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Possible causes for the low prevalence of pediatric urolithiasis
BASIC SCIENCE CME ARTICLE
POSSIBLE CAUSES FOR THE LOW PREVALENCE OF PEDIATRIC UROLITHIASIS OSAMU MIYAKE, KAZUHIRO YOSHIMURA, MASAO TSUJIHATA, TOSHIAKI YOSHIOKA, TAKUO KOIDE, SHIRO TAKAHARA, AND AKIHIKO OKUYAMA
ABSTRACT Objectives. To determine why the incidence of pediatric urolithiasis is less than that of adult urolithiasis, we investigated the difference in inhibition of calcium oxalate (CaOX) crystallization between pediatric and adult urinary macromolecules (UMMs). Methods. Urinary parameters in relation to urolithiasis, the inhibition of CaOX crystallization of original urine and urine from which UMMs (greater than 3 kDa) had been removed, and the inhibition of CaOX crystal growth and aggregation of UMMs alone were measured. These inhibitory activities were compared between children and adults. Results. In the original urine, the inhibition of CaOX crystallization was significantly stronger for children than for adults, but was the same in urine from which the UMMs had been removed. The inhibition of CaOX crystal growth by UMMs alone showed no significant differences between children and adults; their inhibition of CaOX crystal aggregation was significantly stronger for children than for adults. Much more glycosaminoglycan (GAG) was included in pediatric UMMs than in adult UMMs, although there was no difference in UMM concentration between urine from children and urine from adults. Conclusions. The lower incidence of CaOX lithiasis in children may be attributed, among other factors, to the stronger inhibition of CaOX crystal aggregation by pediatric UMMs, which in turn might be affected by the higher concentration of GAGs in children’s urine. UROLOGY 53: 1229–1234, 1999. © 1999, Elsevier Science Inc. All rights reserved.
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any in vitro examinations have been performed to investigate the causes of urolithiasis. In the reports of these studies, the function of various low molecular weight substances (LMWSs) and macromolecules as stone inhibitors or promoters has been discussed, but these substances were almost always extracted from adult urine only and discussed in the context of adult urolithiasis.1– 6 As Ryall7 has pointed out, very few of these studies have examined the roles of urinary macromolecules (UMMs) in pediatric stone disease. The reason for this is that there are few children with idiopathic stone disease, except for patients who have an apparently congenital metabolic disease. In fact, the epidemiologic prevalence
of pediatric urolithiasis is very low compared with that of adult urolithiasis.8,9 Many urologists have wondered why this is so, but hardly any in vitro experiments on pediatric urolithiasis have been conducted at the crystallographic level. We have previously reported that the excretion volume of citrate and magnesium in urine from children is significantly higher than that in urine from adults, and that this difference may be of crucial importance when discussing the reduced incidence of pediatric urolithiasis.10 In the present study, we focused our attention on UMMs and compared their inhibition of calcium oxalate (CaOX) crystallization between children and adults.
From the Department of Urology, Osaka University Medical School; Department of Urology, Sumitomo Hospital; and Department of Urology, Osaka Kosei-nenkin Hospital, Osaka, Japan Reprint requests: Osamu Miyake, M.D., Department of Urology, Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka 565-0871 Japan Submitted: October 12, 1998, accepted (with revisions): December 9, 1998
MATERIAL AND METHODS
© 1999, ELSEVIER SCIENCE INC. ALL RIGHTS RESERVED
We collected urine from 19 boys aged 3 to 8 years (average 5.9) and 18 men aged 25 to 37 years (average 30.7). None had any congenital metabolic disorders or any episodes of urolithiasis. We collected their 24-hour urine samples together with 0.1 mL of 30% sodium azide. The children’s 24-hour urine samples were collected twice or three times and com0090-4295/99/$20.00 PII S0090-4295(99)00004-7 1229
TABLE I. Urinary parameters of 24-hour urine Children Average Urine volume (mL) Body weight (kg) pH Magnesium (mg/dL) Citrate (mg/dL) Uric acid (mg/dL) Calcium (mg/dL) Oxalate (mg/dL) Phosphorus (mg/dL) Creatinine (mg/dL) GAGs (mg/dL) Proteins (mg/dL)
549.7 19.8 6.42 9.90 42.3 54.5 11.6 30.4 92.3 71.4 10.2 4.94
Adults SD
Average
229.2 3.55 0.360 2.96 14.2 13.9 4.92 10.4 27.1 22.9 5.85 2.44
1342 71.3 6.15 5.66 30.4 52.2 17.6 26.3 73.0 134.2 5.06 4.45
SD 390.2 9.25 0.540 1.74 17.1 15.5 14.8 15.3 23.8 50.9 4.70 1.33
P Value
NS* ,0.001† 0.027† NS* 0.0334† NS* 0.0277* ,0.001† 0.0061* NS*
KEY: GAGs 5 glycosaminoglycans; NS 5 not statistically significant. * Unpaired t test. † Mann-Whitney U test.
bined to obtain a sufficient volume of UMMs. The urine volume, pH, magnesium, citrate, uric acid, calcium, oxalate, phosphorus, creatinine, glycosaminoglycans (GAGs), and proteins of all samples were measured. Proteins were measured with a Bio Rad protein assay and GAGs with a modified DMB assay.11
hours (S4). The count at 0 and 4 hours in the control tube (without UMMs) was adopted as C0 and C4. The percentage of growth inhibition (Ig) was then calculated as Ig 5 100 3 [(C4 2 C0) 2 (S4 2 S0)]/(C4 2 C0).
