Carbon dioxide and bicarbonate accumulation in caiman erythrocytes during diving

Crocodilians are semiaquatic reptiles that dive to avoid predators or kill prey by drowning them (e.g. Campbell et al., 2010). The durations of voluntary dives have only been reported in a few species of crocodilians, but appear to be relatively short (10–15 min) compared with their impressive capacity to remain submerged for up to 2 h in laboratory settings (Andersen, 1961; Wright, 1987; Campbell et al., 2010; Rodgers and Franklin, 2017). Thus, voluntary dives are predominately aerobic, with negligible lactate accumulation, although it is likely that underwater foraging or strenuous activities involve substantial anaerobic metabolism (Andersen, 1961; Seymour et al., 1985; Rodgers et al., 2015). Crocodilians exhibit the typical vertebrate ‘dive response’ with a bradycardia, peripheral vasoconstriction and redistribution of blood flows, as well as breath-holding. This is obviously associated with depletion of oxygen stores in lungs and blood, while CO₂ accumulates in tissues and blood. In crocodiles, diving is also associated with a right-to-left shunt, where oxygen-poor blood can bypass the lungs by perfusion of the left aortic arch that emerges from the right ventricle in all crocodilians (White, 1956, 1969; Grigg and Johansen, 1987; Hicks and White, 1992).

As a unique feature amongst vertebrates, the crocodilian haemoglobin allosterically binds HCO₃^–, in addition to CO₂ and an H⁺ upon deoxygenation (Bauer and Jelkmann, 1977; Bauer et al., 1981; Jensen et al., 1998; Berenbrink et al., 2005; Fago et al., 2020; Bautista et al., 2021). Studies have suggested that this unique ability relates to either breath-hold diving or the alkaline tide during digestion (Weber and White, 1986; Weber et al., 2013; Storz, 2019), enhancing CO₂ binding during blood oxygen depletion. However, there are no in vivo data on the partitioning of CO₂ distribution in plasma, red blood cells and haemoglobin of crocodiles. Therefore, the goal of the present study was to address the hypothesis that CO₂ primarily binds to haemoglobin during diving, rather than being accumulated in plasma, as in other vertebrates.

Three spectacled caimans (Caiman crocodilus Linneaus 1758) (1.10–1.75 kg) and five broad-snouted caimans (Caiman latirostris Daudin 1801) (2.07–2.35 kg), of undetermined sex, were donated from Krokodille Zoo (Eskilstrup, Denmark) and transported to Aarhus University a year before experimentation. The animals were held in large aquaria with water at 28°C and a 12 h:12 h day:night cycle with artificial light, and had access to a dry basking platform and a heating lamp for behavioural thermoregulation. They were fed rodents and fish once or twice a week and gained mass in captivity. All animals were habituated to the diving protocol by experiencing submergence in the same container of the experimental procedure five to six times prior to cannulation. The experiments were approved by the Danish Animal Experiments Inspectorate and performed in accordance with the Danish Law for Animal Experimentation.

Animals were individually netted and moved to a surgical table, where the head was covered by a plastic bag containing 2 ml isoflurane. The animal became unresponsive soon after the first inhalation and was placed on a thermal pad to maintain a body temperature of 28±0.5°C, and intubated with an uncuffed 3.0 mm endotracheal tube for artificial ventilation with 1.5–2% isoflurane in air at 1–2 breaths min^–1 and a tidal volume of 30–50 ml kg^–1 (Model SAV04 ventilator, Vetronics, Devon, UK). The skin on the hind leg was cleaned, iodine (Jodopax vet, Pharmaxim, Helsingborg, Denmark) was added, and 2 mg lidocaine (Mylan^®) in saline was injected subcutaneously to induce local analgesia. The femoral artery was exposed through a 3–5 cm incision and cannulated occlusively with polyethylene tubing (PE50: inner diameter 0.58 mm, outer diameter 0.96 mm; Smiths Medical™ Portex™) containing heparinized saline (50 i.u. ml⁻¹; LEO Pharma A/S). The incision was closed with monofilament nylon sutures, and the catheter was secured to the leg using silk sutures. The animal was allowed to regain consciousness during ventilation with air, and then placed in a plastic container (40×40×70 cm, height×width×length) inside a temperature-controlled room at 28°C for recovery.

On the day after surgery, the animal was placed into a custom-build sealed chamber (22×22×112 cm, height×width×length), and the catheter was extended through a hole in the top of the chamber to enable blood sampling from undisturbed animals. The container was half-filled with water (27±0.5°C), allowing spontaneous ventilation, and the animal was left undisturbed for an hour. A 1.5–2.0 ml blood sample was then drawn anaerobically into a heparinized syringe (control condition), after which the animal was submerged by filling the chamber with water (27±0.5°C) to simulate diving. Blood samples were drawn at 18 and 32 min after submergence, and the animal was then given access to air by reducing the water volume in the chamber. At the completion of the study, all animals were euthanized by injecting 400 mg kg⁻¹ pentobarbital (Exagon^® vet 427931) through the catheter.

