Clinica Chimica Acta. 205 ( 1992) IS’- I66 0 1992 Elsevier Scientific Publishers B.V. All rights
157 reserved.
0009-8981/92/$05.00
CCA 05219
Cholinesterases exhibiting aryl acylamidase activity in human amniotic fluid L.D. Jayanthi”, N. Balasubramanianb
and A.S. Balasubramanian”
“Department of Neurological Sciences and hDepartment of Obstetrics and Gynecology, Christian Medical College and Hospital, Vellore 632 004 (India) (Received
16 July 1991; revision
Key words: Cholinesterases;
received
30 September
Aryl acylamidases;
Human
1991; accepted amniotic
4 October
fluid; Neural
1991)
tube defect
Summary
Acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8) in human amniotic fluid were estimated in the presence of selective inhibitors. Amniotic fluid cholinesterases (mixture of acetylcholinesterase and butyrylcholinesterase) purified by procainamide-Sepharose affinity chromatography exhibited aryl acylamidase activity which was sensitive to serotonin inhibition (a property of aryl acylamidases associated with both acetyl- and butyrylcholinesterases) and tyramine activation (shown exclusively by aryl acylamidase associated with butyrylcholinesterase). Tyramine activation was unaffected in the presence of the selective acetylcholinesterase inhibitor BW284C51 whereas it was abolished in the presence of the selective butyrylcholinesterase inhibitor ethopropazine, suggesting the presence of both types of aryl acylamidases in amniotic fluid, one associated with acetylcholinesterase and the other associated with butyrylcholinesterase. Butyrylcholinesterase and the associated aryl acylamidase activity in the affinity purified enzyme was selectively immunoprecipitated by a polyclonal antibody raised against human serum butyrylcholinesterase. Estimation of the activity ratio of acetylcholinesterase to butyrylcholinesterase in a few samples of amniotic fluid showed that this could vary depending on the butyrylcholinesterase arising from contaminating blood in the samples. Gel electrophoresis under non-denaturing conditions and enzyme staining showed that butyrylcholinesterase band was detectable on the gel in all the samples whereas acetylcholinesterase band was below detectable levels in normal samples but visible in samples from pregnancies of neural tube defect fetuses. It is suggested that the use of selective cholinesterase inhibitors along with gel electrophoresis and immunoprecipitation studies may be useful in the assessment of cholinesterase activities in human amniotic fluid. Correspondence to; A.S. Balasubramanian, Hospital, Vellore-632 004, Tamil Nadu,
Dept. of Neurological India.
Sciences, Christian
Medical College &
158
Cholinesterases of vertebrates fall into two categories, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). These two enzymes differ in their substrate specificities and inhibition by selective inhibitors. AChE preferentially hydrolyses acetylchohne or acetyl-~-methylch~line while BuChE hydrolyses butyrylcho~ine preferentially, Bis-(4-ally1 dimethyl ammonium pheny~~-pentane-3one dibromide (BW284C51) is a selective inhibitor of AChE and tetra isopropyl pyrophosphoramide (iso-OMPA) and ethopropazine are selective inhibitors of BuChE. They are widely used in the selective estimation of AChE and BuChE in a mixture of the two enzymes. Excitable tissues and human erythrocytes contain predominantly AChE whereas the human serum contains almost exclusively BuChE. Work from this laboratory [ 1,2] and from other [ 31 has demonstrated that an aryl acylamidase (AAA) (EC 3.5. I, 13) activity sensitive to amines is exhibited by AChE of brain and other sources and BuChE of human serum. Serotonin inhibited the AAA activity of both AChE and BuChE whereas tryamine significantly activated the AAA activity of serum BuChE exclusively [ 13. The human amniotic fluid contains both AChE and BuChE [4,5] and the ratio of AChE to BuChE ranges from 0.02 to 0.26 [ 5 ] . Several reports have indicated that an elevated level of AChE in amniotic fluid is an indicator of spina bifida (menin~omyelocele) and anencephaly, the two major types of neural tube closure defects (NTD) [ 4,s 1. This tinding is rational because most AChE is probably derived from the nervous system f 51. The nervous tissue in NTD is either in direct contact with the amniotic fluid or just covered with a thin membrane f6]. Electrophoretic analysis of amniotic fluid from pregnancies of NTD fetuses showed a faster migrating band corresponding to AChE in addition to the slow moving band corresponding to BuChE [ ‘7-91. Sedimentation studies showed that normal amniotic fluids contain 4s (G,) form of AChE [ lo] and also 10s (G4) form [ 11I. Neural tube defects have been hnked to a disproportional increase in the 10s (GJ form [HI]. Since there is no report showing the presence of AAA activity in human amniotic fluid and its association with amniotic fiuid cholinesterases, we have made an attempt to isolate the amniotic fluid cholinesterases by procainamide-Sepharose affinity chromatography and show the association of AAA activity with the cholinesterases. We have also assessed the AChE and BuChE activities in human amniotic fluid samples from normal and NTD pregnancies in the presence of selective inhibitors and by gel electrophoresis. Materials and Methods Procainamide-Sepharose was prepared by linking procainamide to CNBractivated Sepharose-4B through a IO-carbon spacer arm [ I]. ~rj~cat~on of human serum BuChE by procainamide-Sepharose affinity chromtography and preparation of antibody against purified human serum BuChE were as described earlier [ 11. Electric eel AChE was from Sigma Chemical Co., USA, All other chemicals were prepared or procured as given earlier [ I]. Samples of amniotic fluid were collected during amniocentesis done for routine
