CA2125702A1 - Method for diagnosing osteopenia and determining its severity - Google Patents

Abstract

METHOD FOR DIAGNOSING OSTEOPENIA
AND DETERMINING ITS SEVERITY

ABSTRACT

The present invention relates to a method of determining the severity of diseases such as osteopenia by measuring the concentrations of certain blood constituents and then calculating a bone density coefficient. The blood constituents required for diagnosing osteopenia according to the present invention are calcium, phosphate, estradiol, progesterone, total alkaline phosphatase and an alkaline phosphatase isoenzyme. A bone density-coefficient is calculated using the blood concentrations of these blood constituents. The bone density coefficient can then be used to classify severity of osteopenia in the patient.

Description

WO 93/12255 PC~/US92/10879 212~7~2 , ~

METHOD FOR DL~GNOSING OSTEOPENI~
AND DETERM~NG ITS SEVERITY
Technical Ficld s l~e prcsent ~yention relatcs to ~ethods for diagnos~ng ostcopcr~ia. Morc -pasticul~rly, ~c prc~cn~ rention rclatcs to a method fnr diagnosing osteopenia in a human or animal and determiniag its ~e~rerity ~t cause by mea~urin~ blood scrum levels of specific. p~edete~mined blood constituenL~ and then calculatin6 a ~everity index. Optionally, the ~ge of the human or animal can bc fac~orcd irllo Ihe calLul~lion.

Back~round Or the Inv~ntlon The term "ostcopcnia" as usct hcrein means any decrease in bone mass below the normal. I`he torm "ostcoporosis" as u~et hercin mcans a spccific form of gencral~7.cd osteopenia cha~acterized by a decrease in bone tet~i~y, low bonc ma~, a~d microarchitectural tctcnoration 30 . of bo~e tissue. Os~eoporosi~ can 1ead to ~nhanced bone fra~ility and a con60qu6nt incroase in fracture risk.
Ostcoporosis may be idiopalhic or may occur secondary ~o other ti~eases.
Bone consists primarily of an extracellular malri~
~5 containing ~by wcight) approximately 35~Yo organic and 65%

W093/122S5 2 1 2 ~ 7 ~ 2 PCr/US92/1087~

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inorganic components. The cells of bone represent a minor .
componcnt of the bonc constitucnts, yet they carly oul a maj()l portion of the function of the skeletal system, ~one cells help mainhin serum calcium concentration withill a nam~w rdll~e tO regulate mineral homeostasis while also being responsible for the continuou~ formation alld resurptiun of the extracellular matri~, allowin response of the skeletal system to the mechanical forces re~ulting rrolll physi-:~l ao~ivity.
I`he major organic component of the extracellular o matn~ is collagen. This prolci~l ha~ ;1 rigi~l ru~llike strucn~re and is composed of three alpha chains held to~cther in helical fashion by covalent and nollcovalenl ~ur~cs. Mul~iple collagen molecules foIm tïbrils, and these f~brils in tum are arranged in bundlcs or fibers. It is the bundles or fibers o~ c~)llayen dlat ~5 can be seen in ~e light microscope a& layers or linear a~ays.
Non-collagen componcnts Or b lle ~mpose a very small poltion of the organic matrix of ~e skeleton. This minor fraction consists of pr~leills, glycoproteins, mucopolysaccharidos and lipids. Only a few of these components haYe bcen carcîully i~enlifiell and characlerized.
Two of the proteins eh~t hav~ been i601ated and studied are osteoncctin and osteocalcill. Th~ ~yn~besi~ of osleocalcin i~
vitamin K~dependent. This 6000 dalton protein contains the `
uniquc calcium-binding amiuo aci~l ~amllla-earboxyglutamic acid or Gla. Hence, osteocalcin has also been r¢ferred to in thc litcrature as BGP. o bo~e a1a proleùl. O~teocalcin is syDthesizcd by bone cells. Usteonec~n is pre~ent in bone in the ncxthi~hestconce~a~ionolllvll-collagellc~lmponents. Itis a pmtein with a molecular weight of 32,000 daltons that studies ha~e implicated iD: dle binding of calcium to collagen :
C:alcium and phosphon~s are the main components of the :
inorganic portion of the skelcton. Initially, calciulll an~
pho~phoms ~r~. deposited as amolphou~ salts but later undergo e~ ngements into ~ cry~tallinc stmcturc that resemblcs :. ~: .

3~ hydroxyapatile (Calu(PO~)6tOH)2)~ Se~/eral o~her ion~

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including N~, K, Mg, and 5:03 in v~ing proportions, may l~
found in the sk~letal hydr-~tyapatite. If there h~s been ~ d flnoride intake, ~ere will ~Iso bc F in thc bydroxyapatire.
Althougll considerahle effort has been expendcd in studying the mech~nism of bone mineralizalion. it i.c nnt fully ; ~
understood. Several ~henries have been ~u~gested to cxplain ~ ~ :
All the available data. Onc tllcoly is lhat calcium and phosphorus ion~ are pre.cent in the e~tracellul~r fluid in amounts e~ceeding the solubility product Or [C~] ~ [P]. These o ions a~e kept îîvm pJecipita~ng by inhibitors of c~lcification .cllch as pyrophosphate. Bccause ostcobla~l~ contaln large amounts of alkaline pho.cphatase, it has been specul~ted that the activity of this enz~ne facilitates minerali~alion by cleaving `
thc phosphate gruups, ~hus altenng the C~a:P ratio in thc sites ~
of calcification.
Osleoporwis is the regoll of the gradual tepletion of the -inorganic portion of tho slcelcton ant can b~ caused t~y any number of factors. Osteoporosis can affect men, womcn and even children. The following is a pa~ial lisl of some nf the ~;
catcgories of indivi~luals a~ sk for developing osteoporosis~ d rost-menop~usal women . ;' , ,', '~'".;,' Ciga~ettesmokets lleavy user~ (~f ~Icohnl 2s Users of a variety of d~ug~, such as steroids Female ru~e~s and t~allet dancers M~b m~atho~ers con~ing two ~w calories l~ulin~i~ and anote~
Teen~gers on junk food Peoplcallergic todairyproducts People affectet with cancer Fair ~nd slim ~omen All men and women over the ~ge of 65. ~

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WO g3/122SS PCI~/US92/10879 . ~:

