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11\THIBITION OF CALCIUM OXALATE DIHYDRATE CRYSTALLIZATION BY CHEMICAL MODIFIERS: I. PYROPHOSPHATE AND METHYLENE BLUE GEORGE W. DRACH, ALAN D. RANDOLPH

AND

JOHN D. MILLER

From the Departments of Surgery and Chemical Engineering, University ofArizona, Tucson, Arizona

ABSTRACT

Pyrophosphate and methylene blue are 2 agents observed to inhibit the calcium oxalate monohydrate crystal mass produced in urine or simulated urine. These agents do not produce this effect by inhibition of crystal growth. Information on nucleation rates and studies in the calcium oxalate dihydrate system have not been reported. Mixed suspension mixed product removal crystallizer techniques were used to isolate and analyze effects on calcium oxalate dihydrate crystallization of these 2 inhibitors on kinetics of total crystal mass (MT), linear crystal growth rate (G) and crystal nucleation rate (B 0). Within this system mass and population balances conform to the relationship MT equals 6pkvB 0 r 4 G3 , where p represents crystal density, kv is a volumetric shape factor and r indicates time of :retention of crystals within the crystallizer. Pyrophosphate exerted its inhibitory effect by decreasing the nucleation rate in ratios ranging from 3.4 to 4.2 when compared to uninhibited synthetic urine supersaturated with calcium oxalate. Methylene blue decreases the nucleation rate by ratios of 1.5 to 2.9. Additional inhibitory effects of methylene blue are probably caused by its involvement in the chemical equilibria of the system. Total crystal mass (MT) produced per unit time in the crystallizer was significantly inhibited by both substances. Since growth rate (G) for both inhibitors remained near or actually increased over control rate, inhibition of nucleation must account for most of the inhibition observed. Since nucleation of large quantities (more than 107 nuclei per minute per ml. solution) in crystal systems is known to be related to increased crystal aggregation, it is likely that one effect of inhibition of nucleation is decreased crystal aggregation. Aggregation, however, was not measured in these experiments. Throughout investigations on urinary calculous disease multiple inorganic or organic chemical inhibitors of urinary crystallization have been proposed. 1 A list of some inhibitors is presented in table 1, which also indicates many that are presently unacceptable for use in humans in the United States. Consequently, the number of inhibitors used for therapy is relatively small. Presently, oral phosphates, oral magnesium oxide, oral thiazides and methylene blue are the only agents regularly administered in attempts to inhibit calcium stone formation. 2--4 Allopurinol also has been proposed as an inhibitor of calcium urinary stone formation but its mechanism of action is believed to be owing to decrease in uric acid content of urine rather than direct effects on the calcium ion. 5, 6 Burdette and associates propose that the major mechanism of action of oral phosphate is the resultant increased excretion of pyrophosphate in urine. 7 Marked inhibitory effects of pyrophosphate on in vitro calcium oxalate crystal growth also have been reported by Fleisch, 8 and Sutor and Wooley. 9 The increase in urinary orthophosphate concentration, which also occurs, appears to have no relationship to inhibition of urinary stone formation. 10 Magnesium oxide acts to increase urinary magnesium content with concomitant decrease in urinary calcium content." 7 Th.is beneficially alters the so-called magnesium/calcium ratio and promotes formation of magnesiumoxalate complexes that are more soluble. 11 Hence, calciumoxalate precipitation is inhibited. Administration of oral thiazides results in. decreased urinary secretion of calcium with

