Reply to Boedtkjer and Aalkjaer

reply: We thank Drs. Ebbe Boedtkjer and Christian Aalkjaer (1) for their interest in our paper, for taking the time to express their concerns, and their insightful comments regarding our recently published guidelines for the measurement of vascular function and structure in isolated arteries and veins, published in the American Journal of Physiology-Heart and Circulatory Physiology (2). Here, we will clarify aspects of our methodology regarding the points raised.

In their Letter to the Editor, entitled “The solution to bicarbonate,” Boedtkjer and Aalkjaer expressed potential concerns with the Krebs buffer composition, and specifically the bicarbonate concentration suggested for isometric force studies [see Table 1 in Wenceslau et al. (2)]. Accordingly, the authors pointed out that the use of 14.9 or 19 mM ${\text{HCO}}_3^{-}$ is well below typical plasma concentrations in humans and rodents. Therefore, when these (lower) concentrations of ${\text{HCO}}_3^{-}$ are aerated with a gas mixture containing 5% CO₂, according to the Henderson–Hasselbalch equation, 14.9 mM ${\text{HCO}}_3^{-}$ produces a pH of 7.24 (1). We agree with Boedtkjer and Aalkjaer concerns since this level of acidity will affect tone in many vascular beds (1). However, as we described in the guidelines (2), the ideal buffer for all vascular function is one that limits pH changes to acceptable levels; therefore, it is expected that all buffers, regardless of which buffer recipe is used, whether it be one described in the guidelines or another one of the many adaptations found in the literature, have the pH adjusted when necessary. For instance, the bicarbonate solution at 5% CO₂ should be adjusted with NaOH to pH 7.40. In this scenario, the concentration of ${\text{HCO}}_3^{-}$ will increase with NaOH, as will pH (to 7.40). Therefore, the solution will contain 22 mM ${\text{HCO}}_3^{-}$ and not a final low concentration of ${\text{HCO}}_3^{-}$ (1). Our previous guidelines should have specified this important point, instead of expecting that the reader would add NaOH to the solution. Furthermore, we agree with Drs. Boedtkjer and Aalkjaer that initially adding 22 mM ${\text{HCO}}_3^{-}$ is clearer than adding NaOH to adjust the pH since [${\text{HCO}}_3^{-}$] also has a direct role in regulating vascular tone (3).

Overall, we acknowledge the importance of standardizing buffers and solutions, and the first set of guidelines provides a fundamental starting ground to support the enhancement of rigor and reproducibility (2). We agree with the concerns raised by Boedtkjer and Aalkjaer that recommend the use of a physiological [${\text{HCO}}_3^{-}$] of 22–24 mM with a gas mixture containing 5% or 40 mmHg CO₂ for ex vivo vascular studies, based on the reasons described above and in their letter (1). Any future guidelines will need to be updated to reflect this critical point.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

C.F.W. drafted manuscript; C.F.W. and C.G.M. edited and revised manuscript; C.F.W., C.G.M., S.E., S.K.E., J.A.F., S.G., D.D.G., B.E.I., N.L.K., L.A.M.-L., S.K.S., P.T., A.J.T., S.W.W., and R.C.W. approved final version of manuscript.