In atdition to being Çemale. the three most signific~nt risk factors are poor diet, lack of cxcrcise, and bcing postmenopausal. O~r ri~k factorc whieh are associated with osteoporosis include a Caucasi~n or Oricntal background, a fair complexio~ l a farnily his~ory nf n~teoporosis. ; ~
Osteoporosis po~es a major healdl problcm in thc United ~ ~ :
States, not only for those persuns who are already ~ffected but for indivituals whose diet, life style, ~nd bod~ build make thcrn morc likely to develop~ os~eoport ~is as they age.
IU Pnstmenopausal osteoporosis i6 a comrnon disortcr that results in sub~t~ntial morbid;~y ~1~] moltality. A~ many as 25 million American women suffer from ostcoporosiB~
Osteopcnia encompasse~ ~ group of diseases wi~ diver~e :~
etiologies typified by retuction in bone mass pcr unit volume `
to a le~el below thal which is nececcary for adequate mechanical support ~unction. Prin~ry osteoporo~i~ is an a~
relatcd disordcr that is panicularly common in women and is ~haracterized by decreaset bone mass in ~hc absence of other recognizable causes. Ho-vev~r. osteoporocis nr~alrs in both men and women. In women it is recognized usually at the 5th or6thtccate followin~ men)pause. Ln men n.ctenporosis is often recognized around their 6th or 7~ tecadc. As thc osteopenia progrcsscs, both ~e Iwmber and thickness of the ~rahecular units decrease causing fragility of bonc and an ~5 increasct nsk of fracturcs. Osl~oporosis i~ re~l~onsible for appro~imately 250,000 femoral neck fractures annually ul thc :
United Sta~tes, ant thcse fraclur~s are as~ociate~l with a 2ûY~
mnn~ y rate within 6 months. The risk is particularly hi~h ~unong thc clderly, who alss tend ~o lusc bo~e a~ a resnlt of the .
aging [~rno~ss Wu K., et al., "Bone Resorption Ratcs in Physiological, Senile, ant Postmelu~p~u~al Osteoporo~
Lab Clln. Med, Vol. 69, pg. 81U, (lY67).
lho onset of ostcoporosis may be insi~ious or sudden. t :s followillg lrauma. The most common complaint associated ~ .
~s with osteoporosis is back p~in. This pain may be rel~rred ~ :
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~ ;' ~ .' "- ''' ~', wo ~3~ PCI/US9U10879 2 ~ 7 ~ 2 along the course of involved nerves, particularly down thc postcnor aspecl ~ olle ur bulh l~gs. Evenmally. ~he l~in may spread to the pelvis, the thorax, and the shoulders. In the spine, the venebrac call wmp~ss. and the hack e~n ~ake on a .
s "bent" appearance. Conditions such a~ kyphosis (humpback) or scoliosis may ~;~:ur. If the sp~ne becumes defi-nne(l, other body parts can be affected as well. For example, the ribs can :
be pushcd against ~e pelYis~ or lhe ~tomach can be ~u.ched into the pelvis. In addition to ~pinal problems, osteoporosis can o also lead to fractures ol lb~ hip. wri~l, and rih.c. These fractures can occur with only slight "trauma" and somctimcs can occur with no lraum;l al ~ll. Maze~ B., et al., "Bone :~
Density of the Ratius, Spine, and ~roximal Fcmur in Ostcroporosis," J~ ~JB~ne ancl ~lineral ~esearch, Vnl .~, pgs.
13~18, (1988); Ri~gs B. L., et al., "Involutional ;~ h~
Ostcoporosisn, ~7ew Engl. ~. Me~., Vol~ 314, pg~. 167ff-16~6, (198b). The changes associated with OStOOpOlOSiS are ~mdual so ostcoporosis is oft¢n no~ ted in i~s early ~tages. . ~
It is est~mated from a survey of medical clinics that of : : ::
thoscintiridualslivungtoa~8S. 32qoof ~ewomen~nd 17% ~ ;
of men ~ill fracture hips, weakened by osteoporosis. In atdition to the pain and ~ulf~ing caused by ~he~e fractures, the motletary cost i~ great, ~ccoundng for well over 3.8 billion dollars per ycar in treatmenl ft)r fiactures of l)~teoporosis.
2S Moreover, six to eiBht months following hip fr~cturc, about SO% of ~e osteopor~lic patients a~e in need of a~istanco with activities of d~ly living ant about 25~ roquirc nur~ing homc arc. Only aboul 2596 uf the padents fully recnver. In view of the costs asscciated.with 06teoporosis, as measurcd in ;
tollars ant in human suffcring. u~t~oporosis has increa~in~ly been perceived as a senous and di~ablin~ tisease, warrDnting sub~tantial involvcmcnt on the part or cl~nical inveSligatOrg.
g~vernmental agencies, and phumaceutical indust~ies to develop ~nd evaluatc potential treatmcnts and early d~lection . ~
tecbniques. ~ ::
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WO g3~122~ 1'1~/1.1~i~12/10879 2~X~7~2 Currcn~ s~andard tiagnostic techniqucs ~r~ not effectiYe fnr e~rly ~1ere~.~ion of osteoporosis. Chanxes seen in osteoporosis arc vcry gradual and osteoporosis is oftcn not Aetected in its early ~tages because bone mas~ must he decrca~cd by about 309b to 40~ before it is apparent usi!lg .ctaml~rtl x-ray diagnostic teehnigues. P~eYen~ng osteoporosis by detecting carly bonc loss is far be~ter than identifyin~ tllt disea~e at relatively advanced stages and subsequently ~ttempting to prcvcnt its progression. Once major u deterinratinn h~s oc~ rred and gaps e~ist between the ends otfracturet tr~bccul~r, no currcnt t~atment can bc c~cpected lO
restnre the lost bone. Thus, therapeutic efforts must be directed toward prcvcntion ant early recognition of the progrec.cive di~ea.se so treannent can be instituted bef~re s essentially ir eversible ~tmctural damage ensues. CulTunin~s S.R., et al., "Shnuld Penmenopausal Women Be Screened for Osteoporosis?", /~nn. Int. Med., Vol. 104, pRS. 74~-751, (1986); Courpron P., "Bnne Tis~ne Mechanisms Underlying Osteoporosis," Orthop. Cl~n. North Am., Vol. 12, pg. ~13, (1981): Frost H. M., "Mechanical Determinants of Bone Modelingl" J. U~tabol. Bonc. Dis. Rcl. Res., Vol. 4, p~
217. (1983).
The radiograpbic manifc~tations of osteoporosis rcflect the deficiency t)f the organic mathx and parallel the gross 2s pathologic findings. Thc corticcs are ~in and the trabcculaef~ne and sparse: the skeletal structures are t~erefore more radiolucent th~m normal. Thc tisease proccss c~vcntually afYec~s almo~t all nf the skeletal structures. The principal areas of derminerali~tion ~rc the spine ~nd pclvis, cspecially in l~le femur~l neck and head. Demineraliza~inn is less marked in the sl~ull and e~tten~ides. Althougb u.~eful to tetect brcaks in ~e bone~ orulinarr x-rays are not sensitive enough t-) de.teet osteoporosis until a latge amount of bone tissue has ulreaty becn lost, ~encrally from 25% to 40æ. ~y the lime , .- , ,;~ ~, ~, " ''' -W093/122S5 2 1 2 ~ 7 ~ 2 rcr~us92~ 9 osteoporosis can be identifie~l by X-My techniques, the ;undition is advanced.
~n early dccrcase h~ bone mass can be measured by non-inva.cive assessment of the skeleton by four widely s availablc methods, includi~lg smgle l~hot~n abso~ptometry, dual photon absorptometJy, dual-energy x-ray absolplumetry.
and quantitathe computed lumoglaphy.
A de~ice c~lled a sin~le-photon absorptometer (SPA~ is used to mcasurc bone lllinera~l content, pr~marily in the `~
o folearm an~ ~rAst. l`he heel ~one can also mcasurell using SPA ~ usc the hecl bone i~ ~loughl to l~e a predictor of bone I~ss in the spine. SPA measures prima~ily cortical b()ne.
which is al~o affected by osteoporosis. though not to the ~an~e e~lent as trabecular bone.
The tcchniquc of dual-photon ahsorptometry (DPA) provide~ a measurement of the total cortical and trabe-;ular s mineral content of thc hip a~d spine. DPA uses less radiation lhan convcnlional ~-rays; but, a scan of the spine usill~ DPA
still e~poses the patient to ~ppro~imately one tenth of the ~ rfi, ra~ tion that r~sults from a routino che~t x-ray.
Dual-cnergy ~ray ab~orptometry (nX~) provides mwsu~emen~ of thc amount of bone tissuc in the hip and sp~ne~
This tec~niquc is now uscd r~utinely hecallse it is faster than DPA.
2s Unlikc DrA and DX~. quantitative eomputed tolwgraphy, more commonly called a CAT scan, ca~ ea~ure the densit~ of ci~er bonc or just the trabe.cular portion. CAT
s~;ans unfommately expose patients to highcr doses of luliation than a~y of the othcr techniqucs.
R~diographic abso~ptometry (RA) is a mcthod for UOII- "'' :-,; .. : `, invasive mos~uremcnt of bonc mineral x~ray~ ~f the hant.
Radio~raphs. talcen w~th a standard ~c-ray machinc, are sent to a oentral laborato~ for computer~onlrolled analysls.
Onc of the core problems with all of thesc culTcnt -~
methotologie~ fôr determining whether a patiem i~ sllffering WOg3~1225~i PCI/I1.'i9~/10879 - 212~7~

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from o~teoporosis is ~ha~ the procedures do not ~ive any infonn~tion about the unterlyin~ cause of ~he osteoporosis.
For example, a co nmon callse of postrnenopausal osteoporosis is an estrogen deficit, which x-~ay techlliques c~nnot measure.
S Another key prohlem in advancin~ the medi~
management of the osteopcnic patien~ is that all the cunent methodologies re4uire e~pensive, sophisticated medieal in~tmrnentation to perfolm the bone ~lensity m~asllrements.
Additionally, pati~llts mu.ct be e~tposed to x-rays. Ihis m~s o a general screenin6 of high nsk popula~ n~ ractical due to ~e expcnsc ant u~lavailability of the necessary instn~ncntativ o ~e average clinic. -The prescnt invention generates a new le~cl of interc~
ill screening individuals who arc at ri~k for nsteoporosi~
s because dlis novel dia~nostic procedure provides informatiou about the underlying cause of ~e osteo~enîa~ The present invention also provi~les a ~imple, ~ne~pensi~el and rapi~
method of dete~n~inin~6 bone dcnsity using serum hlood levels of specific, prcdetcnnined blo~d constituents.
In the cli~ical syndromc of osteop~rosls. the reduction ~n bone mass can be nltributed lo osteopenia becausc of dietary ;
d~rlcîency or absorption interfcrcnce of p-utcins or rita~
C~ It can also bc the ~SUIt of a deficient stre~s stimulu~
Os~openi~ can also be caused by osteom~lacia. a failure of 2~ proper mincral~zatîon of osteoi~l resultin~ from bonc calcîum or phosph~ ms deficiency or both. It can also he caused by insufficient absorplî~n from rhe intesti~e due to lack of calciwn or a resistance to ~c actioll Or Vitamin D due to failure of its con~rcrsîoll to Ihe biologically active fonns, 25 hydn)xychl~le-calciferol and 1.25 dihydroxyL:hole-calciferol.
fo~med by thc livcr and kidney. respectively. In addition, it ;
may well be cansed by an abnonnal rate of ~stwlysis due tO
parathyroid honnone stimulation nf osteoclastic activity ;
(ostcocasts are hematopoitic in origin, arising from the 3~ migration of monocytcs to bonc). In reality, most cases of ~ ~-;...' ~'~'";;~,".:"
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WU 93/t2~5S 2 1 ~ ~ 7 ~ ~ ~tUS92/lo879 ,~ ~

osteoporosis, ~hen cal~fully analy~.ed, reveal evidence of the cause.c ~f osteopema.
Chemical analysis uf hlnnd may reveal calcium, phnsphon~s, and aikaline pbospbatasc ~ith~ the nomlal rdnge.
s However, an isoen~yme of alk~line phosphatase may be significantly increa~ed~ Incre~sed bone rcsorptioll ~een in :
osteoporotic patients. which occur.6 as a result of the action of ostenclast~, usually inYolves the di~olueion of both minerals ~nd organic matrib~ ev~lltually leading to incrcased excretion i`
o of urinary hydroxyproline. Serum cstradiol which i~ se~;reled ~Imost entircly by thc wiuy is ~ignificantly decreased in these ~ :
patients. Thisobserva~ionisfurthercorroboratedwh~ has been dcmonstratcd thal e~ogent)ns estrogen therapy in perimenopausal women does delay the onset o~ pos~
5 menopau~al ostcopenia Mu~t expert~ agree that estro~en appear.c to decreasc bone resorption. Weiss N. S.~ ~ al "Docreascd ~isk of Fractu~es of ~e Hip and Lower Fore~
with Postmenopausal Use of Estrogcn," IV. Engl. J, ~ed., Vol. 303, p~s. 1195-1198 (l9~sO) Fttinger B., et al., "Long 20 Term Estrngen Replacement Therapy Pre~rents Bolle Lt~ss and pMcturcg~ Ann. Intern. Med.. Vol. 102, pgs. 319-324, k u belie~ed that blo~l lwelc of calcium are maintainëd wilhmlt increasing calciurn loss from thc bonc as long ~s there are nonnal amounts of estrogen and parathyroid hotmone pre.cent It is reasonably excepted that cstro~en antagollizes the effect of parathyroid hurmoné. F~strogeD deficlency, as seen in peri- and postmenopausal women, rcsults in an ~llcrease in tho sensitivity of bonc lo para~hyMid hormone. This antagoni~tic relationship eventually le~ds to an increase in l~le resorption of the bone and cvlltributes ~o the development of l`ype I Oste~orogis. Type ll Osteoporosis is charactcrized by r- :~
retuc¢d cQlcium absorption, wllich in n~n resul~s in increased secreliun of para~hyroid hormone. Type II Ostcoporosis ~ -:
occurs in olter individuals and is as~ociated with wedge '~

