- Email: [email protected]
Pharmacokinetics of synthetic α-human atrial natriuretic polypeptide in normal men; effect of aging
Regulatory Peptides, 19 (1987) 265-272
Elsevier
265
RPT00636
Pharmacokinetics of synthetic a-human atrial natriuretic polypeptide in normal men; effect of aging M a s a o O h a s h i 1, N o b u a k i F u j i o 1, H a j i m e N a w a t a I , K e n - i c h i K a t o 1, Hisayuki Matsuo 2 and Hiroshi Ibayashi 1 I The Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan and 2The Second Department of Biochemistry Miyazaki Medical College, Miyazaki, Japan
(Received 6 February 1987; revised version received27 May 1987; accepted 10 July 1987)
Summary We reported that plasma human atrial natriuretic polypeptide (hANP) in healthy aged men was significantly higher than that in young men, presumably due to a diminished cellular response to endogenous hANP in the aged subjects (J. Clin. Endocrinol. M e t a b . , 64 (1987) 81). To examine the effect of age on the metabolic clearance rate for hANP, synthetic ~-hANP (2/~g/kg) was administered intravenously to healthy young (n = 6) and aged (n = 4) men. The plasma hANP was measured by a direct radioimmunoassay. The disappearance of ~-hANP from plasma was characterized by a biexponential decay curve, in both groups. There was no difference of the initial phase between young and aged groups (young vs aged; 1.2 + 0.2 min vs 2.9 + 1.0 min), while the second phase of ~-hANP disappearance in the aged group was significantly prolonged compared with that in the young individuals (young vs aged; 17.3 + 3.9 min vs 34.3 + 3.0 min, P < 0.05). We tentatively conclude that the reduced metabolic clearance rates for hANP were responsible, in part, for the high plasma concentrations in the aged men. Aging; a-Human atrial natriuretic polypeptide; Radioimmunoassay; Metabolic clearance rate
Correspondence." M. Ohashi, The Third Department of Internal Medicine,Kyushu University,Faculty of Medicine, Fukuoka 812, Japan.
0167-0115/87/$03.50 © 1987 ElsevierSciencePublishers B.V. (BiomedicalDivision)
266 Introduction
Atrial natriuretic polypeptides (hANPs) have been isolated from human cardiac atria [1,2]. Among the 3 species, ~-hANP was the circulating, biologically most active form. This 28-amino acid peptide has been synthesized and was found to have significant diuretic and natriuretic effects, in human subjects [1]. Immunoreactive ANP was detected in plasma and the level significantly increased following volume-loading with sensitive radioimmunoassay [3,4]. Using a specific radioimmunoassay, we observed high plasma hANP concentrations in the aged male subjects [5]. NaC1 loading also leads to a more enhanced response in plasma hANP, in the aged subjects. The response of plasma cyclic 3"5'-guanosine monophosphate (cGMP), a putative second messenger of hANP, in the aged subjects was indistinguishable from that in the young subjects. This evidence suggests that the increased plasma hANP concentration is associated with a diminished cellular uptake of hANP in the aged subjects [5]. Synthetic ~-hANP infused into mammals is rapidly degraded [6,7]. Although the precise mechanism for the degradation is debatable, it may not depend on renal function [8]. The present study was designed to examine the effect of age on the plasma clearance for hANP.
Materials and Methods
Study protocol Six normal men aged 25-28 years (mean 26.5 years) working in Kyushu University Hospital and 4 men aged 71-77 years (mean 74 years), participated in this study. The elderly men were ambulatory, although they lived in a nursing home. Informed consent was obtained from each individual. All these 10 subjects were normotensive Japanese on no medication, and the physical and laboratory examinations, inclt/ding EKG and chest roentgenogram were normal. The clinical data of the aged subjects are summarized in Table I. Synthetic ~-hANP was obtained from the Peptide Institute (Osaka, Japan) and the purity was checked by high-performance liquid chromatography. The lyophilized peptide (500 #g) was dissolved in 5 ml saline, passed through a Millex-GV filter (0.22 pm) (Millipore Corporation, Bedford, MA, U.S.A.) and stored in 1-ml volumes at 2-4°C. Usually, preparations were tested within two weeks after being solubilized. All subjects were studied in the morning, after an overnight fast in supine position. Two peripheral venous lines were inserted - one for injection of synthetic ~-hANP, one for blood sampling. Synthetic ~-hANP (2 #g/kg b.wt.) was given into one line as a bolus injection, after two basal blood samples had been withdrawn from the opposite venous line. Blood samples were obtained at 2.5, 5.0, 10.0, 15.0, 20.0, 25.0, and 30.0 min after the injection.
