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Succinate and alanine as anaerobic end-products in the diving turtle (Chrysemys picta bellii)
Comparative Biochemistry and Physiology Part B 126 (2000) 409 – 413 www.elsevier.com/locate/cbpb
Succinate and alanine as anaerobic end-products in the diving turtle (Chrysemys picta bellii ) L.T. Buck * Department of Zoology, Uni6ersity of Toronto, 25 Harbord Street, Toronto, Ont., Canada, M5S 3G5 Received 12 November 1999; received in revised form 3 March 2000; accepted 20 March 2000
Abstract The western painted turtle is an extremely anoxia-tolerant vertebrate capable of tolerating blood lactate levels of 150–200 mM. Since lactate increases to such high levels, other fermentation end-products such as succinate and alanine, which have not been previously measured in this species, might also be expected to increase. Therefore, I measured turtle heart, liver, and blood concentrations of lactate, succinate, and alanine following a 28-day anoxic dive at 5°C. Succinate and lactate concentrations increased significantly in all three compartments while alanine increased significantly in the liver only. Lactate was found to accumulate by a similar amount in all three compartments (66.4 – 80.5 mmol g − 1 or ml − 1 in the blood compartment) and was used as a reference to which alanine and succinate concentrations could be compared. Succinate and alanine levels increased by 2 and 0.9% of lactate in liver, approximately 0.3 and 0.04% of lactate in blood, and 0.6 and 0.07% of lactate in heart, respectively. The contribution of each to the total anoxic heat production was calculated and accounted for an additional 1.5% of the previously measured exothermic gap. I conclude that succinate and alanine concentrations do increase in the anoxic turtle but are minor anaerobic end-products. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Lactate; Alanine; Fumarate reductase; Heart; Liver; Blood; Exothermic gap; Anaerobiosis; Chrysemys picta bellii
1. Introduction The pathway of glycogen fermentation to succinate has been well described in invertebrate tissue (Holwerda and de Zwaan, 1979; Hochachka and Somero, 1984). It has also been demonstrated to occur in rat liver (Hoberman and Prosky, 1967) and Taetmeyer (1978) clearly showed the pathway to be present in rabbit papillary muscle. This pathway essentially couples two mitochondrial energy-yielding reactions to those
* Tel.: +1-416-9783506; fax: +1-416-9788532. E-mail address: [email protected] (L.T. Buck)
of glycolysis, resulting in an additional 2 ATP and a partial mitochondrial proton gradient. The proton gradient is generated via the reversal of succinate dehydrogenase to a fumarate reductase with fumarate becoming the terminal electron acceptor and NADH dehydrogenase the proton pump. The functional meaning of this pathway is assumed to be additional energy equivalents and maintenance of a partial mitochondrial membrane gradient. Interestingly, succinate dehydrogenase and fumarate reductase have recently been shown to be two separate enzymes species, capable of differential expression depending on oxygen availability (Tielens and van Hellemond, 1998).
0305-0491/00/$ - see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 3 0 5 - 0 4 9 1 ( 0 0 ) 0 0 2 1 5 - 7
410
L.T. Buck / Comparati6e Biochemistry and Physiology, Part B 126 (2000) 409–413
The western painted turtle (Chrysemys picta bellii ) has been shown to undergo long periods of anoxia, surviving up to 6 months submerged in N2-bubbled water at 3°C and tolerating blood lactate concentrations of 150 – 200 mM (Jackson and Ultsch, 1982; Ultsch and Jackson, 1982; Herbert and Jackson, 1985). In such an anoxia-tolerant species, where blood lactate increases so dramatically, one would expect other metabolic end-products such as succinate and alanine to increase. Hochachka et al. (1975) have examined several diving vertebrates with regard to the anaerobic accumulation of succinate and found that succinate accumulates to a level one-hundredth that of lactate concentrations. This work was based on blood concentrations and as pointed out by the authors may not reflect true tissue concentrations, especially since succinate is not normally permeable to cell membranes and dive durations were relatively short (2 h). Succinate accumulation has also been investigated in the heart and liver of Pseudemys scripta elegans following an anoxic 24-h dive at 22°C (Penney, 1974). Succinate was found to accumulate in the liver but not in the heart. However, the issue as to whether succinate is an important end-product is still inconclusive since in the above study it was not detectable in the aerobic controls (in either liver or heart) and it appears that succinate was successfully measured in only one anoxic animal. Therefore, the importance of the fumarate reductase pathway to long-term anoxic survival is unknown. In this study, the importance of the fumarate reductase pathway to anoxic survival of C. picta bellii is re-evaluated by measuring liver, heart, and blood concentrations of succinate, alanine, and lactate from long-term anoxic turtles. From the measurement of these compounds, an estimate of their respective contributions to anoxic heat flux can be made. This estimate will help explain the large exothermic gap previously observed in turtle hepatocytes (Buck et al., 1993a).
