Strontium Substitution for Calcium in Lithogenesis

Sarah D. Blaschko, Thomas Chi, Joe Miller, Lawrence Flechner, Sirine Fakra,Pankaj Kapahi, Arnold Kahn and Marshall L. Stoller*,†From the Department of Urology, University of California-San Francisco (SDB, TC, JM, LF, MLS), San Francisco, Advanced Light Source,Lawrence Berkeley National Laboratory (SF), Berkeley and Buck Institute for Research on Aging (PK, AK), Novato, California

Purpose: Strontium has chemical similarity to calcium, which enables the replace-ment of calcium by strontium in biomineralization processes. Incorporating stron-tium into human bone and teeth has been studied extensively but little research hasbeen performed of the incorporation of strontium into urinary calculi. We usedsynchrotron based x-ray fluorescence and x-ray absorption techniques to examinethe presence of strontium in different types of human kidney stones.Materials and Methods: Multiple unique human stone samples were obtainedvia consecutive percutaneous nephrolithotomies/ureteroscopies. A portion of eachstone was sent for standard laboratory analysis and a portion was retained forx-ray fluorescence and x-ray absorption measurements. X-ray fluorescence andx-ray absorption measurements determined the presence, spatial distributionand speciation of strontium in each stone sample.Results: Traditional kidney stone analyses identified calcium oxalate, calciumphosphate, uric acid and cystine stones. X-ray fluorescence measurements iden-tified strontium in all stone types except pure cystine. X-ray fluorescence elemen-tal mapping of the samples revealed co-localization of calcium and strontium.X-ray absorption measurements of the calcium phosphate stone showed stron-tium predominately present as strontium apatite.Conclusions: Advanced x-ray fluorescence imaging identified strontium in all cal-cium based stones, present as strontium apatite. This finding may be critical sinceapatite is thought to be the initial nidus for calcium stone formation. Strontium is notidentified by standard laboratory stone analyses. Its substitution for calcium can bereliably identified in stones from multiple calcium based stone formers, which mayoffer opportunities to gain insight into early events in lithogenesis.

Key Words: kidney; urolithiasis; strontium; calcium; spectrometry,

Abbreviations

and Acronyms

FTIR � Fourier transform infraredspectroscopy

Accepted for publication August 24, 2012.Supported by American Federation for Aging

Research, California Urology Foundation and Na-tional Institutes of Health Grants R01 AG031337-01A1, RO1 AG038688, RL1 AAG032113 and P01AG025901-S1 (PK), and K12-DK-07-006 from theMultidisciplinary K12 Urologic Research CareerDevelopment Program (TC), a grant from theAmerican Urological Association Foundation Re-search Scholars Program and Boston ScientificCorp., The Endourological Society and “Friends ofJoe.” The Advanced Light Source is supported bythe Director, Office of Science, Office of BasicEnergy Sciences of the United States Departmentof Energy under Contract DE-AC02-05CH11231.

* Correspondence: Department of Urology,University of California-San Francisco, 400 Par-nassus Ave., A610, San Francisco, California94143 (telephone: 415-476-1611; FAX: 415-476-8849; e-mail: mstoller@urology.ucsf.edu).

† Financial interest and/or other relationshipwith Bard, Cook, Boston Scientific, EM Kineticsand Ravine Group.

x-ray emission

STRONTIUM is a divalent cation that islocated in the same column of the pe-riodic table of the elements as cal-cium. Thus, strontium has chemicalproperties and interactions similar tothose of calcium. Strontium exists as4 stable isotopes (84Sr, 86Sr, 87Sr and88Sr), of which 87Sr is radioactive. Theseisotopes have a known geographic dis-tribution. Thus, evaluating the ratioof the 4 isotopes present in bones or

uroliths can be used to fingerprint

0022-5347/13/1892-0735/0THE JOURNAL OF UROLOGY®

© 2013 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION AND RES

strontium exposure. Additional un-stable radioactive strontium isotopesalso exist. Indeed, 90Sr levels were tracedin milk across the United States in thelate 1950s and early 1960s during theera of nuclear weapon testing.1 Thestrontium-to-calcium ratio in teethand bone has been used to help eval-uate the strontium exposure and geo-graphic mobility of Neolithic Homosapiens.2 Although there is a global

differential distribution of strontium,

http://dx.doi.org/10.1016/j.juro.2012.08.199Vol. 189, 735-739, February 2013

EARCH, INC. Printed in U.S.A.www.jurology.com 735

STRONTIUM SUBSTITUTION FOR CALCIUM IN LITHOGENESIS736

regional evaluation of trace elements present instones in the United States have yielded no differ-ence, which is likely attributable to the geographicmobility of the population.3 The presence of radioac-tive strontium in biomineralization can serve as amarker of radioactivity exposure.4 The radioactiveisotope of strontium has been used as a marker inhuman calcium absorption experiments.5–7

