Origin and Developmental Patterns of Lactase and Other Glycosidases in Sheep Amniotic and Allantoic Fluid M

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Origin and developmental patterns of lactase and other glycosidases in sheep amniotic and allantoic fluid M. Potier, P. Guay, P. Lamothe, D. Bousquet, L. Dallaire and S. B. Melan\l=c;\on Section de Génétique Médicale, Centre de Recherche Pédiatrique, Hôpital Sainte-Justine, Département de Pédiatrie, and *Centre de Recherche en Reproduction Animale, Faculté de Médecine, Université de Montréal, Montréal H3T1C5, Québec, Canada

Summary. Intestinal lactase activity (with its associated cellobiase, 4\x=req-\ methylumbelliferyl-\g=b\-galactosidaseand -\g=b\-glucosidaseactivities) was used as a specific intestinal marker enzyme to study the release of protein and enzymes of intestinal origin in sheep amniotic fluid during gestation. In amniotic fluid, intestinal lactase activity peaked at 66\p=n-\85days of gestation and then decreased with gestation. This enzyme activity was very low or absent in allantoic fluid throughout gestation suggesting that there is no important transfer of amniotic fluid lactase towards the allantoic cavity. Maltase and 4-methylumbelliferyl-\g=a\-glucosidaseshowed no statistically significant variation with gestation in both amniotic and allantoic fluid whereas \g=a\-galactosidaseand N-acetyl-\g=b\-hexosaminidasewhich were first higher in allantoic than in amniotic fluid increased in amniotic fluid to reach allantoic fluid levels near term. Such patterns are consistent with the suggestion that the fetal urine is a source of \g=a\-galactosidaseand N-acetyl-\g=b\-hexosaminidaseactivities and that sheep urine is first accumulated in the allantoic sac via the urachus up to 86\p=n-\90days of gestation and thereafter passes more and more into the amniotic sac.

Introduction

The characterization of fetal enzymes in human amniotic fluid is of interest as an approach to the study of fetal physiology in utero and for prenatal detection of fetal enzymopathies. Previous work indicated that the activities of some amniotic fluid enzymes are related to fetal development. N-Acetyl-ß-hexosaminidase (Sutcliffe, Brock, Robertson, Scrimgeour & Mon- eghan, 1972), amylase (Fernandez de Castro, Usategui-Gomez & Spellacy, 1973), peroxidase (Armstrong, Van Wormer, Dimitt, May & Gideon, 1976) and a-galactosidase (Potier, Dallaire & Melançon, 1974) increase, while a-glucosidase, ß-glucosidase and ß-galactosidase activities decrease with gestation (Butterworth, Broadhead, Sutherland & Bain, 1974; Potier et al, 1975). Other studies have demonstrated that amniotic fluid ß-galactosidase, Af-acetyl-ß-hexosaminidase and arylsulphatase A originate from the fetus and could be used for prenatal diagnosis of GM1 gangliosidosis, GM2 gangliosidosis and metachromatic leukodystrophy, respectively (Lowden, Cutz, Conen, Rudd & Doran, 1973; Desnick, Krivit & Sharp, 1973; Borresen & Van der Hagen, 1973). Sutcliffe (1975) suggested that maternal serum contributes to amniotic fluid enzymes but that specific sources of fetal enzymes include (1) cells and debris in amniotic fluid itself, (2) fetal urine and (3) bronchial, buccal and gastrointestinal secretions. Evidence has been found that the human fetus releases its intestinal content into the amniotic cavity: Benzie & Doran (1975), using