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PCl/US~2/l08~s - 21257~2 .: . .~ , ~ `, fractures of the spine an~l fractures of the hip. A theory recently advanced by BQrnhill, ct al. su~ge~ that eYidence of decrcascd scmln estradiol ~nd increased lymp}locyec allcaline i~
phnsphatase, represents an actiYal~d immune system in s osteopcnic pos~enopau~l women. lhis findin~ suggesls th~t -"uncovered estrogen reccptors" Illay induce an immune reaction which is respon~l~le for one form of ostcopor~)sis.
Barnhill S.~., et al., "Osteoporosis; A Pos~ih1e Autoimmune Etiology," Ann. of Cl~n. Lab. Sci., Vol. 17, pgs. 2~-256.
to (19~7).
In 1987, B~rnhill. et al. propo6ed an autoimlllune etiology for son~e fonns of osteopcni~. In th~l publication, Bamhill, et al. showcd that lymphocyte~deri~ed a1kaline phosph~tase ~vas present Ln thc blood uf 9(1% of severely osteopenic womell. See Barnhill, etal., "Ostcoporosis: A
pos~ible Autoimmune Etiology"l Ann. ~Jf C~in. r~h~ Sci., ~/ol. I
17, pgs. 255-256. ~l987)) lhe concept of a Iyïnphocy~
derived a1kaline pllosphatase is fu ther described by G~iff-tths, et al. Sec Grifftths, J., ct al., "Separation and Identi~lca~ion ~Ikallne Phosphatase Isoenzymes and Iwfonns in Serum of -~ealthy Pcrsons by I~oelect~e Focusing", Clin Chcm, Vol. 32.
ygs. 2171-2177, (1987).
From the ~esearch perspective, osteoporosis can be cl~ssified as either pr~muy or secondary to another disease.
2~ Prima~ osteoporosis is further classifiet as juYenilc. ~ ~- idiopathic, postmenopausal (Type l) and involu~ional ('l'ype Il). It is now ullde~tood that acceleratet bonc loss occur~
with ce~Ation of men6tmation at thc time uf menopause and m wome~: who h~Ye amcn~rrhea as a result of prolactin ~ ~ :
produc}ng p~tuitary tumor, anorcxia nervosa. or inr~nse long~
distance runninR associated with uDdernouri6hment. l~ese situa~ions a~e all accompanied by cstrogen derlciency which is -~
likely to bo a m~or detcnninan~ of the accelerated bonc loss.
Bone loss a~so occllrs when estrogen therapy is Wid~dWII ' ~ ; :

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The Nalional Inslilules of Health 19R4 ~nn~ensus Conference stimulated dlose interested in bone dcnsity with its r~cummendations for "defining persons at tisk, and develnpin~ r safe, effective and low cost strategies for fr~ctur~ protcction."
s See, Office of Medical Application~ ~f Re.ce~rch Narional lnstitute~ of Health, "Osteoporosis: consensus confcrcncc," . - :JAMA. Vol. 252, pgs. 799-802, (19~4). Since 19~S4. t.here h~s been 3 dramatic change in the way physicians view osteopenia. ;
With the development and popularization of highly .~ensirive o tec~ iquss such as DPA and quantita~ive computcd ll)n;~ r~phy. phvsicians are now capable of mea~l~rin~ thP
den!; t~ or dle pro~imal femur ~nd the lumbur Yertcbrac. (For a ~er~eral r s~riew of the me~ods cutrent!y availahle t.l~ me~
~one densi'~, see Avioli, L.V., ed., "Metabolic Bone Disea~c n~ ~1 Clinie~lly Related Disorders". W.B. Saunder~ Cnmpany (1 990)).
Thus, there is new interest in screening ~he individual~
who are at high risk for ostcopen~a. The current methods are ` ` : ~~Y
r~ ely e~pensive a~ld require specialized equipment rn propcrly evaluate a patient. In addition, the current methods of measur~~ ne ~lensity do not give ~ny infonnadon ~n the undorlying cause of the osteopeni~. What is needed is a silllpl~, in~pensive, ~nd r~pid method of de~ermining hone densih~r. In addition, the method should also give infonnation 2~ abou~ e wl~brlying cause of the osteopel~ia.
Summ~r~ of the loYeneioo Tho present invention includes n methot ant systcm for di~gnosing and detenninDg the scvcri~y and underlyin~ callse O~ osteopenia usin6 blood concentratJ.~ns of a specific, predetcrmined set of blood eo~ uellls. The method comprises the steps of ( 1 ) measurin~ ~e seYerity of the disease in a sct of humans or animals with varyi~ severily ~r ~lisease by a standard method, (2) assigning the seventy of disease a ~s numcrical ~alue on a se~eri~ scalc, the scale being from no .:

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-~isease to ~evere di.~ease, (3) measuring the blood concentrations of thc prcdctcm~incd set of blood cons~iluellls in the set of human.~ nr an~snals with va~ying severity of the disease, and then (4) calculatin~ a numcrical relationslliy between ~he ~et of hlc~od c.oncentrations and the severity of tisea~e The pre~ent invention ~Iso comprises a simple and rapid method of detenninin~ seYcrity of osteopenia in a specific p~tient. In a ptEferred embodiment, the method comprises 0 detennining the serum lcv~l of the followin~ scru~
c()nstituenLs: calcillm, l~hn.cph~e, t~tal aUcaline phosphatase, an alk~line pho~phat~se isocnzyme, estradiol, and proges~elvne The alkaline phn~phata~ie is~n~yme is preferable t-lymphocyte delived alkaline phosphatasc or blood, livcr or intestinal ~lkaline phosphata~e i~nen~yme. lhe ~esults of these tests are then introduced into an algond~n, Optionally, thc age of thc p~tient may also be factored into the equation. lhe bone den~ity co~fficient that is calculated by the al~orith eurrela~s tc~ a very high degree to b~ne de~sit~y as measured by ~tandard methots, such as radiographic absorptometry.
qua~ ive computed ~omngraphy, dual photon abso~ptometry and direct measu~ement of bonc tensity. The bone density coe~rlcient that is mea~uted i~ then compaled to an osteopenic sevorinf wah.
2~ Usillg the si~ senlm constituent concentrations, the plesent invention can be used to detcnninc thc ostcopenic slal~
~f ~ patient a~c well a~ give an indication of the undetlyin~
causo of the osteopeniQ. Thc pre~cnt in-rcntion ca~
cunclated t~ any medlod of mea~llring bon~ density simply by recalculating the coeff~cients in the ~Igontb n using a multip~e l~near regrvssiu~ lysis.
Accordingly, it i6 an object of the present in~cntion is to pro~idc a mcthod for dclcm~i~ng the severity of a dl~ease nsing the blood concentradons of a predete~nined set of blood 3S constitucnts, ~'' 2 1 2 ~ 7 ~ ~ PC7~US9U10~s79 ~ ~

'~:~'" `"'`
Anothcr objcct of thc prescnt invcn~ion is co providc a simple and rapid chemical test for diagnosing osteoporosis.
Anoehcr objcct of the present invcntion is to providc a test for osteoporosis which will also give ~for nation aS to the ulldellyillg cause of the ostGopenic condi~ion.
Another object of the present invention is to provide a dia~nostic test for osteoporosis which can bc used to screcn large numbers of mdividuals.
Yct ~nothcr ~bjcct of thc ~rc~cnt invention is to pro~ridc ` -; ``
o a method for diagnosing osteoporosis and determining thP i undcllying cause of thc ostcopcnia wi~out havin~ to subjcct i the patient to radiation. `
These and other obiects, features and ad~ranta~es of the present invention will become apparent after a review of the following detailcd dcsc~iption of thc discloscd cmbodimcnt and the appended clauns.
Brief l~escription of the Figures Figure l is a lincar regression analysis of the correlation ~o between certaul blood cests and l~A.
~igure 2 illustratcs thc discrepancics bctwccn the DPA ~ ~ -me~surcments ant the measurcments obtained using the ~ `
methot of thc prescnt in~lention for indi~itual subjects.
l;igure 3 illustrates the co~elation of the results ~ :
2~ obtained using thc prescnt invendon and DPA.
I;igure 4 demonst~ates the nonnality of values predicted - -by thc prcscnt invc~tion.
~;igure S demonstrates the normality of residual~ ~;
(differencc~ betwcen ljPA measurcmcnts and valucs predicted ~ ~
3a according to the method of the present invention~, as ~; cvidenccd by the arran~emcnt of points alon~e a dia~onal straight lule.
Figure 6 demonst~ates that ~he vasiancc of residuals.
Figure 7 demnr~trate~ the independence nf re~iduals. ~ -~5 Figure 8 temonstr~tes ~t ~j~QI1Y ~11 observet DPA ; ~ -"''"'"-;

."

~- V 7J~ l L~aa ~ l J u~y;z/ l ~7g ~` 21257~2 ` ~

mcasurcments arc prcdicted by the method of the present ~nvention.
Detailed Description The present inYention rcla~es tO a method for tiagnn~in~
diseases in humans or ~nimal~ using blood concentrd~i~n~
spccific, predetem~incd blood constinlents. More specifically, the preserlt invention relates ~o a mcthod for ~lel~c~ g Ihc occurrence of ostcopenia, di~gnosing osteoporo~is and lo dete~ ing its seven~y ~nd underlying causc. n,~ yresel~
invcntion also facilitates the periodic monitonng of specific physiological funcrion~ which may indicate the onsel u~
osteopenia and correlates to bone mineral density ~ `G~"'`''`''':
measurements de~ermined by various standar~l melhuds.
1~1 practicing one sspect of the present invention, the seventy of disease in a .set of humans or anim~ls wilh v~ryillg se~erity of discasc is measured by a stantard method or methods. The measn~emen~ is dlen assi~ned a nulllencal valuc ,~
corresponding to a severity scale. Tlhe 6cale ranges from humans or animals wi~h no diseasc, to humalls or u~imals wid~
sovere discasc. 'rhe scale is preferably a numerical s^ale. ~r example, one cmlld assign a value whi~h corresponds to nomull or slight disense, anodler value which corresponds to modcrate disease and a th~rd Yaluc whicll corrcsponds to 2~ se~cre tiscasc.
Thc concenrratinn of a predctennined s~l oî blood constituents in thc sct of h~mo~s or amimal6 with varying severity of disease is then de~ennined. Acc(~nling lu ~he present invcntion, it is prohrable to measure the blood consdtuent.s in the satne set of human~ or a~ uls in which the scvcriq of disea6e was measuted by the conventional method nr med~ods. ~ : :
After determinin6 the blood concentrations of the ~
predetemull~d sel of blood consdtucnts in thc sct of humans or ~;
3~ animals wi~ vatying scverity of the disease, a m~thematical 2~7a2 PCr/~IS92~108~9 ~nanipulation is perforrned in which rhe numerical Yalue obtained using the con~ention~l di~gnosi~ i~ sct to equal thc re~ul~ of a madlema~ical m~siel ~ ing the set of blood constituen~ measurements. For cxamplc, thc relationship can be made llsing a multil~le linear regression analysis.. lt is lo be understood that other mathcmatical motels could be uscd to determine a correlation hetween the serum constituent concentration~s ~nd ~ stantar~ mcthod of dc~cTminin~ sewtity of the disease. The cnncel-r of nsing mathernatical models to o determille ~ne rel~tionship or ~olTclatiorl is considered to be part of ~epre~en~invention. ~St~nrl~t~ t~ tical analyses that are well known to those of ordinary skill in the art arc then performed ~o delermined the confidence level of the correlation between the di~gnosis by convcntional means and Ih~ set of blood con~iruent.c. ~ese statisdcal analyses include chi-square tests.
An e~ample of practicing one embodiment of the pre6ent invention is a method for diagnosing osteopcnia in a ~lum~ul or ~nimal. lne method prefera~ly utilizes six blood con~tituents. These constituents are calcium, phosphate, total alkalill~ ph~sphatase, an alkaline ~hnsphatase isoenzyme, estradiol, and progesterone. Thc alkaline phosphatasc ~ crlzymes preferred for practicing ~he present invention include Iymphocyte~derived Qlkalinc phosphata~e isoenzyme arl~ bone. Iiver or inte~linal alk~Iine phosphatase isoenzyme~
The present inYention includcs calculatin~ a bonc density 4uodent using lhe afnremenrioned six blood constituent~ by entering the v~lues for thc tcst~ into an aIgorithm that is calcuIated using a mllltiple line~r regression analysis.
Optionally, the age of the patient m~y ~Iso bc incorporated into the ~qu~lion. The bone density cnefflcient ~lerived from the algor~thm allows one to diagnose the osteopenic statc of thc ~tient, includin~ ~e sevcnty of ~e disease.
In addition to diagnosing the osteopenic state of the 3~ human or animal, an indication of the ullderlying c~use ~f ~he .. .. .

