Enzymes in Amniotic Fluid

BRUCE F. WATKINS, Ph. D. and E. W. BERMES, Ph.D.

Departments of Pathology and Biochemistry and Biophysics, Loyola University Medical Center, Maywood, IL 60153

ABSTRACT

The determination of enzyme levels in cell-free amniotic fluid has pro ven useful in assessing fetal maturity and fetal well being, and is being utilized for the prenatal diagnosis of genetic disorders. The activities of amylase, a-galactosidase, phosphatidic acid phosphohydrolase, lysozyme and heat-stable alkaline phosphatase in amniotic fluid increase with gesta tional age and have an established relationship to fetal maturity. The ratio of amniotic fluid diamine oxidase activity to maternal serum activity (am niotic DAO/serum DAO) may be used as an indicator of the degree of rhesus isoimmunization after 28 weeks gestation. Creatine phosphokinase in amniotic fluid is elevated in cases of in utero fetal death and is of diagnostic significance. The prenatal diagnosis of Tay-Sachs disease, Sand- hoffs disease, fucosidosis, GMi-gangliosidosis and I-cell disease have been made from the analysis of appropriate enzymes in cell-free amniotic fluid.

Introduction development of more sensitive methods for enzyme analyses have made possible Since the pioneering work of Bevis2 on routine enzyme assays on amniotic fluid. the spectrophotometric analysis of amni Direct analysis of cell-free amniotic fluid otic fluid, many reports have appeared has several advantages over methods concerning the usefulness of various which involve the growing of cells from biochemical analyses of amniotic fluid in the fluid in culture. In direct analysis, reducing perinatal mortality and morbid smaller volumes of amniotic fluid are ity. Some of these laboratory studies, usually required, the technical difficul aimed at ascertaining fetal status or the ties involved in cell culture are avoided relationship of the fetus to its environ and the results are available much ment, have involved the determination of sooner. the activities of enzymes in the amniotic Some enzymes in amniotic fluid may fluid. result from the steady exchange of The acceptance of transabdominal am water and solute between intra-uterine niocentesis as a safe procedure and the compartments involving, among other * Supported in part by a N IH Research Training less understood pathways, fetal swallow Grant in Clinical Chemistry 5 T01 GM02122-03. ing and urinary excretion. Still others 2 3 1 232 WATKINS AND BERMES may be present in the fluid owing to the stage of gestation.1*31*37 The concen the lysis of cells shed from fetal mem trations of phospholipids have been branes. Even though the origin of most shown to provide valuable information enzymes in amniotic fluid has not been concerning fetal pulmonary maturity, thoroughly delineated, enzyme analysis thus making it possible to predict the of amniotic fluid has proven useful in susceptibility of a newborn to respiratory assessing fetal maturity5,14,30 and fetal distress syndrome.2*4 well being,17,18,44 and is being utilized Many enzymes show a change in their for the prenatal diagnosis of genetic activity at well defined times during ges disorders.6,7,12,19,20,22,28 tation. The activities of amniotic fluid amylase,5 a-galactosidase30 and phos Determination of Fetal Maturity phatidic acid phosphohydrolase14 as a function of fetal maturity have been During the latter half of pregnancy, the studied. Their established relationship to volume and composition of amniotic fluid fetal maturity is shown in figure 1. change significantly. Some of the biochemical changes are sufficiently pro gressive and regular so that they may be A m y l a s e utilized in the determination of the gesta tional age of the fetus and its well being. Amylase activities remain less than 200 The osmolality of amniotic fluid and the I.U. in cell-free amniotic fluid until 36 concentration of creatinine, bilirubin, weeks, at which time abrupt increases are uric acid, and estriol have been related to observed.5 When a value of 250 I.U. was taken as indicative of fetal maturity, the incidence of false positives was 0.5 per ENZYME ACTIVITY IN AMNIOTIC FLUID cent and that of false negatives was 38 percent. When the amylase activity was calculated per unit weight of total amni otic