Serum Nacetyl-beta-Dglucosaminidase (NAG; EC 3.2.1.30) is a hexosaminidase and may be a predictor of vascular injury, e.g., in infant respiratory distress syndrome, pneumonia, bronchopulmonary dysplasia and necrotizing enterocolitis. To estimate the new diagnostic prospects we have modified our urinary NAG assay. In this sensitive colorimetric microassay, VRAGlcNAc was used as a substrate. In the present study the age dependence of serum NAG activity was investigated in newborn babies, infants (124 months), children (218 years) and adults (1980 years). Serum NAG activity was found to be agedependent; it is higher in early childhood (1159 U/l) but decreases to a constant value at the age of 12 years. After the age of 2 years it is similar to adults NAG (1030 U/l). In pediatrics agematched reference ranges must be taken into consideration

Goi G

Horak E

Jung K

Mandic L

O'Brien JS

Oláh VA

Price RG

Pócsi I

Shattuck KE

Clinical Chemistry and Laboratory Medicine (CCLM)

Vargáné Oláh, Anna

Price, R. G.

Csáthy, László

Orszagh, É.

Oláh, Éva

Varga, József

Repository of the Academy's Library

Clin Chem Lab Med 2004;42(3):305–306  2004 by Walter de Gruyter • Berlin • New York 2004/88Short CommunicationAge dependence of serum b-N-acetylhexosaminidase (NAG)activityAnna V. Ola´h1,*, Robert G. Price2, La´szlo´Csa´thy3, E´va Orsza´gh1, E´va Ola´h4 and Jo´zsefVarga51 Department of Clinical Biochemistry and MolecularPathology, University of Debrecen, Medical andHealth Science Center, Debrecen, Hungary2 Division of Life Sciences, King’s College London,London, UK3 Department of Pediatrics, County TeachingHospital, Debrecen, Hungary4 Department of Pediatrics,5 Department of Nuclear Medicine,University of Debrecen, Debrecen, HungaryAbstractSerum N-acetyl-b-D-glucosaminidase (NAG; EC3.2.1.30) is a hexosaminidase and may be a predictorof vascular injury, e.g., in infant respiratory distresssyndrome, pneumonia, broncho-pulmonary dysplasiaand necrotizing enterocolitis. To estimate the newdiagnostic prospects we have modified our urinaryNAG assay. In this sensitive colorimetric micro-assay,VRA-GlcNAc was used as a substrate. In the presentstudy the age dependence of serum NAG activity wasinvestigated in newborn babies, infants (1–24 months), children (2–18 years) and adults (19–80 years). Serum NAG activity was found to beage-dependent; it is higher in early childhood (11–59 U/l) but decreases to a constant value at the ageof 1–2 years. After the age of 2 years it is similar toadults’ NAG (10–30 U/l). In pediatrics age-matchedreference ranges must be taken into consideration.Keywords: age dependence; infant respiratory dis-tress syndrome (IRDS); reference ranges; serum N-acetyl-b-D-glucosaminidase (NAG); vascular injury;VRA-GlcNAc.Lysosomal hexosaminidases such as N-acetyl-b-D-glucosaminidase (NAG, EC 3.2.1.30) exist in a numberof tissues (liver, kidney, spleen and bowel). After tis-sue damage they might be released into the circula-tion. In the last decades NAG isoenzymes were usedmainly for screening Tay-Sachs disease (TSD) (1, 2).*Corresponding author: Anna V. Ola´h, PhD, Department ofClinical Biochemistry and Molecular Pathology, Universityof Debrecen, Medical and Health Science Center, (KBMPI)Debrecen, Nagyerdei krt. 98. 