British Journal of Urology (1992), 70,355-359
01992 British Journal of Urology
ln vitro Inhibition of Struvite Crystal Growth by Aceto hyd roxamic Acid J.A. DOWNEY, J. C. NICKEL, L. CLAPHAM and R. J. C. McLEAN Departments of Urology, Physics, and Microbiology and Immunology, Queen's University, Kingston, Canada
Summary-Struvite (MgN H4P046H,0) crystals were produced by Proteus mirabilis growth in artificial urine, in the presence and absence of the urease inhibitor, acetohydroxamic acid (AHA). In the absence of AHA, struvite crystals assumed an "X-shaped"or dendritic crystal habit due to rapid growth along their 100 axis. When AHA was present, crystal growth, as monitored by phase contrast light microscopy, was greatly slowed, and the crystals assumed an octahedral crystal habit. Scanning electron microscopy revealed that crystals grown in the presence of AHA were pitted on their surface. This pitting was absent in control samples. While most of this inhibition by AHA was due to lowered urease activity, some crystal growth inhibition occurred in struvite produced in the absence of urease activity through N H 4 0 H titration of artificial urine. We conclude that while AHA is primarily a urease inhibitor, it may also disrupt struvite growth and formation directly through interference with the molecular growth processes on crystal surfaces.
Struvite (MgNH4P046H20) calculi arise from urinary tract infections (UTI) by urease-producing organisms such as Proteus mirabilis (McLean et al., 1988). The urease-catalysed hydrolysis of urea generates ammonia, elevates urine pH, and causes the precipitation of Mgz+as struvite (Griffith et al., 1976; Hedelin et al., 1985; McLean et al., 1988). Struvite in situ associates with an organic matrix of host and possibly bacterial origin (McLean et al., 1988) which enables the development and formation of mature struvite calculi and their retention in the urinary tract. Struvite urolithiasis is notorious for its rapid progression, high rate of recurrence, and severe pathological effects on the kidney (Lerner et a/., 1989). The central role of bacterial urease activity in the pathogenesis of this UTI led to the development of a number of urease inhibitors. Acetohydroxamic acid (AHA), an analogue of urea and a competitive inhibitor of urease activity, represents the only inhibitor currently licensed fgr therapeutic use
(Fishbein, 1982; Rosenstein and Hamilton-Miller, 1984; Williams et al., 1984; Rodman et al., 1987; Hess, 1990). It has been quite successful in clinical trials (Williams et al., 1984) but notable side effects of this compound include tremulousness and thrombosis. Animal studies have also shown the possibility of teratogenic and mutagenic side effects (Hess, 1990). As part of our ongoing research into the pathogenesis of P . mirabilis UTI and struvite urolithiasis, we investigated the effects of AHA on struvite crystal development. Apart from its inhibition of urease activity, AHA also had an inhibitory action against struvite crystal growth that was completely independent of its action against urease. These findings suggest that AHA may be doubly effective as a struvite stone inhibitor, in that it interferes with crystal growth as well as blocking urease activity.
Materials and Methods Accepted for publication 16 October 1991
P . mirabilis 2573, serotype 0-6, used in these experiments was originally isolated from a patient
355
356 with struvite urolithiasis and has been deposited with the American Type Culture Collection as strain ATCC 49565 (Beynon et al., in preparation). The experimental protocol used for crystal growth was described in our earlier publications (McLean et al., 1990a and b, 1991). Briefly, it involved growing P. mirabilis in a flask containing 800ml artificial urine (McLean et al., 1990b). Sterile artificial urine was added to the flask from a reservoir at 60 ml/h, and the waste urine removed through a port on the side of the flask. The urine composition was chosen so as to mimic the average 24-h urine composition of human males. The flask volume and rate of addition of fresh urine represented average male bladder capacity and urine production rates. During most experiments, AHA was present in the artificial urine in both the growth flask and reservoir (4 mg/ml). In other experiments, we investigated whether AHA could interfere with struvite crystal growth once crystallisation conditions had been established. This involved inoculating the artificial urine with P. mirabilis and growing the organism in the absence of AHA for 4 h. At this point, sterile AHA was added to the urine in the reservoir (4 mg/ml), and the mixture was gradually introduced into the culture flask (60 ml/ h). Control experiments lacked AHA. Samples were removed at 0,0.5, I , 2, 3,4, 5 , 6, 7 and 24 h after inoculation, and analysed for culture viability, urease activity, ammonia (NH3, NH,+, and total ammonia), pH and optical density (McLean et al., 1990a and b). In addition, samples were examined by phase contrast light microscopy and scanning electron microscopy (SEM), using the equipment and protocols described previously (Clapham et al., 1990; McLean et al., 1990b). We also investigated whether AHA had an effect on struvite crystal growth in addition to its urease inhibiting property. This involved growing struvite crystals in sterile artificial urine through titration with 0.25 M NH,OH added at 60ml/h. This titration protocol with NH,OH yielded approximately the same pH increase kinetics as did P. mirabilis growth (McLean et al., 1991). All crystals produced during these experiments were analysed by powder X-ray diffraction and compared with published values for struvite (McLean et al., 1990a). This experimental protocol allowed us to observe the effects of AHA on dtruvite crystal growth and to ascertain whether any alteration was due to an interference with P. mirabilis viability or urease activity, a buffering of ammonia concentrations or pH, or a direct effect on crystal growth.
