Glycochenodeoxycholic acid inhibits calcium phosphate precipitation in vitro by preventing the transformation of amorphous calcium phosphate to calcium hydroxyapatite

Glycochenodeoxycholic acid inhibits calcium

phosphate precipitation in vitro by preventing

the transformation of amorphous calcium

phosphate to calcium hydroxyapatite.

S M Qiu, … , N K Hong, R S Crowther

J Clin Invest. 1991;88(4):1265-1271. https://doi.org/10.1172/JCI115430.

Calcium hydroxyapatite can be a significant component of black pigment gallstones. Diverse molecules that bind calcium phosphate inhibit hydroxyapatite precipitation. Because glycine-conjugated bile acids, but not their taurine counterparts, bind calcium phosphate, we studied whether glycochenodeoxycholic acid inhibits calcium hydroxyapatite formation. Glycochenodeoxycholic acid (2 mM) totally inhibited transformation of amorphous calcium phosphate microprecipitates to macroscopic crystalline calcium hydroxyapatite. This inhibition was not mediated by decreased Ca2+ activity. Taurocholic acid (2-12 mM) did not affect hydroxyapatite formation, but antagonized glycochenodeoxycholic acid. Both amorphous and crystalline precipitates contained a surface fraction relatively rich in

phosphate. The surface phosphate content was diminish by increasing

glycochenodeoxycholic acid concentrations, and this relationship was interpreted as competition between bile acid and HPO4(-4) for binding sites on the calcium phosphate surface. A phosphate-rich crystal surface was associated with rapid transition from amorphous to crystalline states. These results indicate that glycochenodeoxycholic acid prevents transformation of amorphous calcium phosphate to crystalline hydroxyapatite by competitively inhibiting the accumulation of phosphate on the crystal embryo surface.

Research Article

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Glycochenodeoxycholic

Acid Inhibits Calcium

Phosphate

Precipitation In Vitro by Preventing

the Transformation

of

Amorphous

Calcium Phosphate

to

Calcium

Hydroxyapatite

Sul-Min Qiu,GaryWen, NobuyukiHirakawa, Roger D. Soloway, Nan-KangHong, and Roger S. Crowther

Division of Gastroenterology, Department ofInternal Medicine, UniversityofTexasMedical Branch, Galveston, Texas 77550

Abstract

Calcium hydroxyapatite can be a significant component of blackpigmentgallstones. Diverse molecules that bind calcium phosphate inhibit hydroxyapatite precipitation. Because

gly-cine-conjugatedbile acids, but not their taurine counterparts,

bindcalcium phosphate, we studied whether

glycochenodeoxy-cholic acid inhibits calcium hydroxyapatiteformation.

Glyco-chenodeoxycholic acid (2 mM)totallyinhibited transformation ofamorphouscalciumphosphate microprecipitates to

macro-scopic crystalline calciumhydroxyapatite. This inhibition was notmediatedby decreased

Ca2"

activity. Taurocholic acid

(2-12 mM) didnotaffect hydroxyapatite formation, but

antago-nizedglycochenodeoxycholic acid.Both amorphous and

crys-talline precipitates containedasurfacefraction relatively rich in phosphate. The surface phosphate content was diminished

by increasing glycochenodeoxycholic acid concentrations, and

this relationship wasinterpreted ascompetition betweenbile acid andHPO2- for binding sites on the calcium phosphate

surface.Aphosphate-richcrystal surfacewasassociatedwith

rapid transition from amorphous to crystalline states. These

results indicate that glycochenodeoxycholic acid prevents

transformation of amorphous calcium phosphatetocrystalline hydroxyapatitebycompetitively inhibiting the accumulation of

phosphate on the crystal embryo surface. (J. Clin. Invest. 1991.88:1265-1271.) Keywords:bile salts*biomineralization

*calcification*Fourier transform infraredspectroscopy *

gall-stones

Introduction

Calcium salts ofbilirubin, fatty acids, carbonate, and

phos-phate are important constituents ofall classes of gallstones, although the predominant saltvaries with the type ofstone(1,

2).Thefundamental drive for thesesaltstoprecipitatecanbe

understoodonaphysicochemicalbasisasthesolubility prod-uct fora particular salt beingexceeded. However, many of

Part of this work hasappearedinabstract form(1990.

Gastroenterol-ogy.98:A258).

Dr.QiuisaVisitingScientist from theDepartment of

Hepatobili-arySurgery, FirstAffiliated Hospital,Xian MedicalUniversity, Peo-ple'sRepublicofChina.

Addressreprint requeststo Dr.Crowther, Gastroenterology

Divi-sion,4.106McCullough Building, G-64, UniversityofTexasMedical Branch, Galveston,TX77550.

Receivedfor publication 1I December1990andinrevisedform24 June 1991.

thesespeciesexist in normal bile in supersaturated

concentra-tions,but do notprecipitate.It isbecoming apparent that other componentsof bilecaninteractwith these species in ways that

modify theirprecipitation, and specific modifying factors such

asacidicglycoproteins(3) andbile acids(4) have been identi-fied.

Ourinterest inthepathogenesis ofpigment stones has led

ustostudy factorscapable of influencingtheprecipitationof

calciumhydroxyapatite,

Cal0(OH)2(P04)6,

(HAP),' which, al-though it isarelativelyuncommon gallstone component, can

compriseup to 30%ofthe total massofblackpigmentstones (2). Sutor and Percival (5) demonstrated that human common

ductandgallbladder biles from subjects with cholesterolstones

containedfactorsthatinhibited calciumphosphate precipita-tion, and they proposed that bile salt/phospholipid micelles

wereresponsible for this effect. Hydroxyapatite is formed from amorphous calcium phosphate (ACP) bydissolution and

re-precipitation (6). ACPmay havethe stoichiometry of dical-cium phosphate dihydrate, CaHPO4. 2H20, ortricaldical-cium

phos-phate, Ca3(PO4)2-_XH20,depending _{on the}formation condi-tions. Ithas been shownthatbileacidscanbindtoACPand thatdifferent bile acidshavemarkedly different affinities (7).

