Chronic
fatigue syndrome and mitochondrial
dysfunction
Sarah Myhill,1
Norman E. Booth,2 and John
McLaren-Howard3
1Sarah Myhill
Limited, Llangunllo, Knighton, Powys, Wales
LD7 1SL, UK
2Department of
Physics and Mansfield College, University of
Oxford, Oxford OX1 3RH, UK
3Acumen, PO
Box 129, Tiverton, Devon EX16 OAJ, UK
Received December 2,
2008; Accepted January 12, 2009.
This study aims to
improve the health of patients suffering
from chronic fatigue syndrome (CFS) by
interventions based on the biochemistry
of the illness, specifically the
function of mitochondria in producing
ATP (adenosine triphosphate), the energy
currency for all body functions, and
recycling ADP (adenosine diphosphate) to
replenish the ATP supply as needed.
Patients attending a private medical
practice specializing in CFS were
diagnosed using the Centers for Disease
Control criteria. In consultation with
each patient, an integer on the Bell
Ability Scale was assigned, and a blood
sample was taken for the “ATP profile”
test, designed for CFS and other fatigue
conditions. Each test produced 5
numerical factors which describe the
availability of ATP in neutrophils, the
fraction complexed with magnesium, the
efficiency of oxidative phosphorylation,
and the transfer efficiencies of ADP
into the mitochondria and ATP into the
cytosol where the energy is used. With
the consent of each of 71 patients and
53 normal, healthy controls the 5
factors have been collated and compared
with the Bell Ability Scale. The
individual numerical factors show that
patients have different combinations of
biochemical lesions. When the factors
are combined, a remarkable correlation
is observed between the degree of
mitochondrial dysfunction and the
severity of illness (P<0.001).
Only 1 of the 71 patients overlaps the
normal region. The “ATP profile” test is
a powerful diagnostic tool and can
differentiate patients who have fatigue
and other symptoms as a result of energy
wastage by stress and psychological
factors from those who have insufficient
energy due to cellular respiration
dysfunction. The individual factors
indicate which remedial actions, in the
form of dietary supplements, drugs and
detoxification, are most likely to be of
benefit, and what further tests should
be carried out.
Keywords:
Chronic fatigue
syndrome, myalgic encephalomyelitis,
mitochondria, neutrophils, oxidative
phosphorylation
Chronic Fatigue
Syndrome (CFS) is a multisystem illness
that robs its victims of their health
and their dignity. Two of the most
characteristic and debilitating signs of
CFS are very poor stamina and delayed
post-exertional fatigue. Sometimes the
fatigue is mainly mental, and sometimes
mainly physical. Fatigue is the same as
lack of energy and energy comes from the
basic metabolic process of the oxidation
of food.
A widely-held
hypothesis (A) is that the metabolism of
people with CFS is normal, but the
fatigue and other symptoms are due to
psychological factors. It is
acknowledged that physical fatigue is
lack of energy, but mental fatigue is
considered to be a subjective sensation
characterized by lack of motivation and
of alertness [1],
even though the brain is a major
consumer of resting cellular energy.
Patients may demonstrate negative
illness beliefs that increase the
severity of the symptoms [2,
3]. However, if the metabolism is
functioning properly, the fatigue and
related symptoms must be due to energy
being wasted by the mental and physical
processes of stress, anxiety, tension
and depression. Patients should be able
to be helped, possibly cured by
psychological intervention, e.g.
cognitive behavioural therapy. In order
to explain the post-exertional malaise
an ancillary hypothesis (A′) is needed,
namely deconditioning due to disuse of
muscles. However, hypothesis A′ is not
supported by experiment in many cases as
we will see below.
An alternative
hypothesis (B) is that there is a
metabolic dysfunction with the result
that not enough energy is being
produced. The main source of energy
comes from the complete oxidation of
glucose to carbon dioxide and water. The
digestive system produces glucose,
glycerol and fatty acids, and amino
acids. If there is a problem with the
digestive system, e.g. gut fermentation,
hypochlorhydria or pancreatic
insufficiency, energy production will be
impaired and fatigue may result [4].
These conditions can and should be
tested for. Allergies and thyroid
malfunction can also produce fatigue.
When the digestive
system is functioning properly glucose
and lipids are fed into the blood stream
where, together with oxygen bound to
hemoglobin in erythrocytes (red blood
cells), they are transported to every
cell in the body. In the cytosol of each
cell glucose is broken down in a series
of chemical reactions called glycolysis
into two molecules of pyruvate which
enter the energy-producing organelles
present in most cells of the body, the
mitochondria. Some structural details
and the number of mitochondria per cell
are dictated by the typical energy
requirements; cardiac and skeletal
muscle cells and liver and brain cells
contain the highest numbers. The
mitochondria generate energy by
oxidative metabolism in the form of ATP
(adenosine triphosphate) which when
hydrolysed to the diphosphate, ADP,
releases energy to produce muscle
contractions, nerve impulses and all the
energy-consuming processes including the
chemical energy needed to synthesise all
of the complex molecules of the body [5,
6]. Thus, mitochondrial dysfunction
will result in fatigue and can produce
other symptoms of CFS.