BRITISH JOURNAL OF UROLOGY
Results Growth of P. mirabilis in artificial urine in the absence of AHA resulted in a rapid increase in pH, bacterial numbers (colony forming units, CFU), and urease activity (Fig. 1). When AHA was present, bacterial viability was not affected, but urease activity and pH increase were greatly diminished (Fig. 1). Similar although delayed effects were seen if AHA was added after 4 h growth (data not shown). Crystals produced by P. mirabilis growth or NH,OH titration in the absence of AHA demonstrated the typically dendritic or X-shaped morphologies (crystal habits) of rapid growth along the 100 axis (Figs 2A and B) (Abbona and Boistelle, 1979; McLean et al., 1990b). When AHA was present, crystal growth was delayed and the crystals that did develop exhibited a more 3-dimensional tabular crystal habit typical of slow growth (Figs 3A and B) (Abbona and Boistelle, 1979). Crystal growth was also slowed in crystals produced through NH,OH titration (Fig. 3B). The deleterious effects of AHA on struvite crystal structure were especially evident upon ultrastructural examination by SEM (Fig. 4). Noticeable pitting was observed on struvite crystals produced in the presence of AHA. All crystals produced in this experiment were identified as struvite by X-ray diffraction. No impurities in a concentration greater than 10% were detected by this technique.
Discussion A number of urease inhibiting compounds such as AHA have been developed for use in clinical, nutritional and agricultural applications (Fishbein, 1982; Rosenstein and Hamilton-Miller, 1984; Gould et al., 1986; Mobley and Hausinger, 1989; Hess, 1990). In the case of struvite urolithiasis, urease inhibition is especially important in that ammonia production and pH elevation due to the activity of this enzyme represent the major virulence factors associated with this UTI (Griffith et al., 1976; Burns and Gauthier, 1984; McLean et al., 1988). In light of its urease inhibiting properties, we anticipated that the major effect of AHA inhibition of struvite formation would be due to its lowering of urease activity. Although this did occur, we also found that AHA disrupted struvite formation in a manner quite independent of its action against urease (Figs 3B and 4). As stated before, AHA (CH,CONHOH) is a structural analogue of urea
357
INHIBITION OF STRUVITE CRYSTAL GROWTH
'l
I
-4
i
T/
0
i
I
0
- 0 AHA
-0
Oo5
I 0-0
Control
O - - - -0 AHA
hydrogen bonding are involved in struvite crystal development (Abbona and Boistelle, 1979). AHA is not the only compound that can block struvite growth. Our own work and that of others has shown that naturally occurring urine compounds such as citrate and pyrophosphate are able to inhibit struvite formation through chelating Mg2+,disrupting the hydrogen and ionic bonding of this mineral and coating the surface of struvite crystals, thus preventing further growth (Hedelin et al., 1989; Hugosson et a/., 1990a and b; McLean et al., 1990a, 1991). Although the practical clinical use of AHA is uncertain due to the aforementioned side effects of thrombosis and tremulousness (Hess, 1990), this compound shows a great deal of promise in the control of struvite development by its inhibitory effects against both urease and crystal development. Because of these additional inhibitory effects of the urease inhibitor, AHA, against crystal growth, we would encourage other researchers in the field of developing urease inhibitors to investigate the possibility of their respective test compounds to inhibit struvite crystal growth. The potential clinical implications of developing inhibitors which are doubly effective against both urease and struvite crystal development are very significant.
Acknowledgements
-1
4
9
14
19
24
Time (h)
Fig. 1 pH, cell numbers (log CFU/ml) and urease activity due to P. mirabilis growth in artificial urine in the presence and absence of AHA.
This project was supported by a grant from the Kidney Foundation of Canada. R. J . C. McLean is supported by a Career Scientist Fellowship from the Ontario Ministry of Health and an operating grant from the Natural Sciences and Engineering Research Council of Canada. Electron microscopy was performed at the Faculty of Medicine Electron Microscopy Centre, Queen's University, which is supported by user fees and an operating grant from the Medical Research Council of Canada. We thank Ann Lablans, Janet Clark and Anita Dumanski for their assistance.
References (H2NCONH2). From its structure, it is quite conceivable that AHA, by virtue of its -CO (carbonyl) and -NHOH (hydroxamino) moieties, may interfere with struvite formation through hydrogen bonding to the H 2 0 and PO4 struvite crystal components or possibly through an ionic complexation of Mg2+ to its partially anionic carbonyl moiety. The large quantities of amorphous material and low numbers of crystals present in Fig. 3A suggest that while some precipitation did occur, its molecular re-arrangement into an ordered (crystalline) form was inhibited. Both ionic and
Abbona, F. and Boistelle, R. (1979). Growth morphology and crystal habit of struvite crystals (MgNH,P0,6H20). J . Cryst. Growth, 46,339-354. Beynon, L. M., Dumanski, A. J., McLean, R. J. C. et al. (1992). Capsule structure of Proteus mirabilis (ATCC 49565). J . Bacteriol., 174, in press. Bums, J. R. and Gauthier, J. F. (1984). Prevention of urinary catheter incrustations by acetohydroxamic acid. J . Urol., 132, 455456. Clapham, L., McLean, R. J. C., Nickel, J. C. ef al. (1990). The influence of bacteria on struvite crystal habit and its importance in urinary stone formation. J . Cryst. Growth, 104, 475484. Fishbein, W. N. (1982). Hydroxamic acids as urease inhibitors for medical and veterinary use. In Chemistry and Biology of Hydroxamic Acids, ed. Kehl, H. Pp. 94-103. Basel: Karger.
358
BRITISH JOURNAL OF UROLOGY
Fig. 2 Crystal habits of struvite formed in artificial urine without added A H A through growth and urease activity of P.rnirubilis (A) or NH,OH titration (B). Bar represents 20 pm.
Fig. 3 Crystal habits of struvite formed in the presence of A H A through growth and urease activity of P.mirubilis (A) or NH,OH titration (B). Note the quantity of amorphous material (a) present. Bar represents 40 pm.
Could, W. D., Hagedorn, C. and McCready, R. G . L. (1986). Urea transformation and fertilizer efficiency in soil. Adv. Agron., 40,209. Griffith, D. P., Musher, D. M. and Itin, C. (1976). Urease the primary cause of infection-induced urinary stones. Invest. Urol., 13, 346-350. Hedelin, H., Grenabo, L. and Pettersson, S. (1985). Ureaseinduced crystallization in synthetic urine. J . Urol., 133, 529533.
Hedelin, H., Grenabo, L., Hugosson, J. e t d (1989). The influence of zinc and citrate on urease-induced crystallisation. Urol. Res., 17, 177-180.
Hess, B. (1990). Prophylaxis of infection-induced kidney stone formation. Urol. Res. (Suppl. I ) , 18, S45-S48. Hugosson, J., Grenabo, L., Hedelin, H. et d. (1990a). How variations in the composition of urine influence ureaseinduced crystallization. Urol. Res., 18,413-417. Hugosson, J., Grenabo, L., Hedelin, H. et al. (1990b). Effects of serum, albumen and immunoglobulins in urease-induced crystallization in urine. Urol. Res., 18, 40741 1. Lerner, S. P., Gleeson, M. J. and Griffith, D. P. (1989). Infection stones. J . Urol., 141,753-758. McLean, R. J. C., Downey, J., Clapham, L. e l d. (1990a). Influence of chondroitin sulfate, heparin sulfate, and citrate
359
INHIBITION OF STRUVITE CRYSTAL GROWTH
Fig. 4 Struvite formed in the presence of AHA exhibited pronounced pitting of its surface (A). This was not observed in crystals formed in the absence of AHA (B).
