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Surviving extreme lactic acidosis: the role of calcium lactate formation in the anoxic turtle
Respiratory Physiology & Neurobiology 144 (2004) 173–178
Surviving extreme lactic acidosis: the role of calcium lactate formation in the anoxic turtle Donald C. Jackson∗ Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, RI 02912, USA Accepted 28 June 2004
Abstract During prolonged anoxia at low temperature, freshwater turtles develop high plasma concentrations of both lactate and calcium. At these concentrations the formation of the complex, calcium lactate, normally of little biological significance because of the low association constant for the reaction, significantly reduces the free concentrations of both lactate and calcium. In addition, lactate is taken up by the shell and skeleton to an extent that strongly indicates that calcium lactate formation participates in these structures as well. The binding of calcium to lactate thus contributes to the efflux of lactic acid from the anoxic cells and to the exploitation of the powerful buffering capacity of the shell and skeleton. © 2004 Elsevier B.V. All rights reserved. Keywords: Acid base, buffering, bone; Anoxia, prolonged, survival; Reptiles, turtle; Shell, turtle
1. Introduction The absence of oxygen presents critical challenges to vertebrate organisms because all are ultimately obligate aerobes. Endothermic birds and mammals are particularly vulnerable because of high metabolic requirements of their tissues and poor tolerance of their brains and hearts to even brief periods of anoxia. Ectothermic vertebrates are more resistant and most can probably survive relatively brief exposures to anoxia. Only a few, however, can remain anoxic for very long periods. These facultative anaerobes include freshwater turtles and several species of fish and the ∗
Tel.: +1 401 863 2373; fax: +1 401 863 3352. E-mail address: donald [email protected].
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characteristics that permit their unusual tolerance is of some interest. A major factor limiting duration of anoxia is accumulation of acid end-products and their effect on acid–base balance. Fish such as crucian carp and goldfish avoid this problem by substituting ethanol, an acid–base neutral compound, and CO2 as principle waste products, both of which are readily excreted via the gills (Van Waarde et al., 1993). Turtles, however, like other vertebrates, rely on anaerobic glycolysis for energy production and this leads to lactate and protons as end-products (Hochachka and Mommsen, 1983). They must therefore cope with the metabolic acidosis problem. Plasma lactate concentrations in freshwater turtles, such as the painted turtle, Chrysemys picta, subjected to
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D.C. Jackson / Respiratory Physiology & Neurobiology 144 (2004) 173–178
cold anoxic submergence can reach very high levels, as high as 150–200 mmol l−1 after several months (Ultsch and Jackson, 1982; Jackson et al., 2000). The turtle’s ability to manage this large acid load and remain in a viable acid–base state is a key factor that explains how they live so long without O2 . Shell and skeletal buffers play a major role as has been described in recent reviews (Jackson, 2000, 2002) and, as will be described in this paper, the formation of a complex between calcium and lactate is an integral part of this mechanism.
2. Calcium-binding molecules Calcium is a very reactive ion that binds reversibly to many molecules. Within cells calcium ligands play an important role in regulating the concentration of ionized calcium, the biologically active form. In the plasma, proteins, in particular albumen, are normally the principal ligands. Other calcium-binding molecules, such as EDTA, are valuable laboratory tools for regulating calcium activity in experimental solutions. The strength of the combination between calcium and a ligand is described by the association constant, Ko: Ko (l mol−1 ) =
[CaLigand] [Ca2+ ][ligand]
The values of Ko for organic molecules (Table 1) vary over an extremely wide range. Because a higher value of Ko indicates more avid binding with calcium, the biological importance of a molecule as a calcium ligand is a function of the value of Ko for that molecule. It is notable that the Ko value for lactate is particularly low compared to the other substances shown. The relevant equilibrium reaction for calcium and lactate is as follows:
Fig. 1. Measured association constants, Ko, for the reaction between calcium and lactate at different temperatures. Data from Jackson and Heisler (1982) and unpublished observations.
And the association constant is defined as Ko =
[Ca Lactate+ ] [Ca2+ ][Lactate− ]
Using prepared solutions and a calcium electrode, Ko for this reaction was measured at 3 ◦ C (Jackson and Heisler, 1982) and subsequently at a series of higher temperatures (unpublished observations) (Fig. 1). These values are in general agreement with other published measurements of this constant (Cannan and Kilbrick, 1938; Ghosh and Nair, 1970; Martell and Smith, 1977), but are well below the value of 100 l mol−1 that Williams (1977) defined as the lower limit a reaction must have to achieve biological importance. In the anoxic turtle, however, in contradiction to the statement by Williams, the significance of this reaction is of unquestioned biological importance.
