Estimation of regional metabolism and production of angiotensins

Transcription

1 Br. J. clin. Pharmac. (1989), 28, 15S-113S Estimation of regional metabolism and production of angiotensins in hypertensive subjects M. A. D. H. SCHALEKAMP, P. J. J. ADMIRAAL & F. H. M. DERKX The Department of Internal Medicine I, University Hospital Dykzigt, Erasmus University, Rotterdam, The Netherlands 1 We attempted to collect information on the metabolism and production of angiotensins (ANG) in different vascular beds in humans by constant i.v. infusion of mono-iodinated [1251]-ANG I into subjects with essential hypertension, at the time of renal vein catheterization as part of the diagnostic work-up for renovascular hypertension. 2 Blood samples were taken from the aorta and the renal, antecubital, femoral and hepatic veins. ANG I, ANG II, [1251]-ANG I and [1251]-ANG II were extracted from plasma, separated by h.p.l.c. and quantitated by radio-immunoassay or gamma counting. 3 Our studies so far are restricted to subjects on captopril 5 mg twice daily. Under ACE inhibition (4-6 h after dosing of captopril) no differences in elimination half-life and regional extraction were found between infused [1251]-ANG and unlabelled ANG I. Extraction of arterially delivered [1251]-ANG I was 8% in the kidney, 45% in the forearm, 58% in the leg and 95% in the hepato-mesenteric vascular bed. 4 Measurements of arterial and venous [1251]-ANG II levels indicated that [1251]-ANG I- II conversion had occurred in the cardio-pulmonary vascular bed. 5 ANG I production in the hepato-mesenteric region could be largely accounted for by the renin activity of circulating plasma, but in kidney and limbs only 2-3% of regionally produced ANG I could be accounted for by plasma renin activity. 6 Thus, a major fraction of regionally produced ANG I appears to be formed locally, i.e. not in circulating plasma. Further studies along these lines, also including subjects not on ACE inhibitor treatment, may help to understand better the role of circulating and local renin-angiotensin systems both in the development of hypertension and in its treatment. Such studies may also provide information on differences in ANG I-II conversion between different vascular beds and on possible differences in regional inhibition between different ACE inhibitor compounds. Keywords angiotensin converting enzyme angiotensin metabolism angiotensin conversion local angiotensin production plasma renin Introduction There is little quantitative information on the the rapid ANG I-II conversion and ANG I and II metabolism of plasma angiotensin (ANG) I in degradation occurring in the body. humans. Plasma contains angiotensin converting The classical view is that circulating ANG I is enzyme (ACE) as well as angiotensinase activity, formed by the action of circulating renin on but these activities are by far too low to explain circulating renin substrate and that ANG I is Correspondence: Dr M. A. D. H. Schalekamp, Department of Internal Medicine I, University Hospital Dykzigt, Room H362, Erasmus University, Dr Molewaterplein 4, 315 GD Rotterdam, The Netherlands 15S

2 16S M. A. D. H. Schalekamp, P. J. J. Admiraal & F. H. M. Derkx converted to ANG II at the luminal surface of the vascular endothelium, mainly in the lungs. Animal studies, however, suggest that the rate of metabolism, i.e. conversion and degradation, ofang I may be too high for circulating renin to maintain the actual plasma levels of ANG I and II (Campbell, 1987; Fei et al., 1981). Thus, part of plasma ANG I may be formed by locally acting renin. The source of this locally acting renin is unknown. It is possible that the circulating reninangiotensin (RA) system not only delivers ANG I to the blood vessel wall for conversion to ANG II, but that it also serves to deliver renin for the local production of ANG I. Through binding or uptake of plasma renin by the vessel wall or through local activation of prorenin, the local concentration of renin might reach a level higher than in plasma. Another possibility is that local ANG I formation is due to the action of locally produced rather than systemically delivered renin. There is now strong evidence for the existence of local RA systems in various tissues, including vascular tissues. It has been postulated that a vascular RA system is important for the regulation of vascular tone and the maintenance of hypertension (Dzau, 1986; Swales, 1979; Thurston et al., 1979). Studies using tracer amounts of radiolabelled angiotensins for measuring regional ANG I-II conversion and ANG I and II degradation are scarce, partly because of the instability of some earlier radio-labelled angiotensin preparations and because of the anomalous behaviour of these preparations in vivo. More recent studies using highly purified monoiodinated [1251]-labelled angiotensins