Lipid Hydroperoxide (LOOH) Assay Kit

NWLSS | Northwest Life Science Specialties, LLC.
Lipid Hydroperoxide (LOOH) Assay Kit
NWK-LHP01 $315.00 each

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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.

Advantages of the NWLSS™ Lipid Hydroperoxide Assay:

  • 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.

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.

Test Principle

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.

LHP01 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.

On page 9 of the protocol under “sample preparation - tissues” it says that the tissue should be homogenized in PBS. Looking at the chemical nature of lipid hydroperoxides we wonder if they are sufficiently soluble to go in solution during homogenization or if they will remain within cell debris that sediment easily or associate with membrane fractions and form some micelle-like structures? Shouldn’t we have to centrifuge the homogenate before assaying it?

The lipids are not soluble in PBS. We specifically don't include a centrifuge instruction on page 9 sample processing guidelines because one needs to sample the complete homogenate on page 10, step 4. The lipids will later become solubilized in methanol at step 8. The NWK-LHP01 protocol is specifically designed so that all reagent additions can be performed without separating the supernatant from the homogenate pellet until reading at the very end of the assay.

At point 10 of the assay protocol (page 10) the samples are centrifuged at 10,000 x g for 3-5 minutes. Then at point 11, 12 and 13 a volume of 10ul of the respective sample or control specific solution is added. The protocol does not mention anything about taking off the supernatant after centrifugation. If the 10ul are added to the preparation including pellet, we are wondering what the centrifugation is used for?

The reason of the 10000 x g centrifuge step # 10 is to effectively “sequester” the debris in a tight pellet at the bottom of the tube keeping it separated for the most part from the supernatant fraction for the rest of the assay. If the customer prefers, the supernatant from step 10 can be transferred to other tubes at this point but this would require another set of vials and is not necessary.

Steps 14 and 17 each call for a brief one second vortex to assure mixing. We have found it possible to mix the TCEP and XOF Reagents without fully suspending the pellet. If some of the pellet is suspended it isn’t an issue as both reagents will still be able to react effectively after a quick 1 second mix. In the event that some of the pellet becomes dislodged the whole reaction mix is again spun hard at 10,000 xg (step 19) just before transferring the mix for reading at the end of the assay. In any case, the main reason we do not include instructions to harvest the supernatant after step 10 is to save time and avoid confusion with having to create another large set of tubes for transfer.

For Research Use Only

Catalog Number: NWK-LHP01
Format: 96 Well Competitive ELISA
Sample Requirements: Plasma and tissue homogenates
Specificity: Lipid hydroperoxides
Sensitivity: 0.3 µM LOOH in Rxn mix 1.9 µM LOOH in original sample
Storage and Stability: 6 months from the date of manufacture
Kit Contents: Xylenol Orange: 1 X 8 mL
Iron Reagent: 1 X 8 mL
Catalase Reagent: (ready to use) 1 X 1 mL
TCEP Reagent: (ready to use) 1 X 1 mL
BHT Reagent: 1 Vial
BHT Solvent: 1 X 2.5 mL
Calibrator (9(S)-HPODE) Ready to Use: 1 X 1 mL

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