WOg3/12255 212~i7~32 rcr~ussA~10~7g ~ ~ ;

osteopenia can be detern~ined u~ng the present invention. For e~amplc, by practicing the prcsent invellliu~ s ~lescnbe~
herein, one can deternune whether the osteopenia in a human or an~mal is caused by post-menopausal lack l)f e~tru~en or is caused by some othercondidon, such a6 cancer. This allows the attcndin~ physician to be better al~lo lu pre~cribe the apprcpnate ~eatment for the osteopenia.
I~ivc of the semm tests that are used i~l lbe l)re~ent invention a~e tests that are commonly per~o~ned by clinical o laboratories. The tcst for t-lymphocyte derived alkaline pho~phatase is e~perimental only; however, the test for blood, liYer and intestinal alkalinc phosphotase isoc~ymes ~e also known. The type of test used to detem~1ne the si~ serum constitucnts is not critical to ~e present invention as lunY as s the te~ts give accurate blood concentrations of the constituents bcing meas~

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., .. :
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.

. , -:. . i . ' ~ '" ', ',-W0 93/~2254 2 l 2 ~ 7 a 2 PCI/USI)2~10819 Calcium & ~hosphorus~
Mailltenallce of calcium ant phosphorus homcostasis mvolves the partlcipation of 3 major organs: the small intestine, the kidney, and thc skclcton. and is rc~ulated by s varioUS hormor1e3. C:alcium enters the body through the diet and is absorbed into the circulation from the small intestine.
C:alcium abso ption occurs by two processes, active transport and passive transport. Approximaecly 98~o of the calcium and 85% of the phosphon~s ~ the adult is present in the skeleton o primarily as hydro~cyapatite, ~ which is a crystal lattice compound of calcium, phosphonls9 and hydroxide. Kemain~ng calcium is present in extra ccllular fluid, son~e typcs oP tissue, and skc~etal muscle. ~hosphon~s is combined with lipids, protei-~ carbohydratcs, and o~er or~anic substançes. Of s cri~ical irnportance to calcium homeostasis is the ~act that less than 1% of total skelctal reservoir af calcium is rapidly exchangeable with extracellular fluid. ln additioll to it~
obvious impoltance in skclc~al mincralization, it is ~lso vital for blood coagulation, neuronnolecular ~ lition, maintenance of notmal tone and excitability of skcl~ nd cardiac muscle, and prese~ation of all membrane inte~ ~ and penneability, particularl~ in t~rms of sodium and potas~lum e~cha~lge.
Free or ionized calciurn accounts for 50% of total calcium. About 5% of total calcium is comple~ed wit~
~s variety of anions, particularly phosphate ant citrate. llle rrm~ining 45~o of calcium is bound ~o plasma proteins, Both ioni~et calcium ant calcium compb~es are freely dialyzable, Acidotic and slkalotic conditio~s ad~rerscly affcct the ioni~ed calci~un level in the.blood ln metabolic bone disease such as hyper- or hypoparathyroidism, Pagets of bone, Vitamin D
deficiency, and renal osteodystrophy, calcium and/or phosphorus le~ els are significantly altered, -W093~1225~ 2l2~7a2pcrlus92/lo8~9 l8 ~ ~ `

Se~um Calcium Determination~
In one method of prachcin~ the present inYcntion~ thc procedure for thc determination of calci~ based on the inleraction of the chromogenic agent o-crcsolphthalein s comple~one (Sigma Diagnostic~ Calcium Agent, Sigma Chemical Co., ~it. Louis. MO ) ~hich complexes with calcium ~ u~cation in an alkaline medium to f~rm a purple colored - -complex which h~s absorbance maximum at 575 nm. ~e inten~ity of dle color measur~d at 575 nm i.c tlirectly 0 pr~ nal to ~he calcillm concentration in ~e given ~amplG. -'l'he o-cresolphthalcin complc~t)ne cont~ins hydr(lr.yquinoline wllich prevents interferencc from ma6nesium ions. Rober~svn, W.~., e~ al., "C: alcium Measurements In ~emm and Plasma ^ Total ~nd Inonized," ~ :
1.~ Cri~. ReY. Clin. Lab. Sci., Vol. 11, Pe. 271. (1979); .Sigma .Diagno~lics Product Tnsert: Calcium, Procedure No. 587, -:
Reissued May 1989. The calcium level in ~erum is in the ~ :
range of about g.2 to 11.0 mg/dl which is compo~cd of 3 dis~nct ~ractions.
, Specimen Collcct~on~
In une me~hod of practicing the present invcntion, sen~m or heparinized plasma are suilable for the test. -Anticoagul~nts other than heparm should not be used.
2~
Procedure:
si6m~ Di~gnostic~ Calcium Rea~e~ used wilh Abbott Spcctrulll High Per~om~ance l)iagnostic System for thc quanti~ation of calcium in ~ m. It is generdlly con.cidered good laboratory pr~ctice ~tl run a calibration, linearity, and a quality control pAor to ~e detcnnination of patient sample~
Calcium lc~cls up lo 16 mg~ ~h can be measured by this method. ~:
. .- ..

wo 93~12255 PCIIUS9:ZJ10~79 212~7~2 ~
19 ~ -Serllm ~hosphorus ~Je~erminntlç)n :
ln onc tnethod of pr~cdcing thc prcscnt inYcnti~n, the procedure for the determination of senlm l~hnsT~hon~ is ha~ed on the ultcraction of inorganic phosphorus with ~nmonium ~ ~ -molybdate in the p~esence of sulfuric ac.id. Barnhill S., F:r al., "Osteoporosis; A Possible Autoin~nunc Etio10gy~" Ann. of ~.
Clin~ Lab. Sci., Vol. 17, pg. 255, (l~R7). This reac~ion produces unreduccd phosphomolybdatc cdmp1ex. The absorbance of this complex at 340 nm is direclly ~r~ordonal o t~ ~e inor~anic phosphorus prcscr~t in the given s~mple.
~Sigma l)iagnostics Product Insen Phn.~l~hl~m.~, lnnrganic, ~ :
Procedur~ No. 360-UV. rreviOus Rcvision Dcccmber 1~8~
~eissued luly (1988)). ~lost of the ~hn~phnmc in extracellular fluid is inorganic and in thc rangc of 2.4 to 4.7 mg/d1. ~ n 1s Specimen Collection:
Serum or hepannized plasm~ are l)refe~ed for the tec~
Antitoagulant3 other than hcparin should not be used.
Procedure~
Sigma Chemical Company's phn~r~hnrllc inlrganic rcagcnt can bc u~ed wi~h thc Abbott Spectrum High Performance Diagnostic Sysrem fnr qualificatinn nf the inorganic phosphorus ln scmm. It is cus~omary ~o perform calibration, linearity, and quality con~.ml ~ die~ lr m lhe dctcrmination of te~t 8amplc9. Ph~sphorus leYels up to 12 mgs % can be me~su-ed by thi~ procedllre.
Total Alk~line rkospha~ase~
The demonstration that bone is rich in alk~line phosphatase (ALP~ and th~t nomlal ~en~n contains the same or ~ similar cnzymc 1cd to the sh~dy of serum Al,P ieYels in padent6 widl dis~ase of bone. See, Courpron P.~ "Bon~ Ti~u~
Mechanisms Under1yin~ Osteoporosi~" Or~hop. Clin. North Am., ~'ol. 12. pagc 513, (1981). This i5 especially truc in . ~, ~',';~.
.

wo 93t1225~ PC~/US4;~108~9 2~2~7~2 ~' osteins ~efnlmans, hypelparathyroidism and bone neoplasm.
~LP is ~19O clcYatcd in hcpa~ic tiscase; howeYcr, this can bc di~ingl~ he~ y o~her enrrobor?tive laboratory procedures and ClllliCRI fcaturcs. ~n somc situations, as in ostcop~rosis~
s Al,P per s~ may not be above the reference range; however, thc isocn2yme of ALr is increased. It is wcll understood thal mral ~emm ALP in nonnal subjects consists of isoenzyrnes ~:
contributcd from livcr, bone, fenal, pulmonary~ placcntal and inte~tin~l sources, among others. The isoenzymes and o isoforms of v~rious ds3ue origins` can be fi~ cr separatcd and vi~uali7etl hy ~n Isoelearic Focusing Technique. ~s~ima~ion : ~:
of total ~crum AI,P by isoelectric focusing tcchniques of~ers great ~tendal in the investîgation of metabolic bone diseases.
Serum Alkaline Phnsphatase netermination:
ln one method of practicin~ tbe prescnt invcntion, serum ALP activi~ can ~e measured using various pho~phate esters as substrates. Sigma Chemical Company's alkal~ne phospharase rea~ent me~sllres selum ALP activity by a kinetic method. The reagent for thc tcst conta~ns p-nitrophcnyl phosphaee, carhonate buffer, magnesium ions and mannitol.
Mannitol present in thc rcagent acts as a phosphate acceptor during the enzyme reaction. McComb R.B., et al., Alkaline Pho~hata~e~ Plenum Prcss, Ncw York, (l979); Gundbely.
2s M., ,~lkaljne Phns~hara~e ~nd Osteocal~n. Primer on Is~ ion, Puhli~he~l by Am. Soc. For Bone and ~lineral Kes.
Editor-Mu~ry J. Fa~rus, pgs. 74-76, (1990); Sigma Dia~nostics Producl Insert: Alkaline Phncphat~se tALP), Proeedure No. : :
245, Reissued March 1989; K~plan T.A.,ct al., Clinical ~hQmi~ Sl. Luuis. e.v. Mosby Company, (1987). :
.
Specimen C~llectlon:
ably, sen~ or hepannized plasma is used for the ~otal alkaline phosphatase andalkali~ husphatase isoenzyme - ` 2 1 2 ~ 7 ~ 1/US921111#7Y
~1 .

