Paraoxon ethyl

Manufactured by Merck Group

Sourced in United States

Paraoxon-ethyl is a laboratory reagent used for biochemical research and analysis. It is an organophosphate compound that acts as a cholinesterase inhibitor. Paraoxon-ethyl is commonly utilized in enzymatic assays and other experimental applications requiring a cholinesterase inhibitor. Its core function is to enable the study of cholinergic systems and related biological processes.

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27 protocols using paraoxon ethyl

Acetylcholinesterase inhibitor screening

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Aldicarb (Cas n. 116-06-3) and paraoxon-ethyl (Cas n. 311-45-5) were acquired from Merck. Aldicarb was dissolved in 70% ethanol and paraoxon-ethyl was dissolved in 100% DMSO in a stock concentration of 250 mM and 1M, respectively. The drug stocks were kept at 4°C and used within one month or discarded. Obidoxime was provided by Dstl Porton Down (UK) and dissolved in distilled autoclaved water directly before use. Acetylcholine was purchased from Merck and dissolved in recording buffer (100 mM NaCl, 2.5 mM KCl, 1 mM CaCl₂.2H₂O, 5 mM HEPES, pH 7.3)

Izquierdo P.G., Charvet C.L., Neveu C., Green A.C., Tattersall J.E., Holden-Dye L, & O’Connor V. (2023). Modelling organophosphate intoxication in C. elegans highlights nicotinic acetylcholine receptor determinants that mitigate poisoning. PLOS ONE, 18(4), e0284786.

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Acetylcholinesterase Inhibitors Assay Protocol

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Carbamate (aldicarb) and organophosphates (paraoxon-ethyl, paraoxon-methyl and DFP) were acquired from Merck and dissolved in 70% ethanol and 100% DMSO, respectively. The oximes, obidoxime and 2-pralidoxime, were provided by DSTL Porton Down (UK) and dissolved in distilled autoclaved water. The drug stocks were kept at 4ᵒC, as manufacturer recommended temperature, in a locked cabinet according with standard security protocols. Dissolved compounds were used within one month or discarded.
Acetylthiocholine iodide (ATCh) and 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB) were obtained from Merck (https://www.sigmaaldrich.com/united-kingdom.html) and dissolved in phosphate buffer 0.1 M pH7.4 directly before use.

Izquierdo P.G., O'Connor V., Green A.C., Holden-Dye L, & Tattersall J.E.H. (2021). C. elegans pharyngeal pumping provides a whole organism bio-assay to investigate anti-cholinesterase intoxication and antidotes. Neurotoxicology, 82.

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Paraoxon-Ethyl Inhibition of Cholinesterase

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All required materials and reagents, including PChE standard solutions, Acetylthiocholine iodide (ATCl), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), paraoxon-ethyl, and phosphate buffer saline (PBS) were purchased from Sigma-Aldrich (St. Louis,MO, USA). PChE standard solution (2 μg/mL), ATCl solution (2 wt‰), DTNB solution (0.2 wt‰), and serial-diluted paraoxon-ethyl standard solutions were all prepared with PBS (pH 7.4). We used paraoxon-ethyl as the OP model and spiked paraoxon-ethyl into PChE standard solution and purchased human plasma, respectively. paraoxon-ethyl inhibited PChE enzyme and led to PChE activity decreasing. The assay procedure was as follows: 50 μL PChE standard solution/purchased human plasma were added into centrifuge tubes, and then 50 μL paraoxon-ethyl serial-diluted solutions (0–100 nM) were respectively added into each centrifuge tubes to mix and to incubate. After 15 min incubation at 37 °C, 100 μL ATCl solutions were added into each tube for 15 min incubation (37 °C). In this step, uninhibited PChE enzyme hydrolyzed ATCl and thiocholine was produced. Then, 200 μL DTNB solutions were added to each tube to react with thiocholine producing the yellow 5-thio-2-nitrobenzoate anion (TNB), which was detected using the microplate reader and the LeSSA at 410 nm wavelength [40 (link)–42 (link)].

