Enzyme In Urine Research Articles

Combination Treatment for Severe Forms of Mucopolysaccharidosis, Type I (Hurler Syndrome): Case Report

Background. Hurler syndrome (mucopolysaccharidosis, type I) is a rare hereditary disease with chronic course. The main methods for Hurler syndrome management are hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT). In recent years, combination treatment (ERT administration both before and after HSCT) has shown its efficacy in case of disease progression. Clinical case description. The presented clinical cases demonstrate the efficacy of ERT administration in patients with Hurler syndrome after HSCT: in the first clinical case due to the decrease in alpha-iduronidase activity 2 years after HSCT, in the second clinical case due to the aggravation of the patient's condition (cardiovascular and respiratory systems, hepatomegaly, although the level of enzyme and glycosaminoglycans in the patient's urine remained within normal values). Conclusion. Combination treatment including ERT not only before HSCT, but also in case of clinical state worsening after HSCT, plays significant role in stabilizing the patient's condition, preventing rapid progression of symptoms and development of life-threatening complications (especially cardiovascular ones).

A NIR fluorescent probe for the detection of renal damage based on overrepresentation of alanine aminopeptidase enzyme.

Kidney damage generates changes at the phenotypic and genotypic levels that allow its monitoring using different biomarkers in blood, urine or serum. Among these biomarkers, kidney failure causes the urine overrepresentation of the alanine aminopeptidase (APN) enzyme. Here, we describe the design of a molecular probe (NB-ALA) based on the Nile Blue fluorophore (NB), which can detect the APN enzyme in urine by simple fluorometric measurements.

Detection of ADAM15 in urine from patients with bladder cancer

Cancer is one of the leading causes of death in the United States and Europe. Of the cancers, bladder cancer is the 10th most frequently diagnosed cancer and the 13th most frequently diagnosed cancer in men. There are many studies showing that proteolytic enzymes, e.g. A Disintegrin and Metalloproteinases (ADAMs), play a key role in the development and progression of neoplasms. In this paper, we present the use of chromogenic substrate of ADAM15 for the qualitative determination of specific activity of enzyme in urine of patients with confirmed bladder cancer. In the first step, we optimized the substrate molecule in non-primed positions using combinatorial chemistry. By means of the obtained ABZ-His-Ala-Arg-Gly-ANB-NH2 peptide, we detected ADAM15 activity in urine samples collected from patients diagnosed with bladder cancer. In contrast, we did not observe such activity in urine obtained from healthy volunteers.

Damage to the connective tissue system in patients with post-traumatic osteomyelitis

In 63 patients with suppurative bone process the activity of N-acetyl-fi-D-glucosaminidaze lysosomal enzyme in urine as well as oxyproline and hexuronic acids excretion were evaluated. Significant changes in metabolism of collagen and proteoglycans of osseous organic matrix were detected and it was confirmed by high excretion of their structural components. The more active and vaster the suppurative process was the more quantitative excretion of collagen and glu- cosaminoglycans and higher enzyme activity were. The suggestion was formulated that high excretion of N-acetyl-fl-D-glucosaminidaze in urine noted in the patients with suppurative inflammatory complications was the indicator of pathologic changes in tubular apparatus of kidney. The detection of excretion of the connective tissue metabolites (oxyproline and hexuronic acids) could be used for the diagnostic and prognostic evaluation of the disease severity and for the choice of method for the treatment of suppurative process.

Isolation and evaluation of cow urine micro-organisms for their antibacterial and antifungal potential

Cow urine has tremendous applications in agriculture. It is shown to possess inhibitory activity against many phytopathogenic fungi and bacteria. The present study was conducted to determine antibacterial and antifungal efficacy of cow urine. The well-plate assay showed marked growth of inhibition against Aspergilus niger, Fusarium oxysporum, Xanthomonas auxonopodis, Psedomonas solancearum, Xanthomonas cucurbitae by cow urine as well as microbs isolated from cow urine. The TLC and titrimetric analysis were showed presence of lipase enzyme in the cow urine. Moreover, sterilized cow urine can be utilized as natural growth medium for Pseudomonas fluorescence, Bacillus subtilis, mixture of fungi and yeast. The use of cow urine can be the cost-effective and eco-friendly approach for controlling various diseases of plants.

