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Metabolic responses to experimental diving in adult and fetal weddell seals Murphy, Brian Joseph 1980

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METABOLIC RESPONSES TO EXPERIMENTAL DIVING IN ADULT AND FETAL WEDDELL SEALS by BRIAN JOSEPH MURPHY B.Sc, Memorial Uni v e r s i t y of Newfoundland, St. John's, 1972 M.Sc, Memorial Uni v e r s i t y of Newfoundland, St. John's, 1975 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the THE.--FACULTY OF GRADUATE STUDIES DEPARTMENT OF ZOOLOGY We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March, 1980 ' ( c ) Brian Joseph Murphy I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l m a k e i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e H e a d o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s . t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a 2 0 7 5 Wesbrook Place Vancouver, Canada V6T 1W5 ABSTRACT Metabolic p o t e n t i a l s and a c t i v i t i e s of the heart, lung and brain were examined i n the A n t a r c t i c Weddell s e a l , a species displaying outstanding diving a b i l i t i e s . The a c t i v i t i e s of representative enzymes i n oxidative and fermentative metabolism i n both the adult and near-term fetus are s i m i l a r to those i n homologous organs of the adult ox, but the brain and heart i n both groups of seals contained elevated l e v e l s of l a c t a t e dehydrogenase. The isozyme data ind i c a t e that a l l three organs i n the adult and fetus have the p o t e n t i a l for either l a c t a t e production or u t i l i z a t i o n depending upon metabolic conditions. During experimental diving (up to 30 min) glucose appears to be a c r i t i c a l carbon and energy source for the adult. Glucose i s u t i l i z e d i n a mixed aerobic and anaerobic metabolism. The consequent f a l l i n blood glucose l e v e l s and r i s e i n l a c t a t e l e v e l s are due predominantly to peripheral, hypoperfused tissues, but the c e n t r a l organs influence these metabolite pools as w e l l . The brain of the adult s e a l u t i l i z e s glucose at a rate of approximately 0.3 ymol/gm/hr., releasing 20-25% as l a c t a t e ; t h i s proportion does not change throughout diving-recovery cycles. The lung consumes l a c t a t e and thus diminishes i t s accumulation i n the blood during the dive. One,.and possibly the main, fate of l a c t a t e 14 absorbed by the lung i s oxidation since CO^ i s the only measurable 14 d e r i v a t i v e found i n a o r t i c blood following C-lactate i n f u s i o n into the r i g h t v e n t r i c l e . During recovery, when blood l a c t a t e l e v e l s r i s e above 6 ymol/ml, the b r a i n switches from l a c t a t e release to l a c t a t e uptake at a rate high enough to r e a d i l y support the normal metabolic rate of t h i s organ (about 8 ymol ATP/gm/hr). Enzyme and metabolite measurements suggest that the lung and heart also contribute to l a c t a t e clearance and re-establishment of metabolic homeostasis following diving i n the adult. Blood metabolite analyses suggest l i t t l e involvement of the majority of free amino acids during dive-recovery cycles. Only alanine and glutamine increase throughout both phases and both probably function as waste nitrogen c a r r i e r s . Alanine accumulation could also be caused by anerobic catabolism of protein and/or anaerobic-aerobic t r a n s i t i o n s i n tissues such as s k e l e t a l muscles. Both the p h y s i o l o g i c a l data and metabolite recovery p r o f i l e s i n d i c a t e f e t a l p e r i p h e r a l vasoconstriction and a f u l l y developed diving response as a consequence of maternal d i v i n g . The fetus has apparently adapted to long-duration diving by s i g n i f i c a n t l y increasing i t s blood storage capacity of glucose to beyond that of the mother. Furthermore, these elevated stores appear to be under t i g h t regulation during the l a t t e r stages of experimental diving. X V TABLE OF CONTENTS Page Abstract L i s t of Tables v i - i i L i s t of Figures -x L i s t of Schemes x i i Acknowledgements . ; . x m Chapter I: Introduction 1 Central Organs 4 Substrates for the mammalian heart 5 Heart metabolic biochemistry i n diving animals 8 Cerebral metabolic rate 9 Substrates for the mammalian brain 10 Ci r c u l a t o r y and metabolic consequences of brain hypoxia 12 Brain metabolism i n diving animals 15 Substrates f o r the mammalian lung 17 Heart-lung-brain i n the Weddell seal 22 Fe t a l metabolism 23 Heart metabolism 25 Brain metabolism 25 F e t a l metabolism i n the diving mammal 26 Chapter I I : Materials and Methods 28 Experimental animals and i n vivo manipulation 28 Enzyme extraction and assay 31 Lung s l i c e studies 34 Metabolite assays 35 Amino acid analysis 36 Chapter I I I : Blood Metabolite P r o f i l e s as a Consequence of Diving Introduction Results and Discussion Blood glucose, pyruvate and l a c t a t e p r o f i l e s Free amino acids and glutathione i n r e s t i n g animals Diving p r o f i l e s of amino acids and glutathione Chapter IV: Enzymes of Aerobic and Anaerobic Metabolism i n the Brain, Heart and Lung of the Weddell Seal Introduction Results and Discussion Oxidative enzymes Enzymes of anaerobic g l y c o l y s i s Glucose-6-phosphatase Chapter V: Impact of Diving on Lung Metabolism Introduction Results and Discussion 'In vivo' preparation Tissue s l i c e studies Chapter VI: Impact of Diving and Recovery on Cerebral Metabolism Introduction v i Page Results and Discussion 76 A-V concentration differences 76 Lactate uptake by the brain 80 Chapter VII: Fetal Responses to Maternal Diving 85 (i) Blood metabolite profiles as a consequence of maternal diving 85 Introduction 85 Results and Discussion 87 Blood glucose, pyruvate and lactate profiles 87 Blood amino acid profiles in resting animals 92 Diving profiles of blood amino acids 98 ( i i ) Unusual maternal-fetal glucose concentrations in whole blood of the Weddell seal 99 Introduction 99 Results and Discussion 99 Glucose tolerance test 100 ( i i i ) Enzymes of aerobic and anaerobic metabolism in the three central organs of the fetal Weddell seal 105 Introduction 105 Results and Discussion 106 Enzymes of oxidative metabolism 106 Enzymes of anaerobic glycolysis 107 v i i Page Chapter VIII: Summating Remarks 111 Central organ metabolism as a consequence of diving 111 Recovery 120 Fe t a l responses to maternal diving 125 L i t e r a t u r e Cited 126 Appendix: L i s t of Abbreviations 140 v i i l LIST OF TABLES Table • Page I I I , 1 Whole blood concentrations (uM) of free amino acids i n male Weddell seals, harbor seals and human volunteers 46 II I , 2 S t a t i s t i c a l evaluation of mean differences of amino acid l e v e l s presented i n Table I I I , 1 47 I I I , 3 Mean percentage changes i n whole blood glutamate and glycine as functions of GSH changes during diving-recovery cycles 55 IV, 1 Enzyme a c t i v i t i e s i n brain, heart and lung of the adult Weddell seal 60 14 14 V, 1 production from C-U-lactate and from 14 C-6-glucose by lung s l i c e s of the Weddell seal 71 VI, 1 Whole blood glucose and l a c t a t e concentration gradients across the brain of the Weddell seal before and during diving 77 VII, 1 Mean a r t e r i a l concentrations of amino acid concentrations i n whole blood of the Weddell seal and human feto-maternal p a i r s 93 VII, 2 S t a t i s t i c a l evaluation of mean differences of amino acid concentrations as presented i n Table VII, 1 94 VII, 3 Fetal/maternal r a t i o s for whole blood amino acid concentrations i n the Weddell seal and the human 96 VII, 4 F r a c t i o n a l d i s t r i b u t i o n of glucose between plasma and red blood c e l l s i n maternal and f e t a l Weddell ix Page seal compared with sheep 104 VII, 5 Enzyme a c t i v i t i e s i n brain, heart and lung of the f e t a l Weddell seal 108 X LIST OF FIGURES Figure Page III, 1 Change in glucose concentration in whole ar t e r i a l blood during diving and recovery 40 III, 2 Change in lactate concentration of whole art e r i a l blood during diving and recovery 41 III, 3 Change in pyruvate concentration of whole arterial blood during diving and recovery 42 III, 4 Change in lactate/pyruvate concentration ratio of whole art e r i a l blood during diving and recovery 44 III, 5 Percentage change of alanine levels in whole arte r i a l blood during diving and recovery 50 III, 6 Percentage change of glutamine levels in whole arterial blood during diving and recovery 51 III, 7 Change in glutathione concentration in whole blood during diving and recovery 53 IV, 1 Starch gel electrophoretic separation of lactate dehydrogenase ioszymes in skeletal muscle, lung, red blood cel l s , heart and brain of the Weddell seal 62 14 V, 1 U- C lactate oxidation by the Weddell seal lung 'in vivo' 68 VI, 1 Change in lactate concentration in arterial and epidural blood samples during diving and recovery 82 VI, 2 Lactate concentration changes in arterial and epidural venous blood following lactate infusion 83 x i Page VII, 1 Glucose, lactate and pyruvate concentration changes in maternal and fetal blood during two diving and recovery episodes 88 VII, 2 Glucose, lactate and pyruvate concentration changes in maternal and fetal blood during a diving and recovery episode 89 VII, 3 A plot of glucose concentrations in whole blood of five maternal-fetal pairs 101 VII, 4a Maternal and fetal concentrations of glucose in whole blood following infusion of glucose 103 VII, 4b Maternal glucose and insulin and fetal glucose and insulin in plasma from same glucose tolerance test as shown in VII, 4a 103 VII, 5 Starch gel electrophoretic separation of lactate dehydrogenase isozymes in heart and brain of fetal and adult Weddell seal 110 x i i LIST OF. SCHEMES Scheme Page V, 1 Diagrammatic representation of the Weddell seal c i r c u l a t i o n 67 VIII, 1 Diagrammatic model of some important metabolic int e r a c t i o n s between c e n t r a l blood, brain, lung, heart and other hypoperfused tissues during diving 119 VIII, 2 Diagrammatic model of some of the metabolic int e r a c t i o n s between the brain, lung, heart, l i v e r , kidney and blood during recovery from diving 123 xi±i ACKNOWLEDGEMENTS Thank you, Peter, f o r p u l l i n g the r e p t i l i a n scales from my eyes; comparative biochemistry sure beats r a t l i v e r studies. Your guidance and friendship w i l l forever be appreciated and yes, Peter, you j u s t may have saved me from myself. I further o f f e r my sincere appreciation to Drs. Warren Zapol, Mont Liggins, b i g Mike Snider, Bob Schneider, Bob Creasy and Jesper Qvist and the rest of our An t a r t i c research group. Without your s u r g i c a l and pro f e s s i o n a l help most of t h i s thesis would not have been r e a l i z e d . I also thank the boys from Sea World who did so much f o r both Peter's and my sanity, while we were on the i c e . A very fond greeting i s extended to Peter's cosmopolitan research group. I t ' s been a r e a l joy rubbing shoulders with so may future Nobel Laureates! You were always there to lend a helping hand and that w i l l never be r forgotten by me (nor you, I f e a r ) . I must also thank Mary, f or constantly b a i l i n g me out of the numerous c r i s e s associated with the amino acid analyzer and Fay, f o r not only typing the manuscript but for also correcting my atrocious s p e l l i n g . A vote of thanks i s offered to my examining and supervisory committee for wading through the the s i s ; i t c e r t a i n l y was not an enviable task. F i n a l l y , I must thank a l l my friends here i n Vancouver for helping to make the l a s t f i v e years the most enjoyable of my l i f e . I CHAPTER I Introduction l a Although l i f e i s thought to have evolved from non-living organic matter, i n a reducing atmosphere, most organisms now depend upon oxygen (C^) as the ultimate proton acceptor. However, the degree of dependency d i f f e r s not only from animal to animal but also between d i f f e r e n t tissues of the same animal. I t has now been f i r m l y established that many invertebrates and lower vertebrates (e.g. t u r t l e s ) reduce t h e i r metabolic rates and/or substitute 0^ with other favorable proton disposal sinks i n an e f f o r t to survive periods of low oxygen a v a i l a b i l i t y . F a c u l t a t i v e anaerobes, such as i n t e r t i d a l bivalves, p a r a s i t i c helminths and annelids, sustain extremely lengthy periods of anoxia by modifying and integrating g l y c o l y s i s with other catabolic pathways (e.g. amino acid catabolism), r e s u l t i n g i n the accumulation of succinate, alanine, propionate etc. These adaptations allow f o r substrate-linked ATP production, redox balance and the avoidance of an 0^ debt upon recovery; a l l of which provide the animal with the opportunity to invade habitats that would otherwise prove h o s t i l e . Hochachka (1980) reviews current thinking i n t h i s area. Mammals, on the other hand, display much higher dependencies on 0^ and hence must compensate for hypoxia or anoxia by evoking a wide v a r i e t y of 0^ conserving mechanisms, i f they are to be successful i n e x p l o i t i n g such habitats. Marine mammals o f f e r the perfect example of such an adaptation. These animals probably made the t r a n s i t i o n from a t e r r e s t r i a l to an aquatic existence i n an e f f o r t to secure an e c o l o g i c a l niche safe from the interferences and predation of dry-land competitors. Because of t h e i r diving habit they have evolved large blood volumes with very high hematocrits and have increased muscle myoglobin content, a l l of which 2 provide a large 0^ r e s e r v o i r . However v i t a l these may be to the o v e r a l l s u r v i v a l of the organism they must be of secondary importance when compared to the three components of what i s commonly termed the 'diving response'. This consists of apnea (cessation of breathing), bradycardia (slowing of the heart rate) and peripheral vas o c o n s t r i c t i o n , a l l of which a i d i n profoundly lowering the extraction of 0^ from the blood. Bradycardia leads to a drop i n cardiac output while peripheral v a s o c o n s t r i c t i o n serves to maintain a r t e r i a l blood pressure and r e d i s t r i b u t e blood flow to the most needy tissues which were o r i g i n a l l y thought to include the heart, lung and brain (Scholander, 1940). It i s thought that a large f r a c t i o n of the peripheral c i r c u l a t i o n i s probably r e s t r i c t e d upon i n i t i a t i o n of these cardiovascular adjustments (Irving et^ al., 1942) . Studies u t i l i z i n g microsphere, angiogram and doppler blood flow transducer techniques indicated a more complex s i t u a t i o n . Blood flow determinations i n restrained harbor (Phoca v i t u l i n a ) , elephant (Mirounga  angustuostris) and Weddell seals (Leptonychotes weddelli) suggested that cerebral blood flow (CBF) remains unchanged or decreases s l i g h t l y during a dive (Bron et a l . , 1966; Eisner et a l . , 1966; Kerem et a l . , 1971; Van C i t t e r s et a l . , 1965; Zapol jst _ a l . , 1979). On the other hand, a recent study by Eisner et a l . (1978) demonstrated CBF increases during the l a t t e r stages of diving i n the common harbor seal; s ubstantial CBF increases during simulated diving i n the sea l i o n (Zalophus c a l i f o r n i a n u s ) and the duck (Anas platyrhynchos) have also been reported (Dormer ert al_., 1977; Jones et^ a l . , 1979). In the pinnipeds sampled the myocardial blood flow was found to be reduced to only 7-15% of the pre-dive l e v e l ( B l i x et a l . , 1976; Zapol et a l . , 1979). At present nothing i s known about the a l t e r a t i o n s ( i f any) i n bronchial 3 flow during diving; however, pulmonary flow must decrease since the cardiac output decreases s i g n i f i c a n t l y i n the diving phase. In the Weddell s e a l , at l e a s t , the unaltered CBF implies that the brain enjoys a normal d e l i v e r y of and carbon substrates. Furthermore, a l l other tissues of t h i s A n t a r c t i c seal experience apparent decreases i n absolute flow rates (Zapol et a l . , 1979) which may be consistent with the suggestion that the metabolic rate of the animal a c t u a l l y drops somewhat during diving (Anderson, 1966). In terms of f r a c t i o n a l cardiac output, four organs are i d e n t i f i e d as receiving an increased r e l a t i v e perfusion: the heart, lung, brain and adrenal glands. Peripheral organs such as the kidney, sustain sharp reductions i n absolute perfusion, f r a c t i o n a l cardiac output and hence i n oxygen and nutrient supply. Consequently, these peripheral tissues must sustain lengthy periods of hypoxia and anoxia. For a thorough review on the metabolic or biochemical adjustments involved, r e f e r to Hochachka and Murphy (1979). 4 Central organs Although the p h y s i o l o g i c a l purpose and consequences of the diving response have been treated by many others (see above), remarkably l i t t l e has been done on the metabolic capacities and a c t i v i t i e s of the c e n t r a l organs. However, there are scattered reports on blood metabolite and p h y s i o l o g i c a l parameters i n these mammals, which o f f e r some hints on the metabolic status of the brain, heart and lung as a consequence of diving. Before a dive the C^ content i n the venous c i r c u l a t i o n of the sea mammal increases, presumably for use during the dive episode. The a r t e r i a l p a r t i a l pressure of 0^ (PaC^) f a l l s to 30 mm Hg or lower throughout prolonged diving (Anderson, 1966; Scholander, 1940; Hochachka et a l . , 1977a; Kerem and Eisner, 1973). In the Weddell seal Ba02 asymptotes have been observed to occur i n the range of 25-30 mm Hg (Hochachka et a l . , 1977a). Hochachka's group further reported blood glucose l e v e l s did not f a l l monotonically but rather approached an asymptote i n prolonged simulated di v i n g . The early decreases i n both glucose and 0^ l e v e l s suggest mixed aerobic and anaerobic metabolism while the asymptotic behavior of both i n l a t e r diving implies the i n i t i a t i o n of some type of steady state system, protecting the c e n t r a l organs ( i n p a r t i c u l a r the brain) against hypoglycemia and anoxia. The precise mechanisms have yet to be ascertained. Past studies have not only detected the c l a s s i c washout of l a c t a t e from peripheral tissues into the c e n t r a l c i r c u l a t i o n during the recovery phase (Scholander, 1940) but also demonstrated a 2-4 f o l d increase i n l a c t i c acid i n t h i s c e n t r a l c i r c u l a t i n g blood during the dive (Hochachka e t a l . , 1977a; Davis and Kooyman, 1980). This i n i t i a l increase i n l a c t a t e could be due to anaerobic fermentation i n one or more of the c e n t r a l organs and/or leakage of the 5 anaerobic end-product out of tissues (such as muscles) that have been i s o l a t e d from the main c i r c u l a t i o n . Beyond these previously published insi g h t s very l i t t l e i s known of the ' i n v ivo' metabolic a c t i v i t y of the functioning c e n t r a l organs. Many outstanding questions remain to be answered. For example: 1) what f u e l s were burned by which tissues and at what times during and a f t e r diving? 2) Does cerebral metabolic rate decrease during diving episodes? 3) How prevalent i s i n t e r t i s s u e metabolic cycling? Are there any metabolites cycled, and, i f so, which one(s) and between which tissues? At the onset of t h i s research i t was r e a l i z e d that complete answers to these questions would be impossible but, nevertheless, a beginning could be made to unravel some of the complex int e r a c t i o n s involved. To put such questions i n t h e i r proper perspective, i t i s u s e f u l to b r i e f l y review what i s currently known of the metabolic biochemistry of the three c e n t r a l t i s s u e s . Substrates for the Mammalian Heart The mammalian heart i s considered to be a completely aerobic organ when i n r e s t i n g metabolism, or when only moderately a c t i v e (Neely and Morgan,1974). This f a c t i s further emphasized by the observation that 40% of the t o t a l c e l l u l a r space i s occupied by mitochondria (Fawcett and McNutt, 1969) . When there i s an ample supply of oxygen, the heart w i l l p r e f e r e n t i a l l y oxidize f a t t y acids, mostly i n the form of plasma free f a t t y acids, but i f these are unavailable, i t can burn glucose or l a c t a t e (Most ej: a l . , 1969; Neely et a l . , 1972). In hypoxic or anoxic hearts the preferred f u e l i s glucose. Such a s i t u a t i o n r e s u l t s i n a ten- to twenty-fold increase 6 i n g l y c o l y t i c f l u x (Neely and Morgan, 1974). In the well oxygenated fat-burning heart g l y c o l y s i s i s i n h i b i t e d at four l o c i . F i r s t l y , there appears to be an i n h i b i t i o n of glucose transport by free f a t t y acids. Secondly, the combined e f f e c t s of high l e v e l s of ATP, G6P and decreased l e v e l s of 5'-AMP and inorganic phosphate i n h i b i t glycogen phosphorylase and hexokinase. T h i r d l y , increased concentrations of c i t r a t e and ATP and decreased concentrations of fructose biphosphate, NH* and AMP during f a t t y acid oxidation e s s e n t i a l l y turn off g l y c o l y s i s by i n h i b i t i n g phosphofructokinase. This e f f e c t i v e l y d i r e c t s exogenous glucose into glycogen. And f i n a l l y , high l e v e l s of acetylCoA during f a t t y acid oxidation i n h i b i t the pyruvate dehydrogenase enzyme complex. Fat oxidation, also, appears to convert t h i s enzyme into i t s i n a c t i v e enzymatic form (Neely and Morgan, 1974). During periods of hypoxia and anoxia cardiac glycogenolysis and g l y c o l y s i s are activated by a re v e r s a l of the above e f f e c t s while f a t t y acid oxidation i s retarded. Apparently, hypoxic and anoxic stresses i n the mammalian heart are accompanied by a p a r t i a l r e v e r s a l of the Krebs cycle with a r e s u l t i n g accumulation of succinate and alanine (Penney and Cascarano, 1970; Sanborn et a l . , 1979): malate •• fumarate •• succinate The malate may a r i s e from aspartate through a transamination: Aspartate fe oxaloacetate femalate fe succinate ^glutamate 7 a process that could p a r t i a l l y explain the accumulation of alanine that sometimes occurs during hypoxic or anoxic stress (Hochachka et^ a l . , 1975; F e l i g and Wahren, 1971). C o l l i c u t t and Hochachka (1975) have shown that t h i s e f f e c t i v e "fermentation" of aspartate to succinate i s a very a c t i v e process i n bivalve hearts during anoxia, and there i s also some evidence for i t s contribution to metabolism during the hypoxia associated with diving i n marine mammals (Hochachka et a l . , 1975). Sanborneand associates (1979) using 14 C.