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Chemical definition of the mucopolysaccharidoses
321
Clinica Chimica Acta, 59 (197 5) 321-336 @ Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 6946
CHEMICAL DEFINITION
GEORGE
CONSTANTOPOULOS
Developmental arid Metabolic Bethesda, Md. 20014 (U.S.A.)
(Received
October
OF THE MUCOPOLYSACCHARIDOSES
and ANATOLE Neurology
Branch,
S. DEKABAN NINDS,
National
Institutes
of Health,
23, 1974)
Summary Glycosaminoglycans were isolated from the urines of 46 patients with mucopolysaccharidosis; 11 with Type I (Hurler), 8 with Type II (Hunter), 16 with Type III (Sanfilippo A and B), 9 with Type V (Scheie), one with Type VI (Marateaux-Lamy), and one unclassified. All 46 patients excreted in their urine excessive amounts of dermatan sulfate, heparan sulfate or both. In addition, patients of certain types excreted excessive amounts of chondroitin sulfates A and/or C. There is a trend in each type of the disease towards the same carbazole/orcinol ratio, glucosamine/galactosamine ratio and glycosaminoglycan composition. Molecular weight distribution of the urinary glycosaminoglycans by gel filtration from Sephadex G-ZOO is characteristic for each different type of mucopolysaccharidosis and is distinguished from n’ormal controls and patients without mucopolysaccharidosis. Preparation of elution diagrams from Sephadex G-200 allows an estimation of the composition of the glycosaminoglycans. Practically all heparan sulfate and a sizable part of dermatan sulfate from the urinary glycosaminoglycans of all these patients have been highly degraded. In all the patients in which the specific enzyme defect was demonstrated, the assignment of the type of mucopolysaccharidosis, on the basis of the elution diagrams, was correct. Patients with mucopolysaccharidosis Type V displayed two conspicuously different types of elution patterns, suggesting heterogeneity. Indeed, only a portion of these patients showed a-L-iduronidase deficiency. Carriers had normal urinary glycosaminoglycan output and composition and exhibited normal elution diagrams.
Introduction Mucopolysaccharidoses are inborn errors of the glycosaminoglycan (GAG) or acid mucopolysaccharide metabolism [l]. Recent studies have defined the defects as the deficiency of specific degradative enzymes [2-91 . Abbreviations: AC, chondroitinase-AC;
CPC. cetylpyridinium chloride; CSA and CSC. chondroitin sulfates A and C; Chyase DS, dermatan sulfate; HS, heparan sulfate; GAG, glycosaminoglycan.
322
Excessive excretion of GAG in the urine of the patients (mucopolysacchariduria), is the common feature of the mucopolysaccharidoses and it is utilized as a diagnostic test for these diseases [lO,ll] .In preliminary studies, we have shown that Sephadex G-200 gel filtration of the urinary GAG from patients with mucopolysaccharidoses Types I, II and III (classification of McKusick [12] ) gave elution profiles which are characteristic for each type; moreover, the distribution of molecular weights of the GAG indicated that they represent fragments of the parent molecules [ 13,141. Subsequently, we have extended our work to 46 patients who represent 6 or ‘7 different types of mucopolysaccharidoses. The results confirm and expand the earlier observations and demonstrate the usefulness of this technique in the diagnosis, classification and detection of new types of mucopolysaccharidosis. In addition, elution profiles aid elucidation of the pathogenesis of these disorders. The validity of the method has been confirmed by the identification of the specific enzyme deficiency in a number of the patients investigated. Material
and Methods
During the past eight years, urines from over 150 patients suspected of having mucopolysaccharidosis were examined for GAG; more than one-half of the urines were from patients of our clinical service; the other urines were submitted to us by outside physicians or investigators. Forty-six patients were proven to have one or another type of mucopolysaccharidosis. Thirty-six of these patients were studied by us as inpatients, outpatients or by consultations in the area hospitals. Clinical data and urines of two patients (Cases 32,33) were supplied by Dr H. Moser of Boston, of one patient (Case 45) by Dr A. Cracker of Boston and urines of the remaining seven patients (Cases 1,2,7,11, 22, 24, 28) were submitted to this laboratory by Dr E.F. Neufeld of NIH, for chemical analysis. Subsequently, Dr Neufeld supplied us with confirmatory analysis of the enzymatic defect in these patients. Twenty-four hour urine specimens were kept at 0-4°C without preservatives and if not analyzed within the first two days, stored frozen at -20°C until needed. Isolation of GAG and analytical procedures Glycosaminoglycans were isolated from undialyzed urines by chromatography on Ecteola columns or by precipitation with cetylpyridinium chloride (CPC) [15]. Urines with 24-hour volume <500 ml were diluted to 500 ml with distilled water, prior to isolation. The isolated GAG were routinely analyzed for uranic acid content by the carbazole (Dische) [16] and orcinol [17] methods, which permit calculation of the carbazole/orcinol ratio and by the carbazole (borate) [18] modification which improves the color yield for dermatan sulfate (DS) and gives results closer to the theoretical values for all GAG. The other methods we used for the chemical analysis of the GAG, (total hexosamine, glucosamine/galactosamine ratio, sulfate and N-sulfate content) and the determination of their molecular weight distribution and construction of the elution diagrams, were previously described [14] .
323
Estimation
of GAG composition
GAG from the various types of mucopolysaccharidoses were subjected to fractionation on a Dowex l-X2 column by step-wise elution with increasing concentrations of NaCl, and the individual fractions were analyzed [19] . Susceptibility of the GAG to enzymatic depolymerization with chondroitinase-AC (Chyase AC) and chondroitinase-ABC (Chyase ABC) was examined by measuring the uranic acid content of the released unsaturated disaccharides [20] . The following micro-method was developed for the measurement of the released disaccharides. Two sets of reaction mixtures and a control were prepared in glass tubes 7 mm (i.d.) X 75 mm. The reaction mixture contained 20 ~1 enriched Tris buffer (pH 8.0), test sample containing about 25-50 pg uranic acid, 0.02 units Chyase AC in one set, and 0.1 unit Chyase ABC in the other set; the total volumes were brought up to 100 ~1. The control set lacked the enzyme. The time of incubation was 30 minutes at 37°C for incubation mixtures with Chyase AC and 4 hours at 37°C for mixtures with Chyase ABC. Enzymatic reactions were stopped by boiling for 1 minute, the reaction mixtures were cooled to 0°C and 50 ~1 of 3.0 M NaCl and 0.7 ml of 95% alcohol were added to each tube. The contents were mixed and left at 0--4°C for at least 1 hour. After centrifugation for 15 minutes at 10 000 X g the supernatants containing the released disaccharides were quantitatively removed, evaporated to dryness and analyzed for uranic acid with the carbazole (borate) method. A small amount from each sample was used for electrophoresis [21] and respective patterns before and after enzymatic incubations were compared. Finally, samples were subjected to gel filtrations before and after enzymatic incubations.
Enzyme
assays
a-L-Iduronidase in leukocytes, fibroblasts and tissues of some of the patients was measured with phenyl-cu-L-iduronide as the substrate. Other lysosomal enzymes were measured with the appropriate p-nitrophenyl glycosides [ 221. Phenyl-cu- L-iduronide was a gift of Dr B. Weissmann of the University of Illinois College of Medicine, Chicago, Illinois. Several p-nitrophenyl glycosides were purchased from Sigma and Research Products International Corp. The enzymes Chyase AC and ABC were purchased from Miles Laboratories. GAG standards were kindly supplied by Drs M.B. Mathews, J.A. Cifonelli and L. Roden, University of Chicago. Sephadex G-200 was purchased from Pharmacia Fine Chemicals, Inc. and Ecteola cellulose (ET-11 powder) from Reeve Angel and Co (Clifton, N. J. ). Results Table I summarizes the distribution of patients in various types of mucopolysaccharidosis and gives the output and composition of urinary GAG. From clinical, chemical and enzymatic studies, it was concluded that eleven patients had mucopolysaccharidosis Type I, eight Type II, sixteen Type III (10 were of subtype A and 6 of subtype B), nine Type V (which were further separable into two groups A and B), one Type VI, and one patient whom we were unable to recall for additional studies remained unclassified.
I
AND
Sulfoiduronate
N-Sulf&ase
III (Sanfilippo)
diseases
controls
Mean
+ standard
Type
deviation.
measured
* * * Mucopolysaccharidosis
**
acid
mucopolysaccharidosis
with
* As uranic
Normal
than
Patients
IV
(Morquio)
was not
number
(Dische)
[ 3 11
[3]
the carhazole
B?
In parentheses
with
Arylsulfatase
VI
(Marateaux-Lamy)
a-L-Iduronidasc
V (Scheie) ***
Unclassified
[4,61
sulfatase
[8]
method.
151
investigated.
of
11
115
1
1
9
16
8
11
patients
No.
