Physicochemical characterization of a ouabain isomer isolated from bovine hypothalamus.

Abstract

Recent reports have shown the presence of a ouabain-like inhibitor of Na+/K+-ATPase in humans. We have purified a bovine hypothalamic Na+/K+-ATPase inhibitory factor (HIF) by using affimity chromatography combined with HPLC. This inhibitor has a molecular weight of 584 as determined by ion-spray mass spectrometry, making it isobaric with ouabain. Glycosidase treatment or acid hydrolysis of HIF released only L-rhamnose, the hexose isomer found in ouabain, as detected by chiral GC/MS. Additionally, enzymatically generated desrhamnosyl HIF was found to have a molecular weight of 438, as does ouabagenin, the aglycone of ouabain. HIF and its aglycone were indistinguishable from ouabain and ouabagenin, respectively, by reversed-phase HPLC retention times. However, derivatization with naphthoylimidazole followed by HPLC revealed different retention times for naphthoylation products of HIF and ouabain. Subsequent CD spectroscopy on isolated naphthoylation products of HIF and ouabain confirmed that they were different. This study provides chromatographic and spectroscopic evidence that ouabain and HIF are isomeric cardenolides. The structural difference is presumed to account for the significant differences in biological properties observed for HIF and ouabain.The search for an endogenous inhibitor of Na+/K+-ATPase has been supported by the occurrence of digitalis-like cardiotonic steroids in plants and the presence of a highly conserved binding site for ouabain in mammalian tissues (1). Obstacles faced in attempts to identify such endogenous inhibitors have included the very low concentrations present in mammals and the difficulty ofobtaining stringently purified samples for analysis (2,3). Hamlyn and associates (4, 5) described a ouabain-like inhibitor of Na+/K+-ATPase from human plasma that was indistinguishable from ouabain ( Fig. 1, structure 1) by several criteria including mass spectrometry, immunoreactivity, and biological assays. Their results raised interesting speculation about the ability of humans to synthesize ouabain, previously known only as a plant product. Both portions of ouabain, specifically the highly functionalized steroid ouabagenin (Fig. 1, structure 2) and the sugar L-rhamnose (structure 3), are unprecedented in mammalian biosynthesis. Outside of the plant kingdom, only cardiotonic steroidal aglycones have been isolated from toads (6). In the present study, a Na+/K+-ATPase inhibitor from bovine hypothalamus (7) was purified by an enzyme affinity method coupled with preparative HPLC steps. Microanalytical methods were developed to directly compare the affinitypurified hypothalamic inhibitory factor (HIF) with authentic ouabain. Both HIF and ouabain were shown to have identicalThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. molecular weights and to yield L-rhamnose and isobaric aglycones upon hydrolysis. The stereochemistry of th...

Ouabain is not detectable in human plasma.

“…35 One possible explanation for the discrepant observations from various ouabain assays may be that human endogenous ouabain is a stereoisomer of plant ouabain and is detectable by some antibodies and assay systems 5 -6 but not by others, including our own. However, the NEN ELISA apparently used Ham-lyn's antibody, and in our hands, ouabain was not detected.…”

Structure of 'endogenous ouabain'

“…Naphthoylation of HIF (300 ng) followed by RP-HPLC showed that the major derivatization product from HIF eluted slightly earlier than ouabain pentanaphthoate, the major product from ouabain naphthoylation; moreover, CD spectroscopy revealed that this HIF derivative showed no distinct CD, whereas ouabain pentanaphthoate showed a clear positive CD couplet. Although the molecular ion peak of this HIF derivative was not clear, the product was described as HIF pentanaphthoate (13). Subsequent microscale derivatization of OLC (400 ng, gift from Upjohn Laboratories) also gave the same RP-HPLC and CD (''zero-CD'') profiles as those of HIF pentanaphthoate (14).…”

Bovine Adrenals Contain, in Addition to Ouabain, a Second Inhibitor of the Sodium Pump

“…In fact, a material cross-reacting with antibodies against ouabain, digoxin, and digitoxin and inhibiting the sodium pump circulates in increased concentrations in the blood of patients with low renin essential hypertension (1)(2)(3)(4). There has been an intensive search for such substances that has led to the isolation of an ouabain-like inhibitor of the sodium pump from human plasma (5) and an attempt to isolate an isomer of ouabain from bovine hypothalamus (6). Nanogram scale CD structural analysis indicates that both substances show similar properties but are not identical with ouabain (7).…”