Archives of Gerontology and Geriatrics

28 (1999) 31–36

Human serum pyroglutamyl-b-naphthylamidehydrolyzing activity during development and aging

J.M. Martınez a, M.J. Ramırez a, I. Prieto a, C. Petzelt b,F. Hermoso a, F. Alba a, J.M. Arias Saavedra c, M. Ramırez a,*

a Area de Fisiologıa, Uni6ersidad de Jaen, Edif. 5, Paraje Las Lagunillas, 23071, Jaen, Spainb Experimental Anesthesiology, Department of Anesthesiology and Intensi6e Therapy,

Uni6ersity Hospital Charite, Humboldt Uni6ersity, Spandauer Damm 130, D-14050, Berlin, Germanyc Hospital General de Especialidades Ciudad de Jaen, 23005, Jaen, Spain

Received 27 February 1998; received in revised form 12 August 1998; accepted 21 August 1998

Abstract

Pyroglutamyl-b-naphthylamide hydrolyzing activity (pGluHA) is reported to be capableof removing the amino-terminal pyroglutamic acid residue from peptides (e.g. TRH orGnRH) and artificial substrates. However, its functional role in serum is not yet understood.The aim of the present study was to analyze the activity of pGluHA in human serum duringdevelopment and aging, in an apparently healthy population of 139 men and 148 women. Tomeasure pGluHA we used pGlu-b-naphthylamide as the substrate. Sex differences andage-related changes were observed in men and women. In addition, the developmental profilewas notably different between men and women. In men, activity increased steadily until fullsexual maturity, but did not change substantially after puberty. In women, activity increasedsignificantly in advanced ages but there were no significant changes in the rest of the agegroups tested. Significant sex differences were observed in subjects 46–65 years old, theactivity being higher in men than women. In the total population, a significant directcorrelation was observed between pGluHA and age. Considered independently, men andwomen also showed a highly significant direct correlation between pGluHA and age. Theseresults may reflect changes in the functional status of its circulating substrates duringdevelopment and aging. © 1999 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Pyroglutamyl peptidase; Arylamides; Human serum; Development; Aging

* Corresponding author. Tel.: +34 53 212302; fax: +34 53 212141; e-mail: msanchez@ujaen.es

0167-4943/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved.

PII S0167-4943(98)00123-X

J.M. Martınez et al. / Arch. Gerontol. Geriatr. 28 (1999) 31–3632

1. Introduction

Pyroglutamyl peptidase activity, widely distributed in fluids and tissues, removespyroglutamyl N-terminal residues from peptides and arylamide derivatives in ahighly selective manner (McDonald and Barret, 1986). Pyroglutamyl peptidase-I isa cysteine protease that cleaves on the carboxyl side of pyroglutamyl residues inpeptides such as gonadotropin releasing hormone (GnRH), neurotensin andbombesin (Cummins and O’Connor, 1996). Pyroglutamyl peptidase-II is a metal-loenzyme that catalyzes the degradation of pyroglutamyl-histidyl tripeptides such asthyrotropin releasing hormone (TRH) in both brain (Friedman and Wilk, 1986)and serum (Bauer and Nowak, 1979). Because of the specificity of pyroglutamylpeptidase-II from the degradation of TRH, this enzyme has been named ‘thy-roliberinase’ (Wilk, 1986). The source of circulating serum pyroglutamyl peptidase-II is unknown.

Radioimmunoassays with [14C]pyroglutamyl thyroliberin, thyroliberin (McDon-ald and Barret, 1986), and chromogenic assays with pGlu-His-Pro-b-naphthy-lamide (Friedman et al., 1995) have been used for serum thyroliberinasedeterminations. However, pGlu-b-naphthylamide (pGluNNap) and pGlu-7-(4-methyl)coumarylamide (pGlu-NMec) were claimed not to be useful substrates forthyroliberinase assays (Bauer and Nowak, 1979). Pyroglutamyl-b-naphthylamidehydrolyzing activity (pGluHA) removes amino-terminal pGlu residues from peptideand arylamide derivatives, with TRH, GnRH, neurotensin or gastrins as substrates.However, the actual physiological role of this enzymatic activity in human serum isnot known. Therefore, the present study was done to analyze the influence of sexand age on human serum pGluHA. This information could help us to understandits functional role and the regulatory mechanisms that control this enzymaticactivity. We measured pGluHA, using an arylamide derivative as substrate, invarious age groups from early childhood to old age.

2. Subjects and methods

Blood samples were obtained from 148 girls and women aged 2–87 years and 139boys and men aged 1–92 years who came to the Ciudad de Jaen General Hospitalof Specialities for health screening. The samples were centrifuged for 10 min at3000×g, and the sera were analyzed the same day. Hemolytic, icteric, or turbidsamples were discarded. None of the subjects had any known disease, and routineanalytical determinations were within the range of normality in all cases. Pyroglu-tamyl peptidase activity was assayed as arylamidase activity (McDonald andBarret, 1986).

pGluHA was measured in triplicate in a fluorogenic assay using pGlu-b-naphthy-lamide (pGluNNap) as the substrate, according to the modified method of Schwabeand McDonald (1977): 10 m l of each supernatant was incubated during 120 min at37°C with 1 ml of substrate solution (2.54 mg/100 ml pGluNNap, 10 mg/100 mlDTT, 37.8 mg/100 ml EDTA in 50 mmol/l phosphate buffer, pH 7.4). The reaction

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was stopped by adding 1 ml 0.1 mol/l of acetate buffer, pH 4.2. The amount ofb-naphthylamine released as a result of the enzymatic activity was measuredfluorometrically at an emission wavelength of 412 nm with an excitation wavelengthof 345 nm. This fluorometric method quantifies aminopeptidase activities on theorder of 10−14 mol (Greenberg, 1962). Proteins were quantified in triplicate by themethod of Bradford (1976), using BSA as a standard.

