research article concave urinary crystallines: direct...

Hindawi Publishing CorporationBioinorganic Chemistry and ApplicationsVolume 2013 Article ID 637617 8 pageshttpdxdoiorg1011552013637617

Research ArticleConcave Urinary Crystallines Direct Evidence of CalciumOxalate Crystals Dissolution by Citrate In Vivo

Yun-Feng Shang12 Meng Xu2 Guang-Na Zhang2 and Jian-Ming Ouyang2

1 Yueyang Occupation Technical College Yueyang Hunan 414000 China2 Institute of Biomineralization and Lithiasis Research Jinan University Guangzhou 510632 China

Correspondence should be addressed to Yun-Feng Shang hn-syf163com and Jian-Ming Ouyang toyjmjnueducn

Received 29 August 2013 Revised 30 September 2013 Accepted 17 October 2013

Academic Editor Ian Butler

Copyright copy 2013 Yun-Feng Shang et alThis is an open access article distributed under theCreativeCommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The changes in urinary crystal properties in patients with calcium oxalate (CaOx) calculi after oral administration of potassiumcitrate (K

3cit) were investigated via atomic force microscopy (AFM) scanning electron microscopy (SEM) X-ray powder

diffractometry (XRD) and zeta potential analyzer The AFM and SEM results showed that the surface of urinary crystals becameconcave the edges and corners of crystals became blunt the average size of urinary crystallines decreased significantly andaggregation of urinary crystals was reduced These changes were attributed to the significant increase in concentration of excretedcitrate to 492plusmn118mgL after K

3cit intake from 289plusmn83mgL before K

3cit intake After the amount of urinary citrate was increased

it complexed with Ca2+ ions on urinary crystals which dissolved these crystals Thus the appearance of concave urinary crystalswas a direct evidence of CaOx dissolution by citrate in vivo The XRD results showed that the quantities and species of urinarycrystals decreased after K

3cit intake The mechanism of inhibition of formation of CaOx stones by K

3cit was possibly due to the

complexation of Ca2+ with citrate increase in urine pH concentration of urinary inhibitor glycosaminoglycans (GAGs) and theabsolute value of zeta potential after K

3cit intake

1 Introduction

Nephrolithiasis characterized by renal calculi is a disorderthat has recently shown a trend of rising incidence around theworld Renal calculi mainly contain calcium oxalate (CaOx)crystals [1] However knowledge about its pathogenesis islimited thus far [2 3] and the high recurrence rate is still animportant clinical issue [4]

Although CaOx is supersaturated in urine the formationof renal calculi in healthy people is difficult because of allkinds of inhibitors in urine including citrate magnesiumosteopontin and tyrosine hydroxylase [5] Chemically speak-ing CaOx calculi formation is closely related to with thefollowing factors high concentration of calcium and oxalatein urine nucleation growth and aggregation of CaOx crys-tals and adhesion of calcium oxalate monohydrate (COM)to renal tubular epithelial cells [6] CaOx calculi formationis thought to follow two main routes (slow) formation of

subepithelial plaques at papillary tips and (rapid) formationof intratubular plugs [7]

Potassium citrate (K3cit) is one of the main drugs for

treatment and prevention of renal calculi In July 1985 theUS Food andDrug Administration approved K

3cit as a single

drug to treat CaOx calculi with low urinary citrate and uricacid calculi As a clinical drug K

3cit is widely used for its

advantages such as nontoxicity low price few side effectsand potential for long-term use Studies have shown that afterK3cit intake in patients with chronic renal calculi the occur-

rence rate of new renal calculi was one-fifth of that withoutK3cit intake [8] When patients with renal calculi were orally

administered K3cit after treatment of extracorporeal shock

wave lithotripsy the recurrence rate of renal calculi was 0 in 12months whereas the recurrence rate of patients without K

3cit

intakewas 285 (119875 lt 005) [9]Therefore understanding themechanism of K

3cit has a significant scientific and practical

application for the prevention and treatment of renal calculi

2 Bioinorganic Chemistry and Applications

However little is known about the change in urinarycrystal properties in patients with CaOx calculi after K

3cit

administration After ten years of urinary crystal study wefound that after K

3cit intake crystal depressions emerge

on the surfaces of some urinary crystals in patients withCaOx calculi which is direct evidence that citrate dissolvesCaOx calculi in vivo Based on this previous finding thisstudy examines the crystal depression that emerges on thesurfaces of some urinary crystals especially via atomic forcemicroscope (AFM) The changes in urine citrate and urineGAGs as well as pH and zeta potential were detected and theformationmechanism of concave crystals was discussed Ourresults can shed light on the exploration of preventing renalcalculi formation from a chemical perspective

2 Experiments

21 Reagents and Apparatus Sodium azide (NaN3) and all

other reagents used were all analytically pure All the glass-ware was washed clean with secondary distilled water

