Enzymatic activity of glycogen metabolism in chorionic villi

Placenta 0986), 7, 505-5o9

Enzymatic Activity of Glycogen Metabolism in Chorionic Villi M. C H O W E R S a, D. A B E L I O V I C H b, R. P O T A S H N I K a & N. B A S H A N a,c Paediatric Laboratory, Soroka Medical Centre and Clinical Biochemistry Unit, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheva 841o5, Israel b Cytogenetics Laboratory, Soroka Medical Centre, Beer Sheva, Israel c To whom correspondence should be addressed Paper accepted 8.4.1986

INTRODUCTION The glycogen storage diseases (GSDs) are a group of hereditary disorders caused by a lack of specific enzymes involved in glycogen synthesis or breakdown, and are characterized by the accumulation of abnormal amounts or types of glycogen in the tissues. The incidence of all forms of G S D is about one in 4o ooo. Inheritance is as a rule autosomal recessive, except type IXa which is X-linked. Antenatal diagnosis of several of the GSDs is based on the detection of the enzymatic deficiency in cultured amniotic cells obtained from second-trimester pregnancies (Butterworth and Broadhead, 1977; Besley et al, I983). However, amniocentesis cannot be performed until 16 weeks of pregnancy, and additional time (two to five weeks) is required for cell culture and diagnostic enzyme studies. Thus by the time an affected fetus is diagnosed, abortion is undesirably late. A chorionic villi (CV) biopsy performed some time between 7 and i2 weeks of gestation is a new approach in prenatal diagnosis that enables decisions on pregnancy termination for genetic reasons to be made within the first trimester (Kazy, Rozowsky and Bakherev, 1982; Simoni et al, i983). In view of this new approach, the aim of this study was to determine the presence and levels of glycogen metabolism enzyme activities in CV tissue.

MATERIALS AND METHODS Normal CV tissue from 7 to 12 menstrual week pregnancies was obtained from the placentae of women undergoing voluntary abortion. Placental tissue was immediately placed in sterile Hanks solution. Within I h of abortion, 3 ~ to 4 ~ mg of CV tissue was dissected under stereoscope and separated from maternal cells. After dissection, CV samples were kept at - 7 o ~ for i to 6 weeks, or tested immediately. The karyotype was determined from each CV sample in direct 505

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Placenta (t986), Vol. 7

preparation as described by Simoni et al (1983). Tissue cultures were performed as described by Heaton et al (1984). A fraction of each sample was analysed for glycogen concentration (Johnson, Nash and Fusaro, I963). The remainder of each sample of CV tissue was homogenized in water in a microhomogenizer. T h e cells were ultrasonically disintegrated by applying 2o kilocycles for 20 sec at 4~ The sonicate was centrifuged at i o o o o g for l o m i n at 4~ T h e clear supernatant was incubated with o.I per cent digitonin for 15 min at 4~ and was subsequently used for enzyme assays. Activities of the following CV, liver and muscle enzymes were measured by conventional methods: glucose-6-phosphatase (Steinitz, i967) , ~-glucosidase (Salatsky and Nadler, i973) , amylo-i,6-glucosidase (Steinitz, i967) , total phosphorylase (Stalmans et al, 197o), phosphorylase kinase (Bashan et al, x98 I). Protein concentration was measured according to Lowry et al (i951). The signal-to-noise ratio of each assay was at least three.

RESULTS Twenty-three chorionic villi (CV) samples were examined. T h e CV tissue was dissected in order to separate it from the decidual cells. Karyotype analysis of these samples revealed IZ cases of 46,XY type and i i cases of 46,XX type. All the karyotypes of tissue cultures which were established from the CV were identical to those of the direct cell preparations, and no case with 46,XY/46,XX mosaicism was found. It was therefore concluded that the CV preparations were of fetal origin and were not contaminated with maternal cells. We examined glycogen concentration and the activities of six enzymes of glycogen metabolism which may be deficient in glycogen storage diseases (GSDs): :r amylo1,6-glucosidase, phosphorylase a and b, phosphorylase kinase and glucose-6-phosphatase (Table i). T h e activity of amylo-i,6-glucosidase was found to be of the same order of magnitude as that in liver and muscle. ~-Glucosidase activity is one order of magnitude higher in CV tissues. On the other hand, the activities ofphosphorylase b and of phosphorylase kinase are about one order

Table i.

Biochemicaltissue analysis of chorionic villicompared with liver and muscle Chorionic villi

Glycogen (%) Amylo-i,6-glucosidase(% incorporation/g protein/h) ~-Glucosidase(nmole 4-methylumbelliferone/mg protem/min) Phosphorylase kinase (U phosphorylasea/g protein/min) Phosphorylase+ AMP (#mol/g protem/min) Phosphorylase- AMP (gmol/g proteln/min) Glueose-6-phosphatase(tlmol P/g protem/min)

o.22+o.12 (n = I9) 6.6+2 6 (n =

I3)

1.9+ I.o (n = 23)

1.5+- 1.0 i5.o+3.i

0.5+__0.05

1o.o+2.3

o.12___o.o7

o,o86_+o.ox4

68+I 4

34x+iio

(n= 12)

17o+83

352+Ioo

12)

81 +3 ~

x6I +--85

32A+I6.o(n= 15) z5+z2

Liver (n = IO) Muscle(n = to)

o

(n =

o

(n=3)

The values given are mean + s.d. AMP = adenosine monophosphate.

