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[19] Strategies for measurement of angiotensin and bradykinin peptides and their metabolites in central nervous system and other tissues
[19]
Strategies for Measurement of Angiotensin and Bradykinin Peptides and Their Metabolites in Central Nervous System and Other Tissues Duncan J. Campbell, Anne C. Lawrence, Athena Kladis, and Ann-Maree Duncan
Introduction Whether angiotensin and bradykinin are neuropeptides is a subject of continuing debate. The strength of the evidence for or against such a proposition is dependent on the methodological basis for such evidence. Rather than address this issue directly, in this chapter we describe some of the methodologies we have developed for the measurement of angiotensin and bradykinin peptides and their metabolites in the central nervous system (CNS) and other tissues. In the past, radioimmunoassay (RIA) of angiotensin and bradykinin peptides was based on the use of carboxy (C) terminal-directed antisera. This was due in large part to the ease with which a peptide may be coupled via its amino (N) terminus to carrier proteins for the purpose of immunization. However, for both angiotensin and bradykinin peptides important processing events take place toward the C terminus of the molecule (1-3)- (Figs. 1 and 2). For example, the decapeptide angiotensin I (Ang I) is cleaved between residues 8 and 9 by angiotensin-converting enzyme (ACE, kininase II, EC 3.4.15.1, peptidyl-dipeptidase A) to release angiotensin II (Ang II), and both Ang I and Ang II are cleaved between residues 7 and 8 by a number of endopeptidases to release angiotensin(1-7) [Ang(1-7)]. Both Ang II and Ang(1-7) are bioactive. An alternative pathway of conversion of Ang I to Ang II may involve the sequential cleavage of the two C-terminal residues of Ang I by carboxypeptidase activity (1). Moreover, the nonapeptide bradykinin(1-9) [BK(1-9)] is cleaved between residues 8 and 9 by carboxypeptidases N (kininase I) and M to release bradykinin(1-8) [BK(1-8)], and between residues 7 and 8 by ACE and other endopeptidases to release bradykinin(1-7) [BK(1-7)]. Both BK(1-9) and BK(1-8) are bioactive. When these differentially processed peptides are separated by high-performance liquid chromatography (HPLC), it is of assistance if the peptides of interest 328
Methods in Neurosciences, Volume 23 Copyright 9 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
[19] MEASUREMENTOF ANGIOTENSIN AND BRADYKININ
Mast cell chymase Neutrophil cathepsin G
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Carboxypeptidases Prolylendopeptidase
FIG. 1 Diagrammatic representation of cleavage sites of angiotensin I by different enzymes. Both endopeptidases 24.11 and 24.15 cleave angiotensin I between residues 4 and 5, and between residues 7 and 8; in addition, endopeptidase 24.11 cleaves angiotensin I between residues 2 and 3. After removal of the amino-terminal aspartic acid by aminopeptidase A, the Arg2 residue can be cleaved by aminopeptidase N. For angiotensin II, the sites of cleavage by endopeptidases are the same as those shown for angiotensin I, except that endopeptidases 24.11 and 24.15 do not cleave between residues 7 and 8 of angiotensin II. ACE, Angiotensin-converting enzyme.
can be measured with the same RIA. To this end, we established N terminaldirected RIA for the measurement of angiotensin and bradykinin peptides and their C-terminal truncated metabolites. In previous attempts to raise N terminal-directed antisera to angiotensin peptides, although the peptides were coupled to the carrier protein via the C terminus, the antisera raised were predominantly directed to the C terminus (4, 5). However, Nussberger et al. (4) found that when Asn ~, VaP-Ang II was acetylated at the N terminus and coupled via the C terminus for immunization, they readily achieved N terminal-directed antisera. This result suggests that acetylation of the N terminus of a peptide renders the N terminus more immunogenic. We used this approach to raise N terminal-directed antisera against N-acetylated angiotensin and bradykinin peptide analogs
Endopeptidase24.15
KininaseII (ACE)
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FIG. 2 Diagrammatic representation of cleavage sites of bradykinin by different enzymes. ACE, Angiotensin-converting enzyme.
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(3, 6), with the intention of acetylating peptides extracted from biological samples before RIA with these antisera.
Preparation of Antisera For the preparation of antisera directed against the N terminus of Ang II, angiotensin III (Ang III), and B K(1-9), the following peptides are synthesized: N-acetyl-AspArgValTyrIleHisProPheLys (N-Ac-Lysg-Ang II), N-acetyl-ArgValTyrIleHisProPheLys (N-Ac-Lys8-Ang III), and N-acetyl-ArgProProGlyPheSerProPheLys [N-Ac-Lys9-BK(1-9)]. Peptides are synthesized from tert-butoxycarbonyl-protected amino acids, using an Applied Biosystems (Foster City, CA) 430A automated peptide synthesizer. Acetylation of a-amino groups of Lysg-Ang II, Lys8-Ang III, and Lys9-BK(1-9) is performed on the protected resin before hydrogen fluoride treatment (6). Peptides are coupled to bovine thyroglobulin via the C-terminal lysine residue with glutaraldehyde (7), and antisera are raised in rabbits (8). Six rabbits are immunized with each peptide and the best antiserum against each peptide is subsequently used to establish an RIA.
Description of Radioimmunoassays All peptide concentrations are determined by amino acid analysis, using stocks of approximately 1 mg/ml in 20% (v/v) acetic acid in water, and stored at -30~ Working solutions [1 /~M in lysozyme (1 mg/ml), 10 mM acetic acid] are stored at -30~ and discarded after thawing once. All RIA components are diluted with casein phosphate buffer [casein (1 g/liter), 100 mM sodium phosphate, 10 mM disodium ethylenediaminetetraacetic acid (EDTA), sodium azide (1 g/liter), 154 mM sodium chloride, pH 7.0]. A pH optimum of 7.0 has been shown for each of the three assays described. Initially, the total RIA assay volume was 500 /A (6), but this has since been reduced to 250/~1 to increase sensitivity. Although initially prepared on ice, assays are now prepared at room temperature. Each assay tube contains 50/A of diluted antibody, 50/~1 of tracer (---2500 cpm), 50/~1 of standard or unknown peptide solution, and 100/~1 of buffer. Usually the assays are incubated at 4~ for 48 hr before separation of free from bound radioactivity. For the antibody A41 assay, addition of tracer is delayed t~or 48 hr, and the assay is incubated at 4~ for a further 24 hr before separation of free from bound radioactivity. Separation of free from bound radioactivity with albumin/dextran-coated charcoal is performed using a modification (9) of the method described by
[19] MEASUREMENT OF ANGIOTENSIN AND BRADYKININ
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Herbert et al. (10). Stock dextran-coated charcoal [Norit A charcoal (25 g/liter), dextran T10 (2.5 g/liter), 7.1 mM sodium barbitone, 7.1 mM sodium acetate, adjusted to pH ---7.4 with hydrochloric acid] is stirred with bovine serum albumin (BSA, 10 mg/ml) for 1-24 hr at 4~ and then diluted with 4 vol of 150 mM sodium chloride immediately before use. One milliliter of albumin/dextran-coated charcoal is added to each tube at 4~ and, after standing at 4~ for 10 min, the assay tubes are centrifuged at 5000 g for 10 min at 4~ the supernatants rapidly aspirated, and the charcoal pellets counted. Tracer peptides are iodinated with 125I using chloramine-T (11), and the monoiodinated peptides are purified by HPLC on a C18 column, using a gradient of acetonitrile in 0.1% (v/v) trifluoroacetic acid (TFA), and stored in aliquots at -30~ Tracer peptides can be stored for up to 2 months without deterioration in assay performance. Antibody A41 was raised against N-Ac-Lys9-Ang II. The antibody A41 assay uses N, O-diacetyl-Ang II (Ac-Ang II, acetylated as described below) as standard peptide and mono[~ZSI]iodo-Ac-Ang II as tracer. At a dilution of 1 : 270,000, binding of tracer is approximately 50%, and 50% displacement is obtained with ---8 fmol of Ac-Ang II/tube, with a detection limit of ---0.25 fmol/tube. The within-assay coefficient of variation is 6% and the betweenassay coefficient of variation is 19%. Antibody A41 was initially studied using N-Ac-Lys9-Ang II as standard and 125I-labeled N-Ac-Lys9-Ang II as tracer; however, displacement of 125I-labeled N-Ac-Lys9-Ang II by N-AcLys9-Ang II and Ac-Ang II was not superimposable, with incomplete displacement by Ac-Ang II, indicating that a proportion of the antibody population of A41 was specific for N-Ac-Lys9-Ang II. Consequently, N-Ac-Lys 9Ang II cannot be used as standard for the measurement of Ang II levels in biological samples; instead, Ac-Ang II must be used as standard. The use of 125I-labeled Ac-Ang II as tracer has the advantage that Ac-Ang II produces complete displacement of tracer. Antibody A52 was raised against N-Ac-Lysg-Ang III. The antibody A52 assay uses N-Ac-LysS-Ang III as standard peptide and 125I-labeled N-AcLysS-Ang III as tracer. At a dilution of 1 : 48,500, binding of tracer is approximately 50%; assays with antibody A52 have been performed using a total assay volume of only 500 ~1, and 50% displacement is obtained with --~16 fmol of N-Ac-LysS-Ang III/tube, with a detection limit of ---1.0 fmol/tube. The between-assay coefficient of variation is 12%. In contrast to the antibody A41 assay, displacement of 125I-labeled N-Ac-Lys8-Ang III by N-Ac-Lys 9Ang III is identical to that produced by N,O-diacetyl-Ang III (Ac-Ang III), and this assay can be used to measure Ang III in biological samples. Antibody B24 was raised against N-Ac-Lysg-BK(1-9). The antibody B24 assay uses N-Ac-Lys9-BK(1-9) as standard peptide. 125I-Labeled Tyr 8BK(1-9) is acetylated as described below before purification by HPLC, and
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mono[125I]iodo-Ac-Tyr8-BK(1-9) is used as tracer. At a dilution of 1 : 267,000, binding of tracer is approximately 50%; and 50% displacement is obtained with ---2 fmol of N-Ac-Lys9-BK(1-9)/tube, with a detection limit of---0.05 fmol/tube. The within-assay coefficient of variation is 14.5%. Displacement of nSI-labeled Ac-Tyr8-BK(1-9) by N-Ac-Lys9-BK(1-9) is identical to that produced by N,O-diacetyl-BK(1-9) [Ac-BK(1-9)], and this assay can be used to measure B K(1-9) in biological samples.
