Quantitative determination of glucosamine by column chromatography and radioisotope dilution

AZVALYTICAL

BIOCHEMISTRY

Quantitative

39,

251-257

Determination

Chromatography PHILIP University

(1971)

of

of and

Glucosamine

Radioisotope

by

Column

Dilution’

C. KELLEHER, CHARLES B. HOWARD,’ AND CAROL J. S,\IITH Vermont

College

of Medicine,

Received

May

Burlington,

Vermont

05401

26, 1970

The most commonly employed methods of hexosamine determination are based on the method of Elaon and Morgan (1)) or one of its subsequent modifications (2-7). There are a number of problems in interpreting the results obtained by this method when it is used on proteins (8), on polysaccharides (9,10), or on fractions isolated from protein hydrolyzates (11). Recently, a number of reports appeared in which other methods of analysis of protein-bound hexosamines are described, including gas chromatography (12), ion-exchange chromatography (13), paper electrophoresis and radioisotope dilution (14). This report describes a quantitative method for glucosamine determination based on radioisotope dilution and the column chromatographic procedure for isolation of hexosamines and basic amino acids as described by Kelleher and Smith (15). Evidence is presented that variations in the percentage of radioactively labeled glucosamine degraded during acid hydrolysis of glycoproteins do not affect the results obtained with this method. MATERIALS D- (‘“C,) -Glucosamine (35 &i/mg, New England Nuclear Corporation, Boston, Mass.). D-Glucosamine, n-galact’osamine, n-mannosamine, L-leucine, n-histidine, and L-phenylalanine (Sigma Chemical Co., St. Louis, Missouri). Dowex 50W X8 resin, 20&400 mesh (Sigma Chemical Co.). 2,4,6-Trinitrobenzene-l-sulfonic acid (Eastman Organic Chemicals, Rochester, N. Y., or Sigma Chemical Company). 2,5-Diphenyloxazole (PPO, New England Nuclear Corp.) and 1,4bis[2- (4-methyl-5-phenyloxazolyl) ] benzene (POPOP, New England Nuclear Corp.). ‘Supported ‘Present

by USPHS Grant HD-034S3. address: Walter Reed Army Medical

251

Center,

Washington,

D.

C.

252

EELLEHEFt,

HOWARD,

AND

SMITH

The liquid scintillation mixture was prepared with analytical-grade methyl cellosolve and toluene purchased from Fisher Scientific Co. (Boston, Mass.). Analytical-grade reagents were used in the preparation of the buffers according to the method of Moore and Stein for the chromatography of basic amino acids (16). Samples of hospitalized patients’ plasma were obtained from the clinical laboratory of the Medical Center Hospital of Vermont and samples of normal male human plasma from the Metabolic Unit of the University of Vermont College of Medicine. PROCEDURE

The plasmas were stored at -20” until analyzed. 1 ml aliquots of plasma were mixed with 3 ml 3 N HCI and either 10,500 cpm or 118,125 cpm of D-(14Cs)-glucosamine and heated at 100” for 4 hr in a paraffin bath, conditions found by Winzler (5) and us (17) t,o be adequate for the complete release of glucosamine from plasma glycoproteins. In one set of determinations, 5,309 cpm of D-(l%s)-glucosamine was added to 0.5 ml of plasma. Following digestion, the hydrolyzates were dried in VCLCUO in a desiccator over anhydrous calcium chloride. Ion-exchange chromatography was performed as previously described (15). Synt,hetic mixtures containing L-leucine, L-phenylalanine, L-histidine, n-glucosamine and n-galactosamine or n-mannosamine, each at a concentration of 1 mg/ml, were prepared and 10,500 cpm of D-(I~C&) glucosamine was added. These mixtures were used to test the performance of the columns and the homogeneity of the D-(14Cs)-glucosamine and to determine if the specific activity of the isolated glucosamine peak agreed with the value expected based on the composition of the initial mixture. The dried plasma protein hydrolyzates were dissolved in 2 ml of 0.1 M citrate buffer, pH 5.0, and 1.0 ml of this solution or 0.5 ml of the synthetic mixture was placed on the 1 X 15 cm Dowex 50W X8 column. Column fractions were assayed by the trinitrobenzene sulfonic acid reaction (18) ; the glucosamine was identified by its elution position and the ratio of absorbances at 355 rnp and 475 mp. The amount of glucosamine was determined in each fraction and t’hen the radioactivity present in 0.5 ml aliquots of that portion of the column eluate containing glucosamine was counted using the liquid scintillation mixture described by Prockop and Ebert (19) and a Packard Tri-Carb liquid scintillation spectrometer. The column fractions collected before and after the elution of glucosamine and during washing of the column with sodium hydroxide were pooled separately and the radioactivity present in 0.5 ml aliquots was measured. The specific activity (cpm/pg) of the glucosamine was calculated and

