1. INTRODUCTION
2. CLASSIFICATION AND CHEMISTRY
3. CARBOHYDRATE METABOLISM IN FISH
4. REFERENCES
K. W. Chow
Food and Agriculture Organization
Rome, ItalyJ. E. Halver
University of Washington
Seattle, Washington
1/ Lecture was presented by J. E. Halver
Carbohydrates represent a broad group of substances which include the sugars, starches, gums and celluloses. The common attributes of carbohydrates are that they contain only the elements carbon, hydrogen and oxygen, and that their combustion will yield carbon dioxide plus one or more molecules of Water.
The simplest carbohydrates are the three-carbon sugars which figure importantly in intermediary metabolism and the most complex are the naturally occurring polysaccharides, primarily of plant, origin. In the diet of animals and fish, two classes of polysaccharides are significant:
(a) structural polysaccharides which are digestible by herbivorous species -cellulose, lignin, dextrans, mannans, inulin, pentosans, pectic acids, algic acids, agar and chitin; and(b) universally digestible polysaccharides - principally starch.
Carbohydrates make up three-fourths of the biomass of plants but are present only in small quantities in the animal body as glycogen, sugars and their derivatives. Glycogen is often referred to as animal starch because it is not present in plants. Derived mono-saccharides such as the sugar acids, amino sugars and the deoxysugars are constituents of all living organisms.
2.1 Pentoses
2.2 Hexoses
2.3 Disaccharides
2.4 Oligosaccharides
2.5 Polysaccharides
Carbohydrates are classified generally according to their degree of complexity. Hence, the free sugars such as glucose and fructose are termed monosaccharides; sucrose and maltose, disaccharides; and the starches and celluloses, polysaccharides. Carbohydrates of short chain lengths such as raffinose, stachyose and verbascose, which are three, four and five sugar polymers respectively, are classified as oligosaccharides.
Pentoses are five-carbon sugars seldom found in the free state in nature. In plants they occur in polymeric forms and are collectively known as pentosans. Thus, xylose and arabinose are the constituents of pentosans present in plant fibres and vegetable gums, respectively. As the sugar moieties in nucleic acids and riboflavin, ribose and deoxyribose are indispensable constituents of the life process. D-ribose has the following chemical structure:
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D-Ribose |
The hexoses comprise a large group of sugars. Principal among these are: glucose, fructose, galactose and mannose. While glucose and fructose are found free in nature, galactose and mannose occur only in combined form. The hexoses are divided into aldoses and ketoses according to whether they possess aldehydic or ketonic groups. Thus, glucose is an aldo sugar and fructose is a keto sugar. The presence of aymmetric centres in all sugars with three or more carbon atoms gives rise to stereoisomers. Galactose and mannose are stereoisomers of glucose which, theoretically, is only one of 16 stereoisomers. Because the ketohexoses have only three asymmetric centres, fructose is one of eight stereoisomers. The chemical configurations of the four hexoses mentioned are as follows:
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D-Glucose |
D-Galactose |
D-Mannose |
D-Fructose |
A general phenomenon, known as mutarotation, is observed in a variety of pentoses and hexoses as well as in certain disaccharides. For example, it has been established that two isomers of D-glucose exist, hence requiring an additional asymmetric centre in this sugar. It became apparent that D-glucose and most other sugars have cyclic structures. The position of the hydroxyl group in relation to the ring oxygen characterizes this additional configurations modification. By convention, the positioning of the hydroxyl group on carbon atom 1 on the same side of the structure as the oxygen ring describes a -modification; and, the positioning of the same hydroxyl group on the opposite side of the ring oxygen describes a b -modification.
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a -D-Glucose |
b -D-Glucose |
Carbohydrases, which catalyse the hydrolysis of glycosidic linkages of simple glycosides, oligosaccharides and polysaccharides often exhibit specificity with regard to substrate configuration. As we shall see later, the specificity for enzyme hydrolysis of certain oligosaccharides helps to explain the poor utilization of this class of carbohydrates in fish nutrition.
Sugars containing the aldo or the keto group are capable of reducing copper in alkaline solutions (Fehling's solution) to produce the brick-red colouration of cuprous ions. These sugars are called reducing sugars and the reaction, although not specific for reducing sugars, has use for both qualitative and quantitative determinations.
Glucose is widely distributed in small amounts in fruits, plant juices and honey. It is commercially produced by the acid or enzyme hydrolysis of grain and root starches. Glucose is of special interest in nutrition because it is the end-product of carbohydrate digestion in all non-ruminant animals including fish.
