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Effect of palmitate on hepatic biosynthetic functions at hyperthermic temperatures
Effect
of Palmitate on Hepatic Biosynthetic at Hyperthermic Tempeiatures Fred G. Collins, Frank A. Mitros,
Livers of fasted rats were perfused for 80 min at 37”~43°C. supplemented with lactate, NH&I, and ornithine in the presence or absence of palmitate. Hepatic functional integrity was maintained from 37” to 42°C as assessed by gluconeogenesis, ureogenesis. and 0, consumption. Between 42” and 43°C a sharp decline in biosynthetic function occurred. The ratio of lactate disappearance to glucose formation increased progressively with increasing temperature when compared with the ratio obtained at 37°C. Exogenous palmitate significantly decreased the ratio of lactate disappearance to glucose formation at 43°C. Furthermore, palmitate attenuated the detrimental effects of hyperthermia on gluconeogenesis, ureogenesis. and 0, consumption found in the absence of palmitate. The 3-hydroxybutyrate/acetaacetate ratio progressively decreased as the liver temperature was increased in the presence or absence of palmitate, indicating a more oxidized mitochandrial redox state. Palmitate significantly increased the 3-hydroxybutyratelacetoacetate ratio in the presence of gluconeogenic and ureogenic substrates at all temperatures examined. The data suggest that provision of fatty acid has a protective effect in thermally stressed liver. Moreover, palmitate may substitute for the increased energy requirements of the hyperthermic state.
of thermotherapy as an antineoT HEplasticUSE modality is presently undergoing intense study. A large body of evidence indicates that malignant cells are more sensitive than normal cells to the lethal effects of hyperthermic exposure between 41.5” and 43°C.‘-4 Local or systemic thermotherapy for cancer may result in regression of neoplastic growth; however, significant complications often accompany the therap~.~ Since little is known about the effects of hyperthermia on normal tissue metabolism, the perfused liver mode1 was employed to characterize energy-requiring metabolic pathway function at supranormal temperatures. Gluconeogenesis and ureogenesis are major
Functions
and Joseph L. Skibba
energy-requiring processes in the fasted liver, i.e., 10 moles of ATP to form 1 mole each of glucose and urea.’ Thus, determining the maximal rates of these metabolic processes under the stress of hyperthermia can provide an index of hepatic ATP supply and assess the functional status of the liver. Skibba and Collins’ found a progressive increase in the ratio of lactate disappearance to glucose formation in the perfusate from substrate-supported livers perfused at progressively increasing supernormal temperatures. This finding suggested that hepatic lactate use occurs by an additional pathway during hyperthermia. Moreover, hyperthermia led to a decrease in the ratio of 3-hydroxybutyrate to acetoacetate in control or palmitate-supplemented livers in the absence of other substrates.’ However, the ratio of 3-hydroxybutyrate to acetoacetate remained higher in the palmitatesupplemented livers compared with the controls at all temperatures examined. Since a low ratio of 3-hydroxybutyrate to acetoacetate is indicative of declining hepatic function,’ it was suggested that palmitate may attenuate the detrimental effects of hyperthermia on liver function. Thus, independent alterations in carbohydrate and fatty acid metabolism were observed under different substrate conditions at hyperthermic temperatures in our previous study. Since these pathways interact through the intermediate acetyl-CoA, it was of interest to investigate this common interaction during hepatic hyperthermia. Therefore, the effects of palmitate on substrate-supported gluconeogenesis, ureogenesis, ketogenesis, and 0, consumption at elevated temperatures are addressed in the present paper. MATERIALS
AND
METHODS
Animals From the Departments of Surgery and Pathology, The University of Iowa Hospitals and Clinics, Iowa City, Iowa. Received for publication April 26, 1979. Supported in part by USPHS NCI Grant CA 25790. Address reprinf requests to Dr. Fred G. Collins, Research Service, VA Medical Center, Wood, Wis. 53193. o 1980 by Grune & Stratton, Inc. 0029-0495/80/2906~005$0I.00/0
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Male Sprague-Dawley albino rats (Sprague-Dawley, Madison, Wk.) weighing 175-22.5 g were fed laboratory chow ad lib. and fasted for 24-30 hr prior to perfusion. Materials The enzymes and cofactors necessary for the analysis of perfusate samples were obtained from either Boehringer Mannheim Biochemicals, Indianapolis, Ind., or Sigma
Merabokm,
Vol. 29, No. 6 (June), 1980
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Chemical Co.. St. Louis, MO. Bovine plasma albumin was purchased from Reheis Chemical Co., Phoenix, Ariz. Other reagents were of the highest grade available.
nation. Two to four tissue samples experimental conditions employed.
were obtained
at all
Analytic and Statistical Methods Perfisate The recirculating perfusate consisted of 150 ml KrebsRinger bicarbonate solution” containing 20% (volume/volume) washed, outdated human erythrocytes and 2.5% (weight/volume) bovine plasma albumin, Cohn fraction V. A membrane oxygenator (Sci-Med Life Systems, Inc., Minneapolis, Minn.) was employed for gassing the perfusate with Or + CO, (955).
