Crystal growth of monosodium urate monohydrate

Colloids and Surfaces, Elsevier

Science

CRYSTAL

B.V.,

97-107

97

Amsterdam

-

Printed

GROWTH OF MONOSODIUM

P.D. CALVERT,

R.W.

School of Chemistry (United Kingdom) (Received

14 (1985)

Publishers

FIDDIS

1983;

URATE MONOHYDRATE

and N. VLACHOS

and Molecular

27 September

in The Netherlands

Sciences,

accepted

University

in final form

of Sussex, Brighton 18 September

BNl

9QJ

1984)

ABSTRACT A microscopic method has been used to measure crystal-growth rates for monosodium urate monohydrate from aqueous solution at 37°C. The dependence of the rate on supersaturation obeys a surface-nucleation law, not a screw dislocation type of law. When appropriate corrections were made, the growth rates at high sodium concentrations were in agreement with those in equimolar monosodium urate monohydrate solutions. Extrapolation of these results to the concentration regime in which monosodium urate monohydrate precipitation in human joints leads to gout, suggests that in gout the crystals must grow slowly over a period of years.

INTRODUCTION

Monosodium urate monohydrate, hereafter called sodium urate, is the salt of the singly ionised state of uric acid. It occurs in the form of fine needles in the pH range 6-10. The disease of gout occurs as a result of the precipitation of sodium urate within joint cartilage and the subsequent release of the crystals into the joint space where they cause acute inflammation [l] . The peripheral joints in the fingers and toes are usually affected first, particularly the first joint of the big toe. In man, uric acid is the end product of the breakdown of purines which arise both in the diet and in nucleic acid degradation. In a normal person, the uric-acid pool is about 1 g which is excreted at the rate of about 800 mg per day, mostly in the urine. Uric-acid levels are almost uniform throughout the body fluids, including blood and synovial fluid but are rather lower in saliva and very low in cerebra-spinal fluid and sweat. Some proteins do bind to uric acid in solution but the effect is not large [l] . In the mid-nineteenth century A.B. Garrod introduced the thread test which demonstrated the precipitation of urate crystals on a thread passed through the serum of gouty sufferers and proposed that the disease was caused by crystal precipitation. In the absence of high local concentrations of uric acid or evidence for rapid turnover which might lead to local fluctuations, we could expect gout to be primarily controlled by supersaturation. This is in contrast to bone mineralisation and to phosphate and pyrophosphate precipitation diseases

0166-6622/85/$03.30

0 1985

Elsevier

Science

Publishers

B.V.

98

where concentrations are very dependent on local cellular metabolism and where precipitation may be due to a local failure of control mechanisms. The solubility of uric acid in blood serum (ionic strength 0.14) at 37°C has been measured as 0.4 mM (in clinical units this is 7 mg/lOO ml). The incidence of gout rises rapidly with serum urate level above this saturation concentration reaching above 95% chance of suffering from gout at serum levels above 0.6 rnM [l]. Thus, the initial stage of gout is associated with the appearance of crystals in the joint and this appearance is primarily controlled by the supersaturation. Questions which remain to be answered include why the preferred site of precipitation is joint cartilage and particularly peripheral joint cartilage. It is also unclear why some individuals with particular urate levels contract gout while others have similar levels but are gout-free. One difficulty in this respect is that few people have their serum uric-acid levels measured until they start to be treated for joint disease. Thus detailed information on urate levels over time is rare. It is also a matter of debate whether urate crystals grow during a gout attack. The attack is apparently initiated by the release of crystals from the cartilage into the joint space as a result of some mechanical shock or fracture of the cartilage. The inflammation then builds up in a few hours. The crystals in the joint space may all originate in cartilage or may arise as a result of growth and secondary nucleation from the cartilagederived crystals. We have reviewed the precipitation of urate in relation to gout in more detail elsewhere [l, 21. Wilcox and co-workers [3-71 have made extensive studies of solubility and nucleation of sodium urate. In this work, we have measured crystal-growth kinetics which have also been studied recently using different techniques by Lam Erwin and Nancollas [8] and earlier by Allen et al. [9, lo]. EXPERIMENTAL

