ANALYTICAL

196,

BIOCHEMISTRY

377-384

(19%)

Determination of Serum Cholinesterase Activity by Liquid Chromatography with Electrochemical Detection R. Brent

Miller

Department

Received

and C. LeRoy

of Chemistry

September

Blank

and Biochemistry,

University

of Oklahoma,

Norman,

Oklahoma

73019

28,199O

A sensitive enzymatic assay to measure cholinesterase activity in serum using liquid chromatography with electrochemical detection has been devised and used to examine choline&erase inhibition in mice treated with diisopropyl phosphorofluoridate. Acetylcholine was used as substrate, and a postcolumn reactor containing immobolized choline oxidase converted the enzymatic product, choline, and the internal standard, ethylhomocholine, into the electrochemically active H,O,. The postcolumn reactor also contained acetylcholinesterase to allow the indirect detection of the substrate. Assay optimization included investigations of substrate concentration, buffer pH and ionic strength, enzyme concentration, incubation time, and reaction termination method. The optimized procedure is applicable to samples with activities of 0.11 to 269 clmol/ml/h. Intrasample coefficient of variation for mouse serum samples was 1.7% (n = 12), while intersample coefficient of variation was 8.0% (n = 5). The mean + SE serum cholinesterase activity found for controls and mice treated with diisopropyl phosphofluoridate (6.3 mglkg, ip, 24 h prior) was 168.7 i 6.7 rmol/ml/h and 36.6 + 3.1 pmoll ml/h, respectively (p < 0.001). Q 1991 Academic PI-, IUC.

The nature and occurrence of the various mammalian enzymes which cleave acetylcholine and other choline esters have been nicely summarized in a recent monograph (1). Being collectively referred to as cholinesterases, these enzymes are typically divided into two groups. The first group, with the systematic name acetylcholine acetylhydrolase (EC 3.1.1.7), occurs in neurochemically excitable tissue, neuromuscular junctions, and erythrocytes; it is commonly called acetylcholinesterase (AChE)’ or true cholinesterase. The second 1 Abbreviations used: AChE, acetylcholinesterase; ChE, cholinesterase; DFP, diisopropyl phosphorofluoridate; LCEC, liquid chromatography with electrochemical detection; ACh, acetylcholine; Ch, choline; EHC, ethylhomocholine; ODS, octadecyl silane; ChO, choline oxidase. 0003-2697/91 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form

group, the subject of the current report, has the systematic name acetylcholine acylhydrolase (EC 3.1.1.8) and is found to occur in blood plasma and serum and many tissues of animals. This second family of enzymes, which includes at least 15 distinct isozymes (2), has been referred to as plasma cholinesterase, serum cholinesterase, pseudocholinesterase, butyrylcholinesterase, or, simply, cholinesterase. Throughout the current report, we use the term cholinesterase (ChE) exclusively to refer to the acetylcholine acylhydrolase family of isozymes. The activity of ChE may be influenced by drugs (3), physiological states like age (4), pregnancy (5), and sex (4), genetically determinedconditions (6), andpathological conditions like infectious hepatitis (7). Clinical determinations of ChE activity can provide insight pertaining to these various states. For example, primary liver diseases such as infectious hepatitis or hepatic cirrhosis exhibit lower enzyme activity due to a decreased capacity of the liver, which is responsible for production of the enzyme (7). Improvements in prognosis correspond to increased levels of serum ChE activity. Reduced enzyme activity has also been observed in carcinoma (a), malnutrition (9), genetically atypical individuals (lo), and following treatment with pesticides such as diisopropyl phosphorofluoridate, DFP, a potent inhibitor of acetylcholinesterase and cholinesterase (11). On the other hand, elevated ChE levels have been demonstrated in a variety of disorders including nephrotic syndrome (12) and thyrotoxicosis (13). Hence, we developed an assay to measure the activity of ChE in serum via liquid chromatography with electrochemical detection (LCEC). In the plasma and serum, ChE exhibits substantial activity, whereas AChE does not (14). Thus, acetylcholine (ACh) can reasonably be used as a substrate to determine the activity of ChE in plasma and serum. This activity was assessed by monitoring the amount of choline (Ch) formed during incubation. Both the Ch and the internal standard, ethylhomocholine (EHC), are measured indirectly, following chromatographic separation, through reaction with im377

Inc. reserved.

