Sex differences and age-related changes in human serum aminopeptidase A activity

Clinica Chimica Acta 274 (1998) 53–61

Sex differences and age-related changes in humanserum aminopeptidase A activity

a a a´ ´ ´ ´Jose M. Martınez , Isabel Prieto , Marıa J. Ramırez , Marc deb a a a´Gasparo , Francisco Hermoso , Jose M. Arias , Francisco Alba ,

a ,*´Manuel Ramıreza ´ ´Unit of Physiology, University of Jaen, 23071 Jaen, Spain

bNovartis Pharma, CH-4002 Basel, Switzerland

Received 21 October 1997; received in revised form 17 February 1998; accepted 21 February 1998

Abstract

Given that aminopeptidase A is primarily responsible for cleaving aspartic acid and convertingangiotensin II to angiotensin III, the purpose of the present study was to evaluate the activity ofaminopeptidase A by determination of glutamate aminopeptidase activity (GluAP) and aspartateaminopeptidase activity (AspAP) (reported respectively as aminopeptidase A and angiotensinase Aactivities) in human serum during development and ageing, in an apparently healthy population of139 male and 148 female subjects. To measure GluAP and AspAP we used glutamyl- andaspartyl-2-naphthylamide as substrates. Significant age-related increases were observed in GluAPactivity in males and females and in AspAP activity in females. In males, there were noage-related differences in AspAP activity. A significant correlation was observed between age andGluAP activity in the population analysed as a whole or according to sex. No correlation wasdemonstrated between age and AspAP activity either in the whole population or according to sex.These results may reflect the evolution of the functional status of susceptible circulating substratesduring development and ageing. 1998 Elsevier Science B.V. All rights reserved.

Keywords: Aminopeptidase A; Human serum; Development; Ageing; Sex differences

*Corresponding author. Tel. 1 34 53 212302; fax. 1 4 53 212141; e-mail: [email protected]

0009-8981/98/$19.00 1998 Elsevier Science B.V. All rights reserved.PI I : S0009-8981( 98 )00049-7

´54 J.M. Martınez et al. / Clinica Chimica Acta 274 (1998) 53 –61

1. Introduction

Serum aminopeptidases have been used in clinical chemistry as markers forseveral diseases, and they also play a physiological role in the regulation ofcirculating hormones including angiotensin peptides [1]. Leucyl aminopeptidaseis typically elevated during various liver diseases such as obstructive jaundiceand infectious hepatitis, often exceeding transaminase activities [2] and also ithas been proposed as a marker for plasmacytoid lymphocyte proliferation inacute viral infections [3]. Sanderink et al. [4] found that an isoform of alanylaminopeptidase may be a sensitive marker for liver disease and alcohol abuseand also has been proposed as serum marker for breast and thyroid cancer [5].Serum cystyl aminopeptidase levels increase during normal pregnancy butdecrease in chronic placental insufficiency or premature labour [6].

Circulating glutamyl aminopeptidase (aminopeptidase A) (GluAP) is mostprobably the serum aminopeptidase responsible for the rapid destruction ofangiotensin II [7,8]. In addition, aspartyl aminopeptidase (angiotensinase A;AspAP), adequately distinguished from GluAP [9], also presents a rapid actionon the N-terminal aspartic acid residues of angiotensin analogues, which pointsto a possible physiological role in angiotensin II degradation [10]. It has beenalso described that circulating GluAP, arising from placenta, increase pro-gressively during pregnancy [11], but also shows a significant decrease in casesof pre-eclampsia [12]. On the other hand, blood pressure increases duringdevelopment and ageing, and the nature of this increase differs in women andmen. However, despite their role as angiotensinases, human serum GluAP andAspAP activities have not been investigated during development and ageing. Wemeasured glutamyl aminopeptidase (GluAP: EC 3.4.11.7) and aspartyl amino-peptidase (AspAP: EC 3.4.11.-) activities; previously reported as capable ofhydrolysing the N-terminal aspartic acid residue of angiotensin II [10], usingarylamide derivatives as substrates, in various age groups.

