Luminometric single step urea assay using ATP-hydrolyzing urease

An automatic enzyme kinetic luminometric method for determination of small quantities of urea in biological fluids and in microdialysates is presented. The method is based on the ATP-hydrolyzing urease reaction [urea amidohydrolase (ATP-hydrolyzing); EC 3.5.1.45], monitored by a luciferin-luciferase ATP reaction. The assay range is 100 pmol to 50 nmol with a detection limit of 5 micromol/L in the sample, compared with detection limits of 0.1 mmol/L in earlier spectrophotometric methods. To reduce the non-urea-dependent ATPase activity (v(blank)) and to increase the urea-dependent activity, 1,2-propanediol was included. Assay conditions were optimized by multivariate analysis. Recoveries of urea added to blood dialysate and plasma were 96-103%. No analytical interference of common metabolites, drugs, or other additives was observed. The total CVs (6 days and six concentrations, 1.2-21.8 mmol/L) were 3.6-8.5%. The results obtained with the present assay were highly correlated for dialysate (r = 0.979) and for plasma (r = 0.978) with those obtained by a spectrophotometric kit method with slopes of 1.02-1.03 and intercepts of 0.08-0.23 mmol/L.     

阳极等离子体电解处理高盐废水中的苯酚

采用阳极等离子体电解处理高盐废水中的苯酚.研究了阳极等离子体产生的条件,以及废水中盐的浓度、苯酚的质量浓度和处理时间对废水中COD去除率的影响.实验结果表明,在苯酚质量浓度为0.2g·L^-1,NaCl浓度为0.4mol·L^-1的溶液中,施加90V槽电压,处理10min,苯酚的去除率达100%;处理20min,废水的COD值从0.464g·L^-1降到0.010g·L^-1,COD去除率可达97.8%.探讨了阳极等离子体电解处理高盐废水中苯酚的机理.     

Jack bean urease (EC 3.5.1.5). V. On the mechanism of action of urease on urea, formamide, acetamide, N-methylurea, and related compounds

Acetamide and N-methylurea have been shown for the first time to be substrates for jack bean urease. In the enzymatic hydrolysis of urea, formamide, acetamide, and N-methylurea at pH 7.0 and 38 degrees C, kcat has the values 5870, 85, 0.55, and 0.075 s-1, respectively. The urease-catalyzed hydrolysis of all these substrates involves the active-site nickel ion(s). Enzymatic hydrolysis of the following compounds could not be detected: phenyl formate, p-nitroformanilide, trifluoroacetamide, p-nitrophenyl carbamate, thiourea, and O-methylisouronium ion. In the enzymatic hydrolysis of urea, the pH dependence of kcat between pH 3.4 and 7.8 indicates that at least two prototropic forms are active. Enzymatic hydrolysis of urea in the presence of methanol gave no detectable methyl carbamate. A mechanism of action for urease is proposed which involves initially an O-bonded complex between urea and an active-site Ni2+ ion and subsequently an O-bonded carbamato-enzyme intermediate.     

Retrospective evaluation of urea and creatinine blood levels in calves with diarrhea

Heart myxoma is the most common cardiac neoplasm in adult, even if its biologic profile remains uncertain. The clinicopathologic features of 6 cardiac myxomas in patients ranging in age from 42 to 58 years are described: 5 cases were located in atria, 1 occurred in the right ventricular wall, attached by a thin pedicle to the wall next to the pulmonary artery. Grossly myxomas are generally pedunculated and average 2 to 8 cm in diameter. They appear gelatinous and polypoid, sometimes with areas of hemorrhage. Microscopic examination of specimens of myxomas removed at operation reveals the myxomatous nature of the stroma composed of abundant mucopolysaccharidic matrix, containing stellate or polyhedral cells, singly or in small clusters, and occasional blood vessels. In other cases, the matrix stains more deeply and reticulin fibers and occasional strands of collagen are evident. Immunohistochemical study reveals tumoral positivity for smooth muscle actin cells and for vimentin. In addition, endothelial cells in intramyxomatous vascular channels are positive for factor VIII and CD-34 endothelial markers. Myxomas were diagnosed in patients in whom the symptoms and signs of cardiac tumor may have been attributed to other causes. The clinical pictures produced by cardiac myxomas include non specific manifestations and mechanical interference with cardiac function. The symptoms may simulate a wide variety of other cardiac conditions (mitral valve disease, embolic phemomena, tricuspid valve disease, sudden unexpected death). A wide local excision is needed to assure that the myxoma does not recur.     

