Acid hydrolases in amniotic fluid and chorionic villi at various stages of pregnancy

221

Clinica Chimica Acta, 53 (1974) 221-227 @ Elsevier Scientific Publishing Company,

Amsterdam

- Printed

in The Netherlands

CGA 6407

ACID HYDROLASES IN AMNIOTIC FLUID AND CHORIONIC VARIOUS STAGES OF PREGNANCY

B. HULTBERG,

S. SJ6BLAD

and B. GUSTAV11

Department of Clinical Chemistry and Department Hospital, S-221 85 Lund (Sweden) (Received

February

VILLI AT

of Obstetrics and Gynecology,

University

8, 1974)

Summary A study was made at various stages of pregnancy of five acid hydrolases which occur in amniotic fluid and chorionic villi and which are relevant to serious storage disorders. In amniotic fluid /3-galactosidase and a-mannosidase decreased moderately towards term, while /_I-glucosidase decreased markedly. N-Acetyl-fl-glucosaminidase and @-glucuronidase were relatively unchanged. In chorionic villi N-acetyl-/3-glucosaminidase, /3-galactosidase, and a-mannosidase were substantially decreased towards term, while fl-glucosidase was unchanged and &ducuronidase markedly increased. In both amniotic fluid and chorionic villi the enzyme pattern was approximately the same as that found in liver in a previous study. The findings suggest that these enzyme assays might be useful in the diagnosis of inborn errors prenatally by using amniotic fluid, and early postnatally by using chorionic villi.

Introduction The analysis of amniotic fluid is useful in the prenatal diagnosis of both genetic and non-genetic conditions. Knowledge of the origin and quantitative behaviour of the enzymes (or other proteins) in amniotic fluid would probably increase the diagnostic potential of amniocentesis. The sources of the protein in amniotic fluid are still obscure [ 1,2] . Some genetic disorders are related to a lack or significant decrease in activity of various lysosomal enzymes (acid hydrolases). Such a deficiency leads to failure of intracellular digestion, resulting in storage disorders. At present at least 20 cases of such diseases are known [3] . Tay-Sachs [4] and Pompe’s disease [ 51 have been diagnosed prenatally by assay of the activities of acid hydrolases in amniotic fluid. For early post-

222

natal diagnosis foetal tissues have been used, for example skin (fibroblast cultivation), brain or liver. Recently Wiederschain et al. [6] described a simpler way for making an early postnatal diagnosis, namely the use of a biopsy specimen of the placenta. This paper reports the properties and activity pattern of five acid hydrolases in amniotic fluid and chorionic villi at various stages of pregnancy, and discusses their usefulness for prenatal diagnosis and early postnatally from biopsy specimens of the placenta. The enzymes studied (P-galactosidase, /3-glucosidase, a-mannosidase, @glucuronidase, IV-acetyl-P-glucosaminidase) are all relevant to inborn errors of a serious nature. Material and Methods Amniotic

fluid

Amniotic fluid in 27 women in the 14th to 40th weeks of pregnancy was obtained by amniocentesis. The fluid was centrifuged at 1000 X g for 15 min at +4” ) and the clear supernatant was removed. Enzyme assays were carried out within 1 week of the sampling, during which time the material was stored at -20”. Chorionic

villi

Biopsy specimens of the placenta were obtained from 24 women in the 10th to 12th weeks of pregnancy by vacuum aspiration (12 cases) and at normal term deliveries (12 cases). Free chorionic villi were excised from the placentae, rinsed in physiological saline, and then stored at -20” until analysis which was always done within 1 week. Before analysis the frozen tissue was homogenized in 50 vols of distilled water. Enzyme assays The enzyme

activities were assayed with the following synthetic substrates: 4-methylumbelliferyl-fl-D-galactopyranoside* for the assay of /3-galactosidase (p-D-galactoside galactohydrolase, EC 3.2.1.23), 4-methylumbelliferyl0-D -glucopyranoside* for the assay of fl-glucosidase (0-D -glucoside glucohydrolase, EC 3.2.1.21), 4-methylumbelliferyl-a-D-mannopyranoside* for the assay of (Y-mannosidase (a-D-mannoside mannohydrolase, EC 3.2.1.24), p-nitrophenyl-acetamido-2-deoxy-P-glucopyranoside * for the assay of N-acetyl-fl-glucosaminidase (fl-2-acetylamino-2-deoxy-D-glucoside acetylamino-deoxyglucohydrolase, EC 3.2.1.30), and phenolphthalein glucuronide** for the assay of P-glucuronidase (P-D-glucuronide glucuronohydrolase, EC 3.2.1.31). The enzyme assay methods were carried out as previously described [7] with the following exceptions: 1 M citrate buffer, pH 5.5, was used for incubation in the assay of /3-glucosidase and cu-mannosidase in amniotic fluid and 1 M citrate buffer, pH 5.0, was used for incubation in the assay of P-glucosidase in chor-ionic villi.