159
diagnostic purposes. They were from pregnancies of normal fetuses (not affected by NTD) and also from pregnancies of anencephaly and meningomyelocele (spina bitida) fetuses. The samples collected were centrifuged at 10,000 x g for 5 min to remove cells and other sedimentable matter and dialysed against 20 nmol/l potassium phosphate buffer, pH 7.4 for 4 h at 4°C and used. Enzyme
assays
AChE and BuChE in amniotic fluid were assayed by the method of Ellman et al. [ 121 as described earlier [ 131 using acetylthiocholine and butyrylthiocholine respectively as substrates. Ethopropazine, the selective inhibitor of BuChE was included when AChE was estimated and BW284C5 1, the selective inhibitor of AChE was included when BuChE was estimated. The reaction mixture consisted of 50 ~1 of potassium phosphate buffer (1 mol/l) (pH 7.4), 20 ~1 of the thiocholine substrate (75 mmol/l), 50 ~1 of the selective inhibitor (1 mmol/l), 100 ~1 of 5,5’-dithiobis-(2 nitrobenzoic acid) (10 mmol/l) and 100 ~1 of amniotic fluid in a final assay volume of 500 ~1. The released thiocholine was measured spectrophotometrically at 412 nm. One unit of cholinesterase activity is defined as one pmol of thiocholine formed per minute at 37°C under the given assay conditions. AAA activity was assayed as described earlier [ 141. The reaction mixture contained 50 ~1 of potassium phosphate buffer (1 mol/l) pH 7.4, 250 ~1 of o-nitroacetanilide (10 mmol/l) and 100 ~1 of enzyme in a final assay volume of 500 ~1. Appropriate cholinesterase inhibitors were included in the reaction mixture when AAA activity exhibited by either AChE or BuChE alone was determined. Protein was estimated according to Lowry et al. [ 151. Chromatography
of amniotic fluid on procainamide-Sepharose
affinity column
Amniotic fluid (25 ml) dialysed against 20 mmol/l potassium phosphate buffer, (pH 7.4) was passed through a procainamide-Sepharose column (2 x 1 cm) preequilibrated with the same buffer at 4°C. The column was washed with 100 ml of the same buffer and the enzyme was eluted with procainamide (50 mmol/l) in the buffer. Fractions of 2 ml were collected and assayed for both cholinesterase and AAA activities. Active fractions were dialysed against the buffer to remove procainamide and used for other studies. Immunoprecipitation
To 100 ~1 of procainamide-Sepharose affinity purified enzyme, different concentrations (12.5-100 pg) of the polyclonal antibody raised against human serum BuChE were added. After keeping at 4°C for 12 h with mild shaking the tubes were centrifuged at 10,000 x g for 1 h and the AChE and BuChE activities both in the supernatant and in the immunoprecipitate (resuspended in 20 mmol/l potassium phosphate buffer, pH 7.4) were assayed in the presence of appropriate selective inhibitors. Controls were run without the antibody.
160
P~iyacrylam~d~ gei electr~p~oresis and staining for cho~~~estera~ea~t~~l~iy
Polyacrylamide gel electrophoresis under non-denaturing conditions was done on 7% slab gels in glycine-Tris buffer (50 mmol/l) (pH 8.3). The gels were then preincubated at room temperature for 15 min in potassium phosphate buffer (20 mmol/l) (pH 7.4). Staining for cholinesterase was done by shaking the gel at room temperature for 45 min in a mixture of a-naphthylacetate (20 mg in 2 ml acteone) and Fast Red TR salt (50 mg) in 100 ml potassium phosphate buffer (20 mmolil) (pH 7.4) [ 161. Red bands of cholinesterase activity that appeared on the gel were fixed in 7% acetic acid. For demonstrating AChE or BuChE activity on the gel. the selective inhibitors (10 pmolil) were included during the preincubation and staining of the gel. Results Procainamide-Sepharose
affinity chromatography
Procainamide-Sepharose affinity column could completely bind both AChE and BuChE from the amniotic fluid and both the activities could be eluted by procainamide from the column. In a typical column chromatography using 25 ml of amniotic fluid there was 54% and 55% recovery of AChE and BuChE respectively with 183 and 194 fold increase in enzyme units per mg protein respectively. In confirmation of earlier findings [ 1,2,13,17], the fractions eluted from pro~ainamide-Sepharose column also showed AAA activity co-eluting with cholinesterases. Figure 1 gives the elution profiles of AChE, BuChE and AAA activities from the affinity column. This affinity chromatography method provided a simple and useful method for getting amniotic fluid cholinesterases and AAA in a partially purified concentrated form.
O-06
2 4
6
8 1012 14 1618 2670727476 Fraction number
Fig 1. Procainamide-Sepharose affinity chromatography of human amniotic fluid: Elution profile of AChE ( 0 ), BuChE (0), AAA (A) and protein (A) from the procainamide-Sepharose column. Experimental details and assay procedures are given under Materials and Methods. Arrow indicates the fraction where elution with procainamide was started.
161
A
B uo
100 80 60 40 20 0 Serotonin
(mM )
I1 2 5 Tyromine( mM )
Fig. 2. Inhibition by serotonin and activation by tyramine of AAA activity in a mixture of AChE and BuChE in amniotic fluid: AAA activity was measured in the presence of various concentrations of serotonin (A) and tryamine (B). Enzyme assay procedures are given under Materials and Methods.