determinatioll tests. EDTA. oxalate. citrate alld fluorid~ ~r~
inhibitors ot ALY and are not suitable anticoa6ulants.
Procedure:
Al~alil~e ~ v~plla~ase (.ALP) ~ usli~ r~agelll manufactured by Si~ma Chemical Company i6 used with ~e AbbuU Spe~ l High P~r~rrl~ ~ Dia~no~lic Sy~t~m f~r the quantitation of inor6anic phosphoms in the semm. Serum ALP }Iy~lroly;e~s l~-nilr~phellyl phospllale IU ~-nilruph~ nd 0 inor~anic phosphate. The hydrolysis occurs at alkaline pH and tlle absorb~lce is ~lireelly pruporliullal ~ ALP actiYity of the sorum ~ample. A~ is the usual practice, calibr~tion, linearit~ and qualily eonlrol assays sh-~uld be ~rlivmlell pnt)r to the detennDation of ~est samples. :
~5 ALP levels up to 12~0 U/L can be mcasured by tl pJocedure. Nonnal tanges a~
Infants 50-165 (U/L) Adult 2~70 (U/L~
(~hild 2U-l~U (U~ L) 60 y~ars 30 75 (UIL) Alk~lino Phu~pha~ase Isoenzyme; : ~
Alkaline phosphatase isoenzyme level6 have been : ::
u prhlluily us~d lo aid im ~e lirferenti~ gnosis ~f liver and bone disorders and also to indicate placental growth patte~
Isoelcclric ~o~;u~ g of alkaline pho~phata~e isoenzyme on a p~ecast ~el g*es 12 discemible band6 of diffcrent organ and tissue o-igill. Oî thesc 12 l~ds. Ball~l 10! appcaring at 4.73 30 isoelec~nc point, is o~ placental origin. lt is released by placental syncytiotrophoblast and/or macrophage, alld i~
T~lympho~yte mediated. In the altemative, samples ~or ALY
isocnzymc can bc analyzct by clcctrophorcsis methods which identify mainly liver, bone, and intestinal isoenzymes. The Ciba Coming Alkaline Phospha~se Isoenzyme (AL~) S~stcm WO 93/122~5 2 ~ 2 ~ 7 9 ~ P~/USi92Jl08j9 m~y be usesl for the qualitative and quantita~ive ~Ieterminatinn of ALP isoenzymes in hu~ n serum hy electrophoresis.
The liver i30enzyme ;s the most freq2lcntly ~ncoun~eretl alkaline pho~phatase iso~nzyme in pa~hologically increased s~n~m alkaline phosphatase activi~. Increased li~r isoen2yme is encountercd in a va~iety ~f liver and hepatobiliary disease.s.
se incll~le hepatie cirrhosis9 primary biliary ~:irrhosi.~, congestive ciErhosis, and heya~ic carcinoma. Elevation of the liver isoen7.yme is also a ~ensitive indicator of chol~asis and 0 liv0r infiltration.
Annther o~ the alkaline phospbatase isoenzymes Ihat can be used in practicing lhe present ulvention is Iymphocytc-(lerived ~Ikaline pl~osphatase. The preferre~l me~hod ~ f measuring this isoe~ yme is hy isoelectric focusing ectrophoresis. A commercially available isoclcctric focusing appar~tus that is capable of meacuring alkaline phosphatase enzymes is made by lsoLab, ~c. (ResolYc~-ALP. IsoLah lnc., ~kron, Ohio). On the Re.colve~-AW i~oelectric fucus~ng allp~tatus, the alkaline phosphatasc isoel~yme lha~
~o uset in thc prescnt in~re~ltion resnlves as band 10 in the ~lectrophnretic pattem. In the prcferred bon~ den~ity al~orithm dcfined herei1~below, the value for the band 10 ~Ikaline phosphatase isoenzyme was a~signed 0 if the band ~vere missîng or very wealc, arld I if the band were present.
The e~ectrophoresis ~els can also bc scann~lJ with ~
densitomcter and more ~uandtative values can be assigned to the alka1ine phosphatase isoetlzyme concentrati~Jn. A~s u.s~
herein, Ihe band 10 alk~aline phnsphatase isoenzyme sh~11 bc ~lesignated Iymphocyte-derived all;alinc phosphata~e isoenzymc, orI-Alkp.
It is ro be understood that other m~thods of measuring 1-Alkp could be uscd. ~l~se me~hnds include, b~t are not limited tt), enzyme-Jinked immlmoassay tcchniques (ELISA).
radioimmunoassay techniques ~ffinily c~lumns, and isoelectric ~5 focusing columns. It is ilnportant to note t~at measurcme~ll uf - :` WO 93/1225~ 212 ~ 7 ~ 2 PCl/lJ.'i'92~10819 b~nd 10 on thc RcsolYe~'-ALP apparatus do~ no~ carrel~s~ ~t all wi~h n.steopenia in general. There are many othcr abnonnal conditions which rcsult i~l a l~i~her than normal h~n(l 10 Iymphocyte-derived alkal~ne phosphatase isoenzyme (scc s Barnki~l, et al. and Griffith, et al.. ~llpr~.3.
A number of pathological conditions c~n lgad to an .
elevated Icvel of bone alkaline phospllalase isoenzyme. The highe~t level~ of bone isoenzymes are usually found in Pagets disease. Increased levels arc also encollm~ in nckels. bt)ne o cancer, ns~eomacacia and celiac spn~e. Rerlal disorders cnn also result in increased Icvels. Thesc include renal failure, : .
pnmary hyperparathyroidi~m, secondary hype~parathyroidism induced by long term hemodialysis au~l m~labsoFption. ~ :
~ncreased levels of intestinal alkaline phosphat2se arc encountcrcd in a varicty of diseas~ ur the digeseive tr~cl. . .-'Ihe.~e include intestinal infection and ulcerati~e lesions of thc stomach, duodenusn, small intestu1e alld ~:lulon. . .
Procedurc:
w ln the preferred methcd of practici~g the present invention, the alkalinc ph~sphasas~ i~uenzymes are ~epara~e~
hy el~ctr~phoresis in a buffered agarose system. Aftcr electrophoresis ~c isoenzymes are tetected by incubadng rhe . -gel w1th a fluo~escent compount ~uch as 4-methylumbellifclyl 2s phosphatc, The fluorcscense forllle(l during the intera~tinn is qu~ntitated using a Ci~oa-coming 710 densi~Gmeter at 385 nm.
Total allialinc phosphatase activity of the patiene i.~ reqnit~d to interpret the liver, bone and intestulal ALP iSOe~lZ~llC9, 3U ~.~trogen and Proges~erone:
The estrogcns are steroi~ls lhat have d nIlg con~aining three unsaturated double bonds. The ovary, as well ~s the testcs and adrcnal ~land, has the cal~acity to syn~he~ize e~rngens f~m androgens, androstenedione and testosterone.
3s During thc follicular phasc of tllc mcLl~lru~l cy-;le, ovarian . . ~ .

z~s % 3. ~ ,3 7 ~ ~ PCr/U.~2~19879 secretion represents only one third of total ~strogen production, In contrast to eslr~iol. ~hi~h is seereted almost entir~ly by ~he ovary, most estrone is dcn~cd from penpheral conversion of androstcnedivne and frnm es;radiol mstabolism.
Dllring mennpause, estradiol conc~ntr~ions st~adily llecrease to approxim~tcly 1~ percenl of premenopausal levels. In heallhy poslmenopausal women, the ovaries do nol ~ecrete significant quantities of estro~ens, and virtually all estrogen prodllced is from. peripheral conversion of andr~stule(lione n madc by the adrenal. Eslra~liol is ~resellt in the serum as fc~ vs: :
Follicular Phase ~`~
Early 30-100 ngfL
Late 100400 ng/L
Lut~alE'hasc SO I~)n~
Po,s~nenapausal ~20 ng/L : ; -In men.cmtating t`emales, progcstcronc is ~c~:reled -.~
mainly by the corpus lutellm sf the nYary. I~ is partially : ~:
respunsible fnr eyclic changes in the endometrium ~ t are .
necessary for attachment ~nd grow~h of an embryo.
Progesterone levels are low prior to the L~lid-cycle :
gonadotropin ~urgc. Shortly af~er ~he gonadotr~pin surge, they begin to rise rapidly, reashing peak levels ~luring the rniddle of thc lutcal pha~e. Thereafter, a pto~ressive fall uccurs wtth barely deiectable progcsterone leYels prior r-- ;
menses.
~Ithough proge~terone in l~rge arnounts pro~luces a 2~ ne~ative feedback on gonadu~ropin secretion, it is not the majDr component in the negative feedback system of ov~n~sl steroids. Function of the coryus luleum can be assessed by mea~uring sen~m progesterone concentration. rrogcsterolle is present ~n the senlm as follows:

~ IZ25~ 212 ~ 7 ~ ~, PcrJuss2J~

2~

Follieular Phase 0.1 ~ ng/L
Luteal Ph~se ~ 28.1 nglL
hIid-Lut~al Phase S.7 - 28.1 ng Over 60 Year~ 0.l~ - 0.2 ng/L
Pr~xedure:
In a preferred method of practicin~ the present invention, proge~tcrone measurements can be obt~incd by a radioimmuno-assay method u~ antibu~y coa~ed tubes o (Diagno~tic Products, Lo~ Angles, Califomia~. E~t~adiol mea~ur~ments can be performct ~ a Mi~;rupanicle En~yme Immunoassay (IMX), availahle from Abbott Diagnostics ~bbott Par~
15 Bt~ne Min~ral D~lsity ~BMl)) h~e~su~emen~s:
convcntiondl me~nds of diagnosing osteopenia which may ~e used when pMcticing the p~esellt invemion are are outlined belo~r;
Dual-Photon absorptome~r~ (DPA) is widely u~.d to 2n assess bonc mincral culltent ~nd bone miner~l dcnsity.
Johnston Conrad 1~, et al., "Clinical Use of Rone Oensitomctry~ ew Eng. J. ~Jf ~ed., Vol. 324, pgs. 1105-1109. (199t). I:)P~ u~es tr~nsmission scannillg with ~hotons from a ~adioisotope source, ~uch as 153 (~d, ~h~t emits two 25 ulergy l~eaks, thus allo~ng bone dcnsity lo be measured independcnt of soft ti~sue. DP~ meas~reme~ts are pcrformed lun lumhar spine (Ll-L4) ~nd fcmur (femural neck, Ward-s Trianglc and tro~hanter) and the average determined sep~ately. The ove~all aver~ge o~ both hip alld spine can also 31~ be determuned. BMD can be me~sured by DPA usin~ -Gadolinium a~ the source (I,unar DP3, by Lullar Radialin~
Corporation, Madison, W~sconsin).
The present method employs transmission s~:anning using 44 and 100 KcV photon e~lergies fmm a one Cil53Gd 35 sourcc to alluw computa~on of the ~eral contcnt of bone ~.','.``''~"'.`'`'' .,' ''',,,. .;``", ;,'`,.

