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The absorption of serum albumin and gamma globulin from the knee joint of man and rabbit.

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The Absorption of Serum Albumin and Gamma Globulin
from the Knee Joint of Man and Rabbit
By GERALDP. RODNANAND MARGARET
J. MACLACHLAN
Studies of the rate of clearance of radioactivity from the knee joints of human
subjects free of joint disease and of
normal rabbits following the intra-articular injection of 1131-serum proteins
indicate that the synovial cavity is readily and equally permeable to both serum
albumin and gamma globulin.
Studios deI rapiditate del clearance de
radioactivitate ab le articulation del
g e m in humanos sin morbo articular e
in conilios normal post le injection intraarticular de proteinas seral marcate con
1191 indica gue le cavitate synovial es
preste- e equalmente permeabile pro
albumina seral e globulina gamma.
B
AUER, Short, and Bennett reported studies on the permeability of the
synovium of the rabbit and dog in 1933, utilizing serologic technics for
the detection of a variety of heterologous pr0teins.l They noted the rapid
appearance of egg albumin and horse serum albumin in the joint fluid of
rabbits after intravenous administration of these proteins, and, in the dog,
their prompt removal via lymphatic channels following injection directly
into the knee joint. They failed to detect horse serum globulin in thoracic duct
lymph after intra-articular injection of this protein and concluded that globulins were removed from a normal joint with difficulty, if at all. After reviewing this matter some years later, and adding their own observations on the
removal of colloidal and finely particulate matter from the joint cavity of
the rabbit, Adkins and Davies2 concluded that particles considerably larger
than those of serum globulin passed rapidly into the regional lymphatic
glands.
In the present experiments, we have sought to obtain information concerning the permeability of synovium to protein by examining the clearance of
radioiodine ( 11")-labeled serum albumin and gamma globulin injected into
the knee joints of rabbits and of human subjects free of joint disease. The use
of serum proteins in this regard is supported by the studies of Schmid and
MacNair which indicate that the proteins of normal synovial fluid are identical in character with those of plasma.3 Our findings confirm the observations
of others on the rapid absorption of serum albumin and indicate similar
ready permeability of synovium to serum gamma globulin.
EXPERIMENTAL
PROCEDURE
Iodine (1131)-lubeledproteins.-The Il~l-r&bitserum albumin and gamma globulin used
in this study were obtained from the Department of Pathology, University of Pittsburgh.'
F r o m the Department of Medicine, Uniucrsity of Pittsburgh, Pittsburgh, Pa.
This inuestigution wus supported by a reseurch grunt from the Western Penruylvaniu
Chapter of the Arthritis and Rheumatism Foundation and by research grant B-1287, United
States Public Health Service.
Presented ut the Annual Meeting of the American Rheumatism Association, Washington,
D. C., June, 1959.
'We wish to thank Miss Maria Deichmiller for supplies of these labeled proteins.
152
SERUM ALBUMIN AND Y-GLOBULIN ABSORPTION FROM KNEE
153
P l - h u m a n serum albumin and gamma globulin were purchased from Abbott Laboratories.
The homogeneity of the 1131-hurnan serum protein fractions was checked by adding small
quantities of the labeled albumin or gamma globulin to human serum and then separating
the proteins by means of filter paper electrophoresis. When portions of the paper strip
containing the individual protein fractions were assayed in a well-type scintillation counter,
radioactivity was found to be confined, respectively, to the albumin or gamma globulin
moiety.
Determination of clearance of serum proteins from knee joint following intra-articular injectimN.-Hospitalized patients free of joint and circulatory disease each received 3 ,UC. of
1131-human serum albumin or gamma globulin. The radioactive protein was added to 3
to 5 ml. of either 0.9 per cent NaCl solution or the subject's own plasma, and the mixture,
containing either relatively minute or relatively large doses of protein, was injected into a
suprapatellar bursa. All patients remained at bed rest during the course of study.
Mature New Zealand white rabbits received both homologous and heterologous serum
albumin and gamma globulin. The II"-labeled proteins were diluted with either 0.9 per
cent NaCl solution or rabbit plasma, and 1 ml. of the mixture, containing 1 pc. of radioactivity, was injected into the knee joint via a 25 gage needle inserted directly through
the subpatellar tendon. The animals were confined to their cagcs during the period of study
but were not otherwise restricted in activity.
