Urinary leucine aminopeptidase 3 in population environmentally exposed to airborne arsenic.

INTRODUCTION: Environmental arsenic contamination is a major toxicological problem worldwide due to its carcinogenic and nephrotoxic potential. AIM: The purpose of this observational study was to determine the suspected association between urinary arsenic (uAs) and urinary leucine (or leucyl) aminopeptidase 3 (uLAP3) to evaluate uLAP3 as a candidate biomarker of exposure to airborne arsenic. MATERIALS AND METHODS: A total of 918 adults occupationally and/or environmentally exposed to airborne arsenic were enrolled in the study. Baseline information (age; sex; history of smoking; alcohol, fish and seafood consumption) was gathered. Total uAs concentrations [µg/L] of 918 subjects, as well as the sum of arsenic species (ΣiAs) in 259 subjects, were obtained. Urinary LAP3 was measured by an immune-enzymatic assay using an ELISA kit. Urinary creatinine concentration was assessed with the IB/lAB/1289 research protocol (version II, 2015-09-17). The values of uAs and uLAP3 were recalculated per unit of creatinine. The association between uAs and uLAP3 was assessed using a logistic regression model adjusted for confounders. RESULTS: The study identified a positive correlation between the logarithm of uAs and the logarithm of uLAP3 in the study population (r = 0.1737, p < 0.0000) and between urinary creatinine and uLAP3 concentration not adjusted for creatinine level (r = 0.1871, p < 0.001). In the logistic regression model, there was also an association between increased (≥15 µg/L) uAs and decreased (below the 25th quartile) uLAP3 [OR uLAP3 = 1.22 (95% CI 1.03 to 1.44, p < 0.02)]. CONCLUSIONS: These data suggest that urinary LAP3 may be a potential biomarker of arsenic exposure, which warrants further study.

Sex differences in the excretion levels of traditional and novel urinary biomarkers of nephrotoxicity in rats.

Urinary biomarkers have been used widely in preclinical toxicity studies to detect dysfunctions and injuries of the kidney caused by drugs under development. While they have been well studied for evaluating nephrotoxicity, knowledge of sex differences in excretion levels of urinary biomarkers remains inadequate. We conducted experiments focused on effects of endogenous sex hormones on urinary biomarkers using intact and castrated male and female rats. Comparisons of the urinary biomarker excretion levels between intact male and female rats at 5, 7, 9 and 12 weeks of age revealed higher excretion levels of leucine aminopeptidase (LAP), γ-glutamyl transpeptidase (γGTP), total protein, liver-type fatty acid-binding protein (L-FABP), cystatin C (Cys-C) and ß2-microglobulin (ß2-MG), and lower excretion level of kidney injury molecule 1 (Kim-1), in male rats as compared to female rats. Orchidectomized male rats showed lower urinary excretion levels of alkaline phosphatase (ALP), LAP, γGTP, N-acetyl-ß-D-glucosaminidase (NAG), glucose, total protein, L-FABP, Cys-C, ß2-MG and neutrophil gelatinase-associated lipocalin (NGAL), and higher urinary excretion levels of clusterin (CLU) and Kim-1, than sham-operated male rats. On the other hand, no significant differences in the urinary biomarker excretion levels excluding ALP were observed between ovariectomized and sham-operated female rats. In the present study, we demonstrated the existence of sex differences in excretion levels of urinary biomarkers that are universally used in preclinical toxicity studies, and also that these differences, especially in relation to the urinary excretions of ALP, LAP, γGTP, total protein, L-FABP, Cys-C, and ß2-MG, may closely relate to the endogenous testosterone.

BML-111 Attenuates Renal Ischemia/Reperfusion Injury Via Peroxisome Proliferator-Activated Receptor-α-Regulated Heme Oxygenase-1.

We examine whether BML-111, a lipoxin receptor agonist, inhibits renal ischemia/reperfusion (I/R) injury, and whether peroxisome proliferator-activated receptor-α (PPARα) or heme oxygenase-1 (HO-1) is involved in protective effects of BML-111 on kidney against I/R injury. Rats subjected to renal I/R injury were treated with or without BML-111. Renal histological and immunohistochemical studies were performed. Expressions of phosphorylated p38 mitogen-activated protein kinase (pp38 MAPK), phosphorylated PPARα (pPPARα), and HO-1 were assessed in NRK-52E cells exposed to BML-111. The binding activity of PPARα to peroxisome proliferator-responsive element (PPRE) on HO-1 promoter in the cells was determined. BML-111 treatment resulted in a marked reduction in the severity of histological features of renal I/R injury, and attenuated the rise in renal myeloperoxidase and malondialdehyde, blood urea nitrogen and creatinine, urinary N-acetyl-ß-glucosaminidase, and leucine aminopeptidase levels caused by I/R injury. BML-111 stimulated the renal expressions of pPPARα and HO-1, and cellular messenger RNA (mRNA) and protein expressions of pPPARα and HO-1 which were both blocked by GW6471, a selective PPARα antagonist, and ZnPP-IX, a specific inhibitor of HO-1 pretreatment. The pp38 MAPK inhibitor SB203580 blocked the BML-111-induced expressions of pp38 MAPK, pPPARα, and HO-1 in NRK-52E cells. The binding activity of PPARα to PPRE in nuclear extracts of NRK-52E cells was enhanced by treatment of the cells with BML-111, and was suppressed by GW6471 and SB203580. BML-111 protects the kidney against I/R injury via activation of p38 MAPK/PPARα/HO-1 pathway.

Urinary excretion of brush-border enzymes of the proximal renal tubules in pregnant women with hypertensive disorders.

OBJECTIVES: The aim of our study was to evaluate urinary excretion of three brush border enzymes: gamma-glutamyl transferase, alanine aminopeptidase, and leucyl aminopeptidase in pregnant women with various types of hypertensive disorders. MATERIAL AND METHODS: The study included 120 pregnant women, further subdivided into four groups: 41 women at ≥ 20 weeks gestation with gestational hypertension, 28 women > 20 weeks of pregnancy with preeclampsia, 21 women with chronic hypertension identified > 20 weeks of pregnancy and 30 healthy pregnant controls. RESULTS: No significant differences in urinary levels of all three of the brush border enzymes were found between the groups. Also, there was no correlation between enzyme concentration in the urine and blood pressure values in any of the analyzed groups of pregnant women. CONCLUSIONS: The obtained results suggest no damage to the brush border of the proximal kidney tubules in the early stages of disorders associated with increased blood pressure during pregnancy.

Alterations in the biochemical markers of renal function after sevoflurane anaesthesia.

AIM: This study has been carried out to see whether renal function is acutely altered in patients undergoing sevoflurane anaesthesia. For this purpose, the urinary levels of markers of renal tubular function, namely leucine amino peptidase (LAP), gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and beta-2 microglobulin (beta-2M), and urinary albumin as a predictor of renal glomerular function were measured before and after sevoflurane anaesthesia. METHODS: This study was comprised of 20 patients (11 males and nine females) aged 18-55, who underwent various elective surgical procedures under general anaesthesia. Urine samples of all patients were collected before and 1, 2 and 8 h after the anaesthesia. The levels of LAP, GGT, beta-2M, and albumin were then expressed as factored by urinary creatinine. In all patients, the anaesthesia was maintained with sevoflurane (2% end-tidal) at a high flow-rate (6 L/min). RESULTS: Urinary beta-2M and LAP levels after anaesthesia were unchanged (P > 0.05). While urinary GGT and ALP levels were found elevated in the first hour, LDH levels were higher in the second hour (P < 0.05). They returned to normal levels in the later periods after the anaesthesia. Urinary albumin excretion (UAE) was significantly elevated in the second hour after the anaesthesia (P < 0.001). Although UAE was decreased in the eighth hour after the anaesthesia, it still remained higher than the pre-anaesthesia level (P < 0.001). CONCLUSIONS: These results suggest that a 2% end-tidal concentration of sevoflurane at a high flow-rate (6 L/min) acutely alters renal glomerular function but does not have a significant acute effect on biochemical markers of renal tubular damage.

Early proximal tubule injury in experimental aristolochic acid nephropathy: functional and histological studies.

BACKGROUND: Aristolochic acid (AA), the plant extract of Aristolochia species, is involved in the onset of progressive tubulointerstitial renal fibrosis in humans. Clinical and in vitro findings have previously suggested that the proximal tubule was the target of AA. METHODS: Using a rat model of AA nephropathy, the proximal tubular lesions induced by daily subcutaneous injections of AA for 35 or 5 days were characterized biochemically and histologically. Urinary excretion of proteins, albumin, low molecular weight proteins, N-acetyl-beta-d-glucosaminidase, alpha-glutathione S-transferase, leucine aminopeptidase and neutral endopeptidase (NEP) was determined and related to histological conventional findings and immunostainings of NEP and megalin. RESULTS: In both protocols, an acute phase of release of urinary markers was observed within the first 3 days of AA treatment in parallel with a significant increase of specific AA-related DNA adducts reflecting early tubular intoxication. A dramatic loss of the proximal tubule brush border was histologically confirmed, while the expression of megalin decreased at the damaged apical epithelium (mainly of the S3 segment). CONCLUSION: Proximal tubule injury occurs early after AA intoxication in rats, with a link between specific AA-DNA adduct formation, decreased megalin expression and inhibition of receptor-mediated endocytosis of low molecular weight proteins, bringing in vivo confirmation of previous in vitro studies.

[Urinary enzyme excretion in childhood uropathy]. / Obstruktív uropathiás gyermekek enzimürítése.

INTRODUCTION: Renal tissue, and tubules are rich in enzymes but enzyme measurement in the urine as diagnostic parameters in renal diseases have not yet been generally accepted as routine procedures. AIMS: To study urinary enzyme excretion in childhood obstructive uropathy and compares the diagnostic efficiency of them. METHODS: Excretion of the enzymes alkaline phosphatase (ALP), gamma-glutamyltransferase (gamma-GT), N-acetyl-beta-D-glucosaminidase (NAG) and leucine aminopeptidase (LAP) has been investigated in urine of 34 children suffering from obstructive uropathy and in 31 healthy controls by photometric kinetic analysis using synthetic substrates. RESULTS: Urinary excretion of both enzymes significantly increase--2-10 times higher than normal controls--indicates tubular damage. In this study the following relations were found in specificity and in sensitivity: ALP < LAP < gamma-GT < NAG. CONCLUSIONS: Elevated urinary enzyme excretion may be helpful in identifying upper tract obstruction, which if left untreated may cause progressive renal deterioration. It has a role in helping to determine the surgical correction or can be safely followed without fear of parenchymal damage.

Time course of chronic oral cadmium nephrotoxicity in Wistar rats: excretion of urinary enzymes.

Twelve male and female Wistar rats each received cadmium (as CdCl2) in their diet at concentrations of 0, 10, 50, and 250 ppm for 72 weeks. After 1, 4, 8, 13, 18, 26, 32, 45, 57, and 68 weeks a total of 8 enzymes from different cellular compartments of the nephron were measured. At the end of the study period, the kidneys were examined histopathologically. Concentrations up to and including 50 ppm did not induce any adverse effect. At 250 ppm, growth of male and female animals was markedly retarded. Significantly increased activities of the cytosolic phosphohexose isomerase were excreted by males and females receiving 250 ppm at all timepoints from week 13. The values of the mitochondrial glutamate dehydrogenase were mostly elevated from week 1 to 57, however, due to a wide scatter range, were only occasionally significantly different from control values. The brush border enzymes (gamma-glutamyl transferase, alkaline phosphatase and leucine arylamidase) were not changed in a relevant manner in female rats, while in 250 ppm males the excreted activity of ALP and LAP from week 1 to week 18, and that of GGT during the entire study period were significantly lower than the control values. Excretion of the lysosomal enzymes aryl sulfatase A, beta-galactosidase, and beta-N-acetyl-D-glucosaminidase was at no time influenced in a noteworthy manner. Histopathology after 72 weeks revealed chronic but also acute degenerative changes in the kidneys of 250 ppm males and females. A comparison of published data on persons having undergone high cadmium exposure with the results presented here shows remarkable differences.

Changes of urinary enzyme activity after endoscopic treatment of vesico-ureteric reflux.

The urinary enzymes Gamma Glutamyl Transferase (GGT), Alkaline Phosphatase (ALP), Leucine-Arylamidase (LAS), and Dipeptidyl-Peptidase-IV (DPP-IV) were measured before and after endoscopic treatment of vesico-ureteric reflux (VUR) in two groups of twenty children. Ten patients had undergone successful endoscopic corrective surgery for VUR, another 10 patients had unsuccessful endoscopic intervention. After successful treatment the activity of LAS in the urine did not change, but GGT, ALP and DPP-IV activity in the urine was 2-5 times higher than before treatment (P < 0.03 for all three enzymes). Considerable changes of urinary enzyme activity were not observed following unsuccessful endoscopic treatment. Our data and the literature are contradictory. However, this contradiction might be explained by the differences in urine sampling methods. Our patients received the same chemoprophylactic drug at the time of both urine samplings, a point not considered by other researchers. The extent of increase of enzyme activity after endoscopic treatment of VUR did not reach the level that would permit the use of investigated enzymes for screening, because the observed changes did not exceed the limits of the normal range.

[Changes in urinary enzyme activity following silicon therapy of vesico-ureteral reflux]. / A vizeletenzimek aktivitásváltozása vesicoureteralis reflux szilikonnal végzett kezelésében.

The urinary enzymes Gamma Glutamyl Transferase (GGT), Alkaline Phosphatase (ALP), Leucine-Arylamidase (LAS) and Dipeptidyl-Peptidase-IV (DPP-IV) were measured before and after endoscopic treatment of vesico-ureteric reflux (VUR) in two groups of twenty children's. Ten patients had undergone successful endoscopic corrective surgery for VUR, another 10 patients had unsuccessful endoscopic intervention. After successful treatment the activity of LAS in the urine did not change, but that of GGT, ALP and DDP-IV activity in the urine was 2-5 times higher than before treatment (P < 0.03 for all three enzymes). Considerable changes of urinary enzymes' activity were not observed following unsuccessful endoscopic treatment. Our data and the literature are contradictory. However this contradiction might be explained by the differences in urine sampling methods. Our patients received the same chemoprofilactic drug at the time of both urine sampling, a point not considered by other researchers. The extent of increase of enzyme activity after endoscopic treatment of VUR did not reach the level that would permit the use of investigated enzymes for screening, because the observed changes did not exceed the limits of the normal range.

Urinary excretion of renal brush border membrane enzymes in leprosy patients--effect of multidrug therapy.

Renal function at the brush border membrane level has been studied using characteristic enzymes, such as alkaline phosphatase, leucine-aminopeptidase and gamma-glutamyl transpeptidase. Urinary enzyme studies were performed using leprosy patients, classified on the basis of bacteriological index (BI>3; n=20, BI<3; n=12, BI-ve; n=10) and compared with control subjects (n=10). The role of enzymuria in monitoring WHO-recommended multidrug therapy (MDT) has been evaluated in these patients. A significant increase in the enzyme activities (p<0.01), as well as significant (p<0.01) proteinurea in 24-hour urine samples of both the smear positive groups (BI>3, BI<3) prior to therapy compared to control subjects, indicates proximal tubular functional impairment at brush border membrane level. In the smear negative (BI-ve) group, no significant difference was observed in enzyme activities as compared with the control group. In a follow-up study (BI>3;n=13, BI<3; n=4) the activities of all the enzymes decreased significantly in all the groups when compared to a corresponding untreated group. The follow-up study was not carried out on the smear negative group. The surprising finding was the differential behaviour of r-glutamyl transpeptidase, whose activity increased significantly (p<0.01) even after therapy in BI>3 group when compared with untreated patients. However in a detailed work-up including hepatic and renal function tests, the serum biochemistry was found to be normal both before and after therapy. Urinary excretion of brush border enzymes seems to be related to bacterial load, and their potential in studying the effect of MDT remains unclear.

Urinary leucine aminopeptidase is a more sensitive indicator of early renal damage in non-insulin-dependent diabetics than microalbuminuria.

We measured urinary activity of leucine aminopeptidase (EC 3.4.11.2) and creatinine concentrations (Cr, in mmol) in samples of second morning urine from 25 healthy subjects and 59 non-insulin-dependent diabetic (NIDD) subjects. If NIDD subjects are grouped according to their Alb/Cr ratio into normoalbuminuria (group A, Alb/Cr < 2.8 mg/mmol), microalbuminuria (group B, Alb/Cr 2.8-26.8 mg/mmol), and macroalbuminuria (group C, Alb/Cr > 26.8 mg/mmol), LAP/Cr ratios in all three groups exceeded those for healthy age-matched controls. Moreover, this ratio was higher in group B than in group A. The value for LAP/Cr was clearly abnormal (i.e., exceeded the upper limit of normal, log normal + 2 SD, found in healthy subjects) in 44% of group A. In the first 10-year period Of NIDD, prevalance of abnormal LAP/Cr ratio was 61.3%, whereas that of microalbuminuria was 35.5%. We have also found a LAP/Cr ratio abnormality of 91% in group B. Evidently, LAP/Cr may be increased early in NIDD subjects and be a more sensitive predictor of incipient nephropathy than microalbuminuria.

