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Microenzymatic assays for lysosomal enzymes in primary amniotic fluid cell cultures
325
Clinica Chimica Acta, 105 (1980) 325-334 @ Else~ier/North-HoIIand Biomedical Press
CCA 1442 MICROENZY~ATIC ASSAYS FOR LYSOSOMAL AMNIOTIC FLUID CELL CULTURES
MARILUCI
T. BLADON
* and AUBREY
ENZYMES IN PRIMARY
MILUNSKY
Genetics Division, Eunice Kennedy Shriver Center; and the Department of Pediatrics, Harvard Medical School and the Massachusetts General Hospital, Boston, MA (U.S.A.) (Received
November
21st, 1980)
Summary A study of three Iysosomal enzymes (hexosaminidase, fl-galactosidase and ar-galactosidase) in normal primary amniotic fluid cell cultures using a microenzymatic assay is presented. No difference in enzyme activity was found between primary and amniotic cell cultures in passage number one. A progressive change in the proportions of hexosaminidase A and hexosaminidase B with time was demonstrated in culture. The feasibility of this procedure for the early prenatal diagnosis of disorders due to lysosomal enzyme deficiency is discussed.
Amniotic fluid cell cultures are composed of a heterogeneous population of nucleated cells [ 11. These cells may behave in the following ways: (i) they may float and not attach to the culture flask, (ii) they may attach but rarely divide and give rise to individual colonies or (iii) they may attach and give rise to welldefined colonies, epithelial- or fibroblast-like cell types. The presence of these well-defined colonies (pure or mixed) of the two cell types with varying morphology in the amniotic fluid cell culture suggested that their enzyme activities might differ in relation to their origins and morphology [2]. Kaback and Leonard [3] used normal amniotic fluid cell cultures to analyze epithelial-like, fibroblastic-like and mixed cell type colonies for P-galactosidase,. arylsulfatase-A and P-D-N-acetylglucosaminidase. They found no significant difference in specific enzyme activity in the morphologically different cell types. On the other hand, Gerbie et al. [4] found significantly greater enzyme activity of
* Reprint U.S.A.
address:
Life
Science
Department,
Worcester
Polytechnic
Institute,
Worcester,
MA
01609,
326
histidase in epithelial-like and cystathionine synthetase in fibroblastic-like cell types of amniotic fluid cultures. Earlier work on the quantitative analysis of enzymes required a large number of amniotic cells (on the order of 106) [5,6]. Four to ten weeks of culture were required in order to grow a sufficient number of cells for these assays. Later, Galjaard [ 71 and Hosli [ 81 developed a microtechnique for quantitative enzyme analysis at the single cell level. Their results are based on (i) use of single amniotic cells from the primary culture [7,8], (ii) isolation and dissection of small numbers of specific cell type [Q,lO,ll], and (iii) clones of cells of mainly one cell type [12]. This technique requires only 12-13 days after amniocentesis for the results to be obtained. Depending on the enzyme, the activity may vary greatly between the two cell types 241; hence prenatal diagnosis of some enzyme disorders would produce unreliable results. In addition, Heukels-Dully et al. [13] have shown that the enzyme activity of a given cell varies with its phase of growth in culture. We published the procedure for a microenzymatic fluorescent assay which produces sn average enzyme activity per cell which is proportional to the total number of cells analyzed (100-300 cells) ]14]. All the cells analyzed are in the log growth phase of the cell cycle. The difference in cell types and, consequently, in enzyme activity is averaged out. These factors constitute si~ific~t improvements over other microenzymatic assays. We also reported the enzyme activity values for hexosaminidase in non-porno ~niotic fluid cell cultures and in skin fibroblasts, and the enzyme activity values for LY-and ~-g~actosid~e in skin fibrobl~~. It was shown that the enzyme activity of hexos~inid~e is higher in skin fibrobl~~ that in non-primary amniotic fluid cell cultures. This paper is a continuation of our previous work [14] and will (i) examine hexosaminidase activity in primary amniotic fluid cell cultures, (ii) determine the feasibility of assaying the relatively low activity values of CX-and P-galactosidase activity in amniotic fluid cells using our improved microenzymatic assay and (iii} evaluate the feasibility of using this microassay for the prenatal diagnosis of lysosomal enzyme deficiency disorders within 13 days after obtaining an amniotic fluid sample. Materials and methods
Amniotic fluid cell culture The amniotic fluid cells were obtained during elective abortions performed for non-genetic indications and cultured as follows: (1) Fluids were spun in disposable centrifuge tubes at 1600 X g for 10 min. (2) The supernatant fluid was removed leaving approximately 1.0 ml above the cell pellet. (3) 2.0 ml of 20% FCS in MEM were added to the centrifuge tubes. The total volume of 3.0 ml in each tube was well dispersed using a Pasteur pipette. (4) 0.5 ml of the resuspended cells was pipetted onto a 22-mm square cover glass (Coming No. 1) which was placed in a 35mm petri dish. This step was repeated for six petri dishes. (For a-galactosidase, the entire 3.0-ml sample was placed on one 35mm dish.)
327
(5) The cells were left on the coverglass overnight at 37°C in a 5% COZ atmosphere. (6) 2.0 ml of 20% FCS in MEM were added to each coverslip the next day and the dishes were left undisturbed in the incubator for seven days. (7) After this period, the medium was changed and dishes were examined for cell colonies. (8) The approximate number of cells per colony, as well as the predominant cell type (viz. epithelial or fibrobl~tic), were recorded. (9) If a colony had approximately 100 cells, the dish was used for the microenzymatic assay of the enzyme being studied. (10) The harvest process of the primary amniotic fluid cells was accomplished by using 0.25% trypsin as previously described [14]. An aliquot of the trypsinized cells was transferred to another 35mm petri dish with 2.0 ml of 20% FCS in MEM and left at 37°C in the CO, incubator. This is referred to as Passage No. 1 or P1. The remainder of the trypsinized primary amniotic fluid cells was used for the microenzymatic fluorescence technique. The microenzymatic assay 4.methyl4-Methylumbelliferyl-2-acetamido-2-deoxy~-D-glucopyranoside, umbelliferyl-O-D-galactopyranoside and 4-methylumbelliferyl-cw-D-galactopyranoside were used as substrates for the determination of hexosaminidase (N-acetyl-fl-D-glucosaminidase), /3-galactosidase and a-galactosidase activities, respectively. The enzyme activities were calculated using a standard curve of pure 4-methylumbelliferone (4-MU). The measuring levels of all three lysosomal enzymes presented in this study were within the linear range of their assay conditions. The apparatus used to measure the fluorescence and the procedure for assaying the three enzymes is described elsewhere [ 141. The specific enzyme activity is expressed as picomol of 4-MU released per cell per hour. Experimental
results
Hexosaminidase (N-acetyl-/3-D-glucosaminidase (EC 3.2.1.30)) A total of 19 amniotic fluid samples were assayed for both isozymes of hexosaminidase, isozyme form A {hex A) and isozyme form B (hex B). After obtaining the amniotic fluid, it took between 8 and 13 days to grow the minimum number of cells to perform the assay on the primary cell cultures. Amniotic fluid cells in P, were assayed between 12 and 17 days. Days in culture for passage No. 1 means the total number of days in culture from the plating of the primary cells until the day the cells were assayed. It ,includes the days as primary cell cultures, before the actual splitting of the cells to establish passage No. 1. The enzyme activity of total hexosaminidase includes that due to hex A and hex B. When hexosaiinidase is appropriately heat-treated, hex A is inactivated while hex B is unaffected. The enzyme activities of total and heat-treated hexosaminidase are presented in Tables I and II for primary and P, amniotic fluid cells, respectively. The t-statistic, t, for paired comparisons [ 151 was used to test the following hypothesis: there is no difference in the enzyme activity value of total hexo-
328 TABLE
I
ENZYME Case
ACTIVITY
OF
HEXOSAMINIDASE
Days
No.