The human body processes strontium in much thesame way as calcium, as demonstrated in intestinalabsorption and renal filtration studies.8,9 The kidneysprocess strontium in a manner similar to calcium andstrontium can be administered as a surrogate markerfor calcium. Hypercalciuric stone formers have in-creased strontium absorption compared to normocal-ciuric patients.10 Strontium is absorbed by the gut at arate similar to that of calcium11,12 and calcium absorp-tion testing can be performed with strontium.13 There-fore, strontium has long been used to study calciummetabolism in humans and evaluate differences inmetabolism in patients with and without evidence ofabnormal calcium processing, for example those withosteoporosis.5

Strontium substitutes for calcium in biominer-alization during bone formation, which has beenexploited in osteoporosis studies and in the devel-opment of osteoporosis medications. Strontiumranelate is recognized as a cost saving, effectivemedication for the treatment and prevention ofosteoporosis related fractures14,15 and it may pre-vent mortality in frail individuals at risk for frac-ture.16,17 Strontium ranelate decreases bone resorp-tion and increases bone formation.18 Strontium isincorporated into hydroxyapatite in bone by replac-ing a percent of calcium ions, which causes an ap-parent increase in bone mineral density. This in-crease in density is due to an increase in bonedensity and higher x-ray attenuation when measur-ing strontium.19 Strontium ranelate increases bonevolume and bone trabecular thickness.20 However,some studies suggest that the dimensions of thebone mineral crystals are not increased and stron-tium substituting for calcium in hydroxyapatite isthe major change.21

Although extensive research has been performedon strontium incorporation into bone and strontiumhandling by the kidney, less research has been doneon strontium incorporation in urolithiasis. Stron-tium was suggested to compete with calcium forincorporation in calcium based stones,22 and stron-tium and other trace elements were noted in calciumbased uroliths.23,24 In vitro experiments in nanobac-teria revealed strontium incorporation in early stoneformation,25 but there has been little research onstrontium incorporation into uroliths in in vivo mod-els. We used synchrotron radiation imaging tech-

niques to characterize the incorporation of stron-

tium into kidney stones by evaluating the locationand speciation of strontium in human stone sam-ples. Since strontium can serve as a tagged marker,we postulate that strontium may serve as a partic-ularly valuable tool for quantifying and monitoringcalcium lithogenesis in in vitro and in vivo models.

MATERIALS AND METHODS

Human stone samples were obtained via consecutive per-cutaneous nephrolithotomy and ureteroscopy procedures.A portion of each stone was sent for standard FTIR labo-ratory analysis to 1 of 3 commercial laboratories used byour medical center. A portion of each stone was retainedand evaluated with x-ray fluorescence and x-ray absorp-tion measurements at the Advanced Light Source syn-chrotron radiation facility, Lawrence Berkeley NationalLaboratory, as previously described (S. Blaschko et al,unpublished data). A portion of the whole stones allottedfor synchrotron radiation evaluation were microtome sec-tioned with a diamond blade and evaluated by x-ray flu-orescence mapping to minimize x-ray fluorescence absorp-tion artifacts, which may occur in thicker samples. X-rayfluorescence and x-ray absorption measurements wereused to examine the presence, spatial distribution andspeciation of strontium in each stone sample. Methods ofstone evaluation other than synchrotron radiation basedanalysis cannot provide elemental mapping as well asspeciation of trace elements.

RESULTS

FTIR stone analysis identified composite calciumoxalate, calcium phosphate, uric acid and cystine

Figure 1. X-ray fluorescence elemental mapping demonstratesco-localization of strontium and calcium in different stone types.Photomicrograph shows gross pictures of stones retrieved foranalysis. Calcium and strontium distribution is similar in eachcalcium based stone type. X-ray fluorescence radiation fromcalcium is absorbed more than that of strontium so that samplesurface topology influences final image. Thus, calcium images

show more contrast variation.

ium, i

STRONTIUM SUBSTITUTION FOR CALCIUM IN LITHOGENESIS 737

stone samples. FTIR analysis did not identify thepresence of trace elements and could not identifystrontium in any stone samples. X-ray fluorescenceand x-ray absorption measurements confirmed thesame stone composition identified by FTIR in eachstone sample (data not shown).

X-Ray Fluorescence

X-ray fluorescence elemental mapping of each stonesample demonstrated that strontium is incorporatedinto uroliths with the same specificity and localiza-tion as calcium. Strontium was not found in anystones that did not contain calcium. Strontium co-localization with calcium was clearly noted in themixed composition uric acid and calcium oxalatestone, in which strontium was only found in thecalcium oxalate component (fig. 1). X-ray fluores-cence measurements identified strontium in allstone types except pure cystine, which was expectedbased on the lack of calcium in cystine urolithiasis(data not shown). The co-localization of strontiumwith calcium was also clearly observed in mi-crotome sectioned stone samples, in which thebright spots revealed a high amount of calciumcorresponded to bright spots of localized strontium

Figure 2. A, spatial elemental mapping of 2 microtome sectionindicate where sample x-ray fluorescence spectra were acquireat specific locations on samples reveal small amounts of stront

(fig. 2).

X-ray fluorescence was also done to determine theelements present at specific locations of interestidentified by x-ray fluorescence spatial elementalmapping. This evaluation confirmed the presence ofstrontium in all stone samples except the cystinestone sample. The presence of iron, zinc and leadwas also noted in the calcium stones (figs. 2 and 3).

mples shows co-localization of strontium with calcium. Circlesle bar indicates 100 �m. B, x-ray fluorescence spectra acquiredron, zinc and lead.

