(Clinical Chemistry. 1998;44:239-243.)
© 1998 American Association for Clinical Chemistry, Inc.
|
Endocrinology and Metabolism |
Direct enzyme immunometric measurement of plasma big endothelin-1 concentrations and correlation with indicators of left ventricular function
Cornelia Haug1,a,
Wolfgang Koenig2,
Martin Hoeher2,
Matthias Kochs2,
Vinzenz Hombach2,
Adolf Gruenert1,
and Hans Osterhues2
1
Institute of Clinical Chemistry,
2
Department of Internal Medicine II, University Hospital, Ulm, Germany.
a Address correspondence to this author at: Institute of Clinical Chemistry, University Hospital Ulm, Robert-Koch-Str. 8, D-89070 Ulm, Germany. Fax 0731-5024584; e-mail cornelia.haug{at}medizin.uni-ulm.de.
 |
Abstract
|
|---|
Recent studies have suggested that the plasma concentrations of
endothelin-1, a potent vasoconstrictive peptide, are increased in
patients with congestive heart failure. This study aimed to evaluate a
new direct ELISA for big endothelin-1 (the precursor of endothelin-1),
in comparison with a big endothelin-1 ELISA using plasma sample
extraction, and to investigate whether plasma big endothelin-1
concentrations correlate with indicators of left ventricular function.
The direct ELISA yielded significantly (P <0.001) lower
results than the assay with extracted samples (0.9 ± 0.5 pmol/L
vs 2.7 ± 1.9 pmol/L; n = 90); however, the results of the
two assays were closely correlated (r = 0.86,
P <0.001). Plasma big endothelin-1 concentrations
exhibited a significant (P <0.001) negative correlation
(r = -0.46, r = -0.40) with
the left ventricular ejection fraction and a significant positive
correlation (r = 0.40, P
<0.001; r = 0.36, P <0.01)
with the left ventricular end-diastolic pressure and the left
ventricular end-diastolic (r = 0.42,
r = 0.38, P <0.001) and
end-systolic (r = 0.52, r =
0.47, P <0.001) volume indices. Plasma big
endothelin-1 concentrations were notably greater in patients with New
York Heart Association (NYHA) class II–IV symptoms than in patients
without cardiac disease or in patients categorized to NYHA class I.
These data suggest that plasma big endothelin-1 concentrations,
measured by a direct ELISA, correlate with hemodynamic indicators and
symptoms of left ventricular dysfunction.
|
Introduction
|
|---|
Endothelin is potent vasoconstrictive peptide, originally isolated
from the supernatant of cultured endothelial cells (1).
Until now, three isoforms, endothelin-1, endothelin-2, and
endothelin-3, have been isolated, each consisting of 21 amino acid
residues (2). Endothelin seems to exert its effects
predominantly in a paracrine or autocrine manner, but recent studies
have suggested that endothelin might also act as a circulating hormone
(3). Several studies have demonstrated that plasma
endothelin concentrations are increased in patients with congestive
heart failure (4)(5)(6)(7)(8)(9) and in animal models of heart failure
(10)(11)(12)(13). Recently published studies have shown that the
plasma concentrations of big endothelin-1, the 38-amino acid residue
precursor of endothelin-1, are also increased in patients with
congestive heart failure (14)(15) and seem to
be related to the outcome of patients with severe heart failure
(16). The present study aimed to evaluate a new direct
ELISA for big endothelin-1, in comparison with an ELISA after plasma
sample extraction, and to investigate whether plasma big endothelin-1
concentrations are correlated to hemodynamic indicators and symptoms of
left ventricular dysfunction.
|
Patients and Methods
|
|---|
patients
Ninety patients (61 men and 29 women; mean age, 61.4 years; range,
37–80 years) undergoing cardiac catheterization were investigated.
