Charline Lenaerts

Analytical aspects of marinobufagenin.

Marinobufagenin (MBG), a steroid compound belonging to the bufadienolide cardiac inotropes, is a molecule enjoying a growing interest in the early diagnostic of volume expansion-mediated hypertensive states. This endogenous mammalian cardiotonic and natriuretic bufadienolide (characterized by vasoconstrictive activities) inhibits the α1 isoform of Na(+), K(+)-ATPase, implicating it in series of pathophysiological circumstances such as volume-expansion, essential hypertension and preeclampsia. Indeed, the enhanced production of MBG in preeclamptic patients has been confirmed in several studies, leading us to consider MBG as a biomarker for preeclampsia. The main source for MBG is located in the parotid and skin gland secretions of the toad Bufo marinus in which MBG is the major steroid cardiotonic component. This review emphasizes the key role of analytical development for dosage methods of MBG in biofluids, in the emergence of future perspectives in the diagnostic and therapeutic fields of preeclampsia (e.g. to investigate the biosynthetic origin of MBG and to better understand its implications).

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.

D3. Early prediction of preeclampsia risk assessment: analytical determination for marinobufagenin in pregnant women

Abstract Introduction: Marinobufagenin (MBG), an endogenous bufadienolide cardiac inotrope, is responsible for vasoconstriction and natriuresis [1,2]. Growing interest by its implication in volume expansion mediated hypertensive states such as essential hypertension, congestive heart failure, chronic renal failure, primary aldosterism and preeclampsia (PE). Elevated endogenous MBG levels have been described early in preeclamptic patients [2–5]. Aims: To develop a specific and sensitive analytical MBG assay with special attention for the limit of quantification (LOQ). An algorithm dealing with the MBG plasma levels might be established in the future, in order to help for prediction of the risk for preeclampsia in pregnant women. Method: A LC-MS/MS based assay designed to determine MBG in human plasma is being optimized. The obtained LOQ fully satisfies the need for quantification of MBG plasma levels in pregnancy (nmol/L range). After a validation process, a primary observational clinical study will allow us to confirm previous results observed in preeclampsia. Results and discussion: We had the opportunity to authenticate the presence of MBG by LC-MS/MS in non-pregnant and in early-pregnant women. These pioneering preliminary results that are giving a promising perspective for early preeclampsia risk assessment in pregnant women will be confirmed with the clinical study.

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.