Preparation, validation and use of a vasoactive tryptophan-derived hydroperoxide and relevant control compounds.

The L-tryptophan-derived tricyclic hydroperoxide cis-WOOH was recently identified as a novel and biologically important factor for regulating vascular tone and blood pressure under inflammatory conditions and potentially other cellular redox signaling events. cis-WOOH is highly labile and currently not available commercially. In this protocol, we provide procedures for the synthesis, purification, quantification and characterization of cis-WOOH, its epimer trans-WOOH and their respective alcohols (cis-WOH and trans-WOH). Photo-oxidation of L-tryptophan (L-Trp) results in a mixture containing cis-WOOH and trans-WOOH, which are separated and purified by semi-preparative HPLC. cis-WOH and trans-WOH are then produced by sodium borohydride reduction and purified by semi-preparative HPLC. Characterization of cis-WOOH and trans-WOOH and the reduced alcohol variants is achieved using HPLC, fluorescence, NMR and liquid chromatography-tandem mass spectrometry. The protocol provides instructions for storage and quantification, as well as ways to test the stability of these hydroperoxides in commonly used buffers and media. Finally, we describe examples of how to monitor the formation of cis-WOOH in biological samples. The protocol ensures reasonable yield (11%) and purity (>99%) of cis-WOOH and control compounds in 5-6 d and outlines conditions under which cis-WOOH is stable for several months.

UV-Vis detection of hydrogen peroxide using horseradish peroxidase/copper phosphate hybrid nanoflowers.

A colorimetric platform based on enzyme-inorganic hybrid nanoflowers was used for ultraviolet-visible detection of hydrogen peroxide (H2O2). The hybrid nanoflowers were prepared via incubation at room temperature using horseradish peroxidase (HRP) and copper phosphate (Cu3(PO4)2) as the organic and inorganic components, respectively. HRP/Cu3(PO4)2 hybrid nanoflowers (HRP-HNFs) showed linear H2O2 detection range from 100 nM to 100 µM, moreover, HRP-HNFs exhibited good selectivity for H2O2 detection due to the specificity of HRP. In addition, HRP-HNFs have good reusability, excellent color stability and long-term storage stability, which indicate HRP-HNFs are promising for catalysis and biomedical applications.

Measurements of Hydrogen Peroxide and Oxidative DNA Damage in a Cell Model of Premature Aging.

Reactive oxygen species (ROS) represent a number of highly reactive oxygen-derived by-products generated by the normal mitochondrial respiration and other cellular metabolic reactions. ROS can oxidize macromolecules including lipids, proteins, and nucleic acids. Under physiological condition, the cellular levels of ROS are controlled by several antioxidant enzymes. However, an imbalance between ROS production and detoxification results in oxidative stress, which leads to the accumulation of macromolecular damage and progressive decline in normal physiological functions.Oxidative deterioration of DNA can result in lesion that are mutagenic and contribute to aging and age-related diseases. Therefore, methods for the detection of ROS and oxidative deterioration of macromolecules such as DNA in cells provide important tool in aging research. Here, we described protocols for the detection of cytoplasmic and mitochondria pools of hydrogen peroxide, and the DNA modification 8-oxoguanine, a biomarker of oxidative damage, that are applicable to cell-based studies on aging and other related areas.

Immobilized dye-decolorizing peroxidase (DyP) and directed evolution variants for hydrogen peroxide biosensing.

Immobilized dye-decolorizing peroxidase from Pseudomonas putida MET94 (PpDyP) and three variants generated by directed evolution (DE) are studied aiming at the design of a biosensor for H2O2 detection. Structural properties of the enzymes in solution and immobilized state are addressed by resonance Raman (RR) and surface enhanced RR (SERR) spectroscopy, and the electrocatalytic properties are analyzed by electrochemistry. The wild-type (wt) and 29E4 variant (with E188K and H125Y mutations) represent excellent candidates for development of H2O2 biosensors, since they exhibit a good dynamic response range (1-200 µM H2O2), short response times (2 s) and a superior sensitivity (1.3-1.4 A⋅M-1⋅cm-2) for H2O2, as well as selectivity and long term stability. In contrast to the solution state, 6E10 (with E188K, A142V and H125Y mutations) and 25F6 (with E188K, A142V, H125Y and G129D mutations) variants display much lower activity and are inhibited by high concentrations of H2O2 upon adsorption on an electrode. In terms of sensitivity, the bioelectrodes employing wt PpDyP and 29E4 variant outperform HRP based counterparts reported in the literature by 1-4 orders of magnitude. We propose the development of wt or 29E4 PpDyP based biosensor as a valuable alternative to devices that rely on peroxidases.