Glycine-conjugated dihydroxy bile acids showed the highest affinity, whereasalltaurineconjugates showedalmostno abil-itytobind.

Wehypothesizedthat theeffectsseenby SutorandPercival (5) could be caused by bile acids bindingtocalcium phosphate microprecipitates, thereby preventing furtherprecipitation.To

test this,we studiedthe effects ofa glycine-conjugated

dihy-droxy bile acid, glycochenodeoxycholic (GCDC), anda

tau-rine-conjugated trihydroxy bileacid,taurocholic (TC), which

we predictedtobe active andinactive, respectively, with

re-specttotheinhibitionofhydroxyapatite precipitation.

Methods

Materials.Calciumchloride,dibasic sodiumphosphate, Trizma-HCl,

Trizma-base, calciumhydroxyapatite(typeI),bileacids(sodium salts),

and murexidewerepurchased fromSigmaChemicalCo.,St. Louis,

MO.Diphenylhexatrieneandtetrahydrofuranwereobtained from

Al-drichChemicalCo., Milwaukee,WI.

Stock solutions. Five stock solutionswerepreparedforusein all

experiments: (a)500 mM Tris-HCl and Tris-basebuffer,pH7.50;(b)

100mMcalciumchloride; (c)100mMdibasic sodiumphosphate;(d)

100mMbileacid;and(e)1.5 Msodiumchloride.

Calcium phosphate formation. Solutions containing calcium or

phosphatewereprepared separately.Finalconcentrations in each solu-tionwere:8 mM calciumor8 mMphosphate,50 mM Trisbuffer,0-80 1.Abbreviationsusedin thispaper: ACP, amorphouscalcium

phos-phate; CMC, critical micelleconcentration; FTIR,Fourier transform infrared; GCDC,glycochenodeoxycholic acid; HAP,calcium hydroxy-apatite; It,inductiontime;TC,taurocholic acid.

J.Clin.Invest.

©The American SocietyforClinicalInvestigation,Inc.

0021-9738/91/10/1265/07 $2.00

mMbile acid, andsufficient sodium chloride to let =0.15, in atotal volume of10 ml. Each solution was titrated to pH 7.50, passed through a_0.22-,gmfilter(Micro Separation Inc., Westborough, MA), and equili-brated at370Cfor 20 min. During this time the pH fell from 7.50 to 7.31±0.02. To start thereaction, we mixed equal volumes of calcium-andphosphate-containing solutions, so that the initial concentration of each reactant was 4 mM. These concentrations were used by Sutor and Percival (5) and result inprecipitation within a convenient time scale. The mixed solutions were placed in quartz cuvettes maintained at

370C,and theformation of calcium phosphate was monitored turbidi-metrically at 220 nm (8) (model 4053 spectrophotometer, Biochrom, Cambridge, England). In some experiments an aliquot of 500

.d

was withdrawn after 22 h of incubation and centrifuged for 5 min at 1 1,000 g, and the supernatanttotal calcium and phosphate content was deter-mined.

Determinationof total calciumandphosphate. Total calcium was measured by atomicabsorption spectrophotometry (model 5 100, Per-kin-Elmer Corp., Norwalk, CT) after dilution of samples with 5% tri-chloracetic acid/ 1% lanthanum chloride. Phosphate was determined by

conductivitymeasurementafter ion exchange HPLC (Ionpac AS4A

column, Dionex, Sunnyvale, CA) or by the molybdenum blue method(9).

Fourier transform infrared spectroscopy (FTIR). Calcium- and phosphate-containingsolutions, with or without bile salts, were mixed andplaced in an attenuated total reflectance cell fitted with a zinc selenide crystal (CIRcle cell, Spectra-Tech, Inc., Stamford, CT) and mounted in anFTIRspectrometer (model FTS 60,Digilab,Boston, MA). Spectra were collected atintervals for up to 72 h. The spectrum of areferencesolution, whichconsisted of 50 mM Tris,4mMCa2",and

100mMNaCl,wasmanually subtractedfrom each sample spectrum to disclose the signal from the precipitate. Spectra were not corrected for phosphate because the absorbancefrom precipitates was at least 20 times greater than the absorbance of4 mMphosphate.

Determinationofbileacidcriticalmicelle concentration (CMC).

The CMCs of bile acids were measuredfluorimetrically (10).1 ₄₁of 10 mMdiphenylhexatriene intetrahydrofuranwasaddedtodifferent con-centrations of bile acids dissolved inatotal volumeof 2 ml of Tris-buf-feredsolution,pH 7.5,g=0.15. Tubeswereincubated in the darkat

roomtemperature for 30 min. Fluorescencewasmeasuredat an excita-tionwavelength of 358 nmandan emissionwavelengthof 430nm

(modelLS-5B luminescencespectrometer,Perkin-Elmer Corp.). Effectofbileacids on Ca2" activity. Calcium activitywas

deter-minedwith the calcium-sensitive indicator murexide. The absorbance ofcalcium-containing solutions at540 and 470 nm wasmeasured

againstreference solutions thatwereof thesamecomposition,

includ-ingbile acid whenappropriate,butweredeprivedof calcium(I 1).In this way the effect of any shift in thedyeabsorbance spectrum caused by bileacid-dyeinteractionwasminimized. Thedifference in absor-banceatthetwowavelengthswaslinearlyrelatedtothelogarithmof calciumactivity.

