Fluorescence Differentiation of ATP-Related Multiple Enzymatic Activities in Synovial Fluid as a Marker of Calcium Pyrophosphate Deposition Disease Using Kyoto Green

Abstract

Calcium pyrophosphate deposition disease (CPPD) is a crystal induced inflammation in joints, and causes severe pain in elderly people. The accumulation of pyrophosphate (PPi) in synovial fluid (SF) results from several enzymatic reactions, especially the highly activated e-NPPs, which catalyze the conversion of ATP to PPi. This study demonstrates the detection of relative catalytic activity of 3 enzymes—ecto-nucleotide pyrophosphatase/phosphodiesterases (e-NPPs), tissue nonspecific alkaline phosphatase (TNAP), and ecto-nucleoside triphosphate diphosphohydrolases (e-NTPDases)—using a single molecular sensor called Kyoto Green. Kyoto Green exhibits excellent performance in sensing the catalytic activity of the commercial representatives of the e-NPPs, TNAP, and e-NTPDases, which are ENPP1, PPase, and apyrase, respectively, in both single-enzyme and multi-enzyme assays. Analysis of SF enzymes in 19 SF samples from human and swine revealed moderate activity of e-NPPs, high activity of e-NTPDases, and low activity of TNAP. Our newly developed method for analysis of multiple enzymatic activities using Kyoto Green in biological SF will assist improvement in accuracy of the CPPD prognosis/diagnosis, which will minimize unnecessary medical procedures.

Naturally occurring quercetin and myricetin as potent inhibitors for human ectonucleotide pyrophosphatase/phosphodiesterase 1

“…The screening was performed with a total volume of 200 µl on a white 96-well plate, using the condition described by Yongwattana et al 7 . The assay solution contained 50 mM HEPES buffer (pH 7.4), 10 mM NaCl, 1 mM MgCl 2 , 20 µM Zn(NO 3 ) 2 , 2 µM ATP, 1 µM Kyoto Green and each test compound at 10 µM or maximum soluble concentration in water.…”

“…The most notable member of human NPPs is ENPP1, which is highly expressed in osteoblasts and chrondrocytes, and plays a crucial role in bone mineralization 5 . Moreover, high activity of ENPP1 has also been linked to crystal deposition diseases such as soft tissue calcification and pseudogout, as well as multiple types of cancer, including lung, breast, and ovarian cancer 6 , 7 . The association with these diseases make ENPP1 an attractive target for immunotherapy of various cancers and tissue calcification 8 – 16 .…”

“…Therefore, the measurement of ATP hydrolysis by monitoring its hydrolyzed product, inorganic pyrophosphate (PPi), would be a simpler and more accurate strategy for quantifying ENPP1 activity, without complications due to chemical derivatization and allosteric modulation. For this purpose, sensitive and real-time detection of PPi can be achieved by using Kyoto Green (xanthene dipicolylamine Zn(II) complex), a molecular fluorescent sensor extensively employed for the detection of PPi under physiological conditions and in synovial fluids 7 , 23 – 26 . However, to our knowledge, Kyoto Green has not been leveraged for screening of ENPP1 inhibitors.…”

Fluorescence identification of arthropathic calcium pyrophosphate single crystals using alizarin red S and a xanthene dipicolylamine ZnIIcomplex

“…S4 †). 24,25 For these reasons, it has been employed for the prognosis of pseudogout through the evaluation of the relative enzymatic activity 26 and quantification of pyrophosphate levels 27 in the patient's synovial fluid. In this study, we aimed to utilize the XDZ complex for the detection of the pyrophosphate anion dissolved from the CPPD crystals.…”

“…The selectivity was further enhanced by incorporating a highly specific fluorescent sensor for pyrophosphate based on a xanthene dipicolylamine Zn II (XDZ) complex, which has shown excellent performance in the detection of pyrophosphate and polyphosphate anions in water under physiological conditions. [24][25][26][27] By combining these strategies, we successfully obtained a twostep fluorescence workflow for identifying the arthropathic single crystals of both triclinic calcium pyrophosphate dihydrate (t-CPPD) and monoclinic calcium pyrophosphate dihydrate (m-CPPD).…”