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INTERMEDIARY METABOLISM OF FISHES AND OTHER AQUATIC ANIMALS
UNITED STATES DEPARTMENT OF THE INTERIOR FISH AND WILDLIFE SERVICE
EXPLANATORY NOTE
cooperating agencies and in processed form for economy and io delay in publication. 7
Pr
United States Department of the Interior, Fred A. Seaton, Secretary Fish and Wildlife Service, Amie J. Suomela, Commissioner
INTERMEDIARY METABOLISM OF FISHES AND OTHER AQUATIC ANIMALS
by
M. Gombmann, W. Duane Brown, and A. L. Tappel
U. S. Fish and Wildlife Service Special Scientific Report - Fisheries No. 288 Washington 25, D. C.
December 1958
The Library of Congress catalogue card for this publication is as follows:
Gumbmann, M Intermediary metabolism of fishes and other aquatic ani- mals, by M. Gumbmann, W. Duane Brown, and A. L. Tappel. Washington, U. S. Dept. of the Interior, Fish and Wildlife
Service, 1958. 51 p. illus. 27 cm. (Special scientific report—fisheries, no, 288)
Bibliography : p. 83-51.
1. Fishes—Physiology. 2. Marine fauna. 1. Title. (Series: U.S. Fish and Wildlife Service. Special scientific report: fisheries, no. 288)
[SH11.A335 no. 288} Int 59-58 U.S. Dept. of the Interior. Library
for Library of Congress
The Fish and Wildlife Service series, Special Scientific Report--Fisheries, is catalogued as follows:
U.S. Fish and Wildlife Service. Special scientific report: fisheries. no. 1— Washington, 1949-
no. illus., maps, diagrs. 27 cm.
Supersedes in part the Service’s Special scientific report.
1. Fisheries—Research.
SH11.A335 639.2072 59-60217
Library of Congress (2)
CONTENTS
Introduction . . ee © © © ©
Carbohydrate MCGADOLVSMiepeiien a) el teller 6 ee @ © Tricarboxylic acid cycle and terminal oxidation . Electron transport systems, coenzymes and vitamins Phosphorus metabolism eoeeef © © © © © © 8 ele le Protein and nitrogen metabolism ee « «ee ecee lipid MOCADOVUSM ve) tenet ele er ete 6 elton elle ol te ee Need) for research’ “67s 6 ~« oe ee © ©B Oo wo oO e literature cited eo eee © © © © © © 8 8 ew elle lt FIGURES
Figure
1. Known intermediary metabolism of carbohydrates and elasmobranchs . . e ‘oe © 0 "e608 © 6-0
2. Known intermediary metabolism of carbohydrates ETM Ay ole SO G86 "GO .GHG Oo: oc: 6 00 10
3 Known intermediary metabolism of carbohydrates
he Know intermediary metabolism of carbohydrates Urehitl se 6 © 6 @ ‘6 eo e- 0 @''e @ 6. @) oe
5. Known tricarboxylic acid cycle in teleosts and
oe eeeeeee ©oe°e0 e@ @ © @ © oe e@ ee © @ & @ © e@ @© @ © © © 8 @ e e e © @ e e e eo e@ @ @© © © @ © le
in teleosts Ce en ee ee, in aquatic ee @ @ @ @ in oysters in the sea
eoe@e 8 @ @ @© @
e
elasmobranchs 6- Known tricarboxylic acid cycle in oysters 0 «ee ceececece
ABSTRACT
This paper is a review of current knowledge of intermediary meta= bolism in fish (teleosts and elasmobranchs), shellfish (mollusks and crustaceans), and certain other aquatic animals. All important research relative to the topic is mentioned, although the paper is not a totally exhaustive review. Numerous references to the original literature are included.
The review is organized around the major well-known metabolic divisions: carbohydrate metabolism, including pathways of glycolysis and the phenomenon of rigor mortis; respiratory metabolism and the tri- carboxylic acid cycle; electron transport systems; phosphorus metabolism$ protein metabolism; and lipid metabolism. The information is summarized in figures wherever feasiblee
A section concerning the needs for research is included, and specific suggestions for needed investigations are givene
INTERMEDIARY METABOLISM OF FISHES AND OTHER AQUATIC ANIMALS
al 2 by M. ore W. Duane sels and A. L. Pappe1/
INTRODUCTION
A sizable body of knowledge dealing with composition and physiology of fishes and other aquatic animals is available. There nave been but few studies, however, of intermediary metabolism in these organisms. This paper is e review of tne studies that have appeared in the litera-= ture.
Physiology and composition are discussed where their relationship to intermediary metabolism is indicated. The major differences in compo- sition that distinguish aquatic animals from land animals is pointed out, since such differences suggest deviations in metabolic routes and path- weys.
The animals discussed are limited to fish (teleosts and elasmo- branchs), shellfish (mollusks and crustaceans), sea urchin, and aquatic mammals (dolpnins and wnales). Metabolic studies of the sea urchin have been included because of tne large amount of study given this animal and because the metabolism of the sea urchin appears to resemble that of other marine invertebrates more closely than it does that of higher marine forms. The review is organized around the major well-known meta-= polic divisions: carbohydrete metabolism, including the Embden-Meyerhof pataway, tne phenomenon of rigor mortis, and the hexosemonophosphate snunt; respiratory metabolism and the tricarpoxylic acid cycle; electron- trensport systems of terminal oxidation, including various cofactors;
ljote.--This study was supported in part by funds made available through the Saltonstall-Kennedy Act and administered by means of a collaporative agreement between the U. S. Fish and Wildlife Service and the University of California.
1/ Research Assistant, Department of Food Technology, University of California, Davis, California.
2/ Chemist, U. S. Fish and Wildlife Service.
3/ Associate Professor, Department of Food Technology, University of California, Davis, California.
phosphorus metabolism and the processes of storage and transfer of energy $ protein and nitrogen metabolism, including nitrogenous compounds charac= teristic of aquatic animals; and lipid metabolisn, including fatty acid composition. Where possible, the information is summarized in figures along the lines of conventional metabolic mapse ;
The presentation of compositional analyses and physiological data of aquatic animals is not the concern of this review except in a general Sense where it indicates metabolic pathways and possible unique processes occurring in aquatic animals; tables and charts giving such information have not been included. Many excellent reviews are available where this type of information has been collected, and a few of them might be mentioned heree A recent book giving extensive coverage of the subject, for example, is The Physiology of Fishes by Brown (1957). In a symposium on the bio- chemistry of fish (Williams 1951) are reviews of "The Proteins of Fish" by Hamoir, "The Chemistry and Metabolism of the Nitrogenous Extractives in Fish" by Shewan, and "The Chemistry and Metabolism of Fatsin Fish" by Loverne Hamoir (1955 a) has made an intensive review of fish proteins, including enzymes, respiratory pigments, and contractile proteins.
This review is similar to those in an Annual Review of Biochemistrye It is exhaustive to the point of not missing important research on the intermediary metabolism of aquatic animals, but it does not cite all the work done in this field. By limiting the review to appropriate enzyme researches, it is found that very few enzyme researches of major interest in the present problem were completed before 1945. The survey of litera- ture pertaining to this review was completed in February 1958.
CARBOHYDRATE METABOLISM
The study of carbohydrate metabolism in fish has been confined mainly to compositional analyses of fish tissues and eggse Evidence defining the processes involved in this phase of metabolism is indirect and, for the most part, has been inferred from changes in amounts of : total carbohydrate, glycogen, .and glucose in relation to various physio= logical and environmental factors. Little has been done to characterize the specific enzyme systems and the pathways relating the intermediate products of carbohydrate metabolism.
Immers (1953) followed glycogen metabolism in the stickleback and minnow by measuring changes in glycogen distribution in the liver, gonads, and skin, with regard to sex differentiation and the sexual cycles In the prespawning salmon, the examination of glycogen mobilization indicates that gluconeogensis (the transformation of endogenous fat and protein to carbohydrate) mist occur in order to meet the energy demand required during migration (Fontaine and Hatey 1953). This process mst occur extensively in order to account for the relatively high level of sugar in the blood and the more or less constant reserve of glycogen maintained by these fish during the period of fasting incident to migrations In mammals, es ee of gluconeogensis is dependent upon the pituitary growth honone ee 195), and evidence indicates that the same holds true for fish ae 1957). In the fasting eel that has been previously hypophysectomizeds
2
glycogen reserves progressively decline (Hatey 1951 a bie on injection of glucose, however, the hypophysectomized eel is able to Boas glycogen. In the hypophysectomized, but feeding, fundulus, normal gly= cogen reserves aré found (Pickford 1953),
Glycolysis in the red-colored swim bladder gland of the scup has been demenstrated by Strittmatter et al. (1952). He obtained significant increases in production of lactic acid after addition of glucose and other hexoses to the bisected gland, under both anaerobic and aerobic conditionse De Vincentiis (1952) has measured rates of respiration and glycolysis of retinas of teleosts and of octopuses by manometric measurements, with glu= cose as a substrate. The rate for retinas of octopuses was similar to that found for retinas of teleosts and generally resembled the small values obtained with cold-blooded vertebrates rather than the higher values ob= tained with birds and with mammals. Hishida and Nakano (195) measured the uptake of glycogen and of oxygen in developing ee eggs. Deter— Mination of the respiratory quotient showed that metabolism of carbohy= drate probably begins after the gastrula stage; such a scheme agrees with the glycogen analyses made over this period. Anaerobic glycolysis and lactic acid production also were observede
Evidence of glycolysis occurring in the developing fundulus embryo in relation to regulation of osmotic pressure has been shown by Shanklin (195). That glycolysis supplies the energy necessary for maintaining normal ionic transport and development of the egg was shown by treatment with fluoride and with iodoacetate. These two known glycolytic inhibitors disrupted the normal ionic gradient established across the egg membranes The enzyme catalyzing the conversion of phosphopyruvate to pyruvate has been shown to occur in the muscle of several species of fish (Boyer 1953). The presence of adenosine diphosphate (ADP) and potassium ion is essen= tial for the reaction. The presence of this enzyme in all the species tested indicates the probability that other enzymes of the glycolytic system also are present and functioning.
