Michael Lassman (Presenter)
Merck & Co
Authorship: Derek Chappell, Anita Lee, Omar Laterza, Michael Lassman
Merck & Co, Translational Molecular Biomarkers
| Short Abstract We have a developed and validated a first of its kind immunoaffinity LC/MS assay with sufficient performance characteristics to measure BNP derived fragments as well as “Total BNP” in the plasma of adults diagnosed with heart failure. The following performance characteristics were assessed as part of analytical validation: inter and intra-assay precision, sensitivity, spike recovery, dilution linearity, freeze/thaw stability, and assay recovery. The ratio of active BNP(1-32) to Total BNP in the plasma was found to be low, ranging from below the limit of quantitation to 8%. In addition, we observed that the Total BNP measurement is better correlated (r2=0.89) with the clinical NT-proBNP measurement rather than the more specific IA-LC/MS BNP(1-32) measurement (r2=0.06), leading us to conclude that the accepted clinical based BNP measurements are not indicative of active BNP. |
Long Abstract
Introduction: B-type natriuretic peptide (BNP) is a hormone that is released primarily in response to myocardial stress. Initially synthesized as pre-proBNP(1-134), it is quickly processed into proBNP(1-108), NT-proBNP ¬(proBNP1-76) and the physiologically active peptide BNP(1-32). As NT-proBNP and BNP(1-32) are processed from the same pro-protein, they are produced in equimolar amounts and established anti-body based clinical assays exist for both and are both predictive for heart failure (HF). The analytical concerns with the clinical assays have been well described in the literature and are most likely due to the specificity of the antibodies employed in the assays. In order to address the specificity of the clinical assays, we developed a first of its kind immuno-affinity liquid chromatography mass spectrometry (IA-LC/MS) assay without trypsin digestion, to quantify the endogenous concentrations of five BNP fragments derived from pro-BNP including the active peptide BNP(1-32). This assay also incorporates a parallel digestion step quantify Total BNP, as measured by the proteolytic peptide ISSSSGLGCK, or BNP(18-27). The tryptic fragment, ISSSSGLGCK is conserved in unprocessed proBNP, BNP1-32, as well as in many potential degradation fragments and thus can be used to estimate the sum total of multiple proBNP derived peptides, or “Total BNP.” To our knowledge this is the first assay that can quantitate the endogenous ratio of BNP(1-32) to Total BNP.
Methods: Three anti-BNP antibodies were combined and conjugated to Tosylactivated M-280 Dynabeads for immunoaffinity pulldown. Following the binding and washing steps, the BNP peptides bound to the beads were eluted in 50 µL. The 50 µL sample was then split into 2 aliquots: 30 µL was immediately measured by low flow LC/MS with an ionkey Waters TQS QQQ mass spectrometer for measurement of BNP fragments, and the remaining 20 µL was reduced, alkylated and digested with trypsin. On Day 2, the digested samples were measured by low flow LC/MS with the same ionkey Waters TQS QQQ mass spectrometer. The following assay performance characteristics were assessed: inter-assay precision, intra-assay precision, sensitivity, spike recovery, dilution linearity, freeze/thaw stability, absolute recovery, matrix effect, IA efficiency.
Results: It has been well-established that BNP is inactivated in vivo through cleavage by endogenous proteases such as dipeptidyl peptidase 4 (DPP-4) and neprilysin (NEP). Cleavage of BNP(1-32) by DPP-4 and NEP result in the formation of BNP(3-32) and BNP(5-32) respectively. We suspected these enzymes are among many that could play a role in reducing the stability of BNP both in vivo and ex vivo during blood collection and storage. Therefore, we evaluated the ex vivo stability of BNP(1-32) in both K2EDTA plasma and P800 plasma, which contains a mixture of multiple protease inhibitors, including those that inhibit DPP-4. Separate aliquots were also sent for BNP analysis using the clinical immunoassay platforms. The observed concentration of BNP(1-32) is relatively unchanged after an hour in P800 plasma, but the concentration of BNP(1-32) is reduced even at the nominal zero time point (t=0hr) in K2EDTA plasma, indicating significant and rapid enzymatic cleavage in K2EDTA plasma. The degradation of BNP(1-32) correlates with a similar observed relative increase in BNP(3-32), the likely product of DPP-4 protease activity. Total BNP concentrations were not affected by incubation in either plasma matrix, indicating that Total BNP incorporates a combination of intact and cleaved BNP peptides. Based on the results of this experiment, we chose P800 plasma as the preferred matrix for IA-LC/MS analysis of BNP by mass spectrometry. The clinical immunoassay was not sensitive to the difference in BNP between P800 and EDTA plasma.
Intra-assay precision ranged from 4% to 10%, and 1% to 7% for BNP(1-32) and Total BNP respectively. Inter-assay precision ranged from 6% to 20%, was 7% for both BNP(1-32) and Total BNP. The LOQ of the BNP fragments was determined to be 0.4 pM and 2.2 pM for BNP(1-32) and Total BNP respectively based on a %CV < 20% and a %bias < 5% for the lowest standard point. Of the 11 heart failure patient’s results measured to the date of this abstract, we were able to measure BNP(1-32) in only 8 of the 11 patients samples with concentrations determined to be 0.7 pM to 26.0 pM, while Total BNP values ranged from 4.7 pM to 870.6 pM. BNP(3-32) was measured in 7 of 11 samples with concentrations ranging of 0.6 pM to 35.2 pM. BNP(4-32) was detected in all samples with concentrations ranging from 0.6 pM to 16.7 pM. BNP(5-32) was detected in 10 of 11 patient samples with concentrations ranging from 0.6 pM to 105.9 pM. BNP(5-31) was not detected in any samples.
Conclusions: Here, we add to the published evidence that the clinical immunoassays for BNP are not specific for BNP(1-32) based on the data obtained using the Bayer BNP immunoassay, which is an FDA cleared device approved for the measurement of BNP. The immunoassay was unable to characterize the degradation of BNP(1-32) which occurs in K2EDTA plasma but not in P800 plasma ex vivo as measured by the IA-LC/MS method. Therefore, our findings confirm that routine collection procedures that do not involve addition of protease inhibitors post-collection are not suitable for accurate quantitation of BNP(1-32). Biological variability data from a small number of HF patients showed that BNP(1-32), the peptide that has been demonstrated to exert natriuretic benefit, is less than 8% of the Total BNP measured in all samples for which BNP(1-32) was above the limit of quantitation (n=8). The observation that the Total BNP measurement is better correlated (r2=0.89) with the clinical NT-proBNP measurement versus the more specific IA-LC/MS BNP(1-32) measurement (r2=0.06) is a clear indication that the NT-Pro BNP measurement, which has been demonstrated to have utility in the clinic and in the identification for risk of HF, is not indicative of the circulating concentration of the active BNP(1-32) peptide. Furthermore, we found no significant correlation with any of the fragments. Lastly, the sum of measurable BNP fragments account for approximately 15-30% of the Total BNP pool in circulation. This indicates the presence of more degradation fragments, or more likely, supports the notion that a large amount of unprocessed proBNP remains in circulation. Collectively, these findings may help provide an explanation for the natriuretic paradox; that patients with elevated BNP, as measured by established clinical assays, do not exhibit increased natriuresis. It is not clear why Total BNP (or NT-proBNP) is elevated in patients at risk for HF, but not active BNP(1-32). Increased Total BNP may result from reduced catabolism or from increased synthesis of BNP, but this will require further study.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Merck & Co |
| Board Member | no | |
| Stock | yes | Merck & Co |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |