Standard concentrations were formulated for ETA, ETASO, ETAA, and thiobenzamide (TBZA), by addition to either mobile phase or bovine plasma; analytes were extracted following protein precipitation. The sensitivity of this assay for ETA was 0.25 µg/mL, for ETASO was 0.32 µg/mL, and for ETAA was 0.20 µg/mL. The retention times were 17.5 ± 0.5 min (ETA), 13.0 ± 0.5 min (ETASO), 11.5 ± 0.5 min (TBZA), and 6.3 ± 0.5 min (ETAA) (Figure 1A). The mean ± standard deviation (SD) of the r² for ETA from 15 standard curves (eight in plasma, seven in mobile phase) was 0.98 ± 0.027; ETASO was 0.98 ± 0.033; ETAA was 0.98 ± 0.029. The percent recoveries for ETA, ETASO, ETAA, and TBZA, extracted from spiked mobile phase, ranged from 88%–102%, 88%–102%, 96%–101%, and 85%–99% respectively. The percent recoveries for ETA, ETASO, ETAA, and TBZA, extracted from spiked bovine plasma, ranged from 76%–106%, 88%–102%, 96%–102%, and 84%–105% respectively.

All plasma samples collected from mice were stored at −80 °C after whole-blood separation. The stability of ETA, ETASO, ETAA, and TBZA compounds in stored samples was investigated by extraction of bovine plasma aliquots spiked with 24.9, 27.3, 22.5, and 20.6 µg/mL (0.15 mM, each), respectively. After four months storage at −80 °C there was no degradation of the compounds, determined by peak area (Figure 1B). The stability of ETA suspended in Vet syrup was similarly tested. The age of the suspensions ranged from freshly made to greater than one year. The suspension solutions showed no degradation (Figure 1C,D).

A total of 144 mice were utilized to assess the pharmacokinetics of ETA and its metabolites in wild type and Fmo-knockout mice. Eight time points were examined, ranging from 0.25–3.5 h (Figure 2). Each mouse was gavaged with a single ETA dose of 125 mg/kg. A single 2 h time point of vehicle alone was included as a control for each eight sample time course. The time course was repeated four times for each gender and genotype (C57BL/6J (WT) or Fmo 1/2/4 null (KO)). Vehicle control extracts displayed no interfering peaks (not shown).

Pharmacokinetic profiles of ETA metabolism between WT and Fmo1/2/4 knockout mice are depicted in Figure 2 and Figure 3. All mice had detectable amounts of ETAA in plasma but, levels were low in all mice and were not evaluated further. ETA concentrations were significantly greater in KO mice compared to WT (p < 0.001) (Figure 3).

Primary metabolite (ETASO) concentrations, as shown by GLS regression models, are greater than parent ETA concentrations in WT mice, whereas KO mice exhibited higher concentrations of ETA than ETASO (Figure 3, S2). The model of ETA and ETASO plasma concentrations (Figure 3 and S1) considers ETA and ETASO observations as two separate groups; no consideration is given to the fact that each of these analytes is measured for every sample. Calculating the mean difference from individual mice generates a single value allowing modeling to consider the individual source of the paired observations. ETA-ETASO plasma concentrations in WT (−5.2 µg/mL) and KO (12.4 µg/mL) mice were significantly different (ANOVA, p < 0.001) in agreement with ETA and ETASO GLS regression models (Figure 3, S2), and show that the metabolic shift between ETA and ETASO levels was consistent.

High-performance liquid chromatography (HPLC grade) reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). The vehicle for ETA gavage suspension (Vet syrup®) was from FlavoRx (Washington, DC, USA). ETASO was custom synthesized by Tjaden BioSciences (Burlington, IA, USA). ETAA was a gift from Paul R. Ortiz de Montellano (University of California, San Francisco, CA, USA) [28].

Male and female C57BL/6J mice were obtained from Jackson Laboratory (Sacramento, CA, USA). These mice, utilized as study controls, were delivered at 11 weeks of age to allow mice to acclimate for one week at the Laboratory Animal Resources Center (LARC) (Oregon State University, Corvallis, OR, USA) before study initiation. Fmo1^(−/−)/Fmo2^(−/−)/Fmo4^(−/−) [22,29] mice were backcrossed to WTC57BL/6J mice for eight generations to produce a congenic line. The line was rederived by Harlan Laboratories (Hillcrest, UK) before shipment to Oregon State University. A breeding colony was established and maintained at LARC. Mice were provided pellet diet (Purina 5053) and water ad libitum. Mice were kept on a 12-h light cycle under a controlled temperature between 21–22 °C. The study was approved by the local Institutional Animal Care and Use Committee.

