AMNIOTIC-FLUID ACETYLCHOLINESTERASE AS A POSSIBLE DIAGNOSTIC TEST FOR NEURAL-TUBE DEFECTS IN EARLY PREGNANCY A. D. SMITH
University Department of Pharmacology, Oxford H. S. CUCKLE J. WALD I.C.R.F. Cancer Epidemiology and Clinical Trials Unit, Department of the Regius Professor of Medicine, Radcliffe
Infirmary, Oxford G. M. STIRRAT
Nuffield Department of Obstetrics and Gynæcology, University of Oxford M. BOBROW
Department of Medical Genetics, Oxford Area Health Authority
Occasionally, however, an unaffected pregnancy proa positive result. The frequency of such falsepositives is about 1-5 per 1000 unaffected pregnancies tested, but again the exact rate is not known. Another duces
amniotic-fluid test would be useful to reduce the number of pregnancies misclassified by the A. F. P. test. The cholinesterase activity of cerebrospinal fluid is mainly due to the specific enzyme, acetylcholinesterase (AChE),4,5 which probably originates from the tissues of the brain or spinal cord.6-8 This prompted us to see whether AChE levels might be elevated in the amniotic fluid of pregnancies with fetal open-neural-tube defects. This appeared to be so when 14 amniotic-fluid samples collected in Sweden (6 from pregnancies with neuraltube defects) were analysed in a preliminary study (H. Lagercrantz and A. D. Smith, unpublished). We report here the results of a larger study of amniotic-fluid samples from 77 pregnancies, including 5 misclassified by the A.F.P. test. Methods
Department of Pædiatrics, Karolinska Sweden
Raised levels (≥4·5 munits/ml) of acetylcholinesterase (AChE) activity in amniotic fluid at 14-23 weeks of pregnancy were significantly associated with open fetal neural-tube defects. Out of 72 pregnancies correctly classified by the amniotic-fluid alpha-fetoprotein (A.F.P.) test, 2 of 56 without neuraltube defects and all 16 with open neural-tube defects (8 with anencephaly and 8 with open spina bifida) had raised levels of AChE. Out of 5 pregnancies misclassified by the A.F.P. test (4 without neural-tube defects and 1 with open spina bifida), only 1 was misclassified by the AChE test—namely, one of those without a neural-tube defect. Thus, only 3 of the 77 pregnancies tested were misclassified by the quantitative AChE test. A qualitative test for an isoenzyme of AChE found in cerebrospinal fluid correctly classified these 3 pregnancies. These findings suggest that the analysis of AChE in amniotic fluid may be a useful test in the diagnosis of open neural-tube defects.
Introduction MEASUREMENT of the level of alpha-fetoprotein in amniotic fluid is a widely used diagnostic test for open neural-tube defects (N.T.D.).1-3 The proportion of affected pregnancies with a positive A.F.P. result is
Amniotic-fluid clinic in Oxford
samples collected were
cells, and the
supernatants were stored at 40°C. These had been assayed for A.F.P. by electroimmunodiffusion before storage. The following samples, collected before 1979, which had been taken at 14-23 completed weeks of gestation were retrieved from storage: 1. True-negatives: a random selection of 56 pregnancies without N.T.D., with negative amniotic-fluid A.F.P. results. 2. True-positives: a random selection of pregnancies with open N.T.D., with positive A.F.P. results (8 anencephaly and 8 open spina bifida). 3. False-positives: all 4 pregnancies without N.T.D. but with positive A.F.p. results. 4. False-negatives: the only pregnancy with an open N.T.D. (open spina bifida) but with a negative A.F.P. result. A result of the amniotic-fluid A.F.P. test was taken to be positive if it had a value equal to or greater than 5 standard deviations above the mean for the relevant week of gestation.