GLYCOSAMINOGLYCANS
of experiments
URINARY
12.31
OF
N-Acetyl~-glucosainidase
a-L-Iduronidase
enzyme
II (Hunter)
Deficient
COMPOSITION
I (Hurler)
type
other
ESTIMATED
Mucopolysaccharidosis
OUTPUT
TABLE
5 4
B=
6
A=
B=
A = 10
4.3
4.3
18.0 51.0
29.6
37.4
24.0
50.5
40.5
43.0
(mg/24
7.7
18.5
+
+
1.3
2.8
t 10.4
+ 18.4
i
i
i 12.9
4)
4)
5)
8)
8)
7)**
(
11)
(115)
(
(
(
(
(
(
GAG* h)
k 14.1
Urinary
1.04
1.07
0.44 0.73
0.53
0.69
1.6
2.0
0.90
0.66
+ 0.007
i 0.12
+ 0.06
2 0.07
k 0.15
+ 0.18
+ 0.12
? 0.06
Orcinol
Carbazole
4)
4)
6)
(11)
(43)
(
(
(
(10)
8)
(ll)*‘ (
Estimated
:
<
_
80: _
82:
55
5
46:
60:
:
5:
<
2:
10:
<3:
35
85
20
16
10
:
9
10
:
:
: 10
45:
30
+ CSC)
HS
: 85
(CSA
DS
composition
325
All patients excreted in their urines from 5 to 15 times more GAG than the normal controls or the patients with other diseases. There were not large variations in excretion from day to day {within a few weeks’ time) and the composition of the GAG was consistent in each type of the disease and in the same patient during the several years he was followed-up. Patients with Type I mucopolysaccharidosis (Hurler Syndrome) excreted excessive amounts of DS and heparan sulfate (HS) in their urine in an approximate ratio of 2 : 1. Type II patients (Hunter) also excreted excessive amounts of DS and HS but in a ratio close to 1 : 1. Both Types IIIA and B patients (S~filippo~ excreted large amounts of HS in their urine. It appears that Type V patients (Scheie) could also be separated into two groups: patients in group A excreted excessive amounts of DS and HS in a ratio 55 : 35 while those in group B excreted excessive amounts of DS and chondroitin sulfates A and C (CSA + CSC) in a ratio of 82 : 16. The urinary GAG of the Marateaux-Lamy patient were also composed of DS and CSA + CSC in a ratio of 80 : 20. The ~~b~ole~orcinol ratio, which depends on the GAG composition, was characteristic of each type but with some overlap between Types I and VA, between Type II and normal controls, and between Types VB and VI. The mean 24-hour urinary GAG output, in 115 patients with diseases other than mucopolysaccha~dosis, was 4.3 +_2.8 S.D. (as uranic acid), about the same as 11 normal controls, but with a wider range (l-16 mg per 24 hours). However, DS of HS were not major components of the urinary GAG in any of these patients. The urinary GAG excretion and elution diagrams in a few carriers examined was normal. Table II shows laboratory and clinical data on 11 patients with mucopolysaecharidosis Type I. Patients identified as Cases 1 and 2, 3 and 4, and 5 and 6 were three sets of siblings. The mean GAG-uranic acid excretion in the urine was 43 mg per 24 hours and galactosamine comprized more than two-thirds of the total hexosamine. From the carbazolelorcinol ratio (0.66 ? 0.06), glucosamine/galactosamine ratio, elution pattern and enzymatic analysis [Table VI), it was concluded that the predomin~t GAG were DS and HS at about a ratio of 2 : 1. Clinically, most of the patients, on whom information was available, were in an advanced state of the disease and had mental retardation, corneal opacity, hepatosplenomegaly and skeletal and cardiovascular changes. The urinary GAG from all the 11 patients gave a similar elution pattern by gel filtration on Sephadex G-200 (Fig. 1). Finally, deficiency of cu-L-iduronidase was found in the fibroblasts or tissues available from 8 patients. The laboratory and clinical data from 8 patients with mucopolysaccharidosis Type II are shown in Table III. Patients identified as Cases 14 and 15, and 16, 17 and 18 are two sets of siblings. The mean urinary GAG-uranic acid and carbazole/orcinol ratio were 40.5 mg per 24 hours and 0.9 t 0.12 SD., respectively. These patients excrete in their urine excessive amounts of DS and HS in an approximate ratio of 1 : 1. Mental retardation, organomegaly, cardiovascular and skeletal abnormalities were present in patients, although in a milder degree than in Type I, but no patient had corneal opacity. The elution pattern of the urinary GAG by gel filtration on Sephadex G-200 was characteristic of this type of the disease (Fig. 1).
II
2/2.5
1112 l/l1 l/6
2/F
3/M 4/M
IiS 3/7 114 ?/18
l/l ?/1.5
i( *
*
_----~~--
*
i&fear? 43.00 S.D. 14.15
26 56
35 -
SamPle”
53 63 37 31 15 ml random
samPle*
15 ml random
sample
35 ml random
_~_
248 -
240 131 181
-
-
---.. --_.-. -
(me/g creatioine)
0.66 0.06
0.60 0.65 0.10 0.65
0.55 0.58 0.65 0.65 0.66
0.71
0.76
---
73
++ ++ ++ -
+*+ +++ +++ +++
-
.-
-__..-
Mental retaxd&ion --c
.-__ .__ --..-. .___~_____~_
-
27: -
15: 85 12: 88 23 : 77 26 : 74 -
-
--
Gal NHZ
~..
ii-* ++ 5 -
+++ *++ +t Cf -
-
-
Corneai opacity
MUCOPOLYSACCffARIDOSlS
Or&ml
WITH
Ow/24 h)
PATIENTS Glu NH2
ON II Carbwole
DATA
Total urinary GAG uranic acid’
AND CLINICAL
* Carbazole (Dische). * * Portion of urine available. 1, sibiings.
---.
1llF
6/F 9/F 10/F
5/F 6/M 1 7/M
1
1
? 12.0
l/M
_--
_I__.__-
Age in years onset/ current -_.-._~--
CZlSef Sex
_._.
LABORATORY
TABLE I
____
i*+ ++ ++ -
++ ++ ++ ++
-
-
___
me?zalY
_... ._
++* ++c i+ _-
+++ +++ ++ ++ _-
-
_ ._..-__
Skelefai chan@x
.~.__.-...HepatoSPlUlO-
TYPE
---. -_
++i ++ *
+* *++ ++ f -
-
-
_.-..
Cardio vascular
I I I I
I I I I I
I
I
type
pattern
Gl?l filtration
~__._ _... _ ._-._
Yes
Yes Not examined -
Yes Yes Not examined Yes Yes
Yes
Yes
a-L-Iduronidase deficiency
III
415
18/M
19/M
1
. siblings.
* Carbazole
(Dische).
214.6
3/6
17/M
I
20
40.5
12.86
Mean
S.D.
35
36
37
62
15/M
1.5/11
47
34
3/14
218
14/M
>
1:6
13/M
52
16/M
l/11
12/M
acid*
(mg/24 h)
onset/
ON
urinary
duration
Total
uranic
in
years
DATA
Age
CLINICAL
Sex
AND
CCiX/
LABORATORY
TABLE
99
690
131
92
175
126
75
125
S.D.
Mean
creatinine)
(mzk
GAG
8 PATIENTS
0.12
0.9
0.74
0.83
1.03
1.10
0.82
0.85
0.90
0.92
Orcinol
Carbazole ____
WITH
35
51
65
40
50
68
45
Gal
Glu
: 55 : 32 : 50 : 60 : 35 : 49 : 65
NH2
NH:!
None NOtX
++
None
None
None
+
None
None
+
++
+
rlrone
++
~._~
megaly
SplWlO-
Hepato-
++
I1
None
N0Ile
opacity
C0rlleal
TYPE
++
+++
+++
++
+++
++
+++
dation
retar-
Mental
MUCOPOLYSACCHARIDOSIS
_..
Skeletal
+ + None None +
+ + +
+
+ +
+
+
++ +
vascular
Cardio-
++
changes
Gel
II
II
II
I1
II
II
II
II
type
pattern
filtration
TABLE
>
1
} , siblings.
* Carbazole
35/F
34/F
33/F
32iM
31/F
30/M
1
?/8
?I6
(Dische).
S.D.
16
7.7
24.0
3.4?
20
22
1110
l/l0
26
Mean
18.5
S.D.
36
50.5
Mean
50
47
78
72
40
Random
23
36 Random
58
4115
6111
2/9.6
29/F
IIIB
-
28/M
Type
6/15
1.516
4113
-
24/M
25;M
21/F
4.517
23/F
26/M
219
? 113 ?/15
21,‘M 22/F
IIIA
20/F
Type
ON
S.D.
Mean
S.D.
Mean
sample
sample
~-----.~.~__
75
78
74
82
43
87
200
127
77
104
urine 89
NOW
101 urine
80
creatinine)
24 h)
-
(mg/Iz
(m!Zl
current
onset/
acid*
GAG
urinary
Total
uranic
in
DATA
Age
LABORATORY
years
AND
Sex
IV
Casef
CLINICAL
0.15
1.61
1.52
1.58
1.68
1.88
1.46
1.55
0.18
2.02
2.3
1.86
1.82
1.93
2.33 1.92
1.90
1.93 1.94
2.22
Orcinol
Carbazole ~
16 PATIENTS
84:
94:
89:
89:
so
97:
98:
so
95:
91:
86 -
85:
96: -
88:
Gal
Glu -
3
2
10
5
9
14
16
6
11
11
: 10
:
:
15
4
12
NH2
NH2
WITH
++
++
+
++
++
++
++
-
+‘I+
+++ +++ ++ +++ ++
d&ion
retar-
Mental
None
None
None None None ?
None
None -
None
None
?
?
NOW2
++
2
t
+
+ -
+
+
+ -
c -
None -
+
None -_
megaly
spleno-
Hepato
None
opacity
Corneal
MUCOPOLYSACCHARIDOSIS
IIIA
+
l
NOXE
+ + i
+
+ + + _
+
+ +
changes
Skeletal
TYPE
B
_____~_
+
+ -
F +
-
+
+ -
+
vascular
Cardio-
AND
Gel
IIIB
IIIB
IIXB
111s
IIIB
IIIB
IIIA
IIIA
IIIA
IIIA
IIIA
IIIA
IIIA
IIIA
IIIA
IIIA
type
pattern
filtration
defect
defect
defect
defect
defect
confirmed
confinned
confirmed
confirmed
confirmed
.,,....-. _____.-.- _-.__ _. __-_
Enzymatic
Enzymatic
Enzymatic
Enzymatic
Enzymatic
Comment
V
B
VI
46/M
?
4/14
N.D.
(Dische).
51
18
= non-detectable.
} , siblings.
**
* Carbazole
Unclassified
45/F
Type
22
44
10.4
7/16
44/M
29.6
2113
43/M
29
23
S.D.
3/12
Mean
6/21
18.4
42/F
37.4
S.D.
16
-
60
42
32
29
65
68.0
19.7
61
76
36
-
106
-
0.69
0.05
0.73
0.44
0.53
S.D.