* IN PRIMARY Total
in culture
AMNIOTIC
activity
FLUID Heat-treated
AbF
1
10
2.53(*1.550)
0.86(*0.250)
AbF
2
11
0.85(*0.040)
0.47(*0.000)
AbF
3
13
0.30(*0.000)
0.20(*0.000)
AbF
4
11
0.67(+0.060)
0.43(*0.120)
AbF
5
12
0.28(*0.030)
0.20(*0.007)
AbF
6
12
1.30(+0.200)
0.60(*0.000)
AbF
7
12
0.96(*0.200)
0.27(iO.O10)
AbF
8
13
1.70(*0.420)
0.53(*0.041)
AbF
9
13
1.70(*0.840)
0.50(*0.000)
AbF
10
11
1.80(+0.200)
0.50(i0.070)
AbF
11
11
0.87(+0.470)
0.55(*0.070)
AbF
12
13
0.80(*0.090)
0.27(*0.000)
AbF
13
2.06(+0.730)
0.60(*0.042)
AbF
14
0.54(
0.14(*0.090)
AbF
15
AbF
16
AbF
8 11
iO.070)
0.75(iO.210)
0.25(+0.070)
13
1.25(*0.070)
0.46(*0.060)
17
9
0.85(*0.210)
0.20(~0.000)
AbF
18
13
2.15(*0.210)
0.70(*0.000)
AbF
19
9
1.03(*0.380)
0.40(*0.180)
9
* Enzyme
activity
(k) = standard Each
value
is expressed
in terms
of picomol
of 4-MU
released
CELLS
. cell-’ . h-l.
deviation. represents
the
average
of
3
to
5 readings
of
droplets
containing
cells
from
the
same
case
from
the
same
case
(iS.D.).
TABLE
II
ENZYME
ACTIVITY
OF Days
HEXOSAMINIDASE
* IN
in culture
P1 AMNIOTIC
FLUID
CELLS
Case
No.
AbF
1
15
1.80(+0.140)
0.60(~0.000)
AbF
2
13
0.42(iO.100)
0.15(*0.040)
AbF
3
15
0.72(*0.300)
0.37(*0.090)
AbF
4
13
0.62(*0.020)
0.33(*0.000)
AbF
5
14
0.85(+0.300)
0.27(*0.000)
AbF
6
14
1.50(*0.800)
0.40(*0.000)
AbF
7
14
1.42(*0.120)
0.74(+0.110)
Total
Heat-treated
activity
AbF
8
15
0.40(*0.000)
0.26(iO.O50)
AbF
9
16
0.95(*0.500)
0.50(*0.000)
AbF
10
13
0.63(*0.140)
0.26(+0.110)
AbF
11
17
0.40(*0.000)
0.18(iO.O20)
AbF
12
15
0.75(
AbF
13
12
0.90(*0.140)
0.26(iO.O00)
0.30(*0.170)
iO.070)
AbF
14
13
0.70(*0.520)
0.30(*0.000)
AbF
15
12
0.55(*0.070)
0.20(*0.000)
AbF
16
15
0.65(+0.070)
0.35(*0.070)
AbF
17
12
1.10(*0.000)
0.53(iO.320)
AbF
18
15
1.72(*1.100)
0.45(*0.210)
AbF
19
12
0.65(*0.210)
0.18(iO.O40)
* Enzyme
activity
(*) = standard Each (G.D.).
value
is expressed
in terms
of picomol
of 4-MU
released
. cell-’ . h-l.
deviation. represents
the
average
of
3
to
5 readings
of
droplets
containing
cells
329
saminidase in primary and PI amniotic fluid cells cultured from a given case number. The same hypothesis was tested for heat-treated hexosammidase. For total hexosaminidase, t = 1.73 with 36 degrees of freedom. For heat-treated hexosaminidase, t = 1.45 with 36 degrees of freedom. The 95% confidence interval for t with 36 degrees of freedom is (-2.029, 2.029); therefore, both hypotheses were accepted. Fig. 1 and 2 show percentages of hex A and hex B in primary and PI amniotic fluid cells, respectively, versus the number of days in culture. When the percentages of hex A and hex B in primary amniotic fluid cells were compared to those of PI amniotic cells to ascertain the effect of trypsinization, no difference was found between the two treatments. The t-test was used to test the following hypothesis: trypsinization has no effect on the percentage of hex A (and, consequently, hex B) assayed from amniotic fluid cells obtained from primary and PI cultures from a given case number. The t-test was 0.20 with 36 degrees of freedom. The 95% confidence interval or acceptance region for t with 36 degrees of freedom is (-2.029, 2.029); therefore the hypothesis was accepted. Furthermore, the t-test was used to test the following hypothesis: on the 12th day the percentage of hex A (and, consequently, hex B) is the same for primary and PI amniotic cells obtained from a given case number. The same hypothesis was tested for the 13th day. (The number of days for which primary amniotic fluid cells were cultured was between 8 and 13. The number of days for which PI amniotic fluid cells were cultured was between 12 and 17. Hence, it was only for 12 and 13 days in culture that we could test these hypotheses.) On the 12th day, t = 0.08 with 5 degrees of freedom, and on the 13th day, t = 0.72 with 8 degrees of freedom. The 95% confidence intervals for
100 90 80 70 m P
60 50
B 4 2 2 bE
40 30 20
:: :: 0 II::
. :: ::Ill h HI2
116X B
A
1 1T
:: :: :: :: I
:.
::
::
10
::
::
0
::
I?
?3) DAYS
ii
IN CULTURE
Fig. 1. The percentages of hex A and hex B in primary cell cultures. Where more than one primary amniotic cell culture is cultured for the same number of days. the percentage is the average. (No) specifies the number of cultures analyzed for each day of culture. On each bar is indicated the standard deviation of the mean.
330 100 90
70 m
60
E I
50
ES 4
40
2 I k +S
. .$ in * HEXA
80
30
HEXB
I
.*
PO
.
I
.
1
::
.
.
I
.
,
.
.
.
10
.
a, . . .
.
.
.
0
-
.
::
12,
DAYS
1 .
IN CULTURE
Fig. 2. The percentages of hex A and hex B in PI amniotic cell cultures. Where more than one PI amniotic cell culture is cultured for the same number of days, the percentage is the average. (No) specifies the the number of cultures analyzed for each day of culture. On each bar is indicated the standard deviation of the mean.
t with 5 and 8 degrees of freedom are (-2.571, 2.571) and (-2.306, respectively; therefore, both hypotheses were accepted.