* Purchased from Koch-Light Laboratories, Colnbrook, England. ** Purchased from Sigma Chemical Co., St. Louis, U.S.A.

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Gel filtration was performed with Sephadex G-150 (Pharmacia Fine Chemicals, Uppsala, Sweden) as previously described [7], and isoelectric focusing was performed with commercial equipment (LKB-Produkter, Stockholm, Sweden) as described in the same paper. A volume of 4 ml of supernatant from centrifuged homogenate (100000 X g, 60 min) or centrifuged amniotic fluid was used for experiments with gel filtration and isoelectric focusing. Loss of activity during storage

In enzyme assays of amniotic fluid stored for six months at -2O”, a-manand N-acetyl-P-glucosaminidase lost up to 10% of nosidase, /3-glucuronidase, their original activity, /3-galactosidase, 30-40%, and @-glucosidase 50%. Results pH dependency

In placental tissue the pH dependency of the five acid hydrolases is very similar to that found in liver and fibroblasts [ 7,8] (Fig. 1). In amniotic fluid, however, there are two differences (Fig. 2). Firstly, P-galactosidase exhibits two pH optima, one around pH 4.5 and the other

0

160

1

*

t

< 3

4

5

6

7

I

4

5

I

epH

,

3

4

5

6

7

3

4

5

6

7 PH

Fig. 1. PI-I Dependency of five acid hydrolases in placental tissue obtained from three women in the 10th ) by vacuum aspiration and from three normal term deliveries to 12th weeks of pregnancy (O------O A, fl-galactosidase; B. fl-glucosidase; C, a-mannosidase; D. N-acetyl+glucosaminidase: E, (* -*). p-glucuron.idase. Fig. 2. pH Dependency of five acid hydrolases in amniotic fluid obtained by amniocentesis in three 0) and in three women in the 36th to 40th week women in the 10th to 16th weeks of pregnancy (O*). A, P-galactosidase; B, P-glucosidase; C, or-mannosidase; D, N-acetyl-fl-glucoof pregnancy (*saminidase: E, &glucuronidase.

224

Uil

8-GALACTOSIDASE

u

O-GLLK3SlOASE

I

.

o-MANNOSIOASE

u/t

N.ACETYL-8-GC”COSAM,N,OASE

.

u:,

D- GLUCURO~I~SE

I

Fig. 3. Specific

activity

pattern

of five acid hydrolases

in relation

to gestational

age.

around pH 5.5. The latter probably reflects the @-glucosidase activity which also acts on the 4-methylumbellif~~l-~-g~actoside substrate [9] . Secondly, c+ma.nnosidase exhibits a pH optimum around pH 5.5-6.0, probably depending upon the preponderance, in amniotic fluid, of the a-mannosidase isoenzyme called cr-mannosidase C [ 10.1.

Specific ~etivity ~uttern of acid hydrolyses in autistic

fluid

&Glucuronidase and N-acetyl-P-glucosaminidase activities were relatively unchanged towards term, while /3-galactosidase, a-mannosidase, and p-glucosidase clearly decreased (Fig. 3).

Specific ~ct~uity pattern of acid hydrolases in chorioaic villi The enzyme activity of fl-galactosidase, N-acetyl-@glucosaminidase

and a-mannosidase in chorionic villi decreased towards term, while that of /3-glucuronidase increased and that of P-glucosidase remained largely unchanged (Table I). In chorionie villi the enzyme activities were lower than in liver tissue, with the exception of N-acetyl-fl-glucosaminidase activity, which was higher.

Isoelectric focusing On isoelectric focusing of amniotic fluid and ehorionic villi preponderantly $galactosidase and &lucosidase forms were found at a fow pH (3.8-4.3). This implied the presence of high molecular forms [7]. cw-Mannosidase exhibited a heterogeneous activity between pH 5.0 and pH 7.0 and N-acetyl-fl-glucosaminidase activity was split into the A and B forms, as described earlier [7]. ~-Glucuronidase activity was seen only around pH 3.9-5.0 in amniotic fluid, while in chorionic villi activity was seen also around pH 7.0, as in liver [7].

