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Gml -ganglioside accumulation and β-galactosidase deficiency in a case of Gml -gangliosidosis (Landing disease)
421
CLINICA CHIMICA ACTA
GwGANGLIOSIDE
ACCUMULATION
DEFICIENCY
IN A CASE
G. DACRE>lONT
XNI) J, A. KINT*
Pediatric
Departmerzt*,
(Received
AND fl-GALACTOSIDASE
OF Gwl-GANGLIOSIDOSIS
Rijksuniuersiteit
(LANDING
DISEASE)
Gent (Belgium)
April IO, 1968)
SUMMARY
In a case of “gangliosidosis of the G M1type” (Landing disease) an accumulation of the GMl-ganglioside in the patient’s brain was established. This accumulation apparently
was caused by a /&galactosidase deficiency in the brain, as the activity for only 6.5% of the value found in normal brains.
of
such enzyme accounted
The term “gangliosidoses of the G bn type” (Landing disease) refers to a recently discovered clinical picture which was classified as a thesaurismosis. The most pronounced (Table
biochemical
I). Although
because
feature
is the accumulation
of the so-called
the disease has always been named a lipidosis,
of the glycolipid
nature
of the ganglioside,
one cannot
G&xl-gangliosidel
it must be said that decide a $viori on a
defect in the lipid metabolism. Indeed, in patients with a similar disease, presented by Sacrez and ~011.~and by Seringe and ~011.3, Hers found an absence of a /&galactosidase enzyme, so that it seems more logical to speak of a fault in the carbohydrate metabolism. The present paper is concerned with the preliminary results of the brain analysis while a more extensive study of this case by Hooft and coworkers will be published
later.
MATERIAL
AND METHODS
From the frontal brain of the patient (a girl of 9 months) a biopsy was taken and subsequently frozen quickly and stored at -40~. In order to trace the lesion by chemical analysis, a weighed portion of the gray matter (*zoo mg) was lyophilized, extracted with chloroform-methanol Z/I and I/Z as devised by Suzuki4 and an aliquot of this total lipid extract investigated by thin-layer chromatography. Silicagel G was used as supporting medium for the chromatographic runs. The plates were developed in the following solvent : first, in chloroform-methanol-water 65 : 25 : 4, or chloroform-methanol-14 N ammoniutn hydroxide 14:6 : I up to the edge, and second, in propanol-water 70 :30 or propanol-14 N ammonium *
Director:
Prof. Dr. C. flooft. Clin. Chim.
Acta,
21 (1968) 421-425
DACREMONT,KIN'l
422 TABLE THE
MOST
Stvucfawe
I IMPORTANT ~_~ of
GANGLIOSIDES
AND
the ganglioside
THEIR
~_.
DESIGNATION
-.-~
Ccramide-Glc-Gal--Pu’APu’A-NhNA
ACCORDING
TO
SVENNERHOLM
Suemaerholm designation --.l.“___ -~ GTI
~Gal-NH,-C;al-NAS_k
Ceramidc-Glc-Gal-NANA-NAN.4 L-NH Ccramide-Glc-Gal-.-~A~,~
Grhit -Gal GDlb
LNH,.G&N AN A
Ceramide-Glc-Gal-NANA
G&II
1.G&NH,-Gal Cerxmide-Glc-Gal-NAN A
GM,
I.-Ga!-SH, Ccramide-Glc-Gal--?;IANA ._~ __--. .-__ Abbreviations used : NAN galactosaminc.
