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The inflammatory response to injected microcrystalline monosodium urate in normal hyperuricemic gouty and uremic subjects.

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The Inflammatory Response to Injected Microcrystalline
Monosodium Urate in Normal, Hyperuricemic,
Gouty and Uremic Subjects
By W. WATSON
BUCHANAN,
JAMES R. KLINENBERG
AND J. E. SEEGMILLER
Microcrystalline sodium urate was injected intradermally and subcutaneously
in 15 normal subjects, 12 hyperuricemic
subjects without gout, 14 patients with
gout and 6 uremic subjects, and the
magnitude of the inflammatory response
was determined. The skin reactivity was
markedly diminished in 4 of the 6
uremic patients. The range of skin reactivity was significantly greater in
gouty patients than in normal or hyperuricemic subjects, but there was no significant difference in the mean inflammatory response between these groups of
subjects.
Microcrystallin urato natric esseva injicite per via intradermal e subcutanee in
15 subjectos normal, 12 subjectos hyperuricemic sin gutta, 14 patientes con
gutta, e 6 subjectos uremic. Le magnitude del responsa inflammatori esseva
determinate. Le reactivitate cutanee esseva reducite marcatemente in 4 del 6
patientes uremic. Le variabilitate del
reactivitate cutanee esseva significativemente plus grande in patientes guttose
que in subjectos normal e in subjectos
hyperuricemic, sed nulle significative
differentia esseva constatate inter le mentionate gruppos in le valores medie del
responsa inflammatori.
E
VIDENCE HAS ACCUMULATED that monosodium urate crystals may
play a central role in the genesis of the inflammatory rezction of acute
gouty arthritis.l-O Subcutaneous injection of microcrystalline sodium urate also
produces a substantial range of inflammatory responses in both gouty and
normal subjects. The possibility remains that susceptibility to development of
gouty arthritis might be related to constitutional factors that produce an enhanced inflammatory response to deposits of sodium urate crystals. The present investigation consists of a quantitative evaluation of the magnitude of the
inflammatory response to sodium urate crystals injected subcutaneously and
intradermally in gouty patients, subjects with hyperuricemia and no history of
gout, patients with uremia and normal volunteer subjects.
SUBJECTSAND METHODS
Patients studied included 14 patients with a history of acute gouty attacks with or without tophaceous involvement who were currently being investigated at the National Institute of Arthritis and Metabolic Diseases. Colchicine had been discontinued for at Ieast
5 days before tests were performed and other drugs were discontinued 24 hours before
teats. No patient had received recent corticosteroid or phenylbutazone therapy but uricowrit drugs were continued in some patients. None of the gouty subjects was uremic. Fifteen nornial volunteers served as control subjects. Eight of the normal volunteers were
restudird after they were made hyperuricemic by the oral administration of ribonucleic
x i d 4.0 Gin. per day in divided do-es for 3 days. Four subjects with hyperuricemia but
110 history of gouty arthritis and 6 patients with uremia and hyperuricemia were also
studied.
From the Arthritb and Rheumatism Brunch, National Institute of Arthritis and Metubolic. Diseuses, Notional Institutes of Health, Bethesda, Maryland 20011 4.
361
ARTHRITISAVD RHEUMATISM,VOL. 8, No. 3,
(JUNE),
1965
362
BUCHANAN, KLINENBERG, SEEGMILLER
The microcrystalline sodium urate was prepared (5) by dissolving 8 Gm. of uric acid
in 1,600 ml. of boiling water containing 49 ml. of 1 N NaOH. The p H was adjusted t o
7.2 by the addition of HCl and the solution was slowly cooled with stirring at room temperature and stored overnight at 5" C. This procedure gave crystals with size ranging from
0.3 to 9~ The crystals were then filtered off and dried for 24 hours at 808" C. Crystal aggregates were broken up by passing through a No. 100 sciive of 1 4 0 ~
pore size and fifty
mg. quantities were transferred to vials, sterilized by antoclaving and the vials sealed under
sterilc conditions. Extensive tests showed the sodium urate so prepared was sterile and free
from pyrogens.
