Clinical Chemistry 47: 1962-1966, 2001;
(Clinical Chemistry. 2001;47:1962-1966.)
© 2001 American Association for Clinical Chemistry, Inc.
New Thiocholine Ester Substrates for the Assay of Human Serum Cholinesterase
Magohei Yamada1a,
Yoji Marui2,
Chozo Hayashi2,
Yasuyoshi Miki3 and
Shoji Takemura3
1
International Reagents Co., Ltd., 4-3-2 Takatukadai, Nishiku, Kobe 651-2271, Japan.
2
Laboratory for Clinical Investigation, Osaka University Hospital, 2-15 Yamadaoka, Suita, Osaka 565-0871, Japan.
3
Faculty of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-Osaka 577-0818, Japan.
aAddress correspondence to this author at: 2-5-7 Nakagaito, Daito, Osaka 574-0013, Japan. Fax 81-72-889-7305; e-mail yama53{at}viola.ocn.ne.jp
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Abstract
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Background: Several thiocholine alkanoyl esters were newly synthesized and explored as substrates for the assay of human serum cholinesterase after being subjected to the Ellman reaction (Arch Biochem Biophys 1958;74:443–50 and Arch Biochem Biophys 1959;82:70–7).
Methods: We synthesized thiocholine ester iodides by the method of Renshow et al. (J Am Chem Soc 1938;60:1765–70). We examined solubility in H2O, substrate specificity serum for cholinesterase, (spontaneous) self-hydrolysis, storage stability, and reaction conditions for measurement of the activity of the enzyme.
Results: Isobutyryl and cyclohexane-carboxyl esters showed the best efficiency for the specific and stable assay of human serum cholinesterase. Aqueous solubility of each was >10 mmol/L, and the reactivity with acetylcholinesterase was negligible. For isobutyryl and cyclohexane-carboxyl esters, respectively, spontaneous hydrolysis in the aqueous phase was 
1/25 and 1/175 slower than the enzymatic hydrolysis, and assays with these substrates were linear to 1800 and 3000 U/L, respectively. The Km values of these acylthiocholines with human cholinesterase were almost equivalent (6.9 x 10-3 mmol/L). The substrates were stable in aqueous solution and in the solid state as the iodides for at least 5 years at 5 °C.
Conclusions: The isobutyrate and cyclohexane-carboxylate of thiocholine are suitable for the specific assay of human serum cholinesterase.
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Introduction
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Thiocholine esters such as acetate, propionate, and n-butyrate have been used conventionally as substrates in the assay of human serum cholinesterase (1)(2) with the Ellman reaction (3)(4). The Ellman reaction involves the reaction of 5,5-dithio-bis(2-nitrobenzoic acid) (DTNB) with thiocholine liberated from its esters by enzymatic hydrolysis. The yellow 5-thio-2-nitro-benzoate (TNB) thus formed is detected by colorimetry. Disadvantages, especially for application to automatic measurements, lie in the use of these conventional assay reagents. (a) The assay procedure requires a high dilution of the serum samples (100-fold) (1), whereas the desirable dilution for accurate manual operations would likely be <30-fold, which is also readily applicable to automatic systems. (b) The optimum pH for the enzymatic reaction is 8–8.5, whereas the conventional measurements are usually performed at pH 7.6 to minimize the undesirable self-hydrolysis of the thiocholine esters (1). Using the synthetic substrate, 2,3-dimethoxybenzoylthiocholine, we previously simplified the assay procedure without manipulating the high dilution and reduced self-hydrolysis at the optimum pH of the reaction (5). In the current study, a series of esters were synthesized and their applications as assay reagents for human serum cholinesterase were explored. Two additional esters, thiocholine isobutyrate and cyclohexane-carboxylate, were also explored as suitable substrates for the assay.
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Materials and Methods
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synthesis of thiocholine esters
Thirteen thiocholine ester iodides (1–13 in Table 1
) were synthesized by a modified procedure of Renshow et al. (6).
