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Measurement and Significance of Protein Turnover

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Chapter 2
Measurement and Significance of
Protein Turnover
J.A. Rathmacher
Metabolic Technologies Inc., Ames, Iowa, USA
Introduction
The accretion of body proteins is the net
result of both the synthesis and breakdown
of protein. The dynamic nature of protein
metabolism has been known for 60 years
thanks to the pioneering work of
Schoenheimer and others (Schoenheimer
et al., 1939). Using stable isotopes of amino
acids, they demonstrated that proteins con-
tinually were being broken down and
resynthesized. In addition, they reported
that different organs have different rates of
protein synthesis. The dynamic process by
which body proteins are continually
synthesized and broken down is protein
turnover.
Studies on the growth of body protein
stores and the metabolism of protein have
been a major area of research. For example,
a major reason for this is the dramatic fact
that up to 20–25% of the muscle protein
can be broken down per day early in the
life of humans and farm animals. This rate
slows with age to 1–2% day21in adults.
Rates of synthesis and breakdown are influ-
enced not only by age, but by plane of
nutrition, stress, disease, hormones, exer-
cise and inactivity.
Nomenclature
The nomenclature employed in studying
growth and protein metabolism is rela-
tively straightforward, but it is a worth-
while exercise to define briefly the terms
that will be used in this chapter.
Synthesis. The conversion of amino
acids into proteins by the protein
synthetic apparatus in the cytoplasm in
the cell.
Breakdown. The proteolysis of polypep-
tides by different proteinases within
both the cytoplasmic and lysosomal
compartments of the cell. The terms
breakdown and degradation are used
interchangeably.
Growth. The net accumulation of
protein that occurs when the rate of
synthesis is greater than the rate of
breakdown. The term growth is often
used interchangeably with accretion or
net synthesis.
Wasting. The loss of protein that occurs
when the rate of breakdown is greater
than the rate of synthesis.
Turnover. A general term that involves
both synthesis and breakdown. However,
© CAB
International
2000.
Farm Animal Metabolism and Nutrition
(ed. J.P.F. D’Mello) 25
it is sometimes used to represent break-
down. In this chapter, it will be used to
describe protein metabolism, which
includes both protein synthesis and
protein breakdown.
Fractional synthesis and breakdown.
When quantitating protein synthesis
and breakdown, one often expresses the
rates as per cent per day. This allows
one to compare different organs of
different sizes or different muscles of
different sizes.
Three intrinsic problems are associ-
ated with measuring protein synthesis and
breakdown in body tissues. Details of these
problems can be found reviewed elsewhere
(Bier, 1989). The goal of this chapter will
be to describe the methodology used to
quantitate indirect whole-body protein
turnover and direct measurement of tissue
protein synthesis and breakdown.
One of the key problems in studying
protein synthesis and degradation is that
labelled (radio-labelled or stable isotopes)
amino acids which are to measure their
incorporation into protein, or conversely,
release of amino acids from labelled
proteins, and the true enrichment or
specific activity of the amino acid or
metabolite are difficult to measure in an
experiment. The labelled amino acid has
various states in a cell, summarized in Fig.
2.1. The amino acid can be transported
into a free amino acid pool. Once in the
cell, it can be transported back out,
degraded to other metabolites (depending
on the amino acid) or linked to a specific
aminoacyl-tRNA. From the tRNA pool, it
can be translated into protein by ribosomes
(detailed below) and eventually degraded
to free amino acids (mechanism described
below).
Even though it is known that protein
tissue accretion is influenced by both the
synthesis and degradation of protein,
protein synthesis and degradation are not
always measured simultaneously. However,
mechanisms controlling protein synthesis
and degradation are distinct (Reeds, 1989)
and, therefore, can be influenced indepen-
dently. Often breakdown is ignored,
especially when measuring direct tissue
protein turnover. For example, very signifi-
cant gains in muscle protein can be
increased by decreasing protein break-
down, if protein synthesis remains the
same. A 10% decrease in the fractional
breakdown rate of muscle will result in a
26
J.A. Rathmacher
Fig. 2.1. Pathway of uptake, utilization and reutilization of amino acids for protein synthesis and
breakdown.
Cell membrane
Extracellular pool
Free IC
amino acids tRNA
amino acids Protein
amino acids
Protein
Intracellular pool
AA AA AA-tRNA
Protein breakdown and
AA reutilization
α-OXO-derivative
CO2Etc.
23% increase in the protein accretion rate,
whereas a 10% increase in the fractional
synthesis rate will result in an 11%
increase in the protein accretion rate. One
could theorize even greater increases in
growth if synthesis and degradation were
both affected.
Protein synthesis and breakdown
It is evident that both synthesis and break-
down of proteins are necessary to evaluate
the regulation of protein turnover. A better
understanding of these processes is needed
before we proceed. A brief overview of the
mechanisms involved is presented.
Protein synthesis
Protein synthesis (translation) requires the
coordination of >100 macromolecules
working together. They include DNA,
mRNA, tRNA, rRNA, activating enzymes
and protein factors. Information encoded
in DNA is transcribed into the RNA
molecules, which are responsible for the
synthesis of the individual protein. The
formation of single-stranded mRNA
occurs in the nucleus and is called tran-
scription. The mRNA is transported to the
cytosol, where it associates with ribo-
somes, and the translation of the mRNA
sequence into an amino acid sequence
occurs. There are three phases of pro-
tein synthesis: initiation, elongation and
termination.
Initiation occurs when the mRNA
and ribosome bind. The elongation cycle
proceeds with aminoacyl-tRNA (tRNA
molecules bound to specific amino acids)
assembling on a specific codon on the
mRNA. Many ribosomes can attach to a
single mRNA and translate a protein.
Synthesis is terminated when a stop codon
is encountered. A newly synthesized
protein may undergo post-translational
modifications before it can become a func-
tional protein. The protein synthetic
process is probably regulated in two ways
which can affect the rate of protein
synthesis measured: the amount of RNA
and the rate of translation to form protein.
Protein breakdown
Once a protein is synthesized, it is sub-
ject to breakdown. The mechanism of
protein breakdown involves the hydroly-
sis of an intact protein to amino acids.
Protein breakdown is selective, and spe-
cific proteins degrade within the cell at
widely different rates. There are two gen-
eral mechanisms involved in breakdown,
lysosomal and non-lysosomal systems.
The lysosomal system is characterized by
the following: (i) it is located in lyso-
somes at pH 3–5 and includes the cathep-
tic peptidases (cathepsins B, D, H and L);
(ii) it is involved in degradation of endo-
cytosed proteins; and (iii) it is involved
in bulk degradation of some endogenous
proteins. It is unclear how such a degra-
dation system can produce different half-
lives for different proteins.
A second system is the error-eliminat-
ing system which includes peptidases
located in the cell cytoplasm. This system
is specific for proteins containing errors
of translation (abnormal), short-lived pro-
teins, long-lived proteins and membrane
proteins, and it requires ATP. This system
is the ubiquitin–proteasome pathway
reviewed by Mitch and Goldberg (1996).
Proteins are degraded by this pathway
when ubiquitin binds to the protein. It is
accomplished by three enzymes: (i) the E1
enzyme activates ubiquitin in an ATP-
requiring reaction; (ii) activated ubiquitin
is transferred to E2 carrier protein; and (iii)
this is transferred to the protein, catalysed
by the E3 enzyme. This process is repeated
to form a ubiquitin chain. The ubiquitin-
conjugated proteins are recognized by the
proteasome and degraded within the
proteasome by multiple proteolytic sites.
The peptides are released and degraded in
the cytoplasm.
Another cytoplasmic system is the
calpain system consisting of two iso-
enzymes, µ- and m-calpain. This system is
regulated by Ca2+-binding, autoproteolytic
modification, and its inhibitor, calpastatin
(Emori et al., 1987). It has been hypothe-
sized that the calpain system is involved in
the rate-limiting step of myofibrillar protein
breakdown (Reeds, 1989). The calpains are
Measurement and Significance of Protein Turnover
27
candidates for the disassembly of the
myofibril into filaments. The filament
proteins are then broken down in the cyto-
plasm by other proteolytic enzymes.
The remainder of this chapter will deal
with methodology used to measure protein
synthesis and degradation. Discussion will
be divided into two categories: (i) indirect
and direct measurements of whole-body
protein turnover; and (ii) measurement of
tissue protein metabolism in vivo. Two
methods to measure whole-body protein
turnover include: the [15N]glycine end-
product method; and the [1213C]leucine
constant infusion method. Two methods
commonly used to measure the synthetic
rate of tissue protein directly are the
‘constant infusion’ and ‘flooding dose’
approaches, and these will be discussed in
detail. In addition, the arterio-venous
difference limb balance and tracee release
method will be discussed. The traditional
urinary 3-methylhistidine (3MH) end-
product method will be discussed and con-
trasted with a new compartmental tracer
for measurement of muscle proteolysis for
quantitating the 3MH end-product.
Indirect Measurement of Whole-body
Protein Turnover
The subject of whole-body protein turnover
has been reviewed extensively, and the
following references are suggested reading
Waterlow (1969); Waterlow et al. (1978);
Waterlow (1981); Garlick and Fern (1985);
Bier (1989); Nissen (1991); Wolfe (1992).
Measurements of whole-body protein
turnover have been based on either multi-
compartment or simple three-compartment
models of protein metabolism. Multi-
compartmental models have an advantage
because they produce information on com-
partments with distinct rates of turnover. If
the mass of the compartments is known,
the rates of synthesis and degradation can
be determined. Many methods have been
employed (Waterlow et al., 1978), with
some common assumptions and principles.