Quantitative AChE Assay Each sample was centrifuged at 14 000 g (3 min), and the supernatant was quantitatively assayed for AChE activity without its A.F.P. level or whether it came from an affected pregnancy being known. The method used was that described by Ellman and his colleagues,9 modified to increase the number of samples that can be assayed at one time.’° The hydrolysis of acetylthiocholine (final concentration 1 mmol/1) by amniotic fluid (0-05 ml) was determined in tubes containing 0-1 ml 5,5’-dithiobis(2-nitrobenzoic acid) (final concentration 0.33 mmol/1) and 2.65 ml 50 mmol/1 Na/K phosphate buffer pH 7.0, incubated for 2-3 h at 30 °C. Each sample was divided into two, one volume being incubated with substrate alone and the other with BW284C51 dibromide (final concentration
686 TABLE I-AMNIOTIC-FLUID
IN NON-N.T.D. PREGNANCIES
WITH NEGATIVE AMNIOTIC-FLUID A.F.P. RESULTS
tation. The overall mean AChE level was 2.55 (±S.D. 1.29) munits/ml. The highest AChE level obtained (866 munits/ml) was from a pregnancy which ended in miscarriage about 4 weeks after the amniotic fluid was taken. The next highest AChE level (5-26 munits/ml) was from a pregnancy which ended normally. The remaining 54 AChE levels were less than 4.5 munits/ml. The amniotic-fluid AChE and A.F.P. levels in the 16 N.T.D. pregnancies with positive A.F.P. results (group 2) are shown in table n. The mean AChE level was 9.9 munits/ml for the 8 pregnancies with anencephaly and 7.2 munits/ml for the 8 pregnancies with open spina bifida ; and all values were above 4.5munits/ml. This level, which appeared to separate most effectively the non-N.T.D. from the N.T.D. pregnancies in groups 1 and 2, was provisionally chosen as our cut-off level.
1-5x10 mol/1), a specific inhibitor of AChE, for 5 min before the substrate (0.11 ml) was added. At the end of the incubation period 0.1 ml eserine sulphate (final concentration 2x 10-5 mol/1) was added to inhibit both AChE and non-specific cholinesterase activity. AChE activity, calculated from the difference in optical absorbance (at 412 nm) between the tubes with and without BW284C51, was expressed in units of 1 p.mol substrate hydrolysed per minute. The product (thiocholine) is sensitive to oxidation, and to avoid this we found it necessary to make up all reagents in twice-distilled water which had been boiled in an open beaker to drive off traces of chlorine. AChE estimations were usually done in duplicate but when these differed by more than 50% estimations were done in triplicate. If tests were done in duplicate the arithmetic mean was used for the analyses, but if they were done in triplicate the median was used.
AND A.F.P. IN N.T.D.
PREGNANCIES WITH POSITIVE AMNIOTIC-FLUID A.F.P. RESULTS
The quantitative assay of AChE activity is critically dependent on the concentration of the inhibitor BW284C51: if the concentration is too great some of the non-specific cholinesterase will be inhibited, and if it is too low AChE itself will be incompletely inhibited, resulting, respectively, in falsely high or falsely low estimates of AChE activity. For this reason we also analysed selected samples (20 jjd) qualitatively by gel electrophoresis, which separates AChE from non-specific cholinesterase. Clarke’s simplified procedure," using "tris"/glycine buffers, was followed, except that the electrophoresis in the 6% polyacrylamide gels was continued until the band of bromophenol-blue bound to albumin had reached the end of the gel. Bands containing enzyme activity were revealed by incubation of gels with acetylthiocholine as previously described,1O except that the pre-incubation step in this procedure was carried out in the incubation mixture without substrate, the latter being added to the tubes after 30 min. The gels were incubated with gentle agitation at room temperature until bands of precipitate could be clearly seen (2-16 h). Materials were obtained from the following sources: acetylthiocholine (made up freshly in water) from BDH Chemicals,
AND A.F.P. IN NON-N.T.D.
