Clinical Science (1983) 65,217-225
21 7
CONTROVERSIESLNMEDICLNE
3-Methylhistidine excretion and the urinary
3=methylhistidine/creatinineratio are poor indicators of
skeletal muscle protein breakdown
M. J. RENNIE
AND
D. J. MILLWARD*
Department of Medicine, University College London Medical School, The Rayne Institute, London, and
*Department of Human Nutrition, London School of Hygiene and nopica1 Medicine, London
Basis of the 3-methylhistidine method and its
application
Protein breakdown is more difficult to measure
than protein synthesis, so even imperfect methods
are welcome if they indicate the extent of and
direction of possible changes in the process. We
were therefore initially enthusiastic in embracing
[l-31 the 3-methylhistidine method as a measure
of skeletal muscle protein breakdown.
The basis of the method is that 3-methylhistidine is a constituent of actin and the heavy chain
of myosin in white muscle. Thus the skeletal
muscle mass comprises the largest fraction of
tissue-bound 3-methylhistidine in the whole body.
The amino acid is released on protein breakdown
and is not re-utilized for protein synthesis. It is
not metabolized in man although it is N-acetylated
in the rat [4, 51 and is quantitatively excreted in
urine.
It was suggested (originally by Asatoor &
Armstrong [6], and independently by Young &
Munro [7] who developed the method and checked
many of its assumptions) that the rate of excretion
of the amino acid should be proportional to the
absolute breakdown of 3-methylhistidine-containing proteins, chiefly those in skeletal muscle. Since
creatinine excretion is proportional to skeletal
muscle mass [8] it was suggested that the urinary
3-methylhistidine/creatinine ratio should be proportional to the fractional degradation rate of
myofibrillar protein [7,9]. The method has been
widely used to study the effects of dietary maniCorrespondence: Professor M. J. Rennie, Department of Physiology, University of Dundee,
Dundee DDl 4HN, Scotland, U.K.
pulation, growth, development, ageing, fever,
trauma (both accidental and surgical) [lo-201 and
muscle disease (see later).
A major flaw in the theory of the method
We believe that the characteristics of 3-methylhistidine metabolism outlined above, which are
commonly thought to validate the use of the
method, are necessary for its successful application,
but are not in themselves sufficient. A further
condition has to be fulfilled (one often overlooked):
i.e., not only must skeletal muscle contain most of
the body’s protein-bound 3-methylhistidine, but it
must also contribute most to the excretion. The
tissue contribution to overall excretion is determined not only by the pool size of protein-bound
3-methylhistidine but also by the turnover of that
pool, i.e., the breakdown rate of the proteins. We
believe that in both the rat and man small, rapidly
turning over non-skeletal muscle pools of proteinbound 3-methylhistidine exist (as actin, present in
all cell types), which contribute substantially to
the excretion. If this is so, not only are the urinary
excretion rate and 3-methylhistidine/creatinine
ratio invalid as absolute indices of skeletal muscle
breakdown, but even their usefulness as qualitative
indices is limited. In the worst cases, such as those
set out below, apparent changes indicated in
skeletal muscle breakdown by 3-methylhistidine
excretion are in the opposite direction to those
which actually occur.
Evidence from animal studies
There are now several lines of evidence from
experiments with animals that demonstrate serious
M. J. Rennie and D. J. Millward
Remainder
n
Intestine
_Intestine
Skeletal muscle
Protein-bound
Excretion
pool
FIG. 1. Pool sizes of protein-bound 3-methylhistidine and daily excretion rate for an adult (250 g)
rat. The total pool size per whole body was
83.0 3 . 3 ( s ~ ) p m o l . Intestine pool size was
obtained by direct measurement (1.59 0.10),
and muscle pool size by measurement of the
concentration in skeletal muscle samples and
multiplication by a factor assuming muscle to be
45% of body weight. Urinary excretion was
measured over 5 days and the mean daily excretion was 2.1 1 0.09 pmol. The estimates of the
contributions of skeletal muscle and intestine to
the excretion were made by multiplying the turnover rates of skeletal muscle ( 1 . 5 5 +- O.lO%/day)
and intestinal (29.1 1.4%/day) 3-methylhistidine
by the pool sizes (from [ 231).
*
*
*
*
defects in the assumptions central to the 3-methylhistidine method (Figs. 1,2).
