Clinical Science (1984) 66, 69-78
69
Glycogen content and activities of key glycolytic enzymes in
muscle biopsies from control subjects and patients with
chronic alcoholic skeletal myopathy
F. C . MARTIN, A. J. LEVI*, G . SLAVIN?
AND
T . J. PETERS
Divisions of Clinical Cell Biology and *Clinical Science and the ?Section of Histopathology,
MRC Clinical Research Centre, Harrow, U.K.
(Received 22 November 1982131 May 1983: accepted 25 Jury 1983)
Summary
Introduction
1. The capacity for glycolysisin muscle biopsies
obtained from long-term heavy alcohol drinking
patients has been compared with tissue from
control subjects by assay in vitro of the total
activities of glycogen phosphorylase, phosphofructokinase and fructose 1,6-bisphosphatase,
key regulatory enzymes in the anaerobic glycolytic
pathway.
2. Biopsies from 13 of 22 patients had type I1
fibre atrophy, and the activities of all three enzymes
were reduced in these biopsies, when expressed in
terms of DNA content, the most striking reduction
being in phosphofructokinase activity.
3. The amount of glycogen in the tissue correlated closely with these enzyme activities and was
slightly lower in the most atrophic tissue, when
expressed in terms of DNA content.
4. The activities of acid and neutral a-gluccisidases were similar in biopsies from control
subjects and patients with various severities of
alcohol myopathy.
5. The reduced activities are consistent with a
reduced proportion of type I1 fibre muscle mass in
these patients, and suggest that there may be a
reduced capacity for glycolysis with resultant
reduced lactate production. Whether the changes
in enzyme activities are primary to the selective
atrophy remains to be established.
Alcohol consumption has risen steadily in the U.K.
since 1945 [ l ] , and with this there has been an
increase in alcohol-related illness [2,3] so that
alcohol consumption now accounts for a major
proportion of acute illness seen in general hospitals
[4]. Alcoholic myopathy has been less well recognized than some other forms of tissue damage but
in fact is common among heavy drinkers, usually
in the form of painless proximal muscle wasting
and weakness [5]. Histologically the major feature
is a selective type IIb fibre atrophy [6], which is
reversible with abstention from alcohol [7].
It has been known for some time that there is
a reduced lactic acid response to ischaemic forearm exercise in heavy drinkers [8]. This may be
related to a change in the lactate dehydrogenase
(LDH) isoenzyme pattern [9] or to a reduction in
LDH activity,as has been reported in muscle samples
taken from chronic alcoholics within 1 week of
hospitalization [101. An acquired McArdle’s
syndrome [l 11, i.e. reduction of glycogen phosphorylase activity, has been suggested as an alternative explanation of the reduced ischaemic
lactate response [8] and a previous report based on
morphological assessments suggested that there is
an accumulation of glycogen within the myofibrils
[ 121 of biopsies taken from alcoholics.
This report is concerned with the chemical
determination of glycogen content and the assay
of activities of key enzymes in glycolysis. The
activities of glycogen phosphorylase, which
catalyses the flux generating step for the pathway
as a whole, phosphofructokinase, which catalyses
the main rate-limiting step of glycolysis, and
fructose 1,6-bisphosphatase have been assayed.
The relative activities of the two last-named
Key words: alcohol, fructose 1,6-bisphosphatase,
a-glucosidase, glycogen, glycogen phosphorylase,
glycolysis, muscle diseases, 6-phosphofructokinase,
Correspondence: Dr F. C. Martin, Department
of Medicine, Royal Postgraduate Medical School,
Du Cane Road, London W 12 OHS.
70
F. C. Martin et al.
enzymes are important because a glycolytic rate
amplification mechanism may exist in the form of
a substrate cycle between fructose 6-phosphate
and fructose 1,6-bisphosphate [ 131. Glycogen can
also be hydrolysed by lysosomal acid 1,4amyloglucosidase and possibly by a neutral a-glucosidase
[ 141. A deficiency of the former enzyme has been
reported in association with a reversible myopathy
in hypothyroidism [ 151, a condition also characterized by an accumulation of glycogen and atrophy
of type I1 fibres [16], and also with type I1 glycogen storage disease, Pompe's disease [ 171.
