CLIN. CHEM. 34/7, 1431-1438 (1988)

CLINICALCHEMISTRY, Vol.34, No. 7, 1988 1431

In Vivo 3’P Nuclear Magnetic Resonance Spectroscopyof Bone Mineral for EvaluationofOsteoporosisCharles Eric Brown,1 Joseph H. Battoclettl,2 RavI Srlnlvasan,3 James R.Allaway,4 Jeannine Moore,5 and PeterSlgmann#{176}

The mineral content of stationary bone samples can bequantified by 31Pnuclear magnetic resonance (NMR) spec-troscopy. The assay can be performed in regions of theanatomy that pose problems for absorptiometric techniques,because the mineral content is measured within a selectedvolumewithoutconcern for the geometry of the bone. In vivo31P NMR spectra of the bones in human fingers and wrist arereported. Soft tissue such as marrow and skeletal musclecontributes little to the 31P NMR spectra of human fingersand wrist and thus should not seriously affect the accuracy ofthe mineral assay. 31P NMR spectrometry should provehelpful for confirming rapid bone mineral loss in those at riskand for monitoring response to treatment.

AddItIonal Keyphrases: phosphorus calciumapatite bonemineral osteodystrophy noninvasiveanal-ysis monitoringbone loss

Two major obstacles have impeded efforts toward preven-tion of osteoporosis (1-7). First, the underlying causes andthe most appropriate prophylaxis and treatment still are notwell understood. Second, a method for measuring the pres-ence and extent of osteoporosis is needed, not only for earlydiagnosis but also to assess the effects of treatment. Thework presented here addresses this latter need.

Among the diagnostic procedures currently available formeasurement of bone mineral content are single- and dual-photon absorptiometry, quantitative computed tomography,Compton scattering, measurement of total body calciumwith neutron activation analysis, and determination ofcancellous bone volume from biopsy of the iliac crest (1,3,8-10). Each of these procedures has its technical limitations,and the results tend to correlate poorly with the severity offracture (4, 5, 7, 10-14).

It was concluded in early 1987 that a better analyticalprocedure (i.e., easier, cheaper, with lower radiation expo-sure) was needed (15). Quantitative digital radiography,also called x-ray absorptiometry, has appeared in responseto this need (16).

We have taken the alternative approach of measuring theamount of bone mineral (i.e., apatite) directly on the basis ofits 31P NMR spectrum (7,17-19). The relative peak areas ineach spectrum from apatite and a reference standard (KPF6)

‘Department of Biochemistry, Medical College of Wisconsin,8701 Watertown Plank Road, Milwaukee, WI 53226.

2Medjmi Research Service, Veterans Administration MedicalCenter, Milwaukee, WI 53193; and Department of Neurosurgery,Medical College of Wisconsin.

3Biomedical Engineering Department, Marquette University,Milwaukee, WI 53233.

4Polymer Research Department, Johnson Wax Company, 1525Howe Street, Racine, WI 53403.

5Department of Pharmacology, Medical College of Wisconsin.6Department of Medicine, Medical College of Wisconsin, and

Froedtert Memorial Lutheran Hospital, Milwaukee, WI 53226.Received February 12, 1988; accepted April 1, 1988.

are determined by integration. The ratio of these peak areaswas shown to be linearly related to the weight of hydroxyap-atite within the receiver coil of the NMR spectrometer. Thecoefficient of correlation, r, for linear least-squares regres-sion analysis was found to be between 0.997 and 0.999 (7).

In theory, 31p NMR spectra can be recorded in any part ofthe body. However, the expense of the analysis will bedetermined in large part by the sizes of the magnet andreceiver coil that are required, so measurement of bonemineral content in the extremities would cost less thanmeasurement in the human torso. For this reason, we havebegun our research on analysis of the extremities. Therecent work of Christiansen et al. (20-22) regarding thesuitabifity of the wrist for monitoring mineral metabolismin the clinical setting and of Dias et al. (23) regarding theincidence of osteoporosis in patients with Colle’s fracturessuggests that our current attempts to perform such spectro-scopy with the wrist should have clinical application.

Among the responses to our recommendation that 3’PNMR be used for clinical analysis of bone mineral wasconcern that signals from skeletal muscle and marrowwould affect the accuracy of the mineral assay in vivo (24).Thus, three questions are addressed in the work presentedhere. First, can in vivo 31P NMR spectra of bone mineral bemeasured in the human hand and wrist? Second, cansurface coils be used to record the spectra? Third, what arethe possible contributions to the 31P NMR spectra from softtissues such as skeletal muscle and marrow?

