Clinica Chimica Acta, 200 (1991) 107-118
107
0 1991 Elsevier SciencePublishersB.V.All rights reserved 0009-8981/91/$03.50 ADONIS 000989819100159B
CCA 05024
Pyrophosphate inhibition of Proteus mirabilis-induced struvite crystallization in vitro Robert
J.C. McLean ‘v*,Joe Downey ‘, Lynann Clapham James W.L. Wilson ’ and J. Curtis Nickel ’
3,
I Department of Urology,Queen’sUniversity, Kingston, Ontario, 2 Department of Microbiology and Immunology, Queen’s University, Kingston, Ontario and 3 Department of Physics, Queen’s University, Kingston, Ontario (Canada)
(Received27 December1990;accepted4 March1991) Key words: Urolithiasis; Urine; Proteus mirabilis; Crystal inhibitor; Struvite; Pyrophosphate
Summary Struvite (MgNH,PO, - 6H,O) crystals, the major mineral component of infectious urinary calculi, were produced in vitro by growth of a clinical isolate of Proteus mirabilis in artificial urine. P. mirabilis growth and urease-induced struvite production were monitored by phase contrast light microscopy and measurements of urease activity, pH, ammonia concentrations, turbidity, and culture viability. In the absence of pyrophosphate, struvite crystals appeared within 3-5 h due to the urease-induced elevation of pH and initially assumed a planar or ‘X-shaped’ crystal habit (morphology) characteristic of rapid growth. When pyrophosphate was present, initial precipitation and crystal appearance were significantly impaired and precipitates were largely amorphous. When crystals did appear (usually after 7 or 8 h) they were misshapen or octahedral in shape indicative of very slow growth. X-ray diffraction and Fourier transform infrared spectroscopy (FTIR) of carbonate-apatite identified all crystals as struvite. Trace contaminates (Ca,,(PO,),CO,) or newberyite (MgHPO, * H,O) were produced only in the absence of pyrophosphate. P. mirabilis viability and culture pH elevation were unaffected by the addition of pyrophosphate, whereas urease activity and ammonia concentrations were marginally reduced. Struvite could also be produced chemically by titration of the artificial urine with NH,OH. If pyrophosphate was present during titration, the same inhibitory effect on crystal growth occurred, so it is unlikely that urease inhibition is important. Lowering of pyrophosphate concentration from 13-0.45 pmol/l did not reduce its inhibitory activity so it is unlikely to
Correspondence to: Dr. R.J.C. McLean, Department University, Kingston, Ontario, Canada K7L 3N6.
of Microbiology and Immunology, Queen’s
108
act by chelating free Mg*+. We propose that pyrophosphate inhibits struvite growth principally through direct interference with the chemical mechanisms involved in crystal nucleation and growth, because of its effectiveness at very low concentrations. Introduction
Urinary tract infections (UTI) by urease producing species of bacteria such as Proteus mirubilis often give rise to urinary calculi. These struvite (NH,MgPO, * 6 H,O) and carbonate-apatite (Ca,,,(PO,),CO,) calculi account for only 10% of all urinary stones but often represent a more significant health problem than do conventional metabolic stones [1,2] due to their rapid growth and high rate of recurrence. The role of urease in the pathogenesis of P. mirubilis UT1 and especially struvite urolithiasis has been well-documented [2-41. Bacterial urease activity generates ammonia which in turn increases urine pH, decreases the solubility of Mg*+ and Ca*+, and results in their precipitation as struvite and carbonate-apatite. In addition high ammonia levels from bacterial urease activity can also damage urinary tissues [4]. Although urine supersaturation is an essential prerequisite for urolithiasis, normal urine contains one or more crystallization inhibitors [5,6] which can prevent mineralization. Investigators of metabolic urinary calculi including calcium oxalate urolithiasis have identified several major classes of inhibitory compounds. Included among these inhibitors is pyrophosphate [7-131. This compound is thought to act by either chelating divalent cations such as Ca*+, making them unavailable for precipitation, by interfering with the crystallization process through direct incorporation into and disruption of the crystal lattice, or by coating the crystal surface thereby blocking deposition of additional mineral. In’contrast to the work on metabolic urinary calculi, there has been relatively little work on the role of urine crystallization inhibitors in struvite urolithiasis. Studies to date have suggested that zinc, citrate [14,151 and albumen (H. Hedelin, personal communication) may represent potential inhibitors. As part of our ongoing research into struvite urolithiasis, we have adapted the use of phase contrast light microscopy to investigate the role of various naturally occurring urinary compounds on struvite crystal growth in vitro [16]. This simple approach has allowed us to rapidly screen several compounds for their role as potential crystallization inhibitors, since struvite and other crystals assume a characteristic shape or crystal habit which is influenced directly by growth rate [17,18]. In this paper we report on the ability of pyrophosphate to inhibit P. mirubili.s-induced struvite crystallization in vitro. Materials
and methods
Proteus mirabilis growth and crystal production:
P. mirabilis strain 2573 was
originally isolated from a patient with struvite urolithiasis
[15,16,19] and main-
109