Sakanaka C., Leong P., Xu L., Harrison S.D, & Williams L.T. (1999). Casein kinase Iɛ in the Wnt pathway: Regulation of β-catenin function. Proceedings of the National Academy of Sciences of the United States of America, 96(22), 12548-12552.

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Serum Paraoxonase Activity Measurement

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Serum paraoxonase activity was determined by measuring the hydrolysis of paraoxon to p-nitrophenol and diethyl phosphates in the presence of paraoxonase as a catalyst. Briefly, 10 μL of diluted serum was added to 200 μL of paraoxon-ethyl (Sigma D-9286; Sigma-Aldrich, St. Louis, MO, USA) in a buffer containing 90 mM Tris-HCl, 3.6 mM NaCl, and 2 mM CaCl₂ (pH 8.5). The production of p-nitrophenol at 37 °C was determined by measuring the absorbance at 405 nm using a Microplate reader (Bio-Rad, Hercules, CA, USA). A paraoxonase activity of 1 U/L was defined as the formation of 1 μmol of p-nitrophenol per minute [28 (link)].

Park J.K., Kim J.Y., Moon J.Y., Ahn E.Y., Lee E.Y., Lee E.B., Cho K.H, & Song Y.W. (2016). Altered lipoproteins in patients with systemic lupus erythematosus are associated with augmented oxidative stress: a potential role in atherosclerosis. Arthritis Research & Therapy, 18, 306.

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Paraoxon-ethyl Toxicity Assay

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4 mL of paraoxon-ethyl (Sigma-Aldrich) in water were prepared at different concentrations (10 µM–50 µM). Experiments were performed using 12-well microplates. Five worms were incubated per well and experiments were performed in duplicates. Mortality was followed for 14 days.

Poirier L., Plener L., Daudé D, & Chabrière E. (2020). Enzymatic decontamination of paraoxon-ethyl limits long-term effects in planarians. Scientific Reports, 10, 3843.

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Paraoxonase-I Activity and Total Antioxidant Capacity

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PON-I antioxidant enzyme activity was evaluated in HDL-c isolated by the hydrolysis of diethyl p-nitrophenol phosphate (paraoxon-ethyl) (Sigma-Aldrich, MO, USA). The absorbance was quantified at 405 nm. Enzyme activity was calculated according to the molar extinction coefficient of p-nitrophenol, which is 18,053 (mol/L)^− 1 • cm^− 1. Activity is expressed as nmol p-nitrophenol/dL Apo-A/min [23 (link)]. The total antioxidant capacity assay was measured in plasma by a method based on cupric-reducing antioxidant capacity (CUPRAC) using copper (II) 10 mM and neocuproine reagent 7.5 mM. The reaction mixture was incubated for 20 min at room temperature to quantify non-protein antioxidants and 50 °C to quantify protein antioxidants. The absorbance was determined at 450 nm. A calibration curve was obtained using 2 mM trolox (6-Hydroxy-2, 5, 7,8-tetramethylchromane-2-carboxylic acid) (Sigma-Aldrich, MO, USA) as the standard [24 (link)]. The results are expressed as trolox equivalents (TR-equivalent). The TR-equivalent is defined as the nanomolar concentration of a trolox solution with antioxidant capacity equivalent to a 2.0 mM solution of the substance under investigation.

León-Reyes G., Espino y Sosa S., Medina-Navarro R., Guzmán-Grenfell A.M., Medina-Urrutia A.X., Fuentes-García S., Hicks G.J, & Torres-Ramos Y.D. (2018). Oxidative modifications of foetal LDL-c and HDL-c lipoproteins in preeclampsia. Lipids in Health and Disease, 17, 110.

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Paraoxon Exposure Dosage Protocol

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Paraoxon ethyl (Sigma Chemicals Co., St. Louis, MO, USA) stock solution was prepared at a concentration of 10 mmol/l in anhydrous acetone. Working solution was prepared ex tempore in PBS to a final concentration of 80 nmol/ml. Each mouse received 40 nmol/0.5 ml/day of paraoxon (equivalent to 0.44 mg/kg of body weight) by intraperitoneal (i.p.) injection. Control animals received an equivalent volume of PBS.

