Bioelectronic tongue for the simultaneous determination of urea, creatinine and alkaline ions in clinical samples

Urea and creatinine biosensors based on urease and creatinine deiminase, respectively, covalently immobilized onto ammonium selective electrodes, were included in an array together with sensors sensitive to ammonium, potassium and sodium. Generic sensors to alkaline ions were also included. All the sensors used were of all-solid-state type, employing polymeric membranes and having rather nonspecific response characteristics. A response model based on artificial neural networks was built and tested for the simultaneous determination of urea, creatinine, ammonium, potassium and sodium. The results show that it is possible to obtain a good multivariate calibration model. In this way, the developed bioelectronic tongue was successfully applied to multidetermination of the five species in raw and spiked urine samples. Predicted concentrations showed a good agreement with reference methods of analysis, allowing a simple direct method for determining urea and creatinine in real samples. At the same time, this method permitted to obtain the concentrations of the alkaline interferences (endogenous ammonium, potassium and sodium) without the need of eliminating them.     

A chemiluminescence automatic analyser for the measurement of biological compounds

A compact automated analyser which could analyse constituents in biological fluids with a small sample volume and in a short time has been developed. The instrument was composed of a flow injection analysis system equipped with chemiluminometric detection and an immobilized enzyme column reactor used in combination. Chemiluminescence has high sensitivity, and its reaction proceeds very quickly. Furthermore, an immobilized enzyme column reactor can produce a sufficient amount of hydrogen peroxide from compounds in serum in a short time. When enzymes are used as reagents for the analysis of substances in blood or blood serum, the final signals emitted by different enzyme reactions are usually not only hydrogen peroxide but also ammonia, NAD(P)H and so on. However, the practical chemiluminescence method for ammonia and NAD(P)H has not been established. We have discovered a new practical method for ammonia and NAD(P)H using an enzyme column reactor consisting of both immobilized L -glutamate dehydrogenase and L -glutamate oxidase. The determinations of glucose and uric acid in serum by chemiluminometry after production of hydrogen peroxide by the respective oxidases are presented. A newly chemiluminometric determination of ammonia, NAD(P)H and its applications to other enzymatic analyses that give ammonia and NAD(P)H as a final signal are also described.     

Creatinine metabolism in Cryptococcus neoformans and Cryptococcus bacillisporus.

The pathogenic species of Cryptococcus, C. neoformans and C. bacillisporus, utilized creatinine as a source of nitrogen but not of carbon. Chromatographic and autoradiographic studies suggest that creatinine metabolism in both species involves a single step resulting in the production of methylhydantoin and ammonia. The enzyme responsible for this step, creatinine deiminase, was produced by the cells only in the presence of creatinine in both species. The synthesis of creatinine deiminase was repressed by ammonia in C. neoformans, but not in C. bacillisporus. A possible explanation for this variation, based on the ecological differences between the two species, is discussed. A novel method for measuring creatinine deiminase activity is also described.     

Determination of inorganic phosphorus using immobilized pyruvate oxidase

A new method for the automated analysis of inorganic phosphorus using immobilized enzyme was established. The method was based on the determination of hydrogen peroxide formed by the action of pyruvate oxidase on inorganic phosphate and pyruvate. Since pyruvate oxidase required inorganic phosphate for its stability and therefore had to be kept in a buffer containing inorganic phosphate, it could hardly be used as a reagent in the form of aqueous solution for the determination of inorganic phosphorus. This difficulty was overcome by using immobilized pyruvate oxidase in column form. When the present method was applied to the determination of inorganic phosphorus in serum, it gave perfect linearity of the data up to 0.20 g inorganic phosphorus/L with satisfactory precision, reproducibility, high sensitivity, and accurate recoveries. The immobilized enzyme reactor unit showed enhanced heat stability and good operational stability for a one-month period, during which time it was used over 900 times for analyses. The enzyme column was not affected by organic phosphorus compounds. The results correlated satisfactorily with those obtained by another well-established method.     