High peroxidase-like activity realized by facile synthesis of FeS2 nanoparticles for sensitive colorimetric detection of H2O2 and glutathione.

In the last decades, enzyme mimics have been regarded as strong substitutes to natural enzymes. The construction of biosensors based on these enzyme mimics with competitive catalytic activity and substrate specificity has attracted a lot of research interest. Herein, for the first time, we investigated the capability of nanoscale FeS2 to serve as enzyme mimics. Then, a facile and effective biosensor is fabricated based on its intrinsic peroxidase-like catalytic activity. In the presence of H2O2, FeS2 nanoparticles (NPs) possess high peroxidase-like activity to 3,3',5,5'-tetramethylbenzidine (TMB) oxidation, which can be ascribed to the generation of hydroxyl radicals (·OH) from the H2O2 decomposition catalyzed by FeS2 NPs. As for TMB, the resulting Michaelis-Menten constant (Km) value of FeS2 NPs is found to be about 12 times lower than that of natural horseradish peroxidase (HRP), highlighting the superiority of FeS2 NPs. Based on these intriguing observations, a reliable colorimetric method is then developed for detection of H2O2 and glutathione (GSH) by a simple mix-and-detect strategy. The detection limits of H2O2 and GSH are as low as 0.91 µM and 0.15 µM (3σ/slope), respectively. Moreover, FeS2 NPs can also catalyse the photoluminescence (PL) substrate terephthalic acid (TA) under the assistance of H2O2. This work remarkably extends the utilization of FeS2 NPs in the construction of colorimetric and PL biosensors in the fields of biosensing, environmental monitoring, and medical diagnosis.

Conductive and enzyme-like silk fibers for soft sensing application.

A combination of supercritical carbon dioxide (scCO2) impregnation of pyrrole and sonochemical transformation of permanganate (KMnO4) was used to impart conductive and catalytic properties to silk fibers. The results indicated that the conductivity (from polypyrrole -PPy) and catalytic activities (from manganese dioxide -MnO2) were independent and complementary within the processing parameters used. The enhanced conductivity was attributed to scCO2 preferentially distributing the pyrrole monomers along with the silk internal fibrillar structure and hence, yielding a more linear PPy. The oxidative properties of the PPy-MnO2-silk hybrid showed an enzyme-like behavior for the degradation of hydrogen peroxide (H2O2) with a Km of about 13 mM and specific activity of 1470 ±â€¯75 µmol/min/g. Finally, we demonstrated that the PPy-MnO2-silk hybrid could be used as soft working electrodes for the simultaneous degradation and detection of H2O2.

Effect of Heating on Compositional Characteristics and Oxidative Stability of Crude and Refined Rice Bran Oil.

The compositional characteristics and oxidative stability of rice bran oil were determined by observing the formation of oxidative products and alteration in chemical composition of oils during microwave or oven heating. The values of oxidative indicators such as free acidity, peroxide, p-anisidine, total oxidation, thiobarbituric acid and color values, increased faster in refined oils compared to crude ones during heating. In gas chromatography analysis, the percentages of total saturated, monounsaturated and polyunsaturated fatty acids in the studied oils such as lab extracted crude rice bran oil, lab extracted and refined rice bran oil, crude rice bran oil from commercial mill and refined rice bran oil from commercial mill were: 23.07 to 23.56, 41.15 to 42.38 and 34.38 to 35.88, respectively. The heating caused the reduction of polyunsaturated fatty acids content with increasing saturated fatty acids content, and these changes were greater in refined rice bran oil indicating extensive lipid oxidation occurred in refined oil. The change in triacylglycerol species content as determined by High-performance liquid chromatography, was lower in crude oil; the higher stability of these species in crude oil could have contribution to reduce oxidation. During thermal treatment, the generation of hydroperoxides, their degradation and formation of secondary oxidative products evaluated by Fourier-transform infrared spectroscopy, were lower in crude oils. However, the rate of formation of oxidative products in lab prepared samples was lower compared to that in the samples collected from commercial mill. Under extreme thermal condition, the order of oxidative stability: lab extracted crude rice bran oil > crude rice bran oil from commercial mill>lab extracted and refined rice bran oil > refined rice bran oil from commercial mill. The present results will be useful to oil seed processing mills in refining of rice bran oil for economic feasibility and better marketability.