3. Calcium lactate formation in body fluids
Ca2+ + Lactate− ↔ Ca Lactate+ Table 1 Association constants (Ko) for selected calcium ligands Molecule
Ko (l mol−1 )
EDTA Citrate Malonate Succinate Lactate
5 × 1010 6 × 104 316 100 ∼10–20
In the intracellular compartment, ionized calcium concentration is so low (∼10−7 M) and other calciumbinding molecules are so dominant, that even high intracellular concentrations of lactate are unlikely to have any significant effect on calcium activity, and of course the calcium is far too low in concentration to have any effect on lactate. Likewise, in the extracellular fluid, at usual concentrations of calcium and lactate, calcium lactate formation is also negligible. For example, at
D.C. Jackson / Respiratory Physiology & Neurobiology 144 (2004) 173–178
a plasma lactate of 1 mmol l−1 and ionized calcium of 1.3 mmol l−1 , the concentration of calcium lactate would be about 0.04 mmol l−1 , only about 3% of the total combined calcium, assuming a Ko of 11.7 l mol−1 (Martell and Smith, 1977). This means that under normal conditions the assertion of Williams (1977) referred to above is certainly valid. For calcium lactate formation to bind a significant fraction of either extracellular calcium or lactate, the concentration of one or both of these ions must be abnormally high. Hypercalcemia of sufficient magnitude would be very unusual because calcium concentration is generally regulated within narrow limits. Lactate, on the other hand, can increase markedly in concentration in a variety of states, such as strenuous exercise and breathhold diving, or in various disease states affecting tissue O2 delivery. At plasma lactate concentrations that are commonly observed, in the range of 15–25 mmol l−1 , the Ko for calcium lactate formation predicts that lactate-bound [calcium] would be 20–30% of free [calcium]. This reaction could, therefore, contribute to the hypocalcemia that is a common response during vigorous exercise and in critically ill patients, both conditions in which elevated lactate levels can occur. The role of calcium lactate formation in critically ill patients has been considered but is controversial (Cooper et al., 1992; Aduen et al., 1995). Proponents for this reaction playing a role (Cooper et al., 1992) observed a clear inverse relationship between lactate and ionized calcium (Fig. 2), and simple calculations for the
Fig. 2. Relationship between plasma ionized calcium and lactate concentrations of critically ill patients. Modified from Cooper et al. (1992). Dashed line depicts calculated contribution of calcium lactate formation to the reduction in ionized calcium assuming a Ko value of 11.7 l mol−1 .
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equilibrium reaction confirm a significant contribution from the calcium lactate reaction. The relatively minor contribution of this reaction to the observed relationship, however, and the complexity of other factors influencing ionized calcium in diverse patient populations probably accounts for the negative findings of the other authors (e.g., Aduen et al., 1995). Similarly, hypocalcemia during strenuous exercise with elevated lactate must be at least partially due to calcium lactate formation. In a study of high intensity exercise lasting 42 min, Bouassida et al. (2003) observed a reciprocal relationship between lactate and ionized calcium during and following exercise. The authors attributed the fall in ionized calcium during exercise to renal excretion but over 25% can be predicted from the calcium lactate reaction. Peak lactates were only about 8 mmol l−1 in this study. Lactate may also increase locally in ischemic tissues and exert important effects via calcium-binding. A recent study (Immke and McClesky, 2001) of sensory nerves innervating the heart (in rats) found that a local increase in lactate concentration to 15 mmol l−1 increased the activity of an acid-sensitive ion channel by lowering ionized calcium concentration from 2.0 to 1.71 mmol l−1 . This modest hypocalcemia increased depolarization of these nerves which are thought to mediate anginal chest pain. Two factors make the calcium lactate reaction substantially more significant in the anoxic turtle than in other physiological systems. First, as noted earlier, plasma lactate concentration can rise to very high levels. The second unusual feature of the anoxic turtle is that extracellular concentration of calcium can also be greatly elevated to as high as 50 mmol l−1 (Jackson and Ultsch, 1982). The source of lactate is anaerobic glycolysis, and the source of calcium is mobilization from shell and skeleton together with carbonate for extracellular buffering (Jackson et al., 1999). Hypercalcemia results because renal function, the major means of calcium regulation during bone calcium mobilization, is severely curtailed during anoxia (Warburton and Jackson, 1995). The turtle is operated essentially as a closed system with respect to calcium and lactate, and these solutes are retained and build up within the body fluids, although intracellular ionized calcium is kept in the sub-micromolar range (Bickler, 1998). Because of the elevated plasma concentrations, the reaction between calcium and lactate is very significant in the extracellular compartment. Direct measurements
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at 3 ◦ C, on simple electrolyte solutions and on plasma from anoxic turtles, revealed that more than two-thirds of the total calcium was complexed with lactate when total [lactate] was 145 mmol l−1 and total [calcium] ranged from 34 to 40 mmol l−1 (Jackson and Heisler, 1982). In addition, “free” lactate was calculated to be 115 mmol l−1 and ionized calcium was measured at 12.5 mmol l−1 . These values accord with the measured Ko value for this reaction at 3 ◦ C (∼20 l mol−1 ). Some lactate is also complexed with magnesium which also increases significantly in plasma although the association constant for this reaction is somewhat lower than for calcium lactate. A clear benefit of extracellular calcium lactate formation for the anoxic turtle is that it minimizes the increase in ionized calcium, although even with the lactate binding the animal is still severely hypercalcemic. Another less obvious benefit is the lower “free” lactate concentration in the ECF. Low lactate activity facilitates movement of lactic acid out of cells where it is produced and into the ECF where most of the buffering can occur. Endogenous extracellular buffering is already high in the painted turtle by vertebrate standards due to [HCO3 − ] concentration of about 40 mmol l−1 . In addition, extracellular buffering is supplemented by transfer of carbonates, mainly calcium and magnesium carbonates, from shell and skeleton.
