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Choline levels in plasma of myasthenia gravis patients and normal individuals.

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Choline Levels in Plasma
of Myasthenia Gravis
Patients and Normal
Cathy Stein, BA, Walter Moore, BS,
Daniel Weinreich, PhD, and Richard F. Mayer, M D
Although the role of choline in blood is not well understood, several recent studies have attempted to link blood
choline levels with diseases involving central cholinergic
dysfunction [ l , 4, 61. It is well known that myasthenia
gravis is a disease involving the neuromuscular junction in
man, and it has been reported that a defect in acetylcholine
synthesis might account for the clinical weakness [2]. In the
present investigation, plasma choline levels of patients with
myasthenia gravis were compared with those of normal individuals to determine whether this disease is associated
with differences in free plasma choline concentrations.
Materials and Methods
Twenty patients with chronic myasthenia gravis were studied. All were being followed and treated in the Myasthenia
Gravis Clinic at the University of Maryland Hospital. Fourteen were female. aged 13 to 6 2 years (mean, 37 years), and
6 were male, aged 15 to 81 years (mean, 56 years). All,
except for 2 with localized myasthenia, had generalized
signs and symptoms. Five patients had marked clinical
weakness and 7 were in remission. All were receiving anticholinesterase therapy, either neostigmine, 30 to 150 mg
per day, or pyridostigmine, 60 to 1,200 mg per day, except
2 who were taking prednisone (25 and 100 mg) o n alternate days. Two other patients were also on prednisone
therapy, 20 and 50 mg o n alternate days, and 8 had undergone thymectomy (3 months to 6 years earlier).
Control plasma was collected from laboratory personnel,
12 female, aged 22 to 4 3 years (mean, 28 years) and 12
male, aged 21 to 58 years (mean, 30 years).
Blood (2 ml) was drawn by venipuncture into Vacutainer
glass tubes (Beckton, Dickinson, and Co, NJ) and irnmediately placed on ice. Blood samples were centrifuged at
1,800 rpm for 15 minutes at 4"C, and plasma was transfered to culture tubes (VWR Scientific, MD) for storage at
-20°C. Prior to assay, plasma samples were thawed o n ice,
and the choline in 10 to 20 p1 plasma aliquots was extracted
into 5 vol of formic acid/acetone (15:85 v/v).
Choline in plasma was measured by the method of
Goldberg and McCaman [3] as modified by McCaman
and Stetzler [5]. Briefly, choline was converted to 32Plabeled phosphoryl choline in the presence of y ("P)
From the Department of Pharmacology and Experimental
Therapeutics and the Department of Neurology, University of
Maryland School of Medicine, Baltimore, MD.
Accepted for publication Mar 14, 1978.
Address reprint requests to Ms Stein, Department of Pharmacology and Experimental Therapeutics, University of Maryland
School of Medicine, Baltimore, MD 21201.
Plasma choline concentrations from four experimentalpopulations. Each data point represents the mean value of triplicate determinatiom ofplasna sample from a single individual. Values
of the mean (dashed line) 2 SD (solid 1ine)are indicatedfor
each population.
adenosine triphosphate (New England Nuclear Corp, Boston, MA) and the enzyme choline kinase (ATP:choline
phosphotransferase, EC; Sigma Chemical Co, St.
Louis, MO). Labeled choline phosphate was isolated by
chromatography on microcolumns packed with AG- 1 ionexchange resin (Bio Rad Laboratories, CA). Radioactivity
was measured in a Packard (Model 3320) Tri Carb liquid
scintillation spectrometer at a counting efficiency of 90%
for 32P.Plasma choline values were calculated from internal
standards and reagent blanks, which were carried through
each assay.
Resalts and Discussion
Mean choline values, in nanomoles per milliliter 2 SD
were 10.3 2 3.9 in the 6 myasthenic men and 7.3 2 2.2 in
the 14 myasthenic women. Control values of 10.5 I
2.6 for
12 men and 7.5
1.7 for 12 women were obtained (Figure). The free choline concentration in patients with
myasthenia gravis and in normal individuals did not differ
( p > 0.1).
Because the study did not follow patients temporally,
this conclusion is based on mean values of single samples
and does not reflect individual differences that may occur
during the course of the disease. T h e study did, however,
include patients with a wide range of defects in neuromuscular transmission, ranging from minimal to severe.
Neither patients taking pyridostigmine, neostigmine, or
prednisone therapy nor those who had undergone
thymectomy had altered plasma choline profiles. These
data suggest that plasma choline concentration is under
precise metabolic control.
The plasma choline values obtained for normal individuals are in agreement with those obtained by other investigators [ l , 4, 6, 71. The results also indicate that choline
values obtained for women, both normal and myasthenic,
are lower than for men ( p < 0.01).
Funded by NSF Grant BNS 77-13034 to Dr Weinreich and
Health Professions Capitation Grant (HEW) PH5-035-06307-311
to Mr Moore.
The authors wish to thank Dr Alan M. G o l d b e g for his advice
throughout the study and helpful comments on the manuscript.
We also thank the Potomac Valley Chapter of the Myasthenia
Gravis Society for support.