-aspartate v e r i f i e d t h i s pathway to be operable i n anoxic rabbit heart. Succinate can, of course, also be formed from intermediates i n the f i r s t span of the Krebs cycle. Indeed from recent studies i t i s known that acetyl-CoA i s quickly depleted i n hypoxia, as are c i t r a t e and ketoglutarate (Neely e£ a l . , 1976). In the anoxic bivalve heart ( C o l l i c u t t , 1975) and 14 rabbit p a p i l l a r y muscle (Sanborn et a l . , 1979), the C glutamate that i s taken up i s converted to succinate. This probably occurs v i a the glutamate-alanine transamination: Glutamate^ '-^ketoglutarate : fesuccinyl-CoA fc succinate pyruvate alanine (Hochachka et a l . , 1975; C o l l i c u t t and Hochachka, 1977, Taegtmeyer, 1978; Sanborn et a l . , 1979). From these d i f f e r e n t studies, i t i s t e n t a t i v e l y concluded that when 0^ supply i s l i m i t e d succinate acts as a sink for carbon flowing from both arms of the Krebs cycle. Each span operates u n t i l intermediates (and C^) are l a r g e l y depleted. I t i s now assumed that the second span i s reversed to allow for redox balance and substrate phosphorylation. In theory, both malate dehydrogenase, functioning i n the oxaloacetate > malate d i r e c t i o n , and fumarate reductase, functioning i n the fumarate >succinate 8 d i r e c t i o n , contribute to redox balance. Furthermore, fumarate reduction i s thought to be coupled to a phosphorylation, one mole of ATP being formed/mole of succinate accumulated and under hypoxic conditions when energy production may be at a premium, t h i s small rate of mitochondrial ATP production while probably i n s i g n i f i c a n t i n o v e r a l l energy budget may be l o c a l l y most u s e f u l . Cascarano and h i s coworkers (1976) stated that i t i s fundamental to I | sustaining Ca metabolism of the heart during anoxic s t r e s s . I t has been suggested (Hochachka and Murphy, 1979) that FADH^-linked fumarate reductase could p o s s i b l y form a redox couple with acylCoA dehydrogenase ( f a t t y a c i d oxidation enzyme), linked by the el e c t r o n transport system (ETS): fumarate• —•hydroxyacylCoA If t h i s postulated couple i s operational during hypoxia and anoxia i t would imply that the FAD +-linked dehydrogenase i n the S-oxidation of f a t t y acids i s not a s i g n i f i c a n t control point during the aerobic-anaerobic t r a n s i t i o n . The precise regulatory locus has yet to be ascertained but i t i s believed that the f i r s t step to be "turned o f f " i n 3-oxidation when 0^ i s l i m i t i n g would be the NAD - l i n k e d dehydrogenase (see Hochachka ejt a l . , 1977b). Heart Metabolic Biochemistry i n Diving Animals Unlike the w e l l developed f i e l d b r i e f l y summarized above, there are only scattered reports i n the l i t e r a t u r e on the metabolic biochemistry of 9 the heart i n diving animals. Electrophoretic work and pyruvate saturation k i n e t i c s of l a c t a t e dehydrogenase (LDH) from the hearts of common seals (Phoca v i t u l i n a v i t u l i n a L . ) , beaver (Castor f i b e r ) , pond t u r t l e (Pseudemys  s c r i p t a elegans), eider (Somateria mollissima), the hooded seal (Cystophora  c r i s t a t a ) and several d i f f e r e n t species of whales have been reported ( B l i x et a l . , 1970; Messelt and B l i x , 1976; Altman and Robin, 1969; B l i x and From, 1971; Shoubridge et al., 1976). A l l studies have demonstrated that the isozyme d i s t r i b u t i o n and/or the pyruvate saturation k i n e t i c s i n the hearts of divers approach the muscle type enzyme (LDH-5), implying a greater p o t e n t i a l for anaerobic g l y c o l y s i s (Holbrook eit_ al_., 1975). The hearts of non-diving mammals are known to contain predominantly heart type LDH subunits (Appella and Markert, 1961) . Simon et al_. (1974) showed that there was increasing pyruvate kinase a c t i v i t y i n both the hearts and brains of three d i f f e r e n t seals with increasing diving time. This, they suggested, implies enhanced g l y c o l y t i c p o t e n t i a l of "aerobic t i s s u e s " and may be important i n extending diving time. Kerem et a_l. (1973) found that glycogen concentrations i n the cardiac muscle of the Weddell seal are 2-3 times higher than i n the hearts of t e r r e s t r i a l mammals, again suggesting an enhanced anaerobic capacity. However, these studies merely put the heart of diving animals into perspective and supply l i t t l e information on i t s metabolic status through diving-recovery c y c l e s . Cerebral Metabolic Rate Although the mammalian brain i s a r e l a t i v e l y small organ, i t s basal oxidative metabolic rate (CMRO2) i s high and can account for as much as 20% of the basal metabolic rate of the organism. The human brain u t i l i z e s 0^ at an average rate of about 1.5 umol/gm/min at 37°C, with the more a c t i v e cerebral cortex metabolizing at a s u b s t a n t i a l l y higher rate. Cerebral 10 metabolic rate, also seems to be influenced by the s i z e of the organism, being high i n small animals. The occurrence of s o - c a l l e d "vulnerable areas" i n the b r a i n plus t h i s high oxidative capacity has encouraged the view that the b r a i n i s one of the most 02~dependent organs i n the mammalian body (see Siesjo et a l . , 1976, 1977 for reviews-in t h i s area). Substrates for the Mammalian Brain The unusual oxidative metabolism of the b r a i n i s f i r e d predominantly by glucose upon which the brain by and large i s assumed to have an absolute dependence. The rate of cerebral glucose consumption normally i s regulated by the rate of conversion of fructose-6-phosphate to fructose-1,6-biphosphate by the phosphofructokinase reaction and not by the d e l i v e r y rate at cerebral c a p i l l a r i e s . The rate of consumption can be accelerated s e v e r a l - f o l d when demand increases (e.g. i n hypoxia). In these cases d e i n h i b i t i o n of phosphofructokinase reduces the concentration of both fructose-6-phosphate and i t s metabolic precursor glucose-6-phosphate. Thus, the net e f f e c t i s to increase the rate of glucose phosphorylation by the hexokinase reaction (see Rappaport, 1976). Recent studies show that the rate of glucose uptake by d i f f e r e n t regions of the b r a i n c l o s e l y p a r a l l e l s neural a c t i v i t y (Sokoloff, 1979; P u l s i n e l l i and Duffy, 1979). No substrate can replace glucose i n sustaining brain function i n d e f i n i t e l y . However, a number of other substrates can be u t i l i z e d by the brain, and indeed some of these are also absolutely e s s e n t i a l f or c e r t a i n metabolic processes. Of these, ketone bodies and amino acids are the most important, while l a c t a t e and pyruvate may take on a s i g n i f i c a n c e when blood concentrations are high (Rowe et a l . , 1959; Nemoto elt al. , 1974). 11 The ketone bodies, 3-hydroxybutyrate and acetoacetate, can p a r t i a l l y support brain function when carbohydrate i s i n short supply or cannot be u t i l i z e d . These substrates are generated by the l i v e r and kidney i n starvation and di a b e t i c ketoacidosis, and occur i n high plasma concentrations i n newborn suckling r a t s . They enter the br a i n by simple passive d i f f u s i o n . At very high blood l e v e l s , b r a i n uptake of ketone bodies i s l i m i t e d by metabolic incorporation, which, i n turn, depends on i n t r a c e r e b r a l concentrations of the enzymes involved i n acetoacetate-hydroxybutyrate metabolism (Sokoloff, 1973). Although the concentrations of these enzymes, as well as rates of ketone body consumption, are higher i n brains of neonates than of adults, the s i g n i f i c a n c e of t h i s observation has not been c l a r i f i e d . Of the amino acids, glutamate, glutamine, aspartate, N-acetylaspartate, and GABA ( ot aminobutyric acid) are the predominant amino acids of the mature brain and constitute approximately two-thirds of free a-amino nitrogen. The high concentrations and extensive metabolism of glutamate and i t s d e r i v a t i v e s are key hallmarks of brain metabolism. The product of the a-decarboxylation of glutamic acid i s GABA, which has been reported to be a major i n h i b i t o r y transmitter i n the vertebrate c e n t r a l nervous system (Rappaport, 1976). The large pool of free glutamate i n the brain i s i n equilibrium with the a-ketoglutarate of the Krebs cycle, and aspartate i s i n equilibrium with oxaloacetate. A f t e r i n j e c t i o n of labeled glucose, 70% of the isotope present i n the soluble f r a c t i o n of br a i n i s present i n amino acids, primarily glutamate, glutamine, aspartate, and GABA (Guroff, 1972). Under normal circumstances, l a c t a t e and pyruvate are released from the brain into the c i r c u l a t i o n at rates that account f o r about 10% of the glucose being u t i l i z e d . However, under exceptional conditions, when blood 12 l e v e l s are high (for example, following trauma or ischemia), l a c t a t e and pyruvate can be u t i l i z e d by the brain (Siesjo et a l . , 1976). Lactate and pyruvate as well as other short-chain monocarboxylates (such as acetate, proprionate, and butyrate) cross cerebral c a p i l l a r i e s by a common f a c i l i t a t e d mechanism (Oldendorf, 1973). The l a c t a t e transport capacity, however, i s low and apparently saturates at 3-4 times the normal plasma concentration of la c t a t e ; that presumably i s why l a c t a t e uptake becomes s i g n i f i c a n t only at high blood l a c t a t e l e v e l s . C i r c u l a t o r y and Metabolic Consequences of Brain Hypoxia According to Siesjo (1977) b r a i n hypoxia can be defined as a decrease i n C"2 a v a i l a b i l i t y of such magnitude that there are measurable changes i n metabolism or function of the brai n . Since hypoxia may occur i n prolonged diving (Anderson, 1966; Scholander, 1962 and Ridgeway e_t a l . , 1969) i t i s important to emphasize that hypoxia d i f f e r s from anoxia (or complete ischemia) i n two ways. F i r s t l y , since ti s s u e PO^ i s not reduced to zero, oxidative metabolism continues even i f at a reduced rate. And secondly, since cerebral blood flow (CBF) i s maintained or increased, there i s a continuous supply of glucose for continued anaerobic metabolism. It i s now we l l established that hypoxia i n mammals leads to a compensatory increase i n CBF while hyperoxia leads to a compensatory decrease (Siesjo ejt a l . , 1976; Siesjo and Nordstrom, 1977). I n t e r e s t i n g l y , v a r i a t i o n s i n PaC^ from as high as 2100 mm to as low as 20 mm Hg with PVO2 varying between 57 mm and about 15 mm Hg, do not lead to any changes i n CMRO2 or the energy states as assessed by adenylate l e v e l s . As Siesjo and Nordstrom (1977)argue, t h i s suggests the cytochrome oxidase reaction runs 13 at a constant rate despite rather pronounced v a r i a t i o n s i n t i s s u e 0^ l e v e l s . Mechanisms underlying t h i s obvious close control of 0^ u t i l i z a t i o n i n the face of wide changes i n t i s s u e 0^ a v a i l a b i l i t y are not f u l l y c l a r i f i e d . Another matter that i s unclear i s why b r a i n perfusion varies over PO^ ranges that do not cause changes i n C M R 0 2 . The e a r l i e s t detectable metabolic changes i n hypoxia are increases i n l a c t a t e and pyruvate l e v e l s and a change i n the NADH/NAD+ r a t i o which i s expressed by changes i n the lactate/pyruvate r a t i o . These changes occur at aPaC^of about 50 mm Hg, when CMRO2 i s s t i l l unchanged (see Siesjo and Nordstrom, 1977). At face value, the data imply that both g l y c o l y t i c and aerobic production of ATP are increased with progressive hypoxia, and that the t o t a l energy produced i n terms of umol/ATP/gm/min must presumably r i s e . Why t h i s should be so i s not at a l l understood. If hypoxia i s prolonged a number of metabolic consequences are now w e l l outlined. Thus, l a c t a t e accumulation and glucose u t i l i z a t i o n both remain elevated. Pyruvate elevation i s also sustained and i s thought to ac t i v a t e pyruvate carboxylase catalyzed conversion to oxaloacetate (Mahan et. a l . , 1975) and alanine aminotransferase catalyzed conversion to alanine (Siesjo and Nordstrom, 1977). During maintained hypoxia, there also occurs a r e d i s t r i b u t i o n of carbon i n the Krebs cycle pool as w e l l as a general increase i n s i z e of the pool (Siesjo and Nordstrom, 1977) that i s i n part i n i t i a t e d by pyruvate and i n part by aspartate: pyruvate alanine V Krebs cycle 14 These processes explain r i s i n g alanine, but dropping aspartate l e v e l s coincident with the increase i n Krebs cycle pool s i z e observed at t h i s time. Over a l l , these adjustments are s i m i l a r to those to be anticipated when the Krebs cycle i s activated (Safer and Williamson, 1973), but they occur i n the hypoxic b r a i n presumably due to a gradual decrease i n r e s p i r a t i o n rates and the increasingly reduced state of mitochondrial metabolism. Although i t has been c l e a r l y established that neurological changes occur i n hypoxia, mechanisms underlying the derangement are s t i l l speculative. The c l a s s i c a l "vulnerable regions" include small neurons i n the neocortex and hippocampus, and Purkinje c e l l s i n the cerebellum (Siesjo and Nordstrom, 1977). Recent rather provocative studies imply that incomplete ischemia i s more damaging to the b r a i n than complete ischemia (Nordstrom et a l . , 1976), perhaps because the l a t t e r condition prevents 0^ dependent a u t o l y t i c processes that may damage c e l l membranes. If the mammalian br a i n experiences t o t a l anoxia (e.g. complete ischemia), aerobic energy production drops to zero almost immediately, i n about 4 sec. i n man and i n about 1-2 sec. i n the rat cerebral cortex (Siesjo and Nordstrom, 1977). Concomitant with these events, phosphofructokinase i s activated, probably due to a drop i n ATP and creatine phosphate l e v e l s and a r i s e i n AMP, ADP and P i l e v e l s . This leads to a maximum stimulation of g l y c o l y s i s which ulti m a t e l y r e s u l t s i n complete depletion of glycogen and glucose and the accumulation of l a c t a t e , alanine and succinate (Siesjo and Nordstrom, 1977) . During complete ischemia as i n hypoxia (see above), the pool of Krebs cycle intermediates increases s u b s t a n t i a l l y and there again occurs a s i g n i f i c a n t r e d i s t r i b u t i o n of carbon between the various Krebs cycle 15 components. Key among these are a depletion of 2-ketoglutarate (2-KGA) and oxaloacetate (OXA) and a r e s u l t i n g increase i n succinate. The two decreases observed are probably due to increases i n the concentrations of NADH, H and NH3: 2 KGA + NADH + H + + NH^ " : •glutamate + NAD+ OXA + NADH + H + •malate + NAD+ Succinate may be viewed as an end product of anaerobic metabolism. The i n i t i a l increases i n most of the Krebs cycle pool are probably the r e s u l t of anaplerotic reactions such as CO2 f i x a t i o n at the stage of phosphoenol-pyruvate or pyruvate and a s h i f t i n the alanine aminotransferase reaction. S i g n i f i c a n t portions of these carbon chains are thought to be tapped off v i a the glutamate dehydrogenase reaction. Brain Metabolism i n Diving Animals With respect to diving animals, most of the biochemical work reported on cardiac ti s s u e was also performed on the b r a i n with b a s i c a l l y the same o v e r a l l r e s u l t s , a l l pointing to an increased capacity to sustain periods of hypoxia or anoxia. P h y s i o l o g i c a l studies (Kerem et'al.-, 1973; Eisner et a l . , 1970b; Kerem et a l . , 1971) also have prompted most researchers to i n f e r improved hypoxia tolerance. However, convincing data showing a r e l i a n c e of brain on anaerobic metabolism during diving are hard to come by; i n f a c t , much of the av a i l a b l e information implies a r e l a t i v e l y 'normal' aerobic brain metabolism. According to Bryan and Jones (1980), for example, the NADH/NAD+ r a t i o i n the cerebral hemispheres increases by nearly 40% af t e r 1 minute of apneic asphyxia i n the fowl while i t only increases by 15% i n the duck a f t e r 2 minutes of asphyxia. However, at a given l e v e l of br a i n 16 t i s s u e PC^j both species show the same r e l a t i v e increase i n the NADH/NAD^ r a t i o , implying that both species are equally dependent on an adequate PC^ for the maintenance of oxidative metabolism i n the brain. Moreover, both species show an i s o e l e c t r i c EEG when the NADH/NAD+ r a t i o increases to within 30-40% of maximum (that occurring at death). F i n a l l y , prevention of bradycardia i n ducks during apneic asphyxia by atropine causes the NADH/NAD+ r a t i o to increase at the same rate as i n non-atropinized fowl. The authors, therefore, conclude that 0 2 conserving cardiovascular adjustments are responsible for the increased cerebral tolerance to apneic asphyxia i n ducks, with no s p e c i f i c biochemical mechanisms being involved. However, the non-invasive fluorometric technique used bears information only upon the redox state of the mitochondria and y i e l d s no insight into cytoplasmic ( i . e . g l y c o l y t i c ) events. Along with a large increase i n cerebral blood flow, there automatically occurs a greatly increased d e l i v e r y of glucose to cerebral energy metabolism i n the duck during d i v i n g (or during apneic asphyxia). If aerobic glucose metabolism i s unchanged, what happens to the excess supply of t h i s carbon and energy source remains to be c l a r i f i e d . Is i t fermented? Is i t 'dumped' into the free amino acid pool? Or does i t have some other fate? Only further work w i l l a s c e r t a i n the answers. Other i n d i c a t o r s of the metabolic status of the brain of divers are he l p f u l but, thus far at l e a s t , also equivocal. B l i x (1971), for example found concentrations of creatine phosphate to be s i m i l a r i n seals and sheep. LDH isozyme patterns i n at lea s t 2 cetaceans (Vogl, pers. comm.) imply that both heart and muscle type subunits are involved i n generating LDH holo-enzymes; however, heart type subunits d e f i n i t e l y predominate and o v e r a l l 17 LDH isozyme patterns are therefore not s t r i k i n g l y d i f f e r e n t from those observed i n t e r r e s t r i a l mammals. In t h i s connection, Shoubridge j2t a l . (1976) found a rough c o r r e l a t i o n between the f r a c t i o n of muscle type subunits i n brain LDHs and depth (thus duration?) of diving i n large whales, a c o r r e l a t i o n that i s almost i m p l i c i t l y assumed to occur by most workers i n the f i e l d . A l l the above kinds of data, of course, are ci r c u m s t a n t i a l . Rigorous demonstration of the metabolic status of the brain i n diving mammals must come eit h e r from d i r e c t t i s s u e sampling (thus f a r unavailable) or from AV gradients across the br a i n . Substrates for the Mammalian Lung Eight decades ago i t was suggested that the lung may play useful metabolic role s (Bohr and Henriques, 1897), yet the mapping of metabolic organization i n the mammalian lung has only recently been i n i t i a t e d , and s u r p r i s i n g l y l i t t l e i s known compared to other tissues such as the brain or heart. This s i t u a t i o n stems not only from a general lack of i n t e r e s t by biochemists but also because of the many d i f f i c u l t i e s of working with the lung both In v i t r o and i n vivo. Despite t h i s , some general c h a r a c t e r i s t i c s of the lung's substrate preferences have been documented and deserve a b r i e f d e s c r i p t i o n . Although the rates of 0^ consumption and energy u t i l i z a t i o n are r e l a t i v e l y low compared to the rest of the body (Weber and Visscher, 1969), the many d i f f e r e n t c e l l types of the lung parenchyma (including the a l v e o l a r - c a p i l l a r y interfaces) are considered to be very metabolically ac t i v e (Terney, 1974a). This apparent paradox a r i s e s from the high percentage of i n e r t s t r u c t u r a l lung t i s s u e s . The intense metabolism of the 18 parenchyma was i n i t i a l l y considered to be l a r g e l y fueled by glucose and l i p i d s ( F e l t s , 1964; 0'Neil and Tierney, 1974; Rhoades, 1974); however, within the l a s t few years some compelling evidence i n support of la c t a t e as a p r e f e r e n t i a l pulmonary substrate has been presented (Wolfe et al., 1979; Rhoades et a l . , 1979; Hochachka et a l . , 1977a; Wallace et a l . , 1974). Free f a t t y acids (e.g. palmitate) are considered to be oxidized quite r e a d i l y by lung t i s s u e (Salisbury-Murphy et a l . , 1966; Wang and Meng, 1972; Wolfe et a l . , 1970; Rhoades, 1974). In vivo the f a t t y acids u t i l i z e d by the lung may be derived from n o n e s t e r i f i e d free f a t t y acids as we l l as plasma lipo p r o t e i n s ( F e l t s , 1964). However important these substrates may be to pulmonary energy production t h e i r main function probably l i e s i n the production of surfactants, which are pri m a r i l y composed of phospholipid. Surfactants which l i n e the alveolar surface are responsible for the unusually great surface a c t i v i t y present i n mammalian lungs. Because these important compounds have very short h a l f - l i v e s the pulmonary parenchyma must be continuously involved i n t h e i r biosynthesis. The type II alveolar c e l l s which have been i d e n t i f i e d as among the t i t a n s of l i p i d metabolism (Tierney, 1974a) are now believed to be the main s i t e s of surfactant production and secretion (Klaus,et a l . , 1962, Tierney, 1974a). Although lung function i s apparently not t o t a l l y dependent upon glucose, the substrate probably plays several v i t a l r o l e s . 1. NADPH Generation. NADPH i s required not only for pulmonary synthesis of l i p i d (surfactant) and protein (Tierney, 1974a) but also for the maintenance of reduced glutathione, which may serve to protect against damage to the lungs by oxidants (Tierney eit a l . , 1973) . Most of the pulmonary NADPH i s derived from a very active pentose phosphate cycle, which r e l i e s upon 19 glucose as i t s ultimate substrate (Bassett and Fisher, 1976b; Tierney et a l . , 1973) ; the 'malic' enzyme i s an u n l i k e l y source since i t s a c t i v i t y l e v e l s are so low i n lung ti s s u e (Scholz and Rhoades, 1971). 2. Biosynthesis of L i p i d and Glycoprotein. Two-and three-carbon products (acetylCoA and ^-glycerophosphate) derived from glucose provide intermediates for l i p i d metabolism (Bassett et: a l . , 1974; Godinez and Longmore, 1973). The a-glycerophosphate (aGP) which i s produced from dihydroxyacetone phosphate by the c a t a l y s i s of a very active aGP dehydrogenase (Lee and Lardy, 1965), may be a determinant of the rate of lung l i p i d synthesis and.in p a r t i c u l a r dipalmitoyl l e c i t h i n , a major component of the alveolar surface-active l i n i n g layer ( F e l t s , 1964). I t has also been reported (Yeager and Massaro, 1972) that glucose carbon atoms appear i n pulmonary glycoproteins. 3. Energy Production. Glucose probably fuels most of the pulmonary c e l l types to varying degrees but i s perhaps the major substrate for some of the more anaerobic c e l l s such as type I alveolar e p i t h e l i a l c e l l s (Tierney, 1974a). However, Tierney (1974b) suggested that the i s o l a t e d rat lung can survive for up to 2 hours without any exogenous glucose, implying u t i l i z a t i o n of other substrates such as l i p i d s or amino acids. Parenthetically, a recent study by Scholz and Evans (1977) demonstrated the a b i l i t y of the rat lung to incorporate various amino acids into CO^ and l i p i d s . Yeager and Massaro (1972) determined glucose u t i l i z a t i o n by rabbit lung s l i c e s and found i t to be remarkably high (1.83 mg/g lung/hr). I n t e r e s t i n g l y , only 19% of ^^C i n 14 the glucose that was. consumed appeared i n w n e r e a s 30% appeared i n l a c t a t e , a value s i m i l a r to that observed i n perfused rat lung (Rhoades, 1974) . The elevated l a c t a t e production has also been observed i n lung s l i c e s which were incubated under high 0„ tensions and l a c t a t e l e v e l s (Tierney, 20 1971; Rhoades et: _al., 1978). Presumably, a major f r a c t i o n of the l a b e l l e d glucose carbons were channeled into the pentose phosphate shunt (Bassett and Fisher, 1976a)and into anabolic pathways (Tierney, 1974a). It now appears that the lung not only produces l a c t a t e but also employs the metabolite as a p o t e n t i a l l y important substrate (Searle ej: a l . , 1974; Wallace et a l . , 1974; Wolfe et a l . , 1979; Rhoades et a l . , 1978; Hochachka et a l . , 1977a). Wolfe and associates (1979), u t i l i z i n g perfused rat lung, have proven l a c t a t e to be a good energy substrate which can a c t u a l l y i n h i b i t glucose oxidation by 50% at concentrations as low as 1 mM. A s i m i l a r phenomenon of p r e f e r e n t i a l pulmonary oxidation of l a c t a t e was confirmed by Rhoades e_t a l . (1979). Furthermore, t h i s group demonstrated the incorporation of a s i z a b l e portion of the l a c t a t e carbons into lung l i p i d s . Quite s u r p r i s i n g l y , t h e i r data showed that l a c t a t e concentrations of 5 mM i n the perfusion medium had no s i g n i f i c a n t e f f e c t on the lactate-production even though glucose u t i l i z a t i o n was i n h i b i t e d . They suggested non-carbohydrate sources (e.g. alanine) f u e l i n g the l a c t a t e production under t h e i r conditions. Although l a c t a t e production by the lung was apparently unaltered by the a v a i l a b i l i t y and uptake of l a c t a t e , i t has been shown that t h i s organ exhibits a Pasteur e f f e c t (Bassett and Fisher, 1976b). However, g l y c o l y t i c compensation i n the absence of oxidative metabolism was not considered to be s u f f i c i e n t to maintain ATP content or i t s supply for synthetic a c t i v i t y (Bassett and Fisher, 1976b). On the other hand, Fisher and associates (1972) have demonstrated that the i n t a c t organ remained capable of maintaining f u n c t i o n a l and s t r u c t u r a l homeostasis during rather prolonged periods of hypoxic stress (Fisher et a l . , 1972). Longmore and Mourning (1976) report during hypoxia the rate of l a c t a t e production doubles but only about 50-60% i s apparently derived from glucose. They postulated amino acid catabolism to be the most l i k e l y source of the excess l a c t a t e production. Unfortunately, t h i s postulate has yet to be v e r i f i e d and must await future experimentation. For an organism to withstand hypoxia i t i s advantageous not only to develop e f f i c i e n t anaerobic machinery and redox balancing mechanisms but also to develop i n t e r - t i s s u e cooperation. For example, c e r t a i n end products of anaerobic metabolism from one ti s s u e could be shipped to some less hypoxic tis s u e for conversion ( i . e . reduction) to intermediates which would be u t i l i z e d by the more hypoxic t i s s u e . The lung, of course, i s a prime candidate for a r o l e as an acceptor organ for during hypoxic or even anoxic stress i t probably remains i n a somewhat more aerobic state. Such a r e a l i z a t i o n probably led Cascarano and associates (1976) to postulate that the lungs of r a t s , subject to low tensions of oxygen, w i l l a c t u a l l y take up the anaerobic end product, succinate, produced by other t i s s u e s , and release fumarate and malate for further use i n energy metabolism by the hypoxic heart. This process, they suggested, sets up an electron s h u t t l e between the heart and the lungs. Such a s h u t t l i n g process would be of great advantage to organisms which are routinely exposed to hypoxic or anoxic stress (e.g. the marine mammals). Lung Metabolism i n Diving Mammals Before the 1977 study of Hochachka and associates, pulmonary metabolism i n the marine mammal had been l a r g e l y ignored-, . but t h i s report has provided a very provocative interpretation of the suspected r o l e of the s e a l lung during restrained diving episodes. In working with adult Weddell seals, these investigators focused attention on the impressive a b i l i t y of t h i s 22 animal's lung to absorb s i g n i f i c a n t q u antities of l a c t a t e during the dive phase when the metabolite's concentration i n ce n t r a l whole blood i s 3.0 mM or l e s s . Depending on the animal and the submersion time, between 0.1 and 0.25 ymoles of lactate/ml of blood can be taken up by the lung. The authors also reported low but detectable releases of glucose during t h i s period. They thus, extrapolated that the seal's lung conditions c e n t r a l c i r c u l a t i n g blood by reducing l a c t a t e accumulation through the p r e f e r e n t i a l conversion of l a c t a t e to glucose and by the actual release of glucose into the main bloodstream. However, the ultimate fate of absorbed l a c t a t e was never demonstrated i n t h i s study. Heart-Lung-Brain Metabolism i n the Weddell Seal In Scholander's o r i g i n a l formulation, the diving reflexes were seen to play a primary r o l e i n the conservation of 0^ for the most 02 _dependent tissues . Of these, the heart, lung and br a i n were