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THK AMEHICAN JOURNAL OF CLINICAL PATHOLOGY Vol. 45, No. 3 Copyright © 1906 by The Williams & Wilkins Co. Printed in U.S.A. MACROGLOBULINS AND THE SI A WATER TEST HORACE H. ZINNEMAN, M.D., AND ULYSSES S. SEAL, P H . D . Departments of Medicine and Biochemistry, Minneapolis Veterans Hospital and University of Minnesota School of Medicine, Minneapolis, Minnesota The euglobulin test (Sia water test) has been used routinely as a screening procedure for Waldenstrom's macroglobulinemia. It is well known, however, that 7 S hyperglobulinemia may produce a positive test, and that 19 S macroglobulins with an electrophoretic mobility of /32 or 0i globulins may yield a negative Sia water test.2-3> 5 Macroglobulins of the mobility of 7-globulins give a positive Sia water test. Recently, a patient with Waldenstrom's macroglobulinemia, whose pathologic protein migrated at the speed of 182 (71) globulins, came to our attention. His serum yielded a negative Sia water test. The pathologic macroglobulin was isolated from the serum and studied in greater detail, with the hope of gaining some understanding of the mechanism which may be responsible for the precipitation or the absence of precipitation of these proteins in water. The data and results of this study seem to be worthy of documentation. H E P O H T O F CASE J. C , a 69-year-old Caucasian man, was referred to the Minneapolis Veterans Hospital in October 1963. About 1 year prior to his hospitalization, he had begun to complain of weakness. During the 3 months preceding his admission to this hospital, he had awakened almost every night with his mouth full of blood. He also began to suffer severe epistaxis, of sufficient severity to necessitate repeated blood transfusions. Upon admission, the patient presented as a pale, poorly nourished, Caucasian man, whose skin had multiple petechiae on the trunk as well as on the extremities. There was a healing decubitus ulcer on the right buttock. No significant lymphadenopathy Received, July 27, 1965. Supported in part by U. S. Public Health Service Grant AM 00240. 306 was observed. The heart and lungs were not remarkable upon percussion and auscultation. There was no palpable enlargement of the liver, spleen, or kidneys. The ocular fundi revealed several recent retinal hemorrhages, and the retinal veins were greatly distended. Among the laboratory examinations, the following seemed to be contributory to the case: The urine showed a 1 + proteinuria. The hemoglobin was 8.1 Cm. per 100 ml., the white blood cell count 6150 and platelets 96,000 per cu. mm. The sedimentation rate was 2 mm. in 1 hr., the plasma fibrinogen was measured at 225 mg. per 100 ml., the direct and indirect Coombs tests were negative, and the total serum bilirubin was 0.5 mg. per 100 ml. The blood urea nitrogen (BUN) was 21 mg. per 100 ml., the thymol turbidity 9.7 units, the cephalin cholesterol flocculation 4 + in 24 and 48 hr. The total serum proteins were 13.9 Gm. per 100 ml., and filter paper electrophoresis of the serum showed a prominent sharp protein peak which migrated with the mobility of /32 globulins and which accounted for 74.2 per cent of the total serum proteins. Albumin measured 14.4, ai-globulin 2.3, arglobulin 2.5, and ft-globulin 6.6 per cent. There was only a negligible amount of 72-globulin (Fig. 1). A drop of the serum failed to produce a precipitate in distilled water. The electrophoretic pattern of the urine showed a discrete protein band of the mobility of 72-globulin, a Bence Jones-like protein which accounted for the 1 + protein test of the urine. An attempt to determine the viscosity index of the serum was unsuccessful because its extreme viscosity made the observation of an accurate endpoint impossible. An ultracentrifugal pattern of the serum will be discussed below. It