Determination of the Half-life of Eserine: Evaluation of Laboratory Procedures

Summary
Acetylecholine (Ach) is a neurotransmitter released at the synaptic cleft of the neuromuscular junction whose activity causes muscle contraction. Enzyme Acetylcholinesterase (AChE) is responsible for breaking down ACh and resetting the nerve activity of the neuromuscular junction. AchE inhibitor is therefore used commercially to produce compounds like insecticides that manifest uncontrolled, spastic contractions of muscles resulting in paralysis. The Product Development Division has been asked to evaluate an existing assay for AChE to see if it can be adapted to measure half-life of Eserine indicating length of time the compound will actively demonstrate its potency. The concentration of Eserine which inhibits the activity of AChE by 75% was calculated on a trial basis as 0.1 M. Five serial two-fold dilutions of this solution was prepared, a volume of 30 l taken from each and mixed with 20 l AChE. It was then incubated for 10 minutes and 24 l Acetylthiocholine added to it. Reaction was recorded as a change in optical density over 5 minutes at 405 nm after adding 3000 l of DTNB. This would yield Eserines theoretical half life of 30 minutes. The result so obtained was used to elicit graph of Log % inhibition of AChE over time from which half-life of Eserine was calculated to be 43 minutes and 95% Confidence Interval between 30.72 and 71.67 minutes. This means it takes 43 minutes to reduce the number of original molecules of Eserine by a factor of two. Conclusively, from intra-assay and inter-assay analysis of grouped data, the existing assay was found to be fairly precise and inaccurate and was thus not recommended.

Introduction
The staffs of the Technical Services Laboratory were requested, by the Product Development Division (PDD), to evaluate the suitability of an existing assay for acetylcholinesterase, AChE, for the measurement of the half-lives of putative, novel inhibitors of AChE in solution under conditions of normal laboratory use. An existing AChE inhibitor, Eserine, was chosen as the test-inhibitor to evaluate the assay. There are well-established assays for AChE itself, including monitoring absorbance changes in the presence of substrate analogues such as acetylthiocholine (ATC). In the presence of fixed amounts of enzyme and substrate, the inhibitory effect of any AChE-inhibitor present would be reflected in the change in absorbance. Factors affecting the rate at which solutions of AChE-inhibitor lose their inhibitory activity are likely to include the pH of the solution, its temperature, and possibly its exposure to

light. It is also likely that the potency of the AChE-inhibitor would decline in a logarithmic fashion over time and, thus, regression analysis may provide the best means of quantifying the change in potency and the determination of half-life. The half-life of the inhibitor can be calculated from the slope of the regression of log [enzyme activity in % inhibition] against time in minutes: thus, half-life (minutes) = log2 / -slope. The evaluation of the procedures is as follows: (i) A qualitative and quantitative assessment of the assay procedure is needed. This includes a consideration of the sensitivity of the assay, the means and ease of measurement of the absorbance changes and also the repeatability of the measurements by different operators. (ii) A quantitative measure of the uncertainty of the determined half-life of the test-inhibitor, Eserine, is a central element of the evaluation. This is best expressed as a 95% confidence interval of the half-life which can be deduced from the regression analysis of the data. (iii) The third element, which needs to be considered, is whether the chosen experimental design yields results which were sufficiently accurate and precise for the needs of PDD or whether a more elaborate design is required.

Material and Methods

1) Following assay solutions were used: 0.33 M 5, 5-dithiobis (2-nitrobenzoate) (DTNB) (Sigma) in 0.05M KH2PO4 buffer, pH 7.5, kept at room temperature 10 mg/ml acetylcholinesterase (AChE) (Sigma, Type V-S [Electric Eel], 1000 - 2000 units/mg) 100 M acetylthiocholine (Acetylcholine analogue) (ATC) (Sigma) 0.1 M Eserine (Acetylcholinesterase inhibitor) (Sigma, free base)

2) Treatment of Eserine test-inhibitor: To simulate the breakdown of Eserine, serial two-fold dilutions of a stock solution of 0.1 M Eserine were made and labelled 0, 30, 60, 90 and 120 min. This had the advantage of yielding a theoretical half-life of 30 minutes.

Hence, following concentrations were obtained: Table 1: Serial 2-fold dilution of 0.1 M Eserine corresponding to time labelled. Time (minutes) 0 30 60 90 120 Concentration (M) 0.10000 0.05000 0.02500 0.01250 0.00625

Each sample was assayed in duplicate for its ability to inhibit AChE as a part of a validation procedure within the assay. This would increase precision i.e. the ability of the assay to produce consistent results when performed twice.

