US3275534A - Bioelectric method for the detection of anticholinester-ases - Google Patents
Bioelectric method for the detection of anticholinester-ases Download PDFInfo
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- US3275534A US3275534A US266461A US26646163A US3275534A US 3275534 A US3275534 A US 3275534A US 266461 A US266461 A US 266461A US 26646163 A US26646163 A US 26646163A US 3275534 A US3275534 A US 3275534A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
- C12Q1/46—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase involving cholinesterase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2334/00—O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/16—Phosphorus containing
- Y10T436/163333—Organic [e.g., chemical warfare agents, insecticides, etc.]
Definitions
- This invention relates to a new method for the detection of anticholinesterases. More specifically the present invention provides for the electrical monitoring of cholinesterase catalyzed reactions.
- One of the advantages of the present invention is that it provides an accurate laboratory method for the remote deterimination of micro amounts of a highly toxic agent in a manner similar to the method set forth in the patent issued to Northrop 2,757,132.
- organophosphorous compounds are highly toxic anticholinesterase agents.
- One of the more important enzymes in the higher animals is cholinesterase which is vital for the transmission of nerve impulses for muscle control. Minute amounts of anticholinesterase agents such as the G agents, which are disclosed in Patent 2,926,072, inactivate this enzyme and cause paralysis of the animals muscular system and eventual death from failure of the respiratory system.
- the thiocholine esters which can be used in this process are the acetyl, propionyl, and butyryl thiocholine iodides or the lower alkanoyl esters of ethanthiol trimethyl ammonium iodide.
- the pH of the solution in the concentration cell must be approximately neutral, i.e., in the range of pH from 68 with the preferred pH being 7.4.
- FIG. 1 illustrates the electrical circuit with the vacuum tube volt meter and the recorder connected to the solution.
- FIG. 2 shows the voltage-time curves for the enzymatic hydrolysis of butyrylthiocholine iodide by cholinesterase inhibited by; in case A, no agent; in case B, 0.36 g. per ml.; in case C, 0.50 g. per ml.; in case D, 1.0 g. per ml. of isopropyl methyl phosphonofiuoridate (hereinafter designated as GB), respectively.
- GB isopropyl methyl phosphonofiuoridate
- FIG. 3 shows the calibration plots of AE/At vs. the agent concentration in ig/ml. for four typical anti-cholinesterase agents.
- FIG. 1 the apparatus used to apply a constant low amperage current and to measure the rates of depolarization is shown.
- a -volt battery is applied across two platinum thirnble electrodes, 17 and 19 through a resist ance of a 270-kilohm resistor, 11, and a Z-megohm resistor, 15.
- the circuit is stabilized by means of a Zener diode, 13.
- a reference half cell such as a saturated calomel electrode is shown at 21.
- the solution is contained in a concentration cell, 23, which is constantly stirred and maintained at a constant temperature by means of a standard water jacket or by location in a constant temperature chamber.
- Example 1 Twenty-five ml. of a 1 10- M butyrylthiocholine iodide in 0.1 M aqueous tris(hydroxymethyl)aminomethane solution (pH 7.40) is placed in a 50-ml. beaker, and 1.0 ml. of an aqueous GB sample is added. This sample can contain 1 to 15 ,ug. of GB or the agent which is to be analyzed. The resulting solution is magnetically stirred at a temperature of 25 C., the platinum electrodes and the reference electrode are immersed into the solution and a current of 25 ,MOL is applied across the two platinum electrd-oes by means of the circuit shown in FIG. 1.
- the recorder is switched on, and the resulting potential of the platinum anode vs. the reference electrode is automatically recorded.
- 1.0 ml. of a 1.0 mg. per ml. cholinesterase solution is added to effect the enzymatic hydrolysis of the substrate.
- a method of quantitatively detecting the presence of organophosphorus compounds which comprises:
- a method of quantitatively detecting the presence of organophosphorus compounds which comprises:
- a method of quantitatively detecting the presence of isopropyl methyl phosphonofluoridate which comprises:
- a method of quantitatively detecting the presence of water soluble organophosphorus compounds which comprises:
- a method of quantitatively detecting the presence of water insoluble organophosphorus compounds which comprises:
- organophosphorus compound is 0,0-diethyl O-2-(ethylthio)-ethyl phosphorothionate.