considered the most important. However, i n most p h y s i o l o g i c a l studies, these have been considered separately, i f at a l l (e.g. Kerem and Eisner, 1973) while i n metabolic studies they have usually been lumped together as a "black box" (Scholander, 1962; Hochachka and Murphy, 1979). The only concerted attempt to integrate the metabolism of the d i f f e r e n t c e n t r a l organs has been generated by Hochachka and associates (1977a)> a study discussed i n the preceding section of t h i s chapter. This general lack of i n s i g h t i n t o ' i n vivo' metabolic a c t i v i t i e s of the functioning c e n t r a l organs i s the d i r e c t r e s u l t of the many d i f f i c u l t i e s associated with studying diving mammals. Therefore, advantage was taken of the opportunity to j o i n an i n t e r d i s p l i n a r y group studying the p h y s i o l o g i c a l and metabolic consequences of diving i n 23 the A n t a r c t i c Weddell s e a l . This aquatic mammal offered many us e f u l advantages (e.g. easy capture and maintenance along with many previous f i e l d and laboratory studies by Kooyman and E i s n e r ) . However, the seal's phenomenal diving c a p a c i t i e s provided the most a t t r a c t i v e q u a l i t y . Recent f i e l d studies by Kooyman £t.al. (1980) now indicate that the adult Weddell seal i s capable of breath-hold dives for up to 70 minutes. If any metabolic or biochemical modifications were associated with the heart-lung-brain machine during diving they would c e r t a i n l y be accentuated i n t h i s pinniped. For t h i s study of the Weddell s e a l , a v a r i e t y of techniques were used to approach the problem at various l e v e l s of organization. These included: 14 (1) enzyme p r o f i l e s of a l l three organs, (2) t i s s u e s l i c e studies using C metabolites, (3) arteriovenous concentration d i f f e r e n c e s , (4) i n vivo 14 metabolite infusions using C l a b e l l e d and unlabelled metabolites, and f i n a l l y (5) transient changes of blood metabolite l e v e l s p r i o r to, during, and following d i v i n g . Furthermore, i n c l o s e l y c o l l a b o r a t i v e studies, organ perfusion, heart rate, blood pressure,card iac output, blood gases, and blood pH were simultaneously monitored (Zapol et a l . , 1979; Liggins et a l . , 1980). Thus p h y s i o l o g i c a l parameters could be used to a s s i s t i n i n t e r p r e t i n g biochemical data and v i c e versa. These p h y s i o l o g i c a l parameters w i l l be presented throughout t h i s t h e s i s ; however, for thorough discussions of these phases of the study r e f e r to Zapol'et al_. (1979); Liggins et a l . (1980); Ovist et a l . , (1980). F e t a l Metabolism The mammalian fetus u t i l i z e s the placenta as an avenue through which i t receives a l l substrate and 0 9 requirements from the mother. Maternal-24 derived 0^ i s also considered to be of prime importance for i t f i r e s a very a c t i v e f e t a l catabolism. In a l l species ; thus far studied, f e t a l oxygen -1 -1 consumption ranges from 6 to 9 ml STP. min kg , i n d i c a t i n g a constancy across mammalian species (Battaglia and Meschia, 1976). Therefore, i n an animal l i k e the Weddell seal f e t a l t issues would be a s i t e of comparatively high u t i l i z a t i o n within the maternal organism, due to the normal scaling of 0^ consumption to body s i z e (Coulson et a l . , 1977) . Under normal conditions, i t i s now believed that anaerobic metabolism plays a very minor r o l e i n the generation of energy. Nevertheless i t i s t r a d i t i o n a l l y considered that the mammalian fetus displays a higher tolerance to hypoxemia than that of the mother (Dawes, 1968). In the ovine fetus the p r i n c i p a l exogenous oxidative f u e l s are glucose, l a c t a t e and amino acids. In most mammals- thus far studied, the glucose uptake from the placenta i s inadequate to meet the c a l o r i c requirements of the fetus ( B a t t a g l i a and Meschia, 1976). Under normal conditions glucose uptake i s capable of sustaining 50-70% of f e t a l oxidative metabolism. Unfortunately, the rate of f e t a l gluconeogenesis ( i f any) and i t s regulation has yet to be ascertained. Lactate uptake by the ovine fetus can account for approximately 25% of the t o t a l f e t a l 0^ consumption i f the substrate i s completely catabolized. The amino acids delivered to the u m b i l i c a l c i r c u l a t i o n are u t i l i z e d by the fetus to b u i l d new tissues and, i n part, as f u e l s of oxidative metabolism. At present, no quantative determinations of the i n d i v i d u a l amino acid's c o n t r i b u t i o n to catabolism are a v a i l a b l e ; however, i t i s known that t h e i r r e l a t i v e contribution to energy production 25 increases during maternal hypoglycemia ( B a t t a g l i a and Meschia, 1976). Although most fetuses are capable of free f a t t y acid (FFA) and ketoacid oxidation, i t i s not clear i f these substrates are important sources of energy i n those species whose placenta i s permeable to them. Heart Metabolism Unlike the adult heart, the f e t a l myocardium r e l i e s very l i t t l e on free f a t t y acids but instead consumes predominantly glucose under aerobic conditions (Breuer et a l . , 1967; B a t t a g l i a and Meschia, 1976). The degree to which t h i s holds true depends upon the species and the stage of gestation (Beatty et a l . , 1972; Clark, 1971). This p e c u l i a r i t y of the f e t a l heart i s considered to be due to low carnitine concentrations and low enzyme a c t i v i t i e s i n the 3-oxidation s p i r a l of the f e t a l myocardium (Wittels and Bressler, 1965). Furthermore, f e t a l and neonatal hearts are considered to be r e l a t i v e l y r e s i s t a n t to hypoxia, probably a r e s u l t of increased concentrations of myocardial glycogen (Dawes, 1968; Hoerter, 1976). No relevant enzyme data are yet a v a i l a b l e . Brain Metabolism Glucose i s considered the major f u e l of, at l e a s t , the ovine f e t a l brain; a s i t u a t i o n akin to that of the adult. Recent information suggests that the high rates of cerebral oxygen and glucose u t i l i z a t i o n are a general phenomenon, i r r e s p e c t i v e of s i z e and stage of development ( B a t t a g l i a and Meschia, 1976). However, there are varying • reports regarding the contribution of anaerobic metabolism i n the normal f e t a l b r a i n (Benjamins and McKhann, 1972; Jones et al., 1975). Regarding a l t e r n a t i v e energy 26 sources, i t has been suggested, but not demonstrated, that ketone bodies play an important r o l e i n f e t a l cerebral metabolism of some species, replacing glucose when the carbohydrate supply i s at a premium (Ba t t a g l i a and Meschia, 1976) . F e t a l Metabolism i n the Diving Mammal Pr i o r to these current studies there had been only two p h y s i o l o g i c a l l y orientated reports pertaining to the fetus of the aquatic mammal, c i t e d i n the l i t e r a t u r e . Neither dealt d i r e c t l y with any aspect of the metabolic organization of the fetus. One decade ago, Lenfant's group (1969) studied the re s p i r a t o r y properties of blood i n both the f e t a l and maternal blood of the Weddell s e a l . The pregnant s e a l was found to have higher hemoglobin concentration, hence higher oxygen capacity, than i t s near-term fetus. Qvist et^ a l . (1980), from our group, have v e r i f i e d t h i s phenomenon. This condition i s noteworthy for i t i s the reverse of that i n most mammals (Dawes, 1968). Furthermore, the 0^ a f f i n i t y of the f e t a l s e a l blood i s higher than the maternal blood, a s i t u a t i o n common to most mammals. Therefore the f e t a l seal is capable of extracting more O2 from the maternal blood than i t s t e r r e s t r i a l counterpart at low PaO^ and thus would reduce the requirement f o r uterine blood flow during submergence. Eisner and associates (1970) followed f e t a l heart rate and uterine blood flow i n one asphyxiated gravid Weddell s e a l , paralyzed by doses of s u c c i n y l .choline - (Eisner et a l . , 1970). The authors reported a slow but gradual decline i n f e t a l heart rate (66 beats/ min to 20 beats/min a f t e r 29 minutes) followed by a slow recovery upon resumption of maternal v e n t i l a t i o n . U t i l i z i n g Doppler u l t r a s o n i c blood flow transducers t h i s group also observed an uninterrupted uterine blood 27 flow throughout the en t i r e episode. They concluded that the continuation of blood flow to the fetus during asphyxia served as a defense f o r the fetus by continuing i t s oxygen supply. Strategy of Study Given the opportunity to study i n t a c t Weddell seals, i t was reckoned that a good attempt could be made at unravelling some of the numerous questions r e l a t i n g to the metabolic functionings and cooperation between at l e a s t the three c e n t r a l organs during dive-recovery cycles. To recap the basic questions addressed i n t h i s thesis are as follows: 1) what fuels were burned by each of the three c e n t r a l organs during r e s t , dive and recovery phases? Are the preferences during rest periods s i m i l a r to those of homologous tissues i n comparatively sized t e r r e s t r i a l mammals or have the organs been adapted f o r the diving habit? 2) Does the br a i n experience any increases i n anaerobic g l y c o l y s i s during the dive episode? 3) Are there changes i n cerebral metabolic rate i n response to diving? 4) F i n a l l y , are there any i n t e r - t i s s u e c y c l i n g of metabolites that may be operational during dive-recovery cycles? One of the most l i k e l y candidate i s considered to be l a c t a t e which i s the main anaerobic end product of the more hypoxic t i s s u e s . Since forced diving appears to e l i c i t a maximum diving response ( i . e . profound bradycardia and intense peripheral vasoconstriction) akin to that of long duration diving, i t was hoped that the chosen experimental techniques would shed some l i g h t on the metabolic functionings of the cen t r a l organs during a time when the animal i s preparing f o r the d i s t i n c t p o s s i b i l i t y of extreme taxes on i t s oxygen and substrate supplies. The techniques included: 1) blood metabolite (glucose, l a c t a t e , pyruvate and 27a amino acids) sampling p r i o r to, during and following diving; 2) enzymatic p r o f i l e s of a l l three organs; 3) A-V samplings across the b r a i n and lung; 14 4) i n vivo metabolite i n f u s i o n using C l a b e l l e d and unlabelled l a c t a t e 14 and 5) f i n a l l y t i s s u e s l i c e studies u t i l i z i n g C l a b e l l e d metabolites (glucose and l a c t a t e ) . Although the f e t a l questions were peripheral to the main problems addressed here, i t was i n i t i a l l y hoped that f e t a l a r t e r i a l concentration p r o f i l e s of l a c t a t e i n conjunction with the p h y s i o l o g i c a l data could indicate whether or not the fetus e l i c i t s i t s own diving response, as a consequence of maternal diving. In the process, other f e t a l blood metabolite l e v e l s could be p l o t t e d as functions of rest and dive-recovery cycles and thus compared with the maternal p r o f i l e s . Furthermore, enzymatic p r o f i l e s of the f e t a l heart, lung and b r a i n could be compared to the respective maternal organs. Technical problems precluded any planned measurements of arteriovenous concentration differences across the three organs i n question while time l i m i t a t i o n s prevented the thorough study of any of the peripheral tissues such as s k e l e t a l muscle. 28 CHAPTER II Materials and Methods 28 a Experimental Animals and In Vivo Manipulations Gravid and non-gravid adult Weddell seals (Leptonychotes weddelli), weighing 350-500 kg, were captured near T u r t l e Rock on the Ross Island A n t a r c t i c fast i c e and transported by sled to the Eklund B i o l o g i c a l Laboratory at McMurdo Station. The seals were anesthetized with i n t r a -muscular ketamine hydrochloride (2 mg/kg). Surgery was performed i n a l a t e r a l p o s i t i o n on a mobile operating table. During operations, anesthesia was maintained by spontaneous breathing of 1-4% halothane i n 0^ through a to-and-fro CO^ absorption cannister. Diving experiments were not i n i t i a t e d u n t i l 8-12 hours a f t e r surgery when cont r o l heart rate, Pa0„, PaC0„, and pH had returned to r e s t i n g values. For diving experiments, the adult seal was restrained on a mobile table with the webbing of a cargo net. The mobile table was supported on two tracks, and the seal's head inserted through a sponge rubber gasket into a reinforced wood and p l a s t i c tank f i x e d to the tracks. Ambient temperature i n the laboratory was maintained at 4°C, and the seal's f l i p p e r s and dorsum were frequently bathed with iced seawater. To simulate diving by head submersion, the table was t i l t e d 20° head down and the tank was f i l l e d r a p i d l y with iced seawater. For monitoring metabolite p r o f i l e s i n a r t e r i a l blood of the adult seals, 2 ml samples were drawn from a PE 190 catheter positioned i n the thoracic aorta v i a a f l i p p e r artery. F e t a l blood sampling involved a midline laparotomy i n c i s i o n approximately 25 cm i n length) of the mother, followed by a uterine i n c i s i o n (7.5 cm). The most accessible f l i p p e r was drawn out u n t i l the proximal part of the f l i p p e r was f i r m l y wedged i n the uterine 29 wound. The f e t a l membrane could then be opened without s p i l l a g e of amniotic f l u i d . The f e t a l thoracic aorta was catheterized v i a the l e f t r a d i a l artery. Samples were taken at various times p r i o r to diving, during simulated diving, and during recovery for up to several hours. A l l blood samples were added d i r e c t l y to 2 ml of c h i l l e d 1.4 M p e r c h l o r i c acid and s t i r r e d vigorously. The PCA extracts were then centrifuged and the supernatant solutions were neutralized with 1.4 M KOH or K^CO^- Following another centrifugation to remove the p r e c i p i t a t e d perchlorate s a l t , the extracts e i t h e r were immediately analyzed for unstable metabolites (e.g. pyruvate) or were stored at -80°C i n a Revco freezer u n t i l required. To assess the capacity of the adult lung to metabolize l a c t a t e , a 150 cm long, 8 French diameter, balloon f l o t a t i o n Swan Ganz thermodilution catheter was s u r g i c a l l y introduced into an exposed i n t e r n a l jugular vein at the thoracic i n l e t and advanced i n t o the pulmonary artery with pressure monitoring. The proximal port of the Swan Ganz catheter i n the r i g h t v e n t r i c l e was used as an i n j e c t i o n s i t e , while the d i s t a l port was used for c o l l e c t i n g blood samples from the pulmonary artery. The l a c t a t e i n j e c t a t e or bolus contained "^C-U-lactate (4 uC/1 blood), 2 gm c a r r i e r l a c t a t e to bring concentrations up to about 3-4 mM, and 200 mg Evans blue dye; the in j e c t a t e was made up i n a 15 ml t o t a l volume of normal s a l i n e (9 g/1) at pH 7.4. At 20 second i n t e r v a l s following rapid (5 sec) manual i n j e c t i o n of the l a c t a t e bolus into the r i g h t v e n t r i c l e , 5 ml blood samples were drawn simultaneously from the sampling port i n the pulmonary c i r c u l a t i o n and from the thoracic aorta. The blood samples taken from the pulmonary artery and aorta were added d i r e c t l y to equal volumes of 1.4 M PCA i n stoppered tubes 14 14 containing hyamine hydroxide C0 ? traps. Thus, C0 ? formed from C-lactate 30 during a s i n g l e c i r c u l a t i o n through the lung could be r e a d i l y detected. On removal of the CO^ traps, these blood samples were treated as before, and 14 assayed for dye content, l a c t a t e concentration, and C-lactate r a d i o a c t i v i t y . The marker dye concentration was estimated i n a r b i t r a r y o p t i c a l density units read at 600 nm; l a c t a t e was measured enzymatically, following NAD+ reduction 14 at 340 nm; C-lactate r a d i o a c t i v i t y i n aliquots dissolved i n Aquasol (New England Nuclear, Boston) was determined using a Nuclear Chicago Unilux 2A 14 Liquid S c i n t i l l a t i o n Counter; CO2 trapped i n hyamine hydroxide was taken up i n 10 ml of Aquasol f o r counting. Using the methods of Wolfe et a l . (1979), 14 i A no evidence of blood C-glucose formation from ^ C - l a c t a t e was evident i n these short-term experiments. Thin layer chromatography indicated that no metabolic derivatives of "^C-lactate other than ^^CO^ were released into the blood during the 3 minute time course of these experiments. In t h i s study, r i g h t v e n t r i c u l a r cardiac output was determined i n the r e s t i n g c o n t r o l state, at 10 minutes of a 17 minute dive (just p r i o r to the 14 C-lactate i n j e c t i o n ) immediately a f t e r the end of sampling, and f i n a l l y during recovery from diving. Right cardiac output was estimated by the thermodilution method (Maruschak et^ a l . , 1974). For monitoring cerebral venous metabolites, a PE 50 catheter was introduced v i a a 13 ga s t e e l Tuohy needle into an epidural v e i n i n the c e r v i c a l region and advanced i n an anterior d i r e c t i o n to within a few centimeters of the occiput. In pinnipeds, a cerebral venous blood i s p r i n c i p a l l y drained by these veins without admixture by venous blood from other tissues (King, 1977). In a l l i n vivo experiments, catheters were rou t i n e l y flushed with s a l i n e s o l u t i o n (9 g/1) containing heparin (5,000 units/1) and connected to Statham 31 Model 1280C transducers and a Hewlett Packard 7758B recorder. Heart rate, vascular and i n t r a c a r d i a c pressures and EKG were i n t e r m i t t e n t l y recorded. A r t e r i a l blood pH and gas tensions were determined i n t e r m i t t e n t l y with a Radiometer Model PHM72 blood gas analzyer. Af t e r f u l l recovery from anesthesia (8-12 hours), blood samples were drawn to e s t a b l i s h metabolite l e v e l s i n the con t r o l or r e s t i n g state. This was usually followed by a r e l a t i v e l y short simulated dive (10-20 minutes) and a recovery period. Another simulated dive was performed when the se a l had f u l l y recovered (usually 2-3 hours) as judged by the return to r e s t i n g l e v e l s of pHa, PaO^, PaC^, and heart rate. When the desired i n Vivo experiments were completed, the animal was s a c r i f i c e d with an overdose of anesthetic and the p o s i t i o n of a l l catheters was v e r i f i e d at autopsy. The harbor seals (Phoca v i t u l i n a ) used i n these studies were borrowed from Sea World, San Diego, C a l i f o r n i a . Venous blood samples from the unanesthetized seals were obtained from the extradural vein, catheterized near the posterior end of the animal. Human blood samples were c o l l e c t e d from theantecubical veins of 4 male volunteers by a q u a l i f i e d medical technician. Samples from these two groups were not only treated with 1.4M PCA, as previously described, but some were also added d i r e c t l y to 4 volumes of 3.75% s u l f o s a l i c y c l i c acid (SSA), centrifuged and stored at -20°C f or future analysis. The l a t t e r treatment i s preferred f o r amino acid a n a l y s i s . Enzyme Extraction and Assay Tissues f o r enzyme extraction were excised as quickly as possible a f t e r s a c r i f i c i n g the animal; because of the massive s i z e of the Weddell s e a l , t h i s process took 20-30 minutes. A transmural l e f t v e n t r i c u l a r myocardial 32 sample was obtained from each s e a l . Each sample contained both endocardium and epicardium. The same regions of the b r a i n (anterior cerebral cortex) containing both grey and white matter, and of lung parenchyma ( l e f t lower lobe periphery without major vessels or airways) were sampled i n each s e a l . The samples of heart, lung, and b r a i n were immediately placed i n 0°C Ringers s o l u t i o n and washed several times. A l l further manipulations were at 0°C. Small samples of brai n , heart, and lung were removed, blo t t e d dry on f i l t e r paper, weighed and then homogenized using a Polytron tissue processor (Brinkman Instruments). The homogenization medium was 50 mM imidazole, pH 7.4, 50 mM KC1, with 0.1% Triton-XlOO. The homogenate was well s t i r r e d , then centrifuged i n an RC-2B S o r v a l l centrifuge at 4°C to remove c e l l u l a r debris. The supernatant s o l u t i o n was used to assess the a c t i v i t i e s of several oxidative and g l y c o l y t i c enzymes. For comparative purposes, samples of ox heart, lung and brain were prepared i n i d e n t i c a l manner. A l l enzymes were assayed i n a Unicam SP1800 recording spectrophotometer with a thermostated c e l l holder maintained at 37°C with a Lauda constant temperature bath and c i r c u l a t o r . C i t r a t e synthase was assayed by the method of Srere (1969). Assay conditions were 0.15 mM acetylCoA, 0.5 mM oxaloacetate, i n 50 mM imidazole buffer, pH 7.4, 7.5 mM Mg**, 50 mM K +, at 37°C. The release of CoA was monitored at 412 nm with 0.25 mM DTNB. Glutamate dehydrogenase was assayed by following the oxidation of 0.1 mM NADH at 340 nm. Assay conditions were 2-ketoglutarate, 7 mM; NH^+, 50 mM; and NADH, 0.1 mM; 1 mM ADP was always included to f u l l y a c t i v a t e the enzyme (Smith et a l . , 1975). Imidazole bu f f e r (50 mM), pH 7.4, was used with 7.5 mM Mg*"1", 50 mM K +, at 37°C. J 33 The a c t i v i t y of 3-hydroxybutyrate dehydrogenase was monitored by following the oxidation of NADH at 340 nm. Assay conditions were 0.1 mM I | NADH, 2 mM acetoacetate, i n 50 mM imidazole buffer, pH 7.4, 7.5 mM Mg , 37°C. The c a t a l y t i c a c t i v i t y of 3-hydroxybutyrylCoA dehydrogenase was assayed under i d e n t i c a l conditions except that the substrate was 0.4 mM acetoacetylCoA. The hexokinase assay depended upon coupling the production of glucose-6-phosphate to glucose-6-phosphate dehydrogenase and monitoring NADPH production at 340 nm. Assay conditions were 2.5 mM glucose, 5.0 mM ATP, 7.5 mM Mg , 50 mM K , 0.4 mM NADP , 2 units of glucose-6-phosphate dehydrogenase, 50 mM imidazole, pH 7.4, at 37°C. Measuring phosphofructokinase depended upon coupling the reaction to aldolase, t r i o s e phosphate isomerase, and a-glycerophosphate dehydrogenase. Assay conditions were 1 mM ATP, 1 mM fructose-6-phosphate, 0.1 mM NADH, and excess (over 2 units) aldolase, t r i o s e phosphate isomerase, and a-glycerophosphate dehydrogenase. AMP (1 mM) was included i n the reac t i o n mixture to f u l l y a c t i v a t e the enzyme (Tsai and Kemp, 1975). Imidazole buffer at pH 7.4 and 37°C was used. Pyruvate kinase was assayed by coupling to l a c t a t e dehydrogenase. i/ ++ Assay conditions were 1.0 mM phosphoenolpyruvate, 1.0 mM ADP, 7.5 mM Mg , 50 mM K +, 0.1 mM NADH, and excess l a c t a t e dehydrogenase, i n 50 mM imidazole buffer, pH 7.4, at 37°C. Lactate dehydrogenase a c t i v i t y was assayed by following NADH oxidation. Conditions were 2.5 mM pyruvate, 0.1 mM NADH, i n imidazole buffer (50 mM), pH 7.4, at 37°C. Separation of the l a c t a t e dehydrogenase isozyme i n s k e l e t a l muscle, lung, blood cells,heart and brain of the Weddell seal was 34 accomplished by the use of starch gel electrophoresis. The electrode buffer consisted of 0.05 M dibasic phosphate buffer, pH 7.0 (adjusted with sodium-free c i t r i c a c i d ) . The stationary phase used was 14% (W/V) starch suspended i n a 1:20 d i l u t i o n of the electrode buffer. Depending upon LDH a c t i v i t y , 5-10 ul were placed onto approximately 3 mm square wicks of the f i l t e r paper. These wicks were then placed into s l i t s cut into the starch g e l . Electrophoresis conditions were: 25 m A, 200 v o l t s run for 12 hours at 4°C. The s t a i n medium contained NAD (1 mM), phenazine methosulfate (0.1 mM) , n i t r o blue tetrazolium (1 mM), l a c t i c a c i d (100 mM), Tris-HCl (50 mM) at pH 7.5. Glucose-6-phosphatase was assayed by following the release of inorganic phosphate which was measured according to Nordlie (1971). Assay conditions ++ 't-were 5 mM glucose-6-phosphate, 7.5 mM Mg , 50 mM K , imidazole buffer, pH 7.4, 37°C. A unit of enzyme a c t i v i t y converts 1 pinole substrate to product per minute. A l l enzyme a c t i v i t i e s are expressed i n terms of units/gm wet weight of ti s s u e at 37°C. In a d d i t i o n to the above enzymes, attempts were made to measure glycogen phosphorylase. This seal t i s s u e enzyme was found to be extremely unstable, and we were unable to s t a b i l i z e i t . Drummond (pers. comm.) also found glycogen phosphorylase from diving animals d i f f i c u l t to work with. Lung S l i c e Studies Samples of lung t i s s u e were excised and washed i n c h i l l e d Ringers s o l u t i o n as previously described. Small samples were blotted on f i l t e r paper and weighed to the nearest mg. S l i c e s weighing 25-50 mg were placed 35 i n Ringers s o l u t i o n of the following composition: NaCl (122 mM), KC1 (3 mM), MgSO^ (1.2 mM), C a C l 2 (1.3 mM), KH^PO^ (0.4 mM) and NaHC03 (25 mM), pH 7.8. The 0 2 uptake rate by lung s l i c e s was determined at 37°C using a Gilson 14 14 Oxygraph. The oxidation of C-U-lactate and C-6-glucose by lung s l i c e s was determined using Warburg-type f l a s k s containing a small hyamine hydroxide C0^ trap. S l i c e s were incubated f o r 20-30 minutes i n the presence of l a b e l l e d and c a r r i e r substrates (protocol given below), holding s p e c i f i c a c t i v i t y of glucose and l a c t a t e constant but varying t o t a l concentration. The lung s l i c e experiments were terminated with PCA to stop the reac t i o n 14 and release the CO^ formed. C0 2 c o l l e c t e d i n the hyamine hydroxide traps was counted as described above. Metabolite Assays Lactate and pyruvate concentrations i n PCA extracts were determined by following the reduction of NAD+ or the oxidation of NADH i n the presence of p u r i f i e d l a c t a t e dehydrogenase. Glucose concentrations i n PCA extracts were determined r o u t i n e l y with the hexokinase assay measuring the change i n o p t i c a l density at 340 nm due to NADP+ reduction by glucose-6-phosphate dehydrogenase (Bergmeyer et a l . , 1974). In several extracts, glucose was also determined by the glucose oxidase method, the Benedict assay system and by an automated glucose analyzer with a l i n e a r response over the range of 5-50 umol/ml. A l l three assay procedures yielded the same absolute glucose concentrations. In add i t i o n , whole blood extracts were analyzed by gas l i q u i d chromatography (Albersheim et a l . , 1967) to show that glucose was the only major sugar present i n seal blood; fructose, galactose, mannose, and ribose did not occur i n measurable concentrations. 