tained on tryptic soy agar (Difco Laboratories, Detroit, Mich. USA). It has since been deposited with the American Type Culture Collection as strain ATCC 49565. The experimental protocols used for in vitro production of struvite crystals by this organism were described in earlier publications [15,16]. Briefly, this involved growing P. mirabilis in a continuous culture flask containing 800 ml artificial urine for a period of 24 h. Sterile artificial urine was continuously added to the flask at a rate of 60 ml h-i. The volume of the flask and the rate of addition of urine were chosen so as to approximate the average bladder volume and urine production rate of human males 1201.Samples were removed 0, 0.5, 1, 2, 3, 4, 5, 6, 7, and 24 h after inoculation, examined by phase contrast light microscopy, and monitored for pH, turbidity (OD,a,,), bacterial cell counts, urease activity, and ammonia concentration. A sample (designated as -0.5 h) was also taken from the inoculum for analysis. These biochemical and microbiological analyses were performed so as to determine whether alterations of crystal growth rate could be attributed to a buffering of urine pH, antimicrobial activity, interference with ammonia concentrations, or inhibition of the principal enzyme (urease) responsible for the formation of these struvite calculi. The artificial urine used was our modification [20] of that described by Griffith et al. [3]. For inhibition experiments, the artificial urine was supplemented with sodium pyrophosphate (purchased from the Sigma Chemical Co., St. Louis, MO) at either 6 mg l- ’ or 60 mg 1-i (13.4-134 pmol/l). During control experiments, no pyrophosphate was present. These pyrophosphate concentrations were chosen to reflect normal and excessive concentrations of this compound as found in human urine [13]. A minimum of five replications were conducted under each experimental condition. The influence of pyrophosphate on struvite formation was also measured in the absence of P. mirabilis. This involved titrating 800 ml artificial urine (with or without pyrophosphate) in our in vitro apparatus [16,20] with 0.25 mol/l NH,OH. NH,OH and fresh artificial urine were both added at a rate of 60 ml h-i. This concentration of NH,OH and flow rate gave approximately the same pH and optical density increase kinetics as did inoculation with P. rnirubilis. Samples were removed at 0, 0.5, 1, 2, 3, 4, 5, 6, 7, and 24 h, examined by phase contrast light microscopy, and analyzed for pH, turbidity, and ammonia concentrations. P. mirabilis viability was measured by standard microbiological plate count methods and expressed as colony forming units per ml (CFU ml-‘) [16]. Urease activity (expressed as pg NH, produced ml-’ min-‘1 and culture ammonia concentrations (NH,, NH:, and total ammonia) were analyzed using our modification [15] of the phenol hypochlorite assay of Weatherburn [21]. Direct examination of the samples by phase contrast light microscopy [16] identified the presence and crystal habit(s) of any precipitates present and enabled us to assess the physiological status of P. mirubilis based on cellular motility [16]. Crystal analysis and identification of struvite: All crystals produced during these experiments were identified using X-ray powder diffraction (XRD) and Fouriertransform infrared spectroscopy (FTIR). Crystals were prepared for analysis by
110
harvesting by low speed centrifugation (200 - 300 X g), washing twice in 0.05 mol/l Tris(hydroxymethy1) amino methane (Tris) buffer, pH 8.6, and drying under partial vacuum in a desiccator. For XRD these crystals were ground, and analyzed using a Rigaku ‘miniflex’ X-ray diffractometer as described [15]. FTIR analysis of desiccated crystals mixed with KBr (final concentration 0.5-1.0% (w/w>) was conducted on a Bomem model MB 120 Fourier transform infrared spectrophotometer (Bomem Canada Inc., Quebec, QC, Canada) interfaced with a personal computer. For each specimen, 10 spectra were obtained from 4,000 cm-’ to 400 cm -I. The effective resolution was 4 cm- ‘. In addition, an FTIR spectra was also obtained from the sodium pyrophosphate used in the crystal inhibition experiments. All results obtained were compared to published spectra [22]. Results
P. mirubih 2573 inoculation into artificial urine in the absence of pyrophosphate caused a rapid elevation of urine pH and ammonia levels due to culture urease activity (Fig. 1). Crystals appeared within 4-5 h which typically possessed ‘X-shaped’ or flat planar crystal habits (Fig. 2) characteristic of rapid growth [16,17,19]. Initial growth of P. mirubifis was illustrated by an increase in optical density and in colony counts (Fig. 1). After a period of time (usually 7-24 h), culture optical density diminished (Fig. 1) in an analogous manner to earlier work [15,16]. A contributing factor to this is a reduction in culture viability (due to the high pH and ammonia levels [23]) in that colony counts in the absence of pyrophosphate also were reduced (Fig. 1). When pyrophosphate was present, P. mirabilis growth characteristics were considerably altered. The increase in pH of the artificial urine culture was seemingly unaffected by the addition of pyrophosphate (Fig. 1). However while urease activity and increases in ammonia levels were always evident, these values fluctuated widely and were slightly but not significantly (P > 0.05) lower than control values. This could not be attributed to an interference of pyrophosphate with the phenol-hypochlorite assay [21] (data not shown). P. mirabih grown in the presence of pyrophosphate demonstrated some resistance to the high ammonia levels and alkaline pH. Although at various times through the experiment, bacterial physiological stress became evident by a loss of motility [16] (usually 4-6 h after inoculation), these organisms quickly recovered and became actively motile within 1-2 h. This phenomenon occurred periodically and reproducibly throughout all experimental runs. In contrast P. mirabih motility in the absence of pyrophosphate became irreversibly lost after 4 h. Bacterial viability in the presence of pyrophosphate as expressed by CFU ml-’ was marginally enhanced over the 24-h course of the experiment (Fig. 1). Any precipitation which appeared, consisted largely of amorphous deposits. The first amorphous deposits to be seen often disappeared or became reduced in quantity. Crystal appearance was considerably delayed (up to 24 h after inoculation) and generally involved misshapen or deformed octahedral crystals (Fig. 3).
Control 6.0
Control
o-o
l --- l Pyrophosphots
? >
A-A
4.0
Total
3 0 ;
2.0
I
-1
4
9
14
19
24 Pyrophosphate 6.0, O-O ? >
4.0
3
NH3
l ---0
NH4
A-A
Total
.P i
2.0
I
-1
-1
4
4
9
:.. 9
14
24
o-o
Confrol
. --~0
Pyrophosphats
;.,.:.. 14 Time (h)
19
19
: 24
-1
4
9
o-o
Control
. ---0
pyrophosphde
14
19
22
Time (h)
Fig. 1. Turbidity (measured at 600 nm), pH, urease activity (pg NH, produced ml-’ mm-‘), ammonia concentrations (NH,, NH:, and total ammonia), and growth (log CFU ml-‘) due to Proteus mirabilis growth in artificial urine in the presence (6 mg I-‘) and absence of pyrophosphate. Error bars represent standard error in all cases except urease activity where they represent the 95% probability interval.
