79
Clinica Chimica Acta, 82 (1978) 79-83 @ Elsevier/North-Holland Biomedical Press
CCA 9044
PERICELLULAR GLYCOSAMINOGLYCANS CELLS. A POSSIBLE SOURCE OF ERROR OF MUCOPOLYSACCHARIDOSES
J.J.H.
FORTUIN
Department (Received
IN CULTURED HUMAN IN PRENATAL DIAGNOSIS
* and W.J. KLEIJER
of Cell Biology and Genetics, Erasmus University, Rotterdam
July 15th,
(The Netherlands)
1977)
Normal fibroblasts and amniotic fluid cells, and cells from patients with Mucopolysaccharidosis type I and II, were cultured in the presence of 3sS04. After harvesting by trypsinization, the radioactivity was recovered from the intracellular and pericellular pool of glycosaminoglycans. The amount of incorporated radioactivity in these respective pools was 1 : 2.3 in normal fibroblasts and 1 : 7.2 in control amniotic fluid cells. Incorporation in the pericellular pool was not elevated in cells from patients with Mucopolysaccharidosis type I and II, in contrast to incorporation in the intracellular pool. Studies on different methods of harvesting showed that reliable prenatal analysis can be performed only if the pericellular pool is removed by trypsinization. Amniotic fluid cells from a pregnancy carrying a fetus affected with Hurler’s disease revealed the expected increased level of 3sS04 incorporation if the cells were trypsinized, but the intracellular accumulation of glycosaminoglycans was obscured by the pericellular pool if the cells were harvested by scraping.
Introduction A general feature of nearly all mucopolysaccharidoses (MPS-oses) is the accumulation of sulphated glycosaminoglycans (S-GAG) in tissues and cultured cells from patients [ 11. The studies of Fratantoni et al. [ 21 on 3sS04 incorporation in cultured fibroblasts revealed a defective degradation of S-GAG as the basis of the intracellular storage of this material. Subsequently, deficiencies of specific lysosomal enzymes have been detected for all clinical types of MPS-oses [3]. Parallel to the demonstration of specific enzyme defects, 3sS04 incorpora-
* To whom
correspondence
should be addressed.
80
tion studies are still a valuable and frequently used method in the (prenatal) diagnosis of MPS-oses. In addition to the intracellular (lysosomal) pool of S-GAG, a pericellular fraction of S-GAG has been shown which probably belongs to the cell coat and which can be removed by trypsinization [4-61. In contrast to the intracellular pool, this pericellular fraction seems to be unaffected by the genetic enzyme defects in the MPS-oses. It has therefore been suggested that methods of harvesting cultured cells, whereby the pericellular fraction of S-GAG is not removed, do not allow a reliable diagnosis of MPS-oses [4]. In the present investigation we have compared the 35S04-labeled S-GAG fractions in normal and MPS-osis fibroblasts and amniotic fluid cells harvested by different methods. The necessity of using an adequate method of harvesting is demonstrated by a study of 35S04 incorporation in amniotic fluid cells from a pregnancy in which a fetus affected with MPS-osis type I was detected. Materials and methods Amniotic fluid cells were cultured as described by Galjaard et al. [7]. Subcultures of amniotic fluid cells and of fibroblasts were grown in Eagle’s medium (MME, Difco), supplemented with 15% fetal calf serum, 12 mM NaHC03, 1 mM Na2S04, penicillin and streptomycin (100 I.U. and 0.1 mg/ml respectively). cells were seeded in plastic Petri For experiments on 35S04 incorporation, dishes (Falcon, 35 mm) at a density of 2-3 X 10’ cells per dish and incubated at 37°C in a fully humidified atmosphere of 95% air and 5% CO*. The medium was as described above except for the presence of 5 mM NaHC03, and organic buffers (HEPES, PIPES and BES, 10 mM of each) according to Eagle [ 81, at pH 7.1. The cultures were incubated for two days without 35S04 in order to avoid possible effects of subculture shock [9]. In this period the fibroblast cultures, but not always those of amniotic fluid cells, reached confluency. Subsequently, the cultures were incubated for 5 days