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Articles |
1
Laboratoire de Biologie Hormonale, Hôpital Saint-Louis, 75010 Paris, France.
2
CEA Saclay, Service de Pharmacologie et
d'Immunologie, 91191 Gif/Yvette, France.
3
Biotechnology Department, Roussel-Uclaf, 93235
Romainville, France.
4
Centre d'Investigation Clinique, INSERM, Hôpital
Broussais, 75014 Paris, France.
a Author for correspondence. Fax +33 1 42494280.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Key Words: indexing terms: reference values • endothelins • isopeptides • vasoconstrictors • blood pressure • variation, source of
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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At present, many other physiological effects have been attributed to ET-1 (5), and variations in plasma concentrations of this peptide have been found in certain pathologies, including arterial hypertension (6), renal insufficiency (7), pulmonary hypertension (8), malignant prostatic adenoma (9), and cerebral ischemia (10).
We thought it interesting to develop an assay for big ET-1 because (a) big ET-1 and ET-1 are present in quasi-equimolar plasma concentrations in humans (11); (b) plasma ET-1 is more rapidly cleared than big ET-1 (12), making it preferable to measure the physiological concentrations of the latter; (c) plasma concentrations of ET-1 vary in certain pathologies, and characterization of fluctuations in big ET-1, which is metabolically further upstream, might eliminate possible variations in ECE activity; and (d) assay of big ET-1 as well as ET-1 would allow study of the pharmacological effects of certain ECE inhibitors in in vitro cell models.
Here, we report the plasma values of big ET-1 measured in healthy volunteers with use of a sandwich-type enzyme immunoassay (EIA) that is highly sensitive, specific, and reproducible and constitutes a method for elucidating the physiological, pathophysiological, and clinical significance of big ET-1.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We labeled big ET-1 with 125I, using the lactoperoxidase method, and purified it by HPLC (13). All other reagents used were of the highest grades available.
production of antibodies
Anti-Cys-22-38 human big ET-1 COOH-terminus fragment
polyclonal antibody (anti-P16).
P16 peptide
(NH2-Cys-Val-Asn-Thr-Pro-Glu-His-Val-Val-Pro-Tyr-Gly-Leu-Gly-Ser-Pro-Arg-Ser-COOH)
was produced in our laboratory by solid-phase synthesis in an Applied
Biosystems (Roissy CD6, France) 431-A synthesizer with
9-fluorenylmethyloxycarbonyl-tert-butyl chemistry on a
p-hydroxymethylphenoxymethyl-polystyrene resin, and was
cleaved in the presence of phenol, 1,2-ethanedithiol, trifluoroacetic
acid, and thioanisole. Crude P16 was purified by preparative HPLC with
a linear gradient on a 250 x 9.3 mm Pep'S preparative column
(C18/C2, 15-µm particles; Pharmacia Biotech,
Saclay, France). The linear gradient went from 0% to 60% of solution
B (700 mL/L acetonitrile in distilled water containing 1 mL/L
trifluoroacetic acid) in 120 min at a flow rate of 2 mL/min. A 2-mL
volume of crude peptide solution (10 mg/mL) was injected, and eluted
peptide was detected by measuring absorbance at 214 nm. We checked the
purification with a 250 x 4.6 mm Pep'S analytical column
(C18/C2, 5-µm particles) and a linear
gradient of 0% to 60% solution B over 30 min at a flow rate of 1
mL/min. A single peak was obtained at 214 nm with a retention time of
16 min. The molecular mass of P16 was verified by obtaining the
ion-spray mass spectra of the eluted peptide with an API 100 mass
spectrometer (Perkin-Elmer Sciex, Toronto, Canada).
The P16 obtained was coupled to hemocyanine by the maleimide technique (14), and the product was lyophilized.
