Free amino acids in tay-sachs and normal human brain gray matter

CLINICA

FREE

CHIMICA

327

ACTA

AMINO

ACIDS

IN TAY-SACHS

AND NORMAL

HUMAN

BRAIN

GRAY

MATTER*

ABRAHAM SAIFEK assistance of FRED MAZELIS

JOKMA PALO** with the technical

AND

Biochemistvy Department of the Isaac Albert Reseavch Iwtitzde Center, Brooklw, Few York, 11203 (U.S.A.) (Received

June

Previous

o.f the Kingsbwok

Jewisk Mrdrcal

28, 1908)

studies showed significant

differences

in the amino acid composition

of the ganglioside-peptides in Tay-Sachs disease as compared to normal brain gray matter. The present study was undertaken to determine whether these differences in peptide composition could be related to variations in their free amino acid pools. About

31

amino

acid peaks

were obtained

by means

of automated

ion-exchange

column chromatography of which 27 were identified. The results obtained (moles of amino acid /IOO moles of total amino acids) for autopsied normal brain gray matter agree well with previously published data except for higher values for taurine, threonine and glycine and a lower value for phosphoethanolamine. Comparison of free amino acid levels in Tay-Sachs brains with normals showed significantly decreased values for glutamic acid, aspartic acid and ~-aminobut~ric acid and increased values for alanine, valine, leucine and tyrosine. No significant differences in free amino acid levels were found between samples from frontal, parietal and occipital lobes for either normal or Tay-Sachs brains. The possible relationship of the free amino acid changes in Tay--Sachs disease to ganghoside accumulaiion and to ganglioside-peptide amino acid composition is discussed.

No changes in the amino acid content of serum, urine or cerebrospinal fluid have been found in Tay-Sachs (TS) disease as compared with normals. However, it has been shown by Korey et al.1 using paper chromatography that the free amino acids in biopsy samples of cerebral cortex from normal and TS-cases are different in that glutamic acid, aspartic acid and y-aminobutyric acid are markedly decreased in TS-disease. The characteristic feature of TS-disease is an accumulation of one type of glycolipid material (Gar2-ganglioside) in brain. This glycolipid normally accounts for * Part of the experimental data in this paper was presented at the 20th National Mectinf of the American Association of Clinical Chemists in Washington, D.C. during August 1908. Helsinki, Finland. Send ** I’resent address: Department of Neurology, Universitv of Ilelsinki, reprint requests to Dr. A. Saifer.

P.kLO, SAIFER

328

less than j o& of the total gangliosides

as compared

with about 80 :;, in TS-d&as@.

The cause for this accumulation has not yet been discovered nisms have been proposed by Schneck at al.“.

although

several

mechn-

The amino acid composition of the water-soluble ganglioside-peptide extracted with chloroforn---methanol (2: I, v/v) from frontal lobe gray matter was shown to be different in TS-disease when compared with the normal by Palo and Saifer’. Because it seemed likely that especially large variations in free amino acid levels might alter some aspects of protein metabolism, such as the type of protein formed5, a quantitative column chromatographic study was undertaken to explain at least in part the differences in the amino acid composition of normal and TS ganglioside-peptides, MATERIALS

AND METHOIX

Brains of five TS-patients (ages between I and 3 years) and five other children (ages between I and 5 years) were used for the investigation. The latter group was unaffected by neurological disorders and four of them had died of accidental smoke inhalation and one of pneumonia. The autopsy had been performed in all Casey within 6 h after death and the brains stored at --zcj” until used. The analysis took place within one year in all but two TS-cases which were analyzed after two years of storage. Thin slices from the cortex

of frontal

(in one case also from the parietal

and occipital)

lobes were cut out with a razor blade, avoiding visible blood vessels and any remaining blood was blotted away with damp gauze sponges.

