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Chapter 14. Nutrient Flow and Retention

Chapter 14. Nutrient Flow and Retention

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Halver and Hardy



Fish nutrient metabolism is the sum total of the biochemical reactions

in life processes. Nutrient metabolism pathways outlined in classical biochemistry texts such as those by Lehninger (1979), West et al. (1966), and

Kaneko (1989) for terrestrial animals also apply to poikilothermic animals

such as fish. Fish species vary from carnivores to omnivores to herbivores

living in a wide range of water temperatures, from near 0 to near 40◦ C, with

corresponding abilities to utilize carbohydrate, protein, and lipid sources

for energy and metabolites for anabolic tissue processes. The main nutritional difference between terrestrial and aquatic animals involves the ability

to exploit plants as food sources. Terrestrial animals exploit the ability of

plants to convert the sun’s energy into leaves and seeds, either directly or

indirectly through the food chain. Some fish utilize aquatic plants directly,

but most exploit plankton, both phytoplankton and zooplankton, again

either directly or indirectly through the food chain. There are no fish equivalents of ruminant animals. Thus, fish exhibit subtle differences in genetic

isozymes associated with their habitat, but in general, cellular metabolism

among fish species is similar. In this chapter, the major nutrient pathways

are outlined, and the emphasis is on differences among fish species and

between fish and terrestrial animals.

Most fish live in a heat sink and their metabolism rates are governed by

the water temperature. As a result, most fish have adapted enzyme systems

to poikilothermic environments and their “genetic isozymes,” while very

similar in structure, combine with the same general substrates to accomplish

the digestion, absorption, and subsequent biochemical reactions necessary

for energy generation and metabolite incorporation into cell functions.

Different groups of fish have different abilities to digest and use complex

polysaccharides as energy sources but are well adapted to use proteins and

lipids as a preferred energy source. Warmwater freshwater fish generally can

use more carbohydrate in the diet and herbivorous fish have well-developed

systems to utilize plant material. All fish still need to metabolize proteins for

amino acid sources for anabolism and growth. As a result, all fish studied

have well-developed proteolytic enzyme systems and some have both acidic

and basic tryptic enzymes, in contrast to terrestrial animals. Another feature is the presence of active chitinase in many species with the ability to

digest and utilize the 2-aminoglucose polymer present in the exoskeletons

of insects and crustaceans. Lipid utilization is well developed in most fish,

although certain cyprinids have a limited ability to use high-lipid diets

without generating liver toxicity and obesity. Fish, living in water, also have

the advantage of direct contact with a good solvent for absorption of

14. Nutrient Flow and Retention


excretions. Therefore, fish eliminate most of the nitrogen resulting from

amino acid catabolism and other nitrogenous metabolites into the water as

ammonia from the gills. This is an energy-sparing system that increases the

amount of metabolic energy that fish derive from protein metabolism. As

a result, fish are more efficient in protein utilization than are homeothermic terrestrial animals, which must eliminate nitrogenous waste as urea or

uric acid. More specific information on the above principles can be found

in the chapters on bioenergetics (Chapter 1), proteins (Chapter 3), lipids

(Chapter 4), and digestive physiology (Chapter 7) in this book.


Carbohydrate Metabolism

Digestibility of polysaccharides is poorly developed, although present, in

carnivorous fish. Salmonids and most marine species use protein and lipid

as the major energy and anabolic intermediates for growth. Cowey (1989)

summarized the amylase, pepsin, and trypsin activities in carp and yellowtail,

showing a 10-fold increase in amylase activity in the warmwater carp versus

the marine yellowtail, little, if any, pepsin activity in the neutral stomach of

the carp, but comparable tryptic activity in both species.

Glycogen is still the major carbohydrate storage form in the liver and

muscle of fish, and even during long periods of starvation or migration of

salmonids, little loss of liver or muscle glycogen occurs (French et al., 1983).

This is evidence for gluconeogenesis from amino acids catabolized during

the starvation period.



The classical Emden–Meyerhof pathway of glucose metabolism after the

formation of glucose 1-phosphate exists in fish. After glucose 6-phosphate

is formed, it is converted into fructose 1,6-diphosphate, which is split into

two molecules of glyceraldehyde 3-phosphate and then condensed into two

molecules of pyruvate which can enter the tricaboxylic acid cycle (TCA) as

acetyl-coenzyme A (acetyl-CoA). All enzymes of the cycle have been identified in fish tissues (Tarr, 1972)(Fig. 14.1).

