W. Jessup

1. The kinetics of the depletion of alpha-tocopherol in human low-density lipoprotein (LDL) were measured during macrophage-mediated and cell-free oxidation. The formation of oxidatively modified, high-uptake species of LDL in these systems was not detectable until all of the endogenous alpha-tocopherol had been consumed. 2. Supplementation of the alpha-tocopherol content of LDL by loading in vivo extended the duration of the lag period during which no detectable oxidative modification occurred. 3. The addition of a flavonoid (morin) prevented both alpha-tocopherol consumption and oxidative modification of LDL. 4. The alpha-tocopherol contents of LDLs from a range of individual donors could not be used to predict their relative resistance to oxidation, indicating that other endogenous antioxidants may also be present, and quantitatively significant, in human LDL.

We have reviewed the evidence that amines accumulate in intracellular vesicles of low pH, such as lysosomes and endosomes. There is consequent elevation of intravesicular pH, and inhibition of receptor-ligand dissociation often results from this pH change. We have argued that the capacity for fusion of such vesicles is also reduced by the high pH. We suggest that the variety of effects of amines on membrane flow and macromolecular transport we describe are at least partly due to such reduced fusion (Figs. 1 and 2). We propose that an internal low pH may facilitate heterologous vesicle-vesicle and vesicle-plasma membrane fusion. There is some evidence that clathrin can accelerate phospholipid vesicle fusion in vitro at low pH (Blumenthal et al., 1983) but no direct evidence on the role of intravesicular pH. This idea is consistent not only with the preceding discussion, but also with the fact that the intracellular membrane-bound compartments least involved in fusion events (e.g. mitochondria) are of neutral or alkaline internal pH. Membrane fusion is certainly required for the formation of vesicles at the periphery of the Golgi apparatus, and possibly earlier in the transport and processing of biosynthetic products in the Golgi (Bergeron et al., 1982). Thus the accumulation of amines in the Golgi may be responsible for several effects on the flow of macromolecules along their translocation pathways. The status of the plasma membrane in this view is complex. It might be argued that the pH dictating the fusion step in endocytosis is that of the extracellular fluid, in which case the inhibitory effects of amines on this process are not explained. However, the rapidity of acidification of the newly formed endocytic vesicles allows the possibility that plasma membrane invaginations might temporarily sequester areas which are of lower pH than that of the bulk extracellular fluid even before fusion, since the proton pumping enzyme(s) are probably present on the plasma membrane. Were this the case, then an acid pH could again be a factor determining membrane fusion at the plasma membrane. The inhibition of endocytosis by weak bases thus may again reflect elevation of pH in a sequestered compartment. From the data on the dependence of response on the concentration of amines, we anticipate that most responses involving membrane flow will be biphasic, with inhibitory effects at low amine concentration, giving way to stimulatory ones at higher concentrations. We suggest that the reported dichotomy between different amines in intracellular membrane fusion systems (D'Arcy Hart, 1982) may result from this concentration dependence.(ABSTRACT TRUNCATED AT 400 WORDS)

7-Hydroperoxycholesterols (7OOHs) are intermediates in cholesterol oxidation and potential cytotoxins. A normal-phase HPLC method with UV (205 nm) detection was developed that could resolve 7 alpha OOH, 7 beta OOH, 7-ketocholesterol (7K), and the epimeric 7-hydroxycholesterols (7OHs). 7OOH formation was investigated when LDL was exposed to four different oxidizing systems: Cu2+; Ham's F-10; mouse peritoneal macrophages in Ham's F-10; and a metal-independent peroxyl-radical generating system (AAPH). With all four oxidizing systems, 7OOH (both free and esterified, mostly as the beta-isomer) was the major oxysterol formed at early times, with 7K dominating at later stages (> or = 24 h) in Cu-oxLDL. When LDL was oxidized in the presence of cells there was transfer of free oxysterols from LDL to the cells. Negligible 7OOH, but significant amounts of 7OH, accumulated in the cells suggesting efficient cellular reduction of 7OOH. Lipid extracts from eight plaque samples obtained from patients undergoing carotid endarterectomy were analyzed. Only trace amounts of 7OOH (< 0.02% of total cholesterol) could be detected using this normal-phase HPLC method with UV detection or with a more sensitive reverse-phase method utilizing chemiluminescence detection. 7K was the major 7-oxygenated sterol detected, at least 20-fold in excess of that calculated for 7OOH, followed by 7 beta OH and 7 alpha OH. The trace concentrations of 7OOH in plaque indicate its lability in biological/cellular systems and may signify the ability of cells in the artery wall to metabolize it further.

We have defined the lipid composition of copper-oxidized LDL (Cu-oxLDL) and a macrophage-foam cell model generated by the uptake of this modified lipoprotein. An HPLC method previously developed by our group for the measurement of lipid oxidation products of LDL was extended to permit the analysis of an array of 7-ketocholesteryl esters. Gas chromatography was used for the quantitation of oxysterols (free and esterified) in Cu-oxLDL and their subsequent uptake by macrophages. LDL (1.0 mg protein/ml) was oxidized using Cu(II) (20 microM) for up to 48 h at 37 degrees C. Resident mouse peritoneal macrophages were incubated with 24 h Cu-oxLDL (50 micrograms/ml) for 24 h. In 24 h Cu-oxLDL, cholesterol comprised approximately 50% of total sterols, 7-ketocholesterol comprised approximately 30% with five other oxysterols comprising the remainder (7 alpha- and 7 beta-hydroxycholesterol, cholesterol alpha- and beta-epoxides, and 6 beta-hydroxycholesterol). Macrophages that were incubated with 24 h Cu-oxLDL displayed a profile of oxysterols remarkably similar to that of 24 h Cu-oxLDL itself. The majority of cholesteryl esters and 7-ketocholesteryl esters in Cu-oxLDL and in Cu-oxLDL-loaded macrophages contained fatty acyl chains which are presumed oxidized. This work represents a comprehensive survey of free and esterified oxysterols in Cu-oxLDL and Cu-oxLDL-loaded macrophages and provides a basis for exploring how oxysterols are metabolized by macrophages and authentic human foam cells, and how, in turn, these oxysterols influence cellular metabolism.