Plasma factors affecting the in vitro conversion of high-density lipoproteins labeled with a non-transferable marker

We studied the in vitro conversion of HDL₃ labeled with a radioiodinated diacyl lipid associating peptide (diLAP). DiLAP was previously shown to be nontransferable, which permitted its' use as a reliable marker of HDL particles. DiLAP-labeled HDL₃ was incubated for 23 h at 37°C in human or rat plasma or in reconstituted media containing delipidated plasma and/or lipoproteins and/or partially purified CETP. At the end of the incubations, the samples were adjusted to a density of 1.125 g/ml and ultracentrifuged. The two resulting fractions containing HDL₂ and HDL₃, respectively, were analyzed by gradient gel electrophoresis. Depending upon experimental conditions, diLAP-labeled HDL₃ was converted into HDL_2b- and/or small HDL_3c-like particles. LCAT inhibition and to a lesser extent CETP promoted the formation of small HDL_3c. Reactivation of LCAT led to the disappearance of small HDL_3c. No HDL_3c formed from HDL₂ even in the absence of LCAT activity. When the incubations were performed in the presence of 100 mM thimerosal, which inhibited PLTP but not CETP activity, the conversion of diLAP-labeled HDL₃ into HDL₂ was almost completely blocked. Collective consideration of these data indicates that the formation of small HDL is moderately facilitated by CETP; that small HDL are converted to larger HDL species by LCAT and that the transformation of HDL₃ into HDL₂ is a process which largely depends upon PLTP activity.