Presence of serum and tissue forms of N-acetyl-beta-glucosaminidase in urine from patients with renal disease.

1. The A, B, I1 and I2 forms of N-acetyl-beta-glucosaminidase present in urine, serum, kidney, liver and cerebral spinal fluid were separated on DEAE-cellulose and their presence confirmed by cellogel electrophoresis. The relative activities of each enzyme were determined by integrating the area under the elution peaks. 2. Serum A-form was eluted at a lower molarity of chloride than liver A-form and this was designated the As-form to distinguish it from the A-form of N-acetyl-beta-glucosaminidase found in liver and kidney. 3. The P-form of N-acetyl-beta-glucosaminidase present in the serum of a group of pregnant women was not detectable in urine samples from the same women. 4. Urinary NAG activities were found to be abnormally high in patients with impaired renal function. 5. The activity of both N-acetyl-beta-glucosaminidases A and B increased in pathological urines. The higher the total N-acetyl-beta-glucosaminidase activity excreted the higher the % of activity of the B-form present. 6. In a number of patients with haematuria an A-form similar to the serum As-form was present in the urine.     

The possibility of determining N-acetyl-beta-D-glucosaminidase isoenzymes under alkaline conditions

OBJECTIVES: To have a reliable diagnostic test, the influence of urine pH on the determination of the total activity of N-acetyl-beta-D-glucosaminidase (NAG) and NAG isoenzyme activities was studied. DESIGN AND METHODS: After ultrafiltration and dialysis of the acidic and alkaline urines, the B, A, and A2 forms of NAG were separated by ion-exchange chromatography on DEAE cellulose. RESULTS: A significant decrease in the total activity of NAG in alkaline urines (pH around 8 or higher) was found, which makes this determination unreliable. Analysis of the isoenzymic profiles obtained for weakly acidic and alkaline urines (in the pH range from 5.5. to 10.0) showed that the percent fractions of the individual isoenzyme activities in the total NAG activity and their ratios changed only at pH values above 9.5. CONCLUSIONS: The determination of the denoted isoenzymes of urinary NAG after ultrafiltration, dialysis, and chromatographic separation on DEAE cellulose is reliable in a wide range of alkaline pH values of urine.     

Diagnostic value of urinary N-acetyl-β-d-glucosaminidase, its isoenzymes and the fractional excretion of sodium following renal transplantation

Daily total urine N-acetyl-β-d-glucosaminidase activity, isoenzyme profile and fractional excretion of sodium were measured in 13 consecutive renal transplant patients. Rejection episodes were clinically diagnosed in 12 patients, 11 of whom (92%) showed an increased enzymuria either before or during the onset of clinical signs. The ratio of the two major isoenzymes (A/B) fell during 10 episodes (83%) and in six of these (50%) increased levels of the minor isoenzyme forms were observed. Increased fractional excretion of sodium was associated with nine (75%) of the episodes. Increased fractional excretion of sodium with a raised total enzymuria accompanied by a reduced A/B ratio and an increased proportion of the minor isoenzyme forms occurred in eight (67%) of the rejection episodes. The use of these measurements in the diagnosis of episodes of acute rejection in renal transplantation is discussed.     

Diagnostic value of urinary N-acetyl-beta-D-glucosaminidase, its isoenzymes and the fractional excretion of sodium following renal transplantation

Daily total urine N-acetyl-beta-D-glucosaminidase activity, isoenzyme profile and fractional excretion of sodium were measured in 13 consecutive renal transplant patients. Rejection episodes were clinically diagnosed in 12 patients, 11 of whom (92%) showed an increased enzymuria either before or during the onset of clinical signs. The ratio of the two major isoenzymes (A/B) fell during 10 episodes (83%) and in six of these (50%) increased levels of the minor isoenzyme forms were observed. Increased fractional excretion of sodium was associated with nine (75%) of the episodes. Increased fractional excretion of sodium with a raised total enzymuria accompanied by a reduced A/B ratio and an increased proportion of the minor isoenzyme forms occurred in eight (67%) of the rejection episodes. The use of these measurements in the diagnosis of episodes of acute rejection in renal transplantation is discussed.     

beta-N-acetyl-D-glucosaminidase isoenzymes by chromatofocusing from serum and skin in diabetes

beta-N-Acetyl-D-glucosaminidase and its kinetic characteristics were determined in serum and skin from both diabetes mellitus and impaired glucose tolerance patients and controls. Mean total activity was reduced (p less than 0.05) in the skin of both patient groups. beta-N-Acetyl-D-glucosaminidase isoenzyme expression was investigated using chromatofocusing on PBE-94 coupled with automated enzyme assay. The isoenzyme profiles from serum showed two major forms (A and B) whose ratio varied from 2:1 in controls to 3:1 in diabetics. Moreover, diabetes mellitus and impaired glucose tolerance subjects displayed an intermediate (I) form. The A/B isoenzyme ratio was completely reversed in skin.     