It is difficult to validate these methods
experimentally. Validation often involves
comparing different methods. If com-
parable data under similar circumstances
are obtained, then a method would be con-
sidered valid. This is not always the case.
If two different methods in the same
animal give different conclusions, then one
must be concerned about the conclusions
drawn from each method.
Determination of whole-body protein
turnover employs stochastic analysis.
Stochastic analysis ignores all of the
various pools and components of whole-
body protein turnover, but focuses on the
overall process. This technique uses either
a single bolus or a continuous infusion
technique. Samples are obtained with con-
stant infusion once isotopic equilibrium is
obtained. At isotopic equilibrium, the
various pools become irrelevant in the
stochastic model as sampling and infusion
occur in a central pool.
Indirect methods of whole-body pro-
tein turnover determination are based on
the concept of amino acid flux. Amino acid
flux (Q) is the sum of all pathways of
disposal of amino acids or is equal to the
sum of all pathways of entry into the
amino acid pool. For an essential amino
acid, flux (Q) is Q = incorporation into
protein(s) + irrevocable amino acid cata-
bolism (E) + absorption from diet (D) +
entry from protein catabolism (C) (Fig. 2.2).
For a non-essential amino acid, de novo
synthesis is based on the entry into the free
amino acid pool.
There are some considerations that
should be taken into consideration when
performing such measurements of whole-
body protein turnover: (i) infusion and
sampling sites; (ii) amino acid absorption
and catabolism; and (iii) choice of amino
acid.
Constant infusion of [
15
N]glycine
(end-product method)
Rittenberg and colleagues were the first to
use [15N]glycine to measure whole-body
protein turnover. They administered a
single bolus of [15N]glycine and measured
the urinary 15N decay curve for 3 days.
28
J.A. Rathmacher
They analysed the data using the modified
three-compartment model depicted in Fig.
2.3. Improvements were made to the model
by Waterlow and colleagues (Picou and
Taylor-Roberts, 1969; Waterlow et al.,
1978) by introducing the constant infusion
steady-state approach. This approach
reduces the number of samples and simpli-
fies the mathematics required. The general
concept is depicted in Fig. 2.4. There is a
metabolic pool of N into which amino
acids enter from the diet (I) and from the
breakdown (B) of body protein. N in the
form of amino acids is synthesized into
protein(s) or can be excreted into the urine.
The [15N]glycine model is based on the
following assumptions: (i) the metabolic
pool of N remains constant during tracer
infusions; (ii) [15N]glycine is not recycled;
(iii) the three-pool model is correct; (iv)
exogenous 15N is metabolized in a similar
manner to endogenous and exogenous N;
(v) synthesis and excretion are the major
pathways of N disposal; and (vi) amino
acids from breakdown and the diet are
handled in the same way.
The method involves the administra-
tion of [15N]glycine (intravenously or
orally) at a continuous rate until a plateau
in the 15N enrichment is achieved. This
usually takes 20–40 h. However, the time
to reach plateau is greatly reduced with a
primed constant infusion (Jeevanandam et
al., 1985). Urine samples are taken at the
plateau. The enrichment of [15N]urea is
then determined. At steady state (when the
rate of amino acids entering the metabolic
pool is equal to the rate at which they
leave), the equation for the model is
Q = I + B = S + E. (2.1)
Measurement and Significance of Protein Turnover
29
Protein
S
Free AA pool
BU
E
Excretion
Total body protein
S
Metabolic pool N
B
15N-glycine (F)
E (Urea + non-urea)
Food I
Protein SFree AA pool
B
D
E
Fig. 2.2. Simple model of whole-body protein
metabolism. S = protein synthesis; B = protein
breakdown; D = dietary intake; E = nitrogen
excretion.
Fig. 2.4. Picou and Taylor-Roberts (1969) model of [15N]glycine protein metabolism. I = intake of dietary
protein; F = infusion of [15N]glycine; B = protein breakdown; S = protein synthesis; E = excretion of both
urinary urea and non-urea end-products.
Fig. 2.3. Three-pool model of Rittenberg to describe protein metabolism. S = protein synthesis; B = protein
breakdown; U = urea synthesis; E = nitrogen excretion.
The amino flux (Q) is equal to the infusion
rate (F) divided by the enrichment of urea
or ammonia (d), Q = F d21. Therefore,
synthesis (S) = Q 2E, where E is the total
excretion and breakdown (B) = Q 2I, where
I is intake.
Constant infusion of [
13
C]leucine
The [13C]leucine method was developed
based on common characteristics of the
[15N]glycine method (Golden and Waterlow,
1977). The model is illustrated in Fig. 2.5.
Leucine is an essential amino acid, there-
fore it is not produced in vivo. The first
metabolite of leucine is a-ketoisocaproate
(KIC). Leucine and KIC are intercon-
vertable by a reversible transamination
reaction. Both leucine and KIC have meta-
bolic pools in plasma and inside the cell.
Leucine enters the cell from protein break-
down and leaves through oxidative
disposal (CO2) and non-oxidative disposal
(protein synthesis). These processes take
place in all tissues and may exhibit
different characteristics.
The difference between the leucine
protein turnover model and the [15N]-
glycine model are that the kinetics of the
amino acid are measured directly. The dif-
ficulty with this model is the extrapolation
of leucine kinetics to rates of protein break-
down and synthesis. For a further discus-
sion, the advantages, limitations and
difficulties associated with leucine meta-
bolism can be found elsewhere (Waterlow
et al., 1978; Matthews and Bier, 1983; Bier,
1989). The original model proposed by
Waterlow is illustrated in Fig. 2.6. Leucine
was selected as the essential amino acid of
choice because it is readily available
cheaply in a pure form (L-leucine). In addi-
tion, when leucine is isotopically labelled
as [1213C]leucine or [1214C]leucine, the
label is completely removed as CO2 (the
first irreversible step). The model in Fig.
2.6 is resolved by infusing (constant) label
leucine (L-[1213C]leucine) into the blood
stream until an isotopic steady state is
reached in plasma. The measurements
taken are the dilution of tracer by
unlabelled leucine and the rate of labelled
CO2excretion in the breath. The dilution of
tracer defines the rate of appearance of
leucine in plasma. The labelled CO2excre-
tion divided by the leucine tracer infusion
rate defines the oxidation rate (C). The
breakdown rate (B) in the post-absorptive
rate is equal to Q (leucine flux, leucine
infusion/isotopic enrichment), and syn-
thesis(s) is S = Q 2C. In summary, neither
Q or C protein are measured directly, and B
and S are extrapolated from them. Other
difficulties associated with this simple
model are that the body does not have a
single leucine pool or a single pool of
protein entering and leaving it. In addition,
the leucine tracer is infused into and
sampled from blood, but leucine protein
metabolism occurs within the cell. Leucine
is transanimated (a reversible reaction)
inside cells to KIC. The KIC may suffer one
of three fates: it may be decarboxylated,
30
J.A. Rathmacher
Fig. 2.5. Illustration of metabolism of leucine and a-ketoisotocaproate (KIC).
Leucine
Plasma
Intracellular Leucine
Protein
Oxidation
KIC
KIC
CO2
reaminated to leucine or it may leave the
cell. Because KIC is only found in the cell
from leucine transamination, plasma KIC
reflects the intracellular KIC enrichment
and can be used as an index of intracellular
leucine tracer enrichment. This approach
expands the single-pool model into a four-
pool model. The calculations are made by
substituting the leucine isotopic enrich-
ment for the KIC enrichment (Fig. 2.7).
This approach is commonly called the
‘reciprocal-pool’ approach (measurement
of the tracer in the metabolite opposite to
the infused) (Schwenk et al., 1985). There
are still drawbacks that remain: (i) there are
still multiple intracellular sites in the body,
and it is not known what contribution they
make to plasma KIC, and (ii) there are a
variety of proteins in the body turning over
at different rates.
Finally, Cobelli et al. (1991) designed a
ten-compartment model, which involves
simultaneously infusing dual tracers of
leucine and KIC and measuring the result-
ing four enrichment curves in plasma and
one in expired air. This model accounted
for the complexity of the leucine system,
but the authors failed to adopt a multi-
tissue scheme for slow and fast kinetic
events (liver as compared with muscle).
These data demonstrated that there was no
single intracellular pool for leucine and
KIC. However, this model is mathematically
difficult and is not solved easily. This
model has not been used in an experimental
design where protein metabolism is altered.
Its value may be to test the structural errors
of simpler but commonly used models.
Measurement of Tissue Protein
Metabolism
in Vivo
Currently, protein synthesis can be
measured directly by two approaches
(Garlick et al., 1994; Rennie et al., 1994), i.e.
constant infusion or the ‘flooding dose’ of a
labelled amino acid. The fractional rate of
synthesis of a protein or mixture of proteins
can be measured from an estimate of the
change in incorporation of a labelled amino
acid into protein over time in tissues.
Measurement and Significance of Protein Turnover
31
Protein
S
B
C
ILeucine
Protein
SB
[1213C]KIC
[1213C]Leucine [1213C]Leucine
[1213C]KIC
13CO2Isovaleryl CoA
Tracer Infusion
([1213C]leucine)
Plasma
Intracellular
Fig. 2.6. Original model for leucine kinetics of an
essential amino acid and its relationship to protein
turnover. B = breakdown; S = synthesis (non-
oxidative disposal); I = dietary intake; C =
oxidation. Under steady-state conditions, leucine
flux is Q = I + B = S + C. When I = O (post-
absorptive), B = Q. Whole-body synthesis is
calculated as S = Q 2C.