In order to assess whether the slight reductions in urease activity and ammonia concentrations in the presence of pyrophosphate were responsible for the altered crystal growth, struvite was produced in the absence of urease by titrating artificial urine with 0.25 mol/l NH,OH. As seen in Fig. 4, the chemically produced struvite crystals again exhibited similar misshapen crystal habits in the presence of pyrophosphate as did those produced by P. mirabilis. All crystals produced in the presence and absence of pyrophosphate were identified by XRD as struvite (Fig. 5). No carbonate-apatite nor any other mineral
Fig. 2. Struvite crystals grown in the absence of inhibitor typically assume an ‘X-shaped’ (Fig. 2A) or flat planar (Fig. 2B) crystal habit. Bar represents 25 km.
form was detected by this technique. Slight differences in the mineral types were seen using FTIR (Fig. 6). This may be attributable to a slight increase in the contamination of struvite produced in the absence of pyrophosphate by trace amounts of carbonate-apatite or newberyite (MgHPO, * H ,O). Significant ( > 10%) disruption or contamination of the basic struvite crystal structure did not occur as this would have been indicated by XRD. Similar results were seen at all pyrophosphate concentrations tested (0.45-134 pmol/l). The results shown represent values obtained at a pyrophosphate concentration of 6 mg 1-l (13.4 pmol/l) which represents a normal concentration of this inorganic anion in human urine [13]. Discussion
Identification of effective inhibitors of struvite formation is of paramount importance given the morbidity and mortality associated with the recurrence of these calculi following standard surgical therapy [1,24]. We recently adapted this direct observation technique which employs phase contrast light microscopy so as to rapidly screen and identify compound(s) in urine which might represent potentially effective inhibitors of struvite crystal growth 1161. During these studies, we have investigated the glycosaminoglycans, chondroitin sulphate and heparin sulphate; elevated citrate concentrations [15], and pyrophosphate (this study). Of all
Fig. 3. Struvite appearance in the presence of pyrophosphate is usually delayed by several hours. Typically amorphous deposits (arrows) and octahedral, slow-growing (171 crystals (Fig. 3A) are seen. Crystals often exhibit a misshapen (Fig. 3B) or deformed (Fig. 3C) crystal habit which is evidence of altered growth [29]. Bar represents 100 pm (3A and 3B), 2.5pm (3C).
Fig. 4. Alteration of crystal habit due to pyrophosphate was also evident in chemically-produced struvite (titration of artificial urine with NH,OH). In the absence of pyrophosphate (Fig. 4A), large numbers of crystals were seen which were often ‘X-shaped’ or tabular in appearance. Crystals produced in the presence of pyrophosphate were generally misshapen (Fig. 4B). Bar represents 20 pm.
compounds tested to date, we have found pyrophosphate to represent the potentially most effective crystallization inhibitor. This inhibitory action of pyrophosphate is not unique to struvite formation, in that it has been shown to be effective against urinary calcium oxalate formation [7-131 and even against dental calculus (calcium phosphate) formation [25]. Precipitation and crystallization of a compound from solution is a complex multi step process [18]. Initially the solubility of one or more potential crystals must be exceeded. Once supersaturation occurs, then precipitation can occur. In cases
/ 50
100
I
15.
1
I
t
20’
ZY
30.
35’
28 Fig. 5. X-ray diffraction of misshapen crystals in Fig. 3 identifies them as struvite. Numbers indicate crystallographic axes of struvite. DIFFRACTION
ANGLE
11.5 44
4000
3300
2600
I900
1200
500
CM-I
4000
3300
1900
2600
1200
500
CM-I
Fig. 6. Infrared spectroscopy analysis of struvite crystals produced in the absence (6A) and presence (6B) of pyrophosphate. The peak at 1753 cm-’ (arrow) (Fig. 6A) is possibly due to the contamination of struvite by carbonate-apatite or newberyite [22] in the absence of pyrophosphate.
where solubility is greatly exceeded, an amorphous precipitate is often formed due to its unordered nature. The eventual formation of an ordered structure (i.e. a crystal) from an amorphous precipitate or solution requires nucleation (i.e. the deposition of the first few molecules in an ordered arrangement) and growth through an ordered deposition and binding to the crystal surfaces. This nucleation and growth is influenced by the overall chemistry of the solution [26] and is often enhanced by the presence of biological surfaces such as bacterial cell walls and capsules (referenced in [26-281). In the case of struvite, intermolecular bonding during crystal formation involves ionic interactions between Mg*+, NH: and PO:-, and hydrogen bonding involving the water of crystallization, NH,f and/or
116
PO:- [17]. In addition, struvite formation requires the participation of urease producing bacteria (in this case P. mirubilis) and elevation of pH, NH,, and NH:. As such, there are many instances whereby crystal formation could be disrupted [291. Pyrophosphate inhibition of struvite formation could not be attributed to inhibition of P. mirabih growth as viability expressed as CFU ml-’ was marginally enhanced in the presence of pyrophosphate (Fig. 1). The initial lower concentration of cells in the presence of pyrophosphate was likely due to their reduced concentration in the initial inoculum (represented by the data point at time -0.5 h in Fig. 1). Pyrophosphate slightly inhibited P. mirubilis urease activity and NH, and NH: concentrations (Fig. 1) but not by a statistically significant margin (P > 0.05). Pyrophosphate exerted the same inhibitory effect against chemically produced struvite crystals so it is unlikely that urease inhibition was responsible. Inhibition of struvite could not be attributed to a buffering effect of pyrophosphate against pH as these values were virtually unaffected (Fig. 1). If pyrophosphate acted so as to complex Mg 2+ thereby rendering it unavailable for struvite formation, then diminishing its concentration should result in an elimination of inhibition as more Mg*+ becomes available. Lowering of the pyrophosphate concentration from 6 mg l- ’ to 0.2 mg 1-l (13.4 pmol/l to 0.45 pmol/l) did not alter its inhibitory activity (data not shown) so it is unlikely that Mg2+ complexation is important. During these experiments, struvite crystals in the initial inoculum completely dissolve within 3-5 min of being introduced into the reaction flask [163. Consequently any crystals which arise during the course of this procedure are formed de novo without the benefit of ‘seed’ crystals. As a result the effect of a potential inhibitor such as pyrophosphate can be observed on the nucleation as well as the growth of struvite crystals. Based on our observations, we speculate that the inhibitory action of pyrophosphate against struvite formation arises mainly from its ability to disrupt the ionic and hydrogen bonding necessary for crystal nucleation and development. This is evidenced by the delayed appearance of crystals and its alteration of struvite crystal habit (Figs. 3 and 4). Acknowledgements This project was supported by a grant from the Kidney Foundation of Canada to RJCM, JCN, and JWLW. R.J.C. McLean is supported by a Career Scientist Fellowship from the Ontario Ministry of Health and an operating grant from NSERC. We express our appreciation to Dr. Roy Pottier, Department of Chemistry and Chemical Engineering, Royal Military College of Canada, Kingston, Ontario and Pat Mulligan, Department of Chemistry, Queen’s University for the use of their infrared spectroscopes, and to Ann Lablans, Janet Clark and Anita Dumanski for their enthusiasm and excellent technical assistance. References 1 Lerner SP, Gleeson MJ, Griffith DP. Infection stones. J Ural 1989;141:753-758. 2 McLean RJC, Nickel JC, Cheng K-J, Costerton JW. The ecology and pathogenicity ducing bacteria in the urinary tract. Crit Rev Microbial 1988;16:37-79.
