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 degrees 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 HDL2b- and/or small HDL3c-like particles. LCAT inhibition and to a lesser extent CETP promoted the formation of small HDL3c. Reactivation of LCAT led to the disappearance of small HDL3c. No HDL3c 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.