In vitro Study of Muscle Aerobic Metabolism in Chronic Fatigue Syndrome
Wilhelmina M. H. Behan, MD, FRCPath, FRCP J Holt, PhD David H. Kay, MBChB Pamela Moonic, BSe
Wilhemina M. H. Behan is Senior Lecturer in Pathology , Glasgow University,
Glasgow, Scotland, UK.
Ian J. Holt is Roval Society University Research Fellow, Department
of Biochemical Medicine, Ninewells Medical School, Dundee, Scotland, UK
David H. Kay is House Officer, Western Infirmary, Glasgow, Scotland, UK.
Pamela Moonie is Research Assistant, Department of Biochemical Medicine,
Ninewells Medical School, Dundee,
Scotland, UK
This study was supported entirely by the Persistent Virus Disease Research
Foundation. The patients studied had
previously been investigated with life kind financial assistance of
the Barclay Trust of GIasgow University and the Myalgic
Encephalomyelitis Association. The authors gratitude is also due to
Professor Peter Behan for advice and access to his
patients and to Dr. Ian McKay for the statistical analyses.
Journal of Chronic Fatigue Syndrome, Vol. 5( 1) 1999
1999 by The Haworth Press, Inc. All rights reserved.
ABSTRACT. The purpose of this study was to establish if muscle
acrobic metabolism is normal in chronic
fatigue syndrome (CFS). Myoblast cultures from muscle biopsies of 16
patients with CFS and 10 healthy
controls were established. Micromethods were used to determine the
lactate/pyruvate (L/P) ratio,
respiratory chain function and cytochrome oxidase and latic dehydrogenase
activities. Mitochondrial
DNA (mtDNA) volume was measured and mtDNA rearrangements . The results
showed that myoblasts
from ten of 16 cases of CFS had defects in acrobic metabolism: two
had increased L/P ratios, suggestive of
a defect in oxidative phosphorylation while eight had decreased ratios,
consistent with it deficiency in
pyruvate dehydrogenase. There was it statistically significant broader
range of L/P ratios in the patients’
cultures, compared to controls (p = 0.0 I 1). No mtDNA rearrangernents
were present. This in vitro study
confirms that there is convincing evidence of mild aerobic defects
in skeletal muscle in some cases of CFS.
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INTRODUCTION
The chronic fatigue syndrome (CFS) is a puzzling disorder Of unknown
cause in which the most severe symptom
is that of fatigue, made worse by exercise and accornpanied by myalgia
(1-3). There is considerable debate as to
the nature of the fatigue, since in spite of the fact that muscle appears
to be the tissue most severley involved,
there is no evidence of histological damage (4) or of 'dysfunction
(5- 10). Because of this, a central nervous
system (CNS) effect, i.e., lack of motivation, was considered to be
the major factor. However, phosphorus
nuclear magnetic resonance spectroscopy (3111 NMR) which provides an
excellent method for continuous in vivo
monitoring of intracellular energy metabolism has now demonstrated
conclusiveIv that there is a significant
reduction in exercise capacity in some cases, an observation that points
to a subtle mitochondrial abnormality (11-15).
We decided to exclude any CNS effects in these patients by analyzing
muscle oxidative capacity in
vitro in long-term myoblast cultures. Well-established micrormethods
now offer the potential for detailed examination of acrobic metabolism,
including respiratory chain function. Estimation of latic and pyruvate
production and assays of cytochrome oxiase and latic dehydrogenase activities
in such a system (16). We compared the bioenergetics of muscle cell cultures
from 16 cases of CFS to those of 10 healthy controls. Total mitochondrial
DNA (mtDNA) volume was also measured and two mtDNA rearrangements and one
point mutation sought. Histological, histochemical and ultrastructural
examination was carried out on all biopsies.
MATERIALS
Patients with CFS
Sixteen patients were studied, seven males (aged 25-49 years) nine females
(aged 19-40 years).The diagnose was established according to CDC criteria
(2): they all had overwhelming fatigue, with myalgia, made worse by
neuropsychiatric symptoms, i.e., inability to concentrate, short term
memory loss and disturbance in sleep patterns, present for at least 6 months
up to 7 years (mean 2 years). All cases had begun with an acute febrile
illness, in ten
consisting of a ‘flu-like’ illness, in two each gastroenteritis or
an upper respiratory tract infection, and in one each, shingles or vertigo
(see Table 1). They were all investigated to exclude the many other conditions
which enter the
differential diagnosis of CFS with each patient having a detailed physical
and neurological assessment followed by routine laboratory investigations
and muscle biopsy.