2. Materials and methods These studies were approved by the University Animal Care and Welfare committee and conform to relevant guidelines for the care of experimental animals. Liver, heart, and plasma metabolite samples were obtained from turtles dived in N2 bub-
bled water for 28 days at 5°C as described in (Keiver et al., 1992). Briefly, turtles were obtained in the fall from the supplier (William A. Lemberger Co. Inc, Oshkosk, WI) and held at 15°C in shallow tanks. Temperature was decreased 5°C every 2 days until the experimental temperature of 5°C was reached. Ten turtles were divided into two groups of five, both at 5°C, an anoxic group was placed in wire mesh cages and dived in N2 bubbled water and a control normoxic group placed in a shallow tank with approximately 5 cm of water. The PO2 of the N2-bubbled water was monitored before the experiment, every second day for the first week, and then once weekly to ensure it was 0 torr (or not different from dithionite control). After 28 days the turtles were quickly decapitated by clamping their neck and cutting anterior to the clamp. The plastron was removed with an electric bone saw and tissue samples freeze-clamped with tongs cooled in liquid N2 and blood samples frozen in liquid N2. Samples were stored at − 80°C until assays were performed. Metabolite concentrations were determined in liver, heart, and plasma samples using coupled enzyme assays (Bergmeyer, 1974); for alanine — a glutamate-pyruvate transaminase/lactate dehydrogenase (LDH) linked assay at pH 7.6; for lactate — a LDH/glutamate dehydrogenase linked assay at pH 8.9, and for succinate — a succinyl thiokinase/pyruvate kinase/LDH linked assay. Tissue samples were pulverized in liquid N2 and frozen pieces rapidly placed in a pre-weighed 1.5-ml Eppendorf tube for weight determination. Samples were then immediately acid extracted in 5 vol. 7% perchloric acid, homogenized with an Ultra Turrax homogenizer (Tekmar, Cincinnati, OH.), sonicated 20 s each (Kontes Cell Disrupter, Vineland, NJ), and centrifuged for 10 min at 12 000×g at 4°C. The supernatant was then removed and neutralized with 3 M K2CO3; following a final centrifugation to remove the precipitate (as above), the supernatants were frozen at − 80°C until assay. Frozen plasma samples were assayed directly. All assays were performed with a Perkin-Elmer Lambda 2 UV/Vis spectrophotometer and analyzed with Perkin-Elmer computer spectroscopy software (vers. 3.2). All chemicals were obtained from Sigma (St Louis, MO). Statistics were performed using SYSTAT Version 5 software (Evanston, IL). Data are presented as the mean9 S.E.
L.T. Buck / Comparati6e Biochemistry and Physiology, Part B 126 (2000) 409–413
3. Results Succinate, alanine, and lactate concentrations from liver, heart and blood compartments of control animals were compared to those from animals following a 28-day anoxic dive (Fig. 1). Succinate concentrations increased significantly in all three compartments; liver 0.2490.03 to 1.589 0.12, heart 0.219 0.04 to 0.70 90.12, and blood 0.02 9 0.004 to 0.25 90.01. The control blood succinate concentration was below the suggested detection limit of the assay. All succinate assays were made against a blank containing all reagents except succinyl thiokinase, and a drift-corrected difference was detectable. However, no drift was detectable with any other combination of reagents, coupling enzymes, or sample. Lactate concentra-
Fig. 1. Lactate, succinate, and alanine concentrations in the liver, heart, and blood of control and turtles dived for 28 days in 5°C anoxic water. Values represent the mean and standard error of 4 – 5 measurements each. Asterisks represent values significantly different from corresponding control value (Students t-test P B 0.05). Note lactate concentrations in the three compartments following a 28-day anoxic dive are not significantly different (Tukey’s HSD PB0.05, and assuming 1 ml plasma is approximately 1 g).