4012 Debrecen, HungaryPhone: q36-52-432 283, Fax: q36-52-417 631,E-mail: olaha@jaguar.dote.huFor the previous tests generally plasma or serum isused (2–5), while to detect tubulopathy a urinary sam-ple is used (6–11). Serum NAG may be a possible pre-dictor of vascular injury in diabetes mellitus andhypertension (12, 13). Although hexosaminidaseactivity varies during postnatal development anddepends on feeding, serum NAG has been proposedrecently as a predictor of infant respiratory distresssyndrome (IRDS), broncho-pulmonary dysplasia(BPD) and pneumonia in premature babies (3).Increase in serum NAG has also been suggested asa marker for the early identification of necrotizingenterocolitis (NEC) (14). NAG activity might be deter-mined by fluorometric methods (15), although colori-metric assays present many advantages (16).Our aim was to examine the age dependence ofserum NAG. Because of the limited volume of serum,the urinary NAG assay (8–10) was modified. Thisresulted in a simple and sensitive micro-method,which is applicable to different clinical areas. The Kol-mogorov-Smirnov test was used to test the normalityof distributions, both for NAG enzyme activities andtheir logarithms. Age dependence was tested usingone-way analysis of variance (ANOVA). Dunnet’s testwas used to compare multiple age groups to the adultgroup. No significant difference was found in enzymeactivities between males and females. NAG activitiesshowed a rather large scatter in newborns, with a dis-tribution not significantly different from Gaussian. Inall other age groups NAG activities were log-normallydistributed (Kolmogorov-Smirnov test, p)0.05). WithANOVA we found that NAG is age dependent. As indi-cated by Dunnet’s test, NAG is significantly higherbelow 2 years of age than in adults, and then falls tothe adult value. The reference ranges (95% confidencelevel) are shown in Table 1.Decreased activity of serum total NAG and isoen-zyme A is well known in TSD patients (1, 2). In con-trast, the potential value of increased serum NAG innewborns with IRDS, BPD (3) and NEC (14) was con-firmed. Very limited data can be found for serum hex-osaminidase in newborns and premature babies (4,14). Our NAG results are similar to the values pub-lished earlier (3, 4, 14). In accordance with our results,Lobe et al. (14) showed that NAG activity is independ-ent of sex and is relatively high during the first3 weeks of life. As they have found higher NAG valuesafter the onset of NEC, or pneumonia, we plan tocheck whether NAG could be an early detector forthese inflammatory diseases. Similarly to our new-borns’ range, Shattuck et al. described increased NAGactivity in premature babies at the 1st–8th weeks of306 Ola´h et al.: Age dependence of serum NAGTable 1 Reference ranges of serum NAG isoenzyme activities in various age groups.Age groups Newborns Infants Children Adults-1 month, 1–24 months, 2–18 years, 19–80 years,ns22 ns25 ns24 ns23NAG enzyme Lower limit 11.0 11.5 10.4 10.0activity (U/l) Upper limit 48.1 59.3 28.5 30.0Serum NAG assay (10): 3.3 mmol/l VRA-Glc-NAc substrate (PPR Diagnostics, London, E1W 1AT, UK) was dissolved in citricacid-disodium hydrogen phosphate buffer (0.15 mol/l, pH 4.8) and pre-incubated at 378C for 5 min. Before the enzyme assay,25 ml of serum was diluted with 25 ml of the same buffer to decrease the inhibiting effect of urea (17). To 350 ml of substratesolution, 25 ml diluted serum was added and incubated for 30 min at 378C. The reaction was stopped with 375 ml potassiumhydrogen carbonate-dipotassium carbonate buffer (1.2 mol/l; pH 9.8) and the absorbance (A) was read at 505 nm against thereagent blank in a 1 cm cuvette (Humalyser 2000, Human, Germany). Serum samples were stored at y208C for 1–4 weeks.NAG activity was calculated directly via specific molar absorbance: NAG activity (U/l) s A(505)=28.5=2.life (4). Recently it was suggested that NAG