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A nonequilibrium radioimmunoassay for angiotensin II
JPM Vol. 32, No. 2 October 1994:93-97
A Nonequilibrium Radioimmunoassay for Angiotensin II Susan Paulson, Loukia Verhage, Diane Mayer, Krys Miller, and Grant Schoenhard Pharmacokinetics, Bioanalytical, and Radiochemistry, G.D. Searle & Co., Skokie, Illinois
A method for the measurement of angiotensin II levels in dog plasma is described. The method is similar to previously published assays in that it couples gradient high-performance liquid chromatography (HPLC) with radioimmunoassay (RIA) and requires blood sample collection and processing to plasma in the presence of protease inhibitors. The unique feature of the present method is that it utilized a commerically available angiotensin II RIA run under nonequilibrium conditions. Performing the angiotensin II RIA under nonequilibrium conditions increased RIA sensitivity to allow for a minimal detectable limit of 0.75 pg/mL, a limit of detection not achievable with current commercially available RIAs. This lower limit of detection will now allow for the measurement of circulating levels of angiotensin II. Quality control pools of dog plasma fortified with 4.59-50 pg/mL angiotensin II were assayed and analytical recoveries (ARs) and coefficients of variation (CV) of 72.2%-111% and 3.67%-19.0% were observed for the respective pools.
Keywords: Angiotensin II; Radioimmunoassay;Nonequilibrium.
Introduction Sensitive and specific methods for the quantitation of angiotensin II in human plasma have been described (Nussberger et al., 1985, 1986; Husain et al., 1987; Hermann et al., 1988). These assay methods couple gradient high-performance liquid chromatography (HPLC) with radioimmunoassay (RIA) and require blood sample collection and processing to plasma in the presence of protease inhibitors. The HPLC confers specificity on the assay allowing for the separation of angiotensin II from angiotensin I, angiotensin III, and other endogenous peptides that have the potential for crossreacting with angiotensin II antibodies. The presence of protease inhibitors during sample collection is necessary to prevent the metabolism of angiotensin II to angiotensin III and to prevent the formation of angiotensin II from angiotensin I. The angiotensin II assay method of Nussberger et al. (1985) is sensitive, with a minimal detectable limit of 0.4 pg/mL. However, the investigators used a source of Address reprint requests to Dr. Susan K. Paulson, Pharmacokinetics, Bioanalytical, and Radiochemistry, G.D. Searle & Co., 4901 Searle Parkway, Skokie, Illinois 60077. Received April 13, 1994; revised and accepted June 6, 1994. Journal of Pharmacological and Toxicological Methods 32, 93-97 (1994) © 1994 Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
angiotensin II antibody that is not commercially available. The minimal detection limits of commercially available angiotensin II RIA kits range from 2.5 to 7.5 pg/mL (Tempi and Templ, 1991). The present report describes a method for the measurement of angiotensin II in dog plasma that requires sample collection in the presence of protease inhibitors and also couples gradient HPLC with a commercially available Amersham Angiotensin II RIA kit. The minimal detectable limit of the assay was 0.75 pg/mL and this was achieved by performing the angiotensin II RIA under nonequilibrium conditions. This lower limit of detection will allow for the measurement of circulating low levels of angiotensin II.
Methods Blood Collection Procedure Approximately 3 mL of male Beagle dog blood were collected in ice-cold Na2EDTA Vacutainer tubes containing 150 txL of 0.025 M phenanthroline (Sigma Chemical Co., St. Louis, MO), 0.125 M Na2EDTA, 2 g/L neomycin in 2% EtOH, 30 txL 5 mM Bestatin (Sigma Chemical Co.), and 2.5 mM Chymostatin
1056-8719/94/$7.00
94
(Sigma Chemical Co.) in DMSO; and 12 txL of 0.5 M 8-hydroxyquinoline (Sigma Chemical Co.) and were chilled immediately on ice. Blood cells were separated from plasma by centrifugation at 4°C at 3000 rpm for 15 min. Plasma was fortified with 7 txL of 5% phenylmethyl sulfonyl fluoride (Sigma Chemical Co.) and stored at -20°C until analysis.
Solid-Phase Extraction (SPE) A 1-mL plasma sample was applied to an Analytichem Bond Elut PH 1-cc SPE column (Varian, Sunnyvale, CA) that was preconditioned with 1 mL methanol followed by two 1-mL washes with HPLC grade water (Baxter, McGaw Park, IL). The application of plasma to the SPE column was followed by two 1-mL washes with water and one 1-mL wash with dichloromethane (Baxter, McGaw Park, IL). Angiotensin II was eluted with 2 mL methanol (Baxter, McGaw Park, IL) into a 12 × 75 mm polypropylene culture tube. The methanol fraction was evaporated to dryness with the use of a Savant Speed Vac Concentrator (Savant, Hicksville, NY) and stored at -20°C until HPLC.
