Natriuretic Peptides and Analytical Barriers
Vlad C. Vasile
1
and Allan S. Jaffe
1,2*
BACKGROUND: The natriuretic peptide system is an endo-
crine, autocrine and paracrine system that plays an im-
portant role in the maintenance of cardiovascular ho-
meostasis. Biomarkers based on these peptides are
important diagnostic and prognostic tools for myocardial
function.
CONTENT: Although natriuretic peptides were discovered
more than 2 decades ago, their intricate and complex
biology is associated with important questions not yet
elucidated. The diversity of circulating forms of natri-
uretic peptides, the distinct expression of these forms in
particular patients, and the heterogeneity of heart failure
forms, along with specific assay-related and preanalytic
issues, cause assays to be poorly harmonized.
SUMMARY: This review presents the relevant issues related
to the biology of natriuretic peptides and differences be-
tween assays with immediate implications for clinical
practice.
© 2016 American Association for Clinical Chemistry
Natriuretic peptides assays are essential to the evaluation
and treatment of heart failure (HF)
3
(1). However, there
is relatively poor harmonization of the various assays ow-
ing to the use of different antibodies and different ana-
lytical detectors (2). To understand the use of these as-
says, one needs a basic understanding of natriuretic
peptide physiology and how this physiology interdigi-
tates with the assays.
Basic Physiology
Atrial natriuretic peptide (ANP) and B-type natriuretic
peptide (BNP) were described in the 1980s as circulating
peptides released from the heart and involved in main-
taining cardiorenal homeostasis by diuresis and natriure-
sis (3 ). ANP is produced predominantly in the atrium
and is stored in intracellular granules where it is readily
available for release. BNP is synthesized to a greater ex-
tent in the ventricular myocardium and is stored in only
modest quantities. Thus, BNP requires more time to be
synthesized and released and is a slower reacting peptide.
Both peptides induce diuresis and natriuresis, and reduce
vascular resistance and systemic blood pressure. C-type
natriuretic peptide (CNP) is produced in the central ner-
vous system and vascular endothelium, and acts as a para-
crine regulator with little known implications for the
cardiovascular system. Dendroaspis natriuretic peptide
(DNP) is another member of the natriuretic peptide fam-
ily and urodilantin is a renally secreted cleavage product
of ANP (3 ).
All natriuretic peptides in humans contain a pre-
served ring structure of 17 amino acid residues and form
an intramolecular disulfide bond, which presumably is
responsible for receptor binding. Most BNP assays target
this ring structure for 1 of their epitopes. The receptors,
termed natriuretic peptide receptors (NPRs) are classified
as types A and B. Binding of these guanylyl cyclase-
coupled receptors leads to an increase in cGMP, which
has downstream effects of diuresis and natriuresis, vaso-
dilation, inhibition of the renin-angiotensin-aldosterone
system, enhanced myocardial relaxation, inhibition of fi-
brosis and hypertrophy, promotion of cell survival, and
inhibition of the inflammatory response. NPR type C
lacks guanylyl cyclase activity and is thought to be a clear-
ance receptor (3 ).
Conditions of the heart accompanied by volume and
pressure overload, such as HF and myocardial infarction,
lead to cardiac wall tension and stretch, volume overload,
or ischemia and result in increased production and release
of natriuretic peptides (4). The use of these biomarkers
for diagnostic and prognostic purposes has been delin-
eated (4) and endorsed by guideline authorities for heart
care (5 ). Moreover, recombinant forms of human ANP
(carperitide) and BNP (neseritide) were developed as
therapeutic agents for HF (6, 7). However, clinicians
have lost enthusiasm for these agents due to doubtful
efficacy and safety issues (8 ).
BNP is more stable in vitro and has superior diag-
nostic performance to ANP, thus BNP and BNP-related
peptides have established a role in clinical practice for the
diagnosis of risk stratification and follow-up of patients
with HF (1, 9).
1
Division of Cardiovascular Diseases, Department of Medicine, Rochester, MN;
2
Depart-
mentofLaboratoryMedicineandPathology,MayoClinicCollegeofMedicine,Rochester,
MN.
* Address correspondence to this author at: Cardiovascular Division, Gonda Bldg. 5th
Floor, Mayo Clinic, 200 First St. SW, Rochester, MN 55905. Fax 507-266-0228; e-mail
jaffe.allan@mayo.edu.
Received June 8, 2016; accepted June 30, 2016.
Previously published online at DOI: 10.1373/clinchem.2016.254714
© 2016 American Association for Clinical Chemistry
3
Nonstandard abbreviations: HF, heart failure; ANP, Atrial natriuretic peptide; BNP,
B-type natriuretic peptide; CNP, C-type natriuretic peptide; DNP, Dendroaspis natriuretic
peptide; NPRs, natriuretic peptide receptors.
