T H E AMEIUCAX JOURNAL OF CLINICAL PATHOLOGY
Vol. 49, No. 4
Copyright, © 196S by The Williams & Wilkins Co.
Printed in
U.S.A.
USE OF A TANNIC ACID-CAFFEINE CONCENTRATION PROCEDURE
FOR DETECTING URINARY PROTEINS AND HEMAGGLUTININS
TIBOR J. GREENWALT, M.D., CAREL JAN VAN OSS, D.Sc, AND
EDWIN A. STEANE, B.S.
Milwaukee Blood Center and the Marquette University School of Medicine, Milwaukee,
Wisconsin 53233
The realization that "albuminuria" is a
misnomer and that traces of many plasma
proteins are present in urine has led to
increased interest in methods for concentrating urine. Ultrafiltration, osmotic concentration with dextran, polyvinylpyrrolidone, or Carbowax 20-M, pervaporation,
salting out, alcohol precipitation, and various combinations of these procedures have
been used.4' »• 12 ' 16 ' ^ 25. M j\f one lends itself
readily to routine studies in the clinical
laboratory.
Mejbaum-Katzenellenbogen and her associates 17_19 reported that proteins precipitated
from biologic fluids by tannin can be regenerated from the insoluble protein-tannin
complexes by adding caffeine, which forms
a less soluble compound with tannin. Dobryszycka 6 used this procedure for the
concentration of proteins from normal urine
and detected anti-A and anti-B activity in
the concentrates.
The subject of urinary proteins has been
reviewed recently by others.10, 12 It is our
purpose to describe the use of tannic acid
and caffeine for the concentration of urinary
proteins and to report preliminary observations concerning its usefulness in detecting
monoclonal proteins and anti-A and anti-B
activity in urine.
M A T E R I A L S AND
METHODS
Collection of urine. Voided specimens of
urine, collected in Mason jars without added
preservative, were refrigerated if studied
Received March 10, 1967.
Supported in part by U. S. Public Health Service Grant HE-05006 (HE-11154) from the National
Heart Institute.
Requests for reprints should be addressed to:
T. J. Greenwalt, M. D., American National Red
Cross, National Headquarters, Washington, D. C.
20000.
within 48 hr., but were stored at —25 C.
after that time. Observations published
recently indicate that IgG-globulin and its
chains and fragments are quite stable in
sterile normal urine. 6
Method for concentrating urinary proteins.
Any volume of urine may be used, but 500
ml. were found to be convenient and suitable
for these studies. A 1-ml. sample of each
specimen was tested for gross proteinuria
by the addition of an equal volume of 20 %
trichloracetic acid.
It was not found necessary to dilute the
urine and to remove the mucoids by filtration after precipitation with NaCl, as
recommended by Dobryszycka. 5 However,
more satisfactory and predictable precipitation of proteins occurs if the resistivity of
the sample is adjusted to not less than 75
ohms per cm. with NaCl (0.15 M NaCl = 75
ohms cm. at 20 C). If a conductivity meter
is not available, it is advisable to add 8 Gm.
of NaCl per liter of urine routinely. The
pH is adjusted to 4.7 with hydrochloric
acid and 2.0 ml of 0.1 M tannic acid (molecular weight, 322.22) are added per 500 ml.
The precipitate that forms sediments in the
cold overnight can easily be collected if this
step is performed in a separatory funnel. The
precipitate is separated by centrifugation
and washed three times with cold 0.15 M
NaCl at 4 C. After complete removal of the
final wash solution, 50 mg. of powdered
caffeine are added and the mixture is triturated vigorously for 15 min., preferably in
the cold. The reason for the effervescence
and foaming that occur is unknown. The
insoluble tannic acid-caffeine complex that
precipitates is then separated by hard centrifugation. The authors have used 39,000 X
g for 30 min. in a Sorvall SS3 centrifuge for
maximal yield of supernatant protein concentrate, but lesser forces will suffice. Resus-
472
April 196S
CONCENTRATION OP URINARY PROTEINS
pension of the washed precipitate in 1 to 2
ml. of saline before addition of the caffeine
increases the total recovery but the final dark
brown solution will have a lower concentration of proteins. Additional extractions
of the caffeine-tannate sediment will also
increase the total yield but will result in
further dilution.