N,N-Dimethylamino-ethanethiol hydrochloride (1 mol) was suspended in ether (10-fold the amount of the hydrochloride), and the mixture was stirred with aqueous sodium carbonate (1.2 mol) at room temperature. The ether layer was separated and dried over anhydrous sodium sulfate. The corresponding acid chloride (1.1 mol) was added dropwise under ice-cooling conditions into the stirred ether solution. The separated crystalline ester hydrochloride was filtered and dissolved in water (12-fold the weight of the ester hydrochloride). The solution was alkalized with 100 mmol/L aqueous sodium hydroxide and extracted with ether. The extract was dried over anhydrous magnesium sulfate and stirred with methyl iodide (1.5 mol equivalent to the ester hydrochloride) until no more crystal was precipitated. The product was filtered, dried, and recrystallized from hot water. The melting points and yields of the products are listed in Table 1
. The elemental analysis for carbon, hydrogen, sulfur, and nitrogen were satisfactory (error, within 0.3%). Propionyl- and n-butyryl-thiocholine iodides and starting materials for syntheses were purchased from Aldrich Chemical Co.
reagents for the assay
Reagent materials were obtained from Aldrich Chemical Co. The human serum cholinesterase (EC 3.1.1.8) and acetylcholinesterase (EC 3.1.1.7) preparations were obtained from Sigma Chemical Japan, Ltd. Reagents were prepared as follows. Reagent 1 was a solution of 0.3 mmol of DTNB and 240 mmol of tris(hydroxymethyl)aminomethane in 1 L of water, which was adjusted to pH 8.0 by the addition of maleic acid. Reagent 2 was 6.0 mmol/L thiocholine ester iodide.
general procedure for the assay
A mixture of 2.4 mL of reagent 1 and 0.1 mL of the aqueous solution, containing 13 000 U/L of the enzyme, was preincubated for 5 min at 30 °C; 0.5 mL of reagent 2 was then added to the mixture. The change of the absorbance (
E/min) was measured every minute at 405 nm in a cell with a 1-cm pathlength. The susceptibility of the substrates, expressed in terms of µmol (substrate) · min-1 · L-1 (U/L), was calculated by the equation U/L = (A - B)/
x V/v, where A represents the overall reaction rate (E overall/min), B the reagent blank (E blank/min) attributable to the self-hydrolytic reaction, and the molar absorptivity of TNB at 405 nm (13 300 L/mol = 0.0133 L/µmol). V/v is the ratio between final and sample volumes (3/0.1 = 30). The enzymatic reaction took place at 37 °C.
examination of the stability
The storage stability as the aqueous solution was examined with the use of the Arrhenius relationship between temperature and degradation rate according to Garrett and Carper (7). Aqueous solutions of thiocholine ester iodide samples (5 mmol/L) were kept in appropriate vials at 25 °C, 37 °C, and 50 °C in constant temperature baths. The DTNB solution was prepared separately, and the concentration was 0.1 mmol/L in the 100 mmol/L Tris buffer solution (pH 8.0). Samples of the thiocholine ester solutions were withdrawn from vials at intervals. After acclimatization at room temperature, 0.02 mL of the solution was mixed with 2.5 mL of the DTNB solution and 0.2 mL of serum cholinesterase preparation. The concentration of cholinesterase was chosen to ensure that the absorbance of the total TNB after the enzyme reaction would be as close to 0.5 as possible at time zero. The mixed solution was kept at 37 °C for 1 h, and the absorbance of TNB was measured at 405 nm to estimate the amount of the total thiocholine (At) liberated by the enzyme reaction from the remaining thiocholine ester plus that formed by the thermal degradation (hydrolysis). As the reference, the absorbance of the solution with the DTNB solution added, but not the enzyme preparation, was measured to estimate the amount of thiocholine formed nonenzymatically (As). The rate of degradation was followed for a period of 31 days. The first-order rate constant, k, at each of the temperatures was estimated as the slope of the plot of log (At - As) for the remaining thiocholine esters vs time (h). The values of log k were then subjected to the Arrhenius plot and plotted against the corresponding 1/T (T is the absolute temperature). By extrapolating the linear Arrhenius relationship derived by the regression analysis, the rate constant at 5 °C was estimated, from which the stability of the aqueous substrate solution in terms of t0.8, or the 20% loss time, was calculated.