Sodium-urate solutions were prepared from sodium hydroxide and uric acid heated to 90°C in CO,-free water. The solutions were allowed to cool and then sealed into cells made from 5 X 5 cm glass slides separated by 1 mm and sealed with epoxy resin. Silicone resin is unsatisfactory as it releases acid and changes the pH of the solution. Crystallisation was observed using a polarising microscope with a hot stage which maintained constant temperature to * 0.1” C. Growing crystals were photographed using a camera fitted with a motorised film drive and timer so that pictures could be taken automatically at intervals from minutes to several hours. Growth rates parallel to the (001) axis of individual needle crystals were obtained by measuring from the negatives. Solubilities were measured by saturating solutions with sodium urate, removing crystals by passing the solution through a 0.22~pm filter and determining the urate concentration by UV spectroscopy. Dissolution times of

99

about 2 h were needed to reach equilibrium. Crystallisation to equilibrium was a much slower process. Both we and Wilcox et al. [3] have tried to measure solubility by observing the disappearance of crystals on heating in a hot stage. In our case the heating rate was 1°C min-‘. The two sets of hot stage results are in agreement but underestimate the solubility as the heating rate is too fast for equilibrium to be established, Fig. 1. At 37°C the solubility of sodium urate in water is 7.7 n&f.

5

10 ConcmIk8nul

15 nlM

Fig. 1. Solubility of sodium m-ate. (X ), this work; (o), Allen et al. [9] , ( q), Wicox al. [ 31; (m) Wilcox et al. [ 31 by hot stage; (o), Lam Erwin and Nancollas [8].

et

Studies of crystallisation from large volumes of stirred and unstirred solutions were also carried out. In these cases, where the flasks could not be sterilised and sealed, the solutions were stabilised against bacterial degradation by the addition of a l-mg crystal of thymol. Crystallisation was followed by the UV method in 0.14 M saline solutions or by conductivity in solutions equimolar in sodium and urate. Measurements of pH showed all solutions to be in the range 6.8-7.0. RESULTS

Figure 2 shows the longitudinal growth rate of sodium-urate needles as a function of concentration at 37°C. It can be seen that growth is too slow to measure below twice the saturation concentration. In Fig. 3, the data are

100

I 40

20 CONCENTRATION

Fig. 2. Growth

rates of sodium

urate

60

mM

from equimolar

solution

at 37” C.

2.4-

/

/

_--_I’

8

I

I

I

I

16

24

32

tC-S/S)2

Fig. 3. Growth

rate of sodium

urate

plotted

according

to a dislocation

model.

replotted according to the dislocation controlled growth model (“square law”) as a function of (relative supersaturation)2, (C - S/S)‘. This is clearly not a straight line. Saturation ratio (C/S) is defined as [( [Na+Na”] [urate-])/ where K,, is the solubility product. No correction is made for activity Kspl ‘= coefficients of the ions since the concentrations are small. A reasonably straight line is obtained (Fig. 4) when ln(growth rate) is plotted against l/ln(saturation ratio) according to the surface-nucleation

101

Fig. 4. Growth spread) model.

rates

of sodium

urate

plotted

according

to a surface-nucleation

(birth

and

model (“exponential law”) for growth. There are many theoretical expressions for the growth rate under surface-nucleation control [ll, 121. Davey [ 131 has recently given: G = Kns413 (In C/S)7f6 exp(-iTye2

/3(h57’

WC/S) 1

(1)

where K is a constant, ye is the molecular surface energy, C is the concentration and S is the solubility of the compound. To convert this for 1:l ionic compounds, C and S become the square roots of the concentration and solubility products. n, is the “number of growth units in saturated solution” and, therefore, reflects the decreasing growth rates for constant saturation ratio in low-urate-high-sodium solutions. Fitting our data on this basis, we obtain a correlation coefficient of 0.9994 and a surface energy of 6.9 kJ mole-‘. According to Nielsen and Sijhnel [20], the surface energy can be correlated with the solubility of the compound. Using the unit-cell parameters of Rinaudo and Boistelle [21], we obtain a surface energy for the interface step of 64 mJ me2 which is within the spread of values obtained from nucleation results on compounds of similar solubility [20] . In measuring growth rates from static solutions, one must be particularly concerned about the effects of solute depletion both throughout the solution and at the interface by solute diffusional limitations on growth. General solute depletion was eliminated by only making measurements when very