Materials and Methods

Materials

Hydroxyapatite and potassium hexafluorophosphate[KPF6] were obtained from Bio-Rad Laboratories, Rich-mond, CA 94804, and Aldrich Chemical Co., Milwaukee, WI53233, respectively. Skeletal muscle and marrow were ex-cised from dogs immediately after killing (i.e., remains fromsurgical procedures that had been performed for otherresearch). All experiments with dog tissue and with humanswere performed with appropriate review and, where appro-priate, informed consent.

Quantification of Apatite

The mineral content of each bone sample was determinedfrom the intensity of its 31P NMR spectrum relative to thatof KPF6, as previously described (7, 17-19). In the in vivoexperiments, the KPF6 reference standard was held in closeproximity to the extremity with either a polyethylenesandwich bag or a pair of polyvinyl chloride gloves. 31PNMR spectra were recorded with a Model NT-150 spectrom-eter (Nicolet Instrument Corp., Madison, WI 53711) at aresonance frequency of 60.7 MHz. Each sample was keptstationary in a 3.5-T superconducting magnet with an 85-mm internal bore diameter, and one or more simple pulsesat the resonance frequency were applied with either aHelmholtz coil that surrounded the sample or a saddle-shaped coil that was placed on its surface (7, 17-19). The

1432 CLINICALCHEMISTRY, Vol. 34, No. 7, 1988

time-domain signal was recorded susequent to each pulsefor between 5 ma and 168 ma, depending on the experiment.This signal was apodized by exponential multiplication andthen Fourier transformed to obtain the frequency-domainspectra (7). Chemical shifts are given in parts per million(ppm) from 85% H3P04, which is the accepted method ofpresentation of 31P NMR spectra.

A cross polarization, magic angle sample spinning[CP/MASI 31P probe from the Nicolet Instrument Corp. wasmodified for in vivo spectroscopy of the fingers, hand, andwrist. Its coil was replaced with either of two pairs ofparallel-connected saddle coils. One Helmholtz coil thusproduced had an internal diameter of 55 mm and length of70 mm, whereas the other had an internal diameter of 75mm and length of 55 mm. For surface-coil spectroscopy, theCPIMAS probe was modified with only a single saddle coil(55 mm internal diameter and 70 mm length). Observepulses of 10 us to 60 pa duration were used, depending onthe identity of the probe and sample. The magnetic ffipangle produced is not known, because a reliable 900 pulsewas not possible with our pulse equipment.

Because of the limitations of the superconducting magnetthat is available for our work, a significant proportion of thesample in these larger coils lies outside of the “high-resolution” region of the magnet. Narrow peaks are artifi-cially broadened by the inhomogeneity of the field. Tocircumvent this artifact, we recorded high-resolution invitro spectra, for comparison, using a probe from the NicoletInstrument Corp. with a Helmholtz coil that accommodatesa stationary 20-mm (o.d.) sample tube with about 7 mL ofsample.

Determination of Possible Sources of Interference

Factors that might be expected to interfere with in vivoquantification of bone mineral by 31P NMR spectrometryinclude (a) phosphorylated cytosolic metabolites in softtissue, (b) phosphorylated components of membranes in softtissue, and (c) the protonated mineral forms that arethought to be involved in the deposition of apatite. Thecytosolic metabolites represent only about 5% of the phos-phorus of soft tissue; thus they are not expected to make amajor contribution to spectral intensity on a per-volumebasis. However, the peaks from these metabolites will bequite narrow when the spectra are recorded with a high-resolution magnet. The vast majority of the phosphorus ofsoft tissue is in membranes in the form of phosphate esters.These ester groups are expected to give rise to broadphosphorus resonances, owing to restricted, anisotropic mo-tion. Just how broad the resonances from a particularmembrane will be depends in part on the composition of themembrane (e.g., the relative proportions of the variousphospholipids and sphingolipids) and on the rate of motionof the phosphate ester groups of the different membranecomponents. Although this dependence has been known forsome time, it still appears to be a subject of confusion. Forexample, in recent papers (25-2 7) the broad resonance thatis seen routinely with brain was attributed variously to (a)“membrane phospholipid,” (b) “immobile phosphates,” and(c) “mostly calcium phosphate in the skull and some mem-brane phospholipids.” The terms “immobile phosphates” and“calcium phosphate” are poorly used in this context. Trulyimmobile phosphates would give rise to a powder pattern(28), quite unlike the broad peak observed. Furthermore,not just any mineral form of calcium phosphate will giverise to such a peak. The protonated mineral forms that are

thought to be involved in deposition of apatite give rise toreadily detectable peaks, which are broader than that forapatite. Because the alp NMR spectrum of bone is virtuallyidentical to that of hydroxyapatite, the protonated mineralforms appear to be present in adult mammalian bone in onlyvery low concentrations (7, 18).

