Conformational fingerprinting of angiotensin-converting enzyme in the blood in health and disease

The binding pattern of the set of 16 monoclonal antibodies to the different epitopes on the surface of two domains of angiotensin-converting enzyme, i.e., conformational fingerprinting, allows to reveal in blood the presence of an enzyme that is not produced by lung endothelial cells, but rather by other cells, e.g., Gaucher’s cells and sarcoid granuloma cells. The existence of angiotensin-converting enzyme with changed conformation in the blood of patients with uremia has been shown; this enzyme was characterized by enhanced activity towards angiotensin I and decreased ability to be inhibited by specific inhibitors. The prospects of discovering conformationally changed enzyme in blood of patients with atrial fibrillation have also been discussed.     

Determination of Glucose in Diluted Blood with a Thermal Flow Injection Analysis Biosensor

Abstract

The glucose concentration in diluted whole blood has been measured, using a miniaturized thermal biosensor based on the enzyme thermistor principle. The biosensor is a small flow injection system. A sample volume of 20μl is injected into a flow of 50μl/min. The heat produced when the sample passes the enzyme column is measured with a thermistor connected to a Wheatstone bridge. The enzyme column contains glucose oxidase and catalase co-immobilized on a solid support material. Samples of whole blood usually cause problems in flow-systems. The blood cells tend to block the enzyme column and the back pressure increases. We have tested a superporous agarose material as enzyme support material using tenfold diluted samples of whole human blood. The blood was collected from the finger-tip and diluted with buffer containing an anticoagulant and sodium fluoride. The number of samples possible to inject and the accuracy compared to the Boehringer Mannheim Reflolux have been determined. At least 100 ten-fold diluted blood samples could be injected on a micro-column of superporous agarose. The obtained glucose concentration correlated well with the one obtained with the reference instrument.     

Extraction of free phenols from blood by a liquid membrane enzyme reactor

The development of a liquid membrane enzyme reactor for the extraction of phenols from blood and plasma is described. Phenol permeation across the liquid membrane is studied, the transport of the toxins is linked with an enzymatic reaction. The encapsulation of the enzyme is described in detail. The stability of the multi-emulsion as well as the loss of enzyme activity during encapsulation and treatment axe examined thoroughly. The resulting liquid surfactant membrane emulsion is applied in in-vitro experiments to remove phenols from blood and plasma.     

Distribution model for the intact urokinase and urokinases modified by soluble macromolecules in rat and mouse bodies

The activity of intact urokinase (UK) and urokinases modified by soluble macromolecules (dextran and dextran sulfate sodium) in a mouse body was traced after injection of the 125I-labelled enzymes. The residual fraction of the enzyme in blood can be correlated with the time (t) as follows; Xb = Ae-at + Be-bt + Ce-ct Since a greater than b greater than c, the residual fraction of the enzyme in blood chiefly depends on the magnitude of parameter C. The constant C for modified urokinase was larger than that for urokinase, showing the relative residual in blood was increased by modification of the enzyme. The apparent utilization in 50 min was 13.8% for intact UK, 28.1% for the dextran-UK and 25.2% for the sulfate dextran-UK. Therefore, the apparent utilization of UK in blood was approximately doubled by the modification. Since the half-lives of UK and modified UK in kidney and liver were not long, there were no unacceptable accumulation of the enzymes.     

Single-Molecule Analysis Determines Isozymes of Human Alkaline Phosphatase in Serum

Alkaline phosphatase (ALP) is an important biomarker, as high levels of ALP in blood can indicate liver disease or bone disorders. However, current clinical blood tests only measure the total concentration of ALP but are unable to distinguish enzyme isotypes. Here, we demonstrate a novel and rapid approach to profile various ALP isozymes in blood via a single-molecule-analysis platform. The microarray platform provides enzyme kinetics of hundreds of individual molecules at high throughput. Using these single molecule kinetics, we characterize the different activity profiles of ALP isotypes. By analyzing both healthy and disease samples, we found the single molecule activity distribution of ALP in serum reflects the health status of patients. This result demonstrates the potential utility of the method for improving the conventional ALP test, as well as for analyzing other enzymatic biomarkers, including enzyme isotypes.     