Materials and methods
Subjects
Thirty subjects were studied. Twenty-two
patients, 12 men and 10 women, had consumed at
least 100 g of ethanol daily for at least 3 years.
They had been admitted to hospital because of
physical or psychological problems associated
with their alcohol consumption. Patients with
biochemical evidence of osteomalacia, hypothyroidism, or serum albumin below 30 g/l, or
markedly deranged clotting function which
would have made muscle biopsy unsafe, were
excluded from the study.
Eight control subjects were studied. Three
were volunteers: healthy male hospital workers,
and five (three men and two women) were nonalcoholic inpatients having a muscle biopsy performed as part of the investigation of muscle or
systemic symptoms, and in whom there was no
clinical or histological evidence of neuromuscular
disease, after clinical and laboratory examinations.
Muscle biopsies were obtained from each
subject with a UCH Muscle Biopsy Needle (Needle
Industries Ltd, Studley, Warwickshire, U.K.), by
a standard technique [ 181, from the vastus lateralis
muscle. Biopsies were obtained before breakfast
after an overnight fast with no exercise before the
biopsy. Biopsies for assay of a-glucosidase activities
were obtained from two separate series of male
subjects each satisfying identical clinical criteria to
those above. Biopsies from five patient controls
and 12 alcoholic patients were obtained for acid
maltase assays, and biopsies from eight patient
controls and 17 alcoholic patients were obtained
for neutral a-glucosidase assay.
Each patient gave informed written consent
and the study as a whole was approved by the
Harrow Health Authority Ethical Committee.
Treatment of biopsies
Each biopsy (30-60mg wet weight) was
divided into two portions. One was allowed to
relax at room temperature for 15 min and then
quick frozen in liquid nitrogen for later histological and histochemical assessment as previously
described [12]. Sections were stained with (i)
routine histological stains and evidence sought
of inflammation, necrosis or non-alcoholic muscle
disease, and (ii) myosin Mg*'-ATPase reaction at
pH 9.4 to allow differentiation of fibre types I
and 11. These sections were then studied with a
Magiscan image analysis system [ 191 combined
with an interactive computer program to determine
the median diameters, and the atrophy and hypertophy factors for fibre types I and 11. The atrophy
factor was calculated in a standard way [20] and
gives a numerical weighted expression to the size
and proportion of small fibres (diameter less than
40 ,um) of each type within the biopsy.
The other portion was stored at -180°C in
liquid nitrogen for up to 4 weeks in 0.1 ml of a
homogenizing medium containing triethanolamine
(50 mmol/l), magnesium chloride (2 mmol/l),
dithiothreitol (2 mmol/l) and NazEDTA (1 mmol/l)
adjusted to pH 7.4 with hydrochloric acid (0.1
mol/l). On the day of enzyme assay, each thawed
biopsy specimen was homogenized in 0.2ml of
the homogenizing medium containing 0.1%
deoxycholic acid. This was performed at 0°C by
ten strokes of a ground-glass homogenizer of
0.1 ml capacity [Jencons (Scientific) Ltd, Mark
Road, Heme1 Hempstead, Herts., UX.].
Biochemicals and enzymes
Chemicals and enzymes were obtained from
Sigma London Ltd, unless otherwise indicated.
Enzyme assays
Spectrophotometric methods of assay were
used in which enzyme activity was coupled to the
oxidation or reduction of NAD'or NADP+,and was
followed by the change in absorbance at 340 nm
in a double beam Perkin-Elmer 557 spectrophotometer. All assays were performed at 37°C and
incubates were pre-equilibrated for 2 min in a
water bath before addition of substrate. The rate
of change of absorbance of the assay mixture was
compared with that of a suitable blank incubation
mixture from which substrate had been omitted.
