Development of an immunoassay for endogenous digitalislike factor.

Harris, Douglas W.; Clark, Michael A.; Fisher, Jed F.; Hamlyn, John M.; Kolbasa, Karen P.; Ludens, James H.; DuCharme, Donald W.

doi:10.1161/01.hyp.17.6.936

Cited by 98 publications

(71 citation statements)

References 16 publications

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“…Finally, a specific polyclonal antibody to ouabain cross-reacts strongly with EDLF. 18 Until now we have referred to the inhibitor of the sodium pump we isolated from human plasma as EDLF. We used this general nomenclature because we found our inhibitor to possess many of the properties of the cardiac glycosides.…”

Section: Resultsmentioning

confidence: 99%

Mass spectral characterization of an endogenous digitalislike factor from human plasma.

et al. 1991

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A sodium pump inhibitor has been isolated from human plasma and extensively purified. This material, endogenous digitalislike factor, was examined by a variety of mass spectrometric techniques. A low-resolution fast atom bombardment mass spectrometric analysis of a sample of purified endogenous digitalislike factor revealed a single unique molecular ion in the mass range 100-2,500. The accurate mass was determined to be 585.295 Da in a second highresolution fast atom bombardment mass spectrometric experiment Based on this accurate mass, the elemental composition of endogenous digitalislike factor was determined and found to be identical to the elemental composition of the known cardenolide ouabain. Direct comparison of ouabain and endogenous digitalislike factor by linked scan tandem mass spectrometry, derivatization with acetic anhydride coupled with fast atom bombardment mass spectrometry, and analytical high-performance liquid chromatography failed to reveal any differences. We conclude that the endogenous digitalislike factor isolated from human plasma is ouabain or a closely related isomer. (Hypertension 1991;17:930-935)

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Section: Resultsmentioning

confidence: 99%

Mass spectral characterization of an endogenous digitalislike factor from human plasma.

et al. 1991

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“…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.…”

Section: Antibody Preparationmentioning

confidence: 99%

Ouabain is not detectable in human plasma.

et al. 1994

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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...

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“…4 Briefly, 25 /il assay buffer containing sample or ouabain standard was added to half-area enzyme immunoassay plates (Costar Corp., Cambridge, Mass.) previously coated with a conjugate of ouabain linked to bovine serum albumin via a hexane diamine spacer.…”

Section: Assay Of Samples For Ouabainlike Compoundmentioning

confidence: 99%

“…4 Immunoreactive OLC was evident in extracts of several tissues as well as plasma, with the adrenal gland extracts being particularly enriched. 5 The present studies were carried out to evaluate further the contribution of the adrenal glands to circulating levels of OLC in the rat.…”

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“…The chemical identity of a potent inhibitor of the sodium pump isolated from human plasma recently was reported to be indistinguishable mass spectrometrically, 1 biochemically, 2 pharmacologically, 3 and immunologically 4 from the plant cardenolide ouabain. This purified material subsequently was referred to as ouabainlike compound (OLC), 5 and several lines of evidence were presented which suggested that the OLC originated endogenously.…”

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Rat adrenal cortex is a source of a circulating ouabainlike compound.

et al. 1992

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To determine if the adrenal gland may be the source of plasma-borne ouabainlike compound (OLC) in rats, we 1) measured immunoreactivity expressed as OLC equivalents in extracts from a wide variety of central and peripheral tissues and, for adrenal cortex and medulla, chromatographed the extracts to determine to what extent immunoreactivity in the adrenal was OLC, and 2) measured OLC in the plasma of adrenalectomized and adrenal demedullectomized rats. The highest levels of immunoreactivity were found in adrenal cortex, adrenal medulla, atria, and the pituitary. Based on high-performance liquid chromatographic retention time, immunoreactivity in the adrenal cortex was almost exclusively immunoreactive OLC. Removal of this rich source of OLC from rats resulted in an approximate 50% decrease in circulating levels of OLC by 6 days after removal. Furthermore, although adrenal demedullectomy also caused a decrease in OLC 3 days after surgery, the decline was sustained only with total adrenalectomy, in that plasma levels of OLC in demedullectomized rats 6 days after surgery had returned to levels equal to those of sham controls. Taken together, these findings strongly suggest that the adrenal cortex is a major contributor to circulating OLC in the rat. T he chemical identity and source of an endogenous counterpart to the plant-derived digitalis glycosides have been pursued intently, ever since it was recognized that a high-affinity binding site for these substances existed on the membrane-bound enzyme Na + ,K + -ATPase. Such substances, by virtue of inhibiting the membrane sodium pump (Na + ,K + -ATPase), could play an important role in electrolyte homeostasis in mammalian cells and thereby modulate numerous aspects of cell biology.The chemical identity of a potent inhibitor of the sodium pump isolated from human plasma recently was reported to be indistinguishable mass spectrometrically, 1 biochemically, 2 pharmacologically, 3 and immunologically 4 from the plant cardenolide ouabain. This purified material subsequently was referred to as ouabainlike compound (OLC), 5 and several lines of evidence were presented which suggested that the OLC originated endogenously. Although the hypothalamus has been suggested to be a likely source of an endogenous inhibitor of the sodium pump, 67 a variety of evidence implicates the adrenal gland as well. An inhibitor of Na + ,K + -ATPase possessing biochemical properties strikingly similar to ouabain was extracted from bovine adrenals, 8 and when a variety of tissue extracts were assayed with biochemical and immunological techniques, the level of digitalis-like or ouabainlike factors in adrenal extracts was among the highest. 910 In addition, minced adrenal tissue was found to release an immunoreactive digitalis-like material into a serum-free incubation medium, 11 and adrenalectomy was shown to diminish circulating levels of an immunoreactive digitalis-like factor. 912A highly sensitive, ouabain-specific enzyme-linked immunospecific assay (ELISA) that used a rabbit antiouabain antis...

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Development of an immunoassay for endogenous digitalislike factor.

Cited by 98 publications

References 16 publications

Mass spectral characterization of an endogenous digitalislike factor from human plasma.

Mass spectral characterization of an endogenous digitalislike factor from human plasma.

Ouabain is not detectable in human plasma.

Rat adrenal cortex is a source of a circulating ouabainlike compound.

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