Clinical performance of six different serum tartrate-resistant acid phosphatase assays for monitoring alendronate treatment.

Two forms of tartrate-resistant acid phosphatase (TRACP) circulate in human blood, TRACP 5a derived from inflammatory macrophages and TRACP 5b derived from osteoclasts. We compared the clinical performance of the following TRACP immunoassays for monitoring alendronate treatment in postmenopausal women: 1) TRACP 5b activity using a selective pH; 2) TRACP 5b activity using a selective substrate; 3) Total TRACP activity; 4) Total TRACP protein amount; 5) TRACP 5a activity; 6) TRACP 5a protein amount. TRACP and other bone turnover markers were measured before the start of treatment and at 3 months. Alendronate treatment decreased TRACP values determined with assays 1, 2 and 3, and had no effect on the values determined with assays 4, 5 and 6. Clinical performance of assays 1, 2 and 3 was good, and these assays correlated with each other and with the other bone markers. This study showed that TRACP 5b specific methods are useful for monitoring changes in bone resorption during alendronate treatment, and alendronate treatment does not affect serum TRACP 5a levels.

Inhibition of ERK1/2 activation by phenolic antioxidants protects kidney tubular cells during cold storage.

BACKGROUND: Cold storage of tissues induces reactive oxygen species (ROS), which contribute to cell injury. We have compared different antioxidants in protection of renal tubular cells against hypothermia injury and studied their effect on cold-induced mitogen-activated protein (MAP) kinase activation. METHODS: Cultured renal tubular epithelial cells (LLC-PK1) were stored in University of Wisconsin solution supplemented with compounds tested for 16 hr at 4 degrees C. Release of lactate dehydrogenase and cellular adenosine triphosphate were measured. Activation of MAP kinases was determined by Western blotting. Intracellular ROS were monitored with a fluorescent probe. RESULTS: Cold storage resulted in a substantial loss of cell viability. The simple phenol butylated hydroxyanisol (BHA) most effectively prevented hypothermia-induced cell injury, whereas about 100-fold higher concentration of the polyphenol epigallocatechin gallate (EGCG) was needed, although EGCG most effectively scavenged intracellular ROS elicited by serum withdrawal. The MEK inhibitor U0126 and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenyleneiodonium effectively protected the cells against hypothermia injury. ERK1/2 was rapidly activated during chilling of the cells and this was inhibited by BHA but not by EGCG. CONCLUSION: The results suggest that chilling of renal epithelial cells induces ROS generation by NADPH oxidase, which leads to rapid activation of the MEK-ERK1/2 cascade and initiation of cell injury. This can be prevented by antioxidants.

Tartrate-resistant acid phosphatase 5b (TRACP 5b) as a marker of bone resorption.

Tartrate-resistant acid phosphatase (TRACP) is an enzyme that is expressed in high amounts by bone resorbing osteoclasts, inflammatory macrophages and dendritic cells. Two forms of TRACP circulate in human blood, TRACP 5a derived from macrophages and dendritic cells, and TRACP 5b derived from osteoclasts. Recent data have demonstrated the utility of TRACP 5b as a marker of osteoclast number and bone resorption, and serum TRACP 5a as a marker of inflammatory conditions. This review summarizes the scientific knowledge on the role of TRACP in osteoclastic bone resorption, the mechanism of TRACP 5b generation in osteoclasts and its secretion into the blood circulation, the methodology of measuring TRACP 5b, diagnostic evidence for the use of TRACP 5b as a resorption marker, and characteristics of TRACP 5b compared to other commonly used bone turnover markers.

Effects of proteolysis and reduction on phosphatase and ROS-generating activity of human tartrate-resistant acid phosphatase.

Osteoclasts and macrophages express high amounts of tartrate-resistant acid phosphatase (TRACP), an enzyme with unknown biological function. TRACP contains a disulfide bond, a protease-sensitive loop peptide, and a redox-active iron that can catalyze formation of reactive oxygen species (ROS). We studied the effects of proteolytic cleavage by trypsin, reduction of the disulfide bond by beta-mercaptoethanol, and reduction of the redox-active iron by ascorbate on the phosphatase and ROS-generating activity of baculovirus-generated recombinant human TRACP. Ascorbate alone and trypsin in combination with beta-mercaptoethanol increased k(cat)/K(m) of the phosphatase activity seven- to ninefold. The pH-optimum was changed from 5.4-5.6 to 6.2-6.4 by ascorbate and trypsin cleavage. Trypsin cleavage increased k(cat)/K(m) of the ROS-generating activity 2.5-fold without affecting the pH-optimum (7.0). These results suggest that the protease-sensitive loop peptide, redox-active iron, and disulfide bond are important regulatory sites in TRACP, and that the phosphatase and ROS-generating activity are performed with different reaction mechanisms.