Silvia Baecher (1), Michael Vogeser (1)
Institute of Laboratory Medicine, Hospital of the University of Munich (LMU)
For decades immunoassays were applied in an attempt to quantify the cardiac glycoside ouabain in plasma of patients with cardiovascular conditions. This compound (CAS 630-60-4) was supposed to be of endogenous origin as a counterpart of exogenous cardiac glycosides such as digoxin. Using a fully validated, highly specific and extremely sensitive LC-MS/MS method we were able to show that ouabain cannot be present in relevant concentrations in human plasma - as claimed by immunoassay studies.
Plant derived cardiac glycosides with clinically relevant impact on cardiac output and rhythm have been recognized centuries ago with digoxin and ouabain being the most widely studied molecules. It was found that their pharmacological effect on the heart is mainly due to inhibition of the Na+/K+-ATPase of cardiomyocytes. It is obviously tempting to speculate that endogenously formed compounds might exist which could modulate cardiac functions as the endogenous counterparts of xenobiotic cardiotonic steroids (by analogy to endocannabinoids as the endogenous ligands of cannabinoid receptors identified as the receptors of exogenous cannabinoids).
In 1991 a research group from the University of Maryland and the Upjohn Laboratories reported that ouabain (CAS 630-60-4), which was primarily found in the seed of Stropanthus gratus and the root/bark of Acokanthera ouabaio, seems to be indeed formed in the human body as an endogenous cardiotonic steroid. Immunoassays to quantify this endogenous ouabain in plasma of patients with cardiovascular conditions were developed by few groups. Based on these tests a large number of clinical studies were performed to characterize plasma levels of “endogenous ouabain” in health and disease. Since ouabain is a well defined small molecule we decided in 2011 to develop a LC-MS/MS method for the quantification of plasma ouabain, involving a commercially available stable isotope labelled internal standard. We were able to realize highest sensitivity of this test as verified by analysis of spiked calibrator samples with a lower limit of quantification of 1.7 pmol/L. The method was validated according to the FDA guidelines for the quantification of compounds in biomedical research. However, when analysing a large set of clinical samples from various patients’ populations and healthy individuals, ouabain was not detectable. This result indicates that endogenous ouabain – if it actually exists in the human circulation – would be present in only femtomolar concentration. We consequently investigated the original articles which describe the attempts to characterize compounds which were supposed to be ouabain and had to recognize that indeed there was never a conclusive identification shown for a preparation isolated from human plasma and looked upon as endogenous ouabain. The publication of our results in a peer reviewed journal raised a lively discussion about the non-existence of endogenous ouabain in native human plasma. From our results and this discussion it has to be concluded that more than thousand articles were published in medical journals about a non-existing compound. It seems likely that immunoassays claiming to quantify endogenous ouabain actually cross-react with so far unidentified endogenous compounds. The high specificity of detection realized by LC-MS/MS demonstrated to the scientific community that the hunt for and the identification of hypothetic endogenous cardiotonic compounds has to be re-initiated.
Our work highlights that the high specificity of mass spectrometric methods is of crucial importance for the integrity of results in biomedical research on endogenous small molecules.