- Email: [email protected]
Lysosomal enzymes in motor neurone disease and multiple sclerosis
Clika Chimica Acta, 47 (1973) 397-402 ~3 Elscxrier Scientific Publishing Company,
Amsterdam - Printed in The Netherlands
397
CCA 5838
LYSOSOMAL MULTIPLE
ENZYMES
IN MOTOR
NEURONE
DISEASE
AND
SCLEROSIS
CELIA bl. YATES, HELEN WILSOS MRC Brain Metabolism Unit, Dept. of Pharmacology, I George Swuarc, Edinburgh. EH8 9 JZ (U.K.) AN,,
University of Edinburgh,
D. DAVIDSON
Dept. Medical Xeurology,
Northern General Hospital, Frwy Road, Edinburgh, 5 (U.K.)
(Kcceivcd April 6, 1973)
SUMMARY In nine patients with motor neurone disease the activity of acid phosphatase in cerebrospinal fluid (CSF) was higher (P <0.005) than in nine controls. The activity of this enzyme in CSF from 18 patients with multiple sclerosis did not differ from control values: The activity of arylsulphatase in CSF was the same in the motor neurone, multiple sclerosis and control groups. In the serum, the activities of the two enzymes were similar in the three groups of patients.
ISTRODUCTION An increase in the activity of acid phosphatase has been demonstrated in cerebrospinal fluid (CSF) from one patient with amyotrophic lateral sclerosis and from one with multiple sclerosisl. A tendency for this enzyme to be increased in CSF from Huntington’s chorea patients has also been reported 2. This suggests that lysosomal enzymes may be involved in cell breakdown in central degenerative conditions, as has been shown to be the case in certain peripheral degenerative diseases3T4. The lysosomal enzymes, acid phosphatase and arylsulphatase, have therefore been measured in CSF from patients with motor neurone disease, in which there is progressive neuronal degeneration and in patients with multiple sclerosis in which there is demyelination without marked neuronal loss. METHODS All subjects were in-patients Hospital, Edinburgh.
in the neurological
unit at the Northern
General
Controls Two female and seven male patients who had a peripheral nerve disorder, minor non-progressive central nervous system abnormalities, or who were normal, made up the control group (Table I).
M M
M F F M
IV1 AI
3
1 5 6 7
8 9
15
Transient ischaemic attack 3 weeks previously Congenital mild spastic diplegia Fascia-scapula-peroneal spinal muscular atrophy Migraine _-\nxiety state Post-viral radiculopathq .lnxiety state. Nitrazepam IO mg and diazepam 9 mg daily Mild sensory ncuropathy Peripheral neuropathy. Chlorthalidonc IOO mq, KC1 2.4 g and debrisoquin 30 mg daily
mean j, S.1). 32 z 19 __ ___ * nmoles p-nitrophenol &cased/ml per h. * * nmoles P-nitrocatechol released /ml per h. n.d. = not determined.
2
M
I
6.8
n.d.
7.7 I.9
7.1 I.6 5.7
I.7
2.5
1.j
I.2
7.8
2.4
1.7
2.7
~~~ 4.1 4.1
3.3
0.54
0.27
0.39
Ii 5
680 560
9 15 7 17
830 380 390 520
‘3
0.2j
0.26
IO
0.69
0.41
89 9.5
77 87
78 80
X8 x0
TABLE
M XI
AI iv iv 1;
iv
3 4
5 6 z
9
IN
ASU
SERUM
FRO>1
PATIENTS
\VITH
~__
* nmoles P-nitrophenol released/ml per h. * * nmoles p-nitrocatechol released/ml per h. n.d. = not determined.
NCme 8.0 _k 1.2
8.0
0.49 0.50 0.35 I.23 0.43
2.1 1.9 2.0 0.8 I.1
0.60 i_ 0.26
0.7* 0.65
n.d. 1.2
1.7 & 0.6
0.43 0.60
2.6 2.2
580 + 160
410
570
j&l
530
SK?W% Pvotein (mgiml)
Actiuzty of acid phosphatasr
DISEASE
___ Activity Activity oj- acid of aryl phosphatasr* sulphatasP**
SEUROSE
CSF
MOTOR
N0lle x.0 Phenobarbitone 30 mg, cpanutin ICJOmg X.6 and nitrazapam 10 mg daily 8.2 None IO.0 Prochlorperazine 15 mg and Baclofen 30 mg daily 8.9 None 6.0 Nitrazepam _j mg dail! 6.8 7.5 None
-___
llvugs
CSI;
5.5 2k 13
72
58 58 61 68
49 5.5
32 -to
Age (YY)
ENZYMES
mean + S.D.