36 Amino Acid Analysis Aliquots of the PCA and SSA treated blood samples from the Weddell seals, harbor seals and male volunteers were f i l t e r e d using M i l l i p o r e f i l t e r s (mesh s i z e of 0.4 uM); the PCA samples were adjusted to a pH of approximately 2.2 with concentrated hydrochloric a c i d . One hundred micro-l i t r e portions were then injected into a Beckman C119 amino acid analyzer, packed with a type AA20 c a t i o n i c exchange column. Individual amino acid concentrations were determined by comparison of sample peak areas with those of a standard. No s i g n i f i c a n t differences i n amino acid concentrations between the two extraction procedures were observed. 37 CHAPTER III Blood Metabolite P r o f i l e s as a Consequence Diving 37a INTRODUCTION In studying metabolic events i n animals under st r e s s , i t i s usually advantageous to monitor blood metabolite concentrations p r i o r to, during and following the s t r e s s f u l episode. Itwas j u s t such a t a c t i c that enabled P. F. Scholander (1940) to postulate the probable existence of a s e l e c t i v e blood r e d i s t r i b u t i o n during diving i n aquatic mammals; h i s main, and i n fa c t only, evidence stemmed from observations of a rapid increase of l a c t a t e l e v e l i n the animal's blood upon surfacing. Scholander reasoned that anaerobic g l y c o l y s i s and the concomitant l a c t a t e production would be associated with p e r i p h e r a l va s o c o n s t r i c t i o n . P a r e n t h e t i c a l l y , Irving f i r s t suggested a peripheral v a s o c o n s t r i c t i o n i n 1939 but i t was Scholander who f i r s t offered any tangible proof for such a phenomenon during d i v i n g . Since very few studies have focused upon blood metabolite p r o f i l e s i n diving animals (see Hochachka et a l . , 1975, 1977a) advantage was taken of the opportunity to measure l e v e l s of some of the more important metabolites i n the c e n t r a l c i r c u l a t i o n p r i o r to, during and a f t e r a simulated dive of the Weddell s e a l . The primary aim of these investigations was to monitor glucose, pyruvate and l a c t a t e p r o f i l e s but i n the process a wide spectrum of ninhydrin r e a c t i v e compounds (amino acids and the t r i p e p t i d e glutathione) were also surveyed. Hochachka et aT. (1977a), under i d e n t i c a l experimental conditions to t h i s model, reported s i g n i f i c a n t decreases i n c e n t r a l blood l e v e l s of glucose, along with w e l l defined elevations i n the l a c t a t e p r o f i l e s during simulated d i v i n g . Whereas l a c t a t e l e v e l s rose sharply upon termination of the dive, glucose concentrations usually remained r e l a t i v e l y 38 constant or a c t u a l l y dropped for the f i r s t couple of minutes, followed by abrupt increases i n the whole blood l e v e l s . These r e s u l t s appear to be i n d i r e c t c o n tradiction to those of B l i x and Kjeskshus (unpublished work; see B l i x , 1976) as they not only observed a glucose increase during the forced dive of a harbor s e a l , but also recorded a consistent decline i n the plasma free f a t t y acids. Unpublished work by our group and that of Kooyman and Davis (pers. commun.) on trained harbor seals demonstrated d i s t i n c t i v e drops i n whole blood glucose during routine diving. I t can only be assumed that B l i x and Kjeskshus were observing a harbor s e a l who was mainly sustained by aerobic metabolism. P r i o r to t h i s study, no thorough i n v e s t i g a t i o n of amino acid p r o f i l e s during diving arid recovery cycles had been undertaken. Hochachka and associates (1975) did measure blood l e v e l s of alanine,prior to and a f t e r d i ving i n the harbor s e a l (no sampling was performed during the dive phase). By about 5 minutes i n t o recovery the amino acid had increased to approximately 200% over the predive values, i n d i c a t i n g a s i z a b l e flush-out of the peripheral t i s s u e s . This observation led the authors to postulate the existence of an anaerobic pathway with the amino acid as one of i t s endproducts. 39 RESULTS AND DISCUSSION Blood Glucose, Pyruvate and Lactate P r o f i l e s The present set of data, generated from simulated diving of approximately 10-30 min. predictably demonstrate steady declines i n glucose l e v e l s of the c e n t r a l (or main) c i r c u l a t i o n ; followed by continued decreases or sharp declines during the f i r s t 5-10 min. of recovery, when the t o t a l 60 1. of blood volume i s well-mixed (Fig. I l l , 1). The continued drop i n glucose l e v e l s i n early recovery may i n d i c a t e that glucose i s depleted i n the t o t a l blood volume and not only i n the c e n t r a l blood. If only the l a t t e r occurred, glucose l e v e l s would return to near-normal l e v e l s within the f i r s t minute of recovery as cardiac output rose and f u l l y mixed the blood. (In the above argument, i t i s assumed that there i s a ce n t r a l c i r c u l a t i o n , which i s slowly exchanging with the greater volume of blood trapped i n the peripheral regions of the animals. See Chapter V f o r a thorough discussion.) The eventual increase i n blood glucose i s presumably due to r i s i n g glucagon concentrations. Although no such analyses are a v a i l a b l e f o r the Weddell s e a l , Robin (pers. commun.) observed . the presence of th i s phenomenon-in the common harbor s e a l . In contrast to glucose p r o f i l e s during diving, blood l a c t a t e concentrations c o n s i s t e n t l y r i s e i n a r t e r i a l blood usually from less than 1 umol/ml i n predive states to over 3 umol/ml at the end of the diving period (Fig. I l l , 2). On the other hand, pyruvate l e v e l s can increase, decrease or remain r e l a t i v e l y constant during diving, but following simulated diving,' a large washout of both pyruvate and l a c t a t e i s always observed (Fig. I l l , 3). What was not expected was the large diff e r e n c e 40 F i g . I l l , 1. Change i n glucose concentration of whole a r t e r i a l blood during diving and recovery i n 6 representative seals whose i n i t i a l blood glucose supplies v a r i e d by nearly 2-fold. Duration of dives, standardized to an a r b i t r a r y scale, varied between 10 and 20 minutes. Dive times: ( ."O ), 20 minutes; ( A ) , 20 minutes; ( • ), 10 minutes; ( • ), 15 minutes; ( £ ), 20 minutes; ( AC ) , 20 minutes. 40a F i g . I l l , 1 41 F i g . I l l , 2. Change i n l a c t a t e concentration of whole a r t e r i a l blood during diving and recovery i n 5 representative seals. Dive duration varied between 10 and 20 minutes but was standardized to f a c i l i t a t e comparison on an a r b i t r a r y scale. Dive times: ( • ), 20 minutes; ( A ) , 20 minutes; ( O ) , 10 minutes; ( • ), 20 minutes; ( • ), 15 minutes. 41a Fraction of Recovery Time, Min Dive Time F i g . I l l , 2 42 Fi g . I l l , 3. Change i n pyruvate concentration of whole a r t e r i a l blood during diving and recovery i n 5 represenative seals. Dive duration varied between 10 and 47 minutes, but was standardized to f a c i l i t a t e comparison on an a r b i t r a r y s c a le. Dive times: ( • ), 10 minutes; (-• ), 20 minutes; ( O ), 46 minutes, ( • ), 15 minutes, ( A ), 20 minutes. 42a • 1 1 Fraction of Dive Time Recovery Time, M in F i g . I l l , 3 43 i n the k i n e t i c s of pyruvate and l a c t a t e appearance i n the blood, with the pyruvate washout peak always lagging behind the l a c t a t e washout peak (Fig. 111,4). As a r e s u l t , during early stages of recovery, l a c t a t e concentrations may r i s e well before there i s any measurable change i n pyruvate l e v e l s . This means transient, and very large changes i n lactate/pyruvate r a t i o s may occur i n early recovery periods ( F i g . I l l , 4). Since pyruvate and l a c t a t e are thought to be i n equilibrium i n a l l tissues, the question a r i s e s as to what processes account for t h e i r d i f f e r i n g behaviour during diving and recovery. One p o s s i b i l i t y i s that pyruvate and l a c t a t e form the basis for a kind of i n t e r - t i s s u e hydrogen c y c l i n g mechanism, organs i n d i f f e r e n t redox states exchanging one for the other. This kind of hydrogen s h u t t l i n g mechanism was predicted on t h e o r e t i c a l grounds (Hochachka and Storey, 1975) and has been previously observed i n the perfused mammlian heart during extreme hypoxia (Lee .et a l , 1973). Furthermore, i t i s quite p l a u s i b l e that the high a c t i v i t y l e v e l s of blood l a c t a t e dehydrogenase (Vallyathan et a l . , 1969) may contribute to redox balancing throughout the diving episode. A second p o s s i b i l i t y i s that l a c t a t e and pyruvate exchange rates between tis s u e and blood d i f f e r by a large enough factor to account for the lag i n pyruvate release into the blood. At present, the lack of relevant information p r o h i b i t s ascertainment of the precise s i t u a t i o n . The above data r a i s e further questions pertaining to the source(s) responsible for metabolite changes i n the c e n t r a l blood volume during the dive phase. Peripheral leakage and/or anaerobic fermentation i n one or more of the c e n t r a l organs are l i k e l y candidates. This problem w i l l be addressed and treated i n l a t e r chapters (Chapters V, VI, V I I I ) . 44 F i g . I l l , 4. Change i n lactate/pyruvate concentration r a t i o of whole a r t e r i a l blood during diving and recovery i n 3 representative seals. Duration of dives, standardized to an a r b i t r a r y scale, varied between 10 and 20 min. Dive times: ( • ), 10 min; ( 0 ) 20 min; ( A ) , 15 min. 4 4 a Fig.XII, 4 45 Free Amino Acids and Glutathione i n Resting Animals In an e f f o r t to decipher whether the diving habit i n marine mammals has dictated any a l t e r a t i o n s i n the free amino acid blood pool, blood samples from Weddell and harbor seals (2 phocids with dramatically d i f f e r e n t diving a b i l i t i e s ) and male volunteers were assayed for the more important amino acids. Table I I I , 1 gives absolute concentrations (nmoles/ml whole blood) of the sampled amino acids i n the three d i f f e r e n t groups analyzed. Unfortunately, sampling s i t e s were not consistent i n the three groups; venous blood was c o l l e c t e d from both the harbor seals and the human volunteers while the Weddell s e a l p r o f i l e s were derived from a r t e r i a l samples (see Materials and Methods). I t should be r e a l i z e d that comparisons between a r t e r i a l and venous amino acid pools must be handled with caution for some amino acids display s i g n i f i c a n t a r t e r i o -venous concentration d i f f e r e n c e s , depending on the p o s i t i o n of the catheters ( F e l i g et^ a l . , 1973). Be that as i t may, s t a t i s t i c a l evaluations of the mean differences i n concentrations between a l l three groups (Table I I I j 2) were- performed. Where differences were detected, the human le v e l s were usually the more concentrated, themajor exception being taurine, whose l e v e l s appear to be two f o l d higher i n the harbor s e a l than that of both the human and Weddell s e a l . The metabolic implications of the high taurine concentrations i n harbor s e a l blood are unclear since the precise b i o l o g i c a l functions of t h i s sulphur-containing amino acid have yet to be ascertained (Awapara, 1976). The t e r r e s t r i a l mammal, as represented by the male humans, has s i g n i f i c a n t l y higher concentrations of serine, glutamate, glycine and ornithine than those of both species of seals. Alanine p r o f i l e s 46 Table I I I , 1. Whole blood concentrations (yM) of free amino acids i n male Weddell seals ( a r t e r i a l samples), harbor seals (venous) and human volunteers (venous). Concentrations are presented as means + standard deviations of 3 to 5 subjects. Amino Ac i d Taurine Aspartate Threonine Serine Glutamate Glutamine Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine Lysine Ornithine H i s t i d i n e Arginine Total Branched Chained Amino Acids Total Amino Acid Pool Glutathione 46a Whole Blood Concentration Weddell seal 172.6 + 20.5 26.6 + 5.9 137.2 + 18.5 72.8 + 10.0 97.9 + 18.5 493.3 + 230.0 107.5 + 10.8 83.6 +2.6 167.4 + 16.0 38.4 + 6.5 155.1 + 24.3 18.5 + 2.9 64.6 + 7.2 103.3 + 4.8 28.2 + 1.2 78.4 + 12.6 61.8 + 9.4 391.4 + 42.6 Harbor seal 326.5 + 12.5 35.3 +4.0 99.6 + 25.5 91.3 + 4.8 63.5 +8.6 481.7 + 59.3 178.3 + 34.1 398.4 + 59.8 170.7 + 5.9 51.0 + 2.8 94.1 + 5.8 57.2 + 8.0 53.1 + 3.6 174.4 + 48.2 54.4 + 9.7 74.2 + 16.1 173.5 + 43.9 315.8 + 14.2 (nmol/ml) Human 170.5 + 13.4 35.7 +3.4 133.2 + 23.3 131.9 + 5.8 142.3 + 17.2 556.7 + 109.9 317.3 + 29.0 336.7 + 37.9 170.6 + 15.2 54.4 + 7.2 103.8 +14.0 55.8 + 6.9 55.9 +7.4 158.7 + 30.5 91.8 + 23.4 66.9 + 4.5 99.3 + 34.0 328.7 + 29.5 2769.8 + 156.9 678.0 + 46.6 2145.0+132.0 2692.0+176.0 1535.4 + 127.2 998.2 + 187.4 Table III, 1 47 Table I I I , 2. S t a t i s t i c a l evaluation of mean differences of amino acids concentrations presented i n Table I I I , 1. Subscripts i n d i c a t e the animal with the higher concentration. Abbreviations used: W = Weddell se a l ; , H = Harbor seal; hu = human; N.S. = not s i g n i f i c a n t . 47a Male Male Male Male Weddell vs Male Weddell vs Harbor Harbor vs Male Amino Acid Seal Human Seal Seal Seal Human Taurine N.S. p < .001 (H) p < .001(H) Aspartate N.S. N.S. N.S. Threonine N.S. N.S. N.S. Serine p < .001 (hu) N.S. p < .001(hu) Glutamate p < .01 (hu) p < .01 (W) p < .001 (hu) Glutamine N.S. N.S. N.S. Glycine p < .001(hu) p < .02 (H) p < .01 (hu) Alanine p < .001(hu) p < .001 (H) N.S. Valine N.S. N.S. N.S. Isoleucine p < .02 (hu) p < .05 (H) N.S. Leucine p < .01 (W) p < .001 (W) N.S. Tyrosine p < .001(hu) p < .001 (H) N.S. Phenylalanine N.S. N.S. N.S. Lysine p < .02 (hu) p < .05 (H) N.S. Ornithine p < .01 (hu) p < .01 (H) p < .05 (hu) H i s t i d i n e N.S. N.S. • N.S. Arginine N.S. p < .01 (H) N.S. Total branched chained amino acids N.S. N.S. N.S. Total amino acid pool p < .001(hu) p < .05 (H) N.S. Glutathione p < ,001(W) p < .01 (W) N.S. Table I I I , 2 48 provide the most dramatic d i f f e r e n c e s ; the l e v e l s of th i s metabolically active amino acid (see Goldberg and Chang, 1978) are three f o l d higher i n both the harbor s e a l and human than that of the Weddell s e a l . To assign any d e f i n i t i v e s i g n i f i c a n c e to t h i s f i n d i n g would be quite r i s k y considering the a v a i l a b i l i t y of so l i t t l e information. However, i t has been suggested that free blood alanine i s involved i n an alanine-glucose cycle between the s k e l e t a l muscle and l i v e r i n mammals ( F e l i g , 1973) . According to t h i s model alanine, derived from amnio acid or protein catabolism i n the muscles, i s used i n hepatic gluconeogenesis and c a r r i e s amino groups to the l i v e r f o r d i s p o s i t i o n as urea. If th i s postulated r o l e i s v a l i d for the Weddell s e a l , perhaps the extremely low l e v e l s of alanine could i n d i c a t e an active gluconeogenic process (Young and H i l l , 1973). Since most phocid seals have very low carbohydrate d i e t s ( B l i x , 1976) i t i s i n t u i t i v e l y obvious that most of t h e i r glucose must be derived gluconeogenically. Of course, t h i s does not explain the r e l a t i v e l y high l e v e l s of the amino acid occurring i n harbor s e a l blood since t h i s s e a l i s also a member of the genus Phocidae. The s i t u a t i o n may be more complex and hence must be thoroughly investigated before a precise explanation becomes apparent. Glutathione (GSH) p r o f i l e s , as determined by the Beckman C119 amino acid analyzer, i n d i c a t e that t h i s t r i p e p t i d e i s s i g n i f i c a n t l y more concentrated i n the Weddell s e a l than i n human and harbor s e a l whole blood (see Tables I I I , 1 and I I I , 2). However, the human values are low i n comparison to other l i t e r a t u r e reports; Bernt and 49 Bergmeyer (1974) report whole blood l e v e l s i n the 1000 ymol/ml range. U n t i l more data i s generated i t would be inappropriate to attach much importance to the above f i n d i n g . Therefore, the current data demonstrate that most free amino acids i n mammalian blood remain r e l a t i v e l y constant i n a l l members of the class thus far studied. The differences, as recorded i n t h i s t h e s i s , could be a t t r i b u t e d to sampling s i t e s e l e c t i o n s , f a s t i n g durations and d i f f e r e n t dietary regimes. Diving P r o f i l e s of Amino Acids and Glutathione During restrained diving i n the Weddell s e a l only alanine and glutamine levels inwhole blood increase , (glutamate and glycine also r i s e , but are considered to be involved i n the glutathione changes; t h i s w i l l be discussed l a t e r i n the chapter). Alanine ( F i g . I l l , 5) displays a slow but steady increase throughout the dive, followed by a more rapid r i s e (up to 200% increase over pre-dive levels) early i n the recovery period. Glutamine p r o f i l e s (Fig. I l l , 6) are q u a l i t a t i v e l y s i m i l a r to those of alanine; both amino acids have been implicated to function as waste nitrogen c a r r i e r s , transporting amino nitrogen out of various tissues to the l i v e r , kidney and other s i t e s (Chang and Goldberg, 1978). Since some amino acids are thought to be fermented i n hypoxic muscles i t i s quite possible that both are acting as nitrogen transporters which are flushed-out i n the recovery phase of diving. The alanine increase may also be due to an a c t i v a t i o n of muscle alanine aminotransferase. I t has been postulated that during diving the hypoxic or anoxic muscles w i l l channel pyruvate to l a c t a t e and alanine (Owen and Hochachka, 1974). The alanine and 50 F i g . I l l , 5. Percentage change of alanine concentration i n whole a r t e r i a l blood during diving and recovery i n 4 representative seals. Dive duration varied between 10 and 20 min, but was standardized to f a c i l i t a t e comparison on an a r b i t r a r y scale. Dive times: (0), 10 min; (X),18 min; ( A ) , 20 m i n ; ( p ) , 17 min. 50a F i g . I l l , 5 51 F i g . I l l , 6. Percentage change of glutamine concentration i n whole a r t e r i a l blood during diving and recovery i n three representative seals. Duration of dives, standardized to an a r b i t r a r y scale, varied between 10 and 20 min. Dive times: (0), 10 min; (X), 10 min; ( • ) , 20 min. 51a Rig. 111,6 52 co-substrate oxo-glutarate are produced by the action of alanine aminotransferase on pyruvate and glutamate. The alanine presumably accumulates i n the cytoplasm whereas the oxo-glutarate i s transported to the mitochondria where i t i s thought to be eventually converted to succinate. This pathway not only aids i n balancing redox but also increases energy supply i n the form of GTP (Owen and Hochachka, 1974). Part of the alanine increase associated with recovery could also stem from the sparking of the t r i c a r b o x y l i c acid cycle i n the muscles during anaerobic-aerobic t r a n s i t i o n (Hochachka and Murphy, 1979). Whatever the source, most of the alanine probably i s absorbed by the l i v e r where i t s carbon skeleton i s used for the synthesis of glucose and the nitrogen atoms excreted i n the form of urea ( F e l i g , 1973). Glutamine, on the other hand, may ultimately be u t i l i z e d by the l i v e r , the g a s t r o - i n t e s t i n a l t r a c t (Chang and Goldberg, 1978) and/or catabolized by the kidney (Baruch et a l . , 1976) . Of the ten d i f f e r e n t dive sequences analyzed for glutathione, f i v e displayed rather dramatic drops i n blood concentrations of GSH. These decreases i n the dive phase were usually followed by rapid increases to above pre-dive l e v e l s upon recovery (Fig. I l l , 7). Glutathione, a ubiquitous t r i p e p t i d e (a-glutamyl-cysteinglycine), i s postulated to have at l e a s t four major b i o l o g i c a l r o l e s : 1) i t can conjugate with many d i f f e r e n t substances, priming them f o r eventual excretion (Flohe et a l . , 1974); 2) i t may act as a co-enzyme (Jocelyn, 1972); 3) i t probably functions as an anti-oxidant, protecting membranes and maintaining sulfhydryl-containing i n t r a c e l l u l a r enzymes i n t h e i r active states (Beuther, 1971) and 4) GSH has been postulated to p a r t i c i p a t e i n the 53 Fi g . I l l , 7. Change i n glutathione concentration of whole a r t e r i a l blood during diving and recovery i n 5 d i f f e r e n t seals. Dive duration varied between 10 and 30 min, but was standardized to f a c i l i t i a t e comparison on an a r b i t r a r y scale. Dive times: (0), 20 min; (=p) , 10 min; (A) , 15 min; (•), 22 min; (X), 17min. 53a Dive o e OJ c O JZ -1~> CO 2000 16004 1200 H 800-e 400H r 30 60 .5 1.0 10 20 Fraction of Recovery Time, Min Dive Time Fig. I l l , 7 54 transport of amino acids across c e l l u l a r membranes (Meister, 1973). Although early dive data are sketchy, i t appears that by mid-dive, at the l a t e s t , 80-100% of the GSH had disappeared from the c e n t r a l c i r c u l a t i o n . Erythrocytes, as most other tissues, possess the necessary enzymes needed to convert the t r i p e p t i d e into i t s three amino acid components: glutamate, cysteine and glycine (Palekav et _al., 1974). Table I I I , 3 gives a summary of the mean percentage retentions and disappearances of blood glutamate and glycine as a function of GSH changes i n the c e n t r a l blood volume. Cysteine values are not presented as t h i s amino acid was not detectable by the techniques employed, probably due to the lack of s u l f h y d r y l protection and/or to the r e l a t i v e l y lengthy storage times. During diving only 50-55% of the degraded GSH can be traced to increases i n free blood glutamate and glycine, whereas approximately 35% of the resynthesized GSH appears to be derived from the blood pools of the two amino acids. At the moment, i t i s impossible to account f o r the t o t a l amino acids released from the breakdown of GSH (45-50% unaccountable) and t h e i r subsequent uptake f o r the resynthesis of the t r i p e p t i d e (65% unaccountable). Possibly, a portion of the released amino acids or glutathione, i t s e l f , are stored (or even metabolized) i n some organ or ti s s u e i n the pe r i p h e r a l blood during the dive. Unfortunately no s o l i d p h y s i o l o g i c a l s i g n i f i c a n c e to t h i s set of data can be offered, and furthermore the presence of an ar t e f a c t cannot be ruled out. However, i f t h i s were a manipulative error, one would expect to observe the same phenomenon throughout the e n t i r e study. From the amino acid study i t i s concluded that routine diving episodes (of up to 30 minutes) have very minimal e f f e c t s on the majority of free 55 Table I I I , 3. Mean percentage changes (+ standard errors) i n whole blood glutamate and glycine as functions of GSH changes during diving-recovery c y c l e s . A free amino acid x 100 A GSH Sample sizes = 5 Mean Percentage Change Condition Glutamate Glycine Dive 50.6+4.6 54.2+10.4 Recovery 37.0+3.1 3 5 . 5 + 6 . 2 56 amino acids i n whole blood of the adult Weddell seal; a r e s u l t consistent with that of recent studies on anoxia i n the freshwater t u r t l e , Pseudemys  sc r i p t a elegans and the common g o l d f i s h , Carassius auratus (B. Emmett and E. Shoubridge, pers. commun.). The impressive homeostasis of a l l these animals could be due to a very f i n e l y tuned regulation of the amino acid blood pools or, more l i k e l y , to a lack of dependence on t h i s p o t e n t i a l energy source during transient hypoxic and anoxic episodes. 