LipiRADICAL Green (Lipid Radical Detection Reagent)

Overview

LipiRADICAL Green is a lipid radical-responsive fluorescent dye: an NBD-conjugated nitroxyl radical derivative. It selectively detects lipid radicals, not reactive oxygen radicals. LipiRADICAL Green enables semi-quantification of lipid radicals in biological samples, imaging of cellular lipid radicals, and identification and structural analysis of lipid radicals with an LC/MS system.

Background: Lipid Peroxidation

Lipid peroxidation (LPO) is one of several degradation processes of lipids under oxidative stress. The primary products of LPO are lipid radicals. Two major initiators induce LPO: pro-oxidants, and lipid oxidative enzymes including lipoxygenase (LOX) and cytochrome P450 (CYP).

These reactive aldehydes are cytotoxic and are considered causative factors of organ injury, ferroptosis and ER stress. Understanding the molecular mechanism and physiological relevance of LPO requires methods to detect and quantify lipid radicals, but conventional methods are highly limited. For example, electron spin resonance (ESR) is a major strategy to detect radical products but is not applied to cell-based assays.

Detection Principle

Applications

Specifications

Chemical Structure

Reconstitution & Storage

Application Data

Cell-Based Detection of Lipid Radicals Induced by Diethylnitrosamine (DEN)

Hepa1-6 cells were treated with 1 µM LipiRADICAL Green for 20 min and washed twice with PBS. To induce an LPO signal, the cells were co-treated with LipiRADICAL Green and diethylnitrosamine (DEN), an LPO initiator. Immediately after DEN addition, the cells were observed by confocal microscopy (Ex 458 nm / Em 490-674 nm) for 20 min at 2-min intervals. The LipiRADICAL Green signal from DEN-treated cells clearly increased.

In Vitro Detection of Lipid Radicals Derived from LDL

Purified low-density lipoprotein (LDL, 20 µg protein/mL) was mixed with the pro-oxidant hemin or AAPH and LipiRADICAL Green, and green fluorescence (Ex 470 nm / Em 530 nm) was measured for 60 min at 37°C. Both hemin and AAPH increased green fluorescence in a time-dependent manner, indicating the production of lipid radicals from LDL particles.

Structural Analysis of Lipid Radicals Derived from Arachidonic Acid In Vitro

Arachidonic acid (AA; 500 µM) was incubated with a mixture of the pro-oxidants hemin (10 µM) and AAPH (50 mM) for 60 min. After incubation, 5 µM LipiRADICAL Green was added to the reaction mixture and incubated for 15 min at room temperature. Lipid components were extracted by the Bligh and Dyer method and analyzed by LC-FL/MS-MS.

• Upper panel: Fluorescence chromatogram (Ex 470 nm / Em 530 nm). Each fluorescent peak was further analyzed by MS-MS.
• Lower panel: Product profiles of AA-derived radicals. MS-MS identified 8 full-length AA radicals and 29 truncated radicals. Relative abundances were calculated from each peak area.

Structural Analysis of Lipid Radicals In Vivo

Diethylnitrosamine (DEN, 100 mg/kg body weight), a well-known carcinogen, was injected intraperitoneally into mice; after 1, 4 and 24 hours the mice were anesthetized and received intraperitoneal LipiRADICAL Green (2.5 µmol/kg body weight). To check specificity, OH-Pen, a specific inhibitor of lipid radicals (10 µmol/kg body weight), was injected before LipiRADICAL Green. Livers were removed and homogenized in methanol, lipids were extracted by the Bligh and Dyer method, and the lipid samples were analyzed by LC-FL/MS-MS.

• Left: Lipid radical production was high 4 hours after DEN treatment; 11 lipid radicals were identified.
• Right: Example: the •C₅H₁₁ radical. OH-Pen pre-injection clearly inhibited DEN-induced lipid radical production.

Reference Data: Spectrum & Specificity

Fluorescence Spectrum

LipiRADICAL Green was added to arachidonic acid-lipoxygenase (LOX) mixtures, and fluorescence was observed with 470 nm excitation. Without LOX, the signal was highly quenched (black line). With LOX, green fluorescence (500-650 nm, maximum ~540 nm) was detected in a LOX dose-dependent manner.

Specificity

LipiRADICAL Green was treated with the reagents below, and fluorescence intensity (Ex 470 nm / Em 530 nm) was measured. Reactive oxygen species had little effect on the fluorescence. Green fluorescence was observed only with polyunsaturated lipids (linoleic acid (LA), α-linolenic acid (ALA) or arachidonic acid (AA)) in combination with LOX or the pro-oxidants AAPH and MeO-AMVN.

Research Applications

Publications from the References section, grouped by application area.

References

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