CA1339794C - Enzymatic determination of theophylline - Google Patents

Abstract

The present invention provides a new methodology and test composition for determining the presence of theophylline in test samples. The methodology employs enzymes that utilize or recognize theophylline as a substrate to measure the concentration thereof in samples, including body fluids. This new approach utilizes enzymes as opposed to traditional methods which use antibodies for the recognition of theophylline. The enzymatic approach to theophylline determination is quick, simple and convenient and allows test systems to be made in liquid as well as in dry-chemistry formats. Various protocols, systems or methodologies may be used for assaying and relating the results to the amount of theophylline present. Methods for obtaining theophylline utilizing or recognizing enzymes are also described.

Description

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ENZYMATIC DETE~MTNA~ION OF THEOPHYLLINE

RAC~G~OUND OF THE lNV~NllON

Theophylline is a bronchodilator and respiratory stimulant used in the treatment of patients with asthmatic and allergic conditions. It is also used in the treatment of congestive heart failure and acute pulmonary edema.
10 Benefits, as well as risks, from using this drug directly relate to its serum concentration. In order for the drug to be effective, a concentration of theophylline of about 10-20 mg/L level needs to be maintained in the blood. Theophylline levels of less than 10 mg/L are therapeutically ineffective 15 and levels of more than 20 mg/L may be toxic to the patient.
This toxicity may result in brain damage and death.
Because the therapeutic advantage of the drug lies only within a narrow range of concentrations and because there is a large interpatient difference in drug elimination due to 20 physiological differences, as well as diet or other prescribed drugs, it is important to monitor patients using this drug.

Theophylline has been measured by gas chromatography by 25 Shah, J Pharm Sci 63(8), 1283 (1974) and by a combination of gas chromatography and mass-selective detector by Desage, et al., J Chromat 336(2), 285 (1984). It has been measured 2 1~3~79Ll by high-pressure liquid chromatography by Thompson, et al., J Lab Clin Med 84(4), 584 (1974) and by Naish, et al., Ann Clin Biochem 16(5), 254 (1979). Schack, et al., J Pharm 97, 283 (1949) used an ultraviolet spectrophotometric method for 5 the determination of theophylline. However, because these methods need cumbersome extractions, most clinical approaches today for the determination of theophylline use immunological methods which depend on antibodies for recognizing theophylline in the sample being tested. Such 10 systems involve competitive protein binding where the antibody is the specific binding protein. After the reaction with antibodies takes place, the determination of theophylline varies depending on the particular assay, that is, the assay readout may be turbidimetric, nephelometric, 15 radioactive or colorimetric depending on whether turbidity, radioactivity or color is produced. Examples of these systems are reported by Painter, et al., J Clin Lab Autom 3(3), 179 (1983), Samoszuk, et al., Therp Drug Mon 5(1), 113 (1983), Opheim, et al., Clin Chem 30(11), 1870 20 (1984), Boeckx and Munson, Therp Drug Mon 7(1), 95 (1985), Cook, et al., Res Comm in Chem Path & Pharm 13(3), 497 (1976), and Landesman, et al., Clin Chem 29, 1238 (1983).

Immunological systems have also been reported by Li et 25 al., Clin Chem 27(1), 22 (1981), Davis and Marks, Ther Drug Mon 5(4), 479 (1983), Chang, et al., Clin Chem 28(2), 361 (1982), Hinds, et al., Clin Chem 30(7), 1174 (1984), Jolley, 3 13~97~

et al., Clin Chem 27, 1575 (1983), Morris, et al., Anal Chem 53, 658 (1981), and Tyhach, et al., Clin Chem 27, 1499 (1981).

In addition, enzymes have been used in an enzyme amplification assay, U. S. Patent No. 3,817,837. In this disclosure, enzymes are chemically bound to ligands and these enzyme-bound-ligand combine with receptors. The ligand may be a drug. The specific reaction of the ligand 10 with the receptors gives the specificity to the reaction while the enzyme activity is utilized as a marker for the reaction. Therefore, the enzymes used have no enzymatic recognition of the drug. The use of these approaches are totally different to the presently described methodology 15 which uses enzymes instead of antibodies or ligands for the recognition of theophylline.

It has been also reported in European Patent application number EP86300226.7, Jan 15, 1986, as well as in Clin Chem 20 25, 1370 (1979) that the activity of alkaline phosphatase, which acts by cleaving phosphate groups from a substrate, can be inhibited by theophylline. In this approach, the enzymatic reaction of alkaline phosphatase continues to be that of cleaving the phosphate bonds but this action is 25 interfered with by the presence of theophylline. Again, in this disclosure, the theophylline test produced is one in which theophylline is not enzymatically utilized or changed 4 ~ 3~ 79'i by the enzymatic reaction. By contrast, the present invention teaches that test systems can be produced, using enzymes which utilize or recognize theophylline and use it as a substrate for the quantitation of theophylline in 5 fluids.

No theophylline utilizing enzyme has previously been available or described in the prior art. Moreover, since theophylline is a xanthine derivative, commercially 10 available xanthine oxidases and xanthine dehydrogenases and related enzymes were tried for their ability to utilize theophylline. These attempts were unsuccesful. Althought in humans theophylline is known to be metabolized primarily to 1,3 dimethyl uric acid and also to 1 methyl uric acid and 15 3 methyl xanthine (Cornish, H.H. and Christman, A.A., J Biol Chem 228, 31S (1957), no theophylline utilizing enzymes have been isolated or shown. However, recently three theophylline utilizing or recognizing enzymes have become available from GDS Technology Inc., P.O. Box 473, Elkhart, 20 Indiana 46515. These enzymes were identified as 1. Enzyme T-090, 2. Enzyme T-060 and 3. Enzyme T-040.

These enzymes were used herein for the first time to develop and produce an enzymatic test for the determination 25 of theophylline in samples such as body fluids, food extracts and medicinal compounds and compositions. The 1~$~79 1 tests that resulted from this enzymatic approach are rapid and convenient to perform. The advantages of the enzymatic approaches are 1) unitized reagent or test composition capability, 2) one step addition of sample to reagent or 5 test composition, 3) a liquid system can be made to perform with instrument readout devices, and 4) the reagent or test composition can easily be incorporated into a solid-phase matrix.

BRIEF DESCRIPTION OF THE DRAWING

The figure shown in the drawing depicts absorbance curves for theophylline utilizing enzyme before and after contact with theophylline as further described in Example 3 15 and Example 13.

DESCRIPTION OF THE PREFERRED ENBODIMENTS

The present invention basically consists of a system 20 for the determination of theophylline by means of a theophylline utilizing or recognizing enzyme (or substance containing such enzyme) and optionally including the use of electron carriers. The determination is accomplished by measurement of a signal produced by the reaction of the 25 enzyme with any theophylline present in the sample and converting or correlating the amount of signal generated to the amount of theophylline present in the sample. As used 6 ~ 9~i - herein, the term theophylline utilizing enzyme means an enzyme or substance containing an enzyme which either recognizes or utilizes theophylline to produce a signal which by itself or in conjunction with other reagents or 5 means can be measured using visual, instrumental or other state of the art methodologies.

