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Synovial fluid hyaluronate in rheumatoid arthritis.

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Synovial Fluid Hyaluronate in Rheumatoid Arthritis
By DAVIDHAMERMAN
AND HILDASCHUSTEFI
By several analytic methods, the hyaluronate hexosamine concentration of
synovial fluid was found low in the
joint effusions of rheumatoid arthritis in
comparison to normal synovial fluid. .4n
increase in nonhyaluronate hexosamine
was observed in rheumatoid synovial
fluid of increased volume. Relative viscosity measurements after equilibrating
hexosamine Concentration by dilution revealed few differences between rheumatoid and normal fluid.
Plure methodos analytic monstrava basse
concentrationes de hexosamina hyaluronate in le effusiones de liquido synovial de patientes con arthritis rheumatoide, comparate con normal liquido
synovial. Esseva observate un augment0
de hexosamina non-hyaluronate in rheumatoide liquido synovial de volumine
supranormal. Mesurationes de viscositate
post equilibration del concentration de
hexosamina per dilution non revelava
multe differentias inter liquido rheumatoide e normal.
T
HE CONCENTRATION of hyaluronate in synovial fluid from patients
with rheumatoid arthritis* and in normal synovial fluid has been determined by a number of workers. Using different methods, some workers have
reported a similar concentration,lP2 and others a decreased c ~ n c e n t r a t i o n ~ ~ ~
of hyaluronate in rheumatoid synovial fluid compared with normal fluid.
In this repor:, the hyaluronate concentration of rheumatoid synovial fluid
was determined by several methods described in a previous study of normal
synovial fluid.6 Hyaluronate was precipitated in mucin clots from rheumatoid
synovial fluid by adding acetic acid or a cobalt salt to the fluid, and was
measured indirectly by determining hexosamine in these clots. A part of the
total hexosamine of synovial fluid is not a component of hyaluronate, and it
was shown in the method adopted that no hexosamine other than hyaluronate
hexosamine was precipitated in mucin clots and that no hyaluronate hexosamine remained in the supernatant after a mucin clot was formed. Another
method used to determine hyaluronate in rheumatoid synovial fluid was to
measure the fall in hexosamine after digestion of the fluid with testicular
hyaluronidase and dialysis of the digestion products. Figures for hyaluronate
hexosamine in rheumatoid synovial fluid obtained by a mucin clot method
and by the hyaluronidase digestion method agreed.
Normal and rheumatoid synovial fluids were diluted by adding a buffer
to the fluids until the hyaluronate hexosamine concentration of each fluid,
measured by the hyaluronidase digestion method, was the same. The relative
From the Department of Medicine, Albert Einstein College of Medicine, and the Bronx
Munictpal Hospital Center, New York, N.Y.
This study was supported by Graduate Training Grant 2A-5082 (a) (C), National Institute
of Arthritis and Metabolic Diseases, Nattcnal Institutes of Health, and by a grant from the
New York State Chapter, Arthritis and Rheumatha Foundation.
One of the authors (D.H.) is Markk Scholor in Medtcal Science.
'Synovial fluid obtained from patients with rheumatoid arthritis is more briefly noted
hereafter
RS
rheumatoid synovial fluid.
523
524
DAVID HAMERMAN AND HILDA SCHUSTER
viscosity of these normal and rheumatoid fluids of equivalent hyaluronate
hexosamine concentration was then compared.
METHODS
AND PROCEDURES
.itetkods
Patients with definite rheumatoid arthritis: with active disease at some time in the
course of these studies, were used. All patients were female. Synovial fluid was obtained
from the knee joints. Normal synovial fluid was obtained from the knees of eight deceased
and two living subjects without evidence of joint disease and within the age range of
most of the patients (50 to 66 years).
Samples of synovial fluid for analysis were weighed rather than pipeted, and results
are expressed in terms of grams rather than milliliters of synovial fluid. The density of
rheumatoid synovial fluid was found not to differ from water within the limits of the
precision of the method. Grams and milliliters may therefore be used interchangeably.