Perfusion Technique The techniques employed for in situ perfusion of the liver in which accurate control of the liver temperature can be achieved have been published” and are summarized in this report. The surgical procedure used was that of Hems et al.’ The degree of anesthesia was controlled throughout the operative period with an ether vaporizer. The rat was heparinized (200 U, i.v.) to ensure success of the perfusion technique. The portal vein and the thoracic segment of the inferior vena cava were cannulated and served as the inflow and outflow cannulas, respectively. The maximum period of operative hypoxia never exceeded 2 min. The perfusion was terminated if the liver acquired a blotchy appearance or if 0, extraction was negligible immediately following the initiation of perfusion. The initial 5 ml of perfusate collected from the outflow cannula were discarded. A liver flow rate of I S-20 ml/min was maintained with a hydrostatic pressure of 18 cm. Thermocouple microprobes (Bailey instrument Co.. Saddle Brook, N.J.) were used to monitor and control the liver, cabinet, and perfusate temperatures. Perfusate and cabinet were heated to the desired liver temperature before initiation of the liver perfusion. Liver temperature stabilized at the desired temperature within 5 min of the equilibration period at 37°C and within 20 mitt at 43OC. A 40.min perfusion period was allowed for equilibration of the preparation. All substrates were added to the perfusate by bolus. 38 min into the equilibration period, allowing 2 min for adequate mixing of the substrates throughout the perfusion circuit. The livers were perfused for an additional 80 min after initial sampling at 40 min (0 min of the experimental period). Lactate, NH,CI and ornithine were added to the perfusate ( I50 ml) in aqueous solution at pH 7.4, resulting in perfusate concentrations of IO mM, IO mM, and 2.7 mM, respectively. Sodium palmitate was heated in an aqueous solution at 65OC until the solution was clear. An aqueous solution (55OC) of bovine plasma albumin was vigorously mixed with the palmitate solution to a final albumin concentration of 12% (weight/volume) and the solution, a total volume of 2 ml. was added immediately to the perfusate.” The perfusate concentration of palmitate was 1 mM (1 pmol palmitate/ml perfusate). The molar ratio of free fatty acid (FFA) to albumin in the perfusate was 3 to I. Since bovine plasma albumin (Cohn fraction V) contains a small amount of FFA,” the albumin solution served as a control for all livers perfused in the absence of palmitate. After completion of the perfusion, samples of liver tissue were fixed in buffered formalin for light microscopic exami-
Perfusate samples were obtained at 20-min intervals from 0 min to 80 min of the experimental period. Protein-free filtrates were prepared from the perfusate samples with perchloric acid. Aliquots of these protein-free filtrates were analyzed for glucose.‘4 lactate,15 acetoacetate.‘” 3-hydroxybutyrate,” and urea.” Estimates of the maximal rates of metabolite change were determined by regression analysis as described by Hems et al.’ All samples were analyzed in duplicate. Perfusate O2 consumption was also determined at 20-min intervals during the experimental period. Perfusate 0, content was determined with a Lex 0, Con-TL (Lexington Instruments Corp., Waltham, Mass.). The 0, content of the inflow cannula perfusate remained constant throughout the experimental period; this was determined by periodic sampling of the perfusate from the inflow cannula. Each value for the perfusions represents the mean + SE in rmol/g liver (wet weight). Since liver congestion occurred at hyperthermic temperatures, experimental liver wet weights were extrapolated from the weights of groups of control livers obtained from fasted rats weighing 175-225 g. The reiationship between body weight and liver weight was linear, which agrees with the findings of Peterson et al! The significance of differences between means was determined by Student’s t test or analysis of variance. Split-plot analysis” was applied after analysis of variance to determine the individual ditferences between means. A p value of .-r 0 05 was considered statistically significant. RESULTS
Effect of Hyperthermia and Palmitate on Substrate-Stimulated Biosynthesis Gluconeogenesis from exogenous lactate in the presence of ammonia and ornithine remained above 1 clrnol/g wet weight/min from 37O to 42°C in the presence or absence of palmitate (Fig. 1A). A significant decrease occurred between 42” and 43°C in the presence or absence of palmitate. In the presence of palmitate, the rate of gluconeogenesis at 43°C was significantly greater than the rate in livers perfused without palmitate at the same temperature. The maximal rate of lactate disappearance for livers perfused with or without paimitate occurred at 40°C (Fig. IB). In each case, the rate was significantly higher than the respective 37OC rate. Livers perfused in the presence or absence of palmitate exhibited maximal rates of urea production from ammonia and ornithine at 40” and 41°C, respectively (Fig. 1C). Between 42” and 43”C, a significant decline in ureogenesis
526
COLLINS, MITROS, AND SKIBBA
GLUCOSE FORMED
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UREA FORMED
I-37
39
41
43
37
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41
TEMPERATURE
43
37
39
41
43
PC1
Fig. 1. Effect of hyperthermia and palmitate on substrate-stimulated gluconeogenesis and ureogenesis. Rat livers were perfused as described in Materials and Methods. The maximal rates of metabolic change; (A) glucose formed, (6) lactate consumed, (C) urea formed, after the addition of substrates-lactate (10 mM), NH&I (10 m/W) and ornithine (2.7 m&f) in the presence (0) or absence (0) of 1 mM palmitate are shown. Mean values + SE for four perfusions are shown. Significantly greater than fp < 0.05) the corresponding value obtained in the absence of palmitate by Student’s t test (‘1.
occurred in livers perfused with or without palmitate. Figure 2 shows the ratios of lactate disappearance to glucose formation in the perfusate in the presence or absence of palmitate at liver temper-
0 6OWAcAc TEMP PC,
.SY 37
9
37 .60
39
40
41
42
43
Fig. 3. The effect of hyperthermia and palmitate on ketone body production and the ratio of 3-hydroxybutyrate (SOH) to acetoacetate (AcAc) in the perfused rat liver. Ketone bodies were determined in the same experiments as shown in Fig. 1. The ratios were calculated from the concentration of acetoacetate (shaded area) and 3-hydroxybutyrate (unshaded area) in perfusate samples removed at the end of the 80-min experimental period. Lactate, NH&I, and ornithine were added in the absence (striped bars) or presence (unstriped bars) of palmitate. Mean values from four perfusions are shown.
atures from 37” to 43T. A significant in the ratio was found in the presence tate at 43°C.
decrease of palmi-
E#ect of Hyperthermia on Palmitate-Supported Ketogenesis
I
37
I
I
41 39 Temperature fYJ
1
43
Fig. 2. Effect of hyperthermia and palmitate on the ratio of lactate disappearance to glucose formation. Ratios were determined in the same experiments as shown in Fig. 1. The ratio was calculated from the change in perfusate concentrations of lactate and glucose for the SO-min experimental period. Lactate, NH&I, and ornithine were added in the presence (0) or absence (0) of palmitate. Mean values & SE for four perfusions at each condition are shown. Significantly less than (p < 0.05) the corresponding value determined in the absence of palmitate by Student’s t test (‘1.
Palmitate increased total ketone body production about threefold in the presence of gluconeogenic and ureogenic substrates at all temperatures (Fig. 3). Hyperthermia did not alter total ketone body production in the presence or absence of palmitate. Palmitate significantly increased (p < 0.05) the 3-hydroxybutyrate/acetoacetate ratio at all temperatures studied (Fig. 3). As the liver temperature increased, the 3-hydroxybutyrate/acetoacetate ratio progressively decreased in livers with or without palmitate. However, in the presence of palmitate, the ratio at 43’T was similar to the ratios obtained in the absence of palmitate from 37“ to 41°C.
Effect of Hyperthermia and Palmitate on Substrate-Stimulated 0, Consumption Hepatic O? consumption was stimulated by the addition of lactate, ammonia, and ornithine (Figs. 4 and 5). Palmitate did not affect the stimulation of 0, consumption after 40 min of the experimental period from 37” to 42°C; however, at 43”C, substrate-supported 0,
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consumption was significantly higher in the presence of palmitate (Fig. 4). The time course of 0, consumption at 43°C further illustrates this point (Fig. 5). Palmitate significantly stimulated substrate-supported O2 consumption compared with livers perfused without palmitate at all time points after addition of substrates for gluconeogenesis and ureogenesis. In addition. no stimulation of O2 consumption occurred after addition of lactate, ammonia, and ornithine in the absence of palmitate (Fig. 5). Light Microscop_v of Livers Exposed to Supranormal Temperatures
37
39 41 Temperature toC 1
43
Fig. 4. Effect of hyperthermia on substrate-stimulated 0, consumption 40 mitt into the experimental period. Oxygen consumption was measured at 40 min of the experimental period in the same experiments as described in Fig. 1. Livers were perfused in the presence (0) or absence (0) of palmitata. Lactate 110 m&f). NH&I (10 m&f), and ornithine (2.7 mM) were also present. Significantly greater than (p < 0.05) the corresponding value obtained in the absence of palmitate by split-plot analysis (‘1.
