Triamterene and Renal Stone Formation

0022-5347 /82/1273-0593/$02.00 THE JOURNAL OF UROLOGY

Vol. 127, March

Copyright© 1982 by The Williams & Wilkins Co.

Printed in U.S.A.

TRIAMTERENE AND RENAL STONE FORMATION DONALD J. WHITE*

AND

G. H. NANCOLLASt

From the Department of Chemistry, State University of New York at Buffalo, Buffalo New York

ABSTRACT

We investigated the influence of triamterene (TA), and its metabolites parahydroxytriamterene (PHTA), and parahydroxytriamterene sulfate (PHTAS) on the nucleation and crystal growth of calcium oxalate monohydrate (COM), in supersaturated solution at 37C using a new constant composition technique. The spontaneous precipitation of COM is preceded by induction periods which decrease with increasing supersaturation. The addition of the triamterene seed materials substantially reduces these delay periods and induces the crystal growth of COM. Specific surface area and scanning electron microscopic results indicate that the seed materials act as sources for the heterogeneous nucleation of COM. In addition, the surface of the more crystalline PHTAS appears to offer sites from which COM crystals can develop as well formed rosettes. This evidence suggests that in addition to triamterene renal stone formation, TA and its metabolites may catalyze the precipitation of other stone forming minerals with which urines may be supersaturated. The diuretic triamterene (2,4,7-triamino-6-phenyl pteridine; hereafter, TA) is prescribed chiefly for the treatment of hypertensive disorders. The commonly administered form of TA is Dyazide, used by over 3,000,000 patients in this country. Recently, TA and its metabolites, parahydroxytriamterene (hereafter, PHTA), and parahydroxytriamterene sulfate (hereafter, PHTAS), have been implicated as precursors for renal stone formation in patients undergoing Dyazide treatment. Since the first report of the formation of a TA renal stone, 1 Ettinger and coworkers2 have estimated the incidence of TA stone development in Dyazide users at about 1 in 1500. Almost 0.4 per cent of the renal calculi contained some form of TA with compositions ranging from 100 per cent TA to those containing significant amounts of other renal calculi constituents such as calcium oxalate monohydrate and dihydrate, uric acid, and protein matrix. More than 42 per cent of the TA stones examined by Ettinger contained at least 80 per cent by weight of these common renal stone constituents. The promotion of renal stone formation by TA may involve its spontaneous precipitation in the urinary tract or the participation of TA nuclei in the heterogeneous nucleation or epitaxial growth of other renal stone constituents. TA may also promote the aggregation of small crystallites present in urine. An understanding of the mechanisms of formation of renal calculi in the presence of TA and its metabolites will provide valuable information concerning the nucleation and epitaxial processes which may be involved in stone precipitation. In the present study, a constant composition seeded crystal growth method has been used to investigate the influence of TA and its metabolites on the nucleation and subsequent growth of calcium oxalate from supersaturated solutions. During the experiments, the supersaturation and ionic strength were maintained constant by the addition of solutions containing calcium and oxalate ions, controlled by a specific calcium ion electrode. MATERIALS AND METHODS

Reagent grade calcium chloride dihydrate, potassium oxalate monohydrate, and sodium chloride were used (Fisher ScienAccepted for publication August 31, 1981. Supported in part by Grant No. Rol AM19048 from the National Institute for Arthritic, Metabolic and Digestive Disease, National Institutes of Health. * A Samuel Silbert Fellow, 1980-1981, State University of New York at Buffalo. t Requests for reprints: Department of Chemistry, State University of New York at Buffalo, Buffalo, New York 14214. 593

