Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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0005 The final stage of protein digestion is accomplished

by a wide array of aminopeptidases and di- and

tripeptidases manufactured in the absorptive cells of

the intestinal epithelium. Some of these act in the

lumen of the gut, but most of the activity is located

within the brush-border membrane or within the

cytoplasm of the cells. These are mainly exopepti-

dases, which remove single amino acids from the

N-terminal (aminopeptidases) or the C-terminal

(carboxypeptidases).

0006 Both pepsin and the pancreatic proteolytic enzymes

are synthesized in the form of inactive proenzymes

(also termed zymogens) containing one or more ter-

minal peptide regions which prevent the formation of

the three-dimensional structure that confers catalytic

activity. The terminal peptide of pepsinogen (contain-

ing 42 amino acids) is hydrolyzed by hydrochloric

acid in the stomach. Trypsinogen is activated by the

removal of a hexapeptide from the amino-terminal by

enteropeptidase (formerly known as enterokinase),

an enzyme secreted by the small intestine. Trypsin in

turn activates all the pancreatic proenzymes, includ-

ing itself. Hence the presence of trypsin inhibitors in

some legumes plays a crucial role in reducing the

digestibility of these otherwise valuable sources of

protein.

Absorption

0007 Absorption of the products of digestion involves

active transport into the enterocyte, metabolism

within the cell, and diffusion down a concentration

gradient into the portal circulation. Absorption

occurs throughout the length of the small intestine,

but is confined to the cells in the top third of the

villus.

0008 Amino acids and peptides are transported into cells

by a number of active transport systems, most of

which are linked to the simultaneous entry of sodium

ions. Sodium enters the cell down a chemical concen-

tration gradient; this is maintained by the membrane

‘pump‘ which exchanges sodium for potassium

ions and is fueled by the hydrolysis of adenosine

triphosphate (ATP). The specificity of the amino

acid transport systems in the gut is broadly similar

to that of the systems which are found in other tissues,

particularly the brush border of the kidney tubule,

and this is summarized in Table 2. Evidence from

in vitro studies suggests that small peptides may be

transported more rapidly than free amino acids, but

these studies often have not been carried out under

the conditions which occur in vivo after a normal

meal.

0009After transport across the intestinal wall, amino

acids can be degraded, metabolized to other com-

pounds, including other amino acids, incorporated

into proteins, or released unaltered into the circula-

tion. The rate of protein synthesis in the small intes-

tinal mucosa is amongst the highest of any tissue in

the body. This is no doubt related to the high rate of

cell turnover and the requirement for secretion of

digestive enzymes. These cells also utilize energy at a

high rate, and the amino acid glutamine, supplied

both from the lumen of the gut and from the circula-

tion, is a major fuel. Some of the ammonia which is

formed during the utilization of glutamine is released

into the gut, from where it may subsequently be re-

absorbed and transported to other tissues to be used

for the synthesis of nonessential amino acids.

0010Most of the peptides which are absorbed into the

mucosal cells are hydrolyzed to single amino acids

before being released into the portal circulation.

However, some intact peptides have also been ob-

served to enter the portal circulation, and this has

raised some interest because of the possibility of pep-

tides with hormonal activity being absorbed intact.

tbl0002 Table 2 Intestinal amino acid transport systems

System Sodiumdependence Preferred amino acids

A Yes Alanine, serine, glycine, methionine, proline

L No Leucine, isoleucine, valine, methionine, phenylalanine, tyrosine, tryptophan, histidine

ASCP Yes Alanine, serine, cysteine, proline

Ly Yes Lysine, histidine, arginine, ornithine



Yes Aspartate



Yes Glutamate



No Aspartate, glutamate, cystine

Yes Lysine, arginine, histidine

b Yes b-alanine, taurine

0,þ

No Lysine, leucine

Gly Yes Glycine, sarcosine

N Yes Histidine, glutamine, asparagine

imino Yes Proline

PROTEIN/Digestion and Absorption of Protein and Nitrogen Balance 4855

0011 The circulating concentration of peptides is nor-

mally very low. This may suggest that only small

quantities of peptides are released into the portal

circulation. However, it has also been observed that

when peptides are infused directly into the circulation

they are rapidly metabolized, probably extracellu-

larly, since the plasma concentration of the free

amino acid constituents of the peptides rises almost

immediately. This means that glutamine, an amino

acid which is notoriously unstable during storage in

solution, and sparingly soluble amino acids such as

tyrosine can be supplied in parenteral nutrition mix-

tures in the form of stable and soluble peptides which

are readily available to the tissues.

0012 Very small quantities of whole proteins can also be

absorbed directly from the intestinal lumen, possibly

by diffusion through tight junctions between cells.