4. Calcium lactate formation in bone In the anoxic turtle, a significant fraction (up to 45%) of the total body lactate resides in the shell and skeleton at the end of prolonged anoxic submergences at 3 and 10 ◦ C (Jackson, 1997). At 3 ◦ C, for example, after 4 months of anoxia when plasma lactate was 155 mmol l−1 , shell lactate was about 131 mmol (kg wet weight)−1 and long bone lactate was about 153.5 mmol (kg wet weight)−1 . Although it is not certain what the physical state of lactate is in mineralized tissues, it is likely that a large fraction of this lactate is combined with calcium. The basis for this assumption is two-fold. First, the volume of water into which the lactate could be dissolved in bone is small, and second, calcium is abundant in bone and much of this calcium may be available for binding lactate. Painted turtle shell is about 32% water by weight (Jackson, 1997) but only half or less of this water is
Fig. 3. Estimated concentrations of lactate in intracellular fluid (ICF) in mmol (kg H2 O)−1 , extracellular fluid (ECF) in mmol l−1 , and bone/shell in mmol (kg wet weight)−1 , following 3 months of anoxic submergence at 3 ◦ C. Solid bar represents “free” lactate, open bar represents calcium bound lactate. Shell values assume that all bone water is equilibrated with extracellular lactate. Data adapted from Jackson and Heisler (1982), Jackson and Heisler (1983), and Jackson (1997).
accessible to the circulating extracellular marker PEG (Jackson et al., 1996). Assuming that shell ECF equilibrates with plasma [lactate], and this is the only lactate in the shell, shell lactate concentration at a plasma concentration of 155 mmol l−1 would be only about 25 mmol (kg wet weight)−1 . Even if all the water in the shell were accessible to lactate, this would only increase the shell lactate to about 50 mmol (kg wet weight)−1 , still only 38% of the measured concentration. This strongly suggests that a major fraction of the shell and skeletal lactate exists in combined form. In contrast, in the ECF after long-term anoxia, about 20% of the lactate is in combined form, and in the ICF little if any lactate is in this form because of low intracellular calcium (Fig. 3). The abundance of calcium in the shell and skeleton and its ability to combine reversibly with lactate makes this ion the most likely candidate for lactate sequestration. Over 99% of the total body calcium is found in these structures, and in the painted turtle as much as 20% of this calcium may be associated with carbonate. It should be noted, however, that magnesium also forms a complex with lactate, and this reaction could also contribute to the observed uptake of lactate by bone. Our working hypothesis is that lactic acid enters bone (shell or skeleton) whereupon bone carbonate buffers the protons and calcium (and possibly magnesium) combines with lactate (Jackson et al., 1999). This reaction enables
D.C. Jackson / Respiratory Physiology & Neurobiology 144 (2004) 173–178
bone to act as a sink for lactate because as lactate moves into bone ECF, it complexes with bone calcium according to the Ko and this permits further lactate entry, and so forth. The in vivo kinetics of this exchange are not known but at 10 ◦ C periodic sampling of shell revealed that shell and plasma were close to being equilibrated throughout the 10 days of anoxia as well as during 10 days of recovery when lactate levels returned to normal (Jackson, 1997). The shell is well perfused with blood, even during anoxia (Stecyk et al., 2004) so the opportunity for effective exchange exists. Mineralized structures of other organisms, including vertebrates and invertebrates, also accumulate lactate during periods of anaerobiosis and presumably this also involves complexing between calcium and lactate (Jackson et al., 2001, 2003). The behavior of turtle bone in this regard is, therefore, not unique but its quantitative significance, due to the large mass of bone and the high concentrations of lactate involved, greatly exceeds that of other organisms studied.
5. Calcium lactate formation and the distribution of lactate The first step in the distribution of lactate is its transport out of cells. In most vertebrate cells studied, this is mainly via a monocarboxylate transporter (MCT) that co-transports lactate and proton. An MCT has recently been found to account for most membrane transport in turtle erythrocytes (Warren and Jackson, 2003) and probably plays the same role in other cells of this animal. Previous studies have shown that the equilibrium state for lactate exchange reflects the pH gradient across the membrane, so that if the cell is at lower pH than the ECF, extracellular lactate will exceed intracellular lactate and [lactate]0 /[lactate]i will equal [H+ ]i /[H+ ]0 (Poole and Halestrap, 1993). In turtle skeletal muscle and liver, this pattern of distribution was generally the rule during anoxic submergence at 3 ◦ C (Jackson and Heisler, 1983), and except in the control animals, intracellular lactate was always less than extracellular lactate. The formation of calcium lactate in the extracellular fluid should facilitate this efflux of lactate by reducing the concentrations of “free” lactate. A greater fraction of the cell lactate should, therefore, leave the cells because of the complexing of lactate with extracellular calcium.