After 2 hr of perfusion at 37OC, the hepatocytes appeared normal; however, mild sinusoidal dilatation was observed. Histologic examination of the liver samples disclosed mild portal and central venous congestion, and mild-to-moderate sinusoidal dilatation from 39°C to 41°C. At 42°C moderate-to-severe portal and central venous congestion and sinusoidal dilatation were apparent. These changes became severe at 43OC (Fig. 6). Hepatic cord separation was mild at 42%; however, it was especially noticeable at 43OC, at which point it varied from moderate to severe. The continuum of pathologic changes occurring in the liver perfused at progressively elevated temperatures was attenuated by palmitate. These differences were especially apparent at 43°C (Figs. 6 and 7). DISCUSSION
0
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Time fmin) Effects of pelmitate on the time course of Fig. 5. substrate-stimulated 0, consumption at 43°C. Oxygen consumption was determined in the same experiments as described in Fig. 1. Livers were perfused at 43°C in the presence (*I or absence (0) of palmitate. Lactate (10 m/W), NH&I I10 m&f). and ornithine (2.7 mM) were also present. Significantly greater than fp < 0.05) the corresponding value obtained in the absence of pelmitate by split-plot analysis ( 1. l
A major aim of this study was to assess the effect of exogenous palmitate on substratesupported hepatic biosynthetic processes at elevated temperatures considered useful in thermotherapy of cancer. The data show that palmitate can reduce the detrimental effects of liver perfusion on hepatic metabolic parameters at 43°C. The protective effect of palmitate may be interpreted by present knowledge on hepatic metabolic control at normal temperatures. Menahan and Wieland” reported that the ratio of pyruvate disappearance to glucose formation increased when endogenous liver triglyceride hydrolysis is inhibited, indicating a diversion of C, intermediates from glucose formation to oxidation. Presumably, pyruvate oxidation substituted for fatty acid oxidation under these conditions. in this study, hepatic lactate use was
COLLINS, MITROS, AND SKIBBA
Fig. 6. Liver perfused for 66 min at 43°C with lactate, NH,CI. and ornithine supplementation. Sinusoidal dilatation. erythrocyte congestion, and hepatic cord separation are severe in the centrolobular region. fHematoxylin and eosin x 266).
stimulated at supranormal temperatures, suggesting increased diversion of C, intermediates to oxidation at hyperthermic temperatures. It is generally accepted that hepatic fatty acid metabolism and gluconeogenesis are related.2’-24 Thus, the effects of fatty acid supplementation on hepatic biosynthetic processes at elevated temperatures were investigated. As previously reported, endogeneous ketogenesis was minimal in perfused rat liver; however, palmitate supplementation significantly increased ketone body
Fig. 7. Liver perfused for 80 min at 43°C with lactate, NHJX ornithine. and palmitate supplementation. Severe sinusoidal dilatation, moderate congestion and mild hepatic cord separation are present in the centrolobuler area. (Hematoxylin and eosin x 260).
production8 We now report that addition of pdmitate to the perfusate, supplemented with lactate and ammonia, partially alleviated excess use of Iactate under hyperthermic conditions. Therefore, it appears that lactate provided sufficient C, intermediates to support hepatic energyrequiring processes from 37” to 43°C. Furthermore, at all temperatures, including 43°C acetyl-CoA was derived from palmitatesuppressed C, oxidation. The proportion of pyruvate dehydrogenase in
PALMITATE
IN HEPATIC HYPERTHERMIA
the active form is very low in the fasted state.‘5.26 Among the factors controlling the conversion of pyruvate dehydrogenase to the active state, the mitochondrial NADH/NAD ratio is of major importance.‘7,2x A decrease in the 3-hydroxybutyrate/acetoacetate ratio is indicative of a more oxidized mitochondrial redox state,29 resulting in an increase in the proportion of pyruvate dehydrogenase in the active state. In the presence or absence of palmitate, the 3-hydroxybutyrate/acetoacetate ratio progressively decreased as the temperature was increased from 37” to 43°C. This lends support to the reasoning that pyruvate entrance into the tricarboxylic acid cycle is increased and presumably oxidized at elevated temperatures. In addition, the 3hydroxybutyrate/acetoacetate ratio in the presence of palmitate at 43°C was similar to that found in livers perfused in the absence of palmitate from 37O to 41°C. Thus, it appears that metabolism of exogenous palmitate can suppress C, unit oxidation at supranormal temperatures. Zimmermann et al? followed the effect of varying temperature from lo to 37OC on gluconeogenesis, ketogenesis, and CO, production in perfused livers. A divergence in the Qlo for ketogenesis and CO, production from octanoate occurred between 25” and 37°C. Oxidation of octanoate increased, causing lower rates of ketogenesis, as the liver temperature approached 37OC. The present findings appear to extend this relationship to supranormal temperatures. Hepatic gluconeogenesis from lactate is normally stimulated in the presence of exogenous fatty acid.“m33 Nevertheless, no stimulation of gluconeogenesis from lactate in the presence of palmitate when ammonia and ornithine were also present was found in this investigation. These observations agree with Wojtczak et al.,34 who studied isolated hepatocytes under similar substrate conditions. Since the pathways for gluconeogenesis and ureogenesis in the liver are interrelated,34 37 it has been suggested that competition between common intermediates in the cytosolic and mitochondrial compartments may be related to this finding.34m38 Oxygen consumption is used to assess the functional integrity of perfused livers3’ At 43*C, substrate stimulation of O2 consumption did not occur. However, palmitate significantly en-
529
hanced 0, consumption at 43°C after addition of gluconeogenic and ureogenic substrates. The data suggest that exogenous palmitate can be oxidized at 43”C, providing the necessary energy to partially alleviate deterioration in biosynthetic processes. Maximal rates of gluconeogenesis and ureogenesis can occur simultaneously at normothermic temperatures;7.34 however, the temperature dependency of metabolic reactions dictates that energy metabolism be stimulated as absolute temperature increases. Thus, measurement of these processes provides an excellent test of the functional integrity of the liver at elevated temperatures. Biosynthetic processes were maintained from 37O to 42°C. A sharp decline in biosynthetic function occurred between 42” and 43OC, suggesting a depression in hepatic ATP supply. However, palmitate supplementation partially suppressed hepatic deterioration at 43°C. Presumably, palmitate can supply the ATP required to maintain hepatic integrity under hyperthermic stress. The increased energy requirements for maintenance of biosynthetic processes at elevated temperatures could be explained by several metabolic alternatives. An increase in futile cycling would result in excessive consumption of ATP. Indeed, Williams et a1.j’ reported increased futile cycling in livers perfused at 42°C. An alternative explanation. the uncoupling of oxidative phosphorylation, would result in inefficient generation of ATP. Christiansen and Kvamme4’ found that uncoupling of oxidative phosphorylation was temperature-dependent in liver mitochondria exposed to a temperature of 41”-45°C for 10 min. However. Carlsson et aL4* found no change in ATP, ADP, AMP, and phosphocreatine in the cerebral cortex of rats subjected to whole body hyperthermia (41.8”C for 30 min). Individual enzymes can be inactivated at 40°-430C,43,44 although the inactivation is reversible. Contribution of this factor to hepatic energy metabolism is difficult to predict because of the biochemical complexity of the liver and the fact that most enzymes require much higher temperatures for inactivation. Manipulation of normal tissue metabolism under hyperthermic conditions may lead to improvement in antineoplastic selectivity of
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hyperthermia. To support this concept, additional experiments are planned to examine the role of palmitate as an energy substrate and attenuator of metabolic deterioration at supranormal temperatures.