fluid protein and a value of 65 I.U. per g considered indicative of maturity, the incidence of false positives was 1.5 per cent and the incidence of false negatives was 22.3 percent. This study included a large percentage of patients with tox emia, rhesus incompatibility and diabe tes. Only one case is reported where these conditions affected the amniotic fluid amylase value. In this toxemic pa tient, an amylase activity of 834 I.U. was found at 31 weeks gestation. The lecithin/ spingomyclin (L/S) ratio was 3.04, an in dication of fetal maturity.24 F i g u r e 1. The activities of amniotic amylase • -), a-galactosidase (-----), and phosphatidic The source of amylase activity may be acid phosphohydrolase (...... ) as a function of ges both the fetal pancreas and the salivary tational age. At term, the enzyme activities are greater than 651. U. per gof amniotic fluid protein for glands, and the route of transmission to amylase, 4.3 x 10“3 I.U. per g of amniotic fluid pro the amniotic fluid may be the urine or tein for a-galactosidase and 1.61.U. per gof amniotic fluid protein for phosphatidic acid phosphohydro saliva.46 This is supported by the finding lase. that the isoenzyme pattern of amniotic ENZYMES IN AMNIOTIC FLUID 2 3 3 fluid amylase resembles that of newborn P hosphatidic A c i d urine.41 It has also been reported that the P hosphohydrolase (P A P a s e ) activity of amniotic fluid amylase was un PAPase is an enzyme which catalyzes affected by maternal serum activities as the hydrolytic cleavage of phosphatidic high as 16001.U. in a patient with chronic acid to form 1, 2-diglycerides, which act pancreatitis and a pancreatic pseudo as acceptors of phosphorylcholine in the cyst.41 formation of lecithin in the fetal lung.16 Amylase activity has been determined Since amniotic fluid is inhaled and in the amniotic fluid, after separation of exhaled by the fetus,42 this mechanism suspended cells by centrifugation, by may be responsible for the transport of either a classical procedure using starch both PAPase and lecithin to the fluid sur as substrate or a test using a dye-labeled rounding the fetus. The specific activity substrate.5 of PAPase in amniotic fluid remains con stant at less than 15 nmol of phosphate a-GALACTOSIDASE released per mg of protein per hour be fore 30 weeks gestation. The activity in The activity of a-galactosidase in am creases to its maximum, 100 nmol of niotic fluid has been shown to be a sensi phosphate released per mg per hour, at tive indicator of gestational age. The ac 37 weeks.14 This increase in activity has tivity remains at less than 2 mU per g pro been shown to parallel the increase in tein before 30 weeks after which it rises L/S ratio but to begin earlier. When the rapidly to a range of 8.2 ± 3.9 mU per g value of the L/S ratio was found to be protein at term. Using an activity value of greater than 2, the level of PAPase activ 2.7 mU per g protein as indicative of at ity was greater than 30 nmol phosphate least 33 weeks gestation, 89 percent of released per mg protein per hour. The the patients at less than 33 weeks and 93 determination of this enzyme in the am percent greater than 33 weeks gestation niotic fluid may be of use in the evalua were correctly diagnosed. This criterion tion of fetal lung maturation. for gestional age was found to be correct PAPase activity is assayed by incuba in 60 of 66 cases studied.30 These results tion of phosphatidic acid with amniotic are consistent with those of a larger study fluid at 37° for 40 min and by measuring which included women with rhesus the amount of inorganic phosphate liber isoimmunization.3 ated. The activity is defined as nmol of The a-galactosidase activity can be de phosphace released from the phosphatidic termined fluorimetrically by measuring acid per mg of total amniotic fluid protein the liberation of 4-methylumbelliferone per hour. The enzyme has maximal activ when using 4-methylumbelliferyl-a-D- ity at pH 6.0, and its action is inhibited by galactoside as the substrate. The unit for Mg++, Be++,Tween20 and KF.15 After col a-galactosidase activity is defined as that lection, the samples were chilled in ice amount of enzyme which hydrolyses 1 and centrifuged at 4°. The supernatant nmol of substrate per min at 37° (1 