decrease in urinary supersaturation with calcium. 3 • 12 inhibition of calcium crystal growth by methylene believed to be caused by surface changes. This reaction is concentration-dependent and is partly caused by alteration in the zeta potentials that exist between charged urinary ions. 13 Studies of these inhibitors have concentrated on the effects of the inhibitors on growth of crystals from a static supersaturated solution of simulated or actual urine. 6 ' s, 9 • 13 In addition, all previous studies have been performed in systems that measure calcium phosphate and/or calcium oxalate monohydrate precipitation. 14 ' 15 For example, Meyer and Smith recently reported upon a system for measurement of inhibitors of urinary crystal growth but they used only the calcium oxalate monohydrate system. 16' 17 They reported that crystal growth inhibitors such as magnesium, citrate and pyrophosphate contributed little to the ability of normal urine to inhibit the growth of calcium oxalate crystals. We were puzzled by this observation in view of the fact that we have observed inhibition of urinary calcium oxalate dihydrate crystal total mass formed per unit time 18 from solutions containing pyrophosphate. However, our system differs from others in 2 ways. It measures nucleation rate and growth rate of the crystalline phase in flowing solutions and produces calcium oxalate dihydrate (weddellite) crystals rather than calcium oxalate monohydrate (whewellite). This investigation concerns our observations on the effects of the inhibitors pyrophosphate or methylene blue upon nucleation or growth of weddellite crystals formed from synthetic urine that has been supersaturated with calcium oxalate.

Accepted for publication March 25, Hl77. Read at annual meeting of American Urological Association, Las Vegas, Nevada, May 16-20, 1976. Partial support for this study was obtained from NSF Grant ENG-75-04348.

METHODS

Observations were made in the mixed suspension, mixed product removal (MSMPR) crystallizer system that has been 99

100

DRACH, RANDOLPH AND MILLER TABLE

1. Some potential inhibitors of calcium urolithiasis

Sodium pyrophosphate Methylene blue DL-alanine* MgO Taurine* Tetracycline* Al'+* Heparin* Urea* Zn2 +* Orthophosphate Neutral sodium/potassium phosphate Ethane-1-hydroxy-1, 1-diphosphonate (EHDP)* Citrate* Organic sulfates* Matrix*

Figure 3 presents the nucleation rates observed in the 3 types of solution studied. We note that the major effect of pyrophosphate and of methylene blue is a decrease in the number of nuclei formed per milliliter of supersaturated

100 8060 40

- .- _.

--

,,,,,, __ ,;.,,,- __ _--·--

- - • Blank

Methylene Blue __ - - • Pyrophosphate

/

20

* Not acceptable at this time for therapy of human calcigerous urolithiasis.

described previously. 19• 2 ° Further improvement in this system has resulted in a reproducible method of observation of calcium oxalate dihydrate crystallization, which allows measurement of nucleation rate, growth rate and total crystal mass produced per unit time in a continuously moving solvent that contains 9 times 10-3 molar calcium chloride and 1.5 times 10-3 molar sodium oxalate suspended in a synthetic urine. 20 Observations were made with this continuous-flow crystallizer operating with mean retention times (crystallizer volume divided by flow rate) of approximately 5, 10, 15 and 20 minutes. The periods of time roughly correspond to the amount of time that urine would remain within the kidney and pass through the renal pelvis and ureter to the bladder. Pyrophosphate and methylene blue were selected for our initial investigations because they are known to have major inhibitory effects at small concentrations.'· 13 Addition of small amounts of these chemicals to the simulated system also should have small polyionic effects. For purposes of these studies sodium pyrophosphate was added to simulated urine 20 to produce a final concentration (in the mother liquor) of 0.11 mM. A preliminary experiment with a pyrophosphate level of 0.011 mM. had little effect on calcium oxalate crystallization. Methylene blue was added to provide a mother liquor final concentration of 0.094 mM. At this concentration of methylene blue we observed that it adsorbed on every surface it contacted-feed tank, crystallizer, feed tubing and crystals.

Total Crystal Mass Synthetic Urine

,,.,.'

MT

10 8 6

mg/L.

4

2

~--~---~--~-----, 5.30

14.80

9.95

18.85

Minutes Fm.1

Rate of Growth Synthetic Urine 1.0

.8 .6 .4

Pyrophosphaie

'..,, ... ...... ~ ' " .........-...:. ...... , Blank ,

"' ... k

G

......... ~ ...

....._:: -.;,::.

