Clinica Chimica Acta 443 (2015) 25–28
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Atrial natriuretic peptides in plasma Jens P. Goetze ⁎, Lasse H. Hansen, Dijana Terzic, Nora E. Zois, Jakob Albrethsen, Annette Timm, Julie Smith, Ewa Soltysinska, Solvej K. Lippert, Ingrid Hunter Department of Clinical Biochemistry, Rigshospitalet, Copenhagen, Denmark
a r t i c l e
i n f o
Article history: Received 6 June 2014 Received in revised form 14 August 2014 Accepted 14 August 2014 Available online 23 August 2014 Keywords: ABP BNP CNP Natriuretic peptides Heart failure
a b s t r a c t Measurement of cardiac natriuretic peptides in plasma has gained a diagnostic role in the assessment of heart failure. Plasma measurement is though hampered by the marked instability of the hormones, which has led to the development of analyses that target N-terminal fragments from the prohormone. These fragments are stable in plasma and represent surrogate markers of the actual natriuretic hormone. Post-translational processing of the precursors, however, is revealing itself to be a complex event with new information still being reported on proteolysis, covalent modifications, and amino acid derivatizations. In this mini-review, we summarize measurement of the principal cardiac hormone, e.g. atrial natriuretic peptide (ANP) and its precursor fragments. We also highlight some of the analytical pitfalls and problems and the concurrent clinical “proof of concept”. We conclude that biochemical research into proANP-derived peptides is still worthy of attention and that new biological insight may change our chemical perception of the markers. © 2014 Elsevier B.V. All rights reserved.
1. Introduction “When the right thing can only be measured poorly, it tends to cause the wrong thing to be measured well. And, it is often much worse to have a good measurement of the wrong thing, especially when it is so often the case that the wrong thing will, in fact, be used as an indicator of the right thing, than to have a poor measure of the right thing”. With such precision, the statistician John Tukey expressed his experience with scientific data. Notably, this statement is also valid in the clinical setting, where for instance blood borne markers are often used as surrogate measures of a complex and often poorly understood biology but still reduced to mere numerical numbering. One classic simple example is blood glucose as a measure of diabetes pathophysiology. Natriuretic peptides as markers of the heart failure syndrome should also be considered only as surrogate measures albeit their usefulness in both the diagnosis and monitoring of disease has been documented. From a molecular point of view, John Tukey's statement should also awaken scrutiny. As reviewed below, we now use plasma measurement of peptide fragments from the natriuretic peptide prohormones as markers of the bioactive compounds; to a large extent simply because specific immunoassays are more easily developed for these fragments, which are more stable analytes in plasma. But clinical documentation as to what end of the prohormones is truly the best measure in the heart failure syndrome remains to this day almost unaddressed. ⁎ Corresponding author at: Department of Clinical Biochemistry, Rigshospitalet, Section 3014, Blegdamsvej 9, DK-2100 Copenhagen, Denmark. Tel.: + 45 3545 2202; fax: +45 3545 2880. E-mail address: [email protected] (J.P. Goetze).
http://dx.doi.org/10.1016/j.cca.2014.08.017 0009-8981/© 2014 Elsevier B.V. All rights reserved.
In this mini-review, we first summarize the measurement of the principal cardiac hormone atrial natriuretic peptide (ANP) and its precursor fragments. Then, we highlight on selected analytical pitfalls. For measurement of the related B-type natriuretic peptide in plasma, we refer to earlier reviews on the subject [1–3]. We conclude that further research into plasma measurement of proANP-derived peptides is still worthy of scientific attention. 2. Historical ANP Atrial natriuretic factor, or ANF, was logically named after its primary site of production, the cardiac atria [4]. When infused into animals, atrial tissue extracts elicit prompt natriuresis. The natriuretic factor was identified as a small peptide containing a disulfide bridge (and by many hence renamed ANP), which is essential for receptor binding and physiological effects [5]. In the circulation, ANP circulates without binding to plasma proteins, which is often the case for labile hormones. The ANP gene is also expressed in other tissues [6–8], but this extracardiac production seems to have little impact on plasma ANP concentrations when compared to the cardiac release. The first immunoassay for ANP in plasma was reported in 1985, where the assay was based on antiserum from the Peninsula Laboratories [9]. Although the assay epitope was not defined, antibody detection of ANP1–28 seemed to require the amino-terminal decasequence of the peptide. Plasma concentrations in healthy individuals were (and still are) in the low picomolar range [10]. ANP in plasma stored at −20 °C is, however, unstable which suggests problems in samples handled at room temperature. Accurate ANP measurement in plasma requires delicate handling of the blood sample itself, rapid separation of chelated
26
J.P. Goetze et al. / Clinica Chimica Acta 443 (2015) 25–28
