Ratio of Late to Early Radionuclide Uptake: A Method for Distinguishing Osteoporosis from Osteomalacia in Animal Models 1
Nuclear Medicine
John S. Wilson, M.D.,2 Harry K. Genant, M.D., Robert S. Hattner, M.D., and Paul B. Hoffer, M.D. The ratio of late to early uptake of several radionuclides was examined as a method for distinguishing states of abnormal bone metabolism. Nutritional osteoporosis (secondary hyperparathyroidism) and osteomalacia were produced in young rats and compared to a control group. The ratio of early (3-6 hrs.) to late (4-6 days) uptake of barium-131, nitrate, indium-111 EDTMP, and lead-203 were studied, as was that of strontium-8S chloride, a calcium analogue. Ratios of late to early uptake were found to distinguish osteomalacia from osteoporosis in the models when strontium-8S or barium-131 were used. Barium-131 may be a clinically useful alternative to strontium-8S in the evaluation of metabolic bone disease due to its shorter half-life and lower radiation dose. INDEX TERMS: Osteomalacia 4 [8] .570 • Osteoporosis 4 [8] .560 • Radionuclides. comparative studies • (Skeletal system, other special miscellaneous procedure, 4 [8].1299)
Radiology 126: 185-191, January 1978
usefulness of conventional radionuclide T techniques for the detection, differentiation, and
available terminal phosphorous groups react by chemical absorption to the hydroxyapatite crystal in bone, rather than by chemical substitution for calcium ions (9, 23). Recently, phosphate binding to collagen moieties has been proposed as a possible additional mechanism (25, 31). The short half-life of 99mTc may be an undesirable feature in the study of metabolic bone diseases because early radionuclide uptake (on the order of hours) is probably dependent on factors other than the rate of osteogenesis, such as blood flow, surface binding, and capillary permeability (12,13,36,39,42,43). In the past, studies of bone metabolism have focused on the late uptake of radionuclides to avoid these variable early factors (2,8,28). Autoradiographic studies using calcium-45 (45Ca) demonstrated that several days after initial labeling of bony surfaces intimately in contact with circulating fluids, regions of active bone formation tend to retain the radionuclide, and nonaccreting regions lose the radionuclide (32). An additional factor affecting late radionuclide uptake is a gradual, diffuse labeling of inactive bone not in intimate contact with circulating fluids, known as the diffuse component (8,32). In young growing animals, osteogenesis is the major determinant of late radionuclide retention, whereas the diffuse component is only a minor factor (8).
HE
quantitation of metabolic bone diseases is controversial. Prior to the introduction of the technetium-99 (99mTc) poIyphosphate analogues, extensively used in studies of bone physiology (8, 18, 19, 22, 28, 32, 39, 43) and as agents for external probe-counting (2, 3, 15) or imaging (34, 38) of various skeletal disorders were calcium-47 (47Ca), strontium-85 (85Sr), and fluorine-18 8F). Their use in the clinical evaluation of metabolic bone diseases, however, has been sporadic, and results are varied (2, 15, 31). Furthermore, difficulties encountered in the dosimetry and imaging of these agents have shown them to be currently impractical and undesirable for clinical use (13, 38). The 99mTC poly phosphate analogue bone seekers have recently been used in the evaluation of several metabolic bone disorders (31). Despite nearly ideal dosimetric and imaging characteristics, these compounds suffer two major limitations in this setting: they are not calcium analogues, and the 6-hr. half-life of 99 mTc precludes late imaging (on the order of days). These are important considerations, because in the scintigraphic study of metabolic bone diseases it may be desirable that radionuclide uptake reflect the rate of osteogenesis, a prime factor in such disorders as osteomalacia (5, 17), hyperparathyroidism (17,24,29,30), and hyperthyroidism (1,29). True calcium analogues such as the alkaline earth elements Sr and Ba react by chemical substitution for calcium ions in the hydroxyapatite crystal (23), and their uptake by bone may therefore reflect calcium metabolism (4, 7, 23). The mechanism of phosphate binding to bone, however, is controversial. The most widely accepted hypothesis is that
e
EXPERIMENTAL DESIGN
Our objective was to establish an in vivo method of distinguishing altered rates of bone metabolism (osteogenesis) by using radionuclides that are bone-seeking and that have a half-life sufficient to permit late imaging. By
1 From the Department of Radiology and Section of Nuclear Medicine, University of California School of Medicine, San Francisco. Presented at the Sixty-second Scientific Assembly and Annual Meeting of the Radiological Society of North America, Chicago, III., Nov. 14-19, 1976. 2 Supported in part by NIH Training Grant GM 01272 from the National Institute of General Medical Sciences. ss
185
JOHN S. WILSON AND OTHERS
186
comparing the change in radionuclide uptake over time (late/early uptake ratio) in animal models of two metabolic bone diseases, we hoped to obtain a scintigraphic measure of skeletal metabolic activity that would have clinical utility. In vitro well-counting and tetracycline-labeling techniques were used to substantiate our in vivo results.
TABlE
I:
January
1978
PHYSICAL PROPERTIES OF RADIONUCLIDES STUDIED
Half-life (days)
Radionuclide 85Sr chloride 1318a nitrate lllln EDTMP 203Pb acetate/nitrate
65 11.7 2.82 2.17
Gamma Energy Imaged (keV)
Specific Activity
514 496 173 279
3-30 mCi/mg 3.78 mCi/mg Carrier free Carrier free
MATERIALS AND METHODS
Animal preparations: Approximately 100 threeweek-old Holtzman rats, initially weighing 40 to 50 g, were housed in pairs in a dark room throughout the study. Three models of bone metabolism were established by means of dietary manipulation. Osteomalacia, a state of decreased bone formation (5, 17), was produced in rats by feeding them a diet which was free of vitamin D, and which contained 75.5% ground corn meal, 20% wheat gluten, 3.5 % CaC0 3, and 1% NaCI (13, 16). Nutritional osteoporosis was produced in rats by feeding them the same diet, but with a low level of calcium and a high level of phosphate (0.018% CaC0 3, 1.5% equimolar KH2HOP 4), as described by Steenbock and Herting (40). Supplementary vitamin D (20 IU/wk) was administered orally to prevent the concomitant development of rickets; this form of osteoporosis is considered to result most probably from secondary hyperparathyroidism (16, 20, 21, 33, 35), a state of increased bone formation and bone resorption, with the latter being quantitatively greater (17,20,21,24,29,30). Previous studies have shown that in young rats fed a similar calcium-deficient diet, hypertrophied osteocytes develop and resorption cavities expand (33). In addition, Sevastikoglou (35) demonstrated osteoporosis and hypertrophy of parathyroid glands in young rats on a similar dietary regimen, indicating secondary hyperparathyroidism. A control group (normal state of bone formation) was created by feeding rats the rachitogenic diet mentioned above but with oral vitamin D supplement.
Fig. 1. Quantitative in vivo radionuclide scan with rectangular region of interest outlining the rat knee.
