key: cord-0901959-uxewsyli authors: Janas, Roman M.; Socha, Jerzy; Warnawin, Krzysztof; Rujner, Jolanta title: Further Studies on Aminopeptidase-M in Blood in Children with Cholestatic Liver Diseases and Viral Hepatitis date: 1999 journal: Dig Dis Sci DOI: 10.1023/a:1026626822298 sha: be0d004544c7da98785d19f9b3aaf36745da7afe doc_id: 901959 cord_uid: uxewsyli Aim of this study was to determine and furthercharacterize the serum aminopeptidase-M in children withliver diseases. Based on our new assay, we have showntwo fractions of the enzyme. Activity of the first fraction is expressed in undiluted serumat pH adjusted from 8.5 (pH of storaged serum) to 7.4.Activity of the second fraction (cryptic activity)appears in the serum (pH 7.4) as a result of dilution and/or addition of aniline naphthalene sulfonicacid. In children with Alagille syndrome, extrahepaticbiliary duct atresia, Byler's disease, and acutehepatitis due to hepatitis B virus infection, activities of both fractions are highly elevated ascompared to healthy children or those with chronic viralhepatitis. Moreover, serum aminopeptidase-M seems toreflect other aspects of the pathological process than those reflected by the alanine aminotransferaseand gamma-glutamyltranspeptidase. Due to increasedactivity and broad substrate specificity, the enzymeseems to be also a cofactor of cholestasis andhepatitis. Membrane -bound aminope ptidase -M (EC 3.4.11.2) functions as an ectopeptidase at the cell surface in many tissue s and the re is also a soluble form of this e nzyme in serum and othe r body¯uids (1± 3). Serum aminope ptidase -M activity is marke dly increased in patie nts with chole stasis and was shown to be a use ful marke r of he patobiliary dise ase (4 ± 7). The enzyme is thought to originate from the live r (2) . Howe ver, its multiple mole cular forms pre sent in se rum may arise from othe r organs and tissue s (6, 8) . In he matology, aminope ptidase -M (cluster of dif-fe rentiation 13) is a well-recognize d marker of mye loid cell le uke mia (9 ± 11) . Inte restingly, it has bee n found that aminope ptidase -M on alime ntary or re spiratory tract e pithe lial cells is a receptor for human coronavirus 229E (12) . In a large population of appare ntly healthy humans, the e nzyme activity se ems to e xhibit some variations that are depe nde nt on age , se x, alcohol consumption, smoking, and drug intake (13) . Among the substrate s of aminope ptidase -M, including those involve d in growth and differe ntiation re gulation, phagocytosis, and bacte ricidal and tumoricidal activity (3, 4, 9, 14 ± 16) , both methionine (met) and le ucine (le u) enkephalins are rapidly de grade d afte r the ir administration to humans or animals (17± 28) . In circulation, the e ndoge nous enkephalins se em to be prote cted from de gradation through binding to carrier prote ins (19) . Howeve r, a fre e fraction of the e nke phalins would be susce ptible to the e nzyme. Enkephalins appe ar to be implicate d in chole static live r dise ase and may be an important pathoge nic factor in such complications as pruritus or e nce phalopathy. Inde e d, plasma e nke phalin concentrations are highly elevate d in adult patie nts with chole stasis of diffe ring aetiologie s (7, 29 ± 32) , and in rats with an e xpe rime ntal mode l of acute chole stasis (33, 34) . Accumulating in blood, e nke phalins may cross the blood± brain barrie r and evoke , via the ir incre ase d availability in the central nervous syste m, a syndrome of chronic opiate -receptor stimulation (35, 36) . Preliminary experiments have shown that naloxone or nalme fene , opiate -receptor antagonists, ame liorate d or reve rsed pruritus in patie nts with chronic chole static live r disease and, in some patie nts, pre cipitate d a syndrome that rese mble s the withdrawal reaction of opiate addiction (37, 38) . Recently, we have shown that in childre n with chole stasis of diffe ring e tiologie s incre ase d se rum met-enkephalin occurs concom itantly with highly ele vate d se rum aminope ptidase -M activity and that both parame ters were appare ntly not affe cted