Hexokinase is a glycolytic enzyme that is responsible for the initial activation of a hexose sugar molecule, thus allowing phosphorylation and the subsequent sequence of intermediary steps of glycolysis to proceede Kerly and Leaback (1957) found this enzyme to be active in brain homogen- ates of squids (Sepia officinalis), elasmobranchs (Scyllium canicula and Raia brachyura), teleosts (Seophthalms maximus and Gadus merangulus), and frozs (Rana temporaria). Glucose and fructose were utilized at a high rate in all of the species examined, adenosine triphosphate (ATP) being required for activity. The hexokinase of these aquatic animals resembled the nonspecific type found in mammalian brain in that it attacks both al- doses and ketoses and, in contrast to the hexokinase of yeast, is inhibited
by its reaction producte
The hexosemonophosphate shunt is an important oxidative pathway in carbohydrate metabolism. For fertilized eggs of the sea urchin, Arbacia,
3
it may be considered a short-cut method by which the embryo obtains energy while awaiting the development of other glycolytic and tricarboxylic acid cycle enzymes (Krahl 1956). The existence and relative importance of this pathway in fish has yet to be investigated. A compound formed by the hexosemonophosphate pathway, ribose, is liberated from various ribose— containing compounds by enzymes present in muscle tissue of fishes
Tarr (1952, 1953, and 195) and Tarr and Bissett (195) found that ribose was cleaved from ribonucleic acid, ATP, ribonucleosides, ribomecleo-= tides, and ribose-5=phosphate added to the muscle of certain fish. More recently, using lingcod mscle, Tarr (1955) was able to purify a nonphos= phorolytic nucleosidase capable of hydrolyzing ribonucleosides, such as adenosine, inosine, guanosine, xanthosine, and cytidine. It appears that fish muscle contains a ribonuclease and other enzymes that hydrolyze ribonucleic acid to constituent mononucleosides. These, in turn, may be attacked by a non=phosphorolytic riboside hydrolase enzyme, with the liber— ation of ribose and a base. This degradation process appears to be more rapid in fish than in warm-blooded animals (Hamoir 1955 b).e
Alcohol dehydrogenase, which in mammals is found almost exclusively in the liver, has been isolated and purified from fish liver (Boeri et ale
195h)
ATP is the key compound for trapping the energy released during carbohydrate oxidation in all forms of life and serves as the immediate source of energy for muscular contraction. It therefore is not surprising that considerable interest has been shown the presence and utilization of ATP in fishe Contraction and rigor mortis in fish muscle have been related to post-mortemchanges in amounts of glycogen, ATP, and free sulf= hydryl groups (Noguchi and Yamomoto 1955 a,b; Partmann 1953). These compounds decrease rapidly, reaching a minimum after 9 hours with full rigor and a decrease in pH. Somewhat similar studies of fish muscle were made by Fujimaki and Kojo (1953 a,b), in which glycogen, lactic acid, ammonia, amide nitrogen, and pH were determined in frigate mackerel killed by various methods. Fish that were left to die underwent a rapid decrease of ATP, with a corresponding increase of adenylic acid and inosinic acid within ) hours. Reay and Shewan (199) have also reviewed the immediate post-mortem changes in glycogen and lactic acid content and pH of fish miscle. Compared to mammalian muscle, fish muscle usually contained less glycogen, owing to exhaustion before death, and had a higher pH; these factors increase the speed of onset of rigor mortise
Saito and Hidaka (1955 a,b; 1956 a,b), using purified myosin frac- tions isolated from carp muscle, have studied ATP-ase activity and stab— ility and the post-mortem changes of myosin fraction nitrogen, ATP, and free sulfhydryl groups in carp muscle. In general, ATP=ase activity of the myosin fractions was high and showed a temperature optimum similar to that of rabbit muscle. The decrease of this activity at 25° C. over a period of 10 hours coincided with the decrease in sulfhydryl groupSe Rigor mortis also paralleled the decrease of ATP and sulfhydryl groups and was complete after 6 hours, at which time all ATP had disappearede
h
The main differencesof fish muscle as compared with muscle of land ani- mals are lower glycogen content, initially higher lactic acid content,
and greater ATP-ase activity (Hamoir 1955 b). Steinbach (199) compared ATP-ase activity of muscle-tissue homogenates of swordtail, bluegill, minnow, frog, turtle, mouse, and bird. Measured at 30° C., ATP is split at nearly the same rate by all the homogenates, but at 0° C., wide differ ences occur, with the activity of the fish preparations remaining fairly high. Based on the Q]1o values obtained, the animals fell into three groups: fish, 1,-1.7; bird, mouse, and turtle, 2.0; and frog, intermediate. The relative insensitivity of fish ATP-ase to changes in temoerature suggests that low Q10 values for critical reactions may be an adaption of cold-blooded animals that must survive variable temperatures. Apparently, however,
no correlation exists relating ability to survive in cold temperatures with Qi9 values, since the turtle, minnow, frog, and bluegill all can live at cold temperatures, whereas fish from warmer water (for example, sword= tail) can withstand only a very small decrease in temperature. Davidson and Richards (195) have calculated activation energies for muscle ATP=ase of the crayfish, minnow, and cockroach. At low temperature, the quantit- ative level of ATP=ase activities is in the same order as is the activity of the whole animal in nature. They suggested that ATP-ase activity could be a limiting factor governing activity of these species.
Glycolysis in the oyster has been given considerable attention. As with fish, general composition (fat, protein, and carbohydrate) of this marine invertebrate has been studied (lee and Pepper 1956). Humphrey (1950 b), working with vitreous and nacreous muscle homogenates and extracts of the oyster, demonstrated pyruvic and lactic acid formation from glycogen, glucose, glucose-l-phosphate, glucose=-6-phosphate, and fructose-1,6—diphos- phate added as substrates. He stated that the following intermediates associated with glycolysis were present: ATP, ADP, glucose-l-phosphate, glucose-6—phosphate, fructose—-6—phosvhate, fructose-1,6—-diphosphate, triose phosphate, phosphoglyceric acid, and phosphopyruvic acid. In addition, both vitreous and nacreous muscle are able to synthesize glucose—6—phos- phate from glucose-l-vhosphate and, in the opposite direction, glycogen from glucose-l-phosphate. The formation of fructose-6—phosphate and fruc= tose-1,6—diphosphate from glucose-6-phosphate also occurs. The glycolytic ability of oyster muscle is several hundred times less powerful than is that of rabbit muscle. It may be that glycolysis in the oyster is a slow, continuous process in contrast to glycolysis in mammals, where glycolytic activity is temporary and is brought into operation after muscular contrac= tion (Humphrey 1950 b). Usuki (1956) investigated the effect of the inhib- itors of glycolysis--iodoacetic acid and sodium fluoride--on the activity of oyster gills. The suppression of ciliary action that occurs can be overcome partially by addition of pyruvate and succinate, but not by ad= dition of glucose. This fact suggests that the Embden-Meyerhof pathway
is blocked by these inhibitors.
Also studying the effects of inhibitors, Humphrey (1950 a) demon- strated a decrease in respiration of oyster spermatozoa upon treatment with sulfhydryl-group reactants, such as iodoacetate and phenylmercuric acetate. Compared to glycolytic rates of the sperm of land animals, how=- ever, the glycolytic rates observed are very low, as was shown by the limited production of lactic acid from the breakdown of various hexoses that were added as substrates. Since consumption of oxygen is higher than
5
is that found in other tissues of this animal, the sperm probably depends upon oxidative metabolism almost completely. The respiration of homogen= ates of oyster eggs is stimulated by some of the intermediates of glycoly= sis, such as glycogen, fructose, glucose, glycerol, and glycerophosphate (Cleland 1950). ee
Rather extensive investigation of the metabolism of the eggs of Arbacia eae and other sea urchins has been carried out over a number of years. The emphasis of these studies is on the chemical events taking place with fertilization and the processes by which energy is liberated for cleavage and development. A review of this information covering the period from 1932 to 1949 has been presented by Krahl (1950) and is mainly concerned with the nature and interrelationships of the catalysts by which the Arbacia egg derives energy from foodstuffs. At that time, none of the enzymes of glycolysis had been studied in this speciese The carbohydrate and glycogen content was known (Hutchens et al. 192); however, oxidation of glucose, lactate, 2-glycerophosphate, succi- nate, or butyrate could not be demonstrated with cytolysates of Arbacia eggs (Ballentine 190).
The observations of Cleland and Rothschild (1952) from their study of carbohydrate oxidation of the eggs of the sea urchin, Echinus esculentus, were consistent with the assumption that the glycolytic pathway iS oper-= ative in these animals. Oxygen uptake and lactic acid production of homo= genized eggs were increased by adding glycogen, glucose, and fructose and were stimulated by addition of diphosphopyridine nucleotide (DPN). The phosphorylated intermediates of glycolysis such as glucose-l—phosphate, fructose-6—phosphate, and fructose-1,6—-diphosphate also stimulate the up= take of oxygene Pyruvate and lactate were shown to accumulate in homogen= ized eggs under anaerobic conditions and in the presence of hydrogen cya= nidee Other evidence presented by Cleland and Rothschild for glycolysis includes inhibition of oxygen uptake by fluoride ion and its reversal by pyruvate, and the accumulation of pyruvate after addition of glucose-6- phosphate or hexose-diphosphatee Ycas (1950) reported the existence of phosphoglucomutase, enolphosphopyruvic acid, phosphodihydroxyacetone, enolase, aldolase, and oxoisomerase in eggs of sea urchinse
Krahl et ale (195i a,b) have reported the occurrence of hexokinase, phosphofructokinase, and aldolase in the supernatant and homogenates of Arbacia eggse Evidence that the hexosemonophosphate shunt also is operative was shown by the fact that trinhosphopyridine nucleotide (IPN) was reduced and pentose was formed from glucose-6=phosphate and 6—phos- phogluconate by these egg preparations. Indications are that glucose-6— phosphate can be metabolized by the glycolytic pathway at only. about 5 percent of the rate it can be oxidized by the TPN system. Krahl et al. suggest that the latter system is probably the major pathway for utiliza- tion of carbohydrate in Arbacia eggs. Other glycolytic enzymes present in Arbacia eggs, as shown reduction of the corresponding substrates, are glucose-l-phosphate, phosphoglucomtase; fructose-6-phosphate, hex- ose isomerase; and fructose-1,6-diphosphate, a fructose-1,6-diphosphatase
6
(Krahl et al. 195) b). It is assumed that each of these substrates is connected to glucose—6-phosphate and that its direct oxidation does not occurs
Rothschild (1951) has reviewed the evidence both for Embden= Mey erhot and hexosemonophosphate paths in eggs of sea urchins. Inac~ tivity of the glycolytic pathway in Arbacia eggs also has been suggested by the work of Hutchens et al. (192) and Keltch et al. (1951) in which very limited production of lactate following consumption of carbohydrate was shown to occur. Neither the production nor consumption of lactic acid appeared to be important as an energy-yielding process for the first 2h hourse Recent work by Krahl (1956) establishes more conclusively the relative importance of the hexosemonophosphate shunt to the en= Meyerhof pathway in Arbaciae By determining the ratios of C O> formed by developing eggs and spbryos in the presspce of the substrates glu- cose-1 » glucose-2=C+4, and glucose-6—C4, he found that glucose appears to oxidize by means of the hexosemonophosphate shunt during the early stages of cleavage, the Embden—Meyerhof pathway becoming more important during later development.
