*3.7. Relationships between GSSG/GSH and Lactate/Pyruvate and between Intracellular pH and Lactate Concentration*

Figure 6A shows that the increase in the GSSG/GSH ratio caused by the GXT was significantly linearly related to the augmented lactate/pyruvate ratio (*r* = 0.72, *p* < 0.05). However, this correlation was blunted after training in both the CCT and ECT groups (Figure 6B, *r* = 0.29, *p* = 0.12). Furthermore, Figure 6C demonstrates that the lowered intracellular pH was moderately related to the greater lactate concentration (*r* = −0.50, *p* < 0.05).

**Figure 6.** Correlation analysis between GSSG/GSH, lactate/pyruvate, intracellular pH value and lactate concentration. (**A**) relationship between GSSG/GSH and lactate/pyruvate before interventions, (**B**) relationship between GSSG/GSH and lactate/pyruvate after interventions, and (**C**) the correlation between intracellular pH value and lactate concentration. GSH, glutathione; GSSG, glutathione disulfide.

#### **4. Discussion**

Erythrocyte metabolism includes glycolytic pathways producing both energy and oxidation–reduction intermediates that support O2 transport and antioxidative capacity. This study is the first to demonstrate that both CCT and power-matched ECT not only ameliorate antioxidation capacity in erythrocytes but also significantly increase the flux of anaerobic glycolysis to facilitate oxygen release efficacy. We further elucidated that the reduced oxygen affinity is due to greater lactate synthesis and not to the production of 2,3-BPG. Although ECT did not result in significant improvement in VO2 max, the im-proved VT performance indicates the positive effect ECT has on the aerobic capacity of young and sedentary men.

Several studies have reported that blood GSSG and thus the GSH/GSSG ratio decrease in response to acute exercise, and regular exercise may increase antioxidative capacity [28]. After both CCT and ECT, neither the GSSG/GSH ratio nor GSH decreased due to the

GXT, while enhanced anaerobic glycolysis provided more precursors to activate the PPP. In addition, the downstream X5P and E4P, from the PPP back to glycolysis, also increased after training, thus suggesting significant enhancement of GSH biosynthesis under stress. The linear relationship between GSSG/GSH and lactate/pyruvate was disrupted after training, which may be due to a changed dominance of energy or/and antioxidant production [29]. In addition, the oxidative environment leads to the production of Fe3<sup>+</sup> (met-Hb). To restore Hb function, met-Hb must be reduced mainly by NADH-dependent cytochrome b5 reductase [30].

An interesting aspect of the metabolic pathways is that intracellular pH (pHi) regulates both the glycolytic pathway and the PPP. As with glycolysis, the optimum activity of the enzymes driving the PPP occurs at an alkaline pHi [31]. Generally, the presence of NADPH blocks PPP negative feedback control and shifts metabolism from the PPP to glycolysis, thus increasing the formation of NAD<sup>+</sup> [32]. Although NADH does not directly participate in the reduction of Fe3<sup>+</sup> to Fe2+ in hemoglobin, it has the ultimate responsibility of providing the reducing power needed for such a reaction [33]. Under normal homeostasis in general, and especially in the case of the high glycolytic flux that is required during high-intensity exercise, lactate dehydrogenase oxidizes NADH back to NAD<sup>+</sup> in the conversion of pyruvate to lactate, thereby maintaining necessary levels of the cofactor for the continuation of glycolysis. Cyclists in the high-class group had a higher posttest lactate/pyruvate ratio, which is proportional to NADH/NAD<sup>+</sup> and a marker of glycolytic capacity [34]. Additionally, it has been confirmed that high glucose levels induce in-creases in lactate and 6PG production in vitro and ensure a longer supply of energy sources, preventing the loss of GSH [35].

The blood lactate that was progressively elevated with exercise intensity further reduces local blood pH and thus enhances the Bohr effect to attenuate O2 affinity and facilitate O2 release [17]. In contrast, pulmonary O2 uptake is enhanced, but muscle unloading is hindered with high-affinity hemoglobin [36]. The capillary transit times were very limited; thus, the exchange speed is critical for evaluating the physiological fitness of erythrocytes [37]. Therefore, we developed a novel method for quantifying gas exchange in a constant number of erythrocytes and used it to assess the quality and quantity of O2 releasing capacity.

