Methods compared for determining activity of N-acetyl-beta-D-glucosaminidase in urine without pretreatment of sample: different sensitivity and species effect

N-Acetyl-beta-D-glucosaminidase (NAG) activities in the urine of men and rats were measured with methods recommended as procedures without pretreatment of the urine sample. Four different derivatives of NAG were compared for determination: 4-nitrophenyl; 3,3-dichlorophenylsulfonphthaleinyl; 3-cresolsulfonphthaleinyl, and 2-methoxy-4-(2-nitro-vinyl)phenyl. The conventional test using the 4-nitrophenyl derivative showed the highest activities and correlated very well with the other tests. There are method-dependent differences between NAG activities measured in men and rats due to the different Km values and inhibitory effects by urea.     

Age related changes of N-acetyl-beta-D-glucosaminidase and L-alanine aminopeptidase in mouse kidney, urine and plasma

Age and sex dependent differences of N-acetyl-beta-D-glucosaminidase (NAG) and L-alanine aminopeptidase (AAP) activities in kidney, urine and plasma of male and female mice were studied. The sex difference in NAG activity appeared between 27 and 38 days of age with the manifestation of significant differences in body weight and kidney growth. NAG activity in male kidneys was 3-fold that in females and its urinary level in mature males was over 10-fold higher. Androgenic regulation was found not only in the NAG contents in the kidneys and in the urinary excretion but also in the plasma NAG level, which showed higher in females. On the other hand, AAP activity in kidney, urine and plasma did not show much sex differences. Age related changes in AAP activity were not found except in the kidney and marked androgenic regulation was also not found in AAP. These results indicate that NAG and AAP, which are both urinary enzymes used as indicators of renal lesions, may be regulated differently.     

Enzyme immunoassay for the measurement of 17alpha-estradiol 17-N-acetylglucosaminide in rabbit urine.

17alpha-estradiol 17-N-acetylglucosaminide (17alphaE2 17NAG) is an estrogen metabolite hitherto obtained only in rabbits. To gain insight into this unique conjugate, an enzyme immunoassay (EIA) was established by using antiserum elicited against 3-[3-(1-carboxypropyl)] ether of 17alphaE2 17NAG-bovine serum albumin conjugate; horseradish peroxidase, as a label; and 3,3',5,5'-tetramethylbenzidine, as a chromogen. The method proved to be specific, and the detection range of the assay was 0.20-10.00 ng/ml. A proposed double conjugate, 3-glucuronide of 17alphaE2 17NAG, was synthesized to validate the EIA. The EIA was applied to the determination of the urinary level of 17alphaE2 17NAG in male and female (pregnant and non-pregnant) rabbits with and without beta-glucuronidase-sulfatase preparation from Helix pomatia. The results showed that 17alphaE2 17NAG was mainly excreted as a double conjugate (17alphaE2 17NAG 3-glucuronide and/or 3-sulfate) and that its level varies during pregnancy.     

Urinary N-acetyl- beta-D-glucosaminidase activities in patients with renal disease.

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.     

High-performance liquid chromatographic determination of the polar metabolites of nifedipine in plasma, blood and urine

A relatively simple reversed-phase high-performance liquid chromatographic method for the determination of the polar metabolites of nifedipine in biological fluids is described. After conversion of 2-hydroxymethyl-6-methyl-4-(2-nitrophenyl)pyridine-3,5-dicarboxylic acid 5-methyl ester (IV) into 5,7-dihydro-2-methyl-4-(2-nitrophenyl)-5-oxofuro[3,4-b]pyridine-3-carboxylic acid methyl ester (V) by heating under acidic conditions, V was extracted with n-pentane—dichloromethane (7:3) and analysed on a C₁₈ column with ultraviolet detection. Subsequently, 2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic acid monomethyl ester (III) was extracted with chloroform and analysed on the same system. Limits of determination in blood were 0.1 μg/ml for III and 0.05 μg/ml for IV and V; these limits were two to ten times higher for urine. This inter-assay variability was always less than 7.5%.     