Immediately after blood sampling, haematological parameters and blood gases were measured in the following order. The partial pressure of oxygen in the arterial blood (Pa_O2) was measured using a P_O2 electrode (Radiometer, Copenhagen, Denmark) thermostatted to 27°C. The electrode was flushed with N₂ before the injection of blood and was calibrated using N₂ and humidified air before each measurement. The concentration of oxygen in arterial blood ([O₂]_b) was measured in duplicate as described by Tucker (1967). Arterial pH (pH_a) was measured using a micro pH electrode (Mettler Toledo, Columbus, OH, USA) with the blood sample in a heating block set at 28°C. Haematocrit was measured in duplicate as the fraction of packed erythrocytes after centrifugation (15,322 g, 3 min). The concentration of carbon dioxide in plasma [CO₂]_p was measured using the Cameron method (Cameron, 1971) using a CO₂ electrode (Analytical Sensors and Instruments, Sugar Land, TX, USA) and 20 mmol l⁻¹ NaHCO₃ standards. The remaining blood was centrifuged (2000 g, 3 min) to separate erythrocytes and plasma and stored at −80°C until further analysis.

Erythrocyte intracellular pH (pH_i) was measured by thawing the erythrocytes on ice and placing a pH electrode in the haemolysate using the same setup as for the pH_a measurements (Zeidler and Kim, 1977). Similarly, plasma osmolality, lactate concentration ([lactate]_p) and chloride concentrations were measured in plasma and haemolysates thawed on ice. Osmolality was measured using an osmometer (Model 3320, Advanced Instruments, Inc., Norwood, MA, USA), and chloride concentrations in erythrocytes ([Cl^–]_i) and plasma ([Cl^–]_p) were determined using an MK II Chloride Analyzer 926S (Sherwood Scientific Ltd, Cambridge, UK). Finally, [lactate]_p was measured by colourimetry with the abcam^® L-Lactate Assay kit (ab65331) following the manufacturer's instructions.

The concentration of monomeric haemoglobin in blood ([Hb]) was calculated from the fractional haematocrit using a 25 mmol l⁻¹ intraerythrocytic monomeric haemoglobin concentration typical for vertebrate erythrocytes.

All measured parameters were statistically compared among pre-dive (control), 18 min dive and 32 min dive samples with a mixed-model ANOVA considering individual animals as random effect. Pairwise differences were assessed with a Tukey's honest significant difference test with a Holm correction. The number of replicates decreased with time as a few animals tore out their catheters during diving. The statistical significance level was set at α=0.05, and values are reported as means±1 s.e.m. unless stated otherwise. Data analysis was performed in RStudio v. 1.1.456, and the raw data and R script were deposited in a Github repository (https://github.com/christiandamsgaard/caiman_CO2).

Despite in vitro evidence that crocodilian haemoglobins bind HCO₃^– and CO₂ (Bauer et al., 1981; Bauer and Jelkmann, 1977; Fago et al., 2020; Bautista et al., 2021), the functional implications of this exceptional mechanism for CO₂ transport have not yet been assessed in vivo. Here, we demonstrate that CO₂ primarily accumulates within the erythrocyte during diving and that most of the accumulated CO₂ is bound to haemoglobin. Furthermore, we show that CO₂/HCO₃^– binding is tightly associated with the progressive blood deoxygenation during diving. These findings document a relevance of the deoxygenation-linked CO₂/HCO₃^– binding to haemoglobin during diving in vivo.

As expected, haemoglobin O₂ saturation and Pa_O2 decreased from pre-diving control values, while the animal was at rest and had access to air, to 32 min of submergence (P<0.0001; Fig. 1A,B). In contrast, Pa_CO2 increased over time (P=0.0045; Fig. 1F). These results are in accordance with Harald Andersen's pioneering study on alligators diving for 2 h, where Pa_O2 decreased sharply during the first 10–15 min, followed by a slower rate of decline (Andersen, 1961). Andersen (1961) also showed that Pa_CO2 rose steeply during the first 20–25 min of diving, followed by a subtle increase during the rest of the dive; we report similar changes in the present study. Haematocrit and osmolality did not vary with dive duration (Fig. 1C,L), and fell within the range previously reported for other crocodilian species (Carmena-Suero et al., 1979; Grigg and Cairncross, 1980; Seymour et al., 1985; Pough, 1979).

