Development of a Kinetic Assay for Band 5b Tartrate-resistant Acid Phosphatase Activity in Serum Mamoru Nakanishi, 1* Kousei Yoh, 2 Toshihide Miura, 3 Tatsuya Ohasi, 3 Shiba Kumar Rai, 4 and Kagehiro Uchida 1 Background: Band 5 tartrate-resistant acid phosphatase (TrACP; EC 3.1.3.2) consists of two isoenzymes, bands 5a and 5b, of which band 5b TrACP is considered to be derived from bone. However, no kinetic method for the specific measurement of band 5b TrACP in serum is available. Our aim was to develop a kinetic assay method for the specific measurement of band 5b TrACP in serum. Methods: Band 5b TrACP was measured kinetically in serum as tartrate-resistant fluoride-sensitive heparin- resistant ACP with 2,6-dichloro-4-acetylphenyl phos- phate as substrate at pH 6.6. Results: Heparin inhibited band 5a TrACP but had no effect on band 5b TrACP in serum or in bone extract. The presence of EDTA or ascorbic acid had no effect, but dithiothreitol inhibited enzyme activity. The with- in-run (n  20) and between-run (n  20) CVs of band 5b TrACP activity were 3.3–5.8% and 5.0 –7.3%, respec- tively. The mean  SD values of band 5b TrACP activity in males (n  25) and females (n  57) 20 –29 years of age by this method were 8.0  2.2 U/L and 6.4  1.8 U/L, respectively. The band 5b TrACP value was signifi- cantly higher in females >50 years of age compared with the younger subjects (20 –29 years). The highest band 5b TrACP values were among children younger than 15 years. Conclusions: This kinetic assay is a simple and specific method for the measurement of band 5b TrACP in serum samples and is useful in the evaluation of bone turnover activity. © 2000 American Association for Clinical Chemistry Serum band 5 tartrate-resistant acid phosphatase (TrACP; 5 EC 3.1.3.2) usually is expressed in certain differ- entiated cells of the mononuclear phagocyte system, no- tably osteoclasts and alveolar macrophages (1), and pathologically in Gaucher cells (2) and in the hairy cells of leukemia reticuloendotheliosis (3). Serum band 5 TrACP is one of the most abundant enzymes in osteoclasts (4) and is regarded as a marker (5, 6). Thus, the serum concentrations of band 5 TrACP constitutes a potential index for the bone resorption rate (7, 8). On the basis of the catalytic and ionic properties and carbohydrate content, two isoenzymes of band 5 TrACP, bands 5a and 5b, have been identified (9). Band 5a TrACP is sialylated, whereas band 5b lacks sialic acid. It has been shown that the removal of sialic acid from band 5a by sialidase changes band 5a to 5b (10 ). Increased band 5b TrACP is liberated from osteoclasts during normal phys- iologic bone growth among healthy children and during metastasis of bone (11 ). The presence of band 5b TrACP in Gaucher serum in spite of the presence of band 5a TrACP in the spleen has been reported (10 ). Thus, the measure- ment of band 5b TrACP appears to be a specific marker for osteoclastic activity. However, no kinetic method for the specific measure- ment of band 5b TrACP in serum has been available. Here we report a simple kinetic method for the measurement of this osteoclastic activity marker (band 5b TrACP) by modifying the method we described previously for the 1 FALCO Biosystems Co., 17-1 Nishiarami Tai, Kumiyama-cho, Kuze-gun, Kyoto 613-0036, Japan. 2 Department of Orthopedic Surgery, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 633-8501, Japan. 3 Nitto Boseki Co. Ltd, 1 Shiojima, Fukuhara, Koriyama, Fukusima 963- 8061, Japan. 4 Department of Medical Zoology, Kobe University School of Medicine, 7-5-1 Kusuniki-cho, Chuo-ku 650-0017, Japan. *Author for correspondence. Fax 0774-46-1101; e-mail falconpd@alles.or.jp. Received May 28, 1999; accepted January 21, 2000. 5 Nonstandard abbreviations: TrACP, tartrate-resistant acid phosphatase; TrFsACP, tartrate-resistant fluoride-sensitive acid phosphatase; and DCAPP, 2,6-dichloro-4-acetylphenyl phosphate. Clinical Chemistry 46:4 469 – 473 (2000) Enzymes and Protein Markers 469