I
2
Sex
II
iI1 ill
Patient
LYSOSOMAL I___.
Activity of aryl * sulphatasr
76 i
73 67 72 7x 78 6
.~~ Protein (~~glmt) *+
YATES
400
et al.
Motor neurone disease patients One female and eight male patients with motor neurone disease in which there was clear evidence of anterior horn cell degeneration with moderately severe wasting and fasciculation of the limbs, were investigated (Table II). Mdtiple sclerosis patients Ten female and eight male patients with acute or chronic lesions localised the spinal cord, the brain stem or in both these regions were studied (Table III).
in
CSF About 10-n ml CSF were collected by lumbar puncture from each patient. The CSF samples were stored at 4O and within 6 h after lumbar puncture the cells removed by centrifugation at 3000 rev/min for IO min. The samples were then stored at -20'. The maximum period of storage before estimation was 25 days. Serum Ten ml venous and stored at -20'.
blood was taken
from each patient
and the serum separated
Enzyme assays The
activities
of acid phosphatase
and arylsulphatase
p-nitrophenylphosphate and p-nitrocatechol sulphate, in samples of CSF and serum from each patient2. Protein Protein (Sigma)
was measured
of Lowry
et al.” using
were measured
respectively,
bovine
using
on the same day
albumin,
fraction
V
as standard.
RESULTS
All statistical
comparisons
were made
with Student’s t-test and a value of difference. neurone disease, the activity of acid phos-
P <0.05was taken to indicate a significant In CSF from patients
with motor
phatase was increased (P (0.005) and that of arylsulphatase unaltered as compared to the controls (Tables I and II). However, in patients with multiple sclerosis the activities of both enzymes in the CSF did not differ from control values (Tables I and III). There was no difference in the activity of acid phosphatase in CSF from patients with chronic lesions and those with episodes of demyelination within the previous 4 weeks. Similarly, there was no difference in the activity of this enzyme in CSF from patients with cord or brain stem lesions. In the serum, there was no significant difference between the mean activities of acid phosphatase or arylsulphatase in the three groups of patients (Tables I, II and III). The concentration of protein in CSF and serum did not differ significantly between the three groups. The levels of the two enzymes in CSF and serum did not correlate with protein concentration or the age of the patient.
M F M M F M M F F
M M BI
F F F F F F
Sex
33 39 42 46 46 47 49 5o 50 5’ 53 64
3o 31 32
28
21 23
(YVJ
47
ENZYMES
III
IN
A&D
SERUM
FROM
PATIENTS
WITH
Nitrazepam IO mg night before L.P. None None Nitrazepam 5 mg night before L.P. None Cyclizine 200 mg daily until 28 h before L.P. Orphenadrine 75 mg daily None None None Soluble insulin, protamine Zn insulin None Diazepam 2 mg 8 h before L.P. None None None Nitrazepam 5 mg night before L.P. Inosital nicotinate 1500 mg daily, nitrazepam 5 mg before L.P.
D?Wgs
CSF
* nmoles p-nitrophenol released/ml per h. * * nmoles p-nitrocatechol released/ml per h n.d. = not determined. L.P. = lumbar puncture.
: 9 IO II 12 ‘3 14 15 16 17 I8
Patient
LYSOSOMAL
ThRI,E
1.0
0.4 I.5 1.5 *
5.7 + 1.9
n.d. 0.8 3.2 n.d.
1.6
n.d. 2.6
1.0
0.6 I.7
n.d.
2.0
n.d. n.d.
I.2
::7 5.8 4.5 4.2 8.4 5.0 5.9 7.8 2.7 5.7
-0
8.7
3.7 9.9 5.8 4.0 5.2 6.2
0.8
Activity of aryl * sulphatase * *
SCLEROSIS
Activity of acid phosphatase
CSF
MULTIPLE
0.54 *
0.76 0.41 0.34 0.47
0.41
0.39 0.56 0.71 0.68 0.50 0.73 0.4’
0.82 0.53 0.66 0.33 0.38 0.55
(m&Tlml)
Protein
0.16
Serum
5ro + 170
660 n.d. 660 750 690 440 7oo 370 350 800 540 5oo
360 230 510 370 360 430
Activity of acid phosphatase*
11 + 5
I3 n.d. 7 ‘7
20
II n.d.
