High-performance liquid chromatographic determination of creatine kinase activity influenced by methylglyoxal

Protein glycation has been implicated in the development of diabetic complications and other health disorders, which mainly arise from accumulation of advanced glycation endproducts (AGEs) in vivo. Methylglyoxal (MGO), a typical reactive intermediate carbonyl formed in early glycation process, can react non-enzymatically with N-terminal amino groups on proteins, leading to their inactivation and generation of detrimental AGEs. Recently, it was reported that activity of creatine kinase (CK, EC 2.7.3.2) could be reduced or even eliminated completely after incubation with MGO in vitro. CK activity is usually determined by conventional colorimetric assays. However, these methods are not appropriate for monitoring the influence of MGO on CK activity since MGO can also directly react with creatine, a substrate of CK. In this study, an efficient and much more accurate HPLC approach was established to investigate the effect of MGO on CK activity. Aminoguanidine was utilized to eliminate interference from the undesirable reaction between residual MGO and creatine. It was found that higher concentrations of MGO and longer incubation time for CK and MGO caused more pronounced reduction in CK activity. This HPLC method greatly facilitates acquisition of kinetic data about CK reaction and through further improvement it may be adopted to rapidly screen potential inhibitors of MGO-induced glycation.     

Rapid and quantitative immunological method for the determination of creatine kinase isoenzyme MB activity in serum

Ohne Zusammenfassung

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Rapid and quantitative immunological method for the determination of creatine kinase isoenzyme MB activity in serum

     

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.     

An immobilized immuno-stirrer for the determination of creatine kinase-mb isoenzyme in blood serum

An immobilized immuno-stirrer is described for the determination of creatine kinase-MB isoenzyme in blood serum. The IgG antibodies are immobilized on alkylamine glass beads using glutaraldehyde as cross-linking reagent, and the beads are packed into a rotating porous cell. After incubation with stirring, the CK-M isoenzymes in the blood serum sample are inhibited and are bound to the antibodies inside the stirrer. The residual CK-B isoenzyme activity is then determined spectrophotometrically or electrochemically. The binding capacity of the immuno-stirrer to CK-M isoenzyme was estimated to be 800 Ul^-1 with an average inhibitory efficiency of 97.8%. The within-day and day-to-day coefficients of variation were 5% and 4%, respectively, over a period of 52 days. An immuno-stirrer loaded with antibodies attached to cyanogen bromide-activated cellulose beads was also characterized, but the antibodies were not as stable as on glass beads.     

Bioluminescent flow sensor for the determination of L-(+)-lactate

The amount of L-lactate in biological fluids (serum, plasma and cerebrospinal fluid) was determined by monitoring the reduced form of nicotinamide adenine dinucleotide (NADH) produced by immobilised lactate dehydrogenase (LDH), with bacterial bioluminescent enzymes immobilised on a separate nylon coil. The LDH catalysed the reaction of L-lactate with NAD; this reaction took place in a nylon coil that preceded the coil for the bioluminescent detection. The co-immobilisation of alanine aminotransferase (ALT) with LDH improved the lactate transformation by 117-183%. The response was linear from 0.1 to 50 micron mol l(-1) at 25 degrees C for the LDH - ALT reactor. The intra- and inter-assay coefficients of variation were less than 5% and the recoveries ranged from 93 to 106%. The results agreed well with those obtained with a spectrophotometric method and with the normal reference values.     

Determination of creatine kinase activity using a co-immobilized auxiliary enzyme reactor coupled on-line with a flow injection system

Two flow injection methods (based on spectrophotometric and spectrofluorimetric detection) were developed for the determination of over-all creatine kinase activity. Despite the complexity of the reactions involved (both include three enzyme-catalysed steps), the manifold is very simple because the two auxiliary enzymes which catalyse the two-step indicator reaction are co-immobilized on controlled-pore glass. The features of the proposed methods (calibration ranges between 0.1 and 2.0 and 0.01 and 1.0 U l-1, relative standard deviation 0.93 and 0.53% for the spectrophotometric and spectrofluorimetric methods, respectively) allow the successful determination of the analyte activity in serum samples (recoveries better than 95-105% for both methods).     

