Eliminating Absorbing Interference Using The H-point Standard Addition Method: Case of Griess Assay in The Presence of Interferent Heme Enzymes Such As NOS

Standard calibration methods used to determine trace analytes usually yield significant deviations from the actual analyte value in the presence of interferents in the assay media. These deviations become of particular concern when the concentration of the analyte is low, and when the results are used to draw mechanistic or kinetic conclusions, for instance in enzyme structure-function studies. In these circumstances, the H-point standard addition method (HPSAM) provides superior precision and accuracy. This method is developed here for the case of the spectrophotometric Griess assay used to determine nitrite in various enzymology investigations, such as nitrite determination in studies of nitrite reductases (NiR), or when determining nitrite as a breakdown product of nitric oxide synthesized by NOS enzymes. The results obtained by HPSAM are contrasted with those of the traditional calibration method

Measuring Activity of Endothelial Nitric Oxide Synthase and Nanodisc Complex through Nitrate Production

Nitric oxide is an important bioregulator generated in various regions throughout the body by a family of isozymes referred to as Nitric Oxide Synthases (NOS). Within vascular endothelial cells, nitric oxide is generated from oxygen and arginine (amino acid) by endothelial nitric oxide synthases (eNOS). Within this environment nitric oxide plays a critical paracrine role, mainly anithrombotic and anti-atherosclerotic. This is accomplished by vessel dilation and prevention of platelet and leukocyte aggregation and adherence to the vessel wall. The activity of the eNOS enzyme has been studied within solution and is well understood. However, the impact that the lipid bilayer of endothelial cells has on the activity is not known. To better understand this interaction, we have formed “nanodiscs” to bind to the eNOS. Nanodiscs have two components that combine and self-assemble when added to solution, POPC (a lipid) and MSP1E3D1 (Membrane Scaffold Protein). The nanodiscs help provide a better microenvironment to study the enzyme and its activity. Through reaction with an indicator dye in the Griess reagent system, activity levels, as calculated by nitrate production, reduced dramatically. Over a 50% reduction was seen when calculating specific activity of the eNOS enzyme when bound to nanodiscs. A possible indication that a lipid bilayer restricts activity of the eNOS enzyme.https://engagedscholarship.csuohio.edu/u_poster_2014/1036/thumbnail.jp

Polymerized Hemin as An Electrocatalytic Platform for Peroxynitrite\u27s Oxidation and Detection

Peroxynitrite (ONOO−) constitutes a major cytotoxic agent, implicated in a host of pathophysiological conditions, thereby stimulating a tremendous interest in evaluating its role as an oxidant in vivo. Some of the detection methods for peroxynitrite include oxidation of fluorescent probes, EPR spectroscopy, chemiluminescence, immunohistochemistry, and probe nitration; however, these are more difficult to apply for real-time quantification due to their inherent complexity. The electrochemical detection of peroxynitrite is a simpler and more convenient technique, but the best of our knowledge there are only few papers to date studying its electrochemical signature, or reporting amperometric microsensors for peroxynitrite. Recently, we have reported the use of layered composite films of poly(3,4-ethylenedioxythiophene) (PEDOT) and hemin (iron protoporphyrin IX) as a platform for amperometric measurement of peroxynitrite. The main goal herein is to investigate the intrinsic catalytic role of hemin electropolymerized thin films on carbon electrodes in oxidative detection of peroxynitrite. The electrocatalytic oxidation of peroxynitrite is characterized by cyclic voltammetry. The catalytic current increased as a function of peroxynitrite\u27s concentration, with a peak potential shifting positively with peroxynitrite\u27s concentration. The catalytic efficiency decreased as the scan rate increased, and the peak potential of the catalytic oxidation was found to depend on pH. We show that optimized hemin-functionalized carbon electrodes can be used as simple platforms for peroxinitrite detection and quantification. We report dose–response amperometry as an electroanalytical determination of this analyte on hemin films and we contrast the intrinsic hemin catalytic role with its performance in the case of the PEDOT–hemin as a composite matrix. Finally, we include some work extending the use of simple hemin films for peroxynitrite determination on carbon microfiber electrodes in a flow system

