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The Latex fixation test in the spinal fluid and urine of patients with rheumatoid arthritis.

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The Latex Fixation Test in the Spinal Fluid and
Urine of Patients with Rheumatoid Arthritis
By WILLARD
R. STARNES,
ALEXANDERULLOAAND HOWARD
L. HOLLEY
The finding of an abnormal macroglobu- Plure gruppos de investigatores ha isolin in the serum of patients with rheu- late e characterisate un macroglobulina
matoid arthritis has been isolated and anormal in le sero de patientes con
characterized by several groups of in- arthritis rheumatoide. Esseva studiate
vestigators. A study was undertaken to le question del presentia de iste globustudy other biological fluids as to the lina anormal in altere liquidos biologic.
presence of this abnormal globulin. Liquid0 spinal e urina esseva obtenite
Spinal fluids and urines were obtained ab patientes con arthritis. Le patientes
from patients with arthritis. The pa- esseva dividite in tres gruppos: (1)
tients were divided into three groups: Spondylitis ankylosante; (2) osteoarth(1) ankylosing spondylitis; (2) osteo- ritis; e (3) arthritis rheumatoide, active
arthritis; and (3) rheumatoid arthritis, e non-tractate. Le sero ab patientes con
active, untreated. The serum of patients arthritis rheumatoide esseva positive in
with rheumatoid arthritis gave positive le tests de fixation a latex. Le test de
latex fixation test. Latex fixation test on fixation a latex in le liquid0 spinal ab
the spinal fluids from the three groups omne le tres gruppos esseva negative
gave a negative latex agglutination. The pro agglutination a latex. Le urinas e
urines and spinal fluids were concen- le liquidos spinal esseva concentrate a
trated to a known volume with poly- un cognoscite volumine per medio de
vinyl pyrrolidone (P.V.P.).There was pyrrolidona polyvinylic. Esseva trovate
found an increase in the total protein un augment0 del total hexosas ligate a
bound hexoses in the three latex posi- proteina in le tres concentratos urinari
con positivitate a latex.
tive urine concentrates.
A
BNORMALITIES IN THE electrophoretic pattern of proteins in patients
with rheumatoid arthritis have been identified in serum, synovial fluid
and spinal
It has been reported that in the serum and spinal fluid of
patients with active rheumatoid arthritis, there was an increase in the relative
concentration of the alpha-1, alpha-2, and gamma globulins and a decrease
of the albumin fraction; the tendency being for the alpha globulins to rise
early in the course of the disease, followed by au elevation of the gamma
globulin.* Causes for alterations in an individual protein fraction, however, are
not well known, although there is evidence that thc gamma globulins are increased whenever considerable inflammation or tissue destruction is present or
when the gamma globulin antibody formation is increased.
The rheumatoid factor, a hemoagglutinating substance, has been isolated
or partially isolated and characterized from the serum and synovial fluid of
patients with rheumatoid a r t h r i t i ~By
. ~ means of electrophoresis of those fluids,
by
the factor has been found to migrate within the gamma f r a ~ t i o n However,
.~
~
_
_
_
From the University of Akh:ir:ru Medicul Center und Veterans Administration Hospital,
Birmingham, Ala.
Supported in part by grant (A-3555) from the National institute of Arthritis and Metabolic
Diseases, National Institutes of Health, Bethesda, Md.
364
LATEX FIXATION TEST IN RHEUMATOID ARTHRITIS
365
means of ultracentrifuge studies it has been demonstrated that the factor can
be separated from the normal gamma globulin.G
Since Singer and Plotz6 first applied latex particles to the hemoagglutination
system test for rheumatoid arthritis, numerous other workers have confirmed
the validity of this test in serum.i Others have found it positive in synovial
fluid as well. It was deemed of interest to determine whether the so-called
rheumatoid factor, which apparently determines the results of the agglutinaticn test for rheumatoid arthritis, was also present in other body fluids such
as the cerebrospinal fluid and urine of patients with rheumatoid arthritis.
Quantitative studies of total protein bound hexose content as well as ultracentrifuge characterization of the rheumatoid factor were also conducted.
