Accumulation of technetium-99m methylene diphosphonate

Accumulation of technetium-99m methylene diphosphonate Conditions affecting adsorption to hydroxyapatite Yoichi Okamoto, DDS, a Kanagawa, Japan KANAGAWADENTALCOLLEGE Experiments were carried out to explore the mechanism of technetium-99m methylene diphosphonate (99rnTc-ME)P) adsorption with various calcium compounds including hydroxYapatite powder. 99mTc-MDP adsorption to hydroxyapatite was markedly inhibited by the addition of either pyrophosphate or methylene diphosphonate (MDP). Moreover, adsorbed 99mTc-MDP was partly removed by rinsing with pyrophosphate solution. Adsorption was pH-dependent and was inhibited by univalent cations, adenosine triphosphate solution, and guanosine triphosphate. Adsorption was most apparent to hydroxyapatite and calcium pyrophosphate and was less marked for the other calcium compounds tested. It is suggested that 99mTc-MDPadsorption is affected by the hydroxyapatite crystalline structure and environmental factors such as pH and the presence of phosphates, calcium compounds, and various cations. (ORALSURG ORALMED ORALPATHOLORALRADIO/ENDOD 1995;80"115-9)

Bone scintigraphy with 99mTc-MDP is widely used in the diagnosis of bone diseases. Bone scintigraphy allows evaluation of early changes before they become apparent on radiograph. It is useful for the early detection ofintraosseus disease including the evaluation of malignant tumor metastases: 99rnTc-MDP is considered to accumulate in areas of active bone metabolism such as areas of absorption and resorption. H~ Kanishi suggested two mechanisms for 99mTc-MDP accumulation in bone: chemical adsorption to the surface of hydroxyapatite crystals and actual incorporation into the crystalline structure. 11 This study builds on that of Kanishi by further evaluating the adsorption characteristics of 99mTc-MDP to hydroxyapatite powder and various other calcium compounds.

MATERIAL AND METHODS 99mTc-MDP adsorption to hydroxyapatite Hydroxyapatite powder (100 mg, Japan Chemical Co. Ltd., Tokyo, Japan) was variously added to 2 ml of each of the following solutions: (1) distilled water (control), (2) phosphate solution, and (3) i, 10, and 100 mmol/L pyrophosphate solutions and MDP. Each was mixed with 99mTc-MDP (Nihon Meditphisics Co. Ltd., Tokyo, Japan). After 2 hours the solution was removed, the reaction product was rinsed with 4 ml distilled water, and radioactivity was measured with a well-type scintillation counter (Auto Well Gamma System, Aloka Co. Ltd., Tokyo, Japan). RadioactivaDepartment of Oral and Maxillofacial Radiology, Kanagawa Dental College. Received for publication Jan. 26, 1994; returned for revision Mar. 11, 1994 and Aug. 1, 1994; accepted for publication Feb. 6, 1995. Copyright 9 1995 by Mosby-Year Book, Inc. 0030-4220/95/$3.00 + 0 7/16/64187

ity was also measured 30, 60, 120, 180, and 240 minutes later. To evaluate the effects of rinsing, radioactivity was measured after two 5-second rinses and after three 5-rninute rinses with 10 mmol/L pyrophosphate solution. The rinses were repeated with 100 mmol/L solution.

Effect of pH on 99mTc-MDP adsorption Hydroxyapatite powder (I00 mg) was added to 100 mmol/L phosphate buffer (pH 5.5 to 8.5) and was agitated with 99mTc-MDP for 2 hours. Radioactivity was measured after rinsing was done with 4 ml distilled water.

Effect of univalent and divalent cations on 99mTc-MDP adsorption Hydroxyapatite powder (100 mg) was added to 2 ml univalent cation (10, 50, 100, and 150 mmol/L of both sodium chloride [NaC1] and potassium chloride [KC1]), divalent cation (0.1, 0.5, 1, 5, or 10 mmol/L both of calcium chloride [CaCI2] and magnesium chloride [MgC12]), and distilled water (control); each of these was treated with 99rnTc-MDP (740 Bq) for 2 hours. The reaction product was washed twice with 4 ml of the same concentration of each solution, and radioactivity was then measured. Effect of phosphates on 99mTc-MDP adsorption Hydroxyapatite (100 mg) was added to 2 ml phosphate (0.2, 2, or 5 mmol/L adenosine-5'-monophosphate [AMP], adenosine-5'-diphosphate [ADP], adenosine-5 '-triphosphate [ATP], guanosine-5'triphosphate [GTP], or D-fructose 1,6-diphosphate [FDP]). It was then agitated with 99mTc-MDP for 2 hours. The reaction product was rinsed twice with 4 ml distilled water, and radioactivity was then measured. 115

