International Journal of Urology (2001) 8, 124–127
Original Article
Inhibition of calcium oxalate precipitation by bile salts LUCIANO SASO, ELEONORA GRIPPA, MARIA TERESA GATTO AND BRUNO SILVESTRINI Department of Pharmacology of Natural Substances and General Physiology, University of Rome, Rome, Italy Abstract
Background: Both urinary and biliary stones can contain calcium. Bile salts (BA), which are known to bind Ca2+, are commonly used to dissolve the latter but not the former. Methods: The effect of physiologic BA on calcium oxalate (CaOx) precipitation was evaluated by a recently developed method. Results: The Ca2+ binding properties of BA were confirmed by small but significant decreases in pH observed following addition of CaCl2 to bile acids solutions. More importantly, BA inhibited CaOx precipitation with effective concentrations of approximately 10-3 mol/L. The clinical relevance of the latter observation is presently unknown but it is of note that in the same in vitro assay, the activity of BA appeared comparable to that of citric acid, the most common drug for urolithiasis. Although BA do not reach mmol/L levels in urine, they are known to change the physicochemical properties of this fluid, possibly slowing down the crystal growth process. However, the hypothetical therapeutic use of BA in former stone patients would present at least two major problems: (i) hepatotoxicity and (ii) lithogenic activity, due to hyperoxaluria subsequent to increased intestinal absorption of oxalate. Conclusion: The ability of BA in effectively binding calcium ions and in inhibiting the precipitation of CaOx appears of interest from both a physiopathologic and a pharmacologic point of view, even if it does not currently seem exploitable for prophylactic or therapeutic purposes.
Key words
bile salts/acids, calcium oxalate, nephrolithiasis, stone inhibitors/promoters.
Introduction It is well known that both urinary and biliary stones can contain calcium: the former usually consist of calcium oxalate (CaOx) or phosphate,1 while the latter, which are composed principally of cholesterol monohydrate crystals (cholesterol stones) or the acid salt of calcium bilirubinate (pigment stones),2 often contain nuclei of calcium palmitate, carbonate, or phosphate.2,3 Very rarely, calcium oxalate-phosphate gallstones form.4 The mechanism responsible for the formation of urinary and biliary calcium-containing stones, besides the apparent chemical differences, is somewhat simiCorrespondence: Dr Luciano Saso, Department of Pharmacology of Natural Substances and General Physiology, University of Rome, ‘La Sapienza’, P.le A. Moro 5, 00185 Rome, Italy. Email:
[email protected] Received 10 February 2000; revision 28 June 2000; accepted 8 September 2000.
lar; in both cases, several endogenous substances are known to act as inhibitors5,6 or promoters2,7 by affecting one of the crystallization steps (i.e. nucleation, growth, aggregation and adhesion)8,9 often by interacting with calcium.10–12 Bile salts (BA) supplementation has been used for many years to dissolve gallstones, based on the rationale that these substances are able to make cholesterol soluble,13 without taking into account their calciumbinding properties14 and their important physiologic role in the prevention of calcium-containing gallstones.15 Thus, it is not surprising that BA, with a few exceptions (Geller et al., who described the capability of bile salts to affect the equilibrium solubility of calcium oxalate [CaOx]16 and Rodriguez Rebollo et al., who described the presence of bile pigments in urinary calculi17), have not been studied in relation to calcium nephrolithiasis. Therefore, we decided to evaluate the effect of physiologic BA, i.e. cholate (C), deoxycholate (DC), chenodeoxycholate (CDC) and their conjugates with
Bile salts and calcium stones
glycine (G) and taurine (T), on CaOx precipitation, using an in vitro assay that was recently developed in this laboratory.18
Methods Reagents
Cholate (C), deoxycholate (DC), chenodeoxycholate (CDC), lithocholate (LC) and their glyco-(G) and tauro-(T) conjugates, sodium chloride (NaCl), calcium chloride (CaCl2), sodium oxalate (CaOx), hydrochloric acid (HCl), sodium hydroxide (NaOH) were obtained from Sigma (St Louis, MO, USA). Binding of bile acids to calcium ions
The bile acids C, DC, CDC and LC were dissolved at the concentration of 10-3 mol/L in 0.15 mol/L NaCl and the pH was adjusted at 7 (at 22°C). Then, to 50 mL of these solutions, 0.5 mL of 1 mol/L CaCl2 was added and the pH variations (%) were measured. Effect of pH on the solubility of bile salts in the presence or absence of calcium ions
Bile acids were dissolved at the concentration of 10-3 mol/L in 0.15 mol/L NaCl, the solutions were divided into several 10 mL aliquots and 100 mL of 0.15 mol/L NaCl, containing or not 1 mol/L CaCl2, was added. Then, the pH of two different samples, with and without Ca2+, were adjusted at the same value in the range 2–12, using small volumes of concentrated HCl or NaOH. Samples were incubated at 22°C for 10 min, aliquots of 100 mL were pipetted in duplicate onto 96well microplates (Falcon 3911, Microtest III flexible plates; Becton–Dickinson, Oxnard, CA, USA) and the absorbance of the wells was measured at 595 nm using a microplate reader (model 3550; Bio-Rad, Hercules, CA, USA).
