Elsevier

Clinical Biochemistry

Volume 38, Issue 1, January 2005, Pages 36-45
Clinical Biochemistry

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

https://doi.org/10.1016/j.clinbiochem.2004.08.005Get rights and content

Abstract

Backround and objectives:

There are several potential endogenous digitalis-like factors (EDLF) in mammalian body fluids, and marinobufagenin (MBG) may be the most potent EDLF. Improved assays are needed to confirm the potency of these metabolites. In the present study, we have identified MBG and telocinobufagin (TCB) in human plasma by high-resolution mass spectrometry (MS) and nuclear magnetic resonance (NMR).

Methods and results:

The high-resolution MS analysis revealed the molecular masses of TCB and MBG to be the same as their respective theoretical values. Using a tandem mass spectrometer, the mass–charge ratio for TCB was determined to be 403.2 for the parent ion and 349.2 for the daughter ion. The mass–charge ratio for MBG was m/z 383.2 and m/z 401.2. The NMR study revealed that the signals for MBG and TCB were the same as those obtained by MS analysis. In human blood, MBG and TCB were also identified by liquid chromatography (LC) as well as MS. In the LC/MS assay, proscillaridin A was used as an internal standard. The plasma was pretreated with Sep-Pak C18, and then 50 μL was applied to the C8 high-performance liquid chromatography (HPLC) column. The mean plasma concentration of MBG in healthy volunteers (0.94 ± 0.28 ng/mL) was significantly lower than that in patients undergoing regular hemodialysis (3.81 ± 1.92 ng/mL). The concentration of TCB in the healthy volunteers (1.80 ± 0.55 ng/mL) was also significantly lower than that in patients with terminal renal failure (6.86 ± 4.30 ng/mL).

Conclusion:

These results indicate that the major EDLF is TCB because its plasma concentration is the highest among the reported endogenous digitalis candidates.

Introduction

Bufadienolides and ouabain are the most likely modulators of water and sodium metabolism, and they are commonly referred to as the endogenous digitalis-like factors (EDLFs) or the endogenous sodium pump inhibitors in mammals [1]. Several bufodienolides have been identified in human urine and cataractous eye lens, and in toad skin and salivary glands [2], [3], [4], [5]. Therefore, potentially more than one EDLF exist. Marinobufagenin (MBG, 3-beta,5-dihydroxy-14,15-beta-epoxy-5-beta-bufa-20,22-dienolide, CAS Number: 470-42-8) has been suggested to be the most biologically important EDLF documented thus far, but further studies are needed to confirm this [5], [6], [7]. Bagrov et al. [3] first identified MBG in the mammalian blood by means of mass spectrometry (MS). However, because a large sample amount is required for the MS assay, these investigators subsequently developed an immunoassay for MBG for use in basic and clinical studies [3], [5], [6], [7] and used the term “MBG-like immunoreactivity” (MBG-IR). As shown in a recent review by Schoner [8], EDLF candidates such as 19-norbufalin, MBG, and proscillaridin A are very structurally similar except for the rhamnose residue at the C3 site. In a recent study on the immunoassay for bufalin, an anti-bufalin monoclonal antibody exhibited cross-reactivity with several mimicking bufadienolides [9]. A subtle structural difference such as one or two hydrogens can hardly be a sufficient epitope to raise a specific antibody for MBG. Therefore, the development of a specific assay for MBG other than the aforementioned immunoassay will be necessary to elucidate the physiological role of MBG.

Lichtstein et al. [4], [10] have studied the bufalin-like substances as endogenous sodium pump inhibitors by means of an immunoassay they developed and demonstrated several unique findings in the clinical setting. Again, there are limitations regarding the specificity of this assay system. They have characterized immunochemically identified substances by using high-performance liquid chromatography (HPLC) and confirmed that the retention time is the same as that for the standard (authentic) compound. However, bufadienolides are structurally very similar, and the difference in the retention time in HPLC may not be adequate to characterize the materials.

The recent progress in liquid chromatography–mass spectrometry (LC/MS) has made it possible to study the structure of small molecules with great precision, and this technique is now one of the most sensitive and informative methods available, giving qualitative, quantitative, and structure-specific information. 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 tissue-derived cells [11], as well as both ouabain and digoxin in human plasma by means of the LC/MS technique [12].

In the present study, MBG and another MBG-like bufadienolide, telocinobufagin (TCB, 3-beta,5,14-trihydroxy-5-beta-bufa-20,22-dienolide, CAS Number: 470-42-8), were identified in human plasma by high-resolution MS and nuclear magnetic resonance (NMR). The plasma concentration of TCB was much higher than MBG, and TCB may be the most central endogenous bufadienolide.

Section snippets

Purification of MBG and its related compounds

MBG and its related compounds were purified as follows. MBG powder was purchased from Sigma Aldrich Japan (Tokyo, Japan) as the starting material. All column chromatographic analyses were done under helium gas bubbling to minimize the oxidation of the substances during the procedure, and the elution pattern of MBG and its related compounds in HPLC was monitored by ultraviolet absorption at 300 nm. MBG powder (50 mg) was dissolved in 3 mL of methanol and stored in a dark container in an ice-cold

Isolation and identification of MBG and TCB

In a preliminary LC/MS experiment using commercially available MBG powder, we detected MBG as m/z 401 with its major daughter ion, m/z 365.2, and a similar, unknown compound as m/z 403 with its daughter ion, m/z 349.2. Using low-pressure ODS chromatography, the unknown compound (the m/z 403 component) eluted between 80 and 90 min, and MBG (the m/z 401 component) eluted between 95 and 105 min. These fractions were collected. High-pressure ODS chromatography revealed good isolation of the two

Discussion

This is the first report to provide evidence that TCB exists in the human blood. From an analysis using MS and NMR, the structure of TCB was determined to be quite analogous to that of MBG. MBG had an epoxy group between the 14th and 15th carbon, and TCB a hydroxy group at the 14th carbon, as shown in Fig. 3. The difference in the molecular weight between MBG and TCB was only two mass units.

As an EDLF, ouabain or its isomer has been considered to be the most probable effective candidate of

Acknowledgments

Grants: This work was supported in part by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Science and Culture (Hakuo Takahashi, number 14572191), and Charitable Trust Clinical Pathology Research Foundation of Japan (Yutaka Komiyama).

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