Arterial and intraerythrocytic pH values decreased as the dive progressed (P=0.00012 and P<0.001, respectively; Fig. 1D,E). In contrast, [HCO₃^–]_i and [Cl^–]_i increased (P=0.0014 and P=0.0011, respectively) from pre-diving control values to 32 min into diving (Fig. 1H,K). Additionally, [HCO₃^–]_p and [Cl^–]_p did not differ during experimentation (P=0.11 and P=0.52, respectively; Fig. 1G,J).

The acid–base balance of arterial blood and erythrocytes is depicted in Fig. 2 as a Davenport diagram showing the relationship between [HCO₃⁻] and pH at calculated isopleths for P_CO2. This depiction shows that diving induced an arterial respiratory acidosis, where pH decreased as Pa_CO2 increased following the non-bicarbonate buffer line (shaded areas; Fig. 2). Like arterial blood, the intra-erythrocytic milieu exhibited a respiratory acidosis, but the acidosis was less pronounced – likely owing to the higher intracellular non-bicarbonate buffering capacity of the intracellular environment (Fig. 2) (Jensen et al., 1998).

There was no rise in plasma lactate concentration during diving, as reported previously in the American alligator (Andersen, 1961) and the sea snakes Acalyptophis peronii and Lapemis hardwickii (Seymour and Webster, 1975). In both of these groups, [lactate]_p remains low during the dive but rises abruptly within the first 5–10 min after resuming ventilation and when perfusion of the muscles restored. Notably, pre-diving [lactate]_p in our study were somewhat higher than previously reported for the salt-water crocodile, Crocodylus porosus (Seymour et al., 1985), which likely reflects the manual translocation of the caimans from their resting containers to the diving chamber.

As expected during the simulated dive, the production of CO₂ led to a rise [HCO₃^–] in the blood during diving. In line with our prediction, [HCO₃^–]_p remained stable, while the additional HCO₃^– accumulated within the erythrocyte. Here, the intra-erythrocytic HCO₃^– accumulation is distributed between free HCO₃^– within the cytosol (P=0.0014; Fig. 1D) and HCO₃^– bound to haemoglobin (P=0.002; Fig. 3A). The rise in [Hb–HCO₃^–] during diving appears to be driven by the progressive deoxygenation of the blood (P<0.001; Fig. 3B), which increases the Hb–HCO₃^– affinity. This data provides the first in vivo documentation of deoxygenation-linked HCO₃^– binding in crocodiles. This combined binding of HCO₃^– and H⁺ during deoxygenation enables the simultaneous removal both of H⁺ and free HCO₃^–, and thus increases CO₂ hydration and carrying capacity of crocodilian blood (Jensen et al., 1998).

In addition, physiological responses of voluntarily and forced diving differ, and thus the responses obtained during forced diving could be in part caused, and potentially confounded, by autonomic stress responses (Gaunt and Gans, 1969). Therefore, it is possible that under forced diving, the left-to-right shunt is larger in comparison to a voluntary dive, and as a consequence, the Pa_O2 and Pa_CO2 values in the samples would exhibit lower and higher values, respectively. Additionally, during diving (closed system), shunting would allow poorly oxygenated blood from the right ventricle and ‘well’-oxygenated blood from the left ventricle to mix in the system and reach equilibrium.

Our in vivo data show that the metabolically produced CO₂ is largely bound as HCO₃^– to haemoglobin, as Hb–O₂ saturation fell during the dive; therefore, crocodilians differ from the classic vertebrate pattern, where HCO₃^– accumulates in the plasma upon excretion from the erythrocytes Cl^–/HCO₃^– exchanger (Brauner et al., 2019). The unique crocodilian strategy is not due to impaired activity of the Cl^–/HCO₃^– exchanger, which in fact resembles that of other vertebrates (Jensen et al., 1998; Jensen, 2004). The adaptive value of HCO₃^– binding to haemoglobin versus classic plasma carriage is not entirely clear, but illustrates that either chemical binding of HCO₃^– to the haemoglobin or extrusion to the plasma upon hydration by carbonic anhydrase greatly alleviates the rise in P_CO2. Although the accumulated HCO₃^– is likely to exert allosteric regulation of blood–oxygen binding and thus facilitating tissue oxygen delivery during breath-holding, it is not clear that a similar effect could be achieved through the classic influence of CO₂ and protons. Future studies could address the partitioning of blood CO₂ transport during digestion, where both P_CO2 and blood CO₂ concentration increases during the alkaline tide, while arterial oxygen levels remain high, in contrast to diving where, blood oxygen is depleted.

The authors thank Mr Rene Hedegaard and the staff from the Krokodille Zoo (Eskilstrup, Denmark) for continued assistance in our crocodile studies, and we appreciate the excellent animal care and husbandry by Heidi Meldgaard and Claus Wandborg.