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Developmental patterns of intestinal disaccharidases in human amniotic fluid
Developmental patterns of intestinal disaccharidases in human amniotic fluid l\1ICHEL POTIER, PH.D. SERGE B. MELANCON, M.D. LOUIS DALLAIRE, M.D., PH.D. Montreal, Quebec, Canada A close relationship exists between relative disaccharidase activities (maltase, sucrase, trehalase, palatinase, turanase, lactase, and cellobiase) in amniotic fluid and corresponding jejunal mucosa of five human fetuses (16 to 21 weeks of gestation) suggesting that these intestinal enzymes pass into amniotic fluid. Serial determination of disaccharidase activities in amniotic fluid samples collected between 10 and 42 weeks of gestation showed maximum mean activities at 14 to 17 weeks of gestation and a rapid drop to less than 12 per cent maximum values at about 22 weeks. This drop is probably caused by combined effects of decreased extrusion rate of intestinal disaccharidases and increased reabsorption of the enzymes in swallowed amniotic fluid with fetal development. (AM. J. OBSTET. GYNECOL. 131: 73, 1978.)
of the sources of fetal enzymes in amniotic fluid. Sutcliffe 1 suggested that they include ( 1) cells and debris in amniotic fluid itself, desquamated from fetal tissues, (2) fetal urine, and (3) bronchial, buccal, and gastrointestinal secretions. Fetal enzymes in amniotic fluid could prove useful for prenatal diagnosis of genetic diseases, developmental studies, or assessment of fetal health. Previous work showed that amniotic fluid ,8-galactosidase, N-acetyl-~hexos aminidase, and arylsulfactase A originate from the fetus and could be used for prenatal diagnosis of GM 1 gangliosidosis, G\12 gangliosidosis, and methachromatic leukodystrophy, respectively. 2- 4 The activities of some amniotic fluid enzymes are related to fetal development. N-Acetyl-,8- hexosaminidase, 5 amylase, 6 peroxidase/ and a-ga!actosidase 8 increase, whereas aglucosidase, ,8-glucosidase, and ,8-galactosidase activiLITTLE IS KNOWN
tiPs D'PSt::ttionB· 10 ---- cJprrP::tSP --------- with ··---- o----------·
We have previously reported the presence of fetal From the Section de Geneti
.
0002~9378/78/01131 ~0073$00.40/0
-
©
~
-
1978 The C. V. !-.fosby Co.
intestinal disaccharidases in amniotic ftuid. 10 · 11 Before these er...zymes could be useful for diagnostic purposes or developmental studies, the range of their normal values throughout gestation must be known. The purpose of this paper is to report developmental patterns of disaccharidases between 10 and 42 weeks of gestation.
Material and methods Fetal tissues. Human fetuses used in this work were obtained from therapeutic abortions approved by Certified Hospitals. Fetus I was aborted for social reasons at 16 weeks of gestation by intra-amniotic saline injection. Fetus 2 was aborted by hysterotomy at 19 weeks of gestation after prenatal diagnosis of type 0 GM 2 gangliosidosis. Fetus 3 was aborted by intra-amniotic injection of prostaglandins at 20 weeks of gestation after prenatal diagnosis of Down's syndrome. Fetus 4 was affected with type II mucopolysaccharidosis and was aborted by saline injection at 20 weeks of gestation. Fetus 5. was affected by metachromatic leukodystrophy and was aborted by hysterotomy at 21 weeks of gestation. All diagnoses were verified on aborted fetuses. Fetal tissues (intestine, liver, kidney, skin, amnion, placenta, lung, and bladder) were dissected 1 to 2 hours after abortion. The entire jejunum (or proximal half of the jejunum of fetuses 2 and 5) was washed by injection of a cold 0.154M NaCI solution into the lumen, blotted with filter paper, and stored at -60° C. for less than 2 months.
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Potier, Melancon, and Dallaire
May I, 1978
Am.
J. Obstet. Gynecol.
Table I. Comparison between disaccharidase activities in amniotic fluid and jejunal mucosa of five human fetuses Disaccharidase activity ( U. /Gm. of protein)
I
I
Disaccharidase Maltase Sucrase Trehalase Palatinase Turanase Lactase Ce!!obiase
JM 2.27 (lOOt) 0.97 (42.7) 0.21 (9.4) 0.13 (5.8) 1.68 (70.5) 0.03 ( 1.4) 0.01 (0.4)
526.9 (100) 209.2 ('->9.6) 57.5 (10.9) 35.4 (6.7) 63.3 (12.0) 13.2 (2.5) 2.2 (0.4)
21.0 (100) 7.1 (33.8) 0.60 (2.8) 0.95 (4.6) 1.89 (9.0) 0.25 (1.2) 0.07 (0.3)
168.2 (100) 66.8 (39.7) 16.8 (10.0) 11.1 (6.6) 17.0 (10.1) 4.1 (2.4) 0.61 (0.3)
1.55 (100) 1.14 (73.7) 0.12 (8.1) 0.17 (10.7) 0.51 (32.9) 0.05 (3.2) 0.01 (0.7)
239.6 (100) 92.6 (38.7) 56.8 (23.7) 15.4 (6.5) 37.4 (15.6) 7.8 (3.3) l.l (0.5)
20.2 (100) 9.4 (46.7) 0.6 (3.1) 1.5 (7.6) 3.5 (17.5) 0.4 (2.0) 0.1 (0.4)
298.8 (100) 146.0 (48.8) 31.8 (10.6) 20.9 (7.0) 22.7 (7.6) 8.3 (2.8) !.3 (0.4)
5.10 (100) 2.40 (47.2) NDt
354.0 (100) 115.8 (39.1) ND
0.28 (5.6) 0.56 (11.0) 0.07 (1.5) 0.02 (0.4)
20.8 (6.9) 31.7 (7.7) 13.2 (3.5) 1.9
(0.5)
*AF, An1niotic fluid; jNl, jejunal mucosa. tFigures in parentheses refer to relative disaccharidase activities expressed as percentages of maltase specific activity. tl\Jn 1\.Tn.t riPtP.rrninPrl T.&. .o. .......... '-' ... ..._" ............ .._'-A., ~£..#I
~....,.