The principle of the test method is based on the utilization of theophylline by an enzyme. That is, the 10 enzyme recognizes theophylline as a substrate and changes it to a different compound or product. Schematically, the system can be described as follows:

enzyme theophylline > Product As noted above, the methods disclosed and claimed herein involve detecting any signal produced by contact of theophylline with the theophylline utilizing enzyme which 20 indicates that a specific enzymatic reaction has taken place. The signals produced are measured in a variety of ways as described herein. This process occurs in the presence or absence of added electron carriers, either electron acceptors or electron donors. Examples of electron 25 acceptors are oxygen, nicotinamide adenine dinucleotide (NAD), dichlorophenolindophenol (DCPIP), phenazine methosulfate (PMS), methylene blue, cytochromes, 7 3 ~

ferricyanide J etc. Examples of electron donors are reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), reduced flavin adenine dinucleotide (FADH), etc.

The process of the present invention is otherwise carried out in the usual manner for enzymatic determinations, including optimized pH and temperature ranges in which theophylline utilizing enzymes are active.
10 Moreover, such determinations are preferably carried out in a buffered environment.

In principle, all variants of the theophylline utilizing enzyme which can be used for the enzymatic 15 determination of theophylline fall within the scope of the present invention.

The following represents various test systems wherein theophylline in a test sample can be determined using the 20 methodology disclosed in the present specification:

1) Measuring the decrease of theophylline concentration in the system. In this system, the disappearance of the substrate theophylline can be measured 25 by determining the decrease of absorbance at a wavelength of 272 nm, where theophylline has m~Ximum absorbance. This is shown in Example 1 by using the theophylline enzyme T-060 ~ ~3~79~

and in Example 2 by using the theophylline enzyme T-090.
This change can also be detected by measuring the reflectance, which is the inverse of the absorbance, or 2) Measuring a change of the oxidation-reduction potential of the system in either of two ways, a) measuring a change in the enzyme or enzyme complex system itself as shown in Example 3, wherein the enzyme characteristics change as can be seen by the changes that occur at 10 wavelength 410 nm and 550 nm or measuring changes electrochemically, or b) by a change of an electron carrier added to the system such as, for example, the reduction of ferricyanide to ferrocyanide. Example 4 shows the absorbance change at 410 nm that occurs when ferricyanide 15 changes to ferrocyanide in the presence of the theophylline enzyme T-090 as the reaction takes place. This change can also be measured by spectrophotometric or electrochemical methods, or 3) Measuring the appearance of any product associated 20 with the enzymatic reaction of theophylline. The products produced in this reaction vary with the particular enzyme involved. For example, a theophylline enzyme can react by oxidizing, dehydrogenating or demethylating theophylline.
Example 5 shows the appearance and measurement of 25 formaldehyde when the theophylline enzyme T-040 was used.
Example 6 shows the appearance and measurement of hydrogen peroxide when the theophylline enzyme T-060 was used.

9 ~ 9 ~

The product formation can be further illustrated by the following reactions or processes:

i) When the enzyme is capable of oxidizing theophylline and converts theophylline (1,3 dimethyl xanthine) to 1,3 dimethyl uric acid utilizing oxygen as an electron acceptor. This is diagrammatically shown as follows:
~0 enzyme theophylline + 02 + H20 > 1,3 dimethyl uric acid + hydrogen peroxide In this system, the hydrogen peroxide thus produced can be determined titrimetrically, potentiometrically, polarographically, colorimetrically as well as enzymatically. The enzymatic methods of measuring hydrogen peroxide are preferred since they are not only specific 20 and reliable, but can also be combined in a simple way with the hydrogen peroxide of the above reaction to produce color. For example, a peroxidase method is described in Anal Biochem 105, 389, (1980). Using the theophylline enzyme T-60, Example 6 demonstrates that the hydrogen 25 peroxide formation is proportional to the concentration of theophylline in the sample. Alternatively, the rate of oxygen consumption in accordance with the above general lo ~ ~3~9~

- equation can be measured, for instance, by gas chromatography and depolarization methods. The depolarization method utilizing oxygen electrodes (available from Yellow Spring Instruments, Yellow Spring, 5 OH) is well known and also described in US Patent No.
3,838,011 and in J Appl Physiol 18, 1247 (1963).

ii) When the enzyme uses an acceptor other than oxygen such as ferricyanide, NAD, cytochromes, etc. to oxidize 10 theophylline and produces 1,3 dimethyl uric acid and a reduced acceptor in the following manner:
enzyme theophylline + oxidized acceptor >
1,3 dimethyl uric acid + reduced acceptor In such a system, the product 1,3 dimethyl uric acid can be measured or determined by several methods.
Example 7 describes one in which the absorbance at 292 nm is determined using the theophylline enzyme T-090. At such a 20 wavelength 1,3 dimethy uric acid absorbs optimally. Again, the increase in absorbance was found to be proportionate to the theophylline concentration in the sample.

Moreover, the concentration of theophylline in the sample can be determined using this reaction scheme by measuring the oxidation/reduction state of the electron 1 ~ h ~ 'f 7 9 '1 - acceptors used. Examples 4 and 8 show a test method where ferricyanide is used as an acceptor and is reduced to ferrocyanide by the theophylline enzyme T-090. In Example 4 the decrease of ferricyanide is measured by measuring the 5 decrease in absorbance at 410 nm wavelength as ferricyanide mAx;m~lly absorbs at 410 nm and ferrocyanide has no absorption at that wavelength. It was again found that the decrease in absorbance was proportionate to the concentration of theophylline in the sample. In Example 8, 10 another way of measuring ferrocyanide is shown. In this scheme, the formation of ferrocyanide is measured chemically by using 4,7 diphenyl-1,10 phenanthroline sulfonate by the method described by Avon, M. and Shavit N., Analy Biochem 6, 549 (1963). The Avon method produced color which was 15 measured at 535 nm. The color thus produced was porportionate to the concentration of theophylline in the sample.

Example 9 illustrates the use of another electron 20 acceptor, ferricytochrome c. In this example ferricytochrome c is reduced to ferrocytochrome c. The appearance of ferrocytochrome c is measured by measuring the increase in absorbance at 550 nm wavelength in the presence of the theophylline enzyme T-090. Again, the change in 25 absorbance at 550 nm wavelength was found to be proportionate to the concentration of theophylline in the sample.

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Similarly, one can use other known electron acceptors, such as nicotinamide adenine dinucleotide (NAD), 2,6-dichlorophenolindophenol (DCPIP), phenazine methosulfate 5 (PMS), etc.