mU). fraction has been stored up to three weeks When the ratio of enzymatic activity to at -20° with no loss in activity. the amniotic fluid total protein was used, the relative increase in activity at term was accentuated. After centrifugation of L y s o z y m e s the fluid, the supernatant can be stored at The lysozyme activity in amniotic fluid 4° for two months or at —60° for two years obtained at the time of delivery of a nor with no alteration in activity.30 mal full term infant was found to be in the 2 3 4 WATKINS AND BERMES range of9.3 ± 3.0 mg per 1 by diffusion on constant after 25 weeks and is less than the agarose-gel.10 The activity of lysozyme in activity in the amniotic fluid. amniotic fluid as a function of gestional The activities of DAO in both amniotic age has been studied4, and a progressive fluid and maternal serum from pregnan increase in absolute concentration up to cies complicated by rhesus isoimmuniza 40 weeks gestation has been found. The tion are within normal ranges if the fetus is mean value for lysozyme activity in pa only mildly or moderately affected. In tients with uncomplicated pregnancies cases where the fetus is severely affected, between 14 and 19 weeks gestation was in the level of DAO activity in amniotic fluid the range of 5.1 ± 1.7 fig per ml and rose shows a slight increase, but there is a large to 18.9 ± 4.3 fig per ml at 38 to 40 weeks overlap with the normal range. The ma gestation. ternal serum level of DAO is usually within the normal range, as it is in most complications of pregnancy.43 H e a t S t a b l e A l k a l i n e P h o s p h a t a s e It was found, however, that the ratio of In normal pregnancy, there is an in amniotic fluid DAO activity to maternal crease in the activity of heat stable al serum activity (amniotic DAO/serum kaline phosphatase (HSAP) in amniotic DAO) after 28 weeks gestation was a sig fluid from a range of 1.71 ± 0.185 at 35 nificant indicator of the degree of rhesus weeks gestation to a range of 2.60 ± 0.329 isoimmunization.44 The value for this ratio King-Armstrong units at 38 weeks. In in mild, moderate, and unaffected preg pre-eclampsia, the activity of HSAP is nancies was found to be in the range of significantly higher than normal in the 2.7 ± 1.9. The range found in severely af amniotic fluid with a range of 2.71 ± 0.491 fected pregnancies was 7.7 ± 2.6. The use at 35 weeks and increasing to a range of of this ratio in conjunction with the A450 4.33 ± 1.118 King-Armstrong units at 38 nm peak for bilirubin can provide addi weeks. A temperature of 56° was used for tional information about fetal involve the denaturation of “non-placental” al ment in rhesus isoimmunized pregnan kaline phosphatase.17 Others report a bet cies after the 28th week of gestation. ter correlation if a temperature of 65° is A study of lactate dehydrogenase used for the denaturation.10 (LDH) activity and the ratio of the isoen zymes in amniotic fluid has been made in rhesus isoimmunized pregnancies. The Rhesus Isoimmunization results of this study indicate that there is The activity of diamine oxidase (DAO) no significant difference in either the total in amniotic fluid has been used in the LDH activity or in the isoenzyme dis assessment of fetal well being in preg tribution between sensitized and non nancy complicated by rhesus isoim sensitized pregnancies.21 These results munization.44 In cell-free amniotic fluid are not consistent with an earlier report from normal pregnancies, there is an in that the activity of LDH 5 was uniformly crease in DAO activity from 40 m U per at 9 elevated in the amniotic fluid when the to 12 weeks gestation to a level of 6,000 fetus was severely affected.39 A study of mU per at 28 weeks. The activity remains both acid phosphatase and alkaline phos at approximately this value after 28 weeks phatase activities in sensitized pregnan with only a slight decrease towards term. cies showed no significant changes of the The activity of DAO in maternal serum activities in amniotic fluid from normal also shows an increase from 200 mU per at pregnancies.39 This is also true for all of 9 to 12 weeks gestation to 1,500 mU per at the lysosomal enzymes thus far studied in 25 weeks. The serum activity remains amniotic fluid.3 ENZYMES IN AMNIOTIC FLUID 2 3 5

