Christopher R. Lee, Endogenous ouabain (EO)' in mammals: absence of valid experimental evidence. Researchgate December 2019. DOI: 10.13140/RG.2.2.10144.53765

Summary and conclusions

Physiological experiments involving volume expansion dating from the 1960s indicate the existence of a circulating factor in mammals that inhibits the activity of the sodium-potassium pump (Na,K- ATPase) in the cell membrane. The factor is thought to resemble in its actions the cardioactive steroids including digitalis glycosides and ouabain, which are plant metabolites. Ever since an initial observation in 1989 it has been repeatedly asserted, despite objections on biochemical and analytical grounds, that the mammalian factor is ouabain, termed ‘endogenous ouabain (EO)’. Since these unsubstantiated claims are influencing medical opinion and have likely discouraged re- searchers interested in joining the field, I attempted to challenge the published analytical data in a ResearchGate document (Lee (2018)). The same year Blaustein (2018), one of the main protag- onists, published an autobiographical review which included a Table (Table 2) listing publications he considered to be in favour of the claims, together with the published contradictory results. This document focuses on that table.

I conclude that there is no evidence whatsoever for endogenous ouabain in mammals. Given the claimed enrichment factors, cross contamination is a probable source of the isolated compound, since ouabain is used extensively as a pharmacological tool for these studies. Moreover, ouabain was not shown to be endogenous, whereas suitable stable-isotopic tracer techniques had been introduced decades earlier. There was extensive use of immunoassays, with which positive results are valueless unless validated against a method that meets recognised criteria for specificity. Exceptions are two immunoassay studies conducted as limit tests that gave incontrovertibly negative results. Finally, after a quarter-century, the first validated instrumental method, using LC-MS, demonstrated the absence of significant concentrations of ouabain in the specimens examined (Baecher et al. (2014)). Since I understand that the force of the analytical arguments against EO has not been obvious to all concerned, I provide optional tutorial materiel on specific aspects.

 

1.This affair raises some general issues that could be considered troubling.

Numerous journals have published qualitative and quantitative data that does not meet long-standing validation and reporting criteria. Definitive analytical techniques for steroids including cardiac glycosides were published from the 1960s onwards, but never cited. These include sensitive methods for identification, quantitation and isotopic tracer studies.

 

2. Refutations and other dissident contributions, which may be dicult to write concisely but accurately and with sucient detail when they are multidisciplinary, tend in practice to be excluded from the conventional literature.

 

3. I know of no means whereby the literature concerned can be marked as unreliable, hence not to be cited in future peer-reviewed publications. A damaging historical precedent demonstrates the need for this to be done: a metabolite (the ‘Pink Spot’) in the urine of schizophrenic patients was mis-identified through inadequate analytical techniques, and mistakenly thought to be hallucinogenic. Although the initial finding of 1962 was refuted by means of definitive techniques including mass spectrometry (Boulton et al. (1967); Siegel & Teffte (1971)), it still must (apparently) feature in reviews (Jaskiw et al. (2019)). This spurious affair may have discouraged serious research on the hypothesis that metabolic disturbances might give rise to ‘endogenous hallucinogens’ in the central nervous system (Matthysse & Sugarman (1978)).

 

4. There appears to be a consensus, rarely mentioned nowadays, that cell membrane receptors and transporters can not and do not have endogenous modulators acting at allosteric sites that would be analogous to the targets of heterotropic allosteric modulators of enzymes. This would imply that the searched-for hormone-like modulator of Na,K-ATPase should not exist, but I have not read a convincing rationale for that stance.

 

5. At the origin of this endeavour was the observation that volume expansion influences sodium- potassium transport, a major consumer of ATP. Consequently, one would expect that someone would have carried out a systematic search for the associated metabolic changes. There are a few unconfirmed indications of increased concentrations of plasma lipids, which were at one time thought to alter the lipid-membrane environment and hence the activity of Na,K-ATPase (M Tamura, H Kuwano & Inagami (1985); Tamura et al. (1988)). Such effects would be more in accord with ideas expressed in recent reviews of this enzyme complex than the influence of a putative hormone, but they do not appear to have been investigated further.

 

6. During the late 1970s – early 1980s, preparative biochemical separation practice switched from liquid-liquid countercurrent chromatography to HPLC. As I argued earlier (Lee (2018)), the change may have been largely detrimental to the present endeavour and to one I was once involved in (Lee et al. (1987)). With any new initiatives, it may be preferable to consider returning to the old technique, for which new equipment is available. This remark might also apply to current research aimed at definitively mapping the ‘metabolome’.

 

Preprint submitted to ResearchGate December 9, 2019

 

Baecher S, Kroiss M, Fassnacht M, Vogeser M. No endogenous ouabain is detectable in human plasma by ultra-sensitive UPLC-MS/MS.

