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Clinical association with urinary glycosaminoglycans and urolithiasis
ADULT UROLOGY
CLINICAL ASSOCIATION WITH URINARY GLYCOSAMINOGLYCANS AND UROLITHIASIS ERDAL ERTURK, MAUREEN KIERNAN,
AND
SUSAN R. SCHOEN
ABSTRACT Objectives. To determine the clinical association between urinary glycosaminoglycan (GAG) concentration and kidney stone disease. Methods. Thirty-five patients (14 women and 21 men) with a history of stone disease and 37 controls (13 women and 24 men) were evaluated for urinary GAG concentration. By using a new dye-binding assay, the total GAG concentration in the urine was measured and corrected to urinary creatinine levels (micrograms of GAG per milligram creatinine). Results. The mean urinary GAG concentration in those with stones was significantly lower (31.5 ⫾ 2.6 g GAG/mg creatinine) than in the controls (43.8 ⫾ 3.8 g GAG/mg creatinine, P ⫽ 0.01). Male patients with stones also had a significantly lower mean GAG concentration (26.1 ⫾ 1.8) than did the female patients (39.6 ⫾ 5.3, P ⫽ 0.009). The mean GAG concentration between ureteral (n ⫽ 13) versus renal (n ⫽ 22), single (n ⫽ 19) versus multiple (n ⫽ 16), family history (n ⫽ 11) versus no family history (n ⫽ 24), large (n ⫽ 13) versus small (n ⫽ 20), and the presence (n ⫽ 22) versus absence (n ⫽ 13) of residual stones did not show any significant differences. However, patients with recurrent stone formation (n ⫽ 21) had significantly lower mean GAG levels (26.4 ⫾ 1.6) compared with those with single stone formation (n ⫽ 14; 39.2 ⫾ 5.5, P ⫽ 0.01). Conclusions. Lower urinary GAG levels are more common in patients with stone formation. This may play a more determinant role in male patients and those with recurrent stone formation. UROLOGY 59: 495–499, 2002. © 2002, Elsevier Science Inc.
T
he formation of urinary stones can be explained by the physical and chemical properties of crystals, supersaturation, and lack of inhibitors. Thus, any imbalance can lead to nucleation, growth, and aggregation.1 Inhibitors of crystallization can be divided into two classes: small molecules (citrate and pyrophosphate) and macromolecules (glycoproteins and glycosaminoglycans [GAGs]).2 GAGs and polysaccharides, which are products of degraded proteoglycans, appear in the urine after being filtered through the glomerulus.3 Six GAGs are present in the urine: heparan sulfate, chondroitin sulfate A and C, dermatan sulfate, hyaluronic acid, and keratan sulfate. The major GAG in human urine is chondroitin sulfate.4 Several in vitro models have demonstrated the inhibitory effect of urinary GAGs on calcium oxalate stone for-
mation.5,6 Despite fairly consistent results reported with in vitro studies, significant controversy exists when comparing GAG excretion in patients with stone formation and normal controls. Experiments have revealed either no difference or significantly lower urinary levels of GAGs in patients with stone formation than in the controls.7–11 The aim of this study was to determine the total urinary GAG levels in patients with stone disease and controls using a relatively new, more accurate and reproducible dye-binding assay. Additional analyses were also made to compare the clinical parameters of patients with stones, including the presence of residual stone disease during the study, renal versus ureteral stones, positive family history, and single versus multiple stone episodes in terms of urinary GAG levels.
From the Department of Urology, University of Rochester, Rochester, New York Reprint requests: Erdal Erturk, M.D., Department of Urology, 601 Elmwood Avenue, Box 656, Rochester, NY 14642 Submitted: August 2, 2001, accepted (with revisions): November 28, 2001
MATERIAL AND METHODS
© 2002, ELSEVIER SCIENCE INC. ALL RIGHTS RESERVED
Urine samples were obtained from patients with stone disease and controls during routine clinical visits. None of the patients had had any evidence of urinary tract infection or had undergone any urologic procedures within the past 2 months. 0090-4295/02/$22.00 PII S0090-4295(01)01649-1 495
Urine samples were collected without a preservative and immediately frozen at ⫺70°C. The quantitative assays for GAG were performed with the technique described by Thuy and Nyhan.12 This method depends on measuring a change in the optical density of a mixture of azure A and azure B as a result of binding the dyes with GAG. A stock solution was made by dissolving 10 mg azure A and 10 mg azure B in 100 mL distilled water and diluted 1:10 in water for assays (working solution). Chondroitin sulfate C was used to establish a standard curve, and this was also used for the quantification of GAGs in the urine samples. The assay for total GAG was performed by adding 2.7 mL of the working solution to 100 L of urine or standards and totaling the volume up to 3 mL with water. Absorbance was read at 610 nm on a Varian DMS 200 spectrophotometer using water as a blank. The standard curve was created using chondroitin sulfate C at 1, 2, 4, 6, 8, and 20 g. The standard curve was plotted using regression analysis. Unknown total GAG values were extrapolated using the standard curve. Creatinine was assayed using the principle of the Jaffee reactions.13,14 Results were normalized against creatinine to yield the value in micrograms GAG per milligram creatinine. Thirty-five patients (14 women and 21 men, mean age 47 years, range 24 to 72) with history of stone disease were studied. An attempt was made to exclude those patients who had systemic diseases predisposing them to urolithiasis. Thirtyseven controls (13 women and 24 men, mean age 57 years, range 21 to 89) with no history of urolithiasis, urinary tract infection, or any manipulations of the urinary tract were also studied. Because of the difficulty in finding healthy female controls from the clinic patients, control urine samples were obtained from healthy hospital staff. Twenty-seven patients underwent urologic intervention that included shock wave lithotripsy, ureteroscopy, and percutaneous nephrolithotripsy. Urine samples were obtained in the clinic and were immediately frozen. Clinical stone parameters in relation to urinary GAG included number, location, family history, and current situation of the stone disease. The comparison was also made between the sexes. Statistical analysis was performed with Student’s t test.