In the presence of gross proteinuria 10fold quantities of tannic acid and caffeine are
usually satisfactory. The extraction volume
must be increased to accommodate the
recover}' of larger amounts of protein. Urine
concentrates were stored at — 20 C. before
study.
Electrophoresis. Electrophoresis was carried out on Gelman Sepraphore III cellulose
acetate strips13 in Oxoid modified barbitone
acetate buffer at pH S.6 in a Gelman rapid
electrophoresis chamber at 200 v. for 1}4 hr.
Immunoelectrophoresis. The procedure was
that of Grabar and Williams,8 with microscope slides coated with 1 % Oxoid ionagar
No. 2 in barbitone acetate buffer pH S.6 at
200 v. and 20 to 25 ma. for 3 hr. The pattern
was developed overnight in a humid chamber
at room temperature with the appropriate
antisera. Unwashed, unstained patterns
were photographed with an MP-3 Polaroid
camera.
Titration of anti-A and anti-B activities.
Two volumes of serial doubling dilutions
of serum or urine concentrate obtained
from mothers of infants with hemolytic
disease and controls were mixed with 1
volume of 4% red cells in saline, and then
either 1 volume of saline or 1 volume of
0.5 % bromelin was added. Saline agglutinations were incubated at 23 C. for 30 min.;
bromelin titrations were incubated at 37 C.
for 15 min.; and antiglobulin tests were
incubated at 37 C- for 30 min., followed by
three washings with saline before addition
of the antiglobulin serum. The readings
were macroscopic after centrifugation at
1000 r.p.m. for 2 min. in a Sorvall type M
angle centrifuge.
The bromelin solution was prepared by
thorough mixing of 0.5 Gm. of bromelin
(Takamine) with a small quantity of saline,
dilution to SO ml. and, after filtration
473
through a Millipore filter (pore size 0.47
m/i), adjustment of the volume to 100 ml.
Quantities of 4 ml. were stored at —20 C.
and were thawed only once for use.
Use of saliva for inhibition of anti-A and
anti-B. The method used is a modification
of a technic originally described by Lockyer15
that utilizes titrated saliva for predictable
neutralization of antibodies. Selected saliva
samples have higher neutralizing titers than
commercial preparations from animal
sources. The potencj' of the saliva samples
used for inhibition studies is determined
by using anti-A and anti-B sera adjusted
to have a residual titer of 1:16 to 1:32.
Serial doubling dilutions of boiled saliva
are added to equal volumes of the diluted
antiserum. After 10 min. at 23 C., 2 volumes
of 2% saline suspensions of Ai or B cells
are added and the mixture is incubated at
23 C. for 30 min. and read macroscopically
after centrifugation at 1000 r.p.m. for 2
min. The greatest dilution of saliva giving
complete or almost complete neutralization
is used for calculating the inhibition index
of the saliva, which equals the reciprocal
of saliva titer giving complete inhibition
multiplied by the reciprocal of titer of anti-A
or anti-B serum. For example, a group A
secretor saliva giving an inhibition titer of
1:32 with an anti-A serum of titer 1:16
has an inhibition index of 512, indicating
that the undiluted saliva will neutralize
a serum with an anti-A titer of 1:512 when
equal volumes are mixed.
Ultrafiltration. Filtration was accomplished at a rate of 0.5 to 1.0 ml. per hr.
with a vacuum through a 2-mm. thick membrane prepared from 5 % agarose in saline
on a Gooch filter.28
Protein N determination. The procedure
for digestion and nesslerization was that
described by Johnson" and Minari and
Zilversmit.21
Recovery studies. An anti-D (Rh 0 ) serum
(antiglobulin titer 1:204S) was diluted 1:50
and 1:100 in 0.S5 % saline, in 0.05 M Veronal
acetate buffer, pH 4.67, and in glycine
buffer, pH 3.0. Nitrogen determinations indicated recovery of S0.9 to 94.7% in the
tannic acid-caffeine concentrates. The anti-D
(Rh 0 ) titers of the concentrates ranged from
474
GREENWALT ET
Vol. 49
AL.
anti-X reagents (Fig. 2). The quantity of
protein recovered ranged from 76 mg. to
151 mg. per liter of urine when it was based
on N determinations (Table 1). The values
obtained by refractometry were, on the
average, 30% lower.