Inhibition by serotonin and activation by tyramine of AAA activity associated with cholinesterases of amniotic fluid AAA activity associated with AChE and BuChE is known to be inhibited by serotonin whereas AAA activity associated with BuChE alone is known to be activated by tyramine [l-3]. We examined the effect of serotonin and tyramine on the AAA activity of the affinity purified enzyme taking advantage of the known fact [ 1,2] that selective cholinesterase inhibitors also inhibit selectively the AAA activity associated with each type of cholinesterase. Serotonin at OS-5 mmol/l concentration showed 38-97% inhibition of AAA activity and tyramine at similar concentrations showed 179-307% activation of AAA activity in the absence of selective cholinesterase inhibitors (Fig. 2A and 2B). In the presence of BW284C51, the selective inhibitor of AChE, tyramine at l-2 mmol/l stimulated the AAA activity 340-520% whereas in the presence of ethopropazine, the selective inhibitor of BuChE, tyramine had no effect on AAA activity (Fig. 3). These results suggested that the AAA activity of am-
Tyraminel
mM)
Fig, 3. Effect of various concentrations of tyramine on the AAA activity associated with BuChE and AChE of amniotic fluid: The AAA activity associated with BuChE ( 0) was measured in the presence of BW284C51 (10 pmol/l) and the AAA associated with AChE (0) was measured in the presence of ethopropazine (10 pmolll). Enzyme assay procedures are given under Materials and Methods.
niotic fluid cholinesterases is susceptible to serotonin inhibition and that the AAA associated with BuChE alone is activated by tyramine. Immunoprecipitation of human amniotic fluid BuChE by polyclonal antibody raised against human serum BuChE Polyclonal antibody raised against purified human serum BuChE is known to cross-react specifically with BuChE, but not with AChE [ 11. We examined the immunoprecipitability of human amniotic fluid BuChE by the antibody raised against human serum BuChE. As shown in Fig. 4 BuChE from procainamide-Sepharose affmity purified enzyme could be completely immunoprecipitated by the antibody suggesting immunological similarity of human serum BuChE to human amniotic fluid BuChE. The precipitated BuChE activity was recovered in the immunoprecipitate (Fig. 4). Figure 4 also shows that AChE of amniotic fluid was unaffected by the antibody used and was present almost 100% in the supernatant after immunoprecipitation. The AAA activity present in the immunoprecipitate after 100% precipitation of BuChE was activated by tyramine whereas the AAA in the supernatant was not affected by tyramine confirming that tyramine activation was an exclusive property of the AAA associated with BuChE (results not shown). Estimation of AChE and BuChE activities in human amniotic fluid samples Although AChE and BuChE from different sources preferentially hydrolyse acetylthiocholine and butyrylthiocholine respectively, they also exhibit a low
50 Antibody
100
Fig. 4. i~unoprecipitation of amniotic fluid BuChE by various concentrations of antibody raised against purified human serum BuChE: Activity of BuChE (a) and AChE (0) in the supernatant is shown by solid lines and the activity of BuChE in the i~unoprecipitate is shown by broken line. Details of i~unoprecipitation and determination of BuChE and AChE activity are given under Materials and Methods.
163 TABLE I Ratios of AChE to BuChE activities in human amniotic fluid samples Type of sample
Gestational age (weeks)
AChE/BuChEratio
Normal Normal Normal Normal Normal Spina bifida Spina bifida Spina bifida Anencephaly *Anencephaly *Anencephaly
38 36 30 36 40 40 16 16 36 30 38
0.30 0.30 0.30 0.25 0.30 0.40 0.45 0.49 0.33 0.14 0.11
*Indicates the amniotic fluid samples contaminated with blood.
Fig, 5. Polyacrylamide gel electrophoresis and enzyme staining of cholinesterases in amniotic fluid samples: Lane I, purified BuChE from human serum; Lane 2, standard electric eel AChE (Sigma); Lane 3, eel AChE in presence of BW284CS 1 (10 pm&l); Lane 4. cholinesterases from amniotic fluid of normal pregnancy; Lane 5, amniotic fluid cholinesterases of NTD pregnancy; Lane 6, amniotic fluid cholinesterases of NTD pregnancy in presence of BW284C51 (10 pmolil). The selective AChE inhibitor BW284C51 was added during preincubation and staining of the gel in lanes 3 and 6. The arrow A points to the BuChE band and arrow B points to the AChE band.