Urine crystals were observed using a Philips XL-30environmental scanning electronmicroscope (SEM) at 20 kVafter being covered by an ultrathin layer of gold Atomic forcemicroscopy (AFM) was performed in contact mode withair by using commercial AFM (AutoProbe CP ThermoMi-croscopes USA) Microfabricated silicon nitrite cantilevers(Park Scientific Instruments) were used The 512 times 512 pixelAFM images were obtained using the software (ThermoMi-croscopes Proscan Image Processing Software Version 21)provided with the instrument which can eliminate low-frequency background noise in the scanning direction X-ray diffraction (XRD) results were obtained using a Dmax-120574A X-ray diffractometer (Rigaku Japan) by using Ni-filteredCu-K120572radiation (120582 = 154 A) at a scanning rate of 2∘minminus1

and a scanning range (2120579) from 5∘ to 60∘ The zeta potentialwas determined using a nanoparticle size analyzer fromZetasizer Nano-ZS (Malvern England) in the followingtesting conditions incident beamHe-Ne laser (120582=6330 nm)incident angle 90∘ temperature 250 plusmn 01∘C pH values weremeasured using a PHS-3C precision pH meter (ShanghaiPrecision Scientific Instrument Co Ltd)

22 Collection and Treatment of Stones and ComponentCharacterization The participants in the study included 30randomly selected lithogenic patients (18men and 12 womenmean age = 531 years range = 21sim73 years all of them werefrom the Lithotripsy Center of the First Affiliated Hospital ofJinan University) and 30 randomly selected healthy humanswith no prior history of urinary stones (16 men and 14women mean age = 375 years range = 22sim56 years all ofthem were from the graduates and teachers of Jinan Univer-sity) Urinary stones were collected after surgery disinfectedwith 75 alcohol (AR grade) rinsedwith distilledwater andplaced in a dust-free incubator at 40∘C to dry The urinarystones were then ground into powder by an agate mortar forX-ray diffraction (XRD) characterization which showed thatthe quality fraction of CaOx in stones was between 80 and100 and that the stones contained small amounts of calciumphosphate and uric acid

23 Collection Treatment and Detection of Urine The chan-ges in urinary crystal property in 30 patients (from the same30 patients above) before and after K

3cit intake were studied

for aweek and the dose ofK3cit (in tablet)was set at 2538 gd

None of the patients had gastric intoleranceUrine treatment and urinary crystallite collection were

carried out according to the methods reported in the liter-ature [10ndash14] Fasting morning urine samples were collectedAfter the pH value was detected 2 NaN

3solution (10mLL

urine sample) was added into the urine samples as anantiseptic and zeta potential measurements were takenSubsequently anhydrous alcohol was added into the urinesample (urine ethanol = 3 2) then the urine was stirredand left undisturbed for half an hour to make proteinsdenaturalize and deposit The supernatant was directly usedto detect themicron-sized crystals in urine bymeans of XRDAFM and SEM

XRD Detection About 50 120583L of the urine sample was placedon 12mm times 12mm clean glass slides by using a microsyringeThe glass slides were then dried in an oven at 50 plusmn 5∘C for2 h to volatilize the urine This process was repeated fourtimes The urinary crystals were then treated according toa previously described method [15] to remove the solublefractions of sodium chloride and urea That is the glassslides with urinary crystals were slowly immersed in doubledistilled water at a 45∘ angle of tilt and gently shaken for1min and the soluble fractions (such as sodium chloride andurea) precipitated from urine during drying process couldbe removed After carefully taking out the glass slides waterfrom the edge of the slideswas dried using an absorbent paperand the slides were again dried in a vacuum desiccator for 1 dtomake samples that could be used for XRD characterizationSEM and AFM Detection About 30 120583L of the urine samplewas placed on 10mm times 10mm clean glass slides or a cleanmica sheet by using a microsyringe The glass slides andthe mica sheet were then dried in a vacuum dryer Then asimilar washing process as above was carried out to removethe soluble crystallitesZeta Potential Detection Urine samples were directly pro-cessed for zeta potential detection by using a Zetasizer Nano-ZS nanoparticle size analyzer

24 Detection of Citrate and Glycosaminoglycans (GAGs)Excretion Amount before and after K

3cit Intake Ammonium

metavanadate-assisted catalytic-kinetic spectrophotometrywas performed to determine urinary citrate content [16]and Alcian Blue colorimetric method was used to determineurinary GAGs content [17]

3 Results

31 SEM Data of Urinary Crystals before and after K3cit

Intake Figure 1 shows the SEM images of the urinary crystalsin patients with renal calculi after K

3cit intake for a week

Crystal depressions clearly emerged on the surface of somecrystals in the urine (Figures 1(a) to 1(d)) By contrast thesedepressions rarely occur in urinary crystals before K

3cit

Bioinorganic Chemistry and Applications 3

(a) (b)

(c) (d)

(e) (f)

Figure 1 SEM images of representative urinary crystals from patients with CaOx renal calculi after oral administration of K3cit for one week

Scale bar 20 120583m

intake About one-third of the urinary crystals from the 30patients with CaOx calculi were concave after K

3cit intake

Aside from crystal depressions that emerged on the sur-face of crystals the morphology of urinary crystals showedother changes such as the rounding and smoothing ofthe corners and edges (Figure 1(e)) In addition the sizeof the crystals significantly decreased the proportion ofcalcium oxalate dihydrate (COD) with tetragonal bipyramidincreased (Figure 1(f)) and the aggregation of the crystalsdecreased

