Published online before print August 15, 2005, doi: 10.1161/01.HYP.0000180069.86224.57
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(Hypertension. 2005;46:e9.)
© 2005 American Heart Association, Inc.
Letters to the Editor |
Radioimmunoassays, Ouabain-Like Material, and Ouabain
Renal Laboratory, St Thomas’ Hospital, London, UK
We read with interest the article by Bauer et al1 on the changes in the plasma concentration of a ouabain-like compound associated with vigorous exercise in both humans and dogs. The authors comment on the result that is most striking to us, namely the remarkably high concentrations of the immunoassayable substance achieved without apparent ill effect. Bauer et al1 suggest that this may be a result of either the transient nature of the high concentrations measured, the "slow on-rate in forming the ouabain-Na/K ATPase complex," or by the ouabain-like substance being bound by proteins.
The most remarkable values of ouabain-like compound achieved were those in beagles with a mean "ouabain" concentration of 6882±1436 nmol/L. The raw data are not given but it is reasonable to assume that the concentration in at least one animal reached approximately 8000 nmol/L (4.68 mg/L). At such a ouabain concentration, close to 100% inhibition of any accessible sodium pumps would occur almost instantaneously. Whereas the numbers in the nontrained humans are less dramatic (176±68 nmol/L), it seems likely that the highest concentration seen in this population was in the region of 300 nmol/L (175 µg/L). This would inhibit approximately 50% of the sodium pumps in proximity to the plasma. If the immunoassayable "ouabain" was uniformly distributed throughout the plasma this would correspond to a ouabain "dose" of approximately 0.8 mg in humans and 4.5 mg in beagles. The rapid administration of such an intravenous dose of ouabain would not be expected to be uneventful for the human recipient and even less so in the case of the beagle because the LD50 is around 0.1 mg/kg2 and the animals weighed <20 kg.
If we were to assume a distribution volume equal to the extracellular fluid and make no allowance for binding by sodium pumps, we can calculate the total amount of ouabain secreted by the beagle in 13 minutes from the formula: ECF volume/plasma volumextotal dose in plasma; this is close to 13 mg. Given that significant amounts of endogenously secreted ouabain would have bound to the sodium pumps this is likely to be an underestimate.
Whatever the explanation for these results, they do provide an excellent opportunity to resolve a division that exists in relation to endogenous ouabain. Since the mass-spectrometric identification of ouabain (or closely related substance) in an extract of human plasma,2 there have been those who have questioned its endogenous origins, whereas others, relying principally on radioimmunoassay, have equated the immunoassayable substance with authentic ouabain and use the terms "ouabain-like" and "ouabain" interchangeably. The quantities of the immunoassayable substance seen in these humans and, to a far greater extent in dogs, fall comfortably within the range required for physical characterization by mass spectrometry and, in view of the milligram amounts reported in beagles, proton NMR. If the substance responsible is indeed authentic ouabain the skeptics probably have to revise their position. If not, then the significance of plasma immunoassayable "ouabain" would be called into question.
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References |
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 Top References References |
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- Bauer N, Müller-Ehmsen J, Kramer U, Hambarchian N, Zobel C, Schwinger RHG, Neu H, Kirch U, Grünbaum E-G, Schoner W. Ouabain-like compound changes rapidly on physical exercise in humans and dogs. Hypertension. 2005; 45: 1024–1028.
[Abstract/Free Full Text] - Matthews WR, DuCharme DW, Hamlyn JM, Harris DW, Mandel F, Clark MA, Ludens JH. Mass spectral characterization of an endogenous digitalislike factor from human plasma. Hypertension. 1991; 6: 930–935.
Response
Institute of Biochemistry and Endocrinology, Justus-Liebig University, Giessen, Germany
In their comment to the article of Bauer et al1 on the changes of the plasma concentrations of a ouabain-like compound associated with vigorous exercise in humans and dogs, Hilton and McKinnon are wondering why dogs are apparently healthy although their plasma concentration under exercise exceeds that reported for the LD50 of ouabain. We do agree that ouabain circulating in blood plasma in such high concentrations of about 6 µmol/L should harm dogs and humans. We tested our antibodies for cross-reactivities, which are part of the article, and found them rather specific. We furthermore found a linear correlation between ouabain and the signal (Figure 3 in the Reference 1). Nevertheless, additional unknown compounds may circulate in blood competing with ouabain for ouabain antibodies and the cardiac glycoside receptor site of Na+/K+-ATPase. This is the reason why the compound was called ouabain-like.
The reader should be aware that in addition to the mechanism usually communicated in text books of pharmacology (ie, inhibition of the sodium pump), cardiac glycosides may also use Na+/K+-ATPase as a signal transducer of cardiac glycosides by a mechanism not inhibiting the pump to exert their inotropic effect (for a short review see Reference2). There is a continuing debate on the existence of ouabain and ouabain isomers in mammals:3 Ouabain has been identified by mass spectroscopy and proton NMR in bovine adrenals and hypothalamus; other studies analyzing the nature of the compound without application of proton NMR but mass spectroscopy and other techniques came to the conclusion that the isolated compound is either ouabain (human plasma, PC-12-cell media) or a closely related isomer (human plasma, bovine hypothalamus). No general agreement on the nature of the circulating ouabain-like compound has been reached so far. Interestingly, substances like PST 2238 have been synthesized that bind to the ouabain receptor of Na+/K+-ATPase without inhibiting the pump.4 PST 2238 apparently interferes with the natural circulating ouabain-like compounds at the sodium pump and lowers arterial hypertension in experimental animals.4,5 We may not exclude that additional compounds exist that interact with the cardiac glycoside receptor without inhibiting the pump but acting as a signal transducer2. They may interact with antibodies against ouabain. Certainly, blood plasma from an exercise-stressed mammal would be a good source to isolate this ouabain analog, whose nature should be identified by mass spectroscopy and proton NMR.
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References |
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TopReferencesReferences |
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- Bauer N, Müller-Ehmsen J, Kramer U, Hambarchian N, Zobel C, Schwinger RHG, Neu H, Kirch U, Grünbaum E-G, Schoner W. Ouabain-like compound changes rapidly on physical exercise in humans and dogs. Hypertension. 2005; 45: 1024–1028.
[Abstract/Free Full Text] - Scheiner-Bobis G, Schoner W. A fresh facet for ouabain action. Nature Medicine. 2001; 7: 1288–1289.[CrossRef][Medline] [Order article via Infotrieve]
- Hamlyn JM, Laredo J, Shah JR, Lu ZR, Hamilton BP. 11-Hydroxylation in the biosynthesis of endogenous ouabain: Multiple implications. Ann NY Acad Sci. 2003; 986: 685–693.
[Abstract/Free Full Text] - Ferrari P, Ferrandi M, Tripoldi G, Torielli L, Padoani G, Minotti E, Melloni E, Bianchi G. PST 2238: A new antihypertensive compound that modulates Na,K-ATPase in genetic hypertension. J Pharmacol Exptl Therapeutics. 1999; 288: 1074–1083.
[Abstract/Free Full Text] - Ferrari P, Torielli L, Ferrandi M, Padoani G, Duzzi L, Florio M, Conti F, Melloni L, Vesci L, Corsico N, Bianchi G. PST 2238: A new antihypertensive compound that antagonizes the long term pressor effect of ouabain. J Pharmacol Exptl Therapeut. 1998; 285: 83–94.
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