Preliminary Results of Glycosminoglycans Excretion in Normal and Stone Forming Subjects: Relationship with Uric Acid Excretion

Preliminary Results of Glycosminoglycans Excretion in Normal and Stone Forming Subjects: Relationship with Uric Acid Excretion

0022-5:347 /83/1293-0665$02.00/0 Vol. 129, IVIarch THE ,JOURNAL OF UROLOGY Copyright© 1983 by The Williams & Wilkins Co. Printed in U.S.A. PRELIMI...

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0022-5:347 /83/1293-0665$02.00/0 Vol. 129, IVIarch

THE ,JOURNAL OF UROLOGY

Copyright© 1983 by The Williams & Wilkins Co.

Printed in U.S.A.

PRELIMINARY RESULTS OF GL YCOSMINOGLYCANS EXCRETION IN NORMAL AND STONE FORMING SUBJECTS: RELATIONSHIP WITH URIC ACID EXCRETION R. CAUDARELLA,* F. STEFANI, E. RIZZOLI, N. MALAVOLTA

AND

G. D'ANTUONO

From the University of Bologna, Bologna, Italy

ABSTRACT

In calcium lithiasis, pathogenesis inhibitors have a significant role to play which permits raising of the upper metastability limit in the urine, thus reducing the crystallization processes. The aim of this work is to evaluate glycosaminoglycans excretion and concentration in a group of patients with idiopathic calcium lithiasis, and in a control group for detecting possible differences between the 2 groups. Analysis of our results shows that no significant differences exist between the 24-hour average excretion of glycosaminoglycans in normal and stone forming subjects, but there was a significant difference in the mean concentration values between the 2 groups, either as whole or when separately considered with respect to normal or increased uric acid excretion. Particularly interesting was the correlation study between glycosaminoglycans and uric acid which shows a linear relationship with a positive slope in all groups but in stone formers with hyperuricosuria. In the pathogenesis of urolithiasis, a pivotal role has been attributed to the presence of inhibitors in the urine, which increase the degree of oversaturation necessary to form a crystalline nucleus and prevent its growth once formed. The isolated inhibitors are both organic (urea, citrates) and inorganic (Mg, Zn, pyrophosphates); besides these low molecular weight substances, other organic high molecular weight compounds are present, such as the acid mucopolysaccharides, indicated as inhibitors of calcium oxalate crystallization. 1-'1 Some authors, however, deny this type of mucopolysaccharides action 4• 5 and Foye and associates 1 have even assumed that some mucopolysaccharide may increase the stone's formation. We investigated, by means of cetylpyridinium precipitation, glycosaminoglycans (GAGs) excretion in patients with idiopathic calcium lithiasis and in a control group. MATERIALS AND METHODS

Our study included 37 patients with idiopathic calcium lithiasis (25 males and 12 females, average age 44.1 ± 16.2 years) and a control group of 33 normal subjects (20 males and 13 females, average age 33 ± 10.8 years). All subjects were submitted to metabolic evaluation including determination, in plasma and urine, of calcium, phosphate, uric acid and creatinine. All subjects had normal renal function as evaluated with endogenous creatinine clearance; none had detectable proteinuria or urinary tract infection. Both oxaluria and GAGs were later measured in the urine, which for GAGs determination were collected with thymol crystals as preservative, with the addition of IO ml. of concentrated hydrochloric acid for calcium and oxalate evaluation. The Hallson and Rose method 6 was employed to determine the oxalate and GAGs assay was performed according to the Samuell method; 7 it is based on preliminary precipitation with cetylpyridinium chloride and subsequent reaction with carbazole and sulphuric acid; glycosaminoglycans levels were determined by colorimetric assay of their hexuronic acid content. A pooled normal urine was employed to study the precision of the method. Calcium, phosphate, uric acid and creatinine were evaluated with conventional methods as already described. 8 Accepted for publication December 9, 1982. * Requests for reprints: Istituto de Patologia Speciale Medica II, University of Bologna, Via Massarenti 9, 40138 Bologna, Italy. 665