To clarifSr the uncertainties of these effects, we recordedhigh-resolution 31P NMR spectra (i.e., with the 20-mmprobe) of commercial hydroxyapatite and of freshly excised,but otherwise intact, canine skeletal muscle and marrow.Then, to estimate the contribution of soft tissue to the 31PNMR spectrum of the hand, we compared the total signalintensities from a hand and from freshly excised canineskeletal muscle in the 75-mm (i.d.) Helmholtz coil. The high-resolution spectrum of the hand was simulated by addingtogether the time-domain spectra of these various compo-nents with appropriate weighting factors to approximate themeasured contributions.

Magnet Design

Two-dimensional and three-dimensional finite elementanalysis (29, 30) was used to investigate the feasibility ofconstructing a permanent magnet for a 31P NMR spectrome-ter to be used with the extremities.

Results

In Vivo alp NMR Spectrometry

alp NMR spectra of the fingers (Figure 1) and of the wristand hand (Figure 2) were recorded with the 55-mm (i.d.) and75-mm (i.d.) Helmholtz coils, respectively. The 55-mm coilprovided adequate signal-to-noise with as little as oneacquisition. However, because of the cylindrical shape of theinner bore of our superconducting magnet, the receiver coilfor use with the wrist had to be large enough in diameterand far enough removed from the tune and match capacitorsto permit the hand to pass through. The close proximity ofthe magnet bore to the coil (i.e., 5 mm) required a rakeshield, which loaded the coil. This combination of con-straints caused the sensitivity of the 75-mm coil to besubstantially less than that of the 55-mm coil (e.g., comparethe number of acquisitions in Figures 1 and 2). This problemwith sensitivity was further exacerbated by the fact that thewrist did not ifil the volume within the receiver coil. Inaddition, a significant proportion of the sample (i.e., fingers,hand, or wrist) lies outside of the “high-resolution” region ofthe magnet when these larger receiver coils are used, and sothe narrow peaks from cytosolic metabolites are broadenedby inhomogeneity of the magnetic field. The appearance othe broader peaks from apatite and KPF6 is not affected, butthe relative contributions from cytosolic metabolites andbone mineral cannot be ascertained by direct visual inspec-tion of the spectra. These problems could be overcome byusing a magnet of appropriate geometry (see below).

Use of Surface Coils

alp NMR spectra of a phantom of hydroxyapatite anKPF6 were recorded with the 55-mm (i.d.) saddle-shapedsurface coil (Figure 3A-C). These spectra have a yesimilar appearance but a somewhat lower signal-to-n&ratio than those obtained with the full Helmholtz coil of thesame dimensions (Figure 3D).

Possible Sources of Interference

Phosphorus-containing metabolites dissolved in the cyto-sol of cells in skeletal muscle and marrow exhibit longer

100 0 -DM1CL SHIFT (pFrn

TIME (10.5 ms)

Fig.2. (A Frequency-domainand (C) time-domain invivo31PNMRspectraof (A) wristand (B, C) hand at the level of the palmAll spectra represent the average of 400 auisltions. Only the first 10.5 me of thetime-domain signal is shown. The wrist InAwas surroundedby a solid matrix ofKPF6 in a polyvinylchloride glove. Data from the hand can be compared directlywith the Invitrospectra in Fig.4; the time-domain signal in Cwas apodlzed with aline-broadening factor of only 20 Hz before Fourier transformation to yield thespectrum in B. Because a line-broadening factor of 20Hz willhave littleelect onthe apparent line widths of resonancee fromcytosolucmetabolltes, the apparentabsence of narrowpeaks probablyarises fromInhomogeneitlesin the magneticfield. A 400-Hz line-broadening factor was used to obtain the spectrum in AFIg. 1. In vivo 31P NMR spectra of the fingers of the left hand

surroundedby a solidmatrixof KPF6in a polyvinyl chloride gloveA-C representthe average of one, four, and eight acxiuisitlons,respectively. 0isthe average of eight ecquisitlons with the fingers of a second lndMual. Spectralitegration us represented by the step curves. A 200-Hz line-broadening fectorwes used Inapodizalionof the time-domainsignals

time-domain signals (Figure 4, B and C, left) and thus muchnarrower natural line widths in the frequency-domain spec-tra (Figure 4, B and C, right) than do either cell membranesor bone mineral deposits (7, 18, 31, 32). With a high-resolution magnet, these peaks are readily distinguishedfrom the broad resonance of bone in both the time-domainand frequency-domain spectra (Figure 4A-C). The chemicalshifts of the peaks from cytosolic metabolites are as follows(ref. 33 and references therein): 2,3-diphosphoglyceric acid(marrow) about +3 ppm; phosphocreatine (muscle) -3 ppm;ATP -5, -10, and -20 ppm; and inorganic phosphate about+3 ppm.