Each assay was done in duplicate. Activities are
expressed as m-units, where a m-unit of activity
produces 1 nmol of product/min incubation at
37°C. Activities are generally expressed in terms of
the DNA content of biopsies, which was estimated
by a rapid fluorimetric method [21] with calf
thymus DNA as standard.
Glycolytic enzymes in alcoholic myopathy
6-Phosphofructokinase(EC 2. Z 1.1 1). This assay
was based on a modification by King et al. [22] of
the method of Opie & Newsholme [23].
Fructose 1,6-bisphosphatase(EC3.1.3.11.).This
was based on a modification [22] of the method
of Newsholme & Crabtree [24].
Glycogen phosphorylase (EC2.4.1.1.). This was
based on a method of K. Gohil, D. Jones & R. H. T.
Edwards (unpublished work). The assay mixture
(1 ml) contained potassium phosphate buffer (50
mmol/l, pH7.2), magnesium chloride (1 mmol/l),
AMP (5 mmol/l), glucose 1,6-bisphosphate (50
pmol/l), glycogen (5 mmol of glucose residues/l),
NADF (0.5 mmol/l) and glucose 6-phosphate dehydrogenase (EC 1.1.1.49; 0.6 unit). The conditions
for the assay were established from preliminary
experiments.
Glycogen determination. The method was
modified from that reported by Edwards et al.
[25] in that both acid insoluble and acid soluble
fractions were assayed.
The glycogen was hydrolysed by overnght
incubation with a-amylase (EC 3.2.1.1) and
amyloglucosidase (EC 3.2.1.3) and the released
glucose was assayed by the glucose oxidase-horseradish peroxidase technique of Dahlquist [26].
The total glycogen content of the initial homogenate (both soluble and acid insoluble) was
expressed in terms of glucose residues (pmol).
Acid maltase (a-Bglucosidase) (EC 3.2.1.20).
Muscle samples for this assay (approximately 30 mg
wet weight) were stored in NaCl (0.15 mol/l) at
-2OOC and subsequently homogenized in 3 ml of
ice-cold NaCl (0.15 mol/l) in a 7 ml ground-glass
homogenizer (Kontes Glass Co., Vineland, NJ,
USA.), with ten strokes of the A pestle (loose)
and eight strokes of the B pestle (tight). The assay
was based on the method of Dahlquist [26]. The
first step involved incubation of homogenate
(0.2 ml) with 0.2 ml of sodium maleate-maleic
acid buffer (0.1 mol/l, pH6.0) containing 56
mmol of maltose0 (Merck, Product no.9955289,
Darmstadt D 6100, West Germany). After incubation at 37OC for 1 h, free glucose was assayed as
described above.
In addition a-glucosidase activity was assayed
with 4-methylumbelliferyl a-D-glucopyranoside as
substrate [27]. The biopsies were taken into 3 ml
of sucrose medium containing sucrose (0.25 mol/l),
NaaEDTA (1 mmol/l) and ethanol (20 mmol/l),
at pH7.4. After storage at -2OOC for up to 6
weeks, the biopsies were homogenized in a Dounce
homogenizer as described above for biopsies
obtained for acid maltase assay.
Two a-glucosidaseactivitiescan be demonstrated
in skeletal muscle. One is localized to the endoplasmic reticulum [27] and the other is lysosomal
71
151
10-
5-
0 - r-
PH
FIG. 1. pH-activity curves for maltase
(0)
4-methylumbelliferyl a-D-glycopyranosidase
activities.
and
(0)
[ 171. pH-activity curves for the two activities are
shown'in Fig. 1. There is negligible 4-methylumbelliferyl a-D-glucopyranosidase activity at pH 4.0,
and maltase activity is low at pH 7.5. The maximum
acid maltase activity is approximately 100-fold the
a-D-ghcopyranosidase activity.
