Spin trap agent
DMPO targets free radicals by forming stable adducts with them. It is a spin-trapping reagent used to study radical formation in enzymatic and cellular systems. The compound also has a role as a neuroprotective agent. Incubating lymphocytes with DMPO decreases DNA damage by NiCl₂.

Post-treatment of cells with DMPO attenuated SIN-1-mediated cytotoxicity and ROS generation, restoration of NO levels via increased in eNOS activity and phospho-eNOS levels. Treatment with DMPO alone significantly increased NO levels and induced phosphorylation of eNOS Ser¹¹⁷⁹ via Akt kinase. Transfection studies with wild-type and mutant human eNOS confirmed the dual role of eNOS as a producer of superoxide anion (O₂⁻) with SIN-1 treatment, and a producer of NO in the presence of DMPO [4].
Unlike direct ESR, spin trap methodology depends on the absolute fidelity of the spin trap reaction. Two alternative reactions of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) leading to radical adduct artifacts have been discovered and investigated: inverted spin trapping and the Forrester-Hepburn nucleophilic mechanism. These two alternate pathways to radical adducts are a combination of one-electron oxidation and nucleophilic addition, in either order. In biological systems, serious artifacts have been reported due to the Forrester-Hepburn mechanism, which is initiated by the addition of a nucleophile to DMPO. It has recently been demonstrated that (bi)sulfite (hydrated sulfur dioxide) can react with DMPO via a nonradical, nucleophilic reaction, and it has been further proposed that DMPO/(•)SO(3)(-) formation in biological systems is an artifact and not the result of spin trapping of sulfur trioxide anion radical ((•)SO(3)(-)). The one-electron oxidation of (bi)sulfite catalyzed by horseradish peroxidase (HRP)/hydrogen peroxide (H(2)O(2)) has been reinvestigated by ESR spin trapping with DMPO and oxygen uptake studies to obtain further evidence for the radical reaction mechanism. In the absence of DMPO, the initial rate of (bi)sulfite-dependent oxygen and H(2)O(2) consumption was determined to be half of the initial rate of DMPO/(•)SO(3)(-) radical adduct formation as determined by ESR, demonstrating that, under our experimental conditions, DMPO exclusively forms the radical adduct by trapping the (•)SO(3)(-) [1].
The spin traps substituted with some groups at the 4-position of dimethyl-1-pyrroline N-oxide(DMPO) were compared with DMPO itself regarding their abilities as spin traps and their physical properties. 4,5,5-Trimethyl-1-pyrolline N-oxide (4MDMPO) and 5,5-dimethyl-4-phenyl-1-pyrolline N-oxide (4PDMPO) were synthesized by the Bonnett method, and 5,5-dimethyl-4-hydroxymethyl-1-pyrolline N-oxide (4HMDMPO) was made by a unique method from 2(5H)-furanone. The melting points of 4MDMPO, 4PDMPO and 4HMDMPO were higher than that of DMPO. The magnitude of hydrophilicity was in the order of 4HMDMPO, DMPO, 4MDMPO, and 4PDMPO based on the partition coefficient experiments in a 1-octanol--water system. Several radicals, O2-., HO., .CH3, .CH2OH, .CH(CH3)OH, (CH3)3CO. and H. radicals, were trapped with these DMPO derivatives for comparison with the trapping by DMPO itself. Spin adducts of O2-. with the three DMPO derivatives showed ESR spectra similar to that of DMPO. In spite of the formation of diastereomers arising from spin trapping, the line-width enlargement was very small. The intensities and the decay rates of the spectra of 4MDMPO-O2-, 4PDMPO-O2-, 4HMDMPO-O2- and DMPO-O2- were almost equal. In the trapping of the .OH radical by 4MDMPO, 4PDMPO and 4HMDMPO, the eight-line ESR spectra observed were different from the well-known four-line spectrum of DMPO-OH [2].

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In vitro, DMPO is used to detect free radicals generated by enzymatic reactions, such as acetaldehyde oxidation, and in cellular systems to measure oxidative stress. It is a hydrophilic spin trap that can be used in both in vitro and in vivo studies. DMPO is effective for detecting superoxide and hydroxyl radicals in cell culture.

