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’”

reply: In his Letter to the editor (LTE) of AJP-Cell Physiology, 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 procedures… including chemical derivatization… 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 cell-conditioned 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⁺-K⁺-ATPase inhibition) both before and after the separation steps. The OLC in all these studies co-eluted with authentic ouabain. Finally, bovine adrenocortical cell secretion of the OLC (identified by bioassay, NMR, and MS), but not cortisol, was increased by incubating the cells with rhamnose, suggesting that the OLC, like authentic ouabain, was a rhamnoside (10). Together, these multifarious studies provide compelling evidence that the endogenous compound is, in fact, ouabain.

In his LTE, Vogeser’s disparagement of “massive averaging” (9) of the MS-MS and MS-MS-MS spectra reflects a misunderstanding. Baecher et al. (1) interrogated the output of their ultraperformance liquid chromatography (UPLC) directly with online MS-MS. In this type of liquid chromatography (LC)-MS-MS, the LC runs are so rapid that the mass analyzer only has time (≤6 s) to target one or two product ions and generate a single spectrum. The primary target in Ref. 1 was a single dominant product ion whose m/z (mass/charge) ratio was equivalent to ouabagenin’s. In contrast, Jacobs et al. (9) collected high-pressure LC (HPLC) fractions, and used radio-receptor and radio-immunoassays to identify the fractions that were “ouabain-reactive.” Those fractions were then interrogated, offline, by MS-MS and MS-MS-MS. The offline approach allows the mass analyzer long periods of time (up to several minutes) to generate and average a very large number of replicate spectra from each sample (i.e., “massive averaging”). This not only yields high-quality spectra with minimal noise but, in the case of MS-MS-MS, gives detailed information about multiple prominent fragments of the steroid moiety (e.g., Fig. 4C in Ref. 9 and Supplementary Information in Ref. 8) that cannot be readily obtained by LC-MS-MS instruments.

Vogeser’s implication that the “hypothesis based on (these) qualitative spectroscopic data” is invalid without precise quantitation of the ouabain level is unjustified and misleading. The ion current peaks from the offline MS-MS-MS can be quantitatively compared with ouabain and, when required (8), compared directly with an internal standard (dihydroouabain). Contrast nearly all the OLC studies referenced above (2, 4–11), in which bioassay (Na⁺-K⁺-ATPase binding or inhibition) was used to verify the presence of OLC, with that of Vogeser (1): There is no indication of an effort to determine whether their native plasmas contained any bioassay- or immunoassay-positive OLC; their only measurement was MS (1). Their sample treatment included (1): dilution with 0.1% trifluoroacetic acid, centrifugation, solid phase extraction and evaporation to dryness of the supernate, reconstitution in 5% methanol, and UPLC with online MS-MS. Calibration consisted of MS analysis of “a pool of EDTA plasma from leftover samples (spiked) with a solution of (plant) ouabain in methanol 20%” (1). No original recordings of the calibration or unaltered sample spectra were shown in Ref. 1. Their “method was fully validated” (1), presumably for other steroids, but not optimized for OLC even though ouabain is an unusual, polar steroid. For example, why reconstitute samples in 5% methanol, the solvent used to elute ouabain, when ouabain is water soluble and should stick to the LC column better when dissolved in water than in 5% methanol (Ref. 7 data supplement)? All the “spiked plasmas” and calibration samples involved a step that was different from the native plasma alone. Could the reconstituted samples have contained an OLC that did not stick to the UPLC column, and was washed through before applying the very steep UPLC water-methanol gradient, starting at 5% methanol? Note that the spectrum in Fig. 2 (1) begins at 3.0 min—close to 35% methanol in the gradient. Because of the very rapid (~6 min), steep LC gradient runs, it would be surprising if everything stuck to the column and there was no ion current or even “noise” before 3.0 min.

In summary, Vogeser’s attempt to use a more sensitive MS-MS system to quantify EO levels is commendable. However, while his MS system may be “state-of-the-art,” if the endogenous compound is lost before the MS analysis, the most sensitive MS system will not detect it. The shortcomings of the Baecher et al. study (1) should not detract from the numerous reports that EO is ouabain, that it circulates in human plasma, and that it is a mammalian hormone.

DISCLOSURES

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

AUTHOR CONTRIBUTIONS

M.B. drafted, edited and revised the manuscript, and approved the final version.