Serum-specific aminopeptidase activities were expressed as pmol of substratehydrolyzed per min per mg of protein. Fluorogenic assays were linear with respectto time of hydrolysis and protein content.

For statistical analysis, the subjects were divided into five groups (Table 1). Weused one-way analysis of variance (ANOVA) to analyze differences betweengroups. Post-hoc comparisons were made using Duncan’s test. The linear correla-tion coefficient test was used for relationships between two variables. All compari-sons with P-values below 0.05 were considered significant.

3. Results

Fig. 1 shows the mean values9S.E.M. of specific pGluHA in serum from thedifferent age-groups studied in females and males. Significant age-related changeswere observed in both sexes (PB0.01). In men, the activity increased significantlyin the group of 16–45 years old, and remained unchanged thereafter. In women,activity did not change significantly between 1–6 years and 45–65 years of age.However, pGluHA increased significantly in older women (\66 years old). Sexdifferences were observed in the 46- to 65-year-old group (PB0.01), with highervalues in men than in women. The rest of the age groups did not show sexdifferences. We found a highly significant direct correlation between pGluHA andage for the total population (r=0.265, PB0.001), females (r=0.668, PB0.001)and males (r=0.278, PB0.001).

Table 1Age groups in men and women

Age (years) Men Women

n Mean9S.E.M. n Mean9S.E.M.

1–6 3.390.3618 17 4.790.407–15 11.590.5919 20 11.190.63

26.891.055127.491.2816–45 442546–65 55.491.14 26 53.390.9533 76.290.87\66 3477.391.02

38.190.4614839.090.52139Total

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Fig. 1. Human serum pyroglutamyl-b-naphthylamide hydrolyzing activity (pGluHA) during develop-ment and aging. Line graphs show age-related changes in levels (mean9S.E.M.) of human pGluHAexpressed as pmol of pGlu-b-naphthylamide hydrolyzed per min per mg of protein, in different agegroups of men (n=139) and women (n=148). Tables show significant differences between age groups.* PB0.05. ** PB0.01.

4. Discussion

We found sex differences and age-related changes in human serum pGluHA. Inmales, activity increased steadily until sexual maturity, but did not change substan-tially after puberty. However, the developmental profile of pGluHA was consider-ably different in females. There was a tendency toward a decrease at the age of7–15 years, followed by an increase during sexual maturity and a slight decreaseafter menopause. Activity increased markedly in older men and women. Theseresults demonstrate a different pattern of pGluHA activity in men and women,which could reflect differences in hormonal status during sexual development andaging.

One susceptible substrate for serum pGluHA is the hypophysiotropic hormoneTRH. No major age-related changes have been found in the physiology of thehypothalamic-pituitary-thyroid axis, and thyroid stimulating hormone (TSH) re-lease remains pulsatile. There is a slight age-related decline in serum T3, but valuesusually remain within normal limits. Serum TSH increases slightly with age, butthis too remains within normal limits. The effect of age on the release of TSH byTRH is less clear, but several studies show little change in either sex (Harman et al.,1984).

J.M. Martınez et al. / Arch. Gerontol. Geriatr. 28 (1999) 31–36 35

Another major substrate of hydrolysis by pGluHA is GnRH. In men, as sexualactivity decreases with age, there are only minor physiological changes in thehypothalamic-pituitary-testicular axis. Studies of testosterone economy show thatserum testosterone increase slightly with age. A decrease in the number andresponsiveness of testicular Leydig cells is likely, because serum FSH and LHincrease with age. Smaller pituitary changes are suggested by a decreased go-nadotropic response to GnRH stimulation (Harman and Tsitouras, 1980; Harmanet al., 1982).

The changes in endocrine function are not abrupt in women during spontaneousmenopause. Circulating levels of gonadotropins begin to increase several yearsbefore ovulation ceases, and the production of estrogen and progesterone decreases.The increase in FSH is greater than that of LH, and reflects the lack of feedbackinhibition by estrogen, inhibin, or both (Sherman et al., 1976).

As these results illustrate, TRH shows little change during development andaging in either sex. However, GnRH levels do change with age, and the pattern ofchange during development and aging differs in men and women. Our resultsdemonstrate sex differences in the changes in serum pGluHA, and age-relatedchanges around puberty, menopause and in elderly subjects which suggest thatactivity is influenced by hormonal status. Therefore our results reveal a parallelwith the developmental pattern of GnRH in men and women, and may support arole for human serum pGluHA in the metabolism of GnRH. However, thepotential action of pGluHA on N-terminal pGlu residues other than TRH orGnRH, such as neurotensin or gastrins, should also be taken into account.

Acknowledgements

We thank Karen Shashok for improving the English style of the manuscript.

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