The two hypotheses
are not mutually exclusive. Some
patients may satisfy both. However there
are constraints; the basal metabolic
rate (about 7000 kJ per day) must be
maintained and the first law of
thermodynamics must not be violated.
There is considerable
evidence that mitochondrial dysfunction
is present in some CFS patients. Muscle
biopsies studied by electron microscopy
have shown abnormal mitochondrial
degeneration [7-9].
Biopsies have also found severe
deletions of genes in mitochondrial DNA
(mtDNA), genes that are associated with
bioenergy production [9,
10]. One consequence of
mitochondrial dysfunction is increased
production of free radicals which cause
oxidative damage. Such oxidative damage
and increased activity of antioxidant
enzymes has been detected in muscle
specimens [11].
Some essential compounds (carnitine and
N-acylcarnitine) needed for some
metabolic reactions in mitochondria have
been measured in serum and found to be
decreased in patients with CFS [12,
13]. Both studies found that the
carnitine levels correlated with
functional capacity. Reduced oxidative
metabolism [14-16]
and higher concentrations of xenobiotics,
lactate and pyruvate [17]
have been reported. In one group of
patients a decrease of intracellular pH
after moderate exercise was observed and
a lower rate of ATP synthesis during
recovery was measured [18].
These findings suggest impaired
recycling of ADP to ATP in the
mitochondria.
However, there are
also some similar studies that do not
confirm mitochondrial dysfunction. This
situation is likely due to the different
diagnostic criteria in use. For example,
the Oxford criteria [1],
a definition proposed by psychiatrists,
require only fatigue; “other symptoms
may be present” but are not essential.
The Centers for Disease Control (CDC)
criteria are more selective as they
require an additional four symptoms from
a list of eight [19].
In England in 2007 the National
Institute for Clinical Excellence (NICE)
introduced yet another set of criteria,
fatigue plus one more symptom, for
example “persistent sore throat” [20].
At the other end of the spectrum are
criteria based on studies of patients
with Myalgic Encephalomyelitis (ME) [21-23]
which have culminated in the Canadian
consensus criteria [24];
the Canadian criteria are unlikely to
include patients satisfying only
hypothesis A. Even more confusingly,
both the Canadian and the new NICE
criteria use the term ME/CFS although
their criteria are very different. At
the present time the CDC criteria are
internationally widely used as the
criteria for research purposes despite
their lack of precision [25].
This situation may change in the future
because the Canadian criteria are
gaining wider acceptance and one
charitable research funding agency (ME
Research UK) now requires both the CDC
and Canadian criteria to be used in
research projects that it funds. We use
the term CFS or CFS/ME for the CDC
criteria and ME/CFS for the Canadian
criteria. Our study is aimed to assess
the role of mitochondrial dysfunction
with the primary aim of helping patients
Hypothesis B is
attractive because mitochondrial
dysfunction in various organs offers
possible explanations for many of the
other symptoms of CFS and ME. There is
mounting evidence that the symptoms are
due to dysfunctions on the cellular
level. Abnormalities have been seen in
immune cells [26],
and gene expression studies have
revealed abnormalities in genes
associated with immune cells, brain
cells, skeletal muscle cells, the
thyroid, and mitochondria [27,
28]. A further genetic study
identified seven clinical phenotypes [29].
There seem to be three distinct clusters
of clinical abnormalities that define
CFS [30]:
(a) blood flow and vascular
abnormalities such as orthostatic
intolerance (vascular system), (b)
widespread pain, and high sensitivities
to foods, temperature, light, noise and
odours (central nervous system
sensitization), and (c) fatigue,
exhaustion and brain fog (impaired
energy production). Hypothesis B is that
the lack of energy in the third cluster
originates in the mitochondria of
individual cells. But mitochondrial
dysfunction can also produce
abnormalities (a) and (b) because ATP
produced in each cell by its
mitochondria is the major source of
energy for all body functions.
These observations
from biomedical research into CFS are
very encouraging, but how long do
patients have to wait before there is
some real progress in ameliorating their
symptoms? In a private medical practice
which specializes in CFS the primary
goal is to make the patients feel and
function better. Treatment is started by
making use of the existing biomedical
knowledge to provide a basis of
nutrition, lifestyle management and
pacing. Thyroid, adrenal and allergy
problems are also addressed if they
occur. Most patients improve with these
interventions. However, in many cases
the improvement is not as great as the
patient and doctor would like. When one
of us (SM) became aware of the
commercial “ATP profile” testing package
it was thought that this might be useful
in predicting the level of disability
and identifying any biochemical lesions
that were at fault. The “ATP profile”
testing package, developed by one of us
(JMH), is designed specifically for CFS
and other conditions where energy
availability is reduced. It was found
early on that the “ATP profile” was very
useful in predicting the level of
disability and suggesting the most
likely interventions which would benefit
patients. Tests have now been carried
out on a number of patients and also on
normal, healthy subjects. When collated
the test results show features that were
completely unexpected. Before we report
here on the test procedures and results
we provide a brief summary of how
mitochondria produce energy.