Gnrs
A : N-acetylneuraminic
acid;
Glc
: gluccxe;
Gal
: gala%ose;
Gal-NH,:
hydroxide-water 80 : 8: IZ to IO cm of the start line. Kagi-M&her solution was used as general lipid spray. For further determination of the ganglioside pattern, the gangliosides were isolated by partitioning in 0.88 KC1 and separated by thin-layer chromatography following the method of Suzuk?. After location with I, vapours, the different fractions were scraped off and their NANA content measured with resorcinol reagents. As controls, a case of Tay-Sachs disease as well as autopsy and biopsy material from children of different ages with unspecified diseases were investigated in the same wav. For the enzyme determinations, about 200 mg brain tissue (fresh weight) was used. After lyophilizing, the dry matter (20-50 mg) was suspended with I ml distilled water in a Potter-Elvehjem homogeniser. The ground suspension was then frozen five times in liquid nitrogen and allowed to thaw. After centrifuging at 7000 rev./min during IO min, the supernatant liquid was activated by treating it at 37’ with 20 ,d 10% Triton X-roo. The enzyme assay of Lederberg6 was slightly modified. The incubation mixture contained in a total volume of z ml 4 btrnoles $-nitrophenyl-p-galactopyranoside, 200 ,umoles acetate buffer pH 5.1, and the brain extract. Because of the very low activity in brain tissue as compared to liver, the incubation at 37” had to be carried out for 17 h, Preliminary experiments showed a perfectly linear correlation between the enzyme concentration and the amount of $-nitrophenol liberated. The reaction was stopped by adding I ml 4.9% trichloracetic acid. After centrifuging, I ml 0.5 IV NaOH and 0.6 ml 0.1 M glycine buffer pH 10.0 was added to 1.2 ml of the supernatant. The extinction of the yellow fi-nitrophenol was then measured at 420 m,u in a Beckman DU spectrophotometer. At the same time two blanks were run, in which the substrate and the brain extract were replaced by distilled water respectively. After incubating for 17 h, these were also subjected to analysis.
Clin.Chim.
Acta,
21 (1968) 421-425
GASGLIOSIDOSIS
423
RESULTS
The results of the thin-layer chromatography show an obvious accumulation of one of the gangliosides (Fig. I). Further comparison with a case of Tay-Sachs disease and controls permitted the conclusion that the accumulated ganglioside was of the type GUI (Svennerholm’)
(Fig. 2).
gangliosides A
C
B
Fig. I. A, C, D, E and F: gray and white Solvents: (I) chloroform~methanol~water, water. 80:8: 12.
Other definitions and Wiegandt9) Belgium) vestigated
E
D matter 65 : 25
of this ganglioside
a fraction
(Max Planck
C: : I ; (2)
GCNTI (Kuhn
(Born Bunge Foundation,
Institute,
of he same biopsy obtained
Fig. 2. A : control; B: &I,-gangliosidosis; anol-14 S ammonium hydroxide, I 4 :6
R: gray matter of patient. iv ammonium hydroxide-
are G, (Korey and Gonatasa),
and A, (KlenklO). Dr. Liiwenthal
and Dr. Jatzkewitz
F
of control brains; : 4; (2) propanolLr4
Munich,
identical
Germany)
Antwerp, who in-
results.
Tay-Sachs disease. Solvents propanol-water, 70 : 30.
: (I)
chloroform-meth-
The measurement of the percentage distribution of NANA in the different ganglioside fractions (Table II) shows that 67 96 is present in the GX~Ifraction (normal for gray matter: f 13%). Similar results were obtained in already described cases Clis.
Chim. ilcta,
21 (1968) 421-425
DACREMONT, KINT
424 TABLE
II
GANGLIOSIDEPhTTIlRN IN OF NANh
CASE
OF
GANGLIOSIDOSIS
(;axgliosides
OF
THE
TYPE
Gil,
EXPRESSED
AS
PER
CENT
(Gray matter
(Svennerholm)
(Korey
C;T1
Gl
GDlb
G,
GDia Gw1 Gnrz GA13
G, G, G, G,
G&f, gangliosidosis
6 Gonatas)
G,
Controls* 3.1
1.1
3.1
12.1
8.7 18.0 67.0 1.0
20.7 48.1 13.0 I.5
1.1
1.0
* Average of four control brains (autopsies) of children aged 6 months, tively.
of gangliosidoses of the type Gsn. Suzuki I1 found 74.5% fraction of the gray matter, while Seringe3 gives 60:/,.
zI/~,
4 and g years respec-
of the NANA in the Gm
As can be seen in Table III, the value of the activity of the /Y?-galactosidase in the patients’ brain was only 6.5$/o of the activity in normal gray matter of the controls. TABLE THE
111
,%4LACTOSIDASE
ACTIVITY
OF
CONTROL
PERSONS
AND
OF
THE
PATIENT
Contvol
Sex
Tissue
Activity in units* per mg dry weight
I I day 2 days 2 4 years 3 II years 4 2 years 5 6 L days 4 years 7 Mean value :k deviation
Male Male Male Male Male Male Male
brain gray matter brain gray matter brain gray matter brain gray matter brain gray matter brain white matter brain white matter gray matter white matter gray matter
3.98 3.50 2.27 4.12 3.63 1.62 1.72 3.50 + 1.23 1.67 + 0.05 0.23 ; 0.06
Patient with Landing disease * I unit = amount of enzyme which liberates
I mpmole p-nitrophenol
per hour.
