B-Type Natriuretic Peptide Signal Peptide Circulates in Human Blood
Evaluation as a Potential Biomarker of Cardiac Ischemia
Background— The diagnosis of cardiac necrosis such as myocardial infarction can be difficult and relies on the use of circulating protein markers like troponin. However, there is a clear need to identify circulating, specific biomarkers that can detect cardiac ischemia without necrosis.
Methods and Results— Using specific immunoassay and tandem mass spectrometry, we show that a fragment derived from the signal peptide of B-type natriuretic peptide (BNPsp) not only is detectable in cytosolic extracts of explant human heart tissue but also is secreted from the heart into the circulation of healthy individuals. Furthermore, plasma levels of BNPsp in patients with documented acute ST-elevation myocardial infarction (n=25) rise to peak values (≈3 times higher than the 99th percentile of the normal range) significantly earlier than the currently used biomarkers myoglobin, creatine kinase-MB, and troponin. Preliminary receiver-operating characteristic curve analysis comparing BNPsp concentrations in ST-elevation myocardial infarction patients and other patient groups was positive (area under the curve=0.97; P<0.001), suggesting that further, more rigorous studies in heterogeneous chest pain patient cohorts are warranted.
Conclusion— Our results demonstrate for the first time that BNPsp exists as a distinct entity in the human circulation and could serve as a new class of circulating biomarker with the potential to accelerate the clinical diagnosis of cardiac ischemia and myocardial infarction.
Clinical Trial Registration— URL: http://www.anzctr.org.au. Unique identifier: ACTRN12609000040268.
Received September 17, 2009; accepted May 13, 2010.
Acute coronary syndromes encompass a spectrum of cardiac ischemic events ranging from unstable angina to acute myocardial infarction. However, a significant proportion of patients who present with suspected acute coronary syndromes do not have a cardiac cause for their symptoms or have equivocal findings on history and ECG. This places a heavy emphasis on using circulating biomarker concentrations for accurate diagnosis.1 A number of biomarkers have been proposed for this purpose, including creatine kinase-MB (CK-MB), troponin T, troponin I (TnI), and myoglobin. However, time to detectable or abnormal elevation of plasma cardiac biomarkers in acute myocardial infarction can be 6 to 12 hours, imposing a delay on a precise diagnosis and treatment. Furthermore, myoglobin, CK, and to a lesser degree CK-MB lack specificity and can be secreted from extracardiac sources, especially during trauma or surgery. Accurate early diagnosis of acute myocardial infarction facilitates prompt introduction of effective percutaneous or thrombolytic revascularization and adjunctive anticoagulant and antiplatelet therapy. Such treatments are progressively less effective at reducing mortality and morbidity with each hour of delay in diagnosis and management.2 Given the need for accelerated decision making in this clinical situation, there is considerable interest in the identification of new circulating biomarkers that provide early and specific diagnosis of acute cardiac injury.
Editorial see p 229
Clinical Perspective on p 264
Signal peptides (SPs) perform the function of directing nascent preproproteins through the process of translation into the endoplasmic reticulum (ER) and eventual secretion from biological cells.3 Once this is complete, SPs have been generally thought to be degraded intracellularly.4 There is recent limited evidence, however, that some SPs or their fragments are released from the ER. SPs can be cleaved into N-terminal and C-terminal fragments, which are released into the cytosol of some cells.5 For example, an N-terminal fragment of preprolactin SP is released into the cytosol of bovine pituitary cells.6 Furthermore, the SP of mouse mammary tumor virus Rem protein is released from the ER membrane and accumulates in nucleoli.7 Not only are some SPs released from the ER, but thereafter they may have physiologically important functions beyond directing proteins into specific cellular pathways.8 One example relates to major histocompatability complex I SP fragments, which have been shown to influence self-/non–self-recognition and natural killer cell activity at the cell surface.9 A second example is the N-terminus fragment of preprolactin SP, which interacts with cytosolic calmodulin to modulate intracellular pituitary Ca2+.6 Given this background, we hypothesized that a peptide derived from SP sequences not only may be present in the cell but also could enter the circulation. To this end, we assessed whether the SP sequence of B-type natriuretic peptide (BNP), a peptide hormone primarily released from the cardiac ventricles in response to cardiac wall stretch (strictly transmural pressure) and used as diagnostic plasma marker for suspected cardiac failure,10,11 could yield a circulating peptide.