Mitochondrial
energy metabolism
In each cell
glucose is broken down to pyruvate
with the production of some ATP (2
molecules net per molecule of
glucose). The pyruvate and also
fatty acids enter the mitochondria
of each cell, shown schematically in
, where two
coordinated metabolic processes take
place: the tricarboxylic acid (TCA)
cycle, also known as the Krebs'
citric acid cycle, which produces
some ATP, and the electron transport
chain (ETC, also called the
Respiratory Chain because it uses
most of the oxygen we breathe in)
which regenerates ATP from ADP by
the process of oxidative
phosphorylation (ox-phos).
Altogether some 30-odd molecules of
ATP are produced per molecule of
glucose and these constitute the
main cellular energy packets used
for all life processes. As well as
food and oxygen the metabolic
pathways require all the nutrients
involved in the production of the
large number of enzymes which
control the many biochemical
reactions involved and all the
cofactors needed to activate the
enzymes [31-33].
Most of the enzymes are coded by
nuclear DNA (nDNA) in the cell's
nucleus and a few are coded by mtDNA.
Some of the enzymes rely on other
organs. For example, thyroid hormone
is needed in the TCA cycle. On the
other hand hyperthyroidism can
uncouple the ox-phos process [34],
so a thyroid problem can lead to
fatigue and this can be tested for.
The human body contains typically
less than 100 g of ATP at any
instant, but can consume up to 100
kg per day. Thus the recycling ox-phos
process is extremely important and
it produces more than 90% of our
cellular energy. The main features
and processes are illustrated in a
simplified form in
(further details
can be found in all college-level
textbooks on biochemistry, e.g. [6],
and in secondary school
advanced-level biology textbooks,
e.g. [35]).
The ETC culminates with the protein
complex ATP synthase which is
effectively a reversible stepping
motor in which 3 ATP molecules are
produced from ADP and inorganic
phosphate (Pi) every
revolution [36].
Because of evolutionary history ATP
is made inside the mitochondrial
inner membrane but used outside in
the cytosol where it releases energy
by converting to ADP and Pi.
The Pi as a negative ion is
co-transported back inwards together
with H+, while ADP3−
is transported inwards through the
Translocator protein adenosine
nucleotide translocase (TL or ANT)
in exchange for ATP4−
moving out into the cytosol. There
are potential problems here because
it is known that some specific
molecules (e.g. atractyloside) block
the transfer inwards and certain
others can block transfer outwards [37],
and there is the possibility that
there may be other molecules
including environmental contaminants
which can block transfers.
What happens if
some part of these cellular
metabolic pathways goes wrong? If
the mitochondrial source of energy
is dysfunctional many disease
symptoms may appear [38]
including the symptoms of CFS.
Suppose that the
demand for ATP is higher than the
rate at which it can be recycled.
This happens to athletes during the
100 meters sprint. The muscle cells
go into anaerobic metabolism where
each glucose molecule is converted
into 2 molecules of lactic acid.
This process is very inefficient
(5.2% energy production compared to
the 100% of complete oxidation) and
can last for only a few minutes. The
increased acidity leads to muscle
pain. Also, when the concentration
of ADP in the cytosol increases and
the ADP cannot be recycled quickly
enough to ATP, another chemical
reaction takes place. This becomes
important if there is any
mitochondrial dysfunction. Two
molecules of ADP interact to produce
one of ATP and one of AMP (adenosine
monophosphate). The AMP cannot be
recycled [6]
and thus half of the potential ATP
is lost. This takes some days to
replenish and may account for the
post-exertional malaise symptom
experienced by patients [39-43].
Thus,
mitochondrial dysfunction resulting
in impaired ATP production and
recycling is a biologically
plausible hypothesis, and there is
considerable evidence that it is a
contributory factor in CFS, at least
for a subset of patients. Our study
may be considered to be a test of
this hypothesis.
Participants
Seventy-one
patients, 54 female of average age
47 (range 14 to 75) and 17 male of
average age 52 (range 20 to 86),
were selected from a total of 116
consecutive patients attending a
private medical clinic specializing
in CFS/ME. Patients were excluded
only if they did not meet the CDC
diagnostic criteria for CFS [19]
or if the “ATP profile” test had
been made before they had been seen
clinically. Evaluations, tests and
interventions, where appropriate,
were carried out for diet and sleep
problems, allergies, and thyroid and
adrenal problems. Advice on pacing
was also given. After this stage a
meeting was held with each patient
at which an agreed numerical Ability
was assigned and recorded in the
clinical notes. The integral CFS
Ability Scale [44]
runs from 0 to 10 and is given in
Appendix A. It was proposed to
those patients who had not improved
to an acceptable clinical level
after these interventions that they
have the “ATP profile” test done.
All the participating patients had
scores of 7 or less on the CFS
Ability Scale.