PREGNANCIES WITH POSITIVE AMNIOTIC-FLUID A.F.P. RESULTS
(FALSE POSITIVES) AND IN THE N.T.D. PREGNANCY WITH A NEGATIVE A.F.P. RESULT (FALSE NEGATIVE)
Poole, Dorset; 5,5’-dithiobis(2-nitrobenzoic acid) (recrystallised from water before use) from Boehringer, London; BW284C51 (15,-bis-[4-allyldimethylammoniumphenyl]pentan3-one dibromide) from Wellcome Reagents Ltd., Beckenham, Kent, or from Sigma Chemicals, London; ’Cyanogum 41’ (used to make the polyacrylamide gels) from BDH Chemicals.
Quantitative AChE Assay The amniotic-fluid AChE levels in the 56
pregnancies with negative amniotic-fluid A.F.P. results (group 1) are shown in table i. There did not appear to be a strong relationship between AChE level and ges-
Case Cl of Wald et all and Stirrat et al.," which may have been associated with a fetal nephrotic syndrome. Upon reassay an AChE level of 3. 89 munits/ml was obtained.
687 TABLE IV-SUMMARY OF AMNIOTIC-FLUID RESULTS IN THE
our study according to whether or not they correctly classified by the amniotic-fluid A.F.P. test.
nancies in were
The qualitative AChE
the 3 pregnancies misclassified by the quantitative AChE test and on samples from all the 17 N.T.D. preg-
Table in shows the amniotic-fluid AChE and A.F.P. levels in the 4 non-N.T.D. pregnancies with positive A.F.P. results (group 3) and the N.T.D. pregnancy (open spina bifida) with a negative A.F.P. result (group 4). In group 3, 3 pregnancies were associated with amniotic-fluid AChE levels below the cut-off level, and 1 was associated with a higher level (5-28 munits/ml), but in this pregnancy, which has been described previously (case Clof Wald et al.12 and Stirrat et al.13) the amniotic-fluid-albumin level was also elevated, consistent with a diagnosis of nephrotic syndrome. The open-spina-bifida pregnancy (group 4) was associated with an amniotic-fluid AChE level of 4.8munits/ml-just above the cut-off level. Table iv summarises the AChE results of all the preg-
electrophoresis of amniotic fluid, showing bands of reaction product formed by hydrolysis of acetylthiocholine.
Samples from: (a) Spina-bifida pregnancy. (b) Same as (a) but incubated with the specific inhibitor of AChE (BW284C51 at 10-4 mol/1), showing that band 2 is due to AChE. (c) Pregnancy without a neural-tube defect, showing only a single major band (1), due to non-specific cholinesterase. (d) Pregnancy without a neural-tube defect, showing reaction product on the origin (0), indicating that the AChE was of a different molecular form fron that in band 2. This pregnancy (AChE level 8-6munits/ml) resulted in a miscarriage.
nancies and 17 of the non-N.T.D. pregnancies selected at random. Samples from all 17 pregnancies with neuraltube defects yielded two bands which migrated into the gel-one due to non-specific cholinesterase (band 1. in the figure) and one, which migrated further, due to AChE activity (band 2 in the figure). Band 2 migrated to the same position as the isoenzyme of AChE in samples of human cerebrospinal fluid, suggesting that the amniotic-fluid AChE was of neural origin. None of the samples from any of the 20 pregnancies without neural-tube defects yielded a band which migrated to the position of band 2 although they all yielded one at the position of band 1. One of the samples misclassified by the quantitative AChE test yielded a pronounced band due to AChE activity which remained at the origin of the gel (0 in the figure) instead of migrating towards the anode. This must have been from AChE of larger molecular size or of a different charge from that in cerebrospinal fluid or in amniotic fluid from pregnancies with neural-tube