There may also be some endocytosis of food proteins

by immunologically active cells within the intestinal

epithelium, particularly in the areas known as Peyer’s

patches, and these proteins may subsequently be

transported by lymphocytes to distant sites. The

absorption of intact proteins from maternal milk

makes an important contribution to the development

of passive immunity by neonates. Where it persists

into later life it is likely to be a major factor in the

development of food allergies.

0013 It is now known that some small molecules can be

absorbed from the colon as well as the small intestine,

so that the products of bacterial metabolism can be an

important source of nutrients. In the case of nitrogen-

ous compounds the main process appears to be secre-

tion of urea into the colon, bacterial hydrolysis of this

urea to ammonia, and subsequent absorption of the

ammonia into the body. The ammonia may then be

used for the synthesis of amino acids by transamina-

tion. This process is referred to as urea salvage, since

it results in rather less urea being excreted in the urine

than has been produced by the liver. This may make

an important contribution to the maintenance of ni-

trogen balance when protein intake is low. There is

also some evidence that essential amino acids synthe-

sized by colonic bacteria may also be absorbed into

the body, but the quantitative significance of this

observation is not clear.

Amino Acid Pools

0014 Amino acids are transported in the plasma and are

taken up into tissues by mechanisms similar to the

systems referred to in Table 2. Amino acids are also

present in blood cells, but the interaction of these

amino acids with other tissues may be quite different

from that of plasma amino acids. For example, the

plasma amino acid concentration increases as blood

traverses the gastrointestinal tract after a meal,

whereas the amino acid content of blood cells

decreases.

0015The intracellular free amino acid content varies

between different tissues, and is affected by hormonal

and nutritional influences. However, the intracellular

concentration is greater than the plasma concentra-

tion for most amino acids in most tissues. The distri-

bution ratio (intracellular concentration divided by

plasma concentration) is close to unity for many of

the essential amino acids, particularly in muscle, but

is severalfold higher for some of the nonessential

acids, with a value as high as 70 having been reported

for aspartic acid in liver. The interpretation of changes

in amino acid concentrations in particular situations

is complex. Changes in concentration may result

from changes in outflow to or inflow from other

tissues, or pools of other metabolites in the same or

different tissues, including utilization for protein

synthesis and supply from protein degradation.

0016Only about 2% of the amino acids in the body are

present as free amino acids, the rest being present

as protein. Again there is considerable variation

between different amino acids and different tissues

in the ratio of the free to protein-bound amino

acids, with much lower values generally being found

for essential than nonessential amino acids. Given the

currently accepted values for rates of protein synthe-

sis, it can be shown that in some tissues the entire free

pool of some essential amino acids is incorporated

into protein within seconds, emphasizing the rapidity

with which free amino acids must be resupplied.

Nitrogen Balance

0017The state of protein metabolism in the body as a

whole may be assessed by measuring nitrogen bal-

ance. This is because almost all the nitrogen in the

body is in the form of protein, and the nitrogen con-

tent of a wide range of proteins is relatively constant,

at around 16%. Thus, if the body is in positive nitro-

gen balance, it must be laying down new protein, and

this is normally associated with growth in children or

recovery from illness. Conversely, if the body is in

negative nitrogen balance, it must be losing tissue

protein, either because the diet is inadequate or be-

cause of a pathological response to injury or illness.

Nitrogen balance in an individual will fluctuate from

day to day by a few grams either side of zero, but over

a period of weeks a nongrowing adult will normally

be in zero nitrogen balance.

0018Every protein in the body has a specific functional

role. These roles include the catalytic function of

enzymes, the contractile function of muscle proteins,

the structural role of connective tissue proteins, roles

4856 PROTEIN/Digestion and Absorption of Protein and Nitrogen Balance

in transport, immunological recognition, hormone

receptors, and many others. There is no inert store

of protein to be drawn on in times of need, as exists

for fat (adipose tissue triacylglycerol) and carbohy-

drate (glycogen); any sustained period of negative

nitrogen balance is likely to have functional conse-

quences. Nevertheless, different tissues do lose pro-

teins at different rates when the body goes into

negative nitrogen balance, with muscle protein (in-

cluding the smooth muscle of the gut) being lost in the

early stages while the liver and the heart are relatively

protected.

Measurement of Nitrogen Balance

0019 Nitrogen balance is defined as the difference between

intake and output, and may be formally represented

by the following equation:

Balance ¼ I ðU þ F þ MÞ

where I is nitrogen intake, U is urinary nitrogen

excretion, F is fecal nitrogen excretion, and M is the

sum of all the other routes by which nitrogen is lost

from the body.

