2.5.4. TBARS Concentration

TBARS concentration was analyzed using colorimetry, as described in a previous study [33]. Briefly, 200 μL of plasma was mixed with 200 μL of trichloroacetic acid (TCA; JT Baker, Phillipsburg, NJ, USA), 200 μL of Tris-HCl (Serva Electrophoresis, Heidelberg, Germany) and incubated at room temperature for 10 min. Thereafter, 400 μL of TBARS reagent, including 55 mM thiobarbituric acid (TBA) (from Merck, Darmstadt, Gemany) and 2 M sodium persulfate (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) were added into the tube. Samples were kept to a dry bath in an incubator at 95 ◦C for 45 min and put on ice for 5 min. Subsequently, 400 μL of 75% TCA was added to the tube and mixed well, and the solution was then centrifuged for 5 min at 10,000× *g*. Thereafter, 100 μL of supernatant was loaded onto 96-well plates, and various concentrations of 1,1,3,3-tetraethoxypropane (Aldrich, St. Louis, MO, USA) were used to construct a standard curve, per the procedure described by Boadi et al. [34]. The absorbance was read at 530 nm using a Tecan Infinite M200 microplate reader (Tecan Austria GmbH, Grödig, Austria), and the result was applied to the regression formula to obtain the TBARS concentration.

**Figure 2.** LC MS/MS Chromatograms of (**A**) betaine and choline standards (10 ng/mL each) and (**B**) a plasma sample. Blue: choline; Red: d9-choline; Green: betaine; Gray: d11-betaine.
