Factors affecting the solubility of ammonium acid urate

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Clinica Chimica Acta, 95 (1979) 17-22 @ Elsevier/North-Holland Biomedical Press

CCA 1014

FACTORS AFFECTING

R.C. BOWYER

THE SOLUBILITY

a**, R.K. McCULLOCH

OF AMMONIUM ACID URATE

a, J.G. BROCKIS

b and G.D. RYAN a

a Royal Perth Hospital and b University of Western Australia, Perth (Australia) University of Western Australia, Perth (Australia) (Received

December

4th, 1978)

Summary Evidence is presented that sodium acid mate exists in aqueous solution in a non-colloidal state. The levels of ammonium and urate ions required to precipitate ammonium acid urate have been established for some aqueous media. The effect of pH on the formation product of ammonium acid urate is described. Evidence is presented that there is no epitaxial relationship between this salt and calcium oxalate monohydrate.

Introduction In a previous report on the aetiology of endemic bladder stones of Indonesian children [l], we mentioned the large amounts of ammonium acid urate (NH4U) present in the non-infected stones. Although the solubility of uric acid and sodium acid urate (NaU) has been studied extensively over the last 80 years [2-g], factors affecting the solubility of NH4U remain largely unresearched. Porter [6,7] has studied “flocculation” of NH4U in phosphate buffers on the basis that urate salts, and NaU in particular, exist in solution in a colloidal state. More recently, Pak et al. [8] and Robertson et al. [lo] implicate a role for colloidal urate in the aetiology of calcium oxalate lithiasis. However, in the majority of studies on urate solubility, the possibility of urates forming colloidal sols has either been overlooked or positively discounted [ 5,9]. As a prelude to the study of NH4U solubility we therefore found it necessary to investigate the evidence that urates form colloidal sols in aqueous media. Since many sparingly soluble salts may exist in solution at quite high levels of supersaturation, true solubility values are often misleading from a clinical point of view. Urate salts in particular can exist in solution at concentrations many times the true solubility level for periods of several days [6,8]. More * To whom correspondence should be addressed.

relevant information may be obtained by studying the formation product of such salts, i.e. the levels of ions which result in precipi~tio~ of the salt. Xn this study, conditions under which N&U precipitates from water, urine and other aqueous media are investigated. The effect of pH on the formation product is also established. Finally, because endemic bladder stones often contam significant p~~~ort~ons of calcium oxafate, the poss~b~~~tyof an epitaial relationship between these two salts is discussed, Materials and methods Chemicals used were reagent grade except where indicated. Uric acid (Biochemical grade 3DH) was purified using the method of Porter [6f. NH4U and NaU were prepared by a previously described method [C;]. Water was twice distilled from all-glass apparatus. Urate ~~~~en~at~ons in s~~thet~~~~y prepared solutions were determined spectroscopically [C;j and in urme by the phosphotungstate method (SMA AutoAnalyser Technicon). Ammonia levels were estimated using the ~~d~pl~enol reaction 111 J. Urine was preserved over chloroform. Precipitates were analysed chemi~~ly and by X-ray c~s~o~aphy. ~ondu~tivi~ experiments were performed using a 250~ml glass cell with p~at~nurn electrodes and a Philips P~9~UU~~2 ~ondu~~vit~rne~~ng bridge, Soiutions of NaU were uftrafiItered using an Amicon apparatus with UMlO and UM2 membranes which have a cut-off of ‘10000 and 2000 d&tons, respectively, Dialysis of NaU solutions were performed using Visking dialysis tubing (Union Garbide).

Au. e~pe~rnen~ were debited for solutions of pH &5 at 37°C over a period of 6 h. A stock urate solution was prepared by the dropwise addition of 2 mol/l sodium hydroxide to a suspension of uric acid in the test medium. The pH was maintained at 6.8-7.0 until all the acid had dis~~~~~dand was then adjusted to pH 6.5 with hydrochloric acid. By this means urate solutions up to 20 mmol/l could be prepared. A gradient of urate concentrations was obtained by diluting the urate stock with the or&i& test solution, The addition of the ammonium ion component to this system required the initial p~p~a~on of a series of ~on~n~~d ammonium chloride solutions. These solutions were prepared at such a strength that when a S&u1 atiquot was added to 1 ml of uram solution the desired ~monium ion ~~~~entra~on was obtained. A matrix of tubes conning a range of both ~rnQ~i~rn ion and urale ~~neen~tions was thus provided. After standing for 6 h at 37”C, the minimum concentration of ammonium ion required to initiate precipitation was noted by direct observation for each level of urate ion.

