TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
35,147-l%
(1976)
Characterization of Maternal and Fetal Ovine Plasma Choline&erase1 J. U. BELL' AND G. R. VAN PETTEN Division of Pharmacology and Therapeutics, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N IN4, Canada Received June 19,1975; accepted August 27,1975
Characterization of Maternal and Fetal Ovine PlasmaCholinesterase. PETTEN, G. R. (1976).Toxicol. Appl. Pharmacol. 35, 147-155.Cholinesteraseof maternal and fetal ovine plasma,obtained at 112-l 15 days of gestation,has beencharacterizedand comparedby using various techniquesincluding statistical evaluation of kinetic parameters when using acetyl-, propionyl-, and butyrylthiocholine, gel !?ltration, polyacrylamide-gelelectrophoresis,and sensitivity to various inhibitors. Hydrolysis of acetylthiocholine,the optimum substrate,wasfound to betwo to three timesfaster in fetal plasmathan in maternalplasma,althoughthe Michaelis constantsdid not differ. In both maternal and fetal plasma,gel filtration yieldedamajor andaminor peakof cholinesteraseactivity,whereas electrophoresisyielded six bandsof activity, including three major bands. Thesefindings suggestedquantitative rather than qualitative differences betweenthe maternalandfetal activity. Despitethe quantitative differences, there wasno differencein the maternal and fetal cholinesterase sensitivity to inhibition by eserine,diisopropylffuorophosphate,and dichlorvos. BELL, J. U., AND VAN
Although a physiological role for plasma cholinesterase (pseudocholinesterase; acylcholine acyl hydrolase; EC 3.1.1.S.) has not been defined (Usdin, 1970),it hasbeen reported to play a role in the metabolism of a number of drugs and environmental chemicals (xenobiotics) containing ester linkages (LaMotta and Woronick, 1971; Zsigmond and Downs, 1971; Ecobichon and Stephens, 1973). As the mammalian placenta offers little resistance to the maternal-fetal passageof most xenobiotics .(Villee, 1965; Mirkin, 1973), the ability of the fetus to metabolize such compounds might be an important determining factor in fetal toxicity. In the human, plasma cholinesterasedeterminations have been limited either to cord blood samplesobtained at normal delivery or to blood from prematurely delivered infants (Zsigmond and Downs, 1971; Ecobichon and Stephens, 1973). For technical reasons,enzyme activity in the fetal plasma of laboratory animals can only be determined following sacrifice of the mother and often involves the pooling of samples(Chow and Ecobichon, 1975). The chronically cannulated ovine fetus (Willes et al., 1970) permits blood sampling from the fetus in utero at various gestational ages,providing a good model in which to study and compare maternal and fetal plasma enzymes, such as cholinesterase,which may be involved in the metabolism of maternally derived xenobiotics. This report 1 Supported by Grant No. MA4160 from the Medical Research Councilof Canada. 2 Davies Memorial Fellow. Copyright 0 1976 by Academic Press, Inc. 147 All rights of reproduction in any form reserved. Printed
in Great
Britain
148
BELL
AND
VA&
PETTEN
describes the characterization of maternal and fetal ovine plasma cholinesterase early in the third trimester of gestation (112-115 days) with regard to substrate specificity, kinetic constants, gel filtration, electrophoresis, and sensitivity to various inhibitors. METHODS Under general anesthesia induced and maintained by halothane, silicone rubber cannulas (0.762 mm id.) were placed in the femoral artery and saphenous vein of the ovine fetus by use of the procedure described by Willes et al. (1970), modified so that the cannulas were exteriorized into a cloth bag sewn on the ewe’s flank. During the above procedure a nonoccluding cannula was also placed in the maternal carotid, which permitted simultaneous arterial sampling from the ewe and from her fetus in utero. At least 48 hr were allowed for recovery from the surgery; then maternal and fetal arterial blood samples were collected in heparinized syringes and centrifuged, and the plasma was either used at once or stored in glass vials at -20°C. Plasma cholinesterase activity was measured spectrophotometrically by the method of Ellman et al. (1961) with acetylthiocholine, propionylthiocholine, and butyrylthiocholine as substrates. Incubations were run at 37°C and pH 7.4, and linear, initial reaction velocities were related to a prepared standard curve, with activity presented as micromoles of substrate hydrolyzed per minute per milliliter of plasma. The range of substrate concentration used was 5 x 10m5 to 2 x low3 M. Substrate concentrations and corresponding reaction velocities were fitted to Eq. [l],
rf = (~Inax[WKrl+ PI),
PI
by a least-squares method (Cleland, 1967), assuming homogeneous variance at all velocities. All calculations were performed on a CDC 6400 digital computer using a Fortran program, which provided values for K,,,, V,,,,,, K/V, l/v, and the SE of their estimates. Statistical significance was assessedat the 5 % level of probability using Student’s t test for two means of independent samples. To measure the sensitivity of plasma cholinesterase to inhibition by eserine, diisopropylfluorophosphate (DFP) (Sigma Chemical Co.), and dichlorvos (DDVP) (Shell Oil Company of Canada), 200-~1 samples of plasma were incubated with different concentrations of inhibitor at 37°C for 5 min. The residual cholinesterase activity was then measured by using acetylthiocholine at a concentration of 1 x 10e3 M, which was approximately IO-fold higher than the determined K,. Sephadex G-200 (Pharmacia of Canada Ltd.) was prepared by suspension in an excess of 0.025 M phosphate buffer, pH 7.0, containing 0.02 ‘A sodium azide. Columns (2.5 x 100 cm) were packed and equilibrated at room temperature with the same buffer until a constant flow rate of 12-15 ml/hr was obtained. Plasma samples (3.0-5.0 ml) were applied to the column through a flow adaptor, and ascending buffer flow was used. The protein content of the eluant was continuously recorded at 280 nm with a Uvicord II monitor (LKB Produkter). Polyacrylamide-gel electrophoresis was run, essentially by the procedure described by Smith (1968), with a 7 % running gel and a continuous Tris-glycine buffer system (pH 9.5). A current of 1 mA/tube was increased to a constant 2.5 mA/tube following entry of the sample into the gel. Following the run (approximately 90 min), the gels
MATERNAL-FETAL
OVINE
CHOLINESTERASE
149
were scanned at 280 nm on a recording spectrophotometer to locate the protein peaks. The gels were stained for cholinesterase activity by using the histochemical method of Kamovsky and Roots (1964) and utilizing the optimum substrate as determined from the kinetic studies, acetylthiocholine. Bands of staining were located by scanning the gels on a recording spectrophotometer at 411 nm.