 Clin Chim Acta. 2014 Feb 7;431C:87-92. doi: 10.1016/j.cca.2014.01.038

 

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 cardenolideouabain 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 530pmol/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 ouabainin 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 1.7pmol/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 assumedendogenous counterparts of digoxin in a biomarker discovery approach seems to require distinct analytical techniques.

 

 

Fürstenwerth, H. Comment on: Endogenous Ouabain and Related Genes in the Translation from Hypertension to Renal Diseases. Int. J. Mol. Sci. 2018, 19, 1948. [CrossRef]

Dear Editor,

In their review “Endogenous Ouabain and Related Genes in the Translation from Hypertension to Renal Diseases” [1], Paolo Manunta and coworkers describe a potentially pathogenic role of endogenous ouabain (EO) in various diseases, including renal failure, essential hypertension and heart failure. They assert that EO has significant implications in the pathogenesis of many common diseases. The authors suggest that EO acts as a pro-hypertrophic and growth-promoting hormone, which might lead to cardiac remodeling affecting cardiovascular function and structure. In addition, a possible role of EO in the development of acute kidney injury is hypothesized.

Although it is claimed that EO is identical to plant-derived ouabain, no reference is given to the more than one hundred years of clinical experience with ouabain in the treatment of heart diseases. Ouabain and the related Strophanthus glycoside, k-Strophanthin, by default have been used for more than a century to treat heart diseases. The structurally similar drugs, cymarin and convallatoxin, have also been used [2]. Ouabain is found in both Acokanthera ouabaio and Strophanthus gratus. The glycoside occurring in Strophanthus kombé is k-Strophanthin. Herman Thoms isolated the pure glycosides from S. kombé and S. gratus in 1904 and has unambiguously assigned them with the names k- and g-Strophanthin [3], the latter is referred to as “ouabain” in the English literature. Based on the work of Thomas Fraser, Burroughs, Wellcome & Co in 1886 introduced a S. kombéextract called “Tincture of Strophanthus”. This was sold at seven shillings per ounce. In America, E.R. Squibb and Sons was one of the first suppliers of Strophanthus preparations. Particularly popular was a chocolate-coated tablet of a mixture of Digitalis and Strophanthus extracts, which was sold at 16 cents per one hundred pieces [4]. In 1889, Boehringer Mannheim introduced pure k-Strophanthin to the market. In cooperation with Albert Fraenkel Boehringer, in 1907 introduced a solution of k-Strophanthin for intravenous administration under the brand name “Kombetin”.

In 1904, E. Merck, Darmstadt, commercialized a standardized solution of pure ouabain as “g-Strophanthin crystallisatum nach Thoms”. In 1906, Kali-Chemie also began marketing an ouabain solution under the trade name “Purostrophan”. In 1909, the French physician Henri Vaquez introduced the intravenous application of ouabain (“Ouabain-Arnaud”) in France. In World War I medical personnel in the German army, by order of the ambulance corps, exclusively used ouabain solutions to treat heart failure [5]. The therapeutic profile and the disease profiles for which the use of Strophanthusglycosides is appropriate are documented in many reports on clinical experiences and have been summarized in numerous reviews, mostly in the German literature [6]. As early as the first half of the 20th century distinguished scientists such as Albert Fraenkel, University of Heidelberg, and Ernst Edens, University of Dusseldorf, published monographs [7,8] that document in detail the clinical effects of Strophanthus glycosides. In textbooks ouabain has been praised as “the biggest advance in cardiac therapy since Withering in 1785” [9].

Ouabain-based products, over the decades of their use, have been given as standard medication to millions of heart failure patients. The database of the German Institute for Medical Documentation and Information records more than 20 orally administered ouabain preparations that were used in Germany after 1950. Decades of clinical experience with ouabain provide a yardstick by which all research results and hypotheses related to ouabain have to be measured. Observations at the bedside are more meaningful than speculative hypotheses based on experimental research. It is totally incomprehensible why Manunta et al. do not mention the well-documented clinical experiences with ouabain in their review. It is also incomprehensible that current reports on cardioprotective effects of ouabain are not mentioned either. Current research findings indicate cardio protection is induced by ouabain [10–12]. Lijune Liu and co-workers report that ouabain is beneficial to various stages of heart failure [13]. None of these reports are mentioned by Manunta et al.

Based on historical clinical experiences and current research findings, it can be ruled out that ouabain, that has been used successfully for decades in the treatment of heart failure, will cause heart failure. Equally excluded is that ouabain produces hypertension. In clinical practice ouabain lowers blood pressure [14]. Already Fraser had pointed out that “strophanthin increases the action of the heart without raising blood pressure.”

Although the authors postulate that EO damages the kidney, current findings on renal protective effects of ouabain are not mentioned. Aperia et al. report that ouabain prevents adverse programming of kidney development from negative effects of malnutrition [15,16]. Ouabain (0.1–10 nM) also was found to stimulate proliferation and increase the viability of kidney cells [17].

