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PROGRESS REPORT SUMMARY

PRINCIPAL INVESTIGA TOR OR PROGRAM DIRECTOR

Chest er W. Dougl assAPP LICANT ORGANIZATION

Har var d School of Dent al Medi ci ne TITLE OF PROJ ECT (Repeat title shown in item 1 on first page)

Fl uor i de Exposur e and Ost eosar coma(SEE INSTRUCTIONS)

PROGRESS REPORT SUMMARY

'\NT NUMBERES06 000; . . . 03

9/ 30/ 93 9/ 29/ 94

1. Speci f i cAi ms

The Nat i onal Toxi col ogy pr ogr am ( NTP) f ound Dl equi vocalevi dence" of car ci nogeni ci t y based on t he occur r ence of a smal lnumber ost eosar comas i n mal e r at s f ed doses of hi ghl y f l uor i dat edwat er ! . A s ubcommi t t ee of t he Commi t t ee t o Coor di nat e Envi r onment alHeal t h and Rel at ed pr ogr ams ( CCEHRP) was t hen convened t e anal yzebenef i t s and r i sks of f l uor i de i n dr i nki ng wat er .

An anal ys i s of 12 " f l uor i dat edUl count i es and 21 "non-f l uor i dat ed" count i es wi t hi n t wo SEER si t es used as a basi s f or t heCCEHRP Fi nal Repor t t hat di r ect l y r ef ut ed t he NTP f i ndi ngs 2 Thi sst udy measur ed t he exposur e of i nt er est , i ngest i on of f l uor i dat edwat er , onl y at t he count y l evel , and no at t empt was made t o capt ur et he ef f ect of mi gr at i on. To achi eve t he or i gi nal goal of t heCommi t t ee, t hi s nat i on- wi de hospi t al - based casecont r ol st udy wi l lpr ovi de a mor e sci ent i f i cal l y concl usi ve assessment of r i sk f orost eosar coma f r om i ngest i on of f l uor i dat ed wat er . Speci f i e ai msaddr essed i n t he on- goi ng st udy i ncl ude:

1. To compar e t he compl et e r es i dent i al f l uor i de hi s t or i es of

ost eosar coma pat i ent s ( bot h pr eval ent and i nci dent cases) wi t h t hef l uor i de hi st or i es of hospi t al - based cont r oI s.

2. To compar e t ot al f l uor i de exposur e f r om or al hygi ene pr act i cesbet ween cases and cont r oI s; i ncl udi ng f l uor i de suppl ement s, seI f -appl i ed and pr of essi onal l y appl i ed t opi cal f l uor i des , andpar t i ci pat i on i n school based f l uor i de pr ogr ams.

3. To compar e f l uor i de cont ent i n bone andost eosar coma pat i ent s wi t h t he f l uor i de cont entt oenai l s of hospi t al based cont r ol s.

t oenai l si n bone

of and

4. To cont r ol f or age, gender , and educat i on i n t he compar i son of cases and cont r ol s.

2. STUDI ES AND RESULTS

To accompl i sh t hese ai ms, a r et r ospect i ve and pr ospect i vehospi t al based casecont r ol st udy was pr oposed and i mpl ement ed

PHS 2590 (Rev. 9/91) (Form Page 6) Page ~ (Use continuation pages if nece


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dur i ng t he f i r s t year . The r et r ospeet i ve aspect of t he casecont r ol s t udy col l ected dat a f r om pr eval ent cases and cont r ol s seenat 10 hospi t aI s nat i on- wi de. Cases ar e def i ned as pat i ent sdi agnosed wi t h pr i mar y ost eosar coma f r om 11- 01- 89 t o 12- 31- 92 byt he par t i ci pat i ng or t hopedi e sur geons . Non- cancer ous cont r ol smat ched by age, gender , and di st ance f r om t he hospi t al wer e chosen

f r om t he i n- and out - pat i ent r ost er s of each of t he hospi t al s'sur ger y depar t ment . A 1: 2 r at i o of cases t o cont r ol s was enr ol l ed.