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Fig. 4. Diagram of lactate distribution during anoxic submergence. Lactate efflux from cells is facilitated by calcium lactate formation in ECF and by uptake of lactate by shell and skeleton and calcium lactate formation there. The numbers at the bottom indicate the percentage of total body lactate in each compartment following 3 months anoxia at 3 ◦ C (Jackson, 1997).
The movement of extracellular lactate into shell and skeletal bone further promotes the conditions for cellular efflux and in the process shifts a significant fraction of the total body lactate into bone. The transport mechanism for the exchange between plasma and bone is not fully understood, but preliminary evidence (unpublished observations) suggests that an MCT may play a role here as well, although perhaps a minor role. The complexing of lactate within bone should serve to keep the free lactate concentration in bone ECF low to facilitate continued entry of lactate. The overall effect is a shift of lactate out of the cells and into ECF and bone as depicted in Fig. 4, so that after 3–4 months submergence at 3 ◦ C, only 20% of the total body lactate is in the cells, 36% is in the ECF, and 44% is in bone. Bone directly buffers the 44% that enters its substance, and by release of carbonates into the ECF, contributes further by buffering almost two-third of the lactic acid that remained in the ECF.
6. Conclusion The reaction between calcium and lactate is a relatively weak one but elevations of lactate commonly observed in severe exercise or in critically ill patients can contribute significantly to the hypocalcemia commonly observed in these situations. In the anoxic turtle, however, extraordinarily high lactate levels can bind a large fraction of the extracellular calcium, but because
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calcium is also unusually elevated, a significant fraction of the total lactate is also in combined form. This reduction in the “free” lactate contributes to the efflux of lactic acid from the anoxic cells and into the ECF where buffering power is high. In addition, it is proposed that calcium lactate formation accounts for much of the shell and skeletal lactate accumulation observed in the anoxic turtle. The latter reaction may be significant as well in the mineralized tissues of other organisms experiencing lactic acidosis.
Acknowledgements The author’s research is supported by National Science Foundation (USA) Grant IBN 01-10322.
References Aduen, J., Bernstein, W.K., Miller, J., Kerzner, R., Bhatiani, A., Davison, L., Chernow, B., 1995. Relationship between blood lactate concentrations and ionized calcium, glucose, and acid–base status in critically ill and noncritically ill patients. Clin. Care Med. 23, 246–252. Bickler, P.E., 1998. Reduction of NMDA receptor activity in cerebrocortex of turtles (Chrysemys picta) during 6 weeks of anoxia. Am. J. Physiol. 275, R86–R91. Bouassida, A., Zalleg, D., Zaouali Ajina, M., Gharbi, N., Duclos, M., Richalet, J.P., Tabka, Z., 2003. Parathyroid hormone concentrations during and after two periods of high intensity exercise with and without an intervening recovery period. Eur. J. Appl. Physiol. 88, 339–344. Cannan, R.K., Kilbrick, A., 1938. Complex formation between carboxylic acids and divalent metal cations. J. Am. Chem. Soc. 60, 2314–2320. Cooper, D.J., Walley, K.R., Dodek, P.M., Rosenberg, F., Russell, J.A., 1992. Plasma ionized calcium and blood lactate concentrations are inversely associated in human lactic acidosis. Intens. Care Med. 18, 286–289. Ghosh, R., Nair, V.S.K., 1970. Studies of metal complexes in aqueous solution. I. Calcium and copper lactates. J. Inorg. Nucl. Chem. 32, 3025–3032. Hochachka, P.W., Mommsen, T.P., 1983. Protons and anaerobiosis. Science 219, 1391–1397. Immke, D.C., McClesky, E.W., 2001. Lactate enhances the acidsensing Na+ channel on ischemia-sensing neurons. Nat. Neurosci. 4, 869–870. Jackson, D.C., Heisler, N., 1982. Plasma ion balance of submerged anoxic turtles at 3 ◦ C: the role of calcium lactate formation. Respir. Physiol. 49, 159–174. Jackson, D.C., Ultsch, G.R., 1982. Long-term submergence at 3 ◦ C of the turtle, Chrysemys picta bellii, in normoxic and severely