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ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY

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99mTc-MDP adsorption to various calcium compounds

RESULTS Characteristics of 99mTc-MDP adsorption to hydroxyapatite

99mTc-MDP (740 Bq) was applied separately to calcium carbonate (CaCO3), calcium sulfate (CaSO4), calcium sulfite (CaSO3), calcium phosphite (CaHPO3), calcium pyrophosphate (Ca2P207), and calcium silicate (CaSiO3) in distilled water for 2 hours each and rinsed twice. The radioactivity was then measured. All experiments were done in triplicate, and the mean values were used. All of the data were compared by statistical method by the t test. Values were considered statistically significant when p values were <0.05.

99mTc-MDP adsorption to hydroxyapatite occurred within 30 minutes. Adsorption was markedly inhibited by 100 mmol/L pyrophosphate (Fig. 1). Adsorption with 10 mmol/L or less phosphate did not differ from the control. The adsorption rate in 100 mmol/L phosphate was 34.1%. The adsorption rate in pyrophosphate was 85.3% at a pyrophosphate concentration of 1 retool/L, 19.4% at 10 mmol/L, and 1.4% at 100 mmol/L. The adsorption rate in MDP was 82.2% at an M D P concentration of 1 mmol/L, 22.2% at 10 mmol/L, and 2.6% at 100 mmol/L (Fig. 2). The

ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY Volume 80, Number 1

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40.5%, respectively. These were significantly lower adsorption rates than in the control (p < 0.005). However, the adsorption rate did not differ between the control and <-100 mmol/L NaC1 or KC1 (Fig. 5, A). Also, no difference was seen with <-10 mmol/L CaC12 or MgCle (Fig, 5, B).

Effect of pH on 99mTc-MDP adsorption 99mTc-MDP adsorption to the hydroxyapatite powder was minimized at a pH of 7 to 8. Adsorption increased with an increase or decrease in pH beyond this range (Fig. 4).

Effect of phosphates on 99mTc-MDP adsorption The rate of adsorption to the hydroxyapatite powder was 46.1% in 5 mmol/L ATP, 61.4% in 2 mmol/L ATP, and 58.1% in 5 mmol/L GTP, Thus ATP and GTP both inhibited 99mTc-MDP adsorption to hydroxyapatite. ADP also slightly inhibited adsorption; however, no significant difference was observed between the addition of either AMP or FDP and the control (Fig. 6).

Effect of univalent or divalent cations on 99mTc-MDP adsorption The rates of adsorption to the hydroxyapatite powder in 150 mmol/L NaC1 and KC1 were 50.0% and

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Fig. 7. 99mTc-MDP adsorption to calcium compounds. 99m'I'c-MDP adsorption to calcium compounds The rate of adsorption to calcium compounds was 8.6% for CaCO3, 6.8% for CaSO4, 17.1% for CaSO3, 8.6% for CaHPO3, 88.6% for Ca2P207, and 23.1% for CaSiO3. The rate of adsorption to Ca2P207 was similar to the rate of adsorption to hydroxyapatite, which was 81.4% (Fig. 7). DISCUSSION Kanishi evaluated the mechanisms of 99mTc-MDP accumulation in bone using cultured fetal mouse calvaria, isolated osteoblast-like cells, and collagen sponge. No uptake occurred in the osteoblast-like cells or collagen. In this study 99mTc-MDP adsorption to hydroxyapatite occurred within 30 minutes and was markedly inhibited by pyrophosphate and MDP. Because pyrophosphate resembles MDP in terms of the chemical

structure, competitive inhibition was considered; however, adsorbed 99mTc-MDP was removed by pyrophosphate. Kroesbergen et al. 12 reported that adsorption of 99mTc(Sn)-pyrophosphate to calcium triphosphate is more marked at pH 4.0 than at pH 7.4. An association between pH and 99rnTc-MDP adsorption to bone was shown in this study. Fallon 13 reported acidification of periosteoblast areas during bone absorption. 14 Hence this acidification could explain the mechanism of increased 99mTc-MDP deposition in sites of bone deposition. The tumor pH has been reported to change after radiotherapy 15 and chemotherapy, 16 s o 99mTcMDP uptake should also be increased in these circumstances. In inflammatory lesions affecting bone, the lesions are readily visualized by scintigraphy. This visualization may be possible because of the local acidosis that accompanies inflammation.

ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY Volume 80, Number 1

Adsorption was inhibited by univalent cations but not by divalent cations (Fig. 5). Changes in sodium and potassium concentrations also seem to affect 99mTc-MDP adsorption to bone. Adsorption was inhibited in a dose-dependent fashion by ADP, ATP, and GTP, but only slightly by AMP and FDP. This difference may be associated with differences in the number and locations of the phosphate radical in each case. Among calcium compounds 99mTc-MDP was most markedly and almost equally adsorbed to hydroxyapatite and calcium pyrophosphate. It is concluded that low pH is a major factor in increased uptake of 99mTc-MDP in areas of activity within bone.

Okamoto

6.

7.

8. 9. 10.

The author thanks Prof. I s a m u Kashima, D e p a r t m e n t o f Oral and Maxillofacial Radiology, K a n a g a w a Dental College, for his thoughtful suggestions and advice t h r o u g h o u t this work. T h a n k s are also extended to Dr. H i r o m i W a k a o , Dr. Yoshinori Jinbu, and Dr. Daisuke Kanishi, D e p a r t m e n t of Oral and Maxillofacial Radiology, K a n a g a w a Dental College, for their valuable comments.

11.

REFERENCES

14.

1. Khan RA, Hughes S, Lavender P, Leon M, Spyrou N. Autoradiography of technetium-labelled diphosphonate in rat bone. J Bone Joint Surg 1979;61:221-4. 2. Francis MD, Ferguson DL, Tofe AJ, Bevan JA, Michaels SE. Comparative evaluation of three diphosphonates: in vitro adsorption (C-14 labeled) and in vivo (technetium-99m complexed). J Nucl Med 1980;21:1185-9. 3. Christensen SB, Arnoldi CC. Distribution of 99mTc-phosphate compounds in osteoarthritic femoral head. J Bone Joint Surg 1980;62:90-6. 4. Christensen SB, Krogsgaard OW. Localization of Tc-99m MDP in epiphyseal growth plates of rats. J Nucl Med 1981; 22:237-45. 5. Savelkoul TJF, Visser WJ, Oldenburg S J, Duursma SA. A microautoradiographical study of the localization of 99roTe-

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(Sn)-MDP and 99mTc-MDP in undecalcified sections. Eur J Nucl Med 1986;11:459-62. Bud RS, Hodgson GS, Hare WSC. The relation of radionuclide uptake by bone to the rate of calcium mineralization: I. experimental studies using 45Ca, 32p and 99mTc-MDP. Br J Radiol 1989;62:314-7. Budd RS, Hodgson GS, Hare WSC. The relation of radionuclide uptake by bone to the rate of calcium mineralization: I. patient studies using 99mTc-MDP. Br J Radiol 1989;62:31820. Lausten GS, Christensen SB. Distribution of 99mTc-phosphate compounds in osteonecrotic femoral heads. Acta Orthop Scand 1989;60:419-23. Rohlin M, Larsson A, Hamrnarstrtim L. In vitro interaction between 99Tcm labeled pyrophosphate and rat tissues. Eur J Nucl Med 1978;3:249-55. Subramanian G, Blair RJ, Kallfelz EA, Thomas FD, McAfee JG. 99mTc-MDP (methylene diphosphonate): a superior agent for skeletal imaging [Abstract]. J Nucl Med 1973;14: 640. Kanishi D. 99mTc-MDP accumulation mechanisms in bone. ORAL SURG ORAL MED ORAL PATHOL 1993;75:239-46. Kroesbergen J, van Steijn AM, Gelsema WJ, de Ligny CL. 99mTc-MDP bone scanning agents-II: adsorption of 99mTc(Sn) pyrophosphate complexes on the mineral phase of bone. Int J Nucl Med Biol 1986;12:411-7. Fallon MD. Bone resorbing fluid from osteoclasts is acidic: an in vitro micropuncture study. In: Cohn DV, Fujita T, Potts JT Jr, Talmage RV, eds. Endocrine control of bone and calcium metabolism, 8A. Amsterdam: Elsevier Science Publishers BV, 1984:144-6. Tannok IF, Rotin D. Acid pH and its potential for therapeutic exploitation. Cancer Res 1989;49:4373-84. Ng TC, Majors AW, Vijayakumar S, et al. Human neoplasm pH and response to radiation therapy: P-31 MR spectroscopy studies in situ. Radiology 1989;170:875-8. Smith SR, Martin PA, Edwards RHT. Tumor pH and response to chemotherapy: an in vivo 31p magnetic resonance spectroscopy study in non-Hodgkin's lymphoma. Br J Radiol 1991 ;64:923-8.

Reprint requests: Yoichi Okamoto Department of Oral and Maxillofacial Radiology Kanagawa Dental College 82 Inaoka-cho, Yokosaka, Kanagawa 238, Japan E-mail: MHC00 451 @niftyserve.or.jp