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Comparative Biochemistry and Physiology Part A 125 (2000) 299 – 315
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Review
Living without oxygen: lessons from the freshwater turtle
Donald C. Jackson *
Department of Molecular Pharmacology, Physiology and Biotechnology, Brown Uni6ersity, Box G, Pro6idence, RI 02912, USA
Received 18 October 1999; accepted 20 December 1999
Abstract
Freshwater turtles, and specifically, painted turtles, Chrysemys picta, are the most anoxia-tolerant air-breathing
vertebrates. These animals can survive experimental anoxic submergences lasting up to 5 months at 3°C. Two general
integrative adaptations underlie this remarkable capacity. First is a profound reduction in energy metabolism to : 10%
of the normoxic rate at the same temperature. This is a coordinated reduction of both ATP generating mechanisms and
ATP consuming pathways of the cells. Second is a defense of acid – base state in response to the extreme lactic acidosis
that results from anaerobic glycolysis. Central to this defense is an exploitation of buffer reserves within the skeleton and,
in particular, the turtle’s shell, its most characteristic structure. Carbonates are released from bone and shell to enhance
body fluid buffering of lactic acid and lactic acid moves into shell and bone where it is buffered and stored. The
combination of slow metabolic rate and a large and responsive mineral reserve are key to this animal’s extraordinary
anaerobic capacity. © 2000 Elsevier Science Inc. All rights reserved.
Keywords: Acid – base balance; Anaerobiosis; Anoxia; Buffering; Bone; Lactic acid; Metabolic depression; Metabolic rate; Strong ion
difference; Turtle shell
1. Introduction
Because this review is adapted from an invited
address named in honor of the great Danish
physiologist, August Krogh, it is fitting to begin
with a quotation from his work (Krogh, 1941):
Crocodiles, turtles, and many tortoises, living in
water, dive regularly and many of them are
stated to stay for hours or even days under
This invited review is based upon the Krogh Lecture,
sponsored by the Comparative Physiology Section of the
American Physiological Society, presented at the 1999 Experimental Biology Annual Conference.
* Tel.: +1-401-8632373; fax: + 1-401-8633352.
E-mail address: donald – [email protected] (D.C. Jackson)
water.... It is evident that the metabolism must
be either greatly reduced or become mainly
anaerobic. No really quantitative work on the
diving of reptiles has been made thus far, although some of them should be very suitable
for the purpose.
In the years since Krogh wrote these words, his
predictions have been borne out and we now have
considerable quantitative information on the diving of reptiles. In addition, particular species have
been identified that are indeed ‘‘very suitable for
the purpose.’’ Extensive information is available
on the responses and adaptive mechanisms that
permit long periods of anoxic submergence in the
painted turtle, Chrysemys picta, and the red-eared
1095-6433/00/$ - see front matter © 2000 Elsevier Science Inc. All rights reserved.
PII: S 1 0 9 5 - 6 4 3 3 ( 0 0 ) 0 0 1 6 0 - 4
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D.C. Jackson / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 299–315
slider, Trachemys scripta. My own work, which
will be emphasized in the paper, has largely been
on the former species, in particular the sub-species, C. picta bellii. In its normal range, which
extends into north-central United States and into
southern Canada, this turtle, the western painted
turtle, experiences severe winters when the water
in which they hibernate may be ice-covered for
many months. They therefore face long periods of
apnea under conditions that are very likely
severely hypoxic or anoxic (Ultsch, 1989). Because
of their natural history, this species has been a
logical subject for studies of anoxic tolerance.
Early studies, some even before Krogh’s remarks, documented the capacity for turtles to
survive periods of anoxia (Johlin and Moreland,
1933; Musacchia, 1959; Belkin, 1963; Robin et al.,
1964). Our work (Herbert and Jackson, 1985a)
looked at durations of reversible anoxic submergences over a range of temperatures and found a
marked temperature effect (Fig. 1). Of particular
significance in the present context is the extremely
long anoxic period (12 weeks) from which turtles
can fully recover when studied at 3°C, a temperature close to the conditions under which they
hibernate. In other studies in which recovery was
not monitored, turtles remained alive at this temperature while anoxic for as long as 5 months
(Ultsch and Jackson, 1982), a duration approaching the maximum a turtle could encounter in the
wild.
Turtles, like all other vertebrates, are aerobic
animals that depend ultimately on oxygen for
normal function. But they are also facultative
anaerobes, which means that they possess traits
that permit them to sustain some minimal level of
function with no available oxygen. How can they
survive for such long periods in the absence of
O2? I will discuss two general classes of responses,
metabolic responses and acid–base responses,
which can help to provide a basis for understanding this remarkable performance. My emphasis
will be at the level of whole animal integrative
physiology. A recent review in this journal on the
same general topic focused on the biochemical
basis of metabolic adaptations (Storey, 1996).
2. Metabolic basis for anoxia tolerance
As illustrated in Fig. 1, the length of time an
animal can remain anoxic is inversely related to
its metabolic rate; i.e. the slower its metabolic
rate, the longer the anoxia. A number of factors
combine to produce particularly low metabolic
rate in a cold anoxic turtle. First, the turtle is an
ectotherm and it therefore has a metabolic rate
that is low compared to an endotherm of similar
size, some five to ten times lower even at the same
body temperature. Second, a drop in environmental temperature lowers an ectotherm’s metabolism
even further, usually by two to three times for
each 10°C fall in body temperature (Q10 = 2–3),
and with an even steeper slope at the low end of
the temperature range of most reptiles (Bennett
and Dawson, 1976), including this turtle (Herbert
Fig. 1. Duration of anoxic submergences at various temperatures from which painted turtles (Chrysemys picta bellii ) have been observed
to recover fully. Data are adapted from Herbert and Jackson (1985a).