Determinations of lactate and lactate dehydrogenase activity in serum with the flow injection analysis system involving immobilized enzyme column and chemiluminescence

The methods for the highly sensitive flow injection analysis of lactate and lactate dehydrogenase (LDH) activity in serum using immobilized enzymes in column form and chemiluminescence detection which does not require a blank correction are described. The methods were based on the determination of chemiluminescence formed by the reaction of a luminol-ferricyanide mixture with hydrogen peroxide. This hydrogen peroxide was produced by the lactate oxidase (LOD) reaction from lactate, which was in serum or was produced by the action of LDH in serum. The action of LDH in a flow injection analysis system was performed for 2 min in an incubation coil placed parallel to the substrate-buffer line between the LOD column and the LOD/catalase column. Endogenous lactate in serum was removed by an immobilized LOD/catalase column prior to the action of LDH. The present method gave perfect linearity of the data up to 5.6 mmol/liter for lactate and 1840 IU/liter for LDH activity with satisfactory precision, reproducibility, and accurate reaction recoveries. The results from the lactate and LDH activity correlated satisfactorily with those obtained by other well-established methods.     

Application of creatinine-sensitive biosensor for hemodialysis control

The highly sensitive and selective potentiometric biosensor for creatinine determination has been developed by us earlier. In it, pH-sensitive field effect transistors were used as transducer and immobilized creatinine deiminase (EC 3.5.4.21)--as a biosensitive element. In the work presented, we optimized this biosensor for creatinine analysis in real samples of dialysate in patients with renal failure. The optimized version of biosensor was applied for on-line monitoring of the level of creatinine in the patient's dialysate fluid in the course of dialysis session. High correlation between the biosensor analysis and traditional Jaffe method was demonstrated.     

Properties of Crystalline Creatinine Deiminase from Corynebacterium lilium

The properties of creatinine deiminase (EC 3.5.4.21) were characterized with a crystalline preparation from Corynebacterium lilium ATCC 15990. The molecular weight was determined to be 195,000 by the sedimentation equilibrium method, and the isoelectric point was found to be 4.2 by isoelectric focusing. The enzyme was relatively thermostable and had a broad pH optimum of 7.5 to 10.0. It was specific for creatinine and showed a Km value of 1.27 mm. A compound from creatinine was isolated, with the release of ammonia, and identified as N-methylhydantoin. The enzyme activity was inhibited by heavy metal ions and p-chloromercuribenzoate. The enzyme may be useful in determinations of serum and urinary creatinine.     

Analysis of creatinine in mouse and rat serum by ion exchange high performance liquid chromatography for in vivo studies of renal function

An ion exchange high performance liquid chromatography method was developed for determining creatinine levels in both mouse and rat serum samples. Separation of creatinine from other serum components was achieved in 10 min using a 100 x 4.1-mm, 10 microm strong cation exchange column following acetonitrile precipitation of serum proteins. Incorporation of a guard cartridge placed in-line prior to the analytical column was employed to prevent interference from compounds used in renal disease animal trials. Creatinine levels in normal and diseased animals were accurately determined in the 0.01-10 mg/dL range, and average recovery of the method was approximately 85% for both mouse and rat serum. Addition of 0.5-1.0% acetic acid to the acetonitrile used for protein precipitation significantly improved creatinine recovery to above 97% in mouse serum. The method was used for routine preclinical diagnosis of rat and mouse model renal function, and for the evaluation of renal disease treatment efficacy.     

Enzyme kinetic assays with surface plasmon resonance (BIAcore) based on competition between enzyme and creatinine antibody

A procedure is described which allows the characterization of enzyme by a hybrid approach using an enzyme and an antibody. The presented method is related to the affinity determination of antibodies by the 'affinity in solution' procedure for BlAcore. The antibody is used as an indicator for the concentration of substrate, which is also the antigen. A mixture of enzyme, substrate and antibody is incubated, and an aliquot of this solution is injected periodically into a flowcell containing immobilized substrate, which is bound by the antibody, but not cleaved by the enzyme. The chosen initial concentration of substrate inhibits the binding of antibody to the immobilized substrate by 90%. During the enzymatic reaction, increased amounts of antibody bind to the surface, as the substrate concentration is decreased. With this method, the cleavage of creatinine with creatinine iminohydrolase (6 mU/ml) was monitored for up to 11 h. A recently developed monoclonal antibody against creatinine was used as the indicating protein. For the calculation of enzyme activity, the signals were compared with a calibration curve for inhibition of antibody binding to the chip by creatinine in solution.     