4.o+I.2

o

Chowers et al: Glycogen Metaboh'sm in Chorionic Villi

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of magnitude lower in CV tissue. N o activity of either glucose-6-phosphatase or phosphorylase a was found. T h e following kinetic properties o f C V phosphorylase were found (Table 2): (i) activation by sodium sulphate; (2) inhibition by cysteine; (3) partial stimulation of the b form by adenosine monophosphate (AMP). (The activity of the b form in the presence of A M P was 25 #mol/min/g protein, whereas the a form produced by phosphorylase kinase in the presence of Mg 2+ and adenosine triphosphate (ATP) was 35/~mol/min/g protein). These properties are characteristic of liver phosphorylase found in the adult animal. In order to determine the minimum amount of tissue required for enzyme assays, enzymatic activity was tested as a function of tissue wet weight (Figure i). It is evident that the minimum amount of tissue required for the study of~-glucosidase, phosphorylase or phosphorylase kinase is 5 mg, whereas for amylo-i,6-glucosidase it is about 15 mg. As the amount of tissue obtained from a CV biopsy is about 25 to 40 mg, any one of these enzymes can be assayed in such a biopsy.

DISCUSSION It has been established that prenatal diagnosis for ~-glucosidase and amylo-i,6-glucosidase deficiencies (glycogen storage disease (GSD) types II and I I I ) can be performed on amniotic cells. Enzymatic activity of amylo-i,6-glucosidase and of ~-glucosidase was found in normal chorionic villi (CV) (Table i, Figure I). It is therefore to be expected that CV analysis in the first trimester of pregnancy can be useful for the detection of amylo-i,6-glucosidase and ~glucosidase deficiencies. Indeed, utilizing CV prenatal diagnosis for G S D type I I has been done recently (Besancon et al, i985). On the other hand, glucose-6-phosphatase (which is deficient in G S D I) can normally be found only in liver, kidney and intestinal mucosa (Steinitz, 1967). It is therefore not surprising that this enzyme could not be detected in cultured amniotic cells (Howell and Williams, I983) or in CV (Table i). It follows that the only way to make an antenatal diagnosis o f G S D I by enzyme assay is by performing a liver biopsy. As the phosphorylase b activity found in CV represented (by kinetic parameters) liver enzyme (Table 2), it is conceivable that G S D VI (liver phosphorylase deficiency) can be diagnosed by CV assay. However, this assumption has to be tested on actual cases. Phosphorylase kinase deficiency (type IX G S D ) can appear as a generalized disease (IXc) or may be limited to liver (IXa,b) or muscle (Lederer et al, I98o; Bashan et al, i981; Lerner et al,

Table 2. Effectof sulphate, cysteineand adenosinemonophosphate(AMP) on phosphorylase

activity in chorionic villi (CV) Phosphorylase activity (~tmol/min/gprotein) Addition None 0.4 mol/l Na2SO4 15 mmol/l cysteine

AMP in assay mixture (2 raM)

beforeactivation

after activation

+ + +

o 25 36 I7

35 38 ND ND

Phosphorytase activity was measured in homogenate of CV as described in 'Materials and methods'. Activationwas performedby incubationof CV for x h at 37~ with the addition of io mM Mg2+ and 3 mM adenosine triphosphate (ATP). ND = not determined.

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Placenta (.986), Vol. 7

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blgure 1. ~-Glucosidase,phosphorylase,phosphorylasekinaseand amylo-i,6-glur activitiesas a functionof chorionicvilli (CV)tissue weight.The enzymatic~ctivitiesweremeasuredas describedin 'Materialsand methods'.

1982; Abarbanel et al, I985). From these studies, it is not possible to decide which of the variants is represented by CV. Another parameter for the diagnosis of G S D is the chemical determination of the glycogen content of affected tissue. In our study the mean CV glycogen concentration was o.2i per cent with a range from 0.05 to 0.47 per cent; this is significantly lower than the accepted normal range in liver or muscle. It is reasonable to assume that in enzymatically deficient CV a higher glycogen concentration will be found. Whether this assumption is valid can only be established after testing CV tissue from G S D patients. On the basis of the data presented in this study, it is conceivable that G S D II, III and VI can be diagnosed in CV tissue. This finding is expected to facilitate first-trimester diagnosis of the diseases. However, only the testing of CV enzymes in families at risk, and the correlation to enzymatic activities in fetal organs, will prove the value of CV analysis for the antenatal diagnosis of GSD.