in radioactive medium (1 mM Na235S04, 5 mCi/mmol; Amersham) with one change of medium at the third day. The cells were harvested after rinsing the dishes with saline (3X) either by incubation in 0.25 ml 0.25% trypsin (pig pancreas, l-300), or by scraping with a rubber policeman. A trypsinization period of 15 min was sufficient both for the removal of all 35S04-labeled material from the cell coat of all cell types, and for the detachment of the cells from the dishes. Amniotic fluid cells were not always completely detached but a longer trypsinization period was not applied to avoid possible cell losses. After centrifugation, washing in ice-cold saline and recentrifugation of the cells, the supernatants of both centrifugations were pooled and dialyzed at 4”C, 2 X 24 h against 0.1 M (NH4)#04 and then for 24 h against distilled water. The cell pellets were extracted according to Fratantoni et al. [ 21 in boiling 80% ethanol, dissolved in 0.6 ml 1 M NaOH (15 min at 60°C) and neutralized with 0.6 ml 1 M HCl. In addition to the 35S0,-labeled fractions in the supernates and in the cell pellets, another fraction was isolated from those dishes where the cells had been harvested by scraping. After thorough washing of the dishes, this “dish fraction” was detached by trypsinization.
81
1 ml Samples of the respective solutions of supernate, cell pellet and “dish fraction” were mized with 9 ml scintillation fluid composed of two ml Triton X-100 and 7 ml toluene containing 4 g/ml PPO. The radioactivity was counted in a Packard Tri-Carb 3375 scintillation counter. The protein content of the cell pellets was measured according to Lowry et al. [lo]. The sizes of the total “S-GAG fractions in the supernates, the cell pellets and the dish fractions were expressed as cpm per mg protein determined in the cell pellets. Results and discussion The sizes of different 3sS04-labeled GAG pools in human cells were determined after 5 days incubation with 3sS04 and harvesting of the cells by either trypsinization or scraping. The results of several experiments, carried out under identical conditions, are summarized in Tables I and II. The fibroblast strains used originated from two normal individuals and five patients affected with MPS-osis type IH (Hurler), IS (Scheie) or type II (Hunter). The data for amniotic fluid cells were obtained from 20 different control amniotic fluid cell cultures and one culture from a pregnancy at risk for Hurler’s disease, in which an affected fetus was demonstrated by the deficiency of a-L-iduronidase activity [ 111. After 3sS04 incorporation followed by trypsinization and centrifugation of the cells, two 3sS-GAG pools were measured: the intracellular pool (ICP) in the cell pellet and the pericellular pool (PCP) in the supernatant (Table I). In agreement with the findings of several authors [2,4,5,12] the ICPs in fibroblasts from patients with MPS-oses appeared to be larger (on average 3-4 times) than in normal fibroblasts. A similar difference was found between the ICPs in the amniotic fluid cells from a fetus affected with Hurler’s disease and control amniotic fluid cells. In contrast there was no significant difference between the incorporation of
TABLE
I
INCORPORATION OF 3sS04 BY TRYPSINIZATION
IN DIFFERENT
CELL
FRACTIONS
OBTAINED
AFTER
HARVESTING
Confluent cell cultures were incubated for 5 days in the presence of 5 pCi/mI Na23sS04. harvested by trypsinization and analysed as described in Material and Methods. n. number of separate cell cultures which were analysed. The “Total” values given relate only to fuIIy analysed cultures. Figures are presented as cpm X 10m3 /mg celI protein, Fibroblasts Normal
Intracellular PericeIIular
pool (ICP) Pool (PCP)
Amniotic MPS-osis
Extremes
19
16-27 n=9
63
44-108 n=7
34-57
51
41
?I=9 Total
60
45-76 ?I=9
118
MPS-osis
Normal
Mean
MeaIl
fluid cells
Extremes
Mean
Extremes
9
4-34 n = 20
38 n=l
42-69 n=6
66
35-110 ” = 18
44 n=l
88-162 n=6
76
41-119 n = 18
82 n=l
82
TABLE
II
INCORPORATION BY
OF
=SO4
IN
DIFFERENT
CELL
FRACTIONS
See Table
I for details.
