Radiolabeled cholesteryl ethers: A need to analyze for biological stability before use.

Radiolabeled cholesteryl ethers are widely used as non-metabolizable tracers for lipoproteins and lipid emulsions in a variety of in vitro and in vivo experiments. Since cholesteryl ethers do not leave cells after uptake and are not hydrolyzed by mammalian cellular enzymes, these compounds can act as markers for cumulative cell uptakes of labeled particles. We have employed [3H]cholesteryl oleoyl ether to study the uptake and distribution of triglyceride-rich emulsion particles on animal models. However, questionable unexpected results compelled us to analyze the stability of these ethers. We tested the stability of two commercially available radiolabeled cholesteryl ethers - [3H]cholesteryl oleoyl ether and [3H]cholesteryl hexadecyl ether from different suppliers, employing in vitro, in vivo and chemical model systems. Our results show that, among the two cholesteryl ethers tested, one ether was hydrolyzed to free cholesterol in vitro, in vivo and chemically under alkaline hydrolyzing agent. Free cholesterol, unlike cholesteryl ether, can then re-enter the circulation leading to confounding results. The other ether was not hydrolyzed to free cholesterol and remained as a stable ether. Hence, radiolabeled cholesteryl ethers should be analyzed for biological stability before utilizing them for in vitro or in vivo experiments.

Cationic vesicles from novel bolaamphiphilic compounds.

Effective targeted drug delivery by cationic liposomes is difficult to achieve because of their rapid clearance from the blood circulation. Bolaamphiphiles that form monolayer membrane may provide vesicles with improved stability, as shown for archaeosomes. We investigated a series of bolaamphiphiles with acetylcholine head groups and systematic structural changes in their hydrophobic domain for their ability to form stable nanovesicles. Bolaamphiphiles with two aliphatic chains separated by a short amide midsection produced spherical nanovesicles ranging in diameter from 80 to 120 nm. These vesicles lost their encapsulated material within 24 hours of incubation in phosphate-buffered saline (PBS). Similar bolaamphiphiles with a longer midsection produced a mixture of fibers and more stable nanovesicles. Bolaamphiphiles with ester amide midsection produced only spherical nanovesicles that were stable during incubation in PBS for several days. Vesicles made from bolaamphiphiles with acetylcholine head groups conjugated to the aliphatic chain via the amine were less stable than vesicles made from bolaamphiphiles with head groups conjugated to the aliphatic chain via the acetyl group. Vesicles that were stable in vitro showed good stability in the blood circulation after intravenous administration to mice. These results help in elucidating the bolaamphiphile structures needed to form stable cationic vesicles for targeted drug delivery.

Multiple pathways ensure retinoid delivery to milk: studies in genetically modified mice.

Retinoids are absolutely required for normal growth and development during the postnatal period. We studied the delivery of retinoids to milk, availing of mouse models modified for proteins thought to be essential for this process. Milk retinyl esters were markedly altered in mice lacking the enzyme lecithin:retinol acyltransferase (Lrat(-/-)), indicating that this enzyme is normally responsible for the majority of retinyl esters incorporated into milk and not an acyl-CoA dependent enzyme, as proposed in the literature. Unlike wild-type milk, much of the retinoid in Lrat(-/-) milk is unesterified retinol, not retinyl ester. The composition of the residual retinyl ester present in Lrat(-/-) milk was altered from predominantly retinyl palmitate and stearate to retinyl oleate and medium chain retinyl esters. This was accompanied by increased palmitate and decreased oleate in Lrat(-/-) milk triglycerides. In other studies, we investigated the role of retinol-binding protein in retinoid delivery for milk formation. We found that Rbp(-/-) mice maintain milk retinoid concentrations similar to those in matched wild-type mice. This appears to arise due to greater postprandial delivery of retinoid, a lipoprotein lipase (LPL)-dependent pathway. Importantly, LPL also acts to assure delivery of long-chain fatty acids (LCFA) to milk. The fatty acid transporter CD36 also facilitated LCFA but not retinoid incorporation into milk. Our data show that compensatory pathways for the delivery of retinoids ensure their optimal delivery and that LRAT is the most important enzyme for milk retinyl ester formation.

Macrophage phospholipid transfer protein deficiency and ApoE secretion: impact on mouse plasma cholesterol levels and atherosclerosis.

OBJECTIVE: PLTP and apoE play important roles in lipoprotein metabolism and atherosclerosis. It is known that formation of macrophage-derived foam cells (which highly express PLTP and apoE) is the critical step in the process of atherosclerosis. We investigated the relationship between PLTP and apoE in macrophages and the atherogenic relevance in a mouse model. METHODS AND RESULTS: We transplanted PLTP-deficient mouse bone marrow into apoE-deficient mice (PLTP-/- --> apoE-/-), creating a mouse model with PLTP deficiency and apoE expression exclusively in the macrophages. We found that PLTP-/- --> apoE-/- mice have significantly lower PLTP activity, compared with controls (WT --> apoE-/-; 20%, P<0.01). On a Western diet, PLTP-/- --> apoE-/- mice have significantly lower plasma apoE than that of WT --> apoE-/- mice (63%, P<0.001), and PLTP-deficient macrophages secrete significantly less apoE than WT macrophages (44%, P<0.01). Moreover, PLTP-/- --> apoE-/- mice have significantly higher plasma cholesterol (98%, P<0.001) and phospholipid (107%, P<0.001) than that of WT --> apoE-/- mice, thus increasing atherosclerotic lesions in the aortic arch and root (403%, P<0.001), as well as the entire aorta (298%, P<0.001). CONCLUSIONS: Macrophage PLTP deficiency causes a significant reduction of apoE secretion from the cells, and this in turn promotes the accumulation of cholesterol in the circulation and accelerates the development of atherosclerosis.