The nature of the
test was explained and each patient
agreed (and paid) for the “ATP
profile” test (needing a 3-ml venous
blood sample) to be performed. The
laboratory carrying out the tests (Biolab
Medical Unit,
www.biolab.co.uk) was
blinded to the Ability associated
with any blood sample. As tests were
carried out on more patients, it
became clear that the “ATP profile”
results were providing helpful
information, and patients were asked
to give written, informed permission
for their test data to be used
anonymously. All patients have done
this.
Blood samples
from fifty-three normal, healthy
volunteers were obtained by one of
us (JMH) as Laboratory Director of
Biolab until retirement from that
position in 2007. Biolab obtained
written permission with informed
consent from each volunteer. The
samples from the patient group and
the normal (control) group were
processed in the same way. The
control group consisted of 40
females of average age 36 (range 18
to 63) and 13 males of average age
35 (range 18 to 65).
For both groups
all procedures were consistent with
the Declaration of Helsinki (2000)
of the World Medical Association (www.wma.net)
and this report follows the
guidelines of the International
Committee of Medical Journal Editors
(icmje.pdf available at
www.icmje.org).
Procedures
ATP is present in
cells mainly as a complex with
magnesium and is hydrolysed to the
diphosphate (ADP) as the major
energy source for muscle and other
tissues. ADP conversion to ATP
within mitochondria can be blocked
or partially blocked by some
environmental contaminants.
Specifically, the TL in the
mitochondrial membrane that controls
the transfer of ADP from the cytosol
and ATP to the cytosol may be
chemically inhibited and its
efficiency is also pH dependent.
Changes in acid:base balance,
magnesium status, and the presence
of abnormal metabolic products can
have similar effects to xenobiotic
inhibition of the TL.
A number of
methods have been developed for
assaying ATP. Methods such as
magnetic resonance spectroscopy (MRS)
of 31P require the
patient to be at a facility which is
available only at major hospitals or
research institutes. Biopsies of
skeletal muscles can be taken, but
not of vital organs such as the
heart, brain or liver. Methods using
blood samples (specifically
neutrophils) are relatively
non-invasive and are amenable to
routine testing. In addition the
blood stream reaches almost every
cell in the body and carries much
information concerning what is going
on. The method of measuring ATP used
in the “ATP profile” dates from 1947
when McElroy poured a solution of
ATP onto ground-up firefly tails and
observed bright luminescence and
found that the amount of light
produced was proportional to the ATP
concentration [45].
Thus he showed that the energy
contained in ATP can produce light
and this led the way to the
development of bioluminescent
measurements which can be carried
out routinely and reproducibly with
commercially available biochemical
assay kits and bioluminescence
equipment [46-49].
Light is produced when ATP reacts
with D-luciferin and oxygen in the
presence of Mg2+ and the
enzyme luciferase. When ATP is the
limiting reagent, the light emitted
is proportional to the ATP present.
The
“ATP profile” test
The “ATP profile”
test yields 5 independent numerical
factors from 3 series of
measurements, (A), (B), and (C) on
blood samples (neutrophils). Details
of the measurements made and how the
numerical factors were calculated
are given in
Appendix B. The 3 series are:
- ATP
concentration in the
neutrophils is measured in
the presence of excess
magnesium which is needed
for ATP reactions. This
gives the factor ATP in
units of nmol per million
cells (or fmol/cell), the
measure of how much ATP is
present. Then a second
measurement is made with
just endogenous magnesium
present. The ratio of this
to the one with excess
magnesium is the ATP Ratio.
This tells us what fraction
of the ATP is available for
energy supply.
- The
efficiency of the oxidative
phosphorylation process is
measured by first inhibiting
the ADP to ATP conversion in
the laboratory with sodium
azide. This chemical
inhibits both the
mitochondrial protein
cytochrome a3 (last step in
the ETC) and ATP synthase [50].
ATP should then be rapidly
used up and have a low
measured concentration.
Next, the inhibitor is
removed by washing and
re-suspending the cells in a
buffer solution. The
mitochondria should then
rapidly replete the ATP from
ADP and restore the ATP
concentration. The overall
result gives Ox Phos, which
is the ADP to ATP recycling
efficiency that makes more
energy available as needed.
- The
TL switches a single binding
site between two states. In
the first state ADP is
recovered from the cytosol
for re-conversion to ATP,
and in the second state ATP
produced in the mitochondria
is passed into the cytosol
to release its energy.
Measurements are made by
trapping the mitochondria on
an affinity chromatography
medium. First the
mitochondrial ATP is
measured. Next, an
ADP-containing buffer is
added at a pH that strongly
biases the TL towards
scavenging ADP for
conversion to ATP. After 10
minutes the ATP in the
mitochondria is measured.
This yields the number TL
OUT. This is a measure of
the efficiency for transfer
of ADP out of the cytosol
for reconversion to ATP in
the mitochondria. In the
next measurement a buffer is
added at a pH that strongly
biases the TL in the
direction to return ATP to
the cytosol. After 10
minutes the mitochondria are
washed free of the buffer
and the ATP remaining in the
mitochondria is measured and
this gives the number TL IN.
