Search

Bioelectrochemical membrane-electrodes for O2-sensitive enzymatic flow-through analysis of beta-D-glucose and L-lactate are described. The enzyme-membranes of the biosensors consist of glucose-oxidase or lactate-oxidase molecules cross-linked with glutardialdehyde between two dialysis membranes. The accuracy of the biosensors is demonstrated by electroanalysis of diluted control serum and compared with redox-mediator-free H2O2 detection and photometric methods. Continuous haemoanalysis of uncoagulated blood was carried out, using an intermediate carrier stream with additive systems. Tangential streaming to the miniaturized dialysis chamber with a circular channel minimizes blockage of the pores of the dialysis membrane by erythrocytes, leukocytes or protein. An oxygenator pump for the exchange of gases between the buffered solution of the intermediate carrier and the surrounding atmosphere guarantees a constant oxygen partial pressure within the carrier stream. The pulsations produced by the oxygenator pump are dampened by a miniature pressure balance chamber with an unsignificant dead space volume for protecting the enzyme membrane of the sensor. Glutardialdehyde inhibits growth of microorganisms and any resulting oxygen consumption, so that even in protein-containing measuring solutions enzyme electrodes can be used without interference from microbial contamination. The bioelectrochemical measuring system can therefore also be employed for the electroanalysis of fermentation solutions. For continuous flow-through measurements, it is necessary to change the glucose-oxidase membranes after 100-150 days, and the lactate-oxidase membranes after 3-6 weeks.

New small, light-weight and fast-acting meters for measuring blood glucose have been developed recently. To determine their accuracy and precision we compared Accutrend, Companion 2, Glucometer 3 and One Touch II with the reference glucose oxidase method. For determination of accuracy 150 measurements were performed on two meters of each brand, together with measurement on the Beckman 2 Analyzer, which served as our reference. Capillary blood samples were obtained from patients with type 1 and type 2 diabetes attending our outpatient clinic. All measurements were performed by one experienced technician. Precision in series was determined by 15 measurements of venous EDTA samples. The coefficient of variance was used for statistical analysis. Accuracy was evaluated according to recommendations of the American Diabetes Association and clinically useful criteria such as the error grid analysis. We found that One Touch II performed best overall, followed by Accutrend. Companion 2 and Glucometer 3 showed higher deviations in both accuracy and precision, but nonetheless met the clinical criteria of accuracy and reliability measured by error grid analysis in 87% and 90.5% of measurements, respectively. In conclusion, all four blood glucose meters can safely be used, after proper training, by patients and medical staff for self-measurement of blood glucose.

Three methods of the determination of insulin in serum were optimized in order to determine low concentrations of this analyte reliably (Method I: enzymatic after acidic hydrolysis of inulin; Method II: by anthrone/sulfuric acid reagent; Method III: by cysteine/tryptophan reagent). The interfering reaction of glucose in the methods I and II was avoided by the preincubation of samples with glucose oxidase/catalase reagent. The detection limit of all three methods was below 5 mg/l and the within-run precision amounted to values below 5%. Method I and II gave identical results. In comparison with these two methods, method III resulted in inulin values by about 8% lower, because inulin, depending on the lot used, contained alkali-labile fractions which were destroyed during the sample preparation. Method II is recommended for routine measurements because of its an analytical reliability and its cost-effectiveness.

The immunization of rabbits with a paraoxon-HSA-conjugate resulted in an antibody response with titres of 1:25000 to 1:100000 of antisera dilution and average affinity constants K0 of 10(6) M-1 and heterogeneity indices of 0.8 to 0.9 calculated by means of the Sips equation. The serum cholinesterases and the erythrocyte acetylcholinesterase in immunized rabbits were protected against a stronger inhibition by parenteral application of paraoxon. An increase of blood glucose after paraoxon application to immunized rabbits could not be observed but was detectable in unimmunized animals.

A flow-through glucose-analyser is described based on an oxygen-sensitive enzymatic system. Leak-current-proof welded PTFE-membranes contribute considerably to an easy maintenance of the O2-sensors. Soluble or immobilized glucose oxidase may be used in automatic analysers. As a primary instrument of analysis, the oxygen pO2-electrode lends itself to the measurement of other enzymatic reactions that involve oxygen.

The kinetics of binding the enzymes glucose oxidase, trypsin, and peroxidase and also bovine gamma-globulin to the macroporous carriers dialdehyde cellulose, diazotized amino polystyrene, acrolein-acrylamide copolymer, and isothiocyanate groups carrying CPG-glass was studied. It was found empirically that the increase in protein content and--if there occurred no inactivation of enzyme during binding reaction--of enzymatic activity of the enzyme-carrier complex with the reaction time can be described by saturation curves (first order hyperbolas), which represent straight lines after suitable transformation. Thereby it is possible to calculate protein content or specific activity of the enzyme-carrier complex for any moment during binding reaction. From intercepts of the obtained straight lines with the ordinate and with the abscissa, respectively, one can determine the maximum binding capacity and the maximum specific activity and from their slope the reaction rate can be obtained. If there result no straight lines for plotting enzymatic activity of the enzyme-carrier complex against binding time after transformation, then this is an indication for partial inactivation of the free or already insolubilized enzyme. From kinetic measurements it is concluded that in all cases studied by use the preceding stage in chemical fixation of the enzyme to carrier is an adsorption equilibrium. Limitation of reaction rate by diffusion as in loading an ion exchanger could not be observed.