Radioimmunoassay of hANP The hANP concentration in chilled plasma was measured by radioimmunoassay (RIA). Rabbit anti-~-hANP serum raised against synthetic ~-hANP conjugated to
267 TABLE I Laboratory results in the aged subjects Total protein GOT LDH 7-GTP Thyroxine
6.9-4-0.2g/dl (6.5-8.2) I0.4 :t: 2.2 U/I (8-20) 236.2 4- 7.8 U/I (60-450) 10.6 5:3.5 U/1 ((~60) 7.7 +0.5 pg/dl (4.6-12.2)
BUN Creatinine Na K C1
14.5 4-1.3 mg/dl (8-20) 0.8 +0.1 mg/dl (0.6-2.0) 143.4+ 1.9 mEq/1 (135-150) 4.5 + 0.2 mEq/1 (3.5-5.0) 102.44- 1.6 mEq/1 (90--I10)
GOT, glutamic oxaloacetic acid-transaminase; LDH, lactic acid dehydrogenase; ~,-GTP, ~-glutamyl transpeptidase; BUN, blood urea nitrogen. Values are the mean ± S.D., normal ranges in parentheses.
bovine thyroglobulin was provided by Eiken Chemical Co. Ltd. (Tokyo, Japan). This anti-ct-hANP antiserum was directed mainly against the sequence by the C-terminal in the ct-hANP molecule. The cross-reactivity of the antiserum was as follows; synthetic ~-hANP 100%,/3-hANP 90%, ~-rANP 65%, hANP-(1-26) 0.1%, ANP-(1328) 87%, ANP-(18-28) 87%, atriopeptin I < 0.1%, atriopeptin II 3.8% and atriopeptin III 87%. ~-hANP was iodinated using a lactoperoxidase method and purified by reversed-phase HPLC [3]. The assay mixture contained ['25I]~-hANP (10,000 cpm), rabbit anti-ct-hANP antiserum (final dilution, 1:80,000) and chilled plasma in 0.1 M phosphate buffer (pH 7.5), containing 50 mM NaC1, 0.1% BSA, 0.3 % dextran, 0.1% Triton X-100 and 0.1% NaN3. Antibody-bound and free ct-hANP were separated by addition of anti-rabbit IgG serum. The minimum detectable quantity was 2 pg/tube and the half maximum inhibition was produced by 15 pg/tube ~-hANP. The intra- and interassay coefficients of variations in this assay were 5.6% and 12.7%, respectively.
Gel filtration chromatography of plasma extracts Before gel filtration, plasma samples (1.0 ml) of the normal young subjects who received an i.v. bolus of ~t-hANP, were extracted, using an ODS-silica cartridge (SepPak C18 Waters Associates, Milford, MA, U.S.A.), eluted with 60% (v/v) acetonitrile/0.2% ammonium acetate (pH 4), and then lyophilized. The extracts were dissolved in 100/d of 1 M acetic acid and applied onto a Sephadex G-50 column (0.8 × 35 cm) previously equilibrated in 1 M acetic acid, at room temperature. After gel filtration, each fraction was lyophilized and reconstituted with assay buffer prior to RIA.
Pharmacokinetic analysis Multiexponential analysis of the plasma disappearance of ~-hANP was carried out using the non-linear least-square fitting method [9]. The data was fitted to the biexponential equation, Ct = A e - ' t + Be -at, where t is time, Ct is the plasma concentration of immunoreactive hANP at each time point, and A and ~, and B and/3 are the intercepts and first order rate constants for the first and second disappearance phase, respectively. The metabolic clearance rate (MCR) was calculated as the dose administered divided by the area under the plasma concentration-time curve (AUC),
268 BIT (%)
ON
~
•
\
o\
",,), \. °,
20
\~\
10
% ~ '
/
\x \o
I
I
I
i
i
i
20
40
80
160
320
640
i
1280
pg/ml
Fig. 1. Standard ~-hANP ( 0 ) RIA curves and inhibitory effect of different dilutions of the plasma of a young man given synthetic ~-hANP (2/~g/kg). Plasma at 5.0 rain (©), 10.0 rain (A) and 15.0 min (A) after the injection.
ct-hANP (pg/ml)
o~" 21ag/kg
104
103
i, \ 0 ~\
O ~
•
,g.
-
8 •
o....~.
~
!
,-
.
.
r, 3. 10z o
i 0
i 5
i 10
~ 1
I 20
I 25
i 30
Time (min)
Fig. 2. Decay of plasma ~-hANP concentrations after an i.v. bolus injection of 2.0 #g/kg ~-hANP to normal young (O) and aged (O) men.
269
calculated as AUC = A/a + B/fl for the biexponential analysis. The volume of distribution (Vd) was calculated as Vd = dose administered/(A + B). The volume of distribution at steady state (Vdss) was calculated as Vdss = dose administered
(A/~ 2 + B/fl2)/AUC 2. Results are expressed as the mean + S.E.M. Student's t-test was used for all comparisons. A value of P < 0.05 was considered to have statistical significance.