32 AFM Data of Urinary Crystals before and after K3cit

Intake To further study these concave crystals themorphol-ogy was observed via AFM Figure 2 shows AFM images ofurinary crystals of a patient with CaOx calculi before andafter K

3cit intake for a week and the results show the crystal

depressions

33 XRD Detection of Urinary Crystals before and afterK3cit Intake Figure 3 shows the XRD patterns of urinary

crystals in two patients with CaOx calculi before and afterK3cit intake The number of diffraction peaks significantly

decreased after K3cit intake indicating that the species of

urinary crystals decrease after K3cit intake The intensity

of diffraction peaks also significantly decreased which wasapproximately one-third of that before K

3cit intake This

result indicated that the quality of urinary crystals decreasedafter K

3cit intake The proportion of COM in the urine

decreased after K3cit intake whereas that of COD increased

In detail comparedwith theXRDpatterns beforeK3cit intake

(Figures 3(a) and 3(c)) the diffraction peaks of COM at 119889 =593 365 297 236 and 198 A disappeared (Figure 3(b))[18] whereas the diffraction peaks of COD at 119889 = 618 A(Figure 3(b)) or 309 and 224 A (Figure 3(d)) appearedwhich showed that the amount of COM crystals significantlydecreased after K

3cit intake whereas the relative amount


(a) (b)

Figure 2 AFM images of urinary crystal of one patient with CaOx calculi before (a) and after (b) K3cit intake Image size 3 120583m times 3 120583m

(1010)

(112)

(200)(021)

d = 198d = 236

d = 279

d = 297(101)d = 365

d = 393a

(020)

(303)b (130)

(202)(020)

(211)

d = 316d = 618

(b)

(a)

d = 593

10 20 30 40 502120579 (deg)

Inte

nsity

(au

)

a d = 345

b d = 249

(400) (213)

d = 197d = 248

d = 279

d = 309 d = 224

(130)

(020)

d = 393

(d)

(c)

10 20 30 40 502120579 (deg)

Inte

nsity

(au

)

(211)

(303)

Figure 3 XRD patterns of urinary crystals of two patients with CaOx calculi before ((a) (c)) and after ((b) (d)) K3cit intake 995333 COM f

COD 998771 uric acidX 120573-Ca3(PO4)2

of COD increased This result was consistent with that ofthe SEM data (Figure 1(f)) Moreover the diffraction peaksattributed to uric acid and calcium phosphate disappeared orsignificantly weakened after K

3cit intake

Among the 30 stone patients 21 patients (70) had crys-talluria in which 10 patients had COM as main composition1 patient had COD as main composition and 2 patients hadboth COM and CODThe other compositions were uric acid(UA seven cases) and calcium phosphate (CaP one case)However after K

3cit intake only seven patients had still

crystalluria in which 1 patient had COM 2 patients hadCOD and 4 patients had both COM and COD The othercomposition was urate uric acid and CaP

34 Concentration Differences of Citrate and GAGs in Urinebefore and after K

3cit Intake Before K

3cit intake the citrate

content in urine of patients with CaOx calculi was 289 plusmn83mgL (Figure 4(a)) and increased to 492 plusmn 118mgL (119875 lt001) after K

3cit intake whereas that of the control sample

was 354 plusmn 97mgL After K3cit intake GAGs increased to

1078 plusmn 231mgL (119875 lt 001) from 632 plusmn 113mgL beforeK3cit intake (Figure 4(b)) whereas that of the control sample

was 730plusmn 126mgL Urine pH increased to 642plusmn 045 from601 plusmn 035 before K

3cit intake whereas that of the control

sample was 623 plusmn 036mgL (Figure 4(c))The obtained values were consistent with those of previ-

ous studies For example Trinchieri et al [19] found that theexcretion amounts of citrate in urine of patients with COMand COD calculi were 507 plusmn 264 and 487 plusmn 247mg24 hrespectively (24 h urine volume in healthy control individualswas about 18 L to 2 L whereas the value was 12 L in patientswith renal calculi) Srinivasan et al [20] found that theexcretion amount of citrate in urine of healthy control


0

200

400

600

Before After Healthy

Citr

ate (

mg

L)

lowast

lowast

(a)

3

6

9

12

15

GAG

s (m

gL)

lowast

lowast


(b)

50

55

60

65

70

pH

lowast

lowast


(c)

minus14

minus12

minus10

minus8

minus6

minus4

minus2

0Ze

ta p

oten

tial (

mV

)

lowast

lowast


(d)

Figure 4 Comparison between the properties of urine and urinary crystals from healthy control individuals and patients with CaOx calculibefore and after K

3cit intake (a) Excretion amount of citrate (b) excretion amount of GAGs (c) urinary pH (d) zeta potential (119899 = 30)

individuals (601 plusmn 84mg24 h) was higher than that of uricacid (UA)CaOx and calciumphosphate (CaP) calculi whichwere 415 plusmn 43 429 plusmn 58 and 492 plusmn 52mg24 h respectivelyGu et al [21] randomly divided 252 patients with CaOxcalculi into two groups For the group after K

