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How a Turtle's Shell Helps It Survive Prolonged Anoxic Acidosis

Donald C. Jackson

D. C. Jackson is in the Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, Rhode Island, 02912.

	Abstract

 
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.

	Introduction

Top Introduction Body fluid buffering The turtle's shell and... Conclusions References

 
In 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-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 acidosis, therefore, the turtle must rely principally on endogenous buffering mechanisms.

	Body fluid buffering

Top Introduction Body fluid buffering The turtle's shell and... Conclusions References

 
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 submergence. 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

Top Introduction Body fluid buffering The turtle's shell and... Conclusions References

 
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.

View larger version (81K): [in this window] [in a new window]   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.

View larger version (13K): [in this window] [in a new window]   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).

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 bicarbonate, 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).

View larger version (16K): [in this window] [in a new window]   FIGURE 3. Postulated exchanges between shell/skeleton and blood during anoxic submergence.

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.

View larger version (19K): [in this window] [in a new window]   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).

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 acid-base 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 that bone from other organisms could not store lactate under appropriate conditions.

	Conclusions

Top Introduction Body fluid buffering The turtle's shell and... Conclusions References

 
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 hard-shelled 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 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.

	Acknowledgments

 
My work is supported by National Science Foundation grant IBN-97-28794.

	References

Top Introduction Body fluid buffering The turtle's shell and... Conclusions References

 

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (16) Citing Articles via Google Scholar Google Scholar Articles by Jackson, D. C. Search for Related Content PubMed PubMed Citation Articles by Jackson, D. C.