Fetal Death niocentesis 12 days later, when a mark edly elevated CK activity was found in The diagnosis of fetal death in utero has the amniotic fluid.18 However, the use of been made by the measurement of creatine kinase (CK) activity in amniotic LDH activities to diagnose fetal death is limited by the lack of specificity of this fluid.18,36 Unlike the use of a sharp de enzyme and the possibility of maternal crease in the estriol level in the maternal erythrocyte contamination of the amnio urine,29 this test is independent of gesta tic fluid sample. tional age and does not require serial 24- hour urine collections to confirm the diagnosis. The amniotic fluid CK activity Inborn Errors of Metabolism is elevated above the normal value of 0 to The analysis of enzyme activities in 10 mU per ml (normal maternal serum ac cell-free amniotic fluid has been used in tivity: 15 to 145 mU per ml) within three prenatal diagnosis of several metabolic days after fetal death. In a study of 247 diseases (table I). The use of this normal pregnancies, there were no sam technique has been criticized because ples which exhibited an elevated CK the enzyme activity measured may not be level.18 The activity of CK in amniotic specifically of fetal origin and, therefore, fluid in the case of fetal death showed a the activity may not reflect the true 10-fold to 180-fold increase; maternal biochemical activity in the fetal tissues. serum activities were not elevated. This Experience, however, has shown the use increase in amniotic fluid CK activity is fulness of this procedure, and the follow apparently due to the release of the en ing errors of metabolism have been suc zyme by decomposing fetal tissue. cessfully diagnosed in utero by enzyme analysis of cell-free amniotic fluid. In one case studied, it was found that an elevated level of LDH (2.5 times the Tay- Sachs D isease upper limit of normal) in the presence of a normal CK activity foreshadowed fetal Tay-Sachs disease or GM2-ganglio- death. This was confirmed by repeat am sidosis Type I is the most frequently oc