Affinity chromatography of peptidylarginine deiminase from rabbit skeletal muscle on a column of soybean trypsin inhibitor (Kunitz)-Sepharose

We designed a simple procedure for the purification of peptidylarginine deiminase, which catalyzes the deimination of arginyl residues in protein, from rabbit skeletal muscle using substrate affinity chromatography. Of the immobilized substrate ligands tested, i.e. protamine and soybean trypsin inhibitor (Kunitz) (STI), STI-Sepharose was found to be an effective affinity adsorbent for purification of the enzyme. The specific binding of peptidylarginine deiminase to STI-Sepharose was observed in the presence of calcium ion, and the enzyme could be selectively eluted from the affinity adsorbent by washing with chelator. A 1,800-fold purification with a 50% yield was achieved in the three-step procedure, which involved DEAE-Sephacel ion-exchange and STI-Sepharose affinity chromatography. The purified enzyme was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activity and the recovery were considerably higher than have been obtained by any procedures previously reported. The specific interaction of peptidylarginine deiminase with STI immobilized on Sepharose was also investigated quantitatively by frontal affinity chromatography. In this method, a peptidylarginine deiminase solution was applied continuously to an STI-Sepharose column and the retardation of the elution front was measured as a parameter of the strength of the interaction. The dissociation constant for the enzyme with STI was found to be 2.3 X 10(-7)M. This value was in good agreement with that obtained by kinetic analysis in our previous studies. Peptidylarginine deiminase required millimolar Ca2+ for the binding to STI-Sepharose. The Ca2+ dependence of the enzyme binding was quite similar to that of the enzymatic activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of creatinine in whole blood, plasma, and urine by high-performance liquid chromatography

A sensitive method for the specific determination of creatinine in whole blood, plasma, and urine with high precision and accuracy is described. Samples were deproteinized by addition of acetonitrile and analyzed by high-performance liquid chromatography using a cation-exchange column with a mobile phase of 9% acetonitrile in 40 mM ammonium phosphate (pH 4.0). The recoveries of creatinine added to blood and plasma were almost complete, ranging from 99 to 101%. The coefficients of variation were very small, 1.6% for blood and plasma and 1.5% for urine. Samples can be assayed in 11-min intervals subsequent to the initial injection. As little as 2 microliter of blood or plasma or 0.02 microliter of urine is sufficient for chromatographic analysis. The present method was successfully used for the accurate measurement of small arterial-venous differences of creatinine concentrations in blood across body organs and showed that in the sheep creatinine is produced in the muscles and is excreted by the kidneys. The method is also suitable for routine analysis of creatinine in clinical laboratories.     

Calibrationless determination of creatinine and ammonia by coulometric flow titration

A precise and sensitive working microflow titration procedure was developed to determine creatinine and ammonia in urine samples. This procedure is based on enzymatic conversion of creatinine, gas diffusional membrane separation of the released ammonia into an acid acceptor stream, and coulometric titration of ammonia with hypobromite. The hypobromite is formed after the electrogeneration of bromine in an electrolyte containing 1.0 M NaBr and 0.1 M sodium borate adjusted to pH 8.5. The electrolysis current follows a triangle-programmed current-time course. An amperometric flow detector records the resulting mirror symmetrical titration curves, which show two equivalence points. The analyte concentration is calculated from the time difference between the equivalence points. For quantitative conversion of creatinine and quantitative separation of present and released ammonia no calibration is necessary to get accurate results. Both ammonia/ammonium and creatinine were determined in the range between 2 microM and 2 mM with relative standard deviations between 3.0 and 1.0% (n = 5). High recoveries were obtained for the analysis of diluted urine samples for both creatinine and ammonia.     

Fiber entrapment of naringinase from Penicillium sp. and application to fruit juice debittering

Naringinase from Penicillium sp. was immobilized on cellulose triacetate by the fiber entrapment method. Although the optimum pH (3.7) and optimum temperature (55°C) of the fiber-entrapped enzyme were similar to those of the native form, the immobilized enzyme had better heat stability. Kinetic studies showed that the immobilized enzyme had higher K_m values than its native form. When this immobilized naringinase was successively used in a column reactor for the hydrolysis of ρ-nitrophenyl-α-l-rhamnoside or naringin in a simulated fruit juice system or grapefruit juice, the enzyme column could be operated with satisfactory stability. In addition, when the natural grapefruit juice was recycled through the column reactor, no column blocking or filtering action of the catalyst bed was observed.     