AGGREGATION INHIBITION ASSAY OF UMMS PREPARATION OF UMMS All 24-hour urine samples were filtered through a Whatman No. 1 paper and a 0.22-mm Millipore filter. A 150-mL aliquot was then separated from the samples and used for the crystallization inhibition assay in a whole urine system; the rest was ultrafiltered with a Labomodule (Asahi Kasei, Tokyo, Japan; threshold molecular weight 3 kDa). The filtered samples were divided into urine containing only LMWSs (ULMWSs) and urine containing only macromolecules. The latter was also desalinated with distilled water in a Spectra/Por-1 membrane tube (Spectrum Medical Industries, Houston, Tex), lyophilized, and stored at 220°C until use.
CRYSTALLIZATION INHIBITION ASSAY IN A WHOLE URINE SYSTEM The method reported by Tawashi et al.12 was used for the inhibition assay in a whole urine system. A quarter milliliter of calcium chloride (1 M) was added to 50 mL of filtered original urine or ULMWSs. The pH was adjusted to 5.7, and 2.5 mL of sodium oxalate (5 mM) was added to each of the samples, which were kept at 37°C without shaking. After 3 hours, the particle size of the CaOX crystals was determined with a Coulter Multisizer.
GROWTH INHIBITION ASSAY OF UMMS IN AN ARTIFICIAL URINE SYSTEM The inhibition rate of CaOX crystal growth of the UMMs was also measured with a seed crystal method reported by Robertson et al.,13 with minor modifications. In brief, we produced 10 mL of a metastable solution on CaOX with 14Clabeled oxalate mixed with UMMs at final concentrations of 0.001, 0.01, 0.05, 0.1, 0.2, 0.4, and 1 mg/mL. A quarter milliliter of dispersed CaOX monohydrate crystal solution (100 mg/dL; Wako Chemical, Kyoto, Japan) was seeded onto each sample, and the supernatant counts per minute (cpm) was counted at 0 hours (S0). After incubation at 37°C accompanied by shaking for 4 hours, the supernatant cpm was counted at 4 1230
The inhibition of CaOX crystal aggregation of UMMs was measured with an aggregometer, as reported previously.14 –16 For this study, we used a Hema Tracer 212 (MC Medical, Tokyo, Japan) as the aggregometer. This method is very simple and is theoretically the same as the method reported by Hess et al.17 A 60-mL aliquot of UMM solution (0.5 and 1.0 mg/mL) and a 100-mL aliquot of 7.5 mM CaCl2 (dissolved in 0.05 M Tris-HCl solution containing 0.15 M NaCl, pH 7.4) were put into a cylindrical Pyrex glass cuvette. After the addition of 140 mL of 5 mM Na2OX (dissolved in 0.05 M Tris-HCl solution containing 0.15 M NaCl, pH 7.4) and a small stainless bar stirrer to the cuvette, the solution was stirred at 37°C for 10 minutes. At 10 minutes, stirring was stopped, and spontaneous sedimentation of the CaOX crystals was monitored at 37°C for 15 minutes. Turbidity of distilled water and of the 100 mM CuSO4 solution was set at 100 and 0, respectively. As a control, 60 mL of distilled water was substituted for the UMM solution. The aggregation inhibition rate (Ia) was then calculated as Ia 5 100 3 [1 2 Dturbidity(sample)/Dturbidity(control)].
RESULTS URINARY PARAMETERS The concentrations of urinary parameters were compared between pediatric and adult urine (Table I). Magnesium, citrate, phosphorus, and GAGs were significantly higher in urine from children than adults, and uric acid, oxalate, proteins, and pH showed no differences between the two groups. INHIBITION OF CaOX CRYSTALLIZATION OF ORIGINAL URINE AND ULMWSS IN A WHOLE URINE SYSTEM In the original urine, the particle size was smaller for children (3.667 6 0.632 mm) than for adults UROLOGY 53 (6), 1999
FIGURE 2. Inhibition of CaOX crystal growth of UMMs. Squares denote pediatric UMMs and triangles denote adult UMMs.
FIGURE 1. Average diameter of particles formed in (A) original urine and (B) urine including only LMWSs. Particles were measured in a whole urine system.