on Proteus mirabilis-induced struvite crystallization in uitro. J . Urol., 144, 1267-1271. McLean, R. J. C., Downey, J., Clapham, L. et al. (1990b). A simple technique for studying struvite crystal growth in uitro. Urol. Res., 18. 39-43. McLean, R. J. C., Downey, J., Clapham, L. et al. (1991). Pyrophosphate inhibition of Proteus mirabilis-induced struvite crystallization in uitro. Clin. Chim. Acta, 200, 107-1 18. McLean, R. J. C., Nickel, J. C., Cheng, K.-J. et al. (1988). The ecology and pathogenicity of urease-producing bacteria in the urinary tract. Crif. Reu. Microbiol., 16, 37-19. Mobley, H. L. T. and Hausinger, R. P. (1989).Microbial ureases: significance, regulation, and molecular characterization. Microbiol. Rev., 53, 85-108. Rodman, J. S., Williams, J. J. and Jones, R. L. (1987). Hypercoagulability produced by treatment with acetohydroxamic acid. Clin. Pharmacol. Ther.. 42, 346-350. Rosenstein, I. J. M. and Hamilton-Miller, J. M. T. (1984). Inhibitors of urease as chemotherapeutic agents. Crit. Rev. Microbiol.,11, 1-92.
Williams, J. J., Rodman, J. S. and Peterson, C. M. (1984). A randomized double-blind study of acetohydroxamic acid in struvite nephrolithiasis. N . Engl. J . Med., 311,760-764.
The Authors J . A. Downey, BSc, MSc, Research Associate, Department of Urology. J. C. Nickel, MD, FRCS(C), Associate Professor, Department of Urology. L. Clapham, BSc, PhD, Adjunct Professor, Department of Physics. R. J. C. McLean, BSc, PhD, Assistant Professor, Department of Urology, and Department of Microbiology and Immunology.
Requests for reprints to: R. J. C. McLean, Department of Microbiology and Immunology, Queen’s University, Kingston, Ontario, Canada K7L 3N6.
01992 British Journal of Urology
ln vitro Inhibition of Struvite Crystal Growth by Aceto hyd roxamic Acid J.A. DOWNEY, J. C. NICKEL, L. CLAPHAM and R. J. C. McLEAN Departments of Urology, Physics, and Microbiology and Immunology, Queen's University, Kingston, Canada
Summary-Struvite (MgN H4P046H,0) crystals were produced by Proteus mirabilis growth in artificial urine, in the presence and absence of the urease inhibitor, acetohydroxamic acid (AHA). In the absence of AHA, struvite crystals assumed an "X-shaped"or dendritic crystal habit due to rapid growth along their 100 axis. When AHA was present, crystal growth, as monitored by phase contrast light microscopy, was greatly slowed, and the crystals assumed an octahedral crystal habit. Scanning electron microscopy revealed that crystals grown in the presence of AHA were pitted on their surface. This pitting was absent in control samples. While most of this inhibition by AHA was due to lowered urease activity, some crystal growth inhibition occurred in struvite produced in the absence of urease activity through N H 4 0 H titration of artificial urine. We conclude that while AHA is primarily a urease inhibitor, it may also disrupt struvite growth and formation directly through interference with the molecular growth processes on crystal surfaces.
Struvite (MgNH4P046H20) calculi arise from urinary tract infections (UTI) by urease-producing organisms such as Proteus mirabilis (McLean et al., 1988). The urease-catalysed hydrolysis of urea generates ammonia, elevates urine pH, and causes the precipitation of Mgz+as struvite (Griffith et al., 1976; Hedelin et al., 1985; McLean et al., 1988). Struvite in situ associates with an organic matrix of host and possibly bacterial origin (McLean et al., 1988) which enables the development and formation of mature struvite calculi and their retention in the urinary tract. Struvite urolithiasis is notorious for its rapid progression, high rate of recurrence, and severe pathological effects on the kidney (Lerner et a/., 1989). The central role of bacterial urease activity in the pathogenesis of this UTI led to the development of a number of urease inhibitors. Acetohydroxamic acid (AHA), an analogue of urea and a competitive inhibitor of urease activity, represents the only inhibitor currently licensed fgr therapeutic use
(Fishbein, 1982; Rosenstein and Hamilton-Miller, 1984; Williams et al., 1984; Rodman et al., 1987; Hess, 1990). It has been quite successful in clinical trials (Williams et al., 1984) but notable side effects of this compound include tremulousness and thrombosis. Animal studies have also shown the possibility of teratogenic and mutagenic side effects (Hess, 1990). As part of our ongoing research into the pathogenesis of P . mirabilis UTI and struvite urolithiasis, we investigated the effects of AHA on struvite crystal development. Apart from its inhibition of urease activity, AHA also had an inhibitory action against struvite crystal growth that was completely independent of its action against urease. These findings suggest that AHA may be doubly effective as a struvite stone inhibitor, in that it interferes with crystal growth as well as blocking urease activity.
