Identification of marinobufagenin in plasma as a promising LC-MS assay for preeclampsia risk assessment

Marinobufagenin (MBG) is a bufadienolide compound belonging to the cardiac glycoside class and is present in humans as well as in some plants and other animals. The major source for this compound is located in the parotid and skin gland secretions of some toad species, notably Bufo Marinus species. Endogenous mammalian MBG acts principally as a vasoconstrictive and cardiotonic compound that inhibits the α1 isoform of Na+,K+-ATPase, resulting in hypertension and natriuresis. The enhanced production of MBG has been described in mammals presenting volume expansion-mediated hypertensive states, notably in preeclamptic patients [1-3]. The elevation of endogenous MBG appears prior the development of the main symptoms of preeclampsia (PE), leading us to consider MBG as a potential biomarker for PE. Nowadays, a sensitive and accurate analytical method has become indispensable to assess MBG in as lower level as possible in plasma. A critical threshold value might be established by clinicians in the future, in order to predict the risk for preeclampsia in pregnant women. A MBG standard compound is currently not commercially available. It forced us to develop an effective extraction and purification method of MBG from freshly collected or crystallized toad Bufo Marinus venom. The identity of the compound has been confirmed by different spectral techniques. Currently, only marinobufagenin-like material has been found in humans using two published quantification methods based each on immunoassays. These techniques suffer from a lack of specificity due to cross-reactivity and tend to exhibit high variability at low concentrations [4]. This condition has led us to authenticate the presence of MBG in humans using a more specific and accessible technique. The research work currently presented deals with the optimization of a LC-MS based assay in order to identify and quantify MBG in human plasma. A pre-extraction procedure is needed to concentrate and clean the sample prior to its analysis. The identification of MBG in non-pregnant healthy volunteers as well as in early pregnancy will be demonstrated, giving the clinicians a promising opportunity for early preeclampsia risk assessment in pregnant women. References 1. Lopatin, D.A., et al., Circulating bufodienolide and cardenolide sodium pump inhibitors in preeclampsia. Journal of Hypertension, 1999. 17(8): p. 1179-1187. 2. Agunanne, E., et al., Marinobufagenin Levels in Preeclamptic Patients: A Preliminary Report. American Journal of Perinatology, 2011. 28(7): p. 509-514. 3. Vu, H.V., et al., Involvement of Marinobufagenin in a Rat Model of Human Preeclampsia. American Journal of Nephrology, 2005. 25(5): p. 520-528. 4. Jarvis, Ultra-sensitive analysis of aldosterone in serum using the AB SCIEX Triple QuadTM 6500 LC/MS/MS system, AB SCIEX, 5730212-0

Analysis of marinobufagenin in plasma for preeclampsia risk assessment: a promising early predictive tool

peer reviewedMarinobufagenin (MBG) is a bufadienolide compound belonging to the cardiac glycoside class and is present in humans as well as in some plants and other animals. The major source for this compound is located in the parotid and skin gland secretions of some toad species, notably Bufo Marinus species. Endogenous mammalian MBG acts principally as a vasoconstrictive and cardiotonic compound that inhibits the α1 isoform of Na+,K+-ATPase, resulting in hypertension and natriuresis. The enhanced production of MBG has been described in mammals presenting volume expansion-mediated hypertensive states, notably in preeclamptic patients [1-3]. The elevation of endogenous MBG appears prior the development of the main symptoms of preeclampsia (PE), leading us to consider MBG as a potential biomarker for PE. Nowadays, a sensitive and accurate analytical method has become indispensable to assess MBG levels in plasma as it is expected to be present in picogram/mL concentrations. A diagnostic threshold value might be established by clinicians in the future, in order to predict the risk for preeclampsia in pregnant women. A MBG standard compound is currently not commercially available. It forced us to develop an effective extraction and purification method of MBG from freshly collected or crystallized toad Bufo Marinus venom. The identity of the compound has been confirmed by different spectral techniques. Currently, only marinobufagenin-like material has been found in humans using two published quantification methods based each on immunoassays [4, 5]. These techniques suffer from a lack of specificity due to cross-reactivity and tend to exhibit high variability at low concentrations [6]. This condition has led us to authenticate the presence of MBG in humans using a more specific and accessible technique. The research work currently presented deals with the optimization of a LC-MS based assay in order to identify and quantify MBG in human plasma. A pre-extraction procedure is needed to concentrate and clean the sample prior to its analysis. The identification of MBG in non-pregnant healthy volunteers as well as in early pregnancy will be demonstrated, giving the clinicians a promising opportunity to further assess the use of MBG for early preeclampsia risk assessment in pregnant women. References 1. Lopatin, D.A., et al., Circulating bufodienolide and cardenolide sodium pump inhibitors in preeclampsia. Journal of Hypertension, 1999. 17(8): p. 1179-1187. 2. Agunanne, E., et al., Marinobufagenin Levels in Preeclamptic Patients: A Preliminary Report. American Journal of Perinatology, 2011. 28(7): p. 509-514. 3. Vu, H.V., et al., Involvement of Marinobufagenin in a Rat Model of Human Preeclampsia. American Journal of Nephrology, 2005. 25(5): p. 520-528. 4. Abi-Ghanem, D., et al., A CHEMIFLUORESCENT IMMUNOASSAY FOR THE DETERMINATION OF MARINOBUFAGENIN IN BODY FLUIDS. Journal of Immunoassay and Immunochemistry, 2011. 32(1): p. 31-46. 5. Fedorova, O.V., et al., Endogenous Ligand of α1 Sodium Pump, Marinobufagenin, Is a Novel Mediator of Sodium Chloride-Dependent Hypertension. Circulation, 2002. 105(9): p. 1122-1127. 6. Jarvis, Ultra-sensitive analysis of aldosterone in serum using the AB SCIEX Triple QuadTM 6500 LC/MS/MS system, AB SCIEX, 5730212-0