(4.343 6 0.822 mm) (P 5 0.0109; unpaired t test) (Fig. 1A). In the ULMWSs, no significant difference was recognized between children (4.103 6 1.109 mm) and adults (4.022 6 0.827 mm) (Fig. 1B). Inhibition of CaOX crystallization was stronger for children than for adults in the original urine, but was the same in the ULMWSs. VOLUME OF EXTRACTED UMMS The UMM concentrations in urine from the weight of extracted and freeze-dried UMMs were 0.179 6 0.0788 mg/mL of pediatric UMMs and 0.141 6 0.120 mg/mL of adult UMMs (not significant; unpaired t test). INHIBITION OF CaOX CRYSTAL GROWTH OF UMMS IN AN ARTIFICIAL URINE SYSTEM There were no significant differences in the inhibition of CaOX crystal growth of UMMs alone between children and adults at any UMM concentration (Fig. 2). INHIBITION OF CaOX CRYSTAL AGGREGATION OF UMMS Figure 3 shows examples of the time-course of turbidity. In the buffer including pediatric UMMs UROLOGY 53 (6), 1999
(final UMM concentration 0.1 mg/mL), the turbidity did not change for up to 8 minutes (Fig. 3A), although in the buffer including adult UMMs (0.1 mg/mL), it had already begun to decrease 3 minutes after the stirring was stopped (Fig. 3B). This means that larger aggregated crystals formed faster in the adult UMM buffer than in the pediatric UMM buffer. At a low concentration of UMMs (0.1 mg/mL), the aggregation inhibitory rate was 88.2 6 11.1% for children and 69.1 6 28.6% for adults (P 5 0.0496; Mann-Whitney U test) 5 minutes after the stirring was stopped and 74.4 6 17.4% for children and 46.9 6 24.8% for adults (P 5 0.0011; unpaired t test) at 10 minutes (Fig. 4A). At a higher concentration of UMMs (0.2 mg/mL), the inhibition rate was 95.2 6 9.26% for children and 82.4 6 14.3% for adults (P 5 0.0053; unpaired t test) at 5 minutes and 87.0 6 12.6% for children and 66.4 6 20.2% for adults (P 5 0.0016; unpaired t test) at 10 minutes (Fig. 4B). At any UMM concentration or at any time after the stirring was stopped, pediatric UMMs more strongly inhibited CaOX crystal aggregation than did adult UMMs. GAGS AND PROTEINS IN UMMS The volume of GAGs was 0.164 6 0.0373 mg/mg UMMs in pediatric UMMs and 0.0693 6 0.0206 mg/mg UMMs in adult UMMs (P ,0.001; MannWhitney U test). The volume of proteins was 0.116 6 0.0593 mg/mg UMMs in pediatric UMMs and 0.127 6 0.0482 mg/mg UMMs in adult UMMs (not significant; unpaired t test). COMMENT In North America, the prevalence of pediatric nephrolithiasis is very low compared with that of 1231
FIGURE 3. Inhibition of CaOX crystal aggregation of (A) a child’s UMMs and (B) an adult’s UMMs (UMM concentration 0.1 mg/mL).
adult nephrolithiasis.8,9 The most common mineral content of renal stones is calcium in pediatric, as well as in adult, urolithiasis.8 This suggests that there might be a great difference in the inhibition of CaOX crystallization between pediatric and adult urine, and in a prior study, we investigated LMWSs, in which we found that excretion of citrate and magnesium, well known as important CaOX stone inhibitors, is much higher in pediatric than adult urine.10 It is well known that these substances form citrate-calcium or magnesium-oxalate complexes and thus reduce supersaturation with CaOX. In fact, we demonstrated that the metastable limit of CaOX in pediatric urine is much higher than adult urine.10 In the present study, we focused on UMMs and examined the difference in their inhibition of CaOX crystallization between children and adults. In the case of ULMWSs, no difference in the inhibition of CaOX crystallization was recognized between children and adults. On the other hand, in the original urine, the inhibition of CaOX crystallization was stronger in children than in adults. 1232
FIGURE 4. Inhibition of CaOX crystal aggregation of UMMs at (A) a low concentration (0.1 mg/mL) and (B) a high concentration (0.2 mg/mL). Gray bars denote child, black denote adult.
This means that the inhibition rate of CaOX crystallization is not changed only by the quantity of LMWSs, even though the concentration of citrate and magnesium is much higher. Then what is the meaning of the higher concentration of low molecular weight stone inhibitors in pediatric urine? As aforementioned, it is obvious that inhibitors such as citrate or magnesium reduce supersaturation of CaOX. Another possibility is that the LMWSs may influence the function or the structure of some components of UMMs. In fact, Hess18,19 reported that citrate will affect the structure of Tamm-Horsfall protein, and an increase in citrate concentration in the buffer will make the protein have stronger inhibitory activity by changing the structure and function of the protein. Be that as it may, neither the CaOX crystal growth nor aggregation can be affected by LMWSs alone. Judging from this result, it seems that the discrepancy in the inhibition rate of CaOX crystallization in the original urine of children and adults should arise from differences in the quality or UROLOGY 53 (6), 1999