Results

Calciumphosphateformationinthe absenceofbilesalt. When

theformation of calciumphosphatewasmonitoredby

turbid-ity measurement, a typical curve (Fig. 1) comprised three phases:(1)aninitial steady increaseinturbidity,which

gradu-ally slowed; (2)asuddenrapidincreaseinturbidity,whichwas

initiatedat56.6±4.7min(mean±SD,n=5);and(3)agradual

decline in turbidity aslarge particles sedimented. WhenpH

was monitored, there was an initialrapid fallof - 0.03 pH

units that was probably caused by the formation ofsoluble

Ca2`/HPO'

complexes.Throughouttheremainder ofphase 1

thepHfellafurther 0.01 pHunits,andthen, simultaneously

with the onset of phase 2, the pH of the solution declined sharply(Fig. 1),indicatingthattheeventsofphase2involved

z

0.

E

CD'

I-Time, min

Figure1.Formation ofcalcium phosphate monitored by turbidity and pH.Ca2"and phosphate (4 mM final concentration of each) were mixed togetheratpH7.3,,u=0.15,370C,andformation of

precipi-tate wasmonitored either byturbidity(o)orpH(-) measurements.

precipitationofadditional calciumphosphate, notsimply ag-gregation of existing material. These curves are similar to those

seenby otherinvestigators ( 12).The onset ofrapid

precipita-tion has been associated with the transformation of ACP to

HAPand thetimerequired for thistooccurhas beentermed the induction time

_(Ij)

(12).

Effect

of bile acids on the precipitation ofcalcium phos-phate. GCDC at a concentrationof 0.5 mM reduced

_It

by

- 15 min, but withincreasingGCDC concentrations

It

was

progressively lengthened,until at aconcentration of 2 mM and above, phase 1wasprolongedfor5h, at whichpointthe experi-ment wasterminated(Fig. 2). Additionalexperimentsshowed that when the concentration of GCDCwas2 2mM,therewas

nomarkedincreaseinturbidity andnovisibleprecipitate,even after 4 d(notshown). Measurement of supernatant calcium

0.8

E CD

10

I-0 100 200

Time, min

300

Figure2.Effect ofGCDConcalciumphosphateformation.

Ca2"

and

phosphateweremixedtogetherunder thestandardconditions,either without(o)orwith 0.5mM(-), 1.0mM(o), 1.5mM(n),2.0 mM(A), 4.0 mM(A),8.0mM(o),or12.0mM(x) GCDC;formation of

pre-cipitatewasmonitoredbyturbidity.The_portionsof thecurves

and phosphate concentrations after22 h demonstrated that, as well asincreasing

It,

GCDC also decreased the total amount of

calcium phosphate precipitated. In the absence of GCDC 7.52±0.03

_,mol

of calcium wasprecipitated,andthis was

pro-gressively reduced by 0.5 mM (7.48±0.08), 1.0 mM

(6.76±0.11), 1.5 mM (6.29±0.03), and 2 mM (0.74±0.60)

GCDC.Similar resultswereobtained forphosphate

precipita-tion.Theseexperimentsclearly show that GCDC causes

con-centration-dependent inhibitionofcalcium phosphate

precipi-tation.

The effects of TC were minor compared with those of

GCDC. Therewassomevariation inI,but values were always

within20% ofthecontrol: 0 mM, 55 min;2mM, 55 min;4

mM, 45min;9 mM, 47min;12 mM, 62min.

Theeffect of GCDCand TCin combinationwas also

stud-ied.When a lowconcentration ofGCDC (1.5 mM) was mixed

with increasing concentrations ofTC(2-12 mM),

_I,

decreased

exponentiallyastheamountof TC increased (Fig. 3). Interest-ingly, compared with 1.5 mM GCDC alone, the lowest TC

concentration increased It (164 and 317 min, respectively). Similar effectswereobserved withahigher GCDC

concentra-tion(4 mM) andTC varyingbetween 2 and 12 mM (Table I).

Although TC concentrations of2 and 4 mM plus 4 mMGCDC postponed phase2longer thantheduration oftheexperiment (500 min), the turbidity readings during phase 1 indicateda

fasterprecipitation ratethan when 4 mM GCDC alone was present(TableI).

CMCofbile acids. The CMCs of GCDC and TC were

de-termined to be 2 and 8 mM, respectively. These values are

consistentwith previousreports(13) andwereunaffectedby thepresenceof either4 mMCa2+or 4 mMphosphate.

How-ever,sincethis method isrelativelyinsensitivetosubmicellar

aggregates,itcannotbe ruledoutthat theseionsmayinfluence thesizeornumberof submicellaroligomers. Experimentswere

also performed to study the state ofaggregation of systems

containingbothGCDC and TC. Whenincreasingamountsof TCwereaddedto 1.5 mM GCDC, TCbecameincorporated into mixedspeciesaggregates, and the"CMC"for this mixed

systemwasestimatedtobe 4 mM total bilesalts, but this value will dependupontheGCDC/TC ratio. Thus, the addi-tion of2 mMTCto 1.5 mMGCDCincreased theamountof

system aggregation (eithernumberor sizeofaggregates) and

therebyprovidedmoreeffectiveinhibitionofHAPformation,

resulting in theprolonged

_I,

shown inFig.3.As moreTCwas

added, theaggregatescontainedevergreaterproportionsofTC,

Figure3. Correlationof 400 inductiontime with

GCDC/TCratio.Ca2+ andphosphatewere

S 300. mixedtogetherunder E thestandard conditions

E2

together with 1.5 mM

200 GCDC and various

concentrations of TC

(2-12 mM). Values of_I, X 100 * weremeasured from

plots ofturbidity against

time. Arrow indicates

o. . . I,forasystem

contain-0 2 4 6 8 10 12 14 _{ing 1.5 mM GCDC and} TCconcentratlon,mM noTC.