2. Materials and methods

2.1. Samples

We used blood samples, obtained by venipuncture without additives, from 148females aged 2 to 87 years and 139 males aged 1 to 92 years. All participantswere ambulatory subjects who came to the Hospital General de Especialidades

´‘Ciudad de Jaen’ for health screening. The samples were centrifuged at 48C for10 min at 3000 3 g, and the sera were analysed the same day. When subjectscame to the hospital for health screening, they were required to inform whetheror not they had some known disease or were taking drugs, and were also asked

´J.M. Martınez et al. / Clinica Chimica Acta 274 (1998) 53 –61 55

about alcohol consumption. Blood samples were separated into aliquots fordeterminations of routine biochemical laboratory parameters, aminopeptidaseactivities, total protein content, hormonal parameters and for haematologicalvalues. Haemolytic, icteric, and turbid samples were discarded. The selectedsubjects reported to have no known disease, to be taking no drugs and noalcohol. Routine biochemical laboratory parameters, hormonal levels andhaematological values were within the range of normality in all cases.

2.2. Methods

Glutamyl aminopeptidase (EC 3.4.11.7) (GluAP) and aspartyl aminopeptidase(EC 3.4.11.-) (AspAP) were assayed as arylamidase activities [10]. GluAP wasdetermined in triplicate in a fluorometric assay using Glu-2-naphthylamide(GluNNap) as the substrate according to the method of Tobe et al. [13] modifiedas follows: 25 ml of each serum was incubated during 120 min at 378C with 1ml of the substrate solution (100 mmol / l GluNNap, 1.5 mmol / l BSA, 0.65mmol / l DTT and 50 mmol / l CaCl in 50 mmol / l HCl–Tris, pH 7.4). AspAP2

was also determined fluorometrically in triplicate, with Asp-2-naphthylamide(AspNNap) as the substrate, according to the method of Cheung and Cushman[9] modified as follows: 25 ml of each serum was incubated for 120 min at 378Cwith 1 ml of the substrate solution (100 mmol / l AspNNap 1.5 mmol / l BSA and2 mmol / l MnCl in 50 mmol / l HCl–Tris buffer, pH 7.4). All the reactions were2

stopped by adding 1 ml of 0.1 mol / l acetate buffer, pH 4.2. The amount of2-naphthylamine released as a result of enzymatic activity was measuredfluorometrically at 412 nm emission wavelength with an excitation wavelengthof 345 nm. This rapid fluorometric method permits quantify aminopeptidase

214activities in the order of 10 mole [14]. Proteins were quantified in triplicateby the method of Bradford [15], using BSA as a standard. Serum specificaminopeptidase activities were expressed as pmol of substrate hydrolysed permin per mg of protein. Fluorogenic assays were linear with respect to time ofhydrolysis and protein content.

2.3. Statistical procedures

For statistical analysis, to be able to compare sex, the male and femalesubjects were divided into five age-matched groups (Table 1) which included themain stages of development. We used one-way analysis of variance (ANOVA) toanalyse differences between groups [16]. Post-hoc comparisons were made usingDuncan’s test [17]. Linear correlation coefficients were calculated to testrelationships between two variables [18]. All comparisons with P values below0.05 were considered significant.


Table 1Age groups in males and females

Males Females

Age (years) n mean6SEM (years) n mean6SEM (years)

1–6 18 3.260.36 17 4.760.407–15 19 11.560.59 20 11.160.6316–45 44 27.461.28 51 26.861.0546–65 25 55.461.14 26 53.360.95. 66 33 77.361.02 34 76.260.87Total 139 39.060.52 148 38.160.46

