Flow-injection determination of adenosine and inosine in blood plasma with immobilized enzyme columns connected in series and fluorimetric detection

A rapid and sensitive flow-injection method is described for the enzymatic determination of adenosine and inosine in human blood plama. Teflon columns prepared by packing adenosine deaminase. purine nucleoside phosphorylase, xanthine oxidase, uricase and horseradish peroxidase immobilized chemically on controlled-pore glass beads are connected in series in that order in the flow line. Hydrogen peroxide formed in the enzymatic conversion of adenosine and inosine is measured fluorimetrically after reaction with 3-(p-hydroxyphenyl)propionic acid. Linear calibrations were obtained for 0.5–500 pmol of adenosine or inosine in hte 20 μl sample injected. Necessary deproteination routines are outlined.     

Determination of purine nucleosides and their bases by high-performance liquid chromatography using co-immobilized enzyme reactors

A selective and sensitive assay of inosine, guanosine, hypoxanthine, guanine and xanthine by high-performance liquid chromatography with immobilized enzyme reactors was developed. The separation was achieved on a Capcell Pak C18 column (15 cm x 0.46 cm I.D.) with a mobile phase of 0.1 M phosphate buffer (pH 8.0) containing 7 mM sodium 1-hexanesulphonate and 0.1 mM p-hydroxyphenylacetic acid. The fluorimetric detection of hydrogen peroxide using immobilized peroxidase and p-hydroxyphenylacetic acid was applied to the assay of these compounds, which were oxidized to yield hydrogen peroxide in the presence of immobilized enzyme (purine nucleoside phosphorylase, guanase and xanthine oxidase). Enzyme reactions occurred sufficiently without post-column addition of reagents. Enzymes that catalysed the conversion of purine compounds were co-immobilized on aminopropyl controlled-pore glass packed in stainless-steel tubing. The detection limits were 30-200 pg per injection.     

Construction of a guanine enzyme electrode and determination of guanase in human blood serum with an ammonia gas sensor

The guanine electrode is based on guanase used with an ammonia gas-sensing membrane electrode; immobilization of the enzyme is optimized. Guanine in the range 10^-4–10^-2 M gives a linear potential vs. log(concentration) plot with a response time of 4–1.5 min over the range specified. Guanase (0.12–12 I.U. I^-1) is determined in serum by adding guanine to the sample, and measuring the ammonia evolved with the gas-sensing electrode. Results compare favourably with the xanthine oxidase method.     

Microfabricated bioreactor chips for immobilised enzyme assays

A microfabricated device is reported that has been designed to permit the in situ packing of a section of channel with enzyme immobilised onto controlled pore glass (CPG). It is fabricated from glass and polydimethylsiloxane and to prevent dead volumes, has dedicated channels for packing the reactor. The device has the advantage of being simple in design, the flow through enzyme reactor channel being simply a widened section of the analyte channel. The system is suitable for both hydrodynamic and electro-osmotic pumping, and is designed such that when the packing is exhausted it can be repacked. Controlled pore glass provides a reproducible none swelling, high porosity medium onto which the enzyme could be immobilised. The large particle size meant that it was vital to optimise the immobilisation procedure in order to achieve acceptable enzyme activity. The microfabricated device was developed with two enzymes of different molecular masses; alkaline phosphatase and xanthine oxidase. The pore size of the CPG was found to be very important for xanthine oxidase, where the 697 Å pore size (120-200 mesh) CPG was found to give the highest activity (18-20% activity retained after immobilisation). The microfabricated device was used for the assay of p-nitrophenyl phosphate and hypoxanthine with spectrophotometric detection at 405 and 470 nm, respectively. The limits of detection were 5 and 8 μM, respectively.     

Polyphenols as superoxide dismutase modulators and ligands for estrogen receptors

The capacity of estrogen and stilbene derivatives to modulate the activity of superoxide dismutases in relation with their estrogenic properties has been studied. The properties of trans-resveratrol (3,5,4′-trihydroxystilbene) and its analogues, 4-hydroxystilbene, 4,4′-dihydroxystilbene, 3,5-dihydroxystilbene, 3,5,4′-trimethoxystilbene and 4,4′-dihydroxy-3,5,3′,5′-tetramethylstilbene were compared to 17β-estradiol and its analogues (2-methoxyestradiol, estrone, 2-hydroxyestradiol and 2-methoxyestrone). Measurement of estrogen receptor-β (ER-β) binding capacity was carried out by a receptor competitor assay associated with fluorescence polarisation detection. The superoxide dismutase (SOD) modulation activity was followed with a spectrophotometric assay using the sequence xanthine/xanthine oxidase-2,3-bis[2-methoxy-4-nitro-sulfo-phenyl]-2H-tetrazolium-5-carboxanilide (X/XO-XTT). The structure-activity relationship was different for the two series tested. In the estrogenic series, a compound which does not inhibit SOD, is recognized by the ER-β. In contrast for the stilbenic series both properties are parallel each other.     