Fig. 2.7. The current model of whole-body
synthesis of [1213C]leucine. The infused tracer
reached an equilibrium with intracellular leucine
and KIC. The use of plasma KIC enrichment allows
for the calculation of the total intracellular rate of
appearance and the correct calculation of leucine
oxidation. Breakdown (B) = F/[1213C]KIC
enrichment; oxidation (C) = 13CO2/[1213C]KIC
enrichment; synthesis (S) = B 2C.
Constant infusion
The primed constant infusion method was
pioneered by Waterlow and Stephen (1966)
and further developed extensively by
Garlick (1969). This method aims to set up
a steady state of labelling the amino acid in
plasma and in the intracellular pools of the
body. The method was developed to avoid
the difficulty of measuring both the incor-
poration of the labelled amino acid into
tissue protein and the time course of the
enrichment of the tissue or plasma free
amino acid compared with the rapid
changes of both occurring after a single
injection. In this method, a labelled amino
acid tracer is given by a constant infusion
(with and without a prime) at a rate suffi-
cient to achieve an enrichment of 5–10% of
the tracer amino acid. The infusion con-
tinues for 4–12 h depending on the tissue
or protein of interest. The enrichment of
the free amino acid remains constant for a
substantial portion of the infusion period,
thus the kinetics of the tissue protein
labelling are simple and linear. At the end
of the infusion, tissue samples are taken
and rapidly frozen until they can be
processed further. The enrichment of the
precursor pool amino acid and the enrich-
ment of the labelled amino acid in the
isolated tissue or protein are determined.
The fractional synthetic rate (FSR) can
be calculated by the following equation:
FSR = (E12E0)/[Ep3(t12t0)] 3100
(2.2)
where E0,1 … 4 is the enrichment (tracer/
tracee) of tracer amino acid in the tissue
protein at different times and Epis the
average precursor enrichment during the
same time period that the tissue protein is
being labelled. The true value of the pre-
cursor enrichment is the tRNA molecule,
but this is generally not a practical value to
obtain. Many researchers using this method
have adapted it by using L-[13C]leucine as
their tracer and measuring [13C]KIC as the
precursor enrichment.
An advantage of this method is that it
is applicable to the measurement of proteins
with a slow turnover. In addition, whole-
body protein turnover may be measured at
the same time so that a relationship
between whole-body protein turnover and
tissue protein synthesis may be deter-
mined. The method may be also suitable
for arterio-venous sampling methods.
Flooding dose
The flooding dose technique was developed
to overcome the limitations of the true
precursor enrichment for the calculation of
protein synthesis by the constant infusion
method. Garlick and co-workers developed
this method that was devised originally by
Henshaw et al. (1971). The aim is to ‘flood’
the free amino acid pools, thereby
eliminating the difference between the
intracellular and extracellular (entire pre-
cursors pool) free amino acid enrichments.
This is accomplished by administering the
tracer with a large bolus of tracee. After the
‘flooding dose’, a biopsy of the tissue is
taken and the enrichment determined. The
FSR of tissue protein is determined using
the following formula:
FSR = (eB+ 2eBo)/+0eAdt(2.3)
where eB+ 2eBo is the increase in isotopic
enrichment over time tand +0eAdtis the
area under the curve of the precursor
enrichment versus time. The advantages of
this method include: improved resolution
of precursor enrichment; and shorter
periods of measurement than constant
infusion (10 min flood versus 6 h infusion
in rat; 1–2 h flood versus 4–20 h infusion
in humans). There are concerns that the
flood dose itself may affect protein syn-
thesis and degradation directly or change
amino acid uptake. Also, the large dose of
amino acids may cause a hormonal
imbalance.
Arterio-venous difference
Although the measurement of the direct
incorporation of isotope into protein is the
preferred method to measure protein
synthesis and degradation, it does have
32
J.A. Rathmacher
some disadvantages. The animal is often
killed to collect samples or you must be
able to take biopsies of the tissues, and the
procedures themselves may alter protein
turnover. Therefore, some researchers use
an arterio-venous difference technique to
define the net balances of amino acids. The
balance of any amino acid within an organ
or tissue is the result of the same processes
that apply to the body as a whole, where
amino acid net balance = input 2amino
acid catabolism = protein synthesis 2
proteolysis.
All of the components of the equation
can be determined from the measurements
of amino acid concentration, label uptake
across a tissue, and blood flow. This
method requires the constant infusion of a
tracer amino acid. Both leucine (Pell et al.,
1986) and phenylalanine (Barrett et al.,
1987) tracers have been used, along with
measurements of arterial and venous iso-
tope enrichment, concentrations, metabolic
output and blood flow. The technique
measures the difference between total label
uptake and irrevocable catabolism to pro-
tein synthesis, the difference between net
uptake and irrevocable catabolism to pro-
tein deposition, and the difference between
protein synthesis and deposition to
degradation. Using phenylalanine meta-
bolism of the hindlimb as an example,
(EA3concA3blood flow)
2(EV3concV3blood flow)
= total label uptake (2.4)
and
(concA3blood flow)
2(concV3blood flow)
= net amino acid balance (2.5)
where EAand EVare the isotopic enrich-
ment of a phenylalanine tracer and concA
and concVare the concentration of the
tracee phenylalanine in arterial and venous
blood, respectively. Based on Equations 2.4
and 2.5, protein synthesis = total label
uptake ÷ EA, and protein degradation =
(total label uptake ÷ EA) 2net amino acid
balance.
There are problems with this method.
One is the definition of the isotopic enrich-
ment of protein synthesis and oxidative
compartments. Another problem is the
accurate measurement of amino acid con-
centrations and isotopic enrichments and,
finally, the accurate measurement of blood
flow. However, despite these problems, this
method has great promise in large animals.
Indirect measurement of protein breakdown
The fractional breakdown rate of muscle
protein can be estimated if the fractional
accretion rate of muscle protein is known
(fractional breakdown rate = fractional
synthesis rate 2fractional accretion rate,
FBR = FSR 2FAR) (Millward et al., 1975).
This approach to the study of protein
degradation is somewhat unsatisfactory.
The main problem with this method arises
from the time scale of measurements.
Synthesis is measured over a period of
minutes or hours, while growth is
integrated over the day and measured over
a period of days or months; thus, estima-
tion of protein synthesis will vary over the
course of the day and before and after a
meal. These changes in protein synthesis
could, in turn, grossly over- or under-
estimate the degradation rate.
A more direct approach to measure
muscle degradation is the ‘tracee release
method’ (Zhang et al., 1996). This approach
involves infusing a labelled amino acid to
an isotopic equilibrium and then observing
the isotopic decay in arterial blood and the
muscle intracellular pool. The FBR is cal-
culated as the rate at which tracee dilutes
the intracellular enrichment. This method
can be combined with a tracer incorpora-
tion method in order to measure both the
FSR and FBR in the same study.
Measurement of muscle protein breakdown:
3-methylhistidine metabolism
3-Methylhistidine: historical background
Tallen et al. (1954) were the first to identify
3MH as a component in urine in 1954, but
they were not sure what the source of 3MH
was. The metabolism of 3MH was first
Measurement and Significance of Protein Turnover
33
investigated by Cowgill and Freeberg
(1957) after injecting a radiotracer of 3MH
into the bloodstream of rabbits, rats, chicks
and frogs. The radioactivity was rapidly
excreted in urine; recoveries ranging
between 50 and 90% of the injected
dose were observed. In 1967, 3MH was
identified as a component of actin (Asatoor
and Armstrong, 1967) and of myosin and
actin (Johnson et al., 1967; Hardy et al.,
1970). 3MH is present in the globular head
of the myosin heavy chain (MHC) (Huszar
and Elzinga, 1971) and is localized in the
same area as the ATPase activity and actin-
binding sites. However, there is no
evidence that 3MH is involved in any of
these functions. There is one mole of 3MH
per mole of MHC in the myosin of fast-
twitch, white fibres, but 3MH is absent
in the myosin of the muscle of the
fetus, cardiac muscle and slow-twitch, red
muscle fibres (Huszar and Elzinga, 1971).
Actin also contains this unique amino acid
and it is located at residue 73 of the
polypeptide chain. Unlike myosin, 3MH
is found in all actin isoforms, including
embryonic, smooth and cytoplasmic
isoforms (Cass et al., 1983).
The methyl group is donated to 3MH
by a post-translational event. It has been
shown that S-adenosylmethionine was an
effective methyl donor to histidine of the
nascent polypeptide chains contained in
actin and myosin (Reporter, 1969).
Furthermore, when histidine was used as
the source of labelled amino acid, the
specific activities of histidine and 3MH
were the same in muscle cultures.
Validation of 3-methylhistidine as an index of
muscle protein breakdown
Before 3MH could be used as an index of
myofibrillar protein breakdown, three
assumptions had to be validated, as outlined
by Young and Munro (1978): (i) it does not
charge tRNA and is, therefore, not reutilized
for protein synthesis; (ii) it is excreted
quantitatively in the urine in an identifiable
form; and (iii) the major portion of total
body 3MH is present in skeletal muscle.