Table II. Disaccharidase activities of amniotic fluid during gestation Disaccharidase activity ( U. /Gm. Gestational age (wk.)
Maltase
Sucrase
10-13 14-17 18-21 22-25 26-29 30-33 34-37 38-42
13.3, 27.3 21.8 ± 9.1 (19)* 15.3 ± 8.0 (16) 2.0 ± 2.0 ( 6) 1.7± 1.4( 7) l.l ± 0.9 ( 6) 1.2 ± 0.6 ( 8) 1.3 ± l.l ( 8)
8.5, 11.1 9.2 ± 4.8 (15) 7.0 ± 3.5 (16) l.l ± 1.4 ( 6) 0.72 ± 0.85 ( 7) 0.37 ± 0.44 ( 6) 0.68 ± 0.96 ( 8) 0.75 ± 0.65 ( 8)
Trehalase 0.32, 0.95 0.79 ± 0.49 0.31 ± 0.22 0.09 ± 0.06 0.14 ± 0.11 0.17 ± 0.22 0.20 ± 0.07 0.32 ± 0.28
(21) (16) ( 6) ( 9) ( 6) (12) (12)
of protein) Pawtinase
Lactase
0.95, 1.77 1.54 ± 0.77 (19) 0.92 ± 0.52 (16) 0.02 ± 0.02 ( 6) 0.07 ± 0.06 ( 7) 0.02 ± 0.03 ( 5) 0.04 ± 0.04 ( 8) 0.04 ± 0.02 ( 8)
0.03, 0.52 0.32 ± 0.17 (15) 0.18 ± 0.10 (16) 0.02 ± 0.03 ( 6) 0.006 ± 0.010 ( 7) Q.006 ± 0.000 ( 6) 0.000 ± 0.000 ( 8) 0.001 ± 0.003 ( 8)
*Mean ± S.D. Figures in parentheses refer to the number of amniotic fluid samples used.
Amniotic fluid. Amniotic fluid samples from aborted fetuses were obtained either by transabdominal amniocentesis a fe\v hours before abortion Gust before saline or prostaglandin injection-fetuses 1, 3 and 5) or collected from the intact amniotic sac after hysterotomy (fetuses 2 and 5). All other amniotic fluid samples from 77 pregnancies between 10 and 42 weeks of gestation, terminated for social reasons or at risk for chromosomal anomalies, neural tube defects, or Rh isoimmunization, were obtained by transabdominal amniocentesis as previously described. 12 Samples were centrifuged at 100 x g for l 0 minutes to remove intact cells and debris and stored at 4 o C. for less than 2 months or at -60° C. for periods of time not exceeding 2 years. Enzyme activities were stable under these storage conditions. Fetal age was estimated from menstrual history and measurement of fetal biparietal diameter in utero by ultrasonography . 13 Disaccharidase assay. For disaccharidase assay, the
jejunum was thawed and its mucosa scraped off, homogenized (0.02 Gm. of mucosa per milliliter of \-Vater) and centrifuged at 100 x g for 10 minutes. The supernatant fluid was used. 14 ..A..JI amniotic fluid samples v;ere dialysed at 4° C. for 24 hours against l L. of deionized water just before enzyme assay. This was done to remove glucose in amniotic fluid which gave high blank values in the disaccharidase assay. Disaccharidases in amniotic fluid of fetuses 1, 3, and 5 were concentrated by centrifugation at 105,000 X g for 30 minutes in Rotor 40 of a model L Beckman ultracentrifuge. More than 80 per cent of disaccharidase activities were recovered in the sediment. The supernatant was discarded and the pellet was resuspended in water with 1 : 20 of the initial volume of amniotic fluid. Disaccharidase activities were determined according to the method of Dahlqvist, 15 with the use of maltose, sucrose, trehalose, palatinose, turanose, lactose, and cellobiose as substrates. Enzymes were incubated for 30
Volume 131 Number I
to 120 minutes at 37o C. in a total volume of 0.2 mi. containing 0.058M substrate and O.IM maleate buffer (pH 6). With amniotic fluid, long incubation periods ( 18 hours) were used for lactase and cellobiase assays. Under these assay conditions, the amount of liberated glucose was proportional to the incubation time and the amount of enzyme. One unit of disaccharidase activity corresponded to the hydrolysis of I ~-tmole of substrate per minute. Protein determination. Protein concentration was determined on dialysed samples according to the fluorometric method of Bohlen and associates 16 with bovine serum albumin as standard. Before assay, proteins were dissolved in O.IM NaOH.