The Fmo genotype was confirmed for all knockout mice on arrival at OSU (data not shown). Tested genes included Fmo1, Fmo2, Fmo3 and Fmo4, using primers and PCR conditions already described [23]; the microsomal epoxide hydrolase (MEH) gene was also included as a convenient positive control; use of primers was also described elsewhere [30]. For each mouse, genes were multiplexed together. One reaction contained Fmo2, Fmo4, and MEH (control), whereas the other reaction included Fmo1 and Fmo3 (control). DirectPCR Lysis Reagent (Tail) from Viagen Biotech (Los Angeles, CA, USA) was used to lyse mouse ear punches for DNA extraction. For a 0.2-cm-diameter ear punch the addition of 49 μL DirectPCR Lysis Reagent (Tail) containing 1 μL Proteinase K (PK) solution (20 mg/mL) was sufficient. Tubes were incubated at 55 °C for 2–16 h, with periodic vortexing to assure complete lysis; the crude lysates were then incubated at 95 °C for 10 min, to denature Proteinase K. Lastly, hair was precipitated by centrifuging for 10 s. The supernatant was transferred to a fresh tube, and was centrifuged a second time for 10 s, before transferring the supernatant to a final fresh tube. A 24 µL PCR reaction contained 1 µL of lysed ear supernatant as template.

ETA was ground into fine powder and suspended in Vet Syrup at 12.5 µg/µL with 10 min of sonication and an initial 30 s of vortexing. Suspensions were freshly prepared each morning that mice were gavaged. Typically, nine mice (one per time point, see below) were dosed and harvested in a single morning; all work was completed between 7 am and noon. Four individuals of each sex per time point were collected constituting four replicates for a single sex of a single strain. A single dose of 125 mg ETA/kg body weight was administered orally by gavage (10 µL/g body weight). The drug was vortexed before each gavage to ensure suspension uniformity. For every 8 mice completing a time course replicate, a single mouse was gavaged with vehicle, to allow for monitoring of interfering peaks in HPLC scans. After gavaging, mice were returned to their cages for observation. At designated intervals (0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 h post-gavage for experimental samples, 2 h post-gavage for vehicle control) mice were euthanized by CO₂ asphyxiation and exsanguinated by direct cardiac puncture. Blood was mixed with 33 IU of sodium heparin, to prevent clotting, and stored at room temperature until processed into plasma. Plasma was prepared by centrifugation of whole blood at 2,000 xg for 10 min with subsequent storage at −80 °C until analysis.

Plasma preparations, with volumes ranging between 90–200 µL, were spiked with 9.6 µg/mL TBZA as an internal standard. Samples were extracted at room temperature with three volumes (200 µL) of methanol with vortexing following each addition. The mixture was incubated at room temperature for 10 min, and then centrifuged for 10 min at 4 °C at 17,000 xg. Supernatant was transferred into 1.7-mL microcentrifuge tubes and evaporated to dryness under a stream of compressed nitrogen gas. Residues were resuspended in 100 µL of solvent (H₂O-acetonitrile, 75:25). Samples were centrifuged for 3 min at room temperature at 16,300 xg, to pellet any debris. Clear supernatant was transferred into 2.5-mL Waters (Milford, PA) HPLC vials with 150-µL glass inserts, for analysis.

Stability of analytes was tested in plasma and vet syrup. ETA, ETASO, ETAA, and TBZA were spiked (0.15 mM for each compound) into bovine plasma (Innovative Research, Novi, MI, USA) and 200-µL aliquots were stored at −80 °C for four months before extraction as described above. ETA-Vet syrup suspension (12.5 µg/µL) was extracted as described above with the following modifications: the suspension was diluted 1:1 with water to reduce viscosity; the solution was diluted 150-fold to equal 16.6 µg/mL and placed into HPLC vials for subsequent analysis.

HPLC analysis was performed on a Waters 2695 separations module equipped with a 2,996 photo diode array detector and a Waters dC₁₈ Atlantis^® (5 µm, 3.9 × 150 mm) column. The flow rate was 0.8 mL/min. The mobile phase consisted of 80% H₂O and 20% methanol at a temperature of 35 °C, running isocratically for 20 min. ETA, ETASO, and ETAA were prepared in solvent at concentrations from 0.04 mM to 0.3 mM with TBZA at a constant concentration of 0.1 mM, in order to generate a standard curve with each HPLC run. Peak areas of analytes were determined from tracings collected at 267 nm and were used for calculations of concentrations. A broad spectra range was also collected from 210 nm and 400 nm for each standard to establish their profile, and was collected for all samples to confirm that peaks observed in the 267 nm tracing were not impure mixtures. Quantification of analytes was by comparison to the standards; concentrations of analytes were determined from ratios of analyte to internal standard. Results were analyzed with Empower software (Waters).

Statistical analyses were performed using GraphPad Prism 5 [31]. A one-way analysis of variance (ANOVA) was performed to test for differences in C_max and t_max between treatment groups. Differences in mean ETA, ETASO, and ETAA plasma concentrations between male vs. female and knockout vs. wild type mice were also tested using ANOVA (α = 0.05). Generalized least squares (GLS) regression models of ETA and ETASO plasma concentrations were developed for WT and KO mice in the statistical computing program R [32]. Additive and interactive combinations of time and sex up to the third order were considered for model parameters. Parameters that did not significantly contribute to model predictions (α = 0.05) were removed from consideration, and the ETA and ETASO regression models with the lowest Akaike information criterion (AIC) score were selected for subsequent regression analysis.