D.C. Jackson / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 299–315
Fig. 2. Schematic diagram illustrating factors that contribute
to a drastically lower (to 0.01%) metabolic rate in a cold,
anoxic turtle compared to a euthermic mammal of the same
approximate body size.
and Jackson, 1985b). Third, anoxia and a switch
to anaerobic metabolism produces a further lowering of metabolism to about 10% of the aerobic
rate at the same temperature. Together these factors result in an exceedingly low rate of
metabolism in an anoxic turtle at 3°C that is only
0.5% of its own aerobic metabolism at 20°C and
less than 0.01% of a similar-sized mammal at rest
in a thermoneutral environment (Fig. 2).
2.1. Metabolic depression in anoxic turtles
When a turtle is submerged under anoxic conditions, available oxygen is restricted to what it
carries within its body. Its major oxygen store
resides in its large lungs with a significant additional amount bound to hemoglobin in its blood
(Fuster et al., 1997). If freely diving, a turtle will
routinely return to the surface to refill its oxygen
stores before they fall to critically low levels (Ackerman and White, 1979; Gatten, 1981), but if
denied access to air in anoxic water, either by ice
or by experimental design, oxygen reserves will
become exhausted and cellular metabolism will
shift from aerobic oxidative phosphorylation to
anaerobic glycolysis. This transition to anaerobiosis is associated with a gradual fall in energy
metabolism to about 10 – 15% of the aerobic level
as documented in intact turtles both directly, by
calorimetry (Jackson, 1968) and indirectly, by lactate accumulation (Herbert and Jackson, 1985b).
Metabolic depression of similar magnitude has
also been reported in individual organs: in brain,
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Lutz et al. (1985) estimated from blood lactate
levels that metabolism fell to 5–10% of the aerobic rate; Doll et al. (1994), however, using microcalorimetry on turtle brain slices observed only
a 40% reduction in rate, but brain slices, even
when supplied with oxygen, may already have
suppressed metabolism (Lutz and Nilsson, 1997).
Isolated liver cells from turtles also undergo a
90% reduction in metabolic rate on the basis of
lactate production (Buck et al., 1993).
Our focus at the organ level has been the heart.
The heart is unusual in that its metabolic requirements are largely determined by whole body demand for blood flow. If this demand falls, then
the active metabolic function of the heart, cardiac
output, can be correspondingly reduced. But what
is the intrinsic response of cardiac tissue to anoxic
lack, independent of its reduced obligations to the
rest of the body? To understand this we studied
both perfused working hearts and isolated ventricular muscle strips and exposed the cardiac tissue
to anoxic conditions simulating what it might
experience in vivo. Surprisingly, we observed little
effect of anoxia on the mechanical performance of
perfused hearts (Wasser et al., 1990b; Jackson et
al., 1995), a finding similar to what had been
reported earlier by Reeves (1963). In contrast, we
found a significant fall in contractile force in
ventricular strips exposed to anoxia (Wasser et al.,
1990a), confirming an earlier report on a similar
preparation by Bing et al. (1972). These contradictory results were resolved in a study by Farrell
et al. (1994) on an in situ turtle heart preparation
that performed at levels comparable to peak values observed in intact animals. In this preparation, anoxia caused a 50% reduction in power
output when the heart operated at its maximum
level, but no reduction was observed when performance was submaximal. This suggests that the
failure to observe an anoxic depressive effect in
perfused heart preparations was because they
were not operating at peak levels.
No ambiguity exists, however, about the depressant effect of combined anoxia and lactic
acidosis on cardiac performance, and this combination of stresses is of course the condition that
the animal faces during anoxic submergence. Even
a perfused heart operating well below its peak
power output is weakened significantly by this
duel stress (Wasser et al., 1990b; Jackson et al.,
1995). To examine this further, we studied force–
velocity relations in ventricular strips from turtle
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D.C. Jackson / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 299–315
hearts under controlled conditions of normoxia,
acidosis, anoxia, and combined anoxia/acidosis
(Shi and Jackson, 1997). Strips could be set at
optimal length for maximum force. In this preparation anoxia reduced force by about 50% and
combined anoxia/acidosis lowered force further to
20 – 25% of normoxic force. Interestingly, maximum velocity of shortening was unchanged by
these conditions, although reduced temperature
did slow this rate. The maximum power output of
the heart (the peak product of its force and velocity) of a cold anoxic heart is therefore reduced by
a combination of reduced force due to anoxia and
acidosis and reduced velocity due to low
temperature.
Because overall body metabolic rate and perfusion requirements decrease profoundly during
anoxic submergence, however, it is likely that this
lowered cardiac power is still adequate to supply
the body’s needs and that heart function is not a
limiting factor in anoxic survival. The situation in
vivo is complex, though, because function is suppressed not only by the intrinsic effects of anoxia
and acidosis, but also by reduced venous return
and vagal stimulation. In addition, calcium and
catecholamines, both cardiac activators, are increased in the circulating blood of anoxic turtles,
although their effects on anoxic hearts in vitro are
not dramatic (Wasser et al., 1990a; Shi et al.,
1999). The reduction in heart activity is dramatically illustrated by heart rates as low as one beat
in 10 min in an anoxic turtle at 3°C.
2.2. Metabolic depression of turtles submerged in
aerated water
Reduced metabolism may also be critical for
turtles overwintering in water with high PO2.
Some species, such as softshell turtles (Apalone)
and musk turtles (Sternotherus), have permeable
integuments and can survive indefinitely in aerated water at low temperature (Ultsch et al.,
1984). Other turtles, with heavy calcified shells,
such as Chrysemys or the map turtle (Graptemys
geographica), appear poorly adapted for aquatic
respiration, yet Chrysemys benefits significantly
from aquatic O2 (Ultsch and Jackson, 1982) and
submerged Graptemys can maintain normal blood
acid –base state throughout a cold Vermont winter by selecting well-aerated water for a hibernaculum (Crocker et al., 2000). Although the avenue
of O2 uptake by Graptemys is not certain (it may
involve cloacal and/or buccopharygeal respiration), it is likely that this turtle also depresses its
metabolism to reduce its O2 demands. Recently,
Boutilier et al. (1997) have found that winter frogs
(Rana temporaria) are able to remain aerobic in
hypoxic cold water by reducing their metabolism.
