Alanine Aminopeptidase Activity Research Articles

Low molecular mass proteins and urinary enzymes in amniotic fluid of healthy pregnant women at progressive stages of gestation

Objectives: Amniotic fluid α 1-microglobulin ( α 1-m) and β 2-microglobulin ( β 2-m) levels, as well as N-acetyl-β-D-glucosaminidase (NAG) and alanine aminopeptidase (AAP) activities, were measured in the course of uncomplicated pregnancies to assess their variations as gestation progresses. Design and Methods: Samples were obtained from 141 healthy pregnant women divided into three groups on the basis of gestational stage. Quantitative estimation of proteins was performed immunometrically and enzyme activities were determined spectrophotometrically. Results: It was found that, during pregnancy, α 1-m and β 2-m concentrations as well as AAP activity significantly decrease, although their reduction patterns vary. Controversial results were found for NAG activity: the normalization of values for amniotic fluid creatinine significantly changed the reduction pattern of this enzyme. No statistically significant differences were found between male and female fetuses for amniotic fluid values of the biochemical substances studied. Conclusions: The behavior observed for α 1-m, β 2-m, and AAP might be linked to the progressive development and maturation of fetal renal tubular function. Amniotic fluid total NAG activity seems not to depend only on fetal urinary excretion.

Cisplatin nephrotoxicity and protection by silibinin

The anticancer drug cisplatin is know to have toxic side-effects on different segments of the nephron. The flavonoid silibinin has previously been shown to be protective in models of hepatotoxicity. The aim of the present study was to evaluate, whether silibinin can also ameliorate alterations in renal glomerular and tubular function and tubular morphology induced by cisplatin. In a rat model renal damage was induced by a single injection of cisplatin (5 mg/kg body weight). The protective effects of silibinin were studied in rats that received the flavonoid (200 mg/kg body weight, i.v.) 1 h prior to the administration of cisplatin. Kidney function was monitored by analysing urinary markers of glomerular and tubular function over a period of 11 days. Animals of a second group, with identical treatment, were sacrificed 4 days after drug application for an evaluation of tubular morphology at the light-microscopical level. Administration of cisplatin caused a decline in kidney function within a day following treatment. Symptoms observed were for example decreases in creatinine clearance and increases in proteinuria, in the urinary activity of the proximal tubular enzymes alanine aminopeptidase and N-acetyl-beta-D-glucosaminidase and in renal magnesium wasting. The effects of cisplatin on creatinine clearance and proteinuria were totally prevented by a pretreatment of the animals with silibinin. Impairment of proximal tubular function was ameliorated, that is enzymuria and magnesium wasting was less pronounced. Silibinin alone had no effect on kidney function. Treatment with silibinin distinctly diminished morphological alterations observed in the S3-segment of the proximal tubule 4 days after cisplatin administration. The effects of cisplatin on glomerular and proximal tubular function as well as proximal tubular morphology could totally or partly be ameliorated by silibinin. It is concluded the silibinin can act as a nephroprotectant and it is suggested that it could have beneficial effects on the kidney in clinical settings.

The relationship between enzymuria and kidney enzyme activities in experimental gentamicin nephrotoxicity.

The aim of this study was to investigate the relationship between the urine excretion and kidney activities of enzymes predominantly located in the proximal renal tubule, viz. the lysosomal hydrolase N-acetyl-beta-D-glucosaminidase (NAG) and the predominantly brush border enzymes alanine aminopeptidase (AAP) and gamma-glutamyltransferase (GGT) in an experimental model of gentamicin nephrotoxicity. Groups of six animals received either gentamicin (50 mg/ kg/day by intraperitoneal injection) or saline daily and were killed after 4, 7, 10, or 14 days of treatment. Gentamicin nephrotoxicity was characterized by reduced creatinine clearance rates and increased urinary flow rate and glycosuria, but only on day 14. Structural changes included a similar degree of vacuolation of the renal proximal convoluted tubules (PCT) in all animals sacrificed on days 11 and 14, some evidence of PCT brush border loss, and the presence of protein casts on day 14. Following gentamicin treatment, increased NAG, AAP, and GGT enzymuria were noted at all time points tested. However, while the increases in urine AAP and GGT activity were paralleled by decreased total renal activity, total kidney NAG activity increased on days 4, 7, and 11 before falling back to pretreatment values on day 14. Interestingly, NAG enzymuria was highest in those animals with protein casts in the lumen of the PCT. The results suggest that increased AAP and GGT enzymuria reflect loss of brush border integrity while increased NAG enzymuria is consistent with the autophagic response of the kidney to acute injury.