To clarify the oxygen release efficacy of erythrocytes, we measured the PO2, oxygen release/absorption velocity and acceleration under 0, 1, and 4 mM lactic acid concentrations to mimic resting and near AT conditions, respectively. The PO2 increased (oxygen release) when lactic acid was added. Even with a smaller amount of O2 being supplied to the tissue, the efficacy was enhanced with faster acceleration and velocity, which indicates better efficiency for release. The improved release of oxygen efficacy at 4 mM [lac] might be associated with improved cycling performance before reaching the anaerobic threshold. In addition, a diminished magnitude and velocity of O2 absorption after the acute GXT were noticed. We first speculated that this was a consequence of sufficiently oxygenated Hb after high oxygen demand activity or that this impaired quantity and quality may also be related to increases in oxidative and met-Hb levels after exhaustive exercise [38]. We further demonstrated an enhanced, strong correlation between lactate concentration and oxygen release magnitude and velocity under hypoxic conditions. The O2 affinity of athletes is lower than that of untrained subjects, which is consistent with our results [39]. Slow VO2 kinetics incur a high O2 deficit, usually resulting in poor exercise tolerance [40].

Lactic acid plays a vital indirect role in tissue O2 delivery apart from the direct allosteric interaction of lactate ions with Hb [41]. Lactic acid increases the Bohr shift via acidification as well as via liberation of CO2 [42]. Therefore, the lower affinity of hemoglobin for the O2 of erythrocytes in athletes at rest is maintained by the factor(s) dominating pH and lactate-driven regulation. Under heavy exercise (above the lactic acidosis threshold), acidification of muscle capillary blood by lactic acid accounts for virtually all of the oxygen unloaded from Hb [43].

Erythrocytes must be considered a potential storage site of lactate, storage of which leads to a greater gradient from the interstitial fluid to plasma. This mechanism improves the rate of release from muscle and ameliorates exercise performance [44]. However, a previous study demonstrated that the lactate distribution in erythrocytes and plasma after high-intensity running was not different between trained and untrained subjects. Hence, lactate uptake by erythrocytes cannot or can only in part be seen as a contributor to aerobic athletic performance [45]. Traditionally, a higher Bohr effect is supposed to be related to a higher 2,3-BPG in erythrocytes [46]. However, the generation of 2,3-BPG results in the overall production of ATP per mole of glucose is decreased to zero. Therefore, accumulation of 2,3-BPG leads to decreased production of 2,3-BPG by competitive feedback inhibition of di-phosphoglycerate mutase [47]. The relative ratio of 2,3-BPG synthase to 2,3-BPG phosphatase decreased dramatically with decreasing pH value [48]. The lactate effect even increased after 2,3-BPG depletion [49]. In this study, the presence of large lactate concentrations leading to lower pH values may effectively limit the production of 2,3- BPG [50]. When the downstream enzymes of 2,3-BPG, such as pyruvate kinase and lactate dehydrogenase, maintained higher activities, the enzyme activities of other pathways were significantly repressed [48]. Therefore, this reversed flux of the 2,3-BPG shunt is crucial in maintaining the activities of the latter part of glycolysis and the production of ATP in the latter half of the storage period.

The results in this study clearly presented that, although both ECT and CCT ameliorate the erythrocyte antioxidant and oxygen releasing capacity, thus further delays the anaerobic threshold, yet only ECT has significantly less cardiopulmonary stress without undesirable fatigue or pain impact during the whole training period. Therefore, we suggest that ECT is preferred to those who have exercise intolerance or low physical activity, whereas CCT may be more feasible for those who have general physical activity to increase the ability to cope with the physical demands of daily activity. These findings provide a new suggestion on why ECT is worthy to further developed as a suitable training strategy in cardiopulmonary rehabilitation or the elderly.

A small sample size (*n* = 12 in each group) is a major limitation of this study. However, the results for aerobic capacity and the novel interpretation of the O2 release and antioxidative mechanisms in the metabolic pathways obtained from this investigation have high statistical power (0.862 to 1.000). We speculate that metabolic alteration of erythrocytes generates higher ATP concentrations followed by lactate production. We did not directly detect the ATP concentration because of the fast rate of ATP hydrolysis. Therefore, in this study, we indirectly inferred ATP demand by the lactate/pyruvate ratio. We did not see potential model alterations in Hb affinity as blood traverses the exercising muscles in accordance with local changes in temperature, pHi, or CO2. There are certainly differences between and challenges with in vivo vs. in vitro measurements of O2 dissociation curve dynamics in both the lungs and muscles in response to variables such as temperature, pHi, and 2,3-BPG. A few studies have shown that the additive effects of temperature and pH are responsible for shifting the O2 dissociation curve affinity, especially with prolonged exercise [51]. Additionally, the subjects tended to be young and healthy; thus, further clinical evidence is still required to extrapolate the present results to patients with hemorheological or hemodynamic disorders.