Enzyme immunoassay of beta-hexosaminidase isoenzymes in human urine and renal cortex with monoclonal antibodies

Enzyme immunoassay (EIA) methods with monoclonal antibodies specific for N-acetyl-beta-D-glucosaminidase (NAG) isoenzymes A and B in human urine are presented. The proportion of NAG B obtained with the EIA methods was similar to that found with ion-exchange chromatography. In fresh human control urines, NAG B was found to be approximately 20% of the total NAG activity. A significant correlation was obtained between total NAG activity in human urine assayed with a conventional enzyme substrate method and the total NAG activity obtained as the sum of NAG A and NAG B analyzed with the EIA methods. Total NAG activity with the latter (EIA) methods showed about 30% higher values than found by the enzyme substrate method, which probably was due to inhibitors of NAG activity present in urine did not interfere with the EIA methods. The content of NAG A and NAG B in renal cortex was determined with the EIA methods. NAG B accounted for about 20% of the total NAG activity, which was similar to that found in fresh human urines.     

Preparation of specific antisera to 15alpha-hydroxyestrogen 15-N-acetylglucosaminides.

3-(1-Carboxypropyl) ether derivatives of 15alpha-hydroxyestradiol 15-N-acetylglucosaminide (15alpha-OHE2 15NAG) and 15alpha-hydroxyestriol (E4) 15NAG were synthesized and conjugated with bovine serum albumin. Antisera elicited in rabbits possessed high affinity and specificity for the 15alpha-hydroxyestrogen (15alpha-OHEs) 15NAG, exhibiting no significant cross-reactivity with 15alpha-OHEs and their positional isomers such as 16NAG and 17NAG. Enzyme immunoassay methods developed by using the purified antisera and horseradish peroxidase-labeled antigens were applied to the measurement of 15alpha-OHEs 15NAG and E4 15NAG in normal pregnancy urine. We demonstrated for the first time that the conjugation of N-acetylglucosamine to E4 occurs at the C-15alpha position.     

联合标记物检测在重症感染中合并急性肾损伤的诊断价值

目的:探讨尿中性粒细胞明胶酶相关载脂蛋白(neutrophil gelatinase-associated lipocalin,NGAL)、尿肾损伤分子-1(kidneyinjury molecule-1,Kim-1)、尿N-乙酰-β-D-氨基葡萄糖苷酶(N-acecyl-β-D-glucosaminidase,NAG)、尿微量白蛋白(mALB)在重症感染中合并急性肾损伤的敏感性及临床价值。方法:回顾分析60例在新疆自治区人民医院ICU住院的重症感染合并急性肾损伤(AKI)患者的尿NGAL、Kim-1、NAG及mALB的变化情况。健康体检者20例为对照组。尿NGAL、Kim-1、mALB测定采用酶联免疫法(ELISA)检测,尿NAG测定采用对硝基苯酚(PNP)比色法检测,并以ROC曲线分析其敏感性。结果:AKI组患者尿液中的NGAL、Kim-1、NAG、mALB的测定浓度明显高于对照组,差异具有统计学意义(P<0.001),通过ROC曲线、诊断试验结果显示:尿NGAL、Kim-1曲线下面积分别为0.986、0.956,95%可信区间分别是0.968～1.004、0.910～1.001,较尿NAG、mALB更具有敏感性(P<0.001)。结论:尿NGAL、尿Kim-1的浓度检测对重症感染合并急性肾损伤的诊断更具有敏感性,与NAG、mALB联合检测有助于急性肾损伤的早期监测,对预防急性肾损伤的发生、发展具有重要的临床价值。     

Diurnal variation and reliability of the urine lactate concentration after maximal exercise