We find that the “high-resolution” 31P NMR spectrum ofabout 7 mL of freshly excised, but otherwise intact, dogskeletal muscle exhibits only sharp peaks from phosphozy-lated cytosolic metabolites; the baseline does not exhibit ahump that could be attributed to cell membranes (Figure48). This is true at both 18#{176}Cand 37#{176}C.The phosphorusresonance from membrane probably is too broad to bedetected with these experimental conditions, as was indicat-ed previously by experiments with bovine skeletal muscle(7). (Note that the viability of the muscle tissue used in thisexperiment is demonstrated by high-intensity peaks fromATP and phosphocreatine and that the entire sample re-sides in the “high-resolution” region of our superconductingmagnet.)

Some indication of a broad hump is apparent in thebaseline of the “high-resolution” alp NMR spectrum of

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marrow (Figure 4C). However, this broad resonance is notreadily distinguished from the peaks of ATP, and all of themarrow from the entire length of the femur of a large dog(i.e., a volume of about 6 mL) was needed to detect it. Thespectrum was essentially the same at 18#{176}Cand 37#{176}C.(Again note that the entire sample resides in the “high-resolution” region of our superconducting magnet.)

The in vivo ‘P NMR spectra of bone in fingers, hand, andwrist (Figures 1 and 2) are virtually identical to that ofstationary hydroxyapatite (Figures 3 and 4A). Variations inhomogeneity of the magnetic field over the volume of thesample (e.g., compare the spectra in Figure 4 with those inFigures 1-3) and apodization of the time-domain signal withline-broadening factors ranging from 20 to 400 Hz (e.g.,compare the spectra in Figures 2B and 4 with those inFigures 1, 2A, and 3) have little effect on the line shape ofthe frequency-domain 31P NMR spectrum of apatite andbone. However, the inhomogeneity of the magnetic field inour superconducting magnet over the volume of the fingers,hand, and wrist gives rise to substantial broadening of theinherently narrow resonances from cytosolic metabolites.Regardless of the line-broadening factor used, the 31P NMRspectrum of a freshly excised sample of canine skeletalmuscle that filled the 75-mm (i.d.) Helmholtz coil appearedas a featureless hump about as broad as the peak fromapatite (data not shown). Thus, the spectrum of hand inFigure 28 does not show the narrow resonances of cytosolicmetabolites in spite of the use of only a 20-Hz line-broaden-ing factor. The other time-domain signals recordedwith the 55-mm and 75-mm coils were apodized with line-broadening factors of 200 Hz and 400 Hz to yield thefrequency-domain spectra in Figures 1, 2A, and 3, because

CLINICALCHEMISTRY, Vol. 34, No. 7, 1988 1433

10 I -110-200C*CNIC SHIFT (PPtlI

FIg. 4. lime-domain (left) and frequency-domain(right) 31P NMRspectra of (A) commercial hydroxyapatite, ( freshly excised canIneskeletal muscle, and (C) freshly excised canine marrowThe first 10.5 ma of the time-domain signals is shown; each represents thesummation of 1200 acquisitions. The time-domainsignal in Owes produced byaddingtogetherthose of hydroxyapatite and skeletal muscle such that about 10%of the signal in the frequency-domain spectrum would arise from muscle. Eachtime-domain signal on the left was apodizedwith a 20-Hz line-broadening factoiand then Fourier transformed to obtain the frequency-domain signal on the right.This line-broadening factor is the same as that used to obtain the spectrum cihand in Fig. 2B. The entirevolumeof these in vitrosamples resided in the “highresolution” region of oursuperconductingmagnet, whereas the same is not truefor the in vivo samples. The 31P NMR spectrum of hydroxyapatite coincidescompletely with that of excised bone (7), so these spectra can be compareddirectlywith the Invlvo spectra in Figs. 1 and 2

Fig. 3. 31PNMR spectraofpartially mixed hydroxyapatite and KPF6inapolyethylene bagThe 55-mm diameter probe was used as a surface coil inA-.Cand as a Helmholtzcoil in 0. A and Dare the result of a singleacquisition,whereas Band C representthe average of four and 12 acquIsitions, respectively. Spectral Integration isrepresented by the step ourves. A 200-Hz line-broadening factor was used inapodization of the time-domain signals. The 31PNMAspectrum of hydroxyapatitecoincides completely with that of excised bone (7), so these spectra can becompared directly with the in vivo spectra in Figs. 1 and 2

this provided a better signal-to-noise but did not affect theresolution.

