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The entrance of glucose and other sugars into joints.

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The Entrance of Glucose and Other Sugars into Joints
By MARIANW. ROPES,ALEX F. MULLERAND WALTERBAUER
Entrance of glucose into joints was determined in calves and in patients with
joint disease. In calves under two weeks,
entrance was rapid; delayed in older
calves. In patients with joint disease
there was delay in appearance and slow
subsequent entrance, more marked with
greater severity of disease. The rate with
galactose was the same; that with pentoses and methylated glucoses usually
more rapid. The nature of the mechanism of transport is not apparent.
Esseva determinate le entrata de glucosa in le articulationes de vitellos e de
patientes con morbos articular. In vitellos
de minus que duo septimanas de etate,
le entrata esseva rapide; in vitellos plus
avantiate ill0 esseva retardate. In patientes con morbo articular un retardate
apparition de glucosa e un lente entrata
subsequente de ill0 esseva notate, e
isto esseva plus marcate con plus sever
formas del morbo. In studios con galactosa le mesme comportamento esseva
constatate; con le pentosas e le glucosas
methylate le valores esseva generalmente plus rapide. Le natura del mechanismo de transport0 non es apparente.
T
HE BARRIER between blood and synovial fluid remains poorly characterized and information concerning the transfer of various substances
has accumulated s1owly.l Understanding of the mechanism of exchange of
glucose across the blood-joint barrier and the effect of disease thereon is of
special importance in view of the fact that cartilage is an avascular tissue and
depends for its nutrition largely on synovial fluid. Recent evidence indicates
that substances can reach cartilage from the blood vessels of subchondral
bone,2-5but that the major source of nutrition is the joint fluid.
The glucose concentration in synovial fluid obtained from normal animals
and humans with no evidence of joint disease or with relatively mild inflammation (as in traumatic joint disease and in mild cases of rheumatoid or infectious arthritis) has been shown to be approximately the same as that of
the
With more severe inflammation, however, as in rheumatoid or
infectious arthritis, the sugar concentration in the fluid is reduced, occasionally
to zero in very severely involved joints.
The present investigations were undertaken in order to learn more of the
mechanism of the exchange of glucose between blood and fluid in normal
and diseased joints.
MATERIALS
AND METHODS
Glucose entrance was determined in 47 joints of 19 calves ranging in age from 6 days
to 4 months and in weight from 100 to 350 lbs. The animals were being held for slaughter
From the Department of Medicine, Haroard Medical School and the Medical Seruices
of the Massachwetts General Hospital.
This is publication no. 280 of the Robert W. Looett Memorial for the Study of Cjippling
Disease. Grants in support of these investigations have been received from the Commonwealth Fund, New York City, the National Institute of Arthritis and Metabolic Diseases,
Grani #-2123, National Institutes of Health, United States Public Health SerTuice, and the
La& Research hboratoria, Elf LiUv Co.
496
ENTRANCE OF GLUCOSE AND OTHER SUGARS INTO JOINTS
497
and had been fasting for at least 14 hours. After the calf had been restrained on his side,
specimens of jugular blood and of synovial fluid from the astragalotibial or carpometacarpal
joints were obtained. Sixty to 120 cc. of 25 per cent glucose in water (in 5 cases containing also 3 per cent sodium thiocyanate) were injected into the jugular vein with a
syringe, and subsequently a constant drip of 25 per cent glucose, by means of a polyethylene catheter, was maintained for varying periods of time. The fluid specimens were
withdrawn from the astragalotibial and carpometacarpal joints at regular intervals until
the joint cavities were empty. It was necessary to tap the joints for each specimen.
Definitely hemorrhagic fluids were discarded. Following the cessation of the intravenous
drip, blood specimens were withdrawn from the jugular vein at regular intervals.*
Phloridzin, (1 cc. containing 2.25 to 10 p M ) and dinitrophenol (0.5 to 1.5 cc. containing 2 to 10 p M per cc.) were injected into the selected joints, ( 5 and 18 respectively)
usually from one to 15 minutes before the administration of glucose. In one animsl the
intra-articular injection was made 18 hours before and on one occasion 40 minutes after
the injection of the glucose. In order to compare the effect of other substances that would
cause comparable synovial irritation saline was injected into 5 joints, concentrated turpentine into 1, and orthonitrophenol in the same concentration as dinitrophenol into 3 joints.
The entrance of glucose into the human knee joint was studied in 20 patients with
rheumatoid arthritis (24 tests), one patient with probable rheumatoid arthritis, one patient with gonorrheal arthritis, and three patients with Charcot knees ( 8 tests). In one
of the patients with rheumatoid arthritis and one with Charcot joints, studies were made
before and during treatment with ACTH or cortisone. The patients varied in age from
17 to 67; the duration of disease and effusion and the severity of disease are noted in
table 1. When classified in accordance with the American Rheumatism Association diagnostic criteria,' 13 had classical, 7 definite and 1 probable rheumatoid arthritis.
After the patients had fasted for at least 14 hours, specimens of venous blood and joint
fluid were obtained. The fluid was aspirated with an 18-gauge lumbar puncture needle
which was left in place throughout the experiment. Five per cent glucose in normal saline
solution or water was then injected intravenously by the drip method, 500 cc. being given
in 20 to 25 minutes and the remaining 600 to 1000 cc. more slowly. Blood specimens were
taken from the opposite arm and specimens of synovial fluid from the knee at regular
intervals until the cavity was empty. A separate syringe was used for each sample of fluid.
During the procedure the patient lay at rest with the knee immobile and in as full extension as was possible.
The entrance of sodium thiocyanate was investigated in 10 tests in 8 cases of rheumatoid
arthritis and in 7 tests in 2 patients with Charcot knees in whom the entrance of glucose
had been studied. In these experiments 20 cc. of 5 per cent sodium thiocyanate were
injected intravenously over a period of 2 minutes, usually just prior to the injection of
glucose, but in two cases on separate days. In 12 experiments, blood specimens were
taken under oil to prevent any shift of thiocyanate from cells to plasma.