Differential Diagnosis and Precision Therapy of Two Typical Malignant Cutaneous Tumors Leveraging Their Tumor Microenvironment: A Photomedicine Strategy.

Elevated hydrogen peroxide (H2O2) in biological tissues is generally recognized to be relevant to the carcinogenesis process that regulates the proliferative activity of cancer cells and the transformation of malignant features. Inspired by this observation, it can be hypothesized that imaging H2O2 in the tumor microenvironment (TME) could help diagnose tumor types and malignancy, and even guide precise therapy. Thus, in this study, a noninvasive photomedicine strategy is demonstrated that leverages the different levels of H2O2 in the TME, and two representative skin cancers, malignant melanoma (MM, clinically higher incidence of metastasis and recurrence) and cutaneous squamous cell carcinoma (cSCC, relatively less dangerous), are differentially diagnosed. The working probe used here is one we previously developed, namely, intelligent H2O2 responsive ABTS-loaded HRP@Gd nanoprobes (iHRANPs). In this study, iHRANPs have advantages over ratiometric imaging due to their bimodal imaging elements, in which the inherent magnetic resonance imaging (MR) mode can be used as the internal imaging reference and the H2O2 responsive photoacoustic (PA) imaging modality can be used for differential diagnosis. Results showed that after intravenous injection of iHRANPs, the tumor signals on both MM and cSCC are obviously enhanced without significant difference under the MR modality. However, under the PA modality, MM and cSCC can be easily distinguished with obvious variations in signal enhancement. Particularly, guided by PA imaging, photothermal therapy (PTT) can be precisely applied on MM, and a strong antitumor effect was achieved owing to the excessive H2O2 in the TME of MM. Furthermore, exogenous H2O2 was injected into cSCC to remedy H2O2 deficiency in the TME of cSCC, and an evident therapeutic efficacy on cSCC can also be realized. This study demonstrated that MM can be differentially diagnosed from cSCC by noninvasive imaging of H2O2 in the TME with iHRANPs; meanwhile, it further enabled imaging-guided precision PTT ablation, even for those unsatisfactory tumor types (cSCC) through exogenously delivering H2O2.

Combined calorimetric gas- and spore-based biosensor array for online monitoring and sterility assurance of gaseous hydrogen peroxide in aseptic filling machines.

A combined calorimetric gas- and spore-based biosensor array is presented in this work to monitor and evaluate the sterilization efficacy of gaseous hydrogen peroxide in aseptic filling machines. H2O2 has been successfully measured under industrial conditions. Furthermore, the effect of H2O2 on three different spore strains , namely Bacillus atrophaeus, Bacillus subtilis and Geobacillus stearothermophilus, has been investigated by means of SEM, AFM and impedimetric measurements. In addition, the sterilization efficacy of a spore-based biosensor and the functioning principle are addressed and discussed: the sensor array is convenient to be used in aseptic food industry to guarantee sterile packages.

Comparative studies of mitochondrial reactive oxygen species in animal longevity: Technical pitfalls and possibilities.

The mitochondrial oxidative theory of aging has been repeatedly investigated over the past 30 years by comparing the efflux of hydrogen peroxide (H2 O2 ) from isolated mitochondria of long- and short-lived species using horseradish peroxidase-based assays. However, a clear consensus regarding the relationship between H2 O2 production rates and longevity has not emerged. Concomitantly, novel insights into the mechanisms of reactive oxygen species (ROS) handling by mitochondria themselves should have raised concerns about the validity of this experimental approach. Here, we review pitfalls of the horseradish peroxidase/amplex red detection system for the measurement of mitochondrial ROS formation rates, with an emphasis on longevity studies. Importantly, antioxidant systems in the mitochondrial matrix are often capable of scavenging H2 O2 faster than mitochondria produce it. As a consequence, as much as 84% of the H2 O2 produced by mitochondria may be consumed before it diffuses into the reaction medium, where it can be detected by the horseradish peroxidase/amplex red system, this proportion is likely not consistent across species. Furthermore, previous studies often used substrates that elicit H2 O2 formation at a much higher rate than in physiological conditions and at sites of secondary importance in vivo. Recent evidence suggests that the activity of matrix antioxidants may correlate with longevity instead of the rate of H2 O2 formation. We conclude that past studies have been methodologically insufficient to address the putative relationship between longevity and mitochondrial ROS. Thus, novel methodological approaches are required that more accurately encompass mitochondrial ROS metabolism.