Radionuclide selection: The skeletal uptake of four bone-seeking radionuclides was studied; the physical characteristics of each agent are given in TABLE I. Strontium-85 (8SSr) (New England Nuclear, Boston), a known calcium analogue, was chosen as a standard of comparison for the other agents tested. The late uptake of 85Sr has been considered in large part a manifestation of osteogenesis or mineral accretion (2,7,29). However, the long half-life and high gamma energy of 85Sr have restricted its use in humans to very low dosages (13, 37, 38). Barium-131 (131Ba) nitrate (ICN Pharmaceuticals, Cleveland) was selected because of its physical characteristics and evidence that it is handled by the skeleton in a manner similar to 47Ca(4) and 85Sr (6, 26). An alkaline earth radionuclide, 131Ba with a specific activity of 3.5 to 4 mCi/mg was produced commercially by means of neutron irradiation of enriched 130Ba. Although its decay scheme is complex, resulting in 4 primary gamma-ray emissions, the 11.7-day half-life of 131Ba makes it suitable for late imaging. In addition, previous dosimetric calculations indicate that the radiation dose absorbed by the patient is approximately one-fourth that of 85Sr (37). Indium-111 111n) ethylenediaminetetra phosphonic acid (EDTMP) (Medi Physics, Emeryville, Calif.) was used as a polyfunctional phosphate substitute for the more conventional but short-lived 99mTc compounds. Previous reports indicate active skeletal localization that produces high quality scintigraphic images (41). The 2.8-day half-life of 1111n EDTMP permits comparison of the late uptake of this non-calcium analogue with that of the calcium analogues 85S r and 131Ba. Lead-203 (203Pb) (New England Nuclear) acetate and nitrate were selected because of evidence of high skeletal affinity and excellent imaging characteristics (27). A 2.2-day half-life allows late imaging. Dose calibrations: Radioactivity in one-milliliter syrin~e~ containing the selected radionuclide (ranging in actlvlty from 250 to 750 ,uCi) was counted in an appropriately set dose calibrator. Approximately 4 or 5 weeks after dietary manipulation of the rats was begun, between 200 and 650 ,uCi of the selected radionuclide was injected into the tail veins of lightly anesthetized rats. Following the injection, radioactivity in the syringes was recounted in the dose calibrator to determine the injected radioactivity. The selected radionuclides within the syringes were also counted with a gamma camera before and after the radionuclide injection under conditions identical to those used for in vivo scanning; this permitted calculation of counts/ ,uCi of radionuclide administered.
e
Vol. 126
RATIO OF LATE TO EARLY RADIONUCLIDE UPTAKE
In vivo scanning: Early (3-6 hr) and late (4-6 day) quantitative in vivo imaging was performed using a Pho Gamma scintillation camera (pho/Gamma III, Searle Radiographies, Des Plaines, 111.) with a 4-mm, pin-hole collimator interfaced with a digital computer (DEC RT 11/40, Digital Equipment, Maynard, Mass.). Lightly anesthetized rats were placed in a lead immobilization device that effectively shielded all body parts except the knee region (the distal femur, proximal tibia, and surrounding soft tissues), which was centered 5 em (2 in.) beneath the pin-hole collimator. Two-minute scans of each knee were obtained and the data stored on magnetic discs for analysis at a later time. In vitro counting: A few rats from each group were sacrificed at the end of early scanning and others at the end of late scanning for purposes of in vitro counting of radioactivity. In vitro corroboration of in vivo scanning data was made for all of the rats in the In and Sr groups by means of well-counting. Each distal femur (distal onefourth) was sharply dissected from surrounding soft tissues, dehydrated in acetone, and placed in a counting well for a one-minute determination of radioactivity. Tetracycline labeling: Tetracyclines have been used extensively as markers of new bone formation (5, 10,28, 32). After administration, tetracycline is deposited at sites of active bone formation, resulting in fluorescent bands easily identified under ultraviolet light microscopy. When 2 doses are separated by several days, 2 concentric bands are seen. The degree of separation is generally considered a function of the amount of bone formation which has occurred in the interval (5, 10, 17), although some feel this is controversial in conditions where bone formation and resorption are uncoupled (29). In this study, double tetracycline labels were given to provide a qualitative estimate of new bone formation. A few rats from each group were
187
Nuclear Medicine
given two intraperitoneal doses (8 days apart) of 4 mg demeclocycline hydrochloride. Femora and tibiae were then dissected free of soft tissues and fixed in 50 % alcohol. A high speed, rotary, diamond saw was used to slice unembedded diaphyseal bone. These sections were stained with 0.10% basic fuchsin in 50% ethanol to enhance visualization of osteoid. A final thickness of 50 to 75 J.l was obtained by hand-grinding specimens using the technique of Frost (11). Specimens were viewed under a fluorescent light microscope to assess tetracycline labeling of areas of new bone formation (osteogenesis) (10). Fine detail radiography: Throughout the experiment, fine detail radiography (14) was employed to assess the status of bone disease in the various models. The technique involved the use of Kodak Type M industrial film, 500 milliampere seconds (mAs), 45 kilovolts (kV) and a focus film distance (FFD) of 40 inches. Optical magnification was used to visualize the minute bony architecture of rat femora, pelves, and vertebrae. Data analysis: Using the storage oscilloscope display of the computer, a rectangular region of interest was chosen to quantify early and late skeletal uptake of radionuclide in the knee region (Fig. 2). The early and late uptake was calculated for each rat from decay-corrected counts; the counts ranged from 3,000 to 30,000 counts/ 2-min. scan. The mean per cent of radioactivity in the knee region was determined by averaging decay-corrected counts for right and left knee and dividing by the administered radioactivity expressed in counts (13). In addition, the ratio of mean per cent late uptake to early uptake (late/early uptake ratio) was determined to express the change in uptake over time and to allow individual rats to serve as their own control. The Student's t-test was applied to determine the sta-
Fig. 2. Fine detail radiographs of rat knee, 3 to 4 weeks after dietary manipulation. A. mineralized and free of rachitic changes. B. Rachitic rat shows epiphyseal widening and fraying of metaphysis. C. Osteoporotic rat demonstrates demineralization and loss of trabeculae.