Although the glutathione system is a principal nonenzymic antioxidant system in erythrocytes yet, GSSG is rapidly formed, but it quickly disappears once the oxidative stimulus is interrupted; conversely, S-glutathionylated proteins (PSSGs) may be produced more slowly but are more durable [52]. Therefore, the PSSGs is a worthy parameter for further investigation [53]. In addition, although previously study suggested that exerciseinduced changes in the nonenzymatic glutathione system seem to be more effective in erythrocytes [54]. Nevertheless, many studies have indicated the activity of glutathione peroxidase (GPx) plays a key component in the antioxidant experiment [55], and regular cardiovascular training increased GPx activity in skeletal muscle [56]. Taking together, both PSSGs and the immunoblotting for GPx are very used to supply important information on

the state of this antioxidant network in the future. To assess the reliabilities of biomarkers, metabolites and oxygen releasing capacity to exercise, the subjects (*n* = 5) in a prior study were tested twice at two-day intervals. Results of responses to exercise were highly reproducible from day to day. The intraclass correlation coefficients were from 0.811 to 0.954. Additionally, it requires separate analytical measurements for GSH and GSSG for accurate analysis and specific methodological procedures needed to detect samples [57]. Although the use of classical and well-validated in previous studies [26,58], techniques to perform our measurements, requiring immediate and complex processing of blood samples [59]. This limits the possibility of receiving samples from different centers to be analyzed. In the present study, we tested our participants at the same time of the day and asked them to record their nutritional intake and to maintain the same diet (data not shown). Thus, we assume that our results well represent the physical adaptations after exercise training. Importantly, other more adequate and precise methodologies should be considered in future studies [53].

#### **5. Conclusions**

This study presented evidence that both ECT and CCT simultaneously promote flux into the pentose phosphate pathway and anaerobic glycolysis pathway in response to overcoming accumulated oxidative stress and regulating internal O2 dissociation, respectively. The adaptations of the metabolite process not only increased the synthesis of GSH but also enhanced the production of lactate in glucose metabolism in trained erythrocytes. The lower intracellular pH value related to lactate, instead of 2,3-BPG, ameliorated the O2 release efficacy of erythrocytes under different O2 gradients. In addition, the reduced amount of met-Hb also contributed to the O2 release. The above experimental findings reflect many positive effects of both interventions and provide a novel interpretation of delayed anaerobic threshold by ameliorated O2 release and antioxidative mechanism in the metabolic pathway (Figure 7). Therefore, ECT is a feasible and promising exercise regimen that promotes a less cardiovascular stress way to exercise without undesirable fatigue impact and provides important implications for those who have exercise intolerance.

**Figure 7.** Possible mechanisms of anaerobic glycolytic pathways producing both oxygen releasing and antioxidative capacity caused by eccentric (ECT) and concentric cycling training (CCT). Both CCT and ECT at a given workload enhance an anaerobic glycolysis flux to ameliorate antioxidative capacity in erythrocytes, as well as significantly facilitate oxygen release efficacy. The reduced oxygen affinity is due to greater lactate synthesis and lower intracellular pH, instead of the production of 2,3-BPG. Solid line: positive regulation; dotted line: negative regulation.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2076-392 1/10/2/285/s1, Figure S1: the scheme of testing oxygen release capacity in erythrocytes by using high-resolution respirometry.

**Author Contributions:** Conceptualization, J.-S.W. and Y.-C.H.; methodology, J.-S.W., Y.-C.H., H.-Y.T. and M.-L.C.; formal analysis, J.-S.W. and Y.-C.H.; investigation, Y.-C.H. and H.-Y.T.; writing—original draft preparation, Y.-C.H.; writing—review and editing, J.-S.W., Y.-C.H., H.-Y.T., M.-L.C., C.-Y.H. and K.-M.C.; supervision, J.-S.W.; funding acquisition, J.-S.W. and Y.-C.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Science Council of Taiwan, grant number NSC 108-2314-B-182 -039 -MY3 and 109-2314-B-468 -008 -MY2, and by the Chang Gung Medical Research Program, grant number CMRPD1J0222 and CMRPG2F0193.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of Chang Gung Memorial Hospital in Taiwan (protocol code 201900415A3 and 2019/5/17 approval).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** All data is contained within the article.

**Acknowledgments:** The authors would like to thank the volunteers for their enthusiastic participation and assistance from medical writers, proof-readers and editors.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