Based on the findings of Robinson and Williams6 the ethanol extraction method of Porcellati and Thompson’ was employed. Approximately I g of frozen cortical tissue was weighed out and dispersed in a Potter-Elvehjelm homogenizer in 13 vol. of 75 f’, ethanol. The homogenate was centrifuged at 3000 rev./min for 15 min. The slightly turbid supernatant was transferred into a rotating vacuum evaporator and allowed to evaporate almost to dryness at room temperature. The aqueous residue was taken up in a measured volume (2 x z ml) of water, pooled, transferred to a centrifuge tube and centrifuged at 7500 rev. /nun for 45 min. Usually 300 ,~l of the clear extract was analyzed in each run.

The single column method of Piez and Morris* with the Technicon automated system) was employed. However, two modifications were made. Firstly, methanol was included in the buffer system in order to improve the separation of the acidic amino acids. Secondly, ninhydrin solution was prepared without using 4 N acetate buffer and anllll~Jniawas removed from the solution with acid-washed Permutit. The color produced in the reaction was measured at two wavelengths, 440 m,~4for proline and 570 m&r,for the other amino acids. RESULTS

Twenty-seven amino acid peaks in both normal and TS-samples identified either in this laboratory or elsewhere (Dr. Paul B. Hamilton,

could be personal

AMINO

ACIDS

IN TAY-SACHS

BKAIh’

329

communication). In addition to these, from 3 to 3 unidenti~ed peaks were observed. The first was between cysteic acid and phosphoethanolamine. The second, which was not present in all samples, and the third both appeared between taurine and methionine sulfoxides and the fourth between proline and glycine. In some of the runs, traces of allo-isoleucine were also detected but the peaks were too low and broad to be calculated. Dr. Hamilton reported the presence of urea and asparagine in both normal and TS-samples and he also found peaks in the positions of sarcosine and glucosallline. In this laboratory, urea was eluted together with taurine; glutamine and asparagine with threonine; citrulline with glutamic acid, and a-amino-n-butyric acid with cystine. In most of the runs these could not be calculated separately. Small amounts of cysteic acidandmethionine sulfoxides were calculated and added to cystine and methionine, respectively. In Table I, a comparison is given between the results for the normals and for TS-brains. Two separate extracts were prepared from adjacent areas of the frontal

FREE AXINO ACIDS OF THE GRAY Rloles/~oo

moles

~.

of total

MATTER

FROM

amino acids. .-~

_.___

SORMAI~

____

HUMAN

AND

TAY-S.ACHS

.____~____.

BRAIPi

.-____

_-..

- --. I’hosphoethanolamine ‘I‘aurine

2.71

1.31 I.44 0.66

5.41 6.42

0.79 1.55

4.35-&w

Glutamic lint) * Proline Glycine Alanine*

27.78-42.54 I.70-2.90 4.98-8.40

2 I.92

4.02 0.70

16.52-27.32 1.71-3.57

6.2j

1.11

0.86

2.65-4.99

8.57

I._19

4.75-7.X 6.59-IO.55

1.23 1.42

0.23 0.31

o.9.PI.53 1.00-1.84

I.75

0.55 I.12

0.77 I.58 0.92

0.15

0.,56-0.98 0.59-2.57

1.71

0.73 0.25

o-59-.r.r5

2.11

0.51

1.06-2.44

5.78

0.76-2.56

I.30

7.27-L2.79

6.~9

I .02

7.36-10.06

3.74 3.94

I.01 I .02

2.39-5.09 2.59-5.29

35.16 2.30 6.69 3.82

5.51 0.46 I.28

8.71

Aspartic acid* Thrcunine (plus glutamine and aspara~in~) Serinc

_-

0.67 2.76

I.06

IO.03

(plus urea)

