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An automated method for the assay of N-acetyl-beta-glucosaminidase in human urine is described and a normal range of urinary N-acetyl-beta-glucosaminidase activity is reported. The automated method has been used routinely over a twelve month period in two London hospitals for monitoring the excretion of N-acetyl-beta-glucosaminidase in renal transplant patients. The abnormal elevation of urinary N-acetyl-beta-glucosaminidase provides an early warning of rejection by indicating the presence of renal parenchymal damage. The automated assay of urinary N-acetyl-beta-glucosaminidase may also be used in screening for the presence of renal disease.

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.

Urinary N-acetyl-beta-D-glucosaminidase (NAG) activities were assayed in every urine void throughout 24 hours in 17 normal people and in four patients with renal disease. The variation in NAG activity due to changing rates of urine flow was almost eliminated by factoring enzyme activity by the urinary creatinine concentration. Random samples of urine may thus be used for assay. The results of NAG assay in 36 patients with acute and chronic renal diseases showed that NAG was a sensitive indicator of renal damage. This simple test may be valuable in detecting or monitoring renal disease.

1. Some practical aspects of the assay of urinary N-acetyl-beta-glucosaminidase activity were studied in patients with renal disease. 2. Centrifugation and dialysis of urine were not necessary prior to assay. 3. When enzyme activity was related to urinary creatinine random urine samples could be used. 4. Since preservatives used to prevent bacterial growth may inhibit enzyme activities, samples should be assayed immediately after collection or stored at 4 degrees C or 20 degrees C. 5. Mild visible haemoglobinuria, eg. derived from lysed red blood cells did not interfere with the enzyme assay.

Renal transplant recipients were divided into five categories according to their clinical course from transplantation to their discharge from hospital. Total N-acetyl-beta-D-glucosaminidase (NAG) activity in urine was determined using a chromogenic substrate 2-methoxy-4-(2'-nitrovinyl)-phenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside. The isoenzyme composition of the urine of each patient was determined by semi-automated DEAE-cellulose chromatography. Although raised NAG activity was found in stable transplant patients compared to controls, the level of activity was constant and no change in the isoenzyme profile was found. In reversible rejection there was a marked increase in the intermediate forms, particularly I2 and a concomitant fall in the relative amount of the A-form present but the profile became normal when the patient stabilised. Much more complex patterns were observed in patients who did not respond to treatment. Both the B and I forms were elevated with a fall in the A-form and in one case excretion of the serum As form was observed. The intermediate forms were always increased in rejection.

Urinary N-acetyl-beta-D-glucosaminidase (NAG) activities were measured in 181 patients with renal allografts during a 15-month period. Activities were high immediately after transplantation but decreased rapidly in the absences of complication. Urinary NAG activities increased by 50% or more in relation to 33 of 36 (92%) episodes of acute rejection diagnosed and treated by clinicians during the first 90 days after transplantation. The increase preceded clinical diagnosis in 70% of the cases, the median interval being 1.5 days. NAG activities decreased after treatment of rejection in 90% of the cases. Chronic rejection, renal vein thrombosis, renal artery stenosis, oliguria, hypotension, and the administraion of gentamicin may also cause increased NAG activity. Urinary NAG assay is simple and inexpensive, and is a useful aid to the early diagnosis of rejection of renal transplants. Results must, however, be interpreted by the clinician, bearing in mind other causes for increased activity.

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.

Determination of the urinary excretion of N-acetyl-D-glucosaminidase (NAG) and fibrin degradation products (FDP) made it possible to make a diagnosis of 25 out of 26 acute rejection episodes at least 24 hr before deterioration in renal function occurred. Of the two tests, the estimation of daily urinary NAG is the most practical for routine clinical use. This test alone permitted early diagnosis in 21 out of 26 episodes. In 9 out of 11 episodes in which both estimations showed an increase, the rise in FDP occurred before the rise in NAG. This is consistent with the view that in some forms of rejection, at least, intravascular fibrin deposition occurs first and causes ischaemic damage to renal tubular cells and consequent deterioration in function.

The urinary excretion of N-acetyl-beta-D-glucosaminidase (NAG), beta-galactosidase (GAL), beta-glucosidase (GLU), and alkaline phosphatase (AP) was studied in 83 patients with renal allografts. Thirty of these patients had stable graft function and their urinary enzyme levels provided a range of normal values. Urinary lactic dehydrogenase (LDH) was estimated in 29 normal subjects and in 11 patients with renal allografts. Serum values for the five enzymes were also obtained. Urinary NAG excretion was abnormally high in 16 out of 17 (94%) episodes of acute rejection. The other urinary enzymes were raised less frequently. In nine patients studied before the onset of rejection urinary NAG activity rose up to three weeks before changes in other tests of renal function. Serum enzyme levels were not found to be of value in the diagnosis of rejection.