Research articles

By Dr. Maria Jesus Ramirez-Exposito , Dr. Marce Arrazola , Dr. Jose Manuel Arias de Saavedra , Dr. Rafael Agesta , Dr. Maria Pilar Carrera , Dr. Maria Dolores Mayas , Dr. Jose Manuel Martinez-Martos
Corresponding Author Dr. Jose Manuel Martinez-Martos
Health Sciences, University of Jaen, Campus Universitario Las Lagunillas - Spain E-23071
Submitting Author Dr. Jose Manuel Martinez-Martos
Other Authors Dr. Maria Jesus Ramirez-Exposito
Health Sciences, - Spain

Dr. Marce Arrazola
Pharmacology, - Spain

Dr. Jose Manuel Arias de Saavedra
University Hospital of Jaen, - Spain

Dr. Rafael Agesta
University Hospital of Jaen, - Spain

Dr. Maria Pilar Carrera
Health Sciences, - Spain

Dr. Maria Dolores Mayas
Health Sciences, - Spain


Aminopeptidase N, Aminopeptidase B, Vasopressin-degrading activity, Ageing, Serum, Human

Ramirez-Exposito M, Arrazola M, Arias de Saavedra J, Agesta R, Carrera M, Mayas M, et al. Human serum angiotensinase and vasopressin-degrading activities from childhood to elderly. WebmedCentral AGING 2010;1(9):WMC00736
doi: 10.9754/journal.wmc.2010.00736
Submitted on: 24 Sep 2010 05:15:48 PM GMT
Published on: 24 Sep 2010 07:02:25 PM GMT


The increase of blood pressure with development and ageing has been extensively reported. The aim of this study is to analyse the changes in the angiotensinase and vasopressin-degrading activities in men and women in various age groups. Aminopeptidase N (APN), aminopeptidase B (APB) and vasopressin-degrading activity (AVP-DA) were measured fluorometrically using arylamide derivatives as substrates. All the activities decreased during ageing. The correlation between aminopeptidase activities and age in males and females demonstrated only a highly significant inverse correlation for APB in females. These results could indicate the maintenance or the increase in Ang III and vasopressin in plasma, which may contribute to the increase in blood pressure observed during ageing.


Determination of serum aminopeptidases (AP) is useful in clinical diagnosis for several human diseases. However, in physiological conditions, these enzymes also play a role in the regulation of circulating biologically active peptides. Therefore, hormonal changes in serum may thus be reflected in this enzymatic activity [1].

Development and ageing are characterised by a progressive increase in blood pressure in which plasma renin-angiotensin system (RAS) and arginine-vasopressin (AVP) may play a significant role. It has been described that human serum aminopeptidase A (AP A), the enzyme responsible for the hydrolysis of Ang II to Ang III, increases during development and ageing, and the nature of this increase differs in women and men [2]. Therefore, to better understand the role of AP in blood pressure control, we analysed serum Ang III- and AVP-degrading activities in women and men during development and ageing. We measured aminopeptidase N (APN; EC and aminopeptidase B (APB; EC, reported as capable to hydrolyse Ang III [3], and AVP-degrading activity (AVP-DA; EC [4], using arylamide derivatives as substrates, in various age groups.


We used blood samples, obtained by venipuncture without additives, from 148 females aged 2 to 87 years and 139 males aged 1 to 92 years. All participants were ambulatory subjects who came to the Hospital for health screening. The samples were centrifuged at 4ºC for 10 min at 3000 x g, and the sera were analysed the same day. When subjects came to the hospital for health screening, they were required to inform whether or not they had some known disease, taking drugs and also asked for alcohol consumption. Blood samples were separated into aliquots for determinations of aminopeptidase activities and total protein content. Haemolytic, icteric, and turbid samples were discarded. The selected subjects reported to have no known disease, taking no drugs and no alcohol consumption.