Surface plasmon resonance sensor for heparin measurements in blood plasma

Surface plasmon resonance (SPR) was used as an affinity biosensor to determine absolute heparin concentrations in human blood plasma samples. Protamine and polyethylene imine (PEI) were evaluated as heparin affinity surfaces. Heparin adsorption onto protamine in blood plasma was specific with a lowest detection limit of 0.2 U/ml and a linear window of 0.2-2 U/ml. Although heparin adsorption onto PEI in buffer solution had indicated superior sensitivity to that on protamine, in blood plasma it was not specific for heparin and adsorbed plasma species to a steady-state equilibrium. By reducing the incubation time and diluting the plasma samples with buffer to 50%, the non-specific adsorption of plasma could be controlled and a PEI pre-treated with blood plasma could be used successfully for heparin determination. Heparin adsorption in 50% plasma was linear between 0.05 and 1 U/ml so that heparin plasma levels of 0.1-2 U/ml could be determined within a relative error of 11% and an accuracy of 0.05 U/ml.     

Patterning of immobilized antibody layers via photolithography and oxygen plasma exposure

A novel technique for patterning immobilized antibody layers based upon photolithography and oxygen plasma exposure has been developed. Mouse monoclonal antibodies specific for thiabendazole (a post-harvest fungicide and veterinary anthelmintic) were covalently linked through free amine groups to aminosilanized silicon dioxide films using glutaraldehyde. Immobilized antibody layers were stabilized with sucrose, dehydrated, and stored refrigerated with desiccant. Photolithographic patterning was performed with a positive photoresist with modified bake temperatures and times, selective UV exposure with a contact mask, and aqueous alkaline solubilization of exposed resist. Exposed regions of immobilized antibody were then removed by exposure to a low power, radio frequency oxygen discharge. Residual resist was stripped with acetone. Successful patterning was demonstrated by challenging surfaces with goat anti-mouse antibody conjugated to tetramethylrhodamine isothiocyanate. Sucrose stabilization was necessary for antibody to undergo photoresist processing without loss of binding activity. Challenge with enzyme linked antigen of oxygen plasma exposed antibody layers demonstrated that plasma treatment completely neutralized antibody capture ability. Ellipsometry measurements of oxygen plasma exposed antibody layers indicated complete removal of immobilized antibodies. Fluorescent imaging demonstrated smallest line widths of 2-3 microns.     

A new method for cytodestruction of bladder epithelium using protamine sulfate and urea

Bladder epithelium relies primarily on the presence of a surface glycosaminoglycan (GAG) layer and the structural integrity of cell-cell contact to maintain impermeability to toxic urinary wastes. Previous clinical studies evaluating bladder permeability characteristics in interstitial cystitis patients had indicated that epithelial desquamation occurs after treatment with protamine sulfate (PS) followed by hypertonic urea. The following study was performed using rabbits to further investigate this finding. The urinary bladder was evaluated for optimal treatment conditions for epithelial removal. Protamine sulfate (1 to 10 mg./ml.) and urea (100 to 200 gm./ml.) were instilled into the bladder at volumes ranging from 5 to 60 ml. to that required for near maximum distention. After incubation at room temperature for 15 minutes, the bladders were fixed and evaluated histologically for epithelial removal. The maximum epithelial removal occurred when the bladders were distended, and when PS concentration was 5 to 10 mg./ml. and urea at 200 gm./l. There was greater epithelium removal after repeated treatments. Epithelial cells that were removed were not viable based on Trypan blue staining. There was no significant increase of C14 labeled urea in the plasma after 15 minutes. Rabbits that were followed for 6 weeks after treatment did not show any histological evidence of increased collagen deposition and/or fibrosis. This procedure may have important clinical value since it may remove sufficient bladder epithelium in patients with transitional cell carcinoma to have therapeutic benefit. This offers a realistic option for selective, nontoxic destruction of bladder epithelium.     