Multi-function fibre-optic sensor for the bioluminescent flow determination of ATP or NADH

A multi-function biosensor for the determination of either ATP or NADH using a single bioluminescence-based fibre-optic probe is described. This was made possible by co-immobilizing the firefly luciferase from Photinus pyralis for ATP analysis with the bacterial/oxidoreductase system from Vibrio harveyi for NADH analysis, on the same preactivated polyamide membrane. Compatible analytical conditions with regard to the activity and stability of each bioluminescent system were selected, enabling them to attain their highest performances. It was possible to perform continuous-flow measurements of ATP and NADH over a wide (log-log) linear calibration range with a relative standard deviation of 4.0–4.5% and detection limits of 0.25 pmol ATP and 5 pmol NADH.     

Chemiluminescence flow sensor for the determination of ascorbic acid with immobilized reagents

A novel flow sensor based on chemiluminescence (CL) for the determination of ascorbic acid has been proposed. The analytical reagents, luminol and ferricyanide, were both immobilized on an anion-exchange resin column. The CL signal produced by the reaction between luminol and ferricyanide, which were eluted from the column through sodium phosphate injection, was decreased in the presence of ascorbic acid. The CL emission intensity was linear with ascorbic acid concentration in the range 0.01-0.8 mug ml(-1); the detection limit was 5.5 x 10(-3) mug ml(-1). The whole process, including sampling and washing, could be completed in 1 min with a relative standard deviation of less than 5%. The sensor could be reused more than 100 times and has been applied successfully to the analysis of ascorbic acid in pills and vegetables.     

Amperometric biosensor for the determination of creatine

An amperometric biosensor for the determination of creatine was developed. The carbon rod electrode surface was coated with sarcosine oxidase (SOX) and creatine amidinohydrolase by cross-linking under glutaraldehyde vapour. The SOX from Arthrobacter sp. 1–1 N was purified and previously used for creation of a creatine biosensor. The natural SOX electron acceptor, oxygen, was replaced by an redox mediating system, which allowed amperometric detection of an analytical signal at +400-mV potential. The response time of the biosensor was less than 1 min. The biosensor showed a linear dependence of the signal vs. creatine concentration at physiological creatine concentration levels. The optimal pH in 0.1 M tris(hydroxymethyl)aminomethane (Tris)–HCl buffer was found to be at pH 8.0. The half-life of the biosensor was 8 days in 0.1 M Tris–HCl buffer (pH 8.0) at 20 °C. Principal scheme of consecutively followed catalytic reactions used to design a biosensor for the determination of creatine     

Bioluminescence flow system for determination of branched-chain l-amino acids in serum and urine

A continuous-flow bioluminescence method for determinations of branched-chain l-amino acids in serum and urine is described. Serum can be analyzed directly after simple filtration. Response is linear for 20-2000 pmol in the biological matrix. Leucine dehydrogenase is immobilized onto a nylon coil separated from the reactor coil containing bacterial bioluminescence enzymes. The stability of the immobilized is high (lifetime > two months) and more than 900 samples can be analyzed with the use of a few mg of enzymes. The results obtained agree well with those obtained by ion-exchange chromatography (amino acid analyzer).     

Development of integrated chemiluminescence flow sensor for the determination of adrenaline and isoprenaline

A novel integrated chemiluminescence (CL) flow sensor for the determination of adrenaline and isoprenaline is developed based on the enhancing effect of analytes on CL emission of luminol oxidized by periodate in alkaline solution. The analytical reagents luminol and periodate are immobilized on anion exchange resins, respectively, and packed in a glass tube to construct a reagentless sensor. The proposed sensor allows the determination of adrenaline and isoprenaline over the range from 2.0×10⁻⁸ to 1.0×10⁻⁵ g ml⁻¹ and 2.0×10⁻⁷ to 5.0×10⁻⁵ g ml⁻¹, respectively. The detection limits are 7.0×10⁻⁹ g ml⁻¹ for adrenaline and 5.0×10⁻⁸ g ml⁻¹ for isoprenaline with a relative standard deviation of 1.7% for the 1.0×10⁻⁷ g ml⁻¹ adrenaline (n=11) and 2.1% for 1.0×10⁻⁶ g ml⁻¹ isoprenaline (n=11). The sample throughput was 60 samples h⁻¹. The sensor has been successfully applied to the determination of adrenaline and isoprenaline in pharmaceutical preparations.     