Formalin injected into the left hind paw induced a biphasic nociceptive behaviour. Intraperitoneal injection of DMPO diminished nociceptive behaviors dose-dependently during phase 2 but not phase 1. DMPO (10-100 mg/kg, intraperitoneal injection) has an anti nociceptive effect on formalin induced hyperalgesia in rats [3].

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In vivo, DMPO is used to study oxidative stress in animal models. It can be administered to animals to trap free radicals generated in vivo, which are then detected by EPR spectroscopy. DMPO has also been shown to have neuroprotective effects.

Cell-free assays for DMPO involve incubating the spin trap with a free radical-generating system, such as the xanthine/xanthine oxidase system for superoxide or the Fenton reaction for hydroxyl radicals. The resulting DMPO-radical adducts are then analyzed by EPR spectroscopy to identify the radical species.

BAEC were treated with SIN-1, as a source of peroxynitrite anion (ONOO⁻), and then incubated with DMPO. Cytotoxicity following SIN-1 alone and cytoprotection by adding DMPO was assessed by MTT assay. Levels of ROS and NO generation from HEK293 cells transfected with wild-type and mutant eNOS cDNAs, tetrahydrobiopterin bioavailability, eNOS activity, eNOS and Akt kinase phosphorylation were measured [4].

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In vitro cellular assays for DMPO involve treating cells with the compound and then exposing them to oxidative stress inducers. The formation of DMPO-radical adducts is measured by EPR spectroscopy to quantify the production of specific free radicals within the cells.

5% formalin was injected in the left hind paw after intraperitoneal injection of saline or various doses of DMPO (10 mg/kg, 30 mg/kg, 100 mg/kg). Number of flinches was measured in a 5 minute interval for 1 hour.
Adult male Sprague-Dawley rats (200󰠏300 g) were housed in plastic cages with soft bedding and received free access to food and water. Two rats were housed in each cage, in an environmentally controlled room on a 12 hr light󰠏12 hr dark cycle. All experimental procedures were approved by the Institutional Animal Care and Use Committee at Chungnam National University. Fourty adult rats were divided into 4 groups. One free condition control group and three different experimental groups with different doses of DMPO. Rats were randomly assigned to one of the following treatment groups (n = 10 for each group). The control group (n = 10) were injected with 5 ml of normal saline intraperitoneally 40 minutes before the formalin test. The three experimental groups were injected with 10 mg/kg, 30 mg/kg and 100 mg/kg of DMPO intraperitoneally 40 minutes before performing the formalin test. The intraperitoneal bolus injections were administered in a volume of 5 ml of diluted with saline. After a 5 ml intraperitoneal injection (21 gauge needle) at the right lower abdomen with either saline or DMPO, the rat was placed in a clear, cylindrical, plastic observation chamber. The chamber was then set on a raised wire platform. 40 minutes after intraperitoneal injection, the rats received a 50μl injection of 5% formalin into the plantar of the left hind paw. After formalin injection we counted the total number of spontaneous foot flinches for 5 minutes and recorded it in a 5 minute interval for 60 minutes. Since sedation or anesthesia can affect the mechanical thresholds,4) we performed additional tests in order to see whether DMPO induces sedation or anesthesia. As apposed to analgesia, sedation or anesthesia interferes with posture and righting reflexes. We used a five-point scale for posture and righting reflexes. Five-point scale for posture: 0; normal posture, rearing and grooming; 1; moderate atonia and ataxia. Weight support, but no rearing; 2; weight support, but severe ataxia; 3; muscle tone but no weight support and only small purposive movements; 4; flaccid atonia, fully immobilized with no attempts at movement. Five-point scale for righting reflexes: 0; rat struggles when placed on its side, followed by rapid forceful righting; 1; moderate resistance when the rat is placed on its side, with rapid but not forceful righting; 2; no resistance when the rat is placed on its side, with effortful but ultimately successful righting; 3; insuccessful righting; 4; no movements. Data were presented as means ± standard errors of mean (SEM). Statistical analysis between the control group and experimental group was performed using one-way analysis of variance (ANOVA), followed by Dunnet post hoc tests. Vaules of P ＜ 0.05 were considered statistically significant [3].