To show experimentally that 3MH was
not reutilized for muscle protein synthesis,
3MH was demonstrated not to charge
tRNA. This was accomplished using radio-
labelled 3MH, demonstrating in vitro and
in vivo that 3MH did not charge tRNA of
the rat (Young et al., 1972). However, there
was a high degree of incorporation of label
achieved for histidine and leucine. As a
marker of muscle protein breakdown, 3MH
must be excreted quantitatively in the
urine, i.e. once 3MH is released from actin
and myosin of skeletal muscle it is not
metabolized to any significant extent and is
excreted in the urine. The common
experimental protocol used to confirm
quantitative recovery was to administer
radiolabelled [14C]3MH intravenously and
measure the accumulative recovery in the
urine. [14C-methyl]Nt-methylhistidine was
recovered quantitatively in the urine of rats
after being administered either orally or
intravenously. Ninety-three per cent of the
tracer was recovered in the urine after the
first 24 h, and 98–100% was recovered
after 48 h. Only trace amounts were
recovered in the faeces, and no 14CO2was
recovered in the respired air. Similar
recoveries were confirmed in humans
(Long et al., 1975); 75% during the first
24 h and 95% in 48 h. In addition,
quantitative recoveries were confirmed
with rabbits (Harris et al., 1977), cattle
(Harris and Milne, 1981b) and chickens
(Jones et al., 1986). However, the tracer was
not recovered quantitatively in the urine of
sheep (Harris and Milne, 1980) or pigs
(Harris and Milne, 1981a). More details
will be given about domestic species in the
following sections.
In rats, the radioactivity is distributed
in two compounds, 3MH and N-acetyl-
3MH. The N-acetyl form is the major form
excreted by the rat (Young et al., 1972).
The liver presumably is the site of acetyla-
tion in the rat. The analysis of 3MH from
rat urine requires a hydrolysis step.
Whereas 3MH is the major form excreted in
humans and other species, the N-acetyl
form has been detected in human urine
(4.5%) (Long et al., 1975).
It has been debated whether urinary
3MH is primarily a product of skeletal
muscle protein turnover or whether other
34
J.A. Rathmacher
tissues might contribute a significant
amount to the daily production. Haverberg
et al. (1975) showed that the mixed
proteins in all of the organs sampled
contained detectable levels of bound 3MH.
However, when examining each organ as a
whole, skeletal muscle contained the
majority (98%) of the total amount.
Nishizawa et al. (1977) concluded that the
skin and intestine contributed up to 10%
of the total body pool of 3MH. A study of
humans with short-bowel syndrome
indicated that skeletal muscle was the
major source of urinary 3MH (Long et al.,
1988). In human patients with varying
degrees of infection (Sjölin et al., 1989), it
was concluded that urinary 3MH was a
valid marker of myofibrillar protein break-
down, because it was correlated with the
release of 3MH from the leg. Furthermore,
it was shown later in additional patients
(Sjölin et al., 1990) that there was a signifi-
cant linear relationship between the leg
effluxes of tyrosine, phenylalanine and
3MH and the resulting urinary excretion of
3MH. Therefore, urinary 3MH excretion is
associated with net skeletal muscle protein
breakdown. Our data using the 3MH
kinetic model in portal vein cannulated
swine (van den Hemel-Grooten et al., 1997)
suggest that 3MH production from the
gastrointestinal tract is not increased in
swine fed a protein-free diet. The FBR of
the whole body in swine was 2.16 and 2.56
for controls and those fed a protein-free
diet, respectively, and the percentage from
the gastrointestinal tract was <6% for both
treatments. In conclusion, based on
previous studies, it is reasonable to assume
that changes in 3MH production largely
reflect muscle metabolism.
3-Methylhistidine metabolism in cattle
Cattle, like humans and rats, quantitatively
excrete 3MH in urine. Harris and Milne
(1981b) demonstrated that between 82 and
99% of a [14C]3MH dose was recovered
after 6 days in 21- to 98-month-old non-
lactating cows, steers and a bull. Similarly,
McCarthy et al. (1983) recovered 90% of
the injected tracer dose after 120 h in two
heifers. 3MH is excreted in the urine
unchanged, and occurs in muscle extracts
both in the free form (4–10 nmol g21
muscle) and as a perchloric acid-soluble,
acid-labile form which account for 85% of
the total non-protein-bound 3MH. This
compound was later identified as balenine
(Harris and Milne, 1987), a dipeptide com-
posed of equal molar amounts of b-alanine
and 3MH. Balenine was later identified in
muscle extracts of sheep and pigs. There
appears to be an age-related decline in the
concentration of balenine in muscle, but
this did not produce a measurable change
in the recovery of radioactivity in urine.
3MH is present in whole blood of cattle at
concentrations ranging from 2 to 6 nmol
ml21blood.
The distribution of 3MH in organs has
been determined for cattle (Holstein)
(Nishizawa et al., 1979). Skeletal muscle
contained 93.4% of the total 3MH in the
analysed cattle tissues. The concentration
of 3MH was 3.5106 µmol of 3MH g21
muscle protein or 0.587 µmol g21wet
muscle in growing steers. This value is
used commonly to calculate the protein-
bound pool of muscle 3MH, when deter-
mining the fractional breakdown rate of
skeletal muscle. Other values for the level
of protein-bound 3MH in the muscle of
cattle have been reported: 5.6 µmol g21
protein and 1.8 µmol g21protein. This
variability is of some concern because, in
most studies, the amount of protein-bound
3MH is not determined.
3-Methylhistidine metabolism in sheep
Sheep are unlike cattle in that urinary 3MH
is not a reliable index of muscle protein
breakdown (Harris and Milne, 1980). After
an intravenous dose of 14C-labelled 3MH,
only 25–50% of the label was recovered
after 7 days. The recovery progressively
increased with the age (4 weeks–7 years) of
the animal, becoming almost quantitative
in the older animals after 3 weeks. The
incomplete recovery was not due to excre-
tion in the faeces or elimination as expired
gas, but was related to the presence of a
muscle pool of non-protein-bound 3MH
which was several times larger than the
expected daily urinary excretion. The
Measurement and Significance of Protein Turnover
35
concentration of free 3MH in muscle
ranged from 17 to 120 nmol g21of muscle.
This pool in newly synthesized muscle
tissue was maintained by retention of some
of the 3MH released by breakdown of
muscle protein. Only a small proportion of
the 3MH released from protein breakdown
was available for excretion, and the propor-
tion excreted in the urine increased with
the age of the animal. Another non-protein-
bound component of 3MH in muscle is the
dipeptide, balenine (Harris and Milne,
1987), which comprises on average ;82%
of the total non-protein-bound 3MH in
muscle.
This percentage does not seem to
change as the animal ages, and the same
proportion was seen in cattle muscle. This
dipeptide appeared to be synthesized in
muscle from free 3MH and was not a
terminal product of protein breakdown.
The enzyme system responsible for the
synthesis of the analogous peptides
carnosine and anserine shows a broad
specificity for histidine and histidine
derivatives (Kalyankar and Meister, 1959).
The occurrence of balenine in sheep
muscle should be considered the norm
rather than a rarity. Sheep also have a
much higher concentration of 3MH in
blood than cattle, with values ranging from
17 to 50 nmol ml21of whole blood. The
acid-labile form is present in blood in a
higher proportion than was observed in
cattle, averaging 30% of the total non-
protein-bound 3MH in sheep. The concen-
tration of protein-bound 3MH in sheep was
similar to that in cattle, with an average
concentration from the longissimus dorsi
and leg being 5.8 µmol g21protein. Buttery
(1984) reported a value of 0.6 µmol g21
muscle.
3-Methylhistidine metabolism in pigs
The pig is another species in which the
recovery of radiolabelled 3MH is less than
quantitative (Harris and Milne, 1981a).
From five animals, <21% of the tracer dose
was recovered after 7 days, after which the
recovery was <0.3% day21. The incomplete
recoveries of radiolabelled 3MH were asso-
ciated with the presence of a large pool of
non-protein-bound 3MH in muscle, the
concentration of which increased with age.
The 3MH in the muscle pool was present
as free 3MH, with values ranging from 4 to
8 nmol g21muscle, and as a dipeptide
which constituted >90% of the total non-
protein-bound 3MH. The contribution of
the dipeptide, balenine (Harris and Milne,
1987), increased with age, reaching 99.8%
in older animals, which was 2 µmol of the
total non-protein-bound 3MH g21of
muscle tissue at ;9 months of age. The
concentration in blood was not as high as
in sheep, but was comparable with that in
cattle, with values in pigs ranging from 6 to
19 nmol ml21of blood.
In summary, urinary excretion of 3MH
cannot be used as an index of myofibrillar
protein breakdown in sheep and pigs,
because 3MH is not excreted quantitatively
in the urine. Sheep have elevated levels of
3MH in plasma and muscle, and a dipep-
tide of 3MH is also present at a high
concentration. In pigs, the pool of non-
protein-bound 3MH was maintained and
increased in both established and newly
synthesized tissue by retention of some of
the 3MH released by muscle protein break-
down, only a proportion of which was
available for excretion.
The traditional approach (Fig. 2.8) to
quantitating 3MH production requires
collecting total urinary output for 1–3 days.
The fractional breakdown (day21) rate of
muscle protein rate can be determined by
total urinary 3MH excretion divided by the
total body 3MH muscle pool: 3MH excretion
day21÷ total body 3MH muscle pool. An
estimate of skeletal mass is often difficult to
obtain. The rate of muscle protein break-
down can also be calculated as the rate of
urinary 3MH excretion and its known con-
centration in muscle protein: 3MH excretion
µmol kg21day21÷ 3.63 µmol g21of muscle
protein = B (g of protein kg21of BW day21).
Another approach is to express the data on a
urinary creatinine basis since urinary creati-
nine excretion is a satisfactory estimate of
muscle protein.
