Trace Analysis of Pesticides Using Cholinesterase uman Serum, Rat Liver, Electric Eel, ean Leaf eetle, and White Fringe Beetle M. H. Sadar, S. S. Kuan, and G oG . Guilbault Department of Chemistry, Louisiana State University in New Orleans, New Orleans, La. 70122 A fluorometric method is developed for the determination of organophosphorus and chlorinated pesticides. Advantage is taken of the fact that cholinesterases extracted from five different sources respond differently to various pesticides. Cholinesterase from rat liver and human serum is strongly inhibited by organophosphorus compounds but the chlorinated pesticides have no effect. The enzyme from electric eel is sensitive to chlorinated pesticides only. Cholinesterases from both the bean leaf beetle and white fringe beetle are very strongly inhibited by Paraoxon and DDVP, but Parathion and methyl parathion are very weak inhibitors. There is no interference either from the chlorinated or carbamate pesticides. The substrate N-methyl indoxyl acetate is cleaved by all five different cholinesterases to the highly fluorescent N-methyl indoxyl (Aex = 430 nm, A,, = 510 nm). The various pesticides, when introduced into the reaction system, inhibit the hydrolysis of the substrate catalyzed by cholinesterase causing a decrease in the initial slopes of the fluorescence-time curve, AF/f?lin. This decrease i s a direct measure of the concentration of the inhibitor present.
APPLICATION OF ENZYME catalyzed reactions has found several uses in developing sensitive methods for the trace analysis of toxic compounds and metal ions (1-8). The enzyme catalyzed systems do offer a very high degree of sensitivity, yet lack specificity. By taking advantage of the fact that cholinesterases extracted from different sources respond differently to various inhibitors as reported by O'Brien (9), Dauterman et al., (IO) and many others, an attempt is made to obtain a certain degree of specificity in pesticide analysis. Hence, enzymic methods for the trace analysis of many metals, toxic materials, and pesticides would be extremely useful if a certain degree of specificity can be built into the enzyme system. Guilbault et al. (11) have extracted cholinesterase from two different sources and investigated the inhibition of these enzymes by various pesticides. Cholinesterase from bee body was extremely sensitive to Sevin, DDVP, Parathion, and methyl parathion, but showed little or no inhibition by the six (1) G. E. Mendoza, P. J. Wales, H. A. McLead, and W. P. McKinley, Analyst, (London),93,34 (1968). (2) P. A. Giang and S. A. Hall, ANAL.CHEM., 23, 1830 (1964). (3) D. Mealor and A. Townshend, Talanta, 15, 1371 (1968). (4) G. Voss and H. Geisebuhler, Meded Rijksf. Landbourvwetensch., Gent, 32, 877 (1967). ( 5 ) G. 6. Guilbault and D. N. Kramer, ANAL.CHEM., 34, 1437 (1962). (6) G. G. Guilbault and M. H. Sadar, ibid., 41, 366 (1969). (7) G . G. Guilbault, M. H. Sadar, and M. Zimmer, Anal. Chim. Acta, 44, 361 (1969). (8) A. Townshend and A. Vaughan, Talanta, 16,929 (1969). (9) R. D. O'Brien, Biochern. J., 79,229 (1961). (10) W. C. Dauterman, A. Talens, and K. Van Asperen, J. Ztzst. Physiol., 8, 1-14 (1962). (11) 6. G. Guilbault, S . S. Kuan, and M. H. Sadar, J . Agr. Food Clzem., 18,692 (1970). 1770
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chlorinated pesticides (methoxychlor, Aldrin, DDT, Dieldrin, Lindane, and Heptachlor). On the other hand, cholinesterase extracted from boll weevil showed inhibition by DDVP and very little or no inhibition by the other nine pesticides mentioned above. In the present study, the inhibitory effects of fourteen different pesticides including organophosphorus compounds, carbamates, and chlorinated hydrocarbons on cholinesterase from human serum, rat liver, bean leaf beetle, white fringe beetle, and electric eel is reported. The five different sources of enzymes include human, rodent, insect, and aquatic species, and hence, the comparative inhibition of the same cholinesterase extracted from these different species is of special interest from a biological point of view. Furthermore, some specificity in inhibition was found, permitting the assay of some pesticides in the presence of others. EXPERIMENTAL