Table I. Effect of GCDC and TC in Combination on Calcium Phosphate Formation

Phase 1

[GCDC+TC] precipitation rate It

mM AOD/h min

0+0 0.749 50

4 +0 0.022 >500

4+2 0.025 >500

4 + 4 0.044 >500

4+9 0.055 250

4 + 12 0.076 82

0+ 12 0.872 62

Ca2" andphosphate (4 mM final concentration of each) were mixed together, with or without4 mMGCDC andincreasing concentrations

ofTC,atpH 7.3,j =0.15,

370C;

formationofprecipitatewas

moni-tored by turbidity at 220 nm.Precipitationrateand

Ih

were deter-minedfrom plots of turbidity against time.

andthiswasresponsible for the decreased inhibition seen in Fig. 3. This mechanism also operated when the GCDC

concen-tration was 4 mM and resulted in the attenuated inhibition shown in TableI.

Because potentinhibitionwasachievedatGCDC

concen-trations that approximated the CMC, it seemed likely that small aggregates, perhaps dimers, rather than micelles, were

responsible for this action.To testthispossibility,weexamined the effect ofdehydrocholicacidon HAPformation,since

dehy-drocholic acid does not formmicelles, butcan form dimers

(14).Underconditions ofconstantionic strength, dehydrocho-lic acidcaused modest inhibition ofHAP formation: in the absence ofdehydrocholic acid 4.72±0.08

Mmol

ofphosphate

wereprecipitatedat 24h,and thiswasprogressively reduced by 20mM(4.61±0.09), 40mM(4.21±0.01),60mM(3.81±0.06),

and 80mM(3.40±0.13) dehydrocholicacid.

Effect of bile acidson

Ca2"

activity. Under conditions of

constantionic strength, both TC and GCDC reducedCa2+ ac-tivity,but GCDCwas moreeffective thanwasTC(not shown).

GCDC concentrations <4 mMhadnomeasurable effecton

Ca2+

activity, and GCDC concentrations of4and 20mM

re-ducedactivity by only5%and25%, respectively.Even witha

25% reduction inactivity,thesolutions wouldremainhighly supersaturatedwithrespecttoHAP, and theeffect of GCDC

must be mediated by an alternative mechanism. TC had a

lesser effecton

Ca2+

activity,anda20mMsolution decreased

activitybyonly6%.

Dehydrocholic

acid reducedCa2+

activity

by4% (20 mM), 5% (40 mM), 14% (60 mM),and 20%

(80

mM). There was aclosecorrelation between

Ca2`

activity

in thepresence ofdehydrocholicacid andthe amountofHAP

precipitated

(r2

=0.958,P<0.005).

FTIRstudies

of

calciumphosphate

formation.

To

demon-stratethatphase2wasassociated with theformationofHAP,

weanalyzed the products ofthe reaction in situbyFTIR.Inthe absence ofbile acid, the

precipitation

of calcium

phosphate

caused theintensity of the orthophosphate

(P-O)

absorption

bandat 1,150-950

cm-'

toincrease with atime

dependency

that was verysimilartotheturbiditycurves

produced

inearlier

experments,

except that I wasincreased from 56 to80 min

a)

0 0 co m 0 V) 0 0o

0

co

1.6

1.4

E

CD

CM

.0

I-0 100 200 300

Time, min

Figure 4.Formation of calcium phosphate monitored byFTIR spec-troscopy.Ca2" andphosphate were mixed together under the standard

conditions,except thatthe temperature was 30'C, and precipitation wasmonitored by either FTIR spectroscopy(e)orturbidity (o).

30±1'C, and when a turbidity experiment was repeated at

30'C,the two setsofmeasurementscorrelated exactly (Fig. 4).

Concurrently withthe onsetof phase 2,the IRabsorption spec-trumof the calcium phosphateunderwent aprominent change

(Fig. 5A).Before phase2 the orthophosphate absorption band was relatively symmetrical, indicating little distortion of the phosphateionsfrom an average tetrahedral symmetry, which is

typicalof ACP

(15).

Butbetween 85 and 95min(i.e., 5min after

It),

thephosphate peakbegan totransform intoatriplet (Fig. 5 A), which indicated that certain P-O vibrations were

beingselectively restricted byanewlyforming crystalstructure. By240min the spectrum wasconsistent withthebulkofthe

precipitate beingHAP, asjudgedbycomparison withan

au-thenticHAP standard(Fig. 5 A, inset). Thusphase 2is

asso-ciated withthe emergenceofHAP asthepredominantform of precipitated calcium phosphate. Thepredominance ofHAP

couldexplainthegreatly increasedrateofprecipitationatthat

time,becauseHAPis muchless soluble than are otherforms of calcium phosphate (16).