3cit intake for

one week the average urine pH increased to 681 from 588(119875 lt 0001) and urinary citrate content increased to 196from 078mmol24 h (119875 lt 0001) whereas urinary calciumcontent decreased to 444 from 781mmol24 h

35 Zeta Potential on the Surface of Urinary Crystallinesbefore and after K


3cit intake the zeta

potential of urinary crystals in patients with renal calculiwas minus505 plusmn 312mV and became minus913 plusmn 333mV (119875 lt005) after K

3cit intake whereas the zeta potential of urinary

crystals in healthy control individuals was minus893 plusmn 436mV(Figure 4(d))

4 Discussion

41 Appearance of Concave Crystals and Rounded Crystalsafter K

3cit Intake Whether it was the group which had

hypocitraturia or the group with normocitraturia the citratecontent in urine of patients increased after K

3cit intake But

there was no linear relationship between the increment ofcitrate excretion and the amount of urine citrate before K

3cit

intake The concave urinary crystals appeared in the urinewith a high citrate excretion amount after K

3cit intake

Citrate is one of the most abundant anions in urine andan important inhibitor of calcium calculi [22] An excretion


amount of citrate less than 320mg24 h (about 267mgL)is defined as hypocitraturia which indicates a risk of stoneformation [23]

Figure 4(a) shows that the citrate excretion in urine afterK3cit intake for a week increased to 492 plusmn 118mgL from289 plusmn 83mgL before K

3cit intake Before K

3cit intake 14

cases of the 30 stone patients studied had urinary citrate levellower than 267mgL After K

3cit intake none of them had

urinary citrate level lower than 267mgL (theminimumvaluewas 270mgL) For the healthy controls still 6 cases of the 30persons studied hadurinary citrate level lower than 267mgL

When citrate excretion increases the formation of CaOxcalculi is possibly inhibited through the following mecha-nisms Citric acid is a tricarboxylic acidwith acid dissociationconstants (pKa) of 29 43 and 56 [24] Citrate can complexwith Ca2+ ions to form a chelate with a five- or six-memberedring with a stability constant of 479 times 104 [24] and excellentsolubility Thus increasing citrate excretion in urine canchelate citrate with Ca2+ ions on the surface of CaOx crystalsThis chelation slowly dissolves the crystals which leads tothe emergence of concave crystals (Figures 1(a) to 1(d))Therefore the emergence of concave crystals is direct proofthat citrate chelates with Ca2+ ion to dissolve CaOx crystalsthereby inhibiting CaOx calculi formation

Nine patients of the 30 stone patients studied had urinarycrystallites with concave surface after K

3cit intake Their

citrate excretion amounts were in the region from 510 to725mgL with an average of 631mgL which was higher thanthe average value of citrate excretion amounts after K

3cit

intake (492mgL)

42 Inhibition of K3cit on CaOx Calculi Formation

(1) K3cit makes the tips of the crystals round and smooth

Citrate can maintain a complexation-dissociation equi-librium with the Ca2+ ions on the surface of CaOx crystals inurine especially those surrounding and on the tips of CaOxcrystals These crystals are continuously dissolved because ofthe complexation with citrate At the same time the dissolvedCa2+ ions are continuously deposited on the surface of CaOxcrystals This continuous dissolution-deposition caused themorphology of crystals to be more rounded and blunt afterK3cit intake (Figure 1(e))(2) K3cit inhibits aggregation of urinary crystals

After the tips and edges of crystals became round andsmooth the tendency of crystal aggregation was reducedIn addition the increase in zeta potential absolute value onthe surface of urinary crystals after K

3cit intake (Figure 4(d))

was also favorable to the increase in repulsive force betweencrystals and inhibited the aggregation of urinary crystalsInhibition of aggregationwill affect particle size and thusmayprevent plug formation

(3) K3cit can decrease the size of urinary crystals

The excretion amounts of citrate (Figure 4(a)) and gly-cosaminoglycans (GAGs) (Figure 4(b)) in urine increasedafter K

3cit intake Both citrate and GAGs can chelate with

Ca2+ ions citrate chelates with Ca2+ to form 1 1 complexesand 1120583mol disaccharide unit of chondroitin sulfate (one of

the GAGs) can complex with 0757120583mol Ca2+ [25] Thesechelated compounds were soluble thus the Ca2+ concentra-tion and CaOx saturation in urine decreased and the growthof CaOx crystals was inhibited Therefore the size of urinarycrystals decreased after K

3cit intake

(4) In addition citrate is active in the nucleation phase ofcalcium oxalate and has a profound effect on themorphologyof the crystals formed [26] It retards nucleation at highconcentration Therefore it can explain the reduced numberof crystals in Figure 1

(5)K3cit inhibits the formation ofUAcrystals and reduces

the heterogeneous nucleation process of COM crystals Alot of UA crystals can be found among urinary crystals TheXRD spectra (Figure 3) show that UA quality significantlydecreased after K