0.60
0.52
0.45
0.56
Mean
S.D.
0.66
0.62
0.76
0.74
Orcinol
Carbazole
9 PATIENTS
0.07
ON
Mean
creatinine)
24 h)
GAG
(mg/g
acid*
DATA
(mg/
uranic
urinary
CLINICAL
Total
AND
Mean
8/10
41/M
Group
40/M
7135
39/M
1
10121
5/39
38/F
5131
1
37/M
A
current
onset/
36/F
Group
in
Y‘2?3l3
Age
LABORATORY
TABLE
: 55
: 59
: 70 : 68
NH2
NH2
5:
95
5 : 95
1 : 99
8 : 92
5 : 95
45
41
32
30
Gal
Glu
WITH
NOlIe
_
Non.2
NOIW
NOlltZ
NOIll
NOtlIZ
NCJIle
NIXI‘
NOW
NOM?
dation
retar-
Mental
+ ++ ++ ?
+++ +++ +++ +++ ++
opacity
C0rlleal
MUCOPOLYSACCHARIDOSIS
++
NOIX?
+ + +
++
++
+ + ++ ++
++
++
++
+
changes
+ +++
TYPE
Skeletal
1 WITH
+
V,
++
++
mega1y
spleno-
Hepato-
TYPE
VI,
VB
+
++
VB
NotIe
I?
VI
VB
VB
VA
VA
VA
VA
VA
type
pattern
filtration
Gel
1 UNCLASSIFIED
+++ +
?
+++
+++
+++
+++
Vascular
Cardio-
AND
-
-
NOlTld -
Normal
ND
ND
ND
ND*”
activity
nidase
a-L-Iduro-
330
The common clinical findings of the patients with mucopolysaccharidosis Type III (Sanfilippo) include: later onset than in Types I and II, pronounced mental retardation, absence of corneal opacity, mild joint deformities and slight enlargement of spleen and liver. As shown in Table IV, 16 patients belong to this group and those identified as Cases 30 and 31, 32 and 33, and 34 and 35 were three sets of siblings. The demonstration of two “‘corrective factors” [23] for the fibroblasts from different patients of this type led to eventual separation into two subgroups: Type IIIA deficient in a heparan sulfate sulfatase (heparin sulfamidase) [ 6] and Type IIIB with ~~-acetyl-~-glucos~inid~e deficiency [ 51, but without evident clinical distinguishing features between them. Mean urinary GAG-uranic acid was 50.5 and 24.0 mg per 24 hours for Types IIIA and IIIB, respectively. Mean carbazole/orcinol ratio was 2.02 t 0.18 in the first and 1.61 + 0.15 in the latter. In both types, glucosamine cornprized more than 85% of the total hexosamine and enzymatic analysis of the GAG (Table VI) showed predominance of HS. However, gel filtration patterns were different in each type (Fig. 1). Nine patients with age range between 10 and 39 years when last examined had mucopolysacchariduria and pronounced systemic manifestations but normal mentality (Table V). On the clinical grounds, these patients were considered to fall into Type V mucopolysacch~idosis (Scheie). Except for a generally milder course of the disease in a proportion of these nine patients (Cases 41--44), the involvement of tissues and organs were similar in all. Cases 36 and 37 (siblings) were the original Scheie patients [24] . Cases 38 and 39 are our new patients and they are also siblings. Elution diagrams were constructed for all nine patients. In general, the elution patterns fell into two different categories (Fig. 2), suggesting that we may be dealing with two different groups of patients and tentatively we have designated them as subgroups VA and VB. The mean carbazole/orcinol ratio in group VA was 0.69 ?r 0.07 while in group VB it was 0.53 + 0.05. In group A the ratio of glucosamine/ galactosamine was about 40 : 60 while in the latter it was about 5 : 95. Urinary GAG composition in group VA was: CSA + CSC, DS and HS with a ratio 10 : 45 : 45 and in group VB CSA + CSC, DS and HS 15 : 80 : <5. Patients identified as Cases 36-39 (no material was available in Case 40) had a deficiency of cr-L-iduronidase in their fibroblasts. On the other hand, leukocytes and fibroblasts from patients, Cases 41 and 43 (no material available in Cases 42 and 44) had normal a-L-iduronidase activity (G. Constantopoulos and AS. Dekaban, unpublished observations). One urine was supplied with the information that it belonged to a patient with mucopolysaccharidosis Type VI. As shown in Table V (Case 45), this female patient excreted 18 mg GAG (as uranic acid) per 24 hours. The carbazole/orcinol ratio was 0.44, indicating DS as the major GAG. It was found by enzymatic analysis that 80% of the total GAG are comprized of DS and 20% of CSA + CSC. Only a trace of HS was present. The elution pattern obtained from the urinary GAG of this patient had some resemblance to that of group VB. Finally, patient Case 46, exhibited an elution pattern Type I. This patient had been seen by one of us and at the time we formed the opinion that he was not typical for any known form of mucopolysaccharidosis, but the patient was not available for further study.
331
Composition of urinary GAG Early in the investigation, the urinary GAG from several patients and normal controls were fractiona~d on a Dowex l-X2 column (chloride) by stepwise elution with increasing concentrations of NaCl and the fractions were analyzed [ 191. This method separates the individual GAG but with some overlap. Therefore, the composition of the urinary GAG was estimated from their susceptibility to the Chyases AC and ABC (Table VI). The third column shows uranic acid in the disaccharides released after incubation of the GAG with Chyase AC. From these data it was calculated that CSA + CSC content was within normal range in all types except Types VI3 and VI (Cases 41, 43 and 45). Subsequently, the GAG which were not susceptible to Chyase AC, were incubated with Chyase ABC. The fourth column shows the uranic acid in the disaccharides released, presumably from DS. It appears that DS content is within normal range in Type IIIA, slightly increased in Type IIIB and comprizes about 50% of the total GAG in Types II and VA, 60% in Type I and about 80% in Types VB and VI. The GAG non-susceptible to Chyases AC and ABC represent HS. As shown in the last column, HS comprizes about one-third of the total GAG in Type I, 50% in Types II and VA and over 85% in Types IIIA and IIIB. It was a minor component of the urinary GAG from patients with Type VB and VI. Composition of the urinary GAG was also estimated in all patients from the elution diagrams obtained for the molecular weight distribution by gel filtration from Sephadex G-200 (Fig. 1 and 2). The results were in general agreement with those obtained by enzymatic analysis and have been summarized in Table I. TABLE
VI
COMPOSITION Figures borate
OF
presented method
THE as
URINARY
uonic
acid
GAG %
of
IN MUCOPOLYSACCHARIDOSES
total
urinary
GAG.
Analysis
for
uranic
acid
with
the
carbazole/
[ 181.
-. Case
Mucopoly-
CSA
NO.
saccharidosis
disaccharides
disaccharides
GAG
type
released
released
by
susceptible
Chyase
ABC
DS
+ CSC
i Chyase
by AC
I
after
HS
Chyase
non-
to Chyases AC
AC
and ABC
--. 4
I
7.0
8
--60.0
33.0 31.0
I
8.6
59.0
12
II
2.2
42.0
56.0
18
II
6.0
34.0
61.0
46.0
47.0
19
II
7.0
21
IIIA
7.1
3.9
89.0
27
IIIA
4.0
2.0
94.0
29
IIIA
4.5
1.5
94.0
30
III3
10.0
6.0
84.0
32
IIIB
5.0
4.0
91.0
36
VA
9.0
46.0
41
VB
14.0
18.0
8.0
43
VB
16.0
84.0
TrWX
45
VI
20.0
80.0
Trace
--
45.0
332
Figs 1 and 2 show the molecular weight elution patterns and chemicai reactivities for urinary GAG from the various types of mucopolysaccharidosis and normals, using gel filtration on Sephadex G-200. In the elution diagrams the ordinate represents uranic acid and the abscissa the log of the molecular weight of the GAG. The broken line represents uranic acid measured with the orcinol method and the solid line, uranic acid on the basis of carbazole (Dische) reaction. The carbazole/orcinol ratio is characteristic of each GAG (2.0 for heparan sulfate, 0.4 for dermatan sulfate and about 1.25 for chondroitin sulfates A and C). These data allow an estimation of the composition of the GAG. The 8 panels are representative of the elution patterns from normal
I-
L EyJATE Molecular
wetght
2 X’d
VOLUb$E lb4
(ml)
t 5x1103
Fig. 1. Sephadex G-200 gel filtration elution diagrams for urinary glycosaminoglycans. Samples are from top to bottom: normal controls. mucopolysaccharidosi Type I, Type II, Type IIIA and Type IIIB. The method, described previously 1141, employed a glas column with packed gel 1.06 cm i.d and 86 cm high, which was equilibrated at room temperature with the elution medium 0.025 M N&I. A sofution of GAG about 300 Pg (as uranic acid) in 0.2 ml, was applied and 2.0 ml fractions were collected and were analyzed for uranic acid. Carbazole 1161 and orcinol 1171 methods.
333 3.0
2.0
I.0
B 2.0: r? 0 I.0
Fi G =: z P
TYPE
P
‘7
2.0
$J i 0
I.0 E
2.0
I .o
0 V, 126.5
Molecular
wght
40 ELFATE
50 VOLUM;
2 X IO4
IO4
60
70
(ml) t 5x103
Fig. 2. Sephadex G-200 gel filtration elution diagrams for urinary glycosaminoglycans. Samples are from top to bottom: normal controls, mucopolysaccharidosis Type VA (Scheie). Type V (Group B), and Type VI. Conditions as in Fig. 1.
controls (top panel), six types of mucopolysaccharidosis and one group of patients who may belong to a new type, VB. There were individual variations, but provided that the column was not overloaded, the patterns were distinct. Normal
controls
and patients
with diseases other
than mucopolysacchari-
The curve of the elution diagrams of the urinary GAG from normal controls and patients without mucopolysaccharidosis (Figs 1 and 2, top panels) has a peak corresponding to a molecular weight of about 20 000 and an average molecular weight of about 14 000. The C/O ratio was about 1.0. Mucopolysaccharidosis Type I. The elution diagram of this type is bimodal (Fig. 1, second panel from the top). The peak at left is about 40% of the total GAG, is consisted of DS (C/O about 0.4) and has an average molecular weight about 18 000. The larger hump at right has an average molecular weight of about 6000 and consists of both DS and HS. The C/O ratio is increasing to a maximum of 1.10 to 1.20. Mucopolysaccharidosis Type II. This type of elution diagram (Fig. 1,3rd panel from the top) is also bimodal. The hump at left is smaller and contains mainly DS while the one at right contains both DS and HS with small molecular weight and has a C/O ratio with a maximum of 1.30-1.40, reflecting the higher HS content. dosis.