We estimate on the basis of comparison of the total signalintensity from the hand in Figure 28 with that from freshlyexcised canine skeletal muscle in the same 75-mm Helm-holtz coil that <15% of the signal intensity from 31P in thehand arises from soft tissue. On the basis of anatomicalconsiderations the contribution from soft tissue is expectedto be substantially less in the wrist. We do not expect thespectral contribution from marrow to be very great, becauseall of the marrow from the entire length of the femur of alarge dog was needed when we sought to detect its 31P NMRspectrum. Figure 4D shows a simulation of what the spec-trum of the hand is expected to look like under high-resolution conditions.

Magnet Design

Several different permanent-magnet designs were aia-lyzed by three-dimensional finite element analysis with thegoal of minimizing size and weight. Our current designwould have overall dimensions of 30.5 cm high by 43.2 cmwide by 46 cm deep. Total weight would be 280 kg, 80 kg ofwhich arises from the magnet material. The pole pieces are

U.5 cm in width and 23 cm long and consist of a neodymi-

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DNICL. 54IT IPPNI

urn-iron--boron magnet material 5 cm in thickness. Eachpole piece has a steel face plate, 1.25 cm in thickness, foiimprovement of field homogeneity. The predicted magneticfield at the center of the magnet, without shimming, is 0.40ET (i.e., 4060 G); thus, 31P would resonate at a frequency ol6.9 MHz. A mock-up of the magnet is shown in Figure 5.

Because we want to avoid electrical shimming of thEmagnet, to minimize its cost of operation, the model oIFigure 5 was also analyzed by two-dimensional finite ele-ment analysis to demonstrate the effectiveness of a mechanical ring shim. Two-dimensional finite element analysis wused for computation instead of three-dimensional finielement analysis because elements of much smaller sizebe used with a given size of computer memory. Figureshows flux plots and homogeneity graphs for the magnewith (A) and without (B) a ring shim that is 0.63 cm thicand 1.25 cm wide. The homogeneity factor for the unshimmed magnet is ±484 ppm over a sphere 5 cmdiameter, whereas the much better value of ± 16 ppmobtained with the shimmed magnet. Thus, use of a singlmechanical ring shim effects a 30-fold improvementhomogeneity. Multiple shims should yield even better hmogeneity, and they will be utilized if required.

DiscussIonMeasurement of Bone Mineral Weight by 31P NMR

The weight (i.e., mass) of apatite can be measured directlyfrom the peak area of its 31P NMR spectrum (7,1 7-19). Th#{128}

1434 CLINICALCHEMISTRY, Vol. 34, No. 7, 1988

CLINICAL CHEMISTRY, Vol. 34, No. 7, 1988 1435

relationship between the mass of apatite and the normalizedpeak area (i.e., relative to an internal standard) is linear.The assay also can be performed in vivo, as demonstratedabove, but care must be taken to account for 31P NMRsignals from sources other than apatite.

Although the protonated mineral forms give rise to readi-ly detectable 31P resonances that are broader than that ofapatite, virtually undetectable concentrations of them arepresent in adult bone (7). Thus, interfering signals are morelikely to arise from soft tissues such as skeletal muscle andmarrow.

Soft tissues can give rise to two general types of 31Presonances. The phosphorylated metabolites dissolved in thecytosol give rise to narrow peaks, whereas the phosphateesters in membranes give rise to relatively broad reso-nances. The apparent line-widths of these latter resonancesare determined by the composition of the membrane and therates of motion of its components, and thus they depend onthe identity of the tissue.

The work presented here with freshly excised canineskeletal muscle, in which the viability of the tissue wasdemonstrated by high-intensity peaks from ATP and phos-phocreatine, confirmed our earlier finding (7) that the 31PNMR spectrum of skeletal muscle does not exhibit a broadresonance under our instrumental conditions (Figure 4B),whether at 18#{176}Cor 37 #{176}C.Because claims that skeletalmuscle exhibits a broad resonance (i.e., similar to thatobserved with brain tissue) arose when investigators startedto put whole animals into spectrometers, we would not besurprised if the early in vivo studies had been performedwith some bone apatite in the active volume of the receivercoil.