ACKNOWLEDGMENT The authors thank Dr. Lawrence A. Menahan for his helpful comments on the manuscript, Jean V. Collins for her expert technical assistance, and Mimi Mick for her help in preparation of the manuscript.
REFERENCES 1. Rossi-Fanelli A, Cavaliere R, Mondovi B, et al (eds): Recent Results in Cancer Research, Selective Heat Sensitivity of Cancer Cells. Berlin, Springer-Verlag, 1977 2. Overgaard J: Effect of hyperthermia on malignant cells in vivo. A review and a hypothesis. Cancer 39:2637-2646. 1977 3. Dickson, JA, Suzangar M: A predictive in vitro assay for the sensitivity of human solid tumours to hyperthermia (42°C) and its value in patient management. Clin Oncol 2:141-155, 1976 4. Cavaliere R, Ciocatto EC, Giovanella BC, et al: Selective heat sensitivity of cancer cells. Cancer 20:1351-1381, 1967 5. Pettigrew RJ, Galt JM, Lundgate CM, et al: Clinical effects of whole-body hyperthermia in advanced malignancy. Br Med J 41679-682, 1974 6. Wills EJ. Findlay JM, McManus JPA: Effects of hyperthermia therapy on the liver. II. Morphological observations. J Clin Path01 29:1-IO, 1976 7. Hems R, Ross BD, Berry MN, et al: Gluconeogenesis in the perfused rat liver. Biochem J 101:284-292, 1966 8. Skibba JS, Collins FG: Effect of temperature on biochemical functions in the isolated perfused rat liver. J Surg Res 24:435441, 1978 9. Krebs HA: Formation of ketone bodies in the perfused rat liver, in Staib W, Scholz R (eds): Stoffwechsel der isoliert perfundieten leber. Berlin, Springer-Verlag, 1968, pp 1299141 10. Krebs HA, Henseleit K: Untersuchungen uber die harnstotlbildung im tierkorper. Hoppe Seylers Z Physiol Chem 210:33-66, 1932 11. Collins FG, Skibba JL: Improved in situ rat liver perfusion technique. J Surg Res vol. 28, 1980 12. Krebs HA, Wallace PG. Hems R: Rates of ketone body formation in the perfused rat liver. Biochem J I 12:595600, 1969 13. Spector AA: Fatty acid binding to plasma albumin. J Lipid Res 16:165-179, 1975 14. Huggett AG, Nixon DA: Use of glucose oxidase, peroxidase, and o-dianisidine in determination of blood and urinary glucose. Lancet 2:368-370. 1957 15. Fleischer WR: Enzymatic methods for lactic and pyruvic acids, in MacDonald RP (ed): Standard Methods of Clinical Chemistry. New York, Academic, 1970, pp 245% 259 16. Mellanby J, Williamson DH: Acetoacetate, in Bergermeyer HU (ed): Methods of Enzymatic Analysis. New York, Academic, 1963, pp 454-457 17. Williamson DH, Mellanby J: @-Hydroxybutyrate, in Bergermeyer HU (ed): Methods of Enzymatic Analysis. New York, Academic, 1963, pp 459-461 18. Cracker CL: Rapid determination of urea nitrogen in
serum or plasma without deproteinization. Am J Med Technol33:361-365, 1967 19. Peterson RE, Olson JR, Fujimoto JM: Measurement and alteration of the capacity of the distended biliary tree in the rat. Toxicol Appl Pharmacol36:353-368, 1976 20. Steel RG, Torrie JH: Principles and Procedures of Statistics. New York, McGraw-Hill, 1960, pp 232-241 21. Menahan LA, Wieland 0: The role of endogenous lipid in gluconeogenesis and ketogenesis of perfused rat liver. Eur J Biochem 9: 182-l 88, I967 22. Exton JH: Gluconeogenesis. Metabolism 21:945-990, 1972 23. Parrilla R, Ayuso-Parrilla MS, Williamson JR: Use of endogenous triglycerides to support gluconeogenesis in perfused rat liver. Pfluegers Arch 366:2l l-216. 1976 24. Williamson JR, Rostand SG, Peterson MJ: Control factors affecting gluconeogenesis in perfused rat liver. Effects of 4-pentanoic acid. J Biol Chem 245:2342-3251. 1970 25. Patzelt C, Loffler G. Wieland OH: Interconversion of pyruvate dehydrogenase in the isolated perfused rat liver. Eur J Biochem 33: I 17-l 22, 1973 26. Wieland OH, Lother G, Patzelt C, et al: Regulation of pyruvate dehydrogenase interconversion in liver, in Lundquist F, Tygstrue N (eds): Regulation of hepatic metabolism. New York, Academic, 1974. pp 62-78 27. Wieland 0, von Jagow-Westermann B, Stukowski B: Kinetic and regulatory properties of heart muscle pyruvate dehydrogenase. Hoppe Seylers Z Physiol Chem 350:329334, 1969 28. Denton RM, Randle PJ, Bridge BJ. et al: Regulation of mammalian pyruvate dehydrogenase. Mol Cell Biochem 9~27-53, 1975 29. Williamson DH, Lund P, Krebs HA: The redox state of free nicotinamide-adenine dinucleotide in the cytoplasm and mitochondria of rat liver. Biochem J 103:5 14-527, I967 30. Zimmermann FA, Dietz HG, Sippell WG, et al: Temperaturabhangigkeit von stoffwechselgrossen in der perfundierten rattenleber. Res Expl Med (Berl) 168:57-64, 1976 3 1. Ross BD, Hems R, Freedland A. et al: Carbohydrate metabolism in the perfused rat liver. Biochem J 105:869875. 1967 32. Williamson JR, Browning ET, Scholz R: Control of mechanisms of gluconeogenesis in perfused rat liver. J Biol Chem 244:4607-4616, 1969 33. Williamson JR, Scholz R, Browning ET: Control mechanisms of gluconeogenesis and ketogenesis. II. Interactions between fatty acid oxidation and the citric acid cycle in perfused rat liver. J Biol Chem 244:46 17-4627, 1967 34. Wojtczak AB, Walajtys-Rode EI, Geelen MJH: Interrelations between ureogenesis and gluconeogenesis in
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isolated hepatocytes. The role of anion transport and the competition for energy. Biochem J 170:379-385, 1978 35. Briggs S, Freedland RA: Effect of ornithine and lactate on urea synthesis in isolated hepatocytes. Biochem J I60:205--209, 1976 36. Krebs HA, Lund P, Stubbs M: Interconversions between gluconeogenesis and urea synthesis, in Hanson RW. Mehlman MA (eds): Gluconeogenesis: It’s Regulation in Mammalian Species. New York. Wiley, 1976, pp 2699291 37. Meijer AJ, Gimpel JA, Deleeuw G, et al: Interrelationships between gluconeogenesis and ureogenesis in isolated hepatocytes. J Biol Chem 253:2308-2320. 1978 38. Williamson JR, Meijer AJ, Ohkawa K: Interrelations between anion transport, ureogenesis and gluconeogenesis in isolated rat cells, in Lundquist E, Tygstrup N (eds): Regulation of Hepatic Metabolism. New York, Academic, 1974, pp 457-48 I 39. Ross BD: Perfusion Techniques Oxford. Clarendon, 1972, pp 1355220 40. Williams JF. Cook PC, Matthaei
in Biochemistry. KI, et al: Pyridine