Results
The mean plasma concentration of hANP before the subjects received ~t-hANP was 45.8 + 3.3 pg/ml in the young men and 93.1 + 17.6 pg/ml in the aged men, the higher concentration in the aged subjects being statistically significant (P < 0.05). As shown in Fig. 1, in plasma samples from the subjects given ~-hANP, competition curves in the hANP RIA paralleled the reference standard ~-hANP. ~-hANP in doses of 2 #g/kg were administered to 6 young and 4 aged healthy men. The subsequent disappearance of the atrial peptide could be fitted to a biexT A B L E II Plasma ~-hANP kinetics in normal young and aged m e n
tl/2ct
tl/2fl
(min)
(min)
AUC (ng.min/ml/kg)
MCR (ml/min/kg)
Vd (ml/kg)
(ml/kg)
Young 1 2 3 4 5 6
1.62 1.17 0.77 1.35 0.70 1.57
29.4 23.0 7.1 13.7 6.5 24.0
64.9 92.1 96.3 126.7 120.6 96.9
30.8 21.7 20.8 15.8 16.6 20.6
150.1 55.4 36.2 43.4 23.0 73.4
752.1 281.2 100.1 121.1 58.9 306.5
mean S.E.M.
1.20 0.16
17.3 3.9
99.6 9.1
21.1 2.2
63.6 18.7
270.0 104.8
Aged 1 2 3 4
1.59 2.06 5.70 2.14
26.2 35.6 34.6 40.7
172.7 I 15.2 157.5 158.3
11.6 17.4 12.7 12.6
37.9 108.1 123.8 62.3
157.0 516.3 204.1 316.8
Mean S.E.M.
2.89 0.95
34.3 3.0
150.9 12.4
13.6 1.3
83.0 19.9
298.6 80.0
n.s.
P < 0.05
P < 0.01
P < 0.05
Subject
n.s.
Vdss
n.s.
/1/2, half-time of disappearance for plasma; ct and fl, first and second phases o f disappearance, respectively; A U C , area under the curve; M C R , metabolic clearance rate; Vd, volume o f central compartment; Vdds, volume o f distribution at steady state; n.s., not significant
270
Vo pg / t u be
[12Sl 1-O~-hANP
1
phenol red
1
1
200
i0
20
30
40 Fr.No.
Fig. 3. Sephadex G-50 column chromatography of plasma extracts obtained from normal young subjects given an i.v. bolus of ~-hANP (2/tg/kg). Plasma samples were obtained 2.5 min (O), 5.0 min (O), 10.0 min (A) and 15.0 min {A) after lhe injection.
ponential decay curve in all cases (Fig. 2). The relevant parameters obtained from analyses of the data shown in Fig. 2 are shown in Table II. For young subjects, the first and second phase tl/2 values were 1.2 + 0.2 min and 17.3 + 3.9 min, respectively. For the aged subjects, the first and second phase tl/2 values were 2.9 + 1.0 min and 34.3 + 3.0 min, respectively. Although there was no significant difference in the first-phase tl/2 between young and aged subjects, the slow-phase h/2 of the aged subjects was significantly longer than that of the young subjects (P < 0.05). The metabolic clearance rate of ~-hANP in the aged subjects was also markedly diminished compared with that in the young subjects (aged vs young, 13.6 + 1.3 ml/min/kg vs 21.1 + 2.2 ml/min/kg). Chromatography of the extracts from plasma samples obtained 0.5, 3, 5, 10 min after the infusion of ~-hANP indicated that immunoreactive hANP eluted at the same fraction as [12sI]~-hANP added in buffer and had no small fragment (Fig. 3).
Discussion
The RIA system we used for hANP seems to be specific, sensitive and convenient. Most of ANP RIA systems involve problems in which the plasma level of ANP is overestimated due to the marked non-specific binding to antibody, when the plasma is directly applied to the assay. G-50 column chromatographic pattern (Fig. 3) and the inhibitory effect on c~-hANP-RIA of plasma of the subjects given ~-hANP (Fig. 1) suggest that this assay retains an excellent specificity for ~-hANP. The mean basal plasma immunoreactive hANP concentrations were comparable to the level of hANP in the plasma extract prepared from Sep Pak C1 s reverse phase chromatography [5].
