Hydration of CO2 yields HCO3- via the reaction: CO2 + H2O = H2CO3 = HCO3- + H+ = CO32- + 2H+. (1) Acid-base physiologists traditionally simplify the reaction by omitting the H2CO3 term and lumping all ionic CO2 species into the HCO3- term. The simplified reaction forms the basis for the familiar Henderson-Hasselbalch equation of the CO2-HCO3- buffer system: pH = pKa + log([HCO3-]/(alpha)CO2PCO2), (2) where (alpha)CO2 is the solubility coefficient relating [CO2] and PCO2 (Henry's Law). The apparent pK (pKa) in this equation lacks a rigorous thermodynamic definition. Instead, it is an empirical factor relating pH, the product of (alpha)CO2 and PCO2, and the apparent [HCO3-] (i.e. the sum of all ionic CO2 species). (alpha)CO2 and pKa are sensitive to the temperature, pH and/or the ionic strength of the reaction medium. (alpha)CO2 and pKa of normal mammalian blood plasma have been well defined over a range of temperatures and pH values (e.g. Severinghaus, 1965; Siggaard-Andersen, 1974; Reeves, 1976). These mammalian values are commonly used in analyses of the acid-base status of non- mammalian species, despite evidence that such practices can produce misleading results (Nicol et al. 1983). As an alternative, Heisler (1984; erratum in Heisler, 1986) developed complex equations for (alpha)CO2 (mmol l-1 mmHg-1) (1 mmHg=133.22 Pa) and pKa that are purported to be generally applicable to aqueous solutions (including body fluids) between 0 and 40 °C and incorporate the molarity of dissolved species (Md), solution pH, temperature (T, °C), sodium concentration ([Na+], mol l-1), ionic strength of nonprotein ions (I, mol l-1) and protein concentration ([Pr], g l-1): (alpha)CO2 = 0.1008 - 2.980 × 10–2Md + (1.218 × 10-3Md - 3.639 × 10-3)T - (1.957 × 10-5Md - 6.959 × 10-5)T2 + (7.171 × 10-8Md - 5.596 × 10-7)T3. (3) pKa = 6.583 - 1.341 × 10-2T + 2.282 × 10-4T2 - 1.516 × 10-6T3 - 0.341I0.323 - log{1 + 3.9 × 10-4[Pr] + 10A(1 + 10B)}, (4) where A = pH - 10.64 + 0.011T + 0.737I0.323 and B = 1.92 - 0.01T - 0.737I0.323 + log[Na+] + (0.651 - 0.494I)(1 + 0.0065[Pr]). Experimental validation of these equations has not appeared in the literature to date. We determined the (alpha)CO2 and pKa of blood plasma from Kemp's ridley sea turtles (Lepidochelys kempi Garman) and compared the values with those predicted from Heisler's equations. Blood samples were collected into heparinized syringes from the dorsal cervical sinus of 1- to 2-year- old animals at the National Marine Fisheries Service, Galveston Laboratory, Texas. Separated plasma was obtained by centrifugation of the whole blood samples. (alpha)CO2 was determined gasometrically by equilibrating 2 ml samples of acidified plasma (titrated to pH 2.5 with 1 mol l-1 HCl) in a tonometer with 99.9 % CO2 at 20, 25, 30 or 35 °C. Fresh samples of plasma were used at each temperature. The total CO2 content (CCO2) of plasma was measured in duplicate after 15 min of equilibration, using the methods described by Cameron (1971). The CO2 electrode (Radiometer, type E5036) was calibrated at each temperature using known [HCO3-]. Plasma PCO2 was calculated from the known fractional CO2 content of the equilibration gas, corrected for temperature, barometric pressure and water vapor pressure. Plasma water content was measured by weighing samples of plasma before and after they had been dried at 60 °C to constant weight. (alpha)CO2 was calculated as The quotient of CCO2 and PCO2, taking into account the plasma water content (mean +/− s.e.= 96+/−0.03 %). pKa was determined gasometrically by equilibrating 2 ml samples of plasma in a tonometer with 4.78 or 10.2 % CO2 (balance N2) at 20 or 30 °C. Fresh samples of plasma were used at each temperature and gas concentration. Plasma CCO2 and pH were measured in duplicate. The pH electrode (Radiometer, type G297/G2) was calibrated at each temperature using precision Radiometer pH buffers (S1500 and S1510). Plasma PCO2 was determined as above. pKa was calculated from a rearrangement of the Henderson-Hasselbalch equation (equation 2), assuming CCO2 to be the sum of [HCO3-] and [CO2] (i.e. (alpha)CO2PCO2). Heisler's equations were adapted for use with L. kempi plasma using measured values of the molarity of dissolved species (Md), [Na+] and protein concentration ([Pr]). These parameters were quantified as follows: Md with a vapor pressure osmometer (Precision Systems, model 5004), [Na+] by flame photometry (Jenway, model PFP7) and [Pr] by a standard spectrophotometric method (Sigma kit 541). The average values were Md=0.304+/−0.003 mol l-1, [Na+]=0.141+/−0.004 mol l-1 and [Pr]=28+/−3 g l- 1. The ionic strength of nonprotein ions (I) was assigned a value of 0.150 mol l-1. Computed (alpha)CO2 and pKa values were generated for a wider range of temperature and pH conditions than were used experimentally in order to emphasize the pattern and range of effects of temperature and/or pH.
THE IMMUNOCHEMISTRY OF TOXINS AND TOXOIDS