Characterization of Antisera A complete description of the specificities of the antisera is given elsewhere (3, 6). For all antisera, cross-reactivity studies revealed an absolute requirement for acetylation of the N terminus. For antisera A41 and B24 crossreactivities were 100% for peptides of eight or more residues, 75-80% for peptides of seven residues, and correspondingly less for shorter peptides. For antibody A52, cross-reactivity of Ac-Ang(2-7) was 87.5% of that for AcAng III, with a correspondingly lower cross-reactivity for shorter peptides. In practice, antibody A41 can be used for the measurement of Ac-Ang I, AcAng(1-9), Ac-Ang II, and Ac-Ang(1-7); antibody A52 can be used for the measurement of Ac-Ang(2-10), Ac-Ang(2-9), Ac-Ang III, and Ac-Ang(2-7); antibody B24 can be used for the measurement of Ac-B K(1-9), Ac-B K(1-8), and Ac-BK(1-7).
Acetylation of Peptides The method of acetylation is based on the procedure described by Dobson and Strange (12). Peptides or peptide extracts are taken to dryness in siliconized 13 x 100 mm borosilicate glass tubes, using a vacuum centrifuge (Savant Instruments, Hicksville, NY), then acetylated by sequential addition of 100 /A of water, 10 ~1 of triethylamine, and 5/A of acetic anhydride, with mixing by vortex after each addition. After centrifugation to remove particulate material, the sample is injected directly onto the chromatograph. Alternatively, the acetylated samples may be taken to dryness under vacuum and then dissolved in 120/A of 20% (v/v) acetic acid before centrifugation and injection onto the chromatograph (6). As described below, the acetylation procedure results in the acetylation of residues in addition to the a-amino group of each peptide. We have not identified these other acetylated residues, but they probably include Oacetylation of Try 4 of angiotensin and Ser 6 of bradykinin. In contrast to the N-acetyl group, these O-acetyl groups are labile and can be hydrolyzed by
[19] MEASUREMENT OF ANGIOTENSIN AND BRADYKININ
333
treatment with 10% (v/v) piperidine (3). Samples to be treated with piperidine are taken to dryness following acetylation, then dissolved in 100/xl of 10% piperidine in water and allowed to stand at room temperature for 60 min before evaporation to dryness again, dissolution in 120/zl of 20% acetic acid in water, centrifugation, and injection onto the chromatograph. S e p a r a t i o n of A c e t y l a t e d P e p t i d e s by H i g h - P e r f o r m a n c e Liquid Chromatography All samples are transferred to siliconized microfuge tubes and centrifuged in a microfuge at top speed (15,850 g) for 5 min at room temperature to remove particulate material before the supernatant is injected onto the chromatograph. All separations are performed on a 100 x 4.6 mm Brownlee RP-18 Spheri5 column preceded by a 15 x 3.2 mm RP-18 guard column (Applied Biosystems). The HPLC system consists of two pumps (model 6000A; MilliporeWaters, Milford, MA), an automated gradient controller (model 680; Millipore-Waters), and an injector (Rheodyne, Inc., Cotati, CA) with a 200-/zl sample loop. Solvent A is 0.1% TFA and 0.15 M NaC1 in water; solvent B is 0.1% TFA and 90% acetonitrile in water. Peptides are currently eluted by a linearly increasing gradient of 21-41% solvent B over 30 min, and this may need to be adjusted when the column is changed. The flow rate is 1 ml/min and 0.5-min fractions are collected into 12 x 75 mm borosilicate glass tubes containing 50/zl of protease-free bovine serum albumin (5 mg/ml; (Miles Diagnostics, Kankakee, IL) in water. The solvent blank prepared for assay tubes of the RIA standard curves is 0.5 ml of 31% solvent B in solvent A, added to 50 tzl of bovine serum albumin (5 mg/ml). Fractions and solvent blank tubes are evaporated to dryness under vacuum, and then dissolved in water immediately before RIA. When assayed with one RIA, fractions are dissolved in 120/zl of water and two 50-/zl aliquots taken for RIA of each fraction. When fractions are assayed with more than one RIA, the fractions are dissolved in a correspondingly greater volume of water before RIA. The elution positions of standard angiotensin peptides that were acetylated as described above are shown in Fig. 3A; those that were acetylated and then piperidine treated before HPLC are shown in Fig. 3B. An excellent separation of the different angiotensin peptides is obtained, with N-acetylated peptides (piperidine treated) eluting earlier than N,O-diacetylated peptides. A similar result was obtained for bradykinin peptides (Fig. 4A and B). L a b i l i t y of A c e t y l a t e d P e p t i d e s The first N terminal-directed RIAs we developed were for angiotensin peptides. During the development of these assays we did not suspect that acetyla-
334
III EXTRACELLULAR PROCESSING ENZYMES IN THE CNS 400
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FIG. 3 Elution positions on HPLC of standard angiotensin peptides that were either N,O-diacetylated as described in text (A), or N-acetylated (piperidine-treated) (B). HPLC fractions were assayed by RIA with antibody A41, and data have not been corrected for recoveries or cross-reactivity with the antibody. N,O-Diacetylated peptides are designated Ac-; N-acetylated peptides are designated N-Ac-. AcAng(1-7), N,O-diacetylangiotensin(1-7); Ac-Ang(1-9), N,O-diacetylangiotensin(l-9); Ac-Ang II, N, O-diacetylangiotensin II; Ac-Ang I, N,O-diacetylangiotensin I; N-Ac-Ang(1-7), N-acetylangiotensin(1-7); N-Ac-Ang(1-9), N-acetylangiotensin(l-9); N-Ac-Ang II, N-acetylangiotensin II; N-Ac-Ang I, N-acetylangiotensin I.
tion of residues other than the N terminus was occurring, in that the acetylated products appeared to be completely homogeneous, with an efficiency of acetylation of--~100% (6). However, during subsequent development of the N terminal-directed RIA for bradykinin peptides, it was apparent that the acetylated product was not homogeneous. In Fig. 4A it can be seen that small peaks of immunoreactivity elute in the position ofN-acetylated peptides
[19] MEASUREMENTOF ANGIOTENSIN AND BRADYKININ 35o-
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FIG. 4 Elution positions on HPLC of standard bradykinin peptides that were either N,O-diacetylated as described in text (A) or were N-acetylated (piperidine treated) (B). HPLC fractions were assayed by RIA with antibody B24, and data have not been corrected for recoveries or cross-reactivity with the antibody. N,O-Diacetylated peptides are designated Ac-; N-acetylated peptides are designated N-Ac-. AcBK(1-7), N, O-diacetylbradykinin(1-7); Ac-B K(1-8), N, O-diacetyl-bradykinin(1-8); Ac-BK(1-9), N,O-diacetylbradykinin(1-9); N-Ac-BK(1-7), N-acetylbradykinin(1-7); N-Ac-B K(1-8), N-acetylbradykinin(1-8); N-Ac-B K(1-9), N-acetylbradykinin(1-9).
