Investigation of tubular handling 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 HCO-3. 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 HCO-3 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 HCO-3 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 HCO-3 generates the CO2 (ampholyte effect), the isotope fractionation indicates that CO2 rather than HCO-3 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 HCO-3 reclamation in man (and, by inference, in other mammals as well) works by conversion of HCO-3 to CO2 and reabsorption of CO2.

Determination of carbonate alkalinity and apparent dissociation constants in a multiprotolytic system.

A method for analysing the carbonate system in a multicomponent solultion is presented, which does not need knowledge of the total composition of the system. It is based on two titrations with acid, starting at the same pH, one of the original solution and the other after removal of carbonate species as carbon dioxide and restoration of the pH to the value for the original solution by addition of carbonate-free base. The differential titration curve, obtained by subtracting one titration curve from the other, is associated with the carbonate system. A procedure is proposed for calculating from the differential titration curve the apparent first and second dissociation constants of carbonic acid, total CO(2) and the carbonate alkalinity at the original pH of the solution.

Determination of 18O/16O ratio in inorganic phosphate as an oncogenic marker and indicator for disturbances in phosphate metabolism.

Natural stable isotope fractionation is a potential tool in investigation of metabolic process, since rigid mass balance considerations rule the changes in the isotopic ratio. As the natural changes in 13C/12C ratio of total CO2 between blood and urine served for studying renal bicarbonate reabsorption, studying changes in 18O/16O ratio of phosphate are suggested to investigate deranged phosphate metabolism. The 18O/16O ratio in serum phosphate is constant, determined by the ratio in the environmental drinking water. Therefore, measurements of this ratio in normal individuals, after modifications in phosphate metabolism and in diseases with high alkaline phosphatase activity are proposed. The main purpose of the proposed study is to assess whether measurements of 18O/16O ratio can detect malignant metastases in bones due to deranged phosphate metabolism. An assumption that these determinations might precede other tests for detecting bone metastases and can serve as an oncogenic marker is made.

Delta alkalinity: a simple method to measure cellular net acid-base fluxes.

Acid-base transport across cell plasma membranes is important for cell homeostasis and growth. Current techniques for quantitatively measuring net acid-base fluxes are generally limited by either assumptions concerning properties of intracellular compartments or use of poorly buffered, nonphysiological solutions. We adapted an approach from marine chemistry to quantitate net acid-base changes in standard physiological media that obviates these problems. This method is based on conservation of charge and involves a simple acid titration of the extracellular medium to an end point, the equivalence point, pHe. For standard physiological solutions containing buffers such as bicarbonate and phosphate, pHe exists in the range of pH 4.0-4.7 and is identified as the pH where dpH/dH+ is maximal in an HCl titration. By determining the quantity of H+ required to reach pHe, one can determine precisely total quantity of proton acceptors (alkalinity) present in physiological pH range. Alkalinity (in meq) is a relative measure of the charge capable of interacting with protons. We show that, unlike pH, changes in alkalinity (delta alkalinity) result only from net acid-base changes in medium. Therefore, by monitoring extracellular delta alkalinity associated with cell function, it is possible to quantitate precisely net acid-base fluxes. Moreover, through a second titration procedure, delta alkalinity can be divided into bicarbonate and nonbicarbonate fractions. As an example, we performed the first direct measurement of net Cl(-)-HCO3- exchange in intact human erythrocytes and observed a Cl(-)-HCO3- exchange ratio of 1.01 +/- 0.03. Overall, delta alkalinity measurements are applicable to numerous cell systems, can be performed with solutions containing a mixture of buffers at normal physiological concentrations (e.g., 25 mM HCO3- and 2 mM HPO4(-2), do not require corrections for CO2 diffusion or loss of CO2 from the solution, and avoid assumptions about intracellular or extracellular buffer properties.