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Studies on the nucleation of monsodium urate at 37╨Т┬░C.

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574
STUDIES ON THE NUCLEATION OF MONOSODIUM
URATE AT 37OC
HYON-KI TAK, SHELDON M. COOPER, and WILLIAM R. WILCOX
Factors influencing the nucleation of monosodium urate (NaHU) were studied with supersaturated
solutions of sodium urate at physiologic conditions of
temperature, pH, and ionic strength. Spontaneous nucleation of NaHU did not occur at urate concentrations
of 5 mM (84 mg %) in the presence of 140 mM Na ion.
Addition of synovial fluids from gout patients greatly enhanced nucleation, whereas fluids from degenerative
joint disease patients moderately enhanced nucleation,
and fluids from rheumatoid arthritis patients had only a
slight effect. Hyaluronic acid and purines minimally enhanced urate crystallization, whereas other connective
tissue components had no effect.
There is general acceptance of the concept that
acute gouty arthritis and the development of tophaceous
deposits are the result of deposition of monosodium
urate (NaHU) (1,2). However, the factors that influence
the nucleation and deposition of NaHU are poorly understood. Hyperuricemia alone is apparently not sufficient to guarantee urate crystallization, as evidenced
From the Department of Chemical Engineering, Clarkson
College of Technology; and the Clinical Immunology and Rheumatic
Disease Section, Los Angeles County/University of Southern California Medical Center.
Supported by US Public Health Service Research Grant #
AM 18914.
Hyon-Ki Tak, PhD: Jet Propulsion Laboratory, Pasadena,
California; Sheldon M. Cooper, MD, University of Southern California School of Medicine; William R. Wilcox, PhD: Clarkson College
of Technology.
Address reprint requests to Dr. Sheldon M. Cooper, Clinical
Immunology and Rheumatic Disease Section, Los Angeles County/
University of Southern California Medical Center, OCD 104, 2025
Zonal Avenue, Los Angeles, CA 90033.
Submitted for publication December 13, 1979; accepted in
revised form January 15, 1980.
Arthritis and Rheumatism, Vol. 23, No. 5 (May 1980)
by the fact that only 17% of hyperuricemic individuals
have had an acute attack of gout (3). Since NaHU deposits primarily in connective tissue (4), recent interest
has focused on local factors in connective tissue which
may play a role in the deposition of NaHU. Cartilage
proteoglycans have been shown to enhance the solubility and inhibit crystallization of NaHU, and it has been
proposed that the catabolism of these components results in urate crystallization ( 5 ) . More recent data indicated that while macromolecular aggregates of proteoglycans augment urate solubility, a sustained
enhancement of solubility in sodium buffers is not seen
(6).
Nucleation of NaHU was investigated previously
by means of a temperature-programmed microscope
slide technique in which hot stoichiometric solutions of
monosodium urate in water were sealed on the cavities
of microscope slides with cover glasses and epoxy cement (7). Solubility temperatures were obtained by
slowly heating until all crystals dissolved and nucleation
temperatures were determined by slowly cooling until a
crystal reappeared. Under such conditions nucleation
was enchanced by Ca++,H' and synovial fluid from one
gout patient, whereas synovial fluid from one rheumatoid patient inhibited nucleation.
This experimental technique did not simulate
physiologic conditions. The Na ion concentration was
much lower than the physiologic range and each slide
was subjected to wide temperature variations, which
may have especially influenced the results with synovial
fluids. Improved techniques developed in recent experiments (8,9) have been employed in the present study to
investigate factors that influence the nucleation of
NaHU.
NUCLEATION OF MONOSODIUM URATE
MATERIALS AND METHODS
Materials. Hyaluronic acid, chondroitin-4-sulfate,
chondroitin-6-sulfate, D-glucuronic acid, N-acetyl-D-glucosamine, purine, and uricase (U-9375) were obtained from Sigma
Chemical Company (St. Louis, Missouri).
Synovial fluids were obtained under sterile conditions
from arthrocenteses performed for diagnostic or therapeutic
purposes. Fluids were centrifuged at 800 g for 20 minutes and
the supernatants stored at -20°C. Immediately before use in
the nucleation experiments, the synovial fluids were thawed,
centrifuged at 800 g for 20 minutes, and the supernatants filtered either through a 1.2 pm pore Millipore membrane (Millipore Corp., Bedford, Massachusetts) or sequentially through
a 5 pm and 0.22 pm pore membrane.