GAGs/creatinine ratio was also determined to avoid the mistakes due to incorrect collections of urine. The statistical study was done with Student's t test, correlation coefficient (r) and linear regression. RESULTS

This paper reports only the results of GAGs determination and their relationship with uric acid; the other parameters were reported previously. 8 The GAGs excretion, mean values (µmol./24 hours) with standard deviations, both for renal stone forming and normal subjects, are shown in table 1, which also reports GAGs values according to normal or increased uric acid excretion. There were no significant differences in GAGs output between normal subjects and stone formers, either considering as a whole or separately, according to normal or increased uric acid excretion. Moreover, both normal and stone forming subjects with hyperuricosuria present slightly greater GAGs excretion. The concentration (µmol./1.) of GAGs in the 2 groups are reported in tables 2 and 3. Also in these cases the mean values were considered both "in toto" and separately with respect to the uricosuria values. From these data it is clear that the concentration of GAGs is statistically significantly higher in normal subjects than in stone formers. Considering the stone formers group only, the concentration mean value is lower in patients with hyperuricosuria than in those with normal uric acid excretion (t = 1.51; p < 0.10 N.S.). Furthermore the GAGs/creatinine concentration ratio was within the same range both in normal and stone forming subjects, while being, in the latter group, statistically lower in patients with hyperuricosuria. These last results induced us to consider the problem of the relationship between GAGs and uric acid since literature data has pointed out that the urate can bind or absorb urinary inhibitors. Thus a linear relationship with a positive slope has been observed between daily uric acid and GAGs excretion (r = 0.5; p < 0.01) as well as between their concentration (r = 0.62; p < 0.01) (figs. 1 and 2). Moreover, if 24 hours excretion of GAGs and uric acid are correlated only in normal subjects with hyperuricosuria (fig. 3), this figure is even more significant (r = 0.81; p < 0.001). In stone formers too, a positive linear relation is observed, though less significant, between daily GAGs and uric acid excretion (r = 0.35; p < 0.05). However considering the stone

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CAUDARELLA AND ASSOCIATES

TABLE

1. Urinary GA Gs excretion in healthy subjects and stone

formers*

y

GAGs

=0.06x - 23.61

r = a.a,

.

J.Jmol/24h

Mean Values

60

• 1425

GAGs µmol./die Total

Normouricosuric

Hyperuricosuric

31.56 ± 10.26 30.52 ± 9.29

29.22 ± 6.53 28.54 ± 8.25

34.36 ± 13.16 35.6 ± 10.28

50

Healthy subjects Stone formers

40

* Values are expressed as the mean ± standard deviation. 30

TABLE

2. Urinary GAGs concentration in healthy subjects and stone formers*

20

Mean Values GAGs µmol./1.

10

Healthy subjects Stone formers P value

Total

N ormouricosuric

Hyperuricosuric

29.61 ± 12.26 20.64 ± 10.06 0.0025

29.26 ± 12.61 22.34 ± 12.1 0.05

29.19 ± 12.26 17.97 ± 4.84 0.0025

* Values are expressed as the mean ± standard deviation. TABLE

600

900

1000

1200

1100

URIC ACID ms/24h

FIG. 3. Relationship between urinary GAGs and uric acid excretion in healthy subjects with hyperuricosuria.

3. Urinary GAGs/creatinine concentration ratio*

Healthy subjects Stone formers

Total

Normouricosuric

Hyperuricosuric

2.51 ± 1.03 2.63 ± 1.5

2.58 ± 1.13 2.74 ± 1.58

2.42 ± 0.93 2.13 ± 0.78

GAGs pmol/24h

y =0.03x

60

r

+ 10.54

= o.56

* Values are expressed as the mean ± standard deviation.

..