High-Performance Liquid Chromatography The dried extract was reconstituted in 200 p~L 0.025 M sodium phosphate, pH 7.8/MeOH (90:10, v:v) in preparation for C-18 reversed-phase gradient HPLC. HPLC was performed using two Waters Instruments 590 pumps (Waters Instruments, Milford, MA), a Waters Instruments Model 712 WISP autoinjector, Waters Instruments Temperature Control Module and Column Housing Module, an Applied Biosystems 783A Programmable Absorbance Detector (Applied Biosystems, Foster City, CA), and an ISCO Foxy fraction collector (ISCO, Lincoln, NE). Isolation of angiotensin II fractions was performed using a segmented gradient system on a Nova-Pak C-18 4 ~m 3.9 × 150 mm column (Millipore Corporation, Milford, MA) at 40°C and a flow rate of 1.0 mL/min. A Nova-Pak C-18 Guard Pak precolumn insert (Millipore Corporation, Milford, MA) and a Rheodyne in-line filter preceeded the analytical column in the system. The system began at 100% Mobile phase A (90:10, 0.025 M sodium phosphate, pH 7.8:MeOH, v:v) for 5 min followed by a linear gradient of 55% Mobile phase B (95:5, MeOH:H20). The system was kept at 55% mobile phase B for 5 min after which the column was washed with 100% mobile phase B. The column was re-equilibrated at 100% mobile-phase A prior to each injection. The buffered mobile phase was filtered through a 0.45 Ixm nylon filter (Schleicher & Schuell, Inc., Keene, NH) prior to use. The HPLC fraction containing the angiotensin II was collected (1.4 mL) into polypropylene culture tubes and evaporated to
JPM Vol. 32, No. 2 October 1994:93-97
dryness using a Savant Speed Vac Concentrator. Dried extracts were stored at -20°C until analysis by radioimmunoassay. The angiotensin II HPLC elution position was verified using approximately 100,000-200,000 dpm synthetic [3H]-angiotensin II (Amersham Corporation, Arlington Heights, IL). The [3H]angiotensin I! was injected onto the HPLC and 0.5-rain fractions were collected into 20-mL LSC vials. The tritium was quantirated by liquid scintillation spectroscopy using a TRACOR Mark III liquid scintillation counter (Analytic Inc., Elk Grove Village, IL).
Angiotensin H Radioimmunoassay Equilibrium angiotensin II radioimmunoassay was performed in 12 × 75 mm polypropylene culture tubes as described in the Amersham RIA procedure (Amersham Corporation, Arlington Heights, IL). A standard curve was prepared by serial dilution of synthetic angiotensin II from 200 to 0.75 pg/tube (Sigma Chemical Co). 30,000 dpm (3-[lesI]iodotyrosyl) Angiotensin II-5L-isoleucine) (Amersham Corporation, Arlington Heights, IL) in 50 ~L buffer (50 mM Tris/HC1, pH 7.5 containing 0.3% bovine serum albumin), 100 ~L angiotensin II rabbit antiserum and 100 txL assay buffer were added to the 12 × 75 mm polypropylene test tubes containing sample residues or standards. Contents of the tubes were incubated 15-18 h at 4°C. The reaction was terminated by the addition of 1 mL ice-cold dextrancharcoal suspension. Bound was separated from free ligand by decanting. Radioactivity was determined in a TM Analytic 2250 gammacounter (TM Analytic, Elk Grove Village, IL). Nonequilibrium angiotensin II RIA was performed similar to the equilibrium angiotensin II RIA except the initial 15- to 18-hr incubation at 4°C contained only angiotensin II rabbit antiserum and standards/sample extracts. This incubation was followed by a 5-h incubation of tube contents with 30,000 cpm 125I-angiotensin II at 4°C. The reaction was terminated by the addition of 1 mL ice-cold dextran-charcoal suspension. Bound ligand was separated from free ligand by decanting. Radioactivity was determined in a TM Analytic 2250 gammacounter (TM Analytic, Elk Grove Village, IL).
Statistical Analysis RIA standard curve data were fit to a hyperbolic model. The fit was implemented using the PROC N NLIN procedure in SAS. The minimal detectable limit of the RIA standard curves was defined as the quantity of peptide that displaced tracer 2 standard deviations from zero binding. RIA assay performance was assessed by determining the precision and accuracy of replicate analysis of analyte-fortified quality control (QC) pools.
