Increased blood oxygen affinity during digestion in the snake Python molurus
Department of Zoophysiology, Aarhus University, Building 131, 8000 Aarhus C, Denmark
* Author for correspondence (e-mail: tobias.wang{at}biology.au.dk)
Accepted 5 August 2002
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Summary |
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Key words: blood oxygen binding, oxygen transport, arterial blood gases, acidbase balance, feeding, reptile, snake, Python molurus
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Introduction |
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During exercise, reptiles often experience a metabolic acidosis resulting
from lactic acid production that reduces blood oxygen affinity through the
Bohr effect. The changes in acidbase status are opposite during
digestion, where an alkalinisation of the blood occurs after net secretion of
acid to the stomach lumen (the `alkaline tide'). Nevertheless, all species
studied reduce the pH changes by increasing the partial pressure of
CO2 (PCO2) through a relative
hypoventilation (see Wang et al.,
2001a). Among reptiles, the possible changes of blood oxygen
affinity during digestion have only been studied in Alligator
mississippiensis. In this species, HCO3- binds
directly to the haemoglobin molecule and acts to maintain a virtually constant
blood oxygen affinity during digestion
(Bauer et al., 1981
;
Weber and White, 1986
;
Busk et al., 2000a
).
Pythons exhibit much larger metabolic increments during digestion than
crocodilians, but a similar mechanism is not involved because oxygen binding
of Python haemoglobin is insensitive to HCO3-
(J. Overgaard and R. E. Weber, unpublished data). However, it is possible that
altered acidbase status, the reduction in plasma [Cl-] that
attends the alkaline tide, possible changes in red blood cell volume and the
concentration of phosphates might alter blood oxygen binding during digestion.
In the present study, we examine the effects of digestion on blood
oxygen-binding properties of the snake Python molurus. To do this, we
compare oxygen-binding properties of whole blood from fasting animals with
that of snakes that were 48 h into digestion, because this is the time where
oxygen consumption is maximal (Secor and
Diamond, 1995; Overgaard et
al., 1999
). In addition, we performed simultaneous measurements of
arterial blood gases, acidbase status, plasma ions and haematological
parameters in vivo, which enable a prediction of blood oxygen binding
in vivo. Finally, based on previous determinations of blood flows and
metabolic rate during fasting and digestion
(Secor et al., 2000
), we
predict blood oxygen transport and venous blood gases in vivo.
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Materials and methods |
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Surgical procedure
The snakes were anaesthetised with halothane vapour until they ceased to
exhibit reflexes when pinched. A ventro-lateral incision was made 5-6 cm
anterior to the anus, and the dorsal aorta was exposed for occlusive
cannulation with Bolab catheters (Lake Havasu, AZ, USA) containing heparinised
Ringer solution. The catheter was pushed forward 2-3 cm into the aorta in the
anterior direction, and the vessel was sealed firmly around the catheter with
sutures. The incision was closed, and the catheter was externalised and
secured to the skin with two or three sutures. The surgery normally took less
than 20 min, and the animals spontaneously resumed voluntary breathing. They
were allowed to recover for 24-48 h after surgery. The catheter was flushed
daily to avoid blood clotting. After the experiments had been terminated, all
animals were killed by an overdose of Nembumal (200 mg kg-1).
Experimental protocol
After surgery, the snakes were placed in individual plastic containers kept
in a climatic chamber maintained at 30°C, where they remained for the
entire experiment and had free access to drinking water. After 24-48 h
recovery from surgery, a 1 ml blood sample was taken for determination of
blood gases, haematological parameters and plasma ions in fasting animals. In
addition, 3 ml blood was sampled for construction of whole-blood
oxygen-dissociation curves in vitro. All excess blood cells were
re-infused with an appropriate volume of saline. After blood sampling from
fasting animals, nine snakes were allowed to feed voluntarily on mice or rats
until satiety; these animals consumed 15-35% of their own body mass
(27±2%). Blood sampling for in vivo measurements and in
vitro blood oxygen-dissociation curves was repeated 48 h after feeding,
when the snakes were in a postprandial state. To investigate the effects of
blood sampling, five snakes followed the same protocol but were not allowed to
feed between samplings. These animals served as unfed controls.
Measurements of blood gases and haematological parameters in
vivo
Blood pH was measured using a capillary pH electrode connected to a PHM 73
meter (Radiometer, Copenhagen, Denmark). Total blood oxygen content
(CO2) was measured as described by Tucker
(1967) using the correction
pointed out by Bridges et al.