Bone of control rat appears well
JOHN S. WILSON AND OTHERS
188
tistical significance of the results in comparing rachitic and osteoporotic -to control rats. The correlation between in vivo and in vitro data was assessed by regression analysis. RESULTS
Fine detail radiographs obtained 3 to 4 weeks after dietary manipulation demonstrated the expected skeletal changes in the 3 groups of rats (Fig. 2). Typical radiographic signs of advanced rickets, including metaphyseal fraying and epiphyseal widening, were found in the osteomalacic group. Diffuse osteopenia as evidenced by cortical thinning and dissolution of trabeculae was demonstrated in the osteoporotic group. The skeletal structures of the control rats appeared well mineralized and free of rachitic changes. Tetracycline labeling studies (Fig. 3) revealed a depressed state of new bone formation in the osteomalacic rats by demonstrating vast osteoid formation and scant areas of tetracycline labeling. Conversely, the osteoporotic rats demonstrated numerous Howship's lacunae and resorptive spaces, as well as plentiful bright tetracycline bands. Although some widely separated double bands were observed, they were not as distinct as those in control rats, which showed relatively normal cortical architecture and the expected well-defined tetracycline bands. In addition, the osteoporotic rats showed more sites of osteogenesis than the control rats. The results of quantitative in vivo imaging are given in TABLE II. The amount of early uptake of 85Sr was similar among the three groups, although a small difference was seen with the osteoporotic group. Late imaging, however, demonstrated a striking divergence in the mean per cent uptake of 85Sr. Control rats exhibited 1.85 0/0, rachitic rats 0.83 %, and osteoporotic rats 4.15 % . These results parallel those for the rates of osteogenesis among the 3
January 1978
groups as qualitatively estimated by using tetracycline labeling. Likewise, the ratio of late to early uptake of 85Sr differed significantly among the three groups. Small but statistically significant differences between groups were observed in the early uptake of 1318a, but only the late uptake values and the late/early ratios permitted ready differentiation between the control, rachitic, and osteoporotic rats. The rachitic rats retained the least amount of 1318a and the osteoporotic rats retained the most. This pattern of uptake is very similar to that observed in the rats injected with 85Sr. The three groups of rats injected with 1111n EDTMPfailed to demonstrate, in either early or late uptake, the divergent pattern seen using 85Sr or 1318a. Although small variations in uptake were noted, no major difference was observed which would readily allow a distinction to be made between the 3 groups. Likewise, the early or late uptake of 203Pb nitrate and acetate did not permit differentiation between the models tested, although small, isolated disparities existed (i.e., low early uptake in the rachitic and osteoporotic rats, compared to the control rats). These quantitative in vivo data were well substantiated by data from in vitro well-counting. The correlation coefficient obtained by comparing the in vitro and in vivo 85Sr and 1111n data was 0.93. The regression analysis is displayed in Figure 4. DISCUSSION
The double-label tetracycline studies support the validity of the models tested. Control rats show normal cortical architecture and uniform double labels. In the rachitic model, however, a paucity of tetracycline labeling with no double bands is observed. This implies very little bone formation during the 8-day period between labels, and is to be expected in rickets, a condition in which defects in
Fig. 3. Tetracycline studies with 8-day separation of labels. A. Control rat shows relatively normal cortical architecture and the expected double tetracycline labeling. B. Rachitic rat demonstrates vast osteoid formation with scant tetracycline labeling and no double labeling. C. Osteoporotic rat shows numerous bright tetracycline double labelings, indicating increased osteogenesis.
Vol.
126
RATIO OF LATE TO
TABLE II:
EARLY
85Sr chloride Early Late Late/early 111Ba nitrate Early Late Late/early 1111n EDTMP Early Late Late/early 203Pbacetate/ nitrate Early Late Late/early
Nuclear Medicine
PERCENT SKELETAL UPTAKE OF RADIONUCLIDE IN THEKNEEREGION OF THREE MODELSOF RATS (IN VIVO) Control
Agent
189
RADIONUCLIDE UPTAKE
No. Rats
Mean
Rachitic
±
S.D.
No. Rats
Mean
Osteoporotic
±
S.D.
No. Rats
Mean
±
S.D.
5 4 4
4.01 ± 0.58 1.85 ± 0.07 0.49 ± 0.05
6 4 4
4.31 ± 0.54 0.83* ± 0.10 0.20" ± 0.04
6 4 4
5.05 t ± 0.57 4.15* ± 0.44 0.86" ± 0.04
7 7 7
2.69 ± 0.24 1.46 ± 0.29 0.56 ± 0.07
6 6 6
2.01" ± 0.12 0.60" ± 0.12 0.30* ± 0.04
6 6 6
3.23 ± 0.18 2.32 ± 0.31 O.72 t ± 0.12
7 4 4
2.93 ± 0.40 2.52 ± 0.34 0.89 ± 0.04
7 4 4
3.29 ± 0.48 2.55 ± 0.34 0.78* ± 0.04
7 4 4
2.48~
10
4.05 ± 0.46 4.81 ± 0.61 1.14 ± 0.13
6 3 3
3.05* ± 0.22 2.84* ± 1.05 0.95 ± 0.35
4 3 3
3.10~
6
6
± 0.28 2.22 ± 0.26 0.88 ± 0.04 ± 0.93 3.79 ± 0.12 1.22 ± 0.08
* P < 0.01. r P < 0.02. ~ P < 0.05.
osteogenesis have been well documented (5, 16, 17). These findings are in agreement with the tetracycline studies of Baylink, which showed a 20 % decrease in total osteoblastic matrix formation rate and a 50 % decrease in osteoid mineralization rate in rachitic rats (5). Osteoporotic rats demonstrate extensive heavy tetracycline labeling accompanied by increased resorptive spaces and a general disarray of normal cortical architecture. Widely separated double bands, although present, are somewhat difficult to identify. This could be due to a state of rapid bone formation coupled with rapid resorption primarily in regions of more mature bone. The initial tetracycline label may undergo resorption. This hypothesis is consistent with other studies of secondary hyperparathyroidism, where both increased bone formation and resorption have been observed (17,20,21,24,29,30). If, on the other hand, the rates of bone formation in our model were not increased, heavy tetracycline labeling would not be expected. Therefore, on the basis of tetracycline studies, rachitic rats appear to have decreased bone formation and osteoporotics increased bone formation, relative to controls. In evaluating the in vivo uptake data displayed in TABLE II, a discussion of the 85Sr data will be undertaken first, since the uptake of strontium, a known calcium analogue, is considered a reliable indicator of bone metabolism. It is apparent that control, rachitic, and osteoporotic rats injected with 85Sr demonstrate comparable early uptake, ranging between 4-5 % in the knee region. This similarity may be due to the multiplicity of factors thought to playa role in early radionuclide uptake several hours after administration. These include blood flow, capillary permeability, extraction fraction, and to some extent the rate of bone formation (12, 13,36,39,42,43). Consequently, it is not surprising that a comparison of early uptake values does not permit differentiation of our 3 models of differing bone formation.
Late uptake, however, is markedly divergent among the
3 groups. By our determinations, late uptake is simply a measure of the amount of radioactivity retained in the knee region 4-6 days after administration. The variable factors determining early uptake are minimized. When the late uptake of 85Sr in the 3 models is compared, rachitics retain the least (0.83 %), osteoporotics the most (4.15 %), and controls are in between (1.85 %). These results parallel the findings in our tetracycline studies and are in keeping with our hypothesis that late radionuclide uptake is primarily a function of bone formation when calcium analogues are'used. As shown by Rowland (32) using 45Ca, bony surfaces intimately in contact 'with circulating fluids are transiently labeled with radionuclide shortly after administration. However, several days later, only areas of active bone formation retain this radioactivity. It is interesting to speculate that in these areas of radionuclide retention calcium-analogues are incorporated into recently formed bone matrix and, therefore, are not readily lost. Other metabolically less active areas are apparently unable to incorporate the radionuclide, allowing it to exchange off with time. This is consistent with our finding of increased late radionuclide retention in osteoporotic rats (increased bone formation) and decreased late retention in rachitic rats (decreased. bone formation). The uptake of 131Ba is noted to closely parallel that of 85Sr. Early uptake is comparable among the 3 groups. Late uptake shows the expected divergence. Again, rachitics retain the least radionuclide and osteoporotics 'the most, allowing differentiation of rachitic rats from osteoporotic rats. Although the divergence in the per cent late uptake of 131Ba allows differentiation of the models selected, the ratio of late/early uptake may better serve this purpose. As a measure of the per cent early uptake remaining in the knee 4-6 days later, large differences are observed which allow separation of the 3 models tested. Furthermore,
190
JOHN
S.