quantity of UMMs. Thus, we did additional investigations. In the artificial urine containing only UMMs, however, no difference in the inhibition rate of CaOX crystal growth was found between children and adults. From these results, it can at least be concluded that UMMs alone or LMWSs alone cannot affect the inhibition of CaOX crystal growth. It is definite that crystal growth is one of the steps in renal stone formation, but the process of growth is so slow that crystals cannot become large enough to obstruct the renal tubules and be retained there by this mechanism alone because it takes several minutes for the tubular fluid to pass through the kidney. For this reason, the more critical step is thought to be crystal aggregation. Much experimental data have shown that the inhibition rate of CaOX crystal aggregation of urine from patients with highly recurrent stone formation is less than that of urine from normal subjects.20,21 Our data show that pediatric UMMs have a stronger inhibition of crystal aggregation than adult UMMs, although there was no significant difference in the concentration of the UMMs themselves between pediatric and adult urine. We also found that GAGs were included to a much greater extent in pediatric than in adult UMMs. During the growth phase in animals, the proliferation and differentiation of chondrocytes and the synthesis and excretion of cartilage matrix components such as proteoglycans or collagen are very strong when bone is growing longitudinally. For this reason, the excretion rate of GAGs as metabolites of proteoglycans in children may be much higher than in adults. GAGs have been identified as an important factor related to stone formation. Some studies revealed that there is a significant difference in the excretion level of GAGs between normal subjects and patients with stone formation and suggested a correlation between urinary GAG concentration and urolithiasis.22,23 In addition, Yamaguchi et al.24 demonstrated that heparan sulfate (Hep-S) inhibits the growth and aggregation of CaOX crystals in an artificial urine system. Another report indicated that Hep-S also inhibits the aggregation of CaOX crystal in a whole urine system.25 We have not studied GAG content of pediatric or adult UMMs. Michelacci et al.22 reported that urinary chondroitin sulfates showed a higher proportion of 4-sulfated disaccharide for children than adults, although they did not examine how much Hep-S is included in pediatric urine. Even though they are not conclusive, these findings appear to suggest that a larger volume of GAGs in pediatric UMMs might possibly effect the stronger inhibition of CaOX crystal aggregation of pediatric UMMs. Another major component of UMMs that affects the inhibitory activity is proteins. There was no UROLOGY 53 (6), 1999
difference in the volume of proteins included in UMMs between children and adults, although we did not examine the composition of the proteins themselves. Some of these proteins interact with LMWSs. For this reason, we cannot exclude the possibility that the difference in the inhibition of crystal aggregation of UMMs between children and adults may also arise from the difference between children and adults in the quality or quantity of various proteins. CONCLUSIONS Judging from the finding that the inhibition of CaOX crystallization of pediatric urine did not differ from that of adult urine in the ultrafiltered urine, the crystal inhibitors of LMWSs such as citrate or magnesium cannot reduce the growth or aggregation rate of CaOX crystals by themselves. Pediatric UMMs have a stronger inhibition of CaOX crystal aggregation than adult UMMs, although no difference in the inhibition of CaOX crystal growth was seen between pediatric UMMs alone and adult UMMs alone. It is suggested that the higher content of GAGs may enhance the inhibition of crystal aggregation of pediatric UMMs. Finally, one of the major reasons for the reduced prevalence of pediatric urolithiasis is that pediatric urine features a stronger inhibition of CaOX crystal aggregation. ACKNOWLEDGMENT. To Ms. Yukako Morioka, Ms. Rieko Goto, and Ms. Naoko Matsumoto for their technical assistance. REFERENCES 1. Atmani F, Lacour B, Drueke T, et al: Isolation and purification of a new glycoprotein from human urine inhibiting calcium oxalate crystallization. Urol Res 21: 61– 66, 1993. 2. Atmani F, Lacour B, Jungers P, et al: Reduced inhibitory activity of uronic-acid-rich protein (UAP) in the urine of stone formers. Urol Res 22: 257–260, 1994. 3. Fleisch H: Inhibitors and promoters of stone formation. Kidney Int 13: 361–371, 1978. 4. Nikkila M, Koivula T, and Jokela H: Urinary citrate excretion in patients with urolithiasis and normal subjects. Eur Urol 16: 382–385, 1989. 5. Robertson WG, and Peacock M: Calcium oxalate crystalluria and inhibitors of crystallization in recurrent renal stone-formers. Clin Sci 43: 499 –506, 1972. 6. Robertson WG, Peacock M, Heyburn PJ, et al: Risk factors in calcium stone disease of the urinary tract. Br J Urol 50: 449 – 454, 1978. 7. Ryall RL: Glycosaminoglycans, proteins, and stone formation: adult themes and child’s play. Pediatr Nephrol 10: 656 – 666, 1996. 8. Polinsky MS, Kaiser BA, and Baluarte HJ: Urolithiasis in childhood. Pediatr Clin North Am 34: 683–710, 1987. 9. Reiner RJ, Kroovand RL, and Perlmutter AD: Unusual aspects of urinary calculi in children. J Urol 121: 480 – 481, 1979. 1233