This is a measure of the
efficiency for the transfer
of ATP from the mitochondria
into the cytosol where it
can release its energy as
needed.
The individual
numerical factors
shows scatter
plots (a point for each patient) of
each of the 5 factors vs. CFS
Ability. As we will see later it is
convenient to divide the data from
the 71 patients into 3 categories,
“very severe”, “severe”, and
“moderate”, which have about the
same number of entries (25, 21, and
25). To the right of each scatter
plot we show a stacked projection
histogram for the 3 categories of
Ability, and at the far right a
histogram for the normal controls.
Looking first at the ATP histogram
for the normal controls we see a
well defined minimum value with a
long tail up to a maximum value of
2.89 fmol/cell. The average value is
2.00 ± 0.05 (SEM (Standard Error of
the Mean), n=53) which can be
compared with the measurement, 1.9 ±
0.1 (SEM, n=12), made some 25 years
ago by the same technique in a study
of the energetics of phagocytosis in
neutrophils [51].
The stacked histogram for the
patients and the Ability plot
clearly show that some patients are
in the normal region and some are
below and they split into two groups
with very little overlap. Rather
than comparing the patients with the
average of the normal control group
which is customary, we prefer to
compare with the minimum value of
the control group which is more
cleanly defined. Also, this method
permits us to classify patients as
being in the normal region or being
below the normal region. Clearly
this can be changed easily and all
the numbers are given in
. We therefore
show as a heavy horizontal dashed
line the minimum value of each
factor measured for the controls.
, ATP vs. CFS
Ability, shows that the majority of
the “very severe” and “severe”
patients are below the normal
minimum but very few are below 75%
of this minimum. Note that 3 of the
“very severe” patients are well into
the normal region; they have
problems with one or more of the 4
other factors. Just over 50% of the
“moderate” patients are in the
normal region. There is a small
positive correlation which is
indicated by the “trend” crosses.
There is not a gentle increase in
ATP with Ability, but an increase in
the fraction of patients above the
normal minimum line.
shows ATP Ratio
vs. CFS Ability. The majority of
patients in all 3 categories are
below the normal minimum, and about
1/3 of “moderate” patients are below
75% of the normal minimum. The
correlation with Ability is slightly
negative. Values for the normal
controls are rather tightly grouped
with a minimum of 0.65 and average
of 0.69.
The Ox Phos plot
in
shows a wide
range of values and a strong
positive correlation for this factor
for the patient group. The stacked
projection clearly shows that there
are two groups – above and below the
normal minimum and the upper group
spans a similar range to the
controls. Note the high value for
the sole patient with CFS Ability =
0. This patient also has ATP = 1.26
and ATP Ratio = 0.59 which are not
very far below the normal minima.
The TL OUT plot
of
also shows two
groups with a rather sharp peak in
the stacked projections just above
the normal minimum and this closely
matches the projection of the
control group. Many patients,
particularly the “very severe”, are
far below the normal minimum.
The TL IN plot of
also has a peak
in the normal region. However, some
patients have very low values,
including the patient with CFS
Ability = 0. The product TL OUT × TL
IN is only 0.012 for this patient
who is very severely ill whereas
this product is 0.17 at the normal
minima, a factor of 14 larger. If
just ATP or Ox Phos had been
measured the very severe
mitochondrial dysfunction of this
patient would not have been
detected. Note the strong positive
correlation for TL IN.
Most patients are
below normal in more than one factor
(average [range] is 3.7 [2 to 5] for
“very severe”, 3.5 [2 to 5] for
“severe” and 2.2 [1 to 4] for
“moderate”). Some of the features
are summarised numerically in
Table 1.
|
Table 1
Some features of
the factors
measured in the
“ATP profile”
tests
|
For most of the
factors the percentage of patients
who are in the normal region
increases in going from “very
severe” to “severe” and to
“moderate”. The exception is the ATP
Ratio which gently decreases, but
within the statistical errors is
constant. Both TL IN and the product
TL OUT × TL IN increase by large
factors. For patients in the
“moderate” category the main
influence on their illness appears
to be the ATP Ratio.
Table 1 also illustrates
the importance of measuring more
than one factor. For example, if
only ATP had been measured, 28% of
all the patients would be classified
as normal, and if only Ox Phos had
been measured, 32% of the “very
severe” patients would be classified
as normal.
Correlations
between numerical factors
It is also
helpful to look at correlations
between pairs of numerical factors.
The five most relevant examples are
shown in
.
In the scatter
plots of
the normal
region is the rectangular region in
the upper right corner defined by
the normal minima dashed lines. In
the ATP Ratio vs. ATP plot () most patients are
fairly close to the normal region
apart from the small cluster at ATP
Ratio ~ 0.35.
In the Ox Phos
vs. ATP plot () there are only a few
patients, all “moderate”, in the
normal region for both factors. Some
of the “very severe” and “severe”
patients are in the normal region
for Ox Phos and some are far below.
Note the apparent negative
correlation for the normal controls.
