Farm Animal Metabolism and Nutrition

administration of a dose of labelled 3MH,
but the decay curve of [^14 C]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
[^14 C]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-^2 H 3 ]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

Proteolysis Free blood

Synthesis

3MH-Actin and myosin

(0.60 μmol g
^1 muscle)

3MH

Fig. 2.8.Illustration of 3-methylhistidine (3MH) metabolism.