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UHPLC-MS Analyses of Cardiotonic Steroids in Rat Urine and Investigation of their Hydrolysis by Paraoxonases

Lamichhane, Sabitri
http://orcid.org/0000-0001-7622-9889
http://rave.ohiolink.edu/etdc/view?acc_num=toledo1691105473792078

Abstract Details

2023, Doctor of Philosophy, University of Toledo, Chemistry.
Elevated levels of endogenous cardiotonic steroids (CTS), such as telocinobufagin (TCB) and marinobufagin (MBG), are observed in patients with chronic kidney disease (CKD). The characteristic six-membered δ-lactone ring structure of CTS is critical for binding to the Na+ /K+-ATPase and subsequent signaling. Paraoxonases (PONs) belong to a family of hydrolytic enzymes that can regulate levels of δ-lactone ring structures, like those found in CTS, rendering them inactive. Clinical studies have reported decreased lactonase activity of PONs in CKD patients and its association with cardiovascular morbidity and mortality. PON’s activity is diminished in CKD, yet it is unclear whether it is mechanistically linked to increased CTS levels. This research aimed to enhance the knowledge regarding CTS by exploring their regulation in the body and association with PON enzymes, whose exact physiological substrate(s) is/are still unknown. To bridge these knowledge gaps, this research describes novel methods for extraction, detection, and quantification of CTS using highly specific, sensitive, and accurate analytical techniques, such as ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS) as well as HPLC-photodiode array (PDA). In the first project, a protocol was developed for the extraction, detection and quantification of TCB and MBG. Solid-phase extraction (SPE) with hydrophilic-lipophilic balanced (HLB) cartridges was used for the extraction of CTS from an aqueous solution. The methods were optimized to extract and quantify TCB and MBG from the urine of rats with different genotypes; wild type (WT) and PON3 knockout (KO) subjected to different diets (normal chow and high salt) for 8 weeks. The high salt-treated rats serve as a model for studying CKD, while the wild type rats on normal chow act as controls. The results showed that the urine of SS-PON3 KO rats contained higher CTS levels than urine from SS-WT rats for both control and high-salt samples, suggesting its role in CTS regulation. The second project aimed to investigate the role of the enzymes PON1 and PON3 in the degradation of the TCB. TCB was administered at a dose of 100 µg/kg/day subcutaneously (s.q.) for 4 weeks to male salt-sensitive rats with normal PON1 and PON3 levels (WT), as well as to rats lacking either PON1 or PON3. After 4 weeks, the levels of TCB in urine were measured using gamabufutalin (GBT) as an internal standard and utilizing SPE and UHPLC-MS. The results showed that rats lacking PON1 or PON3 had higher TCB levels in their urine compared to the WT rats. These findings suggest that PON1 and PON3 may play a role in the degradation of TCB in vivo. Additionally, a potential metabolite of TCB, called 3-epitelocinobufagin, was putatively identified based on various data, including its retention time, charge, exact mass, and fragmentation data, which matched information in the literature. In the third project, experiments were conducted to investigate TCB hydrolysis and determine the degradation products in vitro and in vivo. In the in vitro experiments, TCB was incubated individually with recombinant PON1 and PON3 enzymes in a buffer. The conversion of TCB from the lactone form to the hydroxy acid form was monitored. Sample purification using SPE was carried out, and elution fractions from the TCB-PONs mixture and control samples were analyzed using HPLC-PDA and UHPLC-MS. Furthermore, experiments were conducted using rat urine samples to determine the degradation products of TCB in vivo. These urine samples were extracted and analyzed using LC-MS/MS. The potential degradation products were analyzed and compared with those obtained from the in vitro studies and alkaline hydrolysis products. Additionally, some preliminary experiments using UPLC interfaced with ion mobility spectrometry MS (UPLC-IMS-MS) were performed to monitor the TCB and MBG and their degradation products. The research investigated whether TCB is a potential endogenous substrate for PON1 and PON3 using LC-PDA and LC-MS. Understanding this physiological correlation could have implications for future clinical investigations and potential treatments for CKD.
Dragan Isailovic (Committee Chair)
David Kennedy (Committee Member)
Emanuela Gionfriddo (Committee Member)
Jianglong Zhu (Committee Member)
152 p.
Biomedical Research; Chemistry
Cardiotonic Steroids; Paraoxonases; Chronic Kidney Disease; Solid-phase Extraction; UHPLC–MS

Recommended Citations

Citations

  • Lamichhane, S. (2023). UHPLC-MS Analyses of Cardiotonic Steroids in Rat Urine and Investigation of their Hydrolysis by Paraoxonases [Doctoral dissertation, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1691105473792078

    APA Style (7th edition)

  • Lamichhane, Sabitri. UHPLC-MS Analyses of Cardiotonic Steroids in Rat Urine and Investigation of their Hydrolysis by Paraoxonases. 2023. University of Toledo, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=toledo1691105473792078.

    MLA Style (8th edition)

  • Lamichhane, Sabitri. "UHPLC-MS Analyses of Cardiotonic Steroids in Rat Urine and Investigation of their Hydrolysis by Paraoxonases." Doctoral dissertation, University of Toledo, 2023. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1691105473792078

    Chicago Manual of Style (17th edition)

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This open access ETD is published by University of Toledo and OhioLINK.