Preparation of solutions. All solutions were prepared
with distilled water which had been filtered through a 0.22 pm
pore Millipore membrane. Supersaturated solutions are required as a driving force for nucleation. Sodium urate supersaturation can be achieved by mixing equal volumes of one
solution containing a high concentration of urate ion with another solution containing sodium ion and the desired additive.
Supersaturated urate solutions were prepared by dissolving
uric acid (K and K Labs, ICN Life Sciences Group, Plainview, New York) in alkaline solutions (NaOH or KOH), after
which the pH was adjusted to 7.4 and the solution was filtered
to eliminate any undissolved solute. To determine the effect of
sodium ion upon nucleation, equal volumes of uric acid solution of known concentration were mixed, with vigorous stirring, with solutions containing the desired concentration of
NaC1. Purine and all the connective tissue components, except
hyaluronic acid, were dissolved in a NaCl solution. This was
mixed with an equal volume of urate solution; the mixture
was then filtered and stored at 37°C. Solutions containing
hyaluronic acid could not be filtered, so they were added directly to previously filtered solutions of urate and sodium
ions. The sodium ion concentration was maintained at 140
mM in all instances.
In experiments with synovial fluids, uric acid solutions
containing different urate concentrations were prepared. For
urate concentrations up to 2.97 mM * (50 mg%),uric acid was
dissolved in a buffer containing 27 mM NaHCO,, 110 mM
NaCI, and 5mM KC1. For urate concentrations up to 4.16
mM (70 mg%), the buffer contained 3 mM KOH, 2mM
KHC03, 22mM NaHCO,, and 1 lOmM NaC1. For urate concentrations up to 5.35mM (90 mg%), the buffer contained
5mM KOH, 27mM NaCHO,, and IlOmM NaC1. Synovial
fluid at a final concentration of 10%was added to the urate solutions. All nucleation experiments were performed at pH 7.2
to 1.4.
Nucleation technique. Under sterile conditions a drop
of urate solution was placed in the cavity of a preheated microscope slide (6688-D22, A.H. Thomas Company, Philadelphia, Pennsylvania). A coverglass was slid into place over
the cavity and the excess solution was absorbed with porous
paper. Epoxy cement was applied to the edge of the coverglass
to seal in the solution. Slides were stored in a glove box at
37OC and viewed periodically in polarized light with a first order red compensator; a microscope was placed in the glove
* To convert m M to
mg%, divide by 0.0595.
575
box so that only the eyepieces emerged. It was found that if
crystals did not appear within one month, they would not appear after longer storage, so that one month was used as a
convenient time to measure the presence and amount of crystallization. Crystal identity was confirmed by analysis of the
d-spacings obtained from x-ray power diffraction (8,9).
To rule out the possibility of bacterial contamination
and consumption of urate, experiments were also performed
in 40 ml vials. Samples were periodically removed from these
vials and the urate concentration was determined by its absorbance at 265 nm. No evidence of bacterial contamination
was observed, i.e. in the absence of crystallization the urate
concentration remained constant.
RESULTS
Solubility of NaHU. To check our techniques,
we made an independent measurement of the solubility
of monosodium urate. A solution containing 30mM uric
acid in 30mM NaOH was seeded with NaHU crystals
and stirred at 37°C. The urate concentration of the solution was measured periodically until a constant value
of 6.543mM was reached (Figure 1). This compares favorably with previously reported values of NaHU solubility (10,ll). The variability in the reported results
(range 5.41mM to 6.54mM) may be attributed to differences in the way in which final conditions were
achieved.
Effect of sodium ions on the nucleation of
NaHU. To study the role of Na' on the nucleation of
TIME (DAYS)
Figure 1. Solubility of NaHU at 37°C. A stoichiometric solution of
uric acid in NaOH was seeded with NaHU crystals and stirred at
37°C. The final urate concentration was 6.543 mM.
TAK ET AL
576
NaHU, stoichiometric solutions of uric acid in NaOH
(C,,+ = CHU-) were mixed with equal volumes of solutions containing additional Na'. After filtration, duplicate slides were prepared from each solution and stored
in the thermostated glove box. Table 1 shows the average number of crystals and crystal clusters observed for
each solution. A detailed analysis of these and similar
experiments will be reported elsewhere (9); however, it
is important to note that no nucleation was observed
with urate concentration of 5mM, even after 6 months
storage at 37°C. Five millimoles urate greatly exceeds
the supersaturation levels usually encountered in hyperuricemic individuals (1mM urate = 16.8 mg %). Experiments performed in vials confirmed these results and
provided evidence that the urate concentration was
stable, indicating that the lack of nucleation did not result from bacterial degradation of urate.