plasma, and storage at − 80 °C if analysis is to be performed at a later time. From a practical point of view, even more troublesome is the need for plasma extraction prior to immunoassay measurement, as plasma contains unspecific protein interference [11]. While this is achievable in research projects, it is almost impossible in the everyday measurement of ANP in patient plasma. 3. ProANP measurement Given the laborious work with preanalytical sample handling and extraction in ANP measurement, one alternative strategy would be to measure another peptide fragment from the prohormone, which is analogous to C-peptide measurement in assessing insulin production [12]. For proANP, this strategy was first reported in 1989, where antibodies raised against proANP1–30 and proANP79–98 were used [13,14]. Notably, these sequences were deduced from cDNA sequences and not based on identified protein/peptide fragments. Generally, the plasma concentration of the N-terminal fragment(s) is(are) higher than for the bioactive ANP peptide, typically around 4-fold when using the early immunoassays. The preanalytical stability, however, was much better than for ANP, which rapidly led to the concept of a useful marker in heart failure that could be handled by routine laboratories [15]. This resulted in a battery of research assays being introduced by diagnostic companies, most often in the form of ELISAs. The assay validation was often incomplete with little information on specificity and unspecific interference. One consequence was that the molar concentrations between assays varied to an extent that biology itself could not explain. Moreover, ELISA and RIA are not suitable for everyday diagnostics in acute settings, which again only allowed for testing in already collected samples, e.g. clinical studies. Prospective studies were not reported for the proANP peptidology. One automated analysis was reported in 2004, which generated new interest in proANP-derived peptides [16]. This method is based on antibodies raised against the mid-region of proANP, where the dogma was (and still is) that little proteolytic degradation occurs. Hence, this region represents a simple and stable analyte. From this methodology, a series
of clinical reports have been published; essentially the measurement seems to match that of the related proBNP [17,18] with one major difference: Mid-regional proANP concentrations are much higher than that of proBNP-related peptides in non-cardiac patients and thus allow for information on decreased concentrations compared to reference individuals [19]. Notably, this information may be important for instance in obesity and diabetes, where the paradoxical decrease has gained considerable interest in connection to metabolic hormones and their regulation [20,21]. 4. Total measurement of ANP expression One analytical approach in quantitating the total sum of proANPderived peptides in plasma is to measure a fragment that is specifically generated by exogenous proteolysis. This approach is well-established in proteomic identification by mass spectrometry. In brief, plasma is subjected to proteolysis by trypsin for instance, and a particular fragment derived from this process can then be a target for conventional immunoanalyses [22]. One beneficial side effect from this procedure is that unspecific assay interference is eliminated, which allows for a more accurate measurement of the peptide in question [23]. For proANP, we have developed such a methodology, which serves as a specialized analysis in research [24]. The technique is, as for ANP measurement, cumbersome and not applicable for everyday clinical measurement. 5. Post-translational proANP processing Our general understanding of cardiomyocyte peptide processing is a relatively new area of interest. The early data suggested fairly simple processing of the prohormones, typically by endoproteolytic cleavage of the precursor in to an N-terminal fragment and the C-terminal natriuretic hormone (Fig. 1). New technology, however, has documented a more complex processing and harbored new thoughts in to clinical measurement. One early step after translation from mRNA to polypeptide is the cleavage of the signal peptide from the preprohormone. The
Fig. 1. This figure illustrates the human proANP structure, where we postulate that the midregion may be important for granular export and endoproteolytic processing. The Western blot (lower left) shows N-terminal proANP in porcine atrial extract and plasma. Note that different forms of proANP can be differentiated by this method. The gel chromatography on the lower right shows the elution profile of proANP in porcine atrial extract. The blue line depicts immunoreactivity for midregional proANP and the black line for N-terminal proANP (modified from Ref. [24] with permission).
J.P. Goetze et al. / Clinica Chimica Acta 443 (2015) 25–28
dogma so far has been that the signal peptide is degraded immediately after cleavage. New data, however, has identified a fragment from the signal peptide in plasma, which renders also this fragment from the preprohormone eligible for clinical measurement [25]. Although still in its infancy, a new marker related to cardiac ANP expression seems to be at hand. In extension of this discovery, the recent report on the humane proteome suggests that the cleavage of preproANP by the signalase enzyme may not only occur at the predicted site but also further in the prostructure [26]. Whether this is an artifact from degradation or a real biological event that needs to be pursued. After translation, the disulfide bridge is formed. This process can occur spontaneously but may also be enzymatically catalyzed. To what extent this chemical event takes place is still not fully elucidated, but given the two cysteine residues in ANP1–28, only one bridge can be formed. Also dimer forms are formed through intermolecular bridging, but whether this so-called beta-ANP form has a role in human endocrinology and pathophysiology remains unresolved [27]. The endoproteolytic cleavage releases ANP1–28 from the prohormone; an event that is catalyzed by the serine protease Corin [28,29]. Genetic defects in Corin are also implicated in a hypertensive phenotype, which corroborates the need for cleavage of proANP in order to obtain full biological activity [30]. As for other endoproteolytic cleavages, no prohormone convertase (PC) activity has yet been clearly implicated, albeit the PC1 enzyme is expressed in the cardiomyocytes [31]. Exoproteolytic activity may also be involved in the maturation of proANP-derived peptides. However, this trimming also takes place in plasma where for instance DPP-4, an enzyme target for pharmacological inhibition, may theoretically trim the N-termini of both proANP and ANP1–28. Finally, modifications of amino acids may occur, as has been documented for the related proBNP prohormone [32,33]. In fact, when comparing proANP data from different immunoassays, it seems relevant to postulate that glycosylation also is involved in cardiac proANP biosynthesis [34].