The f ol l owi ng hospi t al s pr ovi ded access t o t he pat i ent senr ol l ed as a cas e or cont r ol i n t he r et r os pect i ve aspect of t hest udy:

Hospi t al

Massachuset t s Gener al Hospi t al

# of Pr eval ent Cases/ Cont r ol s#Cases #Cont r ol s Tot al s

45 90 135

Chi l dr en' s Hospi t al - Bost on

Cr ei ght on Uni ver si t y

Chi l dr en' s Hospi t al - DC

Memor i al Sl oan Ket t er i ng

Uni ver si t y of Chi cago

Rush Medi cal Col l ege

Uni ver si t y of Fl or i da

UCLA

Case West er n/ Cl evel and Cl i ni c

18

9

19

27

21

13

27

29

18

226

36

18

38

54

42

26

54

58

36

452

54

27

57

81

63

39

81

87

54

678

Ost eosar coma pat i ent s and t he mat c hed cont r aI s wi t h anypr edi agnos i s hi s tor y of t he known r i sk f ac tor , r adi at i on t her apy,ar e excl uded. l n addi t i on, pat i ent s wi t h a hi st or y of ki dneydi al ys i s ar e exc l uded as t hey chose t o dr i nk dei oni zed wat er f ormedi cal r eas ons . Bot h gender s and aI l r aces ar e i ncl uded f or

st udy.Pr ot ocol and Dat a Col l ec t i on f or t he Ret r ospec t i ve St udy

The pr oj ect Di r ect or , Dr . McGui r e, t r avel ed t e each hospi t alt o r evi ew medi cal r ecor ds and det er mi ne t he el i gi bi l i t y of t he

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pr eval ent cases. Once t he age at di agnosi s, gender , and di st ancef r om t he hospi t al was t r anscr i bed f r om each el i gi bl e case medi calr ecor d, t he pr ocess t o chose t he cor r espondi ng cont r ol s was begun.Each hospi t al ( except f or Chi l dr en' s of DC ) had t he capabi l i t y t ogener at e a comput er pr i nt out of t he i n- andf or out - pat i ent r ost er s.A l i st i ng of t he pat i ent s mat chi ng t he age ( + 5 year s) , gender anddi s t ance f r om t he hospi t al of each of t he cases was r equest ed.Once t he l i st i ng was r ecei ved, si x names wer e sel ect ed at r andomand t hei r medi cal r ecor ds wer e or der ed so t hat abst r act i on of demogr aphi c and medi cal i nf or mat i on coul d be compl et ed. Mor e t hanone r et ur n t r i p t o each hospi t al was necessar y t o r et r i eve t hei nf or mat i on f r om t he medi cal r ecor ds of t he qual i f yi ng mat chedcont r ol s because of t he t i mef r ame needed t o gener at e t he r ost er andor der t he medi cal r ecor ds.

The i nf or mat i on abst r act ed by Dr . McGui r e f r om t he medi calr ecor ds of t he el i gi bl ecases and t he qual i f i ed cont r ol s wasent er ed di r ect l y i nt o a sof t war e pr ogr am devel oped by New Engl and

Resear ch I nst i t ut e ( NERI ) . The dat a wer e t hen downl oaded ont o di sksand mai l ed t o NERI . Resear cher s at NERI pr oceeded t e send a l et t erof expl anat i on of t he r esear ch goal s of t he st udy t o t he el i gi bl eost eosar coma pat i ent s and t he qual i f i ed mat ched cont r ol s. Tel ephonecont act f ol l owed wi t hi n la days f or t he admi ni s t r at i on of t het el ephone quest i onnai r e ( See Appendi x A f or t he r et r ospect i vequest i onnai r e) . A par ent was sur veyed i f t he case/ cont r ol was l esst han 18 year s ol d. Appendi x B r epor t s t he st at us of t hedi spos i t i ons f or t he compl et i on of dat a col l ect i on f r om t hepr eval ent cases and cont r ol s as of t he end of t he Thi r d Quar t er( 6/ 30/ 94) Di s pos i t i ons ar e f i nal f or aI l but t hr ee per cent( 22/ 678) of t he par t i ci pant s. We have yet t o obt ai n weI l wat ersampl es f or t hese r emai ni ng 22 par t i ci pant s. The l ast r emi nder