Reproducibility, For these experiments quadriceps muscle obtained by open biopsy at surgery
for hip replacement was used. The coefficients of
variation (defined as the standard deviation
divided by the mean of five pairs of duplicate
determinations from one homogenate) were:
phosphofructokinase 11%; fructose 1,6-bisphosphatase 8.7%; glycogen phosphorylase 2.9%; acid
maltase 6.0%; neutral a-glucosidase 8.1%;glycogen
7.2%.
Statistical analysis of results
Type I1 fibre atrophy factors were used to divide
the subjects into three groups: controls, alcoholics
with type I1 atrophy (atrophy factor > 150 males,
> 200 females) and alcoholics without atrophy.
For each biopsy the mean of duplicate determinations of enzyme activity and of glycogenestimations
were obtained: the individual results are shown in
dot diagrams, and patient groups were compared
with the Wilcoxon unpaired Rank Sum test, as the
variances of the groups were non-homogeneous.
If no significant difference existed between the
patient control and alcoholic normal group, then
these were combined for comparison with the
alcoholic atrophy group in order to increase the
power of the statistical analysis.
Histological and biochemical results were also
analysed by Spearman's Rank correlation, with a
computerized statistical package 1281. Significance
values (degrees of freedom = number of pairs of
values - 2) were obtained from a table of values
F. C. Martin et al.
72
[ 2 9 ] . Correlations were sought between age, type
I1 atrophy factor, median type I fibre area, median
type I1 fibre area, fractional type I1 fibre area (see
below), activities of glycogen phosphorylase,
phosphofructokinase and fructose 1,6-bisphosphatase and glycogen content expressed relative to
DNA content. The fractional type I1 area was
calculated as the mean type I1 fibre area divided
by the sum of the mean type I and mean type I1
fibre areas. This fraction assumed that the area
= d2/4, where d = mean fibre diameter and that
the proportion of fibre types is equal.
Results
Clinical details and histological studies
Table 1 shows the age and sex distribution of
the three groups obtained from the first series of
30 subjects. There is a significantly lower median
age in the alcoholic normal group compared with
the atrophy group. The difference in ages between
the control group and the alcoholic normal group
was not significant, and when these groups were
combined, then there was no overall difference in
age between this summated normal group and the
alcoholic atrophy group. No biopsy in this series of
patients showed evidence of acute rhabdomyolysis,
but 13 showed type I1 atrophy. Fig.2 shows the
individual results of fibre diameter measurements.
No difference for either types I or I1 existed
between the control and normal alcoholic groups.
A slight drop in the median type I fibre diameter
was seen in those patients with severe type I1
atrophy, but this was not significant for the group
as a whole compared with the other two groups,
taken together or separately. Not surprisingly,
there was a marked reduction in type I1 fibre
TABLE 1. Clinical details and histological studies
Median fibre diameters are given as means 4 SD .
Patient category
Number
(M : F)
8
(6 : 2)
9
(5 : 4)
13
(7 : 6)
Normal controls
Alcoholic normal
Alcoholic atrophy
Age range
[years
(median)]
Type I1
atrophy
factor range
Median fibre diameter (pm)
26-68
(47)
31-58*
(35)
37-65
0-142
56.6
i
5.1
54.0
* 4.7
5-177
58.7
i
9.4
49.8
i
1 7 1-1 235
52.8
i
9.5
36.7
* 8.7
Type I
Type I1
5.6
(54)
*Age less than alcoholic atrophy group (P< 0.05).
Type I fibres
8o
Type I1 fibres
1
1
0
h
E
3 60
ee
0
Q0
0
E:
2
8
a0
80
20
0-I
Controls
Normal
Atrophy
Alcoholic
Controls
Normal
Atrophy
Alcoholic
FIG. 2 . Dot diagrams of individual biopsy results for median fibre diameters in control
subjects and alcoholic patients with or without histological evidence of atrophy. Results
of biopsies from males (e) and females (0)are shown.
