Novel reseach tools for Lipid peroxidation (LPO) LipiRADICAL Green / OH-Pen

• LipiRADICAL Green / OH-Pen:
  Help to understand the molecular mechanism and physiological relevance of LPO
• Features
• Reference data
• Application data
• Reference
• Price

LipiRADICAL Green / OH-Pen:
Help to understand the molecular mechanism and physiological relevance of LPO

"LipiRADICAL Green” is the world’s first lipid radical responsive fluorescent dye. “LipiRADICAL Green” is a green fluorescent dye NBD-conjugated nitroxyl radical-derivative (Below figure). Although this compound contains NBD, the probe ’s fluorescence is highly quenched by intramolecular radical moiety. When the probe reacts with lipid radicals via radical-radical coupling forming a covalent bond to lipids, the fluorescent intensity is drastically recovered. “LipiRADICAL Green" is well validated to selectively detect lipid radicals, not reactive oxygen radicals. “LipiRADICAL Green" enables us to semi-quantification of lipid radicals in biological samples, to image cellular lipid radicals and to identify and analyze the molecular structure of lipid radicals with LC/MS system. “LipiRADICAL Green" is an innovative and powerful tool for LPO research.


"OH-Pen” is also powerful tools to research LPO. This is a unique lipid radical specific inhibitor which will not react with other reactive oxygen species. OH-Pen has similar structure to LipiRADICAL Green; NBD in LipiRADICAL Green was just converted to hydroxyl group.

Go back to index

Features

LipiRADICAL Green

• The reagent itself is highly quenched and shows little fluorescence. After reaction with lipid radicals, it emits green fluorescence in lipid radical dose-dependent manner.
• Ex. / Em. 470 nm / 520-600 nm (max 540 nm); commercial FITC/GFP conditions are compatible.
• Applications
  In vitro detection of lipid radicals by fluorescent detection
  Live cell imaging of lipid radicals by fluorescent microscopy
  Screening of LPO suppressor or anti-oxidant both in vitro and in cellulo
  Structural analysis of lipid radicals by fluorescent-LC/MS-MS etc.
  

OH-Pen

• OH-Pen can be used as LPO suppressor via neutralizing lipid radicals
• OH-Pen is a very stable compound though it is a radical compound and can be stably injected into animals
• OH-Pen neutralizes lipid radicals in situ

Go back to index

Reference data

Fluorescent spectrum

“LipiRADICAL Green” was added to arachidonic acid-lipoxygenase (LOX) mixtures and observed fluorescence excited by 470 nm light. In the absence of LOX enzyme, the fluorescent signal was highly quenched (Black line). In the presence of LOX enzyme, green fluorescence (500-650 nm, maximum ~540 nm) was detected in LOX dose-dependent manner.

Specificity

“LipiRADICAL Green” was treated with the following reagents and fluorescent intensity (Ex 470 nm/Em 530 nm) was observed. All reactive oxygen species had little effects on the fluorescent intensity of “LipiRADICAL Green”. Green fluorescence was only observed under the polyunsaturated lipids (laulic acid (LA), alpha-laulic acid (ALA) or arachidonic acid (AA)) with LOX enzyme or pro-oxidants including AAPH and MeO-AMVN.

Reagents and conditions
LipiRADICAL Green (5 μM) H2O2, ClO-, KO2 for O2-・and ・OH : 0.5 mM Lipids (0.5 mM) with LOX (2.5 μg/ml) , AAPH (10 mM) or MeO-AMVN 50 μM

Go back to index

Application data

Cell-based detection of lipid radicals induced by diethylnitrosamine (DEN)

Hepa1-6 cells were treated with 1 μM of “LipiRADICAL Green” for 20 min and washed twice with PBS. For inducing an LPO signal, the cells were co-treated with diethylnitrosamine (DEN) and “LipiRADICAL Green”, an LPO initiator. Immediately after DEN addition, the cells were observed by confocal microscopy (Ex.458 nm/ Em. 490-674 nm) for 20 min with 2 min interval. The fluorescent signal of “LipiRADICAL Green” from the 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 pro-oxidants hemin or AAPH and “LipiRADICAL Green” and the green fluorescence (Ex. 470 nm/ Em 530 nm) was measured for 60 min at 37℃. Both hemin and AAPH increased green fluorescence indicating the production of lipid radicals from LDL particles in a time-dependent manner.

Structural analysis of lipid radicals derived from arachidonic acid in vitro

Arachidonic acid (AA; 500 μM) was incubated with pro-oxidants hemin (10 μM) and AAPH (50 mM) mixture for 60 min. After incubation, 5 μM of “LipiRADICAL Green” was added to the reaction mixture and incubated for 15 min at RT. Lipid components were extracted by the Bligh and Dyer method and analyzed by the LC-FL/MS-MS technique. (Upper panel) The fluorescent chromatogram is shown (Ex. 470/E. 530 nm). Several fluorescent peaks were observed and each peak was further analyzed by MS-MS. (Lower panel) Product profiles of AA-derived radicals are shown. MS-MS analysis identified a total of 8 full-length AA radicals and 29 truncated radicals. The relative abundances of each radical were calculated from each peak area. Detailed experimental protocol and analytical procedure are described in Ref.5.

Structural analysis of lipid radicals in vivo

A well-known carcinogen, diethylnitrosamine (DEN, 100 mg/kg body weight), was injected intraperitoneally into mice and after 1, 4 and 24 hours, mice were anesthetized. Anesthetized mice then received intraperitoneal injections of “LipiRADICAL Green” (2.5 μmol/kg body weight). To check the specificity of “LipiRADICAL Green”, OH-Pen, a specific inhibitor of lipid radical (Catalog no. #FDV-0042; 10 μmol/kg body weight) was also injected into the mice before “LipiRADICAL Green” injection. The liver was removed from the mice and homogenized with methanol. Lipid solution was extracted from the liver homogenate according to the Bligh and Dyer method. Lipid samples were applied to LC-FL/MS-MS for analysis (Left). After 4 hours of treatment of DEN, there was a high production of lipid radicals. A total of 11 lipid radicals were identified. (Right) An example, a ・C5H11 radical. OH-Pen-preinjection clearly inhibited the production of lipid radicals derived from DEN treatment.

Inhibition of nitrosamine-induced carcinogenesis by OH-Pen

Rats received diethylnitrosamine (DEN, 100 mg/kg body weight), which is a well-known hepatic procarcinogen. Subsequently, rats received OH-Pen (2.5 μmol/kg body weight) by intraperitoneal injection after 1 hour DEN administration. For the acute model and chronic model, livers were dissected after 24 hours and 12 weeks DEN administration, respectively.

(Upper panel) Livers from chronic hepatocelluar carcinoma model and total foci number.

(Middle panel) Quantification of LPO-derived aldehydes in acute model livers.

(Lower panel) Quantification of tissue damage markers.

In all panels, OH-Pen clearly suppressed DEN-induced hepatocellular carcinoma.

Go back to index

Reference

Go back to index

Price

Go back to index