­ lactone, and xylitol (as an internal standard) tatively reduced to the alditol by three re- were reduced. After each reduction, a sam­ ple was removed and analyzed by gic using

1 the aldononitrile method, which precludes 2 any errors due to dehydration products. In Column 8 general, a single reduction converts the lac­ 2000 3 min tones to the alditol in around 90-98% yield. 2°/min to 234 0 Potassium glucuronate and galacturonic acid are not in the lactone form and, conse­ quently, are reduced only to the aldonic acid. The amount of alditol formed is minimal, i.e., about I% or less. During the workup for the subsequent reductions, the aldonic acids are converted into the corresponding aldon­ olactones and readily reduced to the aiditoi. After three reductions, all the samples were converted to their corresponding alditols in 97-102% yield. The standard lactones were checked for purity by converting them to the trimeth­

Glucono·1,5·lactone ylsilyl derivatives and chromatographing Sodium gluconate them as described on the JXR column. Each Mannono·1,4·lactone lactone gave only one peak. E Galactono·1,4·lactone Differential methods applied to syn­ thetic mixtures and natural polysaccha­ 10 15 20 Time, min. rides. Standard solutions containing glucose, galactose, and mannose were prepared and FIG. 5. A series of chromatographic runs demonstrat­ analyzed by the standard aldononitrile and ing the presence of peaks from some aldonolactones fol­ lowing their treatment with acetic anhydride in pyridine. alditol acetate procedures. In a series of par­ Chromatographs (A) is a standard carbohydrate mix­ allel experiments, the standard sugar solu­ ture containing. in order of elution: ribitol (I). arabitol tions were spiked with equimolar amounts (2). xylitol (3). mannitol (5). galactitol (6). glucitol (7). of uronic acids. The alditol acetate method, and inositol (8): (B) glucono-1.5-lactone: peak 4 is de­ employing multiple borohydride reductions, hydrated carbohydrate from glucono-1.5-lactone: (C) sodium gluconate: (D) mannono-1.4-lactone: (E) gal­ consistently gave additive results, whereas actono-IA-Iactone. the data from the aldonitrile method gave .j::.. -0\ TABLE I

EFFECT OF ADDED GI.UCURONO-6,3-I.ACTONE AND GALACTURONIC ACID ON QUANTITATION OF GLUCOSE AND GALACTOSE

Glucose Galactose Glucose G lueurono-6,J-Iaetone Galactose Galaeturonie Sample added added Found" Foundb added acid added Found" Foundb

A 2.1 2.1 2.0 4.1 2.1 2.1 2.3 2.1 B 4.0 4.0 4.1 7.9 4.0 4.0 4.0 3.8 C 5.0 5.0 5.0 9.8 5.0 5.0 5.2 5.0 D 8.0 8.0 7.9 15.7 8.0 8.0 7.9 8.2 E 10.1 10.1 10.0 20.1 10.0 10.1 9.8 10.0

" Aldononitrile procedure. '- b Alditol acetate procedure using one reduction with borohydride. );> () o CO r rn ::c ;:0 .."rn TABLE 2 r o RELATIVE RATIO OF THE COMPONENTS IN POLYSACCHARIDES B-1973 AND B-1459 DERIVED BY THREE DIFFERENT METHODS

Polysaccharide from Arlhrobacter viscosus B-1973 method Polysaccharide from Xalll!lo/l/O/l{lS ca/l/peslris B-1459 method

Alditol Aldononitrile Alditol Aldononitrile Carbohydrate Theoretical acetate acetate Differential Theoretical acetate acetate Differential