Meava

acid (plus citrul-

Cystinc (plus cysteic acid plus waminohutyric acid) Valine* Mcthionine (plus methionine sulfoxide) Cvstathioninc I~olcucine Leucinc* Tyrosine* I’henylaIanine Ethanolamine ;f-Aminobutyric 0rnithine Histidinc Lysi nc H~~nocarnosiRc Arginine Total (molesjroo total)

acid*

* Statistically

significant

3.83

5.92

o.g3 5.02 0.53 1.26

I.OO-2.50 2.33-5.33 0.48-2.94 o.oo-12.67 1.39-2.83 3.50-6.86 0.98-2.00 r.oj-2.53 I.89-5.()I

0.17

0.42-0.84 0.58-0.94

I.49 I.80

o-37

o.rq

3.59

0.44

3.52-4.16

3.90

1.49

7.36 0.32 0.65 I.IO

1.17

5.80-8.98

I.18

r.oo-5.18

0.08

0.20-0.44

3.59 0.56

0.36

0.0%I.04

0.14

0.47-0.83

0.90

0.11

0.75-1.0-j

0.27

0.74-r

I.20

1.47-4.71

0.13 0.29

0.76-1.12

3.09 0.64

0.27

0.2%I.00

‘,.57-1.35

2.08

0.49

I .42-‘2.7.~

.46

O.S6

of 100.03

Total (,mnoles/g of wet weight)

2.64

+3h-6.40

I.75 0.63 0.76

0.94 0.06 moles

.-. -_.-

3 S.E. _ _.. 0.00-3.04 4.37-8.21 1.81-3.61

___

31.91 difference

lg.60

--

roo.oG

.-

at I “/b probability

26.11

28.51 -

level.

CZGL.Chirn. Acta, 22 (1968) 327-334

PALO,

330

S.AIFER

lobe from each brain, and the means represent the mean values of the analyses on these two preparations in each case. The results are expressed as moles of each amino acid per roo moles of total amino acids in order to better demonstrate the observed differences. However, the totals are also given as qoles per g of tissue wet weight. Seven out of 24 listed figures showed statistically significant differences at the I “,, probability level between normals and TS-disease and of these, aspartic acid, glutamic TABLE

11

~cm”les/g

of tissue

Taurine r\spartic acid SAcetylaspartic Threoninc Serine Glutamic

wet weight.

I.45 0.42 acid

(z), 1.37-2.13 (5) (7). 2.87 (z), 1.~7.9*

1.60

(3)

4.3*, 5.5 (2) o.z* (3), “48 (7)> 0.53 (2) 0.75 (7), r.o5* (4). 1.21 (z), r.3vr.s* (3) .~.L-3.9* (3), 3.99 (7)> 10.5. r9.9* (2,

acid

Glutamine C,itrulline l’roline Glycinc hlaninc C!_stlnc Cysteic acid Valine

r.4pr.9* 0.20**

(3). 4.98

lsoleucine Lcucinc l‘yrosinc l’hcnylalanine

1.2s

rr.33

1.2s 2.2.3

I.3 -I.()* (3)> 0.87 (5)

(3) (7)

“.I-o.2*

(3), 0.32

(7)

“.““I (2) ** (0) 1.17 (2) r.o5 (2) ** (‘>) 0.+0.7* (3), I.47 j.5, 4.6* (2)

;J-Guanitlinobutyric acid P-H~tlr”x~-:,-aminobut)-ric Ornithinc Histidine Lysine Homocarnosine i\rgininc

“.“.~~0.07 (2) I’rcscnt (2) 0.21 (7) 0.24 (7) 0.“8* (I), 0.13

1.02

(6)

nihydroxvphenylalaninc Ethanolamine Phosphoethanolarninc Glyccrophosphocthanolaminc a-Amino-z-butyric acid 7’.Aminobutyric acid

acid

L.LO

5-43

1.13 (7), o.+o.i* “..lS~“.74

o.rh*

3.20 2.7’)

0.46

(7)

o.“Xk).j6 (j), 0.17 (7) 0.00 0.31* (H), 0.30* (4) 0.5’-0.0” (5). 1.1-2.5 (2) 0.r.J (7), 0.18 (2) o.r-“.4* (3), 0.37 (7), 0.56 (2) “.“* (3). 0.24 (7) O.“* (,j), 0.25 (7)