t el ephone cal I was made i n J une t o el i ci t t he r emai ni ng sampl es.Appr oxi mat el y 25 per cent of t he cases and cont r ol s used weI l

wat er at some poi nt i n t hei r r esi dent i al hi st or y. Thi s r at e washi gher t han expect ed dur i ng t he pi l ot phase. Har var d School of Dent al Medi ci ne pr oj ect st af f sent l et t er s and vi al s f or weI l wat ersar npl ecol l ect i on t o t he cases/ cont r ol s who r epor t ed weI l wat eruse. The par t i ci pant s ar e pr ovi ded wi t h a post age pai d car dboar dcont ai ner i n whi ch t o r et ur n t he weI l wat er sampl e. Dr . McGui r emeasur es t he l evel of f l uor i de i n each sampl e wi t h t he use of a DR100 Col or i met er .

Expanded Dat a Col l ect i on i n t he Pr ospect i ve st udy

Dur i ng Year 1, ar r angement s had been made t o coor di nat e t hest udy wi t h an expanded dat a col l ect i on ef f or t of t he et i ol ogy of ost eosar coma whi ch wi l l be conduct ed by t he Nat i onal Cancer

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I nst i t ut e ( NCI ) . The f undi ng pr ovi ded f or t oenai l anal ysi s i n Year2 wi l l be car r i edover i n or der t o f aci l i t at e t he col l ect i on of t hese sampl es so t hat t hi s pr ocess can be i nt egr at ed wi t h t he NCIpar t i ci pant i nt er vi ews. We bel i eve t hat t he addi t i on of t he NCIbackqr ound dat a on st udy par t i ci pant s wi l l i ncr ease our abi l i t v t ocont r ol f or al t er nat i ve f act or s i n anal vz i nq t he l i nk bet weenf l uor i des and ost eosar coma.

The pr oj ect Di r ect or and Pr i nci pal I nvest i gat or conduct ed t het r ai ni ng sessi ons f or t he r esear ch nur ses i n Washi ngt on DC at t heend of J anuar y. Wi t h t he ass i s t ance of NCI s t af f , t he f ol l owi ngsubj ect s wer e cover ed dur i ng t he f our day sessi on:

* St udy Obj ect i ves and Tr ai ni ng Obj ect i ves* Dat a Col l ect i on Component s* si t e Coor di nat or Dut i es* I nt er vi ew Requi r ement s f or Cer t i f i cat i on* I nt er vi ewi ng Vi deo* Quest i onnai r e and Q by Q' s* Rol e Pl ays: Communi t y Dyads* Subj ect Dat a For m* Ass i gni ng I D' s* Case and Cont r ol Enr ol l ment and For ms* Recor d of Cont act s and For m* Co- r espondent s Scr een Wor ksheet* I nf or med Consent* Speci men Col l ect i on* pr act i ce Speci men Handl i ng and Label l i ng* si t e Pl an* pr act i ce f or Cer t i f i cat i on* Qual i t y Cont r ol and Edi t i ng

* Repor t i ng t o and Communi cat i ng wi t h pr oj ect St af f * Over al l Revi ew

As of t he end of t he Thi r d Quar t er of Year 2, t he pr ot ocol hasbeen expanded t o adj ust f or t he col l ect i on of an i l i ac cr est bonesampl e and a bl ood sampl e f r om i nci dent cases and a bl ood sampl ef r om t he i nci dent cont r ol s . See Appendi x C f or t he updat edpr ot ocol . The non- t umor pi ece of bone wi l l pr ovi de a st andar di zedanat omi cal posi t i on f r om whi ch a bone sampl e wi l l be t aken f r ombot h t he case and t he cont r ol . The appr oval f or t he r evi s i on f r omeach hospi t al ' s human subj ect r evi ew commi t t ee has been acqui r ed,t he r ecr ui t ment of r esear ch nur ses at each hospi t al has beencompl et ed, and dat a col l ect i on f r om i nci dent cases and cont r ol s hasbegun.