164
hydrolytic activity towards the less preferred substrates. This necessitates the use of selective inhibitors during assay. We estimated AChE and BuChE activities in eleven different amniotic fluid samples and the AChE to BuChE ratios are given in Table I. For five normal amniotic fluid samples (not affected by NTD) the AChE to BuChE ratio ranged from 0.25 to 0.3. For four NTD samples it ranged from 0.33 to 0.49, higher than the value of 0.3. However, two other NTD samples gave low values of 0.11 and 0.14. These two samples with low AChE to BuChE ratios were highly contaminated with blood and therefore the contaminating serum BuChE might have reduced the AChE to BuChE ratios. Polyacrylamide gel electrophoresis Detectability of cholinesterase bands on the gel after electrophoresis followed by enzyme staining was possible only when the amniotic fluid samples used were concentrated by procainamide-Sepharose affinity chromatography. About 25 ml of amniotic fluid available from one normal and one NTD case were concentrated and enzyme activity equivalent to 1.5 units of BuChE from each was subjected to gel electrophoresis and staining (Fig. 5). The NTD sample gave a prominent band corresponding to human serum BuChE and a band of lesser intensity corresponding to electric eel AChE. In the normal sample only the BuChE band was detectable. Use of the selective inhibitor BW284C51 during staining confirmed the identity of the cholinesterase bands (Fig. 5). Discussion The co-elution of AAA activity with cholinesterases in procainamide-Sepharose affinity chromatography of human amniotic fluid supports our earlier findings that cholinesterases exhibit AAA activity [ 1,2]. The eluted AAA was susceptible to inhibition by serotonin, a property exhibited by AAA associated with both AChE and BuChE [ l-31. Moreover tyramine activation of the AAA associated with BuChE in human amniotic fluid indicated the similarity of amniotic fluid BuChE to human serum BuChE [ 11. The immunoprecipitability of amniotic fluid BuChE by a polyclonal antibody raised against human serum BuChE suggested their immunological identity. Further, the electrophoretic mobilities of amniotic fluid AChE and BuChE were similar to those of electric eel AChE and human serum BuChE respectively. These results suggested that the AChE and BuChE of amniotic fluid appears similar to the classical AChE of different sources and human serum BuChE exhibiting amine sensitive AAA activity. There are limitations in estimating AChE and BuChE activities accurately in a mixture of the two using the selective inhibitors, BW284C51 and ethopropazine. At concentrations of lo-100 pmol/l normally used, these inhibitors not only inhibit 100% their respective cholinesterases but also show a minor inhibition of cholinesterase considered to be unaffected by them [ 181. This problem becomes more obvious when the levels of the enzyme activities measured are low such as in human cerebrospinal fluid [ 131 and amniotic fluid. Earlier reports have indicated an elevation of AChE and in some cases elevation of both AChE and BuChE in amniotic fluid of
165
pregnancies with NTD fetuses f 4,5 1. The reported values of AChE in amniotic fluid varied widely because different methods of assay were followed by different investigators [4,5,19-211. We have reported the values in terms of AChE to BuChE ratio [ 221 and the AChE activity was taken as the difference in cholinesterase activity measured in the presence and absence of ethopropazine as inhibitor using acetylthiocholine as substrate and BuChE activity was taken as the difference in choiinesterase activity measured in the presence and absence of BW284C51 as inhibitor using butyrylthiocholine as substrate. The results given in Table I suggest that although the values of AChEiBuChE ratio may be at a higher level in some NTD samples when compared to normals, contamination by blood in the samples can markedly affect the values. It has been reported earlier that contamination of amniotic fluid with maternal or fetal blood invalidates the analysis of AChE activity in amniotic fluid [ 23,241. It appears therefore that a combination of AChE/BuChE ratio determination, gel electrophoresis followed by enzyme staining and determination of enzyme activity after selective immunoprecipitation of BuChE will be helpful in the assessment of AChE and BuChE levels in amniotic fluid. Acknowledgement This work was supported Technology (India).
by a grant from the Department
of Science and
References 1 2
3 4
5 6
1 8 9 IO 11 12
George ST, Balasubramanian AS. The aryl acylamidases and their relationship to cholinesterases in human serum, erythrocyte and liver. Em J Biochem 198 1;121: 177-186. George ST, Balasubramanian AS. The identity of serotonin sensitive aryl acylamidase with acetylcholinesterase from human erythrocytes, sheep basal ganglia and electric eel. Em J Biochem 1980:111:511-524. Tsujita T, Okuda H. Carboxylesterases in rat and human sera and their relationship to serum aryl acylamidases and cholinesterases. Eur J Biochem 1983; 133:2 15-220. Smith AD, Wald NJ, Cuckle HS, Stirrat GM, Bobrow M. Lagercrantz H. Amniotic fluid acetylcholinesterase as a possible diagnostic test for neural tube defects in early pregnancy. Lancet 1979;1.685-688. Chubb IW, Pilowsky PM. Springell HJ, Pollard AC. Acetyl~holinesterase in human amniotic Buid: An index of fetal neural development. Lancet 1979;1:688-690. Rakonczay 2, Brimijoin S. Biochemistry and pathophysiology of the molecular forms of cholinesterases. in: Harris JR, ed Subcellular Biochemistry, Vol. 12. New York: Plenum Pub1 Corp. 1988;335-378. Brock DJH, Hayward C. Gel electrohoresis of amniotic fluid acetylcholinesterase as an aid to the prenatal diagnosis of fetal defects. Clin Chim Acta 1980;108: 13% 141. Buamah PK. Evans L, Ward A.M. Amniotic fluid acetylcholinesterase isoenzyme patterns in the diagnosis of neural tube defects. Clin Chim Acta 1980;102: 147-151. Seller MJ, Cole KJ. Polyacrylamide gel electrophoresis of amniotic fluid cholinesterases: A good prenatal test for neural tube defects. Br J Obstet Gynaecol 1980;87: 1103-I 108. Bonham JR, Atack JR. A neural tube defect specific form of acetylcholinesterase in amniotic Iluid. Clin Chim Acta 1983;135:233-237, Rakonczay Z, Szuts. P, Szabo J. Significance of a~etylcholinesterase molecular forms in prenatal diagnosis of neural tube defects. Acta Physiol Hung 1985;66:280-28 I. Ellman GL, Courtney KD, Andre Jr V. Featherstone RM. A new and rapid calorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 1961;7:88-95.