2.306),
p-GaZactosidase (EC 3.2.1.23) A total of 16 amniotic fluid samples were assayed for /3-galactosidase (P-gal). Table III presents the values obtained with primary and P1 amniotic fluid cells. After obtaining the amniotic fluid, it took at least six days to grow the minimum number of cells to perform the assay. P, amniotic fluid cells were assayed between 10 and 17 days. The t-test was used to test the following hypothesis: there is no difference in the enzyme activity value of p-gal in primary and P1 amniotic fluid cells cultured ‘from a given case number. For 30 degrees of freedom, t = -1.34. The 95% confidence interval for 30 degrees of freedom is (-2.042, 2.042); therefore, the hypothesis was accepted. (EC 3.2.1.22) A total of 15 amniotic fluid samples were assayed for a-galactosidase (a-gal). Table IV presents the enzyme activity values for primary and P, amniotic fluid cells. We were able to assay for a-gal in primary cultures as early as six days after obtaining the fluids. P1 amniotic fluid cells were assayed between 12 and 15 days. The t-test was used to test the following hypothesis: there is no difference in the enzyme activity value of a-gal in primary and P, amniotic fluid cells cultured from a given case number. For 28 degrees of freedom, t = -1.50. The 95% confidence interval for 28 degrees of freedom is (-2.048, 2.048); therefore, the hypothesis was accepted. cx-Galactosidase
331
TABLE
III
ENZYME
ACTIVITY
Case No.
OF
* IN PRIMARY
&GALACTOSIDASE
Primary
amniotic
Enzyme
activity
fluid
cells
AND
PI
AMNIOTIC
FLUID
CELLS
Days
in culture
Pl
Days
Enzyme
in culture
activity
AbF
20
0.050(*0.000)
11
0.050(*0.000)
AbF
21
0.013(*0.000)
13
0.030(*0.000)
‘5
AbF
22
0.010(*0.000)
15
0.070(*0.000)
15
AbF
23
0.013(*0.000)
11
0.033(*0.000)
13
AbF
24
0.026(+0.005)
11
0.033(*0.000)
13
AbF
25
0.010(*0.000)
13
0.020(*0.000)
17
AbF
26
0.010(*0.000)
13
0.031(*0.010)
17
AbF
21
0.050(*0.000)
13
0.130(*0.020)
15
AbF
28
0.020(*0.000)
13
0.06O(iO.O10)
15
AbF
29
0.070(*0.000)
12
0.071(*0.040)
14
AbF
30
0.067(*0.000)
13
0.070(*0.000)
14
AbF
31
0.060(*0.030)
13
0.2OO(iO.O40)
16
AbF
32
0.035(*0.007)
11
0.070(*0.000)
14
AbF
33
0.250(*0.002)
6
0.400(*0.007)
10
AbF
34
0.200(*0.010)
6
0.300(*0.000)
10
AbF
35
0.030(*0.000)
0.070(*0.000)
17
* Enzyme
activity
(k) = standard Each
value
13
is expressed
in terms
of picomol
of 4-MU
released
13
. h-l.
. cell-’
deviation. represents
the
average
of
3
to
5 readings
of
droplets
containing
cells
from
the
same
case
(*S.D.).
TABLE
IV
ENZYME
ACTIVITY
Case No.
OF
wGALACTOSIDASE
Primary
amniotic
Enzyme
activity
fluid
* IN PRIMARY
cells Days
AND
PI
AMNIOTIC
FLUID
CELLS
Days
in culture
Pl in culture
Enzyme
AbF
36
0.0025(iO.O007)
0.0067(*0.0000)
12
AbF
37
0.0022(*0.0000)
12
0.0060(~0.0028)
15
AbF
38
0.0070(*0.0000)
12
0.0090(*0.0000)
15
AbF
39
0.0150(i0.0020)
11
0.0200(*0.0000)
15
AbF
40
0.0010(*0.0000)
9
0.0015(*0.0000)
12
AbF
41
0.0100(*0.0000)
8
0.0180(~0.0040)
14
AbF
42
0.0040(*0.0000)
12
0.0070(*0.0000)
15
AbF
43
0.0150(i0.0012)
12
0.0200(*0.0000)
15
AbF
44
0.0070(*0.0000)
13
0.0090(*0.0020)
15
AbF
45
0.0040(*0.0017)
11
0.0060(~0.0001)
15
AbF
46
0.0040(*0.0000)
13
0.0040(*0.0000)
15
AbF
47
0.0020(*0.0000)
9
0.0040(*0.0013)
13
AbF
48
0.0020(*0.0000)
9
0.0070(*0.0000)
13
AbF
49
0.0090(*0.0020)
9
0.0090(*0.0015)
13
AbF
50
0.0030(*0.0000)
9
0.0060(~0.0000)
13
* Enzyme
activity
(i) = standard Each (kS.D.).
value
is expressed
6
activity
in terms
of picomol
of 4-MU
released
* ceII_’
. h-I.
deviation. represents
the
average
of
3 to
5 readings
of
droplets
containing
cells
from
the
same
case
332
Discussion We have determined the enzyme activity values of three lysosomal enzymes in primary and P1 amniotic fluid cells using a microenzymatic fluorescence assay [ 141. Many metabolic disorders have either quantitative or qualitative lysosomal enzyme changes. A wide variation in lysosomal enzyme activity has been found in cultured skin fibroblasts (normal and diseased) [14] and the variation of activity may be especially great in confluent cultures [16] of normal cells assayed by the macromethods. A confluent culture is made up of a mixture of cells in different stages of the growth phase and, consequently, possessing different enzyme activities [ 131. Since the cultured cells in our assay are in the early log phase period of cell growth, we have eliminated (i) the problem of growth curve-related variations in lysosomal enzyme specific activity [7,13], (ii) the longer period of time required for cultures to attain confluency, and (iii) the variability of specific enzyme activity in confluent cultures. There have been reports on the effect of passage number on specific activity of lysosomal enzymes [17-191. Although only cells in primary culture and P1 were examined (since we were looking for early prenatal diagnosis), we have found no such effect in our microassay system. All three lysosomal enzymes assayed in this study have shown no difference between primary culture and passage number one as demonstrated by the t-test for paired comparisons. The t-test of the results for the hexosaminidase assay indicates no difference between specific enzyme activities of primary and P1 amniotic fluid cell cultures (Tables I and II) at the 5% significance level. This study also confirms the findings of our previous work that the enzyme activity and the percentages of hex A and hex B in amniotic cells are lower than those found in skin fibroblasts [ 141. The average percentage of hex A in primary amniotic cell cultures was 59% (Fig. l), in P1 amniotic cell cultures it was 58% (Fig. 2) and in skin fibroblast cultures it was 73% [ 141. However, an interesting finding in this study was the progressive change of the proportions of the hexosaminidase isozyme forms (hex A and hex B) with time in culture. From Fig. 1 and 2, we see a tendency for the percentage of hex A in cultured cells to be initially higher and to decrease with time. The percentage of hex B tends to start lower and increase with time. In Fig. 2, by the 16-17th day the percentages of hex A and hex B are approximately 55% and 45%, respectively. This is within the normal range of values (5265% and 39-48%, respectively) found for control (nondiseased) amniotic cells in the 5th to 10th passage [ 141. Prior to the 16-17th day, the range of these values is much greater; hence, after this period of time, the proportion of the two isozymes is referred to as Conventional macromethods for prenatal diagnosis seldom yield “fixed”. results earlier than 16-17 days after amniocentesis. This is due, in part, to the larger number of cultured cells required to perform an assay (106-107) [5,6]. Thus, by the time the results are available, the “fixed” proportion of hex A to hex B has already been established. The use of this microenzymatic fluorescence assay not only has the potential for an earlier prenatal diagnosis, but it also permits the study of the progressive change of proportions of the major isozyme forms of hexosaminidase with time in culture. The possibility of such
333