The postexercise urine lactate concentration is a novel valid exercise biomarker, which has exhibited satisfactory reliability in the morning hours under controlled water intake. The aim of the present study was to investigate the diurnal variation of the postexercise urine lactate concentration and its reliability in the afternoon hours. Thirty-two healthy children (11 boys and 21 girls) and 23 adults (13 men and 10 women) participated in the study. All participants performed two identical sessions of eight 25 m bouts of maximal freestyle swimming executed every 2 min with passive recovery in between. These sessions were performed in the morning and afternoon and were separated by 3–4 days. Adults performed an additional afternoon session that was also separated by 3–4 days. All swimmers drank 500 mL of water before and another 500 mL after each test. Capillary blood and urine samples were collected before and after each test for lactate determination. Urine creatinine, urine density and body water content were also measured. The intraclass correlation coefficient was used as a reliability index between the morning and afternoon tests, as well as between the afternoon test and retest. Swimming performance and body water content exhibited excellent reliability in both children and adults. The postexercise blood lactate concentration did not show diurnal variation, showing a good reliability between the morning and afternoon tests, as well as high reliability between the afternoon test and retest. The postexercise urine density and lactate concentration were affected by time of day. However, when lactate was normalized to creatinine, it exhibited excellent reliability in children and good-to-high reliability in adults. The postexercise urine lactate concentration showed high reliability between the afternoon test and retest, independent of creatinine normalization. The postexercise blood and urine lactate concentrations were significantly correlated in all cases, attesting to the validity of urine lactate as an index of anaerobic metabolism. We conclude that urine lactate, after normalization to creatinine, could be used in training practice either in the morning or in the afternoon. Further research is needed to assess the applicability of this novel exercise biomarker.     

Gas chromatographic-negative-ion chemical ionization mass spectrometric determination of a new dihydropyridine calcium antagonist (MPC-1304) and its metabolites in human plasma and urine

A gas chromatographic-negative-ion chemical ionization mass spectrometric method was developed for the determination of a new calcium antagonist, (±)-methyl 2-oxopropyl 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicar☐ylate, and its metabolites in plasma and urine. The sample was extracted with n-hexane-diethyl ether. The dried organic layer was subjected to acetylation: the aqueous layer was acidified and extracted with ethyl acetate, and after the ethyl acetate extract was dried the resulting residue was subjected to methylation. Aliquots of each reactant solution were injected into the gas chromatograph-mass spectrometer, equipped with a chemical ionization source and negative-ion monitoring mode, and analysed by the elected-ion monitoring method using deuterium-labelled internal standards. Detection was limited to 0.02–0.05 ng/ml of plasma and urine for each metabolite. A precise and sensitive assay for the determination of a new dihydropyridine calcium antagonist and its metabolites in plasma and urine was thus established.     

Methodological considerations with the use of urine samples for assessment of mercury excretion and markers of renal damage

Objective:?To provide recommendations for design and analysis of studies using urine specimens to evaluate renal function or mercury excretion in children.

Methods:?An analysis of mercury, albumin, γ-glutamyl transpeptidase (γ-GT) and N-acetyl-β-D-glucosaminidase (NAG) concentrations was carried out.

Results:?Mercury concentration and creatinine-corrected renal markers were higher in daytime compared with overnight samples. Excretion rates increased with urinary flow rate. γ-GT and NAG concentrations decreased with storage time at ?20°C. Differences by age, sex and race were noted.

Conclusions:?We recommend use of these creatinine-corrected markers and collection of timed overnight urine samples, stored at ?70°C, with control for urinary flow rate, age, sex and race in statistical models.     

Activity of alanine aminopeptidase, beta-glucuronidase and N-acetyl-beta-d-glucosaminidase in urine of children with nephrotic syndrome]

Activity of alanine aminopeptidase (AAP), beta-glucuronidase, and N-acetyl-beta-D-glucosaminidase (NAG) in daily urine has been determined in 27 children with nephrotic syndrome, 14 children in remission, and 11 healthy children. It was found, that these enzymes activity is significantly increased in sick children in comparison with healthy ones. Similarly, the activity of AAP and NAG in daily urine is statistically significantly higher in children with remission, than that in healthy children. An assay of these enzymes in the urine may be used in the diagnosis of nephrotic syndrome and in the evaluation of its course.     