by a short-te rm treatment with ursode oxycholic acid (39, 40) . Alternations in the aminope ptidase -M activity as well as the ir re le vance to the situation in chole stasis and viral he patitis are not fully unde rstood yet, and furthe r syste matic studie s see m to be worthwhile . There fore , in this study we attempted to furthe r characterize and compare the serum aminope ptidase -M activity in he althy childre n, childre n with chole static live r dise ase , and childre n with chronic or acute he patitis due to hepatitis B virus (HBV) infe ction. Patients. The protocol of this study was approved by the hospital ethics committe e. The following groups of patie nts were included in the study: group 1, e ight children, 8 ± 14 ye ars old (me an 10 years), with Alagille syndrome (arteriohe patic dysplasia) , alanine am inotransfe rase ( ALAT): Blood Sam p ling. V enous blood was sampled without anticoagulants for the diagnostic examinations; it was possible to use part of e ach sample in this study. Blood samples were centrifuged at 3000g for 15 min at 4°C, and the sera ® ltered through a 0.2-m m ® lte r (Sartorius-Me mbran® lte r, Gottingen, Ge rmany) to exclude all residual cell debris, and stored at 2 30°C. Serum pH was me asured using a pH microelectrode (Sigma Chemical Co., St Louis, Missouri). Assay of Am inop eptid ase-M Activity. Equal aliquots (500 m l) of the sera we re pooled in groups 1± 6, respective ly, and used for characterization of the aminopeptidase-M. The e nzyme activity was also dete rmined in the individual serum samples from patients from groups 1± 6. Dilution of the serum was performed using 25 mmol/liter bisTris± Tris± ace tic acid buffe r with or without 0.15 mol/liter NaCl, at pH range 4.5± 9.0. To maintain the serum pH at a desired leve l, the endogenous HCO 3 2 was hydrolyzed with a negligible volume of 1 mol/liter HCl (® nal concentration 25± 30 mmol/ liter). Resulting CO 2 was remove d under low pressure, and pH was adjusted with a small volume of 1 mol/liter NaO H, HCl, or bisTris± Tris± ace tic acid under the control of a pH microelectrode. The incubation mixture consisted of 100 m l of undiluted or diluted serum and 5 m l of [ 1 25 I] Incubation was performed at 37°C for 0 min (control) to 15 min, and terminate d by acidi® cation with 100 mmol/l ace tic acid. De gradation of the substrates was quanti® e d by the aluminum silicate binding assay as recently described (41) with slight modi® cations. A homogenous suspension (50 mg/ml) of aluminum silicate powder (Lloyd reage nt, Pfaltz and Bauer, Wate rbury, Connecticut) was prepared in 1% acetic acid. Aliquots of 2 ml were adde d to the tubes at the e nd of each incubation period. Afte r vortexing for 1 min, the tubes were centrifuged at 3000g for 10 min. For [ 12 5 I]leu-enkephalin, the supernatants were discarded by aspiration, and the silicate pellets counted for the g -radioactivity. For the [ 3 H]leu-e nkephalin, 1.5-ml aliquots of the supernatants were mixe d with 5 ml aliquots of the scintillation cocktail and counted for the b -radioactivity. Intraand interassay coef® cients of variation we re 4.9% and 10.5% for iodinated and 8.9% and 14.5% for tritiated e nkephalins, respective ly. The e nzyme activity was e xpressed as picograms of degrade d substrate per minute per milliliter whole serum. Intact [ 12 5 I]-or [ 3 H]leu-enkephalin was almost complete ly adsorbed by the aluminum silicate (93 6 3% , me an 6 SEM, N . 30). Reve rse-phase high-performance liquid chromatography showed that silicate pellet-bound radioactivity re pre se nte d the intac t labe le d pe ptide , whereas 85± 90% of the radioactivity appearing in the supernatants was due to [ 1 25 I]-or [ 3 H]tyrosine. Unidenti® e d, two to three amino acid fragme nts constituted about 10 ± 15% of the radioactivity and did not react with the speci® c leu-e nkephalin antibody (data not shown). Effect of Inhibitors an d Oth er Subs tan ces on Am inop eptidase-M. Degradation of the labeled leu-enkephalin by serum aminopeptidase-M was quanti® e d by dete rmining the percentage of intact label in the presence