defects. Discussion The results show that raised amniotic-fluid AChE levels are closely associated with anencephaly and open spina bifida. The quantitative AChE test misclassified only 3 pregnancies out of the 77 studied. Among the 5 selected for study because they had been incorrectly classified by the amniotic-fluid A.F.P. test, only 1 was misclassified by the quantitative AChE test, and this was correctly classified when reassayed. The gel-electrophoresis test was carried out to check the quantitative AChE results; and, although only qualitative, it correctly classified the 3 misclassified by the quantitative AChE test. Clearly both techniques deserve further investigation in a larger study. Raised levels of both AChE and non-specific cholinesterase have been described in amniotic fluid from a pregnancy with anehcephaly.14 Since blood-plasma contains very high levels of non-specific cholinesterase, and since amniotic-fluid samples can be contaminated with blood, we restricted the present study to AChE and its
isoenzymes. Our findings suggest that analysis for AChE may be useful in the diagnosis of neural-tube defects in pregnancies with positive amniotic-fluid-A.F.P. results. A policy of doing AChE tests on patients with positive A.F.P. results may lead to a substantial reduction in the number of unaffected pregnancies which might be terminated if the A.F.P. result alone were used. We cannot yet say what proportion of open N.T.D.S would be missed if such a policy were followed, but the fact that all 17 N.T.D. pregnancies tested had AChE levels above the cutoff suggests that the reduction in the detection-rate may not be great. Our findings also raise the possibility that amniotic-
688 fluid AChE may, by itself, be a better test than amnioticfluid A.F.P. in the diagnosis of open neural-tube defects. However, a very large survey would be needed to investigate this, because the A.F.P. test is associated with only a small proportion of false-positive and false-negative results. We thank Miss L. Bromley, Mr Colin Evans, and Mrs Rosemary Brett for technical assistance, Mrs Nora Jones for collecting clinical details, and Mrs Catherine Harwood for typing the manuscript. This study was supported in part by the R. J. Harris Trusts. H.L. was supported by the Swedish Medical Research Council and the Prenatal-
forskningsamnden. Requests for reprints should be addressed to A. D. S. , University Department of Pharmacology, South Parks Road, Oxford OX1 3QT. REFERENCES
1. 2. 3.
Brock, D. J. H., Scrimgeour, J. B., Nelson, M. M. Clin. Genet. 1975, 7, 163. Kimball, M. E., Milunsky, A., Alpert, E. Obstet. Gynec. 1977, 49, 532. Kleijer, W. J., De Bruijn, H. W. A., Leschot, N. J. Br. J. Obstet. Gynæc.
1978, 85, 512. 4. Reiss, M., Hemphill, R. E. Nature, 1948, 161, 18. 5. Tower, D. B., McEachern, D. Can. J. Res. E. 1949, 27, 105. 6. Chubb, I. W., Goodman, S., Smith, A. D. Neuroscience, 1976, 1, 57. 7. Greenfield, S. A., Smith, A. D. J. Physiol. 1976, 258, 108p. 8. Fuenmayor, T. L., Smith, A. D., Vogt, M. ibid. 1976, 263, 165p. 9. Ellman, G. L., Courtney, K. D., Andres, V., Featherstone, R. M. Biochem. Pharmac. 1961, 7, 88. 10. Chubb, I. W., Smith, A. D. Proc. R. Soc. Lond. B. 1975, 191, 245. 11. Clarke, J. T. Ann. N.Y. Acod Sci, 1964, 121, 428. 12. Wald, N. J., Cuckle, H. S., Boreham, J., et al. Br. J. Obstet. Gynec. 1979,
86, 91. 13. Stirrat, G. M., Turnbull, A. C., Bennett, M. J., Bobrow, M., Wald, N. J., Cuckle, H. S. ibid. (in the press). 14. Chubb, I. W., Springell, H. J., Pollard, A. C. Proc. Aust. physiol. pharmac. Soc. 1978, 9, 114p.
