Reply to “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’”

Abstract

Dr. Vogeser ( 13) raises questions about the identification of endogenous ouabain (EO) in human plasma by mass spectroscopy (MS) and NMR spectroscopy from multiple laboratories including ours. His laboratory reported that they were unable to detect EO in human plasma (1), but serious concerns were raised in my Letter to the Editor of that journal (3) and in a subsequent article (Ref. 7 and data supplement). The basis for the discrepant (negative) results reported in Ref. 1 is unclear; however, the only data are contained in Fig. 2 of Ref. 1, which shows a plasma sample spiked with authentic ouabain. Thus, any endogenous ouabain would have been obscured. Moreover, while Vogeser and colleagues reportedly tested "30 human plasma samples" (1), no data are presented for any of the other 29 samples and only one other sample is shown in their subsequent LTE (12). Below I address the technical issues raised by Vogeser.Regarding the spectroscopic evidence for EO cited in my Table 2A (4), Vogeser criticizes the fact that "various mammalian materials (plasma, adrenal cortex, adrenal cells and conditioned media, hypothalamus, and urine) were submitted to complex sample preparation proceduresѧ including chemical derivatizationѧ to obtain mass or NMR spectra." This statement confuses several issues and is misleading. First, chemical derivatization was used in only a few instances. Second, in most cases, multiple separation steps were required to isolate EO in the purity and amounts sufficient for outright identification. The fact that these isolates, from human, bovine, and rodent sources, treated in so many different ways, and analyzed by both MS and NMR (e.g., see Refs. 10, 11), were all found to contain the same compound (EO) is remarkable and belies Vogeser's concerns. In several studies, three-dimensional MS (MS-MS-MS) collision-induced dissociation revealed ion spectra identical to those of authentic ouabain before and after dissociation of the sugar and, more significantly, after fragmentation of the steroid moiety (7-9) (and see Ref. 7 data supplement). Also, in several of the reports in Table 2A (4), as well as in other studies of human plasma and adrenal cellconditioned media lacking spectral confirmation and therefore not included in Table 2A (e.g., Refs. 2,5,6), ouabain-like compounds (OLC) were detected by bioassay (e.g., Na ϩ -K ϩ -ATPase inhibition) both before and after the separation steps.