Carbon dioxide and bicarbonate accumulation in caiman erythrocytes during diving
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© 2021. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2021) 224, jeb242435. doi:10.1242/jeb.242435
SHORT COMMUNICATION
Carbon dioxide and bicarbonate accumulation in caiman
erythrocytes during diving
Naim M. Bautista1, *, Christian Damsgaard1,2,*,‡, Angela Fago1 and Tobias Wang1,2
ABSTRACT an H+ upon deoxygenation (Bauer and Jelkmann, 1977; Bauer et al.,
The ability of crocodilian haemoglobins to bind HCO3–
has been 1981; Jensen et al., 1998; Berenbrink et al., 2005; Fago et al., 2020;
appreciated for more than half a century, but the functional implication Bautista et al., 2021). Studies have suggested that this unique ability
of this exceptional mechanism has not previously been assessed relates to either breath-hold diving or the alkaline tide during
in vivo. Therefore, the goal of the present study was to address the digestion (Weber and White, 1986; Weber et al., 2013; Storz, 2019),
hypothesis that CO2 primarily binds to haemoglobin, rather than being enhancing CO2 binding during blood oxygen depletion. However,
accumulated in plasma as in other vertebrates, during diving in there are no in vivo data on the partitioning of CO2 distribution in
caimans. Here, we demonstrate that CO2 primarily accumulates plasma, red blood cells and haemoglobin of crocodiles. Therefore,
within the erythrocyte during diving and that most of the accumulated the goal of the present study was to address the hypothesis that CO2
CO2 is bound to haemoglobin. Furthermore, we show that this primarily binds to haemoglobin during diving, rather than being
HCO3– binding is tightly associated with the progressive blood accumulated in plasma, as in other vertebrates.
deoxygenation during diving; therefore, crocodilians differ from the
classic vertebrate pattern, where HCO3– accumulates in the plasma MATERIALS AND METHODS
upon excretion from the erythrocytes by the Cl–/HCO3– exchanger. Experimental animals
Three spectacled caimans (Caiman crocodilus Linneaus 1758)
KEY WORDS: Blood gases, pH, Blood–oxygen affinity, (1.10–1.75 kg) and five broad-snouted caimans (Caiman latirostris
Haemoglobin–bicarbonate binding, Reptile Daudin 1801) (2.07–2.35 kg), of undetermined sex, were donated
from Krokodille Zoo (Eskilstrup, Denmark) and transported to
INTRODUCTION Aarhus University a year before experimentation. The animals were
Crocodilians are semiaquatic reptiles that dive to avoid predators or held in large aquaria with water at 28°C and a 12 h:12 h day:night
kill prey by drowning them (e.g. Campbell et al., 2010). The cycle with artificial light, and had access to a dry basking platform
durations of voluntary dives have only been reported in a few and a heating lamp for behavioural thermoregulation. They were fed
species of crocodilians, but appear to be relatively short rodents and fish once or twice a week and gained mass in captivity.
(10–15 min) compared with their impressive capacity to remain All animals were habituated to the diving protocol by experiencing
submerged for up to 2 h in laboratory settings (Andersen, 1961; submergence in the same container of the experimental procedure
Wright, 1987; Campbell et al., 2010; Rodgers and Franklin, 2017). five to six times prior to cannulation. The experiments were
Thus, voluntary dives are predominately aerobic, with negligible approved by the Danish Animal Experiments Inspectorate and
lactate accumulation, although it is likely that underwater foraging performed in accordance with the Danish Law for Animal
or strenuous activities involve substantial anaerobic metabolism Experimentation.
(Andersen, 1961; Seymour et al., 1985; Rodgers et al., 2015).
Crocodilians exhibit the typical vertebrate ‘dive response’ with a Surgical procedures
bradycardia, peripheral vasoconstriction and redistribution of blood Animals were individually netted and moved to a surgical table,
flows, as well as breath-holding. This is obviously associated with where the head was covered by a plastic bag containing 2 ml
depletion of oxygen stores in lungs and blood, while CO2 isoflurane. The animal became unresponsive soon after the first
accumulates in tissues and blood. In crocodiles, diving is also inhalation and was placed on a thermal pad to maintain a body
Journal of Experimental Biology
associated with a right-to-left shunt, where oxygen-poor blood can temperature of 28±0.5°C, and intubated with an uncuffed 3.0 mm
bypass the lungs by perfusion of the left aortic arch that emerges endotracheal tube for artificial ventilation with 1.5–2% isoflurane in
from the right ventricle in all crocodilians (White, 1956, 1969; air at 1–2 breaths min–1 and a tidal volume of 30–50 ml kg–1 (Model
Grigg and Johansen, 1987; Hicks and White, 1992). SAV04 ventilator, Vetronics, Devon, UK). The skin on the hind
As a unique feature amongst vertebrates, the crocodilian leg was cleaned, iodine (Jodopax vet, Pharmaxim, Helsingborg,
haemoglobin allosterically binds HCO3–, in addition to CO2 and Denmark) was added, and 2 mg lidocaine (Mylan®) in saline was
injected subcutaneously to induce local analgesia. The femoral artery
1
Zoophysiology, Department of Biology, Aarhus University, Aarhus C, Denmark.
was exposed through a 3–5 cm incision and cannulated occlusively
2
Aarhus Institute of Advanced Studies, Aarhus University, 8000 Aarhus C, with polyethylene tubing (PE50: inner diameter 0.58 mm, outer
Denmark. diameter 0.96 mm; Smiths Medical™ Portex™) containing
*Shared first authorship
heparinized saline (50 i.u. ml−1; LEO Pharma A/S). The incision
‡
Author for correspondence (christian.damsgaard@bios.au.dk) was closed with monofilament nylon sutures, and the catheter was
secured to the leg using silk sutures. The animal was allowed to
N.M.B., 0000-0003-0634-0842; C.D., 0000-0002-5722-4246; A.F., 0000-0001-
7315-2628; T.W., 0000-0002-4350-3682 regain consciousness during ventilation with air, and then placed in
a plastic container (40×40×70 cm, height×width×length) inside a
Received 12 February 2021; Accepted 22 March 2021 temperature-controlled room at 28°C for recovery.
1SHORT COMMUNICATION Journal of Experimental Biology (2021) 224, jeb242435. doi:10.1242/jeb.242435
([lactate]p) and chloride concentrations were measured in plasma
List of symbols and abbreviations and haemolysates thawed on ice. Osmolality was measured using an
osmometer (Model 3320, Advanced Instruments, Inc., Norwood,
[Cl–]I intraerythrocytic chloride concentration MA, USA), and chloride concentrations in erythrocytes ([Cl–]i) and
[Cl–]p concentration of chloride in plasma plasma ([Cl–]p) were determined using an MK II Chloride Analyzer
[CO2]b concentration of carbon dioxide in whole blood
[CO2]p concentration of carbon dioxide in plasma
926S (Sherwood Scientific Ltd, Cambridge, UK). Finally, [lactate]p
Hb haemoglobin was measured by colourimetry with the abcam® L-Lactate Assay kit
[Hb] concentration of monomeric haemoglobin in blood (ab65331) following the manufacturer’s instructions.
[Hb–HCO3−] concentration of HCO3− bound to haemoglobin
[Hb–O2] concentration of oxygen bound to haemoglobin Calculations and statistical analysis
[HCO3−]i,app apparent intraerythrocytic bicarbonate concentration The concentration of oxygen bound to haemoglobin ([Hb–O2]) was
[HCO3−]i,free concentration of free intraerythrocytic bicarbonate
calculated by subtracting physically dissolved O2 from [O2]b:
[HCO3−]p plasma bicarbonate concentration
[lactate]p concentration of lactate in plasma
[O2]b concentration of oxygen in arterial blood
½HbO2 ¼ ½O2 b aO2 PaO2 ; ð1Þ
PaCO2 partial pressure of carbon dioxide in the arterial blood
where αO2 is the plasma O2 solubility at 28°C
PaO2 partial pressure of oxygen in the arterial blood
pHa pH of the arterial blood (1.59 µmol l−1 mmHg−1) (Boutilier et al., 1984).
pHi intracellular pH The concentration of monomeric haemoglobin in blood ([Hb])
SHb–O2 fractional haemoglobin oxygen saturation was calculated from the fractional haematocrit using a 25 mmol l−1
αCO2 plasma carbon dioxide solubility intraerythrocytic monomeric haemoglobin concentration typical for
αO2 plasma oxygen solubility vertebrate erythrocytes.