RESULTS Due to reported differences in substrate specificity for cholinesterase in the plasma of various species (Usdin, 1970), the hydrolytic activity of maternal and fetal plasma was measured using acetylthiocholine, propionylthiocholine, and butyrylthiocholine. With this series of substrates, it was found that the Km(Table 1) increased in the order acetyl < propionyl < butyryl for both maternal and fetal plasma and that, for each particular substrate, there was no significant maternal-fetal difference. When maximum
"1
FIG. 1. The effect of increasing substrate concentration from 5 x 10e5to 4 x lo-’ M on the hydrolysis of acetylthiocholine by maternal and fetal ovine plasma at 37°C and pH 7.4.
activities (V,,,) were determined (Table l), it was found that for both maternal and fetal plasma, activity increased in the order butyryl < propionyl < acetyl. However, the hydrolysis of both acetyl- and propionylthiocholine by fetal plasma was two to three times higher (p < 0.05) than with maternal plasma. As a result of the kinetic data obtained with the three thiocholine analogs, acetylthiocholine was chosen as the optimum substrate for the remainder of the study. When the concentration of acetylthiocholine was raised above 2 x low3 M another difference between the maternal and fetal enzyme became apparant (Fig. 1). With the concentration raised 20-fold (from 2 x 10e3 to 4 x lo-’ M), fetal enzyme activity decreased from 0.500 to 0.245 ,nmol/min/ml of plasma, whereas maternal enzyme activity only decreased from 0.282 to 0.218 ~mol/min/ml plasma. Since plasma cholinesterase is reported to exist in a number of polymeric forms with molecular weights ranging as high as 300,000 (LaMotta and Woronick, 1971), an attempt was made to separate these forms by gel titration on Sephadex G-200. Figure 2 shows the elution patterns for protein (percent transmission at 280 nm) and cholinesterase for a maternal and fetal plasma sample. Although there were obvious differences
0.83 * 0.05 (12) 1.52 + 0.09 (6) 8.63 f 3.72 (7)
Maternal 0.89 + 0.05 (10) 1.54 _+ 0.05 (6) 5.48 + 1.01(6)
Fetal
0.148 f 0.023 (12) 0.106 + 0.011 (6) 0.035 f 0.007 (7)
Maternal
0.364 + 0.066 (lO)b 0.267 f 0.038 (6>b 0.041 f 0.010 (6)
Fetal
Maximum velocity (pm01hydrolyzed/min/ml plasma)
a All maternal and fetal plasma samples were obtained between 112 and 115 days of gestation. Values represent the mean + SD of the means of the samples (n). b Fetal valuesignificantlydifferentfrom maternalvalue( p < 0.05).
Acetylthiocholine Propionylthiocholine Butyrylthiocholine
Substrate
Michaelis constant (x low4M)
TABLE 1 HYDROLYSIS OF THI~CHOLINE ESTERS BYMATERNALANDFETALOVINEPLASMA”
z z :: !2
5 s
E F
MATERNAL-FETAL
OVINE
CHOLINESTERASE
151
in the protein elution patterns, the fetal cholinesterase pattern differed only quantitatively from the maternal pattern, with activity in the major fetal peak approximately three times that of the major maternal peak. Polyacrylamide-gel electrophoresis was used in an attempt to separate the components of the cholinesterase activity on the basis of potential charge differences. Figure 3 shows the protein pattern and a schematic diagram of the cholinesterase pattern for maternal and fetal plasma samples. Again, obvious differences were apparent in the protein pattern between maternal and fetal plasma; however, the location of the cholinesterase activity, as indicated by the appearance of brown bands following
TUBE NUMBER
FIG. 2. Gel titration of 4.0-ml aliquots of maternal (A) and fetal (B) ovine plasma on a column (2.5 x 90 cm) of Sephadex G-200. Ascending buffer flow was used and LO-ml fractions were collected. The protein concentration (a) was measured at 280 ML The cholinesterase activity (0) was determined with acetylthiocholine as substrate.