A pooled urine sample was diluted with distilled water ta 2E%, 50% and 75% of its original concentration, The procedure described above was then used to obtain “solubility” curves for the diluted urines.

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Cd)Effect of PH The procedure as described above was used except that the mate concentration was held constant at 8 mmol/l and the pH varied from 5.5 to 8.0. (e) Effect of adding crystalline calcium oxalate (CaC204 . H,O) and solid NH4U The urate concentration of a pooled urine sample was increased as described in section (b) and brought to a pH of 6.5. The urine was then dispensed in duplicate into a series of tubes containing a gradient of ammonium ion concentrations. To one series of tubes 50 ~1 of a CaC204 - Hz0 suspension (1 g/l) was added and to the other, the controls, 50 ~1 of water. The final urate concentration was 8 mmol/l. The tubes containing the crystals were gently inverted every 15 min during the 6-h test period to maintain the crystals in suspension. The same procedure was used for the addition of the NH4U suspension (1 g/l). At the completion of 6 h all tubes were centrifuged (2000 X g for 5 min) and the supernatant was assayed for mate. Results (a) Non-colloidal behaviour of ura te solutions at 3 7°C (i) Conductivity measurements performed with NaU solutions ranging in strength from 4 to 16 mmol/l gave similar readings to solutions of potassium chloride and sodium acetate of equivalent concentration and did not vary over a 16-h period. (ii) Ultrafiltration of a series of NaU solutions ranging in strength from 4 to 16 mmol/l did not result in any significant fractionation of urate concentration between the retentate and ultrafiltrate. (iii) The urate level of a solution of NaU contained in dialysis sacking rapidly decreased during dialysis with distilled water. (iv) Solutions of NaU did not show any Tyndall effect.

16 14' 12 IO' -TgE E

a6.

F e La

42-

b

H4+]

m mot/l

Fig. 1. NH4U solubility curves at 37°C. PH 6.5 and 6 h. 1, pooled 3, water; 4, children’s urine diluted 50%.

adult urine; 2, pooled

~hildren’s

tine;

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(b) NH,U formation product The relation between the levels of ammonium and urate ions which cause precipitation of NHllU has been established for a number of test solutions and the results are expressed as a set of “solubility” curves. These curves were established for a pooled urine from six healthy adult male Australians (Caucasians) (Fig, 1, curve l), a pooled urine from six Australian (Cauc~ian) children aged up to 8 years (Fig. 1, curve 2) and also for water (Fig. 1, curve 3) which served as a control. The effect .on the formation product of adding water to urine is shown in line 4, where it will be observed that with urine dilution, the curve shifts further to the left. This effect is being reported in detail elsewhere (Bowyer et al., in preparation).

The relation between pH and the level of ammonium ion required to precipitate a fixed concentration of urate solution was established for both a pooled adult urine and water (Fig. 2). Analysis of the precipitates showed a variation of composition with pH. Above pH 6.3 the product was NH4U whilst below pH 5.7 for urine and pH 6.0 for water it was uric acid. At intermediate pH both forms of urate were present. (d) Effort of seeding urine (i) CaC204 * H,O. The concentration of ammonium ion needed to precipitate an 8 mmol/l urate solution in a pooled adult urine of pH 6.5 within six hours at 3’7°C was found to be 150 mmol/l. The experiment was repeated with the same sample but this time the urine was seeded with a suspension of CaC204 - Hz0 crystals (1 g/l). The level of ammonium ion that caused precipitation under these conditions was 145 mmol/l. The addition of CaC204 * Hz0 crystals thus caused negligible enh~cement of pr~ipi~tion. (ii) NH~U. In contrast, when the above experiment was repeated with a suspension of NH4U (1 g/l) instead of oxalate, there was a profound enhancement

04 6.5

61)

8.5

ZO

7s

8.0

PH Fig. 2. Effect of pH on the precipitation of NH.+U. 1, urine: 2. water.