27
7. A dark horse in natriuretic peptide measurement As for most peptide hormones, laboratory standardization comes with problems. As different methods measure different epitopes, not one peptide is sufficient for standardization (Table 1). In addition, the use of antibodies pose a constant challenge for laboratories, as each clone will have specific profiles and can only be truly compared with itself. Secondly, the peptide mixture in plasma makes it difficult to choose one “true” peptide form for calibration. This inherent problem has been addressed for proBNP measurement without being successful in generating consensus on an international level [35]. Thus, many laboratories have chosen to harmonize rather than to standardize, which in effect means that we just use the same method. This may seem appealing to the clinicians, as laboratories will then report comparable results from the same method. On an academic level, however, this may lead to diagnostic failure of some patients. Thus, there is a continuous need for specialized laboratories that can provide further molecular information than by just one method if we are to follow the statement by John Tukey. 8. Type 1 and type 2 ANP pathobiology? While laboratories are dedicated to provide quantitative results from proANP measurement, there may be an important aspect in also generating qualitative analyses. In classic terms, a type 1 defect is characterized by reduced protein expression, which can be caused by mutations in the gene or cellular malfunction of the hormone-producing cell. But also qualitative defects with normal protein concentrations and reduced biological effects are known in hormonology. Type 1 and type 2 defects are well-known for many proteins, enzymes, and regulatory peptides. For the cardiac hormones, such thinking may also prove important, and perhaps even help to explain why some patients are relatively asymptomatic from left ventricular dysfunction while others suffer from congestion with a “normal” left ventricular function [36].
6. New assays on the horizon
9. Concluding remarks: what the laboratories need
While immunoassays dominate in peptide measurement, other technologies may also be relevant for clinical testing. Mass spectrometry is, for instance, now developing at a pace where the diagnostic applications are expanding almost every month. Given the molecular complexity of most peptide hormones (and of clinical samples), however, the technology will require some preanalytical step, as for instance antibody grabbing. So-called “targeted mass spectrometry” (using advanced LCMS platforms) and protein profiling (using robust MALDITOF MS platforms) can be combined with antibody capture for specific and absolute quantitation of selected peptides in clinical samples. Another possibility is Western blotting that is able to differentiate between mature and immature forms and thus provide a qualitative aspect into proANP measurement. Notably, Western blotting can today run on semi-automated platforms. Finally, expressing the cognate receptor for ANP in cellular systems and then testing for “receptor binding” without the flaws of antibodies could be a strategy using the Biacore technology.
For now, routine laboratories should have a robust screening analysis for natriuretic peptides, e.g. fragments from the prohormones. Whether it would be proANP or proBNP, both analyses seem comparable in clinical performance for initial heart failure assessment. Laboratories dedicated to serving specialized heart failure clinics must consider having access to analyses for the hormones themselves, as some patients may suffer from lack of prohormone activation, which is not addressed by using prohormone measurements. In this way, new insight of cardiac prohormone processing can be achieved. Finally, a few centers should stay at the analytical front-line with complex analyses for the prohormones and with development of new and more qualitative assessment of the cardiac natriuretic peptide system. 10. Concluding remarks: what the clinicians need Clinicians using the measurements in patients should continuously be informed from the laboratories of analytical pitfalls, in particular
Table 1 Selected immunoassays for ANP gene translational products in human plasma. Assay
Signal peptide
PIA proANP
MR-proANP
ANP-28
Epitope LLD (pmol/L) Reference value (pmol/L) Reference interval (pmol/L) Plasma extraction In vitro stability Reference
16–25 4.6 20.7 (mean) 14.6–34.4 (range) Yes Unknown 25
26–41 34 276 (median) 272–311 (range) Yes Stable 24
98–115 2.1 46.1 (median) 87.2 (97.5% percentile) No Stable 16
124–151 1.0 14.6 (mean) 10.1–24.9 (range) Yes Poor 10
Note: LLD refers to lowest level of detection; different methods for calculating this have been used. The epitopes refer to amino acids in the preproANP structure.