Downloaded from Bioscientifica.com at 09/30/2021 12:34:46PM via free access a special endoscope, observed fetal defaecation in utero in an 18-week fetus, and the presence of intestinal disaccharidases (Potier et al, 1975, 1976) and biliary salts (Délèze, Sidiropoulos & Paumgartner, 1977) has been reported in amniotic fluid. The pregnant ewe is particularly useful for study of the origin of amniotic fluid enzymes because the fetal urine is apparently accumulated in the allantoic cavity early in gestation (Smith, Adams, Borden & Hilburn, 1966; Mellor & Slater, 1971, 1973) while intestinal enzymes are extruded in the amniotic cavity. Lactase is bound to the brush-border membranes of intestinal epithelial cells, constitutes the most active disaccharidase of sheep fetal intestine and, at least in man, is not present in significant quantities in other fetal membranes and tissues (Potier et al, 1978). In mammals, this enzyme is really a hetero-ß-glycosidase since it also exerts its hydrolytic activity on cellobiose and on 4-methylumbelliferyl (or nitrophenyl) derivatives of ß-galactose and ß-glucose (Dahlqvist, 1961; Kraml, Kolinska, Ellenderova & Hirsova, 1972; Schlegel-Haueter, Hore, Kerry & Semenza, 1972; Birkenmeier & Alpers, 1974). In the present study, lactase activity (together with its accompanying cellobiase, 4-methylumbelliferyl-ß-galactosidase and -ß- glucosidase activities) was used as a specific intestinal marker enzyme in sheep amniotic fluid to follow the release of intestinal enzymes and protein into the amniotic cavity during gestation. Other glycosidase activities that are present in the intestine but, unlike lactase, are also distributed in various other fetal tissues, were also studied.

Materials and Methods Animals and surgicalprocedure Thirty-one (31) pregnant Finnish or Suffolk ewes between 49 and 132 days of gestation were used: 17 carried a single fetus, 10 had twins and 5 triplets. They were kept during autumn and winter at the Faculté de Médecine Vétérinaire, Université de Montréal (Saint-Hyacinthe, Québec) and fed alfalfa hay ad libitum. The animals were fasted 24 h before surgery. Routine presurgical procedures were followed. A longitudinal incision of about 20 cm was made in the lower abdomen under local anaesthesia to expose the uterus. Amnion and allantois were then exposed and clearly identified. Amniotic and allantoic fluid samples were obtained by using hypodermic syringes and kept on ice. This procedure allowed for the collection of fetal fluids that were free of blood and tissue exudates. The fluids were centrifuged at 100 g for 10 min to remove intact cells and debris and stored at 60°C until analysed. The amniotic and allantoic — fluids could be stored for 2 months under these conditions with no or negligible losses of enzyme activities. Fetal intestines were dissected immediately after removal of the fetus and kept on ice. Meconium, when present, was carefully removed and stored at —60°C.The entire intestine was washed by injection of a cold 0-154 M-NaCl solution into the lumen, blotted with filter paper and stored at —60°C for less than 2 months.

Enzyme assays For enzyme assays, the intestine was thawed, jejunal mucosa was scraped off, homogenized (0-02 g mucosa/ml water) with a Potter-Elvejhem homogenizer and centrifuged at 100 g for 10 min. The supernatant fluid was used. With fetuses before 70 days of gestation, the intestine was homogenized with the serosa. Thawed amniotic and allantoic fluid samples were dialysed at 4°C for 24 h against 1 litre of deionized water just before enzyme assay. This was done to remove glucose which gave high blank values in the disaccharidase assay. Disaccharidase activities were determined by the method of Dahlqvist (1964) with lactose, cellobiose and maltose as substrates. The substrate concentration was 0-028 m and 0-1 m- maleate buffer (pH 6) was used.

Downloaded from Bioscientifica.com at 09/30/2021 12:34:46PM via free access Activities of -glucosidase (at pH 4-2 and 5-6: EC 3.2.1.20), ß-glucosidase (at pH 5-6: EC 3.2.1.21) and ß-galactosidase (at pH 5-6: EC 3.2.1.23) with artificial substrates, were determined in 0-25 M-acetate buffer with 1 mM-4-methylumbelliferyl-a-glucose (MU-a- glucoside; Koch-Light Laboratories, Colnbrook, England), 5 mM-Mi/-ß-glucoside and 0-66 dim- Mi/-ß-galactoside as substrate (Potier et al, 1975). -Galactosidase (EC 3.2.1.22) activity was assayed with 6 mM-MÍZ-a-galactoside in 0-25 M-acetate buffer (pH 5) (Potier et al, 1974) and N- acetyl-ß-hexosaminidase (EC 3.2.1.52) with 1 mM-MtZ-ß-TV-acetylglucosaminide in 0-04 m- citrate buffer (pH 4-4) (Okada & O'Brien, 1969). One unit (U) of enzyme activity was defined as that amount of enzyme which hydrolysed 1 µ substrate per minute at 37°C.

