Measurement of immunoreactive angiotensin-(I-7) heptapeptide in human blood.
Materials and Methods
The inhibitor solution contained, per liter, 0.2 mol of potassium EDTA (Fluka), 0.01 mol of o-phenanthroline (Sigma) in 25 mL of ethanol, 1.5 mmol of enalkiren (Abbott Laboratories, Abbott Park, IL), and 1 mmol/L of aprotinin (Sigma). The barbitone buffer contained 0.05 mol/L barbitone acetate, pH 8.6 (ionic strength, 0.1; Electran; BDH Chemicals). Isolute PH phenylsilylsilica solid-phase extraction columns (100 mg; 1 mL) were obtained from International Sorbent Technology. The Tris-albumin buffer (Tris, 0.1 mol/L; pH 7.5 at room temperature; Sigma) contained 5 g/L bovine serum albumin (Sigma) and 0.2 g/L sodium azide (Merck). A Vacelute vacuum manifold was obtained from Analytichem. Peptides were either purchased from Peninsula Laboratories or synthesized as referenced (2); they were purified by HPLC, and calibrators were corrected for water and salt content.
Antiserum against Ang-(1-7) heptapeptide was raised in a New Zealand White rabbit by immunization with synthesized Ang-(1-7) coupled to bovine thyroglobulin by the carbodiimide method at a molar incorporation ratio of 63 [+ or -] 24 (2). The antiserum was used in a final dilution of 1:41000 in the Tris-albumin buffer. Radioimmunoassay calibration curves with different angiotensin peptides were established in the Tris-albumin buffer, and the sensitivity of the antiserum against a given peptide was defined as the reciprocal of the concentration of unlabeled peptide that displaced 50% of the [sup.125.I]-labeled Ang-(1-7) bound to the antibody in the absence of any inhibitor. The specificity of the antiserum in discriminating between two peptides was defined as the quotient of their respective sensitivities (2). Taking the reactivity of the antiserum with Ang-(1-7) as 1.00, the cross-reactivities were as follows: Ang-(1-14), 0.00023; Ang-(1-10), 0.00031; Ang-(1-8), 0.004; Ang-(2-10), 0.00026; Ang-(2-8), 0.0008; Ang-(3-8), 0.0001; and Ang-(4-8), 0.000005. The detection limit of the antiserum (2 SD from zero binding) was, under optimal conditions, 0.5 fmol of Ang-(1-7).
The polyethylene glycol solution contained 200 g/L polyethylene glycol (mean molecular weight, 8000; Sigma) dissolved in water containing 0.2 g/L sodium azide. Bovine [gamma]-globulin (1.5 g; Sigma) was dissolved in 100 mL of the Tris-albumin buffer.
Venous blood (5 mL) was drawn into a syringe containing 0.3 mL of inhibitor solution and rapidly transferred to a polypropylene tube in an ice bath.
Two 2-mL aliquots of the inhibitor-containing blood were immediately pipetted into polypropylene tubes, on ice, containing 40 mL of cold absolute ethanol (998 mL/L) and 4 mL of the barbitone buffer. After mixing, the tubes were centrifuged at 3000g and 2[degrees]C for 25 min. The supernatant extract solution was decanted into a Tris-albumin-buffer-coated polypropylene tube containing 0.25 mL of 100 mL/L glycerol to enhance recoveries, dried under an air stream at 37[degrees]C, and then redissolved in 1 mL of bovine serum albumin (5 g/L) solution by sonication (40 W) and vortex-mixing. The latter procedure optimized recoveries and prevented irreversible adsorption of the peptide to the polypropylene. Samples were stored at -70[degrees]C until analyzed.
