Determination of aliphatic amino acids in serum by HPLC with fluorimetric detection using co-immobilized enzyme reactor

A simple and selective method is presented for the determination of aliphatic amino acids such as L-alanine, L-valine, L-isoleucine and L-leucine in serum using HPLC with detection by co-immobilized alanine dehydrogenase/leucine dehydrogenase post-column reactor and fluorimeter. The enzymes were simultaneously immobilized on chitosan beads. The separation was achieved by means of an ods column with elution with phosphate buffer (pH 7.0). The system gave linear responses over two orders of magnitude and detection limits at 1-2muM levels.     

Biosensing based on NADH detection coupled to electrogenerated chemiluminescence from ruthenium tris(2,2'-bipyridine)

A flow injection analysis system for a reduced form of nicotinamide adenine dinucleotide (NADH) was constructed by the electrogenerated chemiluminescence using ruthenium tris(2,2'-bipyridine) complex, and this technique was applied to the determination of ethanol concentration by using an immobilized alcohol dehydrogenase (ADH). The NADH determination was optimum under the following conditions. The flow rate was 1.5 to 2.0 ml/min, and the applied voltage was 1.6 V. The NADH calibration plot showed the linear correlation in the concentration range 10 to 250muM, and the lower detection limit was 10muM. Ethanol was successfully measured in the concentration range 10 ppm to 5% by using ADH-immobilized glass beads.     

Immobilized aminoacylase on porous glass beads

Aminoacylase was immobilized on porous glass by two different coupling methods. One aminoacylase preparation was covalently bound to an alkylaminosilane derivative of porous glass with glutaraldehyde [alkylamino-porous glass-CVB-aminoacylase]; the other aminoacylase derivative was prepared by covalently binding the enzyme to arylaminosilane glass by diazotization [arylamino-porous glass-CVB-aminoacylase]. The enzyme activities of the immobilized aminoacylases were 3.2-13.0 U/ml glass for the former and 1.9-6.8 U/ml glass for the latter. The alkylamino-porous glass-CVB-aminoacylase showed excellent stability at pH 6-9 and at temperatures below 50°C. The derivative could be stored for more than 6 mo without appreciable loss of the activity. Continuous hydrolysis using the alkylamino-porous glass-CVB-aminoacylase packed in column was carried out for 54 days at 37°C, with a calculated half-life of 78 days. It was determined that alkylamino-porous glass-CVB-aminoacylase would be applicable in an industrial preparation of various l-amino acids from their dl forms.     

Enzymatic determination of glucose in a flow system by catalytic oxidation of the nicotinamide coenzyme at a modified electrode

A chemically modified electrode for detection of dihydronicotinamide adenine dinucleotide (NADH) and dihydronicotinamide adenine dinucleotide phosphate (NADPH) is described. Graphite rods were modified by dipping them into solutions of-dimethylamino-1,2-benzophenoxzinium salt (Meldola blue). The modified electrodes were mounted in a flow-through cell in a flow-injection manifold. Samples (50 μl) of pure nicotinamide coenzymes produced strictly linear calibration graphs from 1 μM to 10 mM with a repeatability of 0.2–0.6% RSD. A packed-bed enzyme reactor (210 μl) containing immobilized glucose dehydrogenase was inserted in the manifold for glucose determinations. Oxidized coenzyme was also added to the carrier electrolyte. Straight calibration graphs were again obtained up to 1mM β-d-glucose. The detection limit was 0.25 μM β-d-glucose for 50-μl samples. The electrode was kept at ?50 to 0 m V vs. SCE which was low enough to avoid interferences from ascorbic acid, uric acid or quinones.     

Dual‐enzyme,co‐immobilized capillary microreactor combined with substrate recycling for high‐sensitive glutamate determination based on CE

