Effects of cytochrome P4503A inducer dexamethasone on the metabolism and toxicity of triptolide in rat.

Triptolide (TP), a major active and toxic component of Tripterygium wilfordii, is reported to be converted into four mono-hydroxylated metabolites (m/z 375) by cytochrome P450 (CYP) in vitro, and CYP3A4 was the primary isoform responsible for its hydroxylation. Dexamethasone (DXM), a CYP3A inducer, is frequently combined with TP in clinical therapy. However, the effects of DXM on the metabolism and toxicity of TP are unknown. In this study, the metabolism of TP was investigated in rat liver microsomes pretreated with DXM. The metabolic profile of TP was significantly altered. The V(max) was about 9.58-fold higher than that of vehicle group and the K(m) was about 3.57-fold higher. With DXM, the amount of metabolite M3 was significantly higher than that with no DXM while M1 and M2 were not found, and a new metabolite (m/z 391) was observed. The liver and the kidney toxicity of TP on rat pretreated with DXM were evaluated. We observed that pretreatment with DXM protected against TP hepatotoxicity. No obvious nephrotoxicity was detected on rats treated with TP, whereas the kidney damage was observed in DXM group and the level of toxicity was much reduced with DXM-TP group. This suggested that TP might decrease nephrotoxicity induced by DXM. These studies indicated that DXM had significant impact on the metabolism and the toxicity of TP as a therapeutic agent.

Biotin induced fluorescence enhancement in resonance energy transfer and application for bioassay.

A novel method to significantly enhance fluorescence resonance energy transfer (FRET) signal which occurred from fluoresceine isothiocyanate (FITC) to Dylight 549 was studied in this paper. Streptavidin was labeled with the donor fluorophore FITC and biotinamide was conjugated to the acceptor Dylight 549. When biotinamide bound to streptavidin, FRET would occur from FITC to Dylight 549 while a remarkable fluorescence enhancement of streptavidin-FITC was observed. The fluorescence enhancement of streptavidin-FITC in the presence of biotin was utilized in the FRET system to obtain higher fluorescence signal. Increase of fluorescence intensity of FITC and decrease of Dylight 549 depended on the concentration of competitive biotin. A homogeneous analysis method was established based on the fluorescence recovery of FITC in the FRET system with fluorescence enhancement. This method is highly sensitive and simple to determine the concentration of biotin. The detection limit for biotin was 0.5 nM and the linear range of the assay was 0.8-9.8 nM. The response time is no more than 15 min during the one-step assay due to the high affinity between streptavidin and biotin.

A new role for Fe3+ in TiO2 hydrosol: accelerated photodegradation of dyes under visible light.

The effect of Fe3+ on the photocatalytic activity of TiO2 hydrosol prepared through a low-temperature route has been investigated under visible light irradiation. The total reactive oxygen species (ROS) level and the accumulation of Fe2+ during the photodegradation process were detected to examine the role of Fe3+. In contrastto an aqueous TiO2 dispersion where Fe3+ strongly inhibited the photoactivity of TiO2 via suppressing the reduction of O2 and decreasing the production of ROS, Fe3+ accelerated the photodegradation of all dyes examined in the hydrosol through increasing the yield of oxidative ROS. The influence of the prebound hydroxyl groups on the surface of TiO2 was compared to that of free alcohols in aqueous solution, which revealed the cooperative function of the surface hydroxyl groups. The thoroughly contrary effect of Fe3+ on the photocatalysis of TiO2 hydrosol and TiO2 powder, which are all anatase nanocrystallites but are synthesized with different procedures, was ascribed to the complexation of the hydroxyl groups bound to TiO2 surface with Fe3+. The formation of such complexes has resulted in an altered electron-transfer pathway of the dye-sensitized photocatalysis under visible light irradiation.

[Establishment of the magnetic separation enzyme immunofluorescence method for detecting insulin].

