The overall goal of this experimental method is to determine the function of high density lipoprotein using a biochemical cell-free assay that measures the lipid peroxide content of a specific amount of HDL. This method can help answer key questions in several fields of medicine, such as the role of HDL function in cardiovascular disease metabolic abnormalities. The main advantage of this technique is that it is a cell-free biochemical method that can be used to reliably determine HDL function.
Though this method can provide insight into the role of HDL function in cardiovascular disease and metabolic abnormalities, it can also be applied to other studies of disease, such as inflammatory diseases. To begin this procedure, prepare the working layout for the 96-well plate, including the appropriate controls, samples and replicates, as outlined in the text protocol. Calculate the volume of reagents needed based on the number of samples and replicates.
Using a permanent marker, label tubes for each step in the assay. Next, combine 10 microliters from each sample to create a pooled control sample. Create a second pooled control sample for the clinical laboratory, in order to determine the HDL-C value.
After preparing a large stock of HDL, cryo-preserve several aliquots to minimize freeze thaw cycles. Pool at least 10 different replicates to create a pooled quality control. Then, determine the average value of HDLox as outlined in the text protocol.
Create a second pooled quality control for the clinical laboratory, in order to determine the HDL-C value. To begin, thaw a fresh plasma or serum sample. Mix together equal volumes of plasma and HDL cholesterol precipitating reagent.
Pipette up and down several times to mix well. Centrifuge at a speed between 1000 and 2000 times G for 10 minutes. After this, aspirate the supernatant.
Store the isolated HDL at four degrees Celsius until ready to perform the fluorochrome HDL function assay. If the clinical laboratory's HDL-C value is used to normalize HDLox, quantify the HDL-C from plasma as outlined in the text protocol. To begin, mix cholesterol and HRP, such that the prepared solutions contain five units per milliliter of HRP.
Thaw a vial of fluorochrome reagent and a vial of DMSO to room temperature. After they are thawed, dissolve one milligram of fluorochrome reagent in 200 microliters of DMSO. Store this solution at a temperature below minus 20 degrees Celsius, while protected from light.
Next, add 50 microliters of 1X reaction buffer to the sample plate as a negative control. Add 50 microliters of 20 millimolar hydrogen peroxide working solution as a positive control. After this, set out a fresh round bottom 96-well plate to be used as the preparation plate.
Add 200 microliters of isolated HDL to each well of this preparation plate. Using a multichannel pipette, transfer 50 microliters of HDL from the preparation plate to the experimental plates, as outlined in the text protocol, with the number of experimental plates being equal to the number of replicates in the assay. After this, add 50 microliters of prepared HRP solution to each well.
Incubate at 37 degrees Celsius for 30 minutes, then add 50 microliters of fluorochrome reagent to each well, for a total volume of 150 microliters per well. Mix well and protect from light. Using a fluorescent plate reader, assess the fluorescent readout at 37 degrees Celsius, in the dark, every minute for 120 minutes.
Record the data using appropriate software, then calculate the mean value of fluorescence units as outlined in the text protocol. In this study, a cell-free biochemical fluorometric assay is developed to quantify the HDL lipid peroxide content per milligram of HDL-C. This is accomplished by enzymatic amplification using HRP.
A fluorescent plate reader is then used to measure the fluorescence in the dark, every minute, for 120 minutes. Representative data for a patient known to have dysfunctional HDL is shown here, compared to the pooled control. The dysfunctional HDL is seen to have lipid peroxide levels two-fold higher than those seen in the pooled HDL from healthy participants.
Next, the HDL is isolated and HDLox is determined in 50 healthy subjects and 100 HIV infected patients. The HIV infected patients are seen to have, on average, 60%higher HDL peroxide content per milligram of HDL-C than the healthy subjects. Once mastered, this technique can be done in under six hours if it is performed properly.
While attempting this procedure, it's important to remember to prepare all reagents and controls upfront, be consistent, precise and always include the appropriate experimental control in each experimental plate. After watching this video, you should have a good understanding of how to isolate the HDL, determine the lipid peroxide content of a specific amount of HDL and to depredate our regarding HDL function. Don't forget that working with biospecimens can be extremely hazardous and precautions, such as gloves, should always be taken while performing this procedure.