This pathway allows conversion of glucose into acetyl-CoA and subsequent

generation of the high-energy bonds in adenosine triphosphate (ATP) from

adenosine diphosphate (ADP). The net result from 1 mol of glucose is

36 mol of ATP. Each mole of ATP hydrolyzed in the cell yields 7 kcal of

energy for anabolic and catabolic reactions. The biological efficiency of the


Halver and Hardy

FIG. 14.1

Major enzymes in glycolysis.

aerobic metabolism of glucose can be calculated from the bond energies


C6 H12 O3 + 6O2 −→ 6CO2 + 6H2 O + 686 kcal

Enzymatic glycolysis −→ 36 ATP × 7 kcal −→ 252 kcal

Theoretical biological efficiency −→ 38%

14. Nutrient Flow and Retention


Under anaerobic conditions in the muscle of fish, the glucose is converted into lactate and only 2 mol of ATP per mole is available. The lactate

also requires considerable time to be eliminated from the muscle with adequate oxygen in the water environment. Tarr (1972) listed each of the

enzymes involved in glycolysis and the references for each demonstrated in

fish tissues. Cowey and Walton (1989) discussed the irreversible reactions

and probable regulatory factors in glycolysis and gluconeogenesis and, also,

the range of glycolytic enzymes in trout (Walton and Cowey, 1982). Other

specific enzymes in different fish tissues were more recently identified and

are included in the discussions in Chapters 1 through 5 of this book.


Carbohydrate Synthesis

Carbohydrate synthesis is a very energy-expensive process in fish tissue.

Two moles of pyruvate are needed for the synthesis of 1 mol of glucose. This

uses six high-energy phosphate bonds, but anaerobic conversion of glucose

into pyruvate yields only 2 mol of ATP. Gluconeogenesis does occur in fish,

however, especially during extended periods of starvation or migration when

muscle and liver glycogen must supply the necessary blood glucose for life



Pentose Phosphate Pathway

The pentose phosphate pathway is a minor path for synthesis of ribose

molecules and niacin adenine diphosphate (NADP). The pathway in fish

has been described by Walton and Cowey (1982):

d-Glucose 6-phosphate + 2 NADP + H2 O

−→ d-ribose 5-phosphate + CO2 + 2 NADPH + 2 H+

The reducing equivalents may be used for lipid synthesis. High-carbohydrate

diets in catfish induced increased levels of glucose-6-phosphate dehydrogenase in the liver, indicating stimulated lipogenesis (Likimandi and Wilson,

1982). For more information on lipogenesis see Chapter 4.



Glycogen in liver and muscle is the available glucose storage unit in fish as

in terrestrial animals. Glycogenolysis is one source of glucose, but glycogen


Halver and Hardy

maintenance and hydrolysis to meet metabolic needs are critical to fish

health. Glycogenolysis does not appear to be the major source of liver or

muscle glucose when adequate dietary levels of precursors are present, but it

may be temperature dependent and an emergency strategy before glucose

is made available from the diet or by gluconeogensis from amino acids

from digested protein (Renaud and Moon, 1980; Siebert, 1985; Cowey and

Walton, 1989).


Diet and Carbohydrate Metabolism

Dietary balances may affect carbohydrate metabolism. Cowey et al. (1977)

showed that trout with a 60% protein diet and no digestible carbohydrate

had significantly higher gluconeogenesis than trout fed a low-protein diet

with 50% carbohydrate present. Fasting fish also had a high gluconeogenesis

rate, comparable to that of fish on a high-protein diet. Two other glycolytic

enzymes measured also showed an elevated activity, similar to that in fish fed

the high-protein diet. Buhler and Halver (1961) showed that salmon would

utilize simple hexoses readily and disaccharides well, but polysaccharides

were poorly used in the cold waters that salmon occupy. Digestible energy

values were calculated to be as follows.

Raw starch:

Cooked starch:





1.5–2 kcal/g

2.5–3 kcal

3–3.5 kcal

4.0 kcal

4.0 kcal

0.1 kcal

Digestible energy values for protein and lipids were calculated by Smith

(1989) to be as follows.

Digested protein:

Digested lipid:

5.0 kcal/g

8.0 kcal/g


Lipid Metabolism

An extensive discussion on lipid metabolism can be found in Chapter 4.