Figures
are presented
as cpm
X 10_3/mg
Fibroblasts -
AFTER
HARVESTING
Pellet
Supernate
“Dish”
cell protein. Amniotic
Normal
Total
OBTAINED
SCRAPING
MPS-osis Extremes
Mean
Extremes
Mean
50
47-55
76
57-105
44
n=4
n=4
3
63
4-l
5
17
n=3
n=l
l-5
6
?I=3
tl=l
59-65
93
n=3
n=l
cells MPS-osis
Normal
Mean
10
fluid
16
Extremes 22-77
43
n = 11
n=l
13-19 n=2
9
9-10 n=2
80
77-83 n=2
35S04 in the PCPs of fibroblasts from controls and patients with MPS-oses. The PCPs in amniotic fluid cells varied considerably and were on average 1.6 times as large as in normal fibroblasts. As in fibroblasts, there was no difference between the PCPs in the amniotic fluid cells from a fetus affected with MPS-osis type IH and controls. The size of the PCP in amniotic fluid cells might depend on the conditions of cell cultivation (e.g. degree of confluency) and on the type of amniotic fluid cells involved (epithelioid, fibroblast-like or large cells [ 13]), but so far we have not found any relationship between the size of the PCP and the morphology of the predominant cell type. The results, obtained by addition of the values for incorporated 3sS04 in ICPs and PCPs (Table I) suggest that it will be difficult, or in case of amniotic fluid cell cultures, even impossible to distinguish between normal and MPS-osis cells when the PCP is not sufficiently removed. This suggestion, already made by Neufeld and Cantz [4], was confirmed by the measurement of 35S-GAG pools in cells that were harvested by scraping instead of trypsinization. As shown in Table II, three 35S-GAG pools were found after scraping: a pellet and a supernate fraction obtained after centrifugation of a suspension of the scraped cells, and a minor “dish fraction” which remained attached to the dish after scraping. The “dish fraction” is probably similar to the “undercellular” fraction described by Kresse et al. [5] and may represent the microexudate of cell surface material secreted by cells onto the substrate on which they are grown [ 141. The supernate fraction of scraped cells represented about 20% of the total cellular radioactivity, which is a much smaller fraction than the PCP found after trypsinization. The majority (about 75%) of the total 35S-GAG pool remained in the cell pellet after scraping. Only a slight difference was found between the radioactivity in the pellet fraction in normal and MPS-osis cells, whereas there was no difference at all between the pellet fraction of normal and MPS-osis amniotic fluid cells. This demonstrates that the removal of the PCP by trypsinization is an essential step in the detection of MPS-osis by
83
means of 35S04 incorporation in fibroblasts and especially in amniotic fluid cells. Methods of harvesting, such as dissolution of cells in hyamin, used by Wende1 et al. [12] to facilitate the analysis of small cell numbers, are likely to cause difficulties in the diagnosis of MPS-oses for reasons discussed above. Since micromethods may contribute to a rapid prenatal detection of metabolic diseases [11,15,16] we are now developing a microprocedure for the study of 35S04 incorporation in small numbers of cells, including trypsinization. Acknowledgements The authors are grateful to Mrs. C. Tichelaar-Klepper, Griffioen and Mrs. G.M. Hensing-Wolffers for performing cultures.