Fragmentary information on the carbohydrate metabolism of other aquatic animals is available from studies of a similar nature. ATP= ase activity in heart, skeletal muscle, liver, brain, and kidney tissue of the dolphin also has been exhibited (DuBois et al. 198). Marsh (1952) has investigated rigor mortis in the baleen whale in relation to pH, buffering capacity, formation of lactic acid, and dephosphoryl- ation of ATP, In most respects, post-mortem behavior differed little from that of other animals, The high buffering capacity found may be an adaption to extended anaerobic activity. Attempting to estimate the reserves for anaerobic activity in mscle tissue of a variety of animals, Manery (1935) determined the maximim formation of lactic acid that could be produced. Muscle of seal showed no appreciable difference from miscle of cat, dog, rabbit, frog, and tortoise, in this respecte
That glycolysis proceeds through the Embden=Meyerhof pathway in various tissues of the dolphin has been shown by DuBois et al. (198). Skeletal muscle, brain, liver, kidney, and heart contain the following phosphorylated intermediates of glycolysis: glucose-l-phosphate, glucose- 6-phosphate, fructose-6=-phosphate, triose phosphate, pentosephosphate, phos- phopyruvic acid, and phosphoglyceric acid.
A positive assay for a specific enzyme of glycolysis, glycero- phosphate dehydrogenase, has been obtained from isolated frog skin (Hunter and Hunter 1957).
The mechanism of ATP=ase activity of lobster gusele has been inves= tigated by Clarke and Koshland (1953). Using Ho019, they found that muscle phosphatase hydrolyzes with nucleophilic displacement on the terminal phosphorous atom.
In summarizing present knowledge of carbohydrate metabolism in aquatic animals, we must say that our knowledge is meagere Since the pathways of glycolysis have been known for many years, however, our
7
knowledge of the metabolism of carbohydrates in aquatic animals is more developed than is our knowledge for the other biochemical entities of great importance in metabolism. It seems desirable to summarize present knowledge in the form of diagrams of metabolic pathways, which have found popular use in textbooks of biochemistry such as Outlines of e Chem istry (Nieland and Stumpf 1955) and Dynamic A ects oF Bosh (Baldwin 1952). From these and othet sauetee Phe Sete oY ae tailed information that is available on land animals, plants, and bacteriae In figure 1 is summarized the known intermediary metabolism of carbohy= drates in teleosts and elasmobranchs. Considering the importance of carbohydrate metabolism in supplying energy from carbohydrate foods, it can be seen that our knowledge is very fragmentary. There is no real in- formation on the many metabolic transformations in glycolysis except for that on the formation of pyruvate from phosphopyruvate by pyruvate phos= phoferasee Evidence on the cceurrence of the hexosemonophosphate shunt
is completely lacking. The known intermediary metabolism of carbohydrates in aquatic mammals is shown in figure 2. The identification of five of the important Kmbden—Meyerhof intermediates suggests that this pathway
of metabolism is operative in aquatic mammals, but mich more detailed in=— formation is needed for confirmation. There is no information on the hex osemonophosphate shunt. The known intermediary metabolism of carbohydrates in oysters is shown in figure 3. Here also, the positive identification
of seven important intermediates suggests that the Embden—Meyerhof pathway of glycolysis is operative in the oyster. Much more detailed information, especially identification of the enzymes catalyzing the transformations
in glycolysis, is needed. Again, there is no evidence of the hexose= monophosphate shunt. The known intermediary metabolism of carbohydrates
in the sea urchin is shown in figure . The identification of five im portant enzymes catalyzing reactions in the Hmbden-Meyerhof pathway suggests that this pathway is operative in the sea urchin. Even though the sea urchin has been an aquatic animal of choice for many biochemical studies, our evidence for the existence of this important metabolic pathway is far from complete. It would be very desirable to have positive identification of the occurrence of the intermediates of glycolysis in the sea urchine
The identification of two enzymes of the hexosemonophosphate shunt suggests that this shunt is operative in the sea urchin, but mich more information is needed for a definite conclusion.
TRICARBOXYLIC ACID CYCLE AND TERMINAL OXIDATION
The process of glycolysis, which apparently functions in marine animals, usually results in the formation of pyruvate, which is com- pletely oxidized by the series of enzymatic reactions comprising the trie carboxylic acid cycle. In animals, plants, and bacteria that have been more extensively studied, the acetyl coenzyme A, formed by oxidative decarboxylation of pyruvate, enters the TCA cycle by reacting with oxa= lacetate; and upon completion of one cycle, three molecules of carbon dioxide are produced with the regeneration of oxalacetate. Since the TCA cycle establishes a link between the metabolism of carbohydrate and that of lipids and protein and thereby provides a means for their interconversion and complete oxidation for production of energy » it is important that we have knowledge of this metabolic cycle in marine ani-
malse 8
Glycogen t
1 -7 Protein I = | IE ATP (Ribose nucleic acid, etc.) \ 2 at A p Glucose | ee Echt K (Mannose, Fructose, | y ! ATP Galactose) ‘ Glucokinase l (Hexose monophosphate) =a (ADP) mibose <= —_ = ehunt eee eee Sen Rat 0 -_ eo : ~>ATP (CO) | ‘ ATP-ase | \ ! (ADP) ( Phosphopyruvate) AME Pyruvate phosphoferase i ! (Acetaldehyde) — — —— —— — = eptuate) = eas aE a Lacticyacid u Alcohol | \ dehydrogenase (Ethanol) V
(TCA cycle)
Figure 1. Known intermediary metabelism of carbohydrates in teleosts and elasmobranchs.
Glucose = = === = Glucose-1-phosphate
Glucose-6-phosphate Ih ly
J Fructose-6-phosphate —=_=_=_=—— Pentose phosphate IK
oo=
(Fructose-1, 6-diphosphate)
er lad
Triosephosphate
ue)
SI SS --3>g-->
Phosphoglycerate
SIRS SS
Phosphopyruvate
(TCA cycle)
Figure 2. Known intermediary metabolism of carbohydrates in aquatic mammals.
Glucose-1-phosphate = Glucose
= <é, Aa LAENN 1 se ail SN | oe = NEC | | ATP Glucose-6-phosphate <= \\ N \ ADP ] Fructose-6-phosphate 1 Glucose I h J Fructose-1, 6-diphosphate I) Ni i] Triose phosphate | it 1 Phosphoglycerate | A 1 Phosphopyruvate aK ; 1 Vi — — =a Pyruvate — __ Lactate ——==——" —_ — ~~ —= —> (TCA cycle)
Figure 3.
Known intermediary metabolism of carbohydrates in oysters.
Glycogen
im 1} \ | Hexokinase (Glucose-1-phosphate) <i (Glucose) ADP ATP Phosphoglucomutase | Glucose phosphate | dehydrogenase | TPNt TPNH (Glucose-6-phosphate) ee TPNH Hexoseisomerase l (6-Phosphogluconate) Phosphogluconic dehydrogenase | \ TPNt
(Fructose-6-phosphate)
Phosphofructokinase Fructose-1, 6-diphosphatase i (Fructose-1, 6-diphosphate)
Aldolase Dihydroxyacetonephosphate os
ace
(Glyceraldehyde-3-phosphate) &——-—-—~~
Glycogen (1, 3-Diphosphoglycerate) Glucose i Fructose bo Glucose-1-phosphate i} 3-phosphoglycerate) Fructose-6-phosphate bi diti Fructose-1, 6- Enolase i Bee eee fon diphosphate 1 DPN stimulated 5 Phosphoenolpyruvate t ! 5 ; Nii Glucose-6 “phosphate )_>> Pyruvate --_— Lactate Hexosediphosphate /
V (TCA cycle)
Figure 4. Known intermediary metabolism of carbohydrates in the sea urchin.
Because most of the oxygen used by respiring organisms possessing the TCA cycle is for the oxidation of T0A-cycle intermediates, uptake of oxygen may be used as a measure of the activity of this cycle. MNumer— ous investigations have been undertaken to relate overall oxygen con= Sumption of fish tissues to various physiological factors.
Vernberg and Gray (1953), working with 17 species of marine tele~ osts, found a positive correlation between the activity of each species and the oxygen consumption of excised brain tissue. Based on consumption of oxygen, these fish could be arranged into three groups that related to their activity: active species, characterized by constant swimming move= ments; species of intermediate activity; and sluggish, bottom—dwelling species. The brain tissue of more active fishes, such as menhaden, had a higher Qo, than did bottom dwellers such as the toadfish. In addition, these Qo values could be correlated to various physiological indices of activity such as concentration of hemoglobin, level of blood sugar (Hall and Gray 1929 and Gray and Hall 1930) and ratio of gill area to body weight (Gray 1917). Menhaden, for example, has higher concentration of hemoglobin and level of blood sugar and has 10 times more gill area per gram of body weight than has toadfish. Menhaden and other active fish also have a greater number of immature circulating erythrocytes than have less active species (Dawson 1953). A later camparison of Qo, values of brain, liver, and mscle of the toadfish, scup, and menhaden*again showed a definite correlation between activity of the species and consumption of oxygen for brain tissue; however, this relationship did not hold for liver and muscle tissue (Vernberg 195),3. No positive relationship of activity to body size was noted. The oxygen consumption of excised brain of large= mouth bass was found to be less than was that of slices of rat cortex (Fuhrman et al. 19h).