Improved method for measurement of rhodanese activity using methanethiosulfonate as sulfur donor substrate and its application to human serum.

We have developed a sensitive method for the measurement of rhodanese activity in human serum which is based on the colorimetric method for the determination of thiocyanate produced from methanethiosulfonate and cyanide as substrates. Thiocyanate gives a red complex with ferric ion in an acidic condition. The present method is about 70-fold more sensitive than the conventional method using cyanide and thiosulfate as substrates and correlates well (r = 0.997) with the conventional method in bovine liver rhodanese. Within-run precision of the method is 0.91% for 420 units/l serum and the calibration curve is linear up to 1850 units/l. The normal value for human serum, determined by the present method on 31 healthy persons, was 20.9 +/- 20.0 units/l (mean +/- 2S.D.). Rhodanese activity was clearly elevated in some serum samples which were observed at abnormal values in some biochemical diagnostic tests and showed significant positive correlations with guanase activity (r = 0.728, p less than 0.01) and glutamic-oxalacetic transaminase activity (r = 0.625, p less than 0.01).     

Flow injection determination of xanthine oxidase inhibitory activity and its application to food samples.

The enzyme xanthine oxidase (XOD) has been recognized as a key enzyme causing oxidative injury to tissues by ischemia-reperfusion. For this reason, XOD inhibitor, which effectively suppresses this enzyme, plays an important role in the inhibition of many diseases related to reactive oxygen species (ROS). In order to screen XOD inhibitors rapidly and conveniently, a novel assay using flow injection analysis (FIA) was proposed in the present investigation. To optimize the practical FIA system, we studied the effect of the reagent concentrations and the flow condition on the enzymatic reaction, and then selected the optimum condition as follows: 200-mU/ml XOD concentration, 0.5-mM xanthine concentration, 0.5-ml/min flow rate, and 2-m mixing coil length. Under this condition, a typical XOD inhibitor quercetin was determined in the concentration range 0.1 - 1.5 mM at a sampling frequency of 10 samples/h. Using the optimized FIA method, we determined the XOD inhibitory activity of some food samples: onions, apples and teas, which are the high sources of flavonoids known as the potential XOD inhibitors. Among these samples, tea leaves showed the highest activity, the second was onions and the lowest was apples. Based on the result of the assay, not only quercetin, but also other components in investigated samples, contributed to the XOD inhibitory activity.     

A new chromone, 11-hydroxy-sec-O-glucosylhamaudol from Ostericum koreanum

From the ethyl acetate fraction of the roots of Ostericum koreanum, a new chromone, 11-hydroxy-sec-O-glucosylhamaudol (1) along with the known compounds: four chromones, three coumarins, six phenolic compounds, and three quinic acids were isolated. These compounds were assessed for antioxidant activities in the DPPH radical and superoxide anion radical scavenging assay systems. Among isolates, 4-(2-hydroxy-vinyl)-benzene-1,2-diol (12) showed the most potent DPPH radical scavenging activity (IC(50)=4.80+/-0.62 mug/ml) and superoxide anion radical scavenging activity (IC(50)=11.05+/-0.83 microg/ml) in the xanthine/xanthine oxidase system. The antioxidant activities of 12 were comparable to those of quercetin and luteolin.     

离心超滤质谱法筛选中药复方二妙丸中黄嘌呤氧化酶抑制剂

采用离心超滤质谱分析技术(UF-UPLC/Q-TOF/MS), 结合体外酶活性实验方法, 对中药复方二妙丸提取物中的黄嘌呤氧化酶抑制剂进行了筛选. 体外酶活性实验测得二妙丸水提液对黄嘌呤氧化酶的半数抑制浓度(IC₅₀)为(0.218±0.0034) mg/mL, 表明二妙丸具有较强的黄嘌呤氧化酶抑制活性. 进一步采用离心超滤质谱技术对二妙丸水提物中潜在的黄嘌呤氧化酶抑制剂进行筛选, 从中筛选并鉴定了9种具有潜在黄嘌呤氧化酶抑制活性的化合物, 为开发黄嘌呤氧化酶抑制剂及阐明二妙丸治疗痛风和高尿酸血症的作用机制提供了一定的依据.     

