Letter to the editor: Comments on Blaustein (2018): “The pump, the exchanger, and the holy spirit: origins and 40-year evolution of ideas about the ouabain-Na⁺ pump endocrine system”

to the editor: In a recent article (2), Dr. Blaustein reiterates his hypothesis that a compound found in some plants—ouabain—is identically present also in human circulation, originating from putative endogenous sources. In Table 2A of his article, Dr. Blaustein cites previous articles on several spectroscopic studies. In these studies, various mammalian materials were submitted to complex sample preparation procedures, in part including chemical derivatization to obtain mass or NMR spectra. These spectra were reported compatible with the presence of ouabain in the sample materials. While acceptable for generating hypotheses, these data are far from unequivocal identification of a compound. The main hypothesis generated from this previous work was that ouabain circulates in human blood in concentrations that make a biological function likely. At this point, a hypothesis generated based on qualitative spectroscopic data had to be confirmed by highly accurate and thoroughly validated quantitative spectrometric data.

Atmospheric pressure ionization coupled to triple-stage mass spectrometry (API-MS/MS) is for more than two decades now the technology of choice for the sensitive and specific quantification of well-defined target analytes in complex biological matrices. The technology is widely available in research institutions worldwide now and state-of-the-art in quantitative biomedical analytics. Ouabain pure compound preparation is commercially available. It is an ideal target for API-MS/MS analyses with good ionization properties.

Based on the reports by Blaustein and others, we decided in 2013 to develop a stable-isotope dilution API-MS/MS method for the quantification of ouabain in human plasma. We were able to develop a method of extremely high sensitivity detecting spiked ouabain in human plasma. The method was fully validated according to FDA guidelines and published in the official journal of the International Federation of Clinical Chemistry after peer review (1). Using this method, we did not detect ouabain within the calibration range of the method (1.7–172 pmol/l) in any of the analyzed unspiked human plasma samples.

In his recent review, Dr. Blaustein now declares (2) a very minor region of increased background noise in a MS/MS-chromatogram from our work (4) with a clearly distinct retention time compared with ouabain to represent a putative isomer of ouabain (footnote to Table 2). From an analytical perspective this idea is abstruse. It is beyond all standards of biomedical method validation and scientific work to declare that noise in a published graph of a chromatogram, which is below any reasonable peak detection level, represents a putative compound. Dr. Blaustein bases his assumption on the existence of an ouabain isomer with profoundly different chromatographic properties eluting as a putative peak mainly on a report by Jacobs et al. (3). In this report no human material was investigated. An MS spectrum of a fraction extracted from rat plasma sharing some signals with those found in ouabain pure compound is displayed (Fig. 4 of Ref. 3). No potential conformational structure of that compound is suggested or discussed. It is claimed that applying “massive averaging” quantification was achieved. However, there is absolutely no data reported documenting critical state-of-the-art biomedical method validation. Essential information on how these experiments could be repeated in independent laboratories is not given.

As long as the presence of ouabain from endogenous sources in human blood is not verified by using quantitative state-of-the-art MS technologies [i.e., API-triple-stage tandem mass spectrometry (MS/MS)] with reproducible quantification, applying rigorous method validation by independent bioanalytical laboratories, the hypothesis that “ouabain immunoassays” actually detect endogenous ouabain in human blood cannot be assumed correct.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author.

AUTHOR CONTRIBUTIONS

M.V. drafted the manuscript.