have shown that such preparations can be used for in vivo experiments and such experiments have been performed for measuring intrarenal ANG I-II conversion and ANG II degradation in dogs (Navar et al., 1986; Rosival et al., 1983). We therefore explored the possibility of measuring the rate of metabolism of ANG I in humans by i.v. infusion of mono-iodinated [1251]-ANG I. Regional extraction of ANG I was calculated from measurements of the arterial and venous levels of [1251]-ANG I. From this and from measurements of the concentrations of ANG I, information could be obtained on the rate of ANG I production in different vascular beds. Parallel measurements of [1251]-ANG II provided information on ANG I-II conversion. Such information may help to understand better the role of circulating and local RA systems. The data presented in this paper are part of a more extensive study (Admiraal et al., 1989). Methods Subjects Seven subjects (age years) with essential hypertension were studied at the time they were undergoing renal vein renin sampling followed by renal angiography for diagnostic purposes. The renal arteries and the kidneys of these subjects showed no abnormalities. The subjects were on captopril 5 mg twice daily orally. Studies were performed 4-6 h after the subjects had taken the morning dose of captopril. This time was chosen for technical reasons. At this time there is measurable ANG I-II conversion but ANG I levels are still high, which makes it relatively easy to measure them accurately in small plasma samples. Infusion protocol An indwelling needle for blood sampling was placed in the antecubital vein of the left arm. After insertion of the catheters in the abdominal aorta and the inferior caval vein by the Seldinger technique, the infusion of [1251]-ANG I in the antecubital vein of the right arm was started. [1251]-ANG I was infused for 2 min at a rate of approximately 3. x 16 counts min-' per 1.73 m body surface area. Eight to 1 min after start of the infusion, when the plasma levels of [125I]_ ANG I and [1251]-ANG II had reached a plateau, the first blood samples were taken from the abdominal aorta, the hepatic vein and the antecubital vein of the left arm. In the period between 1 and 2 min after the infusion had been started a second and third series of samples were taken simultaneously from the aorta, the antecubital vein and a renal vein, first from one kidney and then from the other. A sample from the femoral vein was taken shortly before discontinuation of the infusion of [1251]-ANG I. In order to avoid ANG I generation and angiotensin conversion and degradation during blood sampling and handling of the samples, 1 ml blood samples were rapidly drawn (within 7-13 s) into plastic syringes containing.5 ml of an inhibitor solution. For collecting blood samples for angiotensin measurements the following inhibitors were used: 6.25 mm disodium EDTA, 1.25 mm 1,1- phenantroline and 1 nm of the renin inhibitor CGP 29,287 (Ciba Geigy, Basle, Switzerland) (final concentrations in blood). CGP 29,287 is a renin substrate analogue (5% inhibition of plasma renin activity at 1 nm, Wood et al., 1985). For renin measurements 5 ml blood was collected into plastic syringes containing 1,ul sodium citrate (final concentration 13 mm).

3 The plasma concentrations of either [125I]_ ANG I or [1251]-ANG II in the arterial samples taken between 8 and 2 min after the start of the infusion were not significantly different (coefficient of variation less than 4%), and this was also true for the concomitantly collected samples from the antecubital vein. For measuring the elimination half-life of [125I]-ANG I, samples were taken from the aorta 1, 2, 3 and 4 min after the infusion had been stopped. After the last blood sample had been collected the radiocontrast injection for renal angiography was given. Two subjects received a combined infusion of [1251]-ANG I and ANG I. ANG I was infused at a rate of 12 pmol min-1 per 1.73 m2 body surface area. The study protocol was approved by the Hospital Ethics Review Committee. Preparation ofradiolabelled angiotensin Ifor i. v. infusion ANG I was radiolabelled by the chloramine T method (Greenwood et al., 1963). All solutions were made in sterile pyrogene free water. The whole procedure was carried out under aseptic conditions. After separation of the labelled peptide from free 125I-iodide, the peptide was applied to a 1.6 x 1 cm Biogel-P-4 column (2-4 mesh, Bio-Rad, Richmond, CA, USA) and eluted with.5 M acetic acid containing.1 M NaCl and.1% human serum albumin. Fractions of 2.5 ml were collected and counted in a gamma counter. [1251]-ANG I was eluted from the Biogel P-4 column in two separate peaks. The first peak represented mono-iodinated [125I]_ ANG I and the second peak consisted of diiodinated [125I]-ANG I. Fractions from the first peak were