Blood samples were drawn from the femoral artery at the beginning of
cardiac catheterization after informed consent was obtained. The study
was approved by the local ethics committee. Substantial coronary
atherosclerosis, documented by coronary angiography, was found in 76
patients (1 vessel, n = 28; 2 vessels, n = 22; and 3 vessels,
n = 26); mitral valve regurgitation was present in three patients,
and two patients had aortic valve stenosis. Patients were grouped
according to the NYHA (New York Heart Association) classification
[NYHA I: n = 55, mean age (± SD), 60.5 ± 10.0 years; NYHA
II: n = 11, 64.4 ± 12.7 years; NYHA III: n = 12,
64.6 ± 10.3 years; NYHA IV: n = 3, 61.0 ± 16.1
years]. In nine patients (58.4 ± 10.8 years), a cardiac disease
had been excluded. Some patients had a history of hypertension (n
= 41) or hyperlipidemia (n = 39). Patients with renal disease,
liver disease, and diabetes mellitus were excluded from the study. All
patients had normal sinus rhythm at the time of blood sample
collection. Medication, including angiotensin-converting enzyme
inhibitors (n = 31), aspirin (n = 57), ß-adrenergic
antagonists (n = 54), calcium channel blockers (n = 7),
digitalis (n = 7), diuretics (n = 32), and nitrates (n =
40), was discontinued at least 12 h before the investigation.
cardiac catheterization
Left-sided cardiac catheterization was performed with a 6 F
pigtail catheter (Cordis), and a 7 F multipurpose catheter (Cordis) was
used for right-sided cardiac catheterization. Left ventricular ejection
fraction and left ventricular end-diastolic and end-systolic volumes
were evaluated by left ventricular angiography. Cardiac output was
determined by the Fick method. Pulmonary arterial pressure was measured
with standard cardiac catheterization laboratory equipment (Metek).
measurement of plasma big endothelin-1
Blood samples were collected in EDTA-containing tubes and
immediately centrifuged; the plasma was stored at -80 °C. Plasma
big endothelin-1 concentrations were measured with two different
ELISAs: a direct assay without sample extraction, and an assay after
solid-phase extraction. The direct assay (Biomedica) exhibited the
following cross-reactivities as evaluated by serial dilutions of
synthetic peptides and expressed as the mean relative immunoreactivity
compared with big endothelin-1: big endothelin-1 (1–38) 100%, big
endothelin-1 (22–38) <1%, endothelin-1 <1%, endothelin-2 <1%,
and endothelin-3 <1%. According to the manufacturer, the intraassay
CV (determined by repeated measurements of plasma samples) was 4.9%
(1.2 ± 0.06 pmol/L, n = 11) and 3.9% (6.7 ± 0.26
pmol/L, n = 11), respectively; the interassay CV was 6.9%
(1.6 ± 0.11 pmol/L, n = 12) and 6.1% (6.5 ± 0.39
pmol/L, n = 12), respectively; and the detection limit was 0.025
pmol/L. The assay was performed in microwell plates coated with an
immunoaffinity-purified polyclonal rabbit anti-big endothelin antibody.
Calibrator or sample (200 µL) and a monoclonal mouse anti-big
endothelin antibody were added to the wells and incubated for 3 h
at 37 °C. The wells were washed with washing buffer, and 200 µL of
horseradish peroxidase-conjugated anti-mouse IgG antibody was added.
After an incubation period of 1 h at 37 °C and another washing
step, tetramethylbenzidine was added to the wells as a substrate; after
30 min, the reaction was stopped by the addition of 1 mol/L sulfuric
acid. Absorbances were determined with a microtiter plate
spectrophotometer at 450 nm against 620 nm as reference.
For plasma sample extraction, Sep-Pak C18 cartridges
(Waters) were activated by methanol and then acetic acid (40 mL/L).
Plasma samples (2 mL) were acidified with acetic acid (40 mL/L),
centrifuged, and applied to the columns. The absorbed peptide was
eluted with 2 mL of an aqueous solution containing 880 mL/L ethanol and
40 mL/L acetic acid, and the eluate was dried under a stream of
nitrogen (analytical recovery, determined by addition of the cold
peptide: 91.3% ± 7.5%, n = 15, mean ± SD).