and adenine nucleotide ratios and futile substrate cycling in regulation of energy metabolism and proposed hyperthermic regression of neoplasms, in Criss WE, Ono T, Sabine J (eds): Control of Mechanisms in Cancer. New York. Raven. 1976, pp 4255439 41. Christiansen EN, Kvamme E: Effects of thermal treatment on mitochondria of brain, liver and ascites cells. Acta Physiol Stand 76:472-484, 1969 42. Carlsson C, Hagerdal M, Siesjo BK: The effect of hyperthermia upon oxygen consumption and upon organic phosphates, glycolytic metabolites. citric acid cycle intermediates and associated amino acids in rat cerebral cortex. J Neurochem 26:1001-1006, 1976 43. Anson ML, Mirsky AE: The equilibrium between active native trypsin and inactive denatured trypsin. J Gen Physiol 17:393-398, 1934 44. Stearn AE: Kinetics of biological reactions with special reference to enzymatic processes, in Nord FF (ed): Advances in Enzymology, vol 19. New York. Interscience. 1949, pp 25-74
of Palmitate on Hepatic Biosynthetic at Hyperthermic Tempeiatures Fred G. Collins, Frank A. Mitros,
Livers of fasted rats were perfused for 80 min at 37”~43°C. supplemented with lactate, NH&I, and ornithine in the presence or absence of palmitate. Hepatic functional integrity was maintained from 37” to 42°C as assessed by gluconeogenesis, ureogenesis. and 0, consumption. Between 42” and 43°C a sharp decline in biosynthetic function occurred. The ratio of lactate disappearance to glucose formation increased progressively with increasing temperature when compared with the ratio obtained at 37°C. Exogenous palmitate significantly decreased the ratio of lactate disappearance to glucose formation at 43°C. Furthermore, palmitate attenuated the detrimental effects of hyperthermia on gluconeogenesis, ureogenesis. and 0, consumption found in the absence of palmitate. The 3-hydroxybutyrate/acetaacetate ratio progressively decreased as the liver temperature was increased in the presence or absence of palmitate, indicating a more oxidized mitochandrial redox state. Palmitate significantly increased the 3-hydroxybutyratelacetoacetate ratio in the presence of gluconeogenic and ureogenic substrates at all temperatures examined. The data suggest that provision of fatty acid has a protective effect in thermally stressed liver. Moreover, palmitate may substitute for the increased energy requirements of the hyperthermic state.
of thermotherapy as an antineoT HEplasticUSE modality is presently undergoing intense study. A large body of evidence indicates that malignant cells are more sensitive than normal cells to the lethal effects of hyperthermic exposure between 41.5” and 43°C.‘-4 Local or systemic thermotherapy for cancer may result in regression of neoplastic growth; however, significant complications often accompany the therap~.~ Since little is known about the effects of hyperthermia on normal tissue metabolism, the perfused liver mode1 was employed to characterize energy-requiring metabolic pathway function at supranormal temperatures. Gluconeogenesis and ureogenesis are major
Functions
and Joseph L. Skibba
energy-requiring processes in the fasted liver, i.e., 10 moles of ATP to form 1 mole each of glucose and urea.’ Thus, determining the maximal rates of these metabolic processes under the stress of hyperthermia can provide an index of hepatic ATP supply and assess the functional status of the liver. Skibba and Collins’ found a progressive increase in the ratio of lactate disappearance to glucose formation in the perfusate from substrate-supported livers perfused at progressively increasing supernormal temperatures. This finding suggested that hepatic lactate use occurs by an additional pathway during hyperthermia. Moreover, hyperthermia led to a decrease in the ratio of 3-hydroxybutyrate to acetoacetate in control or palmitate-supplemented livers in the absence of other substrates.’ However, the ratio of 3-hydroxybutyrate to acetoacetate remained higher in the palmitatesupplemented livers compared with the controls at all temperatures examined. Since a low ratio of 3-hydroxybutyrate to acetoacetate is indicative of declining hepatic function,’ it was suggested that palmitate may attenuate the detrimental effects of hyperthermia on liver function. Thus, independent alterations in carbohydrate and fatty acid metabolism were observed under different substrate conditions at hyperthermic temperatures in our previous study. Since these pathways interact through the intermediate acetyl-CoA, it was of interest to investigate this common interaction during hepatic hyperthermia. Therefore, the effects of palmitate on substrate-supported gluconeogenesis, ureogenesis, ketogenesis, and 0, consumption at elevated temperatures are addressed in the present paper. MATERIALS
AND
METHODS
Animals From the Departments of Surgery and Pathology, The University of Iowa Hospitals and Clinics, Iowa City, Iowa. Received for publication April 26, 1979. Supported in part by USPHS NCI Grant CA 25790. Address reprinf requests to Dr. Fred G. Collins, Research Service, VA Medical Center, Wood, Wis. 53193. o 1980 by Grune & Stratton, Inc. 0029-0495/80/2906~005$0I.00/0
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Male Sprague-Dawley albino rats (Sprague-Dawley, Madison, Wk.) weighing 175-22.5 g were fed laboratory chow ad lib. and fasted for 24-30 hr prior to perfusion. Materials The enzymes and cofactors necessary for the analysis of perfusate samples were obtained from either Boehringer Mannheim Biochemicals, Indianapolis, Ind., or Sigma
Merabokm,
Vol. 29, No. 6 (June), 1980
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IN HEPATIC HYPERTHERMIA
Chemical Co.. St. Louis, MO. Bovine plasma albumin was purchased from Reheis Chemical Co., Phoenix, Ariz. Other reagents were of the highest grade available.
nation. Two to four tissue samples experimental conditions employed.
were obtained
at all
Analytic and Statistical Methods Perfisate The recirculating perfusate consisted of 150 ml KrebsRinger bicarbonate solution” containing 20% (volume/volume) washed, outdated human erythrocytes and 2.5% (weight/volume) bovine plasma albumin, Cohn fraction V. A membrane oxygenator (Sci-Med Life Systems, Inc., Minneapolis, Minn.) was employed for gassing the perfusate with Or + CO, (955).