Galactose entrance was studied in 7 patients with rheumatoid arthritis, and one patient
with Charcot joints. Studies were carried out with D-xylose in 4 patients with rheumatoid
arthritis and 1 patient with Charcot joints ( 2 tests); fructose in 1 patient with Charcot
joints; L-xylose in 1 patient with rheumatoid arthritis; L-arabinose in 1 patient with
rheumatoid arthritis ( 2 tests); D-arabinose in 2 patients with rheumatoid arthritis; 6methyl glucose in 1 patient with rheumatoid arthritis; and 3-methyl glucose in 2 patients
with rheumatoid arthritis and 1 patient with Charcot joints ( 2 tests). In 4 patients with
rheumatoid arthritis glucose and galactose (each in concentration of 5 per cent) were
given simultaneously. The technic was identical with that used in the case of glucose
~
*The differences between the initial fasting levels of glucose in serum and fluid, often
higher than the differences found in normal human joints,e were generally associated with
abnormally high levels of blood glucose. It was assumed that these elevations of blood
sugar occurred during the process of restraining the animals.
498
ROPES, MULLER, BAUER
except for the fact that usually only 500 to 6001 cc. were given when the pentoses and
the methylated glucoses were used. Most of the data with the pentoses and the methylated
glucoses were collected over a 5 month period on the same patient (case 10,table 1 ) . The
activity of this patient’s disease was seemingly stationary throughout the study period.
Unfortunately, 6-methyl glucose had to b e administered more slowly than the other
sugars since the patient developed a few extrasystoles and complained of vague chest
discomfort.
The removal of glucose from the joint was studied under similar conditions in one
patient with rheumatoid arthritis. The removal of galactose was determined also in the
same patient. After obtaining fasting blood and synovial fluid specimens, 0.75 cc. of 25
per cent glucose or galactose in normal saline was injected into the joint cavity; mixing
was insured by alternate aspiration and reinjection of synovial fluid.
Glucose was determined by the methods of Folins and of Somogyi-Nelson9JO; galactose
and the methylated glucoses by the method of Hagedorn and Jensenll after y e s t
fermentation*; the pentoses either by the method of Hagedorn and Jensenll after yeast
fermentation or by that of Roe and Ricelz; and thiocyanate by the method of Lavietes,
Bourdillon and KIinghoff er.13
Six-methylglucose was synthesized by one of us (A. F. M.) according to Freudenberg
and Hiill,l4 with the following modifications: B-acetyl-3,5-benzylidene-l,2-monoacetone
glucose (1.4) was prepared from 6-acetyl-1, 2-monoacetone glucose ( 1 . 4 ) according to
D. J. Bell.15 The 3-time recrystallized 6-methylglucose (m.p. 143-145 C.) was dissolved
in water and filtered through a Seitz-filter previous to its administration. Three-methylglucose was prepared by Ayerst-McKenna and Harrison Ltd. to whom we are most
grateful for their generous supply. L-xylose was purchased from Hoffman La Roche Pharm.
Indust., Basel, Switzerland; the arabinose from Pfanstiehl Chemical Co., the D-xylose
from Fisher Scientific Co. All these substances were made up in sterile 5 per cent solutions in water, filtered through a Seitz-filter previous to administration, and given intravenously in a constant drip over approximately 30 minutes. They all had been tested
previously in rabbits for possible pyrogenic reactions.
RESULTS
I . Entrance of Glucose and Thiocyanate into Joints of Normal Calves
Characteristic curves depicting the rate of entrance of glucose and thiocyanate into the joints of normal calves are shown in figure 1. The shape of
the curves apparently varied with the age of the calves. In animals less than
150 Ibs. (under 2 weeks of age), glucose appeared in the synovial fluid of
both the astragalotibial and carpometacarpal joints relatively rapidly after
intravenous administration. In older animals, over 150 Ibs. in weight, the entrance of glucose into the astragalotibial joints remained rapid, whereas in the
carpometacarpal joints there was a delay in almost all cases in the appearance
of glucose in the fluid (fig. 1B). Whether the difference in the rate of entrance
into the front and rear joints in older calves is related to the greater trauma
that the front joints suffer, or to other factors, is difficult to say. In both the
young and the older calves thiocyanate entered the joints without any delay
and the concentration rose rapidly. (fig. 1C ) .
*Previous to precipitation by sodium tungstate, the aliquots of plasma and joint fluid
were incubated at room temperature for 20 minutes with a 10 per cent yeast solution.
(Fresh Fleishman’s yeast was washed 10 to 12 times with water in order to remove
non-fermentable reducing substances. This solution was made fresh for each experiment. )
499
ENTRANCE OF GLUCOSE AND OTHER SUGARS INTO JOINTS
8
1
300
ZOO
I
/ /
100
0
20
10
0
40
30
SO
C
0
Lo
M
3
0
10
to
30
40
5C
Fig. 1.-Entrance of glucose and
thiocyanate into joints of normal
calves following intravenous administration.
Abscissa = time in minutes. LR =
left astragalotibial joint; RR = right
astragalotibial joint; LF = left carpometacarpal joint; R F = right carpometacarpal joint. A: 100 Ib. calf;
B and C: 300 Ib. calves. -0and
-0- indicate concentration of, glucose in serum and in synovial fluid;
and x concentration of thiocyanate
in synovinl fluid. In experiment A
only one serum glucose level was obtained so the shape of the curve was
estimated from those of comparable
experiments.
O
W
50
11. Entrance of Glucose into Joints i n Various Joint Diseases
Rheumatoid arthritis.-The
synovial fluid glucose curves obtained after
intravenous injection of glucose in the 24 tests in 20 patients with rheumatoid
arthritis and one with probable rheumatoid arthritis varied markedly as indicated in table 1. The shapes of the curves and the range of variation are shown
by characteristic examples (figure 2 ) . The blood sugar concentration rose
rapidly, and was maintained usually beween 200 and 300 mg./'100 cc. for two
hours. The initial rate of entrance into the joint fluid was slow in all but four
tests' with fluid levels increasing only 25 mg./100 cc. or less in 20 minutes. In
"In one of these, the patient was receiving cortisone intramuscularly and in another
cortisone had been injected intra-articularly 9 days previously.
so0
ROPES, MULLER, BAUER
?
8
1
I
350
e
L
o
,
i
0
2U
40
60
Bo
0
100
120
0
20
40
60
80
100
120
20
40
60
80
100
120
TIME IN M I N U T E S
Fig. 2.-Entrance of glucose into joints of patients with rheumatoid arthritis following intravenous administration.-0and -0- indicate concentration in serum
and synovial fluid respectively. A, B and C represent the same patients as those in
figure 3 (cases 12, 14 and 17 in table 1).
two patients with severe disease there was no alteration for 30 and 50 minutes, respectively (e.g. fig. 2C and case 17, table 1).
