J. clin. Path. (1959), 12, 489.

NEUROCHEMISTRY AND NEUROCHEMISTS*BY

JOHN N. CUMINGSFrom the Department of Chemical Pathology, Institute of Neurology, the National Hospital,

Queen Square, London

A presidential address to a learned society orthe inaugural address of a professor is usuallydevoted to one of three possible themes. It canbe concerned with political considerations, witha historical survey of some subject, or it can bedevoted to a purely scientific problem.We, in our Association, have had in the past

two of these themes, the political and thescientific. I am suggesting combining a historicalapproach with a scientific theme in relation toneurochemistry, and hope to show how in thiscentury progress has been made in this particularfield and how there can now be offered applica-tions of the methods, techniques, and theories ofthe pure neurochemists to clinical medicine andneurology. Therefore the object is rather to showhow basic research has frequently been of valueat a later date in an understanding of diseaseprocesses and subsequently in the treatment ofdisease. Occasionally this order has had to bereversed and this will also be illustrated.During the first 50 years of this decade there

were 59 medical and physiological Nobel prizewinners, and of these some 10 were biochemistsor used biochemical methods in the field ofneurology. Hence one in every six of all theseNobel prize winners was interested in the fieldwhich is under discussion, and some of theseworkers will be mentioned.The chemistry of the nervous system can be

said to have begun with Thudichum. He wasborn in Budingen in Germany in 1828, and diedof a cerebral haemorrhage in London in the year1901. He qualified medically in 1851 at theUniversity of Giessen, but had many otherinterests, one of which was singing, especially thesinging of Italian arias. He was attracted toLieb:g, thereby making his entry into the field ofchemistry. However, like so many others, he hadto flee the country of his birth, and, com.ng toEngland, he married in 1854 Charlotte Dupre, asister of a chemist at the Westminster HospitalMedical School. He discovered urochrome in*The Presidential Address delivered to the Association of Clinical

Pathologists on October 2, 1959.

2Z*

1864, and suggested the name " lutein " for acertain colouring matter in animal tissues, a namelater changed to lipochrome. His work on thechemistry of the brain took place during the years1865 to 1882, and many substances that we nowknow much more about, such as sphingomyelin,kerasin, and cerebronic acid, were described andnamed by him subsequent to their preparationand identification. He measured the specificgravity of brain and also commented on thepresence of such substances as copper in thebrain. His monumental treatise, PhysiologicalChemistry of the Brain, appeared in London in1884. Later in life, after he had retired, he turnedhis attention to food and wine, about which hewrote in 1895 and 1896.As a direct result of his work a chemical

section of the Kaiser Wilhelm Institute wasstarted in Munich in 1928 largely through theactivity of Kraepelin, who had been the first todevise a useful classification of schizophrenia.The name of Thudichum is remembered inanother way, for I have discovered that inGalesburg in the state of Illinois, U.S.A., apsychiatric research department has named itslaboratory after him. It might be worthmentioning now that this Thudichum laboratory,under the direction of Dr. Harold E. Himwich, isthe chief psychiatric biochemical research centrein that state, and had officially given to it in 1957a year's income of some £6,000 more than it isreported our Government gave for psychiatricresearch for the whole of Great Britain.Another milestone was reached in 1937 when

Page, then in New York, but later in Cleveland,published the volume entitled Chemistry of theBrain. He had worked earlier in Germany andknew many of Germany's prominent biochemists.The subject of neurochemistry has now

expanded well beyond the recognition of theseearlier workers, and any general review of thesubject is quite obviously impracticable. I have,therefore, chosen only a few major topics todiscuss.

