二水草酸钙结石碎片愈合的影响因素

BMC Urology

Research article

Bio Med Central

Open Access

Factors affecting calcium oxalate dihydrate fragmented calculi

regrowth

A Costa-Bauzá, JPerelló, BIsern, PSanchis and FGrases*

Address: Laboratory of Renal Lithiasis Research, University Institute of Health Sciences Research (IUNICS), University of Balearic Islands, 07122 Palma of Mallorca, Spain

Email: ACosta-Bauzá-dqufgfo@ps.uib.es; JPerelló-joan.perello@uib.es; BIsern -bernat.isern@uib.es; PSanchis -vdqupsc4@uib.es; F Grases*-fgrases@uib.es* Corresponding author

Published: 05 July 2006BMC Urology 2006, 6:16

doi:10.1186/1471-2490-6-16

This article is available from: http://www.biomedcentral.com/1471-2490/6/16

Received: 16 February 2006Accepted: 05 July 2006

2006 Costa-Bauzá et al; licensee BioMed Central Ltd.

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background: The use of extracorporeal shock wave lithotripsy (ESWL) to treat calcium oxalatedihydrate (COD) renal calculi gives excellent fragmentation results. However, the retention ofpost-ESWL fragments within the kidney remains an important health problem. This study examinedthe effect of various urinary conditions and crystallization inhibitors on the regrowth ofspontaneously-passed post-ESWL COD calculi fragments.

Methods: Post-ESWL COD calculi fragments were incubated in chambers containing syntheticurine varying in pH and calcium concentration: pH = 5.5 normocalciuria (3.75 mM), pH = 5.5hypercalciuria (6.25 mM), pH = 6.5 normocalciuria (3.75 mM) or pH = 6.5 hypercalciuria (6.25 mM).Fragment growth was evaluated by measuring increases in weight. Fragment growth wasstandardized by calculating the relative mass increase.

Results: Calcium oxalate monohydrate (COM) crystals formed on COD renal calculi fragmentsunder all conditions. Under pH = 5.5 normocalciuria conditions, only COM crystals formed(growth rate = 0.22 ± 0.04 µg/mg·h). Under pH = 5.5 hypercalciuria and under pH = 6.5normocalciuria conditions, COM crystals and a small number of new COD crystals formed (growthrate = 0.32 ± 0.03 µg/mg·h and 0.35 ± 0.05 µg/mg·h, respectively). Under pH = 6.5 hypercalciuriaconditions, large amounts of COD, COM, hydroxyapatite and brushite crystals formed (growthrate = 3.87 ± 0. 34 µg/mg·h). A study of three crystallization inhibitors demonstrated that phytatecompletely inhibited fragment growth (2.27 µM at pH = 5.5 and 4.55 µM at pH = 6.5, both underhypercalciuria conditions), while 69.0 µM pyrophosphate caused an 87% reduction in mass underpH = 6.5 hypercalciuria conditions. In contrast, 5.29 mM citrate did not inhibit fragment massincrease under pH = 6.5 hypercalciuria conditions.

Conclusion: The growth rate of COD calculi fragments under pH = 6.5 hypercalciuria conditionswas approximately ten times that observed under the other three conditions. This observationsuggests COD calculi residual fragments in the kidneys together with hypercalciuria and highurinary pH values may be a risk factor for stone growth. The study also showed the effectivenessof specific crystallization inhibitors in slowing calculi fragment growth.

Background

Calcium oxalate dihydrate renal calculi constitute themost prevalent and recurrent type of renal lithiasis [1,2].They are usually associated with hypercalciuria, and onoccasions with urinary pH values above 6.0 [3-7]. The useof extracorporeal shock wave lithotripsy (ESWL) to treatthese renal calculi commonly gives excellent fragmenta-tion results due to their fragility [8]. Nevertheless, theretention of post-ESWL fragments within the kidney is animportant health problem, and a study of calcium stonepatients found only 32% were stone-free 12 months afterESWL [9]. It appears that persistence and growth of frag-ments is common following ESWL [10-14]. In vitro [15-17] and in vivo [9] studies suggest that citrate [9,15,16]and phytate [17] can reduce residual post-ESWL calculifragment growth or agglomeration. Despite those find-ings, however, there is a need for better understanding ofthe factors that contribute to stone growth followingESWL. Such knowledge will assist in designing methodsfor preventing such growth.

The present study belongs to a series examining theregrowth of residual post-ESWL calculi fragments in termsof calculi type, urinary conditions and presence of crystal-lization inhibitors. While a previous study examinedregrowth of calcium oxalate monohydrate (COM) resid-ual post-ESWL calculi fragments [17], the present studyexamined calcium oxalate dihydrate (COD) calculi frag-ments.