Based on all available data it can be ascertained that the mutually exclusive effects of plant derived ouabain and the inhibitor of the Na/K-ATPase observed in mammalian tissues that reacts with ouabain based antibodies, do not support the hypothesis that this inhibitor is identical with ouabain, but favor the interpretation that “endogenous ouabain” is something different.

This conclusion is supported by current analytical findings. Vogeser et al. established a stable-isotope dilution API-MS/MS method for the quantification of ouabain in human plasma [18]. This team developed a method of extremely high sensitivity for 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. Using this method, no ouabain could be detected in unspiked human plasma samples that contained EO levels of 206–665 pmol/l as determined by radioimmunoassays in the laboratory of Paolo Manunta [19].

While some research groups have identified endogenous ouabain in human plasma, others have failed to detect EO [20]. There was a lack of attempts to determine EO in identical samples using different methods by different laboratories. This essential gap has been closed by Vogeser’s experiments. While no EO could be detected by using API-MS/MS, radioimmunoassays performed by Manunta in his laboratory on the same samples indicated EO in substantial concentrations. These findings exemplify that antibody-based radioimmunoassays are often subject to cross reactivity with compounds other than those to which the antibody was raised. Another current example is ionotropin. This substance has been isolated from mammalian tissue. It cross reacts with digoxin-specific antibodies, but has a proposed chemical structure that is not related to digoxin [21]. This inherent methodological disadvantage also applies to the work of Takahashi and coworkers [22]. Their detection of endogenous ouabain, too, is based on a sensitive enzyme-linked immunosorbent assay for ouabain. Based on his findings that endogenous ouabain binds to plasma proteins, Takahashi, in his purification process, uses an acidification step to liberate endogenous ouabain. Contrary to these findings it is well known that ouabain, unlike digitalis glycosides, does not bind to plasma proteins [23]. This fact also confirms that endogenous ouabain is different from ouabain. From LC/MS measurements, Takahashi et al. concluded that the endogenous ouabain “molecule may be one of

the isomers of ouabain, and the molecular size is the same as authentic plant-derived ouabain.” This hypothesis has not been verified by proven analytical methods such as API-MS/MS.

The results of Vogeser’s team are unequivocal: There is no ouabain in human plasma. “Endogenous ouabain” is different from ouabain. Decades of clinical experience with ouabain and current analytical findings refute the hypothesis of “endogenous ouabain”. Time has come to concentrate again on the therapeutic effects of ouabain.

The hypothesis of the existence of “endogenous ouabain” has been subject to a fierce scientific debate [24]. Arguments and findings of scientific experiments must be judged with self-criticism. Science is a set of methods aimed at building a testable body of knowledge open to rejection or confirmation. Therefore, in the end, truth will prevail. For the locals in Africa, Strophanthus was a poison and remedy in one. In the mythology of the tribe of the Wilé in Upper Volta, this plant was sent from paradise to the earth to heal or punish people according to their merit [25]. A clinical reassessment of ouabain offers a unique opportunity to transform this gift from paradise into much needed new treatment options for cardiovascular diseases.

 

References

1. Simonini, M.; Casanova, P.; Citterio, L.; Messaggio, E.; Lanzani, C.; Manunta, P. Endogenous Ouabain and Related Genes in the Translation from Hypertension to Renal Diseases. Int. J. Mol. Sci. 2018, 19, 1948. [CrossRef] [PubMed]

2. Niedner, R. Taschenbuch der Digitalis-Therapie; Georg Thieme Verlag: Stuttgart, Germany, 1961.

3. Gilg, E.; Thoms, H.; Schedel, H. Die Strophanthus-Frage. In Arbeiten aus dem Pharmazeutischen Institut der Universität Berlin; Springer: Berlin/Heidelberg, Germany, 1904; Volume 14, p. 90.

4. Osseo-Asare, A.D. Bitter Roots: The Search for Healing Plants in Africa; The University of Chicago Press:

Chicago, IL, USA, 2014.

5. Dietz, E.; Albert Fraenkel, C.F. Boehringer & Söhne und die intravenöse Strophanthintherapie. In Albert

Fraenkel–Ein Arztleben in Licht und Schatten 1864–1938; Drings, P., Thierfelder, J., Weidemann, B., Willig, F. Ehmann, M., Eds.; Verlag Ecomed: Landsberg, Germany, 2004.

6. Fürstenwerth, H. Ouabain—A Gift from Paradise; Books on Demand: Norderstedt, Germany, 2018; ISBN

978-3748165767.