- organophosphorus compound is diethyl-p-nitrophenyl thiophosphate.
- organophosphorus compound is S-(1,2-dicarbethoxyethyl)- 5O 0,0-dimethyldithiophosphate.
Description
Sept. 27, 1966 D. L. CANNON, JR., ET AL 3,275,534
BIOELECTRIC METHOD FOR THE DETECTION OF ANTICHOLINESTERASES Filed March 19, 1965 HIGH IMPEDENCE VTVM RECORDER 4.0 2 I15 I- 3.0 3 2 g 2.0-
5 '0 CHOLINESTERASE ADDED E VS. S.C.E.,VOLTS A E/A t,MV, SEC.
ZS PER ML OF SOLUTION INVENTORS Paul L. Gannon Jn eggs) David /V. Kramer I: PARATHION WM 24 6d 9 X MALATHION flhg' hn G.
AT ran/v5 Y3.
' the action of the cholinesterase on the substrate.
United States Patent BIOELECTRIC METHUD FOR THE DETECTKON 0F ANTlCHOLINESTER-ASES Paul L. Cannon, in, Harrisburg, Pa., and David N. Kramer, Stevenson, Md, assignors to the United States of America as represented by the Secretary of the Army Filed Mar. 19, 1963, Ser. No. 266,461 Claims. (Cl. 204-11) The invention described herein may be manufactured and used by or for the Government of the United States of America for governmental purposes without the payment to us of any royalty thereon.
This invention relates to a new method for the detection of anticholinesterases. More specifically the present invention provides for the electrical monitoring of cholinesterase catalyzed reactions.
One of the advantages of the present invention is that it provides an accurate laboratory method for the remote deterimination of micro amounts of a highly toxic agent in a manner similar to the method set forth in the patent issued to Northrop 2,757,132.
Some of the organophosphorous compounds are highly toxic anticholinesterase agents. One of the more important enzymes in the higher animals is cholinesterase which is vital for the transmission of nerve impulses for muscle control. Minute amounts of anticholinesterase agents such as the G agents, which are disclosed in Patent 2,926,072, inactivate this enzyme and cause paralysis of the animals muscular system and eventual death from failure of the respiratory system.
It is an object of this invention to detect amounts of these organo-phosphorous anticholinesterase agents in! the order of rnillionths of a gram per milliliter of solution. It is a further object of this invention to detect the above minute amounts of these anticholinesterase agents with an accuracy of about 11%.
It has been proposed (US. Patent 3,049,411) to detect these agents by means of the reaction of cholinesterase with colorless acetates of indophenols. In the absence of an agent, the indophenolester is hydrolyzed to a highly colored compound. The present method is a distinct improvement over this patented method since it gives a quantitative indication of the presence of such agents and is not interfered with by extraneous colored solutions.
The above objects are achieved in this invention by applying a direct current across two platinum electrodes immersed in a buffered approximately neutral aqueous solution of the anticholinesterase compound and a thiocholine ester which also has a half cell or a reference electrode therein. The potential between the positive electrode and the reference electrode is then recorded as a function of time. Cholinesterase is then added to the solution and the time of addition is noted. There will be a change in the recorded potential as a function of time. This potential change per unit of time is the rate of depolarization of the positive electrode caused by This change in potential per unit of time or voltage-time is linearly related to the concentration of the enzyme. In the presence of anticholinesterase the activity of the enzyme is decreased as a function of inhibitor concentration. It has been found that the ratio is inversely proportional to the concentration of the inhibitor. Thus, a relatively large ratio indicates a relatively small amount of inhibitor present in the solution.
The thiocholine esters which can be used in this process are the acetyl, propionyl, and butyryl thiocholine iodides or the lower alkanoyl esters of ethanthiol trimethyl ammonium iodide. The pH of the solution in the concentration cell must be approximately neutral, i.e., in the range of pH from 68 with the preferred pH being 7.4.
In the drawing, FIG. 1 illustrates the electrical circuit with the vacuum tube volt meter and the recorder connected to the solution.