102

few crystals were present so that the amount of solute crystallised was a small fraction of the total when crystallisation is allowed to go to completion. The importance of solute diffusion can be determined by estimating the solute transfer rate over a distance equal to the size of the expected depletion zone [ll]; in this case, this size will be the needle diameter, less than 0.5 I.tm. The potential diffusional transfer over this distance is of the order of 400 times larger than needed for the observed crystal growth rate. Hence, solute diffusion is not a limiting process. Lam Erwin and Nancollas [8] measured growth rates of sodium m-ate by following the depletion of stirred solutions seeded with a known quantity of seed crystals. Their results for solution concentration versus time in individual runs were found to be consistent with the square law, that is dislocation controlled growth. From the initial depletion rates and the size and number of crystals, it is possible to calculate growth rates, as shown in Fig. 5, which are similar to those which we find but the subsequent growth behaviour is in contradiction with the exponential law which we observe. The situation is complicated by the fact that Lam Erwin and Nancollas take a slightly different solubility value from ours and that they plot an integrated form of the growth expression which is not very sensitive to the dependence of the growth rate on supersaturation. To estimate the importance of these

l f l

o” /

l0 /

0

I/

00

.O 0’

/”

r

r/

/

r X

/,.

.

x

.

X 2

6

4

Relative

Supersaturation

5. Growth rate of sodium-urate needles against supersaturation (([Na+l [I-K-l y?/ - 1). (I), This work, 37”C, NaHu; (X ) this work, 37”C, 0.14 M Na’; Wa+l WU-l,,)G (@) this work, 37”C, 0.14 M N a+ corrected; (m) Lam Erwin and Nancollas [8], 37’C,

Fig.

NaHU; (o), Allen et al. [9, Na+ corrected.

lo],

50°C;

0.14

M Na+; (o),

Allen et al. [9, lo],

5O”C, 0.14

M

factors we used an iterative computation to calculate the solution tion as a function of time for various growth laws expressed as: G =A (C/S - l)n

concentra(2)

For our measurements plotted in this way, n = 3.2, whereas Lam Erwin and Nancollas found their data to be consistent with n = 2. In fact, our computations show that the integrated plots used by Lam Erwin and Nancollas give reasonable straight lines for n values from about 1 to 3, but they are clearly curved for higher values of IZ. Thus, the integrated plots are not a very rigorous test for square-law behaviour. In addition, it is possible that the very fragile needle crystals are broken by stirring, so invalidating the assumption of Lam Erwin and Nancollas that the number of growing crystals remains constant. To investigate this, we crystallised solutions containing 2.5 m&f urate and 0.14 A4 sodium at 37°C and pH 7.4. In a quiescent solution no observable crystallisation occurred in 10 days; when stirred with a magnetic stirrer crystallisation was complete in about 24 h, while stirring with a glass rod or one-minute treatments of ultrasonic irradiation every few hours produced crystallisation in 40 h. Microscopy showed the crystals to be broken into lengths of a few micrometers by the ultrasonic irradiation and subsequently to regrow. This effect would explain the initial agreement between the two studies (Fig. 5) and give rise later to more rapid growth in the stirred solutions. Figure 5 also gives the growth rates measured by Allen et al. [9, lo] at 50°C. At equivalent saturation ratios growth is much faster at the higher temperature than at 37°C. Under physiological conditions, sodium-urate crystals grow from 0.14 M sodium solutions rather than equimolar sodium urate. We have investigated this by following growth in unstirred seeded solutions in flasks. The microscopic technique is unsuitable as constant concentration is harder to maintain at the low urate levels required. Seeds were prepared by crystallisation followed by an ultrasonic treatment to break up the fine needles. Small amounts of liquid containing these fragments were passed through 0.22~pm Growth was followed by removing Millipore filters into fresh solution. samples at various times and measuring the crystal length microscopically. A few small crystals were seen which were believed to have nucleated after seeding, but the majority had a roughly uniform length corresponding to growth of the seeds. The resultant rates were slower than those observed for equimolar solutions at the same saturation ratio as is shown in Fig. 5. Following Eqn (l), we correct for n,, the lower values of the m-ate concentration, assuming that attachment of a urate ion to a step site is the limiting interfacial transfer process, we obtain agreement with the data on equimolar crystallisation. A number of organic dyes have been shown to retard crystal growth of sodium urate [9]. We have used conductivity measurements in equimolar solutions to observe the effects of various additives. Figure 6 shows the increase in the time required to 50% crystallisation of a 75 m&f solution on the

LOG( -

x-&A

Fig. 6. Effect of poison Red; (x ), serum albumin.

