The Amplex Red Assay of HDL function in combination with immunoaffinity capture of HDL can detect acute phase HDL in vivo in subjects previously shown to have dysfunctional HDL.
HDL was isolated using immunoaffinity capture as described in Methods from 30 healthy subjects and 30 patients with HIV infection that have previously been shown to have acute phase HDL (Lipids Health Dis 2012; 11: 87). The following different matrices were added in 96 well plates for immunoaffinity capture of HDL: a) purified HDL isolated by ultracentrifugation (5 µg of HDL cholesterol as determined by cholesterol assay), b) apo-B depleted serum (5 µg of HDL cholesterol as determined by cholesterol assay) c) apo-B depleted serum (100 µl) d) plasma (100 µl). In the latter two methods, the fluorescent readout (that corresponds to HDLox) was normalized to the HDL cholesterol concentration (measured by the clinical lab). ApoB depleted serum and plasma was isolated by PEG precipitation and HDL was also isolated by ultracentrifugation as described in methods. The Amplex Red oxidation rate (AROR) as a marker of HDL redox activity (HDLox) was determined as described in Figure 2 and Figure S10. The HIV-infected subjects had significantly higher HDLox (A: 1.66±0.37; B: 1.54±0.32; C: 1.40±0.33; D: 1.32±0.32) compared to the uninfected subjects (A: 1.05±0.28; B: 0.95±0.23; C: 0.81±0.24; D: 0.73±0.24) (p<0.01 for all comparisons).