When alcohol is not swimming in your veins but actually in your experimental subject’s system, you really want to accurately determine its concentration. There are three main methods on the market nowadays: 1) alcohol oxidase (AOD)-based method from Analox, 2) alcohol dehydrogenase (ADH)-based method available from Sigma, and 3) headspace gas chromatography offered by Perkin Elmer. I have used the first two of them in my research, which is focused on the effects of alcohol on brain cells. They are both enzyme-based; however, Analox have certain advantages over the ADH-based kit, and considering the lower cost as compared to gas chromatography, the AOD-based method is, in my opinion, the method of choice.
The AOD assay is based on the oxidization of ethanol by alcohol oxidase in the presence of molecular oxygen. The rate of oxygen consumption is directly proportional to the alcohol concentration. Alcohol concentration can be detected in a variety of tissues: serum, cell culture medium, urine, whole blood, tissue. For accurate measurements from the two latter types of specimen, it is important to precipitate proteins. To do this, simply mix blood or tissue homogenate with 6% perichloric acid, followed by K2CO3, and centrifuge to separate proteins from the supernatant, in which you will determine alcohol level afterward. I have used this method to determine alcohol content in brain tissues with excellent results. Alcohol can also be detected in whole blood using capillaries and a centrifuge to separate erythrocytes and serum in the capillaries. That technique is also very simple. Just fill up a capillary with blood, secure both ends with blue “play-dough” like material (provided) and spin for a few minutes. One thing to remember is to avoid air in the capillary. I have also used this latter method to determine alcohol levels in blood with very good results. Serum, cell culture medium or urine can be used directly, and these materials also worked very well in my hands (except urine, which I did not use in my research). The AOD-based method is very simple, quick and reproducible, with very good sensitivity of 0.1 mM ethanol, and requires very small amount of sample (2.5 – 5.0 uL).
After preparing the pre-warmed enzyme solution in advance, and calibrating with standards, just inject 2.5-10 uL of your unknown sample and the results, displayed in mg/dL or mM, are ready in ~ 30 sec. However, there are several inconveniences which make the use of Analox less enjoyable. Precision of the measurement is limited to the linear range of assay which is rather narrow: 43 mM – 86 mM. To bring your samples to the linear range it is necessary to use more of the sample if lower concentrations are expected, or dilute it – if higher. Thus, you should a priori estimate pretty accurately your concentration or run different volumes of your sample. Considering that the range of working concentrations of alcohol is from 20 mM (~ limit of legal intoxication) to 100 mM (coma), it would be much simpler to have an assay with linearity covering that full range. Analox also requires rather extensive maintenance routines to be performed every month, three months and six months. Another disadvantage is an anachronic interface. At the dawn of the 21st century, we are spoiled with Star Trek-like equipment, and the Analox machine looks like an item from a Ben Franklin museum exhibit. The instrument has a very small display window, not-user friendly commands, lack of (e.g. USB) ports to conveniently transfer data, all of which makes it difficult to work with Analox analyzer. Despite these limitations, it is a very reliable and fast instrument for determination of alcohol concentrations in various samples. Additionally, the Analox analyzer is compatible with other assays for determination of levels of ammonia, cholesterol, creatinine, glucose, glutamine, glycerol, lactate, pyruvate, triglycerides, urea, uric acid, or â-hydroxybutyrate. Thus, Analox analyzer can be a very valuable asset in biochemical, clinical and basic science laboratories.
Instructor
Department of Psychiatry
University of Massachusetts