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JOHN N. CUMINGS

Oxidative and Carbohydrate MetabolinThe brain has been shown to have a high rate

of oxygen utilization. In fact, about 25% of thetotal oxygen used by the body in basal conditionsis utilized by the brain, as was shown relativelyrecently in 1948 by Kety and Schmidt.The brain makes use of glucose almost

exclusively in its metabolism in contrast to otherorgans, and by sampling the arterial and thejugular venous blood it has been shown that therespiratory quotient is for practical purposesunity. Naturally, alterations in degrees ofconsciousness produce some alterations inutilization, and Himwich has made extensiveinvestigations along these lines, and his and otherauthors' work is discussed in his book entitledBrain Metabolism and Cerebral Disorders.Oxygen uptake varies from one tissue to

another, the order of activity increasing fromperipheral nerve to white matter to cerebralcortex. In the same way the trigeminal nucleushas a relatively low activity.The method by which glucose is metabolized

in the brain is very similar to that in otherbody tissues, following the pathway glucose-6-phosphate, fructose-6-phosphate, and fructose-I: 6-diphosphate to 2 molecules of triosephosphate,and finally to pyruvate. There is then aconversion of pyruvate to a 2 C compoundwhich by a complex reaction can enter the citricacid cycle, which has a close connexion with theKrebs cycle.These basic portions of chemical knowledge,

which are related to all body structures, hadbeen obtained by pure chemists such as Krebs, aNobel prize winner in 1953. Hans Adolph Krebswas born in Germany at Hildesheim some 59years ago. After studying medicine he went toBerlin and then returned to Freiberg in ProfessorThannhauser's clinic. He left Germany and cameto Cambridge from whence he went to Sheffieldand then to Oxford. In Cambridge in 1933 heworked on the synthesis of glutamine fromglutamic acid and ammonia, while it was fromSheffield in 1937 that he published his work onthe citric acid cycle. The tricarboxylic acid cycleis now recognized as the major pathway for theoxidation of carbon compounds in the bodygenerally. However, unlike other tissues, thecerebral metabolism of available carbohydrate isnot dependent on any local action of insulin.The work of Krebs embodies earlier theories ofsuch workers as Szent-Gyorgyi and Martius andLopp. Szent-Gyorgyi won his Nobel prize in1937. He was born in Budapest in 1893 and

qualified in 1917. However, he began researchwork as a first-year medical student. After achequered career he finally became Director forthe Institute of Muscle Research at Woods Hole,Massachusetts, where he is at present working.

I must mention two other workers in this fieldof basic science; Carl and Gerty Cori, both bornin Prague in 1896, eventually emigrating toAmerica. They married in 1920, but hadcollaborated in research as class mates earlier.Their work, for which they were joint Nobel prizewinners in 1947, was in relationship to glycogen,an important compound in carbohydratemetabolism.

I will mention very briefly the application ofsome of these various studies in so far as theyaffect us as practising clinical pathologists.John P. Peters was born in 1887 and died in

1955. He was 34 years at Yale, and during thattime he applied the biochemistry of carbohydratesto clinical medicine mainly in the fields ofdisorders of metabolism and more especially todiabetes. He is a joint author of the very well-known book Quantitative Clinical Chemistry.Another person interested in carbohydrate

metabolism who must be mentioned is SirRudolph Peters, who qualified in medicine in1915, and after the first world war went toCambridge and worked with Gowland Hopkins.In 1923 Hopkins offered Peters the chair ofbiochemistry at Oxford, where the laboratory hadbeen started 18 months earlier by BenjaminMoore, who had been the occupant of the chairof biochemistry at Liverpool, the first ever to becreated in England. Here at Oxford at the age of33, Peters continued his pioneer studies, and muchof his earlier work was on oxidation and the SHcompounds. This followed investigations byWarburg and by Meyerhof, two very eminentGerman chemists.Much of the work of Peters was on thiamine,

and few others were interested in this subject atthat time. He is now investigating new problemsof the monofluorocarbon compounds in relation tovitamin deficiency.One of the pupils of Peters is Thompson, who,

while at Oxford with Peters, together withStocken, developed during the last war 2,3-dimer-captopropanol, now known as dimercaprol(B.A.L.). This compound, as is well known, actsas a remover from the body of many heavymetals, including lead and copper. Thompson andPeters were interested in pyruvate metabolism;the pyruvate oxidation system contains highlyreactive thiol (SH) groups. Arsenicals combine

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NEUROCHEMISTRY A ND NEUROCHEMISTS

with these groups and pyruvate metabolism isthereby impaired. Coenzyme A also contains oneof these essential SH groups, and this is the site atwhich B.A.L. competes, thereby offsetting the actionof poisonous substances such as the arsenicals.More recently, Thompson has applied theseprinciples to the question of peripheral neuritisand polyneuritis in man, and he has shown thatin a percentage of cases of polyneuritis the fastingblood pyruvate level may be raised, and furtherthat there is an abnormal rise in blood pyruvateafter a test dose of glucose. He has shown furtherthat, in a proportion of the patients who show thisabnormal pyruvate tolerance test, vitamin Biused therapeutically is able to effect some degreeof cure of the patient and the blood levels ofpyruvate may return to normal.