Relationship between the concentration of ergothioneine in plasma and the likelihood of developing pre-eclampsia

Ergothioneine, an antioxidant nutraceutical mainly at present derived from the dietary intake of mushrooms, has been suggested as a preventive for pre-eclampsia. We analysed early pregnancy samples for a cohort of 432 first time mothers as part of the Screening for Endpoints in Pregnancy (SCOPE, European branch) project to determine the concentration of ergothioneine in their plasma. There was a weak association between the ergothioneine levels and maternal age, but none for BMI. Of these 432 women, 97 went on to develop pre-term (23) or term (74) pre-eclampsia. If a threshold was set at the 90 th percentile of the reference range in the control population (≥ 462 ng/mL), only one of these 97 women (1%) developed pre-eclampsia, versus 97/432 (22.5%) whose ergothioneine level was below this threshold. One possible interpretation of these findings, consistent with previous experiments in a reduced uterine perfusion model in rats, is that ergothioneine may indeed prove protective against pre-eclampsia in humans. An intervention study of some kind now seems warranted

Early cost-effectiveness analysis of screening for preeclampsia in nulliparous women:A modelling approach in European high-income settings

BACKGROUND: Preeclampsia causes substantial maternal and perinatal morbidity and mortality and significant societal economic impact. Effective screening would facilitate timely and appropriate prevention and management of preeclampsia. OBJECTIVES: To develop an early cost-effectiveness analysis to assess both costs and health outcomes of a new screening test for preeclampsia from a healthcare payer perspective, in the United Kingdom (UK), Ireland, the Netherlands and Sweden. METHODS: A decision tree over a 9-month time horizon was developed to explore the cost-effectiveness of the new screening test for preeclampsia compared to the current screening strategy. The new test strategy is being developed so that it can stratify healthy low risk nulliparous women early in pregnancy to either a high-risk group with a risk of 1 in 6 or more of developing preeclampsia, or a low-risk group with a risk of 1 in 100 or less. The model simulated 25 plausible scenarios in a hypothetical cohort of 100,000 pregnant women, in which the sensitivity and specificity of the new test were varied to set a benchmark for the minimum test performance that is needed for the test to become cost-effective. The input parameters and costs were mainly derived from published literature. The main outcome was incremental costs per preeclampsia case averted, expressed as an incremental cost-effectiveness ratio (ICER). Deterministic and probabilistic sensitivity analyses were conducted to assess uncertainty. RESULTS: Base case results showed that the new test strategy would be more effective and less costly compared to the current situation in the UK. In the Netherlands, the majority of scenarios would be cost-effective from a threshold of €50,000 per preeclampsia case averted, while in Ireland and Sweden, the vast majority of scenarios would be considered cost-effective only when a threshold of €100,000 was used. In the best case analyses, ICERs were more favourable in all four participating countries. Aspirin effectiveness, prevalence of preeclampsia, accuracy of the new screening test and cost of regular antenatal care were identified as driving factors for the cost-effectiveness of screening for preeclampsia. CONCLUSION: The results indicate that the new screening test for preeclampsia has potential to be cost-effective. Further studies based on proven accuracy of the test will confirm whether the new screening test is a cost-effective additional option to the current situation

Preeclampsia risk stratification early in pregnancy: Conversion of a promising metabolomics discovery into a LC-MS based clinical assay