3cit intake For example compared with

the XRD pattern before K3cit intake the diffraction peaks

assigned to the (211) and (020) faces of UA at 119889 = 393and 279 A significantly weakened Thus the quality of UAcrystallites in urine remarkably decreased This result wasattributed to the excreted citrate in urine which alkalinizedthe urine (Figure 4(c)) Thus urine pH increased Moreovermost of the UA crystals were turned into urate a muchmore soluble species thereby decreasing the UA content inurine After the amount of UA crystals in urine decreasedthe heterogeneous nucleation process ofCOMinduced byUAcrystals also decreased thereby inhibiting the formation ofCaOx calculi

(6) K3cit converts COM crystals to COD crystals The

SEM data (Figure 1(f)) and XRD analysis (Figures 3(a) and3(b)) show that the content of COM in urinary crystals beforeK3cit intake was higher than that after K

3cit intake whereas

the content of COD after K3cit intake was significantly higher

than that before K3cit intake This result was attributed to

the high affinity of citrate to COM which largely inhibitedthe development of COM [27ndash29] Kok [26] reported thatcitrate steered crystal morphology from the coffin-shapedclassicCOMthroughmore roundedCOMcrystals andfinallyconverted to exclusively COD crystals Wierzbicki et al [30]studied the bonding of citrate with COM crystals by usinga molecular model and calculated the bonding energy ofcitrate with different COM crystal faces thereby indicatingthat citrate could better coordinate withCa2+ on a (101) planeof COM CaOx calculi mainly existed in the form of COMand COD but COD was easier to evacuate from the body inurine than COM [31] Therefore if more COD crystals canbe induced or the transformation of COM to COD can bepromoted renal calculi formation can be prevented and itsrelapse can be delayed

5 Conclusions

Urinary crystal depressions were first observed in patientswith CaOx calculi after K

3cit intake and these depressions

are direct proof of the dissolution of CaOx crystals bycitrate thereby preventing CaOx calculi formationThe causeof crystal depression formation was explained After K

3cit

intake for one week the aggregation of urinary crystalsin patients with CaOx calculi significantly decreased the


species and amount of the crystals decreased COD contentincreased and COM content decreased In addition thezeta potential absolute value on the crystal surface increasedbecause of the increase in urinary citrate and GAGs excretionamount as well as urine alkalization These results inhibitCaOx calculi formation The results in this work will helpshed light on the prevention of kidney stones formation andthe delay of its recurrence

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This research work was supported by the Natural ScienceFoundation of China (81170649 and 30672103)

References

[1] M Y M Fazil and A Salim ldquoClinical risk index in urolithiasisrdquoUrological Research vol 37 no 5 pp 283ndash287 2009

[2] X-Q Yao J-M Ouyang H Peng W-Y Zhu and H-Q ChenldquoInhibition on calcium oxalate crystallization and repair oninjured renal epithelial cells of degraded soybean polysaccha-riderdquo Carbohydrate Polymers vol 90 no 1 pp 392ndash398 2012

[3] S Zhang Z-X Su X-Q Yao H Peng S-P Deng and J-M Ouyang ldquoMediation of calcium oxalate crystal growth onhuman kidney epithelial cells with different degrees of injuryrdquoMaterials Science and Engineering C vol 32 no 4 pp 840ndash8472012

[4] Z Jing G Z Wang J Ning J W Yang G Yan and YFang ldquoAnalysis of urinary calculi composition by infraredspectroscopy a prospective study of 625 patients in EasternChinardquo Urological Research vol 38 no 2 pp 111ndash115 2010

[5] C-C Liu S-P Huang L-Y Tsai et al ldquoThe impact ofosteopontin promoter polymorphisms on the risk of calciumurolithiasisrdquo Clinica Chimica Acta vol 411 no 9-10 pp 739ndash743 2010

[6] H Peng J-M Ouyang X-Q Yao and R-E Yang ldquoInteractionbetween sub-micron COD crystals and African green monkeyrenal epithelial cellrdquoThe International Journal of Nanomedicinevol 7 no 8 pp 4727ndash4737 2012

[7] J C Williams and J A McAter ldquoRetention and growth ofurinary stones insights from imagingrdquo Journal of Nephrologyvol 26 pp 25ndash31 2013

[8] H A Fuselier K Moore and J Lindberg ldquoAgglomerationinhibition reflected stone-forming activity during long-termpotassium citrate therapy in calcium stone formersrdquo Urologyvol 52 no 6 pp 988ndash994 1998

[9] S Tarkan A Aysegul and K Sadettin ldquoEffect of potassiumcitrate therapy on stone recurrence and residual fragmentsafter shockwave lithotripsy in lower caliceal calcium oxalateurolithiasisrdquo Journal of Endourology vol 16 no 3 pp 149ndash1522002

[10] M Robert A-M Boularan O Delbos J Guiter and BDescomps ldquoStudy of calcium oxalate crystalluria on renal andvesical urines in stone formers and normal subjectsrdquo UrologiaInternationalis vol 60 no 1 pp 41ndash46 1998

[11] M Daudon C Hennequin G Boujelben B Lacour andP Jungers ldquoSerial crystalluria determination and the risk ofrecurrence in calcium stone formersrdquo Kidney International vol67 no 5 pp 1934ndash1943 2005