::~

Methylene Blue~

.2

RESULTS

Figure 1 reveals that total crystal mass of calcium oxalate crystals, mg./1., increases as a function of mean residence time in the crystallizer. Note that the presence of either methylene blue or pyrophosphate in the solution produced a significant decrease (t-test indicated significant differences from the blank at the 99 per cent confidence level for inhibition by pyrophosphate and at the 95 per cent confidence level for methylene blue) in the amount of crystal mass formed at any residence time in the crystallizer. Hence, it is clear that methylene blue and pyrophosphate have a direct and definite inhibition of total weddellite crystal mass produced in the simulated urine under the conditions given. Figure 2 reveals the kinetics of growth for crystals within our dihydrate system. It is apparent in our system also that there is no essential difference between the growth rate of calcium oxalate crystals growing in solutions that contain no inhibitor, pyrophosphate or methylene blue. Therefore, it appears that pyrophosphate has no influence on growth rates of either calcium oxalate monohydrate or dihydrate. Meyer and Smith have indicated that pyrophosphate, at least, had no observable or significant inhibition of calcium oxalate monohydrate crystal growth rate. 17 (Crystal growth rate in our MSMPR system is a constrained variable that depends on nucleation/growth kinetics and residence time. This constraint is discussed in more detail in a subsequent section.)

.1 5

10

20

15

Minutes Fm.2

Rate of Nucleation Synthetic Urine 10,000

90

..... ,Blank

8000 6000

........

4000

Methylene Blue

.......................... 81,

........

2000

........ I

"',..



... ....

Pyrophosphate ...., ______ ., _____ _.

1000 ~-------.----r----.------, 5.30 9.95 14.80 18.85

Minutes Fm.3

INHIBITION OF CALCIUM OXALATE DIHYDRATE CRYSTALLIZATION BY CHEMICAL MOilIFIERS

solution. The relatively constant methylene blue line confirms probable concentration dependence of the reaction on methylene blue. That is, as more total crystal mass is produced within the crystallizer, more methylene blue is consumed within the structure and on the surface of the crystals. Therefore, the relative content of methylene blue within the crystallizer solvent decreases with time. This is in keeping with the observation of Rollins and Finlayson, who showed that the effect of methylene blue on inhibition of urinary whewellite crystal formation was concentration-dependent. 13 NUCLEATION AND GROWTH RATE KINETICS

G = kg sa µm. per minute

(1)

For nucleation rate B0

= kn (s -

3o)i no./(mL minute)

=

MT

SUPERSATURATION

Because of the difficulty of measurement (and ambiguous definition) of the crystallization driving forces for stone formation a working definition for supersaturation was developed as follows. A solute mass balance (as the dihydrate, weddellite) around the continuous-flow vessel gives (3)

Subtracting C,, the solubility concentration, gives the working definition of supersaturation, thus (4)

The apparent feed supersaturation, S; equals C; minus Cs, . was obtained by equilibrating the combined feed liquor streams. The slurry concentration (MT) was obtained by weighing a dried filtered sample of product crystals. Some difficulty was encountered in the measurement of slurry density at the longer residence times owing to fouling on the walls of the vessel. This material was carefully and completely removed from the walls, weighed, divided by the total volume · throughout the run and added as a correction to the observed solids concentration measured by filtration. Such corrections were of comparable magnitude to the filtered value at longer retention times (r more than 20 minutes) but represented less than 10 per cent correction at retention times of 5 minutes. Growth (G) and nucleation (B 0) rate kinetics for the simulated urine runs without inhibitor and with pyrophosphate or methylene blue added are given as follows:

=

S; -

s

=

pkv Loo nL3 dL

=

6pkvB0 r 4 G3

Where p is crystal density, kv is the volumetric shape factor, L is particle linear dimension and n(L)dL is the number of crystals per ml. in size range L to L plus dL. The population density distribution n(L) for the MSMPR system takes on the form n(L) equals B0 /G exp(minus L/GT), where -r is the mean retention in the crystallizer. Bringing equations l and 2 to 5 gives S

where s is the calculated supersaturation, .s0 is the metastable supernaturation, and kg and kn are, respectively, growth and nucleation rate constants. The exponents a and i are system parameters that indicate the supersaturation sensitivity of and nucleation, respectively.