378

MILLER

AND BLANK

mobilized choline oxidase to produce the electrochemitally active H,O,. The same postcolumn reactor containing choline oxidase also contains immobilized AChE to allow detection of ACh via similar production of H,O,. This LCEC arrangement is based upon that originally described by Potter et al. (15) with the enzyme immobilization of Yao and Sato (16). A similar approach has recently been reported by Kaneda et al. (17) for the determination of true AChE activity in tissue samples. After optimization of the assay, we investigated the ChE activity in male albino mice treated with DFP. DFP is representative of a number of phosphate ester derived insecticides and is similar in its mode of action to potential chemical warfare agents (11). The action of DFP on both AChE and serum ChE is of particular concern in cases of human exposure since the ChE inhibition persists for at least 15-17 days after moderate inhalation doses (18).

TABLE

1

Preparation of Samplesfor the Determination of Mouse Serum ChE Activity Amount of SolutionAdded (al) Solution

Serum sample

Blank

External standard

Phosphate buffer (0.100 M, pH 7.20) EHC, 8.00 mM Ch, 4.00 mM ACh, 500.00 mM Diluted serum* Water HCIO,, 2.50 M**

500 50 25 25 25

500 50 25 25 25

500 50 25 25 25

‘Reagents were added in order, proceedingfrom the top to the bottom of the table. EHC, ACh, and Ch were stock standards, prepared in 10.0 mM, pH 4.5 acetate buffer. * Diluted serum was prepared by adding 50 al of serum to 200 pl of isotonic saline. ** The HCIOd was added after the sample had been incubated for the prescribed 10 min.

EXPERIMENTAL

Reagents. Acetylcholine chloride, choline chloride, acetylcholinesterase (EC 3.1.1.7), choline oxidase (EC 1.1.3.17), diisopropyl phosphorofluoridate, and sesame oil were all purchased from Sigma Chemical Co., Ltd. (St. Louis, MO); potassium phosphate monobasic was purchased from Fisher Scientific Co. (Fair Lawn, NJ); tetramethylammonium chloride, sodium azide, tris(hydroxymethyl)aminomethane (Tris), and ethylenediaminetetraacetic acid, disodium salt, dihydrate (EDTA) were purchased from Aldrich Chemical Co. Inc. (Milwaukee, WI). Bromoethane and 3-dimethylamino-lpropanol (Aldrich), were employed to synthesize the internal standard, ethylhomocholine bromide (N,N-dimethyl-N-ethyl-3-amino-1-propanol bromide) according to the procedure of Eva et al. (19). Octyl sodium sulfate was purchased from Eastman Kodak Co. (Rochester, NY). All chemicals were of the highest available purity and used without purification. Animals. Adult male mice of the Hsd:ICR albino strain (Harlan Sprague-Dawley, Madison, WI) weighing 25-35 g were used in all experiments. The animals were housed 10 per cage, allowed access to Purina Rat Chow and water ad libitum, and maintained on a 12-h light/dark cycle with lights on at 7 AM. No animals were used in experiments until at least 7 days after receipt from the supplier. Apparatus. The liquid chromatographic system was similar to that previously described (20). The analytical column was a 3.2 mm X 10 cm, 3 pm ODS cartridge unit obtained from Bioanalytical Systems (West Lafayette, IN). The 3.5-cm postcolumn reactor, prepared in-house, contained 60 units of AChE and 60 units of choline oxidase (ChO), according to the description of Yao and Sato (16). The purified AChE and ChO, as well as the