Effects of plasma total ammonia content and pH on urea excretion in Nile tilapia

Nile tilapia (Oreochromis niloticus) were infused with ammonium salts, acid, and base to investigate the effects of changes in arterial plasma total ammonia content (Tamm) and pH (pHa) on plasma urea-nitrogen (urea-N) levels and urea-N excretory fluxes (Jurea-N). The tilapia did not possess a functional hepatic ornithine urea-cycle (no significant carbamyl phosphate synthetase III activity). Infused substances were dissolved in a saline vehicle and injected twice (5 mL kg-1), the first infusion to "prime" the animal and promote a more marked response to the second infusion, given 2.5 h later. The results reported are those of the second infusion. Infusion of 200 mM NH4Cl increased Tamm, reduced pHa, and increased plasma urea-N and Jurea-N. Two hundred mM NH4HCO3 increased Tamm and arterial plasma total CO2 content (TaCO2), reduced pHa, and increased Jurea-N. Fifty mM HCl reduced pHa but had no effects on urea dynamics. Fifty mM NaOH increased pHa, plasma urea-N levels, and Jurea-N. Two hundred mM NaHCO3 increased pHa, TaCO2, plasma urea-N levels, and Jurea-N. Infusion of the saline vehicle was without effect. The results indicate that ammonia loading and plasma alkalosis both stimulate urea excretion in uricolytic fish. The responses to hyperammonemia or alkalosis were not modified when combined with elevated plasma bicarbonate levels.     

Assessment of dialysis adequacy using the dialysate urea monitor: preliminary experience of the dialysate urea monitor

Numerous studies have identified a strong linkage between the delivered dialysis dose (Kt/V) and the survival of hemodialysis (HD) patients. However, the current method used to calculate Kt/V requires multiple blood samples and the process is complex and time consuming. We evaluate the performance of a recently developed on-line monitor (Biostat 1000 dialysate urea monitor, Baxter) that measures the urea concentration in the effluent dialysate and displays Kt/V and nPCR immediately after hemodialysis. To verify the performance of the urea monitor, we selected 21 hemodialysis patients, calculated their Kt/V and nPCR values from blood samples obtained during each hemodialysis, and compared the results with data obtained using the urea monitor. The Kt/V and nPCR values calculated by the urea monitor were both significantly correlated with those obtained using blood samples (R = 0.804, p < 0.001 in Kt/V and R = 0.749, p < 0.001 in nPCR). Our results suggest that the urea monitor may be used for on-line assessment of dialysis adequacy and obviates the need for blood sampling.     

Follow-up of fibrin clot growth in blood plasma

The authors propose a device for following up the kinetics of the fibrin clot enlargement in the plasma. Coagulation is induced by contact activation with a fragment of an arterial wall put into the plasma. Besides the initial time of clot formation, the device helps assess the rate of clot growth. Two phases of contact-induced clotting may be distinguished. The first is slow activation of the clotting system and formation of minor amounts of fibrin, the other is rapid growth of the clot, with the linear increment of the clot size being constant during 15 min.     

Determination of plasma volume and total blood volume using indocyanine green: a short review

Rapid blood and plasma volume measurements gain increasing interest in order to avoid unnecessary blood transfusions. Only the indocyanine green method seems to satisfy the demand for a fast, safe and reproducible bedside method. We summarized older and newer experiences with this method, and also summarized the results for practical application.     