Single‐Molecule Analysis Determines Isozymes of Human Alkaline Phosphatase in Serum

Alkaline phosphatase (ALP) is an important biomarker, as high levels of ALP in blood can indicate liver disease or bone disorders. However, current clinical blood tests only measure the total concentration of ALP but are unable to distinguish enzyme isotypes. Here, we demonstrate a novel and rapid approach to profile various ALP isozymes in blood via a single‐molecule‐analysis platform. The microarray platform provides enzyme kinetics of hundreds of individual molecules at high throughput. Using these single molecule kinetics, we characterize the different activity profiles of ALP isotypes. By analyzing both healthy and disease samples, we found the single molecule activity distribution of ALP in serum reflects the health status of patients. This result demonstrates the potential utility of the method for improving the conventional ALP test, as well as for analyzing other enzymatic biomarkers, including enzyme isotypes.     

An electrophoretic method for the detection of chymotrypsin and trypsin activity directly in whole blood

In biomedical research and clinical diagnostics, it is a major challenge to measure disease‐related degradative enzyme activity directly in whole blood. Present techniques for assaying degradative enzyme activity require sample preparation, which makes the assays time‐consuming and costly. This study now describes a simple and rapid electrophoretic method that allows detection of degradative enzyme activity directly in whole blood using charge‐changing fluorescent peptide substrates. Charge‐changing substrates eliminate the need for sample preparation by producing positively charged cleavage fragments that can be readily separated from the oppositely charged fluorescent substrate and blood components by electrophoresis. Two peptide substrates have been developed for pancreatic α‐chymotrypsin and trypsin. For the first substrate, a detection limit of 3 ng for both α‐chymotrypsin and trypsin was achieved in whole rat blood using a 4% agarose gel. This substrate had minimal cross‐reactivity with the trypsin‐like proteases thrombin, plasmin, and kallikrein. For the second substrate (trypsin‐specific), a detection limit of about 10–20 pg was achieved using thinner higher resolution 20 and 25% polyacrylamide gels. Thus, the new charge changing peptide substrates enable a simple electrophoretic assay format for the measurement of degradative enzyme activity, which is an important step toward the development of novel point‐of‐care diagnostics.     

Polydivinylbenzene/Ethylvinylbenzene Composite Membranes for the Optimization of a Whole Blood Glucose Sensor

A novel ultra thin polydivinylbenzene/ethylvinylbenzene composite membrane has been developed for use as the outer covering barrier in a model amperometric glucose oxidase enzyme electrode. The composite membrane was formed via the cathodic electropolymerization of divinylbenzene/ethylvinylbenzene at the surface of gold sputter coated host alumina membranes, (serving solely as a mechanical support for the thin polymer film). Permeability coefficients were determined for the enzyme substrates, O₂ and glucose, across composite membranes formed with a range of polymer thicknesses. Due to the highly substrate diffusion limiting nature of the composite membrane, it was found that anionic interferents present in blood (such as ascorbate), were effectively screened from the working electrode via a charge exclusion mechanism, in a manner similar to previous findings within our laboratory. The enzyme electrode showed an initial 32% signal drift when first exposed to whole human blood over a period of 2 hours, after which time enzyme electrode responses remained essentially stable. Whole blood patient glucose determinations yielded a correlation coefficient of r²=0.97 in comparison to standard hospital analyses.     

Bacterial homologue of human blood group A transferase

A bacterial version of human blood group A transferase was identified and found to be able to accept five naturally existing H-antigen core structures as good substrates, demonstrating its versatility for synthesis of blood group A antigens. Furthermore, this enzyme was applied in the engineering of bacterial cell surface polysaccharides by remodeling blood group B mimicry into blood group A mimicry.     

