The B-type natriuretic peptide (BNP) is an impor- tant regulator of renal sodium excretion, as well as car- diac function, through the action of BNP-receptors (NPRA and NPRB) coupled to GMP cyclase. Injection of recombinant BNP (Nasiritide) can be used clinically to improve cardiac function in patients with heart fail- ure (HF). In normal hearts, BNP is produced in atrial but not ventricular myocytes as pro-BNP1108 (intact pro-BNP), which is stored in atrial secretory granules.
In situations of atrial dilation or increased wall tension, intact pro-BNP is cleaved by proteases into an inactive N-terminal fragment containing amino acid residues 176 (NT-pro-BNP176) and the bioactive peptide BNP32, which contains the C-terminal 32 amino acids.
Once secreted in circulation, BNP has a very short half-life of 1320 min due to cellular uptake, renal excretion, and inactivation by endothelial-bound neu- tral endopeptidases. In contrast, NT-pro-BNP has a half-life of 2570 min, mostly determined by renal excretion; therefore, it is measured at higher levels in plasma. In the case of renal insufficiency, the levels of BNP and more extensively NT-pro-BNP are elevated.
BNP can be produced by the ventricular myocytes in situations of chronic myocardial stress (stretch and wall tension) and myocardial remodeling associated with AMI and cardiac failure. Therefore, measure- ments of BNP or NT-pro-BNP have been used in the evaluation of patients with AMI and cardiac insuffi- ciency. A good correlation between the levels of natri- uretic peptides and the severity of HF (American Heart Association classes IIV) can be observed, and natriuretic peptides can be in the diagnosis and follow-up of acute and chronic HF. However, these
assays should be complementary and not replace other clinical diagnostic tools for HF diagnosis and therapy.
In general, BNP and NT-pro-BNP are best used when the cause of acute dyspnea is clinically unclear because in patients with obvious HF or very low probability of HF, the predictive values of natriuretic peptide mea- surements are poor [86]. When clinical uncertainty exists, natriuretic peptide values are most helpful if levels are markedly elevated—for example, BNP.500 pg/mL (suggesting HF) or,100 pg/mL (unlikely to be acute HF). Corresponding diagnostic values for NT-pro-BNP are .450 (in those younger than 50 years), .900 (in those 5075 years old), or .1800 pg/mL (in those older than 75 years) for ruling in HF versus ,300 pg/mL for exclusion [87].
Intermediate levels have the lowest predictive values, and the patient may be a candidate for further evalua- tion with specialist assessment and echocardiography depending on the clinical assessment.
Because BNP responds to ventricular wall stress, it can be increased in many other conditions other than cardiac failure, including the following:
1. Other cardiac diseases a. Acute coronary syndrome b. Valvular heart disease
c. Left ventricular hypertrophy with or without arterial hypertension
d. Atrial fibrillation
e. Inflammatory cardiac disease
2. High-output stress (such as tachycardia, hypoxemia, anemia, sepsis, hyperthyroidism,
hyperaldosteronism, Cushing’s syndrome, and advanced liver cirrhosis with ascites)
3. Pulmonary disease and pulmonary vascular conditions (such as embolism, severe pulmonary hypertension, chronic obstructive pulmonary disease, and cor pulmonale)
4. Acute or chronic renal failure (eGFR,60 mL/min/
1.73 m2)
5. Severe neurological disease, such as subarachnoid hemorrhage, stroke, and trauma.
Other useful guidelines for the use of BNP or NT-pro-BNP include the following[88]:
1. Given high physiologic intraindividual variation (B3050%) and analytical imprecision (up to 15%), only changes greater than 40100% compared to the previous result are clinically significant[8991].
2. The test should not be ordered more than once per week because real changes related to
pathophysiology or therapy require time to occur.
3. Patients with chronic HF may have elevated natriuretic peptide levels and acute dyspnea of noncardiac origin.
4. The levels of natriuretic peptide elevations do not differentiate between HF due to left ventricular systolic dysfunction and HF with preserved ejection fraction, although values tend to be lower in
patients with diastolic HF.