13
14
15 16 17 18 19 20 21
22
23 24
Rao RV, Gnanamuthu C, Balasubramanian AS Human cerebrospinal fluid acetyicholinesterase and butyrylcholinesterase. Evidence for identity between the serum and cerebrospinal fluid butyrylcholinesterase. Clin Chim Acta 1989;183:135- 146. George ST, Varghese M, John L, Balasubramanian AS. Aryl acrylamidase activity in human erythrocyte, plasma and blood in pesticide (organophosphates and carbamates) poisoning. Clin Chim Acta 1985;145:1-8. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin-phenol reagent. J Biol Chem 1951;193:265-275. Harris H, Hopkinson DA. Robson EB. Two dimensional electrophoresis of pseudocholinesterase components in normal human serum. Nature (London) l962;194:1294-1298. ~ajumdar R, George ST. Balasubramanjan AS. Serotonjn sensitive aryl acylamidase activity of platelet ac~tylcholinesterase. Biochem Pharmacol 1982;31:2319-2325. Brimijoin S, Hammond P. Butyrylcholinesterase in human brain and acetylcholinesterase in human plasma. Trace enzymes measured by two site immunoassay. J Neurochem 1988;Sl: 1227- 1231. Rasmussen Loft AG. Determination of amniotic fluid acetylcholinesterase activity in the antenatal diagnosis of foetal malformations. The first ten years. J Clin Chem Clin Biochem 1990;8:893-91 I. Dale G, Bonham JR, Lowdon P. Roberts DF. Amniotic fluid acetylcholinesterase and neural-tube defects. Lancet 1979;1:880-881. Hullin DA, Elder GH, Laurence KM, Roberts A, Newcombe RG. Amniotic fluid cholinesterdse measurement as a rapid method for the exclusion of fetal neural-tube defects, Lancet 1981:11;325-327. Rasmussen Loft AG. Mortensen V, Hangaard J, Norgaard-Pedersen B. Ratio of immunochemically determined amniotic fluid acetylcholinesterase to butyrylcholinestera~ in the differential diagnosis of fetal abno~alities. Br J Obstet Gynaecol 1991:98:52-56. Davies P. Gosden C. Brock DJH. Acetylcholinesterase, blood stained amniotic fluids and prenatal diagnosis of neural-tube defects. Lancet 1979;I:1303. Chubb IW, Pilowsky PM, Hodgson AJ. Pollard AC. Acetylcholinesterase in blood-contaminated amniotic fluid. Lancet 1979;1:1148-I 149.
157 reserved.
0009-8981/92/$05.00
CCA 05219
Cholinesterases exhibiting aryl acylamidase activity in human amniotic fluid L.D. Jayanthi”, N. Balasubramanianb
and A.S. Balasubramanian”
“Department of Neurological Sciences and hDepartment of Obstetrics and Gynecology, Christian Medical College and Hospital, Vellore 632 004 (India) (Received
16 July 1991; revision
Key words: Cholinesterases;
received
30 September
Aryl acylamidases;
Human
1991; accepted amniotic
4 October
fluid; Neural
1991)
tube defect
Summary
Acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8) in human amniotic fluid were estimated in the presence of selective inhibitors. Amniotic fluid cholinesterases (mixture of acetylcholinesterase and butyrylcholinesterase) purified by procainamide-Sepharose affinity chromatography exhibited aryl acylamidase activity which was sensitive to serotonin inhibition (a property of aryl acylamidases associated with both acetyl- and butyrylcholinesterases) and tyramine activation (shown exclusively by aryl acylamidase associated with butyrylcholinesterase). Tyramine activation was unaffected in the presence of the selective acetylcholinesterase inhibitor BW284C51 whereas it was abolished in the presence of the selective butyrylcholinesterase inhibitor ethopropazine, suggesting the presence of both types of aryl acylamidases in amniotic fluid, one associated with acetylcholinesterase and the other associated with butyrylcholinesterase. Butyrylcholinesterase and the associated aryl acylamidase activity in the affinity purified enzyme was selectively immunoprecipitated by a polyclonal antibody raised against human serum butyrylcholinesterase. Estimation of the activity ratio of acetylcholinesterase to butyrylcholinesterase in a few samples of amniotic fluid showed that this could vary depending on the butyrylcholinesterase arising from contaminating blood in the samples. Gel electrophoresis under non-denaturing conditions and enzyme staining showed that butyrylcholinesterase band was detectable on the gel in all the samples whereas acetylcholinesterase band was below detectable levels in normal samples but visible in samples from pregnancies of neural tube defect fetuses. It is suggested that the use of selective cholinesterase inhibitors along with gel electrophoresis and immunoprecipitation studies may be useful in the assessment of cholinesterase activities in human amniotic fluid. Correspondence to; A.S. Balasubramanian, Hospital, Vellore-632 004, Tamil Nadu,
Dept. of Neurological India.
