On the structure of endogenous ouabain

Abstract

The ouabain-like sodium pump inhibitor in mammals (so-called "endogenous ouabain") has been considered a subtle structural isomer of ouabain. Its structural investigation, however, has long been hindered by the paucity of sample material. Our recent purification of endogenous ouabain (3 micrograms) from bovine hypothalamus allowed the measurement of its 1H-NMR. The obtained spectrum as well as reexamination of past microscale structural studies on endogenous ouabain led us to identify the purified material as ouabain in an unusual manner. It turned out that the structural analysis had been complicated by a facile ouabain-borate complexation in borosilicate glassware. In retrospect, it is not surprising that the polyhydroxylated ouabain molecule serves as a polydentate ligand to inorganic species. In its physiological environment, ouabain may exist as some unknown complex. The chemical species giving rise to the reported biological activities of hypothalamic inhibitory factor preparations remain to be clarified.

Figure 1

¹H-NMR spectra of 3-μg HIF (A), ouabain (B), an ouabain sample (10 μg) that had been stored in a borosilicate vial as an aqueous solution (C), and ouabain tetrahedral-borates (D). A and B were measured in 10% acetonitrile-d₃ in D₂O, whereas C and D were measured in D₂O. ∗ indicates the peaks arising from either mechanical noise or because of unidentified impurities.

Figure 2

Fluorescence-detected HPLC traces for the naphthoylation product (retention time ≈10 min) from the ouabain sample that had been stored in a borosilicate vial as an aqueous solution (see text) (A), the naphthoylation product from HIF (14) (B), and coinjection of A and B (C).

Figure 3

Structures of the two coordination isomers of ouabain tetrahedral-borates.

Figure 4

¹H-NMR spectra of ouabain borate dissolved in acidic solvent (containing 0.1% TFA) (A), 3-μg HIF, same as Fig. 1A (B), and ouabain trigonal-borate (C). All measurements were carried out in 10% acetonitrile-d₃ in D₂O. ∗ indicates the peaks arising from either mechanical noise or because of unidentified impurities. The spectrum in C also contains signals from a small amount of ouabain.

Figure 5

Expanded ¹H-NMR of ouabain, same as Fig. 1B (A), 3-μg HIF, same as Figs. 1A and 4B (B), and ouabain trigonal-borate, same as Fig. 4C (C). ∗ indicates the peaks arising from either mechanical noise or because of unidentified impurities. The spectrum in C also contains signals from a small amount of ouabain.