ACETYLCHOLINESTERASE IN HUMAN AMNIOTIC FLUID: AN INDEX OF FETAL NEURAL DEVELOPMENT? P. M. PILOWSKY A. C. POLLARD
I. W. CHUBB H. J. SPRINGELL
Centre for Neuroscience Department of Human Physiology, Flinders University of South Australia, Bedford Park, South Australia 5042,
Department of Chemical Pathology, Adelaide Children’s Hospital, Adelaide, South Australia 5006
Acetylcholinesterase (AChE) activity
was detected in amniotic fluid either at 14-23 weeks gestation or amniocentesis sampled by collected at normal parturition. Activity ranged from 0·2 to 8·9 u/l in the earlier samples and from 0·2 to 4·2 u/l in the later samples. Since the fetal but not the maternal serum contained AChE activity, we suggest that the AChE in the amniotic fluid is derived from the fetus. AChE is released from both neural and nonneural tissues, and the release of the enzyme from both sources may diminish as the nervous system matures. The amount of AChE found in amniotic fluid may be affected by abnormalities of the nervous system. Amniotic fluid from two fetuses with spina bifida contained AChE activity: one, with a gestational age of 21 weeks, was in the upper levels of the normal range (4·5 u/l), whereas the other, at 36 weeks, contained 1·9 times more activity than the next highest sample. In a 20-week fetus with anencephaly, the AChE activity in the amniotic fluid was increased 2·8-fold over the activity of the highest "normal" sample.
Introduction SEVERAL reports suggest that acetylcholinesterase (AChE) may be secreted by a variety of excitable celltypes. These include chromaffin and ganglion cel!s,I,2 motor nerves,3,4 central-nervous-system neurons,5.6 and muscle cells in vivo’ or in vitro.8 AChE has been reported to be actively secreted from some cellsl.4.5 by a mechanism dependent on external Ca2+, whereas secretion from muscle appears to be a simple extrusion from the cells Although there are several soluble molecular forms of AChE within these tissues, 1,4,1,1 only one molecular form of the AChE is ever found to be se-
Cytochemical studies have demonstrated a dual distribution of AChE in most cells; there is invariably activity associated with the plasma membrane10 and with endoplasmic reticulum. 2, 10, 11 In the perikarya of neurons, the rough endoplasmic reticulum stains for AChE activity,2,10,11 whereas in axons the enzyme is associated with the smooth endoplasmic reticulum.10,11 AChE is thought to be secreted from both the rough and smooth endoplasmic reticulum. In contrast to adult nerve terminals, immature terminals often contain elements of the endoplasmic reticulum that stain for AChE.12 Moreover, the tubules often appear to have fused with the neuronal plasma membrane.12 Thus, immature nerve terminals might be expected to release more AChE than adult tissues. Measurements of AChE activity in bovine fetal serum seem to support this suggestion. AChE activity is very high early in gestation and then falls, so that by parturition (280 days) the fetal value is still 8 times greater than that found in adults.’3 This may be contrasted with lambs, whose values are also high early in gestation but which reach adult levels by parturition.13 One difference between the nervous systems of these two species is that the bovine sympathetic nervous system is less mature at birth. 14 Since it is known that some proteins from the human fetal circulation, such as ot-fetoprotein, appear in amniotic fluid,15 high circulating levels of AChE in developing fetuses might result in high activities in amniotic fluid. We now report the detection of AChE activity in amniotic fluid at several gestational ages. These results were presented to the meeting of the Australian Physiological and Pharmacological Society in August, 1978.’6,17 Methods Portions of human amniotic fluid were taken from samples either obtained by amniocentesis for routine diagnostic purposes or collected at parturition. They were centrifuged in a Beckman Microfuge for 2-5 min and all samples which contained erythrocytes were discarded. The supernatants were either assayed immediately or stored at -25°C for up to 3 weeks. No losses of activity were observed by freezing for this period. Umbilical-cord blood obtained at parturition was collected in plastic tubes and allowed to clot, and the serum was stored at -25 °C for up to 3 weeks before assay. AChE was measured with 10-3 mol/l acetylthiocholine as substrate and butyrylcholinesterase (BChE), an enzyme derived from the liver and found in adult plasma, with 10x10’’ mol/1 butyrylthiocholine as substrate. Both assays and the
polyacrylamide-gel electrophoresis were performed as previously described.9 a-Fetoprotein was measured with the Laurell Rocket method of immunodiffusion 18 with Behring x-fetoprotein as the standard.