Glycolytic enzymes in alcoholic myopathy
73
TABLE2 . Clinical details and histological studies of the patients and biopsies taken for
or-glucosidase assays
Median fibre diameters are given as means k SD.
Patient category
Number
(all male)
(a) Acid maltase
Normal controls
5
Alcoholic normal
5
Alcoholic atrophy
I
(b) a-Glucosidase
Normal controls
8
Alcoholic normal
4
Alcoholic atrophy
13
Age range
[years
(median)]
Median fibre diameter bm)
Type I
3.3
Type I1
31-52
(44)
37-62
(48)
44-68
(54)
59.8
i
65.4
* 9.9
51.4
i
33-68
(45)
31-48
(41)
37-66*
(5 2)
63.2 i 5.8
60.4 k 6.9
65.5
f
2.9
58.5
58.0
i
7.1
44.5 i 5.6
6.1
57.2
f
3.1
58.6 i 5.3
46.4
f
f
5.6
6.6
*Age distribution of alcoholic atrophy group higher than combined normal groups (P= 0.06).
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Controls
Normal Atrophy
Alcoholic
Controls Normal Atrophy
Alcoholic
FIG. 3 . Dot diagram of individual biopsy results of total glycogen assay, expressed
either in terms of ( a ) DNA or ( b ) wet weight of muscle. Results of biopsies from males
(e) and females ( 0 ) are shown.
diameter in the alcoholic atrophy group compared
with the other groups.
Table 2 shows the corresponding results from
the other two series, which consisted of male
subjects only. The patients from whom biopsies
were taken for acid maltase activity showed no
significant age difference between the groups, but
those from whom the biopsies were obtained for
neutral a-glucosidase assay showed variation in
that the median age of the alcoholic atrophy group
was slightly older. In neither series of biopsies
were there significant differences in the median
type I fibre diameters.
Biochemical studies
Fig. 3 shows the total glycogen content of the
muscle biopsies from the three patient groups.
F. C. Martin et al.
74
There was less total glycogen content per mg of
DNA and slightly more per g wet weight in the
alcoholic atrophy group than in the alcoholic
normal group. No significant difference was found
for either the acid soluble or insoluble glycogen
fractions between the three groups and the proportion which each fraction contributed to the total
glycogen content did not alter significantly between
the three groups.
Fig. 4 shows the results of assays of fructose
1,6-bisphosphatase and phosphofructokinase. For
the former a difference is apparent between males
and females in the atrophy group. On comparing
male subjects only, the median activity in the
atrophy group (0.88 m-unit/pg of DNA) was not
significantly lower than that of the alcoholic
normal or patient controls (1.36 m-units/pg of
DNA). However, for phosphofructokinase, where
no difference between the sexes was apparent, the
activities in the atrophy group were markedly
different from the combined normal groups.
The ratio of phosphofructokinase activity to
fructose 1,6-bisphosphatase activity in the three
groups is shown in Fig. 5. No difference between
the groups is evident when either both sexes or
only males were analysed. The results of glycogen
phosphorylase assays are also shown in Fig. 5. The
activities in the control group and the alcoholic
normal groups were similar but, when combined,
3
they were significantly higher than the atrophy
group. The median activities were 70.6 and 33.2
m-units/pg of DNA respectively. The results of
or-glucosidase assays are shown in Fig. 6. No
difference exists between the groups for either
activity.
Table 3 shows results obtained from correlation
calculations. None of the enzyme activities or
glycogen concentrations correlated closely with
age or type I fibre area. There is a strong positive
correlation of the activities of phosphofructokinase,
glycogen phosphorylase and fructose 1,6-bisphosphatase with each other and with total glycogen
content. Fractional type I1 area correlated with
glycogen phosphorylase, fructose 1,6-bisphosphatase and phosphofructokinase activities. The results
from atrophic biopsies from male alcoholic subjects
were considered separately because between-sex
differences exist in muscle fibre sizes.
Discussion
The enzyme assays, although complex, have been
demonstrated to be reproducible and readily
applicable to portions of needle biopsy samples.