s. PAULSONET AL. ANGIOTENSINII NONEQUILIBRIUMRIA
95
QC pools were prepared at indicated concentrations in plasma. Coefficient of variation (CV) was used as a measure of precision, and analytical recovery (AR) was used as a measure of accuracy. CV =
Standard Deviation x 100 Mean Result
0.090
0.025
ANG I1 ANG
0.020
[[I ^NG
1
[ 0.015
Measured Result AR = x 100 Amount of Analyte Added
0.010
Results
0.005
Sample Collection
0.000
During blood sample collection endogenous angiotensin II has the potential for metabolism to angiotensin III. In addition, new production of angiotensin II from endogenous angiotensin I by proteases present in plasma is possible. To prevent the loss or production of angiotensin II due to in vitro metabolism during sample processing, blood samples were collected in ice-cold tubes containing protease inhibitors as described in the Methods section. To test the effectiveness of this procedure, 3-mL aliquots of dog blood (n = 5) were collected into ice-cold vacutainer tubes and immediately spiked with approximately 200,000 dpm of [3H] angiotensin II. The [3H]angiotensin II-spiked blood samples (n = 5) were then fortified with the protease inhibitors described in the methods and subjected to SPE and HPLC. Recovery of [3H]-angiotensin II after the blood collection procedure, SPE and HPLC was 86.8% ___ 6.52% (Table 1). As a control, ice-cold plasma containing protease inhibitors was spiked with 200,000 dpm of [3H]-Angiotensin II and then immediately subjected to SPE and HPLC. Recovery of [3H]-angiotensin II from control dog plasma containing inhibitor solutions was 86.4%.
HPLC Figure 1 shows the baseline resolution of 100-ng standards of angiotensin I, angiotensin II, and angiotensin III by gradient C-18 reversed-phase HPLC.
0
5 Iime,
I0 minutes
15
20
Figure 1. HPLC chromatogram of Angiotensin I, II, and III standards.
Figure 2. The quantity of angiotensin II that significantly displaced radioligand two standard deviations from zero binding was 6.25 pg/assay tube for the equilibrium RIA and 0.78 pg/assay tube for the nonequilibrium RIA (Table 2). Therefore, minimal detectable limit of the Amersham Angiotensin II RIA was decreased eightfold by performing the assay under nonequilibrium conditions. The assay range of the equilibrium and nonequilibrium Ang II RIAs were 6.25-200 pg/assay tube and 0.78-200 pg/assay tube, respectively.
Angiotensin H in Dog Plasma Plasma angiotensin II levels in dog plasma fortified with known concentrations of angiotensin II are shown in Table 3. The accuracy of the measurement of angiotensin II in dog plasma fortified with 4.59-50.0 pg/mL angiotensin II ranged from 72.2% to 111%. The precision (CV) of the measurement of angiotensin II in dog plasma fortified with 4.59-50.0 pg/mL angiotensin II ranged from 3.67% to 19.0%.
Discussion
Angiotensin H Radioimmunoassay Standard curves for the equilibrium and nonequilibrium angiotensin II radioimmunoassays are shown in Table 1. Recovery of [3H]-Angiotensin II from Dog Blood
Samples Subjected to a Blood Collection Procedure, SPE, and HPLC Described in the Methods Sample Description [3H]Angiotensin II Blood Samples [3H]Angiotensin II Spiked Plasma
No. of Samples
Recovery % (Mean + SD)
5 2
86.8 + 6.52 83.4, 89.4
The measurement of angiotensin II in plasma is accompanied by many challenges. First, circulating levels of the hormone are very low and assay sensitivity must be in the fentomolar range (Nussberger et al., 1985). In addition, angiotensin II must be separated from other peptides in plasma that have similar immunoreactivity (Nussberger et al., 1985; Hermann et al., 1988; Husaid et al., 1987). And, because of the presence of proteases capable of metabolizing the angiotensin peptides, sample collection procedures must be developed that include protease inhibitors during sample collection and processing (Nussberger et al., 1985). Therefore, current assay
96
JPM Vol. 32, No. 2 October 1994:93-97
120
T a b l e 2. A n g i o t e n s i n II E q u i l i b r i u m and N o n e q u i l i b r i u m R I A
Standard C u r v e s (Concentration o f L i g a n d v e r s u s B/Bo%) Angiotensin II Standard (pg/tube) 100
o m
0 0.39 0.78 1.56 3.13 6.25 12.5 25 50 100 200 400
20
11~ o
Nonequilibrium RIA (B/B 0, %) Mean _+ SD
80
80
m
Equilibrium RIA (B/B 0, %) Mean + SD
Anglotensln
II,
pgltube
'°llt
100 + 0 ND ND 96.6 _+ 3.82 92.7 + 3.89 83.3 + 4.19" 71.5 _+ 1.73 56.3 + 3.10 40.9 + 2.64 26.0 +- 1.90 15.4 +- 1.15 ND
100 94.7 93.2 88.4 77.3 59.4 39.4 24.7 15.7 8.84 4.96 2.80
+ 0 + 3.2(/ + 1.28" _+ 2.07 _+ 1.69 + 1.44 + 2.38 + 1.13 + 1.65 -+ 0.261 -+ 0.550 + 0.216
"Designates the angiotensin concentration that displaces radioligand two standard deviations from zero binding. Abbreviations." ND, not done.