definition of units for each, were supplied by Sigma. Hydrogen peroxide production was monitored electrochemically at a platinum electrode with E,pp = 0.50 V vs Ag/AgCl. The mobile phase, prepared fresh every 3 days to avoid bacterial growth, consisted of a 20 mM Tris buffer, pH 7.50, containing 4.6 ml glacial acetic acid, 1.0 mM tetramethylammonium chloride, 200 PM octyl sodium sulfate, 6.0 mM sodium azide, 67 pM EDTA, and 2.0% (vol/vol) acetonitrile. The solution, filtered prior to use through a 0.45-pm Millipore membrane, was typically used at a flow rate of 0.90 ml/min and a pressure of 2000 psi. Procedure. Standard stock solutions of Ch (4.00 mM), EHC (8.00 mM), and ACh (500 mM) were prepared in a weak acetate buffer; the buffer contained 3.0 ml of glacial acetic acid in 1.00 liter of deionized water and was adjusted to a pH of 4.50 with dilute NaOH. These were stored frozen at -80°C. The incubation buffer consisted of 0.100 M phosphate buffer of pH 7.20, prepared by adjusting the pH of 0.100 M KH,PO, with concentrated NaOH. Approximately 500 ~1 of blood was obtained, when required, by cardiac puncture of the mouse. After sitting at room temperature for 15 min, followed by centrifugation at 1OOOgfor 10 min, this yielded approximately 200 ~1 of serum. The serum samples were stored at -80°C until analysis, which occurred within 2 weeks. The stability of ChE during this procedure is of no major concern, since it is reported stable for 30 days at 4°C and several years at -20°C (21). On the day of analysis, an appropriate number of blanks, external standards, and serum samples were prepared according to Table 1, with all items except the

SERUM

CHOLINESTERASE

diluted serum and HClO, being added in advance. Diluted serum was prepared by adding 50 ~1 of serum to 200 ~1 of isotonic saline. The enzyme reaction commenced with the addition of 25.0 ~1 of the diluted serum and was allowed to continue for exactly 10 min at room temperature (22’C). Cessation of enzymatic hydrolysis of ACh was produced by addition of 25.0 ~1 of 2.50 M HClO, and vortexing for ca. 10 s immediately thereafter. Blanks were treated exactly the same as serum samples to correct for any nonenzymatic hydrolysis; however, both blanks and standards received 25 ~1 of water instead of the diluted serum. The samples were filtered through 0.45-pm nitrocellulose filters by low-speed centrifugation for approximately 30 s and collected in 1.5ml polypropylene sample tubes. A 20-~1 aliquot of the filtrate was injected into the LCEC for determination of the choline produced by hydrolysis. The ChE activity, expressed as micromoles of choline produced/ml serum/h, was calculated directly from the amount of product formed: Activity

R

= Tmpie erbmml

- &,a,,~ standard

pmol choline in external (ml serum)(incubation

standard time, h)

I’

where R refers to the ratio of the peak height for Ch to that of EHC! obtained from the chromatograms. The R sa,,,plevalues refer to individual serum samples, while values refer to averages. The the hank and Rextemd standard “pm01 choline”refers to the micromole of Ch contained in a 625-~1 “incubation mixture” for an external standard. The “ml serum” refers to the volume of original serum contained in a 625-~1 sample incubation mixture. Final results are reported as the mean f the standard error of the mean unless otherwise noted. Student’s t test was used to examine statistical significance. The formula for the determination of ChE activity, employing blanks prepared as described in Table 1, corrects for nonenzymatic hydrolysis of the substrate. However, it does not correct for the observed time lag between addition of HClO, and actual reaction termination; in fact, the blank was specifically designed to allow investigation of this phenomena. Correction for the time lag can be achieved by two separate approaches. Since the extrapolated activity of ChE obtained at 0 min using HClO, inactivation, vide infra, represents 8.8% of that obtained for a lo-min incubation, one can simply multiply the individual activities obtained using the above formula by a factor of 0.912 (=l.OOO - 0.088). Alternatively, one could alter the content and treatment of the blanks. In this approach, the blanks would receive 25 ~1 of the diluted serum instead of the prescribed 25 ~1 of water; the blanks so treated would be incubated for the prescribed 10 minprior to the addition of the diluted