Methods

The study used 48 spontaneously-passed post-ESWL frag-ments of COD calculi collected on the day of the ESWLprocedure. Fragment selection proceeded according to thegeneral protocol applied by our laboratory in the study ofall renal stones. This methodology is based on a combina-tion of optical stereomicroscopy, infrared spectrometryand scanning electron microscopy (SEM) equipped withan energy dispersive X-ray analyzer (EDS) [18]. Allselected fragments had a very similar morphology whichwas representative of that observed in the majority ofspontaneously-passed post-ESWL COD calculi fragments.Fragment sizes varied from 2 to 4 mm.

Fragments were not pre-treated, and were placed into fourhermetic flow chambers (3 cm diameter and 4 cm high),with each chamber containing 12 fragments. These cham-bers were then placed into a larger temperature-controlled(37°C) chamber. Each chamber was used to test a differ-ent incubation condition: pH = 5.5 and normocalciuria([Ca total] = 3.75 mM), pH = 5.5 and hypercalciuria ([Catotal] = 6.25 mM), pH = 6.5 and normocalciuria ([Catotal] = 3.75 mM) and pH = 6.5 and hypercalciuria ([Catotal] = 6.25 mM). The duration of all incubations was192 h, except for those under pH = 6.5 hypercalciuric con-

Figure 1Diagram of the experimental flow device used for COD cal-culi crystallization studies. 1. Temperature-controlled cham-ber; 2. Flask containing post-ESWL calculi fragments; 3.

Three-way T mixing chamber for A and B solutions; 4. A and

B solutions for artificial urine; 5. Peristaltic pump.

ditions, which were for 48 h due to the high rate of frag-ment mass increase. The methodology used was similar tothat previously described by Chow et al. [16,19]. Freshlyprepared synthetic urine was introduced into the flowchambers using a multichannel peristaltic pump at a rateof 750 mL/day through the bottom of the flasks. The cal-culi fragments were placed on the porous flask bottom,allowing the entire fragment surface to be exposed to theartificial urine (see Figure 1). This system allows growth ofnew crystals on the fragments. Fragment growth was eval-uated by measuring the difference in weight between thedried fragments before and after the experiment. Theweight of the fragments was measured using a precisionbalance after the fragments had been kept in a desiccatoruntil their weight became constant, indicating completedryness. The mean growth rate of the 12 fragments undereach condition was calculated. The growth of the differentrenal calculi fragments was standardized by calculatingthe relative mass increase in order to avoid the effects thatdifferent surface areas may have on growth rate measure-ments.

Table 1: Composition of synthetic urine

Solution A (mM)Solution B (mM)Na 2SO 4·10H2O 19.34NaH 2PO 4·2H2O 15.45MgSO 4·7H2O 5.93Na 2HPO 4·12H2O

15.64NH 4Cl 86.73NaCl 223.08KCl

162.60

Na 2C 2O 4

0.57

Various volumes of a 1 M calcium solution (prepared by dissolving calcium carbonate with hydrochloric acid) were added to solution A to obtain final calcium concentrations ranging from 3.75–6.25 mM.

The effects of the crystallization inhibitors phytate, pyro-phosphate and citrate were evaluated. The concentrationsused corresponded to the physiological concentrations inurine.

The experimental research has been performed with theapproval of the Bioethics Committee of the University ofBalearic Islands and the research was carried out in com-pliance with the Helsinki Declaration.

Synthetic urine

Synthetic urine supersaturated with calcium oxalate wasprepared using a three-way T mixing chamber containingequal volumes of solutions A and B (compositions shownin Table 1). The pH of both solutions was adjusted eitherto 5.5 or 6.5. Solutions were stored for a maximum of 1week at 4°C. Chemicals of reagent-grade purity were dis-solved in deionized and redistilled water. All solutionswere filtered through a 0.45 µm pore filter before use.Effect of crystal inhibitors

Compounds reported to inhibit crystal formation wereadded to the synthetic urine to the following final concen-trations: 1.32–5.29 mM citrate as a sodium salt (suppliedby Probus), 0.15–4.55 µM phytate as a sodium salt (sup-plied by Sigma), and 11.5–69.0 µM pyrophosphate as asodium salt (supplied by Merck).