After this initial lag, sugar entered the joint fluid, and the subsequent rate
of entrance seemed to show an inverse relationship with the initial difference,
in the fasting state, between the fluid and serum sugar concentrations. The
smaller this initial difference the faster the rate of entrance and the more
clcsely the fluid level approached the serum at the end of two hours when
a tendency toward the establishment of equilibrium was usually apparent. In
general, the fasting difference between fluid and serum sugar and the corresponding lag and slow rate of entrance tended to vary with the severity of
the disease (table 1).In mild cases, as seen in table 1, the fluid sugar concentration tended to be only slightly below the blood level, and similarly, the
rate of entrance of glucose after the initial lag was relatively fast. In contrast,
patients with high initial differences and relatively slow sugar entrances usually had severe arthritis of long duration.
This delayed entrance and slow rise of glucose in the fluid can be partially
reversed by reducing the severity of synovial tissue inflammation as was apparent in studies on the patient with rheumatoid arthritis treated with ACTH.
The lag in entrance was slightly shorter and the subsequent rate of entrance
considerably faster, seven days after the patient was started on ACTH (80
mg. per day). Other signs of decreased inflammation were the reduction in
volume of fluid from 38 to 11 cc., the drop in leukocyte count from 12,800
to 2350, and in neutrophils from 89 to 24 per cent, the improvement in mucin
precipitate, and the slightly increased rate of entrance of thiocyanate and radioactive sodium, NaZ4(given in sodium chloride) .* l6 In a patient with Charcot
*The rate of entrance of radioactive sodium (Na24) was determined in one pationt with
rheumatoid arthritis and one with Charcot joints before and during treatment with ACTH.
In these experiments radioactive sodium ( N H ' ) ~in) sodium chloride (4 to 10 cc.) was
injected intravenously.
These studies were conducted by Dr. Ann Wight Phillips, then Atomic Energy Commission Fellow, presently Assistant in Surgery, Harvard Meclical School.
501
ENTRANCE OF GLUCOSE AND OTHEH SUGARS INTO JOINTS
knees, on the other hand, in whom treatment with ACTH caused no clinical
improvement and no alteration in synovial fluid except an increase in relative
viscosity, the rate of entrance of glucose remained unaltered.
Other joint diseases.-The entrance of glucose into synovial fluid in the three
patients with Charcot joints and the patient with gonorrheal arthritis showed
the same types of curves as in rheumatoid arthritis and the same relationship
between initial fluid sugar levels and the rate of entrance. (table 1).
Entrance of Thiocyanate into Joints in Joint Diseases
Thiocyanate, a readily digusible, nonutilizable substance, entered the
joints differently (fig. 3 ) . There was no initial lag and in all cases the fiuid
levels rose steadily though at different rates. In 5 of the 8 patients with rheumatoid arthritis and in the 2 patients with Charcot joints the concentration had
reached equilibrium in 70 to 110 minutes. The three other rheumatoid cases
difkred only in the slower rate of entry.
No absolute correlation was apparent between the rates of entrance of
thiocyanate and glucose although there was a tendency for thiocyanate equilibrium to be attained sooner in those joints into which glucose entered more
readily. In the cases in which glucose entrance was slow, the rate of thiocyanate entrance was also reduced, though remaining, however, always much
more rapid than that of glucose. (figs. 2 and 3 ) .
I I I . Entrance of Galactose into Joints in Various Joint Viseases
The synovial fluid curves obtained after intravenous injection of galactose
in 6 patients with rheumatoid arthritis and one with Charcot joints were almost identical with those of the glucose experiments (fig. 4). In all cases
there was an initial lag in appearance of galactose in the fluid, similar to that
with glucose, and the' final fluid-serum relationships were the same.
Entrance of Fructose into Joints in Joint Diseuse
The rapidity with which fructose disappeared from the blood stream made
it difficult to reach high blood levels. The curves of entrance into the two
2
0
20
40
60
80
100
120
140
0
20
40
TIME
60
IN
80
I00
120
0
20
40
60
80
100
120
MINUTES
Fig. 3.-Entrance of thiocyanate into joints of patients with rheumatoid arthritis
following intravenous administration. The symbols -X - and -X- indicate concentration in serum and in synovial fluid. A, 3 and C represent the same patients as
those in figure 2 (cases 12, 14 and 17 in table 1).
RA
8. 640228
25. 308128
24. 168891
23. 327100
22. 310162
joints
Charcot
joints
Chamt
joints
Gonococcal
arthritis
RA
RA
RA
RA
RA
Prob.
RA
Charcot
RA
RA
RA
RA
RA
RA
RA
RA
RA
RA
5. 889261
6. 691529
7. 232864
864163
720330
703880
609351
119894
8999
296175
810235
8996
20047
416026
745476
305443
38
RA
54415
4.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
3
1
BA
RA
2. 745536
3. 499948
M
M
M
21
-
-
-
M
60
-
-
F
3
2
2
1
3
3
2
3
3
2
3
2
4
2
3
3
2
2
2
2
2
2
1
2
2
--
3
1
F
F
3
F
M
M
M
1
M
4
2
1
1
2
1
1
4
2
M
F
M
M
M
2
M
M
M
F
F
F
1
2
1
F
Class
M
6
2
0
3
9
0
6
3
2
7
Stage
Sex
67
33
62
61
4
59
40
56
34
47
5
6
5
54
26
4
4
2
5
42
RA
1. 635099
Age
(yrs.)
Disease
Case #
Severitv
Table 1.-Entrance
7
4
3
1
2
3mu.
12
4
2%
1
2
timo.
6
3 mo.
ti
?2
Duration
&usion
(yrs.)
-1 wk.
15
15
1 wk.
9 mo.
15
8
6
59;
5%
7
6
11
11
5
4 wks.
( ? 4 wks.)
P9mo.
?9mo.
7
13
16
15
2
16
4
1
3
1
5
1
7
6
1
12
Duntion
disease
(yrs.)