(compare Fig. 4A and B). N-Acetyl-BK(1-9)represents <10% ofN, O-diacetyl-BK(1-9), but for N, O-diacetyl-BK(1-8) and N, O-diacetyl-BK(1-7) the N-acetylated peptides represent ---20% of the corresponding diacetylated peptides. Our experience with acetylation of bradykinin peptides led us to examine whether angiotensin peptides were also acetylated at residues other than the
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N terminus. As shown in Fig. 3, this was in fact occurring, but the product of acetylation was completely homogeneous. One concern resulting from the data shown in Fig. 3A and B is that N-acetyl-Ang I elutes in the same position as N,O-diacetyl-Ang II. It was therefore necessary to exclude the possibility that the N, O-diacetyl-Ang II peak in acetylated biological samples contains a contribution from N-acetyl-Ang I. This possibility would be of particular concern for samples obtained following administration of an ACE inhibitor, where Ang II levels would be expected to decrease, in association with a large increase in Ang I levels. Evidence that the N,O-diacetyl-Ang II peak does not contain a contribution from N-acetyl-Ang I includes the following: (a) as stated above, when either Ang II or Ang I were acetylated, the product was completely homogeneous (6); (b) when human plasma samples or rat kidney homogenates were spiked with ---1000 fmol of Ang I before extraction, acetylation, and HPLC, there was no increase in the measured amount of Ang II in each sample (6, 13). Nevertheless, we observed that when acetylated samples were inadvertently allowed to stand at room temperature for several hours, partial deacetylation of the N,O-diacetyl-Ang I did occur. For this reason, and because of our experience with acetylated bradykinin peptides, we take particular care to prevent hydrolysis following acetylation. Samples are injected directly onto the chromatograph following acetylation or, when an automatic injector is used, the carousel of the injector is refrigerated to ---3~ For N,O-diacetylated bradykinin peptides (Fig. 4A), it will be noted that Nacetyl-BK(1-8) elutes close to the elution position of N, O-diacetyl-BK(1-9). This is of little concern in practice, however, because BK(1-8) levels in biological samples are much less than those of BK(1-9) (3). Our experience has been that piperidine treatment of acetylated extracts does not result in an appreciable increase in the measured peaks of immunoreactivity in HPLC fractions. For routine assay, samples are N,O-diacetylated as described above. When O-deacetylation may occur, this is corrected for because all estimates of endogenous peptide levels in biological samples are corrected for the recovery of standard peptides from tissue homogenates treated in an identical manner.
Extraction of Peptides from Plasma, Blood, and Tissues Measurement of peptides in biological samples requires the prevention of peptide generation and metabolism during processing of the sample. For the measurement of angiotensin peptides in plasma, trunk blood from decapitated rats or blood obtained by venepuncture from human subjects is collected directly into 0.1 vol of an inhibitor cocktail [50 mM 1,10-phenanthro-
[19] MEASUREMENT OF ANGIOTENSIN AND BRADYKININ
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line, 125 mM EDTA, neomycin sulfate (2 g/liter), and 2% ethanol in water] containing an appropriate renin inhibitor at a concentration sufficient to inhibit renin activity completely (6, 13). The blood is then centrifuged and the plasma immediately extracted with Sep-Pak C~8 cartridges (MilliporeWaters). Each Sep-Pak cartridge is first moistened with 3 ml of methanol and then washed with 10 ml of 1% TFA in water. To minimize nonspecific adsorption, the cartridges are next coated with 1 ml of a 1% polypeptide solution (Polypep; Sigma Chemical Company, St. Louis, MO) and washed again with methanol-water-TFA (80:19:1, v/v/v) and with 1% TFA (14). The sample is then applied and the cartridges washed with 10 ml of 1% TFA- 1% sodium chloride (1 : 1, v/v), then with 2 ml of methanol-water-TFA (30:69: 1, v/v/v), before elution with 6 ml of methanol-water-TFA (80: 19: 1, v/v/v) into a siliconized 13 x 100 mm borosilicate glass tube. The eluate is then cooled to -80~ before evaporation to dryness under vacuum. Cooling of the sample is a precaution against sample loss due to boiling in the vacuum centrifuge. Plasma extracts can then be acetylated and subjected to HPLC. The measurement of circulating levels of bradykinin peptides requires different methodology because of the need to prevent activation of plasma prekallikrein. For the measurement of bradykinin peptides in rat blood, blood is collected from either conscious rats with previously implanted arterial cannulae or from anesthetized rats by needle puncture of the aorta or inferior vena cava. Two milliliters of blood is collected directly into a syringe containing 10 ml of 4 M guanidine thiocyanate (GTC), 1% TFA in water, and the mixture is then homogenized using a Polytron with a 1-cm aggregate (model PT 10-35; Kinematica, Lucerne, Switzerland) operating at maximum speed. The mixture is then centrifuged at 5000 g for 20 min at 20~ and the supernatant extracted by Sep-Pak as described above. Blood levels of angiotensin peptides can also be measured by this method. Tissues are rapidly removed from decapitated rats, weighed, and immediately homogenized in 10 ml of GTC-TFA, sonicated briefly, and then processed as described for blood. The weight of tissue homogenized is no more than 0.5-1.0 g. Higher amounts of tissues result in lower recoveries from the extraction procedure. The use of GTC-TFA for the homogenization of tissues is a modification of the method described by Zingg et al. (15). Extracts of blood and tissue are extracted with diethyl ether before acetylation and HPLC. Each dried extract is dissolved in 1 ml of 0.1 M hydrochloric acid, then extracted twice with 1 ml of diethyl ether, frozen, and taken to dryness again under vacuum. We have previously used 1.0 M hydrochloric acid for this procedure (3, 13, 16), but 0.1 M hydrochloric acid gives equivalent results.
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The blank for each assay was assessed by extraction of 10 ml of GTC-TFA, and these blank extracts were processed as described above and then subjected to HPLC before RIA.
Validation of Methodology Details of the recoveries of peptides extracted by these methods are given elsewhere (3, 6, 13, 16). In general, peptide recoveries approximate 40-50%. A number of different approaches were used to validate the methodology described. 1. Radioimmunoassays of HPLC fractions of blank extracts gave results at or below the detection limit(3, 6). 2. Our identification of individual angiotensin and bradykinin peptides in biological samples was based on criteria additional to their recognition by the appropriate antisera. For N,O-diacetylated angiotensin and bradykinin peptides, the identified peptides were shown to elute in the same position as N,O-diacetylated standard peptides on HPLC. Moreover, in the case of bradykinin peptides, when samples were N-acetylated (piperidine treated), the identified peptides were shown to elute in the same position as N-acetylated standard peptides on HPLC. 3. C terminal-directed antisera raised against Ang II, Ang I, Ang(1-9), and BK(1-9) (3, 9, 13) were able to recognize these peptides when acetylated, although with a reduced efficiency. By using these C terminal-directed antisera we were able to confirm the identity of many of the peaks of immunoreactivity measured in HPLC fractions of biological samples by N terminaldirected RIA (3, 6, 13). 4. We studied the effect of time delay in removal of rat tissues on the measured tissue levels of angiotensin and bradykinin peptides. When a comparison was made between the left and right kidneys homogenized 30 and 90 sec after decapitation, respectively, no difference in angiotensin or bradykinin peptide levels was evident (3, 13). This does not indicate that renal angiotensin and bradykinin peptides are protected from metabolism; rather, the relatively constant level of angiotensin and bradykinin peptides during the delay before homogenization indicates that during this time peptide metabolism approximates the production rate of these peptides in the kidney. 5. To determine the stability of bradykinin peptides in GTC-TFA tissue homogenate, kidney homogenates were divided into two equal portions that were extracted either 1 or 2 hr after homogenization. No difference in the measured levels of bradykinin peptides was evident, indicating that peptides are stable in GTC-TFA tissue homogenates (3). By contrast, for GTC-TFA
[19] MEASUREMENT OF ANGIOTENSIN AND BRADYKININ
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homogenates of blood we did observe a significant decrease in BK(1-9) levels, from 3.00 -+ 0.03 fmol/ml (mean _ 0.03 fmol/ml (mean _+ SEM, n = 6) for homogenates extracted after 1 hr, to 2.3 +__0.3 fmol/ml (p < 0.05) for homogenates extracted after 2 hr (3). 6. In another approach to assess the stability of angiotensin and bradykinin peptides in GTC-TFA tissue homogenates, kidney homogenates were divided into two equal portions, to one of which was added --~1000 fmol of either Ang II, Ang I, or BK(1-9). Subsequent analyses showed no evidence of metabolism of the added peptides, except for a small increase in B K(1-8) levels in the homogenate to which BK(1-9) was added, which represented <1% of the added amount of B K(1-9), again indicating that peptides are stable in GTC-TFA homogenates (3, 13). The same approach was used to show that metabolism of plasma angiotensin peptides did not occur during processing of plasma samples (6). 7. To examine whether homogenization of tissue in GTC-TFA causes an immediate arrest of bradykinin peptide degradation and generation and avoids the possible consequences of activation of prekallikrein, we also measured bradykinin peptide levels in kidneys that were snap-frozen. Following decapitation of each of six rats, the left kidney was immediately homogenized in GTC-TFA at room temperature, and the right kidney immediately clamped between two metal plates that had been cooled to the temperature of liquid nitrogen. The frozen kidney was then pulverized in a metal mortar and pestle that had been cooled to the temperature of liquid nitrogen. The powdered frozen kidney was added to GTC-TFA at 0~ and immediately homogenized by use of a Polytron. Similar bradykinin peptide levels were measured for kidneys that were either immediately homogenized in GTC-TFA at room temperature or rapidly cooled to the temperature of liquid nitrogen before pulverization and homogenization, thus providing further evidence for the efficiency with which bradykinin peptide degradation and generation were arrested by homogenization in GTC-TFA (3). 8. Another aspect of the process of validation was to show that the circulating and tissue levels of angiotensin and bradykinin peptides changed in the manner predicted following administration of an ACE inhibitor (3, 6, 13).