As wouldbe predicted from earlierexperiments, 12 mM

TC hadlittleeffectonthe FTIR spectra, although

_I,

and the

appearanceofthephosphatetripletwereprolongedto - 1 10

min(not shown), which is consistent with the minordelaying effectpreviouslyseenwiththis concentration of TC (Table I). GCDC, however, had considerable concentration-dependent

effectsonthe IR spectraascalciumphosphateprecipitated.At 2 mM, GCDC completely prevented the appearance ofthe

crystalline phosphate triplet, although amorphous material continuedtoform slowly(Fig. 5B).In contrastwiththe

con-trol, wherethelocationofthe maximum absorbance moved progressively to lower wavenumbers throughout the

experi-ment, in the presenceof2 mMGCDC thepeak didnotshiftat

all, whichsuggestsveryeffective inhibition of transformation. Nocrystallinematerialwasdetectedat3d,atwhichpointthe

experimentwasterminated. Thus,theeffect of GCDC is

me-diated by preventingthetransition ofACPmicroprecipitatesto macroscopic crystallineHAP. With 1 mM GCDC,the

phos-phate triplet appeared at 85 min (notshown), and 0.5 mM GCDCaccelerated theappearanceof the_triplet,which became

1.2

1.0

0

_C)

Cu co

D0

0.8

.0

CDen

0.6

-

0.4-

0.2-0.0

1200

K

_0.3

1200 800

0.2

0.1

0.0

800

B

1200

800

Wavenumbers

(Cm-')

Figure 5. Effect of GCDConthe FTIRspectrumof maturing calcium phosphate. Calcium and phosphateweremixed and examinedas

de-scribed inFig.4.Spectrawereobtained(A)inthe absenceofGCDC

at(a)5min,(b) 85min,(c) 95min,(d)240min;or(B)inthe

pres-enceof 2 mMGCDCat(a)1h, (b) 5 h, (c) 48 h, (d) 72 h.Theinset

in Ashows the IRspectrumofstandard HAP. The sharp unresolved peaksat1,075cm-' in Barecaused by GCDC.

visible by 75 min (not shown). Otherwise the precipitation pro-cessdidnotmarkedly differ from the control situation,which

wasagain consistent with earlier observations (Fig. 2).

Effect ofGCDConthestoichiometry ofcalcium phosphate

precipitates.TheFTIRspectrumof controlprecipitatesafter

I,

wasverysimilar, butnotidentical,tothespectrumofthe HAP standard, which suggested that nonapatite material may be

containedintheprecipitate. Precipitateswerecollectedby

fil-tration(0.22

,gm)

30min beforeIt(phase 1)andby centrifuga-tion 30 min after

_I,

(phase 3). Half of each precipitate was

washed with10mlof10mMTrisbuffer, pH 7.50andwashed

and unwashed precipitateswereanalyzedfor calcium and phos-phoruscontent(Table II). Theunwashedphase1 precipitates

allhadCa/Pratios closeto1,which indicated thatmostofthe

phosphatewaspresentas

HPO4'-.

TheCa/Pratio of unwashed

phase 1 precipitates increasedwithincreasingGCDC

concen-tration.Washingthephase1precipitatescaused theCa/Pratio

toincrease, suggestingthat the interior oftheparticleswas

rela-tively less rich in HPO-, although this must bea tentative

interpretation because thesmallamountof materialremaining after washing resulted in relatively large errors. The washed

Table II.EffectofGCDC on Ca/P Ratios (Mean±SD) ofWashed and Unwashed Precipitates

fromPhasesIand 3

Phase I Phase 3

GCDC Unwashed Washed Unwashed Washed

mm

0 1.00± 0.07 1.19±0.14* 1.66±0.02 1.73±0.03*

0.5 1.00±0.06 1.22±0.18* 1.55±0.02 1.69+0.041 1.0 1.05±0.08 1.26 1.67±0.05 1.71±0.05* 1.5 1.05±0.08 ND 1.71±0.03 1.71±0.04* 2.0 1.18±0.05 1.50

Ca2+

andphosphate (4 mM finalconcentration of each) and

increas-ing concentrations of GCDC were mixed together at pH 7.3,u=0.15,

370C.Precipitates were collected both 30 min before (phase 1) and 30 min after (phase 3)I,.Precipitateswereanalyzedeither before or

afterwashingwith 10 mMTris,pH 7.5. Insomecases,onlyone washedphase I sample was assayed because of losses during washing. Where SD values are given, at least three samples were assayed.

Significancebetween washed and unwashed sampleswasassessedby ttest:*NS;$P<0.05;§P<0.005.

Inthecontrolphase 3 precipitates, washingcaused theCa/P ratiotoincrease,which suggested thatasurface layer, relatively rich in phosphate, had been removed from the precipitate. GCDC, 0.5 mM, decreased the Ca/P ratio ofunwashed phase3

precipitates in comparison withthecontrol, but1 and 1.5 mM

GCDC progressively increased theCa/Pratio, until with 1.5 mMGCDCunwashed andwashedprecipitatesdidnotdiffer.

From these data we calculated the Ca/P ratio of the surface fractions removed bywashing from phase 3precipitates,and plotted this ratio against induction time(Fig. 6). AlowCa/P ratio(i.e.,phosphate-rich surfacefraction)wasassociated with

ashort_I,and astheratioapproachedthevalueforHAP(1.67),

verysmallchanges in compositionwerecorrelated with large changes in

_I,.

Discussion

Exceptunderverydiluteconditions(17)theformation ofHAP

proceedsthroughatransitional ACPphase(18) byaprocessof dissolution andreprecipitation (6),rather than

by

solid-phase

Figure 6. Correlation of induction time with the Ca/Pratio of hydroxy-apatitesurface fraction. Thecomposition of the surface fraction of hy-droxyapatites formed in thepresenceof increas-ingconcentrations of GCDC(0-1.5 mM)was calculatedfrom the data in Table II and plotted against values of induc-tiontime obtained from

n turbidity measurements.

transition (19), and the rate ofconversion is proportional to the crystal content of the precipitate (20). The precise structural detail and chemical composition ofboth ACP and its transition products vary considerably (18, 21-23). Our results clearly showthat lowconcentrations (- 2 mM) of GCDC, but not TC,strongly inhibit thetransformation of ACP to HAP. Al-though bile acids reduce

Ca2"

activity (24, 25), and glycine-conjugated bile acids are more effective than taurine conju-gates (25), this is not the mechanism bywhich GCDC pre-vented HAP formation, since inhibition occurred at GCDC concentrations that did not measurably affect

Ca2"

activity.