0020 Nitrogen intake is mainly in the form of protein

and can conveniently be measured by the Kjeldahl

method, which measures virtually all nitrogen except

that bonded to oxygen (e.g., nitrates and nitrites). The

same method is thus also appropriate for measure-

ment of the various components of nitrogen excretion.

Accurate determination of nitrogen balance requires

direct analysis of duplicate portions of diet, although

a reasonable approximation can be made using a

weighed-food record and tables of food composition.

An alternative approach is to feed the subjects a

constant amount of a diet which has been specially

formulated using known quantities of ingredients.

0021 The urine is the major route for excretion of nitro-

gen, so that urinary nitrogen is the major determinant

of nitrogen balance and reliable collection of 24-h

urine specimens is required for measuring nitrogen

balance. Most of the nitrogen in urine is in the form

of urea, which is the end product of amino acid

oxidation. Other nitrogenous compounds in the urine

include ammonia, creatinine, and uric acid, as well as

small quantities of peptides, amino acids, and other

small molecules. The amount of ammonia depends on

the body’s acid–base status and the amount of uric

acid varies with nucleic acid intake. The amount of

creatinine is fairly constant from day to day within a

healthy individual, and shows a good correlation

with muscle mass. When protein intake is low, the

proportion of urea in the urine will decrease, so that

urinary urea content is not a reliable indicator of total

urinary nitrogen.

0022Fecal nitrogen is mainly composed of bacterial cells

from the large intestine, together with mucosal cells

which have been sloughed off from the intestinal

wall, and some remnants of undigested food proteins.

On a normal mixed diet fecal nitrogen amounts to a

fairly constant 8% of nitrogen intake, although on

very-high-fiber diets and particularly those contain-

ing large quantities of legumes this proportion may

increase. In the measurement of nitrogen balance it is

conventional to use a nonabsorbable marker to mark

the beginning and end of the fecal collection, or to use

a continuous fecal marker.

0023The other routes by which nitrogen is lost from the

body include the shedding of hair, nails and dead skin

cells, sweat, saliva, semen, blood lost during menstru-

ation or removed for clinical testing, and the ammo-

nia exhaled in the breath. Dermal losses vary with the

rate of sweating and with protein intake. Many of

these losses are small and hard to measure, so that a

single figure of 0.5 g of nitrogen per day may be taken

to approximate the sum of these miscellaneous routes.

0024The most important use of nitrogen balance is in

evaluating the adequacy of dietary intake. It is the

major criterion which is used to assess quantitative

protein requirements. Such studies have demon-

strated that energy intake is also a major determinant

of nitrogen balance. Measurement of nitrogen bal-

ance is also the basis of many biological methods for

measuring protein quality.

Amino Acid Imbalance and Antagonism

0025It is normally the case that once the minimum require-

ments for total nitrogen and for each essential amino

acid have been met, any surplus protein intake has no

deleterious effect on nitrogen balance. However,

there are reports of the addition of a very large quan-

tity of a single essential amino acid causing a reduc-

tion in the biological value of an otherwise adequate

protein. This has been called amino acid imbalance

and appears to occur only when protein intake is

marginally adequate. It can thus be overcome by

increasing the intake of either total protein or of the

limiting amino acid.

0026The related phenomenon of amino acid antagon-

ism refers to a situation in which the addition of one

amino acid affects the efficiency of utilization of an-

other related amino acid. For example, the addition

of one of the branched-chain amino acids (leucine,

isoleucine, or valine) will impair the utilization of

dietary protein, and this can be overcome by adding

the other two branched-chain amino acids. It is

tempting to speculate that this is because of competi-

tion for membrane transport, or because of increased

activity of the common pathway by which these

PROTEIN/Digestion and Absorption of Protein and Nitrogen Balance 4857

amino acids are oxidized, but no such mechanism has

yet been confirmed.

See also: Amino Acids: Properties and Occurrence;

Determination; Metabolism; Enzymes: Functions and

Characteristics

Further Reading

Alpers DH (1986) Uptake and fate of absorbed amino acids

and peptides in the mammalian intestine. Federation

Proceedings 45: 2261–2267.

Bender DA (1985) Amino Acid Metabolism, 2nd edn.

Chichester: John Wiley.

Bender DA and Bender AE (1997) Nutrition: A Reference

Handbook. Oxford: Oxford University Press.

Calloway DH, Odell ACF and Margen S (1971) Sweat and

miscellaneous nitrogen losses in human balance studies.

Journal of Nutrition 101: 775–786.

Gardner MLG (1984) Intestinal assimilation of intact

peptides and proteins from the diet – a neglected field?

Biological Reviews 59: 289–331.

