Liquid Chromatography Mass Spectrometric Analysis of Ouabainlike Factor in Biological Fluid

Abstract

Ouabainlike factor (OLF), assayed as ouabainlike immunoreactivity (OLI), is a probable endogenous digitalislike factor (EDLF). Liquid chromatography/mass spectrometry (LC/MS) is one of the most highly sensitive tools for obtaining structural information regarding low-molecular weight materials in a target compound, and to measure the concentrations of these materials. We have previously reported that OLI can be isolated from the culture supernatant of the rat pheochromocytoma cell line, PC12, by several reversephase chromatography and LC/MS techniques. The present study was performed to characterize OLF from biological fluids such as plasma and culture supernatant of PC12 cells by LC/MS. The previous applications of LC/MS to OLI in plasma have been limited to structural identification at the final stages of isolation, in which the starting volume of plasma has been over 10 I. In the present study, we tried to minimize the volume of plasma, and to develop a new preclearing step to gain adequate LC/MS characterization using MS/MS analysis. The plasma was acidified, and OLI was purified by ODS column chromatography. OLI in chromatographic fractions from plasma was assayed by a sensitive enzyme-linked immunosorbent assay for ouabain. After Sep-Pak treatment and two rounds of ODS column chromatography, OLI was identified from 80 ml of plasma. The structure of the purified OLI was identical to authentic ouabain and digoxin, as assessed by LC/MS. In conclusion, we identified the chemically or structurally clarified ouabain and digoxin as the circulating form in plasma by LC/MS. (Hypertens Res 2000; 23 Suppl: S21-S27)

A novel endogenous digitalis, telocinobufagin, exhibits elevated plasma levels in patients with terminal renal failure

“…However, the plasma levels of ouabain-like immunoreactivity (0.019 nmol/L = 12 pg/mL) in their study were almost equal to the findings (9 pg/ mL) in our previous enzyme-linked immunosorbent assay (ELISA) [13,20] and LC/MS analysis [12]. Binding proteins for EDLFs have been reported by Schoner's group and by us, and the activity of these proteins may of course affect the results of the assay [21,22].…”

“…It can be usefully applied for the assay of small molecular weight compounds such as steroids, drugs and poisons. We have recently identified ouabain in the culture supernatant from mammalian tissuederived cells [11], as well as both ouabain and digoxin in human plasma by means of the LC/MS technique [12].…”

De Novo Biosynthesis and Radiolabeling of Mammalian Digitalis-Like Factors

“…Although there is evidence to support this idea, most of it has been clouded by suspicion of extra vivo sources (e.g., diet or contamination) (26,28 ) or by certain structural features of the plant-derived cardenolides not consistent with what is known about mammalian steroid biosynthesis (the details are discussed below). Although the fine structural features of DLIFs as a family of compounds have not been definitively determined, there is very persuasive evidence that their structures are remarkably similar to those of the plant-derived digitalis compounds (2,19,29 ). The structure of digoxin, depicted in Fig.…”

Letter to the Editor concerning Baecher et al. (Clin Chim Acta 2014;431:87–92)

“…Given the "objective (of Clin Chim Acta) to publish novel information leading to a better understanding of biological mechanisms of human diseases, their prevention, diagnosis, and patient management", it seems especially important for the journal to get the story 'correct'. Surely, 'proof' that human plasma does not contain ouabain requires far more than an undocumented statement [1], especially when three published reports with MS spectra [4,5,8] and RIA of plasmas provided by Baecher et al show that human plasma does contain ouabain.…”

“…Published HPLC data and MS spectra demonstrating that EO is present in human plasma [2][3][4]8] will not disappear because of [1]. Importantly, key data from bovine tissue and from rodents, including mice with mutated (resistant) Na,K-ATPase ouabain binding sites (e.g.…”