333
~~ucopo~ysaccharidosis Type IIIA. Type IIIA elution diagrams (Fig. 1,4th panel from the top) are consisted of one almost symmetrical hump. The GAG is practically pure HS with a Cl0 ratio up to 3.0 or even higher, and an average molecular weight of about 5000. There is only a small tailing towards the larger molecular weights. Mucopolysaccharidosis Type IIIl3. This curve (Fig. 1, bottom panel) is similar to the Type IIIA diagram with the exception that the C/O ratio is much smaller, only slightly above 2.0. Mucopolysaccharidosis Type VA. Five patients with Type VA (Seheie’s exhibited the same elution pattern shown in Fig. 2 (2nd panel from the top). This curve is not bimodal (as in Type I) and shows considerable shift towards smaller molecular weights. More than 80% of the GAG, consisting of DS and HS, are highly degraded. Average molecular weight is about 8000 and maximum C/O ratio about 1.0. Mucopolysaccharidosis Type VB. The other 4 patients with the diagnosis of mucopolysaccharidosis Type V, exhibited the elution pattern shown in Fig. 2, 3rd panel from the top. More than 80% of the GAG is pure DS of high molecular weight, average >20 000. The C/O ratio is about 0.53 with a maximum of 1.0 in a minor area at the extreme right of the curve. Mucopolysaccharidosis Type VI. The patient designated as mucopolysaccharidosis Type VI, displayed the elution diagram shown in Fig. 2, bottom panel. This curve is bimodal, with almost equal distribution of the GAG in the peaks corresponding to 20 000 and about 5000 molecular weight. A carbazolej orcinol ratio = 0.44 indicates high DS content and a small amount of HS towards the extreme right of the diagram. Uranic acid-containing
Eow molecular
weight compounds
In addition to the GAG, human urine contains large amounts of uranic acid-containing compounds which are not isolated by CPC precipitation or with Ecteola (Cl) column. DiFerrante and co-workers [25] reported that some of this material might be degradation products of the GAG. We measured total urinary uranic acid, polymeric GAG-uronie acid and “degradation products” in 6 patients and we have confirmed their observations. Total urinary uranic acid content ranged from 241 to 760 mg per 24 hours. GAGuronic acid content was from 25 to 88 mg per 24 hours and “de~adation products”-uranic acid 8 to 86 mg per 24 hours. Work is in progress for the chemical ch~ac~r~ation of these uranic acid and hexosamine containing small molecules. Discussion Several investigators have examined the urinary GAG from patients with mucopolysaccharidosis and have attempted to correlate their findings with clinical data. Terry and Linker [ 261 were able to distinguish four forms of the disease on the basis of their urinary GAG excretion pattern. Manley and WiIliams 1273 studied 28 patients with various types of mucopolysaccharidosis and concluded that acetate electrophoresis of the urinary GAG could clearly differentiate only 2 types, the predomin~tly heparan sulfate excretors and the “mixed excretors”. Kaplan 1281 noted four discrete patterns of mucopolysac-
335
chariduria in 46 patients and he found definite clinical correlations. Finally, Gordon and Haust [29] in 23 patients with mucopolysaccharidosis Types I, II and III and Spranger [30] in 51 with all types, independently used chromatography of the urinary GAG on Dowex 1 and found distinct patterns corresponding to the various types of mucopolysaccharidosis as defined with clinical criteria by McKusick et al. [12]. These studies provided additional diagnostic criteria, set the stage for the elucidation of the specific enzyme defect, and provided an abundance of observations which now, after the identification of the enzymatic deficiency, begin to be understood. A shortcoming of all these investigations was the lack of knowledge of the enzymatic deficiency in their patients which necessitated the correlation of their findings with clinical criteria only. The present study confirms and extends many of the findings from the earlier reports, introduces new methodology for the classification of these diseases on the basis of elution diagrams obtained by gel filtration from Sephadex G-200 and provides new information on the structure of the urinary GAG from patients with mucopolysaccharidoses. Moreover the enzymatic defect in a large number of patients has been identified, thus making this work more definitive. In agreement with the results of others, all our patients excreted in their urine excessive amounts of DS, HS or both. We found that patients identified as Cases 41 and 43 (Type VB) and 45 (Type VI), in addition, excreted excessive amounts of CSA + CSC. Reports of mucopolysaccharidosis without mucopolysacchariduria must be viewed with care until a defect in the metabolism of the GAG has been identified as the primary genetic disorder in such patients. The cited chemical data (Tables V and VI) would tend to suggest that Type VB patients could belong to Type VI mucopolysaccharidosis. However, their clinical manifestations differ; patients as described by Marateaux-Lamy (Type VI) were dwarfed and had severe hepatosplenomegaly and skeletal deformities, whereas the patients VB (Cases 41-44) are of normal height and their skeletal and organ involvement is much milder. According to a recent report [ 311, deficiency of sulfatase B was found in patients with Type VI mucopolysaccharidosis. Further work in this area is in progress in our laboratory. Urinary GAG from patients with the same type of the disease tend to have the same carbazole/orcinol ratio and also the same glucosamine/galactosamine ratio and GAG composition. This has been pointed out earlier by Kaplan [28] . From Figs 1 and 2 it is clear that the molecular weight distribution of the urinary GAG is characteristic for each different type of mucopolysaccharidosis and is distinguished from normal controls. Practically all HS and part of the DS in all types of mucopolysaccharidosis have been highly degraded. Knecht et al. [32] initially reported that HS from the urine and the liver of Hurler patients was fragmented. Subsequently, we have confirmed this observation in three types of mucopolysaccharidosis and, in addition, we found that a part of the DS was also highly degraded [13,14]. Here, we found that at least a sizeable portion of the HS and/or DS from all the patients with mucopolysaccharidosis was highly fragmented. None of the normal controls and the patients without mucopolysaccharidosis exhibited this shift in the molecular weight distribution of HS and DS towards the smaller molecular sizes. Regarding the mechanism of
336
the fragmentation of the GAG, one only may speculate that it is the result of the action of non-specific GAG endohydrolases in the tissues. The high N-sulfate content of HS from type IRA patients, which we have reported elsewhere [ 331, may be attributed to their deficiency in N-sulfatase [ 4,6] . The elution diagrams were correlated not only with clinical data but in all patients in which the enzymatic deficiency has been established, with the enzymatic defect as well. Thus, the elution diagrams have been used successfully in this study and may be used by others for the diagnosis of the type of the mucopolysaccharidosis. It is remarkable that urine excretion products are preserving their chemical characteristics for the same patient or type of the disease and for rather a long time, if not for life. This should not be surp~sing because they are the “substrates” for the deficient enzyme(s) and have been altered, only to the extent the metabolic capacity of the organism permits. Needless to say, the definitive understanding of a genetic disorder is the identification of the specific enzyme or protein defect and this must be the final criterion for the establishment of the diagnosis of the type of mucopolysaccharidosis in a patient. On the other hand, the knowledge of the chemistry and fine structure of the partially degraded “substrates” will contribute necessary information for understanding the development and pathogenesis of these diseases. References 1 A. Dorfman 2 3 4 5 6 7 8 3 10 11 12 13 14 15 16 17 18 19 20 21 22 23
and R. Matalon, in J.B. Stanbury, J.B. Wyngaarden and D.S. Fredrickson feds). The Metabolic Basis of Inherited Disease. McGraw-Hill, New York, N.Y., 1372, p. 1218 R. Mat&m and A. Dorfman, Biochem. Biophys. Res. Commun.. 47 (1972) 959. G. Bach, R. Friedman. B. Weissmann and E.F. Neufdd, F’roc. N&l. Acad. Sci. U.S.A., 69 (1372) 2048. H. Kresse and E.F. Neufeld, J. Biol. Chem., 247 (1972) 2164. J.S. O’Brien, F’roc. Natl. Acad. Sci. U.S.A., 69 (1972) 1720. H. Krease, Biochem. Biophys. Res. Common., 54 (1973) 1111. G.V. Coppa. J. Singh. B.L. Nichols and N. DiFerrante. Anal. L&t.. 6 (1973) 225. G. Bach, F. Eisenberg, Jr, M. Cantz and E.F. Neufeld. Proc. Natl. Acad. Sci. U.S.A.. 70 (1973) 2134. C.W. Hall, M. Cantz and E.N. Neufeld, Arch. Biochem. Biophys., 155 (1973) 32. A. Dorfmanand A.E. Lorincz, Proc. Natl. Acad. Sci. U.S.A., 43 (1957) 443. K. Meyer, M.M. Grumbach, A. Linker and P. Hoffman, Proc. Sot. Exp. Biol. Med., 97 (1958) 275. V.A. McKusick, D. Kaplan. D. Wise, W.B. Hanley, S.B. Suddarth, M.E. Sevick and A.E. Maumenee. Medicine, 44 (1965) 445. G. Constantopoulos, Nature, 220 (1368) 583. G. Constantopoulos, AS. Dekaban and W.R. Carroll, Anal. Biochm., 31 (1969) 59. N.M. DiFerrante, Anal. Biochem., 21 (1967) 38. Z. Dische, J. Biol. Chem., 167 (1947) 189. H.A. Brown, Arch. Biochem., 11 (1946) 263, T. Bitter and H.M. Muir, Anal. Biochem., 4 (1362) 330. S. Schiller, G.A. Slaver and A. Dorfman, J. Biol. Chem., 236 (1961) 983. II. Saito, T. Yamagata and S. Suzuki, J. Biol. Chem., 243 (1968) 1536. F. Haruki and J.E. Kirk, Biochim. Biophys. Acta. 136 (1967) 391. J.D. Hocking. R.D. Jolly and R.D. Batt, Biochem. J., 128 (1972) 69,
