Journal of the Royal Society of Medicine Volume 86 September 1993 501

Abnormal glycine metabolism in motor neurone disease:studies on plasma and cerebrospinal fluid

Russell J M Lane MD FRCP1 Rina Bandopadhyay BSc2 Jackie de Belleroche PhD''Academic Unit of Neuroscience, and 2Department of Biochemistry, Charing Cross andWestminster Medical School, London W6 8RF, UK

Keywords: glycine; metabolism; amyotrophic lateral sclerosis

SummaryPlasma amino acid levels were measured followingoral glycine loading in 43 patients with motor neuronedisease (MND), eight normal subjects and 18 neuro-logical disease controls with wasting or spasticityfrom a variety of other causes. Levels at baseline and1.5 h after loading did not differ, but at 4 h, plasmaglycine levels inMND patients remained significantlyhigher than in normal and neurological controls(P< 0.013). Cerebrospinal fluid glycine levels, whichwere maximal at 2.5 h, were also significantly higherin MND patients than neurological controls (P< 0.04).These observations suggest a defect of glycine'housekeeping' in the central nervous system in MNDwhich may be relevant to the pathogenesis of thedisease.

IntroductionIn 1972, Bank and Morrow' described three brothersof Lebanese extraction with a slowly progressivemotor system disorder characterized by spasticity,hyper-reflexia and weakness, with atrophy of theanterior tibial and peroneal muscles, associated withmarked hyperglycinaemia. Bank et aL2 subsequentlyreported a fourth case, also a young man of Lebaneseorigin, who showed optic atrophy and mild cerebellarsigns in addition. Higher cerebral functions andsphincters were intact and sensation largely preserved,although in two cases, mild proprioceptive impairmentwas noted. Plasma glycine levels in these cases werebetween three and 11 times greater than normal, andcerebrospinal fluid (CSF) glycine levels four to fivetimes higher. Plasma glycine levels were examinedin a number of other cases of peroneal muscularatrophy, Friedreich's ataxia and hereditary spasticparaplegia and found to be normal" . Oral glycineloading studies demonstrated a defect in the normalconversion of glycine to serine in two of Bank andMorrow's cases, and also in their asymptomaticmother"'.In a later study, de Belleroche et al.3'4 found that

the levels of several amino acids, including glycine,were increased in the CSF of motor neurone disease(MND) patients, and that there appeared to be arelationship between CSF glycine levels and 'diseaseactivity' as defined on a scale used previously byPatten et al.5. These observations prompted us toexamine changes in plasma and CSF amino acids

following oral glycine loading in MND patients andnormal subjects, and in a number of neurologicaldisease controls with muscular wasting or spasticityfrom other causes.

Patients and methodsInitial studyThirty-four MND patients were investigated in asingle-blinded study (Table 1). These comprised 17with amyotrophic lateral sclerosis (ALS; eight menand nine women, mean age 53.1+11.2), defined as apurely motor system disorder with both upper andlower motor neuron signs and neurophysiologicalevidence of denervation in both upper and lower limbscompatible with anterior horn cell disease; six withprogressive bulbar palsy (PBP; five men and onewoman, mean age 61.5+9.2 years), whose initialsymptoms had been of dysphonia, dysphagia anddysarthria; three cases of presumed primary lateralsclerosis (PLS), with progressive spastic dysarthria,pseudobulbar affect and signs of bilateral pyramidaltract disease without clinical or electrophysiologicalevidence of lower motor neuron involvement orevidence of relevant structural disease in the neuraxisafter detailed neuroradiological investigation6'7; andeight patients with progressive muscular atrophy(PMA; seven men and one woman, mean age 68.1+8.1,having only lower motor neurone involvement onclinical examination, with neurophysiological findingsconsistent with anterior horn cell disease. None hada family history of MND and alternative diagnoseswere excluded by detailed laboratory investigation.Eight normal control volunteers were studied. These

comprised seven spouses of MND patients and onepatient who underwent myelography for possiblecervical radiculopathy, with negative findings (fourmen and four women, mean age 58.9+3.3 years).We also studied 14 neurological disease controls.

Ten had muscular wasting from various causes, threespasticity without wasting, and one had chronic clusterheadache (Table 2).In all instances, renal and hepatic function, as

judged by urinalysis and measurements of plasmaurea, electrolytes, proteins, bilirubin and hepaticenzymes, were within normal limits.