Urinary N-acetyl-beta-D-glucosaminidase isoenzyme activity as measured by fast protein liquid chromatography in patients with nephrotic syndrome

Exactly why N-acetyl-beta-D-glucosaminidase (NAG) excretion is increased in patients with nephrotic syndrome with glomerular lesions is poorly understood. Glomeruli contain less NAG than do proximal tubules. In this study, we have tried to measure the NAG isoenzymes automatically by use of the recently developed fast protein liquid chromatography (FPLC) system, followed by column chromatography on DEAE cellulose (Mono Q). Three isoenzyme peaks--B, I + II, and A--were observed for urine from both healthy subjects and nephrotic patients. The B isoenzyme usually constituted about 10% of the total NAG in healthy controls, 30% in nephrotic patients. In contrast, the proportion of the A isoenzyme was inversely related to that of the B isoenzyme when healthy controls and nephrotic patients were compared. Our system for measuring NAG isoenzymes is reproducible and fast, and it should be useful in further studies.     

N-acetyl-beta-glucosaminidase isoenzymes in serum and urine of patients with diabetes mellitus

The isoenzyme pattern of N-acetyl-beta-glucosaminidase (NAG) in serum and urine was studied in two groups of patients with diabetes mellitus and in 30 control subjects. Total NAG activity was significantly (P less than 0.001) increased in the serum and urine of the 20 diabetics with vascular complications, but was insignificantly increased in the 20 diabetics without vascular complications. Ion-exchange chromatography demonstrated the presence of two major isoenzymes of NAG, A and B. The proportion of isoenzyme A activity always exceeded that of isoenzyme B. The proportion of isoenzyme B in serum of diabetics was lower than in controls; the reverse was true for urine of diabetics. The NAG isoenzymes pattern may provide additional diagnostic information regarding diabetic status and complications of diabetes.     

Semi-automated fluorometric assay for urinary total and B N-acetyl-beta-D-glucosaminidase: analytical investigation

Total N-acetyl-beta-D-glucosaminidase (NAG) and NAG-B isoenzyme determination are determined in order to investigate renal tubular function. Here, we propose a semi-automated adaptation of Sandman's manual method for the Monarch centrifugal analyzer. Fluorescence of the released 4-methylumbelliferone is automatically read and results are directly expressed in nanokatal/l. Mean (1 SD) values expressed in nanokatal/millimole creatinine in urine from healthy female (n = 30) and male (n = 30) subjects were 8.2 (4.0) and 7.8 (2.9) for total NAG and 1.9 (1.4) and 1.5 (0.7) for the NAG-B isoenzyme activity. The assay of six patients with various renal disorders shows definite increase in total NAG and NAG-B isoenzyme activities.     

Early monitoring of human renal transplantations by N-acetyl-beta-D-glucosaminidase isoenzyme activities in urines

Monitoring of variations in N-acetyl-beta-D-glucosaminidase (NAG) urinary activity, following renal transplantation, has been proposed for the early diagnosis of rejection episodes. In this study, the measurement of urinary NAG-B activity was conducted as a complement to total NAG (A + B) measurement, which is normally used alone. Selective measurement of NAG-B activity is carried out after fixation of NAG-A on ion exchanger in test tubes. Results of NAG (A + B) activity confirm that the assay of urinary NAG is a useful indicator of rejection, but a positive correlation between NAG-B and NAG (A + B) activities was observed during the various complications which can occur after transplantation. The specific measurement of this isoenzyme does not, therefore, seem to provide additional information in the early monitoring of human renal transplantations. Apart from rejection episodes, other factors are likely to produce marked NAG-B excretion, e.g. gentamicin therapy.     