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J.P. Goetze et al. / Clinica Chimica Acta 443 (2015) 25–28
that our methods still rely on antibodies that measure an epitope within the primary structure — and not the actual full fragment. Also, the troublesome differences in units must be addressed, where the gold standard is molar concentrations. Finally, patients with an apparent mismatch in symptoms and natriuretic peptide concentrations should be examined further with secondary analyses, preferably at a specialized laboratory with biochemical tools for dissecting the molecular composition in individual patients. In perspective, we foresee that some patients may suffer from inappropriate hormonal response to cardiac disease, whether being quantitative or qualitative. Funding An unrestricted research grant from the Novo Nordisk Foundation. References [1] Nishikimi T, Minamino N, Nakao K. Diverse molecular forms of plasma B-type natriuretic peptide in heart failure. Curr Heart Fail Rep 2011;8:140–6. [2] Goetze JP. B-type natriuretic peptide: from posttranslational processing to clinical measurement. Clin Chem 2012;58:83–91. [3] Clerico A, Vittorini S, Passino C. Circulating forms of the b-type natriuretic peptide prohormone: pathophysiologic and clinical considerations. Adv Clin Chem 2012; 58:31–44. [4] de Bold AJ, Borenstein HB, Veress AT, Sonnenberg H. A rapid and potent natriuretic response to intravenous injection of atrial myocardial extract in rats. Life Sci 1981; 28:89–94. [5] Flynn TG, de Bold ML, de Bold AJ. The amino acid sequence of an atrial peptide with potent diuretic and natriuretic properties. Biochem Biophys Res Commun 1983;117: 859–65. [6] Nakao K, Morii N, Itoh H, Yamada T, Shiono S, Sugawara A, et al. Atrial natriuretic polypeptide in the brain: implication of central cardiovascular control. J Hypertens Suppl 1986;4:S492–6. [7] Vollmar AM. Atrial natriuretic peptide in peripheral organs other than the heart. Klin Wochenschr 1990;68:699–708. [8] Vuolteenaho O, Arjamaa O, Vakkuri O, Maksniemi T, Nikkilä L, Kangas J, et al. Atrial natriuretic peptide (ANP) in rat gastrointestinal tract. FEBS Lett 1988;233:79–82. [9] Gutkowska J, Bourassa M, Roy D, Thibault G, Garcia R, Cantin M, et al. Immunoreactive atrial natriuretic factor (IR-ANF) in human plasma. Biochem Biophys Res Commun 1985;128:1350–7. [10] Burnett Jr JC, Kao PC, Hu DC, Heser DW, Heublein D, Granger JP, et al. Atrial natriuretic peptide elevation in congestive heart failure in the human. Science 1986;231: 1145–7. [11] Cernacek P, Crawhall JC, Levy M. Atrial natriuretic peptide: blood levels in human disease and their measurement. Clin Biochem 1988;21:5–17. [12] Steiner DF. The proinsulin C-peptide—a multirole model. Exp Diabesity Res 2004;5: 7–14. [13] Buckley MG, Sagnella GA, Markandu ND, Singer DR, MacGregor GA. Immunoreactive N-terminal pro-atrial natriuretic peptide in human plasma: plasma levels and comparisons with alpha-human atrial natriuretic peptide in normal subjects, patients with essential hypertension, cardiac transplant and chronic renal failure. Clin Sci (Lond) 1989;77:573–9. [14] Buckley MG, Sagnella GA, Markandu ND, Singer DR, MacGregor GA. Concentrations of N-terminal ProANP in human plasma: evidence for ProANP (1-98) as the circulating form. Clin Chim Acta 1990;191:1–14. [15] Hall C, Aaberge L, Stokke O. In vitro stability of N-terminal proatrial natriuretic factor in unfrozen samples: an important prerequisite for its use as a biochemical parameter of atrial pressure in clinical routine. Circulation 1995;91:911. [16] Morgenthaler NG, Struck J, Thomas B, Bergmann A. Immunoluminometric assay for the midregion of pro-atrial natriuretic peptide in human plasma. Clin Chem 2004; 50:234–6.
[17] Maisel A, Mueller C, Nowak R, Peacock WF, Landsberg JW, Ponikowski P, et al. Mid-region pro-hormone markers for diagnosis and prognosis in acute dyspnea: results from the BACH (Biomarkers in Acute Heart Failure) trial. J Am Coll Cardiol 2010;55:2062–76. [18] Lauridsen BK, Iversen K, Hunter I, Bay M, Kirk V, Nielsen OW, et al. ProANP plasma measurement predicts all-cause mortality in acutely hospitalised patients: a cohort study. BMJ Open 2013;3:e003288. http://dx.doi.org/10.1136/bmjopen-2013003288. [19] Asferg CL, Nielsen SJ, Andersen UB, Linneberg A, Møller DV, Hedley PL, et al. Metabolic rather than body composition measurements are associated with lower serum natriuretic peptide concentrations in normal weight and obese men. Am J Hypertens 2014;27:620–7. [20] Buglioni A, Burnett Jr JC. A gut-heart connection in cardiometabolic regulation. Nat Med 2013;19:534–6. [21] Zois NE, Bartels ED, Hunter I, Kousholt BS, Olsen LH, Goetze JP. Natriuretic peptides in cardiometabolic regulation and disease. Nat Rev Cardiol May 13 2014. http://dx. doi.org/10.1038/nrcardio.2014.64 [Epub ahead of print]. [22] Rehfeld JF, Goetze JP. The posttranslational phase of gene expression: new possibilities in molecular diagnosis. Curr Mol Med 2003;3:25–38. [23] Goetze JP, Kastrup J, Pedersen F, Rehfeld JF. Quantification of pro-B-type natriuretic peptide and its products in human plasma by use of an analysis independent of precursor processing. Clin Chem 2002;48:1035–42. [24] Hunter I, Rehfeld JF, Goetze JP. Measurement of the total proANP product in mammals by processing independent analysis. J Immunol Methods 2011;370: 104–10. [25] Pemberton CJ, Siriwardena M, Kleffmann T, Ruygrok P, Palmer SC, Yandle TG, et al. First identification of circulating prepro-A-type natriuretic peptide (preproANP) signal peptide fragments in humans: initial assessment as cardiovascular biomarkers. Clin Chem 2012;58:757–67. [26] Kim