Chowers etal: Glycogen Metabolism in Chorwnie Vilh

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SUMMARY Glycogen content and six major enzymatic activities involved in glycogen metabolism were analysed in chorionic villi (CV). Glycogen levels were found to be lower than those known to exist "in liver and muscle. Activities o f ~-glucosidase, amylo-l,6-glucosidase, phosphorylase b and phosphorylase kinase were detectable by standard methods. T h e enzymatic activities o f glucose-6-phosphatase and phosphorylase a were undetectable. T h e s e findings suggest that C V biopsies can be useful for first-trimester diagnosis o f glycogen storage disease types H, I l l and VI, but not for type I (glucose-6-phosphatase deficiency).

REFERENCES Abarbanel, J. M., Bashan, N., Potashnik, R. et al 0985) Adult muscle phosphorylase kinase deficiency. Neurology, 36, 560-562. Appleman, M. M., Krebs, E. G. & Fischer, E. H. (1966) Purification and properties of inactive liver phosphorylase. Biochemistry, 5, 2[ol-21o7Bashan, N., Iancu, T. C , Lerner, A. et al (198i) Glycogenosis due to liver and muscle phosphorylase kinase deficiency. Pediatric Research, 15, 299-303. Besancon, A.-M., Casterlau, L., Lieolesco, FI. et ai (J985) Prenatal diagnosis of glycogenosis type II (Pompe's disease) using chorionic villi biopsy. Clinical Genetics, 27, 479-482. Besley, G. T., Cohen, P. T., Faed, M. S. & Woistenholme, J. 0983) Amylo-i,6-glucosidase activity in cultured ceils. A deficiency in type IIl glycogenosis with pro-natal studies. Prenatal Diagnosis, I, 13-19. Butterworth, J. & Broadhead, D. M. (t977) Diagnosis of Pompe's disease in cultured skin fibroblast and primary amniotic fluid cells using 4~ as substrate. Clinica ChimicaActa, 78, 335-342Corl, C. F , Cori, G. T. & Green, A. A. (i943) Crystalline muscle phosphorylase III. Kinetics. Journal of Biological Chemistry, ;r51~ 39~55. Heaton, D. E., Czepu|kowski, B. H., Horwell, E. H. & Coleman, D. V. (1984) Chromosome analysis of first trimester chorionic villus biopsies prepared by a maceration technique. Prenatal Diagnosis, 4, 279-287. Howell, R. R. & Williams, J. C. {1983) The glycogen storage diseases. In The Metabolic Basis of Inherited Diseases (Ed.) Stanbury, J. B, Wyngaarden, J. B., Fredrickson, D. S. etal. pp. 141-166. New York: McGraw-Hill. Johnson, J. A., Nash, J. D. & Fusaro, R. M. 0963) An enzymatic method for the quantitative determination of glycogen. Analytical Biochemistry, 5, 379-387 9 Kazy, Z., Rozowsky, 1. S. & Bakherev, V. A. (I982) Chorionic biopsy in early pregnancy: a method of early prenatal diagnosis for inherited disorders. Prenatal Diagnosis, 2, 39-45. Lederer, B., Van de Werve, G., De Barsy, T. & Hers, H. G. (I98o) The autosomal form of phosphorylase kinase deficiency in man: reduced activity of muscle enzyme. Biochemical and Biophysical Research Communications, 92, 169~74. Lerner, A., lancu, T. C., Bashan, N. et al 0982) A new variant of glycogen storage disease. AmericanJournal of Diseases of Children, 136, 4o6-4to. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (~95i) Protein measurement with the folin phenol reagent. Journal of Biological Chemistry, I93 , 265-275. Poenaru, L., Kaplan, L., Dumez, J. & Dreyfus, J. C. (1984) Evaluation of possible first-trimester prenatal diagnosis in lysosomal disease by trophoblast biopsy. Pediatric Research, xB, to32-to34 . Poenaru, L., Casterlau, L., Choiset, A. et al (t985) Lysosomal hydrolase activity in chorionic villi and embryonic ceils in culture. Human Genetics, 69, 378-379. Salatsky, I. S. & Nadler, H. L. (I973) A fluorimetric assay of alpha-glucosidase and its application in the study of Pompe's disease. Journal of Laboratory and Clinical Medicine, 81, 45o-454 . Simoni, G., Branbati, B., Danesino, C. et a} (~983) Efficient direct chromosome analyses and enzyme determinations from chorionic villi samples in the first trimester of pregnancy. Human Genetics, 63, 349-357. Stalmans, W., De-Wulf, H., Lederer, B. & Hers, H. G. (197o) The effect of glucose and of a treatment by glucocordcoids on the inactivation in vitro of liver glycogen phosphorylase. European Journal oJBiochemistry, 15, 9-~2. Steinitz, K. (i967) Laboratory diagnosis of glycogen diseases. In Advances in Clinical Chemistry (Ed.) Sobntka, H. & Stewart, C. P. pp. 227-354. New York & London: Academic Press.