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Variability of acid hydrolase activities in cultured skin

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Journal of Medical Genetics (1975). 12, 224. Variability of acid hydrolase activities in cultured skin fibroblasts and amniotic fluid cells ELISABET YOUNG, P. WILLCOX, A. E. WITFIELD, and A. D. PATRICK
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Journal of Medical Genetics (1975). 12, 224. Variability of acid hydrolase activities in cultured skin fibroblasts and amniotic fluid cells ELISABET YOUNG, P. WILLCOX, A. E. WITFIELD, and A. D. PATRICK From the Department of Chemical Pathology, Institute of Child ealth, London, WC1N 1E Summary. The specific activities of lysosomal hydrolases in cultured skin fibroblasts and amniotic fluid cells showed wide and unpredictable variations between cultures, which may lead to difficulty in differentiating normal, heterozygous, and homozygous cells. owever, the variability for a given culture was similar for all enzymes assayed, so that a clearer differentiation of a relative deficiency of a given enzyme uld be obtained by expressing its activity in ratio to that of another enzyme. Activity ratios were particularly useful in the evaluation of enzyme levels in cultured amniotic fluid cells. Results of their application to tests of pregnancies at risk for metachromatic leudystrophy, Krabbe's leudystrophy, GM1-gangliosidosis, and GM2-gangliosidosis (Sandhoff variant) are presented. The increasing use of cultured cells for the diagnosis and prenatal detection of inherited metabolic disorders has led to an awareness of the extent to which the enzymic activity of these cells may be affected by culture nditions. The type of medium (Beutler et al, 1971; Ryan et al, 1972), the p of the medium (Ryan et al, 1972), the ncentration of fetal calf serum (Kittlick et al, 1973), and other culture materials-such as amphotericin B and trypsin (Uhlendorf, 197)-have all been shown to influence the levels of enzymic activity of cultured cells. The enzyme ntent of cells might also be augmented by endocytosis of enzymes present in the serum nstituents of culture media (ors-cayla et al, 1968). Enzymic activities are also influenced by the state of the cultures at the time they are harvested for assay; these effects are particularly evident with respect to cell density (Leroy et al, 197; Ryan et al, 1972), time after feeding (Ryan et al, 1972) or subculture (Cristofalo et al, 1967; Okada et al, 1971), age of the culture (olliday, 1972; Butterworth et al, 1973), and the phase of growth (Pan and Krooth, 1968). It is essential, therefore, that a laboratory intending to use cultured cells in diagnosis should, whereever possible, attempt to ntrol these effects in order to establish ranges of enzymic activity on which reliable diagnosis can be based. Differences Received 3 August in the enzyme levels of cultured cells reported from individual laboratories appear to be mainly due to variations in the culture nditions employed; differences may also be due to variations in assay procedures and in the use of protein ncentration or other index to which specific activities are referred (Russell, 1969; Kaback and owell, 197; Leroy et al, 197). Even when all the above variables are ntrolled by standard practice, it is often found that the range of activity of cultures is very wide, particularly for amniotic fluid cells (Gerbie et al, 1972) and skin fibroblasts (Milunsky et al, 1972). In view of these nsiderations and because of our interest in the prenatal diagnosis of lysosomal storage disorders, we have studied the variability of acid hydrolase activities of cultured amniotic fluid cells and fibroblasts. Our objective was to determine the ranges of enzymic activity obtained under our particular culture nditions and to investigate means by which the wide ranges of activity found between different cultures might be reduced. These findings have been applied to the interpretation of results of prenatal tests for metachromatic leudystrophy, Krabbe's leudystrophy, GM1-ganglosidosis, and GM2-gangliosidosis (Sandhoff variant). Our experience in the prenatal diagnosis of Tay- Sachs disease has been reported elsewhere (Ellis et al, 1973). 