Other workers have investigated consumption of oxygen with respect to acclimatization to temperature (Freeman 1950 and Peiss and Field 1950). Freeman held goldfish at temperatures ranging from h® to 37.59 C. for 1 to 2 weeks, after which time the influence of this thermal acclimatization on the rate of oxygen consumption of excised brain tissue was noted. Uptake of oxygen measured at a given temperature was highest for fish acclima- tized at the lowest temperatures. This finding supports the idea that the brain probably plays a major role in determining the level of re con sumption of fish, as indicated above by the work of Vernberg (195h). ; Peiss and Field demonstrated adaptation to cold by comparing the respira= tory activity of minced or sliced brain and liver preparations of Arctic cod, which lives normally at -1.5° to 2.0° G., to that of golden orfe, which lives at 259 C. Uptake of oxygen,measured over a temperature range of 0° to 25°, increased with temperature. As was found by Freeman (1950) however, respiratory activity showed an inverse relationship to the ene vironmental temperature to which the animal is acclimatized; that is, — uptake of oxygen at any given temperature was greater for the preparations of Arctic cod than for those of golden orfe. Similar results were obtained with the md sucker Gillichthys mirabilis acclimatized to high and low temperature (Wells 1935). In addition, the temperature coefficient of brain and liver of Arctic cod remained essentially constant over the ehe= tire experimental range of temperatures, whereas it increased sharply 2 the orfe tissues from 0° to 109 C, Novikov (1954) found a low activation energy for the dehydrogenation system of minced carp muscle. By meas of the Thundberg technique of methylene blue reduction, he compared the
13
succinic dehydrogenating ability of muscle tissue from carp and frog over a temperature range of 10° to 309 G, The activity of the fish muscle was constant within this range, whereas that of the frog muscle showed an average Q10 of approximately 1.7. It appears that the two factors——a relatively high oxygen consumption and a low temperature coefficient for respiratory enzymes of fish, especially at low temperatures—=are of value in metabolic adaptation to temperature and also indicate an important difference in these enzymes of fish compared to those of warmblooded animals, Such adaptation also has been noted by Steinbach (1919) for ATR» ase activity of cold= and warm-blooded animals,
Of the enzymes of the TCA cycle involved in the uptake of oxygen observed in these experiments, the succinoxidase system probably pre= deminates, owing to its relative stability; however, numerous other enzym atic pathways account for some of this activity. Succinoxidase activity has been observed in homogenized goldfish gill by noting the increase in consumption of oxygen following the addition of succinate. Inhibition studies with mercuric chloride showed this activity to be dependent upon the presence of sulfhydryl groups (Sexton and Russel 1955}. Umemmra (i982 asb, and c) investigated the respiratory enzymes of carp by comparing the dehydrogenase activities of lateral red and white muscle, liver, and kidney homogenates. Using the Thunberg technique and succinate or malate as substrates, he found that red mscle showed five to eight times the activ- ity of ordinary white muscles; the activities of liver and kidney were un= affected by these substrates. Succinic and cytochrome oxidases also demonstrated in these tissues were found to be most active in red mscle. Thus the oxidative and reductive activity of the lateral red muscle of fish far surpasses that of ordinary mscle and resembles that of heart muscle in these respects. Measurements of succinic dehydrogenase and cytochrome oxidase also have been made on a variety of homogenized tissues of the toadfish (Lazarow and Cooperstein 1951). Heart muscle was by far the most active, followed by liver and kidney tissue. Compared with corres= ponding preparations of rat tissues, approximately the same order of activ ity was shown; however, the absolute activity of rat heart was three times that of toadfish hearte
Homogenized eggs of the fish, Oryzias latipes, can oxidize several substrates of the TCA-cycle (Hishida and Nakano Th). The rate of up=
take of en is stimlated by addition of citrate, succinate, malate, pineieste, and pyruvate. ATP was found to have a stimulatory effect on the oxidation of any of these substrates other than succinate. Endo- genous oxygen uptake and succinoxidase activity imcrease as development progressese
The citric acid content of fish muscle decreases after death and is found to accumulate upon addition of pyruvic or oxalacetic acids (Ya= mada and Suzuki 1950). As their work indicated, this is probably not the result of microbial action but of the presence of the condensing enzyme system, Yamada and Suzuki (1951) also reported a malomic acid decomposing enzyme, present in carp-muscle homogenate, that decomposes malonie acid into carbon dioxide and acetic acide
Ui
The respiration of other aquatic animals, including marine inverte= brates, also has been investigated to some extent. Oxygen consumption of Seven species of marine invertebrates, scallops, and crustacea was com pared with regard to their environmental temperature (Fox 1936 and Fox and Wingfield 1937). Measurements of oxygen consumption have also been made on the fiddler crab (Démeusy 1957) and on numerous species of other crustaceans, including the kelp crab (Weymouth et al 19h). By means of staining techniques, using neotetrazolium and tetrazolium, Nayar and Parameswaran (1955) detected succinic dehydrogenase in neurosecretory cells of the thoracic ganglion of the crab.
Humphrey (1947) has studied endogenous respiration and succinoxidase activity of homogenates of oyster muscle. The large increase in respira= tion observed when succinate was added indicates that oyster muscle pos= Sesses an active system for succinate metabolism. The inhibitory effects of certain narcotics on respiration of oyster—mscle homogenate also were studied (Humphrey 19))8).
Jodrey and Wilbur (1955) have demonstrated that a major portion of the TCA cycle is present in the oyster. Specific assays for aconitasey, isocitric dehydrogenase, succinic dehydrogenase, malic dehydrogenase, cytochrome oxidase, and fumarase were made, using homogenates or acetone powders of oyster tissue. With the exception of aconitase, positive re- Sults were obtained for these TCA=-cycle enzymes; however, their activities were considerably lower than in mammalian tissue. Of possible special significance in the process of carbonate deposition is oxalacetate de= carboxylase activity, which was found to be more than 100 times greater in oyster mantel than in mouse liver (Jodrey and Wilbur 1955). The effects of several TCA-cycle acids on the respiration of strips of mantel tissue also were studied. Succinate, malate, and oxalacetate brought about sig= nificant increases in consumption of oxygen; pyruvate had only slight effect; and citrate was without effect. Heavy-metal catalysis in oyster mantel is indicated by strong inhibition of respiration by cyanide; how ever, the inability of methylene blue to reverse this inhibition suggests that the cytochrome system may not be of major importance in respiration of this tissue. Inhibition of oxygen uptake by the metal-complexing agent, diethyldithiocarbamate, indicates the presence of a metal respiratory catalyst, perhaps copper (Jodrey and Wilbur 1955).
Evidence for the occurrence of the TCA cycle in oyster eggs was shown by Cleland (1950); preparations of homogenized oyster eggs could bring about the complete oxidation of pyruvate. Uptake of oxygen was also stimlated by glutamate and various TCA intermediates: succinate, citrate, a-keto-glutarate, fumarate, and malate (Cleland 1951).
Consumption of oxygen by oyster spermatozoa is increased by the ad= dition of certain a-keto acids such as q-ketoglutaric, oxalacetic, pyruvic and isocitric acids and amino acetic acia (Humphrey 1950 a and Humphrey and Jeffrey 195l:). No significant effect on respiration was noted, however, when other TCA-cycle intermediates were used as substrates. To overcome possible problems of permeability, esters of these acids were tried, The
15
ethyl esters of succinate, malate, citrate, and butyrate; glucose penta- acetate; glucosamine; and hippuric acid all increased consumption of oxygene Apparently, esters are able to penetrate the cell more easily. It would appear that the TCA cycle is operative in oyster spermatozoa, since most of the intermediates of the TCA cycle are able to stimlate respiration. Malonate, mainly considered to be an inhibitor of succinate dehydrogenase, was found to cause a significant increase in uptake of oxygen. There is evidence in other animals (Glass 1951) that malonate may serve aS an uncoupling agent for oxidative phosphorylation. If this effect of malonate also occurs with oyster spermatozoa, a reduction in the efficiency of oxidative phosphorylation would account for the increas= ed consumption of oxygen. A small amount of information about the TCA cycle in a few other aquatic animals is also available; this information is summarized below.
The demonstration of the TCA cycle in sea-urchin eggs (Arbacia) has been confined to the study of their ability to oxidize pyruvate, oxalace- tates a-ketoglutarate, and succinate and to the study of the occurrence of oxidative phosphorylation (Krahl 1950).
Goldinger and Barron (19)6) determined the rate of disappearance of preformed pyruvate from intact unfertilized and fertilized eggs of Arbaciae Fertilized eggs were over six times as active as were unferti- Tized eggse Krahl et al. (192) observed a similar pattern for the rate of disappearance of added pyruvate to cytolysates of unfertilized and fertilized eggs except that the breakdown of the egg structure in prepar- ing the cytolysates raised the rate for unfertilized eggs closer to that of the level of fertilized eggs. The fate of pyruvate in these studies was not determined. Changes in succinic dehydrogenase activity of sea- urchin see in relation to the process of fertilization also were studied
(Hida 1957).
Evidence presented by Cleland and Hocbecht tg (2992) So a rence of the enzymes of the TCA cycle in the sea-urchin egg esculentus) aneludess (1) significant stimlation of uptake Of oxygen by an addition of citrate, a-ketoglutarate, succinate, fumarate, malate, and glutamate; (2) inhibition of endogenous uptake of oxygen by malonate and fluoroacetate3; and (3) complete oxidation of added pyruvate. take of oxygen of sea-urchin spermatozoa has been measured (Wicklund 195).
Succinoxidase activity has been demonstrated in mitochondrial pre- parations of lobster leg and claw nerves (Foster 1956)
Baldwin (1938), in a study of the respiratory metabolism of Helix pomaties has ees the hepatopancreas to resemble liver tissue of ver te= raves closely. Succinate, lactate, a-glycerophosphate, and alone ereased the rate of respiration. Succinic acid from the marine snail was
isolated and identified (Ackermann 1955).
tabolic Even though the frog is not strictly an aquatic animal, me
studies made on the frog are of interest here. Hunter and Bante Oi obtained positive assays for succinic, malic, citric Ceo 9 ce a-ketoglutaric dehydrogenases and fumarases A comparison of the ac
16
of respiratory enzymes in tissues of dolphin and rat was made by DuBois
et ale (198), using homogenized liver, kidney, brain, skeletal muscle, and cardiac mscle, Assays for cytochrome oxidase, succinic dehydrogenase, and malic dehydrogenase demonstrated the presence of these enzymes in dolphin tissues, although their activities were found to be lower than were those in corresponding rat tissues. A slower rate of metabolism in dolphin is indicated.
Our knowledge of the tricarboxylic acid cycle in aquatic animals is very incomplete. The presently available information can be summar= ized in two figures. Nigure 5 summarizes the known information on the tricarboxylic acid cycle in teleosts and elasmobranchs. For the tele= osts and elasmobranchs, identification of three important enzymes also suggests that this cycle may be operative. In figure 6 is summarized our present knowledge of the tricarboxylic acid cycle in oysters. For the oyster, there is sufficient information in the form of the identification of four of the important enzymes catalyzing the transformation of inter= mediates of the tricarboxylic acid cycle to suggest that this cycle is operative in these aquatic animals. In the oyster and in the large groups teleosts and elasmobranchs, much more detailed knowledge is required be- fore we will know if they have a tricarboxylic acid cycle that is the same as that of land animals.