of increasing concentrations of speci® e d substances. The exte nt of the label degradation in the control probes, no substance added (35± 40% degradation afte r a speci® e d incubation period) was take n as 100% relative activity. Before the assay, the inhibitors to be teste d were preincubated with the serum at ; 20°C for 15 min. The inhibitory constants (K i ) and Michaelis constant (K M ) towards unlabeled leu-e nkephalin were determined as previously described (39) . The following substances used in the present study we re purchased from Sigma: 1,10-phe nanthroline, bestatin, puromycin, leu-e nkephalin, me t-e nkephalin, bovine serum albumin, 8-aniline naphthalene sulfonic (ANS) acid, taurocholic acid, ursodeoxycholic acid, and cholic acid. Other chemicals we re of analytical grade. Statis tical An alysis. V alues are e xpressed as means 6 SE M unless otherwise speci® ed. The Mann-Whitne y U test was used to dete rmine the signi® cance of differences betwe en means; P , 0.05 was considered signi® cant. During storage , serum pH rise s to 8.5. The e ffect of pH on aminope ptidase -M activity in undilute d se rum pools from groups 1± 6 is shown in Figure 1 . The pH curve s were symme tric and dynamic with an optimum at about 6.5± 7.3 for both iodinate d and tritiate d le ue nkephalin. The same data were obtaine d in the e xpe rime nts with dilute d se ra. The increase in serum aminope ptidase -M activity as a result of dilution with 25 mmol/lite r buffe r, pH 7.4, is shown in Figure 2a ,b. As shown in Figure 2a , the e nzyme activity increased from 45 6 5 (undilute d se rum, pH 7.4) up to maxim al value s of 880 6 90 pg/min/ml (80-fold dilute d serum; value s are means 6 SEM) . There were no statistically signi® cant diffe re nce s in the aminope ptidase -M activity be twe e n he althy childre n and those with chronic active he patitis due to HBV infe ction (P . 0.05) . As shown in Figure 2b , the e nzyme activity in the undilute d se ra, pH 7.4, from childre n with Alagille syndrome , extrahe patic biliary duct atre sia, Byler' s dise ase , and acute he patitis due to HBV infe ction was: 411 6 55, 480 6 39, 198 6 25, and 300 6 69 pg/min/ml, re spe ctive ly. In the sample s dilute d 50-to 80-fold, the activity re ache d maximal value s of 6100 6 800, 6900 6 950, 2150 6 280, and 3880 6 410 pg/m in/ml, re spe ctive ly. Negligible dilution, up to 1.5-fold, was without effe ct (Figure 2a,b) . The same data were obtaine d in the e xpe r- ime nts whe re buffe re d saline , pH 7.4, se rved as a se rum dilue nt (data not shown) . The effect of the incre asing concentrations of ANS acid on aminope ptidase -M activity is shown in Figure 3 . In the undilute d and 5-and 30-fold dilute d sera, the e nzyme activity incre ase d about twice in the prese nce of 5, 1 and 0.1 mmol/lite r ANS acid, respe ctively. For any dilution, an optim al conce ntration (C) of the ANS acid may be estimate d from the following e mpirical formula: 5/A 5 C (in millimole s pe r liter), whe re A is the -fold dilution of the serum. Highe r ANS acid conce ntrations were inhibitory, but the e nzyme activity did not decrease below its basal le vel (no ANS acid adde d). Inhibition and substrate spec-i® city data of serum aminope ptidase -M in the se rum pools in groups 1± 6 are shown in Table 1 . In both undilute d and dilute d se ra, puromycin, be statin, and amastatin inhibite d the e nzyme with K i value s of about 70 m mol/lite r, 1.8 m mol/lite r, and 70 nmol/lite r, re spective ly. In the prese nce of ANS acid, the enzyme se nsitivity to the inhibitors was change d to a certain de gre e; howe ver, pote ncy and se le ctivity range s of the inhibitors re maine d unchange d. The K M value , de te rmined with leu-e nke phalin as substrate , was 359 6 62 m mol/lite r in both undilute d and dilute d se ra from groups 1± 6 ( Table 1) . Aminope ptidase -M activity in the individual sera from groups 1± 6 was evaluate d unde r diffe re nt assay conditions, and the re sults are shown in Table 2 . In the undilute d sample s with pH 8.5, the e nzyme activity was statistically signi® cantly unde