Analysis Note

Positive Control13-hydroperoxyoctadecanoic acid

7. 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).﹤div class="Bio_doc_image">Figure 2: Typical standard curve using the spectrophotometric method.﹤/div>﹤div class="Bio_doc_image">Figure 3: Typical standard curve using the Glass 96-Well Plate method.﹤/div>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)/slopee. Calculate the concentration of hydroperoxide in the original sample as shown below:﹤div class="Bio_doc_image">Figure 4: Calculate the concentration of hydroperoxide﹤/div>

Components

FTS Reagent 1, FTS Reagent 2, Lipid Hydroperoxide Standard, Extract R, Triphenylphosphine, and a user protocol.

General description

A 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.

Legal Information

CALBIOCHEM is a registered trademark of Merck KGaA, Darmstadt, Germany

Other Notes

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.

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. Methods68, 133.Mihaljevic, B., et al. 1996. Free Radic. Biol. Med.21, 53.Porter, N.A., et al. 1995. Lipids30, 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.

Packaging

1 kit in Glass bottle

Preparation Note

1. 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 l 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:﹤div class="Bio_doc_image">Table 1: Lipid Hydroperoxide Standard﹤/div>

Any 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.

Principle

The 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).

Specifications

Assay Time: 1.5 h

Storage and Stability

Upon arrival store the Lipid Hydroperoxide Standard at -80°C. The remaining components of the kit should be stored at 4°C.

Warning

Toxicity: Multiple Toxicity Values, refer to MSDS (O)