Glycine loading testThe method of Bank and Morrow' was followed.After an overnight fast (12 h), subjects were weighedand a baseline fasting blood sample (10 ml) was takenfor amino acid analysis. L-glycine (Analar, screenedby the Pharmacy Quality Control Unit at CharingCross Hospital as fit for human consumption) was

Correspondence to: Dr R J M Lane, Regional NeurosciencesCentre, Charing Cross Hospital, Fulham Palace Road,London W6 8RF, UK

502 Journal of the Royal Society of Medicine Volume 86 September 1996

Table 1. Plasma and cerebrospinal fluid (CSF) amino acid measurements in motor neurone disease patients. Patients studiedin initial and additional series and timing of CSF sampling

ALS PBP PLS PMA Total

PAAInitial 17 6 3 8 34Additional 6 0 0 3 9

Total (23) (6) (3) (11) (43)CSFTime (hours)* 1.5 2.5 4 1.5 2.5 4 1.5 2.5 4 1.5 2.5 4Initial 1 1 1 1 1 1 6Additional 3 2 5 1 1 1 13

Total (4) (3) (5) (1) (1) (2) (2) (1) (19)

*Total time: six= 1.5 h; seven=2.5 h; six=4 h

Table 2. Plasma and cerebrospinal fluid (CSF) amino acidmeasurements in neurological disease controls. Diagnoses andCSF sampling times

No. of Timepatients Diagnosis (hours)

Initial series (14)Patients with 1 Kennedy syndromewasting (10) 1 early onset SMA

1 late onset SMA1 dermatomyositis1 distal SMA 1.52 post-polio1 Kugelberg-Welander1 gold neuropathy251 polyneuropathy

Patients with 2 cervical myelopathy 1.5spasticity (3) 1 multiple sclerosis

Other (1) 1 cluster headache

Additonal series(12) 1 Paraneoplastic 1 .5

syndrome1 OPCA 41 Multiple system 2.5

atrophy1 PSP 4

CSF only 1 OPCA 2.53 OPCA 41 Motor neuropathy 2.51 FSH dystrophy 41 Multiple sclerosis 1.51 Multiple sclerosis 2.5

OPCA=Olivopontocerebellar atrophy; PSP=progressivesupranuclear palsy; FSH=facioscapulohumeral

dissolved in orange squash and water to give a stocksolution containing 84 g glycine/L. Subjects weregiven 3.6 ml/Kg (0.3 g/Kg) of this solution, andsubsequent blood samples were taken at 1.5 and 4 hafter the glycine load.All blood samples were taken into tubes containing

ethylenediaminetetra-acetic acid and placed immedi-ately into ice. The samples were coded before transportto the laboratory for analysis. The code was brokenonly on completion of the initial phase of the study.

Additional seriesAnalysis of the data from the initial study indicatedthat plasma glycine levels at 4 h, but not at earlier

time points, were significantly higher in MNDpatients than normal control subjects (see below). Afurther nine MND patients (six ALS and three PMA)and four neurological controls were therefore giventhe oral glycine loading test, sampling at 4 h only(Tables 1 and 2).

Amino acids in cerebrospinal fluidCSF amino acids were measured at either 1.5, 2.5 or4 h after glycine load in some of the MND patientsand neurological controls in the initial and additionalseries, and in several additional MND cases andneurological controls not included in the plasmastudies. Overall, six MND patients and four neuro-logical controls were sampled at 1.5 h, seven MNDand four controls at 2.5 h and six MND and sixcontrols at 4 h (Tables 1 and 2).

Amino acid analysisBlood samples were centrifuged at 2000xg for 20 minat 4°C. Plasma was removed to avoid contaminationfrom the buffy coat and stored at -70°C. The sampleswere deproteinized with sulphosalycylic acid (45 g/ml),left overnight at 0°C for 1 h and centrifuged at10 000xg for 5 min at 4°C. The supernatant was thenre-centrifuged and the supernatant analysed. CSFsamples were processed in a similar fashion asdescribed previously3'4.Amino acid analysis was carried out using an LKB

415 Alpha Plus analyser using a sodium citrate bufferwith a pH gradient of 3.2-6.25, ninhydrin as the colourreagent and norleucine as the internal standard.

Statistical analysisPlasma amino acids in the MND patients werecompared to those of the normal control subjects usingStudent's t-test (one-tailed, unpaired). Values for theneurological disease controls were not normallydistributed, however, and comparisons were madeusing the Mann-Whitney non-parametric statistic.CSF amino acid levels were normally distributed andcompared by Student's t-test, as above.