Three New Zealand rabbits received monthly intradermal injections of 0.5 mg of the immunogen, combined with complete Freund's adjuvant in the first injection and with incomplete Freund's adjuvant in the succeeding ones. Serum was obtained from blood drawn from the ear vein of the rabbits and tested by RIA. Whole and diluted serum samples (100 µL) were incubated with 125I-labeled big ET-1 (10 000 dpm) in a total volume of 0.5 mL of phosphate-buffered saline, pH 7.2, containing 5 g/L bovine serum albumin (BSA). After overnight incubation at 4 °C, the mixture was mixed with 0.1 mL of charcoal-treated human serum, and the bound radioactivity was precipitated with 1 mL of sheep anti-rabbit antiserum (PR 100 99980; Cis Bio International, Gif/Yvette, France). The radioactivity of the pellets obtained after centrifugation (3000g, 15 min, 4 °C) was measured in an automated gamma counter (LKB Wallac 1277 Gamma Master Counter; Pharmacia Biotech). The polyclonal antibodies were purified by affinity chromatography on EAH Sepharose 4B gel (cat. no. 17-0569-01; Pharmacia Biotech) coupled to the P16 peptide by the maleimide technique (14), followed by membrane ultrafiltration (on YM10 membrane; Amicon, Epernon, France). Protein concentrations were then determined according to Bradford's method, with Coomassie Blue dye.
Anti-ET-1 monoclonal antibody.
To prepare the immunogen,
we coupled ET-1 to BSA by the glutaraldehyde method (15).
Five six-week-old female BALB/c mice received subcutaneous injections
of immunogen (100 µg) dissolved in saline and emulsified in an equal
volume of complete Freund's adjuvant. Booster injections in incomplete
Freund's adjuvant were given intraperitoneally at 4-week intervals.
The mice were bled through the retro-orbital sinus 10 days after the
third injection. Three days before cell fusion, the mice received an
intravenous injection of 200 µg of immunogen in 200 µL of saline.
SP2/0 myeloma cells were grown in RPMI-1640 medium supplemented with, per liter, 2 mmol of glutamine, 1 mmol of sodium pyruvate, 100 kIU of penicillin, 50 mg of streptomycin, 20 µmol of 8-azaguanine, and 100 mL of heat-inactivated FCS. The day before fusion, the cells were cultured in the same medium but without 8-azaguanine and with 200 mL/L FCS ("RPMI-1640 20%FCS").
Just before fusion, the SP2/0 cells were washed twice with RPMI-1640 alone. At the same time, the selected donor mouse was killed, the spleen was removed, and the spleen cells were harvested by repeated 10-mL injections of RPMI-1640 medium alone.
SP2/0 and spleen cells were mixed in a 2:1 cellular ratio and centrifuged (100g, 10 min). Fusogen was prepared by melting 1 g of polyethylene glycol 4000 (cat. no. 807490; Merck, Chelles, France) at 61 °C for 1 h and mixing with 1 mL of RPMI-1640 alone. This solution was then kept at 37 °C until fusion.
Fusion was performed at 37 °C by dropwise addition of 1 mL of the fusogen for 1 min to the cell mixture pellet. The cell suspension was immediately diluted by addition of 5 mL of RPMI-1640 over 2 min, followed by 20 mL of same medium added over 2 min. The cells were centrifuged (100g, 10 min) and the pellet was carefully resuspended in RPMI-1640 20%FCS. The fused cell suspension was distributed in 24-well plates at 5 x 101 cells per well. The next day, 1 mL of medium was replaced by 1 mL of selecting medium (RPMI-1640 20%FCS containing 5.8 µmol/L azaserine and 0.1 mmol/L hypoxanthine). The selecting medium was renewed on days 4 and 7 after fusion. Culture in selecting medium was maintained for 2 weeks after fusion, after which the azaserine was removed from the culture medium and the hypoxanthine was progressively eliminated. Culture supernatants growing hybridomas were tested by ELISA (Mouse Hybridoma Screening Kit, cat. no. 1110225; Boehringer Mannheim, Mannheim, Germany) and RIA to detect anti-ET-1 antibodies after 30% confluence was reached. Culture supernatants (100 µL) were incubated with 125I-labeled big ET-1 (10 000 dpm) in a total volume of 0.5 mL of phosphate-buffered saline, pH 7.2, containing 5 g/L BSA. After overnight incubation at 4 °C, the mixtures were incubated for 15 min with 1 mL of preprecipitated sheep anti-mouse immunoglobulin (cat. no. A231; UCB Bioproducts, Braine L'Alleud, Belgium), and the bound radioactivity was separated by centrifugation (3000g, 15 min, 4 °C). Radioactivity of the pellets was measured in an automated gamma counter.
Hybrid cells from hybridoma-positive wells were cloned by limiting dilution in 96-well microplates in RPMI-1640 20%FCS supplemented with 100 mL/L BM-Condimed H1 additive (cat. no. 1088947; Boehringer) to support cell growth and then were subtyped with the Mouse Hybridoma Subtyping Kit (cat. no. 1183117; Boehringer).