RIethionine Cystathioninc

3.07 _

I.21

1

0.30 0.28

11.2”

r.75+ 0.10’ 0.74 2.13

I .2L

o.,j

I

O.L() 0.50 0.20 0.24

r.15 O.jL

0.03

-

(7), 2.0,

r.jr

(9)

(7)

Total liorey rt aL1, (4) = G erritsen + (I) : .kbraham et al.“, (2) = ‘l‘allanl”, (3) Rrcnton et al.“, (6) = Perry rf aZ.14, (7) ~~ Prcnsky & hlo~rr’~, (8) == Shimizu zawa & Sane’“. * Cerebral cortex (other data arc for mixed white and gray matter). ** Present in the cerebral cortex of a h?;permethionincmia case. 4 Sot included in the total pmoks.

& Waisman’:‘, (5) ~~ it al.‘&, (9) _m lian;l-

AhIISO ACIDS Ih’ TAY-SACHS

BKAIS

331

acid and ~-aminobutyric acid were decreased in TS-cases and alanine, valine, leucine and tprosine were increased. The mean values for some other amino acids also showed marked differences, especially that for cystathionine, but, because of wide variations, they were not significantly different. In Table II, the mean values for the normal brain are expressed as /smoles per g of tissue wet weight, together with previous data reported in the literature. i\‘acetylaspsrtic acid, ~hydroxypllenylalanine, gl~~ceropl~ospl~oethanolamine, ?f-guanidinobutyric acid and j?-hydroxy-y-aminobutyric acid were not found in the present study unless some of the unidentified peaks represent these compounds. On the other hand these amino acids have been reported in the mixed white and gray matter of whole brain while the present study dealt only with gray matter, as did the investigation by Robinson and Williams6 who reported separate data for gray and white matter. The latter in~lest~~ators were also unable to find these compounds in the cerebral cortex. Most of the previous data are taken from the paper by Okumura et aL9 as summarized by Tallan lo. This was a study on various parts of single human brain from an autopsy after the accidental death of a healthy rg-year old male and in Table II only the data for the frontal lobe are presented. The values reported by Brenton et ~~2E.11 are also for whole brain tissue from the frontal lobe, as is the data of Prensky and Moser12for a4-day old child. The data of Gerritsen and Waismanr3 are for the brain of a z-year old child with leukemia and only cortex or sub-cortex was analyzed. Korey Et al.1 studied temporal lobe cortex taken by biopsy from two children with undifferentiated mental retardation. The data published by Perry et al.‘” were for gray matter but the patient died of hypernlethioninemia and cannot be considered as a normal case. Shimizu et aLz5, Kanazawa and SanorG and Abraham et ~1.‘~ also studied the free amino acids of gray matter. The values for taurine and threonine show the greatest differences as compared with previous published data. Age may be a factor for the great difference in taurine values and the high figure for threonine can be explained on the basis that the peak contained some glutamine and small amounts of asparagine. The taurine peak also contained some urea. Glycine is somewhat increased in the present series as compared with the earlier data, and phosphoethanolamine is half of the only value previously published. The rest of the present data agree well with either the results of Robinson and William9 or with the other data. In particular, the total numer of ;lmoles per g of tissue wet weight is remarkably similar in the two studies where it has been reported. DISCUSSION

There are increases in the concentration of several amino acids coincident with the development of the brain from the fetal or neonatal state to that of the adult. This has been shown to be true at least for aspartic acid, glutamic acid, glutamine, y-aminobutyric acid, leucine and isoIeucine in both animal and human brainlo. On the other hand, the values for taurine and phosphoethanolamine decrease with increasing age and tyrosine also decreases in early stages of growth in mammalian brainlo. The age groups in the present study were similar so that the observed differences in the values for aspartic acid, glutamic acid, y-aminobutyric acid and leucine between the

PALO,

332

SAII;EK

normal and TS-brains are not likely to be due to age differences. However, the high taurine value in the normals, as compared with previous published data, might be explained on this basis. An intensive search of the literature revealed no data on the possible presence of urea in human brain although it has been found in rat, frog and chicken brain by Buniatian and Davtian I8. Citrulline reported in animal brain \~a‘; thought to be of hepatic

origin.