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Brief Relevant Molecular Biology of Natriuretic
Peptides
ANP
ANP is synthesized as a 151-amino acid prepropeptide,
preproANP, which is stored in atrial intracellular gran-
ules (10 ). PreproANP is secreted and cleaved to the ma-
ture peptide, ANP, in response to atrial stretch, angioten-
sin II, endothelin, or sympathetic-mediated stimulation.
PreproANP is cleaved to proANP, which is processed by
the convertase corin into 2 circulating peptides, ANP
1–28 and the N-terminal fragment, NT-proANP 1–98
(11). NT-proANP is subsequently cleaved into 3 frag-
ments with biological importance. ProANP 1–30 is the
long-acting natriuretic peptide, proANP 31– 67 has va-
sodilation properties, and proANP 79 –98 or kaliuretic
peptide, is involved in potassium excretion. NT-proANP
and its cleavage derivatives are all found in circulation.
Another member of the ANP family, urodilantin can be
isolated from human urine and increases diuresis (12).
BNP
BNP is synthesized as a 134-amino acid preproBNP pre-
cursor, which is cleaved to the 108-amino acid proBNP
by removal of a 26-amino acid signal peptide (13 ).A
peptide fragment of preproBNP containing amino acid
residues 17–26 is present in normal individuals and pa-
tients with acute MI. It has been proposed as a biomarker
of cardiac ischemia. Another peptide of the signal residue
containing amino acid residues 16 –25 may be a bio-
marker of ischemia (14).
BNP is encoded by an early response gene allowing
transcription to reach maximal levels rapidly (15). Intra-
cellular BNP storage is minimal and it is synthesized
de novo when needed and released by ventricular
cardiomyocytes.
The processing of proBNP forms an N-terminal
proBNP (NT-proBNP) fragment with amino acid resi-
dues 1–76 and a C-terminal region active BNP with
amino acid residues 77–108 (16 ). Because of proteolysis
of proBNP, BNP and NT-proBNP are produced in a
stoichiometric ratio of 1:1. NT-proBNP has no known
biologic activity. Despite the 1:1 stoichiometric ratio, the
molar plasma concentration of NT-proBNP is higher
than the concentration of BNP likely because NT-
proBNP has slower clearance from the circulation (17 ).
ProBNP in nonprocessed form is found in healthy
individuals and patients with HF (18). ProBNP is post-
translationally modified in patients with HF by
O-glycosylation at several threonine and serine residues
within the N-terminal region (amino acid residues
1–76), but not within the BNP-portion of proBNP
(amino acid residues 77–108) (19). It appears that NT-
proBNP is glycosylated in the central region (amino acid
residues 28 –56), while the C-terminus region of the mol-
ecule (amino acid residues 61–76) is not post-
translationally modified (19). ProBNP, however, is gly-
cosylated both in the central region and in the region
located close to the cleavage site, specifically at amino
acid residues 63–76. This region can be blocked to site-
specific antibodies because of steric impediment due to
glycosylation (20). The degree of glycosylation of both
proBNP and NT-proBNP is highly individual. It is also
known to inhibit the activity of furin on the 76–78
amino acid cleavage site (21 ).
The diversity of circulating proBNP-derived pep-
tides is explained in part by proBNP processing, which
occurs immediately before, or at the time of release. The
processing is facilitated by prohormone convertases. Fu-
rin and corin are convertases currently thought to be
proBNP-processing enzymes. The evidence is indirect
from in vitro studies, which demonstrate the formation
of BNP 1–32 (by furin) and BNP 4–32 (by corin). Since
corin produces a short BNP form, BNP 4 –32, this en-
zyme is unlikely to be the only one responsible for the
processing of proBNP, suggesting that furin also plays a
role (22).
Glycosylation residues in the region of the proBNP
molecule close to the cleavage site inhibit the processing
of proBNP. In in vitro studies, both furin- and corin-
mediated processing of proBNP are suppressed by
O-glycans located at Thr71. It appears that only proBNP
molecules not glycosylated at Thr71 can be processed
into BNP and NT-proBNP, probably due to access of
enzyme to the unbound cleavage site. Inhibition of pro-
cessing via glycosylation might be a pathologic phenom-
enon found in HF.
Degradation Fragments of BNP in Circulation
Although initially it was thought that there were only 2
circulating fragments of BNP, BNP 1–32 and NT-
proBNP 1–76, contemporary data have challenged this
concept recently (23). Mass spectrometry analyses
showed only minute amounts of circulating BNP 1–32.