POISON -0-

on crystallisation

CONCENTRATION Neutral

PM)

red

half-times,

7*10-*

M NaHU,

37°C.

(a),

Neutral

addition of a cationic dye of molecular weight 289, Neutral Red or a protein of molecular weight 66,000, bovine serum albumin. The serum albumin has some effect whilst that of the dye is quite dramatic at a roughly similar weight concentration. On adsorption, the protein probably denatures and covers many surface sites. We also found that both 0.1% polyethyleneimine, a positively charged polymer, and 0.01% heparin, a negatively charged polymer, were effective growth poisons. A 4% addition of synovial fluid also increased the crystallisation time four-fold. By ultrafiltration, we showed that this effect was due to the macromolecular components of the synovial fluid. The effect of growth poisons in such crystallising systems is complicated since, at high concentrations, they may act either on the crystal surface or by complexing ions in solution. In addition, inclusion of the compounds in the growing crystals will lead to their being depleted from the solution. The retardation of growth kinetics by serum albumin is constant (at 4X ) from 2. 10m3% to 10% protein concentrations. Controlled urate activities in synovial fluid are hard to produce. If we assume the same behaviour for the macromolecular components of synovial fluid as for serum albumin, we do not expect large reductions in growth rates in synovial fluid.

105 DISCUSSION

The accord of urate crystallisation with the exponential-growth law is of particular interest for two reasons. Firstly, such growth kinetics from solution are relatively rare and dislocation control is far more commonly observed. Secondly, we need to extrapolate from the observed growth data in order to estimate growth rates under physiological conditions, where supersaturations are much lower. Surface-nucleation controlled growth is common in melt growth of large molecules [ 151. Davey [13] discusses growth of similar molecules from concentrated organic solution where nucleation control also occurs. KDP and ADP growth from aqueous solution show nucleation control on (101) faces [ 161, and there is a recent report of nucleation control in aqueous solutions of CaF, and SrF, [22], but otherwise clear-cut examples of nucleation control seem rare except for urate and some proteins [14]. In the case of urate, it seems clear that growth would be faster if a dislocation mechanism was available; possibly the needle shape of the crystals prevents this in that dislocations may rapidly grow out through the side faces. If we adopt the nucleation control mechanism, it is possible to extrapolate the growth rates to the supersaturation range observed in gout patients. These rates are given in Table 1 for cases corresponding to moderately (9 mg or 0.6 mM) and severely (14 mg or 0.8 mM) gouty serum levels. It can be seen that, at 37°C the times are unreasonably long. Two modifying factors should be considered. Firstly, it is possible that growth may convert to a dislocation mechanism. There seem to be no clear examples of such changes

TABLE

1

Extrapolated Surfacenucleation control (exponential law) Concentration mM Rate, pm min.’ Rate, Mm ye’

growth

rate for mate

crystallisation

in gout

Temperature 37°C

32°C

0.4 0 0

0.6 10-11 5.10+

Dislocation control (square law)

37°C

Concentration, Rate, pm y-’