The gamma radiation of the knee joint was measured by means of a collimated scintillation probe kept at a fixed position immediateIy above the infrapatellar (in the case of
the rabbits) or suprapatellar (in the case of the human subjects) ligament of the flexed
joint. The area of detection of radioactivity encompassed by the probe at the level of the
knee joint was 17 cm. in diameter. It thus included in the case of the rabbit the prolongation of the synovial membrane of the knee about the tendon of the extensor digitorurn Zongus.
Duplicate counts were obtained a t intervals following the injection of radioactive protein,
and the values corrected for background activity and natural decay of isotope. The results
are expressed in figures 1 to 4 in terms of the change in radioactivity of the knee joint as a
percentage of the amount present in the joint immediately following injection.
Determination of appearance of serum proteins in peripheral blood following intraarticular injection of knee.-In 6 patients free of joint disease, the amount of Ilal-human
serum albumin or gamma globulin injected into the knee joint was increased to 40 pc.
Samples of serum and of the various ,protein fractions obtained by filter paper electrophoresis were assayed for radioactivity a t intervals beginning 15 minutes after injection. In
a similar manner, the serum protein fractions of 5 rabbits were analyzed for radioactivity
following the intra-articular administration of 12 pc. of 1131-rabbit serum albumin or
gamma globulin.
OBSERVATIONS
Clearance of Serum Proteins from the Knee Joint of the Rabbit
Serum albumin.-Both rabbit and human serum albumin, diluted in saline
solution, and injected in the amount of 0.1 mg., were rapidly removed from
the knee (table 1; fig. 1).An average of 25 per cent of the original radioactivity remained in the joint after 24 hours. At 48 hours, the clearance of
radioactivity was nearly complete. After this time the low level of radioactivity of the joint and the circulation of radioactive albumin in the blood
prevented any further accurate estimation of the amount of local, i.e., noncirculating, radioactivity. The peripheral blood was found to contain nondialyzable radioactivity as early as 15minutes after the intra-articular injection
of 1131-serum albumin. This activity was present in the albumin fraction
obtained by electrophoresis of the serum proteins.
Serum gamma globulin.-The radioactivity of 0.03 to 0.6 mg. of salinediluted 1131-serumgamma globulin was absorbed from the knee joint of the
154
RODNAN AND MACLACHLAN
Table 1.-The
Clearance of Llsl-Serum Proteins from the Knee Joint following
Intra-articular Iniection in Normal Rabbits
% of original radioactivity of
Type and
amount of
protein injected
No. of
animals
studied
RSA, 0.1 mg.
HSA, 0.1 mg.
4
6
60 k 6*
81 2 6
25 + 5
28 ir 9
9+4
8t3
RSGG, 0.03 mg.
HSGG, 0.6 mg.
5
5
74 46
70 I
3
24 t 4
34 t 4
10 -+ 2
16 c 2
RSGG, 10 mg.
RSGG, 10 mg.
150
U. hyaluronidase
5
57 k 8
16 F 1
6-tl
5
58 t 8
16 F 4
8-+3
+
4 hr.
joint, hours after injection
24 hr.
48 hr.
RSA = rabbit serum albumin, HSA = human serum albumin, RSGG
gamma globulin, HSGG = human serum gamma globulin.
"Mean and standard deviation.
12 hr.
4311
=
rabbit serum
rabbit at a rate which did not differ significantly from that of albumin (table
1; fig. 2 ) . The clearance of the globulin was virtually complete at 72 hours.
There was again little, if any, difference between the rate of removal of the
heterologous and the homologous protein. The peripheral blood was found
to contain increasing amounts of radioactivity beginning 15 minutes after
injection of the labeled protein. This radioactivity was confined to the gamma
globulin fraction.
When the total amount of gamma globulin injected into the knee was
sharply increased by administering the tracer dose of labeled protein in 1
inl. of rabbit plasma instead of saline solution, there was no significant
clifference in the rate of removal of radioactivity from that observed followI'3'-SERUM ALBUMIN
AMOUNT OF PROTEIN. 0.1 mg
II3'-SERUM GAMMA GLOBULIN
AMOUNT PROTEIN INJECTED
VOLUME INJECTATE: I ml.
0A.m
- RABBIT S A
OQOoAV-HUMAN
SA
RABBIT S G G - 0 0 3 m q
HUMAN S G G - 0 6 mg
VOLUME INJECTATE
OAT.4
OAPO 0
HOURS AFTER INTRA-ARTICULAR INJECTION
OF 1 1 3 1 - s ~
I ml
-RSGG
-HSGG
HOURS AFTER INTRA-ARTICULAR INJECTION
OF 1131
-
SGG
Fig. 1.-The clearance of rabbit and human P1l-serum albumin from the knee
joint of the rabbit, as determined by surface counting technic.