Estrogen-progesterone ratio of blood plasma in prolonged pregnancy

The buoyant densities in Cs2SO4 of the double-stranded RNA from the bacteriophage phi 6 and the virus-like particles of Helminthosporium maydis and Penicillium chrysogenum were determined by a Taylor series expansion and by the position and slope of the gradient relative to the isopycnic position (hinge point method). Buoyant densities for the three types of double-stranded RNA calculated by the two methods were, respectively, 1.6089 and 1.6083, 1.6065 and 1.6059 and 1.6057 and 1.6050.     

Micro-methods for serial determinations of elastin metabolism parameters in blood plasma and serum

Micro-methods are described for the serial determinations of elastin metabolism related parameters in human serum or plasma samples: serum elastase activity, elastase inhibitor titers and elastin peptide conc-s. These methods were used in an epidemiological study (the EVA study, " Etude du vieillissement vasculaire") carried out in Nantes, west of France on several thousand individuals. Blood samples obtained from the first-1389 individuals (574 males, 815 females) were used for the determinations. In order to carry out this large number of determinations previously described procedures had to be modified. These methods used for the large serial determinations of the above mentioned three elastin metabolism related serum (or plasma) parameters are described because of their potential interest for serial clinical investigations.     

Susceptibility testing of Cryptococcus neoformans using the urea broth microdilution method

An urea broth microdilution method to assay the susceptibility of Cryptococcus neoformans to antifungal drugs was newly developed. Using this method, urease activity of the fungus was measured instead of the viability by checking colony development. The urease activities were indicated by colour changes in optical density at 545 nm. The end point in this assay was considered as 99% inhibitory concentration. When we measured antifungal activities of the three drugs against 16 isolates of Cr. neoformans using this assay method, mean minimum-inhibitory concentrations (MICs) of fluconazole, itraconazole and terbinafine were 2.0 micrograms ml-1, 0.008 microgram ml-1 and 0.25 microgram ml-1 respectively. This assay method resulted in higher sensitivity in MICs of the three antifungal drugs than the broth microdilution method recommended by the Committee for Laboratory Standards of the Japanese Society for Medical Mycology. The results obtained using this assay method support the more effective evaluation of antifungal substances in susceptibility testing of Cr. neoformans.     

Binding of apoB-containing lipoproteins from unfractionated human blood sera to immobilized LDL receptor

Binding of apoB-containing lipoproteins from unfractionated human blood sera to the immobilized bovine receptor of low density lipoproteins (LDL receptor) was studied. Peroxidase-labeled anti-human apoB antibodies were used to evaluate the lipoprotein binding. The equilibrium dissociation constant (Kd) of the interaction between apoB-containing lipoproteins from unfractionated human sera from healthy donors and the immobilized LDL receptor varied from 1 to 20 microg apoB/ml. To describe the binding of lipoproteins to the LDL receptor, a parameter of relative binding affinity (RBA) was used. RBA is inversely related to value of Kd and equal to unity for the standard serum. The RBA values for the binding of apoB-containing lipoproteins from unfractionated sera to the immobilized LDL receptor were found to correlate with the RBA values for the binding of isolated VLDL (r = 0.76, p < 0.001) and fail to correlate with the RBA values for the binding of isolated LDL. The RBA values for the binding of apoB-containing lipoproteins from unfractionated sera correlated with the RBA values for the binding of apoE-containing lipoproteins from unfractionated sera (r = 0.92, p < 0.001) and with values of triglyceride concentration in the sera (r = 0.93, p < 0.001). The RBA values for the binding of apoB-containing lipoproteins from sera of patients with FDB whose LDL were unable to bind to the LDL receptor did not significantly differ from the RBA values for the normal sera. However, the removal of VLDL from the normal sera significantly decreased the RBA values for the binding of apoB-containing lipoproteins from unfractionated sera. The results indicate that the different binding of apoB-containing lipoproteins to the immobilized LDL receptor mainly depended on the different binding of VLDL and not of LDL.