RESULTS Stone analysis was available for 22 patients (63%). Of the 35 patients with stone disease, 18 had calcium oxalate or mixed phosphate stones; 2 were struvite and 2 were uric acid stones. There were 13 ureteral stones and 22 renal stones. Eighteen patients (51%) had undergone a metabolic evaluation and were receiving treatment. The mean urinary GAG levels in patients with stone formation was significantly lower (31.5 ⫾ 2.6) compared with the controls (43.8 ⫾ 3.8, P ⫽ 0.01) (Fig. 1). However, the mean GAG levels (26.0 ⫾ 1.6) in male patients with stone disease (n ⫽ 21) were significantly lower (P ⫽ 0.009) than those (39.6 ⫾ 5.3) of female patients with stone disease (n ⫽ 14). When the GAG levels were compared separately in the male and female patients with stone disease and controls, male patients with stones had more pronounced lower levels than did controls (26.0 ⫾ 1.6 versus 48.4 ⫾ 5.7, P ⫽ 0.0009); no differences were found between female patients with stone disease and controls (39.6 ⫾ 5.3 versus 39.2 ⫾ 5.9) (Fig. 2). Also no difference 496
FIGURE 1. Patients with history of stone disease demonstrated lower levels of mean urinary GAG concentration (P ⫽ 0.01).
FIGURE 2. Male patients with stone disease had significantly lower urinary GAG concentrations compared with female patients (P ⬍0.005). No difference was found between female patients with stone disease and female controls.
was found in the GAG levels between male (48.3 ⫾ 5.7) and female (39.2 ⫾ 5.1) controls. Additional comparisons revealed no significant differences in the GAG concentrations among ureteral (28.6 ⫾ 2.7) versus renal (33.3 ⫾ 3.7), family history (37.4 ⫾ 7.7) versus no family history (28.8 ⫾ 2.0), single (28.7 ⫾ 5.5) versus multiple (38.9 ⫾ 1.6), small size (less than 1 cm) (29.6 ⫾ 3.2) versus large (greater than 1 cm) (34.8 ⫾ 4.9), and between the presence (33.1 ⫾ 3.7) or absence (28.7 ⫾ 3.2) of residual stones. However, when patients with recurrent stone formation were compared with those with single stone episodes, the mean GAG levels were significantly lower (26.4 ⫾ 1.6 versus 39.2 ⫾ 5.5, P ⫽ 0.012). The mean GAG levels were similar in patients with single stone formation and controls (39.2 and 43.9, P ⫽ 0.51). The same number of women with stone disease had single stone episodes (n ⫽ 7) as multiple stone episodes (n ⫽ 7). Although more male patients had multiple stone episodes (n ⫽ 14) than single (n ⫽ 7), it was not significant (P ⫽ 0.32) (Table I). UROLOGY 59 (4), 2002
TABLE I. Patient characteristics Characteristic Ureteral stones Renal stones Family history of stones No family history of stones Single stone Multiple stones Small stones Large stones Residual stones No residual stones Single stone episode Multiple stone episodes
Patients (n) 13 22 11 24 19 16 20 13 22 13 14 21
Mean Urinary GAG Levels (g GAG/mg Creatinine) 28.6 27.4 37.4 28.8 28.7 38.9 29.6 34.8 33.1 28.7 39.2 26.4
⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾
3 4 7 2 2 5 3 5 4 3 2* 6*
P Value 0.46 0.12 0.24 0.36 0.42 0.01
KEY: GAG ⫽ glycosaminoglycan. * Patients with multiple stone episodes had significantly lower urinary GAG levels.
