The measurement of urinary mucopolysaccharides

..\NALYTICAL

The

21, !&-lo6

BIOCHEMISTRY

Measurement

of Urinary

NICOLA Departvnrnt

(1967)

M. DI

Mucopolysaccharides FERRANTEl

of Biochemistry, Baylor University Houston, Texas ‘?‘?‘025 Received

March

College

of Medicine,

28, 1967

Quaternary ammonium salts (QSN+) , introduced as precipitants for various polyanions between 1953 and 1955 (1)) were used to precipitate urinary acid mucopolysaccharides (RIPS) in 1956 (2). Since then, the original method or its many modifications have been widely used for the isolation and measurement of MPS present in normal urine (3-6) or urine of patients with various diseases (7-17). In the last few years, however, it has been claimed that such precipitation techniques afford values either too low (18, 19) or too high (17). The purpose of this paper is: (1) to analyze some of the factors which influence the precipitation of urinary MPS with QSN+ and to compare the efficiency, as precipitants, of cetylpyridinium chloride (CPC), cetyltrimethylammonium bromide (CETAB), and 5-aminoacridine HCI (5 aa HCI) ; and (2) to describe an alternate method for the measurement of urinary MPS and to compare its results with t’hose obtained wit,h the precipitation method. MATERIALS

AND

METHODS

Twenty-four hour urine samples were obtained from apparently healthy individuals of both sexes under 40 years of age. No preservative was used; the containers, kept at 4°C during the collection period, were frozen upon arrival at the laboratory and kept frozen until they were anaIyzed. Each urine specimen was measured and duplicate 25 ml aliquots were precipitated with CPC (K & K Laboratories, Inc.), CETAB (Eastman Kodak Co.), and 5 ax HCl (K & K Laboratories, Inc.), or used for the alternate method. Recovery experiments were performed by adding various amounts of standard MPS to 25 ml aliquots of urine of known MPS content. The precipitation method used was a simplified version of the original one published in 1956 (2) : 25 ml aliquots of urine (in duplicate) were precipitated with appropriate volumes of aqueous solutions of the precipitant,s; after 12 hr at 4”C, the precipitate was collected by centrifuga’ Retnbli~heii

Investigator

of the

American

Heart 9s

Association.

MEASUREMENT

OF

URINARY

MUCOPOLYSACCHARIDEP

99

tion, washed twice with 95% ethanol containing 10% potassium acetate, and dissolved in 5 ml of water. On aliquots of this solution, the borate modification (20) of the carbazole reaction (21) was performed, and the results obtained, reported to the total volume of the urine sample. were expressed as mg hexuronic acid/24 hr. Repetitive experiments with increasing amounts of the three precipitants established that 0.5 ml of 10% CPC, 2 ml of 5% CETAB, and 10 ml of 0.5% 5 aa HCl (w/v. aqueous solutions) were sufficient to obtain complete precipitation of MPS present in or added to 25 ml of urine. In the alternate method for measurement of urinary MPS, 25 ml of urine was applied to a 1 X 15 cm column of ECTEOLA2 modified cellulose, suspended in 0.9% NaCI. The column was washed with 100 ml of 0.9% NaCl in order to remove urinary pigments, the urinary mucoprotein of Tamm and Horsfall (22), and any other protein which might have been present in the urine. Subsequently, it was eluted with NaCl (0.5, 1.0, 2.0, and 4.0M), four aliquots of 5 ml each being collected for every NaCl solution. The borate carbazole (20) or the anthrone reaction was performed on aliquots of each fraction, and the results were expressed as mg hexuronic acid, or mg galactose/24 hr. RESULTS

The analyses of the crude urinary MPS precipitated with QSN+ and the analytical methods used have been published (5). Figures 1, 2, and 3 illustrate the effect of temperature, salt concentration, and toluene on the extent of precipitation of urinary MPS. The results of the recovery tests, performed at 24 and 4’C, are presented in Table 1, while Table 2 compares the values obtained for the same urine specimens with the precipitation and column methods. DISCUSSION

The data summarized in Figure 1 and Table 1 clearly indicate that 5 aa HCl is the least efficient of the three precipitants used, at least under the experimental conditions described. While its ability to precipitate MPS added to urine is quite satisfactory, its ability to precipitate urinary MPS (possibly more depolymerized and less sulfated than the standard preparations) is inferior to that of QSN+. Although 5 aa HCl is the only reagent which precipitates keratan sulfate from aqueous solutions (Fig. 2), CPC and CETAB afford a much better recovery of keratan sulfate when this is dissolved in urine rather than in water (Table 1) ’ ECTEOLA (epichlorohydrin triethanolamine-cellulose, Schleicher and Schuell. lot 1285, 0.4 meq/gm capacity) was washed with 1.0 N NaOH, 1.0 N HCI, distilled water, 95% and absolute alcohol, and dried with acetone prior to use.

