Date sent:      	Sun, 6 Aug 2000 

American Journal of Medicine
Vol 108, Issue 9, Pages 695-782

Enhanced sensitivity of the peripheral cholinergic vascular response in
patients with chronic fatigue syndrome

Vance A. Spence PhDa, Faisel Khan PhD, and Jill J. F. Belch MDa 

Section of Vascular Medicine and Biology, University Department of
Medicine, Ninewells Hospital and Medical School, Dundee, Scotland, UK

Received 19 July 1999; revised 1 March 2000; accepted 1 March 2000.
Available online 27 July 2000.


Article Outline

Patients with chronic fatigue syndrome have a variety of clinical
symptoms, with associated physical, psychological, and social
disability. The syndrome is poorly understood; the etiology and
pathophysiology are not known.

Cholinergic mechanisms are important in the control of peripheral
skin perfusion [1 and 2] and, in part, are regulated by endothelial
cells [3]. We have developed a method for assessing endothelial
function in which acetylcholine is delivered transdermally across
intact skin using iontophoresis [4], and the vascular responses are
measured by laser Doppler imaging [5]. These techniques have been
used in many patients with cardiovascular disease, in whom
endothelial dysfunction and impaired vasodilatation are common. We
have now applied this methodology to investigate cholinergic
activity in patients with chronic fatigue syndrome to see if the
reported evidence of central nervous system cholinergic sensitivity
in these patients [6] is widespread.


Subjects and methods

Patients were randomly selected from 420 members of a local chronic
fatigue syndrome/myalgic encephalomyelitis self-help group. We
enrolled 22 patients (7 men and 15 women; mean [p/m SD] age 45 p/m 9
years, range 26 to 59) who fulfilled the Centers for Disease Control
criteria for chronic fatigue syndrome [7]. All patients had a
principal complaint of fatigue, exacerbated by physical and mental
activity, for more than 6 months (illness duration 8 p/m 5 years,
range 1 to 15). No other medical cause of fatigue was identified,
and none of the patients had a history of vascular disease. We also
enrolled 22 age-matched (p/m 1 year) and sex-matched control subjects.
None of the control subjects and 13 of the patients with chronic fatigue
syndrome took medications. Four patients with chronic fatigue syndrome
were taking amitryptilline (10 mg to 20 mg at night for pain control), 3
were taking low doses of a serotonin reuptake inhibitor, and 7 were taking
nonsteroidal anti-inflammatory drugs (which were stopped for at least 10
days before the study date). All participants gave written informed
consent. The local committee on medical research ethics approved the
study.


MEASUREMENT OF SKIN BLOOD FLOW AND DRUG DELIVERY

We used a laser Doppler imager (Moor Instruments, Axminster, United
Kingdom) that scans a low-power laser beam over the skin surface.
The scanner was set to a spatial resolution of 100x100 pixels and
a scan speed of 4 ms/pixel. Moving blood in the microvasculature
causes a Doppler shift, which is processed to build a color image of
blood flow (termed "erythrocyte flux") in arbitrary perfusion units.

Iontophoresis was used to transport drugs in solution across intact
skin. We used a protocol similar to that used in our previous
studies [4 and 8]. In brief, a circular iontophoresis electrode
(inner diameter 20 mm) was attached midway on the volar aspect of
the forearm. A reference electrode was placed around the wrist to
complete the circuit. Both electrodes were connected to an
iontophoresis controller, which provided a direct current for drug
delivery.

Studies were performed in a temperature-controlled room (22 C to
23 C). Following a 20-minute equilibration period, baseline skin
perfusion was measured at the volar aspect of the dominant forearm
over the iontophoresis electrode. Using a 0.1-mA anodal current, a
1% solution of acetylcholine chloride (Sigma Chemical, St. Louis,
Missouri) was iontophoresed for 10 seconds to achieve a dose of 1
mC/cm2. The duration was increased to 20 seconds for a dose of 2
mC/cm2, to 40 seconds for a dose of 4 mC/cm2, and to 80 seconds for
a dose of 8 mC/cm2. Scans were performed for 2 minutes between
doses. At a different site, we used a 0.1-mA cathodal current to
iontophorese sodium nitroprusside (David Bull Laboratories, Warwick,
United Kingdom) to give doses of 1, 2, 4, and 8 mC/cm2. Scans were
performed for 4 minutes between doses.