pooled, sterilized by filtration through a.22 p.m Millipore membrane (Waters, Millford, MA, USA) and stored at -2 C until use. The specific radioactivity of the [1251]-ANG I preparation was approximately 3.6 x 16 counts min-' pmol-1 (74 kbq pmol-). To obtain information about the homogeneity of mono-iodinated [125I]- ANG I, a sample of the pooled fractions of the first peak was injected into the h.p.l.c. column. The h.p.l.c. elution profile of the radioactive material indicated that 99% consisted of monoiodinated [125I]-ANG I. Extraction, separation and quantitation of angiotensins Solid phase extraction cartridges (Sep-Pak C18, Waters) were conditioned with 4 ml methanol and equilibrated twice with 4 ml of cold water. Thawed plasma samples (2 ml) were applied to Local production of angiotensins 17S the cartridges. After washing with two times 4 ml of cold water the bound peptides were eluted with 2 ml methanol and collected into conical polypropylene tubes. The methanol extracts were evaporated at 4 C. Separation of the peptides in the methanol extracts was performed by h.p.l.c. using the method of Nussberger et al. (1985, 1986). We used a reversed phase Nucleosil C18 steel column of 25 x 4.6 mm and 1.m particle size equipped with a direct-connect guard column (Alltech, Eke, Belgium). Mobile phase A was.85% ortho-phosphoric acid containing.2% sodium azide (ph 2.33). Mobile phase B was methanol. The flow was 1 ml min71 and the working temperature was 45 C. The column was calibrated with pure 125I-labelled standards of ANG I, ANG II, ANG III and some of their metabolites as well as with their unlabelled counterparts. The vacuum dried plasma extracts were dissolved in 1,ul h.p.l.c. solvent (65% A/35% B), centrifuged and injected into the h.p.l.c. system. Elution was performed as follows: 65% A/35% B from -9 min followed by a linear gradient to 45% A/ 55% B until 18 min. The eluate was collected in.5 min fractions into polystyrene tubes coated with BSA. The concentrations of [1251]-ANG I and [1251]- ANG II in the h.p.l.c. fractions were measured in the gamma counter. The fractions containing ANG I and ANG II were pooled separately. They were neutralized and evaporated at 4 C. The concentrations of ANG I and ANG II were measured by radio-immunoassay. Details on these assays and on the anti-angiotensin antibodies are presented elsewhere (Admiraal et al., 1989). Measurement ofplasma renin activity Plasma renin activity (PRA) was measured by incubating plasma at 37 C, at ph for, 15, 3 and 6 min (Derkx et al., 1978). A small rise of ph by less than.2 was observed after 6 min of incubation. In order to prevent ANG I-II conversion, ANG I degradation, proreninrenin conversion and bacterial growth, the following mixture of inhibitors was added to plasma before incubation (35 pu1 of inhibitor solution per ml plasma): 5 mm disodium EDTA, 3.4 mm 8-hydroxyquinoline sulphate, 2.4 mm phenylmethylsulphonyl-fluoride, 2.2 nm aprotinin and 1 g 1-1 neomycin sulphate (final concentrations in plasma). These inhibitors do not interfere with the reaction of renin with substrate (Derkx et al., 1986). ANG I that was generated during incubation was quantitated by radio-immunoassay (Derkx

4 18S M. A. D. H. Schalekamp, P. J. J. Admiraal & F. H. M. Derkx et al., 1978). ANG I generation in the PRA assay was linear in the first 3 min of incubation, but in some samples the rate of ANG I generation was somewhat lower in the following 3 min. Only the first linear part of the ANG I generation curve was used for calculating PRA. The recovery of renin or ANG I added to plasma prior to assay was better than 98%. PRA is expressed as pmol ANG I 1-1 plasma min-' of incubation. The normal level of PRA in our laboratory is 12.7 pmol 1L min' (geometric mean), range pmol 1-1 min-. This is in close agreement with the results of the so called antibody trapping technique (Poulsen & Jorgensen, 1974). -C._ c 5r F 1 = 2C ( 1 Aorta Results Regional extraction of angiotensin I Separation patterns of radiolabelled angiotensins in extracts of plasma from subjects who received an i.v. infusion of ['251]-ANG I were in close agreement with the patterns obtained with mixtures of standard angiotensin peptides and allowed us to perform separate measurements of the plasma levels of [125I]-ANG I and [125I]_ ANG II as well as their unlabelled counterparts. Figure 1 shows an example of the separation of radiolabelled angiotensins from a plasma sample }L U~~~~~~~~~~~~~~~~~.4 ll ( DO - Antecubital vein DO _ C) Renal vein 4C C O * 2c 1C F L ~**~****** ~III Time (min) Figure 1 H.p.l.c. separation of [1251]-labelled angiotensins in plasma of a patient who received a constant i.v. infusion of [1251]-ANG I. ANG (1-1) = ANG I = 1Asp-2Arg-3Val-lTyr-5Ile-6His-7Pro-8Phe-9His- " Leu. 1 = [1251]-ANG (1-8), 2 = [125I1-ANG (2-1), 3 = [1251]-ANG (1-1).

