Figure 2. Integrated approach for the analysis of morphological LDL transitions [modified from ref. (29)].

Single-donor human LDLs (0.5 mg/ml apoB, 20 mM Na phosphate, pH 7.5) were incubated at 85°C, and the time course of the increase in the particle size was monitored by turbidity (A). Aliquots taken at various stages, from 0 (intact LDLs) to 6 (fully denatured), were analyzed by (A) native PAGE, (B) SEC, and (C) negative-stain EM. SEC identified three peaks: I, intact size LDLs (~22 nm); II, enlarged lipoproteins (~40 nm); III, much larger particles (≥100 nm). Native PAGE resolved bands corresponding to peaks I and II, and EM of isolated peak II suggested LDL dimerization (A). Negative-stain EM showed progressive increase in the number of fused (violet arrows, stage 2) and ruptured LDLs (orange arrows, stages 5 and 6) and their aggregates. Similar changes in the particle size and morphology were observed upon lipolysis, sample aging, prolonged ultracentrifugation, or chemical denaturation of LDLs (18, 29).