3) Assay of acetylcholinesterase: Blank: The spectrophotometer was initially zeroed at 405 nm against a cuvette containing 3000 l DTNB and 24 l ATC. AChE (0% inhibition): 20 l AChE and 24 l ATC were added to a cuvette in room temperature. The spectrophotometer was zeroed and the reaction was started by adding and mixing 3000 l DTNB. The absorbance change at 405 nm was recorded on a flatbed recorder running at 1 cm/min. Absorbance changes were recorded for 5 minutes for subsequent calculation of the rate of absorbance change (absorbance units/min). The assay was carried out in duplicate, and the mean value of absorbance was calculated. AChE plus test-inhibitor (100% inhibition): 20 l AChE and 30 l 0.1 M Eserine stock were mixed in a cuvette and allowed to stand for 10 min; this was followed by the addition of 24 l ATC. The spectrophotometer was zeroed and the reaction was started by adding and mixing 3000 l DTNB. The absorbance change at 405 nm was recorded as before. The assay was carried out in duplicate, and the mean value of absorbance was calculated. This test solution was prepared to ensure that the results were due to Eserine decay and not because of DTNB and AChE decay.

4) Monitoring the inactivation of the test-inhibitor The AChE plus test-inhibitor assay was carried in duplicates using the serially-diluted Eserine test solutions (Table 1). These were dilutions of a 0.01 l Eserine previously found to give approximately 75% inhibition of AChE. Care was taken to ensure that the equilibration periods between the inhibitor and enzyme were similar for each test solution.

5) Analysis of the data and calculation of the half-life of the Eserine testinhibitor solution Change in rate of AChE inhibition with each serially diluted test solution was monitored for duplicate samples and mean calculated. Logs of the mean percent AChE inhibition were calculated and plotted against the simulated 'sampling time' (0, 30, 60, 90, 120 minutes). The line of best fit for the regression and the 95% confidence interval for the slope were determined using MINITAB version 15. The point estimates and 95% confidence intervals for the half-life of the test-inhibitor solutions were calculated using the equation: Half-life = log2 / -slope.

Results
Data for linear Regression analysis was obtained from the graphs from flatbed recorder. Mean of Absorbance Rate was calculated, percentage inhibition was also calculated using the formula: % Inhibition = 100 [(Abs rate 0.1M Eserine sample Abs rate 100% inhibition) / (Abs rate 0% inhibition Abs rate 100% inhibition) X 100] Log of value so obtained from above equation was used to plot linear regression line using the table drawn in next page. (Table 2)

Table 2: Calculation of % Inhibition using mean absorbance rate. Experiment Absorbance Absorbance Mean of % Rate, Rate, Absorbance Inhibition Sample 1 Sample 2 Rate No AChe, 0 0 0 100 no Eserine AChe, no 24.00 22.00 23.00 0 Eserine 0 min, 0.1 3.50 2.50 3.00 86.96 M Eserine 30 min, 0.1 6.00 8.00 7.00 69.57 M Eserine 60 min, 0.1 11.00 12.00 11.50 50.00 M Eserine 90 min, 0.1 18.00 17.00 17.50 23.91 M Eserine 120 min, 20.00 20.00 20.00 13.04 0.1 uM Eserine Log % Inhibition 1.94 Time (min) 0

1.84

30

1.70

60

1.38

90

1.12

120

Figure 1: Graph of Change in absorbance rate with concentration of Eserine

Change in Rate of AChe inhibition with serial 2-fold dilution of Eserine
Rate ( Absorbance at 405 nm per minute)
20

15

10

0.00

0.02

0.04 0.06 Concentration of Eserine (M)

0.08

0.10

Using the data from Table 2, a linear regression line and a curve of 95% confidence Interval were estimated to analyse the significance of the data. Figure 2: Linear Regression line with 95% Confidence Interval
Linear Regression line with 95% Confidence Interval

Equation of best fit i.e Log % inhibition Y = 2.016 - 0.007000 Time (min) 2.2

Log % inhibition (arbitrary unit)

2.0 1.8 1.6 1.4 1.2 1.0 0 20 40 60 Time (min) 80 100 120

Regression 95% CI S R-Sq R-Sq(adj) 0.0835065 95.5% 94.0%

The R-squared value of 95.5% indicates significant extent to which variation in inhibition of AChE can be explained in terms of time. It also helps us to predict what results might look like when certain variable is altered. The p-value of the slope coefficient is 0.004 (less than 0.05) which implies that we can reject the null hypothesis i.e. value of slope is significantly different to 0 and there is a significant effect of time on Eserines inhibitory activity. We can now estimate its half life by quantifying its inhibitory activity using data from Figure 2. In Log % inhibition equation: Y = 2.02 0.00700 Time (min); slope = -0.00700 Half life = Log (2) / (-slope) = 0.301/-(-0.007) = 0.301/0.007 = 43 minutes To determine the line of best fit within which we can be 95% sure that the true value lies, following data are used: Degrees of Freedom (df) = 3 (i.e n-2 =5-2 =3)

