Although the literature included relatively little evidence regarding the role of different cholesteryl esters. Determining the plasma profile of unesterified fatty acids is normally problematic due to the direct influence of diet on plasma fatty acid content. Using a single diet for both WT and fat-1 animals made it possible for plasma content analysis to be used as a reliable indicator of endogenous PUFA metabolism. Notably, the diet does not contain longer chain PUFAs and any circulating lipids longer than 18 carbons must come from endogenous metabolism of shorter PUFAs. Our study highlighted a significant difference in the omega-6/omega-3 ratio between unesterified plasma lipids and other previously reported tissues. In particular, our results showed a marked increase for unesterified EPA compared to unesterified DHA in the plasma of fat-1 mice, which could be explained, in part, by a retroconversion of DHA to EPA. The concomitant elevation of the DHA-containing phospholipids, however, suggests that EPA might be a preferred substrate for the hydrolytic activity of phospholipase A2 in plasma, rather than DHA. These observations point to a differential metabolism for EPA and DHA, which could explain the diverse physiological effects previously reported for these two omega-3 PUFAs. On the other hand, DHA-containing phospholipids and lysophospholipids may play roles in the cellular membrane properties and in the transport of DHA to other tissues. Lysophospholipids containing DHA do, in fact, appear to be best suited as carriers of this essential fatty acid to eye and brain tissues, where it modulates the membrane fluidity of synaptic vesicles and displays neuroprotective properties. Notably, our untargeted analysis highlights a wide increase in other lysophospholipid species, which comports with a previous report showing that dietary supplementation with omega-3s can dynamically regulate plasma lysoPC. The untargeted lipidomic analysis also showed a significant decrease in triacylglycerols and cholesterol. Such a decrease is in accordance with the fact that the fat-1 phenotype is resistant to metabolic syndrome, obesity, and liver steatosis. Significantly, the unexpected decrease in cholesterol-sulfate in the plasma of fat-1 mice may also be linked with some of the protective effect of omega-3s. Cholesterol sulfate is present in lipoproteins, and it has been found in atherosclerotic lesions of the human aorta, where it plays a role in platelet adhesion, possibly determining the prothrombotic potential of atherosclerotic lesions. Cholesterol sulfate is also found to be particularly enriched in DHA-rich cellular membranes, where it seems to modulate the lipid raft formation. One might reasonably speculate that the observed decrease in the circulating levels of cholesterol sulfate might indicate the effect of possible sequestration caused by the DHA-rich cellular membranes in fat-1 mice.

However, the 128.1 antibody localized to a CD63-positive late endosomal/lysosomal compartment one hour after internalization, where it presumably is degraded. In contrast to 128.1, the mouse monoclonal antibody MEM-189 is processed like the ligand. It is endocytosed at a slightly slower rate than transferrin and the 128.1 antibody in hCMEC/D3 cells, which is likely attributable to the lower-affinity binding profile of the antibody. However, once internalized, the antibody is processed through the endocytic pathway with transcytosis and recycling kinetics comparable to that of transferrin. The 128.1 and MEM-189 antibodies target overlapping epitopes, as shown by a competition ELISA, which lowers the probability that the MEM-189 local epitope on TfR is solely responsible for its transcytosis potential, although it cannot be excluded. Even antibodies with closely overlapping epitopes can demonstrate fundamental functional differences, as illustrated for the CD20 antibodies GA101 and rituximab. Intracellular degradation of TfR antibodies after endocytosis has been described by others, and the 128.1 antibody follows those examples in terms of lysosomal localization and degradation. Furthermore, incubation of murine lymphoma cells with full IgGs against the TfR has been shown to even downregulate surface expression of the TfR. In contrast, Yu et al. have demonstrated that in vivo, antibodies against the transferrin receptor are transported into the brain to an extent inversely correlated to their binding affinity. We compared the bivalent affinities of the different TfR antibodies by direct ELISA and observed a similar correlation with regard to transcytosis potential. A low-affinity antibody, LT-71 was capable of shuttling through hCMEC/ D3 monolayer, albeit after low uptake, while the highaffinity antibodies 128.1 and 13E4 remain inside the cells. However, two antibodies with intermediate binding affinities demonstrated significantly different sorting behavior: while MEM-189 was efficiently transcytosed and recycled, MA712 was incapable of leaving the endothelial cells. It is unlikely that the small difference in binding affinity should be responsible for the striking difference in transcytosis potential. The transferrin:transferrin receptor complex undergoes dramatic conformational changes upon endosomal acidification, which are partly driven by iron release from the ligand, but which are accompanied by significant changes in the TfR conformation. These conformational changes might be responsible for the observed pH-dependence of several TfR antibodies. In addition, histidine residues in antibody CDR could contribute to pH-dependent target binding. The mechanism of transferrin transcytosis, especially which sorting events determine routing for basolateral transcytosis or apical recycling, have not yet been investigated. In polarized cells, TfR is known to be recycled via recruitment of the adaptor protein AP1B in the recycling endosome.