ACTIVITY

379

serum, and the perchloric acid would be added immediately after the addition of the serum. This second approach is recommended for routine analyses. However, for simplicity and consistency, the former method, i.e., multiplication of individual results by a factor of 0.912, was employed in the current investigation.

RESULTS

General assay optimization. Optimization of enzyme incubation parameters, discussed below, actually occurred in a cyclical process. Initial investigations, not reported here, of the effect of each of the parameters on the observed activity resulted in the final ChE analysis conditions reported above under Experimental. Using the optimized conditions, we investigated each parameter to both verify its establishment as the optimum and to derive the additional information presented. Reaction termination. In order to terminate the enzymatic hydrolysis of ACh, an appropriate reagent or condition must be applied to the reaction in progress. Three enzyme inactivation procedures, namely, (i) addition of physostigmine, (ii) addition of HClO,, and (iii) heating in a water bath were investigated and assessed for their effectiveness. Initial investigations indicated some delay between the applied termination condition and the actual cessation of serum ChE activity. Each termination condition was, therefore, examined using various incubation times. The extrapolation of these results was then employed to determine the interval between the application of the terminating condition and the achievement of actual termination. One of the easiest methods which can be employed for reaction termination simply involves addition of a deproteinizing reagent. The selection of HClO, as the deproteinizing agent also enhances preservation of the sample following incubation by lowering the pH and, thereby, reducing nonenzymatic hydrolysis of the substrate. Preliminary investigations employed 25 ~1 of 10.0, 2.5, and 1.25 M HCIO,. The 1.25 M HCIO, did not adequately stop the enzymatic reaction and was, hence, discarded. The two higher concentrations both effectively acidified the reaction mixture, achieving measured pH values below 2.0. However, the chromatograms resulting from the use of 10.0 M HClO, were undesirable for two reasons. First, samples inactivated by this higher concentration of HClO, produced extensive solvent fronts, obscuring the early eluting Ch peak. Secondly, the chromatographic resolution between the Ch and EHC peaks in such samples was decreased by approximately 10% when compared to samples inactivated with 2.5 M HCIO,. Using 25 ~1 of 2.5 M HCIO, for inactivation, the measured activity as a function of incubation time was determined, as shown in Fig. 1. These results indicate that enzyme inactivation is achieved

380

MILLER

AND

Choline (56 max)

0’0

10 Incubation

20 Time

(min)

FIG. 1. Choline measured as a function of incubation time following inactivation by perchloric acid (D), heating (0), and physostigmine (0). Perchloric acid inactivation employed the addition of 25 ~1 of 2.50 M HClO,. Heating incorporated immersion of the incubation mixture in a 70 + 2°C water bath for 5 min. Physostigmine inactivation used 25 ~1 of 10.0 mM physostigmine. All results presented represent the mean + SE for at least four separate determinations.

BLANK

incubation. The stock solutions of physostigmine were prepared daily and protected from light; lack of degradation was assured through a lack of observation of the distinctive pink color of rubrescerine. The resulting ChE activity, determined as a function of incubation time, is also illustrated in Fig. 1. The lack of linearity in these results obtained with physostigmine is not currently understood. Additionally, it was felt that the presence of solutions having such a high concentration of physostigmine would be a possible biological hazard. Thus, reaction termination by physostigmine was eliminated. Buffer pH and ionic strength. The pH optimization of the ChE assay was investigated by using various solutions of 0.100 M phosphate buffer. Used in prior colorimetric assays (24,25), this buffer material exhibited favorable physical properties over the investigated pH range of 5.50-8.50 at increments of 0.50 pH units. All buffers were prepared by taking a solution of 0.100 M KH,PO, and adjusting to the desired pH with concentrated NaOH. Examination of the results, shown in Fig. 2, yields an optimum of 114.6 + 0.1 pmol/ml/h at pH 7.00. The activity was considerably less at lower pH values. However, only a small and insignificant decrease in activity, to 114.1 + 1.4 pmol/ml/h, was obtained using the separately investigated pH value of 7.20. Thus, pH