Healthy Controls
At the time the muscle biopsy was taken, ten of the patients were accompanied by a friend or relative who also underwent a biopsy. The study was approved by the Ethics Committee of the Institute of Neurological Sciences Glasgow. these control Subjects were all healthy; none took any unusual physical activity but none were sedentary. The group comprised seven females (17-60 years) and three males (37 to 43 years).
Myoblast Cultures
Needle muscle biopsies (three cores) were taken from the right or left
vastus lateralis muscle, under local anesthetic (2% lignocaine), using
a muscle biopsy needle, as described (17). Two cores were placed immediately
in
Dulbecco's modified Eagle's rnedium (DMFM) and myoblast culturesinitiated
within four hours of biopsy. Initially the culture medium consisted of
DMEM supplemented with 30% foctal claf serum and 1% chick embryo extract
(CEE)
with uridasc. On the second pass, the scrum concentration was decreased
to 20% and CEE omitted: on subsequent passes the medium was 90% DMEM, 10%
seruim, with uridase.
METHODS
Aerobic Capacity
Pruvatic production was determined by measuring the oxidation of nicotinamide
dehydrogenase (NADH) in the presence of lactic dehydrogenase (1.DH) in
RPMl 1640 medium after 48 hours incubation with cultured cells, and
lactate production was measured similarly, except that the reduction
of NAD was determined in the presence of hydrazine hydrate 0.1% (v/v),
the methods were adapted from Noll (18). The respiratory capacity of' intact
patient-derived and control cell lines was measured by incubating cells
in medium without glucose (RPMl 1640 Life Technologies) in an oxygen electrode
chamber with stirring at a concentration of 5 x 106 cells/ml. Tile rate
of oxygen which allows the contribution of specific respiratory chain cornp1cxcs
is to be asscessed, was measured as described (19) and modified (20). All
oxygen consumption rates were expressed as fmol 02/min/cell. Cytochrorne
C oxidase (COX) activity was measured as described (21).
DNA Analysis
DNA was extracted from a 20 mm3 cube of tissue snap frozen in liquid
nitrogen followed by restriction endonuclease digestion, electrophoresis
and Southern blotting (22), The muscle samples were screened for rearrangements
of mtDNA and for the point mutation at nucleotide pair 3243 and two specific
deletions of mtDNA were sought using ploymerase chain reaction (PCR). Four
primer sets were used: 11, 12; Milo A and Milo B; EDL and EDR;. And D1
and D3 (sequences are given in Table 2). Mito A and B amplify a 121 bp
fragment of mtDNA that lies upstream of the deletion and thus is representative
of total cellular rntDNA. 11 Ind 12 amplify a band of 122 bp that lies
inside the deleted
area and can therefore only be generated from wild-type mtDNA Whcrc
the mtDNA 4977 deletion is present, primers EDI and EDR yield a product
of 193 bp. Similarly, D I and D3 amplify a band 42.3 bp long only when
the deletion of mtDNA 7436 is present.
Histological, Histocheinical and Ultrastructural Studies
The remaining part of the muscle cores was orientated on a cork disc to form a cylinder 25 mrn2 in cross-section, surrounded by Tissue-Tek and frozen in arcton, precooled in liquid nitrogen. Sections (5 *m) were then prepared and stained as described (17). For electron microscopic examination, the specimens were left at 4o C overnight. Then, after washing and treatment with 1% OsO4, the tissue was dehydrated using graded alcohols, impregnated with Araldite resin and polymerised in fresh resin mixture overnight at 60 o C. Sections (80-90 nm),Were cut, stained in the conventional manner using uranium and lead and 6xamined in a Philips CM10 electron microscope.
RESULTS
The results are shown in Figure l. Myoblast cultures from the group
Of patients with CFS showed a mild defect in energy
metabolism compared to normal controls, when assayed for the ratio
of lactate/pyruvate (L/P) production. This difference is not statistically
significant but what is statistically significant is the wider dispersal
of results in the CFS group (p= 0.011 by the F varianceratio or F-test)
which is interpreted to mean that the patients comprise a hetero-geneous
group in which more than one mild energy defect may be present.LactatelPyruvate
Ratio This is shown in Figure I with the actual values and descriptive
statistics in Table 3. Analysis of the L/P ratio from the cultured muscle
cells showed a range from 24 to 39 in the controls, compared with 14 to
49 in the patients' cells. There was no suggestion that the two populations
were distributed round different means or different medians but the patient
group was spread more widely than controls. If we adopt the null hypothesis
that the two populations have the same variance and assume that they are
normally distributed,
then the difference between the groups is statistically significant
(p=0.011 by the variance-ratio or F test).