DISCUSSION
The ,&galactosidase activity found in normal brains shows a general agreement with the results of Hayra 12. The ganglioside accumulation in the patient’s brain is apparently due to a severe deficiency of a /3-galactosidase, as a result of which the velocity of the breakdown to gangliosides with less sugar molecules is greatly diminished. It is noteworthy that in our case of Landing disease no absolute absence of the enzyme was found, which seems to be in contrast with the results of Hers, as mentioned by Seringe and coworkers 3. The discrepancy could be explained by the prolonged time of our enzyme assay which allowed us to show some possibly very weak residual activity. The question concerning the specificity of glucosidases (e.g. /3-glucosidase, pClin. Chim.
Acta,
21
(1968) 421-425
GANGLIOSIDOSIS
4%
galactosidase) has received much attention during the last few years. Hayra and coworkers12 give some arguments according to which several specific cerebroside ,%galactosidases should exist. The paper of Brady13 also implicates the presence of several b-galactosidases. The actual state of our knowledge seems to agree with the existence of a series of /?-galactosidases in the different organs examined, each active only toward one or a few natural substrates, but all active toward some unnatural substrates such as nitrophenyl glycosides. As the enzyme defect in the diseases of Fabry and Gaucher also concerns ,&galactosidase, the different specificity of the enzyme will cause a variable accumulation. Likewise, one can postulate that the GM1ganglioside accumulation is due to a deficiency of a specific G%n-ganglioside$-galactosidase. It is our intention to begin a detailed investigation into this specificity and the presence of isoenzymes of P-galactosidases. ACKNOWLEDGEMENT
The authors are greatly concerning the diagnosis.
indebted
to Prof. Hooft for his valuable
suggestions
REFERENCES I J. S. O'BRIEN, M. B. STERN, B. H. LANDING, J. Ii.O'BRIEN AND G. N. DONNELL, Diseases
Childhood,
109 (1965)
Am.
1.
338.
2 R. SACRES, J. G. JUIF, J. M. GIGONNET
AND J. E. GRUNER, Pediatric,22 (1967) 143. 3 P.SERINGE,B.PLAINFOSSE,F.LAUTMANN, J.LORILLOUX,G.CALAMY,J.P.BERRYAND J.M. WATCHI, (Sem. H@. Paris) Ann. Pt!diat. (Paris), 44 (1968) 685/P 165. 4 I(. SUZUKI, &je .%i.,3 (1964) 1227. 5 L. SVENNERHOLM, Biochim. Bioflhys. Acta, 24 (1957) 604. 6 J. LEDERBERG, J. Bacterial., 60 (1950) 381. 7 L. SVENNERHOLM, J. Lipid Res., 5 (1964) 145. 8 S. R. KOREY i~~~ J. GONATAS, J. Neuropathol. Expl. Xeurol., 22 (1963) 56. 9 K. KUHN AND H. WIEGANDT, Chem. Ber., 96 (1963) 866. IO E. KLENK AND W. KUNAU, Hoppe Seylers Z. Physiol. Chem., 335 (1964) 275. II K. SUZUKI,~~ S. M. ARONSON AND B. W.VOLK(E&.), S~hingolipidoses,grdInternational Symposium on Sphingolipidoses, 1965. Pergamon Press,New York, 1966. 12 A. K. H~YRA, D. M. BOWEN, Y. KISHIMOTO AND S. RADIM, J. Lipid Res., 7 (1966) 379. 13 R. 0. BRADY, 4. E. G.~L,R. M. BRADLEY, E. MARTENSSON, A. L. WARSH.%\V AND L. LASTER. New Engl. J. Med., 276 (1967) 1163. Clin. Chim.