WILSON AND OTHERS
imaging variables such as age, blood volume, and size of bone studied are eliminated, and an animal or patient can serve as his own control. These factors may be important in the clinical setting where per cent uptake in an individual may be meaningless, but the dynamic change in uptake may be a useful expression of metabolic activity. Thus, when 85Sr or 1318a are used, a low late/early uptake ratio appears to reflect decreased bone formation, and a high uptake ratio reflects increased bone formation. Since similar information is derived from either agent, 1318a may have greater potential in the cllnlcal study of metabolic bone disease, because of its shorter half-life and improved dosimetry. The skeletal uptake values of' the non-calcium analogues IIlln and 203Pb do not parallel those of 85S r and 1318a. Although small isolated discrepancies in early or late uptake are observed among the 3 models, there is no consistent relationship between the degree of radionuclide uptake and the rate of bone formation as estimated by our tetracycline studies. Consequently, the uptake of IlIln or 203Pb does not permit differentiation of our models of osteomalacia and osteoporosis. One may speculate that this
IN VIVO VERSUS IN VITRO KNEE UPTAKE
January
1978
is due to the non-calcium analogue properties of these agents and their failure to be incorporated selectively at sites of active bone formation. Instead,they may label bone indiscriminately. In the case of 203Pb, late radionuclide retention is greater than early among all three models, possibly indicating prolonged residence at sites of initial uptake and, unlike 85Sr and 131aa, an inability to exchange off these areas with time.
SUMMARY Two models of altered bone metabolism representing decreased and increased bone formation were compared to a control group. The late uptake and late/early uptake ratios of the alkaline earth elements 1318a and 85Sr were found to reflect ratios of bone formation qualitatively estimated by tetracycline studies, and permitted differentiation of the 3 models studied. The uptake of IIIln and 203Pb did not appear useful for this purpose. Ratios of late/early uptake of 1318a and 85Sr provide a dynamic measure of skeletal metabolic activity, allowing differentiation of our animal models of osteomalacia and osteoporosis. The lower radiation exposure and the shorter half life of 1318a make it a clinically promising alternative to the longer-lived 85S r.
6.0
•
5.0
~I
~I
/
I ,
4.0
w
• I•
~
-c ~
e,
=>
3.0
I.
w w
,.:'1•
z
~ ~
Z w
u Q::
I .... •
..-'
2.0
w
c,
1.0
~/
r> 0.93
•
o
1.0 2.0 3.0 PERCENT KNEE UPTAKE IN VITRO
Fig. 4. Regression analysis demonstrating high coefficient of correlation between in vivo and in vitro results.
REFERENCES 1. Adams P, Jowsey J: Bone mineral metabolism in hyperthyroidism: an experimental study. Endocrinology 81:735-740. Oct 1967 2. Bauer GH: The use of radionuclides in orthopaedics. Part IV. Radionuclide scintimetry of the skeleton. J Bone Joint Surg 50A: 1681-1709, Dec 1968 3. Bauer GH, Wendeberg B: External counting of 47Ca and 85Sr in studies of localized skeletal lesions in man. J Bone Joint Surg 41A:558-580. Aug 1959 4. Bauer GH. Carlsson A, Lindquist B: A comparative study on the metabolism of 14°Baand 45Ca in rats. Biochem J 83:535-542, Aug 1956 5. Baylink D, Stauffer M, Wergedal J. et al: Formation. mineralization and resorption of bone in vitamin D-deficient rats. J Clin Invest 49: 1122-1134, 1970 6. Bligh PH, Taylor DM: Ccmparative studies of the metabolism of strontium and barium in the rat. Biochem J 87~612-618, 1963 7. Cohn, SR, Lippincott SW, Gusmano EA, et al: Comparative kinetics of 47Ca and 85Sr in man. Radiat Res 19:104-119, 1963 8. Ellsasser JC, Barnham JE, Marshall JH: Comparative kinetics and autoradiography of 45Ca and 133Ba in ten-year-old beagle dogs: the diffuse component distribution throughout the skeleton. J Bone Joint Surg 51A:1397-1412, Oct 1969 9. Francis MD: The inhibition of calcium hydroxyapatite crystal growth by polyphosphonates and polyphosphates. Calc Tiss Res 3: 151-162,31 March 1969 10. Frost HM, Villanueva AR, Roth H, et al: Tetracycline bone labeling. J New Drugs 1:206-216, Sep-Oct 1961 11. Frost HM: Preparation of thin undecalcified bone sections by rapid manual method. Stain Tech 33:273-277, Nov 1958 12. Garnett ES, Bowen BM, Coates G, et al: An analysis of factors which influence the local accumulation of bone-seeking radiopharmaceuticals. Invest RadioI10:564-568, Nov-Dec 1975 13. Genant HK, Bautovich GJ, Singh M, et al: Bone-seeking radionuclides: an in vivo study of factors affecting skeletal uptake. Radiology 113:373-382, Nov 1974
Vol. 126
RATIO OF LATE TO EARLY RADIONUCLIDE UPTAKE
14. Genant HK, Doi K, Rossman K, et al: Fine detail radiography: theoretical and practical considerations. Presented at the First International Workshop on Bone Morphometry, Ottawa, Canada, 28-31 Mar 1973 15. Harris F, Hoffenberg R, Black EG: The radio-isotope osteogram in rickets. Clin Sci 28:75-82, 1965 16. Harris LJ: Vitamin 0 and bone. [In]: Bourne GH, ed: The Biochemistry and Physiology of Bone. New York, Academic Press, 1956, pp 581-622 17. Harris WH, Heaney RP: Skeletal renewal and metabolic bone disease. N Engl J Med 280:193-201, 253-259, Jan 1969; 303-311, Feb 1969 18. Harrison GE, Carr TE, Sutton A: Distribution of radioactive calcium, strontium, barium and radium following intravenous injection into a healthy man. International J Radiat Bioi 13:235-247, 1967 19. Harrison GE, Howells GR, Pollard J: Comparative uptake and elution of 45Ca, 85Sr, 133Baand 223Ba in bone powder. Calc Tiss Res 1:105-113, Jul1967 20. Harrison M, Frazer R: Bone metabolism in rats studied with stable strontium. J Endocrinol 21: 191-196, Nov 1960 21. Harrison M, Frazer R: Bone structure and metabolism in calcium-
Nuclear Medicine
John S. Wilson, M.D.,2 Harry K. Genant, M.D., Robert S. Hattner, M.D., and Paul B. Hoffer, M.D. The ratio of late to early uptake of several radionuclides was examined as a method for distinguishing states of abnormal bone metabolism. Nutritional osteoporosis (secondary hyperparathyroidism) and osteomalacia were produced in young rats and compared to a control group. The ratio of early (3-6 hrs.) to late (4-6 days) uptake of barium-131, nitrate, indium-111 EDTMP, and lead-203 were studied, as was that of strontium-8S chloride, a calcium analogue. Ratios of late to early uptake were found to distinguish osteomalacia from osteoporosis in the models when strontium-8S or barium-131 were used. Barium-131 may be a clinically useful alternative to strontium-8S in the evaluation of metabolic bone disease due to its shorter half-life and lower radiation dose. INDEX TERMS: Osteomalacia 4 [8] .570 • Osteoporosis 4 [8] .560 • Radionuclides. comparative studies • (Skeletal system, other special miscellaneous procedure, 4 [8].1299)