There is some indication of a broad resonance fromphospholipids in marrow (Figure 4C). However, all of themarrow that could be surgically removed from the entirelength of the femur of a large dog (i.e., a volume of about 6mL) was required in order to detect its 31P NMR spectrum.This is much more marrow than is expected to reside in thewrist, yet the number of pulses needed to obtain a usablesignal-to-noise ratio with all this marrow was severalfoldgreater than that needed with an equal volume of bone. A‘broad resonance from marrow, if it exists, is not expected tointerfere significantly with the analysis of apatite. A recent

Fig. 5. Mock-up of a permanent magnet that could be used for in vivoanalysis of bone mineral density of the extremities by 31 NMRspectromeftyThe x- and zexes indicate the upper right quadrant of the magnet that isrepresented in the flux plots in Fig. 6

report (31) of high-field ‘P NMR spectra of human marrowdid not indicate the presence of a broad peak.

Although our experiments with canine tissue do notpermit an exact indication of the contribution of soft tissue(e.g., muscle and marrow) to the 31P NMR spectrum ofhuman wrist or heel, they do suggest that interferingresonances should arise primarily from dissolved cytosolicmetabolites (i.e., primarily phosphocreatine, ATP, and 2,3-diphosphoglyceric acid). These yield much narrower peaksthan does apatite under high-resolution conditions, and sotheir spectral contribution could be ascertained by simplevisual inspection or computed deconvolution of the frequen-cy-domain spectra (e.g., compare the spectra in Figure 4,right). The much shorter time-domain signal of apatitecompared with those of the cytosolic metabolites (Figure 4,left) also would permit the use of maximum entropy calcula-tions (27). In any case, the contributions from soft tissues inanatomical sites such as the wrist and heel are expected tobe relatively small. More will be said of this below.

Assay Requirements

The obvious question then arises of how bone loss mightbe monitored by 31P NMR. The answer depends in part onthe manner in which the loss occurs (4). The Office of HealthTechnology Assessment reported (4):

Two broad hypotheses have been proposed for the pathogenesis ofprimary osteoporosis. The first is that skeletal mass achieved atmaturity is the critical determinant for development of clinicalosteoporosis later in life. An underlying assumption of this theory isthat the rate of bone loss is not significantly increased in personsdestined to become osteoporotic. . . . Bone loss is considered a nor-mal manifestation of the aging process. The second theory ofpathogenesis is that those determined to become osteoporotic havesignificantly increased rates of bone loss by comparison with age-and sex-matched controls. The factors responsible for increasedrates of bone loss in affected individuals may be the same as thoseoperative during the aging process but are exaggerated. These twotheories are not mutually exclusive, and presently it is not possibleto state which one of these variables (i.e., bone mass at maturity oraccelerated bone loss in later life) is more important.

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Fig. 6. Homogeneity graphs (left) and flux plots (i.e., magnetic fieldlines) (right) in the transverse midplane of the permanent magnet (A)wIth a 0.63 cm thickby 1.25 cm wide mechanical ring shim, and (B)without mechanical ring shimsBecause of symmetry and mirroring,only the upper right quadrant of each fluxplot is shown. See Fig. 5 for the orientation of this quadrantand the relativelocations of the magneticpole piece, steel face plate, fluxreturn, and airgap of themagnet. During the measurement, the wrist or heel would be centered atcoordinates x = 0, z = 0. The homogeneity plots (Jell)indicate a 30-foldlnrovement with the shim

0

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If accelerated bone loss in later life is found to play animportant role in the etiology of osteoporosis, then serialtests will have to be run on individuals to find metabolicchanges (34, 35), as is done with the clinical monitoring ofother progressive diseases. A primary requirement of suchserial tests is that the accuracy and precision of the assaymust be sufficiently good to detect small changes that occurover time. The “relative” weight of apatite at a defined locusin a given bone structure would likely be obtained withbetter accuracy and precision than would calculated valuesof density that require separate measurement of (a) wqightof apatite and (b) volume of bone. The error associated withmaking two measurements, instead of just one, would beavoided.

If”screening” with a single measurement in midlife (i.e.,one measurement of bone mass at maturity) becomes accept-ed as a valid indicator of possible risk in the patient’s laterlife, however, it generally is assumed that determination ofdensity will be needed to permit a correlation betweenindividuals and a reference population. Therefore, we inves-tigated alternative approaches that might be used to mea-sure mineral density by 31P NMR.