In all examples shown, one can also measure the change in reflectance as reflectance is inversely proportionate to absorbance.

The measurement of the change produced by the transferring of electrons in the above reaction is by no means limited to the spectrophotometric or reflectance methods. It is well known to use potentiometric, 15 fluorescent or electrochemical methods to measure the transfer or change of electrons in oxidation-reduction reactions. For example, Reed and Hawkredge have shown an electron transfer reaction of cytochrome c at silver elctrodes in Anal Chem 59, 2334 (1987) which can be used 20 with this invention to measure the change of cytochrome c that occurs. Also, ferrocene or ferrocene derivatives have been used as electron acceptors for electrochemical methods as reported in the US Patent No. 4,545,382. These acceptors can also be used in the present enzymatic theophylline 25 measurement and the change measured electrochemically.
Also, the change of ferricyanide to ferrocyanide can be determined by measuring the change in current using platinum 1 3 g a ~ 7 9 electodes as has been established and reported in Anal Chem 36, 343 (1964).

iii) when the enzyme is capable of demethylating 5 theophylline by cleaving either one or both methyl groups.
When one methyl group is cleaved, it produces either l-methyl xanthine or 3-methyl xanthine along with 1 mole of formaldehyde. When both methyl groups are cleaved, it produces xanthine and 2 moles of formaldehyde. The reaction 10 is shown below:

enzyme theophylline + NADPH + 02 >
1 methyl xanthine + formaldehyde +NADP
and/or 3 methyl xanthine + formaldehyde + NADP
and/or xanthine + formaldehyde + NADP

In the above reaction, NADPH is shown as electron donor.
However, there are other electron donors, such as NADH or FADH, which can also be used. The formaldehyde reaction product can be measured by customary and already known chemical, enzymatic, or electrochemical methods. Example 5 25 shows one method of measuring formaldehyde when the 14 ~, 3 .~ 7 ~ ~

~ theophylline enzyme T-040 was used. As indicated in the Example, the formaldehyde thus produced was proportionate to the concentration of theophylline in the sample.

The reaction products xanthine or methyl xanthine can also be measured as indicated below:

xanthine oxidase xanthine (or methyl xanthine) + 02 uric acid (or methyl uric acid) + H202 The hydrogen peroxide (H202) formed can be measured by various methods as mentioned earlier, while uric acid or 15 methyl uric acid can be measured, for example, by determining the increase in absorbance at 292 nm wavelength or colorimetrically as described in Clin Chem 26, 227 (1980).

The decrease of NADPH or NADH can also be measured spectrophotometrically or fluorometrically by customary methods as described in Anal Biochem 12, 357 (1965). The decrease of FADH can be measured by measuring the decrease at 450 nm wavelength as shown in J Biol Chem 246, 2371 25 (1971).

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iv) other products produced by the enzymatic recognition of theophylline and measured by customary methods such as spectrophotometric, electrochemical or chromatographic or alternatively by a decrease in 5 theophylline concentration as in Example 1.

In addition to the liquid test reactions disclosed herein, the test reagent compositions and devices of the present invention can contain state of the art additives and 10 adjuvants which are advantageous to the reaction, such as, for example, buffers, suspending agents, thickening agents, color enhancers, surfactants, and so forth.

Moreover, in addition to the liquid reagent test 15 systems described previously, the compositions of the present invention can advantageously be incorporated into solid carriers or matrices. Such a configuration or format is referred to in the art as dry-chemistry or solid state test device formats. The most common matrix is paper;
20 however, other bibulous materials such as polymers, clays, gels and so forth may be utilized. Basically the reagent composition is incorporated or impregnated into the matrix and dried. In use, the device is either dipped into or contacted with the sample being tested. The signal 25 generated in the device by the reaction of theophylline with the test composition containing inter lia the theophylline utilizing enzyme can then be detected and quantified using 7 ~

state of the art techniques, such as, for example, visual comparison to a color chart, reflectance spectrophotometry, and so forth. Example 10 shows the device resulting from impregnating a filter paper with the theophylline enzyme 5 T-O90 and cytochrome c. The change in color with increasing concentration of theophylline can be read semi-quantitatively by visual inspection or quantitatively by existing reflectance measuring instruments.
Alternatively, the change in electron transfer in a solid 10 matrix can be measured electrochemically.

In summary, all the examples mentioned above and described below show that this enzymatic approach for the 15 determination of theophylline allows the production of an easy and convenient test format not only in a liquid system but also in a solid matrix test device.

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EXAMPLF~

The following examples are intended for illustration of the present invention and are not intended to limit the scope thereof.

In all the examples given herein, the enzyme activity 10 is defined as 1 ~mole of theophylline utilized per minute at 30~ C. temperature unless specified otherwise.

F~XAMPT.~ 1 The assay mixture contained 0.05 M potassium phosphate buffer pH 7.0 and 0.4 u/ml of the theophylline enzyme T-060. To 2 ml assay mixture, 100 ~l of a sample containing theophylline at several concentrations was added in separate cuvettes. The reaction was carried out in a Gilford 20 spectrophotometer with 10 mm light path cuvette at 30~ C. A
decrease in optical density at 272 nm was observed after 30 minutes which was proportionate to the theophylline concentration in the sample.

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Theophylline concentration Decrease in OD at 272 nm 40 mg/L .067 20 mg/L .033 10 mg/L .017 ~mple 2 The assay mixture contained 50 ~moles/ml potassium phosphate buffer at pH 7.5 and 1.8 u/ml of the theophylline enzyme T-0~0 and 25 nmoles/ml of cytochrome c. To 0.5 ml assay mixture, 25 ~l of a sample containing theophylline at 15 the following concentrations were added in separate cuvettes. The reaction was carried out in a Gilford spectrophotometer with 10 mm light path cuvette at 30~ C.
After 20 minutes, the decrease in optical density at 272 nm was recorded which was proportionate to the theophylline 20 concentration in the sample.

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Theophylline Concentration Decrease in OD at 272 nm 40 mg/L 0.065 20 mg/L 0.031 10 mg/L 0.015 ~x~m In this example, the theophylline enzyme T-090 was used. As shown in the Figure, Curve A describes the absorbance spectra of the theophylline enzyme T-090 at a concentration of 1.8 u/ml in 0.05 M potassium phosphate 15 buffer at pH 7.5. Curve B shows the absorbance spectra of the same enzyme in the presence of theophylline at the concentration of 2 mg/L under the same conditions.

As can be seen, theophylline caused the increase in 20 absorbance at 417.5 and 550 nm wavelength. These changes in absorptions are used as a basis for the determination of theophylline concentration in a sample.

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~;3mpl ~ 4 In this example, the theophylline enzyme T-090 was used with potassium ferricyanide as an acceptor. The 5 potassium ferricyanide changed to ferrocyanide in the presence of the enzyme when theophylline was added. The formation of ferrocyanide can be measured by measuring the decrease in optical density at 410 nm.