Materials and Methods Accepted for publication 16 October 1991
P . mirabilis 2573, serotype 0-6, used in these experiments was originally isolated from a patient
355
356 with struvite urolithiasis and has been deposited with the American Type Culture Collection as strain ATCC 49565 (Beynon et al., in preparation). The experimental protocol used for crystal growth was described in our earlier publications (McLean et al., 1990a and b, 1991). Briefly, it involved growing P. mirabilis in a flask containing 800ml artificial urine (McLean et al., 1990b). Sterile artificial urine was added to the flask from a reservoir at 60 ml/h, and the waste urine removed through a port on the side of the flask. The urine composition was chosen so as to mimic the average 24-h urine composition of human males. The flask volume and rate of addition of fresh urine represented average male bladder capacity and urine production rates. During most experiments, AHA was present in the artificial urine in both the growth flask and reservoir (4 mg/ml). In other experiments, we investigated whether AHA could interfere with struvite crystal growth once crystallisation conditions had been established. This involved inoculating the artificial urine with P. mirabilis and growing the organism in the absence of AHA for 4 h. At this point, sterile AHA was added to the urine in the reservoir (4 mg/ml), and the mixture was gradually introduced into the culture flask (60 ml/ h). Control experiments lacked AHA. Samples were removed at 0,0.5, I , 2, 3,4, 5 , 6, 7 and 24 h after inoculation, and analysed for culture viability, urease activity, ammonia (NH3, NH,+, and total ammonia), pH and optical density (McLean et al., 1990a and b). In addition, samples were examined by phase contrast light microscopy and scanning electron microscopy (SEM), using the equipment and protocols described previously (Clapham et al., 1990; McLean et al., 1990b). We also investigated whether AHA had an effect on struvite crystal growth in addition to its urease inhibiting property. This involved growing struvite crystals in sterile artificial urine through titration with 0.25 M NH,OH added at 60ml/h. This titration protocol with NH,OH yielded approximately the same pH increase kinetics as did P. mirabilis growth (McLean et al., 1991). All crystals produced during these experiments were analysed by powder X-ray diffraction and compared with published values for struvite (McLean et al., 1990a). This experimental protocol allowed us to observe the effects of AHA on dtruvite crystal growth and to ascertain whether any alteration was due to an interference with P. mirabilis viability or urease activity, a buffering of ammonia concentrations or pH, or a direct effect on crystal growth.
BRITISH JOURNAL OF UROLOGY
Results Growth of P. mirabilis in artificial urine in the absence of AHA resulted in a rapid increase in pH, bacterial numbers (colony forming units, CFU), and urease activity (Fig. 1). When AHA was present, bacterial viability was not affected, but urease activity and pH increase were greatly diminished (Fig. 1). Similar although delayed effects were seen if AHA was added after 4 h growth (data not shown). Crystals produced by P. mirabilis growth or NH,OH titration in the absence of AHA demonstrated the typically dendritic or X-shaped morphologies (crystal habits) of rapid growth along the 100 axis (Figs 2A and B) (Abbona and Boistelle, 1979; McLean et al., 1990b). When AHA was present, crystal growth was delayed and the crystals that did develop exhibited a more 3-dimensional tabular crystal habit typical of slow growth (Figs 3A and B) (Abbona and Boistelle, 1979). Crystal growth was also slowed in crystals produced through NH,OH titration (Fig. 3B). The deleterious effects of AHA on struvite crystal structure were especially evident upon ultrastructural examination by SEM (Fig. 4). Noticeable pitting was observed on struvite crystals produced in the presence of AHA. All crystals produced in this experiment were identified as struvite by X-ray diffraction. No impurities in a concentration greater than 10% were detected by this technique.