The initial studies showing the inade-
quacy of 3MH as an index of muscle
protein breakdown required the intravenous
36
J.A. Rathmacher
administration of a dose of labelled 3MH,
but the decay curve of [14C]3MH in plasma
was not characterized. An alternative
approach to quantitating urinary 3MH
would be to describe the isotopic decay of
a tracer of 3MH in plasma by a compart-
mental mathematical model. A compart-
mental model for swine or sheep must
include a compartment for 3MH meta-
bolism other than excretion into a urinary
compartment. Swine not only have a large
pool of free 3MH in muscle but also a large
metabolic ‘sink’ of 3MH in the form of
balenine. Likewise, sheep excrete ;15% of
3MH in the urine, with the remainder
being retained in muscle as the dipeptide
balenine. Hence a compartmental model
describing the metabolism of 3MH in these
two species must incorporate these meta-
bolic differences as compared with humans,
cattle, rats and rabbits.
Isotope model of 3MH kinetics
Urinary 3MH had been used in cattle and
humans (Fig. 2.8) as an index of muscle
protein breakdown but was invalid for use
in swine and lambs. 3MH is produced in
these species but is not excreted quantita-
tively in the urine. Previously, in validat-
ing urinary 3MH as an index of muscle
proteolysis, researchers have injected
[14C]3MH intravenously and recovered the
tracer in urine, but have never described its
decay in plasma. 3MH is a histidine
residue with one methyl group attached to
the tau-nitrogen on the imidazole ring. To
understand the metabolism of 3MH, we
have used a deuterated molecule of 3MH,
in which the three hydrogens of the methyl
group have been replaced with three
deuterium atoms, therefore the tracer is 3
mass units heavier than the natural
occurring 3MH and can be detected by
gas chromatography–mass spectrometry
(Rathmacher et al., 1992b). In constructing
the three-compartment model, we kept in
mind the known physiology of 3MH. It has
been established that there are pools of
3MH in plasma, in other extracellular fluid
pools, within muscle and in other tissues.
The primary fate of 3MH in humans, cattle
and dogs is into the urine (model exit from
compartment 1), but in sheep and swine
there is a balenine pool in muscle that
accumulates over time (model exit from
compartment 3).
The 3MH kinetic model was developed
from the need to measure muscle proteo-
lysis directly in growing lambs. However,
the problem was that 3MH was a valid
muscle protein turnover method in cattle
and humans but invalid in sheep and
swine. Model development proceeded by
the strategy of developing the model first in
sheep and swine and then validating the
model in cattle and humans. Our basic
experimental design involves the follow-
ing: (i) an intravenous bolus dose of tracer
(3-[methyl-2H3]methylhistidine); (ii) sampl-
ing (blood, urine and muscle tissue); (iii)
3MH isolation by ion-exchange chromato-
graphy; (iv) t-butyldimethylsilyl derivatiza-
tion; (v) analysis by gas chromatography–
mass spectrometry; and (vi) compartmental
Measurement and Significance of Protein Turnover
37
Free blood
Proteolysis
Synthesis
3MH-Actin and myosin
(0.60 µmol g21 muscle)
3MH
Fig. 2.8. Illustration of 3-methylhistidine (3MH) metabolism.
modelling using the SAAM/CONSAM pro-
gram (Berman and Weiss, 1978; Boston et
al., 1981).
We have conducted a series of
studies that propose that 3MH metabo-
lism in humans (Rathmacher et al., 1995),
cattle (Rathmacher et al., 1992a), dogs
(Rathmacher et al., 1993a), swine
(Rathmacher et al., 1996) and sheep
(Rathmacher et al., 1993c) can be defined
from a single bolus infusion of a stable
isotope, 3-[methyl-2H3]methylhistidine.
Following the bolus dose of the stable
isotope tracer, serial blood samples and/or
urine were collected over 3–5 days. A
minimum of three exponentials were
required to describe the plasma decay
curve adequately. The kinetic linear-time-
invariant models of 3MH metabolism in
the whole animal were constructed by
using the SAAM/CONSAM modelling
program. Three different configurations of
a three-compartment model are described:
(i) a simple three-compartment model for
humans, cattle and dogs, in which plasma
kinetics (3-[methyl-2H3]MH/3MH) are
described by compartment 1 and which
has one urinary exit from compartment 1;
(ii) a plasma–urinary kinetic three-com-
partment model with two exits for sheep (a
urinary exit out of compartment 1 and a
balenine exit out of tissue compartment 3);
and (iii) a plasma three-compartment
model with an exit out of tissue compart-
ment 3 in swine. The kinetic parameters
reflect the differences in the known physi-
ology of humans, cattle and dogs, com-
pared with sheep and swine that do not
quantitatively excrete 3MH into the urine.
Steady-state model calculations define
masses and fluxes of 3MH between the
three compartments, and importantly the
de novo production of 3MH. The de novo
production of 3MH for humans, cattle,
dogs, sheep and swine are 3.1, 6.0, 12.1,
10.3 and 7.2 µmol kg21day21, respectively.
The de novo production of 3MH as
calculated by the compartmental model
was not different when compared with
3MH production as calculated via tradi-
tional urinary collection. Additionally, the
data suggest that steady-state compartment
masses and mass transfer rates may be
related to fat-free mass and muscle mass in
humans and swine, respectively.
Species comparison of 3MH kinetics
Figures 2.9 and 2.10 and Tables 2.1 and 2.2
are a summary of the efforts to model 3MH
metabolism using a three-compartment
model in humans, cattle and dogs which
quantitatively excrete 3MH in urine, as
compared with sheep and swine which do
not. Figure 2.10 is a comparison of model
structures between the species. The
diversity of models between humans, cattle
and dogs, and sheep and swine reflects
differences in known physiology. In each
species, the tracer is injected into compart-
ment 1 which, based on the size (volume
and mass), is similar to plasma and extra-
cellular water space. Compartment 1 was
the sampling compartment and the com-
partment from which the steady-state
calculations were initiated. All models for
each species can be resolved by sampling
only plasma. The exception is sheep,
which, as shown in Fig. 2.10, required the
sampling of both plasma and urine.
However, the sheep model can be resolved
from the plasma kinetics of 3MH if the rate
of exit from compartment 1 is fixed. From
the steady-state calculations, the de novo
production of 3MH was obtained into com-
partment 3 for humans, cattle and dogs and
into compartment 2 for sheep and swine.
The de novo production of 3MH could be
placed as an entry into compartment 2 and
an identical rate calculated. The compart-
ment identity of compartments 2 and 3 is
intracellular pools of 3MH. The metabolic
form of 3MH in these compartments may
not be identical nor is the identity of com-
partments 2 or 3 for one species the same
for another species (i.e. cattle versus
sheep). The models also depict differences
in the route by which 3MH exits the
system. In humans, cattle and dogs, 3MH is
excreted quantitatively in the urine as
illustrated by the exit from compartment 1.
This urinary exit has been confirmed by
comparison of urinary excretion of 3MH
and model calculated values (Fig. 2.11).
Whereas sheep excrete only 15% of total
38
J.A. Rathmacher
daily 3MH produced in the urine, swine
excrete 1.5% day21. Therefore, accurate
determination of 3MH production in sheep
and swine requires an exit out of the
system from compartment 3. This exit
accounts for appreciable loss of 3MH into a
balenine ‘sink’ which turns over slowly or
not at all during the time frame of the
study.
Representative plasma decay curves
following a single dosing of 3-[methyl-
2H3]methylhistidine tracer are illustrated in
Fig. 2.9. In general, each species exhibited
a similar exponential decay, characterized
by rapid decay over the first 2–3 h,
followed by a slower decay up to 12 h and
a steady-state decay over the remainder of
the study. The decays of the tracer are
representative of the models used.
Humans, cattle and dogs exhibit very
similar decays, while sheep and swine are
very different.
Measurement and Significance of Protein Turnover
39
Cattle
P
> 0.25
14
12
10
8
6
4
2
0
3-methylhistidrie production
(µmol kg21 day21)
HumansDogs
Model Urine
P
> 0.25
P
< 0.25
Fig. 2.9. Daily 3-methylhistidine production expressed as µmol kg21day21for dogs, cattle and humans as
calculated from urinary excretion and by a three-compartment model of 3-methylhistidine production.
*There was no mean difference between urinary and model 3-methylhistidine production for cattle and
humans,
P
> 0.25.
Table 2.1. Comparison of 3-methylhistidine kinetic parameters.
Species
Parameter Cattle Humans Dogs Swine Sheep
Animals,
n
39 4 5 20 40
Urinary 3MH loss, % of total 100 100 100 1 17
Fractional transfer rate, min21a
L2,1 0.18 0.08 0.11 0.23 0.21
L1,2 0.06 0.06 0.06 0.09 0.08
L3,2 0.003 0.009 0.006 0.014 0.007
L2,3 0.002 0.002 0.008 0.006 0.005
L0,3 NA NA NA 0.0009 0.0004
L0,1 0.006 0.004 0.02 NA 0.0003
aFractional transfer rate (Li,j) from compartment j to i.
NA, not applicable.
Table 2.1 lists the model parameters
and the fractional transfer rates (Li,j from
compartment j to i). The fractional standard
deviation of the parameters ranges from 5
to 50% and, in general, L2,1, L1,2 and L0,1 or
L0,3 are solved with a higher precision than
40
J.A. Rathmacher
1.000
0.100
0.010
0.001
Tracer to tracee ratio
0 1 2 3 4 5
Cattle, human and dog model
Observed Calculated
(A)
Time (min 3 1000)
0.100
0.010
0.001
Tracer to tracee ratio
0 1 2 3 4 5
Sheep model
Observed Calculated
(B)
Time (min 3 1000)
1.000
0.100
0.010
0.001
Tracer to tracee ratio
0 1 2 3 4 5 6
Swine model
Observed Calculated
(C)
Time (min 3 1000)
Fig. 2.10. Disappearance of tracer, 3-[2H3-methyl]methylhistidine, as the ratio of 3-[2H3-methyl]methylhisti-
dine to 3-methylhistidine in plasma as described by a three-compartment model of 3-methylhistidine (see
Fig. 2.11). Symbols (l) represent observed data, and the line ([) represents best fit generated by the model.