1. Hexosamine determinations on synooial fluid and mucin clots.-The method of measuring total hexosamine in synovial fluid, the technic of forming mucin clots by adding
acetic acid or a complex cobalt salt to synovial fluid, and the method of analyzing for
hexosaniine in mucin clots have been reported in detail." One additional method to precipitate hyaluronate hexosaniine in mucin clots from rheumatoid synovial fluid was studied.
Synovial fluid (0.3Gm. to 0.4 Gm. ) was weighed in Pyrex test tubes ( 13 by 100 mm. )
and diluted 1:l with tap water. A mucin clot was formed in the cold by adding a volume
of glacial acetic acid equal to 1 per cent of the total volume of diluted synovial fluid.
2. Hyaluronidase digestion of synoviol fluid and mucins.-This
was carried out exactly
as previously described." Hexosamine of a dialyzed control less hexosamine of a hyaluronidase-treated dialyzed sample was a measiire of hyaluronate hexosamine.
Procedures
1. Herosomine of mucin clots.-By
contrast with the findings in nornial synovial fluid,
all the methods used to precipitate hyaluronate hexosamine in mucin clots from rheumatoid synovial fluids did not give the same results (table 1). There was always less
hexosamine in mucins precipitated with glacial acetic acid from synovial fluids diluted
1:5, and more hexosaniine in mucins precipitated with the cobalt salt. These differences
were due to incomplete precipitation of hyaluronate hexosamine by the former method,
and to precipitation of proteins containing nonhyaluronate hexosamine by the latter
method. The failure o f glacial acetic acid to precipitate all the hyaluronate hexosamine
from fluids diluted 1:5 was shown by the considerable loss of hexosamine following
hyaluronidase digestion and didysis of these mucin supernatants (table 1). Precipitation
by the cobalt salt of protcins containing nonhyaluronate hexosamine was shown by the
high nitrogen to hexosainine ratios of these mucins and by the failure of streptococcal
hyaluronidase to digest and render dialyzable all the hexosamine in these mucins. Almost
all the hexosamine in mucins precipitated by glacial acetic acid from fluids diluted 1:1
was digested by hyaluronidase and rendered dialyzable. No hexosamine was lost after
hyaluronidase digestion and dialysis of these mucin supernatants. This method was therefore used to determine hyaluronate hexosamine in rheumatoid synovial fluids by the
mucin clot technic.
2. Hyaluronidase digestion of synovial fluid.-Incubation
of synovial fluid at pH 4.8
with testicular hyaluronidase and subsequent dialysis removed that part of the total hexosamine that was hyaluronate hexosaniine. Table 1 shows that in normal synovial fluid
a figure for hyaluronate hexosamine obtained by this method agreed closely with hexosamine
of any mucin. In rheumatoid synovial fluids hyaluronate hexosamine obtained by the
method of hyaluronidase digestion agreed only with the hexosamine of rnucins precipitated
by glacial acetic acid from fluids diluted 1:l with water.
Rheumatoid synovial fluids were examined for the presence of hyaluronidase inhibitors,'
and none were found. Table 2 shows that testicular hyaluronidase completely digested and
rendered dialyzable the hexosamhe of chondroitin sulfate or a mucin solution (as a source
of hyaluronate ) in rheumatoid synovial fluid.
525
SYNOVIAL FLUID HYALURONATE I N RHEUMATOID ARTHRmS
TABLE1.-Studies
on the Specificitu of Methods to Determine Hyaluimate Hexosamine
in Rheumatoid Synooial Fluids’
Results of hyaluronidase digestion and dialysis
HyalurMucin
Control
Total hx. onate h r . molar ratio
hx.
mg./Gm. ma./Gm.
N./hx.
mg./Grn.
