Cell culture system for determining the cholesterol efflux potential for serum
The present invention relates to a cell culture system to provide a tool for assessing the potential of a patient's serum for preventing the accumulation of cholesterol in arteries (i.e. serum efflux potential) that leads to atherosclerosis and to screen new drug compositions being developed to reduce the accumulation of cholesterol or enhance the clearance of cholesterol from the vessel wall. The present invention combines two individual assays, which are two different cholesterol assays: an assay measuring scavenger receptor class B type I (SR-BI)-mediated cholesterol efflux and an assay measuring ATP binding cassette protein 1 (ABCA1)-mediated cholesterol efflux, and uses them in parallel to test human and animal sera for their potential to stimulate efflux, as mediated by either of the two receptors described above.
1. A method of assessing the ability of serum to mediate cell cholesterol efflux comprising the steps of:
a) exposing at least one first cell sample and at least one second cell sample to said serum;
b) assaying said first cell sample for contribution of scavenger-receptor class B type 1 (SR-BI) to serum mediated cholesterol efflux from said first cell sample;
c) assaying said second cell sample for contribution of ATP binding cassette protein I (ABCA1) to serum mediated cholesterol efflux from said second cell sample; and
d) determining the ability of said serum to mediate cholesterol efflux from said first and second cell sample by measuring said contribution of SR-BI to cholesterol efflux and said contribution of ABCA1 to the cholesterol efflux.
2. The method of claim 1 wherein said step b) for determining contribution of SR-BI to cholesterol efflux comprises the steps of:
i) providing a cell sample lacking SR-BI and a cell sample expressing high levels of SR-BI;
ii) contacting the cells of i) with said serum;
iii) determining a first amount of cholesterol released from said cell sample lacking SR-BI and a second amount of cholesterol released from said cell sample expressing high levels of SR-BI; and
iv) determining the contribution of SR-BI to cholesterol efflux by subtracting said first amount of cholesterol from said second amount of cholesterol.
3. The method of claim 2 wherein said cholesterol is radiolabeled in said first cell sample and said step of determining a first amount of cholesterol and a second amount of cholesterol measures said radiolabeled cholesterol.
4. The method of claim 1 wherein said step c) for determining contribution of ABCA1 to cholesterol efflux comprises the steps of:
i) providing a cell sample lacking ABCA1 and a cell sample which expresses high levels of ABCA1;
ii) contacting the cells of i) with said serum;
iii) determining a first amount of cholesterol released from said cell sample lacking ABCA1 and a second amount of cholesterol released from said cell sample expressing high levels of ABCA1; and
iv) determining the contribution of ABCA1 to cholesterol efflux by subtracting said first amount of cholesterol from said second amount of cholesterol.
5. The method of claim 4 wherein said cholesterol is radiolabeled in said second cell sample and said step of determining a first amount of cholesterol and a second amount of cholesterol measures said radiolabeled cholesterol.
6. The method of claim 1 wherein said serum is human serum.
7. The method of claim 1 wherein high density lipoprotein subfraction 3 (HDL 3 ) is added to said serum before step a).
8. The method of claim 1 wherein apolipoprotein AI (Apo AI) is added to said serum before step a).
9. The method of claim 1 wherein high density lipoprotein subfraction 3 (HDL 3 ) and apolipoprotein AI (Apo AI) are added to said serum before step a).
10. The method of claim 1 wherein said serum is treated with a reagent to precipitate Apo B containing lipoproteins in said serum before step a) and centrifuged to remove said precipitate from said serum.
11. The method of claim 10 wherein the reagent is selected from the group consisting of polyethylene glycol, polysaccharide, sodium phosphotungstate and tetracycline.
12. A method for determining efficacy of a composition for potentiating release of cholesterol in a patient comprising the step of:
a) extracting serum from said patient,
b) administering to said patient an effective amount of said composition and extracting serum from said patient, and
c) determining cell cholesterol efflux of the serum obtained in step a) and the serum obtained in step b) using the method of claim 1 whereby an increase in efflux of cholesterol after administration of said composition indicates efficacy of said composition for potentiating release of cholesterol in said patient.
13. A method of determining a cholesterol-related condition in a patient comprising the steps of:
obtaining a first sample of serum from a normal subject and a second sample of serum from a patient;
comparing cell cholesterol efflux mediated by said serum samples using the method of claim 1 wherein increased cholesterol efflux mediated by said second sample relative to said first sample indicates that said patient has a cholesterol-related condition selected from the group consisting of Alagille Syndrome, hypercholesterolemia and atherosclerosis.