Enzymes. Cholinesterase (electric eel) Type 111, Sigma Chemical Co., activity 1.087 p M units per mg. (One unit will hydrolyze 1 pmole of acetylcholine per minute at pH 8.0 and at 37 "e.) A stock solution of 0.092 mg/ml of protein was prepared in triply distilled H20. Cholinesterase from bean leaf beetle and white fringe beetle was prepared, purified, and assayed according to a method reported previously (12). The bean leaf beetles were collected at a farm of the Entomology Department of Louisiana State University in Baton Rouge; the white fringe beetles were collected at the U. S. Department of Agriculture farm in Gulfport, Miss. These insects were reared in the laboratory by the usual procedure. Cholinesterase from rat was extracted and purified as previously described (13). The liver was obtained from Sigma Chemical Co. as the acetone powder. Cholinesterase from human serum was prepared and purified as follows: The human serum obtained from a blood bank was vigorously mixed with butanol according to the method of Morton (14) to release the ChE from undesirable lipoprotein into true solution. The mixture was kept at 4 "C overnight. The upper lipid layer was removed by suction and the lower portion containing ChE was fractionated with ammonium sulfate at 0-20, 20-40, 40-60, 60-80, and 80-100% saturation. The fractions of 40-60 and 60-80 saturation, which had the highest specific activity, were combined, then dialyzed against O.O5M, pH 7.0, phosphate buffer. The dialysate was further chromatographed on a Sephadex G-100 column. The fractions of high enzyme activity were combined and then were freeze dried. The overall purification was about 600-fold. Methods of protein estimation, electrophoretic analysis, and characterization of cholinesterase were the same as previously described. (12) G. G. Guilbault, M. H. Sadar, S. S. Kuan, and D. Casey, Anal. Chim. Acta, October 1970. (13) G. 6.Guilbault, M. H. Sadar, S . S. Kuan, and D. Casey, Anal. @him.Acta, 51, 83 (1970). (14) R. K. Morton, Nature, 166,1092(1950).
ANALYTICAL CHEMISTRY, VOL. 42, NO. 14, DECEMBER 1970
Substrate. N-Methyl indoxyl acetate was obtained from ISOLAB, Inc., Drawer 4350, Akron, Ohio. A stock solution (10-2M) of the substrate was prepared in methyl cellosolve. Buffers. Phosphate buffer, O.lM, pH-7.0 was used. Apparatus. All fluorescent measurements were made with an Aminco Bowman spectrofluorometer (SPF) grating instrument. A constant temperature of 25 "C was maintained with a thermoelectric cooler. The rate of production of Nmethyl indoxyl was measured by setting A, = 430 nm and ,A, = 510nm. Pesticides. All pesticide solutions were prepared in dioxane. The pesticides were of +99% purity and were obtained from Polyscience Corporation, Evanston, Ill., except for DDVP which was obtained from the Food and Drug Administration, Perrine, Fla., and Paraoxon which was provided by American Cyanamid Company. Procedure. Three ml of 0.1M phosphate buffer, pH 7.0, 0.1 ml of 10-ZMN-methyl indoxyl acetate, 0.1 ml of dioxane (solvent for pesticides) and 0.1 ml of enzyme solution are placed in a fluorometric cell and the increase in fluorescence with time was recorded. The initial slope of this curve is the blank or uninhibited rate, AF*/At. In the next step, the cell contains all the above except dioxane which is replaced by 0.1 ml of the known pesticide solution. The initial slope of this curve obtained in the presence of a known concentration of a particular pesticide is measured. The new value of AF/At should be less than AF*/At if the enzyme is inhibited by the pesticide. The procedure is repeated with different concentrations of pesticide. The percentage inhibition is calculated as follows: AF*/At - AF/At = AF*/At AF*/At = initial slope with no pesticide (only 0.1 ml dioxane)
present in the fluorometric cell. AF/At = initial slope with a certain concentration of pesticide
present. The percentage inhibition is plotted us. the concentration of the inhibitor present and from the plot, the Z50value (concentration of the pesticide to inhibit 5 0 x of the enzymic activity) is found. RESULTS AND DISCUSSION
Purification of Enzymes. The purity of the isolated enzymes, human serum, rat liver, bean leaf beetle, and white fringe beetle and their characterization was done as described previously (12,13). The rate of hydrolysis of N-methyl indoxyl acetate by these partially purified preparations is shown in Table I. The data in Table I indicate that the specific activity of the preparations from animal tissues were much higher than those from insects. To classify the cholinesterase from different sources, these preparations were characterized by their reactivity toward different choline esters (Table 11). Cholinesterases from both insects hydrolyzed acetylcholine chloride much faster than butyryl choline chloride and had very little reactivity against benzoyl choline. This indicates that they are acetylcholinesterase. In contract to insect cholinesterase, serum cholinesterase appeared to be pseudo cholinesterase since this enzyme rapidly hydrolyzed butyrylcholine and benzoyl choline. The rat liver cholinesterase was more difficult to judge since this preparation hydrolyzed both acetyl and butyryl choline at approximately the same rate and also hydrolyzed benzoyl choline ester. Therefore, this preparation may contain two types of cholinesterases. The disk electrophoretic pattern of bean leaf beetle and white fringe beetle cholinesterases shows only one protein and one enzyme band indicating their high purity. The cholines-