Galactose 1.00 1.15 1.02 1.02 Glucose 1.00 1.09 1.00 1.00 2.0 3.06 2.07 2.07 Mannose 1.00 2.0 2.0 2.00 2.00 0.09 1.0 0.99 Mannuronic acid 1.00 1.00 Galaeturonic acid 0.13 URONIC ACID DETERMINATION IN CARBOHYDRATE MIXTURES 417 the same results from both the spiked and hydrolyzed by the method of Saeman (32) unspiked solutions. Table I summarizes data while polysaccharide NRRL B- I973 was from one such experiment. Xylitol was added hydrolyzed by the method of Sioneker (3 I). as an internal standard to each of the five The differential method gives for Poly­ samples (A-E). Each sample contained in­ saccharide NRRL B-1973 a lowered man­ creasing amounts of glucose and galactose nuronic acid figure (1.0 vs I.I4). This is in in addition to equimolar amounts of glucu­ better agreement with the theoretical ratios. rono-6,3-lactone and galacturonic acid. The The figures for xanthan are well within the solutions were reduced one time only with reported ranges. Values for a commercial a large excess of sodium borohydride to con­ sample varied by a factor of 15%. The whole trast the results obtained with a lactone and cells and cell debris commonly found in com­ an acid after one reduction and after three mercial zanthan gum samples probably ac­ reductions. Reduction of the lactone to glu­ count for this discrepancy. citol increased the glucitol found by a factor A differential method for the estimation of 2. In contrast, galacturonic acid was re­ of neutral and acidic sugars has been de­ duced by a single reduction only to the al­ scribed. The aldononitrile procedure gives an donic acid. Galactonic acid is essentially de­ accurate estimate of the neutral sugars only stroyed by acetic anhydride and pyridine, because the acidic sugars are destroyed (Fig. thus no phantom peaks appear. When two 2). The alditol acetate procedure accurately additional reductions were performed, the quantitates the sum of the neutral sugars and results with galacturonic acid were the same the acidic sugars. The alditol acetate method as with the lactone. can quantitate both neutral and acidic sug­ Table 2 represents the results obtained ars because the multiple reductions convert from two natural polysaccharides utilizing even those nonlactone-forming uronic acids the procedure outlined under Materials and into aldonic acids that, in turn, readily form Methods. The hydrolysate chromatograms lactones and/or esters during the removal of B- 1459 and B-1973 readily point out the of borate by methanol. These lactones or presence of the uronic acids. For example, esters are then converted to alditols by sub­ the hydrolysate from B- I459 treated to form sequent borohydride reductions. Conse­ the aldononitrile acetates shows only two quently, the difference between the two peaks, 5 and 6 (Fig. 3), in equal proportions. methods gives an estimate of the acidic sug­ However, when the hydrolysate is treated to ars present. form the alditol acetate derivatives, peaks 4 and 6 (Fig. 4) appear in the ratio of 2:3. REFERENCES The hydrolysate from B-1973, when treated I. Sioneker. J. H. (1972) Melhods Carbohydr. Chem. to form the aldononitrile acetates, shows two 6, 20-24. peaks, 6 and 7 (Fig. 3), in equal portions. 2. Albersheim. Poo Nevins. D. J., English, P. D., and However, when the hydrolysate is treated to Karr, A. (1967) Carbohydr. Res. 5, 340-345. form the alditol acetate derivatives, three 3. Jones. T. M., and Albersheim, P. (1972) Plam peaks, 4, 5, and 6 (Fig. 4) appear in equal Physiol. 49, 926-936. 4. Selvendran, R. Roo March, J. F., and Ring, S. G. proportions. The polysaccharide from Xan­ (1979) Anal. Biochem. 96, 282-292. thomonas campestris NRRL B-1459 (30) 5. Easterwood. Y. M., and Huff, B. J. L (1969) Sven. contains a repeating unit of 2-g1ucose, 2­ Papperslidn. 72, 768-772. mannose, and I-glucuronic acid; polysac­ 6. Dmitriev. B. A., Backinowsky, L Y., Chizhov, charide from Arthrobacter viscosus NRRL O. Soo Zolotarev, B. M., and Kochetkov, N. K. (1971) Carbohydr. Res. 19,432-435. B- I973 contains a repeating unit of I-ga­ 7. Seymour, F. R., Chen. E. C. M., and Bishop, S. H. lactose, I-glucose, and I-mannuronic acid (1979) Carbohydr. Res. 73, 19-45. (3 I). Polysaccharid~ NRRL B- I459 was 8. Mawhinney, T. P., Feather, M.S., Barbro, G. J., 418 JACOB LEHRFELD

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