N-acetyl-beta-D-glucosaminidase is a lysosomal enzyme made up of two isoenzymes (A and B). It has been used extensively as a marker for kidney damage. However, its estimation in urine has not been standardized. We have established a method for the estimation and separation of NAG isoenzymes by ion-exchange chromatography. In 19 experiments done so far, this method has given reproducible results. The significance of this method is that with a single experiment, one can estimate total as well as individual isoenzyme activity. Furthermore, urine constituents do not appear to interfere in this assay

Hussain, A N

Javed, M H

Waqar, M A

English

eCommons@AKU

eCommons@AKUDepartment of Biological & Biomedical Sciences Medical College, PakistanMarch 1992Estimation and separation of N-acetyl-beta-D-glucosaminidase isoenzymes in urineM H. JavedAga Khan UniversityA N. HussainAga Khan UniversityM A. WaqarAga Khan UniversityFollow this and additional works at: https://ecommons.aku.edu/pakistan_fhs_mc_bbsPart of the Biochemistry CommonsRecommended CitationJaved, M. H., Hussain, A. N., Waqar, M. A. (1992). Estimation and separation of N-acetyl-beta-D-glucosaminidase isoenzymes inurine. Journal of Pakistan Medical Association, 42(3), 64-66.Available at: https://ecommons.aku.edu/pakistan_fhs_mc_bbs/479ESTIMATION AND SEPARATION OF N-ACETYL-B-D-GLUCOSAMINIDASE ISOENZYMES IN URINEPages with reference to book, From 64 To 66 Masood H. Javed, Abida N. Hussain, Mohammad A. Waqar ( Department of Biochemistry, The Aga Khan UniversityMedical college, Karachi. ) ABSTRACT N-acetyl-B-D-glucosaminidase is a lysosomal enzyme made up of two isoenzymes (A and B). It hasbeen used extensively as a marker for kidney damage. However, its estimation in urine has not beenstandardized. We have established a method for the estimation and separation of NAG isoenzymes byion-exchange chromatography. In 19 experiments done so far, this method has given reproducibleresults. The significance of this method is that with a single experiment, one can estimate total as wellas individual isoenzyme activity. Furthermore, urine constituents do not appear to interfere in this assay(JPMA 42: 64,1992).INTRODUCTION The monitoring of N-acetyl-B-D-glucosaminidase (NAG; EC 3.2.1.30) in urine is one of the mostpromising, early non-invasive tests of renal injury1-4. During the last decade, great efforts have beenmade to develop rapid, reliable and cost-effective methods for assay of NAG in urine. All the methodscurrently used involve incubation of the enzyme with a phenolic N-acetyl-glucosaminide and can bedivided into two groups. The first group utilizes endpoint methods5-7 in which an alkaline buffer isadded to stop the enzyme reaction and to develop the colour of the released chromogenic phenolmolecule. In contrast, the second group includes several recently developed kinetic rate assays, orcontinuous methods8-10, in which the substrate incorporates a phenolic aglycon with a low pICa valuefor the free phenol. When the aglycone is released at pH 4.8-6.25, a colour develops, obviating the needfor addition of alkaline-stopping buffer. NAG is now being used in clinical nephrology to detect earlychanges of renal functions1-4. but unfortunately it has not been used in our country. In Pakistan,clinicians are mostly recommending the classical tests, i.e., creatinine clearance or plasma urea toassess the kidney functions11,12. We have standardized a method for the estimation of total NAGactivity and its isoenzymes in urine.PATIENTS AND METHODS Normal urine was taken from the clinical laboratory Of the Aga Khan University Hospital (AKUR),Karachi. The urine of the kidney stone patients was taken directly from the patients in the kidney ward.Some urine samples were also taken from the patients just before and after lithotripsy treatment fromthe lithotripsy section of the surgery department. The urine samples were centrifuged for about 10 minat 5000 rpm and used as such, or frozen at -70°C. Some of the urine samples were also dialyzed against10 mm sodium phosphate buffer, pH 7.0, for about 24 hrs. NAG-A from human uterus forstandardizing the DEAE-column was partially purified according to the method described elsewhere13.DEAE-Sepharose column chromatographyOne ml urine sample was applied on a minicolumn of DEAE-Sepharose (1.5x3 cm) pre-equilibratedwith 10 mm sodium phosphate buffer, pH 7.0. The flow rate of the column was about 40 mI/hr. Thecolumn was washed with 10 ml equilibrating buffer. The NAG-A was then eluted with 0.3 m NaCl inequilibrating buffer. 