Investigation of tubular handing of bicarbonate in man. A new approach utilizing stable carbon isotope fractionation

Two alternative mechanisms have been proposed for tubular reabsorption of bicarbonate: (a) H⁺ secretion and CO₂ reabsorption and (b) direct reabsorption of HCO₃^-. In an attempt to differentiate between the two mechanisms, the present study utilized the natural abundance of stable carbon isotopes (¹³C, ¹²C) in the urinary total CO₂. This novel methodology used mass spectrometric analysis of ¹³C/¹²C ratios in urinary total CO₂ under normal conditions and during acetazolamide treatment. Blood and respiratory CO₂ were analyzed to yield reference values. The results demonstrate that alkaline urine is preferentially enriched with ¹³C relative to the blood. It is suggested that this fractionation results from reaction out of isotopic equilibrium in which HCO₃^- converts to CO₂ during the reabsorption process in the distal nephron. The presence of carbonic anhydrase in the proximal nephron results in rapid isotopic exchange between CO₂ and HCO₃^- and keeps them in isotopic equilibrium. The ratio of urinary ¹³C/¹²C increases strikingly after acetazolamide administration and consequent inhibition of carbonic anhydrase in the proximal tubule. Although it is possible that in the latter case high HCO₃^- generates the CO₂ (ampholyte effect), the isotope fractionation indicates that CO₂ rather than HCO₃^- is reabsorbed. In contrast, at low urinary pH and total CO₂ values, the carbon isotope composition approaches that of blood CO₂. This indicates rapid CO₂ exchange between urine and blood, through luminal membrane highly permeable to CO₂. These results could be anticipated by a mathematical model constructed to plot ¹³C concentration of urinary total CO₂. It is concluded that the mechanism of HCO₃^- reclamation in man (and, by inference, in other mammals as well) works by conversion of HCO₃^- to CO₂ and reabsorption of CO₂.