Introduction

Natriuretic peptides (NP) are key proteins that improve and regulate circulation. They are essential proteins that act on blood vessels causing them to widen or dilate.[1] These peptides are expressed predominantly by cardiomyocytes of the mixed secretory-contractile phenotype in atrial and ventricular walls. In addition to regulating blood pressure, NPs inhibit cardiac hypertrophy and remodeling.[2] In patients with decompensated heart failure due to volume overload, a treatment-induced drop in wedge pressure is often accompanied by a rapid drop in NP levels. Measurement of NP levels might thus assist with hemodynamic assessment and subsequent treatment titration.[3] NPs can also be found in the kidneys and can cause this essential organ to excrete more water and salt.[4] 

The b-type natriuretic peptide (BNP) was initially identified in porcine brain tissue in 1988 and was previously referred to as the “brain natriuretic peptide.” Later, researchers determined that the primary site of its release is cardiac ventricular cells, particularly in response to ventricular distention.[5] Following this discovery, abundant literature emerged regarding utilizing BNP as a potential biomarker to measure the presence and severity of heart failure.[6]

B-type natriuretic peptide (BNP) is a member of a family of natriuretic peptides (NP) that include atrial natriuretic peptide (ANP), C-type natriuretic peptide, D-type natriuretic peptide, and urodilatin.[7] Pro-BNP is a 108–amino acid precursor protein of BNP found in the ventricles and atria of the heart. Human ANP and BNP are encoded by genes located on chromosome 1.[8] In situations of volume overload and myocardial muscle stretching, proBNP is upregulated at the genomic level.[9]

A small amount of BNP is found in the cytoplasmic granules of myocytes. Upon stimulation for production and release, proBNP is cleaved by corin to equimolar amounts of the biologically active BNP hormone and the biologically inactive amino-terminal proBNP (NT-proBNP), which is also released into circulation as the unprocessed precursor protein.[8] When the effects of this peptide are evaluated on vascular smooth muscle cells, cardiac fibroblasts, and myocytes, this peptide did not appear to have any biological activity.[10]

The C-type natriuretic peptide is synthesized primarily in the central nervous system. It is also found in vascular endothelial cells and may play a role in the local regulation of vascular tone.[11] Unlike ANP and BNP, the human gene encoding CNP, NPPC, is not located on chromosome 1 but on chromosome 2. In normal human subjects, the mean CNP concentration is very low (1 fmol/mL).[12] It is elevated in patients with heart failure, although to a much lower extent than ANP and BNP. The clearance of CNP-22 in human plasma is very rapid, with a calculated half-life of 2.6 minutes.[13]

The natriuretic peptide system consists of 3 receptors: natriuretic peptide receptor-A (NPR-A), natriuretic peptide receptor-B (NPR-B), and natriuretic peptide receptor-C (NPR-C).[14] The human NPR-A gene is located on chromosome 1q21–22 and consists of 22 exons and 21 introns within 16 kilobases. The human NPR-B gene is located on chromosome 9p12–21, and the murine version, Npr2, is located on chromosome 4. The human NPR-C gene is located on chromosome 5p13–14 and contains 8 exons and 7 introns spanning more than 65 kilobases.[15] The natriuretic peptide receptors NPR-A and NPR-B catalyze the synthesis of a classic intracellular second messenger, cyclic guanosine monophosphate or cGMP. cGMP binds to proteins such as cGMP-dependent protein kinases, cGMP-binding phosphodiesterases, and cyclic nucleotide-gated ion channels.[16] NPR-C controls the concentrations of NPs through receptor-mediated internalization and degradation. The NPR-A to NPR-C ratio plays a role in regulating the biological activity of natriuretic peptides (NP).[17]

Pathophysiology

BNP is a 32–amino acid peptide hormone with a molecular weight of 3472 daltons.[6] The molecule consists of an N-terminal tail of 9 amino acids, a C-terminal tail of 6 amino acids, and a 17-member ring closed by a disulfide bond between two cysteine residues. Eleven amino acids found in the ring are conserved across the family of natriuretic peptides.[18] BNP binds to natriuretic peptide receptors A and C, causing the loss of water and electrolytes, vasodilation, and inhibition of the renin-angiotensin-aldosterone axis (RAAS).[19] BNP is cleared from the circulation by neutral endopeptidases. The first two amino acids from the N-terminal sequence (serine and proline) are removed by dipeptidyl peptidase IV very soon after BNP is released into circulation.[7]