Measurement of Bone Mineral Density by NMR-CoiIDesign

A measure of in vivo mineral density could, in theory, beobtained by a combination of NMR spectroscopy and imag-ing (7). The mass of bone mineral within the receiver coilwould be measured by 31P NMR spectroscopy, and thevolume of bone would be determined by proton magneticresonance imaging via the same coil. The density would becalculated from the mineral mass and bone volume. Becauseboth the mass of bone mineral and the volume of the bonewould be measured without moving the patient’s extremity,errors in the calculated bone mineral density could beminimized. Furthermore, the exact orientation of the wristor heel within the instrument would be known. However,the resolution of the image obtained might not be sufficientto provide the needed accuracy and precision of the mea-sured volume and, thus, of the calculated mineral density.An instrument with imaging capabilities would be lessroutine to operate and substantially more costly to buildthan a simple spectrometer. A “high-resolution MR imag-ing” system for use with the wrist (36) and “bone-marrowimaging” of the spine (37) have been described.

Alternatively, one could define the volume to be analyzedwithin a given structure and then measure the amount ofmineral within that volume. For example, the processusstyloideus of the ulna has a rather high proportion oftrabecular bone with a relatively thin cortical layer, and itis covered primarily by just a layer of skin in thin women(i.e., those at highest risk of developing osteoporosis). Asurface coil of appropriate dimensions could be placed suchthat the signal from material in the active volume of the coilwould arise almost exclusively from bone mineral ratherthan soft tissue. The volume measured and the response ofthe apatite within that volume to the “observe” pulse wouldbe determined by the dimensions of the coil. This approachwould provide a measure of density in a defined volume withminimal contribution from phosphorus in soft tissue. Areceiver coil of appropriate shape might also be used in asimilar fashion with the posterior and lateral aspects of thecalcaneus, and possibly also with the trochanter.

Our demonstration of the use of a surface coil to measureapatite (Figure 3) indicates the technical feasibility of such

1436 CLINICALCHEMISTRY, Vol. 34, No. 7, 1988

an approach. However, one must keep in mind the inherentcharacteristic of surface coils that sensitivity to resonancesignals is not constant over the volume of the sample. Inaddition, the ifip angle produced by a given pulse and, thus,the magnitude of the signal intensity obtained varies like-wise as a function of the distance from a surface coil. Signalintensities will depend on placement of the surface coil andreference standard relative to each other and to the boneunder investigation. This is quite different from Helmholtzand solenoid coils, which produce more uniform fields withintheir active volumes. However, reproducible placement ofthe surface coil over a given bone structure and the use ofcalibration curves will permit accurate and precise mea-surement of both relative” weight and density of apatite.

Magnet Design

The spectra in Figure 4 indicate that the 31P resonancesfrom apatite and soft tissues can be distinguished quitereadily with a “high-resolution” magnet. The contributionsfrom naturally “broad” and “narrow” resonances can bedistinguished in either the time-domain or frequency-do-main spectra.

As indicated above, however, the superconducting magnetavailable for our use does not have a sufficiently large borediameter to permit routine work with human extremities.Because the high-resolution region of the magnet is smallerthan a hand or wrist, the resonances from cytosolic metabo-lites in soft tissue appear very broad (e.g., Figure 2B).Furthermore, the close proximity of the magnet bore to thereceiver coils for in vivo use seriously compromises sensitiv-ity.

Large-bore magnets that would be capable of reasonablyhigh-resolution spectrometry are available in magnetic res-onance imagers. However, such instruments are not readilyavailable for research purposes. Modification of these instru-ments to perform 31P NMR spectroscopy of solid sampleswould be somewhat expensive, and their use for clinicaldetection of osteoporosis would not be cost effective. There-fore, we have designed a magnet that would be sufficientlyinexpensive to build and operate for our research.

The O.4-T magnet we have designed fits on a desk top(Figure 5). With a single mechanical ring shim, the fieldexhibits a homogeneity of better than ± 16 ppm over asphere, located at the center of the air gap, that is 5 cm indiameter. This suffices to record the broad resonance ofapatite but is not adequate to resolve the inherently narrowpeaks of cytosolic metabolites. Better resolution can beachieved with additional ring shims or electrical shim coils,or both, but these would add to the cost of construction.Therefore, we have considered the feasibility of using aninexpensive, medium-resolution version of the magnet inFigure 5 for analysis of the extremities.

Two main considerations are involved. First is the likelycontribution from soft tissue to the change over time in the31P signal intensity. Second is the possibility of makingreceiver coils of the appropriate geometry to minimizespectral contributions from soft tissues.