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6 ilos M. A. D. H. Schalekamp, P. J. J. Admiraal & F. H. M. Derkx Table 2 PRA and plasma ANG I concentrations across different vascular beds PRA ANGI ANG I Subject Aorta Aorta Vein Liver and gut Kidney Forearm Leg (pmol 1- min-m ) (pmol l-) (pmol l-) (pmol l1-) (pmol 1-) (pmol 1-) Mean Mean values are geometric means. The normal concentration of PRA is 12.7 pmol 1-1 min-1 (range pmol 1-1 min-1, n = 72). The normal concentration of ANG I is 2.9 pmol 1-1 (range pmoll-',n = 18). taken at the time the plasma levels of [1251]-ANG I and [125I]-ANG II had reached a plateau during constant i.v. infusion of [1251]-ANG I. An important assumption of our study is that the rate of metabolism of radiolabelled ANG I in our subjects was not different from the metabolism of unlabelled ANG I. Results of combined [1251]-ANG I and ANG I infusions are summarized in Table 1. During infusion of the two peptides the regional vein-to-artery ratios for labelled and unlabelled ANG I were not different. After discontinuation of the infusion the [1251]-ANG I and ANG I concentrations in the aorta fell monoexponentially. The elimination half-life in subject 1 was 32 s for [1251]-ANG I and 3 s for unlabelled ANG I. In subject 2 the half-life values were 28 and 35 s respectively. PRA and angiotensin levels vary widely among subjects with essential hypertension. This was also the case in our subjects. The levels of PRA and the plasma levels of [125I]-ANG I and endogenous ANG I in the aorta and in the veins of the kidney, forearm, leg and liver during infusion of [1251]-ANG I are presented in Table 2. From measurements of the arterial and venous plasma levels of [1251]-ANG I it was calculated that 8 (2) %, was extracted by the kidney, 45 (4) % by the forearm, 58 (4) % by the leg and 95 (1) % by the hepato-mesenteric vascular bed (mean, s.e. mean) (see Table 3). We did not measure the [1251]-ANG I levels in mixed venous plasma but an approximation was made by assuming an overall extraction of arterially delivered [1251]-ANG I of 6%. The mixed venous level downstream the site where the infused [125I]-ANG I enters the central venous compartment can then be obtained by dividing the infusion rate by the cardiac output, which was taken to be 3 1 plasma min-1 per 1.73 m2 body surface area, and by adding to this quotient 4% of the arterial level of [125I]-ANG I. In this way it was calculated that approximately 3- Table 3 Regional extraction of [1251]-ANG I and venous concentration of regionally produced ANG I Subject Extraction of [1251]-ANG I Concentration of regionally produced ANG I in venous plasma Liver and gut Kidney Forearm Leg Liver and fut Kidney Forearm Leg (%) (%) (%) (%) (pmol 1- ) (pmol l-1) (pmol 1-) (pmol 1-1) Mean Mean values are arithmetic means for extraction of [1251]-ANG I and geometric means for the venous concentration of regionally produced ANG I.