of urease-pro-
117 3 Griffith DP, Musher DM, Itin C. Urease the primary cause of infection-induced urinary stones. Invest Urol 1976;13:346-350. 4 Jones BD, Lockatell CV, Johnson DE, Warren JW, Mobley HLT. Construction of a urease-negative mutant of Proteus mirabilis: analysis of virulence in a mouse model of ascending urinary tract infection. Infect Immunol 1990;58:1120-1123. 5 Hedelin H, Grenabo L, Pettersson S. The effects of urease in undiluted human urine. J Urol 1986;136:743-745. 6 Ryall RL, Hibberd CM, Mazzachi BC, Marshall VR. Inhibitory activity of whole urine: a comparison of urines from stone formers and healthy subjects. Clin Chim Acta 1986;154:59-68. 7 Baumann JM, Ackermann D, Affolter B. The influence of hydroxyapatite and pyrophosphate of the formation product of calcium oxalate at different pHs. Urol Res 1989;17:153-155. 8 Conte A, Rota P, Genestar C, Grases F. The relation between orthophosphate and pyrophosphate in normal subjects and in patients with urolithiasis. Urol Res 1989;17:173-175. 9 Grases F, Genestar C, Conte A, March P, Costa-Bauza A. Inhibitory effect of pyrophosphate, citrate, magnesium and chondroitin sulphate in calcium oxalate urolithiasis. Br J Urol 1989;64:235237. 10 Grases F, Gil JJ, Conte A. Urolithiasis inhibitors and calculus nucleation. Urol Res 1989;17:163-166. 11 Robertson WG, Peacock M, Nordin BEC. Inhibitors of the growth and aggregation of calcium oxalate crystals in vitro. Clin Chim Acta 1973;43:31-37. 12 Ryall RL, Harnett RM, Marshall VR. The effect of urine, pyrophosphate, citrate, magnesium and glycosaminoglycans on the growth and aggregation of calcium oxalate crystals in vitro. Clin Chim Acta 1981;112:349-356. 13 Wilson JWL, Werness PG, Smith LH. Inhibitors of crystal growth of hydroxyapatite: a constant composition approach. J Urol 1985;134:1255-1258. 14 Hedelin H, Grenabo L, Hugosson J, Pettersson S. The influence of zinc and citrate on urease-induced urine crystallisation. Urol Res 1989;17:177-180. 15 McLean RJC, Downey J, Clapham L, Nickel JC. Influence of chondroitin sulfate, heparin sulfate, and citrate on Proteus mirabilis-induced struvite crystallization in vitro. J Ural 1990;144:1267-1271. 16 McLean RJC, Downey J, Clapham L, Nickel JC. A simple technique for studying struvite crystal growth in vitro. Urol Res 1990;18:39-43. 17 Abbona F, Boistelle R. Growth morphology and crystal habit of struvite crystals (MgNH,PO,. 6HzO). J Cryst Growth 1979;46:339-354. 18 Kern R. Crystal growth and adsorption. In: Sheftal NN., ed. Growth of crystals, Vol. 8. New York: Consultants Bureau, 1969;3-23. 19 Clapham IL, McLean RJC, Nickel JC, Downey J, Costerton JW. The influence of bacteria on struvite crystal habit and its importance in urinary stone formation. J Cryst Growth 1990;104:475-484. 20 McLean RJC, Nickel JC, Noakes VC, Costerton JW. An in vitro study of infectious kidney stone genesis. Infect Immun 1985;49:805-811. 21 Weatherburn MW. Phenol-hypochlorite reaction for determination of ammonia. Anal Chem 1967;39:971-974. 22 Hesse A, Sanders G. Atlas of infrared spectra for the analysis of urinary concretements. Stuttgart, FRG: Georg Thieme Verlag, 1988: 23 Visek WJ. Ammonia: its effects on biological system, metabolic hormones and reproduction. J Dairy Sci 1984;67:481-498. 24 Boyce WH. Surgery of urinary calculi in perspective. Urol Clin N Am 1983;10:585-594. 25 Mallatt ME, Beiswanger BB, Stookey GK, Swancar JR, Hennon DK. Influence of soluble pyrophosphate on calculus formation. J Dent Res 1985;64:1159-1162. 26 Mann S. Mineralization in biological systems. Struct Bond 1983;54:125-174. 27 Mclean RJC, Beveridge TJ. Metal binding capacity of bacterial surfaces and their ability to form mineralized aggregates. In: Ehrlich HL, Brierley CL., eds. Microbial mineral recovery. New York: McGraw Hill, 1990;185-222. 28 Beveridge TJ. Role of cellular design in bacterial metal accumulation and mineralization. Annu Rev Microbial 1989;43:147-171. 29 Sangwal K. Growth and dissolution of crystals. In: Sangwal K, ed. Etching of crystals theory, experiment, and application. Amsterdam: North-Holland Publishing Company, 1987;43-86.