12
6 n.d. 4 n.d.
6 n.d. n.d. 14 n.d.
12
Activity of aryl sulphatase**
81
:;
;: 83 70 93 77 73
86 n.d. 94
*
17
(ms Iml)
Protein
TATES
402
ct a/.
The increase in activity of acid phosphatase in CSF from patients with motor neurone disease was not accompanied by a raised activity in the serum, suggesting that the source of this increase was the central nervous system. Since age and the activity of acid phosphatase were not correlated in the control group, the age tlistribution of which partially overlapped that of the motor neurone disease group, the increased activitv of this enzvme in CSF from the motor neurone disease patients is unlikely to be age related. An elevated activity of acid phosphatase has been reported in amyotrophic lateral sclerosisl. Histologically normal anterior horn cells of tlrc, spinal cord have been shown to contain about ten times more acid phosphatasc activity than the neighbouring neuropil”. In motor neurone disease, there is a loss of acid phosphatase activity from the degenerating anterior horn ~11s~ which coultl account for the increased activity of this enzyme found in the lumbar CSF. The lack of an increase in arylsulphatase activity ma)’ be due to the presence, in CSF, of inorganic ions known to inhibit human brain arylsulphatase H,9. Of interest is the ohservation by \\:ender and Wenclewski lo that the level of arvlsulphatase in CSF \cax not increased even in cases of damage to the blood brain barrier, although this produced elevations in the activity of acid phosphatase~~~~~ and other lv.~sonial cn zymcsl~-l~. In multiple sclerosis, myelin degeneration has been associated with increased levels of acid hydrolases, including acid phosphatase, in the white matter15. Contrar! to a previous report on one patient’, no increase in acid phosphatase activitv was found in lumbar CSF from patients with brain stem or cord lesions. This is possibl! because in multiple sclerosis, unlike motor neurone disease, cell breakdown with consequent enzyme release, is not marked Is. Another possibly significant factor is tlic difference in duration of the two diseases, motor neurone disease being rapidl!, progressive
and multiple
sclerosis
much more prolonged16.
REFBRENCISS I K. GUZOWSKI AN,) J, KXOBLOCH, Avch. ~‘rurol. (Chic.), X (1963) 105. z C. M. YTATES, B. E. .4. MMXL, D. DAVIDSON, 1~. G. MURRAY, H. \+::ILSOS >zsu 1. .\. I’~LLAR, czin. Ckim. Acta, 44 (1973) 139. 3 D. I?. PAGE THOMAS, in DIKGLE ASI> FELL (&is.), Z.ysosonzrs in Biology nurl Pathology, \.ol. 2, North-Holland, Amsterdam, 1969, p. 87. .+ N. C. KAR ASD C. WI. PEARSON, Clin. Chiun. Acta, 40 (1972) 341. 5 0. H. LOWRY, N. J. ROSEBROV~H, X. L. F~RR ATL) R. J. RANI~ALL, ,I. Rzoi. Chrw., 193 (19.5’) 265. 6 H. B. HIRSCH, J. 1Veuvochcun., 15 (1968) 123. 7 N. ROBIXSON, Acta n:europathol, 7 (1966) 101. 8 H. E. HIRSCH, J. Nruvochem., 16 (1969) 1147. 9 B. C. HARINATH AND E. ROBIP~S, J. h:wrochem., IX (1971) 237. IO WI. WENDER AND A. WE~~CLEWSKI in J. SAYK (Ed.), .5ymposiztwz iiber dw Zcrebuospilzalfliissigheit, Veb Gustav Fischer Vcrlag, Jena, 1966, p. T.+j. 11 K. COLLIXG AND R. ROSSITER, Canad. 1. Rrs., 28 (1950) 56. 12 E. C. SHUTTLEWORTH .WD N. ALLEN, A~~uvology, IX (1968) 534. ‘3 N. ALLEK AND E. REAGAN, ilvch. Neuvol., II (1964) 144. rq E. KOVACS, ,I. Pediatrics, 46 (1955) 691. 13 A. II. .L\RSTILA,P. RIEKKIKEX, U. K. RINSE ASD L. LAITISEN, Euvop. Seuvol., 9 (197.3) I. 16 XV. R. RRAIS, Diseases qf thr ~Vevuous System. Oxford l’niv. Press, 1962, pp. 433, 631.