Cytosine deaminase that hydrolyzes creatinine to N-methylhydantoin in various cytosine deaminase-forming microorganisms

Creatinine deimination has been newly detected in the following various cytosine deaminase-forming microorganisms: Escherichia coli, Proteus mirabilis, Pseudomonas aureofaciens, Pseudomonas chlororaphis and Pseudomonas cruciviae. All these microorganisms, except for E. coli, formed cytosine deaminase in a constitutive or repressive way. P. putida 77 and E. coli showed highly increased formation of creatinine deiminase in the presence of creatinine and cytosine. Throughout serial DEAE-Sephacel and Sephacryl S-300 column chromatographies, the cytosine deaminases of these microorganisms, except for that of P. ovalis, were found to hydrolyze both creatinine and cytosine at comparable rates. No concrete evidence was obtained for the presence of any other protein that hydrolyzed creatine and/or cytosine than the cytosine deaminases in the three test microorganisms randomly selected for investigation.Different from P. putida 77, none of the test microorganisms degraded N-methylhydantoin; neither N-methylhydantoin amidohydrolase nor N-carbamoylsarcosine amidohydrolase was formed in the presence of creatinine in these microorganisms. As a result, the wide occurrence of cytosine deaminases in microorganisms was found to be related to the wide distribution of those microorganisms which hydrolyze creatinine to N-methylhydantoin without further degradation.     

Certified reference materials (GBW09170 and 09171) of creatinine in human serum

Creatinine is the most widely used clinical marker for assessing renal function. Concentrations of creatinine in human serum need to be carefully checked in order to ensure accurate diagnosis of renal function. Therefore, development of certified reference materials (CRMs) of creatinine in serum is of increasing importance. In this study, two new CRMs (Nos. GBW09170 and 09171) for creatinine in human serum have been developed. They were prepared with mixtures of several dozens of healthy people's and kidney disease patient's serum, respectively. The certified values of 8.10, 34.1 mg/kg for these two CRMs have been assigned by liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) method which was validated by using standard reference material (SRM) of SRM909b (a reference material obtained from National Institute of Standards and Technology, NIST). The expanded uncertainties of certified values for low and high concentrations were estimated to be 1.2 and 1.1%, respectively. The certified values were further confirmed by an international intercomparison for the determination of creatinine in human serum (Consultative Committee for Amount of Substance, CCQM) of K80 (CCQM-K80). These new CRMs of creatinine in human serum pool are totally native without additional creatinine spiked for enrichment. These new CRMs are capable of validating routine clinical methods for ensuring accuracy, reliability and comparability of analytical results from different clinical laboratories. They can also be used for instrument validation, development of secondary reference materials, and evaluating the accuracy of high order clinical methods for the determination of creatinine in human serum.     

Evaluation of two alternative metabolic pathways for creatinine degradation in microorganisms

The microbial decomposition of creatinine was found to proceed mainly via N-methylhydantoin or creatine as the first degradation product. Either N-methylhydantoin or urea or both were detected as metabolites derived from creatinine in various microorganisms, and creatinine deiminase and creatinine amidohydrolase activities were detected concomitantly. N-Methylhydantoin hydrolase and N-carbamoylsarcosine amidohydrolase were found to be formed inducibly in the presence of creatinine or N-methylhydantoin. Three microorganisms which decompose creatinine in different ways were screened from soil. Pseudomonas putida 77 rapidly metabolized creatinine solely via N-methylhydantoin. Degradation of creatinine proceeded with both creatine and N-methylhydantoin as the first degradation products at the same time in Pseudomonas sp. H21. Pseudomonas sp. 0114 was found to metabolize creatinine mainly via creatine and to also metabolize N-methylhydantoin. Changes in the metabolites of creatinine during a cultivation or enzyme reaction were found to be closely related to the enzyme activities of interest which are regulated by creatinine or N-methylhydantoin in different ways depending on the microbial strain.     