[12] M Daudona and P Jungers ldquoClinical value of crystalluria andquantitative morphoconstitutional analysis of urinary calculirdquoNephron Physiology vol 98 no 2 pp p31ndashp36 2004

[13] Z-J Huang J-J Li J-Y He and J-MOuyang ldquoStudy on nano-and microcrystallites in the urines of calcium oxalate stoneformersrdquo Spectroscopy and Spectral Analysis vol 30 no 7 pp1913ndash1917 2010

[14] M-X Zhao X-J Xu and J-M Ouyang ldquoComparative researchon nanocrystals and crystallites in urines of healthy persons andstone patientsrdquo Journal of Jinan University Natural Science andMedicine Edition vol 28 no 5 pp 486ndash490 2007

[15] Y-F Shang L Kuan W-Y Zhu and J-M Ouyang ldquoWashingmethod an accurate detection method of urinary nanocrystal-lites to eliminate interference of soluble components in urinerdquoAdvanced Materials Research vol 781ndash784 pp 932ndash935 2013

[16] S J Peng and S F Ding ldquoDetermination of citric acid byactivation catalytic reaction system of potassium bromate-bromophenol blue-vanadium (V) dynamic spectrophotomet-ricrdquoChinese Journal of Health Laboratory Technology vol 21 no8 pp 1845ndash1846 2011

[17] P Whiteman ldquoThe quantitative determination of glycosamino-glycans in urine with Alcian Blue 8GXrdquo Biochemical Journalvol 131 no 2 pp 351ndash357 1973

[18] M King W F McClure and L C Andrews Powder DiffractionFile Alphabetic Index Inorganic PhasesOrganic Phases Interna-tional Center for Diffraction Data Newtown Square Pa USA1992

[19] A Trinchieri C Castelnuovo R Lizzano and G Zanetti ldquoCal-cium stone disease a multiform realityrdquo Urological Researchvol 33 no 3 pp 194ndash198 2005

[20] S Srinivasan P Kalaiselvi R Sakthivel V Pragasam V Muthuand P Varalakshmi ldquoUric acid an abettor or protector in cal-cium oxalate urolithiasis Biochemical study in stone formersrdquoClinica Chimica Acta vol 353 no 1-2 pp 45ndash51 2005

[21] X-A Gu X-Y Li and C-T Sun ldquoPotassium citrate preventscalcium oxalate stone formation in clinical researchrdquo ChineseMedical Journal vol 36 no 8 pp 22ndash23 2001

[22] D J Kok ldquoClinical implications of physicochemistry of stoneformationrdquo Endocrinology and Metabolism Clinics of NorthAmerica vol 31 no 4 pp 855ndash867 2002

[23] Y-S Ha D-U Tchey H W Kang et al ldquoPhosphaturia as apromising predictor of recurrent stone formation in patientswith urolithiasisrdquo Korean Journal of Urology vol 51 no 1 pp54ndash59 2010

[24] J A Dean andHMcGraw ldquoCitric acidrdquo in Langersquos Handbook ofChemistry pp 5ndash46 McGraw-Hill 13th edition 1985

[25] H A Fuselier and D M Ward ldquoUrinary Tamm-Horsfallprotein increased after potassium citrate therapy in calciumstone formersrdquo Urology vol 45 no 6 pp 942ndash946 1995

[26] D J Kok Inhibitors of Crystallization I Calcium Oxalate inBiological Systems CRC Press 1995

[27] M L Weaver S R Qiu J R Hoyer W H Casey G HNancollas and J J de Yoreo ldquoInhibition of calcium oxalatemonohydrate growth by citrate and the effect of the backgroundelectrolyterdquo Journal of Crystal Growth vol 306 no 1 pp 135ndash145 2007


[28] S Guo M D Ward and J A Wesson ldquoDirect visualizationof calcium oxalate monohydrate crystallization and dissolutionwith atomic force microscopy and the role of polymeric addi-tivesrdquo Langmuir vol 18 no 11 pp 4284ndash4291 2002

[29] V Fischer K Landfester and R Munoz-Espı ldquoStabilization ofcalcium oxalate metastable phases by oligo(L-glutamic acid)effect of peptide chain lengthrdquo Crystal Growth and Design vol11 no 5 pp 1880ndash1890 2011

[30] AWierzbicki C S Sikes J D Sallis J DMadura E D StevensandK LMartin ldquoScanning electronmicroscopy andmolecularmodeling of inhibition of calcium oxalate monohydrate crystalgrowth by citrate and phosphocitraterdquo Calcified Tissue Interna-tional vol 56 no 4 pp 297ndash304 1995

[31] J M Ouyang X Q Yao Z X Su and F Z Cui ldquoSimulation ofcalcium oxalate stone in vitrordquo Science in China B vol 46 no 3pp 234ndash242 2003

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Analytical Methods in Chemistry

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Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


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Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


However little is known about the change in urinarycrystal properties in patients with CaOx calculi after K