411
tions increased significantly in all three compartments; liver 2.659 0.27 to 69.09 7.13, heart 1.609 0.23 to 83.79 5.91, and blood 7.549 1.12 to 88.09 9.79. The only significant increase in alanine concentration was found in the liver.
4. Discussion The ratio of the absolute concentration change of succinate to alanine in the liver is approximately 2:1; that is, a ratio which would be expected from an active fumarate reductase pathway (Hochachka et al. 1975). Heart undergoes a threefold increase in succinate concentration and a small but insignificant change in alanine concentration. One would expect a clear relationship in a tissue that typically has twice the mitochondrial volume density and cytochrome oxidase activity of liver (Else and Hulbert, 1981); this may be due to differential metabolite washout as emphasized by Hochachka et al. (1975). In these experiments the degree to which succinate or alanine enters the much larger blood compartment is unknown; however, an approximation based on concentrations relative to lactate can be made (since there is no significant difference in lactate concentrations among the compartments, Tukey’s HSD PB 0.05). In the blood and heart compartments the change in [succinate] is 0.3 and 0.6% that of lactate, respectively; and alanine accumulates to 0.04 and 0.07%, respectively. In the liver, succinate and alanine accumulated to their highest levels, 2 and 0.9% of the lactate concentration, respectively. Thus, it appears that a greater fraction of the succinate and alanine pools are retained by the liver than by the heart. These data could be interpreted to indicate that the succinate and alanine concentrations in the heart are underestimated, and that tissue washout is greater in this tissue (also, partially due to being a more highly perfused tissue than liver). The relative percentage values obtained for succinate are similar to those obtained by Hochachka et al. (1975) in blood samples (1% of lactate) from the sea turtle, sea lion, seal, and porpoise, and of Penney (1974) from turtle liver (3% of lactate). Regardless of the uncertainty about the absolute tissue succinate levels, it is clear from the relative comparison that succinate is not a major anaerobic end-product, but could fulfil a mitochondrial maintenance function in the absence of oxygen.
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L.T. Buck / Comparati6e Biochemistry and Physiology, Part B 126 (2000) 409–413
Table 1 Estimated contribution of succinate and alanine to the total heat production of turtle hepatocytes
Metabolite flux (mmol h−1 g−1) Caloric equivalent (kJ mol−1) Heat flux (mW g−1) Contribution to total heat production (%)c
Lactate
Succinate
Alanine
Glucose
4.2a −77.7a −93 36
0.084 −136 to −152b −3.2 to −3.5 1.2–1.3
0.038 −32 to −40b −0.34 to −0.42 0.13–0.16
17.1a −3.1b −14.7 6
a
Values from Buck et al. (1993a). Values from Gnaiger (1983). c Percentage based on heat production of anoxic hepatocytes of 260 mW g−1 at 25°C, from Buck et al. (1993a). b
Rotermund and Demos (1990) have observed, from ultrastructural studies, mitochondrial swelling in the hearts from P. scripta following a 2-week anoxic dive at 5°C. While not providing evidence for anoxic mitochondrial metabolism, this observation is consistent with a decrease in mitochondrial membrane gradients. Therefore, a partial mitochondrial gradient maintained by fumarate reductase activity could cause mitochondrial swelling and could permit a more rapid transition to oxidative metabolism when conditions allow. Relatively few studies of anaerobic metabolism in anoxia-tolerant species have found a good correlation between direct and indirect calorimetric measurements of heat production (Hardewig et al., 1991; Schmidt et al., 1996). Predominantly direct measures give higher values for heat production than indirect measures. This discrepancy has been termed the ‘exothermic gap’ and has largely been ascribed to unaccounted for sources of heat