In vitro metabolites of di-2-ethylhexyl adipate (DEHA) as biomarkers of exposure in human biomonitoring applications

Abstract

Di-2-ethylhexyl adipate (DEHA) is a common plasticizer used in food packaging. At high doses, DEHA can cause adverse health effects in rats. Although the potential for human exposure to DEHA is high, no DEHA specific biomarkers are identified for human biomonitoring. Using human liver microsomes, we investigated the in vitro phase I metabolism of DEHA and its hydrolytic metabolite mono-2-ethylhexyl adipate (MEHA) and, for comparison purposes, of the analogous di-2-ethylhexyl phthalate (DEHP) and its hydrolytic metabolite mono-2-ethylhexyl phthalate. We unequivocally identified MEHA, a DEHA specific biomarker, and adipic acid, a nonspecific biomarker, using authentic standards. On the basis of their mass spectrometric fragmentation patterns, we tentatively identified two other DEHA specific metabolites: mono-2-ethylhydroxyhexyl adipate (MEHHA) and mono-2-ethyloxohexyl adipate (MEOHA), analogous to the oxidative metabolites of DEHP. Interestingly, although adipic acid was the major in vitro metabolite of DEHA, the analogous phthalic acid was not the major in vitro metabolite of DEHP. Our preliminary data for 144 adults with no known exposure to DEHA suggests that adipic acid is also the main in vivo urinary metabolite, while MEHA, MEHHA, and MEOHA are only minor metabolites. Therefore, the use of these specific metabolites for assessing the exposure of DEHA may be limited to highly exposed populations.

Figure 1

Chromatographic separation of DEHA metabolites detected in a human liver microsomes suspension of DEHA after 5 h of incubation at 37 °C. MEOHA, mono-2-ethyl oxohexyl adipate; MEHHA, mono-2-ethylhydroxyhexyl adipate; MEHA, mono-2-ethyl-hexyl adipate.

Figure 2

Mass spectrometric fragmentation of (A) adipic acid, (B) MEOHA, (C) MEHA, and (D) MEHHA formed after in vitro phase I metabolism of DEHA (left) and ¹³C₆-DEHA (right) using human liver microsomes. Structures shown are for only one of the potential isomers.

Figure 3

Time dependent formation of phase I in vitro metabolites of MEHP (A), MEHA (B), DEHP (C), and DEHA (D) with human liver microsomes. Error bars represent the standard deviation (N = 3). MEHHA and MEOHA were quantified using MEHA. DEHA and DEHP levels were not monitored.