tific). Solutions were prepared using triply distilled, deionized water, and were filtered twice (0.22-µm. Millipore filters) before use. The filters were prewashed in order to remove any residual wetting agents or surfactants. Solutions were analyzed (0.1 per cent) by the passage of aliquots through an ion exchange resin (Dowex 50), in the hydrogen form, and titrating the eluted acid with sodium hydroxide. An ionic strength of 0.15 M was maintained throughout the experiments by the addition of sodium chloride. Samples of TA, PHTA and PHTAS were kindly provided by Dr. B. Ettinger. Their purity was confirmed by thin layer chromatography, infra-red spectroscopy (Perkin Elmer Model 467), and x-ray power diffraction (Phillips XRG 3000 diffractometer, copper Ka radiation). The specific surface areas, SSA, of the triamterene seed materials, measured by a single point nitrogen BET absorption method (Quantasorb, Quantachrome Corporation), were 0.62, 4.72, and 5.65 m. 2g.- 1 for TA, PHT A and PHTAS, respectively. Crystallization experiments were made in a 400-ml. magnetically stirred Pyrex double walled reaction cell maintained at 37.0 ± O.lC. Nitrogen gas, saturated with respect to 0.15 M NaCl at the temperature of the reaction vessel, was bubbled continuously through the solutions during the experiments. The pH was monitored using a glass electrode (Corning No. 47602A) and the calcium ion activity was measured by means of a specific calcium ion selective electrode (Radiometer Model Ca72112) coupled with a thermal electrolytic silver/silver chloride reference electrode. 3 The latter was immersed in 4.0 M KCl solution, saturated with AgCl, in a thermostated limb of the electrode assembly, and was separated from the working solution by an intermediate liquid junction containing 0.15 M sodium chloride. Errors in emf due to changes in the liquid junction potential through leakage of salt bridge solution were therefore eliminated. In a typical seeded crystal growth experiment, the calcium electrode was standardized in calcium chloride solutions by successive additions of aliquots of standard CaCh solutions to 0.15 M sodium chloride. Subsequently, supersaturated solutions were prepared by the slow addition of potassium oxalate solution. The crystallization reactions were initiated by the addition of weighed amounts of TA material to the supersaturated solution in the reaction cell. The constant composition method was also used to investigate spontaneous calcium oxalate precipitation in the absence of added seed crystals. Following the onset of precipitation, the calcium ion activity was maintained by the addition of calcium chloride and potassium oxalate titrant solutions, delivered simultaneously by a pair of piston

594

WHITE AND NANCOLLAS

driven 10 ml. burets, controlled by a Metrohm Herisau pH-stat (Model Combitrator 3D, Brinkman Instrument Co.). Periodically, aliquots of reaction mixture were withdrawn, rapidly filtered, (0.22-µ.m. Millipore filters), and the filtrates analyzed for calcium by Atomic Absorption (Perkin Elmer Model 503) in order to verify the constancy (to <1.0 per cent) of the calcium concentrations. In addition, the solid phases, dried at room temperature in a desiccator, were investigated by infrared, xray diffraction and by scanning electron microscopy (ISi Model Super II). Measurements of particle density and size distributions were made by the transferral of 1.0-ml. aliquots of reaction mixture to 50 ml. of 0.15 M sodium chloride solution saturated with respect to calcium oxalate monohydrate (COM). Immediate determinations were made with an Elzone particle distribution analyzer (Model 111LTSCD1ACD S/N 72462, Particle Data Inc.) by the passage of 500 µ.l. volumes through a calibrated orifice.

experiments, supersaturated solutions of calcium oxalate were prepared by the rapid mixing of calcium chloride and sodium oxalate solutions of equal volumes. The moles of COM precipitated, plotted in figure 1 as a function of time for constant supersaturation, show the excellent reproducibility of the results. It can be seen that precipitation was preceded by induction periods of duration shown in the table, increasing with decreasing supersaturation. Throughout the experiments, the solid phases formed were shown, by x-ray analysis, to consist entirely of COM. Moreover, the appreciable specific surface areas (e.g., 13.09 m.2g.-1 Expt. 273, table) reflect the formation of a large number of very small particles at these relatively high supersaturations. At lower supersaturations, the precipitation was preceded by longer induction periods and the solid phases had markedly smaller SSA (specific surface area) values (Expts. 275, 280, table). The rates of titrant addition at the ends of the induction periods, were used to calculate effective rates of crystallization which can be represented by Equation 2:

RESULTS AND DISCUSSION

The activities of the ionic species in the solutions were calculated from mass balance and electroneutrality expressions as described previously, taking into account the formation of the oxalate ion-pairs, CaOx and Naox-.4 • 5 Activity coefficient corrections were made by successive approximations for the ionic strength using the extended form of the Debye-Hiickel equation proposed by Davies. 6 At 37C, the thermodynamic solubility product of COM, 2.28 X 10-9 mol. 21.-2 , determined by allowing crystallization experiments to proceed to equilibrium, agreed well with the value, 2.20 X 10-9 mol. 21.-2 , reported previously. 5 The crystallization experiments are summarized in the table, in which the supersaturation, S, is defined by Equation 1; S

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300

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TIME (min) Fm. 1. Spontaneous crystallization of COM. Moles of COM precipitated plotted against time: 0, Expt. 273; 0, Expt. 274; 6., Expt. 275; D, Expt. 279; '11, Expt. 280.

Crystallization of COM at 37C At End of Experiments Exp. No.