Jackson AA (1995) Salvage of urea-nitrogen and protein

requirements. Proceedings of the Nutrition Society 54:

535–547.

Waterlow JC, Garlick PJ and Millward DJ (1978) Protein

Turnover in Mammalian Tissues and the Whole Body.

Amsterdam: North Holland.

Webb KE (1986) Amino acid and peptide absorption from

the gastrointestinal tract. Federation Proceedings 45:

2268–2271.

Yudilevitch DL and Boyd CAR (eds) Amino Acid Transport

in Animal Cells. Manchester: Manchester University

Press.

Synthesis and Turnover

P W Emery, King’s College London, London, UK

Background

0001 It is self-evident that growth must involve the synthe-

sis of new protein. Equally, there are situations, usu-

ally associated with illness or undernutrition, when

the organism loses protein, indicating that a mechan-

ism must also exist for degrading tissue proteins.

However, not until the 1940s was there direct evi-

dence that the processes of protein synthesis and deg-

radation continue to occur in healthy nongrowing

adult animals which are neither gaining nor losing

weight. The amino acid tyrosine, labeled with the

stable isotope

N, was fed to an adult rat, and after

10 days only half the isotope had been excreted in the

urine while most of the rest had been incorporated

into tissue proteins. Protein turnover includes both

the synthesis of protein from amino acids and the

breakdown of protein to amino acids. Much experi-

mental work over the last 40 years has been devoted

to measuring the rates at which these two processes

occur and identifying the factors which modulate

them. It is now clear that both processes occur con-

tinuously and simultaneously, and it is estimated that

an adult man synthesizes and degrades about 300 g of

protein each day, which is three or four times as much

as is consumed in the diet.

Mechanism of Protein Synthesis

0002The major biochemical pathways by which protein is

synthesized in cells are now well established. Infor-

mation specifying the primary structure is carried in

the sequence of base pairs in the DNA molecules

in the nucleus. This information is transcribed by

the formation of short-lived molecules of messenger

ribonucleic acid (mRNA) with base sequences com-

plementary to those of the deoxyribonucleic acid

(DNA). The mRNA moves out of the nucleus into

the cytosol, where it associates with ribosomes to

allow translation of the message as a sequence of

amino acids. Every three bases on the mRNA specify

a single amino acid which then binds to the ribosome

in association with a specific molecule of transfer

RNA (tRNA). A peptide bond is formed to join the

new amino acid to the growing polypeptide chain,

and the deacylated tRNA is released from the ribo-

some.

0003Even when the completed polypeptide has been

released from the ribosome it may undergo consider-

able posttranslational modification. Some amino acid

residues may be hydroxylated or methylated, while

others may form covalent links with other parts of

the molecule as the protein adopts a stable three-

dimensional structure. Nonprotein molecules, par-

ticularly carbohydrates, may be added. Finally, parts

of the protein may be removed altogether, possibly

after being transported to a different compartment of

the cell, to allow activation of an inactive precursor or

to facilitate secretion of export proteins.

0004Regulation of the rate of protein synthesis within a

given cell occurs at two levels. There is specific con-

trol of the types of protein synthesized, which occurs

mainly by controlling the transcription of particular

genes. There is also general control of the total

amount of protein synthesized, which occurs mainly

at the level of translation. This includes regulation of

the number of ribosomes present in the cell, and

hence its capacity for protein synthesis, as well as

regulation of the efficiency of translation, which

4858 PROTEIN/Synthesis and Turnover

appears to be achieved largely by changes in the rate

of initiation of peptide synthesis, although elongation

and termination may also be modulated under some

circumstances.

Protein Degradation

0005 The mechanisms by which proteins are broken down

are much less well understood. A large number

of proteolytic enzymes have been identified, with

differing substrate specificities and conditions for op-

timal function. This may reflect the complexity and

variety of the substrates on which they operate, and

may also indicate a degree of cooperative activity,

since once a protein is committed to degradation it

appears to be broken down to its constituent amino

acids very rapidly.

0006 Many of the proteolytic enzymes appear to operate

within a specific organelle, the lysosome. This pro-

vides the acidic environment in which enzymes such

as the cathepsins are optimally active. Lysosomes can

engulf and degrade large structures such as whole

mitochondria, but individual soluble proteins can

also enter the lysosome from the cytosol.

0007 Lysosomes are believed to be responsible for the

majority of protein degradation, particularly

in tissues such as the liver where the overall rate of

protein turnover is high. However, there are also

a number of proteinases which operate in the

cytosol, and are active at neutral or even alkaline

pH. These include the following: the calpains,

which are activated by calcium ions and may be

particularly active in muscle; a large multicatalytic

proteinase sometimes called a proteosome; and a

series of enzymes which commit proteins to degrad-

ation by binding them to a 76-amino-acid polypep-

tide called ubiquitin.