This shows that for normal subjects
there is a compensatory mechanism,
i.e. if ATP is high Ox Phos is low
and vice versa. This is expected
because the ATP concentration is a
major factor in the control of the
rate of the ox-phos process and the
energy supply is adjusted to meet
the energy demand. There is no
obvious evidence for this effect in
the patient group.
In the Ox Phos
vs. TL OUT plot () only 6 (all
“moderate”) patients are in the
normal region of both factors. Note
that as a function of TL OUT there
are 2 groups, a distinct narrow band
to the right of the vertical dashed
line and a spread-out group to the
left of this line. Looking
vertically, note that for the first
group the Ability of patients
(indicated by the 3 categories) is
correlated with their value of Ox
Phos. There is a large spread in
this factor and again there appears
to be two groups roughly divided by
the horizontal normal minimum line.
Some of the patients in all 3
categories are above this line, but
they have problems with one or more
of the other factors. Some of the
“very severe” patients have Ox Phos
lower than the normal minimum by an
order-of-magnitude.
In the Ox Phos
vs. TL IN plot () there are many more
patients in the normal region for
both factors, but also many with
very low values of one or both
factors.
In the TL IN vs.
TL OUT plot () there are two
clusters, one in the upper right
corner which is the normal region
for both factors, and another well
below it at TL IN ~ 0.1. Several
patients are far below the normal
minimum for both factors.
In the
biochemical methods used we might
expect some correlation between the
TL factors and Ox Phos because they
are closely coupled and interacting
parts of the ADP to ATP reconversion
cycle. However, the plots indicate
that the biochemical methods used
can separate the Ox Phos and TL
factors and measure them
individually.
To our knowledge
this is the first time that such
detailed effects have been observed.
The Mitochondrial
Energy Score
The biochemical
measurements in the “ATP profile”
separate the energy generation and
recycling processes into 5 steps. As
in any multistep process, for
example electrical power production
or an assembly line, the efficiency
of the overall process is the
product of the efficiencies of the
individual steps. Any suggestion of
relative weighting is irrelevant; it
only results in an overall
normalization factor. The product of
ATP and ATP Ratio is the cellular
concentration of ATP complexed with
magnesium and this is the available
energy supply of ATP. Ox Phos is the
efficiency of the ETC which converts
ADP into ATP. However, for the
recycling of ADP to make more energy
available the Translocator protein
must efficiently have its binding
site facing out to collect ADP (TL
OUT) and alternately facing in (TL
IN) to efficiently transmit ATP from
the mitochondria into the cytosol
where its energy can be used.
We have found it
useful to calculate the product of
the five factors, the overall
mitochondrial energy-producing
relative efficiency, and call it the
Mitochondrial Energy Score. We just
multiply the 5 factors together for
each patient and each control. The
minimum value for the controls is
0.182 fmol/cell. We have chosen this
as our normalisation point so we
divide all the Energy Scores (for
both patients and controls) by this
value. Thus all controls have
Mitochondrial Energy Score ≥ 1.00.
A scatter plot of
the Energy Score for each patient at
each value of CFS Ability and each
control is shown in
. The horizontal
dashed line indicates the minimum
value for the normal controls and
this is our normalization value of
1.00. Only one of the 71 patients
has an Energy Score > 1 (namely 1.25
for one of the patients with Ability
= 7). However this patient has 2 of
the 5 factors below the normal
minima.
Note the high
degree of correlation between Energy
Score and CFS Ability and this is
independent of where the mean or
minimum of normal subjects is. It is
natural to believe that the CFS
Ability of patients is more likely
to depend upon mitochondrial
dysfunction than vice versa, so we
should really plot CFS Ability vs.
Energy Score. However, the Ability
was measured first, and
shows
convincingly that mitochondrial
dysfunction is a major risk factor,
and this has not been demonstrated
before. Also shown in
is the best
straight line fit to all 71 entries.
The fit is good, but there is no
reason that the relationship should
be a straight line.
Table 2 gives the
parameters of the fit. The Standard
Error in the slope of the fitted
straight line is so small that the
probability P of the null
hypothesis (i.e. that the slope is
zero) is extremely small, P
<0.001, when computed from the
Student's t-distribution [52].
The 99.9% confidence interval is
0.092 < β < 0.174 where β is the
true slope and this lower limit is
still several Standard Errors above
zero.
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Table 2
Parameters of
straight line
fit to
Mitochondrial
Energy Score
data
|
In
the Energy
Scores are plotted as a function of
the age of each participant. It is
believed that mitochondria play a
major role in the aging process [31,
33] so there is the possibility
that the younger mean age of the
control group as compared to the
patient group may influence our
results. We have looked at the age
dependence of all 5 factors and see
no effect, and this is not
surprising in view of the wide
spread in values of each factor. The
Energy Score is a more reliable
measure of mitochondrial
dysfunction. In
there is no
evidence for age dependence in the
control group but the maximum age is
only 65 years (points for three
controls have been omitted because
their Energy Score is more than 2.00
with a maximum of 2.83). There are
six patients of age > 70, one “very
severe” with Ability = 2, one
“severe” with Ability = 3, and four
“moderate” who all have Ability = 4.