Perfusion Technique The techniques employed for in situ perfusion of the liver in which accurate control of the liver temperature can be achieved have been published” and are summarized in this report. The surgical procedure used was that of Hems et al.’ The degree of anesthesia was controlled throughout the operative period with an ether vaporizer. The rat was heparinized (200 U, i.v.) to ensure success of the perfusion technique. The portal vein and the thoracic segment of the inferior vena cava were cannulated and served as the inflow and outflow cannulas, respectively. The maximum period of operative hypoxia never exceeded 2 min. The perfusion was terminated if the liver acquired a blotchy appearance or if 0, extraction was negligible immediately following the initiation of perfusion. The initial 5 ml of perfusate collected from the outflow cannula were discarded. A liver flow rate of I S-20 ml/min was maintained with a hydrostatic pressure of 18 cm. Thermocouple microprobes (Bailey instrument Co.. Saddle Brook, N.J.) were used to monitor and control the liver, cabinet, and perfusate temperatures. Perfusate and cabinet were heated to the desired liver temperature before initiation of the liver perfusion. Liver temperature stabilized at the desired temperature within 5 min of the equilibration period at 37°C and within 20 mitt at 43OC. A 40.min perfusion period was allowed for equilibration of the preparation. All substrates were added to the perfusate by bolus. 38 min into the equilibration period, allowing 2 min for adequate mixing of the substrates throughout the perfusion circuit. The livers were perfused for an additional 80 min after initial sampling at 40 min (0 min of the experimental period). Lactate, NH,CI and ornithine were added to the perfusate ( I50 ml) in aqueous solution at pH 7.4, resulting in perfusate concentrations of IO mM, IO mM, and 2.7 mM, respectively. Sodium palmitate was heated in an aqueous solution at 65OC until the solution was clear. An aqueous solution (55OC) of bovine plasma albumin was vigorously mixed with the palmitate solution to a final albumin concentration of 12% (weight/volume) and the solution, a total volume of 2 ml. was added immediately to the perfusate.” The perfusate concentration of palmitate was 1 mM (1 pmol palmitate/ml perfusate). The molar ratio of free fatty acid (FFA) to albumin in the perfusate was 3 to I. Since bovine plasma albumin (Cohn fraction V) contains a small amount of FFA,” the albumin solution served as a control for all livers perfused in the absence of palmitate. After completion of the perfusion, samples of liver tissue were fixed in buffered formalin for light microscopic exami-
Perfusate samples were obtained at 20-min intervals from 0 min to 80 min of the experimental period. Protein-free filtrates were prepared from the perfusate samples with perchloric acid. Aliquots of these protein-free filtrates were analyzed for glucose.‘4 lactate,15 acetoacetate.‘” 3-hydroxybutyrate,” and urea.” Estimates of the maximal rates of metabolite change were determined by regression analysis as described by Hems et al.’ All samples were analyzed in duplicate. Perfusate O2 consumption was also determined at 20-min intervals during the experimental period. Perfusate 0, content was determined with a Lex 0, Con-TL (Lexington Instruments Corp., Waltham, Mass.). The 0, content of the inflow cannula perfusate remained constant throughout the experimental period; this was determined by periodic sampling of the perfusate from the inflow cannula. Each value for the perfusions represents the mean + SE in rmol/g liver (wet weight). Since liver congestion occurred at hyperthermic temperatures, experimental liver wet weights were extrapolated from the weights of groups of control livers obtained from fasted rats weighing 175-225 g. The reiationship between body weight and liver weight was linear, which agrees with the findings of Peterson et al! The significance of differences between means was determined by Student’s t test or analysis of variance. Split-plot analysis” was applied after analysis of variance to determine the individual ditferences between means. A p value of .-r 0 05 was considered statistically significant. RESULTS
Effect of Hyperthermia and Palmitate on Substrate-Stimulated Biosynthesis Gluconeogenesis from exogenous lactate in the presence of ammonia and ornithine remained above 1 clrnol/g wet weight/min from 37O to 42°C in the presence or absence of palmitate (Fig. 1A). A significant decrease occurred between 42” and 43°C in the presence or absence of palmitate. In the presence of palmitate, the rate of gluconeogenesis at 43°C was significantly greater than the rate in livers perfused without palmitate at the same temperature. The maximal rate of lactate disappearance for livers perfused with or without paimitate occurred at 40°C (Fig. IB). In each case, the rate was significantly higher than the respective 37OC rate. Livers perfused in the presence or absence of palmitate exhibited maximal rates of urea production from ammonia and ornithine at 40” and 41°C, respectively (Fig. 1C). Between 42” and 43”C, a significant decline in ureogenesis
526
COLLINS, MITROS, AND SKIBBA
GLUCOSE FORMED
LACTATE CONSUMED
60
UREA FORMED
I-37
39
41
43
37
39
41
TEMPERATURE
43
37
39
41
43
PC1
Fig. 1. Effect of hyperthermia and palmitate on substrate-stimulated gluconeogenesis and ureogenesis. Rat livers were perfused as described in Materials and Methods. The maximal rates of metabolic change; (A) glucose formed, (6) lactate consumed, (C) urea formed, after the addition of substrates-lactate (10 mM), NH&I (10 m/W) and ornithine (2.7 m&f) in the presence (0) or absence (0) of 1 mM palmitate are shown. Mean values + SE for four perfusions are shown. Significantly greater than fp < 0.05) the corresponding value obtained in the absence of palmitate by Student’s t test (‘1.
occurred in livers perfused with or without palmitate. Figure 2 shows the ratios of lactate disappearance to glucose formation in the perfusate in the presence or absence of palmitate at liver temper-
0 6OWAcAc TEMP PC,
.SY 37
9
37 .60
39
40
41
42
43
Fig. 3. The effect of hyperthermia and palmitate on ketone body production and the ratio of 3-hydroxybutyrate (SOH) to acetoacetate (AcAc) in the perfused rat liver. Ketone bodies were determined in the same experiments as shown in Fig. 1. The ratios were calculated from the concentration of acetoacetate (shaded area) and 3-hydroxybutyrate (unshaded area) in perfusate samples removed at the end of the 80-min experimental period. Lactate, NH&I, and ornithine were added in the absence (striped bars) or presence (unstriped bars) of palmitate. Mean values from four perfusions are shown.
atures from 37” to 43T. A significant in the ratio was found in the presence tate at 43°C.
decrease of palmi-
E#ect of Hyperthermia on Palmitate-Supported Ketogenesis
I
37
I
I
41 39 Temperature fYJ
1
43
Fig. 2. Effect of hyperthermia and palmitate on the ratio of lactate disappearance to glucose formation. Ratios were determined in the same experiments as shown in Fig. 1. The ratio was calculated from the change in perfusate concentrations of lactate and glucose for the SO-min experimental period. Lactate, NH&I, and ornithine were added in the presence (0) or absence (0) of palmitate. Mean values & SE for four perfusions at each condition are shown. Significantly less than (p < 0.05) the corresponding value determined in the absence of palmitate by Student’s t test (‘1.
Palmitate increased total ketone body production about threefold in the presence of gluconeogenic and ureogenic substrates at all temperatures (Fig. 3). Hyperthermia did not alter total ketone body production in the presence or absence of palmitate. Palmitate significantly increased (p < 0.05) the 3-hydroxybutyrate/acetoacetate ratio at all temperatures studied (Fig. 3). As the liver temperature increased, the 3-hydroxybutyrate/acetoacetate ratio progressively decreased in livers with or without palmitate. However, in the presence of palmitate, the ratio at 43’T was similar to the ratios obtained in the absence of palmitate from 37“ to 41°C.
