Sulpho-conjugation of ethanol in humans in vivo and by individual sulphotransferase forms in vitro

Abstract

We studied whether ethanol is sulphonated in humans with the perspective of using the urinary excretion of ethyl sulphate after ethanol consumption as a biomarker for SULT (sulphotransferase) activity. We developed a sensitive and selective HPLC-MS/MS method for determining ethyl sulphate in urine. Ten volunteers received a low dose of ethanol (0.1 g/kg of body mass). In general, excretion of ethyl sulphate was maximal in the first or second hour after dosage. Within 8 h, 2.5-6.8 micromol of ethyl sulphate was excreted. A 5-fold increase in the dose of ethanol led to an increase in the amount of ethyl sulphate excreted within 8 h (28-95 micromol) and the presence of this metabolite in urine for at least 24 h. Since ethyl sulphate was still being excreted for a substantial period after the elimination of ethanol, it might be used as a medium-time biomarker for preceding ethanol consumption. We have expressed previously all human SULT forms identified in Salmonella typhimurium. Ethanol sulphonation was studied in cytosolic preparations of these strains. The highest activities were observed with SULT1A2, 1B1 and 1C2, followed by 1A3. Activities were markedly lower with SULT1E1, 1A1 and 2A1, and were negligible with SULT1C1, 2B1a, 2B1b and 4A1. If the expression levels in tissues are additionally taken into account, SULT1A3 might be the predominant form for the sulphonation of ethanol in vivo, although a robust estimate requires further studies. With this limitation, urinary ethyl sulphate excretion appears very promising as a biomarker for SULT activity in vivo.

Figure 1. Negative electrospray ionization mass spectra of ethyl sulphate (upper trace) and 2-propyl sulphate (lower trace)

Two main ions, the molecular ion (m/z=125 and 139 respectively) and HSO₄⁻ (m/z=97) were formed under the conditions selected (cone voltage, 25 V; source temperature, 80 °C; desolvation temperature, 350 °C). They were then used for detecting ethyl sulphate and 2-propyl sulphate in the MRM mode.

Figure 2. HPLC–MS/MS chromatograms for detecting ethyl sulphate and 2-propyl sulphate

(A) Blank urine was spiked with 20 μM ethyl sulphate and 10 μM 2-propyl sulphate respectively; (B) a blank urine taken before consumption of ethanol spiked with the internal standard 2-propyl sulphate; (C) a urinary sample of the same subject collected between 2 and 3 h after ethanol intake (0.1 g/kg of body mass). Peaks are labelled with the retention time and the absolute area under the peak. In all cases, 20 μl of sample (equivalent to 10 μl of urine) was injected. Upper traces in each panel show ethyl sulphate (m/z for molecular and daughter ions 125 and 97 respectively); lower traces in each panel show 2-propyl sulphate (m/z for molecule and daughter ions 139 and 97 respectively).

Figure 3. Time course of the urinary excretion of ethyl sulphate after consumption of 0.1 g of ethanol per kg of body mass

Ethanol was consumed as sparkling wine within 5 min. Total urine was collected in time intervals indicated by the width of the bars. The bars indicate the means of all subjects and repeat experiments presented in Table 2. The symbols represent the individual subjects and experiments.

Figure 4. Time course of the urinary excretion of ethyl sulphate after consumption of 0.5 g of ethanol per kg of body mass

Ethanol was consumed as sparkling wine within 5 min. Total urine was collected in time intervals indicated by the width of the bars. The bars indicate the means of both subjects and repeat experiments (see Table 2). Open and solid circles: subject 6, first and second experiment respectively; open and solid squares: subject 10, first and second experiment respectively.