hypoxic water. II. Extracellular ionic responses to extreme lactic acidosis. J. Exp. Biol. 96, 29–43. Jackson, D.C., Heisler, N., 1983. Intracellular and extracellular acid–base and electrolyte status of submerged anoxic turtles at 3 ◦ C. Respir. Physiol. 53, 187–201. Jackson, D.C., Toney, V.I., Okamoto, S., 1996. Lactate distribution and metabolism during and after anoxia in the turtle, Chrysemys picta bellii. Am. J. Physiol. 271, R409–R416. Jackson, D.C., 1997. Lactate accumulation in the shell of the turtle, Chrysemys picta bellii, during anoxia at 3 and 10 ◦ C. J. Exp. Biol. 200, 2295–2300. Jackson, D.C., Goldberger, Z., Visuri, S., Armstrong, R.N., 1999. Ionic exchanges of turtle shell in vitro and their relevance to shell function in the anoxic turtle. J. Exp. Biol. 202, 513–520. Jackson, D.C., Crocker, C.E., Ultsch, G.R., 2000. Bone and shell contribution to lactic acid buffering of submerged turtles Chrysemys picta bellii at 3 ◦ C. Am. J. Physiol. 278, R1564–R1571. Jackson, D.C., 2000. How a turtle’s shell helps it survive prolonged anoxic acidosis. News Physiol. Sci. 15, 181–185. Jackson, D.C., Wang, T., Koldkjaer, P., Taylor, E.W., 2001. Lactate sequestration in the carapace of the crayfish Austropotamobius pallipes during exposure in air. J. Exp. Biol. 204, 941–946. Jackson, D.C., 2002. Hibernating without oxygen: physiological adaptations of the painted turtle. J. Physiol. London 543 (3), 731–737. Jackson, D.C., Andrade, D.V., Abe, A.S., 2003. Lactate sequestration by osteoderms of the broad-nose caiman, Caiman latirostris, following capture and forced submergence. J. Exp. Biol. 206, 3601–3606. Martell, A.E., Smith, R.M., 1977. Critical Sability Constants. In: Other Organic Ligands, vol. 3. Plenum Press, New York, p. 28. Poole, R.C., Halestrap, A.P., 1993. Transport of lactate and other monocarboxylates across mammalian plasma membranes. Am. J. Physiol. 264, C761–C782. Stecyk, J.A.W., Overgaard, J., Farrell, A.P., Wang, T., 2004. ␣Adrenergic regulation of systemic peripheral resistance and blood flow distribution in the turtle Trachemys scripta during anoxic submergence at 5 and 21 ◦ C. J. Exp. Biol. 207, 269–283. Ultsch, G.R., Jackson, D.C., 1982. 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. Van Waarde, A., van den Thillart, G., Verhagen, M., 1993. Ethanol formation and pH-regulation in fish. In: Hochachka, P.W., Lutz, P.L., Sick, T., Rosenthal, M., van den Thillart, G. (Eds.), Surviving Hypoxia: Mechanisms of Control and Adaptation. CRC Press, Boca Raton, pp. 157–170. Warburton, S.J., Jackson, D.C., 1995. Turtle (Chrysemys picta bellii) shell mineral content is altered by exposure to prolonged anoxia. Physiol. Zool. 68, 783–798. Warren, D.E., Jackson, D.C., 2003. Lactate transport in turtle erythrocytes. FASEB J. 17, A423. Williams, R.J.P., 1977. Calcium chemistry and its relation to calcium binding. In: Wasserman, R.H., Corradino, R.A., Carafoli, E., Kretsinger, R.H., MacLennan, D.H., Siefal, F.L. (Eds.), Calcium Binding Proteins and Calcium Function. North Holland, New York.
Surviving extreme lactic acidosis: the role of calcium lactate formation in the anoxic turtle Donald C. Jackson∗ Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, RI 02912, USA Accepted 28 June 2004
Abstract During prolonged anoxia at low temperature, freshwater turtles develop high plasma concentrations of both lactate and calcium. At these concentrations the formation of the complex, calcium lactate, normally of little biological significance because of the low association constant for the reaction, significantly reduces the free concentrations of both lactate and calcium. In addition, lactate is taken up by the shell and skeleton to an extent that strongly indicates that calcium lactate formation participates in these structures as well. The binding of calcium to lactate thus contributes to the efflux of lactic acid from the anoxic cells and to the exploitation of the powerful buffering capacity of the shell and skeleton. © 2004 Elsevier B.V. All rights reserved. Keywords: Acid base, buffering, bone; Anoxia, prolonged, survival; Reptiles, turtle; Shell, turtle
1. Introduction The absence of oxygen presents critical challenges to vertebrate organisms because all are ultimately obligate aerobes. Endothermic birds and mammals are particularly vulnerable because of high metabolic requirements of their tissues and poor tolerance of their brains and hearts to even brief periods of anoxia. Ectothermic vertebrates are more resistant and most can probably survive relatively brief exposures to anoxia. Only a few, however, can remain anoxic for very long periods. These facultative anaerobes include freshwater turtles and several species of fish and the ∗
Tel.: +1 401 863 2373; fax: +1 401 863 3352. E-mail address: donald [email protected].