Progress of pancreatitis disease biomarker alpha amylase enzyme by new nano optical sensor

A new nano optical sensor binuclear Pd-(2-aminothiazole) (urea), Pd(atz,ur) complex was prepared and characterized for the assessment of the activity of alpha amylase enzyme in urine and serum samples for early diagnosis of Pancreatitis disease. The assessment of alpha amylase activity is carried out by the quenching of the luminescence intensity of the nano optical sensor binuclear Pd(atz,ur) complex at 457nm by the 2-chloro-4-nitrophenol (2-CNP) which produced from the reaction of the enzyme with 2-chloro-4-nitrophenyl-α-d-maltotrioside (CNPG3) substrate. The remarkable quenching of the luminescence intensity at 457nm of nano Pd(atz,ur) doped in sol-gel matrix by various concentrations of the 2-CNP was successfully used as an optical sensor for the assessment of α-amylase activity. The calibration plot was achieved over the concentration range 8.5×10−6 to 1.9×10−9molL−1 2-CNP with a correlation coefficient of (0.999) and a detection limit of (7.4×10−10molL−1). The method was used satisfactorily for the assessment of the α-amylase activity over activity range (3–321U/L) in different urine and serum samples of pancreatitis patients. The assessment of the alpha amylase biomarker by the proposed method increases its sensitivity (96.88%) and specificity (94.41%) for early diagnosis of pancreatitis diseases.

Alterations in Circulatory and Renal Angiotensin-Converting Enzyme and Angiotensin-Converting Enzyme 2 in Fetal Programmed Hypertension

Antenatal betamethasone treatment is a widely accepted therapy to accelerate lung development and improve survival in preterm infants. However, there are reports that infants who receive antenatal glucocorticoids exhibit higher systolic blood pressure in their early adolescent years. We have developed an experimental model of programming whereby the offspring of pregnant sheep administered clinically relevant doses of betamethasone exhibit elevated blood pressure. We tested the hypothesis as to whether alterations in angiotensin-converting enzyme (ACE), ACE2, and neprilysin in serum, urine, and proximal tubules are associated with this increase in mean arterial pressure. Male sheep were administered betamethasone (2 doses of 0.17 mg/kg, 24 hours apart) or vehicle at the 80th day of gestation and delivered at term. Sheep were instrumented at adulthood (1.8 years) for direct conscious recording of mean arterial pressure. Serum and urine were collected and proximal tubules isolated from the renal cortex. Betamethasone-treated animals had elevated mean arterial pressure (97+/-3 versus 83+/-2 mm Hg; P<0.05) and a 25% increase in serum ACE activity (48.4+/-7.0 versus 36.0+/-2.7 fmol/mL per minute) but a 40% reduction in serum ACE2 activity (18.8+/-1.2 versus 31.4+/-4.4 fmol/mL per minute). In isolated proximal tubules, ACE2 activity and expression were 50% lower in the treated sheep with no significant change in ACE or neprilysin activities. We conclude that antenatal steroid treatment results in the chronic alteration of ACE and ACE2 in the circulatory and tubular compartments, which may contribute to the higher blood pressure in this model of fetal programming-induced hypertension.