restimate d (P , 0.005) as compare d to the same sample s with pH adjuste d to the physiological le vel. Maximal enzyme activity was observe d in the samples that were dilute d 80-fold. The se rum aminope ptidase -M activity was statistically signi® cantly highe r in childre n with cholestasis and acute he patitis as compare d to he althy childre n or those with chronic active hepatitis (P , 0.005) . We atte mpte d to de te rmine and characte rize the e xpre ssion and prope rtie s of se rum aminope ptidase -M activity in childre n with live r diseases and in he althy childre n. We have shown the pre sence of two fractions of the enzyme . Activity of the ® rst fraction (basic activity) is e xpre ssed in the undilute d or ne gligibly dilute d se rum at pH 7.4. Activity of the se cond fraction (cryptic activity) appe ars in the serum as a re sult of dilution and/or addition of ANS acid. In childre n with chole stasis and in childre n with acute he patitis due to HBV infection, both fractions of the e nzyme are highly elevate d as compare d to he althy childre n or those with chronic active he patitis due to HBV infection. Since enkephalins are among the well-characte rize d, natural aminope ptidase -M substrate s (2, 3, 17, 19 ± 28) , in our assay we use d radiolabe le d le ue nkephalin at a concentration close to the peptide leve l in circulation. [ 3 H]Le u-e nkephalin is biologically indistinguishable from the native pe ptide whe reas substitution of the iodine see mingly in¯ue nced the substrate ± e nzyme inte raction, re sulting in a slightly de crease d rate of hydrolysis ( Figure 1 ). The enzyme pH optimum was narrow, about 6.5± 7.3, and the activity dramatically decreased outside that range . After one free ze± thaw cycle , the pH of se rum increases to 8.5 due to ne ar comple te CO 2 evaporation. To avoid a se rious unde re stimation of the enzyme activity (20 ± 24, 26, 39, 40) , se rum pH must be carefully adjuste d to 7.4 or, alte rnative ly, to the enzyme pH optim um. Maintaining the serum pH may be achie ve d by dilution of se rum with an appropriate buffe r. Howe ver, as we have shown, a dilution is anothe r factor affe cting the serum aminope ptidase -M activity. Whe the r pH plays a role in in vivo re gulation of the e nzyme activity re mains to be determined. Aminope ptidase -M activity dynamically increased as a re sult of dilution of the serum with simple buffer or buffe red saline . The plate au of the activity was found in the sample s dilute d 50-to 80-fold, where as a negligible dilution, up to 1.5-fold, was without e ffe ct. In he althy childre n and childre n with chronic active he patitis due to HBV infe ction, enzyme activity incre ase d from 70 (undilute d serum) to 900 pg/min/m l (80-fold dilute d serum). In childre n with Byler' s disease, the re spective value s were 240 and 2000 pg/min/ml. The highe st incre ase s were found in childre n with Alagille syndrome (from 360 to 6300 pg/min/m l), e xtrahe patic biliary duct atresia (from 430 to 6600 pg/min/ml) , and those with acute he patitis due to HBV infection (from 300 to 4000 pg/min/ml) . In many re ports (6, 7, 11, 13, 19, 23, 27, 33) , including ours (39, 40) , serum dilution was not take n into account as a factor that may affect aminope ptidase -M activity. An increase of e nzyme activity as a result of dilution sugge sts that a large portion of the enzyme molecule s is not hydrolytically active and is prese nt in the se rum as a cryptic fraction. This may be due to prese nce of the enzyme natural inhibitor( s) that dissociate as a result of dilution. If this assumption is true , the n an increase of the se rum aminope ptidase -M in chole stasis and acute he patitis is not due to a de® cie ncy of such an inhibitor since both fractions of the enzyme are highly e levate d. Activation of the e nzyme may occur in vivo. Re cently, Martine z and coworke rs ( 20, 26) have shown that serum aminope ptidase -M activity in rats is capable of changing rapidly in re sponse to environmental e xpe rie nce . Howeve r, the mechanism of e nzyme activation as a re sult of se rum dilution re