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3. SI GNI FI CANCE

When r est r i ct ed t o mal es under 20 wi t h ost eosar coma i n t heSEER st udy i n t he CCEHRP Fi nal Repor t , t he r at e of ost eosar comai ncr eased 79% i n t he f l uor i dat ed count i es and decr eased 4% i n t henon- f l uor i dat ed count i es f r om 1973- 80 t o 1981- 87. Ti me- t r endanal yses wer e per f or med and t he com: mi t t ee concl uded t hat f i ndi ngwas not s i gni f i cant . However , as was s tat ed pr evi ous l y, t hemeasur e of exposur e, dur at i on of i ngest i on of f l uor i dat ed wat er ,was onl y at t he count y l evel and no at t empt was made t o capt ur e t heef f ect of t he mi gr at i on. Cases i n t he pi l ot s t udy casecont r olst udy3 ( pr i mar i l y I owans) I i ved i n an aver age of 1. 7 di f f er ent t ownspr i or t o t he di agnosi s. Thi s f i ndi ng weakens concl usi ons based ont he assumpt i on on no mi gr at i on. The CCEHRP r epor t says DDTot heext ent t hat mi gr at i on i s a f act or , i t i s l i kel y t o di mi ni sh t hesensi t i vi t y of a geogr aphi c cor r el at i on st udy t o det ect possi bl eexcess r i sk . . . Fai l ur e t o obser ve exposur e- di sease associ at i on canbe at t r i but ed . . t o a com: muni t yl evel st udy . . Thi s i s especi al l y t r ue

f or mal i gnanci es wi t h a ver y l ow i nci dence such as ost eosar coma. "Li nkage of f l uor i de i ngest i on and cancer i ni t i at i on coul d

r esul t i n a l ar ge- scal e def l uoi r i dat i on of muni ci pal wat er syst emsunder t he Del aney cl ause. ( one aspect of t hi s EPA cl ause pr event st he addi t i on of car ci nogeni c agent s t o t he nat i on' s wat er suppl y) .Al t hough t he SEER s t udy showed equi vocal evi dence, t he pi l ot st udyr esul t s f or t hi s gr ant showed no l i nkage of t he i nges t i on of f l uor i dat ed wat er t o t he occur r ence of ost eosar coma. Ther ef or e, ani ncor r ect i nf er ence i mpl i cat i ng syst emat i c f l uor i de car ci nogeni ci t yand i t s r emoval f r om our wat er syst ems woul d be det r i ment al t o t heor al heal t h of most Amer i cans, par t i cul ar l y t hose who cannot af f or dt o pay i ncr eas i ngl y expensi ve r est or at i ve dent al car e.

Addi t i onal l y, a gr eat er under s t andi ng of t he et i ol ogy of ost eosar coma may pr event ot her s f r om exper i enci ng i t .

4. PLANS

Dat a Co1l ect i on f or Year 3

Pr eval ent Cases and Cont r ol s: Al I pr eval ent cases and cont r ol swer e accr ued i n t he f i r st year . Because of hi gher pr eval ence t hanexpect ed, t he wor k t o compl et e t he gat her i ng aI l weI l wat er sampl eswas ext ended i nt o year 2. By t he end of t he 4t h Quar t er of Year 2,f i nal di sposi t i ons wi l l have been i ssued, t he anal ysi s wi l l havebeen cor npl et edand a r nanuscr i pt submi t t ed f or publ i cat i on.