0.4 0

mM

27” c

0.8

0.4

0.6

0.8

6.10.’ 0.3

2.10-13 9*10-@

2.10-7 0.1

2.1O-5 8

0.6 15-80

0.8 100-500

0.4 1o-7 0.05

0.6

0.8

10-S 5

1o-4 52

106 with concentration, but for melt growth there are examples where damage to growing crystals has led to an increase in growth rate and a switch to a dislocation mechanism [17, 181. Such damage could arise from the mechanical action of moving the joint. Secondly, it should be noted that joint temperatures do not necessarily remain at 37°C. Detailed local-temperature maps do not seem to be available for the human body. There are some measurements on temperatures of knee and ankle joints, giving 33 and 29”C, respectively, [ 191 and our fairly crude measurements of toe joint temperatures [2] showed that temperatures as low as 26°C could be reached when inactive in cool surroundings. Such temperatures bring the extrapolated growth rates into reasonable regime (Table 1) for growth over a period of years. This would suggest a model for gout where nucleation and growth occurred slowly within the cartilage and, occasional release of these crystals into the synovial space gave rise to an inflammatory response and gout attack but no further crystal growth. It is of interest that treatments which reduce serum uric-acid levels frequently precipitate attacks since the initial stages of solubilisation may loosen crystal deposits within the cartilage surface. CONCLUSIONS

Monosodium urate monohydrate crystallises from aqueous solution in the form of fine needles. These needles extend at a rate which depends on supersaturation according to the surface nucleation control or “birth and spread” model, Eqn (1). Growth rates observed in physiological saline correlate with those measured at similar supersaturations in equimolar solutions. Extrapolation of these rates to urate concentrations observed in gout leads to impossibly low predicted growth rates at 37°C. Correction for the lower temperatures of the peripheral joints which are affected by gout suggests that the crystals found in gouty cartilage would take several years to grow. ACKNOWLEDGEMENT

We would

like to thank

Professor

I. Gutzow

for his helpful

comments.

REFERENCES 1 2 3 4 5 6 7

P.A. Dieppe and P.D. Calvert, Crystals and Joint Disease, Chapman and Hall, London, 1983. R.W. Fiddis, N. Vlachos and P.D. Calvert, Ann. Rheum. Dis. Supp. 1, 42 (1983) 12. W.R. Wilcox, A. Khalaf, A. Weinberger, I. Kippen and J.R. Klinenberg, Med. Biol. Eng., 10 (1972) 522. A.A. Khalaf and W.R. Wilcox, J. Cryst. Growth, 20 (1973) 227. I. Kippen, J.R. Klinenberg, A. Weinberger and W.R. Wilcox, Ann. Rheum. Dis, 33 (1974) 313. W.R. Wilcox and A.A. Khalaf, Ann. Rheum. Dis., 34 (1975) 332. H.K. Tak, S.M. Cooper and W.R. Wilcox, Arthritis Rheum., 23 (1980) 574.

107 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

C.-Y. Lam Erwin and G.H. Nancollas, J. Cryst. Growth, 53 (1981) 215. D.J. Allen, G. Milosovich and A.M. Mattocks, Arthritis Rheum., 8 (1965) 1123. D.J. Allen, G. Milosovich and A.M. Mattocks, J. Pharm. Sci., 54 (1965) 383. A.E. Nielsen, Kinetics of Precipitation, Pergamon, Oxford, 1964. M. Ohara and R.C. Reid, Modeling Crystal Growth Rates from Solution, Prentice Hall, Englewood Cliffs, NJ, 1973. R.J. Davey in E. Kaldis (Ed.), Current Topics in Materials Science, Vol. 8, North Holland, Amsterdam, 1982. R.W. Fiddis, R.A. Longman and P.D. Calvert, J. Chem. Sot. Faraday Trans. 1, 75 (1979) 2573. D.R. Uhlmann in L.L. Hench and S.W. Freman (Eds.), Advances in Nucleation and Crystallisation of Glasses, Am. Ceram. Sot, Colombus, OH, 1971, p. 91. W.J.P. Van Enckevort, R. Janssen-Van Rosmalen and W.H. Van der Linden, J. Cryst. Growth, 49 (1980) 502. I. Gutzow, J. Cryst. Growth, 42 (1977) 15. D.E. Ovsienko, G.A. Alfintsev, G.P. Chemerinsky, S. Budurov and N. Stoichev, J. Cryst. Growth, 60 (1982) 107. J.L. Hollander, E.K. Stoner, E.M. Brown and P. DeMoor, J. Clin. Invest., 30 (1951) 701. A.E. Nielsen and 0. Sohnel, J. Cryst. Growth, 11 (1971) 233. C. Rinaudo and R. Boistelle, J. Cryst. Growth, 57 (1982) 432. A.E. Nielsen and J.M. Toft, J. Cryst. Growth, in press.