W() 9~122~C ~ g2l1o879 in~ependent of soft tissue thi kness. Bone mineral dcnsity.
expressed in ~lcm2~ is derivetl by di~ ling bone mineral ~;umenl [P~MC) by the pro3ected area of the scanned bo~
Peppler W.W., et al.. "Total B~dy Bone Mineral and l.ean Body Mass by Dual-~hoton Abso;ptomctry: 1. Thcory a~
Measllremcnt Procedure," C~lLclP, I.f.~ Int., Vol. 33. P6.
3~3. (19~1); Shipp C., et al., "Precision of Dual~Phot~
Absorptometry," CALCIP, Is~ue lnt.. Vol. 42, pgs. 287-~Y2, (1988). Dllnng spine scans, lhe d&tector move3 in a rectilinear ~o pattem at a rate of Smrn/sec a~ l ~ith scan linP.. ~ 4.5 nun apart.
Thc BMC ~n~ BMD are calculated in lumbar Yertcbrac 1 through 4 (~ncludin~ intcn~erlebral discs) with a software version sup~lied by Lunar Kadiation Colporation of Madiso Wiscons~
During femur mea~urements, the scanncr moYcs at a rate of 2.5 ml;~/sec and a step dislance of 2..~ mm. The ~M(~`
an~l BMD of the. neck~ Ward's Triangle a~d trochanteric re6ionE of dlc proxuual fcmur ar~ calculatet1 us-ng a femur soflware ver~ion supplied by Lunar Radiation Corporation.
lhe femor~l neek region of intere~ (RO~) i.c that band about 1.5 ~In w~de acr~ss the neck of the bone pe~pendicular to the neutral axis with the lo~vest density. Wards TriangIe is de~ ~ as a .~quare ROI (about 1.5 x l.5 cm) with the low~st density wi~in the pro~imal femur regil)n. Wartl's Triangle is predo -lin~ntly trabecular bone and contains the Ica~t amount of bone mineraI within thc lleck r~gion. Carter n.R., et al., "R~Ialionshi~ P~et~een Loadin6 History ~nd l:cmoral C'ancellou6 Bone Architechlre~ J. Biumechan~ , Vol. 22, pgs.
23l-2~4, (1989). Tn Ihe pro~imal femur, tho ROI is usually ~n the area 1.5 cm widc across the eIllire femnral neck~
Additional region.c are de~med by the ~oftware in thc lower density Ward'~ TriEmg1c region, and in Ihe region of the greater trochanter. Bone ln~s in the pro~imal felnur begins in the Ward's Iriangle region and proceeds outward rrum there.
3s (Brow~ D., et al.. "Mechanical Property Distributions in tbe wo 93/~22~ 2 1 ~ ~ ~ 3 2 P~tUS92/10879 Cancellous Bonc of thc Human rroximal l~emur," Act.
nrrhop S~.fand., vol 51, pgs. 429-~37, ~19~0). This makes thc rcgion an carly indica~or of bone loss, but ~he higher vanan~e in measuring it, compared to the neck re~ion, make~
s the latter zone, a better discriminator. However, Ward's Triangle is the lowest de.nsity area at the point where the neck and greater trochanter mcct, a pnmary hip fracture site. This operational ROI may not co~respond e~ac~ly to the anatomic Ward's Triangle region but docs p~o~ide a rcpcaeable n measuremen~. The w~dth of the neck ROI and the size of the Ward`s Tnan~le ROI arc actually proportional to the measured size of ~e femaral neclc. It has been shown that the dcnsity of the Ward's Triangle area is substatltially reduced in hip fracture patients compared with age matched contrDls.
lS (Vo~c G. P., et al., "Femoral Necl; rractunn~ its Rclationship tn Radiogrnphic Bone nensity," J. Gerontol., Vol. 20, pgs.
300-305, (l965?
DPA measur~ments have a precision of 1-3% and the scan can be completed in about 20 minutcs. Bone density of 2n ver~ebrae c~rre.lates well with risk for veltebral fracture, and bone density of areas in ~he proximal ~cmur colTelates wcll wlth risk for hip fracture. ~PA has a low radiation dose (c.ll~
mrcm to ~kin, dnd 2 mrem to ma~row). DPA has appr~x~n1ate SenSitiYity of spine and hip of 50% and 53%, respectively, at 95% spcif1city (i.e. ~o of fracture sases below 5~percentile~.
Moreover, I:)PA is not su~ject to systematic er~o~ introduced by Yanable osteoid and variab1c marrow.
Rone density can also be measured by radiographic absorptometry. Radiographic absorptometry is a method of ~u measur~ng bone den.~iry which is well known to those of ordinary skill in the ~rt. Other methods of mcasuling bone density include qua~titanve computed tomograE~hy and ~irect me~surement of bone density.
D~r~el measurement of bone densi~y can be o?~tained hy 3~ mea~unng the bone density ul cadavers. Thus, another method !
21~7~2 %8 of determining thc corrclation bctween the blood concentratiorls of ce.lt~in predet~nine(l hlnnd cnn.c~imenls and bone density is to mcasurc thc bonc dcnsity of a sct of e~d~vefs from which blood concemrArinn.~ nf ~ le~errninell blond constituents are known. Thc multiple linear regression analysis can then be pe~folmed and a ~one density cnefflcient rclatio~lship can easily be detemined.
ln correlating the bone den~ity me~.cllrement~ to concentration of bloot constitucnts, blood concentrations of ~o calcium, phosphate, total alkalin~ phosph~rase. An alkaline phosphata~e isoenzymc c~tradiol, and progesterone are measu~ed. Liver, ~one and intestinal is~en~ e.c can he u~ed.
Band 10 alkalinc phosphatase isvenzyme. A m~thematical rela~ionship ~.lween the cnncentrations of blood cons~iments ~nd bonc dcrl~ity, as measuredby radiographie ~bsorptometry or nther stand~rd method ~f measuring bone density, is dctcrmincd by pcrforming a multiple linear regression analysis w~th the fnllnwing m~del-zu Bone ~n~ily euerrleielll =bo ~ ~lCa ~ ~2P ~ ~,E2 + b~P~
bsAlkp I bCI-Alkp ~ b7(CaCa) ~ bg(PP) I b~( 2~ 2) blo(P~ ~ P~) + ~Il(All~pAlkp) ~ l l2~CaP) ~ bl~(CaE2) +
bl4(CaPg) Ibl~(Ca~lkp) I b~ al-Alkp) + bl7(~2 bl8(PPg) + blg(PAlkp) ~ ~2U(PI-AlkP~ ~ b2l~E2P~
~5 ~22(~;2Alkp) I b23(E~l-A~ 24(P~All;p) I b2s(~gl-Alkp) ~26(AlkpI-Alkp) where:
emm cnncenmtion nf calcium P - Semrn concentr~tion of phosphoms ~2_ Serum concentrationofe~trddiol PG = S~rum concentration of pro~ester~ne Alkp ~ Selum eoneen~aliun o~ lolal alkalule phosphatase I Alkp - Serum concentration of alkaline phosphatase iSoellzyme.

~' -?

wo g~/l22s5 212 ~ 7 ~ 2 Pcr/ussa/los~

~9 ~ :