Fig. 2.-The clearance of rabbit and human 1131-serumgamma globulin from the
knee joint of the rabbit.
155
SEAUM ALBUMIN AND y-GLOBULIN ABSORPTION FROM KNEE
ing the injection of more minute quantities of the protein (table 1; fig. 3).
Inclusion of 150 U. of hyaluronidase ( Wydase) in the intra-articular injection
of the gamma globulin appeared to have no effect upon the rate of clearance
of the radioactive protein from the joint.
the Human
Serum albumin.-In seven subjects the tracer dose of P3l-human serum
albumin was mixed in 3 to 5 ml. of each individual’s plasma, the approximate
amount of serum albumin injected ranging from 90 to 160 mg. Five ather
individuals were given minute quantities of I131-albumin, 0.1 to 0.25 mg., in
3 to 4 ml. of 0.9 per cent NaCl solution. Both the small and large doses of
albumin were removed from the joint at a remarkably similar rate (table 2),
averages of 37 and 40 per cent of original radioactivity being absorbed at
24 hours. Appreciable quantities of radioactivity remained at the site of injection after 72 hours, however. The serum albumin fraction of the peripheral
blood of two patients was found to contain radioactivity within 15 minutes
after injection of the I1”-protein into the suprapatellar bursa.
Serum gamma globulin.-Six patients, all free of joint disease, received
the tracer dose of 1131-humanserum gamma globulin mixed in 3 to 4 ml. of
plasma. The total amount of gamma globulin given in the intra-articular
injection was approximately 30 to 40 mg. The radioactivity of the knee declined at a rate remarkably similar to that following the injection of 11”albumin (table 3; fig. 4). At 24 hours, an average of 61 per cent remained in
the joint; by 48 hours the average amount left was 43 per cent of the original
dose. Approximately one-fourth of the radioactivity was still present at the
site of injection after 96 hours. Radioactive gamma globulin was detected
in the peripheral blood of four patients within 15 minutes after the intraarticular injection of the 1131-gamma globulin, and increased steadily in
amount for a period of 48 hours after the intra-articular injection.
Clearance of Serum Proteins from the Knee joint of
Table 2.--The Clearance of Z131’-€ZumanSerum Albumin from the Knee Joint
f o l l m i n g Intra-articular Znjection in Human Subjects Free of Joint Disease
Subject
number, age
and sex
% of original radioactivity of joint,
hours after injection
4 hr.
24 hr.
48 hr.
72 hr.
a. Injection of 0.1 to 0.25 mg. albumin in 3 t o 4 ml. of 0.9% NaCl solution
1,35, M
94
72
52
38
2,34, F
64
49
3,62, F
76
41
28
4,24, F
83
65
44
32
5,34, F
98
73
55
42
Mean and S.D.
88 4 7
63 2 1 1
46 rt 9
b. Injection of 90 to 160 ing. albumin in 3 to 5 ml. of plasma
1,41, F
56
29
2, 36, F
77
60
29
3,35, F
96
72
59
4,65, M
68
63
43
5,% F
70
48
33
28
6,58, M
93
61
7,60,F
92
62
45
36
Mean and S.D.
83 -t- 11
60 & 6
40 3z 10
156
RODNAN AND MACLACHLAN
Table 3.-The Clearance of IIS1-HumanSerum Gamma Globulin from the Knee
Joint following Intra-articular Injection in Human Subjects
Free of Joint Disease
% of original radioactivity of joint,
hours after injection
Subject
number, age
and sex
24 hr.
4 hr.
72 hr.
48 hr.
96
Injection of 30 to 40 mg. gamma globulin in 3 to 4 ml. of plasma
1, 35, F
90
74
54
45
2,19, F
82
65
52
34
3,26, F
82
63
43
31
82
60
41
28
4,74, M
5,40, M
80
52
39
27
6,39, F
71
50
30
20
Mean and S.D. 81 +- 5%
61 2 8%
43 -t 8%
31 & 7%
RABBIT SERUM GAMMA GLOBULIN
t3I-
hr.