Sample
After
76TRU
T. Hyd.
hx.
ma./Gm.
After
Hx.
75TRU
FIX.
rendered S. Hyd. rendered
dialyzable
hx.
dialyzable
%
mg./Gm.
%
~
Synovial fluid
GL mucin
Z 10% A. mucin
f co. mucin
GI. sup?.
10% A. sup’t.
co. sup’t.
1.60
Synovial fluid
GI. mucin
p G1’. m u c h
S 10% A. mucin
co. mucin
? G1. sup’t.
Gl’. sup’t.
10% A. sup’t.
co. 3upY.
.84
Synovial fluid
1
2
a
f
16.1
266
240
.067
.027
20.4
.228
16.8
253
.024
.021
.120
.124
.27
-20
-28
.a2
.43
1.37
89
80
.030
.028
R8
92
8
1
.031
88
.177
.122
.166
.117
4
A24
.421
32
88
.313
.227
.069
.066
26.1
.299
.964
294
204
220
.222
.om
78
72
74
.211
42
76
68
62
69.4
10% A. mucin
19.2
24.6
co. mucin
.64
61.6
.606
.240
.241
274
.la4
.189
.210
.210
21
7
6
6
384
37
.014
82
76
.058
GB
.lo3
G1’. sup’t.
236
.05Y
.OQ2
206
co. sup%.
.236
365
.l54
10% A. sup?.
74
18.1
19.4
-51
.49
.b4
g GI’.mucin
S
1.18
1.22
1.26
1.23
219
.076
18
7
7
*Abbreviations : hx.. hexosamine; N.. nitrogen; GI.. g!aeial acetic acid added to synovial fluid diluted
1:5; GI,., glacial acetic acid added to synovial fluid dilutpd 1:l: 10% A., 10% acetic acid; Co., cobalt
salt; sup’t.. supernatant; T. Hyd., teaticular hyaluronidase; S. Hyd.. streptococcal hyaluronidase; TRU.
turbidity reducing units.
TABLE?.-Digestion
b y Testiculur Hyuluroniduse of Substrutes Added to
Rheumatoid Synotrial Fluid
Hx. of substrate digested by T. Hyd.
and rendered dia!yzable
Substrate
(%)
Chondroitin sulfate
Chondroitin sulfate
rheumatoid S.F.
Normal mucin
Noniial mucin + rheumatoid S.F.
+
97
95
89
81
kXJLTS
1. Herosumine in rheumatoid synoviul fluid.-The total hexosamine and
lryaluronate hexosamine in normal fluids and in rheumatoid synovial fluids
are compared in table 3. Hyaluronate hexosamine in rheumatoid fluids was
determined by hexosamine analyses of mucin clots formed with acetic acid.
In some rheumatoid fluids the technic of hyaluronidase digestion was also
used to measure hyaluronate hexosamine. In all rheumitoid effusions whose
volume” was slightly or considerably greater than normal, the hyaluronate
‘Figures for the volume of synovial fluid obtained from the knees of patients with rheumatoid arthritis should be regarded as approximate. A slight inaccuracy in the estimation
of the volume can arise from failure to withdraw completely all the fluid from a joint.
528
DAVID HAMERMAN AND HILDA SCHUSTER
TABLE3.-Synoviol
Fluid Hynhrronate Herosumfne Levels in Patients
with Rhcumutoid Arthritis
Hyaluronate hx.
mg./Gm. S.F.
Total
Age
Normal
hx.
Knee mg./Gm.
:0-66
Mucin
GL
1.41
-c.20+
2
44
16
12
.14
1.07
6
<1
13
1.77
<1
14
66
Rt.
1.22
10%A.
1.26
GI.
61
Rt.
Lt.
Rt.
Rt.
1.17
lOO/,A.
32
3/67
Rt.
1.36
GI.
.18
11/66
Rt.
1.00
lOO/,A.
.29
Lt.
1.37
CP.