Simple fats are digested by lipases, releasing fatty acids and glycerol. The

fatty acids are degraded 2 carbon units at a time in classical β-oxidation steps,

14. Nutrient Flow and Retention

FIG. 14.2

Nutrient flow and ATP production.



Halver and Hardy

yielding acetyl-CoA for transport into the TCA cycle to generate high-energy

phosphate bonds in ATP. The primary oxidation sequence is as follows:






Fatty acid plus ATP and S–CoA forms fatty acid–S–CoA.

Dehydration forms β-dehydro fatty acid–CoA.

Hydration forms β-hydroxy fatty acid–CoA.

Oxidation forms β-keto fatty acid–CoA.

The lysis by CoA–SH forms new fatty acid–CoA and acetyl–CoA.

Then further β-oxidation in the steps above is needed to yield more acetylCoA. Each cycle reduces the carbon chain of the fatty acid by two carbons.

As an example, palmitic acid (C16 ) forms palmityl-CoA and yields eight

acetyl-CoA molecules. In the process, seven reduced flavin adenine dinucleotide (FADH) and seven reduced diphosphopyridine nucleotide

(DPNH) molecules are generated. The net energy gain is then as follows.

(a) Palmitic acid to palmityl-CoA - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - → −2 ATP

(b) 7 Palmityl-CA yields 8 acetyl-CoA

(i) 7 FADH to 7 FP (7 × 2) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - → +14 ATP

(ii) 7 DPNH to 7 DPN (7 × 3) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - → +21 ATP

(c) 8 Acetyl-CoA to 16 CO2 + 8 HOH

+ 8 CoASH (8 × 12) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - → +96 ATP

Estimated biological yield:

129 ATP × 7 kcal = 903 kcal

But complete oxidation of palmitic acid to CO2 and H2 O is 2340 kcal.

Hence the biological efficiency is 903/2340, or 39%. The biological efficiency of fatty acid cellular oxidation is thus approximately the same as that

for digestible carbohydrates or lipids and, as shown later, for proteins as well.

Several vitamins are involved in the various steps of oxidation: pantothenic acid in CoA in step (1); riboflavin as flavin adenine diphosphate in step

(2); biotin and ascorbic acid in step (3); riboflavin, niacin, and pyridoxine

in step (4); pantothenic acid again in step (5); and lipoic acid during the

TCA cycle that follows utilization of the “active acetate” to generate ATP for

cell metabolic energy demands (see Fig. 14.2).


Odd-Chain-Length Fatty Acid Oxidation

Odd-Chain-Length fatty acids are present in small amounts in fish skin

slime and on exoskeleton surfaces of insects and crustaceans. These serve as

bactericidal agents when degraded by β-oxidation to three-carbon propionic

acid, which cannot be degraded by most bacteria. Metabolism of these fatty

acids involves removing one carbon atom from the fatty acid and thus generating an even-chain-length fatty acid that can then be used by β-oxidation

for energy production. Specific enzymes for demethylation similar to those

14. Nutrient Flow and Retention


found in terrestrial animals have been identified in fish (Nicolaides and

Laves, 1958; Gurr and Harwood, 1991).


Electron Transfer Cascade

In oxidation–reduction reactions electrons are transferred from the electron donor to an oxidant or electron acceptor. There are four main classes

of these enzymes.

(1) Pyridine dehydrogenases catalyze reversible transfer of electrons from the

substrate to niacin adenine dinucleotide (NAD) or NAD phosphate to form


(2) The flavin dehydrogenases contain flavin mononucleotide (FMN) or

dinucleotide (FAD). The most important flavin oxidases are succinic

dehydrogenase and NADH dehydrogenases.

(3) Iron–sulfur proteins have from two to eight atoms of iron and a comparable

number of acid-labile sulfur atoms where the iron atoms undergo Fe2+ to

Fe3+ changes as electrons are transferred.

(4) Cytochromes in series transfer electrons from the flavoproteins to oxygen.

These contain iron–porphyrin complexes and can undergo reversible Fe2+

to Fe3+ reactions. Electron transport from NADH to oxygen involves a

decrease in free energy, which can be conserved by phosphorylation of ADP

to ATP to yield the high-energy bonds readily available as an energy source

for anabolism and catabolism in tissues. Copper-containing cytochromes

also occur in the pathway, with the copper available for electron transfer

from the Cu+ to the Cu2+ state. Molecular oxygen can be used in the system

also to hydroxylate organic compounds. Dioxygenases insert two oxygen

atoms into the substrate and monooxygenases insert only one atom of

oxygen. Cytochrome P450 is a common active cofactor in enzymatic

hydroxylations. The schematic cascade of oxidative phosphorylation was

succinctly illustrated by West et al. (1966) in their textbook of biochemistry

for medical students.