an event has been speculated on by others [19], but, for the first time, we can now demonstrate its occurrence by means of our sensitive microtechnique. Any effect of trypsinization on the specific enzyme activity of hexosaminidase [13] and on the changing proportions of its isozyme is ruled out by comparing primary and P1 cells with 12 and 13 days in culture (Fig. 1 and 2). The t-test indicated no difference in specific enzyme activity nor, hence, in the proportion of the two isozymes for cells cultured 12 or 13 days. The average enzyme activities of /3-galactosidase (p-gal) in amniotic fluid cells found in this study were lower (0.08 picomol of 4-MU released per cell per hour) than those obtained from skin fibroblasts (0.24 picomol of 4-MU released per cell per hour) [14]. In the present study, the t-test indicates no difference in the specific enzyme activity of p-gal in primary and P1 amniotic fluid cells. This microenzymatic fluorescence assay is sensitive enough to detect the enzyme activity values of a-gal, which are much lower than those obtained for the P-gal assay. As in the case of p-gal, the e-gal activity in amniotic fluid cell cultures shows lower values than those obtained with skin fibroblasts. Also, no difference in enzyme activity was found between primary and P1 amniotic fluid cells. The average a-gal activity for primary and P1 amniotic fluid cells was 0.0074 picomol of 4-MU released per cell per hour, compared to 0.015 picomol of 4-MU released per cell per hour for skin fibroblasts. We conclude that there is no difference in enzyme activity between primary and P1 amniotic cells for all three lysosomal enzymes studied. This microenzymatic fluorescence assay is sufficiently sensitive to allow detection of enzyme activities in amniotic cells in culture as early as 6 days after obtaining the,samples. In addition, our equipment is much less expensive than that used by others (Galjaard et al. [7-g]. Precaution is necessary for the proper interpretation of the assay of hex A and hex B because their ratio is not “fixed” until around the 15th day in culture. Therefore, prenatal diagnoses determined before a “fixed” ratio is attained could be erroneous. Acknowledgements This study was supported by grants from the U.S.P.H.S. (HD-0551509) and the National Foundation for the March of Dimes. We are grateful to Maureen L. Lo and J. Bell for their help in setting up primary amniotic fluid cell cultures. We also wish to thank Ms. Marsha L. Pawlina for her assistance in the typing of this manuscript. We wish to acknowledge partial support in the preparation of this manuscript from N.I.H. Grant No. ROl AGO 1212-01. References 1 Hoehn. H.. Rodriquez. M.L.. Norwood. T.H. and Maxwell, C.L. (1978) Mosaicism in amniotic fluid cell cultures: classification and significance. Am. J. Med. Genet. 2, 253-266 2 Van der Veer, E., Kleijer, W.J.. De Josselin de Jon& J.E. and Galjaard, H. (1978) Lysosomal enzyme activities in different types of amniotic fluid cells measured by microchemical methods, combined with interference microscopy. Hum. Genet. 40. 285-292
334
3 Kaback. MM. and Leonard. of Chicago Press, Chicago
C.O. (19721
in Antenatal
Diagnosis
(Dorfman,
A., ed.), pp. 81, University
4 Gerbie. A.B., Melancon, S.B., Ryan, C. and Nadler, H.L. (1972) Cultivated epithelial-like cells and fibroblasts from amniotic fluid: their relationship to enzymatic and cytologic analysis. Am. J. Obstet. Gynecol. 114, 314-320 5 Niermeijer, M.F.. Sachs, E.S., Jahodova, M.. Tichelaar-Klepper, C., Kleijer, W.J. and Galjaard, H. (19761 Prenatal diagnosis of genetic disorders. J. Med. Genet. 13, 182-194 6 Milunsky. A. (19761 Current concepts in genetics: prenatal diagnosis of genetic disorders. New Engl. J. Med. 295,377-380 7 Galiaard. H. (19721 Possibilities of quantitative histochemical analysis in prenatal detection and genetic classification of hereditary metabolic disorders. Histol. Cytochem. 185-186 8 Hash P. (1974) in Birth Defects (Motulsky, A.G. and Lentz. W.. eds.). pp. 226-233, Excerpta Medica, Amsterdam 9 Galiaard. H., Van Hoogstraten. the quantitative cytochemical 409-429
J.J.. De Josselin de Jong, J.E. and Mulder. M.P. (1974) Methodology of analysis of single or small numbers of cultured cells. Histochem. J. 6,
IO Gabaard, H.. Mekes, M., De Josselin de Jong. J.E. and Niermeijer, M.F. (19731 A method for rapid prenatal diagnosis of glycogenesis II (Pompe’s disease). Clin. Chim. Acta 49, 361-375 E. and Vlek-Noot. C. (1974) The use of 11 Galjaard. H., Hoogeveen. A., Keijrer. W.. De Wit-Verbeek. quantitative cytochemical analysis in rapid prenatal detection and somatic cell genetic studies of metabolic diseases. Hlstocbem. J. 6. 491-509 12 Galjaard, H., Niermeijer, M.F., Hahnemann, N.. Mohr, J. and Sorensen, S.A. (1974) An example of rapid prenatal diagnosis of Fabry’s disease using microtechniques. Clin. Genet. 5, 368-377 13 Heukels-Dully. M.J. and Niermeijer. M.F. (1976) Variation in lysosomal enzyme activity during growth in culture of human fibroblasts and amniotic fluid cells. Exp. Cell Res. 97, 304-312 14 Bladon, M.T. and Milunsky, A. (1978) Use of microtechniques for the detection of lysosomal enzyme disorders: Tay-Sachs disease, GMl-gangliosidosis and Fabry disease. Clln. Genet. 14. 359-366 15 Li, J.C.R. (1968) in Statistical Inference I. pp. 108, Edward Bros. Inc.. Ann Arbor, MI 16 Mllunsky, A., Spielvogel, C. and Kanfer, J.N. (1972) Lysosomal enzyme variations in cultured normal skin fibroblasts. Life Sci. 11, part II, 1101-1107 17 Butterworth. J., Sutherland, G.R., Broadhead. D.M. and Bain. A.D. (19731 Lysosomal enzyme levels in human amniotic fluid cells in tissue culture. Life Sci. 13, 713-722 18 Sutherland, G.R., Butterworth. J.. Broadhead, D.M. and Bain, A.D. (1974) Lysosomal enzyme levels in human amniotic fluid cells in tissue culture. II. a-Galactosidase, @-galactosidase and o-arabinosidase. Clin. Genet. 5.351-355 19 Butterworth, J., Sutherland, G.R.. Broadhead, D.M. and Bain, A.D. (1974) Lysosomal enzyme levels in human amniotic fluid cells in tissue culture. Clin. Genet. 5, 356-362 20 Swallow, D.M., Stokes, D.C., Corney, G. and Harris, H. (1974) Differences between the N-acetyl hexosaminidase isozymes in serum and tissues. Ann. Hum. Genet. 37, 287-302
Clinica Chimica Acta, 105 (1980) 325-334 @ Else~ier/North-HoIIand Biomedical Press
CCA 1442 MICROENZY~ATIC ASSAYS FOR LYSOSOMAL AMNIOTIC FLUID CELL CULTURES
MARILUCI
T. BLADON
* and AUBREY
ENZYMES IN PRIMARY
MILUNSKY
Genetics Division, Eunice Kennedy Shriver Center; and the Department of Pediatrics, Harvard Medical School and the Massachusetts General Hospital, Boston, MA (U.S.A.) (Received
November
21st, 1980)
Summary A study of three Iysosomal enzymes (hexosaminidase, fl-galactosidase and ar-galactosidase) in normal primary amniotic fluid cell cultures using a microenzymatic assay is presented. No difference in enzyme activity was found between primary and amniotic cell cultures in passage number one. A progressive change in the proportions of hexosaminidase A and hexosaminidase B with time was demonstrated in culture. The feasibility of this procedure for the early prenatal diagnosis of disorders due to lysosomal enzyme deficiency is discussed.