Effect of Hyperthermia and Palmitate on Substrate-Stimulated 0, Consumption Hepatic O? consumption was stimulated by the addition of lactate, ammonia, and ornithine (Figs. 4 and 5). Palmitate did not affect the stimulation of 0, consumption after 40 min of the experimental period from 37” to 42°C; however, at 43”C, substrate-supported 0,
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IN HEPATIC HYPERTHERMIA
consumption was significantly higher in the presence of palmitate (Fig. 4). The time course of 0, consumption at 43°C further illustrates this point (Fig. 5). Palmitate significantly stimulated substrate-supported O2 consumption compared with livers perfused without palmitate at all time points after addition of substrates for gluconeogenesis and ureogenesis. In addition. no stimulation of O2 consumption occurred after addition of lactate, ammonia, and ornithine in the absence of palmitate (Fig. 5). Light Microscop_v of Livers Exposed to Supranormal Temperatures
37
39 41 Temperature toC 1
43
Fig. 4. Effect of hyperthermia on substrate-stimulated 0, consumption 40 mitt into the experimental period. Oxygen consumption was measured at 40 min of the experimental period in the same experiments as described in Fig. 1. Livers were perfused in the presence (0) or absence (0) of palmitata. Lactate 110 m&f). NH&I (10 m&f), and ornithine (2.7 mM) were also present. Significantly greater than (p < 0.05) the corresponding value obtained in the absence of palmitate by split-plot analysis (‘1.
After 2 hr of perfusion at 37OC, the hepatocytes appeared normal; however, mild sinusoidal dilatation was observed. Histologic examination of the liver samples disclosed mild portal and central venous congestion, and mild-to-moderate sinusoidal dilatation from 39°C to 41°C. At 42°C moderate-to-severe portal and central venous congestion and sinusoidal dilatation were apparent. These changes became severe at 43OC (Fig. 6). Hepatic cord separation was mild at 42%; however, it was especially noticeable at 43OC, at which point it varied from moderate to severe. The continuum of pathologic changes occurring in the liver perfused at progressively elevated temperatures was attenuated by palmitate. These differences were especially apparent at 43°C (Figs. 6 and 7). DISCUSSION
0
20
40
60
80
Time fmin) Effects of pelmitate on the time course of Fig. 5. substrate-stimulated 0, consumption at 43°C. Oxygen consumption was determined in the same experiments as described in Fig. 1. Livers were perfused at 43°C in the presence (*I or absence (0) of palmitate. Lactate (10 m/W), NH&I I10 m&f). and ornithine (2.7 mM) were also present. Significantly greater than fp < 0.05) the corresponding value obtained in the absence of pelmitate by split-plot analysis ( 1. l
A major aim of this study was to assess the effect of exogenous palmitate on substratesupported hepatic biosynthetic processes at elevated temperatures considered useful in thermotherapy of cancer. The data show that palmitate can reduce the detrimental effects of liver perfusion on hepatic metabolic parameters at 43°C. The protective effect of palmitate may be interpreted by present knowledge on hepatic metabolic control at normal temperatures. Menahan and Wieland” reported that the ratio of pyruvate disappearance to glucose formation increased when endogenous liver triglyceride hydrolysis is inhibited, indicating a diversion of C, intermediates from glucose formation to oxidation. Presumably, pyruvate oxidation substituted for fatty acid oxidation under these conditions. in this study, hepatic lactate use was
COLLINS, MITROS, AND SKIBBA
Fig. 6. Liver perfused for 66 min at 43°C with lactate, NH,CI. and ornithine supplementation. Sinusoidal dilatation. erythrocyte congestion, and hepatic cord separation are severe in the centrolobular region. fHematoxylin and eosin x 266).
stimulated at supranormal temperatures, suggesting increased diversion of C, intermediates to oxidation at hyperthermic temperatures. It is generally accepted that hepatic fatty acid metabolism and gluconeogenesis are related.2’-24 Thus, the effects of fatty acid supplementation on hepatic biosynthetic processes at elevated temperatures were investigated. As previously reported, endogeneous ketogenesis was minimal in perfused rat liver; however, palmitate supplementation significantly increased ketone body
Fig. 7. Liver perfused for 80 min at 43°C with lactate, NHJX ornithine. and palmitate supplementation. Severe sinusoidal dilatation, moderate congestion and mild hepatic cord separation are present in the centrolobuler area. (Hematoxylin and eosin x 260).
production8 We now report that addition of pdmitate to the perfusate, supplemented with lactate and ammonia, partially alleviated excess use of Iactate under hyperthermic conditions. Therefore, it appears that lactate provided sufficient C, intermediates to support hepatic energyrequiring processes from 37” to 43°C. Furthermore, at all temperatures, including 43°C acetyl-CoA was derived from palmitatesuppressed C, oxidation. The proportion of pyruvate dehydrogenase in
PALMITATE
IN HEPATIC HYPERTHERMIA
the active form is very low in the fasted state.‘5.26 Among the factors controlling the conversion of pyruvate dehydrogenase to the active state, the mitochondrial NADH/NAD ratio is of major importance.‘7,2x A decrease in the 3-hydroxybutyrate/acetoacetate ratio is indicative of a more oxidized mitochondrial redox state,29 resulting in an increase in the proportion of pyruvate dehydrogenase in the active state. In the presence or absence of palmitate, the 3-hydroxybutyrate/acetoacetate ratio progressively decreased as the temperature was increased from 37” to 43°C. This lends support to the reasoning that pyruvate entrance into the tricarboxylic acid cycle is increased and presumably oxidized at elevated temperatures. In addition, the 3hydroxybutyrate/acetoacetate ratio in the presence of palmitate at 43°C was similar to that found in livers perfused in the absence of palmitate from 37O to 41°C. Thus, it appears that metabolism of exogenous palmitate can suppress C, unit oxidation at supranormal temperatures. Zimmermann et al? followed the effect of varying temperature from lo to 37OC on gluconeogenesis, ketogenesis, and CO, production in perfused livers. A divergence in the Qlo for ketogenesis and CO, production from octanoate occurred between 25” and 37°C. Oxidation of octanoate increased, causing lower rates of ketogenesis, as the liver temperature approached 37OC. The present findings appear to extend this relationship to supranormal temperatures. Hepatic gluconeogenesis from lactate is normally stimulated in the presence of exogenous fatty acid.“m33 Nevertheless, no stimulation of gluconeogenesis from lactate in the presence of palmitate when ammonia and ornithine were also present was found in this investigation. These observations agree with Wojtczak et al.,34 who studied isolated hepatocytes under similar substrate conditions. Since the pathways for gluconeogenesis and ureogenesis in the liver are interrelated,34 37 it has been suggested that competition between common intermediates in the cytosolic and mitochondrial compartments may be related to this finding.34m38 Oxygen consumption is used to assess the functional integrity of perfused livers3’ At 43*C, substrate stimulation of O2 consumption did not occur. However, palmitate significantly en-
529
hanced 0, consumption at 43°C after addition of gluconeogenic and ureogenic substrates. The data suggest that exogenous palmitate can be oxidized at 43”C, providing the necessary energy to partially alleviate deterioration in biosynthetic processes. Maximal rates of gluconeogenesis and ureogenesis can occur simultaneously at normothermic temperatures;7.34 however, the temperature dependency of metabolic reactions dictates that energy metabolism be stimulated as absolute temperature increases. Thus, measurement of these processes provides an excellent test of the functional integrity of the liver at elevated temperatures. Biosynthetic processes were maintained from 37O to 42°C. A sharp decline in biosynthetic function occurred between 42” and 43OC, suggesting a depression in hepatic ATP supply. However, palmitate supplementation partially suppressed hepatic deterioration at 43°C. Presumably, palmitate can supply the ATP required to maintain hepatic integrity under hyperthermic stress. The increased energy requirements for maintenance of biosynthetic processes at elevated temperatures could be explained by several metabolic alternatives. An increase in futile cycling would result in excessive consumption of ATP. Indeed, Williams et a1.j’ reported increased futile cycling in livers perfused at 42°C. An alternative explanation. the uncoupling of oxidative phosphorylation, would result in inefficient generation of ATP. Christiansen and Kvamme4’ found that uncoupling of oxidative phosphorylation was temperature-dependent in liver mitochondria exposed to a temperature of 41”-45°C for 10 min. However. Carlsson et aL4* found no change in ATP, ADP, AMP, and phosphocreatine in the cerebral cortex of rats subjected to whole body hyperthermia (41.8”C for 30 min). Individual enzymes can be inactivated at 40°-430C,43,44 although the inactivation is reversible. Contribution of this factor to hepatic energy metabolism is difficult to predict because of the biochemical complexity of the liver and the fact that most enzymes require much higher temperatures for inactivation. Manipulation of normal tissue metabolism under hyperthermic conditions may lead to improvement in antineoplastic selectivity of
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COLLINS, MITROS, AND SKIBBA
hyperthermia. To support this concept, additional experiments are planned to examine the role of palmitate as an energy substrate and attenuator of metabolic deterioration at supranormal temperatures.