COMMENT Using a new quantitative assay, the total GAG concentration in the urine was measured. Before this, it had been shown that spot tests for the determination of GAG levels resulted in significant variability.15 The results we obtained were also corrected to the urinary creatinine levels, which has been shown to make the assay more reliable.16 This assay is not validated for infected urine samples, fresh versus frozen samples, or different temperatures. Thuy and Nyhan12 in describing their method performed validation assays for volume, NaCl, sodium phosphate buffers, pH, and recovery from spiked samples. They also performed intra and interassay variation.12 The GAGs that were included in the assay were dermatan sulfate, keratan sulfate, heparan sulfate, chondriotin-6-sulfate A, C, and hyaluronic acid. When compared with normal controls, the GAG levels were significantly lower in the patient population with stones. When urine aliquots are used, the correction of the GAG levels to creatinine measurements has been reported to be more reliable, because there is diurnal variability in the GAG excretion.16 We did not analyze 24-hour urine collections, which might have made the assay more reliable. The urine samples in this study were all obtained in the morning. Of the 35 patients with stones, 22 had their stones analyzed. Of the 22 patients, 18 had calcium oxalate and/or mixed phosphate stones, 2 had struvite stones, and 2 had uric acid stones. Only 51% of the patients in this study had documented calcium oxalate stones. Limiting the study population to only those patients with calcium oxalate stones could have resulted in more reliable findings. The stone analysis was not known before the GAG assay in our study. Although the published data indicating the inhibitory effect of urinary GAGs on stone disease are abundant,9,17 the focus was on calcium UROLOGY 59 (4), 2002
oxalate crystal nucleation and propagation.16,18,19 Clinical evidence has shown that in general a reduction of stone disease may also be associated with higher urinary GAG levels. In vitro studies have documented the inhibition of heterogeneous calcium oxalate nucleation.20 –22 Another proposed mechanism is that reduced GAG levels may result in proximal tubular epithelial cell injury, thus creating the potential for spontaneous crystal nucleation.23 When the groups were separated according to sex and a comparison was made, no difference in urinary GAG concentration was found between the female patients with stone formation and the female controls. However, a significant difference was found between the male patients with stone formation and the male controls. Other studies also did not find an association between lower urinary GAG levels and stone disease in female patients with stones.24 Conversely, the higher incidence of urolithiasis in the male population may, in part, be explained by this finding. No significant difference in the urinary GAG levels was found between female control patients (39.2 ⫾ 5.1) and male control patients (48.4 ⫾ 5.7, P ⫽ 0.29). In the present study, however, the age difference between the patients with stone disease (46.8 ⫾ 2.6 years) and the controls (55.9 ⫾ 2.9 years) was significant (P ⫽ 0.021). When the mean age was compared between male patients with stones (54.4 ⫾ 2.8 years) and male controls (62.5 ⫾ 3.1 years), a difference was noted, but it did not reach significance (P ⫽ 0.06). The mean age between the female patients with stones (35.3 ⫾ 3.1 years) and female controls (40.1 ⫾ 2.8 years) was also not statistically significant (P ⫽ 0.283). Thus, the study may have had different findings if the ages had been more comparable. It is also conceivable that if the patients with stone formation had been older, the 497
difference in the GAG levels would have been more pronounced. It is well documented in published reports that the pediatric population generally has higher levels of urinary GAG concentrations.25,26 This may also account for the relatively low incidence of urolithiasis in the pediatric population. Reports have investigated the clinical activity of stone disease and its association with urinary GAG concentrations. Despite the mixed results in published studies, we found that patients with recurrent stone formation tended to have significantly lower urinary GAG concentrations. Twenty-one of our patients (60%) were patients with recurrent stone formation, 14 (40%) had single stone episodes. No difference was found in the urinary GAG levels when comparing stone location (kidney versus ureter), stone size, presence of residual stones, and family history of urolithiasis. A recent finding indicates that shock wave lithotripsy can lead to a transient rise in urinary GAG excretion.27 Twentythree patients (66%) in our study group had undergone shock wave lithotripsy treatment. However, we were careful not to obtain urine samples from any patients within 2 months of their procedure. Additionally, in the stone study group, 5 (14%) underwent ureteroscopy, 3 (9%) percutaneous nephrolithotripsy, and 7 (20%) no treatment. It is not known how urinary GAGs affect crystal nucleation. The inherent acidic properties of the molecules has been suggested as the mechanism of action and the degree of sulfation was determined to be significant.22 It is also known that high urinary oxalate concentrations exhibit adverse affects in terms of GAG inhibitory action.28 Urinary GAGs may also have an affect on heterogeneous nucleation induced by uric acid in calcium oxalate stone genesis.29 Nonsteroidal anti-inflammatory drugs increase urinary GAG secretion and may potentiate or induce their inhibitory effects.30 These factors underscore the complexity of the interaction between urinary GAGs and clinical urolithiasis. The elucidation of the cause and effect of urinary GAGs and urolithiasis remains a challenge. However, long-term clinical trials with therapeutic agents have revealed that manipulating urinary GAGs may have a beneficial role in preventing urolithiasis.31 Studies have been performed in an attempt to identify which of the GAGs may play a role in this function. With atomic force microscopy, it was shown that chondroitin sulfate delayed maturation of calcium oxalate stones and dermatan sulfate inhibited nucleation in vitro.18 In another study, sodium pentosan polysulfate was found to have a high affinity to bind to calcium oxalate stones and thus exerted a potential inhibitory effect on crystal aggregation.32 498