Remarkably, Graptemys sustains a predominantly
aerobic metabolism with a blood PO2 of only
about 1 Torr (Crocker et al., 2000).
2.3. Energy status of anoxic turtles
Although the metabolic rate of anoxic turtles
decreases dramatically, the decrease is a carefully
controlled process that has little effect on the
energy status of the cells. It is important to consider metabolic rate in two ways: first, as the rate
at which energy is released by intermediary
metabolism and stored in high energy bonds of
ATP; i.e. ATP production rate; second, as the rate
at which an organism utilizes ATP to carry out
vital cellular functions such as synthesis, transport, and contraction; i.e. ATP consumption rate.
A number of studies have found that cellular ATP
concentrations remain constant during anoxia in
turtles, demonstrating that these rates are
matched. Kelly and Storey (1988), for example,
sampled various tissues from anoxic turtles and
found that ATP concentration and total adenylates fell somewhat after 1 h anoxia at 20°C in
Pseudemys (= Trachemys) scripta, but returned to
normal after 5 h anoxia. Energy charge ([ATP]×
0.5[ADP]/([ATP] + [ADP]+[ADP])) was constant throughout in all tissues examined. Constant
ATP during anoxia has also been found in studies
of specific organs and cells, including brain (Lutz
et al., 1985), heart (Wasser et al., 1990a), and liver
(Buck et al., 1993).
The metabolism of the anoxic turtle, therefore,
undergoes a coordinated downregulation of ATP
synthesis and ATP hydrolysis (Fig. 3), and the
close matching between these processes maintains
relatively constant cell levels of ATP. Three important fundamental questions arise from this
observation: First, what regulatory mechanisms
produce the profound drop in ATP production
when this animal switches from aerobic to anaerobic metabolism? Second, how can 90% of the
cellular ATP-requiring processes be suspended
and the stability and viability of the cells preserved? Third, how is the metabolic reorganization of the animal coordinated?
D.C. Jackson / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 299–315
Depressed anaerobic ATP production in turtles
has been termed the ‘reverse Pasteur effect’ because the classical effect of low oxygen on the
glycolytic pathway is activation rather than suppression (Hochachka, 1986). The term may be
misleading, however, because even with an overall
metabolic depression of 90%, glycolytic flux must
increase somewhat over the aerobic rate because
of low ATP yield of this pathway. The full aerobic oxidation of a mole of glucose gains only
5 – 6% of the generated moles of ATP from glycolysis alone. Nonetheless, the failure of glycolysis to
increase in activity to sustain aerobic-level ATP
synthesis must require some controlling factors.
Major work on this problem has been carried out
by Storey and his colleagues, and has been summarized in thorough reviews of the subject (Storey
and Storey, 1990; Storey, 1996). Three general
anoxia-induced alterations of glycolytic enzymes
have been described by these authors. First, alteration of enzyme activity via phosphorylation or
dephosphorylation; second, reversible binding of
enzymes to cellular macromolecules or organelles;
third, allosteric regulation of enzyme function by
specific metabolites. In addition, although protein
303
synthesis is usually found to be severely depressed
in anoxic tissues, selective proteins are expressed,
including members of the heat shock family
(Chang et al., 2000) and also possibly glycolytic
enzymes (Hochachka et al., 1996). In contrast to
these findings, Brooks and Storey (1993) observed
sustained or even increased protein synthesis during exposure to nitrogen gas at 6°C and the
appearance of specific protein, perhaps of the heat
shock family, in animals recovering from anoxia.
Matched to the fall in ATP production is a
parallel fall in ATP hydrolysis, the process that
pays for energy-requiring processes of the cells.
Because the anoxic tissues function with only 10%
of the aerobic energy input, drastic reductions in
the various forms of cell work must occur. Studies
on brain in particular, but also on liver cells, have
documented reductions in several key processes
that drive ATP utilization: transmembrane ion
leakage, electrical activity, and protein synthesis.
In the language of the field, these have been
referred to, respectively, as channel arrest, spike
arrest, and translational arrest (Hochachka et al.,
1996). Channel arrest is a reduction in the number
of functional ion channels and/or in the probabil-
Fig. 3. Constant cellular ATP levels of anoxic turtles indicates that a coordinated decrease occurs in both ATP production and ATP
utilization when a turtle makes the transition from a normoxic to an anoxic state.
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D.C. Jackson / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 299–315
Fig. 4. Cellular processes of isolated turtle hepatocytes that
require ATP all decrease when the cells are made anoxic, and
the overall cumulative decrease ( :90%) is close to the decrease observed in the whole animal (Jackson, 1968; Herbert
and Jackson, 1985b) and in the total metabolic rate of hepatocytes (Buck et al., 1993). Data shown are adapted from
Hochachka et al., 1996).
ity that ion channels are open. This slows ion
leakage and the dissipation of ion gradients and
less cell work is required to maintain these gradients via ATP-dependent ion pumps. Evidence for
downregulation of Na+ channels in brain (PérezPinón et al., 1992) and liver cells (Buck et al.,
1993), for K+ channels in brain (Chih et al.,
1989), and for Ca2 + in brain (Bickler and Buck,
1998) have been reported. The reduced channel
activity in brain contributes in turn to what Sick
et al. (1998) called spike arrest, a reduction in
brain electrical activity. Also contributing to spike
arrest are increased levels of the inhibitory neurotransmitter, GABA (Nilsson and Lutz, 1991), reduced sensitivity of the NMDA-type glutamate
receptor (Buck and Bickler, 1998), and an apparently overall regulating action of adenosine (Lutz
and Nilsson, 1997). Doll et al. (1993) attributed
all the reduction in anoxic brain ATP expenditure
for ion pumping to spike arrest, since they could
detect no significant decrease during anoxia in
whole cell ionic conductance in turtle pyramidal
neurons. Finally, reduced protein synthesis, translational arrest, has been observed both in anoxic
turtle hepatocytes (Land et al., 1993) and in
anoxic turtle heart (Bailey and Driedzic, 1996),
although the upregulation of specific proteins,
such as stress proteins in heart (Chang et al.,
2000) and possibly glycolytic enzymes in liver cells
(Hochachka et al., 1996) has been reported.