ACKNOWLEDGMENT The authors thank Dr. Lawrence A. Menahan for his helpful comments on the manuscript, Jean V. Collins for her expert technical assistance, and Mimi Mick for her help in preparation of the manuscript.
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serum or plasma without deproteinization. Am J Med Technol33:361-365, 1967 19. Peterson RE, Olson JR, Fujimoto JM: Measurement and alteration of the capacity of the distended biliary tree in the rat. Toxicol Appl Pharmacol36:353-368, 1976 20. Steel RG, Torrie JH: Principles and Procedures of Statistics. New York, McGraw-Hill, 1960, pp 232-241 21. Menahan LA, Wieland 0: The role of endogenous lipid in gluconeogenesis and ketogenesis of perfused rat liver. Eur J Biochem 9: 182-l 88, I967 22. Exton JH: Gluconeogenesis. Metabolism 21:945-990, 1972 23. Parrilla R, Ayuso-Parrilla MS, Williamson JR: Use of endogenous triglycerides to support gluconeogenesis in perfused rat liver. Pfluegers Arch 366:2l l-216. 1976 24. Williamson JR, Rostand SG, Peterson MJ: Control factors affecting gluconeogenesis in perfused rat liver. Effects of 4-pentanoic acid. J Biol Chem 245:2342-3251. 1970 25. Patzelt C, Loffler G. Wieland OH: Interconversion of pyruvate dehydrogenase in the isolated perfused rat liver. Eur J Biochem 33: I 17-l 22, 1973 26. Wieland OH, Lother G, Patzelt C, et al: Regulation of pyruvate dehydrogenase interconversion in liver, in Lundquist F, Tygstrue N (eds): Regulation of hepatic metabolism. New York, Academic, 1974. pp 62-78 27. Wieland 0, von Jagow-Westermann B, Stukowski B: Kinetic and regulatory properties of heart muscle pyruvate dehydrogenase. Hoppe Seylers Z Physiol Chem 350:329334, 1969 28. Denton RM, Randle PJ, Bridge BJ. et al: Regulation of mammalian pyruvate dehydrogenase. Mol Cell Biochem 9~27-53, 1975 29. Williamson DH, Lund P, Krebs HA: The redox state of free nicotinamide-adenine dinucleotide in the cytoplasm and mitochondria of rat liver. Biochem J 103:5 14-527, I967 30. Zimmermann FA, Dietz HG, Sippell WG, et al: Temperaturabhangigkeit von stoffwechselgrossen in der perfundierten rattenleber. Res Expl Med (Berl) 168:57-64, 1976 3 1. Ross BD, Hems R, Freedland A. et al: Carbohydrate metabolism in the perfused rat liver. Biochem J 105:869875. 1967 32. Williamson JR, Browning ET, Scholz R: Control of mechanisms of gluconeogenesis in perfused rat liver. J Biol Chem 244:4607-4616, 1969 33. Williamson JR, Scholz R, Browning ET: Control mechanisms of gluconeogenesis and ketogenesis. II. Interactions between fatty acid oxidation and the citric acid cycle in perfused rat liver. J Biol Chem 244:46 17-4627, 1967 34. Wojtczak AB, Walajtys-Rode EI, Geelen MJH: Interrelations between ureogenesis and gluconeogenesis in
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isolated hepatocytes. The role of anion transport and the competition for energy. Biochem J 170:379-385, 1978 35. Briggs S, Freedland RA: Effect of ornithine and lactate on urea synthesis in isolated hepatocytes. Biochem J I60:205--209, 1976 36. Krebs HA, Lund P, Stubbs M: Interconversions between gluconeogenesis and urea synthesis, in Hanson RW. Mehlman MA (eds): Gluconeogenesis: It’s Regulation in Mammalian Species. New York. Wiley, 1976, pp 2699291 37. Meijer AJ, Gimpel JA, Deleeuw G, et al: Interrelationships between gluconeogenesis and ureogenesis in isolated hepatocytes. J Biol Chem 253:2308-2320. 1978 38. Williamson JR, Meijer AJ, Ohkawa K: Interrelations between anion transport, ureogenesis and gluconeogenesis in isolated rat cells, in Lundquist E, Tygstrup N (eds): Regulation of Hepatic Metabolism. New York, Academic, 1974, pp 457-48 I 39. Ross BD: Perfusion Techniques Oxford. Clarendon, 1972, pp 1355220 40. Williams JF. Cook PC, Matthaei
in Biochemistry. KI, et al: Pyridine
and adenine nucleotide ratios and futile substrate cycling in regulation of energy metabolism and proposed hyperthermic regression of neoplasms, in Criss WE, Ono T, Sabine J (eds): Control of Mechanisms in Cancer. New York. Raven. 1976, pp 4255439 41. Christiansen EN, Kvamme E: Effects of thermal treatment on mitochondria of brain, liver and ascites cells. Acta Physiol Stand 76:472-484, 1969 42. Carlsson C, Hagerdal M, Siesjo BK: The effect of hyperthermia upon oxygen consumption and upon organic phosphates, glycolytic metabolites. citric acid cycle intermediates and associated amino acids in rat cerebral cortex. J Neurochem 26:1001-1006, 1976 43. Anson ML, Mirsky AE: The equilibrium between active native trypsin and inactive denatured trypsin. J Gen Physiol 17:393-398, 1934 44. Stearn AE: Kinetics of biological reactions with special reference to enzymatic processes, in Nord FF (ed): Advances in Enzymology, vol 19. New York. Interscience. 1949, pp 25-74