I nci dent cases and cont r ol s: I nci dent cases and cont r ol s wi l laccr ue t hr oughout t he ent i r e per i od of year 3. Resear ch nur ses ateach hospi t al ar e pr ef or r ni ng t he f ol l owi ng dut i es:

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1.. assi st t he or t hopedi csur geon i n gai ni ng i nf or medconsent f r om t he i nci dent cases,

2. l ocat e qual i f i ed cont r ol s f r om t he depar t ment alroster,

3. admi ni st er t he quest i onnai r es t o and col l ect t oenai lsampl es f r om t he el i gi bl e cases and qual i f i edcont r ol s ( See Appendi x D f or t he Pr ospect i veQuest i onnai r e)

4. col l ect t he t umor sl i ce, bl ood speci men, and i l i accrest bone bi opsy f r om t he case at t he t i me of t hesur ger y,

5. col l ect t he cont r ol aut opsy bone6. col l ect cont r ol bl ood speci men7. pr epar e aI l t he pat hol ogy speci mens f or t r anspor t at i on8. send t he t oenai l sampl es f or anal ysi s9. send t he quest i onnai r e d~ t a f or anal ys i s

The pr oj ect Di r ect or and Pr i nci pal I nvest i gat or wi l l wor k wi t hNCI s t af f as t hey t r avel t o each hos pi t al t o moni t or 1.) t her el i abi l i t y of t he dat a, 2) t he pr ocess by whi ch" t he cont r ol s ar echosen and 3) pr ovi de gui dance on t he col l ect i on of speci mens; andmoni t or t he col l ect i on of dat a at each hospi t al weekl y.

MI NORI TY RESEARCH TRAI NI NG PROTOCOL

Dur i ng t he past year , Dr . Da Si l va has become an i nt egr al par t of t he pr oj ect . He has wor ked cl osel y wi t h t he Har var d per sonnel ,NERI and NCI . Dr . Da Si l va wor ked di r ect l y wi t h Har var d per sonneland NCI t o i ncor por at e hi s r esear ch pr ot ocol i nt o t he pr ospect i vepor t i on of t he pr oj ect . He assi s t ed i n t he devel opment of t he

i nt er vi ew quest i onnai r e t o ensur e t hat t he speci f i e ai ms of hi spor t i on of t he pr oj ect woul d be addr essed:

1. To det er mi ne i f an associ at i on exi s t s bet ween hi ghersoci oeconomi c st at us and hi gher f l uor i de exposur e.

2. To det er mi ne i f an associ at i on exi s t s bet ween hi ghersoci oeconomi c st at us and t he r i sk f or ost eosar coma and i f t hi s r el at i onshi p i s medi at ed t hr ough hi gh f l uor i deexposur e.

Dr . Da Si l va has al so been i nvol ved i n f ol l ow up wi t h NERI t oi dent i f y shor t f al l s i n dat a acqui s i t i on. He made a s i t e vi s i t ,wi t h Dr . MCGui r e, t o Washi ngt on D. C. ' s Chi l dr en' s Hospi t al . Dur i ng

t he s i t e vi s i t he l ear ned t he on s i t e pr ot ocol f or i dent i f yi ngmat ched cont r ol s and assi st ed i n obt ai ni ng t he r emai ni ng necessar ycont r ol s.

Dr . Da Si l va par t i ci pat ed, al ong wi t h ot her Har var d per sonnel ,i n t he t r ai ni ng sessi on hel d at NCI f or t he Si t e Coor di nat or s who

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wi l l be i nvol ved i n t he pr ospec t i ve por t i on of t he s tudy. Hepar t i ci pat ed i n aI l aspect s of t he t r ai ni ng i ncl udi ng t he consentr equi r ement s, dat a col l ect i on component s, r ol e pl ayi ng andcer t i f i cat i on as an i nt er vi ewer . Dr . Da Si l va wi l l al so ser ve asan addi t i onal cont act per son f or t he i ndi vi dual s at each of t hesi t es and wi l l assi st i n t he r esol ut i on of any pr obl ems t hat ar i se.

l n r esponse t o a subopt i mal acqui si t i on of par t i ci pat i ngcont r ol s f r om UCLA Medi cal Cent er , Dr . Da Si l va made a si t e vi si tt o updat e dat a on pr evi ousl y i dent i f i ed cont r ol s, cont act t hem andr equest t hei r consent t o par t i ci pat e i n t he t el ephone i nt er vi ewconduct ed by NERI .