Cerebra LipidsThe brain contains some 12% to 15% by wet

weight of lipid, or 50% to 60% by dry weight.This fatty material was found by Thudichum toconsist largely of cholesterol, sphingomyelin, andcerebroside. Neutral fat is also present but invery small quantities. The early workers usedwhole human brain and separated the substanceson a macro basis with characterization of thecomponent constituents. Thierfelder, forinstance, at the beginning of this century, isolatedone of the cerebrosides (cerebron), and 50 yearslater Chatagnon in Paris, by fresh experimentalmethods, obtained a substance which gavecrystalline appearances and infra-red spectro-scopic findings which were similar to those ofThierfelder's original preparation, which was stillin existence and with which it was compared.

It is perhaps correct to state that the largestamount of work done on these substances in thehuman brain has taken place in the last 10 years.Nevertheless, in the 1920s Klenk, Remkamp,Deuel, and others were separating andcharacterizing many of these compounds on apurely chemical basis. Considering very brieflyone or two compounds, one can say that Gmelinin 1826 found that cholesterol in the brain had thesame formula and configuration as cholesterol in

KSphingomyePhosphosphingosides

? Cephalin

SphingolipidCerebroside

-Glycosphingosides-Ganglioside

gall-stones, but it was later found that theesterified form of cholesterol did not exist in anyappreciable amount in normal brain tissue.

Sphingomyelin, so named by Thudichum, waslater found to contain a fatty acid which isprobably the C 24 lignoceric acid, but it may alsobe stearic or nervonic acid. Sphingomyelin isclosely related to cerebroside as can be seen inFig. 1, from which it can be observed thatsphingosine is a component part of not onlysphingomyelin and cerebroside but also ofganglioside and possibly of a substance calledcephalin B.Klenk and his co-workers around 1940 found a

compound in the brain which they namedsubstance X, later renamed ganglioside, and it wasshown that it contained neuraminic acid,hexosamine-probably a galactosamine-and ahexose radicle. There is still a good deal ofcontroversy about the exact composition ofganglioside, for slightly varying views from thoseof Klenk have been expressed by bothSvennerholm and Bogoch in the last two years.Klenk is a pure chemist and has built up atCologne a famous department full of enthusiasticworkers who are, however, also interested indisease processes.Thannhauser and his colleagues in recent years

have made important contributions to thechemistry of sphingomyelin and of theplasmalogens, originally described by Feulgen inmuscle. He has had much to do with work onvarious diseases, such as Gaucher's disease andxanthomatosis, while working in America.The school in Canada led by Rossiter had

investigated the brains of normal infants andadults, estimating the relative amounts ofsphingomyelin, cerebroside, cholesterol, andlecithin found in the white and grey matter, andtheir results have been confirmed by Brante inSweden and also by the group working with me,all within the past 10 years. Just recently, wehave been able to add, in an even more extensiveseries, the results of similar estimations of thevarious lipids in brains from the foetus, the full-term infant, the young infant, and the child.

elin = Fatty Acid + Sphingosine + Phosphorylcholine

B = Fatty Acid + Sphingosine + Phosphorus + ?

e = Fatty Acid + Sphingosine + Hexose

Neuraminic Acid= Fatty Acid + Sphingosine + KHexosamine

L. HexoseFIG. 1.-The sphingolipids.

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JOHN N. CUMINGS

and his colleagues have investigated as recently as1951. Proteolipid is an important part of themyelin sheath, and Finean has represented themyelin sheath as being made up of alternate layersof lipid and of protein.