Results Disaccharidase activities in amniotic fluid of our five fetuses, expressed per gram of protein, were 8- to 250fold lower than in corresponding jejunal mucosa (Table I). However, despite this large variation from one fetus to the other, when amniotic fluid and jejunal disaccharidase activities were expressed as a percentage of maitase activity, the relative values were similar for each disaccharidase. The effect of fetal development on disaccharidase activities in amniotic fluid was studied between 10 and 42 weeks of gestation. Results were arbitrarily divided into eight groups of 4 week periods and mean and standard deviation were computed for each group (Table II). Maximum mean values were found in the amniotic fluids at 14 to 17 weeks of gestation. Disaccharidase activities then dropped simultaneously to less than 12 per cent maximum values after 21 weeks of gestation. All disaccharidase activities remained low thereafier with the exception of trehalase, which increased from 22 to 25 weeks to term. Disaccharidase assays in mixtures of amniotic fluid samples from early and late pregnancies indicated that the drop of disaccharidase actiVIties at midpregnancy is not caused by inhibitors or activators. To examine other possible sources of disaccharidase activities in amniotic fluid, we assayed maltase, sucrase, trehalase, palatinase, and lactase activities in maternal and fetal serum, and in homogenates (I 0 or 20 per cent in water) of fetal liver, renal cortex, skin, amnion, and bladder. Their activities never exceeded 6 per cent of those found in jejunal mucosa and under our standard assay conditions, sucrase and palatinase activities were absent or barely detectable in aU tissues except the intestine.
Comment The results presented in this paper are consistent with the suggestion that disaccharidase activities of
Intestinal disaccharidases in amniotic fluid
75
amniotic Huid originate hom the fetal intestine. This conclusion is also supported by previous results showing identical physical and kinetic properties for amniotic Huid and intestinal disaccharidases. 1° Comparison between disaccharidase activities (per gram of protein) in jejunal mucosa and amniotic Huid of our fetuses suggests that as much as I :2 per ,·ent of total amniotic fluid protein comes from the fetal intestine. Since the bulk of protein in amniotic Huid originates from the maternal serum, 1 the fetal intestine could be a major source of fetal protein in amniotic fluid. Although four of our five fetuses suffered from various genetic diseases. their disaccharidase activities in amniotic fluid and jejunal mucosa were approximately in the range of previously reported value> lor similar gestional ages."· 14 · 17 • 1 ~ However, the mean activity on a large group of such patients could differ from the normal mean. Since disaccharidase activities in letai intestine increase or remain stable throughout gestation. 1 '1· 17 • '"' the cause(s) of the rapid fali of these er1lvme activities in amniotic fluid at about 22 weeks of gestation (fable II) is unknown. Available information suggests that both reabsorption and extrusion of disacch;nidases are influenced by fetal development. Arnnioti< fluid protein and enzymes are cleared in swallowed amniotic fluid. 19 As the fetus develops, swallowing imTeases and could overtake extrusion of disaccharidase. On the other hand, the striking correlation between the rapid accumulation of meconium in human fetal intestine 14 and the fall of disaccharidase activities at 22 weeks suggests that extrusion stops or is markedly decreased at this period of gestation. Extrusion of ciisaccharidases could also be diminished by increased permeabilitv of intestinal mucosa to intestinal protein in the lumen or decreased release of disaccharidases into dte lumen. More information is needed on the physioio>gv of meconium formation and on changes of the permeability of intestinal mucosa during fetal deveiopmem before any conclusion can be reached. Unlike other amniotic fluid disaccharidases. trehalase activity increases from 22 weeks to term (Table II). There are several possibilities to explai 11 rhis discordant finding. First. we have found that trehalase activity in near term amniotic fluid sample:; was in a soluble form since it was not sedimented when centrifuged at 105,000 X g for 30 minutes, contrarilY to trehalase and other disaccharidases before :l2 weeks, v;hich \Vere sedimented. 10 i~~lthough the physical state of trehalase in intestinal lumen after 22 weeks is unknown. it is possible that, due to the Jarge arnounts of meconium in fetal intestine during this period, 14 only soluble trehalase could pass into the amni<1tic cavity whereas other bound disaccharidases (presumably to
76
Potier, Melancon, and Dallaire