At the time of injection 50 mg. of microcrystalline sodium urate was suspended in 0.5 ml.
of 5 per cent dextrose in water. For these studies 5 per cent dextrose in water was used
for suspending the crystals since 50 per cent glycerol used in earlier tests produced in
some cases a slight inflammatory response by itself and the crystals were less effectively
suspended in isotonic saline. After cleansing the subject's forearm with alcohol, 0.1 ml. of
the suspension (containing 10 mg. of sodium urate) was injected at 2 sites intradermally
and at 2 additional sites subcutaneously using a #23 needle attached to a 1.0 ml. Lurelock
syringe. For control purposes, 0.1 ml. of 5 psr cent dextrose in water was injected intratlermally and subcutaneously. Blood was drawn prior to the injections and serum uric acid
was determined by an enzymatic spectrophotoinetric method.8
The inflammatory response in the skin was maximal at 24 hours and this was the time
selected for its measurement. An objective index of the magnitude of the inflammatory response (tables 1 4 ) was obtained by averaging the minimum and maximum diameter of the
areas of erythema ( E ) and of induration ( I ) . In addition the temperature ( T ) of the
inflamed area was measured by using a skin thermocouple (Yellow Spring Instrument
Company) and was recorded as the difference in temperature ( A'C) between the area of
the skin reaction and an area of adjacent uninvolved skin. Although varying degrees of
tenderness were noted to accompany the inflammatory response, they were not included
in this study because of their subjective nature. The skin reactivity (fig. 1) was an arbitrary number indicating the magnitude of inflammatory response in which the relative
contribution of the three objective measurements have been equalized by appropriate
weighting. The number was obtained by adding the numerical mean values for erythema
( E ) , twice the value for induration ( I ) , and three times the temperature value ( T ) , since
the mean numerical values obtained for the latter two measurements were roughly ?4
and 1/3 the value for the erythema respectively (table 1).
RESULTS
Intradermal or subcutaneous injection of microcrystalline sodium urate produced a measurable skin reaction in all of the normal, hyperuricemic and
gouty subjects. Injection of the suspending media (0.1 ml. 5 per cent dextro'se
in water) did not prosduce a skin reaction in any of the subjects tested. Biopsy
of skin reactions in two subjects shomwed an acute inflammatory reaction with
exudation of polymorphonuclear leuko'cytes.
The skin reactivity of 15 normal volunteer subjects is shown in table 1. A
comparison of the mean values for skin reactivity obtained in 12 patients with
hyperuricemia (table 2 ) and in 14 patients with gout (table 3 ) showeld no
significant diflerence at the 5 per cent l e d by the " T test, while the mean
for the 6 uremic patients (table 4) was significantly lowe'r ( P < 0.01). There
was no correlation between the skin reactivity and the duration of the arthritis. Furthermore, there was no significant difference between those patients
with tophaceous gout and patients with no tophi. All values expressed are the
mean values of duplicate determinations. The duplicates were in very good
363
h I 0 N O S O D I U M URATE CRYSTALS AND INFLAMATION
Table 1.-The
Inflammatory Responsz to Microcrystalline Sodium
Urate in Normal Human Subjects
Skin Reactivity
Intradermal Injection
Serum
Urate
Subject
Sex
Age
1
F
Id
2
M
3
6’
1
M
18
1x
32
6
M
21
6
7
M
M
19
26
8
M
18
9
10
M
M
34
21
M
M
M
11
12
13
14
15
44
46
59
43
39
M
M
Mean
SD of duplicate
determinations
3O.5
~-__--___
EmIndura- Temp.
A
mg./
1 0 0 ml.
thcma
cm .
tion
cm.
.j.2
5.3
3.4
4.5
(4.7)
4.5
(5.4)
6.3
6.1
(6.4)
6.2
(6.9)
6.2
6.0
(5.6)
4.6
4.1
6.5
5.4
6.2
3.x
3.8
1.9
1.1
(1.8)
1.5
(1.3)
0.6
1.1
(1.3)
0.6
(1.2)
1.5
0.9
(1.0)
3.3
2.6
1.4
2.8
0.5
0.9
1.3
1.o
1.0
(1.0)
0.5
(0.5)
0.4
1.2
(1.2)
0.6
(0.7)
0.4
1.0
0.8
0.3
0.5
(0.7)
0.8
(1.0)
0.0
1.5
(1.3)
0.8
(1.0)
.n
0.7
0.3
1.0
(0.8)
0.5
0.5
0.5
1.0
1.0
5.4
1.8
0.8
0.7
0.19
0.14
0.16
1
(0.9)
0.9
0.6
0.8
Subcutaneous Injection
Eythema
cm.
“C
0.5
-
Induration
cm.
Temp.