10. Miyake O, Yoshimura K, Yoshioka T, et al: High urinary excretion level of citrate and magnesium in children: potential etiology for the reduced incidence of pediatric urolithiasis. Urol Res 26: 209 –213, 1998. 11. Jong JGN, Weavers RA, and Sambeek RL: Measuring urinary glycosaminoglycans in the presence of protein: an improved screening procedure for mucopolysaccharidoses based on dimethylmethyleneblue. Clin Chem 38: 803– 807, 1992. 12. Tawashi R, Consineau M, and Denis G: Calcium oxalate crystal growth in normal urine: role of contraceptive hormones. Urol Res 12: 7–9, 1984. 13. Robertson WG, Peacock M, and Nordin BEC: Inhibitors of the growth and aggregation of calcium oxalate crystals in vitro. Clin Chim Acta 43: 31–37, 1973. 14. Yoshida T: Studies on calcium oxalate crystallizationpropensity in human urine. (1) Basal studies for the measurement using an aggregometer. Jpn J Urol 78: 1693–1700, 1987. 15. Yoshida T: Studies on calcium oxalate crystallizationpropensity in human urine. (2) Clinical studies using an aggregometer. Jpn J Urol 78: 1701–1706, 1987. 16. Ebisuno S, Miyata K, Kohjimoto Y, et al: Inhibitory activity of urinary macromolecule upon calcium oxalate crystal aggregation using an aggregometer. Jpn J Urol 84: 339 – 344, 1993. 17. Hess B, Nakagawa Y, and Coe FL: Inhibition of calcium
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oxalate monohydrate crystal aggregation by urine proteins. Am J Physiol 257: F99 –F106, 1989. 18. Hess B: Tamm-Horsfall glycoprotein—inhibitor or promoter of calcium oxalate monohydrate crystallization process? Urol Res 20: 83– 86, 1992. 19. Hess B: Tamm-Horsfall glycoprotein and calcium nephrolithiasis. Miner Electrolyte Metab 20: 393–398, 1994. 20. Koide T, Takemoto M, Itatani H, et al: Urinary macromolecular substances as natural inhibitors of calcium oxalate crystal aggregation. Invest Urol 18: 382–386, 1981. 21. Kok DJ, Papapoulos SE, and Bijvolet OLM: Crystal agglomeration is a major element in calcium oxalate urinary stone formation. Kidney Int 37: 51–56, 1990. 22. Michelacci YM, Glashan RQ, and Schor N: Urinary excretion of glycosaminoglycans in normal and stone forming subjects. Kidney Int 36: 1022–1028, 1989. 23. Nikkila MT: Urinary glycosaminoglycan excretion in normal and stone forming subjects: significant disturbance in recurrent stone formers. Urol Int 44: 157–159, 1989. 24. Yamaguchi S, Yoshioka T, Utsunomiya M, et al: Heparan sulfate in the stone matrix and its inhibitory effect on calcium oxalate crystallization. Urol Res 21: 187–192, 1993. 25. Suzuki K, and Ryall RL: The effect of heparan sulfate on the crystallization of calcium oxalate in undiluted, ultrafiltered human urine. Br J Urol 78: 15–21, 1996.
UROLOGY 53 (6), 1999
POSSIBLE CAUSES FOR THE LOW PREVALENCE OF PEDIATRIC UROLITHIASIS OSAMU MIYAKE, KAZUHIRO YOSHIMURA, MASAO TSUJIHATA, TOSHIAKI YOSHIOKA, TAKUO KOIDE, SHIRO TAKAHARA, AND AKIHIKO OKUYAMA
ABSTRACT Objectives. To determine why the incidence of pediatric urolithiasis is less than that of adult urolithiasis, we investigated the difference in inhibition of calcium oxalate (CaOX) crystallization between pediatric and adult urinary macromolecules (UMMs). Methods. Urinary parameters in relation to urolithiasis, the inhibition of CaOX crystallization of original urine and urine from which UMMs (greater than 3 kDa) had been removed, and the inhibition of CaOX crystal growth and aggregation of UMMs alone were measured. These inhibitory activities were compared between children and adults. Results. In the original urine, the inhibition of CaOX crystallization was significantly stronger for children than for adults, but was the same in urine from which the UMMs had been removed. The inhibition of CaOX crystal growth by UMMs alone showed no significant differences between children and adults; their inhibition of CaOX crystal aggregation was significantly stronger for children than for adults. Much more glycosaminoglycan (GAG) was included in pediatric UMMs than in adult UMMs, although there was no difference in UMM concentration between urine from children and urine from adults. Conclusions. The lower incidence of CaOX lithiasis in children may be attributed, among other factors, to the stronger inhibition of CaOX crystal aggregation by pediatric UMMs, which in turn might be affected by the higher concentration of GAGs in children’s urine. UROLOGY 53: 1229–1234, 1999. © 1999, Elsevier Science Inc. All rights reserved.
M
any in vitro examinations have been performed to investigate the causes of urolithiasis. In the reports of these studies, the function of various low molecular weight substances (LMWSs) and macromolecules as stone inhibitors or promoters has been discussed, but these substances were almost always extracted from adult urine only and discussed in the context of adult urolithiasis.1– 6 As Ryall7 has pointed out, very few of these studies have examined the roles of urinary macromolecules (UMMs) in pediatric stone disease. The reason for this is that there are few children with idiopathic stone disease, except for patients who have an apparently congenital metabolic disease. In fact, the epidemiologic prevalence
of pediatric urolithiasis is very low compared with that of adult urolithiasis.8,9 Many urologists have wondered why this is so, but hardly any in vitro experiments on pediatric urolithiasis have been conducted at the crystallographic level. We have previously reported that the excretion volume of citrate and magnesium in urine from children is significantly higher than that in urine from adults, and that this difference may be of crucial importance when discussing the reduced incidence of pediatric urolithiasis.10 In the present study, we focused our attention on UMMs and compared their inhibition of calcium oxalate (CaOX) crystallization between children and adults.