Calcium-citrate complexation

Owing to the high concentration of citrate used, and con-sidering its ability to complex with calcium ions, a cal-cium supplement was used in experiments involvingcitrate ions in order to achieve the same calcium oxalatesupersaturation value as occurs in the absence of citrate. Itmust be noted that a decrease in supersaturation wouldimply a decrease in the crystallization rate that could notbe assigned to inhibitory effects. The amount of calciumions added was potentiometrically calculated using a cal-cium-selective electrode (Ingold) and a potentiometer(Crison 2002). Calcium standards in the presence andabsence of citrate were prepared using synthetic urine as amatrix. The activity of free calcium ions must be the samein the presence and absence of citrate. Therefore, the cal-cium concentration was increased by 0.15 mM per 0.53

Figure 2COM crystal formation on a post-ESWL COD renal calculus fragment following a 192 h incubation in normocalciuric (3.75 mM) and normooxaluric (0.28 mM) synthetic urine (pH =

5.5).

mM increase in the citrate concentration. The levels ofphytate and pyrophosphate used were so low that thedecrease in free calcium concentration was negligible, asdetermined potentiometrically. Consequently, it was notnecessary to add a calcium supplement to the solutionscontaining phytate and pyrophosphate.

Results

Under all four incubation conditions, COM crystals werefound to form on COD calculi fragments (Figures 2, 3, 4,5). New COD crystal formation represented the minority(less than 50%) of crystal formation under pH = 5.5hypercalciuric (Figure 3) and pH = 6.5 normocalciuric

Figure 3COM and COD crystal formation on post-ESWL COD renal calculi fragments following a 192 h incubation in hypercalciu-ric (6.25 mM) and normooxaluric (0.28 mM) synthetic urine

(pH = 5.5).

Figure 4COM, COD and HAP crystal formation (arrows) on post-ESWL COD renal calculi fragments following a 192 h incuba-tion in normocalciuric (3.75 mM) and normooxaluric (0.28

mM) synthetic urine (pH = 6.5).

(Figure 4) conditions. In contrast, large amounts of CODcrystals formed under pH = 6.5 hypercalciuric conditions(Fig 5a,c). Significant amounts of hydroxyapatite (HAP)and brushite (BRU) crystals formed under pH = 6.5 hyper-calciuria conditions (Figure 5a,b), but not under otherconditions.

The mean growth rates of COD calculi fragments underthe four incubation conditions are summarized in Table2. While growth rates of 0.22–0.35 µg/mg·h wereobserved under the majority of conditions, the rate wasapproximately ten times greater (3.87 ± 0.43 µg/mg·h)under pH = 6.5 hypercalciuria conditions.

The effects of three known crystallization inhibitors(phytate, pyrophosphate and citrate) were investigated(Figures 6 and 7). We found that addition of 2.27 µM phytate to the pH = 5.5 hypercalciuria incubation, and theaddition of 4.55 µM phytate to the pH = 6.5 hypercalciu-ria incubation completely inhibited the COD calculi frag-ment mass increase. Addition of lower phytateconcentrations (less than 2.27 µM), mainly inhibitedCOM crystal formation. The addition of 69.0 µM pyro-phosphate to the pH = 6.5 hypercalciuria incubationresulted in an 87% reduction of calculi fragment massincrease. The addition of 5.29 mM citrate to the pH = 6.5hypercalciuria incubation had no effect on the COD cal-culi fragment mass increase.

Discussion

The present study found that in normocalciuric/nor-mooxaluric urine at pH = 5.5, only new COM crystalsformed on COD calculi fragments. At the same pH, newCOD crystals only formed under hypercalciuric condi-

Figure 5a) HAP and BRU crystal, b) COM and HAP crystal and c) COD and HAP crystal formation on post-ESWL COD renal calculi fragments following a 48 h incubation in hypercalciuric (6.25 mM) and normooxaluric (0.28 mM) synthetic urine (pH

= 6.5).

tions, in addition to COM crystals (Figure 3). Low phytateconcentrations (less than 2.27 µM), inhibited this COMcrystal formation. At pH = 6.5, COD crystals formedunder normocalciuric conditions (Figure 4), while underhypercalciuria conditions the calcium phosphates HAPand BRU also formed with the COD crystals. These find-ings are consistent with several clinical observations

Figure 6Increase in the relative weight of post-ESWL COD renal cal-culi fragments incubated for 196 h in normooxaluric (0.28 mM) synthetic urine at pH = 5.5 in the absence or presence of phytate. Values represent mean ± SEM for12 fragments. a . Normocalciuric urine ([Ca total] = 3.75 mM). b . Hypercalciu-

ric urine ([Ca total] = 6.25 mM).

showing that COM calculi are generally associated with alack of crystallization inhibitors and COD calculi withhypercalciuria and high urinary pH values [3-6]. It isimportant to emphasize that the growth rate of COD cal-culi fragments was similar under all conditions exceptunder pH = 6.5 hypercalciuria, conditions under whichthe growth rate was approximately ten times that observedunder other conditions (see Table 2). These findings sug-gest that the presence of COD calculi fragments in the kid-neys together with hypercalciuria and high urinary pHvalues (around 6.5) are a high risk factor for stone devel-opment. These data suggest that a COD calculus fragmentof only 5 mg could become a 45 mg fragment in just 3months under these conditions.