APN, APB and AVP-DA were measured fluorometrically as previously described [2] using Ala-, Arg- and Cys-ß-naphthylamide as substrates. Ten microliters of each supernatant was incubated during 30 min at 25ºC with 1 ml of the substrate solution (2.14 mg/100 ml Ala-ß-naphthylamide or 3.35 mg/ 100 ml Arg-ß-naphthylamide or 5.63 mg/100 ml Cys-ß-naphthylamide), 10 mg/100 ml bovine serum albumin (BSA), and 10 mg/100 ml dithiothreitol (DTT) in 50 mM of phosphate buffer, pH 7.4, for APN and APB; and 50 mM HCl-Tris buffer, pH 6, for AVP-DA. All the reactions were stopped by adding 1 ml of 0.1 mol/L acetate buffer, pH 4.2. The amount of ß-naphthylamine, released as a result of enzymatic activity, was measured fluorometrically at 412 nm emission wavelength with an excitation wavelength of 345 nm. This rapid fluorometric method permits quantify aminopeptidase activities in the order of 10-14 mole. Serum specific aminopeptidase activities were expressed as pmol of substrate hydrolysed per min per mg of protein. Fluorogenic assays were linear with respect to time of hydrolysis and protein content.

 For statistical analysis, to be able to compare sex, the male and female subjects were divided into five age-matched groups (Table 1) which included the main stages of development. We used one-way analysis of variance (ANOVA) to analyse differences between groups. Linear correlation coefficients were calculated to test relationships between two variables. All comparisons with P values below 0.05 were considered significant.


Values (mean ± SEM) of specific aminopeptidase activities in serum of the different age groups in female and male subjects are showed in Table 2. Significant age-related changes were observed in males for APN (P < 0.05), in females for APB (P < 0.05) and in females for AVP-DA (P< 0.05). In general, all the activities decreased during ageing and reached statistical significance in the groups of 46-65 and > 66 years old. Sex differences were observed only for AVP-DA in the group of 16-45 years old (P<0.05), being higher in males than in females. In the whole population, according to the linear model of the regression analysis, APB (r = -0.14, P < 0.01) and AVP-DA (r = -0.13, P < 0.02) showed a significant inverse correlation with age, whereas no correlation was observed for APN (r= -0.10, P < 0.08). The correlation between AP activities and age in males and females considered separately demonstrated a highly significant inverse correlation for APB in females (r=-0.24. P<0.0001), whereas no correlation were observed for APN or AVP-DA. No correlation were demonstrated in males, but a borderline inverse correlation was found for AVP-DA (r=-0.15, P=0.06).


Blood pressure, particularly systolic blood pressure, tends to increase progressively with age. Blood pressure increases more in males than in females among young people, but later increases more notably with age in females and values in elderly males and females are similar. This behaviour is similar in several international populations, as described by the Hypertension optimal treatment (HOT) [5] and other studies [6-8].  In the RAS, plasma renin activity and Ang II decline progressively with age. The decrease in plasma renin activity is exponential, being very rapid in early childhood and continuing into old age [9]. The contractile response to Ang II decrease also with age [10]. This age-related profile is the opposite of the one described previously for APA activity [2]. Therefore, the inverse correlation between peptide level and peptide-hydrolysing activity suggests a relation between high breakdown capacity and low level of substrate, which in turn supports a role for human plasma APA activity as an angiotensinase. However, these observations appear incompatible with the mentioned tendency of blood pressure to increase with age and for Ang II-induced contractility to decline [2]. Therefore, additional factors must be involved in this phenomenon, which could include changes with ageing that result in gradually increasing rigidity of vessels and the role of other vasoactive peptides such as Ang III or AVP.

Increased levels of APA [2] imply a high metabolism of Ang II and consequently an increase in the formation of Ang III, which is further converted to Ang IV by APB and/or APN. APN can lower blood pressure when is intracerebroventricularly infused in spontaneously hypertensive rats, probably as a result of the increased metabolism of Ang III. Pretreatment with the Ang II antagonist losartan significantly attenuated this hypotensive effect [11]. However, it has been suggested recently that a central component might be responsible for the sympathetic hyperactivity observed at early stages of hypertension [12]. In fact, hyperactivity of the brain RAS has been implicated in the development and maintenance of high blood pressure [13,14]. In the brain RAS, Ang III exhibits the same affinity for type 1 and type 2 Ang II receptors than Ang II, and both peptides cause similar increases in vasopressin release and blood pressure [15-17]. The inhibition of AlaAP induces vasopressin release by increasing the half-life of brain Ang III [3]. On the other hand, while ageing-related decreases in AVP concentration have been found in rat brain [18], an increase has been noted in rat [18] and human [19] plasma. This may be due in part, to the here reported decreasing AVP-DA during ageing.