A comprehensive study of the effect of anoxia
on various energy-requiring functions in isolated
turtle hepatocytes was conducted by Hochachka
and his colleagues. The results of their studies,
adapted from Hochachka et al. (1996), are summarized in Fig. 4. Interestingly, the overall decrease in hepatocyte ATP hydrolysis, estimated
from the sum of the various components, closely
matches total hepatocyte metabolic reduction
(Buck et al., 1993) and whole animal metabolic
reduction (Jackson, 1968).
The coordination of metabolic events leading to
a matched decrease in ATP production and utilization, long-term survival in an hypoxic state,
and eventual recovery is not well understood. One
proposal that has been put forward (Hochachka
et al., 1996) suggests that anoxia-tolerance in turtles and in other facultative anaerobes is a twophase process: first, a defense phase during which
ATP supply and consumption are suppressed in a
controlled fashion, as just described; and second,
a rescue phase in which selective proteins, including transcription and elongation factors, molecular chaperones, and glycolytic enzymes, are
produced to enable the cells to function during
anoxia. A heme-based oxygen sensing mechanism,
analogous to that described for the regulation of
erythropoietin, has been proposed as the trigger
for these responses (Hochachka et al., 1996).
The unraveling of the molecular and cellular
basis for anoxia tolerance in turtles, and in other
organisms, is an active and exciting aspect of this
field that will doubtless lead to findings of general
biological significance in the coming years. For
recent more detailed accounts of this work on
anoxic turtles, the reader is referred to Hochachka
et al. (1996), Storey (1996), Hochachka (1997),
Lutz and Nilsson (1997), Bickler and Buck (1998).
2.4. Ad6antages of reduced metabolism
The submerged anoxic turtle is to a great extent
a closed system, exchanging only a few substances
(oxygen if available, CO2, and water) with the
surrounding water. At 3°C, loss of CO2 to the
water allows blood PCO2 to remain normal or
even fall during anoxia thereby avoiding the respiratory acidosis that is characteristic of submergence at higher temperatures. A net uptake of
water from the surroundings also occurs as indicated by an increase in body mass during longterm submergence (Ultsch and Jackson, 1982).
D.C. Jackson / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 299–315
Major regulated exchanges, however, that are crucial for normal homeostasis, including feeding
and pulmonary gas exchange do not occur, and
renal function is either lost or severely curtailed
(Warburton and Jackson, 1995; Jackson et al.,
1996). As a consequence, the submerged anoxic
turtle must support its metabolic needs throughout its submergence time with on-board energy
reserves. In addition, metabolic end-products,
other than CO2, remain within the body and must
be tolerated by the animal. For an anoxic turtle,
the principle metabolic storage molecule is glycogen, and the principle end-product is lactic acid.
The length of time a turtle can remain anoxic may
therefore be critically dependent on glycogen depletion and/or on lactate accumulation. Adaptations that can delay either of these processes
reaching critical limits may extend underwater
time.
Reduced metabolism is clearly a useful adaptation in this regard. By slowing the rate at which
glycogen is utilized by 10-fold, the turtle may
extend the anoxic period by that magnitude. Furthermore, painted turtles have large glycogen reserves residing principally in liver and skeletal
muscle (Daw et al., 1967; Wasser et al., 1991).
Based on estimated anaerobic metabolic rate
(Herbert and Jackson, 1985b) and body glycogen
stores, we calculate that a turtle could sustain
anoxic periods of about 5.5 months at 3°C before
glycogen became depleted. It is unlikely that a
305
turtle in nature would ever be subjected to continuous anoxia for that long, so that glycogen reserves may not be the limiting factor for anoxic
survival, assuming that the turtle begins the winter with an adequate supply.
Lactic acid, on the other hand, may pose a
more significant threat to the animal. Lactate is
produced by the reduction of pyruvate in order to
regenerate NAD+ for sustaining flux through the
glycolytic pathway. Protons are a product of ATP
hydrolysis and, in a situation such as anoxic
submergence in which ATP levels are maintained,
protons and lactate ions are produced in equal
amounts (Hochachka and Mommsen, 1983) making lactic acid, in effect, the overall end-product
of anaerobic metabolism. Even though metabolic
depression slows the rate at which lactate is produced and accumulates within the body, very high
concentrations are reached during long periods of
anoxia. Plasma concentrations as high as 200 mM
were measured after 5 months of submergence at
3°C (Ultsch and Jackson, 1982), and concentrations between 100 and 150 mM were reached after
3 months anoxia (Jackson and Heisler, 1982; Herbert and Jackson, 1985a), a submergence duration
from which these turtles can fully recover (Herbert and Jackson, 1985a). Despite these high lactate concentrations, that are far in excess of what
other animals are able to endure, the painted
turtles are able to maintain a viable blood pH in
the range of 7.0 or higher (Fig. 5). Clearly the
Fig. 5. Blood pH and plasma lactate concentration during 125 days of anoxic submergence in the eastern painted turtle (Chrysemys
picta picta). Data are re-drawn from Ultsch et al., 1999.
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D.C. Jackson / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 299–315
painted turtle possesses specialized mechanisms
for dealing with this enormous acid load. The
balance of this paper will address these
mechanisms.
3. Acid–base adaptations of anoxic turtles
3.1. Body fluid buffering
Lactic acid is a relatively strong acid (approximately pK 4.0) that at physiological pH (approximately 7.0) is about 99.9% dissociated to protons
and lactate ions (i.e. [lactate]/[lactic acid] : 1000/
1). During anoxic submergence, turtles neither
excrete lactate nor can they convert it to other,
less toxic, compounds. To avoid life-threatening
acidosis, therefore, the anoxic turtle must neutralize the generated protons using endogenous
buffering. The first line of defense against
metabolically produced fixed acid is by intracellular and extracellular buffering.
