Download How a Turtle`s Shell Helps It Survive Prolonged Anoxic

How a Turtle’s Shell Helps It Survive Prolonged
Anoxic Acidosis
Donald C. Jackson
Anoxic turtles accumulate high levels of lactate in blood. To avoid fatal acidosis, turtles exploit
buffer reserves in their large mineralized shell. The shell acts by releasing calcium and magnesium
carbonates and by storing and buffering lactic acid. Together with profound metabolic
depression, shell buffering permits survival without oxygen for several months at 3°C.
n contrast to our own limited ability for breath-held diving,
many aquatic vertebrates can remain submerged for remarkably long periods of time. For example, time-depth records of
free-ranging Southern elephant seals have documented deep
dives lasting for over an hour (4). Even more impressive feats are
achieved by some air-breathing lower vertebrates, whose
underwater times can extend for months. In many of these animals, including both amphibians and reptiles, direct oxygen
uptake from the water enables full aerobic function, particularly
at low temperature; but some freshwater turtles can survive for
extraordinarily long periods in the absence of ambient oxygen
by relying fully on anaerobic metabolism. A prime example is
the painted turtle, Chrysemys picta, a freshwater species whose
range extends into the northern U.S. and southern Canada. Its
natural winter habits, which can subject it to continuous submergence for months in ice-covered ponds, may require this
animal to sustain function for long periods despite severely
hypoxic or anoxic conditions. Laboratory studies have revealed
that turtles can recover fully from experimental submergences
lasting at least three months in oxygen-free water at 3°C (5).
How is the turtle able to do this? The object of this review is to
discuss the mechanisms that permit anoxic survival of this duration, with a particular focus on acid-base aspects.
A key adaptation underlying the anaerobic performance of
Chrysemys is a profound metabolic depression to ~10% of its
aerobic rate at the same temperature. Because the turtle is
ectothermic (“cold-blooded”), even its aerobic metabolic rate
is only a small fraction of that of a similar-sized euthermic
mammal or bird. But when it is deprived of oxygen at winter
temperatures, the combined depressant effects of low temperature and anoxia further reduce the turtle’s estimated ATP production to a rate <0.01% of a similar-sized aerobic rat at rest
in a thermally neutral environment. Under these conditions,
the turtle’s heart rate can be as low as 1 beat every 5–10 min.
Remarkably, despite the profound metabolic reduction, cellular ATP levels and energy state remain virtually unchanged
in the anoxic turtle, revealing a coordinated, and as yet poorly
understood, downregulation in both anaerobic ATP production (13) and cellular ATP utilization (6). The anoxic turtles are
somehow able to sacrifice a large fraction of the energy-
D. C. Jackson is in the Department of Molecular Pharmacology, Physiology,
and Biotechnology, Brown University, Providence, Rhode Island, 02912.
0886-1714/99 5.00 © 2000 Int. Union Physiol. Sci./Am.Physiol. Soc.
requiring activities of the cells and still retain function. Protein
breakdown and synthesis are severely curtailed, and ion
channels are downregulated, leading to a reduction in the ATP
required for powering ion pumps (1, 2, 12).
The turtle’s low anaerobic metabolism has clear significance for extending the period of anoxia. Low metabolism
diminishes the rates at which both stored substrates such as
glycogen are consumed and acid endproducts such as lactic
acid are produced. Either of these processes, the depletion of
glycogen or the developing lactic acidosis, could define the
limits to anoxic survival in this animal, and the turtles have
made provisions to lessen these risks in both cases.
With regard to glycogen, the painted turtle possesses a
large liver (4% of body mass) with high glycogen content
(8–10%) plus additional stores of glycogen in skeletal muscle and heart. These reserves could support anaerobic utilization at the observed glycolytic rate for ~5.5 months,
close to the maximum apneic submergence it would ever
face naturally. Glycogen depletion, therefore, would not
ordinarily represent a problem unless a turtle begins a hard
winter with low reserves.
The production and accumulation of lactic acid, however,
is clearly a serious threat, and its management by the anoxic
turtle is a major challenge that it faces. Lactic acid is a relatively strong acid (pK ~4), so its buildup in the body can
result in a metabolic acidosis of enormous proportions. Even
though the rate at which lactic acid is produced is slow due
to metabolic depression, the time scale over which it accumulates may be great. As a result, plasma concentrations as
high as 200 mM have been observed after experimental
anoxic periods approaching five months in duration (14).