0008 There also exist extracellular proteinases which de-

grade specific extracellular proteins such as collagen.

0009 The control of protein degradation may be exer-

cised at both a general and a specific level, as with

protein synthesis. Some of the factors that affect the

rate of protein degradation are intrinsic to the protein

substrate itself, for example, increasing size and

acidic nature favor more rapid degradation. At least

some protein degradation appears to require energy.

There also appears to be a requirement for continuing

protein synthesis, although this may simply reflect the

fact that the enzymes which degrade proteins are, of

course, proteins themselves. However, this associ-

ation may at least partly explain the observation

that rates of protein synthesis and degradation often

change in the same direction in response to external

influences.

Measurement of Protein Synthesis and

Breakdown

0010Having identified the pathways by which proteins are

synthesized and degraded it is desirable to measure

the rates at which these processes occur. Such meas-

urements can be made in vivo, in humans or experi-

mental animals, or in vitro, in perfused or incubated

tissues or organs, cell cultures, or cell-free systems.

Unfortunately there are quantitative differences be-

tween the results obtained from different systems,

which limits the usefulness of in vitro measurements.

For example, rates of protein synthesis measured in

incubated muscle preparations tend to be lower, and

rates of protein breakdown are always considerably

higher than those observed in vivo. This occurs des-

pite careful attention being paid to factors which

are known to affect the viability of the model such

as tissue oxygenation, muscle integrity, stretch and

electrical stimulation, as well as nutrient supply

and hormonal environment. Hence such preparations

are always in net negative protein balance, whereas

in vivo muscle switches between negative balance, in

the postabsorptive state, and positive balance after a

meal.

0011The ultimate aim is to be able to measure the

turnover rates of individual proteins. This can be

done in vitro, and it may soon be possible to make

such measurements in vivo as well. However, for

technical reasons the methods that are currently

available for use in vivo are only able to give reliable

data for turnover rates in whole tissues or even the

whole body. Nevertheless, such data do provide a

useful insight into the mechanisms by which nutri-

tionally significant quantities of protein are gained or

lost, and such measurements are being made increas-

ingly frequently in both clinical and agricultural

research.

Measurement of Whole-Body Protein

Turnover

0012The measurement of protein turnover necessitates a

model which is simple enough to analyze fully but can

be reasonably interpreted in terms of what happens in

a living animal or person. The model used most com-

monly is the two-pool model, shown in Figure 1. All

the protein in the body is represented by one pool,

while the other pool represents the total amount of

one particular free amino acid (both intracellular and

extracellular). The flux of that amino acid (Q)is

defined as the sum of all the processes by which the

amino acid leaves the free pool:

Q ¼ O þ S þ M

PROTEIN/Synthesis and Turnover 4859

In this equation O is the rate at which the amino acid

is oxidized, S is the rate at which it is incorporated

into protein, and M is the rate at which it is metabol-

ized to other compounds. Measurements are nor-

mally made in a steady state, when the amino acid

pool is neither expanding nor contracting, so that flux

is also equal to the sum of all the processes by which

the amino acid enters the pool:

Q ¼ I þ B þ N

where I is the intake of the amino acid from dietary

protein, B is the rate at which it is released by protein

breakdown, and N is the rate at which it is synthe-

sized. Judicious choice of amino acid allows these

equations to be simplified: for essential amino acids,

N ¼0, and several of the amino acids have no meta-

bolic fates other than oxidation, so that M ¼0. Diet-

ary intake can be controlled during the period of

measurement, and is often set to zero. Thus, if the

rates of flux and oxidation can be measured, the rates

of incorporation of amino acid into protein (i.e., pro-

tein synthesis) and release from protein (i.e., protein

degradation) can be calculated. Knowledge of the

proportion of this amino acid in tissue proteins then

allows the overall rates of protein synthesis and

degradation to be calculated.

0013 There are two commonly used methods for meas-

uring amino acid flux. One involves the administra-

tion of a single dose (d) of an amino acid (often

glycine) labeled with

N and measurement of the

cumulative urinary excretion of label in an end

product of amino acid metabolism, usually either

urea or ammonia, over a period of up to 60 h. The

flux is calculated from the following equation:

Q ¼ dðE=etÞ

In this equation e is the cumulative excretion of the

labeled end product and E is the total excretion rate

of that end product. Oxidation rate can be estimated

from the rate of excretion of urea nitrogen expressed

as a proportion of the nitrogen flux. This method is

noninvasive and therefore particularly suitable for

certain clinical situations. However, from a theoret-

ical point of view it has major disadvantages in that

the long period necessary to obtain a representative

collection of labeled end product allows some isotope

to reenter the free amino acid pool after incorpor-

ation into rapidly turning over proteins, leading to

an underestimate of flux. In addition, the fact that

very different values for flux are obtained using

different end products even in the same experiment

suggests that the end products may not actually be

derived from the same pool as the precursors for

protein synthesis. This therefore casts doubt on the

validity of the method, particularly when comparing

different clinical and metabolic conditions.