Influence of ochratoxin A treatment on the activity of membrane bound enzymes in rat brain regions.

Ochratoxin A is a mycotoxin produced by Aspergillus ochraceus and is a natural contaminant of mouldy food. We examined the neuroactive potential of ochratoxin A by measuring the changes in the activities of several membrane bound, cytoplasmic and lysosomal enzymes in the brain of adult female rats, following subchronic application of ochratoxin A. The activities of both soluble and membrane bound fractions of ecto-5'nucleotidase, ecto-Ca2+/Mg2+ATPase, alanine aminopeptidase, gamma-glutamyl transferase, as well as activities of lactate dehydrogenase and of N-acetyl-beta-D-glucosaminidase were followed. Biochemical effects were examined in cerebral cortex, cerebellum and hippocampus. The results obtained showed physiologically significant alterations in the activity of enzymes tested. The changes were found to be time-dependent and regionally selective. Compared to controls, statistically significant increases in gamma-glutamyl transferase were observed in all three brain regions, while in the case of alanine aminopeptidase activities differed with regard to region, the highest increase being observed in hippocampus. Ecto-Ca2+/Mg2+ATPase and ecto-5'nucleotidase showed distinct changes lasting for 20 days of treatment, while increase in the activities of N-acetyl-beta-D-glucosaminidase and lactate dehydrogenase were visible only at the beginning of the treatment. By the end of the trial the activities of almost all enzymes returned back to normal values.

Comparison of Halothane, Enflurane and Isoflurane Kidney Effects through Alanine Aminopeptidase/Urine Creatinine Values

The kidney effects of halothane, enflurane and isoflurane were evaluated by using the ratio of urinary excretion of alanine aminopeptidase (AAP) to urine creatinine. Thirty patients in ASA class 1 or 2 were studied. None had renal disease nor received nephrotoxic drugs. Groups 1, 2 and 3 received halothane, enflurane and isoflurane respectively. Creatinine and AAP activities in urine spot tests, serum creatinine and BUN levels were determined preoperatively and on the first and second postoperative days. Urine AAP activity and AAP/urine creatinine values increased significantly on the first and second postoperative days compared with the preoperative values in all groups (P < 0.05). The present study did not reveal any significant difference in the kidney effects of halothane, enflurane and isoflurane through AAP/creatinine in spot urine values.

A non-invasive approach to differential diagnosis of urinary tract infections using lactate dehydrogenase isoenzyme profile, N-acetyl-β-D-glucosaminidase and alanine aminopeptidase

Lactate dehydrogenase (LDH) and its isoenzyme profile, Nacety-β-d-glucosaminidase (NAG) and alanine aminopeptidase (AAP) enzyme activities were measured in 21 cases with well documented acute untreated urinary tract infections (UTI) and compared with a group of 68 normal healthy subjects matched for age and sex. Urinary NAG, AAP and LDH total activities were highly elevated (3–30 fold for NAG, 2–10 fold for AAP and 2–50 fold for LDH over upper limit of normal) in upper tract infections. In lower tract infections there was a marginal elevation in NAG and LDH while AAP activity was very high; though there was no overlap. Further, the isoenzyme profile of LDH showed predominance of LD-5 isoenzyme in all cases of pyelonephritis, while it was completely absent in all the cases of lower tract infections. We conclude that urinary NAG and LDH-5 isoenzyme would serve as specific non-invasive parameters for differential localization of urinary tract infections.