debris of brush border membranes) remain in the intestine. If this is the case, increasing activity of amniotic fluid trehalase would be the reflection of increasing trehalase activity in the fetal intestine itself. 14 Second, the trehalase activity after 22 weeks could come from a different source than the intestine. Among the fetal organs examined (at 16 to 21 weeks of gestation), the kidney cortex showed some trehalase activity although at a much lower level than in the intestine (less than 3 per cent). However, this enzyme could develop later during gestation and it is known that fetal micturition increases with gestation. 20 Work is in progress in our laboratory to clearly define the origin of amniotic fluid trehalase near term by comparing physical and kinetic properties of trehalase to fetal intestine, kidney, and amniotic fluid. The physiologic role of disaccharidases in amniotic fluid is not clear. These enzymes have been implicated in both degradation and transglycosidation reactions of disaccharides and oligosaccharides21 and could participate in the biosynthesis of complex carbohydrates. However, it is possible that amniotic fluid disaccharidases are also implicated in a more general physiologic process involving other intestinal protein and enzymes which could be recirculated in the fetal intestine via the
REFERENCES 1. Sutcliffe, R. G.: The nature and origin of the soluble protein in human amniotic fluid, Bioi. Rev. 50: 1, 1975. 2. Lowden, J. A., Cutz, E., Conen, P. E., Rudd, N., and Doran, T. A.: Prenatal diagnosis of GM 1 gangliosidosis, N. Engl.]. Med. 288: 225, 1973. 3. Desnick, !t J., Krivit, W., a_11d Sharp, M. L.: In utero diagnosis of Sandhoffs disease, Biochem. Biophys. Res. Commun. 51: 20, 1973. 4. Borresen, A.-L., and Van der Hagen, C. B.: Metachromatic leucodystrophy. II. Direct determination of arylsulfatase A activity in amniotic fluid, Clin. Genet. 4: 442, 1973. . 5. Sutcliffe, R. G., Brock, D. ]. M., Robertson, ]. G., Scrimgeour, J. B., and Moneghan, J. M.: Enzymes in amniotic fluid: A study of specific activity patterns during pregnancy,]. Obstet. Gynaecol. Br. Commonw. 79: 895, 1972. 6. Fernandez de Castro, A., Usategui-Gomez, M., and Spellacy, W. N.: Amniotic fluid amylase, AM. J. OBSTET. GYNECOL. 116: 931, 1973. 7. Armstrong, D., Van Wormer, D., Dimmitt, S., May, P., and Gideon, W. P.: The determination of peroxidase in amniotic fluid, Obstet. Gynecol. 47: 593, 1976. 8. Potier, M., Dallaire, L., and Melancon, S. B.: Amniotic fluid a-galactosidase activity: An index of gestational age, Gynecol. Invest. 5: 306, 1974. 9. Butterworth, J., Broadhead, D. M., Sutherland, G. R., and Bain, A. D.: Lysosomal enzymes of amniotic fluid in relation to gestational age, AM. J. OssTET. GYNEcoL. 119: 821, 1974. 10. Potier, M., Dallaire, L., and Melancon, S. B.: Occurrence and properties of fetal intestinal glycosidases (disaccharidases) in human amniotic fluid, Bioi. Neonate 27: 141, 1975.
May l. 197H
Am. J. Obstet. Gvnecol.
amniotic fluid. Lev and Oriic 22 demonstrated intestinal absorption and transport of an enzyme (horseradish peroxidase) instiiiated into rat amniotic cavity. Reabsorption of intestinal protein might play a role in the regulation of biosynthesis andior degradation processes of these proteins or could represent a mechanism for the recyciing of intestinal protein by the intestine. The importance of such mechanisms for normal fetal development remains to be established. A possible clinical application of these findings is in the detection of obstruction of the lower part of fetal intestine at 14 to 18 weeks on the basis of low or absent disaccharidase activities in amniotic fluid. However, it should be pointed out that much more information about the normal biology of intestinal disaccharidases is necessary before the information obtained in this study can be applied clinically. We greatly appreciate the technical assistance given by Mrs. Marie-Josee Code. Addendum We have recently reported a deficiency of disaccharidase activity in the amniotic fluid of a fetus born with anal imperforation (Potier, M., Dallaire, L., and Melan{:on, S. B.: Lancet 2:982, 1977.).
ll. Potier, M., Melancon, S. B., and Dallaire, L.: Fetal intestinal disaccharidases in human amniotic fluid, Biomedicine [Express] 25: 167, 1976. 12. Dallaire,~., Potier, M., Melancon, S. B., and Patrick, J.: Fetomaternal aminoacid metabolism. J. Obstet. Gynaecol. Br. Commonw. 81: 761, 1974. 13. Campbell, S.: Ultrasonic fetal cephalometry during the second trimester of pregnancy. J. Obstet. Gynaecol. Br. Commonw. 77: 1057, 1970. 14. Dahiqvist, A., and Lindberg, T.: Developtnent of the intestinal disaccharidase and alkaline phosphatase activities in the human fetus, Clin. Sci. Mol. Med. 30: 517, 1966. 15. Dahlqvist, A.: Method for assay of intestinal disaccharidases. Anal. Biochem. 7: 18, 1964. 16. Bohlen, P., SteLT!, S., Dairman, W., and Udenfriend; S.: Fluorometric assay of protein in the nanogram range. Arch. Biochem. Biophys. 155: 213, 1973. 17. Antonowicz, I., Chang, S. K., and Grand, R. C.: Development and distribution of lysosomal enzymes and disaccharidases in fetal intestine, Gastroenterology 67: 51, 1974. 18. Auricchio, S., Rubino, A., and Mi.irset, G.: Intestinal glycosidase activities in the human embryo, fetus and newborn, Pediatrics 35: 944, 1965. 19. Gitlin, D., Kumate, J., Morales, C., Noriega, L.. and Arevalo, N .: The turnover of amniotic fluid protein in the human conceptus, AM. J. 0BSTET. GYNECOL. 113: 632, 1972. 20. Jeffcoate, T. 1~. A., and Scott, j. S.: Polyhydramnios and oligohydramnios, Can. Med. Assoc. J. 80: 77, 1959. 21. Karlson, P.: Glycosides, oligosaccharides, polysaccharides, in Introduction to Modern Biochemistry, New York, 1968, Academic Press, Inc., p. 314. 22. Lev, R., and Orlic, D.: Protein absorption by the intestine of the fetal rat in utero, Science 177: 522. 1972.
