Overview

  • Product name

    Lipid Hydroperoxide (LPO) Assay Kit
  • Detection method

    Colorimetric
  • Sample type

    Plasma, Cell culture extracts, Tissue Extracts
  • Assay type

    Quantitative
  • Range

    0.25 nM - 5 nM
  • Species reactivity

    Reacts with: Mammals, Other species
  • Product overview

    Abcam's Lipid Hydroperoxide (LPO) Assay Kit (ab133085) measures the hydroperoxides directly utilizing the redox reactions with ferrous ions. Hydroperoxides are highly unstable and react readily with ferrous ions to produce ferric ions. The resulting ferric ions are detected using thiocyanate ion as the chromogen. Since this method relies on the measurement of ferric ions generated during the reaction, ferric ions present in the sample are a potential source of error. Also, many biological samples contain hydrogen peroxide which readily reacts with ferrous ions to give an over-estimation of lipid hydroperoxides. These problems are easily circumvented by performing the assay in chloroform.


     


    An easy to use, quantitative extraction method was developed to extract lipid hydroperoxides into chloroform and the extract is directly used in the assay. This procedure eliminates any interference caused by hydrogen peroxide or endogenous ferric ions in the sample and provides a sensitive and reliable assay for lipid peroxidation.

  • Notes

    Quantification of lipid peroxidation is essential to assess the role of oxidative injury in pathophysiological disorders. Lipid peroxidation results in the formation of highly reactive and unstable hydroperoxides of both saturated and unsaturated lipids. Traditionally, lipid peroxidation is quantified by measuring malondialdehyde (MDA) and 4-hydroxy nonenal (4-HNE), the degradation products of polyunsaturated fatty acids (PUFAs) hydroperoxides.

     

    Sensitive colorimetric assays have been developed to measure these aldehydes. However, these assays are non-specific and often lead to an over-estimation of lipid peroxidation. There are important additional problems in using these by-products as indicators of lipid peroxidation. The by-product formation is highly inefficient and varies according to the transition metal ion content of the sample. Only hydroperoxides derived from PUFAs give rise to these by-products. For example, 4-HNE is formed only from omega-6 PUFA hydroperoxides and is catalyzed by transition metal ions like ferrous.

     

    Decomposition of hydroperoxides derived from abundant cellular lipids such as cholesterol and oleic acid does not produce MDA or 4-HNE. These factors can lead to an under-estimation of lipid peroxidation. MDA is also produced in ng/ml concentrations by the platelet enzyme thromboxane synthase during whole blood clotting and platelet activation. This leads to gross over-estimation of lipid peroxidation. Estimation of lipid hydroperoxide levels range from 0.3-30 µM depending on the method used. However, direct methods of estimation suggest that the concentration in normal human plasma is approximately 0.5 µM. Given the limitations of the indirect methods, direct measurement of hydroperoxides is the obvious choice.

    Related products

    Review the oxidative stress marker and assay guide, or the full metabolism assay guide to learn about more assays for metabolites, metabolic enzymes, mitochondrial function, and oxidative stress, and also how to assay metabolic function in live cells using your plate reader.

  • Platform

    Microplate reader

Properties

Associated products

Images

  • Typical standard curve obtained using lipid hydroperoxide standard provided in the kit.

Protocols

References

This product has been referenced in:

  • Hou L  et al. Inhibition of NADPH oxidase by apocynin prevents learning and memory deficits in a mouse Parkinson's disease model. Redox Biol 22:101134 (2019). Read more (PubMed: 30798073) »
  • Naderi M  et al. Dopaminergic dysregulation and impaired associative learning behavior in zebrafish during chronic dietary exposure to selenium. Environ Pollut 237:174-185 (2018). Read more (PubMed: 29482023) »
See all 4 Publications for this product

Customer reviews and Q&As

1-2 of 2 Abreviews or Q&A

Answer


Regarding ab133085, we do not have data for the collection, freezing and storage of plant tissue samples for 6 months at -80ºC. From your customer’s protocol, they did a good job of collecting, freezing and storing their tissues, but they are incorrect with the statement, “I have always been under the impression that -80ºC storage would stop everything from degrading.”

Storing samples at -80ºC will decrease the rate of degradation, but it does not stop it. For example, our labs have worked with a purified enzyme that would degrade after 4 weeks of storage at -80ºC. Even storing it at liquid nitrogen temperatures did not stop the degradation but it did extend the life of the enzyme for several months.

The plant samples may have oxidized in the -80ºC freezer, so the samples may have more/less lipid hydroperoxides. The samples may still be used to gather data for comparison purposes within the group of samples stored for that period of time, but may not be compared with samples freshly collected. The customer would have to perform a test to see if and/or how much change in the levels of lipid hydroperoxides occurred due to the storage conditions. (Test for lipid hydroperoxides on freshly collected samples and the same samples stored for 6 months at -80ºC.)

As for ab118970, again we have not tried using this kit with frozen samples from plants; therefore we cannot guarantee what the success rates would be of such sample’s assays. We can however say that plant samples should be compatible with the assay.

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Answer


Unfortunately we have not tested this kit using plant tissues so we cannot guarantee that it will work. This kit is only guaranteed to work in human samples.

In addition, for optimal results we would suggest following the protocol booklet exactly. When preparing your samples, did you extract the lipid hydroperoxidase and freeze these.? If not I would not recommend using this kit. If you did, then your samples have still be frozen for 6 months and we recommend that lipid peroxidases are stable for at least one month. We have not tested this kit using samples frozen for 6 months so cannot predict whether this will work or not but it is possible that after 6 months, the lipid hydroperoxidases will have degraded and therefore will no longer be detectable using this kit so I would not recommend doing this. I suggest using fresh samples if at all possible.

Unfortunately, we do not have any other lipid hydroperoxidase kit in the catalogue or any kit which has been guaranteed to quantify oxidative damage in plant tissues. I would recommend looking at http://www.biocompare.com which has an excellent life science search facility.

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