Two biologically active isomers of dihydroouabain isolated from a commercial preparation.
Two biologically active isomers of dihydroouabain isolated from a commercial preparation.
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从商业制剂中分离出二氢哇巴因的两种生物活性异构体。
DOI：
10.1016/s0304-4165(99)00153-1
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
ValdesJr,R
中科院分区：
文献类型：
--
作者：
Qazzaz,HM;El-Masri,MA;Stolowich,NJ;ValdesJr,R
Ouabain 是一种植物来源的强心苷，可抑制 Na+,K+-ATP 酶（钠泵；NKA）的催化活性。二氢哇巴因是哇巴因的内酯环还原衍生物，通常用作钠泵拮抗剂。假定市售二氢哇巴因是均质的。我们现在报道二氢哇巴因制剂含有两种成分，每种成分具有不同的抑制钠泵活性的效力。我们使用反相 HPLC 色谱法、紫外分光光度法、电喷雾电离质谱法 (ESI-MS)、核磁共振 (NMR) 光谱法和两种独立的生物测定法来表征这些化合物。两个二氢哇巴因级分（Dho-A 和 Dho-B）经过 3 分钟色谱分离，在 196 nm 处具有最大紫外吸光度，分别占母液二氢哇巴因的 37% 和 63%。每种成分抑制猪大脑皮层NKA的摩尔效力相差4.4倍（Dho-A，IC50=7.13±0.8μM；Dho-B，IC50=1.63±0.12μM）。相对效力分别为哇巴因的 9% 和 40%。观察到类似的 NKA 磷酸化模式。质谱 (ESI-MS) 和断裂模式与 Dho-A 和 Dho-B 一致，它们是具有相同分子量 (587 Da) 的异构体，并且各自具有六个羟基、一个脱氧己糖部分和一个内酯环。此外，NMR 光谱通过仅在分配给 H-21 和 H-22 的两组质子共振处显示明显不同的化学位移，揭示了 Dho-A 和 Dho-B 之间的结构差异。 ESI-MS和NMR结果证实内酯环C20处存在异构现象。我们的结果证明存在两种分子形式的二氢哇巴因，每种分子形式都具有不同的生物效力。这些发现强调了表征二氢哇巴因商业制剂纯度的重要性。它还为研究最近在哺乳动物中报道的内源性哇巴因样因子的代谢提供了可能的分子模型。
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, IC50=7.13±0.8 μM; Dho-B, IC50=1.63±0.12 μM). 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.