0014The other method for measuring protein turnover

is to give a constant infusion of labeled amino acid (at

a rate i) and to measure the isotopic enrichment of the

amino acid in the plasma when this reaches a steady-

state value (L

max

), at which time isotope is entering

and leaving the plasma pool at the same rate. The flux

is then calculated from

Q ¼ i=L

max

is expressed as the ratio of plasma enrichment to

infusate enrichment. The most commonly used amino

acid is leucine labeled with

C in the carboxyl group

so that the oxidation rate can be obtained by measur-

ing the rate at which

C-labeled carbon dioxide

(

) is excreted in the breath. By giving priming

doses of labeled amino acid and bicarbonate, steady-

state labeling of both plasma leucine and breath

can be reached very quickly and the whole pro-

cedure can be completed in 4 h, although the need to

take repeated blood samples is considered a practical

disadvantage. The major theoretical limitation is that

the enrichment of free amino acids in the plasma may

not be the same as that at the site of protein synthesis,

which is likely to be intracellular and will thus vary

between different tissues anyway. The enrichment of

the intracellular amino acid pool will be lower than

that of the plasma pool, so that this method tends to

underestimate the true flux rate. When

C-leucine is

being used as the tracer the enrichment of its transmi-

nation product, a-ketoisocaproic acid, in plasma is

often taken as an estimate of the enrichment of intra-

cellular free leucine.

Measurement of Protein Synthesis in

Individual Tissues

0015The rate of protein synthesis in an individual tissue

can be measured directly by administering a labeled

amino acid and measuring the amount of the

Free

amino

acids

Protein

fig0001 Figure 1 Two-pool model for investigating whole-body protein

turnover. I, intake of amino acid from dietary protein; S, protein

synthesis; B, protein degradation; O, amino acid oxidation; N,

synthesis of amino acid; M, metabolism to other compounds.

4860 PROTEIN/Synthesis and Turnover

labeled amino acid that has been incorporated into

the proteins within the tissue after a given time. The

enrichment of the precursor pool for protein synthesis

during the incorporation time must also be measured

in order to calculate the rate of protein synthesis. This

can be problematical since the enrichment of the free

amino acid pool following administration of a tracer

dose of labeled amino acid follows rather complex

kinetics, and erroneous conclusions have been drawn

from experiments in which the enrichment of the

precursor pool was not measured.

0016 One way of overcoming this problem is again to

give a constant infusion of the labeled amino acid, so

that the free amino acid enrichment remains constant

for a period of several hours. Since the practical ap-

proach is similar this can conveniently be combined

with measuring whole-body protein turnover. The

major limitation of this technique is again that the

enrichment of free amino acids reaches different

steady-state values in the plasma and within the

cells. It is not clear which value best reflects the

precursor pool for protein synthesis, since there is

some evidence that extracellular amino acids may be

incorporated directly into proteins. Furthermore, in

some tissues which contain proteins that turn over

very rapidly, there is the possibility of some reentry

of label from protein after several hours of infusion.

0017 A method which largely overcomes these problems

involves givinga verylarge single dose oflabeled amino

acid. This effectively floods all the free amino acid

pools, so that they reach virtually the same level of

isotopic enrichment, and this value remains constant,

or declines slowly and linearly for an adequate period,

after which the tissue sample is taken. This period is

usually 10 min in rats or mice and 90 min in humans.

0018 The practical drawback to both these methods for

measuring protein synthesis in tissues is the need to

take a sample of the tissue. This can be achieved in

animals which can be killed at the end of the experi-

ment, but studies in humans are restricted to those

tissues which can safely be sampled by percutaneous

biopsy (such as muscle) or those which can be sampled

during scheduled surgical operations.

0019 An alternative approach which obviates the need

for tissue sampling involves sampling arterial and

venous blood simultaneously during a constant infu-

sion of a labeled amino acid. The decrease in amino

acid enrichment between arterial and venous blood

samples reflects the rate at which free amino acid is

being produced in the tissue, and at steady state, for

an amino acid that is not synthesized in that tissue,

this is assumed to equate with the rate of release by

protein degradation. This value can then be sub-

tracted from the net difference in amino acid concen-

tration across the tissue to calculate the rate of amino

acid incorporation into tissue protein, so long as the

amino acid has no other metabolic fate within that

tissue. This method has been used to measure protein

synthesis and degradation in muscle using deuterated

or tritiated phenylalanine as the tracer. However

there are practical limitations, including the difficulty

of measuring small differences in isotopic enrichment,

measuring blood flow accurately, and reliably sam-

pling venous blood from a single tissue.