Identification of fendiline metabolites in human urine

After oral application of 14C labelled fendiline, 13 metabolites of this drug could be identified in human urine. Only traces of parent fendiline were excreted in the urine. The main pathway of metabolism is hydroxylation of phenyl groups with subsequent glucuronidation and sulphation. On the other hand, oxidative dealkylation occurs with the amino group remaining at the 3,3-diphenylpropyl moiety and p-hydroxyacetophenone being formed almost entirely from the 1-phenylethyl group.     

Changes in N-acetyl-beta-D-glucosaminidase activity in the urine and urinary albumin excretion in magnesium deficient rats

To discover the details of the effects of magnesium (Mg) deficiency on kidney function, the course of changes in N-acetyl-beta-D-glucosaminidase (NAG) activity in the urine and in urinary albumin excretion were examined in rats fed a Mg-deficient diet. NAG activity in the urine and urinary albumin excretion in rats fed the Mg-deficient diet significantly increased from 7 d until the end of the feeding period. We suggest that Mg-deficient diet rapidly induces kidney function insufficiency.     

探讨尿视黄醇结合蛋白对高血压早期肾损害的作用研究

目的:探讨尿视黄醇结合蛋白(RBP)、尿N-乙酰-β-D-氨基葡萄糖苷酶(NAG)、尿胱蛋白酶抑制剂C(Cystatin C)的测定对原发性高血压早期肾损害诊断的敏感性及临床意义.方法:选择2011年4月～2011年11月在新疆自治区人民医院肾病科住院的原发性高血压患者69例作为观察组,另设健康体检者30例为对照组.尿RBP、Cystatin C测定采用酶联免疫法(ELISA)检测,尿NAG测定采用对硝基苯酚(PNP)比色法检测.结果:观察组中尿RBP、NAG、CystatinC的测定含量明显高于正常对照组(P＜0.001),通过ROC曲线、诊断试验结果显示:尿RBP的曲线下面积为0.962,95％可信区间为0.921～0.983,较尿NAG、Cystatin C更具有敏感性(P＜0.001).结论:检测尿RBP有助于高血压肾损害的早期监测,对预防高血压性肾病的发生、发展具有重要的临床价值.     

Use of genital inspection and female urine tests to detect oestrus in captive Asian elephants

Captive Asian elephant (Elephas maximus) populations are decreasing due to low birth rates compared to wild elephants. Improving oestrous detection in female elephants is required to ensure successful mating in captive and semi-captive herds. Responsive behaviours of eight semi-captive bull elephants to the uro-genital area (genital inspection test) or urinary pheromones (urine test) of 14 female elephants throughout the oestrous cycle were evaluated. Weekly blood samples were collected for 27 consecutive months (14 months for the genital inspection test and 13 months for the urine test) from female elephants to characterize the patterns of circulating progestagen. Responsive behaviours of bulls were compared between females in the follicular versus the luteal phase of the cycle. The sensitivity and specificity of the genital inspection test were 65% and 68%, while those of the urine test were 52% and 61%, respectively. The bulls showed significantly higher “genital inspection”, “flehmen from genital area” and “trunk on back” behaviours during the genital inspection test, and “flehmen” behaviours during the urine test in oestrous than in non-oestrous females. In sum, this study showed that monitoring sexual behaviours of Asian elephant bulls towards females or their urine can be used to detect the oestrous period. Although the sensitivity and specificity of both tests were not as high as expected, still, these methods appear to be more efficient at detecting oestrous than traditional methods based on mahout estimations of female receptivity. The use of genital inspection and urine tests may lead to more successful matings and thus to creating self-sustaining populations of captive elephants in range countries.     

滇西并流河谷区土壤酶活性化学计量学特征与环境因子的关系

横断山河谷区具有极高的景观异质性,气候与植被类型多样化程度较高.为探讨土壤C、N、P、S四种生物元素在滇西怒江、澜沧江、金沙江及元江并流河谷区的区域循环特征,在各河谷的森林、草地、农田中分别取浅层(0～ 10 cm)土样,测定了土壤中C、N、P、S的循环酶,即β-葡萄糖苷酶(BG)、N-乙酰-β-D-氨基葡萄糖苷酶(N...     