.49
.61
Rt.
1.31
Gl’.
.06
Lt.
1.43
GI.
56
9/67
Lt.
1.44
GI..
4/67
Rt.
1.40
1.08
GI’.
G1’.
1.14
G1’.
G1’.
1.26
1.23
1.26
1.65
GI’
.42
.43
4/66
8/66
11/67t
12/67#
Bre.
1.69
*.la*
Duration
of
RheumVolume arthritis.
atoid
T.Hyd.
ml.
years Nodules Factor
Rei.
9/66
Nor.
Swe.
WaL
16
27
6
+
10
+
+
+
0
0
0
0
30
68
44
+
-I-
0
63
4/67
3/67
GL
GL
o
-I-
0/6
40
0/11
I 4
10
12
o
+
.64
20
2
+
+
.72
.64
10
2.5
+
.66
.40
.K6
10
6
1.5
0
2
o
+
0/6
0
0
f
~
3/61
Gor.
69
Lt.
Cin.
36
9/67
6/67
.30
4-
Rt.
Lt.
<1
<1
Pol.
4
6/67
Bau.
64
Rt.
1.89
GI’.
15
f
8/67
Ali.
26
Lt.
1.69
Gl’.
1.04
<1
0/6
0
9/67
Pan.
67
Rt.
G1’.
1.34
<I
14
+
+
10/67
Sus.
28
Lt.
G1’.
1.37
<I
7
0
0
GI.
1.32
<1
0/7
0
0
11167
Cil.
Lt.
1.64
Rt.
.84
2
.37
~
26
~~
64
Gl’.
.28
Summaw
11
No. of fluids
Volume S.F. mL
Rheumatoid
Arthritis
H x . mg./Gm. S.F.
Normal
Hx. mg./Gm. S.F.
.27
>5
0
30
3
8
26
<1
Total
1.29
1.22
1.66
Hyate
.43
.27
Total
1.69
1.41
Hrate
1.30
*These 5gures are the mean value of 8 normal cases & the standard deviation.
1Th-e h r e s are the mean value of 10 normal cases f the standard deviation.
#Thhpatient was on oral cortisone at the time of these studies.
hexosamine concentration was reduced by a third to a fifth of normal. The
total hexosamine concentration of these rheumatoid fluids was usually only
slightly less than normal. The total hexosamine and hyaluronate hexosamine
concentration were normal in fluids of a volume of 1ml. or less obtained from
the knees of some patients with rheumatoid arthritis.
Fluids of greater than normal volume and low hyaluronate hexosamine concentration were obtained from patients who complained of stiff and painful
knees. X-rays of these knees showed increased soft-tissue density in the
527
SYNOVIAL FLUID HYALURONATE IN RHEUMATOID ARTHRITIS
suprapatellar recess." Synovial fluids of normal volume and normal hyaluronate
hexosamine concentration were obtained from patients who had minor or
no complaints referable to the knees but whose disease was nonetheless tictive. In one patient (Cin., table O), however, normal fluid was obtained at
a time when the disease had spontaneously remitted. Several months prior
to this, when the disease was active and there were complaints of pain and
stiffness in the knees, fluid of slightly increased volume and very low hyaluronate hexosamine concentration was obtained.
2. Relative viscosity of normal and rheumatoid synouial fltiids.-The relative viscosity of eight fluids of greater than normal volume and of low
hyaluronate hexosamine concentration from patients with rheumatoid arthritis
was compared with the viscosity of eight fluids from normal subjects. Prior
to these studies, the hyaluronate hexosamine concentration of each fluid was
reduced to about 0.25 mg./Gm. by adding a buffer (0.1 M NaCl and 0.1 M
NaHC03. pH 8.1) to the fluids, and the fluids were then dialyzed in this
buffer. The hyaluronate hexosamine concentration of each fluid was determined by the method of hyaluronidase digestion. Relative viscosity of the
fluids was measured in an Ostwald viscosimeter in a constant temperature
bath at 37°C. Table 4 shows that at an equivalent hyaluronate hexosamine
concentration some rheumatoid fluids had a relative viscosity equal to that
of the normal fluids, but slightly lower values were more frequently noted.
