Farm Animal Metabolism and Nutrition

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 = (E 1 
E 0 )/[Ep(t 1 
t 0 )]  100

(2.2)

where E0,1 ... 4is the enrichment (tracer/
tracee) of tracer amino acid in the tissue
protein at different times and Ep is 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-[^13 C]leucine as
their tracer and measuring [^13 C]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+
eBo)/∫+ 0 eAdt (2.3)

where eB+
eBois the increase in isotopic

enrichment over time tand ∫+ 0 eAdtis 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