224 J Med Genet: first published as /jmg on 1 September Downloaded from on 4 September 218 by guest. Protected by Variability af acid hydrolase activities in cultured skin fibroblasts and amniotic fluid cells 225 Materials uman albumin-fraction V, 4-nitrocatechol sulphate and p-nitrophenylphosphate were obtained from Sigma (London) Chemical Co Ltd; 4-methylumbelliferyl substrates, bovine brain cerebrosides and p-nitrophenyl palmitate from Koch-Light Laboratories Ltd; Medium 199 and fetal bovine serum from Flow Laboratories; and trypsin from Wellme Research Laboratories. Methods Cell culture. Skin fibroblasts and amniotic fluid cells were cultured in Medium 199, ntaining 15%/O fetal bovine serum; penicillin 1,g/ml; streptomycin 1,ug/ml, and added glutamine (1 mmol/l) in pyrogenfree distilled water. The medium was sterilized by Millipore filtration. Cultures were incubated at 37 C in an atmosphere of 5%/ C2 :95%O air, fed twice weekly, and harvested 24 h after feeding. Cells were detached with a. 1 solution of trypsin in.1 mol/l sodium citrate, p 7.8, ntaining.1 mol/l NaCl. Skin fibroblast cultures. A skin biopsy (1-2 mm3) was cut into several pieces and placed on a glass slide, which was then inverted in a Pyrex Petri dish (5 mm diameter) and vered with 5 ml of culture medium. Approximately 4 weeks later the pieces of skin were discarded and the fibroblast outgrowths trypsinized from the slide, which was then removed. Culture was ntinued in the dish until a monolayer was formed; the cells were then subcultured. Amniotic fluid cell cultures. Amniotic fluid was centrifuged in sterile nical polycarbonate tubes at 4 g for 1 min at room temperature. The cell pellet was resuspended gently in.3 ml culture medium and added to 5 mm Pyrex Petri dishes, each dish receiving the cells from approximately 5 ml of amniotic fluid. Culture medium was added and the dishes were left undisturbed for 3 to 4 days, after which they were fed twice weekly. After 2 weeks, the lonies were trypsinized to disperse the cells, which were then allowed to grow to form a monolayer before being subcultured. Cells harvested by trypsinization were washed with.9% (w/v) NaCl solution and homogenized in ice-ld water using a small hand-operated glass homogeniser of the Potter type. The homogenate was frozen in solid CO2/methanol and stored at 15 C. - Enzyme assays. Whole homogenates of cultured cells were used in all assays. Incubation temperature was 37 C. All assay mixtures ntained.1% human albumin. N-acetyl-,B-D-hexosaminidase was assayed acrding to the method of Brett et al (1973);,B-galactosidase acrding to the method of Young et al (1972); galactocerebrosidase acrding to the method of Bowen and Radin (1969), using the modified substrate solution described by Radin and Arora (1971); and acid esterase acrding to the method of Young and Patrick (197), using p-nitrophenyl palmitate as substrate and 1,ul of cell homogenate (3-4,g protein) in a final volume of 15 dtl. Acid phosphatase activity was measured in a mixture ntaining.1 mol/l acetate buffer, p 5. (5 p1); 11 mmol/l p-nitrophenyl phosphate (5 pl) and 5 1l of homogenate (5-25,ug protein). After 3 min incubation the reaction was stopped by the addition of 1 mol/l Tris-Cl, p 9. (3,lI). After centrifugation, 2,ul of the clear supernatant fluid was added to 1 mol/l Tris- C1, p 9. (2,l), and the absorbance measured immediately at 4 nm. Arylsulphatase A activity was measured in a mixture ntaining.75 mol/l acetate buffer-.137 mol/l NaCl, p 4.9 (1 1); 4.4 mmol/l sodium pyrophosphate (1,ul); 61 mmol/l 4-nitrocatechol sulphate (25 ti), and 3 pl of homogenate (1-5 tog protein). Activity was estimated for the incubation period 2-7 min. At these times the reaction was stopped by the addition of 1 mol/l NaO (4,ul) and, after centrifugation, the absorbance was measured at 5 nm. a-galactosidase activity was measured in a mixture