Phenylsilylsilica cartridges were primed with 1 mL of methanol followed by 1 mL of water and 2 mL of Tris-albumin buffer. The extracted sample aliquots (1 mL) were thawed, kept on ice, and diluted with 3 mL of cold Tris-albumin buffer. After centrifugation at 3000g and 2[degrees]C for 5 min, supernatants were drawn through the activated cartridges by a vacuum manifold. Columns were rinsed twice with 3 mL of water. Retained angiotensins were eluted with 0.8 mL of methanol into polypropylene tubes coated with the Tris-albumin buffer. The methanol was evaporated under an air stream at 37[degrees]C, the dry extract was redissolved in 0.25 mL of ice-cold Tris-albumin buffer, and Ang-(1-7) was quantified by subsequent radioimmunoassay.
LABELING OF ANG-(1-7) HEPTAPEPTIDE
Ang-(1-7) heptapeptide was labeled with [sup.125.I] by the chloramine-T method (3). Specific activity of the detection angiotensin was 1500-2200 pCi/fmol, i.e., ~1-2 fmol of angiotensin provided 2000 cpm.
Anti-Ang-(1-7) antiserum (0.25 mL) and the extract solution (0.25 mL) were added together with 0.025 mL of detection-labeled Ang-(1-7) heptapeptide (2000 cpm). For calibration curves, increasing amounts of Ang-(1-7) heptapeptide (0-1000 fmol) were added to 0.25 mL of Tris-albumin buffer, rather than the extract solution, and 0.025 mL of 0.04 mol/L o-phenanthroline in 30 mL/L ethanol was added to correct for extracted o-phenanthroline. For volume correction, 0.025 mL of 30 mL/L ethanol was added to the unknown samples. Calibration and unknown samples were incubated for 72 h at 4[degrees]C. Antibody-bound and free Ang-(1-7) heptapeptides were separated by polyethylene glycol precipitation, and bovine [gamma]-globulin solution (0.075 mL) was added to all samples to facilitate subsequent precipitation of the antibody-bound peptide by mixing with 0.75 mL of 200 mL/L polyethylene glycol. After centrifugation for 20 min at 4000g and 4[degrees]C, both the precipitate and supernatant were counted in a well-type gamma counter. The hormone concentration of the blood extract was read from the calibration curve, and plasma concentrations were calculated taking the hematocrit into account.
Recovery. Recoveries were determined by supplementing the blood of three different volunteers with 5, 10, or 15 fmol of Ang-(1-7) heptapeptide, respectively (n = 10 each). The endogenous concentrations (mean [+ or -] SD of triplicate measurements) of this peptide were 2.7 [+ or -] 0.2, 6.1 [+ or -] 0.7, and 5.0 [+ or -] 0.2 pmol/L, respectively. Before ethanol extraction, 5, 10, or 15 fmol of exogenous Ang(1-7) in 0.05 mL of Tris-albumin buffer was added to 10 2-mL aliquots of the blood of one volunteer, and total Ang-(1-7) was then measured. The difference between total and endogenous peptide was expressed as a percentage of the added (exogenous) Ang-(1-7). Subsequent results were not corrected for recoveries.
Imprecision. Imprecision was defined by CVs. Between-assay imprecision was determined by measuring the Ang-(1-7) heptapeptide concentrations of three blood samples in six consecutive independent assays (n = 6). Within-assay imprecision was determined by repeated measurements of three different blood samples in a single assay (n = 10).
Accuracy. The accuracy and the absence of nonspecific interferences in the radioimmunoassay were determined by serially diluting pooled final-blood extracts with the Tris-albumin buffer (1:2, 1:4, and 1:8 by volume). In another experiment, we tested for possible interferences caused by the extraction procedure by extracting water samples (water blank; n = 6) and analyzing them for Ang-(1-7).
Healthy human subjects. Eight healthy male volunteers (ages, 21-44 years) came to the hospital after an overnight fast. They were placed in a supine position, and intravenous cannulas were inserted into an antecubital vein of each arm: one for infusion and one for blood sampling. Five consecutive infusions of saline and Ang-(1-7) (Clinalfa) at increasing doses (0, 2, 4, 8, 20 pmol*[kg.sup.-1]*[min.sup.-1]) were administered for 30 min each (0.5 mL/min). The doses were separated by 30-min washout infusions with saline. Blood samples were drawn during the final 3 min of each dosing.