All measurements and data analyses were blinded to patient status
and were performed by a researcher who was unaware whether the
subject was a patient or a control.


STATISTICAL ANALYSIS

Results are expressed as mean ± SD. Differences between patients
with chronic fatigue syndrome and control subjects were compared
using two-way analysis of variance (ANOVA) for repeated measures.
The null hypothesis was rejected at P <0.05.


Results

Baseline skin erythrocyte flux was similar in patients with chronic
atigue syndrome (22 p/m 7 perfusion units) and control subjects (20 p/m 5
perfusion units, P = 0.23). The vascular responses to acetylcholine were
significantly greater in patients with chronic fatigue syndrome than in
control subjects at all 4 doses (P = 0.01, ANOVA; Figure 1). In contrast,
the vascular responses to sodium nitroprusside (Figure 2) were not
significantly different between the groups of subjects.

[Figure 1. Skin vascular responses to iontophoresis of
     acetylcholine in patients with chronic fatigue syndrome (n = 22) and
     control subjects (n = 22). Vascular responses were significantly
     enhanced in patients with chronic fatigue syndrome (P = 0.01, ANOVA).
     P values on the graph refer to posthoc testing. Error bars represent
     p/m 1 SD.]

[Figure 2. Skin vascular responses to iontophoresis of sodium
     nitroprusside in patients with chronic fatigue syndrome (n = 22) and
     control subjects (n = 22). There were no significant differences
     between the groups. Error bars represent p/m 1 SD.]

Excluding the 4 patients taking amitryptilline did not alter the
results. The vascular response to acetylcholine was still
significantly greater in the patients with chronic fatigue syndrome
(22 p/m 7, 60 p/m 26, 105 p/m 28, 127 p/m 23, and 141 p/m 21 perfusion
units) than in the control subjects (19 p/m 5, 37 p/m 24, 69 p/m 36, 95
p/m 35, and 112 p/m 30 perfusion units, P = 0.001).


Discussion

The results of this study show enhanced cholinergic activity in the
peripheral microcirculation of patients with chronic fatigue
syndrome. This enhancement was specific for acetylcholine; the
vascular responses to sodium nitroprusside were similar in patients
with chronic fatigue syndrome and in control subjects.

We could not determine why the patients with chronic fatigue
syndrome have acetylcholine supersensitivity in the skin
microcirculation. Recent evidence in mice treated with
cholinesterase inhibitors suggests that cholinergic stimulation
promotes changes in the genes regulating acetylcholine metabolism
[9] and that such changes are facilitated by stress [10].
Whether such changes of cholinergic tone apply to the metabolism of
acetylcholine in vascular endothelium, however, remains speculative.
There are several ways that the acetylcholine-endothelial pathway
could account for cholinergic supersensitivity. Acetylcholine
produces vasodilatation through a sequence of events beginning with
its binding to muscarinic receptors on the endothelial cell surface.
This activates G proteins, promoting the conversion of l-arginine to
nitric oxide, which diffuses into the smooth muscle cells and
stimulates guanylate cyclase to produce cyclic GMP, thereby causing
relaxation. As vascular responses to sodium nitroprusside were not
enhanced in patients with chronic fatigue syndrome, we suggest that
the enhanced vasodilator activity to acetylcholine is situated at
the muscarinic M3 receptor or along the muscarinic receptor/vascular
smooth muscle pathway.