I
I
0
10
I
20
I
I
I
30
40
50
1
I
60
Time (days)
FIG. 2. Loss of label in protein-bound 3-methylhistidine in skeletal muscle and intestine, and free
3-methylhistidine in urine over 60 days after a
single injection of [ methy1-14C]methionine in 250 g
adult female rats. Values are the means for groups
of four rats at 2, 5, 15, 30, 45 and 60 days (data
from [22, 231). See the text for discussion of the
observed cross-over of labelling values.
in the methyl pool. We attempted to overcome this
problem by the measurement of the synthesis rate
of 3-methylhistidine in the steady state, when it
lhmover of skeletal muscle 3-methylhistidine
should be equal to the degradation rate [21-231.
Evidence collected from isotopic labelling
We recognize that other potentially serious
studies in rats indicates that the turnover of 3problems remain; these include the assumption of
methylhistidine in skeletal muscle is too slow to
the steady state, the problem of the actual value of
account for more than about half of the excretion
precursor labelling, and the assumption that
rate [21-241. The component of protein turnover
methylation rates of histidine are comparable with
of interest here is breakdown, the measurement of
protein synthetic rates. The first of these is not a
which is beset by difficulties well illustrated by the
serious problem in fed adult rats in which the
attempts of Nishizawa’s group to measure 3growth rate is near zero, so that a steady state
methylhistidine turnover in muscle [ 2 5 ] . In that
would, in the fed state, be likely to lead to an
work, often cited as showing that non-skeletal
overestimate of protein synthesis, and thus of
muscle sources account for only 17% of the excredegradation; this would result in the apparent
tion, no net loss (i.e. no apparent turnover) of
turnover of skeletal muscle 3-methyhistidine
labelled 3-methylhistidine in skeletal muscle was
being artificially high, leading to an understateobserved after pulse-labelling with [m~ethyl-’~C]
- ment of the discrepancy between the low turnover
methionine, possibly due to persistence of the label
in muscle and the excretion rate. Uncertainty of
3-Methylhistidine and muscle breakdown
precursor labelling could have been a source of
error in the first labelling study [21] if S-adenosylmethionine, the precursor, was compartmented in
sarcoplasm. We have no evidence that this was in
fact the case, and we believe it was not; if so, our
initial measurements would remain valid. Nevertheless, in our recent studies [22-241 we have
adopted a different technique, using incorporation
of [14C]histidine, which, more likely than not,
labels a common pool for both presumptively
methylated and non-methylated histidine incorporated into muscle protein.
Let us now deal with the last problem, frequently raised by critics of our conclusions. Everyone agrees that for an unchanging composition of
muscle the overall turnover rates of 3-methylhistidine, histidine and the methyl group substituent, must be identical, since demethylation
apparently does not occur. In other words, the
methylation rate of histidine (e.g. in actin) is equal
to the rate of protein (i.e. actin) synthesis. What
about the possibility of a lag between protein
synthesis and histidine methylation? If the processes were not synchronous the mismatch would
give rise to a falsely low labelling ratio in 3-methylhistidine to histidine after pulse injection of
[I4C]histidine, especially noticeable soon after
injection of the tracer. This is, of course, a testable
hypothesis, and we have checked to see whether a
lag in the labelling ratio exists. The turnover of
3-methylhistidine in skeletal muscle protein was
35% of the overall rate of mixed muscle protein
synthesis irrespective of whether it was measured
1 h or 4-5 h after injection of tracer [23,24]. This
confirms the results of others, who have shown no
differences between rates of methylation and
overall protein synthesis [26,27].
A further possible criticism of our measurements
is that they have been made on one group of
muscles (i.e. those of the hindlimbs), which may
be unrepresentative of the entire musculature.. We
feel that this is unlikely, since the fibre-type distribution of leg muscles is similar to that of the
whole-body musculature in rodents [28], and
measurements of protein synthesis in rat quadratus
lumborum and psoas, muscles of the trunk, have
given values similar to those obtained for leg
muscles [29].