(1979
). Total CO2
content of the plasma (CplCO2) was measured
according to Cameron (1971
),
and plasma bicarbonate concentration ([HCO3-]) was
calculated as
[HCO3-]=CplCO2-(PCO2
CO2),
using an
CO2 (CO2 solubility in blood)
value of 0.0366 mmol l-1
(Heisler, 1984
). Arterial
PCO2 (PaCO2)
in vivo was calculated on the basis of the
HendersonHasselbalch equation:
PaCO2=CplCO2/[
CO2(1+10(pH-pK'))].
The apparent pK' for Python plasma at 30°C was calculated
using the rearranged HendersonHasselbalch equation on the basis of
CplCO2, and the pH that was measured in the
tonometers at known PCO2 levels. Correlation
analysis of these data resulted in the following relationship between
pK' and pH: pK'= -0.0763xpH+6.7283 (r=0.37); This
value is in good agreement with that predicted on the basis of plasma ion
composition (Heisler,
1984
).
Arterial PO2 (PaO2) was measured at 30°C with an E5046-0 O2 electrode (Radiometer, Copenhagen), and haematocrit (Hct) was determined as the fractional red blood cell volume after spinning blood in capillary tubes at 12000 revs min-1 for 3 min. Plasma chloride concentration ([Cl-]) was measured using a CMT 10 chloride titrator (Radiometer, Copenhagen), and potassium and sodium concentrations of the plasma ([K+] and [Na+], respectively) were determined by flame photometry (FLM3 Flame photometer, Radiometer, Copenhagen). Osmolality was determined by freezing point depression (Knauer semimicro osmometer; Berlin, Germany).
Construction of blood oxygen-dissociation curves
The freshly collected blood was divided in two Eschweiler tonometers (Kiel,
Germany) at 30°C. Each oxygen-dissociation curve (ODC) was constructed
in vitro at a constant PCO2 (1.87 kPa
and 4.67 kPa) from measurements of blood oxygen content at various
PO2 levels
(Tucker, 1967). Gas mixtures
were prepared by Wösthoff gas-mixing pumps (Bochum, Germany) and
humidified in glass flasks. Initially, blood was equilibrated to a high
PO2 (30% O2) for 35 min to measure
oxygen-carrying capacity (full saturation). The
PO2 of the gas mixture was then reduced in
steps. Blood was equilibrated to each oxygen level for a minimum of 35 min
before measurements, and blood CO2 was always
determined in duplicate. Haemoglobin-bound oxygen ([HbO2]) was
calculated as:
[HbO2]=CO2(PO2
O2),
where
O2 is the oxygen solubility in blood at
30°C (Christoforides and Hedley-Whyte,
1969
). Haemoglobin-oxygen saturation was calculated as
[HbO2] relative to the oxygen capacity measured at 30%
O2. Each ODC was established on the basis of three to four points,
with HbO2 saturations between 20% and 80%. The results were plotted
in Hill plots (log(Y/1Y) vs
logPO2), where Y is the fractional
HbO2 saturation, and a linear regression was applied to the
individual data. Values for cooperativity (n) were estimated as the
slopes of the regression lines, and P50 was determined as
the PO2 where
log(Y/1Y)=0.
In all blood samples used for determination of oxygen-binding properties in vitro, we measured Hct, pH, CplCO2 and [NTP] (the concentration of organic phosphates in the blood) at 30% air (approximately at half saturation). [NTP] was determined spectrophotometrically using a Sigma kit (no. 366) (Sigma-Aldrich, Vallensbæk Strand, Denmark). Total haemoglobin concentration ([Hb4]) was assumed to be equal to [HbO2] values at full saturation (30% O2), and red blood cell haemoglobin concentration ([Hb4]RBC) was calculated using Hct and [Hb4].
In vivo blood oxygen affinity (P50) and
cooperativity (n) values were estimated from the Bohr factor
(=
logP50/
pH), and arterial pH was
determined for each individual animal at 0 h and 48 h. From these values and
the PO2 measured in vivo,
HbO2 saturation in vivo could be estimated using the Hill
equation: HbO2 saturation =
(100(PO2/P50)n)/(1+(PO2/P50)n).
Statistical analysis
The results from 0 h and 48 h measurements were compared using paired
t-tests (two-tailed) using P0.05 as the level of
statistical significance between treatments. All results are presented as
means ± 1 S.E.M.
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Results |
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Although arterial pH remained constant after feeding, digestion was associated with a significant increase in both [HCO3-] and PaCO2. Thus, PaCO2 increased by approximately 0.7 kPa after feeding, and [HCO3-] increased by approximately 6 mmol l-1. Plasma osmolality increased significantly in the fed animals and was accompanied by a proportional increase in plasma [Na+] (Table 1) and a visually detectable `milky' appearance of the plasma during digestion. Both plasma [Cl-] and plasma [K+] decreased significantly during the postprandial period.
Blood oxygen-binding characteristics and haematological
variables in vitro
Mean values for oxygen-binding properties and haematological properties at
2% and 5% CO2 are listed in
Table 2 for both fed animals
and unfed controls. High PCO2 reduced pH and
oxygen affinity in all individuals (Table
2). The calculated Bohr effects () increased significantly
following feeding, but remained unchanged in unfed control animals
(Table 2).
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Blood obtained from digesting animals had a significantly higher pH at any given PCO2 in vitro, indicating an alkaline tide in vivo (Tables 1, 2). There was also a small, but significant, increase in pH at 2% CO2 after 48 h in the unfed control animals, but this was not observed at 5% CO2 (Table 2). Blood oxygen affinity was significantly increased at 48 h during the postprandial period with a reduction in P50 from 4.67 kPa to 3.42 kPa and 5.36 kPa to 4.43 kPa at 2% and 5% CO2, respectively (Table 2). There were no changes in P50 with time in the unfed control animals.
The reductions in haematocrit and [Hb4] observed in vivo were accompanied by similar reductions in the blood samples used for in vitro studies (Tables 1, 3). [Hb4]RBC in vitro was significantly reduced from 4.8±0.1 mmol l-1 to 4.5±0.1 mmol l-1 after feeding, while there were no significant changes in the unfed control animals (Table 3). There was a significant decrease in the [NTP]/[Hb4] ratio from 2.42±0.15 in fasting animals to 2.21±0.18 after feeding (Table 3). The [NTP]/[Hb4] ratio also decreased with time in the unfed control animals, although this reduction was not significant (Table 3).
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The influence of [NTP]/[Hb4] and [Hb4]RBC on blood oxygen affinity is depicted in Fig. 1, which shows the calculated logP50 for each animal at a pH of 7.6 (obtained from the individual Bohr effects).
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Discussion |
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Plasma osmolality increased significantly after digestion. The levels of
amino acids and nutrients increase during digestion and might contribute to
the increase in osmolality (Secor and
Diamond, 1997), but plasma osmolality and plasma [Na+]
levels also tended to increase in unfed control animals
(Table 1). Thus, it appears
that the surgical procedure and/or blood sampling contribute to the increase
in osmolality.
Earlier studies on Python, using either chronically cannulated
animals (Overgaard et al.,
1999) or heart puncture of very large individuals
(Secor et al., 2001
), have
reported a decrease in Hct following digestion. Our study shows similar
reductions in both postprandial and unfed control animals, suggesting that
this decrease results from blood sampling. Nevertheless, assuming a blood
volume of 7% of body mass and removal of 5 ml blood kg-1, blood
sampling alone can only account for a reduction in Hct from 19.2% to
approximately 17.8%. Hence, other processes must contribute to the changes in
Hct, which is in apparent conflict with plasma osmolality. Recent studies show
that large fluid shifts account for the increased intestinal mass
(Starck and Beese, 2001
), and
pronounced water shifts between intra- and extracellular stores may occur
during digestion. Clearly, this aspect needs further investigation. The
decrease in Hct contrasts with the marked postprandial increases observed in
dogs and amphibians (Kurata et al.,
1993
; Wang et al.,
1995
; Busk et al.,
2000a
); however, Hct does not change during digestion in
alligators (Busk et al.,
2000b
). Increased oxygen-carrying capacity of the blood would be
beneficial when metabolic demands are high, and increased Hct often occurs
during exercise in mammals and other vertebrates
(Nikinmaa, 1990
).
As in previous studies on Python and other ectothermic
vertebrates, PaO2 was not affected by digestion
(Wang et al., 1995;
Overgaard et al., 1999
; Busk
et al., 2000a
,
2000b
). In contrast, using
heart puncture, Secor and Diamond
(1995
) reported that
PaO2 decreases from 16.0 kPa to approximately
3.3 kPa following digestion in Python, but this sampling technique is
likely to yield a mixture of arterial and venous blood. Hence, in spite of the
relative hypoventilation that characterises the postprandial state
(Overgaard et al., 1999
;
Secor et al., 2000
),
Python maintains a high PaO2 during
digestion.