Amino Acid Metabolism

Fish and all animals need a constant source of amino acids for tissue

protein synthesis and for synthesis of other compounds associated with

metabolism, including hormones, neurotransmitters, purines, and metabolic enzymes. Amino acids are also catabolized to supply metabolic energy.

Amino acids for these purposes are supplied by the diet and, also, by turnover

of amino acids in tissue proteins, which occurs constantly. Free amino acid

levels in tissues are relatively low; most amino acids in the body are


Halver and Hardy

components of tissue proteins. Thus, the need for a constant supply of

amino acids for metabolic needs is a reality of life. Compared to rats, fish

appear to be more dependent on dietary amino acids to supply metabolic

needs. Cowey and Luquet (1983) estimated that catabolism of tissue proteins supplies about 50% of the amino acids used in metabolism; whereas

in rats this figure approaches 70% (Millward et al., 1976). Additional details

are reported in Chapter 6.

As mentioned earlier, fish excrete nitrogen from amino acid catabolism

mainly as ammonia, rather than as urea or uric acid, as is the case in mammals or birds, respectively. Synthesis of urea and uric acid from ammonia

consumes energy, thus mammals and birds derive less metabolic energy

from amino acid catabolism than do fish. This special facility of fish to

utilize amino acids as energy sources may explain why it is difficult to feed

diets under laboratory conditions that yield much more than 55% protein

(nitrogen) retention, whereas in other animals, nitrogen retention under

ideal conditions is higher (National Research Council, 1974). Ammonia

represents 70–90% of the total nitrogenous wastes in fish (Mommsen and

Walsh, 1992), with 5–15% excreted as urea, depending on the species of

fish (Dosdat et al., 1996). Between 80 and 90% of all nitrogen excretion in

fish occurs through the gills.

Amino acid catabolism occurs mainly in the liver as a part of nutrient

metabolism via deamination reactions, i.e., aminotranferase and deaminase

reactions in the cytosol and mitochondria, respectively. Each amino acid has

a specific route of catabolism involving enzyme-catalyzed reactions with specific end products. This subject has not been studied extensively in fish; it is

assumed that pathways and reactions identified in animals also exist in fish.

Cowey and Walton (1989) summarized the enzymes and catabolic pathways

of amino acid degradation for each amino acid, and this information still

holds today. The first step in the catabolism of amino acids is the removal

of the amino group (transdeamination) to intermediate compounds that

can be further metabolized in the TCA cycle to yield energy or used as

substrates for synthesis of other compounds. The amino group, containing nitrogen, is transferred to α-ketoglutarate to form glutamic acid. This

reaction involves cytoplasmic transaminases. The deaminated product can

then be metabolized through oxidative pathways to yield energy. The net

biological efficiency of converting protein to energy is about 40%.

Glutamic acid is then transported into mitochondria, is deaminated by

glutamate dehydrogenase, and the resulting NH4 + leaves the mitochondria

(Walsh, 1997). It is hypothesized that NH4 + participates in an ammonia

“shuttle” involving cytoplasmic glutamine synthetase to produce glutamine,

which is then transported into the mitochondria, where it is converted to

NH3 . This proposed mechanism exists in the kidney of elasmobranches

and aids in the establishment of a chemiosmotic gradient needed for ATP

14. Nutrient Flow and Retention


production. More detailed descriptions of specific amino acid metabolism

can be found in Chapter 6.

Nearly all nitrogen excretion in fish occurs in the gills. The amino group

(NH2 ) removed from glutamic acid in the liver yields α-ketoglutarate and

NH3 , which is then carried by the circulatory system to the gills, where it is

excreted by direct diffusion from blood to water, direct diffusion of NH3 ,

and/or functional Na+ /NH4 + exchange. Which mechanism is dominant

depends on whether the fish involved is a marine or a freshwater species. In

freshwater species, NH3 diffusion is the main mechanism of excretion. Most

teleost fish are ammoniotelic, yet a portion of their nitrogen excretion is in

the form of urea. Elasmobranchs, coelacanths, and several other teleost fish

living in specialized environments are ureotelic, and the majority of their

nitrogen excretion is as urea (Walsh, 1997). Elasmobranchs and coelacanths

are ureosmotic fish and can maintain osmoregulatory balance by maintaining a high level of urea in their tissues. In spiny dogfish (Squalus acanthias)

more than 90% of nitrogen excretion is as urea excreted by the gills (Wood

et al., 1995). Urea is much less toxic than ammonia, but at the levels found

in some ureosmotic fish, urea can precipitate proteins. Fish counteract this

by having high concentrations of trimethylamine oxide (TMAO), which

counteracts the effects of urea and stabilizes proteins (Somero, 1986).

