Next-Level Natriuretic Peptide Measurement

Measurement of natriuretic peptides or their molecular precursors has been implemented in everyday assessment of heart failure. Today, the most used markers are B-type natriuretic peptide (BNP) and N-terminal proBNP (NT-proBNP) (1). From a diagnostic standpoint, the 2 markers seem equal in their performance when evaluating possible systolic heart failure. In addition, measurement of peptides stemming from the related atrial natriuretic peptide (ANP) gene seem non-inferior, albeit that reliable methods for measuring ANP in plasma remain to be automated. Since the introduction of natriuretic peptide measurement, heart failure has been classified into heart failure with either preserved (HFpEF) or reduced (HFrEF) left ventricular systolic function, and the prognostic value of natriuretic peptide measurement in the latter condition still remains a matter of controversy (2). Furthermore, new therapy modalities have been introduced over the last decade which may impact on interpretation of natriuretic peptide measurements. Treatment with sacubitril/valsartan aims at, at least partially, increasing endogenous natriuretic peptide signaling. In addition to increasing the concentration of the endogenous hormones, altered processing of their proforms has also been suggested. This challenges the choice of analyte for disease monitoring. In this issue of Clinical Chemistry, Hélène Nougué and her colleagues report on the different molecular forms of proBNP-derived peptides measured by immunological analyses (3). The authors use mass spectrometry to quantify peptide forms and compare these data with the immunological measurements. Besides determining different lengths of the peptides (intact proBNP, NT-proBNP, and BNP), they are also able to evaluate the effect of glycosylation on the immunological measurements—namely a specific glycosylation at position 44 in the propeptide (where NT-proBNP antibodies bind). The authors conclude that glycosylation does interfere with NT-proBNP immunological measurement, but NT-proBNP still seems to be the most robust marker of overall BNP expression. Since the discovery of the natriuretic peptides, mass spectrometry as an analytical tool has advanced to allow for new insight into “small” peptide chemistry. Earlier methods for characterizing peptide forms were largely chromatographic in nature followed by immuno-based measurement. This old technique, however, has some inherent problems as characterization of the eluted peptides still relies on the immunoassays and their epitope-specific binding. This explains why it took >15 years before the complex and variable glycosylation of natriuretic propeptides was acknowledged (4). Glycosylated proforms were not measured when using immunoassays, as the carbohydrate group(s) blocked the recognition of the epitope by the antibody which had been generated against non-glycosylated peptide forms. Looking back, some larger forms were occasionally noted on chromatograms, but the actual biochemical modifications were not identified (5). Hence, the terms “big BNP” and “big-big BNP” were sometimes used, as has been common practice for many other peptide hormones over the years. With the use of mass spectrometry (LC-MS/MS), however, this limitation has been overcome. On the other hand, mass spectrometry in terms of peptidomics is still far from perfect, as insufficient sensitivity still limits the technology (6). To improve the clinical value of a biomarker is a multifaceted task. Obviously, the marker should be specific for the condition in question. Furthermore, a form compatible with the laboratory phase of measurement and refractory to preanalytical degradation should be identified. The latter has in fact been an argument for measuring NT-proBNP in the first place. Moreover, proforms in circulation often fluctuate less than their bioactive end products; one well-known example is C-peptide vs insulin concentrations in diabetes mellitus. In the case of natriuretic peptides, there is also another challenge. When it comes to “classic” heart failure with reduced left ventricular ejection fraction, both BNP and NT-proBNP perform well; so well that improvement in terms of clinical accuracy is hard to obtain. In cohort studies, receiver operating curve (ROC) analyses of their performance often yield AUC (area-under-the curve) values >0.85, and from there, further improvement may be difficult. Many clinical laboratories already use mass spectrometry. At present, it is not feasible to use LC-MS/MS on a daily basis for natriuretic peptides that circulate in the low-picomolar concentration range, but other aspects may still deserve exploration. For instance, already diagnosed patients with highly increased concentrations may benefit from a qualitative evaluation of the peptides. It seems plausible that such a holistic “map of the forms” could contain relevant information for further prognostication and even for therapy monitoring (7). Thus, specialists within heart failure, either HFrEF or HFpEF, could get an extended laboratory report for choosing treatment strategy and follow-up. With the current focus on HFpEF, the biomarkers are challenged once again. HFpEF comes with several issues in terms of BNP and NT-proBNP concentrations. One major issue is the fact that conditions associated with development of HFpEF also independently affect their circulating concentrations. Obesity has for long been known to associate with decreased concentrations, as has diabetes mellitus (8). Genetics and race also play a role in terms of natriuretic peptide expression and their circulating concentrations (9). Taken together, when it comes to the use of established biomarkers in a new disease entity, caution is needed to prevent confounders. From the laboratory point of view, we may even need to change the way we report the results. Are the test results to be used for HFrEF or HFpEF? Should our suggested cutoff values be related to the primary question behind the measurement? And should we work on coupling patient measurements to other factors, e.g., adjustment for body mass index, diabetes, and perhaps time of day? With the new electronic health care platforms, digital integration of clinical data and biochemical results certainly holds a promise for more personalized interpretation and conclusions. With automation of this process, clinicians can then be spared having to remember different cutoff values for various patient categories, which otherwise may introduce misinterpretation and even lead to mistakes. Peptide glycosylation is not constant but most likely also regulated in terms of the underlying organ disease and how we intervene with pharmacotherapy. Glycosylation of natriuretic peptides is a much more complex biological process than uniform modification of one particular amino acid. We have shown that even bioactive natriuretic peptides (for example, ANP) are glycosylated; This modification influences receptor activation, but the proportion of ANP with this modification still awaits in-depth quantification in health and in disease (10, 11). Biological changes in proBNP glycosylation have also been reported in obese patients (12). Whether gender, race, diabetes mellitus, or other factors influence glycosylation will be most interesting to elucidate. Also, diurnal effects could be relevant—and could perhaps partly explain the marked circadian biology of natriuretic peptides (13). Of note, almost no information is available on changes in glycosylation in response to pharmacotherapy. For therapies that directly target endogenous peptides, e.g., sacubitril/valsartan as in the present study, it is obvious that glycosylation changes should be assessed, but other types of therapy are also likely to affect glycosylation. Modified natriuretic peptides with better safety profiles—as compared to the native forms—look promising as therapy of both heart failure and essential hypertension (14). Strategies to mimic the effects of natriuretic peptides, as for instance with a soluble guanylate cyclase stimulator, have also been introduced (15). When exploring the therapeutic possibilities for these and future drugs, the biochemical aspects of endogenous natriuretic peptides should be evaluated including changes in glycosylation and bioactivation/bioactivity. Basically, using natriuretic peptides for clinical assessment may one day move from considering them as continuous markers to also including a qualitative map, thereby taking natriuretic peptide measurement to the next level towards personalized medicine. BNP, B-type natriuretic peptide: NT-proBNP, N-terminal proBNP; ANP, atrial natriuretic peptide; HFpEF, heart failure with preserved ejection fraction. All authors confirmed they have contributed to the intellectual content of this paper and have met the following 4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. No potential conflicts of interest.