Occupational exposure to lead, kidney function tests, and blood pressure

In the present study we examined sensitive biochemical markers of kidney function and damage in 166 workers exposed to lead and in 60 control workers. The objective was to investigate the chronic renal toxicity of lead and its possible correlation with arterial pressure. Diastolic arterial pressure was higher in the exposed group (p < 0.05), but the two groups did not differ in systolic pressure. Median activity of urinary N-acetyl-beta-D-glucosaminidase was higher in the exposed group (p < 0.001), and correlated with blood lead levels (p < 0.001) and duration of exposure (p < 0.001), but not with arterial pressure. The other indicators studied, gamma-glutamyl-transpeptidase and alanine-aminopeptidase activity, urine albumin, and total urine protein, were not higher than in the control group and were not correlated with blood lead, duration of exposure, or arterial pressure.

The hepatobiliary disease marker serum alanine aminopeptidase predominantly comprises an isoform of the haematological myeloid differentiation antigen and leukaemia marker CD-13/gp150

In clinical chemistry, determination of serum alanine aminopeptidase (AAP) levels has been found to be useful for detecting or confirming biliary obstructions from either intra- or extrahepatic disorders. In haematology, ‘gp150’ is a surface-expressed protein molecule, recognised by monoclonal antibodies belonging to the so-called ‘Cluster of Differentiation’ (CD-) 13, which has independently been found to be a useful marker of myeloid leukaemia in addition to providing potential prognostic value. The current report links these two independent research streams and provides evidence that the hepatobiliary disease marker, serum AAP, predominantly comprises a circulating isoform of ‘gp150’. Thus, a (CD-13) monoclonal antibody raised to, and specifically reactive with, cell surface myeloid ‘gp150’ is able to specifically and almost completely block serum (or plasma) AAP activity otherwise observed in its absence. This holds true for serum (or plasma) derived both from normal individuals or from patients suffering hepatic dysfunction, with or without associated biliary obstruction. In the case of patients with obstructive jaundice, raised levels of AAP are observed, which fall to near normal levels following preincubation with this monoclonal antibody. In addition, data are presented to support the view of varying isoforms of AAP within flowing blood. Finally, preliminary data is provided on AAP activity in cases of leukaemia. These studies should thus prove of use to clinical laboratories investigating the involvement of AAP activity in various pathological processes.

Enzyme Activities of Urinary Alanine Aminopeptidase (AAP) and N‐Acetyl‐β‐D‐Glucosaminidase (NAG) in Healthy Dogs

The activity of the urinary enzymes alanine aminopeptidase (AAP; EC 3.4.11.2) and N-acetyl-beta-D-glucosaminidase (NAG; EC 3.2.1.30) was measured after eliminating distorting factors in ten healthy dogs on three consecutive days in order to determine inter- and intra-individual variability. All the animals were being housed and fed in the same way. The urine (2nd morning urine) was collected between 8:30 and 10:00 a.m. by means of ultrasound-controlled cystocentesis. Our comparative measurements of native and gel-filtered urine showed an increase in enzyme activity in all of the samples in the case of AAP and in most of the samples in the case of NAG, thus proving the existence in the dog of AAP and NAG urinary enzyme inhibitors. The large inter- and intra-individual AAP and NAG fluctuation ranges were reduced considerably by relating enzyme activity to urine creatinine concentration. The provisional upper limit of the AAP reference range is 6.3 U/g creatinine (90% percentile), that of NAG 6.2 U/g creatinine (90% percentile). The AAP and NAG enzyme activities remained constant in gel-filtered samples kept at 4 degrees C for at least 5 days, in those kept at -18 degrees C for at least 4 weeks.

