Fluorescence determination of soluble pyrophosphate levels in synovial fluid as a marker of pseudogout using middle point of quantification concept and molecular sensor

Nattha Yongwattana^a, Nutsara Mekjinda^a, Wannee Thepsing^a, Supasara Ounsuk^a, Pravit Wongkongkatep^a, Tulyapruek Tawonsawatruk^b, Itaru Hamachi^c, Akio Ojida^d, Jirarut Wongkongkatep^a,?

ABSTRACT: Pseudogout is a type of joint inflammations caused by deposition of calcium pyrophosphate (CaPPi) crystals in the affected joint. As Ca²⁺ is abundant in the synovial fluid (SF), high levels of soluble PPi in the SF could be one of the key factors that contribute to CaPPi formation in the joint and may serve as a biomarker for pseudogout. Here, we developed and applied an artificial molecular sensor to selective fluorescent detection of soluble PPi in SF of the arthritis patients. The sensor employed xanthene as a fluorophore and the Dpa/Zn(II) as two specific binding sites for PPi. When titrated with serially diluted aqueous PPi solutions, the sensor displayed high sensitivity and exhibited the detection limit of 0.01 µM. The effect of salt concentration was normalized via the concept of Middle Point of Quantification (MPOQ) firstly proposed in this study. The performance of this sensor was also further validated by testing with SF samples extracted from eight clinical patients. The results revealed that six patients had the PPi levels in the range of 60 and 200 µM, indicating moderate likelihood of having pseudogout. Hence, our new method for determining the soluble PPi levels in SF shows promise as a robust, sensitive, and accurate diagnostic tool for the pseudogout.

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^a	Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok 10400 Thailand
^b	Department of Orthopedics, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400 Thailand
^c	Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510 Japan
^d	Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582 Japan

* Corresponding author, E-mail: jirarut.chu@mahidol.ac.th

Received 2 Nov 2019, Accepted 25 Feb 2020

Fluorescence determination of soluble pyrophosphate levels in synovial fluid as a marker of pseudogout using middle point of quantification concept and molecular sensor

Nattha Yongwattanaa, Nutsara Mekjindaa, Wannee Thepsinga, Supasara Ounsuka, Pravit Wongkongkatepa, Tulyapruek Tawonsawatrukb, Itaru Hamachic, Akio Ojidad, Jirarut Wongkongkatepa,?

Nattha Yongwattana^a, Nutsara Mekjinda^a, Wannee Thepsing^a, Supasara Ounsuk^a, Pravit Wongkongkatep^a, Tulyapruek Tawonsawatruk^b, Itaru Hamachi^c, Akio Ojida^d, Jirarut Wongkongkatep^a,?