of the sources of fetal enzymes in amniotic fluid. Sutcliffe 1 suggested that they include ( 1) cells and debris in amniotic fluid itself, desquamated from fetal tissues, (2) fetal urine, and (3) bronchial, buccal, and gastrointestinal secretions. Fetal enzymes in amniotic fluid could prove useful for prenatal diagnosis of genetic diseases, developmental studies, or assessment of fetal health. Previous work showed that amniotic fluid ,8-galactosidase, N-acetyl-~hexos aminidase, and arylsulfactase A originate from the fetus and could be used for prenatal diagnosis of GM 1 gangliosidosis, G\12 gangliosidosis, and methachromatic leukodystrophy, respectively. 2- 4 The activities of some amniotic fluid enzymes are related to fetal development. N-Acetyl-,8- hexosaminidase, 5 amylase, 6 peroxidase/ and a-ga!actosidase 8 increase, whereas aglucosidase, ,8-glucosidase, and ,8-galactosidase activiLITTLE IS KNOWN
tiPs D'PSt::ttionB· 10 ---- cJprrP::tSP --------- with ··---- o----------·
We have previously reported the presence of fetal From the Section de Geneti
.
0002~9378/78/01131 ~0073$00.40/0
-
©
~
-
1978 The C. V. !-.fosby Co.
intestinal disaccharidases in amniotic ftuid. 10 · 11 Before these er...zymes could be useful for diagnostic purposes or developmental studies, the range of their normal values throughout gestation must be known. The purpose of this paper is to report developmental patterns of disaccharidases between 10 and 42 weeks of gestation.
Material and methods Fetal tissues. Human fetuses used in this work were obtained from therapeutic abortions approved by Certified Hospitals. Fetus I was aborted for social reasons at 16 weeks of gestation by intra-amniotic saline injection. Fetus 2 was aborted by hysterotomy at 19 weeks of gestation after prenatal diagnosis of type 0 GM 2 gangliosidosis. Fetus 3 was aborted by intra-amniotic injection of prostaglandins at 20 weeks of gestation after prenatal diagnosis of Down's syndrome. Fetus 4 was affected with type II mucopolysaccharidosis and was aborted by saline injection at 20 weeks of gestation. Fetus 5. was affected by metachromatic leukodystrophy and was aborted by hysterotomy at 21 weeks of gestation. All diagnoses were verified on aborted fetuses. Fetal tissues (intestine, liver, kidney, skin, amnion, placenta, lung, and bladder) were dissected 1 to 2 hours after abortion. The entire jejunum (or proximal half of the jejunum of fetuses 2 and 5) was washed by injection of a cold 0.154M NaCI solution into the lumen, blotted with filter paper, and stored at -60° C. for less than 2 months.
73
74
Potier, Melancon, and Dallaire
May I, 1978
Am.
J. Obstet. Gynecol.
Table I. Comparison between disaccharidase activities in amniotic fluid and jejunal mucosa of five human fetuses Disaccharidase activity ( U. /Gm. of protein)
I
I
Disaccharidase Maltase Sucrase Trehalase Palatinase Turanase Lactase Ce!!obiase
JM 2.27 (lOOt) 0.97 (42.7) 0.21 (9.4) 0.13 (5.8) 1.68 (70.5) 0.03 ( 1.4) 0.01 (0.4)
526.9 (100) 209.2 ('->9.6) 57.5 (10.9) 35.4 (6.7) 63.3 (12.0) 13.2 (2.5) 2.2 (0.4)
21.0 (100) 7.1 (33.8) 0.60 (2.8) 0.95 (4.6) 1.89 (9.0) 0.25 (1.2) 0.07 (0.3)
168.2 (100) 66.8 (39.7) 16.8 (10.0) 11.1 (6.6) 17.0 (10.1) 4.1 (2.4) 0.61 (0.3)
1.55 (100) 1.14 (73.7) 0.12 (8.1) 0.17 (10.7) 0.51 (32.9) 0.05 (3.2) 0.01 (0.7)
239.6 (100) 92.6 (38.7) 56.8 (23.7) 15.4 (6.5) 37.4 (15.6) 7.8 (3.3) l.l (0.5)
20.2 (100) 9.4 (46.7) 0.6 (3.1) 1.5 (7.6) 3.5 (17.5) 0.4 (2.0) 0.1 (0.4)
298.8 (100) 146.0 (48.8) 31.8 (10.6) 20.9 (7.0) 22.7 (7.6) 8.3 (2.8) !.3 (0.4)
5.10 (100) 2.40 (47.2) NDt
354.0 (100) 115.8 (39.1) ND
0.28 (5.6) 0.56 (11.0) 0.07 (1.5) 0.02 (0.4)
20.8 (6.9) 31.7 (7.7) 13.2 (3.5) 1.9
(0.5)
*AF, An1niotic fluid; jNl, jejunal mucosa. tFigures in parentheses refer to relative disaccharidase activities expressed as percentages of maltase specific activity. tl\Jn 1\.Tn.t riPtP.rrninPrl T.&. .o. .......... '-' ... ..._" ............ .._'-A., ~£..#I
~....,.