Blank runs:

These kinetics by themselves do not readily show the effect of inhibitors on crystallization parameters such as growth and nucleation rate because these parameters are a complex function of supersaturation. Supersaturation in an MSMPR crystallizer is itself a dependent variable. Thus, complete mass and population balances must be solved, with the given kinetics, to ascertain the effects of such kinetics on process variables. Fortunately, relationships that satisfy mass and population balances are relatively simple for the MSMPR crystallizer. 19 Thus, a mass balance becomes

C; - C

The observations of nucleation inhibitory effects of methylene blue and pyrophosphate were quantified further by regressing these data to growth and nucleation kinetics models. 19 For growth rate

101.

=

S; -

6pkvk0 r'kg3

S3a

(s - s0)1

or symbolically s equals F(s). Equation 6 must" be solved by trial-and-error to determine supersaturation and, hence, the levels of growth and nucleation in the system. Table 2 summarizes calculated values of growth rate, nucleation rate and supersaturation at 2 residence times for the 3 systems: blank, pyrophosphate or methylene blue inhibitors. A representative value of Ci equals 245 mg.fl. (as weddellite) was used for these calculations. This value of feed concentration was used in most of the experimental kinetic runs. 20 Table 3 shows the ratio, blank/inhibitor, of these 3 dependent quantities at 2 retention times. Clearly, the effect of pyrophosphate is to inhibit nucleation (3-to-4-fold, depending on absolute supersaturation level). As a necessary consequence of the mass balance constraint for an MSMPR crystallizer this inhibition leads to higher supersaturations (less crystal surface area to relieve the supersaturation) and, thus, correspondingly higher values of growth rate. This represents a classical example of true nucleation inhibition. 19 Methylene blue also results in lower but constant nucleation inhibition (table 2), lower solids concentration and slightly higher growth rate. However, a much lower supersaturation is calculated. Referring to the working definition of supersaturation

and the response of MT (fig. 1) it is apparent that the only way smaller supersaturations could be calculated with equal feed concentrations and lower solids concentrations would be if the solubility concentration Cs were much larger. This was, indeed, found to be the case (C, equals 157 mg.fl. for methylene blue versus 107 mg.IL for both blank and pyrophosphate). Thus, methylene blue enters into the chemical equilibria of the system and, as such, is not simply a nucleation inhibitor even though nucleation rate is, indeed, reduced to a constant level. DISCUSSION

B0 = exp(-4.5)s2· 9 G = exp(-12.6)s2 · 55

(r2 (r2

= 0.85) = 0.85)

Sodium pyrophosphate (0.11 mM.) B0 = exp(-7.8) (s -35.3) 3 · 5 G = exp(-37.1)s7 · 7

(r2 = 0.68) (r2 = 0.93)

Methylene blue (0.094 mM.) B0 = exp(8.l)s0 •0 G = exp(-13.5)s3 · 1

(r2

= 0.84)

Pyrophosphate and methylene blue have direct and measurable inhibitory effects on the nucleation of calcium oxalate dihydrate crystallization from a supersaturated solution of simulated urine. The effect of pyrophosphate is believed 'w occur solely by nucleation inhibition. Methylene blue results are partly owing to nucleation inhibition but are compounded by other changes in system equilibria. There were no significant effects of either substance on the linear growth rate of weddellite crystals. However, the end result of inhibition of nucleation was significant enough to decrease the total amount of crystal formed per unit time with both inhibited

102

DRACH, RANDOLPH AND MILLER

TABLE

2. Calculated values of supersaturation, growth and nucleation for weddellite using experimental nucleation/growth rate kinetics [C; = 245 mg.fl.] T

Nucleation (No./ml.min.) Growth (µm./min.) Supersaturation (mg./1.)

TABLE

=

7 Mins.

Pyrophosphate Methylene blue

=

15 Mins.

Blank

Pyrophosphate

Methylene Blue

Blank

Pyrophosphate

Methylene Blue

9,512 0.56 111

2,272 0.78 120

3,294 0.71 70

4,891 0.31 88.3

1,442 0.39 110

3,294 0.32 54

3. Ratio of calculated nucleation, growth and supersaturation [blank/inhibitor] T =

T

7Mins.