An ultrasensitive fluorescence sensor for determination of trace levels of copper in blood samples

A novel SBA-15-based fluorescent sensor, SBA-PI: mesoporous SBA-15 structure modified with iminostilbene groups, was designed, synthesized, and characterized by Fourier transform-infrared spectroscopy (FT-IR), ultraviolet–visible spectroscopy, field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy (EDS), thermogravimetric analysis (TGA), low-angle X-ray diffraction techniques (low-angle XRD), and N₂ adsorption–desorption techniques. The SBA-PI as a sensor with a selective behavior for detection of Cu²⁺ comprises iminostilbene carbonyl as the fluorophore group. The SBA-PI sensor displays an excellent fluorescence response in aqueous solutions and the fluorescence intensity quenches remarkably upon addition of Cu²⁺. Other common interfering ions even at high concentration ratio showed either no or very small changes in the fluorescence intensity of SBA-PI in the absence of Cu²⁺. A limit of detection of 8.7 × 10⁻⁹ M for Cu²⁺ indicated that this fluorescence sensor has a high sensitivity and selectivity toward the target copper (II) ion. The fabricated Cu²⁺ sensor was successfully applied for the determination of the Cu²⁺ in human blood samples without any significant interference. With the selective analysis of Cu²⁺ ions down to 0.9 nM in blood, the sensor is a promising and a novel detection candidate for Cu²⁺ and can be applied in the clinical laboratory. A reversibility and accuracy in the fluorescence behavior of the sensor was found in the presence of I^¯ that was described as a masking agent for Cu²⁺.

Graphical abstract

     

Spectrophotometric and fluorimetric enzymatic determination of serum creatine kinase mb isoenzyme by using immuno-inhibition

Two fully enzymatic methods, colorimetric and fluorimetric, are reported for the determination of creatine kinase (EC 2.7.3.2) MB isoenzyme after immune-inhibition with the use of goat anti-human CK-M IgG antibodies. The residual creatine kinase activity is assayed by using hexokinase and glucose-6-phosphate dehydrogenase systems; the resulting NADPH is determined spectrophotometrically by reaction with p-iodonitro-tetrazolium violet in the presence of diaphorase as an intermediate electron carrier, or fluorimetrically by coupling the NADPH with resazurin/diaphorase to form resorufin. Both assays take only 12 min and require only 100 or 25μl of serum. The calibration plots of enzyme activities are linear up to 580 and 435 U l^-1 of CK-MB in serum for the spectrophotometric and fluorimetric assays, respectively, the coefficients of variation being 2.3% and 4.6%, and the recovery values 103% and 100% respectively. The results correlate very well with those obtained by the Helena electrophoresis—fluoridensitometric methods. As little as 4 U l^-1 and 2 U l^-1 CK-MB could be measured reproducibly.     

Spectrophotometric simultaneous determination of creatinine and creatine by flow injection with reagent injection

The reactions of creatinine with picric acid and of creatine with 1-naphthol and biacetyl, both in an alkaline medium, have been used to develop a flow injection method for the simultaneous determination of creatinine and creatine, respectively. The sample containing both analytes is continuously merged with a picrate stream and mixed through the reactors; the coloured stream passes through a flow cell in a spectrophotometer set at 520 nm, recording a continuous signal proportional to the creatinine concentration. The mixture of the reagents 1-naphthol and biacetyl is inserted into the stream by use of the injection valve, which results in a peak (superimposed on the continuous signal), proportional to the creatine concentration. Linear calibration graphs for both analytes were obtained up to 30 mg l^–1 with relative standard deviations <2%, and a sampling rate of 42 measurements h^–1. The method was applied to the determination of creatinine and creatine in broth cube samples.     

