Development, validation and utilisation of experimental assays for detection and quantification of endogenous cardiotonic steroids

Fobian, Dannie (2020). Development, validation and utilisation of experimental assays for detection and quantification of endogenous cardiotonic steroids. University of Birmingham. Ph.D.

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Abstract

Digoxin is commonly used for the treatment of patients with atrial fibrillation (AF) with/without heart failure (HF). However, in clinical practice the response to digoxin therapy is variable. The primary cellular target of digoxin, the Na+/K+ ATPase pump, is shared with a group of hormones called cardiotonic steroids (CTS). CTS are elevated in patients with cardiovascular disease. Differing levels of endogenous CTS in individual patients may explain the varied response to digoxin and susceptibility to adverse drug reactions. Thus validated methodologies to measurements of levels and effects of CTS are necessary.

In this thesis two methodologies were developed 1) a cell-based assay for assessment of effects of CTS and 2) an ouabain competitive ELISA with aim to use for patients from the RATE control Therapy Evaluation in permanent Atrial Fibrillation (RATE-AF) trial.

HEK293t cell-based assay developed were able to measure consistently changes in sodium sensitive fluorescents using SBFI sodium fluorescent dye. Changes were observed between different CTS were observed in IC50 values and maximal effects on sodium sensitive fluorescents and when calibrated to sodium levels. Extraction methodology was able to be optimised to extract potential cardiotonic steroids out of spiked human plasma. Competitive ELISA methodology developed and validated for detecting of ouabain levels from spiked and extracted samples. Some cross-reactivity with the primary antibody was observed from digoxin.

Significant lowered intracellular Na+ levels was observed in HEK293t cells when applied with serum from patients with type 2 diabetes, heart failure, myocardial infarction and with a number of unexpected hospitalisations due to AF or HF. Lastly, increased levels of endogenous ouabain were detected in more severe HF (NYHA IV vs NYHA II) and in unexpected hospitalisations caused by AF or HF. Furthermore, endogenous ouabain concentrations correlated with AF (mEHRA) and HF (NYHA class) disease severity. The results presented in this thesis suggests endogenous CTS to have an important role in HF and potentially also AF. Thus, further work with CTS and the potential of these assays to be used to measure biomarkers could help better patient stratification.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):