Leucine aminopeptidase enzymuria: quantification of renal tubular damage following extracorporeal shock wave lithotripsy.

To evaluate the extent of renal parenchymal injury following high energy shock wave (HESW) application we measured the urine levels of two renal tubular brush border enzymes: leucine aminopeptidase (LAP) and gamma-glutamyltranspeptidase (GGT) the day before, 24 hours and 7 days after extracorporeal shock wave lithotripsy (ESWL) in 23 patients. All patients had caliceal stones and were treated with a Dornier MPL 9000 lithotriptor under sedoanalgesia. Creatinine concentration of each sample together with total shock wave effect (TSWE) values were also assessed and comparatively evaluated with enzymuria levels. Our results indicated a statistically significant rise in urinary excretion of both enzymes after 24 hours following lithotripsy (p < 0.05). All these values returned to normal limits within 7 days after ESWL (p > 0.05). Transient tubular damage due to HESW was found to be related to TSWE values assessed in our group.

[The role of urinary peptide hydrolase in the laboratory diagnosis of urological diseases]. / Rol' peptidgidrolazy mochi v laboratornoi diagnostike urologicheskikh zabolevanii.

The authors determined activity of leucine arylamidase (LA) or microsomal aminopeptidase locating in tubular cell microsomes and as a specific enzyme indicating parenchymal damage in the urine of 28 healthy subjects and 187 patients with nephroliths (103), renal injury (13 contusions, 11 rupture) before and after extracorporeal lithotripsy. Changes in LA were followed up spectrophotometrically. LA levels in healthy controls, nephrolithiasis patients free of pyelonephritis or with it in remission were similar, elevated in latent course and significantly elevated in complicated by inflammation nephrolithiasis, renal injury and in patients with associated pyelonephritis after lithotripsy. The highest LA activity was recorded in patients with renal injury and after lithotripsy with latent or active inflammation before lithotripsy. LA urinary content may serve indication of inflammation in the kidneys, parenchymal involvement. It is a helpful adjuvant diagnostic method in urology.

Tubular damage in microalbuminuric patients with primary glomerulonephritis and diabetic nephropathy.

Tubular damage as suggested by enzymuria and tubular proteinuria is a recognized feature of glomerulonephritis (GN) with clinical proteinuria and both incipient and overt diabetic nephropathy (DN). However, little is known about the presence of tubulopathy in patients with primary GN, microalbuminuria [albumin excretion (AER) 30-300 mg/d] and microhematuria. Three groups were studied. The GN group comprised 17 (2 F) patients with biopsy-proven GN with microalbuminuria. The DN group comprised 35 (14 F) patients with incipient diabetic nephropathy with AER 30-300 mg/d, and controls comprised 38 (15 F) normal subjects with normal AER < 30 mg/d. Serum creatinine, albuminurinuria, transferrinuria, and markers of tubular damage such as urinary excretion of N-acetyl-glucosaminidase (NAG), leucine aminopeptidase (LAP), gamma-glutamyl transferase (gGT), and retinol binding protein (RBP) were measured. GN and DN had comparable degrees of albuminuria, transferrinuria, and LAP excretion, and these were significantly higher than controls. Serum creatinine was significantly higher in GN than DN and controls. DN had significantly higher NAG and RBP, and lower gGT than GN and controls. In both GN and DN groups, both glomerular proteins correlated with each other and NAG correlated significantly to LAP and gGT. Albuminuria correlated to tubular enzymuria in GN group but not in patients with DN. The results suggest that tubular damage is less marked in microalbuminuric patients with GN than those with DN despite similar degree of glomerular proteinuria. The pattern of tubulopathy is also different in the two groups, indicating differences in the pathogenesis of tubular damage in these two clinical settings.

[Urinary excretion of leucine aminopeptidase after extracorporeal shock wave lithotripsy]. / Excrétion urinaire de la leucine aminopeptidase après lithotriptie extra-corporelle par ondes de choc.