100

within approximately 0.7 min following the addition of HClO,. One of the most commonly employed ChE inactivation procedures utilizes heating of the incubation mixture in a water bath (22). In these investigations, a beaker was filled with distilled water and maintained at 70 + 2°C on a hot plate. The reaction mixture, contained in a 1.5ml polypropylene tube, was inactivated by immersion in the beaker for 5 min. The resulting activity, measured as a function of the incubation time, is also presented in Fig. 1. Extrapolation of the results indicated that this approach required approximately 3.1 min between the initial immersion of the incubation mixture and subsequent inactivation of the enzyme. Aside from this relatively long inactivation time, this approach suffers from being both time consuming and awkward in comparison to the simple addition of HClO, described above. Therefore, heat inactivation was eliminated from further consideration. Blockade of the ChE enzyme was also attempted as a reaction termination condition. In these investigations, 25 ~1 of 10.0 mM physostigmine, a reversible cholinesterase inhibitor (23), was added to the mixture following

80 Activity (pmol/mL/hr)

60

40 5.0

7.0

6.0

8.0

PH FIG. 2. Mouse serum choline&erase activity as a function of pH. All results presented represent the mean f SE for at least four separate determinations.

SERUM

CHOLINESTERASE

7.20 was selected to ensure maximum buffering capacity while maintaining a high activity for the assay. The effect of the ionic strength of the buffer was investigated at pH 7.20 by employing various phosphate concentrations between 1.00 and 300 mM. The results of this experiment are shown in Fig. 3. Despite the optimum of 114.2 f 0.5 pmol/ml/hr obtained with 0.050 M phosphate, a 9.1% decrease using 0.100 M phosphate, to an activity of 104.0 f 1.1 pmol/ml/hr, was accepted in order to retain a higher degree of buffering capacity. Using the pH 7.20, 0.100 M phosphate buffer, a measured pH value of 7.16 was achieved in the actual incubation mixture. The linearity of the observed Enzyme concentration. ChE activity with respect to the amount of added original mouse blood serum was investigated to include a range from 0.625 to 100.0 ~1. Retaining a constant volume of 625 ~1 for the assay, the buffer volume was decreased to compensate for serum volumes greater than 25.0 ~1. For serum volumes less than 25.0 ~1, dilutions of the original serum with isotonic saline were performed. As indicated in Fig. 4, the measured activity is directly proportional to the amount of serum added throughout the range of 0.625 to 10.0 ~1. At values above 20.0 ~1, the results clearly deviate from linearity. Based upon these results, an original serum volume of 5.0 ~1, contained

381

ACTIVITY

Activity (pmollhr)

0

25

50

pL

Serum

75

100

Added

FIG. 4. Mouse serum cholinesterase activity measured as a function of the amount of serum added to the incubation mixture. All results presented represent the mean +- SE for at least four separate determinations.

within tion.

a 25.0~~1 aliquot,

was selected for routine

utiliza-

Incubation time. The ChE reaction progress was monitored at various incubation times up to and including 40 min. The enzymatic production of Ch was completely linear for the first 20 min of incubation, as indicated in Fig. 5. Thus, an incubation time of 10 min was selected for the routine application of the developed assay. Substrate concentration. The effect of increasing ACh concentrations on the observed activity was systematically examined. Simple linear regression of the resulting Lineweaver-Burk plot, shown in Fig. 6, yielded values for K,,, and V,, of 1.90 mM and 138.4 pmol/ml/h, while the weighted regression of Wilkinson (26) yielded values of 2.09 mM and 144.8 pmol/ml/h. Based upon the K,,, values obtained, a substrate concentration of 20.0 mM was selected for routine analyses.

