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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.

Therapeutic hypothermia (HT) is a currently accepted treatment for neonatal asphyxia and is a promising strategy in adult stroke therapy. We previously reported that acute administration of docosahexaenoic acid (DHA) triglyceride emulsion (tri-DHA) protects against hypoxic-ischemic (HI) injury in neonatal mice. We questioned if co-treatment with HT and tri-DHA would achieve synergic effects in protecting the brain from HI injury. Neonatal mice (10-day old) subjected to HI injury were placed in temperature-controlled chambers for 4 h of either HT (rectal temperature 31–32°C) or normothermia (NT, rectal temperature 37°C). Mice were treated with tri-DHA (0.375 g tri-DHA/kg bw, two injections) before and 1 h after initiation of HT. We observed that HT, beginning immediately after HI injury, reduced brain infarct volume similarly to tri-DHA treatment (~50%). Further, HT delayed 2 h post-HI injury provided neuroprotection (% infarct volume: 31.4 ± 4.1 vs. 18.8 ± 4.6 HT), while 4 h delayed HT did not protect against HI insult (% infarct volume: 30.7 ± 5.0 vs. 31.3 ± 5.6 HT). HT plus tri-DHA combination treatment beginning at 0 or 2 h after HI injury did not further reduce infarct volumes compared to HT alone. Our results indicate that HT offers similar degrees of neuroprotection against HI injury compared to tri-DHA treatment. HT can only be provided in tertiary care centers, requires intense monitoring and can have adverse effects. In contrast, tri-DHA treatment may be advantageous in providing a feasible and effective strategy in patients after HI injury.

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