Reagentless chemiluminescence flow sensor for the determination of riboflavin in pharmaceutical preparations and human urine

A novel continuous-flow sensor based on chemiluminescence (CL) detection was developed for the determination of riboflavin at pg ml(-1) levels by the immobilization of the reagents. It was found that the CL intensity from the oxidation between luminol and periodate could be enhanced in the presence of riboflavin. The increase of CL emission was correlated with the riboflavin concentration in the range from 0.04 to 200 ng ml(-1), and the detection limit was 0.02 ng ml(-1) (3s). Considering the effective reaction ions, luminol and IO4- was immobilized on anion-exchange resin. The system could produce an evident CL signal by water as eluant and it was also shown that the flow sensor could greatly improve the selectivity and sensitivity for determination of riboflavin with a high signal-to-noise ratio. A complete analysis, including sampling and washing, could be performed in 0.5 min with a relative standard deviation of less than 3.0%. The flow sensor was applied successfully to the determination of riboflavin in pharmaceutical preparations and human urine samples.     

Modification of the anion exchange chromatographic method for separation of creatine kinase isoenzymes. its use for measurement of an abnormal creatine kinase

Summary Serial blood samples from dogs with pacing induced atrial tachycardia and from patients after different catheterization procedure showed consistently the presence of a new subband creatine kinase (CK) located between MM and MB. This fraction was called MX. An electrophoretic procedure on cellulose acetate with fluorometric scanning has ruled out the possibility of this MX fraction being either an artefact or adenyl kinase. Chromatography by continuous buffer gradient elution ion-exchange method confirmed this finding. The original method led to the finding of this MX and MB fractions in the same eluate. This method has been modified: Sample layered on sephadex were eluted with 100 and 165 mmol/liter NaCl for separation and subsequent quantification of MM and MX, while 200 mmol/liter NaCl separated the MB fraction if present. Good recoveries (88–106%) of total CK activity from the column were achieved.

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Zusammenfassung Serienweise entnommene Blutproben von Hunden, bei denen mittels eines Schrittmachers eine Vorhoftachykardie induziert wurde, und von Patienten nach verschiedenen Katheterisierungsverfahren wiesen regelmäßig eine neue Kreatinkinase-Bande auf, die zwischen MM und MB lag. Diese Fraktion wurde als MX bezeichnet.Die elektrophoretische Trennung auf Celluloseacetatfolie mit anschließender fluorimetrischer Auswertung schloß die Möglichkeit aus, daß diese MX-Fraktion ein Artefakt oder Adenylkinase war. Dieses Ergebnis wurde auch durch Ionenaustauschchromatographie unter Verwendung eines kontinuierlichen Puffergradienten bestätigt.Die ursprüngliche Methode trennte die MX- und MB-Fraktion nicht voneinander und wurde daher folgendermaßen abgeändert: Die auf Sephadex aufgetragenen Proben wurden mit 100 und 165 mMol NaCl/l eluiert, um MM und MX zu trennen und anschließend quantitativ zu bestimmen, während 200 mMol NaCl/l die eventuell vorhandene MB-Fraktion abtrennte. Mit dieser Methode wurden gute Wiederauffindungsraten (88–106%) der gesamten Kreatinkinaseaktivität von der Säule erzielt.

     

A novel PVC membrane sensor for determination of loratadine in pharmaceutical compounds and blood serum

A novel PVC membrane-selective electrode based on loratadine-tetraphenyl borate ion-pair was prepared for the determination of loratadine potentiometrically. This electrode exhibits a Nernstian slope of 59.1 ± 0.3 mV decade^-1 for loratadine in a concentration range of 5.0 × 10^-6?1.0 × 10^-2 M at pH 2.2 with a detection limit of 2.9 × 10^-6 M. The potential of the electrode is very stable and exhibits good reproducibility with very fast response time (??3 s). The selectivity of the proposed electrode towards some cations and organic compounds was tested and the selectivity coefficients were calculated. The electrode was successfully applied to the determination of loratadine in tablets and blood samples.