Sciences, Christian
Medical College &
158
Cholinesterases of vertebrates fall into two categories, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). These two enzymes differ in their substrate specificities and inhibition by selective inhibitors. AChE preferentially hydrolyses acetylchohne or acetyl-~-methylch~line while BuChE hydrolyses butyrylcho~ine preferentially, Bis-(4-ally1 dimethyl ammonium pheny~~-pentane-3one dibromide (BW284C51) is a selective inhibitor of AChE and tetra isopropyl pyrophosphoramide (iso-OMPA) and ethopropazine are selective inhibitors of BuChE. They are widely used in the selective estimation of AChE and BuChE in a mixture of the two enzymes. Excitable tissues and human erythrocytes contain predominantly AChE whereas the human serum contains almost exclusively BuChE. Work from this laboratory [ 1,2] and from other [ 31 has demonstrated that an aryl acylamidase (AAA) (EC 3.5. I, 13) activity sensitive to amines is exhibited by AChE of brain and other sources and BuChE of human serum. Serotonin inhibited the AAA activity of both AChE and BuChE whereas tryamine significantly activated the AAA activity of serum BuChE exclusively [ 13. The human amniotic fluid contains both AChE and BuChE [4,5] and the ratio of AChE to BuChE ranges from 0.02 to 0.26 [ 5 ] . Several reports have indicated that an elevated level of AChE in amniotic fluid is an indicator of spina bifida (menin~omyelocele) and anencephaly, the two major types of neural tube closure defects (NTD) [ 4,s 1. This tinding is rational because most AChE is probably derived from the nervous system f 51. The nervous tissue in NTD is either in direct contact with the amniotic fluid or just covered with a thin membrane f6]. Electrophoretic analysis of amniotic fluid from pregnancies of NTD fetuses showed a faster migrating band corresponding to AChE in addition to the slow moving band corresponding to BuChE [ ‘7-91. Sedimentation studies showed that normal amniotic fluids contain 4s (G,) form of AChE [ lo] and also 10s (G4) form [ 11I. Neural tube defects have been hnked to a disproportional increase in the 10s (GJ form [HI]. Since there is no report showing the presence of AAA activity in human amniotic fluid and its association with amniotic fiuid cholinesterases, we have made an attempt to isolate the amniotic fluid cholinesterases by procainamide-Sepharose affinity chromatography and show the association of AAA activity with the cholinesterases. We have also assessed the AChE and BuChE activities in human amniotic fluid samples from normal and NTD pregnancies in the presence of selective inhibitors and by gel electrophoresis. Materials and Methods Procainamide-Sepharose was prepared by linking procainamide to CNBractivated Sepharose-4B through a IO-carbon spacer arm [ I]. ~rj~cat~on of human serum BuChE by procainamide-Sepharose affinity chromtography and preparation of antibody against purified human serum BuChE were as described earlier [ 11. Electric eel AChE was from Sigma Chemical Co., USA, All other chemicals were prepared or procured as given earlier [ I]. Samples of amniotic fluid were collected during amniocentesis done for routine
159
diagnostic purposes. They were from pregnancies of normal fetuses (not affected by NTD) and also from pregnancies of anencephaly and meningomyelocele (spina bitida) fetuses. The samples collected were centrifuged at 10,000 x g for 5 min to remove cells and other sedimentable matter and dialysed against 20 nmol/l potassium phosphate buffer, pH 7.4 for 4 h at 4°C and used. Enzyme
assays
AChE and BuChE in amniotic fluid were assayed by the method of Ellman et al. [ 121 as described earlier [ 131 using acetylthiocholine and butyrylthiocholine respectively as substrates. Ethopropazine, the selective inhibitor of BuChE was included when AChE was estimated and BW284C5 1, the selective inhibitor of AChE was included when BuChE was estimated. The reaction mixture consisted of 50 ~1 of potassium phosphate buffer (1 mol/l) (pH 7.4), 20 ~1 of the thiocholine substrate (75 mmol/l), 50 ~1 of the selective inhibitor (1 mmol/l), 100 ~1 of 5,5’-dithiobis-(2 nitrobenzoic acid) (10 mmol/l) and 100 ~1 of amniotic fluid in a final assay volume of 500 ~1. The released thiocholine was measured spectrophotometrically at 412 nm. One unit of cholinesterase activity is defined as one pmol of thiocholine formed per minute at 37°C under the given assay conditions. AAA activity was assayed as described earlier [ 141. The reaction mixture contained 50 ~1 of potassium phosphate buffer (1 mol/l) pH 7.4, 250 ~1 of o-nitroacetanilide (10 mmol/l) and 100 ~1 of enzyme in a final assay volume of 500 ~1. Appropriate cholinesterase inhibitors were included in the reaction mixture when AAA activity exhibited by either AChE or BuChE alone was determined. Protein was estimated according to Lowry et al. [ 151. Chromatography
of amniotic fluid on procainamide-Sepharose
affinity column
Amniotic fluid (25 ml) dialysed against 20 mmol/l potassium phosphate buffer, (pH 7.4) was passed through a procainamide-Sepharose column (2 x 1 cm) preequilibrated with the same buffer at 4°C. The column was washed with 100 ml of the same buffer and the enzyme was eluted with procainamide (50 mmol/l) in the buffer. Fractions of 2 ml were collected and assayed for both cholinesterase and AAA activities. Active fractions were dialysed against the buffer to remove procainamide and used for other studies. Immunoprecipitation
To 100 ~1 of procainamide-Sepharose affinity purified enzyme, different concentrations (12.5-100 pg) of the polyclonal antibody raised against human serum BuChE were added. After keeping at 4°C for 12 h with mild shaking the tubes were centrifuged at 10,000 x g for 1 h and the AChE and BuChE activities both in the supernatant and in the immunoprecipitate (resuspended in 20 mmol/l potassium phosphate buffer, pH 7.4) were assayed in the presence of appropriate selective inhibitors. Controls were run without the antibody.