CONCLUSIONS We studied the urinary GAG concentration of patients with stone disease and normal controls. Patients with stone formation had lower urinary GAG levels, which was more pronounced in patients with recurrent stone formation and in male patients. The higher stone incidence in the male population may, in part, be explained by the lack of an inhibitor theory. REFERENCES 1. Robertson WG, Peacock M, Marshall RW, et al: Saturation inhibition index as a measure of the risk of calcium oxalate stone formation in the urinary tract. N Engl J Med 294: 249 –252, 1976. 2. Worcester EM: Inhibitors of stone formation. Semin Nephrol 16: 474 – 486, 1996. 3. Hesse A, Wusel H, and Vahlensieck W: Significance of glycosaminoglycans for the formation of calcium oxalate stones. Am J Kidney Dis 17: 414 – 419, 1991. 4. Angell AH, and Resnick MI: Surface interaction between glycosaminoglycans and calcium oxalate. J Urol 141: 1255–1258, 1989. 5. Fujisawa M, Arima S, and Yachiku S: A study of inhibitory effect of chondroitin polysulfate on stone formation of calcium oxalate. Jpn J Urol 83: 1647–1654, 1992. 6. Matsuma A, and Takeuchi M: Inhibitory factor of calcium oxalate crystal growth in urinary macromolecules. Jpn J Urol 81: 883– 888, 1990. 7. Trinchieri A, Mandressi A, Luongo P, et al: Urinary excretion of citrate, glycosaminoglycans, magnesium and zinc in relation to age and sex in normal subjects and in patients who form calcium stones. Scand J Urol Nephrol 26: 379 –386, 1992. 8. Akinci M, Esen T, Kocak T, et al: Role of inhibitor deficiency in urolithiasis: rationale of urinary magnesium, citrate, pyrophosphate and glycosaminoglycans determinations. Eur Urol 19: 240 –243,1991. 9. Nesse A, Garbossa G, Romero MC, et al: Glycosaminoglycans in urolithiasis. Nephron 62: 36 –39, 1992. 10. Grases F, Llompart I, Conte A, et al: Glycosaminoglycans and oxalocalcic urolithiasis. Nephron 68: 449 – 453, 1994. 11. Sidhu H, Hemal AK, Thind SK, et al: Comparative study of 24-hour urinary excretion of glycosaminoglycans by renal stone formers and healthy adults. Eur Urol 16: 45– 47, 1989. 12. Thuy LP, and Nyhan WL: A new quantitative assay for glycosaminoglycans. Clin Chim Acta 212: 17–26, 1992. 13. Pennock CA: A modified screening test for glycosaminoglycan excretion. J Clin Pathol 22: 379 –380, 1969. 14. Gold EW: The quantitative spectrophotometric estimation of total sulfated glycosaminoglycan levels: Formation of soluble Alcian blue complexes. Biochim Biophys Acta 683: 408 – 415, 1981. 15. Brimble A, Pennock C, and Stone J: Results of a quality assurance exercise for urinary glycosaminoglycan excretion. Ann Clin Biochem 27: 133–138, 1990. 16. Michelacci YM, Glashan RQ, and Schor N: Urinary excretion of glycosaminoglycans in normal and stone forming subjects. Kidney Int 36: 1022–1028, 1989. 17. Nikkila MT: Urinary glycosaminoglycan excretion in normal and stone-forming subjects: significant disturbance in recurrent stone formers. Urol Int 44: 157–159, 1989. 18. Shirane Y, Kurokawa Y, Miyashita S, et al: Study of inhibition mechanisms of glycosaminoglycans on calcium oxalate monohydrate crystals by atomic force microscopy. Urol Res 27: 426 – 431, 1999. UROLOGY 59 (4), 2002
19. Borghi L, Meschi T, Guerra A, et al: Effects of urinary macromolecules on the nucleation of calcium oxalate in idiopathic stone formers and healthy controls. Clin Chim Acta 239: 1–11, 1995. 20. Worcester EM: Inhibitors of stone formation. Semin Nephrol 16: 474 – 486, 1996. 21. Cao LC, Boeve ER, de Bruijn WC, et al: Glycosaminoglycans and semisynthetic sulfated polysaccharides: an overview of their potential application in the treatment of patients with urolithiasis. Urology 50: 173–183, 1997. 22. Sallis JD: Glycosaminoglycans as inhibitors of stone formation. Miner Electrolyte Metab 13: 273–277, 1987. 23. Chan VS, and Li MK: Quantitative analysis on the localization of chondroitin sulfate proteoglycan in renal tissues of patients with calcium nephrolithiasis. Urol Res 26: 271– 274, 1998. 24. Ryall RL, Harnett RM, Hibberd CM, et al: Urinary risk factors in calcium oxalate stone disease: comparison of men and women. Br J Urol 60: 480 – 488, 1987. 25. Akcay T, Konukoglu D, and Dincer Y: Urinary glycosaminoglycan excretion in urolithiasis. Arch Dis Child 80: 271–272, 1999. 26. Suzuki K, Mayne K, Doyle IR, et al: Urinary glycosaminoglycans are selectively included into calcium oxalate crys-
UROLOGY 59 (4), 2002
tals precipitated from whole human urine. Scanning Microsc 8: 523–530, 1994. 27. Winter P, Schoeneich G, Ganter K, et al: Extracorporeal shock wave lithotripsy and glycosaminoglycans in urine. Int Urol Nephrol 30: 113–121, 1998. 28. Cao LC, Deng G, Boeve ER, et al: Does urinary oxalate interfere with the inhibitory role of glycosaminoglycans and semisynthetic sulfated polysaccharides in calcium oxalate crystallization? Eur Urol 31: 485– 492, 1997. 29. Conte A, Roca P, Genestar C, et al: Uric acid and its relationship with glycosaminoglycans in normal and stoneformer subjects. Nephron 52: 162–165, 1989. 30. Brundig P, Borner RH, Haerting R, et al: Glycose aminoglycane excretion and concentration in the urine of patients with frequently recurrent calcium-oxalate lithiasis prior to and following Diclofenac-Na therapy. Urol Res 18: 21–24, 1990. 31. Fellstrom B, Backman U, Danielson B, et al: Treatment of renal calcium stone disease with the synthetic glycosaminoglycan pentosan polysulphate. World J Urol 12: 52–54, 1994. 32. Senthil D, Subha N, Saravanan N, et al: Influence of sodium pentosan polysulphate and certain inhibitors on calcium oxalate crystal growth. Mol Cell Biochem 156: 31–35, 1996.