Table II. Disaccharidase activities of amniotic fluid during gestation Disaccharidase activity ( U. /Gm. Gestational age (wk.)
Maltase
Sucrase
10-13 14-17 18-21 22-25 26-29 30-33 34-37 38-42
13.3, 27.3 21.8 ± 9.1 (19)* 15.3 ± 8.0 (16) 2.0 ± 2.0 ( 6) 1.7± 1.4( 7) l.l ± 0.9 ( 6) 1.2 ± 0.6 ( 8) 1.3 ± l.l ( 8)
8.5, 11.1 9.2 ± 4.8 (15) 7.0 ± 3.5 (16) l.l ± 1.4 ( 6) 0.72 ± 0.85 ( 7) 0.37 ± 0.44 ( 6) 0.68 ± 0.96 ( 8) 0.75 ± 0.65 ( 8)
Trehalase 0.32, 0.95 0.79 ± 0.49 0.31 ± 0.22 0.09 ± 0.06 0.14 ± 0.11 0.17 ± 0.22 0.20 ± 0.07 0.32 ± 0.28
(21) (16) ( 6) ( 9) ( 6) (12) (12)
of protein) Pawtinase
Lactase
0.95, 1.77 1.54 ± 0.77 (19) 0.92 ± 0.52 (16) 0.02 ± 0.02 ( 6) 0.07 ± 0.06 ( 7) 0.02 ± 0.03 ( 5) 0.04 ± 0.04 ( 8) 0.04 ± 0.02 ( 8)
0.03, 0.52 0.32 ± 0.17 (15) 0.18 ± 0.10 (16) 0.02 ± 0.03 ( 6) 0.006 ± 0.010 ( 7) Q.006 ± 0.000 ( 6) 0.000 ± 0.000 ( 8) 0.001 ± 0.003 ( 8)
*Mean ± S.D. Figures in parentheses refer to the number of amniotic fluid samples used.
Amniotic fluid. Amniotic fluid samples from aborted fetuses were obtained either by transabdominal amniocentesis a fe\v hours before abortion Gust before saline or prostaglandin injection-fetuses 1, 3 and 5) or collected from the intact amniotic sac after hysterotomy (fetuses 2 and 5). All other amniotic fluid samples from 77 pregnancies between 10 and 42 weeks of gestation, terminated for social reasons or at risk for chromosomal anomalies, neural tube defects, or Rh isoimmunization, were obtained by transabdominal amniocentesis as previously described. 12 Samples were centrifuged at 100 x g for l 0 minutes to remove intact cells and debris and stored at 4 o C. for less than 2 months or at -60° C. for periods of time not exceeding 2 years. Enzyme activities were stable under these storage conditions. Fetal age was estimated from menstrual history and measurement of fetal biparietal diameter in utero by ultrasonography . 13 Disaccharidase assay. For disaccharidase assay, the
jejunum was thawed and its mucosa scraped off, homogenized (0.02 Gm. of mucosa per milliliter of \-Vater) and centrifuged at 100 x g for 10 minutes. The supernatant fluid was used. 14 ..A..JI amniotic fluid samples v;ere dialysed at 4° C. for 24 hours against l L. of deionized water just before enzyme assay. This was done to remove glucose in amniotic fluid which gave high blank values in the disaccharidase assay. Disaccharidases in amniotic fluid of fetuses 1, 3, and 5 were concentrated by centrifugation at 105,000 X g for 30 minutes in Rotor 40 of a model L Beckman ultracentrifuge. More than 80 per cent of disaccharidase activities were recovered in the sediment. The supernatant was discarded and the pellet was resuspended in water with 1 : 20 of the initial volume of amniotic fluid. Disaccharidase activities were determined according to the method of Dahlqvist, 15 with the use of maltose, sucrose, trehalose, palatinose, turanose, lactose, and cellobiose as substrates. Enzymes were incubated for 30
Volume 131 Number I
to 120 minutes at 37o C. in a total volume of 0.2 mi. containing 0.058M substrate and O.IM maleate buffer (pH 6). With amniotic fluid, long incubation periods ( 18 hours) were used for lactase and cellobiase assays. Under these assay conditions, the amount of liberated glucose was proportional to the incubation time and the amount of enzyme. One unit of disaccharidase activity corresponded to the hydrolysis of I ~-tmole of substrate per minute. Protein determination. Protein concentration was determined on dialysed samples according to the fluorometric method of Bohlen and associates 16 with bovine serum albumin as standard. Before assay, proteins were dissolved in O.IM NaOH.