A total of 15 mg was adequate for the three assays
and glycogen determinations, in duplicate. The
reduced activities of phosphofructokinase and
glycogen phosphorylase in atrophic tissue provide
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Controls Normal Atrophy
Alcoholic
Controls Normal Atrophy
Alcoholic
FIG. 4. Dot diagram of individual biopsy results of ( a ) fructose 1,6-bisphosphatase and
( b ) 6-phosphofructokinase activity. Results of biopsies from males (e) and females (0)
are shown.
Glycolytic enzymes in alcoholic myopathy
(a1
75
(6)
h
-4
z
CI
0
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0
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Controls Normal
Controls Normal
Atrophy
Atrophy
Alcoholic
Alcoholic
FIG. 5. Dot diagram of individual biopsy results of ( a ) the ratio of 6-phosphofructokinase to fructose 1,6-bisphosphatase activity and (b) glycogen phosphorylase activity.
Results of biopsies from males (*)and females ( 0 ) are shown.
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Controls
Normal
Atrophy
Controls
Alcoholic
Normal
Atrophy
Alcoholic
FIG. 6. Dot diagram of individual biopsy results fo I ) acid maltase and (b) neutral
a-glucosidase activity. Results are from male patients only.
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alcoholics [8] and may play a role in the pathogenesis of alcoholic myopathy.
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expressed in terms of wet or dry weight, total
protein or non-collagen protein or DNA content.
However, in a condition where the major patho-
F. C. Martin et al.
76
TABLE 3. Spearman S rank correlations between histomorphornetric data, and results of biochemical analyses are shown f o r all alcoholic patients and f o r male alcoholic patients with atrophy
Correlation
coefficient
No. of
pairs
6-phosphofructokinase
fructose 1,6-bisphosphatase
total glycogen (per mg of DNA)
fructose 1,6-bisphosphatase
total glycogen (per mg of DNA)
total glycogen (per mg of DNA)
0.71
0.76
0.43
0.7 1
0.62
0.46
17
17
17
18
18
18
< 0.01
glycogen phosphorylase
fructose 1,6-bisphosphatase
6-phosphofructokinase
0.98
0.98
0.87
5
5
< 0.05
< 0.05
5
0.06
Parameters correlated
P
All alcoholic patients
Glycogen phosphorylase
Glycogen phosphorylase
Glycogen phosphorylase
6-Phosphofructokinase
6-Phosphofructokinase
Fructose 1,6-bisphosphatase
Male alcoholics with atrophy
Fractional type I1 fibre area
Fractional type I1 fibre area
Fractional type I1 fibre area
vs
vs
vs
vs
vs
vs
vs
vs
vs
logical change is loss of myofibrillary protein,
there may be spurious elevation of concentration
or activity when expressed in terms of any of
those reference parameters except DNA, which
probably does not alter in the biopsies we studied
as there was no evidence of necrosis or an inflammatory cellular infiltrate.
Glycogen
The amount of glycogen detected in the patient
controls (approx. 750 pmol/g dry weight) was
higher than that reported by Edwards er al. [25]
in normal quadriceps muscle biopsies (249 pmol/g
dry weight) but they measured only the acid
insoluble component, which we found to be
approximately half of the total. It is closer to
values reported by Essen et al. [30],who found a
value of 359 pmol/g dry weight from type I1 fibres
and 355 pmol/g dry weight from type I fibres.
Suominen et al. [ 101 have previously reported
glycogen content in the muscles of alcoholics to be
similar to that in control subjects. Our results illustrate the difference in interpretation depending on
the reference parameter chosen. The slight increase
in glycogen per g wet weight in the alcoholic
atrophy group contrasts with the decrease when
expressed per mg of DNA. A relative maintenance
of glycogen content in the presence of a reduced
amount of myofibrillary mass would be consistent
with these results and also with the ultrastructural
appearance of apparent glycogen accumulation
[121.