The sample residues were dissolved in assay buffer (250 µL), and big
endothelin-1 concentrations were measured by another commercially
available ELISA (Johnson and Johnson). Cross-reactivity (the
immunoreactivity relative to big endothelin-1, based on the
concentration giving 50% B/Bmax; data provided by the
manufacturer) was big endothelin-1 100%, big endothelin-1 (22–38)
<0.012%, endothelin-1 <0.028%, endothelin-2 <0.006%, endothelin-3
<0.006%; intraassay CV was 6.7% (17.8 ± 1.2 pmol/L, n =
14; established in the present study by repeated measurements of a
pooled plasma sample) and 5.9% (41.0 ± 2.4 pmol/L, n = 18);
interassay CV (evaluated by the manufacturer by repeated measurements
of calibrator solutions) was 9.5% (48.0 ± 4.5 pmol/L, n =
22) and 9.0% (97.5 ± 8.7 pmol/L, n = 22); detection limit
of the assay was 3 pmol/L, corresponding to 0.38 pmol/L in the original
plasma sample (eightfold concentration of the samples during the
extraction procedure). Calibrators and samples (100 µL) were
incubated in microtiter wells precoated with anti-big endothelin-1
(22–38) rabbit IgG (18 h at 4 °C). After a washing step,
horseradish peroxidase-labeled Fab' fragment of anti-big endothelin-1
(1–21) rabbit IgG was added and incubated at 37 °C for 30 min.
Tetramethylbenzidine was added as a substrate; after an incubation of
15 min, the reaction was stopped by addition of 1 mol/L sulfuric acid,
and the resulting color was read at 450 nm in a microtiter plate
spectrophotometer.
statistical analysis
Results are expressed as means ± SD. The correlation between
plasma big endothelin-1 concentrations, measured by the two different
assays, and hemodynamic variables was evaluated by linear regression
analysis. Results from the two ELISA systems were compared by means of
the two-sample t-test. Plasma big endothelin-1
concentrations in patients categorized according to the NYHA
classification were evaluated by one-way analysis of variance, followed
by the Newman–Keuls test.
|
Results
|
|---|
The direct big endothelin-1 ELISA yielded significantly
(P <0.001) lower results than the ELISA with extracted
samples (0.9 ± 0.5 pmol/L vs 2.7 ± 1.9 pmol/L, n =
90); however, the results obtained with the two different assay systems
were closely correlated (r = 0.86; P
<0.001) (Fig. 1
).
View larger version (16K):
[in this window]
[in a new window]
|
Figure 1. Correlation between plasma big endothelin-1
concentrations, measured by a direct ELISA and by an ELISA with plasma
sample extraction (n = 90).
|
|
Plasma big endothelin-1 concentrations exhibited a significant
negative correlation with the left ventricular ejection fraction, and a
significant positive correlation with the left ventricular
end-diastolic pressure, the left ventricular end-diastolic and
end-systolic volume indices, and the mean and end-diastolic pulmonary
arterial pressure. Data are shown in Table 1
and in Figs. 2
and
3. No significant correlation was observed with cardiac index or
systolic, diastolic, or mean blood pressure values.
View larger version (13K):
[in this window]
[in a new window]
|
Figure 2. Correlation between plasma big endothelin-1
concentrations, measured by a direct ELISA, and the left ventricular
ejection fraction (LVEF; n = 90).
|
|
In accordance with the significant correlation between plasma big
endothelin-1 concentrations and hemodynamic indicators of left
ventricular function, a significant increase of plasma big endothelin
concentrations was observed in patients with symptoms of moderate and
severe heart failure (direct ELISA: patients without cardiac disease,
0.5 ± 0.1 pmol/L; NYHA I, 0.7 ± 0.2 pmol/L; NYHA II,
1.1 ± 0.5 pmol/L; NYHA III, 1.2 ± 0.6 pmol/L; and NYHA IV,
2.1 ± 0.7 pmol/L; ELISA after sample extraction: patients without
cardiac disease, 1.8 ± 0.3 pmol/L; NYHA I, 2.2 ± 1.0
pmol/L; NYHA II, 3.2 ± 1.6 pmol/L; NYHA III, 4.1 ± 1.9
pmol/L; and NYHA IV, 6.0 ± 2.3 pmol/L); significances and data
are shown in Fig. 4
.