hydrolysis of acetylthiocholine, was similar for both with the possible exception of band 3. Using staining intensity as an indicator of acetylthiocholine hydrolysis, there were three major bands of cholinesterase activity (bands 1, 3, and 4) in both maternal and fetal plasma, with the greatest activity occurring in fetal band 3 and maternal band 4. Minor activity occurred at maternal bands 5 and 6 and fetal bands 2 and 6, with trace activity detectable at maternal band 2 and fetal band 5. Since plasma cholinesterase should be sensitive to inhibition by both eserine and organophosphate esters (Augustinsson, 1961), inhibition curves were established for eserine, diisopropylfluorophosphate (DFP), and dichlorvos (DDVP). Examination of the inhibition curves (Fig. 4) disclosed that with maternal plasma cholinesterase, the 150 values for eserine, DFP, and DDVP were 3.0 x lo-*, 1.1 x 10m6, and 2.0 x 10m6M, respectively, whereas with fetal plasma cholinesterase they were 3.0 x lo-*, 1.4 x IO+,
152
BELL
-
2 E. $i :x ::: .:. I
::: ::::::g:: ff g;;;i ;:;: :.:.:.:.:.: ($1::g$$$ :.:;:.a>::::: 23
AND
iz$ ,:.:.:. ;Q ,:,:,:, ::y:: 4
VAN
PETTEN
.~,~.~:.~,~.~.~.~.~.~.~..~.‘.~.’ :::;. ~.~.~.~.~.~.~.~.~.~,~.~. .~.~.~,~.~.~.~.~.~.\~ .:..A’. ,~.~.~.~,~.~.~.~.~,~:.~,~ .:. ,~.~.~.~,~.~,~,~.~.~.~.‘.~.~.‘.‘.’. 6
+
5
3. Polyacrylamide-gel electrophoretic patterns for maternal (A) and fetal (B) plasma, showing the protein scan profile (top) measured at 280 mn and a schematic diagram of the choline&erase pattern (bottom) measured at 411 mn. The shading is designed to indicate major, minor, and trace staining density of the bands. Sample application was at the cathode with migration toward the anode. FIG.
and 1.7 x 10m6M, respectively. It was of interest to note that the hydrolysis of acetylthiocholine by both maternal and fetal plasma was approximately 100 times more sensitive to inhibition by eserine than by either of the organophosphates. DISCUSSION
Since the computer program used in this study provides SE of the fitted kinetic constants, it was possible to analyze whether Eq. [l] satisfactorily described our data. With acetylthiocholine as the substrate, it was noted that the SE of the fitted constants K,,, and V,,,,, averaged, respectively, 6.8 and 1.8 % for both maternal and fetal plasma. Since these SE were less than 10 % of the actual values, the use of Eq. [l] to describe the experimental data was justified according to the criteria established by Cleland (1967). In addition, the kinetic data obtained with the thiocholine analogs (Table 1) confirmed the findings of Augustinsson (1959) that acetyl- and propionylcholine are hydrolyzed by adult ovine plasma at a greater rate than is butyrylcholine. Although there was no
MATERNAL--FETAL
OVINE
CHOLINESTERASE
153
loo-
B
80-
p P g s
40-
P-
O-
FIG. 4. The inhibition of maternal (A) and fetal (B) ovine plasma choline&erase by eserine, DFP, and DDVP. Plasma samples (200 ~1) were incubated with the inhibitor at the indicated concentrations for
5 min at 37”C, and residual activity was assayed with acetylthiocholine at a final concentration of 1 X 1o-3 M.
signicant maternal-fetal difference in the K,,,values for the individual substrates, confirming a similar situation reported for the human (Ecobichon and Stephens, 1973), the observation that fetal plasma hydrolyzed acetylthiocholine two to three times faster than maternal plasma prompted further characterization and comparison of the maternal and fetal cholinesterase. There have been a number of reports in the literature
showing the protein concentration of fetal plasma to be lower than that found in the adult (Ecobichon and Stephens, 1973; Chow and Ecobichon, 1975). Although this was confirmed in the present study (Fig. 2) it was apparent from both Figs. 2 and and 3 that the bulk of the protein present in the plasma was not related to cholinesterase and as such should not be considered in the expression of enzyme activity. In an attempt to separate the cholinesterase activity into components possessing different molecular weights, plasma samples were fractionated by gel filtration on Sephadex G-200 (Fig. 2). In both maternal and fetal plasma, two peaks of cholinesterase activity (one major and one minor) were observed having similar elution volumes,
suggesting that the cholinesterase of both maternal and fetal plasma could be of a similar molecular weight. If isoenzymes of cholinesterase are responsible for the hydrolytic activity it should be possible to separate them on the basis of their electrical charge using electrophoresis. Although a separation was achieved (Fig. 3), the maternal and fetal patterns appeared to differ only in relative activity but not in band position. Thus, the gel filtration and electrophoretic data both suggest that maternal and fetal cholinesterase are qualitatively similar but quantitatively different. Further evidence for this was provided by the inhibition studies with eserine and the organophosphates DFP
154
BELL AND VANPETTEN