Beta-N-acetylhexosaminidase in the urine, kidney and serum of bromobenzene-intoxicated mice.

beta-N-Acetylhexosaminidase isoenzymes were separated from the kidney, serum and urine of normal mice and mice intoxicated with bromobenzene, using DEAE-cellulose chromatography. Both mouse serum and urine showed hexosaminidase profiles similar to the human counterparts with the presence of B (basic), I (intermediate) and A (acidic) isoenzymes. A notable feature was the presence of a high proportion of an intermediate form in mouse urine which is not always present in human urine. Hexosaminidase activity increased significantly in urine of mice intoxicated with bromobenzene. Its increase was time-dependent and due to kidney damage with a release in the urine of hexosaminidase A, I and, in higher proportion, B. No significant differences were observed in mouse kidney and serum profiles following intoxication with bromobenzene. The total activity of hexosaminidase, using 4-methylumbelliferyl-2-acetamido-2-deoxy-beta-D-glucopyranoside as substrate, did not increase in the serum of mice intoxicated with bromobenzene. Both hexosaminidase activity and the isoenzyme pattern in urine can be used as indicators of kidney damage by bromobenzene intoxication.     

The significance of microproteinuria for the diagnosis of kidney involvement in hypertensive disease and secondary forms of arterial hypertension]

N-acetyl-beta-glucosaminidase (NAG) activity, the concentrations of microalbumin (MA) and B2-microglobulin (B2-MG) were measured in urine of 50 healthy subjects and 200 patients suffering from arterial hypertension (AH) with preserved renal function, including patients with essential hypertension (EH), stages I and II, chronic pyelonephritis (CPN), chronic glomerulonephritis (CGN) and vasorenal hypertension (VRH). The healthy subjects, the patients with stage II EH, and those with secondary forms of AH demonstrated significant differences in NAG activity in urine. A positive correlation (r = +0.53; p < 0.03) was discovered between systolic AP and NAG activity in urine of EH patients. The concentration of MA in urine of CGN and VRH patients was significantly higher than that in the healthy subjects, EH and CPN patients. The patients with CPN and VRH showed significantly higher levels of B2-MG in urine.     

Urinary lysosomal enzyme excretion after renal allotransplantation.

Three urinary lysosomal enzymes, beta-glucuronidase (beta-Gluc), beta-galactosidase (beta-Gal) and N-acetyl-beta-D-glucosaminidase (NAG), were measured in twenty-one renal allograft recipients to evaluate their role in the diagnosis and prediction of rejection episodes, and in the prediction of eventual graft outcome. A fluorometric assay using methylumbelliferone substrates was used to measure the three enzymes in morning urine samples and enzyme activity was defined in terms of urine creatinine concentration. Urinary NAG levels increased significantly in 13/16 first rejection episodes and 4/4 instances of acute tubular necrosis and graft infarction. In 5 of the 16 first rejection episodes the NAG was predictive of the rejection. NAG was not useful in diagnosing second or subsequent rejections and beta-Gluc and beta-Gal were of little value in assessing any component of renal transplant pathology. As a prognostic index of eventual graft outcome, the peak urinary NAG was particularly encouraging. It correlated strongly with deterioration in graft function as time passed such that only 2/10 patients with peak NAG greater than 1400 Units had normal serum creatinines at 6 months post transplantation. Conversely 4/4 patients with peak NAG levels less than 700 Units had normal serum creatinine at that time. In our series the measurement of urinary NAG was a useful adjunct to the diagnosis of first rejections but appears to be more valuable in predicting graft outcome.     

Influence of pigments and pH of urine on the determination of N-acetyl-beta-D-glucosaminidase activity with 2-methoxy-4-(2'-nitrovinyl)-phenyl-N-acetyl-beta-D-glucosaminide

The influence of urinary pigments and urine pH on the spectrophotometric determination of N-acetyl-beta-D-glucosaminidase (NAG; EC 3.2.1.30) activity with 2-methoxy-4-(2'-nitrovinyl)-phenyl-N-acetyl-beta-D-glucosaminide as a substrate was studied. The investigation was performed with human and rabbit urine samples. It was found that alkaline urine pH values influenced NAG activity in two ways: 1) NAG activity decreased due to enzyme instability with pH increase, and 2) NAG activity increased because of the contribution of urinary pigments to absorbance of 2-methoxy-4-(2'-nitrovinyl)-phenol (MNP) at 505 nm. It was shown that besides the maximum (I) in the range of 350-360 nm of the absorption spectra of alkaline urine, there was a maximum (II) in the range of 380-460 nm. With the increase of pH, maximum II was shifted toward higher wavelengths and contributed to MNP absorption (5-90%). On the other hand, the maximum of MNP absorption was shifted toward lower wavelengths (495-400 nm) with increasing pH. Two procedures to eliminate the influence of urinary pigments are presented. The justification of applying a correction to the values of NAG activity in human and rabbit urine (a model system for studying the toxic effects of cadmium) was discussed.     