From the Department of Urology, Osaka University Medical School; Department of Urology, Sumitomo Hospital; and Department of Urology, Osaka Kosei-nenkin Hospital, Osaka, Japan Reprint requests: Osamu Miyake, M.D., Department of Urology, Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka 565-0871 Japan Submitted: October 12, 1998, accepted (with revisions): December 9, 1998
MATERIAL AND METHODS
© 1999, ELSEVIER SCIENCE INC. ALL RIGHTS RESERVED
We collected urine from 19 boys aged 3 to 8 years (average 5.9) and 18 men aged 25 to 37 years (average 30.7). None had any congenital metabolic disorders or any episodes of urolithiasis. We collected their 24-hour urine samples together with 0.1 mL of 30% sodium azide. The children’s 24-hour urine samples were collected twice or three times and com0090-4295/99/$20.00 PII S0090-4295(99)00004-7 1229
TABLE I. Urinary parameters of 24-hour urine Children Average Urine volume (mL) Body weight (kg) pH Magnesium (mg/dL) Citrate (mg/dL) Uric acid (mg/dL) Calcium (mg/dL) Oxalate (mg/dL) Phosphorus (mg/dL) Creatinine (mg/dL) GAGs (mg/dL) Proteins (mg/dL)
549.7 19.8 6.42 9.90 42.3 54.5 11.6 30.4 92.3 71.4 10.2 4.94
Adults SD
Average
229.2 3.55 0.360 2.96 14.2 13.9 4.92 10.4 27.1 22.9 5.85 2.44
1342 71.3 6.15 5.66 30.4 52.2 17.6 26.3 73.0 134.2 5.06 4.45
SD 390.2 9.25 0.540 1.74 17.1 15.5 14.8 15.3 23.8 50.9 4.70 1.33
P Value
NS* ,0.001† 0.027† NS* 0.0334† NS* 0.0277* ,0.001† 0.0061* NS*
KEY: GAGs 5 glycosaminoglycans; NS 5 not statistically significant. * Unpaired t test. † Mann-Whitney U test.
bined to obtain a sufficient volume of UMMs. The urine volume, pH, magnesium, citrate, uric acid, calcium, oxalate, phosphorus, creatinine, glycosaminoglycans (GAGs), and proteins of all samples were measured. Proteins were measured with a Bio Rad protein assay and GAGs with a modified DMB assay.11
hours (S4). The count at 0 and 4 hours in the control tube (without UMMs) was adopted as C0 and C4. The percentage of growth inhibition (Ig) was then calculated as Ig 5 100 3 [(C4 2 C0) 2 (S4 2 S0)]/(C4 2 C0).
AGGREGATION INHIBITION ASSAY OF UMMS PREPARATION OF UMMS All 24-hour urine samples were filtered through a Whatman No. 1 paper and a 0.22-mm Millipore filter. A 150-mL aliquot was then separated from the samples and used for the crystallization inhibition assay in a whole urine system; the rest was ultrafiltered with a Labomodule (Asahi Kasei, Tokyo, Japan; threshold molecular weight 3 kDa). The filtered samples were divided into urine containing only LMWSs (ULMWSs) and urine containing only macromolecules. The latter was also desalinated with distilled water in a Spectra/Por-1 membrane tube (Spectrum Medical Industries, Houston, Tex), lyophilized, and stored at 220°C until use.
CRYSTALLIZATION INHIBITION ASSAY IN A WHOLE URINE SYSTEM The method reported by Tawashi et al.12 was used for the inhibition assay in a whole urine system. A quarter milliliter of calcium chloride (1 M) was added to 50 mL of filtered original urine or ULMWSs. The pH was adjusted to 5.7, and 2.5 mL of sodium oxalate (5 mM) was added to each of the samples, which were kept at 37°C without shaking. After 3 hours, the particle size of the CaOX crystals was determined with a Coulter Multisizer.
GROWTH INHIBITION ASSAY OF UMMS IN AN ARTIFICIAL URINE SYSTEM The inhibition rate of CaOX crystal growth of the UMMs was also measured with a seed crystal method reported by Robertson et al.,13 with minor modifications. In brief, we produced 10 mL of a metastable solution on CaOX with 14Clabeled oxalate mixed with UMMs at final concentrations of 0.001, 0.01, 0.05, 0.1, 0.2, 0.4, and 1 mg/mL. A quarter milliliter of dispersed CaOX monohydrate crystal solution (100 mg/dL; Wako Chemical, Kyoto, Japan) was seeded onto each sample, and the supernatant counts per minute (cpm) was counted at 0 hours (S0). After incubation at 37°C accompanied by shaking for 4 hours, the supernatant cpm was counted at 4 1230
The inhibition of CaOX crystal aggregation of UMMs was measured with an aggregometer, as reported previously.14 –16 For this study, we used a Hema Tracer 212 (MC Medical, Tokyo, Japan) as the aggregometer. This method is very simple and is theoretically the same as the method reported by Hess et al.17 A 60-mL aliquot of UMM solution (0.5 and 1.0 mg/mL) and a 100-mL aliquot of 7.5 mM CaCl2 (dissolved in 0.05 M Tris-HCl solution containing 0.15 M NaCl, pH 7.4) were put into a cylindrical Pyrex glass cuvette. After the addition of 140 mL of 5 mM Na2OX (dissolved in 0.05 M Tris-HCl solution containing 0.15 M NaCl, pH 7.4) and a small stainless bar stirrer to the cuvette, the solution was stirred at 37°C for 10 minutes. At 10 minutes, stirring was stopped, and spontaneous sedimentation of the CaOX crystals was monitored at 37°C for 15 minutes. Turbidity of distilled water and of the 100 mM CuSO4 solution was set at 100 and 0, respectively. As a control, 60 mL of distilled water was substituted for the UMM solution. The aggregation inhibition rate (Ia) was then calculated as Ia 5 100 3 [1 2 Dturbidity(sample)/Dturbidity(control)].