Following systemic secretion, the natriuretic peptides activate transmembrane guanylate cyclases on the surface endothelial cells, thereby increasing intracellular levels of cyclic guanosine monophosphate (cGMP), leading to vasodilation. Other systemic effects include diuresis and natriuresis, resulting in lowered blood pressure.[20] The peptides have also demonstrated the ability to antagonize adverse pathways that are over-activated in the setting of heart failure, for example, RAAS, which has anti-diuretic effects, and the transforming growth factor-beta pathway, which increases cardiac remodeling and fibrosis.[21]

Expression of the BNP gene is a feature of both atrial and ventricular myocytes. In the normal heart, the main site of BNP expression is in the atrial regions. Ventricular BNP gene expression increases drastically in cardiac diseases affecting the ventricles, such as heart failure (HF).[22] Observing ventricular BNP gene expression in ventricular disease may have given rise to the common statement that BNP is predominantly a ventricular hormone. However, atrial and ventricular myocytes differ considerably concerning their endocrine phenotypes, and it is reasonable to expect significant differences in peptide storage and secretion patterns.[23] Atrial granules contain both intact precursors and biosynthetic end products, that is, bioactive ANP-28 and BNP-32. In contrast, normal ventricular myocytes do not seem to express such granules, and normal ventricular myocytes do not contain proBNP-derived peptides.[24]

The estimated half-life of BNP is about 20 minutes, whereas NT-proBNP has a half-life of 120 min; this difference explains why NT-proBNP serum levels are approximately six times higher than BNP levels, even though both molecules are released in equimolar proportions.[25] Like BNP, NT-proBNP concentrations vary by age; the normal threshold for those under 50 years of age is 450 pg/mL, while it is 900 pg/mL for those greater than 50 years of age.[26] The clinical importance of measuring one peptide over the other has not yet been demonstrated.

BNP production in cardiomyocytes is significantly increased by inflammatory cytokines such as interleukin-1-beta, an effect that is markedly increased when fibroblasts are present; inflammatory cytokines directly stimulate BNP production in the heart independent of hemodynamics.[27] This phenomenon is not seen with ANP. In clinical practice, plasma BNP levels will be higher in patients with myocarditis than expected solely from the hemodynamics.[28]

Specimen Requirements and Procedure

The b-type natriuretic peptide (BNP) is labile and must be collected into tubes containing EDTA and tested ideally within four hours of collection. Serum samples and citrated or heparinized plasma cannot be used. BNP can be stored for up to 24 hours at 2^oC to 8^oC for some assays.[29] BNP is degraded by contact activation of the blood coagulation system and can be stabilized by storage under frozen conditions or by adding kallikrein protease inhibitors.[30] In contrast, NT-proBNP is much more stable than BNP and can be kept at refrigerated temperatures for three days. NT-proBNP can be tested in serum and plasma collected in either heparin or EDTA. When EDTA plasma is used, NT-proBNP concentration is about 10% lower than for serum or lithium heparin.[31] 

Testing Procedures

Both BNP and NT-proBNP levels can be tested through standard blood draws. There are also point-of-care BNP assays, which take approximately fifteen minutes for results and are often helpful in emergent settings.[32] BNP and NT-proBNP are determined by several different immunoassays using antibodies directed to different epitopes on the antigen molecules.[33]

Before introduction into clinical practice, BNP and NT-proBNP assays must be characterized with respect to the following pre-analytical and analytical issues:[34]

Pre-Analytical

a. Effect of storage time and temperatureb. Influence of different anticoagulantsc. Influence of gel separator tubesd. Plastic blood collection tubes are necessary for BNP; for NT-proBNP, either glass or plastic is acceptable

Analytical

a. Identification of both antibody recognition epitopesb. Cross-reactivity characteristics with related natriuretic peptides, including NT-pro ANP, ANP, CNP, BNP, and glycosylated and nonglycosylated NTproBNP and proBNPc. Identification of interferences from heterophile antibodies, rheumatoid factors, and human antimouse antibodiesd. Description of calibration material used, how the material was defined, and the concentration value assignede. Clarification of dilution responses