confirmed the clinical diagnosis of Waldenstrom's macroglobulinemia. A bone marrow aspirate was only sparsely cellular. A subsequent March 1966 MACROGLOBULINS AND SfA W A T E R TEST 307 serum to human serum demonstrated not •duly the presence of a small amount of contaminating albumin, but also antiglobulin, in addition to IgM (yil\ I -globulin). The material was then separated by means of chromatography on DEAE Sephadex A-50, using an 0.0175 JVI phosphate buffer of pH 6.3 (Fig. 3). This procedure separated the material into 3 components: IgM' (YIMmacroglobulin) eluted first (fractions 4 to 7), followed by a small peak of as-macroglobulins (fractions 9 to 11), and finally the contaminating albumin (fractions 23 to 33). Immunoelectrophoresis of fractions 4 to 7 resulted in a single precipitation line which corresponded to IgM (7iM-globulin) (Fig. ^2 H Pi <*Z <*\ Alb. FIG. 1. Elccfcrophoretic pattern of the serum of u patient with macroglobulinemia. The pathologic inacroglobiilin is migrating with the electrophoietic mobility of 02-globulins. bone marrow biopsy showed a uniform field of large lymphocytoid cells (Fig. 2). Roentgenograms of the chest and long bones failed to show radiolucent bone defects. In accordance with his wish, the patient was returned to his home state for care and further study. He is receiving 10 mg. of prednisone daily, and the most recent report indicated total serum proteins to be 9.3 Gm. per 100 nil. (albumin 4.0, globulins 5.3 C!m. per 100 ml.), hemoglobin was 12.4 Gm. per 100 ml. The patient was gaining weight, noticed less bleeding of the oral cavity, and there was no additional epistaxis. M A T E R I A L S , METHODS, AND RESULTS Fifteen milliliters of the serum were subjected to gel filtration on a column of Sephadex G-200. Immunoelectrophoresis of the frontally eluted peak with rabbit anti- Ultracentrifugation studies of the IgM fraction were performed for the determination of the sedimentation coefficient. An analytic ultracentrifuge (Spinco model E) was used for this procedure. Two peaks were noted which corresponded to those seen in the macroglobuhns of the whole serum (Fig. 5), one 19 S, the smaller one 24 S. Addition of mercaptoethanol to 0.01 M and incubation for 18 hr. at 4 C. resulted in a single peak of 6.3 S (Fig. 6). The material was then studied for its carbohydrate content. Sialic acid was assayed by the method of Warren,14 and fucose,1 hexose,15 and hexosamine9 were determined as described by Winzler.16 The macroglobulin was thus found to contain 1.7 per cent hexose, 2.2 per cent hexosamine, 0.97 per cent sialic acid, and 1.1 per cent fucose. For quantitative estimation of amino acids, the macroglobulin was subjected to acid hydrolysis" and analyzed by ion exchange chromatography on columns of Amberlite resins by means of an automatic amino acid analyzer, as described by Spademan and associates12 (Spinco Model 120). The results from the analysis of the macroglobulins from J. C., whose serum was negative in the Sia water test, and C. K., whose serum yielded a positive Sia water test, are recorded in Table 1, together with the amino acid composition of IgG and macroglobulins reported elsewhere. There is good agreement between the values obtained by others and those of the macro- 308 Vol. 45 ZINNEMAN AND SEAL FIG. 2. The photomicrograph of the bone marrow biopsy of patient J. C. illustrates uniform infiltration with lymphocytoid cells, typical of Waldenstrom's macroglobulinemia. X 400. globulin under discussion. There were significant deviations from the amino acid composition of normal IgG. Incubation of the whole serum at 37 C. with Vibrio Cholerae filtrate in 0.2 N acetate buffer at pH 5.5 for 45 hr. resulted in complete removal of bound sialic acid. After this procedure, the serum reacted intensely positive with the Sia water test. Paper electrophoresis