Lipid peroxidation inhibition capacity assay for antioxidants based on liposomal membranes

Abstract

An in vitro antioxidant assay has been developed to better reflect the in vivo conditions of antioxidants interacting with membrane and lipid surfaces. The lipid peroxidation inhibition capacity (LPIC) method measures the ability of both lipophilic and hydrophilic antioxidants to protect a lipophilic fluorescent probe 4, 4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid, incorporated in the membrane, from 2,2'-azobis(2-amidinopropane)hydrochloride generated radicals in the surrounding aqueous solution. Antioxidant activities of test compounds were measured either after they were mixed with preformed liposomes (LPIC(Mixed)) or after they were incorporated into liposomes (LPIC(Inco)) as they were made. The results were analysed to determine how the method of mixing and the structures of the antioxidants influenced their protection of the membrane from free radical attack. The LPIC(Mixed) values were larger than the LPIC(Inco) values for a range of 12 structurally diverse antioxidant compounds. However, there was no linear correlation between the lipophilicities, as measured by their partition coefficient, log P and either LPIC(Inco) or LPIC(Mixed) values. A strong correlation was found between LPIC(Inco) and LPIC(Mixed) values.

Free radical scavenging and cytoprotective activities of phenolic antioxidants

“…The analysis of flow cytometric data was carried using CXP data analysis software (Beckman Coulter, Miami, FL [11]. This may be due to higher incorporation of antioxidants during the making of the vesicles, and therefore more activity, being achieved by the LPI-C Inco method.…”

“…The LPIC assay thus measures the ability of antioxidants to protect a substrate embedded in a lipid membrane. Hence, the LPIC assay is suitable to study the effectiveness of antioxidants against membrane lipid peroxidation, which is an important mechanism in causing oxidative stress to the cells [11].…”

“…The antioxidants were either incorporated into liposomes (LPIC Inco ) or mixed with preformed DOPC-unilamellar vesicles (LPIC Mixed ) and the LPIC assay was performed as previously described [11]. Briefly, in the final assay mixtures (2 mL of total volume), the fluorescent probe C 11 -BODIPY (2.4 lM) was used as a target of free radical damage, AAPH (40 mM) as a peroxyl radical generator and Trolox as a control standard.…”

“…However, many phenolic antioxidants such as flavonoids and phenolic acids have a high lipophilicity and therefore will partition into a hydrophobic phase more than into water. The lipid peroxidation inhibition capacity (LPIC) assay [11] uses a fluorescent 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (C 11 -BOD-IPY) probe dissolved into the membrane phase of the liposome. This assay simulates the ORAC assay but in a liposomal form that is sensitive to the membrane partitioning behavior of antioxidants.…”

Polyphenolic phytochemicals – just antioxidants or much more?

“…Flavonoid aglycones should be hydrophobic enough to incorporate into cell membranes and lipoprotein lipids and interfere with lipid peroxidation. Flavonoid aglycones have log P values of~2 (similar to many synthetic drugs) and cinnamic acids values of~1 [44]. Circulating PPs, however, are predominantly conjugated and the log P of the glucuronyl residue is À 2.3 (calculated using Chemdraw ), so metabolic conjugates of PPs would have log P values around zero or negative.…”