An Efficient Electrochemical Sensor Driven by Hierarchical Hetero-Nanostructures Consisting of RuO2 Nanorods on WO3 Nanofibers for Detecting Biologically Relevant Molecules.

By means of electrospinning with the thermal annealing process, we investigate a highly efficient sensing platform driven by a hierarchical hetero-nanostructure for the sensitive detection of biologically relevant molecules, consisting of single crystalline ruthenium dioxide nanorods (RuO2 NRs) directly grown on the surface of electrospun tungsten trioxide nanofibers (WO3 NFs). Electrochemical measurements reveal the enhanced electron transfer kinetics at the prepared RuO2 NRs-WO3 NFs hetero-nanostructures due to the incorporation of conductive RuO2 NRs nanostructures with a high surface area, resulting in improved relevant electrochemical sensing performances for detecting H2O2 and L-ascorbic acid with high sensitivity.

Nanodot-Directed Formation of Plasmonic-Fluorescent Nanohybrids toward Dual Optical Detection of Glucose and Cholesterol via Hydrogen Peroxide Sensing.

Hybrid nanoparticles (NPs) have emerged as an important class of nanomaterials owing to their integrated enhanced properties and functionality. In this study, we have developed an effective nanodot templating strategy for the in situ formation of surfactant-free nanohybrids with unique plasmonic-fluorescent properties. A bright photoluminescent biodot synthesized from serine and histamine biomolecular precursors (Ser-Hist dot) was first engineered to have rich functional groups on the nanosurface capable of anchoring Ag+ ions via electrostatic interaction. Upon UV irradiation, free electrons could transfer from the photoexcited Ser-Hist dot to the Ag+ ions, facilitating the in situ growth of AgNPs. The resulting nanohybrid system (Bio@AgNPs) exhibits distinct characteristic surface plasmon resonance absorbance and highly quenched PL intensity due to the inner filter effect. Furthermore, the Bio@AgNP nanohybrid retains its redox capability, enabling hydrogen peroxide sensing via AgNP etching, which in turn empowers a dual colorimetric and fluorescent detection of glucose and cholesterol in complex biological samples (i.e., synthetic urine and human plasma) with high selectivity and sensitivity. This finding reveals a new effective and facile method for the preparation of highly functional hybrid nanomaterials for dual-mode detection of hydrogen peroxide-producing species and/or reactions.

Anti-Cancer Effects of Sulfasalazine and Vitamin E Succinate in MDA-MB 231 Triple-Negative Breast Cancer Cells.

Aim: Sulfasalazine (SSZ) displayed anti-cancer activities. Vitamin E succinate (VES) could inhibit cell growth in various cancer cells. However, chemical therapies were often not useful for triple-negative breast cancer cells (TNBCs) treatment. Here, this study investigated the anti-cancer effects and the mechanisms on TNBCs under combination treatment with SSZ and VES. Methods: Cell viability was analyzed by using the MTT assay. The H2O2 levels were determined by using lucigenin-amplified chemiluminescence method. In addition, caspase and MAPs signals were studied by using western blotting. Results: Low-dose VES antagonized the SSZ-induced cytotoxicity effects while high-dose VES promoted the SSZ-induced cytotoxicity effects on TNBCs. In addition, SSZ alone treatment activated both caspase-3 and ERK signals, however, VES alone treatment only activated JNK signals. On the other hand, activation of caspase-3, JNK, and ERK were found in SSZ plus VES-treated cells. Conclusion: Combined SSZ and VES has synergistic or antagonistic cytotoxic effects depending on VES concentration. In addition, different cytotoxic signals are induced on SSZ-treated, VES-treated and SSZ plus VES-treated cells.