MS, Pinto SM, Getnet D, Nirujogi RS, Manda SS, Chaerkady R, et al. A draft map of the human proteome. Nature 2014;509(7502):575–81. [27] Sugawara A, Nakao K, Morii N, Yamada T, Itoh H, Shiono S, et al. Synthesis of atrial natriuretic polypeptide in human failing hearts. Evidence for altered processing of atrial natriuretic polypeptide precursor and augmented synthesis of beta-human ANP. J Clin Invest 1988;81:1962–70. [28] Yan W, Sheng N, Seto M, Morser J, Wu Q. Corin, a mosaic transmembrane serine protease encoded by a novel cDNA from human heart. J Biol Chem 1999;274: 14926–35. [29] Wu F, Wu Q. Corin-mediated processing of pro-atrial natriuretic peptide in human small cell lung cancer cells. Cancer Res 2003;63:8318–22. [30] Dries DL, Victor RG, Rame JE, Cooper RS, Wu X, Zhu X, et al. Corin gene minor allele defined by 2 missense mutations is common in blacks and associated with high blood pressure and hypertension. Circulation 2005;112:2403–10. [31] Beaubien G, Schäfer MK, Weihe E, Dong W, Chrétien M, Seidah NG, et al. The distinct gene expression of the pro-hormone convertases in the rat heart suggests potential substrates. Cell Tissue Res 1995;279:539–49. [32] Schellenberger U, O'Rear J, Guzzetta A, Jue RA, Protter AA, Pollitt NS. The precursor to B-type natriuretic peptide is an O-linked glycoprotein. Arch Biochem Biophys 2006; 451:160–6. [33] Tonne JM, Campbell JM, Cataliotti A, Ohmine S, Thatava T, Sakuma T, et al. Secretion of glycosylated pro-B-type natriuretic peptide from normal cardiomyocytes. Clin Chem 2011;57:864–73. [34] Hunter I, Alehagen U, Dahlström U, Rehfeld JF, Crimmins DL, Goetze JP. N-terminal pro-atrial natriuretic peptide measurement in plasma suggests covalent modification. Clin Chem 2011;57:1327–30. [35] Luckenbill KN, Christenson RH, Jaffe AS, Mair J, Ordonez-Llanos J, Pagani F, et al. Crossreactivity of BNP, NT-proBNP, and proBNP in commercial BNP and NT-proBNP assays: preliminary observations from the IFCC Committee for Standardization of Markers of Cardiac Damage. Clin Chem 2008;54:619–21. [36] Hunter I, Goetze JP. Next generation natriuretic peptide measurement. Adv Clin Chem 2012;58:45–8.
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Atrial natriuretic peptides in plasma Jens P. Goetze ⁎, Lasse H. Hansen, Dijana Terzic, Nora E. Zois, Jakob Albrethsen, Annette Timm, Julie Smith, Ewa Soltysinska, Solvej K. Lippert, Ingrid Hunter Department of Clinical Biochemistry, Rigshospitalet, Copenhagen, Denmark
a r t i c l e
i n f o
Article history: Received 6 June 2014 Received in revised form 14 August 2014 Accepted 14 August 2014 Available online 23 August 2014 Keywords: ABP BNP CNP Natriuretic peptides Heart failure
a b s t r a c t Measurement of cardiac natriuretic peptides in plasma has gained a diagnostic role in the assessment of heart failure. Plasma measurement is though hampered by the marked instability of the hormones, which has led to the development of analyses that target N-terminal fragments from the prohormone. These fragments are stable in plasma and represent surrogate markers of the actual natriuretic hormone. Post-translational processing of the precursors, however, is revealing itself to be a complex event with new information still being reported on proteolysis, covalent modifications, and amino acid derivatizations. In this mini-review, we summarize measurement of the principal cardiac hormone, e.g. atrial natriuretic peptide (ANP) and its precursor fragments. We also highlight some of the analytical pitfalls and problems and the concurrent clinical “proof of concept”. We conclude that biochemical research into proANP-derived peptides is still worthy of attention and that new biological insight may change our chemical perception of the markers. © 2014 Elsevier B.V. All rights reserved.
1. Introduction “When the right thing can only be measured poorly, it tends to cause the wrong thing to be measured well. And, it is often much worse to have a good measurement of the wrong thing, especially when it is so often the case that the wrong thing will, in fact, be used as an indicator of the right thing, than to have a poor measure of the right thing”. With such precision, the statistician John Tukey expressed his experience with scientific data. Notably, this statement is also valid in the clinical setting, where for instance blood borne markers are often used as surrogate measures of a complex and often poorly understood biology but still reduced to mere numerical numbering. One classic simple example is blood glucose as a measure of diabetes pathophysiology. Natriuretic peptides as markers of the heart failure syndrome should also be considered only as surrogate measures albeit their usefulness in both the diagnosis and monitoring of disease has been documented. From a molecular point of view, John Tukey's statement should also awaken scrutiny. As reviewed below, we now use plasma measurement of peptide fragments from the natriuretic peptide prohormones as markers of the bioactive compounds; to a large extent simply because specific immunoassays are more easily developed for these fragments, which are more stable analytes in plasma. But clinical documentation as to what end of the prohormones is truly the best measure in the heart failure syndrome remains to this day almost unaddressed. ⁎ Corresponding author at: Department of Clinical Biochemistry, Rigshospitalet, Section 3014, Blegdamsvej 9, DK-2100 Copenhagen, Denmark. Tel.: + 45 3545 2202; fax: +45 3545 2880. E-mail address: [email protected] (J.P. Goetze).
http://dx.doi.org/10.1016/j.cca.2014.08.017 0009-8981/© 2014 Elsevier B.V. All rights reserved.