How can a turtle experience lactate levels of this magnitude and still maintain a viable body fluid pH? The answer is
that the turtle uses internal buffering mechanisms similar in
principle to those that are well known from other organisms
but in an extreme form not found in other vertebrates. In
addition, it utilizes a previously unknown mechanism
whereby it sequesters lactic acid in its shell and bone during
the anoxic episode. During anoxic submergence, the turtle is
in most respects a closed system, exchanging little with its
environment. Some extrapulmonary gas exchange occurs,
and the turtles take up water from the surroundings, but no
pulmonary ventilation or feeding occurs, and we have found
no evidence for urinary excretion. To counteract metabolic
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I
acidosis, therefore, the turtle must rely principally on
endogenous buffering mechanisms.
Body fluid buffering
The major extracellular buffer, bicarbonate, is found in particularly high concentration in freshwater turtles; in Chrysemys, plasma bicarbonate concentration is ~40 mM, some
1.5–2 times that of most other vertebrates. In addition, as first
reported by H. Smith in 1929, this turtle and its close relatives have unusually high concentrations of bicarbonate in
peritoneal fluid (~80 mM) and pericardial fluid (~120 mM).
The extracellular fluids thus provide considerable capacity
for buffering a fixed acid such as lactic acid, and direct measurements on turtles confirm that bicarbonate in all of these
fluids participates in buffering an acid load.
Although the volumes of the peritoneal and pericardial fluids are large compared with other vertebrates (~16 ml/kg and
2 ml/kg, respectively) and bicarbonate levels are high, their
contributions to overall buffering of a large acid load, relative
to the entire extracellular compartment, are nonetheless
rather minor. Could there be some other advantage these turtles enjoy by having their viscera and heart bathed in highly
alkaline solutions? This question is unresolved.
Lactate distributes preferentially to the extracellular fluid in
anoxic turtles; lactate concentrations of liver and skeletal
muscle, expressed as mmol/kg cell water, are only ~60% of
extracellular values (10). Both intracellular and extracellular
fluids share in the buffering process, however, and the fall in
pH observed is similar in both compartments. Yet it is obvious that when lactate levels rise to the very high values
reported (100–200 mM), the intrinsic capacity of both compartments should be overwhelmed. The observation, though,
is that both blood pH and intracellular pH remain at viable
values. At 3°C, for example, blood pH falls from its control
value of ~8.0 to ~7.0 after three months of anoxic submer182
News Physiol. Sci. • Volume 15 • August 2000
gence. Over the same period, however, plasma lactate
concentration has risen by ~150 mM, far in excess of the
concentration of extracellular buffers. Plasma bicarbonate
concentration has fallen to <5 mM, and the pericardial and
peritoneal fluids are similarly depleted of their bicarbonate.
Clearly, the normal extracellular capacity has been supplemented by recruitment of buffers from elsewhere in the body.
The predominant source of these additional buffers is the turtle’s characteristic structural feature: its shell.
The turtle’s shell and its contribution to extracellular
buffering
The bone-like shell of Chrysemys accounts for ~32% of the
body mass of the animal; the portion of its skeleton not incorporated into the shell represents an additional 5.5%. Together,
these bony tissues are more than three times the skeletal mass
of a similar-sized mammal or nonchelonian reptile. Besides
the obvious roles it plays as a protective armor and an anchoring site for muscles, the turtle’s shell is also the major mineral
reservoir of the body. Over 99% of the total body calcium,
magnesium, and phosphate, over 95% of the carbon dioxide,
and over 60% of the body’s sodium reside in the shell and
bone. The shell and bone of the turtle, like the skeletons of
other vertebrates, are perfused with blood and participate in a
variety of exchange processes with the extracellular fluid. Of
particular importance to the anoxic turtle is the potential acid
buffering by these shell minerals. Recent work from my laboratory reveals that the shell and bone contribute to buffering
lactic acid in two ways: first, carbonate buffers are released
from shell and bone into the extracellular fluid where the
buffering takes place, and second, lactic acid enters the shell
and bone and is sequestered there. These mechanisms are
illustrated schematically in Fig. 1.