shows that the loss of sialic acid resulted in a retardation of the anodebound migration of the macroglobulin. The migration of the other major globulin fractions also was greatly retarded. Albumin, which does not contain sialic acid, showed no change in migration (Fig. 7). When normal human serum was subjected to the same experiment, it did not form a precipitate in distilled water, although the mobility of the major glycoprotein fractions was retarded. DISCUSSION A comparison of the macroglobulin of the patient (Table 1) with another pathologic human macroglobulin as described by Madema and associates,4 the macroglobulin of C. K., one of our patients with macroglobulmemia, and a normal human serum macroglobulin as analyzed by Svartz, 13 reveals that the 4 macroglobulins contain relatively more arginine and less tyrosine than did 3 IgG (7 S 7-globulins) which were analyzed in this laboratory, and IgG studied by Svartz.13 I t should be emphasized at this point that the amino acid composition of normal and abnormal macroglobulins is similar, whether or not they produce a March .1966 309 MACROGLOBULINS AND SIA WATER TEST 3.0 o M 2.0 o c o < :> 3 i i i—i—i—i—r~r- 15 20 25 40 Tuba Number F I G . 3. Isolation of the a, M-(/32 M) macroglobulin by chromatography of DEAE-Sephadex A-50. The 7iM-globulin was elated with the first peak, followed by« 2 M-macroglobulins. The third peak contained other serum proteins. F J G . 4. Immunoelectrophoresis of the protein of the first peak from the DEAE-Sephadex A-50 column resulted in a single line of precipitation with antihuman serum rabbit antiserum. T h e anode is on the right, the cathode on the left. positive Sia water test. A mean value of 4 "rheumatoid factor" (RF) macroglobulins, which were analyzed by Svartz,13 seems to indicate that they occupy a place somewhere between the other macroglobulins and the IgG. The 4 R F macroglobulins of Svartz were in such close agreement that it was deemed permissible to use a mean value. As with the other macroglobulins, the R F globulins contained less tyrosine than IgG, but their arginine content was comparable to that of IgG. Reisner and Franklin 8 were able to demonstrate heterogeneity of the 19 S macroglobulin molecule. By means of chromatography at pH 8, 6, and 5, they separated 3 distinct subunits which varied in amount in the individual normal and pathologic macroglobulins. The differences between IgG and the monomers derived from the sulfhydryl cleavage of macro- 310 ZINNEMAN AND SEAL TIG. 5. Ultracentrifugal pattern of the whole serum. The 19 S microglobulins ;ire seen as a "self-sharpening peak." A smaller peak represents 24 S globulins. globulins are probably complex and involve differences in carbohydrate content. The carbohydrates of myeloma proteins, particularly sialic acid, seem to be linked with the rate of their electrophoretic migration.r' Although sialic acid is an important factor in the electrophoretic mobility of normal globulins, it obviously is not the only one. Both albumin and prealbumin have a greater negative charge and migrate at a faster rate than the glycoproteins, yet they contain no sialic acid. Other factors, including the free carboxylic end-groups of aspartie and glutamic acid, as well as bound anions, are instrumental in the electrophoretic migration of proteins. 