Synthetic human BNPsp (17-26), (Tyr)BNPsp (17-26), and (Cys) BNPsp (17-26) were synthesized by Mimotopes (Melbourne, Australia) and were confirmed as >95% pure by mass spectrometry (MS). All other synthetic peptides were purchased from the Peptide Institute (Osaka, Japan) or Sigma-Aldrich (St. Louis, Mo).
BNPsp (17-26) Assay Development
Specific antibodies to BNPsp (17-26) for use in immunoassay were developed according our previous protocols.12–14 Briefly, synthetic BNPsp (17-26) coupled to bovine BSA was injected subcutaneously every 4 weeks until adequate titer and sensitivity levels were obtained.
BNPsp and Cardiac Marker Assays
The cross-reactivity of BNPsp antiserum with other endogenous peptides was negligible (Table I in the online-only Data Supplement). For the BNPsp assay, all sample extracts, radioactive trace, and standard and antiserum solutions were diluted in radioimmunoassay buffer.12 The assay incubate consisted of 100 μL extracted sample or standard [0 to 640 pmol/L of BNPsp (17-26) peptide] combined with 100 μL antiserum H13–3 and 100 μL iodinated (Tyr)BNPsp (17-26) (4000 to 6000 cpm). Tubes were incubated for 24 hours at 4°C, and then free BNPsp and bound BNPsp were separated by solid-phase second-antibody method (donkey anti-rabbit Sac-Cel, Immunodiagnostic Systems, Boldon, UK). Sac-Cel (1 mL) diluted in 5% dextran solution (final Sac-Cel concentration, 5%) was added to each tube; the solution was vortexed and incubated at room temperature for 30 minutes. Tubes were centrifuged at 2800g for 10 minutes at 20°C and decanted, with the pellet counted in a Gammamaster (LKB, Uppsala, Sweden). Assessment of hemolysis indicated that BNPsp immunoreactivity in assay was not altered up to a hemoglobin concentration of 8 g/L. Assessment of lipolysis indicated that BNPsp immunoreactivity was not altered by plasma lipid content up to 6g/L.
N-terminal prohormone BNP (NT-proBNP) concentrations in human plasma were determined on a commercial Roche Elecsys 2010 system (proBNP, Roche Diagnostics, Indianapolis, Ind). CK-MB and myoglobin measurements were all determined by commercial assays (Abbott, Abbott Park, Ill) by the Clinical Biochemistry section of Canterbury Health Labs (Christchurch Hospital, New Zealand) according to our previously described protocols.12,14,15 Troponin I was determined by a late-generation assay (Abbott Architect) with a 99th percentile cutoff of 0.03 μg/L.
Human Plasma Sample Collection
Human plasma samples were obtained from 6 patient groups (healthy volunteers and patients with cardiac catheterization, ST-elevation myocardial infarction [STEMI], chronic renal failure, congestive heart failure, and thyroid disease; see Table II in the online-only Data Supplement), and cardiac tissue samples obtained from those undergoing heart transplant surgery were procured in accordance with ethics protocols approved by local ethics committees (Canterbury, Auckland) of the Ministry of Health, New Zealand. Explant cardiac tissue donations were obtained from 10 patients enrolled in the Green Lane Hospital (Auckland, New Zealand) cardiac transplant program. Written consent was obtained before surgery in each case. Atrium and ventricular tissue samples (≈10 g of each) were collected from explant hearts, washed in cold saline, immediately frozen at −80°C, and stored until tissue extraction and analysis. Extracts of human plasma were prepared for the measurement of BNPsp immunoreactivity with solid-phase C18 cartridges, and cardiac tissue extracts from explant human hearts were prepared as previously reported.13,14 All participants gave informed consent before recruitment, and all investigations conform to the principles of the Declaration of Helsinki.