In this mini-review, we first summarize the measurement of the principal cardiac hormone atrial natriuretic peptide (ANP) and its precursor fragments. Then, we highlight on selected analytical pitfalls. For measurement of the related B-type natriuretic peptide in plasma, we refer to earlier reviews on the subject [1–3]. We conclude that further research into plasma measurement of proANP-derived peptides is still worthy of scientific attention. 2. Historical ANP Atrial natriuretic factor, or ANF, was logically named after its primary site of production, the cardiac atria [4]. When infused into animals, atrial tissue extracts elicit prompt natriuresis. The natriuretic factor was identified as a small peptide containing a disulfide bridge (and by many hence renamed ANP), which is essential for receptor binding and physiological effects [5]. In the circulation, ANP circulates without binding to plasma proteins, which is often the case for labile hormones. The ANP gene is also expressed in other tissues [6–8], but this extracardiac production seems to have little impact on plasma ANP concentrations when compared to the cardiac release. The first immunoassay for ANP in plasma was reported in 1985, where the assay was based on antiserum from the Peninsula Laboratories [9]. Although the assay epitope was not defined, antibody detection of ANP1–28 seemed to require the amino-terminal decasequence of the peptide. Plasma concentrations in healthy individuals were (and still are) in the low picomolar range [10]. ANP in plasma stored at −20 °C is, however, unstable which suggests problems in samples handled at room temperature. Accurate ANP measurement in plasma requires delicate handling of the blood sample itself, rapid separation of chelated
26
J.P. Goetze et al. / Clinica Chimica Acta 443 (2015) 25–28
plasma, and storage at − 80 °C if analysis is to be performed at a later time. From a practical point of view, even more troublesome is the need for plasma extraction prior to immunoassay measurement, as plasma contains unspecific protein interference [11]. While this is achievable in research projects, it is almost impossible in the everyday measurement of ANP in patient plasma. 3. ProANP measurement Given the laborious work with preanalytical sample handling and extraction in ANP measurement, one alternative strategy would be to measure another peptide fragment from the prohormone, which is analogous to C-peptide measurement in assessing insulin production [12]. For proANP, this strategy was first reported in 1989, where antibodies raised against proANP1–30 and proANP79–98 were used [13,14]. Notably, these sequences were deduced from cDNA sequences and not based on identified protein/peptide fragments. Generally, the plasma concentration of the N-terminal fragment(s) is(are) higher than for the bioactive ANP peptide, typically around 4-fold when using the early immunoassays. The preanalytical stability, however, was much better than for ANP, which rapidly led to the concept of a useful marker in heart failure that could be handled by routine laboratories [15]. This resulted in a battery of research assays being introduced by diagnostic companies, most often in the form of ELISAs. The assay validation was often incomplete with little information on specificity and unspecific interference. One consequence was that the molar concentrations between assays varied to an extent that biology itself could not explain. Moreover, ELISA and RIA are not suitable for everyday diagnostics in acute settings, which again only allowed for testing in already collected samples, e.g. clinical studies. Prospective studies were not reported for the proANP peptidology. One automated analysis was reported in 2004, which generated new interest in proANP-derived peptides [16]. This method is based on antibodies raised against the mid-region of proANP, where the dogma was (and still is) that little proteolytic degradation occurs. Hence, this region represents a simple and stable analyte. From this methodology, a series
of clinical reports have been published; essentially the measurement seems to match that of the related proBNP [17,18] with one major difference: Mid-regional proANP concentrations are much higher than that of proBNP-related peptides in non-cardiac patients and thus allow for information on decreased concentrations compared to reference individuals [19]. Notably, this information may be important for instance in obesity and diabetes, where the paradoxical decrease has gained considerable interest in connection to metabolic hormones and their regulation [20,21]. 4. Total measurement of ANP expression One analytical approach in quantitating the total sum of proANPderived peptides in plasma is to measure a fragment that is specifically generated by exogenous proteolysis. This approach is well-established in proteomic identification by mass spectrometry. In brief, plasma is subjected to proteolysis by trypsin for instance, and a particular fragment derived from this process can then be a target for conventional immunoanalyses [22]. One beneficial side effect from this procedure is that unspecific assay interference is eliminated, which allows for a more accurate measurement of the peptide in question [23]. For proANP, we have developed such a methodology, which serves as a specialized analysis in research [24]. The technique is, as for ANP measurement, cumbersome and not applicable for everyday clinical measurement. 5. Post-translational proANP processing Our general understanding of cardiomyocyte peptide processing is a relatively new area of interest. The early data suggested fairly simple processing of the prohormones, typically by endoproteolytic cleavage of the precursor in to an N-terminal fragment and the C-terminal natriuretic hormone (Fig. 1). New technology, however, has documented a more complex processing and harbored new thoughts in to clinical measurement. One early step after translation from mRNA to polypeptide is the cleavage of the signal peptide from the preprohormone. The
Fig. 1. This figure illustrates the human proANP structure, where we postulate that the midregion may be important for granular export and endoproteolytic processing. The Western blot (lower left) shows N-terminal proANP in porcine atrial extract and plasma. Note that different forms of proANP can be differentiated by this method. The gel chromatography on the lower right shows the elution profile of proANP in porcine atrial extract. The blue line depicts immunoreactivity for midregional proANP and the black line for N-terminal proANP (modified from Ref. [24] with permission).
J.P. Goetze et al. / Clinica Chimica Acta 443 (2015) 25–28
dogma so far has been that the signal peptide is degraded immediately after cleavage. New data, however, has identified a fragment from the signal peptide in plasma, which renders also this fragment from the preprohormone eligible for clinical measurement [25]. Although still in its infancy, a new marker related to cardiac ANP expression seems to be at hand. In extension of this discovery, the recent report on the humane proteome suggests that the cleavage of preproANP by the signalase enzyme may not only occur at the predicted site but also further in the prostructure [26]. Whether this is an artifact from degradation or a real biological event that needs to be pursued. After translation, the disulfide bridge is formed. This process can occur spontaneously but may also be enzymatically catalyzed. To what extent this chemical event takes place is still not fully elucidated, but given the two cysteine residues in ANP1–28, only one bridge can be formed. Also dimer forms are formed through intermolecular bridging, but whether this so-called beta-ANP form has a role in human endocrinology and pathophysiology remains unresolved [27]. The endoproteolytic cleavage releases ANP1–28 from the prohormone; an event that is catalyzed by the serine protease Corin [28,29]. Genetic defects in Corin are also implicated in a hypertensive phenotype, which corroborates the need for cleavage of proANP in order to obtain full biological activity [30]. As for other endoproteolytic cleavages, no prohormone convertase (PC) activity has yet been clearly implicated, albeit the PC1 enzyme is expressed in the cardiomyocytes [31]. Exoproteolytic activity may also be involved in the maturation of proANP-derived peptides. However, this trimming also takes place in plasma where for instance DPP-4, an enzyme target for pharmacological inhibition, may theoretically trim the N-termini of both proANP and ANP1–28. Finally, modifications of amino acids may occur, as has been documented for the related proBNP prohormone [32,33]. In fact, when comparing proANP data from different immunoassays, it seems relevant to postulate that glycosylation also is involved in cardiac proANP biosynthesis [34].
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7. A dark horse in natriuretic peptide measurement As for most peptide hormones, laboratory standardization comes with problems. As different methods measure different epitopes, not one peptide is sufficient for standardization (Table 1). In addition, the use of antibodies pose a constant challenge for laboratories, as each clone will have specific profiles and can only be truly compared with itself. Secondly, the peptide mixture in plasma makes it difficult to choose one “true” peptide form for calibration. This inherent problem has been addressed for proBNP measurement without being successful in generating consensus on an international level [35]. Thus, many laboratories have chosen to harmonize rather than to standardize, which in effect means that we just use the same method. This may seem appealing to the clinicians, as laboratories will then report comparable results from the same method. On an academic level, however, this may lead to diagnostic failure of some patients. Thus, there is a continuous need for specialized laboratories that can provide further molecular information than by just one method if we are to follow the statement by John Tukey. 8. Type 1 and type 2 ANP pathobiology? While laboratories are dedicated to provide quantitative results from proANP measurement, there may be an important aspect in also generating qualitative analyses. In classic terms, a type 1 defect is characterized by reduced protein expression, which can be caused by mutations in the gene or cellular malfunction of the hormone-producing cell. But also qualitative defects with normal protein concentrations and reduced biological effects are known in hormonology. Type 1 and type 2 defects are well-known for many proteins, enzymes, and regulatory peptides. For the cardiac hormones, such thinking may also prove important, and perhaps even help to explain why some patients are relatively asymptomatic from left ventricular dysfunction while others suffer from congestion with a “normal” left ventricular function [36].
6. New assays on the horizon
9. Concluding remarks: what the laboratories need
While immunoassays dominate in peptide measurement, other technologies may also be relevant for clinical testing. Mass spectrometry is, for instance, now developing at a pace where the diagnostic applications are expanding almost every month. Given the molecular complexity of most peptide hormones (and of clinical samples), however, the technology will require some preanalytical step, as for instance antibody grabbing. So-called “targeted mass spectrometry” (using advanced LCMS platforms) and protein profiling (using robust MALDITOF MS platforms) can be combined with antibody capture for specific and absolute quantitation of selected peptides in clinical samples. Another possibility is Western blotting that is able to differentiate between mature and immature forms and thus provide a qualitative aspect into proANP measurement. Notably, Western blotting can today run on semi-automated platforms. Finally, expressing the cognate receptor for ANP in cellular systems and then testing for “receptor binding” without the flaws of antibodies could be a strategy using the Biacore technology.