Using of Telomerase Enzyme in Urine as a Non invasive Marker for Cancer Bladder Detection

Background: Urinary bladder cancer is one of the major health problem all over the world. Cystoscopy remains the gold standard for identifying bladder cancer but it is invasive and expensive, therefore, a simple, non invasive test for detecting bladder cancer would be helpful. Several biomarkers for bladder cancer have been used, but no single marker has been accurate and conclusive. Aim: The current study aimed to measure telomerase enzyme in urine as a useful non invasive marker for detection of bladder cancer. Methods : Forty eight patients ( 39 males and 9 females) were included, They are complaining of urinary symptoms and undergo cystoscopy with biopsy of bladder lesions and histopathological examination. They were divided into groups: Group I: 16 patients ( 11 males and 5 females) have benign urologic conditions. Group II: 32 patients (28 males and 4 females) have proven bladder cancer patients underwent transurethral resection of bladder tumor or cystoscopy with biopsy of bladder lesions. Also, 15 apparently healthy volunteers with matched age and sex with patients were served as a control group. All subjects were submitted to laboratory estimation of the following in urine: urinary creatinine, urine cytology, telomerase enzyme in urine by telomerase PCR and complete urine examination. Results : The results of this study revealed that a highly significant increase in the frequency of cytolological positive cases for tumor cells in malignant group than each of benign group and healthy subjects, while no significant difference was detected between benign group and healthy subjects. The frequency of telomerase in urine was significantly higher in malignant group than each of benign group and healthy subjects, while no significant difference was detected between benign group and healthy subjects. The telomerase activity has sensitivity of 90.6% for diagnosis of cancer bladder with 93.7% for specificity and PPV was 96.6%, NPV was 83.3% and diagnostic accuracy of 91.6%. While, urine cytology gives a sensitivity of 68.8%, specificity of 87.5%, PPV of 91.6%, NPV 58.3% and diagnostic accuracy of 75%. When combined tests were used the sensitivity raised to 96,8%, and specificity reached to 100%, PPV was 96.6%, NPV was 94.1% and diagnostic accuracy increased to 97.9%. Conclusion: the urinary assay of telomerase could be used as non invasive test to identify the bladder cancer patients and distinguish them from normal subjects and patients with benign tumor of urinary bladder. The low cost of this test may help to be implicated as non invasive screening of bladder cancer.

Using of Telomerase Enzyme in Urine as a Non invasive Marker for Cancer Bladder Detection

: Urinary bladder cancer is one of the major health problem all over the world. Cystoscopy remains the gold standard for identifying bladder cancer but it is invasive and expensive, therefore, a simple, non invasive test for detecting bladder cancer would be helpful. Several biomarkers for bladder cancer have been used, but no single marker has been accurate and conclusive.

ADAM 12 Cleaves Extracellular Matrix Proteins and Correlates with Cancer Status and Stage

ADAM 12 is a member of a family of disintegrin-containing metalloproteases that have been implicated in a variety of diseases including Alzheimer's disease, arthritis, and cancer. We purified ADAM 12 from the urine of breast cancer patients via Q-Sepharose anion exchange and gelatin-Sepharose affinity chromatography followed by protein identification by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Four peptides were identified that spanned the amino acid sequence of ADAM 12. Immunoblot analysis using ADAM 12-specific antibodies detected an approximately 68-kDa band identified as the mature form of ADAM 12. To characterize catalytic properties of ADAM 12, full-length ADAM 12-S was expressed in COS-7 cells and purified. Substrate specificity studies demonstrated that ADAM 12-S degrades gelatin, type IV collagen, and fibronectin but not type I collagen or casein. Gelatinase activity of ADAM 12 was completely abrogated by zinc chelators 1,10-phenanthroline and EDTA and was partially inhibited by the hydroxamate inhibitor Marimastat. Endogenous matrix metalloprotease inhibitor TIMP-3 inhibited activity. To validate our initial identification of this enzyme in human urine, 117 urine samples from breast cancer patients and controls were analyzed by immunoblot. The majority of samples from cancer patients were positive for ADAM 12 (67 of 71, sensitivity 0.94) compared with urine from controls in which ADAM 12 was detected with significantly lower frequency. Densitometric analyses of immunoblots demonstrated that ADAM 12 protein levels were higher in urine from breast cancer patients than in control urine. In addition, median levels of ADAM 12 in urine significantly increased with disease progression. These data demonstrate for the first time that ADAM 12 is a gelatinase, that it can be detected in breast cancer patient urine, and that increased urinary levels of this protein correlate with breast cancer progression. They further support the possibility that detection of urinary ADAM 12 may prove useful in the development of noninvasive diagnostic and prognostic tests for breast and perhaps other cancers.