Many of the symptoms of chronic fatigue syndrome, such as
temperature sensitivity, gastrointestinal difficulties, problems
with sleep, and orthostatic intolerance, are consistent with altered
cholinergic activity. Our findings of enhanced
cholinergically-mediated vasodilatation are related only to the
vasoactive properties of acetylcholine within the endothelium, which
are distinct from its action as a neurotransmitter. Nevertheless,
the response of skin microvessels to acetylcholine is typical of
blood vessels elsewhere in the body. Thus, our findings might have
important implications for features of chronic fatigue syndrome that
involve vascular integrity.

The patients in this study were heterogeneous and had a diverse
range of symptoms and severity of presentation. We did not examine
the potential effects of duration of illness or overall health at
the time of testing. A study that used more specific criteria might
identify differences in cholinergic sensitivity among different
groups of patients with chronic fatigue syndrome, as has been seen
for immune function [11 and 12].


Acknowledgements

We are grateful to the Myalgic Encephalomyelitis Association Moray
Firth group and Tenovus Scotland for their support. We thank Jenny
Cooper for her technical assistance.


References

 1. F. Khan and J.D. Coffman, Enhanced cholinergic cutaneous
    vasodilation in Raynaud's phenomenon. Circulation 89 (1993), pp.
    1183-1188.
 2. J.D. Coffman and R.A. Cohen, Cholinergic vasodilator mechanism in
    human fingers. Am J Physiol 252 (1987), pp. H594-H597.
 3. J.G. Parnavelis, W. Kelly, G. Burnstock et al., Ultrastructural
    localisation of ChaT in vascular endothelial cells in rat brain.
    Nature 316 (1985), pp. 724-725.
 4. F. Khan, S.J. Litchfield, M. McLaren et al., Oral L-arginine
    supplementation and cutaneous vascular responses in patients with
    primary Raynaud's phenomenon. Arthritis Rheum 40 (1997), pp. 352-357.
 5. F. Khan, I.R. Greig, D.J. Newton et al., Skin blood flow after
    transdermal S-nitrosothio-acetylglucose. Lancet 350 (1997), pp.
    410-411.
 6. A. Chaudhuri, T. Majeed, T. Dinan and P.O. Behan, Chronic fatigue
    syndrome: a disorder of central cholinergic transmission. J Chron
    Fatigue Synd 3 (1997), pp. 3-16.
 7. International Chronic Fatigue Syndrome Study Group, K. Fukuda,
    S.E. Strauss, I. Hickie et al., The chronic fatigue syndrome: a
    comprehensive approach to its definition and study. Ann Intern Med 121
    (1994), pp. 953-959.
 8. F. Khan, N.C. Davidson, R.C. Littleford et al., Cutaneous
    vascular responses to acetylcholine are mediated by a
    prostacyclin-dependent mechanism in man. Vasc Med 2 (1997), pp.
    82-86.
 9. D. Kaufer, A. Friedman, S. Seidman and H. Soveq, Acute stress
    facilitates long-lasting changes in cholinergic gene expression.
    Nature 393 (1998), pp. 373-377.
10. R.M. Salposky, The stress of Gulf War syndrome. Nature 393
    (1998), pp. 308-309.
11. I.S. Hassan, B.A. Bannister, A. Akbar et al., A study of the
    immunology of chronic fatigue syndrome: correlation of immunologic
    parameters to health dysfunction. Clin Immunol Immunopathol 87 (1998),
    pp. 60-67.
12. A.C. Mawle, R. Nisenbaum, J.G. Dobbins et al., Immune responses
    associated with chronic fatigue syndrome: a case control study. J
    Infect Dis 175 (1997), pp. 136-141.

Correspondence should be addressed to Faisel Khan, PhD,
Section of Vascular Medicine and Biology, University Department of
Medicine, Ninewells Hospital and Medical School, Dundee, DD1 9SY
Scotland, United Kingdom

--------
(c) 2000 ScienceDirect - an Elsevier Science B.V. registered trademark.