Measurement of Protein Degradation

0020The method outlined above produces values for the

rate of protein degradation as well as protein synthe-

sis. This is important since there are no other methods

for measuring protein degradation in single tissues

directly. Other methods based on isotopic labeling

are limited by the problem of reutilization of amino

acids after release from protein. Thus many workers

have concluded that the most reliable estimate of

protein breakdown is obtained from the difference

between protein synthesis and the net rate of change

of the protein mass of the tissue. This involves

sequentially killing closely matched groups of animals

over a period of days, and thus cannot give any infor-

mation about short-term fluctuations in protein

degradation.

0021One approach to estimating protein degradation

specifically in skeletal muscle involves 3-methylhisti-

dine. This amino acid is formed by post translational

modification of certain histidine residues in the myo-

fibrillar proteins actin and myosin, and once it is

released from degraded protein it cannot be reutilized

for protein synthesis. 3-Methylhistidine is not metab-

olized, except for N-acetylation in certain species,

including the rat; assessment of its production rate,

from measurement of urinary excretion, should there-

fore indicate the rate of myofibrillar protein break-

down. Unfortunately, it is now known that many

other tissues, including smooth muscle and skin, con-

tain significant quantities of actin, and can make

appreciable contributions to 3-methylhistidine excre-

tion, and this has led to erroneous conclusions about

muscle protein degradation in certain circumstances

such as fasting. Some useful data have been derived

from measurements of arteriovenous differences in

3-methylhistidine across muscles, but results based

solely on measurement of urinary excretion of

3-methylhistidine should be interpreted with extreme

caution.

Rates of Protein Synthesis

0022Whole-body protein turnover has been measured in a

number of species. Values (per kg of body weight per

PROTEIN/Synthesis and Turnover 4861

day) reported for mature animals range from 40 g in

the mouse to 3 g in the cow and 4–5 g in humans

(Table 1). Both synthesis and degradation are consid-

erably increased in young growing animals, with

values of 12 g kg

1

day

1

being reported for prema-

ture infants, falling to 7 g kg

1

day

1

in 1-year-old

children.

0023 Whole-body protein turnover represents the sum of

the contributions from many different tissues. The

contribution of each tissue depends on the fractional

rate of protein synthesis in the tissue and on the

proportion of the body’s protein mass present in

that tissue. Table 2 shows representative data for a

number of tissues in the rat; isolated values are avail-

able for several other species, including humans but

these do not yet form a comprehensive picture.

0024 It should be noted that protein turnover in different

tissues is controlled independently. Thus a simple

measurement of whole-body protein turnover may

fail to detect changes in opposite directions in two

different tissues, such as muscle and liver.

Regulation of Protein Synthesis and

Degradation

0025 The rates of both protein synthesis and degradation

change in response to nutritional and other environ-

mental influences. For example, muscle protein syn-

thesis increases rapidly after a meal, as the tissue goes

temporarily into positive balance, then subsequently

declines as amino acids are mobilized during the post-

absorptive phase. There is a similar cycle of positive

and negative balance with feeding and fasting at the

level of the whole body, although the changes in

protein synthesis and degradation are more equivo-

cal. Whole-body protein degradation is suppressed by

feeding, but the rate of whole-body protein synthesis

does not always increase. The magnitude of the

changes in protein balance is affected by the habitual

diet, so that people who are habituated to a high-

protein diet lose more protein during the postabsorp-

tive period. This has important implications for the

estimation of protein requirements, which will appear

to be higher in people who are accustomed to a high

protein intake.

0026It is possible that the concentrations of free amino

acids within the cell have an influence on the rate of

protein synthesis, but this view is hard to reconcile with

the observation that the Michaelis constants (K

)of

the amino acyl tRNA ligases are generally well below

the intracellular amino acid concentrations, so that the

tRNAs are normally fully charged. Moreover, intracel-

lular amino acid concentrations have been observed

to increase in starvation, when the rate of protein

synthesis is known to go down. Thus it is likely that

other factors such as hormones play a more important

role in regulating protein synthesis.