These four patients have Energy
Scores which are below the average
(0.42) for this Ability so there may
well be a decrease with increasing
age. On the other hand there is a
33-year old patient who also has
Ability = 4 and is well below the
average. Excluding the six patients
of age ≥ 70 slightly improves the
straight line fit R2
= 0.677) but has negligible effect
on the other parameters or our
conclusions.
The “ATP profile”
results indicate mitochondrial
dysfunction of the neutrophils in the
patients in our cohort, and moreover the
degree of dysfunction is strongly
correlated with the severity of their
illness. Neutrophils are the major
effector cells of the immune system and
the observed mitochondrial dysfunction
is bound to have a deleterious effect on
this system. We note that increased
apoptosis of neutrophils has been
observed previously in people with CFS [26].
Mitochondria are important functional
parts of almost all human cells but we
cannot assert from the present study
that the mitochondria in other cells are
dysfunctional to the same degree; human
biology provides energy to vital organs
at the expense of less important parts.
However, dysfunction in heart muscle
cells and in central nervous system
cells could explain respectively the
vascular and central sensitization
clusters of clinical abnormalities
mentioned in the Introduction. Thus, our
results strongly suggest that the
immediate cause of the symptoms of CFS/ME
is mitochondrial dysfunction.
We cannot
overemphasize the importance of a
careful diagnosis using the CDC criteria
[19],
or even better the Canadian criteria
which more precisely describe the
symptoms [24].
(An abridged version designed for health
professionals, patients and carers is
available at
www.mefmaction.net/Patients/Overviews/tabid/122/Default.aspx).
It is doubtful that patient selection
with less selective criteria would yield
the high degree of correlation observed
here.
The ways that the
individual factors in the Mitochondrial
Energy Score behave show that not all
patients are affected in the same way.
This may be due to the heterogeneous
nature of the precipitating agents or to
variations in the way patients react to
them. The results indicate specific
biochemical lesions and some of these
may be amenable to ameliorative
intervention. Mitochondria need all of
their essential vitamins, minerals,
essential fatty acids and amino acids to
function properly [31-33].
From the clinical point-of-view of
helping patients this is very important;
the typical time-consuming hit-or-miss
protocol can be replaced by
interventions based on biochemical
information and understanding [53].
An analysis of the
outcomes of interventions being carried
out (see
www.drmyhill.co.uk) will be
the subject of a future publication.
We have demonstrated
the power and usefulness of the “ATP
profile” test in confirming and
pin-pointing biochemical dysfunctions in
people with CFS.
Our observations
strongly implicate mitochondrial
dysfunction as the immediate cause of
CFS symptoms. However, we cannot tell
whether the damage to mitochondrial
function is a primary effect, or a
secondary effect to one or more of a
number of primary conditions, for
example cellular hypoxia [30],
or oxidative stress including excessive
peroxynitrite [54-58].
Mitochondrial dysfunction is also
associated with several other diseases
and this is not surprising in view of
the important role of mitochondria in
almost every cell of the body, but this
fact appears to have been recognised
only in recent years [34,
38,
59,
60].
The observations
presented here should be confirmed in a
properly planned and funded study. The
biochemical tests should be done on CFS
patients after, as well as before,
appropriate interventions and possibly
on patients with other disabling fatigue
conditions. It would also be good to
confirm the biochemical test results in
a second (perhaps government-supported)
laboratory.
We acknowledge
helpful comments from Dr. Derek
Pheby and Dr. Neil Abbot.
Appendix A – The Bell
CFS Ability scale
This scale is a
useful and sensitive measure of the
level of activity and ability to
function of patients with CFS/ME [44].
It is similar to the Energy Index
Point Score (ElPS™,
www.cfsviraltreatment.com)
[61].
It runs from 0 to 10 with:
-
0.
Severe
symptoms on a continuous
basis; bedridden constantly;
unable to care for self.
-
1.
Severe
symptoms at rest; bedridden
the majority of the time. No
travel outside of the house.
Marked cognitive symptoms
preventing concentration.
-
2.
Moderate
to severe symptoms at rest.
Unable to perform strenuous
activity. Overall activity
30–50% of expected. Unable
to leave house except
rarely. Confined to bed most
of day. Unable to
concentrate for more than 1
hour per day.
-
3.
Moderate
to severe symptoms at rest.
Severe symptoms with any
exercise; overall activity
level reduced to 50% of
expected. Usually confined
to house. Unable to perform
any strenuous tasks. Able to
perform desk work 2–3 hours
per day, but requires rest
periods.
-
4.
Moderate
symptoms at rest. Moderate
to severe symptoms with
exercise or activity;
overall activity level
reduced to 50–70% of
expected. Able to go out
once or twice per week.
Unable to perform strenuous
duties. Able to work sitting
down at home 3–4 hours per
day, but requires rest
periods.
-
5.