Phosphorimetric microassay for succinyltrialanyl-p-nitroanilide-hydrolyzing enzyme activity in human serum

A phosphorimetric assay of succinyltrialanyl-p-nitroanilide-hydrolyzing enzyme activity in human serum is described. p-Nitroaniline formed enzymatically is extracted with ether and determined phosphorimetrically in an ether/ethanol glass. The method is precise and very sensitive, requiring as little as 5 μl of human serum. The limit of detection is 10 pmol.     

In vitro anti-inflammatory and xanthine oxidase inhibitory activity of Tephrosia purpurea shoot extract

The methanolic extract of Tephrosia purpurea (Leguminosae) shoots was evaluated in-vitro for its anti-inflammatory and xanthine oxidase inhibitory activity. Anti-inflammatory activity was measured by the Diene-conjugate, HET-CAM and beta-glucuronidase methods. The enzyme inhibitory activity was tested against isolated cow milk xanthine oxidase. The average anti-inflammatory activity of T. purpurea shoot extract in the concentration range of 1-2 microg/mL in the reacting system revealed significant anti-inflammatory activities, which, as recorded by the Diene-conjugate, HET-CAM and beta-glucuronidase assay methods, were 45.4, 10.5, and 70.5%, respectively. Screening of the xanthine oxidase inhibitory activity of the extract in terms of kinetic parameters revealed a mixed type of inhibition, wherein the Km and Vmax values in the presence of 25 to 100 microg/mL shoot extract was 0.20 mM/mL and 0.035, 0.026, 0.023 and 0.020 microg/min, while, for the positive control, the Km and Vmax values were 0.21 mM/mL and 0.043 microg/min, respectively. These findings suggest that T. purpurea shoot extract may possess constituents with good medicinal properties that could be exploited to treat the diseases associated with oxidative stress, xanthine oxidase enzyme activity and inflammation.     

Flow injection analysis for glucose and urea with enzyme reactors and on-line dialysis

A flow injection system for glucose and urea determination is described. The glucose determination uses immobilized glucose oxidase in a reactor designed to give 100% substrate conversion. The hydrogen peroxide formed is converted to a coloured complex with 4-aminophenazone and N,N-dimethylaniline. The coupling is catalysed by a reactor containing immobilized peroxidase. The coloured complex is measured in a flow-through spectrophotometric cell. Urea is converted to ammonia in a reactor with immobilized urease and detected with an ammonia gas membrane electrode. Proteins and other interfering species from serum samples are removed in an on-line dialyzer. Calibration curves are linear for glucose in the range 1.6 × 10^-4–1.6 × 10^-2 M and for urea in the range 10^-4–10^-1 M. The samples are 25 μl for glucose determination and 100 μl for urea determination. Linear ranges can be changed by varying the sample sizes. The effects of the dialyser, enzyme reactors and detectors on dispersion are evaluated.     

Simultaneous assay of hypoxanthine, xanthine and allopurinol by high-performance liquid chromatography and activation of immobilized xanthine oxidase as an enzyme reactor

A selective and sensitive assay of substrates (hypoxanthine, xanthine and allopurinol) of xanthine oxidase by reversed-phase liquid chromatography coupled with the use of immobilized enzyme reactors is described. These compounds were oxidized by immobilized xanthine oxidase and produced hydrogen peroxide, which was determined fluorometrically using immobilized peroxidase and p-hydroxyphenylacetic acid. The detection limits of hypoxanthine, xanthine and allopurinol were approximately 50, 120 and 130 pg per injection, respectively. Immobilized xanthine oxidase inhibited by oxipurinol during the assay was reactivated by 2,6-dichlorophenolindophenol and could be used for a long period without a significant activity loss. These methods were applied to plasma and urine samples.     

An ultramicro assay for human plasma prekallikrein activity by phosphorimetry

A highly sensitive method for the phosphorimetric assay of prekallikrein in human blood plasma is described. Prekallikrein is converted to kallikrein (active form) by reaction at 0°C with actin. p-Nitroaniline, formed enzymatically from H-d-prolyl-l-phenylalanyl-l-arginyl-p-nitroanilide, is extracted with ether and determined phosphorimetrically in a mixture of ether and ethanol. The method is precise and highly sensitive, requiring as little as 0.25 μl of human blood plasma. The limit of detection for p-nitroaniline formed enzymatically is 5 pmol.     