A new method for high‐sensitive determination of glutamate was developed and evaluated based on CE by using dual‐enzyme co‐immobilized capillary microreactor combined with substrate recycling. The capillary microreactor was prepared by covalently co‐immobilizing glutamate dehydrogenase (GDH) and glutamic pyruvic transaminase (GPT) on the inner surface of a capillary and was characterized by SEM, ultraviolet‐visible spectroscopy, and fluorescence spectroscopy. The GDH‐GPT co‐immobilized capillary microreactor showed great stability and reproducibility. The apparent K_m for glutamate with GDH‐GPT coupled reaction was determined to be 0.61±0.06 mM but 2.56±0.24 mM when only GDH was immobilized. Glutamate determination was based on on‐column monitoring UV absorption at 340 nm of the reaction product reduced nicotinamide adenine dinucleotide, of which peak area was directly related to the glutamate concentration. The response of the present co‐immobilized GDH‐GPT assay for glutamate is greatly enhanced over single enzyme system, and a 15.7‐fold improvement in sensitivity was obtained. The detection limit of the proposed method is 0.15 μM glutamate (S/N=3). Selectivity for glutamate is good over most of the 20 amino acids. Finally, this method was successfully applied to determine the glutamate content in rat plasma and serum samples.     

Preparation and properties of immobilized pig kidney aminoa cylase and optical resolution of N-acyl-dl-alanine

Aminoacylase (EC 3.5.1.14) was immobilized into DEAE-Sephadex A-25 by ion-exchange absorption for optical resolution of N-acyl-dl-alanine. The effects of pH, temperature, and Co²⁺ concentration on the activity of free and immobilized enzymes were in vestigated along with the operational and the thermal stability of the immobilized enzyme. The immobilized enzyme retained high catalytic activity. The optimum pH and temperature for the hydrolysis of N-acyl-l-alanine in the dl-isomer mixture were 8.0 and 65°C, respectively. Co²⁺ was an activator for the immobilized enzyme in a similarroleas for the free enzyme. Nosignificant loss of activity was observed for at least 300 h of continuous operation. The yield of l-alanine was about 70% of the theoretical yield. The immobilized aminoacylase column decayed over a very long period of operation, but could be completely reactivated by regeneration.     

Enzymic determination of ammonia in food by flow injection

Ammonia in food samples was determined by its reaction in an immobilised enzyme reactor containing glutamate dehydrogenase (GIDH) in a flow injection system, by measuring the decrease in the absorbance of ultraviolet radiation by reduced nicotinamide adenine dinucleotide (NADH). There was a linear relationship (r = 0.9995) between peak height and ammonia concentration over the range 0.05-0.6 mM. The detection limit was 0.005 mM for an injection volume of 19 microliters. Sampling frequency was 60 h-1 and the precision was better than 1.09% for 11 successive assays. The interference effect of urea and ascorbic acid at concentrations greater than 100 mg per 100 g of product should be taken into account. The interference caused by glycine, creatinine and amino acids is negligible. Only a 20% loss in the activity of the GIDH column was observed after 500 determinations during a 3-month period.     

Enzymatic Determination of Ammonia in Lake Water Using a Semi-Automatic Analyser

Abstract

An enzymatic method was developed for the determination of ammonia concentrations in lake water. Lake water samples containing ammonia were mixed with a glutamate dehydrogenase (GIDH), reduced nicotinamide adenine dinucleotide phosphate (NADPH) and 2-oxoglutarate. The decrease in the absorbance intensity caused by the disappearance of NADPH by this reaction was measured at 340 nm. There was a linear relationship (r = 0.9997) between peak height and ammonia concentration over the range 0–29 μM. The detection limit was 0.29 μM for a sample volume of 250 μl. Interference of amino acids and urea at concentrations of 50 mg l^?1 was negligible. Good agreement was found between the enzymatic method and indophenol blue colorimetry.     

Flow-injection determination of 3-hydroxybutyrate in serum with an immobilized 3-hydroxybutyrate dehydrogenase reactor and fluorescence detection

A flow-injection system with an immobilized enzyme reactor is proposed for the determination of 3-hydroxybutyrate. 3-Hydroxybutyrate dehydrogenase is immobilized on aminated poly(vinyl alcohol) beads and packed into a stainless-steel column (4 cm x 4 mm I.D.). Serum is diluted and filtered. Sample solution (20 mul) is injected into the carrier stream [4mM NAD(+) in glycine buffer (pH 9.3)]. The NADH formed is detected at 465 nm (excitation at 340 nm). The calibration graph is linear for 0.7-500muM 3-hydroxybutyrate; the detection limit is 0.5muM.     