1 ml fraction was taken. The NAG activity was determined in alternate fractions.NAG activityThe NAG activity in fractions after DEAE-Sepharose chromatography was measured according to themethod described by Severint et al14 by incubating (37°C, 15 min) 0.1 ml fraction with 0.9 ml of 20mm citrate/phosphate buffer (pH 4.4) containing 0.1 mm methylumbelliferyl glucosaminide. To stopthe reaction, we added 2 ml 0.5 M glycine buffer (pH 10.4). The fluorescence of 4-methylumbelliferone (4 MU) was measured with Jasco spectrofluorimeter at excitation and emissionwave lengths of 368 and 448 nm, respectively and corrected for blank fluorescence. To calibrate themethod, we used freshly prepared 4 MU standards (in 0.2 M sodium carbonate) of knownconcentration. One unit of NAG activity was defined as umole of 4 MU produced/mm under assayconditions. Total NAG activity was calculated by adding the activities of NAG-A and NAG-B.RESULTS The results of DEAE-Sepharose chromatography of one typical experiment is shown in Figure 1.This figure shows that NAG-B does not bind with the column while NAG-A is eluted by 0.3 M NaCl asa single peak. We have estimated the NAG activity in 19 samples of urine from normals and patients.In all samples NAG-A was always eluted at almost same place. Only two samples (which were fromkidney stone patients) showed NAG-B. In five normal urine samples, only NAG-A was present.Dialysis of urine has shown no effect on the elution profile of NAG-A. When we calibrated the columnwith known amount of NAG-A, we found that the activity in normal urine samples was 2.55± 1.29 U/Land in stone patients sample was 18.25 U/L (Table).DISCUSSION Increased urinary NAG level is a sensitive biochemical marker of tubular insult and the estimation ofurinary NAG in foreign countries is being used in clinical nephrology to detect early changes of renalfunction1-4. However, in Pakistan it has not yet been applied for assessing renal functions. Differentlaboratories are using various methods for its estimation in urine5-10. Each method has its own meritsand demerits. For example, p-nitrophenyl N-acetyl-B-D-glucosaminide is a reliable substrate for mostNAG determinations; but because of the colour of the p-nitrophenolate ion is similar to that of urine,pre- treatment of each urinary specimen by gel filtration has been recommended5. In Japan, Yagi et al9have synthesized 3, 4- dinitrophenyl N-acetyl-B-Dglucosaminide for direct (continuous) assay of NAG.However, this substrate is not yet available in the market. Moreover, it is sparingly soluble in water andthe reagent mixture is slightly unstable after dissolution10. Our method for the estimation of NAG inurine has been found highly reproducible and reliable. There is no problem of interference ofendogenous low-molecular mass activators or inhibitors present in human urine15, because these havebeen removed in DEAE-exchange chromatography. The results (Table) clearly showed that kidneyshave been damaged by stones because normal urine samples have very low NAG activity as comparedto stone patients. The main problem is that it is a time consuming method and also it involvesspectrofluorimeter which is a costly equipment and usually not available in many pathologylaboratories. The increased total NAG activity has been associated with renal damage but this methodalone cannot explain the mechanism of damage. A more convenient method is necessary, such asanalysis of NAG isoenzymes in urine14-16. It has been shown that NAG-B increased in urine inmembrane-proliferative glomerulonephritis, focal glomerulosclerosis but not in proliferativeglomerulonephritis or membranous glomerulonephritis. Analysis of NAG isoenzymes has beenperformed using DEAE-cellulose column chromatography14. Very recently Shibasaki et al16 haveseparated the NAG isoenzymes using automated fast protein liquid chromatography system. HPLC hasalso been used for estimation and separation of isoenzymes of NAG17. But these methods are veryexpensive. Using linear gradient of NaCl (0-0.3 