The assay mixture contained 50 ~moles/ml of potassium phosphate buffer, 1.8 u/ml of theophylline enzyme, and 1.43 ~moles/ml of potassium ferricyanide. To 0.35 ml assay mixture, 50 ~l of a sample containing theophylline at the following concentrations was added in separate cuvettes.
15 The reaction was carried out in a Gilford spectrophotometer with 10 mm light path cuvette at 30~ C. After 30 minutes, the decrease in optical density at 410 nm wavelength was measured which was proportionate to the concentration of theophylline in the sample.

2 ~ 7 -3 ~

Theophylline Concentration Decrease in OD at 410 nm 40 mg/L 0.141 30 mg/L 0.109 20 mg/L 0.076 10 mg/L 0.047 ~x~mpl e In this example, the theophylline enzyme T-040 was used. In the presence of NADPH or NADH, this enzyme demethylated theophylline and produced xanthine and/or 1 and/or 3 methyl xanthine and formaldehyde. The formaldehyde 15 was measured by a known chemical method as reported by Nash, Biochem J 55, 416-421 (1953). The formaldehyde production was proportionate to the concentration of theophylline in the sample.

The assay mixture contained 50 ~moles/ml of Tris-HCL
buffer pH 8.0, 1 ~mole/ml of NADPH and 10 nmoles/ml of semicarbazide. To 2.0 ml of assay mixture in a test tube, 2.5 units of the theophylline enzyme T-040 was added. After the reaction mixture was shaken at 30~ C for 15 minutes, the 25 reaction was stopped by adding 0.6 ml of 20% zinc sulfate, 0.66 ml of saturated barium hydroxide and allowing to stand 10 minutes at room temperature. The tubes were centrifuged at 8,000 g for 10 minutes. To 1.0 ml of supernatant, the following additions were made; 0.4 ml of Nash reagent (150 g ammonium acetate and 2 ml acetyl acetone in 500 ml of deionized water) and the tubes incubated at 60~C in a water 5 bath for 30 minutes. Absorbance was immediately read at 415 nm wavelength.

Theophylline Concentration Decrease in OD at 415 nm 180 mg/L 1.481 40 mg/L 0.269 20 mg/L 0.140 10 mg/L 0.075 15 ~x~mrl e 6 In this example, the theophylline enzyme T-060 was used which produced 1,3 dimethyl uric acid and hydrogen peroxide in the presence of theophylline. The hydrogen 20 peroxide was thus measured by a known modified Trinder's method as described by Fossati et al., Clin Chem 26, 227 (1980).

The assay mixture contained 50 ~moles/ml potassium 25 phosphate, pH 7.5, 5 ~moles/ml of 3,5-dichloro-2-hydroxy benzene sulfonate hydrochloride (DHBS), 1 ~mole/ml 4-aminoantipyrine, 5.0 u/ml of horseradish peroxidase and 23 ~ Ji ~ ~3 Li 0.7 u/ml of the theophylline enzyme T-060. To 0.7 ml assay mixture, 50 ~1 of a sample cont~;n;ng theophylline at the following concentrations was added in separate cuvettes.
The reaction was carried out in a Gilford spectrophotometer 5 with 10 mm light path cuvette at 37~ C. After 20 minutes, the increase in optical density at 510 nm was measured.

Theophylline Concentration Increase in OD at 510 nm 40 mg/L 0.172 20 mg/L 0.089 10 mg/L 0.045 ~ 3 3 ~
~x~mE)l e 7 In this example, the theophylline enzyme T-090 was 5 used with cytochrome c. In the presence of theophylline the reaction produced 1,3 dimethyl uric acid. This product was measured at 292 nm which is the wavelength of maximum absorbance for 1,3 dimethyl uric acid.

The assay mixture contained 50 ~moles/ml potassium phosphate buffer at pH 7 . 5 , 1.8 u/ml of the theophylline enzyme T-090 and 25 nmoles/ml of cytochrome c. To 0.5 ml assay mixture, 25 ~l of a sample containing theophylline at the following concentrations was added in separate cuvettes.
15 The reaction was carried out in a Gilford spectrophotometer with 10 mm light path cuvette at 30~ C. After 20 minutes, the increase in optical density at 292 nm was recorded which was proportionate to the theophylline concentration in the sample.

Theophylline Concentration Increase in OD at 292 nm 40 mg/L 0.162 30 mg/L 0.125 20 mg/L 0.087 10 mg/L 0.036 ~x~mE'l ~ ~

In this example, the theophylline enzyme T-090 was used with potassium ferricyanide as an acceptor which produces potassium ferrocyanide in the presence of theophylline. The ferrocyanide thus produced is measured chemically by using 4,7 diphenyl-1,10 phenanthroline 15 sulfonate as described by Avon and Shavit in Anal Biochem 6, 549 (1963).

The assay mixture contained 50 ~moles/ml of potassium phosphate buffer at pH 7.5, 1.8 u/ml of the theophylline 20 enzyme T-090, and 1.43 ~moles/ml of potassium ferricyanide.
To 0.35 ml assay mixture, 50 ~l of a sample containing theophylline at the following concentrations was added.
The reaction was carried out in a Gilford spectrophotometer with 10 mm light path cuvette at 30~ C for 30 minutes. From 25 the above, 50 ~l of assay mixture was mixed with 35 ~l of deionized water and 150 ~l of color producing reagent. The color producing reagent contains 1 M sodium acetate, 0.066 M

26 ~ 3 !~ 3 ~

citric acid, .00055 M ferrichloride in 0.1 M acetic acid, and 83.3 ~g of 4,7-diphenyl-1,10 phenanthroline. After 6 minutes the absorbance was measured at 535 nm wavelength.
The absorbance is proportionate to the concentration of 5 theophylline.

Theophylline Concentration Increase in OD at 535 nm 40 mg/L 0.319 30 mg/L 0.241 20 mg/L 0.171 10 mg/L 0.080 5 mg/L 0-045 ~x~m~le 9 In this example, the theophylline enzyme T-090 was used with ferricytochrome c as an acceptor which produces 20 1,3 dimethyl uric acid and ferrocytochrome c in the presence of theophylline. The formation of ferrocytochrome c is measured by the increase in absorbance at 550 nm wavelength.

The assay mixture contained 50 ~moles/ml of potassiom 25 phosphate buffer at pH 7.5, 5 u/ml of the theophylline enzyme T-090, and 0.25 nmoles/ml horse ferricytochrome c.

27 ~3~97~

- To 0.5 ml assay mixture, 25 ~l of sample containing theophylline at the following concentrations was added in separate cuvettes. The reaction was carried out in a Gilford spectrophotometer with 10 mm light path cuvette at 5 30~ C. After 15 minutes, when the reaction was complete, the increase in optical density was measured. As in the other examples, the absorbance has a linear relationship to the concentration of theophylline in the sample.