273t 274t 275t 279t 280t 261 262 263t 267 269 264 265 266 268 270 271 272 276 277 278

Seed Type

S"ed Cone. (mg.1.-•)

Tea X 104 (moler')*

Induction Time

232.7 174.0 202.0 197.3 154.0 214.0 194.7 193.3 228.7 213.3 170.7 168.0 84.7 92.0 109.3

5.05 4.54 4.04 5.05 4.04 5.05 4.54 4.04 4.04 5.05 5.05 4.54 4.04 5.05 5.05 4.54 4.04 4.04 4.04 4.04

52 120 288 39. 344 -08 83 125 2 4 9

PHTAS PHTAS PHTAS PHTAS PHTAS PHTA PHTA PHTA PHTA TA TA TA (272)§ (264)§ (269)§

•Tc.= To, in the supersaturated solutions. t Spontaneous precipitation experiments. t Expt. 263. Ground PHTAS seed material. § Grown solid phases from these expts.

s %Growth

11

5 4 20 180 8 3 2

1.52 1.28 1.04 1.52 1.04 1.52 1.28 1.04 1.04 1.52 1.52 1.28 1.04 1.52 1.52 1.28 1.04 1.04 1.04 1.04

10.4 25.7 34.8 34.1 33.2 27.5 31.6 32.2 28.3 29.6 37.2 14.4 50.9 48.4 42.0

Rate of COM Growth X 104 mole min.-• (mg. COM)- 1

Specific Surface Area (m.2g.-')

4.971 1.501 0.687 3.221 0.446 2.420 2.000 1.750 0.752 2.600 3.460 3.500 0.732 3.201 6.600 2.912 1.301 1.001 0.781 1.201

13.09 2.45 1.66 10.00 2.20 -49.00 4.13 6.54 2.87 6.34 9.45 13.48 10.91 12.13 5.62 19.01 10.02 5.02 10.04 6.92

595

TRIAMTERENE AND RENAL STONE FORMATION

R _ dTca _ k -~-

/j. 2

(2)

gS

In Equation 2, Tea is the molar calcium concentration, R the rate of crystal growth, kg the rate constant and s a function of the surface area of the crystals. It was found that Equation 2 satisfactorily represented the growth rates of COM, normalized for the specific surface area of the final grown phase. The calculated rate constant for crystal growth was 8.57 x 103 1 mole- 1min.- 1m.-2 • In order to investigate the effectiveness of TA in inducing the crystallization of COM, a number of experiments were made in which the metastable supersaturated solutions of COM were inoculated with the TA seed material. The results of the experiments are summarized in the table and plots of the moles of calcium oxalate precipitated as a function of time are shown in figure 2. It can be seen that the induction periods preceding the crystallization of COM were considerably shorter than those in the corresponding spontaneous precipitation experiments (fig. 1). This indicates that TA offers favorable sites for the nucleation and subsequent growth of COM. In these experiments, the slow wetting rate of the TA seed crystals was probably a factor in the measured induction times. This is clearly illustrated by

FIG. 4. Scanning electron micrograph of TA seeded Expt. 271 (X5000).

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FIG. 3. Particle distribution analysis of TA induced COM crystallization for Expt. 270; total counts plotted against the particle size: D, initial; 0, final.

FIG. 5. PHTA induced COM crystallization. Moles of COM precipitated plotted against time: 0, Expt. 264; 0, Expt. 268; D, Expt. 265; 6., Expt. 266.

the particle distribution data in figure 3, in which the number of particle counts per channel is plotted against the size of the particles. Immediately following inoculation, very few particles are detected in the distribution analysis. This agrees with the experimental observation that the TA seed particles tended to float initially on the surface of the supersaturated solution and sedimentation did not take place until the precipitation had commenced. As precipitation of COM proceeded, particle sedimentation was observed, and the solution became progressively more turbid with an increase in particle density from 1.3 x 107 to 8.9 X 107 particles 1-1 in the first hour of reaction. The resultant change in the particle data profile is shown in figure 3. Infrared and x-ray analysis of the grown phases again confirmed the exclusive formation of COM. The relatively large increases in SSA during the experiments shown in the table resulted from the formation of the small COM crystallites. The nucleation and growth of these microcrystals at the TA surface is shown in the scanning electron micrographs for experiment 271 in figure 4. In all cases, the COM was associated with the dispersed TA fibers and no separate crystallites of spontaneously precipitated oxalate could be observed in any of the samples examined. The considerable degree of aggregation of