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0 1991 Elsevier SciencePublishersB.V.All rights reserved 0009-8981/91/$03.50 ADONIS 000989819100159B
CCA 05024
Pyrophosphate inhibition of Proteus mirabilis-induced struvite crystallization in vitro Robert
J.C. McLean ‘v*,Joe Downey ‘, Lynann Clapham James W.L. Wilson ’ and J. Curtis Nickel ’
3,
I Department of Urology,Queen’sUniversity, Kingston, Ontario, 2 Department of Microbiology and Immunology, Queen’s University, Kingston, Ontario and 3 Department of Physics, Queen’s University, Kingston, Ontario (Canada)
(Received27 December1990;accepted4 March1991) Key words: Urolithiasis; Urine; Proteus mirabilis; Crystal inhibitor; Struvite; Pyrophosphate
Summary Struvite (MgNH,PO, - 6H,O) crystals, the major mineral component of infectious urinary calculi, were produced in vitro by growth of a clinical isolate of Proteus mirabilis in artificial urine. P. mirabilis growth and urease-induced struvite production were monitored by phase contrast light microscopy and measurements of urease activity, pH, ammonia concentrations, turbidity, and culture viability. In the absence of pyrophosphate, struvite crystals appeared within 3-5 h due to the urease-induced elevation of pH and initially assumed a planar or ‘X-shaped’ crystal habit (morphology) characteristic of rapid growth. When pyrophosphate was present, initial precipitation and crystal appearance were significantly impaired and precipitates were largely amorphous. When crystals did appear (usually after 7 or 8 h) they were misshapen or octahedral in shape indicative of very slow growth. X-ray diffraction and Fourier transform infrared spectroscopy (FTIR) of carbonate-apatite identified all crystals as struvite. Trace contaminates (Ca,,(PO,),CO,) or newberyite (MgHPO, * H,O) were produced only in the absence of pyrophosphate. P. mirabilis viability and culture pH elevation were unaffected by the addition of pyrophosphate, whereas urease activity and ammonia concentrations were marginally reduced. Struvite could also be produced chemically by titration of the artificial urine with NH,OH. If pyrophosphate was present during titration, the same inhibitory effect on crystal growth occurred, so it is unlikely that urease inhibition is important. Lowering of pyrophosphate concentration from 13-0.45 pmol/l did not reduce its inhibitory activity so it is unlikely to
Correspondence to: Dr. R.J.C. McLean, Department University, Kingston, Ontario, Canada K7L 3N6.
of Microbiology and Immunology, Queen’s
108
act by chelating free Mg*+. We propose that pyrophosphate inhibits struvite growth principally through direct interference with the chemical mechanisms involved in crystal nucleation and growth, because of its effectiveness at very low concentrations. Introduction
Urinary tract infections (UTI) by urease producing species of bacteria such as Proteus mirubilis often give rise to urinary calculi. These struvite (NH,MgPO, * 6 H,O) and carbonate-apatite (Ca,,,(PO,),CO,) calculi account for only 10% of all urinary stones but often represent a more significant health problem than do conventional metabolic stones [1,2] due to their rapid growth and high rate of recurrence. The role of urease in the pathogenesis of P. mirubilis UT1 and especially struvite urolithiasis has been well-documented [2-41. Bacterial urease activity generates ammonia which in turn increases urine pH, decreases the solubility of Mg*+ and Ca*+, and results in their precipitation as struvite and carbonate-apatite. In addition high ammonia levels from bacterial urease activity can also damage urinary tissues [4]. Although urine supersaturation is an essential prerequisite for urolithiasis, normal urine contains one or more crystallization inhibitors [5,6] which can prevent mineralization. Investigators of metabolic urinary calculi including calcium oxalate urolithiasis have identified several major classes of inhibitory compounds. Included among these inhibitors is pyrophosphate [7-131. This compound is thought to act by either chelating divalent cations such as Ca*+, making them unavailable for precipitation, by interfering with the crystallization process through direct incorporation into and disruption of the crystal lattice, or by coating the crystal surface thereby blocking deposition of additional mineral. In’contrast to the work on metabolic urinary calculi, there has been relatively little work on the role of urine crystallization inhibitors in struvite urolithiasis. Studies to date have suggested that zinc, citrate [14,151 and albumen (H. Hedelin, personal communication) may represent potential inhibitors. As part of our ongoing research into struvite urolithiasis, we have adapted the use of phase contrast light microscopy to investigate the role of various naturally occurring urinary compounds on struvite crystal growth in vitro [16]. This simple approach has allowed us to rapidly screen several compounds for their role as potential crystallization inhibitors, since struvite and other crystals assume a characteristic shape or crystal habit which is influenced directly by growth rate [17,18]. In this paper we report on the ability of pyrophosphate to inhibit P. mirubili.s-induced struvite crystallization in vitro. Materials
and methods
Proteus mirabilis growth and crystal production:
P. mirabilis strain 2573 was
originally isolated from a patient with struvite urolithiasis
[15,16,19] and main-
109
tained on tryptic soy agar (Difco Laboratories, Detroit, Mich. USA). It has since been deposited with the American Type Culture Collection as strain ATCC 49565. The experimental protocols used for in vitro production of struvite crystals by this organism were described in earlier publications [15,16]. Briefly, this involved growing P. mirabilis in a continuous culture flask containing 800 ml artificial urine for a period of 24 h. Sterile artificial urine was continuously added to the flask at a rate of 60 ml h-i. The volume of the flask and the rate of addition of urine were chosen so as to approximate the average bladder volume and urine production rate of human males 1201.Samples were removed 0, 0.5, 1, 2, 3, 4, 5, 6, 7, and 24 h after inoculation, examined by phase contrast light microscopy, and monitored for pH, turbidity (OD,a,,), bacterial cell counts, urease activity, and ammonia concentration. A sample (designated as -0.5 h) was also taken from the inoculum for