Methods In The Assessment Of Alcohol Abuse
Ethanol is a flammable, volatile colorless liquid which is also known popularly as as ethyl alcohol, alcohol, and drinking alcohol (National Institute of Standards and Technology 2008). Ethanol has many uses in industry, but it is also historically consumed by humans as antiseptic and disinfectant (McDonnell and Russell 1999), ethanol-based fuels (Pimentel 2003), and alcoholic drinks. The intake of alcohol, affects almost every system in the body, but most damage can be observed in the brain, digestive system, the liver and the lungs. In the brain, major neurotransmitters like the opioid systems, dopamine, serotonin and gamma-amino-butyric acid (GABA) and their interactions are most affected (Chastain 2006). Being volatile, alcohol can be inhaled during ingestion and freely diffused into the airways (George, et al. 1996). This property became the basis for the breath test for estimating levels of blood alcohol (Hlastala 1998). Changes in levels of important enzymes were based on the effect of alcohol on hepatic functions and characteristics (Purohit and Brenner 2006) and other diseases (Purohit, Khalsa and Serrano, 2005). Liver damage due to alcohol has been attributed to increased oxidative stress which could be due to alterations in the liver's antioxidant defense mechanism. Chronic ethanol intake was found to decrease cytosolic and mitochondrial glutathione/glutathione peroxidase-1 activities by 40% and 30%, respectively thereby promoting the oxidative modification of liver proteins (Bailey, et al. 2006) (Cunningham and Bailey 2001). The long-term mean daily intake of 61.6 g of ethanol increases the frequency for fatty liver and alcoholic hepatitis (Savolainen, et al. 2007).
Ethanol transfers from the blood into the air sacs in the lungs, and thus, consumption can be routinely determined by what is known as the breath test (Hlastala 1998). The breath alcohol concentration predicts the blood alcohol concentration at certain time points. The development of breath testing instruments ("breathalyzers") has resulted in a low-cost, accurate, rapid and painless ethanol quantification. The level of alcohol exhaled is then multiplied with a factor to get an approximation of blood alcohol. However, many factors can affect the breathalyzer results; among these is diabetes which can increase blood alcohol levels due to ketogenic reactions in the body (Berg, Tymoczko and Stryer 2002).
In emergency cases where a breath test is not suitable, ethanol concentrations can be determined from blood, urine and saliva samples. Quantitative ethanol detector (QED) kits have been developed that use saliva as sample. It was found that ethanol concentrations measured from blood using alcohol dehyrogenase method and saliva using a QED kit did not differ significantly (Smolle, et al. 1999). QED kits can also measure blood ethanol levels that are comparable to concentration measured by gas chromatography (Biwasaka 2001).
However, being volatile, ethanol has a short half-life in body fluids. Thus other biomarkers, mostly proteins, that are found in serum or blood, are used to measure ethanol effects and chronic alcoholism (Table 1) (Niemela 2007).
Gamma-glutamyltransferase (GGT) is a glycoprotein that increases in serum as a result of ethanol intake (Conigrave, et al. 2003; Hietala, et al. 2005). GGT catalyzes the transfer of the gamma-glutamyl moiety of glutathione to various peptide acceptors, and is possibly the most widely used biomarker to detect excessive alcohol consumption. Serum GGT responds to even low amounts of ethanol, and this should be accounted for when defining reference levels. GGT activity is measured by monitoring changes in the absorbance of substrate residue after reaction of GGT substrate (commonly gamma-glutamyl-para-nitroanilide) (Walker, Hall and Hurst 1990). Substrate solubility may be enhanced by using substrate derivatives to improve GGT activity, although most laboratories prefer to use unsubstituted substrate based on established clinical experience. Serum GGT is preferred over whole blood because hemoglobin interferes with the absorbance. Although ethanol intake and serum GGTactivity are correlated, there is variation in the sensitivity of the clinical materials, exceeding those for other markers and showing dependence on gender of the patient (Hietala, et al. 2005) and obesity.
Mean corpuscular volume (MCV) measures red blood cell size, and is increased in heavy drinkers (Morgan, et al. 1981). This marker is suitable for monitoring long-term drinking habits in patients that do not show signs of alcoholism (Koivisto, et al. 2006). However, MCV does not respond rapidly to abstinence and is more suitable for selected conditions like foetal screening. MCV also should be interpreted with caution in patients with deficiency the B vitamins and folic acid.
Carbohydrate-deficient transferrin (CDT) with increasing usage due to its highly specific nature is a relatively new marker. Transferrin biosynthesis occurs in the liver. It is involved in iron transport. Moderate to heavy ethanol consumption impairs the incorporation of sialic acid (carbohydrate) moieties and increases the desialylated isoforms of transferrin in biological fluids (Stibler 1991). Thus, disialotransferrin was set to be the primary target molecule for the measurement. Due to lack of standardization in the protocol, a working group was formed to standardize procedures for CDT analysis. In the absence of a mass spectrometric reference, HPLC should be used as the reference method. Furthermore, it was recommended that CDT be expressed in relative terms (% CDT to total CDT). Monoclonal antibodies to human CDT and automation has allowed for single-step analysis of CDT (Jeppsson, et al. 2007).
After heavy ethanol intake, serum triglycerides and free fatty acid ethyl esters (FAEEs) concentrations are markedly increased. FAEEs are formed by the esterification of free fatty acids with ethanol and glyceride trans-esterification (Moore, et al. 2003). FAAEs can be detected in serum and erythrocytes even after 24 hours of drinking and are quantified by gas chromatography and mass spectrometry (Kulig, Beresford and Everson 2006). Although not stable in blood samples, the FAEEs can be detected in hair samples and are stable after months. FAAE was incorporated from sebum to distal hair sections, although variables like cosmetics can affect the process (Wurst, et al. 2004). FAEEs can also be detected in the meconium of neonates, which make FAEEs concentration a suitable measure of alcohol exposure in neonates, which could increase the risk of fetal alcohol syndrome (Moore, et al. 2003). A total FAEE concentration that is greater than 10000 ng/g in the meconium could be an indication of alcohol exposure during pregnancy. The potential of neonatal FAEE measurement in hair for alcohol exposure of infants has been explored in the diagnosis of fetal alcohol spectrum disorder (FASD) (Caprara, Klein and Koren 2006).
Other biomarkers can also be used like the modification of native proteins by metabolites of ethanol, increased concentration of ethanol metabolites, and increased liver enzyme activities (Niemela 2007). Levels of liver enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are elevated in alcoholics, and thus, are used in assessing alcohol intakes (Niemela 2007). While ALT is a specific liver marker, the selective increase in serum AST could be due to mitochondrial damage in liver tissue, skeletal muscle injury or alcoholic cardiomyopathy.