Possible Mechanism of Egress of Free Cholesterol from the Arterial Wall

Abstract

SPERRY¹ 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 Wright² 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. Murphy⁴ 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 together⁶. 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 3,000g 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, and removed from the same necropsy and prepared as described, were used for subsequent incubation experiments. One segment was first heated at 56° C for 30 min to inactivate any LCAT enzyme from the arterial wall and the other two were untreated. Incubations for 6 h at 37° C were then carried out in the following conditions: 5 ml. aliquots of serum (untreated) were incubated with arterial segments (untreated); a similar untreated aliquot of serum was incubated with an inactivated arterial segment, and an aliquot of inactivated serum was incubated with an untreated arterial segment. Incubations were carried out in small sealed Erlenmeyer flasks to which oxygen had been added. The flasks were placed in a Dubnoff shaker to mix the serum and arterial intima. At the end of incubation arterial segments were carefully lifted and any serum adhering to the intima was allowed to drain off for 15 min. The total and free cholesterol concentrations were then determined by the method of Zak et al.⁷ on all serum samples for all the conditions described. The data on initial LCAT activity could be used as a measure of the expected decrease in free cholesterol with incubation of serum alone, and any increase of total and free cholesterol in the serum after incubation with arterial segments could be taken as a measure of egress of cholesterol from the arterial wall (Table 1). The decrease in free cholesterol when serum was incubated alone (A) occurs without any significant change in total cholesterol, the slight difference being compatible with a less than 2% error in the technique. Furthermore, the method of inactivation of the LCAT enzyme of the serum was dependable, the decrease in free cholesterol being negligible (B). Whenever arterial segments were incubated with serum, there was an increase in the mean total cholesterol concentration (C, D, E) regardless of prior inactivation. When serum was inactivated before incubation with the arterial wall (C) there was no change in the concentration of free cholesterol, which increased, however, after incubation of untreated serum regardless of any prior inactivation of the arterial wall (D, E). The lower part of Table 1 shows that there was a similar egress of total and free cholesterol from the arterial wall after incubation with untreated serum. The negligible increase of free cholesterol in the inactivated serum (C-B) is significantly different from the other two conditions at the P< 0.001 level. But there are no significant differences in the increase in total cholesterol for all the groups. Thus when LCAT enzyme is absent from the incubation medium, only cholesterol ester seems to leave the arterial wall, whereas when LCAT enzyme is present, virtually only free cholesterol seems to leave the arterial wall.