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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.
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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.,
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11. Spackman, D . H . : Instruction Manual, Amino
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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
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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.