L3,2 and L2,3. Table 2.2 compares the com-
partment masses and mass transfer rates between compartments for each species.
Also listed is the de novo production rate
Measurement and Significance of Protein Turnover
41
Tracer
Cattle, dog and human model
(A)
1
Extracellular
fluid
2
Tissue
pool
3
Tissue
pool
Urine 3MH
Skeletal
muscle
de
novo
Tracer
Sheep model
(B)
Extracellular
fluid
Tissue
pool
Tissue
pool
Urine 3MH
Skeletal
muscle
de
novo
Balenine
Tracer
Swine model
(C)
1
Extracellular
fluid
2
Tissue
pool
3
Tissue
pool
Skeletal
muscle
de
novo
Balenine
Fig. 2.11. Schematic of the three-compartment models used to analyse the kinetics of distribution, metabolism
and
de novo
production of 3-methylhistidine (3MH). The tracer, 3-[2H3-methyl]methylhistidine (D3-3MH), was
injected into compartment 1. Sampling was performed from compartment 1.
De novo
production of 3MH
and the exit from the system are dependent on the physiology of the species.
42
J.A. Rathmacher
Table 2.2. Steady-state compartment masses and mass transfer rates of a three-compartment model of
3-methylhistidine (3MH) metabolism.
Parameter Human Cattle Swine Sheep Dogs
n
4392040 5
Plasma 3MH, µM2.9 8.6 10.4 36.9 21.8
M1, nmol kg21a 603 807 1,110 5,308 3,227
M2, nmol kg21912 2,291 2,857 12,483 7,973
M3, nmol kg217,938 8,079 6,151 17,017 9,261
R21, nmol kg21min21b 51 101 247 944 319
R12, nmol kg21min2153 105 247 946 329
R32, nmol kg21min217.9 5.8 37 81 56
R23, nmol kg21min219.6 9.9 32 77 56
R01, nmol kg21min212.2 4.1 NA 1.4 9
R03, nmol kg21min21NA NA 5.0 5.8 NA
3MH productionc, µmol kg21day213.1 6.0 7.2 10.3 12
aMi= compartment mass I.
bRij = mass transfer rate from compartment j and i.
c3MH production was obtained from the model.
calculated by the model in Table 2.2. An
important feature of these models is the
description of 3MH metabolism within the
body. The significance of mass transfer
rates and compartment sizes is not fully
understood. However, the model para-
meters and mass transfer rates may explain
the failure of sheep and swine to
excrete 3MH quantitatively in the urine
(Rathmacher and Nissen, 1992). Three
mechanisms may explain the failure of
sheep and swine to excrete 3MH: (i) 3MH
transport between the compartments limits
the excretion of 3MH; (ii) 3MH is
reabsorbed avidly by the kidney; and (iii)
enzymatic conversion of 3MH to balenine
is enhanced. In comparing the data from
Tables 2.1 and 2.2, the low rate of 3MH
excretion in sheep and swine is not due to
impaired transfer of 3MH out of and
between compartments. Cattle appear to
have a slower exchange of 3MH between
tissues despite near quantitative urinary
excretion. The most likely reason for
sequestering of 3MH in sheep and swine is
that the kidneys are very efficient in
conserving 3MH, which in turn increases
the compartment size and plasma concen-
tration, and through mass action could
increase the synthesis of balenine.
The models described represent a
framework and methodological approach
describing steady-state 3MH kinetics in the
whole animal and constitutes a working
theory for testing by further experimenta-
tion using designs which alter muscle
protein breakdown. The rate of 3MH
production is an important tool in under-
standing the regulation of muscle protein
degradation. The advantages of these
models are that: (i) they do not necessitate
quantitative urine collection (plasma
model); (ii) they reduce error due to the
frequency of plasma sampling versus the
infrequency of urine collection in other
models; (iii) they are more quantitative and
measure the total production rate indepen-
dently of the determination of free or
conjugated forms; (iv) they give informa-
tion about pool size and transfer rates; (v)
they establish a relationship to muscle
mass; (vi) they provide a method for direct
measurement of muscle proteolysis in
swine and sheep; and (vii) they do not
require restraint of the animals for long
periods.
Significance of Protein Turnover
Since isotopes were first used by
Schoeheimer and his colleagues in 1940,
many aspects of protein turnover have
been described. The methods described in
Measurement and Significance of Protein Turnover
43
Table 2.3. Tabulation of results from protein turnover studies in farm animals.
Model Species Response Reference
3MH kinetic model Swine Myofibrillar proteolysis was increased by van den Hemel-Grooten
27% in protein-deficient barrows; no
et al.
(1995)
direct relationship between myofibrillar
proteolysis and
in vitro
proteinase activity
3MH kinetic model Swine Myofibrillar proteolysis was not different van den Hemel-Grooten
from controls swine during the protein
et al.
(1998)
refeeding period
3MH kinetic model Cattle There was a 20% decrease in 3MH Rathmacher
et al
. (1993b)
production in the trenebolone acetate-
implanted cattle, but when combined with
an oestrogen implant the decrease was
prevented
3MH kinetic model Dogs In terms of post-surgical nutrition, meeting Rathmacher
et al
. (1993a)
the protein requirement is critical in
minimizing muscle protein catabolism, and
hyper-supplementation of both energy and
protein has little affect
Urine 3MH Cattle FBR was increased in feed-restricted cattle Jones
et al.
(1990)
Urine 3MH Cattle No effect on muscle protein breakdown Hayden
et al.
(1992)
Arterio-venous Sheep Hepatic protein synthesis was maintained Pell
et al.
(1986)
at the expense of muscle
Whole-body protein Muscle protein synthesis contributes to
turnover–leucine 50% of whole body protein synthesis
Arterio-venous Sheep Whole-body and hind-limb protein Harris
et al.
(1992)
Whole-body protein synthesis is increased with food intake.
turnover–leucine Leucine oxidation increased with food
intake
[15N]Glycine Cattle Whole-body protein synthesis and Wessels
et al.
(1997)
degradation increase when the limiting
amino acid is given
[15N]Glycine Pigs Increased protein accretion with lysine Salter
et al.
(1990)
supplementation was due to a greater
increase in protein synthesis
Whole-body protein Cattle Steroid-implanted steers had a greater Lobley
et al.
(1985)
turnover–leucine increase in synthesis and reduced amino
acid oxidation
Whole-body protein Cattle Bovine somatotropin increased the FSR in Eisemann
et al
. (1989)
turnover–leucine/ muscle and the small intestine
constant infusion
Flooding dose Sheep The FSR was, in decreasing order, intestine, Attaix
et al.
(1988)
liver and muscle
Flooding dose Sheep Changes in nutrients at weaning enhance Attaix
et al.
(1992)
protein synthesis without any specific
effect on small intestine site
Constant infusion Swine FSR was higher in ractopamine-fed pigs as Bergen
et al.
(1989)
compared with controls
Constant infusion Swine Testosterone levels had no effect on muscle Skjaerlund
et al
. (1994)
growth or turnover
Constant infusion/ Sheep Protein synthesis increases with intake in
flooding dose muscle, skin and liver Lobley
et al.
(1992)
this chapter have been used increasingly in
farm animals to improve the production of
muscle for meat and the relationship
between whole-body protein turnover and
muscle protein turnover. Table 2.3
summarizes some of these findings.
44
J.A. Rathmacher
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... Starting oven temperature was 100°C and was held for 0.65 min; the temperature was increased 30°C/min to 250°C and held for 10 min. The mass spec- Precursor-product principle was utilized to calculate fractional synthesis rate (FSR) as given in elsewhere (Dänicke, Böttcher, Simon, & Jeroch, 2001;Ekmay, Salas, England, Cerrate, & Coon, 2013) F I G U R E 1 Average temperature and relative humidity (RH) for two grow-out houses at hot season (a, b) and cool season (c, d Degradation rate was measured using the following expression as follow (Rathmacher, 2000;Young & Munro, 1978): ...
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... There has been considerable interest among animal biochemists in the evaluation of futile cycles to energy expenditures such as Na + /K + transport and protein turnover (Baldwin et al., 1980). Protein turnover is defined as the continuous breakdown and replacement of cellular proteins and is influenced by age, nutrition, stress, disease, hormones, and exercise (Rathmacher, 2000). An idea of minimum estimates of whole body protein synthesis at the height of productive performance is given by Lobley (2003), i.e., 4000 g/d for dairy cows, 2700 g/d for steers, 650 g/d for pigs, 400 g/d for dairy goats, 300 g/d for man, and 250 g/d for sheep: the rate of protein synthesis greatly exceeds normal protein intake. ...
... As to degradation, the muscles with majority in slow-twitch fibre presented significantly greater FDR than those with major fast-twitch or mixed fibre did in not only the postweanling pigs (the current study) but also broilers (Tesseraud et al., 1996a). In addition, the methodology adopted in the current study for FDR based on the difference between FSR (measured in 40 min) and FAR (measured over weeks) might be somewhat unsatisfactory (Rathmacher, 2000) because the calculation rested on the assumption that FSR was constant over the time when FAR was determined while protein synthesis would vary with age and then result in underestimate in degradation. ...
