# Urinary Levels of Free 2,5-Hexanedione in Italian Subjects Non-Occupationally Exposed to n-Hexane
## 4 Discussion  
The use of free 2,5-HD as a biomarker to assess the occupational exposure to n-hexane was preferred, by some authors, over the total 2,5-HD, after acidic hydrolysis of urine during the quantitative analysis. This latter analytical phase, which covers other nontoxic metabolites in 2,5-HD in addition to the fact that the toxic effects have been linked to the free 2,5-HD itself [36; 37], appears to make free 2,5-HD a better biomarker of exposure and neurotoxic risk. Some organizations proposed free 2,5-HD as a biomarker of occupational exposure to n-hexane and established limit values for this metabolite. The Japan Society of Occupational Health (JSOH), adopted a biological limit value of 0.3 mg/g creatinine [38]. Since 2003, the American Conference of Government Industrial Hygienists (ACGIH), instead, adopted a biological exposure index (BEI) equal to 0.4 mg/L [39] that was changed to 0.5 mg/L, on the basis of more recent studies,  
Figure 3: Correlation between the levels of free and total 2,5-HD. in 2019 [40]. On the contrary with what was established by JSOH, ACGIH reported that no correction for creatinine concentration was recommended because it does not give a significant improvement in the exposure-excretion relationship [31]. The JSOH and ACGIH limit values could be considered substantially superimposable if a mean value of creatinine of 1.69 g/L is taken into account [41]. In occupational medicine, the comparison of biological monitoring data in the exposure assessment with the occupational limit value is essential to evaluate the probability of occurrence of health effects and the need of a reduction of exposure, as the comparison with the background level of a biomarker, could be very useful to health professionals to assess the exposure that is likely to be occupational and not from the general environment. Levels at or above the levels of biomarker in the fluids of the general population will have a high probability of resulting from occupational exposure. The aim of the present study is to contribute to the definition of the background urinary levels of free 2,5-HD in the Italian general population. Many studies report the levels of total 2,5-HD in the urine of non-occupationally exposed subjects quantified after acidic hydrolysis of urine [23; 26; 42; 43] or they quantified free 2,5-HD in urine of occupationally exposed subjects [22; 44; 45] but the studies that analyzed the levels of free 2,5-HD in general population are very few [21; 46]. Moreover they were carried out on a small number of subjects or the analytical method used had a very high limit of detection. In these studies the levels of total 2,5-HD in urine from non-occupationally exposed subjects varied from about 100 ug/L to 1 mg/L [42]; instead, a detectable amount of free 2,5-HD was not found [21; 46]. Free 2,5-HD represents a small percentage of the total. In a study where 2,5-HD was measured in the urine of workers in the shoe industry, the urinary levels of free metabolite were in the range of 30 to 5590 ug/L with a mean value equal to 790 ug/L and represented the 14.2% of the total [45]. A lower ratio between free and total 2,5-HD was found by [PERSON] et al. [23] and [PERSON] et al. [24] equal to 8% and 10%, respectively. A ratio equal to 10% was reported, as well, by JSOH that established a biological limit value of 0.3 mg/g creatinine for free 2,5-HD and 3 mg/g creatinine for total 2,5-HD [38]. In another study, the amount of free 2,5-HD was about one-fifth of the total 2,5-HD, although the percentage of free to total 2,5-HD varied from 0.4% to 28% in relation to the individual variability [47]. This large variability could be due to the different n-hexane metabolic rates or different factors that can affect its excretion such as ethnicity, gender, cigarette smoke or age. Some authors reported a less active form of cytochrome P450 2E1 (CYP 2E1) in the Asian population compared to the Caucasian population [48] or reported that CYP 2E1 levels decrease with age [49].