Kinetic studies

Activity curves of the glycosidases with pH were determined in the conditions described for the enzyme assay except that the pH of the incubation medium was varied from 4-0 to 6-8 by 0-4 unit increments for lactase (EC 3.2.1.23), cellobiase (EC 3.2.1.21) and maltase (EC 3.2.1.20), from 3-8 to 6-6 by 0-2 unit increments for M£/-ß-glucosidase, -ß-glucosidase, - - galactosidase and -ß-galactosidase (a 0-05 M-cacodylate buffer was used at pH values above 5-8 instead of the 0-25 M-acetate buffer) and from 2-6 to 5-8 by 0-4 unit increments for N- acetyl-ß-hexosaminidase. Michaëlis-Menten constants (Km) were determined for each enzyme under the conditions of the assay except that the substrate concentration was varied. The maximum substrate concentration was that of the standard assay procedure and initial velocity of the enzyme reaction was determined at each substrate concentration using at least a 10-fold substrate concentration range. The Km values were obtained from reciprocal plots of initial velocity of enzymic reaction versus substrate concentration (Lineweaver & Burk, 1934).

Protein determinations

Protein concentrations were determined on dialysed samples using the fluorometric method of Bohlen, Stein, Dairman & Udenfriend (1973) with bovine serum albumin as standard. Before assay, proteins were dissolved in 0-1 M-NaOH.

Results

The possibility of bias related to the distribution of twins and triplets throughout gestation was examined, but there was no positive correlation between enzyme activities in amniotic and allantoic fluid with litter size at any of the gestational periods.

Disaccharidase activities in sheepfetaljejunum and meconium Disaccharidase activities in jejunal mucosa and meconium are presented in Table 1. The data were arbitrarily divided into four groups according to the gestational age of the fetuses and median enzyme activity and range of values are given for each group. Preliminary studies indicated that only lactase, cellobiase and maltase activities are detected in sheep fetal intestine. Other disaccharidase activities tested included sucrase, trehalase, palatinase, turanase and gentiobiase. Jejunal lactase and cellobiase activities increased about 6- fold during gestation while maltase activity was highest at 66-85 days of gestation and decreased thereafter. Disaccharidase activities in meconium at 106-132 days of gestation were 55- to 190-fold lower than those of jejunal mucosa of corresponding gestational ages. Similar disaccharidase activities were found in the meconium collected from either the jejunum or the colon.

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Downloaded from Bioscientifica.com at 09/30/2021 12:34:46PM via free access Properties ofamniotic, allantoicfluid andjejunal enzymes The pH optima and Michaëlis-Menten constants (Km) of the glycosidases are shown in Table 2. The lactase, cellobiase, ß-galactosidase and ß-glucosidase activities were more than 80% sedimentable in sheep amniotic fluid samples centrifuged at 105 000 g for 30 min. Similar results were also obtained with homogenates of fetal sheep jejunum, in contrast to results with maltase, a-glucosidase (at pH 4-2 and 5-6), a-galactosidase and N-acetyl-ß-hexosaminidase activities which remained in the supernatant fraction. Table 2. Kinetic properties of glycosidases in amniotic fluid, allantoic fluid, and jejunum of sheep fetuses at 86-105 days of gestation

Amniotic fluid Allantoic fluid Jejunum Enzyme pH optimum Km (mu) pH optimum Km (ihm) pH optimum Km (min)