ELECTRON TRANSPORT SYSTEMS, COENZYMES AND VITAMINS
The aerobic transport of hydrogen and electrons to oxygen is accom plished by a chain of several carrier systems, beginning with dehy drogen- ases, which act directly on their substrates, followed by flavoproteins, and finally, the cytochromes. Some dehydrogenases are able to catalyze the reaction of hydrogen removed from the substrate with molecular oxy= gen. Other dehydrogenases, however, are linked to oxygen through the cytochrome system. Many require pyridine nucleotides, such as DPN and TPN, as coenzymes, and for others, the nature of the coenzyme is still unknom. Also included in this chain are flavoproteins, some of which are capable of dehydrogenating substrates directly, whereas others trans- port electrons from pyridine nucleotides to the cy tochrome systems These engymes require the prosthetic group, flavin adenine dinucleotides
The probability that the coenzymes of the hydrogen-transport system are in fish is indicated by the results of numerous vitamin analyses of fish tissues, since many of the vitamins have been found to be coenzymes or precursors of coenzymes. Nicotinamide, which is incorporated in the pyridine nucleotides, has been measured in various tissues of fish (Hi- gashi and Hirai 196 and Ghosh et al. 1951). Assays of commercially important marine species for riboflavin, the precursor of flavin ees tides and flavin adenine dinucleotide, have been made by Sautier a ) and by Higashi and Iseki (192 and 198). The distribution of these a5) vitamins in fish also has been reported by Umemra (1951 g)° Stamey ve > Karrick (1955), Joshi et al. (1953), Braekkan et aie (1955 Porn Sis (1956). DPN and flavin have been determined in the eggs of zias ee shida and Nakano 195) and in the eggs of the sea urchin, Arbacia (sr
1950) 17
(Pyruvate)
Condensing enzyme
Malic dehydrogenase
(Malate) \ rN iS ay SS ~ > (Fumarate) &=
Figure 5. Known tricarboxylic acid cycle in teleosts and elasmobranchs.
CO, complete (Malonate) ae CO,+ H,O N decarboxylase NS (Pyruvate) N \ N * ee ae SAV (Oxalacetate) (Citrate) ? i . (isocitrate) (DPNH) (TPN) \" , Isocitric (TPNH) : dehydrogenase Malic | dehydrogenase (c€ - Ketoglutarate)
i
v]
Ve
(Succinate)
~ = O . => (Fumarate) — Succinoxidase
Electron transport
| | | system | "2 ' Cytochrome oxidase H,O
Figure 6. Known tricarboxylic acid cycle in oysters.
The cytochrome system is widespread in nature--occurring in micro=— organisms, higher plants, and animals-—and has been studied a great deal in fish. Comparison of the amounts of cytochrome c in red and white muscle of several species of fish shows red muscle to be a much richer source (Matsuura and Hashimoto 195) and Huys 195). Crystalline cytochrome c from heart muscle of the bonito (Katsuwonus vagans, Lesson) and tunny (Thynnus alalunga, Gmelin) has been prepared (Hagihara et ale 1957 and Matsuura and Hashimoto 1955). Several cytochromes (a, a3 > b, and c) from the octopus (Octopus wulgaris lam.) have been identified (Ghiretti-Magaldi et al. 1957). Paléus (195) compared the amino acid composition of cytochrome c from salmon, beef, and chicken. He found it to be similar for these three cytochromes excenvt for the presence of three histidine groups per mole in beef and chicken cytochrome c and only two per mole in that of salmon. Attempts to isolate cytochrome c from oyster muscle (Humohrey 197) and sea-urchin (Arbacia) eggs (Yoas 1950 and Krahl 1950) have not been successful, indicating that it is present in extremely small amounts, if at all. In contrast to the eggs, sea urchin sperm does contain considerable cytochrome ¢ (Ball and Meyer hof 1910). Ycas (1950) presents spectral evidence for the presence of cytochrome a and b in Arbacia eggs. Other marine invertebrates contain— ing cytochromes are horseshoe crab, squid, and lobster (Ball and Meyer- hof 190). Baldwin (1938) was unable to detect any cytochrome in Helix pomatia by spectroscopic meanse
Terminal oxidative processes occurring in the oyster have been investigated extensively by Humphrey (196 and 1917). A study of in= hibitors showed that the respiration of whole homogenate is strongly inhibited by cyanide, selenite, and arsenate. Azide, arsenite, pyro= phosphate, and iodoacetate also inhibit. Inhibitions by cyanide and azide suggested the presence of cytochrome oxidase, which was confirmed by an assay using ascorbic acid as a reductant. A correlation may be made between the very low concentration of cytochrome found in oyster muscle and the lack of oxygen-transporting pigment in the blood with the low metabolism of the organism. Umemura (1951 b and c) found red muscle of fish to contain greater cytochrome oxidase activity than did liver, kidney, and ordinary muscle. In the toadfish, the concentration of cytochrome oxidase is highest in the heart, kidney, and liver. Al- though this distribution is similar to that found in the rat, the absol- ute activity of cytochrome oxidase in the toadfish is only one-third as much (Lazarow and Cooperstein 1951). During development of Oryzias eggs, cytochrome oxidase activity was found to increase over an 8-day period (Hishida and Nakano 195), An enzyme similar to cytochrome oxidase of other animal tissues is present in Arbacia eggs (Krahl 1950 and Yeas 1950). The presence of cytochrome oxidase has been demonstrated in parti- cles isolated from octopus muscle (Ghiretti-Magaldi et al. 1957), in oyster muscle (Humphrey 197), and in horseshoe crab, lobster, and squid (Ball and Meyerhof 190). Foster (1956) found cytochromeoxidase to be concentrated in mitochondria from lobster leg and claw nerves and from
squid giant fiber nerveSe
20
The vitamin, thiamine, is the precursor of thiamine pyrophosphate or cocarboxylase which is the coenzyme for decarboxylation reactions such as those catalyzed by pyruvic and a-ketoglutaric dehydrogenases. Thiamine assays of fish tissue often have been included in surveys of the vitamin content of fish (Higashi 198, Stansby 1953, Joshi et al. 1953, Hishida and Nakano 195, Braekkan et al. 1955, and Braekkan 1956).
An enzyme found only in fish and invertebrates, thiaminase, splits thiamine into pyrimidine and thiazole moieties (Krampitz and Woolley 19h and Hamoir 1955 b). The distribution of thiaminase among marine inverte= brates and marine vertebrates (Tatarskaya et al. 195) and fresh-water fishes (Chaet and Bishop 1952) has been studied. Sealock and White (199) have made inhibition studies of this enzyme obtained from carp viscera. Deolalkar and Sohonie (195) found the thiaminase in fresh-water fish to be remarkably different from that found in brackish- or salt-water species with respect to activity measured over a wide range of pH. At least two thiaminases are indicated in brackish- and salt-water fish: one with an optima activity in the alkaline range and the other with an optimum active ity in the acid range. Attempts to fractionate these enzymes were un= succesful, although the presence of a dialyzable coenzyme was demonstratede Kaminishi (1951) found thiaminase of shellfish and that of fish to be Similar and the respective apo=- and coenzyme components to be interchange= able. Tatarskaya et al. (1955) have made quantitative estimations of co= thiaminase and have investigated some co-thiaminase-like compounds (Ta= tarskaya 1952); however, the presence or absence of specific thiaminase coenzymes in living cells is not known. A rather complete review of thiaminase has been made by Lee (198) and Yudkin (1949). Harris (1951) has tabulated the occurrence of this enzyme in many fish, mollusks, mussels, clams, lobsters, shrimp, starfish, frogs, toads, and some warm blocded animals and has listed the tissues in which it is found.
Fish possess an enzyme system capable of hydrolyzing folic acid, a vitamin that has coenzymatic function (Jacobsohn and Deodata de Azevedo 1949). The cobalt-containing vitamin, vitamin Bj9, which is required for many reactions of intermediary metabolism, has been reported to occur in shellfish and in red and white fish muscle by Hashimoto et ale (1953). Other workers reporting vitamin Bj in fish tissue include Peeler et al. (1951), Hausmann and Mulli (1952), Karrick (1955), Braekkan et al. (1955)5 and Braekkan (1956). Pantothenic acid, which is a precursor of the im- portant acetyl group carrier, coenzyme A, has been determined in red and white mscle of tunafish by Braekkan et al. (1955).
Reference to these and other vitamins found in fish is given by Stansby (1953) and Browm (1957). A definite coenzymatic role has not as yet been established for many of these vitaminse
Many enzymes are metal containing or metal activated. Iron, for example, is found in the porphyrin moiety of the cytochromes, Magnesium is involved in various kinase systems where phosphate is transferred from ATP to substrate. Some of the common metals directly involved in inter= mediary metabolism—-such as iron, copper, magnesium, potassium, calcium, and cobalt—have been measured in fish and marine invertebrate tissues (Coulson et al. 193), Umemra 1951 c, Kruchakova 1952, Hausmann and Muli 1952, and Stansby 1953). A general discussion of the mineral composition
of fishes may be found in The Physiology of Fishes (Brown 1957). 21
PHOSPHORUS METABOLISM
In considering phosphorus metabolism in fish, attention will be focused on the occurrence of acid-soluble phosphate esters on enzymes such as phosphatases and phosphorylases, and on the process of oxidative phosphorylation. Since phosphorus is of primary importance in carbo= hydrate metabolism and in energy production and storage through the forma= tion of acid-soluble esters, such as hexosephosphate, adenylic acid, and creatine phosphate, it is important to learn if these compounds of phos= phorus occur in fish. Considering the overall intermediary metabolism of energy production in animals, glycolysis yields one-fourth of the energy made available by oxidation of glucose to carbon dioxide and water, and the rest is trapped in the TCA cycle and accompanying terminal oxidative pathways. The only means known by which this energy may be trapped is by phosphorylation reactions resulting in the formation of ATP. Since the Site for nearly all such phosphorylation reactions is along the electron= oxygen transfer system that transfers hydrogen or electrons from substrate to molecular oxygen, it is most important to examine the evidence for the occurrence of oxidative phosphorylation in marine animals. Also, since some esterification of inorganic phosphates can take place on a substrate level, in which case oxygen is not required, evidence for the occurrence of this reaction is also of considerable importance. One example of this type of reaction is found in the TCA cycle, where 1 mole of ADP is ester= ified when 1 mole of succinic acid is formed from succinyl coenzyme A breakdown.