T =

15Mins.

Nucleation

Growth

Supersaturation

Nucleation

Growth

Supersaturation

4.2 2.9

0.72 0.79

0.93 1.6

3.4 1.5

0.79 0.97

0.80 1.6

effect of 2 inhibitor compounds, pyrophosphate and methylene blue, on calcium oxalate dihydrate crystallization in simulated urine was a reduction in crystal nucleation rate. Growth rates and supersaturation levels also were affected slightly because of mass balance constraints and solubility changes. A mechanism of stone formation owing to particle aggregation (which is a strong function of nucleation rate) is suggested by these results. The presence of matrix in urine also might have a significant role in the formation and stability of such aggregates.

systems even though growth rates were forced to a higher REFERENCES level owing to mass balance constraints. Since the composition of simulated urine used is similar to that of human urine 1. Finlayson, B.: Renal lithiasis in review. Urol. Clin. N. Amer., there is reason to believe that the effects of these inhibitory 1: 181, 1974. 2. Smith, M. J. V. and Boyce, W. H.: Allopurinol and urolithiasis. substances in urine are similar to that shown in this system. J. Urol., 102: 750, 1969. The failure of Meyer and Smith to show effects of pyrophos3. Thomas, W. C., Jr.: Clinical concepts ofrenal calculous disease. phate on urinary calcium oxalate crystal growth (although in J. Urol., 113: 423, 1975. the whewellite system) seems to support our present observa4. Drach, G. W.: Urolithiasis. In: Current Therapy. Edited by H. tions.17 Aggregation of crystals is strongly related to nucleaF. Conn, Philadelphia, W. B. Saunders Co., p. 552, 1976. tion rate and may, therefore, also be inhibited by pyrophos5. Coe, F. L. and Kavalach, A.G.: Hypercalciuria and hyperuricophate or methylene blue. 21 suria in patients with calcium nephrolithiasis. New Engl. J. Almost all past investigations of urinary crystallization of Med.,291: 1344, 1974. calcium oxalate have depended upon non-nucleating systems 6. Meyer, J. L., Bergert, J. H. and Smith, L. H.: The epitaxially in which seed crystals were added to supersaturated solutions. induced crystal growth of calcium oxalate by crystalline uric acid. Invest. Urol., 14: 115, 1976. Therefore, only growth rather than growth and nucleation 7. Burdette, D. C., Thomas, W. C., Jr. and Finlayson, B.: Urinary were measured. The MSMPR crystallization system provides supersaturation with calcium oxalate before and during orthothe only presently known method for simultaneous investigaphosphate therapy. J. Urol., 115: 418, 1976. tion of nucleation and growth kinetics in systems applied to H.: Some new concepts on the pathogenesis and the the study of urinary stone disease. Some observations of 8. Fleisch, treatment ofurolithiasis. Urol. Int., 19: 372, 1965. inhibition of stone growth may have been thwarted previously 9. Sutor, D. J. and Wooley, S. E.: Growth studies of calcium because of inability to effectively measure nucleation rates. oxalate in the presence of various compounds and ions-part There are certain limitations that must be placed upon II. Brit. J. Urol., 42: 296, 1970. these observations. These include the fact that several authors 10. Thomas, W. C., Jr.: Effectiveness and mode of action of orthophosphates in patients with calcareous renal calculi. Trans. have reported that the most common nucleus of calcium Amer. Clin. Climatol. Ass., 83: 113, 1971. oxalate stone is whewellite, whereas the most common layering growth crystal is weddellite. 22· 23 Hence, inhibitors of 11. Oreopoulos, D. G., Walker, D., Akroitis, D. J., Roncari, D. A. K., Husdan, H., Symvoulidis, A., Deveber, G. A., Rapoport, weddellite may not necessarily alter whewellite nucleation A. and Reid, D. B. W.: Excretion of inhibitors of calcification processes in the formation of human urinary stone. In addiin urine. Part I. Findings in control subjects and patients tion, the role of matrix in nucleation of human urinary stone with renal stones. Canad. Med. Assoc. J., 112: 827, 1975. disease remains unclear. Recent reports have indicated the 12. Yendt, E. R. and Cohanim, M.: Ten years' experience with the fact that the matrix of stone formers may indeed be qualitause of thiazides in the prevention of kidney stones. Trans. tively different and may provide binding sites for the initial Amer. Clin. Climatol. Assc., 85: 65, 1973. deposition of urinary calcium on a proteinaceous structure. 24• 25 13. Rollins, R. and Finlayson, B.: Mechanism of prevention of calcium oxalate encrustation by methylene blue and demonSince our system included only simulated urine and contained strations of the concentration dependence of its action. J. no matrix, it is possible that the presence of matrix would Urol., 110: 459, 1973. alter greatly the kinetics of a system such as ours. 14. Gill, W. B., Silvert, M. A. and Roma, M. J.: Supersaturation We are presently working upon methods for the analysis of levels and crystallization rates of calcium oxalate from urines human urine by the MSMPR crystallization system. The of normal humans and stone formers determined by 14C26 methodology is similar to that used by Rose, in which a oxalate technique. Invest. Urol., 12: 203, 1974. small proportion of urine is added to a large proportion of 15. Pak, C. Y. and Holt, K.: Nucleation and growth of brushite simulated urine and the effect of this addition is quantitated. and calcium oxalate in urine of stone-formers. Metabolism, 25: 665, 1976. Preliminary results with this type of system indicate that nucleation and, to a lesser extent, growth rates can be 16. Meyer, J. L. and Smith, L. H.: Growth of calcium oxalate crystals. I. A model for urinary stone growth. Invest. Urol., markedly altered by the addition of small amounts of urine to 13: 31, 1975. large amounts of simulated urine. In view of the fact that 17. Meyer, J. L. and Smith, L. H.: Growth of calcium oxalate several orally administered stone inhibitors produce their crystals. II. Inhibition by natural urinary crystal growth effects by subsequent alteration in the composition of urine, inhibitors. Invest. Urol., 13: 36, 1975. this method is to be used in the future to assess inhibitory 18. Miller, J. D.: Crystallization kinetics of calcium oxalate in effects of orally ingested modifiers of crystallization. simulated urine. Thesis. University of Arizona Graduate College, Tucson, 1976. Therefore, this study revealed that the major significant