FIG. 2 shows the voltage-time curves for the enzymatic hydrolysis of butyrylthiocholine iodide by cholinesterase inhibited by; in case A, no agent; in case B, 0.36 g. per ml.; in case C, 0.50 g. per ml.; in case D, 1.0 g. per ml. of isopropyl methyl phosphonofiuoridate (hereinafter designated as GB), respectively.
FIG. 3 shows the calibration plots of AE/At vs. the agent concentration in ig/ml. for four typical anti-cholinesterase agents.
In FIG. 1, the apparatus used to apply a constant low amperage current and to measure the rates of depolarization is shown. A -volt battery is applied across two platinum thirnble electrodes, 17 and 19 through a resist ance of a 270-kilohm resistor, 11, and a Z-megohm resistor, 15. The circuit is stabilized by means of a Zener diode, 13. A reference half cell such as a saturated calomel electrode is shown at 21. The solution is contained in a concentration cell, 23, which is constantly stirred and maintained at a constant temperature by means of a standard water jacket or by location in a constant temperature chamber.
Example 1 Twenty-five ml. of a 1 10- M butyrylthiocholine iodide in 0.1 M aqueous tris(hydroxymethyl)aminomethane solution (pH 7.40) is placed in a 50-ml. beaker, and 1.0 ml. of an aqueous GB sample is added. This sample can contain 1 to 15 ,ug. of GB or the agent which is to be analyzed. The resulting solution is magnetically stirred at a temperature of 25 C., the platinum electrodes and the reference electrode are immersed into the solution and a current of 25 ,MOL is applied across the two platinum electrd-oes by means of the circuit shown in FIG. 1. The recorder is switched on, and the resulting potential of the platinum anode vs. the reference electrode is automatically recorded. At zero time, 1.0 ml. of a 1.0 mg. per ml. cholinesterase solution is added to effect the enzymatic hydrolysis of the substrate.
Under operating conditions without the addition of GB, addition of cholinesterase to the magnetically stirred solution of the thiocholine ester caused a rapid hydrolysis of the substrate to thiocholine and butyric acid. The thiocholine produced causes a depolarization of the electrodes and a reduction in the potential of the system. This is illustrated by the aforementioned curves of FIG. 2. By means of predetermined calibration plots of the type set forth in FIG. 3 one can obtain the amount of agent present in the original sample. In a manner similar to Blaedel and Hicks (Analytical Chemistry, vol. 34, No. 3, March 1962, pages 388-394), one can calibrate the recorder to give a direct readout in micrograms of the agent. The temperature must be closely controlled to give the optimum results as far as accuracy is concerned.
We have found that the above procedure works well with GB and Systox (0,0-diethyl-O-[Z-(ethylthio)-ethyl] phosphorothionate). However, for compounds such as parathion (diethyl-p-nitrophenyl thiophosphate) and malathion (S-[1,2-dicarbethoxyethyl]-0,0 dimethyl-dithiophosphate), we have found that these compounds must be preincubated wtih the enzyme for about ten minutes to give sensitive and accurate results. Likewise, with GB or Systox a short incubating period of 3-5 minutes made the process more sensitive to smaller amounts of these agents.
We have also found that with parathion and malathion and other water insoluble agents a cosolvent had to be used to get these agents into the solution We have found that an aliphatic oxygenerated solvent such as glycerol, ethylene glycol, diethylene glycol, diethylene glycol monoethyl ether, ethylene glycol monomethyl ether were excellent cosolvents to use in our process Best results were obtained with the use of ethylene glycol monomethyl ether. It was found that up to about 1% of the aliphatic oxygenated solvent could be tolerated in the final reaction mixture without giving erratic results.
Our method can be applied to other agents or inhibitors such as GA, GD, paraoxon, malaoxon, and DFP with equally good results.
We claim:
1. A method of quantitatively detecting the presence of organophosphorus compounds which comprises:
(a) applying a low direct current across a negative and a positive electrode immersed in a stirred buffered aqueous solution of said compound and a lower alkanoyl thiocholine ester having a reference electrode therein,
(b) recording the potential between said positive electrode and said reference electrode,
(c) adding cholinesterase to said solution,
(d) recording the ratio of the change in potential between said positive electrode and said reference electrode with respect to the change in time from the addition of said cholinesterase, said ratio being inversely proportional to the concentration of said compound in said buffered solution.