RESULTS URINARY PARAMETERS The concentrations of urinary parameters were compared between pediatric and adult urine (Table I). Magnesium, citrate, phosphorus, and GAGs were significantly higher in urine from children than adults, and uric acid, oxalate, proteins, and pH showed no differences between the two groups. INHIBITION OF CaOX CRYSTALLIZATION OF ORIGINAL URINE AND ULMWSS IN A WHOLE URINE SYSTEM In the original urine, the particle size was smaller for children (3.667 6 0.632 mm) than for adults UROLOGY 53 (6), 1999
FIGURE 2. Inhibition of CaOX crystal growth of UMMs. Squares denote pediatric UMMs and triangles denote adult UMMs.
FIGURE 1. Average diameter of particles formed in (A) original urine and (B) urine including only LMWSs. Particles were measured in a whole urine system.
(4.343 6 0.822 mm) (P 5 0.0109; unpaired t test) (Fig. 1A). In the ULMWSs, no significant difference was recognized between children (4.103 6 1.109 mm) and adults (4.022 6 0.827 mm) (Fig. 1B). Inhibition of CaOX crystallization was stronger for children than for adults in the original urine, but was the same in the ULMWSs. VOLUME OF EXTRACTED UMMS The UMM concentrations in urine from the weight of extracted and freeze-dried UMMs were 0.179 6 0.0788 mg/mL of pediatric UMMs and 0.141 6 0.120 mg/mL of adult UMMs (not significant; unpaired t test). INHIBITION OF CaOX CRYSTAL GROWTH OF UMMS IN AN ARTIFICIAL URINE SYSTEM There were no significant differences in the inhibition of CaOX crystal growth of UMMs alone between children and adults at any UMM concentration (Fig. 2). INHIBITION OF CaOX CRYSTAL AGGREGATION OF UMMS Figure 3 shows examples of the time-course of turbidity. In the buffer including pediatric UMMs UROLOGY 53 (6), 1999
(final UMM concentration 0.1 mg/mL), the turbidity did not change for up to 8 minutes (Fig. 3A), although in the buffer including adult UMMs (0.1 mg/mL), it had already begun to decrease 3 minutes after the stirring was stopped (Fig. 3B). This means that larger aggregated crystals formed faster in the adult UMM buffer than in the pediatric UMM buffer. At a low concentration of UMMs (0.1 mg/mL), the aggregation inhibitory rate was 88.2 6 11.1% for children and 69.1 6 28.6% for adults (P 5 0.0496; Mann-Whitney U test) 5 minutes after the stirring was stopped and 74.4 6 17.4% for children and 46.9 6 24.8% for adults (P 5 0.0011; unpaired t test) at 10 minutes (Fig. 4A). At a higher concentration of UMMs (0.2 mg/mL), the inhibition rate was 95.2 6 9.26% for children and 82.4 6 14.3% for adults (P 5 0.0053; unpaired t test) at 5 minutes and 87.0 6 12.6% for children and 66.4 6 20.2% for adults (P 5 0.0016; unpaired t test) at 10 minutes (Fig. 4B). At any UMM concentration or at any time after the stirring was stopped, pediatric UMMs more strongly inhibited CaOX crystal aggregation than did adult UMMs. GAGS AND PROTEINS IN UMMS The volume of GAGs was 0.164 6 0.0373 mg/mg UMMs in pediatric UMMs and 0.0693 6 0.0206 mg/mg UMMs in adult UMMs (P ,0.001; MannWhitney U test). The volume of proteins was 0.116 6 0.0593 mg/mg UMMs in pediatric UMMs and 0.127 6 0.0482 mg/mg UMMs in adult UMMs (not significant; unpaired t test). COMMENT In North America, the prevalence of pediatric nephrolithiasis is very low compared with that of 1231
FIGURE 3. Inhibition of CaOX crystal aggregation of (A) a child’s UMMs and (B) an adult’s UMMs (UMM concentration 0.1 mg/mL).
adult nephrolithiasis.8,9 The most common mineral content of renal stones is calcium in pediatric, as well as in adult, urolithiasis.8 This suggests that there might be a great difference in the inhibition of CaOX crystallization between pediatric and adult urine, and in a prior study, we investigated LMWSs, in which we found that excretion of citrate and magnesium, well known as important CaOX stone inhibitors, is much higher in pediatric than adult urine.10 It is well known that these substances form citrate-calcium or magnesium-oxalate complexes and thus reduce supersaturation with CaOX. In fact, we demonstrated that the metastable limit of CaOX in pediatric urine is much higher than adult urine.10 In the present study, we focused on UMMs and examined the difference in their inhibition of CaOX crystallization between children and adults. In the case of ULMWSs, no difference in the inhibition of CaOX crystallization was recognized between children and adults. On the other hand, in the original urine, the inhibition of CaOX crystallization was stronger in children than in adults. 1232
FIGURE 4. Inhibition of CaOX crystal aggregation of UMMs at (A) a low concentration (0.1 mg/mL) and (B) a high concentration (0.2 mg/mL). Gray bars denote child, black denote adult.