Degenerative Disorders of the Central Nervous System

Degenerative Disorders of the Central Nervous System

Purification and characterization of a neutral endopeptidase-like enzyme from human urine.

The aims of this study were to purify and characterize a neutral endopeptidase-like enzyme (NEP-like) in human urine and propose a rapid, sensitive and specific assay for this enzyme using the fluorogenic substrate Abz-FDQ-EDDnp, where Abz = O-aminobenzoic acid and EDDnp = N-(2,4-dinitrophenyl)ethylenediamine. Soluble urinary NEP was purified from human urine using a DEAE-cellulose Cellex D column and gel filtration on an AcA-44 column. NEP-like activity was assayed by its ability to hydrolyse bradykinin (BK) and the fluorogenic substrates Abz-BKQ-EDDnp and Abz-FDQ-EDDnp. The Km was determined using Abz-FDQ-EDDnp as a substrate. The hydrolysis products of BK and Abz-FDQ-EDDnp were analysed by high-performance liquid chromatography (HPLC). The mol. wt was estimated by polyacrylamide gel electrophoresis and the enzyme analysed by Western blot using the antibody obtained from purified recombinant NEP expressed in Pichia pastoris yeast. The NEP-like was purified from human urine until homogeneity and presented a mol. wt of 94000. The substrate Abz-FDQ-EDDnp was selectively hydrolysed at the F-D bond by NEP-like and by recombinant NEP. For this substrate, the NEP-like activity was maximal at pH 7.0, although a small peak of activity was observed at pH 8.0, and the determined Km was 14 microM. The enzymatic activity was inhibited by thiorphan and phosphoramidon. In Western blot analysis, NEP-like reacted strongly with a polyclonal antibody for NEP. A NEP-like enzyme was purified from human urine. Based on the mol. wt of the isolated NEP-like enzyme, it was concluded that this enzyme was produced in the kidney. In the kidney, this enzyme may cleave the kinins filtered through the glomerulus and also the kinins produced in the distal nephron. An internally quenched fluorogenic peptide, Abz-FDQ-EDDnp, was selectively hydrolysed by NEP-like and by recombinant NEP.

Crystallization, preliminary X-ray analysis and Patterson search of a new aspartic protease isolated from human urine.

Aspartic protease (EC 3.4.23) make up a widely distributed class of enzymes in animals, plants, microbes and, viruses. In animals these enzymes perform diverse functions, which range from digestion of food proteins to very specific regulatory roles. In contrast the information about the well-characterized aspartic proteases, very little is known about the corresponding enzyme in urine. A new aspartic protease isolated from human urine has been crystallized and X-ray diffraction data collected to 2.45 A resolution using a synchrotron radiation source. Crystals belong to the space group P2(1)2(1)2(1). The cell parameters obtained were a = 50.99, b = 75.56 and c = 89.90 A. Preliminary analysis revealed the presence of one molecule in the asymmetric unit. The structure was determined using the molecular replacement technique and is currently being refined using simulated annealing and conjugate gradient protocols.