Results Disaccharidase activities in amniotic fluid of our five fetuses, expressed per gram of protein, were 8- to 250fold lower than in corresponding jejunal mucosa (Table I). However, despite this large variation from one fetus to the other, when amniotic fluid and jejunal disaccharidase activities were expressed as a percentage of maitase activity, the relative values were similar for each disaccharidase. The effect of fetal development on disaccharidase activities in amniotic fluid was studied between 10 and 42 weeks of gestation. Results were arbitrarily divided into eight groups of 4 week periods and mean and standard deviation were computed for each group (Table II). Maximum mean values were found in the amniotic fluids at 14 to 17 weeks of gestation. Disaccharidase activities then dropped simultaneously to less than 12 per cent maximum values after 21 weeks of gestation. All disaccharidase activities remained low thereafier with the exception of trehalase, which increased from 22 to 25 weeks to term. Disaccharidase assays in mixtures of amniotic fluid samples from early and late pregnancies indicated that the drop of disaccharidase actiVIties at midpregnancy is not caused by inhibitors or activators. To examine other possible sources of disaccharidase activities in amniotic fluid, we assayed maltase, sucrase, trehalase, palatinase, and lactase activities in maternal and fetal serum, and in homogenates (I 0 or 20 per cent in water) of fetal liver, renal cortex, skin, amnion, and bladder. Their activities never exceeded 6 per cent of those found in jejunal mucosa and under our standard assay conditions, sucrase and palatinase activities were absent or barely detectable in aU tissues except the intestine.
Comment The results presented in this paper are consistent with the suggestion that disaccharidase activities of
Intestinal disaccharidases in amniotic fluid
75
amniotic Huid originate hom the fetal intestine. This conclusion is also supported by previous results showing identical physical and kinetic properties for amniotic Huid and intestinal disaccharidases. 1° Comparison between disaccharidase activities (per gram of protein) in jejunal mucosa and amniotic Huid of our fetuses suggests that as much as I :2 per ,·ent of total amniotic fluid protein comes from the fetal intestine. Since the bulk of protein in amniotic Huid originates from the maternal serum, 1 the fetal intestine could be a major source of fetal protein in amniotic fluid. Although four of our five fetuses suffered from various genetic diseases. their disaccharidase activities in amniotic fluid and jejunal mucosa were approximately in the range of previously reported value> lor similar gestional ages."· 14 · 17 • 1 ~ However, the mean activity on a large group of such patients could differ from the normal mean. Since disaccharidase activities in letai intestine increase or remain stable throughout gestation. 1 '1· 17 • '"' the cause(s) of the rapid fali of these er1lvme activities in amniotic fluid at about 22 weeks of gestation (fable II) is unknown. Available information suggests that both reabsorption and extrusion of disacch;nidases are influenced by fetal development. Arnnioti< fluid protein and enzymes are cleared in swallowed amniotic fluid. 19 As the fetus develops, swallowing imTeases and could overtake extrusion of disaccharidase. On the other hand, the striking correlation between the rapid accumulation of meconium in human fetal intestine 14 and the fall of disaccharidase activities at 22 weeks suggests that extrusion stops or is markedly decreased at this period of gestation. Extrusion of ciisaccharidases could also be diminished by increased permeabilitv of intestinal mucosa to intestinal protein in the lumen or decreased release of disaccharidases into dte lumen. More information is needed on the physioio>gv of meconium formation and on changes of the permeability of intestinal mucosa during fetal deveiopmem before any conclusion can be reached. Unlike other amniotic fluid disaccharidases. trehalase activity increases from 22 weeks to term (Table II). There are several possibilities to explai 11 rhis discordant finding. First. we have found that trehalase activity in near term amniotic fluid sample:; was in a soluble form since it was not sedimented when centrifuged at 105,000 X g for 30 minutes, contrarilY to trehalase and other disaccharidases before :l2 weeks, v;hich \Vere sedimented. 10 i~~lthough the physical state of trehalase in intestinal lumen after 22 weeks is unknown. it is possible that, due to the Jarge arnounts of meconium in fetal intestine during this period, 14 only soluble trehalase could pass into the amni<1tic cavity whereas other bound disaccharidases (presumably to
76
Potier, Melancon, and Dallaire