Anti-A and anti-B activity in urine concentrates. In an early survey 200-ml. urine
samples from parous women of unselected
ABO groups were concentrated. JNTO anti-A
or anti-B activity was found in the urine
concentrates of 21 (including seven of
group 0 ) with AB-compatible husbands;
1:128 to 1:512. Electrophoretic patterns revealed loss of the ai-globulins (Fig. 1).
RESULTS
Urines of normal subjects. Electrophoresis
and immunoelectrophoresis of urine concentrates from 12 normal subjects regularly
established the presence of 7-globulins and
somewhat more albumin. In one concentrate, precipitin lines were observed in the
)3- and a-globulin zones. In no instance was
any reaction in the globulin zones observed
on immunoelectrophoresis with anti-K or
TABLE 1
COMPARISON
OF A N T I - A AND A N T I - B T I T E R S IN SERA AND U R I N E CONCENTRATES
INFANTS
WITH
ABO
HEMOLYTIC
DISEASE
AND G R O U P
0
FROM M O T H E R S
OF
CONTROLS
Reciprocal of Titer
Serum
Subject*
Sal n e t
Group 0 controls
1. K. F .
2. J. L.
3. M. L.
4. R. B .
5. T. B .
6. C. B .
7. P . L.
8. B . N .
9. J. C.
10. P. C.
Mothers of infants
with ABO H D N | |
11. B . C.
12. D. C.
13. J. C.
14. V. G.
15. F . B .
16. A. B .
17. R. B .
18. D. W.
19. P . II.
20. G. K.
Urine concentrate
HDNt
O-A
O-A
O-A
O-B
O-A
O-A
O-B
O-B
O-A
O-B
+ Saliva
4- bromelin§
Rromelin
Mp. of p r o t e i n /
liter of urine
Anti-A 1
Anti-A 1
Anti-B
Anti-A 1
Anti-B
256
512
250
512
04
512
256
128
64
128
12S
250
256
12S
16
512
250
04
64
64
512
128
04
128
128
250
04
256
128
256
128
32
128
128
16
250
64
04
256
64
0
2
0
0
0
0
0
4
2
0
0
1
32
0
0
0
10
8
1
0
N . D.
N. D.
141
76
131
151
116
121
No precipitate
92
250
512
1024
512
1024
250
250
128
04
250
04
128
512
128
04
250
1024
1024
04
128
512
512
1024
128
4090
512
04
04
128
1024
04
04
128
512
128
04
1024
4090
512
512
10
04
32
0
128
0
0
0
8
32
4
S
64
8
2
0
04
512
16
32
139
135
11s
S7
114
Anti-B
ss
128
29
120
119
* All groups O and all female except Nos. 5 and 10. Controls had no known exposure to A and B antigens.
f H D N = hemolytic disease.
t At 23 C.
§ Neutralized with saliva before incubation with bromelin a t 37 C. Antiglobulin titers were similar.
|| All had previously delivered one or more babies with ABO H D N diagnosed by this laboratory.
April 1968
CONCENTRATION OF URINARY PROTEINS
one of three group 0 women with ABincompatible husbands, but no history of
hemolytic disease of the newborn, had
anti-A activity (1:4 antiglobulin). Antibody
was detected in the urines of seven of the
17 group 0 women who had borne infants
with ABO hemolytic disease; 1:16 was the
NORMAL HUMAN SERUM
CONCENTRATED
AFTER 1:500 DILUTION
I"! FIG. I. Comparison of the electrophoretic patterns of a normal serum before and after dilution
in saline and reconcentration. Note loss of <*iglobulin zone.
475
liighest antiglobulin titer. All had had affected pregnancies within the preceding
2 years.
In a second stud}', concentrates prepared
from 1000 ml. of urine from group 0 patients
all contained anti-A or anti-B activity or
both. The findings in the women who had
infants with ABO hemolytic disease were
not distinctive. Three group 0 males who
had been stimulated with group A soluble
blood group-specific substances in past years
had urinary anti-A titers of 1:12S, 1:512,
and 1:1024, and anti-B titers of 1:32,
1:8, and 1:32, respectively. Only antiglobulin titers are reported here, but most
samples also had weaker grades of saline
agglutinating activity. In Table 1 are summarized the most pertinent serologic data
obtained in comparison of the sera and urine
concentrates prepared from 500-ml. urine
samples of 10 normal group O subjects and
10 women who had infants with ABO hemolytic disease within 2 years. The saline and
bromelin titers after ultrafiltration of the
sera are listed in Table 2. In control studies,
saline-agglutinating human anti-M, anti-D
(RHo), and anti-Ai were not detectable,
whereas incomplete anti-D (RH 0 ) was present in the filtrates of serum through agarose
membranes. Three of the 10 control sera
Normal Serum
ANTh k
Urine Concentrate
Normal Serum
ANTI-X
PATIENT * 4 4
Urine Concentrate
PATIENT
*47
Fin. 2. Immunoelectrophoresis of concentrated normal urine. Normal whole human serum has been
run in the upper well of each slide in parallel with the urine concentrate placed in the lower well. Note
that the precipitin lines formed by the whole serum with anti-x and anti-X are absent in the urine concentrate.