The relative viscosity of mucins precipitated from rheumatoid synovial fluids
and dissolved in buffer was lower than the viscosity of normal mucin solutions of equivalent hvaluronate hexosamine concentration (table 4).
It was recognized that normal synovial fluids required a greater dilution
TABLE4.--Relrrtiue Viscositu of Normal and Rheumatoid Synouial Fluids of Similar
Hyaltwonate Hexosamine Concentration
Dissolved mucins
Diluted, dialyzed wnovial fluid
Hy. hx.
-
ma./Gm.
vrel.8
.23
4.9
4.9
1a
Normal
.28
.28
.24
.94
26
.26
3/67
K.2
Much
Clot
GI.
lOO/,A.
co.
Hy. hx.
mg./Gm.
prel..
.27
28
.28
3.4
25
.26
.27
1.6
4.8
6.7
K.6
5.6
6.1
4.9
6.1
Lt.
27
6.4
Gor.
Lt.
.26
6.6
3/67
Nor.
Rt.
.21
4.8
4/67
swc.
Lt.
26
4.6
9/67
GI'.
1OO/,A.
co.
4/67
Cin.
Rt.
Lt.
.26
.20
.21
4.7
6.0
6.2
Rt.
25
4.8
Lt.
11/57
Cil.
C1'.
co.
-31
.31
1.6
3.0
1.4
3.3
*greI. ir the rehtive viscosity, or ratio of the flow time in seconds, of synovial fluid or mucin to the
flow time of buffer (0.1 Y NaCI, 0.1 M NaHCOa) in an Oatwald viscosimeter at 87' C.
528
DAVID HAMERMAN AND HILDA SCHUSTER
TABLES.-Effects of Zntra-Articular Znjection of Hydrocortisone or Saline on
Rheumtoid Sunoijial Fluid
Hyaluronate hx. mg./Gm.
Volume Total hx.
Total N.
Knee
ml. mg./Gm. much
T. Hyd.mg./Gm.
11/56
Swe.
Rt.
Rt.
Lt.
Lt.
30
8
60
60
1.00
Lt.
40
12
1.37
1.68
GL’.
20
1.43
1.40
GI’.
.96
1.17
1.31
lO%A.
10%A.
IO%A.
1OPJoA.
.29
.47
.43
-41
Diluted. dialyzed S.P.
Hy. hx.
mg./Gm.
qrel.
Intra-articular
injection
none
6.9
4.6
6.6
7.3
7 days after steroid
none
7 days after NaCl
4/57
Swe.
Lt.
3/61
Gor.
7
G1’.
Rt.
Rt.
Lt.
Lt.
10
1
6
7
.61
1.00
7.7
6.1
.24
.27
4.6,
6.1?
none
5 days after steroicl
.66
-11
.64
-67
7.7
6.7
.27
.26
6.4
6.7
7 days after steroid
GI’.
~
~~~
4/61
Cin.
.49
1.08
~
1.40
1.46
1.08
.99
GI’.
GL’.
GI’.
G1’.
~
.65
.01
.40
.34
.66
.89
.30
-26
6.8
6.1
6.8
1.4
none
~~
.26
.24
.20
-21
none
4.7
4.3
6.0
6.2
7 days after steroid
none
7 days after NaCl
~
+The relative viscosity of this synovial fluid before dilution and dialysis was 11.
!The relative viscosity of this synovial fluid before dilution and diiilysis was 603.
with buffer than did rheumatoid fluids to bring the hyaluronate hexosamine
concentration to 0.25 mg./Gm., and as a result the final protein concentration
of the normal fluids was only a sixth of that of the rheumatoid fluids. The
possibility that a low protein concentration might decrease the relative viscosity of the normal fluids apart from changes in the hyaluronate was tested.
