only a few weeks before term, disaccharidases are fully developed in the human fetal intestine as early as 10 weeks.165 The fetal kidney contains only trehalase and some maltase activities in detectable quantities. The intestinal mucosa contains disaccharidases able to hydrolyze a variety of substrates.
Origin of amniotic fluid disaccharidases
Disaccharidase activities in AF apparently originate from the fetal intestine and the kidney.130, 131, 134, 135, 137 The kidney disaccharidases (mainly trehalase) are detected in the AF at a later stage of gestation than the intestinal enzymes132 and in fetuses affected with renal pathologies.166, 167 Maltase activity of AF originates exclusively from the fetal intestine.168 The amount of each disaccharidase released into the AF seems to be dictated by their relative sensitivities to proteolytic digestion in vivo.168
Intestinal microvilli of fetal origin have been characterized in AF after purification by Ca2+ precipitation of contaminating organelles followed by differential centrifugation of the microvillar membranes.169, 170 In the purified preparation, the specific activities of the intestinal microvillar marker enzymes maltase and sucrase increased about 77‐fold over those in cell‐free AF. AF microvilli contain typical enzymes of intestinal microvilli, including maltase, sucrase, trehalase, alkaline phosphatase, and γ‐glutamyl transferase, and their morphology detected by electron microscopy resembles that of vesiculated intestinal microvilli. Jalanko et al.171 also reported the presence of vesicles in AF, which they concluded originate in the fetal intestine. Prenatal detection of genetic diseases due to a deficiency of a protein expressed in these membranes or associated with abnormal morphology of microvilli seems feasible, although such diagnoses have not been described for many years. Transport system activities expressed in these membranes can also be assayed by measuring the uptake of radioactive substrates. Na+‐dependent glucose transport, inhibitable by phlorizin, was demonstrated in microvilli purified from AF, suggesting that transporter systems can be assayed in these membranes (Figure 3.2). There is evidence that trehalase activity also could originate from the fetal kidney, at least in pathologic situations. Several fetuses with proven intestinal obstructions had normal trehalase activity, despite the fact that the other disaccharidases were almost completely deficient.166, 172, 173 In addition, high trehalase activity (relative to the other disaccharidases) was found in the AF of fetuses with renal anomalies such as polycystic kidney disease166 and congenital nephrotic syndrome.173 In their study on the origin of α‐glucosidase activity in human AF, Poenaru et al.174 concluded that both renal and intestinal α‐glucosidases were present.
Figure 3.2 The uptake of 3H‐glucose in microvilli prepared from (a) fetal intestinal mucosa and (b) amniotic fluid.
Isoelectric focusing revealed that the intestinal form of trehalase (pI54.60) was present in AF samples collected before 21 weeks, whereas only the renal form (pI54.24) was present in samples obtained later in pregnancy.175 In one fetus affected with polycystic kidney disease, the renal form of trehalase was markedly increased in the AF. In another fetus with intestinal obstruction, the intestinal form of trehalase, as well as other disaccharidase activities, was reduced in the AF. However, no systematic study on the clinical usefulness of AF trehalase for the detection of fetal renal anomalies has yet been conducted.
Other microvillar enzyme activities
Peptidases such as γ‐glutamyl transpeptidase, leucine aminopeptidase, aminopeptidase M, and alkaline phosphatase are no longer used for the prenatal diagnosis of cystic fibrosis (CF) (see Chapter 15). Given the intestinal origin of microvillar enzymes, efforts have been made to establish a diagnosis of intestinal obstruction,176, 177 megacystis‐microcolon intestinal hypoperistalsis syndrome,178, 179 and even fetal anal incontinence in spina bifida.180
Development of amniotic fluid disaccharidases
During the second trimester, 10–20 percent of the total proteins of AF appear to be of intestinal origin.181 Developmental patterns of AF disaccharidases have been studied by Potier et al.130 and Antonowicz et al.131 With the exception of trehalase, all disaccharidase activities vary approximately in parallel during pregnancy, being present from 10 weeks, reaching maximum values around 15–18 weeks, and dropping rapidly to low values after 22 weeks (Figure 3.3). In contrast, trehalase activity increased after 22 weeks.
Figure 3.3 A comparison of the various disaccharidase activities in amniotic fluid between 14 and 42 weeks of gestation, showing the different patterns of trehalase activity. All activities are expressed as a percentage of the mean activity at 14–17 weeks of gestation. — ‐ — ‐ Maltase, ‐ ‐ ‐ sucrase, – – – trehalase, —— palatinase, . . . lactase.
The drop of disaccharidase activities at around 22 weeks is due to increasing fetal swallowing with age and accumulation of large quantities of meconium in the fetal intestine.182, 183 Similar developmental patterns were observed for intestinal alkaline phosphatase and trypsin.184, 185 Results of studies on animal models support the conclusion that there is a relationship between meconium accumulation in the fetal intestine and the rapid fall of disaccharidase activities in the AF.182
Clinical use of amniotic fluid disaccharidases
The AF disaccharidases have been used for the prenatal detection of fetal intestinal obstruction on the basis of low or absent activities in the AF.166, 186 Van Diggelen et al.186 described a fetus with anal atresia at 17 weeks of gestation, and Dallaire and Perreault176 studied 16 fetuses between 16 and 20 weeks with different types of intestinal obstruction, with or without ventral wall defects and chromosomal syndromes. In all of these cases, disaccharidase activities were abnormally low. The exclusively intestinal hydrolases maltase, sucrase, palatinase, and an intestinal form of alkaline phosphatase were the best markers for detecting intestinal obstruction.172 For the disaccharidase test to give valid results, the intestinal obstruction should hamper normal release of disaccharidases into the amniotic cavity. With multiple intestinal atresia the intestinal obstruction is complete, whereas with Hirschsprung disease the intestinal obstruction may be incomplete, thus permitting normal AF disaccharidase activities.187
Low or absent disaccharidase activities, as well as alkaline phosphatase and α‐glutamyl transferase186, 188–191 have been reported in fetuses affected with CF. These fetuses seem to be unable to release their intestinal content normally into the amniotic cavity. It is important that the disaccharidase assay be performed no later than the 20th week because, after this period, some normal AF samples were found to have very low