Mechanism 1: supplemental buffers released from shell. This
contribution of bone to acid-base balance in the turtle was
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FIGURE 1. The shell acts in two ways to buffer lactic acid. First, carbonates (such as calcium carbonate) are released into the extracellular fluid (ECF), and second, lactic acid is stored in the shell and buffered there.
initially deduced from observations of progressive increases
over time in the plasma concentrations of both calcium and
magnesium during anoxia. Quite high levels are reached in
extreme cases, up to 50 mM calcium and 20 mM magnesium
(9). As shown in Fig. 2A, the rise in these concentrations also
correlated with blood acidity. Direct measurement of shell
concentrations revealed a significant fall in magnesium but not
in calcium; shell calcium is so high that even the large release
was not sufficient to permit detection (15).
To verify the role of the shell in this process, we have studied powdered shell incubated in vitro in solutions at a range
of pH values using a procedure that allowed us to hold pH
constant by automatic titration with HCl (8). We found that
the shell alkalinized the solution, requiring acid titration for
pH-stat control, and that the amount of titrated acid required
increased as solution pH fell. This is consistent with the
hypothesis that shell buffers help neutralize circulating acids
and that their release during anoxia is a passive consequence
of acid demineralization of shell and bone. In addition, we
found that calcium and magnesium release in vitro were also
direct functions of acidity and bore a relationship to pH that
resembled the in vivo results (Fig 2B). Note also that phosphate was not mobilized from the incubated shell in vitro,
consistent with our finding that plasma phosphate does not
change during anoxia in vivo. If phosphate is not released,
what then is the buffer anion accompanying the divalent
cations? From measurements of evolved carbon dioxide from
incubated shell (8) and of significant loss of total carbon
dioxide from in vivo shell (15), we deduced that carbonate
serves this function, just as it does in mammalian bone (3).
On the basis of these results, we conclude that the primary
mineral of shell and bone, calcium phosphate, is not broken
down during anoxic acidosis in the turtle but that calcium
and magnesium carbonates are.
A simple model of this buffer release mechanism illustrates
the points already made and also introduces other aspects of
this overall process (Fig. 3A). The triggering event is the production and release into the extracellular fluid of lactic acid.
The first line of defense is the preexisting extracellular bicar-
bonate, but the developing acidemia causes the mobilization
of calcium and magnesium carbonates from shell and bone.
The carbonate supplements the extracellular fluid buffering,
and the divalent ions balance the lactate charge. At low temperature, the carbon dioxide generated by the titration of carbonate diffuses out of the animal into the surrounding water,
with the result that blood PCO2 remains unchanged or even
falls somewhat from the value observed during air breathing.
A significant portion of the mobilized calcium (as much as
two-thirds of the total at high lactate levels) complexes
reversibly with lactate to form Ca-Lactate+ (9). A similar reaction presumably also occurs between magnesium and lactate. These reactions, which are of negligible importance
under most physiological states because of the low concentrations of the reactants, are of great importance in this situation and substantially minimize the increase in the ionized
forms of calcium and magnesium. Nonetheless, rather large
increases in free calcium, up to 12.5 meq, do occur, the consequences of which have not been fully explored. Other
plasma ion concentrations also change significantly during
the course of anoxia: potassium concentration rises from
2.5 meq to as high as 10 meq, and chloride concentration
falls from ~80 meq to as low as 40–50 meq. These changes,
like the changes in calcium and magnesium, can be viewed
as compensatory to the metabolic acidosis by balancing the
increased lactate and minimizing the change in the strong
ion difference (9). The potassium presumably derives from
the intracellular compartment, whereas the fall in chloride
concentration may be due largely to dilution by water uptake
from the surroundings, manifested by an increase in body
weight during submergence (14). Plasma sodium concentration does not change significantly during long-term
anoxia, but we have recently found that bone and shell
release a sizable amount of sodium, so that this release may
serve to stabilize sodium concentration in the face of the
body fluid dilution. Despite the uptake of water, plasma
osmolality increases after three months of anoxia at 3°C by
~100 mosmol/kgH2O (from 250 to 350 mosmol/kgH2O), primarily due to the increases in lactate and calcium (9).
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FIGURE 2. A: relationship between plasma calcium and magnesium and blood pH during anoxic submergence at 3°C (adapted from Ref. 5); B: relationship
between bathing medium calcium, magnesium, and phosphate and solution pH after 2 h incubation of powdered turtle shell (adapted from Ref. 8).
FIGURE 3. Postulated exchanges between shell/skeleton and blood during anoxic submergence.
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solution. The stoichiometry was such that we concluded that
each lactate ion was, in effect, entering (or leaving) the shell
accompanied by a proton. This confirms that this exchange
has important acid-base consequences to the animal and
represents a second way by which the turtle exploits the
enormous buffering potential of its shell.