Indirect evidence for the occurrence of oxidative phosphorylation in develcping fish eggs (Oryzias latipes) was reported by Ishida (1951). Inhibition of growth and devatopnenteith a simmltaneous increase of oxygen consumption occurred upon treatment with 2,l)-dinitrophenol (DNP). Since this compound is a powerful decoupling agent of oxidative phospho= rylation in other animals, it may be assumed that phosphoryation is an essential process for the activity of developing fish eggs. Later, oxi- dative phosphorylation was actually measured in homogenized eggs of devel- oping Oryzias latipes. Complete inhibition of phosphorylation but not succinate oxidation with DNP addition was observed (Hishida and Nakano
195h4) 6
Oxidative phosphorylation by a cell-free partimlate system pre= pared from unfertilized eggs of the sea urchin (Arbacia) has been shown to be greatly stimlated by the addition of the TCA=cycle intermediates, a-ketoglutarate, oxalacetate and succinate. Apparently the generation of high-energy phosphate bonds in Arbacia eggs is coupled with the func- tioning of the TCA cycle as is the case in mammalian liver or kidney (Keltch et al. 1950). There is also evidence,however, of a second phos= phorylating mechanism in unfertilized Arbacia eggs in addition to the phosphorylation coupled to the oxidation of TCcA-cycle intermediates (Clowes et al. 1951 and Keltch et al. 1951). The uptake of phosphorus in cell-free homogenates with o-ketoglutarate or pyruvate as substrate is completely suppressed by 10-5M dinitrocresol (DNC). But with hexose diphosphate as a substrate, phosphorylation is 63 percent resistant to DNC addition. Indications are that DNC-resistant phosphorylation is
22
apparently coupled with anaerobic disposal of hexose diphosphate, since phosphate uptake in a nitrogen atmosphere with or without DNC and with hexose diphosphate and pyruvate as snbstrate is approximately equal to that under aerobic conditions in 10-4M DNC. Cleland (1950) reported the formation of high-energy phosphate during respiration of homogenized oyster eggs. Anaerobiosis, cyanide poisoning, or DNP depleted the eggs
of high-energy phosphate. Lobster nerve mitochondrial preparations have been shown to be capable or carrying out oxidative phosphorylation (Foster 1956); P/O ratios in excess of 1.0 were obtained.
Browne et al. (1950) and later Longley (1955), using histochemical methods to study the presence and distribution of alkaline phosphatase in the kidney of various marine teleosts and of other aquatic animals, demonstrated the presence of this enzyme in aglomerular as well as glomer= ular fish. Alkaline phosphatase was believed to function in the kidneys of glomerular vertebrates in the process of glucose resorption; however, its presence in aglomerular fish constitutes evidence against this theorye
Phosphorylase, which catalyzes the reaction: glucose=l-phosphate > glucose # HOPO3=, has been obtained in extracts of lobster tail muscle (Cowgill and Cori 1955). An inactive form of the enzyme was found that on incubation, changed to phosphorylase b(inactive in the absence of adenosine monophosphate) and then to phosyhorylase a.
Two enzymes of phosphorus metabolism found in oyster eggs are acid phosphatase and apyrase (Cleland 1951). Compositional studies on these eggs showed the presence of fructose phosphates, phosphoglyceric acid, adenylic acid, and ATP (Cleland 1950). Suzuki (195) compared the phos- phorus compounds of red and white mscle of fish. Although red mscle contained more total phosphorus than did white, white mscle had a higher concentration of acid-soluble and inorganic phosphoruse
The possibility of assimilation of inorganic phosphorus from water by fish was investigated by Shekhanova (1956). Carp was found to take up HP320)= with activity being located mainly in the gills and scales. Some P32 was found in all other organs including blood and digestive tracte The various tissues of the carp, arranged in descending order according to rate of uptake of P32 are gills, fins, heart, liver, bladder, verte~ brae, and miscle (Kirpichnikov et al. 1956).
PROTEIN AND NITROGEN METABOLISM
The metabolism of proteins includes a vast number of diversified processes giving rise to a large array of nitrogenous substancese Cellu= lar structure and organization are primarily dependent upon proteins as is the regulation of chemical activity of the cell by proteins in the form of enzymes. The overall scheme may be summarized as follows? an amino acid pool is formed through the breakdown of food proteins, tissue cata= bolism, and the synthesis of nonessential amino acids. From this pool,
23
new protein is synthesized for growth and repair as also are essential non=protein substances such as coenzymes, hormones, creatine, and choline --to mention a few. Excess amino acids in this pool are deaminated; some, the glucogenic amino acids, are converted to glucose, while others, the ketogenic amino acids, give rise to ketones. The end products of many are yet unknown. Thus in addition to synthetic functions, amino acids may serve as a source of energy. Hutchens et al. (192) found that certain samples of eggs of the sea urchin, Arbacia, consumed little or no carbohydrate in early stages of development, during which cell division proceeds most rapidly. Processes involving the oxidation of protein or protein breakdown products could account for the observed uptake of oxy= gene Indeed, in some cases, ammonia production agreed with oxygen con— sumed for complete oxidation of protein. Ammonia formed from the process of deamination is eliminated either unchanged or in less toxic forms as urea or uric acide Protein metabolism, therefore, is essentially the metabolism of amino acids and includes the pathways connecting their interconversion and those leading to the synthesis of other nitrogenous compounds, including nitrogenous waste products excreted by the animal.
Detailed information of protein metabolism, as outlined above, is almost nonexistent for fish, being limited almost completely to warm= blooded animals and to microorganisms. By means of compositional studies, however, possible relationships of amino acids to other metabolites found in fish may be initially formulated, using information known for other animals. Geiger (198) has reported the protein content of the muscle of many species of fish and has compared the various fractions to mammal— ian mscle. Included are proteins of fish blood, collagens, and proteins isolated from fish sperm and from the female reproductive organs of fishe Analyses of fish protein by many workers have shown over 20 of the common amino acids to be present (Baertich and Weber 197, Geiger 1948, Dunn et ale 199, Kakimoto et al. 1953, Goncalves 1952, Master and Magar 195h, Sugimura et al. 195), and Eastoe 1957). Compared to beef muscle, fish muscle has higher amount of glutamic acid (Jarpa 1950). The nature and function of the red muscle of fish have been given much considerations Although it resembles heart muscle in certain respects, it has essen- tially the same amino acid composition as has white miscle (Matsuura et ale 1955). Studies of the extractive nitrogen of certain shellfish and squid muscle have shown generally higher amounts of amino nitrogen to be present than are found in vertebrate muscle (Simidu et al. 1953
and Endo et al. 195h)6
i-amino acid oxidase, which catalyzes the oxidation of amino acids to the corresponding a-keto acids and ammonia, occurs in marine inverte= brates (Roche et al. 1952). Differing from the I-amino acid oxidase found in many vertebrates, however, this enzyme in invertebrates is capable of catalyzing the oxidation of basic amino acids, especially arginine, Tissue extracts of hepatopancreas and digestive tract of annelids, gephyriens, mollusks, crustaceans, and echinoderms were able to oxidize I-histidine, Ieleucine, I-tryptophane, L-ornithine, and I-citrulline in addition to
2h
arginine, Arginine metabolism occurs by a process different from that common to vertebrates in that o-keto- §=guanidovaleric and y-guanidobuty= ric acids are formed. Glycine, Dieserine, I=-glutamic, l-aspartic, and i-proline failed to be metabolized by these extracts. Umemura (1951 b
and ¢) determined amino acid oxidase in various tissue homogenates of carpe The activity of red muscle was generally lower than was that of liver and kidney; white muscle showed little or no activity. Of special significance was the oxidation of glutamic acid and alanine by red muscle, liver, and kidney tissue, since the resulting keto compounds, a-ketoglutaric and pyruvic acids, are able to enter into the TCA cycle for production of energy or for synthesis of fatty acid and of carbohydrate. Thus a link
is established between these two important intermediary pathways and pro- tein metabolism in fish.
In the areas of nitrogenous excretion and nitrogenous composition of tissues, the comparative aspects of protein metabolism for fish are best known. Three substances characterize most of the nitrogenous ex cretion of fish: ammonia, urea, and trimethylamine oxide (TMA oxide). For both marine and fresh-water teleosts and for aquatic invertebrates, the predominant end product of protein metabolism is ammonia; for elasmo— branchs the predominant end product is urea (Baldwin 1952). Suyama and Tokuhiro (195) a and b) determined the distribution of urea in various tissues of sharks and rays. For the general distribution of urea in fish and shellfish, refer to Shewan (1951). Although ammonia, which is con- stantly formed during deamination processes, is toxic and must be elimin= ated rapidly, a provision for the storage of it for future use in synthesis of amino acids is found in the formation of the amides of glutamic and aspartic acids--glutamine and asparagine, respectively. The presence of asparaginase, reported to exist in liver and other tissues of fish (Ter= reine 193), indicates the possibility of such a store for amino groups
in asparagines
Although arginase, a key enzyme in the ornithine cycle, is found in fish liver, evidence indicates that this elaborate mechanism for produc— tion of urea from ammonia does not exist in teleosts (Baldwin 1952). The addition of ornithine and ammonia and a source of energy, such as lactate, to slices of fish liver causes no production of urea in teleosts, though it does in animals possessing the ornithine cycle. Since arginine occurs in considerable amounts in most proteins, the presence of arginase can account for the production of urea in fish. Matsuura et al. (1953) found that white muscle of various kinds of fish contained little or no arginasee It was present, however, in red muscle. Similar studies with elasmo= branch tissues (Connell 1955) showed arginase activity to be absent in the skeletal muscle of the common skate, thornback skate, and cuckoo ray but not in the white skeletal and red lateral band muscle of dogfish (Scyliorhinus caniculus).The dogfish (Squalus suckleyi) has also been re- por © contain arginase in voluntary muscle er and Dauphinee 192k). Apparently, for rays, extrahepatic regulation of urea cannot occur in the muscle as it does in other elasmobranchs (Cannell 1955).