H. mess% u. Weismann, M. Cant.? C.W. Hall and E.F. Neufeld, Biochem. Biophys. Res. Commun (1971) 892.
24 G. ConstantoPoulos, A.& Dekaban and H.G. Scheie, Am. J. Ophthalmol., 72 (1971) 25 N. DiFerrante. G. Neri. M.E. Neri and W.E. Hogsett, ifI, CONI. Tissue yes., 1 (1972) 26 K. Tenty and A. Linker, Proc. Sot. Exp. Biol. Med., 115 (1964) 394. 27 G. Manley and U. Williams. J. Clinin. Pathol., 22 (1369) 67. 28 D. Kaplan, Am. J. Med., 47 (1969) 721. 29 B.A. Gordon and D.M. Haust, Clin. Biochem., 3 (1970) 203. 30 J.W. Stranger, 2 Kinderheilk, 108 (1970) 17.
,
42
1106. 93.
31 D.A. 8tumPf. J.H. Austin. A.C. Cracker and M. LaFrance, Am. J. Dis. Child., 126 (1973) 747. 32 J. Knecht, A. Cifonelli and A. Dorfman, J. Biol. Chem.. 240 (1965) 4140. 33 A.L. Stone. G. Constantopoulos. S.M. Sotsky and A.S. Dekaban, Biochim. Biophys. Acta, 222 (1970) 79.
Clinica Chimica Acta, 59 (197 5) 321-336 @ Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 6946
CHEMICAL DEFINITION
GEORGE
CONSTANTOPOULOS
Developmental arid Metabolic Bethesda, Md. 20014 (U.S.A.)
(Received
October
OF THE MUCOPOLYSACCHARIDOSES
and ANATOLE Neurology
Branch,
S. DEKABAN NINDS,
National
Institutes
of Health,
23, 1974)
Summary Glycosaminoglycans were isolated from the urines of 46 patients with mucopolysaccharidosis; 11 with Type I (Hurler), 8 with Type II (Hunter), 16 with Type III (Sanfilippo A and B), 9 with Type V (Scheie), one with Type VI (Marateaux-Lamy), and one unclassified. All 46 patients excreted in their urine excessive amounts of dermatan sulfate, heparan sulfate or both. In addition, patients of certain types excreted excessive amounts of chondroitin sulfates A and/or C. There is a trend in each type of the disease towards the same carbazole/orcinol ratio, glucosamine/galactosamine ratio and glycosaminoglycan composition. Molecular weight distribution of the urinary glycosaminoglycans by gel filtration from Sephadex G-ZOO is characteristic for each different type of mucopolysaccharidosis and is distinguished from n’ormal controls and patients without mucopolysaccharidosis. Preparation of elution diagrams from Sephadex G-200 allows an estimation of the composition of the glycosaminoglycans. Practically all heparan sulfate and a sizable part of dermatan sulfate from the urinary glycosaminoglycans of all these patients have been highly degraded. In all the patients in which the specific enzyme defect was demonstrated, the assignment of the type of mucopolysaccharidosis, on the basis of the elution diagrams, was correct. Patients with mucopolysaccharidosis Type V displayed two conspicuously different types of elution patterns, suggesting heterogeneity. Indeed, only a portion of these patients showed a-L-iduronidase deficiency. Carriers had normal urinary glycosaminoglycan output and composition and exhibited normal elution diagrams.
Introduction Mucopolysaccharidoses are inborn errors of the glycosaminoglycan (GAG) or acid mucopolysaccharide metabolism [l]. Recent studies have defined the defects as the deficiency of specific degradative enzymes [2-91 . Abbreviations: AC, chondroitinase-AC;
CPC. cetylpyridinium chloride; CSA and CSC. chondroitin sulfates A and C; Chyase DS, dermatan sulfate; HS, heparan sulfate; GAG, glycosaminoglycan.
322
Excessive excretion of GAG in the urine of the patients (mucopolysacchariduria), is the common feature of the mucopolysaccharidoses and it is utilized as a diagnostic test for these diseases [lO,ll] .In preliminary studies, we have shown that Sephadex G-200 gel filtration of the urinary GAG from patients with mucopolysaccharidoses Types I, II and III (classification of McKusick [12] ) gave elution profiles which are characteristic for each type; moreover, the distribution of molecular weights of the GAG indicated that they represent fragments of the parent molecules [ 13,141. Subsequently, we have extended our work to 46 patients who represent 6 or ‘7 different types of mucopolysaccharidoses. The results confirm and expand the earlier observations and demonstrate the usefulness of this technique in the diagnosis, classification and detection of new types of mucopolysaccharidosis. In addition, elution profiles aid elucidation of the pathogenesis of these disorders. The validity of the method has been confirmed by the identification of the specific enzyme deficiency in a number of the patients investigated. Material
and Methods
During the past eight years, urines from over 150 patients suspected of having mucopolysaccharidosis were examined for GAG; more than one-half of the urines were from patients of our clinical service; the other urines were submitted to us by outside physicians or investigators. Forty-six patients were proven to have one or another type of mucopolysaccharidosis. Thirty-six of these patients were studied by us as inpatients, outpatients or by consultations in the area hospitals. Clinical data and urines of two patients (Cases 32,33) were supplied by Dr H. Moser of Boston, of one patient (Case 45) by Dr A. Cracker of Boston and urines of the remaining seven patients (Cases 1,2,7,11, 22, 24, 28) were submitted to this laboratory by Dr E.F. Neufeld of NIH, for chemical analysis. Subsequently, Dr Neufeld supplied us with confirmatory analysis of the enzymatic defect in these patients. Twenty-four hour urine specimens were kept at 0-4°C without preservatives and if not analyzed within the first two days, stored frozen at -20°C until needed. Isolation of GAG and analytical procedures Glycosaminoglycans were isolated from undialyzed urines by chromatography on Ecteola columns or by precipitation with cetylpyridinium chloride (CPC) [15]. Urines with 24-hour volume <500 ml were diluted to 500 ml with distilled water, prior to isolation. The isolated GAG were routinely analyzed for uranic acid content by the carbazole (Dische) [16] and orcinol [17] methods, which permit calculation of the carbazole/orcinol ratio and by the carbazole (borate) [18] modification which improves the color yield for dermatan sulfate (DS) and gives results closer to the theoretical values for all GAG. The other methods we used for the chemical analysis of the GAG, (total hexosamine, glucosamine/galactosamine ratio, sulfate and N-sulfate content) and the determination of their molecular weight distribution and construction of the elution diagrams, were previously described [14] .
323
Estimation
of GAG composition
GAG from the various types of mucopolysaccharidoses were subjected to fractionation on a Dowex l-X2 column by step-wise elution with increasing concentrations of NaCl, and the individual fractions were analyzed [19] . Susceptibility of the GAG to enzymatic depolymerization with chondroitinase-AC (Chyase AC) and chondroitinase-ABC (Chyase ABC) was examined by measuring the uranic acid content of the released unsaturated disaccharides [20] . The following micro-method was developed for the measurement of the released disaccharides. Two sets of reaction mixtures and a control were prepared in glass tubes 7 mm (i.d.) X 75 mm. The reaction mixture contained 20 ~1 enriched Tris buffer (pH 8.0), test sample containing about 25-50 pg uranic acid, 0.02 units Chyase AC in one set, and 0.1 unit Chyase ABC in the other set; the total volumes were brought up to 100 ~1. The control set lacked the enzyme. The time of incubation was 30 minutes at 37°C for incubation mixtures with Chyase AC and 4 hours at 37°C for mixtures with Chyase ABC. Enzymatic reactions were stopped by boiling for 1 minute, the reaction mixtures were cooled to 0°C and 50 ~1 of 3.0 M NaCl and 0.7 ml of 95% alcohol were added to each tube. The contents were mixed and left at 0--4°C for at least 1 hour. After centrifugation for 15 minutes at 10 000 X g the supernatants containing the released disaccharides were quantitatively removed, evaporated to dryness and analyzed for uranic acid with the carbazole (borate) method. A small amount from each sample was used for electrophoresis [21] and respective patterns before and after enzymatic incubations were compared. Finally, samples were subjected to gel filtrations before and after enzymatic incubations.
Enzyme
assays
a-L-Iduronidase in leukocytes, fibroblasts and tissues of some of the patients was measured with phenyl-cu-L-iduronide as the substrate. Other lysosomal enzymes were measured with the appropriate p-nitrophenyl glycosides [ 221. Phenyl-cu- L-iduronide was a gift of Dr B. Weissmann of the University of Illinois College of Medicine, Chicago, Illinois. Several p-nitrophenyl glycosides were purchased from Sigma and Research Products International Corp. The enzymes Chyase AC and ABC were purchased from Miles Laboratories. GAG standards were kindly supplied by Drs M.B. Mathews, J.A. Cifonelli and L. Roden, University of Chicago. Sephadex G-200 was purchased from Pharmacia Fine Chemicals, Inc. and Ecteola cellulose (ET-11 powder) from Reeve Angel and Co (Clifton, N. J. ). Results Table I summarizes the distribution of patients in various types of mucopolysaccharidosis and gives the output and composition of urinary GAG. From clinical, chemical and enzymatic studies, it was concluded that eleven patients had mucopolysaccharidosis Type I, eight Type II, sixteen Type III (10 were of subtype A and 6 of subtype B), nine Type V (which were further separable into two groups A and B), one Type VI, and one patient whom we were unable to recall for additional studies remained unclassified.