The diagnostic value of urinary enzyme measurements in hypertension

N-Acetyl-beta-D-glucosaminidase (NAG), beta-D-galactosidase, alkaline phosphatase (ALP) and leucine aminopeptidase (LAP) were assayed in the urine of 100 normal and 112 hypertensive subjects. Age-related urinary activities for these enzymes in the normotensive control subjects are presented. A new procedure for the assay of urinary ALP using 2-methoxy-4-(2'-nitrovinyl)phenyl (MNP) phosphate is described. Thirty-five of the hypertensive patients were considered to have primary renal disease. The urinary activity of NAG was increased in 27 (77%) of these patients and the detection of primary renal disease was not enhanced by measurements of the other urinary enzymes. Testing the urine both for NAG activity and protein, led to the detection of 91% of these patients. The assay procedures described are simple to perform and can be carried out in outpatient clinics. The measurement of urinary NAG activity is a cheap and reliable method for detecting renal disease in hypertensive patients but maximum diagnostic yield is achieved when proteinuria is determined as well.     

Predictive value of urinary N-acetyl-beta-D-glucosaminidase (NAG), alanine-aminopeptidase (AAP) and beta-2-microglobulin (beta 2M) in evaluating nephrotoxicity of gentamicin

Concentrations of N-acetyl-beta-D-glucosaminidase (NAG), alanine-amino-peptidase (AAP) and beta-2-microglobulin (beta 2M) were determined daily in the urine of 28 patients treated with gentamicin (2-3 mg . kg-1 . day-1) for a mean of 15 days. All had normal renal function. Increased activity in NAG and AAP was observed for all patients, either immediately or after 2 or 3 days of treatment. The results were compared with serum creatinine concentrations and urinary beta 2M levels. This study indicates a relationship between the nephrotoxicity of gentamicin and initial urinary enzymic activity(NAGi) prior to any treatment. The degree of NAG response during the first ten days of treatment appeared as a second prognostic factor. Renal failure was observed for one out of the 12 patients with normal NAGi (NAGi less than 200 mumol/day). Seven of them showed a marked enzyme activity response (greater than 1500 mumol/day) with an increase in beta 2M activity. Eleven out of the 16 patients with elevated NAGi (NAGi greater than 200 mumol/day) developed renal failure and showed an elevated maximal response. The concentration of AAP appears to be of little prognostic value. The variation in individual maximal urinary enzyme responses observed among the 28 patients during the first ten days of treatment points to the existence of individual sensitivities to gentamicin, the exact mechanism of which remains unclear.     

Expression modes of urinary N-acetyl-beta-D-glucosaminidase in patients with chronic renal insufficiency

BACKGROUND: Urinary N-acetyl-beta-D-glucosaminidase (NAG) activity has emerged as potentially useful early marker of renal tubular injury. This activity is usually evaluated in random urine samples and is related to urinary creatinine concentration. Reports about the lack of correlation between NAG activity of 24-h urines and activity of random urine samples in some clinical and experimental situations led us to study the correlation existing between different procedures for expressing urinary NAG in patients with chronic renal insufficiency. METHODS: Thirty samples of 24-h urine and 30 random urine samples from chronic renal insufficiency patients were collected. The activity of urinary NAG was examined fluorimetrically. RESULTS: The following correlations were observed: (1) r = 0.431 (P = 0.017) for activity in random urine samples and total activity in 24-h urines); (2) r = 0.281 (P = 0.005) for activity in random samples and activity, expressed as U/l, in 24-h urines. CONCLUSIONS: The data show that collection of urine excreted over the whole day and evaluation of total daily excretion of NAG seems the method of choice, at least for patients with chronic renal insufficiency.     