1.4
1.6
1.3
1.8
(1.6)
0.3
(0.4)
0.4
0.6
(0.5)
1.2
(1.5)
1.0
1.5
a
“C
2.7
3.1
2.7
3.9
(8.7)
2.3
(2.0)
1.3
2.0
(2.1)
3.0
(3.3)
2.8
2.9
(3.1)
3.1
3.0
2.6
3.5
2.5
(0.9)
1.4
1.2
2.7
1.3
0.7
1.0
(1.3)
1.0
1.0
0.8
1.0
1.0
2.8
1.2
0.9
0.36
0.13
0.20
1.o
0.8
1.o
1.3
(1.0)
0.8
(1.0)
0.0
0.8
(0.8)
0.8
(0.5)
0.8
~
Results obtained in repeat studies a t a later date a-e indicated by ( ) and ape not included in
the calculation of the mean.
Table 2.-The
Inflammatory Response to hficrocrystalline Sodium Urate
in Hyperuricemic Subjects witlaout Gout
Skin Reactivity
Subject
Sex
Age
1
2
M
M
%
5
6
7
8
9
M
M
E’
M
M
M
M
in
M
11
12
M
40
42
51
42
18
32
21
19
26
18
34
26
30.8
4
M
Mean
SD of duplicate
determinations
Sercm
Urate
ma./
100ml.
S.3
7.2
Intradermal Injection
Subcutaneous Injection
~ _ _ ~ - . - _ _ _
Emthema
cm.
Induration
cm.
Temp.
a
Erythema
cm.
Induration
em.
Temp.
“C
1.0
1.2
0.4
0.8
1.0
0.9
0.6
0.4
1.0
3.3
2.7
2.0
3.0
2.5
3.7
2.1
1.4
1.8
3.2
2.6
3.1
1.0
2.2
1.4
2.0
1.5
1.6
0.4
0.3
0.4
1.4
0.8
1.0
0.5
0.5
0.8
0.8
1.n
1.5
1.0
0.3
1.0
0.5
1.0
1.3
n
“C
7.4
8.9
7.2
7.0
8.3
9.6
8.7
7.1
7.0
2.9
3.0
1.7
1.6
3.2
1.2
1.6
0.7
1.0
0.5
1.3
0.7
0.4
0.7
0.3
0.5
0.5
0.3
1.0
0.5
0.5
0.5
1.0
1.0
0.5
1.0
7.9
1.6
0.7
0.6
2.6
1.2
0.9
0.21
0.10
0.18
0.46
0.23
0.18
8.4
0.4
agreement as shown by the low value for the standard deviation of duplicate
determinations.
Although there was variation in the skin reactivity of 5 normal ITolunteer
subjects a n d 2 gouty subjects who had repeated skin tests performed at inter-
BUCHANAN, KLINENBERG, SEEGMILLER
Table 3.-The
Inflammatory Response to Microcrystalline Sodium
Urate in Patients with Gout
Skin Reactivity
Tophi
Present
Subject
Sex
Age
1
2
3
M
YeS
Yes
YeS
Yes
7
R
M
52
71
56
71
53
29
65
49
9
M
52
Yes
10
11
12
13
14
M
M
M
57
61
49
55
63
Yes
Yes
No
NO
No
4
5
6
F
M
M
M
M
M
M
M
Xean
S D of duplicate
determinations
N O
No
Yes
YeS
55.9
Serum
Urate
mg./
100ml.
_-
Intradermal Injection
Subcutaneous Injection
Induration
em.
Temp.
'C
Erythema
cm.
1.0
1.5
1.2
1.7
1.2
0.4
0.5
1.2
(1.4)
1.5
(1.7)
0.8
0.4
0.9
0.4
1.3
1.0
0.5
1.0
1.0
1.8
0.5
1.0
1.0
(1.0)
2.0
(1.5)
1.5
0.5
1.3
0.0
1.8
0.4
2.2
1.9
3.3
3.3
3.0
2.3
2.6
(2.5)
3.2
(3.4)
4.0
1.7
3.4
2.2
4.2
0.2
1.4
1.6
1.6
1.4
0.7
1.4
1.6
(1.5)
2.1
(2.2)
1.7
0.4
1.5
0.8
2.2
0.5
0.3
1.0
0.8
1.8
1.5
1.0
1.5
(1.8)
2.5
(2.5)
2.5
0.5
1.8
0.5
2.5
2.1
1.0
1.1
2.7
1.3
1.3
0.27
0.24
0.15
0.29
0.24
0.33
Erythema
cm.