TABLE 2
S T U D I E S OF A N T I - A AND A N T I - B IN U R I N E CONCENTRATES AND ULTHAFILTRATES OF SERUM FROM T H E
SAME S U B J E C T S AS T H O S E IN T A B L E 1; U R I N E CONCENTRATES N E U T R A L I Z E D WITH SALIVA
AS DESCRIBED IN THE T E X T
Neutralized Urine
Concentrate
Ultrafiltrate of Serum
Subjects
HDN*
Group 0 Controls
1. K. F .
2, J. L.
3. M . L.
4. R. B .
5. T. B .
6. C. B .
7. P . L.
S. B . N .
9. J. C.
10. P . C.
Mothers of infants with ABO H D N
11. B . C.
12. D . C.
13. J. C.
14. F . G.
15. F . B .
16. A. B .
17. R. B.
IS. D . W.
19. P . H .
20. G. K .
Sa ine
Bromelin
Bromelin
Anti-A
Anti-B
Anti-A
Anti-B
Anti-A
Anti-B
2
2
2
1
1
Qnsj
2
16
2
16
2
2
16
0
2
Qns
4
S
2
1
256
128
64
64
16
Qns
16
32
16
128
32
32
12S
2
16
Qns
16
16
4
16
0
0
0
0
0
0
4
0
0
0
0
4
0
0
0
16
4
0
0
0
4
4
1
8
64
2
2
4
4
32
1
2
1
16
2
1
S
32
16
16
64
64
64
128
512
16
16
2
64
256
16
32
32
32
64
16
256
256
64
32
8
16
16
0
16
0
0
0
8
16
2
4
32
0
0
0
32
12S
2
10
O-A
O-A
O-A
O-B
O-A
O-A
O-B
O-B
O-A
O-B
* H D N = hemolytic disease,
f Qns = q u a n t i t y not sufficient.
TABLE 3
S T U D I E S OF P A T I E N T S W I T H MYELOMATOSIS AND MACROGLOBULINEMIA
Urine Concentrate*
Patient
Serum M-spike
Proteinuria
Immunoelectrophoresis
Electrophoresis
Anti-IgG
1.
4.
5.
7.
9.
10.
20.
22.
23.
27.
31.
32.
43.
44.
47.
M. 0 .
H. H.
A. C.
A. F .
C. C.
A. G.
O. B .
J . S.
G. N .
E. II.
A. H.
O. 0 .
A. K.
R. S.
I. IT.
IgG
IgG
IgG
IgG
IgA
IgG
IgG
TgM
IgG
IgG
IgM
IgG
IgG
IgG
0
0
Trace
0
+ ++
0
+++
0
0
0
0
0
++
0
0
+
T-spike
0
0
T-spike
IgA spike
0
T-spike
T-spike
7-spike
T-spike
T-spike
0
0
T-spike
* Blank spaces mean t h a t procedure was not done.
476
Anti-lgA
Anti-IgM
Anti-K
Anti-X
Spike
0
Spike
++
—
+
+
+
Trace
0
+
++
++
++
++
+
0
++
-
+
—
+
+
-
(+)
0
++
(+)
April 1968
CONCENTRATION OF URINARY PROTEINS
contained anti-A and anti-B antibodies not
neutralized by soluble blood group-specific
substances in saliva, whereas such antibodies
occurred in seven of 10 sera from the ABO
hemolytic disease group. In Table 2 the results of the saliva inhibition studies of anti-A
and anti-B in urine concentrates are also
given. Pooled group 0 cells were used in
studying every preparation, in order to rule
oiit the possibility of nonspecific reactions.
Studies in patients 'with paraproteinemias.