57 CHAPTER IV Enzymes of Aerobic and Anaerobic Metabolism i n the Brain, Heart and Lung of the Weddell Seal 57a INTRODUCTION In any comprehensive metabolic study i t i s sometimes us e f u l to explore the 'enzymatic p o t e n t i a l s ' of the tissues or organs under i n v e s t i g a t i o n . However, the mapping of enzyme t i t e r s w i l l , at the best, provide a gross index of the complex metabolic organization i n the t i s s u e . Invariably, such enzymatic assays are performed under optimal experimental conditions of substrate, cofactors, pH, a c t i v a t o r concentra-t i o n , etc. A more v a l i d and revealing procedure would be to mimic the precise ' i n vivo' environment under the d i f f e r e n t conditions to which the enzyme i s exposed; unfortunately t h i s s t i l l remains a formidable task. Without such data one i s compelled to extrapolate from optimal a c t i v i t i e s to actual ' i n vivo' rates. Such conjecture may render the analysis meaningless, e s p e c i a l l y i f the enzyme i s near equilibrium or non-regulatory, for such enzymes do not accurately mirror the pathway's p o t e n t i a l . Nevertheless, a c a r e f u l s e l e c t i o n of enzymes to be tested can allow for v a l i d i n s i g h t s i n t o metabolic d i r e c t i o n s and p o t e n t i a l s . For example Cr.abtree and Newsholme (1972) have demonstrated that g l y c o l y t i c rates can be d i r e c t l y correlated with the a c t i v i t i e s of phosphorylase, hexokinase (HK) and phosphofructokinase (PFK). Simon and Robin (1971, 1972) observed a close r e l a t i o n s h i p between pyruvate kinase (PK) a c t i v i t y and l a c t a t e production during anaerobiosis and cytochrome oxidase a c t i v i t y with basal oxygen consumption. More recently, Guppy and co-workers (1979) have demonstrated that the tuna white muscle, a very intense anaerobic t i s s u e , contains not only unusually high a c t i v i t i e s of phosphorylase and l a c t a t e dehydrogenase (LDH) but also a-glycerophosphate dehydrogenase, 58 * m a l a t e dehydrogenase and glutamate o x a l o a c e t a t e transaminase-, i m p l y i n g h i g h a n a e r o b i c p r o d u c t i o n of energy v i a g l y c o l y s i s w i t h a v e r y e f f i c i e n t mechanism to b a l a n c e redox. Simon and a s s o c i a t e s (1979) r e p o r t a c t u a l i n c r e a s e i n muscle PK a c t i v i t i e s d u r i n g p r o l o n g e d submergence o f the f r e s h w a t e r t u r t l e (Pseudemys s c r i p t a ) . To r e c a p i t u l a t e , i t i s r e a l i z e d t h a t t h e r e a r e many i n h e r e n t l i m i t a t i o n s a s s o c i a t e d w i t h the e x t r a c t i o n of r e l e v a n t m e t a b o l i c i n f o r m a t i o n from maximal v e l o c i t y d a t a of enzymes; but i f used ( w i t h c a r e and r e s t r a i n t ) i n c o n j u n c t i o n w i t h o t h e r m e t a b o l i c parameters a c l e a r e r p i c t u r e of the o v e r a l l m e t a b o l i c s t a t e of the t i s s u e w i l l p r o b a b l y emerge. The f o l l o w i n g study was performed i n an attempt t o p l a c e some ground work f o r the e v e n t u a l e l u c i d a t i o n of the m e t a b o l i c p r o f i l e s o r p o t e n t i a l s o f the h e a r t , l u n g and b r a i n i n the e x p e r i m e n t a l a n i m a l . Some key enzymes of b o t h g l y c o l y s i s and o x i d a t i v e m e t a bolism were measured; a l l a r e compared to homologous enzymes of a c o m p a r a b l y - s i z e d t e r r e s t r i a l mammal ( o x ) . 59 RESULTS AND DISCUSSION Oxidative Enzymes Four mitochondrial marker enzymes were assayed to measure the oxidative capacities of the s e a l heart, lung and brain: g-hydroxybutrylCoA dehydrogenase and g-hydroxybutyrate dehydrogenase, functioning i n g-oxidation of f a t t y acids and ketone body metabolism, res p e c t i v e l y ; c i t r a t e synthase, catalyzing the entry of acetylCoA carbon into the Krebs cycle and thought to represent an important c o n t r o l s i t e (Tischlev j 2 t a l . , 1977); and glutamate dehydrogenase, a key regulatory enzyme (Srere, 1969), catalyzing the entry of glutamate carbon i n t o the Krebs cycle. As the heart, lung and b r a i n d i f f e r greatly i n metabolic organization (Siesjo and Nordstrom, 1977; Neely and Morgan, 1974; Tierney, 1974a), t h e i r enzyme a c t i v i t y p r o f i l e s also d i f f e r greatly (Table IV, 1). However, the a c t i v i t i e s of these mitochondrial marker enzymes i n s e a l heart, lung, and b r a i n are s i m i l a r or somewhat lower than those measured under i d e n t i c a l • conditions i n homologous ox t i s s u e s . P a r e n t h e t i c a l l y B a l l a n t y n e (personal communica-tion) found cardiac c a r n i t i n e palmitoyl-transferase, an enzyme considered to be rate l i m i t i n g i n the oxidation of f a t t y acids (Pande and Blanchaer, 1971), to be s i m i l a r i n a c t i v i t y l e v e l s to that occurring i n bovine heart. These r e s u l t s imply that when oxygen i s a v a i l a b l e , a l l three organs can sustain oxidative metabolism at rates per gm. t i s s u e that w i l l be s i m i l a r to other mammalian species. This i s not s u r p r i s i n g since a vigorous C^-based metabolism i s w e l l documented i n seals (Ashwell-Erickson and Eisner, 1977; G a l l i v a n and Ronald, 1979). Enzymes of Anaerobic G l y c o l y s i s Hexokinase, phosphofructokinase, and pyruvate kinase, which are a l l 60 Table. IV, 1., Enzyme a c t i v i t i e s i n brain, heart and lung of. the. Weddell seal and ox. Values are. means, with, ranges i n parentheses, expressed as umole substrate converted/min/g wet tis s u e at 37°C, pH 7.4, and saturating l e v e l s of substrates, cofactors or coenzymes.. Detailed assayed conditions are presented i n Chapter I I . Sample sizes f o r the se a l and bovine assays were 6 and 2, re s p e c t i v e l y . *n = 1; almost i d e n t i c a l values obtained from two cetaceans (W. Vogl, unpublished data). 60a Brain Heart Lung Enzymes Seal Ox Seal Ox Seal Ox C i t r a t e Synthase 17.8 + 2.5 (15.6 - 20.5) 16.8 (15.4 - 18.1) 28.8 + 6.9 (18.8 - 35.3) 61.7 (45.8 - 79.9) 1.52 + 0.9 (0.8 - 1.7) 6.6 (6.4 - 6.9) Glutamate Dehydrogenase 7.5 + 1.6 (5.7 - 8.6) 4.5 4.4 + 1.3 (3.5 - 6.5) 2.8 (2.4 - 3.2) 0.90 + 0.4 (0.6 - 1.7) 0.9 3 - hydroxybutrate dehydrogenase 0.40 + 0.10 (.28 - 0.5) 0.3 (1.-9 - 3.6) 2.42 + 0.7 (1.9 - 3.6) 2.8 1.7 +1.0 (0.5 - 2.5) 0.2 3-hydroxybutyryl CoA dehydrogenase 3.4 + (0.3) (3.2 - 3.7) -16.0 + 3.5 (12.5 - 20.5) -1.2 + 0.4 (0.5 - 2.5) -Hexokinase 5.2 + 1.8 (3.0 + 5.7) 1.3 (1.3 - 1.4) 2.0 + 0.9 (1.2 - 3.4) 2.0 (2.8 - 3.0) 1.7 + 0.8 (1.1 - 2.9) 2.2 (2.2 - 2.1) Glucose 6-phosphatase * 0.61 - 0.48 - 0.72 -Phospho-fructokinase 8.6 + 2.8 (5.3 - 12.3) 8.6 (9.2 - 9.0) 16.7 + 5.9 (9.9 - 24.1) 14.0 (13.9 - 14.0) 3.7 + 1.3 (2.3 - 5.4) 4.7 (4.2 - 5.2) Pyruvate kinase 167.3 + 11.1 (157 - 350) 196 (194 - 198) 217.5 +51.5 (183.3 - 294.0) 133.1 '(128 - 137.9) 45.6 + 17.7 (21.6 - 66.3) 98.0 (94.4 - 101.6) Lactate dehydrogenase 228.8 (200 - 350) 128.2 (125.8 - 130.6) 1032.0 + 45.7 (1013 - 1050) 556.0 (508 - 604) 69.6 + 30.7 (50.6 - 107.8) 91.9 (79.8 - 104.0) 61 p o t e n t i a l regulating s i t e s i n anerobic g l y c o l y s i s (Scrutton and Utter, 1968), and l a c t a t e dehydrogenase, catalyzing the terminal step i n g l y c o l y s i s , were measured to assess the p o t e n t i a l f or anaerobic g l y c o l y s i s i n these organs. In the seal brain, hexokinase and l a c t a t e dehydrogenase occur at 4- and 2-fold higher l e v e l s than i n the ox brain, r e s p e c t i v e l y , while pyruvate kinase and phosphofructokinase occur at s i m i l a r concentrations i n the ox and s e a l b r a i n (Table IV, 1). In the s e a l lung, these four g l y c o l y t i c enzymes occur at l e v e l s s i m i l a r to those i n the ox lung. In the heart of both species hexokinase and phosphofructokinase occur at s i m i l a r l e v e l s . In contrast, the a c t i v i t i e s of seal heart PK and LDH are 1.5- to 2-fold higher than i n the ox heart, the l a t t e r occurring at lev e l s of about 1000 umoles product/min/gm at 37°C. This i s the highest concentration of LDH of any comparatively sized vertebrate heart thus f a r studied. I n t e r e s t i n g l y , electrophoretic studies show that i n a l l three organs both heart- and muscle-type subunits of LDH are synthesized; thus multiple isozymes occur i n a l l three organs ( F i g . IV, 1). In the heart and brain, the heart-type subunits have higher a c t i v i t y than the muscle-type subunits. Nevertheless, a l l three organs c l e a r l y have the p o t e n t i a l e i t h e r f o r l a c t a t e production, catalyzed most e f f e c t i v e l y by muscle-type l a c t a t e dehydrogenase, or for l a c t a t e u t i l i z a t i o n , catalyzed most e f f e c t i v e l y by heart-type l a c t a t e dehydrogenase- (Holbrook et. a l . , 1975) . Emp i r i c a l l y t h i s i s indicated by the r a t i o of pyruvate reductase a c t i v i t y to l a c t a t e oxidase a c t i v i t y , which i s s t r i k i n g l y higher for the s k e l e t a l muscle l a c t a t e dehydrogenases (Fig. IV, 1). Similar r e s u l t s have been generated from other diving animals including seals (see Chap. I). 62 F i g . IV, 1. Starch gel electrophoretic separation of l a c t a t e dehydrogenase isozymes in s k e l e t a l muscle (M), lung (L), red blood c e l l s (RBC), heart (H), and brain (B) of the Weddell s e a l . Electrophoresis conditions: 25 mA, 200 v o l t s , 12 hours at 4°C, anode at the top, o r i g i n marked with an arrow. The subunit composition of each isozyme i s shown on the r i g h t . The numbers below r e f e r to the r a t i o of pyruvate reductase a c t i v i t y to l a c t a t e oxidase a c t i v i t y at pH 7.4 at saturating coenzyme and substrate concentrations, assayed at 37°C. 62a 63 Glucose-6-Phosphatase Glucose-6-phosphatase catalyzes the terminal step i n the formation of glucose e i t h e r from t r i o s e precursors or glycogen (Scrutton and Utter, 1968). Int e r e s t i n g l y , the enzyme occurs i n a l l three organs of the Weddell seal (Table IV, 1). The r a t i o of glucose-6-phosphatase to hexokinase a c t i v i t i e s , which may supply an i n d i c a t i o n of the p o t e n t i a l for glucose release vs. glucose phosphorylation, i s highest for the lung (0.4) and lowest (about 0.1) for the brain. These r e s u l t s may explain two previous fi n d i n g s . F i r s t l y , i t has been reported that the seal lung can release small amounts of glucose into the blood (Hochachka et^ a l . , 1977a), a process that would necessitate glucose-6-phosphatase function. And secondly, the Weddell seal heart metabolically may serve as a glucose-storage organ (Kerem et a l . , 1973) since i t accumulates huge amounts of glycogen which could be mobilized and released as glucose under hypoxic s i t u a t i o n s . Although glucose release by the heart has not been demonstrated, i f i t occurs, i t too would require glucose-6-phosphatase function. The occurrence of s i g n i f i c a n t a c t i v i t i e s of glucose-6-phosphatase i n brain t i s s u e i s i n agreement with recent studies by Anchors and coworkers (1977) . The brain i s usually regarded as having an absolute dependence upon glucose as a substrate and, as discussed below, the seal brain also u t i l i z e s i t (see Chapter VI). However, infrequently and only when blood l a c t a t e l e v e l s are high, the seal brain appears to release measurable amounts of glucose into the blood (P. W. Hochachka and B. Murphy, unpubl. data). The phenomenon may be common to marine and t e r r e s t r i a l mammals a l i k e since the same enzyme p r o f i l e s emerges from studies of the heart, lung, and brain i n two species of whales (Vogl, 1979) as well as from 64 nervous t i s s u e and nerve c e l l l i n e s of t e r r e s t r i a l species (Anchors et a l . , 1977). From the above enzyme p r o f i l e s , i t i s t e n t a t i v e l y concluded that the p o t e n t i a l f o r anaerobic g l y c o l y s i s i n the seal brain and heart may be somewhat greater than that of the t e r r e s t r i a l species, while oxidative p o t e n t i a l i s unchanged or s l i g h t l y reduced; however, diff e r e n c e s , where found, are rather modest. Such a s i t u a t i o n may imply quite a d i f f e r e n t l e v e l of biochemical adaptation, which could include k i n e t i c modifications of c e r t a i n p i v o t a l regulatory enzymes. The enzyme data presented unequivocally indicate that a l l three organs are capable of using or releasing both glucose and l a c t a t e . With t h i s i n mind, i t was decided to monitor organ arterio-venous (A-V) concentration differences of both metabolites i n the i n t a c t animal; t h i s could provide insi g h t s into glucose and l a c t a t e metabolism from r e s t i n g , diving and recovery phases. Such information would also be h e l p f u l i n ascertaining c e n t r a l organ contributions to the observed metabolite changes i n the main c i r c u l a t i o n during diving episodes (see Chapter I I I ) . 65 CHAPTER V Impact of Diving on Lung Metabolism 65a INTRODUCTION Since the metabolic and biochemical organization of the mammalian lung has, u n t i l recently, been l a r g e l y ignored, i t i s understandable that the present knowledge of pulmonary metabolism i n diving mammals i s i n an expanding phase. Recent and innovative ' i n vivo' lung research (Wolfe et a l . , 1979; Rhoades et a l . , 1978, Hochachka et a l . , 1977a) have uncovered some revealing i n s i g h t s into the metabolic status of t h i s organ i n both the t e r r e s t r i a l and aquatic mammals (see Chapter I for a general review of mammalian lung metabolism) . According to Hochachka e_t a l . (1977a) the Weddell s e a l lung i s capable of using or r e l e a s i n g both l a c t a t e and glucose. The i n i t i a l studies showed that A-V concentration gradients are small and therefore a more powerful approach was needed to ascertain the metabolic fate of c i r c u l a t i n g l a c t a t e or glucose a r r i v i n g at the lung. That i s why i n these studies emphasis was placed on isotope experiments; i t was reckoned that more d e f i n i t i v e data could be generated by u t i l i z i n g r a d ioactive tracers i n both ' i n vivo' preparations 14 and tissue s l i c e incubations. C - l a c t a t e was used todeduce the 14 14 metabolic fate of absorbed l a c t a t e while C - l a c t a t e and C - glucose competition experiments were u t i l i z e d to compare r e l a t i v e lung preferences for the two substrates. 66 RESULTS AND DISCUSSION "In Vivo' Preparation These experiments e s s e n t i a l l y consisted of tracing metabolic derivatives 14 (e.g. C O 2 ) of C-lactate infused into the blood on the afferent side of the lung. A bolus of "^C-lactate (about 4 uC/1 blood) plus dye and c a r r i e r l a c t a t e (whose f i n a l concentration was about 3 mM, equivalent to what the lung might experience towards the end of 15-20 minute dive periods) was r a p i d l y i n j e c t e d into the r i g h t v e n t r i c l e a f t e r 10 minutes of simulated diving. Blood samples were taken every 20 seconds at two ports representing r i g h t (pulmonary artery) and l e f t (aorta) sides of the c i r c u l a t i o n (Scheme 14 V - l ) . Most of the C-lactate (as w e l l as the unlabelled bolus) traversed the pulmonary c i r c u l a t i o n simultaneously with the tracking dye (Fig. V, 1). A small f r a c t i o n of the l a c t a t e bolus r a p i d l y traversed the pulmonary c i r c u l a t i o n so that at the time of the f i r s t two samples (at 20 and 40 14 seconds), s i g n i f i c a n t amounts of C-lactate were already present i n a r t e r i a l blood. At the same time (20-40 seconds a f t e r i n j e c t i o n ) although 14 the C-lactate and absolute l a c t a t e concentrations were decreasing i n 14 the pulmonary c i r c u l a t i o n , CO2 had already appeared i n a o r t i c blood and could only have been generated by lung metabolism since at t h i s time 14 the lung was the only t i s s u e (other than blood) to have received C-lactate. 14 The majority of the CO2 pulse appeared on the l e f t side of the heart, peaking at about 35 seconds a f t e r i n j e c t i o n ; the smaller i n i t i a l peak 67 Scheme V - l . Diagrammatic representation of the Weddell s e a l c i r c u l a t i o n , i n d i c a t i n g blood sampling ports. RV, r i g h t v e n t r i c l e ; RA, r i g h t atrium; PA, pulmonary artery; PV, pulmonary vein; LA, l e f t atrium; LV, l e f t v e n t r i c l e . Blood flow rates from Zapol et a l . (1979). Bronchial c i r c u l a t i o n assumed to be s i m i l a r to t e r r e s t r i a l mammals (Nagarshi, 1974). The diagram emphasizes two (coronary and cerebral) and possibly three (bronchial) ' f a s t - c i r c u l a t i o n pathways' for tracer to return to i n j e c t i o n s i t e , as w e l l as numerous 'slow c i r c u l a t i o n pathways'. 67a Diaphramatic Sphincter CORONARY BRAIN LIVER KIDNEYS MUSCLE Cardiac Outputs PREDIVE: 40 L /n DIVE: 6 L/min ~~j LA j LV |  Bronchial Circulation OTHER TISSUES AND ORGANS PREDIVE: 1000 ml/min DIVE: 140 ml/min PREDIVE: 300 ml/min DIVE: 300 ml/min PREDIVE: 1000 ml/min . DIVE: 40 ml/min PREDIVE: 4000 ml/min DIVE: 360 ml/min PREDIVE: 8000 ml/min DIVE: 500 ml/min Scheme V - l 68 F i g . V, 1. U- C l a c t a t e oxidation by the Weddell seal lung i n vivo. M u l t i p l e openings i n r i g h t v e n t r i c u l a r i n j e c t i o n port of 14 Swan-Ganz catheter assured well-mixed introduction of C l a c t a t i bolus. Rapid manual i n j e c t i o n was performed a f t e r a stable bradycardia (heart rate of 15 beats/min) was established at 10 min i n t o a 17 min simulated dive. At 20-s i n t e r v a l s simultaneous 5-ml blood samples were withdrawn from pulmonary artery (PA) and aorta and treated as described i n Chapter I I . Cardiac output i n t h i s experimental s e a l (350-kg male) was 24 1/min before diving and decreased to 4 1/min at 10 min i n t o dive. Q u a l i t a t i v e l y s i m i l a r r e s u l t s were obtained i n a preliminary experiment with another seal during entry into a simulated dive. 68a Seconds F i g . V,l 69 of CC>2 i n the pulmonary a r t e r i a l blood probably arr i v e d v i a the coronary and/or bronchial c i r c u l a t i o n s (see Scheme V - l ) . Consistent with t h i s i n t e r p r e t a t i o n and predicted by i t i s the observation that 14 the o s c i l l a t i o n s i n blood CC^ l e v e l s on the l e f t and r i g h t sides of the heart i n i t i a l l y are about 90° out of phase with each other. This s i t u a t i o n holds for about the f i r s t 100 seconds. Nevertheless, for the duration of the experiment (7 minutes, of which only the f i r s t 280 seconds 14 are shown i n F i g . V, 1), i t appears that lung oxidation of C-lactate exceeded l a c t a t e oxidation by any other organs, a process which would 14 maintain the co n s i s t e n t l y higher l e v e l s of CO2 i n the aorta than i n the PA blood. The mean pulmonary-to-thoracic aorta c i r c u l a t i o n time (time for traversing from the r i g h t to the l e f t side) i s about 30-40 seconds under simulated diving conditions, with heart rate reduced from 55 to 15 beats/min and cardiac output reduced from about 24 to 4 £/min. Thus a complete c i r c u l a t i o n time i s estimated to be about 60-80 seconds (Fig. V, 1) . Since the average cardiac output i s 6 Z-/min (Zapol et a l . , 1979), i t can be estimated that a " c e n t r a l " blood volume of 8 Z exchanges slowly with the rest of the 60 t blood volume, which i s presumably pooled i n the venous system. Unfortunately, the present data do not allow for a concrete estimation of the exchange rate between the two blood pools. Nevertheless, t h i s rate i s assumed to be r e l a t i v e l y low since the l a c t a t e p r o f i l e s change so dramatically immediately upon recovery from the dive episodes (see F i g . I l l , 2). Tissue S l i c e Studies The above experiments supply d i r e c t ' i n vivo' evidence that a major metabolic fate of l a c t a t e taken up by the s e a l lung i s complete oxidation. 70 Further evidence i n d i c a t i n g that l a c t a t e i s a good substrate f o r th i s organ comes from ti s s u e s l i c e experiments. Using a i r - e q u i l i b r a t e d Krebs-Henseleit Ringer s o l u t i o n , the Q0^ for lung t i s s u e was found to be s i m i l a r to that reported for other mammals (Wallace eJ: a l . , 1974), at 30.19 y l 02/hr/lOO mg wet weight (n=14; standard error = + 1.63), and remained stable f o r over an hour. Lung s l i c e s were found to oxidize 14 14 C-lactate at r e l e v a t i v e l y high rates but C-6-glucose at s u b s t a n t i a l l y reduced rates. Increasing glucose concentration from 1 to 10 mM caused a three-fold increase i n the rate of oxidation, while increasing l a c t a t e concentration from 1 to 10 mM increased oxidation rate by nearly f i v e - f o l d (Table V, 1A). Lactate oxidation exceeded glucose oxidation rates at a l l substrate concentrations, but t h i s was accentuated at high l e v e l s . The maximum oxidation rates compared quite c l o s e l y with those observed i n perfused rat lung (Wolfe et a l . , 1979; Rhoades et a l . , 1979); i n t e r a c t i n g e f f e c t s of glucose and la c t a t e were modest (Table V, IB) , i n contrast to the perfused rat lung, i n which p h y s i o l o g i c a l l e v e l s of l a c t a t e (1-10 mM) 14 i n h i b i t g l y c o l y s i s up to 60%. In part, the differences between C-U-lactate 14 and C-6-glucose oxidation may be a r t e f a c t u a l because the pyruvate 14 dehydrogenase reaction sequence allows CO2 production from uniformly-14 l a b e l l e d l a c t a t e but not from C-6-glucose while complete oxidation i n 14 the Krebs c y c l e releases CQ^ from them both. From the a v a i l a b l e data i t i s tempting to speculate that there would have been very l i t t l e t e l e o l o g i c a l need for the sea mammal to improve upon the pulmonary metabolic equipment i n the t r a n s i t i o n from a t e r r e s t r i a l to an aquatic environment. I t , l i k e other mammalian lungs, w i l l burn l a c t a t e i n preference to glucose; an arrangement that may be of 71 Table V, 1. """^ CC^  production from """^C-U-lactate and from 14 C-6-glucose by lung s l i c e s of the Weddell s e a l . Conditions are given i n Materials and Methods. In part A, glucose and la c t a t e concentrations are varied independently, while i n part 14 B, both are varied simultaneously. The rate of CO^ release i s expressed i n nmoles/hour/gm wet weight of ti s s u e at 37°C. Number of experiments given i n brackets. S p e c i f i c a c t i v i t y of glucose and l a c t a t e was constant under a l l conditions. A l l data are means + S.E. C0 2 from: 14 14 Conditions C-6-glucose C-U-lactate 10 mM glucose 247.8 + 62.0 (.6) 5 mM glucose 122.2 +16.6 (.5) 1 mM glucose .63.3 + 8.1 C6) 10 mM l a c t a t e 531.5 + 71.8 (6) 5 mM l a c t a t e 396.5 + 35.3 (6) 1 mM l a c t a t e 201.3 +25.7 (6) 10 mM glucose 1 mM l a c t a t e 204.0 + 23.2 197.8 + 15.2 (11) (12) 1 mM glucose 10 mM lacate 50.1 + 7.0 848.4 + 74.7 (12) (11) Table V, 1 72 paramount importance during a prolonged dive, for not only w i l l t h i s preserve v i t a l blood glucose stores for the c e n t r a l nervous system but w i l l also a id i n mopping,up' a p o t e n t i a l l y hazardous end product. However, i t should be r e a l i z e d that these experiments were performed without any thoracic compression, which the animal w i l l i n e v i t a b l y encounter upon diving to deep depths (e.g. 500 m for the Weddell s e a l ) . Thoracic compression could not only a l t e r bronchial and perhaps pulmonary blood flow but i t may d r a s t i c a l l y diminish the ale v o l a r oxygen stores. However, i t can be safe l y assumed that the lung does have an important function during the normobaric recovery period. When large quantities of l a c t i c acid are washed out of peripheral t i s s u e s , g l y c o l y s i s w i l l l i k e l y be dampened and the lung w i l l p r i m a r i l y burn the anaerobic end product. This coupled with an increased heart rate (M. Snider, pers. commun.) and hyperventilation may allow f o r a greater o x i d i z i n g capacity of the lung during t h i s c r i t i c a l phase when blood l a c t a t e l e v e l s can leap to over 20 umol/ml (Kooyman, pers. commun.). If these l e v e l s were unchecked they could pose serious buffering and oxygen loading problems for the organism. Since no pulmonary A-V concentration gradients were measured i n t h i s study i t was impossible to determine the lung's c o n t r i b u t i o n to metabolite changes i n the c e n t r a l blood volume during simulated d i v i n g . Nevertheless, i f the findings of Hochachka et a l . (1977a) are r e a l , i t can be t e n t a t i v e l y concluded that t h i s organ cannot contribute to the observed increases i n l a c t a t e l e v e l s during d i v i n g . In f a c t , one can extrapolate that an A-V concentration gradient of +0.1 umol of lactate/ml whole blood (Hochachka et al_. , 1977a) by a 4 kg. lung w i l l 73 e f f e c t i v e l y decrease l a c t a t e blood l e v e l s at a rate of 0.5 mmol/min., assuming a cardiac output of 5 Z/min. Such an arrangement also has the added advantage of sparing v i t a l glucose stores for other organs (e.g. the b r a i n ) . 74 CHAPTER VI Impact of Diving and Recovery on Cerebral Metabolism 74a INTRODUCTION It i s commonly thought that the mature mammalian br a i n uses glucose as i t s major substrate (Sokoloff, 1973) and oxygen as the terminal proton acceptor (Siesjo et a l . , 1976). Any s u b s t a n t i a l i n t e r r u p t i o n i n the flow of these two substrates could s p e l l grave consequences to the brain and i n e v i t a b l y to the organism. In t e r r e s t r i a l species, hypoglycemia re s u l t s i n the metabolism of endogenous substrates (Siesjo et a l . , 1976) while reductions i n the mean PaO^ (down to about 15 mm Hg) cause concomitant increases i n the cerebral blood flow (CBF), e f f e c t i v e l y maintaining a constant 0^ d e l i v e r y rate. Such an 0^ compensatory mechanism has been observed i n the duck (Anas platyrhynchos), the sea l i o n (Zapophus  Californianus) and most recently i n the harbor s e a l (Phoca v i t u l i n a ) during routine diving (Jones et a l . , 1979; Dormer et a l . , 1977; Eisner et a l . , 1978). However, other studies on the harbor s e a l and the northern elephant seal (Mirounga a n g u s t i r o s t r i s ) indicated l i t t l e or no CBF changes throughout routine and long term diving (Bron et a l . , 1966; Kerem et a l . , 1971; Van C i t t e r s et _al. , 1965), whilst that of the grey seal (Halichoerus grypus) showed a marked reduction (Dormer et a l . , 1977). At the time of t h i s study, p a r a l l e l work by Zapol et a l . (1979) demonstrated that CBF remained unaltered during simulated diving (of the Weddell seal) when Pa02 l e v e l s f e l l to about 25 mm Hg. Since these low tensions are considered to be hypoxic to the mammalian br a i n (Seisjo et a l . , 1976) one would i n t u i t i v e l y expect an increased CBF and/or an increased dependence upon anaerobic g l y c o l y s i s . P r i o r to t h i s study there were no a v a i l a b l e data concerning the anaerobic versus aerobic contributions to cerebral energy metabolism nor 75 on substrate preferences of the b r a i n under normal states and dive-recovery episodes. Some i n d i r e c t estimates suggested an activated r e l i a n c e on anaerobic g l y c o l y s i s , i n f e r r e d from substrate ( i . e . glycogen) storage data (Kerem et a l . , 1973), enzyme p r o f i l e s (Simon e^ t a l . , 1974) and electrophoretic patterns ( B l i x et a l . , 1970; Messelt and B l i x , 1976; Altman and Robin, 1969; B l i x and From, 1971). However two physiologic investigations did provide more tangible evidence. Ridgeway _et a l . (1969) i n measuring 0^ tensions i n expired a i r samples from a trained bottlenose porpoise (Tursiops truncatus) extrapolated that towards the end of a routine dive the porpoise b r a i n was by necessity, deriving most of i t s energy requirements anaerobically; the simulated diving experiments of Kerem and Eisner (1973) showed marked increases i n cerebral l a c t a t e production i n the l a t t e r stages of a prolonged harbor s e a l dive (20 minutes), again strongly suggesting an a c t i v a t i o n of anaerobic g l y c o l y s i s . I t was reasoned that assaying A-V differences for glucose and l a c t a t e i t would be possible to ascertain: 1) i f there i s any increase i n cerebral anaerobic metabolism as a function of low PaO^ l e v e l s associated with d i v i n g . 