In various freshwater turtles, as first reported
by Smith (1929), extracellular buffering of lactic
acid is particularly effective due to high concentrations of bicarbonate. In the western painted
turtle and in related freshwater species, plasma
[HCO−
3 ] is in the range of 35 – 45 mM whereas
values for other reptiles are rarely above 30 mM
and are often below 20 mM (Ultsch and Jackson,
1996). Furthermore, chelonian species including
the painted turtles also have specialized pericardial and peritoneal fluids, bathing the heart and
abdominal viscera, respectively, that have even
higher concentrations of HCO−
3 : approximately
80 mM in the peritoneal fluid and 120 mM in the
pericardial fluid (Smith, 1929; Jackson and Silverblatt, 1974). During acidosis, whether imposed
by gastric infusion of dilute HCl (Jackson, 1969)
or by prolonged anoxic submergence at low temperature (Murdaugh et al., 1962; Jackson and
Ultsch, 1982; Jackson and Heisler, 1984), the
[HCO−
3 ] in these fluid compartments falls as [lactate] rises. The specialized pericardial and peritoneal fluids of Trachemys scripta elegans are not
affected, however, by briefer (4 h) anoxic submergences at 24°C (Jackson and Silverblatt, 1974) so
exchange kinetics may be slow.
Buffering by intracellular fluids of the painted
turtle, as in other organisms, is mainly by non-bicarbonate buffers and [HCO−
3 ] is lower than in
the ECF, at about 10 mM (Jackson and Heisler,
1983). A recent measurement of non-bicarbonate
buffering capacity in cardiomyocytes of C. picta
−1 −1
l
cell water
bellii was 22.3 mmol HCO−
3 pH
(Shi et al., 1997), a value that conforms to published values for various tissues from other organisms (Pörtner, 1990). Lactate, in the anoxic turtle,
distributes preferentially to extracellular fluid
leading to cell lactate concentrations in muscle
and liver that are generally B50% of extracellular
concentrations (Jackson and Heisler, 1983). This
distribution conforms generally to a transmembrane equilibrium of the undissociated acid form
as predicted by the pH difference between the two
compartments. Despite the lower levels of intracellular lactate, however, pH falls by similar magnitudes in the cells and in the blood (Jackson and
Heisler, 1983). Assuming that the distribution of
the associated protons is the same as the lactate,
this indicates that the effective buffering in the
extracellular compartment is superior. Effective
buffering includes the endogenous buffers, such
HCO−
3 , as well as any supplemental buffering
contributed by exchanges with other body
compartments.
The magnitude of the acid load experienced by
the turtle after several months of anoxia at 3°C
far exceeds the endogenous buffering capacity of
the extracellular fluid. Even the high levels of
HCO−
3 possessed by this animal are not adequate
to deal with acid levels that can reach 150–200
mM. Keeping blood pH near 7.0 requires the
contribution of additional buffering from elsewhere in the body. The predominant source of
this additional buffering, as will now be discussed,
is the shell and skeleton of the turtle.
3.2. The turtle’s shell
The shell of the turtle is its characteristic structural feature. Its primary function as a protective
enclosure for the animal is a familiar one and one
that has served it well during the some 200 million
years of its existence in essentially its present form
(Pough et al., 1996). The shell also serves as an
insertion site for muscle and connective tissue.
These well-known roles of the shell require a
structure that is rigid and strong, but not one that
is necessarily metabolically active or that is in
dynamic exchange with the extracellular fluid. But
like bone, the turtle’s shell is a dynamic structure;
it is perfused with blood, it grows and remodels,
and it serves as the major reservoir for the body’s
minerals (Zangerl, 1969).
D.C. Jackson / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 299–315
The shell of the painted turtle, Chrysemys picta
bellii, is large and accounts for about 32% of the
animal’s body mass. Included in this shell mass
are elements of the skeleton (vertebrae and ribs)
that are fused to the shell. Skeletal elements separate from the shell (limb girdles and long bones
and skull) represent a further 5.5% of the body
mass (unpublished observations). The total mass
of bony tissue, therefore, is over 35% of total
body mass, some three times the bone mass of a
similar sized lizard or mammal (Calder, 1984).
The composition of shell is similar to bone and
within these mineralized tissues reside over 95% of
the body’s calcium, magnesium, phosphate, and
CO2, and over 60% of the body sodium (Jackson,
1999). In a recent study (Jackson et al., 2000) the
concentration of these elements and the percent of
water and ash were determined in the shell and
hindlimb bones of Chrysemys (Table 1).
The minerals of the turtle’s shell and bone
represent an enormous potential buffer reserve for
this animal, and these are exploited during anoxia
in two ways. First, mineral buffers, principally in
the form of carbonates, are released into the
extracellular fluid where they supplement existing
buffering in that compartment. Second, lactic acid
is taken up by shell and bone where the protons
are buffered by carbonate and the lactate is stored
for the duration of the anoxic period.
3.3. Mechanism 1: release of carbonate buffers
Exploitation of bone carbonates as supplemental buffers is a response to sustained acidosis that
has been described in humans and other mammals
(Irving and Chute, 1932; Burnell, 1971; Bettice,
1984; Bushinsky and Lechleider, 1987), and in a
variety of invertebrates (DeFur et al., 1980; Byrne
and McMahon, 1991). Its occurrence in the submerged turtle at 3°C was originally indicated by a
progressive increase in the plasma concentrations
of calcium and magnesium (Jackson and Ultsch,
1982). This study produced results that were initially quite puzzling. Plasma lactate concentrations increased to very high levels, 150 – 200 mM,
but analyses of what we regarded as the major
plasma ions (Na+, K+, Cl−, and HCO−
3 ) failed
to satisfy ion balance and we were left with a
large ‘cation gap.’ However, measurements of total Ca2 + and Mg2 + revealed large increases in
these elements, to concentratons as high as 50 and
35 mM, respectively, that almost completely ac-
307
counted for the gap. This result was surprising
and not anticipated because concentrations of
Ca2 + and Mg2 + generally remain very constant
and deviations from normal, especially in Ca2 + ,
can lead to serious disruptions of excitable tissue
function.