7 4% of the [125I]-ANG I delivered to the cardiopulmonary circulation was extracted by this vascular bed. Regional angiotensin I-II conversion The [1251]-ANG II/[1251]-ANG I plasma concentration ratios in the antecubital and femoral veins were.68 (.19) and.83 (.24) (s.e. mean) respectively, compared with a ratio of.34 (.6) in arterial plasma (P <.5). The ["'2I]-ANG II/ [1251]-ANG I plasma concentration ratio in the renal vein was.27 (.3), which was not different from the ratio in the aorta. The ratio in the hepatic vein was.4 (.2), which was lower than the ratio in the aorta (P <.1). These results suggest that in the limbs a larger fraction of the arterially delivered [125I]-ANG I was converted to ['25I]-ANG II than in the renal and hepato-mesenteric vascular beds. Part, if not all, of the cardio-pulmonary metabolism of [1251]_ ANG I was likely to be due to [1251]-ANG I-II conversion, since the plasma level of [1251]-ANG II measured in the aorta was always higher (by a factor of 1.5 or more) than the levels in the renal, antecubital, femoral and hepatic veins. Regional production of angiotensin I In spite of the rapid extraction of arterially delivered ANG I the arterial and venous plasma levels of ANG I across the kidneys and the limbs showed little difference. The vein-to-artery ANG I concentration ratio was 1. (.7) across the kidney,.99 (.4) across the forearm, and 1. (.4) across the leg. ANG I concentration in the hepatic vein was significantly lower than in the aorta. The vein-to-artery ANG I concentration ratio across the hepato-mesenteric vascular bed was.53 (.6). Since the regional extraction of arterially delivered ANG I was known, it was possible to calculate the venous concentration of regionally produced ANG I. This is shown in Table 3. It appeared that 4-9% of ANG I in the renal, antecubital, femoral and hepatic veins did not originate from arterial blood but from regional production. Discussion Circulating ANG I and II are rapidly metabolized by the action of peptidases. ANG I is converted to ANG II, the biologically active end-product of the RA system, and both ANG I and II are degraded into smaller inactive peptides. The Local production of angiotensins ll1s term metabolism is used here to denote both conversion and degradation. In our patients on captopril treatment the extraction of arterially delivered [1251]-ANG I by the kidney, the limbs and the hepato-mesenteric vascular bed was not different from the extraction of arterially delivered unlabelled ANG I. Also the elimination half-life was not different for the two peptides. This supports the validity of using our measurements of [125I]-ANG I metabolism for estimating the metabolism and production of endogenous ANG I, at least in subjects on ACE inhibitor treatment. With such treatment ANG I metabolism is mainly due to degradation. Apparently there is little or no difference in the rate of degradation between labelled and unlabelled ANG I. Whether the conversion rates of the two peptides are also similar has to be established by measurements in subjects not on ACE inhibitor treatment. During a single passage of blood a large fraction of arterially delivered [1251]-ANG I was metabolized in the vascular beds. Regional extraction ranged from approximately 45 to 95%. With exception of the hepato-mesenteric vascular bed, where the plasma level of ANG I in the hepatic vein was lower than in the aorta, there was little arterio-venous difference in ANG I. Thus, the rapid metabolism of ANG I was matched by a high rate of production. Circulating levels of PRA were not sufficient to account for this high rate of ANG I production. The blood transit time in the kidney and in the limbs is 1 s or less (Ladefoged et al., 1967; Wegener, 1981). From this and from the data presented in Tables 2 and 3 it can be calculated that the contribution of PRA to the production ofang I in these vascular beds was less than 2-3%. The calculated 2-3% contribution of PRA is indeed a maximum value because it is possible that part of the regionally produced ANG I is metabolized before it can reach the veins. The blood transit time in the hepato-mesenteric vascular bed is 2-25 s (Wegener, 1981) so that ANG I production in this region could be largely accounted for by circulating levels of PRA. Our study provides only semi-quantitative information on ANG I-II conversion in the different vascular beds, because the rate of metabolism of ANG II was not measured. Assuming that in the absence of conversion the venous recovery of [1251]-ANG I, expressed as a percentage of arterial [1251]-ANG I, is equal to the recovery of [125I]-ANG II (equal fractional degradation), our measurements of the arterial and venous [125I]-ANG II/[125I]-ANG I plasma concentration ratios would indicate that most of the ANG I-II conversion had occurred in the