1569-9048/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2004.06.020
characteristics that permit their unusual tolerance is of some interest. A major factor limiting duration of anoxia is accumulation of acid end-products and their effect on acid–base balance. Fish such as crucian carp and goldfish avoid this problem by substituting ethanol, an acid–base neutral compound, and CO2 as principle waste products, both of which are readily excreted via the gills (Van Waarde et al., 1993). Turtles, however, like other vertebrates, rely on anaerobic glycolysis for energy production and this leads to lactate and protons as end-products (Hochachka and Mommsen, 1983). They must therefore cope with the metabolic acidosis problem. Plasma lactate concentrations in freshwater turtles, such as the painted turtle, Chrysemys picta, subjected to
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D.C. Jackson / Respiratory Physiology & Neurobiology 144 (2004) 173–178
cold anoxic submergence can reach very high levels, as high as 150–200 mmol l−1 after several months (Ultsch and Jackson, 1982; Jackson et al., 2000). The turtle’s ability to manage this large acid load and remain in a viable acid–base state is a key factor that explains how they live so long without O2 . Shell and skeletal buffers play a major role as has been described in recent reviews (Jackson, 2000, 2002) and, as will be described in this paper, the formation of a complex between calcium and lactate is an integral part of this mechanism.
2. Calcium-binding molecules Calcium is a very reactive ion that binds reversibly to many molecules. Within cells calcium ligands play an important role in regulating the concentration of ionized calcium, the biologically active form. In the plasma, proteins, in particular albumen, are normally the principal ligands. Other calcium-binding molecules, such as EDTA, are valuable laboratory tools for regulating calcium activity in experimental solutions. The strength of the combination between calcium and a ligand is described by the association constant, Ko: Ko (l mol−1 ) =
[CaLigand] [Ca2+ ][ligand]
The values of Ko for organic molecules (Table 1) vary over an extremely wide range. Because a higher value of Ko indicates more avid binding with calcium, the biological importance of a molecule as a calcium ligand is a function of the value of Ko for that molecule. It is notable that the Ko value for lactate is particularly low compared to the other substances shown. The relevant equilibrium reaction for calcium and lactate is as follows:
Fig. 1. Measured association constants, Ko, for the reaction between calcium and lactate at different temperatures. Data from Jackson and Heisler (1982) and unpublished observations.
And the association constant is defined as Ko =
[Ca Lactate+ ] [Ca2+ ][Lactate− ]
Using prepared solutions and a calcium electrode, Ko for this reaction was measured at 3 ◦ C (Jackson and Heisler, 1982) and subsequently at a series of higher temperatures (unpublished observations) (Fig. 1). These values are in general agreement with other published measurements of this constant (Cannan and Kilbrick, 1938; Ghosh and Nair, 1970; Martell and Smith, 1977), but are well below the value of 100 l mol−1 that Williams (1977) defined as the lower limit a reaction must have to achieve biological importance. In the anoxic turtle, however, in contradiction to the statement by Williams, the significance of this reaction is of unquestioned biological importance.
3. Calcium lactate formation in body fluids
Ca2+ + Lactate− ↔ Ca Lactate+ Table 1 Association constants (Ko) for selected calcium ligands Molecule
Ko (l mol−1 )
EDTA Citrate Malonate Succinate Lactate
5 × 1010 6 × 104 316 100 ∼10–20
In the intracellular compartment, ionized calcium concentration is so low (∼10−7 M) and other calciumbinding molecules are so dominant, that even high intracellular concentrations of lactate are unlikely to have any significant effect on calcium activity, and of course the calcium is far too low in concentration to have any effect on lactate. Likewise, in the extracellular fluid, at usual concentrations of calcium and lactate, calcium lactate formation is also negligible. For example, at
D.C. Jackson / Respiratory Physiology & Neurobiology 144 (2004) 173–178
a plasma lactate of 1 mmol l−1 and ionized calcium of 1.3 mmol l−1 , the concentration of calcium lactate would be about 0.04 mmol l−1 , only about 3% of the total combined calcium, assuming a Ko of 11.7 l mol−1 (Martell and Smith, 1977). This means that under normal conditions the assertion of Williams (1977) referred to above is certainly valid. For calcium lactate formation to bind a significant fraction of either extracellular calcium or lactate, the concentration of one or both of these ions must be abnormally high. Hypercalcemia of sufficient magnitude would be very unusual because calcium concentration is generally regulated within narrow limits. Lactate, on the other hand, can increase markedly in concentration in a variety of states, such as strenuous exercise and breathhold diving, or in various disease states affecting tissue O2 delivery. At plasma lactate concentrations that are commonly observed, in the range of 15–25 mmol l−1 , the Ko for calcium lactate formation predicts that lactate-bound [calcium] would be 20–30% of free [calcium]. This reaction could, therefore, contribute to the hypocalcemia that is a common response during vigorous exercise and in critically ill patients, both conditions in which elevated lactate levels can occur. The role of calcium lactate formation in critically ill patients has been considered but is controversial (Cooper et al., 1992; Aduen et al., 1995). Proponents for this reaction playing a role (Cooper et al., 1992) observed a clear inverse relationship between lactate and ionized calcium (Fig. 2), and simple calculations for the
Fig. 2. Relationship between plasma ionized calcium and lactate concentrations of critically ill patients. Modified from Cooper et al. (1992). Dashed line depicts calculated contribution of calcium lactate formation to the reduction in ionized calcium assuming a Ko value of 11.7 l mol−1 .