In Table 3 are presented the results obtained
with the urine concentrates of 13 patients
with myelomatosis and two with Waldenstrom's macroglobulinemia. In the four patients with gross proteinuria, Bence Jones
proteins were detected by the conventional
heat test, but there were no diagnostic urinary findings in the unconcentrated urine
477
specimens of the others. In six patients without gross proteinuria, M-peaks were found
in the urine concentrates. In all instances
where immunoelectrophoretic studies with
anti-K and anti-A were performed, the findings were interpreted as indicating the exclusive presence or predominance of
monoclonal proteins because precipitin arcs
developed with only one of these antisera or
because the reaction with one serum was
much stronger (Fig. 3). Simultaneous
studies with whole human serum to control
the reactions of all reagents greatly increased the confidence in these interpretations.
In one of the two patients with macroglobulinemia (J. S.) a specific precipitin
line was demonstrated in the urine concentrate with anti-IgM, and in the other
ANTIkappa
lambda
I9G
WHOLE SERUM
CONCENTRATE OF
NORMAL URINE *8.B.N.
F I G . 3. Immunoelectrophoresis of urine concentrates from two patients with myeloma. In patient
N o . 44 the reaction is only shown with anli-X, whereas in patient N o . 47 the reaction with anti-x is much
stronger.
478
GREENWALT ET
(A. H.) no studies with anti-IgM were
made but a 19S peak was seen on ultracentrifugation of the urine concentrate.
DISCUSSION
The proteins in the urine were at one time
loosely referred to as "albumin." The modern investigation of proteins in normal
urine may have started in 1951, when Rigas
and Heller24 demonstrated a colloid with
the electrophoretic mobility of albumin.
More recently Grieble and associates10 stated
that 19 proteins that may originate in the
plasma have been described in normal urine.
In addition there may be uroproteins other
than the mucoproteins of Tamm and Horsfall that have no antigenic relationship to
any plasma proteins. Even IgM and a2macroglobulin have been found in normal
urine. 1,10,22 It is therefore not surprising
to have found a protein that behaved serologically like IgM in the urine concentrate
of patient J. S., and another identified as a
19S protein on ultracentrifugation in patient
A. H. (Table 3). Both of these patients had
macroglobulinemia. The authors have also
seen macroglobulins in concentrates of normal urine on ultracentrifugation.
Fragments of immunoglobulins of approximately IS to 3S on ultracentrifugation
and with estimated molecular weights of
from 10,000 to 35,000 have been reported
in normal urine. 1 " 3 ' 7 - "• 20, 2 3 , 2 6 ' 2 7 , 29~31 Poliomyelitis virus-neutralizing activity and the
ability to precipitate diphtheria toxoid have
been attributed to protein molecules of
small size found in urine concentrates. 14,20
From normal urine, Berggard and Edelman 3
have isolated light chains (Y L ) having the
same thermosolubility properties and the
same spectrofluorometric behavior as L
chains obtained from IgG globulins and
Bence Jones proteins.
Large volumes of normal urine must be
used to achieve the concentration necessary to detect and identify components
other than albumin and 7-globulins. In
concentrates prepared from 500 ml. of
normal urine the authors have only occasionally seen precipitin arcs in zones other than
the albumin and 7-globulin ones. Thus far,
no specific precipitin patterns have been
AL.
Vol. 49
seen with anti-K and anti-X reagents, although they would be expected with more
intensely concentrated preparations. It was
not our purpose to study and identify trace
proteins excreted in normal urine; the goal
was to find the critical degree of concentration needed to differentiate normal subjects from patients with dysproteinemias.
Various circumstances prevented study of
each specimen with all of the technics listed
in Table 3, but the data nevertheless indicate that tannic acid-caffeine concentrates
from 500 ml. of urine add diagnostic information not otherwise readily obtained
in patients with myelomatosis and macroglobulinemia and no clinical proteinuria.
Useful information can be gained by 50- to
100-fold concentration of the urine in some
patients with mild clinical proteinuria. For
example, in patient I. H. (No. 47, Table 3)
the 7 spike and the reactions with anti-K
and anti-X were not obtained with the unconcentrated urine.