Moderate
symptoms at rest. Moderate
to severe symptoms with
exercise or activity;
overall activity level
reduced to 70% of expected.
Unable to perform strenuous
duties, but able to perform
light duty or desk work 4–5
hours per day, but requires
rest periods.
-
6.
Mild to
moderate symptoms at rest.
Daily activity limitation
clearly noted. Overall
functioning 70% to 90%.
Unable to work full time in
jobs requiring physical
labour (including just
standing), but able to work
full time in light activity
(sitting) if hours flexible.
-
7.
Mild
symptoms at rest; some daily
activity limitation clearly
noted. Overall functioning
close to 90% of expected
except for activities
requiring exertion. Able to
work full-time with
difficulty.
-
8.
Mild
symptoms at rest. Symptoms
worsened by exertion.
Minimal activity restriction
noted for activities
requiring exertion only.
Able to work full time with
difficulty in jobs requiring
exertion.
-
9.
No
symptoms at rest; mild
symptoms with activity;
normal overall activity
level; able to work
full-time without
difficulty.
-
10.
No
symptoms at rest or with
exercise; normal overall
activity level; able to work
or do house/home work full
time without difficulty.
Appendix B – The
“ATP profile” tests
The “ATP profile”
tests were developed and carried out
at the Biolab Medical Unit, London,
UK (www.biolab.co.uk),
where one of us (JMH) was Laboratory
Director until retirement in 2007.
Blood samples in 3-ml heparin tubes
were normally received, tested and
processed within 72 hours of
venepuncture. We briefly describe
here the 3 series of measurements,
(A), (B) and (C) and how the 5
numerical factors are calculated.
(Step-by-step details can be
obtained by contacting JMH at
acumenlab@hotmail.co.uk
).
Neutrophil cells
are separated by Histopaque™ density
gradient centrifugation according to
Sigma® Procedure No. 1119 (1119.pdf
available at
www.sigmaaldrich.com).
Cell purity is checked using optical
microscopy and cell concentration is
assessed using an automated cell
counter. Quantitative bioluminescent
measurement of ATP is made using the
Sigma® Adenosine 5′-triphosphate
(ATP) Bioluminescent Somatic Cell
Assay Kit (FLASC) according to the
Sigma® Technical Bulletin No. BSCA-1
(FLASCBUL.pdf). In this method ATP
is consumed and light is emitted
when firefly luciferase catalyses
the oxidation of D-luciferin. The
light emitted is proportional to the
ATP present, and is measured with a
Perkin-Elmer LS 5B Fluorescence
Spectrometer equipped with a
flow-through micro cell. Sigma® ATP
Standard (FLAA.pdf) is used as a
control and as an addition-standard
for checking recovery. Similar kits
are available from other providers,
e.g. the ENLITEN™ ATP Assay System
(Technical Bulletin at
www.promega.com), and
dedicated instruments are now
available, e.g. Modulus Luminescence
Modules (see Application Note
www.turnerbiosystems.com/doc/appnotes/PDF/997_9304.pdf).
(A). ATP is first
measured with excess magnesium added
via Sigma® ATP Assay Mix giving
result a. This is the first factor,
the concentration of ATP in whole
cells, ATP = a in units of nmol/106
cells (or fmol/cell).
The measurement
is repeated with just the endogenous
magnesium present by using analogous
reagents produced in-house without
added magnesium, giving result b
in the same units. The ratio, c
= b/a, is the
second factor, the ATP Ratio.
(B). In order to
measure the ADP to ATP conversion
efficiency via the ox-phos process,
the ATP (with excess magnesium)
result, a, is used and then the
conversion is inhibited in the
laboratory with sodium azide for 3
min and result d is
obtained (also with excess
magnesium). The laboratory inhibitor
is then removed by washing with
buffered saline and the mitochondria
should rapidly replete (again 3 min)
the ATP supply from ADP. This gives
result e in the same units.
The conversion efficiency Ox Phos is
(C). In order to
measure the effectiveness of the
Translocator (TL) in the
mitochondrial membrane the cells are
ruptured and the mitochondria are
trapped onto pellets of an affinity
chromatography medium doped with a
low concentration of atractyloside.
This immobilises the mitochondria
while the other cell components are
washed away. The buffers used then
free the mitochondria leaving the
atractyloside on the solid support
that plays no further part in the
method. The mitochondrial ATP
concentration is measured giving
result g in units of pmol/million
cells. For the next measurement some
pellets are immersed in a buffer
(which acts as an artificial cytosol)
containing ADP at pH = (5.5 ± 0.2)
which biases the TL towards
scavenging ADP to be converted to
ATP in the mitochondria. After 10
min the ATP is measured again,
giving result h in the same
units. The factor TL OUT is the
fractional increase in ATP
For the next
measurement pellets are immersed in
a buffer not containing ADP and the
TL is biased away from ADP pickup
and towards ATP transfer into the
artificial cytosol at pH = (8.9 ±
0.2) After 10 min the mitochondrial
ATP is again measured giving result
k, and the factor TL IN is
the fractional decrease
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