High-Performance Liquid Chromatography
Plasma and cardiac tissue extracts were dried under air, reconstituted in 60% acetonitrile/0.1% trifluoroacetic acid, and subjected to size-exclusion high-performance liquid chromatography (HPLC) on a Superdex G75 Superose column (Pharmacia Biotech, Uppsala, Sweden), followed by reverse-phase (RP) HPLC. Fractions were collected as previously described,13,14 dried, reconstituted, and resubjected to BNPsp immunoassay.
MS Analysis of BNPsp
Endogenous human BNPsp (17-26) that was immunopurified from plasma was analyzed by matrix-assisted laser desorption/ionization time of flight (online-only Data Supplement). All MS spectra were acquired in positive-ion mode with 800 to 1000 laser pulses per sample spot. A maximum of 6 precursor ions of each sample spot were selected for MS/MS collision-induced fragmentation analysis. Modifications of endogenous BNPsp were analyzed by liquid chromatography multistage mass spectrometry (LC-MS3) (LTQ-OrbitrapXL MS, Thermo Scientific, San Jose, Calif). Eluting peptides were monitored by a full mass scan using the linear ion trap in a mass range from m/z 400 to 1400. The predicted m/z value of the doubly charged peptide was selected as the exclusive precursor mass triggering subsequent scan events.
Results are presented as mean±SD. Comparison of means was carried out with paired, 2-tailed Student t test when appropriate. Statistical differences between various biomarker peak times were determined by Wilcoxon signed-rank test. Assessment of multiple markers within individuals and regional NT-proBNP/BNPsp measurements from multiple regional venous sites was carried out with a nonparametric Friedman test followed by Tukey multiple-comparisons test with ranked sums. Analysis of data from independent samples was carried with the Kruskal-Wallis test followed by the Dunn method for multiple comparisons. Relational analyses of plasma hormone concentrations using Spearman rank-order correlation testing and receiver-operating characteristic curve analysis were carried out with SPSS version 17 (SPSS Inc, Chicago, Ill). In all analyses, a value of P<0.05 was considered significant.
Identification of Endogenous BNPsp Immunoreactivity in Human Plasma
BNPsp contains 26 amino acids (Figure 1A). We generated an antibody (H13–3) directed toward the C-terminal 10 amino acids of BNPsp, ie, BNPsp (17-26), and used it to establish a sensitive, specific radioimmunoassay (Figure 1B). This assay had a mean zero binding of 29±1%, sample detection limit of 5.0±0.6 pmol/L, ED50 of 161±8 pmol/L, and a working range of 80 to 320 pmol/L in which the intra-assay coefficient of variation was 5.4%. Interassay coefficients of variation were 13.6% at 130 pmol/L and ≈12.8% at 44pmol/L. Cross-reactivity assessment of the antisera showed no detectable interference with other relevant peptides or with medications commonly used in cardiovascular disorders (Table I in the online-only Data Supplement). Initial assessment confirmed that BNPsp immunoreactivity was present in human plasma from peripheral blood collected into Na3 EDTA and that it diluted in parallel with the synthetic standard curve (Figure 1B). The level of immunoreactive BNPsp present in normal human venous plasma obtained from 125 healthy volunteers was 14.1±4.7 pmol/L (range, 7 to 25 pmol/L): immunoreactivity was detected in every sample. Correlation analysis of plasma BNPsp with concomitant NT-proBNP levels, gender, and body mass index revealed no significant association, but there was a weak, although statistically significant, inverse correlation with age (r=−0.23, P<0.01; Figure 1C).
Immunoreactive BNPsp Is Released From the Human Heart Into the Circulation
Given that BNP is predominantly a cardiac peptide,16,17 we anticipated that like its congener NT-proBNP, immunoreactive BNPsp levels would be higher in blood draining the heart (ie, in cardiac coronary sinus samples) than in blood supplying the heart (ie, arterial samples). Indeed, coronary sinus plasma contained significantly higher concentrations of immunoreactive BNPsp than simultaneously drawn arterial plasma samples (18.9±3.6 versus 15.9±3.7 pmol/L; P<0.00001; n=50; Figure 1D). Statistically significant differences in immunoreactive BNPsp concentrations were also observed across the head and neck, kidney, and lower limb (Figure 1D). Immunoreactive BNPsp appeared to be cleared across the liver (P<0.05). In contrast, NT-proBNP levels were clearly and solely elevated in cardiac coronary sinus plasma (117.8±117.3 versus 84.9±89.7 pmol/L, coronary sinus versus arterial respectively; P<0.00001; n=50), and there was evidence of renal clearance of NT-proBNP (P<0.001; Figure 1D).