ResultsPlasma amino acid levelsThe mean ages of the MND patients in the initialseries and the normal controls did not differ sig-nificantly. Baseline amino acid levels in the MNDpatients did not differ from levels in the normalcontrol subjects and were within the range of valuesreported by others8.

Journal of the Royal Society of Medicine Volume 86 September 1993 503

Following oral glycine loading, normal controlsubjects showed a large rise in plasma glycine ofmorethan 10-fold above baseline at 1.5 h, as noted by Bankand Morrow1, with significant increases in threeother amino acids, serine, alanine and threonine inkeeping with the known metabolic fate of glycine. Nochanges in the levels of other amino acids wereobserved. No differences in the levels of glycine or anyother amino acids were found between MND patientsand normal controls at this time point, whetherconsidering MND patients as a whole or clinical sub-groups. However, at 4 h, glycine levels in the ALS,PBP and PLS patients were found to be significantlygreater than normal controls: (normal control glycinelevel 670.6± 32.3: ALS group 1003.6± 85.0, P< 0.023;PBP group 1139.18±131.7 P<0.001 and PLS group1093±132.4 P< 0.001, respectively). Glycine levels inthe PMA patients were also higher (919.4±192.9), butthe difference was not quite significant at the 0.05level; the variance of values in this group wasconsiderably larger.

Additional seriesFour hour time-point data from the additional nineMND patients were subsequently included in theanalysis. The grouped data confirmed the observationthat 4 h plasma glycine levels were significantlygreater in ALS, PBP and PLS patients than normalcontrols (P< 0.05, < 0.001 and < 0.001, respectively),while data from the PMA group again showed nosignificant difference (Figure 1).

Comparison with neurological disease controlsThere was no significant difference in 4 h plasmaglycine levels between the normal and neurologicaldisease control groups. Levels in the combinedALS/PBP/PLS patient group were significantly higherthan the combined normal plus neurological diseasecontrols (P<0.013, Figure 1).Figure 2 shows the individual data points for 4 h

glycine levels in the MND sub-groups, normal controlsand neurological disease controls. None of theneurological disease controls had baseline or 1.5 h

2000 -

0E

0).0

Cu

Ecoco)Cu

1000 AI

* 0

0* 0

0

0

00

0

1 : II* I

*.

0

00

00

0

cont PMA ALS PBP PLS

* (a)

* (b)

0

;(c)

1S

Neurocont

Figure 2. Individual data points for patients and controlsshown in Figure 1. Common values for more than one caseare shown as a single point. Patient (a) had 'late onset spinalmuscular atrophy'; patient (b) multiple system atrophy; and(c) Kennedy syndrome

amino acid levels outside normal control values. At 4 h,while 8/23 ALS cases, and 15/32 MND patients hadplasma glycine levels of > 1000 Amol/L, only threeneurological controls showed abnormally increased 4 hglycine levels [patients (a), (b) and (c), Figure 2].Patient (a) had 'late onset spinal muscular atrophy',(b) had multiple system atrophy and patient (c)had X-linked spinobulbar neuronopathy (Kennedysyndrome).We examined the possibility that the higher 4 h

glycine levels in the MND patients might reflectreduced absorption rates. We did not observe lowerglycine levels in the MND patients at 1.5 h, as mighthave been anticipated on this basis and studies onseveral individual ALS cases, measuring half-hourlyplasma glycine levels after loading, confirmed thatthe peak glycine level occurred at 1.5 h1. The sig-nificantly higher 4 h glycine levels in the MNDpatients must therefore reflect a reduced rate ofglycine clearance from the blood in MND.

100

P< 0.001T P< 0.001

)5 L <0.0130E*0'._

0cu

coCDlL

Allcont26

Figure 1. Four hourplasma glycine levels (means and SEM)in. normal controls (cont); progressive muscular atrophy(PMA): amyotrophic lateral sclerosis (ALS); progressive bulbarpalsy (PBP); andprimary lateral sclerosis (PLS) patients. Thefigures indicate the number ofpatients and subjects studied.The sixth and seventh panels show combined values for ALS,PBP and PLS cases compared to all normal and neurologicaldisease controls. Values in motor neurone disease patientswere higher than normal or neurological controls at thesignificance levels shown