Cryoprotective Effect of l-Carnitine on Motility, Vitality and DNA Oxidation of Human Spermatozoa

Successful cryopreservation for human spermatozoa markedly influences the reproductive outcomes of assisted reproductive technologies. But in spite of its usefulness, cryopreservation significantly decreases sperm quality. l-carnitine has been found to improve the quality of spermatozoa in selected cases with male infertility. Here, we examined the efficacy of l-carnitine in improving sperm motility and vitality and reducing sperm DNA oxidation during cryopreservation. Semen samples from infertile patients (n = 22) were collected and analysed. Cryopreservation medium supplemented with l-carnitine was mixed with the semen at a ratio of 1 : 1 (v/v). The final l-carnitine concentration in each cryovial was 0.5 mg ml−1 per 5 × 106 cell ml−1. Controls were cryopreserved without addition of l-carnitine. After 24 h of cryopreservation, thawed sperm samples were analysed for motility, vitality and DNA oxidation. Sperm vitality was assessed by the eosin–nigrosin test, while sperm DNA oxidation was measured by flow cytometry. Addition of l-carnitine significantly improved sperm motility and vitality (P \u3c 0.05) compared with the control. The flow cytometry experiment showed no statistical difference (P \u3e 0.05) in the levels of DNA oxidation between samples and controls. In conclusion, l-carnitine improves human sperm motility and vitality, but has no effect on sperm DNA oxidation after cryopreservation

Electron Transfer Catalysis of The Hydrogenolysis of Acyl Dicarbonyl Cyclopentadienyliron Complexes by Tributyltin Hydride

The conversion of acyl dicarbonyl cyclopentadienyliron compounds Cp(CO)2Fe(COR) into aldehydes is not straightforward. It is shown here, mainly from electrochemical results, that the hydrogenolysis of the metal acyl bond can be efficiently achieved by trialkytin hydrides under very mild conditions, according to a chain reaction process initiated by an electron transfer to the acyl complex. The expected aldehyde is formed together with the heterobinuclear iron-tin complex

Nanostructured Poly(3,4-Ethylenedioxythiophene)–Metalloporphyrin Films: Improved Catalytic Detection of Peroxynitrite

We investigated in this paper the sensing performance of inherently conductive polymer, poly(3,4-ethylenedioxythiophene) (PEDOT), functionalized with hemin (iron protoporphyrin) as an electrocatalytic reporter. The sensing platform is prepared by electrodeposition of a composite film of hemin–PEDOT on a 30-μm diameter carbon fiber electrode (CFE). The polymerized films were characterized by field emission scanning electron microscopy (FESEM), which pointed to nanostructured films with tortuous pores. The electrocatalytic oxidation of peroxynitrite was characterized by cyclic voltammetry as well as other electrochemical methods. The catalytic current is proportional to the analyte\u27s concentration. Optimized hemin–PEDOT modified CFEs were utilized for the first time to detect ONO2-, with a response time down to 5 s and a limit of detection as low as 200 nM as evidenced by amperometry. Our hemin–PEDOT modified CFEs have a sensitivity of 13 nA/μM, ca.130 times higher than the bare CFE. More work is underway using other metalloporphyrins as electrocalalysts to improve the detection limit, the selectivity, and to further miniaturize these hemin–PEDOT modified electrodes

Electron Transfer Catalysis of The Hydrogenolysis of Acyl Dicarbonyl Cyclopentadienyliron Complexes by Tributyltin Hydride

The conversion of acyl dicarbonyl cyclopentadienyliron compounds Cp(CO)2Fe(COR) into aldehydes is not straightforward. It is shown here, mainly from electrochemical results, that the hydrogenolysis of the metal acyl bond can be efficiently achieved by trialkytin hydrides under very mild conditions, according to a chain reaction process initiated by an electron transfer to the acyl complex. The expected aldehyde is formed together with the heterobinuclear iron-tin complex