100

NICOLA

M.

DI

~ER~NT~

4C

24C

100

60

60

40

20

0~

: PC 5aa CETAB

C PC 5aa CETAB

FIG. 1. Effect of temperature on precipitation of urinary MPS with CPC, CETAl3, or 5 aa; 25 ml aliquots of the same urine were precipitated at 4 and at 24°C with an appropriate amount of each precipitant.

0

I .04

1 .08

+-a

Kefotansulfate,

m-0

Heparin,

I mg CCPC

o-2

Heparin,

1 mg

O-O

Chondroitinsulfote,

I .I2

I .I6

I 0.2 NaCI,

1 mg + 500 + 500 Img I 0.3

+500 I 0.4

I 0.5

M FIG. 2. Effect of salt concentration on precipitation of standard MPS with CPC or 5 aa; keratan sulfate dissolved in water or in salt solutions was not precipitated with QSS+.

l’dEAfXJFtEMENT

OF

UBINABY

MUCOPOLYSACCHARIDES

101

These data and those of Pedrini and co-workers, who isolated keratan sulfate from the urine of some patients with Morquio’s disease (12) using CETAB, do not agree with Muir’s opinion that QSN+ do not precipitate urinary keratan sulfate (23). Therefore, it is difficult to understand the higher amounts of urinary MPS that Muir and co-workers claim to obtain from normal or pathological urine using 5 aa as the precipitant (18, 19). One must speculate that their method (which consists of digestion of the urine with pancreatin, dialysis, precipitation of MPS with 5 aa HCl, removal of the precipitant, and determination, by weight, of the dry material obtained) may also measure material other than MPS. The unusual behavior of the keratan sulfate-QSN” complex deserves comment. These complexes seem to be soluble both in water and in

t I

I 0

FICA 3. Effect of addition MPS with CPC.

035

I .070 TOLUENE,

I 105

I 140

ml

of toluene to 25 ml of urine on precipitation

at 4°C of

aqueous salt solutions, but they coprecipitate in the presence of insoluble complexes formed by QSN+ with MPS and with the Tamm and Horsfall mucoprotein, resulting in fairly satisfactory recovery of the keratan sulfate. The keratan sulfate-5 aa HCI complex is insoluble, at least in part, in distilled water and even more insoluble in salt solutions. However, the recoveries of keratan sulfate dissolved in nondialyzed urine are much lower with 5 aa HCl than with QSN*. Although QSN+ seem to be better precipitants for MPS than 5 aa HCI, several factors may influence their performance. The data presented emphasize the unfavorable effect of toluene and the necessity of performing the precipitation of MPS at 4°C for at least

102

NICOLA

M.

DI

FERRANTE

12 hr. The washings of the QSN+-MPS complex with 95% ethallU1 containing 10% potassium acetate also require particular care. While the recoveries of sulfated MPS added to urine do not seem to vary with the duration or the temperature of the ethanol washes, the recoveries of

(St,andard

MPS

TABLE 1 Recovery Tests at 24 and 4°C added to 25 ml urine of known Hsy;oo”pic

MPS added to urine

Chondroitin

sulfate

1 w

Heparin,

1 mg

Keratan 0.75

sulfate mg

Hyaluronate, 0.5 mg

Precipitant

CPC CETAB 5 aa CPC CETAB 5 as CPC CETAB 5 aa CPC CETAB 5 aa

ga!actose added, pg

160

3’30

173

163

MPS

content)

pg found

% reco”uy

24”

4”

240

168 157 155 294 320 310 91 109 60

161 165 161 298 307 355 141 138 60 142 133 132

105 98 97 92 100 97 52.5 63 34.6

4’ 100

103 100 93 95 111 81.5 80 34.6 87 81.5 81

Chondroitin sulfate, crude, from bovine nasal septa. Hexuronic acid 16%, S 2.9%. Heparin, commercial, from beef lungs. Hexuronic acid 32%. Hexosamine (nitrous acid) 25.6%. Keratan sulfate, from bovine nasal septum. N 1.85. S 1.16, galactose 0.77, hexosamine 1.00. Hyaluronate, from umbilical cord. Hexuronic acid 32.6$&

hyaluronate indicated in Table 1 and those of “desulfated” chondroitin sulfate published previously (5) were achieved only when a complete precipitation of the MPS from the ethanol wash was allowed to take place overnight at 4°C. One cannot be too emphatic in discouraging the dialysis of urine prior to precipitation. There are several indications that a large amount of urinary MPS is lost during dialysis (17, 24). It is probable that heated cellophane tubings may retain some of the material that would dialyze t,hrough normal tubings (18) ; however, the complete absence of salts seems to influence in a negative fashion the efficiency of all the precipitants tested. Thompson and Castor (17) have proposed a rather elaborate method for the precipitation of urinary MPS with CPC. Their results indicate a range of normal values lower than those previously reported and refute t.he existence of a difference in values between males and females (2, 25-