View larger version (36K):
[in this window]
[in a new window]
|
Figure 4. Plasma big endothelin-1 concentrations, measured by a
direct ELISA and by an ELISA after sample extraction, in patients
without cardiac disease (C; n = 9) and in patients categorized
according to the NYHA classification (NYHA I, n = 55; II, n =
11; III, n = 12; and IV, n = 3; *, P <0.05;
**, P <0.01; ***, P <0.001 vs C or
NYHA I).
|
|
|
Discussion
|
|---|
In the present study, plasma big endothelin-1 concentrations were
measured by a new direct ELISA and by an ELISA performed after plasma
sample extraction. The direct assay yielded significantly lower results
than the assay with sample extraction. These differences might in part
be explained by different sources of peptide calibrator and big
endothelin-1 antibodies or by differences in the detection of big
endothelin-1 bound to plasma proteins (a recently published study
suggested a plasma protein binding of ~65% (17)).
However, a close correlation between the results obtained with the two
different assay systems was observed, suggesting that alterations in
circulating plasma big endothelin-1 concentrations are reflected by
both assays similarly.
Previous studies have demonstrated that plasma endothelin-1
concentrations are increased in patients with congestive heart failure,
and a significant correlation between plasma endothelin-1
concentrations and indicators of left ventricular function, such as
left ventricular ejection fraction, and with pulmonary arterial
pressure values was observed
(4)(7)(9). This study adds further
evidence that plasma concentrations of the precursor peptide big
endothelin-1 are also correlated to indicators of left ventricular
function and to pulmonary arterial pressure, as well as to the
symptomatic status of left ventricular dysfunction.
Until now, the physiological relevance of circulating endothelin
remains unclear, because it is believed to act primarily as a paracrine
or endocrine factor, and several authors have suggested that increased
plasma concentrations may represent spillover from a locally increased
endothelin production. However, previous studies have suggested that
endothelin might also act as a circulating hormone (3),
and that increased circulating endothelin in patients with congestive
heart failure may represent a part of the neurohumoral compensatory
response and thus may contribute to the maintenance of the circulation,
along with other neurohormones such as norepinephrine, angiotensin, and
vasopressin (8)(18). In support of this view
are the observations of increased circulating endothelin concentrations
in response to acute hemodynamic stress induced by upright posture
(8). Most of the above-mentioned studies, demonstrating
increased plasma endothelin concentrations in patients with congestive
heart failure, have been performed with radioimmunoassays
cross-reacting with big endothelin (10–38%) (4)(5)(6)(7)(8)(9). Wei
et al. (9) have demonstrated, by gel filtration
chromatography, that circulating plasma endothelin in patients with
severe congestive heart failure consisted of ~62% big endothelin
compared with 0% in healthy subjects. These data suggested that
endothelin synthesis and release may be increased in congestive heart
failure. Pacher et al. (14)(16) and Kiowski et
al. (15) found increased big endothelin-1 concentrations,
determined by a radioimmunoassay after Sep-Pak C18
extraction, in patients with congestive heart failure. Pacher et al.
(16) also have demonstrated that plasma big endothelin-1
concentrations are strongly related to survival in patients with
chronic heart failure. Because the direct big endothelin-1 assay can
easily be performed in a routine laboratory, it might become a tool in
the diagnosis or management of left ventricular dysfunction and
pulmonary hypertension.
View larger version (14K):
[in this window]
[in a new window]
|
Figure 3. Correlation between plasma big endothelin-1
concentrations, measured by a direct ELISA, and the mean pulmonary
arterial pressure (n = 42).
|
|
|
References
|
|---|
-
Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988;332:411-415.
[Medline]
[Order article via Infotrieve]
-
Inoue A, Yanagisawa M, Kimura S, Kasuya Y, Miyauchi T, Goto K, Masaki T. The human endothelin family: three structurally and pharmacologically distinct isopeptides predicted by three separate genes. Proc Natl Acad Sci U S A 1989;86:2863-2867.
[Abstract/Free Full Text]
-
Lerman A, Hildebrand FL, Aarhus LL, Burnett JC. Endothelin has biological actions at pathophysiological concentrations. Circulation 1991;83:1808-1814.