Stability of Endogenous BNPsp in Human Plasma
The stability of endogenous BNPsp immunoreactivity in blood and plasma and during its subsequent extraction is an important analytic prerequisite. The addition of aprotinin (a serine protease inhibitor) to venous collection tubes containing Na3 EDTA had no effect on measured BNPsp levels in venous blood collected from 5 patients undergoing clinically indicated diagnostic cardiac catheterization (Figure I in the online-only Data Supplement). However, whole blood collected into Na3 EDTA and stored at room temperature for 24 hours before centrifugation gave plasma immunoreactive BNPsp levels ≈75% higher than in samples centrifuged immediately after venesection. This effect was completely blocked by storing whole blood at 4°C. The observed extraction efficiency of synthetic BNPsp (17-26) from plasma was ≈70%.
MS Characterization of BNPsp (17-26) and Modified BNPsp (17-26) in Human Plasma
Given that immunoreactive BNPsp concentrations in plasma were elevated soon after the onset of symptoms in patients with STEMI (see below), we collected 30 mL peripheral venous blood from each of the 50 patients (cumulative volume, 1.5 L) who presented to Christchurch Hospital with STEMI within 4 hours of symptom onset. After solid-phase extraction and immunoaffinity purification, separation of purified immunoreactive BNPsp by RP-HPLC revealed 3 major components, one of which eluted close to a synthetic BNPsp (17-26) marker (Figure 2A). Analysis of the BNPsp plasma components by matrix-assisted laser desorption/ionization tandem time-of-flight MS and manual interpretation of collision-induced dissociation fragment spectra revealed a common core sequence of LHLAFLGGRS in all detected BNPsp (17-26) derivatives (Figure IIA and IIB in the online-only Data Supplement). The alternate elution times of this core sequence were found to be related to differential modifications of the peptide (Figure 2C), ranging in weight from 28 to 151 Da. In all collision-induced dissociation MS/MS spectra of modified peptides, only the detectable b ions (product ion of an amide bond cleavage containing the N-terminus) were shifted, whereas y ions (product ion of an amide bond cleavage containing the C terminus) from y2-y9 were constant (Figure IIC in the online-only Data Supplement), indicating that the modification resides on the N-terminal leucine. To gain further insights into the nature of some of these modifications, we conducted high-resolution LC-MS3 experiments on collision-induced dissociation derived b2 ions of modified BNPsp (17-26) using the high-energy collision cell of a LTQ-OrbitrapXL MS. The histidine immonium ion (m/z 110.07127) gave the strongest signal in all MS3 spectra, confirming that the histidine side chain was unmodified. Under high-energy collision conditions for MS3 of the b2 ion, the most abundant peak carrying the modification was identified as an a1-type ion ie, the leucine immonium ion plus the mass adduct. In some cases, a leucine immonium ion (m/z 86.09643) was detectable in addition to the a1 ion, indicating that in these cases the leucine side chain was not involved in the modification. Therefore, an N-terminal modification is most likely. Elemental composition analysis on either the MS2 b2 ions or MS3 a1 ions with a mass error of <1 mmu revealed clear chemical formulas of CO, C2H2O2, C3H2O2, and C3H4O2 for the mass adducts relative to the unmodified peptide of 28, 58, 70, and 72 Da, respectively (Table III in the online-only Data Supplement). The elemental compositions of these detected modifications are consistent with formylation (CO) and modifications of amino groups by glyoxal (C2H2O2) and methylglyoxal (C3H4O2). Taken together, these results confirmed that our immunoassay detects authentic endogenous BNPsp (17-26) and the modified forms of BNPsp in human cardiac tissue and blood.