and DDVP (Fig. 4), where inhibition constants for each inhibitor were similar in both maternal and fetal plasma. Although Ecobichon and Comeau (1973) found DFP to be a more potent inhibitor of plasma cholinesterasethan DDVP in a variety of mammals, the 150 values they obtained for the goat were quite similar to our findings, where DFP and DDVP were nearly equipotent. The observation of inhibition of activity with high concentrations of substrate (Fig. 1) may indicate that the major fetal isoenzyme was sensitive to inhibition by substrate (or product), whereasthe major maternal isoenzyme was not. Although no kinetic studies are available on cholinesterase of fetal ovine plasma, there are a number of reports of cholinesterasemeasurementsin the plasma of other mammals during the perinatal period. In the human, Ecobichon and Stephens (1973) found activity to increasefrom 28 weeksto levels at term (37-40 weeks) which were 50 to 60 % of adult levels. Their values in the term neonate agreed with those of Lehmann et al. (1957) and Zsigmond and Downs (1971) who reported newborn activities which were 50 and 77 ‘A, respectively, of the adult values. Plasmacholinesteraseactivity in the fetal guinea pig was reported to increase from low values at day 56 of gestation to values at birth which were approximately 50 ‘A of the adult levels (Chow and Ecobichon, 1975). Our results, which show the activity of 112-115-day fetal ovine plasma to be two to three times higher than maternal plasma, markedly contrast with the findings reported in both the human and the guinea pig. The data on the guinea pig reported by Chow and Ecobichon (1975)raisean interesting point regarding interpretation of human data. They observedthat there was a significant fall in perinatal plasma cholinesteraseactivity coincident with birth. Since the human developmental study by Ecobichon and Stephens(1973) involved prematurely delivered neonatesat various gestational ages(from 26 weeks), the possibility must be raised that the low values were causedin someway by the birth process.This illustrates the value of the chronically cannulated fetus, where sampling is possiblein utero without sacrifice of either the mother or the fetus, as a model for following the development of components of the blood during the last trimester of gestation. In addition, it is known that the guinea pig is generally very well developed at birth, so that late in gestation, e.g. day 57 to term at day 68, the guinea pig fetus may be more similar to neonates of other speciessuch as the sheepand the human. From these studies, it becomesevident that much further investigation is required at different gestational ages in order that the role of speciesvariation in fetal toxicity studiesinvolving estertype xenobiotics can be more accurately assessed. ACKNOWLEDGMENTS The authors expressappreciationto Shell Oil CanadaLtd. for the gift of analytical grade diclilorvos. REFERENCES AUGUSTINSSON, K.-B. (1959).Electrophoresisstudieson blood plasmaesterases. I. Mammalian plasmata.Acta Chem.Stand. 13, 571-592. AUGUSTINSSON, K.-B. (1961). Multiple forms of esterases in vertebrate blood plasma.Ann.
N. Y. Acad. Sci. 94, 844-860. CHOW, A. Y. K. AND ECOBICHON, D. J. (1975). Perinatal developmentof cavian plasma,
hepatic and renal esterases. Biol. Neonate25,23-30.
MATERNAL-FETALOVINE
CHOLINESTERASE
155
CLELAND, W. W. (1967). The statistical analysisof enzyme kinetic data. Adtan. Enzymol. 29, l-32. ECOBICHON. D. J. AND COMEAU, A. M. (1973).Pseudocholinesterases of mammalianplasma: Physicochemicalproperties and organophosphateinhibition in eleven species.Toxicol. Appl. Pharmacol. 24,92-100. ECOBICHON, D. J. AND STEPHENS, D. S. (1973).Perinatal developmentof humanblood esterases.Clin. Pharmacol. Ther. 14,41-47. ELLMAN,G.L.,COURTNEY, K.D., ANDRES, V. ANDFEATHERSTONE, R.M.(1961). Anew and rapid calorimetric determination of acetylcholinesterase activity. B&hem. Pharmacol. 7, 88-95. KARNOVSKY, M. J. AND RENTS, L. (1964).A “direct coloring” thiocholine methodfor cholinesterases. J. Histochem. Cytochem. 12, 219-221. LAMOTTA, R. V. AND WORONICK, C. L. (1971). Molecular heterogeneity of human serum cholinesterase.Clin. Chem. 17, 135-144. LEHMANN, H., COOK, J. AND RYAN, E. (1957).Pseudocholinesterase in early infancy. Proc. Roy. Sot. Med. 50,147-150. MIRKIN, B. L. (1973).Drug distribution in pregnancy. In Fetal Pharmacology (L. 0. Boreus,
Ed.), pp. l-28. Raven Press,New York. SMITH,I. (1968).Techniquesof discelectrophoresis.In Chromatographic and Electrophoretic Techniques (I. Smith, Ed.), Vol. 2, pp. 365-388.IntersciencePublishers,New York. USDIN, E. (1970). Cholinesterases. In Anticholinesterase Agents: International Encyclopedia of Pharmacology and Therapeutics (A. G. Karczmar, Ed.), Section 13, Vol. 1, pp. 60-122. PergamonPress,Oxford. VILLEE, C. A. (1965).Placentaltransfer of drugs.Ann. N. Y. Acad. Sci. 123,237~240. WILLES, R. F., VAN PETTEN, G. R. AND TRUELOVE, J. F. (1970).Chronic exteriorization of cannulasand ECG electrodesfrom the ovine fetus.J. Appl. Physiol. 28,248-250. ZSIGMOND, E. K. AND DOWNS, J. R. (1971).Plasmacholinesterase activity in newbornsand infants. Can. Anaesth. Sot. J. 18.278-285.
