Objective: Phosphatidylethanol (PEth) is an abnormal phospholipid that is formed and accumulated in mammalian cells that have been exposed to ethanol. PEth has been proposed as a marker of ethanol abuse. This study was conducted to investigate the concentration of PEth in blood and organs obtained during the autopsy of alcoholics. In addition, we performed experiments on rat tissues and human blood to evaluate the effect of various storage conditions on PEth concentrations.
Methods: Human tissues and blood from alcoholics and controls were obtained at autopsy and frozen at −20°C until extraction. Blood from healthy donors was incubated with ethanol for 24 hr and thereafter either extracted directly or stored at room temperature, stored at 4°C, frozen at −20°C, or frozen in liquid nitrogen and stored at −80°C before extraction. Rats were given intraperitoneal injections of ethanol and then killed, either while still intoxicated or when sober. Rat organs were homogenized and extracted directly, after a period of storage, and/or after freezing at −20°C. PEth concentration was analyzed using HPLC and verified by mass spectrometry.
Results: In all rat organs studied, PEth was formed during freezing at −20°C with ethanol present. PEth concentrations of 9 to 205 μmol/liter were observed in the blood obtained at autopsy. The highest value was found in the case with the highest blood alcohol concentration (114 mmol/liter) at the time of death. In the experiments on human blood stored with ethanol present, PEth concentrations were not affected after 72 hr at 4°C or after freezing in liquid nitrogen and storage at −80°C for up to 144 hr but were slightly elevated after 24 hr at room temperature and at −20°C. PEth was found in all organs obtained from the cadavers of alcoholics. Storage of organs at 4°C for 24 hr with ethanol present had no effect on the PEth concentration. The PEth concentration was unaffected when no ethanol was present at the time of freezing.
Conclusions: The rat experiments indicated that the very high PEth concentrations found in the organs of the alcoholics were probably largely formed while the organs were frozen at −20°C. Our data suggest that tissue material from bodies that were exposed to ethanol must be stored properly to obtain reliable results from subsequent analysis for PEth. Tissue should not be frozen at −20°C but instead stored refrigerated until extraction, preferably within hours of autopsy, or frozen in liquid nitrogen and stored at −80°C. Blood samples that contain ethanol can be stored refrigerated for up to 72 hr or frozen in liquid nitrogen and stored at −80°C without affecting PEth levels.