Cardiac Tissue Levels and Molecular Forms of Immunoreactive BNPsp
Atrial and ventricular extracts from explant cardiac tissue contained immunoreactive BNPsp, the concentrations of which, however, were much lower than corresponding BNP and NT-proBNP levels (Figure 3A). Average atrial BNPsp concentrations (1.74±0.85 pmol/g) were significantly higher than ventricular BNPsp concentrations (1.09±0.41 pmol/g; P<0.05; ratio, 1.5:1), but the atrial/ventricular ratio for BNP and NT-proBNP was much higher, ≈10:1 for both. No statistical correlation was found between ventricular BNPsp and NT-proBNP concentrations (r=−0.55, P=0.09; Figure 3B). RP-HPLC and size-exclusion HPLC analyses confirmed that cardiac tissue extracts contain low-molecular-weight BNPsp species ≈1 kDa in size (Figure 3C).
BNPsp Is an Early Rising Biomarker of Cardiac Ischemia
Having established that immunoreactive BNPsp is present in the human heart and peripheral circulation and that the cardiac coronary sinus is a source of the circulating peptide, we sought to determine the potential utility of circulating BNPsp as a diagnostic biomarker of acute cardiac ischemia resulting in myocardial infarction. In patients with documented STEMI whose symptom onset was <4 hours before presentation to Christchurch Hospital, peripheral venous plasma concentrations of BNPsp were markedly elevated 4 to 5 hours after symptom onset but fell thereafter, returning to the normal range within 10 hours (Figure 4A). Average peak levels were ≈6-fold higher than the average observed in normal health and >3-fold beyond the 99th percentile upper limit of normal (25 pmol/L). Of the 25 STEMI patients, 19 had TnI >0.03 μg/L at presentation, and 23 had TnI >0.03 μg/L at 1 hour after presentation. Peak levels of BNPsp tended to correlate positively with simultaneously measured CK-MB (r=0.28, P=0.128) and myoglobin (r=0.31, P=0.105). There was no significant association between peak BNPsp and peak TnI nor between peak BNPsp and peak NT-proBNP. The time to peak BNPsp after symptom onset was significantly earlier than that for myoglobin (P<0.05) and CK-MB, troponin I, and NT-proBNP (all P<0.01; Figure 4A). In contrast to BNPsp, plasma NT-proBNP rose steadily over a much longer time course, reaching peak levels ≈76 hours after symptom onset (Figure 4A).
Average venous BNPsp levels in patients with chronic renal disease were significantly higher than the mean in healthy volunteers but were nevertheless still within the normal range (Figure 4B). Patients with hyperthyroidism or hypothyroidism and congestive heart failure had mean BNPsp levels no different from normal subjects. Receiver-operating characteristic curve analysis of BNPsp levels 5 hours after chest pain onset to detect STEMI (n=25) compared with normal control subjects (n=125) and patients with thyroid disease (n=11), chronic renal failure (n=34), and congestive heart failure (n=10) generated an area under the curve of 0.97 (P<0.001), a sensitivity of 88%, and a specificity of 92% at 25 pmol/L (99th percentile of the upper limit of the normal range; Figure 4C). Receiver-operating characteristic curve analysis of TnI at the same time point gave an area under the curve of 0.99 (P<0.001), a sensitivity of 96%, and a specificity of 95% with a 99th percentile cutoff of 0.03 μg/L.
Our results are the first demonstration of a defined signal sequence as a distinct, separate entity within the circulation in humans. Whereas Christofferson et al18 reported that the SP of apolipoprotein M is retained along with the entire apolipoprotein M protein in the circulation of humans and that this uncleaved SP helps prevent renal filtration of apolipoprotein M through hydrophobic association with circulating lipoproteins, they did not detect the apolipoprotein M SP as a distinct, separate entity. We found no evidence on immunoassay or HPLC for the SP fragment BNPsp (17-26) in human plasma to be associated with proBNP (1-108), ie, as a component of preproBNP (1-134). Nevertheless, we cannot exclude the existence of such an association because our BNPsp (17-26) assay is designed to detect free carboxyl terminal residues in position 26 of the SP, which requires cleavage of proBNP (1-108). Future work with assays directed toward the amino terminal region of preproBNP (1-134) is needed to definitively answer whether it also exists in the circulation.
