Silvia Baecher1, Matthias Kroiss2, Martin Fassnacht3, Michael Vogeser4. 1. Institute of Laboratory Medicine, Hospital of the University of Munich, Marchioninistr. 15, 81377 Munich, Germany. Electronic address: silvia.baecher@med.uni-muenchen.de. 2. Department of Internal Medicine I and Comprehensive Heart Failure Center, University Hospital, University of Würzburg, Oberdürrbacher Strasse 6, 97080 Würzburg, Germany. 3. Department of Internal Medicine I and Comprehensive Heart Failure Center, University Hospital, University of Würzburg, Oberdürrbacher Strasse 6, 97080 Würzburg, Germany; Department of Internal Medicine IV, Hospital of the University of Munich, Ziemssenstr. 1, 80336 Munich, Germany. 4. Institute of Laboratory Medicine, Hospital of the University of Munich, Marchioninistr. 15, 81377 Munich, Germany.
Abstract
BACKGROUND: The presence of a binding site for cardiac glycosides, such as digitoxin and digoxin, in the sodium-potassium-ATPase, stimulated attempts to isolate endogenous cardiotonic steroids. Using immunoassays, clinical studies found the cardenolide ouabain to be secreted endogenously in response to exercise and untreated hypertension and to be correlated with severity of clinical conditions such as kidney failure and dilated cardiomyopathy. The assays used were not standardized and the mean concentrations of endogenous ouabain reported for healthy controls ranged from 60 to 530 pmol/l. None of these immunoassays is available any more. Therefore, the aim of this study was to develop a highly specific and reliable method for measurement of ouabain in human plasma based on isotope dilution liquid chromatography tandem-mass spectrometry (ID-LC-MS/MS). METHOD: An ultra-sensitive and specific ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed which applied solid phase extraction of plasma for sample preparation. RESULTS: The method was comprehensively validated and had a lower limit of quantification of 1.7 pmol/l. However, despite this very low detection limit ouabain was not observed in plasma samples from patients with and without heart failure. CONCLUSION: Our results suggest that immunoassays previously used to quantify assumed endogenous ouabain detected compounds which are not structurally identical with ouabain. Cross reactivity of structurally related compounds of endogenous origin may cause these discrepancies between immunological and mass spectrometric analyses. Conclusive characterization of assumed endogenous counterparts of digoxin in a biomarker discovery approach seems to require distinct analytical techniques.
BACKGROUND: The presence of a binding site for cardiac glycosides, such as digitoxin and digoxin, in the sodium-potassium-ATPase, stimulated attempts to isolate endogenous cardiotonic steroids. Using immunoassays, clinical studies found the cardenolide ouabain to be secreted endogenously in response to exercise and untreated hypertension and to be correlated with severity of clinical conditions such as kidney failure and dilated cardiomyopathy. The assays used were not standardized and the mean concentrations of endogenous ouabain reported for healthy controls ranged from 60 to 530 pmol/l. None of these immunoassays is available any more. Therefore, the aim of this study was to develop a highly specific and reliable method for measurement of ouabain in human plasma based on isotope dilution liquid chromatography tandem-mass spectrometry (ID-LC-MS/MS). METHOD: An ultra-sensitive and specific ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed which applied solid phase extraction of plasma for sample preparation. RESULTS: The method was comprehensively validated and had a lower limit of quantification of 1.7 pmol/l. However, despite this very low detection limit ouabain was not observed in plasma samples from patients with and without heart failure. CONCLUSION: Our results suggest that immunoassays previously used to quantify assumed endogenous ouabain detected compounds which are not structurally identical with ouabain. Cross reactivity of structurally related compounds of endogenous origin may cause these discrepancies between immunological and mass spectrometric analyses. Conclusive characterization of assumed endogenous counterparts of digoxin in a biomarker discovery approach seems to require distinct analytical techniques.