3cit

administration After ten years of urinary crystal study wefound that after K

3cit intake crystal depressions emerge

on the surfaces of some urinary crystals in patients withCaOx calculi which is direct evidence that citrate dissolvesCaOx calculi in vivo Based on this previous finding thisstudy examines the crystal depression that emerges on thesurfaces of some urinary crystals especially via atomic forcemicroscope (AFM) The changes in urine citrate and urineGAGs as well as pH and zeta potential were detected and theformationmechanism of concave crystals was discussed Ourresults can shed light on the exploration of preventing renalcalculi formation from a chemical perspective

2 Experiments

21 Reagents and Apparatus Sodium azide (NaN3) and all

other reagents used were all analytically pure All the glass-ware was washed clean with secondary distilled water

Urine crystals were observed using a Philips XL-30environmental scanning electronmicroscope (SEM) at 20 kVafter being covered by an ultrathin layer of gold Atomic forcemicroscopy (AFM) was performed in contact mode withair by using commercial AFM (AutoProbe CP ThermoMi-croscopes USA) Microfabricated silicon nitrite cantilevers(Park Scientific Instruments) were used The 512 times 512 pixelAFM images were obtained using the software (ThermoMi-croscopes Proscan Image Processing Software Version 21)provided with the instrument which can eliminate low-frequency background noise in the scanning direction X-ray diffraction (XRD) results were obtained using a Dmax-120574A X-ray diffractometer (Rigaku Japan) by using Ni-filteredCu-K120572radiation (120582 = 154 A) at a scanning rate of 2∘minminus1

and a scanning range (2120579) from 5∘ to 60∘ The zeta potentialwas determined using a nanoparticle size analyzer fromZetasizer Nano-ZS (Malvern England) in the followingtesting conditions incident beamHe-Ne laser (120582=6330 nm)incident angle 90∘ temperature 250 plusmn 01∘C pH values weremeasured using a PHS-3C precision pH meter (ShanghaiPrecision Scientific Instrument Co Ltd)

22 Collection and Treatment of Stones and ComponentCharacterization The participants in the study included 30randomly selected lithogenic patients (18men and 12 womenmean age = 531 years range = 21sim73 years all of them werefrom the Lithotripsy Center of the First Affiliated Hospital ofJinan University) and 30 randomly selected healthy humanswith no prior history of urinary stones (16 men and 14women mean age = 375 years range = 22sim56 years all ofthem were from the graduates and teachers of Jinan Univer-sity) Urinary stones were collected after surgery disinfectedwith 75 alcohol (AR grade) rinsedwith distilledwater andplaced in a dust-free incubator at 40∘C to dry The urinarystones were then ground into powder by an agate mortar forX-ray diffraction (XRD) characterization which showed thatthe quality fraction of CaOx in stones was between 80 and100 and that the stones contained small amounts of calciumphosphate and uric acid

23 Collection Treatment and Detection of Urine The chan-ges in urinary crystal property in 30 patients (from the same30 patients above) before and after K

3cit intake were studied

for aweek and the dose ofK3cit (in tablet)was set at 2538 gd

None of the patients had gastric intoleranceUrine treatment and urinary crystallite collection were

carried out according to the methods reported in the liter-ature [10ndash14] Fasting morning urine samples were collectedAfter the pH value was detected 2 NaN

3solution (10mLL

urine sample) was added into the urine samples as anantiseptic and zeta potential measurements were takenSubsequently anhydrous alcohol was added into the urinesample (urine ethanol = 3 2) then the urine was stirredand left undisturbed for half an hour to make proteinsdenaturalize and deposit The supernatant was directly usedto detect themicron-sized crystals in urine bymeans of XRDAFM and SEM

XRD Detection About 50 120583L of the urine sample was placedon 12mm times 12mm clean glass slides by using a microsyringeThe glass slides were then dried in an oven at 50 plusmn 5∘C for2 h to volatilize the urine This process was repeated fourtimes The urinary crystals were then treated according toa previously described method [15] to remove the solublefractions of sodium chloride and urea That is the glassslides with urinary crystals were slowly immersed in doubledistilled water at a 45∘ angle of tilt and gently shaken for1min and the soluble fractions (such as sodium chloride andurea) precipitated from urine during drying process couldbe removed After carefully taking out the glass slides waterfrom the edge of the slideswas dried using an absorbent paperand the slides were again dried in a vacuum desiccator for 1 dtomake samples that could be used for XRD characterizationSEM and AFM Detection About 30 120583L of the urine samplewas placed on 10mm times 10mm clean glass slides or a cleanmica sheet by using a microsyringe The glass slides andthe mica sheet were then dried in a vacuum dryer Then asimilar washing process as above was carried out to removethe soluble crystallitesZeta Potential Detection Urine samples were directly pro-cessed for zeta potential detection by using a Zetasizer Nano-ZS nanoparticle size analyzer

24 Detection of Citrate and Glycosaminoglycans (GAGs)Excretion Amount before and after K

3cit Intake Ammonium

metavanadate-assisted catalytic-kinetic spectrophotometrywas performed to determine urinary citrate content [16]and Alcian Blue colorimetric method was used to determineurinary GAGs content [17]