2. A method of quantitatively detecting the presence of organophosphorus compounds which comprises:
(a) applying a weak direct current across a negative and a positive electrode immersed in a neutral aqueous solution of said compound and a lower alkanoyl thiocholine ester which has a reference electrode therein,
(b) recording the potential between said positive electrode and said reference electrode,
() adding cholinesterase to said solution,
((1) recording the ratio of the change in potential between said positive electrode and said reference electrode with respect to the change in time from the addition of said cholinesterase, said ratio being inversely proportional to the concentration of said compound in said neutral solution.
3. A method of quantitatively detecting the presence of isopropyl methyl phosphonofluoridate which comprises:
(a) applying a direct current of low amperage across a negative and a positive electrode immersed in a stirred buffered approximately neutral aqueous solution of said phosphonofluoridate and a lower alkanoyl thiocholine ester having a reference electrode therein,
(b) recording the potential between the positive electrode and said reference electrode,
(c) adding cholinesterase to said solution,
' ((1) recording the ratio of the change in potential between said positive electrode and said reference electrode with respect to the change in time from the addition of said cholinesterase, said ratio being inversely proportional to the concentration of said phosphonofluoridate in said buffered solution.
4. A method of quantitatively detecting the presence of water soluble organophosphorus compounds which comprises:
(a) applying a weak direct current across a negative and a positive electrode immersed in a stirred aqueous addition of said cholinesterase, said ratio being inversely proportional to the concentration of said organophosphorus compound in said solution. 5. A method as set forth in claim 4 in which the organophosphorus compound is isopropyl methyl phosphonofluoridate.
6. A method of quantitatively detecting the presence of water insoluble organophosphorus compounds which comprises:
(a) applying a weak direct current across a negative and a positive electrode immersed in a stirred aqueous solution containing said compound, tris(hydroxymethyl)aminomethane, butyrylthiocholine iodide, and up to about 1% of the total solution of a solvent selected from the group consisting of glycerol, ethylene glycol, diethylene glycol, diethylene glycol monoethyl ether, and ethylene glycol monomethyl ether, said solution being adjusted to a pH range of 7.0 to 8.0 and having a reference electrode therein,
(b) recording the potential between said positive electrode and said reference electrode,
(c) adding cholinesterase to said solution,
((1) recording the ratio of the change in potential between said positive electrode and said reference electrode with respect to the change in time from the addition of said cholinesterase, said ratio being inversely proportional to the concentration of said organophosphorus compound in said solution.
7. A method as set forth in claim 6 in which the solvent is ethylene glycol monomethyl ether.
8. A method as set forth in claim 7 in which the organophosphorus compound is 0,0-diethyl O-2-(ethylthio)-ethyl phosphorothionate.
9. A method as set forth in claim 7 in which the organophosphorus compound is diethyl-p-nitrophenyl thiophosphate.
10. A method as set forth in claim 7 in which the organophosphorus compound is S-(1,2-dicarbethoxyethyl)- 5O 0,0-dimethyldithiophosphate.
References Cited by the Examiner UNITED STATES PATENTS 2,757,132 7/1956 Northrop 204195 2,926,072 2/1960 Kramer et al. 23232 3,049,411 8/1962 Gelman et al 23232 OTHER REFERENCES Blaedel et al.: Analytical Chemistry, vol. 34, No. 3, March 1962, pp. 388394.
JOHN H. MACK, Primary Examiner.
T. H. TUNG, Assistant Examiner.
Claims (1)
1. A METHOD OF QUANTITATIVELY DETECTING THE PRESENCE OF ORGANOPHOSPHROUS COMPOUND WHICH COMPRISES: (A) APPLYING A LOW DIRECT CURRENT ACROSS A NEGATIVE AND A POSITIVE ELECTRODE IMMERSED IN A STIRRED BUFFERED AQUEOUS SOLUTION OF SAID COMPOUND AND A LOWER ALKANOYL THIOCHOLINE ESTER HAVING A REFERENCE ELECTRODE THEREIN, (B) RECORDING THE POTENTIAL BETWEEN SAID POSITIVE ELECTRODE AND SAID REFERENCE ELECTRODE, (C) ADDING CHLORINESTERASE TO SAID SOLUTION, (D) RECORDING THE RATIO OF THE CHANGE IN POTENTIAL BETWEEN SAID POSITIVE ELECTRODE AND SAID REFERENCE ELECTRODE WITH RESPECT TO THE CHANGE IN TIME FROM THE ADDITION OF SAID CHLORINESTERASE, SAID RATIO INVERSELY PROPORTIONAL TO THE CONCENTRATION OF SAID COMPOUND IN SAID BUFFERED SOLUTION.