1. Aquilonius S-M, Eckernas S-A: Choline therapy in Huntington
chorea. Neurology (Minneap) 27:887-889, 1977
2. Desmedt JE: Presynaptic mechanisms in myasthenia gravis, in
Third International Symposium on Myasthenia Gravis. Ann
N Y Acad Sci 135:209-246, 1966
3. Goldberg AM, McCaman RE: An enzymatic method for the
determination of picomole amounts of choline and acetylcholine, in Hanin 1 (ed): choline and Acetylcholine: Handbook
of Chemical Assay Methods. New York, Raven, 1974
4 . Hanin I, Kopp U, Zahniser NR, et al: Acetylcholine and
choline in human plasma and red blood cells: a gas
chromatograph/rnass spectrometric evaluation, in Jenden DJ
(ed): Cholinergic Mechanisms and Psychopharmacology. New
York, Plenum, 1977
5. McCaman RE, Stetzler J: Radiochemical assay for ACh: modifications for sub-picomole measurements. J Neurochem
28:669-671, 1977
6. Stavinoha WB, Modak AT, Bowden C L Acetylcholine and
choline in the blood of normal individuals and psychiatric patients. SOCNeurosci Abstr 3:416, 1977
7. Wurtman RJ, Hirsch MJ, Growdon JH: Lecithin consumption
raises serum-free-choline levels. Lancet 2:68-69, 1977
Multiple Sclerosis
and House Dogs
controls shared this characteristic. T h e differences were
not statistically significant for all dogs but were for indoor
dogs. T h e conclusion may be not that indoor dogs are associated with MS but that indoor dog owners make friends
with outdoor dog owners, or at least are more likely to
remember them fourteen years later. Other interpretations
may suggest themselves.
As Kurland and Brian pointed out [4], once the controls
are chosen, their recollection of events nineteen to fourteen years previously may not be the same as that of a
patient who had a very important life event occur at the end
of that period. Most patients who have had MS for this
length of time have suffered some mental changes [5].
Questioning another source in each case, e.g., a parent,
may have been worthwhile. Also, it may be noted that the
interviewers were not unbiased, as they apparently knew
whether they were interviewing a patient or a control [23.
The finding that MS is related to events in the five years
preceding the onset of the illness seems inconsistent with
the popular hypothesis that MS is acquired in childhood
and has a latency of ten or more years [I]. Were the subjects asked about exposure to pets at different ages (0to 5,
5 to 10 years, and so forth) and at multiple periods of time
before onset? Certainly there is much to be said for publishing negative as well as positive results [31, and many
possible variations are not tabulated in this study.
1. Alter M: Is multiple sclerosis an age-dependent host response
to measles? Lancet 1:456-457, 1976
2. Feinstein AR: Clinical biostatistics: XX. The epidemiologic
trohoc, the ablative risk ratio, and "retrospective" research.
Clin Pharmacol Ther 14:291-307, 1973
3. Gordon RS: Clinical trials. N Engl J Med 298:400-401, 1978
4. Kurland L, Brian D: Multiple sclerosis and canine pets. Ann
Neurol 3:97-99, 1978
5 . McAlpine D: Symptoms and signs, in McAlpine D, Lumsden
CE, Acheson ED (eds): Multiple Sclerosis, A Reappraisal. Baltimore, Williams & Wilkins, 1972, pp 180-184
Robert Karis, M D
The hypothesis of Cook, Natelson, Levin, et a1 (Ann
Neurol 3:141-143, 1978) concerning the association between house dogs and multiple sclerosis (MS) provides an
interesting exercise in statistical reasoning. Kurland and
Brian [ 4 ] in the same issue mentioned several possible
shortcomings of this study. The following points bear emphasis.
T h e control subjects were named by the patients. What
possible biases are there in asking a patient to name a friend
whom he knew before the onset of his illness (on the average, fourteen years ago in this study)? Whatever may be
said in favor of this technique, these are certainly not randomized controls, as would be more acceptable hospital
and neighborhood controls [2]. Forty patients with MS admitted to dog ownership (presumably at any time prior to
the onset of their illness), as did 34 of the controls. Thirtynine patients with MS had an indoor dog, but only 29 of the
From the Department of Neurology, St. Louis University School
of Medicine, 1221 S Grand Ave, St. Louis, MO 63104.
S. D. Cook, MD, B. H. Natelson, MD, B. E. Levin, MD,
P. S. Chavis, MD, and P. C. Dowling, MD
W e agree with Dr Karis in his concern about possible artifacts in the association found between dogs and MS. Problems inherent in retrospective epidemiological studies such
as ours have been commented on by us and others [2-51.
Clearly, further studies using various types of controls, carried out in other geographic areas, and employing written
rather than telephone communications are both desirable
and necessary in order to determine the significance of our
data. Based o n this paper and recent information from the
Faroe Islands showing a close temporal relationship between an epidemic of animal neurological disease and subsequent multiple sclerosis (Lancet 1:605-606, 1978), it
may also be important to inquire about exposure to animals
with a neurological illness similar to that produced by
canine distemper virus. Only a small percentage of dogs in
Notes and Letters
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level, patients, myasthenia, choline, norman, plasma, gravis, individual
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