Theophylline Concentration Increase in OD at 550 nm 40 mg/L 0-454 30 mg/L 0.361 20 mg/L 0.264 10 mg/L 0.109 5 mg/L 0.055 mE)1~? 1 () Ten by ten mm square filter paper was impregnated with a solution containing the theophylline enzyme T-090 at various concentrations. For example, 50 u of the theophylline enzyme T-090 in 0.05 M phosphate buffer, pH 7.5. This 25 solution also contained 1 ~mole/ml of ferricytochrome c.
The paper was dipped in the above solution and air dried.
When 50 ~l of serum containing different concentrations of 2 8 ~ r ~ t~ 9L

theophylline, 5-40 mg/L, was added to the filter paper, increasingly deeper shades of pink appeared corresponding to the increasing theophylline concentration. The grada-tion of pink color allowed the estimation of the different theophylline concentrations.

SUPPLEMENTARY DISCLOSURE

In the foregoing description, we have described a method for determining theophylline in a sample and, more particularly, a method for determining theophylline which uses a theophylline utilizing enzyme. Such an enzyme utilizes or recognizes theophylline and uses it as a sub-strate for the quantitation of theophylline in samples.

The present invention contemplates the measurement or quantitation of theophylline concentration using these theophylline utilizing or recognizing enzymes. Examples of these enzymes, namely, theophylline dehydrogenase, theo-phylline oxidase, and theophylline demethylase are used todemonstrate the efficacy of the method, test composition and test device of the present invention for the measure-ment of theophylline in samples such as body fluids, food extracts, and medicinal compounds and compositions.

The present invention also contemplates a process of obtaining theophylline utilizing or recognizing enzymes from microbial sources which react with theophylline as a substrate and produce a product.

The present invention contemplates a method or sys-tem for the determination of theophylline by means of a theophylline utilizing or recognizing enzyme or enzymes (or substance containing such enzyme or enzymes) and optionally including the use of electron carriers, as well as a test composition and test device containing or employing such enzyme or enzymes. The present invention also contemplates a method of selecting organisms and finding, isolating, and purifying these enzymes from such organisms. The determin-ation of theophylline using these enzymes is accomplished by measurement of a signal produced by the reaction of the enzyme with any theophylline present in the sample and converting or correlating the amount of signal generated to the concentration of theophylline present in the sample.
As used herein, the term "theophylline utilizing enzyme"
means an enzyme or substance containing an enzyme which either recognizes or utilizes or reacts with theophylline to produce a signal which by itself or in conjunction with other reagents or means can be measured using visual, instrumental, or other state-of-the-art methodologies and that can be used to measure the concentration of theophyl-line.

The methods disclosed and claimed herein involve ~, ,~

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detecting any signal produced by contact of theophyllinewith the theophylline utilizing enzyme which indicates that a specific enzymatic reaction has taken place and showing that these signals are directly proportional to the concen-tration of theophylline in a sample.

A method of obtaining theophylline utilizing or rec-ognizing enzymes is also disclosed herein (and is described in detail hereinafter) and can be used to obtain such enzymes.

The following represents various test systems where-in theophylline in a test sample can be determined using the methodology disclosed in the present specification:

1) Measuring the decrease of theophylline concen-tration in the system. In this system, the disappearance of the substrate theophylline can be measured by determin-ing the decrease of absorbance at a wavelength of 272 nm, where theophylline has maximum absorbance. This is shown in Example 11 by using a theophylline oxidase and in Example 12 by using a theophylline dehydrogenase. However, this decrease is common to all reactions involving theo-phylline utilizing enzymes. This change can also be de-tected by measuring the reflectance, which is the inverseof the absorbance; or 31 ~c-~3~9'i 2) Measuring a change of the oxidation-reduction potential of the system in either of two ways, (a) measuring a change in the enzyme or enzyme complex system itself, as shown in Example 13, wherein the enzyme spec-trum, such as an absorption spectrum, changes in the pres-ence of theophylline, as can be seen by the changes that occur at wavelength 410 nm and 550 nm or measuring changes electrochemically, or (b) by a change of an electron car-rier added to the system such as, for example, the reduc-tion of ferricyanide to ferrocyanide. Example 14 shows theabsorbance change at 410 nm that occurs when ferricyanide changes to ferrocyanide in the presence of the theophylline dehydrogenase enzyme as the reaction takes place. This change can also be measured by spectrophotometric or elec-trochemical methods; or 3) Measuring the appearance of any product associ-ated with the enzymatic reaction of theophylline. The pro-ducts produced in this reaction vary with the particular enzyme involved. For example, an enzyme can recognize theophylline sufficiently and react by oxidizing, dehydro-genating or demethylating theophylline. Example 15 shows the appearance and measurement of formaldehyde when theo-phylline demethylase was used. Example 16 shows the ap-pearance and measurement of hydrogen peroxide when theo-phylline oxidase was used.

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The product formation can be further illustrated by the following reactions or processes:

i) When the enzyme is capable of oxidizing theo-phylline and converts theophylline (1,3 dimethyl xanthine)to 1,3 dimethyl uric acid utilizing oxygen as an electron acceptor. This is diagrammatically shown as follows:

theophylline oxidase theophylline + ~2 + H2O > 1,3 dimethyl uric acid + hydrogen peroxide In this system, the hydrogen peroxide thus produced can be determined titrimetrically, potentiometrically, pol-argraphically, colorimetrically as well as enzymatically.
The enzymatic methods of measuring hydrogen peroxide are preferred since they are not only specific and reliable, but can also be combined in a simple way with the hydrogen peroxide of the above reaction to produce color. For ex-ample, a peroxidase method is described in Anal. Biochem., 105, 389, (1980). Using theophylline oxidase, Example 16 demonstrates that the hydrogen peroxide formation is pro-portional to the concentration of theophylline in thesample and can, therefore, be used for the quantitation of theophylline. Alternatively, the rate of oxygen consump-tion in accordance with the above general equation can be measured, for instance, by gas chromatography and depolar-ization methods. The depolarization method utilizing oxy-gen electrodes (available from Yellow Spring Instruments, ~-c~ ~

3 3 ~ r ~ 4 Yellow Spring, OH) is well known and also described in U.S.
Patent No. 3~838~011 and in J. Appl. Physiol., 18~ 1247 ( 19 6 3 ) ~

ii) When the enzyme uses an acceptor other than oxygen such as ferricyanide, NAD, cytochromes, etc. to oxi-dize theophylline and produces 1, 3 dimethyl uric acid and a reduced acceptor in the following manner:

theophylline dehydrogenase theophylline + oxidized acceptor > 1, 3 dimethyl uric acid + reduced acceptor In such a system, the product 1, 3 dimethyl uric acid can be measured or determined by several methods. Example 17 describes one in which the absorbance at 292 nm is de-termined using theophylline dehydrogenase. At such wave-length, 1, 3 dimethyl uric acid absorbs optimally. Again, the increase in absorbance was found to be proportionate to the theophylline concentration in the sample and can, therefore, be used for the quantitation of theophylline.

