Journal of Lipid Research, Vol 28, 1166-1176, Copyright © 1987 by Lipid Research, Inc.
Effect of cholesterol enrichment on 12-hydroxyeicosatetraenoic acid metabolism by mouse peritoneal macrophages
SN Mathur and FJ Field
Department of Internal Medicine, University of Iowa, Iowa City 52242.
The metabolism of 12-hydroxyeicosatetraenoic acid (12-HETE) was
investigated in mouse peritoneal macrophages enriched in cholesterol by
incubation with acetylated low density lipoproteins. After incubating with
labeled arachidonic acid, cholesterol-rich cells released more 12- HETE
into the medium than unmodified macrophages. With time, however, 12-HETE
decreased in the medium of both cell preparations suggesting re- uptake of
this monohydroxyfatty acid and perhaps further metabolism. When control
macrophages were incubated with radiolabeled 12-HETE for 2 hr, almost 70%
of the cell-associated 12-HETE label was incorporated into phospholipids.
In contrast, in cholesterol-rich cells, only 31% of the 12-HETE label was
incorporated into phospholipids. Bee venom phospholipase completely
hydrolyzed the label, suggesting that the monohydroxyfatty acid was
esterified at the sn-2 position of the phospholipid. In cholesterol-rich
cells, 69% of the 12-HETE was diverted into neutral lipids. Two major
neutral lipids were identified in cholesterol-rich macrophages. One neutral
lipid band which migrated with an Rf value of 0.34 contained the
hydroxylated fatty acid esterified to a glyceride. The other neutral lipid
band having an Rf value of 0.49 contained cholesterol and by further
analysis was found to contain predominantly cholesteryl-12-HETE. The
labeled fatty acids in these two neutral lipids were mostly oxidized
products of 12-HETE in contrast to the native 12-HETE observed in the
phospholipids. Cholesterol-rich macrophages released 25% more products of
12-HETE metabolism than control macrophages. Two major products were
observed in the medium which eluted in the area of a standard di-HETE,
LTB4, on high performance liquid chromatography (HPLC) analysis. We propose
that the reincorporation of 12-HETE into these neutral lipids and the
increased capacity for further metabolism of this biologically potent
hydroxyfatty acid could be a mechanism by which the cholesterol-rich
macrophage maintains its membrane function, and regulates the amount of
12-HETE in the pericellular space.
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