- - -o-.-
40
'
0
~
"
1 O0
NoileeluilibnumRIA equdibnumRIA
200
300
F i g u r e 2, A n g i o t e n s i n II N o n e q u i l i b r i u m and E q u i l i b r i u m R I A Standard Curves. (B) represents the a m o u n t o f radiolabeled ang i o t e n s i n II bound. Bo represents radiolabeled a n g i o t e n s i n II b o u n d in the p r e s e n c e o f zero concentration o f unlabeled angiotensin II.
methods for angiotensin II in plasma couple a separation procedure (HPLC) with a highly sensitive RIA and include protease inhibitors during sample collection and processing. Such a sensitive and specific method for the quantitation of angiotensin II in plasma was developed by Nussberger et al. (1985). The method reported here is similar to previously published assays in that it couples a separation procedure (HPLC) with a highly sensitive RIA. And, as other investigators observed (Nussberger et al., 1985; Hermann et al., 1988; Husain et al., 1987), we also found that angiotensin II was stable during blood sample processing. The disadvantage of current highly sensitive angiotensin II assay methods (Nussberger, 1985) is that the antibody utilized is not commercially available. In the present report a modification of a commercially available angiotensin II RIA is described that allows for the measurement of angiotensin II in the fentomolar range. This low limit of detection will allow for the measurement of low circulating levels of the hormone. This
improvement in angiotensin II RIA sensitivity was obtained by performing the assay under nonequilibrium conditions. Using nonequilibrium conditions the Amersham angiotensin II RIA sensitivity was increased eightfold. The nonequilibrium conditions were obtained by using sequential addition of ligand and radioligand. The use of nonequilibrium conditions to improved sensitivity of RIAs is an established technique that has been successful for other ligands (Rodbard et al., 1971; Zettner and Duly, 1974). Another modification of previous angiotensin II assays (Nussberger et al., 1985; Hermann et al., 1988)
T a b l e 3, P l a s m a A n g i o t e n s i n II L e v e l s in Control D o g P l a s m a Pools and D o g P l a s m a Pools Fortified with a K n o w n C o n c e n t r a t i o n o f A n g i o t e n s i n II Sample Description
n A.
Angiotensin II (pg/mL)
SD
CV (%)
AR (%)
0.64
10.7
NA
Experiment I
Control plasma
6
Control plasma + 4.59 pg/mL Angiotensin II
6
10.8
0.72
Control plasma + 12.45 pg/mL Angiotensin II
5
15.0
2.85
B.
5.99
6.63 19.0
105 72.2
Experiment II
Control plasma
3
ll.4
0.42
3.67
NA
Control plasma + 5 pg/mL Angiotensin II
3
15.4
1.29
8.39
80.1
Control plasma + 50 pg/mL Angiotensin II
3
62.1
6.65
Dilution QC Pool control plasma + 50 pg/mL Angiotensin II
3
66.8
2.92
Abbreviations: NA, not applicable.
10.7
4.37
101
111
S. PAULSON ET AL. ANGIOTENSIN II NONEQUILIBRIUM RIA
used in the present assay was the addition of a chloroform wash prior to elution of the analyte in the SPE procedure. The chloroform wash eliminated a contamination that cross-reacted with the angiotensin antibody (data not shown). In addition, the chloroform was provided for a cleaner sample free of particulates to inject onto an HPLC system. In conclusion, a method for measurement of angiotensin II in the fentomolar range using a commercial RIA kit is reported.
References Hermann K, Ganten D, Unger T, Bayer C, Lang R E (1988) Measurement and characterization of angiotensin peptides in plasma. Clin Chem 34(6): 1046-1051.
97
Husain A, DeSilva P, Speth R, Merlin Bumpus R (1987) Regulation of angiotensin II in rat adrenal gland. Circ Res 60:64~648. Nussberger J, Brunner DB, Weber B, Brunner R (1985) True versus immunoreactive angiotensin II in human plasma. Hypertension 7(Supple I):I- 1-1-7. Nussberger J, Brunner D, Waeber B, Brunner H (1986) Specific measurement of Angiotensin metabolites and in vitro generated Angiotensin II in plasma. Hypertension 8:476~82. Rodbard D, Ruder H J, Vaitukaitis J, Jacobs H S (1971) Mathematical analysis of kinetics of radioligand assays: Improved sensitivity obtained by delayed addition of labeled ligand. J Clin Endocr 33:343355. SAS Institute Inc. (1985) SAS User Guide, Version 5 Edition, SAS Institute Inc., City. Tempi E, Templ H (1991) Quantitation of circulating Angiotensin II using commercial immunoassays. Horm Res 36:78-79. Zettner A, Duly P E (1974) Principles of competitive binding assays (saturation analyses). II. Sequential saturation. Clin Chem 20:5-14.
