title: Two biologically active isomers of dihydroouabain isolated from a commercial preparation.
abstract: Ouabain is a plant-derived cardiac glycoside that inhibits the catalytic activity of Na(+),K(+)-ATPase (sodium pump; NKA). Dihydroouabain, a derivative of ouabain with a reduced lactone ring, is commonly used as a sodium pump antagonist. It has been assumed that commercially available dihydroouabain is homogeneous. We now report that preparations of dihydroouabain contain two components each with a different potency for inhibition of sodium pump activity. We used reverse-phase HPLC chromatography, UV spectrophotometry, electrospray ionization-mass spectrometry (ESI-MS), nuclear magnetic resonance (NMR) spectroscopy and two independent bioassays to characterize these compounds. The two dihydroouabain fractions (Dho-A and Dho-B) resolved by 3 min chromatographically, had UV absorbance maxima at 196 nm, and comprised 37% and 63% of the stock dihydroouabain, respectively. The molar potency of each component for inhibition of NKA from porcine cerebral cortex differed by 4. 4-fold (Dho-A, IC(50) = 7.13 +/- 0.8 microM; Dho-B, IC(50) = 1.63 +/- 0.12 microM). The relative potencies were 9% and 40% of those of ouabain, respectively. A similar pattern for phosphorylation of NKA was observed. Mass spectrometry (ESI-MS) and fragmentation patterns are consistent with Dho-A and Dho-B being isomers of identical molecular mass (587 Da) and each with six hydroxyl groups, a deoxyhexose sugar moiety and a lactone ring. Furthermore, NMR spectroscopy revealed structural differences between Dho-A and Dho-B by displaying noticeably different chemical shifts at only two groups of proton resonances assigned to H-21 and H-22. The ESI-MS and NMR results confirm the presence of the isomerism at C20 of the lactone ring. Our results demonstrate the existence of two molecular forms of dihydroouabain, each with a different biological potency. These findings underscore the importance of characterizing the purity of dihydroouabain commercial preparations. It also provides possible molecular models for investigating the metabolism of endogenous ouabain-like factors recently reported in mammals.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Louisville, School of Medicine, Louisville, KY 40292, USA.
authors.forename: H M
authors.initials: HM
authors.lastname: Qazzaz
authors.forename: M A
authors.initials: MA
authors.lastname: El-Masri
authors.forename: N J
authors.initials: NJ
authors.lastname: Stolowich
authors.forename: R
authors.initials: R
authors.lastname: Valdes
mesh_terms: D002851:Chromatography, High Pressure Liquid; D007536:Isomerism; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D015394:Molecular Structure; D010042:Ouabain / Q000031:analogs & derivatives* / Q000737:chemistry / Q000302:isolation & purification; D010766:Phosphorylation; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors* / Q000737:chemistry; D012996:Solutions; D013056:Spectrophotometry, Ultraviolet
chemical_list: D012996:Solutions; C000977:dihydroouabain; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase