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437639 | Lipid Hydroperoxide (LPO) Assay Kit

437639
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      Overview

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      437639-1KIT
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          Description
          OverviewA sensitive and reliable assay kit for the measurement of the hydroperoxides from any sample containing lipid hydroperoxides, directly utilizing the redox reactions with ferrous ions. Quantification of lipid peroxidation is essential to assess the role of oxidative injury in pathophysiological disorders.
          Catalogue Number437639
          Brand Family Calbiochem®
          Materials Required but Not Delivered A plate reader or spectrophotometer with a 500 nm filter
          An adjustable pipettor
          A source of pure water. Glass distilled water or HPLC-grade water is acceptable
          Deoxygenated chloroform and methanol (see Reagent Preparation)
          A reusable glass 96 well plate
          References
          ReferencesMorrow, J.D. and Roberts, L.J. 1997. Prog. Lipid Res. 36, 1.
          Halliwell, B. 1996. Free Radic. Res. 25, 57.
          Hoffman, S.W., et al. 1996. J. Neurosci. Methods 68, 133.
          Mihaljevic, B., et al. 1996. Free Radic. Biol. Med. 21, 53.
          Porter, N.A., et al. 1995. Lipids 30, 277.
          Esterbauer, H., et al. 1991. Free Radic. Biol. Med. 11, 81.
          Janero, D.R., 1990. Free Radic. Biol. Med. 9, 515.
          Pryor, W.A., et al. 1990. Free Radic. Biol. Med. 8, 541.
          Cross, C.E., et al. 1987. Ann. Intern. Med. 107, 526.
          Yamamoto, Y., et al. 1987. Anal. Biochem. 160, 7.
          Warso, M.A. and Lands, W.E.M. 1984. Clin. Physiol. Biochem. 2, 70.
          Diczfalusy, U., et al. 1977. FEBS Lett. 84, 271.
          Bligh, E.G. and Dyer, W.J. 1959. Can. J. Biochem. Physiol. 37, 911.
          Product Information
          Detection methodColorimetric
          Form100 Tests
          FormatCuvette or 96-well plate
          Kit containsFTS Reagent 1, FTS Reagent 2, Lipid Hydroperoxide Standard, Extract R, Triphenylphosphine, and a user protocol.
          Positive control13-hydroperoxyoctadecanoic acid
          Applications
          Biological Information
          Assay range0.25-5 nmol hydroperoxide per assay tube
          Assay time1.5 h
          Sample TypeTissues, cultured cells, plant materials, foods, biological fluids, etc.
          Physicochemical Information
          Dimensions
          Materials Information
          Toxicological Information
          Safety Information according to GHS
          Safety Information
          Product Usage Statements
          Intended useThe Calbiochem® Lipid Hydroperoxide (LPO) Assay Kit is designed to measure lipid hydroperoxides in any sample type containing detectable levels (e.g. cells, tissues, plant material, biological fluids).
          Storage and Shipping Information
          Ship Code Blue Ice Only
          Toxicity Multiple Toxicity Values, refer to MSDS
          Hazardous Materials Attention: Due to the nature of the Hazardous Materials in this shipment, additional shipping charges may be applied to your order. Certain sizes may be exempt from the additional hazardous materials shipping charges. Please contact your local sales office for more information regarding these charges.
          Storage +2°C to +8°C
          Storage ConditionsUpon arrival store the Lipid Hydroperoxide Standard at -80°C. The remaining components of the kit should be stored at 4°C.
          Avoid freeze/thaw Avoid freeze/thaw
          Do not freeze Ok to freeze
          Packaging Information
          Transport Information
          Supplemental Information
          Kit containsFTS Reagent 1, FTS Reagent 2, Lipid Hydroperoxide Standard, Extract R, Triphenylphosphine, and a user protocol.
          Specifications

          Documentation

          SDS

          Title

          Safety Data Sheet (SDS) 