The fractional haemoglobin O2 saturation, SHb–O2, was found as
[Hb–O2] relative to [Hb]:
Experimental procedure ½HbO2
On the day after surgery, the animal was placed into a custom-build SHbO2 ¼ : ð2Þ
½Hb
sealed chamber (22×22×112 cm, height×width×length), and the
catheter was extended through a hole in the top of the chamber to The partial pressure of CO2 in the arterial blood, PaCO2, was
enable blood sampling from undisturbed animals. The container calculated from [CO2]p, the plasma CO2 solubility
was half-filled with water (27±0.5°C), allowing spontaneous (37.6 µmol l−1 mmHg−1; Boutilier et al., 1984), pHa and the CO2
ventilation, and the animal was left undisturbed for an hour. A dissociation constant ( pK′=6.78−0.0817×pHa) for alligator plasma
1.5–2.0 ml blood sample was then drawn anaerobically into a (Jensen et al., 1998) by rearranging the Henderson–Hasselbalch
heparinized syringe (control condition), after which the animal was equation:
submerged by filling the chamber with water (27±0.5°C) to
simulate diving. Blood samples were drawn at 18 and 32 min ½CO2 p
after submergence, and the animal was then given access to air by PaCO2 ¼ 0 : ð3Þ
aCO2 ð1 þ 10pHa pK Þ
reducing the water volume in the chamber. At the completion of the
study, all animals were euthanized by injecting 400 mg kg−1 The plasma bicarbonate concentration, [HCO3−]p, was calculated
pentobarbital (Exagon® vet 427931) through the catheter. by subtracting physically dissolved CO2 from [CO2]p:
Blood analysis ½HCO3 p ¼ ½CO2 p aCO2 PaCO2 : ð4Þ
Immediately after blood sampling, haematological parameters and
blood gases were measured in the following order. The partial The apparent erythrocytic bicarbonate concentration
pressure of oxygen in the arterial blood (PaO2) was measured using a ([HCO 3−]i,app) was calculated from [HCO3−]p using previously
PO2 electrode (Radiometer, Copenhagen, Denmark) thermostatted determined HCO3− Donnan distribution ratios, r, across the
to 27°C. The electrode was flushed with N2 before the injection of erythrocyte membrane that were corrected for pHa and SHb–O2
blood and was calibrated using N2 and humidified air before each (Jensen, 2004):
Journal of Experimental Biology
measurement. The concentration of oxygen in arterial blood ([O2]b) ½HCO3 i;app ¼ r ½HCO3 p ; ð5Þ
was measured in duplicate as described by Tucker (1967). Arterial
pH ( pHa) was measured using a micro pH electrode (Mettler where r=13.9−1.68×pHa and 5.60−0.507×pHa for fully oxygenated
Toledo, Columbus, OH, USA) with the blood sample in a heating and fully deoxygenated blood, respectively, and we weighted the
block set at 28°C. Haematocrit was measured in duplicate as the slopes and intercepts based on SHb–O2. We also calculated [HCO3−]i,app
fraction of packed erythrocytes after centrifugation (15,322 g, based on whole-blood [CO2] measurements, but because the low
3 min). The concentration of carbon dioxide in plasma [CO2]p was sensitivity of present-day CO2 electrodes reduces the signal-to-noise
measured using the Cameron method (Cameron, 1971) using a CO2 ratio of directly determined HCO3− Donnan distribution ratios, we
electrode (Analytical Sensors and Instruments, Sugar Land, TX, adopted to this derived approach to obtain [HCO3−]i,app.
USA) and 20 mmol l−1 NaHCO3 standards. The remaining blood The concentration of free erythrocytic bicarbonate ([HCO3−]i,free)
was centrifuged (2000 g, 3 min) to separate erythrocytes and plasma was calculated from the measured Donnan distribution ratio of [Cl–]
and stored at −80°C until further analysis. across the erythrocyte membrane:
Erythrocyte intracellular pH ( pHi) was measured by thawing the
erythrocytes on ice and placing a pH electrode in the haemolysate ½Cl i
½HCO3 i;free ¼ ½HCO3 p : ð6Þ
using the same setup as for the pHa measurements (Zeidler and ½Cl p
Kim, 1977). Similarly, plasma osmolality, lactate concentration
2SHORT COMMUNICATION Journal of Experimental Biology (2021) 224, jeb242435. doi:10.1242/jeb.242435
The concentration of Hb-bound HCO3− ([Hb–HCO3−]) was mixed-model ANOVA considering individual animals as random
determined by subtracting [HCO3−]i,app and [HCO3−]i,free: effect. Pairwise differences were assessed with a Tukey’s honest