63,750
400
( 2 ) 1.250
4,200
150
4,950
ti,9nn
2,650
b,7no
9,500
-_
31.600
16,850
27,400
23,600
33,000
33,750
58,100
26
-
(4)
96
10
38
n
0
22
fi8
61
-
62
64
86
81
71
88
fi2
73
40
70
93
--
so
-
22
52
48
27
24
Neutm
phils %
10,900
(3) 25,550
17,450
( 2 wks. before)
4,925
7.200
(2) 2.350
11,400
8.m
GLUCOSE
3,800
(1) 2 0 . ~ 2 5
Leukocytes
percu.mm.
Synovial fluid
Initial glucose
85
89
105
04
99
85
96
92
77
74
95
88
99
01
103
84
71
76
77
$9
82
77
78
114
82
89
91
57
inn
Difference
between serum
and fluid sugar
at two hours
(mn./100 ce.)
Maximum
serum
sugar
(mg./
100 ec.)
19
78
78
6
84
55
75
75
82
43
47
47
38
14
18
3
6
8
4
43
in0
85
88
75
96
64
65
59
81
69
21
18
97
0
64
53
73
71
81
78
34
44
49
30
14
25
32
39
42
21:
12
15
14
n
5
9
11
10
3
0
1
4
7
4
1
18
n
0
0
0
0
0
0
110
8
0
n
5
64
13
11
0
5
0
3
11
2
10
5
5
6
23
0
8
1
25
n
10
1
200
13
12
25
10
25
2
6
6
35
10
8
67
35
24
10
15
210
2
12
168
32
34
--36
119 (90min)
65
25
74
15
132 (98min)
235
205
104
265 (90 min)
52
40
n
6
31
-26
18
40 (40min)
30
55 (90min)
25
275
270
170
360
291
225
165
387
400
264
255
235
200
290
283
250
300
230
345
270
280
313
216
166
260
300
225
235
195
Y
i
X
*B
sr
z
'd
"E
K
3
Increase in
fluid serum
concentration
0
ui
with Various Joint Diseases
in 10
in.20
serum
fluid difference min.
min.
(mg./100 ce.)
(ms./lOO ec.)
-
of Sugars into Knee Joints of Patients
4
5
4
40
47
5
54
33
49
6
5
5
67
6
6
6
6
5
6
3
6
67
RA
RA
RA
RA
RA
RA
RA
Charcot
joints
RA
RA
RA
RA
Charcot
joints
Charcot
joints
RA
RA
RA
RA
RA
CA
RA
Charcot
joints
23. 327100
23. 327100
10. 720330
10. 720330
10. 720330
26. 758916
10. 720330
2. 745536
10. 720330
23. 327100
640228
720330
609351
415026
2
1
1
1
6
1
1
7
1
9
0
6
0
3
3
M
M
M
M
F
F
F
F
F
F
F
M
M
1
2
2
2
2
2
2
-
-
-
2
1
2
1
F
3
3
-
M
:j
4
1
1
1
F
F
M
F
M
M
M
-
-
--
1
8
3
3
2
3
3
2
3
S
3
--
3
2
2
2
2
2
4
4
17
1
4
4
2
4
4
17
15
17
I
-
-
-
-
(I.)
150
81
78
-10
94
48
46
40
0
80
--
0
--
fi
0
5
2
0
IF
3
4
9
53
5
11
25
2n
20
40
18
30
11
40
10
24
0.7
1.7
13
27
55
5
14
7
33
12
0
12
10
28
15
24
0
13
6
0.1
0.7
14
1
1
3
8
23
3
0
0
4
2
3
4
33
20
5
89
49
9
9
15
87
86
76
50
-
69
88
93
77
-
83
96
91
230
320
148
188
10
159
241
246
175
216
234
5
5
143
177
242
242
178
143
217
105
95
128
69
345
110 (5)
135
115 ( 5 )
0
-5
38
-11
-28
57 (89 min)
-7
-24
-23
0.7
1.0 ( l 0 0 m i n )
42
26
12
107
85
-35
10
7 (90 min)
-8
0 (85 min)
0
237 (90min)
30 (5)
90 (105 min)
48 (5)
usual occupation o r self-care; IV. incapacitated, largely o r wholly; little o r no
self-care.
The patients a r e listed in order of increasing difference between the initial
serum and fluid glucose levels since this represents one indication of increasing
severity i n the joints aspirated. ( I ) After intra-articular cortisone: (2) on ACTH;
(3) on cortisone; (4) repeated on same day; (5) glucose & galactose measured
together.
-
__
-
-
L-ARABINOSE
3
8.800
8,200
D-ARABINOSE
3
7,950
2
17,500
62
6-METHYL GLUCOSE
3
10,500
4s
3-METHYL GLUCOSE
3 mo.
11,950
3
17
( R ) 400
( L ) 350
( R ) 150
-
3
17
17
15
-
4
13
GO
4
93
38
83
-
90
90
G9
650 ( R )
0
750 ( R ) 12
450 (1,) FRUCTOSE
(R)
(L)
L-XYLOS E
7.550
6
2
7
-
3
2
4
GALACTOSE
23.150
16,250
8,550
39.400
6.800
11.250
12,100
360
(1 wk. before)
D-XYLOSE
4,050
3,700
2%
11
? 9 mo.
6
6 mo.
1
1
3 mo.
7
3
7
8
11
? 9 mo.
18
1
1
1
T h e stage of disease is determined by the degree of presumably irreversible
involvement rather than activity. Stage I is characterized only by subchondral
hone atrophy; stage 11, by cartilaginous and cortical destruction; stage I11 by
deformities such as ulnar deviation and subluxation; stage IV, hy fibrow or bony
ankylosis. Nodulss and significant muscle atrophy must not be present i n stage I.
The functional classes are: I, ability to carry on all usual duties without handicap;
TI, adequate for normal activities. despite handicap of discomfort or limited motion in one or more joints; 111, limited only to few or none of the duties of
8.
10.
12.
19.
5. 889261
7. 232864
11. 703888
13. 119894
16. 810235
17. 8996
20. 745476
22. 310162
8
cn
3
M
I
3
i
8
4
r
0
504
ROPES, MULLER, BAUER
0
20
40
60
100
80
o
120
20
40
60
eo
ioo
120
TIME IN M l N U TES
Figure 4.-Entrance of glucose and galactose into joints of patients with rheumatoid arthritis following intravenous administration. -0and -0- indicate concentration of glucose in serum and in synovial fluid; - - and -A- concentration of
galactose in serum and in synovial fluid. A and I3 represent individual patients
(cases 17 and 7 in table 1).
a
knees of a patient with Charcot joints (fig. 5 ) , suggested that there was a
lag in the entrance of fructose similar to that observed with glucose and galactose, but the observed concentrations in fluid and serum were extremely low.