2) If there i s any change i n substrate preferences during diving and recovery episodes. 3) If cerebral metabolism could account for glucose and l a c t a t e concentration changes i n the c e n t r a l c i r c u l a t i o n throughout the dive phases. 76 RESULTS AND DISCUSSION A-V Concentration Differences By simultaneous sampling of a r t e r i a l blood i n the aorta and epidural vein close to occiput (King, 1977) i t was found that glucose uptake by the brain during simulated diving (0.4 umole/ml) i s somewhat higher than during c o n t r o l , r e s t i n g states (Table VI, 1). P a r e n t h e t i c a l l y predive estimates (.28 umole/ml) are i n the same range as those reported for the t e r r e s t r i a l mammalian br a i n (Maker et a l . , 1976). Only a small f r a c t i o n (about 1/11) of the t o t a l b r a i n hexokinase a c t i v i t y (Table IV, 1) would be needed to sustain these rates of glucose metabolism. The change i n the A-V concentration gradient during diving could be caused by changes i n uptake or by a reduction i n blood flow to the brain. However, p a r a l l e l microsphere studies of organ flow showed that brain perfusion, at l e a s t i n 8-12 minutes of diving, i s l a r g e l y unchanged or a c t u a l l y r i s e s somewhat (Zapol, e^ t a l . , 1979). Thus, the 140% increase i n the glucose concentration gradient (Table VI, 1) probably somewhat underestimates changes i n glucose uptake by the b r a i n during simulated diving. The estimate i s nevertheless i n s t r u c t i v e , f or i f b r ain oxygen supplies were l i m i t i n g during diving, glucose uptake would presumably increase by up to 18-fold due to the energetic i n e f f i c i e n c y of anaerobic g l y c o l y s i s ; s i m i l a r l y , the f r a c t i o n of glucose appearing as l a c t a t e should r i s e . The estimated 140% increase i n glucose uptake, therefore, indicates that the brain's dependence upon anaerobic g l y c o l y s i s does not r i s e greatly during simulated diving of up to 20 minutes duration, despite Pa0„ l e v e l s (as low as 25 mm Hg) which may be hypoxic to non-diving 77 Table VI, 1. Whole blood glucose and l a c t a t e concentration gradients across brain of the Weddell s e a l before and during simulated d i v i n g . Values are means + SE, expressed as the difference i n umol/ml between a r t e r i a l and venous blood samples drawn simultaneously. Maximum metabolic rate sustained by glucose catabolism i s calculated i n terms of umol ATP/g/min assuming a flow rate of 0.6 ml/g/min i n the control predive state and 0.75 ml/g/min during diving. Numbers i n parenthesis i n table r e f e r to number of samples. Duration of dives i n minutes shown i n square brackets. Cerebral Arteriovenous Differences Rate of „ - Lactate Glucose % Glucose ATP Seal No. Condition Production Uptake Fermented Formation 9 Predive 0.13 + 0.039(5) 0.38 + 0.042(5) 17 6.9 17 Predive 0.18 + 0.064(6) 0.26 + 0.031(6) 34 3.9 Dive Q L ( T | 0.24 + 0.125(2), 0.46 + 0.030(2) 23 10.8 18 Predive 0.13 + 0.120(3) 0.26 + 0.141(3) 25 4.4 !Dive [ l 5 ] 0.13 + 0.115(3) 0.30 + 0.109(3) 22 6.3 18 Predive 0.13 + 0.052(4) 0.25 + 0.106(3) 26 4.2 Dive [j2(T| 0.31 + 0.071(4) 0.50 + 0.038(3) 31 9.5 20 Predive 0.14 + 0.122(4) 0.26 + 0.092(3) 26. 9 3.6 Dive (3°J. 0.11 + 0.087(4) 0.34 + 0.119(4) 16 7.8 Predive mean 0.14 + 0.032(20) 0.28 + 0.033(21) 25. 0 4.7 Dive mean 0.16 + 0.068(12) 0.40 + 0.051(12) 20. 0 8.3 Table VI, 1 78 mammals (Siesjo and Nordstrom, 1977). This was confirmed by l a c t a t e measurements which estimate that the l a c t a t e released by the b r a i n accounts for 20-25% of the cerebral glucose uptake under both co n t r o l and diving conditions (Table VI, 1). In the r a t , the brain releases l a c t a t e (5-15% of absorbed glucose) because of l i m i t e d pyruvate dehydrogenase function (Cremer and Teal, 1974) and i t i s assumed a s i m i l a r mechanism operates i n the s e a l brain. Because of the higher t o t a l l a c t a t e dehydrogenase a c t i v i t i e s (Table IV, 1) and r e l a t i v e l y more muscle-type isozyme ( F i g . IV, 1) a large f r a c t i o n of the pyruvate pool may be diverted to l a c t a t e i n the seal brain. The routine and apparently "wasteful" release of l a c t a t e by the brain may therefore represent a minor cost of increasing i t s anaerobic p o t e n t i a l somewhat. Since the mean Pa02 l e v e l s never f e l l below 20-30 mm Hg i n any of the Weddell seals studied, i t would be informative to follow cerebral metabolism i n the animal during prolonged diving (50-70 min.) when a r t e r i a l 0^ tensions may dip below 20 mm Hg. Eisner and associates (1970b) suggest Weddell seals have greater cerebral tolerance for low 0^ than t e r r e s t r i a l mammals. Furthermore, harbor seals, apparently, show increased cerebral V-A l a c t a t e content towards the end of prolonged diving (Kerem and Eisner, 1973). I t can only be surmised that prolonged 'hypoxic' diving i n the Weddell s e a l would also be accompanied by a greater r e l i a n c e on anaerobic metabolism with a possible increase i n CBF. However, Kerem and Eisner (1973) report no such compensatory increases i n harbor s e a l CBF even at Pa02 l e v e l s approaching 10 mm Hg. Since the mean blood flow to the seal brain was measured, the glucose and l a c t a t e gradients allow estimation of metabolic rates i n terms of umol 79 ATP/gm/min that can be sustained with glucose as the carbon and energy source (Table VI, 1). The calculated metabolic rates sustainable by glucose catabolism (assuming complete oxidation) are somewhat lower than for brain metabolism i n man and other mammals (Siesjo and Nordstrom, 1977). This may be expected from the s c a l i n g e f f e c t s of body s i z e since b r a i n metabolic rates of large mammals are reported to be lower than i n small-sized species (Siesjo and Nordstrom, 1977). The estimates of cerebral metabolic rates do not take into consideration the flow of glucose carbon into glycogen, the pentose cycle, the free amino acid pool, or other metabolic pathways, and t h i s may explain the apparent differences between the diving and control states (Table VI, 1). Under some conditions, glucose incorporation into the free amino acid pool accounts for a large f r a c t i o n of the glucose uptake by the brain i n t e r r e s t r i a l mammals (Siesjo and Nordstrom, 1977). Thus, the higher estimated values for cerebral metabolic rates during diving (about 1.8 f o l d increases) may merely r e f l e c t an increased pooling of glucose carbon i n non-oxidative metabolic pathways. Are these metabolic rates high enough to cause s i g n i f i c a n t depletion of blood glucose reserves during diving? The answer i s evident i n a simple set of c a l c u l a t i o n s . I f one assumes an average A-V concentration gradient across the brain of about 0.4 umol/ml, and an average blood flow of about 700 ml/kg/min, then a 500 gm Weddell s e a l b r a i n can take up glucose at a rate of 0.14 mmol/min, or about 3 mmoles/20 min dive. Assuming an 8 1 volume exchanging slowly with t o t a l blood volume during diving (see Chapter V), t h i s rate of glucose uptake would lead to an o v e r a l l concentration change of 0.37 umol/ml blood over a 20 minute dive. This 80 glucose u t i l i z a t i o n rate would decrease blood glucose l e v e l s by less than 0.05 ymol/ml when the t o t a l blood volume was w e l l mixed. Complex mixing would be expected very early i n the recovery process since cardiac output tends to overshoot prediving control l e v e l s , reaching values as high as 60 1/min during the f i r s t minute of recovery (M. Snider, unpubl. data). Although i n fermenting some glucose to l a c t a t e , the se a l b r a i n i s not unusual, i t i s unusual i n the high f r a c t i o n (20-25%) that appears to be fermented, so the question arises as to whether b r a i n l a c t a t e production rate i s high enough to s i g n i f i c a n t l y increase blood l a c t a t e l e v e l s during diving. The same kind of c a l c u l a t i o n can be made as above to demonstrate that b r a i n anaerobic g l y c o l y s i s can lead to an o v e r a l l increase i n blood l a c t a t e concentration of only 0.18 ymol/ml over a 20 minute diving period, assuming 8 1 of c i r c u l a t i n g blood; t h i s increase would be less than 0.03 ymol/ml when the blood was f u l l y mixed i n recovery. I f these c a l c u l a t i o n s are correct, they imply that during routine diving periods (of about 20 minutes duration) b r a i n metabolism on i t s own does not markedly a l t e r t o t a l blood pools of glucose or l a c t a t e . For t h i s reason, and because lung metabolism alone contributes to opposite changes i n blood glucose and l a c t a t e l e v e l s , i t i s evident that these two organs cannot account f o r the rather marked changes i n both these metabolites during diving (Chapter I I I ) . I t can be, therefore, assumed that such metabolite a l t e r a t i o n s are due to peripheral leakage and, possibly, cardiac metabolism (see Chapter VIII for a discussion on cardiac metabolism). Lactate Uptake by the Brain In addition to being capable of releasing l a c t a t e , the Weddell s e a l brain can also consume i t . Routine l a c t a t e A-V measurements upon recovery 81 show a net cerebral uptake of t h i s anaerobic end product whenever the mean a r t e r i a l concentration reaches c r i t i c a l l e v e l s of about 7 umol/ml (Fig. VI, 1). This f i n d i n g was v e r i f i e d by means of a l a c t a t e i n f u s i o n experiment (Fig. VI, 2). For experimental d e t a i l s r e f e r to Materials and Methods. In both cases, once t h i s l a c t a t e concentration i s surpassed, the brain vigorously consumes the substrate generating an A-V gradient of up to 1.25 ymol/ml (Figs. VI, 1 and VI, 2). The capacity for either l a c t a t e production or l a c t a t e uptake i s consistent with the occurence i n seal b r a i n of high l e v e l s of l a c t a t e dehydrogenase, k i n e t i c a l l y w e l l suited for b i - d i r e c t i o n a l function (Fig. IV, 1, Table IV, 1). If a l l the l a c t a t e consumed were f u l l y oxidized, i t could support a metabolic rate of 9 ymol/ml ATP/gm/min, assuming that brain blood flow were normal. This value i s equal to, or greater than, that sustainable by glucose metabolism and indicates that l a c t a t e metabolism under these conditions can r e a d i l y supply a l l of the energy demands of the brain. However, at present, there are no experimental estimates on the ultimate fate(s) of the absorbed l a c t a t e . The experiments described i n t h i s chapter pointedly demonstrate that, during simulated diving of 30 minutes or l e s s , the Weddell seal brain derives ample supplies of glucose and oxygen from a r t e r i a l blood to meet i t s demanding energy requirements. Whether adjustments i n the CBF could p e r f e c t l y compensate for the possible decreasing l e v e l s of both oxygen and glucose during longer duration diving s t i l l remains unclear and, unfortunately, beyond the scope of the t h e s i s . However, i t has been shown that t h i s b rain w i l l absorb and probably oxidize l a c t a t e when a r t e r i a l concentrations reach a c r i t i c a l l e v e l of approximately 7 ymol/ml. Such an a b i l i t y w i l l l i k e l y a id i n the speedy removal of the acidic,anaerobic 82 F i g . VI, 1. Change i n l a c t a t e concentration i n a r t e r i a l and epidural venous blood samples during d i v i n g and recovery i n seal showing an unusually large l a c t a t e washout. Dive time: 20 min. 82a F i g . VI, 1 83 Fi g . VI, 2. Lactate concentration changes i n a r t e r i a l and epidural venous blood following i n f u s i o n of 176g of l a c t a t e . Lactate was infused i n normal s a l i n e at pH 7.3 at a rate of about 100 ml/min. 83a Lac ta te Infusion A r t e r i a l 0 5 10 15 Time (min) F i g . VI,2 84 end-product during the recovery period. I t i s also expected that, i f for any reason (e.g. peripheral leakage) a r t e r i a l l a c t a t e concentrations reach i n t o l e r a b l e l e v e l s during d i v i n g , cerebral metabolism may switch from a glucose to a l a c t a t e based oxidative metabolism (assuming i s a v a i l a b l e ) . 85 CHAPTER VII F e t a l Responses to Maternal Diving 85 a "I sometimes wonder why i t i s that, when a pregnant whale dives, and the fetus i n her womb begins to f e e l that awful weight and the slowing pulse, the l i t t l e thing i s not expelled l i k e a popping cork. Perhaps, when the oxygen l e v e l drops, i t too begins to "dive" i n i t s own small way, to obey the automatic signals i n her blood." - From the Year of the Whale, V i c t o r B. Scheffer, Charles Scribner's Sons, New York, 1979 -(i) Blood Metabolite P r o f i l e s as a Consequence of Maternal Diving INTRODUCTION In o v e r a l l o u t l i n e the general metabolic consequences of diving (e.g. peripheral organs sustained by anaerobic metabolism, .leading to lac t a t e accumulation and i t s eventual release into the c i r c u l a t i o n ) have been appreciated, at l e a s t , since the c l a s s i c studies of Scholander (1940) four decades ago. What has not been c l a r i f i e d at a l l , however, i s the metabolic status of the seal fetus during maternal diving. There are two p o s s i b i l i t i e s : e i t h e r the fetus simply tolerates the consequences of the maternal dive or i t too evokes the above diving responses. With respect to bradycardia, d i r e c t measurements implicate the l a t t e r strategy. Thus, soon a f t e r the maternal bradycardia i s e l i c i t e d , a f e t a l bradycardia develops, heart rates t y p i c a l l y f a l l i n g from about 90 to 30 beats/min by the end of a maternal dive (Liggins et a l . , 1980). Although absolute flow rates are not known, Liggins et al. (1980) demonstrated that the placenta probably receives an increased f r a c t i o n of the maternal cardiac output during diving and that the mean a r t e r i a l pressure of the fetus remains r e l a t i v e l y constant (91 to 96 mm Hg) throughout t h i s s t r e s s f u l episode 86 (Liggins et a l . , 1980). The implications are, therefore, that the diving response i s already developed i n the l a t e term fetus. In that event, s i m i l a r metabolic p r o f i l e s should be obtained i n the f e t a l c i r c u l a t i o n as i n the maternal system. With t h i s i n mind a r t e r i a l blood p r o f i l e s of l a c t a t e , pyruvate and glucose, a wide spectrum of amino acid and glutathione i n feto - maternal pa i r s of the Weddell seal p r i o r to, during and following normobaric diving were monitored. 87 RESULTS AND DISCUSSION Blood Glucose, Pyruvate and Lactate P r o f i l e s In only 3 of 6 fetal-maternal pa i r s u t i l i z e d did the fetus show good recovery, with heart rate, PO^, PCC^, and blood pH quickly returning to normal l e v e l s following diving (Liggins e£ a l . , 1980). These 3 pa i r s were used i n 4 d i f f e r e n t diving experiments summarized i n Figs. VII, 1 and VII, 2. The o v e r a l l pattern i s s i m i l a r i n both. Except i n one short dive (Fig. VII, 1), maternal blood glucose l e v e l s t y p i c a l l y decrease s l i g h t l y during simulated diving. During recovery, glucose l e v e l s are gradually restored presumably at the expense of body stores of glycogen ( p a r t i c u l a r l y the l i v e r ) , and because of activated gluconeogenic processes. Concomitant with the f a l l i n maternal blood glucose, there i s seen a consistent r i s e i n blood l a c t a t e (Figs. VII, 1 and VII, 2). However, t h i s r i s e i s modest compared with the large increase that i s observed i n recovery due to l a c t a t e wash-out from peripheral tissues (Figs. VII, 1 and VII, 2). These patterns for the pregnant s e a l are s i m i l a r to those noted for adult males (See Chapter I I I ) . F e t a l blood glucose l e v e l s d i f f e r from maternal ones i n two important regards. F i r s t l y , f e t a l blood glucose concentrations are always s u b s t a n t i a l l y higher than i n maternal blood; t h i s rather unusual glucose gradient w i l l be throughly discussed l a t e r i n t h i s chapter. A second important difference between f e t a l and maternal glucose metabolism i s evident i n the response to diving. In the fetus, unlike the mother, blood glucose l e v e l s t y p i c a l l y r i s e somewhat as a consequence of d i v i n g . In one 20-minute dive (Fig. VII, 1, dive I I ) , the blood glucose 88 Fig. VII, 1. Glucose, lactate and pyruvate concentration changes in maternal and fetal a r t e r i a l blood during two experimental dives. This maternal-fetal pair was used 3 hours earlier in a glucose tolerance test, and glucose levels prior to dive I were s t i l l f a l l i n g . Dive I was 10 minutes; dive II was 19 minutes long. F = fetus; M = mother. 88a Dive I D i v e II Minutes F i g . VII,1 89 Fig. VII, 2. Glucose, lacatate and pyruvate concentration curves in a 20 min dive of another maternal-fetal pair, indicating an essentially identical pattern to the 19 minute dive in Fig. VII, 1. In a l l three dives in Fig. VII, 1 and 2, fetal and maternal P02, PC02 and blood pH values quickly returned to normal after diving. 89a Minutes F i g . VII, 2 90 curve drops i n i t i a l l y but then swings upwards again well before the end of the diving period. In other mammals, the fetus i s known to mobilize l i v e r glycogen under s t r e s s f u l conditions, a process leading to increased blood glucose l e v e l s (Shelly, 1973). However, i t i s not known i f a s i m i l a r process i s activated i n the s e a l fetus during maternal d i v i n g . There i s no doubt however, that blood glucose regulation of the fetus during diving i s regulated independently of that i n the mother; otherwise, i t would be d i f f i c u l t to understand how f e t a l l e v e l s could be r i s i n g at the same time as maternal concentrations are f a l l i n g ( F ig. VII, 1, dive I I , for example). Ei t h e r metabolic factors or perfusion changes i n the placenta could lead to the observed glucose p r o f i l e s , but l i t t l e information i s av a i l a b l e on t h e i r r e l a t i v e contributions. Despite evident differences i n glucose handling, there i s a s t r i k i n g s i m i l a r i t y betwen f e t a l and maternal p r o f i l e s of blood l a c t a t e and pyruvate (Figs. VII, 1 and 2). In the fetus, as i n the mother, blood l a c t a t e tends to increase during the dive, although the f e t a l response lags somewhat behind the maternal one. Then, following the dive, there occurs the usual wash-out of l a c t a t e and pyruvate from peripheral tissues and hence the recovery "spikes" i n l a c t a t e and pyruvate concentrations i n the blood. Again, the f e t a l response lags behind the maternal one. Peak concentrations are usually, but not always, lower i n the fetus; moreover, i n the recovery process, the p l a c e n t a l gradients for l a c t a t e and pyruvate can be reversed. Of course, a r i s e i n l a c t a t e l e v e l s during recovery i s not conclusive evidence of peripheral v a s o c o n s t r i c t i o n i n the fetus for t h i s may simply r e f l e c t transplacental e q u i l i b r a t i o n . However, when these metabolic data' are coupled to the p h y s i o l o g i c a l p r o f i l e s (decreased cardiac output, 91 unaltered mean a r t e r i a l blood pressure plus the well-sustained d i a s t o l i c pressure during the long i n t e r v a l s between f e t a l heart beats) they appear to be i n t e r n a l l y consistent with peripheral v a s o c o n s t r i c t i o n i n the fetus during maternal diving. Another important outcome of t h i s study concerns the l a c t a t e and pyruvate p r o f i l e s throughout diving and recovery. In metabolic terms the importance stems from d i r e c t i o n s rather than magnitudes of change, for while l a c t a t e l e v e l s r i s e continuously during diving, pyruvate l e v e l s i n f a c t can f a l l . That i s , l a c t a t e production may occur simultaneously with pyruvate u t i l i z a t i o n , a r e s u l t consistent with a pyruvate-lactate based hydrogen s h u t t l i n g system between organs that vary i n anoxia tolerance. Such a process has been reported i n extremely hypoxic perfused heart preparations (Lee et a l . , 1973) and on t h e o r e t i c a l grounds, was predicted to be involved i n the extended hypoxia tolerance of diving animals (Hochachka and Storey, 1975). The process can continue into post-diving recovery as w e l l , which may be why the pyruvate wash-out peaks sometimes occur a f t e r the l a c t a t e wash-out peaks (Fig. VII, 1 for example). I t i s noted that t h i s pattern has also been observed i n the adult males of the species (see Chapter I I I ) . The p h y s i o l o g i c a l implications of the data stem mainly from the recovery patterns, for the easiest way to explain the l a c t a t e and pyruvate wash-out curves i s to assume f e t a l p eripheral v a s o c o n s t r i c t i o n during the dive. P h y s i o l o g i c a l p r o f i l e s (Liggins e_t a l . , 1980) are, indeed, consistent with t h i s i n t e r p r e t a t i o n . Thus, the metabolic and p h y s i o l o g i c a l data taken together, ind i c a t e that the two key components of the diving response (bradycardia and peripheral vasoconstriction) are both developed 92 i n the near-term s e a l fetus, and appear to be an i n t e g r a l part of the f e t a l adaptational responses to maternal diving. Blood Amino Acid P r o f i l e s i n Resting Animals Whole blood concentrations (ymoles/ml) of amino acids i n r e s t i n g Weddell seal and human feto-maternal pairs are presented i n Table VII, 1, with a s t a t i s t i c a l evaluation of the more obvious mean differences between the two groups given i n Table VII, 2. A comparison of the amino acid p r o f i l e s indicates outstanding differences between the two groups of mammals f o r , at l e a s t , alanine. The human mother and fetus display 5 and 2 f o l d higher concentrations, r e s p e c t i v e l y , than t h e i r s e a l counterparts. R e c a l l a s i m i l a r q u a l i t a t i v e pattern associated with the adult males of the two species. (The possible s i g n i f i c a n c e of t h i s trend has been discussed i n Chapter III.) Table VII, 1 also suggests sub s t a n t i a l taurine differences (p. <.001) between the two groups. These differences stem from the feto-maternal (human) l e v e l s of taurine as recorded by Velazguez et a l . (1976) since t h e i r data i n d i c a t e concentrations of the amino acid are an order of magnitude lower than those presented by F e l i g et_ a l . (1973) and i n Table VII, 1. Although both F e l i g and I assayed male volunteers, i t i s d i f f i c u l t to believe that sex or pregnancy differences could cause such huge v a r i a t i o n s . Furthermore, a l l three groups employed the s u l f o s a l i c y c l i c acid method of deproteinization, i n which cysteine was not protected against oxidation. Cysteine can be oxidized to c y s t e i c acid, cysteine s u l f i n i c acid and perhaps hypotaurine (Awapara, 1976); none appear to co-elute with taurine standards. Taurine can a r i s e by the decarboxylation of c y s t e i c acid and/or the oxidation of 93 Table VII, 1. Mean a r t e r i a l concentrations (+ standard deviations) of amino acid . • ... i n whole blood of the Weddell seal and human, feto-maternal p a i r s . The human sample s i z e (as assayed by Velazguez et a l . , 1976) was 8 while that of the seal varied between 4 to 7. Human f e t a l pool p r o f i l e s i n the venous c i r c u l a t i o n are also presented. This author's c a l c u l a t i o n s of t o t a l pool s i z e s from Velazguez's data are marked by an (*). Whole Blood Concentrat Weddell Seal ernal F e t a l Amino Acid Taurine Aspartate Threonine Serine Glutamate Glutamine Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine Lysine Ornithine H i s t i d i n e Arginine Total Branched Chained Pool Total Pool Glutathione Mat 176.0 + 37.6 30.8 + 9.9 122.4 + 22.1 101.3 + 38.3 128.0 + 28.8 359.1 + 147.0 124.3 + 18.3 87.2 + 29.2 130.9 + 34.3 27.6 + 8.9 94.4 + 16.6 24.6 ± 5.7 48.9 + 7.6 110.0 + 32.2 46.1 + 7.8 59.4 + 14.9 45.0 + 9.5 222.7 + 52.6 1807.0 + 172.0 1341.4 + 539.6 133 .6 + 37. 4 63 .0 + 10. 3 152 .2 + 24. 3 141 .3 + 37. 0 195 .0 + 63. 0 363 .0 + 85. 0 252 .3 + 89. 5 214 .6 + 77. 6 217 .2 + 45. 3 35 .0 + 7.0 147 .3 + 31. 2 51 .5 + 4.8 71 .4 + 16. 5 126 .1 + 33. 6 63 .3 + 10. 5 94 .4 + 14. 5 69 .9 + 10. 2 407 .2 + 46. 6 \529 .0 + 195 J 605 .0 + 334 J (nmol/ml) Human Maternal F e t a l F e t a l (venous) 18 .0 + 4.0 61 .0 + 9.0 53 .0 + 11 .0 32 .0 + 7.0 31 .0 + 5.0 50 .0 + 8. 0 175 .0 + 31.0 205 .0 + 28.0 266 .0 + 39 .0 91 .0 + 18.0 142 .0 + 21.0 177 .0 + 24 .0 116 .0 + 15.0 184 .0 + 28.0 123 .0 + 16 .0 380 .0 + 61.0 428 .0 + 65.0 340 .0 + 61 .0 148 .0 + 28.0 101 .0 + 15.0 145 .0 + 18 .0 319 .0 + 68.0 455 .0 + 73.0 428 .0 + 75 .0 107 .0 + 18.0 131 .0 + 18.0 140 .0 + 22 .0 56 .0 + 11.0 65 .0 + 9.0 80 .0 + 13 .0 71 .0 + 14.0 83 .0 + 14.0 96 .0 + 15 .0 50 .0 + 10.0 61 .0 + 10.0 59 .0 + 11 .0 63 .0 + 9.0 48 .0 + 8.0 68 .0 + 10 .0 139 .0 + 18.0 147 .0 + 21.0 174 .0 + 17 .0 35 .0 + 6.0 89 .0 + 17.0 83 .0 + 14 .0 83 .0 + 12.0 93 .0 + 9.0 150 .0 + 12 .0 37 .0 + 9.0 107 .0 + 12.0 98 .0 + 11 .0 234* 1920* 279* 2431* 316* 2530* 94 Table VII, 2. S t a t i s t i c a l evaluation of mean differences of amino acid: concentrations ( a r t e r i a l pools) presented i n Table VII, 1. Subscripts in d i c a t e the animal with the higher concentration. W = Weddell seal; hu = human; N.S. = not s i g n i f i c a n t . 