significant difference test with a Holm correction. The number of
½HbHCO3 ¼ ½HCO3 i;app ½HCO3 i;free : ð7Þ
replicates decreased with time as a few animals tore out their
All measured parameters were statistically compared among catheters during diving. The statistical significance level was set at
pre-dive (control), 18 min dive and 32 min dive samples with a α=0.05, and values are reported as means±1 s.e.m. unless stated
A 125 B 100 C 30
100
75 a 25
a
PaO2 (mmHg)
SHb–O2 (%)
75
[Hct] (%)
a a
50 20 a
50 b
b
b
c 25 15
25
0 0 10
D 7.7 E 7.4 F 40
a
7.6
PaCO2 (mmHg)
a 7.3 a 30 b
ab
pHa
pHi
7.5 b
b
7.2 20 a
7.4 c
7.3 7.1 10
G 30 H 25 I 15
[HCO3−]i,free (mmol l−1)
[HCO3−]p (mmol l−1)
[Lactate] (mmol l−1)
25 20
b b
10
a a
a
20 15 a a
a a
5
15 10
10 5 0
J 120 K 120 L 350
Journal of Experimental Biology
Osmolality (mOsm kg−1)
a
a a 325
[Cl−]p (mmol l−1)
[Cl−]i (mmol l−1)
b
110 100 b
a a
300 a
a
100 80
275
90 60 250
Pre-dive 18 min 32 min Pre-dive 18 min 32 min Pre-dive 18 min 32 min
Fig. 1. The effect of diving time on blood acid-base status in Caiman sp. Data were collected from blood samples at pre-diving state, and at 18 and 32 min
diving. (A) Arterial haemoglobin oxygen saturation (SHb–O2); (B) arterial PO2 (PaO2; mmHg); (C) haematocrit %; (D) arterial blood pH; (E) intraerythrocytic pH;
(F) arterial PCO2 (mmHg); (G) plasma bicarbonate concentration [(HCO3–)]p (mmol l–1); (H) intraerythrocytic bicarbonate concentration [HCO3–]i (mmol l–1);
(I) lactate concentration (mmol l l–1); (J) plasma chloride concentration (mmol l l–1); (K) intraerythrocytic chloride concentration (mmol l l–1); and (L) osmolality
(mOsm kg–1). Coloured points and lines represent individual animals, and black points and error bars represent means±1 s.e.m. Different letters indicate
statistically significant pairwise differences between time points as tested by a mixed-model ANOVA.
3SHORT COMMUNICATION Journal of Experimental Biology (2021) 224, jeb242435. doi:10.1242/jeb.242435
otherwise. Data analysis was performed in RStudio v. 1.1.456, and 22.5 Arterial
30 20
the raw data and R script were deposited in a Github repository
(https://github.com/christiandamsgaard/caiman_CO2). Erythrocyte
RESULTS AND DISCUSSION 20.0
32 min dive
Despite in vitro evidence that crocodilian haemoglobins bind HCO3– and 18 min dive
CO2 (Bauer et al., 1981; Bauer and Jelkmann, 1977; Fago et al., 2020;
Bautista et al., 2021), the functional implications of this exceptional
[HCO3−] (mmol l−1)
mechanism for CO2 transport have not yet been assessed in vivo. Here, 17.5
32 min dive
we demonstrate that CO2 primarily accumulates within the erythrocyte Pre-dive
during diving and that most of the accumulated CO2 is bound to 18 min dive
haemoglobin. Furthermore, we show that CO2/HCO3– binding is
tightly associated with the progressive blood deoxygenation during 15.0
diving. These findings document a relevance of the deoxygenation-
linked CO2/HCO3– binding to haemoglobin during diving in vivo. 10
Oxygen and acid/base status during diving 12.5
As expected, haemoglobin O2 saturation and PaO2 decreased from Pre-dive
pre-diving control values, while the animal was at rest and had
access to air, to 32 min of submergence (PSHORT COMMUNICATION Journal of Experimental Biology (2021) 224, jeb242435. doi:10.1242/jeb.242435
A 30
Author contributions
Conceptualization: N.M.B., C.D., A.F., T.W.; Methodology: N.M.B., C.D., T.W.;
Formal analysis: N.M.B., C.D.; Investigation: N.M.B., C.D.; Resources: T.W.; Data
curation: C.D.; Writing - original draft: N.M.B., C.D.; Writing - review & editing:
[Hb−HCO3−] (mmol l−1)
N.M.B., C.D., A.F., T.W.; Visualization: N.M.B., C.D.; Supervision: A.F., T.W.; Project
b administration: A.F.; Funding acquisition: A.F., T.W.
20
a,b
Funding
a This work was funded by the Danish Council for Independent Research (Det Frie
Forskningsråd | Natur og Univers), the Carlsberg Foundation (CF18-0658), the
10 European Union’s Horizon 2020 research and innovation program under the Marie
Skłodowska-Curie grant agreement (no. 754513), and The Aarhus University
Research Foundation.
Data availability
0 All raw data and computer code are available from GitHub at https://github.com/
Pre-dive 18 min 32 min christiandamsgaard/caiman_CO2
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