Study of the regional plasma levels of NT-proBNP revealed only 1 site of clear secretion, namely the cardiac coronary sinus. This observation underscores the cardiovascular utility of NT-proBNP measurement and suggests that the heart is the only significant contributor to circulating NT-proBNP levels. In contrast, we observed significant elevations in circulating BNPsp levels across the heart, head/neck, kidney, and lower limbs. That the heart should be a significant contributor to circulating BNPsp is understandable; the other putative sites of secretion are less so. However, it should be noted that in humans, both the pituitary and kidney contain significant amounts of preproBNP RNA transcript, at levels only 10 to 100 times less than cardiac levels.10 This pattern of human BNP tissue expression contrasts with results from animal studies that report cardiac BNP expression to be 1000 times higher than any other organ.10,11,17 Thus, it is conceivable that the head and kidney could contribute to BNPsp secretion in humans. However, it is unclear as to why this should be apparent for BNPsp but not NT-proBNP. Additional studies are required to address this issue, along with how the lower limbs might potentially contribute to circulating BNPsp levels.
The generation of BNPsp (17-26) from the 26–amino acid SP is consistent with previous reports that have outlined a 2-step proteolysis of SPs in the ER membrane.7–9 Thus, in the case of preproBNP, cleavage of the SP from the propeptide by signal peptidase followed by further cleavage at the midregion hydrophobic core by SP peptidase generates the C-terminus peptide beginning with residue Leu.17 However, high-resolution MS on MS2 and MS3 fragment ions suggested that the N terminus of Leu17 carried differential modifications ranging in molecular weight from 28 to 151 Da. Elemental composition analyses of low-mass ions revealed clear chemical formulas for some of these modifications that are identical to the chemical compositions of modifications of ε-amino groups of lysines by glyoxal and methylglyoxal forming Nε-carboxymethyl-lysine and Nε-carboxyethyl-lysine, respectively. Both modifications can be either a product of the decomposition of protein glycations referred to as advanced glycation end products or a direct reaction of amino groups with reactive carbonyl and dicarbonyl compounds generated by oxidative degradation of sugars and lipid peroxidation.19 An increased level of advanced glycation end products and similar nonenzymatic protein modifications is associated with conditions such as diabetes mellitus, atherosclerosis, inflammatory conditions, and cardiac ischemia/reperfusion.20 It has been shown that oxidative stress resulting from ischemic injuries in rat hearts and elevated levels of peroxynitrite can be a source of rapid generation of methylglyoxal and glyoxal and nonenzymatic protein modifications.21,22
The majority of immunopurified BNPsp (17-26) from STEMI patients was N-terminally modified. Nonenzymatic protein modifications by compounds generated in the process of oxidative stress may result in a very complex profile of different modifications as we have observed for BNPsp (17-26).23,24 Further analyses of the BNPsp (17-26) modifications are required to properly characterize the origin of nonenzymatic peptide modifications of BNPsp (17-26) in plasma.
The rise in plasma BNPsp in patients with documented STEMI occurred significantly earlier than all other current markers of acute myocardial infarction (myoglobin, CK-MB, TnI). Our preliminary receiver-operating characteristic curve analysis suggests that plasma BNPsp has potential in the diagnosis of acute coronary syndromes, although it is obviously limited by the fact that it comprised only 25 STEMI patients. Further study in a larger “all comers” acute coronary syndromes cohort that includes potential false-positive/negative patients is clearly required to determine BNPsp assay specificity in cardiac ischemia and acute coronary syndromes. Although peak circulating BNPsp levels did not significantly associate with peak CK-MB, myoglobin, or TnI levels in our STEMI cohort, the sample size was small and hence additional study is needed. Why circulating BNPsp levels should peak so early after STEMI is unclear but may relate to the comparatively smaller weight of this signal sequence (Mr 1070 versus Mr 15 000 to 40 000 for the other 3 markers of acute myocardial infarction noted above). Consistent with this line of reasoning is the observation that BNPsp was cleared much more quickly from the circulation than myoglobin, TnI, and CK-MB; its approximated clearance time was 15 minutes. Such a pathophysiological profile suggests that BNPsp may be located within the myocardial cells adjacent to the outer membrane or that intracellular stores of the peptide are subject to a rapid release mechanism on ischemic challenge.