The data presented in Table 3 do not
establish the nature of the monoclonal
proteins in the urine concentrates. It is
likely that some of the urinary M-peaks
were produced by L chains or their dimers,
but similar patterns would have been found
by concentrating intact monoclonal immunoglobulins excreted in the urine. No
attempt was made to clarify this point. The
reaction of the urinary globulins of some
patients 'wasnot an "all or none" reaction with
one of the two antisera but was distinctly
stronger with either anti-K or anti-X. The
authors are confident in interpreting this
type of pattern as the excretion of predominantly monoclonal proteins because whole
normal serum was run in parallel with each
antiserum. It was not surprising that this
occurred, because considerable amounts of
normal immunoglobulins persist in the sera
of many patients, along with the M-proteins,
and therefore their fragments may also be
found in urine concentrates.
Dobryszycka 5 was able to detect anti-A
and anti-B in tannin-caffeine concentrates
of normal urine. Antibody activity of other
specificities has been found associated with
0.9S to 1.2S globulin fragments (approximate
molecular weight 10,000 to 13,000) isolated
April 1968
CONCENTRATION OF URINARY PROTEINS
from the urine of persons being actively
immunized by the corresponding antigens.
i4,20,23 i t j s possum that similar fragments
of blood group antibodies may be spilled
in the urine during periods of increased
production in response to specific stimuli.
It is also possible that larger amounts of
7S antibodies are excreted when this is the
predominant antibody molecule that is being
produced. In either case mothers, while
they are carrying infants destined to have
ABO hemolytic disease, and perhaps for
some time after the pregnancy, might be
expected to excrete antibody proteins in
urine more regularly and in larger quantities
than "normal" controls. The authors' earliest studies demonstrated anti-A or anti-B
or both activity in all 1000-fold urine concentrates studied. Concentrates of 500-fold
appear to be near the critical range for
distinguishing the "immunized" population
from the "nonimmunized." Unfortunately
the distinction is not absolute. Predictably,
some group 0 persons without known exposure to the A or B antigens had urinary
isoantibody activity. The presence of the
antibody protein cannot be related to the
total quantity of protein in the urine concentrate. The trend does have some value in
the antenatal prediction of hemolytic disease, but has the same drawback as all
previous attempts to predict ABO hemolytic
disease on the basis of the quality of anti-A
and anti-B in maternal serum. All of these
observations have been retrospective, utilizing cooperative women whose infants
had ABO hemolytic disease established in
the laboratory. More studies are indicated
in order to determine whether the distinction is sharper during active immunization.
Studies of group A and group B donors before and .after immunization with soluble
blood group-specific substances were not
conclusive; however, no such studies with
group 0 patients have been done.
SUMMARY
Urinary proteins were precipitated with
tannic acid and were then regenerated by
adding caffeine, which forms an insoluble
tannate complex. Anti-A and anti-B activity was demonstrated in concentrates
479
prepared from 500 ml. of urine. Seven of
10 mothers of infants with ABO hemolytic
disease had urinary antibodies, whereas
only three of 10 group O controls had detectable antibodies. Similar concentrates of
urine from patients with myelomatosis and
macroglobulinemia yielded information on
electrophoretic analysis not obtainable without concentration.
Acknowledgments.
Technical assistance was
provided by Juliet Lord, Anna Scheinman,
C a t h r y n McConnell, and J a n e t Leu. D r s . A. V.
Pisciotta, G. Becker, J . A. Libnoch, and B . H .
Dessel furnished the urine and blood samples t h a t
made these studies possible.
REFERENCES
1. Berggard, I . : Studies on the plasma proteins
in normal human urine. Clin. Chim. Acta,
6: 413-429, 1961.
2. Berggard, I . : On a •y-globulin of low molecular
weight in normal human plasma and urine.
Clin. Chim. Acta, 6: 545-549, 1961.
3. Berggard, I., and Edelman, G. M . : Normal
counterparts to Bence Jones proteins: free
L polypeptide chains of human y-globulin.
Proc. N a t . Acad. S c , 49: 330-337, 1963.
4. Boyce, W. H . , Garvey, F . K . , and Norflcot,
C. M . , J r . : Proteins and other biocolloids
of urine in health and in calculous disease.
I . Electrophoretic studies a t p l l 4.5 and
8.6 of those components soluble in molar
sodium chloride. J. Clin. Invest., 33:
1287-1297, 1954.
5. Dobryszycka, W.: Proteins of the normal
urine. I. Use of tannin and caffeine for concentration of proteins of the normal urine.
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