AND
APPLIED
PHARMACOLOGY
35,147-l%
(1976)
Characterization of Maternal and Fetal Ovine Plasma Choline&erase1 J. U. BELL' AND G. R. VAN PETTEN Division of Pharmacology and Therapeutics, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N IN4, Canada Received June 19,1975; accepted August 27,1975
Characterization of Maternal and Fetal Ovine PlasmaCholinesterase. PETTEN, G. R. (1976).Toxicol. Appl. Pharmacol. 35, 147-155.Cholinesteraseof maternal and fetal ovine plasma,obtained at 112-l 15 days of gestation,has beencharacterizedand comparedby using various techniquesincluding statistical evaluation of kinetic parameters when using acetyl-, propionyl-, and butyrylthiocholine, gel !?ltration, polyacrylamide-gelelectrophoresis,and sensitivity to various inhibitors. Hydrolysis of acetylthiocholine,the optimum substrate,wasfound to betwo to three timesfaster in fetal plasmathan in maternalplasma,althoughthe Michaelis constantsdid not differ. In both maternal and fetal plasma,gel filtration yieldedamajor andaminor peakof cholinesteraseactivity,whereas electrophoresisyielded six bandsof activity, including three major bands. Thesefindings suggestedquantitative rather than qualitative differences betweenthe maternalandfetal activity. Despitethe quantitative differences, there wasno differencein the maternal and fetal cholinesterase sensitivity to inhibition by eserine,diisopropylffuorophosphate,and dichlorvos. BELL, J. U., AND VAN
Although a physiological role for plasma cholinesterase (pseudocholinesterase; acylcholine acyl hydrolase; EC 3.1.1.S.) has not been defined (Usdin, 1970),it hasbeen reported to play a role in the metabolism of a number of drugs and environmental chemicals (xenobiotics) containing ester linkages (LaMotta and Woronick, 1971; Zsigmond and Downs, 1971; Ecobichon and Stephens, 1973). As the mammalian placenta offers little resistance to the maternal-fetal passageof most xenobiotics .(Villee, 1965; Mirkin, 1973), the ability of the fetus to metabolize such compounds might be an important determining factor in fetal toxicity. In the human, plasma cholinesterasedeterminations have been limited either to cord blood samplesobtained at normal delivery or to blood from prematurely delivered infants (Zsigmond and Downs, 1971; Ecobichon and Stephens, 1973). For technical reasons,enzyme activity in the fetal plasma of laboratory animals can only be determined following sacrifice of the mother and often involves the pooling of samples(Chow and Ecobichon, 1975). The chronically cannulated ovine fetus (Willes et al., 1970) permits blood sampling from the fetus in utero at various gestational ages,providing a good model in which to study and compare maternal and fetal plasma enzymes, such as cholinesterase,which may be involved in the metabolism of maternally derived xenobiotics. This report 1 Supported by Grant No. MA4160 from the Medical Research Councilof Canada. 2 Davies Memorial Fellow. Copyright 0 1976 by Academic Press, Inc. 147 All rights of reproduction in any form reserved. Printed
in Great
Britain
148
BELL
AND
VA&
PETTEN
describes the characterization of maternal and fetal ovine plasma cholinesterase early in the third trimester of gestation (112-115 days) with regard to substrate specificity, kinetic constants, gel filtration, electrophoresis, and sensitivity to various inhibitors. METHODS Under general anesthesia induced and maintained by halothane, silicone rubber cannulas (0.762 mm id.) were placed in the femoral artery and saphenous vein of the ovine fetus by use of the procedure described by Willes et al. (1970), modified so that the cannulas were exteriorized into a cloth bag sewn on the ewe’s flank. During the above procedure a nonoccluding cannula was also placed in the maternal carotid, which permitted simultaneous arterial sampling from the ewe and from her fetus in utero. At least 48 hr were allowed for recovery from the surgery; then maternal and fetal arterial blood samples were collected in heparinized syringes and centrifuged, and the plasma was either used at once or stored in glass vials at -20°C. Plasma cholinesterase activity was measured spectrophotometrically by the method of Ellman et al. (1961) with acetylthiocholine, propionylthiocholine, and butyrylthiocholine as substrates. Incubations were run at 37°C and pH 7.4, and linear, initial reaction velocities were related to a prepared standard curve, with activity presented as micromoles of substrate hydrolyzed per minute per milliliter of plasma. The range of substrate concentration used was 5 x 10m5 to 2 x low3 M. Substrate concentrations and corresponding reaction velocities were fitted to Eq. [l],
rf = (~Inax[WKrl+ PI),
PI
by a least-squares method (Cleland, 1967), assuming homogeneous variance at all velocities. All calculations were performed on a CDC 6400 digital computer using a Fortran program, which provided values for K,,,, V,,,,,, K/V, l/v, and the SE of their estimates. Statistical significance was assessedat the 5 % level of probability using Student’s t test for two means of independent samples. To measure the sensitivity of plasma cholinesterase to inhibition by eserine, diisopropylfluorophosphate (DFP) (Sigma Chemical Co.), and dichlorvos (DDVP) (Shell Oil Company of Canada), 200-~1 samples of plasma were incubated with different concentrations of inhibitor at 37°C for 5 min. The residual cholinesterase activity was then measured by using acetylthiocholine at a concentration of 1 x 10e3 M, which was approximately IO-fold higher than the determined K,. Sephadex G-200 (Pharmacia of Canada Ltd.) was prepared by suspension in an excess of 0.025 M phosphate buffer, pH 7.0, containing 0.02 ‘A sodium azide. Columns (2.5 x 100 cm) were packed and equilibrated at room temperature with the same buffer until a constant flow rate of 12-15 ml/hr was obtained. Plasma samples (3.0-5.0 ml) were applied to the column through a flow adaptor, and ascending buffer flow was used. The protein content of the eluant was continuously recorded at 280 nm with a Uvicord II monitor (LKB Produkter). Polyacrylamide-gel electrophoresis was run, essentially by the procedure described by Smith (1968), with a 7 % running gel and a continuous Tris-glycine buffer system (pH 9.5). A current of 1 mA/tube was increased to a constant 2.5 mA/tube following entry of the sample into the gel. Following the run (approximately 90 min), the gels
MATERNAL-FETAL
OVINE
CHOLINESTERASE
149
were scanned at 280 nm on a recording spectrophotometer to locate the protein peaks. The gels were stained for cholinesterase activity by using the histochemical method of Kamovsky and Roots (1964) and utilizing the optimum substrate as determined from the kinetic studies, acetylthiocholine. Bands of staining were located by scanning the gels on a recording spectrophotometer at 411 nm.