5,10 In the case of pathologic macroglobuhns, the rate of migration does not seem to have a direct relation to the content of sialic acid;2, 5' 10 however, most pathologic macroglobuhns which do not give a positive euglobulin test (Sia water test) are of the mobility of /3iand 02-globulins.2, '• 7 The hexose and hexosamiiie content of the macroglobuhn Vol. 45 PIG. 6. Superimposition of simultaneous ultracentrifugal runs of the isolated macroglobulin with (above) and without (below) addition of merciiptoethanol. It may be seen that the 19 S and 24 S peaks were reduced to one 0.3 S component by the sulfhydryl compound. under discussion was comparable to that of normal IgG, but the hexose was considerably less than the values reported for normal serum macroglobuhns0 and the macroglobulin of patient C. K. (Table 2). The sialic acid content, however, was 6 times that of IgG and 20 per cent greater (1 per cent) than the sialic acid of the macroglobulin of patient C. K. (0.8 per cent), whose serum gave a positive Sia water test and whose macroglobuhns migrated with 72-globulins. The fucose content also was increased when compared with IgG, but was identical with the fucose of the macroglobulin of patient C. K. The macroglobuhn of the patient migrated electrophoretically with the mobility of 7i- ((32-)globulin, its sialic acid content was high, and it seems likely that it was partially responsible for the increased solubility of this protein in water and hence the negative Sia water test. Incubation of the whole serum with neuraminidase not only retarded the electrophoretic migration of the macroglobulin, but also changed the behavior of March .1966 MACROGLOBULINS AND SIA WATER 311 TEST TABLE 1 AMINO ACID COMPOSITION OP SEVERAL M I C R O G L O B U L I N S IN COMPARISON WITH 7 S Y - G L O B U L I N AND RHEUMATOID FACTOR MACKOGLOBULIN* jVlacroglobulins Amino Acid 7 S 7-Globulinf .Lysine 1-list.idinc Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cystine/" Valine Methionine Isoleucinc Leucine Tyrosine Phenylalanine G.92 2.32 3.16 7.98 0.89 7.81 10.44 6.41 3.94 3.67 3.11 8.71 1.04 2.27 8.50 6.36 4.28 RF Macroglobulin Svartz" Normal Svartz" Pathologic Madema4 Pathologic C. K. Pathologic J. C. 10.5 3.S 6.3 8.3 5.1 5.0 11.8 3.7 2.4 5.0 3.2 S.3 0.5 1.9 8.0 2.6 4.9 7.1 1.5 7.0 10.7 S.7 S.9 11.2 1.6 4.2 5.1 6.S7 2.32 5.S9 9.47 7.3S 5.72 11.14 6.23 4.47 4.95 3.S6 8.77 1.06 3.93 S.37 4.91 4.66 6.16 1.9S 6.45 S.3S 7.41 S.1S 11.99 4.44 4.44 4.65 3.44 7.64 1.16 3.24 7.S3 4.52 5.09 5.57 2.25 4.1 8.85 7.0 7.1 12.0 5.3 3.3 4.6 2.5 0.57 2.95 8.2 3.47 4.92 10.2 1.3 3.3 9.2 4.5 6.4 * T h e values arc given as percentage by weight, f Mean of three 7 S y-globulius from our laboratory. 11 serum -, 1(1 c ' I £erum C In CM b . c • I K/et» •**•* ••""'' a '"* F I G . 7. Incubation with neuraminidase and removal of all bound sialic acid resulted not only in the appearance of a positive Sia water test, b u t also a retardation of the electrophoretic mobility of the •microglobulin. The electrophoretic mobilities of the on-, <*2-, and /3-groups of globulins were also changed. this serum in the euglobulin test. A dense precipitation appeared immediately where none could be elicited prior to the treatment with neuraminidase. Normal human serum failed to yield a precipitate in distilled water either before or after removal of sialic acid. In its isoliited state, however, this macro- globulin gave a positive euglobulin test, although its electrophoretic mobility remained that of a YI- (/3>-) globulin. When it was added to normal human serum in the proportion which prevailed in the original serum, it failed to give a positive Sia water test. It seems that the sialic acid content 312 Z I N N E M A N AND TABLE 2 T H E CARBOHYDRATE C O M P O N E N T S O F 7 S 7 - G L O B U LIN COMPARED WITH THE MACROGLOBULINS OF J. C , Vol. 45 SEAL Acknowledgment. Technical assistance was given by Mrs. Gay Goodsell and Miss J a n e t Johnson. THE P R O P O S I T U S WITH A N E G A T I V E S I A T E S T AND OF C. K., W H O S E SERUM HAD A P O S I TIVE S I A T E S T * Macroglobulin Carbohydrate Hexose Hexosamine Fucose Sialic acid 7 S 7-Globulin l.G 1.17 0.34 0.15 J. C. C.K. (Positive Sia Test) (Negative Sia Test) 4.Go 1.3 1.2 0.8 1.7 2.2 1.1 0.97 * T h e values are given as percentage by weight. of the macroglobulin, although instrumental in the production of the Sia water