A range of molecules and ions, including proteoglycans (26); phytates (27);

_Mg2e,

Sr2",

and

Zn2`

(28); ATP(29); pyro-phosphate (28, 30); casein (31); andphospholipids(32),inhibit

HAP formation. Williams and Sallis (33) proposed that the

minimum requirement forstronginhibition by anionic species

is thepossession of one phosphate group and one otheracidic

group,which may be either phosphate or carboxylic acid.

Mole-culeswith multiplecarboxylicgroupsalsoinhibit,but the min-imumnumberofgroupsrequiredisunknown (34).Inhibition ofHAPformationbyGCDCappearsreasonable because in the

aggregated state GCDC shouldmimicapolycarboxylated

mole-cule. The needfor multiple carboxylgroups suggeststhat the action ofsubmicellar concentrations ofGCDCrequiresdimers

orhigher multimers. That GCDC caused potent inhibition at 2

mM,aconcentrationatwhichveryfew micelles would be antic-ipated,alsosuggestssubmicellar multimers may betheactive species. Experiments withdehydrocholic acid confirmed that

partial inhibition could becausedby bile aciddimers, but80

mM dehydrocholic acid was much less effective than 2 mM

GCDC, and, unlike GCDC, the effect ofdehydrocholic acid

wasassociatedwithreduced

Ca2"

activity. The effect of dehy-drocholic acidon

Ca2"

activitywas unexpected.

Taurodehy-drocholicacid does not affect

Ca2"

activityasmeasuredbythe

Ca2"-sensitive

electrode (35),andthereforethe presentresults

may reflect our use of different methodology for measuring

Ca2+ activity (1 1).Itisunknownwhether thegreaterinhibiting

powerof GCDC derivesfrom its ability to form higher

mul-timers,orfromspecificstructuraldifferences thatmay enhance theability of GCDC dimers tointeract with HAP.

Dehydro-cholicacid differs from GCDCintwoimportantways: thering

substituents are equatorial keto groups rather than axial

hy-droxylgroups, and the carboxyl-bearingside-chain is shorter. The presenceof hydroxylgroups inGCDCmaybeimportant,

since citric acid isaninhibitorofHAPformation, althoughits dehydroxyl and dehydro derivatives are inactive (36). The

current data do not allow a definite conclusionto be

drawn

about thesize oftheGCDCaggregaterequiredtoinhibitHAP

formation.

Williams and Sallis (12) proposed that most inhibitors

could bedivided intotwoclasses: type Iactprincipally by

bind-ingto HAPcrystalembryosandpoisoning them;type II func-tion bydecreasing the labilityof ACP. Bothtypes

delay

HAP

formation, but only type I inhibitors reduce the amount of HAPformed(12). Because GCDC both delayed and reduced theproductionofHAP,thisbileacid isatype I inhibitor. This

conclusion is consistentwithourobservation that GCDC hasa

higheraffinityforHAPthanforACP(Qiu, unpublished

obser-vations).

Both ACP andHAP containHPO2--richsurfacefractions that can beremovedbywashing (18). The difference in

Ca/P

ratios between unwashed and washed

hydroxyapatites

was a

1.8-

1.7-

1.6-1.5

1.4

1.3-c 0 7.

O

Lo

0 O

.2

a.

. *

C)

1.21

-25 75 125

function of GCDCconcentration, and the amount of surface phosphateintheprecipitatewas reduced by GCDC

concentra-tions >0.5 mM. The amount of surfacephosphate could be reducedeitherbystabilization,so that it could not be removed

bywashing,orbyinhibition oftheinitial accumulation of acid

phosphateontheparticle surface.The general upward trend in

theCa/P ratio of unwashed precipitates withincreasing GCDC

concentration supports the second interpretation. We have

shown that GCDCbindingto HAPis inhibitedby

HPO4-,

but not byH2PO4?,suggestingthat GCDC andHPO-compete for commonbinding sites(37). Such competition for binding sites onnewly formedHAPembryos should poisonthe crystal

sur-face. This competition is reflected in our observation that a

phosphate-richsurfacefraction iscorrelated with a shortI.At

lowGCDC concentrations, the competition may be enough

onlytoslow therateof crystal growth,but athigher

concentra-tions, becauseofan increase in either the sizeor numberof

aggregates,thedegreeofpoisoningmaybeso great that growth

ispreventedandthe HAP embryosredissolvebefore they

con-stituteasufficient fractionof the total calciumphosphate

pre-cipitatetobe detected by FTIR. Tothe observerthisprocess

wouldappear asstabilization ofthe amorphous state.

Itis unknown why GCDCshows greaterbindingthan TC toACP (7) and HAP(38). However, because all the glycine-conjugated bile acids investigated, as well as unconjugated deoxycholic acid, bound to amuch greater extentthan any

taurineconjugate,acarboxylicacidgroup mayfavor binding (7). Inaddition, glycocholic acid boundtoACP(7) and HAP

(38) less well than did theglycine-conjugated dihydroxy bile

acids,thus theineffectivenessofTCmaybe relatedtobothits

conjugationanditshydroxylationpattern.Theincreased affin-ity of

Ca2"

forglycine conjugatesovertaurine conjugates has beenattributedto moreextensivehydrationof the latter (25).