94a Amino Acid Taurine Aspartate Threonine Serine Glutamate Glutamine Glycine Alanine Valine Isoleucine Leucine Tyrosine P heny 1 a 1 an ine Lysine Ornithine H i s t i d i n e Arginine Total Branched pool Total pool Glutathione Gravid Weddell seal vs Gravid Human Fet a l Weddell seal vs Fet a l Human p <.001(W) N.S. N.S. N.S. N.S. N.S. N.S. p <.OOl(hu) N.S. p < .OOl(hu) p < .05 (W) p < .001(hu) N.S. N.S. N.S. p < .01 (hu) N.S. p < .001(W) p < .ooi(w) p < .01 (hu) N.S. N.S. N.S. P < .ooi(w) p < .001(hu) p < .001(W) p < .001(hu) p < .001(W) N.S. p < .01 (W) N.S. p < .02 (hu) N.S. p < .OOl(hu) Table VII, 2 95 hypotaurine. However, cysteine samples treated i n exactly the same manner as described i n Materials and Methods showed formation of only c y s t e i c acid, which i s eluted from the column w e l l before taurine. The above differences w i l l probably be traced to the presence of te c h n i c a l a r t e f a c t s i n one or more groups' procedures. There are other, not so dramatic, v a r i a t i o n s of whole blood amino acids between the two d i f f e r e n t groups (Tables VII, 1 and 2). On the maternal side, the human displays higher l e v e l s of i s o l e u c i n e , tyrosine, and h i s t i d i n e (p <.001, .001 and .01 respectively) while the Weddell s e a l contains s l i g h t l y more leucine (p <.05). On the other hand, the amino acid blood pools of the fetuses appear to be more v a r i a b l e . Beside alanine, the human pool i s more concentrated with respect to threonine (p <.01), is o l e u c i n e (p <.001), ornithine (p <.02) and arginine (p <.001), while the f e t a l s e a l exhibits s i g n i f i c a n t l y higher concentrations f o r aspartate (p <.001), glycine (p <.001), v a l i n e (p <.001), leucine (p <.001) and phenylalanine (p <.01). Measurements of transplacental gradients i n d i c a t e that the majority of amino acids are more concentrated on the f e t a l side of the placenta i n both groups of animals (see Table VII, 3). Only taurine l e v e l s are lower i n the f e t a l seal while glycine and phenlyalanine appear to be reduced i n the a r t e r i a l c i r c u l a t i o n of the f e t a l human. (The t r i p e p t i d e , glutathione, also demonstrates a reverse gradient i n the s e a l ) . The Weddell seal displays higher r a t i o s ( f e t a l a r t e r i a l pool/maternal a r t e r i a l pool) f o r aspartate, glycine, alanine, v a l i n e , tyrosine, phenylalanine and the t o t a l branched chained amino acids; human r a t i o s are elevated for taurine, ornithine and arginine. The s i g n i f i c a n c e s of these r e s u l t s , 96 Table VII, 3. Fetal/maternal ratios for whole blood amino acid concentrations in the Weddell seal and the human. Values are calculated from the data in Table VII, 1. The seal ratios are derived from the sampling of art e r i a l blood on both sides of the placenta whereas the human ratios are expressed as fetal a r t e r i a l or venous versus maternal ar t e r i a l levels. Human ratios are computed from values taken from Velazguez et^ al.. (1976) . Subscripts used: a = arte r i a l sample v = venous sample Amino Acid WEDDELL SEAL Fetal(a):Maternal(a) HUMAN Fetal(a):Maternal(a) Fetal(v):Maternal(a) Taurine 0.76 3.38 2.94 Aspartate 2.03 0.97 1.56 Threonine 1.25 1.17 1.52 Serine 1.40 1.56 1.95 Glutamate 1.52 1.59 1.06 Glutamine 1.01 1.13 0.90 Glycine 2.03 0.71 0.98 Alanine 2.47 1.43 1.34 Valine 1.66 1.22 1.31 Isoleucine 1.25 1.16 1.42 Leucine 1.56 . 1.17 1.35 Tyrosine 2.08 1.22 1.18 Phenylalanine 1.45 0.76 •1.08 Lysine 1.15 1.06 1.25 Ornithine 1.58 2.54 2.37 Arginine 1.56 2.89 2.65 H i s t i d i n e 1.59 1.12 1.81 Tot a l Branched Chained Amino Acids 1.83 1.19 1.35 Tot a l pool Glutathione 1.40 0.45 1.26 1.32 97 i f any, have yet to be ascertained, although they may be r e f l e c t i o n s of d i e t s , f a s t i n g times and d i f f e r e n t growth requirements of the two groups. The general gradient favoring the f e t a l c i r c u l a t i o n i s e a s i l y explained by the fac t that during gestation the f e t a l growth requirements increase exponentially, therefore the mother must respond by supplying a l l the precursors (e.g. e s s e n t i a l and some non-essential amino acids)needed for both anabolism and catabolism. I t i s now thought that t h i s gradient i s upheld by act i v e or f a c i l i t a t e d transport mechanism for most of the amino acids (Berry et aJ.. , 1975). At t h i s juncture, i t should be noted that a proper comparison between maternal and f e t a l blood pools would involve the sampling of maternal a r t e r i a l and f e t a l venous concentrations since nutrients are transported from the maternal uterine artery, v i a the placenta, into the venous c i r c u l a t i o n of the fetus. However, te c h n i c a l r e s t r i c t i o n s i n th i s study dictated catheter placement i n the f e t a l f l i p p e r artery (Liggins et al., 1980). Table VII, 3 demonstrates the differences between the two sampling procedures i n human p a i r s . Aspartate, threonine, serine, glutamate, glutamine, glycine and h i s t i d i n e pool sizes appear a l t e r e d to some extent between the f e t a l a r t e r i a l and venous blood. Of course, these probably vary with the p h y s i o l o g i c a l state of the mother and fetus. In summary, th i s study was performed to provide added metabolic descriptions i n f e t a l aquatic mammals. Some differences i n the amino acid pools of r e s t i n g , feto-maternal p a i r s of Weddell seals and humans were found; however, there i s an o v e r a l l , general s i m i l a r i t y between the two species. Discrepancies i n a r t e r i a l pools (e.g. alanine) and trans-98 placental gradients are probably due to d i f f e r e n t dietary regimes and f e t a l growth requirements. Diving P r o f i l e s of Blood Amino Acids Unfortunately, t h i s segment of the study i s much too sketchy to extract d e f i n i t i v e c h a r a c t e r i s t i c s of the f e t a l amino acid pools during simulated diving of the pregnant s e a l . I t i s noted that the majority of the free amino acids i n f e t a l whole blood remain r e l a t i v e l y constant throughout the dive phase, which i s not too s u r p r i s i n g i n view of the amino acid p r o f i l e s of the adult seals (Chapter I I I ) . Of the three d i f f e r e n t feto-maternal p a i r s sampled, there appears to be detectable elevations i n both alanine and glutamine l e v e l s during, at l e a s t , the recovery phase. Both amino acids displayed post-dive peak l e v e l s of about 1.4-1.6 f o l d higher than those of the predive. Again, t h i s appears to be consistent with the adult observations. Actual diving p r o f i l e s were too scattered to decipher r e a l trends. U n t i l more rigorous studies are performed, i t would be inappropriate to consider alanine and glutamine p r o f i l e s as further evidence of a f u l l y developed diving response i n the f e t a l s e a l . Curiously, not one fetus displayed the unique glutathione p r o f i l e s associated with some of the gravid and male adults. Furthermore, when the maternal glutathione was hydrolyzed, there were no observable increases i n the f e t a l blood pools of either glutamate nor glycine. Amongst other things, t h i s suggested 1) a very t i g h t and s e l e c t i v e c o n t r o l of amino acid flow across the seal placenta; 2) maternal (or adult) changes i n glutathione blood l e v e l s during diving-recovery cylces were not a r e s u l t of sampling a r t e f a c t s (see Chapter I I I ) . 99 ( i i ) Unusual Maternal-fetal Glucose Concentrations In Whole Blood of the  Weddell Seal INTRODUCTION It i s usual for f e t a l whole blood glucose l e v e l s to be lower than, and to f l u c t u a t e with, maternal l e v e l s . That i s , a favourable concentration gradient i s maintained to allow f o r the " f a c i l i t a t e d t r a n s f e r " of glucose across the placenta from mother to fetus (Shelley, 1973; Dawes and Shelley, 1968). This s i t u a t i o n i s apparently reversed i n the Weddell s e a l . The concentration of glucose i n maternal whole blood i s lower than that i n f e t a l blood during at l e a s t three metabolic states: i n the r e s t i n g , f a s t i n g state, i n simulated diving, and i n recovery from diving (see Chapter VII, i ) . The explanation for t h i s consistent r e v e r s a l of glucose concentration gradients between maternal and f e t a l blood was explored i n the present study. RESULTS AND DISCUSSION At the outset of these experiments, i t was considered important to test f or the presence of sugars other than glucose i n the f e t a l and maternal blood. For example, fructose i s not only present i n most cetaceans (Comline and S i l v e r , 1974) but also occurs i n the blood of most mammalian fetuses ( B a t t a g l i a and Meschia, 197 6). Glucose was normally determined by following the change i n o p t i c a l density at 340 nm due to NADP+ reduction by glucose-6-phosphate dehydrogenase (see Materials and Methods). However, i n several f e t a l and maternal extracts 100 glucose was determined also by the glucose oxidase method, the Benedict assay system and by an automated glucose analyzer with a l i n e a r response over the range of 5-50 ymol/ml. A l l three assay procedures yielded the same fetal-maternal differences and the same absolute glucose concentrations. To exclude the presence of an unusual sugar, whole blood extracts were analyzed by gas l i q u i d chromatography (Albersheim et a l . , 1967). These studies showed that glucose was the only major sugar present i n maternal and f e t a l blood; fructose, galactose, mannose, and ribose did not occur i n measurable concentrations. Under re s t i n g and fasted conditions ( i n c a p t i v i t y f o r at l e a s t 12 hours), maternal whole blood glucose concentrations ranged between 4-6 ymol/ml compared to 5-8 ymol/ml i n f e t a l whole blood, with an average gradient across the placenta of approximately 2 ymol/ml. (This i s a c t u a l l y an underestimation since a r t e r i a l samples were used.) Similar l e v e l s were found i n samples drawn from undisturbed seals asleep on i c e . These data may r e f l e c t e i t h e r an active gluconeogenic process or a low rate of glucose turnover. Although the f e t a l concentrations were consistently higher than maternal ones, the two values were not unrelated and tended to f l u c t u a t e i n unison (Fig. VII!'., 3). Glucose Tolerance Test To better understand how maternal and f e t a l blood glucose l e v e l s are regulated, a glucose load (1.5 gm/kg of estimated maternal body weight) was infused over a period of 20 min into the maternal c i r c u l a t i o n of 3 pregnant seals. In a l l 3 animals, the maternal concentration of glucose i n both whole blood and plasma rose to values that exceeded those i n the 101 Fi g . VII, 3. A plot of glucose concentrations i n whole blood of f i v e maternal-fetal p a i r s . The d i f f e r e n t symbols r e f e r to d i f f e r e n t p a i r s . Blood samples were taken before, during and a f t e r simulated diving. The highest glucose values were obtained a f t e r intravenous in f u s i o n of glucose. 101a 141 12 10 8 6 4 2! o n % yi e o ta 0 ° 0 6 8 10 12 14 F-glucose, >jmol/ml F i g . VII, 3 102 fetus throughout the i n f u s i o n and for a short period a f t e r i t was completed, but within 20-40 min f e t a l blood glucose l e v e l s were again higher than i n the mother (Figs. VII, 4A, 4B). The concentration of i n s u l i n increased sharply i n the plasma of both mother and fetus approximately 15 min a f t e r the r i s e i n glucose l e v e l s ( F ig. VII, 4B). In the fetus, the concentrations of plasma glucose and i n s u l i n followed s i m i l a r patterns but i n the mother, i n s u l i n l e v e l s remained elevated f o r at le a s t 5 hr a f t e r glucose i n f u s i o n although glucose concentrations were f a l l i n g . ( I n s u l i n and plasma glucose p r o f i l e s compiled by Dr. G. C. Liggins.) The reason f o r the unusual r e l a t i o n s h i p of f e t a l to maternal glucose content of whole blood becomes apparent when plasma glucose concentrations are compared (Fig. VII, 4B; Table VII, 4). In common with other mammals, glucose l e v e l s are higher i n maternal plasma than i n f e t a l plasma and glucose moves from maternal to f e t a l blood down a concentration gradient. The high glucose content of f e t a l whole blood r e l a t i v e to the mother arises from several f a c t o r s . F i r s t , the feto-maternal gradient of plasma glucose concentrations i s small (Table VII, 4). Secondly, the r a t i o of red cell:plasma concentration of glucose i n mother i s unusually low whereas that of fetus i s s i m i l a r to other species (Table VII, 4). F i n a l l y , the hematocrits of both mother and f e t u s • i s high (approximately 60% and 70% respectively) which has the e f f e c t of increasing the r a t i o of plasma:whole blood concentration of glucose, e s p e c i a l l y i n the mother. Whereas a high haematocrit increases the 0^ carrying capacity of the blood, i t c l e a r l y reduces the glucose carrying capacity although to a le s s e r degree i n the fetus because of the greater entry of glucose into f e t a l red c e l l s . The sustained hyperglycaemia a f t e r glucose loading 103 F i g . VII, 4a. Maternal (0) and f e t a l (. • ) concentrations of glucose i n whole blood following intravenous i n f u s i o n of glucose (1.5 g per kg). Time zero indicates the end of the 20 minute period of i n f u s i o n . F i g . VII, 4b. Maternal glucose (0) and i n s u l i n ( A ) and f e t a l glucose ( • ) and i n s u l i n ( A ) concentrations i n plasma from the same glucose tolerance test as shown i n 4a. 103a i i i i i i . i ^ • .-- • i -20 O 20 40 60 120 180 240 300 E c - 2 0 0 20 4 0 60 120 180 240 300 Minutes F i g . VII, 4a, 4b 104 Table VII, 4. F r a c t i o n a l d i s t r i b u t i o n of glucose between plasma and red blood c e l l s i n maternal and f e t a l Weddell seal compared with the sheep. 104a Glucose ( umo 1 m l _ 1) Ratio plasma: Whole Red red Species Condition blood Plasma c e l l s c e l l s Weddell seal (adult) Weddell seal (fetus) Sheep (adult) Sheep (fetus) Pre-infusion 3.1 60 min post-infusion 13.5 Pre-infusion 60 min post-infusion Post-prandial 3.5 15.8 7 30 0.5 2.5 6 2.4 22.5 12.9 2.5 0.3 0.7 0.2 14 12 2.5 1.7 8.3 3.5 Table VII, 4 105 (Figs. VII, 4A, 4B) suggests that the rate of glucose turnover i n the adult seal i s low. Likewise, the small gradient of plasma glucose concentrations across the placenta may suggest a low turnover rate i n the fetus. The data derived from these studies may r e f l e c t a very important adaptation of the f e t a l seal as a consequence of the maternal d i v i n g habit. The high glucose stores of the f e t a l blood are viewed as a buffering system protecting the fetus against periods of hypoglycemia and hypoxia, possibly associated with the well recorded, long duration diving of the gravid Weddell seal (Eisner e^ t a l . , 1968) . ( i i i ) Enzymes of Aerobic and Anaerobic Metabolism i n the Three Central  Organs of the F e t a l Weddell Seal INTRODUCTION It was reasoned that a determination of maximum pot e n t i a l s of some important enzymes associated with g l y c o l y t i c anaerobosis and the aerobic oxidation of carbohydrates, fats and ketone bodies would lay a foundation for future metabolic work on the heart, lung and brain of the f e t a l Weddell seal (see Chapter IV for the r a t i o n a l e behind t h i s reasoning). Before t h i s study there were no known reports on enzymatic p r o f i l e s of any f e t a l t i s s u e i n any of the marine mammals; however such a thorough p l o t t i n g was considered to be out of the scope of t h i s thesis and thus must await future analyses. Since no companion ' i n vivo' data were generated, r e s u l t s were compiled with some i n t u i t i v e speculation as to t h e i r metabolic 106 s i g n i f i c a n c e . The focus of t h i s study centered upon two main questions: 1) What are the metabolic preferences of the heart, lung and brain? 2) Is the f e t a l enzymatic machinery primed for both high aerobic and anaerobic fluxes? It i s now w e l l established that the t e r r e s t r i a l mammalian fetus not only exhibits r e l a t i v e l y high oxygen consumption rates ( B a t t a g l i a and Meschia, 1976) but also possesses a marked tolerance to hypoxemia (Dawes, 1968). I t was reckoned that the hypoxia tolerance may be more emphasized and thus observable on the enzyme a c t i v i t y l e v e l i n the fetus of aquatic mammals. RESULTS AND DISCUSSION The data i n t h i s section were derived from 2 to 5 near-term Weddell seals, ages unknown. Presumably, these fetuses were s t i l l developing at the time of experimentation and, therefore, i t i s possible that there may be some resultant scatter i n the a c t i v i t y p r o f i l e s . Enzymes of Oxidative Metabolism Four mitochondrial marker enzymes associated with oxidative pathways of the f e t a l heart, lung and b r a i n were used: g-hydroxybutrylCoA dehydro-genase and g-hydroxybutryrate dehydrogenase, functioning i n g-oxidation of f a t t y acids and ketone body metabolism respectively; c i t r a t e synthase, catalyzing the entry of acetylCoA carbon into the Krebs cycle and thought to represent an important c o n t r o l s i t e ( T i s c h l e r et a l . , 1977) and glutamate dehydrogenase, a key regulatory enzyme (Srere, 1969), catalyzing the entry of glutamate carbon into the Krebs cycle. As the heart, lung 107 and brain d i f f e r greatly i n metabolic organization (Siesjo and Nordstrom, 1977; Neely and Morgan, 1974; Tierney, 1974a), t h e i r enzyme p r o f i l e s also show s i g n i f i c a n t differences (Table VII, 5). Organ by organ comparisons indic a t e that the a c t i v i t i e s of oxidative enzymes i n the f e t a l heart, lung and brain are very s i m i l a r to those i n homologous tissues of the adult s e a l (Refer to Tables VII,' 5 and IV, 1). 3-hydroxybutyrylCoA dehydrogenase i n the f e t a l heart appears to be the only exception, displaying about a two f o l d increase over the adult s e a l (32.5 vs. 16.0 pmole/min/g). Since t h i s enzyme was assayed i n only two animals, I f i n d i t d i f f i c u l t to attach much s i g n i f i c a n c e to the data. Otherwise, i t i s concluded that the near-term fetus of the Weddell s e a l has developed i t s aerobic c a p a b i l i t i e s to the same extent as the mature adult. However, without supporting ' i n vivo' data i t i s impossible to assume s i m i l a r metabolic preferences and regulation. These enzymatic p o t e n t i a l s may be merely developed i n preparation f o r extrauterine existence. Furthermore, i t i s i n t e r e s t i n g to note that the f e t a l heart appears to exhibit highly developed f a t catabolic enzymes when i t i s thought that mammalian, f e t a l myocardium consumes predominantly glucose under aerobic conditions ( B a t t a g l i a and Meschia, 197 6). Such a s i t u a t i o n may r e f l e c t t h i s preparation f o r post-f e t a l l i f e or could a c t u a l l y be a v a l i d index of the aerobic metabolism of these beasts. Enzymes of Anaerobic G l y c o l y s i s Hexokinase, phosphofructokinase and pyruvate kinase, which are a l l p o t e n t i a l regulatory s i t e s i n g l y c o l y s i s (Scrutton and Utter, 1968), and l a c t a t e dehydrogenase, catalyzing the terminal step i n g l y c o l y s i s were 108 Table VII, 5. Enzyme a c t i v i t i e s i n brain, heart and lung of the Weddell seal fetus expressed i n terms of pmoles substrate converted/ min/gm wet t i s s u e weight at 37°C, pH 7.4, and saturating l e v e l s of substrates, cofactors and coenzymes. See Methods and Materials (Chapter II) for det a i l e d assay conditions. Three to f i v e f e t a l seals were sampled and the values given are averages + S.D., with the range of values i n brackets below, ^indicates values derived from 2 animals. 108a C i t r a t e Synthase Glutamate dehydrogenase (3-hydroxybutyrate dehydrogenase g-hydroxybutyrl CoA dehydrogenase Hexokinase Phosphofructokinase Pyruvate kinase Lactate dehydrogenase Brain 17.40+ 0.70 (16.90 - 17.80) 5.10 + 2.90 (2.40 - 8.30) 0.28 + 0.08 (0.20 - 0.36) 3.60 + 1.20 (1.90 - 4.40) 3.50+ 1.90 (2.10 - 5.70) 6.90* (4.80 - 8.90) 115.10 + 11.10 (102.40 - 127.70) 167.00*-(150.00 - 187.00) Enzyme A c t i v i t y  Heart 25.00 + 11.90 (16.50 - 42.00) 3.46 + 1.70 (1.65 - 5.38) 2.20 + 1.00 (1.20 - 3.20) 32.50* (26.50 - 37.20) 2.50 + 0.80 (1.40 - 3.00) 10.50 * (8.90 - 12.00) 228.00 + 58.00 (145.00 - 257.00) 957.00 + 192.30 (747.00 1125.40) L u n s 1.10 + 0.20 (0.92 - 1.27) 0.50 + 0.21 (0.36 - 0.80) 1.50 + 0.90 (0.84 - 2.10) 2.30* (3.40 - 1.20) 0.97 + 0.38 (0.28 - 1.40) 3.70* (2.70 - 4.60) 40.60 + 5.50 (35.40 - 46.30) 41.00 + 19.30 (21.70 603.00) Table VII, 5 109 measured to q u a l i t a t i v e l y assess the p o t e n t i a l f or anaerobic and aerobic g l y c o l y s i s i n these organs. Here again, enzymatic p r o f i l e s approaching those of the adult (Tables VII, 5 and IV, 1) were found thus i n d i c a t i n g the possible operational existence of a s i m i l a r metabolic organization to that of the adult organs. Electrophoretic evidence ( F i g . VII, 5) points to modest elevations i n muscle-type subunits of LDH i n both the heart and brain , i n d i c a t i n g a possible adaptation to anaerobosis i n these f e t a l t i s s u e s . Nevertheless both organs c l e a r l y have the p o t e n t i a l e i t h e r f or l a c t a t e production, catalyzed most e f f e c t i v e l y by muscle-type l a c t a t e dehydrogenase, or for l a c t a t e u t i l i z a t i o n , catalyzed most e f f e c t i v e l y by heart-type l a c t a t e dehydrogenase (Holbrook et a l . , 1975). To r e c a p i t u l a t e , these enzyme p r o f i l e s i n d i c a t e that, at l e a s t , the brain and heart may have some improved anaerobic p o t e n t i a l as indexed by LDH subunit d i s t r i b u t i o n ; however, such differences are subtle and probably do not r e f l e c t any improved anaerobic capacity of the c e n t r a l organs i n the aquatic mammal's fetus as compared to the fetuses of other t e r r e s t r i a l species. At t h i s point, i t should be re-emphasized that d e f i n i t i v e explanations regarding the metabolic preferences and regulation of the three c e n t r a l organs of the fetus must await more thorough ' i n vivo' studies. 110 Fi g . VII, 5. Starch gel electrophoretic separation of heart (H) and brain (B) of the f e t a l and adult Weddell s e a l . Electrophoresis conditions: 25 mA; 200 V; 12 h at 4°C; anode at top; o r i g i n marked with an arrow. Subunit composition of each isozyme i s shown on r i g h t . Subscripts f_ and a_ r e f e r to f e t a l and adult r e s p e c t i v e l y . 