precipitate, is not the only factor responsible for this phenomenon. One of the patently visible effects of incubation with neuraminidase is the change of electrophoretic mobilities of the glycoproteins of the a- and |3-globulin groups in the serum of the patient (Fig. 7) as well as in normal serums. Thus, it may not be unreasonable to assume that the euglobulin test depends not only on the sialic acid content of this macroglobulin, but also on its interaction with other sialic acid-containing glycoproteins in the serum. SUMMARY The serum of a patient with Waldenstrom's macroglobulinemia failed to yield a positive Sia water test (euglobulin test). I t is known that macroglobulins which migrate electrophoretically with the 72 globulins usually have a positive Sia water test, whereas this test is likely to be negative in macroglobulins of greater electrophoretic mobility. The macroglobulin under discussion belongs within the second group as a 02- ("yi-) globulin. Further study of this protein and comparison with the results of others suggest that this difference in water solubility is not likely to be associated with the amino acid composition of the protein but possibly is associated with the quantity of the sialic acid component and its interaction with other sialic acid-containing glycoproteins. REFERENCES 1. Dische, Z., and Shettles, L. B . : A specific color reaction of methylpentoses and a spectrophotometric micromethod for their determination. J. Biol. Cheni., 175: 595003, 1948. 2. Laurell, C. B . , Laurell, H . , and Waldenstrom, J.: Glycoproteins in serum from patients with myeloma, macroglobulinemia and related conditions. Am. J. Med., 22: 24-36, 1957. 3. Laurell, C. B . , and Waldenstrom, J . : Sera with exceptional appearance and the euglobulin reaction as screen test. Acta med. scandinav., Suppl. 367, 97-100, 1961. 4. Madema, E . , Van Der Shaff, P . C , and Huisman, T . H . J . : Investigations on t h e amino acid composition of a macroglobulin and a cryoglobulin. J . L a b . & Clin. Med., 45: 261-269, 1955. 5. Miiller-Eberhard, H. J., and Kunkel, H . G.: The carbohydrate of 7-globulin and myeloma proteins. J. E x p . Med., 104: 253-269, 1956. 6. Norberg, R . : Carbohydrate content of normal 19S globulins. Clin. chim. Acta, 9: 89-90, 1964. 7. Ratcliff, P . , Soothill, J. F . , and Stanworth, D . R . : Physicochemical and immunological studies of pathological serum macroglobulins. Clin. chim. Acta, 8: 91-10S, 1903. 8. Reisner, C. A., and Franklin, E . C.: Studies of mercaptoethanol-dissociated normal human 19 S 7-globulin and pathologic macroglobulins from patients with macroglobulinemia. J. Immunol., 87: 654-664, 1961. 9. Rimington, C : Seromucoid and bound carbohydrate of serum proteins. Biochem. J., 34: 931-940, 1940. 10. Schultze, H. E . : Influence of bound sialic acid on electrophoretic mobility of human serum proteins. Arch Biochem. it Biophys., Suppl. 1, 290-294, 1962. 11. Spackman, D . H . : Instruction Manual, Amino Acid Analyzer, Model 120B. Palo Alto: Spinco Division, Beckman I n s t r u m e n t s , Inc., 1962. 12. Spackman, D . H . , Stein, W . H . , and Moore, S.: Automatic recording a p p a r a t u s for use in the chromatography of amino acids. Anal. Chem., 30: 1190-1206, 1958. 13. Svartz, N . , Lilijamaa, J., and Ericsson, B . : Amino acid analyses of the rheumatoid factor macroglobulin. Acta med. scandinav., 172: 767-768, fasc. 6, 1962. 14. Warren, L . : T h e thiobarbituric acid assay of sialic acids. J. Biol. Chem., 284: 19711975, 1959. 15. Weimer, H . E . , and Moshin, J. R,.: Serum glycoprotein concentrations in experimental tuberculosis of guinea pigs. Am. Rev. T u b e r c , 68: 594-602, 1953. 16. Winzler, R. J . : Determination of serum glycoproteins. In Glick, D . : Methods of Biochemical Analysis, Vol. 2. New York: Interscience Publishers, 1955, p p . 279-311.