High-Density Lipoprotein-Specific Phospholipid Efflux Assay

We developed a surrogate assay to measure HDL-mediated removal of coronary artery plaque lipids to assess coronary artery disease risk. The current challenge is to develop a simple, rapid and robust HDL lipid influx assay for both research and clinical applications. To begin, add all the required contents into a glass under a chemical fume hood, and vortex the lipid mixture briefly to mix the solutions.

Dry the mixture in a glass tube under a gentle stream of nitrogen for one hour to form a dry lipid film at the bottom of the tube. For coating calcium silicate hydrate, insert a weighing paper cone into the glass tube containing the dried lipids and add 80 milligrams of calcium silicate hydrate powder into the tube using the cone to direct the powder to the base. Immediately add 2 milliliters of normal saline to the tube and cover the top with Parafilm.

Vortex the glass tube by hand to dislodge the bulk of the lipid from the bottom. Place the glass tube into a hole made in a styrofoam platform attached to a vortex and secure the tube to the vortex platform using reinforced tape. Vortex the glass tube for 10 minutes and confirm that no lipid remains on the wall of the tube.

Transfer the contents of the glass tube to a 15-milliliter conical plastic tube using a 1-milliliter pipette. Wash the glass tube with 3 milliliters of saline and transfer the wash into the same 15-milliliter tube. Next, place the 15-milliliter plastic tube containing lipid-coated calcium silicate hydrate into a centrifuge and spin at 935G for 2 minutes at 4 degrees Celsius.

Use a long and fine pipette tip to aspirate the supernatant. Starting from the top of the supernatant, glide the pipette tip slowly along the side of the tube while aspirating. Tip the tube slightly during aspiration and leave approximately 200 microliters of saline above the pellet to avoid disturbing it.

Then, add saline to bring the total volume to 5 milliliters. Repeat the centrifugation and supernatant removal four times. After the final wash, add saline to bring the total volume to 2.5 milliliters Pipette 75 microliters of saline into each well of a 0.3 milliliter 96-well plate according to the number of wells needed for the sample set.

Vortex the 15-milliliter plastic stock tube containing LC-CSH three times for 10 seconds each. Using a single well pipette, dispense 50 microliters of LC-CSH along the right side of the saline containing wells. After three wells, repeat the vortexing protocol before dispensing into the next three wells.

Rotate the 96-well plate by 180 degrees so that the left side of the wells is now to the right. Pipette 25 microliters of plasma or serum samples along the right side of each well. For the positive control wells, add 25 microliters of reference standard human normal lipidemic plasma or serum.

Next, seal the 96-well plate tightly with adhesive film and incubate the sealed plate in the dark for one hour at 37 degrees Celsius and 1, 200 revolutions per minute in a thermomixer. After incubation, remove the plate from the thermomixer and place it on ice. Centrifuge the plate for two minutes at 935G at 4 degrees Celsius to pellet the donor particles and stop the transfer reaction.

Now, carefully remove the adhesive film from the 96-well plate without disturbing the LC-CSH pellet. Transfer 50 microliters of supernatant from the reaction plate into the wells of a black 96-well flat bottom polystyrene plate using a multi-well pipette. Fill a reservoir with normal saline and add 50 microliters of saline to each well.

using a multi-well pipette. Prepare 1%Triton X-100 in a 125-milliliter plastic Erlenmeyer flask. Fill a reservoir with the prepared 1%Triton X-100, and add 100 microliters of the same solution at room temperature into each well.

Mix by gently pipe heading up and down two to three times with a multi-well pipette and pop any bubbles using air from a 3-milliliter transfer pipette. Finally, measure lissamine rhodamine fluorescence using a fluorometer with excitation at 540 nanometers and emission at 600 nanometers. A standard curve was generated using serially diluted LRHPE labeled LC-CSH and showed a highly linear fluorescence response.

In the plasma concentration curve study, increasing volumes of pooled human plasma were dispensed in triplicate wells alongside saline controls. The plasma dose response curve showed excellent linearity between PE, afflux and plasma volume with a strong correlation in the 15-35 microliter range. Our simple and rapid protocol, which predicts incident cardiovascular disease risk, can be used for both research and clinical diagnostic studies.

Our findings demonstrate that our assay can elucidate the role of both cellular and extracellular plaque lipids in cardiovascular disease.