Lipid Hydroperoxide Assay

Oxidative damage to lipids (lipid peroxidation) has been found to play an important role in various disease and aging processes. During the early stages of lipid peroxidation, lipid hydroperoxides (LOOH) are formed as a result of fatty acidoxidation by any species sufficiently reactive to abstract hydrogen from a methylene group, to form a carbon-centered radical that reacts with molecular oxygen to form the lipid hydroperoxide. LOOH can be used as a biomarker to detect and quantify early stage lipid peroxidation.

Introduction

Lipids are a varied group of water insoluble compounds which function as energy storage molecules, structural components of biological membranes, enzyme cofactors, intracellular messengers, and other critical biological functions. All lipids are derivatives of fatty acids. Fatty acids are carboxylic acids containing aliphatic chains of 4-36 carbons. Saturated fatty acids contain no carbon-carbon double bonds, monounsaturated fatty acids have one -C=C- bond while polyunsaturated fatty acids (PUFA) contain 2 or more -C=C- bonds. Lipid hydroperoxides (LOOH) are the result of oxidation of fatty acids by any species sufficiently reactive to abstract hydrogen from a methylene group, forming a carbon-centered radical that reacts with molecular oxygen to form the lipid peroxide. Lipid peroxidation occurs in two distinct steps; initiation and propagation.

Initiation: Allylic hydrogens (methylene hydrogen with an adjacent carbon-carbon double bond) possess a weakened H-C bond and as such are especially prone to abstraction, forming a carbon centered radical. The carbon-centered radical can then react with molecular oxygen to produce the lipid peroxyl radical. This radical in-turn abstracts hydrogen to form a lipid hydroperoxide.

Propagation: The lipid peroxyl radical can then abstract hydrogen from another lipid molecule. The resulting radical (LO" or L" ) can then continue a free radical chain reaction. Iron chelates (DNA-Fe, ATP-Fe, etc.), heme iron (hemoglobin, myoglobin, cytochrome c, etc.) and oxidized and reduced copper all react with LOOH to form LOO" , facilitating propagation of lipid peroxidation.

Method:

The NWLSS™ Lipid Hydroperoxide Assay is based on the reaction of LOOH with ferrous iron to form ferric iron and the subsequent reaction of ferric iron with 3,32 -Bis[N,N bis (carboxymethyl)aminomethyl]-o-cresolsulfo-nephthalein disodium salt (Xylenol Orange reagent) to form a chromagen (XOF complex)with measurable absorbance at 560 nm.

LHP 01 Reaction

The slope of the regression of A₅₆₀ vs concentration of LOOH is equal to the molar absorption coefficient of the XOF complex thus providing the basis for data analysis.

Method Improvements:

The NWLSS™ method minimizes possible errors associated with:

• Variable LOOH Recovery: A standard is supplied for use as an internal calibrator to correct for variable recovery of LOOH. This may also be used to generate a standard curve if one is desired.
• Sample H₂O₂: Catalase enzyme is supplied to negate possible H₂O₂ contribution.
• Sample iron content: Reductant (TCEP) is supplied to negate contributions from endogenous iron content.