Traditional Henderson–Hasselbalch approach
The analysis of the parameters of the traditional Henderson–Hasselbalch approach showed that exercise significantly decreased pH, reflecting a rapid and progressive acidification of the plasma with increasing exercise intensity. One might assume that during exercise, and even earlier during hypoxic exercise, acidosis was primarily caused by Lac
−. The Lac
− anion builds-up mainly in actively exercising muscle and is transported out of the cell by monocarboxylate transporters (Gladden
2004; Cairns and Lindinger
2008). A fast transport of Lac
− across the plasma membrane is important for muscle function and for maintaining muscle pH despite the high H
+ and Lac
− load that develops during intense exercise (Kowalchuk and Scheuermann
1995; Cairns and Lindinger
2008). The present study shows that at peak exercise intensity the plasma concentration of Lac
− did not differ between normoxia and hypoxia. This finding is in accordance with Kato et al. who found that peak values of plasma Lac
− did not differ between normoxia and hypoxia (Kato et al.
2004). In hypoxia, the peak power output was reduced by ~26%, what is in line with previous studies (Fulco et al.
1998; Kato et al.
2004). This finding suggests that rather than the inspired oxygen fraction (FiO
2) the intracellular Lac
− levels and transmembrane ion fluxes determined intracellular [H
+] and led to voluntary fatigue and termination of exercise. However, which of these factors contribute the most to muscle fatigue still remains controversial (Lindinger et al.
1995; Gladden
2004; Cairns and Lindinger
2008; Morales-Alamo et al.
2015).
According to the traditional approach, BE represents a non-respiratory (metabolic) component of acid–base status. The increase in plasma Lac
− during exercise we observed showed a close inverse correlation with BE and HCO
3
− (not shown), indicating that Lac
− was an important contributor to exercise-induced metabolic acidosis. However, it has previously been suggested that the exercise-induced increase in Lac
− is not the only contributor to exercise-induced acidosis (Sejersted et al.
1982; Lindinger et al.
1992; Lindinger and Heigenhauser
2008).
Despite the higher plasma concentrations of Lac
− at T100 and T200, pH values remained significantly higher in hypoxia than in normoxia. This finding indicates that in hypoxia compensating mechanisms averted a stronger plasma acidification. According to the traditional approach, the lower
PCO
2 values caused by hypoxia-induced hyperventilation counteracted the severity of exercise-induced metabolic acidosis. In hypoxia and at high altitude, respiratory alkalosis might be favourable (West
2000; Samaja et al.
2003; Winslow
2007; Mollard et al.
2008). However, at an exercise intensity of 200 W, pH values were about the same in normoxia and hypoxia, with
PCO
2 values being lower and Lac
− values being higher in hypoxia. These findings reflect the rapid and intense respiratory response that occurs during intense exercise (Davies et al.
1986; Stringer et al.
1992; Wasserman et al.
2014). Kato et al. showed similar changes for pH and
PCO
2 as in the present study (Kato et al.
2004). The authors suggested a pH-dependency of muscle Lac
−-release which has been described for both, respiratory alkalosis and HCO
3
−-induced metabolic alkalosis (Davies et al.
1986; McLellan et al.
1988; LeBlanc et al.
2002). Thus, alkalinizing plasma by hyperventilation could have modified the Lac
−-shift from exercising muscle into circulating blood (Davies et al.
1986; Lindinger et al.
1992; Kato et al.
2004).
However, while the traditional approach sufficiently describes the changes of some variables many other aspects of acid–base homeostasis that are considered in the modified physicochemical approach are not taken into account.
Modified physicochemical approach
According to the Stewart approach, acid–base status is determined by three independent variables, i.e. PCO2, SID and A
tot
−. Only if at least one of these variables changes, the dependent variables, i.e. pH, HCO3
− and CO3
2−, may be altered.
