Non-carbonic buffer power of whole blood is increased in experimental metabolic acidosis: An in-vitro study

Non-carbonic buffer power (βNC) of blood is a pivotal concept in acid-base physiology as it is employed in several acid-base evaluation techniques, including the Davenport nomogram and the Van Slyke equation used for Base excess estimation in blood. So far, βNC has been assumed to be independent of metabolic acid-base status of blood, despite theoretical rationale for the contrary. In the current study, we used CO2 tonometry to assess βNC in blood samples from 10 healthy volunteers, simultaneously analyzing the electrolyte shifts across the red blood cell membrane as these shifts translate the action of intracellular non-carbonic buffers to plasma. The βNC of the blood was re-evaluated after experimental induction of metabolic acidosis obtained by adding a moderate or high amount of either hydrochloric or lactic acid to the samples. Moreover, the impact of βNC and pCO2 on the Base excess of blood was examined. In the control samples, βNC was 28.0 ± 2.5 mmol/L. In contrast to the traditional assumptions, our data showed that βNC rose by 0.36 mmol/L for each 1 mEq/l reduction in plasma strong ion difference (p < 0.0001) and was independent of the acid used. This could serve as a protective mechanism that increases the resilience of blood to the combination of metabolic and respiratory acidosis. Sodium and chloride were the only electrolytes whose plasma concentration changed relevantly during CO2 titration. Although no significant difference was found between the electrolyte shifts in the two types of acidosis, we observed a slightly higher rate of chloride change in hyperchloremic acidosis, while the variation of sodium was more pronounced in lactic acidosis. Lastly, we found that the rise of βNC in metabolic acidosis did not induce a clinically relevant bias in the calculation of Base excess of blood and confirmed that the Base excess of blood was little affected by a wide range of pCO2.

The primary role of transmembrane electrolytes shifts as the means by which intracellular buffers act upon plasma can be easily demonstrated using Stewart's physicochemical approach (Stewart, 1981) to compare CO2 titration of isolated plasma and the plasma phase of whole blood. Note that both fluids in this thought experiment consist only of a single compartment, which is necessary for applying Stewart's theory. According to this theory, pH of two fluids can only differ if at least one of the three independent variables (pCO2, strong ion difference, or ATOT) differs. Provided that the two studied fluids originate from the same donor and that plasma weak non-volatile acids (albumin, phosphates, and organic acids) do not cross the RBC membrane, their ATOT cannot differ. Neither pCO2 can differ as it is the variable we manipulate. Therefore, the higher resilience of blood plasma to pH fluctuations, attributable to the presence of intracellular buffers, can only be mediated by alterations of its SID through transmembrane shifts of electrolytes.
Correspondingly, it has been shown in-silico that suppression of the chloride shift markedly decreases βNC of blood. At the same time, the usual difference between pH/[HCO3 -] curves of oxygenated and deoxygenated blood, a footprint of the Haldane effect, disappears (O'Neill and Robbins, 2017). This implies that were it not for this electrolyte shift, the action of intracellular buffers would not be detectable in plasma.

Part II: Hemolysis quantification
In preliminary experiments, hemolysis was quantified in 11 aliquots that underwent dilution and tonometry as described in Methods. The variables we analyzed were free plasma hemoglobin and [K + ] rise compared to fresh blood. The percentage of hemolyzed RBC was calculated assuming zero Supplementary Material 2 free plasma hemoglobin in fresh blood and intracellular [K + ] of 90 mmol/L (Beilin et al., 1966). It should be noted that K + is released from RBCs even without cell lysis and, therefore, the estimation of hemolysis by [K + ] rise represents the upper limit of the hemolysis that could have occurred.

Part III: Accuracy analysis of the collected data
For a given pH value, the ratio between the associated and dissociated form of plasma weak acids and, therefore, the amount of charge they expose is constant, determined by the difference between We defined [HCO3 -]7.2 in the same manner as SID7.2 (see Methods) and explored the relationship between these two variables using linear regression with a shared slope and individual intercept for each volunteer.
Good agreement between SID7.2 and [HCO3 -]7.2 was confirmed ( Figure S4). The slope of the regression line shared between volunteers was 1.008 (95%CI: 0.974 to 1.043), i.e., not different from one (p=0.64). The mean difference between the two variables was -15.9±1.5 mEq/L, representing the net charge carried by plasma weak acids (albumin, phosphate, and organic acids) at pH=7.2 together with Mg 2+ , which was not incorporated in our SID calculation. Thus, [HCO3 -]7.2 could have been used as the quantitative measure of the degree of metabolic acidosis in our study if we had chosen to use a bicarbonate-centered measure instead of SID7.2.