Basic metabolomics research has uncovered that combinations of blood borne metabolites can risk-stratify women early in pregnancy according to their risk of developing pre-eclampsia later in their pregnancy. Since then, a company has been established which is dedicated to translating this finding into a tool for health care providers and pregnant women. A targeted approach is being developed whereby ca. 40 metabolites are (semi-) quantified using liquid chromatography-tandem mass spectrometry. An update on the method development progress as well as an overview of the clinical studies lined-up to verify and validate the pre-eclampsia risk stratification test will be discussed

Elaboration of a quantification assay of marinobufagenin in human plasma: a novel approach based on response factor quantification by LC-MS/MS

Marinobufagenin (MBG) is a bufadienolide cardiac inotrope implicated in the early diagnosis of volume expansion-mediated hypertensive states such as preeclampsia (PE). Endogenous MBG is an inhibitor of the α1 isoform of Na+,K+-ATPase with vasoconstrictive and cardiotonic properties,resulting in hypertension and natriuresis. Elevated endogenous MBG levels have been described in pregnant mammals and especially in preeclamptic patients [1-3]. The rise of endogenous MBG seems to appear prior the development of the main symptoms of PE, leading us to consider MBG as one of the potential biomarkers for PE. This demonstrates the need for a sensitive analytical method to detect MBG in plasma at low levels. Currently, the enhanced production of MBG in preeclamptic patients has been described using poor-specific immunoassays based on the detection of marinobufagenin-like material [4,5]. These techniques suffer from a lack of specificity due to cross-reactivity and tend to exhibit high variability at low concentrations [6]. Moreover, the two studies that recorded MBG plasma levels in preeclampsia compared to normal pregnancy are in marked discrepancy concerning the values of MBG plasma concentration. Our aim is to develop a specific and sensitive analytical MBG assay by LC-MS/MS with special attention to 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. As the major source for MBG is located in the parotid glands of the Bufo marinus toad, we developed a purification method from toad venom in order to get pure MBG standard. The identity of the compound was confirmed using TLC-MS and HPLC-MS/MS. A very sensitive and specific LC-MS/MS based assay is now being optimized in order to determine MBG in human plasma. The assay is preceded by an extraction step of the plasma on SLE cartridges. Using 5α-dihydrotestosterone-d3 as internal standard for response factor calculation, the obtained LOQ fully satisfies the need for quantification of MBG plasma levels in pregnancy (nmol/L range). This assay allowed us to confirm the identity of MBG in a woman plasma, before and during pregnancy

PREECLAMPSIA RISK STRATIFICATION EARLY IN PREGNANCY: LEVERAGING A PROMISING METABOLOMICS DISCOVERY INTO A LC-MS BASED CLINICAL ASSAY

Objectives Unbiased metabolite biomarker discovery has revealed that combinations of blood-borne metabolites have the potential to predict preeclampsia accurately at ca. 15 weeks of gestation (Kenny et al., 2010). Our aim is to deploy a dedicated translational effort bringing the merits of de-novo biomarker research to health care providers and patients. Methods To exploit the widespread availability of quadrupole mass spectrometers (QqQ-MS) in clinical laboratories world-wide, a platform migration to QqQ-MS was performed. To support the further refinement of the metabolites-based pre-eclampsia prediction algorithm, and the technical and clinical validation of the test, access to appropriate patient samples from prospective cohorts is warranted. A public-private partnership involving supranational government funding, clinicians and dedicated small and medium sized companies, collaborated to establish a pregnancy biobank. Results Thus far, a simple metabolite extraction and a targeted LC-QqQ -MS approach using stable isotope labelled metabolites for relative quantification has been developed. The (semi-) quantitative analysis of circa 40 metabolites with very disparate physicochemical characteristics is achievable in a single 10 minute run. The company is engaged in 2 dedicated public-private initiatives, i.e. SCreening fOr Pregnancy Endpoints (SCOPE) (North et al, 2011) and IMproved Pregnancy Outcomes by Early Detection (IMPROvED) (Navaratnam et al, 2013). These collaborations are instrumental for the further clinical and technical validation of the test under development. Conclusion Development of a dedicated translational effort to further exploit newly discovered biomarkers relevant to the prediction of preeclampsia has already resulted in an analytical method ready for testing in clinically relevant patient cohorts. Public-private biobanking efforts have proved to be a cost-efficient means enabling both additional basic biomarker research and progression of biomarkers to market

Relationship between the concentration of ergothioneine in plasma and the likelihood of developing pre-eclampsia