Lactase N.D. N.D. 4-8 17-1 Cellobiase N.D. N.D. 4-4 6-0 ß-Galactosidase N.D. N.D. 5-4 0-07 ß-Glucosidase N.D. N.D. 5-2- 5-6 0-50 Maltase 5-6 4-4 5-6 2-7 -Glucosidase 4-2 0-82 5-6 010 -Galactosidase 4-2 7-4 4-4-4 8-6 yV-acetyl- ß-hexosaminidase 4-6 0-87 4-6 0-79

N.D. = not determined because of low enzyme activity. * A broad pH-activity curve was obtained with maximum activity between the pH values indicated. Developmental patterns ofglycosidases in amniotic and allantoicfluid The effect of fetal development was determined for each glycosidase activity in both amniotic and allantoic fluid (Table 3). Large variations of enzyme activities were observed as shown by the wide range of activities found at each gestation period. In amniotic fluid, lactase, cellobiase and ß-glucosidase activities were maximum at 66-85 days of gestation and decreased toward term. ß-Galactosidase in amniotic fluid also showed a similar pattern except that equal values of median activity were found at 66-85 and 86-105 days of gestation. Kruskal-Wallis tests (Siegel, 1956) performed for each of these enzymes indicated that medians calculated for each gestational period were taken from different populations (P < 0-05). These results demonstrate that the variation of median enzyme activities for lactase, cellobiase, ß-galactosidase and ß- glucosidase with gestation do not represent merely chance variations but signify genuine population differences. Among other glycosidases studied the statistical test applied does not specify which medians were statistically different from the others but it justifies our conclusion that the observed variation with gestational age is significant. The variation of maltase activity in amniotic fluid was not statistically significant (P > 0-05). In allantoic fluid, lactase, cellobiase, ß-glucosidase and ß-galactosidase activities were very low or barely detectable. Protein concentration in allantoic fluid was higher than in amniotic fluid but significantly decreased with gestation (P < 0-02) and became similar to that of amniotic fluid near term.

Discussion The development of lactase activity in sheep fetal intestine resembled that of the fetal calf and pig (Sprague et al, 1963; Toofanian, Hill & Kidder, 1974). Lactase is detected in early stages of development and gradually increases until term. In other species, it is only in late gestation that lactase activity rapidly increases to newborn levels (Alvarez & Sas, 1961; Dahlqvist & Lindberg, 1966). Development of cellobiase approximately followed that of lactase in accordance with the