Serum was added to diluted and dialyzed normal synovial fluids, but no
increase in the viscosity of these fluids was observed.
3. Effects of intra-articular injection of hydrocortisone.-Synovial fluid was
obtained from the knees of some patients with rheumatoid arthritis prior to
and one week after the intra-articular injection of 37.5 mg. (one and one-half
ml.) of hydrocortisone. In two cases synovial fluid was simultaneously withdrawn from the opposite knee before and after intra-articular injection of
saline. Intra-articular injection of hydrocortisone led to these results (table 5 ) :
( 1) reduction in the volume of synovial fluid of 70 to 90 per cent; ( 2 ) a rise
in the concentration of hyaluronate hexosamine in the fluid of 20 to 50 per
cent; and ( 3 ) a slight reduction of total nitrogen of the order of 20 per cent,
or no change. * These changes in volume, hyaluronate hexosamine concentration and total nitrogen were not observed in synovial fluid obtained after
*Despite this small change in total nitrogen of synovial fluids, intra-articular hydrocortiqone injection led to a marked decrease in the nitrogen to hevosamine ratio of mucins
formed with the cobalt salt. High ratios were consequences of the fact that proteins (chiefly
alpha-, globulins) containing nonhyaluronate hexosamine precipitated in mucins fornied
in rheumatoid fluid with the cobalt salt. Steroid injection led to changes in synovial
fluid which resulted in decreased precipitation of nonhyaluronate hewosamine by the
cobalt salt, and hence to lower nitrogen to hexosamine ratios of these mucins. These
changes in the fluid were: an increase in hyaluronate hexosamine concentration; a slight
decrease in the concentration of alpha-, glohulins and a slight increase in the albumin
concentration?
SYNOVIAL FLUID HYALURONATE IN RHEUMATOID ARTHRITIS
529
intra-articular injection of saline. Hydrocortisone did not bring about <any
change in the relative viscosity of synovial fluid as shown by similar viscosities
of all fluids diluted to an equivalent hyaluronate hexosamine concentration
and dialyzed (table 5 ) .
DISCUSSION
A hyaluronate hexosamine in rheumatoid synovial fluid was determined in
several ways. Hyaluronate hexosamine was completely precipitated in mucin
clots by adding glacial acetic acid to synovial fluid. Hyaliironate hexosamine
in rheumatoid synovial fluid was digested by testicular hyaluronidase and
rendered dialyzable. The results obtained by these methods generally agreed.
The average concentration of hyaluronate hexosamine in rheumatoid fluid
of greater than normal volume was 0.4 mg./Gm. or less compared with 1.4
mg./Gm. in fluids from normal subjects. The volume of most of these rheumatoid fluids was much greater than normal and thus the total amonnt of
hyaluronate hexosamine in the fluids exceeded normal, as has been previously
noted.'J Hyaluronate hexosamine constituted only about 30 per cent of the
total hexosamine of these rheumatoid fluids instead of 85 per cent as was
the case in the normal fluids. This large amount of nonhyaluronate hexosamine
in these rheumatoid fluids is presumably caused by an increased concentration of plasma proteins containing hexosamine.
Some criteria can be offered for abnormal and normal synovial fluids from
patients with rheumatoid arthritis. .4bnormal fluids are those of increased
volume and low hyaluronate hexosamine concentration. These changes in
the fluid presumably result from inflammation of the synovialis of the knee.
By contrast, normal fluids from patients with rheumatoid arthritis are of
normal volume and normal hyaluronate hexosamine concentration, and inflammation of the synovialis in these knees is presumed to be slight or absent.