According with the present results, there is no change or even a decrease in APN, APB and AVP-DA during ageing. Therefore, presumably due to their reduced metabolism, Ang III and AVP would maintain or increase their levels in plasma, which may contribute to the increase in blood pressure observed during ageing in male and female through central nervous system mechanisms. Our results also showed a significant decrease of APB activity in female but not in male at the age of 46-65. However, blood pressure in female is similar or lower than in male. This discrepancy could be explained by the existence of a light (non significant) decrease on male APB, which could explain this similar blood pressure in female and male. The significant decrease on APB activity in female may be also related with other factors. In fact, it has been described an important influence of sex steroids on several serum aminopeptidase activities [1], and this female age-group (46-65) is characterized by important changes in sex hormone levels due to menopause.


Supported by Junta de Andalucía through PAIDI BIO-296.


1. Martínez JM, Ramirez MJ, Prieto I, Alba F, Ramírez M. Sex differences and in vitro effects of steroids on serum aminopeptidase activities. Peptides 1999; 19: 1637-1640.
2. Martínez JM, Prieto I, Ramirez MJ, de Gasparo M, Hermoso F, Arias JM, Alba F, Ramirez M. Sex differences and age-related changes in human serum aminopeptidase A activity. Clin Chim Acta 1998; 274: 53-56.
3. Réaux A, de Mota N, Zini S, Cadel S, Fournier-Zalusky MC, Roques BD, Corvol P, Llorens-Cortes C. PC18 a specific aminopeptidase N inhibitor, induces vasopressin release by increasing the half-life of brain angiotensin III. Neuroendocrinology 1999; 69: 370-376.
4. Itoh C, Nagamatsu A. An aminopeptidase activity from porcine kidney that hydrolyzes oxytocin and vasopressin: purification and partial characterization. Biochim Biophys Acta 1995; 1243: 203-208.
5. Suarez C, Cucala M, Coca A, Ruilope LM. Spanish contribution to the HOT (Hypertension Optimal Treatment) study. Final results. Spanish Investigators in the HOT study. Med Clin (Barc) 1999; 113: 361-365.
6. Martínez E, Puras A, Escribano J, Sánchis C, Carrión L, Artigao M, Divison JA, Masso J, Vidal A, Fernández JA. Angiotensin-converting enzyme (ACE) gene polymorphisms, serum ACE activity and blood pressure in a Spanish-Mediterranean population. J Human Hypertens 2000; 14: 131- 135.
7. Banegas JR, Rodríguez-Artalejo F, De la Cruz Troca JJ. Blood pressure in Spain: distribution, awareness, control, and benefits of a reduction in average pressure. Hypertension 1998; 32: 998-1002.
8. Banegas JR, Villar Álvarez F, Pérez de Andrés C, Jimenez García Pascual R, Gil López E, Muñiz García J, Juane Sánchez R. An epidemiological study on cardiovascular risk factors in 35-64 years old Spanish population. Rev Sanid Hig Pública (Madr.) 1993; 67: 419-445.
9. Michel JB, Heudes D, Michel O, Poiterin P, Philippe M, Scalbert E, Corman B Levy BI. Effect of chronic Ang I-converting enzyme inhibition on aging processes. II. Large arteries. Am J Physiol 1994;  267: 124-R 135.
10. Jung YS, Lee S, Shin HS. Effects of age on angiotensin II response and antagonistic activity of losartan in rat aorta and liver. Arch Pharmacol Res 1996; 19: 462-468.
11. Wright JW, Hamilton TA, Harding JW. Anomalous effects of losartan on aminopeptidase-induced reductions of blood pressure in SHR. Brain Res Bull 1995;  36: 69-74.
12. Palatini P. Sympathetic overactivity in hypertension: a risk factor for cardiovascular disease. Curr Hypertens Rep 2001; 3 Suppl 1:3-9.
13. Averill DB, Matsumura K, Ganten D, Ferrario CM. Role of area postrema in transgene hypertension. Hypertension 1996; 27: 591-597.
14. Davisson RL, Yang G, Beltz TG, Cassell MD, Johnson AK, Sigmund CD. The brain renin-angiotensin system contributes to the hypertension in mice containing both the human renin and human angiotensinogen transgenes. Circ Res 1998; 83: 1047-1058.
15. Reaux A, Iturrioz X, Vazeux G, Fournie-Zaluski MC, David C, Roques BP, Corvol P, Llorens-Cortes C. Aminopeptidase A, which generates one of the main effector peptides of the brain renin-angiotensin system, angiotensin III, has a key role in central control of arterial blood pressure. Biochem Soc Trans 2000; 28: 435-440.
16. Chen CY, Huang WC. Pressor and renal effects of intracerebroventricularly administered angiotensins II and III in rats. Kidney Blood Press Res 2000; 23: 95-105.
17.Reaux A, Fournie-Zaluski MC, Llorens-Cortes C. Angiotensin III: a central regulator of vasopressin release and blood pressure. TIEM 2001; 12: 157-162.
18. Terwel D, Markerink M, Jolles J. Age-related changes in concentrations of vasopressin in the central nervous system and plasma of male Wistar rat. Mech Aging Dev 1992; 65: 127-136.
19. Osi I, Kieldsen SE, Aakesson I. Evidence of age-related variation in plasma vasopressin of normotensive men. Scand J Clin Lab Invest 1985; 45: 263-268.