MEASUREMENT

OF

URINARY

103

MUCOPOLYSACCHARIDES

27). Losses of urinary MPS during dialysis and incomplete precipitation of the nondialyzable material are factors which may explain their results. In their work, the precipitation with CPC was performed at room temperature and at extremely low salt concentration (approximately 0.004 M Na$O,). Under such circumstances, the addition of TABLE 2 Measurement of Urinary Muwpolysaccharide@ (Comparison between precipitation and column methods and between values obtained in males and females. Results are mg hexuronic acid/24 hr.) Test

Mean

f

S.D.

Statistiwl X9.“@? (2 S.D.)

P

Male Column (16)a Precipitation (16) Female Column (15) (15) Precipitation Column, male femaIe Precipitation, male female


n Figures in parentheses are number o* cases analyzed.

more salt to the supernatant and failure to observe further precipitation at room temperature do not constitute evidence for an earlier ,complete precipitation. The fact that the presence of toluene did not influence their result,s may be due to its removal during vacuum concentration and dialysis of the urine. Data collected in this laboratory indicate that CPC precipitates MPS of smal1 molecular weight. As an example, a recent case of Hurler’s disease, studied with the CPC precipitation method and with the ECTEOLA column method, had 54 mg of total MPS in 200 ml of urine. Dialysis of this material overnight, against several changes of distilled water, resulted in the recovery of only 9 mg of MPS inside the dialysis membrane. This observation, unusual as it may be in its quantitative aspects, demonstrates that QSN’ precipitate not only the MPS extracted’ from tissues and added to urine (19) but also urinary MPS of molecular size small enough to pass through a dialysis membrane. Moreover, the heparin preparation used in the recovery tests (M.W. 11,000) dialyzes readily through nonheated cellophane tubings. Nevertheless, it is precipitated quantitatively from urine or appropriate salt solutions by QSN’ (Table 1, Fig. 2) but not from saIt solutions below 0.04 &I (Fig. 2). Obviously,

104

NICOLA

M.

DI

FERRANTE

when the depolymerization of the MPS reaches extreme values, the products are not precipitated by QSN’, a fact recognized long ago and used for a simple turbidimetric measurement of MPS and hyaluronidase activity (28). The reported failure of CPC to precipitate at room temperature, low salt concentration, and in absence of the Tamm and Horsfall mucoprotein that fraction of the urinary MPS which had passed through a dialysis membrane (17) is not in contrast with my findings, but merely reiterates the adverse effect of dialysis prior to precipitation of the urine sample. The data presented indicate that optimal results may be obtained with the precipitation method only if complete precipitation of the MPSQSN+ complex and of the MPS from the ethanol washes are allowed to take place. Thus, approximately two days elapse before the results of a simple test are obtained. The column method has been developed to obviate these inconveniences. Its results are in good agreement with those obtained with the precipitation method; recovery tests performed with chondroitin sulfate, keratan sulfate, a mixture of the two, and a crude cornea1 extract gave recoveries ranging between 90 and 112%. The presence of toluene, urinary protein, or urinary sediment does not interfere with the column assay but it does interfere with the precipitation methods, either by preventing the complete precipitation of the MPS or by making the final precipitate incompletely soluble in water. One hundred milliliters of normal human urine may be processed with a 1 X 15 cm column of ECTEOLA; 500 ml of urine requires a 2 X 25 cm column. Therefore, the chances of overloading a column of standard dimension are fairly remote even with a 25 ml urine aliquot of abnormally high MPS content. The urinary MPS are eluted from the column with 0.5 and 1.0 M NaCI. For the measurement of total urinary MPS, the method may be shortened further by eluting the column only with 2.0 M NaCl; on the other hand, for the fractionation of urinary MPS and the measurement of different fractions, elution may be performed with NaCl solutions of gradually increasing molarity (5). Despite the high salt concentrations of the eluting solutions, no depression of color was observed when the borate-carbazole reaction was performed. Finally, it is worthy of mention that both methods, as performed in t,his laboratory, confirm the significant difference in the excretion values of males and females (2,25-27) (Table 2). SUMMARY

