Several lines of evidence suggest that oxidatively modified low density lipoprotein (LDL) is atherogenic and that antioxidants may protect LDL against oxidation. In addition, cigarette smoking is known to induce oxidant stress. We have examined the effect of ingestion of the antioxidants D,L-ar-tocopherol (vitamin E) and 0-carotene and of smoking on the resistance of LDL against copper-mediated oxidation. Six healthy nonsmoking volunteers ingested 1,000 IU/day D,L-a-tocopherol acetate for 7 days. After vitamin E ingestion concentrations of or-tocopherol in plasma and LDL increased 3.0-and 2.4-fold, respectively. Simultaneously, the oxidation resistance of LDL was elevated significantly (+41%), and the rate of oxidation was decreased significantly (-19%). The increase in o-tocopherol content of LDL and the increase in resistance time were highly correlated (r,=0.89, p=0.014). Eight weeks after termination of the vitamin E intake, o-tocopherol concentrations in plasma and LDL and oxidation resistance of LDL had returned to baseline values. In smokers (n=46), plasma levels of vitamin C (-26%) and concentrations of 0-carotene (-44%, -43%) and total carotenoids (-23%, -29%) in plasma and LDL, respectively, were significantly lower compared with nonsmokers (n=23). No differences were found in o-tocopherol content of LDL and the susceptibility of LDL to lipid peroxidation in both groups. Supplementation of a group of smokers in a 14-week randomized, double-blind, placebo-controlled intervention trial witĥ -carotene resulted in a 16.6-and 5.0-fold increase of LDL /^-carotene and total carotenoid content, respectively. Similar increases were found in plasma without changes in other vitamins. Comparison of the changes in the length of the resistance time between the /J-carotene group (R=23) and the placebo group (n=23) showed no significant differences. We conclude that Ingestion of vitamin E strongly protects LDL against oxidative modification, whereas ^-carotene is not effective, and that smoking does not alter the oxidation resistance of LDL. (Arteriosclerosis and Thrombosis 1992;12:554-562) KEY WORDS • low density lipoprotein oxidation • vitamin E • /3-carotene • cigarette smoking E levated plasma levels of low density lipoprotein (LDL) are associated with an accelerated development of atherosclerotic lesions, which is one of the main causes of coronary artery disease.

Previous studies showed that both endothelial and Kupffer cells contain specific recognition sites of oxidized low-density lipoprotein (OxLDL), in addition to recognition sites which recognize OxLDL and acetylated LDL (AcLDL). We have determined the binding characteristics of the recognition sites for OxLDL on Kupffer cells and endothelial cells (OxLDL-specific binding-site) in comparison to the recognition site for AcLDL on endothelial cells, which recognizes both AcLDL and OxLDL (Ac/OxLDL binding site). The capacity of Kupffer cells to bind OxLDL (Bmax. = 779 ng of 125I-OxLDL/mg of cell protein; Kd = 6 micrograms/ml) was comparable to the binding-capacity of endothelial cells (Bmax. = 803 ng of 125I-OxLDL/mg of cell protein; Kd = 5 micrograms/ml). The effect of net charge of modified LDL on its affinity for the recognition sites on Kupffer and endothelial cells was evaluated using competition studies. The affinity of AcLDL for the Ac/OxLDL binding site was greatly increased from 460 micrograms/ml to 4 micrograms/ml with increasing extent of modification and thus net charge. The Ac/OxLDL binding-site on endothelial cells also displayed an increased affinity towards LDL with an increasing degree of oxidation. The affinity of OxLDL for the Ac/OxLDL binding-site appeared to be about 4-fold higher than that of AcLDL with a similar extent of modification. At higher degrees of oxidation of LDL, the affinity for the OxLDL-specific site on endothelial and Kupffer cells was also strongly enhanced; the OxLDL-specific binding-site possesses a higher affinity for mildly oxidized LDL as compared with the Ac/OxLDL binding-site. It is concluded that recognition of OxLDL by both the OxLDL-specific binding-site and the Ac/OxLDL binding-site on liver endothelial and Kupffer cells depends on the net negative charge of modified LDL. The similarity in binding pattern of these binding sites makes it likely that the newly described 95 kD OxLDL binding protein on Kupffer cells [Y. B. De Rijke and Th. J. C. van Berkel, J. Biol. Chem. (1994), 269, 824-827] contains a recognition site with similar structural elements as described earlier for scavenger receptors.

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The nature of the liver binding site which is responsible for the initial recognition and clearance of chylomicron-remnants and P-migrating very-low-density lipoprotein (P-VLDL) is under active dispute. We have investigated the effect of the 39-kDa receptor-associated protein (RAP) on the recognition site for activated a,-macroglobulin and P-VLDL on rat liver parenchymal cells in vivo and in vitro in order to analyze whether both substrates are recognized and internalized by the same receptor system. Radiolabelled trypsin-activated a,-macroglobulin (a,M-T) was cleared rapidly by the liver (maximal uptake of 80.8 ? 1 .O% of the injected dose). Prior injection of 5, 15, or 50 mg gluthathione-S-transferase-linked RAP (GST-RAP)& rat reduced the liver uptake to 62.2 -+ 2.3 %, 59.3 2 1.1 %, or 2.9 ? 0.1 % of the injected dose, respectively. Concurrently the serum decay was strongly delayed after injection of 50 mg GST-RAP/kg rat but this did not affect the serum decay and liver uptake of '"I-P-VLDL. Binding studies with isolated liver parenchymal cells in vitru demonstrated that the binding of 'ZSI-a,M-T was 98% inhibited by GST-RAP with an IC,, of 0.3 pg/ml (4.2 nM), whereas the binding of '*'I-P-VLDL and "'7-p-VLDL + recombinant apolipoprotein E (rec-apoE) was unaffected by GST-RAP up to 50 pg/ml (700 nM). Also, the cell association and degradation of a,M-T was blocked by RAP, while the association and degradation of P-VLDL and P-VLDL + rec-apoE were not influenced. The inhibitory effect of RAP on the cell association and degradation of a,M-T lasted for 1-2 h of incubation at 37 "C. The binding of the radioiodinated RAP to isolated liver parenchymal cells was highly efficiently coupled to lysosomal degradation. Upon in vivo injection into rats, '*'I-labeled RAP is rapidly cleared from the serum and taken up by the liver, which is also coupled to efficient degradation. Since RAP blocks binding of all known ligands to the a,-macroglobulin receptor/low-density lipoprotein receptor-related protein (the a,Mr/LRP) and at high concentrations the binding to the LDL receptor, we conclude that the initial binding and internalization of P-VLDL by rat liver parenchymal cells is not mediated by the a,Mr/LRP. The properties of binding of P-VLDL to rat liver parenchymal cells points to an apoE-specific recognition site for lipoprotein remnants which differs from the a,Mr/LRP, proteoglycans and the LDL receptor and is tentatively called the lipoprotein remnant receptor.Keywords: lipoprotein remnants ; low-density-lipoprotein-receptor-related protein : liver; receptor-associated protein (39 kDa).Chylomicrons and very-low-density lipoprotein (VLDL) interact with lipoprotein lipase after entering the blood circulation. This interaction leads to hydrolysis of most of their triacylglycerols (Redgrave and Small, 1979). During this process, the apo-