ntaining.2 mol/l acetate buffer, p 4.4 (5 tj); 1 mmol/l 4-methylumbelliferyl-cc-D-galactopyranoside in.1 mol/l acetate buffer, p 4.4 (1 pl), and 5 [1I of homogenate (1-3,ug protein). After 3 min incubation the reaction was stopped by the addition of.25 mol/l glycine-nao, p 1.4 (1. ml) and the liberated 4-methylumbelliferone measured fluorimetrically. a-glusidase activity was measured in a mixture ntaining.1 mol/l acetate buffer, p 4. (1,ul); 2 mmol/i 4-methylumbelliferyl-a-D-glupyranoside (1 1I) and 25!d of homogenate (2-3,ug protein). After 3 min incubation the reaction was stopped and fluorescence measured as for a-galactosidase above. The protein ntent of the supematant fluid obtained after centrifuging a sample of homogenate at 2 g for 5 min was determined by the method of Lowry et al (1951), modified for small volumes. For all homogenates, supernatant protein ncentration was directly proportional to total protein. Results and discussion Ranges of acid hydrolase activities of cultured amniotic fluid cells assayed at various stages of subculture from 1 to 5 are shown in Table I. Wide ranges of activity were found for all the enzymes assayed. No rrelation between enzymic activity and gestational age was found for any enzyme. Table I also shows that similarly wide ranges were obtained when the activities of a particular subculture (send) were mpared. In further experiments, some individual cultures assayed at every stage of subculture from 1 to 5 showed little variation in activities, while in others there was up to a 1-fold variation between subcultures. This variation might be due to the presence of different cell types of differing enzymic nstitution at various stages of culture. Comparison of epithelioid and J Med Genet: first published as /jmg on 1 September Downloaded from on 4 September 218 by guest. Protected by 226 Young, Willx, Whitfield, and Patrick TABLE I ACID YDROLASE ACTIVITIES OF CULTURED AMNIOTIC FLUID CELLS -Galactosidase Galactocerebrosidase Arylsulphatase A 6-exosaminidase Number of cultures Subculture assayed Total number of assays Activity range * * t t * * * * Mean ± SD 2.8 ± ± ± ± ± ± ± ±3.87 * Specific activity expressed as Mmol substrate hydrolysed per mg protein per hour. t Specific activity expressed as nmol substrate hydrolysed per mg protein per hour. TABLE II ACID YDROLASE ACTIVITIES OF REPLICATE CULTURES OF AMNIOTIC FLUID CELLS Culture ClueSub- P-Galacto- cerebro- Aryl- Ol-exos- exosid- Acid Acid Acid a-galacto- culture sidase*- c idaeb sulphatase-a* aminidase amndase ( Phosphatase Maltaset Esterase* sidase* A56 a b c d A61 a b c d A63 a b c d * Specific activity is expressed as Mmol substrate hydrolysed per mg protein per hour. t Specific activity is expressed as nmol substrate hydrolysed per mg protein per hour. fibroblast-like cells derived from amniotic fluid has shown both similarities and specific differences between their respective enzyme ntents (Gerbie et al, 1972; Kaback and Leonard, 1972). Such a division into two cell types would appear to be an over-simplification, since many more types may be present in amniotic fluid cell cultures (Uhlendorf, 197). Furthermore, skin fibroblast cultures which are normally mposed of a single cell type, show wide variation in enzymic activity (Milunsky et al, 1972). Quintuplet cultures derived from a single skin biopsy and treated identically were found to vary nearly 1-fold with respect to the activities of several lysosomal hydrolases. We carried out a similar study to determine the variation in enzymic activity of cultures derived from a single specimen of amniotic fluid. In three separate experiments in which cultures were initiated in quadruplicate from a single fluid and treated identically, there was an approximate 2-fold variation in enzymic activity between the cultures (Table II). Cells obtained from the fluid of each amniotic sac of a twin pregnancy were also cultured. The cultures showed markedly different growth characteristics, one being mposed mainly of fast-growing fibroblast-type cells and the other slow-growing epithelioid cells. J Med Genet: first published as /jmg on 1 September Downloaded from on 4 September 218 by guest. Protected by U, (A m m z cn U qut w4 U Variability of acid hydrolase activities in cultured skin fibroblasts and amniotic fluid cells.4) C,o x 444) :3 V tv - S ra O O x = -k Sc 4:1. - a, - +I s ie;od,o =o9bo ( +1 I._ 4) 4) tt: 4) 4) L_E 4I.2 ci -Ce - D~4t X ) 11 - too in o-o 666 enf('c'4 CQU_ +1 a... a %Q CS 2 OdX mo k4tv 4 cis4)~ 4).4 I _ A4c 1 ' ciss. +1 I tooooo OMVNMM -n o 4. 