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

Nattha Yongwattana,Jirarut Wongkongkatep,Tulyapruek Tawonsawatruk,Itaru Hamachi,Nutsara Mekjinda,Akio Ojida

doi:10.3390/molecules25051116

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.

Highlights

Articular cartilage calcification is a phenomenon that causes severe pain and inflammation in the joints of elderly individuals, overlapping broadly with osteoarthritis
Www.mdpi.com/journal/molecules hydrolyzed via two pathways: 1) through ecto-nucleotide pyrophosphatase/phosphodiesterases (e-NPPs), forming people. The accumulation of pyrophosphate (PPi) which can be subsequently hydrolyzed to Pi via the activated tissue nonspecific alkaline phosphatase (TNAP); and 2) though e-NTPDase, which converts ATP to adenosine 5’-diphosphate disodium (ADP) and adenosine 3’-monophosphate (AMP)
After efflux from the chondrocyte via the multipass membrane ANKH protein, ATP is hydrolyzed via two pathways: 1) through e-NPP, forming PPi which can be subsequently hydrolyzed to Pi via the activated TNAP; and 2) though e-NTPDase, which converts ATP to ADP and AMP simultaneously (Figure 1a)

Summary

Introduction

Articular cartilage calcification is a phenomenon that causes severe pain and inflammation in the joints of elderly individuals, overlapping broadly with osteoarthritis. Among the calcium salts that cause articular cartilage calcification, calcium pyrophosphate dihydrate (CPP) and calcium phosphate are found in over 20% of population over 60 years of age [1], and in 60% of patients considering joint arthroplasty [2]. The deposition of CPP crystal in joints is referred to as calcium pyrophosphate deposition disease (CPPD), known as pseudogout until recently [3]. The most accurate indicator of CPPD is the positive birefringence of the CPP crystals in synovial fluid. We began by verifying the fluorescent signal of Kyoto Green toward various molecules that are commonly found in biological fluids.

Methods

Results

Conclusion