499
CLINICAL ASSOCIATION WITH URINARY GLYCOSAMINOGLYCANS AND UROLITHIASIS ERDAL ERTURK, MAUREEN KIERNAN,
AND
SUSAN R. SCHOEN
ABSTRACT Objectives. To determine the clinical association between urinary glycosaminoglycan (GAG) concentration and kidney stone disease. Methods. Thirty-five patients (14 women and 21 men) with a history of stone disease and 37 controls (13 women and 24 men) were evaluated for urinary GAG concentration. By using a new dye-binding assay, the total GAG concentration in the urine was measured and corrected to urinary creatinine levels (micrograms of GAG per milligram creatinine). Results. The mean urinary GAG concentration in those with stones was significantly lower (31.5 ⫾ 2.6 g GAG/mg creatinine) than in the controls (43.8 ⫾ 3.8 g GAG/mg creatinine, P ⫽ 0.01). Male patients with stones also had a significantly lower mean GAG concentration (26.1 ⫾ 1.8) than did the female patients (39.6 ⫾ 5.3, P ⫽ 0.009). The mean GAG concentration between ureteral (n ⫽ 13) versus renal (n ⫽ 22), single (n ⫽ 19) versus multiple (n ⫽ 16), family history (n ⫽ 11) versus no family history (n ⫽ 24), large (n ⫽ 13) versus small (n ⫽ 20), and the presence (n ⫽ 22) versus absence (n ⫽ 13) of residual stones did not show any significant differences. However, patients with recurrent stone formation (n ⫽ 21) had significantly lower mean GAG levels (26.4 ⫾ 1.6) compared with those with single stone formation (n ⫽ 14; 39.2 ⫾ 5.5, P ⫽ 0.01). Conclusions. Lower urinary GAG levels are more common in patients with stone formation. This may play a more determinant role in male patients and those with recurrent stone formation. UROLOGY 59: 495–499, 2002. © 2002, Elsevier Science Inc.
T
he formation of urinary stones can be explained by the physical and chemical properties of crystals, supersaturation, and lack of inhibitors. Thus, any imbalance can lead to nucleation, growth, and aggregation.1 Inhibitors of crystallization can be divided into two classes: small molecules (citrate and pyrophosphate) and macromolecules (glycoproteins and glycosaminoglycans [GAGs]).2 GAGs and polysaccharides, which are products of degraded proteoglycans, appear in the urine after being filtered through the glomerulus.3 Six GAGs are present in the urine: heparan sulfate, chondroitin sulfate A and C, dermatan sulfate, hyaluronic acid, and keratan sulfate. The major GAG in human urine is chondroitin sulfate.4 Several in vitro models have demonstrated the inhibitory effect of urinary GAGs on calcium oxalate stone for-
mation.5,6 Despite fairly consistent results reported with in vitro studies, significant controversy exists when comparing GAG excretion in patients with stone formation and normal controls. Experiments have revealed either no difference or significantly lower urinary levels of GAGs in patients with stone formation than in the controls.7–11 The aim of this study was to determine the total urinary GAG levels in patients with stone disease and controls using a relatively new, more accurate and reproducible dye-binding assay. Additional analyses were also made to compare the clinical parameters of patients with stones, including the presence of residual stone disease during the study, renal versus ureteral stones, positive family history, and single versus multiple stone episodes in terms of urinary GAG levels.
