No transketolase abnormalities in Wernicke-Korsakoff patients

Journal ~{/'ttw ;Veumh~.t~al ,~,ct,'m'c.~,I{)t~( lq91 ) 88-9() 1991 Elsevier Science Publishers B.V. All rights rc,,crved 0022-510X/91/$113.5(I

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No transketolase abnormalities in Wernicke-Korsakoff patients Ben A. Blansjaar

t,

Rcns Zwang : and Bert G. Blijcnbcrg :

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( Received 22 t:cl',ruais. 1091) (Revised. rcccivctl 22 May. ltlOl) (..\cceplcd 28 June. 1901) Key words: l'ranskctolasc: Wcrnickc-Korsakoff syndrome: Ion-exchange chromatography: lsoclectric focusing

Summao'

l'ranskctolasc isocnzymc patterns of Wcrnickc-Korsakoff patients, relatives, alcoholics and controls, obtained by isoclcclric focusing of purified crythrocylc transkctolasc, wcrc all identical. After a series of invcsligations published by several author,', liulc evidence remains to support the hypothesis of an inborn transkctolasc abnormality in Wcrnickc-Korsakoff patients.

lnt r o d u c t i o n

The degree of vulnerability of alcoholics to the W c r n i e k c - K o r s a k o f f syndrome appears to vary. Differences in individual susceptibility on a biochemical basis have long been suspected (Lishman 1990). Blass and Gibson (1t,~77) claimed that they had discovered an inborn abnormality in the thiamine-dependent enzyme transkctolasc (EC 2.2.1. I.) in 4 patients with the Wcrnickc-Korsakoff syndrome. Transketolasc extracted from tissue-cultured fibroblasts from these patients had a lower affinity for thiamine pyrophosphatc than transkctolasc from control cells. They suggested that an inborn enzymatic protein abnormality could constitute a predisposition to the Wernicke-Korsakoff syndrome according to Garrod's concepts (Garrod 1928, 1931: Childs 1970). The fact that the WcrnickeKorsakofl" syndrome is a rare complication of alcoholism (Blansjaar ct al. 1987) made their suggestion platlsiblc. This fascinating hypothesis, which has been widely accepted (Wilson and Madison 1980: Victor ct al. 1989: l.ishman 1990), started a series of investigations, the results of which were disappointing (Tatc and Nixon 1987). Nixon c t a l . (1984) reported a failure to reproduct the results of Blass :,nd Gibson with crythroeytc

(orrcspondeme to." B.A. Blansjaar. P.C. Joris, St. Joris~cg 2. 2612 (;A l)clf/, -l'hc Ncthcrl.,nds. Tel.: (33) 015-607607: Fax: (33) 01512O272.

transkctolase. Apparent K,,, values for thiamine disphosphate wcrc similar for W c r n i c k c - K o r s a k o f f patients and controls. However, in the same publication they reported having flmnd different patterns of transketolase isoenzymcs in W e r n i c k e - K o r s a k o f f patients and controls, using ion-exchange chromatography, isoelectric fl~cusing and staining (Kaczmarek and Nixon 1983). "Fhcir experiments showed five transkctolase isoenzymc patterns with a constant central part of three bands and four variable bands. One pattern was flmnd more frequently in Wernicke-Korsakoff patients. After examining bloodsamples taken from healthy individuals, Kaufmann ct al. (1987) reported that the technique described by Kaczmarek and Nixon (1983) produced unreliable results. The aim of the present study was to compare transketolase isoenzyme patterns of Wernicke-Korsakoff patients with that of their relatives, alcoholics and healthy subjects.

Materials and methods Patients and controls

Five patients with alcoholic Korsakoff syndrome were studied (4 men, 1 woman, aged 46-63 years, mean 58 years). Three patients were admitted to an alcohol clinic, two lived in a nursing home. All had a history of prolonged alcohol abuse, severe memory impairment without major loss of other intellectual

N9

abilities and a history of past episodes of either acute confusion or Wcrnickc's encephak)pathy and peripheral neuropathy. The patients were detoxified and given vitamins if necessary. They had not taken any alcoholic bcvcragc for at least 3 weeks and showed only minor abnormalities in haematological and biochemical tests. Neuropsyehological tests showed severe a n t e m g r a d c a n d retrograde mcmoz'y defcct, and slight impairment of perceptual and conceptual functions, Differences between IQ and MQ, measured with Raven's Standard Progressive Matrices and the Wechsler Memory Scale, ranged l'rona 19 to 26 points, with a mean of 22. The Korsakoff patients were compared with 6 of their first and second degree relatives, 4 patients with severe alcohol dependency and 8 healthy individuals. The relatives of the Korsakoff patients were all in good health. The alcohol-dependent patients had historics of prolonged alcohol abuse comparable to the histories of the Korsakolf patients. They were admitted to an alcohol clinic, were dctoxificd and had no alcoholic beverage for at least 3 weeks. Haematological and biochemical tests showed only minor abnormalities. Neuropsychological tests showed no memory impairment. Methods The methods f o r sample preparation, isoelectrie l+ocusing and staining of crythrocytc transketolase described by Kaczmarck and Nixon (19831 and Kaufmann et al. (19871 were modified. Hcparinizcd blood (10 m l ) w a s collected from each subject. Erythrocytes wcrc separated by centrifugation for 10 rain at 22(~)x j~ at 10 ° C, washed 3 times with cold NaCI (0.9%:). suspended in an equal volume of double distilled water and stored at - 8 ( 1 ° C until needed for further use, but not longer than one month. The hemolysate was dialyzed against a buffer (20 m m o l / I Tris-HCl, pH 8.2) and afterwards cleared of cell debris by ccntrifugation at 166011 x J4 for 2(I rain. Bcforc purification by ion-exchange chromatography the samples werc diluted with double distilled water to

