Trace Elements and Urinary Stone Formation: New Aspects of the Pathological Mechanism of Urinary Stone Formation

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THEJOURNAL OF UROLOGY Copyright O 1991 by AMERICANUROLOGICAL ASSOCIATION, INC

Voi. 1-15, 93-96, January 1991 Printed in U.S.A.

TRACE ELEMENTS AND URINARY STONE FORMATION: NEW ASPECTS OF T H E PATHOLOGICAL MECHANISM OF URINARY STONE FORMATION J. HOFBAUER, I. STEFFAN, K. HOBARTH, 6. VUJICIC, H. SCHWETZ, G. REICH 0 . ZECHNER

AND

From the Department of Urology, Institute of Analytical Chemistry, University of Vienna, Vienna, Austria, and Institute for Testing of Materials, Glattbrugg, Switzerland

ABSTRACT

The urinary stone, serum and 24-hour urine concentrations of 14 trace elements were determined in urinary stone patients by inductively coupled plasma atomic-emission spectroscopy. The data obtained for 25 active stone patients and 32 whose last stone episode had occurred at least 12 months previously were compared with those of 25 healthy individuals. Urinary nickel, manganese and lithium excretion, and serum nickel, manganese and cadmium concentrations were statistically significantly lower for active stone patients compared to those with previous stone episodes and healthy individuals. No difference in the concentrations of trace elements could be found, however, for patients with previous stone episodes and healthy individuals. Nickel, manganese, lithium and cadmium could be of significance in the pathological mechanism of stone formation, not from mineralogical or crystallographic viewpoints but for the smooth flow of enzymatic reactions in biological systems. KEYWORDS: urinary calculi; spectrometry, x-ray emission

Despite years of intensive research the pathological mechanism of urinary stone formation remains largely unexplained and, thus, cannot be used in the prophylaxis of stone formation. Initial conclusions concerning stone formation had been anticipated from more precise methods of analysis. In addition to the well known main components of stones, much attention was paid to trace elements. After proof of their presence in urinary stones had been furnished, their possible influence on urinary stone formation in vitro was investigated from mineralogical and crystallographical viewpoints. The in vitro trials demonstrated inhibitory as well as stimulating influences of individual trace elements on the crystallization Unfortunately, the predominantly unphysiologically high concentrations do not permit any definite conclusion concerning the in vivo effect of trace elements on stone formation. Meyer and Angino demonstrated, on the other hand, that the physiological concentration of individual trace elements in urine has no influence on crystal growth of calcium oxalate or calcium phosphate stones.1° The trace element concentrations in various categories of stones also are stated by others as being extremely variable."-l8 The reason for this is probably the use of different analytical methods for their determination. In addition, no definite statement can be made regarding the period before stone formation. It also should be considered that the importance of trace elements is not mainly a mineralogical or crystallographic problem but, rather, it is to be found in the smooth running of vital functions in biological systems. Essential trace elements serve as cofactors for enzymes, without which enzymatically controlled reaction chains cannot ocTherefore, we determined these elements in urinary stone patients not only in urinary stones but in 3 different areas: stones. urine and serum. MATERIALS AND METHODS

The trace element concentration in stones, serum and 24hour urine of 25 active stone patients (group 1, 13 men and 12 women 52.8 18 years old) was determined. The stone episode occurred a maximum of 2 weeks before the study. Stone analysis Accepted for publication June 14, 1990.

+

was performed with x-ray diffractometry. Urinary stones were either passed spontaneously or removed operatively. Serum and urine were sampled as soon as possible after stones had been procured, and precautions were taken to ensure that the urine was sterile and no medication had been given (on the average 1to 2 weeks after stones had been obtained). The patients were on a normal diet without any limitations. Group 2 consisted of 26 healthy controls (15 men and 11 women 37 + 13 years old) who had no history of stone episodes and whose urine had no pathological findings. Group 3 consisted of 32 patients (21 men and 11women 49.1 f 17 years old) who had already had urinary stones but were free of calculi a t examination. The last stone episode had occurred a t least 12 months previously. The 14 trace elements analyzed included lithium (Li), aluminum (Al), titanium (Ti), chromium (Cr), manganese (Mn), iron (Fe), cobalt (Co), nickei (Ni), copper (Cu), zinc (Zn), strontium (Sr), molybdenium (Mo), cadmium (Cd) and lead (Pb). The determinations were performed with inductively coupled plasma atomic-emission spectrometry (ICP-AES). A simultaneous sequential ARL (3580-ICP) spectrometer was used and wavelengths without interference were selected. Stray light effects were eliminated by means of background correction. The sample aspiration rate was 1.4 ml. per minute, which was kept constant by means of a peristaltic pump. Measuring design parameters: ARL-3580 ICP. A 1 m. Paschen-Runge spectrometer (simultaneous and sequential) with 1,080 grooves per mm., 1 to 4 order and 39 simultaneous channels was used. The device included a Himmelwerk, free running HF-generator with a Fassel type torch and mass flow controlled argon gas flows (1. per minute, outer 12, intermediate 0.8, carrier 1). The observation height was 15 mm. above the coil. Data were processed on a DEC LS-11 computer. U r i n e a n d s e r u m samples. Two different methods were applied to decompose the samples. Wet Combustion in an Open Container: A 20 ml. urine sample was diluted with 5 ml. nitric acid and 2 ml. hydrogen peroxide and evaporated. The treatment was repeated with 5 ml. nitric acid. The final volume for determination with the ICP-AES unit was 10 ml. Serum samples were decomposed by the reaction of 5 ml. serum with 3 ml. nitric acid and 1 ml.