production, such as undescribed metabolic end-products or pH buffering reactions. An exothermic gap has been described in the anoxiatolerant turtle hepatocyte preparation by Buck et al. (1993b) and it was postulated that alternate anaerobic end-products were the source of the missing heat production. Ethanol (crucian carp) and succinate are the only known alternative anaerobic end-products (besides lactate) known to occur in vertebrates. Succinate is the logical alternative anaerobic end-product in the turtle since ethanol would accumulate in and be toxic without a gill to excrete it. In the present study the contribution succinate makes to the total anaerobic heat production can be estimated if the percentage increase of succinate and alanine relative to lactate during anoxia are assumed to reflect accurately their tissue concentrations. Then these values can be used to determine an anoxic rate of
production and an estimation of the heat produced by their accumulation in turtle hepatocytes. The total heat flux from anoxic hepatocytes at 25°C has been previously measured to be 260 mW g − 1 and the contribution of lactate production and glycogen hydrolysis was show to comprise 42% of this amount, or 109.2 mW g − 1 (Buck et al., 1993b). Shown in Table 1 are the calculated anoxic rates of succinate and alanine accumulation and their estimated contribution to the total anoxic heat flux. Using caloric equivalents of − 136 and −93 kJ mol − 1 for succinate and alanine, respectively (Gnaiger, 1983; Gnaiger and Kemp, 1990); a combined additional 1.46% or 3.8 mW g − 1 of the total heat production can be accounted for. In conclusion, succinate and alanine are minor anaerobic end-products in the anoxic turtle and the previously observed exothermic gap in anoxic turtle hepatocytes cannot be accounted for by their accumulation. References Bergmeyer, H.U., 1974. Methods in Enzymatic Analysis. Academic Press, New York. Buck, L.T., Hochachka, P.W., Scho¨n, A., Gnaiger, E., 1993a. Microcalorimetric measurement of reversible metabolic suppression induced by anoxia in isolated hepatocytes. Am. J. Physiol. 265 (34), R1014 – R1019. Buck, L.T., Land, S.C., Hochachka, P.W., 1993b. Anoxia tolerant hepatocytes: a model system for the study of metabolic suppression. Am. J. Physiol. 265 (34), R49 – R56. Else, P.L., Hulbert, A.J., 1981. Comparison of the ‘mammal machine’ and the ‘reptile machine’: energy production. Am. J. Physiol. 240, R3 – R9. Gnaiger, E., 1983. Heat dissipation and energetic efficiency in animal anoxibiosis: economy contra power. J. Exp. Zool. 228, 471 – 490.
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Gnaiger, E., Kemp, R.B., 1990. Anaerobic metabolism in aerobic mammalian cells. Information from the ratio of calorimetric heat flux and respirometric oxygen flux. Biochim. Biophys. Acta 1016, 328– 332. Hardewig, I., Addink, A.D.F., Grieshaber, M.K., Portner, H.O., Van Den Thillart, G., 1991. Metabolic rates at different oxygen levels determined by direct and indirect calorimetry in the oxyconformer Sipunculus nudus. J. Exp. Biol. 157, 143–160. Herbert, C.V., Jackson, D.C., 1985. Temperature effects on the responses to prolonged submergence in the turtle Chrysemys picta bellii. II. Metabolic rate, blood acid base and ionic changes, and cardiovascular function in aerated and anoxic water. Physiol. Zool. 58 (6), 670–681. Hoberman, J.D., Prosky, L., 1967. The evidence of reduction of fumarate to succinate in perfused rat liver under conditions of reduced O2 tensions. Biochim. Biophys. Acta 148, 392–399. Hochachka, P.W., Somero, G.N., 1984. Biochemical Adaptations. Princeton University Press, Princeton, NJ. Hochachka, P.W., Owen, T.G., Allen, J.F., Whittow, G.C., 1975. Multiple end products of anaerobiosis in diving vertebrates. Comp. Biochem. Physiol. 50B, 17–22. Holwerda, D.A., de Zwaan, A., 1979. Fumarate reductase of Mytilus edulis L. Mar. Biol. Lett. 1, 33–40. 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. Extracel-
.
