Cholesterol Quantification using a Spectrophotometric Assay

By Matthew Smith and Chris Haggard Main Source: BioVision I. Introduction: The structure of cholesterol combines the backbone of a steroid and an alcohol and is found in the bloodstream (Fig. 1). This important biomolecule maintains membrane fluidity and acts as a precursor in the production of Vitamin D and hormones. Recent studies have correlated high cholesterol levels (in conjunction with an increased low-density lipoprotein concentration) with atherosclerosis and myocardial infarction (Weinruch 2007). Low cholesterol levels (hypocholesterolemia) indicate a weakening immune system (Shor Posner G 1993). Clinicians and scientists measure cholesterol content of human serum to monitor patient health and prevent cholesterol related diseases. Using a commercial colorimetric assay, the purpose of this experiment is to determine the cholesterol content in a lab sample. Each group will prepare a cholesterol standard calibration curve: absorbance at 570nm vs. standard concentration, which will be used to measure the content of cholesterol in the lab sample in mg/dL. Figure 1. Structure of cholesterol The product of our coupled chemical reaction is a substance that will fluoresce or absorb light at a specific wavelength. In this experiment, the product will be resorufin, which exists in a 1:1 ratio with cholesterol absorbs light at 570nm. The chemical reaction proceeds as follows: Cholesterol + O2 -> cholest-4-ene-3-one + H2O2 (1) H2O2 + cholesterol probe -> resorufin (max at 570nm) (2) These reactions take place when catalyzed by enzymes (cholesterol oxidase for reaction (1) and peroxidase for reaction (2)) which prevents them from spontaneously forming resorufin from cholesterol. It is important to supply either enough enzymes or enough time to allow all of the substrate to perform the coupled reaction. At the end of this lab, each lab group will have fifteen data points; six points to create the standard curve (run in duplicate) and one point for the unknown (run in triplicate). Analyzing the unknown cholesterol concentration from absorbance requires a line of best fit. It is possible to transfer the six absorbance values from the excel spreadsheet into a x-y scatter plot, and a line of best fit can be determined. While the line of best fit is an equation to determine y (the absorbance), it is possible to change the equation to solve for x. Example: If , . If we are given an absorbance of 0.5, II. Solutions and Reagents to be used: from BioVision, Kit K603-100
Component Product # Volume Label Cholesterol Probe (lypophilized) K603-100-1 1 Vial P Cholesterol Esterase (lypophilized) K603-100-2 1 Vial CE Enzyme Mix (lypophilized) K603-100-3 1 Vial E Cholesterol Reaction Buffer K603-100-4 25 mL White Color Dimethylsulfoxide (DMSO:anhydrous) K603-100-5 0.4mL Brown Color Cholesterol Standard ((5 g/ l) K603-100-6 100 l Yellow Color Unkown #1 (X mg/dL) N/A 200L Pink Unkown #2 (Y mg/dL) N/A 200L Blue Unkown #3 (Z mg/dL) N/A 200L Green

III. Storage and Handling: Store all components of the kit at -20C and protect from light. Allow all reagents to come to room temperature and centrifuge vials before use. Once the cholesterol probe, the cholesterol esterase, and the enzyme mix have all been mixed, they should be used within two months. IV. Instructions for the Instructors: Preparing the Reagents: Cholesterol Probe: Dissolve in 220L of anhydrous dimethylsulfoxide (DMSO). Aliquot and store at -20C. The shelf life is two months, and it should be protected from light. Cholesterol Esterase: Dissolve in 220L of Cholesterol Reaction Buffer before use. Aliquot and store at -20C; the shelf life is two months. Enzyme Mix: Dissolve in 220L of Cholesterol Reaction Buffer before use. Aliquot and store at -20C; the shelf life is two months. Reaction Mix: Add 704L of cholesterol reaction buffer, 32L of cholesterol probe, 32L of enzyme mix, and 32L of cholesterol esterase to a vial. You should use a vial that can contain 1.5mL of solution, yet be small enough to facilitate easy micropipette use (<1mL plastic vial). This is the Reaction Mix. V. Protocol for the Students: Step 1: Dilute the cholesterol standard to 0.5g/L by adding 20L of cholesterol standard to 180L of cholesterol reaction buffer. This will create 200L of the 0.5g/L solution. (Use a vial to store this standard) Step 2: Mix the 200L solution well to ensure complete mixing. Add 0, 4, 8, 12, 16, and 20L of the new standard solution into each microwell (0L in well #1, 4L in well #2, etc.). Perform this step in duplicate (using different wells). Step 3: Add Cholesterol Reaction Buffer to each well in order to have each well contain 50L of solution (add 50L of buffer into well #1, 46L of buffer into well #2, etc.) You have now generated a 0, 2, 4, 6, 8, and 10g/well standard. Step 4 (Using the Unknown Sample): Add 50L of the unknown sample into an empty well (Perform this step in triplicate using different wells). Write down which color you are using, as you will need this information to confirm your result with the instructors. The unknown has been previously diluted, so do not worry about adding anything else except for the Reaction Mix. Step 5: Add 50L of the Reaction Mix to each of the respective wells. Be sure not to cross-contaminate wells when pipetting. Step 6: Incubate the 96-well plate for 37C for 60 minutes under aluminum foil. Protect the solution from light. Step 7: Measure the absorbance at 570nm wavelength using a microplate reader. Ask your TA for details on how to operate the instrument. Correct the back ground by subtracting the absorbance for the control value from all subsequent absorbance results. Calculate the concentration of your unknown using the calibration curve you created with your standards. Below is a typical graph for absorbance vs. concentration (Figure 2). If one of your data points is too high or too low you may remove it to ensure R2>0.99 (Figure 3). Use the Qtest

to do this. R2 value can be determined by creating a trendline for your graph and selecting the display of R2. Figure 2. Example of calibration curve of cholesterol Figure 3. Example of calibration curve of cholesterol rejecting one data point to obtain a better R2 value. VI. Safety Information: Gloves, protective clothing, and eyewear should all be worn during this procedure and we recommend that all chemicals are handled with caution. The cholesterol reaction buffer that is used may cause irritation if it comes in contact with the skin. The hazards of the cholesterol probe have not been thoroughly investigated. The cholesterol probe is not listed by the NTP, IARC, or OSHA. DMSO is harmful if swallowed, inhaled, or absorbed through the skin. DMSO can cause irritation and in case of contact, wash skin or eyes for 15 minutes. Vomiting should be induced upon ingestion. If DMSO is inhaled move to fresh air quickly. This chemical is also not listed by the NTP, IARC, or OSHA. The enzyme mix, cholesterol esterase, and cholesterol standard are all proved to be nonhazardous. Clean up all spills using appropriate protective equipment and methods. Dispose of spills and trash in an organic waste bin. For more information, check out the available MSDS information for each reagent used (www.hazard.com/msds) IX. Reference: Shor, Posner G. et al. Hypocholesterolemia is associated with immune dysfunction in early human immunodeficiency virus-1 infection. American Journal of Medicine. Volume 94, Issue 5. May 1993. Released by Pub Med. Viewed February 15 2009. http://www.ncbi.nlm.nih.gov/pubmed/7605397 Weinrauch, Larry A. Heart Attack Medical Encyclopedia. Medline Plus. United States National Library of Medicine. Updated March 2007. Viewed February 15, 2009