3. Results

Values (mean6SEM) of specific aminopeptidase activities in serum of thedifferent age groups in female and male subjects are presented in Fig. 1. Thestatistical differences between means are indicated in Table 2. Significantage-related changes were observed in males (P , 0.05) and females (P , 0.001)for GluAP activity and in females ( p , 0.05) for AspAP activity. For GluAPactivity, the largest increases were observed in males and females aged 16–45and . 66 years. For AspAP activity, the largest increases were observed infemales . 66 years old. Sex differences were observed for GluAP in the groupof 16–45 years old (P , 0.01) and for AspAP in the group of 46–65 years old(P , 0.001), both activities being higher in males than in females. No sexdifferences were seen in the other age groups. In females the highest values forboth activities were observed in subjects over 66 years old. In males, the highestvalues for GluAP were also found in subjects over 66 years old, but nodifferences were observed for AspAP. ln the whole population, according to thelinear model of the regression analysis, GluAP activity showed a significantdirect correlation with age (r 5 0.278, P , 0.001), whereas no correlation wasobserved for AspAP (r 5 0.102, P 5 0.082). The correlation between APactivities and age in males and females separately demonstrated a significantdirect correlation for GluAP in males (r 5 0.178, P , 0.05), and highlysignificant in females (r 5 0.449, P , 0.001). For AspAP, no correlation wasobserved in males (r 5 0.031, P 5 0.7) and a borderline direct correlation wasfound in females (r 5 0.152, P 5 0.063).

4. Discussion

Aminopeptidase A appears to be a membrane-bound enzyme widely distribut-ed in the body, with especially high levels in the kidney [19,20]. Although this


Fig. 1. Age-related changes in mean6S.E.M. levels of human serum glutamate aminopeptidase(GluAP) and aspartate aminopeptidase (AspAP) activities, expressed as pmol of Glu- andAsp-2-naphthylamide respectively hydrolyzed per min per mg of protein in the different agegroups of males and females. See Table 2 for explanation of the statistical analysis.

activity is also present in vascular circulation, its origin is not totally understood.It has been suggested that this enzyme becomes soluble in plasma via autolysisof membrane-bound aminopeptidase A [7]. The only aminopeptidase previouslyreported to be specific for dicarboxylic amino acids is glutamate aminopeptidase(GluAP; aminopeptidase A; EC 3.4.11.7), which readily removes the N-terminal

1Asp residue from L-Asp -angiotensin II [10]. GluAP does not hydrolyse 2-naphthylamide derivatives of amino acids other than aspartic and glutamic acids.This specificity also applies to the hydrolysis of true peptide bonds such as thosein angiotensin analogs [9]. An aspartate aminopeptidase with low activity on2-naphthylamide derivatives may also be partially responsible for the hydrolysis


Table 2Statistical differences of glutamate aminopeptidase (GluAP) and aspartate aminopeptidase(AspAP) activities between age groups in serum of males and females

Sex Males Females

Enzymatic Age 7–15 16–45 46–65 . 66 7–15 16–45 46–65 . 66activity

a a bGluAP 1–6 NS NS NS NS NSa a b7–15 NS NS NS

b16–45 NS NS NSb46–65 NSaAspAP 1–6 NS NS NS NS NS NS NS

7–15 NS NS NS NS NS NS16–45 NS NS NS NS

a46–65 NS

Comparisons between means were made using Duncan’s multiple range test.a P , 0.05.b P , 0.001.

of N-terminal Asp-residues of peptides. Therefore, with AspNNap as thesubstrate, the release of 2-naphthylamine may be due to the action of at least twodifferent enzyme activities (aminopeptidase A activity). Because of its spe-cificity and its rapid action on N-terminal aspartic acid residues of angiotensinanalogs, AspNNap hydrolysing activity may also act as an angiotensinase [9].The present results demonstrate a different profile for GluAP and AspAPactivities in males and females, which supports the existence of two enzymeswith presumably different properties and roles in the regulation of susceptiblesubstrates.

We found sex differences and age-related changes in aminopeptidase Aactivity. For GluAP we observed a progressive increase during development andageing, which was more prominent in females. In both, females and males therewas a rapid increase after puberty ( . 15 years) and in elderly subjects ( . 65years). The profile of, AspAP activity is also different in females and males,essentially after 45 years of age. Sanderink et al [21] reported higher values forAlaAP in males than in females, and also noted sex differences duringdevelopment, i.e., an inverse correlation with age in males but not in females.These authors also found low levels of AlaAP in males (10%) and high levels infemales (15%) among hypertensive subjects. AlaAP (AP M; EC.3.4.11.2) hasalso been proposed as an angiotensinase [7]. However, the present resultssuggest a more specific action of aminopeptidase A, particularly GluAP, in themetabolism of circulating angiotensin II during development and ageing.Interestingly, AspAP activity did not correlate significantly with age or with sex;


therefore, such results do not support its putative role as an angiotensinase inhuman serum.