80

60

40

20

0 1 5 2.5 4

Hours

Figure 3. Levels ofglycine and one of its metabolites, alanine(mean and SEM) in the cerebrospinal fluid (CSF) in motorneurone disease (MND) patients and neurological controls.Baseline values (0 h) were taken from previous observations;glycine levels in MNDpatients were significantly higher thanneurological controls (P<0.025)3'4. Values at 1.5, 2.5 and4 h after oral glycine loading represent data from severalindividual cases sampled on a single occasion (see text). CSFglycine and alanine levels at 2.5 h peak were significantlyhigher in MND patients (P<0.04). o = Control glycine;* =motor neurone disease (MND); =control alanine;*=MND alanine; *=P<0.04

P<O

T

1400 -

- 1200 -

E 1000-0)* 800-

a600ECo(X 400

200 -

o

8 11

504 Journal of the Royal Society of Medicine Volume 86 September 1993

CSF amino acid levelsFigure 3 shows changes in CSF levels of glycine andits metabolite, alanine following oral glycine loadingin MND patients and neurological disease controls.The mean values and standard errors at 1.5, 2.5 and4 h after glycine loading were derived from severaldifferent patients, each sampled at one time point.Baseline (time 0) values were taken from the earlierpublished study3'4.

It was previously reported from this laboratory thatbaseline CSF glycine levels were some 60% higher inMND patients than controls (P< 0.025)34. Levels ofboth glycine and alanine rose progressively in MNDpatients and neurological controls after loading,peaking at 2.5 h. Levels of both glycine and alaninewere significantly higher in MND patients thancontrols at 2.5 h (P< 0.04) and remained well abovebaseline values even 4 h after glycine ingestion.Levels in MND patients were not significantly higherthan neurological controls at this time point, however.

DiscussionPlasma amino acid levels in the baseline fastingsamples from MND patients did not differ fromnormal controls. Glutamate levels in normal subjectswere similar to those reported by others89 but wewere unable to confirm the significantly higherplasma glutamate levels in MND patients noted bysome groups8 9. Our main finding was that plasmaglycine levels 4 h after oral loading were significantlygreater in MND patients than normal control subjects,and that this appeared to be due to a reduced rate ofglycine clearance from the plasma. We were unableto demonstrate a clear correlation between 'diseaseactivity' or any other clinical parameter and plasmaglycine levels or clearance rates, but in general levelswere higher in cases with upper neurone signs andparticularly with bulbar symptoms.These data could have resulted from reduced glycine

uptake by wasted muscles in the MND patients, ordefects in renal or hepatic handling of the glycineload. However, four-hour glycine levels in theneurological disease controls, which included caseswith muscular wasting and spasticity due to varietyof other causes, were not significantly different fromthe normal controls; the reduced glycine clearanceobserved in the MND cases was not therefore simplya reflection ofthe general metabolic effects ofcachexiaor disuse. Furthermore, all patients had normal renaland hepatic function on conventional screening tests.Three neurological control cases did show abnormallyhigh glycine levels at 4 h, but one ofthese patients hadlate onset SMA, and another spinobulbar neurono-pathy; these are also forms of 'motor neurone disease'.CSF glycine levels, previously found to be signific-

antly higher in MND patients than controls atbaseline3 4, increased sixfold in the MND cases and4-fold in neurological controls at 2.5 h following glycineloading, indicating substantial transport across theblood-brain barrier, the peak CSF levels occurring1 h later in CSF than in plasma. Levels of glycine andits metabolite, alanine, were higher in the MNDpatients than in neurological controls, and significantlyhigher at 2.5 h (P< 0.04). The time at which CSFglycine levels return to baseline has not been estab-lished; they were still more than four times higherthan baseline values 4 h after glycine ingestion.These observations may be of relevance to the