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Downloaded from Bioscientifica.com at 09/30/2021 12:34:46PM via free access theory that both activities are exerted by the same enzyme (Dahlqvist, 1961; Kraml et al, 1972; Schlegel-Haueter et al, 1972; Birkenmeier & Alpers, 1974). The development of sheep intestinal maltase resembles that of the bovine (Toofanian et al, 1974) and horse (Roberts, 1972) fetus. Bovine and equine maltase activity was very low compared to lactase but was approximately stable throughout gestation. The presence of disaccharidase activities in the meconium is predictable since meconium contains desquamated mucosal cells (Timiras, 1972). Disaccharidase activities in fetal sheep meconium were 55- to 190-fold lower than in jejunal mucosa, indicating that these enzymes are considerably degraded, presumably by pancreatic proteases present in meconium (Eggermont, 1966), and/or that large amounts of protein from swallowed amniotic fluid have been incorporated into meconium. Intestinal lactase and its accompanying cellobiase, 4-methylumbelliferyl-ß-galactosidase and -ß-glucosidase activities are detected in sheep amniotic fluid throughout gestation (Table 3). Amniotic fluid lactase exhibited several properties of intestinal lactase. First, the enzyme showed a high degree of specificity for lactose as compared to Mt/-ß-galactoside and M(7-ß-glucoside, and cellobiose is hydrolysed at a 6-5-fold lower rate than lactose (Wallenfels & Fisher, 1960; Doell & Kretchmer, 1962; Asp & Dahlqvist, 1968). Second, lactase is present in amniotic fluid with its accompanying cellobiase, ß-galactosidase and ß-glucosidase activities, all tightly bound to sedimentable material (presumably debris of brush-border membranes). As expected, the developmental patterns of these glycosidase activities were similar, showing a peak of activity at 66-105 days of gestation and then decreasing until term. Third, amniotic fluid lactase and cellobiase showed broad pH-activity curves and high apparent Km similar to those found in the intestine (Table 2). The developmental pattern of lactase activity in sheep amniotic fluid resembled the pattern displayed by this enzyme in human amniotic fluid (Potier et al, 1978). Although sheep intestinal lactase activity increased with gestation (Table 1), amniotic fluid lactase peaked just before mid- gestation and then dropped to low values as meconium accumulated in the fetal colon (from 85 days of gestation in sheep). Available information suggests that this drop is caused by combined effects of decreased extrusion rate of intestinal disaccharidases and reabsorption of the enzyme in swallowed amniotic fluid with fetal development (Potier et al, 1978). The accumulation of meconium in the fetal intestine during the second half of gestation probably constitutes a physical barrier to the extrusion of intestinal content into amniotic cavity. On the other hand, it is known that the bulk of amniotic fluid protein and enzymes are cleared by fetal swallowing and that deglutition increases with gestation (Gitlin, Kumate, Morales, Noriega & Arevalo, 1972). It is possible that at mid-gestation swallowing could overtake extrusion of intestinal lactase. More work is needed to define the relative importance of each of these physiological factors. Intestinal enzymes and protein are released into the amniotic cavity whereas urinary enzymes are released into the allantoic and amniotic cavity. It was found that a-galactosidase and A^-acetyl-ß-hexosaminidase activities, which were first higher in the allantoic than in the amniotic fluid at early gestation, increased in amniotic fluid to reach similar values in both sacs near term. Such patterns would be consistent with the suggestion that fetal urine is a source of a- galactosidase and /Y-acetyl-ß-hexosaminidase activities and that sheep fetal urine is first accumulated in the allantoic sac via the urachus up to a gestational age of 86-90 days and thereafter passes more and more into the amniotic sac. This is thought to be due to the occlusion of the urachus and increasing potency of the urethra (Smith et al, 1966; Mellor & Slater, 1971, 1973). The protein concentration also followed a similar pattern being about 6-fold more elevated in allantoic than in amniotic fluid at 49-65 days and reaching approximately equal concentrations in both sacs at 106-132 days. However, the exact contribution of maternal serum a-galactosidase and TV-acetyl-ß-hexosaminidase to amniotic and allantoic fluid is difficult to evaluate. The presence of maternal serum TY-acetyl-ß-hexosaminidase has been documented in human amniotic fluid (Potier, Boire, Dallaire & Melançon, 1977; Geiger, Navon & Arnon,

Downloaded from Bioscientifica.com at 09/30/2021 12:34:46PM via free access 1978) and could represent an important proportion of total activity in sheep amniotic and allantoic fluid. In conclusion, the paper reports activities of intestinal lactase and other glycosidases in sheep allantoic and amniotic fluid during gestation. No apparent transfer of amniotic fluid lactase into the allantoic cavity was observed, perhaps because lactase is particulate and thus is unlikely to be transferred through a cellular membrane. The developmental pattern of lactase activity, with its associated cellobiase, ß-galactosidase and ß-glucosidase activities, in amniotic fluid was markedly different from that of other glycosidases. These results indicate that there are sources of amniotic fluid glycosidases other than the fetal intestine. The fetal urine, via the allantoic cavity and urachus before 86-90 days of gestation and directly by the urethra thereafter, is probably the main source of N-acetyl-ß-hexosaminidase and a-galactosidase activities in sheep amniotic fluid, although the maternal serum could also contribute. These data could be useful to determine the origin of a given amniotic fluid protein or enzyme by comparing its developmental pattern with those of glycosidases of known origin.