Dr . Da Si l va has al so been t r ai ned i n t he pr ot ocol f or t hemeasur ement of f l uor i de i n weI l wat er sampl es at Har var d. He hasassi st ed i n t he f Ol l ow- up on mi ssi ng weI l wat er sampl es. Thus

"r educi ng t he number of mi ssi ng dat a poi nt s. He has al so beeni nvol ved i n dat a t r ansmi ssi on of wat er sampl e f l uor i de l evel s t oNERI and i n dat a er r or checki ng.

Det er mi nat i on of f l uor i de exposur e f r om r esi dent i al hi st or y

Pr eval ent and I nci dent al Cases and Cont r ol s: The f l uor i del evel s of t he muni ci pal i t i es suppl yi ng wat er t o t he cases andcont r ol s ar e l i st ed i n t he CDC Fl uor i dat i on Census i n ppm. Year of f l uor i dat i on i s al so l i s t ed. However , we have f ound t hat manyci t i es and t owns ar e not l i s t ed i n t he Census, even t hough t heyhave f l uor i dat ed wat er . Pr obl em t owns wer e i dent i f i ed i n year 1and t he Depar t ment of Heal t h i n t he f ol l owi ng st at es wer e cal l ed t o

obt ai n a s t at ewi de l i s t of t he f l uor i de l evel s f or aI l c i t i es andt owns i n t he s tat e: Ar i zona, Ar kansas , Cal i f or ni a, Col or ado,Connect i cut , Del awar e, Fl or i da, Geor gi a, I l l i noi s, I ndi ana, I owa,Kansas, Kent ucky, Loui si ana, Mai ne, Mar yl and, Massachuset t s, NewHampshi r e, Ohi o, Okl ahoma, Pennsyl vani a, Rhode I sl and, Sout hCar ol i na g Texas, Tennessee, Ver mont g and Washi ngt on.

Wat er sampl es t ubes wi l l cont i nue t o be pr ovi ded by mai l t ot he par t i ci pant s i n t he pr ospect i ve st udy who use weI l wat er . Thepar t i ci pant s wi l l be r equest ed t o r et ur n a weI l wat er sampl e f oranal ysi s at t he Har var d School of Dent al Medi ci ne. Response r at et o dat e has been appr oxi mat el y 70% f or weI l wat er sampl ecol l ect i on. We expect " t hi s t o i mpr ove, however i n over 30

i nci dences t he weI l i n quest i on has been shut of f .Col l ec t i on of t oenai l sampl es and det er mi nat i on of f l uor i deconcent r at i on

I nci dent cases and cont r oI s: The r esear ch nur ses wi l l col l ect

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a set of t oenai l cl i ppi ngs f r om aI l 10 t oes f r om aI l i nci dentpar t i ci pant s. The cl i ppi ngs wi l l be cl eaned i n dei oni zed wat er ateach hospi t al . Thi s pr ocedur e i s hi ghl y ef f ect i ve at r emovi ngsuper f i ci al cont ami nat i on. The cl i ppi ngs wi l l t hen be sent t o t hel abor at or y of Dr . J . St even Mor r i s at t he Uni ver si t y of Mi ssour iResear ch React or . The case cont r ol pai r s wi l l be anal yzed i n t hesame assay r un by neut r on act i vat i on anal ysi s; t he speci mens wi l lbe l abel ed by I D number onl y, and t he posi t i on of t he case andcont r ol speci men wi l l var y r andoml y. Af t er exposur e t o t her malneut r ons, gamma emi ssi ons f r om t he 2 0F i sot ope ( hal f - l i f e 11. 03seconds) ar e count ed and, wi t h t he use of a cer t i f i ed s t andar d,conver t ed t o wei ght of f l uor i de. The coef f i ci ent of var i at i on f ort hese measur ement s on separ at e nai l speci mens f r om t he same per son( whi ch i ncl udes bi ol ogi cal var i at i on and t echni cal er r or ) i s 10- 15%.