A large quantity of anti-ET-1 monoclonal antibody was produced in ascites by intraperitoneal injection of 0.5 mL of incomplete Freund's adjuvant and 10 days afterward by injection of between 5 and 10 x 106 hybrid cells in 0.5 mL of RPMI-1640 medium without additive. The antibody preparation was purified by HPLC with a HiTrap Protein G column (cat. no. 17-0404-01; Pharmacia Biotech), followed by membrane ultrafiltration as above. Protein concentrations were then determined by Bradford's method with use of Coomassie Blue dye.
biacore tests
BIAcore (Pharmacia Biosensor, Saint Quentin-en-Yvelines, France)
is an analytical system for real-time biomolecular interaction
analysis, in which the binding of analytes to surface-immobilized
ligands is directly observed.
To visualize the affinity of each antibody, we immobilized big ET-1 (35 µL of a 100 mg/L solution diluted in 10 mmol/L acetate buffer, pH 4.5) on the sensor chip (gold film with carboxylated dextran on a glass support) of the BIAcore apparatus, using the Amine Coupling Kit (cat. no. BR-1000-50, Pharmacia Biosensor). We then separately injected 35 µL of dilute rabbit serum or of hybrid cell supernatant at a rate of 5 µL/min. Each regeneration was carried out with 5 µL of 100 mmol/L HCl.
We also tested the anti-P16/big ET-1/anti-ET-1 complex in the BIAcore system by immobilizing purified anti-P16 (35 µL of a 70 mg/L protein solution in 10 mmol/L acetate buffer, pH 4.5), using the Amine Coupling Kit. We then injected 35 µL of a 50 mg/L big ET-1 solution diluted in 10 mmol/L Hepes buffer containing 150 mmol/L NaCl (cat. no. 22-0512-44; Pharmacia Biosensor) at a rate of 5 µL/min. Finally, we injected 4 µL of anti-ET-1 antibody purified from ascites fluid.
acetylcholinesterase-labeled anti-et-1
Acetylcholinesterase (AchE) was purified from the electric eel
Electrophorus electricus by affinity chromatography
(16). The tetrameric form of the enzyme was used to label
the antibody. The characteristics of this preparation have been
described elsewhere (17)(18). AchE activity
was measured by the colorimetric method of Ellman (19), 1
Ellman unit being defined as the quantity of enzyme that produces an
increase of 1 absorbance unit at 25 °C in 1 min in 1 mL of medium in
an optical pathlength of 1 cm. This corresponded to ~8 ng of enzyme.
F(ab')2 fragments were obtained from purified anti-ET-1 antibody by treatment with pepsin in acidic medium (20). We then obtained Fab' fragments by reduction of F(ab')2 in the presence of 10 mmol/L 2-mercaptoethylamine. The derived fragments were subsequently covalently coupled to AchE that had been pretreated with succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (cat. no. M5525; Sigma Aldrich Chimie, Saint-Quentin-Fallavier, France), as previously described (21).
population studies
Healthy subjects of both sexes were recruited for the study at the
Broussais Hospital Clinical Investigations Center (Paris) according to
the following criteria: ages 18–35 years, nonsmokers, normotensive
(blood pressure <140/90 mmHg), and normal results for physical
examination and routine biological measurements. In addition, the women
were investigated during the first phase of their menstrual cycle and
did not take oral contraceptives. Volunteers gave their informed
written consent to participate in the study. The protocol was approved
by the "Comité consultatif de protection des personnes se
prêtant à des recherches biomédicales" (Cochin
Hospital, Paris, France).
Twenty-two fasting subjects (11 men and 11 women) were admitted at 0800 h on the day of the study. Venous blood was drawn after 1 and 2 h of rest in the supine position—i.e., at 0900 (Su1 h) and 1000 (Su2 h)—and after having been standing for 1 h at 1100 (St1 h). Blood samples were collected in tubes containing an enzyme inhibitor (aprotinin) and EDTA. Each sample was immediately centrifuged, separated into 2.2-mL aliquots, and frozen at -20 °C until assay.
effects of posture on plasma big et-1 concentrations
Extraction of big ET-1 from plasma.
After
thawing the plasma samples, we acidified 2 mL of sample with 3 mL of
dilute acetic acid (40 mL/L) in distilled water, then placed the
solution in a Sep-Pak C18 chromatography column (cat. no.