However, many of the patients from whom normal synovial fluid was obtained
showed signs of disease activity, such as inflammation of other joints, subcutaneous nodules and an elevated erythrocyte sedimentation rate. The finding
of normal synovial fluid in patients with disease activity supports the view
of Egelius, Jonsson and SundbladlO that connective tissues are affected only
at some sites and not involved diffusely by the inflammatory process of rheumatoid arthritis.
Studies reported in this paper have compared the relative viscosity of
synovial fluids from normal subjects with the viscosity of fluids of increased
volume and low hyaluronate concentration from patients with rheumatoid
arthritis. The approach used was first to bring the hyaluronate hexosamine
concentration of all the fluids to the same level (0.25 mg./Gm.) by diluting
them with a buffer. The method of hyaluronidase digestion was used to determine accurately the hyaluronate hexosamine concentration. Comparison
of the relative viscosities of normal and rheumatoid fluids of equivalent hyaluronate hexosamine concentration revealed similar values, or a slight reduction in the viscosity of some rheumatoid fluids. These results suggest that
the state of polymerization of hyaluronate is similar in normal and in rheumatoid fluids or that it is only slightly lower in some rheumatoid fluids. These
530
DAVID HAMERMAN AND HILDA SCHUSTER
findings, which differ from those of other workers1Js4 who have reported that
hyaluronate is incompletely polymerized in rheumatoid synovial fluid, must
be considered in relation to the conditions employed in this study. First, the
methods of measuring hyaluronate are not the same as those of other workers, and thus, the finding of figures for hyaluronate hexosamine in normal
synovial fluid which are higher than those reported by others necessitated
a considerably greater dilution of these fluids than of the rheumatoid fluids
to reduce the hyaluronate hexosamine concentration to 0.25 mg./Gm. Second, the fact that dilution of all the fluids to this concentration of hyaluronate
hexosamine minimized the effects of interaction of hyaluronate molecules which
occurs at higher concentrations.
It must be emphasized that measurements of relative viscosity of synovial
fluids may not reveal information about the shape of hyaluronate molecules
or about a state of the hyaluronate which depends on its conibination with
part of the protein of synovial fluid." Changes in synovial fluid which come
about as a result of changes in shape or in this state of hyaluronate can be
detected by viscosity determinations at varying velocity gradients. When normal and rheumatoid fluids have been compared in this way, changes in the
physical properties of hyaluronate in rheumatoid synovial fluids have been
noted."1".13
These changes in hyaluronate have been thought to impair the effectiveness
of rheumatoid fluids as a lubricant between joint ~ u r f a c e s . ~The
~ J ~low concentration of hyaluronate in rheumatoid effusions may also contribute to an
inadequate lubricating action of these fluids. The particular suitability of
synovial fluid as a lubricant in the joint has been related14 to the properties of
hyaluronate when it is present in synovial fluids at a concentration of 0.2
Gm./100 ml. and above." At this concentration, hyaluronate molecules overlap, chains interpenetrate, and fluids possess special viscous and elastic properties not shared by dilute fluids. The concentration of hyaluronate is almost
always greater than 0.2 Gm./lOO ml. in synovial fluid from normal joints, but
is considerably less than this in fluids from inflamed joints of patients with
rheumatoid arthritis.
SUMMARY
1. Methods were studied to determine accurately hyaluronate concentration of synovial fluid from patients with rheumatoid arthritis. In one method,
hyaluronate was precipitated in mucin clots by adding glacial acetic acid
to weighed aliquots of rheumatoid fluid and was measured indirectly by
determining hexosamine in these clots.
2. These clots contained all the hyaluronate hexosamine of synovial fluid,
and the hexosamine of these clots was almost completely digested and rendered
dialyzable by streptococcal hyaluronidase. Nonhyaluronate hexosamine remained in the supernatant and was not digested by hyaluronidase.
3. In a second method, hyaluronate in rheumatoid fluids was measured by
the fall in hexosamine following hyaluronidase digestion and dialysis. Figures
*This concentration of hyaluronate corresponds to :I hyaluronate hexosamine concentration of 0.08 Gm./100 ml., assuming 40 per cent hexosamine in hyaluronate.