The role of various factors on the precipitation of urinary mucopolysaccharides has been investigated. With quaternary ammonium salts as precipitants, temperature, salt concentration, and presence of toluene

MDASUREWENT

OF

URINARY

105

MUCOPOLYSACCHARIDES

influence the amount of material precipitated. Under optimal conditions, quaternary ammonium salts are more efficient precipitants than 5-aminoacridine HCl. As an alternate method for the measurement of urinary mucopolysaccharides, an aliquot of urine is passed over a column of ECTEOLA modified cellulose, and the mucopolysaccharides, eluted with NaCl solutions of increasing molarity, are measured as hexuronic acid or galactose. The two methods yield comparable results and confirm previously reported differences in the excretion of urinary mucopolysaccharides by males and females. ACKNOWLEDGMENTS This investigation was supported by United States Public Health Grant AM 10811 and by a Grant-In-Aid of the American Heart Association. I wish to thank Miss Lynn Schwarz for technical assistance. REFERENCES 1. Scorn, E. J., Meth. Biochem. Anal. 8, 145 (1960). 2. DI FERRANTE, N., AND RICH, C., J. Lab. Cl&z. Med. 48,491 (1956). 3. DI FERRANTE, N., AND RICH, C., Clin. C&K Acta 1,519 (1956).

4. RICH, C., DI %RRANTE, N., ?LND ARCHIBALD, R. M., J. La.b. Clin. Med (1957). 5. DI FERRANTE,

N., J. Lab.

Clin.

Med.

61, 633 (1963). J. W.,

6. TELLER, W. M., BURKE, E. C., ROSEVEAR, Clin. Med. 59, 95 (1962).

50,

686

AND MCKENZIE, B. F., J. Lab.

7. DI FERRANTE, N., J. Clin. Invest. 36, 1516 (1957). 8. DI FERRANTE, N., ROBBINS, W. C., AND RICH, C., J. Lab. Clin. Med. 50, 897 (1967). 9. MEYER, K., GRUMBACH, M. M., LINKER, A,, AND HOFFMAN, P., Proc. Sot. Exptl. BioZ. Med. 97, 275 (1958). 10. MEYER, K., HOFFMAN, P., LINKER, A., GRUMBACH, M. M., AND SAMPBON, P., Proc. Sot. Exptl. BioZ. Med. 102, 587 (1959). 11. TELLER, W. M., ROSEVEAR,J. W., AND BUREE, E. C., Proc. Sot. Exptl. BioZ. Med. 108, 276 (1961). 12. PEDRINI, V., LENZI, L., AND ZAMBOITI, V., PTOC. Sot. Exptl. BioZ. Med. 110, 847 (1962). 13. TERRY, K., AND LINKER, A., Proc. Sot. Ezptl. BioZ. Med. 115, 394 (1964). 14. MAYES, J., AND ~NSEN, R. G., Proc. Sot. Exptl. BioZ. Med. 112, 927 (1966). 15. NAQEL, D. A., J. Bone Joint Surg. 47, 1176 (1965). 16. MELCHIOR, J. C., CLAUSEN, J., AND DYGWE, H. V., CZin. Pediatrics 4, 468 (1965). 17. THOMPSON, G. R., AND CASTOR, C. W., J. Lab. CZin. Med. 68, 617 (1966). 18. MUIR, H., MIWWOCH, U., AND BITTER, T., Arch. Disease Childhood 38, 358 m33). 19. MUIR, H., Intern. Rev. Connective Tissue Res. 2, 141 (1964), 20. BITTER, T., AND MUIR, H. M., Anal. Biochem. 4, 330 (1962). 21. DISCHE, Z., J. BioZ. Chem. 167, 189 (1947). 22. TAMM, I., AND HORIFALL, F. L., JR., Proc. Sot. Exptl. BtiZ. Med. 74, 108 (1950). 23. Mum, H., St. Andrews, Scotland, 1964, personal communication.

106

24.

NICOLA

M.

DI

FERRANTE

J. S., Ja., AND BOYCE, W. H., in “High Molecular Weight Substances in Human Urine,” p. 111. Thomas, Springfield, Ill., 1961. 25. KERBY, G. P., J. Clin. Invest. 34, 1738 (1955). 26. MORARD, J. C., GHATA, J. F., SALAMIN, L., AND AZERAD, E., Compt. Rend. Sot. KING,

Biol. 154, 276 (1960). 27. GALLETTI, F., AND STANCARI, V., La Clinica 28. DI FERRANTZ, N., J. Biol. Chem. 220, 303

(Bologna)

(1956).

1’7, 283 (1957).