37
25
24
22
20
15
24 2 6%
HUMAN SERUM GAMMA GLOBULIN
AMOUNT OF PROTEIN 10 mq
AMOUNT OF PROTEIN 30-40mg
VOLUME INJECTATE
VOLUME INJECTATE 3-4 ml
*A789
OOAVO
I ml
- CONTROL STUDY
- HYALURONIOASE
(150 units Included
in injection)
Y
,I
I
O f
4
:
41.9
;2
HOURS AFTER INTRA-ARTICULAR INJECTION
OF
-RSGG
1
Of;
I
24
i 8
;2
916
HOURS AFTER INTRA-ARTICULAR INJECTION
OF 113'-HSGG
Fig. 3.-The clearance of Pf-rabbit serum gamma globulin from the knee joint
of the rabbit with and without simultaneous administration of hyaluronidase.
Fig. 4.-The clearance of Pl-hurnan seruni gamma globulin from the knee
joints of 6 patients free of rheumatic disease.
DISCUSSION
It would appear from this study that the synovium of the knee joint of
man and of rabbit is as readily permeable to serum gamma globulin as it is
to serum albumin. The discrepancy in the rate of clearance of these proteins
observed between rabbit and man may be more apparent than real, however,
and can be accounted for by a difference in intra-articular hydrostatic pressure resulting from the injection of the 1131-protein-containingfluid. Edlund
has shown that high initial pressures following intra-articular injection lead
to an increased absorption of fluid and of hem~globin.~
In the present experiments, the injection of 1 ml. into the knee joint of the rabbit produced visible
distention and considerabIe increase in intra-articular pressure, whereas in-
SERUM ALBUMIN AND Y-GLOBULIN ABSORPTION FROM KNEE
157
jection of 3 to 5 ml. of fluid into the human knee had relatively little effect.
The rate of clearance of 1131-serum albumin from the joints of normal subjects reported herein is less rapid than that observed by Ahlstrom and Kuipers
and their respective co-workers5p6in studies of individuals with active rheumatoid arthritis. There is relatively little difference, however, when the absorption of the protein is compared to that noted in patients with inactive joint
disease or those with active arthritis who have received an intra-articular
injection of hydrocortisone,j circumstances in which the permeability of the
synovium may be considered as having been restored to a more normal state.
SUMMARY
1. The synovium of the knee of man and of rabbit is readily permeable to
homologous I1"-serurn aIbumin and gamma globulin, and there appears to
be little difference in the rate of absorption of these two proteins following
their intra-articular injection.
2. Radioactively labeled albumin and gamma globulin appear in the
peripheral blood within 15 minutes after intra-articular injection of the
respective proteins.
3. Under the conditions of the present experiments, absorption of both
serum albumin and gamma globulin from the knee joint of the rabbit is
virtually complete within 72 hours after intra-articular injection. In the case
of absorption from the joints of human subjects free of joint disease, appreciable quantities of radioactivity (approximately one-third of the amount injected) remain in sits as long as 72 hours after intra-articular injection.
4. The simultaneous intra-articular administration of hyaluronidase appears
to have had no effect upon the rate of clearance of gamma globulin from the
knee joint of the rabbit.
REFERENCES
1. Bauer, W., Short, C. L., and Bennett, G.
A,: The manner of removal of proteins from normal joints. J.ExPer*Md.
57:419, 1933.
2. Adkins, E. W. D., and Davies, D. V.:
Absorption from the joint cavity. Quart.
J.Exper.Physio1. 30~147, 1940.
3. Schmid, K., and MacNair, M. B.: Characterization of he proteins of certain
postmortem human synovial fluids. J.
Clin.Invest. .37:708, 1958.
4. Edlund, T.: Studies on absorption of colloids and fluid from rabbit knee joints.
Acta physiol. scandinav. Supplementum
62, 1949.
5. Ahlstrom, Gedda p. o.,and &&erg, H.:
Disappearance of radioactive serum
albumin from joints in rheumatoid arthritis. Acta Rheum. scandinav. 2~129,
1956.
6. K ~ i ~ e r sR., K. W., F r a n k C., and
Robert, W. N.: Effects of antirheuinatic drugs on the resorption of 1131labeled albumin from the synovial fluid.
Acta Rheum. scandinav. 231, 1956.
Gerald P . Rodnan, M.D.,Assistant Professor of Medicine,
University of Pittsburgh, School of Medicine, Pittsburgh, Pa.
Margaret J . Maclachlan, M.D.,lnstructor in Medicine, University of Pittsburgh, School of Medicine, Pittsburgh, Pa.
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