0027However, it is still possible that the concentration

of one or another amino acid may affect protein

turnover. Tryptophan is the amino acid which is gen-

erally present in the lowest concentrations in intracel-

lular free amino acid pools, at least in mammalian

tissues, and there is evidence from in vitro studies

using rat liver cells that changes in tryptophan supply

can affect the rate of protein synthesis by affecting the

aggregation of ribosomes into polysomes (the form in

which they are active). However, the circumstances in

which such large changes in tryptophan concentra-

tion would occur in vivo are not clear.

0028In vitro studies have also suggested that the

branched-chain amino acids, or leucine alone, may

regulate protein synthesis in skeletal muscle, and that

the deaminated metabolite of leucine, a-ketoisoca-

proic acid, may regulate protein degradation. How-

ever, injection of a large quantity of leucine has been

tbl0001 Table 1 Representative values for whole-body protein turnover

(i.e., synthesis or degradation) rates in adult animals

Species Protein turnover (g kg

1

body weight day

1

)

Mouse 40

Rat 20

Rabbit 10

Sheep 6

Human 4–5

Cow 3

tbl0002 Table 2 Contributions of tissues to whole-body protein synthesis (average values for 100 g rats)

Tissue Protein content (% of wholebody) Fractional synthetic rate (% per day) Absolute synthesis (% of wholebody)

Whole body 100 34 100

Muscle 45 17 23

Skin 10 64 19

Liver 4 90 12

Gut 4 100 11

Bone 3 90 8

Remainder 34 26 27

4862 PROTEIN/Synthesis and Turnover

shown to have no effect on muscle protein synthesis

in vivo.

0029 Recent evidence has indicated that the rate of

protein synthesis in muscle correlates with the intra-

cellular concentration of glutamine, particularly in

circumstances where both are reduced, including

injury and infection. However, increasing muscle glu-

tamine concentration has not been shown to alter the

balance between protein synthesis and degradation.

0030 Of the many hormones that are known to affect

protein turnover, insulin has perhaps received the

most attention. Studies in rats show that insulin in-

creases protein synthesis, and this may be the major

mechanism by which food intake affects protein me-

tabolism. In contrast, insulin’s main action in humans

appears to be to decrease protein degradation. Glu-

cocorticoids depress muscle protein synthesis, and

thyroid hormones increase protein breakdown. Tes-

tosterone and its synthetic derivatives appear to exert

their anabolic effects either by increasing protein

synthesis or by decreasing protein degradation,

depending on concentration.

0031 The rate of protein synthesis in muscle is affected

by physical activity. Exercise causes muscles to hyper-

trophy by increasing the rate of protein synthesis.

During normal growth an increase in the length of

long bones tends to stretch the muscles which are

attached to them, and this stimulates muscle protein

synthesis, resulting in the growth of the muscle.

Factors that Depress Protein Synthesis

0032 One of the major reasons for studying protein turn-

over in relation to nutrition is to investigate the mech-

anisms by which growth rate is reduced or by which

lean body mass is lost in situations of undernutrition

or disease. It is perhaps not surprising that retard-

ation or even cessation of growth is associated with

a reduction in the rate of protein synthesis. There is

now considerable evidence that the net loss of lean

body mass in adults suffering from chronic diseases

such as cancer as well as primary muscle-wasting

diseases is also caused by a decrease in protein

synthesis. Protein breakdown appears not to be in-

creased, and may even be decreased in many tissues as

a secondary response to decreased protein synthesis.

This has major implications for therapy, which

should be directed towards augmenting protein syn-

thesis rather than suppressing protein breakdown.

0033 Several factors appear to be involved in the depres-

sion of protein synthesis. Patients with chronic dis-

eases often have inadequate intakes of protein and

energy, and this is likely to depress protein turnover.

Lack of physical activity, particularly in bedridden

patients, is another important factor. Changes in

conventional hormones are likely to be involved, as

are cytokines such as the interleukins and tumor ne-

crosis factor. These are peptides produced by the cells

of the immune system in response to infection and

injury which, apart from their immediate local action,

also appear to have systemic effects, including sup-

pressing muscle protein synthesis. This presumably

evolved as an adaptive response to acute injury or

infection, when it would be more important to insure

the supply of substrates for such processes as gluco-

neogenesis and acute-phase protein synthesis than to

synthesize muscle proteins. However, this now

appears as a maladaptive response to chronic disease,

since prolonged or severe muscle wasting is associ-

ated with considerable morbidity and mortality. At

the cellular level there is some evidence that these

cytokines, and indeed some conventional hormones,

may act by modulating the production of eicosanoids.

In this context prostaglandin F

appears to increase

the rate of protein synthesis while prostaglandin E

promotes protein breakdown.

See also: Amino Acids: Properties and Occurrence;

Exercise: Muscle; Nucleic Acids: Properties and

Determination; Physiology; Protein: Functional

Properties