CABA Neurotrunsmission Bruin Rewrrch Bulletin, Vol. 5, Suppl. 2, pp. 765-768. Printed in the U.S.A.

Cerebrospinal Fluid Norepinephrine and Free y-Aminobutyric Acid in

Amyotrophic Lateral Sclerosis

BENJAMIN RIX BROOKS

Department of Neurology Johns Hopkins University School of Medicine

Baltimore, MD 21205

MICHAEL G. ZIELGER

Department of Medicine and Pharmacology University of Texas Medical Branch

Galveston, TX 77.550

C. RAYMOND LAKE

Laboratory of Clinical Science, National Institutes of Mental Health National Institutes of Health, Bethesda, MD 21205

JAMES H. WOOD

Division of Neurosurgery University of Pennsylvania School of Medicine

Philadelphia, PA 19104

S. J. ENNA

Departments of Pharmacology, Neurobiology and Anatomy University of Texas Medical School of Houston

Houston, TX 77025

AND

W. K. ENGEL

Neuromuscular Disease Section, Medical Neurology Branch National Institute of Neurological and Communicative Disorders and Stroke

Bethesda, MD 20014

BROOKS, B. R., M. G. ZIELGER, C. R. LAKE, J. H. WOOD, S. J. ENNA AND W. K. ENGEL. Cerebrospinalfluid norepinephrine andfree yaminobuytric acid in amyotrophic lateral sclerosis. BRAIN RES. BULL. 5: Suppl. 2,765-768, 1980.- Amyotrophic lateral sclerosis (ALS) is a rapidly progressive degenerative neurological disease of unknown etiol- ogy. As the first step in evaluating systematically neurotransmitter metabolism in ALS, we measured norepinephrine (NE) in plasma and cerebrospinal fluid (CSF) by radioimmunoassay and y-aminobutyric acid (GABA) in CSF by radioreceptor assay of 34 patients with ALS, 31 patients with myopathies but no central nervous system disease and 167 patients with various neurological disorders. CSF GABA was significantly decreased in ALS patients compared to myophathy bcO.05) and neurological @<O.Ol) patients. However CSF NE was significantly increased (p<O.Ol) with respect to either compari- son group. This dichotomy in neurotransmitter concentrations could result from a single neurotoxin leading to neuronal degeneration and a decrease in glutamic acid decarboxylase. The resultant decrease in GABA which is inhibitory on noradrenergic neurons could lead to the observed elevation in CSF NE due to activation of tyrosine hydroxylase.

Amyotrophic lateral sclerosis y-Aminobutyric acid (GABA) Norepinephrine Cerebrospinal fluid

765

766 BROOKS ET AL.

AMYOTROPHIC lateral sclerosis (ALS) is a rapidly pro- gressive degenerative neurological disease affecting primarily motor neurons. The etiology of amyotrophic lateral sclerosis is unknown [22]. Chemical abnormalities of central nervous system metabolism in ALS are substantiated by the lowered concentrations of metabolites of dopaminergic neurons [4] and cyclic nucleotides in the cerebrospinal fluid (CSF) of ALS patients compared with other neurologic disease con- trol patients [Xl. Such chemical changes, however, may be secondary to the pathologic changes in ALS rather than be related etiologically to the observed degeneration. As the first step in systematic~ly evaluating neurotransmitter metabolism directly in amyotrophic lateral sclerosis we measured norepinephrine (NE) in plasma and cerebrospinal fluid (CSF) by radioenzymatic assay and free y-amino- butyric acid (GABA) in CSF by radioreceptor assay of patients with ALS and neurological disease controls.

METHOD

Patients reported in this study were hospitalized at the National Institute of Neurological Communicative Disorders and Stroke for investigation of nerve or muscle disease. Of the 34 patients with ALS 1 I were female, 23 were male; they had a mean age of 43 2 3 (SEM) years and had symptoms of their disease for 36 2 7 months (range 6-152 months). Two of the patients had a clinical picture compatible with a diag- nosis of progressive muscular atrophy, but none were from Guam or Kii peninsula. Of the 34 patients with ALS, 11 received medication during the study including ben- zodiazepines (4 patients), chloral hydrate [ 11, amitriptyline 121, anti-hype~ensive agents [ZJ, chlo~hen~amine [l], and replacement thyroid 111. These medications did not have any apparent effect on NE or GABA levels.