We have shown that soft tissue contributes about 15% othe signal intensity of the 31P NMR spectrum of the humanhand. The other 85% arises from bone. By way of compari-son, the wrist has a much smaller proportion of soft tissueand, thus, the spectral contribution will be proportionatelysmaller. If we assume that soft tissue contributes 5% of thespectral intensity of wrist, then the volume of soft tissuewould have to change at an annual rate of 20% to produce

CLINICALCHEMISTRY,Vol. 34, No. 7, 1988 1437

an apparent change of 1% per year in the intensity of the 31PNMR spectrum. Such a large change in soft tissue massshould be readily apparent to the physician on the basis ofappearance and weight of the patient. Furthermore, such alarge change in soft tissue could be detected in the extrem-ities on the basis of the absolute intensity of its ‘H NMRspectrum (38). The receiver coil of the spectrometer could bedouble tuned-and-matched to the resonance frequencies ofboth ‘H and 31P nuclei, and the proton signal would berecorded at the same time that the phosphorus spectrumwas obtained. The area under the peak in this broad-line 1Hspectrum would provide a measure of the amount of tissuein the receiver coil.

The contribution of soft tissue to 31P NMR spectra of theextremities could be reduced further by appropriate probedesign. The experimental results presented above indicatethat both surface coils and Helmholtz pairs could be de-signed such that a minimum of soft tissue would be presentin the active volume. These could be placed on or around, forexample, the processus styloideus of the radius or ulna, orthe posterior or lateral aspects of the calcaneus. The large,accessible air gap of the magnet in Figure 5 was chosen tofacilitate the use of such coil designs.

Conclusions

Bone is a complex structural composite of a fibrousorganic matrix and an inorganic filler. The inorganic compo-nent, primarily apatite, makes up about 45% of the wetweight of mature cortical bone, whereas the organic sub-stances, mainly collagen, account for about 35%. The re-maining 20% is water (39). Mechanical strength (4, 5, 34,35, 39-47) is determined by a complex interplay among (a)the amount of mineralized tissue present in the bone, (b) theextent of mineralization of the organic matrix, (c) thechemical composition and extent of crosslinking of theorganic matrix, (d) architectural aspects of the bone, (e)fatigue from repeated loading of the bone, (/) the rate anddirection of deformation of the bone during trauma, and (g)possibly other factors. Thus, it is a gross oversimplificationto attempt a predictive correlation of fracture risk based ononly one parameter, namely density (4,5,10,34,35,39-47).

One must keep in mind that proper deposition of collagenis a prerequisite of mineralization and, thus, a decrease inbone mineralization during later life could arise in part fromlimitations in the ability topss this protein. The activi-ties of the enzymes involved in synthesis and utilization ofcollagen decrease with age and are affected by hormoneconcentrations and other factors (e.g., refs. 48-50 and refer-ences therein). This could also result in a decreased rate ofrepair of microfractures caused by normal use.

Whereas some have considered a fracture to be “essentialto the diagnosis of osteoporosis-or even diagnostic of it,”Nordin (35) has stated that “this practice is not onlyundesirable but positively misleading.” It has come to berealized that fracture risk is a continuous variable thatincreases as bone density declines, but the relation is not asimple linear one. Thus, if the amount of mineral in a boneis reduced, “osteoporosis must be present (discounting therare case of osteomalacia), and the main problem is to definethe standard against which this reduction should be mea-sured” (35). Although support has been given to the notionof administering estrogen, as replacement therapy, to manyif not most postmenopausal women without prior or concom-itant bone-mineral analysis (51, 52), Wasnich et al. (53)have calculated that indiscriminate administration of estro-

gen would not be cost effective. Notelovit.z (34) has written,“When one considers the physiology of bone formation andloss, it soon becomes apparent that there is much more topostmenopausal osteoporosis than estrogen replacement.The rate of postmenopausal bone loss needs to be monitoredand modulated. Central to the entire issue is the fact thatbone is a living organ and needs to be treated as such.”

In vivo 31P NMR spectrometry permits the homeostasis ofapatite to be measured in living bone. A measure of relativeweight loss and of density of mineral should be possible.Correction can be made for the spectral contributions fromsoft tissues when high-resolution instrumentation is usedor, in the case of the extremities, practicably minimized byappropriate choice of receiver coils.

Use of 31P NMR would not be limited solely to detection ofosteoporosis in the elderly. Difficulties with mineral metabo-lism in childhood and during space travel also could bemonitored. Smaller receiver coils for use with children, andalternative magnet and coil designs for assessing the rapidloss of mineral from load-bearing bones and atrophy ofskeletal muscle during space travel (ref. 54 and referencestherein) are possible.

This work was made possible by The Retirement ResearchFoundation and S. C. Johnson & Son, Inc. Magnet design was doneas part of J. H. Battocletti’s Merit Review funding at the MilwaukeeVAMC. The assistance of Daniel P. Collins, William K. Genthe,Richard E. Halbach, and Donald H. Westermann in reviewing themanuscript is greatly appreciated.

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