Det er mi nat i on of f l uor i de cont ent i n bone

About one cm~ of r esect ed bone f r om t he i nci dent ost eosar coma

t umor sur gi cal s i t e of pat i ent s wi l l be anal yzed f or f l uor i decont ent NMR i magi ng and spect r oscopy. Col l ect i ng t he r esect edspeci men wi l l not i n any way al t er t he t ype of sur gi cal pr ocedur et hat gi ven pat i ent wi l l r ecei ve. Addi t i onal l y, whi l e t he i nci dentcase i s under gener al anest hesi a f or t he t r eat ment of t he sar comal esi on, an i l i ac cr est bone bi opsy wi l l be obt ai ned. At a poi nt 2cma 4 cm i nci si on wi l l be made. The sof t t i ssue wi l l be r et r act edt o expos e t he out er cor t ex of t he i l i um. The t r ochar i s t henposi t i oned so t hat t he needl e wi l l acqui r e a t r ansi l i ac bi cor t i calcor e bi opsy wi t h a di amet er of appr oxi mat el y 1 cm. Hemost asi s wi l lbe achi eved by el ect r ocoagul at i on or mi nor bl eeder s. The sof tt i ssues ar e al l owed t o f al l back i nt o pl ace. The wound i s cl osedi n a r out i ne f ashi on. The bone bi ops y f r om t he i nci dentost eosar coma pat i ent wi l l be anal yzed f or f l uor i de cont ent . Thenon- t umor pi ece of bone wi l l pr ovi de a st andar di zed anat omi calposi t i on f r om whi ch a bone sampl e wi l l be t aken f r om bot h t he caseand t he cont r ol . Cadaver i l i ac crest bone sampl es ( t he cont r olbone) mat ched by age and gender wi l l al so under go f l uor i deanal ysi s. Speci mens wi l l be st or ed at - 60 0 C and t hen t r anspor t edt a MGH i n dr y i ce unt i l r eady f or use.

The speci men wi l l be cl eaned of adher i ng sof t t i ssue andper i ost eum f ol l owi ng br i ef t hawi ng, pl aced under l i gui d ni t r ogen,and gr ound t o a f i ne powder wi t h a l ar ge cer ami c mor t ar and pest aI . The r esul t i ng powder wi l l be l yophi l i zed over ni ght at r oomt emper at ur e, and t hen ext r act ed wi t h chl or of or m- met hanol . Thi st r eat ment wi l l r emove most of t he wat er and l i pi d, pr oduci ng a dr ypowder whi ch i s easy t o spi n at hi gh speed. The mi l dness of t het r eat ment i nsur es t hat no chemi cal al t er at i on of t he mi ner aI wi l loccur . l n our exper i ence, gr ound but ot her wi se unt r eat ed speci mensof t en cannot be spun above 1. 5 kHz, whi ch woul d be suf f i ci ent f or

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f l uor apat i t e, but i ns uf f i ci ent f or cal ci um f l uor i de- t ypeconst i t uent s. Lyophi l i zi ng yi el ds a past y mat er i al t hat can beusual l y be coaxed above 2. 5 kHz. Removal of t he maj or i t y of t hel i pi d i s r equi r ed t o pr oduce a dr y, f r ee f l owi ng powder whi ch candynami cal l y bal ance i t sel f as t he spi nner appr oaches speeds above6 kHz. Speci mens wi l l be equi l i br at ed t o l abor at or y t emper at ur eand humi di t y f or 24 hr pr i or t o NMR anal ysi s.