4 t ~ ~ 4 o~ '+ ~ 6 - U In+- NU)n - 4) 4) C~~~~U, ) 41 Go w r. u a E a C3, 4)4.4.1 as P. U P4 * +I r, s4 4w ca Ui) e W C) 4 :Y C) as *B (44 z z 4 U4 A4,.-I COO u 14 (A.4).5 44) 4 Ex M 4 I I I I I I li I 1 , t1~o~ o 4) i CN 1' 4.6 Cf 4)4 4-4 C4 4 4( o 4 C; m 4f~~~~~~~~~~~t as liii 4 u en ~c -*e~~~ 44 ~~~~~~~~~~~Cq.- e ~X 44' 4) ' * ~~~~~~~~~~~ Li.I '' -- a, 444 Cq en C6t.roZ4 4.. : 4 L O ax go 4 Ca, 1w GoU U I.2 S.I 44 C' 4)-o '- be i u 227 C C: e to 1 n .S._ o la J Med Genet: first published as /jmg on 1 September Downloaded from on 4 September 218 by guest. Protected by 228 Young, Willx, Whitfield, and Patrick Enzymic activities of the cells harvested after two zygote. All the diagnoses were nfirmed at birth subcultures also showed marked differences, the by enzymic analysis of leucytes prepared from slow-growing cells having approximately twice the rd blood. Both fetuses tested for Krabbe's leudystrophy were nsidered normal; this was n- activity of the fast-growing cells. All the above findings nfirm that current techniques and knowledge of cell culture do not permit to term. Activity ratios were particularly useful in firmed at birth in one case, the other is proceeding strict ntrol over the developmental patterns of one prenatal test for Krabbe's leudystrophy in lysosomal enzymes in cultured cells. Nevertheless, which a very low specific activity but normal activity results showed that while the activities of individual ratio were found. Results of these tests are summarized in Table IV. Enzymic activity ratios have enzymes varied widely between cultures, the degree of variation of activity for a given culture was similar been used with advantage in postnatal diagnosis for all enzymes assayed. Therefore, it appeared (den Tandt and Schaberg, 1973; all and Neufeld, that a clearer differentiation of a relative deficiency 1973), but their use in the prenatal detection of in the activity of a particular enzyme might best be lysosomal storage disorders has not been reported obtained by expressing that activity as a ratio to that previously. of another enzyme. Experiments in which similarly cultured skin fibroblasts derived from ntrol intending to offer a service in screening pregnancies The importance of ntrol work by a laboratory subjects, patients affected with a variety of lysosomal at risk for metabolic disorders cannot be overstated. The ability to diagnose the diseased n- storage disorders and parents of these patients were mpared, showed that wide ranges of activity were dition in leucytes or cultured skin fibroblasts is again found for ntrol cultures, although the variability was less than that found for amniotic cell noses there is a need for absolute differentiation be- not sufficient since in the majority of prenatal diag- cultures. When these results were expressed as tween homozygous and heterozygous levels of enzymic activity. There must be no possibility that activity ratios the variability was nsiderably reduced (Table III). In all cases, activity ratios were an exceptionally low heterozygous level of activity is useful in distinguishing homozygous, heterozygous interpreted as indicating an affected fetus. Similarly, a high residual activity in affected cells must and ntrol cultures, and were particularly helpful in differentiating heterozygotes for metachromatic not be interpreted as a low heterozygous level. leudystrophy. The only discrepancy was a result obtained for the father of a patient with many variables, it is essential that a laboratory Since the enzyme level of cultured cells is affected by Krabbe's leudystrophy. The