No endogenous ouabain is detectable in human plasma by ultra-sensitive UPLC-MS/MS

Area of Science:

• Biochemistry
• Analytical Chemistry
• Cardiology

Background:

• Cardiac glycosides like digoxin bind to sodium-potassium-ATPase, prompting research into endogenous cardiotonic steroids.
• Previous immunoassays suggested endogenous ouabain secretion in conditions like hypertension and heart failure, with high variability in reported concentrations.

Purpose of the Study:

• To develop a highly specific and reliable method for measuring ouabain in human plasma using isotope dilution liquid chromatography tandem-mass spectrometry (ID-LC-MS/MS).

Main Methods:

• Developed an ultra-sensitive and specific ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method.
• Employed solid-phase extraction for plasma sample preparation.

Main Results:

• The validated method achieved a lower limit of quantification of 1.7 pmol/l.
• Ouabain was not detected in plasma samples from patients with or without heart failure, despite the method's high sensitivity.

Conclusions:

• Immunoassays previously used likely detected compounds structurally different from ouabain, possibly due to cross-reactivity.
• Discrepancies between immunoassay and mass spectrometry results highlight the need for distinct analytical techniques in biomarker discovery.