A spectrophotometric procedure for DNA assay with a micro-sequential injection lab-on-valve meso-fluidic system

A micro-sequential injection spectrophotometric procedure for DNA assay was developed based on the employment of a lab-on-valve (LOV) meso-fluidic analytical system. A small amount of crystal violet solution (10 μl) was de-colored inside the flow cell of the LOV at the presence of 5 μl λ-DNA/HindIII within a certain pH range, and the absorbance decrease of crystal violet solution at 591 nm was measured via optical fibers and was employed as the basis of quantification. A uni-variant approach was adopted for the optimization of experimental parameters, including buffer pH, concentration and volume of crystal violet solution, reaction time and sample/reagent loading flow rates. A linear calibration graph was obtained within 0.2-6.0 μg ml⁻¹, along with a detection limit of 0.07 μg ml⁻¹. The procedure was applied for the determination of λ-DNA/HindIII in synthetic samples in comparison with a documented procedure.     

A colorimetric method for the determination of guanine and xanthine and its application to the assay of xanthine oxidase

Diazotized sulfanilic acid couples with guanine and xanthine to form a stable orange color (λ_max 460). The reaction is reproducible and linear over a wide range of guanine and xanthine concentrations (5–100 μg). Twelve purine and pyrimidine derivatives do not interfere. Thymine interferes to a small extent (5%). Guanine is directly determined in DNA and RNA acid hydrolyzates prior to chromatographic separation. The results are comparable with these obtained by uv method after PC separation. The method is also applicable to the assay of xanthine oxidase activity.     

Differential kinetic analysis and flow injection analysis: Part I. The trans-1,2-diaminocyclohexanetetraacetate complexes of magnesium and strontium

The flow injection principle is used in simultaneous differential kinetic analysis for magnesium and strontium ions in solution. The method is based on the acid dissociation of the trans-1,2-diaminocyclohexanetetraacetate complexes of magnesium and strontium ions and on the use of copper(II) ion as a scavenger. A 200-μl sample is used for a two-point kinetic assay; special consideration is given to optimizing the flow parameters. At sampling rates of 60 samples per hour, high reproducibility of measurement and relatively low reagent consumption can be achieved. The limiting factor of the method is that a rather pronounced difference must exist between the rates of reaction of the metal complexes.     

Exploitation of reversed micelles as a medium in mimetic peroxidase-catalyzed fluorogenic reaction between hydrogen peroxide and l-tyrosine

The peroxidase activity of mimetic enzyme, iron tetrasulfonatophthalocyanine (FeTSPc), was characterized in reversed micelles of hexadecyltrimethylammonium bromide (CTAB) formed in n-heptane-n-pentanol solution (2:1, V:V). The assay is based on its catalytic effect on the oxidation reaction of l-tyrosine (l-tyr) by hydrogen peroxide. The influences of environmental factors, such as the water content, CTAB concentration and pH, on the peroxidase activity of FeTSPc were investigated. It was observed that the reaction rate was distinctly enhanced in CTAB reversed micelles as compared with the rate in aqueous solution. Under optimum conditions, application of the FeTSPc-catalyzed fluorescence system in reversed micelles to the determination of H(2)O(2) and FeTSPc led to a highly sensitive system compared with that in aqueous solution, permitting detection limits of 5x10(-9) mol l(-1) H(2)O(2) and 2.3x10(-9) mol l(-1) FeTSPc. The advantages and limitations of employing the reversed micellar media in such mimetic peroxidase-catalyzed fluorescent detection schemes were discussed.     

Discovery of xanthine oxidase inhibitors from a complex mixture using an online,restricted-access material coupled with column-switching liquid chromatography with a diode-array detection system

To find potential lead compounds for antigout drug discovery, an automated online, restricted-access material coupled with column-switching liquid chromatography with a diode-array detection (RAM–LC–DAD) system was developed for screening of xanthine oxidase (XO) inhibitors and their affinity rankings in complex mixtures. The system was first evaluated by analyzing a mixture of six compounds with known inhibition of XO. Nonspecific binding to the denatured XO was investigated and used as the control for screening. Subsequently, the newly developed system was applied to screening of a natural product, Oroxylum indicum extract, and four compounds which could specifically interact with XO were found and identified as oroxin B, oroxin A, baicalin, and baicalein. The results were verified by a competitive binding test using the known competitive inhibitor allopurinol and were further validated by an inhibition assay in vitro. The online RAM–LC–DAD system developed was shown to be a simple and effective strategy for the rapid screening of bioactive compounds from a complex mixture.

Arabinofuranose disaccharide analogs as inhibitors of Mycobacterium tuberculosis

Several octyl 5-O-(α-d-arabinofuranosyl)-α-d-arabinofuranoside disaccharide analogs substituted at the 5-position of the non-reducing end sugar were synthesized and tested in vitro against Mycobacterium tuberculosis (M.tb.), Mycobacterium avium complex (MAC) as well as in a cell free assay system for arabinosyltransferase acceptor/inhibitor activity. A few compounds showed interesting inhibitory activity in the cell free assay as well as against the whole microorganism in vitro.