Interfering Factors

BNP and NT-proBNP levels may be affected by specific comorbidities, such as chronic renal failure, type 2 diabetes mellitus, obesity, and acute coronary syndrome (ACS).[23] Levels are higher in patients with renal failure, diabetes, and ACS and lower in patients with obesity.[7] Decreasing renal function with age may be partly responsible for the increase in BNP/NT-proBNP. In the absence of heart failure, the concentration of BNP/NT-proBNP increases in patients with a decreasing glomerular filtration rate.[35] It is also possible that elderly subjects who appear healthy may have declining cardiac function not detectable by current noninvasive techniques.[36]

The body mass index (BMI) is another variable influencing the reference intervals for BNP and NT-proBNP. Patients with a BMI in the obese range have lower BNP and NT-proBNP concentrations than patients with a normal or underweight BMI. There have been several theories as to the mechanism for these observations.[37] Natriuretic peptide receptors are found in high concentrations in adipose tissues, suggesting that BNP is degraded. However, this does not account for the obesity effect seen for NT-proBNP, which is not degraded by these receptors.[38] Therefore, comparisons of levels among patients are less meaningful than tracking the peptide trajectory of an individual patient.[39]

All immunoassays for BNP and NT-proBNP are heterogeneous, using a separation step between bound and free antibodies; there are no problems with photometric interferents such as hemoglobin, bilirubin, or lipemia.[40] The cross-reactivity of assays for BNP and NT-proBNP towards proBNP is an important consideration since it is now known that proBNP also circulates in the blood of patients with heart failure.[41] Because proBNP is the precursor protein to both BNP and NT-proBNP, the epitopes used to raise antibodies for these latter two peptides are also present in the proBNP peptide structure.[42] 

proBNP also exists in the blood of heart failure patients as an O-linked glycoprotein. There are seven potential glycosylation sites from the center of the molecule (from the non-BNP amino acid sequences).[43] There have been studies that have tested the cross-reactivity of proBNP towards commercial BNP and NT-proBNP assays. For BNP, the cross-reactivity ranged from 17% to 38% for glycosylated proBNP and 5% to 14% for non-glycosylated proBNP.[44] For NT-proBNP, the cross-reactivity ranged from 29% to 249% for non-glycosylated proBNP but with no cross-reactivity towards glycosylated proBNP.[45]

The relative composition of blood with regard to the various BNP, NT-proBNP, proBNP, and glycosylated forms present in the blood of patients with heart failure is not known.[46] Therefore, comparing the results of different markers and different assays of the same marker will be difficult.[8] The clinical significance of the presence of HAMA and heterophile antibodies is diminished for BNP and NT-proBNP assays; mild false-positive results do not have significant clinical implications.[47]

Results, Reporting, and Critical Findings

BNP and NT-proBNP levels increase in patients with cardiac disease due to myocardial stress and volume overload.[42] Patients with a BNP elevation of over 100 pg/mL should be assessed further for the signs and symptoms of cardiac disease.[48] Though BNP is traditionally used in the diagnosis of left ventricular systolic function, it can be elevated through other processes as well; for example, right ventricular failure, acute myocardial infarction, congenital heart disease, and valvular disease.[6] 

Other situations in which natriuretic peptides are elevated include any disease that increases blood volume and thus wall stress, such as sepsis, anemia, renal dysfunction, Cushing syndrome, hyperaldosteronism, hypertension with left ventricular hypertrophy, and cirrhosis.[49] In healthy individuals, BNP levels vary by age and genetic sex; levels increase with age and are higher on average in females than males.[34]

Circulating levels of natriuretic peptides increase after acute myocardial infarction; the extent of the increase is related to the size of the infarct.[50] Patients with smaller infarcts tend to have a monophasic increase in plasma BNP, peaking at 20 hours after the onset of symptoms; on the other hand, those with larger infarcts, lower ejection fraction, and clinical signs of heart failure may present a further peak at five days after admission.[51]

A value of less than 100 pg/mL for BNP and less than 300 pg/mL for NT-proBNP makes the diagnosis of heart failure less likely in an acutely dyspneic patient.[52] Elevated levels of natriuretic peptides are associated with poor long-term prognosis and have an adverse impact on long-term mortality in patients with heart failure and various other conditions such as coronary artery disease and atrial fibrillation.[53]

