Influence of age, BMI and seasonal effects on ethyl glucuronide concentration in hair

Objectives: Ethylglucuronide in head hair (HEtG) has become the most accredited marker to prove chronic alcohol abuse, although some warnings about the general applicability of this determination have been recently raised. We recently published conclusions about the reliability of utilizing alternative keratin sources (pubic, axillary or chest hair) whenever head hair is not available [1]. In this study, we investigated some of the other potential discriminating factors, in order to understand the possible sources of individual variability and to regulate and standardize HEtG determinations in the forensic context. Our objective was to evaluate the HEtG level distribution in the selected population, as classified according to personal and objective parameters, including (i) age, (ii) body mass index, and (iii) season of hair growth, not to their alcohol consumption. The experimental results were interpreted by statistical analysis, on the assumption that large population datasets will provide similarly-shaped distributions, leveling off the specific contribution of individual alcohol consumption. Materials and Methods: Hair samples were collected from medical commissions for driving licences located in Northern Italy. These medical commissions examine a broad range of drivers requesting to undertake a medical examination to obtain the renewal of their suspended or expired licence. HEtG was determined by HPLC-MS/MS operating in SRM mode. Results: The non parametric Kruskal-Wallis test was applied to compare HEtG level distributions, for all samples showing a measurable concentration (>10 pg/mg; N=575 for age evaluation; N=225 for BMI evaluation). A significant distribution difference based on age was observed. The percentage of hair samples classified as positive ranged from 2.9% in the 18-30 years old group (HEtG median=17.0 pg/mg, IQR=9) to 22.0% in the group over 40 years old (HEtG median=30.0 pg/mg, IQR=41). In contrast, BMI appears not to produce statistically significant differences among the groups, although the uneven dimension of examined populations prevents definitive conclusions at present. Lastly, the season of hair growth was evaluated. Hair samples (N=816) were initially selected on the basis of their length (between 1-3 cm) and period of collection (March, June, September and December), and subsequently divided into four groups, basically corresponding to hair growth in winter, spring, summer and autumn. Seasonal trends were observed, with HEtG peak levels in winter and minimum levels in summer (percentage of positive samples in winter 2009, summer 2010 and winter 2010 were, respectively, 22.14%, 8.93% and 16.50%), suggesting, as possible sources of HEtG concentration bias, differences in sweating, frequency of hair washing or alcohol intake, depending on the sampling season. Conclusion: While the trustworthiness of HetG determination to assess chronic alcohol consumption and distinguish heavy from social drinkers is unquestionable, further studies are needed to understand the real degree of biological inter-individual variation and to confirm or exclude any possible source of bias, leading to more realistic uncertainty factors and possibly differentiated cut-off levels.