METHODS
Three groups of adult male patients were studied: ( 1) Seven patients with the diamosis
of ankylosing spondylitis; ( 2 ) ten patients with the diagnosis of rheumatoid arthritis, active,
untreated; and (3)three patients with the diagnosis of osteoarthritis. The latex agglutination was positive only in the patients with rheumatoid arthritis. Serum and cerebrospinal
fluid latex agglutinations were performed by the method of Singer and Plotzo and by the
Hyland RA-Test." The urine, 250 ml., was dialyzed against running tap water for eight
hours and then concentrated to 1.0 ml. with polyvinyl pyrrolidone (P.V.P.)for 24 hours
at 5 C. The slide latex agglutination test was performed on 1:20 and 1:40 dilutions of the
urine concentrates. All urines were tested for albumin prior to dialysis.
Ten to 15 ml. of cerebrospinal fluid were dialyzed with P.V.P. to a volume of 0.1 ml.
Total urine concentrate proteins were determined by the Biuret method.8 Total protein
bound hexose levels were determined by the Thymol-H,SO, meihod.0 Urine electrophoretic
studies were performed on a Spinco Model R electrophoretic apparatus using Verona1
buffer p H 8.6 0.05 mol. Ultracentrifuge studies were performed on a Model E Spinco
ultracentrifuge.
RESULTSAND DISCUSSION
Table 1gives the results found in serum and cerebrospinal fluid. All cerebrospinal fluids were found to give negative agglutinations on both the latex agglutination and Hyland RA-Tests. Apparently, the rheumatoid factor is of
such a high molecular weight that it does not pass the blood brain barrier.
Karcher and associatesl0 suggested that in certain neurologic disease processes
where there is an increase in gamma globulin, this particular globulin originates in the meningeal envelope, other globulins possibly arising from the serum.
Ten specimens of urine of patients with rheumatoid arthritis were concentrated and three were found to give positive latex agglutinations (table 2).
This finding indeed is interesting because, if we assume that the rheumatoid
factor is a macroglobulin which apparently does not pass the choroid plexus, it
is difficult to visualize how the same globulin could appear in the urine of
individuals who did not have any detectable kidney disease. As shown in
table 2, the three patients exhibiting the positive urine titers had relatively
high serum titers. It is also of interest to note that the thirteen patients gave
negative routine urine tests for albumin which were confirmed on electro"Hyland Laboratories, Los Angeles, Calif.
366
STARNES, ULLOA AND HOLLEY
Table 1.-Latex
Firation liter of Serum and Cerebrospinal Fluid
Patients with Rheumatoid Arthritis
Rheumatoid Arthritin
Spondylitm
Serum
CSF
L !-)
c
(-1
(-1
(-1
(-1
(-1
(-1
(--!
L
(-)
(-1
K (-)
E (-1
H (-)
D (-1
Serum
CSF
L 1:2560
CI 1:5120
(-)
(-)
(-!
M 1:2560
c
H
1:1280
1:1280
G 1:1280
V 1:680
H 1:320
K 1:655,360
of
Osteonrthritis
Serum
CSY
-
c (-1
(-)
C" ( - )
(-1
L (-1
(-)
(-)
(-)
(-)
(-i
(-1
(-1
F 1:20,480
~ _ _ _( - ) _ _ - ~
Table 2.-Latex Firation Titer and Total Protein of Serum and Urine Concentrates
of Patients with Rheumatoid Arthritis
~
M.
K.
F.
Normal
Serum
Urine'
1:2560
1 :855,360
1:20,480
+
+
+-
-
T. P. (me. 56)
360
280
360
80
'Performed by Slide Test.
phoretic studies of the same specimens. Patient M. had a total protein content
of 360 mg. per cent; F., 360 mg. per cent; and K., 280 mg. per cent on the urine
concentrates.
As reported previously, the serum euglobulin fraction in patients with
rheumatoid arthritis showed an increase in total protein bound hexose content." The present study, as shown in table 3, indicates that the total protein
bound hexose content in the urine concentrates was also markedly increased.
Comparatively, the percentage of total protein bound hexose in the urine total
protein of these three patients was increased over the average of ten normal
subjects.
The ultracentrifuge studies of the urine protein concentrates revealed a
globulin with a mobility of O.QS, but not the presence of the characteristic 19s
globulin usually associated with the rheumatoid factor. This finding, however,
may be related to the low concentration of the macroglobulins in urine or other
agglutinating substances which would give a positive test.