We thank Mrs Marie-Josée Code for her excellent technical assistance and Doctor M. Rouleau, Faculté de Médecine Vétérinaire, for helpful criticism of the manuscript. This work is supported by Grant MA-5163 from the Medical Research Council of Canada.

References

Alvarez, . & Sas, J. (1961) ß-Galactosidase changes in Délèze, G., Sidiropoulos, D. & Paumgartner, G. (1977) the developing intestinal tract of the rat. Nature, Determination of bile acid concentration in human Lond. 190, 826-827. amniotic fluid for prenatal diagnosis of intestinal Armstrong, D., Van Wormer, D., Dimitt, S., May, P. & obstruction. Pediatrics, Springfield 59, 647-650. Gideon, W.P. (1976) The determination of peroxi- Desnick, R.J., Krivit, W. & Sharp, M.L. (1973) In utero dase in amniotic fluid. Obstet. Gynec. 47, 593-598. diagnosis of Sandhoff s disease. Biochem. Biophys. Asp, N.G. & Dahlqvist, A. (1968) Rat small-intestinal ß- Res. Commun. 51, 20-24. galactosidases, separation by ion-exchange Doell, R.G. & Kretchmer, N. (1962) Studies of small- chromatography and gel filtration. Blechern. J. 106, intestine during development. I. Distribution and 841-845. activity of ß-galactosidase. Biochim. Biophys. Acta Benzie, R.J. & Doran, T.A. (1975) The "fetoscope". A 62, 353-362. new clinical tool for prenatal genetic diagnosis. Am. Eggermont, E. (1966) Enzymic activities in meconium J. Obstet. Gynec. 121, 460-464. from human fetuses and newborn. Biol. Neonat. 10. Birkenmeier, E. & Alpers, D.H. (1974) Enzymatic 266-271. properties of rat lactase-phlorizin hydrolase. Fernandez de Castro, ., Usategui Gomez, M. & Biochim. Biophys. Ada 350, 100-112. Spellacy, W.N. (1973) Amniotic fluid amylase. Am. Bohlen, P., Stein, S., Dairman, W. & Udenfriend, S. J. Obstet. Gynec. 116, 931-936. (1973) Fluorometric assay of protein in the Geiger, ., Navon, R. & Arnon, R. (1978) Hexos- nanogram range. Archs Biochem. Biophys. 155, 213- aminidase A in amniotic fluid of Tay-Sachs Fetuses. 220. Clin. Chem. 24, 1131-1133. Borresen, A.-L. & Van der Hagen, C.B. (1973) Gitlin, D., Rumate, J., Morales, Noriega, L. & Arevalo, Metachromatic leucodystrophy. II. Direct deter¬ N. (1972) The turnover of amniotic fluid protein in mination of arylsulfatase A activity in amniotic fluid. the human conceptus. Am. J. Obstet. Gynec. 113, Clin. Genet. 4, 442-446. 632-645. Butterworth, J., Broadhead, D.M., Sutherland, G.R. & Kraml, J., Kolinska, J., Ellenderova, D. & Hirsova, D. Bain, A.D. (1974) Lysosomal enzymes of amniotic (1972) ß-Glucosidase (phlorizin hydrolase) activity fluid in relation to gestational age. Am. J. Obstet. of the lactase fraction isolated from the small Gynec. 119,821-828. intestinal mucosa of infant rats, and the relationship Dahlqvist, A. (1961) Intestinal carbohydrases of a new¬ between ß-glucosidases and ß-galactosidases. born pig. Nature, Lond. 190, 31-32. Biochim. Biophys. Ada 258, 520-530. Dahlqvist, A. (1964) Method for assay of intestinal Line weaver. H. & Burk, D. (1934) The determination of disaccharidases./Inalyt. Biochem. 7, 18-25. enzyme dissociation constants. /. Am. Chem. Soc. Dahlqvist, A. & Lindbergh, T. (1966) Development of 56, 658-666. the intestinal disaccharidase and alkaline Lowden, J.A., Cutz, E., Conen, P.E., Rudd, N. & Doran, phosphatase activities in the human fetus. Clin. Sci. T.A. (1973) Prenatal diagnosis of GM1 ganglio¬ 30,517-528. sidosis. N. Engl. J. Med. 288, 225-228.