160
P~iyacrylam~d~ gei electr~p~oresis and staining for cho~~~estera~ea~t~~l~iy
Polyacrylamide gel electrophoresis under non-denaturing conditions was done on 7% slab gels in glycine-Tris buffer (50 mmol/l) (pH 8.3). The gels were then preincubated at room temperature for 15 min in potassium phosphate buffer (20 mmol/l) (pH 7.4). Staining for cholinesterase was done by shaking the gel at room temperature for 45 min in a mixture of a-naphthylacetate (20 mg in 2 ml acteone) and Fast Red TR salt (50 mg) in 100 ml potassium phosphate buffer (20 mmolil) (pH 7.4) [ 161. Red bands of cholinesterase activity that appeared on the gel were fixed in 7% acetic acid. For demonstrating AChE or BuChE activity on the gel. the selective inhibitors (10 pmolil) were included during the preincubation and staining of the gel. Results Procainamide-Sepharose
affinity chromatography
Procainamide-Sepharose affinity column could completely bind both AChE and BuChE from the amniotic fluid and both the activities could be eluted by procainamide from the column. In a typical column chromatography using 25 ml of amniotic fluid there was 54% and 55% recovery of AChE and BuChE respectively with 183 and 194 fold increase in enzyme units per mg protein respectively. In confirmation of earlier findings [ 1,2,13,17], the fractions eluted from pro~ainamide-Sepharose column also showed AAA activity co-eluting with cholinesterases. Figure 1 gives the elution profiles of AChE, BuChE and AAA activities from the affinity column. This affinity chromatography method provided a simple and useful method for getting amniotic fluid cholinesterases and AAA in a partially purified concentrated form.
O-06
2 4
6
8 1012 14 1618 2670727476 Fraction number
Fig 1. Procainamide-Sepharose affinity chromatography of human amniotic fluid: Elution profile of AChE ( 0 ), BuChE (0), AAA (A) and protein (A) from the procainamide-Sepharose column. Experimental details and assay procedures are given under Materials and Methods. Arrow indicates the fraction where elution with procainamide was started.
161
A
B uo
100 80 60 40 20 0 Serotonin
(mM )
I1 2 5 Tyromine( mM )
Fig. 2. Inhibition by serotonin and activation by tyramine of AAA activity in a mixture of AChE and BuChE in amniotic fluid: AAA activity was measured in the presence of various concentrations of serotonin (A) and tryamine (B). Enzyme assay procedures are given under Materials and Methods.
Inhibition by serotonin and activation by tyramine of AAA activity associated with cholinesterases of amniotic fluid AAA activity associated with AChE and BuChE is known to be inhibited by serotonin whereas AAA activity associated with BuChE alone is known to be activated by tyramine [l-3]. We examined the effect of serotonin and tyramine on the AAA activity of the affinity purified enzyme taking advantage of the known fact [ 1,2] that selective cholinesterase inhibitors also inhibit selectively the AAA activity associated with each type of cholinesterase. Serotonin at OS-5 mmol/l concentration showed 38-97% inhibition of AAA activity and tyramine at similar concentrations showed 179-307% activation of AAA activity in the absence of selective cholinesterase inhibitors (Fig. 2A and 2B). In the presence of BW284C51, the selective inhibitor of AChE, tyramine at l-2 mmol/l stimulated the AAA activity 340-520% whereas in the presence of ethopropazine, the selective inhibitor of BuChE, tyramine had no effect on AAA activity (Fig. 3). These results suggested that the AAA activity of am-
Tyraminel
mM)
Fig, 3. Effect of various concentrations of tyramine on the AAA activity associated with BuChE and AChE of amniotic fluid: The AAA activity associated with BuChE ( 0) was measured in the presence of BW284C51 (10 pmol/l) and the AAA associated with AChE (0) was measured in the presence of ethopropazine (10 pmolll). Enzyme assay procedures are given under Materials and Methods.
niotic fluid cholinesterases is susceptible to serotonin inhibition and that the AAA associated with BuChE alone is activated by tyramine. Immunoprecipitation of human amniotic fluid BuChE by polyclonal antibody raised against human serum BuChE Polyclonal antibody raised against purified human serum BuChE is known to cross-react specifically with BuChE, but not with AChE [ 11. We examined the immunoprecipitability of human amniotic fluid BuChE by the antibody raised against human serum BuChE. As shown in Fig. 4 BuChE from procainamide-Sepharose affmity purified enzyme could be completely immunoprecipitated by the antibody suggesting immunological similarity of human serum BuChE to human amniotic fluid BuChE. The precipitated BuChE activity was recovered in the immunoprecipitate (Fig. 4). Figure 4 also shows that AChE of amniotic fluid was unaffected by the antibody used and was present almost 100% in the supernatant after immunoprecipitation. The AAA activity present in the immunoprecipitate after 100% precipitation of BuChE was activated by tyramine whereas the AAA in the supernatant was not affected by tyramine confirming that tyramine activation was an exclusive property of the AAA associated with BuChE (results not shown). Estimation of AChE and BuChE activities in human amniotic fluid samples Although AChE and BuChE from different sources preferentially hydrolyse acetylthiocholine and butyrylthiocholine respectively, they also exhibit a low
50 Antibody
100
Fig. 4. i~unoprecipitation of amniotic fluid BuChE by various concentrations of antibody raised against purified human serum BuChE: Activity of BuChE (a) and AChE (0) in the supernatant is shown by solid lines and the activity of BuChE in the i~unoprecipitate is shown by broken line. Details of i~unoprecipitation and determination of BuChE and AChE activity are given under Materials and Methods.