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Results
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Reaction and self-hydrolysis rates of the synthesized thiocholine esters were examined by the general procedure described above at pH 8.0. As shown in Table 2
, isobutyrate (no. 2) exhibited a relatively high reactivity and a low self-hydrolysis. The presence of the secondary 
-carbon atom in the aliphatic acyl moiety seems appropriate for the enzymatic reaction with lower self-hydrolysis. This also applies to the 2-ethylbutyryl (no. 4) and 2-methylbutyryl (no. 5) derivatives. Electron-donating and medium-sized steric effects of secondary alkyl groups may contribute to the low self-hydrolysis.
Pivaloyl (no. 1) and adamantanecarboxyl (no. 11) derivatives showed a very low reactivity. This may be attributable to a severe hindrance at the tertiary alkyl groups that interferes with the approach of the enzyme molecule to the thioester linkage, as well as the linkage of water to the acylated enzyme. With the succinyl derivative (no. 3), the enzymatic reactivity decreased and the self-hydrolysis increased. The negatively charged carboxylate group in the neighborhood of the thioester bond may accelerate the self-hydrolysis.
In the series of cycloalkanethiocarboxylates (nos. 6–10) in Table 2
, the reactivity increased with the number of the ring carbons up to six. The cyclohexanethiocarboxyl (no. 9) derivative exhibited the highest reactivity and the lowest self-hydrolysis rate in this series. Cycloheptanethiocarboxylate (no. 10) showed a very low reactivity. The dimensions of the cycloheptane ring appear to be intolerable for the enzyme reaction.
Although the reactivities of the 2-ethylbutyryl (no. 4) and 2-methylbutyryl (no. 5) derivatives did not differ significantly from that of isobutyrylthiocholine (no. 2), their aqueous solubility was lower than that of isobutyryl thiocholine as shown in Table 2
. Thus, the isobutyrate (no. 2) and cyclohexanecarboxylate (no. 9) derivatives of thiocholine were selected for detailed examinations.
Table 3
shows the results for reactivities (E) of isobutyryl- (no. 2) and cyclohexanecarboxyl- (no. 9) thiocholines within solutions of human serum cholinesterase (EC 3.1.1.8; 13 000 U/L) and acetylcholinesterase (EC 3.1.1.7; 13 000 U/L), with the propionyl (no. 14) and n-butyryl (no. 15) compounds as references. The reactivity of isobutyryl and cyclohexanecarboxyl compounds with acetylcholinesterase was negligible (almost equal to their rate of self-hydrolysis), demonstrating that these substrates are specific to serum cholinesterase. However, the propionyl- and n-butyryl-thiocholines showed some reactivity with acetylcholinesterase.
The effects of pH variations between 7.0 and 9.0 on the reactivity and the rate of self-hydrolysis were examined for the isobutyrate (no. 2) and cyclohexane-carboxylate (no. 9) derivatives. The maximum reactivity was observed at pH 8–8.25 (100%), as shown in Fig. 1
. The reactivity of the blank reaction (self-hydrolysis) was <5% in the same pH range. Thus, the selection of pH 8.0 in the standard procedure is justified. The effects of pH variations between 7.0 and 8.5 for the propionate (no. 14) are shown in Table 4
. Whereas the enzymatic and the self-hydrolytic reactivities were much higher in this pH range, the pH dependence was somewhat similar to those of the isobutyrate (no. 2) and cyclohexanecarboxylate (no. 9).
The influence of the buffer concentration was examined for the reactivity of isobutyrate (no. 2) and cyclohexanecarboxylate (no. 9) in the range between 10 and 1000 mmol/L. The maximum reactivity was 100 mmol/L at pH 8.0 and lower at increased concentrations (70% in 1000 mmol/L).
The Km value was calculated from the plot of E values against the reciprocals of the concentrations for the substrate. The Km for both the isobutyrate and cyclohexane-carboxylate derivatives was 6.9 x 10-3 mmol/L.
Various concentrations of the human serum cholinesterase were assayed by the general procedure. Very good linearities (r >0.999) were obtained for isobutyrate (no. 2) and cyclohexane-carboxylate (no. 9) substrates up to 1800 and 3000 U/L, respectively.