Figure 3. X-ray fluorescence spectra of 4 calcium containing stonesshows strontium, iron, zinc and lead in each sample. Low lead levels

ed sad. Sca

were also observed, which are not visible in this presentation.

STRONTIUM SUBSTITUTION FOR CALCIUM IN LITHOGENESIS738

X-ray Absorption Near Edge Spectroscopy

Absorption studies allowed for the determination ofthe speciation of the type of strontium incorporatedinto the uroliths by fitting the measured stone sam-ple x-ray absorption curve against the spectra ofknown samples. This analysis showed that 80% ofthe strontium was present as strontium apatite and20% was present as strontium carbonate (fig. 4).

DISCUSSION

Although prior studies have demonstrated the pres-ence of trace elements in calcium based stones, in-formation on the spatial distribution of these ele-ments is mostly lacking.3,22,24 In prior studies thatprovide the spatial distribution of strontium, reso-lution is fivefold lower26 and strontium speciation isnot provided.26,27 Our evaluation of strontium inurolithiasis highlights the advantages of synchro-tron radiation compared with traditional FTIR stoneanalysis and other methods of elemental mapping.X-ray fluorescence elemental mapping provides in-formation about the localization of calcium and traceelements present throughout each sample (figs. 1and 2). FTIR vaporizes each sample during process-ing and, thus, obfuscates elemental mapping. X-rayfluorescence spectra analysis provides the elementalcomposition of stones at areas of interest with spe-cific identification of trace elements, which are notreported with FTIR analyses (fig. 3). Although FTIR

Figure 4. X-ray absorption near edge spectroscopy of calciumoxalate stone sample 4 allowed for speciation of strontium typeby fitting stone sample x-ray absorption curve against curve ofknown samples. Analysis revealed that strontium present was

80% strontium apatite and 20% strontium carbonate.

can identify the primary components present inmixed composition stones, the identification andspeciation of trace elements available with x-rayabsorption analysis are lacking (fig. 4). Trace ele-ments, including those demonstrated by this syn-chrotron radiation analysis, have a role in lithogen-esis28 and in stabilizing calcium oxalate dihydrate.29

The presence of strontium has correlated with thepresence of zinc in uroliths26 and the study of thecritical role of zinc in stone formation is promising(T. Chi et al, unpublished data). However, additionalstudies must be completed before recommendinganalysis of trace elements in 24-hour urine analyses.

X-ray fluorescence analysis clearly shows thatstrontium co-localizes with calcium in calcium basedstone formation. In addition, the kidneys processstrontium similarly to calcium.9 The amount of stron-tium and apatite in kidney stones is correlated.28

These findings validate the use of strontium as amarker for studying calcium lithogenesis in vivo andin vitro. Moreover, radioactive and nonradioactivestrontium may serve as a marker depending on priorstrontium exposure in in vivo models. Future studiesare warranted with tagged strontium as a marker forcalcium in in vitro and in vivo experiments.

Our x-ray absorption studies show that in humankidney stones 80% of strontium appears as stron-tium apatite and 20% appears as strontium carbon-ate. Since hydroxyapatite is thought to serve as thenidus for stone formation and more trace elementsare found in apatite stones than in other stonetypes,3 the presence of strontium apatite indicatesthat strontium may be useful for studying the eventsof lithogenesis and stone enlargement. In support ofthis, strontium was present during early stone for-mation in nanobacteria studies25 and more stron-tium is found in the kidney stones of older patientsthan in the kidney stones of younger patients.30

Strontium has been demonstrated to interact inother hydroxyapatite biomineralization processes.Strontium in the form of strontium ranelate inter-acts with hydroxyapatite. In many situations it canincrease bone mineral density and decrease frac-tures in high risk individuals. It is not surprising tofind a similar incorporation of strontium into neph-rolithiasis in calcium based urinary stones. The in-corporation of strontium in calcium stone formingpatients who receive strontium ranelate for osteopo-rosis is an area of research open to study. Thispopulation likely has de facto tagged uroliths due toregular strontium administration. We hypothesizethat these patients would have a higher amount ofstrontium incorporation during stone formation thanpatients who do not receive strontium ranelate but theimplication of this is yet unknown. Strontium apatiteand calcium apatite have approximately the same

hardness since they are categorized as apatite, level 5

STRONTIUM SUBSTITUTION FOR CALCIUM IN LITHOGENESIS 739

on the Mohs hardness scale. Adding strontium to cal-cium stones may affect the brittleness of the stonestructure due to differences in the atomic size of stron-tium and calcium, causing differences in chemicalbonds. The administration of strontium at set timepoints in in vitro and in vivo models may be used tostudy the interaction of strontium apatite in earlystone formation. Additionally, strontium may beadded to new therapeutics to possibly aid in the tar-geting of developing kidney stones.

CONCLUSIONS

Strontium is present in the human diet in trace

amounts that vary with geographic location. Stron-

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