Fructose is the only important ketohexose and is found in the free state alongside glucose in ripening fruits and honey. Combined with glucose it forms sucrose. Fructose is somewhat sweeter than sucrose and is produced in increasing quantities commercially as a sweetener.
Galactose occurs in milk in combination with glucose. It is also present in oligo-saccharides of plant origin, in combination with both glucose and fructose.
Mannose is present in some plant polysaccharides collectively termed mannans.
Disaccharides are condensation products of two molecules of monosaccharides. Sucrose is the predominant disaccharide occurring in the free form and is the principal substance of sugar cane and sugar beet. It is also formed during germination of legume seeds. Other common disaccharides are maltose and lactose. Maltose is a dimer of glucose, and lactose is a copolymer of galactose and glucose. The two molecules of glucose in maltose are held together in an a -1,4 glycosidic linkage whereas the two hexose entities of galactose are linked at the b -1,4 position. Glucose and fructose are combined in an a -1,2 linkage in sucrose. The abbreviated name of sucrose is D-Glu-(a, 1® 2)-D-Fru.
a -Maltose
b -Lactose
Sucrose
The Oligosaccharides raffinose, stachyose and verbascose are present in significant quantities in legume seeds. Raffinose, which is the most widespread among the three, consists of one molecule of glucose linked to a molecule of sucrose at the a -1,6 position. Its abbreviated chemical name is a -D-Gal (1® 6) -a - D -Glu - (1® 2) - b -D-Fru. Further chain elongation at the galactose end with another galactose molecule will yield stachyose. These galactose-galactose linkages are all at the a-l,6 position and digestion of these Oligosaccharides by animals requires a highly specific enzyme not elaborated by the animals themselves but by certain bacteria present in the animals guts. The gradual disappearance of oligosaccharides from the cotelydons of legume seeds during germination is part of an intricate process beginning with uptake of water by the seed. This uptake of moisture releases gibberellic acid which in turn activates the DNA in the seed, thereby triggering the life cycle of the plant. The DNA directs the production of a -galactosidase which is required for the hydrolysis of these Oligosaccharides. Any interference of the DNA transcription process blocks enzyme production and will be evidenced by continued senescence of the seed and persistence of oligosaccharides in the seed cotelydons.
The polysaccharides represent a large group of complex carbohydrates which are condensation products of undetermined numbers of sugar molecules. The various subgroups are rather ill-defined and there is a lack of agreement on their classification. Most polysaccharides are insoluble in water. Upon hydrolysis with acids or enzymes they eventually yield their constituent monosaccharides.
Starch is a high molecular weight polymer of D-glucose and is the principal reserve carbohydrate in plants. Most starches consist of a mixture of two types of polymers, namely; amylose and amylopectin. The proportion of amylose and amylopectin is generally one part of amylose and three parts of amylopectin. Enzymes capable of catalyzing the hydrolysis of starch are present in the digestive secretions of animals and fish within their cells. The a-amylases which are found virtually in all living cells cleave the a -D-(1® 4) linkages at random and bring about an eventual total conversion of the starch molecule into the reducing sugars. The principal a -amylases of animal origin are those produced in the salivary gland and the pancreas. Starch is insoluble in water and is stained blue by iodine.
Glycogen is the only complex carbohydrate of animal origin. It exists in limited quantities in liver and muscle tissues and acts as a readily available energy source.
Dextrins are intermediate compounds resulting from incomplete hydrolysis or digestion of starch. The presence of a -D-(1® 6) linkages in amylopectin and the inability of a -amylase to cleave these bonds give rise to low molecular weight carbohydrate segments called limit dextrins. These residues are acted upon primarily by acidophilic bacteria in the digestive tract.
Cellulose is made up of long chains of glucose units held together by b -D-(1® 4) linkages. The enzymes which cleave these linkages are not ordinarily present in the digestive secretions of animals and fish although some species of shellfish are believed to elaborate cellulase, the enzyme which catalyzes the hydrolysis of cellulose. Cellulase producing micro-organisms present in the gut of herbivorous animals and fish impart to their host animals the ability to utilize as food the otherwise indigestible cellulose.
Other complex polysaccharides in common occurrence are the hemicelluloses and pentosans. Hemicellulose represents a group of carbohydrates including araban, xylan, certain hexosans and polyuronides. These substances are generally less resistant to chemical treatment and undergo some degree of enzymatic hydrolysis during normal digestive processes. Pentosans are polymers of either xylose or arabinose as constituents of plant structural material and vegetable gums, respectively.