51910; Millipore Corp, Bedford, MA) pretreated with 5 mL of methanol, 5
mL of distilled water, and 5 mL of dilute acetic acid (40 mL/L) in
distilled water. Next, we washed each column with 3 mL of distilled
water, followed by 3 mL of dilute ethanol (250 mL/L) in distilled
water, and eluted big ET-1 twice with two applications of 1 mL of
dilute acetic acid (40 mL/L) in ethanol (860 mL/L). The combined
eluates were evaporated at 37 °C, then reconstituted with 0.5 mL of
EIA buffer [0.1 mol/L phosphate buffer, pH 7.4, containing 0.15 mol/L
NaCl, 1 g/L BSA, 0.1 g/L sodium azide, and 1 mL/L P20 surfactant (cat.
no. BR-1000-54; Pharmacia Biosensor)].
To check the extraction yield of the Sep-Pak column, we included in each batch a plasma sample with a known concentration of big ET-1 (obtained by addition of big ET-1 to a plasma treated with dextran charcoal and thus stripped of peptides). The extraction yield was between 85% and 100% and was taken into account when calculating the measured concentrations of big ET-1.
Big ET-1 assay.
In preparation for the assay,
Maxisorp plates, 96-well (cat. no. 469949; Nunc, Roskilde, Denmark),
were passively coated at room temperature for 18 h with a 10 mg/L
solution of polyclonal anti-P16 antibodies in 0.05 mol/L phosphate
buffer, pH 7.4 (200 µL/well). After five washings with washing buffer
(distilled water containing 0.5 mL/L Tween 20), the wells were
saturated with 300 µL of EIA buffer. The plates were then stored at
4 °C until use.
On the day of the assay, the wells were again washed five times with 300 µL of washing buffer, and 100 µL of the concentrated extracts or of big ET-1 calibrators was added (concentration range: 0, 1.56, 3.12, 6.25, 12.5, 25, 50, and 100 ng/L) to the wells. We then added to each well 100 µL of AchE-labeled anti-ET-1 antibody (diluted to 10 Ellman units). After overnight incubation and 10 washings with 300 µL of washing buffer per well, we added to each well 200 µL of Ellman reagent substrate: 0.75 mmol/L acetylthiocholine iodide (A5751; Sigma) and 0.25 mmol/L 5–5'-dithiobis-2-nitrobenzoate (D8130; Sigma) in 0.1 mol/L phosphate buffer, pH 7.4, containing 14.5 mmol/L NaCl. After incubation for 30 min, the absorbance of the plates was read at 410 nm with a spectrophotometer.
statistical analysis
Where applicable, we used the nonparametric tests of Mann–Whitney
and Wilcoxon.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The dissociation constant (Kd) was
determined by competitive-binding studies to be 7.2 x
10-11 mol/L. The cross-reactivity of the anti-P16 antibody
with big ET-2 and big ET-3 for 50% inhibition of the
binding with 125I-labeled big ET-1 were both <0.01%.
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Hybrid 1B3 monoclonal anti-ET-1 antibody was selected for the assay,
its absorbance read by ELISA in the culture supernatant being >1.
After subcloning this clone three times, we determined that it secreted
IgG1 
immunoglobulins. Titers of this ascites-derived antibody were
measured by RIA fixation of 125I-labeled big ET-1. The
dilution of the ascites-derived 1B3 antibody that fixed 50% of the
labeled big ET-1 was 1:100 000. Finally, Fig. 2
confirms the fixation of the 1B3 antibody to big ET-1
immobilized on the BIAcore sensor chip. Its Kd
was determined to be 4.4 x 10-9 mol/L, and its
cross-reactivities with big ET-2 and big ET-3 were respectively 22%
and <0.02%.
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Successive phases of the sandwich assay—immobilization of the anti-P16
antibody on the surface of the BIAcore sensor-chip, recognition of big
ET-1, and finally, immobilization of 1B3—could be observed in real
time, thereby demonstrating the feasibility of this assay (Fig. 3
).
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analytical evaluation
Figure 4
represents a typical EIA curve obtained with the big ET-1
calibrators (0–100 ng/L; see above). The blank value was defined as
the absorbance obtained for the reagents only, the sample having been
replaced with EIA buffer; averaging 10 results showed this to be 0.029
A ± (SD 0.004 A). We calculated the least
detectable dose by adding 3 SD to the average value obtained for the
blank; this corresponded to 0.04 A, or 1.16 ng/L on the
calibration curve.