RESULTS Due to reported differences in substrate specificity for cholinesterase in the plasma of various species (Usdin, 1970), the hydrolytic activity of maternal and fetal plasma was measured using acetylthiocholine, propionylthiocholine, and butyrylthiocholine. With this series of substrates, it was found that the Km(Table 1) increased in the order acetyl < propionyl < butyryl for both maternal and fetal plasma and that, for each particular substrate, there was no significant maternal-fetal difference. When maximum
"1
FIG. 1. The effect of increasing substrate concentration from 5 x 10e5to 4 x lo-’ M on the hydrolysis of acetylthiocholine by maternal and fetal ovine plasma at 37°C and pH 7.4.
activities (V,,,) were determined (Table l), it was found that for both maternal and fetal plasma, activity increased in the order butyryl < propionyl < acetyl. However, the hydrolysis of both acetyl- and propionylthiocholine by fetal plasma was two to three times higher (p < 0.05) than with maternal plasma. As a result of the kinetic data obtained with the three thiocholine analogs, acetylthiocholine was chosen as the optimum substrate for the remainder of the study. When the concentration of acetylthiocholine was raised above 2 x low3 M another difference between the maternal and fetal enzyme became apparant (Fig. 1). With the concentration raised 20-fold (from 2 x 10e3 to 4 x lo-’ M), fetal enzyme activity decreased from 0.500 to 0.245 ,nmol/min/ml of plasma, whereas maternal enzyme activity only decreased from 0.282 to 0.218 ~mol/min/ml plasma. Since plasma cholinesterase is reported to exist in a number of polymeric forms with molecular weights ranging as high as 300,000 (LaMotta and Woronick, 1971), an attempt was made to separate these forms by gel titration on Sephadex G-200. Figure 2 shows the elution patterns for protein (percent transmission at 280 nm) and cholinesterase for a maternal and fetal plasma sample. Although there were obvious differences
0.83 * 0.05 (12) 1.52 + 0.09 (6) 8.63 f 3.72 (7)
Maternal 0.89 + 0.05 (10) 1.54 _+ 0.05 (6) 5.48 + 1.01(6)
Fetal
0.148 f 0.023 (12) 0.106 + 0.011 (6) 0.035 f 0.007 (7)
Maternal
0.364 + 0.066 (lO)b 0.267 f 0.038 (6>b 0.041 f 0.010 (6)
Fetal
Maximum velocity (pm01hydrolyzed/min/ml plasma)
a All maternal and fetal plasma samples were obtained between 112 and 115 days of gestation. Values represent the mean + SD of the means of the samples (n). b Fetal valuesignificantlydifferentfrom maternalvalue( p < 0.05).
Acetylthiocholine Propionylthiocholine Butyrylthiocholine
Substrate
Michaelis constant (x low4M)
TABLE 1 HYDROLYSIS OF THI~CHOLINE ESTERS BYMATERNALANDFETALOVINEPLASMA”
z z :: !2
5 s
E F
MATERNAL-FETAL
OVINE
CHOLINESTERASE
151
in the protein elution patterns, the fetal cholinesterase pattern differed only quantitatively from the maternal pattern, with activity in the major fetal peak approximately three times that of the major maternal peak. Polyacrylamide-gel electrophoresis was used in an attempt to separate the components of the cholinesterase activity on the basis of potential charge differences. Figure 3 shows the protein pattern and a schematic diagram of the cholinesterase pattern for maternal and fetal plasma samples. Again, obvious differences were apparent in the protein pattern between maternal and fetal plasma; however, the location of the cholinesterase activity, as indicated by the appearance of brown bands following
TUBE NUMBER
FIG. 2. Gel titration of 4.0-ml aliquots of maternal (A) and fetal (B) ovine plasma on a column (2.5 x 90 cm) of Sephadex G-200. Ascending buffer flow was used and LO-ml fractions were collected. The protein concentration (a) was measured at 280 ML The cholinesterase activity (0) was determined with acetylthiocholine as substrate.