INHIBITION OF CALCIUM OXALATE DIHYDRATE CRYSTALLIZATION BY CHEMICAL MODIFIERS

19. Randolph, A. D. and Larson, M. A.: Theory of Particulate Processes: Analysis and Techniques of Continuous Crystallization. New York: Academic Press, 1971. 20. Miller, J. D., Randolph, A. D. and Drach, G. W.: Observations calcium oxalate crystallization kinetics in simulated J. Urol., 117: 342, 1977. 21. Walton, A. G.: The Formation and Properties of Precipitates. New York: Interscience Publishers, 1967. 22. J. S.: Structure and composition of urinary calculi. J. Ul9: 82, 1973. 23. Bastian, H. P. and Gebhardt, M.: The varying composition of

103

the nucleus and peripheral layers of urinary calculi. UroL Res., 2: 91, 1974. 24. Foye, W. 0., Hong, H. S., Kim, C. M. and Prien, E. L, Sr.: Degree of sulfation in mucopolysaccharide sulfates in normal and stone-forming urines. Invest. Urol., 14: 33, 1976. 25. Spector, A. R., Gray, A. and Prien, E. L., Jr.: Kidney stone matrix. Differences in acidic protein composition. Invest. Urol., 13: 387, 1976. 26. Rose, M. B.: Renal stone formation. The inhibitory effect of urine on calcium oxalate precipitation. Invest. Urol., 12: 428, 1975.

Inhibition of calcium oxalate dihydrate crystallization by chemical modifiers: I. Pyrophosphate and methylene blue.

0022-5347/78/11!!1-0099$02.00/() Vol. 119, Printed in THI! JOURNAL OF UROLOGY Copyl'ight © l.978 by The Williams & Wilkins Co. 11\THIBITION OF CALC...
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