Acta,
21 (1968) 421-425
CLINICA CHIMICA ACTA
GwGANGLIOSIDE
ACCUMULATION
DEFICIENCY
IN A CASE
G. DACRE>lONT
XNI) J, A. KINT*
Pediatric
Departmerzt*,
(Received
AND fl-GALACTOSIDASE
OF Gwl-GANGLIOSIDOSIS
Rijksuniuersiteit
(LANDING
DISEASE)
Gent (Belgium)
April IO, 1968)
SUMMARY
In a case of “gangliosidosis of the G M1type” (Landing disease) an accumulation of the GMl-ganglioside in the patient’s brain was established. This accumulation apparently
was caused by a /&galactosidase deficiency in the brain, as the activity for only 6.5% of the value found in normal brains.
of
such enzyme accounted
The term “gangliosidoses of the G bn type” (Landing disease) refers to a recently discovered clinical picture which was classified as a thesaurismosis. The most pronounced (Table
biochemical
I). Although
because
feature
is the accumulation
of the so-called
the disease has always been named a lipidosis,
of the glycolipid
nature
of the ganglioside,
one cannot
G&xl-gangliosidel
it must be said that decide a $viori on a
defect in the lipid metabolism. Indeed, in patients with a similar disease, presented by Sacrez and ~011.~and by Seringe and ~011.3, Hers found an absence of a /&galactosidase enzyme, so that it seems more logical to speak of a fault in the carbohydrate metabolism. The present paper is concerned with the preliminary results of the brain analysis while a more extensive study of this case by Hooft and coworkers will be published
later.
MATERIAL
AND METHODS
From the frontal brain of the patient (a girl of 9 months) a biopsy was taken and subsequently frozen quickly and stored at -40~. In order to trace the lesion by chemical analysis, a weighed portion of the gray matter (*zoo mg) was lyophilized, extracted with chloroform-methanol Z/I and I/Z as devised by Suzuki4 and an aliquot of this total lipid extract investigated by thin-layer chromatography. Silicagel G was used as supporting medium for the chromatographic runs. The plates were developed in the following solvent : first, in chloroform-methanol-water 65 : 25 : 4, or chloroform-methanol-14 N ammoniutn hydroxide 14:6 : I up to the edge, and second, in propanol-water 70 :30 or propanol-14 N ammonium *
Director:
Prof. Dr. C. flooft. Clin. Chim.
Acta,
21 (1968) 421-425
DACREMONT,KIN'l
422 TABLE THE
MOST
Stvucfawe
I IMPORTANT ~_~ of
GANGLIOSIDES
AND
the ganglioside
THEIR
~_.
DESIGNATION
-.-~
Ccramide-Glc-Gal--Pu’APu’A-NhNA
ACCORDING
TO
SVENNERHOLM
Suemaerholm designation --.l.“___ -~ GTI
~Gal-NH,-C;al-NAS_k
Ceramidc-Glc-Gal-NANA-NAN.4 L-NH Ccramide-Glc-Gal-.-~A~,~
Grhit -Gal GDlb
LNH,.G&N AN A
Ceramide-Glc-Gal-NANA
G&II
1.G&NH,-Gal Cerxmide-Glc-Gal-NAN A
GM,
I.-Ga!-SH, Ccramide-Glc-Gal--?;IANA ._~ __--. .-__ Abbreviations used : NAN galactosaminc.