A Nonequilibrium Radioimmunoassay for Angiotensin II Susan Paulson, Loukia Verhage, Diane Mayer, Krys Miller, and Grant Schoenhard Pharmacokinetics, Bioanalytical, and Radiochemistry, G.D. Searle & Co., Skokie, Illinois
A method for the measurement of angiotensin II levels in dog plasma is described. The method is similar to previously published assays in that it couples gradient high-performance liquid chromatography (HPLC) with radioimmunoassay (RIA) and requires blood sample collection and processing to plasma in the presence of protease inhibitors. The unique feature of the present method is that it utilized a commerically available angiotensin II RIA run under nonequilibrium conditions. Performing the angiotensin II RIA under nonequilibrium conditions increased RIA sensitivity to allow for a minimal detectable limit of 0.75 pg/mL, a limit of detection not achievable with current commercially available RIAs. This lower limit of detection will now allow for the measurement of circulating levels of angiotensin II. Quality control pools of dog plasma fortified with 4.59-50 pg/mL angiotensin II were assayed and analytical recoveries (ARs) and coefficients of variation (CV) of 72.2%-111% and 3.67%-19.0% were observed for the respective pools.
Keywords: Angiotensin II; Radioimmunoassay;Nonequilibrium.
Introduction Sensitive and specific methods for the quantitation of angiotensin II in human plasma have been described (Nussberger et al., 1985, 1986; Husain et al., 1987; Hermann et al., 1988). These assay methods couple gradient high-performance liquid chromatography (HPLC) with radioimmunoassay (RIA) and require blood sample collection and processing to plasma in the presence of protease inhibitors. The HPLC confers specificity on the assay allowing for the separation of angiotensin II from angiotensin I, angiotensin III, and other endogenous peptides that have the potential for crossreacting with angiotensin II antibodies. The presence of protease inhibitors during sample collection is necessary to prevent the metabolism of angiotensin II to angiotensin III and to prevent the formation of angiotensin II from angiotensin I. The angiotensin II assay method of Nussberger et al. (1985) is sensitive, with a minimal detectable limit of 0.4 pg/mL. However, the investigators used a source of Address reprint requests to Dr. Susan K. Paulson, Pharmacokinetics, Bioanalytical, and Radiochemistry, G.D. Searle & Co., 4901 Searle Parkway, Skokie, Illinois 60077. Received April 13, 1994; revised and accepted June 6, 1994. Journal of Pharmacological and Toxicological Methods 32, 93-97 (1994) © 1994 Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
angiotensin II antibody that is not commercially available. The minimal detection limits of commercially available angiotensin II RIA kits range from 2.5 to 7.5 pg/mL (Tempi and Templ, 1991). The present report describes a method for the measurement of angiotensin II in dog plasma that requires sample collection in the presence of protease inhibitors and also couples gradient HPLC with a commercially available Amersham Angiotensin II RIA kit. The minimal detectable limit of the assay was 0.75 pg/mL and this was achieved by performing the angiotensin II RIA under nonequilibrium conditions. This lower limit of detection will allow for the measurement of circulating low levels of angiotensin II.
Methods Blood Collection Procedure Approximately 3 mL of male Beagle dog blood were collected in ice-cold Na2EDTA Vacutainer tubes containing 150 txL of 0.025 M phenanthroline (Sigma Chemical Co., St. Louis, MO), 0.125 M Na2EDTA, 2 g/L neomycin in 2% EtOH, 30 txL 5 mM Bestatin (Sigma Chemical Co.), and 2.5 mM Chymostatin
1056-8719/94/$7.00
94
(Sigma Chemical Co.) in DMSO; and 12 txL of 0.5 M 8-hydroxyquinoline (Sigma Chemical Co.) and were chilled immediately on ice. Blood cells were separated from plasma by centrifugation at 4°C at 3000 rpm for 15 min. Plasma was fortified with 7 txL of 5% phenylmethyl sulfonyl fluoride (Sigma Chemical Co.) and stored at -20°C until analysis.
Solid-Phase Extraction (SPE) A 1-mL plasma sample was applied to an Analytichem Bond Elut PH 1-cc SPE column (Varian, Sunnyvale, CA) that was preconditioned with 1 mL methanol followed by two 1-mL washes with HPLC grade water (Baxter, McGaw Park, IL). The application of plasma to the SPE column was followed by two 1-mL washes with water and one 1-mL wash with dichloromethane (Baxter, McGaw Park, IL). Angiotensin II was eluted with 2 mL methanol (Baxter, McGaw Park, IL) into a 12 × 75 mm polypropylene culture tube. The methanol fraction was evaporated to dryness with the use of a Savant Speed Vac Concentrator (Savant, Hicksville, NY) and stored at -20°C until HPLC.