The main purpose of this study is to investigate the effects of dietary lysine deficiency on protein turnover of porcine muscles. There were 18 LYD three-breed-crossing postweanling barrows from six litters cannulated with gastric tubes through the esophagus at approximate 10 kg of body weight and allocated into three treatment groups. When their body weights reached over 12 kg, one group was sacrificed for determining the initial protein masses of m. masseter, m. longissimus dorsi, m. adductor and m. biceps femoris from the right body side. The others received a diet containing 100% or 61.4% (calculated values) of the lysine requirement (NRC, 1998) multiplied by 1.103 for a period of 17 days. Daily feed provision was computed for each pig according to body weight at the same day. All pigs were infused a flooding dose of [H]-phenylalanine to determine the fractional protein synthesis rates (FSR) of the aforementioned muscles in the end. Their four muscles from the right body side were also dissected for measuring the fractional rates of protein accretion (FAR). As for protein degradation, fractional rates (FDR) were calculated by differences between synthesis and accretion. Results showed that the lysine deficiency resulted in, significantly (p
Thesis
Primary goals of this thesis were to: 1) examine the in vivo digestion of macronutrients from conventional or alternative feed ingredients used in practical diets of juvenile gadoids (Atlantic cod and haddock), 2) document growth potential of fish at the juvenile grower phase given varying levels of dietary protein and energy and 3) assess the potential of in vitro pH-Stat methods for rapid screening protein quality of feed ingredients, specifically for gadoids. All primary research questions were linked to and built upon one another with the goal of gaining a better understanding of protein and energy utilization of juvenile grower phase gadoids. Studies showed that cod and haddock have a high capacity to utilize a wide range of dietary feed ingredients, such as fish meals, zooplankton meal, soybean products (meal, concentrate and isolate) and wheat gluten meal. New dietary formulations for gadoids may also utilize pulse meals, corn gluten meal, canola protein concentrate and crab meal. Digestibility data in this thesis is currently the only research that examined both in vivo and in vitro macronutrient digestibility of a large number and wide range of individual ingredients, specifically for gadoids. This is essential to gain new knowledge on protein and energy utilization as well as for least-cost ration formulations and effective substitution of ingredients into new formulations. Data has demonstrated a dietary digestible protein/digestible energy (DP/DE) ratio of 30 g DP/MJ DE is required for gadoids during the juvenile phase (<100 g) to ensure maximum somatic tissue growth, high digestibility, maximum nitrogen and energy retention efficiency and minimal excessive liver growth. Preliminary nutrient requirement studies together with an applied nutritional approach has identified that feeds for juveniles farmed in the Western North Atlantic should contain 50-55% crude protein, <12% fat and <17% carbohydrate. Data in this thesis is currently the first aimed at development and application of an in vitro closed-system pH-Stat assay for rapid screening protein quality of test ingredients that is ‘species-specific’ to gadoids. It is demonstrated that in vitro results generally reflected results obtained through conventional in vivo protein digestibility methods. Studies resulted in the first generation of a ‘gadoid-specific’ proteolytic enzyme extraction method and in vitro closed-system pH-Stat assay which may be useful to investigate protein digestion, absorption and metabolism of gadoids and further development of their feeds.
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A total of 32 Piétrain×(Landrace×Large White) barrows were used to test the effect of reducing the dietary crude protein (CP) level on performance and composition of gain in piglets from 12 to 27 kg. Four diets based on corn, wheat, barley and soybean meal providing 1.01 g of ileal digestible lysine per MJ of NE (net energy) were prepared. Diet 1 was formulated without free amino acids (AA), while in diets 2–4, CP level was progressively reduced and free AA were supplemented according to the ideal protein concept. The CP levels were 224, 204, 184, 169 g/kg for diets 1, 2, 3 and 4, respectively. Piglets were housed individually and had free access to feed and water. The feed intake was measured daily. At the end of the experiment, animals were slaughtered and body composition was measured. The 12 control piglets were slaughtered at the beginning of the experiment and the composition of gain (water, ash, protein lipid, AA and energy) was calculated according to the comparative slaughter technique. Feed intake was the lowest (P<0.05) with diet 1 (959 g/day) and similar for diets 2–4 (1049 g/day, on average). Nevertheless, average daily gain of the animals did not differ between treatments (666 g/day, on average). Reducing CP level resulted in a numerically reduced water consumption and urine production, but no effect on the occurrence of diarrhea was observed. The N excretion over the total experiment was reduced by 42% from diet 1 to diet 4. At identical ME (metabolizable energy) intakes (2.52 MJ/day per kg BW0.60), protein and lipid composition of gain (18.9 and 13.0 g/day per kg BW0.60, on average) were not affected by the diet. Energy gain (0.94 MJ/day per kg BW0.60) and heat production (1.58 MJ/day per kg BW0.60) were also not different between treatments. The amino acid pattern of deposited protein was not affected by dietary CP level, except that histidine deposition was significantly smaller with diets 3 and 4. These results indicate that a reduction of dietary CP level by up to 55 g/kg in association with adequate AA supplementation does not affect the level of performance and body composition of piglets and contributes to lower N excretion.
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Our objectives were to examine the effect of an i.p. injection of a flooding dose of l-phenylalanine (Phe) containing l-[ring-(2)H(5)]Phe on time courses of physiologic responses, the tracer Phe enrichments, and fractional protein synthesis rates (FSR) in plasma, visceral organs, and muscles. In a randomized complete block design, 5 blocks of 5 littermate piglets were weaned at 16 d of age and injected i.p. with a flooding dose of l-Phe (1.5 mmol/kg body weight) on d 8 postweaning under fed conditions. Tissues were collected at 15, 30, 45, 60, and 75 min postinjection. Plasma glucose concentration increased (cubic effect, P < 0.05) from 4.8 preinjection to 5.8 mmol/L 15 min postinjection and returned to preinjection levels thereafter. Plasma insulin concentration did not change (P > 0.05) over time. Plasma Phe concentration increased logarithmically (P < 0.05) from 85 to 711 micromol/L and reached 95% of the maximum concentration 48 min postinjection, but no changes (P > 0.05) in tissue contents of other free amino acids were observed. The Phe free pools in plasma, visceral organs, and muscles were evenly enriched (32.3 +/- 1.4 mol%) with l-[(2)H(5)]Phe 15 min after the i.p. injection. The FSR in visceral organs did not change (P > 0.05), whereas plasma and muscle protein FSR decreased (P < 0.05) over time. We conclude that the i.p. injected tracer Phe rapidly distributed into plasma and intra- and extracellular spaces, and was effective for measuring FSR in visceral organs, but not in plasma and muscles of pigs.
Article
The major N-containing polymer compounds in the body include protein, RNA, and DNA. The endogenous gastrointestinal secretions as well as the portal-drained visceral and peripheral immune responses are basic physiological functions. Elevated endogenous secretions and immune activities, as affected by developmental stages, diets, and management factors, decrease the availability of dietary nutrients for peripheral muscle synthesis and deposition. Measurements of in vivo protein, RNA, and DNA synthesis rates associated with the viscera, peripheral immune cells, and skeletal muscles should, in principle, be the sensitive biochemical and cellular endpoints for studying factors affecting nonruminant nutrition, metabolism, and growth. The selection of stable isotope tracers for precursors, routes of tracer delivery, and mass spectrometric analyses of tracer enrichments are the major methodological considerations. To measure in vivo protein, RNA, and DNA synthesis rates, oral feeding with heavy water (2H2O), and continuous infusion of [U-13C]glucose and [15N]Gly intravenously for labeling the sugar moieties ribose and deoxyribose and de novo purine base synthesis have been established. Flooding doses of tracer Phe, for example, L-[ring-2H5]Phe, via the i.p. route are reliable and cost-effective for measuring in vivo protein synthesis rates, especially for the viscera in small nonruminants. Therefore, measurements of the major N-containing polymer synthesis rates in the viscera, the peripheral immune cells, and muscles through oral feeding with 2H2O and/or i.p. flooding doses of Phe tracers are the emerging tools for studying nonruminant nutrition, metabolism, and growth under research and field test conditions.
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Rates of protein synthesis (PS) and turnover are more rapid during the neonatal period than during any other stage of postnatal life. Vitamin A and lactoferrin (Lf) can stimulate PS in neonates. However, newborn calves are vitamin A deficient and have a low Lf status, but plasma vitamin A and Lf levels increase rapidly after ingestion of colostrum. Neonatal calves (n = 6 per group) were fed colostrum or a milk-based formula without or with vitamin A, Lf, or vitamin A plus Lf to study PS in the jejunum and liver. l-[(13)C]Valine was intravenously administered to determine isotopic enrichment of free (nonprotein-bound) Val (AP(Free)) in the protein precursor pool, atom percentage excess (APE) of protein-bound Val, fractional protein synthesis rate (FSR) in the jejunum and liver, and isotopic enrichment of Val in plasma (APE(Pla)) and in the CO(2) of exhaled air (APE(Ex)). The APE, AP(Free), and FSR in the jejunum and liver did not differ significantly among groups. The APE(Ex) increased, whereas APE(Pla) decreased over time, but there were no group differences. Correlations were calculated between FSR(Jej) and histomorphometrical and histochemical data of the jejunum, and between FSR(Liv) and blood metabolites. There were negative correlations between FSR(Liv) and plasma albumin concentrations and between FSR(Jej) and the ratio of villus height:crypt depth, and there was a positive correlation between FSR(Jej) and small intestinal cell proliferation in crypts. Hence, there were no effects of vitamin A and Lf and no interactions between vitamin A and Lf on intestinal and hepatic PS. However, FSR(Jej) was correlated with histomorphometrical traits of the jejunum and FSR(Liv) was correlated with plasma albumin concentrations.