Crystallization inhibitors can notably retard calculi frag-ment development. The present study found that phytateat concentrations found in normal human urine [20]totally prevented COD calculi fragment growth. In addi-

Table 2: Mean growth rates (relative mass increase, µg/mg·h) of post-ESWL COD renal calculi fragments under different incubation conditions. All incubations contained 0.28 mM oxalate. Results are expressed as mean ± SEM (n = 12).

pH = 5.5

pH = 6.5

[Ca total] = [Ca total] = [Ca total] = [Ca total] = 3.75 mM6.25 mM3.75 mM6.25 mM0.22 ± 0.04*

0.32 ± 0.03*

0.35 ± 0.05*

3.87 ± 0.43

* p

tion, pyrophosphate also reduced the COD calculi frag-ment mass increase. In contrast, citrate did not show anysignificant crystallization inhibitory capacity under theconditions investigated. This finding appears to be in dis-agreement with previous reports showing that high citrateconcentrations reduced the growth rate of stones by morethan 50% [16]. However, this apparent discordance maybe because the stated growth rate reduction must beassigned to citrate complexation with calcium, whichresults in an important reduction in the relative calciumoxalate supersaturation. Thus, it must be considered thatin the present study a calcium supplement was added toobtain the same calcium oxalate supersaturation valuefound in the absence of citrate. Interestingly, while citratedid not inhibit calcium salt crystallization on COD crys-tals in the present study, it was shown to inhibit calciumsalt crystallization on glycoproteins and organic matter[21,22]. These observations again highlight the specificityof crystallization inhibitors.

Conclusion

The growth rate of COD calculi fragments under pH = 6.5hypercalciuria conditions was approximately ten timesthat observed under other conditions. This observationsuggests COD calculi residual fragments in the kidneystogether with hypercalciuria and high urinary pH valuesmay represent an important risk factor for stone develop-ment. In addition, the study found that specific crystalli-zation inhibitors can effectively reduce calculi fragmentgrowth.

Competing interests

The author(s) declare that they have no competing inter-ests.

Authors' contributions

AC conceived the study and participated in the search andselection of COD calculi fragments. JP performed the crys-tallization studies under hypercalciuria conditions. BI per-formed the crystallization studies under normocalciuriaconditions and pH = 5.5. PS performed the crystallizationstudies under normocalciuria conditions and pH = 6.5.FG participated in the study design and coordination, and

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Figure 7Increase in the relative weight of post-ESWL COD renal calculi fragments following incubation in normooxaluric (0.28 mM) synthetic urine at pH = 6.5. Values represent the mean ± SEM for 12 fragments. a . Incubation in normocalciuric urine ([Ca total] = 3.75 mM) for 192 hours in the absence or presence of phytate. b . Incubation in hypercalciuric urine ([Ca total] = 6.25 mM) for 48 hours in the absence or presence of phytate. c . Incubation in hypercalciuric urine ([Ca total] = 6.25 mM) for 48 hours in the absence or presence of pyrophosphate. d . Incubation in hypercalciuric urine ([Ca total] = 6.25 mM) for 48 hours

in the absence or presence of citrate.

assisted in preparation of the manuscript. All authors readand approved the final manuscript.

Acknowledgements

P.S. is grateful to the Spanish Ministry of Education, Culture and Sport for an FPU program fellowship. B.I. is grateful to the Conselleria d'Innovació i Energia del Govern de les Illes Balears for a fellowship. Financial support from the Conselleria d'Innovació i Energia, Govern Balear (Grant PROIB-2002GC1-04) and from the Spanish Ministry of Science and Technology (project BQU 2003-01659) is gratefully acknowledged.

5. 6. 7. 8. 9.

References

1. 2. 3.

Krepinsky J, Ingram AJ, Churchill DN: Metabolic investigation ofrecurrent nephrolithiasis: compliance with recommenda-tions. Urology 2000, 56:915-920.