DETERMINATION

OF

GLUCOSAMINE

253

the plasma concentration of glucosamine derived by isotope dilution. The plasma glucosamine concentration was measured on three separate samples from each subject. Statistical analysis was performed using standard methods with p < 0.05 as the level of significance. The significance of differences between glucosamine levels in t’he normal subjects and patients was determined by analysis of variance. Linear regression analysis was employed to test for significance of the relationship between the amount of radioactively labeled glucosamine altered during hydrolysis and the calculated serum protein-bound glucosamine concentration. RESULTS

Chromatographic analysis of the synthetic mixture revealed that glucosamine was well separated from leucine, phenylalanine, histidine, and galactosamine as previously reported (15). Mannosamine was eluted at the same position as galactosamine. Recovery of glucosamine added to the column was 100.276, 102.9% and 102.8% in 3 experiments. The D-(14C6) -glucosamine used in this study was eluted symmetrically with the chemically determined glucosamine peak and the specific activity of the glucosamine was constant throughout the column fractions in which glucosamine was present. Over 98% of the radioactivity recovered in the column eluate was present in the glucosamine peak. The remaining radioactivity was evenly distributed throughout the elution pattern and less than 1% of the radioactivity was present in the final sodium hydroxide wash. The specific activity (cpm/pg) of the glucosamine isolated from the synthetic mixture was within 5% of the expected value in duplicate determinations. Human plasma protein-bound glucosamine levels as determined by this method are summarized in Table 1. The specific activities of the glucosamine were determined in the 3 peak fractions and these values were used to calculate the plasma glucosamine concentrations. The variance in glucosamine concentrations among the subjects, considered as a whole, was 25 times greater (p < 0.001) than the variance among replicate determinations on each subject. The difference between levels in the normal group and in the patient group was not statistically significant; however, the mean glucosamine concentration of the hospitalized patients was higher than that of the normal male group. These results agree with the serum glucosamine concentrations determined by the Elson-Morgan reaction (5,20). The purity of the eluted plasma glucosamine was indicated by the constant specific activity (cpm/mg) of the glucosamine present in consecutive column fractions collected within individual plasma glucosamine peaks. The same glucosamine concentration was found in three

254

KELLEHER,

HOWARD,

AND

TABLE; 1 Plasma Protein-Bound Glucosamine Concentrations Hospitalized Patients Determined by Column Dilution Method Subject No.

Sample 1

NORMAL

SMITH

in Normal Male Subjects Chromatographic-Isotope

number 2

GROUP:

Individual average

3

glucosamine

(mg/lOO

ml)

96 92 119 10s 94

91 99 100 127 110 97

91 96 98 121 106 92

88

87

85 98

91

92

92 101 117 100 84 80

Mean 8.1). S.E. of Mean PATIENT

GROITP:

glucosamine

and

(mg/lOO

Zkll.9

+2.7 ml)