The full implications of these high circulating
Ca2 + and Mg2 + levels are still not clear, but two
factors appear to blunt any potentially negative
impact on cell function. First, Ca2 + and Mg2 +
both complex with lactate; in the case of Ca2 + , a
cationic complex, CaLact+, is formed (Ghosh and
Nair, 1970). Although the association constant
for this reaction is relatively low (:20 l mol − 1)
and complex formation is insignificant at normoxic levels of the reactants, complex formation
is very significant under conditions of prolonged
anoxic acidosis, when as much as two-thirds of
the total Ca2 + can be bound in this way (Jackson
and Heisler, 1982). As a consequence, ionized
calcium, the active form, is much lower in concentration than total Ca2 + and the effective gradient
for Ca2 + entry into cells is lessened. A second
factor that permits the turtle to tolerate high
circulating Ca2 + concentrations is that Ca2 + entry into cells is inhibited during anoxia. In cardiac
cells, low extracellular pH reduces Ca2 + influx
(Orchard and Kentish, 1990); in brain cells, Ca2 +
channels, such as the NMDA receptor, are downregulated, thereby protecting the cells from the
damaging elevation of intracellular Ca2 + that occurs in anoxic mammalian cells (Bickler and
Buck, 1998) even at normal extracellular concentrations. Nonetheless, plasma ionized Ca2 + of
anoxic turtles can be many times higher than
control values (12.5 mM compared with 1.0 mM),
and further studies on possible effects of, and
adaptations to, this unusual hypercalcemic condition are certainly needed.
Because shell and bone are the turtle’s major
sites for Ca2 + and Mg2 + storage, it is logical to
postulate that the elevated plasma concentrations
of these elements during anoxia derive from these
sources. In addition, various lines of direct experimental evidence support this idea. First, analysis
of shell composition revealed a significant decrease in [Mg2 + ], although not in [Ca2 + ], during
anoxia at 3°C (Warburton and Jackson, 1995).
Calcium is some 50 times more abundant than
Mg2 + in shell and bone, but estimated Ca2 +
release from these structures is only two to three
times as great (Jackson and Heisler, 1982; Herbert
308
Water
(%)
Ash
(%)
Organic
(%)
Shell
27.89 0.6
44.79 0.8
27.490.4
Bone
33.69 1.1
41.09 1.0
25.4 9 0.2
a
b
[P]
(mmol g−1 ash−1)
[Mg]
(mmol g−1 ash−1)
[Na]
(mmol g−1 ash−1)
[K]
(mmol g−1 ash−1)
[CO2]b
(mmol g−1 ash−1)
9420 9116
4800 9 32
228.0 96.6
362.29 8.3
10.79 1.4
1347 9 67
10 3609186
5138 9 38
196.092.4
369.19 11.2
17.89 0.9
[Ca]
(mmol g−1 ash−1)
All data, except shell CO2 concentration, is from Jackson et al. (2000).
Unpublished data.
–
D.C. Jackson / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 299–315
Table 1
Shell and bone composition from control painted turtles (Chrysemys picta bellii ) at 3°Ca
D.C. Jackson / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 299–315
309
Fig. 6. Shell sodium concentration in mmol kg − 1 ww − 1 and plasma sodium and chloride concentrations in mmol l − 1 of painted turtles
(Chrysemys picta bellii ) submerged in anoxic water for up to 125 days. The top trace shows the absence of any change in shell sodium
during 125 days of submergence in turtles in normoxic water. Data are from Jackson et al. (2000).
and Jackson, 1985a. Therefore, the relative
change in shell Mg2 + is much easier to detect.
Second, in vitro incubation of shell powder in
simulated extracellular solution (Jackson et al.,
1999) resulted in significant release of both Ca2 +
and Mg2 + , in proportions similar to in vivo observations, and in direct proportion to solution
acidity. Phosphate, the major anion of bone and
shell in turtles, was not released in significant
amounts from incubating shell powder in vitro,
nor did plasma concentration of phosphate increase during prolonged anoxia in vivo (Jackson
et al., 2000). This indicates that the major structural mineral of bone, calcium phosphate, is not
broken down under these conditions, and that
another buffer anion is released instead.
Carbonate, as noted above, is the major anion
released from bone under acidotic conditions in a
variety of organisms. In turtle, several experimental observations confirm a similar role in prolonged acidosis in this animal. First, total CO2
concentration of shell decreases significantly during anoxia in vivo at 3°C (Warburton and Jackson, 1995). Second, shell powder incubated in
vitro releases CO2 in proportion to solution acidity and with a stoichiometry that indicates divalent carbonate as the molecular form (Jackson et
al., 1999). In mammalian bone, CO2 is thought to
exist in three different sites (Rey et al., 1989): as
carbonate substituted for hydroxyl ion in hydroxyapatite, as carbonate substituted for phosphate
in hydroxyapatite (the major site), and in a
rapidly exchanging fraction, possibly as carbonate
on the surface of bone crystals or as bicarbonate
in the hydration shell of the crystals. It is the
rapidly exchanging fraction that is thought to
participate in buffering responses. Our observatons on turtle shell in vitro (Jackson et al., 1999)
suggest that the exchangeable CO2 in this structure is in the form of carbonate and not bicarbonate. This is also supported by an earlier
observation that the shell possesses no heat-labile
CO2 (Warburton and Jackson, 1995); in mammalian studies where a heat-labile CO2 fraction is
found, this is taken as evidence for bicarbonate
release (Poyart et al., 1975).
In addition to calcium and magnesium carbonates, turtle shell also releases sodium ions, presumably also accompanied by carbonate (Jackson
et al., 2000). As shown in Fig. 6, shell [Na+] fell
progressively throughout 4 months of anoxic submergence at 3°C; however, no change in shell
[Na+] was observed in turtles submerged for 4
months in aerated water. The participation of
310
D.C. Jackson / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 299–315
Fig. 7. Schematic model described putative exchanges between
blood and shell (and bone) during prolonged anoxia in the
turtle Chrysemys picta bellii. In mechanism 1 (to the left),
calcium, magnesium, and sodium carbonates move from the
shell to the blood in response to lactic acidosis and thereby
supplement extracellular buffering capacity. In mechanism 2
(to the right) lactic acid enters the shell and is buffered and
sequestered there.