Potential Problems Many different methods for the extraction of peptides from tissue have been described. In the present studies, blood and tissues were homogenized in GTC-TFA because GTC is a potent chaotropic agent that rapidly denatures
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EXTRACELLULAR PROCESSING ENZYMES IN THE CNS
proteins and arrests enzymatic activity, and 1% TFA, by lowering the pH of the sample, also inactivates many proteolytic enzymes. However, it is important to be aware of the possibility that the extraction method may modify some peptides. For example, oxidation of methionine residues may occur, and Smyth (17) has described a strategy to overcome this problem. Other possibilities are the deacetylation of O-acetylated residues (18) and the deamidation of asparagine and glutamine residues by exposure to acid (19). Fortunately these potential problems do not apply to angiotensin and bradykinin peptides. We chose to homogenize fresh tissue immediately because such an approach inactivates proteolytic enzymes and thus prevents peptide generation and metabolism during subsequent processing of the sample. Many authors freeze tissues before subsequent homogenization and extraction of peptides. We have avoided the freezing of tissue because of the potential for peptide generation and metabolism during freezing and thawing. An example of the problems that may occur is shown by our study of the effect of different methods of extraction of rat hypothalami on the measured levels of angiotensin peptides (16). Angiotensin II levels in rat hypothalami are low (--~18 fmol/ g wet weight), and Ang(1-7) levels are below the minimum detectable (<6 fmol/g). However, if hypothalami that had been frozen in liquid nitrogen were allowed to thaw before homogenization in GTC-TFA, the measured levels of Ang(1-7) were --~100 fmol/g. A similar result was obtained when frozen tissue was boiled in 0.1 M hydrochloric acid (16). It is important to use an HPLC protocol that produces optimal separation of the peptides of interest because it is not possible to predict whether other immunoreactive material may elute close to the peptides of interest. This problem is illustrated by our studies of angiotensin peptides in plasma of rats administered the ACE inhibitor perindopril. Shown in Fig. 5 are HPLC profiles for plasma angiotensin peptides of a control rat (Fig. 5A) and a rat administered perindopril (4.2 mg/kg/day) in the drinking water for 7 days (Fig. 5B). Note that ACE inhibition was associated with large increases in Ang(1-7) and Ang I levels, with a reduction in Ang II levels. Fractions were not collected for measurement of Ang(1-9) levels in these experiments. Angiotensin-converting enzyme inhibition was associated with the appearance of a peak of immunoreactivity in fraction 45. The identity of this peak is not known but it is probably related to the markedly increased Ang I levels. These profiles emphasize the need to optimize HPLC conditions to achieve maximal separation of cross-reacting peptides because such an approach ensures maximal separation of other "unanticipated" peaks of immunoreactivity from the peaks of interest. Becuase of the need to differentiate authentic peptide peaks from such "unanticipated" peaks, we always assay individual fractions rather than pool fractions for RIA.
341
[19] MEASUREMENT OF ANGIOTENSIN AND BRADYKININ 100
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FIG. 5 Characterization of angiotensin peptides in plasma of a control rat (A) and a rat administered perindopril (4.2 mg/kg/day) in the drinking water for 7 days (B). Plasma samples were extracted, N,O-diacetylated, run on HPLC, and angiotensin peptides measured by RIA with antibody A41. Fractions containing N, O-diacetylangiotensin(1-9) were not assayed in this experiment. Ac-Ang(1-7), N, O-diacetylangiotensin(1-7); Ac-Ang II, N, O-diacetylangiotensin II; Ac-Ang I, N, O-diacetylangiotensin I.
Advantages and Disadvantages of Methodology The advantages of the methodology described are as follows: (a) the combined use of HPLC and N terminal-directed antisera enables the simultaneous RIA of a number of peptides of interest. Moreover, determination of the ratio of the C terminal-truncated metabolites to the parent peptide gives important information concerning the activity of these pathways of peptide metabolism in vivo. For example, the Ang II/Ang I ratio in plasma or tissues
342
III EXTRACELLULARPROCESSING ENZYMES IN THE CNS is a sensitive indicator of Ang I conversion to Ang II at these sites in oioo; (b) an excellent separation of acetylated peptides is achieved by HPLC; (c) the RIAs are sensitive and robust. The N terminal-directed RIAs for the angiotensin peptides have sensitivities equivalent to the most sensitive described, and the N terminal-directed bradykinin RIA is severalfold more sensitive than the most sensitive described. Disadvantages of the methodology include the following: (a) the methodology is time consuming and tedious. To some extent this can be overcome by the automation of HPLC and of RIA; (b) precautions are necessary to prevent the deacetylation of labile O-acetyl groups. Alternatively, acetylated samples could be routinely treated with piperidine before HPLC; (c) acetylation may result in a lower peptide recovery than might be achieved if samples are not acetylated before HPLC; (d) the N terminal-directed RIA cannot be used to measure N terminal-extended peptides. Thus, while the antibody B24 assay is ideal for studies in the rat, where BK(1-9) is the predominant kinin peptide, this assay cannot be used to measure Lys~ which may be the predominant kinin peptide in humans (20).
Acknowledgments The studies reported here were supported by grants from the National Health and Medical Research Council of Australia, and by the National Heart Foundation of Australia.
References 1. D. J. Campbell, in "The Renin-Angiotensin System" (J. I. S. Robertson and M. G. Nicholls, eds.), p. 23.1. Gower Medical Publishing, London, 1993. 2. J. W. Ryan, Am. J. Physiol. 257, L53 (1989). 3. D. J. Campbell, A. Kladis, and A.-M. Duncan, Hypertension (Dallas) 21, 155 (1993). 4. J. Nussberger, G. R. Matsueda, R. Re, and E. Haber, J. Immunol. Methods 56, 85 (1983). 5. J. R. Stockigt, H. M. Shizgal, and W. F. Ganong, Int. Congr. Ser. Excerpta Med. 241, 290 (1972). 6. A. C. Lawrence, G. Evin, A. Kladis, and D. J. Campbell, J. Hypertens, 8, 715 (1990). 7. H. M. Geysen, S. J. Barteling, and R. H. Meloen, Proc. Natl. Acad. Sci. U.S.A. 82, 178 (1985). 8. J. Vaitukaitis, J. B. Robbins, E. Nieschlag, and G. T. Ross, J. Clin. Endocrinol. Metab. 33, 988 (1971).
[19] MEASUREMENTOF ANGIOTENSIN AND BRADYKININ
343
D. J. Campbell and A. Kladis, J. Hypertens. 8, 165 (1990). 10. V. Herbert, K.-S. Lau, C. W. Gottlieb, and S. J. Bleicher, J. Clin. Endocrinol. Metab. 25, 1375 (1965). 11. W. M. Hunter and F. C. Greenwood, Nature (London) 194, 495 (1962). 12. P. R. M. Dobson and P. G. Strange, in "Methods in Enzymology" (L. Birnbaumer and B. O'Malley, eds.), Vol. 109, p. 827. Academic Press, Orlando, FL, 1985. 13. D. J. Campbell, A. C. Lawrence, A. Towrie, A. Kladis, and A. J. Valentijn, Hypertension (Dallas) 18, 763 (1991). 14. K. Hermann, R. E. Lang, T. Unger, C. Bayer, and D. Ganten, J. Chromatogr. 312, 273 (1984). 15. H. H. Zingg, D. Lefebvre, and G. Almazan, J. Biol. Chem. 2,61, 12956 (1986). 16. A. C. Lawrence, I. J. Clarke, and D. J. Campbell, J. Neuroendocrinol. 4, 237 (1992). 17. D. G. Smyth, Anal. Biochem. 136, 127 (1984). 18. M. E. Goldman, M. Beaulieu, J. W. Kebabian, and R. L. Eskay, Endocrinology (Baltimore) 112, 435 (1983). 19. A. I. Smith and R. A. Lew, this volume [7]. 20. F. Alhenc-Gelas, J. Marchetti, J. Allegrini, P. Corvol, and J. Menard, Biochim. Biophys. Acta 677, 477 (1981). .
