Possible Mechanism of Egress of Free Cholesterol from the Arterial Wall

SPERRY 1 was the first to demonstrate in vitro esterification of cholesterol, reporting that incubation of human serum and plasma at 37° C for 3 days decreased free cholesterol without changing total cholesterol. He also showed that esterification of free cholesterol during incubation could be inhibited by heating serum to 55°–60° C for 1–2 h, and suggested that this procedure resulted in enzyme inactivation. Glomset and Wright 2 showed a similar inhibition of cholesterol esterification after heating serum for 30 min at 56° C; this has been confirmed by others 3,4 as well as by us. There is evidence that the heat-labile enzyme is lecithin–cholesterol–acyl–transferase (LCAT) which seems to react preferentially with high density lipoproteins to catalyse the transfer of fatty acids from the 2-position of lecithin to the hydroxyl group of free cholesterol. Murphy 4 has demonstrated an equilibrium between free cholesterol of serum and that of the red blood cells, and shown that the flux of free cholesterol from cells to serum increases as the free cholesterol of the serum becomes esterified during incubation. If there was a similar equilibrium between free cholesterol of the serum and of the arterial tissue, the rate of esterification of free cholesterol might be an important factor in similarly allowing more cholesterol to leave the arterial wall. To test this hypothesis segments of human iliac arteries which appeared from gross examination to be free of atherosclerosis were removed at autopsy in sterile conditions, washed with cold saline, stripped of adventitia and the wall turned inside out, so that the intimal surface was then external, and the ends were sewn together 6 . These arterial segments were used immediately or stored frozen and dried in sterile tubes until needed. Fasting blood samples were drawn from healthy volunteers, without the use of anticoagulants, defibrinated and centrifuged at 3,000 g for 10 min. The serum was removed and used immediately for all incubation procedures. The serum was first separated into two large aliquots, one of which was heated at 56° C for 30 min to inactivate LCAT enzyme. Samples of both untreated and inactivated serum were incubated for 6 h at 37° C. The decrease in serum free cholesterol concentration recorded after incubation was a measure of LCAT activity. Inactivation of serum by heating always prevented a decrease in the free cholesterol during incubation. Three iliac arterial segments, equal in size and area,