When the distribution of the L/P ratios is examined further, it is apparent that the results can be divided into three groups: increased, normal and decreased, compared to healthy controls.
Muscle cells from two patients (P 3 and 14) had increased ratios, of 47 and 49. While these are much less than are found in patients with typical mitochondrial myopaithies, nonetheless they are in keeping with a defect in mitochondrial respiration. A second group of 8 cases (P4-9 and 13) had mildly decreased ratios, from 14 to 23. These results raise the possibility of a defect in pyrLivate dehydrogenase (PDH) and are strongly against any defect in oxidative phosphorylation. The final group of six cases all fell into the normal range.
The patients and healthy controls did not differ significantly in the amount of lactate produced. There was, however, sonic indication of a difference in the pyruvate concentrations which were increased in the patients' cultures (p=0.053 by the t-test; p = 0.042 by the Mann-Whitney test).
Minor, non-specific abnormalities were found in muscle biopsies of the
group with decreased L/P ratios
(P2, P4-9 and P13). This group comprised 4 females, aged 40-44 years,
with disease durations from 9
months to 3 years and 4 males, aged 24, 25, 34 and 42 years, with disease
duration from 7 months to 5
years. Paracrystalline mitochondrial inclusions were detectable in
the muscle biopsy of P5, while increased
numbers of pleomorphic milochondria with proliferation of cristae,
were found in two others (P8 and 13).
Increased lipid deposition was conspicuous in three cases (P2, 5 and
6). Focal atrophy of type 2 fibres was
also detected in five of the eight (PS, 7, 8, 9, 13). No ragged red
fibres were present and the enzyme stains
were within normal limits.
In contrast, the two cases with increased L/P ratios (P3 and 14) had no abnormalities on muscle biopsy. One of the six cases with a normal LIP ratio showed plentiful, pleomorphic mitochondria on ultrastructure, associated with mild non-specific atrophy. Similar atrophy was present in another (PI1); the other biopsies in this group were normal. No abnormalities were detected in biopsies from the control group.
Respiratory Chain Function
Measurements of respiratory chain function in intact and permeabilised cells showed no significant difference between patients with CFS and healthy controls. Measurements of LDH or COX activity revealed no difference between the two groups. Although growth curves revealed sonic variation in the generation time between the different cell lines, similar variation was seen in control and patient-derived cell lines (data not shown). Thus, there was no suggestion that patient-derived cell lines with low- L/P ratios were growing more slowly than those of controls.
Mitochondrial DNA
Quantitative analysis of mitochondrial DNA levels demonstrated a small
but insignificant increase in
patients with CFS, compared to healthy controls. None of the patients
or disease controls had deletions in
MtDNA4977 or mtDNA7436by PCR or any other large scale rearrangements
by Southern blotting.
Moreover, all were negative for the mtDNA mutation at np3243 associated
with MELAS.
DISCUSSION
The results reported here are in agreement with the recent specific
and sensitive exercise studies (11-15,23) in demonstrating that mild mitochondrial
abnormalities are present in CFS, and they also support the view that this
is a heterogeneous syndrome, with different defects in aerobic metabolism
detectable, All our cases
conformed to the strict CDC diagnostic criteria and none had previously
had a diagnosis of a mitochondrial disorder.
This is the first study in which continuous, long-term assessment of
muscle respiration has been made. The most informative test of those carried
out proved to be assessment of the cells' redox state, derived by comparing
the steady, state levels of lactate and pyruvate in the spent medium and
obtaining the L/P ratio. Our
results show that the L/P ratios were spread significantly more widely
(p = 0.011) in the samples from patients with CFS compared to controls.
There was no evidence that the two populations were distributed around
different means or
medians and therefore the patient group was the more heterogeneous.
Further analysis of the data indicated that the CFS results could be divided
into three categories: muscle cells from tw6 patients had increased ratios,
suggestive of
mild mitochondrial dysfunction, while eight had decreased ratios, raising
the possibility of pyruvate dehydrogenase (PDH) deficiency. The remaining
six myoblast cultures had a ratio within the same range as the controls.
High L/P ratios are seen when respiratory chain function is defective.
We did not find such a defect by enzymatic assays of respiratory chain
complexes in the patient group but these tests are less specific. An earlier
study by one of us, on
cells in which partially duplicated mitochondrial DNA was known to
be present, also revealed no decrease in respiratory chain function on
enzymatic assays although a greatly increased L/P ratio based on measurement
of lactate and
pyruvate production by these cells clearly indicated mitochondrial
dysfunction with increased dependence on glycolytic ATP production (25).