SUMMARY
1. Seven patients with ankylosing spondylitis, nine patients with rheumatoid
arthritis, active, and three patients with osteoarthritis gave negative latex
fixation test on the cerebrospinal fluid.
2. Of the thirteen patients examined with rheumatoid arthritis and high
serum titers, three gave positive latex fixation tests on urine protein concentrates.
3. All urine concentrates were examined electrophoretically and found to be
within normal limits. There was an increase in the total protein bound hexose
content of the urine protein concentrates of the patients with rheumatoid arthritis.
367
LATEX FIXATION TEST IN RHEUMATOID ARTHRITIS
___
Table 3.-Carbohydrate and Protein Content of Urine Concentrates
Name
M.
F.
Total Protein
mg. %
T.P.R.H.*
mg. 90'
Percent T.P.U.H.'
of T.P.t
360
360
280
28.8
70.8
44.4
8%
20%
16%
8.8
11%
K.
Normal
( Av. 10 samples)
80
'Total Protein bound hexose (T.P.B.H.).
tTotal protein (T.P.).
4. Ultracentrifuge studies on the latex positive urine concentrates revealed a
component with a mobility of 0.9s.
REFERENCES
1. Ropes, M. W., Perlmann, G. E., Kaufman, D., and Bauer, W.: The electrophoretic distribution of proteins in
plasma in rheumatoid arthritis. J.
Clin. Invest. 33~311,1954.
2. Perlmann, G.E., Ropes, M. W., Kaufman, D., and Bauer, W.: The elertrophoretic patterns of proteins in synovial fluid and serum in rheumatoid
arthritis. J. Clin. Invest. 33:319, 1954.
3. Starnes, W.K.,Hammack, W. J., Ulloa,
A., Holley, H. L., and Hill, S. R.:
Studies on serum and spinal fluid
protein patterns in patients with
rheumatoid arthritis spondylitis. Clin.
Res. 6:1W, 1958.
4. Svartz, N., Carlson, A., Schlosamann,
K., and Ehrenberg, A.: Isolation of the
rheumatoid factor. Acta med. scandinav. 160:87, 1958.
5. Kranklin, E. C., Holman, H. R., MullerEberhard, H. I., and Kunkel, H. J.:
An unusual protein component of
high molecular weight in the serum of
certain patients with rheumatoid arthritis. J. Exper. Med. 105425, 1937.
6. Singer, J. M., and Plotz, C. M.: The
latex fixation test. I. Application to
the serologic diagnosis of rheumatoid
arthritis. Ani. 1. Med. 21:888, 1956.
7. Holley, H. L., Ulloa, A,, Henry, M.,
and Johnson, M. L.: A comparison
of the latex fixation-whole serum,
latex fixation-euglobulin fraction, and
bentonite flocculation test in the laboratory diagnosis of rheumatoid arthritis. Am. J. hf. Sc. 237345, 1959.
8. Fister, H. J.: Manual of standardized
procedures for spectrophotometric
chemistry. New York, Standard Scientific Supply Corp., 1960, p. MA.
9. Shetlar, M. R., and Masters, Y. F.: Use
of thymol-sulfuric acid reaction for
determination of carbohydrates in
biological materials. Ann. Chern. 29:
402, 1957.
10. Karcher, D.,and Lowenthal, A.: L'origine des proteines de liquide cephalorachidien. Clin. Chim. Acta. 3:78,
1958.
11. Starnes, W. R., Ulloa, .4., and Holley,
H. L.: Glycoprotein studies on the
rheumatoid factor. Am. J. Med. 27:
326, 1959.
Willurd R. Starnes, M.S., C,hief Biochemist, Metabolic Laboratories, Veterans Administration Hospital, Birmingham, .4h.
Alexander Ulloa, LM.D., lmtructor in Medicine, University of
Alabama Medical Cater, Birmingham, Ah., and Trainee of
the National Imtitute of Arthritis and Metabolic Diseases,
National lnstitutes of Health, Bethesdu, M d .
Howard L. Holley, M.D.,Professor of Medicine and Director
of the Section of Arthritis and Rheumatism, University of
Alubamu Medical Center and Veterans Administration Hospital, Birmingham, A h .
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