Robinson

and Williams6

detected

citrulline

only in

the white matter of human brain but did not speculate about its origin. Various opinions have been expressed in earlier papers about the possible effects of storage on the level of different amino acids in brain tissue and post mortem autolysis has been particularly discussed. It has been assumed that increases, esprcially in the simplest amino acids, may be a sign of structural changes leading to liberaenzymes 1 but certainly after several hours of refrigeration tion of proteolytic to which the body is exposed, the brain should be cooled sufficiently to retard autolysis according to Himwich et ~1.‘~. Although no significant post mortem changes have been reported for some single compounds, q., cystatlGonine*5 or homocarnosine*6, and although amino acid levels in biopsy specimens have not been shown to be significantly different from those in autopsy samples as determined by paper chromatcrgraphy8, the fact remains that free amino acid levels may be changed byproteolytic activity in the immediate post mortem period as well as during subsequent storagel’. In a recent extensive study of free amino acids in human brain tissue, Mardens alld. Van Sande (personal comlnunicati[~n) showed marked differences in autopsy as compared to biopsy samples. Except for taurine, phenylalanine, y-aminobutyric acid and lysinc, the autopsy data of these investigators check well with the values reported here. In the present study, an experiment was done where a three-year old bioll>! sample was analyzed and the results were compared with a twn-year old post mortt,m specimen taken from tlte same patient. There was excellent agreement hctwecn tlrci two series of results except for glycine and serine which were considerably increas~~d in the autopsy samples from 6.7 mole (jC, to 8.4 mole 1;) and from 6.6 mole :;a to 7.0 mole 00 respectively. This would imply that, at least after a certain period of time in storage, no appreciable changes take place in brain tissue and thus that the two group)-; of post mortem brain specimens analyzed in the present study are comparable Lvitll each other. Nevertheless, further investigation is needed to determine the exact nature ot the autolytir processes in brain tissue if the free amino acid valucbs are to br-k utilized in neurochemical research. It has been stated by Korey et ~i.1 that o(*currences during the biopsy procedure and events thereafter, tend to lower the amino acid concentrations. ~~)ncentrati~~ns of the same free amino acid in different anatoIrlica1 regions of the central nervous system (cerebrum, cerebellum, spinal cord) have been found to be similar, with hut a few exceptions, by Robinson and Willia.msG. In most studies on the free amino acids of human brain, either frontal or occipital lobe of the cerebrum has been utilized. In the present study, only the data obtained for frontal lobe were compared with each other but another study was undertaken to determine possible differences between the various lobes of the same brain. Duplicate analyses for frontal, parietal and occipital lobes of the same individual, however, revealed no significant differences either in the normal or in the TS-group. Only cortex was analyzed in the present study and contamination of the sanples with white matter was strictly avoided. This was due to the fact that in several

AMINO

ACIDS IiiTAY-SACHS

studies on various to be different

333

BRAIN

amino acids, the level of an individual

between

gray and white matter,

compound

e.g., cystathionine

has been found

has a higher level

in human white matter as compared with gray matter according to Shimizu15. In several of the earlier studies mixed gray and white matter has been analyzed, which makes difficult the comparison between the results of various workers. Korey et al.1 studied three TS-patients who represented different stages (early, moderately late and late) of the disease. The reported values for the free amino acids in biopsy samples of cerebral cortex, as determined by paper chromatography, showed tremendous variations between the patients, from 0.03 to 0.11 A!mole/roo mg of tissue wet weight for alanine and from 0.03 to 0.47 pmole/roo mg of tissue wet weight for glycine. In the late stage of the disease, only 7 different amino acids were found and the total amount of amino acids per IOO mg of tissue wet weight was considerably decreased. On the other hand, the highest total amount was reported for the patient at the moderately late stage of the disease. The values for temporal cortex (with no microscopic pathology) taken by biopsy from two other children with undifferentiated mental retardation threonine (0.02 pmole/roo for aspartic

show much better consistency with identical values for mg of tissue wet weight) and with the greatest variation

acid (from 0.12 to 0.79 pmole/roo

mg of tissue

wet weight).