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lular ionic responses to extreme lactic acidosis. J. Exp. Biol. 96, 29 – 43. Keiver, K.P., Weinberg, J., Hochachka, P.W., 1992. Roles of catecholamines and corticosterone during anoxia and recovery at 5°C in turtles. Am. J. Physiol. 263, R770 – R774. Penney, D.G., 1974. Effects of prolonged diving anoxia on the turtle, Pseudemys scripta elegans. Comp. Biochem. Physiol. 47A, 933 – 941. Rotermund, A.J., Demos, G., 1990. Effects of induced cold torpor and hypoxia upon cytochrome oxidase (COX) activity, phospholipid content, and ultrastructure in cardiac mitochondria of the fresh water turtle, Pseudemys scripta. Physiologist 33(4), A-109, Abstr. 79.9. Schmidt, H., Wichmann, A., Lamprecht, I., Zerbst-Boroffka, I., 1996. Anaerobic metabolism in the leech (Hirudo medicinalis L.): direct and indirect calorimetry during severe hypoxia. J. Comp. Physiol. 166, 205 – 214. Taetmeyer, H., 1978. Metabolic responses to cardiac hypoxia, increased production of succinate by rabbit papillary muscles. Circ. Res. 43 (5), 808 – 815. Tielens, A.G.M., van Hellemond, J.J., 1998. The electron transport chain in anaerobically functioning eukaryotes. Biochim. Biophys. Acta 1365, 71 – 78. 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.
Succinate and alanine as anaerobic end-products in the diving turtle (Chrysemys picta bellii ) L.T. Buck * Department of Zoology, Uni6ersity of Toronto, 25 Harbord Street, Toronto, Ont., Canada, M5S 3G5 Received 12 November 1999; received in revised form 3 March 2000; accepted 20 March 2000
Abstract The western painted turtle is an extremely anoxia-tolerant vertebrate capable of tolerating blood lactate levels of 150–200 mM. Since lactate increases to such high levels, other fermentation end-products such as succinate and alanine, which have not been previously measured in this species, might also be expected to increase. Therefore, I measured turtle heart, liver, and blood concentrations of lactate, succinate, and alanine following a 28-day anoxic dive at 5°C. Succinate and lactate concentrations increased significantly in all three compartments while alanine increased significantly in the liver only. Lactate was found to accumulate by a similar amount in all three compartments (66.4 – 80.5 mmol g − 1 or ml − 1 in the blood compartment) and was used as a reference to which alanine and succinate concentrations could be compared. Succinate and alanine levels increased by 2 and 0.9% of lactate in liver, approximately 0.3 and 0.04% of lactate in blood, and 0.6 and 0.07% of lactate in heart, respectively. The contribution of each to the total anoxic heat production was calculated and accounted for an additional 1.5% of the previously measured exothermic gap. I conclude that succinate and alanine concentrations do increase in the anoxic turtle but are minor anaerobic end-products. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Lactate; Alanine; Fumarate reductase; Heart; Liver; Blood; Exothermic gap; Anaerobiosis; Chrysemys picta bellii
1. Introduction The pathway of glycogen fermentation to succinate has been well described in invertebrate tissue (Holwerda and de Zwaan, 1979; Hochachka and Somero, 1984). It has also been demonstrated to occur in rat liver (Hoberman and Prosky, 1967) and Taetmeyer (1978) clearly showed the pathway to be present in rabbit papillary muscle. This pathway essentially couples two mitochondrial energy-yielding reactions to those
* Tel.: +1-416-9783506; fax: +1-416-9788532. E-mail address: [email protected] (L.T. Buck)
of glycolysis, resulting in an additional 2 ATP and a partial mitochondrial proton gradient. The proton gradient is generated via the reversal of succinate dehydrogenase to a fumarate reductase with fumarate becoming the terminal electron acceptor and NADH dehydrogenase the proton pump. The functional meaning of this pathway is assumed to be additional energy equivalents and maintenance of a partial mitochondrial membrane gradient. Interestingly, succinate dehydrogenase and fumarate reductase have recently been shown to be two separate enzymes species, capable of differential expression depending on oxygen availability (Tielens and van Hellemond, 1998).