AIM: To establish a convenient and sensitive magnetic separation enzyme immunofluorescence (MEIF) method for detecting human insulin. METHODS: Two monoclonal antibodies (mAbs) were conjugated with FITC and alkaline phosphatase (AP) respectively, which were incorporated magnetic solid phase separation. Magnetic beads were coupled with sheep anti-FITC antibody as solid phase and 4-Methylumbelliferyl-phosphoric acid (4-MUP) was used as substrate to set up MElF for detecting insulin. RESULTS: The sensitivity of this assay was 2.0 microIu/mL, the linear range was from 0 microIu/mL to 188.52 microIu/mL, and the intra-assay variation and inter-assay variation were 4.3%-5.2% and 2.6%-9.5%, respectively. The recovery rate of dilution was 92.6%-117% and the recovery rate of accession was 106%-121%.The result of the assay correlated well with that of magnetic enzyme chemiluminescence immunoassay system. CONCLUSION: The MEIF for detecting insulin is low at cost, sensitive, specific and stable, which can be widely used in clinical immune detection.

Study the oxidative injury of yeast cells by NADH autofluorescence.

Autofluorescence has an advantage over the extrinsic fluorescence of an unperturbed environment during investigation, especially in complex system such as biological cells and tissues. NADH is an important fluorescent substance in living cells. The time courses of intracellular NADH autofluorescence in the process of yeast cells exposed to H(2)O(2) and ONOO(-) have been recorded in detail in this work. In the presence of different amounts of H(2)O(2) and ONOO(-), necrosis, apoptosis and reversible injury are initiated in yeast cells, which are confirmed by acridine orange/ethidum bromide and Annexin V/propidium iodide staining. It is found that intracellular NADH content increases momently in the beginning of the apoptotic process and then decreases continually till the cell dies. The most remarkable difference between the apoptotic and the necrotic process is that the NADH content in the latter case changes much more sharply. Further in the case of reversible injury, the time course of intracellular NADH content is completely different from the above two pathways of cell death. It just decreases to some degree firstly and then resumes to the original level. Based on the role of NADH in mitochondrial respiratory chain, the time course of intracellular NADH content is believed to have reflected the response of mitochondrial redox state to oxidative stress. Thus, it is found that the mitochondrial redox state changes differently in different pathways of oxidative injury in yeast cells.

Online kinetic studies on intermediates of laccase-catalyzed reaction in reversed micelle.

Using water/AOT/n-octane reversed micelle as the medium, the optical signal of the reactive intermediate of laccase-catalyzed oxidation of o-phenylenediamine, which was indetectable in aqueous solutions, was successfully captured. Thus online kinetic studies of the intermediate were accomplished. Two-way kinetic spectral data were acquired with stopped-flow technique. By resolving the data with global analysis software, both the kinetic curves and the absorption spectra of the components involved in the reaction process were simultaneously obtained. The whole reaction in the reversed micelle was proved to be composed of two successive steps, an enzymatic generation of the intermediate and a following nonenzymatic decay of the intermediate. A consecutive first-order kinetic model of the whole reaction was confirmed. The influences of microenvironmental factors of the medium (such as the pH value of the water pool and the water/AOT ratio) on the detection of the intermediate were also investigated.

Study on the influence of potassium iodate on the metabolism of Escherichia coli by intrinsic fluorescence.

Intrinsic fluorescence, in particular, has the advantage over the extrinsic fluorescence of an unperturbed environment during investigation, especially in complex systems such as biological cells and tissues. Potassium iodate may restrain bacteria growth as well as it acts as an additive in the salt. The influence of potassium iodate (KIO3) on the metabolism of Escherichia coli (E. coli) is investigated for the first time with the intrinsic fluorescence of tryptophan (Trp) and reduced nicotinamide adenine dinucleotide (NADH). We found that potassium iodate may restrain the growth of E. coli as a bacteriostatic agent. When the potassium iodate concentration was below 1.32 mmol/L, the intensity of tryptophan fluorescence decreased linearly whereas the NADH fluorescence did not change. When the KIO3 concentration was over 1.32 mmol/L, the fluorescence of tryptophan and NADH increased a little and their fluorescence intensity decreased when KIO3 was over 6.67 mmol/L. And the bacteria could not continue growing if the KIO3 was over 6.67 mmol/L.We could conclude that potassium iodate has great inhibiting effects on the growth of E. coli through the pathway of protein synthesis and respiratory chain.