Moreover, the concentration of theophylline in the sample can be determined using this reaction scheme by measuring the oxidation/reduction state of the electron acceptors used. Examples 14 and 18 show a test method where ferricyanide is used as an acceptor and is reduced to 30 ferrocyanide by theophylline dehydrogenase. In Example 14, the decrease of ferricyanide is determined by measuring the 34 ~33979~

decrease in absorbance at 410 nm wavelength as ferricyanide maximally absorbs at 410 nm and ferrocyanide has no absorp-tion at that wavelength. It was again found that the de-crease in absorbance was proportionate to the concentration of theophylline in the sample. In Example 18, another way of measuring ferrocyanide is shown. In this scheme, the formation of ferrocyanide is measured chemically by using 4,7 diphenyl-l,10 phenanthroline sulfonate by the method described by Avon, M. and Shavit N., Analy. Biochem., 6, 549 (1963). The Avon method produced color which was measured at 535 nm. The color thus produced was propor-tionate to the concentration of theophylline in the sample.

Example 19 illustrates the use of another electron acceptor, ferricytochrome c. In this example ferricyto-chrome c is reduced to ferrocytochrome c. The appearance of ferrocytochrome c is measured by measuring the increase in absorbance at 550 nm wavelength in the presence of theo-phylline dehydrogenase. Again, the change in absorbance at 550 nm wavelength was found to be proportionate to the con-centration of theophylline in the sample.

Example 15 shows one method of measuring formalde-hyde when theophylline demethylase was used. As indicated in the Example, the formaldehyde thus produced was propor-tionate to the concentration of theophylline in the sample and can, therefore, be used for the quantitation of theo-phylline.

Other products produced by the enzymatic recognitionof theophylline and measured by customary methods such as spectrophotometric, electrochemical or chromatographic or alternatively by a decrease in theophylline concentration are illustrated by Example 11.

Example 20 shows the device resulting from impreg-nating a filter paper with theophylline dehydrogenase andcytochrome c. The change in color with increasing concen-tration of theophylline can be read semi-quantitatively by visual inspection or quantitatively by existing reflectance measuring instruments. Alternatively, the change in elec-tron transfer in a solid matrix can be measured electro-chemically. In the above reaction, cytochrome c can be replaced by other indicators such as NBT, MTT, etc.

In summary, all the examples mentioned above and described below show that this enzymatic approach for the determination of theophylline allows the production of an easy and convenient test format not only in a liquid system but also in a solid matrix test device.

The microbial enzymes used in the method of the pre-sent invention were obtained as follows. Using the follow-ing procedures, surprisingly, micro-organisms were found A

36 1 3~7~'1 which contain enzymes which recognize theophylline suffic-iently and utilize theophylline as a substrate.

Approximately two hundred and fifty micro-organisms were tested for the presence of theophylline utilizing or recognizing enzymes. Each micro-organism was streaked on a plate consisting of a sterile media composed of 0.1% pur-ines, such as theophylline, salt solution, (salt solution containing per liter: 6.8 g KH2PO4, 7.1 g Na2HPO4, 0.2 g 10 MgSO4-7H2O, 0.1 mg MnC12 4H2O, 0.2 mg FeSO4 7H2O, 0.2 mg (NH4)2SO4, 2.0 mg CaC12, 1 gm NH4Cl, adjusted to pH 6.8 using potassium hydroxide) and 2% agarose. The plates were incubated at 30~C for 48 to 72 hours. The micro-organisms which grew on these plates were transferred to 250 mL
erlenmeyer flasks containing 50 mL of media. This media contained 0.1% purines, such as theophylline, 1% yeast extract, and salt solution. The flasks were placed in a shaker and stirred at 200 rpm at 30~C. The micro-organisms thus grown served as an inoculum for 2.8 L flasks contain-ing 1 L of growth media of the same composition as men-tioned above. The 2.8 L flasks were shaken at 30 C for 36 to 48 hours at 200 rpm. The cells were harvested by cen-trifugating the grown media at 10,000 g for 30 minutes.
The cells were suspended in 0.1 M potassium phosphate buf-fer, pH 7.0, containing 0.1 mM EDTA at the concentration of1 g per 10 mL of buffer. The cells were broken using a french press at 15,000 psi. The supernatant was collected ., 37 3 ~7~

by centrifugation at 15,000 g for 30 minutes. The superna-tant, also referred to as crude extract (Sl), thus obtained from each micro-organism was checked for theophylline rec-ognizing or utilizing enzyme activity by using the follow-ing assay procedures.

ASSAY FOR THEOPHYLLINE OXIDASE

Reaction 1 Enzyme Theophylline + ~2 > 1,3 dimethyl uric acid + H2O2 In a cuvette containing 0.5 mL of buffer, 0.1 potas-sium phosphate, pH 7.0, and 0.1 mM theophylline, 25 ~L of crude enzyme (Sl) was added and the decrease of absorbance at 273 nm wavelength was measured. As theophylline has a maximum absorption at 273 nm wavelength, the decrease of absorption at 273 nm indicates the presence of theophylline recognizing or utilizing enzymes as depicted in Reaction 1.

The reaction was also confirmed by simultaneously following the increase in absorbance at 293 nm where the reaction product 1,3 dimethyl uric acid typically absorbs.

Furthermore, as Reaction 1 produced hydrogen perox-ide, and as the measurement of H2O2 confirms the presence of theophylline oxidase, the crude extract (Sl) was tested ~ 3 ~3 ~'7 ~

for theophylline oxidase activity using an H2O2 assay as follows:

The assay mixture contained 0.1 M potassium phos-phate, pH 7.5, 10 mM theophylline, 14 mM phenol, 0.015% 4-aminoantipyrine, and 18 U/mL horseradish peroxidase. The assay was run at 37~C. The increase in absorbance was measured at 500 nm wavelength. The enzyme activity was calculated as the formation of 1 ~mole of H2O2 per minute at 37~C.

~ ODs00/minute x total assay volume x dilution Activity U/mL =
5.33 x sample volume This assay provided the quantitative measurement of theophylline oxidase in crude extracts prepared from the various organisms.

ASSAY FOR THEOPHYLLINE DEHYDROGENASE

Theophylline recognizing or utilizing enzymes were shown to be present by using other assays. When crude ex-tract samples were found which showed the utilization of theophylline by exhibiting a decrease in absorbance at 273 nm or increase in absorbance at 293 nm wavelength but did not produce hydrogen peroxide, they were checked for theo-7 ~ ~

phylline dehydrogenase activity (i.e., oxidation of theo-phylline in the presence of an electron acceptor other than oxygen). Various electron acceptors such as potassium fer-ricyanide, NAD, and cytochrome c, were used in assaying crude extracts for dehydrogenase activity. The theophyl-line dehydrogenase reactions are depicted in Reaction 2.