Na+ in the shell buffering response agrees with
earlier observations in mammals where bone Na+
release is an important regulatory response to
both acidosis and hyponatremia (Forbes, 1960;
Burnell, 1971). The release of Na+ from powdered shell in vitro, in contrast, was inconsistent
and unrelated to incubating solution pH (Jackson
Fig. 8. Plasma strong ion difference (SID), the sum of the
concentrations of strong cations (Na+, K+, Ca2 + , and Mg2 + )
minus the sum of the concentrations of strong anions (Cl− and
lactate−) under control normoxic conditions at 3°C (top
trace) and during 12 weeks of anoxic submergence at the same
temperature (middle trace). The bottom trace depicts the SID
that would have resulted from the observed increases in
plasma [lactate−] had no changes in other strong ions occurred. The vertical lines therefore represent the magnitude of
the compensatory increases in K+, Ca2 + , and Mg2 + concentrations and decreases in Cl− concentration. Data are from
Jackson and Heisler, 1982).
et al., 1999). The loss of shell Na+ in vivo is
significant and, based on the magnitude of the fall
in concentration and the mass of shell, should
elevate extracellular [Na+] by about 39 mmol l − 1
(Jackson et al., 2000). Paradoxically, though,
plasma [Na+] in this same study actually decreased significantly, and although in previous
studies plasma [Na+] has generally remained unchanged (e.g. Jackson and Heisler, 1982), an increase in [Na+] has never been observed. The
whereabouts of the sodium leaving the shell is
therefore uncertain and must await further study.
A simplified model for mechanism 1 (Section
3.3) is presented in Fig. 7. The initiating event in
this exchange process is the addition of lactic acid
to the extracellular fluid (ECF). The first line of
defense, and the principal one at low levels of
lactic acid, is the endogenous buffers of the ECF,
chiefly HCO−
3 . As lactate levels rise, however, the
developing acidosis causes a progressive dissolution of shell and bone minerals, specifically calcium, magnesium and sodium carbonates. The
carbonates titrate the protons producing CO2 that
at 3°C is mostly lost through non-pulmonary
pathways to the surrounding water. Calcium released into the ECF reacts with lactate and up to
two thirds may bind in this way. ECF levels of
both Ca2 + and Mg2 + increase markedly, but
Na+ levels either remain unchanged or paradoxically fall.
The impact of the released minerals from turtle
shell and bone can best be appreciated in terms of
their effect on the strong ion difference (SID) of
plasma. Strong ion difference, a term popularized
by Stewart (1981) in his analysis of acid–base
physiology, is the difference in concentration between the strong cations (Na+, K+, Ca2 + , Mg2 + )
and the strong anions (Cl−, lactate−). In turtle
plasma, SID is normally about 44 meq l − 1 (Herbert and Jackson, 1985a) and electroneutrality is
satisfied by ‘weak’ anions, primarily HCO−
3 and
titratable groups on protein. Because the defense
of a viable acid–base state requires maintenance
of SID in the positive range, the challenge facing
a turtle experiencing long-term anoxic submergence is clear. An increase in the concentration of
lactate to 150–200 meq l − 1 with no changes in
other plasma strong ions would produce a highly
negative SID (Fig. 8). All available buffers would
be fully titrated and severe, probably fatal,
acidosis would ensue. But release of shell and
bone buffers helps counteract this by providing
D.C. Jackson / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 299–315
Fig. 9. Lactate concentration of shell and plasma of painted
turtles (Chrysemys picta bellii ) sampled at intervals during 9
days of anoxic submergence and 9 days of recovery at 10°C
(Jackson, 1997).
carbonate to titrate the protons and by providing
strong cations (Ca2 + , Mg2 + , and Na+) to sustain
a positive SID. Additional plasma ion changes,
apparently unrelated to shell function, that contribute to the defense of SID include increased
ECF [K+] and decreased ECF [Cl−].
3.4. Mechanism 2: uptake of lactic acid by shell
and bone
The second way that the shell and skeleton of
the anoxic turtle contribute to acid – base balance
is by sequestering lactic acid. Unlike the buffer
release mechanism, the lactate storage mechanism
has not previously been described in other organisms. The first evidence for its occurrence was
obtained when shell powder, collected from submerged anoxic turtles previously injected with
14
C-labeled lactate, was found to exhibit significant 14C activity. Subsequent systematic investigation, in which lactate was measured by both 14C
activity and by conventional enzymatic assay, revealed a substantial and uniform distribution of
lactate throughout the entire shell of the turtles
during anoxia at 20°C (Jackson et al., 1996).
More recently, the phenomenon has been documented at 10 and 3°C as well (Jackson, 1997). In
the study at 10°C, turtles were submerged in
anoxic water for 9 days and then allowed to
311
recover with access to air for an additional 9 days.
Blood and shell samples, taken periodically
throughout this period, revealed a parallel rise in
lactate in both plasma and shell during submergence, and then a parallel fall in lactate during
recovery (Fig. 9). In a more recent study at 3°C
(Jackson et al., 2000), bone was shown to accumulate lactate as well, to an even greater extent
than shell, during 4 months of anoxic
submergence.
The extent to which this mechanism contributes
to management of total body lactate is considerable. After 4 months anoxia at 3°C, shell and
bone concentrations reached 135.6 and 163.6
mmol g − 1 ww − 1, respectively, when plasma lactate concentrations averaged 155.2 mmol l − 1
(Jackson et al., 2000). Based on estimated lactate
distribution in the body fluids, the size of body
fluid compartments, and the mass of the shell and
skeleton, we estimated that 47% of the total body
lactate resided in the shell and bone at this time.
A similar fraction of body lactate was calculated
to be stored in shell and bone after 9 days anoxia
at 10°C.