Induration
cm.
Temp.
7.6
7.4
8.7
6.1
7.4
7.6
5.9
8.4
(6.9)
9.7
(9.7)
5.9
7.5
8.7
5.2
4.5
1.4
2.7
1.4
3.1
3.4
0.6
2.2
1.9
(1.4)
2.5
(1.7)
1.9
0.4
2.7
0.4
4.1
7.2
A
A
"C
_ _ _ _ _ _ _ _ - ~
Results obtained in reoeat studies a t a later date a r e indicated by ( ) and a r e not included in the
calculation of the mean.
Table 4.-The
Inflammatory Response to Microcrystalline Sodium
Urate in Uremic Patients
Skin Reactivity
Subject
Sex
Age
1
2
3
4
5
6
M
70
39
74
37
M
M
M
M
M
Mean
SD of duplicate
determinations
P value*
44
71
55.8
Blood
Urea
Nitrogen
mg./
100ml.
Serum
Urate
mg./
100ml.
46
54
3R
71
110
126
74.1
Intradermal Injection
Em-
Subcutaneous Injection
Temp.
"C
Erythema
cm.
Induration
em.
Temp.
thema
cm.
Induration
em.
4.8
7.2
7.9
7.4
7.6
7.3
0.3
0.4
0.7
0.0
0.0
0.0
0.3
0.3
0.3
0.0
0.0
0.0
0.0
1.0
0.5
0.0
0.0
0.0
0.0
2.0
1.0
0.0
0.0
0.0
0.8
0.8
2.2
0.2
0.4
0.6
0.0
1.5
1.0
0.0
0.0
0.0
7.0
0.2
0.2
0.3
0.5
0.8
0.4
,022
<0.01
.017
<0.01
.14
<0.05
.15
<O.Ol
A
.13
N.S.
A
"C
0.0
<0.05
*Compared with normal subjects (table 1) using T test for sienificance.
vals ranging from 1 to 4 weeks after their initial skin test (tables 1 and 3 ) ,
there was very good correlation (coefficient of correlation > 0.93) between
the two tests in all instances except for erythema in the intradermal tests. The
lower degree of correlation (0.53) for erythema in the intradermal tests probably reflects the difficulty in injecting material intradermally.
The magnitude of the inflammatory response in individual subjects showed
a considerable variation among members of each group studied, but the range
of values in the gouty group is significantly greater than in any other group
tested (fig. 1).Furthermore, three patients with gout had a greater skin re-
365
MONOSODIUM URATE CRYSTALS AND INFLAMATION
.
.
.
. ..
.
. _ _.._ _
.
.
..
... .
. .
. _ __
. .
...
1
~
I
!!!
..
.
. ...
. .
.
..
.
*.
-+-
--__
0.
0 .
Hyper:
Gout
iricemio
Uremia
Normal
Hyper:
Gout
uricemia
Uremia
Fig. 1.-Inflammatory response in arbitrary units (see Methods) 24 hours after
the injectio'nof 10 mg. of microcrystalline sodium urate.
activity with both intradermal and subcutaneous injections than any of the
subjects who did not have gout. Four of the six uremic patients had markedly
depressed skin reactivity.
DISCUSSION
Previous studies have shown that the subcutaneous injection of crystals o€
sodium urate or sodium orotate produces a localized inflammatory reaction"^^
and when injected into a normal knee joint may result in an acute self-limiting
arthritis simulating
In the present study we have considered whether
or not patients who develop acute gout have a n altered sensitivity to suspensions of sodium urate crystals. An enhanced tissue reaction in patients who
have had acute gouty arthritis might afford an explanation as to why only 10
to 15 per cent of the total hyperuricemic population develop acute gout.g No
difference was found in the mean skin reactivity between normal subjects,
hyperuricemic subjects, and patients with chronic gouty arthritis. The range
of the skin reactivity, however, was significantly greater in the gouty patients
and three of the fourteen gout patients developed a substantially greater degree of inflammatory reaction to injected urate crystals than was seen in the
normal or hyperuricemic groups. This suggests that an enhanced reactivity to
sodium urate crystals may play a role in the pathogenesis of acute gouty
arthritis in some but not all of the patients with gout.