Amsterdam - Printed in The Netherlands
397
CCA 5838
LYSOSOMAL MULTIPLE
ENZYMES
IN MOTOR
NEURONE
DISEASE
AND
SCLEROSIS
CELIA bl. YATES, HELEN WILSOS MRC Brain Metabolism Unit, Dept. of Pharmacology, I George Swuarc, Edinburgh. EH8 9 JZ (U.K.) AN,,
University of Edinburgh,
D. DAVIDSON
Dept. Medical Xeurology,
Northern General Hospital, Frwy Road, Edinburgh, 5 (U.K.)
(Kcceivcd April 6, 1973)
SUMMARY In nine patients with motor neurone disease the activity of acid phosphatase in cerebrospinal fluid (CSF) was higher (P <0.005) than in nine controls. The activity of this enzyme in CSF from 18 patients with multiple sclerosis did not differ from control values: The activity of arylsulphatase in CSF was the same in the motor neurone, multiple sclerosis and control groups. In the serum, the activities of the two enzymes were similar in the three groups of patients.
ISTRODUCTION An increase in the activity of acid phosphatase has been demonstrated in cerebrospinal fluid (CSF) from one patient with amyotrophic lateral sclerosis and from one with multiple sclerosisl. A tendency for this enzyme to be increased in CSF from Huntington’s chorea patients has also been reported 2. This suggests that lysosomal enzymes may be involved in cell breakdown in central degenerative conditions, as has been shown to be the case in certain peripheral degenerative diseases3T4. The lysosomal enzymes, acid phosphatase and arylsulphatase, have therefore been measured in CSF from patients with motor neurone disease, in which there is progressive neuronal degeneration and in patients with multiple sclerosis in which there is demyelination without marked neuronal loss. METHODS All subjects were in-patients Hospital, Edinburgh.
in the neurological
unit at the Northern
General
Controls Two female and seven male patients who had a peripheral nerve disorder, minor non-progressive central nervous system abnormalities, or who were normal, made up the control group (Table I).
M M
M F F M
IV1 AI
3
1 5 6 7
8 9
15
Transient ischaemic attack 3 weeks previously Congenital mild spastic diplegia Fascia-scapula-peroneal spinal muscular atrophy Migraine _-\nxiety state Post-viral radiculopathq .lnxiety state. Nitrazepam IO mg and diazepam 9 mg daily Mild sensory ncuropathy Peripheral neuropathy. Chlorthalidonc IOO mq, KC1 2.4 g and debrisoquin 30 mg daily
mean j, S.1). 32 z 19 __ ___ * nmoles p-nitrophenol &cased/ml per h. * * nmoles P-nitrocatechol released /ml per h. n.d. = not determined.
2
M
I
6.8
n.d.
7.7 I.9
7.1 I.6 5.7
I.7
2.5
1.j
I.2
7.8
2.4
1.7
2.7
~~~ 4.1 4.1
3.3
0.54
0.27
0.39
Ii 5
680 560
9 15 7 17
830 380 390 520
‘3
0.2j
0.26
IO
0.69
0.41
89 9.5
77 87
78 80
X8 x0
TABLE
M XI
AI iv iv 1;
iv
3 4
5 6 z
9
IN
ASU
SERUM
FRO>1
PATIENTS
\VITH
~__
* nmoles P-nitrophenol released/ml per h. * * nmoles p-nitrocatechol released/ml per h. n.d. = not determined.
NCme 8.0 _k 1.2
8.0
0.49 0.50 0.35 I.23 0.43
2.1 1.9 2.0 0.8 I.1
0.60 i_ 0.26
0.7* 0.65
n.d. 1.2
1.7 & 0.6
0.43 0.60
2.6 2.2
580 + 160
410
570
j&l
530
SK?W% Pvotein (mgiml)
Actiuzty of acid phosphatasr
DISEASE
___ Activity Activity oj- acid of aryl phosphatasr* sulphatasP**
SEUROSE
CSF
MOTOR
N0lle x.0 Phenobarbitone 30 mg, cpanutin ICJOmg X.6 and nitrazapam 10 mg daily 8.2 None IO.0 Prochlorperazine 15 mg and Baclofen 30 mg daily 8.9 None 6.0 Nitrazepam _j mg dail! 6.8 7.5 None
-___
llvugs
CSI;
5.5 2k 13
72
58 58 61 68
49 5.5
32 -to
Age (YY)
ENZYMES
mean + S.D.