hydrolysis of acetylthiocholine, was similar for both with the possible exception of band 3. Using staining intensity as an indicator of acetylthiocholine hydrolysis, there were three major bands of cholinesterase activity (bands 1, 3, and 4) in both maternal and fetal plasma, with the greatest activity occurring in fetal band 3 and maternal band 4. Minor activity occurred at maternal bands 5 and 6 and fetal bands 2 and 6, with trace activity detectable at maternal band 2 and fetal band 5. Since plasma cholinesterase should be sensitive to inhibition by both eserine and organophosphate esters (Augustinsson, 1961), inhibition curves were established for eserine, diisopropylfluorophosphate (DFP), and dichlorvos (DDVP). Examination of the inhibition curves (Fig. 4) disclosed that with maternal plasma cholinesterase, the 150 values for eserine, DFP, and DDVP were 3.0 x lo-*, 1.1 x 10m6, and 2.0 x 10m6M, respectively, whereas with fetal plasma cholinesterase they were 3.0 x lo-*, 1.4 x IO+,
152
BELL
-
2 E. $i :x ::: .:. I
::: ::::::g:: ff g;;;i ;:;: :.:.:.:.:.: ($1::g$$$ :.:;:.a>::::: 23
AND
iz$ ,:.:.:. ;Q ,:,:,:, ::y:: 4
VAN
PETTEN
.~,~.~:.~,~.~.~.~.~.~.~..~.‘.~.’ :::;. ~.~.~.~.~.~.~.~.~.~,~.~. .~.~.~,~.~.~.~.~.~.\~ .:..A’. ,~.~.~.~,~.~.~.~.~,~:.~,~ .:. ,~.~.~.~,~.~,~,~.~.~.~.‘.~.~.‘.‘.’. 6
+
5
3. Polyacrylamide-gel electrophoretic patterns for maternal (A) and fetal (B) plasma, showing the protein scan profile (top) measured at 280 mn and a schematic diagram of the choline&erase pattern (bottom) measured at 411 mn. The shading is designed to indicate major, minor, and trace staining density of the bands. Sample application was at the cathode with migration toward the anode. FIG.
and 1.7 x 10m6M, respectively. It was of interest to note that the hydrolysis of acetylthiocholine by both maternal and fetal plasma was approximately 100 times more sensitive to inhibition by eserine than by either of the organophosphates. DISCUSSION
Since the computer program used in this study provides SE of the fitted kinetic constants, it was possible to analyze whether Eq. [l] satisfactorily described our data. With acetylthiocholine as the substrate, it was noted that the SE of the fitted constants K,,, and V,,,,, averaged, respectively, 6.8 and 1.8 % for both maternal and fetal plasma. Since these SE were less than 10 % of the actual values, the use of Eq. [l] to describe the experimental data was justified according to the criteria established by Cleland (1967). In addition, the kinetic data obtained with the thiocholine analogs (Table 1) confirmed the findings of Augustinsson (1959) that acetyl- and propionylcholine are hydrolyzed by adult ovine plasma at a greater rate than is butyrylcholine. Although there was no
MATERNAL--FETAL
OVINE
CHOLINESTERASE
153
loo-
B
80-
p P g s
40-
P-
O-
FIG. 4. The inhibition of maternal (A) and fetal (B) ovine plasma choline&erase by eserine, DFP, and DDVP. Plasma samples (200 ~1) were incubated with the inhibitor at the indicated concentrations for
5 min at 37”C, and residual activity was assayed with acetylthiocholine at a final concentration of 1 X 1o-3 M.
signicant maternal-fetal difference in the K,,,values for the individual substrates, confirming a similar situation reported for the human (Ecobichon and Stephens, 1973), the observation that fetal plasma hydrolyzed acetylthiocholine two to three times faster than maternal plasma prompted further characterization and comparison of the maternal and fetal cholinesterase. There have been a number of reports in the literature
showing the protein concentration of fetal plasma to be lower than that found in the adult (Ecobichon and Stephens, 1973; Chow and Ecobichon, 1975). Although this was confirmed in the present study (Fig. 2) it was apparent from both Figs. 2 and and 3 that the bulk of the protein present in the plasma was not related to cholinesterase and as such should not be considered in the expression of enzyme activity. In an attempt to separate the cholinesterase activity into components possessing different molecular weights, plasma samples were fractionated by gel filtration on Sephadex G-200 (Fig. 2). In both maternal and fetal plasma, two peaks of cholinesterase activity (one major and one minor) were observed having similar elution volumes,
suggesting that the cholinesterase of both maternal and fetal plasma could be of a similar molecular weight. If isoenzymes of cholinesterase are responsible for the hydrolytic activity it should be possible to separate them on the basis of their electrical charge using electrophoresis. Although a separation was achieved (Fig. 3), the maternal and fetal patterns appeared to differ only in relative activity but not in band position. Thus, the gel filtration and electrophoretic data both suggest that maternal and fetal cholinesterase are qualitatively similar but quantitatively different. Further evidence for this was provided by the inhibition studies with eserine and the organophosphates DFP
154
BELL AND VANPETTEN