94

H O F B A U E R AN1D ASSOCIATES

hydrogen peroxide, and repeating the treatment after addition of 5 ml. nitric acid. The volume of the measuring solution was 5 ml. Combustion in a CEM Microwave Decomposition Apparatus (MDS-ID): A 20 ml. urine or 5 ml. serum sample was diluted with 5 ml. nitric acid and decomposed for 3 minutes a t 50% of machine power, then for 1 minute a t 100% and finally for 10 minutes a t 70%. The final volume corresponded to that in method 1. Kidney stone samples. Kidney stones (1to 2 gm.) were dried for 3 hours a t 100C, ground and dried for 3 more hours a t 100C, and aliquots subsequently were obtained. Then, 5 ml. aqua regia were added to the samples and evaporated. In the case of phosphate stones the proper amount of hydrogen peroxide (30%) required for complete decomposition was added. The final volume was 10 ml. Scandium was used as an internal standard to correct varying acid and sample concentrations. Data were evaluated statistically by the Wilcoxon rank sum test (significance level 0.05). RESULTS

Our method of analysis permitted evidence of almost all trace elements under investigation in every individual urinary stone. Concentrations were only sporadically below the limit of detection (see table). In contrast to the numerous prior reports, we were unable to find any statistically significant differences in concentration for a particular trace element for different stone categories. Even additional investigations of 25 further urinary stones provided no evidence of differences. Similarly, it was not possible to establish either statistically significant correlations between urinary stone concentration and urinary concentration for any of the 1 4 trace elements investigated, or correlations between serum and urinary stone concentrations. However, significant differences were calculated for all 3 groups for Ni, Mn, Li and Cd concentrations in serum and urine. Figure 1 shows the differences in urine concentrations among the 3 groups and figure 2 shows the values for the serum concentrations. There is a statistically highly significant difference between groups 1 and 2, and between groups 1 and 3, yet groups 2 and 3 do not differ as far as the trace elements referred to are concerned. Ni in serum is the only exception. For this element there is also a difference between groups 2 and 3, since the concentrations in group 3 are elevated compared with group 2. However, this could be traced back to the somewhat younger

subjects in group 2. As reported in the literature concentrations of some trace elements are remarkably constant throughout most of life. There is no tendency for decrease or accumulation to occur with age.21a22Nevertheless, it is possible that at this time too little information about this exists. However, since groups 1and 3 are comparable in sex and patient age, and these elements nonetheless differ significantly, it is unlikely that the somewhat younger control group represents a source of error. More likely, it may be assumed that the difference between the patients and the healthy controls will become even clearer, since the control group exhibits lower concentrations than group 3. DISCUSSION

As techniques for the analysis of trace elements improve, they gain in significance for the evaluation and necessity of regular processes in biological systems. However, the knowledge about trace elements is poor. T o date only the significance in the crystallographic and mineralogical aspects of urinary stone formation has been investigated. Our ,results show that when sensitive methods of analysis are used, almost all trace elements under investigation are found in urinary stones as long as the latter are present in urine. The concentrations in urinary stones vary considerably and regularity concerning the various categories of stones cannot be established. The same is true for the concentrations in urine. This statement is contradictory to numerous earlier r e p o r t ~ . l ~ ,We l ~ - explain ~~ this fact by the unknown interval for stone formation and stone growth. Because of the minimal concentrations of trace elements compared with the main components of urinary stones, the influence on the crystallization process in urine appears to be of secondary importance. The inhibitory and/or stimulating effect of individual trace elements demonstrated in vitro may have originated by the use of higher concentrations physiologically present. This opinion is also held by Meyer and Angino.l0Being aware of the previous reports on trace elements and urinary stone formation, we aimed a t extending the investigation to 3 separate areas-serum, urine and urinary stones. This had already been tried by Joost aqd Tessadri and no correlation was found.17 To us, however, it seems that the small number of analyses, in most of which the limit of detection was not achieved, are not able to prove anything definite. Inductively coupled plasma atomic-emission spectroscopy represents a suitable, precise method for ultra trace analysis, which when a

Trace element concentr ation i n urinary stones , ,. rL.