It is also interesting to note that the number of
NaHU clusters increased as the sodium ion concentration increased, and that clusters were found in all solutions that had crystals if the Na+ concentration exceeded 100mM. A typical cluster is shown in Figure 2.
Table 1. Effect of additional Na+ on the nucleation of NaHU at 37°C
Urate and
NaOH (M)*
0.0 15
0.0 I2
0.010
Additional
NaCI(M)*
Average
no.
clusters/
slide
0
0.02
0.04
0.06
0.12
0.14
3
200
178
99
75
I64
0
0
0.02
0.04
0.06
0.08
0.13
0
0
0
0
0
0
0
3
0
0.04
0.06
0.08
0.10
0.12
0.14
0.005
Average
no.
crystals/
slide
0
0.06
0.10
0.16
6
10
57.5
111
0
0.5
2.5
9.5
25
0
0
1
3
6
17.5
21
0
0
0
0
2
5
0
0
0
0
Equal volumes of stoichiometric solutions of uric acid in NaOH
were mixed with solutions containing additional Na+.
Figure 2. A cluster of NaHU crystals viewed with polarized light.
These negatively birefringent crystal clusters appear to be similar to
those found in tophaceous deposits (X 100).
Cluster formation usually started 1 or 2 days after slide
preparation, and the clusters became well-developed
within 2 or 3 days.
Influence of synovial fluids on nucleation of
NaHU. Supersaturated solutions of uric acid (see Materials and Methods) were prepared and synovial fluids
from patients with gout, degenerative joint disease, and
rheumatoid arthritis were added to a final concentration
of 10%.Three to six microscope slides were prepared for
each urate concentration and stored for 1 month at
37"C, at which time they were examined for crystals.
Initially the fluids were added to urate solutions of 3545 mg%. If crystals were found, the urate concentration
was decreased; if no crystals were seen, the urate concentration was increased. In the absence of synovial
fluid, a urate concentration of 5.06mM was required for
nucleation. Addition of synovial fluids from gout patients induced nucleation of NaHU at all urate concentrations tested. Synovial fluids from degenerative joint
disease patients moderately enhanced nucleation,
whereas rheumatoid arthritis fluids had only a minimal
enhancing effect. The results are shown in Table 2.
Cell counts and chemical analyses of the synovial fluids studied in Table 2 are presented in Table 3.
Gouty fluids 1, 2, and 4 were from NaHU crystal-positive, acutely inflamed joints, while fluid 3 was a crystalnegative aspirate from a patient with chronic tophaceous gout. The white blood cell counts indicate that
NUCLEATION OF MONOSODIUM URATE
577
Table 2. Eflect of the addition of synovial fluids on the nucleation of NaHU at 37OC*
~
Urate
concentration
mg%
mM
85
80
5.06
4.76
70
4.16
60
50
No
synovial
fluid
X
0
0
0
0
0
0
0
3.57
2.97
40
2.38
30
1.79
RA
1
2
3
0
0
0
0
0
0
0
0
0
~
DJD
4
5
xo
x
X
xo
o o
x
0
xo
~
0
0
0
0
0
0
0
1
0
2
~~~
Gout
3
1
X
X
2
3
4
X
x x
o o x
x x
o o o x x x x
x x x
X
*Synovial fluids at a final concentration of 10% were added to sodium urate solutions (35-45 mg%).
Three to six microscope slides were prepared for each solution and observed for crystals after one month.
X = if crystals were observed, the urate concentration was lowered 0 = if no crystals were observed, the
urate concentration was raised; XO = some slides had crystals whereas others did not at the urate concentration shown.
the inflammatory nature of the gouty and rheumatoid
fluids was comparable. Chemical analyses were performed on the supernatants of samples that had been
thawed and centrifuged at 800 g. Urate levels were
slightly higher in the gouty fluids, but the additional
urate contributed by the fluids is negligible in comparison to the urate already in the solution (relative
concentrations 0.95 mg% to 40 mg%). No further information in these results helps in explaining the differences in nucleation behavior.
To further explore the enhanced nucleation by
synovial fluids from gouty patients, we performed additional experiments using the microscope slide technique
at 22”C, pH 7.4, in buffers containing 0.14M NaC1.