High-performance liquid chromatographic detection of L- and D-amino acids by use of immobilized enzyme electrodes as detectors

Two enzyme electrodes based on immobilized L- and D-amino acid oxidases give specific responses to L- and D-amino acids, respectively. They are used as amperometric detectors for high-performance liquid chromatography, by splitting the flow after elution from the column and detecting D-isomers in one line, L-isomers in the other. The detection limit is about 2 pmol for some amino acids (methionine, tyrosine, leucine, and phenylalanine). The procedure is useful for the specific detection of L- and D-amino acids without complicated pretreatment. The electrodes retain most of their original activities after repetitive use for one month.     

Studies on Immobilization of Aminoacylase From Aspergillus Oryzae on Macroporous Carriers

Synthesized macroporous cross-linked copolymers of methyl acrylate-divinyl benzene (MA-DVB), acrylamide-N,N'-methylenebisacrylamide (AAM-BIS) and their functionalized products were used for immobilization of aminoacylase from Aspergillus oryzae. Effects of the carrier properties on the activity of immobilized aminoacylase were investigated and effects of substrate concentration, pH, phosphate buffer concentration and temperature on the immobilized aminoacylase were compared with those of the soluble aminoacylase. A column of immobilized aminoaoylase was prepared and used for continuous resolution of N-acyl-DL-methionine; the operational stability of immobilized enzyme was also investigated.     

Monitoring the preparation of NAD analogs by reversed-phase high-performance liquid chromatography

Summary Pig brain NAD glycohydrolase immobilized on Affi-Gel 10 or nylon 6 was used for the conversion of NAD into 3-acetylpyridine adenine dinucleotide (APAD) or 3-aminopyridine adenine dinucleotide (AAD). A reversed-phase chromatographic system consisting of a C₁₈ Resolve column and phosphate buffer (pH 6.2)-methanol as the mobile phase was used to monitor the production of APAD and AAD.     

Site-specific immobilization of flavin adenine dinucleotide on indium/tin oxide electrodes through flavin adenine amino group

A Mannich-type reaction was used to attach flavin adenine dinucleotide (FAD) covalently to aminosilane derivatized indium/tin oxide-coated glass plates. The aminosilane was activated with formaldehyde to give an intermediate that attached specifically to the adenine amino group of FAD. The presence of the intermediate also was demonstrated by coupling hydroquinone to the formaldehyde activated support. The immobilized FAD and hydroquinone were characterized by cyclic or differential pulse voltammetry. The immobilized FAD was shown to reduce the overpotential for NADH oxidation by 180 mV. In keeping with results for FAD on glassy carbon, FAD attached to indium/tin oxide at the adenine amino group did not lead to reconstitution of activity with apoglucose oxidase. On leave from University of Madras, India.     

Photoelectrochemical reaction of chlorophyll immobilized with liquid crystal on metal surface

Chlorophyll was immobilized with liquid crystal on a platinum surface to prepare a photoexcitable electrode. Liquid crystals such as N-(p-methoxybenzylidene)-p-butylanil-ine were found to be effective on the photoexcitation of the immobilized chlorophyll. Such a chlorophyll-liquid crystal electrode produced photocurrent when it was coupled with a solution of nicotinamide adenine dinucleotide and exposed to light. The electron transfer accompanied by the photoelectrochemical reaction is discussed.     

Hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry method for the simultaneous determination of l‐valine,l‐leucine,l‐isoleucine,l‐phenylalanine,and l‐tyrosine in human serum

l ‐Valine, l ‐leucine, l ‐isoleucine, l ‐phenylalanine, and l ‐tyrosine are important proposed biomarkers for the early detection and diagnosis of type 2 diabetes. A simple and selective hydrophilic interaction chromatography with tandem mass spectrometry method was developed for the simultaneous determination of these amino acids in human serum, using stable isotope‐labeled amino acids as internal standards. Chromatographic separation was carried out on a Syncronis HILIC column (150 mm × 2.1 mm, 5 μm) with the column temperature of 35°C and a mobile phase consisted of acetonitrile/120 mM ammonium acetate (89:11, v/v), and the run time was 11.0 min. The mass spectrometric analysis was performed using a QTRAP 5500 mass spectrometer coupled with an electrospray ionization source in positive ion mode. As these five amino acids are endogenous compounds in serum, we used the corresponding stable isotope‐labeled amino acids to evaluate the matrix effect and recovery in serum. The matrix effect was 98.7–107.3%, and the recovery was 92.7–102.3%. Calibration curves spiked unlabeled amino acids in water were linear over the range of 0.200–100 μg/mL. The accuracy, inter‐, and intraday precision were below 10.2%. Analytes were stable during the study. This assay method has been validated and applied to the early diagnosis research of type 2 diabetes.     