A model for the lactate uptake mechanism is shown in
Fig. 3B. According to this scheme, lactate generated from
anaerobic glycolysis moves into the shell and bone accompanied by hydrogen (note that exchange of two lactates for
one carbonate is an alternative and equivalent mechanism to
the one illustrated). The lactate is sequestered in the shell,
possibly combined with calcium, and the hydrogen is
buffered by carbonate. The molecular carbon dioxide produced by the acid titration moves into the extracellular fluid
and then out of the animal into the surrounding water via
extrapulmonary routes. The unique feature of this mechanism
as depicted is that it segregates a large fraction of accumulated lactate, buffers the associated protons, and, because the
carbon dioxide is lost, has no effect whatsoever on the acidbase balance of the extracellular fluid. This large component
of the lactic acid burden of the body is essentially invisible to
the general body fluids. When the anoxic period is over, lactic acid is released from the shell and reutilized (11).
Might this lactate sequestration mechanism be important in
other organisms experiencing increases in circulating lactate?
Perhaps, but it is unlikely to be as significant as in the anoxic
turtle for three reasons. First, as just noted, the shell and bone
of the turtle are an unusually large fraction of body mass, so
the capacity for uptake greatly exceeds that of other animals.
Second, the time scale over which lactic acidosis can occur in
submerged turtles can be extremely prolonged (up to months),
thereby permitting substantial uptake despite the inherently
slow kinetics of bone exchange. Finally, the levels of lactate
reached in the anoxic turtle are extraordinarily high compared
with other circumstances. Lactate production during intense
exercise, in contrast, is an acute phenomenon lasting just seconds or minutes and resulting in circulating lactate levels that
are at most 20–30 mM. Furthermore, the clearance of blood
lactate during recovery may occur too rapidly for significant
bone participation. Nevertheless, there is no reason to think
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The release of mineral buffers from the shell and bone of the
anoxic turtle is similar in most respects to the well-known role
of mammalian bone in responding to metabolic acidosis (3).
Both calcium and sodium are released with primarily carbonate (or bicarbonate) as the associated anion. Because kidney
function is usually operational during chronic acidosis in
mammals, the mobilized calcium is excreted and does not
build up in the blood as it does in the turtle. The control of the
demineralization process in mammalian bone is thought to be
a combination of a passive equilibrium process, as appears to
be the case in the turtle, and an active cellular process (3). The
contribution of cellular mechanisms to breakdown of shell
and bone in the anoxic turtle is uncertain (15).
Mechanism 2: lactic acid uptake and release by shell and
bone. The storage of lactate in bone has apparently not previously been described as a significant contributor to the
management of a lactate load. We discovered it serendipitously when we tested 14C activity of shell powder generated
during dissection of anoxic turtles treated with 14C-labeled
lactate (11). The high values prompted a systematic investigation that revealed a significant and progressive uptake of
lactate into turtle shell during anoxia (7). In a study at 10°C,
for example, shell lactate (meq/kg wet weight) rose in parallel with plasma lactate (meq/l) during anoxia and then
declined in parallel during recovery (Fig. 4). Similar observations were made during anoxic submergence at both 3 and
20°C. The total lactate that accumulated in shell and bone,
which was a function both of the concentrations reached and
the unusually large mass of these structures, amounted to
almost 50% of the total body lactate after nine days anoxia at
10°C or three months anoxia at 3°C.
An important issue is how this accumulation of lactate in
shell and bone contributes to acid-base balance of the body
fluids of the turtle. If lactate enters the bone in exchange for
chloride, for example, or is accompanied by a strong cation,
such as sodium or potassium, this mechanism would be acidbase neutral and would not ameliorate the impact of the lactic acid on body fluid acid-base status. To clarify this issue,
we turned again to the in vitro preparation (8) and found that
lactate entering shell from the bathing solution alkalinized
the solution and lactate moving from the shell acidified the
bury themselves in anoxic mud at the bottoms of ponds.
Should anoxic conditions prevail, and even if they should
persist for several months, Chrysemys and its near relatives
are well equipped with the necessary adaptations to survive
the winter and to fully recover when warm weather returns.
My work is supported by National Science Foundation grant IBN-9728794.
References
that bone from other organisms could not store lactate under
appropriate conditions.