Gnrs
A : N-acetylneuraminic
acid;
Glc
: gluccxe;
Gal
: gala%ose;
Gal-NH,:
hydroxide-water 80 : 8: IZ to IO cm of the start line. Kagi-M&her solution was used as general lipid spray. For further determination of the ganglioside pattern, the gangliosides were isolated by partitioning in 0.88 KC1 and separated by thin-layer chromatography following the method of Suzuk?. After location with I, vapours, the different fractions were scraped off and their NANA content measured with resorcinol reagents. As controls, a case of Tay-Sachs disease as well as autopsy and biopsy material from children of different ages with unspecified diseases were investigated in the same wav. For the enzyme determinations, about 200 mg brain tissue (fresh weight) was used. After lyophilizing, the dry matter (20-50 mg) was suspended with I ml distilled water in a Potter-Elvehjem homogeniser. The ground suspension was then frozen five times in liquid nitrogen and allowed to thaw. After centrifuging at 7000 rev./min during IO min, the supernatant liquid was activated by treating it at 37’ with 20 ,d 10% Triton X-roo. The enzyme assay of Lederberg6 was slightly modified. The incubation mixture contained in a total volume of z ml 4 btrnoles $-nitrophenyl-p-galactopyranoside, 200 ,umoles acetate buffer pH 5.1, and the brain extract. Because of the very low activity in brain tissue as compared to liver, the incubation at 37” had to be carried out for 17 h, Preliminary experiments showed a perfectly linear correlation between the enzyme concentration and the amount of $-nitrophenol liberated. The reaction was stopped by adding I ml 4.9% trichloracetic acid. After centrifuging, I ml 0.5 IV NaOH and 0.6 ml 0.1 M glycine buffer pH 10.0 was added to 1.2 ml of the supernatant. The extinction of the yellow fi-nitrophenol was then measured at 420 m,u in a Beckman DU spectrophotometer. At the same time two blanks were run, in which the substrate and the brain extract were replaced by distilled water respectively. After incubating for 17 h, these were also subjected to analysis.
Clin.Chim.
Acta,
21 (1968) 421-425
GASGLIOSIDOSIS
423
RESULTS
The results of the thin-layer chromatography show an obvious accumulation of one of the gangliosides (Fig. I). Further comparison with a case of Tay-Sachs disease and controls permitted the conclusion that the accumulated ganglioside was of the type GUI (Svennerholm’)
(Fig. 2).
gangliosides A
C
B
Fig. I. A, C, D, E and F: gray and white Solvents: (I) chloroform~methanol~water, water. 80:8: 12.
Other definitions and Wiegandt9) Belgium) vestigated
E
D matter 65 : 25
of this ganglioside
a fraction
(Max Planck
C: : I ; (2)
GCNTI (Kuhn
(Born Bunge Foundation,
Institute,
of he same biopsy obtained
Fig. 2. A : control; B: &I,-gangliosidosis; anol-14 S ammonium hydroxide, I 4 :6
R: gray matter of patient. iv ammonium hydroxide-
are G, (Korey and Gonatasa),
and A, (KlenklO). Dr. Liiwenthal
and Dr. Jatzkewitz
F
of control brains; : 4; (2) propanolLr4
Munich,
identical
Germany)
Antwerp, who in-
results.
Tay-Sachs disease. Solvents propanol-water, 70 : 30.
: (I)
chloroform-meth-
The measurement of the percentage distribution of NANA in the different ganglioside fractions (Table II) shows that 67 96 is present in the GX~Ifraction (normal for gray matter: f 13%). Similar results were obtained in already described cases Clis.
Chim. ilcta,
21 (1968) 421-425
DACREMONT, KINT
424 TABLE
II
GANGLIOSIDEPhTTIlRN IN OF NANh
CASE
OF
GANGLIOSIDOSIS
(;axgliosides
OF
THE
TYPE
Gil,
EXPRESSED
AS
PER
CENT
(Gray matter
(Svennerholm)
(Korey
C;T1
Gl
GDlb
G,
GDia Gw1 Gnrz GA13
G, G, G, G,
G&f, gangliosidosis
6 Gonatas)
G,
Controls* 3.1
1.1
3.1
12.1
8.7 18.0 67.0 1.0
20.7 48.1 13.0 I.5
1.1
1.0
* Average of four control brains (autopsies) of children aged 6 months, tively.
of gangliosidoses of the type Gsn. Suzuki I1 found 74.5% fraction of the gray matter, while Seringe3 gives 60:/,.
zI/~,
4 and g years respec-
of the NANA in the Gm
As can be seen in Table III, the value of the activity of the /Y?-galactosidase in the patients’ brain was only 6.5$/o of the activity in normal gray matter of the controls. TABLE THE
111
,%4LACTOSIDASE
ACTIVITY
OF
CONTROL
PERSONS
AND
OF
THE
PATIENT
Contvol
Sex
Tissue
Activity in units* per mg dry weight
I I day 2 days 2 4 years 3 II years 4 2 years 5 6 L days 4 years 7 Mean value :k deviation
Male Male Male Male Male Male Male
brain gray matter brain gray matter brain gray matter brain gray matter brain gray matter brain white matter brain white matter gray matter white matter gray matter
3.98 3.50 2.27 4.12 3.63 1.62 1.72 3.50 + 1.23 1.67 + 0.05 0.23 ; 0.06
Patient with Landing disease * I unit = amount of enzyme which liberates
I mpmole p-nitrophenol
per hour.