However,in thepresentsituation bile acidbinding isnot

occur-ringtoisolated

Ca2"

ions insolution,but rathertotheHAP

surface, which consists ofregionsofalternating positive and

negativepolarity. Therefore, ifadjacentanionicgroupsofthe

bile acid aggregate are not arrayed in a mannerthatbrings

themintoappositionwithregionsofpositive polarity, theywill likely interact withnegativelychargedareas and be repelled. Underthisscenario,GCDCaggregates possessanionicgroups

thatmirror the spacing ofcalcium ionsontheHAPsurface,

whereas the sulphonate groups of TC aggregates are

mis-aligned.Inexperimentswith mixedTC/GCDCsolutions,TC

was incorporated into GCDCaggregates and attenuated the

inhibitionofHAPformation. This effect isprobably causedby increasingamounts ofTCinducingprogressively severe

mis-alignmentofanionicgroups. Theobservation that the action of

agivensubmicellarconcentration ofGCDCcanbeaugmented

by smallamountsof TCsufficientto causeincreased bile acid

aggregation indicates that the deleterious effects of small amounts of TCareinsufficienttooffset theadvantage of in-creasedaggregation.

Theability ofmixedTC/lecithinmicellestoinhibit calcium phosphate formation (5) isprobablycausedbybindingtoACP

orHAPnuclei initiatedbythe

lipid

phosphategroups.TC and othertaurine-conjugated bile

acids,

aswellas

GCDC,

inhibit

theprecipitationof calcium bilirubinate(4, 39). Thefact that

somebile acidsinhibit

precipitation

of onlysomecalcium salts (TCcaninhibit calciumbilirubinate,but notHAP)again sug-geststhatinhibition ofcalcium saltprecipitationdoesnot neces-sarilyoccur asaresult of direct actiononthecommon

cation,

calcium,in solution.In someinstancesinhibitionmaybe

me-diatedby specificinteractions between the bile acid and the

anion, either in solution or in a microprecipitate. In other cases,thetarget maybecalcium ionsincorporated intoa

crys-talembryo. Thespacingof these calcium ions isafunction of

crystaltype, sosmalldifferencesin thestructuresofaggregates

ofdifferent bile saltsmay meanthattheanionicgroupsinany

givenbile saltaggregate areoptimally alignedtointeract

prefer-entiallywith the calcium ionsofaparticularcrystal. If this is

true,changesin the bileacidcompositionofbileovertimemay

affecttherelativeproportionsof the various calcium salts that will belaid down in developing gallstones. This hypothesis couldpartially explainthedifferences incompositionobserved inmoving fromthecenter tothesurface inmanygallstones (2).

Finally, weemphasizethat the effect of bile acidson the

precipitationofcalcium saltscannotbeconsidered in isolation, butmusteventuallybeintegratedintoamorecomprehensive model inwhich the roleofothercomponentsofbile, suchas

phospholipids (32)andglycoproteins(3), and thepresenceof distinct microenvironments within the gallbladder (2) are

takenintoaccount.

WewishtothankAlice Cullu for editorialassistance,and Dr. Nancy Alcockforperformingthe totalcalciumdeterminations.

Thisstudywas supportedin part bygrant AM-16549 from the National Institutes of Health and grants from the Sealy and Smith Foundation.

References

1.Womack,N.A.,R.Zeppa,and G. L.Irvin. 1963. The anatomy of

gall-stones. Ann.Surg.157:670-686.

2.Crowther,R.S.,and R.D.Soloway.1990.Pigment gallstone pathogenesis:

fromman tomolecules. Semin. Liver Dis. 10:171-180.

3.Shimizu, S.,B.Sabsay,A.Veis,J. D.Ostrow,R. V.Rege,and L.G.Dawes.

1989. Isolationofanacidicproteinfrom cholesterolgallstones,which inhibits the

precipitationof calcium carbonate in vitro. J.Clin.Invest.84:1990-1996. 4.Angelico, M.,S. C. DeSanctis,C.Gandin,and D. Alvaro.1990.

Spontane-ousformation ofpigmentaryprecipitatesinbilesalt-depletedratbile and its

prevention bymicelle-formingbile salts.Gastroenterology.98:444-453.

5.Sutor,D.J.,and J. M.Percival. 1976. Presenceorabsenceof inhibitors of

crystal growthinbile.1.Effectof bileontheformation of calciumphosphate,a

constituent ofgallstones.Gut.17:506-510.

6.Eanes,E.D.,J. D.Termine,and M. U.Nylen.1973.Anelectron

micro-scopic studyof the formationofamorphouscalciumphosphateandits

transfor-mationtocrystallineapatite.Calcif Tissue Res.12:143-158.

7. Van DerMeer, R.,andH. T. DeVries. 1985. Differentialbindingof

glycine-andtaurine-conjugatedbile acidstoinsolublecalciumphosphate.

Bio-chem.J. 229:265-268.

8.Crowther,R.S.,S.-M.Qiu,G._Wen,N.Alcock,N.-K.Hong,and R. D.

Soloway.1990.Aturbidimetricstudyof theprecipitationof calciumphosphate.

Gastroenterology.98:A245.(Abstr.)

9.Crouch,S.R.,andH.V.Malmstadt. 1967.Amechanisticinvestigationof

molybdenum blue method for determination ofphosphate. Anal. Chem. 39:1084-1089.

10.Chattopadhyay,A.,andE. London.1984.Fluorimetricdetermination of critical micelle concentrationavoidinginterference fromdetergent charge.Anal. Biochem. 139:408-412.

11.Lichtenberg, D.,E.Werker,A.Bor,S.Almog,and S. Nir. 1988. Precipita-tionof calciumpalmitatefrom bilesalt-containing dispersions. Chem. Phys. Lipids.48:231-243.

12.Williams, G.,andJ. D. Sallis. 1982. Structural factorsinfluencingthe

abilityofcompoundstoinhibithydroxyapatiteformation. Cakif Tissue Int. 34:169-177.