No,

Stone Chemistry

1 2 3 4 5 6 7 8 9 10

Whewellite-apatite Whewellite-wheddellite Uric acid Apatite-struvite Apatite-struvite Apatite-struvite Whewellite Uric acid Apatite Whewellite Wheddellite-apatite Apatite Whewellite Whewellite-apatite Whewellite-uric acid Apatite Whewellite Whewellite Whewellite-uric acid Whewellite-wheddellite Whewellite-uricacid Whewellite-uricacid Wheddellite Apatite

Trace Elements (ue.lem.) .,-,. Mo

11

12 13 14 15 16 17 18

1s

20 21 22 23 24

25 Oxalatp

Ni

Li

1.6780 1.7944 0.1333 1.2188 2.54455 3.08421 0.3961 0.5433 0.7954 1.8225 0.5051 1.3778 0.6590 0.0200 15.6953 0.33957 0.0193 0.0599 0.1768 0.5895 0.4226 0.4938 16.626 22.59 0.7591 0.5440 0.1816 0.0295 0.2697 1.4154 0.5312 0.7509 8.0183 0.3301 0.4676 0.9107 0.7309 1.3155 4.2522 0.1375 0.5416 0.6452 1.6760 1.56790 16.339 5.4108 160.81 0.1173 0.2374 12.19 126.27 5.9700 1.6197 1.5914 0.0824 1.4989 2.9250 0.0410 10.827 51.104 26.722 0.8240 0.8126 0.7878 3.8310 0.3963 0.9810 0.2851

Zn 379.25 371.28 5.04457 304.11 561.19 10.004 113.066 2.8747 168.31 205.91 4.3681 841.00 45.509 770.34 93.231 137.11 193.591 -

662.19 5987.4 39.398 35.013 1333.1 554.84 731.50

Sr

Cd

Pb

A1

Fe

26.342 32.538 39.643 10.372 2.63957 21.2376 8.2373 25.357 135.33 0.0687 11.930 273.19 0.0453 24.666 21.437 128.35 54.098 2.0967 6.1713 0.4366 67.5767 24.1338 54.8688 150.195 32.957 0.6390 0.0335 1.4057 3.3171 25.315 153.21 0.0093 19.050 7.1266 17.69 2.0095 51.480 64.175 168.78 0.0338 3.16 5.6785 18.311 133.02 0.0885 17.863 11.267 22.210 6.5133 43.071 66.875 189.50 182.49 0.6345 25.783 16.867 49.361 8.8907 0.3069 23.426 47.567 142.49 343.42 0.0363 13.469 6.4449 11.214 51.2225 39.6157 9.14399 40.4344 628.22 167.07 483.99 7.2680 14.302 88.118 1381.4 8.1113 867.97 178.58 1388.2 6.53840.0173 7.8768 18.976 50.187 56.693 0.0082 6.4052 10.499 26.390 9.0512 65.637 521.82 190.72 0.4073 32.514 31.061 38.847 0.8934 3.2977 66.332 419.34 175.75 0.3091 153.95 64.113 38.357

Cr 1.65 9.35 7.3242 3.673 1.355 1.394 33.944 3.215 1.498 5.089 0.433 0.564 0.689 0.96 10.49 1.499 3.669 24.15 -

191.20 1.456 3.346 77.407 2.5373 4.1315

Mn

Cu

Co

0.5944 11.667 0.7099 0.6230 3.8601 0.1555 3.31541 0.54052 0.9203 4.8112 0.3570 0.7700 39.486 0.3317 2.3709 20.463 0.1655 4.83467 22.7088 0.91915 0.1747 0.585 0.0441 0.2322 4.7395 0.3612 1.4668 17.327 2.2627 0.1532 0.8795 0.1030 0.2017 3.0217 0.0828 0.9552 9.7521 0.9216 0.3636 4.1559 0.1816 2.1048 13.492 0.5735 0.1599 3.8831 0.1409 0.53834 6.46218 0.20039 5.1304 81.547 1.1522 0.2381 9.3238 0.7525 29.517 122.19 0.6592 4.2784 0.0345 0.2677 19.325 0.2014 9.4271 17.821 27.297 0.5630 9.6361 0.4311 1.7833 13.559 1.8622