Only IO

different amino acids were detected in both of these normal samples. Nevertheless, the general conclusions reached by these workers were confirmed in the present study, namely, that the group of glutamic acid, glutamine, y-aminobutyric acid and aspartic acid is markedly decreased in TS-disease and that there is a decrease in the content of the free amino acid pool. In addition, the present data showed that alanine, leucine and tyrosine were increased in TS-disease. In a previous study published from this laboratory”, the amino acid composition of water-soluble ganglioside-peptide isolated from TS-brain gray matter was shown to be significantly different from that of normal human brain. Glycine, alanine and threonine were decreased and valine, leucine, isoleucine, tyrosine, half-cystine, methionine and aspartic acid were increased. The observed differences were statistically significant at the I 9',p robability level. It has been suggested by Korey et al.1 that the decrease in the content of the free of TS-patients may limit protein synthesis tides show structural changes between the some selectivity so that not all of those

amino pool and indeed, normal and amino acids

in the central nervous system the data on ganglioside-pepTS-brains. However, there is which are present in higher

amounts in the free amino acid pool are also increased in the gangliosideepeptide, nor are all those compounds having decreased levels in a free form present in lower concentrations in the ganglioside-peptide. This is true of alanine in the first group and of aspartic acid in the second. No y-aminobutyric acid was found in the gangliosidepeptide and alanine showed the most significant difference. The profile and concentrations of the cerebral free amino acids reflect the presence of neural disease and the values found in TS-disease are among the lowest reportedl. Amino acid metabolism, particularly of the glycogenic amino acids, is of great importance to the proper functioning of the central nervous system, although little is known about the connection between abnormal amino acid metabolism and cerebral dysfunction. It has been established that cells can accumulate a “pool” of amino acids at concentrations much higher than those of the extracellular environment by means of a process of active transporP. It is this intracellular pool which

334 provides

the critical

constituents

in formatiol~

of the enzymes

and the protein-

containing structural elements of brain tissue. Since TS-disease results in the accumulation of large amounts of gangliosides, whose molecular structure is particularly, suited to take part in membrane formationzl, it can be assumed that this would alter membrane

permeability

and therefore

the transport

of amino acids and enzymes into

and out of neuronal celW. Such alterations would result in the formation of different peptides in TS-disease as was found by Palo and Saifer’, as well as in other types of gangliosidoses. A different amino acid pool would also give rise to other compounds and the catabolism of the diverse peptides to other active metabolites which ultimately enter into carbohydrate or lipid metabolic reactions. However, the study of neurological diseases of infants, e.g., Tay-Sachs, Niemann-Pick, etc., is complicated by its occurrence in a developing brain associated with maturation changes and other effects secondary

to the primary

pathology

of the disease.

ACKNOWLEDGEMENTS

This investigation was supported by grants from the John A. Hartford Foundation, the National Tay-Sachs Association and by U.S. Public Health Service Grant NB-0~85 C-16 from the National Institute of Neurological Diseases and Blindness. Dr. J. Palo was the grateful recipient of a Fulbright Travel Grant. The authors gratefully acknowledge the valuable discussions of Dr. Larry, S&neck, DuPont

Director Institute,

of Pediatrics and the advice of Dr. Paul B. Hamilton of the Alfred I. Wilmington, Delaware, concerning the identification of some of

the amino acids present in the extracts. We also wish to express our appreciation and typing the manuscript.

to Mrs. Lillian

Salowitz

for editing