Turnover of non-skeletal muscle 3-methylhistidine
(Fig. 1 )
There is now solid evidence, again from isotopic
studies, that small pools of 3-methylhistidine in
non-skeletal muscle tissues of the rat do in fact
turn over sufficiently rapidly to contribute disproportionately to whole-body excretion. Our
219
initial measurements of turnover in skin and intestine suggested that although turnover in these
tissues was faster than in skeletal muscle, they did
not appear to be major sources of urinary 3methylhistidine [21]. It now appears that we
initially underestimated 3-methylhistidine turnover
in non-skeletal muscle tissues, particularly in
intestine, most likely as a result of precursor compartmentation. We applied the histidine labelling
method [22-241, coupled with accurate measurements of overall turnover rates of mixed protein
by the large-dose phenylalanine method [30], to
the measurements of intestinal 3-methylhistidine
turnover in adult rats (Fig. 1). By these methods
turnover of 3-methylhistidine in intestine is 20
times faster (at 29%/day) than in skeletal muscle,
so even though intestine contains only 2% of the
whole-body pool, it accounts for over 20% of the
excretion. Independently, other workers [311 have
recently come to similar conclusions after showing
that, on the basis of 3-methylhistidine release from
perfused intestine and hemicorpus preparations,
intestine could produce two-thirds as much
3-methylhistidine as skeletal muscle [31]. It seems
to us that there is every reason to expect that other
smooth musclecontaining tissue (e.g. skin, the
vascular bed and the lungs), together with the wide
variety of non-muscle cells containing actin,
could account for the 20-30% of 3-methylhistidine
excretion not due to skeletal muscle and intestine.
Time course of urine and tissue 3-methylhistidine
labelling (Fig.2)
The existence of substantial non-muscle sources
of 3-methylhistidine is also indicated by the time
course of the labelling of urinary and tissue 3methylhistidine after a pulse of [methyl-’4C]methionine. We initially analysed the urinary
decay curve in terms of the contributions from
various pools [21], and it appears that by frankly
stating that our analysis was not likely to produce
reliable absolute estimates, we caused some confusion in the minds of our critics [32]. In fact the
importance of the data lies not in the magnitude
of the pool sizes obtained from an analysis of the
curve, but in the fact that the urinary labelling
falls to much lower levels than in either muscle or
intestinal 3-methylhistidine [23,24]. This can
only be explained on the basis of substantial nonmuscle sources.
One criticism of our work is that we have
reported a variety of measured values for the rates
of turnover of 3-methylhistidine and the contributions to urinary excretion (e.g. [21-241) so that
there is inconsistency in our published data. Such
an inconsistency has been suggested between our
M. J. Rennie and D. J. Millward
220
20c
150
x
0
*
m
0
2 100
0
L
04
l
*
5
K
PI
5a
0
I
1
lV 5
FIG. 3. Total body 3-methylhistidine production
( 0 , i.e. urinary excretion plus the change in the
free pools of carcass, gut, skin and plasma) and
skeletal muscle protein degradation (a) in groups
of six to eight rats over 4 days of fasting. Values
are means. Error bars for 3-methylhistidine production represent 1 SE. The errors in muscle
protein degradation are difficult to assess as they
are the combined errors for the protein synthetic
rate estimated in individual rats, and the rate of
wasting of muscle protein estimated from the
mean differences in protein content in muscles of
rats killed on successive days; however, they are
unlikely to exceed 12% of the mean value. (Values
from Cotellessa e t al. [ 3 6 ).)
values for the relative labelling of urinary, muscle
and intestinal 3-methylhistidine (Fig. 1) and the
pool size and turnover data (Fig. 2) which we have
used to calculate the extent of tissue contributions.
The mean values for the data points in Fig. 2
between 30 and 6 0 days imply the existence of a
rapidly turning-over, but as yet unassigned, pool
which is much larger than that indicated by the
data in Fig. 2. These differences are taken to
indicate a lack of scientific rigour in our arguments
and are used as justification for their rejection.
We believe this to be unwarranted and it displays a
disregard for the difficulties involved in the
measurement of absolute rates of protein turnover.
In the case of the above example, for instance, it
must be recognized that there are considerable
errors (as shown in Fig. 2) for the actual values
for the labelling of the three pools of 3-methylhistidine; taken together with theoretical considerations relating t o the kinetics and mechanism
of protein breakdown [21,24] these factors suggest that it is fruitless t o make any attempt to fit
the data to a rigid quantitative scheme. However,
this caveat does not influence our main conclusions. The general concurrence of information
from different studies should in itself be seen to
provide strong support for our views.
Taking all the isotopic evidence together we
believe that it is consistent with the existence of
physiologically important contributions to the
whole-body production of 3-methylhistidine from
fast turning-over, non-skeletal muscle pools; the
results cannot be explained simply as methodological artifacts. There is certainly no justification
for arbitrarily increasing our measured values for
rat muscle 3-methylhistidine turnover and consequently raising the presumed skeletal muscle
contributions, as adopted by one advocate of the
case for 3-methylhistidine excretion [32].