Radiology 126: 185-191, January 1978
usefulness of conventional radionuclide T techniques for the detection, differentiation, and
available terminal phosphorous groups react by chemical absorption to the hydroxyapatite crystal in bone, rather than by chemical substitution for calcium ions (9, 23). Recently, phosphate binding to collagen moieties has been proposed as a possible additional mechanism (25, 31). The short half-life of 99mTc may be an undesirable feature in the study of metabolic bone diseases because early radionuclide uptake (on the order of hours) is probably dependent on factors other than the rate of osteogenesis, such as blood flow, surface binding, and capillary permeability (12,13,36,39,42,43). In the past, studies of bone metabolism have focused on the late uptake of radionuclides to avoid these variable early factors (2,8,28). Autoradiographic studies using calcium-45 (45Ca) demonstrated that several days after initial labeling of bony surfaces intimately in contact with circulating fluids, regions of active bone formation tend to retain the radionuclide, and nonaccreting regions lose the radionuclide (32). An additional factor affecting late radionuclide uptake is a gradual, diffuse labeling of inactive bone not in intimate contact with circulating fluids, known as the diffuse component (8,32). In young growing animals, osteogenesis is the major determinant of late radionuclide retention, whereas the diffuse component is only a minor factor (8).
HE
quantitation of metabolic bone diseases is controversial. Prior to the introduction of the technetium-99 (99mTc) poIyphosphate analogues, extensively used in studies of bone physiology (8, 18, 19, 22, 28, 32, 39, 43) and as agents for external probe-counting (2, 3, 15) or imaging (34, 38) of various skeletal disorders were calcium-47 (47Ca), strontium-85 (85Sr), and fluorine-18 8F). Their use in the clinical evaluation of metabolic bone diseases, however, has been sporadic, and results are varied (2, 15, 31). Furthermore, difficulties encountered in the dosimetry and imaging of these agents have shown them to be currently impractical and undesirable for clinical use (13, 38). The 99mTC poly phosphate analogue bone seekers have recently been used in the evaluation of several metabolic bone disorders (31). Despite nearly ideal dosimetric and imaging characteristics, these compounds suffer two major limitations in this setting: they are not calcium analogues, and the 6-hr. half-life of 99 mTc precludes late imaging (on the order of days). These are important considerations, because in the scintigraphic study of metabolic bone diseases it may be desirable that radionuclide uptake reflect the rate of osteogenesis, a prime factor in such disorders as osteomalacia (5, 17), hyperparathyroidism (17,24,29,30), and hyperthyroidism (1,29). True calcium analogues such as the alkaline earth elements Sr and Ba react by chemical substitution for calcium ions in the hydroxyapatite crystal (23), and their uptake by bone may therefore reflect calcium metabolism (4, 7, 23). The mechanism of phosphate binding to bone, however, is controversial. The most widely accepted hypothesis is that
e
EXPERIMENTAL DESIGN
Our objective was to establish an in vivo method of distinguishing altered rates of bone metabolism (osteogenesis) by using radionuclides that are bone-seeking and that have a half-life sufficient to permit late imaging. By
1 From the Department of Radiology and Section of Nuclear Medicine, University of California School of Medicine, San Francisco. Presented at the Sixty-second Scientific Assembly and Annual Meeting of the Radiological Society of North America, Chicago, III., Nov. 14-19, 1976. 2 Supported in part by NIH Training Grant GM 01272 from the National Institute of General Medical Sciences. ss
185
JOHN S. WILSON AND OTHERS
186
comparing the change in radionuclide uptake over time (late/early uptake ratio) in animal models of two metabolic bone diseases, we hoped to obtain a scintigraphic measure of skeletal metabolic activity that would have clinical utility. In vitro well-counting and tetracycline-labeling techniques were used to substantiate our in vivo results.
TABlE
I:
January
1978
PHYSICAL PROPERTIES OF RADIONUCLIDES STUDIED
Half-life (days)
Radionuclide 85Sr chloride 1318a nitrate lllln EDTMP 203Pb acetate/nitrate
65 11.7 2.82 2.17
Gamma Energy Imaged (keV)
Specific Activity
514 496 173 279
3-30 mCi/mg 3.78 mCi/mg Carrier free Carrier free
MATERIALS AND METHODS
Animal preparations: Approximately 100 threeweek-old Holtzman rats, initially weighing 40 to 50 g, were housed in pairs in a dark room throughout the study. Three models of bone metabolism were established by means of dietary manipulation. Osteomalacia, a state of decreased bone formation (5, 17), was produced in rats by feeding them a diet which was free of vitamin D, and which contained 75.5% ground corn meal, 20% wheat gluten, 3.5 % CaC0 3, and 1% NaCI (13, 16). Nutritional osteoporosis was produced in rats by feeding them the same diet, but with a low level of calcium and a high level of phosphate (0.018% CaC0 3, 1.5% equimolar KH2HOP 4), as described by Steenbock and Herting (40). Supplementary vitamin D (20 IU/wk) was administered orally to prevent the concomitant development of rickets; this form of osteoporosis is considered to result most probably from secondary hyperparathyroidism (16, 20, 21, 33, 35), a state of increased bone formation and bone resorption, with the latter being quantitatively greater (17,20,21,24,29,30). Previous studies have shown that in young rats fed a similar calcium-deficient diet, hypertrophied osteocytes develop and resorption cavities expand (33). In addition, Sevastikoglou (35) demonstrated osteoporosis and hypertrophy of parathyroid glands in young rats on a similar dietary regimen, indicating secondary hyperparathyroidism. A control group (normal state of bone formation) was created by feeding rats the rachitogenic diet mentioned above but with oral vitamin D supplement.
Fig. 1. Quantitative in vivo radionuclide scan with rectangular region of interest outlining the rat knee.