Urease, commonly found in plant and invertebrate tissue (Baldwin 1952), has also been detected in the blood and mscle of shark (Ferguson= Wood 1950). Part of the ammonia excreted by fish may arise from action of this enzyme on urea. Urease activity of the sea urchin was followed
25
during development from eggs to adults by Brookbank and Whiteley (195)). ene oxidase, uricase, allantoinase, and allantoicase also were found € 688Se
Much speculation exists concerning the origin and significance of TMA oxide in fish muscle and blood. Formerly believed to be present only in marine fish (Kutscher and Ackermann 1933 and Baldwin 1952), it has been shown to occur in si icant but smaller amounts in fresh-water species (Cook 1931, Reay 1938, and Lintzel et al. 1939). Reay found that elasmo- branchs contain more TMA oxide than do teleosts. Suyama and Tokuhiro (1954 b) also studied the distribution of TMA oxide in elasmobranchs,
In contrast to the blood of elasmobranchs, the blood of marine tele= osts has no appreciable TMA oxide (Norris and Benoit 195). Norris and Benoit also reported the oxide to be of general occurrence in marine Grus= acea,which includes the lobster (Reay 1938). For a more detailed comparison between various fresh= and salt-water fish, see the review on nitrogenous extractives in fish by Shewan (1951). As was pointed out by Shewan, en- vironment, seasonal variation, size, and age all can affect the TMA oxide content. His work shows that different mscles also have different TMA oxide content; the tissue forming the dark lateral line of both herring and tunny have about half the content of the rest of the skeletal msclee Similar differences also were found between the white and red muscles of fish by Endo and Shimidu (1955). The content of TMA and TMA oxide of numerous species of fish occurring near Bombay has been reported by Joshi et al. (1953) TMA oxide has been found in the skin of the octopus (As- ano and Sato 195), but in shellfish muscles, the amounts are relatively low (Simidu et al. 1953).
TMA oxide may be the end product of protein metabolism, but enzymes capable of oxidizing TMA in TMA oxide, such as occur to mammals, are ap= parently lacking (Shewan 1951). The difference in TMA oxide content be- tween marine and fresh-water species is reflected in other organisms used as food by these animals. Fresh-water sooplankton contain a very low concentration of the oxide, whereas marine plankton have levels comparable to other marine species (Reay 1938). Feeding experiments carried out with young salmon (Benoit and Norris 1945) indicate that the origin of TMA oxide is exogenous in this species. When diets free from TMA axide were fed, little or no TMA oxide was found, but some retention occurred when the oxide was included in the diet. TMA oxide reductase activity in the dark mscle of albacore and frigate mackerel has been demonstrated by the reduction of aseptically added TMA oxide (Kawabata 1953} Immediately after catch, large amounts. of free trimethylamine were found in the dark muscle; these amounts increased on incubation. No increase in free tri- methylamine occurred in white muscle, nor was there any reduction of added TMA oxide. The reaction was found to be sensitive to both cyanide and heat. Nickerson et al. (1950) found only slight increases in TMA content of sterilized fish ea ereeg raga Mee! tot en ee naturally occurring TMA oxide reductasee Since elasmobrar - pecially high concentrations of TMA oxide in all body fluids and tissueSy, the base probably serves as an osmoregulatory factor (Reay 1938 and eer 1951). TMA oxide therefore may not be entirely of exogenous origin an
26
may play an important role in ammonia detoxification and transmethylation reactions (Nikkila 1951 and Suyama and Tokuhiro 195) b).
Both non-protein arginine and creatine may serve as phosphate accept= ors; these amino acids are found in muscle tissue mainly in the form of phosphate esters (phosphagens). Just as arginine phosphate appears to be limited to invertebrates, only a trace of creatine being found in Crustacea and other shellfish (Kutscher and Ackermann 1933), creatine phosphate is limited to the vertebrates. In loach mscle and eggs, creatine was the major nitrogenous compound extracted (Marushima 19h)
The synthesis of creatine in mammals is believed to be associated with arginine from which an amidine group is furnished. This association is shown by the work of Bloch and Schoenheimer (19))1), who fed rats argi- nine labelled isotopically in the amidine nitrogen and found a high con= centration of labelled nitrogen in the amidine group of creatine. Whether or not this amidine transfer occurs in fish is not known.
Other guanidine compounds found in fish mscle--such as octopine, methyl guanidine, arcaine, agmatine, and glycocyamine (Kutscher and Acker= mann 1933 and Kakimoto 195):)—are of unknown significance. Bacterial action may be responsible for the presence of some of these products measured in fish tissue. Agmatine, for example, can be formed by an arginine decarbo- xylase of bacterial origin (Baldwin 1952).
Two widely distributed imidazole bases in vertebrates, anserine and carnosine, have been found in the mscle tissue of numerous fish (Reay et ale 193, Marushima 19), and Shewan 1951). Yudaev (1950), however, found neither carnosine nor anserine in fresh-water species, although histidine Was present in considerable amounts. Since the fish containing histidine had no fB-—alanine, histidine did not arise from carnosine or anserine in
this casée
Jones (195) and 1955) has reported an enzyme, anserinase, occurring in codling muscle, that is capable of splitting anserine into f-alanine and l-methylhistidine, Analyses of extracts of codling muscle showed that before spoilage had occurred, l-methylhistidine and f-alamine in= creased, The same results were obtained with sterile homogenates and cell- free aqueous mscle extracts with added anserine. ILater, a partially purified anserinase was obtained by dialysis and acetone fractionation of celi-free mscle pape it underwent complete heat inactivation and apparently was Zn’7 activated.
Various betaines have been found in fish muscle; however, little is know of their formation or function (Shewan 1951). It is possible that they take part in biological methylations. Since betaines are wide— spread among plants, many of them may originate directly from the food supply of an animal,
Ackermann (1955) has identified by means of paper chromatography various nitrogenous compounds such as taurocyamine, homarine, choline, large amounts of lysine, and other amino acids in the marine snail.
27
The enzyme cholinesterase, which is closely associated with the propagation of brain and nerve impulses, has been measured in homogenates of a central nervous system and other organs of fish (Laurent 1952 a and D)e
LIPID METABOLISM
Consideration of the lipid metabolism of aquatic animals must in- clude the metabolism of triglycerides, fatty acids derived from the tri- glycerides, the compound lipids such as phospholipids, and other lipid- soluble compounds such as hydrocarbons and sterols. Our present knowledge of lipid metabolism in animals is that free fatty acids are first con= verted into acyl coenzyme A derivatives, which then are rapidly degraded, two carbon atoms at a time, into acetyl CoA. Acetyl CoA enters the meta- bolic pool, and it may be utilized in a large number of synthetic reactions, The metabolism of carbohydrates and certain amino acids also may contribute to the metabolic pool of acetyl CoA. Since each step in the oxidation of fatty acids is reversible, their synthesis may proceed by the condensation of two carbon units. In general, our knowledge of lipid metabolism is still far from complete. This lack of knowledge is especially true for fish and other aquatic animals. There is essentially no information avail- able on specific intermediary pathways of lipid synthesis and oxidation in aquatic animals. Studies to date on these animals have been chiefly concerned with compositional analyses and selective modifications of dietary fat. A review of this information on fish has been presented by
Lovern ( 1951) ®
The fatty acids of fish, like those of land animals, are all straight chain, are monocarboxylic, and contain an even number of carbon atoms, Al-= though the entire series is not present in any given species, a wide range of homologous fatty acids containing 12 to 28 carbon atoms are found in fish fats, whereas the majority of fatty acids of land animals and plants contain 16 te 18 carbon atoms. The maximm possible degree of unsatur- ation for any given fatty acid increases with chain length. Mixtures of 22 carbon atoms, for example, have been found containing one, five, and Six double bonds (Lovern 19/,2) and two, three, and four double bonds (Bau dart 19)8).
Cod=liver oil has a composition that may be regarded as being typical or average for marine species of fish (Lovern 1942). It is approximately 8h percent unsaturated, the unsaturation occurring mainly in fatty acids containing 18 and 20 carbon atoms and in some containing 16 and 22 carbon atoms. The saturated portion is composed primarily of palmitic, with smaller amounts of myristie and stearic acids. Since triglycerides in fish tend to be completely heterogenous, and since a large number of individ=- ual fatty acids are present—for example,about 20 in cod-liver oil (Ban= dart 1948)--the triglyceride composition of fish fat is extraordinarily complex (Harper and Hilditch 1937 and Hilditch and Terleski 1937). No compositional investigation that is based on content of glyceride is prac- tical with present analytical methods.
28
In addition to triglycerides, fatty acids esterified with such com pounds as chimyl, batyl, and selachyl alcohols are found in shark-liver oils. These alcohols are ether esters formed from higher normal fatty alcohols with one of the e¢-hydroxyl groups of glycerol (Lovem1951).
} Baldwin and Parks (193) have reported on the low-temperature crys= talization of glycerides and the occurrence of some of the 18= and 20= carbon-atom acids in menhaden oil. The separation and characterization of several highly unsaturated fatty acids in sardine oil have been de= scribed by Toyama and Tsuchiya (1953 a, b, c, d and e) and Tutiya (190). Toyama and Shimooka (1953 b and 195) have made ultraviolet absorption Measurements of conjugated acids obtained by alkali isomerization of highly unsaturated acids from sardine oil.
Many other investigations have been made of the composition of fats in fish. Pathak and Ojha (1957) have compared the fat of two species of indian fresh-water fish to that of some British fresh-water fish and of the herring with regard to component fatty acids. A comparison was also made of the lipids of an Arctic fish to those of the guppy (Wilber 199). The naturally occurring eicosapentaenoic acids have been shown consider=- able interest. Their presence has been reported by Toyama and Tsuchiya (1935 ¢ and e), Tutdya (190), and Toyama and Shimooka (1953 a) in sardine oil, Matsuda (192) in bonito oil, Baldwin and Parks (193) in menhaden oil, Tsuchiya and Kato (1950) in herring oil, Tsuchiya and Okubo (1952) in fish—-liver oil, and Abu-Nasr and Holman (195)) in cod-liver oil. Whit= cutt and Sutton (1956) have isolated and determined the structure of an eicosapentaenoic acid from South Africa pilchard oil. These acids are not restricted to lipids of fish, however, as their occurrence also has been reported in ox-liver lipid (Klenk and Dreike 1955), ox-adrenal lipid (Herb et al. 1951), and butter fat (Shorland and Johannesson 1951).
From the many studies of composition, it is apparent that all fats of aquatic animals possess an exceptional variety of fatty acids, mainly unsaturated. The question arises as to what extent the complex mixture of fatty acids found in most fish is due to the ingestion of fat of that type or to modifications of ingested fat by the fish itself. Fresh= Water animals possess one broad type of fat, which distinctly differs from that of marine species (Hilditch and Lovern 1936 and Lovern 1937 and 1942). The difference between these two types of fats in fish is shown mainly in the frequency of distribution of the unsaturated fatty acids; that is, there is a shift from relatively greater amounts of the un- saturated acids of shorter chain length of 16 and 18 carbon atoms in fresh-water species to larger amounts of the longer chain acids of 20 and 22 carbon atoms in marine fish. The total amount of 16-carbon- atom acids is also found to be less in marine fish. This same pattern in composition of fat is found in the fat of marine and of fresh-water plankton crustaceans (Lovern 1935), which are the main food link between the aquatic plant and animal kingdoms. It thus would seem that the na- ture of the fat of fish is due almost exclusively to the ingestion of fat of the same type. Further supporting evidence for this view are the re- sults of feeding experiments with the fresh-water eel (Lovern 1938).