From the Department of Urology, University of Rochester, Rochester, New York Reprint requests: Erdal Erturk, M.D., Department of Urology, 601 Elmwood Avenue, Box 656, Rochester, NY 14642 Submitted: August 2, 2001, accepted (with revisions): November 28, 2001
MATERIAL AND METHODS
© 2002, ELSEVIER SCIENCE INC. ALL RIGHTS RESERVED
Urine samples were obtained from patients with stone disease and controls during routine clinical visits. None of the patients had had any evidence of urinary tract infection or had undergone any urologic procedures within the past 2 months. 0090-4295/02/$22.00 PII S0090-4295(01)01649-1 495
Urine samples were collected without a preservative and immediately frozen at ⫺70°C. The quantitative assays for GAG were performed with the technique described by Thuy and Nyhan.12 This method depends on measuring a change in the optical density of a mixture of azure A and azure B as a result of binding the dyes with GAG. A stock solution was made by dissolving 10 mg azure A and 10 mg azure B in 100 mL distilled water and diluted 1:10 in water for assays (working solution). Chondroitin sulfate C was used to establish a standard curve, and this was also used for the quantification of GAGs in the urine samples. The assay for total GAG was performed by adding 2.7 mL of the working solution to 100 L of urine or standards and totaling the volume up to 3 mL with water. Absorbance was read at 610 nm on a Varian DMS 200 spectrophotometer using water as a blank. The standard curve was created using chondroitin sulfate C at 1, 2, 4, 6, 8, and 20 g. The standard curve was plotted using regression analysis. Unknown total GAG values were extrapolated using the standard curve. Creatinine was assayed using the principle of the Jaffee reactions.13,14 Results were normalized against creatinine to yield the value in micrograms GAG per milligram creatinine. Thirty-five patients (14 women and 21 men, mean age 47 years, range 24 to 72) with history of stone disease were studied. An attempt was made to exclude those patients who had systemic diseases predisposing them to urolithiasis. Thirtyseven controls (13 women and 24 men, mean age 57 years, range 21 to 89) with no history of urolithiasis, urinary tract infection, or any manipulations of the urinary tract were also studied. Because of the difficulty in finding healthy female controls from the clinic patients, control urine samples were obtained from healthy hospital staff. Twenty-seven patients underwent urologic intervention that included shock wave lithotripsy, ureteroscopy, and percutaneous nephrolithotripsy. Urine samples were obtained in the clinic and were immediately frozen. Clinical stone parameters in relation to urinary GAG included number, location, family history, and current situation of the stone disease. The comparison was also made between the sexes. Statistical analysis was performed with Student’s t test.
RESULTS Stone analysis was available for 22 patients (63%). Of the 35 patients with stone disease, 18 had calcium oxalate or mixed phosphate stones; 2 were struvite and 2 were uric acid stones. There were 13 ureteral stones and 22 renal stones. Eighteen patients (51%) had undergone a metabolic evaluation and were receiving treatment. The mean urinary GAG levels in patients with stone formation was significantly lower (31.5 ⫾ 2.6) compared with the controls (43.8 ⫾ 3.8, P ⫽ 0.01) (Fig. 1). However, the mean GAG levels (26.0 ⫾ 1.6) in male patients with stone disease (n ⫽ 21) were significantly lower (P ⫽ 0.009) than those (39.6 ⫾ 5.3) of female patients with stone disease (n ⫽ 14). When the GAG levels were compared separately in the male and female patients with stone disease and controls, male patients with stones had more pronounced lower levels than did controls (26.0 ⫾ 1.6 versus 48.4 ⫾ 5.7, P ⫽ 0.0009); no differences were found between female patients with stone disease and controls (39.6 ⫾ 5.3 versus 39.2 ⫾ 5.9) (Fig. 2). Also no difference 496
FIGURE 1. Patients with history of stone disease demonstrated lower levels of mean urinary GAG concentration (P ⫽ 0.01).
FIGURE 2. Male patients with stone disease had significantly lower urinary GAG concentrations compared with female patients (P ⬍0.005). No difference was found between female patients with stone disease and female controls.
was found in the GAG levels between male (48.3 ⫾ 5.7) and female (39.2 ⫾ 5.1) controls. Additional comparisons revealed no significant differences in the GAG concentrations among ureteral (28.6 ⫾ 2.7) versus renal (33.3 ⫾ 3.7), family history (37.4 ⫾ 7.7) versus no family history (28.8 ⫾ 2.0), single (28.7 ⫾ 5.5) versus multiple (38.9 ⫾ 1.6), small size (less than 1 cm) (29.6 ⫾ 3.2) versus large (greater than 1 cm) (34.8 ⫾ 4.9), and between the presence (33.1 ⫾ 3.7) or absence (28.7 ⫾ 3.2) of residual stones. However, when patients with recurrent stone formation were compared with those with single stone episodes, the mean GAG levels were significantly lower (26.4 ⫾ 1.6 versus 39.2 ⫾ 5.5, P ⫽ 0.012). The mean GAG levels were similar in patients with single stone formation and controls (39.2 and 43.9, P ⫽ 0.51). The same number of women with stone disease had single stone episodes (n ⫽ 7) as multiple stone episodes (n ⫽ 7). Although more male patients had multiple stone episodes (n ⫽ 14) than single (n ⫽ 7), it was not significant (P ⫽ 0.32) (Table I). UROLOGY 59 (4), 2002
TABLE I. Patient characteristics Characteristic Ureteral stones Renal stones Family history of stones No family history of stones Single stone Multiple stones Small stones Large stones Residual stones No residual stones Single stone episode Multiple stone episodes
Patients (n) 13 22 11 24 19 16 20 13 22 13 14 21
Mean Urinary GAG Levels (g GAG/mg Creatinine) 28.6 27.4 37.4 28.8 28.7 38.9 29.6 34.8 33.1 28.7 39.2 26.4
⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾
3 4 7 2 2 5 3 5 4 3 2* 6*
P Value 0.46 0.12 0.24 0.36 0.42 0.01
KEY: GAG ⫽ glycosaminoglycan. * Patients with multiple stone episodes had significantly lower urinary GAG levels.