8 112S M. A. D. H. Schalekamp, P. J. J. Admiraal & F. H. M. Derkx cardio-pulmonary vascular bed, presumably in the lungs, and that some conversion had also occurred in the limbs. Our study adds new evidence to previously published data supporting the hypothesis that a major fraction of ANG I in circulating plasma is produced locally rather than in circulating plasma itself. The local production of ANG I may depend on locally synthesized renin but binding of plasmaderived renin to the vessel wall or uptake of plama renin by vascular tissue with subsequent local production of ANG I is another possibility. Further studies along these lines may reveal abnormal local metabolism or production of angiotensins in certain pathological conditions including hypertension. Abnormal angiotensin production might be independent, at least in part, of abnormal secretion of renin by the kidneys. Such studies will also provide information on differences in ANG I-1I conversion between different vascular beds and on possible differences in regional inhibition between different ACE inhibitor compounds. References Admiraal, P. J. J., Derkx, F. H. M., Danser, A. H., Pieterman, H. & Schalekamp, M. A. D. H. (1989). Metabolism and production of angiotensin I in different vascular beds in subjects with hypertension. Hypertension, (in press). Campbell, D. J. (1987). Circulating and tissue angiotensin systems. J. clin. Invest., 79, 1-6. Derkx, F. H. M., Wenting, G. J., Man in 't Veld, A. J., Verhoeven, R. P. & Schalekamp, M. A. D. H. (1979). Control of enzymatically inactive renin under various pathological conditions: implications for the interpretation of renin measurements in peripheral and renal venous plasma. Clin. Sci., 54, Derkx, F. H. M., Stuenkel, C., Schalekamp, M. P. A., Visser, W., Huisveld, I. H. & Schalekamp, M. A. D. H. (1986). Immunoreactive renin, prorenin, and enzymatically active renin in plasma during pregnancy and in women taking oral contraceptives. J. clin. Endocrinol. Metab., 63, Dzau, V. J. (1986). Significance of the vascular reninangiotension pathway. Hypertension, 8, Fei, D. T. W., Scoggins, B. A., Tregear, G. W. & Cochlan, J. P. (1981). Angiotensin I, II and III in sheep. A model of angiotensin production and metabolism. Hypertension, 3, Greenwood, F. C., Hunter, W. M. & Glover, J. S. (1963). The preparation of 131I labeled human growth hormone of high specific radioactivity. Biochem. J., 89, Ladefoged, J. & Pedersen, F. (1967). Renal blood flow, circulation times and vascular volume in normal man measured by the intra-arterial injectionexternal counting technique. Acta Physiol. Scand., 69, Navar, L. G., Rosivall, L., Carmines, P. K. & Oparil, S. (1986). Effects of locally formed angiotensin II on renal hemodynamics. Fed. Proc., 45, Nussberger, J., Brunner, D. B., Waeber, B. & Brunner, H. R. (1985). True versus immunoreactive angiotensin II in human plasma. Hypertension, 7 (suppl. I), 1-1-I-7. Nussberger, J., Brunner, D. B., Waeber, B. & Brunner, H. R. (1986). Specific measurement of angiotensin metabolites and in vitro generated angiotensin II in plasma. Hypertension, 8, Poulsen, K. & Jorgensen, J. (1974). An easy radioimmunological microassay of renin activity, concentration and substrate in human and animal plasma and tissue based on angiotensin I trapping by antibody. J. clin. Endocrinol. Metab., 39, Rosivall, L., Rinder, D. F., Champion, J., Khosla, M. C., Navar, L. G. & Oparil, S. (1983). Intrarenal angiotensin I conversion at normal and reduced blood flow in the dog. Am. J. Physiol., 245, F48- F415. Swales, J. D. (1979). Arterial wall or plasma renin in hypertension. Clin. Sci., 56, Thurston, H., Swales, J. D., Bing, R. F., Hurst, B. C. & Marks, E. S. (1979). Vascular renin-like activity and blood pressure maintenance in the rat. Hypertension, 1, Wegener,. H. (1981). Kontrastmittel. In Whole body computerized tomography, ed Wegener,. H., pp Basel: Karger. Wood, J. M., Gulati, N., Forgiarini, P., Fuhrer, W. & Hofbauer, K. G. (1985). Effects of a specific and long acting renin inhibitor in the marmoset. Hypertension, 7, Discussion Hans R. Brunner (Lausanne): You have presented some very nice studies. I am sure you would agree that it is important to be sure that the radiolabel stays on the angiotensin that you infuse. About 15 years ago, I believe, it was shown that when [125 ]-labelled angiotensin was injected, in vivo, very rapid uncoupling of the label occurred. At that time it was thought that either 14C- or 3H-labelled ANG II should be used for in vivo infusion. What evidence do you have that [1251]-labelled ANG I stays intact throughout the experiment?

9 Maarten A. D. H. Schalekamp (Rotterdam): More recent experiments with purified monoiodinated [1251I]-ANG I in animals have demonstrated that this preparation can be used for in vivo studies of ANG I metabolism (Rosivall et Local production of angiotensins 113S al., 1983). Furthermore, as I have mentioned, our studies showed no difference in regional extraction between labelled and unlabelled ANG I. Thus we had no evidence for rapid uncoupling of the radiolabel from intact ANG I.