Hydration of CO2 yields HCO3via the reaction:
formula
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:
formula
where αCO2 is the solubility coefficient relating [CO2] and (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 αCO2 and and the apparent [HCO3] (i.e. the sum of all ionic CO2 species).
Hydration of CO2 yields HCO3via the reaction:
formula
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:
formula
where αCO2 is the solubility coefficient relating [CO2] and (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 αCO2 and and the apparent [HCO3] (i.e. the sum of all ionic CO2 species).
αCO2 and pKa are sensitive to the temperature, pH and/or the ionic strength of the reaction medium. αCO2 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 αCO2 (mmol l−1 mmHg−1) (1mmHg=133.22Pa) 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], gl−1):
formula
formula
where
formula
and
formula
Experimental validation of these equations has not appeared in the literature to date.

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 (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±0.003mol 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:

Fig. 1.

Experimentally determined values of aCO2 (A) and pKa (B) for the plasma of Lepidochelys kempi. Data points are means ± standard deviation (N=3–9). The absence of error bars indicates that the standard deviation was smaller than the data symbol. Dashed lines give the predicted values of αCO2 and pKa computed from equations 5 and 6 (see text).

Fig. 1.

Experimentally determined values of aCO2 (A) and pKa (B) for the plasma of Lepidochelys kempi. Data points are means ± standard deviation (N=3–9). The absence of error bars indicates that the standard deviation was smaller than the data symbol. Dashed lines give the predicted values of αCO2 and pKa computed from equations 5 and 6 (see text).

formula
The measured values varied inversely with temperature, from 0.0474 mmol l−1 mmHg−1 at 20°C to 0.0363mmol l−1 mmHg−1 at 35°C. The predicted relationship between αCO2 and temperature (equation 5) provided an excellent description of the experimentally determined data (Student’s t-test, P<0.01). αCO2 data for L. kempi differed only slightly (⩽0.0015mmol l−1 mmHg−1 from published mammalian values (Severinghaus, 1965; Siggaard-Andersen, 1974; Reeves, 1976). Such differences introduce relatively minor errors in calculations of [CO2] (e.g. less than 0.06mmol l−1 at a of 40mmHg).
Fig. 1B gives the measured values of pKa together with the predicted values computed from the following adaptation of Heisler’s generalized equation:
formula
The measured pKa valsues decreased as either temperature or pH increased. The appropriate adaptation of Heisler’s equation (equation 6) provided a good description of the measured data (Student’s t-test, P<0.05). The pKa values for L. kempi differed markedly from published mammalian values, not only in absolute value but also in sensitivity to temperature and pH (Table 1). The differences in pKa are sufficiently large to confound analyses of the effects of temperature or pH on turtle plasma [HCO3]. Consequently, the use of mammalian-derived αCO2 and pKa values cannot be recommended for analyses of the acid–base status of sea turtles.
Table 1.

Comparison of pKa values for Lepidochelys kempi (see text) with the mammalian-derived values of Severinghaus (1965), Siggaard-Andersen (1974) and Reeves (1976) 

Comparison of pKa values for Lepidochelys kempi (see text) with the mammalian-derived values of Severinghaus (1965), Siggaard-Andersen (1974) and Reeves (1976)
Comparison of pKa values for Lepidochelys kempi (see text) with the mammalian-derived values of Severinghaus (1965), Siggaard-Andersen (1974) and Reeves (1976)

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.

Cameron
,
J. N.
(
1971
).
Rapid method for determination of total carbon dioxide in small blood samples
.
J. appl. Physiol.
31
,
632
634
.
Heisler
,
N.
(
1984
).
Acid–base regulation in fishes
. In
Fish Physiology
, vol.
X
(ed.
W. S.
Hoar
and
D. J.
Randall
), pp.
315
401
.
New York
:
Academic Press
.
Heisler
,
N.
(
1986
).
Buffering and transmembrane ion transfer processes
. In
Acid–Base Regulation in Animals
(ed.
N.
Heisler
), pp.
3
47
. Amsterdam: Elsevier Science Publishers.
Nicol
,
S. C.
,
Glass
,
M. L.
and
Heisler
,
N.
(
1983
).
Comparison of directly determined and calculated plasma bicarbonate concentration in the turtle Chrysemys picta belli at different temperatures
.
J. exp. Biol.
107
,
521
525
.
Reeves
,
R. B.
(
1976
).
Temperature-induced changes in blood acid–base: pH and in a binary buffer
.
J. appl. Physiol.
40
,
752
761
.
Severinghaus
,
J. W.
(
1965
).
Blood gas concentrations
. In
Handbook of Physiology, Respiration
(ed.
W. O.
Fenn
and
H.
Rahn
), pp.
1475
1487
.
Washington, DC
:
American Physiological Society
.
Siggaard-Andersen
,
O.
(
1974
).
The Acid–Base Status of the Blood.
Baltimore
:
William & Wilkins
.