a hemoglobin ctmcentration of 1 rnrnol Fe,,/I. After ion-exchange chromatography (l)EAE-TrisacD'l M. Pharmacia-LKB) to remove hemoglobin, transketolase activity was nearly quantitatively recovered. All fractions containing erythrocyte transketohtsu activity (Smeets et al. 19711, wcre concentrated (Kaufmann c t a l . 1987) to an end w~lumc ot 30 ~.l. The concentrated eluate was stored at - N() ('. lsoelectric focusing was carried out on agarosc gel,, (0.5 × l I()x 24(1 mm, 15~ agarose FIr. . LKB; 3c4 Ampholine pH 3.5-111, LKB), electrode ,,olutions u.,,cd were cathode (1.5 M N a O H , anode 0.5 M Acetic ',told. Focus conditions were 15(10 V, 15(1 mA and 15 W. ]''he gel was preh~cused for 3(1 rain before I ,el sample was applied 30 mm from the anode. Samples were focused fi~r another 60 rain at 10+'('. The pi-I gradient was checked against a set of colored l[ii: markers (LKB). After isoelectric fi)cusing the fractions containing transketolase activity was transferred to a nitrocellulose membrane using Southern blotting. A nitrocellulose membrane (0.45 .urn. Probind 45 LKB) was placed on the gel and covered by 5 sheets ot d~' filter paper, a glass plate and a weight of (k5 kg. After l(I rain the paper sheets were replaced by 3 new ~,hccts. which were left for a furthc~ 1(1 rain to complete the transfer. The nitrocellulose membrane ~,as incubated m a fresh staining mixturc (Kaufmann el :Lt. 19871 for 30-40 rain at 37 '' ('.

Results We examined bloodsamples of 5 Korsakoff patients+ 6 of their relatives, 4 alcoholics and ,~ controls. Reproducible transketolase patterns consisting of many bands were obtained with the modified technique described above. In our judgment, apart from concentration differences, all patterns were identical. [Zig. 1 shows transkctolase patterns of two randomly chosen individuals of each group.

(A3

(~ 7.3__

7.3--

8.3--

pI

pI

t

t 1

2

-~-

~

s

6

:,

e

Fig. I. I.~rythrocyte transkeh+la++c pattern++ o l W c r m c k c - K o r s a k o l f patient+, (3, 41. t'm,t and second degree rekttivc> ol the.,c pattcnls <1. 2) alcoholic control~ (5, 61 and healthy control++ (7, 8). All pattern', are identical apart from concentration clfflt:rcnce>,.

Keferences Hlacs and (;ibon

(1977)

10 have found

cl;limcd

law in Korsddf

cnqmatic

pow\ the individual i\ had

protein

on the assumption

tcnc~ of tran>kctolasc Hl;i\s

and

Michxll+Mcntcn

( 1977)

that thcw

an

constant for thiamin skin

crythrocytc

of

rcportd

grcatl!~. Mukhcrjcc

ct

original

aI.

incrcascd

diphosphate of

fihrohlasts

(

Wcrnickc-

k,,,

;th \\cll as fibrohlast

(iihson’b

syndiffcr-

indicate the esis-

and

found

Kc~r~;ik~~l~l’patic‘nb. 1lowc\xr. tlilfcrcd

prcdis-

isocnrymcs.

(iihson

tran\kc’tcdasc from hum;ln

ahnormality

to the Wcrnickc-Korsakoff

arc genetically dctcrmincd

cnccs

ct al. (IW!)

hctwcen transkcto-

patient\ and controls. ‘I‘hc hyothcds

that an inborn drcmc

and Nixon

diffcrcnccs

laluc4

of

transkctolasc

1987) confirmed

Ul;15\

;~iici

hIat\.

hut their result\ \howcd much lower /i,,, v;~lucs

~)vc~;ill. 2nd intcrmcdiatc Wcrnickc

Kor5ahoff

cspcrinicnt

with

fihro-

values for alcoholic5 \bithout

y ntlrcwc.

which ix not compati-

hIC with the h\:pothcuis of 21 gcnctic predisposition. ‘l‘;11122nd biison ( lLH7) pointed oul that hcc;lusc OI

rn;~n> potential

Michxcli+Mcntcn thiamin

and

artifacts. for

conztanf

the

mcasurcmcnt

human

of

tr;lnskctolasc

diphosphatc i\ not \rcc rcliahlc.

ICi\ori cl al. ( IUN) scCmcrl to have made ;I stronger c;i,w

with

the ilctcction

of transkctol~ise

anot her niCthoc1. Aocluctric

OIO~IC;II wcahncsscs (K;iufm~inn :\lthough Southern c;iticw5 improved

et al. 1987).

blotting and sonic minor modifi-

t hc rcliahility

wrihccl I>! K;ic/m;lrck

variant\ tl>

focusing. dcspitc mcthod-

of t hc mcthotls

dc-

and Nixon ( IWJ) anti Kaufmann

ct ~1. ( 1057). comparison

of the l’c\v do~cn transkcto-

Ixsc pdttcrns u’c ohtain~d rcmaincd ;I matter of cuhjcctiw

~iiclgiw~it.

It i\ possihlc

that the rcproducihlc

pattern of trans-

kctolaxc fractionx \\c isol;ited contain\ transkctolasc isoc’n/\.nics. 1lowebcr. this pattern is in our judgment Idcntlc;rl

in Korsakoff

holic~hwithout

patients.

their

rclatiws.

alto-

memory dcfccts and healthy individuals.

IVc 1h*:rctorc conclude that no cvidcncc was found ot AI\ inborn tranxkctol;w ;thnormalit\: in Wernickc Korwhofl’

patients.