Table I. Purity of the Partially Purified Cholinesterase Preparations White Bean Rat fringe leaf Enzyme source liver beetle beetle Protein (pg/ml) 236 146 163 Enzyme activity (units) 42.5 11.2 3.56 Specific activity 180 77 22
Human serum 172 27.3 158
Table 11. Reactivity of Partially Purified Preparations against Different Substrates Enzyme source (,uM/min/ml) Bean White Rat fringe leaf Human Substrate liver beetle beetle serum Acetylcholine chloride 12.2 11.5 4.6 12.1 Propionyl choline chloride 12.0 13.0 7.2 19.5 Butyrylcholine chloride 11.0 3.5 1.4 23.0 Benzoylcholine chloride 2.5 0.2 ... 13.0
terases from human serum and rat liver, however, have one enzyme band and two protein bands but the major protein band corresponds to enzyme band. Stability of Enzyme Preparation. The stability of the enzyme solutions was determined by noting the rate of hydrolysis of N-methyl indoxyl acetate every 24 hours. Cholinesterase (electric eel) from Sigma is stable and its solution, kept at 4 "C, can be utilized for many weeks without appreciable loss in activity. The enzymes from human serum and rat liver are more stable than those from bean leaf beetle and white fringe beetle, but less stable than electric eel. The nonfluorescent ester, N-methyl indoxyl acetate, is cleaved by each of the enzymes to the highly fluorescent Nmethyl indoxyl under conditions reported previously (IS).
I
Nonfluorescent
I
CB, Highly fluorescent
The rate of production of the highly fluorescent N-methyl indoxyl is measured. Inhibition by Various Pesticides. Since the effectiveness of various pesticides differs from one living creature to another, it was decided to investigate the inhibition of a certain enzyme-cholinesterase-extracted from different sources. In the present study, each of the five different sources has its own biological importance. The study includes human, as well as another mammal, rat, two insects, and an aquatic species, electric eel. We wanted to look at the comparative inhibition of these enzymes and establish if a more specific enzyme system could be obtained (15) G. G. Guilbault, M. H. Sadar, R. Glazer, and C. Skou, Anal. Lett. 1(6), 365 (1968).
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Figure 1. Plot of inhibition of rat liver cholinesterase by various pesticides. Three-minute preincubation 0 0
Paraoxon DDVP
Parathion
INHIBITOR ( M x that could be useful for the analysis of some of these pesticides. Using a 3-minute preincubation time, some of the results obtained for the inhibition of rat liver, human serum, electric eel, and white fringe beetle cholinesterases by various pesticides, are indicated in Figures 1-4. The inhibition curves for bean leaf beetle are similar to those for white fringe beetle (Figure 4). The comparative inhibition by Paraoxon of the two insect cholinesterases (Le., bean leaf beetle and white fringe beetle) is shown in Figure 5. All five are inhibited by DDVP but rat liver cholinesterase is most strongly inhibited. Paraoxon is the most potent inhibitor of all. Electric eel cholinesterase is not inhibited by any of the most widely used
organophosphorus compounds ; DDVP inhibits at high concentration, probably because it contains a chlorinated group. Although not terribly sensitive, electric eel cholinesterase is totally specific for chlorinated and carbamate insecticides. A comparison of Z60values (concentration of the pesticide necessary to cause a 50% inhibition of the enzyme activity) of all the five enzymes is listed in Table 111. As little as 8.0 10-8M and 1.8 X 10-7M Paraoxon and DDVP, respectively, produce a 50% inhibition of rat liver cholinesterase. Both the bean leaf beetle and the white fringe beetle are totally specific for organophosphorus compounds, but are much less sensitive than rat liver cholinesterase. Sevin interferes in the assay of organophosphorus com-
+
Figure 2. Plot of inhibition of human serum cholinesterase by various pesticides. Three-minute preincubation o Paraoxon DDVP
0
0
Parathion
A Methyl parathion
lNHl6lTOR 1772
ANALYTICAL CHEMISTRY, VOL. 42, NO. 14, DECEMBER 1970
(Mx IOm6)
00
c 7
70t Figure 3. Plot of % inhibition of electric eel cholinesterase by various pesticides. Three-minute preincubation Aldrin Dieldrin Sevin rn Methoxychlor A Methyl parathion 0 0
Figure 4. Plot of inhibition of white fringe beetle by various pesticides. Three-minute preincubation 0
Paraoxon
0
DDVP
Q
rn Parathion A
Methyl parathion
INHIBITOR
Pesticides
(M x
IOq7)
Table 111. ZG0Values (moles per liter), 3 Minutes Incubation Rat liver Human serum Electric eel Bean leaf beetle 1.8 x 1 0 - 7 ~ 10.0 x i o - 7 ~ 3000.0 X 10-?M 10.0 x 10-7M 0.8 x 1 0 - 7 ~ 2.4 x 1 0 - 7 ~ X 3.0 x 1 0 - 7 ~ 30.0 x 1 0 - 7 ~ 15.0 x 1 0 - 7 ~ X 100.0 x 1 0 - 7 ~
DDVP Paraoxon Parathion Methyl parathion 100.0 x 10-7~4 300.0 x 1 0 - 7 ~ . . .a ... Aldrin ... ... Dieldrin Sevin 300.0 x 1 0 - 7 ~ ... Methoxychlor ... ... ... Mirex ... ... Heptachlor Lindane Xb ... DDT X X a . . . No inhibition. X Does not reach 5 0 z inhibition. .