Strategies for Measurement of Angiotensin and Bradykinin Peptides and Their Metabolites in Central Nervous System and Other Tissues Duncan J. Campbell, Anne C. Lawrence, Athena Kladis, and Ann-Maree Duncan
Introduction Whether angiotensin and bradykinin are neuropeptides is a subject of continuing debate. The strength of the evidence for or against such a proposition is dependent on the methodological basis for such evidence. Rather than address this issue directly, in this chapter we describe some of the methodologies we have developed for the measurement of angiotensin and bradykinin peptides and their metabolites in the central nervous system (CNS) and other tissues. In the past, radioimmunoassay (RIA) of angiotensin and bradykinin peptides was based on the use of carboxy (C) terminal-directed antisera. This was due in large part to the ease with which a peptide may be coupled via its amino (N) terminus to carrier proteins for the purpose of immunization. However, for both angiotensin and bradykinin peptides important processing events take place toward the C terminus of the molecule (1-3)- (Figs. 1 and 2). For example, the decapeptide angiotensin I (Ang I) is cleaved between residues 8 and 9 by angiotensin-converting enzyme (ACE, kininase II, EC 3.4.15.1, peptidyl-dipeptidase A) to release angiotensin II (Ang II), and both Ang I and Ang II are cleaved between residues 7 and 8 by a number of endopeptidases to release angiotensin(1-7) [Ang(1-7)]. Both Ang II and Ang(1-7) are bioactive. An alternative pathway of conversion of Ang I to Ang II may involve the sequential cleavage of the two C-terminal residues of Ang I by carboxypeptidase activity (1). Moreover, the nonapeptide bradykinin(1-9) [BK(1-9)] is cleaved between residues 8 and 9 by carboxypeptidases N (kininase I) and M to release bradykinin(1-8) [BK(1-8)], and between residues 7 and 8 by ACE and other endopeptidases to release bradykinin(1-7) [BK(1-7)]. Both BK(1-9) and BK(1-8) are bioactive. When these differentially processed peptides are separated by high-performance liquid chromatography (HPLC), it is of assistance if the peptides of interest 328
Methods in Neurosciences, Volume 23 Copyright 9 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
[19] MEASUREMENTOF ANGIOTENSIN AND BRADYKININ
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FIG. 1 Diagrammatic representation of cleavage sites of angiotensin I by different enzymes. Both endopeptidases 24.11 and 24.15 cleave angiotensin I between residues 4 and 5, and between residues 7 and 8; in addition, endopeptidase 24.11 cleaves angiotensin I between residues 2 and 3. After removal of the amino-terminal aspartic acid by aminopeptidase A, the Arg2 residue can be cleaved by aminopeptidase N. For angiotensin II, the sites of cleavage by endopeptidases are the same as those shown for angiotensin I, except that endopeptidases 24.11 and 24.15 do not cleave between residues 7 and 8 of angiotensin II. ACE, Angiotensin-converting enzyme.
can be measured with the same RIA. To this end, we established N terminaldirected RIA for the measurement of angiotensin and bradykinin peptides and their C-terminal truncated metabolites. In previous attempts to raise N terminal-directed antisera to angiotensin peptides, although the peptides were coupled to the carrier protein via the C terminus, the antisera raised were predominantly directed to the C terminus (4, 5). However, Nussberger et al. (4) found that when Asn ~, VaP-Ang II was acetylated at the N terminus and coupled via the C terminus for immunization, they readily achieved N terminal-directed antisera. This result suggests that acetylation of the N terminus of a peptide renders the N terminus more immunogenic. We used this approach to raise N terminal-directed antisera against N-acetylated angiotensin and bradykinin peptide analogs
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FIG. 2 Diagrammatic representation of cleavage sites of bradykinin by different enzymes. ACE, Angiotensin-converting enzyme.
330
III
EXTRACELLULAR PROCESSING ENZYMES IN THE CNS
(3, 6), with the intention of acetylating peptides extracted from biological samples before RIA with these antisera.
Preparation of Antisera For the preparation of antisera directed against the N terminus of Ang II, angiotensin III (Ang III), and B K(1-9), the following peptides are synthesized: N-acetyl-AspArgValTyrIleHisProPheLys (N-Ac-Lysg-Ang II), N-acetyl-ArgValTyrIleHisProPheLys (N-Ac-Lys8-Ang III), and N-acetyl-ArgProProGlyPheSerProPheLys [N-Ac-Lys9-BK(1-9)]. Peptides are synthesized from tert-butoxycarbonyl-protected amino acids, using an Applied Biosystems (Foster City, CA) 430A automated peptide synthesizer. Acetylation of a-amino groups of Lysg-Ang II, Lys8-Ang III, and Lys9-BK(1-9) is performed on the protected resin before hydrogen fluoride treatment (6). Peptides are coupled to bovine thyroglobulin via the C-terminal lysine residue with glutaraldehyde (7), and antisera are raised in rabbits (8). Six rabbits are immunized with each peptide and the best antiserum against each peptide is subsequently used to establish an RIA.
Description of Radioimmunoassays All peptide concentrations are determined by amino acid analysis, using stocks of approximately 1 mg/ml in 20% (v/v) acetic acid in water, and stored at -30~ Working solutions [1 /~M in lysozyme (1 mg/ml), 10 mM acetic acid] are stored at -30~ and discarded after thawing once. All RIA components are diluted with casein phosphate buffer [casein (1 g/liter), 100 mM sodium phosphate, 10 mM disodium ethylenediaminetetraacetic acid (EDTA), sodium azide (1 g/liter), 154 mM sodium chloride, pH 7.0]. A pH optimum of 7.0 has been shown for each of the three assays described. Initially, the total RIA assay volume was 500 /A (6), but this has since been reduced to 250/~1 to increase sensitivity. Although initially prepared on ice, assays are now prepared at room temperature. Each assay tube contains 50/A of diluted antibody, 50/~1 of tracer (---2500 cpm), 50/~1 of standard or unknown peptide solution, and 100/~1 of buffer. Usually the assays are incubated at 4~ for 48 hr before separation of free from bound radioactivity. For the antibody A41 assay, addition of tracer is delayed t~or 48 hr, and the assay is incubated at 4~ for a further 24 hr before separation of free from bound radioactivity. Separation of free from bound radioactivity with albumin/dextran-coated charcoal is performed using a modification (9) of the method described by
[19] MEASUREMENT OF ANGIOTENSIN AND BRADYKININ
331
Herbert et al. (10). Stock dextran-coated charcoal [Norit A charcoal (25 g/liter), dextran T10 (2.5 g/liter), 7.1 mM sodium barbitone, 7.1 mM sodium acetate, adjusted to pH ---7.4 with hydrochloric acid] is stirred with bovine serum albumin (BSA, 10 mg/ml) for 1-24 hr at 4~ and then diluted with 4 vol of 150 mM sodium chloride immediately before use. One milliliter of albumin/dextran-coated charcoal is added to each tube at 4~ and, after standing at 4~ for 10 min, the assay tubes are centrifuged at 5000 g for 10 min at 4~ the supernatants rapidly aspirated, and the charcoal pellets counted. Tracer peptides are iodinated with 125I using chloramine-T (11), and the monoiodinated peptides are purified by HPLC on a C18 column, using a gradient of acetonitrile in 0.1% (v/v) trifluoroacetic acid (TFA), and stored in aliquots at -30~ Tracer peptides can be stored for up to 2 months without deterioration in assay performance. Antibody A41 was raised against N-Ac-Lys9-Ang II. The antibody A41 assay uses N, O-diacetyl-Ang II (Ac-Ang II, acetylated as described below) as standard peptide and mono[~ZSI]iodo-Ac-Ang II as tracer. At a dilution of 1 : 270,000, binding of tracer is approximately 50%, and 50% displacement is obtained with ---8 fmol of Ac-Ang II/tube, with a detection limit of ---0.25 fmol/tube. The within-assay coefficient of variation is 6% and the betweenassay coefficient of variation is 19%. Antibody A41 was initially studied using N-Ac-Lys9-Ang II as standard and 125I-labeled N-Ac-Lys9-Ang II as tracer; however, displacement of 125I-labeled N-Ac-Lys9-Ang II by N-AcLys9-Ang II and Ac-Ang II was not superimposable, with incomplete displacement by Ac-Ang II, indicating that a proportion of the antibody population of A41 was specific for N-Ac-Lys9-Ang II. Consequently, N-Ac-Lys 9Ang II cannot be used as standard for the measurement of Ang II levels in biological samples; instead, Ac-Ang II must be used as standard. The use of 125I-labeled Ac-Ang II as tracer has the advantage that Ac-Ang II produces complete displacement of tracer. Antibody A52 was raised against N-Ac-Lysg-Ang III. The antibody A52 assay uses N-Ac-LysS-Ang III as standard peptide and 125I-labeled N-AcLysS-Ang III as tracer. At a dilution of 1 : 48,500, binding of tracer is approximately 50%; assays with antibody A52 have been performed using a total assay volume of only 500 ~1, and 50% displacement is obtained with --~16 fmol of N-Ac-LysS-Ang III/tube, with a detection limit of ---1.0 fmol/tube. The between-assay coefficient of variation is 12%. In contrast to the antibody A41 assay, displacement of 125I-labeled N-Ac-Lys8-Ang III by N-Ac-Lys 9Ang III is identical to that produced by N,O-diacetyl-Ang III (Ac-Ang III), and this assay can be used to measure Ang III in biological samples. Antibody B24 was raised against N-Ac-Lysg-BK(1-9). The antibody B24 assay uses N-Ac-Lys9-BK(1-9) as standard peptide. 125I-Labeled Tyr 8BK(1-9) is acetylated as described below before purification by HPLC, and
332
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E X T R A C E L L U L A R PROCESSING ENZYMES IN THE CNS
mono[125I]iodo-Ac-Tyr8-BK(1-9) is used as tracer. At a dilution of 1 : 267,000, binding of tracer is approximately 50%; and 50% displacement is obtained with ---2 fmol of N-Ac-Lys9-BK(1-9)/tube, with a detection limit of---0.05 fmol/tube. The within-assay coefficient of variation is 14.5%. Displacement of nSI-labeled Ac-Tyr8-BK(1-9) by N-Ac-Lys9-BK(1-9) is identical to that produced by N,O-diacetyl-BK(1-9) [Ac-BK(1-9)], and this assay can be used to measure B K(1-9) in biological samples.