Comparison of urinary N-acetyl-beta-D-glucosaminidase and urinary fibrin degradation products for diagnosis of rejection after renal transplantation

A comparison was made of two established tests to see which was the better indicator of rejection after renal transplantation. Daily urine samples were assayed for excretion of N-acetyl-beta-D-glucosaminidase (NAG) and fibrin degradation products (FDP). Twenty-five rejection episodes were studied in 19 patients. Both indicators tended to move in parallel. NAG was more often elevated in rejection, namely in 96% of rejection episodes as against 76% for FDP, FDP had the advantage of fewer false positive results and when it correctly indicated rejection its increase above the baseline was relatively greater than for NAG, NAG and FDP can both provide early warning of rejection. NAG is preferred for its ease of assay. Assays are performed daily and are of practical value in clinical management. The results of assays must be interpreted in conjunction with all relevant information.     

2型糖尿病患者尿微量蛋白、尿酶检测的临床意义

目的探讨2型糖尿病患者尿微量白蛋白（mAlb）、N-乙酰--βD-氨基葡萄糖苷酶（NAG）检测的临床意义。方法对101例2型糖尿病患者（根据尿蛋白定性的有无分为尿蛋白定性阴性组51例和尿蛋白定性阳性组50例）和70例健康体检者（正常对照组）采用免疫比浊法,使用全自动生化分析仪检测尿mAlb水平;采用速率法检测尿NAG水平;尿蛋白定性采用干化学法检测。结果尿蛋白定性阴性组、尿蛋白定性阳性组尿mAlb水平、尿NAG活性均明显高于正常对照组（均P〈0.01）;尿蛋白定性阴性组尿mAlb、尿NAG与尿mAlb＋尿NAG的阳性率分别为41.18%、37.25%、62.75%,尿蛋白定性阴性组尿mAlb、尿NAG阳性率均明显低于尿mAlb＋尿NAG的阳性率（均P〈0.05）。结论尿蛋白定性阴性不能除外糖尿病患者早期肾损伤,尿mAlb、尿NAG联合检测对早期发现糖尿病肾病具有重要意义。     

Urinary N-acetyl-beta-D-glucosaminidase and aminopeptidase N in the diagnosis of graft rejection after live donor renal transplantation

An evaluation of the usefulness of urinary N-acetyl-beta-D-glucosaminidase (NAG) and aminopeptidase N (AAP) measurements in the diagnosis and prediction of acute and chronic renal allograft rejection was made. Enzyme activities were measured in 2,745 morning spot urine samples from 53 consecutive live donor renal allograft recipients up to 180 days after transplantation. Reference ranges of urinary enzyme activities in 14 recipients with normal graft function were higher than those established in a carefully selected group of healthy controls. 89 and 91% of 76 clinically diagnosed acute rejection episodes (ARE) in the remaining 39 graft recipients were accompanied by sharp increase over baseline of NAG and AAP respectively. All rejection episodes occurring in the early period after transplantation were characterised by high enzymuria. AAP was more sensitive than NAG as the magnitude of its increase over baseline was more, while NAG was more specific with less number of false positive elevations. Both enzymes were found to be equally good prognostic indices of graft loss and chronic graft deterioration. Regular monitoring of urinary NAG and AAP activities throughout the post transplant period would thus be valuable in (a) diagnosis and prediction of ARE in the early as well as late post operative period and (b) prediction of eventual graft outcome.     

Improved kinetic rate assay of urinary N-acetyl-beta-D-glucosaminidase with 2-chloro-4-nitrophenyl-N-acetyl-beta-D-glucosaminide as substrate

We have improved the kinetic rate assay method for determining N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30; NAG) activity in urine with use of the synthetic substrate, 2-chloro-4-nitrophenyl-N-acetyl-beta-D-glucosaminide (CNP-NAG), reported previously (Clin Chem 1988;34:2140-2). To increase the solubility of this substrate, we used crown ether (15-crown-5-ether) and ethylene glycol. In addition, we used for the standard solution NAG from human placenta, with specificity corresponding to that of human urine, so that values obtained with the CNP-NAG method and a p-nitrophenyl-NAG method ("MEI Assay NAG") correlated almost completely (r = 0.995, n = 29). Reference values for urinary NAG activity determined by the CNP-NAG method were established for untimed urine specimens from 674 healthy volunteers. The normal reference interval (mean +/- 2 SD) for NAG: 1.6-15.0 (mean 4.9) U per gram of creatinine.