Conclusions
The acid-base status of a submerged anoxic turtle at 3°C is
essentially pure nonrespiratory (metabolic) acidosis, so slow in
its development that it can be considered a chronic state.
Because the turtle, unlike a patient with metabolic acidosis,
lacks either active respiratory compensation or renal acid
excretion, it must rely entirely on endogenous buffering
reserves to counteract the acidosis. These reserves are so plentiful and effective, however, that the turtle can sustain a viable
acid-base condition and survive with circulating lactate
concentrations approaching 200 mM. The contribution of the
turtle’s shell and bone is crucial in this regard, and it is no exaggeration to state that the prolonged periods of anoxia observed
would not be possible without the shell’s involvement.
The extreme anoxic acidoses described in this article were
produced under experimental conditions in the laboratory,
and it is uncertain whether turtles ever experience conditions
this severe in nature. If aquatic PO2 is adequate, these hardshelled turtles can extract enough dissolved oxygen from the
water to significantly reduce their reliance on anaerobic
metabolism; however, oxygen can become depleted in natural aquatic environments, and turtles have been reported to
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FIGURE 4. Plasma and shell lactate concentrations during 9 days of anoxic
submergence at 10°C and during 9 days of recovery from anoxia (from Ref. 7).
1. Bickler PE and Buck LT. Adaptations of vertebrate neurons to hypoxia and
anoxia: maintaining critical Ca2+ concentrations. J Exp Biol 201:
1141–1152, 1998.
2. Buck LT and Hochachka PW. Suppression of Na+/K+ ATPase activity and a
constant membrane potential in hepatocytes during anoxia: evidence in
support of the channel arrest hypothesis. Am J Physiol Regulatory Integrative Comp Physiol 265: R1020–R1025, 1993.
3. Bushinsky DA. Acidosis and bone. Miner Electrolyte Metab 20: 40–52, 1994.
4. Campagna C, LeBoeuf BJ, Blackwell SB, Crocker DE, and Quintana F. Diving behavior and foraging location of female southern elephant seals from
Patagonia. J Zool (Lond) 236: 55–71, 1995.
5. Herbert CV and Jackson DC. Temperature effects on the responses to prolonged submergence in the turtle Chrysemys picta bellii. I. Blood acidbase and ionic changes during and following anoxic submergence. Physiol Zool 58: 655–669, 1985.
6. Hochachka PW, Buck LT, Doll CJ, and Land SC. Unifying theory of
hypoxic tolerance: molecular/metabolic defense and rescue mechanisms
for surviving oxygen lack. Proc Natl Acad Sci USA 93: 9493–9498, 1996.
7. Jackson DC. Lactate accumulation in the shell of the turtle, Chrysemys
picta bellii, during anoxia at 3 and 10°C. J Exp Biol 200: 2295–2300, 1997.
8. Jackson DC, Goldberger Z, Visuri S, and Armstrong RN. Ionic exchanges
of turtle shell in vitro and their relevance to shell function in the anoxic
turtle. J Exp Biol 202: 513–520, 1999.
9. Jackson DC and Heisler N. Plasma ion balance of submerged anoxic turtles at 3°C: the role of calcium lactate formation. Respir Physiol 49: 159174, 1982.
10. Jackson DC and Heisler N. Intracellular and extracellular acid-base and
electrolyte status of submerged anoxic turtles at 3°C. Respir Physiol 53:
187–201, 1983.
11. Jackson DC. Toney VI, and Okamoto S. Lactate distribution and metabolism during and after anoxia in the turtle, Chrysemys picta bellii. Am J
Physiol Regulatory Integrative Comp Physiol 271: R409–R416, 1996.
12. Pek M and Lutz PL. Role for adenosine in channel arrest in the anoxic turtle brain. J Exp Biol 200: 1913–1917, 1997.
13. Storey KB. Metabolic adaptations supporting anoxia tolerance in reptiles: recent advances. Comp Biochem Physiol B Biochem Mol Biol 113:
23–35, 1996.
14. Ultsch GR and Jackson DC. Long-term submergence at 3°C of the turtle,
Chrysemys picta bellii, in normoxic and severely hypoxic water. I. survival,
gas exchange and acid-base status. J Exp Biol 96: 11–28, 1982.
15. Warburton SJ and Jackson DC. Turtle (Chrysemys picta bellii) shell mineral
content is altered by exposure to prolonged anoxia. Physiol Zool 68:
783–798, 1995.