Other evidence from animal studies
The question of most importance for confident
use of the method is not whether muscle is the
sole source or not of 3-methylhistidine but whether
changes in urinary 3-methylhistidine give a useful
(i.e. reliable) indication of at least the direction of
changes in skeletal muscle breakdown. In some
cases the methzd does appear to give a reasonable
qualitative description of changes in muscle protein
breakdown. For example, in protein deficiency
there is a fall in 3-methylhistidine excretion [33]
and also in skeletal muscle protein breakdown
[34]. Also, in rats treated with large doses of corticosterone we have confirmed the previously reported [35] transient increase in 3-methylhistidine
excretion, and this is synchronous with a parallel
increase in skeletal muscle protein breakdown
(Odedra el al. [35a]). However, circumstances also
exist in which the output of 3-methylhistidine alters
in the opposite direction to the change in skeletal
muscle protein breakdown. Such paradoxical increases (Fig. 3) in 3-methylhistidine excretion are
observed in young starving rats, in which measurements of skeletal muscle protein content and
protein synthetic rate indicate a fall in both muscle
protein synthesis and breakdown over the first 3
days of starvation, during which time 3-methylhistidine production (i.e. excretion and the increase in
the body free pool of 3-methylhistidine) almost
doubles [36]. Results from current research indi-
3-Methylhistidine and muscle breakdown
cate that the increase in whole-body 3-methylhistidine production is partly due to an increase in
the rate of breakdown of 3-methylhistidinecontaining proteins in gut tissues. At the time of
maximum output of 3-methylhistidine from the
body, it is possible to measure a significant net
output of 3-methylhistidine in blood draining the
gut but there is no detectable efflux of 3-methylhistidine from leg muscle (L. Cotellessa, P. W.
Emery & M. J. Rennie, unpublished results). In
these circumstances interpretation of increased
3-methylhistidine excretion as an increase in
skeletal muscle protein breakdown would be totally
misguided.
Evidence from studies in man
The relative contribution of muscle to the lean
body mass is greater in man than in the rat. Thus
it may have been expected that problems of interpretation of 3-methylhistidine excretion data
would be less in human studies. There is growing
evidence that this is not so. For example, Afting
and co-workers showed that there was considerable residual excretion of 3-methylhistidine and
creatinine in a paralysed patient, who on later
autopsy was declared to have no detectable skeletal
muscle [37]. The 3-methylhistidine excretion
could have accounted for between 20 and 80%of
the range of excretion in normal subjects. There
are many difficulties in the interpretation of these
values and we do not wish to use them to derive a
‘correct’ value for the contribution of skeletal
muscle to the excretion rate of 3-methylhistidine;
rather, we believe this kind of information to be
highly suggestive of a significant contribution of
non-skeletal muscle pools of 3-methylhistidine,
thereby ruling out the possibility of the method
providing a definitive measure of muscle protein
breakdown in man.
The most obvious application of the 3-methylhistidine method is to circumstances in which
there is gross muscle wasting. It is not surprising,
therefore, that the method was rapidly taken up
for studies of muscle protein breakdown in a
variety of muscle diseases, most notably Duchenne
muscular dystrophy. Many groups of workers have
now shown that although the daily excretion of
3-methylhistidine is lower than normal in affected
children, the urinary 3-methylhistidine/creatinine
ratio is elevated [38-441. This has been interpreted
as showing that the fractional breakdown of myofibrillar protein is elevated, with the absolute rate
of breakdown (i.e. grams of muscle protein
degraded per unit time) being depressed because of
a similar muscle mass.
221
However, we cannot accept the validity of the
3-methylhistidine/creatinine ratio as an expression
of fractional breakdown in circumstances of reduced muscle mass. There is evidence from
measurement of the excretion of creatine, creatinine and guanidino compounds that the rate of
synthesis by liver and kidney of creatine (which
becomes trapped in muscle as creatine phosphate)
is controlled to match the muscle mass in patients
with wasting diseases [45]. Thus if the loss of lean
tissue is largely confined to muscle such a loss will
cause a disproportionately greater fall in the
excretion of creatinine than of 3-methylhistidine,
assuming that a constant amount of 3-methylhistidine is contributed from, non-skeletal muscle
sources. It can be calculated, for example, that
with the muscle mass only 25% of normal, the
3-methylhistidine/creatinine ratio would double
without any alteration in turnover rate of any
muscle proteins at the time of measurement of the
urine 3-methylhistidinelcreatinine ratio.