Recently a study has been carried out on thewater-soluble proteins of the brain. Portions of

.cerebral white matter in 5 to 10 g. amounts wereextracted with 10 to 20 ml. of 10% sucrose, andsimilar amounts of cerebral cortex extracted with5 to 10 ml. quantities of sucrose. The extractswere concentrated, and the proteins presentexamined by starch electrophoresis. Differentareas of the brain have been selected, and so farnine portions of the frontal lobe, seven from theparietal, and 10 from the occipital lobe have beenexamined with some slight differences in patternbeing observed. The results are illustrated inFigs. 2 and 3. The patterns found do not appearto bear any relation to the proteins of the serum,which in some instances were put up in parallelwith the brain extracts.

Interesting results, however, have been obtainedin cases of suspected cerebral oedema. A well-

FIG. 2.-Starch electrophoresis of water-soluble proteins of normalcerebral white matter. F=frontal, O=occipital, P=parietallobes.

In recent years cerebral metabolism has beeninvestigated extensively. The modern trend is toconsider everything from the dynamic aspect, andtherefore investigators using radioactive isotopeshave followed the metabolic processes of many ofthese substances. In the animal it has beenshown by Waelsch and his colleagues that very m.alittle, if any, build-up of cholesterol takes place inthe adult brain. This may also be true forphospholipids, but the position regarding thesesubstances is not yet finalized.The cerebral lipids do not, of course, constitute

the whole of the cerebral substance. Water,electrolytes, amino-acids, and mucopolysacchar-ides are also present, but these must be supportedand held together, and in part this is done byvarious protein substances, some of which areproteolipids. Total proteins constitute some 40%of the dry weight of the brain or 10% by wetweight. Knowledge of most of these proteins isextremely meagre, but the neurokeratin of the

FIG. 3.-Starch electrophoresis of water-soluble proteins of normalolder writers may be the proteolipid which Folch cerebral cortex. F=frontal,O=occipital,P=parietal lobes.

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NEUROCHEMISTRY A ND NEUROCHEMISTS

marked band of protein has been demonstrated inthe same region that albumin appears in a serumpattern in starch electrophoresis (Figs. 4 and 5).This albumin band has been found even inportions of brain which on naked-eye examinationwere not definitely oedematous. Macroscopicdiagnosis of cerebral oedema is notoriouslydifficult as has been shown previously byhistological examination as well as by chemicaltechniques, and this method appears to be adelicate one for its demonstration.Much work has been done on the amino-acid

composition of the various proteins, and Richterin England and Waelsch in America haveworked extensively on this subject. Amino-acidsare being metabolized continuously as has beenshown by isotope studies, and some of them, suchas glutamic acid, play a very important part ingeneral metabolic processes, as was mentionedearlier when discussing the work of Krebs.

wRP LP

FIG. 5.-Oedematous right parietal and non-oedematous left parietal cortex. Starchelectrophoresis.

FIG. 4.-Oedematous right parietal and non-oedematous left parietal white matter.Starch electrophoresis.

Brain tissue contains both glutamic acid and itsamide glutamine. There is no evidence to suggestthat glutamic acid can replace glucose as a source

of fuel. It can react with ammonia to formglutamine or be oxidized by glutamic dehydro-genase with the formation of ammonia. The laterstages of such a cycle include a ketoglutaric acid,and this has a connexion with the Krebs cycle. Ithas been suggested that some of these pathwaysmay be involved in hepatic coma.

Returning now to the lipids, the next links in thechain that should be considered are the experi-ments in 1948 on Wallerian degeneration by thegroup led by Rossiter. They cut the sciaticnerve in animals and at various stages ofdegeneration examined chemically the distal partof the nerve as well as control nerves. It was

found that at first water increased in amountwhile total phospholipids decreased. Neutral fatdecreased but later returned to normal levels.

RPG

LP

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JOHN N. CUMINGS

The components of myelin-sphingomyelin,cerebroside, and free cholesterol-decreased, butesterified cholesterol from being absent in theintact and normal nerve increased appreciably.Proteins, probably proteolipid or neurokeratin,also decreased. Here then was experimentalevidence relating to demyelination. Recent papers

by this group have been concerned with variousassociated enzyme changes.Some workers have applied these techniques

to the various disease processes. It has, forinstance, been shown in work which I did in 1953that demyelination in multiple sclerosis followeda chemical pattern which in many ways was verysimilar to that found in the cut sciatic nerve.There is in multiple sclerosis in the activeplaques, but not in chronic lesions, an increase ofwater and a loss of the myelin lipids, together witha well-marked increase in esterified cholesterol.One of Klenk's pupils, Dr. Debuch, has shown