5. Cutoffs for the use of NT-pro-BNP have been published by the International Collaborative for NT- proBNP Study (ICON)[87]:
a. For monitoring therapy and risk stratification in HF, it is reasonable to collect a baseline sample and a second prior to discharge for risk
assessment and monitor therapeutic
effectiveness. Patients are at lower risk with reductions greater than 50%. These
measurements identify those in need of more aggressive management. For example, the threshold with the best balance of sensitivity and specificity for 1-year mortality is approximately 1000 pg/mL for NT-pro-BNP. An admission NT- pro-BNP concentration greater than 5180 pg/mL is strongly predictive of death by 76 days.
b. If estimated glomerular filtration rate is less than 60 mL/min/1.73 m2, a higher threshold (e.g., NT-pro-BNP,1200 pg/mL) is best for exclusion of HF.
c. For risk stratification in pulmonary embolism, values below 1000 pg/mL indicate good prognosis. Persistent elevations of NT-pro-BNP greater than 7500 pg/mL after 24 hr or less than 50% decrease indicate right ventricular
dysfunction and a poor prognosis.
d. For risk stratification in ACS, values less than 1115 pg/mL indicate a high probability for recovery of left ventricular function. Values greater than 1170 pg/mL for men and greater than 2150 pg/mL for women identify high-risk patients.
False-negative results—that is, normal natriuretic peptide levels despite the presence of HF—can be observed in the following situations:
1. Obesity, diuretics, ACE inhibitors,β-blockers, aldosterone receptor blockers, and aldosterone antagonists can reduce levels and decrease sensitivity for HF.
2. Patients with end-stage HF can have low levels of circulating natriuretic peptides.
3. Lack of elevation despite acute hemodynamic compromise and/or severe acute pulmonary congestion can occur in patients who present within the first hour of the onset of pulmonary edema caused by acute mitral regurgitation, in patients with preserved ejection fractions, and in those with constrictive pericarditis without intrinsic heart disease.
144 9. CHALLENGES IN ROUTINE CLINICAL CHEMISTRY ANALYSIS
Pre-Analytical Considerations
Due to poor stability in serum, BNP assays should be performed in EDTA anticoagulated plasma and samples should not be stored for more than 4 hr at room temperature. NT-pro-BNP measurements can be performed in either serum or plasma, and the sample is stable for at least 72 hr at room temperature.
Serum samples show approximately 10% higher levels of NT-pro-BNP compared to EDTA-anticoagulated samples[92].
Analytical Issues
Like troponin, natriuretic peptide measurements are immunoassays and suffer from the same limitations discussed in Chapter 6. There is considerable heteroge- neity in the exact peptides that are measured by the various BNP and NT-pro-BNP assays [93]. “BNP”
assays typically measure intact pro-BNP1108 in addi- tion to BNP32, although it is unclear if this affects the diagnostic ability of BNP assays. In plasma, BNP32
quickly loses the N-terminal three amino acids to yield BNP332, which may not be fully measured with some BNP assays. No other proteolytic fragments appear to circulate in patients with HF[93,94].
All “NT-pro-BNP” assays measure NT-pro-BNP176 and pro-BNP1108. However, intact NT-pro-BNP176 has not been detected in circulation; instead, several other N-proteolytic fragments originating from cleav- age at both the N-terminus and the C-terminus of NT- pro-BNP1108 can be present in plasma and more extensively in serum. For this reason, the best assays for pro-BNP should be directed at the central amino acids. Current U.S. Food and Drug Administration- licensed assays use a sandwich immunoassay approach, with the capture antibody directed against amino acids 121 and the detection antibody directed against amino acids 3950.
Glycosylation of the central portion of pro-BNP has been described, which may affect interaction with anti- bodies directed against amino acids 3950 and signifi- cantly increase renal excretion. Oligomerization of pro- BNP peptides can occur through leucine zipper motifs at the N-terminus of pro-BNP [95], potentially hiding or exposing epitopes and changing the half-life and therefore the levels of measured BNP or NT-pro-BNP.
CONCLUSIONS
Proteins and enzymes are useful biomarkers, and only the most commonly assayed proteins in clinical laboratory medicine were discussed in this chapter.
Enzyme assays are subject to a variety of interferences
depending on the particular approach used for prod- uct detection, whereas immunoassays for biomarkers suffer from common pitfalls, including interference by heterophilic antibodies, rheumatoid factors, autoanti- bodies, hemolysis, lipemia, and fibrin particles and bubbles. Occasionally, falsely elevated levels of protein biomarkers or enzymes occur due to complexes with immunoglobulins, the most common examples being macroamylase and macro-CK. As with all other tests, particular attention to each manufacturer’s specifica- tions for pre-analytical and analytical test characteris- tics is essential to avoid errors and explain discrepancies with other clinical parameters.
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