Characterization of lactococci and lactobacilli isolated from semihard goats' cheese

SummarySeveral strains ofLactococcus lactissubsp.lactis, Lactobacillus caseiandLactobacillus plantarumisolated from traditional goats' cheese have been studied for titratable acidity, proteolysis in milk and enzymic activities. Aminopeptidasc activities were measured with whole cells and cells permeabilized with Triton X-100. Caseinolytic activity was investigated using electrophoresis in polyacrylamide gel with sodium dodecyl sulphate.Lc. lactissubsp.lactishad a level of proteolytic activity in skim milk greater than that ofLb. casei, while this activity inLb. plantarumwas very low. Alanine aminopeptidase activity was almost non-existent for all strains tested, while lysine aminopeptidase activity appeared to be of fundamentally intracellular origin. Leucine aminopeptidase activity was also greater in cells that had been permeabilized than in whole cells forLb. caseiandLb. plantarum. Lc. lactissubsp.lactisleucine aminopeptidase activity was greater in whole cells. No significant hydrolysis of casein was found withLb. caseiI FPL 725 andLb. plantarumIFPL 722 permeabilized with Triton X-100 after 24 h incubation with whole bovine casein.

Experimental studies on the elevation of urinary enzyme activities and its pathogenesis in acute renal failure

The urinary activities of N-acetyl-beta-D-glucosaminidase (NAG), gamma-glutamyl transpeptidase (gamma-GTP) and alanine aminopeptidase (AAP) are known to elevate markedly in initial phase of clinical acute renal failure (ARF). This study was performed to clarify the pathophysiological mechanism of the activation of these enzymes using experimental postischemic reperfusion ARF in rats. The relation between the levels of the lysosomal enzymes and lipid peroxidation induced by oxidant stress in these animal models was the main focus of this study. Renal ischemia was made by clamping renal artery for 30 minutes to create a complete ischemia and reflow. Catheterized urine was collected to measure changes of the activities of NAG. gamma-GTP and AAP from 60 to 480 minutes after reperfusion of the kidney. The activities of renal tissue glutathione peroxidase (GSH-Px), NAG and gamma-GTP, and the values of renal contents of glutathione (GSH) and malondialdehyde (MDA) were measured in each sample. It is already known that GSH redox cycle plays an important role in removing various hydroperoxides induced by oxidant stress, generating oxidated GSH from GSH in scavenging process. In order to confirm if GSH plays an important role in intrinsic anti-oxidant system in this model, buthionine sulfoximine (BSO) which is gamma-glutamylcysteine synthetase inhibitor, was administered intraperitoneally to decrease renal GSH contents before the procedure renal ischemia. The following results were obtained; 1) urinary activities of NAG, gamma-GTP and AAP were elevated markedly in GSH depleted rats compared with controls, 2) renal tissue activities of NAG were higher in BSO administered rats than controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Sulfated bile acids inhibit Na(+)-H+ antiport in human kidney brush-border membrane vesicles

Patients with obstructive jaundice are at a high risk for acute renal failure after surgery. Direct toxic membrane effects of bile acids or bilirubin have been discussed as possible causes. Therefore, we investigated the influence of bile acids and conjugated bilirubin on Na(+)-H+ antiport and ion permeabilities in brush-border membrane vesicles isolated from the human kidney. Brush-border membrane vesicles were prepared by Mg2+ precipitation. These were highly purified as estimated from the 14-fold enrichment in the specific activity of alanine aminopeptidase. The pH-sensitive dye acridine orange was used to study the properties of proton uptake under different conditions. The brush-border membrane vesicles from human kidney cortex exhibited Na+ and K+ conductances, which were small compared with H+ conductance. Furthermore, these membranes possess an Na(+)-H+ antiporter that is sensitive to amiloride. Various bile acids (30 microM) had no significant effect on Na(+)-H+ antiport. However, the addition of sulfated bile acids resulted in a significant inhibition (greater than 50%) of the Na(+)-H+ antiporter. A nonspecific effect of sulfated bile acids on the vesicles was excluded by the use of ionophores to determine vesicle integrity and to estimate the various ion conductances. Therefore specific inhibition of the human renal Na(+)-H+ antiporter by sulfated bile acids occurs. This could result in an impaired cellular pH regulation and might play a role in postoperative acute renal failure in patients with obstructive jaundice.

How should we measure activity of alanine aminopeptidase in urine?

Journal Article How should we measure activity of alanine aminopeptidase in urine? Get access K Jung, K Jung Search for other works by this author on: Oxford Academic Google Scholar G Jähnchen G Jähnchen Search for other works by this author on: Oxford Academic Google Scholar Clinical Chemistry, Volume 36, Issue 1, 1 January 1990, Pages 177–178, https://doi.org/10.1093/clinchem/36.1.177 Published: 01 January 1990

Alanine aminopeptidase in serum: biological variations and reference limits.