IV. The Entrance of D-xylose, L-xylose, D-nmbinose, L-arnbinose, 3-methyl
Glucose and &methyl Glucose into Joints
In general, the curves of entrance of D-xylose, L-xylose, D-arabinose,
L-arabinose, 3-methyl glucose and 6-methyl glucose were alike (figs. 6, 7 and
8
“t_
-t
0
0
10
-- --_- -_---
*r
-;
20
A*
;
>
-*
!
RK
30
40
50
60
TIME IN MINUTES
Fig. 5.-Entrance of fructose into joints of a patient with Charcot joints (case
23 in table 1). and * indicate concentration in serum and in synovial fluid. RK =
right knee and LK = left knee.
*
505
ENTRANCE OF GLUCOSE AND OTHER SUGARS INTO JOINTS
,-,
B
300
300
z- \
I
0
20
40
60
80
100
0
120
\
20
40
60
80
100
120
TIME I N MINUTES
Fig. 6.-Entrance of pentoses into joint of a patient with rheumatoid arthritis following intravenous administration ( 5 separate experiments in case 10 in table 1).
0 and 0 indicate concentration of D-xylose in serum and in synovial fluid;
and
concentration of L-xylose in serum and in synovial fluid;
and concentration of D-arabinose in serum and in synovial fluid; A and concentration of
L-arabinose in serum and in synovial fluid (the solid lines represent one experiment,
the dotted lines a second experiment two months later).
. .
I
J
140
TIME
IN MINUTES
Figure 7.-Entrance of 3-methyl glucose, 6-methyl glucose and glucose into joint
of a patient with rheumatoid arthritis following intravenous administration. (3
separate experiments in case 10 in table 1).
0 and indicate concentration of 3-methyl glucose in serum and in synovial fluid;
8 and o concentration of 6-methyl glucose in serum and in synovial fluid; 0 and
o concentration of glucose in serum and in synovial fluid.
ROPES, MULLER, SAUER
0
20
40
60
80
100
120
TIME
0
I
I
I
I
20
40
60
80
I
100
I
120
I N MINUTES
Fig. %-Entrance of glucose, 3-methyl glucose, D-xylose and thiocyanate into
joints of a patient with Charcot joints following intravenous administration. (A indicates right knee and B left knee of case 23 in table 1).
0 and o indicate concentration of glucose in serum and in synovial fluid; 0 and
0 concentration of 3-methyl glucose in serum and in synovial fluid;
and 0 concentration of D-xylose in serum and in synovial fluid; and x and X concentration
of thiocyanate in serum and synovial fluid.
8). Following intravenous administration the sugars appeared relatively
rapidly in the joint fluid. The rate of entrance varied from patient to patient
and was relatively slow in the patient with Charcot joints in whom the entrance of thiocyanate also was relatively slow (fig. 8 ) . The reproducibility of
the experiments is well shown in figure 6. The two entrance studies done on
the same patient with L-arabinose are, for practical purposes, superimposable
even though they were done at approximately a two month interval. There
were some differences in the rates of entrance of the individual sugars that
are of interest when compared with their transport across other barriers. For
example, the entrance of L-arabinose was slightly less rapid than that of Darabinose. In the case of xylose, D-xylose entered the joint at essentially the
same rate as L-xylose in 3 cases, but with a lag in 3 others.
V. The Effect of Simultaneous Administration of Glucose and Galactose on
Their Entrance into Joints in Rheumutoid Arthritis Patients
In order to determine whether there was any evidence of competition of
sugars entering the joint simultaneously, glucose and galactose were given
intravenously at the same time. In the case of galactose there was almost no
difference in the time of appearance or rate of entrance when it was given
alone or with glucose (table 2, fig. 9). However, the entrance of glucose was
delayed in 3 of 4 patients when glucose was given with galactose. In one of
these patients, however, the subsequent rate of entrance of glucose was rapid.
V l . The Effect of Enzyme lnhibitors on Entrance of Glucose into Joints in
Nomull Calves
Enzyme inhibitors that could be injected into joints, dinitrophenol and
phloridzin, were utilized in the studies in calves to determine whether they
ENTRANCE OF GLUCOSE AND OTHER SUGARS INTO JOINTS
507
1 - 0 0 4
G
c,
350
t
I
Fig. 9.-Entrance of glucose and galactose into joint of a patient with rheumatoid arthritis following intravenous administration (case 16 in table 1).
Abscissa = time in minutes. A: glucose injected alone; B: galactose injected alone;
C: glucose and galactose injected simultaneously. 0 and 0 indicate concentration of
glucose in serum and synovial fluid; and A and A concentration of galactose in
serum and in synovial fluid.
altered the entrance of glucose. Other substances that might cause comparable
synovial irritation, orthonitrophenol, saline and concentrated turpentine,
were used for comparison.
In almost all the joints into which the various test substances had been
injected the initial rate of entrance of glucose was more rapid than in the
uninjected control joints. In many instances the shapes of the curves of entrance resembled in all other respects those in the control joints. However,
in the majority of the dinitrophenol joints (13 out of 18) there was a definite
reduction in the rate of entrance occurring between 5 and 35 minutes after
injection of glucose (fig. lo).* A similar lessening in the rate of entrance oc'A reduction in rate of entrance was amsidered significant only when the slope of
entrance decreased by 2.2% degrees. The measurements were made by an observer who
had no knowledge as to which joints had been injected with the various substances).
508
ROPES, MULLER, BAUER
Table Z.--Effect of Simultaneous Adminbtration of Glucose and Gakzctose on
Entrance of Sugars into Synovial Fluid
-___
Case No.
-___
- ..
Initial serumfluid glucose
difference
(mg./100 cc.)
745476
78
78
52
703880
5
39
8
810235
60
53
74
889261
-
Sugar entrance
Final
at appearfluid sugar
Appearance
time
ance time
concentration
(min.)
(mg./100 cc.) (mg./100 cc.)