Amniotic fluid cell cultures are composed of a heterogeneous population of nucleated cells [ 11. These cells may behave in the following ways: (i) they may float and not attach to the culture flask, (ii) they may attach but rarely divide and give rise to individual colonies or (iii) they may attach and give rise to welldefined colonies, epithelial- or fibroblast-like cell types. The presence of these well-defined colonies (pure or mixed) of the two cell types with varying morphology in the amniotic fluid cell culture suggested that their enzyme activities might differ in relation to their origins and morphology [2]. Kaback and Leonard [3] used normal amniotic fluid cell cultures to analyze epithelial-like, fibroblastic-like and mixed cell type colonies for P-galactosidase,. arylsulfatase-A and P-D-N-acetylglucosaminidase. They found no significant difference in specific enzyme activity in the morphologically different cell types. On the other hand, Gerbie et al. [4] found significantly greater enzyme activity of
* Reprint U.S.A.
address:
Life
Science
Department,
Worcester
Polytechnic
Institute,
Worcester,
MA
01609,
326
histidase in epithelial-like and cystathionine synthetase in fibroblastic-like cell types of amniotic fluid cultures. Earlier work on the quantitative analysis of enzymes required a large number of amniotic cells (on the order of 106) [5,6]. Four to ten weeks of culture were required in order to grow a sufficient number of cells for these assays. Later, Galjaard [ 71 and Hosli [ 81 developed a microtechnique for quantitative enzyme analysis at the single cell level. Their results are based on (i) use of single amniotic cells from the primary culture [7,8], (ii) isolation and dissection of small numbers of specific cell type [Q,lO,ll], and (iii) clones of cells of mainly one cell type [12]. This technique requires only 12-13 days after amniocentesis for the results to be obtained. Depending on the enzyme, the activity may vary greatly between the two cell types 241; hence prenatal diagnosis of some enzyme disorders would produce unreliable results. In addition, Heukels-Dully et al. [13] have shown that the enzyme activity of a given cell varies with its phase of growth in culture. We published the procedure for a microenzymatic fluorescent assay which produces sn average enzyme activity per cell which is proportional to the total number of cells analyzed (100-300 cells) ]14]. All the cells analyzed are in the log growth phase of the cell cycle. The difference in cell types and, consequently, in enzyme activity is averaged out. These factors constitute si~ific~t improvements over other microenzymatic assays. We also reported the enzyme activity values for hexosaminidase in non-porno ~niotic fluid cell cultures and in skin fibroblasts, and the enzyme activity values for LY-and ~-g~actosid~e in skin fibrobl~~. It was shown that the enzyme activity of hexos~inid~e is higher in skin fibrobl~~ that in non-primary amniotic fluid cell cultures. This paper is a continuation of our previous work [14] and will (i) examine hexosaminidase activity in primary amniotic fluid cell cultures, (ii) determine the feasibility of assaying the relatively low activity values of CX-and P-galactosidase activity in amniotic fluid cells using our improved microenzymatic assay and (iii} evaluate the feasibility of using this microassay for the prenatal diagnosis of lysosomal enzyme deficiency disorders within 13 days after obtaining an amniotic fluid sample. Materials and methods
Amniotic fluid cell culture The amniotic fluid cells were obtained during elective abortions performed for non-genetic indications and cultured as follows: (1) Fluids were spun in disposable centrifuge tubes at 1600 X g for 10 min. (2) The supernatant fluid was removed leaving approximately 1.0 ml above the cell pellet. (3) 2.0 ml of 20% FCS in MEM were added to the centrifuge tubes. The total volume of 3.0 ml in each tube was well dispersed using a Pasteur pipette. (4) 0.5 ml of the resuspended cells was pipetted onto a 22-mm square cover glass (Coming No. 1) which was placed in a 35mm petri dish. This step was repeated for six petri dishes. (For a-galactosidase, the entire 3.0-ml sample was placed on one 35mm dish.)
327
(5) The cells were left on the coverglass overnight at 37°C in a 5% COZ atmosphere. (6) 2.0 ml of 20% FCS in MEM were added to each coverslip the next day and the dishes were left undisturbed in the incubator for seven days. (7) After this period, the medium was changed and dishes were examined for cell colonies. (8) The approximate number of cells per colony, as well as the predominant cell type (viz. epithelial or fibrobl~tic), were recorded. (9) If a colony had approximately 100 cells, the dish was used for the microenzymatic assay of the enzyme being studied. (10) The harvest process of the primary amniotic fluid cells was accomplished by using 0.25% trypsin as previously described [14]. An aliquot of the trypsinized cells was transferred to another 35mm petri dish with 2.0 ml of 20% FCS in MEM and left at 37°C in the CO, incubator. This is referred to as Passage No. 1 or P1. The remainder of the trypsinized primary amniotic fluid cells was used for the microenzymatic fluorescence technique. The microenzymatic assay 4.methyl4-Methylumbelliferyl-2-acetamido-2-deoxy~-D-glucopyranoside, umbelliferyl-O-D-galactopyranoside and 4-methylumbelliferyl-cw-D-galactopyranoside were used as substrates for the determination of hexosaminidase (N-acetyl-fl-D-glucosaminidase), /3-galactosidase and a-galactosidase activities, respectively. The enzyme activities were calculated using a standard curve of pure 4-methylumbelliferone (4-MU). The measuring levels of all three lysosomal enzymes presented in this study were within the linear range of their assay conditions. The apparatus used to measure the fluorescence and the procedure for assaying the three enzymes is described elsewhere [ 141. The specific enzyme activity is expressed as picomol of 4-MU released per cell per hour. Experimental
results
Hexosaminidase (N-acetyl-/3-D-glucosaminidase (EC 3.2.1.30)) A total of 19 amniotic fluid samples were assayed for both isozymes of hexosaminidase, isozyme form A {hex A) and isozyme form B (hex B). After obtaining the amniotic fluid, it took between 8 and 13 days to grow the minimum number of cells to perform the assay on the primary cell cultures. Amniotic fluid cells in P, were assayed between 12 and 17 days. Days in culture for passage No. 1 means the total number of days in culture from the plating of the primary cells until the day the cells were assayed. It ,includes the days as primary cell cultures, before the actual splitting of the cells to establish passage No. 1. The enzyme activity of total hexosaminidase includes that due to hex A and hex B. When hexosaiinidase is appropriately heat-treated, hex A is inactivated while hex B is unaffected. The enzyme activities of total and heat-treated hexosaminidase are presented in Tables I and II for primary and P, amniotic fluid cells, respectively. The t-statistic, t, for paired comparisons [ 151 was used to test the following hypothesis: there is no difference in the enzyme activity value of total hexo-
328 TABLE
I
ENZYME Case
ACTIVITY
OF
HEXOSAMINIDASE
Days
No.