analysis. These biochemical and microbiological analyses were performed so as to determine whether alterations of crystal growth rate could be attributed to a buffering of urine pH, antimicrobial activity, interference with ammonia concentrations, or inhibition of the principal enzyme (urease) responsible for the formation of these struvite calculi. The artificial urine used was our modification [20] of that described by Griffith et al. [3]. For inhibition experiments, the artificial urine was supplemented with sodium pyrophosphate (purchased from the Sigma Chemical Co., St. Louis, MO) at either 6 mg l- ’ or 60 mg 1-i (13.4-134 pmol/l). During control experiments, no pyrophosphate was present. These pyrophosphate concentrations were chosen to reflect normal and excessive concentrations of this compound as found in human urine [13]. A minimum of five replications were conducted under each experimental condition. The influence of pyrophosphate on struvite formation was also measured in the absence of P. mirabilis. This involved titrating 800 ml artificial urine (with or without pyrophosphate) in our in vitro apparatus [16,20] with 0.25 mol/l NH,OH. NH,OH and fresh artificial urine were both added at a rate of 60 ml h-i. This concentration of NH,OH and flow rate gave approximately the same pH and optical density increase kinetics as did inoculation with P. rnirubilis. Samples were removed at 0, 0.5, 1, 2, 3, 4, 5, 6, 7, and 24 h, examined by phase contrast light microscopy, and analyzed for pH, turbidity, and ammonia concentrations. P. mirabilis viability was measured by standard microbiological plate count methods and expressed as colony forming units per ml (CFU ml-‘) [16]. Urease activity (expressed as pg NH, produced ml-’ min-‘1 and culture ammonia concentrations (NH,, NH:, and total ammonia) were analyzed using our modification [15] of the phenol hypochlorite assay of Weatherburn [21]. Direct examination of the samples by phase contrast light microscopy [16] identified the presence and crystal habit(s) of any precipitates present and enabled us to assess the physiological status of P. mirubilis based on cellular motility [16]. Crystal analysis and identification of struvite: All crystals produced during these experiments were identified using X-ray powder diffraction (XRD) and Fouriertransform infrared spectroscopy (FTIR). Crystals were prepared for analysis by
110
harvesting by low speed centrifugation (200 - 300 X g), washing twice in 0.05 mol/l Tris(hydroxymethy1) amino methane (Tris) buffer, pH 8.6, and drying under partial vacuum in a desiccator. For XRD these crystals were ground, and analyzed using a Rigaku ‘miniflex’ X-ray diffractometer as described [15]. FTIR analysis of desiccated crystals mixed with KBr (final concentration 0.5-1.0% (w/w>) was conducted on a Bomem model MB 120 Fourier transform infrared spectrophotometer (Bomem Canada Inc., Quebec, QC, Canada) interfaced with a personal computer. For each specimen, 10 spectra were obtained from 4,000 cm-’ to 400 cm -I. The effective resolution was 4 cm- ‘. In addition, an FTIR spectra was also obtained from the sodium pyrophosphate used in the crystal inhibition experiments. All results obtained were compared to published spectra [22]. Results
P. mirubih 2573 inoculation into artificial urine in the absence of pyrophosphate caused a rapid elevation of urine pH and ammonia levels due to culture urease activity (Fig. 1). Crystals appeared within 4-5 h which typically possessed ‘X-shaped’ or flat planar crystal habits (Fig. 2) characteristic of rapid growth [16,17,19]. Initial growth of P. mirubifis was illustrated by an increase in optical density and in colony counts (Fig. 1). After a period of time (usually 7-24 h), culture optical density diminished (Fig. 1) in an analogous manner to earlier work [15,16]. A contributing factor to this is a reduction in culture viability (due to the high pH and ammonia levels [23]) in that colony counts in the absence of pyrophosphate also were reduced (Fig. 1). When pyrophosphate was present, P. mirabilis growth characteristics were considerably altered. The increase in pH of the artificial urine culture was seemingly unaffected by the addition of pyrophosphate (Fig. 1). However while urease activity and increases in ammonia levels were always evident, these values fluctuated widely and were slightly but not significantly (P > 0.05) lower than control values. This could not be attributed to an interference of pyrophosphate with the phenol-hypochlorite assay [21] (data not shown). P. mirabih grown in the presence of pyrophosphate demonstrated some resistance to the high ammonia levels and alkaline pH. Although at various times through the experiment, bacterial physiological stress became evident by a loss of motility [16] (usually 4-6 h after inoculation), these organisms quickly recovered and became actively motile within 1-2 h. This phenomenon occurred periodically and reproducibly throughout all experimental runs. In contrast P. mirabih motility in the absence of pyrophosphate became irreversibly lost after 4 h. Bacterial viability in the presence of pyrophosphate as expressed by CFU ml-’ was marginally enhanced over the 24-h course of the experiment (Fig. 1). Any precipitation which appeared, consisted largely of amorphous deposits. The first amorphous deposits to be seen often disappeared or became reduced in quantity. Crystal appearance was considerably delayed (up to 24 h after inoculation) and generally involved misshapen or deformed octahedral crystals (Fig. 3).
Control 6.0
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-1
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.P i
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I
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-1
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19
19
: 24
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14
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22
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Fig. 1. Turbidity (measured at 600 nm), pH, urease activity (pg NH, produced ml-’ mm-‘), ammonia concentrations (NH,, NH:, and total ammonia), and growth (log CFU ml-‘) due to Proteus mirabilis growth in artificial urine in the presence (6 mg I-‘) and absence of pyrophosphate. Error bars represent standard error in all cases except urease activity where they represent the 95% probability interval.