BNP 1–32 is found in patients with advanced HF in only
very low levels (14 ). In blood samples from HF patients
multiple N- and C-truncated fragments of BNP are pres-
ent such as BNP 1–32, BNP 3–32, BNP 4 –32, BNP
5–32, BNP 5–31, BNP 1–26, and BNP 1–25 (24). The
variety of such fragments is believed to be the result of
proteolysis by dipeptidyl peptidase IV (DPP IV), which
forms BNP 3–32 and neprilysin (NEP), which forms
BNP 5–32. BNP can also be proteolyzed by insulin-
degrading enzyme (IDE), but not by NEP and IDE im-
plying that another enzyme is involved in cleavage (25 ).
BNP 4–32 may be the result of corin activity (21). An-
other protease, peptidyl arginine aldehyde protease, can
also degrade BNP, and meprin was shown to lyse BNP in
animal models but not in humans (26 ). Due to BNP
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instability in EDTA plasma even at low temperatures,
protease inhibitors in high concentrations should always
be used (23 ).
ProBNP and Cross-Reactivity
Intact proBNP is found in healthy individuals and pa-
tients with HF. Immunoassays used in clinical practice to
detect BNP show extensive cross-reactivity with
proBNP. The extent of cross-reactivity is different for
various assays and different forms of glycosylated or non-
glycosylated proBNP. Notably, only BNP and not the
precursor proBNP has been shown to have a natriuretic
response in patients with HF (27 ). ProBNP is resistant to
proteolysis and inactivation by human kidney mem-
branes (28). The role of proBNP in healthy volunteers
and patients with HF may be a physiologic or a patho-
logic process. ProBNP is a poor stimulator of guanylyl
cyclase. There are differential cGMP activating proper-
ties of BNP forms and, notably, proBNP 1–108 and
NT-proBNP 1–76 have reduced cGMP activity in vitro
(29). Some speculate that unprocessed proBNP is a cir-
culating “reserve” of BNP since in vivo cleavage of
proBNP produces BNP. Glycosylation of Thr71 in
proBNP has inhibitory effects since it prevents proteoly-
sis by both furin and corin. Only proBNP that is not
glycosylated at Thr71 can form active BNP1–32.
ProBNP Circulating Forms in Acute and
Chronic HF
The highest percentage of glycosylated proBNP is pres-
ent in patients with chronic HF compared to patients in
the acute decompensated and nonacute decompensated
groups (30 ). Another interesting finding is that furin
activity but not its concentration is greater in the acute
HF group than in the chronic HF group (30 ). Perhaps
there is a differential mechanism of proBNP processing
in disease progression in HF patients with increased BNP
production in the acute decompensated group of patients
with fluid overload. In the chronic HF group, proBNP
degradation will not occur since there is less acute fluid
overload, implying that there might be regulatory mech-
anisms responsible for rapid increases in plasma BNP by
degradation of the processing-susceptible proBNP.
These findings demonstrate that there is a different na-
triuretic peptide spectrum in patients with different
forms of HF. Thus, assays that detect glycosylated and
nonglycosylated forms of proBNP might provide addi-
tional diagnostic information.
Immunoassays
There is a multitude of immunodetection platforms for
natriuretic peptides that are used in clinical practice. All
detect multiple forms and resulting interpretation is chal-
lenging. It is important to keep in mind both this fact and
also the type of HF syndrome involved. Moreover, it
appears that BNP and NT-proBNP have the same renal
extraction fraction in humans, but there is marked ex-
traction of NT-proBNP across the skeletal muscle (31 ).
Additionally, there are handling differences associated
with various assays. Analytical characteristics of currently
available commercial assays for natriuretic peptides are
presented in Table 1. The location of epitopes on BNP
and NT-proBNP molecules is depicted in Fig. 1.
NT-proBNP ASSAYS
All NT-proBNP assays use the same antibodies and cal-
ibrators. These are distributed by Roche, thus there is an
advantage of unifying these detection platforms. There
are only minor differences between the commercial NT-
proBNP assays with variation across methods that is
⬍10%. Despite this standardization, however, assay har-
monization remains incomplete (32 ). The International
Collaborative of NT-proBNP Study involving 1256 pa-
tients proposed a cutoff value for NT-proBNP assays
below or above 125 ng/L. A value ⱖ300 ng/L was opti-
mal with the existing assays for the exclusion of acute HF
(33, 34 ) . This study included a blend of chronic and
acute HF patients. Reference limits also vary by age and
sex of patients analyzed (35 ).