Demonstration of the Presence of Cyclic Inositol Phosphohydrolase in Human Urine

Cyclic inositol phosphohydrolase (cIPH), cleaves the cyclic bond of cyclic inositol monophosphate (cIP) to yield inositol monophosphate. In this communication, we demonstrate the presence of cIPH in human urine. cIPH was measured in the 24-h urine samples of both male and female hospital patients. cIPH released per day ranged from 0 to 243 units in men (n= 16) and from 15 to 346 units in women (n= 18). Release of cIPH activity was not related to renal function as measured by creatinine clearance. HPLC ion-exchange chromatography or HPLC gel filtration of ammonium sulfate precipitate yielded a distinct cIPH peak with an apparent molecular weight of 40 kDa on gel filtration. This is the first demonstration of the presence of this enzyme in human urine. The large variation (over 20-fold) in the excretion of this protein suggests that it may have physiological and/or pathological significance.

AN IMPROVED KINETIC METHOD FOR MEASUREMENT OF URINARY ALANINE AMINOPEPTIDASE (AAP) ACTIVITY AS A NEPHROTOXIC MARKER

This study examines the effect of various factors that influence alanine aminopeptidase (EC 3.4.11.2; AAP) activity in untreated urine samples, using L-alanine- p -nitroanilide as substrate. An improved kinetic method for assay of AAP activity without pretreatment of urine samples was obtained. The results show that the final concentration of 6 mmol/ L L-alanine- p -nitroanilide and 325 mmol/ L tris(hydroxymethyl)aminomethane (pH 7.90 at 30 C) and the sample/ final reaction volume ratio of 1/ 25 to be optimum. The within-run coefficient of variation (CV) was 1.40%; the between-run CV averaged 3.11%. Reference values were established for AAP activity of untimed urine samples obtained from 810 healthy persons. The normal reference interval (mean 1 SD) was 0.81-38.64 (12.76 6.88) U/ g of creatinine. Freezing the untreated urine sample resulted in a considerable loss of AAP activity. However, when urinary AAP activity is measured by the present method, there is no need for urine and reagent blanks; assay of the enzyme in urine is possible using one reagent solution. Therefore, the present method is suitable for use with various automated analyzers. Finally, the present method requires only 30muL of untreated urine samples, so it is suitable for evaluating of nephrotoxicity in human (especially neonates and infants) and small animal models such as rats.

Telomerase activity in bladder cancer, bladder washings and in urine

With only a few exceptions, the ribonucleoprotein telomerase has been found in malignant, but not in benign tissues. Telomerase is thus a potentially new diagnostic marker. Carcinoma of the urinary bladder is the most frequent malignant tumor of the urinary tract and, after prostatic carcinoma, the second most common malignancy of the genitourinary system. In order to evaluate the diagnostic capabilities of telomerase in bladder carcinomas, four cell lines derived from human urothelial carcinomas of the bladder, 75 tissue samples from bladder carcinomas, eight tissue samples of normal bladder urothelium, 40 bladder washings and 30 urine samples were examined for telomerase activity. The four cell lines derived from urothelial carcinoma of the bladder (F975, 582, SCaBER, UM-UC-3) all exhibited high telomerase activity and were thus used as positive controls. Telomerase activity was found in nearly all (96%) tissue samples obtained from histologically confirmed urothelial carcinoma of the bladder. None of the normal tissue samples examined showed telomerase activity. Telomerase activity was similarly found in 73% of bladder washings in patients with histologically confirmed bladder carcinoma. There were no false positive results. The determination of telomerase activity in bladder washing samples thus represents a new diagnostic method for detection of tumor cells in rinsing media. Because of the early inactivation or degradation of telomerase there was no detection of the enzyme in native urine in the present study.

RK7-kallikrein in rat kidney and urine

Rat Kidney is reported to contain the rK7-kallikrein gene expressed at a high mRNA level. However, there is no direct evidence that the rK7 enzyme is present in kidney or urine. We have analyzed rat kidney and urine for rK7-like enzyme activity, using distinctive properties of purified rK7 enzyme (hydrolysis of specific substrate, inhibition by SBTI). Rat urine and kidney cortex contain substantial levels of rK7-like enzyme activity. Nearly 40% of the enzyme in urine is in an active form while nearly all of the rK7 in kidney cortex is in the inactive form. Treatment with low salt diet and mineralocorticoid increases rK7 in both kidney cortex and urine approximately 2-fold. The percentage of active enzyme in urine increases markedly on this regimen.