Study and application of perturbation of peroxynitrite on peroxidase-oxidase oscillation.

Peroxynitrite was one of the important reactive oxygen species (ROS) which have been focused for many years. Peroxynitrite is an extremely strong and reactive oxidant which can cause many diseases linked to inflammatory processes and autoimmune diabetes, etc. ROS can significantly react with peroxidase and nicotinamide adanine dinucleotide (NADH) which is linked to numerous biological processes. NADH, NAD and horseradish peroxidase (HRP) were included in peroxidase-oxidase (PO) oscillation, dissolved oxygen was concerned with the oscillation and many ROS intermediates came into being in PO oscillation which was sensitive to the perturbation of ROS. The influence of peroxynitrite (ONOO(-)) on this oscillation system was investigated. It was found that the oscillation amplitude increased when the system was perturbed with peroxynitrite. There was a linear relationship between the increment ratio in the oscillation amplitude and perturbing peroxynitrite concentration in the range 2.50 x 10(-8) to 1.56 x 10(-6) mol/L. And further experimental results revealed that amplitude increasing may be caused by the effect of peroxynitrite on HRP. Based on this phenomenon, a highly sensitive method for the determination of peroxynitrite was developed.

A new kinetic method for quantification phenoxyl free radicals.

In this work, a new kinetic method was proposed for quantification phenoxyl radicals generated in enzyme reaction. Instead of direct detecting the spectral signals of phenoxyl radicals, a molecular probe, the reduced form of nicotinamide adenine dinucleotide (NADH), was employed to indicate the formation of phenoxyl free radicals. It was found that the reactions of NADH and phenoxyl radicals are very fast, but can be followed by using stopped-flow fast scanning spectrophotometric technique. The initial rate of accelerated-oxidation of NADH represents the reactivity of phenoxyl free radical, which is proportional in a certain range to the initial concentration of the parent chlorophenols of the radicals. With this method, the phenoxyl radicals generated in oxidation reaction of chlorophenols (2-CP; 4-CP; 2,4-DCP; 2,4,6-TCP and 2,3,4,6-Tetra-CP) with hydrogen peroxide, catalyzed by horseradish peroxidase, were investigated. The method is highly sensitive. Phenoxyl radicals generated from as low as 1x10(-8)M 2,4-DCP, for example, can be readily detected with the proposed method. The results show that the reactivity of various phenoxyl radicals are in the following order: 2,4-DCP>4-CP>2-CP>2,4,6-TCP>2,3,4,6-Tetra-CP. A mechanism is proposed to explain the possible pathway of the probe reaction. The feasibility of this method was assessed by the determination of enzymatic generation of phenoxyl radicals in lake water samples.

Preparation of photosensitized nanocrystalline TiO(2) hydrosol by nanosized CdS at low temperature.

A new method was developed for the fabrication of CdS-TiO(2) semiconductor nanoparticles as visible-light-excitable photocatalyst at low temperatures. Nanosized CdS acting as an effective and stable sensitizer was incorporated into TiO(2) by microemulsion-mediated solvothermal hydrolyzation followed by acidic peptization of the precipitate under 70 °C. The new method avoided the calcination or other pyrochemical treatments involved in traditional preparations, and thus eliminated the unwanted agglomeration of nanoparticles or the oxidation of CdS by oxygen. Compared to traditional methods, it was highly simplified, bypassing those miscellaneous steps like filtration, sintering, milling and redispersion in solutions. The crystal structure, configuration, element composition, as well as the light-absorption properties of the obtained CdS-TiO(2) hydrosol were characterized in detail. The hydrosol consisting of uniform and small crystalline particles of about 2 nm in diameter was thermodynamically stable and showed good dispersibility. The photocatalytic activity of the 'coupled' material was confirmed through the photocatalytic degradation of methylene blue (MB) dye under visible light irradiation, and the cooperative photocatalytic mechanism is discussed.

Spectral and kinetic studies on the interaction between oxyhaemoglobin and peroxynitrite: a comparison with hydrogen peroxide.