Characterization of Antisera A complete description of the specificities of the antisera is given elsewhere (3, 6). For all antisera, cross-reactivity studies revealed an absolute requirement for acetylation of the N terminus. For antisera A41 and B24 crossreactivities were 100% for peptides of eight or more residues, 75-80% for peptides of seven residues, and correspondingly less for shorter peptides. For antibody A52, cross-reactivity of Ac-Ang(2-7) was 87.5% of that for AcAng III, with a correspondingly lower cross-reactivity for shorter peptides. In practice, antibody A41 can be used for the measurement of Ac-Ang I, AcAng(1-9), Ac-Ang II, and Ac-Ang(1-7); antibody A52 can be used for the measurement of Ac-Ang(2-10), Ac-Ang(2-9), Ac-Ang III, and Ac-Ang(2-7); antibody B24 can be used for the measurement of Ac-B K(1-9), Ac-B K(1-8), and Ac-BK(1-7).
Acetylation of Peptides The method of acetylation is based on the procedure described by Dobson and Strange (12). Peptides or peptide extracts are taken to dryness in siliconized 13 x 100 mm borosilicate glass tubes, using a vacuum centrifuge (Savant Instruments, Hicksville, NY), then acetylated by sequential addition of 100 /A of water, 10 ~1 of triethylamine, and 5/A of acetic anhydride, with mixing by vortex after each addition. After centrifugation to remove particulate material, the sample is injected directly onto the chromatograph. Alternatively, the acetylated samples may be taken to dryness under vacuum and then dissolved in 120/A of 20% (v/v) acetic acid before centrifugation and injection onto the chromatograph (6). As described below, the acetylation procedure results in the acetylation of residues in addition to the a-amino group of each peptide. We have not identified these other acetylated residues, but they probably include Oacetylation of Try 4 of angiotensin and Ser 6 of bradykinin. In contrast to the N-acetyl group, these O-acetyl groups are labile and can be hydrolyzed by
[19] MEASUREMENT OF ANGIOTENSIN AND BRADYKININ
333
treatment with 10% (v/v) piperidine (3). Samples to be treated with piperidine are taken to dryness following acetylation, then dissolved in 100/xl of 10% piperidine in water and allowed to stand at room temperature for 60 min before evaporation to dryness again, dissolution in 120/zl of 20% acetic acid in water, centrifugation, and injection onto the chromatograph. S e p a r a t i o n of A c e t y l a t e d P e p t i d e s by H i g h - P e r f o r m a n c e Liquid Chromatography All samples are transferred to siliconized microfuge tubes and centrifuged in a microfuge at top speed (15,850 g) for 5 min at room temperature to remove particulate material before the supernatant is injected onto the chromatograph. All separations are performed on a 100 x 4.6 mm Brownlee RP-18 Spheri5 column preceded by a 15 x 3.2 mm RP-18 guard column (Applied Biosystems). The HPLC system consists of two pumps (model 6000A; MilliporeWaters, Milford, MA), an automated gradient controller (model 680; Millipore-Waters), and an injector (Rheodyne, Inc., Cotati, CA) with a 200-/zl sample loop. Solvent A is 0.1% TFA and 0.15 M NaC1 in water; solvent B is 0.1% TFA and 90% acetonitrile in water. Peptides are currently eluted by a linearly increasing gradient of 21-41% solvent B over 30 min, and this may need to be adjusted when the column is changed. The flow rate is 1 ml/min and 0.5-min fractions are collected into 12 x 75 mm borosilicate glass tubes containing 50/zl of protease-free bovine serum albumin (5 mg/ml; (Miles Diagnostics, Kankakee, IL) in water. The solvent blank prepared for assay tubes of the RIA standard curves is 0.5 ml of 31% solvent B in solvent A, added to 50 tzl of bovine serum albumin (5 mg/ml). Fractions and solvent blank tubes are evaporated to dryness under vacuum, and then dissolved in water immediately before RIA. When assayed with one RIA, fractions are dissolved in 120/zl of water and two 50-/zl aliquots taken for RIA of each fraction. When fractions are assayed with more than one RIA, the fractions are dissolved in a correspondingly greater volume of water before RIA. The elution positions of standard angiotensin peptides that were acetylated as described above are shown in Fig. 3A; those that were acetylated and then piperidine treated before HPLC are shown in Fig. 3B. An excellent separation of the different angiotensin peptides is obtained, with N-acetylated peptides (piperidine treated) eluting earlier than N,O-diacetylated peptides. A similar result was obtained for bradykinin peptides (Fig. 4A and B). L a b i l i t y of A c e t y l a t e d P e p t i d e s The first N terminal-directed RIAs we developed were for angiotensin peptides. During the development of these assays we did not suspect that acetyla-
334
III EXTRACELLULAR PROCESSING ENZYMES IN THE CNS 400
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FIG. 3 Elution positions on HPLC of standard angiotensin peptides that were either N,O-diacetylated as described in text (A), or N-acetylated (piperidine-treated) (B). HPLC fractions were assayed by RIA with antibody A41, and data have not been corrected for recoveries or cross-reactivity with the antibody. N,O-Diacetylated peptides are designated Ac-; N-acetylated peptides are designated N-Ac-. AcAng(1-7), N,O-diacetylangiotensin(1-7); Ac-Ang(1-9), N,O-diacetylangiotensin(l-9); Ac-Ang II, N, O-diacetylangiotensin II; Ac-Ang I, N,O-diacetylangiotensin I; N-Ac-Ang(1-7), N-acetylangiotensin(1-7); N-Ac-Ang(1-9), N-acetylangiotensin(l-9); N-Ac-Ang II, N-acetylangiotensin II; N-Ac-Ang I, N-acetylangiotensin I.
tion of residues other than the N terminus was occurring, in that the acetylated products appeared to be completely homogeneous, with an efficiency of acetylation of--~100% (6). However, during subsequent development of the N terminal-directed RIA for bradykinin peptides, it was apparent that the acetylated product was not homogeneous. In Fig. 4A it can be seen that small peaks of immunoreactivity elute in the position ofN-acetylated peptides
[19] MEASUREMENTOF ANGIOTENSIN AND BRADYKININ 35o-
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FIG. 4 Elution positions on HPLC of standard bradykinin peptides that were either N,O-diacetylated as described in text (A) or were N-acetylated (piperidine treated) (B). HPLC fractions were assayed by RIA with antibody B24, and data have not been corrected for recoveries or cross-reactivity with the antibody. N,O-Diacetylated peptides are designated Ac-; N-acetylated peptides are designated N-Ac-. AcBK(1-7), N, O-diacetylbradykinin(1-7); Ac-B K(1-8), N, O-diacetyl-bradykinin(1-8); Ac-BK(1-9), N,O-diacetylbradykinin(1-9); N-Ac-BK(1-7), N-acetylbradykinin(1-7); N-Ac-B K(1-8), N-acetylbradykinin(1-8); N-Ac-B K(1-9), N-acetylbradykinin(1-9).