DISCUSSION
The ,&galactosidase activity found in normal brains shows a general agreement with the results of Hayra 12. The ganglioside accumulation in the patient’s brain is apparently due to a severe deficiency of a /3-galactosidase, as a result of which the velocity of the breakdown to gangliosides with less sugar molecules is greatly diminished. It is noteworthy that in our case of Landing disease no absolute absence of the enzyme was found, which seems to be in contrast with the results of Hers, as mentioned by Seringe and coworkers 3. The discrepancy could be explained by the prolonged time of our enzyme assay which allowed us to show some possibly very weak residual activity. The question concerning the specificity of glucosidases (e.g. /3-glucosidase, pClin. Chim.
Acta,
21
(1968) 421-425
GANGLIOSIDOSIS
4%
galactosidase) has received much attention during the last few years. Hayra and coworkers12 give some arguments according to which several specific cerebroside ,%galactosidases should exist. The paper of Brady13 also implicates the presence of several b-galactosidases. The actual state of our knowledge seems to agree with the existence of a series of /?-galactosidases in the different organs examined, each active only toward one or a few natural substrates, but all active toward some unnatural substrates such as nitrophenyl glycosides. As the enzyme defect in the diseases of Fabry and Gaucher also concerns ,&galactosidase, the different specificity of the enzyme will cause a variable accumulation. Likewise, one can postulate that the GM1ganglioside accumulation is due to a deficiency of a specific G%n-ganglioside$-galactosidase. It is our intention to begin a detailed investigation into this specificity and the presence of isoenzymes of P-galactosidases. ACKNOWLEDGEMENT
The authors are greatly concerning the diagnosis.
indebted
to Prof. Hooft for his valuable
suggestions
REFERENCES I J. S. O'BRIEN, M. B. STERN, B. H. LANDING, J. Ii.O'BRIEN AND G. N. DONNELL, Diseases
Childhood,
109 (1965)
Am.
1.
338.
2 R. SACRES, J. G. JUIF, J. M. GIGONNET
AND J. E. GRUNER, Pediatric,22 (1967) 143. 3 P.SERINGE,B.PLAINFOSSE,F.LAUTMANN, J.LORILLOUX,G.CALAMY,J.P.BERRYAND J.M. WATCHI, (Sem. H@. Paris) Ann. Pt!diat. (Paris), 44 (1968) 685/P 165. 4 I(. SUZUKI, &je .%i.,3 (1964) 1227. 5 L. SVENNERHOLM, Biochim. Bioflhys. Acta, 24 (1957) 604. 6 J. LEDERBERG, J. Bacterial., 60 (1950) 381. 7 L. SVENNERHOLM, J. Lipid Res., 5 (1964) 145. 8 S. R. KOREY i~~~ J. GONATAS, J. Neuropathol. Expl. Xeurol., 22 (1963) 56. 9 K. KUHN AND H. WIEGANDT, Chem. Ber., 96 (1963) 866. IO E. KLENK AND W. KUNAU, Hoppe Seylers Z. Physiol. Chem., 335 (1964) 275. II K. SUZUKI,~~ S. M. ARONSON AND B. W.VOLK(E&.), S~hingolipidoses,grdInternational Symposium on Sphingolipidoses, 1965. Pergamon Press,New York, 1966. 12 A. K. H~YRA, D. M. BOWEN, Y. KISHIMOTO AND S. RADIM, J. Lipid Res., 7 (1966) 379. 13 R. 0. BRADY, 4. E. G.~L,R. M. BRADLEY, E. MARTENSSON, A. L. WARSH.%\V AND L. LASTER. New Engl. J. Med., 276 (1967) 1163. Clin. Chim.
Acta,
21 (1968) 421-425