98

102

114

105

74 175 112 171 141

7s 183 127 153 135

73 153 124 134 133

75 170 121 153 136 127 f34.4

Mean S.D. S.E. of Mean

IkS.1

plasma samples from a single individual using either 5,300 cpm and 0.5 ml pla:ma, or 118, 125 cpm and 1.0 ml plasma, indicating that the results of this method are not affected by a lo-fold difference in D- (‘“C,) glucosamine to plasma volume ratio. Figure 1 illustrates the relationship between the amount of D-(14C6)glucosaminc that was degraded and the measured plasma glucosamine concentration. The percentage of the added D- (“C,) -glucosamine altered sufficiently during acid hydrolysis and evaporation to dryness so that it was not eluted with the glucosamine peak during chromatography was variable. Preferential loss either of the added radioactively labeled glucosamine or of the protein-bound glucosamine would yield falsely high or falsely low values, respectively, for the plasma protein-bound glucosamine levels. Our results do not reveal if equal percentages of added D- (W,) -glucosamine and protein-bound glucosamine are degraded but the results can be analyzed to show if variable alteration of the added D- (14C6)-glucosamine correlates with the differences in measured plasma glucosamine.

DETERMINATION

GLUCOSAMINE

OF GLUCOSAMINE

CONCENTRATION

255

(mg/lOOml)

FIG. 1. Correlation of calculated plasma glucosamine concentration versus amount of n-(“CQ-glucosamine degraded during acid hydrolysis of plasma proteins. 0 150 nanograms of n-(lCe)-glucosamine was added to each plasma sample prior to hydrolysis. The amount of n-(“G)-glucosamine degraded was determined from the total amount of radioactivity present in the Dowex 50W X8 column eluate outside of the glucosamine-containing fractions. Line 1, least-squares regression line of calculated glucosamine concentration on amount of n-(“G)-glucosamine degraded. L&e 2, least-squares regression line of amount of n-(“CJ-glucosamine degraded on calculated glucosamine concentration.

Model II linear regression analysis showed no statistically significant correlation (r = -0.2214, p > 0.1) between the amount of D-(‘~C&)glucosamine altered and the calculated plasma glucosamine levels. The coefficient of determination (r2 = 0.0490) indicated that less than 5% of the observed variation in plasma glucosamine concentrations was explained by differences in the amount of D- (Ws)-glucosamine altered. D- (‘“C,) -Glucosamine added after hydrolysis but before evaporation of the HCl showed no chromatographic evidence of degradation. Recovery of the radioact’ivity added to the Dowex 5OW X8 columns was between 78 and 93% in the 15 plasma samples from 5 individuals. Radioactivity counting of the resin present in the sample application

256

KELLEHER,

HOWARD,

AND

zone showed that no detectable radioactivity after the sodium hydroxide wash.

SMITH

remained

on the column

DISCUSSION

The number of modifications that have been made in the ElsonMorgan method for hexosamine determination indicate that there is no completely satisfactory solution to the problems inherent in this method, and for this reason other methods for t’he analysis of hexosamines have been developed. Except when measuring free hexosamines, these methods involve hydrolysis of the hexosamine-containing compound, with subsequent measurement of the liberated hexosamine. Radioisotope dilution offers a convenient way of measuring the amount of a material present in mixtures such as biological fluids or incorporated into high molecular weight compounds since it allows correction for losses or chemical alteration during the isolation procedure. In order for isotope dilution methods to yield satisfactory quantitative results, the fractional loss of the isotopically labeled material must be the same as the material present in the biological sample. This requirement is fulfilled when both the added radioactively labeled substance and the substance that is being measured arc both present in the same physicochemical state. When t’he substance to be measured is incorporated into a macromolecule, such as glucosamine into glycoproteins, selective losses of either the isotopically labeled tracer or of material initially present might occur under conditions used to release the bound substance. Our results indicate that the percentage of added D- (14C6)glucosamine that was destroyed during hydrolysis of the plasma proteins was variable, but that’ there was no correlation between the amount of radioactively labeled glucosamine altered and the measured amount’ of plasma glucosamine. This indicat’es that the ratio of D-(I%~) -glucosamine to protein-bound glucosamine altered during the procedure remained constant with a limited range. The constancy of the ratio of D- (“CL)glucosamine: protein-bound glucosamine altered is further supported by the fact that a lo-fold variation in the ratio of the ~-(~~Gj-glucosamine: protein-bound glucosamine did not affect the calculated plasma glucosamine concentration. The agreement of our calculated glucosamine level with those measured by other methods suggest t’hat any differences in the percentage of the radioactively labeled glucosamine and proteinbound glucosamine altered are small. This method permits measurement of glucosamine present in biological compounds without interference by other chromogens and allows correction for degradation of glucosamine during hydrolysis. The hydrolysis procedure used in this study has proved satisfactory for plasma glyco-