The relative concentrations of BNPsp (17-26) we observed in myocardium and plasma raise intriguing questions about secretion of the peptide. The facts that first there was no significant relationship between cardiac BNPsp and BNP/NT-proBNP concentrations and second the atrial concentrations of BNPsp were only 1.7 times higher than ventricular BNPsp (compared with an atrial/ventricular ratio of ≈10 times for BNP and NT-proBNP) suggest that the storage and secretion of BNPsp differ substantially from that for BNP/NT-proBNP. It is currently considered that secretion of BNP and NT-proBNP occurs by the regulated, storage granule–based pathway in cardiac atria and by constitutive secretion in the ventricles.10,25 Release of these storage granules is dictated largely by alterations in cardiac transmural pressure11,26 but contributed to by factors such as local myocardial hypoxia and pH, as well as hormones like endothelin-1 and angiotensin II.27–29 However, mechanisms governing BNPsp levels in the circulation of normal, healthy individuals remain unknown. It is known that the SP sequence of a preproprotein encodes for cellular secretion or storage of that protein, but not all secreted proteins in the circulation meet this “classic” criteria because they have no discernible SP motif.30 This is true for established markers of cardiac necrosis such as CK-MB and myoglobin, both of which appear in the circulation in normal health. How “nonclassic” secretion pathways such as membrane blebbing or shedding, endosomal recycling, or active membrane transportation might relate to BNPsp secretion is an obvious target for future in vitro studies. Furthermore, the role of the ER and the unfolded-protein response (a cytoprotective mechanism in the face of impending cellular ischemia)31 in channeling or controlling BNPsp secretion also needs to be considered.
We provide the unique observation that BNPsp (17-26) exists in the circulation of healthy individuals and could be used as a marker of cardiac ischemia/infarction. Further studies in a prospective patient group with recent onset of chest pain with multiple confounding diagnoses are now required to determine the diagnostic specificity of BNPsp in cardiac ischemia, especially compared with other putative markers of ischemia such as ischemia-modified albumin, free fatty acids, glycogen phosphorylase isoenzyme BB, and high-sensitivity troponin.
We thank the technical staff of Canterbury Health Laboratories and Endolab, Christchurch Hospital, New Zealand, for assistance with biomarker assays and the research nursing staff of the Cardioendocrine Research Group for assistance with patient blood sampling.
Sources of Funding
This work was supported by the Health Research Council of New Zealand (grant 07/114) and the National Heart Foundation of New Zealand (grant 1351). Dr Pemberton is the recipient of a Health Research Council of New Zealand Sir Charles Hercus Research Fellowship, and Dr Richards holds the National Heart Foundation of New Zealand Professorial Chair in Cardiovascular Studies.
The University of Otago, Christchurch, New Zealand, has filed a patent application on the composition and diagnostic/prognostic use of BNPsp measurement in acute cardiovascular disorders. Drs Pemberton, Richards, Nicholls, and Yandle are listed as inventors on this application. The other authors report no conflicts.
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The clinical diagnosis of acute coronary syndromes relies heavily on circulating diagnostic biomarkers such as troponin. However, delays in detectable changes in circulating troponin, combined with their absence in ischemia short of infarction, result in clinical uncertainty in a significant number of patients presenting with suspected acute coronary syndromes. Thus, identification of novel biomarkers that may provide early information on acute myocardial infarction and cardiac ischemia is of major importance. We provide here the identification of a novel potential biomarker of acute coronary syndromes, namely a peptide fragment derived from the signal peptide region of B-type natriuretic peptide (BNPsp). BNPsp is present as a distinct peptide in explant human cardiac tissue and is secreted into the circulation in normal health. Furthermore, detectable elevations in BNPsp were observed in ST-elevation myocardial infarction patients significantly earlier than myoglobin, creatine kinase-MB, and troponin. BNPsp thus presents as a novel class of potential biomarker in acute coronary syndromes, and further studies to determine its assay specificity and diagnostic potential in the complete spectrum of acute coronary syndromes are clearly warranted.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.109.909937/DC1.