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of precipitation. Whereas the 8 mmof/l mate solution in both urine and urine seeded with CaC204 *Hz0 was stable to levels of ammonium as high as 145 mmol/l, in the presence of solid NW4U the solution was unstable to levels of ammonium ion as low as 10 mmolll. Discussion The behaviour of urate salts in solution is unusual in that they can exist in a state of high sup~~satu~tion for prolonged periods. While this may be explained by very sluggish kinetics of nucleation and crystal growth, it has been suggested that solutions of urate salts are colloidal. We have subjected solutions of NaU of varying concentrations to the standard tests for establishing colloidal properties and our findings suggest that NaU is present in true solution and not in a colloidal state, In studying the precipitation of NH4U from aqueous solutions, it was found necessary to make observations over a standard period of time as precipitation is t~e~ependent” We chose 6 h as this is about the time urine stays in the bladder overnight. Results have been expressed in the form of “solubility” curves (Fig. 1). Any combination of urate and ammonium ion concentration that gives an intercept in the region to the right of the curve results in precipitation of NH4U within 6 h. From Fig. 1, it is observed that subs~tially higher concentrations of ammonium and urate ions are required to cause precipitation in urine than in water. Solubility studies on different dilutions of the same urine show that the degree of “pro~ction” afforded, compared with water, is propo~ion~ to the urine content. From these results it is apparent that very high urinary levels of both urate and ammonium ions must occur before precipitation of NH,U will begin, Similar conclusions were reached by Teotia and Sutor [lZ] under different experimental conditions. Ex~e~rnen~ on diluted urines suggest that urine of low osmolality but overloaded with ammonium and urate ions is particularly prone to NH4U precipitation. The effect of pH on precipitation is shown in Fig. 2. For this study the mate level was held cons~t at 8 mmol/l and the pH and ammonium ion concentrations varied. Both for urine and water, higher levels of ammonium ion are required to initiate precipitation as the pH is decreased, At pH below 5.7 in the presence of amn~onium ion concentrations up to 2~0 mmol/l, the predominant precipitate is uric acid which is only slightly soluble in the und~socia~d form. At higher pH a greater proportion of urate anion is present in solution, requiring lower levels of ammonium ion to exceed the NH4U formation product. The theoretical dist~bution of uric acid and urate anion in an 8 mmolfl solution is shown in Fig. 3. Our investigation into an epitaxial relationship between NH,U and calcium oxalate shows that seeding of supersaturated soIutions of NH,U with crystals of CaC204 - Hz0 has no si~ific~t effect. In con~~t, seeding with solid NH,U results in heavy deposition of NH.JJ from solution. Although high levels of both ammonium and urate ions are required for initial precipitation, once solid N&U is present, precipitation continues at much lower levels of supematuraLion.

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6-

2,’ ,’

I‘ Uric acid concentration

,I’

0-r 4

5

6

7

1 .9

PU Fig. 3. Distribution of uric acid and urate ion concentrations as a function of pH for an 8 mmol/l uric acidlurate solution. -, uric acid concentration; - - - - - -, urate concentration.

Acknowledgements The authors would like to acknowledge financial support for this work from the Royal Perth Hospital Research Foundation, the Australian Kidney Foundation and TVW Telethon Research Foundation. We would also like to thank Dr. E. Maslen of the University of Western Australia for X-ray c~s~o~aphy analyses and Professor A, Posner of the Unive~i~ of Wes~~ Australia for helpful discussions. References 1 Thslut, K., Rizal, A., Brockis, J.G., Bowyer, R.C., Taylor, T.A. and Wisniewski, Z.S. (1976) Br. J. Ural. 48,617-621 2 Seegmiller. J.E. (1974) in Disease of Metabolism (Bandy, P.K. and Rosenbery, L.E.. eds.). pp. 656661, Saunders, Philadelphia 3 Kippen, 1.. Khnenberg, J.R., Weinberger, A. and Wilcox, W. (1974) Ann. Rheum. Dis. 33, 313-318 4 Wilcox. W.R., Khalaf, A., Weinberger, A. and Klinenberg, J.R. (1972) Med. Biol. Eng. 10. 522-531 5 DeVries. A. and Sperling, 0. (1976)in Scientific Foundations of Urology (Williams. E.D.. ed.), pp. 297-299, Year Book Medical Publishers, Chicago 6 Porter, P. (1963) Res. Vet. Sci. 4, 580-591 1 Porter, P. (1963) Res. Vet. Sci. 7, 692-602 8 Pak, C.Y.C., Waters, O., Arnold, L., Holt, K., Cox, C. and Barilla. D. (1977) J. Clin. Invest. 59, 426431 9 Finlayson. B. and Smith, A. (1974) J. Chem. Eng. Data 19,94-97 10 Robertson, W.G., Knowles. F. and Peacock, M. (1976) in Urolithiasis Research (Fleisch. H.. Robertson, W.G., Smith, L.H. and Vahlensieek, W., eds.), pp. 331-334, Plenum Press, New York 11 Gips. C.H., Reitsma, A. and Wibbens-Alberts, M. (1970) Clin. Chim. Acta 29,501-505 12 Teotia. M. and Sutor. D.J. (1971) Br. J. Ural. 43, 381-386