Because ammonia is toxic, it must be excreted relatively rapidly to prevent tissue accumulation. The plasma ammonia concentration is related

to the protein intake (Kaushik, 1980; Jobling, 1981; Kikuchi et al., 1991)

and begins to rise 3–8 hr following a meal, depending on the species of fish,

protein intake, and quality of protein in the meal (Dosdat et al., 1996). Freshwater species (trout) had higher levels of plasma nitrogen following a meal

(6.5 mg/liter) compared to marine species (sea bass, sea bream, and turbot),

which were uniformily lower (3.5 mg/liter). In contrast, plasma urea concentrations in marine species were not related to dietary protein intake

and were seven to eight times higher in marine species than in freshwater

species (44–59 vs 6.5–7 mg/liter, respectively). Maximum nitrogen excretion occurred 3–5 hr following a meal in small fish (ca. 10 g), compared to

5–8 hr following a meal in large fish (100 g). Dosdat et al. (1996) reported

that between 30 and 38% of ingested nitrogen was excreted within 24 hr

in trout, sea bass, and sea bream, but for turbot, the value was only 21% of

ingested nitrogen.


Effect of Diet on Intermediary Metabolism

The dietary lysine intake influences the fractional rates of protein synthesis and degradation in liver and muscle of rainbow trout (Garzon et al.,

1994). Fish fed lysine-deficient diets exhibited increased rates of protein


Halver and Hardy

degradation without significant changes in protein synthesis rates in the

liver, while in the muscle lysine deficiency caused significant increases in

both the protein degradation and the protein synthesis rates. Other published studies confirm that various amino acid transferases exhibit a higher

activity in fish fed high-protein diets compared to fish fed low-protein diets

but this effect is not uniform (summarized by Cowey and Walton, 1989).

There is little effect of dietary protein intake on the activities of amino

acid-catabolizing enzymes.


Measuring Protein Accretion and Degradation

New and sensitive methods have been developed to measure protein synthesis and degradation rates in tissues using a flooding dose of [3 H]phenylalanine, administered by injection (Garlick et al., 1980). Using this approach,

researchers have developed a number of insights into the metabolic processes associated with protein accretion in tissues. As summarized by

Houlihan (1991), “It is well established that the amount of protein synthesized exceeds the amount retained as growth.” This amount varies with

feed (protein) intake, fish size, and species and also varies among tissues.

Carter et al. (1993a) found a relationship among protein intake, protein synthesis, and protein retention efficiency (protein gain/protein intake rate)

in grass carp, but the efficiency of protein growth was determined by the

rate of protein degradation. Faster-growing fish had lower rates of protein

degradation, higher retention of synthesized protein, higher RNA activity,

and a lower capacity for synthesis and variable rates of protein synthesis.

Houlihan et al. (1988) reported that fractional rates of protein synthesis

differed among tissues, with liver > gills > intestine > spleen > ventricle

> stomach > gonads > white muscle in cod. White muscle in cod had the

highest protein retention efficiency and accounted for 40% of the total body

protein accretion per day, an observation also reported by Facuconneau et al.

(1995) for rainbow trout. Houlihan et al. (1986) ranked rainbow trout tissues

in terms of fractional protein synthesis and degradation rates as follows: gills

> ventricle > red muscle > white muscle. However, the ranking based upon

total amounts of protein synthesized per day was as follows: white muscle =

gills > red muscle > ventricle. They reported that 76% of the protein synthesized in white muscle appeared as growth, compared to only 4% for

gills. Recently, the possibility of estimating protein (muscle) synthesis rates

in fish by measuring tissue levels of mRNA associated with myosin synthesis has been introduced (Overturf and Hardy, 2001). If a similar method

can be developed to measure protein (muscle) degradation rates, this may

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Chapter 14. Nutrient Flow and Retention

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