I
AND
Sulfoiduronate
N-Sulf&ase
III (Sanfilippo)
diseases
controls
Mean
+ standard
Type
deviation.
measured
* * * Mucopolysaccharidosis
**
acid
mucopolysaccharidosis
with
* As uranic
Normal
than
Patients
IV
(Morquio)
was not
number
(Dische)
[ 3 11
[3]
the carhazole
B?
In parentheses
with
Arylsulfatase
VI
(Marateaux-Lamy)
a-L-Iduronidasc
V (Scheie) ***
Unclassified
[4,61
sulfatase
[8]
method.
151
investigated.
of
11
115
1
1
9
16
8
11
patients
No.
GLYCOSAMINOGLYCANS
of experiments
URINARY
12.31
OF
N-Acetyl~-glucosainidase
a-L-Iduronidase
enzyme
II (Hunter)
Deficient
COMPOSITION
I (Hurler)
type
other
ESTIMATED
Mucopolysaccharidosis
OUTPUT
TABLE
5 4
B=
6
A=
B=
A = 10
4.3
4.3
18.0 51.0
29.6
37.4
24.0
50.5
40.5
43.0
(mg/24
7.7
18.5
+
+
1.3
2.8
t 10.4
+ 18.4
i
i
i 12.9
4)
4)
5)
8)
8)
7)**
(
11)
(115)
(
(
(
(
(
(
GAG* h)
k 14.1
Urinary
1.04
1.07
0.44 0.73
0.53
0.69
1.6
2.0
0.90
0.66
+ 0.007
i 0.12
+ 0.06
2 0.07
k 0.15
+ 0.18
+ 0.12
? 0.06
Orcinol
Carbazole
4)
4)
6)
(11)
(43)
(
(
(
(10)
8)
(ll)*‘ (
Estimated
:
<
_
80: _
82:
55
5
46:
60:
:
5:
<
2:
10:
<3:
35
85
20
16
10
:
9
10
:
:
: 10
45:
30
+ CSC)
HS
: 85
(CSA
DS
composition
325
All patients excreted in their urines from 5 to 15 times more GAG than the normal controls or the patients with other diseases. There were not large variations in excretion from day to day {within a few weeks’ time) and the composition of the GAG was consistent in each type of the disease and in the same patient during the several years he was followed-up. Patients with Type I mucopolysaccharidosis (Hurler Syndrome) excreted excessive amounts of DS and heparan sulfate (HS) in their urine in an approximate ratio of 2 : 1. Type II patients (Hunter) also excreted excessive amounts of DS and HS but in a ratio close to 1 : 1. Both Types IIIA and B patients (S~filippo~ excreted large amounts of HS in their urine. It appears that Type V patients (Scheie) could also be separated into two groups: patients in group A excreted excessive amounts of DS and HS in a ratio 55 : 35 while those in group B excreted excessive amounts of DS and chondroitin sulfates A and C (CSA + CSC) in a ratio of 82 : 16. The urinary GAG of the Marateaux-Lamy patient were also composed of DS and CSA + CSC in a ratio of 80 : 20. The ~~b~ole~orcinol ratio, which depends on the GAG composition, was characteristic of each type but with some overlap between Types I and VA, between Type II and normal controls, and between Types VB and VI. The mean 24-hour urinary GAG output, in 115 patients with diseases other than mucopolysaccha~dosis, was 4.3 +_2.8 S.D. (as uranic acid), about the same as 11 normal controls, but with a wider range (l-16 mg per 24 hours). However, DS of HS were not major components of the urinary GAG in any of these patients. The urinary GAG excretion and elution diagrams in a few carriers examined was normal. Table II shows laboratory and clinical data on 11 patients with mucopolysaecharidosis Type I. Patients identified as Cases 1 and 2, 3 and 4, and 5 and 6 were three sets of siblings. The mean GAG-uranic acid excretion in the urine was 43 mg per 24 hours and galactosamine comprized more than two-thirds of the total hexosamine. From the carbazolelorcinol ratio (0.66 ? 0.06), glucosamine/galactosamine ratio, elution pattern and enzymatic analysis [Table VI), it was concluded that the predomin~t GAG were DS and HS at about a ratio of 2 : 1. Clinically, most of the patients, on whom information was available, were in an advanced state of the disease and had mental retardation, corneal opacity, hepatosplenomegaly and skeletal and cardiovascular changes. The urinary GAG from all the 11 patients gave a similar elution pattern by gel filtration on Sephadex G-200 (Fig. 1). Finally, deficiency of cu-L-iduronidase was found in the fibroblasts or tissues available from 8 patients. The laboratory and clinical data from 8 patients with mucopolysaccharidosis Type II are shown in Table III. Patients identified as Cases 14 and 15, and 16, 17 and 18 are two sets of siblings. The mean urinary GAG-uranic acid and carbazole/orcinol ratio were 40.5 mg per 24 hours and 0.9 t 0.12 SD., respectively. These patients excrete in their urine excessive amounts of DS and HS in an approximate ratio of 1 : 1. Mental retardation, organomegaly, cardiovascular and skeletal abnormalities were present in patients, although in a milder degree than in Type I, but no patient had corneal opacity. The elution pattern of the urinary GAG by gel filtration on Sephadex G-200 was characteristic of this type of the disease (Fig. 1).
II
2/2.5
1112 l/l1 l/6
2/F
3/M 4/M
IiS 3/7 114 ?/18
l/l ?/1.5
i( *
*
_----~~--
*
i&fear? 43.00 S.D. 14.15
26 56
35 -
SamPle”
53 63 37 31 15 ml random
samPle*
15 ml random
sample
35 ml random
_~_
248 -
240 131 181
-
-
---.. --_.-. -
(me/g creatioine)
0.66 0.06
0.60 0.65 0.10 0.65
0.55 0.58 0.65 0.65 0.66
0.71
0.76
---
73
++ ++ ++ -
+*+ +++ +++ +++
-
.-
-__..-
Mental retaxd&ion --c
.-__ .__ --..-. .___~_____~_
-
27: -
15: 85 12: 88 23 : 77 26 : 74 -
-
--
Gal NHZ
~..
ii-* ++ 5 -
+++ *++ +t Cf -
-
-
Corneai opacity
MUCOPOLYSACCffARIDOSlS
Or&ml
WITH
Ow/24 h)
PATIENTS Glu NH2
ON II Carbwole
DATA
Total urinary GAG uranic acid’
AND CLINICAL
* Carbazole (Dische). * * Portion of urine available. 1, sibiings.
---.
1llF
6/F 9/F 10/F
5/F 6/M 1 7/M
1
1
? 12.0
l/M
_--
_I__.__-
Age in years onset/ current -_.-._~--
CZlSef Sex
_._.
LABORATORY
TABLE I
____
i*+ ++ ++ -
++ ++ ++ ++
-
-
___
me?zalY
_... ._
++* ++c i+ _-
+++ +++ ++ ++ _-
-
_ ._..-__
Skelefai chan@x
.~.__.-...HepatoSPlUlO-
TYPE
---. -_
++i ++ *
+* *++ ++ f -
-
-
_.-..
Cardio vascular
I I I I
I I I I I
I
I
type
pattern
Gl?l filtration
~__._ _... _ ._-._
Yes
Yes Not examined -
Yes Yes Not examined Yes Yes
Yes
Yes
a-L-Iduronidase deficiency
III
415
18/M
19/M
1
. siblings.
* Carbazole
(Dische).
214.6
3/6
17/M
I
20
40.5
12.86
Mean
S.D.
35
36
37
62
15/M
1.5/11
47
34
3/14
218
14/M
>
1:6
13/M
52
16/M
l/11
12/M
acid*
(mg/24 h)
onset/
ON
urinary
duration
Total
uranic
in
years
DATA
Age
CLINICAL
Sex
AND
CCiX/
LABORATORY
TABLE
99
690
131
92
175
126
75
125
S.D.
Mean
creatinine)
(mzk
GAG
8 PATIENTS
0.12
0.9
0.74
0.83
1.03
1.10
0.82
0.85
0.90
0.92
Orcinol
Carbazole ____
WITH
35
51
65
40
50
68
45
Gal
Glu
: 55 : 32 : 50 : 60 : 35 : 49 : 65
NH2
NH:!
None NOtX
++
None
None
None
+
None
None
+
++
+
rlrone
++
~._~
megaly
SplWlO-
Hepato-
++
I1
None
N0Ile
opacity
C0rlleal
TYPE
++
+++
+++
++
+++
++
+++
dation
retar-
Mental
MUCOPOLYSACCHARIDOSIS
_..
Skeletal
+ + None None +
+ + +
+
+ +
+
+
++ +
vascular
Cardio-
++
changes
Gel
II
II
II
I1
II
II
II
II
type
pattern
filtration
TABLE
>
1
} , siblings.
* Carbazole
35/F
34/F
33/F
32iM
31/F
30/M
1
?/8
?I6
(Dische).
S.D.
16
7.7
24.0
3.4?
20
22
1110
l/l0
26
Mean
18.5
S.D.
36
50.5
Mean
50
47
78
72
40
Random
23
36 Random
58
4115
6111
2/9.6
29/F
IIIB
-
28/M
Type
6/15
1.516
4113
-
24/M
25;M
21/F
4.517
23/F
26/M
219
? 113 ?/15
21,‘M 22/F
IIIA
20/F
Type
ON
S.D.
Mean
S.D.