While the first generation of NT-proBNP immuno-
assays uses polyclonal antibodies to amino acids 1–21
and 39 –50, the second generation uses monoclonal an-
tibodies (MAb) recognizing the central region of NT-
proBNP: amino acids 22–28 and 42– 46 (27–31) . Many
studies have demonstrated the negative effect of glycosyl-
ation for antibody recognition to the middle area of the
NT-proBNP molecule. The NT-proBNP immunoassays
currently available show important cross-reactivity with
nonglycosylated proBNP and do not detect glycosylated
NT-proBNP and proBNP peptides owing to the pres-
ence of O-glycans in the epitopes recognized by the an-
tibodies in the nonglycosylated form. Clinicians should
be aware that these detection platforms underestimate
the concentration of circulating biomarker considerably.
Some studies suggest that the underestimation might be
up to 10-fold (19, 35). Some recommend that future
NT-proBNP platforms use deglycosylation of blood
samples before assay. Perhaps more robust deglycosyla-
tion explains why some patients such as those with renal
failure have discordantly high values for NT-proBNP. If
alternative assays for NT-proBNP are developed, they
will need to assess carefully the extent to which the anti-
bodies are influenced by glycosylation.
BNP ASSAYS
All BNP assays use different antibodies and materials for
peptide detection; thus there is a paucity of standardiza-
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Table 1. Analytical characteristics of commercially available MR-proANP, BNP, and NT-proBNP assays per the manufacturer.
Capture antibody Detection antibody Standard material FDA
a
cleared-yes/no/claim
Assay BNP
Abbott Architect,
AxSYM iSTAT
NH
2
terminus and part
of the ring structure
(Scios), murine
MAb, aa 5–13
COOH terminus,
murine MAb,
aa 26–32
Synthetic BNP 32 Assist in diagnosis of HF;
assess severity of
disease
Alere
b
Triage BNP
NH
2
terminus and part
of the ring structure
(Scios), murine
MAb, aa 5–13
BNP (Biosite),
murine
omniclonal AB,
epitope not
characterized
Recombinant BNP Aid in diagnosis and
severity assessment of
HF; risk stratification of
patients with ACS and
HF; FDA cleared
Beckman Coulter
b
Access, Access 2, DxI
BNP (Biosite), murine
Omniclonal AB,
epitope not
characterized
NH2 terminus
and part of the
ring structure
(Scios), murine
MAb, aa 5–13
Recombinant BNP Diagnosis HF; assess
severity HF; risk ACS;
risk HF
Siemens (Bayer) ACS
180, Advia Centaur,
Advia Centaur CP
COOH terminus
(BC–203) (Shionogi),
murine MAb,
aa 27–32
Ring structure
(KY-hBNPII)
(Shionogi),
murine MAb
Synthetic BNP Aid in diagnosis and
assessment of severity
of HF; predict survival
and likelihood of
future HF in ACS
patients
Siemens (Dade Behring)
Dimension VISTA,
Dimension ExL
Ring structure (KY-
hBNPII) murine
MAb, aa 14–21
COOH terminus
(BC-203),
murine MAb,
aa 27–32
Synthetic BNP 32 Aid in diagnosis and
assessment of severity
of HF; predict survival
and likelihood of
future HF in ACS
patients; pending FDA
clearance
Shionogi COOH terminus (BC–
203), murine MAb,
aa 27–32
Ring structure
(KY-hBNPII),
murine MAb
Synthetic BNP Not FDA cleared
Tosoh ST AIA-PACK
BNP
COOH terminus (BC-
203), murine MAb,
aa 27–32
Ring structure
(KY-hBNPII),
murine MAb
Synthetic BNP Not FDA cleared
Assay 1–76 NT-proBNP
Alere Triage NT-proBNP Murine MAb, aa 27–31 Sheep MAb, aa
42–46
Synthetic NTproBNP
1–76
Aid in diagnosis of HF;
risk stratification of
patients with ACS
and HF; assessment of
increased risk of
cardiovascular events
and mortality in
patients at risk for HF
who have stable CAD;
not currently available
in the US
bioMerieux NT-
proBNP1 VIDAS
NH
2
terminus
polyclonal sheep
AB, aa 1–21
Central molecule,
polyclonal
sheep AB, aa
39–50
Synthetic NTproBNP
1–76
Diagnosis HF
NT-proBNP2 VIDAS Murine MAb, aa 27–31 Sheep MAb, aa
42 −46
Synthetic NTproBNP
1–76
Not FDA cleared
Mitsubishi Chemical
PATHFAST
NH
2
terminus
polyclonal sheep
AB, aa 1–21
Central molecule,
polyclonal
sheep AB, aa
39–50
Synthetic NTproBNP
1–76
Aid diagnosis of CHF;
assess severity CHF;
risk stratification in
ACS and stable CAD
Continued on page 54
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tion and reports show differences of up to 50% across
various BNP detection platforms, even when the same
antibodies are used on equipment (2).