* IN PRIMARY Total
in culture
AMNIOTIC
activity
FLUID Heat-treated
AbF
1
10
2.53(*1.550)
0.86(*0.250)
AbF
2
11
0.85(*0.040)
0.47(*0.000)
AbF
3
13
0.30(*0.000)
0.20(*0.000)
AbF
4
11
0.67(+0.060)
0.43(*0.120)
AbF
5
12
0.28(*0.030)
0.20(*0.007)
AbF
6
12
1.30(+0.200)
0.60(*0.000)
AbF
7
12
0.96(*0.200)
0.27(iO.O10)
AbF
8
13
1.70(*0.420)
0.53(*0.041)
AbF
9
13
1.70(*0.840)
0.50(*0.000)
AbF
10
11
1.80(+0.200)
0.50(i0.070)
AbF
11
11
0.87(+0.470)
0.55(*0.070)
AbF
12
13
0.80(*0.090)
0.27(*0.000)
AbF
13
2.06(+0.730)
0.60(*0.042)
AbF
14
0.54(
0.14(*0.090)
AbF
15
AbF
16
AbF
8 11
iO.070)
0.75(iO.210)
0.25(+0.070)
13
1.25(*0.070)
0.46(*0.060)
17
9
0.85(*0.210)
0.20(~0.000)
AbF
18
13
2.15(*0.210)
0.70(*0.000)
AbF
19
9
1.03(*0.380)
0.40(*0.180)
9
* Enzyme
activity
(k) = standard Each
value
is expressed
in terms
of picomol
of 4-MU
released
CELLS
. cell-’ . h-l.
deviation. represents
the
average
of
3
to
5 readings
of
droplets
containing
cells
from
the
same
case
from
the
same
case
(iS.D.).
TABLE
II
ENZYME
ACTIVITY
OF Days
HEXOSAMINIDASE
* IN
in culture
P1 AMNIOTIC
FLUID
CELLS
Case
No.
AbF
1
15
1.80(+0.140)
0.60(~0.000)
AbF
2
13
0.42(iO.100)
0.15(*0.040)
AbF
3
15
0.72(*0.300)
0.37(*0.090)
AbF
4
13
0.62(*0.020)
0.33(*0.000)
AbF
5
14
0.85(+0.300)
0.27(*0.000)
AbF
6
14
1.50(*0.800)
0.40(*0.000)
AbF
7
14
1.42(*0.120)
0.74(+0.110)
Total
Heat-treated
activity
AbF
8
15
0.40(*0.000)
0.26(iO.O50)
AbF
9
16
0.95(*0.500)
0.50(*0.000)
AbF
10
13
0.63(*0.140)
0.26(+0.110)
AbF
11
17
0.40(*0.000)
0.18(iO.O20)
AbF
12
15
0.75(
AbF
13
12
0.90(*0.140)
0.26(iO.O00)
0.30(*0.170)
iO.070)
AbF
14
13
0.70(*0.520)
0.30(*0.000)
AbF
15
12
0.55(*0.070)
0.20(*0.000)
AbF
16
15
0.65(+0.070)
0.35(*0.070)
AbF
17
12
1.10(*0.000)
0.53(iO.320)
AbF
18
15
1.72(*1.100)
0.45(*0.210)
AbF
19
12
0.65(*0.210)
0.18(iO.O40)
* Enzyme
activity
(*) = standard Each (G.D.).
value
is expressed
in terms
of picomol
of 4-MU
released
. cell-’ . h-l.
deviation. represents
the
average
of
3
to
5 readings
of
droplets
containing
cells
329
saminidase in primary and PI amniotic fluid cells cultured from a given case number. The same hypothesis was tested for heat-treated hexosammidase. For total hexosaminidase, t = 1.73 with 36 degrees of freedom. For heat-treated hexosaminidase, t = 1.45 with 36 degrees of freedom. The 95% confidence interval for t with 36 degrees of freedom is (-2.029, 2.029); therefore, both hypotheses were accepted. Fig. 1 and 2 show percentages of hex A and hex B in primary and PI amniotic fluid cells, respectively, versus the number of days in culture. When the percentages of hex A and hex B in primary amniotic fluid cells were compared to those of PI amniotic cells to ascertain the effect of trypsinization, no difference was found between the two treatments. The t-test was used to test the following hypothesis: trypsinization has no effect on the percentage of hex A (and, consequently, hex B) assayed from amniotic fluid cells obtained from primary and PI cultures from a given case number. The t-test was 0.20 with 36 degrees of freedom. The 95% confidence interval or acceptance region for t with 36 degrees of freedom is (-2.029, 2.029); therefore the hypothesis was accepted. Furthermore, the t-test was used to test the following hypothesis: on the 12th day the percentage of hex A (and, consequently, hex B) is the same for primary and PI amniotic cells obtained from a given case number. The same hypothesis was tested for the 13th day. (The number of days for which primary amniotic fluid cells were cultured was between 8 and 13. The number of days for which PI amniotic fluid cells were cultured was between 12 and 17. Hence, it was only for 12 and 13 days in culture that we could test these hypotheses.) On the 12th day, t = 0.08 with 5 degrees of freedom, and on the 13th day, t = 0.72 with 8 degrees of freedom. The 95% confidence intervals for
100 90 80 70 m P
60 50
B 4 2 2 bE
40 30 20
:: :: 0 II::
. :: ::Ill h HI2
116X B
A
1 1T
:: :: :: :: I
:.
::
::
10
::
::
0
::
I?
?3) DAYS
ii
IN CULTURE
Fig. 1. The percentages of hex A and hex B in primary cell cultures. Where more than one primary amniotic cell culture is cultured for the same number of days. the percentage is the average. (No) specifies the number of cultures analyzed for each day of culture. On each bar is indicated the standard deviation of the mean.
330 100 90
70 m
60
E I
50
ES 4
40
2 I k +S
. .$ in * HEXA
80
30
HEXB
I
.*
PO
.
I
.
1
::
.
.
I
.
,
.
.
.
10
.
a, . . .
.
.
.
0
-
.
::
12,
DAYS
1 .
IN CULTURE
Fig. 2. The percentages of hex A and hex B in PI amniotic cell cultures. Where more than one PI amniotic cell culture is cultured for the same number of days, the percentage is the average. (No) specifies the the number of cultures analyzed for each day of culture. On each bar is indicated the standard deviation of the mean.
t with 5 and 8 degrees of freedom are (-2.571, 2.571) and (-2.306, respectively; therefore, both hypotheses were accepted.