In order to assess whether the slight reductions in urease activity and ammonia concentrations in the presence of pyrophosphate were responsible for the altered crystal growth, struvite was produced in the absence of urease by titrating artificial urine with 0.25 mol/l NH,OH. As seen in Fig. 4, the chemically produced struvite crystals again exhibited similar misshapen crystal habits in the presence of pyrophosphate as did those produced by P. mirabilis. All crystals produced in the presence and absence of pyrophosphate were identified by XRD as struvite (Fig. 5). No carbonate-apatite nor any other mineral
Fig. 2. Struvite crystals grown in the absence of inhibitor typically assume an ‘X-shaped’ (Fig. 2A) or flat planar (Fig. 2B) crystal habit. Bar represents 25 km.
form was detected by this technique. Slight differences in the mineral types were seen using FTIR (Fig. 6). This may be attributable to a slight increase in the contamination of struvite produced in the absence of pyrophosphate by trace amounts of carbonate-apatite or newberyite (MgHPO, * H ,O). Significant ( > 10%) disruption or contamination of the basic struvite crystal structure did not occur as this would have been indicated by XRD. Similar results were seen at all pyrophosphate concentrations tested (0.45-134 pmol/l). The results shown represent values obtained at a pyrophosphate concentration of 6 mg 1-l (13.4 pmol/l) which represents a normal concentration of this inorganic anion in human urine [13]. Discussion
Identification of effective inhibitors of struvite formation is of paramount importance given the morbidity and mortality associated with the recurrence of these calculi following standard surgical therapy [1,24]. We recently adapted this direct observation technique which employs phase contrast light microscopy so as to rapidly screen and identify compound(s) in urine which might represent potentially effective inhibitors of struvite crystal growth 1161. During these studies, we have investigated the glycosaminoglycans, chondroitin sulphate and heparin sulphate; elevated citrate concentrations [15], and pyrophosphate (this study). Of all
Fig. 3. Struvite appearance in the presence of pyrophosphate is usually delayed by several hours. Typically amorphous deposits (arrows) and octahedral, slow-growing (171 crystals (Fig. 3A) are seen. Crystals often exhibit a misshapen (Fig. 3B) or deformed (Fig. 3C) crystal habit which is evidence of altered growth [29]. Bar represents 100 pm (3A and 3B), 2.5pm (3C).
Fig. 4. Alteration of crystal habit due to pyrophosphate was also evident in chemically-produced struvite (titration of artificial urine with NH,OH). In the absence of pyrophosphate (Fig. 4A), large numbers of crystals were seen which were often ‘X-shaped’ or tabular in appearance. Crystals produced in the presence of pyrophosphate were generally misshapen (Fig. 4B). Bar represents 20 pm.
compounds tested to date, we have found pyrophosphate to represent the potentially most effective crystallization inhibitor. This inhibitory action of pyrophosphate is not unique to struvite formation, in that it has been shown to be effective against urinary calcium oxalate formation [7-131 and even against dental calculus (calcium phosphate) formation [25]. Precipitation and crystallization of a compound from solution is a complex multi step process [18]. Initially the solubility of one or more potential crystals must be exceeded. Once supersaturation occurs, then precipitation can occur. In cases
/ 50
100
I
15.
1
I
t
20’
ZY
30.
35’
28 Fig. 5. X-ray diffraction of misshapen crystals in Fig. 3 identifies them as struvite. Numbers indicate crystallographic axes of struvite. DIFFRACTION
ANGLE
11.5 44
4000
3300
2600
I900
1200
500
CM-I
4000
3300
1900
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Fig. 6. Infrared spectroscopy analysis of struvite crystals produced in the absence (6A) and presence (6B) of pyrophosphate. The peak at 1753 cm-’ (arrow) (Fig. 6A) is possibly due to the contamination of struvite by carbonate-apatite or newberyite [22] in the absence of pyrophosphate.
where solubility is greatly exceeded, an amorphous precipitate is often formed due to its unordered nature. The eventual formation of an ordered structure (i.e. a crystal) from an amorphous precipitate or solution requires nucleation (i.e. the deposition of the first few molecules in an ordered arrangement) and growth through an ordered deposition and binding to the crystal surfaces. This nucleation and growth is influenced by the overall chemistry of the solution [26] and is often enhanced by the presence of biological surfaces such as bacterial cell walls and capsules (referenced in [26-281). In the case of struvite, intermolecular bonding during crystal formation involves ionic interactions between Mg*+, NH: and PO:-, and hydrogen bonding involving the water of crystallization, NH,f and/or
116
PO:- [17]. In addition, struvite formation requires the participation of urease producing bacteria (in this case P. mirubilis) and elevation of pH, NH,, and NH:. As such, there are many instances whereby crystal formation could be disrupted [291. Pyrophosphate inhibition of struvite formation could not be attributed to inhibition of P. mirabih growth as viability expressed as CFU ml-’ was marginally enhanced in the presence of pyrophosphate (Fig. 1). The initial lower concentration of cells in the presence of pyrophosphate was likely due to their reduced concentration in the initial inoculum (represented by the data point at time -0.5 h in Fig. 1). Pyrophosphate slightly inhibited P. mirubilis urease activity and NH, and NH: concentrations (Fig. 1) but not by a statistically significant margin (P > 0.05). Pyrophosphate exerted the same inhibitory effect against chemically produced struvite crystals so it is unlikely that urease inhibition was responsible. Inhibition of struvite could not be attributed to a buffering effect of pyrophosphate against pH as these values were virtually unaffected (Fig. 1). If pyrophosphate acted so as to complex Mg 2+ thereby rendering it unavailable for struvite formation, then diminishing its concentration should result in an elimination of inhibition as more Mg*+ becomes available. Lowering of the pyrophosphate concentration from 6 mg l- ’ to 0.2 mg 1-l (13.4 pmol/l to 0.45 pmol/l) did not alter its inhibitory activity (data not shown) so it is unlikely that Mg2+ complexation is important. During these experiments, struvite crystals in the initial inoculum completely dissolve within 3-5 min of being introduced into the reaction flask [163. Consequently any crystals which arise during the course of this procedure are formed de novo without the benefit of ‘seed’ crystals. As a result the effect of a potential inhibitor such as pyrophosphate can be observed on the nucleation as well as the growth of struvite crystals. Based on our observations, we speculate that the inhibitory action of pyrophosphate against struvite formation arises mainly from its ability to disrupt the ionic and hydrogen bonding necessary for crystal nucleation and development. This is evidenced by the delayed appearance of crystals and its alteration of struvite crystal habit (Figs. 3 and 4). Acknowledgements This project was supported by a grant from the Kidney Foundation of Canada to RJCM, JCN, and JWLW. R.J.C. McLean is supported by a Career Scientist Fellowship from the Ontario Ministry of Health and an operating grant from NSERC. We express our appreciation to Dr. Roy Pottier, Department of Chemistry and Chemical Engineering, Royal Military College of Canada, Kingston, Ontario and Pat Mulligan, Department of Chemistry, Queen’s University for the use of their infrared spectroscopes, and to Ann Lablans, Janet Clark and Anita Dumanski for their enthusiasm and excellent technical assistance. References 1 Lerner SP, Gleeson MJ, Griffith DP. Infection stones. J Ural 1989;141:753-758. 2 McLean RJC, Nickel JC, Cheng K-J, Costerton JW. The ecology and pathogenicity ducing bacteria in the urinary tract. Crit Rev Microbial 1988;16:37-79.