0305-0491/00/$ - see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 3 0 5 - 0 4 9 1 ( 0 0 ) 0 0 2 1 5 - 7
410
L.T. Buck / Comparati6e Biochemistry and Physiology, Part B 126 (2000) 409–413
The western painted turtle (Chrysemys picta bellii ) has been shown to undergo long periods of anoxia, surviving up to 6 months submerged in N2-bubbled water at 3°C and tolerating blood lactate concentrations of 150 – 200 mM (Jackson and Ultsch, 1982; Ultsch and Jackson, 1982; Herbert and Jackson, 1985). In such an anoxia-tolerant species, where blood lactate increases so dramatically, one would expect other metabolic end-products such as succinate and alanine to increase. Hochachka et al. (1975) have examined several diving vertebrates with regard to the anaerobic accumulation of succinate and found that succinate accumulates to a level one-hundredth that of lactate concentrations. This work was based on blood concentrations and as pointed out by the authors may not reflect true tissue concentrations, especially since succinate is not normally permeable to cell membranes and dive durations were relatively short (2 h). Succinate accumulation has also been investigated in the heart and liver of Pseudemys scripta elegans following an anoxic 24-h dive at 22°C (Penney, 1974). Succinate was found to accumulate in the liver but not in the heart. However, the issue as to whether succinate is an important end-product is still inconclusive since in the above study it was not detectable in the aerobic controls (in either liver or heart) and it appears that succinate was successfully measured in only one anoxic animal. Therefore, the importance of the fumarate reductase pathway to long-term anoxic survival is unknown. In this study, the importance of the fumarate reductase pathway to anoxic survival of C. picta bellii is re-evaluated by measuring liver, heart, and blood concentrations of succinate, alanine, and lactate from long-term anoxic turtles. From the measurement of these compounds, an estimate of their respective contributions to anoxic heat flux can be made. This estimate will help explain the large exothermic gap previously observed in turtle hepatocytes (Buck et al., 1993a).
2. Materials and methods These studies were approved by the University Animal Care and Welfare committee and conform to relevant guidelines for the care of experimental animals. Liver, heart, and plasma metabolite samples were obtained from turtles dived in N2 bub-
bled water for 28 days at 5°C as described in (Keiver et al., 1992). Briefly, turtles were obtained in the fall from the supplier (William A. Lemberger Co. Inc, Oshkosk, WI) and held at 15°C in shallow tanks. Temperature was decreased 5°C every 2 days until the experimental temperature of 5°C was reached. Ten turtles were divided into two groups of five, both at 5°C, an anoxic group was placed in wire mesh cages and dived in N2 bubbled water and a control normoxic group placed in a shallow tank with approximately 5 cm of water. The PO2 of the N2-bubbled water was monitored before the experiment, every second day for the first week, and then once weekly to ensure it was 0 torr (or not different from dithionite control). After 28 days the turtles were quickly decapitated by clamping their neck and cutting anterior to the clamp. The plastron was removed with an electric bone saw and tissue samples freeze-clamped with tongs cooled in liquid N2 and blood samples frozen in liquid N2. Samples were stored at − 80°C until assays were performed. Metabolite concentrations were determined in liver, heart, and plasma samples using coupled enzyme assays (Bergmeyer, 1974); for alanine — a glutamate-pyruvate transaminase/lactate dehydrogenase (LDH) linked assay at pH 7.6; for lactate — a LDH/glutamate dehydrogenase linked assay at pH 8.9, and for succinate — a succinyl thiokinase/pyruvate kinase/LDH linked assay. Tissue samples were pulverized in liquid N2 and frozen pieces rapidly placed in a pre-weighed 1.5-ml Eppendorf tube for weight determination. Samples were then immediately acid extracted in 5 vol. 7% perchloric acid, homogenized with an Ultra Turrax homogenizer (Tekmar, Cincinnati, OH.), sonicated 20 s each (Kontes Cell Disrupter, Vineland, NJ), and centrifuged for 10 min at 12 000×g at 4°C. The supernatant was then removed and neutralized with 3 M K2CO3; following a final centrifugation to remove the precipitate (as above), the supernatants were frozen at − 80°C until assay. Frozen plasma samples were assayed directly. All assays were performed with a Perkin-Elmer Lambda 2 UV/Vis spectrophotometer and analyzed with Perkin-Elmer computer spectroscopy software (vers. 3.2). All chemicals were obtained from Sigma (St Louis, MO). Statistics were performed using SYSTAT Version 5 software (Evanston, IL). Data are presented as the mean9 S.E.