Both L/P ratios and direct measurement of respiratory chain function in
our patient cohort were
normal. In theory, an increased L/P ratio could be due to a decrease
in pyruvate production rather than an increase in lactate, but this was
shown not to case by analyzing the actual amounts of lactate and pyruvate
produced.
The L/P ratio in cultured cells has been used extensively in studies
of Infants with severe metabolic defects (16,26-29) and we have used it
for Detailed investigation of two severe infantile cases of mitochondrial
myopathy and NARP
(neuropathy, ataxia, retinitis pigfmentosa), respectively. In the former,
the ratio was more than 200, associated with a marked decrease in respiratory
chain activities (complexes I and IV) (Holt and Young, unpublished studies)
while in NARP, the L/P ratio was approximately 100 (Hold and Harding, unpublished).
Clearly we would not have predicted L/P ratios over 100, similar to such
severe infantile cases, in the patients with CFS but there is no doubt
that the assay was working as expected.
Eight of our cases (P 2, 4-9 and 13) had L/P ratios lower than any in
the control group. Analysis of all their results makes it unlikely that
they had a defect in oxidative phosphorylation but raises the possibility
of PDH deficiency. In the remaining six cases, the cells revealed no abnormalities
in L/P ratios, production of lactate - and pyruvate, or activity of cytochrome
oxidase and the other complexes. Therefore, despite fulfilling the agreed
criteria, these cases appear to lack a measurable metabolic defect, at
least with regard to the tests applied in this study. A defect cannot be
completely excluded,
however, because of the remote possibility that the satelite cells
from which the cultures were derived, did not express the deficiency: it
has been shown previously that even severe mitochondrial defects may be
restricted to certain muscle fibres only (30). The clinical features of
the patients in these different subgroups did not differ: all complained
of the abrupt onset, after an infectious illness, of severe fatigue with
rnyalgia, made worse by exercise. Defective aerobic metabolism is suggested
but the picture is unlike that in typical mitochondrial disorders where
the patients are symptom-free until they
exercise. Similarly, none of our cases hud ragged-red fibres on biopsy,
one of the defining characteristics of rnitochondrial myopathics, However,
nine of the 16 cases did show mild non-specific ultrastructural abnormalities
in their biopsies: interestingly enough, seven of these belonged to the
group with low L/P ratios in whom the possibility of PDH deficiency was
raised: the changes seen-rare paracrystalline inclusions, numerous pleomorphic
mitochondria with proliferation of cristae and increased lipid, are all
reported in this condition (24). Two of the six cases with L/P ratios in
the normal range also showed changes: increased lipid and plcornorphic
mitochondria in one, and atrophy of Type 2 fibres
in the other. No abnormalities were present in the remainder, including
the two cases with increased L/P ratios. However, even in otherwise typical
mitochondrial disorders, morphological changes may be minimal or absent,
No ultrastructural abnormalities were present in any of the muscle biopsies
from the, healthy controls. We postulated originally that CFS might be
due to a mitochondrial disorder because of the mitochondrial proliferation
and pleoniorphism identified in a series of cases (17), the same changes
as are, seen in some mitochondrial myopathies. Although these observations
have not
been confirmed (3 1), there is strong support for a mitochondrial role
in CFS based on the functional studies discussed here as well as the work
of Zhang and colleagues, in describing unusual mtDNA deletions in a typical
case (32), the presence of the common mtDNA4977 deletion in two of eight
cases examined (33) and significantly low levels of serum acylcarnitine
reported (34).
There are convincing similarities between CFS and mitochondrial disorders: for instance, the fact that different aerobic defects may be present is typical of the latter where the relationships between the distribution and expression of abnormal mtDNA and the biochemical lesions, are complex and heterogeneous: the same disease manifestations frequently correlate with different enzyme defects (35). Both may be precipitated by viruses (1,3,36) and by exercise (37,38). It is striking how many patients with CFS are described as very fit before onset of the disorder, indeed some are accomplished athletes while others have recently taken up intense exercise. Toxins have been implicated in both: in CFS, the fish poison, ciguatera toxin produces a syndrome identical to CFS in 2% of those affected (39). Leber's hereditary optic neuropathy (LHON) may masquerade as tobacco-alcohol induced amblyopia (40) and a mitochondrial ribosomal RNA mutation has been described in association with aminoglycoside-induced deafness (41).' Recovery may occur in both diseases: in LHON, the eyesight may improve and in benign cytochrome.C oxidase deficient myopathy of infancy, completerecovery can be expected (42) as is also the case in a proportion of those with CFS, especially when precipitated by ciguatera toxin (39). What the final pathogenetic mechanism in CFS may be is unclear: a novel recent hypothesis is that cell membrane abnormalities, specifically of the ion channels, may be involved (43).
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