2758 The Journal of Rheumatology 2009; 36:12; doi:10.3899/jrheum.090098 Personal non-commercial use only. The Journal of Rheumatology Copyright © 2009. All rights reserved. Mercaptopyruvate Inhibits Tissue-Nonspecific Alkaline Phosphatase and Calcium Pyrophosphate Dihydrate Crystal Dissolution JOVILV.A. KANNAMPUZHA, JINDRAH. TUPY, and KENNETH P.H. PRITZKER ABSTRACT. Objective. The enzymatic activities of tissue-nonspecific alkaline phosphatase (TNAP) including capacity to inhibit calcium pyrophosphate dihydrate (CPPD) crystal dissolution are known to be inhibited by endogenous amino acids, notably cysteine. As cysteine is recognized as a strong TNAP inhibitor, we investigated whether cysteine-related metabolites such as mercaptopyruvate (MPA) could show similar enzyme inhibition effects and, if so, whether these effects might be synergistic with cysteine at approximate physiologic concentrations of the amino acids. Methods. We studied the inhibitory effects of MPA as well as MPA and cysteine combined in equimolar concentrations on TNAP’s phosphatase, inorganic pyrophosphatase, and CPPD crystal dissolution activities. Kinetic parameters V max ,K M , concentration for 50% inhibition (I 50 ), inhibitor constant (K I ), and specific activities calculated from initial velocity, Eadie-Hofstee, Simple, Dixon, and secondary plots were used to assess enzyme inhibition. Results. MPAsignificantly inhibited TNAP’s phosphatase and pyrophosphatase activities at 10× and 100× physiological concentrations. In the presence of calcium [Ca 2+ ] and [Mg 2+ ] = 1 mM, MPA inhibited uncompetitively TNAP’s phosphatase activity and inhibited noncompetitively its pyrophosphatase activity. CPPD crystal dissolution activity was also inhibited. Cysteine and MPA together in equimolar concentrations inhibited TNAP enzyme activities and CPPD crystal dissolu- tion much more effectively than MPAor cysteine alone, reducing CPPD dissolution to 38% of con- trols at approximate physiologic inhibitor concentrations. Conclusion. Endogenous amino acids like cysteine and its derivative MPA have the capacity to inhibit TNAP activities at physiologic concentrations. Downregulation of their inhibiting concentra- tion in the cartilage interstitial fluid environment may provide a therapeutic avenue to controlled dis- solution of CPPD crystal deposition in tissues. (First Release Nov 1 2009; J Rheumatol 2009;36: 2758–65; doi:10.3899/jrheum.090098) Key Indexing Terms: ALKALINEPHOSPHATASE MERCAPTOPYRUVATE AMINO ACIDS CALCIUM PYROPHOSPHATE ARTHRITIS CARTILAGE From the Department of Pathology and Laboratory Medicine, Mount Sinai Hospital; and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada. J.V.A. Kannampuzha, HBSc, MSc; J.H. Tupy, MSc; K.P.H. Pritzker, MD, FRCPC, Professor, University of Toronto. Address correspondence to Dr. K.P.H. Pritzker, Pathology and Laboratory Medicine, Mount Sinai Hospital, 600 University Avenue, Room 6-500, Toronto, Ontario M5G 1X5, Canada. E-mail: kpritzker@mtsinai.on.ca Accepted for publication July 6, 2009. Calcium pyrophosphate dihydrate crystal deposition disease (CPPDD), a common form of arthropathy in the elderly, is characterized by calcium pyrophosphate dihydrate (CPPD) crystal deposition in the articular joints. Symptoms of CPPDD include joint swelling and pain in the knees, wrists, ankles, and other joints, and in some cases result in severe disability. Also referred to as pseudogout, CPPDD can be initially misdiagnosed as gouty arthritis, rheumatoid arthri- tis, and osteoarthritis because of similar symptoms. CPPD crystal deposition is restricted to articular connec- tive tissues, preferentially affecting fibrocartilage, hyaline cartilage, and tissues such as synovium and intervertebral disc, which undergo fibrocartilaginous metaplasia. CPPD crystals are very insoluble 1-3 . Therapy for CPPDD remains nonspecific, for example, nonsteroidal antiinflammatory drugs (NSAID) such as indomethacin or analgesics. Better understanding of the disease mechanisms related to CPPD crystal formation and dissolution appears to be a prerequi- site to the development of novel specific treatment options. Tissue-nonspecific alkaline phosphatase (TNAP) prefer- entially hydrolyzes various monophosphate esters at alka- line pH and pyrophosphates at a physiological pH = 7.4 4-6 . Little is known about the physiological function of TNAP in most tissues except that the bone isoenzyme has long been thought to have a role in normal skeletal mineralization. TNAP is located extracellularly, tethered to the outer cell membrane by a phosphatidylinositol linkage. It is present on the cell membrane of adult articular chondrocytes, particu- larly in the mid and deep zones of articular cartilage. www.jrheum.org Downloaded on April 19, 2022 from