175
equilibrium reaction confirm a significant contribution from the calcium lactate reaction. The relatively minor contribution of this reaction to the observed relationship, however, and the complexity of other factors influencing ionized calcium in diverse patient populations probably accounts for the negative findings of the other authors (e.g., Aduen et al., 1995). Similarly, hypocalcemia during strenuous exercise with elevated lactate must be at least partially due to calcium lactate formation. In a study of high intensity exercise lasting 42 min, Bouassida et al. (2003) observed a reciprocal relationship between lactate and ionized calcium during and following exercise. The authors attributed the fall in ionized calcium during exercise to renal excretion but over 25% can be predicted from the calcium lactate reaction. Peak lactates were only about 8 mmol l−1 in this study. Lactate may also increase locally in ischemic tissues and exert important effects via calcium-binding. A recent study (Immke and McClesky, 2001) of sensory nerves innervating the heart (in rats) found that a local increase in lactate concentration to 15 mmol l−1 increased the activity of an acid-sensitive ion channel by lowering ionized calcium concentration from 2.0 to 1.71 mmol l−1 . This modest hypocalcemia increased depolarization of these nerves which are thought to mediate anginal chest pain. Two factors make the calcium lactate reaction substantially more significant in the anoxic turtle than in other physiological systems. First, as noted earlier, plasma lactate concentration can rise to very high levels. The second unusual feature of the anoxic turtle is that extracellular concentration of calcium can also be greatly elevated to as high as 50 mmol l−1 (Jackson and Ultsch, 1982). The source of lactate is anaerobic glycolysis, and the source of calcium is mobilization from shell and skeleton together with carbonate for extracellular buffering (Jackson et al., 1999). Hypercalcemia results because renal function, the major means of calcium regulation during bone calcium mobilization, is severely curtailed during anoxia (Warburton and Jackson, 1995). The turtle is operated essentially as a closed system with respect to calcium and lactate, and these solutes are retained and build up within the body fluids, although intracellular ionized calcium is kept in the sub-micromolar range (Bickler, 1998). Because of the elevated plasma concentrations, the reaction between calcium and lactate is very significant in the extracellular compartment. Direct measurements
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at 3 ◦ C, on simple electrolyte solutions and on plasma from anoxic turtles, revealed that more than two-thirds of the total calcium was complexed with lactate when total [lactate] was 145 mmol l−1 and total [calcium] ranged from 34 to 40 mmol l−1 (Jackson and Heisler, 1982). In addition, “free” lactate was calculated to be 115 mmol l−1 and ionized calcium was measured at 12.5 mmol l−1 . These values accord with the measured Ko value for this reaction at 3 ◦ C (∼20 l mol−1 ). Some lactate is also complexed with magnesium which also increases significantly in plasma although the association constant for this reaction is somewhat lower than for calcium lactate. A clear benefit of extracellular calcium lactate formation for the anoxic turtle is that it minimizes the increase in ionized calcium, although even with the lactate binding the animal is still severely hypercalcemic. Another less obvious benefit is the lower “free” lactate concentration in the ECF. Low lactate activity facilitates movement of lactic acid out of cells where it is produced and into the ECF where most of the buffering can occur. Endogenous extracellular buffering is already high in the painted turtle by vertebrate standards due to [HCO3 − ] concentration of about 40 mmol l−1 . In addition, extracellular buffering is supplemented by transfer of carbonates, mainly calcium and magnesium carbonates, from shell and skeleton.
4. Calcium lactate formation in bone In the anoxic turtle, a significant fraction (up to 45%) of the total body lactate resides in the shell and skeleton at the end of prolonged anoxic submergences at 3 and 10 ◦ C (Jackson, 1997). At 3 ◦ C, for example, after 4 months of anoxia when plasma lactate was 155 mmol l−1 , shell lactate was about 131 mmol (kg wet weight)−1 and long bone lactate was about 153.5 mmol (kg wet weight)−1 . Although it is not certain what the physical state of lactate is in mineralized tissues, it is likely that a large fraction of this lactate is combined with calcium. The basis for this assumption is two-fold. First, the volume of water into which the lactate could be dissolved in bone is small, and second, calcium is abundant in bone and much of this calcium may be available for binding lactate. Painted turtle shell is about 32% water by weight (Jackson, 1997) but only half or less of this water is
Fig. 3. Estimated concentrations of lactate in intracellular fluid (ICF) in mmol (kg H2 O)−1 , extracellular fluid (ECF) in mmol l−1 , and bone/shell in mmol (kg wet weight)−1 , following 3 months of anoxic submergence at 3 ◦ C. Solid bar represents “free” lactate, open bar represents calcium bound lactate. Shell values assume that all bone water is equilibrated with extracellular lactate. Data adapted from Jackson and Heisler (1982), Jackson and Heisler (1983), and Jackson (1997).