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0.01 mol/L sodium oxalate (pH 6.0) was added and the plate was incubated for 15 min at 22°C. Finally, the absorbance of each solution was determined at 595 nm as described and the inhibitory activity was calculated with the formula a = 1–AX/A0, where AX is the absorbance of the CaOx solution in the presence of the substance X and A0 is the absorbance of the CaOx solution without it. Each activity versus concentration curve was analyzed statistically according to Tallarida and Murray19 to determine the effective concentration 50 (EC50).
Results When Ca2+ was added to bile acids (10-3 mol/L in 0.15 mol/L NaCl, pH 7 at 22°C) to a ratio 10 : 1, small but significant decreases in pH were observed (Fig. 1). When calcium ions were added to BA (10-3 mol/L in 0.15 mol/L NaCl) to a ratio 10 : 1 and the pH was adjusted at different values in the range 2–12, no significant (DC), small (C and CDC) or marked (LC) differences in the turbidity of the solutions were observed (Fig. 2). For C, DC and CDC, a sudden change of turbidity was observed below pH 6. The effect of Ca2+ on the solubility of LC (10-3 mol/L), which yielded turbid solutions at all pH values, was pH dependent; in particular, outside the pH range of 4–8, a marked increase of turbidity was observed (Fig. 2). Bile salts inhibited CaOx precipitation with effective concentrations (EC50) of approximately 10-3 mol/L (Fig. 3).
Effect of bile salts on calcium oxalate precipitation
The effect of C, DC, CDC and their conjugates on CaOx precipitation was evaluated by an in vitro assay recently developed in this laboratory:18 each BA was dissolved at its maximum solubility in 0.01 mol/L CaCl2 (pH 6.0 at 22°C) and serially diluted with the same solution. Then, aliquots of 50 mL were loaded onto a 96-well microplate, an equal volume of
Bile Salts Fig. 1 Binding of bile acids to calcium ions. The pH variations of cholate (C), deoxycholate (DC), chenodeoxycholate (CDC) and lithocholate (LC).
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L Saso et al. a 1.0
Discussion
0.8 0.6 0.4 0.2
Absorbance A595 nm
0.0 b 1.0
c
0.8 0.6 0.4 0.2 0.0 d 1.0
e
0.8 0.6 0.4 0.2 0.0 2
4
6
8 10 12
2
4
6
8 10 12
pH
Effective concentration 50 (mol/L)
Fig. 2 Effect of pH on the solubility of bile salts in the presence or absence of calcium ions. 10-2 mol/L Ca2+; 10-3 mol/L bile salt; 10-3 mol/L bile salt plus 10-2 mol/L Ca2+. CTRL, control; C, cholate; DC, deoxycholate; CDC, chenodeoxycholate; LC lithocholate.
Bile salts
Fig. 3 Effect of () bile salts cholate (C), deoxycholate (DC), chenodeoxycholate (CDC) and their glycoand tauro-conjugates on calcium oxalate precipitation.
Bile salts effectively bind calcium ions (Fig. 1), a phenomenon that affects the solubility of selected BA at specific pH values (Fig. 2) and these physiologic substances can inhibit the precipitation of calcium oxalate (CaOx), the most common component of urinary stones. The small but significant decreases in pH observed following addition of CaCl2 to solutions containing bile acids (Fig. 1) indicated the binding of calcium ions to predominantly undissociated BA20 and subsequent release of H+. By this method, which is extremely simple and applicable in all laboratories, we confirmed the well known stronger affinity for Ca2+ of deoxycholate (DC), chenodeoxycholate (CDC) and lithocholate (LC) compared to cholate (C).14 In Fig. 2, the sudden change of turbidity observed for C, DC and CDC at pH 6, was due to the precipitation of these substances at pH £ pKa, as reported by Beher.20 As expected,21 at the concentration examined (10-3 mol/L), LC yielded turbid solutions at all pH values (Fig. 2). In addition, we noted that Ca2+ caused a marked precipitation of the Ca2+-LC complexes outside the pH range of 4–8, which is the normal range of variation of the human urinary pH.22 Bile salts inhibited CaOx precipitation with effective concentrations (EC50) of approximately 10-3 mol/L (Fig. 3); their mechanisms of action is unknown but probably did not involve only calcium binding,14,23 and we speculate that poisoning of the crystallization process played a role, as previously reported for calcium phosphate.24 The clinical relevance of this phenomenon is presently obscure but the activity of BA appeared comparable to that of citric acid (EC50 = 3.9 ¥ 10-3 mol/L),18 the most common drug against urolithiasis.25 Although BA do not reach mmol/L levels in urine (the normal level, which is < 10 mm,26,27 increases less than 100-fold in patients with hepatobiliary diseases27,28 or supplemented with oral BA29), they are known to change the physicochemical properties of this fluid,30 possibly slowing down the crystal growth process. However, the hypothetical therapeutic use of BA in stone-forming patients would present at least two major problems: these substances are known to be (i) hepatotoxic when used at high doses (which would be necessary to yield high urinary concentrations) for prolonged periods of time;31 and (ii) lithogenic, like other calcium sequestrant agents which cause hyperoxaluria by increasing intestinal absorption of oxalate,32 one of the major risk factors for calcium urolithiasis.8
Bile salts and calcium stones
In conclusion, we found that BA inhibit calcium oxalate precipitation in vitro, a property that appears of interest from both a physiopathologic and pharmacologic point of view even if does not seem currently exploitable for prophylactic or therapeutic purposes.
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