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1. The effects on growth performance, energy and nitrogen retention, and leucine metabolism of a subcutaneous combined implant of 140 mg trenbolone acetate (TBA)+20 mg oestradiol-17β (OE) have been examined in Hereford × Friesian beef steers (280–520 kg). Comparisons were made both with the same animals before implantation and with untreated control animals maintained under similar physiological and nutritional conditions. 2. Over a 10 week period the implanted steers showed an improvement in rate of live-weight gain (LWG) of 0.5–0.6 with an even greater proportional increase in N retention compared with control animals. Total energy retention was unaffected and thus the ratio, protein energy: total energy gain was 0.43 for implanted steers compared with 0.26 for untreated animals. 3. Estimates of protein synthesis and protein oxidation were obtained from the specific radioactivities of blood free-leucine and exhaled carbon dioxide during continuous infusions of [1- ¹⁴ C]leucine. Whole-body protein synthesis, based on metabolic size, and amino acid fractional oxidation remained similar for control steers throughout the experiment. Steroid-treated steers showed a slight decline in synthesis which was significant (P < 0.05) at week + 5 post-implant while amino acid oxidation was significantly lower at weeks +2 (P < 0.01) and + 5 (P < 0.05) compared with control animals. The ratio, protein deposition: protein synthesis was 0.05 for control animals but 0.08–0.10 for steroid-treated animals after implantation. 4. There was a slight decrease in urinary N T -methylhistidine elimination after implantation which suggested that muscle protein degradation may be reduced although the estimated decrease was insufficient to account for the total improvement in growth rate and N retention. 5. The results suggest that for both control and treated steers, less than 0.5 of total urine N elimination was derived directly from tissue catabolism of protein and amino acids. 6. The combined action of the exogenous steroids in the promotion of protein gain, primarily through a decrease in total protein degradation with little alteration of total energy retention, is compared with present understanding of the role of the endogenous sex hormones.
Chapter
The dynamic nature of cellular proteins was demonstrated over 40 years ago (Schoenheimer et al., 1939). But it was not until 1967–1972 that the measurement of protein turnover in vivo received systematic attention, notably by Wa-terlow’s group (Waterlow and Stephen, 1967; Picou and Taylor-Roberts, 1969; Garlick, 1969; Garlick and Marshall, 1972). It is now generally accepted that cellular proteins are always subject to continual degradation even when protein is neither gained nor lost from the body.
Article
1. Total body protein turnover was studied in six elderly patients. 2. During the study they were fed by continuous infusion of a liquid formula through a nasogastric tube. l-[1-14C]Leucine and [15N]glycine were infused at a constant rate for 30 h. The labelled glycine was infused into the intragastric line; the labelled leucine was given either by this route or intravenously. 3. The specific radioactivity of free leucine in plasma and the rate of output of 14CO2 in expired air both reached a plateau at 10 h, and remained constant until the end of the infusion at 30 h. 4. The 15N abundance in urinary urea and total N was very similar. In neither was a plateau reached by 30 h but in four out of the six patients the abundance in urinary NH4+ had attained a plateau by the end of the infusion. 5. Flux rates and rates of protein synthesis were calculated in four ways: (A) from the specific radioactivity of plasma leucine at plateau; (B) from the proportion of dose excreted as 14CO2 at plateau; (C) from the final rates of 15N excretion in urea or total urinary N; (D) from the final or plateau rates of 15N excretion in urinary NH4+. 6. On average, the estimates of synthesis rate obtained by methods B and C agreed closely; those given by methods A and D were lower. Methods A, B and D ranked the individual patients in almost identical order. 7. The comparison of methods makes it possible to examine the validity of the assumptions on which the different methods depend. Method B is probably theoretically the most satisfactory and, of the methods used in this work, probably gives the best estimate of the absolute turnover rate. The other methods can be used for comparative purposes. 8. The results suggest that the rate of protein turnover is reduced in the elderly, compared with younger subjects.
Article
Whole body 3-methylhistidine (3MH) production rates and proteinase activities in porcine skeletal muscles were studied during a protein-free feeding period and subsequent realimentation. Out of 54 castrated male pigs (35 kg on day 0), six pigs were slaughtered on day 0, and 48 were randomly divided between six dietary treatment groups. During the 14 day protein-free feeding period, three isocaloric diets were provided on a restricted basis: Control (C), protein-free, carbohydrate-rich (PF/CH) or protein-free, fat-rich (PF/FAT). On day 14, eight pigs per treatment group were slaughtered. During the seven day realimentation period, all remaining pigs received the control diet at a restricted level and this formed the other three treatment groups: C–C, PF/CH–C and PF/FAT–C. On day 21, these pigs were slaughtered. Measurement of the 3MH production rate was performed during the last three days of each period. Feeding either one of the PF diets stopped the growth rate and increased the 3MH production rate, whereas proteinase and inhibitor activities in skeletal muscles were not influenced. During realimentation, the growth rate and feed efficiency were higher for both PF–C treatments than for the C–C treatment only during the first three days. The 3MH production rate, as well as proteinase and inhibitor activities in muscles, were not different between treatment groups. These data suggest that compensatory growth occurred only during the first three days of the realimentation period, but pigs did not fully compensate.
Article
The aim of the present study was to estimate the contribution of the gastrointestinal (GI) tract in relation to whole-body 3-methylhistidine (3MH) production in growing pigs and, secondly, to determine whether this contribution changes during a protein-free feeding period in pigs.Seven crossbred barrows with an initial weight of approximately 32 kg, were used in a cross-over design study. The experiment consisted of two dietary treatments (control diet or protein-free diet) and two experimental periods, which were 14 days in duration. The pigs were surgically cannulated with an indwelling catheter placed in the portal vein (P) and carotid artery (A). Diets were offered at a feeding level of 2.5 times energy for maintenance in four portions at 6 h intervals each day. Data were analysed with analysis of variance with dietary treatment and experimental period as the main effects.The experimental period had no effect on any of the measured parameters. Difference in 3MH production rate as measured from P and A sampling and calculated from 3MH kinetics, using a published compartmental computer model, did not differ significantly between treatments. The contribution of the GI tract to the whole-body 3MH production was less than 6% and was not altered by the dietary treatments. Fractional breakdown rate (FBR, % day−1) was significantly higher for the protein-free group than for the control group (2.56 and 2.16% day−1, respectively).These results indicate that the contribution of the GI tract to the whole-body 3MH production is not substantial and this contribution is not influenced by a protein-free feeding period. Therefore it is concluded that increased 3MH production during a protein-free feeding period does not originate from an increased protein degradation in the GI tract but mainly from increased breakdown of skeletal muscle.ZusammenfassungDer Beitrag des Gastrointestinaltraktes zur 3-Methylhistidin-Produktion im gesamten Körper von wachsende SchweinenIn der vorliegenden Untersuchung wird der Beitrag des Gastrointestinaltraktes (GI) an der Bildung von 3-Methylhistidin (3MH) im gesamten Körper von wachsenden Schweinen ermittelt und inwieweit sich dieser Anteil bei Fütterung von proteinfreien Diäten verändert.Als Versuchstiere dienten sieben Kreuzungs-Kastraten mit einem Anfangsgewicht von 32 kg in einem cross-over-Versuch. Der Versuch umfaßte zwei vierzehntägige Perioden mit der Fütterung einer Kontrolldiät und einer proteinfreien Diät. Die Schweine erhielten je ein Katheter in die Portalvene (P) und in die Carotisarterie (A). Die Rationen enthielten das 2,5-fache der Energie für Erhaltung und wurden viermal täglich in 6-stündigen Intervallen verabreicht. Die Versuchsperiode hatte keinen Einfluß auf die gemessenen Parameter. Die Differenz zwischen den 3MH-Produktionsraten der P- und AProben und die 3MH-Kinetik, berechnet mit Hilfe eines kompartimentellen Computermodells, zeigte keine Unterschiede zwischen den zwei Ernährungsregimen. Der Beitrag des GI an der gesamten 3MH-Bildung lag unter 6% und wurde durch die Ernährung nicht beeinflußt. Die fraktionelle Abbaurate (FBR, %/d) war in der proteinfreien Ration signifikant höher als in der Kontrolldiät (2,56%/d und 2,16%/d).Aus den Ergebnissen geht hervor, daß der Beitrag des GI zur gesamten Körper-3MH-Produktion gering ist und nicht durch eine proteinfreie Ration beeinflußt wird. Ein Anstieg in der 3MH-Produktion durch die Ernährung mit einer proteinfreien Diät stammt also nicht aus einem erhöhten Proteinabbau im GI sondern aus einer erhöhten Proteinabbaurate der Skelettmuskulatur.
Article
Actin and myosin, the contractile proteins of skeletal muscle, are methylated following peptide bond synthesis, with production of Ntau-methylhistidine (3-methylhistidine, 3-MeHis). During intracellular breakdown of these proteins, the 3-MeHis is released and excreted in the urine. Studies on tissue distribution of 3-MeHis and on its qunatitative excretion following administration to rats and to man show that urinary output of this amino acid provides a reliable index of the rate of myofibrillar protein breakdown in the musculature of intact rats and human subjects. Estimates of the fractional rate of muscle protein breakdown based on 3-MeHis data are consistent with rates computed by other techniques. By this technique, it has been shown that the fractional rate of muscle protein breakdown is not significantly different in the elderly as compared with young adults. However, since muscle mass is less in the elderly, it makes a smaller contribution to whole body protein breakdown with aging in humans. Output of 3-MeHis diminishes in growing rats and obese human subjects with protein or energy restriction, though the initial response of myofibrillar protein breakdown in growing rats to protein and protein-energy restriction differs. Measurement of 3-MeHis excretion has also proved useful in exploring the effects of physical and thermal trauma on the rate of muscle useful in exploring the effects of physical and thermal trauma on the rate of muscle protein breakdown.