Studies on steroids : CLXV. Determination of isomeric catechol estrogens in pregnancy urine by high-performance liquid chromatography with electrochemical detection

A method for the determination of 2- and 4-hydroxylated estrone and estradiol in pregnancy urine by high-performance liquid chromatography with electrochemical detection (HPLC-ECD) is described. The urine catechol estrogens were deconjugated, purified by adsorption on alumina, and subjected to HPLC-ECD. Two pairs of isomeric catechol estrogens were distinctly separated on a μBondapak C₁₆ column with acetonitrile-0.5% ammonium dihydrogen phosphate (pH 3.0). The amounts of these four compounds were satisfactorily determined with a quantitation limit of 1 ng using 4-hydroxy-16-oxoestradiol 17-acetate as an internal standard. The validity of the present method for the determination of urine catechol estrogens was verified by the recovery test.     

Urinary aminopeptidase activities as early and predictive biomarkers of renal dysfunction in cisplatin-treated rats

This study analyzes the fluorimetric determination of alanyl- (Ala), glutamyl- (Glu), leucyl-cystinyl- (Cys) and aspartyl-aminopeptidase (AspAp) urinary enzymatic activities as early and predictive biomarkers of renal dysfunction in cisplatin-treated rats. Male Wistar rats (n = 8 each group) received a single subcutaneous injection of either saline or cisplatin 3.5 or 7 mg/kg, and urine samples were taken at 0, 1, 2, 3 and 14 days after treatment. In urine samples we determined Ala, Glu, Cys and AspAp activities, proteinuria, N-acetyl-β-D-glucosaminidase (NAG), albumin, and neutrophil gelatinase-associated lipocalin (NGAL). Plasma creatinine, creatinine clearance and renal morphological variables were measured at the end of the experiment. CysAp, NAG and albumin were increased 48 hours after treatment in the cisplatin 3.5 mg/kg treated group. At 24 hours, all urinary aminopeptidase activities and albuminuria were significantly increased in the cisplatin 7 mg/kg treated group. Aminopeptidase urinary activities correlated (p<0.011; r²>0.259) with plasma creatinine, creatinine clearance and/or kidney weight/body weight ratio at the end of the experiment and they could be considered as predictive biomarkers of renal injury severity. ROC-AUC analysis was made to study their sensitivity and specificity to distinguish between treated and untreated rats at day 1. All aminopeptidase activities showed an AUC>0.633. We conclude that Ala, Cys, Glu and AspAp enzymatic activities are early and predictive urinary biomarkers of the renal dysfunction induced by cisplatin. These determinations can be very useful in the prognostic and diagnostic of renal dysfunction in preclinical research and clinical practice.     

Purification and characterization of cold-active L-glutamate dehydrogenase independent of NAD(P) and oxygen

L-Glutamate dehydrogenase (GLDH) independent of NAD(P) and oxygen was first obtained from the psychrotrophic bacterium Aeromonas sp. L101, originally isolated from the organs of salmon (Oncorhynchus keta). GLDH was purified by a series of chromatography steps on DEAE-Sepharose, Superdex 200pg, Q-Sepharose, CM-Sepharose, and Phenyl-Sepharose. The purified protein was determined to have a molecular mass of 110 kDa and a pI of 5.7. Maximum activity was obtained at 55 degrees C and pH 8.5. The activity of GLDH at 4 and 20 degrees C was 38 and 50%, respectively, of that at 50 degrees C. GLDH was coupled to cytochrome c and several redox dyes including 1-methoxy-5-methylphenazinium methylsulfate (1-Methoxy PMS), 2, 6-dichlorophenylindophenol (DCIP), 9-dimethylaminobenzo[alpha]phenoxazin-7-ium chloride (meldola's blue), 3,3'-[3,3'-dimethoxy-(1,1'-biphenyl)-4, 4'-diyl]-bis[2-(4-nitrophenyl)-5-phenyl-2H tetrazolium chloride] (nitroblue tetrazolium; NBT), and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H tetrazolium (INT). The presence of NAD(P) and oxygen gave no oxidation activity to GLDH. Spectroscopic profile and ICP data indicated a b-type cytochrome containing iron.