271 The non-specific binding reported in the above-mentioned paper was completely eliminated in the present direct RIA. Also, the present RIA system for hANP is most useful for metabolic clearance studies, because significant losses during the plasma extraction would not have to be considered. The plasma decay for synthetic ct-hANP was fitted to the biexponential disappearance curve. The rapid phase tl/2 of the aged subjects tended to be prolonged compared with that in the young subjects, however, there was no statistical signifiance, and the value was much the same as reported by Tang et al. [6] and Yandle et al. [7]. The slow phase tl/2 of the aged subjects was signficantly inhibited. It is accepted that the rapid phase of peptide disappearance represents both distribution and metabolism, whereas the slow component reflects primarily metabolism of the peptide. At least 3 factors may be responsible for the metabolism of plasma hANP: (1) hepatorenal extraction; (2) receptor-mediated internalization; (3) degradation with plasma factors. Murthy et al. [8] stated that the in vivo disappearance of ANP from plasma is probably due to binding to receptors in the cells since the in vitro incubation of ANP with rat plasma caused only a slight loss in immunoreactivity in the first 5 min. From the observations that hepatectomy and nephrectomy caused no major prolongation of the disappearance rate, they suggested that these two organs may not be the primary sites involved in the removal of this peptide from the circulation [8]. There has been no documentation on the presence of hANP specific degrading enzyme in the plasma. In the gel filtration analysis, only a trace amount of smaller fragments were identified and most of the infused ct-hANP remained intact in the plasma of men given ~-hANP. These findings support the view that the administered ~-hANP does not undergo proteolytic degradation in the plasma. The receptor for ANP in cultured smooth muscle cells was found to bind specifically to ANP and was subsequently internalized into the cells, much like events seen with receptors for insulin, epidermal growth factor and low-density lipoprotein [10-12]. This evidence supports the hypothesis that most of the circulating ANP could be metabolized by receptor-mediated degradation. If this postulation is valid, then the prolonged second phase T1/2 may be due to decreased receptor functions in the aged subjects. However, we could not neglect the possibility that the relatively high concentration of endogenous plasma hANP may influence the degradation of infused synthetic ~-hANP in the aged subjects. In summary, a diminished degrading capacity of synthetic ~-hANP was evident in the healthy aged men and such an alteration may contribute to the high plasma concentration of hANP in aged subjects.
Acknowledgements We wish to thank Dr. S. Higuchi in the Department of Pharmacy, Kyushu University Hospital for the helpful discussions and for a computed analysis of the data, M. Ohara for critical comments, and Y. Mori for the excellent secretarial services.
272
References 1 Kangawa, K. and Matsuo, H., Purification and complete amino acid sequence of ~-human atrial natriuretic polypeptide (~-hANP), Biochem. Biophys. Res. Commun., 118 (1984) 131-139. 2 Kangawa, K., Fukuda, A. and Matsuo, H., Structural identification offl and y-human atrial natriuretic polypeptide, Nature (London), 313 (1985) 397-400. 3 Miyata, A., Kangawa, K., Toshimori, T., Hatoh, T. and Matsuo, H., Molecular forms of atrial natriuretic polypeptides in mammalian tissues and plasma, Biochem. Biophys. Res. Commun., 129 (1985) 248-255. 4 Lang, R.E., Tholken, H., Ganten, D., Luft, F.C., Ruskoako, H. and Unger, T., Atrial natriuretic factor - a circulating hormone stimulated by volume loading, Nature (London), 314 (1985) 264-266. 5 Ohashi, M., Fujio, N., Nawata, H., Kato, K., Ibayashi, H., Kangawa, K. and Matsuo, H., High plasma concentrations of human atrial natriuretic polypeptide in aged men, J. Clin. Endocrinol. Metab., 64 (1987) 81-85. 6 Tang, J., Webber, R.J., Chang, D , Chang, J.K., Kiang, J. and Wei, E.T., Depressor and natriuretic activities of several atrial peptides, Regul. Peptides, 9 (1984) 53-59. 7 Yandle, T.G., Richard, A.M., Nicholls, M.G., Cuneo, R., Espiner, E.A. and Livesey, J.H., Metabolic clearance rate and plasma half life of alpha-human atrial natriuretic peptide in man, Life Sci., 38 (1986) 1827-1833. 8 Murthy, K.K., Thibault, G., Schiffrin, E.L., Garcia, R., Chartier, L., Gutkowska, J., Genest, J. and Cantin, M., Disappearance of atrial natriuretic factor from circulation in the rat, Peptides, 7 (1986) 241-246. 9 Wagner, J.G., Linear pharmacokinetic equations allowing direct calculation of many needed pharmacokinetic parameters from the coefficients and exponents of polyexponential equations which have been fitted to the data, J. Pharmacokinet. Biopharmacol. 4 (1976) 443-467. 10 Goldstein, J.L. and Brown, M.S., The low-density lipoprotein pathway and its relationship to atherosclerosis, Annu. Rev. Biochem., 46 (1977) 897-930. 11 Gorden, P., Carpentier, J.-L., Freychet, P. and Orci, L., Internalization of polypeptide hormones. Mechanism, intracellular localization and significance, Diabetologia, 18 (1980) 263-274. 12 Hirata Y., Tomita, M., Yoshimi, H. and Ikeda, M., Specific receptors for atrial natriuretic factor (ANF) in cultured vascular smooth muscle cells of rat aorta, Biochem. Biophys. Res. Commun., 125 (1984) 562-568.