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The intra- and interassay reproducibilities (expressed in ng/L) were studied with two plasma samples. The intraassay reproducibility was as follows: for sample 1, the mean ± SD = 4.6 ± 0.4, CV = 8.5%; for sample 2, 16.2 ± 1.1, CV = 6.8% (n = 10 each). Interassay reproducibility was 4.2 ± 0.7, CV = 16.6%, and 16.3 ± 1.4, CV = 8.5%, respectively (n = 10 each).
A plasma with a high big ET-1 concentration (17.3 ng/L) was diluted with plasma that had been treated with dextran charcoal to be free of big ET-1. The big ET-1 mean concentrations determined in this dilution test were, for plasma diluted 1:2, 1:4, and 1:8, 18.1, 16.9, and 19.3 ng/L, respectively (n = 10).
We also loaded with increasing concentrations of big ET-1 a dextran
charcoal-treated plasma (big ET-1-free) and analyzed these samples for
big ET-1. The analytical recovery was between 94% and 105% (Table 1
).
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Possible interference from ET-1, big ET-2, and big ET-3 was evaluated by determining the concentrations of those peptides that (under the same conditions used to assay big ET-1) resulted in the same absorbance values obtained for big ET-1 at both 50 ng/L and 1.2 ng/L. Cross-reactivity for ET-1 was undetectable at both concentrations; big ET-2 and big ET-3 cross-reactivities were <0.4% and <0.1%, respectively, at the two concentrations.
values for healthy human subjects
As reported in Table 2
, the mean values determined for big ET-1 were very close to
each other, between 1.78 and 1.87 ng/L, regardless of the posture and
sex of the subjects; the minimum and maximum concentrations encountered
were respectively 1.05 and 3.13 ng/L. Any differences between big ET-1
concentrations found in women and men in the supine and standing
positions were not significant (two-tailed, unpaired Mann–Whitney
nonparametric test). Similarly, we found no significant difference
between big ET-1 concentrations with respect to position (Su1 h, Su2 h,
and St1 h; two-tailed paired Wilcoxon's nonparametric test).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This EIA is also quite sensitive, detecting 0.12 pg of big ET-1 per well, which is equivalent to 1.16 ng/L of the concentrated extract. This sensitivity is comparable with that previously described by Suzuki et al. (11), 0.2 pg/well.
The method is also specific, the cross-reactivities found with ET-1, big ET-2, and big ET-3 being either undetectable or very small. Our results show that 1B3, which is an antibody to both ET-1 and big ET-1, is in fact raised against the loop fragment of ET-1 (the only part of the peptide's sequence that differs among the three forms of big ET).
The intra- and interassay reproducibility tests carried out with plasma samples, either directly or after extraction, provided results in the range of values generally obtained by "sandwich" assay measurement of very low concentrations of this analyte (12).
The results of the dilution and loading tests carried out during the validation study of our big ET-1 immunoassay argue against interference by other substances. Indeed, the peptides that are structurally close to big ET-1 had very weak cross-reactivities in the EIA system.
This study of 22 healthy subjects of both sexes between ages 18 and 35 has allowed us to determine reference values for big ET-1 in two different postures (supine and standing). Mean plasma concentrations of big ET-1 appeared to be below those previously reported by Suzuki et al. (women: 5.7 ± 1.6 ng/L, men: 5.2 ± 1 ng/L) (12) and 3.2 ± 0.5 ng/L (11). However, the populations in whom they assayed big ET-1 were older than our subjects (11)(12).
Comparison of the plasma concentrations of big ET-1 determined with the EIA indicated no significant difference between male and female subjects, thereby confirming others' results (12). On the other hand, we demonstrated the absence of any influence of postural position on the circulating concentrations of big ET-1 in healthy subjects and showed that concentrations of big ET-1 remained unchanged for each subject whatever the posture. Thus, unlike renin, secretion of big ET-1 in humans is not influenced by a change in the subject's posture. This observation is noteworthy for pharmacological development aimed at evaluating the medicinal effects of various concentrations of big ET-1 and ET-1.
In conclusion, the assay of big ET-1 we developed, in association with that of ET-1 (22), will certainly lead to improved clarification of the physiological and clinical roles of this family of peptides and permit pharmacological study of the actions of ECE inhibitors, especially in vitro in human endothelial cells (23)(24).
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 1ß: development of two enzyme immunometric assays (EIA) using acetylcholinesterase and application to biological media. J Immunol Methods 1989;123:193-209.
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