SYNOVIAL FLUID HYALURONATE IN RHEUMATOID ARTHRITIS
531
for hyaluronate hexosamine obtained by this method generally agreed with
those of the mucin clot technic.
4. The average hyaluronate hexosamine concentration of rheumatoid synovial fluid whose volume exceeded normal was 0.4 mg./Gm. The average
hyaluronate hexosamine concentration of synovial fluid from normal subjects was 1.4 mg./Gm. Hyaluronate hexosamine constituted only 30 per cent
of the total hexosamine of rheumatoid synovial fluid. By contrast, hyaluronate
hexosamine constituted 85 per cent of the total hexosamine of normal synovial
fluids.
5. The hyaluronate hexosamine concentration of some of the rheumatoid
and normal fluids was brought to 0.35 mg./Gm. by diluting the fluids with
a buffer. Comparison of the relative viscosities of these fluids then revealed
similar values, or a slight reduction in viscosity of some rheumatoid fluids.
It is suggested that the state of polymerization of hyaluronate in normal and
in rheumatoid fluids is similar, or only slightly lower in some rheumatoid
fluids.
REFERENCES
9. Sandson, J. and Hamerman, D.: Paper
electrophoresis of synovial fluid in
rheumatoid arthritis. Data to be published.
10. Egelius, N., Jonsson, R. and Sundblad,
L.: Studies of hyaluronic acid in
acid in synovial fluid. Acta SOC. med.
rheumatoid arthritis. Ann. Rheumat.
upsal. 58:113, 1953.
Dis. 15357, 1956.
Ropes, M. W. and Bauer, W.: Synovial
Fluid Changes in Joint Disease. Cam- 11. Ogston, A. G. and Stanier, J. E.:Further
observations on the preparation and
bridge, Harvard University Press,
composition of the hyaluronic acid
1953.
complex of ox synovial fluid. Biochem.
Bollet, A. J.: The intrinsic viscosity of
J. 52149, 1952.
synovial fluid hyaluronic acid. J. Lab.
12. Blumberg, B. S. and Ogston, A. G.:
Clin. & Med. 48:721, 1956.
The protein complexes of hyaluronic
Hamerman, D. and Schuster, H.: HyaIacid from various sources. Presented
uronate in normal human synovial
at the Fourth Interim Session, Amerifluid. J. Clin. Invest. 37:57, 1958.
can Rheumatism Association, Dec.
Ropes, M. W., Bennet, G. A,, Cobb, S.,
1957.
Jacox, R. and Jessar, R. A.: Proposed
diagnostic criteria for rheumatoid ar- 13. Johnston, J. P.: Viscosity of nonnal and
pathological human synovial fluids.
thritis. Bull. Rheumat. Dis. 7~121,
Biochem. J. 59.4333, 1955.
1956.
Dorfman, A.: The action of serum on 14. Blumberg, B. S. and Ogston, A. G.:
Physicochemical studies on hyaluronic
hyaluronidase. Ann. New York Acad.
acid. I n Ciba Foundation Symposium
Sc. 52:1098, 1950.
on Chemistry and Biology of MucoLewis, R. W.: The Joints of the Expolysaccharides. Boston, Little, Brown
tremities. Springfield, Ill., Charles C
& Co., 1958.
Thomas, 1955.
1. Ragan, C. and Meyer, K.: The hyaluronic acid of synovial fluid in rheumatoid arthritis. J. Clin. Invest. 28:
56, 1949.
2. Sundblad, L.: Studies on hyaluronic
3.
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8.
David Hamerman, M.D., Assistant Professor of Medicine,
Albert Einstein College of Medicine, New York, N.Y.
Hilda Schuster, B.S., Albert Einstein College of Medicinc,
New Ymk, N.Y.
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