13.Roda, A.,A.F.Hofmann,andK.J._Mysels.1983. Theinfluenceof bile saltstructureonself-associationin aqueous solutions.J.Biol.Chem. 258:6362-6370.

14.Small, D. M. 1971. The physical chemistry of cholanic acids. In The Bile Acids, Volume I. P. P. Nair and D. Kritchevsky, editors. Plenum Press, New York.249-356.

15.Termine, J. D., and D. R. Lundy. 1974. Vibrational spectra of some phosphate salts amorphous to X-ray diffraction. Calcif Tissue Res. 15:55-70.

16.Nancollas, G. H. 1984. Crystallization in bile. Hepatology(Baltimore).

4:169S-172S.

17.Boskey, A. L., and A. S. Posner. 1976. Formation of hydroxyapatite at low

supersaturation.J.Phys. Chem.80:40-45.

18.Termine, J. D., andE.D. Eanes. 1972.Comparativechemistryof amor-phousandapatitic calcium phosphate preparations.CalcifTissueRes.

10:171-197.

19.West, V. C. 1971.Observations on phase transformation ofaprecipitated calcium phosphate.CalcifTissue Res.7:212-219.

20. Eanes, E. D., andA.S.Posner. 1965.Kinetics and mechanismof

conver-sionof noncrystalline calcium phosphatetocrystallinehydroxyapatite. Trans.

N.Y.Acad.Sci.28:233-241.

21. Cheng, P.-T., and K. P. H. Pritzker. 1983. Solution Ca/P ratio affects

calciumphosphate crystal phases.CalcifTissueInt. 35:596-601.

22. Eanes, E. D., and J. L. Meyer. 1977. The maturation ofcrystalline calcium phosphates in aqueous suspensions at physiologic pH. Calcif Tissue Res. 23:259-269.

23. Tung, M.S., and W. E. Brown. 1983. An intermediate state in hydrolysis of amorphous calcium phosphate. Calcif Tissue Int. 35:783-790.

24. Moore, E.W., L.Celic, and J. D. Ostrow. 1982. Interactions between ionized calcium and sodium taurocholate: bile salts are important buffers for prevention ofcalcium-containinggallstones.Gastroenterology. 83:1079-1089.

25.Rajagopalan, N., and S.Lindenbaum. 1984. Counter-ion binding by bile acidsolutions.Hepatology (Baltimore).4:110S-1 14S.

26.Blumenthal, N. C., A. S. Posner, L. D. Silverman, and L. C. Rosenberg. 1979.Effect ofproteoglycans on in vitro hydroxyapatite formation.CalcifTissue Int.27:75-82.

27. Van Den Berg, C. J., L. F.Hill, and S. W. Stanbury. 1972. Inositol phos-phates andphytic acid as inhibitorsofbiological calcification in the rat. Clin. Sci.

(Oxf).43:377-383.

28. Root, M. J. 1990.Inhibition of the amorphous calcium phosphate phase

transformation reaction by polyphosphates and metal ions.Calcif Tissue Int. 47:112-116.

29.Termine, J. D., and K. M. Conn. 1976. Inhibition ofapatite formation by phosphorylated metabolites and macromolecules.Calcif Tissue Res. 22:149-157.

30.Fleisch, H., and S. Bisaz. 1962. Isolation from urine of pyrophosphate, a calcificationinhibitor. Am. J. Physiol. 203:671-675.

31.Termine, J. D., R. A. Peckauskas, and A. S. Posner. 1970. Calcium phos-phateformation invitro II. Effects of environment on amorphous-crystalline transformation. Arch. Biochem.Biophys. 140:318-325.

32.Wuthier, R. E., and E. D. Eanes. 1975. Effect of phospholipids on the transformation of amorphous calcium phosphate to hydroxyapatite in vitro. Cal-cif Tissue Res. 19:197-210.

33.Williams, G., and J. D. Sallis. 1979. Structure-activity relationship of inhibitors of hydroxyapatite formation. Biochem. J. 184:181-184.

34. Hay, D. I., E. C. Moreno, and D. H. Schlesinger. 1979. Phosphoprotein-inhibitors of calcium phosphateprecipitationfromsalivarysecretions. Inorg. Perspect.Biol.Med. 2:271-285.

35. Moore, E. W.,A.F. Hofmann, C. D. Schteingart, and H.-T. Ton-Nu. 1990.High-affinity premicellarCa++-bindingtobile salts:II. Structure-activity relationships. Hepatology (Baltimore). 12:998. (Abstr.)

36. Tew, W. P., C. Mahle, J.Benavides,J. E.Howard,and A. L. Lehninger. 1980.Synthesis and characterization of phosphocitric acid,apotent inhibitorof hydroxyapatite crystal growth. Biochemistry. 19:1983-1988.

37.Crowther,R.S.,N.-K.Hong,S.-M.Qiu,and R. D.Soloway.1991. Glyco-chenodeoxycholic acid inhibits calcium hydroxyapatite formation by competeti-vely inhibiting HP04- binding to the particle surface. Gastroenterology.

100:A733.(Abstr.)

38.Qiu, S.-M., R. S. Crowther, and R. D. Soloway. 1991. The ability of bile acidstoinhibit calciumhydroxyapatiteformation is reflected in their apatite

bindingaffinity.Gastroenterology. 100:A787.(Abstr.)

39. Hong, N.-K., R. D.Soloway, R. S.Crowther, F.Liu,S.-M.Qiu, H. Guo,

N.Alcock, and J.-G. Wu. 1990.Chenodeoxycholateconjugatesare more

effec-tivethantaurocholate ininhibitingtheprecipitation of calcium bilirubinate.