We studied the effect of various factors on the biological variation of alanine aminopeptidase (AAP, EC 3.4.11.2) in serum in a population of 2178 apparently healthy subjects and in subjects taking specific drugs. To measure AAP, we used an automated assay, with alanyl-4-nitroanilide as substrate. AAP activity concentrations were significantly higher in males than in females in all age groups between 10 and 55 years. The highest mean AAP values were found for children between 10 and 14 years. In male heavy smokers, AAP values were 8% higher than in moderate or nonsmokers (P less than 0.001); in females, this effect was less pronounced. AAP concentrations were higher in subjects consuming over 44 g of ethanol a day than in those consuming less (P less than 0.01 for men, P less than 0.05 for women). The use of oral contraceptives increased AAP values by 12% (P less than 0.001). Hypolipidemic drugs caused lower values for AAP in men (P less than 0.001). Subjects showing induction of gamma-glutamyltransferase (EC 2.3.2.2) by anticonvulsant drugs had also higher AAP activities (by 23%). Taking these data into account, we established reference limits for AAP in serum.

Enzymuria in neonates during treatment with tobramycin or ceftazidime.

Aminoglycosides have a well-known nephrotoxic potential in adults whereas renal side effects in neonates receiving these drugs are rare. Several studies have, however, shown that neonates receiving gentamicin or tobramycin invariably develop increases in the urinary activity of enzymes originating from renal tubular cells, notably alanine aminopep-tidase (AAP) and TV-acetyl-beta-D-glucosaminidase (NAG).14 Although the clinical importance of enzymuria remains to be determined, it has been considered to be an early sign of renal damage.1,5,6 Because of the unique antibacterial spectrum of the aminoglycosides it has not been possible until recently to study enzymuria in neonates receiving aminoglycosides with a control group receiving other antibacterial agents. With the introduction of beta-lactam antibiotics with improved activity against Gram-negative rods this has now become possible. The aim of the present study was to compare the urinary activities of AAP and NAG in neonates during treatment with tobramycin or ceftazidime, a new beta-lactam antibiotic with a broad antibacterial spectrum.

Behaviour of several enzymes of fetal chick intestine in tissue culture

The behaviour of several enzymes is described of the fetal chick duodenum in tissue culture in a defined medium free of serum and hormones. During culture the activity of sucrase, maltase, alanine aminopeptidase, and gamma-glutamyltransferase is raised in tissue explants, whereas the activity of other enzymes (dipeptidyl peptidase IV, leucine amino-peptidase, alkaline phosphatase) remains constant. After culture, depending on the enzyme, a varying amount of activity is found in the medium, a part of which can be sedimented by ultracentrifugation. Sucrase is subject to the strongest increase in activity during culture and thus should represent a sensitive marker for investigating maturation processes in the fetal intestine and their disturbances.

Raised Thyrotrophin‐Releasing Hormone, Pyroglutamylamino Peptidase, and Proline Endopeptidase Are Present in the Spinal Cord of Wobbler Mice but Not in Human Motor Neurone Disease

The Wobbler mouse (wr) is a mutant that exhibits loss of anterior horn cells in the spinal cord and brainstem and subsequent muscle wasting, particularly of the forelimbs and neck. The wr mice, 2-3 months of age, were found to have increased levels of immunoreactive-thyrotrophin-releasing hormone (ir-TRH) in the spinal cord and pons and medulla, but not in other CNS areas. This increase was observed in dorsal and ventral cord and at cervical, thoracic, and lumbar levels and was confirmed by HPLC to be authentic TRH. The levels of immunoreactive-somatostatin, -neurotensin, and -substance P were not raised in the CNS of wr mice. The activities of two peptidases capable of degrading TRH, pyroglutamylaminopeptidase (PGAP, EC 3.4.11.8) and proline endopeptidase (PEP, EC 3.4.21.26), and the level of 5-hydroxyindoleacetic acid were also raised in the spinal cord of 2-3-month-old wr mice although the activities of alanine aminopeptidase and lactate dehydrogenase and the level of 5-hydroxytryptamine were not. Increased spinal cord levels of ir-TRH and PGAP and PEP activities were not observed in the 1-month-old wr mice. In addition, a pilot study using spinal cord obtained at autopsy from three patients with motor neurone disease and 12 control subjects indicated no increase in spinal cord ir-TRH, PGAP, or PEP in human motor neurone disease.