225 TABLE I ACID HYDROLASES

IN CHORIONIC

VILLI AND LIVER

AII figures given as mean + S.D. Activity expressed as U/g protein. @Galactosidase

@Glucosidase

a-Mannosidase

N-Acetyl-flglucosaminidase

fl-Glucuronidase

3.52 f 1.15 1.65 t 0.28 4.19 ? 3.29

0.288 0.247 0.631

2.35 k10.52 1.06 f 0.28 5.18 f 5.15

133.8 f 58.5 43.2 f 8.5 13.3 t 5.49**

1.06 r 0.26 1.78 f 0.48 4.32 f 1.76

Chotionic viIIi: 10-12 weeks, ” = 12 38-41 weeks, n = 12 Liver *

f 0.110 f 0.040 + 0.31

* Control values of a-galactosidase, a-mannosidase, N-acetvl-P-glucosaminidase, and P-glucuronidase were obtained from a study by Lundquist a:ld &kerman [151 (n = 24). The values of P-glucosidase are unpublished data (n = 6). ** Analysis of N-acetyl-P-glucosaminidase in liver was carried out in acetate buffer, while citrate buffer was used in analysis of chorionic vilIi (see Discussion).

Gel filtration High molecular forms of fl-galactosidase and /3-glucosidase were seen on gel filtration. Thus much of the activities was excluded from the gel and some also eluted earlier than the monomer forms of the enzymes [7,9]. The other enzymes showed elution volumes similar to those found in liver preparations [ 71. Discussion The five acid hydrolases studied showed definite activities in amniotic fluid and chorionic villi. The isoenzyme patterns were principally the same as previously found in liver [ 71. In previous studies [6,11--131 complex changes of enzyme activity were found in amniotic fluid as gestation proceeded. In the present study three of the enzymes @-galactosidase, a-mannosidase, /3-glucosidase) decreased, while the other two (/3-glucuronidase, N-acetyl-fl-glucosaminidase) were relatively unchanged. On comparison of the activities of acid hydrolases in amniotic fluid in the present investigation with those found in plasma in a previous study [14], P-glucuronidase and N-acetyl-/3-glucosaminidase showed about the same activity, while fl-galactosidase and a-mannosidase activities were higher in plasma. At term, /3-glucosidase activity in plasma was the same as that in amniotic fluid. But these results may depend upon inhibitors and activators in plasma or amniotic fluid. Hence no conclusions can be drawn regarding the placental circulation or the source of the enzymes. With the exception of N-acetyl-P-glucosaminidase, the activities of the enzymes investigated were lower in chorionic villi than in liver. The assay of N-acetyl-&glucosaminidase in liver was performed with acetate buffer [15]. Incubation with citrate buffer doubles the activity. Nonetheless the N-acetyl-/3glucosaminidase activity was still somewhat lower in liver. Complex time patterns of acid hydrolases in placental tissue have been

226

reported [6,16,17]. We found a decrease in /3-galactosidase, a-mannosidase, and N-acetyl-fl-glucosaminidase towards term, while /3-glucosidase remained unchanged, and fl-glucuronidase increased. Thus, the activity of a-mannosidase and fl-galactosidase in chcrionic villi as well as in amniotic fluid decreased, while P-glucuronidase increased. However, because of the disparity of /3-glucosidase and N-acetyl-/3-glucosaminidase activity in chorionic villi and amniotic fluid it is not possible to draw any conclusions about the origin of the lysosomal enzymes in amniotic fluid. N-Acetyl-Pglucosaminidase activity decreased in chorionic villi and did not change much in amniotic fluid, while /3-glucosidaseshowed the opposite pattern. The best time to obtain viable amniotic cells for culture and prenatal diagnosis seems to be the 15th week of pregnancy [18]. A biochemical diagnosis requires about 2 weeks owing to the time necessary for growth of a suitable number of cells. Assaying enzyme activities in amniotic fluid, however, permits a diagnosis within 1 day, The enzyme activities in amniotic fluid are low, but should allow detection of an enzyme deficiency in about the 15th week of pregnancy, which has proved possible in N-acetyl-/3-glucosaminidase A deficiency (Tay-Sachs disease) [ 41 and a-glucosidase deficiency (Pompe’s disease) [51. Our finding of relatively high enzyme activity in chorionic villi indicates that this tissue can be used in the diagnosis of specific enzyme deficiencies immediately after birth. However, the chorionic villi must be carefully separated from the decidual tissue, and thoroughly rinsed in physiological saline to avoid contamination with maternal plasma. In this study we found that the distribution of the enzyme activities in chorionic villi differed from that previously found in plasma [13] , which indicates that the chorionic villi activities measured were intrinsic. In this investigation we studied five enzymes relevant to inborn lysosomal storage disease, i.e. /3-galactosidase (relevant to GM1 -gangliodosis), /3-glucosidase (Gaucher’s disease), a-mannosidase (mannosidosis), N-acetyl-/3-glucosaminidase (Tay-Sachs disease and its variants), and /3-glucuronidase (/3-glucuronidase deficiency, a new disease described by Sly et al. [19] ). It seems probable that these diseases could be diagnosed prenatally from amniotic fluid and early postnatally from placental biopsy instead of from the foetus. Acknowledgements The present study was supported by grants from the Medical Faculty, University of Lund. Skilful technical assistance was given by Mrs. Sonja Stolpe. References D.R.

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