Blood pressure, particularly systolic blood pressure, tends to increase pro-gressively with age [22–24]. Blood pressure increases more in males than infemales among young people, but later increases more notably with age infemales, and values in elderly males and females are similar. In the renin-angiotensin system, plasma renin activity and angiotensin II decline pro-gressively with age [23–27]. The decrease in plasma renin activity is exponen-tial, being very rapid in early childhood and continuing into old age. Thecontractile response to Ang II decrease also with age [28]. This age-relatedprofile is the opposite of the one we found in the present study for aminopeptid-ase A activity. Therefore, the inverse correlation between peptide level andpeptide-hydrolysing activity suggests a relation between high breakdowncapacity and low level of substrate, which in turn supports a role for humanplasma aminopeptidase A activity as an angiotensinase. However, these observa-tions appear incompatible with the above mentioned tendency of blood pressureto increase with age and for Ang II-induced contractility to decline. Therefore,additional factors must be involved in this phenomenon which could includechanges with ageing that result in gradually increasing rigidity of vessels. Inaddition, the existence of sex differences and the fact that the most significantchanges were observed after puberty and in elderly subjects suggest thathormonal status is involved in aminopeptidase A activity, which is also inagreement with the results of Sanderink et al [21] for human serum AlaAPactivity. Moreover, as previously suggested, aminopeptidase activities may beinfluenced by sexual hormones during development and ageing [20]. Theinvolvement of the hormonal status on aminopeptidase activity is also inagreement with recent in vitro results which demonstrate a direct influence ofcholesterol and steroid hormones on aminopeptidase activities, being GluAP andAspAP specifically increased by the presence of high levels of cholesterol in themedium of incubation [29].

In summary, the present study demonstrates sex differences and an age-relatedincrease in human serum aminopeptidase A activity, which contrasts with thewell known decrease in renin and angiotensin II with age. However, we shouldtake into account that the correlational results obtained in the present work donot indicate a causal relationship among increased aminopeptidase A activity,reduced plasma peptide level, and increased blood pressure with age. In anycase, our results provide additional evidence for a role of these enzymes(essentially GluAP) in regulating serum levels of angiotensin peptides. Giventhat GluAP is probably the serum enzymatic activity responsible for the rapidmetabolism of circulating angiotensin II, which exhibits a major role in theregulation of the arterial pressure, it is important to establish its behaviour inserum, by the use of a rapid and sensitive method, in normal male and female


subjects during development and ageing. Moreover, the potential activity ofaminopeptidase A in N-terminal Asp residues other than angiotensin II, such ascholecystokinin (26–33) octapeptide or cholecystokinin (26–29) tetrapeptide,should be taken into account as well.

Acknowledgements

We thank Karen Shashok for improving the use of English in the manuscript.

References

[1] Sanderink GJ, Artur Y, Siest G. Human aminopeptidases. A review of the literature. J ClinChem Clin Biochem 1989;26:795–807.

[2] Plaquet R, Ledeme N, Vincent-Fiquet O et al. Automatic determination of leucin amino-peptidase in human serum. Clin Chim Acta 1973;46:91–103.

[3] Pancheva-Haschen R, Haschen RJ. Serum leucine aminopeptidase for monitoring viralinfections with plasmacytoid reaction. Enzyme 1986;36:179–86.

[4] Sanderink G-J, Artur Y, Paille F et al. Clinical significance of a new isoform of serum alanineaminopeptidase; relationship with liver disease and alcohol consumption. Clin Chim Acta1989;179:23–32.

[5] Severini G, Gentilini L, Tirelli C. Diagnostic evaluation of alanine aminopeptidase as serummarker for detecting cancer. Cancer Biochem Biophys 1991;12:199–204.

[6] Gunther R, Landgraf R, Koppe I. Cystine aminopeptidase and oxytocin in the plasma ofpregnant patients with premature labor. Zentralbl Gynakol 1985;107:1178–85.