Clinical Significance

Heart Failure

The strongest indication for BNP measurement is distinguishing between cardiogenic and non-cardiogenic causes of dyspnea in an emergent setting. Dyspnea is a symptom in nearly all cases of HF exacerbation. However, it is not specific to HF and may result from other pathologic processes.[54] The sensitivity of BNP in HF is approximately 97%. Therefore a normal BNP level (less than 100 pg/mL) virtually excludes heart failure and should prompt a search for noncardiac causes of dyspnea. An elevated level may indicate the presence of cardiac disease and the need for further cardiac workup, such as echocardiography, to determine the etiology of the patient's symptoms.[32] Furthermore, short-term elevations in hospitalized patients with HF have correlated with longer hospital stays.

BNP can also be a useful prognostic marker in patients with known chronic heart failure; elevated levels are found in patients with higher New York Heart Association functional class scores and correlate with mortality, morbidity, and recurrent hospital admission.[20][55] Furthermore, in hospitalized patients with heart failure, short-term elevations in BNP have been shown to predict increased hospital stays.[56]

Acute Coronary Syndrome

Left ventricular dysfunction due to cardiac remodeling is a significant cause of mortality following myocardial infarction (MI). BNP monitoring can help assess prognosis following MI because it becomes elevated in patients with post-MI left ventricular dysfunction with ejection fractions less than 40%.[57]

Limitations

While BNP is a useful tool in assessing prognosis in patients with cardiac disease, it has not been well established that BNP-guided therapy offers improved outcomes compared to symptom-guided therapies.[54] Therefore, knowledge of BNP levels may predict outcomes but does not yet dictate treatment in the chronic management of heart failure. For this reason, BNP should not be used as a stand-alone test when guiding therapy in cardiac disease. Furthermore, while elevated BNP levels are highly sensitive to heart failure, they are not specific to its cause; elevated BNPs may be seen in various cardiac and noncardiac diseases, as previously mentioned. Therefore, evaluating abnormal BNP levels with further cardiac testing is necessary.[57] Particular care is needed when interpreting an elevated BNP in patients with multiple comorbidities, such as those with renal failure, to identify the cause of the elevation.[19]

Relevance 

Heart failure is a significant and growing cause of morbidity and mortality in the United States; it is the leading cause of hospitalization in those aged 65 and older and is responsible for 10% of all healthcare expenditures.[56] Many symptoms of heart failure exacerbations are not specific to the disease process; therefore, identifying a biomarker that could assist in the diagnosis process was critical. BNP measurement is a highly sensitive, low-cost, and rapid test that can be utilized in hospitals to assist in diagnosing heart failure.[58]

Quality Control and Lab Safety

For non-waived tests, laboratory regulations require, at the minimum, analysis of at least two levels of control materials once every 24 hours. If necessary, laboratories can assay QC samples more frequently to ensure accurate results. Quality control samples should be assayed after calibration or maintenance of an analyzer to verify the correct method performance.[59] To minimize QC when performing tests for which manufacturers’ recommendations are less than those required by the regulatory agency (such as once per month), the labs can develop an individualized quality control plan (IQCP) that involves performing a risk assessment of potential sources of error in all phases of testing and putting in place a QC plan to reduce the likelihood of errors.[60]

Westgard multi-rules are used to evaluate the quality control runs. If a run is declared out of control, investigate the system (instrument, standards, controls, etc.) to determine the cause of the problem. Do not perform any analysis until the problem has been resolved.[61]

Consider all specimens, control materials, and calibrator materials as potentially infectious. Exercise the usual precautions required for handling all laboratory reagents. Disposal of all waste material should be in accordance with local guidelines. Wear gloves, a lab coat, and safety glasses when handling human blood specimens. Place all plastic tips, sample cups, and gloves that come into contact with blood in a biohazard waste container.[62] Discard all disposable glassware into sharps waste containers. Protect all work surfaces with disposable absorbent bench top paper, discarded into biohazard waste containers weekly or whenever blood contamination occurs. Wipe all work surfaces weekly.[63]

Enhancing Healthcare Team Outcomes

From clinicians on through nursing staff, pharmacists, and other ancillary staff, all healthcare practitioners should understand BNP values commensurate with their function in providing patient care. This is particularly true for those in emergent settings who may encounter patients in need of prompt intervention for cardiac-related events and conditions.