For now, routine laboratories should have a robust screening analysis for natriuretic peptides, e.g. fragments from the prohormones. Whether it would be proANP or proBNP, both analyses seem comparable in clinical performance for initial heart failure assessment. Laboratories dedicated to serving specialized heart failure clinics must consider having access to analyses for the hormones themselves, as some patients may suffer from lack of prohormone activation, which is not addressed by using prohormone measurements. In this way, new insight of cardiac prohormone processing can be achieved. Finally, a few centers should stay at the analytical front-line with complex analyses for the prohormones and with development of new and more qualitative assessment of the cardiac natriuretic peptide system. 10. Concluding remarks: what the clinicians need Clinicians using the measurements in patients should continuously be informed from the laboratories of analytical pitfalls, in particular
Table 1 Selected immunoassays for ANP gene translational products in human plasma. Assay
Signal peptide
PIA proANP
MR-proANP
ANP-28
Epitope LLD (pmol/L) Reference value (pmol/L) Reference interval (pmol/L) Plasma extraction In vitro stability Reference
16–25 4.6 20.7 (mean) 14.6–34.4 (range) Yes Unknown 25
26–41 34 276 (median) 272–311 (range) Yes Stable 24
98–115 2.1 46.1 (median) 87.2 (97.5% percentile) No Stable 16
124–151 1.0 14.6 (mean) 10.1–24.9 (range) Yes Poor 10
Note: LLD refers to lowest level of detection; different methods for calculating this have been used. The epitopes refer to amino acids in the preproANP structure.
28
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that our methods still rely on antibodies that measure an epitope within the primary structure — and not the actual full fragment. Also, the troublesome differences in units must be addressed, where the gold standard is molar concentrations. Finally, patients with an apparent mismatch in symptoms and natriuretic peptide concentrations should be examined further with secondary analyses, preferably at a specialized laboratory with biochemical tools for dissecting the molecular composition in individual patients. In perspective, we foresee that some patients may suffer from inappropriate hormonal response to cardiac disease, whether being quantitative or qualitative. Funding An unrestricted research grant from the Novo Nordisk Foundation. References [1] Nishikimi T, Minamino N, Nakao K. Diverse molecular forms of plasma B-type natriuretic peptide in heart failure. Curr Heart Fail Rep 2011;8:140–6. [2] Goetze JP. B-type natriuretic peptide: from posttranslational processing to clinical measurement. Clin Chem 2012;58:83–91. [3] Clerico A, Vittorini S, Passino C. Circulating forms of the b-type natriuretic peptide prohormone: pathophysiologic and clinical considerations. Adv Clin Chem 2012; 58:31–44. [4] de Bold AJ, Borenstein HB, Veress AT, Sonnenberg H. A rapid and potent natriuretic response to intravenous injection of atrial myocardial extract in rats. Life Sci 1981; 28:89–94. [5] Flynn TG, de Bold ML, de Bold AJ. The amino acid sequence of an atrial peptide with potent diuretic and natriuretic properties. Biochem Biophys Res Commun 1983;117: 859–65. [6] Nakao K, Morii N, Itoh H, Yamada T, Shiono S, Sugawara A, et al. Atrial natriuretic polypeptide in the brain: implication of central cardiovascular control. J Hypertens Suppl 1986;4:S492–6. [7] Vollmar AM. Atrial natriuretic peptide in peripheral organs other than the heart. Klin Wochenschr 1990;68:699–708. [8] Vuolteenaho O, Arjamaa O, Vakkuri O, Maksniemi T, Nikkilä L, Kangas J, et al. Atrial natriuretic peptide (ANP) in rat gastrointestinal tract. FEBS Lett 1988;233:79–82. [9] Gutkowska J, Bourassa M, Roy D, Thibault G, Garcia R, Cantin M, et al. Immunoreactive atrial natriuretic factor (IR-ANF) in human plasma. Biochem Biophys Res Commun 1985;128:1350–7. [10] Burnett Jr JC, Kao PC, Hu DC, Heser DW, Heublein D, Granger JP, et al. Atrial natriuretic peptide elevation in congestive heart failure in the human. Science 1986;231: 1145–7. [11] Cernacek P, Crawhall JC, Levy M. Atrial natriuretic peptide: blood levels in human disease and their measurement. Clin Biochem 1988;21:5–17. [12] Steiner DF. The proinsulin C-peptide—a multirole model. Exp Diabesity Res 2004;5: 7–14. [13] Buckley MG, Sagnella GA, Markandu ND, Singer DR, MacGregor GA. Immunoreactive N-terminal pro-atrial natriuretic peptide in human plasma: plasma levels and comparisons with alpha-human atrial natriuretic peptide in normal subjects, patients with essential hypertension, cardiac transplant and chronic renal failure. Clin Sci (Lond) 1989;77:573–9. [14] Buckley MG, Sagnella GA, Markandu ND, Singer DR, MacGregor GA. Concentrations of N-terminal ProANP in human plasma: evidence for ProANP (1-98) as the circulating form. Clin Chim Acta 1990;191:1–14. [15] Hall C, Aaberge L, Stokke O. In vitro stability of N-terminal proatrial natriuretic factor in unfrozen samples: an important prerequisite for its use as a biochemical parameter of atrial pressure in clinical routine. Circulation 1995;91:911. [16] Morgenthaler NG, Struck J, Thomas B, Bergmann A. Immunoluminometric assay for the midregion of pro-atrial natriuretic peptide in human plasma. Clin Chem 2004; 50:234–6.
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