Marinobufagenin extraction from Rhinella marina toad glands: Alternative approaches for a systematized strategy.

Marinobufagenin is a bufadienolide compound detected mainly in skin and parotoid gland secretions of Rhinella marina (L.) toad. Bufadienolides regulate the Na+ /K+ -ATPase pump by inhibiting the cardiotonic steroid dependent-site and act as cardiac inotropes with vasoconstrictive properties. Marinobufagenin and other bufadienolides, such as telocinobufagin and bufalin, are thought to be found endogenously in mammals in salt-sensitive hypertensive states such as essential hypertension, congestive heart-failure, and preeclampsia. The role of marinobufagenin as antimicrobial agent and its cytotoxic potential have also been recognized. The particular interest around marinobufagenin prompts us to consider the Rhinella marina toad venom as a possible source for molecules with pharmacological and/or diagnostic potential. In this article, two different approaches of extraction and purification of marinobufagenin from Rhinella marina (L.) venom are studied: (i) Preparative thin-layer chromatography combined to mass spectrometry and/or ultraviolet detection and (ii) solid-phase extraction coupled with fractionation on high-performance liquid chromatography. Different chromatographic conditions are tested for each approach. The solid-phase extraction combined with high-performance liquid chromatography fractionation approach was preferred as it offered a greater yield, was less time-consuming and allowed us to selectively isolate marinobufagenin. Both protocols aim to provide efficient and convenient methods for toad venom extraction, based on an easily automatable and systematized strategy.

Revealing of endogenous Marinobufagin by an ultra-specific and sensitive UHPLC-MS/MS assay in pregnant women.

Marinobufagenin (MBG) is a bufadienolide cardiac inotrope implicated in volume expansion-mediated hypertensive states including essential hypertension and preeclampsia (PE). Endogenous MBG is an inhibitor of the α1-isoform of Na+,K+-ATPase with vasoconstrictive and cardiotonic properties, causing hypertension and natriuresis. Elevated endogenous MBG-like material levels have been described by immunoassays in salt-sensitive pregnant and preeclamptic rats as well as in preeclamptic human patients. The rise of endogenous MBG-like material appears prior the development of the main symptoms of PE, leading us to consider MBG as one of the potential biomarkers for PE. The weak specificity and the high variability of the published immunoassays gives no certification about endogenous MBG existence. This led us to set-up a highly specific and sensitive analytical method to detect MBG in plasma at low levels relying on liquid chromatography combined to mass spectrometry (UHPLC-MS/MS) with recording of 7 highly specific MRM transitions for MBG. Pure MBG standard used in the method development was obtained by purification from the Bufo marinus toad venom. d3-25-hydroxyvitamin D3 was used as internal standard. An increasing organic gradient with mobile phase A and B composed of 97:3 (v/v) H2O: MeOH and 50:45:5 (v/v/v) MeOH:IPA:H2O at pH 4.5 respectively was used on a Pursuit 3 PFP column (100 mm × 3 mm; 3 µm) to allow elution and separation of the plasmatic compounds. Chromatographic analyses of plasma samples were preceded by a precipitation of proteins pretreatment. The developed UHPLC-MS/MS assay has been applied to early-pregnant women plasma samples allowing us to investigate MBG plasma levels. Thanks to the high specificity of the assay we were able to authenticate and certify the presence of endogenous MBG in early-pregnant women plasma with the use of the 7 selected specific mass transitions. These pioneering preliminary results are giving a promising perspective for early preeclampsia risk assessment in pregnant women.

PP089. Analytical aspects of marinobufagenin and its applications in the diagnosis of preeclampsia.

INTRODUCTION: Marinobufagenin (MBG), a bufadienolide cardiac inotrope, enjoys a growing interest in the early diagnosis of volume expansion-mediated hypertensive states such as preeclampsia (PE). This endogenous mammalian vasoconstrictive compound, is a selective inhibitor of the α1 subunit of Na+,K+-ATPase, leading to hypertension and natriuresis. Enhanced production of MBG has been described in preeclamptic patients prior the development of hypertension and proteinuria, leading to consider MBG as a biomarker for PE [1-3]. However, the role of MBG as a biomarker remains to be fully understood as well as his biosynthetic pathway. The need has arisen for an accuracy and sensitive analytical method of MBG plasma levels in order to further investigate the implications of MBG in PE, and to help to establish a diagnosis for this syndrome. OBJECTIVE: Our aim is to develop a sensitive and robust analytical MBG dosage method allowing quantifications as low as possible. A critical threshold value may be established in order to discriminate normal pregnant from preeclamptic women. METHODS: Nowadays, the MBG standard compound is not commercially available. It forced us to develop an extraction method of MBG from the crystallized toad Bufo Marinus venom. A pre-extraction step on rat and human plasma is performed in order to clean and to concentrate samples. Several liquid chromatography (LC) strategies coupled with different detection methods are considered and performed. RESULTS AND DISCUSSION: Pure MBG has been successfully extracted from the crystallized toad venom. The identity of the compound has been confirmed by different spectral techniques. The pre-extraction step from plasma samples spiked with MBG has been carried out through a solid phase extraction HLB cartridge with an extraction yield of 88%. Preliminary reversed-phase LC method allows quantifications of MBG between 6µg/mL and 11µg/mL.