Picric acid methods greatly overestimate serum creatinine in mice: more accurate results with high-performance liquid chromatography

The creatinine levels of blood and urine from humans, rats, and mice were measured by high-performance liquid chromatography. These were compared to the alkaline picrate analysis of creatinine performed by standard colorimetric, kinetic, and AutoAnalyzer techniques. For human serum and urine the values obtained using the HPLC technique gave good agreement with four out of five alkaline picrate techniques. For black or white mice, the serum creatinine concentration was 8.7 +/- 0.4 microM by HPLC but 44.9 +/- 1.9 microM by the lowest alkaline picrate method. Mouse urine creatinine concentrations were 3.24 +/- 0.19 mM by HPLC and 4.59 +/- 0.39 mM by the nearest alkaline picrate method. Rat serum creatinine concentrations analyzed by HPLC were about half the values obtained by AutoAnalyzer. Mouse and rat samples seemed to have substances which gave nonspecific color and thus interfered with the analysis of creatinine by the alkaline picrate methods. While the alkaline picrate analysis of creatinine was adequate for human samples, it was necessary to use HPLC to accurately measure rodent creatinine. The fractional excretion of creatinine was determined by measuring creatinine in mouse urine and plasma by both the kinetic and HPLC methods and comparing these values to urine and plasma inulin. Using the kinetic method, creatinine was cleared at 43 +/- 3% of the rate of inulin. Using the HPLC method, creatinine was cleared at 170 +/- 11% of the rate of inulin.     

Use of a bioreactor consisting of sequentially aligned L-glutamate dehydrogenase and L-glutamate oxidase for the determination of ammonia by chemiluminescence

A chemiluminometric method for the automated flow injection analysis of ammonia is described. The essence of the invention is the use of a bioreactor consisting of both immobilized L-glutamate dehydrogenase (GLDH) and L-glutamate oxidase (GLXD), which are sequentially aligned in this order in a minicolumn measuring 2.0 X 20 mm. The unidirectional constant flow of liquid through the column reactor minimizes the reversed diffusion of the solutes so that the following sequence of reactions is ensured. Thus, ammonia to be determined is first transformed by GLDH into L-glutamate, which then produces hydrogen peroxide by GLXD. Hydrogen peroxide in the effluent from the column is then determined by its chemiluminescence upon admixing with luminol and potassium ferricyanide. The present method gives linearity of the standard curve for ammonia up to 1.0 mM. It is at least 100 times more sensitive than the conventional method for ammonia assay using ultraviolet absorption measurement.     

Immobilization of enzymes on Spheron: 2. β-Amylase — The development of a column reactor—separator

The titanium-chelation method has been used to immobilize β-amylase (1,4-α-d-glucan maltohydrolase, EC 3.2.1.2) on to Spheron. On various grades of Spheron, protein coupling yields of 56–76% were obtained with barley and sweet-potato β-amylases. The specific enzymic activities of the immobilized enzymes fell in the range 3.7–7.6% of those of the soluble enzymes. The immobilized enzymes were more stable than the soluble, especially in the presence of l-cysteine and serum albumin. The presence of cysteine and serum albumin brought about increases in activity in the preparations, presumably by regenerating essential thiol groups in the enzyme which had been oxidized during the operations. Maltose could be separated from amylopectin and other large polysaccharides by chromatography on Spheron P100, and a system was developed in which maltose, produced by hydrolysis of amylopectin applied in pulses to a column of immobilized β-amylase, was separated from starting material and by-products on a second column of Spheron P100.     

Automated determination of orotic acid, uracil and pseudouridine in urine by high-performance liquid chromatography with column switching

A column-switching high-performance liquid chromatographic method, requiring no sample preparation apart from filtration, is described for quantification of urinary orotic acid, uracil and pseudouridine. The analyses were carried out using a reversed-phase octadecylsilane-bonded column for sample clean-up and a cation-exchange column for separation; 5–20 ]sml samples of urine were directly analysed, and more than 100 samples could be analysed consecutively. Each sample required only 30 min. Detection limits of these compounds were 5 pmol. Creatinine-related urinary uracil excretion was lowest in the newborn period (17.3 ± 14.4 μmol/g of creatinine). A patient with partial ornithine transcarbamylase deficiency and his mother usually excreted a high level of uracil during the period of normal orotic acid excretion and normal serum ammonia level.