[7] Wright JW, Harding JW. Brain angiotensin receptor subtypes AT1, AT2 and AT4 and theirfunctions. Regul Pept 1995;59:269–95.

[8] Ahmad S, Ward PEJ. Role of aminopeptidase activity and the regulation of the pressoractivity of circulating angiotensins. Pharm Exp Therap 1990;252:643–50.

[9] Cheung HS, Cushman DW. A soluble aspartate aminopeptidase from dog kidney. BiochimBiophys Acta 1971;242:190–3.

[10] McDonald JK, Barret AJ. Mammalian Proteases, a Glossary and Bibliography Vol 2:Exopeptidases. New York: Academic Press, 1986;23–100.

[11] Mizutani S, Okano K, Hasegawa E et al. Aminopeptidase A in human placenta. BiochimBiophys Acta 1981;678:168–70.

[12] Mizutani S, Yamada R, Kurauchi O. Serum aminopeptidase A (AAP) in normal pregnancyand pregnancy complicated by pre-eclampsia. Arch Gynecol 1987;240:27–31.

[13] Tobe H, Kojima F, Aoyagi T. Purification by affinity chromatography using amastatin andproperties of aminopeptidase A from pig kidney. Biochim Biophys Acta 1980;459–468.

[14] Greenberg LJ. Fluorimetric measurement of alkaline phosphatase and aminopeptidase214activities in the order of 10 mole. Biophys Res Commun 1962;9:430–5.

[15] Bradford MM. A rapid and sensitive method for the quantification of microgram quantities ofprotein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–54.

[16] Box GEP, Hunter WG, Hunter JS. Statistics for experimenters. New York: Wiley, 1978.[17] Duncan AJ. Quality control and industrial statistics. 4th edition. Homewood, Illinois:

Richard, D. Irwin. Inc., 1974.


[18] Draper N, Smith H. Applied regression analysis. 2nd edition. New York: Wiley, 1981.[19] Lodja Z, Gossrau R. Study on aminopeptidase A. Histochemistry 1980;67:267–90.

´ ´[20] Ramırez M, Arechaga G, Sanchez B et al. Developmental and ageing changes ofaminopeptidase activities in selected tissues of the rat. Experientia 1993;49:300–3.

[21] Sanderink GJ, Artur Y, Schiele F et al. Alanine aminopeptidase in serum: biologicalvariations and reference limits. Clin Chem 1988;34:1422–6.

[22] Rowland M, Roberts J. NCHS Advance data, No. 84, Vital and Health Statistics of theNational Center for Health Statistics. US Department of Health and Human Services.Washington, DC, Oct 8, 1982.

[23] Michel JB, Heudes D, Michel O. Effect of chronic Ang I-converting enzyme inhibition onageing processes. II. Large arteries. Am J Physiol 1994;267:124–135.

[24] Duggan J, Nussberger J, Kilfeather S et al. Aging and human hormonal and pressorresponsiveness to angiotensin II infusion with simultaneous measurement of exogenous andendogenous angiotensin II. Am J Hypertens 1993;6:641–7.

[25] Meade TW, Imeson JD, Gordon D et al. The epidemiology of plasma renin. Clin Sci1983;64:273–80.

[26] Van Acker KJ, Scharpe SL, Deprettere AJR. Renin-angiotensin-aldosterone system in thehealthy infant and child. Kidney Int 1979;16:196–203.

[27] Duggan J, Kilfeather S, O’Brien E. Effects of ageing and hypertension on plasma angiotensinII and platelet angiotensin II receptor density. Am J Hypertens 1992;5:687–93.

[28] Jung YS, Lee S, Shin HS. Effects of age on angiotensin II response and antagonistic activityof losartan in rat aorta and liver. Arch Pharmacol Res 1996;19:462–8.

´ ´[29] Martınez JM, Prieto I, Ramırez MJ et al. Cholesterol and steroids action on aminopeptidases.Biochem Soc Trans 1997;25:113S.

Sex differences and age-related changes in human serum aminopeptidase A activity

Documents

Transcript of Sex differences and age-related changes in human serum aminopeptidase A activity