The most frequent method used clinically is the
“sandwich” assay that uses 2 antibodies specific for 2
distantly located epitopes of the BNP peptide. One of
these antibodies is always specific for the intact cysteine
ring, which is thought to be the active form, while the
other antibody recognizes either the C-terminus of the
peptide (Abbott AxSYM and Architect, Shionogi IRMA)
or for the N-terminus (Alere Triage and Beckman Ac-
cess). Assays using antibodies specific to the C-terminus
will not detect BNP molecules that are degraded in this
region, whereas assays using antibodies specific to the
N-terminus of BNP molecule will not measure peptides
processed in the N-terminus region. The “Single Epitope
Sandwich” Immunoassay (SES-BNP™) designed by
HyTest and implemented by ET healthcare is different.
The assay uses 1 MAb (24C5) specific to the relatively
stable ring fragment of the BNP molecule (epitope 11–
Table 1. Analytical characteristics of commercially available MR-proANP, BNP, and NT-proBNP assays per the manufacturer.
(Continued from page 53)
Capture antibody Detection antibody Standard material FDA
a
cleared-yes/no/claim
Nanogen LifeSign
DXpress Reader
Monoclonal (mouse)
and polyclonal
(goat) Abs
Polyclonal sheep
AB
Synthetic NTproBNP
1–76
Diagnosis HF
Ortho Clinical
Diagnostics Vitros ECi
NH2 terminus
polyclonal sheep
AB, aa 1–21
Central molecule,
polyclonal
sheep AB, aa
39–50
Synthetic NTproBNP
1–76
Aid diagnosis of CHF;
risk stratification of
ACS and CHF; risk
assessment of CV
events and mortality in
patients at risk for HF
with stable CAD;
assess severity in HF
Radiometer AQT90
FLEX NT-proBNP
NH2 terminus
polyclonal sheep
AB, aa 1–21
Central molecule,
polyclonal
sheep AB, aa
39–50
Synthetic NTproBNP
1–76
Diagnosis HF; risk
stratification of
patients with ACS and
HF; not FDA cleared
Response Biomedical
RAMP
Murine MAb, aa 27–31 Central molecule,
polyclonal
sheep AB, aa
39–50
Synthetic NTproBNP
1–76
Diagnosis HF; assess
severity HF
Roche NT-proBNP I
Elecsys, E170
NH2 terminus
polyclonal sheep
AB, aa 1–21
Central molecule,
polyclonal
sheep AB, aa
39–50
Synthetic NTproBNP
1–76
Diagnosis HF; assess
severity HF; risk ACS;
risk HF
NT-proBNP II Elecsys,
E170
MAb, aa 27–31 Sheep MAb, aa
42–46
Synthetic NTproBNP
1–76
Treatment monitoring in
LVD
Siemens (Dade Behring)
Dimension RxL, Stratus
CS, Dimension VISTA,
Dimension EXL with LM
NH2 terminus
monoclonal sheep
AB, aa 22–28
Central molecule,
Sheep MAb, aa
42–46
Synthetic NTproBNP
1–76
Aid in the diagnosis of
CHF and assessment
of severity; risk
stratification of
patients with ACS and
HF
Siemens (DPC) Immulite
1000, 2000 2500
NH2 terminus
polyclonal sheep
AB, aa 1–21
Central molecule,
polyclonal
sheep AB, aa
39–50
Synthetic NTproBNP
1–76
Not FDA cleared
Assay MR-proANP
Thermo Fisher Scientific
KRYPTOR
Polyclonal sheep AB,
aa 50–72 of NT-
proANP
Monoclonal rat
AB, aa 73–90 of
NT-proANP
Synthetic NTproANP
50–90
Not FDA cleared
a
FDA, US Food and Drug Administration; CHF, congestive heart failure; ACS, acute coronary syndrome; CV, cardiovascular; CAD, coronary artery disease; LVD, left ventricular
dysfunction; aa, amino acid; AB, antibody; MR-proANP, midregional pro-ANP.
b
Both the Alere and Beckman systems use the same 2 antibodies but due to their different assay formats, designation of the monoclonal and omniclonal antibodies as capture and
detection antibody is not absolute. (Adapted with permission from IFCC-International Federation of Clinical Chemistry and Laboratory Medicine, website http://www.ifcc.org.)
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