On a typically high-fat marine diet (herring), there was appreciable modification of eel fat in the direction of the dietary fate
29
In addition to dietary effects on the composition of the fat of fish, Lovern (1951) has noted specific modifications of deposited fat by small groups of closely related species. This situation is not unlike that found in land animals in which the nature of the dietary fat is super- imposed upon a characteristic fat deposited by the species. Two types of changes may occur before deposition: (1) selective hydrogenation or dehydrogenation and (2) alteration of the chain length of one or more fatty acids by shortening or lengthening processese
A physiologically reversible process of saturation and desaturation of fats is known to occur in mammals, as demonstrated by feeding experi= ments with mice in which saturated and unsaturated fatty acids containing deuterium were used (Rittenberg and Schoenheimer 1937 and Schoenheimer and Rittenberg 1936). For fish, a detailed fatty acid analysis of herring made over periods of fasting, feeding, and spawning was compared to the fat of Calanus finmarchicus, the principal organism in the herring diet (Lovern 1951). By following changes in the degree of unsaturation of any individual fatty acid it was observed, for the most part, that the com- position of herring fat tended to approach that of Calanus finmarchicus during the period of active feeding but gradually returned to the com position characteristic of the fasting state. The results of these stud- ies indicate that fish probably possess enzyme systems capable of selective hydrogenation or dehydrogenation of different groups of fatty acids simi- lar to those systems found in mammals,
Evidence of another specific modification of ingested fat by fish and by other aquatic animals-—-that of shortening or lengthening processes affecting fatty acids—-also is based on studies of composition (Lovern 1951). Since the fats of most marine fish are made up of roughly equal amounts of 16-, 18-, and 20-carbon=atom acids, decreased amounts of l)- and 22=carbon-atom acids, and little or no 2)j-carbon—atom acids, it is possible to define an "average" composition. Large deviations from this “Naverage" distribution of fatty acids appear to be an indication of a progressive shifting or alteration of some fatty acids, two carbon atoms at a time, into others of different length. Examples of such transforma= tions are found in the toothed whale (Lovern 192), in which chain short= ening occurs, and in the liver fats of sharks and related fishes, in which chain lengthening occurs. Another example is found in the liver fatty acids of the catfish (Anarrhicas lupus), which bear evidence of simultaneous chain lengthening and chain shortening (Lovern 1937)
Ingested fats are absorbed either directly in a finely divided e= mulsified form or as the free fatty acids after hydrolysis by lipasese The effect of lipolysis on absorption of fat in fish is not known; hovw= ever, fish do possess very active lipases in their gastro-intestinal : tract. As was shown by Lovern and Morton (1939), rapid post-mortem build= up of free fatty acids from lipids in the intestinal tract of halibut occurs, the content of free fatty acid rising to over 20 percent ina period of 5 to 10 minutes.
lipases are found rather widespread throughout other tissues and organs of fish, as in other forms of life. Falk et al. (1927) and Noyes et al. (1927) obtained extracts of whole trout and trout eggs that possess
30
lipase action toward various simple esters such as phenylacetate. ¢ triacetate, ethyl acetate, and others. Lipase activity als ee ee a in the spleen, heart, kidney, testes and liver of carp (Kernot and Hills 1932). Schmidt-Nielsen and Stene (1938) found "lipase activity" ins almon muscle that was absent in boiled muscle but that could be restored upon treatment with sodium sulfate. It generally is held that before oxidation of fats can occur, the fat must be hydrolized to free fatty acids. Thus these lipases probably perform an essential operation preliminary to the oxidation of fats,
The phospholipids of several aquatic animals have been investigated: the lecithins, cephalins, and phosphatidic acids of the heart of a ror— qual (Balaenoptera borealis); the brain cephalins of the sperm whale (Phys= ter catodon) and of pollock (Theragra chalcogramma); the egg lecithins and cephalins of pollock (Theragra chalcogramma)3; and the cephalins, leci- thins and phosphatidic acids of unfertilized and fertilized eggs of crab (Paralithodes camtschatica) (Igarashi et al. 1956, Zama and Igarashi 1956,
ahd Zama and Katada 1956 and 1957).
Recent compositional studies by Lovern's group have included the fractionation of the various classes of lipids extracted from cod flesh (Garcia et al. 1956). Analysis of the various fractions showed thef ollow= ing lipids and amounts to be presents lecithin 35 percent, waxes and alcohols 13 percent, free cholesterol 8 percent, phosphatidyl ethanol= amine 7 percent, free fatty acids 6 percent, cholesterol esters 5 percent, triglycerides 3 percent, inositol lipids 2 percent, and unidentified lipids 21 percent. Similar results were found for the lipids of haddock tissue (Olley and Lovern 195):). Triglycerides, hydrocarbons, c holesterol esters, free alcohols, and esterified alcohols have been separated by means of silica-gel chromatography from extracts of haddock flesh (Lovern 1956 a). Determination of the fatty acid components of the cholesterol ester, tri- glyceride, and total lecithin fractions showed them to be characteric of each class of lipids.
The phospholipid fractions from haddock and from cod flesh have been investigated in more detail by Olley (1956) and Lovern (1956 b). Studies of the distribution of lecithin in sardine revealed the brain to be the richest source, followed by heart mscle (Matsumato 1950). Red mscle in the sardine was found to contain more than twice the amount of lecithin present in white mscle.
The sterols, cholic acid and cholesterol, have been identified in the bile of the Spanish tunny-fish (Castells 1953). A new sterol, isolated by chromatographic techniques and comprising about 50 percent of the sterols found in the oyster and in the clam, has been identified to be 2h-methylene-cholesterol (Idler and Fagerlund 1955). Cleland (1950) has determined the contents of phospholipid and neutral fat in unferti- lized oyster eggs and has found lipase to be present (1951).
NEED FOR RESEARCH As this review shows, there is little knowledge of intermediary
metabolism of aquatic animals. This lack of information is very undesir= able in studies in the scientific disciplines of biochemistry, biology ys
31
and nutrition. Especially for comparative purposes, a thorough knowledge of the intermediary metabolism of aquatic animals would be invaluable. There would be great value in the application of this knowledge to better commercial use of these marine resources.
Research study of the intermediary metabolism of aquatic animals is a fertile field for further seientitic effort. Almost none of the research reviewed herein has been directed toward the objective of de- termining the pathways of intermediary metabolism, and the small amount of information that is available applies to this objective only super= ficiallye Future studies should be directed to two broad objectives: Firstly, extensive research will be required to map out the overall path- ways of intermediary metabolism in aquatic animals, especially the fish. Secondly, intensive research will be required to determine the exact mech— anism of each metabolic transformation.
We believe that the overall need for future research may be outlined as follows:
1. Enzymes know to occur in pathways of general intermediary meta=— bolism should be identified and their activities measured. The identifica— ‘tion of a particular enzyme in the homogenates of tissue of the aquatic animal under study will require specific assays in which the reaction sys=- tem consists of known chemical compounds. It will be preferable to have the enzymes either fractionated from the major portion of the protein and interfering enzymes or purified until all extraneous material has been removed,
2e The intermediate compounds of metabolism should be studied in such a way as to show the sequence in which they are formed. Several ways of carrying out this study are known. Radiolabeled reaction com ponents may be added to the whole aquatic animal, to a given tissue, or to a tissue preparation; and after reaction, the products may be separ= ated on filter paper and an autoradiograph developed (Bassham et al. 1953)e The sequential transformation of metabolites also may be established by adding a reactant to the tissue or tissue preparation of the aquatic ani= mal and then separating the products by filter-paper chromatography to determine the next member in the metabolic sequence (Avron and Biale 1957).
3. After enzymes and the metabolic intermediates have been identi=- fied, the pathways of intermediary metabolism should be checked at the whole=animal level, using radioactive or mass isotope tracers. This re= search can best be patterned after that which has already been done with land animals, plants, and bacteriae The amount of carbohydrate metabolized by the Enbden=Meyerhof pathway and the amount metabolized by the hexose= monophosphate shunt, for example, can be determined zie studies of forma= tion of GOs following glucose-1-C™™* and glucose=6=C-+ injection into fishe
le As our knowledge of the pathways of intermediary metabolism de- velops, attempts should be made to integrate this knowledge with that of the nutrition and physiology of aquatic animals. There are a variety of important questions in the nutrition and physiology of aquatic animals that should be answered at the level of intermediary metabolism. How, for example, are the characteristic highly unsaturated fatty acids of fish
32
metabolized for energy conversion and to intermediates for synthetic re- actions? Does the "red mscle" of fish function primarily as a mscle, or does it have some properties in common with liver and other organs (Braekkan 1956)?
Experience in the past has shown that sound fundamental research of this kind opens wide fields to applied research, which then leads to new industrial applications or aids in the solution of long-standing industrial problems.
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Ackermann, De : 1955. The action of homarine, taurocyamine, choline, lysine and other amino acids as well as succinic acid in marine snail. Z. Physiol.
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Asano, M., and H. Sato : 195). Biochemical studies on octopus. I. Trimethylamine and tri- methylamine oxide contents of octopus. Tohoku J. Agr. Research 5, 191-195. Biol. Ab. 31, 389k.
Avron, M., and J. B, Biale 1957. Metabolic processes in cytoplasmic particles of the avocado fruit. III. The operation of the tricarboxylic acid cycle. Plant Physiol. 32, 100-105.
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Baldwin, E, 1938. On the respiratory metabolism of Helix pomatia. Biochem. Je 32, 1225-12376
Baldwin, E. 1952. Dynamic aspects of biochemistry. Second Kd., Cambridge Univ.
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Baldwin, We Hey and L. Ee. Parks 1943. The body oil from menhaden (Brevoortia annus) A. Low tempera= ture crystallization of glycerides. B. some o e 18 and 20 carbon atom acids. Oil and Soap 20, 101-10h.
Ball, Ee G,.and Ba Je Meyerhof 1940. On the occurrence of iron=porphyrin compounds and succinic de- hydrogenase in marine organisms possessing the copper blood pigment hemocyanin, J. Biol. Chem, 134, 183-L9h.
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Ballentine, Re
1940. Analysis of the changes in respiratory activity accompanying the Aa meme of marine eggs. J. Cellular Com. Physiol. 1s a md e
Bassham, Je Aey Ae Ae Benson, and M. Calvin 1953. Isotope studies in photosynthesis. J. Chem. Educ. 305 274-2830
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