that a substance, lysocephalin, possesses someproperties similar to those of lysolecithin. It hasbeen suggested that this might well producedemyelination locally within the myelin sheath,and this type of enzymic action has beenproposed as a possible cause of demyelination inmultiple sclerosis. Thompson and his colleaguesare pursuing this aspect.Very similar lipid chemical findings have been

obtained in Schilder's disease or the so-calledsudanophilic type of diffuse sclerosis. It has notyet been possible to demonstrate the factor thatcauses this demyelination, whether in fact it isenzymic in nature and whether it also has agenetic basis. The results that have been obtainedin both multiple sclerosis and sudanophilicdiffuse sclerosis are illustrated in Tables I and II,

where it will be seen that there is a very greatincrease in the amount of esterified cholesterol,and this is absolutely characteristic of these twodiseases. The deposition of esterified cholesterolin histological sections stained by Scharlach Rstain red, as can be seen in Fig. 6.Turning very briefly to the group of diseases

known as the lipidoses, there is abundant evidencefrom the work of Thannhauser, Klenk, and myown group that some of the various substanceswhich have been described by the chemists areincreased in amount in these diseases.Thannhauser and his associates found inNiemann-Pick's disease affecting the peripheralorgans that there was an increased content ofsphingomyelin in these organs, and Klenk and Ihave both shown in patients in whom the nervoussystem is involved that there is an increasedcontent of sphingomyelin and of cholesterol in

TABLE ICEREBRAL LIPIDS IN MULTIPLE SCLEROSIS

Normal White Demyelinated White

Total phospholipid 5-8 1 8Sphingomyelin 2-4 0 5Total cholesterol 4*1 2-8Esterified 0-3 17Ganglioside .. 10Water .. 69-0% 80-4%

Results in g.l100 g. fresh tissue except ganglioside, which is ing./100 g. dry tissue.

TABLE IICEREBRAL LIPIDS IN BIOPSY SPECIMENS OF

SUDANOPHILIC DIFFUSE SCLEROSIS

Cerebral White Cerebral Cortex

Total phospholipid 4-1 3 35Total cholesterol 2-4 1-21Esterified ,, 0-8 0-17Hexosamine 0-39 0-59Ganglioside . 1-5Water 77-8% 82-6%

Results in g./100 g. fresh tissue except ganglioside and hexosamine,which are in g. 100 g. dry tissue.

the brain itself. We have also found an increasedamount of ganglioside in the cerebral cortex.

In amaurotic family idiocy Klenk and hisassociates in 1942 obtained raised levels ofganglioside in the brains of patients, and theyfound that the level of neuraminic acid, that is ofganglioside, showed an increase of even as muchas five to 10 times the normal, and this increaseoccurred almost exclusively in the cerebral cortexwhere the ganglion cells can be seen by histologyto be full of a P.A.S.-positive material.During the past few years the application of

these basic chemical facts has been used in clinicalpathology in the field of neurology. Chemicalinvestigations of the brains of patients dyingfrom multiple sclerosis, from lipidoses, and fromencephalitis have been made by me, anddistinctive patterns obtained in these differentdiseases. This knowledge has been utilized in theexamination of cerebral biopsy material.Numerous patients with progressive mentaldisturbances have been examined by clinicianswithout a definite diagnosis being possible,although some of the conditions are known to befamilial. After all the facts have been given tothe parents and permission obtained, a smallsample of cerebral tissue, about 1 cm. cube, wasremoved. My histological colleagues haveshared these pieces of tissue with me, and I haveexamined some 74 such specimens. A positivediagnosis has been made on 51 occasions bychemical examination, and in only one case havethe histological and biochemical opinions been in

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NEUROCHEMISTRY AND NEUROCHEMISTS

complete disagreement. It is not possible to givethe complete details, but one or two examples willdemonstrate the type of findings that have beenobtained. (I must mention that no clinical defecthas resulted, nor have we been able as yet toobtain post-mortem confirmation of our opinions,apart from three cases, and these patients diedmore than six months after operation.)A patient with a diagnosis of amaurotic family

idiocy arrived at histologically and chemicallyshowed typical histological lesions in the cerebralcortex, and these can be seen in Figs. 7 and 8.The chemical examination revealed a markedincrease of ganglioside in the absence of anyevident loss of phospholipid or of demyelination(Table III).

