2.5.5. PC Concentration

PC concentration was analyzed using an enzyme-linked immunosorbent assay with a commercial assay kit (BioVision, Milpitas, CA, USA). Briefly, 100 μL of plasma was mixed with 10 μL of streptozocin solution, and the mixture was incubated at room temperature for 15 min before being centrifuged for 5 min; the supernatant was then transferred to a new tube. Subsequently, 100 μL of DNPH was added to each sample, which was then vortexed and incubated at room temperature for 10 min. Thereafter, 30 μL of TCA was added to each sample, which was then put on ice for 5 min, and centrifuged for 2 min. Then, the supernatant was removed, 500 μL of cold acetone was added to each tube, and the solution was placed in a sonicating bath for 30 s. Subsequently, samples were placed at –20 ◦C for 5 min before being centrifuged for 2 min, and the acetone was then carefully removed. These steps were repeated twice with 500 μL of cold acetone added. Finally, 200 μL of guanidine solution was added, and the solution was sonicated and incubated at 60 ◦C for 15 min. The 100 μL sample was loaded onto 96-well plates and read by the Tecan Infinite M200 microplate reader (Tecan Austria GmbH, Grödig, Austria) at an absorbance of 375 nm. The result was applied to the regression formula to obtain the PC concentration.

#### *2.6. Statistical Analysis*

Statistical analysis was performed using SPSS version 22.0 (IBM, Armonk, NY, USA). Data are expressed as mean ± standard deviation (SD). A paired *t* test was used to compare the variables of time to exhaustion, peak oxygen consumption, HRmax, and AHR between the trials. A 2 (trials: betaine and placebo) × 3 (time points: Pre, Post-0, and Post-3) two-way repeated-measures analysis of variance was used to compare the variables of apoptosis, MTP decline, TBARS, and PC. When an interaction was noted, least significant difference post hoc tests were performed to determine where the difference occurred. The significance level was set at *p* < 0.05.