This means that the inhibition rate of CaOX crystallization is not changed only by the quantity of LMWSs, even though the concentration of citrate and magnesium is much higher. Then what is the meaning of the higher concentration of low molecular weight stone inhibitors in pediatric urine? As aforementioned, it is obvious that inhibitors such as citrate or magnesium reduce supersaturation of CaOX. Another possibility is that the LMWSs may influence the function or the structure of some components of UMMs. In fact, Hess18,19 reported that citrate will affect the structure of Tamm-Horsfall protein, and an increase in citrate concentration in the buffer will make the protein have stronger inhibitory activity by changing the structure and function of the protein. Be that as it may, neither the CaOX crystal growth nor aggregation can be affected by LMWSs alone. Judging from this result, it seems that the discrepancy in the inhibition rate of CaOX crystallization in the original urine of children and adults should arise from differences in the quality or UROLOGY 53 (6), 1999
quantity of UMMs. Thus, we did additional investigations. In the artificial urine containing only UMMs, however, no difference in the inhibition rate of CaOX crystal growth was found between children and adults. From these results, it can at least be concluded that UMMs alone or LMWSs alone cannot affect the inhibition of CaOX crystal growth. It is definite that crystal growth is one of the steps in renal stone formation, but the process of growth is so slow that crystals cannot become large enough to obstruct the renal tubules and be retained there by this mechanism alone because it takes several minutes for the tubular fluid to pass through the kidney. For this reason, the more critical step is thought to be crystal aggregation. Much experimental data have shown that the inhibition rate of CaOX crystal aggregation of urine from patients with highly recurrent stone formation is less than that of urine from normal subjects.20,21 Our data show that pediatric UMMs have a stronger inhibition of crystal aggregation than adult UMMs, although there was no significant difference in the concentration of the UMMs themselves between pediatric and adult urine. We also found that GAGs were included to a much greater extent in pediatric than in adult UMMs. During the growth phase in animals, the proliferation and differentiation of chondrocytes and the synthesis and excretion of cartilage matrix components such as proteoglycans or collagen are very strong when bone is growing longitudinally. For this reason, the excretion rate of GAGs as metabolites of proteoglycans in children may be much higher than in adults. GAGs have been identified as an important factor related to stone formation. Some studies revealed that there is a significant difference in the excretion level of GAGs between normal subjects and patients with stone formation and suggested a correlation between urinary GAG concentration and urolithiasis.22,23 In addition, Yamaguchi et al.24 demonstrated that heparan sulfate (Hep-S) inhibits the growth and aggregation of CaOX crystals in an artificial urine system. Another report indicated that Hep-S also inhibits the aggregation of CaOX crystal in a whole urine system.25 We have not studied GAG content of pediatric or adult UMMs. Michelacci et al.22 reported that urinary chondroitin sulfates showed a higher proportion of 4-sulfated disaccharide for children than adults, although they did not examine how much Hep-S is included in pediatric urine. Even though they are not conclusive, these findings appear to suggest that a larger volume of GAGs in pediatric UMMs might possibly effect the stronger inhibition of CaOX crystal aggregation of pediatric UMMs. Another major component of UMMs that affects the inhibitory activity is proteins. There was no UROLOGY 53 (6), 1999
difference in the volume of proteins included in UMMs between children and adults, although we did not examine the composition of the proteins themselves. Some of these proteins interact with LMWSs. For this reason, we cannot exclude the possibility that the difference in the inhibition of crystal aggregation of UMMs between children and adults may also arise from the difference between children and adults in the quality or quantity of various proteins. CONCLUSIONS Judging from the finding that the inhibition of CaOX crystallization of pediatric urine did not differ from that of adult urine in the ultrafiltered urine, the crystal inhibitors of LMWSs such as citrate or magnesium cannot reduce the growth or aggregation rate of CaOX crystals by themselves. Pediatric UMMs have a stronger inhibition of CaOX crystal aggregation than adult UMMs, although no difference in the inhibition of CaOX crystal growth was seen between pediatric UMMs alone and adult UMMs alone. It is suggested that the higher content of GAGs may enhance the inhibition of crystal aggregation of pediatric UMMs. Finally, one of the major reasons for the reduced prevalence of pediatric urolithiasis is that pediatric urine features a stronger inhibition of CaOX crystal aggregation. ACKNOWLEDGMENT. To Ms. Yukako Morioka, Ms. Rieko Goto, and Ms. Naoko Matsumoto for their technical assistance. REFERENCES 1. Atmani F, Lacour B, Drueke T, et al: Isolation and purification of a new glycoprotein from human urine inhibiting calcium oxalate crystallization. Urol Res 21: 61– 66, 1993. 2. Atmani F, Lacour B, Jungers P, et al: Reduced inhibitory activity of uronic-acid-rich protein (UAP) in the urine of stone formers. Urol Res 22: 257–260, 1994. 3. Fleisch H: Inhibitors and promoters of stone formation. Kidney Int 13: 361–371, 1978. 4. Nikkila M, Koivula T, and Jokela H: Urinary citrate excretion in patients with urolithiasis and normal subjects. Eur Urol 16: 382–385, 1989. 5. Robertson WG, and Peacock M: Calcium oxalate crystalluria and inhibitors of crystallization in recurrent renal stone-formers. Clin Sci 43: 499 –506, 1972. 6. Robertson WG, Peacock M, Heyburn PJ, et al: Risk factors in calcium stone disease of the urinary tract. Br J Urol 50: 449 – 454, 1978. 7. Ryall RL: Glycosaminoglycans, proteins, and stone formation: adult themes and child’s play. Pediatr Nephrol 10: 656 – 666, 1996. 8. Polinsky MS, Kaiser BA, and Baluarte HJ: Urolithiasis in childhood. Pediatr Clin North Am 34: 683–710, 1987. 9. Reiner RJ, Kroovand RL, and Perlmutter AD: Unusual aspects of urinary calculi in children. J Urol 121: 480 – 481, 1979. 1233
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