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In vivo animal studies for DMPO involve administering the compound to animals, often via intraperitoneal injection, and then collecting tissues or bodily fluids for EPR analysis. This approach is used to study oxidative stress in various disease models.

Pharmacokinetic properties of DMPO are not well characterized, as it is a research reagent rather than a drug. The compound is cell-permeable and hydrophilic, suggesting it can be distributed throughout the body. It is stable under recommended storage conditions.

DMPO is not associated with significant toxicity at the concentrations used for spin trapping. The compound is considered safe for use in research applications. Standard laboratory safety practices apply when handling the compound.

[1]. Ranguelova K, Mason RP. The fidelity of spin trapping with DMPO in biological systems. Magn Reson Chem. 2011;49(4):152-158.

[2]. Konaka R, Kawai M, Noda H, Kohno M, Niwa R. Synthesis and evaluation of DMPO-type spin traps. Free Radic Res. 1995;23(1):15-25.

[3]. Reversal of SIN-1-induced eNOS dysfunction by the spin trap, DMPO, in bovine aortic endothelial cells via eNOS phosphorylation. Br J Pharmacol. 2014 May;171(9):2321-34.

[4]. Britigan BE, Hamill DR. Effect of the spin trap 5,5 dimethyl-1-pyrroline-N-oxide (DMPO) on human neutrophil function: novel inhibition of neutrophil stimulus-response coupling? Free Radic Biol Med. 1990;8(5):459-70.

[5]. The spin trap 5,5-dimethyl-1-pyrroline N-oxide inhibits lipopolysaccharide-induced inflammatory response in RAW 264.7 cells. Life Sci. 2012 Mar 10;90(11-12):432-9.

[6]. Antinociceptive Effect of Intraperitoneally Administered 5,5-dimethyl-1-pyrroline N-oxide on Formalin Induced Nociception in Rats. Korean Journal of Anesthesiology 2008;54(3):S35-S39.

5,5-Dimethyl-1-pyrrololine N-oxide is a 1-pyrrololine nitroketone (1-pyrrololine N-oxide) compound, formed by the N-oxidation of 5,5-dimethyl-1-pyrrololine. It can be used as a spin trapping agent to study free radicals generated by the enzymatic oxidation of acetaldehyde. In addition, it also possesses neuroprotective and spin-trapping properties. Its structure is similar to that of 5,5-dimethyl-1-pyrrololine.

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DMPO is a research reagent with no clinical applications or regulatory approvals. It is widely used as a spin trap for the detection and identification of free radicals in chemical, biochemical, and biological systems. Its role as a neuroprotective agent makes it a subject of interest in neuroscience research.

C6H11NO

113.16

113.084

3317-61-1

1774

Colorless to light yellow solid if <25°C; liquid if >29°C; Melting Point: 25-29 °C

1.0±0.1 g/cm3

274.2±0.0 °C at 760 mmHg

25-29 °C(lit.)

130.7±11.5 °C

0.0±0.5 mmHg at 25°C

1.478

-1.14

0

1

0

8

127

0

CC1(C)CCC=[N+]1[O-]

VCUVETGKTILCLC-UHFFFAOYSA-N

InChI=1S/C6H11NO/c1-6(2)4-3-5-7(6)8/h5H,3-4H2,1-2H3

2,2-dimethyl-1-oxido-3,4-dihydropyrrol-1-ium

5,5-Dimethyl-1-pyrroline-N-oxide; 3317-61-1; 5,5-Dimethyl-1-pyrroline N-oxide; DMPO; 5,5-Dimethyl-1-pyrroline-N-oxide; 5,5-Dimethyl-1-pyrroline-1-oxide; 2,2-Dimethyl-3,4-dihydro-2H-pyrrole 1-oxide; 2,2-dimethyl-1-oxido-3,4-dihydropyrrol-1-ium; MFCD00005279;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O: ≥ 100 mg/mL (883.70 mM)
DMSO: 100 mg/mL (883.70 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (22.09 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (22.09 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (22.09 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)