Enzymes
Reduced levels of glycogen phosphorylase in
the muscle of patients with acute alcoholic rhab-
< 0.01
0.07
< 0.01
< 0.01
0.05
domyolysis has been previously reported and
proposed as the explanation for the observed
reduction of forearm lactic acid production during
ischaemic exercise [8]. The reduced activity we
report in the atrophic biopsies is consistent with
this finding. There is a higher level of glycogen
phosphorylase in normal type I1 fibres than type I
fibres. The observed drop in specific activity is
associated with a selective atrophy of these fibres,
and conclusions about the activity per unit mass of
type I1 tissue remain uncertain. Phosphofructokinase activity in type I1 fibres is approximately
twice that in type I [30].The activity we report for
the patient control group is slightly higher than that
reported in quadriceps muscle of adultsundergoing
elective surgery [22]. The difference is partly
explained by the higher incubation temperature,
37'C, used in the present study, compared with
25°C in that report. The enzyme activity found
in this study is similar to that reported by Opie &
Newsholme in their original work with this assay
method [23]. The reduction in median phosphofructokinase activityin the atrophy group compared
with that in patient controls was similar in magnitude to that for glycogen phosphorylase activity.
The activity of fructose 1,6-bisphosphatase we
report for the patient controls is rather lower than
that of King et al. [22] but similar to that reported
by Newsholme (0.95 pmol min-' g-' wet weight)
[3 11. The reduction in fructose 1,6-bisphosphatase
activity in the atrophic biopsies compared with
that in patient controls was less than that for
glycogen phosphorylase or phosphofructokinase,
but the activity did correlate closely with that of
the other two enzymes.
Reduced values of phosphofructokinase and
fructose 1,6-bisphosphatase in both the rectus
and vastus muscles of severely ill surgical patients
Glycolytic enzymes in alcoholic myopathy
has been reported [22]. No morphometric analyses
of these biopsies were made, but it is possible that
a type I1 atrophy was present in these patients.
No reduction in either a-glucosidase activity
was found in association with type I1 atrophy in
alcoholics. This contrasts with the single case report
of Hurwitz et al. [ 151 and the study of McDaniel
et al. [32], both of which showed a fall in acid
maltase activity associated with a reversible
myopathy of hypothyroidism. In a detailed study
of a case of Pompe’s disease, an increased activity
of hepatic neutral a-glucosidase was reported
[33], perhaps as an adaptation to the lack of acid
maltase. We have found no such change.
Kiessling et al. [34] found reduced activities of
both lactate dehydrogenase and triose phosphate
dehydrogenase in biopsies from chronic alcoholics,
and also showed that the reduced enzyme activities
correlated with the presence of type I1 atrophy.
Neither reduced activity was thought by these
authors to be implicated in the pathogenesis of the
myopathy. Suominen et al. [lo] found reduced
activity of both hexokinase and lactate dehydrogenase in the muscle of chronic alcoholics. The
activities had returned to control levels after 7 days
of abstinence. In a previous series of patients with
alcoholic myopathy and type I1 fibre atrophy, we
found no evidence of reduced lactate dehydrogenase activity [6].
The results reported here suggest that there may
be a reduced capacity for glycogenolysis in the
proximal muscle of heavy drinkers. Such a reduction may be secondary to a loss of type I1
fibre tissue by an undetermined mechanism.
Alternatively reduction in such a capacity might
lead to a metabolically induced disuse atrophy.
Further experiments are clearly needed to investigate the effects of ethanol and its metabolites on
the control mechanisms of glycolysis.
Acknowledgments
We are grateful to Dr R. F. G . J. King for advice on
phosphofructokinase and fructose 1,6-bisphosphatase assays, Dr D. Jones for advice on the glycogen
estimation, P. Ward for technical assistance with
histochemistry, Ms C. Dork for statistical advice
and Mrs M. Moriarty for secretarial assistance. The
support of the Vera Levi Medical Research
Memorial Fund is gratefully acknowledged.
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