accessible to the circulating extracellular marker PEG (Jackson et al., 1996). Assuming that shell ECF equilibrates with plasma [lactate], and this is the only lactate in the shell, shell lactate concentration at a plasma concentration of 155 mmol l−1 would be only about 25 mmol (kg wet weight)−1 . Even if all the water in the shell were accessible to lactate, this would only increase the shell lactate to about 50 mmol (kg wet weight)−1 , still only 38% of the measured concentration. This strongly suggests that a major fraction of the shell and skeletal lactate exists in combined form. In contrast, in the ECF after long-term anoxia, about 20% of the lactate is in combined form, and in the ICF little if any lactate is in this form because of low intracellular calcium (Fig. 3). The abundance of calcium in the shell and skeleton and its ability to combine reversibly with lactate makes this ion the most likely candidate for lactate sequestration. Over 99% of the total body calcium is found in these structures, and in the painted turtle as much as 20% of this calcium may be associated with carbonate. It should be noted, however, that magnesium also forms a complex with lactate, and this reaction could also contribute to the observed uptake of lactate by bone. Our working hypothesis is that lactic acid enters bone (shell or skeleton) whereupon bone carbonate buffers the protons and calcium (and possibly magnesium) combines with lactate (Jackson et al., 1999). This reaction enables
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bone to act as a sink for lactate because as lactate moves into bone ECF, it complexes with bone calcium according to the Ko and this permits further lactate entry, and so forth. The in vivo kinetics of this exchange are not known but at 10 ◦ C periodic sampling of shell revealed that shell and plasma were close to being equilibrated throughout the 10 days of anoxia as well as during 10 days of recovery when lactate levels returned to normal (Jackson, 1997). The shell is well perfused with blood, even during anoxia (Stecyk et al., 2004) so the opportunity for effective exchange exists. Mineralized structures of other organisms, including vertebrates and invertebrates, also accumulate lactate during periods of anaerobiosis and presumably this also involves complexing between calcium and lactate (Jackson et al., 2001, 2003). The behavior of turtle bone in this regard is, therefore, not unique but its quantitative significance, due to the large mass of bone and the high concentrations of lactate involved, greatly exceeds that of other organisms studied.
5. Calcium lactate formation and the distribution of lactate The first step in the distribution of lactate is its transport out of cells. In most vertebrate cells studied, this is mainly via a monocarboxylate transporter (MCT) that co-transports lactate and proton. An MCT has recently been found to account for most membrane transport in turtle erythrocytes (Warren and Jackson, 2003) and probably plays the same role in other cells of this animal. Previous studies have shown that the equilibrium state for lactate exchange reflects the pH gradient across the membrane, so that if the cell is at lower pH than the ECF, extracellular lactate will exceed intracellular lactate and [lactate]0 /[lactate]i will equal [H+ ]i /[H+ ]0 (Poole and Halestrap, 1993). In turtle skeletal muscle and liver, this pattern of distribution was generally the rule during anoxic submergence at 3 ◦ C (Jackson and Heisler, 1983), and except in the control animals, intracellular lactate was always less than extracellular lactate. The formation of calcium lactate in the extracellular fluid should facilitate this efflux of lactate by reducing the concentrations of “free” lactate. A greater fraction of the cell lactate should, therefore, leave the cells because of the complexing of lactate with extracellular calcium.
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Fig. 4. Diagram of lactate distribution during anoxic submergence. Lactate efflux from cells is facilitated by calcium lactate formation in ECF and by uptake of lactate by shell and skeleton and calcium lactate formation there. The numbers at the bottom indicate the percentage of total body lactate in each compartment following 3 months anoxia at 3 ◦ C (Jackson, 1997).
The movement of extracellular lactate into shell and skeletal bone further promotes the conditions for cellular efflux and in the process shifts a significant fraction of the total body lactate into bone. The transport mechanism for the exchange between plasma and bone is not fully understood, but preliminary evidence (unpublished observations) suggests that an MCT may play a role here as well, although perhaps a minor role. The complexing of lactate within bone should serve to keep the free lactate concentration in bone ECF low to facilitate continued entry of lactate. The overall effect is a shift of lactate out of the cells and into ECF and bone as depicted in Fig. 4, so that after 3–4 months submergence at 3 ◦ C, only 20% of the total body lactate is in the cells, 36% is in the ECF, and 44% is in bone. Bone directly buffers the 44% that enters its substance, and by release of carbonates into the ECF, contributes further by buffering almost two-third of the lactic acid that remained in the ECF.
6. Conclusion The reaction between calcium and lactate is a relatively weak one but elevations of lactate commonly observed in severe exercise or in critically ill patients can contribute significantly to the hypocalcemia commonly observed in these situations. In the anoxic turtle, however, extraordinarily high lactate levels can bind a large fraction of the extracellular calcium, but because
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calcium is also unusually elevated, a significant fraction of the total lactate is also in combined form. This reduction in the “free” lactate contributes to the efflux of lactic acid from the anoxic cells and into the ECF where buffering power is high. In addition, it is proposed that calcium lactate formation accounts for much of the shell and skeletal lactate accumulation observed in the anoxic turtle. The latter reaction may be significant as well in the mineralized tissues of other organisms experiencing lactic acidosis.
Acknowledgements The author’s research is supported by National Science Foundation (USA) Grant IBN 01-10322.
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