TABLE I

Enzyme Activity in Cell-free Amniotic Fluid from Patients with Inborn Errors of Metabolism

D isease Enzyme Normal Affected

Tay-Sachs disease7 Hexosaminidase Total: 658 nmol/hr/ml Total: 529 nmol/hr/ml A: 4.2-35.6% total on A: none detected acrylamide gel A: 5.7-32% total on A: 8.1-10.2% heat fractionation Tay-Sachs disease2 8 Hexosaminidase Total: 283-592 nmol/hr/ml Total: 350-726 nmol/hr/ml A: 11-26% total A: 3-8% total Sandhoff's disease^ Hexosaminidase Total: 580-1020 nmol/hr/ml Total: 12.5 nmol/hr/ml A: 11-30% total A: 20% Fucosidosis2^ ct-L-Fucosidase 41-48.4 nmol/hr/ml 3.0-5.5 nmol/hr/ml a-D-Mannosidase 5-10 nmol/hr/ml 7.8 -1 2 . 0 nmol/hr/ml Pompe's disease2 2 a-1,4 Glucosidase 3.1-7.7 mol/min/g Unchanged GM ]_-gangliosidosis1 9 B-Galactosidase 23.3-61.3 nmol/hr/ml 0 . 0 I-Cell disease1 2 3-Glucuronidase 0.33 nmol/min/ml 1.67 nmol/min/ml Arylsufatase A 0 . 2 nmol/min/ml 0.83 nmol/min/ml a-Galactosidase 0.06 nmol/min/ml 0.148 nmol/min/ml 3 -Galactosidase 0.003 nmol/min/ml 0.016 nmol/min/ml Hexosaminidase 6.26 nmol/min/ml 54.7 nmol/min/ml Acid phosphatase 0.92 nmol/min/ml 0.97 nmol/min/ml a-Glucosidase 1.13 nmol/min/ml 1.35 nmol/min/ml 2 3 6 WATKINS AND BERMES curring ganglioside storage disease at 37°. The total activity of hexosa known. The disease occurs predomi minidase in the affected pregnancies nantly among individuals of Ashkenazi- was normal or slightly elevated. The Jewish ancestry, and the number of percentage of total activity owing to known cases is in the thousands. The hexosaminidase A was determined by a onset of symptoms occurs in the first six heat denaturation method and found to months of life and the disease runs a fatal be less than 8 percent (normal: 11 to 26 course. The patient usually expires from percent). If acrylamide gel electro bronchopneumonia by three years of phoresis is used to separate the hexo age.26 The detection of heterozygous in saminidase fractions, the gels can dividuals by serum hexoaminidase assay be quantitated by fluorimetric scanning is possible,8,27,47 and there is a need for a after incubation with the 4-methyl- rapid method for in utero screening in umbelliferyl -/3 - D - N - acetylglucosa- high-risk pregnancies. mine. By use of this method, no The enzymatic defect in Tay-Sachs hexosaminidase A activity was detected disease is a deficiency in hexosaminidase in the cell-free amniotic fluid of affected A.26 This hexosaminidase component is fetuses.7 separable from the B form by elec The work that has been done on the trophoresis,8 by heat dénaturation of prenatal diagnosis od Tay-Sachs disease hexosaminidase A,28,47 and by pH inacti seems to demonstrate that direct analysis vation of hexosaminidase A.33 The pre of cell-free amniotic fluid is as reliable as natal diagnosis of Tay-Sachs disease by other methods, and may be the method of enzymatic assay of cell-free amniotic choice as the diagnosis can be made on the fluid has been made and compared with same day as the amniocentesis is per methods using both cultured and uncul formed. tured amniotic fluid cells. The reliability of diagnosis made using cell-free fluid S a n d h o f f ’s D i s e a s e has proved to be as good as those made Sandhoffs disease is a variant of Tay- from analysis of cultured cells. The dif Sachs disease which results from the lack ference in value between controls and af of both /3-N-acetyl-hexosaminidase A and fected fetuses is smaller for this method B activities. The clinical course of the dis than that obtained with uncultured cells ease is similar to classical Tay-Sachs dis and is much less than that obtained with ease except that it has occurred only in cultured cells.28 The use of acrylamide families with no Jewish ancestry. The dis gel electrophoresis for fractionation of ease is diagnosed by finding a reduction of the hexosaminidase has been found to activity of both A and B isoenzymes to differentiate better between controls and about 10 percent of normal in leukocytes affected fetuses.7 or serum.27 In a study of 15 high-risk pregnancies, The diagnosis of Sandhoff’s disease has five diagnoses of Tay-Sachs disease were been made in utero by the demonstration made in utero on the basis of deficient of marked deficiencies of hexosamin hexosaminidase A activity in the cell-free idase A and B activities in cell-free amni amniotic fluid.28 The total hexosa otic fluid.6 In one case the total minidase activity was determined by hexosaminidase activity in the affected measuring the amount of liberated pregnancy was only 12.5 nmol of 4- 4-methylumbelliferone upon incubation methylumbelliferyl-/3- N -acetylglucosa- of the amniotic fluid with 4-methyl- mine hydrolyzed per ml per hr compared umbelliferyl- /3- D- N-acetylglucosamine to a control value mean of 842 nmol per ENZYMES IN AMNIOTIC FLUID 2 3 7 ml per hr. The diagnosis was confirmed crease slightly near term from a value of 3 after termination of the pregnancy by nmol substrate hydrolysed per min per g demonstration of deficient activity of protein to a value of 10 nmol substrate both hexosaminidase A and B in the fetal hydrolysed per min per g protein. Storage tissues. of the amniotic fluid for six months at -20° caused a loss in activity of 30 to 40