3.1 Digestion, Absorption and Storage
3.2 Other Factors Affecting Metabolism
3.3 Energy Transformation
Much of the carbohydrates that enter the diets of animals, including fish, is of plant origin. Carnivorous fish like the Atlantic salmon and the Japanese yellowtail, therefore, deal with little carbohydrate,. Indeed, experiments have shown that these species are ill-equipped to handle significant quantities of raw carbohydrate, in their diets. On the other hand, omnivores such as the common carp and the channel catfish are able to digest fair amounts of carbohydrates in their diets. The grass carp, a herbivore, subsists primarily on a vegetarian diet.
The ability of animals to assimilate starch depends on their ability to elaborate amylase. All species of fish have been shown to secrete a -amylase. It has also been demonstrated that activity of this enzyme was greatest in herbivores. In carnivores such as the rainbow trout and sea perch, amylase is primarily of pancreatic origin whereas in herbivores the enzyme is widespread throughout the entire digestive tract. In Tilapia mossambica the pancreas has been shown to be the site of greatest amylase activity followed by the upper intestine. Although the digestion of starch and dextrin by the carnivorous rainbow trout was shown to decrease progressively as levels of the carbohydrates were increased beyond the 20 percent level, the fish could effectively utilize up to 60 percent glucose, sucrose or lactose in the diet. This demonstrates that, contrary to earlier belief, carnivorous fish are capable of efficiently utilizing simple carbohydrate as a primary energy source.
The crystalline structure of starch appears also to influence its attack by amylase as evidenced by the two-fold increase in metabolizable energy content of fully cooked (gelatinized) maize in feeding trials with channel catfish. Rainbow trout have also been shown to have a higher tolerance for carbohydrate (present as wheat starch) in the diet when it was cooked. The process of gelatinization involves both heat and water. If an aqueous suspension of starch is heated, the granules do not change in appearance until a certain critical temperature is reached. At this point some of the starch granules swell and simultaneously lose their crystallinity. The critical temperature is that at which hydrogen bonds of the starch molecule loosen to permit complete hydration, leading to a phenomenon known as "swelling".
Alpha-amylase, promotes a more or less random fragmentation of the starch molecule by hydrolyzing at the a -D-(l® 4) glucosidic bonds in the inner and outer chains of the compound. The result of complete hydrolysis of the amylose component are maltose and D-glucose, while the amylopectin component is reduced to maltose, D-glucose and branched limit dextrins. As a consequence of these action patterns by a -amylase on starch, other enzymes are needed for complete hydrolysis of starch to D-glucose in fish. In this regard, it has been demonstrated that even the carnivorous sea bream possess the ability to digest maltose. On the other hand, cellulase and a -galactosidase have not been shown to be secreted by fish although cellulase of bacterial origin is present in the gut of most species of carps. The lack of a -galactosidase may partly explain the poor response by fish to dietary soybean meal which contains significant levels of the galactosidic oligosaccharides raffinose, and stachyose. As has been pointed out earlier, these oligosaccharides do undergo enzymatic hydrolysis during the germination process to yield galactose and sucrose. It would, therefore, appear that the nutritive value of soybean meal will be enhanced if the bulk of this indigestible starch is first transformed. This can be achieved by soaking the beans for 48 hours prior to processing for meal production. It should also be pointed out that the nutritive value of pulses and other legume seeds can likewise be improved for fish since oligosaccharides constitute a large portion of the carbohydrates in legume seeds.
Data on glucose absorption by fish are scanty. Work with goldfish has shown that active transport of glucose is coupled with Na+ transport as in most mammals. It is generally believed that absorption takes place on the mucosal surface of intestinal cells. The mono-saccharides which result from carbohydrate digestion consist primarily of glucose, fructose, galactose, mannose, xylose and arabinose. Although the rates of absorption of these sugars have been determined for many land mammals, similar information for fish is not available.
Glucose does not appear to be a superior energy source for fish over protein or fat although digestible carbohydrates do spare protein for tissue building. Also, unlike in mammals, glycogen is not a significant storage depot of energy despite evidence of an active and reversible Emden - Meyerhoff pathway in fish. The more efficient metabolism of amino acids over glucose for energy could be due to the ability of fish to excrete nitrogenous waste as ammonia from their gills without the high cost of energy in converting the waste to urea.
