Clinical and Technical Review - Tecomedical

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20 Measurement of BAP and Osteocalcin in cell culture • DMEM or equivalent commercial medium • Fetal bovine serum or calf serum or serum substitutes (FCS free medium) • L-Glutamine or Ascorbat • Antibiotic Additionally, 50 nM Vitamin D3 is necessary to generate measurable quantities of osteocalcin. The cells can be grown in serum containing media, but osteocalcin must be harvested from tissue culture supernatant that is serumfree, because the antibody will also detect this analyte in serum. Bone alkaline phosphatase is membrane-bound and it has to be measured in culture from cell lysate. Cell culture media should not be treated with ion chelators like EDTA or citrate because of an inhibition of the BAP enzyme activity. After the incubation the cell layers should be washed twice with saline and scraped into TMN buffer solution (20 mM Tris-HCl, pH 7.4; 2 mM MgCl2; 150 mM NaCl) uising a stir stick. The number of cells has to be determined (e.g. Coulter cell counter Model F) before the cells become solubilized by the addition of Triton X-100 to a final concentration of 1 %. Centrifugate the samples at 70,000g for 60 min and examine the aliquots of the supernatant for alkaline phosphatase activity. Sheep and human cells will be fine for this purpose. References for BAP, PICP, OSTEOCALCIN in cell culture Kaspar D, Seidl W, Neidlinger-Wilke C, Ignatius A, Claes L (2000) Dynamic cell stretching increases human osteoblast proliferation and CICP synthesis but decreases osteocalcin synthesis and alkaline phosphatase activity. J Biomech 33, 45-51. Martínez ME, Medina S, Del Campo MT, et al. (1998) Effect of polyethylene on osteocalcin, alkaline phosphatase and procollagen secretion by human osteoblastic cells. Calcif Tissue Int 62, 453-456. Measurement of CICP / PICP in cell culture The assay is suitable for the measurement of cell culture supernatant from cells producing type-I collagen. The METRA ® CICP Test can be used for the determination of the collagen production level of skin fibroblasts and bone cells. It is recommended, that the supernatant of these cultures contains serum-free medium. Bovine serum in the medium may contain bovine CICP, which cross-reacts with the antibody in the kit. This would result in erroneously increased values. In a confluating cell culture at an optimal collagen production level, the amount of CICP in the supernatant is approximately 20 – 50 ng/ml or slightly below a normal human serum sample. Dilute the cell culture supernatant 1:12 (like a normal serum sample) and calculate the final concentration according to the dilution. If the CICP concentration of the 1:12 dilution is below the detection limit it is possible to choose a 1:6-dilution. Sell S, Gaissmaier C, Fritz J et al. (1998) Different behavior of human osteoblast-like cells isolated from normal and heterotopic bone in vitro. Calcif Tissue Int 62, 51-59. Siggelkow H, Niedhart C, Kurre W, et al. (1998) In vitro differentiation potential of a new human osteosarcoma cell line (HOS 58). Differentiation 63, 81-91. Siggelkow H, Rebenstorff K, Kurre W, et al. (1999) Development of the osteoblast phenotype in primary human osteoblasts in culture: comparison with rat calvarial cells in osteoblast differentiation. J Cell Biochem 75, 22-35.
Bone marker - Reference values in animals BAP in Dogs Serum / Plasma: < 1 year 56.3 ± 9.8 U/L 1 – 2 years 10.7 ± 4.5 U/L 2 – 3 years 7.0 ± 2.5 U/L 3 – 7 years 6.7 ± 3.6 U/L > 8 years 7.0 ± 2.9 U/L Reference: Allen LC et al. (2000) A comparison of two techniques for the determination of serum bone-specific alkaline phosphatase activity in dogs. Res Vet Sci 68, 231-235. BAP in Cats Serum: < 2 years 10 – 70 U/L > 2 years 2 – 15 U/L Reference: DeLaurier A, Jackson B, Ingham K, Pfeiffer D, Horton MA, Price JS. (2002) Biochemical markers of bone turnover in the domestic cat: relationships with age and feline osteoclastic resorptive lesions. J Nutr 132, 1742S-4S. BAP in Horses Serum: 12.2 – 25.5 U/L Plasma: 12.6 – 22.7 U/L Reference: Hoekstra K et al. (1999) Comparison of bone mineral content and biochemical markers of bone metabolism in stall vs. pasture-reared horses. Equine Exercise Phys Equine Vet J 30, 601-604. BAP in goats Serum: 12 ± 4 U/L Reference: Liesegang A, Risteli J, Wanner M (2005) The effects of first gestation and lactation on bone metabolism in dairy goats and milk sheep. Bone. Dec 16; [Epub ahead of print] BAP in sheep Serum: 13 ± 4 U/L Reference: Liesegang A, Risteli J, Wanner M (2005) The effects of first gestation and lactation on bone metabolism in dairy goats and milk sheep. Bone. Dec 16; [Epub ahead of print] BAP in porcine Reference: Liesegang A et al. (2002) Influence of a Vegetarian Diet Versus a Diet with Fishmeal on Bone in Growing Pigs. J. Vet. Med. A 49, 230-238 21
Page 1 and 2: Author: Peter Haima, Ph Clinical an
Page 3 and 4: Figure 1: Soloway 0 = Patients with
Page 5 and 6: Bone Resorption Osteoclast: Large m
Page 7 and 8: Bone Turnover - Biochemical Marker
Page 9 and 10: Bone marker and their behavior in v
Page 11 and 12: Biomarker according to NIH Osteoart
Page 13 and 14: Cartilage Synthesis - Biochemical M
Page 15 and 16: Cartilage Biomarker and their behav
Page 17 and 18: Bonemarker normal values for childr
Page 19: Specific recommendations BAP Osteoc
Page 23 and 24: Cross reactivity - different specie

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