A cell-free assay for detecting HDL that is dysfunctional in preventing the formation of or inactivating oxidized phospholipids

Mohamad Navab,Susan Y Hama,Greg P Hough,Ganesamoorthy Subbanagounder,Srinivasa T Reddy,Alan M Fogelman

doi:10.1016/s0022-2275(20)31582-0

Mohamad Navab, Susan Y Hama + Show 4 more

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https://doi.org/10.1016/s0022-2275(20)31582-0

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Abstract

We have developed a novel and rapid cell-free assay of the ability of HDL to prevent the formation of or inactivate oxidized phospholipids. HDL was tested for its ability to inhibit the oxidation of LDL, or inhibit the oxidation of l-α-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC) by hydroperoxyoctadecadienoic acid (HPODE), or inactivate oxidized PAPC (Ox-PAPC). In each case the fluorescent signal generated in the presence of the test substances and the test HDL was determined. As little as 2.5 μg of normal human HDL cholesterol significantly inhibited the fluorescent signal generated by Ox-PAPC; results did not differ regardless of whether the HDL was prepared by gel electrophoresis, fast protein liquid chromatography, or dextran sulfate precipitation. HDL from each of 27 patients with coronary atherosclerosis failed to inhibit the fluorescent signal generated by a control LDL, whereas HDL from each of 31 matched normal subjects with the same levels of HDL cholesterol significantly inhibited the signal. Results from an established cell-based assay (Navab, M., S. Hama, J. Cooke, G. M. Anantharamaiah, M. Chaddha, L. Jin, G. Subbanagounder, K. F. Faull, S. T. Reddy, N. E. Miller, and A. M. Fogelman. 2000. —Navab, M., S. Y. Hama, G. P. Hough, G. Subbanagounder, S. T. Reddy, and A. M. Fogelman. A cell-free assay for detecting HDL that is dysfunctional in preventing the formation of or inactivating oxidized phospholipids.

Highlights

We have developed a novel and rapid cell-free assay of the ability of HDL to prevent the formation of or inactivate oxidized phospholipids
To determine whether the signal generated by the interaction of oxidized PAPC (Ox-PAPC) with DCFH was dose dependent, Ox-PAPC was incubated with DCFH at the indicated concentrations and fluorescence intensity was determined after 2 h of incubation at room temperature
The values for each assay of the coculture supernatants were plotted on the y axis for each concentration of PAPC plus hydroperoxyoctadecadienoic acid (HPODE) (Fig. 2A–C) or Ox-PAPC (Fig. 2D–F)

Summary

Introduction

We have developed a novel and rapid cell-free assay of the ability of HDL to prevent the formation of or inactivate oxidized phospholipids. HDL from the patients failed to inhibit the fluorescent signal generated from PAPC plus HPODE (10 of 10 patients) whereas HDL from matched controls (8 of 8 patients) significantly inhibited the fluorescent signal We conclude that this new assay has the potential to allow widespread testing of the hypothesis that HDL that is dysfunctional in preventing the formation or inactivating oxidized phospholipids may play an important role in the development of atherosclerosis.—Navab, M., S. We report here a novel and rapid test of HDL function that does not require cells but that gives results highly comparable to those of the previously described cell-based assay

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Conclusion