N-acetyl-B-D-glucosaminidase isoenzymes in the diagnosis of poisoning and kidney diseases.

Damaged lysosomes from renal tubular cells are, to the greatest degree, the source of activity of NAG in urine. Besides this, the enzyme can appear as a result of degranulation of granulocytes (PMN), active infection of the urinary system as also from serum as a result of glomerular filtration during damage to the glomerular basement membrane. For the purpose of explaining the source of origin of the enzyme in urine, NAG was separated into isoenzymes from the kidneys, granulocytes and serum for comparison with isoenzymes in physiological and pathological urines after ethylene glycol poisoning, and glomerulonephritis, respectively. The separation was made by column ion-exchange chromatography on DEAE-52 cellulose and by electrophoresis in 7% polyacrylamide gel. In addition, the thermostability of isolated isoenzymes was compared.

Poststatin, a novel inhibitor of bradykinin-degrading enzymes in rat urine

Incubation of bradykinin with rat urine resulted in the successive degradation of bradykinin to bradykinin-(1–8), bradykinin-(1–7) and bradykinin-(1–6). In contrast, in rat plasma, bradykinin was degraded via either bradykinin-(1–8) or bradykinin-(1–7) to bradykinin-(1–5). Phosphoramidon (1 mM) partially inhibited the degradation of bradykinin by rat urine, as well as the conversion of bradykinin-(1–7) to bradykinin-(1–6). D,L-2-Mercaptomethyl-3-guanidinoethylthiopropanoic acid (1 mM) and captopril (1 mM) did not have a significant effect on any of the degradation steps in rat urine. In contrast, all of the degradation steps in urine, namely, from bradykinin to bradykinin-(1–8), from bradykinin-(1–8) to bradykinin-(1–7) and from bradykinin-(1–7) to bradykinin-(1–6), were markedly inhibited by poststatin (1 mM), even though this compound was reported originally to be a novel inhibitor of post-proline cleaving enzyme. Poststain (1 mM) did not inhibit the degradation of bradykinin in rat plasma. These results indicate that poststatin is an effective inhibitor of kinin-degrading enzyme in rat urine.

"VRA-GlcNAc": novel substrate for N-acetyl-beta-D-glucosaminidase applied to assay of this enzyme in urine

We describe a new chromogenic substrate and assay for determining N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30; NAG) activity in normal and pathological human urine. The novel substrate, ammonium 5-[4-(2-acetamido-2-deoxy-beta-D-glucopyranosyloxy)-3- methoxyphenylmethylene]-2-thioxothiazolidin-4-one-3-ethan oate (VRA-GlcNAc), is prepared by condensation of vanillyl N-acetyl-beta-D-glucosaminide and rhodanine-3-ethanoic acid. The phenol released by enzyme action has an intense absorption peak at 492 nm (epsilon = 37,000 L.mol-1.cm-1). The Km for NAG in urine was 0.5 mmol/L at pH 4.5. The substrate solution may be stored at 4-7 degrees C for one week without any increase in the substrate blank. For optimal assay conditions, we used a substrate concentration of 3 mmol/L and a 15-fold sample dilution at pH 4.5-5.0, with measurement at 505 nm, not significantly different from that at 492 nm. The sensitivity of the assay with VRA-GlcNAc as substrate is twice that of the MNP-GlcNAc [2-methoxy-4-(2'-nitrovinyl)-phenyl GlcNAc] procedure (Clin Chim Acta 1982;124:195-204) and can detect low amounts of minor NAG isoenzymes. This assay is an improvement on previously available colorimetric methods and could be easily incorporated into kits based on either an enzyme calibrant or the molar absorptivity of the phenol.