[Abstract/Free Full Text]
-
Cody RJ, Haas GJ, Binkley PF, Capers Q, Kelley R. Plasma endothelin correlates with the extent of pulmonary hypertension in patients with chronic congestive heart failure. Circulation 1992;85:504-509.
[Abstract/Free Full Text]
-
Lerman A, Kubo SH, Tschumperlin LK, Burnett JC. Plasma endothelin concentrations in humans with end-stage heart failure and after heart transplantation. J Am Coll Cardiol 1992;20:849-853.
[Abstract]
-
McMurray JJ, Ray SG, Abdullah I, Dargie HJ, Morton JJ. Plasma endothelin in chronic heart failure. Circulation 1992;85:1374-1379.
[Abstract/Free Full Text]
-
Rodeheffer RJ, Lerman A, Heublein DM, Burnett JC. Increased plasma concentrations of endothelin in congestive heart failure in humans. Mayo Clin Proc 1992;67:719-724.
[Web of Science][Medline]
[Order article via Infotrieve]
-
Stewart DJ, Cernacek P, Costello KB, Rouleau JL. Elevated endothelin-1 in heart failure and loss of normal response to postural change. Circulation 1992;85:510-517.
[Abstract/Free Full Text]
-
Wei CM, Lerman A, Rodeheffer RJ, McGregor CGA, Brandt RR, Wright S, et al. Endothelin in human congestive heart failure. Circulation 1994;89:1580-1586.
[Abstract/Free Full Text]
-
Cavero PG, Miller WL, Heublein DM, Margulies KB, Burnett JC. Endothelin in experimental congestive heart failure in the anesthetized dog. Am J Physiol 1990;259:F312-F317.
[Abstract/Free Full Text]
-
Margulies KB, Hildebrand FL, Lerman A, Perrella MA, Burnett JC. Increased endothelin in experimental heart failure. Circulation 1990;82:2226-2230.
[Abstract/Free Full Text]
-
Teerlink JR, Löffler BM, Hess P, Maire JP, Clozel M, Clozel JP. Role of endothelin in the maintenance of blood pressure in conscious rats with chronic heart failure. Acute effects of the endothelin receptor antagonist Ro 47–0203 (Bosentan). Circulation 1994;90:2510-2518.
[Abstract/Free Full Text]
-
Sakai S, Miyauchi T, Sakurai T, Kasuya Y, Ihara M, Yamaguchi I, et al. Endogenous endothelin-1 participates in the maintenance of cardiac function in rats with congestive heart failure. Marked increase in endothelin-1 production in the failing heart. Circulation 1996;93:1214-1222.
[Abstract/Free Full Text]
-
Pacher R, Bergler-Klein J, Globits S, Teufelsbauer H, Schuller M, Krauter A, et al. Plasma big endothelin-1 concentrations in congestive heart failure patients with or without systemic hypertension. Am J Cardiol 1993;71:1293-1299.
[Web of Science][Medline]
[Order article via Infotrieve]
-
Kiowski W, Sütsch G, Hunziker P, Müller P, Kim J, Oechslin E, et al. Evidence for endothelin-1-mediated vasoconstriction in severe chronic heart failure. Lancet 1995;346:732-736.
[Web of Science][Medline]
[Order article via Infotrieve]
-
Pacher R, Stanek B, Hülsmann M, Koller-Strametz J, Berger R, Schuller M, et al. Prognostic impact of big endothelin-1 plasma concentrations compared with invasive hemodynamic evaluation in severe heart failure. J Am Coll Cardiol 1996;27:633-641.
[Abstract]
-
Brunner F, Stessel H, Watzinger N, Löffler BM, Opie LH. Binding of endothelin to plasma proteins and tissue receptors: effects on endothelin determination, vasoactivity, and tissue kinetics. FEBS Lett 1995;373:97-101.
[Web of Science][Medline]
[Order article via Infotrieve]
-
Stewart DJ. Endothelin in cardiopulmonary disease: factor paracrine vs neurohumoral. Eur Heart J 1993;14:48-54.
[Abstract/Free Full Text]
Top
Abstract
Introduction