2.306),
p-GaZactosidase (EC 3.2.1.23) A total of 16 amniotic fluid samples were assayed for /3-galactosidase (P-gal). Table III presents the values obtained with primary and P1 amniotic fluid cells. After obtaining the amniotic fluid, it took at least six days to grow the minimum number of cells to perform the assay. P, amniotic fluid cells were assayed between 10 and 17 days. The t-test was used to test the following hypothesis: there is no difference in the enzyme activity value of p-gal in primary and P1 amniotic fluid cells cultured ‘from a given case number. For 30 degrees of freedom, t = -1.34. The 95% confidence interval for 30 degrees of freedom is (-2.042, 2.042); therefore, the hypothesis was accepted. (EC 3.2.1.22) A total of 15 amniotic fluid samples were assayed for a-galactosidase (a-gal). Table IV presents the enzyme activity values for primary and P, amniotic fluid cells. We were able to assay for a-gal in primary cultures as early as six days after obtaining the fluids. P1 amniotic fluid cells were assayed between 12 and 15 days. The t-test was used to test the following hypothesis: there is no difference in the enzyme activity value of a-gal in primary and P, amniotic fluid cells cultured from a given case number. For 28 degrees of freedom, t = -1.50. The 95% confidence interval for 28 degrees of freedom is (-2.048, 2.048); therefore, the hypothesis was accepted. cx-Galactosidase
331
TABLE
III
ENZYME
ACTIVITY
Case No.
OF
* IN PRIMARY
&GALACTOSIDASE
Primary
amniotic
Enzyme
activity
fluid
cells
AND
PI
AMNIOTIC
FLUID
CELLS
Days
in culture
Pl
Days
Enzyme
in culture
activity
AbF
20
0.050(*0.000)
11
0.050(*0.000)
AbF
21
0.013(*0.000)
13
0.030(*0.000)
‘5
AbF
22
0.010(*0.000)
15
0.070(*0.000)
15
AbF
23
0.013(*0.000)
11
0.033(*0.000)
13
AbF
24
0.026(+0.005)
11
0.033(*0.000)
13
AbF
25
0.010(*0.000)
13
0.020(*0.000)
17
AbF
26
0.010(*0.000)
13
0.031(*0.010)
17
AbF
21
0.050(*0.000)
13
0.130(*0.020)
15
AbF
28
0.020(*0.000)
13
0.06O(iO.O10)
15
AbF
29
0.070(*0.000)
12
0.071(*0.040)
14
AbF
30
0.067(*0.000)
13
0.070(*0.000)
14
AbF
31
0.060(*0.030)
13
0.2OO(iO.O40)
16
AbF
32
0.035(*0.007)
11
0.070(*0.000)
14
AbF
33
0.250(*0.002)
6
0.400(*0.007)
10
AbF
34
0.200(*0.010)
6
0.300(*0.000)
10
AbF
35
0.030(*0.000)
0.070(*0.000)
17
* Enzyme
activity
(k) = standard Each
value
13
is expressed
in terms
of picomol
of 4-MU
released
13
. h-l.
. cell-’
deviation. represents
the
average
of
3
to
5 readings
of
droplets
containing
cells
from
the
same
case
(*S.D.).
TABLE
IV
ENZYME
ACTIVITY
Case No.
OF
wGALACTOSIDASE
Primary
amniotic
Enzyme
activity
fluid
* IN PRIMARY
cells Days
AND
PI
AMNIOTIC
FLUID
CELLS
Days
in culture
Pl in culture
Enzyme
AbF
36
0.0025(iO.O007)
0.0067(*0.0000)
12
AbF
37
0.0022(*0.0000)
12
0.0060(~0.0028)
15
AbF
38
0.0070(*0.0000)
12
0.0090(*0.0000)
15
AbF
39
0.0150(i0.0020)
11
0.0200(*0.0000)
15
AbF
40
0.0010(*0.0000)
9
0.0015(*0.0000)
12
AbF
41
0.0100(*0.0000)
8
0.0180(~0.0040)
14
AbF
42
0.0040(*0.0000)
12
0.0070(*0.0000)
15
AbF
43
0.0150(i0.0012)
12
0.0200(*0.0000)
15
AbF
44
0.0070(*0.0000)
13
0.0090(*0.0020)
15
AbF
45
0.0040(*0.0017)
11
0.0060(~0.0001)
15
AbF
46
0.0040(*0.0000)
13
0.0040(*0.0000)
15
AbF
47
0.0020(*0.0000)
9
0.0040(*0.0013)
13
AbF
48
0.0020(*0.0000)
9
0.0070(*0.0000)
13
AbF
49
0.0090(*0.0020)
9
0.0090(*0.0015)
13
AbF
50
0.0030(*0.0000)
9
0.0060(~0.0000)
13
* Enzyme
activity
(i) = standard Each (kS.D.).
value
is expressed
6
activity
in terms
of picomol
of 4-MU
released
* ceII_’
. h-I.
deviation. represents
the
average
of
3 to
5 readings
of
droplets
containing
cells
from
the
same
case
332
Discussion We have determined the enzyme activity values of three lysosomal enzymes in primary and P1 amniotic fluid cells using a microenzymatic fluorescence assay [ 141. Many metabolic disorders have either quantitative or qualitative lysosomal enzyme changes. A wide variation in lysosomal enzyme activity has been found in cultured skin fibroblasts (normal and diseased) [14] and the variation of activity may be especially great in confluent cultures [16] of normal cells assayed by the macromethods. A confluent culture is made up of a mixture of cells in different stages of the growth phase and, consequently, possessing different enzyme activities [ 131. Since the cultured cells in our assay are in the early log phase period of cell growth, we have eliminated (i) the problem of growth curve-related variations in lysosomal enzyme specific activity [7,13], (ii) the longer period of time required for cultures to attain confluency, and (iii) the variability of specific enzyme activity in confluent cultures. There have been reports on the effect of passage number on specific activity of lysosomal enzymes [17-191. Although only cells in primary culture and P1 were examined (since we were looking for early prenatal diagnosis), we have found no such effect in our microassay system. All three lysosomal enzymes assayed in this study have shown no difference between primary culture and passage number one as demonstrated by the t-test for paired comparisons. The t-test of the results for the hexosaminidase assay indicates no difference between specific enzyme activities of primary and P1 amniotic fluid cell cultures (Tables I and II) at the 5% significance level. This study also confirms the findings of our previous work that the enzyme activity and the percentages of hex A and hex B in amniotic cells are lower than those found in skin fibroblasts [ 141. The average percentage of hex A in primary amniotic cell cultures was 59% (Fig. l), in P1 amniotic cell cultures it was 58% (Fig. 2) and in skin fibroblast cultures it was 73% [ 141. However, an interesting finding in this study was the progressive change of the proportions of the hexosaminidase isozyme forms (hex A and hex B) with time in culture. From Fig. 1 and 2, we see a tendency for the percentage of hex A in cultured cells to be initially higher and to decrease with time. The percentage of hex B tends to start lower and increase with time. In Fig. 2, by the 16-17th day the percentages of hex A and hex B are approximately 55% and 45%, respectively. This is within the normal range of values (5265% and 39-48%, respectively) found for control (nondiseased) amniotic cells in the 5th to 10th passage [ 141. Prior to the 16-17th day, the range of these values is much greater; hence, after this period of time, the proportion of the two isozymes is referred to as Conventional macromethods for prenatal diagnosis seldom yield “fixed”. results earlier than 16-17 days after amniocentesis. This is due, in part, to the larger number of cultured cells required to perform an assay (106-107) [5,6]. Thus, by the time the results are available, the “fixed” proportion of hex A to hex B has already been established. The use of this microenzymatic fluorescence assay not only has the potential for an earlier prenatal diagnosis, but it also permits the study of the progressive change of proportions of the major isozyme forms of hexosaminidase with time in culture. The possibility of such