I
2
Sex
II
iI1 ill
Patient
LYSOSOMAL I___.
Activity of aryl * sulphatasr
76 i
73 67 72 7x 78 6
.~~ Protein (~~glmt) *+
YATES
400
et al.
Motor neurone disease patients One female and eight male patients with motor neurone disease in which there was clear evidence of anterior horn cell degeneration with moderately severe wasting and fasciculation of the limbs, were investigated (Table II). Mdtiple sclerosis patients Ten female and eight male patients with acute or chronic lesions localised the spinal cord, the brain stem or in both these regions were studied (Table III).
in
CSF About 10-n ml CSF were collected by lumbar puncture from each patient. The CSF samples were stored at 4O and within 6 h after lumbar puncture the cells removed by centrifugation at 3000 rev/min for IO min. The samples were then stored at -20'. The maximum period of storage before estimation was 25 days. Serum Ten ml venous and stored at -20'.
blood was taken
from each patient
and the serum separated
Enzyme assays The
activities
of acid phosphatase
and arylsulphatase
p-nitrophenylphosphate and p-nitrocatechol sulphate, in samples of CSF and serum from each patient2. Protein Protein (Sigma)
was measured
of Lowry
et al.” using
were measured
respectively,
bovine
using
on the same day
albumin,
fraction
V
as standard.
RESULTS
All statistical
comparisons
were made
with Student’s t-test and a value of difference. neurone disease, the activity of acid phos-
P <0.05was taken to indicate a significant In CSF from patients
with motor
phatase was increased (P (0.005) and that of arylsulphatase unaltered as compared to the controls (Tables I and II). However, in patients with multiple sclerosis the activities of both enzymes in the CSF did not differ from control values (Tables I and III). There was no difference in the activity of acid phosphatase in CSF from patients with chronic lesions and those with episodes of demyelination within the previous 4 weeks. Similarly, there was no difference in the activity of this enzyme in CSF from patients with cord or brain stem lesions. In the serum, there was no significant difference between the mean activities of acid phosphatase or arylsulphatase in the three groups of patients (Tables I, II and III). The concentration of protein in CSF and serum did not differ significantly between the three groups. The levels of the two enzymes in CSF and serum did not correlate with protein concentration or the age of the patient.
M F M M F M M F F
M M BI
F F F F F F
Sex
33 39 42 46 46 47 49 5o 50 5’ 53 64
3o 31 32
28
21 23
(YVJ
47
ENZYMES
III
IN
A&D
SERUM
FROM
PATIENTS
WITH
Nitrazepam IO mg night before L.P. None None Nitrazepam 5 mg night before L.P. None Cyclizine 200 mg daily until 28 h before L.P. Orphenadrine 75 mg daily None None None Soluble insulin, protamine Zn insulin None Diazepam 2 mg 8 h before L.P. None None None Nitrazepam 5 mg night before L.P. Inosital nicotinate 1500 mg daily, nitrazepam 5 mg before L.P.
D?Wgs
CSF
* nmoles p-nitrophenol released/ml per h. * * nmoles p-nitrocatechol released/ml per h n.d. = not determined. L.P. = lumbar puncture.
: 9 IO II 12 ‘3 14 15 16 17 I8
Patient
LYSOSOMAL
ThRI,E
1.0
0.4 I.5 1.5 *
5.7 + 1.9
n.d. 0.8 3.2 n.d.
1.6
n.d. 2.6
1.0
0.6 I.7
n.d.
2.0
n.d. n.d.
I.2
::7 5.8 4.5 4.2 8.4 5.0 5.9 7.8 2.7 5.7
-0
8.7
3.7 9.9 5.8 4.0 5.2 6.2
0.8
Activity of aryl * sulphatase * *
SCLEROSIS
Activity of acid phosphatase
CSF
MULTIPLE
0.54 *
0.76 0.41 0.34 0.47
0.41
0.39 0.56 0.71 0.68 0.50 0.73 0.4’
0.82 0.53 0.66 0.33 0.38 0.55
(m&Tlml)
Protein
0.16
Serum
5ro + 170
660 n.d. 660 750 690 440 7oo 370 350 800 540 5oo
360 230 510 370 360 430
Activity of acid phosphatase*
11 + 5
I3 n.d. 7 ‘7
20
II n.d.
