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 [^15 N]glycine end-
product method; and the [1^13 C]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 [^15 N]glycine to measure whole-body
protein turnover. They administered a
single bolus of [^15 N]glycine and measured
the urinary^15 N decay curve for 3 days.28 J.A. Rathmacher