Oxygen-binding characteristics
Blood oxygen affinity of fasting Python is within the range of the
42 snake species summarised by Pough
(1977), including the family
Boidae. The Bohr effect of fasting and digesting Python
(Table 2) was slightly lower
than the mean of -0.44 reported for nine snake species by Pough
(1980
). The Bohr effect of
Python increased during the postprandial period
(Table 2). This may be
attributed to relative changes between plasma pH and red blood cell pH
(pHe and pHi, respectively) following digestion. The
decreased red blood cell content of phosphates (a non-diffusable anion) found
in this study (Table 3) will
increase
pHi/
pHe as well as pHi
with elevated pHe (Wood et al.,
1978
). Thus, it is possible that pHi changed more for a
given change in pHe during digestion. In addition, decreased [NTP],
together with increased pHi, may have enhanced the specific effect
of CO2 on oxygen affinity
(Duhm, 1976
). This would
increase the apparent Bohr effect in spite of the decrease in red blood cell
[NTP].
Blood oxygen affinity increased markedly during digestion. This effect was
present at both 2% and 5% CO2 levels
(Table 2) and also when the
P50 of each individual animal was estimated at a pH of 7.6
using their respective Bohr effects. The increased affinity was associated
with a decrease in the [NTP]/[Hb4] ratio and a decreased
haemoglobin concentration within the red blood cells
(Fig. 1 and
Table 3). Both of these
variables are likely to contribute to the increased oxygen affinity, as a
multiple linear regression analysis showed a positive correlation with
logP50
(logP50=0.134x[Hb4]RBC+0.112x[NTP]/Hb4]+0.595;
r2=0.62). This regression analysis, however, does not
exclude the possibility that other factors, such as red blood cell pH or
[Cl-], may contribute. The specific effect of
[NTP]/[Hb4] on the logP50 of 0.112 was lower
than the values of approximately 0.2 reported for other snakes
(Johansen and Lykkeboe, 1979;
Ragsdale et al., 1995
;
Herman and Ingermann, 1996
).
An influence of cell volume on blood oxygen affinity has been demonstrated in
a number of vertebrates. It is generally attributed to reduced interactions
between phosphate and haemoglobin molecules but may also be caused by reduced
haemoglobinhaemoglobin interactions
(Nikinmaa, 1990
). In
Python molurus, it seems that reduced haemoglobinhaemoglobin
interactions may indeed have a marked effect on blood oxygen affinity. Thus,
haemoglobin solutions with an [Hb4] of 0.05 mmol l-1
have a P50 of approximately 1.5 kPa at saturating
phosphate concentrations (J. Overgaard and R. E. Weber, unpublished data),
which is a considerably higher affinity than reported here for intact red
blood cells, where the [Hb4] is much higher.
Very little is known about the factors that regulate red blood cell
phosphate concentrations in ectothermic vertebrates, but humoral factors are
likely candidates (Tetens and Lykkeboe,
1981; Nikinmaa,
1990
). In garter snakes Thamnophis sirtalis, it has been
shown that progresterone increases [NTP]/[Hb4] ratio and reduces
oxygen affinity (Ragsdale et al.,
1993
). Catecholamines are known to decrease [NTP]/[Hb4]
in teleost red blood cells (Nikinmaa,
1990
), but this mechanism has not been studied in reptiles, and it
seems unlikely that digestion is associated with increased levels of
circulating catecholamines (Wang et al.,
2001b
). In several vertebrate species, it is well established that
the increased blood oxygen affinity during hypoxia correlates with reductions
in [NTP]/[Hb4] (Nikinmaa,
1990
; Weber and Jensen,
1988
). The low venous oxygen levels during digestion may be
involved in the regulation of [NTP]/[Hb4] ratio in Python
but is unlikely to result from oxygen limitation to the red blood cell, as red
blood cells maintain their [NTP]/[Hb4] ratio when kept at low
oxygen tension in vitro (Tetens
and Lykkeboe, 1981
).
Prediction of blood oxygen affinity and oxygen transport in
vivo
P50 and cooperativity (n) were estimated at
in vivo pH (Table 4) using the variation of P50 and n with pH for each
snake. The estimated in vivo oxygen dissociation curves (ODCs) for
fasted and digesting snakes are illustrated in
Fig. 2. The
P50 estimated at in vivo pH decreased in all nine
animals after feeding by an average of approximately 1.07 kPa, while
cooperativity decreased slightly (Table
4). The predicted saturation of arterial blood remained high
during digestion and in control animals
(Table 4).