There were two control groups: 31 patients hospitalized with myopathies without CNS disease who had a mean age of 29 jl 2 which was lower than the patients with ALS and a larger group of patients [I371 with a variety of chronic neurologic diseases inciuding n~uropathies, CNS tumors, epilepsy, and cerebral arterio-venous malformations who had a mean age of 44 c 11, which was not different from patients with ALS.

CSF was obtained from patients who were recumbent overnight through lumbar puncture performed at approx- imately 9:00 a.m. The 12th to 16th ml of CSF removed was employed since the spinal sub~chnoid space gradient for NE plateaus above 12 ml. CSF was collected in a poly- propylene tube containing 10 mg ascorbic acid and placed on ice until transferred to a -70°C freezer. Blood for determi- nation of plasma NE was drawn from an antecubi~l vein prior to lumbar puncture with the patient recumbent. CSF GABA was measured by radioreceptor assay 181 and CSF and plasma NE was measured by radioenzymatic methods [25, 26, 271. The accuracy and reliability of these methods was previously evaluated by comparison with results ob- tained using gas chromatographic-mass spectroscopic tech- niques f8,25]. CSF concentratjons of NE and GABA found in our control groups were similar to those reported in other studies [7, 8, 13, 141, although the GABA levels among the patients with mixed neurologic disease are higher than re- ported for normal volunteers.

RESULTS

Patients with ALS had 180 i: 34 pmoYm1 GABA in their CSF, significantly less than a smali group of patients without

TABLE 1 NEUROCHEMICAL ALTERATlONS IN CSF AND I’LASMA

ALS myopathy Neuroloyic disorders

GABA in CSF 180 + 29”f 250 ” 2.Y 322 f 36 pmoleiml (18) (14) (137)

NE in CSF 365 -r. 44*i 219 t 21 251 2 14

pg/ml (34) (31) (136)

NE in plasma 455 I 59** 277 t 36 309 + 19

pgml (16) (19) (86)

Levels of GABA (in picomoleiml) in CSF, NE (in pgiml) in CSF and plasma of patients with ALS, controls who have myopathy without nervous system disease, or neurologic controls with central nervous system diseases unrelated to ALS. Values are shown i SE&f with the number of patients in parentheses.

*signi~cantiy different from mixed neurologic patients by two taited Student’s t-test.

Ssignificantly different from patients with myopathy by two tailed Student’s t-test.

b

FIG. 1. The relations~p between NE and GABA in the CSF of 18 patients with ALS. There is a sign&ant inverse reIationship be-

tween levels of the neurotransmitters (r=-0.50, pqO.05).

CNS disease (pcO.05) or a larger group of patients with di- verse neurologic disorders @<O.Ol) (Table 1). Of the I8 patients tested, 16 had GABA concentrations below the mean of the larger control group.

Notably, the ALS patients have CSF concentrations of NE higher than either control group (p<O.Ol) which corre- lated negatively with GABA (r= -0.50, Fig. 1). High concen- trations of CSF NE and GABA were present only in patients with ALS, but not in controls (r= -0.03).

A blood sample was withdrawn from 16 patients with ALS just prior to lumbar puncture for NE ~t~nation. Patients with ALS had plasma NE concentrations sign% cantly higher than NE concentrations under similar condi- tions from the two control groups tp<O.Ol, Table 1). The blood pressure of patients with ALS (132 + 3187 t 3 mm Hg) was higher than that of patients with myopathy

CSF NE AND GABA IN ALS 767

TABLE 2

COMPARISON OF BEDRIDDEN AND AMBULATORY ALS PATIENTS

Bedridden (14) Ambulatory (20)

CSF NE (pgiml) 595 2 W 204 + 20 Plasma NE (pgiml) 658 * 119* 363 + 49 CSF GABA (pmoleiml) 149 + 50 205 + 33

134 + 6 1302 4 Blood pressure (mm Hg)

912 5 84? 4

Pulse 89+ 4 782 6

Levels of NE in CSF and plasma (in pgiml) GABA in CSF (in pmoleiml) blood pressure and pulse rate in 14 patients with severe ALS who were bedridden and 20 ALS patients who could ambulate.

*significantly @<0.05) different from ambulatory ALS patients.

(117 t 3/74 ? 2 mm Hg; pcO.01) and slightly higher than patients with other neurologic diseases (128 * 2183 + 1 mm Hg). The pulse rate of ALS patients (86 + 3) was slightly higher than that of patients with other neurologic disease (83 + 1) and correlated with plasma NE (~~0.61, p<O.OS). The amount of NE in blood drawn prior to lumbar puncture correlated with NE in CSF (r =0.51, p ~0.05). Those patients with the highest cerebrospinal fluid NE had the highest plasma NE. This is similar to the relationship between NE concentrations in plasma and CSF seen in other subjects without ALS [25].