At least three gluconic acid forming enzymes were identified in cell-free extracts of Aspergillus niger: glucose oxidase (EC 1.1.3.4), a glucose dehydrogenase (EC 1.1.99.10), and an enzyme or a mixture of enzymes catalyzing the cleavage of 6-phosphogluconate into gluconate and inorganic phosphate. 2,6-dichlorphenolindophenol was one of the hydrogen acceptors in vitro of the glucose dehydrogenase. Some properties of this enzyme (Km values, pH-dependence, substrate and hydrogen acceptor specificity), as determined in cell-free extracts, were found to be in good agreement with properties described in literature for a glucose dehydrogenase which has been purified from Aspergillus oryzae. The formation of Pi from 6-phosphogluconate and other phosphate esters was found to have an optimum between pH 7 and 8 , and another below pH 4. This suggests that it is catalyzed by an alkaline and an acid phosphomonoesterase (EC 3.1.3.1, 3.1.3.2), both enzymes exhibiting only low substrate specificity. The influence of extraction and assay buffers on the activity of gluconate forming enzymes was investigated. Loss of activity during preparation of cell-free extracts, as calculated from loss of activity storage of cell-free extracts at 4 degrees C, was found to be lower than 4%. Purified glucose oxidase added before homogenization was found in the extract almost quantitatively.

Glucose oxidase from Penicillium notatum was immobilized by covalent, adsorptive or ion exchange attachment to insoluble carriers. The yields of immobilization using Spherons, DEAE-Sephadex, DEAE-cellulose and porous glass carriers are compared. Methods used for the estimation of kinetic parameters Km and kcat are described and results obtained for GOD in solution and in immobilized form are given. Investigations about the dependence of the enzymatic activity on pH, temperature and storage serve for the further characterization of the GOD-derivatives. There are no significant changes in the functional behaviour of the enzyme due to immobilization on selected carriers. The results are discussed with regard to application of carrier bound GOD in enzyme reactors.

The subcutaneous glucose concentration was continuously monitored in the neck of healthy unanaesthetized dogs employing a miniaturized glucose oxidase/H2O2 sensor. Under steady state conditions it amounted to approximately 50% of the circulating level. Induced alterations (oral or intravenous glucose loads) were mirrored with a delay, which was mainly determined by the sensor characteristics. Minor alterations in circulating glucose concentration (e.g. from 4.5 up to 7.5 mmol/l within 60 min) as induced by the oral load do not alter the concentration ratio between interstitial glucose and glycoemia.

The construction and operation of a measuring device containing an enzyme electrode is described. Glucose oxidase is enclosed in an acrylamide film by means of photopolymerization. The maximal increase of the anodic current of the H2O2 being formed in the enzyme reaction serves as measuring signal. The device gives for diluted serum and blood correlation coefficients above 0,99 according to the methods given in the German Pharmacopeia 7. The measuring time for each specimen is roughly 90 s, the serial variation coefficient 2%.

Both, the differing definitions of glucoamylase activity and the different determination of the fragment, glucose, preclude a comparison of literature data. Specific enzymatic determination of the fragment, glucose, with an enzyme electrode is proposed. The contribution describes the manufacture of an enzyme electrode from a commercial pO2-electrode and immobilized GOD. The measurement may be carried out by kinetic determination of utilized oxygen or oxygen peroxide formed. The continuous measurement proposed for serial experiments is made via a calibration curve, whose regression coefficient was determined to be 0.999. Depending on the enzyme activity, the reaction time is between 15 s and 3 min.

Earlier studies showed that an antibacterial lactoperoxydase system in the saliva is activated by the application of a hydrogen paroxide-generating combination of enzymes: amyloglucosidase and glucose oxydase. The object of the present study was to investigate the effect of these enzymes in a test and control group of 29 subjects. The results showed the plaque-inhibiting effect of the enzyme paste. An influence on the gingiva could not be demonstrated.

Iron and copper ions, in a concentration greater than 10(-4) M, inhibit the indicator reaction of a glucose oxidase-peroxidase reagent for the enzymatic determination of glucose, when weakly complexing buffers or buffer-free reaction media are used. The addition of EDTA and other complexing agents or, time-dependently, the buffer ions themselves reverse the inhibition to a great extent. The discussed mechanism of inhibition is based on the assumption that the metal ions share in the re-oxidation of the co-enzyme of glucose oxidase.

The physiological effects attributed to honey are described and discussed. These effects are to a great extent generalizations from experimental and clinical observations. In some cases the effects attributed to honey are based on tests which are no longer acceptable. Most of the statements about physiological effects caused by eating honey cannot be supported by scientific methods. Under German food law these are designated as "misleading". Honey is comparable to sugar in its nutritive value. Other nutrients like protein, minerals and vitamins are present in such small amounts that they have no nutritional importance.

A reaction kinetic method is described for the determination of glucose in biological fluids. Glucose is oxidized to gluconic acid and NAD is reduced to NADH in the presence of glucose dehydrogenase. The rate of formation of NADH is followed in a LKB Reaction-Rate-Meter. Together with the analogue read-out, the rate of reaction is determined with a newly developed kinetic calculator. The specifications of the calculator are given. The new method has the advantage of a high rate of throughput and good reproducibility.

Results obtained with the commercial test-set "GOD-UV" (Dr. Haury, München) are reported. This method permits the determination of serum-glucose within 3 minutes. The procedure is satisfactory in normal and hyperglycaemic patients. In the hypoglycaemic state, however, there is a danger of misinterpretation. The glucose oxidase-UV is compared with the hexokinase reaction.