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|
In conjunction with previously published data on blood flows and rates of
oxygen consumption, our data on oxygen-binding properties and arterial blood
gases permit analysis of systemic oxygen delivery during fasting and
postprandial conditions. In Python, systemic blood flow
(sys) increases from a
fasting value of 19 ml kg-1 min-1 to 85 ml
kg-1 min-1 during digestion of a meal equivalent to 25%
of body mass, while oxygen consumption increases from 0.8 ml O2
kg-1 min-1 to 7.2 ml O2 kg-1
min-1 (Secor et al.,
2000
); however, this study did not include measurements of blood
flow in the carotid and vertebral arteries, so we have used a
sys of 25 ml
kg-1 min-1 and 100 ml kg-1 min-1
in fasting and digesting snakes, respectively, for predictions of oxygen
transport. Assuming a blood oxygen-binding capacity of 4 mmol l-1
O2 for both fasting and digesting snakes, we calculated venous
HbO2 saturation and PO2 using
sys, our predicted arterial
HbO2 saturation (Table
4) and the in vivo blood ODCs. This analysis predicts
mean venous HbO2 saturations of 58% and 14% in fasting and
digesting animals, respectively, with corresponding venous
PO2 values of 5.2 kPa and 1.6 kPa, respectively
(Fig. 2). It seems, therefore,
that Python molurus extracts oxygen extremely efficiently during
digestion, and, because our analysis predicts mixed venous oxygen levels, it
must be expected that some tissues extract even more oxygen. No measurements
of venous blood gases during digestion in Python appear to exist,
but, given our predictions, it would certainly be worthwhile obtaining these
measurements in future studies. Secor and Diamond
(1995
) report a
PO2 of approximately 3.3 kPa in blood obtained
by cardiac puncture, indicating that venous PO2
does indeed reach low values during digestion. In comparison, pulmonary
PaO2 of the Savannah monitor Varanus
exanthematicus is approximately 3.3 kPa during maximal exercise on a
treadmill (Hopkins et al.,
1995
), and PvO2 decreases to
approximately 2.7 kPa in hard-working human muscles (Pedersen et al.,
1999).
In Python, the rate of O2 uptake
(O2) increases
with meal size, and values in excess of 20 ml O2 kg-1
min-1 have been reported (Secor
and Diamond, 1997
). Our estimated venous oxygen levels at a
O2 of 7.2 ml
O2 kg-1 min-1 leave very little room for
further oxygen extraction (Fig.
2). Hence, to sustain a
O2 of 20 ml
O2 kg-1 min-1, the snakes must increase
sys to values three times
greater than those measured by Secor et al.
(2000
). Alternatively, Hct,
and thus blood oxygen-carrying capacity, should be increased to 60%. This
seems unlikely given that all studies on Python have documented
slight decreases in Hct following feeding
(Overgaard et al., 1999
;
Secor et al., 2001
;
Table 1).
The increased blood oxygen affinity in Python during conditions of
elevated metabolic rate seems at odds with the general view that a
right-shifted dissociation curve would favour unloading of oxygen in the
tissues. In fact, as originally proposed by Weber and White
(1986), the specific effect of
HCO3- on haemoglobin oxygen binding in crocodilians
compensates for the alkaline tide, such that blood oxygen affinity remains
virtually unchanged during digestion (Busk
et al., 2000b
). Furthermore, Pough
(1980
) suggested that reptiles
possess a low blood oxygen affinity to compensate for low capillary density
and large diffusion distances. The low estiamted
PvO2 values during digestion
(Fig. 2) do not, however,
indicate that unloading is impaired. Furthermore, diffusion distances in
gastrointestinal organs have not been reported in reptiles, so it is possible
that unloading is adequately ensured in these organs. We have previously shown
that there is no anaerobic contribution to the SDA response in
Python, as plasma concentration of lactic acid does not increase
(Overgaard et al., 1999
). The
increased affinity during digestion may, therefore, relate to the loading of
O2 within the pulmonary circulation. It is possible that saturation
of pulmonary venous blood is reduced during digestion because of the decreased
pulmonary transit time (caused by the elevated pulmonary blood flow) and the
greatly reduced systemic PvO2. This effect may
be particularly important for reptilian lungs that have limited diffusive
capacity (Hopkins et al.,
1995
; Glass,
1991
). Under these circumstances, an increased blood oxygen
affinity would enhance the PO2 gradient across
the lung epithelium and, thereby, increase oxygen saturation.
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Acknowledgments |
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References |
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