83
Clinica Chimica Acta, 94 (1979) 83--E% @ Elsevier~North-Holland Biomedical Press
CCA 10184
GLYCOSIDASES
IN SERUM OF CYSTIC FIBROSIS PATIENTS
L. CASOLA a**, G. DI MATTE0 G. MASTELLA b
a, M. ROMAN0
a, B. RUTIGLIANO
a and
a International institute of Genetics and Biophysics, C.N.R., Via Marconi 10, 80125 Napoli {Italy) and b Cystic Fibrosis Clinic, Children’s Hospital “Alessandri”, Verona (Italyj (Received November 24th, 1978)
summary In a study of eight glycosidases in serum samples from 72 cystic fibrosis patients, 85 cystic fibrosis parents and 34 healthy and diseased controls, significant elevations of mean e-glucosidase levels were found in cystic fibrosis patients. All other glycosidases did not show any significant change. Mean cu-glucosidase levels in obligate heterozygotes were the same as in control individu~s. Moreover, cr-glucosidase levels in cystic fibrosis patients correlated with the degree of clinical impairment as measured by the Schwachman score.
Introduction A number of reports on the morphology and biochemists of lysosomes in cystic fibrosis (CF) cells has appeared. An increase in the number and size of these subcellular structures [l] and the presence of metachromatic granules of possible lysosomal origin [ 2,3], have been reported in diseased cells. Biochemical analysis of cultured fibroblasts, lymphoid cells and skin biopsies from CF patients, heterozygotes and normal controls has revealed increased total acid mucopolysacch~des [4,5] and possible differences in the level of some lysosomal enzymes [6--191. In spite of this work, however, the role of lysosomes in the etiopathogenesis of cystic fibrosis is as yet undefined. Recently [ 201 it has been claimed, on the basis of an altered isoelectrofocusing pattern of liver cu-L-fucosidase, that sialylation of this enzyme as well as of other lysosomal enzymes might be altered in cystic fibrosis. It is known [Zl] that lysosomal glycosidases are also present in extracellular fluids and that a liver recognition system functions to maintain a very low level of these enzymes in the blood, Since the plasma clearance of nearly all glycoproteins is * To whom correspondence should be addressed.
84
dependent on previous removal of terminal sialic acid [22], one might expect that a general defect in sialylation, as recently postulated, would result in quantitative and qualitative changes in some glycosidases. We are engaged in a program to study biochemical differences in glycoprotein biosynthesis and catabolism between normal and CF cells. In this report, we have focused on the quantitative analysis of some serum acid glycosidases of lysosomal origin. Materials and methods Study subjects included 72 patients with CF from the Cystic Fibrosis Center in Verona, 85 of their parents and a control group including healthy children, diseased children and adults. Cystic fibrosis patients were clinically assessed by the Schwachman-Kulczycki score [23]. Venous blood (usually 5 ml) was collected and allowed to clot in plastic tubes. Serum was separated by centrifugation at 2000 X g for 10 min at 4” C and frozen in at least two aliquots at -20” C. Enzyme assays Conditions of assay for nine hydrolases are given in Table I. Substrates were purchased from Koch Light Ltd. In all enzyme assays, diluted serum samples were incubated at 37°C with 50 ~1 of substrates in 0.2 M buffer for the appropriate length of time after which 2.0 ml of 0.2 M potassium carbonate were added to stop the reaction and to develop the fluorogen. Common to all enzyme assays was that the pH of the incubation mixture was the pH optimum for the enzyme and the substrate used. In all assays, the enzyme activity measured was directly proportional to the incubation time and to the volume of serum in the incubation mixture. Blanks were carried throughout this study to control for non-enzymatic hydrolysis of the substrate which was always negligible. The fluorescence of released 4-methylumbelliferone was measured in a Turner spectrophotofluorimeter, model 