163 TABLE I Ratios of AChE to BuChE activities in human amniotic fluid samples Type of sample
Gestational age (weeks)
AChE/BuChEratio
Normal Normal Normal Normal Normal Spina bifida Spina bifida Spina bifida Anencephaly *Anencephaly *Anencephaly
38 36 30 36 40 40 16 16 36 30 38
0.30 0.30 0.30 0.25 0.30 0.40 0.45 0.49 0.33 0.14 0.11
*Indicates the amniotic fluid samples contaminated with blood.
Fig, 5. Polyacrylamide gel electrophoresis and enzyme staining of cholinesterases in amniotic fluid samples: Lane I, purified BuChE from human serum; Lane 2, standard electric eel AChE (Sigma); Lane 3, eel AChE in presence of BW284CS 1 (10 pm&l); Lane 4. cholinesterases from amniotic fluid of normal pregnancy; Lane 5, amniotic fluid cholinesterases of NTD pregnancy; Lane 6, amniotic fluid cholinesterases of NTD pregnancy in presence of BW284C51 (10 pmolil). The selective AChE inhibitor BW284C51 was added during preincubation and staining of the gel in lanes 3 and 6. The arrow A points to the BuChE band and arrow B points to the AChE band.
164
hydrolytic activity towards the less preferred substrates. This necessitates the use of selective inhibitors during assay. We estimated AChE and BuChE activities in eleven different amniotic fluid samples and the AChE to BuChE ratios are given in Table I. For five normal amniotic fluid samples (not affected by NTD) the AChE to BuChE ratio ranged from 0.25 to 0.3. For four NTD samples it ranged from 0.33 to 0.49, higher than the value of 0.3. However, two other NTD samples gave low values of 0.11 and 0.14. These two samples with low AChE to BuChE ratios were highly contaminated with blood and therefore the contaminating serum BuChE might have reduced the AChE to BuChE ratios. Polyacrylamide gel electrophoresis Detectability of cholinesterase bands on the gel after electrophoresis followed by enzyme staining was possible only when the amniotic fluid samples used were concentrated by procainamide-Sepharose affinity chromatography. About 25 ml of amniotic fluid available from one normal and one NTD case were concentrated and enzyme activity equivalent to 1.5 units of BuChE from each was subjected to gel electrophoresis and staining (Fig. 5). The NTD sample gave a prominent band corresponding to human serum BuChE and a band of lesser intensity corresponding to electric eel AChE. In the normal sample only the BuChE band was detectable. Use of the selective inhibitor BW284C51 during staining confirmed the identity of the cholinesterase bands (Fig. 5). Discussion The co-elution of AAA activity with cholinesterases in procainamide-Sepharose affinity chromatography of human amniotic fluid supports our earlier findings that cholinesterases exhibit AAA activity [ 1,2]. The eluted AAA was susceptible to inhibition by serotonin, a property exhibited by AAA associated with both AChE and BuChE [ l-31. Moreover tyramine activation of the AAA associated with BuChE in human amniotic fluid indicated the similarity of amniotic fluid BuChE to human serum BuChE [ 11. The immunoprecipitability of amniotic fluid BuChE by a polyclonal antibody raised against human serum BuChE suggested their immunological identity. Further, the electrophoretic mobilities of amniotic fluid AChE and BuChE were similar to those of electric eel AChE and human serum BuChE respectively. These results suggested that the AChE and BuChE of amniotic fluid appears similar to the classical AChE of different sources and human serum BuChE exhibiting amine sensitive AAA activity. There are limitations in estimating AChE and BuChE activities accurately in a mixture of the two using the selective inhibitors, BW284C51 and ethopropazine. At concentrations of lo-100 pmol/l normally used, these inhibitors not only inhibit 100% their respective cholinesterases but also show a minor inhibition of cholinesterase considered to be unaffected by them [ 181. This problem becomes more obvious when the levels of the enzyme activities measured are low such as in human cerebrospinal fluid [ 131 and amniotic fluid. Earlier reports have indicated an elevation of AChE and in some cases elevation of both AChE and BuChE in amniotic fluid of
165
pregnancies with NTD fetuses f 4,5 1. The reported values of AChE in amniotic fluid varied widely because different methods of assay were followed by different investigators [4,5,19-211. We have reported the values in terms of AChE to BuChE ratio [ 221 and the AChE activity was taken as the difference in cholinesterase activity measured in the presence and absence of ethopropazine as inhibitor using acetylthiocholine as substrate and BuChE activity was taken as the difference in choiinesterase activity measured in the presence and absence of BW284C51 as inhibitor using butyrylthiocholine as substrate. The results given in Table I suggest that although the values of AChEiBuChE ratio may be at a higher level in some NTD samples when compared to normals, contamination by blood in the samples can markedly affect the values. It has been reported earlier that contamination of amniotic fluid with maternal or fetal blood invalidates the analysis of AChE activity in amniotic fluid [ 23,241. It appears therefore that a combination of AChE/BuChE ratio determination, gel electrophoresis followed by enzyme staining and determination of enzyme activity after selective immunoprecipitation of BuChE will be helpful in the assessment of AChE and BuChE levels in amniotic fluid. Acknowledgement This work was supported Technology (India).
by a grant from the Department
of Science and
References 1 2
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5 6
1 8 9 IO 11 12
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