More concrete reasons for doubting the interpretations based on the urinary ratio values is
provided by measurements of synthesis rate of
mixed muscle protein taken in needle biopsy
samples after infusions of L-[ l-’3C]leucine [44]. In
normal children who are growing, skeletal muscle
protein synthesis ought to be greater than in adult
men, but we found the rate in mixed muscle from
boys with Duchenne muscular dystrophy (n = 11,
aged 5-18 years) to have a mean value only onethird of the adult value [44,46]. It is apparent
both from clinical inspection and from measurement of the muscle mass on the basis of creatinine
excretion and % measurements that muscle mass
in these children does not fall markedly beyond
the age of 5 or 6 years [47]. Thus an elevated rate
of muscle protein breakdown can only be reconciled with the lack of ongoing muscle wasting if
muscle protein synthesis is elevated. We have
examined our methods for measurement of muscle
protein synthesis in these children and can find no
easily identifiable error which would justify the
threefold correction necessary to increase the
value obtained for dystrophic muscle to the
normal value in adult men.
Particular criticisms of values for protein synthesis obtained in children with muscle disease have
been levelled: these include the possible interference in the method from collagen-synthesizing
cells and our assumption that the labelling of the
precursor pool for muscle-protein synthesis was
similar to that of blood a-ketoisocaproate. In
answer to the first criticism we must reemphasize
that our measurements were made on alkalisoluble protein extracted from muscle biopsy
samples after gross dissection of fat and connective
M. J. Rennie and D. J. Millward
222
tissue from the frozen sample. In any case there is
evidence that the synthesis of collagen by muscle
taken from affected patients proceeds faster than
the synthesis of other classes of protein [48]. AS
far as the precursor pool is concerned we have
made a limited number of measurements of the
labelling of the free pool of leucine and a-ketoisocaproate in muscle biopsy samples. The labelling
of the free amino acids from muscle was about
85% of the labelling in plasma and was very similar
to the labelling of the a-ketoisocaproate in blood;
the labelling of the a-ketoisocaproate in blood and
muscle were identical [48,49].
We therefore feel that the discrepancy between
the measured rate of muscle protein degradation
(as the difference between net wasting and protein
synthesis) and the assumed rate inferred from the
3-methylhistidine/creatinine ratio can only be
reconciled if the 3-methylhistidine/creatinine ratio
is not a good index of muscle protein breakdown.
In addition to the effect of reduced muscle mass
discussed above, we have some reason to think
that the turnover in non-skeletal muscle tissue in
children with Duchenne muscular dystrophy is
more rapid than usual [44], and this would exacerbate the elevation of the urinary 3-methylhistidine/
creatinine ratio.
On the basis of studies in a variety of neuromuscular disases (see Table 1) as well as in cancer
and malnutrition we have to come to the conclusion that a depression of protein synthesis is the
main cause of wasting in man, and that protein
breakdown in muscle is not elevated, despite the
occurrence of an elevated 3-methylhistidine/
creatinine urinary ratio. That these elevations in
the ratio are artifactual is strongly suggested by
TABLE
recent information obtained by Rothig et al. [SO].
They showed that by correcting the measured excretion of 3-methylhistidine and creatinine (on the
basis of the excretion of these substances from
their patient without detectable skeletal muscle)
there was no longer any evidence of an elevation in
the ratio measured in urine from patients with a
variety of muscle diseases [SO]. This is strong corroborative evidence of the artifactual nature of the
elevation in the ratio.