Reaction 2 theophylline dehydrogenase theophylline + electron > 1,3 dimethyl uric acid acceptor + reduced dye a) Potassium Ferricyanide as an electron acceptor If the crude extract (Sl) contains theophylline de-hydrogenase and potassium ferricyanide is used as an elec-tron acceptor, the potassium ferricyanide will be converted to potassium ferrocyanide. The potassium ferrocyanide is detected and measured by measuring the decrease in absor-bance (or optical density) at 410 nm wavelength.

The assay mixture contained 0.05 M potassium phos-phate buffer at pH 7.0, 50 ~L of crude extract (Sl), and 1.40 mM potassium ferricyanide. The reaction was carried out in a Gilford spectrophotometer with 10 mm light path at 30 C. The decrease in absorbance at 410 nm indicates the presence of theophylline dehydrogenase activity. Dehydro-genase activity was quantitated by the following calcula-~ 3~ 7 g ~
tion.

~OD410/minute x total reaction volume x dilution Activity U/mL =
1.0 x volume of sample (mL) b) NAD as an alternate electron acceptor The method was the same as above except that the re-action mixture contained 1 mM NAD instead of potassium fer-ricyanide. The reaction was followed at 30~C and the ap-pearance of NADH was measured by following the increase in absorbance at 340 nm wavelength. The dehydrogenase activ-ity was quantitatively calculated as:

~OD340/minute x total reaction volume x dilution Activity U/mL =
6.22 x volume of sample (mL) c) Cytochrome c as an alternate electron acceptor The method was the same as above except the reaction mixture contained 25 nmoles cytochrome c instead of potas-sium ferricyanide. The reaction was followed at 30 C and the appearance of reduced cytochrome was detected and fol-lowed by measuring the increase in absorbance at 550 nm wavelength. The dehydrogenase activity was quantitatively calculated as:

4~ 3 7 9 '~
~ OD550/minute x total reaction volume x dilution Activity U/mL =
20.0 x volume of sample (mL) In all cases the unit activity of the enzyme was defined as: 1 unit of enzyme utilizes 1 ~mole of substrate (theophylline) per minute of 1 ~mole of product is formed per minute under the given assay conditions.

THEOPHYLLINE DEMETHYLASE ACTIVITY

As the presence of theophylline utilizing or recog-nizing enzyme in the crude extract does not have to only involve oxidation reactions as shown in Reactions 1 and 2, other assays were performed in the crude extract. Reaction 3 shows an example of other enzymatic reactions that can also take place and which can be used in the measurement of theophylline in the sample.

20 Reaction 3 Theophylline demethylase 25 Theophylline + NADH > xanthine or + HCHO + NAD
(NADPH) l or 3 methyl - (NADP) xanthine where theophylline is demethylated in the presence of NADH
OR NADPH and is converted to methylxanthine or xanthine with formation of formaldehyde and NAD.

3 ~3 7 g ~

The crude extracts were tested for the presence of theophylline demethylase using the following procedure:

The assay mixture consists of 0.05 M Tris-HCl, pH

8.0, buffer containing 0.4 mM NADH and 10 mM theophylline.
The decrease in absorbance due to NADH utilization and the formation of NAD was measured at 340 nm wavelength at 30 C
(~OD sample). A blank rate was determined where the same assay mixture was used without theophylline (~OD blank).
The difference between these two absorbancies, i.e., (~OD
sample - ~OD blank)/minute, is proportionate to the activ-ity of theophylline demethylase present in the crude ex-tract. Theophylline demethylase activity was calculated as:

(~~Dsample ~ ~~Dblank)/min x total reaction vol-ume (mL) x dilution Activity U/mL =
6.22 x sample volume (mL) Crude extracts (Sl) from over 22 micro-organisms showed theophylline utilizing or recognizing activity.
Most of these crude extracts were identified to contain oxidase, dehydrogenase, or demethylase activity. However, three micro-organisms used theophylline as a substrate and showed a decrease in absorption at 273 nm wavelength but could not be specifically identified to carry out oxida-tion, demethylation, or dehydrogenation suggesting that ~ <~ 7 ~ -1 these samples contained some other theophylline recognizing or utilizing enzymes which could be used for determining the concentration of theophylline.

Three micro-organisms were selected whose crude ex-tracts (Sl) showed the most activity/mL and which belonged to one class of microbial enzyme, i.e. oxidase, dehydrogen-ase, or demethylase. The micro-organisms producing maximum demethylase activity, maximum oxidase activity, and maximum dehydrogenase activity were selected, denominated T-040, T-060 and T-090, respectively, and were deposited on August 10, 1990 at NORTHERN REGIONAL RESEARCH LABORATORY, Peoria, IL, where they were given the deposit numbers NRRL B-18697, NRRL B-18698, and NRRL B-18699, respectively.

PURIFICATION OF ENZYMES

The enzymes - theophylline demethylase from T-040 organism, theophylline oxidase from T-060 organism, and theophylline dehydrogenase from T-090 organism - were isolated and partially purified. The partially purified enzymes demethylase, oxidase, and dehydrogenase were ident-ified as T-040, T-060, and T-090, respectively, and called as a group theophylline utilizing enzymes which were used in determining the concentration of theophylline in a given sample.

,, ;.~

44 1 ~i3~

The enzymes were partially purified using well esta-blished biochemical techniques. The same common method, as described below, was used.

Step 1) Crude extracts were prepared by the procedure described earlier. The method involved growing respective organisms in 50 L media, collecting the organisms by centrifugation, suspending the organisms in a buffer medium (10 mM potassium phosphate buffer, pH 7.5, containing 2 mM
EDTA), breaking the cells by homogenization using a french press or Menton-Gaulin homogen-izer, and collecting the crude extract by cen-trifugation.

Step 2) DEAE ion-exchange chromatography: the crude extracts were checked for the respective en-zyme activity. A column of 5 x 100 cm with a bed volume of approximately 2 L capacity was packed with Pharmacia DEAE-Sepharose resin, which was previously equilibrated with 10 mM
potassium phosphate, pH 7.5, containing 2 mM
EDTA. The crude extract, approximately 5 liters, was diluted to the same ionic strength with deionized water and charged to the above DEAE column. After loading the enzyme, 10 L of equilibrating buffer was passed through the * Trade-mark column. Subsequently, the column was eluted with 6 liters of equilibrating buffer contain-ing 50 mM NaCl, 100 mM NaCl, 150 mM NaCl, and 200 mM NaCl as a step-wise gradient. The frac-tions were collected in 20 mL volumes in an LKB
fraction collector. Each fraction was checked for enzyme activity and the protein cont~nt was determined by measuring OD2go. The fractions with maximum specific activity, activity/mL .
mg protein/mL were pooled, and rechecked for activity. Demethylase activity appeared in fractions when the column was eluted with eluting buffer containing 100 mM NaCl, oxidase activity appeared in fractions when the column was eluted with eluting buffer containing 150 mM NaCl, and dehydrogenase activity appeared in fractions when the column was eluted with elut-ing buffer containing 50 mM NaCl.