Clinical observations have shown that patients with uremia and hyper-
366
BUCHANAN, KLINENBERG, SEEGMILLER
uricemia rarely develop acute gouty arthritis and that patients with gout
who develop uremia frequently have a lessening of the symptoms of their
gouty arthritis. This might be due in part to a lessening of their tissue reactivity to sodium urate crystals. This study shows that four out of six patients with uremia had, in fact, a significantly diminished cutaneous inflammatory response to injected sodium urate crystals, and the patients with the
liighest blood urea nitrogen concentration tended to show less skin reactivity.
Uremic patients also show a diminished local exudative cellular response.1°
Other factors such as altered solubility of urate in uremic body fluids or the
diminished life expectancy of uremic patients may contribute to the failure of
patients with primary renal disease to deposit clinically apparent tophi.
SUMMARY
Microcrystalline sodium urate wai injected intradermally and subcutaneously in 15 normal subjects, 12 hyperuricemic subjects without gout, 14 patients
with gout, and 6 uremic patients. There was no statistically significant difference in the mean inflammatory response
e\Toked by sodium urate between
groups of normal subjects, and patients with hyperuricemia or gouty arthritis.
The range of skin reactivity was significantly greater in gouty patients than
in normal or hyperuricemic subjects. Three of the fourteen gouty patients developed an inflammatory response to injected sodium urate crystals that was
greater than was obsened in the normal group, while four of the six patients
with uremia showed a significantly diminished inflammatory response to injected sodium urate crystals.
ACKNOWLEDGMENTS
We wich to thank Dr. Sol Katz of the Mount Alto Veterans Administration Hospital,
Washington, D. C. for allowing us to study the six patients with uremia. We are also
grateful to Dr. Seymour Geisser and Dr. Williain O'Brien of the National Institute of
Arthritis and Metabolic Disease who provided assistance in the stTtistica1 analyses presented in this paper.
REFERENCES
1. Garrod, A. B.: A Treatise on Gout and
Rheumatic Gout: Rheumatoid Arthritis. 3rd ed. London, Longmans,
Green & Co., 1876, 88 pp.
2. Howell, R. R., and Seegmiller, J. E.:
Inflammatory response to injected
sodium urate. Eighth Interim Scientific Session of the Amer. Rheum. Assoc., December, 1961.
3. Seegmiller, J. E., Howell, R. R., and
Malawista, S. E.: The inflammatory
reaction to sodium nrate. J.A.M.A.
180:469, 1962.
4. Faires, J. S., and McCarty, D. J,, Jr.:
Acute arthritis in man and dog pro-
duced by intrasynovial injection of
sodium urate crystals. Clin. Res. 9:
329, 1961.
5. Faires, J. S., and McCarty, D. J., Jr.:
Acute arthritis in man and dog after
intrasynovial injection of sodium urate
crystals. Lancet 2:682, 1962.
6. Seegmiller, J. E., and Howell, R. R.:
The old and new concepts of acute
gouty arthritis. Arth. & Rheumat. 5:
616, 1962.
7 . Malawista, S. E., and Seegmiller, J. E.:
The inflammatory response to injected
microcrystalline monosodium urate in
man: The effect of pretreatment with
MONOSODIUM URATE CRYSTALS AND I N F L A M A T I O S
colchicine on the magnitude of the
response. (Submitted)
8. Liddle, L., Seegmiller, J. E., and Laster,
L.: The enzymatic spectrophotometric
method for determination of uric acid.
J. Lab. Clin. Med. 54:903, 1959.
9. Hauge, M., and Harvald, B.: Heredity
in gout and hyperuricemia. Acta
Med. Scandinav. 152:472, 1955.
10. Perillie, I?. E., Nolan, J. P., and Finch,
S. C.: Studies of the resistance to infection in diabetes mellitus: Local
exudative cellular response. J. Lab.
Clin. Med. 59:1008, 1962.
W . Watson Buchanan, M.D., University D q a r t m e n t of Medicine, Gardiner Institute, Western Infirmary, Glasgow W 1,
Scotlund; (Formerly U.S.P.H.S. Fellow, National Institute of
Arthritis and Metabolic Diseases, Bethesdu, Maryland.)
James R. Klinenberg, M.D., Resident Physician, Private Medical Service, T h e Johns Hopkins Hospital, Baltimore, Maryland;
(Formerly Clinical Associate, National Institute of Arthritb
and Metubolic Diseuses, Bethesdu, Maryland.)
J . E . Seegmiller, M.D., Senior Incestigator, National Institute
of
367
Arthritis and Metabolic Diseases, Bethesda, Maryland.
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