Discussion A number of urease inhibiting compounds such as AHA have been developed for use in clinical, nutritional and agricultural applications (Fishbein, 1982; Rosenstein and Hamilton-Miller, 1984; Gould et al., 1986; Mobley and Hausinger, 1989; Hess, 1990). In the case of struvite urolithiasis, urease inhibition is especially important in that ammonia production and pH elevation due to the activity of this enzyme represent the major virulence factors associated with this UTI (Griffith et al., 1976; Burns and Gauthier, 1984; McLean et al., 1988). In light of its urease inhibiting properties, we anticipated that the major effect of AHA inhibition of struvite formation would be due to its lowering of urease activity. Although this did occur, we also found that AHA disrupted struvite formation in a manner quite independent of its action against urease (Figs 3B and 4). As stated before, AHA (CH,CONHOH) is a structural analogue of urea
357
INHIBITION OF STRUVITE CRYSTAL GROWTH
'l
I
-4
i
T/
0
i
I
0
- 0 AHA
-0
Oo5
I 0-0
Control
O - - - -0 AHA
hydrogen bonding are involved in struvite crystal development (Abbona and Boistelle, 1979). AHA is not the only compound that can block struvite growth. Our own work and that of others has shown that naturally occurring urine compounds such as citrate and pyrophosphate are able to inhibit struvite formation through chelating Mg2+,disrupting the hydrogen and ionic bonding of this mineral and coating the surface of struvite crystals, thus preventing further growth (Hedelin et al., 1989; Hugosson et a/., 1990a and b; McLean et al., 1990a, 1991). Although the practical clinical use of AHA is uncertain due to the aforementioned side effects of thrombosis and tremulousness (Hess, 1990), this compound shows a great deal of promise in the control of struvite development by its inhibitory effects against both urease and crystal development. Because of these additional inhibitory effects of the urease inhibitor, AHA, against crystal growth, we would encourage other researchers in the field of developing urease inhibitors to investigate the possibility of their respective test compounds to inhibit struvite crystal growth. The potential clinical implications of developing inhibitors which are doubly effective against both urease and struvite crystal development are very significant.
Acknowledgements
-1
4
9
14
19
24
Time (h)
Fig. 1 pH, cell numbers (log CFU/ml) and urease activity due to P. mirabilis growth in artificial urine in the presence and absence of AHA.
This project was supported by a grant from the Kidney Foundation of Canada. R. J . C. McLean is supported by a Career Scientist Fellowship from the Ontario Ministry of Health and an operating grant from the Natural Sciences and Engineering Research Council of Canada. Electron microscopy was performed at the Faculty of Medicine Electron Microscopy Centre, Queen's University, which is supported by user fees and an operating grant from the Medical Research Council of Canada. We thank Ann Lablans, Janet Clark and Anita Dumanski for their assistance.
References (H2NCONH2). From its structure, it is quite conceivable that AHA, by virtue of its -CO (carbonyl) and -NHOH (hydroxamino) moieties, may interfere with struvite formation through hydrogen bonding to the H 2 0 and PO4 struvite crystal components or possibly through an ionic complexation of Mg2+ to its partially anionic carbonyl moiety. The large quantities of amorphous material and low numbers of crystals present in Fig. 3A suggest that while some precipitation did occur, its molecular re-arrangement into an ordered (crystalline) form was inhibited. Both ionic and
Abbona, F. and Boistelle, R. (1979). Growth morphology and crystal habit of struvite crystals (MgNH,P0,6H20). J . Cryst. Growth, 46,339-354. Beynon, L. M., Dumanski, A. J., McLean, R. J. C. et al. (1992). Capsule structure of Proteus mirabilis (ATCC 49565). J . Bacteriol., 174, in press. Bums, J. R. and Gauthier, J. F. (1984). Prevention of urinary catheter incrustations by acetohydroxamic acid. J . Urol., 132, 455456. Clapham, L., McLean, R. J. C., Nickel, J. C. ef al. (1990). The influence of bacteria on struvite crystal habit and its importance in urinary stone formation. J . Cryst. Growth, 104, 475484. Fishbein, W. N. (1982). Hydroxamic acids as urease inhibitors for medical and veterinary use. In Chemistry and Biology of Hydroxamic Acids, ed. Kehl, H. Pp. 94-103. Basel: Karger.