[Evaluation of scavenging activity of hydrogen peroxide in different origins of Liropes Radix by HPLC-UV-CL system].

The hydrogen peroxide generation system was used to analyze the scavenging activity of hydrogen peroxide by Liropes Radix from different origins by HPLC-UV-CL. The UV-CL fingerprints of Liropes Radix from different origins were evaluated,and the HPLC-UV and LC-CL fingerprints were systematically analyzed and evaluated. The results showed that the ether fractions of Liriope spicata var. prolifera and L. muscari had good scavenging activity of hydrogen peroxide,and the total activity of different origins varied greatly,while the similar samples had similar activities. The total antioxidant activity of L. muscari is higher than that of L. spicata var.prolifera. The similarity analysis of the two fingerprints was carried out by two different analytical methods. The chemical fingerprints and the active fingerprints have different characteristics. The contribution of each fingerprint to the total peak area and total activity is also different. There are significant differences between the two different fingerprint clustering results.

De Novo Iron Oxide Hydroxide, Ferrihydrite Produced by Comamonas testosteroni Exhibiting Intrinsic Peroxidase-Like Activity and Their Analytical Applications.

Natural enzyme mimics have attracted considerable attention due to leakage of enzymes and their easy denaturation during their storage and immobilization procedure. Here in this study, for the first time, a new iron oxide hydroxide, ferrihydrite - Fe1.44O0.32 (OH) 3.68 magnetic nanoparticles were synthesized by bacterial strain named Comamonas testosteroni. The characterization of the produced magnetic nanoparticles was confirmed by transmission electron microscopy (TEM), Fourier-transform spectroscopy (FTIR), X-ray diffraction (XRD), and magnetization hysteresis loops. Further, these extracted nanoparticles were proven to have biogenic magnetic behavior and to exhibit enhanced peroxidase-like activity. It is capable of catalyzing the oxidation of 3, 3', 5, 5'-Tetramethylbenzidine (TMB) by H2O2 to produce blue color (typical color reactions). Catalysis was examined to follow Michaelis-Menton kinetics and the good affinity to both H2O2 and TMB. The K m value of the Fe1.44O0.32 (OH) 3.68 with H2O2 and TMB as the substrate was 0.0775 and 0.0155 mM, respectively, which were lower than that of the natural enzyme (HRP). Experiments of electron spin resonance (ESR) spectroscopy proved that the BMNPs could catalyze H2O2 to produce hydroxyl radicals. As a new peroxidase mimetic, the BMNPs were exhibited to offer a simple, sensitive, and selective colorimetric method for determination of H2O2 and glucose and efficiently catalyze the detection of glucose in real blood samples.

3D hierarchical hollow hydrangea-like Fe3+@ɛ-MnO2 microspheres with excellent electrochemical performance for dopamine and hydrogen peroxide.

Construction a sensor to accurately detect dopamine (DA) and hydrogen peroxide (H2O2) is meaningful due to their close relation to the health of organisms. In this work, one-step hydrothermal method was employed to synthesize hierarchical hollow hydrangea-like Fe3+@ɛ-MnO2 microspheres constructed by interconnected nanosheets, and the growth mechanism of the microspheres (nFe/nMn = 0.6) was also investigated in detail. The material was used to construct an electrochemical sensor for DA and H2O2 detection with the linear range of 0.02-78 µmoL-1 and 0.000133-5.19 mmoL-1, respectively. The detection limit and sensitivity for DA and H2O2 are 5 and 50 nmoL-1 (S/N = 3), 7034.1 and 242.6 µA m(mol·L-1)-1 cm-2, respectively. Furthermore, the sensor was successfully applied to the detection of DA and H2O2 in serum and urine samples, indicating a potential value of this work in the pharmaceutical and environmental fields.

A Chemiluminescent Method for the Detection of H2O2 and Glucose Based on Intrinsic Peroxidase-Like Activity of WS2 Quantum Dots.