Reductive Decomposition of A Diazonium Intermediate by Dithiothreitol Affects The Determination of NOS Turnover Rates

Accurate determination of nitrite either as such or as the breakdown product of nitric oxide (NO) is critical in a host of enzymatic reactions in various settings addressing structure–function relationships, as well as mechanisms and kinetics of molecular operation of enzymes. The most common way to quantify nitrite, for instance in nitric oxide synthase (NOS) mechanistic investigations, is the spectrophotometric assay based on the Griess reaction through external standard calibration. This assay is based on a two-step diazotization reaction, in which a cationic diazonium derivative of sulfanilamide is formed as intermediate before the final absorbing azo-product. We show that this intermediate is very sensitive to reducing agents that may be transferred from the reaction media under investigation. The interaction of this vital intermediate with the reducing agent, dithiothreitol (DTT), which is widely used in NOS reactions, is characterized by both electrochemical and spectroscopic means. The effect of DTT on the performance of external calibration, both in sample recovery studies and in actual NOS reactions, is presented. Finally an alternative method of standard additions, which partially compensates for the accuracy and sensitivity problems of external calibration, is proposed and discussed

Human Sperm DNA Oxidation, Motility and Viability in The Presence of l-Carnitine During in Vitro Incubation and Centrifugation

In vitro incubation and centrifugation is known to decrease human sperm quality. In the human body, besides its antioxidant effects, l-carnitine (LC) facilitates the transport of activated fatty acids from the cytosol to the mitochondrial matrix. In this study, we investigated the effect of LC on human sperm motility, viability and DNA oxidation after incubation and centrifugation, following the sperm preparation protocols of assisted reproduction. Normozoospermic semen samples (n = 55) were analysed according to the World Health Organization (WHO) guidelines. LC concentrations that are not toxic to spermatozoa as determined by sperm motility and viability were standardised after 2 and 4 h of incubation at 37 °C. Semen samples to which the optimal LC concentrations were added were also centrifuged for 20 min at 300 g and analysed for sperm motility, viability and DNA oxidation. Sperm motility was improved at 0.5 mg ml−1 LC after incubation and centrifugation with 5 × 106 sperm ml−1. Higher concentration of LC (50 mg ml−1) significantly decreased sperm motility and viability. LC did not alter the baseline of sperm DNA oxidation during both incubation and centrifugation. In conclusion, LC may enhance sperm motility following incubation and centrifugation, while it might not affect sperm viability and DNA oxidation

Manganese Oxide/Hemin-Functionalized Graphene as a Platform for Peroxynitrite Sensing

Peroxynitrite (ONOO−, PON) is a powerful oxidizing agent generated in vivo by the diffusion-limited reaction of nitric oxide (NO) and superoxide (O2˙−) radicals. Under oxidative stress, cumulated peroxynitrite levels are associated with chronic inflammatory disorders and other pathophysiological conditions. The accurate detection of peroxynitrite in biological systems is important, not only to understand the genesis and development of diseases, but also to explore and design potential therapeutics. Herein, a manganese oxide/hemin-modified graphene interface is explored as a platform for peroxynitrite amperometric detection. Hemin-functionalized reduced graphene oxide was further modified with manganese oxide nanoparticles to provide a composite material with catalytic activity toward the electrochemical oxidation of peroxynitrite. The morphology of the composite material was characterized using scanning electron microscopy, energy dispersive X-ray analysis, X-ray photoelectron spectroscopy, and UV-Vis absorption measurements. We investigated the electrocatalytic oxidation of peroxynitrite on graphite electrodes modified with the composite material using cyclic voltammetry and amperometry. The results showed that the incorporation of manganese oxide nanoparticles into graphene/hemin material enhances the catalytic detection of peroxynitrite compared to graphene/hemin alone