Development of a Kinetic Assay for Band 5b Tartrate-resistant Acid Phosphatase Activity in Serum

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Abstract
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 phosphate 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 within-run (n = 20) and between-run (n = 20) CVs of band 5b TrACP activity were 3.3–5.8% and 5.0–7.3%, respectively. 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 significantly 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.