Extracorporeal shock-wave lithotripsy (ESWL) is the treatment of choice in the majority of urinary calculi. In experimental and clinical research it has been shown that the method has short-term deleterious effects on renal function. Several factors, such as urinary tract infection (UTI), the site of the stone, pre-existing renal abnormalities, the number and intensity of the shock waves may influence the extent of the damage. The aim of this study was to measure the excretion of the renal microsomal enzyme, leucinaminopeptidase (MLAP) as expression of renal damage following ESWL. Our study shows that there was a significant elevation in urinary excretion of MLAP immediately after ESWL, especially in patients with UTI. These results indicate that UTI may be one of the risk factors for aggravation of renal damage after ESWL. However, these changes are transient and expressed less than in patients with light renal trauma arising because of other external influence.

Tubulopathy with macroalbuminuria due to diabetic nephropathy and primary glomerulonephritis.

Tubular damage is a recognized feature of both overt diabetic nephropathy and glomerulonephritis. However, the pattern and mechanism of tubular damage in the two clinical settings remain unclear. Two groups of patients with macroalbuminuria (albuminuria > 300 mg/day) were studied. Group 1 comprised 41 patients with biopsy proven primary glomerulonephritis and group 2 comprised 28 patients with clinical diabetic nephropathy due to insulin dependent diabetes mellitus. Serum creatinine, creatinine clearance, glomerular proteinuria (albuminuria and transferrinuria), markers of tubular damage such as urinary excretion of lysosomal enzyme (N-acetyl glucosaminidase), brush border enzymes (leucine aminopeptidase and gamma-glutamyl transferase) and retinol binding protein (tubular protein) were measured. Both groups were comparable in serum creatinine, creatinine clearance, glomerular proteinuria and excretion of N-acetyl-glucosaminidase. However, a significantly higher degree of tubular brush border enzymuria and a lower level of tubular proteinuria were seen in group 1 than in group 2. In group 1, albuminuria correlated to tubular enzymuria and tubular proteinuria. However, there was no correlation in diabetic patients between parameters of glomerular and tubular damage or dysfunction. The data presented suggested that the pattern of tubulopathy is different in patients with comparable degree of macroalbuminuria due to diabetic nephropathy and glomerulonephritis. Moreover, in diabetic nephropathy contrary to glomerulonephritis, markers of tubular damage are unrelated to glomerular proteinuria. This may suggest different mechanisms of tubular damage in the two clinical settings. We recommended that in all patients with proteinuria, particularly those with diabetic nephropathy, markers of renal tubular damage may be useful in monitoring the course of their disease.

Diagnostic significance of urinary enzymes for diabetes mellitus and hypertension.

In order to evaluate tubular damage in diabetic patients, the activity of renal proximal tubule derived enzymes excreted in 24-hour urine were recorded in 5 groups as follows: (i) 48 noninsulin-independent diabetic patients with normal renal function and a urinary albumin excretion rate within the normal range; (ii) 45 noninsulin-dependent diabetic patients with normal renal function and a high urinary albumin level; (iii) 26 noninsulin-dependent diabetic patients with renal failure; (iv) 40 patients with essential hypertension and normal renal function, and (v) 48 normal control subjects. Regardless of whether cases were noninsulin-dependent diabetics with normal or high urinary albumin excretion rate or cases with renal dysfunction, urinary dipeptidyl aminopeptidase IV and N-acetyl-beta-D-glucosaminidase excretions were significantly higher than in healthy subjects, and urinary gamma-glutamyl transpeptidase excretion was significantly lower than in healthy subjects. No significant changes in urinary enzyme excretions showed specific variations in the essential hypertensive patients. These results suggest that there is tubular damage in the early stages of noninsulin-dependent diabetic patients with normal renal function and normal urinary albumin excretion rate. Detection of urinary excretion of dipeptidyl aminopeptidase IV, N-acetyl-beta-D-glucosaminidase and gamma-glutamyl transpeptidase may be especially useful for the early diagnosis of diabetic nephropathy.

Electrophoretically mediated microanalysis of leucine aminopeptidase in complex matrices using time-resolved laser-induced fluorescence detection.

Leucine aminopeptidase, a clinically significant enzyme, was assayed in complex biological samples using a new technique termed electrophoretically mediated microanalysis. The assay was performed in capillary electrophoresis columns using time-resolved laser-induced fluorescence detection. Human serum, human urine, and Escherichia coli supernatant samples were assayed using this method. Results for serum and urine were within the ranges of expected values found in the literature. A low concentration of 6 x 10(-13) M enzyme in buffer was detected using this method. A detection limit (3 sigma) of 400 enzyme molecules in buffer was determined.