3 Results

31 SEM Data of Urinary Crystals before and after K3cit

Intake Figure 1 shows the SEM images of the urinary crystalsin patients with renal calculi after K

3cit intake for a week

Crystal depressions clearly emerged on the surface of somecrystals in the urine (Figures 1(a) to 1(d)) By contrast thesedepressions rarely occur in urinary crystals before K

3cit


(a) (b)

(c) (d)

(e) (f)




3cit intake





depressions






3cit intake This








(a) (b)


(1010)

(112)

(200)(021)

d = 198d = 236

d = 279

d = 297(101)d = 365

d = 393a

(020)

(303)b (130)

(202)(020)

(211)

d = 316d = 618

(b)

(a)

d = 593

10 20 30 40 502120579 (deg)

Inte

nsity

(au

)

a d = 345

b d = 249

(400) (213)

d = 197d = 248

d = 279

d = 309 d = 224

(130)

(020)

d = 393

(d)

(c)

10 20 30 40 502120579 (deg)

Inte

nsity

(au

)

(211)

(303)




3cit intake
















0

200

400

600


Citr

ate (

mg

L)

lowast

lowast

(a)

3

6

9

12

15

GAG

s (m

gL)

lowast

lowast


(b)

50

55

60

65

70

pH

lowast

lowast


(c)

minus14

minus12

minus10

minus8

minus6

minus4

minus2

0Ze

ta p

oten

tial (

mV

)

lowast

lowast


(d)




3cit intake for








4 Discussion




3cit intake But


3cit


3cit intake






3cit intake 14






3cit intake Their


3cit

intake (492mgL)












3cit intake









3cit intake whereas




5 Conclusions




3cit






Acknowledgment


References










































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


(a) (b)

(c) (d)

(e) (f)




3cit intake





depressions






3cit intake This








(a) (b)


(1010)

(112)

(200)(021)

d = 198d = 236

d = 279

d = 297(101)d = 365

d = 393a

(020)

(303)b (130)

(202)(020)

(211)

d = 316d = 618

(b)

(a)

d = 593

10 20 30 40 502120579 (deg)

Inte

nsity

(au

)

a d = 345

b d = 249

(400) (213)

d = 197d = 248

d = 279

d = 309 d = 224

(130)

(020)

d = 393

(d)

(c)

10 20 30 40 502120579 (deg)

Inte

nsity

(au

)

(211)

(303)




3cit intake
















0

200

400

600


Citr

ate (

mg

L)

lowast

lowast

(a)

3

6

9

12

15

GAG

s (m

gL)

lowast

lowast


(b)

50

55

60

65

70

pH

lowast

lowast


(c)

minus14

minus12

minus10

minus8

minus6

minus4

minus2

0Ze

ta p

oten

tial (

mV

)

lowast

lowast


(d)




3cit intake for








4 Discussion




3cit intake But


3cit


3cit intake






3cit intake 14






3cit intake Their


3cit

intake (492mgL)












3cit intake









3cit intake whereas




5 Conclusions




3cit






Acknowledgment


References










































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


(a) (b)


(1010)

(112)

(200)(021)

d = 198d = 236

d = 279

d = 297(101)d = 365

d = 393a

(020)

(303)b (130)

(202)(020)

(211)

d = 316d = 618

(b)

(a)

d = 593

10 20 30 40 502120579 (deg)

Inte

nsity

(au

)

a d = 345

b d = 249

(400) (213)

d = 197d = 248

d = 279

d = 309 d = 224

(130)

(020)

d = 393

(d)

(c)

10 20 30 40 502120579 (deg)

Inte

nsity

(au

)

(211)

(303)




3cit intake
















0

200

400

600


Citr

ate (

mg

L)

lowast

lowast

(a)

3

6

9

12

15

GAG

s (m

gL)

lowast

lowast


(b)

50

55

60

65

70

pH

lowast

lowast


(c)

minus14

minus12

minus10

minus8

minus6

minus4

minus2

0Ze

ta p

oten

tial (

mV

)

lowast

lowast


(d)




3cit intake for








4 Discussion




3cit intake But


3cit


3cit intake






3cit intake 14






3cit intake Their


3cit

intake (492mgL)












3cit intake









3cit intake whereas




5 Conclusions




3cit






Acknowledgment


References










































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


0

200

400

600


Citr

ate (

mg

L)

lowast

lowast

(a)

3

6

9

12

15

GAG

s (m

gL)

lowast

lowast


(b)

50

55

60

65

70

pH

lowast

lowast


(c)

minus14

minus12

minus10

minus8

minus6

minus4

minus2

0Ze

ta p

oten

tial (

mV

)

lowast

lowast


(d)




3cit intake for








4 Discussion




3cit intake But


3cit


3cit intake






3cit intake 14






3cit intake Their


3cit

intake (492mgL)












3cit intake









3cit intake whereas




5 Conclusions




3cit






Acknowledgment


References










































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





3cit intake 14






3cit intake Their


3cit

intake (492mgL)












3cit intake









3cit intake whereas




5 Conclusions




3cit






Acknowledgment


References










































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





Acknowledgment


References










































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

research article concave urinary crystallines: direct...

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