G m1-Gangliosidosis percent.13 GMi-gangliosidosis is an hereditary au P o m p e ’s D is e a s e tosomal recessive mucopolysaccharidosis in which lysosomes are overloaded with Pompe’s disease is a fatal disorder componds requiring /3-galactosidase for caused by a deficiency of the lysosomal their degradation. In tissue samples from enzyme acid a-1, 4-glucosidase thus re patients with the disease, there is an over sulting in the accumulation of glycogen in all decrease in /3-galactosidase and a-L- the tysosomes of affected tissues. arabinosidase activity.25 The activity of Symptoms manifest themselves during both of these enzymes in cell-free amni- the first few months of life and the disease otic fluid from normal pregnancies has is characterized by cardiac enlargement been reported.3 and muscular weakness.11 The in utero The prenatal diagnosis of GMi- diagnosis of Pompe’s disease has been re gangliosidosis has been made by ported in the literature on the basis of a showing the absence of /3-galactosidase deficiency of a-1, 4-glucosidase in cell- activity in cell-free amniotic fluid at 14 free amniotic fluid.23 However, the same weeks gestation.19 Amniotic fluid from investigators found normal activity in the normal pregnancies has sufficient amniotic fluid from another affected /3-galactosidase activity to be measured pregnancy.22 by a fluorimetric assay. A range of 23.3 Glycogen is degraded in lysosomal to 61.3 nmol per ml per hr was found structures by two isoenzymes. These two for normal amniotic fluid in the period isoenzymes have different pH optima at 4 of 14 to 20 weeks gestation. The amni and 7. The isoenzyme absent in Pompe’s otic fluid from the affected pregnancy disease has an optimum at pH 4 and af was found to have a complete absence of fected patients show normal activity of /3-galactosidase activity. The activity of the isoenzyme with an optimum of pH hexosaminidase in the same amniotic 7.11 The enzyme found in the amniotic fluid sample was measured and a value of fluid from both normal and affected 590 nmol per ml per hr (normal: 244 to 644 pregnancies shows an optimum at pH 6, nmol per ml per hr) was found. This sec the same as found for an isoenzyme pres ond enzyme served as an internal control ent in the kidney of both normal and af for the analysis. The diagnosis of GMi- fected patients,34 The kidney may be the gangliosidosis was confirmed by his source of the amniotic isoenzyme. The tologic examination of the fetus after ter enzymes have similar properties except mination of the pregnancy at 17 weeks.19 that the amniotic isoenzyme is inacti A fluorimetric assay using 4-methyl- vated by heating at 45° while the kidney umbelliferyl - /3 - D - galactopyranoside as isoenzyme is stable at this temperature. substrate has been used for the measure The highest activity of the amniotic fluid ment of /3-galactosidase activity in amni enzyme is found in that fraction otic fluid.13 In normal amniotic fluid, the sedimented with the lysosomes by ultra enzyme exhibits two pH optima at 4.5 and centrifugation,35 apparently indicating 5.9. The enzyme activity was found to in that the enzymatic activity resulted from 2 3 8 WATKINS AND BERMES the lysis of cells present in the amniotic tissues but increased activity in the body fluid. However, cultured and uncultured fluids of affected patients.45 amniotic fluid cells have revealed an The activity of acid hydrolase enzymes isoenzyme similar to that found in liver in the amniotic fluid of a fetus afflicted cells and not the kidney type enzyme. with I-Cell disease has been studied, and The amnion was ruled out as the source the prenatal diagnosis of I-Cell disease of this isoenzyme.34 has been made.12 The activities of acid Although the prenatal diagnosis of phosphatase and a-glucosidase were Pompe’s disease cannot be made by the found to be normal. The activities of direct analysis of cell-free amniotic fluid, /3-g.lucuronidase, arylsulfatase A, it can be made by determination of en a-galactosidase, /3-galactosidase, and zyme levels in uncultured as well as cul hexosaminidase were all significantly tured amniotic fluid cells.9 elevated. The normal values in amniotic fluid for acid phosphatase,3,38

F u c o s i d o s i s a-glucosidase3,35,38 /3-glucuronidase,3'13 a-galactosidase,30 y3-galactosidase19 and Fucosidosis is transmitted by an au hexosaminidase3,38 have been reported tosomal gene and results in the absence elsewhere. In that study of the affected of activity of the enzyme a-L-fucosidase. pregnancy, a normal amniotic fluid sam The prenatal diagnosis of fucosidosis by ple from a pregnancy of the same gesta measurement of the activity of a-L- tional age was used as an external control fucosidase in cell-free amniotic fluid has and the analysis of acid phosphatase been reported.20 The enzyme activity in served as an internal control. fluid from an affected twin pregnancy at 14 and 18 weeks gestation was only 10 percent of the activity of a control sample. References