High-Performance Liquid Chromatography The dried extract was reconstituted in 200 p~L 0.025 M sodium phosphate, pH 7.8/MeOH (90:10, v:v) in preparation for C-18 reversed-phase gradient HPLC. HPLC was performed using two Waters Instruments 590 pumps (Waters Instruments, Milford, MA), a Waters Instruments Model 712 WISP autoinjector, Waters Instruments Temperature Control Module and Column Housing Module, an Applied Biosystems 783A Programmable Absorbance Detector (Applied Biosystems, Foster City, CA), and an ISCO Foxy fraction collector (ISCO, Lincoln, NE). Isolation of angiotensin II fractions was performed using a segmented gradient system on a Nova-Pak C-18 4 ~m 3.9 × 150 mm column (Millipore Corporation, Milford, MA) at 40°C and a flow rate of 1.0 mL/min. A Nova-Pak C-18 Guard Pak precolumn insert (Millipore Corporation, Milford, MA) and a Rheodyne in-line filter preceeded the analytical column in the system. The system began at 100% Mobile phase A (90:10, 0.025 M sodium phosphate, pH 7.8:MeOH, v:v) for 5 min followed by a linear gradient of 55% Mobile phase B (95:5, MeOH:H20). The system was kept at 55% mobile phase B for 5 min after which the column was washed with 100% mobile phase B. The column was re-equilibrated at 100% mobile-phase A prior to each injection. The buffered mobile phase was filtered through a 0.45 Ixm nylon filter (Schleicher & Schuell, Inc., Keene, NH) prior to use. The HPLC fraction containing the angiotensin II was collected (1.4 mL) into polypropylene culture tubes and evaporated to
JPM Vol. 32, No. 2 October 1994:93-97
dryness using a Savant Speed Vac Concentrator. Dried extracts were stored at -20°C until analysis by radioimmunoassay. The angiotensin II HPLC elution position was verified using approximately 100,000-200,000 dpm synthetic [3H]-angiotensin II (Amersham Corporation, Arlington Heights, IL). The [3H]angiotensin I! was injected onto the HPLC and 0.5-rain fractions were collected into 20-mL LSC vials. The tritium was quantirated by liquid scintillation spectroscopy using a TRACOR Mark III liquid scintillation counter (Analytic Inc., Elk Grove Village, IL).
Angiotensin H Radioimmunoassay Equilibrium angiotensin II radioimmunoassay was performed in 12 × 75 mm polypropylene culture tubes as described in the Amersham RIA procedure (Amersham Corporation, Arlington Heights, IL). A standard curve was prepared by serial dilution of synthetic angiotensin II from 200 to 0.75 pg/tube (Sigma Chemical Co). 30,000 dpm (3-[lesI]iodotyrosyl) Angiotensin II-5L-isoleucine) (Amersham Corporation, Arlington Heights, IL) in 50 ~L buffer (50 mM Tris/HC1, pH 7.5 containing 0.3% bovine serum albumin), 100 ~L angiotensin II rabbit antiserum and 100 txL assay buffer were added to the 12 × 75 mm polypropylene test tubes containing sample residues or standards. Contents of the tubes were incubated 15-18 h at 4°C. The reaction was terminated by the addition of 1 mL ice-cold dextrancharcoal suspension. Bound was separated from free ligand by decanting. Radioactivity was determined in a TM Analytic 2250 gammacounter (TM Analytic, Elk Grove Village, IL). Nonequilibrium angiotensin II RIA was performed similar to the equilibrium angiotensin II RIA except the initial 15- to 18-hr incubation at 4°C contained only angiotensin II rabbit antiserum and standards/sample extracts. This incubation was followed by a 5-h incubation of tube contents with 30,000 cpm 125I-angiotensin II at 4°C. The reaction was terminated by the addition of 1 mL ice-cold dextran-charcoal suspension. Bound ligand was separated from free ligand by decanting. Radioactivity was determined in a TM Analytic 2250 gammacounter (TM Analytic, Elk Grove Village, IL).
Statistical Analysis RIA standard curve data were fit to a hyperbolic model. The fit was implemented using the PROC N NLIN procedure in SAS. The minimal detectable limit of the RIA standard curves was defined as the quantity of peptide that displaced tracer 2 standard deviations from zero binding. RIA assay performance was assessed by determining the precision and accuracy of replicate analysis of analyte-fortified quality control (QC) pools.