To obtain more reference values, we also collected venous blood from healthy hospital staff members in a supine position (ages, 24-62 years). Results were not corrected for recovery losses.
Recoveries. When 5, 10, and 15 mg of Ang-(1-7) were added to blood, the recoveries were 57% [+ or -] 13% (n = 10), 52% [+ or -] 5% (n = 10), and 50% [+ or -] 8% (n = 10), respectively.
Precision. Between-assay CVs for three different blood samples were 12% (9.1 [+ or -] 1.1 pmol/L; n = 6),10% (9.4 [+ or -] 0.98 pmol/L; n = 6), and 7.6% (20.6 [+ or -] 1.8 pmol/L; n = 6). Within-assay CVs for three different blood samples were 20% (3.9 [+ or -] 0.77 pmol/L; n = 10), 7.7% (18.3 [+ or -] 1.4 pmol/L; n = 10), and 3.5% (26.8 [+ or -] 0.95 pmol/L; n = 10).
Accuracy. Assay accuracy was demonstrated by the linearity of Ang-(1-7) concentrations measured in serially diluted blood extracts (Fig. 1A). Water blanks were consistently below the detection limit of the assay.
Detection limit. Despite the somewhat better sensitivity of the antiserum, the lowest point of the calibration curve was set at an Ang-(1-7) concentration of 1 pmol/L, which is well beyond 2 SD of the zero binding [[sup.125.I]-labeled Ang-(1-7) bound to antibody in the absence of unlabeled peptide].
Reference values. Plasma Ang-(1-7) concentrations in healthy subjects were 5.1 [+ or -] 2.0 pmol/L (mean [+ or -] SD; n = 28; range, 1.0-9.5 pmol/L). During infusion of Ang-(1-7), plasma Ang-(1-7) concentrations increased linearly in relation to the infused dose from a baseline of 4.7 [+ or -] 0.9 pmol/L to 10.4 [+ or -] 0.7, 20.6 [+ or -] 3.0, 37.9 [+ or -] 4.5, and 79.1 [+ or -] 8.4 pmol/L (mean [+ or -] SE) at 2, 4, 8, and 20 pmol*[kg.sup.-1]*[min.sup.-1], respectively (Fig. 1B).
The Ang-(1-7) heptapeptide induces distinct effects in the brains, kidneys, and coronary arteries of laboratory animals. Ferrario et al. (1) originally described several effects of Ang-(1-7) in the brain, such as vasopressin release (and potential blood pressure regulatory functions), and increased prostaglandin [E.sub.2] release from isolated rabbit vasa deferentia was observed after Ang-(1-7) administration (4). Other studies in anesthetized rats provided evidence for diuretic and natriuretic actions and increases in the glomerular filtration rate by Ang-(1-7) (5). Hilchey and Bell-Quilley (6) found that the natriuretic action of Ang-(1-7) was associated with prostaglandin [I.sub.2] release in isolated rat kidneys. Porcine coronary artery rings are dilated by Ang-(1-7), and this effect can be enhanced by the inhibition of angiotensin-converting enzyme and attenuated by kinin [B.sub.2]-antagonism and NO-synthase inhibition, suggesting that bradykinin and [N.sub.2]O may be involved (7). All of these findings await confirmation in human subjects.
[FIGURE 1 OMITTED]
Methods for the measurement of the Ang-(1-7) in plasma and tissues have been established both without (8) and with specific HPLC isolation of this heptapeptide (9,10). The generation and metabolization of angiotensin peptides after specimen sampling is a major problem, and enzyme inhibition before peptide measurement is crucial (11-13). We have therefore established a simple method for the measurement of ir-Ang-(1-7) in blood, based on an optimized enzyme-inhibitor cocktail in the sampling syringe, immediate ethanol precipitation of proteins, and subsequent solid-phase extraction of angiotensin peptides on phenylsilylsilica before radioimmunoassay.