Value of slope coefficient = -0.007 S.D associated with slope coefficient = 0.0008802 (from minitab raw data) Critical value for t at 3 degrees of freedom = 3.182 (from Cambridge statistical table) Therefore, 95% Confidence Interval for the Slope = -0.007 (0.0008802 x 3.182) = -0.007 0.0028 Now, Table3: Calculation of magnitude of 95% Confidence Interval for the Slope -0.007 + 0.0028 = -0.0042 Half life = log 2 / -slope = 0.301 / -(-0.0042) = 0.301 / 0.0042 = 71.67 minutes -0.007 - 0.0028 = -0.0098 Half life = log2 / -slope = 0.301 / -(-0.0098) = 0.301 / 0.0098 = 30.72 minutes

There is 95% probability that the half life of Eserine lies between 71.67 minutes and 30.72 minutes. Theoretically, the half life should have been approximately equal to 30 minutes; the possible reasons for discrepancies will be discussed later.Also the following group results were obtained under similar laboratory condition using the same solution: Group 1: Regression line Y = 1.863 0.0038 X; R-squared = 0.970 (97%) Therefore, Half life = log 2 / -slope = 0.301 / - (-0.0038) = 79.2 minutes Group 2: Regression line Y = 1.919 0.0066 X; R-squared = 0.970 (97%) Therefore, Half life = 0.301 / - (-0.0066) = 45.6 minutes Group 3: Regression line Y = 1.815 0.0054 X; R-squared = 0.990 (99%) Therefore, Half life = 0.301 / - (-0.0054) = 55.74 minutes

Group 4: Regression line Y = 2.147 0.0134 X; R-squared = 0.808 (80.8%) Therefore, Half life = 0.301 / - (- 0.0134) = 22.46 minutes The mean of half lives so obtained from 5 sets of data = (43 + 79.2 + 45.6 + 55.74 + 22.46) / 5 = 49.2 minutes. This is the best estimate of half life of Eserine. Standard Error of these values was 9.24 and standard deviation was 20.67 (obtained using minitab 15) indicating fairly acceptable precision for repeatability of the experiment although not accurate compared to expected value of 30 minutes. % error = [(Actual value expected value)/ expected value] x 100 = (43 30 / 30) x 100 = 43.3 % % error relative to Standard Deviation = S.D / actual value x 100 = 20.67 / 43 x 100 = 48.07 %

Discussion
The existing assay of AChE to see if it is adapted to calculate half life of Eserine yielded a result of 43 minutes with 95% Confidence Interval lying between 30.72 minutes and 71.67 minutes. The half life so obtained is 0.70% more than expected value of 30 minutes with % error of 43.3%. AChE catalyses the breakdown of ATC into acetate and thiocholine. Thiocholine reacts with DTNB to produce a yellow compound which was monitored at 405 nm. Measurements of very small volumes of AChE, Eserine and ATC were used; this measurement might not have been precise and is an experimental limitation. Experimenter error and error inherent to experimental set-up might have caused the results to skew. Percentage error relative to Standard Deviation was fairly high implying systemic error of the method. There might also have been various sources of random error like change in temperature of DTNB causing the recording to curve which will affect the deriving of data from the graph recording, exposure of Eserine solution to light and change in pH of the solution. Equipment variation might have also caused differences in group data. The results of these group data produced mean value of 49.20 minutes and the results were not constant from one group to another although no change was induced experimentally. Therefore this assay is not reproducible between operators.

The values of half lives obtained vary fairly largely between each measurement with a standard error or 9.24 showing low precision of the method. Accuracy is often not applicable to biological systems because the true value itself is obscure and varies between different methods. Our results thus vary to existing presumption that the assay would yield a half-life of Eserine 30 minutes using 0.1M Eserine producing 75% inhibition.

Recommendation to the Product Development Division

Eserine was used as a convenient test substance. A value of 75% inhibition was chosen so that we were producing sufficient inhibition, the gradual loss of which would produce a reasonably large increase in enzyme activity. The evaluation of this procedure hence needs to be revised to be applied to any other test inhibitor and is not recommended until modifications for improvements are made. A more elaborate design is required by improving the experimental procedures. Eserine was theoretically decayed every 30 minutes by diluting it down and obtained value compared to this result. A more precise method could include natural decay of Eserine. But then this would be unrealistic as the half life could vary from any seconds to days or years. Although analogue compound was used, it would also be experimentally precise to use ACh itself.

References
Information, calculation and analysis were deduced and put together using practical session handouts, lecture booklet and minitab version 15.