7. Fraenkel, A.; Thauer, R. Strophanthin-Therapie; Verlag von Julius Springer: Berlin, Germany, 1933.

8. Edens, E. Die Digitalisbehandlung [Digitalis treatment], 3rd ed.; Verlag Urban&Schwarzenberg: Berlin-München, Germany, 1948.

9. Eichholtz, F. Lehrbuch der Pharmakologie [Textbook of Pharmacology], 5th ed.; Springer Verlag: Berlin/Heidelberg, Germany, 1947.

10. Lagerstrom, C.F.; McElroy, D.D.; Taegtmeyer, H.; Walker, W.E. Improved recovery of cardiac function after hypothermic ischemic storage with ouabain. J. Thorac. Cardiovasc. Surg. 1988, 96, 782–788. [PubMed]

11. Morgan, E.E.; Li, Z.; Stebal, C.; Belliard, A.; Tennyson, G.; Salari, B.; Garlid, K.D.; Pierre, S.V. Preconditioning by subinotropic doses of ouabain in the Langendorff perfused rabbit heart. J. Cardiovasc. Pharmacol. 2010, 55, 234–239. [CrossRef] [PubMed]

12. Wu, J.; Li, D.; Du, L.; Baldawi, M.; Gable, M.E.; Askari, A.; Liu, L. Ouabain prevents pathological cardiac

hypertrophy and heart failure through activation of phosphoinositide 3-kinase α in mouse. Cell Biosci. 2015,

5, 64. [CrossRef] [PubMed]

13. Liu, L.; Wu, J.; Kennedy, D.J. Regulation of Cardiac Remodeling by Cardiac Na(+)/K(+)-ATPase Isoforms. Front. Physiol. 2016, 7, 382. [CrossRef] [PubMed]

14. Fürstenwerth, H. Ouabain and Endogenous Ouabain-Dr. Jekyll and Mr. Hyde of Cardiac Glycosides? Br. J. Med. Med. Res. 2015, 8, 477–484. [CrossRef]

15. Khodus, G.R.; Kruusmägi, M.; Li, J.; Liu, X.L.; Aperia, A. Calcium signaling triggered by ouabain protects

the embryonic kidney from adverse developmental programming. Pediatr. Nephrol. 2011, 26, 1479–1482. [CrossRef] [PubMed]

16. Li, J.; Khodus, G.R.; Kruusmägi, M.; Kamali-Zare, P.; Liu, X.L.; Eklöf, A.C.; Zelenin, S.; Brismar, H.; Aperia, A. Ouabain protects against adverse developmental programming of the kidney. Nat. Commun. 2010, 1, 1–7. [CrossRef] [PubMed]

17. Li, J.; Zelenin, S.; Aperia, A.; Aizman, O. Low doses of ouabain protect from serum deprivation-triggered apoptosis and stimulate kidney cell proliferation via activation of NF-kappaB. J. Am. Soc. Nephrol. 2006, 17, 1848–1857. [CrossRef] [PubMed]

18. Baecher, S.; Kroiss, M.; Fassnacht, M.; Vogeser, M. No endogenous ouabain is detectable in human plasma by ultra-sensitive UPLC-MS/MS. Clin. Chim. Acta 2014, 431, 87–92. [CrossRef] [PubMed]

19. Blaustein, M.P. Letter to the Editor concerning Baecher et al. (Clin Chim Acta 2014;431:87-92). Clin. Chim. Acta2015, 448, 248–249. [CrossRef] [PubMed]

20. Lewis, L.K.; Yandle, T.G.; Hilton, P.J.; Jensen, B.P.; Begg, E.J.; Nicholls, M.G. Endogenous ouabain is not ouabain. Hypertension 2014, 64, 680–683. [CrossRef] [PubMed]

21. Chasalow, F.; Pierce-Cohen, L. Ionotropin is the mammalian digoxin-like material (DLM). It is a phosphocholine ester of a steroid with 23 carbon atoms. Steroids 2018, 136, 63–75. [CrossRef] [PubMed]

22. Komiyama, Y.; Nishimura, N.; Munakata, M.; Mori, T.; Okuda, K.; Nishino, N.; Hirose, S.; Kosaka, C.; Masuda, M.; Takahashi, H. Identification of endogenous ouabain in culture supernatant of PC12 cells.J. Hypertens. 2001, 19, 229–236. [CrossRef] [PubMed]

23. Kramer, P.; Köthe, E.; Scheler, F. Uraemic and normal plasma protein binding of various cardiac glycosides under “in vivo” conditions. Eur. J. Clin. Investig. 1974, 4, 53–58. [CrossRef] [PubMed]

24. Kaaja, R.J.; Nicholls, M.G. Does the hormone “endogenous ouabain” exist in the human circulation? Biofactors2018, 44, 219–221. [CrossRef] [PubMed]

25. Leuenberger, H. Gesund durch Gift; Deutsche Verlagsanstalt: Stuttgart, Germany, 1972.

 

 

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