COMMENT Using a new quantitative assay, the total GAG concentration in the urine was measured. Before this, it had been shown that spot tests for the determination of GAG levels resulted in significant variability.15 The results we obtained were also corrected to the urinary creatinine levels, which has been shown to make the assay more reliable.16 This assay is not validated for infected urine samples, fresh versus frozen samples, or different temperatures. Thuy and Nyhan12 in describing their method performed validation assays for volume, NaCl, sodium phosphate buffers, pH, and recovery from spiked samples. They also performed intra and interassay variation.12 The GAGs that were included in the assay were dermatan sulfate, keratan sulfate, heparan sulfate, chondriotin-6-sulfate A, C, and hyaluronic acid. When compared with normal controls, the GAG levels were significantly lower in the patient population with stones. When urine aliquots are used, the correction of the GAG levels to creatinine measurements has been reported to be more reliable, because there is diurnal variability in the GAG excretion.16 We did not analyze 24-hour urine collections, which might have made the assay more reliable. The urine samples in this study were all obtained in the morning. Of the 35 patients with stones, 22 had their stones analyzed. Of the 22 patients, 18 had calcium oxalate and/or mixed phosphate stones, 2 had struvite stones, and 2 had uric acid stones. Only 51% of the patients in this study had documented calcium oxalate stones. Limiting the study population to only those patients with calcium oxalate stones could have resulted in more reliable findings. The stone analysis was not known before the GAG assay in our study. Although the published data indicating the inhibitory effect of urinary GAGs on stone disease are abundant,9,17 the focus was on calcium UROLOGY 59 (4), 2002
oxalate crystal nucleation and propagation.16,18,19 Clinical evidence has shown that in general a reduction of stone disease may also be associated with higher urinary GAG levels. In vitro studies have documented the inhibition of heterogeneous calcium oxalate nucleation.20 –22 Another proposed mechanism is that reduced GAG levels may result in proximal tubular epithelial cell injury, thus creating the potential for spontaneous crystal nucleation.23 When the groups were separated according to sex and a comparison was made, no difference in urinary GAG concentration was found between the female patients with stone formation and the female controls. However, a significant difference was found between the male patients with stone formation and the male controls. Other studies also did not find an association between lower urinary GAG levels and stone disease in female patients with stones.24 Conversely, the higher incidence of urolithiasis in the male population may, in part, be explained by this finding. No significant difference in the urinary GAG levels was found between female control patients (39.2 ⫾ 5.1) and male control patients (48.4 ⫾ 5.7, P ⫽ 0.29). In the present study, however, the age difference between the patients with stone disease (46.8 ⫾ 2.6 years) and the controls (55.9 ⫾ 2.9 years) was significant (P ⫽ 0.021). When the mean age was compared between male patients with stones (54.4 ⫾ 2.8 years) and male controls (62.5 ⫾ 3.1 years), a difference was noted, but it did not reach significance (P ⫽ 0.06). The mean age between the female patients with stones (35.3 ⫾ 3.1 years) and female controls (40.1 ⫾ 2.8 years) was also not statistically significant (P ⫽ 0.283). Thus, the study may have had different findings if the ages had been more comparable. It is also conceivable that if the patients with stone formation had been older, the 497
difference in the GAG levels would have been more pronounced. It is well documented in published reports that the pediatric population generally has higher levels of urinary GAG concentrations.25,26 This may also account for the relatively low incidence of urolithiasis in the pediatric population. Reports have investigated the clinical activity of stone disease and its association with urinary GAG concentrations. Despite the mixed results in published studies, we found that patients with recurrent stone formation tended to have significantly lower urinary GAG concentrations. Twenty-one of our patients (60%) were patients with recurrent stone formation, 14 (40%) had single stone episodes. No difference was found in the urinary GAG levels when comparing stone location (kidney versus ureter), stone size, presence of residual stones, and family history of urolithiasis. A recent finding indicates that shock wave lithotripsy can lead to a transient rise in urinary GAG excretion.27 Twentythree patients (66%) in our study group had undergone shock wave lithotripsy treatment. However, we were careful