A general method for detecting protease activity via gelation and its application to artificial clotting

A modular system for detecting protease activity via enzyme-triggered gel formation is described. Protease-specific recognition sequences are utilized to achieve enzyme specificity. Artificial blood clotting is demonstrated by activating endogenous thrombin to trigger gelation in fibrinogen-deficient blood plasma.     

Utilization of adsorption-immobilized urease in gas-diffusion flow injection

A gas-diffusion flow-injection system for the assay of urea is reported. Urea is enzymatically converted to ammonia, which is determined spectrophotometrically by detecting the change in absorbance of a mixed pH indicator. The enzyme is immobilized by addition of perfluoroalkyl chains to the free amine groups of the enzyme and then adsorption of this modified enzyme on a polytetrafluoroethylene gas-diffusion membrane. The method is applicable in the range 0.1–500 mM urea. The procedure was used in comparison assays using blood serum samples.     

Part I. A specific electrode for urea.

A specific simple enzyme stirrer electrode is described for the assay of urea in blood serum. The enzyme is placed directly on a magnetic stirrer and held in place with a nylon net. The enzyme stirrer both stirs the solution and effects an enzymatic transformation, permitting the direct assay of a substrate such as urea. Potassium, Na⁺ , NH₄⁺ and other organic and inorganic species present in blood do not interfere. Linear curves are obtained from 5· 10^-2M to 1· 10^-4M urea with slopes close to Nernstian, 0.95 pH/decade. Urea in blood was assayed with an accuracy of 1.8% and a precision of 2.0% with immobilized urease in the stirrer. The stirrers were used for 15 weeks and over 500 assays with excellent results.     

Enzyme coated glass pH-electrode: Its fabrication and applications in the determination of urea in blood samples

A new enzyme coated electrode for the determination of urea in blood samples has been developed. It is based on the encapsulation of urease enzyme in the porous silicate matrix by the sol-gel technique on a glass electrode for the purpose of sensing urea in blood samples. Various parameters like the effect of pH, selection of a suitable buffer of appropriate concentration and interference of common substances in blood samples have been evaluated to optimize the conditions for the determination of urea. The electrode can be used for the determination of urea in the concentration range 0.03-30.0 mM in a solution. The detection limit of the present enzyme-coated electrode is found to be 52 μg/ml of urea. The relative standard deviation for the electrode-to-electrode reproducibility is found to be 2.4% for the determination of 0.1 mM of urea (six replicate electrodes). Sol-gel matrix containing immobilized enzyme was stable for about 25 days at ∼4 °C with 80% urease activity. Urea content in various clinical blood samples has been estimated using this electrode and the results are found to be in good agreement with the standard clinical methods as reported in the literature.     

壳聚糖固定化血管紧张素转化酶及其性质

以壳聚糖微球为载体, 戊二醛为交联剂固定化血管紧张素转化酶, 研究了酶固定化的最优条件和固定化酶的性质. 结果表明, 在戊二醛质量分数为2.5%、给酶量为8 mg/mL时, 固定化酶的比活性最大, 为0.085 U/g. 固定化酶在40~50 ℃, pH在7~9之间有最大活性, 其米氏常数Km为2.39 mmol/L. 同时, 固定化酶具有良好的稳定性, 可重复利用.     

Anwendung der Affinitätschromatographie an NAD-Sepharose zum Nachweis und zur Reinigung vonmyo-Inosit-1-phosphat-Synthase in Erythrocyten von Hühnern und inLemna gibba

With the help of affinity chromatography on NAD-Sepharose it was possible to findmyo-inositol-1-phosphate synthase (EC 5.5.1.4) in blood of anaemic chicken and to obtain a relatively active preparation of the enzyme. In the blood of normal chicken, which had no reticulocytes, no activity of this enzyme could be detected. The same method was also successfully applied for the concentration of an enzyme of the same specificity fromLemna gibba (duckweed).

