Ouabain is not detectable in human plasma.

Abstract

An enzyme-linked immunosorbent assay is described for the measurement of ouabain in human plasma. This assay is specific for ouabain, strophanthidin, and ouabagenin, with other steroids, including digoxin and vasopressor hormones, exhibiting negligible cross-reactivity. Assay sensitivity was 0.06 nmol/L if 1 mL plasma was extracted and less than 0.005 nmol/L when 20 mL plasma was analyzed. Extracted plasma samples showed ouabainlike immunoreactivity that diluted in parallel with the ouabain standard curve. Repeated extraction and assay of single plasma samples, however, did not produce consistent results in the assay. suggested that ouabain is secreted by the adrenal glands and circulates in plasma in humans. This raises the question of whether ouabain might be the, or one of the, digoxin-like compounds thought to play a role in the regulation of sodium balance, arterial pressure, and vascular smooth muscle tone under normal circumstances and in disorders such as essential hypertension, pregnancy-induced hypertension, cardiac failure, and chronic renal failure. Uncertainty surrounding the involvement of ouabain in the regulation of blood pressure and electrolyte balance should be resolved in part by the availability of a reliable method for measuring ouabain concentrations in plasma. Published enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay methods can measure picomolar concentrations of ouabain. 56 We describe an alternative ELISA method based on the methodology of Lewis et al 7 that is sensitive and precise and compare the results obtained by assay of plasma extracts with those obtained after extraction and high-performance liquid chromatographic (HPLC) fractionation. Methods BuffersPhosphate-buffered saline (PBS) solution (60 mmol/L Na 2 HPO 4 , 40 mmol/L NaH 2 PO 4 , 150 mmol/L NaCl, pH 7.0) containing 1% bovine serum albumin and 1% thiomersal was used to reconstitute extracted samples and to dilute standards. PBS (pH 7.4) containing 1% gelatin and 0.05% Tween 20 was used for diluting antibody, and PBS (pH 7.4) containing 0.6% Tween 20 was used for washing microtitre plates. Antibody PreparationAntibody raising procedures were approved by the Animal Ethics Committee, Christchurch School of Medicine. Ouabain dialdehyde was coupled to C6-ovalbumin following the method of Harris et al. 5 The resulting solution was freeze-dried. Ouabain-ovalbumin (10 mg) was suspended in 5 mL 0.9% saline and emulsified with 5 mL Freund's complete adjuvant. Each of two New Zealand White rabbits received a subcutaneous injection of the ouabain-ovalbumin solution with boosters at monthly intervals for 2 months. Blood samples were collected into EDTA tubes through a small incision on a marginal ear vein 2 weeks after the booster injections. Immunoreactivity to ouabain was detected in EDTA plasma from both rabbits. Plasma from the second bleed of one rabbit (Gil-2) was diluted 1:100 with assay buffer and stored in 100-/J.L aliquots at -20°C. Conjugate PreparationPreparation of thyroglobulin-ouabain conjugate...

Structure of 'endogenous ouabain'

“…(1999) used for the detection of endogenous ouabain. Many previous contradictory results regarding the detection, purification, and biological studies of endogenous ouabain need to be reviewed in the light of such possibilities (24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34).…”

Endothelial Modulation of Ouabain-lnduced Contraction and Sodium Pump Activity in Aortas of Normotensive Wystar-Kyoto and Spontaneously Hypertensive Rats

“…However, the possible involvement of vascular endothelium has not yet been elucidated although an important endothelial dys function associated with hypertensive disease has been reported [12], Therefore, the aim of the present work was to investi gate any involvement of vascular endothelium in the responses to ouabain in vessels from WKY and SHR, as well as the possible mechanisms involved. This work is particularly interesting due to the recent description of enhanced plasma levels of sodium pump inhibitors in SHR and also in hypertensive patients, which appears to be ouabain [13,14], This assumption, however, needs additional confirmation because some researchers have some doubts about the existence of ouabain in the human plasma [15], In this paper, the influence of endothelium on oua bain-induced contractions was analyzed in aortas of SHR and WKY. Additionally, the changes in sodium pump activity caused by endothelium removal were studied in both types of vessels by analyzing K+-induced relaxations and 86Rb+ uptake.…”

Endogenous Ouabain Is Not Ouabain

“…This experience suggests that the characteristics of any antibody raised against such a substituted steroid needs to be investigated thoroughly to avoid confusion as to what the resulting immunoassay is actually measuring. In this regard, 3 groups (2 from the United States and 1 from Christchurch, New Zealand) that published their data in the same year (1994) were unable to detect immunoreactive ouabain after high-performance liquid chromatography separation of human plasma, [21][22][23] raising the possibility that EO as measured by available immunoassays did not represent authentic ouabain.…”