With these conditions, spontaneous nucleation of
NaHU was always found at urate concentration of 60
mg% or greater, but seen in only some slides at 50 mg%
(Table 4). Synovial fluids from 3 gouty patients were
thawed, centrifuged at 800g, and sequentially filtered
through 5pm and 0.22pm Millipore filters. Fluids 1 and
Table 3. Analyses of synovial fluids used in nucleation experiments*
Ionized
Ca
(mEq/L)
Total
protein
(gmW
WBCt
PMNt
Diagnosis
(X 103)
%
RA 1
RA 2
RA 3
RA 4
RA 5
17.5
13.2
32.5
18.0
40.6
65
ND*
71
49
66
5.5
4.7
4.3
2.2
5.1
7.8
7.1
7.9
7.7
8.1
1.59
1.56’
1.34
1.49
1.76
7.1
6.1
5.4
4.2
5.2
2.2
2.4
3.0
2.5
1.9
DJD 1
DJD 2
DJD 3
0.6
ND
0.7
9
ND
ND
6.3
6.5
5.5
7.9
7.4
8.1
1.34
1.56
1.54
4.1
3.8
4.0
2.4
1.8
2.8
Gout I
Gout 2
Gout 3
Gout 4
10.8
21.5
2.7
48.4
72
59
34
81
9.5
8.3
1.3
7.7
1.5
7.5
6.6
7.7
1.53
1.53
1.59
1.49
3.4
5.7
4.9
4.3
2.1
3.1
3.0
2.2
* Chemical analyses were performed on the supernatants of samples that had been thawed and centrifuged at 800g.
t White blood cell counts (WBC) and determination of percent polymorphonuclear leukocytes (PMN%)
were performed on freshly obtained fluids.
$ ND = not determined.
TAK ET AL
578
Table 4. Effect of addition of gouty synovial fluids on the nucleation of NaHU at 22°C.
__
Urate
Gout 1
Gout 2
Gout 3
concenNo
tration
synovial
UriCaSe
UriCaSe
(mg%)
fluid
Untreated Dialyzed
dialyzed Untreated dialyzed Untreated Dialyzed
-.
60
50
40
30
20
10
-~
__ -
.-
-
X
xo
X
0
0
0
0
X
X
X
xo
0
0
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
0
0
0
0
0
0
0
0
xo
0
X
X
-
* Three slides were prepared at each urate concentration. After 48 hours the slides were examined for the
presence (X)or absence (0) of crystals (XO = some slides at that urate concentration had crystals, whereas
others did not). The positive slides usually demonstrated crystals by 48 or 72 hours. In those samples Eontaining uricase, the long-term effect of uricase on formed crystals was not determined.
2 were from crystal-positive, acutely inflamed joints,
while fluid 3 was a crystal-negative aspirate from a patient with chronic tophaceous gout.
Aliquots of the fluids were either dialyzed
against 0.14M NaCl or digested with uricase and then
dialyzed. Uric acid was not detectable by Caraway colorimetric analysis in dialyzed or digested and dialyzed
samples. Table 4 shows the urate concentration where
crystals were observed when these samples were added
to supersaturated urate solutions.
Effect of connective tissue components on nucleation of NaHU. In order to test the influence of connective tissue components on sodium urate nucleation,
solutions were first prepared by dissolving uric acid in a
stoichiometric NaOH solution and the pH was adjusted
to 7.4 with 0.1M KOH or HCI. Each connective tissue
component except the hyaluronic acid was dissolved in
the appropriate NaCl solution. Warmed NaCl and urate
solutions were mixed, filtered, and stored in vials at
37°C.
Solutions containing hyaluronic acid could not
be filtered through 0.22 pm membranes, so they were
added directly to solutions containing urate and sodium
ions. These solutions usually showed a colloidal dispersion in a strong light beam after 1 day. A week later
this had settled. A similar result was obtained when
hyaluronic acid was dissolved in distilled water, with
identical appearance of the colloidal particles under the
microscope.
Crystals were observed with hyaluronic acid and
5mM urate, but not with 1.2 or 0.6mM urate. Chondroitin-4 and 6-sulfate, D-glucuronic acid, N-acetyl-D-glucosamine, Type I and Type I1 collagen did not induce
nucleation of 5mM urate solutions. The results are summarized in Table 5.