Radionuclide selection: The skeletal uptake of four bone-seeking radionuclides was studied; the physical characteristics of each agent are given in TABLE I. Strontium-85 (8SSr) (New England Nuclear, Boston), a known calcium analogue, was chosen as a standard of comparison for the other agents tested. The late uptake of 85Sr has been considered in large part a manifestation of osteogenesis or mineral accretion (2,7,29). However, the long half-life and high gamma energy of 85Sr have restricted its use in humans to very low dosages (13, 37, 38). Barium-131 (131Ba) nitrate (ICN Pharmaceuticals, Cleveland) was selected because of its physical characteristics and evidence that it is handled by the skeleton in a manner similar to 47Ca(4) and 85Sr (6, 26). An alkaline earth radionuclide, 131Ba with a specific activity of 3.5 to 4 mCi/mg was produced commercially by means of neutron irradiation of enriched 130Ba. Although its decay scheme is complex, resulting in 4 primary gamma-ray emissions, the 11.7-day half-life of 131Ba makes it suitable for late imaging. In addition, previous dosimetric calculations indicate that the radiation dose absorbed by the patient is approximately one-fourth that of 85Sr (37). Indium-111 111n) ethylenediaminetetra phosphonic acid (EDTMP) (Medi Physics, Emeryville, Calif.) was used as a polyfunctional phosphate substitute for the more conventional but short-lived 99mTc compounds. Previous reports indicate active skeletal localization that produces high quality scintigraphic images (41). The 2.8-day half-life of 1111n EDTMP permits comparison of the late uptake of this non-calcium analogue with that of the calcium analogues 85S r and 131Ba. Lead-203 (203Pb) (New England Nuclear) acetate and nitrate were selected because of evidence of high skeletal affinity and excellent imaging characteristics (27). A 2.2-day half-life allows late imaging. Dose calibrations: Radioactivity in one-milliliter syrin~e~ containing the selected radionuclide (ranging in actlvlty from 250 to 750 ,uCi) was counted in an appropriately set dose calibrator. Approximately 4 or 5 weeks after dietary manipulation of the rats was begun, between 200 and 650 ,uCi of the selected radionuclide was injected into the tail veins of lightly anesthetized rats. Following the injection, radioactivity in the syringes was recounted in the dose calibrator to determine the injected radioactivity. The selected radionuclides within the syringes were also counted with a gamma camera before and after the radionuclide injection under conditions identical to those used for in vivo scanning; this permitted calculation of counts/ ,uCi of radionuclide administered.
e
Vol. 126
RATIO OF LATE TO EARLY RADIONUCLIDE UPTAKE
In vivo scanning: Early (3-6 hr) and late (4-6 day) quantitative in vivo imaging was performed using a Pho Gamma scintillation camera (pho/Gamma III, Searle Radiographies, Des Plaines, 111.) with a 4-mm, pin-hole collimator interfaced with a digital computer (DEC RT 11/40, Digital Equipment, Maynard, Mass.). Lightly anesthetized rats were placed in a lead immobilization device that effectively shielded all body parts except the knee region (the distal femur, proximal tibia, and surrounding soft tissues), which was centered 5 em (2 in.) beneath the pin-hole collimator. Two-minute scans of each knee were obtained and the data stored on magnetic discs for analysis at a later time. In vitro counting: A few rats from each group were sacrificed at the end of early scanning and others at the end of late scanning for purposes of in vitro counting of radioactivity. In vitro corroboration of in vivo scanning data was made for all of the rats in the In and Sr groups by means of well-counting. Each distal femur (distal onefourth) was sharply dissected from surrounding soft tissues, dehydrated in acetone, and placed in a counting well for a one-minute determination of radioactivity. Tetracycline labeling: Tetracyclines have been used extensively as markers of new bone formation (5, 10,28, 32). After administration, tetracycline is deposited at sites of active bone formation, resulting in fluorescent bands easily identified under ultraviolet light microscopy. When 2 doses are separated by several days, 2 concentric bands are seen. The degree of separation is generally considered a function of the amount of bone formation which has occurred in the interval (5, 10, 17), although some feel this is controversial in conditions where bone formation and resorption are uncoupled (29). In this study, double tetracycline labels were given to provide a qualitative estimate of new bone formation. A few rats from each group were
187
Nuclear Medicine
given two intraperitoneal doses (8 days apart) of 4 mg demeclocycline hydrochloride. Femora and tibiae were then dissected free of soft tissues and fixed in 50 % alcohol. A high speed, rotary, diamond saw was used to slice unembedded diaphyseal bone. These sections were stained with 0.10% basic fuchsin in 50% ethanol to enhance visualization of osteoid. A final thickness of 50 to 75 J.l was obtained by hand-grinding specimens using the technique of Frost (11). Specimens were viewed under a fluorescent light microscope to assess tetracycline labeling of areas of new bone formation (osteogenesis) (10). Fine detail radiography: Throughout the experiment, fine detail radiography (14) was employed to assess the status of bone disease in the various models. The technique involved the use of Kodak Type M industrial film, 500 milliampere seconds (mAs), 45 kilovolts (kV) and a focus film distance (FFD) of 40 inches. Optical magnification was used to visualize the minute bony architecture of rat femora, pelves, and vertebrae. Data analysis: Using the storage oscilloscope display of the computer, a rectangular region of interest was chosen to quantify early and late skeletal uptake of radionuclide in the knee region (Fig. 2). The early and late uptake was calculated for each rat from decay-corrected counts; the counts ranged from 3,000 to 30,000 counts/ 2-min. scan. The mean per cent of radioactivity in the knee region was determined by averaging decay-corrected counts for right and left knee and dividing by the administered radioactivity expressed in counts (13). In addition, the ratio of mean per cent late uptake to early uptake (late/early uptake ratio) was determined to express the change in uptake over time and to allow individual rats to serve as their own control. The Student's t-test was applied to determine the sta-
Fig. 2. Fine detail radiographs of rat knee, 3 to 4 weeks after dietary manipulation. A. mineralized and free of rachitic changes. B. Rachitic rat shows epiphyseal widening and fraying of metaphysis. C. Osteoporotic rat demonstrates demineralization and loss of trabeculae.
