Methylmalonic acid and propionylcarnitine (C3) were measured by LC-MS/MS, as elsewhere reported [6 (link),48 (link)]. Briefly, cells were homogenized in 500 μL of cold methanol. The supernatant was separated from proteins and cell debris by centrifugation at 14,000× g for 20 min at 4 °C. The supernatant was used for the detection of methylmalonic acid and C3. The analyses were performed using an API 4000 triple quadrupole mass spectrometer (Applied Biosystems-Sciex, Toronto, ON, Canada) coupled with a 1100 series Agilent high-performance liquid chromatograph (Agilent Technologies, Waldbronn, Germany). Metabolites concentrations were normalized to the protein content, and calculated based on 1 mg protein for each cellular extract and expressed as µM [49 (link)]. Data, reported as the mean of six replicates ± standard error of the mean (SEM), were elaborated using GraphPad Prism version 7.0a. Differences between treatments were considered significant at a value lower than 0.05.
1100 series agilent high performance liquid chromatograph
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The 1100 series Agilent high-performance liquid chromatograph is a laboratory instrument used for the separation, identification, and quantification of chemical compounds in a liquid sample. It is capable of performing high-resolution, high-sensitivity analyses.
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5 protocols using 1100 series agilent high performance liquid chromatograph
1
Quantification of Methylmalonic Acid and C3
2
Dried Blood Spot Metabolite Profiling
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Serum (10 µl) of each sample was transferred on a filter paper card and dried overnight to obtain a dried blood spot (DBS) at room temperature (Ruoppolo et al., 2018 ). The metabolites were extracted from DBS and esterified as previously described (Ruoppolo et al., 2015 ). Finally, the metabolite mixtures were resuspended in 300 μl of acetonitrile/water (70:30) containing 0.1% formic acid. The subsequent LC‐MS/MS analysis was carried out using an 1,100 series Agilent high‐performance liquid chromatograph (Agilent Technologies) and API 4000 triple quadrupole mass spectrometer (Applied Biosystems‐Sciex), by Precursor Ion Scan of 85 Da fragments. Quantitative analysis was performed by ChemoView v1.2 software (Ruoppolo et al., 2014 ; Scolamiero et al., 2014 ).
3
Measurement of Serum Linoleic Acid by LC-MS/MS
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LA was measured by LC-MS/MS, as elsewhere reported [30 (link)]. Briefly, a volume of 500 μL of cold methanol was added to each serum sample. The supernatant was collected and dried under nitrogen. The dried supernatant was dissolved in methanol containing labeled standards at a concentration of 10 microM. The analyses were performed using an API 4000 triple quadrupole mass spectrometer (Applied Biosystems-Sciex, Toronto, ON, Canada) coupled with an 1100 series Agilent high-performance liquid chromatograph (Agilent Technologies, Waldbronn, Germany). The MS/MS analysis of LA was performed by using a Multiple Reaction Monitoring (MRM) experiment (Q1 89.00; Q3 59.00) [31 (link)].
4
Quantifying Ferrihydrite Reduction and Sulfate Dynamics
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The reduction of ferrihydrite was assessed by measuring the Fe(II) in the suspensions by acid extraction and analysis by the ferrozine method (Stookey 1970 ) using HEPES-buffered ferrozine reagent (Sørensen 1982 (link)) as described by O'Loughlin et al. (2019) . Sulfate reduction was monitored by measuring sulfate concentration using the method of Kolmert et al. (2000) (link), scaled for 0.5 mL of sample. Briefly, 0.5 mL of sample was combined with 0.5 mL of conditioning reagent and 30 mg of BaCl2 powder was added and the mixture was immediately mixed (vortexed) for 30 s. Immediately after mixing, the mixture was poured into a microcuvette and the absorbance at 430 nm was measured; the detection limit was 0.2 mM SO42-. Aqueous phase Sb concentrations were determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES) using a PerkinElmer 4300DV instrument. Measurement of the Sb emission line at 217.582 nm in radial view mode provided a detection limit of 5 μM. An Agilent 1100 series high performance liquid chromatograph (HPLC) was used to determine the concentrations of acetate, lactate, and propionate as described by Kwon et al., 2014 , Kwon et al., 2014 . The pH of aqueous solutions was measured using a Semi Micro pH electrode (Thermo Scientific Inc.) calibrated with NIST-traceable pH standards to a precision of 0.01 pH units.
5
Testosterone Metabolism Assay in HepG2
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After treatment with DCF ± TNF-cells were washed with PBS and incubated at 37ºC with testosterone dissolved in Williams'E medium without phenol red. After 2h, medium was collected and CYP3A4 activity was measured using a high performance liquid chromatography equipment (Agilent 1100 series high performance liquid chromatograph equipped with an autosampler and Agilent 1100 series fluorescence and UV detectors) with two solvents, acetic acid (0.1%) and acetonitrile, as previously (Aninat et al., 2006) .
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