Currently, researchers are looking for nanomaterials with peroxidase-like activity to replace natural peroxidase enzymes. For this purpose, WS2 quantum dots (WS2 QDs) were synthesized via a solvothermal method, which improved the mimetic behavior. The resulting WS2 QDs with a size of 1⁻1.5 nm had a high fluorescence emission, dependent on the excitation wavelength. WS2 QDs with uniform morphology showed a high catalytic effect in destroying H2O2. The peroxidase-like activity of synthesized nanostructures was studied in H2O2 chemical and electrochemical reduction systems. The mimetic effect of WS2 QDs was also shown in an H2O2⁻rhodamine B (RB) chemiluminescence system. For this aim, a stopped-flow chemiluminescence (CL) detection system was applied. Also, in order to confirm the peroxidase-like effect of quantum dots, colorimetry and electrochemical techniques were used. In the enzymatic reaction of glucose, H2O2 is one of the products which can be determined. Under optimum conditions, H2O2 can be detected in the concentration range of 0⁻1000 nmol·L-1, with a detection limit of 2.4 nmol·L-1. Using this CL assay, a linear relationship was obtained between the intensity of the CL emission and glucose concentration in the range of 0.01⁻30 nmol·L-1, with a limit of detection (3S) of 4.2 nmol·L-1.

Hydroperoxides derived from marine sources: origin and biological activities.

Hydroperoxides are a small and interesting group of biologically active natural marine compounds. All these metabolites contain a group (R-O-O-H). In this mini-review, studies of more than 80 hydroperoxides isolated from bacteria, fungi, algae, and marine invertebrates are described. Hydroperoxides from the red, brown, and green algae exhibit high antineoplastic, anti-inflammatory, and antiprotozoal activity with a confidence of 73 to 94%. Hydroperoxides produced by soft corals showed antineoplastic and antiprotozoal activity with confidence from 81 to 92%. Metabolites derived from sea sponges, mollusks, and other invertebrates showed antineoplastic and antiprotozoal (Plasmodium) activity with confidence from 80 to 90%.

Detection of choline and hydrogen peroxide in infant formula milk powder with near infrared upconverting luminescent nanoparticles.

Choline is an essential nutrient for the growth and development of the baby, and therefore it is often added to infant formula. In this paper, a novel sensor for choline determination in infant formula is developed based on upconverting nanoparticles (UCNPs) with near infrared luminescence. UCNPs-based detection can avoid the interference of background fluorescence from complex samples, and thus provide high selectivity and sensitivity. It was observed that in the presence of Fe3+, polyacrylic acid coated UCNPs were quenched to 3% of its original intensity. The degree of quenching was among the best for UCNPs. Hydrogen peroxide could oxidize Fe2+ to Fe3+, which caused quenching of the upconversion luminescence. A new H2O2 detection method was thus established. In addition, choline could be hydrolyzed to betaine by choline oxidase, and at the same time produced H2O2, which also caused luminescence quenching through Fe2+ oxidation. Therefore, selective choline sensing was achieved.

Thyroid state affects H2O2 removal by rat heart mitochondria.

We investigated the effects of thyroid state on the mechanisms underlying rat heart mitochondrial capacity to remove H2O2 produced by an exogenous source. The removal rates were higher in the presence of respiratory substrates independently from thyroid state and were higher in hyperthyroid than in hypothyroid preparations. The thyroid state-linked changes in H2O2 removal rates, mirrored those in H2O2 release rates, showing that endogenous and exogenous H2O2 do not compete for the removing system. Mitochondrial content of coenzyme Q9 and Q10 was lower in hypothyroidism and higher in hyperthyroidism suggesting that the thyroid state-linked changes in the rates of H2O2 production are due to changes in the ubiquinone mitochondrial content. The rates of H2O2 removal in the presence of antioxidant enzyme inhibitors indicated that the contribution of each antioxidant is dependent on the thyroid state. This was supported by enzymatic activity measurements. Pharmacological inhibition also showed that the overall percentage contribution of the enzymatic processes, as well as that of non-enzymatic processes, is not affected by thyroid state. Cytochrome levels, inferred by light emission measurements, and western blot determination of cytochrome c, were lower in hypothyroid and higher in hyperthyroid preparations supporting the idea that the levels of reducing compounds were modified in opposite way by the changes in thyroid state. Further support was obtained showing that the whole antioxidant capacity, which provides an evaluation of capacity of the systems, different from cytochromes, assigned to H2O2 scavenging, was lower in hyperthyroid than in hypothyroid state.