430 with excitation set at 365 nm and emission at 445 nm. All enzyme activities are reported as nmoles of substrate transformed per ml of serum under the conditions stated in Table I. a-Glucosidase activity was also measured using maltose or glycogen as substrate. The reaction mixture contained 0.004 M maltose, 0.05 M potassium acetate buffer pH 4.0 and 200 ~.tl of dialyzed serum in a final volume of 1.0 ml. After incubation for 120 min at 37”C, the reaction was stopped by heating the mixture in a boiling water bath for 1 min. After centrifugation, the glucose content of the clear supernatant was determined [ 241. The formation of glucose by ol-glucosidase action on glycogen was determined in a similar way with 2% glycogen as substrate in 0.1 M potassium acetate buffer, pH 4.0 containing 0.2 M KCl. cu-Amylase was assayed by the Smith and Roe procedure [ 25 1. Results Serum samples collected heterozygotes were analyzed tions of all these glycosidases Table II clearly demonstrate
from normal, cystic fibrosis homozygotes and for several glycosidases. Since serum concentrado not vary with age and sex [ 261, the results of the absence of any significant difference among
TABLE I
Substrate
4-MUor-L-fucopyranoside 4-MUa-D-mannopyranoside 4-MU-2-acetamide-2~eoxy-~-D~ucopYr~o~de 4-MU-2-acetamide-2deoxyQ-Dgalactopyranoside 4-MU-&D-glucuronide trihydrate 4-MUQ-D-galactopyranoside PMU-o-D-glucopyranoside 4MUsulphate potassium salt 4-MU-phosphate
Concentration (mM)
2.0 10.0 2.0 1.0 5.0 1.0 2.0 10.0 4.0
Enzyme
a-L-Fucosidase o-Mannosidase ~-~-Acetyl~uco~~id~ p-N-Acetulgalacto~inidase @Glucuronidase &Galactosidase o-Glucosidase Arylsulphatase Acid Phosphatase
CONDITIONS OF ENZYME ASSAY
Citrate Acetate Acetate Acetate Acetate Acetate Acetate Acetate Acetate
Buffer
5.4 4.0 4.5 4.5 4.0 4.0 4.5 5.8 5.0
PD
1 : 10 1:5 1 : 10 1:5 1:5 1:5 1:5 1:5 1 : 10
Serum dilution
10 30 30 30 30 60 60 30 10
Incubation time (mm)
86 TABLE
II
ACTIVITY AND
OF
SEVEN
GLYCOSIDASES
IN
SERUM
FROM
CYSTIC
FIBROSIS
PATIENTS,
FAMILIES
CONTROLS
Enzyme
Activity
of
enzymes
Controls
CF patients
(N
= 72)
CF parents
(N
= 8 5)
(N=35) Mean
f S.D.
Mean
? S.D.
P
Mean
k S.D.
P
wL-Fucosidase
25.32
? 5.54
29.31
? 4.71
N.S.
14.36
k 2.36
N.S.
a-Mannosidase
6.38
? 0.87
6.68
+ 0.62
N.S.
4.97
+ 0.79
N.S.
fl-N-Acetylglucosaminidase
6.67
+ 0.72
5.28
r 0.32
N.S.
5.31
+ 0.52
N.S.
p-N-Acetylgalactosiminidase
2.32
+ 0.26
2.02
? 0.12
N.S.
2.06
? 0.22
N.S.
17.09
r 2.97
17.13
r 2.99
N.S.
? 2.19
N.S.
P-Galactosidase
0.88
+ 0.18
0.62
t 0.12
N.S.
0.53
f 0.09
N.S.
Arylsulphatase
0.35
* 0.03
0.33
h 0.03
N.S.
0.31
f 0.02
N.S.
P-Glucuronidase
P values,
as determined
by
Student’s
t-test:
N.S..
not
13.22
significant.
the enzyme levels in cystic fibrosis patients, heterozygotes and controls. For comparative purposes, we have also assayed the activity of serum acid phosphatase, an acid hydrolase of lysosomal origin. Unlike the glycosidases, the serum level of this enzyme is significantly higher in children as compared to adults (42.53 + 4.08 vs. 22.19 ? 1.15, P < 0.001). However, when the cystic fibrosis groups are compared with their age-matched controls, no significant difference in this enzyme activity is found in either children (37.47 + 3.33 vs. 42.53 ? 4.08) or adults (22.18 * 1.15 vs. 19.73 or,1.64). Table III shows that the serum level of a-glucosidase in the cystic fibrosis patients is significantly higher (3.86 ? 0.40 vs. 0.79 5 0.06, P < 0.001)than in the control group. The enzyme level in the heterozygote group is not significantly different from that of the control group. In view of the fact that the activity of lysosomal cu-glucosidase can be greatly stimulated by monovalent and divalent cations [ 271, the possibility was considered that the higher level of this enzyme in cystic fibrosis serum could be due to a higher electrolyte concentration in cystic fibrosis as compared to normal serum. However, addition of various amounts of KC1 to normal serum failed to bring about any increase in its cr-glucosidase activity.