The sensitivity of our antiserum and its low cross-reactivity of <1% with other angiotensin peptides made it possible to reliably measure low endogenous Ang-(1-7) concentrations in blood even without additional HPLC. Our reference values for ir-Ang-(1-7) in human subjects (1.0-9.5 pmol/L of plasma) are only slightly higher than the 1.0 [+ or -] 0.7 pmol/L measured in human plasma after HPLC by Lawrence et al. (9). We also found lower plasma concentrations of Ang-(1-7) in supine volunteers (1.9 [+ or -] 1.6 pmol/L; range, 0.3-4.8 pmol/L; n = 9) after solid-phase extraction and HPLC as described previously for the specific measurement of angiotensin peptides (11), but modified by blood collection on additional renin inhibitor (12). Ferrario et al. (14) found plasma Ang-(1-7) concentrations of 22.9 [+ or -] 8.8 pmol/L in healthy volunteers. Botelho et al. (8) found plasma ir-Ang-(1-7) concentrations of 33 [+ or -] 20 ng/L in healthy male Wistar rats, which appear to be considerably higher than our human results. However, Campbell et al. (15) found Ang-(1-7) concentrations in plasma from male Sprague-Dawley rats that were fivefold higher than those found by Lawrence et al. (9) for human plasma. In addition to the species differences, sampling techniques and nonspecific cross-reactions may account for the higher values obtained by Botelho et al. (8). The specific cross-reactivities of the antisera appear comparable, with even slightly better specificity for the Brazilian antiserum (8).
Precision validation for the described new assay was performed at low Ang-(1-7) concentrations and provided results similar to or markedly better than established angiotensin measurements (11). Within the physiological range, the CV decreased from 20% to 4% when blood Ang-(1-7) concentrations increased from 4 to 27 fmol in 2-mL samples. The good precision is based on both a reliable extraction procedure with constant recoveries and efficient and rapid enzyme-inhibiting procedures that do not interfere with the radioimmunoassay by sensitive and specific antibodies. A clear linearity of Ang-(1-7) concentrations measured in serially diluted blood extracts confirmed the accuracy of the new method (Fig. 1A). Another indication of the accuracy of our method is provided by the dose-response curve of infused Ang-(1-7) in healthy volunteers (Fig. 1B), whose blood pressure and heart rates remained unchanged throughout the infusions. Very similar plasma Ang 11 concentrations were obtained when Ang-(1-8) was infused at the same rates in humans (16) and dogs (17). Interestingly, plasma Ang-(1-7) increased 2.8, 4.0, 4.2, and 3.7 pmol/L per pmol of Ang-(1-7) infused per kilogram per minute for infusion rates of 2, 4, 8, and 20 pmol*[kg.sup.-1]*[min.sup.-1], respectively [i.e., the smallest increase in plasma Ang-(1-7) occurred at the lowest infusion rate]. This would be compatible with a feedback suppression by Ang-(1-7) of renin secretion and, hence, a decrease of endogenous Ang-(1-7) during infusion of exogenous Ang-(1-7). Such a phenomenon was documented previously for endogenous and exogenous Ang-(1-8) (16), but not for Ang-(1-7). However, such an interpretation of our infusion results remains hypothetical because no renin measurements were performed in the present study.
In conclusion, we present a reliable assay for ir-Ang-(1-7) in human plasma based on efficient inhibition of enzymes that generate or degrade Ang-(1-7) after blood sampling, extraction in ethanol, and a sensitive and specific antiserum.
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JUERG NUSSBERGER,* DORETTE B. BRUNNER, JUERG A. NYFELER, LILLY LINDER, and HANS R. BRUNNER
Hypertension Division CHUV, CH-1011 Lausanne, Switzerland.
* Author for correspondence. Fax 41-21-314 0761; e-mail firstname.lastname@example.org.
Received October 20, 2000; accepted February 1, 2001.
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|Title Annotation:||Endocrinology and Metabolism|
|Author:||Nussberger, Juerg; Brunner, Dorette B.; Nyfeler, Juerg A.; Linder, Lilly; Brunner, Hans R.|
|Date:||Apr 1, 2001|
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