Published Online
on October 14, 2008

A more recent version of this article appeared on November 1, 2008

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Original Article

Detection of Endogenous B-Type Natriuretic Peptide at Very Low Concentrations in Patients with Heart Failure

Eric E. Niederkofler¹; Urban A. Kiernan¹; Jessica O'Rear²; Santosh Menon³; Syed Saghir³; Andrew A. Protter⁴; Randall W. Nelson¹ and Ute Schellenberger^5,6

¹ Intrinsic Bioprobes, Inc. Tempe, AZ;
² Scios Inc., Mountain View, CA;
³ Christ Hospital, Cincinnati, OH;
⁴ Medivation Inc. San Francisco, CA;
⁵ Scios Inc., Mountain View, CA; Medivation Inc. San Francisco, CA

⁶ E-mail: uschellenberger{at}gmail.com

Background—The myocardium secretes B-type natriuretic peptide (BNP) in response to stimuli associated with heart failure (HF). However, high immunoreactive-BNP (iBNP) levels in patients with HF are associated with a paradoxical lack of natriuretic response. We hypothesized that commercially available assays for iBNP do not reflect the bioactivity of the natriuretic peptide system, since they measure both unprocessed, inactive proBNP and mature BNP 1-32. Here, we describe an assay for the detection of bioactive BNP 1-32 and confirm very low concentrations in plasma from HF patients.

Methods and Results—We developed a quantitative mass spectrometry immunoassay (MSIA) to capture endogenous BNP peptides using high affinity antibodies. Bound BNP and its truncated fragments were detected by matrix assisted laser desorption ionization—time of flight (MALDI—TOF) mass spectrometry based on their predicted masses. MSIA revealed rapid in-vitro degradation of BNP 1-32 in plasma, which requires plasma collection in the presence of high protease inhibitor concentrations. In 11 of 12 HF patients BNP 1-32 was detectable, ranging from 25 to 43 pg/ml. Several degraded forms of BNP were also detected at similarly low levels. In contrast, parallel measurements of iBNP using the Biosite assay ranged from 900 to 5000 pg/ml.

Conclusions—Detection of endogenous BNP 1-32 requires special preservation of plasma samples. MSIA technology demonstrates that HF patients have low levels of BNP 1-32. Commercially available iBNP assays over-represent biological activity of the natriuretic peptide system because they cannot distinguish between active and inactive forms. This observation may, in part, explain the "natriuretic."

Key Words: circulation • diagnosis • heart failure • natriuretic peptides • mass spectrometry

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