DISCUSSION
Solubility of a crystalline solid in solution is defined as its concentration in equilibrium with crystals of
the substance. Nucleation, or the birth of a new crystal,
requires enormous surface energy per mole and, thus,
crystals may not form even though the concentration
greatly exceeds solubility (12). The latter point is emphasized by the present experiments in which solutions
containing 5mM urate (84 mg%) and physiologic concentrations of Na ion did not nucleate after 6 months
storage at 37°C. This helps to explain why hyperuricemia alone is not sufficient for the development of
gout and supports the concept that other factors must be
acting in vivo to enhance the nucleation of NaHU.
Several theories have been advanced to explain
the phenomenon of NaHU nucleation in gout. Simkin
has proposed that the preferential localization of gout to
the base of the big toe occurs because of transient increases in the concentration of urate as a result of water
leaving the joint space more rapidly than urate in resolving synovial effusions (13). However, as has been
pointed out by McCarty (14), this theory could not account for the formation of urate crystals only in selected
individuals, nor is it consistent with our in vitro experiments which indicate that solutions of sodium urate
which are 10-fold more concentrated than that encountered in hyperuricemic individuals can be maintained
for long periods without evidence of crystal formation.
Katz has reported an increased level of uronic
acid in the sera of gout patients when compared to
asymptomatic hyperuricemic individuals and controls
(15). He has hypothesized that if the high levels of
serum polysaccharides in gout patients reflect turnover
of connective tissue, deposition of urate crystals may re-
NUCLEATION OF MONOSODIUM URATE
579
Table 5. Effect of connective tissue components on NaHU nucleation at 140mM Na'
Additive* (conc)
Urate (mM)
Purine (50 mg/100 ml)
5
Days after
preparation
0.6
5
60
60
75
5
15
No nucleation
5
90
No nucleation
5
90
No nucleation
60
I .2
60
5
14
Hyaluronic acid
(6.5 mg/100 ml)
60
I .2
Chondroitin-4-sulfate
(100 mg/100 ml)
Chondroitin-6-sulfate
(100 mg/100 ml)
D-glucuronic acid
(100 mg/100 ml)
N-acetyl-D-glucosamine
(100 mg/100 ml)
Observationt
No nucleation
Crystal clusters on
bottom of vial
No nucleation
Heterogeneous nucleation on the wall
of the vials
Same as above
No nucleation
No nucleation
No nucleation
7
* Concentrations are shown in parentheses.
t Solutions were stored in 40 ml vials.
sult from the association of accelerated connective tissue metabolism and supersaturation of these tissues
with urate. Alvsaker has approached this question by
studying the plasma urate binding by a1-d-globulins in
gout patients and their kindred and has reported that
this population has decreased binding capacity (16).
These studies suggest that there may be genetically determined differences in the plasma and connective tissues of gout patients which in effect raise the urate concentration and promote NaHU nucleation.
The most interesting finding to emerge from this
study is that synovial fluids from gouty patients greatly
enhanced the nucleation of NaHU from supersaturated
solutions, whereas fluids from degenerative joint disease
and rheumatoid arthritis patients enhanced nucleation
to a considerably lesser degree. For several reasons, it is
unlikely that the enhancement found with the gout
fluids results from the seeding of the supersaturated
urate solutions with crystals. All gouty fluids showed no
crystals after filtration, and the three fluids tested at
lower urate concentrations (20 mg%) did not cause
spontaneous nucleation at these concentrations (Table
4). Intentional seeding of urate solutions (20 mg%) with
small NaHU crystals resulted in the formation of larger,
easily visible NaHU crystals. Furthermore, uricase digested and dialyzed fluids, in which uric acid was not
detectable, also induced nucleation and gave results that
were similar to untreated samples.
Since all the synovial fluids enhanced NaHU nucleation to some degree, it would appear that a synovial
fluid component is responsible for this effect, and that
this substance may be present in significantly different
concentrations in synovial fluids of gout patients. Although the experiments reported here do not allow us to
identify this component or components, we can definitely say that it is not uric acid and that it is present in
synovial fluids from both acutely inflamed and chronically involved gouty joints. The results with the dialyzed and untreated synovial fluid samples suggest that
the component is nondialyzable. The slight difference
found at 20 mg% uric acid with gouty fluid 1 (Table 4)
most likely results from the increased volume in the dialyzed samples and a dilution effect upon the enhancing
component, rather than a loss of the enhancing substance during dialysis.