Ti 1.0079 0.3807 0.05401 0.3072 1.7153 0.3873 1.41393 0.1454 0.5670 2.0842 0.2091 0.8115 4.5368 2.8711 1.2361 0.1680 0.42202 14.406 0.3412 31.949 0.6522 0.3655 29.102 8.3476 2.1290

TRACE E L E M E N T S AND URINARY STONE FORMATION L i in URINE

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Mn in SERUM

SERUM

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FIG. 2. Serum concentration of Mn, Ni and Cd in active stone patients (group 11, healthy controls (group 2) and patients with history of urolithiasis (group 3).

simultaneous sequential apparatus is used has the advantage of requiring smaller sample quantities for multiple analysis.23 We also were able to demonstrate a high percentage of all 14 trace elements in serum, urine and urinary stones. Variable normal values in the literature led us t o set up a separate healthy control group. A correlation between trace element content in serum and urine, serum and urinary stones or urine and urinary stones could be found in the group of active stone patients. A highly significant difference was noted, however, in the excretion of urinary Ni, Mn and Li, as well as in serum concentrations of Ni, Mn and Cd between urinary stone patients and the healthy control group. This interesting observation caused us to perform the same investigation for patients with a history of stones but who were without stones for 1year. Of the 14 trace elements investigated exactly the same elements were of importance-Ni, Mn and Li in urine, and Ni, Mn and Cd in serum. This third group differed highly significantly from the patient group but not from the healthy controls. These results permit the assumption that trace elements may be of significance in the formation of urinary stones. To date little has been known about the biological significance of these trace elements. However, there are statements in the literature that,

for example, Mn is supposed to be of central importance as a coenzyme for the synthesis of the mucopolysaccharides."O Similarly, a reverse relationship to calcium absorption in the gut has been reported.20This and many other aspects still must be investigated to find a possible connection between trace elements and urinary stone formation, and to clarify the pathological mechanism of urinary stone formation. Accordingly, this study should not be regarded as an attempt for a solution but as a stimulus to intensive basic research in this field. REFERENCES

1. Hesse, A,, Berg, W., Schneider, H. J. and Hienzsch, E.: A contri-

bution to the formation mechanism of calcium oxalate urinary calculi. I. Stabilising urinary constituents in the formation of weddellite. Urol. Res., 4: 125, 1976. 2. Hesse, A,, Berg, W., Schneider, H. J. and Hienzsch, E.: A contribution to the formation mechanism of calcium oxalate urinary calculi. 11. In vitro experiments concerning the theory of the formation of whewellite and weddellite urinary calculi. Urol. Res., 4: 157, 1976. 3. Berg, W., Hesse, A. and Schneider, H. J.: A contribution to the formation mechanism of calcium oxalate urinary calculi. 111. On the role of magnesium in the formation of oxalate calculi. Urol. Res., 4: 161, 1976.