358
BRITISH JOURNAL OF UROLOGY
Fig. 2 Crystal habits of struvite formed in artificial urine without added A H A through growth and urease activity of P.rnirubilis (A) or NH,OH titration (B). Bar represents 20 pm.
Fig. 3 Crystal habits of struvite formed in the presence of A H A through growth and urease activity of P.mirubilis (A) or NH,OH titration (B). Note the quantity of amorphous material (a) present. Bar represents 40 pm.
Could, W. D., Hagedorn, C. and McCready, R. G . L. (1986). Urea transformation and fertilizer efficiency in soil. Adv. Agron., 40,209. Griffith, D. P., Musher, D. M. and Itin, C. (1976). Urease the primary cause of infection-induced urinary stones. Invest. Urol., 13, 346-350. Hedelin, H., Grenabo, L. and Pettersson, S. (1985). Ureaseinduced crystallization in synthetic urine. J . Urol., 133, 529533.
Hedelin, H., Grenabo, L., Hugosson, J. e t d (1989). The influence of zinc and citrate on urease-induced crystallisation. Urol. Res., 17, 177-180.
Hess, B. (1990). Prophylaxis of infection-induced kidney stone formation. Urol. Res. (Suppl. I ) , 18, S45-S48. Hugosson, J., Grenabo, L., Hedelin, H. et d. (1990a). How variations in the composition of urine influence ureaseinduced crystallization. Urol. Res., 18,413-417. Hugosson, J., Grenabo, L., Hedelin, H. et al. (1990b). Effects of serum, albumen and immunoglobulins in urease-induced crystallization in urine. Urol. Res., 18, 40741 1. Lerner, S. P., Gleeson, M. J. and Griffith, D. P. (1989). Infection stones. J . Urol., 141,753-758. McLean, R. J. C., Downey, J., Clapham, L. e l d. (1990a). Influence of chondroitin sulfate, heparin sulfate, and citrate
359
INHIBITION OF STRUVITE CRYSTAL GROWTH
Fig. 4 Struvite formed in the presence of AHA exhibited pronounced pitting of its surface (A). This was not observed in crystals formed in the absence of AHA (B).
on Proteus mirabilis-induced struvite crystallization in uitro. J . Urol., 144, 1267-1271. McLean, R. J. C., Downey, J., Clapham, L. et al. (1990b). A simple technique for studying struvite crystal growth in uitro. Urol. Res., 18. 39-43. McLean, R. J. C., Downey, J., Clapham, L. et al. (1991). Pyrophosphate inhibition of Proteus mirabilis-induced struvite crystallization in uitro. Clin. Chim. Acta, 200, 107-1 18. McLean, R. J. C., Nickel, J. C., Cheng, K.-J. et al. (1988). The ecology and pathogenicity of urease-producing bacteria in the urinary tract. Crif. Reu. Microbiol., 16, 37-19. Mobley, H. L. T. and Hausinger, R. P. (1989).Microbial ureases: significance, regulation, and molecular characterization. Microbiol. Rev., 53, 85-108. Rodman, J. S., Williams, J. J. and Jones, R. L. (1987). Hypercoagulability produced by treatment with acetohydroxamic acid. Clin. Pharmacol. Ther.. 42, 346-350. Rosenstein, I. J. M. and Hamilton-Miller, J. M. T. (1984). Inhibitors of urease as chemotherapeutic agents. Crit. Rev. Microbiol.,11, 1-92.
Williams, J. J., Rodman, J. S. and Peterson, C. M. (1984). A randomized double-blind study of acetohydroxamic acid in struvite nephrolithiasis. N . Engl. J . Med., 311,760-764.
The Authors J . A. Downey, BSc, MSc, Research Associate, Department of Urology. J. C. Nickel, MD, FRCS(C), Associate Professor, Department of Urology. L. Clapham, BSc, PhD, Adjunct Professor, Department of Physics. R. J. C. McLean, BSc, PhD, Assistant Professor, Department of Urology, and Department of Microbiology and Immunology.
Requests for reprints to: R. J. C. McLean, Department of Microbiology and Immunology, Queen’s University, Kingston, Ontario, Canada K7L 3N6.