might bea marker of oxidative stress, e.g., in diabetic micro-angiopathy, as it correlates with plasma malondial-dehyde in diabetes mellitus type 2 (18). Althoughserum NAG is not a specific marker for vascular dis-eases, it may contribute to the early diagnosis of thediseases mentioned above.References1. Natowicz MR, Prence EM. Heterozygote screening for Tay-Sachs disease: past successes and future challenges. CurrOpin Pediatr 1996;8:625–9.2. Saifer A, Rosenthal AL. Rapid test for detection of Tay-Sachs disease heterozygotes and homozygotes by serumhexosaminidase assay. Clin Chim Acta 1973;43:417–21.3. Goi G, Bairati C, Massaccesi L, Lombardo A, Bonafe L,Zanardo V, et al. Lysosomal enzymes in preterm infantswith bronchopulmonary dysplasia: a potential diagnosticmarker. Clin Chim Acta 1998;278:23–34.4. Shattuck KE, Richardson CJ, Rassin DK, Lobe TE. Devel-opment of serum hexosaminidase activity in infants. BiolNeonate 1986;49:126–31.5. Goi G, Besozzi M, Bairati C, Gaugnellini E, LombardoA,Tettamanti G. Preparation of a stable liquid material forcalibration and quality control for lysosomal enzymes inplasma. Eur J Clin Chem Biochem 1992;30:595–8.6. Jung K, Hempel A, Gru¨tzmann KD, Hempel RD, SchreiberG. Age dependent excretion of alanine aminopeptidase,alkaline phosphatase, gamma-glutamyltransferase and N-acetyl-b-D-glucosaminidase in human urine. Enzyme1990;43:10–6.7. Price RG. Urinary enzymes, nephrotoxicity and renal dis-ease. Toxicology 1982;23:98–134.8. Ola´h VA, Csa´thy L, Varga J, Po´csi I, Price RG. Referenceranges for urinary N-acetyl-b-D-glucosaminidase inhealthy children determined with three colorimetricmethods. Ann Clin Biochem 1994;31:87–8.9. Horak E, Hopfer SM, Sunderman FW Jr. Spectrophoto-metric assay for urinary N-acetyl-b-D-glucosaminidaseactivity. Clin Chem 1981;27:1180–5.10. Po´csi I, Csa´thy L, Ola´h VA, Price RG. Assay of N-acetyl-b-D-glucosaminidase in urine from neonates: compari-son of two colorimetric methods using MNP-GlcNAc andVRA-GlcNAc as substrates. Ann Clin Biochem1992;29:292–5.11. Osborne J. Urinary excretion of N-acetyl-b-D-glucosa-minidase in children. Arch Dis Child 1980;55:719–21.12. Mandic L, Filipovic D. Changes of isoenzymes of serumN-acetyl-b-D-glucosaminidase in relation to differenttypes of diabetes. Biochem Mol Biol Int 1998;45:545–54.13. Goi G, Fabi A, Lorenzi R, Lombardo A, Tettamanti G, Bur-lina AB, et al. Serum enzymes of lysosomal origin asindicators of metabolic control in diabetes: comparisonwith glycated hemoglobin and albumin. Acta DiabetolLat 1986;23:117–25.14. Lobe TE, Richardson CJ, Rassin DK, Mills R, SchwartzMZ. Hexosaminidase: a bio-chemical marker for necro-tizing enterocolitis in preterm infant. Am J Surg1984;147:49–52.15. O’Brien JS, Okada S, Chen A, Fillerup DL. Tay-Sachs dis-ease. Detection of heterozygotes and homozygotes byserum hexosaminidase assay. N Engl J Med1970;283:15–20.16. Price RG. Measurement of N-acetyl--D-glucosaminidaseand its isoenzymes in urine: methods and clinical appli-cations. Eur J Clin Chem Biochem 1992;30:693–705.17. Boudiou MT, Bourbouze R, Bernard M, Percheron F, Per-ez-Gonzalez N, Cabezas JA. Inhibition of A and B N-ace-tyl-b-glucosaminidase urinary isoenzymes by urea. ClinChim Acta 1985;149:67–73.18. Skrha J, Hilgertova´ J. Relationship of serum N-acetyl-b-D-glucosaminidase activity to oxidative stress in diabe-tes mellitus, Clin Chim Acta 1999;282:167–74.Received February 17, 2003, accepted January 23, 2004

Age dependence of serum beta-N-acetylhexosaminidase (NAG) activity

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