TABLE IIICEREBRAL LIPIDS IN BIOPSY SPECIMENS FROM

AMAUROTIC FAMILY IDIOCY

Cerebral Cerebral NormalWhite Cortex Cortex

Total phospholipid 5-88 3 70 35-4 5Total cholesterol 3-41 1 11 1-0-1-5Esterified 0 0 0Hexosamine 0-36 0-70 0-50-7Ganglioside .. 2-3 1-01-5Water .. 75-1% 81-9%, 82-85%

Results in g./100 g. fresh tissue except ganglioside and hexosamine,which are in g. 100 g. dry tissue.

Metachromatic leucodystrophy, which isfamilial, shows an altered appearance of themyelin pattern, for in the white matter there ismetachromasia as illustrated in Fig. 9, while thechemical findings are seen in Table IV. It is tobe noted that there is a marked increase ofhexosamine in the white matter, while thereis also evidence of a moderate degree ofdemyelination.

TABLE IVCEREBRAL LIPIDS IN BIOPSY SPECIMENS OF

METACHROMATIC LEUCODYSTROPHY

Cerebral Cerebral NormalWhite Cortex White

Total phospholipid 3-12 3 77 70-8 0Total cholesterol 1-28 0 93 40-5 0Esterified ,, 011 0-14 0Hexosamine 0-48 0-74 0-2Ganglioside .. 1-4Water .. 8 1-0% 83-3% 70-74%

Results in g./ 100 g. fresh tissue except ganglioside and hexosamine,which are in g.I 100 g. dry tissue.

It must be mentioned that metachromaticdeposits occur in the kidney, and examination ofthe urine reveals such metachromatic material,and this was indeed found in this patient.

Lastly, subacute leucoencephalitis of theinclusion body or Van Bogaert type shows some

slight but definite chemical findings, and areshown in Table V. There is only slightdemyelination without the presence of cholesterolester but with a slight increase in hexosaminelevels.

TABLE VCEREBRAL LIPIDS IN BIOPSY SPECIMENS OF

SUBACUTE ENCEPHALMS

White Cortex

Total phospholipid 5-4 4 0Total cholesterol 2-7 1-2Esterified 0-05 0-02Hexosamine 0-51 0-63Ganglioside .. 10Water .77-5% 81-1%

Results in g./100 g. fresh tissue except ganglioside and hexosamine,which are in g./100 g. dry tissue.

Table VI shows the actual number of caseswhich have been examined chemically, and thegroups of diseases into which they fall.

TABLE VI74 CEREBRAL BIOPSIES EXAMINED CHEMICALLY

nomaltyAbnormal N

Condition Abnorslity but Not NoDigotc D.ua Abnrmalit

Normal .. 26Amaurotic family idiocy 10 5Metachromatic leucodys-

trophy .. 9Multiple sclerosis . .Sudanophilic diffuse

sclerosis .. .. 4Subacute encephalitis .. 6Organic dementia .. 8Miscellaneous .. . 4

AcetylcholineHunt and Taveau in 1906 were the first to

demonstrate that acetylation of choline (asubstance known to Thudichum and obtainedoriginally from phosphatide in bile, hence itsname) resulted in a product of greatly increasedpharmacological activity. Loewi demonstrated in1921 that liberation of a substance now known asacetylcholine, but called by him " Vagusstoff,"was present in the perfusing fluid of a frog's heartafter stimulation of the vagus nerve. Sir HenryDale in 1914, 1929, and 1933 very ably describedthe various effects of acetylcholine, and in thelast paper of 1933 introduced the terms"cholinergic " and "adrenergic." Loewi andDale shared the Nobel prize in 1936. In 1911Dale, in association with Barger, was the first toisolate histamine from animal tissues. However,he is best known for his work on acetylcholine,which was later followed by work on neostigmine.Considerable knowledge is now available