In the present study, SIDapp decreased during both normoxic and hypoxic exercise, reflecting metabolic acidosis, of which Lac− seemed the main contributor. Simultaneously, plasma concentrations of inorganic strong ions increased, ultimately leading to an increase in SIDinorganic, thus alleviating the acidifying effect of Lac−. However, quantitatively, the increase in SIDinorganic could not fully compensate for the increase in Lac− as reflected by the decrease in pH. Notably, while at the level of peak exercise intensity, Lac− values were about the same in normoxia and hypoxia, SIDinorganic and SIDapp were significantly higher in normoxia. This indicates that during normoxia a more pronounced alkalinizing process had occurred, ultimately contributing to higher pH values. In combination with the higher PCO2 values that were observed in normoxia, the results indicate that in situations of insufficient respiratory compensation, non-respiratory mechanisms, e.g. ion-shifts, significantly affect acid–base regulation.
Another independent variable that affects pH as well as other dependent variables of the Stewart approach is
A
tot
−. Main contributors to
A
tot
− are albumin, globulins and [P
i]. Several different formulas have been applied to calculate
A
tot
− in a clinically feasible way (Figge et al.
1991; Constable
2001; Staempfli and Constable
2003; Lloyd
2004). In the present study,
A
tot
− was calculated according to the equation from Lloyd (
2004). The results show that both an increase in albumin and [P
i] caused a significant increase in
A
tot
−. Although albumin concentrations were not different between normoxia and hypoxia, the contribution of albumin to
A
tot
− was ~20% higher in hypoxia. This might be explained by the almost linear relationship between the ionic charge of albumin and plasma pH (Figge et al.
1991,
1992; Fogh-Andersen et al.
1993; Figge
2009). Because pH values were higher in hypoxia, the negative ionic charge of albumin had increased and in turn elevated the albumin fraction of
A
tot
−.
The contribution of [Pi] to A
tot
− was ~30% greater in normoxia than in hypoxia. The phosphoric ionic system is not as pH dependent as albumin. Because of their trivalent structure, the phosphorous ions have different dissociation equilibria, whose titration curves follow a triphasic course. Therefore, exercise-induced changes in the concentration of [Pi] rather than pH changes determined the contribution of [Pi] to A
tot
− in the present study.
Evidence suggests that beside albumin, globulines and [P
i], also other weak and strong ions alter acid–base homeostasis during exercise (Forni et al.
2006; McKinnon et al.
2008). Particularly, amino acids, intermediates of the Krebs cycle, tricarboxylic acids and ammonia are released into the blood and may affect acid–base balance (Sewell et al.
1994; Wagenmakers
1998; Casas et al.
2001; Kato et al.
2004). As most of these substances are organic acids, it is plausible that their anions also contributed to both the observed increase in
A
tot
− and the decrease in SID, thus generating an additional acidifying load. It is generally accepted that determination of
A
tot
− is sufficiently precise for clinical purposes, when it is calculated from the net charge of albumin, globulines and [P
i]. However, by applying this mathematical “shortcut”, the otherwise unmeasured anions could be missed and their contribution to acid–base behaviour remain uncertain. Based on the work of Stewart, Figge, Fencl and Mydosh (Figge et al.
1991,
1992), Kellum proposed a method to quantify unmeasured ions in the context of a modified physicochemical approach (Kellum et al.
1995), which is now referred to as the SIG. By calculating SID
app and SID
eff, the remainder represents unmeasured ions that contribute to acidosis if SIG is >0, or to alkalosis if SIG results in negative values. In the present study, SIG increased significantly during exercise in normoxia and hypoxia. However, at the level of peak exercise intensity, SIG was significantly higher in normoxia, indicating a higher plasma concentration of unmeasured anions. These findings could be explained by an alkalosis-related and altered release of ammonia and organic acids into the plasma during hypoxia (Casas et al.
2001; Kato et al.
2004; McKinnon et al.
2008).
In the present study, plasma Lac
− increased faster in hypoxia than in normoxia. This faster release of Lac
− probably blunted intracellular acidosis and thus the release of organic acids and ammonia. Kato et al. reported lower plasma ammonia levels during exercise in hypoxia (FiO
2 = 0.12) when compared to normoxia (Kato et al.
