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 vitroand
in vivothat 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 [^14 C]3MH intravenously and
measure the accumulative recovery in the
urine. [^14 C-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^14 CO 2 was
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 other34 J.A. Rathmacher