          Certificates of Analysis

          TitleLot Number
          437639

          References

          Reference overview
          Morrow, J.D. and Roberts, L.J. 1997. Prog. Lipid Res. 36, 1.
          Halliwell, B. 1996. Free Radic. Res. 25, 57.
          Hoffman, S.W., et al. 1996. J. Neurosci. Methods 68, 133.
          Mihaljevic, B., et al. 1996. Free Radic. Biol. Med. 21, 53.
          Porter, N.A., et al. 1995. Lipids 30, 277.
          Esterbauer, H., et al. 1991. Free Radic. Biol. Med. 11, 81.
          Janero, D.R., 1990. Free Radic. Biol. Med. 9, 515.
          Pryor, W.A., et al. 1990. Free Radic. Biol. Med. 8, 541.
          Cross, C.E., et al. 1987. Ann. Intern. Med. 107, 526.
          Yamamoto, Y., et al. 1987. Anal. Biochem. 160, 7.
          Warso, M.A. and Lands, W.E.M. 1984. Clin. Physiol. Biochem. 2, 70.
          Diczfalusy, U., et al. 1977. FEBS Lett. 84, 271.
          Bligh, E.G. and Dyer, W.J. 1959. Can. J. Biochem. Physiol. 37, 911.
          User Protocol

          Revision19-August-2009 RFH
          Form100 Tests
          FormatCuvette or 96-well plate
          Detection methodColorimetric
          StorageUpon arrival store the Lipid Hydroperoxide Standard at -80°C. The remaining components of the kit should be stored at 4°C.
          Intended useThe Calbiochem® Lipid Hydroperoxide (LPO) Assay Kit is designed to measure lipid hydroperoxides in any sample type containing detectable levels (e.g. cells, tissues, plant material, biological fluids).
          BackgroundQuantification of lipid peroxidation is essential for assessing 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-hydroxynonenal (4-HNE), the degradation products of polyunsaturated fatty acid (PUFA) 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 byproducts as indicators of lipid peroxidation. The byproduct formation is highly inefficient and varies according to the transition metal ion content of the sample. Only hydroperoxides derived from PUFA give rise to these byproducts. For example, 4-HNE is formed only from ω-6 PUFA hydroperoxides and is catalyzed by transition metal ions like ferrous ions. 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 can lead to a 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 ~0.5 µM. Given the limitations of the indirect methods; direct measurement of hydroperoxides is the obvious choice.
          Principles of the assayThe Calbiochem® Lipid Hydroperoxide (LPO) Assay Kit measures lipid hydroperoxides directly utilizing redox reactions with ferrous ions (see below). 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.