Sugar
galactose
glucose
galactose
+glucose
galactose
glucose
galactose
+glucose
galactose
glucose
galactose
55
40
55
85
20
20
30
20
20
30
20
85
+glucose
galactow
glucose
galactose
+glucose
12
23
--
18
5
20
8
28
6
12
17
25
12
12
13
4
5
37
17
10
10
20
40
59
53
58
77
85
97
70
105
104
85
115
13
66
140
54
91
time
(min.)
(120)
(105)
(120)
( 105 )
(85)
(85)
(85)
(85)
( 100)
(100)
(100)
(103)
(90)
-
(90)
(100)
(100)
500
400
2
300
0
200
$2
0
100
n
0
10
20
30
40
50
60
TiME I N
0
10
LO
30
40
50
60
70
MiNUTES
Fig. 10.-Entrance of glucose into joints of normal calves following intravenous
administration.
A. Dinitrophenol was injected into left carpometacarpal joint (LF) before administration of glucose. B. Dinitrophenol was injected into right carpometacarpal joint
( R F ) before administration of glucose. 0 and 0 indicate concentration of glucose
in serum and synovial fluid. The vertical arrows indicate time at which intravenous
glucose was started; horizontal arrows indicate duration of injection.
so9
ENTRANCE OF GLUCOSE AND OTHER SUGARS INTO JOINTS
curred in only 5 of 37 control joints, in 1 of 5 saline, in the 1 turpentinc, in 1
of 5 phloridzin and 1 of 3 orthonitrophenol joints. The occurrence of the
marked change in the slope of entrance of glucose was significantly more
frequent in the dinitrophenol joints than in the others. (using the chi-squared
test p < .01)
VII. Exit of Glucose and Other Sugars from Joints
In order to aid in the interpretation of the lag in the entrance of gIucose
and galactose into joints, these sugars were injected on separate occasions
into the knee joint of one of the patients with rheumatoid arthritis in whom
glucose and galactose entrances were exceptionally slow (fig. l l ) , table 1,
Case 17). During 100 minutes after intra-articular injection of glucose the
fluid level fell 95 mg./100 cc. whereas in 2 hours the galactose level decreased
only approximately 15 mg./100 cc.
Comparison of the breakdown of glucose and galactose in vitro by joint
fluid cells was obtained by incubating uncentrifuged fluid containing approximately 9000 cells with glucose and with galactose at 37 C. In the case of
glucose the content had fallen from 100 mg./100 cc. to 50 mg. after 24 hours.
In the galactose experiment there was no decrease in the galactose concentralion after 22 hours’ incubation.
In the present experiments the fluid glucose level did not begin to fall for
10 to 20 minutes after the blood level fell, the lag being of the same magnitude
as the delay in entrance. Similar relationships between blood and fluid glucose
curves, with the resulting phenomenon of fluid level above blood, occur in
various joint effusions after the oral administration of glucose. Cajori, Crouter,
and PembertonlT reported that in three of four patients investigated, the
synovial fluid glucose concentration was above that of the blood between 35
and 85 minutes following the oral administration of 100 grams of glucose. In
this laboratory,’ synovial fluid glucose levels ranging as high as 67 mg./100 cc.
2001
8
A
I
e0”
ci
I
2ool
I
$h
F
IS0
1
I
I
20
40
60
0
0
20
40
60
80
100
I20
TIME
0
IN
I
I
I
80
100
120
MINUTES
Fig. 11.-Removal of glucose and galactose from joint of a patient with rheumatoid arthritis.
A. glucose injected into joint. B. galactose injected into joint. 0 and 0 indicate
concentration of glucose in serum and synovial fluid; indicates concentration of
galactose in synovial fluid.
510
ROPES, MULLER, BAUER
Table 3.-Concentration
of Glucose in Serum and Synovial Fluid
in Nonfasting Patients*
Glucose (mg./100 cc.)
Case
Disease
342755
rheumatoid arthritis
6760
25615
347 182
42840
358373
374295
rheumatoid arthritis
rheumatoid arthritis
gonococcal arthritis
rheumatic fever
gonococcal arthritis
rheumatoid arthritis
(L)
(R)
Fluid
Serum
102
103
101
139
99
143
124
150
81
81
96
117
67
113
96
83
'Serum and fluid were obtained 3 to 4 hours after patients had eaten.
above the blood level have been observed on eight occasions when effusions
of various types were aspirated 3 to 4 hours after the patients had eaten meals
containing approximately 100 grams of carbohydrate (table 3 ) . In the case
of the pentoses, also, the fluid level remained higher than the corresponding
blood level when the latter was falling. With the methylated sugars the concentration in the joint fluid never stayed above that in the blood in the few
experiments in which it was observed.
DISCUSSION
AND SUMMARY
It is evident that in calves over 2 weeks old and in patients with various
joint diseases there is a delay in the appearance of glucose in the joint fluid
following intravenous administration in contrast to the rapid appearance of
a readily diffusible substance such as thiocyanate. The rate of entrance of
glucose after this initial lag is also slower than that of thiocyanate. In general,
the more severe the joint inflammation, the greater the initial lag and the slower the subsequent entrance of glucose. There is a similar though less marked
tendency to reduction in the rate of entrance with greater severity of disease
in the case of thiocyanate.
Whatever the mechanism of the slow entrance of glucose, it plays an important role in determining the joint fluid sugar level. The difference between
blood and fluid sugar concentrations in the fasting state is relatively constant
for any one joint. It is low in normal and traumatic joints and increased in
more severely inflamed j0ints.l Whenever the equilibrium between entrance,
utilization and removal is disturbed in any way, this blood-fluid sugar difference tends to be re-established. In normal and traumatic joints it is presumably restored fairly quickly, as indicated by the patient with Charcot knee (case
23 table 1) in whom the fluid and blood levels were equal two hours after
intravenous injection of glucose. In many rheumatoid and infectious joints, on
the other hand, the restoration is retarded and the greater the fasting difference between blood and fluid glucose, the slower is the restoration (case 17
table 1) .
The cause of the difficulty in entrance of glucose is not obvious. It is not
associated with a general decreased rate of entrance of other substances. On
ENTRANCE OF GLUCOSE AND OTHER SUGARS INTO JOINTS
511
the contrary, the permeability to proteins is greatest in the severely inflamed
joints into which entrance ok sugar is slowest. Furthermore, the slow rate of
appearance of glucose in joint fluid is not merely a consequence of increased
utilization in the joint as indicated by the similar rate of entrance of galactose.