TABLE
III
(Y-GLUCOSIDASE
Group
LEVELS
N
IN
CYSTIC
or-Glucosidase
(mean CF
patients
FIBROSIS
CF
parents
3.86
f 0.40
0.79
f 0.06
P
0.85
? 0.05
0.79
? 0.06
Clinica Chimica Acta, 94 (1979) 83--E% @ Elsevier~North-Holland Biomedical Press
CCA 10184
GLYCOSIDASES
IN SERUM OF CYSTIC FIBROSIS PATIENTS
L. CASOLA a**, G. DI MATTE0 G. MASTELLA b
a, M. ROMAN0
a, B. RUTIGLIANO
a and
a International institute of Genetics and Biophysics, C.N.R., Via Marconi 10, 80125 Napoli {Italy) and b Cystic Fibrosis Clinic, Children’s Hospital “Alessandri”, Verona (Italyj (Received November 24th, 1978)
summary In a study of eight glycosidases in serum samples from 72 cystic fibrosis patients, 85 cystic fibrosis parents and 34 healthy and diseased controls, significant elevations of mean e-glucosidase levels were found in cystic fibrosis patients. All other glycosidases did not show any significant change. Mean cu-glucosidase levels in obligate heterozygotes were the same as in control individu~s. Moreover, cr-glucosidase levels in cystic fibrosis patients correlated with the degree of clinical impairment as measured by the Schwachman score.
Introduction A number of reports on the morphology and biochemists of lysosomes in cystic fibrosis (CF) cells has appeared. An increase in the number and size of these subcellular structures [l] and the presence of metachromatic granules of possible lysosomal origin [ 2,3], have been reported in diseased cells. Biochemical analysis of cultured fibroblasts, lymphoid cells and skin biopsies from CF patients, heterozygotes and normal controls has revealed increased total acid mucopolysacch~des [4,5] and possible differences in the level of some lysosomal enzymes [6--191. In spite of this work, however, the role of lysosomes in the etiopathogenesis of cystic fibrosis is as yet undefined. Recently [ 201 it has been claimed, on the basis of an altered isoelectrofocusing pattern of liver cu-L-fucosidase, that sialylation of this enzyme as well as of other lysosomal enzymes might be altered in cystic fibrosis. It is known [Zl] that lysosomal glycosidases are also present in extracellular fluids and that a liver recognition system functions to maintain a very low level of these enzymes in the blood, Since the plasma clearance of nearly all glycoproteins is * To whom correspondence should be addressed.
84
dependent on previous removal of terminal sialic acid [22], one might expect that a general defect in sialylation, as recently postulated, would result in quantitative and qualitative changes in some glycosidases. We are engaged in a program to study biochemical differences in glycoprotein biosynthesis and catabolism between normal and CF cells. In this report, we have focused on the quantitative analysis of some serum acid glycosidases of lysosomal origin. Materials and methods Study subjects included 72 patients with CF from the Cystic Fibrosis Center in Verona, 85 of their parents and a control group including healthy children, diseased children and adults. Cystic fibrosis patients were clinically assessed by the Schwachman-Kulczycki score [23]. Venous blood (usually 5 ml) was collected and allowed to clot in plastic tubes. Serum was separated by centrifugation at 2000 X g for 10 min at 4” C and frozen in at least two aliquots at -20” C. Enzyme assays Conditions of assay for nine hydrolases are given in Table I. Substrates were purchased from Koch Light Ltd. In all enzyme assays, diluted serum samples were incubated at 37°C with 50 ~1 of substrates in 0.2 M buffer for the appropriate length of time after which 2.0 ml of 0.2 M potassium carbonate were added to stop the reaction and to develop the fluorogen. Common to all enzyme assays was that the pH of the incubation mixture was the pH optimum for the enzyme and the substrate used. In all assays, the enzyme activity measured was directly proportional to the incubation time and to the volume of serum in the incubation mixture. Blanks were carried throughout this study to control for non-enzymatic hydrolysis of the substrate which was always negligible. The fluorescence of released 4-methylumbelliferone was measured in a Turner spectrophotofluorimeter, model 