Alanine aminopeptidase in serum: automated optimized assay, and effects of age, sex, smoking, and alcohol consumption in a selected population.

In the diagnosis of chronic (as opposed to acute) liver diseases, combinations of indicators are needed to improve specificity. Alanine aminopeptidase (AAP; microsomal aminopeptidase, EC 3.4.11.2) activity in serum reportedly is a very sensitive indicator of intrahepatic cholestasis and biliary obstruction; it is also particularly useful in diagnosing chronic liver disease when combined with an indicator of hepatocyte damage such as aspartate aminotransferase or alanine aminotransferase. We optimized the assay of AAP in serum, automated the assay by using a centrifugal analyzer, then used this automated assay to determine activity in 202 individuals, ages one to 73 years. The preliminary results were analyzed in terms of the effects of age, sex, smoking, and alcohol consumption on AAP activity in serum. Striking sex-related differences were observed: AAP activity in males declined 2.5 times more rapidly with age than did that in females; indeed, activity in adult females remained essentially constant. Moreover, AAP values were higher in men who smoked than in those who did not, the difference being of borderline significance by analysis of covariance (p = 0.0865) but significant by partial correlations (p = 0.02). No similar differences were seen for women smokers and non-smokers. When the effects of other variables were held constant, alcohol consumption alone did not significantly correlate with AAP activity in men or women.

Comparison of n-acetyl-β- d-glucosaminidase and alanine aminopeptidase activities for evaluation of microangiopathy in diabetes mellitus

The activities of urinary n-acetyl-β- d-glucosaminidase (NAG) and alanine aminopeptidase (AAP) were measured in 207 diabetic patients and 57 healthy controls, and the relationship of these enzymes to different stages of diabetic microangiopathy was studied. Diabetics with clinical proteinuria had higher urinary NAG and AAP (17.7 ± 1.9 and 42.8 ± 4.9 U/g creatinine, mean ± SE, respectively) than healthy controls (1.8 ± 0.1 and 10.0 ± 0.4) or diabetics without proteinuria. Among diabetics without proteinuria, NAG excretion in those with retinopathy was slightly higher than in those without (6.4 ± 0.5 v 5.4 ± 0.4), and AAP in those with retinopathy was significantly higher than in those without (23.0 ± 1.5 v 17.4 ± 0.8, P < 0.01). Urinary albumin measured by radioimmunoassay and lysozyme in diabetics with retinopathy but without proteinuria was higher than those without retinopathy ( P < 0.001 and P < 0.01). The increase in albumin was the greatest in diabetics with long duration of the disease (≥8 years); however, NAG and AAP increased more significantly in those with high hemoglobin A 1c than in patients with long duration. Although urinary NAG correlated moderately with AAP ( r = .70, P < 0.001), the coefficient of correlation between AAP and albumin was greater than that between NAG and albumin in diabetics without proteinuria and in healthy subjects ( r = .47 and P < 0.001 v r = .32 and P < 0.001). These results indicate that urinary NAG and AAP can be used to diagnose the onset and to follow the development of diabetic vascular complications. AAP appears to be more useful for this purpose.

A stable liquid control material for urinary enzyme assays

A stable liquid control material for urinary enzyme assays was prepared by addition of ethylene glycol (30% v/v) or glycerol (30% v/v) to centrifuged urine, and pH adjustment to 7.0. Tested for 375 days, the material exhibited stable β- N-acetylglucosaminidase, alanine aminopeptidase, γ-glutamyltransferase and lactate dehydrogenase activities, at both 4 and −20°C. The material further has been successfully evaluated in an external quality control program.