2004), while other authors showed increased ammonia levels after exercise in normoxia (Sewell et al.
1994; Casas et al.
2001). Alkalinisation of blood by hypoxia and exercise-induced respiratory alkalosis might have caused a change in transmembrane transport of ammonia and organic acids. The impact of this organic compound on acid–base homeostasis is not yet fully clarified. Several clinical and experimental studies suggest these organic compounds to be organic acids as well as ketone bodies and metabolic intermediates of the intracellular cycles of glucose and fatty acid metabolism (Forni et al.
2006; Moviat et al.
2008). McKinnon et al. investigated this compound using liquid chromatography and enzyme assays (Forni et al.
2006; McKinnon et al.
2008). They found that beside the well-known exercise-induced lactic acidosis, increased plasma concentrations of α-ketoglutarate, citrate, isocitrate and malate contributed to the acidic load (McKinnon et al.
2008). Although the SIG does not determine the origin of all contributing anions, they may be quantified and thus allow a more precise description of the acid–base changes. However, the SIG has its limitations. SIG itself represents a sum of competing acidifying or alkalinizing ions, which possibly could extinguish each other’s impact on acid–base changes. Thus, SIG allows the calculation of the net effect of unmeasured ions without describing their specific nature. Another weakness of SIG is that its calculation requires many different variables, whose errors in measurements can magnify and falsify the validity of SIG. Nevertheless, calculating SIG is more accurate than calculating the traditional anion gap (Kellum et al.
1995; Forni et al.
2006).
In the present study, exercise significantly increased plasma albumin, what is in line with previous studies that attributed this finding to an exercise-induced reduction in plasma volume (Novosadová
1977; Iwato et al.
1993; Haskell et al.
1997; Kargotich et al.
1998; Alis et al.
2015). At the level of peak exercise intensity, there was no significant difference in the albumin concentration between normoxia and hypoxia. Likewise, the reduction in plasma volume did not differ at peak exercise intensity but was significantly higher in hypoxia at T100W and T200W. These findings were paralleled by changes in haematocrit, indicating that plasma volume contraction occurred earlier during exercise in hypoxia than in normoxia, respectively.
The observed increase in albumin concentration was higher than what has previously been attributed to exercise-induced plasma volume contraction or exercise-induced albumin losses (Hansen et al.
1994; Haskell et al.
1997). However, albumin was not the only contributor to the increase in
A
tot
−, as confirmed by the independent increase in SIG. In fact, plasma volume contraction could have contributed to the increase in inorganic ions (Table
2), which in turn resulted in an increase in SID
inorganic. However, during exercise, K
+ and Ca
2+ are added to plasma which also additionally contributes to an increase in SID
app and SID
inorganic. In fact, the degree of plasma volume contraction and the increase in SID
inorganic correlated well, suggesting that the exercise-induced decrease in plasma volume contributed significantly to the increase in SID
inorganic. During exercise a complex shift of ions, water, and CO
2 takes place between different compartments (i.e. intracellular space, interstitial space, red blood cells and plasma) which is determined by intracellular hydrolysis of phosphocreatine, glycolysis, CO
2 production, intracellular Lac
− and H
+ accumulation and release of these products and K
+ into extracellular fluids, where RBC plays a crucial role in handling and distributing these products (Sejersted et al.
1982; Medbø and Sejersted
1985; Lindinger et al.
1992; Gladden
2004; Cairns and Lindinger
2008). Thus, these processes result from the efforts of the cell to satisfy energy demands and prevent cellular damage. With respect to the complexity of these processes and the number of physiologically active compartments, changes in plasma acid–base status are net effects and difficult to interpret in terms of cause and origin.
Regarding the very complex mechanisms of acid–base changes during exercise and hypoxia, in the present study, the modified physicochemical approach offered a more detailed and precise view on the different variables of acid–base control as did the traditional Henderson–Hasselbalch approach.