96

HOFBAUER AN D ASSOCIATES

4. Hesse, A., Dietze, H. J., Berg, W. and Hienzsch, E.: Mass spectrometric trace element analysis of calcium oxalate uroliths. Eur. Urol., 3: 359, 1977. 5. Elliot, J. S. and Eusebio, E.: Calcium oxalate solubility: the effect of trace metals. Invest. Urol., 4: 428, 1967. 6. Eusebio, E. and Elliot, J. S.: Effect of trace metals on the crystallization of calcium oxalate. Invest. Urol., 4: 431, 1967. 7. Sutor, D. J. and Wooley, S. E.: Growth studies of calcium oxalate in the presence of various compounds and ions-11. Brit. J. Urol., 42: 296, 1970. 8. Sutor, D. J.: Growth studies of calcium oxalate in the presence of various ions and compounds. Brit. J. Urol., 41: 171, 1969. 9. Welshman, S. G. and McGeown, M. G.: A quantitative investigation of the effects on the growth of calcium oxalate crystals on potential inhibitors. Brit. J . Urol., 44: 677, 1972. 10. Meyer, J. L. and Angino, E. E.: The role of trace metals in calcium urolithiasis. Invest. Urol., 14: 347, 1977. 11. Wandt, M. A. E., Pougnet, M. B. A. and Rodgers, A. L.: Analysis of urinary calculi by inductively coupled plasma atomic emission spectroscopy: new insight into stone structure. In: Urolithiasis and Related Clinical Research. Edited by P. 0. Schwille, L. H. Smith, W. G. Robertson and W. Vahlensieck. New York: Plenum Press, p. 699, 1985. 12. Wandt, M. A. E., Pougnet, M. A. B. and Rodgers, A. L.: Determination of calcium, magnesium and phosphorus in human stones by inductively coupled plasma atomic-emission spectroscopy. Analyst, 109: 1071, 1984. 13. Wandt, M. A. E. and Pougnett, M. A. B.: Simultaneous determination of major and trace elements in urinary calculi by microwave-assisted digestion and inductively coupled plasma atomic emission spectrometric analysis. Analyst, 111: 1249, 1986. 14. Scott, R., East, B. W., Janczyszyn, J., Boddy, K. and Yates, A. J.: Concentration and distribution of some minor and trace elements in urinary tract stones: a preliminary study. Urol. Res., 8: 167, 1980. 15. Durak, I., Yasar, A., Yurtarslani, Z., Akpoyraz, M. and Tasman, S.: Analysis of magnesium and trace elements in urinary calculi by atomic absorption spectrophotometry. Brit. J. Urol., 62: 203, 1988. 16. Levinson, A. A,, Nosal, M., Davidman, M., Prien, E. L., Sr., Prien, E. L., Jr. and Stevenson, R. G.: Trace elements in kidney stones from three areas in the United States. Invest. Urol., 15: 270, 1978. 17. Joost, J , and Tessadri, R.: Trace element investigations in kidney stone patients. Eur. Urol., 13: 264, 1987. 18. Schneider, H. J. and Hesse, A,: Spurenelemente in Harnsteinen: Lithium. Zeitschrift. Urol. Nephrol., 64: 759, 1971. 19. Pethes, G.: The need for trace element analyses in the animal sciences. In: Elemental Analysis of Biological Material: Current Problems and Techniques With Special Reference to Trace Elements. Vienna: IAEA, Technical Reports Series No. 197, pp. 3-18, 1980. 20. Hambidge, K. M.: The need for trace element analyses in the medical sciences. In: Elemental Analysis of Biological Material: Current Problems and Techniques With Special Reference to

Trace Elements. Vienna: IAEA, Technical Reports Series No. 197, pp. 19-28, 1980. 21. Schroeder. H. A.. Balassa. J . J. and Tiuton, I. H.: Abnormal trace metals in man- nickel.'^. Chron. is., 15: 51, 1962. 22. Schroeder, H. A,, Balassa, J. J. and Tipton, I. H.: Essential trace metals in man: manganese. A study in homeostasis. J. Chron. Dis., 19: 545, 1966. 23. McLeod, C. W., Worsfold, P. J. and Cox, A. G.: Simultaneous multi-element analysis of blood serum by flow injection-inductively coupled plasma atomic-emission spectrometry. Analyst, 109: 327,1984. EDITORIAL COMMENTS The authors demonstrate that significant differences in the serum concentration and urinary excretion of various trace elements exist between control and stone patients. The role of these trace elements in the pathogenesis of stone formation is as yet unclear. It would be helpful to know how much daily variation exists for trace element excretion in a single patient, and whether excretion is altered by urinary obstruction. Measurements obtained 1to 2 weeks after an acute stone episode may be misleading. In examining the various figures it is interesting to note that the mean urinary excretion or serum concentration of trace elements for the nonstone-bearing controls usually falls somewhere between the levels of the active stone patients and those with a history of urolithiasis. This is contrary to what would be expected. If decreased levels of trace elements are truly important in the pathogenesis of calculous formation one would logically expect active stone patients to have the lowest levels, patients with a history of urolithiasis to have intermediate levels and the control group to have the highest levels. The reasons for this discrepancy are unclear. John R. Burns Division of Urology University of Alabama Birmingham, Alabama The authors have made the interesting observation that certain trace elements are significantly lower in the blood and urine of active stone patients compared to normal subjects and metabolically inactive stone patients. Unfortunately, they have made only observations concerning the decreased levels of these trace elements and have not been able to correlate these findings significantly with any etiological or pathogenetic mechanisms that would explain why trace elements might have a role in stone formation. Are they implying that these trace elements may have a role in certain metabolic functions that might determine excretion of specific stone-forming risk factors? Further correlation between trace elements and stone-forming risk factors must be made before one can conclude that trace elements have a significant role in stone formation. Glenn M. Preminger Division of Urology University of Texas Southwestern Medical Center Dallas, Texas