Elsevier
265
RPT00636
Pharmacokinetics of synthetic a-human atrial natriuretic polypeptide in normal men; effect of aging M a s a o O h a s h i 1, N o b u a k i F u j i o 1, H a j i m e N a w a t a I , K e n - i c h i K a t o 1, Hisayuki Matsuo 2 and Hiroshi Ibayashi 1 I The Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan and 2The Second Department of Biochemistry Miyazaki Medical College, Miyazaki, Japan
(Received 6 February 1987; revised version received27 May 1987; accepted 10 July 1987)
Summary We reported that plasma human atrial natriuretic polypeptide (hANP) in healthy aged men was significantly higher than that in young men, presumably due to a diminished cellular response to endogenous hANP in the aged subjects (J. Clin. Endocrinol. M e t a b . , 64 (1987) 81). To examine the effect of age on the metabolic clearance rate for hANP, synthetic ~-hANP (2/~g/kg) was administered intravenously to healthy young (n = 6) and aged (n = 4) men. The plasma hANP was measured by a direct radioimmunoassay. The disappearance of ~-hANP from plasma was characterized by a biexponential decay curve, in both groups. There was no difference of the initial phase between young and aged groups (young vs aged; 1.2 + 0.2 min vs 2.9 + 1.0 min), while the second phase of ~-hANP disappearance in the aged group was significantly prolonged compared with that in the young individuals (young vs aged; 17.3 + 3.9 min vs 34.3 + 3.0 min, P < 0.05). We tentatively conclude that the reduced metabolic clearance rates for hANP were responsible, in part, for the high plasma concentrations in the aged men. Aging; a-Human atrial natriuretic polypeptide; Radioimmunoassay; Metabolic clearance rate
Correspondence." M. Ohashi, The Third Department of Internal Medicine,Kyushu University,Faculty of Medicine, Fukuoka 812, Japan.
0167-0115/87/$03.50 © 1987 ElsevierSciencePublishers B.V. (BiomedicalDivision)
266 Introduction
Atrial natriuretic polypeptides (hANPs) have been isolated from human cardiac atria [1,2]. Among the 3 species, ~-hANP was the circulating, biologically most active form. This 28-amino acid peptide has been synthesized and was found to have significant diuretic and natriuretic effects, in human subjects [1]. Immunoreactive ANP was detected in plasma and the level significantly increased following volume-loading with sensitive radioimmunoassay [3,4]. Using a specific radioimmunoassay, we observed high plasma hANP concentrations in the aged male subjects [5]. NaC1 loading also leads to a more enhanced response in plasma hANP, in the aged subjects. The response of plasma cyclic 3"5'-guanosine monophosphate (cGMP), a putative second messenger of hANP, in the aged subjects was indistinguishable from that in the young subjects. This evidence suggests that the increased plasma hANP concentration is associated with a diminished cellular uptake of hANP in the aged subjects [5]. Synthetic ~-hANP infused into mammals is rapidly degraded [6,7]. Although the precise mechanism for the degradation is debatable, it may not depend on renal function [8]. The present study was designed to examine the effect of age on the plasma clearance for hANP.
Materials and Methods
Study protocol Six normal men aged 25-28 years (mean 26.5 years) working in Kyushu University Hospital and 4 men aged 71-77 years (mean 74 years), participated in this study. The elderly men were ambulatory, although they lived in a nursing home. Informed consent was obtained from each individual. All these 10 subjects were normotensive Japanese on no medication, and the physical and laboratory examinations, inclt/ding EKG and chest roentgenogram were normal. The clinical data of the aged subjects are summarized in Table I. Synthetic ~-hANP was obtained from the Peptide Institute (Osaka, Japan) and the purity was checked by high-performance liquid chromatography. The lyophilized peptide (500 #g) was dissolved in 5 ml saline, passed through a Millex-GV filter (0.22 pm) (Millipore Corporation, Bedford, MA, U.S.A.) and stored in 1-ml volumes at 2-4°C. Usually, preparations were tested within two weeks after being solubilized. All subjects were studied in the morning, after an overnight fast in supine position. Two peripheral venous lines were inserted - one for injection of synthetic ~-hANP, one for blood sampling. Synthetic ~-hANP (2 #g/kg b.wt.) was given into one line as a bolus injection, after two basal blood samples had been withdrawn from the opposite venous line. Blood samples were obtained at 2.5, 5.0, 10.0, 15.0, 20.0, 25.0, and 30.0 min after the injection.