.
OC
100o.o x
I
c
400.0 X 10-7M
X X X X ... X X X 0 X X X 0 5 0 z inhibition at concentration >lO-aM. 10-7~ 670.0 X lC-7M 700.0 X lW7M 1400.0 x 1 0 - 7 ~
White fringe beetle 50.0 X lW7M 4 . 5 x 10-'M 150.0 X 1 k 7 M 450.0
x
10-7~
X X X
X X X X X
ANALYTICAL CHEMISTRY, VOL. 42, NO. 14, DECEMBER 1970
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Figure 5. Comparison of the % inhibition of bean leaf and white fringe beetle cholinesterase by Paraoxon
Bean leaf beetle o White fringe beetle pounds using rat liver cholinesterase at a concentration greater than 5 X 10-7M. Human serum cholinesterase is a better choice for assay of the organophosphorus pesticides than bean leaf beetle or white fringe beetle because not only is Sevin not an interference, but it is also more sensitive. Using rat liver cholinesterase, 10-*M to 2 X IIO-eM Paraoxon and DDVP can be assayed with an accuracy of about 5 %. As mentioned earlier, Parathion, methyl parathion, and Sevin will interfere at concentrations greater than 10-7M. Interference from Sevin and methyl parathion can be eliminated by dilution of the sample to very low concentrations. Human serum is also good for the assay of Paraoxon and DDVP, since both carbamates and chlorinated hydrocarbons do not interfere. It is, however, less sensitive than rat liver cholinesterase. Phosphorothionates are not strong inhibitors of esterases, but their oxygen analogs are very potent inhibitors (Id). The I R spectra of both Parathion and methyl parathion had a 0
II
very distinct peak corresponding to -P-. Separation on TLG showed less than 1% impurity but the magnitude of the I60 values is not in agreement with this fact. In the case of human serum cholinesterase, for example, the Z50of Parathion is about 6.2 times higher than that of Paraoxon but in the case of rat liver, it is 37.5times higher. In any case, it can be concluded that inhibition is not only due to paraoxon as an impurity but to Parathion as well. Since both Parathion and methyl parathion, available commercially and in common use,
(16) R. M. Hollingsworth, T. R. Fukoto, and R. L. Metcalf, J. Agr. Food Chern., IS, 235 (1967).
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are of 99% purity, the data of inhibition by these pesticides is presented. Figure 3 shows that electric eel cholinesterase is inhibited by some chlorinated hydrocarbons and a carbamate, Sevin. Out o f fourteen pesticides tried, dieldrin, Aldrin, methoxychlor, and Sevin are good inhibitors. Two other chlorinated pesticides, namely, lindane and DDT, are very weak inhibitors. Organophosphorus compounds which are very potent inhibitors of cholinesterase from almost all other sources are not very potent inhibitors of electric eel cholinesterase. From the results of this study, it can be concluded that in addition to sensitivity, some specificity in the assay of pesticides using enzymic methods has been achieved. ACKNOWLEDGhlENT
The authors wish to thank Dr. L. D. Newsom, Entomology Department, Louisiana State University, Baton Rouge, for providing the bean leaf beetles used in this study, and Mr. F. J. Bartlett of the U. S. Department of Agriculture, Gulfport, Miss., who provided the white fringe beetle. We would also like to thank Mr. R.M.Smith for his assistance in the experiments described. RECEIVED for review May 15, 1970. Accepted September 9, 1970. Financial support of the National Institutes of Health (Grant No. ES-00426-01), and the Louisiana Community Studies Pesticides Project, Department of Pharmacology, Louisana State University Medical Center supported by Contract No. PH21-2016 with the Division of Community Studies, Office of Pesticides and Products Safety, Bureau of Foods, Pesticides, and Product Safely, Food and Drug Administration, Public Health Service, Department of Health, Education, and Welfare, Chamblee, Ga., is gratefully acknowledged.
ANALYTICAL CHEMISTRY, VOL. 42, NO. 14, DECEMBER 1970