Activity (pmol/mL/hr)

0.10

Phosphate

0.20

Concentration

0.30

(M)

FIG. 3. Mouse serum choline&erase activity as a function of buffer molarity. Buffer pH was 7.20. All results presented represent the mean + SE for at least four separate determinations.

Assay detection limit, linear dynamic range, and reproducibility. The detection limit of the current LCEC assay was determined through consideration of signals obtained for pure Ch samples and peak-to-peak chromatographic noise. Using a signal value of three times noise as the detection limit, 3.05 pmol of Ch could be determined. This corresponds to a ChE activity value of

382

Activity (pmol/mL)

MILLER

AND

BLANK

134.4 f 2.3 pmol/ml/h (mean f SD, n = 12), or a coefficient of variation of 1.7%. The reproducibility between animals may be estimated by the results obtained for control mice in the DFP treatment experiment described below. The five control mice yielded a serum ChE activity of 158.7 f 12.7 (mean + SD) for a coefficient of variation of 8.0%. Cholinesterase activity following DFP treatment. Mice acutely treated with DFP, 6.3 mg/kg, ip, 24 h prior to sacrifice, exhibited tremor and readily recognizable parasympathomimetic symptoms including diarrhea, excessive salivation, and lacrymation (28). The control group did not exhibit any of these characteristics. The LCEC determination of the serum ChE levels in these two groups yielded chromatograms which are typified by those shown in Fig. 7. As seen in Table 2, the DFP treated animals had ChE activities which were only 22% of the controls. The mean value for the controls in this table corresponds to 44.1 nkat/ml serum.

4o

DISCUSSION 20 Incubation

40 Time

FIG. 5. Mouse serum cholinesterase activity bation time. All results presented represent least four separate determinations.

(min) as a function of incuthe mean + SE for at

0.11 pmol/ml/h using the procedure described for routine analysis. This is one to two orders of magnitude lower than the detection limits reported for commonly employed spectrophotometric procedures (9,27). It should also be noted that the LCEC detection limit stated here is relatively conservative. It could easily be lowered an additional factor of 10 to 20 by simply doubling the reaction time and/or amount of original serum employed and increasing the injection volume to 100 ~1. None of these suggested additional measures would substantially alter the precision or accuracy of the procedure. The linear dynamic range of the prescribed procedure was determined by consideration of the range in which the LCEC exhibits a linear response to Ch. The lowest detected value of Ch, as described, corresponds to 0.11 pmol/ml/h. The largest amount of injected Ch attempted, 7.45 nmol, also showed a response directly proportional to concentration. This upper limit for Ch corresponds to a ChE activity of 269 pmol/ml/h. Thus, the assay should exhibit linearity from at least 0.11 to 269 pmol/ml/h, or over a range of approximately three decades. The reproducibility of this procedure is indicated by results obtained with repetitive determinations of the same pooled serum sample; these yielded an activity of

The LCEC method outlined offers a simple, moderately rapid, and cost-effective technique to obtain ChE activity in serum samples. Most significantly, the ap-

l/Activity (Fmol/mL/hr)

l/C31

(mM)

FIG. 6. Lineweaver-Burk plot showing effect of substrate concentration on observed mouse serum cholinesterase activity. All results presented represent the mean + SE for at least four separate determinations.