DETERMINATION

OF

257

GLUCOSAMINE

proteins, but optimal hydrolysis conditions would have to be determined for each type of material. Galactosamine, the other hesosamine found in vertebrates, may also be det,ermined by this method since it is well separated from glucosamine and amino acids by the chromat’ographic procedure used (15). SUMMARY

A specific method for the quantitative determination of proteinbound glucosamine using radioisotope dilution and ion-exchange chromatography is described. Plasma glucosamine levels of a group of normal male humans and hospitalized patients arc reported. Evidence is presented that variable preferential destruction of either the added isotopically labeled glucosamine or the protein-bound glucosamine is not’ a significant factor in the results obtained. ACKNOWLEDGMENTS We wish to thank Dr. H. methods used in this report assistance.

I,. McCrorey and Mrs.

for Edith

his atlvicc regarding the Howard for her expert

statistical technical

REFERENCES

1. ELSON, L. A., AND MORGAN, W. 2. BLIX, G., Acta Chem. Scund. 2, 3. BOAS, N. F., J. Biol. Chem. 204, 4. R~NDLE, C. J. M.. AND Mo~c.4~. 5. WINZLER, R. J., in “Methods of 6. 7. S.

9. 10. 11. 12.

13.

T. J., Biochem. J. 27, 467 (1948). 553 (1953). W. T. J.. Biochem. J. Biochemical Analysis” pp. 292-294. Interscience, New York. 1955. CESSI, C., AND PILIEGO, F., Biochem. J. 77, 508 (1960). CHENC, P. T. H., J. Invest. Dematol. 51, 4S4 (1%8). IMMERS, J., AND VASSEUR, E., Nature 165, 898 (1950). APPLEGARTH, D. A., AND BOZOIAN, G., Nature 215, 1352 OGSTON, A. G., Anal. B&hem. 8, 337 (1964). CHOTINER, G., SMITH, J. CT., JR.. AND DAVIDSON, E. il., (1968). RADHAKRISHNAMURTHY, B., DALFERES, E. R., JR., AND Biochem. 17, 545 (1966). BRENDEL, K.. ROSZEL, N. O., WHEAT, R. W., AND DAVIDSON,

1824 (1933).

61, 586 (1955). (D. Glicb, ed.),

Vol.

2,

(1967). Anal.

Biochem.

BERENSON,

G.

E. A., Anal.

26,

146

S., Ancd. Biochem.

18, 147 (1967). 14. GRAHAM, E. R. B.. AND XEUB~RCER, A., Biochem. J. 109, 645 (1965). 15. KELLEHER, P. C., AND SMITH, C. J., J. Chromatog. 34, 7 (1968). 16. MOORE, S., AND STEIN, W. H., J. Bid. Chem. 192, 663 (1951). 17. KELLEHER, P. C., AND SMITH. C. J., Biochim. Biophys. Acta 201, 76 (1970). 18. GALAMBOS, J. T., AND SHAPIRO. R., Anal. &o&em. 15, 334 (1966). IF). PROCKOP, D. J., AND EBERT, P. S., Anal. Biochem. 6, 263 (1963). 20. MOSCHIDES. E., STEFANINI, M.. MAcaLrNI, S. I., SND KISTNER, S. A.. J. Clin. Invest. 37, 127 (1958),