Bioluminescent flow sensor for L-phenylalanine determination in serum

A highly sensitive and rapid bioluminescent flow sensor was developed for the determination of the content of L-phenylalanine (Phe) in serum by monitoring the reduced form of nicotinamide adenine dinucleotide (NADH), produced by immobilized phenylalanine dehydrogenase (PheDH), with bacterial bioluminescent enzymes immobilized on a separate nylon coil. The L-PheDHs extracted from Bacillus badius, Bacillus sphaericus and Rhodococcus sp. M 4 were investigated and the performances of the three immobilized L-PheDH's were analysed. The B. badius reactor was found to give higher transformation rate and better sensitivity; the response was linear from 1 to 100 microM at 25 degrees , with a detection limit of 10 pmoles (0.5 microM). The intra- and inter-assay coefficients of variation were less than 5% and recoveries ranged from 90 to 101%. The results agreed well with those obtained with a chromatographic method for the Phe determination in serum and with the normal reference values.     

Highly sensitive biotin-labelled hybridization probe

A simple chemical method for introducing biotin into nucleic acids has been developed for the synthesis of nonisotopic hybridization probes. The method is based on the reaction of biotin hydrazide with amino residues of nucleic acids by using glutaraldehyde as a bifunctional coupling reagent. Biotin-labelled deoxyribonucleic acid (DNA) was detected by the use of alkaline phosphatase-labelled avidin, and alkaline phosphatase activity was measured by colorimetric and chemiluminescence methods. The chemiluminescence method using the nicotinamide adenine dinucleotide phosphate (NADP)/alcohol/alcohol dehydrogenase/microperoxidase/isoluminol system gave the highest sensitivity. A few picograms of lambda-phage DNA coated on a microtiter plate well could be detected by this method.     

Chemiluminometric flow-injection method for determination of free l-malate in wine with co-immobilized malate dehydrogenase/NADH oxidase

A flow-injection system with a co-immobilized malate dehydrogenase/reduced nicotineamide adenine dinucleotide (NADH) oxidase reactor and a chemiluminometer is described for the determination of free l-malate in wine. Malate dehydrogenase and NADH oxidase were co-immobilized on poly(vinyl alcohol) beads and packed into a stainless-steel column (5 cm x 4 mm i.d.). The hydrogen peroxide produced was detected chemiluminometrically via a luminol-hexacyanoferrate(III) reaction. The calibration graph was linear from 3 x 10(-7) M to 2.5 x 10(-4) M (the linear correlation coefficient was 0.9998); the detection limit (signal-to-noise ratio, 3) was 8 x 10(-8) M. The sample throughput was 30 h(-1) without carryover. The ractor was renewed every 2 weeks.     

Molecular emission cavity analysis: Part 28. Monitoring of Alcohol Dehydrogenase-catalyzed Reactions

Ethanol in the presence of yeast alcohol dehydrogenase at pH 8 is oxidized by nicotinamide adenine dinucleotide to acetaldehyde. The aldehyde is treated with sodium sulphite and the sulphite addition compound formed is monitored by the S₂ emission generated in a hydrogen-based flame. The detection limit is 10 ng of ethanol in the 5-μl injection, with linear response up to 240 ng. Mercury(II) (50–175 pg/5 μl) and silver (75–225 pg/5 μl), both of which inhibit the enzyme, can be determined indirectly.     

Thallium interactions in biological fluids. A potentiometric investigation of dimethylthallium complex equilibria with some typical amino acids

This paper concerns dimethylthallium (DMT) complex equilibria with sulphur- containing amino acids, glycine also being considered as a reference for simple amino acids. Formation constants for DMT complexes with -cysteine (Cys), -penicillamine (Pen), N- acetyl- -cysteine (Acy), N-acetyl- -penicillamine (Ape) and glycine (Gly) were determined under physiological conditions using glass electrode potentiometry. It is concluded that, as expected from the HSAB theory, the coordination of DMT by the carboxylate anion is insignificant. Binding of DMT to the amino and thiol groups involves logarithmic stability constants of 1.2 and 2.6, respectively. When these two groups are engaged in the same chelate ring, no additional effect is noted with respect to the sum of their individual contributions.