Apart from genetic adaptation, climatic factors also play an important role in carbohydrate metabolism in fish. Acclimation in fish, in essence, reflects enzyme acclimation, since the animal's ability to survive depends largely upon its ability to carry out normal metabolic functions. Some enzymes for metabolic acclimation show good compensation while others do not. The enzymes associated with energy liberation (enzymes of glycolysis, pentose shunt, tricarboxylic acid cycle, electron transport and fatty acid oxidation) exhibit temperature compensation whereas, those enzymes dealing largely with the degradation of metabolic products show poor or reverse compensation (see Table 1).
Table 1 Enzymes Subject to Metabolic Acclimation 1/
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Enzymes exhibiting compensation |
Enzymes exhibiting reverse or no compensation |
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phosphofructokinase |
catalase |
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aldolase |
peroxidase |
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lactic dehydrogenase |
acid phosphatase |
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6-phosphogluconate dehydrogenase |
D-amino acid oxidase |
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succinic dehydrogenase |
Mg-ATP ase |
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malic dehydrogenase |
choline acetyl transferase |
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cytochrome oxidase |
acetylcholine esterase |
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succinate-cytochrome C reductase |
alkaline phosphatase |
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NAD-cytochrome C reductase |
allantoinase |
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aminoacyl transferase |
uricase |
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Na-K-ATPase |
amylase |
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protease |
lipase |
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malic enzyme |
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glucose-6-phosphate dehydrogenase |
1/Adapted from: Comparative Animal Physiology, edited by C.L. Prosser, 1973
It is interesting to note that two key enzymes involved in carbohydrate metabolism, amylase and glucose-6-phosphate dehydrogenase, together with an enzyme involved in fat digestion, lipase, show no temperature compensation. It is not certain if this is in any way connected with the cessation of feeding by fish at low temperatures. The molecular mechanism of thermal acclimation are not well understood and may consist of changes in synthesis or amounts of a given enzyme. Differences in kinetics, changes in the proportion of isoenzymes suitable for particular temperatures, and changes in co-factors such as lipids, co-enzymes, or other factors such as pH and ions may be important in the animal's adjustment to temperature changes.
Despite species differences in the tolerance of dietary carbohydrates it is generally believed that the principal end-product of carbohydrate digestion, glucose, is metabolized in a manner prevailing in all cells, i.e., via the reversible Emden-Meyerhoff pathway. In this pathway, glucose has only one principal fate: phosphorylation to glucose-6-phosphate. The major metabolic transformations are depicted as follows:
Reversible arrows show reaction step or steps catalyzed by same enzymes in both directions.
Broken arrows show reactions over many intermediate steps.
Paired solid arrows show different enzymes involved in the two directions of the reaction.
(Adapted from: Principles of Biochemistry, by A. White, et al., 1978)
All transformations proceed with a loss of free energy. Thus, the formation of two moles of lactate from glucose-6-phosphate occurs with free energy change of D Go = -22000 cal/mole. The net result is the formation of four molecules of ATP. A functional reversal of this transformation can only occur via a different sequence requiring the input of six ATP molecules per mole of glucose-6-phosphate recovered.
Cells do not store glucose or glucose-6-phosphate. The readily available storage form is glycogen which is made from glucose-1-phosphate by one pathway and returned by another. Although in mammalian cells glucose-6-phosphate is transformed into fatty acids, such transformation does not appear to take place in fish. Studies with the common carp indicate that the precursor for lipogenesis is citrate formed when amino acids are actively metabolized through the tricarboxylic acid cycle.
The major form of utilizable energy in all cells is ATP. In most cells this energy currency is generated by the oxidation of NADH by the mitochondrial electron-transport systems. The reductants of NAD+ for this process are intermediates derived from the TCA cycle and fatty acids. The energy yield from glucose in a respiring system may be summarized in the following sequence of reactions:
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Reaction |
ATP Yield |
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1. glucose® fructose-1,6-diphosphate |
-2 |
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2. 2 triose phosphate® 2,3-phosphoglyceric acid |
+2 |
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3. 2 NAD+® 2 NADH® 2 NAD+ |
+6 |
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4. 2 phosphoenol pyruvate® 2 pyruvic acid |
+2 |
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5. 2 pyruvic acid® 2 acetyl CoA + 2 CO2 |
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2 NAD+® 2 NADH® 2 NAD2 |
+6 |
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6. 2 Acetyl CoA® 4 CO2 |
+24 |
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Overall: |
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C6H12O6 + 6O2® 6 CO2 + 6 H2O |
+38 |
Prosser, C.L. (ed.),1973 Comparative animal physiology. Philadelphia, W.B. Saunders Company, 1011 p. 3rd ed.
White, A., et al., 1978 Principles of biochemistry. New York, McGraw-Hill Book Company, 1492 p. 6th ed.
