Human Bone Alkaline Phosphatase Isoforms

Sammanfattning: Determination of serum total alkaline phosphatase (ALP) is frequently requested in clinical routine, mostly to estimate skeletal and hepatobiliary status. In this respect, clarification of the various ALP isoenzymes and isoforms contributing to the total ALP activity could be valuable in daily medical decision making. The general aim of this thesis was to investigate methodological, metabolic, and clinical aspects of bone ALP (BALP) isoforms in human bone and mineral metabolism. BALP is a glycoprotein and functions as an ectoenzyme attached to the osteoblast cell membrane by a glycosyl-phosphatidylinositol (GPI) anchor. The precise function of BALP is not known, however, there is evidence that BALP is necessary for initiating bone mineralization.A weak anion-exchange high-performance liquid chromatography (HPLC) assay was developed for the determination of BALP and liver ALP (LALP) isoforms. Six peaks with ALP activity were separated and quantified in serum from healthy individuals: B/I, a minor fraction composed of bone (70%) and intestinal (30%) ALP, and two major BALP isoforms B 1 and B2, and three LALP isoforms. Reference intervals were reported for healthy children, adolescents, and adults (range 7-65 years). In healthy adults the BALP isoforms, B/I, Bl, and B2, contributed to 4, 16, and 37%, respectively, of the total ALP activity. Bone samples were prepared from human femora in order to characterize and investigate the origin of these BALP isoforms found in serum. Cortical bone had about 2-fold higher activities of Bl compared with B2, and trabecular bone had about 2-fold higher activities of B2 compared with Bl. Treatment with GPI -specific phospholipase C did not influence the activities or retention times of Bland B2. Thus, the biochemical differences between Bland B2 are likely to be due to different glycosylation patterns, rather than the presence of GPI cell membrane anchor fragments.Decreased B I activity was observed after I week of IGF-I administration, and after I month of GH therapy, followed by an increase after 3 months. B2 was not influenced by IGF-I administration, but was similarly increased after 3 months of GH therapy. It was proposed that the initial decrease of B 1 could be an effect of endocrine IGF-I action mediated by GH. Different responses of B 1 and B2 during IGF-I and during GH therapy suggest different regulations of these BALP isoforms in vivo. Differences of BALP isoforms in metastatic bone disease were found, as well as discrepant effects of clodronate on different skeletal sites indicated by the location of bone pain. Patients with skeletal metastases and healthy males had B2 activities corresponding to 75% and 35% of the total ALP activity, respectively.Taken together, the BALP isoforms B I and B2 can be used as early indicators of pharmacological efficacy and, possibly provide information relating to specific bone compartments. Future investigations have to elucidate if they also reflect different stages in osteoblast differentiation during osteogenesis where one isoform is presented before the other during extracellular matrix maturation.