Radioimmunoassay of hANP The hANP concentration in chilled plasma was measured by radioimmunoassay (RIA). Rabbit anti-~-hANP serum raised against synthetic ~-hANP conjugated to
267 TABLE I Laboratory results in the aged subjects Total protein GOT LDH 7-GTP Thyroxine
6.9-4-0.2g/dl (6.5-8.2) I0.4 :t: 2.2 U/I (8-20) 236.2 4- 7.8 U/I (60-450) 10.6 5:3.5 U/1 ((~60) 7.7 +0.5 pg/dl (4.6-12.2)
BUN Creatinine Na K C1
14.5 4-1.3 mg/dl (8-20) 0.8 +0.1 mg/dl (0.6-2.0) 143.4+ 1.9 mEq/1 (135-150) 4.5 + 0.2 mEq/1 (3.5-5.0) 102.44- 1.6 mEq/1 (90--I10)
GOT, glutamic oxaloacetic acid-transaminase; LDH, lactic acid dehydrogenase; ~,-GTP, ~-glutamyl transpeptidase; BUN, blood urea nitrogen. Values are the mean ± S.D., normal ranges in parentheses.
bovine thyroglobulin was provided by Eiken Chemical Co. Ltd. (Tokyo, Japan). This anti-ct-hANP antiserum was directed mainly against the sequence by the C-terminal in the ct-hANP molecule. The cross-reactivity of the antiserum was as follows; synthetic ~-hANP 100%,/3-hANP 90%, ~-rANP 65%, hANP-(1-26) 0.1%, ANP-(1328) 87%, ANP-(18-28) 87%, atriopeptin I < 0.1%, atriopeptin II 3.8% and atriopeptin III 87%. ~-hANP was iodinated using a lactoperoxidase method and purified by reversed-phase HPLC [3]. The assay mixture contained ['25I]~-hANP (10,000 cpm), rabbit anti-ct-hANP antiserum (final dilution, 1:80,000) and chilled plasma in 0.1 M phosphate buffer (pH 7.5), containing 50 mM NaC1, 0.1% BSA, 0.3 % dextran, 0.1% Triton X-100 and 0.1% NaN3. Antibody-bound and free ct-hANP were separated by addition of anti-rabbit IgG serum. The minimum detectable quantity was 2 pg/tube and the half maximum inhibition was produced by 15 pg/tube ~-hANP. The intra- and interassay coefficients of variations in this assay were 5.6% and 12.7%, respectively.
Gel filtration chromatography of plasma extracts Before gel filtration, plasma samples (1.0 ml) of the normal young subjects who received an i.v. bolus of ~t-hANP, were extracted, using an ODS-silica cartridge (SepPak C18 Waters Associates, Milford, MA, U.S.A.), eluted with 60% (v/v) acetonitrile/0.2% ammonium acetate (pH 4), and then lyophilized. The extracts were dissolved in 100/d of 1 M acetic acid and applied onto a Sephadex G-50 column (0.8 × 35 cm) previously equilibrated in 1 M acetic acid, at room temperature. After gel filtration, each fraction was lyophilized and reconstituted with assay buffer prior to RIA.
Pharmacokinetic analysis Multiexponential analysis of the plasma disappearance of ~-hANP was carried out using the non-linear least-square fitting method [9]. The data was fitted to the biexponential equation, Ct = A e - ' t + Be -at, where t is time, Ct is the plasma concentration of immunoreactive hANP at each time point, and A and ~, and B and/3 are the intercepts and first order rate constants for the first and second disappearance phase, respectively. The metabolic clearance rate (MCR) was calculated as the dose administered divided by the area under the plasma concentration-time curve (AUC),
268 BIT (%)
ON
~
•
\
o\
",,), \. °,
20
\~\
10
% ~ '
/
\x \o
I
I
I
i
i
i
20
40
80
160
320
640
i
1280
pg/ml
Fig. 1. Standard ~-hANP ( 0 ) RIA curves and inhibitory effect of different dilutions of the plasma of a young man given synthetic ~-hANP (2/~g/kg). Plasma at 5.0 rain (©), 10.0 rain (A) and 15.0 min (A) after the injection.
ct-hANP (pg/ml)
o~" 21ag/kg
104
103
i, \ 0 ~\
O ~
•
,g.
-
8 •
o....~.
~
!
,-
.
.
r, 3. 10z o
i 0
i 5
i 10
~ 1
I 20
I 25
i 30
Time (min)
Fig. 2. Decay of plasma ~-hANP concentrations after an i.v. bolus injection of 2.0 #g/kg ~-hANP to normal young (O) and aged (O) men.
269
calculated as AUC = A/a + B/fl for the biexponential analysis. The volume of distribution (Vd) was calculated as Vd = dose administered/(A + B). The volume of distribution at steady state (Vdss) was calculated as Vdss = dose administered
(A/~ 2 + B/fl2)/AUC 2. Results are expressed as the mean + S.E.M. Student's t-test was used for all comparisons. A value of P < 0.05 was considered to have statistical significance.