debris of brush border membranes) remain in the intestine. If this is the case, increasing activity of amniotic fluid trehalase would be the reflection of increasing trehalase activity in the fetal intestine itself. 14 Second, the trehalase activity after 22 weeks could come from a different source than the intestine. Among the fetal organs examined (at 16 to 21 weeks of gestation), the kidney cortex showed some trehalase activity although at a much lower level than in the intestine (less than 3 per cent). However, this enzyme could develop later during gestation and it is known that fetal micturition increases with gestation. 20 Work is in progress in our laboratory to clearly define the origin of amniotic fluid trehalase near term by comparing physical and kinetic properties of trehalase to fetal intestine, kidney, and amniotic fluid. The physiologic role of disaccharidases in amniotic fluid is not clear. These enzymes have been implicated in both degradation and transglycosidation reactions of disaccharides and oligosaccharides21 and could participate in the biosynthesis of complex carbohydrates. However, it is possible that amniotic fluid disaccharidases are also implicated in a more general physiologic process involving other intestinal protein and enzymes which could be recirculated in the fetal intestine via the
REFERENCES 1. Sutcliffe, R. G.: The nature and origin of the soluble protein in human amniotic fluid, Bioi. Rev. 50: 1, 1975. 2. Lowden, J. A., Cutz, E., Conen, P. E., Rudd, N., and Doran, T. A.: Prenatal diagnosis of GM 1 gangliosidosis, N. Engl.]. Med. 288: 225, 1973. 3. Desnick, !t J., Krivit, W., a_11d Sharp, M. L.: In utero diagnosis of Sandhoffs disease, Biochem. Biophys. Res. Commun. 51: 20, 1973. 4. Borresen, A.-L., and Van der Hagen, C. B.: Metachromatic leucodystrophy. II. Direct determination of arylsulfatase A activity in amniotic fluid, Clin. Genet. 4: 442, 1973. . 5. Sutcliffe, R. G., Brock, D. ]. M., Robertson, ]. G., Scrimgeour, J. B., and Moneghan, J. M.: Enzymes in amniotic fluid: A study of specific activity patterns during pregnancy,]. Obstet. Gynaecol. Br. Commonw. 79: 895, 1972. 6. Fernandez de Castro, A., Usategui-Gomez, M., and Spellacy, W. N.: Amniotic fluid amylase, AM. J. OBSTET. GYNECOL. 116: 931, 1973. 7. Armstrong, D., Van Wormer, D., Dimmitt, S., May, P., and Gideon, W. P.: The determination of peroxidase in amniotic fluid, Obstet. Gynecol. 47: 593, 1976. 8. Potier, M., Dallaire, L., and Melancon, S. B.: Amniotic fluid a-galactosidase activity: An index of gestational age, Gynecol. Invest. 5: 306, 1974. 9. Butterworth, J., Broadhead, D. M., Sutherland, G. R., and Bain, A. D.: Lysosomal enzymes of amniotic fluid in relation to gestational age, AM. J. OssTET. GYNEcoL. 119: 821, 1974. 10. Potier, M., Dallaire, L., and Melancon, S. B.: Occurrence and properties of fetal intestinal glycosidases (disaccharidases) in human amniotic fluid, Bioi. Neonate 27: 141, 1975.
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Am. J. Obstet. Gvnecol.
amniotic fluid. Lev and Oriic 22 demonstrated intestinal absorption and transport of an enzyme (horseradish peroxidase) instiiiated into rat amniotic cavity. Reabsorption of intestinal protein might play a role in the regulation of biosynthesis andior degradation processes of these proteins or could represent a mechanism for the recyciing of intestinal protein by the intestine. The importance of such mechanisms for normal fetal development remains to be established. A possible clinical application of these findings is in the detection of obstruction of the lower part of fetal intestine at 14 to 18 weeks on the basis of low or absent disaccharidase activities in amniotic fluid. However, it should be pointed out that much more information about the normal biology of intestinal disaccharidases is necessary before the information obtained in this study can be applied clinically. We greatly appreciate the technical assistance given by Mrs. Marie-Josee Code. Addendum We have recently reported a deficiency of disaccharidase activity in the amniotic fluid of a fetus born with anal imperforation (Potier, M., Dallaire, L., and Melan{:on, S. B.: Lancet 2:982, 1977.).
ll. Potier, M., Melancon, S. B., and Dallaire, L.: Fetal intestinal disaccharidases in human amniotic fluid, Biomedicine [Express] 25: 167, 1976. 12. Dallaire,~., Potier, M., Melancon, S. B., and Patrick, J.: Fetomaternal aminoacid metabolism. J. Obstet. Gynaecol. Br. Commonw. 81: 761, 1974. 13. Campbell, S.: Ultrasonic fetal cephalometry during the second trimester of pregnancy. J. Obstet. Gynaecol. Br. Commonw. 77: 1057, 1970. 14. Dahiqvist, A., and Lindberg, T.: Developtnent of the intestinal disaccharidase and alkaline phosphatase activities in the human fetus, Clin. Sci. Mol. Med. 30: 517, 1966. 15. Dahlqvist, A.: Method for assay of intestinal disaccharidases. Anal. Biochem. 7: 18, 1964. 16. Bohlen, P., SteLT!, S., Dairman, W., and Udenfriend; S.: Fluorometric assay of protein in the nanogram range. Arch. Biochem. Biophys. 155: 213, 1973. 17. Antonowicz, I., Chang, S. K., and Grand, R. C.: Development and distribution of lysosomal enzymes and disaccharidases in fetal intestine, Gastroenterology 67: 51, 1974. 18. Auricchio, S., Rubino, A., and Mi.irset, G.: Intestinal glycosidase activities in the human embryo, fetus and newborn, Pediatrics 35: 944, 1965. 19. Gitlin, D., Kumate, J., Morales, C., Noriega, L.. and Arevalo, N .: The turnover of amniotic fluid protein in the human conceptus, AM. J. 0BSTET. GYNECOL. 113: 632, 1972. 20. Jeffcoate, T. 1~. A., and Scott, j. S.: Polyhydramnios and oligohydramnios, Can. Med. Assoc. J. 80: 77, 1959. 21. Karlson, P.: Glycosides, oligosaccharides, polysaccharides, in Introduction to Modern Biochemistry, New York, 1968, Academic Press, Inc., p. 314. 22. Lev, R., and Orlic, D.: Protein absorption by the intestine of the fetal rat in utero, Science 177: 522. 1972.