(compare Fig. 4A and B). N-Acetyl-BK(1-9)represents <10% ofN, O-diacetyl-BK(1-9), but for N, O-diacetyl-BK(1-8) and N, O-diacetyl-BK(1-7) the N-acetylated peptides represent ---20% of the corresponding diacetylated peptides. Our experience with acetylation of bradykinin peptides led us to examine whether angiotensin peptides were also acetylated at residues other than the
336
III
EXTRACELLULAR PROCESSING ENZYMES IN THE CNS
N terminus. As shown in Fig. 3, this was in fact occurring, but the product of acetylation was completely homogeneous. One concern resulting from the data shown in Fig. 3A and B is that N-acetyl-Ang I elutes in the same position as N,O-diacetyl-Ang II. It was therefore necessary to exclude the possibility that the N, O-diacetyl-Ang II peak in acetylated biological samples contains a contribution from N-acetyl-Ang I. This possibility would be of particular concern for samples obtained following administration of an ACE inhibitor, where Ang II levels would be expected to decrease, in association with a large increase in Ang I levels. Evidence that the N,O-diacetyl-Ang II peak does not contain a contribution from N-acetyl-Ang I includes the following: (a) as stated above, when either Ang II or Ang I were acetylated, the product was completely homogeneous (6); (b) when human plasma samples or rat kidney homogenates were spiked with ---1000 fmol of Ang I before extraction, acetylation, and HPLC, there was no increase in the measured amount of Ang II in each sample (6, 13). Nevertheless, we observed that when acetylated samples were inadvertently allowed to stand at room temperature for several hours, partial deacetylation of the N,O-diacetyl-Ang I did occur. For this reason, and because of our experience with acetylated bradykinin peptides, we take particular care to prevent hydrolysis following acetylation. Samples are injected directly onto the chromatograph following acetylation or, when an automatic injector is used, the carousel of the injector is refrigerated to ---3~ For N,O-diacetylated bradykinin peptides (Fig. 4A), it will be noted that Nacetyl-BK(1-8) elutes close to the elution position of N, O-diacetyl-BK(1-9). This is of little concern in practice, however, because BK(1-8) levels in biological samples are much less than those of BK(1-9) (3). Our experience has been that piperidine treatment of acetylated extracts does not result in an appreciable increase in the measured peaks of immunoreactivity in HPLC fractions. For routine assay, samples are N,O-diacetylated as described above. When O-deacetylation may occur, this is corrected for because all estimates of endogenous peptide levels in biological samples are corrected for the recovery of standard peptides from tissue homogenates treated in an identical manner.
Extraction of Peptides from Plasma, Blood, and Tissues Measurement of peptides in biological samples requires the prevention of peptide generation and metabolism during processing of the sample. For the measurement of angiotensin peptides in plasma, trunk blood from decapitated rats or blood obtained by venepuncture from human subjects is collected directly into 0.1 vol of an inhibitor cocktail [50 mM 1,10-phenanthro-
[19] MEASUREMENT OF ANGIOTENSIN AND BRADYKININ
337
line, 125 mM EDTA, neomycin sulfate (2 g/liter), and 2% ethanol in water] containing an appropriate renin inhibitor at a concentration sufficient to inhibit renin activity completely (6, 13). The blood is then centrifuged and the plasma immediately extracted with Sep-Pak C~8 cartridges (MilliporeWaters). Each Sep-Pak cartridge is first moistened with 3 ml of methanol and then washed with 10 ml of 1% TFA in water. To minimize nonspecific adsorption, the cartridges are next coated with 1 ml of a 1% polypeptide solution (Polypep; Sigma Chemical Company, St. Louis, MO) and washed again with methanol-water-TFA (80:19:1, v/v/v) and with 1% TFA (14). The sample is then applied and the cartridges washed with 10 ml of 1% TFA- 1% sodium chloride (1 : 1, v/v), then with 2 ml of methanol-water-TFA (30:69: 1, v/v/v), before elution with 6 ml of methanol-water-TFA (80: 19: 1, v/v/v) into a siliconized 13 x 100 mm borosilicate glass tube. The eluate is then cooled to -80~ before evaporation to dryness under vacuum. Cooling of the sample is a precaution against sample loss due to boiling in the vacuum centrifuge. Plasma extracts can then be acetylated and subjected to HPLC. The measurement of circulating levels of bradykinin peptides requires different methodology because of the need to prevent activation of plasma prekallikrein. For the measurement of bradykinin peptides in rat blood, blood is collected from either conscious rats with previously implanted arterial cannulae or from anesthetized rats by needle puncture of the aorta or inferior vena cava. Two milliliters of blood is collected directly into a syringe containing 10 ml of 4 M guanidine thiocyanate (GTC), 1% TFA in water, and the mixture is then homogenized using a Polytron with a 1-cm aggregate (model PT 10-35; Kinematica, Lucerne, Switzerland) operating at maximum speed. The mixture is then centrifuged at 5000 g for 20 min at 20~ and the supernatant extracted by Sep-Pak as described above. Blood levels of angiotensin peptides can also be measured by this method. Tissues are rapidly removed from decapitated rats, weighed, and immediately homogenized in 10 ml of GTC-TFA, sonicated briefly, and then processed as described for blood. The weight of tissue homogenized is no more than 0.5-1.0 g. Higher amounts of tissues result in lower recoveries from the extraction procedure. The use of GTC-TFA for the homogenization of tissues is a modification of the method described by Zingg et al. (15). Extracts of blood and tissue are extracted with diethyl ether before acetylation and HPLC. Each dried extract is dissolved in 1 ml of 0.1 M hydrochloric acid, then extracted twice with 1 ml of diethyl ether, frozen, and taken to dryness again under vacuum. We have previously used 1.0 M hydrochloric acid for this procedure (3, 13, 16), but 0.1 M hydrochloric acid gives equivalent results.
338
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EXTRACELLULAR PROCESSING ENZYMES IN THE CNS
The blank for each assay was assessed by extraction of 10 ml of GTC-TFA, and these blank extracts were processed as described above and then subjected to HPLC before RIA.
Validation of Methodology Details of the recoveries of peptides extracted by these methods are given elsewhere (3, 6, 13, 16). In general, peptide recoveries approximate 40-50%. A number of different approaches were used to validate the methodology described. 1. Radioimmunoassays of HPLC fractions of blank extracts gave results at or below the detection limit(3, 6). 2. Our identification of individual angiotensin and bradykinin peptides in biological samples was based on criteria additional to their recognition by the appropriate antisera. For N,O-diacetylated angiotensin and bradykinin peptides, the identified peptides were shown to elute in the same position as N,O-diacetylated standard peptides on HPLC. Moreover, in the case of bradykinin peptides, when samples were N-acetylated (piperidine treated), the identified peptides were shown to elute in the same position as N-acetylated standard peptides on HPLC. 3. C terminal-directed antisera raised against Ang II, Ang I, Ang(1-9), and BK(1-9) (3, 9, 13) were able to recognize these peptides when acetylated, although with a reduced efficiency. By using these C terminal-directed antisera we were able to confirm the identity of many of the peaks of immunoreactivity measured in HPLC fractions of biological samples by N terminaldirected RIA (3, 6, 13). 4. We studied the effect of time delay in removal of rat tissues on the measured tissue levels of angiotensin and bradykinin peptides. When a comparison was made between the left and right kidneys homogenized 30 and 90 sec after decapitation, respectively, no difference in angiotensin or bradykinin peptide levels was evident (3, 13). This does not indicate that renal angiotensin and bradykinin peptides are protected from metabolism; rather, the relatively constant level of angiotensin and bradykinin peptides during the delay before homogenization indicates that during this time peptide metabolism approximates the production rate of these peptides in the kidney. 5. To determine the stability of bradykinin peptides in GTC-TFA tissue homogenate, kidney homogenates were divided into two equal portions that were extracted either 1 or 2 hr after homogenization. No difference in the measured levels of bradykinin peptides was evident, indicating that peptides are stable in GTC-TFA tissue homogenates (3). By contrast, for GTC-TFA
[19] MEASUREMENT OF ANGIOTENSIN AND BRADYKININ
339
homogenates of blood we did observe a significant decrease in BK(1-9) levels, from 3.00 -+ 0.03 fmol/ml (mean _ 0.03 fmol/ml (mean _+ SEM, n = 6) for homogenates extracted after 1 hr, to 2.3 +__0.3 fmol/ml (p < 0.05) for homogenates extracted after 2 hr (3). 6. In another approach to assess the stability of angiotensin and bradykinin peptides in GTC-TFA tissue homogenates, kidney homogenates were divided into two equal portions, to one of which was added --~1000 fmol of either Ang II, Ang I, or BK(1-9). Subsequent analyses showed no evidence of metabolism of the added peptides, except for a small increase in B K(1-8) levels in the homogenate to which BK(1-9) was added, which represented <1% of the added amount of B K(1-9), again indicating that peptides are stable in GTC-TFA homogenates (3, 13). The same approach was used to show that metabolism of plasma angiotensin peptides did not occur during processing of plasma samples (6). 7. To examine whether homogenization of tissue in GTC-TFA causes an immediate arrest of bradykinin peptide degradation and generation and avoids the possible consequences of activation of prekallikrein, we also measured bradykinin peptide levels in kidneys that were snap-frozen. Following decapitation of each of six rats, the left kidney was immediately homogenized in GTC-TFA at room temperature, and the right kidney immediately clamped between two metal plates that had been cooled to the temperature of liquid nitrogen. The frozen kidney was then pulverized in a metal mortar and pestle that had been cooled to the temperature of liquid nitrogen. The powdered frozen kidney was added to GTC-TFA at 0~ and immediately homogenized by use of a Polytron. Similar bradykinin peptide levels were measured for kidneys that were either immediately homogenized in GTC-TFA at room temperature or rapidly cooled to the temperature of liquid nitrogen before pulverization and homogenization, thus providing further evidence for the efficiency with which bradykinin peptide degradation and generation were arrested by homogenization in GTC-TFA (3). 8. Another aspect of the process of validation was to show that the circulating and tissue levels of angiotensin and bradykinin peptides changed in the manner predicted following administration of an ACE inhibitor (3, 6, 13).