Mrs. G. Olijhoekamniotic fluid cell
References 1 Dorfman. A. and Matalon. R. (1972) in The Metabolic Basis of Inherited Disease (Stanbury. J.B., Wijngaarden. J.B. and Fredrickson, D.S., eds.). p. 1218. McGraw-Hill. New York 2 Frantantoni, J.C., Hall, C.W. and Neufeld. E.F. (1968) Proc. Natl. Acad. Sci. U.S.A. 60. 699-706 3 Neufeld, E.F.. Lim. T.W. and Shapiro. L.J. (1975) Ann. Rev. Biochem. 44.367-376 4 Neufeld, E.F. and Cants, M. (1973) in Lysosomes and Storage Diseases (Hers, H.G. and van Hoof, F., eds.), P. 261, Academic Press, New York and London 5 Kresse, H.. van Ffgura, K.. Buddecke, E. and Fromme. H.G. (1976) Hoppe-Seyler’s Z. Physiol. Chem. 366.929-S41 6 Kraemer. P.M. and Smith, D.A. (1974) Biochem. Biophys. Res. Commun. 66, 423-430 7 Galjaard. H.. Mekes. M.. de Josselin de Jong, J.E. and Niermeijer. M.F. (1973) Clin. Chim. Acta 49, 361-375 8 Eagle, H. (1971) Science 174,500-503 9 Heukels-Dully, M.J. and Niermeijer, M.F. (1976) Exptl. Cell Res. 97, 304-312 10 Lowry. O.H.. Rosebrough. N.J., Farr. A.L. and Randall, R.J. (1951) J. Biol. Chem. 193.266-275 11 Kleijer. W.J.. Sachs, E.S. and Niermeijer. M.F. (1975) Histochem. J. 7, 496498 12 Wendel, U.. Riidiger. H.W. and Passarge. E. (1974) Monatscbr. Kinderbeflk. 122.23-30 13 Hoehn. H.. Bryant, E.M.. Karp. L.E. and Martin, G.M. (1974) Pediatr. Res. 8. 746-754 14 Weiss. L., Paste, G.. MacKearnin. A. and Willett, K. (1975) J. Cell Biol. 64.136-146 15 Gahaard. H., Hoogeveen. A., Keijzer. W.. de Wit-Verbeek. E. and Vlek-Noot, C. (1974) Histochem. J. 6.491-509 16 Gahaard, H.. Sachs, E.S.. Kleijer, W.J. and Nienneijer, M.F. (1975) in Early Diagnosis and Prevention of Genetic Diseases (Went, L.N., Vermeij-Keers. Chr. and van der Linden, A.G.J.M.. eds.), p. 82. Leiden University Press, Leiden
Clinica Chimica Acta, 82 (1978) 79-83 @ Elsevier/North-Holland Biomedical Press
CCA 9044
PERICELLULAR GLYCOSAMINOGLYCANS CELLS. A POSSIBLE SOURCE OF ERROR OF MUCOPOLYSACCHARIDOSES
J.J.H.
FORTUIN
Department (Received
IN CULTURED HUMAN IN PRENATAL DIAGNOSIS
* and W.J. KLEIJER
of Cell Biology and Genetics, Erasmus University, Rotterdam
July 15th,
(The Netherlands)
1977)
Normal fibroblasts and amniotic fluid cells, and cells from patients with Mucopolysaccharidosis type I and II, were cultured in the presence of 3sS04. After harvesting by trypsinization, the radioactivity was recovered from the intracellular and pericellular pool of glycosaminoglycans. The amount of incorporated radioactivity in these respective pools was 1 : 2.3 in normal fibroblasts and 1 : 7.2 in control amniotic fluid cells. Incorporation in the pericellular pool was not elevated in cells from patients with Mucopolysaccharidosis type I and II, in contrast to incorporation in the intracellular pool. Studies on different methods of harvesting showed that reliable prenatal analysis can be performed only if the pericellular pool is removed by trypsinization. Amniotic fluid cells from a pregnancy carrying a fetus affected with Hurler’s disease revealed the expected increased level of 3sS04 incorporation if the cells were trypsinized, but the intracellular accumulation of glycosaminoglycans was obscured by the pericellular pool if the cells were harvested by scraping.
Introduction A general feature of nearly all mucopolysaccharidoses (MPS-oses) is the accumulation of sulphated glycosaminoglycans (S-GAG) in tissues and cultured cells from patients [ 11. The studies of Fratantoni et al. [ 21 on 3sS04 incorporation in cultured fibroblasts revealed a defective degradation of S-GAG as the basis of the intracellular storage of this material. Subsequently, deficiencies of specific lysosomal enzymes have been detected for all clinical types of MPS-oses [3]. Parallel to the demonstration of specific enzyme defects, 3sS04 incorpora-
* To whom
correspondence
should be addressed.