mains to be de te rmine d. Addition of ANS acid, capable of disrupting weak bonds, incre ase d serum aminope ptidase -M activity in a dose -de pe nde nt manne r ( Figure 3 ). Howe ver, the mechanisms of incre asing enzyme activity by ANS acid and by dilution se em to be diffe rent, and both factors appe ar to act inde pe nde ntly on the e nzyme structure , on re gulatory factors, or both. O ur inhibition and speci® city data have shown substantially the same characte ristics of the e nzyme both in the undilute d and dilute d sera de te rmined with or without the pre sence of ANS acid. O ur data sugge st that a re liable aminope ptidase -M assay may be performed using a picomolar conce ntration of the e nke phalin labe le d with iodine or tritium, adde d at a ne gligible volum e to an aliquot of the undilute d se rum with pH adjuste d to the physiological leve l. Maximal e nzyme activity may be de te rmine d using 50-to 80-fold-dilu te d se rum. Hemolysis must be avoide d since e rythrocyte s contain an abundance of e nke phalin-de grading activity (40) . Use of E DTA plasma, phosphate , citrate , or barbital buffers marke dly decrease s the e nzyme activity. For the monitoring of aminope ptidase -M activity, a varie ty of substrate s (6 ± 9, 11, 13, 18 ± 26, 28, 33, 42) and technique s may be applie d (7± 11, 20 ± 22, 24, 27, 42) ; howe ver, we have shown he re that a se nsitive , fast, and inexpe nsive alum inum silic ate binding assay is an e xce lle nt method for detection of degradation of 3 H-or 125 Ilabe le d enkephalins. Chole stasis is associate d with an accumulation of e nkephalins and othe r opiate peptide s in blood that may contribute to pathoge nesis of pruritus or encephalopathy (7, 29 ± 39) . Whe the r the opioid syste m is alte re d in acute viral he patitis remains to be de te rmined. Although the origin of e nkephalins in cholestasis is unknown (35) , it see ms that an impairm ent of the he patic degradation of these pe ptide s doe s not occur (40) . The refore, an increase of serum aminope ptidase -M in chole stasis may be speculate d to be a home ostatic atte mpt to preve nt unlim ited accumulation of opiate pe ptide s in circulation. Howe ver, due to increase d activity, the e nzyme see ms to cause substantial change s in the turnove r of othe r, biologically active peptide s in the blood (43) . There fore , we suggest that the e nzyme is not only a marker but also a cofactor of the disease. The source of se rum aminope ptidase -M was not prove n, but the e nzyme is thought to originate from the live r (2), where it is a he patocyte -membrane -bound metallope ptidase virtually abse nt in the he patocyte cytosol. The e nzyme may re¯ect othe r pathways or aspe cts of the pathological proce ss than those re¯ected by se rum ALAT or g GTP. Inde ed, our patie nts with Byle r's disease (se e Mate rials and Me thods) had a normal le vel of the se rum g GTP, moderate ly increased ALAT, and high le vels of aminope ptidase -M. In contrast, childre n with active chronic he patitis had e levate d leve ls of g GTP and ALAT but normal aminope ptidase -M activity. Among our six patie nts with acute he patitis due to HBV infe ction (ALAT . 600 units/lite r), two patie nts had mode rately elevate d g GTP and highly incre ase d aminope ptidase -M activity whe reas four othe r patie nts e xhibite d high incre ase of both parame ters. Aminope ptidase -M in the serum e xhibits a high amplitude of change s both in chole stasis and acute viral he patitis. Howeve r, its physiological and pathophysiological role , as well as its re levance to the situation in chole static live r dise ase s and viral he patitis will re quire furthe r studie s. Our data se e m to provide ne w, important insights for furthe r studie s on a role of this e nzyme. 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We thank Dr. H. Falk, Falk Foundation e .V. Freiburg, Germany, for a trave l grant funded to R.M.J., as a result of which we were able to present part of the present data at the X International Congress of Liver Diseases, O ctober 19 ± 21, 1995, Basel, Switzerland.