Perhaps the most direct evidence that urinary
3-methylhistidine is not a valid index of skeletal
muscle production of 3-methylhistidine is from
recent measurements we have made in comparing
urinary excretion of 3-methylhistidine with the
production from the leg of 3-methylhistidine,
measured by estimation of the difference between
arterial and femoral venous 3-methylhistidine concentration with concomitant measurement of leg
blood flow. As shown by others [51], surgical
trauma results in an increased excretion of 3methylhistidine, which falls during postoperative
nutritional support, and we confirmed this. Despite
this increase in whole-body production rate, the
efflux of 3-methylhistidine from legs of patients
studied 1 day after major abdominal surgery
showed a depression by 50% when compared with
values obtained before surgery (Fig. 4). The
patients were on a meat-free diet before surgery
and were sustained by total parenteral nutrition
after surgery. All measurements were made after
overnight fast. Tyrosine efflux from muscle, an
indication of net amino acid balance, increased as
a result of the operation, in contrast with the fall
in 3-methylhistidine. These results together indicate that there was an increase in the net loss of
1. Muscle protein synthetic rate in healthy adult men and patients with wasting
diseases of muscle
All patients were studied in the fed state. Muscle mass in all patients appeared to be
steady over periods up to 2 years on the basis of 24 h creatinine excretion and 40K
measurements. For methods see references [44,49 J .Values for myotonic dystrophy are
from [ 4 6 ] and unpublished work of R.C. Griggs, M.J. Rennie, D. Halliday & R.H.T.
Edwards.
Skeletal
10’X Urinary
Muscle mass
muscle protein 3-methylhistidine/ (%of normal)
synthetic rate creatinine molar
ratio
Normal men, fed (n = 7)
Normal men, fasted 18 h ( n = 7)
Boys with Duchenne muscular dystrophy
(n = 9)
Adult limb girdle dystrophy (n = 1)
quadriceps
calf
Myotonic dystrophy, adults (n = 6)
0.198 f 0.055
0.098 t 0.043
0.055 t 0.033
0.120
0.020
0.086 f 0.041
19.0
17.6
49.1
* 2.4
f
k
1.9
8.5
26.2
27.2
f
7.8
96.2
12.5
30.6 k 15.9
57
58
* 21.0
3-Methylhistidine and muscle breakdown
0 0*.
\
/
%
%
0
--a
0
0
223
We have also shown that in malnourished and
cachectic patients, and in patients with acute
infection, 3-methylhistidine efflux is depressed
from normal when tyrosine efflux is elevated [52].
0
.
' P < 0.01
Conclusions
P < 0.01
9
a, P<O.Ol
p<0.01,=
\
/
\
\
/
0
\
'U'
'
/
'n, \\
0
0
< 0.01
\
\
0
,'P
\
P
< 0.1
*
FIG. 4. Femoral venous-arterial differences of
tyrosine and (b) 3-methylhistidine and ( c ) 3methylhistidine excretion in 10 surgical patients
studied before, 1 day after and 4-6 days after
(a)
major abdominal surgery. Values are means. Values
for the changes between the pre-operative and
immediately postoperative samples are significantly
different (P< 0.01) by paired one-tailed t-test.
The postoperative values are also significantly
different except for the excretion values, which
just fail to attain significance by paired t-test
(P= 0.1). Leg blood flow measured by plethysmography was not significantly different on the three
occasions. For methods see [ 5 11.
amino acids from muscle as a result of surgery and
since myofibrillar protein breakdown apparently
decreased, protein synthesis must have also decreased to allow for net proteolysis. After 3-6 days
of total p a r e n t e d nutrition all of the values
became normal, i.e. 3-methylhistidine efflux rose,
tyrosine efflux from the leg fell and the wholebody 3-methylhistidine production rate fell. In all
circumstances the urinary 3-methylhistidine/creatinine ratio showed changes in the same direction as
changes in total 3-methylhistidine output.
These last-mentioned studies of leg exchange and
urine output of 3-methylhistidine provide the
most unequivocal demonstrations to date of
the discrepancy between urinary excretion and
skeletal muscle protein degradation. We believe
that such demonstrations, taken with the evidence
previously presented above, leave little room for
doubt that the use of 3-methylhistidine production in urine or of the urinary 3-methylhistidinel
creatinine ratio as indices of skeletal muscle
protein breakdown should be discontinued. However, we believe that the measurement of arteriovenous differences of 3-methylhistidine across
skeletal muscle can be used to provide useful
information about the regulation of myofibrillar
protein breakdown, and this method deserves
wider use.
Acknowledgments
This work was supported by the MRC, Muscular
Dystrophy Association Inc., Muscular Dystrophy
Group of Great Britain, Abbott Laboratories Inc.,
The Stanley Thomas Johnson Foundation and The
Nuffield Foundation. M.J.R. is a Wellcome Senior
Lecturer. We are grateful for the help and encouragement of our colleagues R. C. Griggs, D. Halliday, K. Lundholm, P. Emergy, P. C. Bates and
R. H. T. Edwards.
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