Interaction between oxyhaemoglobin and peroxynitrite was studied using stopped-flow rapid-scan spectrophotometry. The influence of pH, peroxynitrite concentration and temperature on the pseudo-first-order rate constants was studied and the activation energy calculated. The kinetic curve for the oxyhaemoglobin-peroxynitrite reaction showed that a fast reaction occurred in the initial seconds, followed by a slow process of decrease in absorbance. The biphasic reaction kinetics of oxyhaemoglobin with peroxynitrite or hydrogen peroxide demonstrated the existence of an intermediary species. For the first time a rapid-scan stopped-flow spectrophotometry study is presented, yielding spectral and kinetic data of the reaction.

Fluorometric assay of tiopronin based on inhibition of multienzyme redox system.

In this paper, a simple and sensitive fluorimetric method for the determination of tiopronin (N-(2-mercaptopropionyl)-glycine) is proposed. The method is based on the strong inhibitory effect of tiopronin on the multienzyme redox system of hemoglobin, nicotinamide adenine dinucleotide (NADH) and H(2)O(2), in which the intrinsic fluorescence of NADH was employed as the detection signal. The calibration graph is linear in the range 6.13 x 10(-7) to 6.13 x 10(-6) M with a detection limit of 1.65 x 10(-7) M and the relative standard deviation of 2.02%. Kinetics in the pseudo-first-order conditions was investigated by stopped-flow spectrofluorometry and the inhibition mechanism of tiopronin was verified of the competitive type.

Study on the effect of thiamine on the metabolism of yeast by intrinsic fluorescence.

Thiamine in living human bodies exists mainly as diphosphate, which works as a co-enzyme of the sugar metabolism system (active vitamin B1). Thiamine deficiency brings many clinically significant problems, such as dysphoria, quadriplegia and dyspepsia. Intrinsic fluorescence has an advantage over the extrinsic fluorescence of an unperturbed environment during investigation, especially in complex systems such as biological cells and tissues. Cellular fluorescence provides a sensitive index of the functional state of a living cell (1). Different amounts of thiamine were added to culture medium and the fluorescence of tryptophan and NADH from yeast was determined. When the thiamine concentration was greater than 0-0.16 microg/mL, the intensity of tryptophan fluorescence increased linearly, whereas the NADH fluorescence decreased. When the thiamine concentration was above 0.24 microg/mL, the fluorescence of tryptophan and NADH was almost unchanged. We concluded that low thiamine concentration in culture medium had a large effect on the growth of Saccharomyces cerevisiae and possible reasons are discussed.

Determination of myoglobin based on its enzymatic activity by stopped-flow spectrophotometry.

A new method has been developed for the determination of myoglobin (Mb) based on its enzymatic activity for the oxidation of o-phenylenediamine (OPDA) with hydrogen peroxide. Stopped-flow spectrophotometry was used to study the kinetic behavior of the oxidation reaction. The catalytic activity of Mb was compared to other three kinds of catalyst. The time dependent absorbance of the reaction product, 2,3-diamimophenazine (DAPN), at a wavelength of 426 nm was recorded. The initial reaction rate obtained at 40 degrees C was found to be proportional to the concentration of Mb in the range of 1.0 x 10(-6) to 4.0 x 10(-9)mol L(-1). The detection limit of Mb was found to be 9.93 x 10(-10)mol L(-1). The relative standard deviations were within 5% for the determination of different concentrations of Mb. Excess of bovine serum albumin (BSA), Ca(II), Mg(II), Cu(II), glucose, caffeine, lactose and uric acid did not interfere.

Electrochemical DNA sensing based on gold nanoparticle amplification.

A hybridization signal-amplified method based on a gold nanoparticle-supported DNA sequence for electrochemical DNA sensing has been investigated by cyclic voltammetry, differential-pulse voltammetry, and atomic-force microscopy (AFM). Quantitative analysis showed that the peak current increment (DeltaIp) is linearly dependent on the concentration of the gold nanoparticle-supported DNA sequence Au2 over the range 0.51-8.58 pmol L(-1). AFM results indicated that the extent of surface hybridization was dependent on the concentration of the gold-nanoparticle-supported DNA sequence. Moreover, a new pair of peaks, which might arise from the special configuration of the gold-nanoparticle-supported DNA sequence, appeared in the cyclic voltammogram after hybridization. Although quite sensitive, this DNA sensing surface was not easily regenerated, so this kind of amplified method was suitable for disposable DNA sensors and chip-based gene diagnosis sensors.