Ergothioneine, an antioxidant nutraceutical mainly at present derived from the dietary intake of mushrooms, has been suggested as a preventive for pre-eclampsia (PE). We analysed early pregnancy samples from a cohort of 432 first time mothers as part of the Screening for Endpoints in Pregnancy (SCOPE, European branch) project to determine the concentration of ergothioneine in their plasma. There was a weak association between the ergothioneine levels and maternal age but none for BMI. Of these 432 women, 97 went on to develop pre-term (23) or term (74) PE. If a threshold was set at the 90th percentile of the reference range in the control population (≥462 ng/ml), only one of these 97 women (1%) developed PE, versus 96/397 (24.2%) whose ergothioneine level was below this threshold. One possible interpretation of these findings, consistent with previous experiments in a reduced uterine perfusion model in rats, is that ergothioneine may indeed prove protective against PE in humans. An intervention study of some kind now seems warranted

Role of nitrogen lewis basicity in boronate affinity chromatography of nucleosides. Anal

Urinary modified nucleosides have a potential role as cancer biomarkers, and most of the methods used in their study have utilized low-pressure phenylboronate affinity chromatography materials for the purification of the cisdiol-containing nucleosides. In this study, a boronate HPLC column was surprisingly shown not to trap the nucleosides as would be expected from experience with the classic Affigel 601 resin but showed only partial selectivity toward cis-diol groups while other groups exhibited better retention. In aprotic conditions, trapping of nucleosides was possible; however, the selectivity toward cis-diol-containing compounds was lost with the Lewis basicity of available nitrogens being the main determinant of retention. The experimental findings are compared to and confirmed by DFT calculations. Modified nucleosides are naturally occurring modifications of the "normal" nucleosides. They have various roles within many nucleic acids but are mainly found in transfer RNA. They are excreted from the body via the urine as they cannot be salvaged; moreover, some are toxic when allowed to accumulate. Many past reports have investigated the modified nucleosides as potential cancer biomarkers and indicate considerable promise. [1][2][3][4][5] The methodologies used in these studies are wide ranging; however, since the introduction of boronate affinity chromatography as a ribonucleoside-selective cleanup step, on Affi-Gel 601 (Bio-Rad), utilized by Gehrke et al., 1,2 most research employed this off-line cleanup step process in the analysis. The subsequent identification/quantification of the ribonucleosides was almost exclusively carried out via RPLC-UV methods. More recently, some CE-UV methods have also been developed. [6][7][8][9] The further potential/ demand to obtain unambiguous identification via mass spectrometric detection led to the development of some off-line boronate chromatography GC/MS procedures. 3,5,10 However, the most natural choice for the analysis of the prepurified urinary nucleosides analysis is found in LC-MS. 11 Yet, the development of LC-MS procedures for urinary nucleosides only advanced 12 when electrospray mass spectrometry (ESI-MS) became available. Past studies by our group have considered the cleanup samples prior to ESI-MS analysis, 13 the optimization of the detection conditions, 14 comparison of various mass spectrometric methods, 15 and identification of the excreted nucleosides. 16,17 Other groups have taken advantage of mass spectrometry in the study of these compounds

A machine learning algorithm to differentiate bipolar disorder from major depressive disorder using an online mental health questionnaire and blood biomarker data.

The vast personal and economic burden of mood disorders is largely caused by their under- and misdiagnosis, which is associated with ineffective treatment and worsening of outcomes. Here, we aimed to develop a diagnostic algorithm, based on an online questionnaire and blood biomarker data, to reduce the misdiagnosis of bipolar disorder (BD) as major depressive disorder (MDD). Individuals with depressive symptoms (Patient Health Questionnaire-9 score ≥5) aged 18-45 years were recruited online. After completing a purpose-built online mental health questionnaire, eligible participants provided dried blood spot samples for biomarker analysis and underwent the World Health Organization World Mental Health Composite International Diagnostic Interview via telephone, to establish their mental health diagnosis. Extreme Gradient Boosting and nested cross-validation were used to train and validate diagnostic models differentiating BD from MDD in participants who self-reported a current MDD diagnosis. Mean test area under the receiver operating characteristic curve (AUROC) for separating participants with BD diagnosed as MDD (N = 126) from those with correct MDD diagnosis (N = 187) was 0.92 (95% CI: 0.86-0.97). Core predictors included elevated mood, grandiosity, talkativeness, recklessness and risky behaviour. Additional validation in participants with no previous mood disorder diagnosis showed AUROCs of 0.89 (0.86-0.91) and 0.90 (0.87-0.91) for separating newly diagnosed BD (N = 98) from MDD (N = 112) and subclinical low mood (N = 120), respectively. Validation in participants with a previous diagnosis of BD (N = 45) demonstrated sensitivity of 0.86 (0.57-0.96). The diagnostic algorithm accurately identified patients with BD in various clinical scenarios, and could help expedite accurate clinical diagnosis and treatment of BD