80
tion studies are still a valuable and frequently used method in the (prenatal) diagnosis of MPS-oses. In addition to the intracellular (lysosomal) pool of S-GAG, a pericellular fraction of S-GAG has been shown which probably belongs to the cell coat and which can be removed by trypsinization [4-61. In contrast to the intracellular pool, this pericellular fraction seems to be unaffected by the genetic enzyme defects in the MPS-oses. It has therefore been suggested that methods of harvesting cultured cells, whereby the pericellular fraction of S-GAG is not removed, do not allow a reliable diagnosis of MPS-oses [4]. In the present investigation we have compared the 35S04-labeled S-GAG fractions in normal and MPS-osis fibroblasts and amniotic fluid cells harvested by different methods. The necessity of using an adequate method of harvesting is demonstrated by a study of 35S04 incorporation in amniotic fluid cells from a pregnancy in which a fetus affected with MPS-osis type I was detected. Materials and methods Amniotic fluid cells were cultured as described by Galjaard et al. [7]. Subcultures of amniotic fluid cells and of fibroblasts were grown in Eagle’s medium (MME, Difco), supplemented with 15% fetal calf serum, 12 mM NaHC03, 1 mM Na2S04, penicillin and streptomycin (100 I.U. and 0.1 mg/ml respectively). cells were seeded in plastic Petri For experiments on 35S04 incorporation, dishes (Falcon, 35 mm) at a density of 2-3 X 10’ cells per dish and incubated at 37°C in a fully humidified atmosphere of 95% air and 5% CO*. The medium was as described above except for the presence of 5 mM NaHC03, and organic buffers (HEPES, PIPES and BES, 10 mM of each) according to Eagle [ 81, at pH 7.1. The cultures were incubated for two days without 35S04 in order to avoid possible effects of subculture shock [9]. In this period the fibroblast cultures, but not always those of amniotic fluid cells, reached confluency. Subsequently, the cultures were incubated for 5 days in radioactive medium (1 mM Na235S04, 5 mCi/mmol; Amersham) with one change of medium at the third day. The cells were harvested after rinsing the dishes with saline (3X) either by incubation in 0.25 ml 0.25% trypsin (pig pancreas, l-300), or by scraping with a rubber policeman. A trypsinization period of 15 min was sufficient both for the removal of all 35S04-labeled material from the cell coat of all cell types, and for the detachment of the cells from the dishes. Amniotic fluid cells were not always completely detached but a longer trypsinization period was not applied to avoid possible cell losses. After centrifugation, washing in ice-cold saline and recentrifugation of the cells, the supernatants of both centrifugations were pooled and dialyzed at 4”C, 2 X 24 h against 0.1 M (NH4)#04 and then for 24 h against distilled water. The cell pellets were extracted according to Fratantoni et al. [ 21 in boiling 80% ethanol, dissolved in 0.6 ml 1 M NaOH (15 min at 60°C) and neutralized with 0.6 ml 1 M HCl. In addition to the 35S0,-labeled fractions in the supernates and in the cell pellets, another fraction was isolated from those dishes where the cells had been harvested by scraping. After thorough washing of the dishes, this “dish fraction” was detached by trypsinization.