not to obtain urine samples from any patients within 2 months of their procedure. Additionally, in the stone study group, 5 (14%) underwent ureteroscopy, 3 (9%) percutaneous nephrolithotripsy, and 7 (20%) no treatment. It is not known how urinary GAGs affect crystal nucleation. The inherent acidic properties of the molecules has been suggested as the mechanism of action and the degree of sulfation was determined to be significant.22 It is also known that high urinary oxalate concentrations exhibit adverse affects in terms of GAG inhibitory action.28 Urinary GAGs may also have an affect on heterogeneous nucleation induced by uric acid in calcium oxalate stone genesis.29 Nonsteroidal anti-inflammatory drugs increase urinary GAG secretion and may potentiate or induce their inhibitory effects.30 These factors underscore the complexity of the interaction between urinary GAGs and clinical urolithiasis. The elucidation of the cause and effect of urinary GAGs and urolithiasis remains a challenge. However, long-term clinical trials with therapeutic agents have revealed that manipulating urinary GAGs may have a beneficial role in preventing urolithiasis.31 Studies have been performed in an attempt to identify which of the GAGs may play a role in this function. With atomic force microscopy, it was shown that chondroitin sulfate delayed maturation of calcium oxalate stones and dermatan sulfate inhibited nucleation in vitro.18 In another study, sodium pentosan polysulfate was found to have a high affinity to bind to calcium oxalate stones and thus exerted a potential inhibitory effect on crystal aggregation.32 498
CONCLUSIONS We studied the urinary GAG concentration of patients with stone disease and normal controls. Patients with stone formation had lower urinary GAG levels, which was more pronounced in patients with recurrent stone formation and in male patients. The higher stone incidence in the male population may, in part, be explained by the lack of an inhibitor theory. REFERENCES 1. Robertson WG, Peacock M, Marshall RW, et al: Saturation inhibition index as a measure of the risk of calcium oxalate stone formation in the urinary tract. N Engl J Med 294: 249 –252, 1976. 2. Worcester EM: Inhibitors of stone formation. Semin Nephrol 16: 474 – 486, 1996. 3. Hesse A, Wusel H, and Vahlensieck W: Significance of glycosaminoglycans for the formation of calcium oxalate stones. Am J Kidney Dis 17: 414 – 419, 1991. 4. Angell AH, and Resnick MI: Surface interaction between glycosaminoglycans and calcium oxalate. J Urol 141: 1255–1258, 1989. 5. Fujisawa M, Arima S, and Yachiku S: A study of inhibitory effect of chondroitin polysulfate on stone formation of calcium oxalate. Jpn J Urol 83: 1647–1654, 1992. 6. Matsuma A, and Takeuchi M: Inhibitory factor of calcium oxalate crystal growth in urinary macromolecules. Jpn J Urol 81: 883– 888, 1990. 7. Trinchieri A, Mandressi A, Luongo P, et al: Urinary excretion of citrate, glycosaminoglycans, magnesium and zinc in relation to age and sex in normal subjects and in patients who form calcium stones. Scand J Urol Nephrol 26: 379 –386, 1992. 8. Akinci M, Esen T, Kocak T, et al: Role of inhibitor deficiency in urolithiasis: rationale of urinary magnesium, citrate, pyrophosphate and glycosaminoglycans determinations. Eur Urol 19: 240 –243,1991. 9. Nesse A, Garbossa G, Romero MC, et al: Glycosaminoglycans in urolithiasis. Nephron 62: 36 –39, 1992. 10. Grases F, Llompart I, Conte A, et al: Glycosaminoglycans and oxalocalcic urolithiasis. Nephron 68: 449 – 453, 1994. 11. Sidhu H, Hemal AK, Thind SK, et al: Comparative study of 24-hour urinary excretion of glycosaminoglycans by renal stone formers and healthy adults. Eur Urol 16: 45– 47, 1989. 12. Thuy LP, and Nyhan WL: A new quantitative assay for glycosaminoglycans. Clin Chim Acta 212: 17–26, 1992. 13. Pennock CA: A modified screening test for glycosaminoglycan excretion. J Clin Pathol 22: 379 –380, 1969. 14. Gold EW: The quantitative spectrophotometric estimation of total sulfated glycosaminoglycan levels: Formation of soluble Alcian blue complexes. Biochim Biophys Acta 683: 408 – 415, 1981. 15. Brimble A, Pennock C, and Stone J: Results of a quality assurance exercise for urinary glycosaminoglycan excretion. Ann Clin Biochem 27: 133–138, 1990. 16. Michelacci YM, Glashan RQ, and Schor N: Urinary excretion of glycosaminoglycans in normal and stone forming subjects. Kidney Int 36: 1022–1028, 1989. 17. Nikkila MT: Urinary glycosaminoglycan excretion in normal and stone-forming subjects: significant disturbance in recurrent stone formers. Urol Int 44: 157–159, 1989. 18. Shirane Y, Kurokawa Y, Miyashita S, et al: Study of inhibition mechanisms of glycosaminoglycans on calcium oxalate monohydrate crystals by atomic force microscopy. Urol Res 27: 426 – 431, 1999. UROLOGY 59 (4), 2002
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