          Figure 1: Reduction/Oxidation Reactions with Ferrous Ions


          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 that readily reacts with ferrous ions to give an over-estimation of lipid hydroperoxides. Performing the assay in chloroform can circumvent these issues.
          An easy to use, quantitative extraction method was developed to extract lipid hydroperoxides in chloroform; this extract is used directly 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.
          Materials provided• FTS Reagent 1 (Kit Component No. KP31630): 1 vial, 4.5 mM ferrous sulfate in 0.2 M HCl, ready to use
          • FTS Reagent 2 (Kit Component No. KP31631): 1 vial, 3% ammonium thiocyanate in methanol, ready to use
          • Lipid Hydroperoxide Standard (Control) (Kit Component No. KP31632): 1 vial, 50 µM 13-HpODE (13-hydroperoxyoctadecanoic acid) in ethanol, ready to use
          • Extract R (Kit Component No. KP31633): 1 vial, crystalline solid
          • Triphenylphosphine (Kit Component No. KP31634): 1 vial, crystalline solid
          Materials Required but not provided A plate reader or spectrophotometer with a 500 nm filter
          An adjustable pipettor
          A source of pure water. Glass distilled water or HPLC-grade water is acceptable
          Deoxygenated chloroform and methanol (see Reagent Preparation)
          A reusable glass 96 well plate
          Precautions and recommendations Please read these instructions carefully before beginning this assay.
          It is recommended to take appropriate precautions when using the kit reagents (i.e., lab coat, gloves, eye goggles, etc.), as some of them can be harmful.
          Pipetting Hints
          a. When pipetting chloroform or Chloroform:Methanol only polypropylene- or teflon-based pipet tips should be used.
          b. Before pipetting each reagent, equilibrate the pipet tip in that reagent (i.e. fill the tip and gently expel the contents, repeat several times).
          c. Do not expose the pipet tip to the reagent(s) already in the test tube.
          • CAUTION: Extract R is acidic and hence direct contact should be avoided. If exposed, wash thoroughly with cold water. Excess reagent can be disposed of in sanitary sewer drains after dilution in 10 volumes of water.
          • NOTE: Use of triphenylphosphine is optional and in most cases this is not necessary. See "Interferences" for details.
          PreparationAny sample containing lipid hydroperoxides is suitable for this assay. Tissues, cultured cells, plant materials, foods, and biological fluids such as plasma can be used in the assay. Most tissues contain peroxidases (e.g., glutathione peroxidase) that effectively reduce endogenous lipid hydroperoxides to their corresponding alcohols. Peroxidase activity decreases hydroperoxide concentrations to extremely low or undetectable levels in normal tissues. Even under oxidative stress conditions, hydroperoxide production by free radicals must overwhelm the peroxidase defenses before a detectable increase in lipid hydroperoxide concentration will occur. Measurement of lipid hydroperoxides provides a snapshot of the lipid peroxidation level at the time of the assay. Integrated values of lipid peroxidation (lipid peroxidation over time) can be determined more reliably by measuring the 8-isoprostane levels. Tissues, plant materials, and foods should be homogenized in HPLC-grade water or in buffer containing no transition metal ions before use. Cultured cells should be sonicated in HPLC-grade water or in medium containing no transition metal ions before use. Samples should be assayed immediately upon collection. If samples cannot be assayed fresh, then the lipid hydroperoxides should be extracted and the extracts should be stored at -80°C. The extracted lipid hydroperoxides are stable for at least one month at -80°C. Lipid hydroperoxides must be extracted from the sample with chloroform before performing the assay. The standard Bleigh and Dyer13 extraction protocol is not reproducible and hence not suitable for quantitative analysis. In this kit, a deproteination procedure is combined with the extraction of lipid hydroperoxides to achieve quantitative extraction of lipid hydroperoxides. This extraction step ensures the elimination of nearly all interfering substances from the sample. The following is a typical extraction procedure using plasma as the sample: 1. Aliquot a known volume of sample (e.g., 500 µl plasma) into a glass test tube (12 x 75 mm). 2. Add an equal volume of Extract R-saturated methanol (500 µl for this example) to each tube and vortex. 3. Add 1 ml cold deoxygenated chloroform to each tube and mix thoroughly by vortexing. 4. Centrifuge the mixture at 1500 x g for 5 min at 0°C. 5. Collect the bottom chloroform layer by carefully inserting a pasteur pipet along the side of the test tube. Transfer the chloroform layer to another test tube and store on ice. CAUTION: Avoid collecting the middle protein layer or the upper water layer along with the chloroform layer. Any water carried over to the assay tube will interfere with color development. It is not necessary to collect all of the chloroform layer; 700 µl will be sufficient.
          Reagent preparation1. Extract R: This vial contains a crystalline solid used for extraction of samples. Prepare a saturated solution of Extract R as follows: Weigh about 100 mg Extract R into a tube and add 15 ml methanol; vortex thoroughly for ~2 min. The methanol will become cloudy and most of the solid remains undissolved. Use the Extract R-saturated methanol within 2 h. 2. Triphenylphosphine: This vial contains crystalline triphenylphosphine. Weigh 2.6 mg triphenylphosphine and dissolve in 1 ml Chloroform:Methanol Mixture (see below for preparation) to prepare a 10 mM solution. Keep the solution, tightly closed, on ice and use within 12 h. 3. Chromogen: Prepare Chromogen by mixing equal volumes of FTS Reagent 1 and FTS Reagent 2 in a test tube and vortex. Prepare ONLY enough Chromogen for the number of samples being assayed; each tube requires 50 µl Chromogen. NOTE: Prepare the Chromogen IMMEDIATELY PRIOR TO USE. 4. Glass 96-Well Plate (optional): The glass plate must be cleaned before each use. Clean the plate with warm soapy water, and then rinse it with HPLC-grade water, followed by rinsing with acetone. NOTE: Do not clean the plate with abrasive agents. The plate must be completely dry before use in the assay. 5. Chloroform: Deoxygenate ~100 ml chloroform (not supplied) by bubbling nitrogen through the solvent for at least 30 min. Cool part of the deoxygenated chloroform to 0°C and store it on ice for extraction of the samples. Two volumes chloroform are required for the extraction of one volume sample. 6. Methanol: Deoxygenate approximately 100 ml methanol (not supplied) by bubbling nitrogen through the solvent for at least 30 min. 7. Chloroform:Methanol Mixture: Mix two volumes deoxygenated chloroform with one volume deoxygenated methanol. The solvent mixture is ready for use in the assay. Approximately 1 ml Chloroform:Methanol Mixture is needed for each assay tube. 8. Lipid Hydroperoxide Standards: Store the Lipid Hydroperoxide Standard at -80°C and keep it on ice during the experiment. Prepare 24 clean test tubes (glass or polypropylene) by labeling them A-H, in triplicate. Aliquot the Lipid Hydroperoxide Standard and Chloroform:Methanol Mixture to each tube as described in the table below:

          Table 1: Lipid Hydroperoxide Standard

          Detailed protocolNote:

          Store the Lipid Hydroperoxide Standard at -80°C and keep it on ice during the experiment.
          The final volume of the assay is 1 ml in all tubes.
          It is not necessary to use all the reagents at one time. However, a standard curve must be run simultaneously with each set of samples.
          If the concentration of lipid hydroperoxides in the sample is not known or if it is expected to be beyond the range of the standard curve, it is recommended to assay the sample at several dilutions.
          It is recommended that the samples and standards be assayed in triplicate.
          Prepare the Chromogen immediately prior to addition to the tubes.
          The background absorbance (abs. of Std. A) should be ≤0.3.

          1. Prepare Lipid Hydroperoxide Standards as described in the Reagent Preparation section above.
          2. Add 500 µl chloroform extract of each sample to appropriately labeled glass test tubes. Avoid any transfer of water from the extract.

          NOTE: The volume of extract used for the assay can be changed depending on the concentration of hydroperoxide. However, it is also necessary to adjust the volume of Chloroform:Methanol Mixture so that the final volume is 950 µl before the addition of the chromogen.

          3. Add 450 µl Chloroform:Methanol Mixture to each sample tube.
          4. Add 50 µl freshly prepared Chromogen to each tube (Lipid Hydroperoxide Standards and samples) and vortex; mix well. Close the tubes tightly with polypropylene caps.
          5. Incubate at room temperature for 5 min.
          6. There are two methods of completing this assay, either using a spectrophotometer to test each sample individually (6a. below) or by using the Glass 96-Well Plate and a plate reader (6b. below).
          a. Measure the absorbance of each tube at 500 nm using either glass or quartz 1 ml cuvettes. Use the Chloroform:Methanol Mixture for a blank if the spectrophotometer requires it.
          OR
          b. Transfer 300 µl from each tube to designated wells of the Glass 96-Well Plate following the configuration shown in the sample plate layout below. While there is no specific pattern required for using the wells on the plate, a typical layout of standards and samples, measured in triplicate, is shown in the sample plate layout. To avoid evaporation from the wells, cover the plate with aluminum foil as the aliquots of each sample are dispensed. NOTE: Do not use plastic plate covers, the solvent will dissolve the cover. Do not shake the plate on the table top, it will scratch the bottom of the plate. Read the absorbance at 500 nm using a 96-well plate reader.

          NOTE: The color is stable for 5 h. If the tubes are not tightly capped, evaporation of solvent will result in a change in the absorbance.
          Calculations7. Calculating the Results: a. Calculate the average absorbance for each Lipid Hydroperoxide Standard and sample. b. Subtract the average absorbance of Lipid Hydroperoxide Standard A from itself and all other Lipid Hydroperoxide Standards and samples. c. Plot the corrected absorbance of Lipid Hydroperoxide Standards (from step b above) as a function of final hydroperoxide value from the table (see Reagent Preparation, Lipid Hydroperoxide Standards above). See below for typical standard curves using the spectrophotometric method (figure 2) and the Glass 96-Well Plate method (figure 3).

          Figure 2: Typical standard curve using the spectrophotometric method.

          Figure 3: Typical standard curve using the Glass 96-Well Plate method.

          d. Calculate the hydroperoxide values of the sample tubes (HPST) using the equation obtained from the linear regression of the standard curve substituting corrected absorbance values for each sample. HPST (nmol) = (sample absorbance - y-intercept)/slope e. Calculate the concentration of hydroperoxide in the original sample as shown below:

          Figure 4: Calculate the concentration of hydroperoxide

          Assay Range0.25-5 nmol hydroperoxide per assay tube
          Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
          Interactive Pathways™ is a trademark of EMD Chemicals, Inc.