Quant i t at i ve 19 p MAS NMR spect r a of an accur at el y wei ghedsampl e of each powder ed speci men wi l l be obt ai ned on a Br uker MSL-400 NMR spect r omet er oper at i ng at a f i el d st r engt h of 9. 4 T and a19

p f r equency of 376 MHz. Spi nni ng speeds wi l l be pr eci sel y 7. 00kHz so t hat t he di st r i but i on of si gnal i nt ensi t i es among t he bot hbef or e and af t er NMR anal ys i s t o check f or pos si bl e wat erabsor pt i on. Quant i t at i on wi l l be per f or med bycompar i son of t her eagent gr ade CaP 2 st andar d. CaF 2 i s chosen as a pr i mar y st andar dbecause i t may be accur at el y det er mi ned by gr avi met r y al one. Dat apr ocessi ng wi l l be per f or med of f l i ne f r om t he spect r omet er usi ng

t he NMRI sof t war e package f r omNew Met hods Resear ch ( Syr acuse, Ny) RePl l i cat e anal yses wi l l be per f or med f or each speci men on t wodi f f er ent days.

The f i gur e of 7 kHz r epr esent s t he appr oxi mat e maxi mumspi nni ng speed achi evabl e by our equi pment . As shown byKr ei nbr i nk, et a1 31 t hi s i s mor e t han adequat e t o f ul l y nar r owapat i t i c f l uor i de, and suf f i ci ent t o nar r ow CaF2- t ype r esonancesenough f or r easonabl e quant i t at i on. Al t hough combi ned MAS/ mul t i pl epul se met hods 4 pr ovi de t he shar pest , pot ent i al l y most usef ul ,r esonances, t her e i s of t en a l oss i n t ot al si gnal / noi se r at i o, andso we wi l l avoi d t hese t echni ques because of t hei r somet i mesquest i onabl e absol ut e quant i t at i ve accur acy.

pr i or t o t he st ar t of t hi s as pect of t he st udy, we wi l lchar act er i ze t he quant i t at i ve accur acy of our met hods by bl i ndanal ys i s of a ser i es of wei ghed mi xt ur es of hydr oxyapat i t e,pr evi ousl y char act er i zed 5 f l uor apat i t e, and r eagent gr ade CaF

2 A

standar d s et of condi t i ons and i ns t r ument s et t i ngs wi l l bedevel oped. Sampl es of t hese mi xt ur es and occasi onal bone sampl eswi l l be sent t o commer ci al l abor at or i es f or f l uor i de det er mi nat i onby di ssol ut i on and pot ent i omet r i c anal ysi s i n or der t o est abl i sh acompar i son bet ween NMR and convent i onal met hods. Occasi onal boneNMR and pot ent i omet r i c anal yses wi l l be checked by t he met hod of st andar d addi t i ons usi ng r eagent gr ade CaF

2

Repor t Wr i t i ng

Af t er t he r et ur n of aI l t he obt ai nabl e weI l wat er sampl es f r omt he pr eval ent cases and cont r ol s, dat a col l ect i on f or t her et r ospect i ve aspect of t he st udy wi l l be compl et e. Anal ysi s wi l l

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commence i mmedi at el y. By t he end of t he 4t h Quar t er of Year 2, at he anal ysi s of t he r el at i onshi p bet ween exposur e t o f l uor i de andost eosar coma wi l l be compl et e and a manuscr i pt submi t t ed f orpubl i cat i on. One publ i cat i on i s compl et e and has been accept ed f orpubl i cat i on. BBTheGenet i c Ri sk Fact or s f or Bone and Sof t Ti ssue Tumor s" has been accept ed f or publ i cat i on i n t he Sur gi cal Oncol ogy

Cl i ni cs of Nor t h Amer i ca. The def i ni t i ve dat a however , wi l l accr uef r om t he f i ndi ngs of t he pr ospect i ve st udy.

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fluoride bone cancer 7

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