M) for the elution of NAG from DEAE-column, NAGhas been differentiated into B-, I- and A-forms14,16. We have also used the linear gradient of NaCl (0-0.5 M) for the elution of NAG from human uterus cytosol (Figure 2)and we have obtained almost identical results. In the present study our aim was not to differentiate theNAG isoenzymes. The aim was only to determine the activity of NAG in urine samples of variouskidney diseases. After establishing the method for NAG activity and its isoenzymes in urine, we wouldlike to apply this methodology for the assessment of renal disease and for monitoring the secondaryinvolvement of the kidney in disorders such as diabetes, hypertension and rheumatoid arthritis. Themethod may also be applied in testing renal functions in the screening of new drugs.ACKNOWLEDGEMENTS We thank Dr. Jamsheer Talati, Department of Surgery, The Aga Khan University Hospital (AKUH) andstaff of the clinical laboratory of AKUH for providing urine samples.REFERENCES 1. Price, R.G. Urinary enzymes, nephrotoxicity and renal diseases. Toxicology, 1982; 22:99-134.2. Stolarclc, I., Howey, J.E.A. and Fraser, CO. Biological variation of urinary NAG: Practical andclinical implications, din. Chem., 1989; 35:560-63.3. Jung IC, Hempel, A., Grutzmann, K.D., Hempel, RD. and Schreiber, 0. Age-depend. ent excretion ofalanine aminopeptidase, alkaline phoaphatase, gammaglutamyltranaferase and NAG in human urine.Enzyme. 1990; 43:10-16.4. Price, R.G., Pocsi, 1., Richardson, AC and Whiting, P.H. Rate assays of NAG: aome problems andfundsrnental principles. dIm. Chem., 1990; 36:1259-60,5. Honk, E., Hopfer, S.M. and Sunderman, F.W. Jr. Spectrophotometric assay for urinary N-acetyl-beta-D- glucosaminidase activity. dim. dhem., 1981; 27:1180-5.6. Yuen, CT., Price, ItO., dhattagoon, L., Richardson. A.C and Prsill, P.F. dolorimetric assays for N-acetyl-beta-D. gslactoaidasein human urineusing newly-developed omeganitrostycyl substrates, dlidhim. Acta, 1982; 124:195-204.7. Pocsi, I., Taylor, S.A., Richardson, AC, Aamlid, ICH., Smith, B.V. and Price, R.G., "VRA-GlcNAc":Novsl substrate forNAG applied to assay of thisenzyme in urine. Clin. dhem., 1990; 36:1884-88.8. Makise, J., Salto, P., Obuchi, M. Kinetic rate assay of urinary NAG with 2-chloro-4 nitrophenyl-N-acetyl-B-D-glucosaminide ass substrate. dIm. dhem., 1988; 34:2140-43.9. Yagi, T., Hisada, it and Shibita, H. 3, 4-dinitropheny N-acetyl- B-D-glucosaminide, a syntheticsubstrate for direct spectrophotometric assay of NAG or N-acetyl-B-Dhexosaminidase. Anal.Biochem., 1989; 183:245-249.10. Makise, 3., Saito, E., Obuchi, M., Kanayama, M., Hsrskawa, K and Yoshida, K. Improved kineticrate sssay of NAG with 2-chloro-4- nitrophenyl-N-acetyl-B-Dglucosaminide as substrate. dim. chem.,1990; 36:319-22.11. Lente, F.V. and Suit, P. Assessment of renal functions by serum creatinine and creatinine clearance:Glomerular filtration rate estimated by four procedures. Clin. dhem., 1989; 35:2326-30.12. Murphy, I.E., Preuss, HG. and Henry, J.B. Evaluation of renal function and water, electrolyte andacid-base balance in clinical diagnosis and management by laboratory methods, byJ.B. Henry.Philadelphia, Saunders, 1984, pp. 118-32.13. Javed, M.H., Hussain, AN. and Waqar, M.A NAG isoenzyme-A in human ovarian cyst adenoma.Biochem. Soc. Trans., 1991; 19:213S-213S14. Severini, G., Aliberti, L.M. and Girsisme, MD. NAG isoenzymes in serum and urineof patients withdiabetes mellitus. dim. dhem., 1988; 34:2430-32.15. Konings, C.H. and Mulder, c. Effect of gel filtration on urinary NAG activitymessured by the m-cresolsulfonphthaleinyl procedure. dIm. dhem., 1990; 36:402-3.16. Shibasaki, T., Gami, H., Ishinoto, F. and Miyahara. T. Urinary N- acetyl-B-D. glucosaminidaseisoenzyme activity as measured by fsst protein liquid chromatography in patientswithnephroticsyndrome. dli dhem., 1990; 36:102-3.17. Nicot, 0., Lachatre, 0., Gonnet, C, Dupuy, J.L. and Valette, J.P. Rapid assay of NAG isoenzymes inurine by ion-exchange chromatography. dIm. dhem., 1989; 33:1796-1800.

Estimation and separation of N-acetyl-beta-D-glucosaminidase isoenzymes in urine

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Estimation and separation of N-acetyl-beta-D-glucosaminidase isoenzymes in urine

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