s. PAULSONET AL. ANGIOTENSINII NONEQUILIBRIUMRIA
95
QC pools were prepared at indicated concentrations in plasma. Coefficient of variation (CV) was used as a measure of precision, and analytical recovery (AR) was used as a measure of accuracy. CV =
Standard Deviation x 100 Mean Result
0.090
0.025
ANG I1 ANG
0.020
[[I ^NG
1
[ 0.015
Measured Result AR = x 100 Amount of Analyte Added
0.010
Results
0.005
Sample Collection
0.000
During blood sample collection endogenous angiotensin II has the potential for metabolism to angiotensin III. In addition, new production of angiotensin II from endogenous angiotensin I by proteases present in plasma is possible. To prevent the loss or production of angiotensin II due to in vitro metabolism during sample processing, blood samples were collected in ice-cold tubes containing protease inhibitors as described in the Methods section. To test the effectiveness of this procedure, 3-mL aliquots of dog blood (n = 5) were collected into ice-cold vacutainer tubes and immediately spiked with approximately 200,000 dpm of [3H] angiotensin II. The [3H]angiotensin II-spiked blood samples (n = 5) were then fortified with the protease inhibitors described in the methods and subjected to SPE and HPLC. Recovery of [3H]-angiotensin II after the blood collection procedure, SPE and HPLC was 86.8% ___ 6.52% (Table 1). As a control, ice-cold plasma containing protease inhibitors was spiked with 200,000 dpm of [3H]-Angiotensin II and then immediately subjected to SPE and HPLC. Recovery of [3H]-angiotensin II from control dog plasma containing inhibitor solutions was 86.4%.
HPLC Figure 1 shows the baseline resolution of 100-ng standards of angiotensin I, angiotensin II, and angiotensin III by gradient C-18 reversed-phase HPLC.
0
5 Iime,
I0 minutes
15
20
Figure 1. HPLC chromatogram of Angiotensin I, II, and III standards.
Figure 2. The quantity of angiotensin II that significantly displaced radioligand two standard deviations from zero binding was 6.25 pg/assay tube for the equilibrium RIA and 0.78 pg/assay tube for the nonequilibrium RIA (Table 2). Therefore, minimal detectable limit of the Amersham Angiotensin II RIA was decreased eightfold by performing the assay under nonequilibrium conditions. The assay range of the equilibrium and nonequilibrium Ang II RIAs were 6.25-200 pg/assay tube and 0.78-200 pg/assay tube, respectively.
Angiotensin H in Dog Plasma Plasma angiotensin II levels in dog plasma fortified with known concentrations of angiotensin II are shown in Table 3. The accuracy of the measurement of angiotensin II in dog plasma fortified with 4.59-50.0 pg/mL angiotensin II ranged from 72.2% to 111%. The precision (CV) of the measurement of angiotensin II in dog plasma fortified with 4.59-50.0 pg/mL angiotensin II ranged from 3.67% to 19.0%.
Discussion
Angiotensin H Radioimmunoassay Standard curves for the equilibrium and nonequilibrium angiotensin II radioimmunoassays are shown in Table 1. Recovery of [3H]-Angiotensin II from Dog Blood
Samples Subjected to a Blood Collection Procedure, SPE, and HPLC Described in the Methods Sample Description [3H]Angiotensin II Blood Samples [3H]Angiotensin II Spiked Plasma
No. of Samples
Recovery % (Mean + SD)
5 2
86.8 + 6.52 83.4, 89.4
The measurement of angiotensin II in plasma is accompanied by many challenges. First, circulating levels of the hormone are very low and assay sensitivity must be in the fentomolar range (Nussberger et al., 1985). In addition, angiotensin II must be separated from other peptides in plasma that have similar immunoreactivity (Nussberger et al., 1985; Hermann et al., 1988; Husaid et al., 1987). And, because of the presence of proteases capable of metabolizing the angiotensin peptides, sample collection procedures must be developed that include protease inhibitors during sample collection and processing (Nussberger et al., 1985). Therefore, current assay
96
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120
T a b l e 2. A n g i o t e n s i n II E q u i l i b r i u m and N o n e q u i l i b r i u m R I A
Standard C u r v e s (Concentration o f L i g a n d v e r s u s B/Bo%) Angiotensin II Standard (pg/tube) 100
o m
0 0.39 0.78 1.56 3.13 6.25 12.5 25 50 100 200 400
20
11~ o
Nonequilibrium RIA (B/B 0, %) Mean _+ SD
80
80
m
Equilibrium RIA (B/B 0, %) Mean + SD
Anglotensln
II,
pgltube
'°llt
100 + 0 ND ND 96.6 _+ 3.82 92.7 + 3.89 83.3 + 4.19" 71.5 _+ 1.73 56.3 + 3.10 40.9 + 2.64 26.0 +- 1.90 15.4 +- 1.15 ND
100 94.7 93.2 88.4 77.3 59.4 39.4 24.7 15.7 8.84 4.96 2.80
+ 0 + 3.2(/ + 1.28" _+ 2.07 _+ 1.69 + 1.44 + 2.38 + 1.13 + 1.65 -+ 0.261 -+ 0.550 + 0.216
"Designates the angiotensin concentration that displaces radioligand two standard deviations from zero binding. Abbreviations." ND, not done.