110a I l l CHAPTER VIII Summating Remarks 111a Central Organ Metabolism as a Consequence of Diving Previous to these studies no concerted e f f o r t had been applied to u nravelling the metabolic p r o f i l e s of the three c e n t r a l organs (brain, lung and heart) during diving and recovery i n marine mammals. O r i g i n a l l y , the diving response was thought to conserve 0^ for a l l three organs (Scholander, 1940). Since then most of the relevant reports found i n the l i t e r a t u r e (see Introduction) have strongly emphasized 1 suspected' anaerobic potentials i n both the b r a i n and heart of aquatic mammals. However, none of these unequivocally demonstrated the existence of such a metabolic p o t e n t i a l nor conditions under which i t may be activated. This thes i s , i n concert with other current ' i n vivo' studies attempts to c l a r i f y the s i t u a t i o n . A revised integrated modus operandi during diving and recovery cycles can be pieced together, with s p e c i a l emphasis on metabolic functions i n the heart, lung and brain. The data presented i n t h i s thesis implicate free blood glucose as a c r i t i c a l carbon and energy source f o r the Weddell s e a l during simulated diving, when p h y s i o l o g i c a l responses are maximally evoked. Although glycogen may be mobilized, a precise contribution of endogenous substrates to metabolism has not yet been quantified f o r any tiss u e i n any diving mammal. Of the three c e n t r a l organs, the br a i n i s considered to be the major glucose absorber, u t i l i z i n g approximately 3 mmoles of the carbohydrate over a 20 min dive period (assuming a 500 g brain with a CBF of 700 ml/kg/ min). Predictably, the peripheral tissues (e.g. s k e l e t a l muscles) appear responsible for quite a large f r a c t i o n of the t o t a l depletion, observed during the dive and into the f i r s t 10-15 min of recovery when the en t i r e 112 blood volume i s remixed (see F i g ; I I I , 1) • The a v a i l a b l e data also demonstrate steady increases of l a c t a t e l e v e l s , concomitant with the glucose decreases, i n the c e n t r a l bloood volume during diving periods of up to 50 min. Although most of t h i s l a c t a t e probably originates from anaerobic metabolism of the peripheral ti s s u e s , some (1500 umoles/20 min dive) i s d i r e c t l y derived from cerebral metabolism. At f i r s t glance t h i s rather high output of l a c t a t e , which accounts for 25% of absorbed glucose by the brain would seem to intimate cerebral aerobic metabolism during di v i n g , as predicted by other groups (see Chapter I ) . Although i t i s s t i l l p ossible that cerebral metabolism may, by necessity, increase i t s dependency on anaerobic-derived energy, most of the present metabolic and physiologic measurements do not substantiate t h i s postulate. F i r s t l y , cerebral enzyme p r o f i l e s i n d i c a t e very modest po t e n t i a l s for increased fluxes through the g l y c o l y t i c pathway to l a c t a t e as compared to other mammals. Secondly, the cerebral A-V differences f or both glucose and l a c t a t e , associated with forced diving of 10-30 minutes i n the Weddell s e a l , i n d i c a t e very l i t t l e a c t i v a t i o n of cerebral anaerobic g l y c o l y s i s despite Pa02 l e v e l s below 30 mm Hg, a condition considered hypoxic to non-diving animals (Siesjo and Nordstrom, 1977). Furthermore, there was no observed compensatory increase i n CBF (Zapol et a l . , 1979), as would be expected i f 0^ supply were severly l i m i t i n g . This phenomenon has also been observed i n harbor seals and northern elephant seals (Bron et a l . , 1966; Kerem et; ad., 1971; Van C i t t e r s et a l . , 1965). Nevertheless, i t i s stressed that the data of t h i s thesis were mainly derived from simulated dives l a s t i n g no longer than 30 min, and 113 thus, i t remains impossible to predict whether the b r a i n would remain i n a metabolic state akin to that of the pre-dive throughout longer and strenuous diving episodes. As for the lung and heart, a l l a v a i l a b l e evidence suggests that diving metabolism remains l a r g e l y oxidative, u t i l i z i n g exogenous l a c t a t e as a primary carbon and energy source. The evidence, i n support of the t e r r e s t r i a l , mammalian lung functioning as a l a c t a t e absorber and burner, i s undeniably strong (see Introduction). Recent work by Hochachka et a l . (1977a) has demonstrated the a b i l i t y of the Weddell seal lung to generate a l a c t a t e arteo-venous concentration gradient of 0.1 to0.25 ymol/ml during a forced dive. This same study also pointed to the a b i l i t y of the lung to release 0^ into the c e n t r a l c i r c u l a t i o n for up to 50 minutes of simulated diving; thus, i t i s probable that the organ receives enough 0^ to f i r e i t s own lactate-based oxidative metabolism for extended periods of apnea. The radiotracer work, reported i n t h i s t h e s i s , on both the i n t a c t Weddell seal preparation and on the animal's lung s l i c e s unequivocally i l l u s t r a t e pulmonary capacity can oxidize l a c t a t e i n preference to glucose. Similar trends have also been detected i n harbor seal lung s l i c e s (B. Murphy and B. Emmett, unpublished data). I t i s , therefore, t e n t a t i v e l y concluded that although t h i s metabolic property of the lung i s ubiquitously observed throughout the class Mammalia, i t probably holds more fun c t i o n a l s i g n i f i c a n c e f or the aquatic mammals who routinely face extended periods of hypoxia (with the concomitant increases of l a c t a t e and decreases of glucose i n t h e i r c i r c u l a t o r y systems and t i s s u e s ) . Unfortunately, d e f i n i t i v e ' i n vivo' experimentation has ' yet to be 114 attempted on the s e a l heart. However, a l l a v a i l a b l e data imply myocardial uptake of l a c t a t e during diving and recovery episodes. This conclusion i s based on several observations: 1) heart work during the diving phase i n , at l e a s t , the Weddell s e a l probably remains supported by oxidative metabolism. This stems from measurements of blood flow, cardiac output and a r t e r i a l pressure (Zapol et a l . , 1979), a l l of which indi c a t e a c l o s e l y matched work load and coronary blood supply (R. B r i l l , pers. commun.). It can be further argued, even i f the heart does r e l y somewhat on anaerobic g l y c o l y s i s , i t s l a c t a t e output would be minimal due to i t s d r a s t i c a l l y reduced work rate. 2) Whereas oxidative metabolism i n the mammalian heart may be f i r e d by a v a r i e t y of substances (glucose, f a t t y acids, l a c t a t e ) , l a c t a t e i s known to be p r e f e r e n t i a l l y u t i l i z e d whenever concentrations r i s e above normal (Mochizuki et. a l . , 1978, L i u and Spitzer, 1978). 3) The Weddell s e a l heart LDH a c t i v i t y i s the highest amongst a l l comparatively sized mammals thus far studied and i s k i n e t i c a l l y b i f u n c t i o n a l to act as a c a t a l y s t f or l a c t a t e oxidation. The isozyme d i s t r i b u t i o n pattern of the heart LDH i s quite s i m i l a r to that occuring i n the b r a i n and lung and since t h i s study has demonstrated the c a p a b i l i t y of both to take up l a c t a t e , there i s no reason why the heart cannot also metabolize i t . To r e c a p i t u l a t e , the present data imply that a l l three c e n t r a l organs remain i n an oxidative metabolic state throughout diving episodes associated with mean PaC^ l e v e l s near 25 mm Hg. Blood glucose supplies appear to be p r e f e r e n t i a l l y u t i l i z e d by the b r a i n and non-specified peripheral tissues with the lung and probably the heart absorbing and -1 115 o x i d i z i n g l a c t a t e produced by cerebral and peripheral ti s s u e metabolism. This metabolic cooperation between the three c e n t r a l organs thus aids i n maintaining reasonably low l e v e l s of the a c i d i c endproduct (of anaerobosis)in the ce n t r a l c i r c u l a t i o n while sparing l i m i t e d blood glucose supplies for cerebral u t i l i z a t i o n during subsequent di v i n g . In comparing the data of t h i s thesis with that of the c l o s e l y r e l a t e d investigations of Hochachka et a l . (1977a) i t was noticed that the blood glucose p r o f i l e s of the dive phases d i f f e r e d rather dramatically. Whereas Hochachka's group (1977a) rou t i n e l y observed glucose asymptotes midway into 40-50 min dives, when l e v e l s f e l l to 3.8-4.5 ymol/ml, the present data (see F i g . I l l , 1) do not c l e a r l y i n d i c a t e such trends; however, the blood concentrations of glucose never did drop below 3 ymol/ml (Fig. I l l , 1). Glucose asymptotes may have been detected, i n t h i s study, i f blood sampling had been more frequent during the l a t t e r stages of the simulated dives. P a r e n t h e t i c a l l y , p h y s i o l o g i c a l parameters from the two studies ind i c a t e the occurrences of 0^ asymptotes near PaO^ values of 25 mm Hg. (Liggins et a JL ., 1980; Hochachka et a l . , 1977a; J. Qvist, pers. commun.). Although no p a r t i c u l a r glucose concentration appears to i n i t i a t e a steady state mechanism one would assume i f the dive times of the present study had been extended, a drop i n the blood glucose l e v e l s (below 3.0 ymol/ml) would have c e r t a i n l y resulted i n the a c t i v a t i o n of compensatory mechanisms. For example the average adult seal (500 kg; 60 1 blood) stores about 300 mmoles of free blood glucose, of which about 60 mmoles are u t i l i z e d during 20 min dives ( f i n a l glucose depletion about 1 ymol/ml blood). I f the same depletion rate continued over a 70 min simulated dive, over 200 mmoles of glucose would be u t i l i z e d ; 116 a state which would surely render the organism hypoglycemic. I t i s therefore not too s u r p r i s i n g that blood glucose l e v e l s , following 50-70 min of voluntary diving i n the f i e l d (samples supplied by G. L. Kooyman) were found to be approximately 3 ymol/ml. The suspected compensations could take a v a r i e t y of forms: 1) Metabolic compensation. In t h i s scheme the metabolic a c t i v i t y of c e r t a i n organs or tissues could be reduced i n response to the lowered glucose l e v e l s and/or Pa0 2 l e v e l s . The s u b s t a n t i a l drops i n a o r t i c blood temperatures, glucose and oxygen l e v e l s during voluntary dives, i n excess of 50 min, as reported by Kooyman et a l . (1980) could be e a s i l y explained by the above strategy. On the other hand, a recent study by G a l l i v a n and Ronald (1979) suggests that free diving episodes of another phocid, the harp s e a l (Phoca groenlandica) were not associated with any detectable depression of metabolism. Since companion metabolite studies were not performed by G a l l i v a n and Ronald (1979), the above proposal remains e s s e n t i a l l y untested. 2) Gluconeogensis and glycogenolysis. Although blood perfusion to the l i v e r and kidney are reduced, i t i s s t i l l not t o t a l l y r e s t r i c t e d from reaching these organs. For example, i n the adult Weddell seal the kidney receives 3% of the t o t a l cardiac output during diving and, i n terms of absolute blood flow, receives more blood than the heart (Zapol et a l . , 1979) . Therefore, i t i s p l a u s i b l e f or both organs to p a r t i c i p a t e i n not only glycogenolysis but also gluconeogensis. These processes could be activated by the low glucose and/or the high catecholamine l e v e l s c i r c u l a t i n g during diving (Exton and Park, 1967; Exton et a l . , 1970). Other organs such as the heart and lung may also prove to be s i g n i f i c a n t 117 i n glucose homeostasis. If the copious, myocardial glycogen deposits (60-120 umol/g wet weight) were to be mobilized and released into the c i r c u l a t i o n , the c e n t r a l blood could see up to 150 mmols of glucose assuming an average cardiac weight of 1.2 kg (see Kerem et a l . , 1973). This process could be made possible by the s i g n i f i c a n t glucose-6-phosphatase a c t i v i t i e s occuring i n the heart (see Chapter IV). E a r l i e r studies (Hochachka et: a l . , 1977a) have considered the gluconeogenic c a p a b i l i t i e s of the Weddell seal lung and have postulated 14 i t s r o l e as a glucose supplier during diving. However, following C-la c t a t e i n f u s i o n , i t was not possible to detect any release of l a b e l l e d glucose from the lung. Furthermore, the lung stores small quantities of glycogen and, hence, i t i s t e n t a t i v e l y eliminated as a s i g n i f i c a n t glucose source f o r the c e n t r a l c i r c u l a t i o n . The most important metabolic function of the lung during the dive phase i s now assumed to be the oxidation and anabolism of l a c t a t e to compounds such as l i p i d s . 3) Alternate f u e l supplies. It i s possible that some tissues switch f u e l sources throughout a dive i n response to lowered l e v e l s of blood glucose. Although blood l e v e l s of free f a t t y acids and ketone bodies have yet to be monitored throughout prolonged diving, i t i s improbable that any organism would burn highly reduced f a t or ketone bodies i n preference to glucose under low oxygen conditions associated with diving at the time of the glucose asymptotes (Hochachka et a l . , 1977a). Tissue or blood-derived amino acids could play p i v o t a l r o l e s f o r i t has been demonstrated that animals such as salmon r e l y quite heavily on amino acid catabolism when carbohydrate stores are depleted (Idler and Clemens, 1959). More s p e c i f i c a l l y , Hochachka and associates (1975) proposed a 118 scheme involving anaerobic protein catabolism i n the musculature of marine mammals during diving. (This could explain why the s k e l e t a l muscles of these animals have r e l a t i v e l y low concentrations of both glycogen and f a t ; see B l i x , 1976.) Whether the c e n t r a l organs have the p o t e n t i a l s for t h i s proposed set-up and i t s a c t i v a t i o n during low-glucose stress are unknown. If any of these f u e l sources are to aid i n the generation of energy (ATP), substrate phosphorylations must be i n t e g r a l hallmarks of the a l t e r n a t i v e catabolic pathways. This c r i t e r i o n alone would reduce the p o t e n t i a l l i s t of substrates to amino acids (see Hochachka et. a l . , 1975). 4) C i r c u l a t o r y adaptations. As previously stated, the phocid s e a l may pool as much as 85% of i t s t o t a l blood volume i n the venous system as a r e s u l t of the diving response. A s i g n i f i c a n t portion of t h i s pooled blood i s apparently stored i n a post-diaphragmatic enlargement of the c r a n i a l portion of the abdominal vena cava, known as the hepatic sinus and presumably, t h i s r e s e r v o i r can be slowly added to the c e n t r a l blood volume by means of a muscular caval sphincter (Harrison and Kooyman, 1968). Herein l i e s the a t t r a c t i o n of t h i s system, for t h i s 'leaking blood' could not only supply fresh glucose stores but also more 0^ and, thus could account f o r the steady-state or asymptotic behaviour of one or both substrates. Despite t h i s rather a t t r a c t i v e explanation, i t i s conceivable that the Weddell s e a l could u t i l i z e combinations of the above strategies i n response to lowered blood glucose and/or 0^ l e v e l s . The following i s a tentative diagramatic model of the metabolic status of the more important components during simulated diving. This encompasses most of the relevant data now a v a i l a b l e . 119 Scheme VIII, 1. Diagramatic model of some of the more important metabolic interactions between the c e n t r a l blood pool, brain, lung, heart and other hypoperfused ti s s u e s during simulated d i v i n g . Where possible (e.g. heart), f a t t y a c i d catabolism may remain operational throughout the aerobic phases of d i v i n g . BRAIN Glucose •Lac t a t e + C0„ + H„0 1 I L . HEART Glycogen V G-l-P -•G-6-P j Glucose(?) Pyruvate^— H 20 + C O , / - • L a c t a t e 1.4 \ CENTRAL BLOOD \ Glucose VOLUME — Lactate• I o fD B < H H H zziz GLUCOSE, 0 2 Pooled Venous Blood I LACTATE | PYRUVATE C0 2 + H 20 LUNG Anabolic Pathways (?) Lactate >C0 2 + H 20 •GLUCOSE AMINO ACIDS ALANINE SUCCINATE HYPOPERFUSED TISSUES 120 Although not included i n the above model, i t i s important to re-emphasize the possible existence of i n t e r - t i s s u e c y c l i n g of other metabolites besides that of glucose and l a c t a t e . R e c a l l from Chapters III and VII that pyruvate p r o f i l e s do not always follow those of l a c t a t e ; i n f a c t , pyruvate can be decreasing while l a c t a t e i s s t e a d i l y increasing during the dive phase. This could be merely a r e f l e c t i o n of transport discrepancies or may stem from an i n t e r - t i s s u e hydrogen cycle, i n which organs of d i f f e r e n t redox states exchange pyruvate and l a c t a t e . Since data on glutathione p r o f i l e s are so e r r a t i c , no further speculations regarding any metabolic implications of t h i s phenomenon are advanced (see Chapter I II for a discussion). Recovery The metabolic s i t u a t i o n i n the recovery period appears to be better elucidated and perhaps represents a simpler working system. Upon surfacing, there i s an immediate remixing of p a r t i a l l y trapped venous blood with that of the c e n t r a l c i r c u l a t i o n ; concomitant with t h i s washout come large spikes of l a c t a t e , alanine and glutamine from the peripheral tissues and organs. Presumably, the brain, lung and, most l i k e l y , the heart absorb and oxidize l a c t a t e i n preference to glucose at t h i s time (see Mochizuki et^ a l . , 1978 and L i u and Spitzer, 1978 f o r discussions on the mammalian heart). Consequently, a l l three c e n t r a l organs could derive a large percentage of t h e i r energy and carbon needs d i r e c t l y from t h i s anaerobic endproduct. I f basic metabolic features observed i n the mammalian class hold for the aquatic mammals, i t can be predicted that many more tissues and organs w i l l a c t i v e l y 'co-operate' i n c l e a r i n g the system of l a c t a t e . The mammalian l i v e r , kidney (Scrutten and Utter, 1966) and some muscle f i b e r s (McLane and Holloszy, 1979) exhibit 121 de novo synthesis of glucose and glycogen from l a c t a t e . Gluconeogenesis i n at l e a s t the l i v e r i s further sparked by rapid and s i g n i f i c a n t increases i n blood glycogen^during recovery (Robin, pers. commun.). Glucose stores i n the blood may be further replenished by the action of the high l e v e l s of c i r c u l a t i n g catecholamines, since these hormones not only activ a t e glycogen breakdown i n the l i v e r and the muscles ( P i l k i s et al.., 1978) but also apparently stimulate gluconeogenesis i n the l i v e r . Recent studies by Davis and Kooyman (1979) on the harbor s e a l , i n d i c a t e that 29% of the t o t a l blood l a c t a t e i s converted to blood glucose i n the recovery phase of a routine 10 min dive (forced), whilst 8-26% i s apparently oxidized. Since t h e i r methods do not allow for tr a c i n g of l a c t a t e and glucose beyond the blood volume i t can only be assumed that the remainder of the l a c t a t e i s e i t h e r converted to glycogen or to other metabolites such as amino acids. As previously mentioned, glutamine and alanine pools i n the a o r t i c blood are also s u b s t a n t i a l l y elevated i n the recovery phase. P a r e n t h e t i c a l l y , the r i s e s i n both amino acids during diving are l i k e l y caused by peripheral leakage. The increases may be the r e s u l t of anaerobic catabolism of p r o t e i n and carbohydrate i n the peripheral tissues (see Chapter I I I and Hochachka et a l . , 1975); whereas the alanine increases could be further implicated with the sparking of the t r i c a r b o x y l i c acid cycle during the anaerobic-aerobic t r a n s i t i o n s , associated with recovery i n such tissues as the muscles (Hochachka and Murphy, 1979). Regardless of t h e i r o r i g i n s , both metabolites are l i k e l y absorbed by the l i v e r and kidney where they may serve as precursors to gluconeogenesis ( F e l i g , 1973; Hochachka and Murphy, 1979). Furthermore, alanine increases may cause consistent increases i n plasma glucagon 122 concentration (Muller et. a l . 1970) and thus be i n d i r e c t l y responsible for the increased gluconeogenesis from l a c t a t e and glutamine and of course alanine. In view of the above considerations i t i s not s u r p r i s i n g that blood glucose l e v e l s quickly return to normal and, i n f a c t , a c t u a l l y overshoot control concentrations. In the phocids these mechanisms may be p a r t i c u l a r l y u s eful because of t h e i r low carbohydrate d i e t ( B l i x , 1976). The above data are summarized i n the following diagrammatic model. 123 Scheme VIII, 2. Diagrammatic model of some of the metabolic interactions between the brain, lung, heart, l i v e r , kidney and blood during recovery from simulated diving. BRAIN Anabolic Pathways y r c o 2 + H 2 ° v Glucose •Lactate TOTAL BLOOD VOLUME ucose + Lactate ^ — J G T L JAlanine; l a c t a t e ; glutaminej LUNG Glucose-\ \ — • A n a b o l i c Pathways C0 2 + H 20 Anabolic, Lactate. • Pathways' to Lactate — Alanine Glutamine -•C0 2, H 20, |Glucose! -•Urea Gluconeogenic Organs  (e.g. l i v e r , kidney) 124 In summary, the present data on r e l a t i v e l y short-term, simulated diving (10-30 min) i n the Weddell s e a l i n d i c a t e cooperative metabolic interactions between the heart, lung and b r a i n . These i n t e r a c t i o n s , which probably contribute to extending diving duration, depend upon how enzyme pot e n t i a l s are used, and not on the development of any new or q u a l i t a t i v e l y d i f f e r e n t enzymic machinery. Indeed, only a few modest adjustments i n enzyme l e v e l s seem to c o r r e l a t e with the observed metabolic organization. In t h i s sense, the biochemical strategies associated with extending hypoxia tolerance appear to d i f f e r fundamentally from those u t i l i z e d by many, more p r i m i t i v e animal anaerobes (Hochachka, 1980). It i s stressed that these i n t e r p r e t a t i o n s are based on forced diving episodes, l a s t i n g up to 30 minutes, when metabolism of the three c e n t r a l organs i s not oxygen l i m i t e d . I f oxygen were to become se r i o u s l y l i m i t i n g the above key i n t e r a c t i o n s would break down. How the s e a l deals with such emergencies remains a question for the future. However, t h i s would probably not a l t e r the present i n t e r p r e t a t i o n of the recovery process. 125 F e t a l Responses to Maternal Diving Companion studies performed by Liggins et a l . , (1980) have demonstrated that, upon i n i t i a t i o n of a maternal dive, the fetus responds by developing a s u b s t a n t i a l bradycardia (90 beats/min to 34 beats/min a f t e r 4 min and to 27 beats/min at the end of d i v i n g ) . This decline i n heart rate i s accompanied by a retention i n the mean a r t e r i a l blood pressure and a well-sustained d i a s t o l i c pressure during the longer i n t e r v a l s between f e t a l heart beats. These p h y s i o l o g i c a l parameters when coupled to our metabolic blood p r o f i l e s ( i . e . l a c t a t e and pyruvate recovery peaks) strongly suggest the existence of a peripheral v a s o c o n s t r i c t i o n and thus the development of a f e t a l diving response. Thus the f e t a l s e a l appears to have a w e l l developed oxygen conserving mechanism akin to that i n the adult. However, the maternal 'message' s i g n a l l i n g onset of diving has yet to be ascertained (Liggins et: a l . , 1980) . The f e t a l studies also demonstrated an unusual feto-maternal gradient of blood glucose concentrations, the f e t a l concentrations being higher than the maternal. This appears to be a unique biochemical adaptation of the Weddell s e a l , ensuring ample substrate supply f o r the fetus during prolonged maternal diving. During short-term diving, glucose p r o f i l e s i n the a r t e r i a l pool of the fetus f l u c t u a t e somewhat with a s l i g h t increase i n l e v e l s towards the end of the episode. This may be due to the a c t i v a t i o n of f e t a l gluconeogenic processes and to the continued p l a c e n t a l perfusion of maternal blood (see Eisner et a l . , 1970 and Liggins et a l . , 1980 f o r discussions r e l a t i n g to placental perfusion during maternal asphyxia and di v i n g ) . 126 Although blood alanine and glutamine p r o f i l e data are sketchy recovery spikes can, nevertheless, be deciphered, suggesting washouts from previously, vasoconstricted peripheral t i s s u e s . A q u a l i t a t i v e l y s i m i l a r response was also observed i n both male adults and pregnant females (see Chapter I II for a thorough discussion). Otherwise, there were no detectable changes i n the rest of the amino acid blood pool during or a f t e r maternal simulated d i v i n g . Again i t i s noted that t h i s set of data was generated from r e l a t i v e l y short term diving (10-30 min for the gravid s e a l ) ; whether t h i s p o t e n t i a l l y u s e f u l pool remains i n a steady state condition throughout long term diving (up to 70 min) remains a question f o r future research. I t has been suggested that i n periods of maternal hypoglycemia the mammalian fetus w i l l increase i t s catabolism of amino acids ( B a t t a g l i a and Meschia, 1976). In r e s t i n g animals amino acid concentration gradients across the placenta favor, mainly, the f e t a l c i r c u l a t i o n ; a r e s u l t consistent with other mammalian species thus f a r studied. This phenomena i s due to the high f e t a l demands for catab o l i c and anabolic substrates and because of the fetus' i n a b i l i t y to synthesize some non-essential amino acids (Palou et^ al., 1977). Alanine gradients are the most dramatic (1:3 r a t i o across the placenta), r e f l e c t i n g the low l e v e l s i n the c i r c u l a t i o n of the adults. Our enzyme data alone, do not constitute a good i n t e r p r e t a t i v e basis for the understanding of the metabolic preferences and i n t e r r e l a t i o n s h i p s of the three c e n t r a l organs of the fetus as a consequence of maternal diving. Nevertheless, the enzymatic machinery appears to be f u l l y developed and thus the enzyme studies are consistent with the fetus behaving i n a s i m i l a r fashion to that of the adult i n a l l three states associated with 127 diving. Glucose and oxygen are probably the main substrates f i r i n g the cerebral production of ATP i n both normal and diving states while the heart may r e l y on a mixed aerobic and anaerobic metabolism depending upon the mean a r t e r i a l oxygen tension. 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P h y s i o l . 47: 968-973. 140 Appendix L i s t of Abbreviations 140 a Acetyl CoA: acetyl-S-coenzyme A 5'AMP, ADP, ATP: adenosine 5'-mono-,di-,triphosphate A-V: arterio-venous CaCl^: calcium ch l o r i d e CBF: cerebral blood flow CO2: carbon dioxide yC: microcurie DTNB: 5,5'-dithiobis-(2-nitrobenzoic acid) FAD: f l a v i n adenine dinucleotide FADH2: reduced FAD GABA: gamma aminobutyric acid cxGP: a-glcerophosphate GSH: reduced glutathione GSSG: oxidized glutathione GTP: guanosine 5'-triphosphate G-l-P: glucose-l-phosphate G-6-P: glucose-6-phosphate HK: hexokinase hydroxyacylCoA: hydroxyacyl-S-coenzymeA KC1: potassium chloride K^CO^: potassium carbonate 2-KGA: 2-ketoglutarate KH^PO^:" potassium phosphate, monobasic KOH: potassium hydroxide LDH: l a c t a t e dehydrogenase 141 MgSO^: magnesium s u l f a t e NAD: nicotinamide adenine dinucleotide NADH: reduced NAD NADP: nicotinamide adenine dinucleotide phosphate NADPH: reduced NADP NaHCO^: sodium bicarbonate OXA: oxaloacetate P: p r o b a b i l i t y PaC^: a r t e r i a l p a r t i a l pressure of oxygen PaCC^: a r t e r i a l p a r t i a l pressure of carbon dioxide PCA: per c h l o r i c acid PFK: phosphofructokinase P^: inorganic phosphate PK: pyruvate kinase RBC: red blood c e l l S.D.: standard deviation S.E.: standard error STP: standard temperature and pressure SSA: s u l f o s a l i c y c l i c acid PUBLICATIONS Murphy,B.J . and M.E.Brosnan. 1976. 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Metabol ic sources of heat and power in tuna muscles. I. Muscle f i n e s t r u c t u r e . J . Exp. B i o l . 82:289-301. L i g g i n s , G . C . , Q v i s t , J . , P.W. Hochachka, B . J . Murphy, R.K. Creasy, R.C. Schneider , M.T. Snider and W.M. Zapol . 1980. Fetal card iovascu la r and metabol ic responses to simulated d iv ing in the Weddell s e a l . J . App l . P h y s i o l . Submitted. Murphy,B.J . and P.W. Hochachka. 1980. Free amino ac id blood l eve ls of the WEddell s e a l . In Preparat ion. 

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