L.T. Buck / Comparati6e Biochemistry and Physiology, Part B 126 (2000) 409–413
3. Results Succinate, alanine, and lactate concentrations from liver, heart and blood compartments of control animals were compared to those from animals following a 28-day anoxic dive (Fig. 1). Succinate concentrations increased significantly in all three compartments; liver 0.2490.03 to 1.589 0.12, heart 0.219 0.04 to 0.70 90.12, and blood 0.02 9 0.004 to 0.25 90.01. The control blood succinate concentration was below the suggested detection limit of the assay. All succinate assays were made against a blank containing all reagents except succinyl thiokinase, and a drift-corrected difference was detectable. However, no drift was detectable with any other combination of reagents, coupling enzymes, or sample. Lactate concentra-
Fig. 1. Lactate, succinate, and alanine concentrations in the liver, heart, and blood of control and turtles dived for 28 days in 5°C anoxic water. Values represent the mean and standard error of 4 – 5 measurements each. Asterisks represent values significantly different from corresponding control value (Students t-test P B 0.05). Note lactate concentrations in the three compartments following a 28-day anoxic dive are not significantly different (Tukey’s HSD PB0.05, and assuming 1 ml plasma is approximately 1 g).
411
tions increased significantly in all three compartments; liver 2.659 0.27 to 69.09 7.13, heart 1.609 0.23 to 83.79 5.91, and blood 7.549 1.12 to 88.09 9.79. The only significant increase in alanine concentration was found in the liver.
4. Discussion The ratio of the absolute concentration change of succinate to alanine in the liver is approximately 2:1; that is, a ratio which would be expected from an active fumarate reductase pathway (Hochachka et al. 1975). Heart undergoes a threefold increase in succinate concentration and a small but insignificant change in alanine concentration. One would expect a clear relationship in a tissue that typically has twice the mitochondrial volume density and cytochrome oxidase activity of liver (Else and Hulbert, 1981); this may be due to differential metabolite washout as emphasized by Hochachka et al. (1975). In these experiments the degree to which succinate or alanine enters the much larger blood compartment is unknown; however, an approximation based on concentrations relative to lactate can be made (since there is no significant difference in lactate concentrations among the compartments, Tukey’s HSD PB 0.05). In the blood and heart compartments the change in [succinate] is 0.3 and 0.6% that of lactate, respectively; and alanine accumulates to 0.04 and 0.07%, respectively. In the liver, succinate and alanine accumulated to their highest levels, 2 and 0.9% of the lactate concentration, respectively. Thus, it appears that a greater fraction of the succinate and alanine pools are retained by the liver than by the heart. These data could be interpreted to indicate that the succinate and alanine concentrations in the heart are underestimated, and that tissue washout is greater in this tissue (also, partially due to being a more highly perfused tissue than liver). The relative percentage values obtained for succinate are similar to those obtained by Hochachka et al. (1975) in blood samples (1% of lactate) from the sea turtle, sea lion, seal, and porpoise, and of Penney (1974) from turtle liver (3% of lactate). Regardless of the uncertainty about the absolute tissue succinate levels, it is clear from the relative comparison that succinate is not a major anaerobic end-product, but could fulfil a mitochondrial maintenance function in the absence of oxygen.