Downloaded from Bioscientifica.com at 09/30/2021 12:34:46PM via free access Mellor, DJ. & Slater, J.S. (1971) Daily changes in absorption of carbohydrates in the equine small amniotic and allantoic fluid during the last three intestine. Ph.D. thesis, University of Bristol. months of pregnancy in conscious, unstressed ewes, Schlegel-Haueter, S., Hore, P., Kerry, K.R. & Semenza, with catheters in their foetal fluid sacs. J. Physiol., G. (1972) The preparation of lactase and glucoamyl- Lond. 1 , 573-604. ase of rat small intestine. Biochim. Biophys. Ada Mellor, DJ. & Slater, J.S. (1973) Variations in the 258,506,519. distribution of glucose and fructose within chron¬ Siegel, S. (1956) Non-parametric Statistics, pp. 184- ically catheterized sheep conceptuses during the last 193. McGraw-Hill Book Co., New York. two months of pregnancy. Res. Vet. Set. 15, 317— Smith, F.G., Adams, F.M., Borden, M. & Hilburn, J. 321. (1966) Studies of renal function in the intact fetal Okada, S. & O'Brien, J.S. (1969) Tay-Sachs disease: lamb. Am. J. Obstet. Gynec. 96, 240-243. generalized absence of a ß-D-N-acetylhexo- Sprague, J.I., Ullrey, D.E., Waddfll, D.G., Miller, E.R., saminidase component. Science, N.Y. 165, 698-700. Zutaut, C.L. & Hoefer, J.A. (1963) Intestinal lactase, Potier, M., Dallaire, L. & Melançon, S.B. (1974) alkaline and acid phosphatase in the swine fetus and Amniotic fluid-galactosidase activity: An index of newborn pig. J. Anim. Sci. 11, 121-124. gestational age. Gynecol. Invest. 5, 306-310. Sutcliffe, R.G. (1975) The nature and origin of the Potier, M., Dallaire, L. & Melançon, S.B. (1975) soluble protein in human amniotic fluid. Biol. Rev. Occurrence and properties of fetal intestinal 50, 1-33. glycosidases (disaccharidases) in human amniotic Sutcliffe, R.G., Brock, DJ.M., Robertson, J.G., fluid. Biol. Neonate. 27, 141-152. Scrimgeour, S.B. & Moneghan, J.M. (1972) En¬ Potier, M., Melançon, S.B. & Dallaire, L. (1976) Fetal zymes in amniotic fluid: a study of specific activity intestinal disaccharidases in human amniotic fluid. patterns during pregnancy. J. Obstet. Gynaec. Br. Biomedicine Express 25, 167-169. Commonw. 79, 895-901. Potier, M., Boire, G., Dallaire, L. & Melançon, S.B. Timiras, P.S. (1972) Development of gastrointestinal (1977) N-Acetyl-ß-hexosaminidase isoenzymes of tract. In Developmental Biology and Aging, pp. 220- amniotic fluid and maternal serum. Their relevance to 225. Macmillan, New York. prenatal diagnosis of the GM2 gangliosidoses. Clin. Toofanlan, F., Hill, F.W.G. & Kidder, D.E. (1974) The Chim.Acta 76, 309-315. development of the intestinal disaccharidase activities Potier, M., Melançon, S.B. & Dallaire, L. (1978) in the fetal and newborn calf. Res. Vet. Sci. 16, 375— Developmental patterns of intestinal disaccharidases 381. in human amniotic fluid. Am. J. Obstet. Gynec. 131, Wallenfels, K. & Fisher, J. (1960) Lactase-hydrolysing 73-76. enzymes. X. The lactase from calf s intestine. Z. Roberts, M.C. (1972) Studies on the digestion and Physiol. Chem. 321, 223-245.

Received 15 November 1978

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