Although it has been reported that chondroitin-4
sulfate decreases the solubility of NaHU (17), we were
unable to identify any synovial fluid components which,
when added to supersaturated urate solutions, s i p &
cantly enhanced NaHU nucleation. Hyaluronic acid, at
a concentration approximately one-tenth the normal synovial fluid level, appeared to cause heterogeneous nucleation of NaHU only at high urate concentration. The
concentration of hyaluronic acid in these experiments
(6.5 mg%) approximates the final concentration of
hyaluronic acid in the synovial fluid experiments. However, it is difficult to attribute the enhancing effect seen
with the gouty synovial fluids to hyaluronic acid, since
we would expect equal or greater enhancement with the
degenerative joint disease fluids which are known to
have higher hyaluronic acid concentrations (1 8).
Caution must be exercised in relating the signifi-
TAK ET AL
580
cance of our observations to the in vivo nucleation of
NaHU. After filtration, none of the synovial fluids included in the study demonstrated spontaneous nucleation of NaHU after prolonged storage. Synovial fluids
from degenerative joint disease and some rheumatoid
arthritis patients also enhanced nucleation of NaHU
from supersaturated urate solutions, although to a considerably lesser degree than gouty synovial fluids. In
spite of these considerations, our results provide further
evidence that the nucleation of NaHU is dependent
upon many factors, only one of which is the level of hyperuricemia. Differences in the composition of synovial
fluids and connective tissues of gouty patients, perhaps
genetically determined, must be considered in the pathogenesis of the disease.
ACKNOWLEDGMENTS
We wish to thank Mrs. Liddia Balisi for excellent
technical assistance and Ms. Nancy Jones for preparing the
manuscript.
REFERENCES
1. Seegmiller JE: The acute attack of gouty arthritis. Arthritis Rheum 8:714-725, 1965
2. Wyngaarden JB: The etiology and pathogenesis of gout,
Arthritis and Allied Conditions. Edited by JL Hollander
and DJ McCarty, Jr. Philadelphia, Lea & Febiger, 1972,
pp 1071-1111
3. Hall AP, Barry PE, Dawber TR, McNamara PM: Epidemiology of gout and hyperuricemia: a long-term population study. Am J Med 42:27-37, 1967
4. Sokoloff L: The pathology of gout. Metabolism 6:23&
243, 1957
5 . Katz WA, Schubert M: The interaction of monosodium
urate with connective tissue components. J Clin Invest
49: 1783- 1789, 1970
6. Perricone E, Brandt KD: Enhancement of urate solubility
by connective tissue. I. Effect of proteoglycan aggregates
and buffer cation. Arthritis Rheum 21:453460, 1978
7. Wilcox WR, Khalaf AA: Nucleation of monosodium
urate crystals. Ann Rheum Dis 34:332-339, 1975
8. Tak HK: Influence of additives on crystallization of urates. Ph.D. dissertation. Potsdam, New York, Clarkson
College of Technology, 1978
9. Tak HK, Wilcox WR, Cooper SM: Crystallization of
monosodium urate and calcium urate at 37°C. J Colloid
Interface Science, in press.
10. Khalaf AA: Nucleation and solubility of monosodium
urate in relation to gouty arthritis. Ph.D. dissertation.
University of Southern California, 1973
I I . Finlayson B, Smith A: Stability of first dissociable proton
of uric acid. J Chem Eng Data 19:94-97, 1974
12. Strickland-Constable RF: Kinetics and Mechanism of
Crystallization from the Fluid Phase. London, Academic
Press, 1968
13. Simkin PA: The pathogenesis of podagra. Ann Intern
Medicine 86:23&233, 1977
14. McCarty DJ: The gouty toe-a multifactorial condition
(editorial). Ann Intern Med 86:234-236, 1977
15. Katz WA: Deposition of urate crystals in gout: altered
connective tissue metabolism. Arthritis Rheum 18
( s u P P ~ )1-756,
: ~ ~ 1975
16. Alvsaker JO: Genetic studies in primary gout: investigations on the plasma levels of the urate-binding al-a2globulin in individuals from two gouty kindred. J Clin Invest 47:1254-1261, 1968
17. Laurent TC: Solubility of sodium urate in the presence of
chondroitin-4-sulfate. Nature 202: 1334, 1964
18. Ropes MW, Bauer W: Synovial fluid changes in disease.
Cambridge, Harvard University Press, 1953
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