Grases F, Costa-Bauzá A, Ramis M, Montesinos V, Conte A: Recur-rence of renal lithiasis. Scand J Urol Nephrol 2003, 37:482-486. Daudon M, Reveillaud RJ: Whewellite and weddellite: toward adifferent etiopathogenesis. The significance of morphologi-cal typing of calculi. Nephrologie 1984, 5:195-201.

10.

Galan JA, Conte A, Llobera A, Costa-Bauzá A, Grases F: A compar-ative study between etiological factors of calcium oxalatemonohydrate and calcium oxalate dihydrate urolithiasis.Urol Int 1996, 56:79-85.

Asplin JR, Lingeman J, Kahnoski R, Mardis H, Parks JH, Coe FL: Met-abolic urinary correlates of calcium oxalate dihydrate inrenal stones. J Urol 1998, 159:664-668.

Parent X, Boess G, Brignon P: Calcium oxalate lithiasis. Rela-tionship between biochemical risk factors and crystallinephase of the stone. Prog Urol 1999, 9:1051-1056.

Grases F, Costa-Bauzá A, Ramis M, Monstesinos V, Conte A: Simple classification of renal calculi closely related to their micro-morphology and etiology. Clin Chim Acta 2002, 22:29-36.

Cecchetti W, Tasca A, Zattoni F, Villi G, Levorato CA, Pagano F: Five years experience in experimental laser lithotripsy. Eur Urol1993, 24:185-189.

Cicerello E, Merlo F, Gambaro G, Maccatrozzo L, Fandella A, BaggioB, Anselmo G: Effect of alkaline citrate therapy on clearanceof residual renal stone fragments after extracorporeal shockwave lithotripsy in sterile calcium and infection nephrolithi-asis patients. J Urol 1994, 151:5-9.

Delvecchio FC, Preminger GM: Management of residual stones.Urol Clin North Am 2000, 27:347-354.

BMC Urology 2006, 6:1611.

Zanetti G, Montanari E, Mandressi A, Guarneri A, Ceresoli A, MazzaL, Trinchieri , Pisani E, Mazza L: poreal shockwave lithotripsy in renal stone treatment.Long-term results of extracor-12. Endourol J Zanetti G, Seveso M, Montanari E, Guarneri A, Del Nero A, Nespoli 1988, 2:163-171.

R, Trinchieri A: 13. lithotripsy. Yu CC, Lee YH, Huang JK, Chen MT, Chen KK, Lin AT, Chang LS: J UrolRenal stone fragments following shock wave 1997, 158:352-355.

Long-term stone regrowth and recurrence rates after extra-14.

corporeal shock wave lithotripsy.Carr LK, D'A Honey J, Jewett MA, Ibanez D, Ryan M, Bombardier C: Br J Urol 1993, 72:688-691. New stone formation: a comparison of extracorporeal shockwave lithotripsy and percutaneous nephrolithotomy.15.

1996, Suzuki K, Tsugawa R, Ryall RL: 155:1565-1567.

J Urolcitrate (CG-120) of calcium oxalate crystal growth on to kid-Inhibition by sodium-potassiumney stone fragments obtained from extracorporeal shock16. wave lithotripsy.Chow K, Dixon J, Gilpin S, Kavanagh JP, Rao PN: Br J Urol 1991, 68:132-137.

growth of residual fragments in an in vitro model of calciumCitrate inhibitst

17. oxalate renal stones.Costa-Bauzá A, Perelló J, Isern B, Grases F: Kidney Int 2004, 65:1724-1730.

on residual lithiasis after shock wave lithotripsy.An experimental study18. 33: Urol Res 2005,Grases F, Garcia-Ferragut L, Costa-Bauzá A: 51-56.

renal calculi. A new insight.19.

Chem Chow K, Dixon J, Gilpin S, Kavanagh JP, Rao PN: 1998, 1:187-206.

Recent Res Devel in Pure & Applied AnalAnalytical study ofdevelopment of a method for simultaneous production ofA stone farm:multiple calcium oxalate stones in vitro.20.

32: Urol Res 2004,Grases F, Simonet BM, Vucenik I, Prieto RM, Costa-Bauzá A, March55-60.

JG, Shamsuddin AM: istered inositol hexaphosphate (IP(6) or phytate) in humans.Absorption and excretion of orally admin-21. BioFactors Grases F, Isern B, Perelló J, Costa-Bauzá A: 2001, 15:53-61.

teins in calcium oxalate crystal development.The role of glycopro-22.

94: BJU Int 2004,Grases F, Isern B, Perelló J, Costa-Bauzá A: 177-181.

matter in calcium oxalate lithiasis.10:1534-1538.

Role of the organic Fron Biosci 2005,Pre-publication history

The pre-publication history for this paper can be accessedhere:

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