pathogenesis ofMND. There is increasing interest in

the concept that certain neurodegenerative disordersmay result from excessive activity of potentiallyneurotoxic excitatory amino acid neurotransmitterssuch as glutamate10'11. Glutamate analogues suchas N-oxylamino-L-alanine (BOAA, the neurotoxinresponsible for lathyrism) and domoic acid (implicatedin the encephalopathic syndrome resulting from theingestion of contaminated mussels from the PrinceEdward Island estuary in Canada) are toxic toneurones12-14. Glutamate acts at several receptorsub-types in the nervous system, notably N-methyl-D-aspartate (NMDA) receptors, but the action ofglutamate at the NMDA receptor appears to bedependent on glycine15"16, which is principally storedin and released from the interneurones. Glycine bindsto a strychnine-insensitive allosteric modulator siteon the NMDA receptor, and greatly potentiates theexcitatory action of glutamate15l6. Our preliminaryobservations on abnormal glycine metabolism inMND17"18 led us and others19 to suggest that adisturbance of glycine regulation at synaptic levelmight lead to excessive neuronal stimulation andexcitotoxic damage via the NMDA receptor. Theassociation of serious neurological disorders such asseizures, encephalopathy and mental retardation ininfantile ketotic and non-ketotic hyperglycinaemicsyndromes2, and the cases of motor system disorderwith hyperglycinaemia described by Bank andcolleagues1' 2, support this hypothesis. We have alsorecently described a further case of isolated hyper-glycinaemia, with high CSF glycine levels, charac-terized at presentation by cerebellar dysfunction, mildpyramidal tract signs and cognitive impairment20;48 months after presentation this patient haddeveloped muscular atrophy and bulbar palsy withspasticity, suggestive of motor neurone disease. Post-mortem findings in this case will be reportedseparately.Any hypothesis concerning the pathogenesis ofMND must address the issue of the selectivevulnerability of the upper and lower motor neurones,and the regions of the neuraxis affected in the disease.While NMDA receptors are widely distributed andfound in high concentrations in areas relativelyunaffected in the disease, such as the substantiagelatinosa, hippocampus and cerebellum (de Belleroche,personal observations), the highest tissue levels ofglycine are found in sites of predilection, in thebrainstem and spinal cord, where it functions as aneurotransmitter21; and within the cord, glycine isconcentrated in the region of the anterior horns21.We would suggest that the reduced glycine clearancerate demonstrated in MND patients, and the resultingincreased CSF glycine concentrations, reflect a failureof normal glycine uptake mechanisms in the centralnervous system, possibly resulting in local excess ofsynaptic glycine and neuronal damage mediated bythe NMDA receptor.The average person probably ingests between 2-4 g

of glycine daily, compared to the loading dose in thisstudy of some 18-20 g, but the neuraxis is clearlyexposed to significant amounts of exogenous glycineon a chronic basis. Although animal studies indicatethat most central nervous system glycine is derivedfrom glucose via serine22, there is also a flux ofexogenous glycine into nervous tissue22. Most of thisglycine presumably enters the general metabolic poolin the neuroglia, but factors regulating glycineconcentration and activity at synaptic level are not

Journal of the Royal Society of Medicine Volume 86 September 1993 505

fully elucidated. Glycine is known to be removed, andthus inactivated, by a high affinity uptake system23,and its site of action at the NMDA receptor iscompetitively blocked by kynurenate (itself derivedfrom tryptophan), which is released by surroundingglial cells, and is thought to act as an endogenousglycine antagonist15. Raised CSF glycine levels mightresult from failure of such systems. Such a defect maynot be specific to glycine, however, since it has alsobeen previously reported that glutamate levels riseexcessively and remain elevated longer than normalfollowing glutamate loading in MND patients9.

It is noteworthy that glutamate, glycine andaspartate share similar high affinity uptake mechan-isms23. Malessa et al.24 reported that concentrationsof aspartate and glutamate were reduced in allregions of the spinal cord in ALS, and that glycinelevels were reduced in the lumbar ventral anddorsal horns, in keeping with a defect in re-uptakemechanisms, while the levels of other transmitterssuch as GABA were unchanged. Intriguingly, it wasrecently shown that MND patients do indeed showa marked impairment of the high-affinity glutamateuptake mechanism in synaptosomes derived from thespinal cord, motor and somatosensory cortex, comparedto normal control subjects and patients with someother neurodegenerative diseases26. Further studieson the control of factors regulating excitotoxic trans-mitters might provide some insight into pathogenesisand potential therapeutic approaches in MND.

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21 Berger SJ, Carter JG, Lowry OH. The distribution ofglycine, GABA, glutamate and aspartate in rabbit spinalcord, cerebellum and hippocampus. J Neurochem 1977;28:149-58

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24 Malessa S, Leigh NP, Bertel 0, Sluga E, Hornykiewicz 0.Amyotrophic lateral sclerosis: glutamate dehydrogenaseand transmitter amino acids in the spinal cord. J NeurolNeurosurg Psychiat 1991;54:984-8

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26 Rothstein JD, Martin LJ, Kuncl RW. Decreasedglutamate transport by the brain and spinal cord inamyotrophic lateral sclerosis. New Engl J Med 1992;326:1464-8

(Accepted 12 November 1992)