In the above experiments, galactose was shown not to disappear rapidly after
intra-articular injection and was not readily fermented in vitro by synovial
fluid cells. Further suggestion that the slow entrance of glucose was not determined solely by utilization was furnished by the findings in a patient (table
1, case 17) in whom synovectomy, with the removal of a large amount of
proliferated tissue, had been performed following glucose entrance studies.
Two years later, at which time there had been no reaccumulation of the-fluid
and no clinically apparent proliferation of the synovial tissues, 1.5 cc. of fluid
was withdrawn and found to contain only 8 mg. of glucose/100 cc., approximately the same concentration present before synovectomy 2 years previously.
The absolute polymorphonuclear leukocyte counts in the two fluids were 36,490
cells before operation and 11,630 cells 2 years later.
In order to determine whether structural specificity, particularly stereospecificity, plays a role in the transport of sugars from blood to synovial fluid,
the studies with the stereoisomers of xylose and arabinose and with 3- and
6-methyl glucose were undertaken. The rate of appearance of these sugars
was in general more rapid than that of glucose and galactose. The isomers
were alike except in 3 of the tests with D-xylose in which there was a lag in
entrance similar to that seen with glucose. The results are suggestive of the
possibility of specificity in transfer, but more definite evidence is needed.
The relative rates of transfer of various hexoses and pentoses across the
intestinal, renal, red cell and joint barriers are strikingly similar, if one compares an increasing order of absorption in intestine, kidney and red cell with
a decreasing order in the joint. D-galactose, D-glucose, and fructose are
transferred across the intestine^,^"^^ red cells25-20and probably rena127-2e
barriers more rapidly than the stereoisomers of xylose or arabinose or the
methylated glucoses, with few exceptions, whereas at the joint barrier there
is apparently delayed entrance of D-galactose, D-glucose and fructose as compared with the others.
Recent investigators suggest that the C1conformation of the pyranose ring
is essential in a monosaccharide if it is to react with the constituent in the red
cell surface which is involved in the movement of sugars through the membrane.30
The specificity in the effect of insulin on the transfer of sugars into tiss u e ~ the
, ~ ~sugars responsive to insulin being galactose, glucose, D-xylose
and L-arabinose, is different than that suggested in the joint. Furthermore, the
apparent variations in the rates of transfer of different sugars into joints are
in contrast to the results of studies of the absorption of sugars from the peritoneal cavity32 and the passage of sugars from blood to liver33 in both of
which the rate was the same for all sugars tested.
Further suggestion of a possible specificity of the mechanism of transfer
of glucose across the blood-joint barrier is given by the results obtained with
Sl.2
ROPES, MULLER, RAVER
simultaneous administration of glucose and galactose. The marked delay in
appearance time of glucose in three of the joints when injected intravenously
simultaneously with galactose, in contrast to the absence of any lag in entrance of galactose, suggests competition of galactose for the mechanism by
which glucose enters the joint, but further ,evidence is necessary. Competition
of different monosaccharides has been found in the intestine,'" kidney,34 and
red cell.25
The significant decrease in the slope of the curve of glucose entrance in
joints into which dinitrophenol had been injected suggests a possible specific
effect of dinitrophenol. If this represents an inhibition of phosphorylation,
the mechanism of the decreased transport could be either a decrease in the
energy required for the transport, or injury of cells so that they can no longer
transport sugar actively.
The results of these experiments suggest the possibility that the mechanism
for transfer of glucose from blood to joint fluid may be specific and selective,
as in the intestine, renal tubule and red cell, but the evidence is not conclusive.
The possibility of an enzymatically controlled transport, so clearly discussed
in the case of the red cell membrane by Rosenberg and Wilbrandt,35 the
activity of which is reduced in the presence of joint inflammation, or of an
association-dissociation phenomenon, such as that hypothesized by Shannon
and Fisher36 in the case of the renal tubule must be considered. In any case,
one can agree with Rosenberg and Wilbrandt,"5 discussing entrance into the
red cell, that . . . the complexity of the mechanism involved in glucose
penetration may seem surprising. Apparently it has to be considered as an inherited remnant, indicating the general biological occurrence of the phenomena discussed here."
"
SUMMARY
1. In calves less than two weeks old, following glucose administration intravenously, glucose enters the astragalotibial and carpometacarpal joints
rapidly, at essentially the same rate as thiocyanate.
2. In older calves, there is a delay in the appearance of glucose in the carpometacarpal joints.
3. In patients with rheumatoid arthritis and other joint diseases there is a
delay in the appearance of glucose in the joint fluid and the subsequent rate
of entrance is slow, in contrast to relatively rapid appearance and subsequent
entrance of thiocyanate.
4. The degree of the initial lag and the reduction in subsequent rate of
entrance increase with greater severity of disease.
5. The rate of entrance of galactose and probably fructose is the same as
that of glucose.
6. The stereoisomers of xylose and arabinose, and glucose methylated in the
3 and in the 6 position, in most cases, enter into the joint fluid rapidly.
7 . The nature of the mechanism of transport is not apparent.
ACKNOWLEDGMENTS
The authors wish to express their appreciation to Dr. E. G. L. Bywaters (now at
Canadian Red Cross Memorial Hospital, Taplow, Bucks, England) for the active part
ENTRANCE OF GLUCOSE AND OTHER SUGARS INTO JOINTS
513
he played in the initial phase of the study; to the many other physicians in the Arthritis
Group who have aided in these investigations; to Miss Elsie C. Rossmeisl, Mrs. R. Barbara
Peabody, Miss Elizabeth Manning, Miss Katherine L. Brine, Miss Dorothy Woodhams
and Miss Phoebe h e y , for their assistance.
REFERENCES
1. Ropes, M. W., and Bauer, W.: Synovial
Fluid Changes in Joint Disease. Cambridge, Harvard University Press,
1953.
2. Ingelmark, B. 0. E.: The nutritive supply and nutritional value of synovial
fluid. Acta orthscandinav. 20~144,
1950.
3. Ekholm, R.: Articular cartilage nutrition. How radioactive gold reaches
the cartilage in rabbit knee joints.
Acta Anat. (Suppl. 15) pg. 1, 1951.