The pseudo first-order degradation (hydrolysis) rate constants in the aqueous buffer solution (pH 8) for the thiocholine ester iodides at various temperatures are listed in Table 5
. The log k value and t0.8 (20% loss time) at 5 °C were estimated from the log k values at higher temperatures. With storage at 5 °C, the 20% loss time of the isobutyryl (no. 2) and cyclohexanecarboxyl (no. 9) analogs was 5–6 years. Because the amount of substrate reagents used for the clinical assay was selected to greatly exceed that of the enzyme, the 20% loss would be of no significance for practical measurements. We also confirmed that there is no difference in the melting point or the substrate efficiency of the aqueous solution of solid samples for isobutyryl and cyclohexanecarboxyl thiocholines between those stored for 10 years at 5 °C and those that were freshly prepared.
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Discussion
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Isobutyryl- and cyclohexanecarboxyl-thiocholines exhibit a moderate reactivity with serum cholinesterase preparations at pH 8.0 with a high selectivity against acetylcholinesterase and a sufficient ratio over spontaneous hydrolysis. Propionyl- and n-butyryl-thiocholines are much more susceptible to enzymatic hydrolysis than the branched alkanoyl thiocholines, as shown in Table 2
. The reactivity of nonbranched alkanoyl thiocholines is too high for accurate measurement unless the serum sample is highly diluted.
In the assay method of Dietz et al. (1), the concentration of the human serum cholinesterase is measured at pH 7.6 with propionylthiocholine as the substrate. As shown in Table 4
, the reactivity at pH 7.6 was 5% lower. At pH 7.0, it was 10% lower than that at pH 8.0. The self-hydrolysis at pH 7.6 was >50% lower than that at pH 8.0. The Dietz method carried out at the lower-than-optimum pH may be compromised by the decrease in self-hydrolysis. The ratios of the enzymatic reactivity vs self-hydrolysis of propionyl- and n-butyryl-thiocholines were among the highest (even under nonoptimum conditions). Those of isobutyrate and cyclohexane-carboxylate at pH 8 came next, as shown in Table 2
.
Isobutyryl- and cyclohexanecarboxyl-thiocholines cover concentration ranges of serum cholinesterase approximately two- to threefold broader than propionyl-thiocholine (1). In addition, they are very stable in the solid state as iodide esters and in aqueous solution, making them directly utilizable as assay reagents for at least 5 years at 5 °C. In contrast, the t0.8 time for the propionyl ester iodide solution is estimated at 2 years. Because of the moderate reactivity that does not require high dilution of the serum samples, the isobutyrate and cyclohexanecarboxylate derivatives are favored for the serum cholinesterase assay, especially under automated conditions.
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References
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Dietz AA, Rubinstein HN, Lubrano T. Colorimetric determination of serum cholinesterase and its generic variants by the propionylthiocholine-dithio-bis-(nitrobenzoic acid) procedure. Clin Chem 1973;19:1309-1313.[Web of Science][Medline]
[Order article via Infotrieve]
-
Dietz AA, Rubinstein HN. Standardization of the Ellman reaction. Clin Biochem 1972;5:136-138.[Web of Science][Medline]
[Order article via Infotrieve]
-
Ellman GL. A colorimetric method for determining low concentrations of mercaptans. Arch Biochem Biophys 1958;74:443-450.[Web of Science][Medline]
[Order article via Infotrieve]
-
Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys 1959;82:70-77.[Web of Science][Medline]
[Order article via Infotrieve]
-
Yamada M, Marui Y, Hayashi C, Takemura S. Benzoylthiocholine derivatives as substrates for pseudocholinesterase: synthesis and application. Chem Pharm Bull (Tokyo) 1987;35:1491-1496.[Medline]
[Order article via Infotrieve]
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Renshow RR, Dreisbach PF, Ziff M, Gerrn D. Thioesters of choline and -methylcholine and their physiological activity: onium compounds. J Am Chem Soc 1938;60:1765-1770.
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Garrett ER, Carper RF. Prediction of stability in pharmaceutical preparations. I. Color stability in a liquid multisulfa preparation. J Am Pharm Assoc 1955;44:515-519.
Top
Abstract
Introduction
, enzymatic reaction; 
, self-hydrolysis. Compound no. 9 (cyclohexane-carboxylate): •, enzymatic reaction; 
, self-hydrolysis.