Results
The mean plasma concentration of hANP before the subjects received ~t-hANP was 45.8 + 3.3 pg/ml in the young men and 93.1 + 17.6 pg/ml in the aged men, the higher concentration in the aged subjects being statistically significant (P < 0.05). As shown in Fig. 1, in plasma samples from the subjects given ~-hANP, competition curves in the hANP RIA paralleled the reference standard ~-hANP. ~-hANP in doses of 2 #g/kg were administered to 6 young and 4 aged healthy men. The subsequent disappearance of the atrial peptide could be fitted to a biexT A B L E II Plasma ~-hANP kinetics in normal young and aged m e n
tl/2ct
tl/2fl
(min)
(min)
AUC (ng.min/ml/kg)
MCR (ml/min/kg)
Vd (ml/kg)
(ml/kg)
Young 1 2 3 4 5 6
1.62 1.17 0.77 1.35 0.70 1.57
29.4 23.0 7.1 13.7 6.5 24.0
64.9 92.1 96.3 126.7 120.6 96.9
30.8 21.7 20.8 15.8 16.6 20.6
150.1 55.4 36.2 43.4 23.0 73.4
752.1 281.2 100.1 121.1 58.9 306.5
mean S.E.M.
1.20 0.16
17.3 3.9
99.6 9.1
21.1 2.2
63.6 18.7
270.0 104.8
Aged 1 2 3 4
1.59 2.06 5.70 2.14
26.2 35.6 34.6 40.7
172.7 I 15.2 157.5 158.3
11.6 17.4 12.7 12.6
37.9 108.1 123.8 62.3
157.0 516.3 204.1 316.8
Mean S.E.M.
2.89 0.95
34.3 3.0
150.9 12.4
13.6 1.3
83.0 19.9
298.6 80.0
n.s.
P < 0.05
P < 0.01
P < 0.05
Subject
n.s.
Vdss
n.s.
/1/2, half-time of disappearance for plasma; ct and fl, first and second phases o f disappearance, respectively; A U C , area under the curve; M C R , metabolic clearance rate; Vd, volume o f central compartment; Vdds, volume o f distribution at steady state; n.s., not significant
270
Vo pg / t u be
[12Sl 1-O~-hANP
1
phenol red
1
1
200
i0
20
30
40 Fr.No.
Fig. 3. Sephadex G-50 column chromatography of plasma extracts obtained from normal young subjects given an i.v. bolus of ~-hANP (2/tg/kg). Plasma samples were obtained 2.5 min (O), 5.0 min (O), 10.0 min (A) and 15.0 min {A) after lhe injection.
ponential decay curve in all cases (Fig. 2). The relevant parameters obtained from analyses of the data shown in Fig. 2 are shown in Table II. For young subjects, the first and second phase tl/2 values were 1.2 + 0.2 min and 17.3 + 3.9 min, respectively. For the aged subjects, the first and second phase tl/2 values were 2.9 + 1.0 min and 34.3 + 3.0 min, respectively. Although there was no significant difference in the first-phase tl/2 between young and aged subjects, the slow-phase h/2 of the aged subjects was significantly longer than that of the young subjects (P < 0.05). The metabolic clearance rate of ~-hANP in the aged subjects was also markedly diminished compared with that in the young subjects (aged vs young, 13.6 + 1.3 ml/min/kg vs 21.1 + 2.2 ml/min/kg). Chromatography of the extracts from plasma samples obtained 0.5, 3, 5, 10 min after the infusion of ~-hANP indicated that immunoreactive hANP eluted at the same fraction as [12sI]~-hANP added in buffer and had no small fragment (Fig. 3).
Discussion
The RIA system we used for hANP seems to be specific, sensitive and convenient. Most of ANP RIA systems involve problems in which the plasma level of ANP is overestimated due to the marked non-specific binding to antibody, when the plasma is directly applied to the assay. G-50 column chromatographic pattern (Fig. 3) and the inhibitory effect on c~-hANP-RIA of plasma of the subjects given ~-hANP (Fig. 1) suggest that this assay retains an excellent specificity for ~-hANP. The mean basal plasma immunoreactive hANP concentrations were comparable to the level of hANP in the plasma extract prepared from Sep Pak C1 s reverse phase chromatography [5].