Mean
sample
sample
~-----.~.~__
75
78
74
82
43
87
200
127
77
104
urine 89
NOW
101 urine
80
creatinine)
24 h)
-
(mg/Iz
(m!Zl
current
onset/
acid*
GAG
urinary
Total
uranic
in
DATA
Age
LABORATORY
years
AND
Sex
IV
Casef
CLINICAL
0.15
1.61
1.52
1.58
1.68
1.88
1.46
1.55
0.18
2.02
2.3
1.86
1.82
1.93
2.33 1.92
1.90
1.93 1.94
2.22
Orcinol
Carbazole ~
16 PATIENTS
84:
94:
89:
89:
so
97:
98:
so
95:
91:
86 -
85:
96: -
88:
Gal
Glu -
3
2
10
5
9
14
16
6
11
11
: 10
:
:
15
4
12
NH2
NH2
WITH
++
++
+
++
++
++
++
-
+‘I+
+++ +++ ++ +++ ++
d&ion
retar-
Mental
None
None
None None None ?
None
None -
None
None
?
?
NOW2
++
2
t
+
+ -
+
+
+ -
c -
None -
+
None -_
megaly
spleno-
Hepato
None
opacity
Corneal
MUCOPOLYSACCHARIDOSIS
IIIA
+
l
NOXE
+ + i
+
+ + + _
+
+ +
changes
Skeletal
TYPE
B
_____~_
+
+ -
F +
-
+
+ -
+
vascular
Cardio-
AND
Gel
IIIB
IIIB
IIXB
111s
IIIB
IIIB
IIIA
IIIA
IIIA
IIIA
IIIA
IIIA
IIIA
IIIA
IIIA
IIIA
type
pattern
filtration
defect
defect
defect
defect
defect
confirmed
confinned
confirmed
confirmed
confirmed
.,,....-. _____.-.- _-.__ _. __-_
Enzymatic
Enzymatic
Enzymatic
Enzymatic
Enzymatic
Comment
V
B
VI
46/M
?
4/14
N.D.
(Dische).
51
18
= non-detectable.
} , siblings.
**
* Carbazole
Unclassified
45/F
Type
22
44
10.4
7/16
44/M
29.6
2113
43/M
29
23
S.D.
3/12
Mean
6/21
18.4
42/F
37.4
S.D.
16
-
60
42
32
29
65
68.0
19.7
61
76
36
-
106
-
0.69
0.05
0.73
0.44
0.53
S.D.
0.60
0.52
0.45
0.56
Mean
S.D.
0.66
0.62
0.76
0.74
Orcinol
Carbazole
9 PATIENTS
0.07
ON
Mean
creatinine)
24 h)
GAG
(mg/g
acid*
DATA
(mg/
uranic
urinary
CLINICAL
Total
AND
Mean
8/10
41/M
Group
40/M
7135
39/M
1
10121
5/39
38/F
5131
1
37/M
A
current
onset/
36/F
Group
in
Y‘2?3l3
Age
LABORATORY
TABLE
: 55
: 59
: 70 : 68
NH2
NH2
5:
95
5 : 95
1 : 99
8 : 92
5 : 95
45
41
32
30
Gal
Glu
WITH
NOlIe
_
Non.2
NOIW
NOlltZ
NOIll
NOtlIZ
NCJIle
NIXI‘
NOW
NOM?
dation
retar-
Mental
+ ++ ++ ?
+++ +++ +++ +++ ++
opacity
C0rlleal
MUCOPOLYSACCHARIDOSIS
++
NOIX?
+ + +
++
++
+ + ++ ++
++
++
++
+
changes
+ +++
TYPE
Skeletal
1 WITH
+
V,
++
++
mega1y
spleno-
Hepato-
TYPE
VI,
VB
+
++
VB
NotIe
I?
VI
VB
VB
VA
VA
VA
VA
VA
type
pattern
filtration
Gel
1 UNCLASSIFIED
+++ +
?
+++
+++
+++
+++
Vascular
Cardio-
AND
-
-
NOlTld -
Normal
ND
ND
ND
ND*”
activity
nidase
a-L-Iduro-
330
The common clinical findings of the patients with mucopolysaccharidosis Type III (Sanfilippo) include: later onset than in Types I and II, pronounced mental retardation, absence of corneal opacity, mild joint deformities and slight enlargement of spleen and liver. As shown in Table IV, 16 patients belong to this group and those identified as Cases 30 and 31, 32 and 33, and 34 and 35 were three sets of siblings. The demonstration of two “‘corrective factors” [23] for the fibroblasts from different patients of this type led to eventual separation into two subgroups: Type IIIA deficient in a heparan sulfate sulfatase (heparin sulfamidase) [ 6] and Type IIIB with ~~-acetyl-~-glucos~inid~e deficiency [ 51, but without evident clinical distinguishing features between them. Mean urinary GAG-uranic acid was 50.5 and 24.0 mg per 24 hours for Types IIIA and IIIB, respectively. Mean carbazole/orcinol ratio was 2.02 t 0.18 in the first and 1.61 + 0.15 in the latter. In both types, glucosamine cornprized more than 85% of the total hexosamine and enzymatic analysis of the GAG (Table VI) showed predominance of HS. However, gel filtration patterns were different in each type (Fig. 1). Nine patients with age range between 10 and 39 years when last examined had mucopolysacchariduria and pronounced systemic manifestations but normal mentality (Table V). On the clinical grounds, these patients were considered to fall into Type V mucopolysacch~idosis (Scheie). Except for a generally milder course of the disease in a proportion of these nine patients (Cases 41--44), the involvement of tissues and organs were similar in all. Cases 36 and 37 (siblings) were the original Scheie patients [24] . Cases 38 and 39 are our new patients and they are also siblings. Elution diagrams were constructed for all nine patients. In general, the elution patterns fell into two different categories (Fig. 2), suggesting that we may be dealing with two different groups of patients and tentatively we have designated them as subgroups VA and VB. The mean carbazole/orcinol ratio in group VA was 0.69 ?r 0.07 while in group VB it was 0.53 + 0.05. In group A the ratio of glucosamine/ galactosamine was about 40 : 60 while in the latter it was about 5 : 95. Urinary GAG composition in group VA was: CSA + CSC, DS and HS with a ratio 10 : 45 : 45 and in group VB CSA + CSC, DS and HS 15 : 80 : <5. Patients identified as Cases 36-39 (no material was available in Case 40) had a deficiency of cr-L-iduronidase in their fibroblasts. On the other hand, leukocytes and fibroblasts from patients, Cases 41 and 43 (no material available in Cases 42 and 44) had normal a-L-iduronidase activity (G. Constantopoulos and AS. Dekaban, unpublished observations). One urine was supplied with the information that it belonged to a patient with mucopolysaccharidosis Type VI. As shown in Table V (Case 45), this female patient excreted 18 mg GAG (as uranic acid) per 24 hours. The carbazole/orcinol ratio was 0.44, indicating DS as the major GAG. It was found by enzymatic analysis that 80% of the total GAG are comprized of DS and 20% of CSA + CSC. Only a trace of HS was present. The elution pattern obtained from the urinary GAG of this patient had some resemblance to that of group VB. Finally, patient Case 46, exhibited an elution pattern Type I. This patient had been seen by one of us and at the time we formed the opinion that he was not typical for any known form of mucopolysaccharidosis, but the patient was not available for further study.
331
Composition of urinary GAG Early in the investigation, the urinary GAG from several patients and normal controls were fractiona~d on a Dowex l-X2 column (chloride) by stepwise elution with increasing concentrations of NaCl and the fractions were analyzed [ 191. This method separates the individual GAG but with some overlap. Therefore, the composition of the urinary GAG was estimated from their susceptibility to the Chyases AC and ABC (Table VI). The third column shows uranic acid in the disaccharides released after incubation of the GAG with Chyase AC. From these data it was calculated that CSA + CSC content was within normal range in all types except Types VI3 and VI (Cases 41, 43 and 45). Subsequently, the GAG which were not susceptible to Chyase AC, were incubated with Chyase ABC. The fourth column shows the uranic acid in the disaccharides released, presumably from DS. It appears that DS content is within normal range in Type IIIA, slightly increased in Type IIIB and comprizes about 50% of the total GAG in Types II and VA, 60% in Type I and about 80% in Types VB and VI. The GAG non-susceptible to Chyases AC and ABC represent HS. As shown in the last column, HS comprizes about one-third of the total GAG in Type I, 50% in Types II and VA and over 85% in Types IIIA and IIIB. It was a minor component of the urinary GAG from patients with Type VB and VI. Composition of the urinary GAG was also estimated in all patients from the elution diagrams obtained for the molecular weight distribution by gel filtration from Sephadex G-200 (Fig. 1 and 2). The results were in general agreement with those obtained by enzymatic analysis and have been summarized in Table I. TABLE
VI
COMPOSITION Figures borate
OF
presented method
THE as
URINARY
uonic
acid
GAG %
of
IN MUCOPOLYSACCHARIDOSES
total
urinary
GAG.
Analysis
for
uranic
acid
with
the
carbazole/
[ 181.
-. Case
Mucopoly-
CSA
NO.
saccharidosis
disaccharides
disaccharides
GAG
type
released
released
by
susceptible
Chyase
ABC
DS
+ CSC
i Chyase
by AC
I
after
HS
Chyase
non-
to Chyases AC
AC
and ABC
--. 4
I
7.0
8
--60.0
33.0 31.0
I
8.6
59.0
12
II
2.2
42.0
56.0
18
II
6.0
34.0
61.0
46.0
47.0
19
II
7.0
21
IIIA
7.1
3.9
89.0
27
IIIA
4.0
2.0
94.0
29
IIIA
4.5
1.5
94.0
30
III3
10.0
6.0
84.0
32
IIIB
5.0
4.0
91.0
36
VA
9.0
46.0
41
VB
14.0
18.0
8.0
43
VB
16.0
84.0
TrWX
45
VI
20.0
80.0
Trace
--
45.0
332
Figs 1 and 2 show the molecular weight elution patterns and chemicai reactivities for urinary GAG from the various types of mucopolysaccharidosis and normals, using gel filtration on Sephadex G-200. In the elution diagrams the ordinate represents uranic acid and the abscissa the log of the molecular weight of the GAG. The broken line represents uranic acid measured with the orcinol method and the solid line, uranic acid on the basis of carbazole (Dische) reaction. The carbazole/orcinol ratio is characteristic of each GAG (2.0 for heparan sulfate, 0.4 for dermatan sulfate and about 1.25 for chondroitin sulfates A and C). These data allow an estimation of the composition of the GAG. The 8 panels are representative of the elution patterns from normal
I-
L EyJATE Molecular
wetght
2 X’d
VOLUb$E lb4
(ml)
t 5x1103
Fig. 1. Sephadex G-200 gel filtration elution diagrams for urinary glycosaminoglycans. Samples are from top to bottom: normal controls. mucopolysaccharidosi Type I, Type II, Type IIIA and Type IIIB. The method, described previously 1141, employed a glas column with packed gel 1.06 cm i.d and 86 cm high, which was equilibrated at room temperature with the elution medium 0.025 M N&I. A sofution of GAG about 300 Pg (as uranic acid) in 0.2 ml, was applied and 2.0 ml fractions were collected and were analyzed for uranic acid. Carbazole 1161 and orcinol 1171 methods.