50

GAGs

y

pmol/24h

=0.03x

+

12.06

r = o.s

40

60

50

30

40

20 30

10 20

0

10

400

200

600

800

URIC ACID 100

200

300

400

500

600

700

800

900

1000

FIG. 1. Relationship between urinary GAGs and uric acid excretion in healthy subjects. GAGs

Y =0.03x

).lmol/1

r = o.s2

mg/24h

1100

URIC ACID ms/24h

+ 7.36

FIG. 4. Relationship between urinary GAGs and uric acid excretion in stone formers with normouricosuria. GAGs p moi/1

y

=0.03x + 4.26

r :;: 0.11

60

60

50 50

40 40

30

30

20

20

10

10

10

20

30

40

50

60

70

60

90

100

110

URIC ACID mg/ctl

FIG. 2. Relationship between urinary GAGs and uric acid concentration in healthy subjects.

forming group separately according to normal or increased uric acid excretion, the behavior observed was different; in fact, while the stone formers with normal uric acid excretion show a positive linear relationship between GAGs and uric acid excretion (r = 0.56; p < 0.01) (fig. 4) as well as between their concentration (r = 0.71; p < 0.01) (fig. 5), patients with hyperuricosuria do not show this kind of relation but even an opposite tendency.

0 10

20

30

40

50

60

70

80

URIC ACID m9/ctl

FIG. 5. Relationship between urinary GAGs and uric acid concentration in stone formers with normouricosuria. DISCUSSION

In idiopathic calcium lithiasis pathogenesis inhibitors have a significant role which permits rising of the upper metastability limit in urine, thus reducing the crystallization processes. According to some authors, 9 • 10 patients with idiopathic calcium stones would tend to have reduced inhibitors activity compared to normal subjects, when assayed in vitro, while others 11 could

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GYCOSAMINOGL YCANS AND URIC ACID SECRETION

not confirm this observation. According to Robertson and associates.1 the reduction in inhibitors activity should be related to a decrease of acid mucopolisaccharides in about 50 per cent of cases. In these patients, most of whom show, besides the higher calcium and oxalate excretion, a tendency to hyperuricosuria, the real concentration of acid mucopolysaccharides would be reduced by absorption of a part of them on the surface of colloidal particle of urate. 12 Due to this reduction of GAGs in the urine of patients with idiopathic calcium lithiasis, a lower amount is available to prevent the calcium oxalate crystals growth and aggregation and a reduced inhibitory activity is determined in the urine. 12 Analysis of our results shows that non-significant differences exist between the mean excretion of GAGs in the normal and stone-forming subjects, either as a whole or when separately considered, according to normal or increased uric acid excretion. However subjects with hyperuricosuria (stone forming as well as normal subjects) show a tendency to slightly higher excretion, though this figure does not reach statistical significance. To better evaluate this behavior, GAGs concentration was studied in both groups always evaluating the excretion values of uric acid. A significant statistical difference in the average concentration values was observed between normal people and stone-formers both when considered as a whole (t = 3.26; p < 0.0025) and separately with respect to the presence or not of increased uric acid excretion (t = 1.71: 0.05 < p < 0.1; t = 3.42: p < 0.0025). Thus the lack of a significant difference in GAGs excretion between the 2 groups seems to be explained by the different urinary volume, greater in stone formers (1.24 ± 0.43 ml./minute) than in normal subjects (0.67 ± 0.21 ml./minute). Particularly interesting was the correlation study between GAGs and uric acid excretion. In fact it is possible to observe a linear relationship with a positive slope between GAGs and uric acid excretion in normal subjects both when considered as a whole (fig. 1) and particularly when hyperuricosuric subjects were evaluated (fig. 3). On the contrary, 2 different courses can be observed in the group of stone formers: while patients with uric acid excretion within normal range show a positive linear correlation between the 2 parameters (r = 0.56; p < 0.01) (fig. 4), those with increased uric acid excretion show an opposite tendency. This result is also confirmed by the study of GAGs and uric acid concentration relationship (figs. 5 and 6) and by the comparison of GA Gs concentration mean values in patients with normal or increased uric acid excretion (tables 2 and 3). Stone formers with hyperuricosuria show a lower GAGs concentration (unlike normal subjects where no modifications are observed) probably due not only to a dilution effect tied up with greater urinary volume, but also to a real decrease in their concentration, as the lower GAGs/creatinine ratio in this group of patients would suggest (tables 2 and 3). Should this reduction in GAGs concentration be related to a link or absorption the urate, this cannot be affirmed by the results of the present study. This study of the relationship between GAGs and uric acid would however suggest that normal and stone forming subjects behave differently when a hyperuricosuric condition is present.