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concerning the synthesis and destruction ofacetylcholine after its release at nerve endings andthe conception of an acetylcholine cycle has beenput forward. It was soon clear from experimentalprocedures that an enzyme must be present toconvert acetylcholine into some less active form.Such an enzyme, cholinesterase, which has beenfound to be widely distributed in the body, canin fact break down acetylcholine to acetic acidand choline. Two main types of such an enzymehave been found and these have been called, in1943, true and false cholinesterase, or specific ornon-specific cholinesterase. Nachmansohn, whoproposed the latter term in 1945, was also an ableworker on acetylcholine.These two cholinesterases have been found to

act on different substrates, and are present invarying amounts in different organs and areas.True cholinesterase was found by Nachmansohnin 1939 to be located more in the cerebral cortexthan in the cerebral white matter, while pseudo-cholinesterase was present to a greater extent inthe white matter, as in fact was shown by Ordand Thompson in 1952.

After release of acetylcholine at the nerveendings and its hydrolysis some process mustthen be present for its resynthesis. Quastel andhis co-workers in 1936 suggested such a process,and it has since been proved that acetyl-coenzyme A provides the acetyl group which istransferred to choline by the action of theenzyme choline acetylase. Acetyl-coenzyme A isprobably derived from pyruvate, which links thismechanism, therefore, with the glucose cycle.Only one or two applications of these findings

to clinical neurology will be mentioned. Muchbiochemical work on epilepsy has been doneat Montreal led by Elliott. Elliott and Towerin 1952 estimated bound acetylcholine andcholinesterase in normal portions of brain and inepileptogenic areas of brain, and found that in thelatter there was a marked reduction of boundacetylcholine. Further work both by Elliott inMontreal and by Tower in Bethesda has followed,but it is too early to state its final practical value.Elliott has recently reported on y amino-butyricacid which is present in brain tissue, and has saidthat both glutamate and y amino-butyric acid maybe " action substances " of neurophysiologicalimportance, and that from his experimentalresults there may be a balance of effects of thesetwo substances, and that they may determineneuronal activity.The other condition I wish to mention is ginger

paralysis. This is a form of motor neuritis firstseen in the United States in 1930 following the

drinking of a beverage containing an extract ofJamaica ginger. This was later found to containtri-ortho-cresyl phosphate (T.O.C.P.). Variousdegenerative lesions were found in axis cylindersand in the myelin sheath in not only theperipheral neuritis mentioned but also afterpoisoning by T.O.C.P. present in other substancessuch as apiol and in certain cooking oils. Thehen can be used as an experimental animal, andwork has been done since 1950 on this subject bya number of experimenters. Earl and Thompsonin 1952 showed that T.O.C.P. was a selectiveinhibitor of pseudocholinesterase in nervous tissueand in plasma but that true cholinesterase wasunaffected. Certain new organophosphorusinsecticides are also similarly dangerous, forparathion and mipafox both cause plasma anderythrocyte cholinesterases to be lowered. Theselast compounds do not, however, produceparalysis or degenerative lesions of nerve.

It is perhaps of interest to notice here thatamongst some of these substances recentlyproduced is one for locust control, andexperimental work on this has just been publishedshowing the effect of such substances oncholinesterases in the nerve cells of locusts.So far an attempt has been made to show that

our understanding of disease processes hasfollowed a fairly definite pattern, and hasstemmed from basic chemical work. Applicationof such basic knowledge has been applied tosuitable patients with varying diseases in order toelucidate the cause and the nature of thepathological lesion involved. However, we nowcome to a group of disorders in which the reverseprocedure has been followed. In patients withsome diseases, abnormal chemical findings werefirst discovered, and, following this, basicchemical knowledge had to be determined. In1908 Sir Archibald Garrod delivered the Croonianlectures at the Royal College of Physicians andhis subject was " Inborn Errors of Metabolism."There are now known to be a number of suchdisorders, and one or two of them will bementioned and an indication as to how progresshas been made in our knowledge of these diseases.

In 1934 Folling found in certain imbeciles inOslo that there was an excess in the urine of asubstance, phenylpyruvic acid. He detected thiswith the use of a weak ferric chloride solution. Itwas soon found that the disease, phenylketonuria,was associated with a rare recessive gene inhomozygous form. Various physical differencesfrom the normal are present in these children aswell as a varying degree of mental impairment.The patients are often blonde, the stature and

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Xt4.r * 43

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Fia. 6.-.Sudanophilic diffuse sclerosis. Scharlach R, x 40.