Patients with ALS who have predominantly bulbar, cer- vical or lumbar disease do not differ with respect to their NE or GABA concentrations. However, patients with disease so severe that they were bedridden had the highest CSF and plasma NE as well as a trend toward elevated blood pres- sure, elevated pulse rate and lowered GABA in their CSF (Table 2).

DISCUSSION

Patients with ALS have decreased concentrations of GABA and increased concentrations of NE in CSF. There was a significant negative correlation between GABA and NE in CSF and a positive correlation between NE in CSF and plasma. The correlations suggest that these neurochemi- cal derangements may be caused by the same CNS defect. Electrical stimulation of the human cerebellum can also ele- vate NE [24] and depress GABA levels in CSF [23]. A possible explanation for these findings is predicated by re- ports that some neurostimulants may cause neuronal degen- eration [ 16,171 leading to a decrease in glutamic acid decar- boxylase, the enzyme which catalyzes the synthesis of GABA. Such a toxic effect would tend to lower GABA levels, which in turn might cause an increase in tyrosine hydroxylase activity, the rate limiting enzyme in the synthe- sis of NE [19]. Hence, an increase in tyrosine hydroxylase activity, and NE turnover, may be secondary to the loss of GABA which is inhibitory on noradrenergic neurons [5].

Thus, a single agent could cause the neurochemical changes observed in ALS patients in this study.

Bedridden ALS patients had higher CSF and plasma NE than ambulatory patients. In normal subjects a short bed rest lowers [14] and hospitalization further lowers plasma NE [13]. Motor activity increases the amount of 3-meth- oxy-4-hydroxyphenylglycol, the major brain metabolite of NE, in CSF [18]. Thus decreased activity alone tends to lower peripheral and central NE but bedridden ALS patients have higher CSF NE concentrations, so their disease ap- pears to enhance NE release despite their immobility. These patients tend to have the lower GABA concentrations so neurochemical abnormalities in ALS were most striking in those most severely affected by the disease. There is a de- crease in GABA levels in the CSF of patients with other degenerative CNS diseases such as Alzheimer’s disease and Huntington’s chorea [7]. However, in Huntington’s chorea plasma NE is low 1201 and CSF concentrations of NE are not elevated [25-271.

The elevated systolic and diastolic blood pressure and heart rates in these ALS patients are probably related to the high levels of plasma NE. This increase in sympathetic ner- vous system activity may also explain some peculiar aspects of the disease. One hundred years ago Charcot noted that patients with ALS did not form bedsores [3] and others have confirmed the observation that bedsores form in other dis- eases which paralyze while sparing sensory function, but not in ALS [ 111. Since sympathetic nerve stimulation can pre- vent arterioles from becoming flaccid and compressible, Forrester hypothesized that this freedom from bedsores was due to increased sympathetic tone and proposed that; patients with ALS should show other evidence of increased sympathetic tone [lo]. Furakawa and Toyokura [ 121 recently noted that on plethysmographic analysis ALS patients have a rigid monophasic pattern of blood flow through their fin- gers. In addition, patients with ALS are known to have a high incidence of non-atherosclerotic angiopathy in periph- eral arteries [21]. These phenomena may all be the result of increased sympathetic nervous system activity with enhanced NE discharge.

The increase in peripheral NE may be mediated by the change in central NE since plasma NE correlates with NE in CSF and central noradrenergic nerves terminate near sym- pathetic preganglionic nerves 121. The decrease in the in- hibitory transmitter GABA could enhance discharge of noradrenergic and other nerves [ 191. There are drugs which might increase GABAergic and decrease noradrenergic ac- tivity which might normalize neurotransmitter activity in patients with ALS. A controlled trial could determine if such agents might be helpful in ALS.

ACKNOWLEDGEMENTS

Supported in part by NHLBI Grant HL 21817, USPHS Research Career Development Award NS-00335 (S.J.E.), Teacher- Investigator Development Award NS-00385 (B.R.B.) and grants from the Pharmaceutical Manufacturers Association, Huntington’s Chorea Foundation and Merck Sharp and Dohme. We thank Saratha Balachandran, Sandra Henriksen and Elaina Mann for their assist- ance.

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