412
L.T. Buck / Comparati6e Biochemistry and Physiology, Part B 126 (2000) 409–413
Table 1 Estimated contribution of succinate and alanine to the total heat production of turtle hepatocytes
Metabolite flux (mmol h−1 g−1) Caloric equivalent (kJ mol−1) Heat flux (mW g−1) Contribution to total heat production (%)c
Lactate
Succinate
Alanine
Glucose
4.2a −77.7a −93 36
0.084 −136 to −152b −3.2 to −3.5 1.2–1.3
0.038 −32 to −40b −0.34 to −0.42 0.13–0.16
17.1a −3.1b −14.7 6
a
Values from Buck et al. (1993a). Values from Gnaiger (1983). c Percentage based on heat production of anoxic hepatocytes of 260 mW g−1 at 25°C, from Buck et al. (1993a). b
Rotermund and Demos (1990) have observed, from ultrastructural studies, mitochondrial swelling in the hearts from P. scripta following a 2-week anoxic dive at 5°C. While not providing evidence for anoxic mitochondrial metabolism, this observation is consistent with a decrease in mitochondrial membrane gradients. Therefore, a partial mitochondrial gradient maintained by fumarate reductase activity could cause mitochondrial swelling and could permit a more rapid transition to oxidative metabolism when conditions allow. Relatively few studies of anaerobic metabolism in anoxia-tolerant species have found a good correlation between direct and indirect calorimetric measurements of heat production (Hardewig et al., 1991; Schmidt et al., 1996). Predominantly direct measures give higher values for heat production than indirect measures. This discrepancy has been termed the ‘exothermic gap’ and has largely been ascribed to unaccounted for sources of heat production, such as undescribed metabolic end-products or pH buffering reactions. An exothermic gap has been described in the anoxiatolerant turtle hepatocyte preparation by Buck et al. (1993b) and it was postulated that alternate anaerobic end-products were the source of the missing heat production. Ethanol (crucian carp) and succinate are the only known alternative anaerobic end-products (besides lactate) known to occur in vertebrates. Succinate is the logical alternative anaerobic end-product in the turtle since ethanol would accumulate in and be toxic without a gill to excrete it. In the present study the contribution succinate makes to the total anaerobic heat production can be estimated if the percentage increase of succinate and alanine relative to lactate during anoxia are assumed to reflect accurately their tissue concentrations. Then these values can be used to determine an anoxic rate of
production and an estimation of the heat produced by their accumulation in turtle hepatocytes. The total heat flux from anoxic hepatocytes at 25°C has been previously measured to be 260 mW g − 1 and the contribution of lactate production and glycogen hydrolysis was show to comprise 42% of this amount, or 109.2 mW g − 1 (Buck et al., 1993b). Shown in Table 1 are the calculated anoxic rates of succinate and alanine accumulation and their estimated contribution to the total anoxic heat flux. Using caloric equivalents of − 136 and −93 kJ mol − 1 for succinate and alanine, respectively (Gnaiger, 1983; Gnaiger and Kemp, 1990); a combined additional 1.46% or 3.8 mW g − 1 of the total heat production can be accounted for. In conclusion, succinate and alanine are minor anaerobic end-products in the anoxic turtle and the previously observed exothermic gap in anoxic turtle hepatocytes cannot be accounted for by their accumulation. References Bergmeyer, H.U., 1974. Methods in Enzymatic Analysis. Academic Press, New York. Buck, L.T., Hochachka, P.W., Scho¨n, A., Gnaiger, E., 1993a. Microcalorimetric measurement of reversible metabolic suppression induced by anoxia in isolated hepatocytes. Am. J. Physiol. 265 (34), R1014 – R1019. Buck, L.T., Land, S.C., Hochachka, P.W., 1993b. Anoxia tolerant hepatocytes: a model system for the study of metabolic suppression. Am. J. Physiol. 265 (34), R49 – R56. Else, P.L., Hulbert, A.J., 1981. Comparison of the ‘mammal machine’ and the ‘reptile machine’: energy production. Am. J. Physiol. 240, R3 – R9. Gnaiger, E., 1983. Heat dissipation and energetic efficiency in animal anoxibiosis: economy contra power. J. Exp. Zool. 228, 471 – 490.
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