*3.4. Target Metabolite Analysis of Glycolysis and Pentose Phosphate Pathway Intermediates*

At baseline, the acute GXT showed greater glucose consumption and was accompanied by a series of unchanged levels of downstream metabolites until G3P (Figure 4A). The GSSG/GSH ratio significantly increased following an acute GXT, indicating an accumulation of oxidative stress. In addition, 2,3-BPG was depleted after the GXT.

Both glycolysis and the PPP were upregulated due to exercise training. Although no change was observed in the glucose level, the downstream metabolites were markedly elevated even under resting conditions. Interestingly, although 2,3-BPG was lower after training, the end product of glycolysis, lactate, was dramatically increased. The PPP flux was also facilitated, as evidenced by a decrease in 6PG and a constant GSSG/GSH ratio. Furthermore, the higher levels of Ru5P and GSH suggested a significant enhancement of GSH biosynthesis under stress. X5P and E4P are intermediates between the PPP and glycolysis, which was dramatically enhanced in both training groups.

In addition, the level of met-Hb was significantly increased by the acute GXT compared to the rest among the three groups, whereas the extent of elevation was diminished after training. The intracellular pH in erythrocytes did not change relative to the GXT before training, whereas both CCT and ECT lowered the pH value after 6 weeks (Figure 4B).

**Figure 4.** The target metabolite analysis of glycolysis and the pentose phosphate pathway intermediates in erythrocytes in ECT and CCT. (**A**) Levels of metabolites in pentose phosphate pathway and glycolytic pathway (*n* = 5) (**B**) the intracellular pH of erythrocytes. **Pre**, pre-intervention; **Post**, post-intervention; R, at rest; M, immediately after a GXT; \* *p* < 0.05, R vs. M; † *p* < 0.05, Pre vs. Post. Values were mean ± SEM.