Detection of DNA with Catalytic Beacons Based on Peroxidase-oxidase Oscillating Reaction.

This paper aims at investigating a new method for the detection of DNA with catalytic beacons based on peroxidase-oxidase (PO) oscillation and analytic pulse perturbation technique. Two DNAzymes were constructed by the binding of specific DNA sequence with hemin or by the hybridization of target DNA with the catalytic beacon. Both DNAzymes possessed peroxidase-like activity and perturb the PO oscillator reaction when they were added into the oscillation system. The period and amplitude of oscillation increased significantly by both DNAzymes, which implied the decrease in the average rate of consumption of oxygen in solution, i.e., the decrease of the average rate of NADH oxidation. The results provide a new sensitive method for DNA detection and molecular recognition.

A new method for determination of uric acid by the lactic acid-acetone-BrO(3)(-)-Mn(2+)-H(2)SO(4) oscillating reaction using the analyte pulse perturbation technique.

A new analytical method for the determination of uric acid (UA) by the perturbation of UA on the Belousov-Zhabotinsky oscillating reaction is proposed. The method is based on the linear relationship between the changes in the oscillating period and the concentration of UA. The calibration curve is linear over the range of 2.0 x 10(-5) to 5.0 x 10(-4)M, with a detecting limit of 3.28 x 10(-6)M. The method features good precision (R.S.D.: 3.59%) and excellent throughput (10samplesh(-1)). The possible mechanism of the perturbation of UA on the oscillating reaction is discussed.

Studies on the photodegradation of Rhodamine dyes on nanometer-sized zinc oxide.

The nanometer-sized ZnO was prepared through the sol-gel method. Its average particle diameter, determined by TEM, was 20-30 nm. The specific surface area was determined to be 22 m2 g(-1) by BET. The photodegradation mechanism of Rhodamine dyes on nanometer-sized ZnO was studied by dynamic molecular spectra, and the results showed that the photodegradation of Rhodamine dyes obeyed the rules of a pseudo first-order kinetic reaction. The rate constant k of the degradation of Rhodamine B (RB) and butyl-Rhodamine (BR) were 0.0128 and 0.0154 min(-1), respectively, and the half period t(1/2) were 60 and 52 min, respectively. The photodegradation reaction conditions were optimized. After intermixing with silver, the photodegradation efficiency was greatly improved. A life-span test showed that nanometer-sized ZnO had a long life-span.

Resonance light scattering spectroscopy study of interaction between gold colloid and thiamazole and its analytical application.

In this paper, we used resonance light scattering (RLS) spectroscopy to study the interaction between thiol-containing pharmaceutical-thiamazole and gold colloid. At pH 5.2, the resonance light scattering spectrum of gold nanoparticles has a maximum peak at 555 nm and the RLS intensity is enhanced by trace amount of thiamazole due to the interaction between thiamazole and gold colloid. The binding of colloidal gold to thiamazole results in ligand-induced aggregation of colloidal gold, which was characterized by RLS spectrum, ultraviolet-visible (UV-Vis) spectrum, and transmission electron microscopy (TEM). Based upon the study, we proposed a highly sensitive, gold colloid-based assay using RLS spectrum to detect pharmaceuticals for the first time. The mechanism of binding interaction between Au colloid and thiamazole was also discussed.

Study and analytical application of inhibitory effect of captopril on multienzyme redox system.

Multienzyme redox system is the most important biological oxidation process in cellular respiration chain. Nicotinamide adanine dinucleotide redox state (NADH/NAD) can be directly control site in numerous biological processes linked to rhythm, senescence, cancer and death. Therefore, it is very important to investigate the influencing factor of this multienzyme redox system. We have already found a strong inhibitory effect of captopril on this enzyme-catalyzed reaction and further experimental results revealed that this inhibitory action belonged to competitive type. Based on this phenomenon, we developed a highly sensitive spectroflourimetry method for the determination of captopril in pharmaceuticals.