The diagnosis was confirmed after deliv 1. BERMES, E. W., J r . and CROLLA, L.: Determi ery by demonstration of an absence of nation of uric acid, creatinine, total protein, and osmotality in amniotic fluid as a measure a-L-fucosidase activity in white blood of fetal maturity. Amniotic Fluid. Natelson, S., cells obtained from the identical twins. Scommegna, A., and Epstein, M. B., eds. New The activity of the enzyme a-D-man- York, John Wiley and Sons, pp. 95-99, 1974. 2. Bevis, D. C.: Composition of liquor amnii in nosidase was measured in the same amni haemolytic disease of newborn. Lancet 2:443, otic fluid as an internal control, and its 1950. activity was found to be slightly elevated. 3. B u t t e r w o r t h , J., B r o a d h e a d , D. M., S u t h e r l a n d , G. R., and B a i n , A. D.: Lysosomal enzymes of amniotic fluid in rela I-Ce l l D isease tion to gestational age. Amer. J. Obstet. Gynecol. 119:821-828, 1974. I-Cell disease is a mucopolysaccha 4. C h e r r y , S. H ., F i l l e r , M., an d H a r v e y , H .: Lysozyme content of amniotic fluid. Amer. J. ridosis without mucopolysacchariduria Obstet. Gynecol. 116:639-642, 1973. and is classified as Type II 5. D e C a s t r o , A. F., U s a t e g u i-Go m e z , M., and mucolipidosis. It is characterized by the SPELLACY, W. N.: Amniotic fluid amylase. Amer. J. Obstet. Gynecol. 226:1931-1936, presence of coarse cytoplasmic inclu 1973. sions in cultured fibroblasts and an 6. D e s n ic k , R. J., K r iv t t , W., and S h a r p , H. L,: Hurler-like syndrome. It is an autosomal In utero diagnosis of SandhofPs disease. Biochem. Biophys. Res. Commun. 51:20-26, recessive trait and death usually occurs 1973. between one and nine years of age.40 7. F r i e d l a n d , J ., P e r l e , G., S a i f e r , A ., Most lysosomal enzymes, with the excep SCHNECK, L., and V OLK , B. W.: Screening for Tay-Sachs disease in utero using amniotic tion of /3-glucosidase and acid phos fluid. Proc. Soc. Exp. Biol. Med. 23 6:1 29 7- phatase, show decreased activity in the 1298, 1971. ENZYMES IN AMNIOTIC FLUID 2 3 9

8. F r i e d l a n d , J., S c h n e c k , L., S a i f e r , A., 24. N e l s o n , G. H.: Lecithin concentration of am Po r u f a r , M., and VOLK, V. W.: Identification niotic fluid as an index to fetal maturity. Amnio of Tay-Sachs disease carriers by acrylamide gel tic Fluid. Natelson, S., Scommegna, A., and electrophoresis. Clin. Chim. Acta 28:397-400, Epstein, M. B., eds. New York, John Wiley and 1970. Sons, pp. 221-236, 1974. 9. G a l j a a r d , H., M e k e s , M ., D e J o s s e l in D e - 25. O’B r ie n , J.: G Mi-gangliosidoses. The Meta JONG, J. E., and N ie r m e ij e r , M . F.: A method bolic Basis of Inherited Disease, 3rd ed. Stan- for rapid prenatal diagnosis of glycogenosis II bury, J., Wyngarden, J., and Fredrickson, D., (Pompe s Disease). Clin. Chim. Acta 49:361- eds. New York, McGraw-Hill, pp. 639-662, 375, 1973. 1972. 10. H a n k ie w ic z , J. and SwiERCZEK, E.: Lysozyme 26. O’B r ie n , J. S.: Tay-Sachs disease and juvenile in human body fluids. Clin. Chim. 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