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| Application Number | Priority Date | Filing Date | Title |
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| US266461A US3275534A (en) | 1963-03-19 | 1963-03-19 | Bioelectric method for the detection of anticholinester-ases |
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| US266461A US3275534A (en) | 1963-03-19 | 1963-03-19 | Bioelectric method for the detection of anticholinester-ases |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3451901A (en) * | 1966-10-11 | 1969-06-24 | Gulton Ind Inc | Method of detecting nerve gases |
| US3506544A (en) * | 1964-10-09 | 1970-04-14 | Magna Corp | Method of determining microbial populations,enzyme activities,and substrate concentrations by electrochemical analysis |
| US3539455A (en) * | 1965-10-08 | 1970-11-10 | Leland C Clark Jr | Membrane polarographic electrode system and method with electrochemical compensation |
| US3659193A (en) * | 1969-12-24 | 1972-04-25 | Comp Generale Electricite | Apparatus including initial electrode charge maintaining means for measuring the concentration of an electrolyte |
| US4072576A (en) * | 1975-10-06 | 1978-02-07 | Ab Kabi | Method for studying enzymatic and other biochemical reactions |
| US4891104A (en) * | 1987-04-24 | 1990-01-02 | Smithkline Diagnostics, Inc. | Enzymatic electrode and electrode module and method of use |
| US4935106A (en) * | 1985-11-15 | 1990-06-19 | Smithkline Diagnostics, Inc. | Ion selective/enzymatic electrode medical analyzer device and method of use |
| US4946651A (en) * | 1985-11-15 | 1990-08-07 | Smithkline Diagnostics, Inc. | Sample holder for a body fluid analyzer |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US2757132A (en) * | 1944-12-06 | 1956-07-31 | John H Northrop | Method of electrochemical analysis |
| US2926072A (en) * | 1959-04-06 | 1960-02-23 | David N Kramer | Detection of g agents |
| US3049411A (en) * | 1959-02-11 | 1962-08-14 | Gelman Charles | Enzymatic method for detection of anticholinesterases |
-
1963
- 1963-03-19 US US266461A patent/US3275534A/en not_active Expired - Lifetime
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US2757132A (en) * | 1944-12-06 | 1956-07-31 | John H Northrop | Method of electrochemical analysis |
| US3049411A (en) * | 1959-02-11 | 1962-08-14 | Gelman Charles | Enzymatic method for detection of anticholinesterases |
| US2926072A (en) * | 1959-04-06 | 1960-02-23 | David N Kramer | Detection of g agents |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3506544A (en) * | 1964-10-09 | 1970-04-14 | Magna Corp | Method of determining microbial populations,enzyme activities,and substrate concentrations by electrochemical analysis |
| US3539455A (en) * | 1965-10-08 | 1970-11-10 | Leland C Clark Jr | Membrane polarographic electrode system and method with electrochemical compensation |
| US3451901A (en) * | 1966-10-11 | 1969-06-24 | Gulton Ind Inc | Method of detecting nerve gases |
| US3659193A (en) * | 1969-12-24 | 1972-04-25 | Comp Generale Electricite | Apparatus including initial electrode charge maintaining means for measuring the concentration of an electrolyte |
| US4072576A (en) * | 1975-10-06 | 1978-02-07 | Ab Kabi | Method for studying enzymatic and other biochemical reactions |
| US4935106A (en) * | 1985-11-15 | 1990-06-19 | Smithkline Diagnostics, Inc. | Ion selective/enzymatic electrode medical analyzer device and method of use |
| US4946651A (en) * | 1985-11-15 | 1990-08-07 | Smithkline Diagnostics, Inc. | Sample holder for a body fluid analyzer |
| US4891104A (en) * | 1987-04-24 | 1990-01-02 | Smithkline Diagnostics, Inc. | Enzymatic electrode and electrode module and method of use |
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