Bone of control rat appears well
JOHN S. WILSON AND OTHERS
188
tistical significance of the results in comparing rachitic and osteoporotic -to control rats. The correlation between in vivo and in vitro data was assessed by regression analysis. RESULTS
Fine detail radiographs obtained 3 to 4 weeks after dietary manipulation demonstrated the expected skeletal changes in the 3 groups of rats (Fig. 2). Typical radiographic signs of advanced rickets, including metaphyseal fraying and epiphyseal widening, were found in the osteomalacic group. Diffuse osteopenia as evidenced by cortical thinning and dissolution of trabeculae was demonstrated in the osteoporotic group. The skeletal structures of the control rats appeared well mineralized and free of rachitic changes. Tetracycline labeling studies (Fig. 3) revealed a depressed state of new bone formation in the osteomalacic rats by demonstrating vast osteoid formation and scant areas of tetracycline labeling. Conversely, the osteoporotic rats demonstrated numerous Howship's lacunae and resorptive spaces, as well as plentiful bright tetracycline bands. Although some widely separated double bands were observed, they were not as distinct as those in control rats, which showed relatively normal cortical architecture and the expected well-defined tetracycline bands. In addition, the osteoporotic rats showed more sites of osteogenesis than the control rats. The results of quantitative in vivo imaging are given in TABLE II. The amount of early uptake of 85Sr was similar among the three groups, although a small difference was seen with the osteoporotic group. Late imaging, however, demonstrated a striking divergence in the mean per cent uptake of 85Sr. Control rats exhibited 1.85 0/0, rachitic rats 0.83 %, and osteoporotic rats 4.15 % . These results parallel those for the rates of osteogenesis among the 3
January 1978
groups as qualitatively estimated by using tetracycline labeling. Likewise, the ratio of late to early uptake of 85Sr differed significantly among the three groups. Small but statistically significant differences between groups were observed in the early uptake of 1318a, but only the late uptake values and the late/early ratios permitted ready differentiation between the control, rachitic, and osteoporotic rats. The rachitic rats retained the least amount of 1318a and the osteoporotic rats retained the most. This pattern of uptake is very similar to that observed in the rats injected with 85Sr. The three groups of rats injected with 1111n EDTMPfailed to demonstrate, in either early or late uptake, the divergent pattern seen using 85Sr or 1318a. Although small variations in uptake were noted, no major difference was observed which would readily allow a distinction to be made between the 3 groups. Likewise, the early or late uptake of 203Pb nitrate and acetate did not permit differentiation between the models tested, although small, isolated disparities existed (i.e., low early uptake in the rachitic and osteoporotic rats, compared to the control rats). These quantitative in vivo data were well substantiated by data from in vitro well-counting. The correlation coefficient obtained by comparing the in vitro and in vivo 85Sr and 1111n data was 0.93. The regression analysis is displayed in Figure 4. DISCUSSION
The double-label tetracycline studies support the validity of the models tested. Control rats show normal cortical architecture and uniform double labels. In the rachitic model, however, a paucity of tetracycline labeling with no double bands is observed. This implies very little bone formation during the 8-day period between labels, and is to be expected in rickets, a condition in which defects in
Fig. 3. Tetracycline studies with 8-day separation of labels. A. Control rat shows relatively normal cortical architecture and the expected double tetracycline labeling. B. Rachitic rat demonstrates vast osteoid formation with scant tetracycline labeling and no double labeling. C. Osteoporotic rat shows numerous bright tetracycline double labelings, indicating increased osteogenesis.
Vol.
126
RATIO OF LATE TO
TABLE II:
EARLY
85Sr chloride Early Late Late/early 111Ba nitrate Early Late Late/early 1111n EDTMP Early Late Late/early 203Pbacetate/ nitrate Early Late Late/early
Nuclear Medicine
PERCENT SKELETAL UPTAKE OF RADIONUCLIDE IN THEKNEEREGION OF THREE MODELSOF RATS (IN VIVO) Control
Agent
189
RADIONUCLIDE UPTAKE
No. Rats
Mean
Rachitic
±
S.D.
No. Rats
Mean
Osteoporotic
±
S.D.
No. Rats
Mean
±
S.D.
5 4 4
4.01 ± 0.58 1.85 ± 0.07 0.49 ± 0.05
6 4 4
4.31 ± 0.54 0.83* ± 0.10 0.20" ± 0.04
6 4 4
5.05 t ± 0.57 4.15* ± 0.44 0.86" ± 0.04
7 7 7
2.69 ± 0.24 1.46 ± 0.29 0.56 ± 0.07
6 6 6
2.01" ± 0.12 0.60" ± 0.12 0.30* ± 0.04
6 6 6
3.23 ± 0.18 2.32 ± 0.31 O.72 t ± 0.12
7 4 4
2.93 ± 0.40 2.52 ± 0.34 0.89 ± 0.04
7 4 4
3.29 ± 0.48 2.55 ± 0.34 0.78* ± 0.04
7 4 4
2.48~
10
4.05 ± 0.46 4.81 ± 0.61 1.14 ± 0.13
6 3 3
3.05* ± 0.22 2.84* ± 1.05 0.95 ± 0.35
4 3 3
3.10~
6
6
± 0.28 2.22 ± 0.26 0.88 ± 0.04 ± 0.93 3.79 ± 0.12 1.22 ± 0.08
* P < 0.01. r P < 0.02. ~ P < 0.05.
osteogenesis have been well documented (5, 16, 17). These findings are in agreement with the tetracycline studies of Baylink, which showed a 20 % decrease in total osteoblastic matrix formation rate and a 50 % decrease in osteoid mineralization rate in rachitic rats (5). Osteoporotic rats demonstrate extensive heavy tetracycline labeling accompanied by increased resorptive spaces and a general disarray of normal cortical architecture. Widely separated double bands, although present, are somewhat difficult to identify. This could be due to a state of rapid bone formation coupled with rapid resorption primarily in regions of more mature bone. The initial tetracycline label may undergo resorption. This hypothesis is consistent with other studies of secondary hyperparathyroidism, where both increased bone formation and resorption have been observed (17,20,21,24,29,30). If, on the other hand, the rates of bone formation in our model were not increased, heavy tetracycline labeling would not be expected. Therefore, on the basis of tetracycline studies, rachitic rats appear to have decreased bone formation and osteoporotics increased bone formation, relative to controls. In evaluating the in vivo uptake data displayed in TABLE II, a discussion of the 85Sr data will be undertaken first, since the uptake of strontium, a known calcium analogue, is considered a reliable indicator of bone metabolism. It is apparent that control, rachitic, and osteoporotic rats injected with 85Sr demonstrate comparable early uptake, ranging between 4-5 % in the knee region. This similarity may be due to the multiplicity of factors thought to playa role in early radionuclide uptake several hours after administration. These include blood flow, capillary permeability, extraction fraction, and to some extent the rate of bone formation (12, 13,36,39,42,43). Consequently, it is not surprising that a comparison of early uptake values does not permit differentiation of our 3 models of differing bone formation.
Late uptake, however, is markedly divergent among the
3 groups. By our determinations, late uptake is simply a measure of the amount of radioactivity retained in the knee region 4-6 days after administration. The variable factors determining early uptake are minimized. When the late uptake of 85Sr in the 3 models is compared, rachitics retain the least (0.83 %), osteoporotics the most (4.15 %), and controls are in between (1.85 %). These results parallel the findings in our tetracycline studies and are in keeping with our hypothesis that late radionuclide uptake is primarily a function of bone formation when calcium analogues are'used. As shown by Rowland (32) using 45Ca, bony surfaces intimately in contact 'with circulating fluids are transiently labeled with radionuclide shortly after administration. However, several days later, only areas of active bone formation retain this radioactivity. It is interesting to speculate that in these areas of radionuclide retention calcium-analogues are incorporated into recently formed bone matrix and, therefore, are not readily lost. Other metabolically less active areas are apparently unable to incorporate the radionuclide, allowing it to exchange off with time. This is consistent with our finding of increased late radionuclide retention in osteoporotic rats (increased bone formation) and decreased late retention in rachitic rats (decreased. bone formation). The uptake of 131Ba is noted to closely parallel that of 85Sr. Early uptake is comparable among the 3 groups. Late uptake shows the expected divergence. Again, rachitics retain the least radionuclide and osteoporotics 'the most, allowing differentiation of rachitic rats from osteoporotic rats. Although the divergence in the per cent late uptake of 131Ba allows differentiation of the models selected, the ratio of late/early uptake may better serve this purpose. As a measure of the per cent early uptake remaining in the knee 4-6 days later, large differences are observed which allow separation of the 3 models tested. Furthermore,
190
JOHN
S.