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FIG. 8.-Amaurotic family idiocy. Scharlach R, x 400.

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NEUROCHEMISTRY A ND NEUROCHEMISTS

head measurements may be reduced, andtemperamentally they are docile. The sweat mayalso be excessive and contain phenylpyruvic acid.Having discovered a chemical abnormality in

the patient, other workers in the past decadeproceeded to determine the metabolic pathwaysthat could be involved to produce thisabnormality. The fundamental disorder appearsto be that the inborn error is one concerned witha failure to metabolize phenylalanine along thenormal pathways. In the normal subjectphenylalanine is changed in the body into tyrosineby means of an enzyme, phenylalaninehydroxylase. In the patient with this disease thisstep is not possible, and phenylalanine circulatesin the blood and is disposed of as phenylpyruvicacid and phenyllactic acid. There also appearsto be an abnormality involving indoles andtryptophane. An attempt has recently been madeto treat these patients by dietary restriction ofphenylalanine, but this should be done in the firstfew weeks of life. The patients are given acasein hydrolysate which is freed of phenylalanine,tyrosine, and tryptophane.

Acute porphyria is another condition in whichabnormal metabolic pathways have been shown toexist by Rimington. This condition is sometimesassociated with a polyneuritis, and I have had theopportunity of seeing and examining a number ofsuch neurological patients.The third condition I wish to mention is

hepatolenticular degeneration, which is nowregarded as due to an inborn error of metabolism,for this disease appears to be associated with adisordered copper metabolism. Again the initialobservations were made in patients with thisdisease, for it was discovered by Rumpel thatcopper and silver appeared to be deposited in thetissues. Although this observation was made in1913, and further observations along very similarlines were made in 1929 and 1930, it was not untilan extensive investigation was made in 1948 thatits full significance was appreciated. There is alsoa considerably increased urinary excretion ofcopper, and the blood level of copper is low. Asubstance, caeruloplasmin, which, it has beensuggested, may act as a copper transporter, wasshown in 1952 by Gitlin and Scheinberg to beabsent or only present in minimal amounts in theblood. Two monographs have recently been

written on this subject, so that it is obviouslyimpossible to discuss it in full. Further, althoughsome of the chemical abnormalities in the diseasehave been discovered, the actual mechanismwhich causes these changes had not yet beenfound. However, in 1948, as a result of thefinding of increased copper deposition, asuggestion was made that B.A.L., a substancementioned earlier, might be an effective form oftherapy. This indeed is the case, for remissionsof considerable periods of time have resulted fromits use in many patients. Wilson considered thatpatients with this disease died within four toseven years after the onset of symptoms, and,although older patients have been known to livelonger, the younger patients have died sooner. Ihave investigated 35 patients and personallytreated 20 patients with B.A.L., and Table VII

TABLE VIIRESULTS OF INVESTIGATION OF 35 CASES OF

HEPATOLENTICULAR DEGENERATION

Copper metabolism, abnormal (blood or urine) 34Treatment (under personal supervision)ByB.A.L. .20

and penicillamine .. 11Follow-up period

Up to 2 years.8From 2 to 4 years. . 9Morethan4. 3

Initial improvement after B.A.L. .18Contact lost with patient..4Patients died after initial successful treatment .. 2

shows the results obtained. One of those treatedis alive after eight years, and a number lived atleast five years. As far as is known, two havemarried, and a few have been able to work again.Only two are known to have died, and each ofthese developed a severe cirrhosis of the liver; aportal caval shunt operation was performed andlater both died in hepatic coma. The coppercontent of the liver in both cases, and the brain inone case, was within normal limits, therebydemonstrating the effectiveness of the treatment asfar as the copper deposits were concerned.Neurologically, too, they were still improvedbefore death, but the cirrhosis of the liver hadadvanced relentlessly.

This review is very incomplete, only a fewsubjects having been mentioned, but it may bethat it demonstrates the evolving pattern of thebiochemistry of the nervous system and the valueof basic research when applied to clinical practice.

3A

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