Al-Barazie R.M., Bashir G.H., Qureshi M.M., Mohamed Y.A., Al-Sbiei A., Tariq S., Lammers W.J., al-Ramadi B.K, & Fernandez-Cabezudo M.J. (2018). Cholinergic Activation Enhances Resistance to Oral Salmonella Infection by Modulating Innate Immune Defense Mechanisms at the Intestinal Barrier. Frontiers in Immunology, 9, 551.

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Spectrophotometric Assay for Serum PON-1 Activity

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Serum PON‐1 activity was measured spectrophotometrically using an automated analyzer (Cobas Mira, Roche diagnostic, Basel, Switzerland), and an enzymatic method already validated in horses.12 Briefly, 6 μL samples were incubated at 37°C with 89 μL of distilled water and 100 μL of reaction buffer (glycine buffer 0.05 mM, pH 10.5 containing 1 mM of paraoxon‐ethyl, purity >90% [Sigma‐Aldrich, Saint Louis, Missouri], and 1 mM of CaCl₂). The rate of hydrolysis of paraoxon to p‐nitrophenol was measured by monitoring the increase in absorbance at 504 nm using a molar extinction coefficient of 18.050 L/mol/cm⁻¹ as previously suggested.2 The unit of PON‐1 activity expressed as U/mL is defined as 1 nmol of p‐nitrophenol formed per minute under the assay conditions.

Ruggerone B., Paltrinieri S., Giordano A., Scavone D., Nocera I., Rinnovati R., Spadari A., Scacco L., Pratelli P, & Sgorbini M. (2020). Paraoxonase‐1 activity evaluation as a diagnostic and prognostic marker in horses and foals. Journal of Veterinary Internal Medicine, 34(2), 949-954.

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Paraoxonase-1 Activity Quantification

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The paraoxonase-1 (PON-1) activity toward paraoxon was determined by evaluating the hydrolysis of paraoxon to p-nitrophenol and diethylphosphate catalyzed by the enzyme [7 (link),56 (link)]. Equally diluted CML-treated HDL3 (20 μL, 2 mg/mL), was added to 180 μL of paraoxon-ethyl (Sigma Cat. No. D-9286) containing solution (90 mM Tris-HCl/3.6 mM NaCl/2 mM CaCl2 (pH 8.5)) with or without CIGB-258. The PON-1 activity was then determined by measuring the initial velocity of p-nitrophenol production at 37 °C, as determined by measuring the absorbance at 415 nm (microplate reader, Bio-Rad model 680; Bio-Rad, Hercules, CA, USA).

Cho K.H., Kim J.E., Nam H.S., Kang D.J, & Na H.J. (2022). Anti-Inflammatory Activity of CIGB-258 against Acute Toxicity of Carboxymethyllysine in Paralyzed Zebrafish via Enhancement of High-Density Lipoproteins Stability and Functionality. International Journal of Molecular Sciences, 23(17), 10130.

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Determination of PON-1 Activity in HDL3

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The paraoxonase-1 (PON-1) activity toward paraoxon was determined by evaluating the hydrolysis of paraoxon to p-nitrophenol and diethylphosphate catalyzed by the enzyme [24 (link)]. Equally diluted HDL₃ (20 μL, 2 mg/mL) was added to 180 μL of paraoxon-ethyl (Sigma Cat. No. D-9286) containing asolution (90 mM Tris-HCl/3.6 mM NaCl/2 mM CaCl₂ (pH 8.5)) with either OSO or SO (final 1, 2, 4%). The PON-1 activity was then determined by measuring the initial velocity of p-nitrophenol production at 37 °C, as determined by measuring the absorbance at 415 nm (microplate reader, Bio-Rad model 680; Bio-Rad, Hercules, CA, USA).

Cho K.H., Kang D.J., Nam H.S., Kim J.H., Kim S.Y., Lee J.O, & Kim B.J. (2021). Ozonated Sunflower Oil Exerted Protective Effect for Embryo and Cell Survival via Potent Reduction Power and Antioxidant Activity in HDL with Strong Antimicrobial Activity. Antioxidants, 10(11), 1651.

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