271 The non-specific binding reported in the above-mentioned paper was completely eliminated in the present direct RIA. Also, the present RIA system for hANP is most useful for metabolic clearance studies, because significant losses during the plasma extraction would not have to be considered. The plasma decay for synthetic ct-hANP was fitted to the biexponential disappearance curve. The rapid phase tl/2 of the aged subjects tended to be prolonged compared with that in the young subjects, however, there was no statistical signifiance, and the value was much the same as reported by Tang et al. [6] and Yandle et al. [7]. The slow phase tl/2 of the aged subjects was signficantly inhibited. It is accepted that the rapid phase of peptide disappearance represents both distribution and metabolism, whereas the slow component reflects primarily metabolism of the peptide. At least 3 factors may be responsible for the metabolism of plasma hANP: (1) hepatorenal extraction; (2) receptor-mediated internalization; (3) degradation with plasma factors. Murthy et al. [8] stated that the in vivo disappearance of ANP from plasma is probably due to binding to receptors in the cells since the in vitro incubation of ANP with rat plasma caused only a slight loss in immunoreactivity in the first 5 min. From the observations that hepatectomy and nephrectomy caused no major prolongation of the disappearance rate, they suggested that these two organs may not be the primary sites involved in the removal of this peptide from the circulation [8]. There has been no documentation on the presence of hANP specific degrading enzyme in the plasma. In the gel filtration analysis, only a trace amount of smaller fragments were identified and most of the infused ct-hANP remained intact in the plasma of men given ~-hANP. These findings support the view that the administered ~-hANP does not undergo proteolytic degradation in the plasma. The receptor for ANP in cultured smooth muscle cells was found to bind specifically to ANP and was subsequently internalized into the cells, much like events seen with receptors for insulin, epidermal growth factor and low-density lipoprotein [10-12]. This evidence supports the hypothesis that most of the circulating ANP could be metabolized by receptor-mediated degradation. If this postulation is valid, then the prolonged second phase T1/2 may be due to decreased receptor functions in the aged subjects. However, we could not neglect the possibility that the relatively high concentration of endogenous plasma hANP may influence the degradation of infused synthetic ~-hANP in the aged subjects. In summary, a diminished degrading capacity of synthetic ~-hANP was evident in the healthy aged men and such an alteration may contribute to the high plasma concentration of hANP in aged subjects.
Acknowledgements We wish to thank Dr. S. Higuchi in the Department of Pharmacy, Kyushu University Hospital for the helpful discussions and for a computed analysis of the data, M. Ohara for critical comments, and Y. Mori for the excellent secretarial services.
272
References 1 Kangawa, K. and Matsuo, H., Purification and complete amino acid sequence of ~-human atrial natriuretic polypeptide (~-hANP), Biochem. Biophys. Res. Commun., 118 (1984) 131-139. 2 Kangawa, K., Fukuda, A. and Matsuo, H., Structural identification offl and y-human atrial natriuretic polypeptide, Nature (London), 313 (1985) 397-400. 3 Miyata, A., Kangawa, K., Toshimori, T., Hatoh, T. and Matsuo, H., Molecular forms of atrial natriuretic polypeptides in mammalian tissues and plasma, Biochem. Biophys. Res. Commun., 129 (1985) 248-255. 4 Lang, R.E., Tholken, H., Ganten, D., Luft, F.C., Ruskoako, H. and Unger, T., Atrial natriuretic factor - a circulating hormone stimulated by volume loading, Nature (London), 314 (1985) 264-266. 5 Ohashi, M., Fujio, N., Nawata, H., Kato, K., Ibayashi, H., Kangawa, K. and Matsuo, H., High plasma concentrations of human atrial natriuretic polypeptide in aged men, J. Clin. Endocrinol. Metab., 64 (1987) 81-85. 6 Tang, J., Webber, R.J., Chang, D , Chang, J.K., Kiang, J. and Wei, E.T., Depressor and natriuretic activities of several atrial peptides, Regul. Peptides, 9 (1984) 53-59. 7 Yandle, T.G., Richard, A.M., Nicholls, M.G., Cuneo, R., Espiner, E.A. and Livesey, J.H., Metabolic clearance rate and plasma half life of alpha-human atrial natriuretic peptide in man, Life Sci., 38 (1986) 1827-1833. 8 Murthy, K.K., Thibault, G., Schiffrin, E.L., Garcia, R., Chartier, L., Gutkowska, J., Genest, J. and Cantin, M., Disappearance of atrial natriuretic factor from circulation in the rat, Peptides, 7 (1986) 241-246. 9 Wagner, J.G., Linear pharmacokinetic equations allowing direct calculation of many needed pharmacokinetic parameters from the coefficients and exponents of polyexponential equations which have been fitted to the data, J. Pharmacokinet. Biopharmacol. 4 (1976) 443-467. 10 Goldstein, J.L. and Brown, M.S., The low-density lipoprotein pathway and its relationship to atherosclerosis, Annu. Rev. Biochem., 46 (1977) 897-930. 11 Gorden, P., Carpentier, J.-L., Freychet, P. and Orci, L., Internalization of polypeptide hormones. Mechanism, intracellular localization and significance, Diabetologia, 18 (1980) 263-274. 12 Hirata Y., Tomita, M., Yoshimi, H. and Ikeda, M., Specific receptors for atrial natriuretic factor (ANF) in cultured vascular smooth muscle cells of rat aorta, Biochem. Biophys. Res. Commun., 125 (1984) 562-568.