# Urinary Levels of Free 2,5-Hexanedione in Italian Subjects Non-Occupationally Exposed to n-Hexane
## 1 Introduction  
Occupational exposure to n-hexane occurs in different industrial processes for the production of paints, glues, polyethylene and polypropylene foils, plastics, rubbers and tires where it is used as solvent [1]. Its worldwide diffusion started after the use of benzene as a solvent was banned. n-Hexane is considered a ubiquitous pollutant. It is present not only in various industrial materials [2; 3; 4; 5] but also in home-use solvents [6] and it is emitted from furniture, paint or building/furnishing materials [7]. Experiments carried out on the emission at room temperature from particleboard showed that n-hexane was the most abundant compound among detected volatile organic compounds except formaldehyde [8]. A study carried out in three Italian cities has confirmed the continued presence of this solvent in air with average concentrations (indoor and outdoor) ranged between 8.4 and 15.2 ug/m\({}^{3}\)[9]. Likewise, another study carried out in two campaigns (summer and winter) in about 140 rooms of office buildings across Europe, measured indoor concentrations of n-hexane up to 9.1 and 8.6 ug/m\({}^{3}\), respectively [10]. n-Hexane is present also as a component of gasoline [11; 12]. Since 1980, the increasing use of unleaded gasoline with a high-octane rating has led to a growing content of n-hexane in gasoline, which now stands at around 3%. The main route of absorption is via inhalation. The metabolic pathway of the n-hexane in humans consists of a series of oxidative steps (Figure 1) which lead to the formation of several metabolites including 2,5-hexanedione (2,5-HD). This metabolite appears to be responsible for the polyneuropathy due to the exposure to n-hexane [13; 14]. It is excreted through the urine after conjugation with sulfate anion or glucuronic acid or as free 2,5-HD. Total 2,5-HD was used as a biomarker for the monitoring of workers exposed to n-hexane [15; 16]. It was given by the sum of the free and the 2,5-HD obtained by the conversion of other metabolites of n-hexane (4,5-dihydroxy-2-hexanone and 5-hydroxy-2-hexanone) after acid hydrolysis of the urine sample [17; 18]. Total 2,5-HD was not only detected in the urine of workers occupationally exposed to n-hexane, but was detected, in lower concentrations, in the urine of subjects of the general population by several studies [19; 20; 21; 22; 23; 24; 25; 26]. The knowledge of the urinary levels of this metabolite determined in conditions of non-occupational exposure and of the confounding factors that may affect them, is essential for interpreting results of biological monitoring in the exposure assessment. In the occupational field the comparison between the data from biological monitoring in workers and the background levels in non-occupationally exposed subjects is informative about the existence of exposures greater than the general population. The importance of background values is demonstrated by the growing number of studies carried out on the general population all over the world [27; 28; 29]. Figure 1: Metabolic pathway of n-hexane in humans. Since 2005, the Italian Society of Reference Values (SIVR) defined ranges of reference values in the urine of non-occupationally exposed subjects for many biomarkers. SIVR reported for total 2,5-HD a range (5 th-95 th percentiles) between 100 and 760 ug/L [30]. In the last few years, total 2,5-HD no longer appeared to be the best biomarker of exposure to n-hexane, although it appears correlated with airborne n-hexane, because of the analytical procedure to quantify it, that involves an acidic hydrolyzation, also converts non-toxic metabolites into 2,5-HD and the yield varies with the hydrolysis conditions and the laboratory performances. Free 2,5-HD seems to be more reliable than total in order to assess the exposure to n-hexane [18; 23; 31].