333 3.0
2.0
I.0
B 2.0: r? 0 I.0
Fi G =: z P
TYPE
P
‘7
2.0
$J i 0
I.0 E
2.0
I .o
0 V, 126.5
Molecular
wght
40 ELFATE
50 VOLUM;
2 X IO4
IO4
60
70
(ml) t 5x103
Fig. 2. Sephadex G-200 gel filtration elution diagrams for urinary glycosaminoglycans. Samples are from top to bottom: normal controls, mucopolysaccharidosis Type VA (Scheie). Type V (Group B), and Type VI. Conditions as in Fig. 1.
controls (top panel), six types of mucopolysaccharidosis and one group of patients who may belong to a new type, VB. There were individual variations, but provided that the column was not overloaded, the patterns were distinct. Normal
controls
and patients
with diseases other
than mucopolysacchari-
The curve of the elution diagrams of the urinary GAG from normal controls and patients without mucopolysaccharidosis (Figs 1 and 2, top panels) has a peak corresponding to a molecular weight of about 20 000 and an average molecular weight of about 14 000. The C/O ratio was about 1.0. Mucopolysaccharidosis Type I. The elution diagram of this type is bimodal (Fig. 1, second panel from the top). The peak at left is about 40% of the total GAG, is consisted of DS (C/O about 0.4) and has an average molecular weight about 18 000. The larger hump at right has an average molecular weight of about 6000 and consists of both DS and HS. The C/O ratio is increasing to a maximum of 1.10 to 1.20. Mucopolysaccharidosis Type II. This type of elution diagram (Fig. 1,3rd panel from the top) is also bimodal. The hump at left is smaller and contains mainly DS while the one at right contains both DS and HS with small molecular weight and has a C/O ratio with a maximum of 1.30-1.40, reflecting the higher HS content. dosis.
333
~~ucopo~ysaccharidosis Type IIIA. Type IIIA elution diagrams (Fig. 1,4th panel from the top) are consisted of one almost symmetrical hump. The GAG is practically pure HS with a Cl0 ratio up to 3.0 or even higher, and an average molecular weight of about 5000. There is only a small tailing towards the larger molecular weights. Mucopolysaccharidosis Type IIIl3. This curve (Fig. 1, bottom panel) is similar to the Type IIIA diagram with the exception that the C/O ratio is much smaller, only slightly above 2.0. Mucopolysaccharidosis Type VA. Five patients with Type VA (Seheie’s exhibited the same elution pattern shown in Fig. 2 (2nd panel from the top). This curve is not bimodal (as in Type I) and shows considerable shift towards smaller molecular weights. More than 80% of the GAG, consisting of DS and HS, are highly degraded. Average molecular weight is about 8000 and maximum C/O ratio about 1.0. Mucopolysaccharidosis Type VB. The other 4 patients with the diagnosis of mucopolysaccharidosis Type V, exhibited the elution pattern shown in Fig. 2, 3rd panel from the top. More than 80% of the GAG is pure DS of high molecular weight, average >20 000. The C/O ratio is about 0.53 with a maximum of 1.0 in a minor area at the extreme right of the curve. Mucopolysaccharidosis Type VI. The patient designated as mucopolysaccharidosis Type VI, displayed the elution diagram shown in Fig. 2, bottom panel. This curve is bimodal, with almost equal distribution of the GAG in the peaks corresponding to 20 000 and about 5000 molecular weight. A carbazolej orcinol ratio = 0.44 indicates high DS content and a small amount of HS towards the extreme right of the diagram. Uranic acid-containing
Eow molecular
weight compounds
In addition to the GAG, human urine contains large amounts of uranic acid-containing compounds which are not isolated by CPC precipitation or with Ecteola (Cl) column. DiFerrante and co-workers [25] reported that some of this material might be degradation products of the GAG. We measured total urinary uranic acid, polymeric GAG-uronie acid and “degradation products” in 6 patients and we have confirmed their observations. Total urinary uranic acid content ranged from 241 to 760 mg per 24 hours. GAGuronic acid content was from 25 to 88 mg per 24 hours and “de~adation products”-uranic acid 8 to 86 mg per 24 hours. Work is in progress for the chemical ch~ac~r~ation of these uranic acid and hexosamine containing small molecules. Discussion Several investigators have examined the urinary GAG from patients with mucopolysaccharidosis and have attempted to correlate their findings with clinical data. Terry and Linker [ 261 were able to distinguish four forms of the disease on the basis of their urinary GAG excretion pattern. Manley and WiIliams 1273 studied 28 patients with various types of mucopolysaccharidosis and concluded that acetate electrophoresis of the urinary GAG could clearly differentiate only 2 types, the predomin~tly heparan sulfate excretors and the “mixed excretors”. Kaplan 1281 noted four discrete patterns of mucopolysac-
335
chariduria in 46 patients and he found definite clinical correlations. Finally, Gordon and Haust [29] in 23 patients with mucopolysaccharidosis Types I, II and III and Spranger [30] in 51 with all types, independently used chromatography of the urinary GAG on Dowex 1 and found distinct patterns corresponding to the various types of mucopolysaccharidosis as defined with clinical criteria by McKusick et al. [12]. These studies provided additional diagnostic criteria, set the stage for the elucidation of the specific enzyme defect, and provided an abundance of observations which now, after the identification of the enzymatic deficiency, begin to be understood. A shortcoming of all these investigations was the lack of knowledge of the enzymatic deficiency in their patients which necessitated the correlation of their findings with clinical criteria only. The present study confirms and extends many of the findings from the earlier reports, introduces new methodology for the classification of these diseases on the basis of elution diagrams obtained by gel filtration from Sephadex G-200 and provides new information on the structure of the urinary GAG from patients with mucopolysaccharidoses. Moreover the enzymatic defect in a large number of patients has been identified, thus making this work more definitive. In agreement with the results of others, all our patients excreted in their urine excessive amounts of DS, HS or both. We found that patients identified as Cases 41 and 43 (Type VB) and 45 (Type VI), in addition, excreted excessive amounts of CSA + CSC. Reports of mucopolysaccharidosis without mucopolysacchariduria must be viewed with care until a defect in the metabolism of the GAG has been identified as the primary genetic disorder in such patients. The cited chemical data (Tables V and VI) would tend to suggest that Type VB patients could belong to Type VI mucopolysaccharidosis. However, their clinical manifestations differ; patients as described by Marateaux-Lamy (Type VI) were dwarfed and had severe hepatosplenomegaly and skeletal deformities, whereas the patients VB (Cases 41-44) are of normal height and their skeletal and organ involvement is much milder. According to a recent report [ 311, deficiency of sulfatase B was found in patients with Type VI mucopolysaccharidosis. Further work in this area is in progress in our laboratory. Urinary GAG from patients with the same type of the disease tend to have the same carbazole/orcinol ratio and also the same glucosamine/galactosamine ratio and GAG composition. This has been pointed out earlier by Kaplan [28] . From Figs 1 and 2 it is clear that the molecular weight distribution of the urinary GAG is characteristic for each different type of mucopolysaccharidosis and is distinguished from normal controls. Practically all HS and part of the DS in all types of mucopolysaccharidosis have been highly degraded. Knecht et al. [32] initially reported that HS from the urine and the liver of Hurler patients was fragmented. Subsequently, we have confirmed this observation in three types of mucopolysaccharidosis and, in addition, we found that a part of the DS was also highly degraded [13,14]. Here, we found that at least a sizeable portion of the HS and/or DS from all the patients with mucopolysaccharidosis was highly fragmented. None of the normal controls and the patients without mucopolysaccharidosis exhibited this shift in the molecular weight distribution of HS and DS towards the smaller molecular sizes. Regarding the mechanism of
336
the fragmentation of the GAG, one only may speculate that it is the result of the action of non-specific GAG endohydrolases in the tissues. The high N-sulfate content of HS from type IRA patients, which we have reported elsewhere [ 331, may be attributed to their deficiency in N-sulfatase [ 4,6] . The elution diagrams were correlated not only with clinical data but in all patients in which the enzymatic deficiency has been established, with the enzymatic defect as well. Thus, the elution diagrams have been used successfully in this study and may be used by others for the diagnosis of the type of the mucopolysaccharidosis. It is remarkable that urine excretion products are preserving their chemical characteristics for the same patient or type of the disease and for rather a long time, if not for life. This should not be surp~sing because they are the “substrates” for the deficient enzyme(s) and have been altered, only to the extent the metabolic capacity of the organism permits. Needless to say, the definitive understanding of a genetic disorder is the identification of the specific enzyme or protein defect and this must be the final criterion for the establishment of the diagnosis of the type of mucopolysaccharidosis in a patient. On the other hand, the knowledge of the chemistry and fine structure of the partially degraded “substrates” will contribute necessary information for understanding the development and pathogenesis of these diseases. References 1 A. Dorfman 2 3 4 5 6 7 8 3 10 11 12 13 14 15 16 17 18 19 20 21 22 23
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