4. -: Nutrition of articular cartilage. Acta
Anat. 24~323,1955.
5. Brodin, H.: Paths of nutrition in articular cartilage and intervertebral
discs. Acta orthscandinav. 24:177,
1955.
6. Ropes, PVI. W., Kossmeisl, E. C., and
Bauer, W.: The origin and nature of
normal human synovial fluid. J . C h .
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7. -, Bennett, G. A., Cobb, S., Jacox, R.
and Jessar, R.: 1958 revision of diagnostic criteria for rheumatoid arthritis. Bull. Rheumat. Dis. 9:175, 1958.
8. Folin, 0.:TWQrevised copper methods
for blood sugar determination. J.Biol.
Chem. 82:83, 1929.
9. Nelson, N.: A photometric adaptation
of the Somogyi method for the determination of glucose. J.Biol.Chem.
153:375, 1944.
10. Somogyi, M.: Notes on sugar determination. J.Biol.Chem. 19519, 1952.
11. Hagedorn, H. C., and Jensen, B. N.:
In Peters, J. P. and van Slyke, D. D.:
Quantitative Clinical Chemistry. Vol.
11, Methods. Baltimore, Williams &
Wilkins, 1932, p. 471.
12. Roe, J. H., and Rice, E. W.: h photometric method for the determination
of free pentoses in animal tissues.. J.
Bio1,Chem. 173307, 1948.
13. Lavietes, P. H., Bourdillon, J., and
Klinghoffer, K. A.: The volume of
the extracellular fluids of the body.
J.Clin.Invest. 15261, 1936.
14. Freudenberg, K., and Hiill, G . : 2,6 Di
Methyl Glucose. Berichte d.Dcutsch.
Chem.Gesellsch. 74-B:237, 1941.
15. Bell, D. J.: Crystalline 6-methyl glucose. A new synthesis. J.Am.Chem.
SOC.p. 859, 1936.
16. Ropes, M. W., and Phillips, A. W.: Unpublished data.
17. Cajori, F. A., Crouter, C . Y., and Pemberton, R.: The physiology of synovial
fluid. A.M.A.Arch.Int.Med. 37~92,
1926.
18. Cori, C. F.: The fate of sugar in the
animal body. I The rate of absorption of hexoses and pentoses from
the intestinal tract. J.Biol.Chem. 66:
691, 1925.
19. Wilbrandt, W., and Laszt, L.: Untersuchungen uber die Ursachen der
selecktiven Resorption der Zucker
aus dem Darm. Biochem. Zeitsch.
259:398, 1933.
20. Verzar, F., and McDougall, E. J.: Absorption from the Intestine. New
York, Longmans, Green & Co., 1936.
21. Westenbrink, H. G. K.: Ober die
Spezifitat der Resorption einiger Monosen aus d t m Darme der Ratte und
der Taube. Arch.Neere.Physio1. 21:
433, 1936.
22. CsBky, T. Z.: Ober die Rolle der
Struktur des Glucosemolekuls bei
der Resorption aus dem Dunndarm.
Zeitsch. physiolog. Chemie. 2?7:47,
1942.
23. Campbell, P. N., and Davson, H.: Absorption of 3-methyl glucose from
the small intestine of the rat and the
cat. Biochem.J. 43:426, 1948.
24. Hele, M. P.: Phosphorylation and absorption of sugars in the rat. Nature
166:86, 1950.
25. Wilbrandt, W.: Die Wirkung des Phlorizins auf die Permeabilitiit der menschlichen Erythrocyten fur Glukose
und Pentosen. Helvet. Physiol. Pharmacol. Acta. S:CG4, 1947.
26. Wilbrandt, W.: Die Permeabilitiit der
roten Blutkorperchen fur einfache
Zucker. Arch. gesamte Physiol. 241:
302, 1938.
27. Hober, R.: Uber die Ausscheidung
514
28.
29.
30.
31.
ROPES, MULLER, BAUER
von Zuckern durch die isolierte
Froschniere. Pfliiger’s Arch. gesamte
Physiol. 233181, 1934.
Hamburger. H. J.: Weitere Untersuchungen iiber die Permeabilitat
der Glomeriilusmembran fiir Stereoisomere Ziicker, mit besonderer Beriicksichtigung von Galactose. Biochem.Zeitsch. 128:185, 1922.
Campbell, P. N., and Young, F. G.:
Metabolic studies with 3-m~thyl
glucose. 1. Its fate in the animal
body. Bi0chem.J. 52:439, 1952.
LeFevre, P. G., and hlarshall, J. K.:
Conformational specificity in a biological transport system. Am.J.Physiol. 194333, 1958.
Goldstein, M. S., Henry, W. L., Huddlestun, B., and Levine, R.: Action
of insulin on transfer of sugars across
cell barrirrs: common chemical con-
32.
33.
34.
35.
36.
figuration of substances responsive to
action of the hormone. Am.J. Physiol.
173:207, 1953.
Cori, C. F., and Goltz, H. L.: Rate of
absorption of hexoses and pentoses
from peritoneal cavity. Proc.Soc.
Euper.Biol.& Med. 623:122, 1925.
Cori, C. F., and Goltz, H. L.: The permeability of liver and muscles for
hexoses and pentoses. Proc.Soc.Exper.
Biol.& Med. a, 23124, 1925.
Shannon, J. -4.: The tubular reabsorption of xylose in the normal dog. Am.
J. Physiol. 122:775, 1938.
Rosenberg, T., and Wilbrandt, W.:
Enzymatic processes in cell memlirane
penetration. 1nt.Rev.Cytol. 1:65,1952.
Shannon, J. A., nnd Fisher, S.: The
renal tubular reabsorption of glucose
in the normal dog. Am.J.Physio1. 122:
765, 1938.
Walter Bauer, M.D., Chief of Medical Services, Mussachrcsetts
General Hospital; lackson Professor of Clinicd Medicine,
Harvarcl Medico1 School, Boston, Mass.
Alex F . hluller, M.D., Mhdicin Adjuvant Clinique therapeutique, Universifb tle Gc-eva, Geneua, Switzcrlarul.
Maruin W. Ropes, M.D.,Associate Physician in Mdicine,
Massachusetts Generul Hospital; Assistant Clinical Professor
of Medicine, Harvurtl Medical School, Boston, Mass.
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