Calc.ulation of the mathematica~ model utili~ed the Systat~' ~tatistical packagc (Sy~tat: lnc., I:~vanston~ IL). Thc ~ ~ :
mulriple linear regression ~nalysis is an iterative proces~ which calculatcs ~he correct cocfflcients so that the result of the s algori~hm using the blo~d ~oncentrations correlates ~o a high degree with the result of the bone densi~y measurement by r~diogIaphic absorptometry.
For diagnosing ostcopcnia9 thc gcncral forrn of the pre~erred algorithm ~ha~ is used in the present invention is as o follow~
Bonedensitycoefficients ~O + ~IC~ ~ ~2P ~ b3E2 +
b4Pg ~SAlkp ~ b61~Alkp ~ ~7(CaCa) ~ hg~PP) + hg(E2P~2) I blo(Pg + Pg) + bll(AlkpAlkp) + bl2(C~P) + hl3(C~E2) +
s bl4(CaPg) ~bls(CaAlkp) ~ bl6(Cal-Alkp) ~ bl7(PE2) ~18(PPg) + blg(PAlkp) + b2o(pI Alkp) ~ b21(E2Pg) +
b22(E2Alkp) t- b23(E21-Al~cp) t b24tpgAlkp) ~ b2s(P
Alkp) + b26(Alkpl-Alkp) whete:
bo is a constant enlm conc~m~dan of calcium P - Serum conccntr~tion of phosphoNs P,2 = .Serum concentration of estradiol PG - Serum conccntration of progcstcronc 2~ Alkp = Sen~n conceMradon of total alkal~e phosphatase l-Alkp = Scrum concentration of alkaline phosphatasc isoenzy~e.
~t i~ tn he lmderst~tl That eoncen~tion of l-Alkp can be replaced with serum coflcentration of liYcr, bonc or in~estinal derived alkaline phospha~ase i~oenzymes.
In practicing the present invention, the ~alues obt~ined from lhe patierlt fvr the six blood constituent concen~ralions are inserted into the algori~m as u~dicated. The ma~herna~cal 3S lllallipUlatiOIl iS pCr~Ol;lllCd. ThC n su~ g numb~r is called a r, wo g3,.22~ 2 1 ~ ~ 7 ~ 2 P~:T/11!i92/10879 ::
3() bone density coefficient and is then pla~e~ h~ severity ~cale.
ThiS results In a bone density probability quotien~ which ~ :
COlTClatCS to a high de~ree to bulle ~lell~hy as measured hy - ~ -radiographic methods.
As seen ill the ~l~oril~Lm that is considered part nf rhe present invention, there ~re a number of coefflcients which arc a part of the algorith~ is to be under~tood ~hat the.se coefficients can chan,6e if the bone tensity coefflcient is :~
corrclated tO a different l~lelhc1d of ~letelmining bone den~ity.
n Thus, if bone density i~ measured usi~g du~l photon absolptomct~y, arld ~ne wanlcd lo oorrelate the bone den~ity coefficient to the results o~ Che dual photon abso~ptometly. the overall relationship of ~e tests ill the algorilhM would be the : ~:
same or similar as disclosed herein ~ut the coefflcients could be dif~ercnt~
ln its preferred embodimcnt, the bone density probability quoticnt is assigned to olle u~ the following diagnostic categories:
7.0 Nonnal to mild osteopenia Moterate osteopenia :
Severe osteopenia In addition, the menopausal status may be detesmined 2s based on the lesults of the estradiol and ~ esterone levels into one of the follow-n~3 diagnostic cate,6ones:
Probable Pre-menopausal Probable ~eri~ nopausal 3u Pro~able Post.menopausal Thus, accnrding to the present inventio~, using blood concentrations of certain blood con~tituents, onc can not only ~liagno~e bone den~ but can al~ nbtain an indication of the underlying cause of the osteopeni~.

~ ~ ~'`

W~ 3/lZ2S5 Y(:,'l'/IJ';92/1118~9 This inYcntion is further illu~trated by ~ lullowing examples, which ~re n~t to be construed in any way as imposing l~itations upon the scope ~crenf. To lhe ~;untrary.
il is tn he cl~arly understood that resort may ~e had to various other embodimcnts, modifications, and equi~ al~nt~ the~eof which, af~er reading the descnption here~n, may ~uggest themsclvc~ ~o those sl;illcd in thc art withoul ~l~parting from Ihe .~llirit of the present invention and/or the scope of the appended claims. ~ :
E~ample I
In the following e~ample, the bone density coef~cient ~l~olithm was correlated with bone dcnsity as measured by radiographic abso~p~ometry. The coefficients urere ca~culated by correl~ting the blood concentration of the indical~l blood constituen~ in 27 pa~ient~ to the results of bone density in those patients ~s mca~urcd ~ radiographic absorp~ t~y.
The bone den~ity algnnthm which ~esults is as follows:
0 ~ (~g.9~5)(~ (7g.370)tp) 1 (34.076)~) + (-9.216)(Pgj I (~0.600)(Alkp) + (-57.855)(I-Alkp) ~ (0.926)~C~Ca) +
(-2.735)(PP) ~ (-0.272)(E2E2) ~ (-U.()64)(P~Pg) +
(0.004)(~1kpAlkp) t (-4.029)(CaP) ~ (-3.156)(CaE2) ~
(1.172)(CaPg) ~ (-0.0~1)(CaAlkp) l (8.2~)(CaI-Alkp) +
2s (1.906)(PE2) ~ (-0.138)(I'PK) ~ (^O.Ol~)(PAlkpl +
(-6.710)(PI-AlkE-) I (0.193)(E2Pg) I ~-O.U4~ 2Alkp) +
(-1.825)(E21-~lkp) ,wherein:, ...................... ' Ca = Seruun concen~ration nf calcium P = ~erum conce~ltration of phosphorus E2 - Sem~ oncen~ation of estrad1O1 PG ~ ~;en~m concerltration of progesterone ~ -:
Alkp ~ Serunl collcenlralion uf total aLl~aline phn~phatase ~:

WOY~JlZ~i5 PCr~US92/108ig `- 2~2~7~2 ~ ~

I-Alkp - ~emm concentration of serum Iymphycyte-deriYed aL~aline phosplla~sc i~ ~yme. ~ ~:
The bone dc~ y coefrlu~ !h~t is oblained from the s bone density algoritl~n is assi~ned to one of three c~tegoncs:
t;roup 1 no~nalto mildo~teopenia Group II mo~ler~ u~teopenia ~roup 111 ~evere osteopellia ~.
C;roup 111~ approximately 78.5 c Group II s approxim~tcly 100 ~ Group I
It has been determined that the bone densaty coefficicnt calculated accordillg to the prc~ vention places pa~ient.c in s the sarne groups as radio~raphic abso~ptometry measurements for bone density. Thus. ~e pre~ent inven~ion pro~ide~ a ~afe, economical and accurate me~od for diagnosing osteopenia.
Bonc density quotients deri~d prd~:ticing Ihe present invention using ~e si1~ biochemical serum constituents were found to strongly cor~elate with bonc ~l~nsity measurement~
from radiographic absorptometry. lt is to be understood that thc p~cferred embodimenl u~ilizes all six of the biochemic~l senun constituents listed hereinabove. How0ver, if any fiYe of thc scmm constituents are u~ l then included in Ihe 2s bone density al~orithm, a bone density quo~ient is obtaincd which does not corn:lalc as well a~ when sLl~ tes~s are u.~ed.
It is contemp1ated as part of the present inveneion that any five of thc bio~l1e~ rum ~ nS~imeMs can be u.sed tn determine the osteopenic state of a patient. For example, if ~enLm calciurn is rcmoved from the bolle d~lsily algoritbm, ~hen the algoritlun predictive value ~or moderate or severe o~tcopcnia is appro~nately 66% althou~h the predictive v~lue for nonn~l ~o mild osteopenia is 100% lf pro~esterone is removed from thc bonc dcn~ity al~rit}un, thcn thc algori~
predic~ive vallle for moderate or severe osteopenia of W0 93t122~S 2 1 2 ~ 7 ~ 2 ~'C ~ YZ/~11879 ~ppro~imately 7S% and a prediclive value ~r nu~nal 1~) mild osteopenia of approx~mately Y4%. lf alkaline phosphatase is re~llove~ e b~ d~n~ily al~ h~ hen the al~orithm predictive value for mod~rate or seYere osleopenia of ~pr~ im~ely 83% and ~ predictiYe vdlue for no~mal lo mild osteopenia of approximately 85~o. If estratiol is removed ~ . :
froll~ lh~ bolle ~lensi~y ~Ig~ hm, Lhen the al~vrithm p~edicrive value for moderate or ~evere osteopenia of approximately G7% au~l ~ pr~di~;liv~ v-dlue îur ~ mlal lu mil~ openia of o approximately 85~/o. However, it has been det~nnined that wl~ dll ~i~ le~l~ arc illl;lulle~l in Lh~ bu~ nsily algs~rithm.
the predictive value ~or moderate and seYere ~steopenia is 100% alld d~e prediclive Yalue ~r nortnal 1~ lild u~ p~nia is 1()U%. All ot the predietive ~alues may vary sli~htly with s different patient populations.
Example II ~ -'llle followin~ semm cons~ituents are measwed in a 37 year old WOIl~
~o ~ .
Calcium ................................ 9.5 mg/dL
Phosphate..... ~......................... S.O m~/dL
Estradiol................... ............ 2 y~/luL
~rogesterone................ ............ 0.2 n~/mL ;
2~ Alkalhle Pllosphatase............ 80 U/L .
ALkaline phosph~tase isoenzyme .. O (ne~ative) 'f he above blood chemi6tty values we~e used to calculate a bone density coef~l~ienl u~ing lllc followill~ al~ori~un~
~
O ~ (~8.9~5)(Ca) ~ (79.370)(P) ~ (34.076)(E2) ~ (-9.216)(P~
(-0.600)(Alkp) I (-57.855)~1-Alkp) I (~ 26)(~:a~a) (-2.735)(PP) ~ (^0.272)(E2E2) ~ (-0.064~(PgP~) + I
~0.004)(ALlcpAlkp~ 4.029)~CaP) ~ (-3.1 56)(CaE2) 3~ 72)(carg) + (-0.031)(CaAlkp) ~ (8.238)(Cal-Alkp) : : , 2 ~ 2 5 7 ~ ~ PClJUS92~08~9 , (I.~O~)(PE2) ~ (-0.138)(F'P~ O.t)l8)(PAlkp) +
(-6.710)(PI-~lkp) + (0.1~3)~ P~) + (-0.048)(~2Alkp) (-1.825)~1~21-Alkp) where:
s Ca - Sen~m concentration of calcium P = Serum concen~ration of phosphorus E2 = Semm concen~ration of estradi~)l PG - Serum concentr~do~ of pro~esterone Allcp = ~eru~n concentradoD of total alkaline phosphatase :
Alkp = Se~um concentration o`f serum Iymphycyte-dcriYe~
al~aline pho6phat~ ocnzymc.
A bone density cocfficient of 101.2 is caleulated for thi~
patient. ll~i~ ~;oefficient falls within Group I in the followi s severi~y scale:
Group m s 78.5 ~ Group II S 100 ~ up 1 Group 1 normal to mild ~steopenia ao Group Il moderateosteopenia .
Group lIl scvereosteopelu~
This patient has nonnal to mild ~sleopenia. When the patient's bone density is measured by radio~raphiL: :
2s abso~ptomehy, she is found to havc nonnal bone den~ity.
Ex~MPLE III
~hc foll~wing .~emm constituent~ are mea~ured in a 47 year old woman: .
Calciurn ........................ 9.5 mgldL
Phosphat~........................ 5.0 mg/dL
E~tradiol........................ 2 pglmL
Progestcrone.......... ,.......... U.2 nglml Alkaline Phosphatase............. 80 U/L

2 1 ~ i~ 7 3 2 P~CT/US9Z/1~87~

Alkaline phosl-h~tas~ i~oen7yme .. ~........ l (positive) I'he valucs an~ inserled ~n'o the bone density al~orilhm recited ul E~ample I and a bone density euefficient of ~4.4 i~
calculated. l~is falls in Grm~p II, indicating a modera~e osteopenia. Wtlen the patient's bone dc~ y was measured by radio~aphic abso~ptometry, ~he was ~ound to bc moderalely osteopellic.
o b:X~MPiE IV
l~e followin~ scm~ ;on~n~ents are measurcd in a 47 ycar old wuman who hag beerl dia~osed wilh br~ an cer:

Calcium ..................... ~.. 9.0 m~!tdL
1~ Phos~hate.................... ... 4.0 n7 '~1L
Estradiol.................... ... 0 pg/mL : : :
l~to6e~t~ronc ............... ... 0 n~/mL ; . :~
Allcaline Phosphatase ..................... 60 1 IIL ,~
Alkal1ne phosphatasc isoenzyme ............ 1 (positivc~ :
The values arc inserted in~o the bone density algorithm :
recited in E~ample ~ and a bone density coefrl~;ienl nf 69.9 is calculated. TJ~is falls in Group III indicating a se~ere ostcopenia illsured ~y another me~od. : :
2~s " "", ~, , EXAMPLE V :
The following exampl~ ~hows the co~relation between thc ~ctbo~l o~ the present invention alld Dual Photon A)sorptometry (DPA) as a tool for dia~nosing and ~o detennining lhe sevelity of osteoporo~is in ~ par~iclllar : --individual. 2W femnle subjccts ~p~esent;ng a cross section of ages and mcnopau~al stan~ were evaluatet. llhcsc specific Age ~roups were represented u~ follows;

. ,~

~ 212 ~ i ~ 2 Y~ -I/U!i!lZ/lU1~79 36-4~ 2~

s 56-65 ~n 16-85 2~
85 and uvcr 26 Bach subjcct comp~et~d an Osteoporosis Data QuestioMai~e Foml, which inelu~ed detailed information on personal histor~ family history, gynecolo~ic histoly, medicnl history, surgical hi~u~ry, and dn~g history, wi~h special rc~rcnccs tohormone tre~tment.
Three nl~es of hlond wete collected from each subject via Ycnipuncture. One tube was sent to Bamhill-MetPath Laboraln~ie in ~avann~h, Georgia for analysis of serum calcium, phosphorous, and tot~l alkaline phosph~se. The second mbe was senl t.o Wnn~ Therapeutio~, ~c. in Sava~
Gcorgia for dctcmlination of Iymphocyte alkal~ne phosphatase ~dli2ing the Isoelectnc Fn~u~sing Method. ~e third tube was scnt to MctPath Laboratories in Teterboro, New lersey for evaluatinn nf e~tradiol and progeslerone by immunoassay.
Data from all ~hree sources w~re then subjected eo ~tatistical .s ~naly~i~. At the time of ~he analysis of da~. ~ach subject's chart W~IS critic~lly reviewed for reIe~ant clinical infonnation, The ~nne Mineral Dens~ty (BMD) of each subject was also mcasu~cd b~ Dual-Photon Absorptometry using ~
~',ad~linium a~ a source (using Lwlar DP3, Lunar R~liati~n ~o Colporation, Madison, Wisconsin). DPA measurements were perfonned on the Lumbar Spillc (L1-L4) alld the hlp.
specifically the femoral neck, W~rd's Iriangle, and the ~chanten~ lls. BMD was m~asurcd in gm/cm2 for cach re~ion noted, and ~e fracn~re sisk was detem~ined.

$~`` ``` ` ' ~ - ` `` ~

--` 2~ 2~7~2 The BMD measurements were then ag~ matched, and adjusted for sex, age, ethnic group, and weight. From the individual values obtained, avera~e BMD for hip and spine was calculated. The overall average BMD for both hip and spine was also computed.
A mathematical al~orithrn correlating the results using th~ present invention with DPA was determined with the reswltant data as shown in Figure 1.
Figure 2 illustrates the discrepancies behNeen the DPA measurements ~nd the measurements obtained using the method of the present invention for individual subjects. ...
The amount of the discrepancy (the residual) for any -patient is the vertical distance between the points for that patient on the DPA and the present invention lines.
Figure 3 ili:~st~es the appropr.iatcn~ss of usin~ tlle present invention as a prediction of DPA measurements. ~`IOt~
the ahsence of any tendency for points to bc consistently abovc or consistcntly bclow the ~lia~onal ~raight line, as gre~ter DPA rneasurements are considered. Additionally, there is 'Ul .
ab~encc of any ~cndency ~r pOiIll~; al~mg any vertical line to be : :more widespread Ihan are points sllon~ any ~ther vcrtical lin~
Finally, notc ~at ~c ~enerally elong~ted shape fo~med by rhe :
poin~s is consistent with a strong lincar correlation bet~e~
DPA and ~e present inY~ntion~
Figure 4 demonstrates ~he normality of values predicletl .
by the prescnt invenlion (the horizonral scale~. The vertical scale sho~s the of ~o normnlizcd ~cale tz) scorcs v-dlues dctermincd accor~ling to the present invention as ~ould be expected from the assumption of thc present invention values 30 for~nin~ a normally distribu~d collecrion of numbers.
lhe closer ~he dots are to dle straight line, the ~reater is the consistency belw~ell what is o ~s~.rved about the present in~ention values and vh~t is expectcd about thcm f~ol~ e assumption of rheu fonnLng a normally distri~uted collection ::
3s of numbeirs .

:. g ~ , WU 93/12255 2 ~ ~ ~ 7 ~ ~cr/us9z/lon7s ~ig~ S dcmonstraec~ the normality of residuals (the difference hetween the observed nPA measurement and the valuc prcdiratcd by present invention), as cvidenced by the a~angements of p~ints along the diagonal straight line. I he s horizontal scale shows values of rcsiduals The vcltical sc~l~
shows ~he residuals' nonnalized scale (~) sGOreS as would be cxpcctct from the assumption that thc residuals ~or no~Tnally distribused collection of numbers.
Thc clo~cr tbc dot~ are to the strai~ht line, the ~reater is IU ~he enn.sisleney l~.tween what is obse~ed about the residuals Qnt what i~ c~pcctcd about ~cm from the assumptioll of tl~ei fo~ming a nnm ~lly distri~uted colleclion of numbers.
FiguFe 6 demonstra~cs that the variance of rcsiduals ~ays Ihe .came regardless of the value calculated according to the present inYention in~olvcd in the residuals' computation This homl~geneity nf varianee appears in the uniform density of ~e dots in ~ rectangle fo~mcd by re~iduals between -0.17 and ~0.17 and hy the l)resent invendon besween ~0.4 and +1.0 Moreover, there is no disccmable tendency for points along any vertical line s~ be more widespread than are poin~s alon~
any o~er ~ertical Imc.
Figure 7 demnn~trates the independence of residuals and the v~lues predicted by thc prcsent invention. Thc horizontal scales shows the values predicted by the present invention.
The vertic~l scale shows thc r~situal's studentized scale (t) scores.
When therc i9 DO rcladon~hip between ~aluec prcdicted by the present inventinn and the residuals involving those predicted values (that is, ~hen there is indcpcndcnce between resi~lu~ls ~nd predicted preseM inventinn vallles) dle following criteria 6hould be met:
1. There should be no discernable p~ttern (such as a straighl or curved lille~ tu the dots.

~v ~" ~ 2 ~ 2 ~ 7 ~ US92.~10879 2. About half the dots should appear in thc top half of thc ~rapll.
Pigure 8 ~ulullslra~es ~at virtually ~11 observed DPA
s measurements are 6atisfactonly predictet by thc method of the present inv~nliun. Thehorizonfal ~cale sh-)w.~ values predicted by the method o~ the present inve~tion. Thc ~cr~ical scale shows Cook's ~lislanees between ob~e~ed T~PA measureme.nts and the predictinns of those measufetnents by thc mcthod of 0 the present inv~ iun Thus. the closer Ihe dnr~ ~re to ~ height of Cook - 0, the more accur~te is the method of thc present ention. Becau~ Cook's distances lend to fnrrn a collection of numbers fi~ting an F-statistical distribution, the two ou~lier points at heights l~lween 10 and 20 are not thollg'nl to be problematic in view of dle large si~e of the sEImplc.
It should bc ull~lerstood, of course, that the foregoing relates only to preferred embodiments of the present in~cntion ant that numerous Ino~ c~tions or altera~it n~ may be made there~n without departing from the spint ~nd the scopc of the invcn~ion as set forth ill Ibe appended claim~.

Claims (11)

Claims What is claimed is:

1. A method of providing a diagnostic system for diagnosing osteoporosis and determining the severity of osteoporosis in a human or animal comprising the steps of:
a. measuring the severity of osteoporosis in a se?
of humans or animal with varying severity of osteoporosis by a standard method, b. assigning the severity of disease a numerical value on a severity scale, the scab being from no disease to severe disease;
c. measuring the blood concentrations of a preddetermined act of blood constituents in the set of humans or animals with varying severity of the disease; and d. determining a numerical relationship between the set of blood concentrations and the severity of disease

2. The method of Claim 1. wherein the numerical relationship is calculated using multiple linear regression analysis.

3. The method of Claim 1, wherein the predetermined set of blood constituents comprises calcium, phosphate, total alkaline phosphatase, alkaline phosphatase isoenzyme, estradiol and progesterone.

4. The method of Claim 3, wherein the age of the human or animal is factored into the numerical relationship.

5. The method of Claim 3 wherein the alkaline phosphatase isoenzyme is selected from the group consisting of lymphocyte-derived alkaline phosphatase, liver alkaline phosphatase, bone alkaline phosphatase and liver alkaline phosphatase.

6. The method of Claim 1, wherein the standard method of measuring severity of osteoporosis is measuring bone density by radiographic absorptometry, quantitative computed tomography, dual photon absorptometry or direct measurement of bone density.

7. The method of Claim 6, wherein the standard method of measuring severity of osteoporosis is dual photon absorptometry.

8. A method of determining the diagnosing osteopenia in a human or animal comprising the steps of:
a. measuring the concentrations of a predetermined set of blood constituents in the blood of the human or animal, b. determining a bone density coefficient using the numerical relationship of Claim 1; and c. determining the severity of osteoporosis in the human or animal by comparing the bone density coefficient to the severity scale of Claim 1.

9. The method of Claim 8, wherein the predetermined set of blood constituents comprises calcium, phosphate, total alkaline phosphatase, alkaline phosphatase isoenzyme, estradiol and progesterone.

10. The method of Claim 9, wherein the alkaline phosphatase isoenzyme is selected from the group consisting of lymphocyte-derived alkaline phosphatase, liver alkaline phosphatase, bone alkaline phosphatase and liver alkaline phosphatase.

11. The method of Claim 8, wherein the age of the human or animal is factored into the numerical relationship.