The entry of lactate into shell and bone does
not, however, prove that these structures are contributing to lactic acid buffering. Lactate cannot
enter the shell alone; it must either be accompanied by a cation or must exchange with another
anion in order to maintain electroneutrality. The
identity of this other ion is crucial for understanding the acid–base significance of lactate entry. If,
for example, a strong cation such as Na+ enters
with lactate, or a strong anion such as Cl− leaves
as lactate enters, then the lactate sequestration
will have no impact on extracellular acid–base
status. This is because the process will not effect
the ECF strong ion difference (see above). If,
however, lactate enters with an H+ or if two
lactates enter in exchange for a carbonate, then
this would have great significance for the ECF
acid–base status. Both indirect and direct evidence support the latter possibility. The indirect
evidence is that no strong ion among the major
ECF ions appears to be a likely candidate. It is
known that calcium, magnesium, and sodium all
leave the shell during anoxia, and that ECF calcium and magnesium concentrations rise significantly. Also, ECF [Cl−] falls sharply during
anoxia making it unlikely that Cl− serves as a
counterion for lactate. The direct evidence comes
from in vitro incubation of either shell powder in
312
D.C. Jackson / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 299–315
solutions with high lactate, and of shell powder
containing high lactate in solutions without lactate (Jackson et al., 1999). These experiments
reveal that lactate entry into shell alkalinizes the
bathing solution whereas lactate release from shell
acidifies the solution. This supports the proposition that protons, in effect, accompany lactate
movement, and that this mechanism of lactate
sequestration exploits the buffering capacity of
the shell and bone.
A simple model for this lactate uptake mechanism is depicted in Fig. 7. As in mechanism 1, the
first step is the addition of lactic acid to the ECF.
Assuming that lactate entry into the shell is a
passive process, elevation of ECF [lactate] will
initiate diffusion of lactate into the shell and this
will continue as further lactate is produced anaerobically and enters the ECF. The equilibrium
state between the two compartments is not certain, but it seems to be approached when the shell
concentration in mmol kg − 1 ww − 1 approximates
the plasma lactate concentration in mmol l − 1
(Jackson, 1997, 1999). As depicted by the model,
each lactate is accompanied, in effect, by a proton, and the proton is buffered within the shell
substance by carbonate. The CO2 produced
thereby diffuses back into the ECF and is lost by
diffusion to the surrounding water. The physical
state of lactate within the shell is uncertain, but it
probably exists primarily in combined form, most
likely complexed with Ca2 + , as has already been
described to occur in the ECF. The high concentration of lactate and the small volume of shell
water (only : 30% of shell mass) makes it unlikely that lactate could exist in simple solution;
the estimated concentration would have to be too
high. The mechanism as described occurs in the
skeleton of the turtle as well as in the shell
(Jackson et al., 2000).
A remarkable feature of this mechanism is that
the lactic acid sequestered in this fashion may
have no impact whatever on extracellular acid –
base state. Both the lactate and proton are processed within the shell itself. The only product
coming out (other than water) is CO2, and at low
temperature this is lost to the environment and
ECF PCO2 is the same as or even lower than the
normoxic value. Because, as already noted, shell
and bone lactate storage can account for over
45% of the total body lactate, this mechanism
greatly minimizes the effect of anaerobic acid
production on body fluid acid – base status.
It is likely that other organisms employ this
same mechanism under circumstances when circulating lactate is elevated. In non-chelonian vertebrates, however, its quantitative importance is
almost certainly less for the following reasons.
First, no vertebrates other than turtles have a
comparable mass of mineralized tissue. As noted
earlier, skeletal mass in mammals and lizards of
similar size to the painted turtle have only one
third the bone mass. Second, no other vertebrate
experiences or can tolerate extracellular lactate
concentrations as high as observed in the turtle,
nor are elevated lactate levels sustained for such
extended periods of time. The kinetics of bone
exchange are generally slow so that acute elevations of lactate, such as those that occur during
and following strenuous exercise, may not provide
adequate time for significant lactate uptake by
bone. More promising candidates for utilizing this
mechanism to a significant degree are invertebrates, such as crustaceans and mollusks, which
possess large shells or exoskeletons with abundant
calcium carbonate. Many of these organisms also
experience hypoxia-induced elevations of lactate,
and mobilization of calcium carbonate to the
hemolymph for supplemental buffering is well-established. In addition, in a recent study (Jackson,
D.C., Wang, T. and Taylor, E.W., unpublished
observations), lactate uptake by the exoskeleton
of the freshwater crayfish, Austropotamobius pallipes, was observed during emersion hypoxia.
3.5. O6erall contribution of shell and bone to
lactic acid buffering
The two buffering mechanisms occurring in turtle shell and bone-carbonate release and lactic
acid uptake-may together account for as much as
70% of the total lactic acid buffering in the anoxic
turtle. This calculation refers to the acid–base
state that exists after 3 months of anoxic submergence at 3°C. About two thirds of the shell and
bone contribution is contributed by uptake and
buffering of lactic acid within the shell and bone.
The balance, buffer release, is estimated from the
calculated increases in the extracellular concentrations of calcium and magnesium (Jackson, 1997).
The calculation of the buffer release mechanism,
however, did not take into account the possible
contribution of sodium carbonate release, so it
may actually be an underestimate. An accurate
quantitative assessment of the whole body picture
D.C. Jackson / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 299–315
is further complicated by uncertainty regarding
possible changes in body fluid compartment volumes during anoxia.
Clearly, though, the mineralized tissues are crucial to the successful survival of turtles during
prolonged anoxic submergence. Not only is the
turtle shell a protective shield against the hazards
of life and a structural framework for muscle
attachments, but it also is a enormous reserve of
mineral that the turtle can tap to counteract
acidosis during extended periods of anoxia. The
combination of this enormous capacity to neutralize metabolic acids with an extremely low
metabolic rate that slows the release of these acids
helps explain how the turtle can endure such long
periods in the absence of oxygen.
Acknowledgements
The author’s work is supported by the National
Science Foundation (USA). The author thanks
Malia Schwartz for her careful reading of the
manuscript.
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