430 with excitation set at 365 nm and emission at 445 nm. All enzyme activities are reported as nmoles of substrate transformed per ml of serum under the conditions stated in Table I. a-Glucosidase activity was also measured using maltose or glycogen as substrate. The reaction mixture contained 0.004 M maltose, 0.05 M potassium acetate buffer pH 4.0 and 200 ~.tl of dialyzed serum in a final volume of 1.0 ml. After incubation for 120 min at 37”C, the reaction was stopped by heating the mixture in a boiling water bath for 1 min. After centrifugation, the glucose content of the clear supernatant was determined [ 241. The formation of glucose by ol-glucosidase action on glycogen was determined in a similar way with 2% glycogen as substrate in 0.1 M potassium acetate buffer, pH 4.0 containing 0.2 M KCl. cu-Amylase was assayed by the Smith and Roe procedure [ 25 1. Results Serum samples collected heterozygotes were analyzed tions of all these glycosidases Table II clearly demonstrate
from normal, cystic fibrosis homozygotes and for several glycosidases. Since serum concentrado not vary with age and sex [ 261, the results of the absence of any significant difference among
TABLE I
Substrate
4-MUor-L-fucopyranoside 4-MUa-D-mannopyranoside 4-MU-2-acetamide-2~eoxy-~-D~ucopYr~o~de 4-MU-2-acetamide-2deoxyQ-Dgalactopyranoside 4-MU-&D-glucuronide trihydrate 4-MUQ-D-galactopyranoside PMU-o-D-glucopyranoside 4MUsulphate potassium salt 4-MU-phosphate
Concentration (mM)
2.0 10.0 2.0 1.0 5.0 1.0 2.0 10.0 4.0
Enzyme
a-L-Fucosidase o-Mannosidase ~-~-Acetyl~uco~~id~ p-N-Acetulgalacto~inidase @Glucuronidase &Galactosidase o-Glucosidase Arylsulphatase Acid Phosphatase
CONDITIONS OF ENZYME ASSAY
Citrate Acetate Acetate Acetate Acetate Acetate Acetate Acetate Acetate
Buffer
5.4 4.0 4.5 4.5 4.0 4.0 4.5 5.8 5.0
PD
1 : 10 1:5 1 : 10 1:5 1:5 1:5 1:5 1:5 1 : 10
Serum dilution
10 30 30 30 30 60 60 30 10
Incubation time (mm)
86 TABLE
II
ACTIVITY AND
OF
SEVEN
GLYCOSIDASES
IN
SERUM
FROM
CYSTIC
FIBROSIS
PATIENTS,
FAMILIES
CONTROLS
Enzyme
Activity
of
enzymes
Controls
CF patients
(N
= 72)
CF parents
(N
= 8 5)
(N=35) Mean
f S.D.
Mean
? S.D.
P
Mean
k S.D.
P
wL-Fucosidase
25.32
? 5.54
29.31
? 4.71
N.S.
14.36
k 2.36
N.S.
a-Mannosidase
6.38
? 0.87
6.68
+ 0.62
N.S.
4.97
+ 0.79
N.S.
fl-N-Acetylglucosaminidase
6.67
+ 0.72
5.28
r 0.32
N.S.
5.31
+ 0.52
N.S.
p-N-Acetylgalactosiminidase
2.32
+ 0.26
2.02
? 0.12
N.S.
2.06
? 0.22
N.S.
17.09
r 2.97
17.13
r 2.99
N.S.
? 2.19
N.S.
P-Galactosidase
0.88
+ 0.18
0.62
t 0.12
N.S.
0.53
f 0.09
N.S.
Arylsulphatase
0.35
* 0.03
0.33
h 0.03
N.S.
0.31
f 0.02
N.S.
P-Glucuronidase
P values,
as determined
by
Student’s
t-test:
N.S..
not
13.22
significant.
the enzyme levels in cystic fibrosis patients, heterozygotes and controls. For comparative purposes, we have also assayed the activity of serum acid phosphatase, an acid hydrolase of lysosomal origin. Unlike the glycosidases, the serum level of this enzyme is significantly higher in children as compared to adults (42.53 + 4.08 vs. 22.19 ? 1.15, P < 0.001). However, when the cystic fibrosis groups are compared with their age-matched controls, no significant difference in this enzyme activity is found in either children (37.47 + 3.33 vs. 42.53 ? 4.08) or adults (22.18 * 1.15 vs. 19.73 or,1.64). Table III shows that the serum level of a-glucosidase in the cystic fibrosis patients is significantly higher (3.86 ? 0.40 vs. 0.79 5 0.06, P < 0.001)than in the control group. The enzyme level in the heterozygote group is not significantly different from that of the control group. In view of the fact that the activity of lysosomal cu-glucosidase can be greatly stimulated by monovalent and divalent cations [ 271, the possibility was considered that the higher level of this enzyme in cystic fibrosis serum could be due to a higher electrolyte concentration in cystic fibrosis as compared to normal serum. However, addition of various amounts of KC1 to normal serum failed to bring about any increase in its cr-glucosidase activity.
TABLE
III
(Y-GLUCOSIDASE
Group
LEVELS
N
IN
CYSTIC
or-Glucosidase
(mean CF
patients
FIBROSIS
CF
parents
3.86
f 0.40
0.79
f 0.06
P
0.85
? 0.05
0.79
? 0.06