596

WHITE AND NANCOLLAS

the precipitated COM during the experiments is also shown in figure 4. The results of experiments using PHT A and PHT AS seed materials are summarized in the table. Plots of the moles of calcium oxalate precipitated as a function of time for PHTA and PHTAS are shown in figures 5 and 6 respectively. The observed induction periods preceding the crystallization of COM were again considerably shortened as compared with the spontaneous crystallization experiments (fig. 1) and the increase in SSA in the initial stages of the reaction followed the same trend as that observed in the TA seeded experiments (table). It can be seen from a comparison of the results of Expts. 263 and 267 in the table that grinding of PHTAS seed markedly reduced the induction period and increased the subsequent rate of COM crystallization even though the initial specific surface area of the seed samples was unchanged. Particle distribution analysis and scanning electron micrographs are shown in figures 7 and 8 for PHT A and figures 9 and 10 for PHT AS, respectively. The particle distributions, following the precipitation of COM on the PHT A seed fibres, shown in figure 7, suggest the formation of significant numbers of small particles within a narrow size range. The excellent repro-

ducibility of the particle distribution measurements is also demonstrated in figure 7, where the initial PHTA distributions are found to be identical for Expts. 268, and 264, while the final samples both show an increase in the number of small particles.

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Fm. 6. PHT AS induced COM crystallization. Moles of COM precipitated plotted against time: D, Expt. 269; 0, Expt. 261; 0, Expt. 262; !:::., Expt. 263; V, Expt. 267.

Fm. 8. Scanning electron micrographs of PHTA seeded Expt. 266 (A. xl0,000; B. X7000).

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2

4 6 8 PARTICLE SIZE (microns)

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Fm. 9. Particle distribution analysis of PHT AS induced COM crystallization.% total counts plotted against size (µm.). Expt. 267; A., final; !:::., initial. Expt. 269: 11111, final; D, initial.

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Fm. 11. COM crystallization induced by grown phases. Moles COM precipitated plotted against time. 0, Expt. 276; 'v, Expt. 277; Q, Expt. 278.

Fm. 10. Scanning electron micrographs of PHT AS seeded. Expt. 263: A, initial sample (X5000); B, final grown phase (X5000).

In contrast to the PHT A experimental

particle size :::·::·p·c:;n.:~;::c·;,'-~ 267 and 269, using PHTAS seeds, size of the ne1,+,,..:,,_, increased during 0L,ctLili1lle( electron c,,•,,m•<>r>hQ lil figure of TA and

of the This may but it is difficult to separate the effects of and aggregation in the later stages of the ex1peJrrn1e1rts. A number of ex1pe1rm1e11ts 276-278) were made in which the grovm collected at the end of ex·pe1rm1e11ts shown in the were used as seed in subsequent COM crystallization constant com1::ioi,1ti1on studies. It can be seen in figure 11 that the induction periods were appreciably shorter than those obtained for the triamterene seed materials indicating that the COM present in the grown phases promoted crystallization. The crystal growth processes in these experiments, as distinct from those in which additional nucleation ~CCCVO'VHUUC..,,

took place on triamterene seed surfaces, resulted in little change in specific surface area. In conclusion, all three of the triarnterene materials tested to initiate the nucleation and of COM in solutions supersaturated with respect to this mineral phase. The results suggest that nucleation of C01VI on the surface of the TA seed materials is followed crystal growth of this phase. In contrast to the results for TA and PHT A in which considerable agg-regation was observed during the experiments, the PHT AS seed crystals appeared to induce the growth of COJVI crystals of quite different morphology. This difference in behaviour is not surprising in view of the strikingly different morphologies of triamterene seed materials. Many dyazide patients have urines which may be supersaturated with respect to TA and its metabolites possibly resulting in the spontaneous nucleation and precipitation of these phases. The crystallites may grow and into "triamterene" stones, or m.ay act as nucleation sites the grnwth of other stone-forming minerals. The results of the present work indicate that COM may readily nucleate on the surface of triamterene and its metabolites with PHT AS probably being the most effective in inducing the of this vVe thank Dr, Bruce Ettinger for nrn,1,mn Peter Muehlbauer surface rneasu:rerr1ents, and Steven Zavvacki for data measurements. REFERENCES

1. Ettinger, B., Weil, E., Mandel, N. S. and

2. 3. 4. 5. 6.

S,: Trian1.tereneinduced Ann. Intern. lvied., 745, 1979. Ettinger, B., N. and Sorge!, F.: Triamter-ene nephrolithiasis. In press. Hamed, H. S.: The electromotive forces of uni-univalent halides in concentrated aqueous solutions. J. Amer. Chem. Soc., 51: 416, 1929. Nancollas, G. H.: Interactions in Electrolyte Solutions. Amsterdam: Elsevier Publishing Co., 1966. Tomazic, B., and Nancollas, G. H.: The kinetics of dissolution of calcium oxalate hydrates. J. Crystal Growth, 46: 355, 1979. Davies, C. W.: Ion Association. London: Butterworths, 1962.