2.5.2. MTP Decline in Lymphocyte

The MitoProbe™ JC-1 Assay Kit (Molecular Probes, Eugene, OR, USA) was applied to detect the MTP decline in lymphocyte. After being gently mixed, 200 μL of whole blood sample was transferred to a conical polypropylene test tube, and 4 mL of lysing buffer (Thermo Fisher Scientific, Waltham, MA, USA) was added. The solution was incubated at room temperature for 7 min before being centrifuged at 1500× *g* for 5 min at 20 ◦C, and the supernatant was then discarded. Subsequently, 3 mL of PBS (Corning®, Corning, NY, USA) was added to wash the cells, the solution was centrifuged for 5 min, and the supernatant was then discarded. Lymphocytes were suspended with 500 μL of PBS by using a MitoProbeTM JC-1 assay kit, and then subjected to a dry bath in an incubator (MD-01N, Major Science, Taoyuan, Taiwan) at 37 ◦C for 15 min. Finally, samples were gently drawn and subjected to analysis by the BD FACSCaliburTM flow cytometry (Becton Dickson, San Jose, CA, USA).

#### 2.5.3. Concentrations of Plasma Betaine and Choline

To determine the blood concentrations of betaine and choline, d11-betaine and methyl-d9-choline were used as internal standards. Betaine hydrochloride, the standard of betaine, and d11-betaine were obtained from Chem Service (West Chester, PA, USA) and Cambridge Isotope Laboratories (Tewksbury, MA, USA), respectively. Choline chloride, the standard of choline, and methyl-d9-choline were purchased from Sigma-Aldrich (St. Louis, MO, USA). Initially, 30 μL of plasma was mixed with 90 μL of internal standard solution (10 ng/mL d11-betaine and d9-choline in methanol) in a microcentrifuge tube before being vortexed for 5 min and subsequently centrifuged at 5350× *g* for 10 min at 4 ◦C. The supernatants (60 μL) were placed in an LC-MS vial, and then analyzed using an AB SCIEX API 2000 liquid chromatography tandem-mass spectrometry system (Sciex Division of MDS, Toronto, ON, Canada). The analysis was performed using an Agilent 1260 Infinity Binary high-performance liquid chromatography system (Agilent Technologies, Santa Clara, CA, USA) equipped with an integrated degasser (G1322A), a pump (G1312C), an autosampler (G1329B), and a thermostat (G1330B). Analytes were chromatographically separated using an Waters XBridge BEH Amide column (100 mm × 2.1 mm, 3.5 μm) with a gradient mobile phase comprising (A) 0.1% formic acid (Sigma-Aldrich, St. Louis, MO, USA) in H2O, and (B) 0.1% formic acid in acetonitrile (Duksan, Seoul, Korea) under linear-gradient conditions (Period, A:B-%, *v*/*v*: 0–0.5 min, 30:70; 0.5–4 min, 30:70; 4–4.1 min, 50:50; 4.1–8 min, 30:70) at a flow rate of 400 μL/min; this procedure was a modification of that described by Bruce et al. [32]. The column was maintained at 28 ◦C. The monitoring conditions for multiple reactions are presented in Table 1, and Figure 2A,B present the LC MS/MS chromatograms of the standards and one blood sample.



DP, declustering potential; FP, focusing potential; EP, entrance potential; CE, collision energy; CXP, collision cell exit potential.