GAGs J-! mol/1

y;;;-Q.0003 )(

60

.,. 18.13

i" = M.S

50

40

30

20

10

w

~

w

ro

~

URIC ACID

mg/di

Fm. 6. Relationship between urinary GAGs and uric acid concentration in stone formers with hyperuricosuria. REFERENCES 1. Foye, W. 0., Hong, H. S., Kim, C. M. and Prien, E. L., Sr.: Degree

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3.

4.

5.

6. 7. 8.

9.

10.

11.

12.

of sulfation in mucopolysaccharide sulfates in normal and stoneforming urines. Invest. Urol., 14: 33, 1976. Gardner, G. L. and Doremus, R. H.: Crystal growth inhibitors in human urine. Effect on calcium oxalate kinetics. Invest. Urol., 14: 478, 1978. Robertson, W. G., Peacock, M., Marshall, R. W., Marshall, D. H. and Nordin, B. E. C.: Saturation inhibition index as a measure of the risk of calcium oxalate stone-formation in the urinary tract. N. Engl. J. Med., 294: 249, 1976. Gill, W. B. and Roma, M. J.: Determinants of supersaturation levels and crystallization rates of calcium oxalate from urines of normal humans and stone-formers: effects of nondialyzable materials. J. Surg. Res., 21: 45, 1976. Ito, H. and Shimizaki, J.: High molecular weight substances which inhibit calcium oxalate crystal growth. Four studies on the role of urinary pepsinogen and mucopolysaccharide as inhibitors of calcium oxalate crystal growth. Jpn. J. Urol., 69: 1155, 1978. Hallson, P. C. and Rose, G. A.: A simplified and rapid enzymatic method for determination of urinary oxalate. Clin. Chim. Acta, 55: 29, 1974. Samuell, C. T.: A study of glycosaminoglycan excretion in normal and stone-forming subjects using a modified cetylpyridinium chloride technique. Clin. Chim. Acta, 117: 63, 1981. Caudarella, R., Stefani, F., Rizzoli, E., Buli, P. and D'Antuono, G.: Determinazione enzimatica del l'ossaluria: esperienze preliminari in un gruppo di pazienti con litiasi calcica. 54th Congresso Nazionale della Soc. Ital. di Urologia, Bologna 1981. Baumann, J. M., Bisaz, S., Felix, R., Fleisch, H., Ganz, U. and Russell, R. G. G.: The role of inhibitors and other factors in the pathogenesis of recurrent calcium-containing renal stones. Clin. Sci. Mol. Med., 53: 141, 1977. Robertson, W. G. and Peacock, M.: Calcium oxalate crystalluria and inhibitors of crystallization in recurrent renal stone formers. Clin. Sci., 43: 499, 1982. Rose, M. B.: Renal stone formation. The inhibitory effect of urine on calcium oxalate precipitation. Invest. Urol., 12: 428, 1975. Robertson, W. G., Knowles, C. F. and Peacock, M.: In: Urolithiasis Research. Edited by H. Fleisch, W. G. Robertson, L. H. Smith and W. Valhensieck. New York, London: Plenum Press, p. 331, 1976.