- - -o-.-
40
'
0
~
"
1 O0
NoileeluilibnumRIA equdibnumRIA
200
300
F i g u r e 2, A n g i o t e n s i n II N o n e q u i l i b r i u m and E q u i l i b r i u m R I A Standard Curves. (B) represents the a m o u n t o f radiolabeled ang i o t e n s i n II bound. Bo represents radiolabeled a n g i o t e n s i n II b o u n d in the p r e s e n c e o f zero concentration o f unlabeled angiotensin II.
methods for angiotensin II in plasma couple a separation procedure (HPLC) with a highly sensitive RIA and include protease inhibitors during sample collection and processing. Such a sensitive and specific method for the quantitation of angiotensin II in plasma was developed by Nussberger et al. (1985). The method reported here is similar to previously published assays in that it couples a separation procedure (HPLC) with a highly sensitive RIA. And, as other investigators observed (Nussberger et al., 1985; Hermann et al., 1988; Husain et al., 1987), we also found that angiotensin II was stable during blood sample processing. The disadvantage of current highly sensitive angiotensin II assay methods (Nussberger, 1985) is that the antibody utilized is not commercially available. In the present report a modification of a commercially available angiotensin II RIA is described that allows for the measurement of angiotensin II in the fentomolar range. This low limit of detection will allow for the measurement of low circulating levels of the hormone. This
improvement in angiotensin II RIA sensitivity was obtained by performing the assay under nonequilibrium conditions. Using nonequilibrium conditions the Amersham angiotensin II RIA sensitivity was increased eightfold. The nonequilibrium conditions were obtained by using sequential addition of ligand and radioligand. The use of nonequilibrium conditions to improved sensitivity of RIAs is an established technique that has been successful for other ligands (Rodbard et al., 1971; Zettner and Duly, 1974). Another modification of previous angiotensin II assays (Nussberger et al., 1985; Hermann et al., 1988)
T a b l e 3, P l a s m a A n g i o t e n s i n II L e v e l s in Control D o g P l a s m a Pools and D o g P l a s m a Pools Fortified with a K n o w n C o n c e n t r a t i o n o f A n g i o t e n s i n II Sample Description
n A.
Angiotensin II (pg/mL)
SD
CV (%)
AR (%)
0.64
10.7
NA
Experiment I
Control plasma
6
Control plasma + 4.59 pg/mL Angiotensin II
6
10.8
0.72
Control plasma + 12.45 pg/mL Angiotensin II
5
15.0
2.85
B.
5.99
6.63 19.0
105 72.2
Experiment II
Control plasma
3
ll.4
0.42
3.67
NA
Control plasma + 5 pg/mL Angiotensin II
3
15.4
1.29
8.39
80.1
Control plasma + 50 pg/mL Angiotensin II
3
62.1
6.65
Dilution QC Pool control plasma + 50 pg/mL Angiotensin II
3
66.8
2.92
Abbreviations: NA, not applicable.
10.7
4.37
101
111
S. PAULSON ET AL. ANGIOTENSIN II NONEQUILIBRIUM RIA
used in the present assay was the addition of a chloroform wash prior to elution of the analyte in the SPE procedure. The chloroform wash eliminated a contamination that cross-reacted with the angiotensin antibody (data not shown). In addition, the chloroform was provided for a cleaner sample free of particulates to inject onto an HPLC system. In conclusion, a method for measurement of angiotensin II in the fentomolar range using a commercial RIA kit is reported.
References Hermann K, Ganten D, Unger T, Bayer C, Lang R E (1988) Measurement and characterization of angiotensin peptides in plasma. Clin Chem 34(6): 1046-1051.
97
Husain A, DeSilva P, Speth R, Merlin Bumpus R (1987) Regulation of angiotensin II in rat adrenal gland. Circ Res 60:64~648. Nussberger J, Brunner DB, Weber B, Brunner R (1985) True versus immunoreactive angiotensin II in human plasma. Hypertension 7(Supple I):I- 1-1-7. Nussberger J, Brunner D, Waeber B, Brunner H (1986) Specific measurement of Angiotensin metabolites and in vitro generated Angiotensin II in plasma. Hypertension 8:476~82. Rodbard D, Ruder H J, Vaitukaitis J, Jacobs H S (1971) Mathematical analysis of kinetics of radioligand assays: Improved sensitivity obtained by delayed addition of labeled ligand. J Clin Endocr 33:343355. SAS Institute Inc. (1985) SAS User Guide, Version 5 Edition, SAS Institute Inc., City. Tempi E, Templ H (1991) Quantitation of circulating Angiotensin II using commercial immunoassays. Horm Res 36:78-79. Zettner A, Duly P E (1974) Principles of competitive binding assays (saturation analyses). II. Sequential saturation. Clin Chem 20:5-14.