12
6 n.d. 4 n.d.
6 n.d. n.d. 14 n.d.
12
Activity of aryl sulphatase**
81
:;
;: 83 70 93 77 73
86 n.d. 94
*
17
(ms Iml)
Protein
TATES
402
ct a/.
The increase in activity of acid phosphatase in CSF from patients with motor neurone disease was not accompanied by a raised activity in the serum, suggesting that the source of this increase was the central nervous system. Since age and the activity of acid phosphatase were not correlated in the control group, the age tlistribution of which partially overlapped that of the motor neurone disease group, the increased activitv of this enzvme in CSF from the motor neurone disease patients is unlikely to be age related. An elevated activity of acid phosphatase has been reported in amyotrophic lateral sclerosisl. Histologically normal anterior horn cells of tlrc, spinal cord have been shown to contain about ten times more acid phosphatasc activity than the neighbouring neuropil”. In motor neurone disease, there is a loss of acid phosphatase activity from the degenerating anterior horn ~11s~ which coultl account for the increased activity of this enzyme found in the lumbar CSF. The lack of an increase in arylsulphatase activity ma)’ be due to the presence, in CSF, of inorganic ions known to inhibit human brain arylsulphatase H,9. Of interest is the ohservation by \\:ender and Wenclewski lo that the level of arvlsulphatase in CSF \cax not increased even in cases of damage to the blood brain barrier, although this produced elevations in the activity of acid phosphatase~~~~~ and other lv.~sonial cn zymcsl~-l~. In multiple sclerosis, myelin degeneration has been associated with increased levels of acid hydrolases, including acid phosphatase, in the white matter15. Contrar! to a previous report on one patient’, no increase in acid phosphatase activitv was found in lumbar CSF from patients with brain stem or cord lesions. This is possibl! because in multiple sclerosis, unlike motor neurone disease, cell breakdown with consequent enzyme release, is not marked Is. Another possibly significant factor is tlic difference in duration of the two diseases, motor neurone disease being rapidl!, progressive
and multiple
sclerosis
much more prolonged16.
REFBRENCISS I K. GUZOWSKI AN,) J, KXOBLOCH, Avch. ~‘rurol. (Chic.), X (1963) 105. z C. M. YTATES, B. E. .4. MMXL, D. DAVIDSON, 1~. G. MURRAY, H. \+::ILSOS >zsu 1. .\. I’~LLAR, czin. Ckim. Acta, 44 (1973) 139. 3 D. I?. PAGE THOMAS, in DIKGLE ASI> FELL (&is.), Z.ysosonzrs in Biology nurl Pathology, \.ol. 2, North-Holland, Amsterdam, 1969, p. 87. .+ N. C. KAR ASD C. WI. PEARSON, Clin. Chiun. Acta, 40 (1972) 341. 5 0. H. LOWRY, N. J. ROSEBROV~H, X. L. F~RR ATL) R. J. RANI~ALL, ,I. Rzoi. Chrw., 193 (19.5’) 265. 6 H. B. HIRSCH, J. 1Veuvochcun., 15 (1968) 123. 7 N. ROBIXSON, Acta n:europathol, 7 (1966) 101. 8 H. E. HIRSCH, J. Nruvochem., 16 (1969) 1147. 9 B. C. HARINATH AND E. ROBIP~S, J. h:wrochem., IX (1971) 237. IO WI. WENDER AND A. WE~~CLEWSKI in J. SAYK (Ed.), .5ymposiztwz iiber dw Zcrebuospilzalfliissigheit, Veb Gustav Fischer Vcrlag, Jena, 1966, p. T.+j. 11 K. COLLIXG AND R. ROSSITER, Canad. 1. Rrs., 28 (1950) 56. 12 E. C. SHUTTLEWORTH .WD N. ALLEN, A~~uvology, IX (1968) 534. ‘3 N. ALLEK AND E. REAGAN, ilvch. Neuvol., II (1964) 144. rq E. KOVACS, ,I. Pediatrics, 46 (1955) 691. 13 A. II. .L\RSTILA,P. RIEKKIKEX, U. K. RINSE ASD L. LAITISEN, Euvop. Seuvol., 9 (197.3) I. 16 XV. R. RRAIS, Diseases qf thr ~Vevuous System. Oxford l’niv. Press, 1962, pp. 433, 631.