SERUM

CHOLINESTERASE

preach offers an order of magnitude (or more) decrease in detection limits compared to commonly employed colorimetric procedures. The linear dynamic range and detection limits are comparable to those reported for radiochemical (29) and flow injection (30) methods. The analysis is based upon the reaction of ChE with ACh to produce Ch. The results of the assay may thus be alternatively expressed as micromoles of ACh hydrolyzed/ ml serum/h. The procedure requires only 5 ~1 of original serum sample. The current chromatographic throughput of 5 samples per hour could be increased, if necessary, to approximately 12 samples per hour by simply eliminating the AChE in the preparation of the postcolumn reactor for the LCEC setup. This would eliminate the large chromatographic peak observed for the substrate, ACh, and allow injections to be made every 5 min instead of every 12 min. The procedure is highly reproducible and routinely yields less than 2% variation between replicate samples and 8% between animals. Various factors affecting the measurement of enzyme activity were evaluated in developing this assay. The pH optimum of 7.00 and the optimum phosphate buffer strength of 0.050 M were slightly compromised in the selection of the values of 7.20 and 0.100 M, respectively, to be used for routine analysis. This compromise, however, decreases the maximal observed activity by only 9%, while offering considerably improved buffering capacity. The measured K, values for mouse serum cholinesterase were 1.90 and 2.09 mM, respectively, by direct analysis of the Lineweaver-Burk plot and by the

DFP Treated

Control 100

.a

nAmp 50

H

-2

i Time, min

i2

383

ACTIVITY

TABLE Effect of DFP on Mouse Group

n

Control DFP

5 5

2 Serum ChE Activity” Activity

(rmol/ml/h)

+ SE

158.7 -t 5.7 36.6 + 3.1*

o The DFP group received 6.3 mg/kg DFP, ip, 24 h prior to sample removal by cardiac puncture. * P < 0.001, DFP compared to controls, Student’s t test.

weighted analysis of Wilkinson (26). All currently available methods for the determination of serum or plasma ChE activity, in fact, do not measure the activity of a single enzymatic entity. Indeed, there are multiple isozymes which contribute to the measured ChE activity in these methods (2). Thus, their treatment as a single entity for kinetic analysis is not strictly correct. Nonetheless, the measured values offer a means of comparison between alternative techniques. In that regard, the currently obtained K,,, values are reasonably comparable to that of 1.8 mM reported by Foldes et al. (31) for human serum ChE activity using the same substrate. The effect of DFP on mouse serum ChE activity was quite profound. Animals treated with 6.3 mg/kg, ip, 24 h prior to sacrifice yielded ChE activities which were only 22% of control values. This was a somewhat greater reduction than that reported by Martin (32), who found that a single dose of DFP, 1 mg/kg, iv, produced a reduction in mouse serum ChE to 35% of that of controls when measured 8 h later and a reduction to 75% of that of controls when measured 24 h later. This difference between the current results and those of Martin (32), of course, could be explained by either the mode of injection or the dose employed. On the other hand, studies employing rats appear to provide more comparable reductions in serum ChE. For example, Davison (33) reported that 0.5 mglkg DFP inhibited rat serum ChE in a manner that exhibited exponential recovery to 50% of control values at 72 h following treatment. Extrapolation of this data indicates that the ChE levels were approximately 21% of controls at 24 h following DFP treatment. The same study by Davison reported a reduction of true cholinesterase, or AChE, in rat brain to ca. 20% of controls at 24 h following the described treatment with DFP. By comparative inference, rats treated with DFP, 1.0 mg/kg, im, shown by Glow and Richardson (34) to produce reductions in AChE to 30% of that of controls, would be expected to exhibit serum ChE values 24 h after treatment which were approximately 30% of that of controls, again comparable to the current results.

Time, min

FIG. 7. LCEC chromatograms obtained from typical control DFP pretreated animals for the determination of mouse serum line&erase activity. Conditions stated in text.

ACKNOWLEDGMENTS and cho-

The authors thank Douglas lumn LCEC reactor containing

J. Turk AChE

for preparation and ChO. These

of the postcoinvestigations

384

MILLER

AND

were supported by the following: Department of Chemistry and Biochemistry, University of Oklahoma; Biomedical Research Grant No. 2507 RR07078-14 from NIH/DHHS; and a grant from Great Plains Laboratories, Inc., Norman, OK.

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