81
1 ml Samples of the respective solutions of supernate, cell pellet and “dish fraction” were mized with 9 ml scintillation fluid composed of two ml Triton X-100 and 7 ml toluene containing 4 g/ml PPO. The radioactivity was counted in a Packard Tri-Carb 3375 scintillation counter. The protein content of the cell pellets was measured according to Lowry et al. [lo]. The sizes of the total “S-GAG fractions in the supernates, the cell pellets and the dish fractions were expressed as cpm per mg protein determined in the cell pellets. Results and discussion The sizes of different 3sS04-labeled GAG pools in human cells were determined after 5 days incubation with 3sS04 and harvesting of the cells by either trypsinization or scraping. The results of several experiments, carried out under identical conditions, are summarized in Tables I and II. The fibroblast strains used originated from two normal individuals and five patients affected with MPS-osis type IH (Hurler), IS (Scheie) or type II (Hunter). The data for amniotic fluid cells were obtained from 20 different control amniotic fluid cell cultures and one culture from a pregnancy at risk for Hurler’s disease, in which an affected fetus was demonstrated by the deficiency of a-L-iduronidase activity [ 111. After 3sS04 incorporation followed by trypsinization and centrifugation of the cells, two 3sS-GAG pools were measured: the intracellular pool (ICP) in the cell pellet and the pericellular pool (PCP) in the supernatant (Table I). In agreement with the findings of several authors [2,4,5,12] the ICPs in fibroblasts from patients with MPS-oses appeared to be larger (on average 3-4 times) than in normal fibroblasts. A similar difference was found between the ICPs in the amniotic fluid cells from a fetus affected with Hurler’s disease and control amniotic fluid cells. In contrast there was no significant difference between the incorporation of
TABLE
I
INCORPORATION OF 3sS04 BY TRYPSINIZATION
IN DIFFERENT
CELL
FRACTIONS
OBTAINED
AFTER
HARVESTING
Confluent cell cultures were incubated for 5 days in the presence of 5 pCi/mI Na23sS04. harvested by trypsinization and analysed as described in Material and Methods. n. number of separate cell cultures which were analysed. The “Total” values given relate only to fuIIy analysed cultures. Figures are presented as cpm X 10m3 /mg celI protein, Fibroblasts Normal
Intracellular PericeIIular
pool (ICP) Pool (PCP)
Amniotic MPS-osis
Extremes
19
16-27 n=9
63
44-108 n=7
34-57
51
41
?I=9 Total
60
45-76 ?I=9
118
MPS-osis
Normal
Mean
MeaIl
fluid cells
Extremes
Mean
Extremes
9
4-34 n = 20
38 n=l
42-69 n=6
66
35-110 ” = 18
44 n=l
88-162 n=6
76
41-119 n = 18
82 n=l
82
TABLE
II
INCORPORATION BY
OF
=SO4
IN
DIFFERENT
CELL
FRACTIONS
See Table
I for details.
Figures
are presented
as cpm
X 10_3/mg
Fibroblasts -
AFTER
HARVESTING
Pellet
Supernate
“Dish”
cell protein. Amniotic
Normal
Total
OBTAINED
SCRAPING
MPS-osis Extremes
Mean
Extremes
Mean
50
47-55
76
57-105
44
n=4
n=4
3
63
4-l
5
17
n=3
n=l
l-5
6
?I=3
tl=l
59-65
93
n=3
n=l
cells MPS-osis
Normal
Mean
10
fluid
16
Extremes 22-77
43
n = 11
n=l
13-19 n=2
9
9-10 n=2
80
77-83 n=2
35S04 in the PCPs of fibroblasts from controls and patients with MPS-oses. The PCPs in amniotic fluid cells varied considerably and were on average 1.6 times as large as in normal fibroblasts. As in fibroblasts, there was no difference between the PCPs in the amniotic fluid cells from a fetus affected with MPS-osis type IH and controls. The size of the PCP in amniotic fluid cells might depend on the conditions of cell cultivation (e.g. degree of confluency) and on the type of amniotic fluid cells involved (epithelioid, fibroblast-like or large cells [ 13]), but so far we have not found any relationship between the size of the PCP and the morphology of the predominant cell type. The results, obtained by addition of the values for incorporated 3sS04 in ICPs and PCPs (Table I) suggest that it will be difficult, or in case of amniotic fluid cell cultures, even impossible to distinguish between normal and MPS-osis cells when the PCP is not sufficiently removed. This suggestion, already made by Neufeld and Cantz [4], was confirmed by the measurement of 35S-GAG pools in cells that were harvested by scraping instead of trypsinization. As shown in Table II, three 35S-GAG pools were found after scraping: a pellet and a supernate fraction obtained after centrifugation of a suspension of the scraped cells, and a minor “dish fraction” which remained attached to the dish after scraping. The “dish fraction” is probably similar to the “undercellular” fraction described by Kresse et al. [5] and may represent the microexudate of cell surface material secreted by cells onto the substrate on which they are grown [ 141. The supernate fraction of scraped cells represented about 20% of the total cellular radioactivity, which is a much smaller fraction than the PCP found after trypsinization. The majority (about 75%) of the total 35S-GAG pool remained in the cell pellet after scraping. Only a slight difference was found between the radioactivity in the pellet fraction in normal and MPS-osis cells, whereas there was no difference at all between the pellet fraction of normal and MPS-osis amniotic fluid cells. This demonstrates that the removal of the PCP by trypsinization is an essential step in the detection of MPS-osis by
83
means of 35S04 incorporation in fibroblasts and especially in amniotic fluid cells. Methods of harvesting, such as dissolution of cells in hyamin, used by Wende1 et al. [12] to facilitate the analysis of small cell numbers, are likely to cause difficulties in the diagnosis of MPS-oses for reasons discussed above. Since micromethods may contribute to a rapid prenatal detection of metabolic diseases [11,15,16] we are now developing a microprocedure for the study of 35S04 incorporation in small numbers of cells, including trypsinization. Acknowledgements The authors are grateful to Mrs. C. Tichelaar-Klepper, Griffioen and Mrs. G.M. Hensing-Wolffers for performing cultures.
Mrs. G. Olijhoekamniotic fluid cell
References 1 Dorfman. A. and Matalon. R. (1972) in The Metabolic Basis of Inherited Disease (Stanbury. J.B., Wijngaarden. J.B. and Fredrickson, D.S., eds.). p. 1218. McGraw-Hill. New York 2 Frantantoni, J.C., Hall, C.W. and Neufeld. E.F. (1968) Proc. Natl. Acad. Sci. U.S.A. 60. 699-706 3 Neufeld, E.F.. Lim. T.W. and Shapiro. L.J. (1975) Ann. Rev. Biochem. 44.367-376 4 Neufeld, E.F. and Cants, M. (1973) in Lysosomes and Storage Diseases (Hers, H.G. and van Hoof, F., eds.), P. 261, Academic Press, New York and London 5 Kresse, H.. van Ffgura, K.. Buddecke, E. and Fromme. H.G. (1976) Hoppe-Seyler’s Z. Physiol. Chem. 366.929-S41 6 Kraemer. P.M. and Smith, D.A. (1974) Biochem. Biophys. Res. Commun. 66, 423-430 7 Galjaard. H.. Mekes. M.. de Josselin de Jong, J.E. and Niermeijer. M.F. (1973) Clin. Chim. Acta 49, 361-375 8 Eagle, H. (1971) Science 174,500-503 9 Heukels-Dully, M.J. and Niermeijer, M.F. (1976) Exptl. Cell Res. 97, 304-312 10 Lowry. O.H.. Rosebrough. N.J., Farr. A.L. and Randall, R.J. (1951) J. Biol. Chem. 193.266-275 11 Kleijer. W.J.. Sachs, E.S. and Niermeijer. M.F. (1975) Histochem. J. 7, 496498 12 Wendel, U.. Riidiger. H.W. and Passarge. E. (1974) Monatscbr. Kinderbeflk. 122.23-30 13 Hoehn. H.. Bryant, E.M.. Karp. L.E. and Martin, G.M. (1974) Pediatr. Res. 8. 746-754 14 Weiss. L., Paste, G.. MacKearnin. A. and Willett, K. (1975) J. Cell Biol. 64.136-146 15 Gahaard. H., Hoogeveen. A., Keijzer. W.. de Wit-Verbeek. E. and Vlek-Noot, C. (1974) Histochem. J. 6.491-509 16 Gahaard, H.. Sachs, E.S.. Kleijer, W.J. and Nienneijer, M.F. (1975) in Early Diagnosis and Prevention of Genetic Diseases (Went, L.N., Vermeij-Keers. Chr. and van der Linden, A.G.J.M.. eds.), p. 82. Leiden University Press, Leiden