Potential Problems Many different methods for the extraction of peptides from tissue have been described. In the present studies, blood and tissues were homogenized in GTC-TFA because GTC is a potent chaotropic agent that rapidly denatures
340
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EXTRACELLULAR PROCESSING ENZYMES IN THE CNS
proteins and arrests enzymatic activity, and 1% TFA, by lowering the pH of the sample, also inactivates many proteolytic enzymes. However, it is important to be aware of the possibility that the extraction method may modify some peptides. For example, oxidation of methionine residues may occur, and Smyth (17) has described a strategy to overcome this problem. Other possibilities are the deacetylation of O-acetylated residues (18) and the deamidation of asparagine and glutamine residues by exposure to acid (19). Fortunately these potential problems do not apply to angiotensin and bradykinin peptides. We chose to homogenize fresh tissue immediately because such an approach inactivates proteolytic enzymes and thus prevents peptide generation and metabolism during subsequent processing of the sample. Many authors freeze tissues before subsequent homogenization and extraction of peptides. We have avoided the freezing of tissue because of the potential for peptide generation and metabolism during freezing and thawing. An example of the problems that may occur is shown by our study of the effect of different methods of extraction of rat hypothalami on the measured levels of angiotensin peptides (16). Angiotensin II levels in rat hypothalami are low (--~18 fmol/ g wet weight), and Ang(1-7) levels are below the minimum detectable (<6 fmol/g). However, if hypothalami that had been frozen in liquid nitrogen were allowed to thaw before homogenization in GTC-TFA, the measured levels of Ang(1-7) were --~100 fmol/g. A similar result was obtained when frozen tissue was boiled in 0.1 M hydrochloric acid (16). It is important to use an HPLC protocol that produces optimal separation of the peptides of interest because it is not possible to predict whether other immunoreactive material may elute close to the peptides of interest. This problem is illustrated by our studies of angiotensin peptides in plasma of rats administered the ACE inhibitor perindopril. Shown in Fig. 5 are HPLC profiles for plasma angiotensin peptides of a control rat (Fig. 5A) and a rat administered perindopril (4.2 mg/kg/day) in the drinking water for 7 days (Fig. 5B). Note that ACE inhibition was associated with large increases in Ang(1-7) and Ang I levels, with a reduction in Ang II levels. Fractions were not collected for measurement of Ang(1-9) levels in these experiments. Angiotensin-converting enzyme inhibition was associated with the appearance of a peak of immunoreactivity in fraction 45. The identity of this peak is not known but it is probably related to the markedly increased Ang I levels. These profiles emphasize the need to optimize HPLC conditions to achieve maximal separation of cross-reacting peptides because such an approach ensures maximal separation of other "unanticipated" peaks of immunoreactivity from the peaks of interest. Becuase of the need to differentiate authentic peptide peaks from such "unanticipated" peaks, we always assay individual fractions rather than pool fractions for RIA.
341
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FIG. 5 Characterization of angiotensin peptides in plasma of a control rat (A) and a rat administered perindopril (4.2 mg/kg/day) in the drinking water for 7 days (B). Plasma samples were extracted, N,O-diacetylated, run on HPLC, and angiotensin peptides measured by RIA with antibody A41. Fractions containing N, O-diacetylangiotensin(1-9) were not assayed in this experiment. Ac-Ang(1-7), N, O-diacetylangiotensin(1-7); Ac-Ang II, N, O-diacetylangiotensin II; Ac-Ang I, N, O-diacetylangiotensin I.
Advantages and Disadvantages of Methodology The advantages of the methodology described are as follows: (a) the combined use of HPLC and N terminal-directed antisera enables the simultaneous RIA of a number of peptides of interest. Moreover, determination of the ratio of the C terminal-truncated metabolites to the parent peptide gives important information concerning the activity of these pathways of peptide metabolism in vivo. For example, the Ang II/Ang I ratio in plasma or tissues
342
III EXTRACELLULARPROCESSING ENZYMES IN THE CNS is a sensitive indicator of Ang I conversion to Ang II at these sites in oioo; (b) an excellent separation of acetylated peptides is achieved by HPLC; (c) the RIAs are sensitive and robust. The N terminal-directed RIAs for the angiotensin peptides have sensitivities equivalent to the most sensitive described, and the N terminal-directed bradykinin RIA is severalfold more sensitive than the most sensitive described. Disadvantages of the methodology include the following: (a) the methodology is time consuming and tedious. To some extent this can be overcome by the automation of HPLC and of RIA; (b) precautions are necessary to prevent the deacetylation of labile O-acetyl groups. Alternatively, acetylated samples could be routinely treated with piperidine before HPLC; (c) acetylation may result in a lower peptide recovery than might be achieved if samples are not acetylated before HPLC; (d) the N terminal-directed RIA cannot be used to measure N terminal-extended peptides. Thus, while the antibody B24 assay is ideal for studies in the rat, where BK(1-9) is the predominant kinin peptide, this assay cannot be used to measure Lys~ which may be the predominant kinin peptide in humans (20).
Acknowledgments The studies reported here were supported by grants from the National Health and Medical Research Council of Australia, and by the National Heart Foundation of Australia.
References 1. D. J. Campbell, in "The Renin-Angiotensin System" (J. I. S. Robertson and M. G. Nicholls, eds.), p. 23.1. Gower Medical Publishing, London, 1993. 2. J. W. Ryan, Am. J. Physiol. 257, L53 (1989). 3. D. J. Campbell, A. Kladis, and A.-M. Duncan, Hypertension (Dallas) 21, 155 (1993). 4. J. Nussberger, G. R. Matsueda, R. Re, and E. Haber, J. Immunol. Methods 56, 85 (1983). 5. J. R. Stockigt, H. M. Shizgal, and W. F. Ganong, Int. Congr. Ser. Excerpta Med. 241, 290 (1972). 6. A. C. Lawrence, G. Evin, A. Kladis, and D. J. Campbell, J. Hypertens, 8, 715 (1990). 7. H. M. Geysen, S. J. Barteling, and R. H. Meloen, Proc. Natl. Acad. Sci. U.S.A. 82, 178 (1985). 8. J. Vaitukaitis, J. B. Robbins, E. Nieschlag, and G. T. Ross, J. Clin. Endocrinol. Metab. 33, 988 (1971).
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D. J. Campbell and A. Kladis, J. Hypertens. 8, 165 (1990). 10. V. Herbert, K.-S. Lau, C. W. Gottlieb, and S. J. Bleicher, J. Clin. Endocrinol. Metab. 25, 1375 (1965). 11. W. M. Hunter and F. C. Greenwood, Nature (London) 194, 495 (1962). 12. P. R. M. Dobson and P. G. Strange, in "Methods in Enzymology" (L. Birnbaumer and B. O'Malley, eds.), Vol. 109, p. 827. Academic Press, Orlando, FL, 1985. 13. D. J. Campbell, A. C. Lawrence, A. Towrie, A. Kladis, and A. J. Valentijn, Hypertension (Dallas) 18, 763 (1991). 14. K. Hermann, R. E. Lang, T. Unger, C. Bayer, and D. Ganten, J. Chromatogr. 312, 273 (1984). 15. H. H. Zingg, D. Lefebvre, and G. Almazan, J. Biol. Chem. 2,61, 12956 (1986). 16. A. C. Lawrence, I. J. Clarke, and D. J. Campbell, J. Neuroendocrinol. 4, 237 (1992). 17. D. G. Smyth, Anal. Biochem. 136, 127 (1984). 18. M. E. Goldman, M. Beaulieu, J. W. Kebabian, and R. L. Eskay, Endocrinology (Baltimore) 112, 435 (1983). 19. A. I. Smith and R. A. Lew, this volume [7]. 20. F. Alhenc-Gelas, J. Marchetti, J. Allegrini, P. Corvol, and J. Menard, Biochim. Biophys. Acta 677, 477 (1981). .