and DDVP (Fig. 4), where inhibition constants for each inhibitor were similar in both maternal and fetal plasma. Although Ecobichon and Comeau (1973) found DFP to be a more potent inhibitor of plasma cholinesterasethan DDVP in a variety of mammals, the 150 values they obtained for the goat were quite similar to our findings, where DFP and DDVP were nearly equipotent. The observation of inhibition of activity with high concentrations of substrate (Fig. 1) may indicate that the major fetal isoenzyme was sensitive to inhibition by substrate (or product), whereasthe major maternal isoenzyme was not. Although no kinetic studies are available on cholinesterase of fetal ovine plasma, there are a number of reports of cholinesterasemeasurementsin the plasma of other mammals during the perinatal period. In the human, Ecobichon and Stephens (1973) found activity to increasefrom 28 weeksto levels at term (37-40 weeks) which were 50 to 60 % of adult levels. Their values in the term neonate agreed with those of Lehmann et al. (1957) and Zsigmond and Downs (1971) who reported newborn activities which were 50 and 77 ‘A, respectively, of the adult values. Plasmacholinesteraseactivity in the fetal guinea pig was reported to increase from low values at day 56 of gestation to values at birth which were approximately 50 ‘A of the adult levels (Chow and Ecobichon, 1975). Our results, which show the activity of 112-115-day fetal ovine plasma to be two to three times higher than maternal plasma, markedly contrast with the findings reported in both the human and the guinea pig. The data on the guinea pig reported by Chow and Ecobichon (1975)raisean interesting point regarding interpretation of human data. They observedthat there was a significant fall in perinatal plasma cholinesteraseactivity coincident with birth. Since the human developmental study by Ecobichon and Stephens(1973) involved prematurely delivered neonatesat various gestational ages(from 26 weeks), the possibility must be raised that the low values were causedin someway by the birth process.This illustrates the value of the chronically cannulated fetus, where sampling is possiblein utero without sacrifice of either the mother or the fetus, as a model for following the development of components of the blood during the last trimester of gestation. In addition, it is known that the guinea pig is generally very well developed at birth, so that late in gestation, e.g. day 57 to term at day 68, the guinea pig fetus may be more similar to neonates of other speciessuch as the sheepand the human. From these studies, it becomesevident that much further investigation is required at different gestational ages in order that the role of speciesvariation in fetal toxicity studiesinvolving estertype xenobiotics can be more accurately assessed. ACKNOWLEDGMENTS The authors expressappreciationto Shell Oil CanadaLtd. for the gift of analytical grade diclilorvos. REFERENCES AUGUSTINSSON, K.-B. (1959).Electrophoresisstudieson blood plasmaesterases. I. Mammalian plasmata.Acta Chem.Stand. 13, 571-592. AUGUSTINSSON, K.-B. (1961). Multiple forms of esterases in vertebrate blood plasma.Ann.
N. Y. Acad. Sci. 94, 844-860. CHOW, A. Y. K. AND ECOBICHON, D. J. (1975). Perinatal developmentof cavian plasma,
hepatic and renal esterases. Biol. Neonate25,23-30.
MATERNAL-FETALOVINE
CHOLINESTERASE
155
CLELAND, W. W. (1967). The statistical analysisof enzyme kinetic data. Adtan. Enzymol. 29, l-32. ECOBICHON. D. J. AND COMEAU, A. M. (1973).Pseudocholinesterases of mammalianplasma: Physicochemicalproperties and organophosphateinhibition in eleven species.Toxicol. Appl. Pharmacol. 24,92-100. ECOBICHON, D. J. AND STEPHENS, D. S. (1973).Perinatal developmentof humanblood esterases.Clin. Pharmacol. Ther. 14,41-47. ELLMAN,G.L.,COURTNEY, K.D., ANDRES, V. ANDFEATHERSTONE, R.M.(1961). Anew and rapid calorimetric determination of acetylcholinesterase activity. B&hem. Pharmacol. 7, 88-95. KARNOVSKY, M. J. AND RENTS, L. (1964).A “direct coloring” thiocholine methodfor cholinesterases. J. Histochem. Cytochem. 12, 219-221. LAMOTTA, R. V. AND WORONICK, C. L. (1971). Molecular heterogeneity of human serum cholinesterase.Clin. Chem. 17, 135-144. LEHMANN, H., COOK, J. AND RYAN, E. (1957).Pseudocholinesterase in early infancy. Proc. Roy. Sot. Med. 50,147-150. MIRKIN, B. L. (1973).Drug distribution in pregnancy. In Fetal Pharmacology (L. 0. Boreus,
Ed.), pp. l-28. Raven Press,New York. SMITH,I. (1968).Techniquesof discelectrophoresis.In Chromatographic and Electrophoretic Techniques (I. Smith, Ed.), Vol. 2, pp. 365-388.IntersciencePublishers,New York. USDIN, E. (1970). Cholinesterases. In Anticholinesterase Agents: International Encyclopedia of Pharmacology and Therapeutics (A. G. Karczmar, Ed.), Section 13, Vol. 1, pp. 60-122. PergamonPress,Oxford. VILLEE, C. A. (1965).Placentaltransfer of drugs.Ann. N. Y. Acad. Sci. 123,237~240. WILLES, R. F., VAN PETTEN, G. R. AND TRUELOVE, J. F. (1970).Chronic exteriorization of cannulasand ECG electrodesfrom the ovine fetus.J. Appl. Physiol. 28,248-250. ZSIGMOND, E. K. AND DOWNS, J. R. (1971).Plasmacholinesterase activity in newbornsand infants. Can. Anaesth. Sot. J. 18.278-285.