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31. Mitt.:W. Woloszczuk undO. Hoffmann-Ostenhof, Z. physiol. Chem. (im Druck).     

Conducting organic salt amperometric glucose sensor in continuous-flow monitoring using a wall-jet cell

A simplified method for preparing tetrathiafulvalene?7,7,8,8-tetracyanoquinodimethane-based amperometric enzyme electrodes is described. The electrode is shown to be suitable for the routine monitoring of blood glucose levels. Using the wall-jet cell geometry, the steady-state mode gave more consistent results than the flow-injection mode because of variability in the blood matrix from patient to patient. The results show that, for the wall-jet cell geometry, due consideration must be given to the orientation of the enzyme electrode with respect to the jet inlet. High precision (<2% r.s.d.) and accuracy are then feasible.     

Liquid chromatography with electrochemical detection for the determination of choline and acetylcholine in plasma and red blood cells. Failure to detect acetylcholine in blood of humans and mice

An assay is described for the measurement of choline in plasma and red blood cells using liquid chromatography, an enzyme reactor and electrochemical detection after a simple sample pretreatment. The intra-assay coefficient of variation for choline was 6.2 and 3.8% in plasma and in red blood cells, respectively. Using this method we have re-investigated the presence of acetylcholine in blood constituents. We were not able to demonstrate acetylcholine with a limit of detection of 10 pmol per ml of plasma or per ml of red blood cells.     

Detection of Adrenaline in Blood Plasma as Biomarker for Adrenal Venous Sampling

An amperometric bi‐enzyme biosensor based on substrate recycling principle for the amplification of the sensor signal has been developed for the detection of adrenaline in blood. Adrenaline can be used as biomarker verifying successful adrenal venous sampling procedure. The adrenaline biosensor has been realized via modification of a galvanic oxygen sensor with a bi‐enzyme membrane combining a genetically modified laccase and a pyrroloquinoline quinone‐dependent glucose dehydrogenase. The measurement conditions such as pH value and temperature were optimized to enhance the sensor performance. A high sensitivity and a low detection limit of about 0.5–1 nM adrenaline have been achieved in phosphate buffer at pH 7.4, relevant for measurements in blood samples. The sensitivity of the biosensor to other catecholamines such as noradrenaline, dopamine and dobutamine has been studied. Finally, the sensor has been successfully applied for the detection of adrenaline in human blood plasma.     

Some aspects of the angiotensin-converting enzyme for leukemias

Russian Chemical Bulletin - The present article shows the diagnosis of different type leukemias in children by the activity of angiotensin-converting enzyme in blood serum to be not promising....     

Optimization of the Design and Operating Conditions of an Amperometric Biosensor for Glutamate Concentration Measurements in the Blood Plasma

Today, the concentration of glutamate in the patient′s blood is an important indicator in medical diagnostics; therefore, it is necessary to have a simple, accurate, and fast tool to obtain the data. Here, a recently developed amperometric glutamate-sensitive biosensor was optimized to improve its characteristics. The platinum disk electrode was used as a transducer. As a bioselective element we used the enzyme glutamate oxidase, covalently crosslinked with bovine serum albumin by glutaraldehyde. Circumstances of enzyme immobilization on the transducer‘s surface were optimized (enzyme and glutaraldehyde concentrations and immobilization duration). To test the ability of this biosensor to work in biological environments containing complex biological substances, the influence of the working solution was investigated (concentration of the working buffer, its temperature, presence of the protein in the analyzed sample). The linear range of biosensor was from 5 to 600 μM of glutamate and the sensitivity was 150–200 nA/mM. Measurements of glutamate concentrations in the blood plasma were performed by biosensor and glutamate dehydrogenase assay. The linear correlation between the methods was found in a range of 50.4–182.5 μM (R²=0.99). Thus, it has been shown that the developed biosensor makes it possible to measure the concentration of glutamate in blood plasma.