of urease-pro-
117 3 Griffith DP, Musher DM, Itin C. Urease the primary cause of infection-induced urinary stones. Invest Urol 1976;13:346-350. 4 Jones BD, Lockatell CV, Johnson DE, Warren JW, Mobley HLT. Construction of a urease-negative mutant of Proteus mirabilis: analysis of virulence in a mouse model of ascending urinary tract infection. Infect Immunol 1990;58:1120-1123. 5 Hedelin H, Grenabo L, Pettersson S. The effects of urease in undiluted human urine. J Urol 1986;136:743-745. 6 Ryall RL, Hibberd CM, Mazzachi BC, Marshall VR. Inhibitory activity of whole urine: a comparison of urines from stone formers and healthy subjects. Clin Chim Acta 1986;154:59-68. 7 Baumann JM, Ackermann D, Affolter B. The influence of hydroxyapatite and pyrophosphate of the formation product of calcium oxalate at different pHs. Urol Res 1989;17:153-155. 8 Conte A, Rota P, Genestar C, Grases F. The relation between orthophosphate and pyrophosphate in normal subjects and in patients with urolithiasis. Urol Res 1989;17:173-175. 9 Grases F, Genestar C, Conte A, March P, Costa-Bauza A. Inhibitory effect of pyrophosphate, citrate, magnesium and chondroitin sulphate in calcium oxalate urolithiasis. Br J Urol 1989;64:235237. 10 Grases F, Gil JJ, Conte A. Urolithiasis inhibitors and calculus nucleation. Urol Res 1989;17:163-166. 11 Robertson WG, Peacock M, Nordin BEC. Inhibitors of the growth and aggregation of calcium oxalate crystals in vitro. Clin Chim Acta 1973;43:31-37. 12 Ryall RL, Harnett RM, Marshall VR. The effect of urine, pyrophosphate, citrate, magnesium and glycosaminoglycans on the growth and aggregation of calcium oxalate crystals in vitro. Clin Chim Acta 1981;112:349-356. 13 Wilson JWL, Werness PG, Smith LH. Inhibitors of crystal growth of hydroxyapatite: a constant composition approach. J Urol 1985;134:1255-1258. 14 Hedelin H, Grenabo L, Hugosson J, Pettersson S. The influence of zinc and citrate on urease-induced urine crystallisation. Urol Res 1989;17:177-180. 15 McLean RJC, Downey J, Clapham L, Nickel JC. Influence of chondroitin sulfate, heparin sulfate, and citrate on Proteus mirabilis-induced struvite crystallization in vitro. J Ural 1990;144:1267-1271. 16 McLean RJC, Downey J, Clapham L, Nickel JC. A simple technique for studying struvite crystal growth in vitro. Urol Res 1990;18:39-43. 17 Abbona F, Boistelle R. Growth morphology and crystal habit of struvite crystals (MgNH,PO,. 6HzO). J Cryst Growth 1979;46:339-354. 18 Kern R. Crystal growth and adsorption. In: Sheftal NN., ed. Growth of crystals, Vol. 8. New York: Consultants Bureau, 1969;3-23. 19 Clapham IL, McLean RJC, Nickel JC, Downey J, Costerton JW. The influence of bacteria on struvite crystal habit and its importance in urinary stone formation. J Cryst Growth 1990;104:475-484. 20 McLean RJC, Nickel JC, Noakes VC, Costerton JW. An in vitro study of infectious kidney stone genesis. Infect Immun 1985;49:805-811. 21 Weatherburn MW. Phenol-hypochlorite reaction for determination of ammonia. Anal Chem 1967;39:971-974. 22 Hesse A, Sanders G. Atlas of infrared spectra for the analysis of urinary concretements. Stuttgart, FRG: Georg Thieme Verlag, 1988: 23 Visek WJ. Ammonia: its effects on biological system, metabolic hormones and reproduction. J Dairy Sci 1984;67:481-498. 24 Boyce WH. Surgery of urinary calculi in perspective. Urol Clin N Am 1983;10:585-594. 25 Mallatt ME, Beiswanger BB, Stookey GK, Swancar JR, Hennon DK. Influence of soluble pyrophosphate on calculus formation. J Dent Res 1985;64:1159-1162. 26 Mann S. Mineralization in biological systems. Struct Bond 1983;54:125-174. 27 Mclean RJC, Beveridge TJ. Metal binding capacity of bacterial surfaces and their ability to form mineralized aggregates. In: Ehrlich HL, Brierley CL., eds. Microbial mineral recovery. New York: McGraw Hill, 1990;185-222. 28 Beveridge TJ. Role of cellular design in bacterial metal accumulation and mineralization. Annu Rev Microbial 1989;43:147-171. 29 Sangwal K. Growth and dissolution of crystals. In: Sangwal K, ed. Etching of crystals theory, experiment, and application. Amsterdam: North-Holland Publishing Company, 1987;43-86.