WILSON AND OTHERS
imaging variables such as age, blood volume, and size of bone studied are eliminated, and an animal or patient can serve as his own control. These factors may be important in the clinical setting where per cent uptake in an individual may be meaningless, but the dynamic change in uptake may be a useful expression of metabolic activity. Thus, when 85Sr or 1318a are used, a low late/early uptake ratio appears to reflect decreased bone formation, and a high uptake ratio reflects increased bone formation. Since similar information is derived from either agent, 1318a may have greater potential in the cllnlcal study of metabolic bone disease, because of its shorter half-life and improved dosimetry. The skeletal uptake values of' the non-calcium analogues IIlln and 203Pb do not parallel those of 85S r and 1318a. Although small isolated discrepancies in early or late uptake are observed among the 3 models, there is no consistent relationship between the degree of radionuclide uptake and the rate of bone formation as estimated by our tetracycline studies. Consequently, the uptake of IlIln or 203Pb does not permit differentiation of our models of osteomalacia and osteoporosis. One may speculate that this
IN VIVO VERSUS IN VITRO KNEE UPTAKE
January
1978
is due to the non-calcium analogue properties of these agents and their failure to be incorporated selectively at sites of active bone formation. Instead,they may label bone indiscriminately. In the case of 203Pb, late radionuclide retention is greater than early among all three models, possibly indicating prolonged residence at sites of initial uptake and, unlike 85Sr and 131aa, an inability to exchange off these areas with time.
SUMMARY Two models of altered bone metabolism representing decreased and increased bone formation were compared to a control group. The late uptake and late/early uptake ratios of the alkaline earth elements 1318a and 85Sr were found to reflect ratios of bone formation qualitatively estimated by tetracycline studies, and permitted differentiation of the 3 models studied. The uptake of IIIln and 203Pb did not appear useful for this purpose. Ratios of late/early uptake of 1318a and 85Sr provide a dynamic measure of skeletal metabolic activity, allowing differentiation of our animal models of osteomalacia and osteoporosis. The lower radiation exposure and the shorter half life of 1318a make it a clinically promising alternative to the longer-lived 85S r.
6.0
•
5.0
~I
~I
/
I ,
4.0
w
• I•
~
-c ~
e,
=>
3.0
I.
w w
,.:'1•
z
~ ~
Z w
u Q::
I .... •
..-'
2.0
w
c,
1.0
~/
r> 0.93
•
o
1.0 2.0 3.0 PERCENT KNEE UPTAKE IN VITRO
Fig. 4. Regression analysis demonstrating high coefficient of correlation between in vivo and in vitro results.
REFERENCES 1. Adams P, Jowsey J: Bone mineral metabolism in hyperthyroidism: an experimental study. Endocrinology 81:735-740. Oct 1967 2. Bauer GH: The use of radionuclides in orthopaedics. Part IV. Radionuclide scintimetry of the skeleton. J Bone Joint Surg 50A: 1681-1709, Dec 1968 3. Bauer GH, Wendeberg B: External counting of 47Ca and 85Sr in studies of localized skeletal lesions in man. J Bone Joint Surg 41A:558-580. Aug 1959 4. Bauer GH. Carlsson A, Lindquist B: A comparative study on the metabolism of 14°Baand 45Ca in rats. Biochem J 83:535-542, Aug 1956 5. Baylink D, Stauffer M, Wergedal J. et al: Formation. mineralization and resorption of bone in vitamin D-deficient rats. J Clin Invest 49: 1122-1134, 1970 6. Bligh PH, Taylor DM: Ccmparative studies of the metabolism of strontium and barium in the rat. Biochem J 87~612-618, 1963 7. Cohn, SR, Lippincott SW, Gusmano EA, et al: Comparative kinetics of 47Ca and 85Sr in man. Radiat Res 19:104-119, 1963 8. Ellsasser JC, Barnham JE, Marshall JH: Comparative kinetics and autoradiography of 45Ca and 133Ba in ten-year-old beagle dogs: the diffuse component distribution throughout the skeleton. J Bone Joint Surg 51A:1397-1412, Oct 1969 9. Francis MD: The inhibition of calcium hydroxyapatite crystal growth by polyphosphonates and polyphosphates. Calc Tiss Res 3: 151-162,31 March 1969 10. Frost HM, Villanueva AR, Roth H, et al: Tetracycline bone labeling. J New Drugs 1:206-216, Sep-Oct 1961 11. Frost HM: Preparation of thin undecalcified bone sections by rapid manual method. Stain Tech 33:273-277, Nov 1958 12. Garnett ES, Bowen BM, Coates G, et al: An analysis of factors which influence the local accumulation of bone-seeking radiopharmaceuticals. Invest RadioI10:564-568, Nov-Dec 1975 13. Genant HK, Bautovich GJ, Singh M, et al: Bone-seeking radionuclides: an in vivo study of factors affecting skeletal uptake. Radiology 113:373-382, Nov 1974
Vol. 126
RATIO OF LATE TO EARLY RADIONUCLIDE UPTAKE
14. Genant HK, Doi K, Rossman K, et al: Fine detail radiography: theoretical and practical considerations. Presented at the First International Workshop on Bone Morphometry, Ottawa, Canada, 28-31 Mar 1973 15. Harris F, Hoffenberg R, Black EG: The radio-isotope osteogram in rickets. Clin Sci 28:75-82, 1965 16. Harris LJ: Vitamin 0 and bone. [In]: Bourne GH, ed: The Biochemistry and Physiology of Bone. New York, Academic Press, 1956, pp 581-622 17. Harris WH, Heaney RP: Skeletal renewal and metabolic bone disease. N Engl J Med 280:193-201, 253-259, Jan 1969; 303-311, Feb 1969 18. Harrison GE, Carr TE, Sutton A: Distribution of radioactive calcium, strontium, barium and radium following intravenous injection into a healthy man. International J Radiat Bioi 13:235-247, 1967 19. Harrison GE, Howells GR, Pollard J: Comparative uptake and elution of 45Ca, 85Sr, 133Baand 223Ba in bone powder. Calc Tiss Res 1:105-113, Jul1967 20. Harrison M, Frazer R: Bone metabolism in rats studied with stable strontium. J Endocrinol 21: 191-196, Nov 1960 21. Harrison M, Frazer R: Bone structure and metabolism in calcium-