333
an event has been speculated on by others [19], but, for the first time, we can now demonstrate its occurrence by means of our sensitive microtechnique. Any effect of trypsinization on the specific enzyme activity of hexosaminidase [13] and on the changing proportions of its isozyme is ruled out by comparing primary and P1 cells with 12 and 13 days in culture (Fig. 1 and 2). The t-test indicated no difference in specific enzyme activity nor, hence, in the proportion of the two isozymes for cells cultured 12 or 13 days. The average enzyme activities of /3-galactosidase (p-gal) in amniotic fluid cells found in this study were lower (0.08 picomol of 4-MU released per cell per hour) than those obtained from skin fibroblasts (0.24 picomol of 4-MU released per cell per hour) [14]. In the present study, the t-test indicates no difference in the specific enzyme activity of p-gal in primary and P1 amniotic fluid cells. This microenzymatic fluorescence assay is sensitive enough to detect the enzyme activity values of a-gal, which are much lower than those obtained for the P-gal assay. As in the case of p-gal, the e-gal activity in amniotic fluid cell cultures shows lower values than those obtained with skin fibroblasts. Also, no difference in enzyme activity was found between primary and P1 amniotic fluid cells. The average a-gal activity for primary and P1 amniotic fluid cells was 0.0074 picomol of 4-MU released per cell per hour, compared to 0.015 picomol of 4-MU released per cell per hour for skin fibroblasts. We conclude that there is no difference in enzyme activity between primary and P1 amniotic cells for all three lysosomal enzymes studied. This microenzymatic fluorescence assay is sufficiently sensitive to allow detection of enzyme activities in amniotic cells in culture as early as 6 days after obtaining the,samples. In addition, our equipment is much less expensive than that used by others (Galjaard et al. [7-g]. Precaution is necessary for the proper interpretation of the assay of hex A and hex B because their ratio is not “fixed” until around the 15th day in culture. Therefore, prenatal diagnoses determined before a “fixed” ratio is attained could be erroneous. Acknowledgements This study was supported by grants from the U.S.P.H.S. (HD-0551509) and the National Foundation for the March of Dimes. We are grateful to Maureen L. Lo and J. Bell for their help in setting up primary amniotic fluid cell cultures. We also wish to thank Ms. Marsha L. Pawlina for her assistance in the typing of this manuscript. We wish to acknowledge partial support in the preparation of this manuscript from N.I.H. Grant No. ROl AGO 1212-01. References 1 Hoehn. H.. Rodriquez. M.L.. Norwood. T.H. and Maxwell, C.L. (1978) Mosaicism in amniotic fluid cell cultures: classification and significance. Am. J. Med. Genet. 2, 253-266 2 Van der Veer, E., Kleijer, W.J.. De Josselin de Jon& J.E. and Galjaard, H. (1978) Lysosomal enzyme activities in different types of amniotic fluid cells measured by microchemical methods, combined with interference microscopy. Hum. Genet. 40. 285-292
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3 Kaback. MM. and Leonard. of Chicago Press, Chicago
C.O. (19721
in Antenatal
Diagnosis
(Dorfman,
A., ed.), pp. 81, University
4 Gerbie. A.B., Melancon, S.B., Ryan, C. and Nadler, H.L. (1972) Cultivated epithelial-like cells and fibroblasts from amniotic fluid: their relationship to enzymatic and cytologic analysis. Am. J. Obstet. Gynecol. 114, 314-320 5 Niermeijer, M.F.. Sachs, E.S., Jahodova, M.. Tichelaar-Klepper, C., Kleijer, W.J. and Galjaard, H. (19761 Prenatal diagnosis of genetic disorders. J. Med. Genet. 13, 182-194 6 Milunsky. A. (19761 Current concepts in genetics: prenatal diagnosis of genetic disorders. New Engl. J. Med. 295,377-380 7 Galiaard. H. (19721 Possibilities of quantitative histochemical analysis in prenatal detection and genetic classification of hereditary metabolic disorders. Histol. Cytochem. 185-186 8 Hash P. (1974) in Birth Defects (Motulsky, A.G. and Lentz. W.. eds.). pp. 226-233, Excerpta Medica, Amsterdam 9 Galiaard. H., Van Hoogstraten. the quantitative cytochemical 409-429
J.J.. De Josselin de Jong, J.E. and Mulder. M.P. (1974) Methodology of analysis of single or small numbers of cultured cells. Histochem. J. 6,
IO Gabaard, H.. Mekes, M., De Josselin de Jong. J.E. and Niermeijer, M.F. (19731 A method for rapid prenatal diagnosis of glycogenesis II (Pompe’s disease). Clin. Chim. Acta 49, 361-375 E. and Vlek-Noot. C. (1974) The use of 11 Galjaard. H., Hoogeveen. A., Keijrer. W.. De Wit-Verbeek. quantitative cytochemical analysis in rapid prenatal detection and somatic cell genetic studies of metabolic diseases. Hlstocbem. J. 6. 491-509 12 Galjaard, H., Niermeijer, M.F., Hahnemann, N.. Mohr, J. and Sorensen, S.A. (1974) An example of rapid prenatal diagnosis of Fabry’s disease using microtechniques. Clin. Genet. 5, 368-377 13 Heukels-Dully. M.J. and Niermeijer. M.F. (1976) Variation in lysosomal enzyme activity during growth in culture of human fibroblasts and amniotic fluid cells. Exp. Cell Res. 97, 304-312 14 Bladon, M.T. and Milunsky, A. (1978) Use of microtechniques for the detection of lysosomal enzyme disorders: Tay-Sachs disease, GMl-gangliosidosis and Fabry disease. Clln. Genet. 14. 359-366 15 Li, J.C.R. (1968) in Statistical Inference I. pp. 108, Edward Bros. Inc.. Ann Arbor, MI 16 Mllunsky, A., Spielvogel, C. and Kanfer, J.N. (1972) Lysosomal enzyme variations in cultured normal skin fibroblasts. Life Sci. 11, part II, 1101-1107 17 Butterworth. J., Sutherland, G.R., Broadhead. D.M. and Bain. A.D. (19731 Lysosomal enzyme levels in human amniotic fluid cells in tissue culture. Life Sci. 13, 713-722 18 Sutherland, G.R., Butterworth. J.. Broadhead, D.M. and Bain, A.D. (1974) Lysosomal enzyme levels in human amniotic fluid cells in tissue culture. II. a-Galactosidase, @-galactosidase and o-arabinosidase. Clin. Genet. 5.351-355 19 Butterworth, J., Sutherland, G.R.. Broadhead, D.M. and Bain, A.D. (1974) Lysosomal enzyme levels in human amniotic fluid cells in tissue culture. Clin. Genet. 5, 356-362 20 Swallow, D.M., Stokes, D.C., Corney, G. and Harris, H. (1974) Differences between the N-acetyl hexosaminidase isozymes in serum and tissues. Ann. Hum. Genet. 37, 287-302