galactocerebrosidase activity of his skin fibroblasts nsistently fell exhaustive investigation into the enzymology of attempting prenatal diagnoses should nduct an within the normal range whether related to protein amniotic fluid cells cultured acrding to a strictly or to f-galactosidase (or other enzyme). Normal ntrolled practice. The enzymic activities of values for galactocerebrosidase activity had previously been found for leucytes prepared from ferent workers cannot be directly mpared, nor cultured amniotic fluid cells determined by dif- several specimens of the father's blood. Blood can results obtained for other cell types be applied group matching nfirmed parenthood. to the evaluation of activities of cultured amniotic Present experience suggests that the use of activity fluid cells (Brock, 1973). It has been shown that ratios rather than specific activities will be particularly helpful in the evaluation of enzyme levels in may vary greatly between subcultures. Therefore, the enzymic activities of amniotic fluid cell cultures amniotic fluid cells. As found for skin fibroblasts, when monitoring a pregnancy at risk, the enzyme the wide ranges of specific activity of individual assay should be carried out on at least two different subcultures. A ntrol culture of amniotic enzymes of ntrol cultures were narrowed nsiderably when expressed in ratio to the activity of fluid cells should also be processed using identical fi-galactosidase. The clearer differentiation obtained has been of practical use in prenatal diagnosis. amniotic fluids can be stored in liquid nitrogen and culture nditions. Uncultured cells from ntrol Pregnancies at risk for either metachromatic removed for culture when required, thus easing the leudystrophy, Krabbe's leudystrophy, GM1- problem of the availability of ntrol* cultures gangliosidosis, or GM2-gangliosidosis (Sandhoff (Niermeijer et al, 1973). variant) were monitored by enzymic analysis of cultured amniotic fluid cells. The fetuses at risk for We thank the Camilla Samuel Fund and the Joint Research Board of the ospital for Sick Children Great metachromatic leudystrophy or Sandhoff disease were nsidered normal; in the case of GM1- Ormond Street and the Institute of Child ealth for gangliosidosis the fetus was predicted to be a hetero- generous financial support. J Med Genet: first published as /jmg on 1 September Downloaded from on 4 September 218 by guest. Protected by Variability of acid hydrolase activities in cultured skin fibroblasts and amniotic fluid cells REFERENCES Beutler, E., Kuhl, W., Trinidad, F., Teplitz, R., and Nadler,. (1971). B-Glusidase activity in fibroblasts from homozygotes and heterozygotes for Gaucher's disease. American Journal of uman Genetics, 23, Bowen, D. M. and Radin, N. S. (1969). Cerebroside galactosidase: a method for determination and a mparison with other lysosomal enzymes in developing rat brain. Journal of Neurochemistry, 16, Brett, E. M., Ellis, R. B., aas, L., Ikonne, J. U., Lake, B. D., Patrick, A. D., and Stephens, R. (1973). Late onset GM2-gangliosidosis. Clinical, pathological and biochemical studies on eight patients. Archives of Disease in Childhood, 48, Brock, D. J.. (1973). Biochemical studies on amniotic fluid cells. In Antenatal Diagnosis of Genetic Disease, ed. by A. E.. Emery, pp Churchill Livingstone, London. Butterworth, J., Sutherland, G. R., Broadhead, D. M., and Bain, A. D. (1973). Lysosomal enzyme levels in human amniotic fluid cells in tissue culture. I. a-glusidase and,-glusidase. Life Sciences, 13, Cristofalo, V. J., Parris, N., and Kritchevsky, D. (1967). Enzyme activity during the growth and ageing of human cells in vitro. Journal of Cellular Physiology, 69, Ellis, R. B., Ikonne, J. U., Patrick, A. D., Stephens, R., and Willx P. (1973). Prenatal diagnosis of Tay-Sachs disease. Lancet, 2, Gerbie, A. B., Melann, S. B., Ryan, C., and Nadler,. L. (1972). Cultivated epithelial-like cells and fibroblasts from amniotic fluid: their relationship to enzymatic and cytologic a
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