Determination of the Constants of the Henderson–Hasselbalch Equation, αCO₂ and pKa, in Sea Turtle Plasma

We determined the αCO₂ 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. αCO₂ was determined gasometrically by equilibrating 2ml samples of acidified plasma (titrated to pH2.5 with 1mol 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 of plasma was measured in duplicate after 15min of equilibration, using the methods described by Cameron (1971). The CO₂ electrode (Radiometer, type E5036) was calibrated at each temperature using known [HCO₃⁻]. Plasma 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. αCO₂ was calculated as the quotient of and taking into account the plasma water content (mean ± S.E.= 96±0.03%). pKa was determined gasometrically by equilibrating 2ml 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 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 was determined as above. pKa was calculated from a rearrangement of the Henderson–Hasselbalch equation (equation 2), assuming to be the sum of [HCO₃⁻] and [CO₂] (i.e. ⁠).

Heisler’s equations were adapted for use with L. kempi plasma using measured values of the molarity of dissolved species (M_d), [Na⁺] and protein concentration ([Pr]). These parameters were quantified as follows: M_d 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 M_d=0.304±0.003mol l⁻¹, [Na⁺]=0.141±0.004mol l⁻¹ and [Pr]=28±3 gl ⁻¹. The ionic strength of nonprotein ions (I) was assigned a value of 0.150mol l⁻¹. Computed αCO₂ 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.

Fig. 1A gives the measured values of αCO₂ (mmol l⁻¹ mmHg⁻¹) together with the predicted values computed from the following adaptation of Heisler’s generalized equation:

The present study demonstrates that Heisler’s generalized equations for αCO₂ and pKa (equations 3 and 4) are applicable for use with reptile blood. The appropriate adaptations of Heisler’s equations (equations 5 and 6) provided good descriptions of the experimentally determined data for L. kempi. Moreover, equation 6 also provided a good description of the pKa values measured by Nicol et al. (1983) for the freshwater turtle Chrysemys picta at 10 and 20°C, although not at 30°C. We conclude that Heisler’s (1984) generalized equations provide better estimates of the αCO₂ and pKa of reptile blood than do classical mammalian-derived values.

This study was conducted under US Fish and Wildlife Service Endangered and Threatened Species Permit no. PRT-676379. This work was partially supported by Grant no. NA89AA-D-SG139 from the National Marine Fisheries Service of the National Oceanic and Atmospheric Administration (NOAA) to the Texas Sea Grant College Program. The views expressed are those of the authors and do not necessarily reflect the views of NOAA or any of its sub-agencies.