Source(s) of Funding

Andalusian Goverment through PAIDI BIO296

Competing Interests

Nothing to declare


This article has been downloaded from WebmedCentral. With our unique author driven post publication peer review, contents posted on this web portal do not undergo any prepublication peer or editorial review. It is completely the responsibility of the authors to ensure not only scientific and ethical standards of the manuscript but also its grammatical accuracy. Authors must ensure that they obtain all the necessary permissions before submitting any information that requires obtaining a consent or approval from a third party. Authors should also ensure not to submit any information which they do not have the copyright of or of which they have transferred the copyrights to a third party.
Contents on WebmedCentral are purely for biomedical researchers and scientists. They are not meant to cater to the needs of an individual patient. The web portal or any content(s) therein is neither designed to support, nor replace, the relationship that exists between a patient/site visitor and his/her physician. Your use of the WebmedCentral site and its contents is entirely at your own risk. We do not take any responsibility for any harm that you may suffer or inflict on a third person by following the contents of this website.

1 review posted so far

Posted by Dr. Chow KM on 07 Apr 2011 03:18:57 AM GMT

0 comments posted so far

Please use this functionality to flag objectionable, inappropriate, inaccurate, and offensive content to WebmedCentral Team and the authors.


Author Comments
0 comments posted so far


What is article Popularity?

Article popularity is calculated by considering the scores: age of the article
Popularity = (P - 1) / (T + 2)^1.5
P : points is the sum of individual scores, which includes article Views, Downloads, Reviews, Comments and their weightage

Scores   Weightage
Views Points X 1
Download Points X 2
Comment Points X 5
Review Points X 10
Points= sum(Views Points + Download Points + Comment Points + Review Points)
T : time since submission in hours.
P is subtracted by 1 to negate submitter's vote.
Age factor is (time since submission in hours plus two) to the power of 1.5.factor.

How Article Quality Works?

For each article Authors/Readers, Reviewers and WMC Editors can review/rate the articles. These ratings are used to determine Feedback Scores.

In most cases, article receive ratings in the range of 0 to 10. We calculate average of all the ratings and consider it as article quality.

Quality=Average(Authors/Readers Ratings + Reviewers Ratings + WMC Editor Ratings)