20 Step 3) Concentration of enzymes: each enzyme was con-centrated using ultrafiltration method with a membrane cut off of 10,000 molecular weight (PM-10 membrane from Amicon). The concentrated enzymes were checked for activity/mL (10-20 U/mL), protein, and specific activity (0.5 -2.0 U/mg protein). The enzymes were stored at -20~C in a freezer in small portions and were * Trade-mark ' 13.~73~

used for the estimation of theophylline in samples in a manner similar as shown in the following examples.

EXAMPLES

The following examples are intended for illustration of the present invention and are not intended to limit the scope thereof.

In all the examples given herein, the enzyme activ-ity is defined as 1 ~mole of theophylline utilized per mlnute at 30~C temperature unless specified otherwise.

The assay mixture contained 0.05 M potassium phos-phate buffer, pH 7.0, and 0.4 u/mL of a theophylline util-izing or recognizing enzyme, such as theophylline oxidase.
To 2 mL assay mixture, 100 ~L of a sample containing theo-phylline at several concentrations was added in separate cuvettes. The reaction was carried out in a Gilford spec-trophotometer with 10 mm light path cuvette at 30 C. A
decrease in optical density at 272 nm was observed after 30 minutes which was proportionate to the theophylline concen-tration in the sample.

47i~.P~ 7 ~ ~

Theophylline coneentration Decrease in OD at 272 nm 40 mg/L .067 20 mg/L .033 10 mg/L .017 The assay mixture contained 50 ~moles/mL potassium phosphate buffer at pH 7.5 and 1.8 u/mL of a theophylline utilizing or recognizing enzyme, such as theophylline dehy-drogenase and 25 nmoles/mL of cytochrome e. To 0.5 mL
assay mixture, 25 ~L of a sample eontaining theophylline at the eoneentrations below were added in separate euvettes.
The reaction was earried out in a Gilford spectrophotometer with 10 mm light path cuvette at 30~C. After 20 minutes, the decrease in optieal density at 272 nm was recorded which was proportionate to the theophylline concentration in the sample.

Theophylline concentration Decrease in OD at 272 nm 40 mg/L 0.065 20 mg/L 0.031 10 mg/L 0.015 48 133979'I

In this example, theophylline dehydrogenase was used. As shown in the Figure, Curve A describes the absor-bance spectra of theophylline dehydrogenase at a concentra-tion of 1.8 u/mL in 0.05 M potassium phosphate buffer at pH
7.5. Curve B shows the absorbance spectra of the enzyme in the presence of theophylline at the concentration of 2 mg/L
under the same conditions.

As can be seen, theophylline caused the increase in absorbance at 417.5 and 550 nm wavelength. These changes in absorptions are used as a basis for the determination of theophylline concentration in a sample.

In this example, theophylline dehydrogenase was used with potassium ferricyanide as an acceptor. The potassium ferricyanide changed to ferrocyanide in the presence of the enzyme when theophylline was added. The formation of fer-rocyanide can be measured by measuring the decrease in optical density at 410 nm.

The assay mixture contained 50 ~moles/mL of potas-sium phosphate buffer, 1.8 u/mL of theophylline enzyme, and 1.45 ~moles/mL of potassium ferricyanide. To 0.35 mL assay .~.

~ ~ 3 .~ 7.~ '~

mixture, 50 ~L of a sample containing theophylline at the concentrations below was added in separate cuvettes. The reaction was carried out in a Gilford spectrophotometer with 10 mm light path cuvette at 30 C. After 30 minutes, the decrease in optical density at 410 nm wavelength was measured which was proportionate to the concentration of theophylline in the sample.

Theophylline concentration Decrease in OD at 410 nm 40 mg/L 0.141 30 mg/L 0.109 20 mg/L 0.076 10 mg/L 0.047 In this example, theophylline demethylase was used.
In the presence of NADPH or NADH, this enzyme demethylated theophylline and produced xanthine and/or 1 and/or 3 methyl xanthine and formaldehyde. The formaldehyde was measured by a known chemical method as reported by Nash, Biochem.
J., 55, 416-421 (1953). The formaldehyde production was proportionate to the concentration of theophylline in the sample.

The assay mixture contained 50 ~moles/mL of Tris-HCl , ~

7 ~ ~
buffer at pH 8.0, 1 ~mole/mL of NADPH and 10 nmoles/mL of semicarbazide. To 2.0 mL of assay mixture in a test tube, 2.5 units of theophylline demethylase was added. After the reaction mixture was shaken at 30~C for 15 minutes, the reaction was stopped by adding 0.6 mL of 20% zinc sulfate, 0.66 mL of saturated barium hydroxide and allowing the mixture to stand 10 minutes at room temperature. The tubes were centrifuged at 8,000 g for 10 minutes. To 1.0 mL of supernatant, the following additions were made: 0.4 mL of Nash reagent (150 g ammonium acetate and 2 mL acetyl acetone in 500 mL of deionized water) and the tubes incu-bated at 60~C in a water bath for 30 minutes. Absorbance was immediately read at 415 nm wavelength.

15 Theophylline concentration Decrease in OD at 415 nm 180 mg/L 1.481 40 mg/L 0.269 20 mg/L 0.140 10 mg/L 0.075 In this example, theophylline oxidase was used which produced 1,3 dimethyl uric acid and hydrogen peroxide in the presence of theophylline. The hydrogen peroxide was measured by a known modified Trinder's method as described 51 ~. 3 ~) ~ 7 ,~ ~

by Fossati et al., Clin. Chem., 26, 227 (1980).

The assay mixture contained 50 ~moles/mL potassium phosphate, pH 7.5, 5 ~moles/mL of 3,5-dichloro-2-hydroxy benzene sulfonate hydrochloride (DHBS), 1 ~mole/mL 4-aminoantipyrine, 5.0 u/mL of horseradish peroxidase and 0.7 u/mL of theophylline oxidase. To 0.7 mL assay mixture, 50 ~L of a sample containing theophylline at the following concentrations was added in separate cuvettes. The reac-tion was carried out in a Gilford spectrophotometer with 10mm light path cuvette at 37~C. After 20 minutes, the in-crease in optical density at 510 nm was measured.

Theophylline concentrationIncrease in OD at 510 nm 40 mg/L 0.172 20 mg/L 0.089 10 mg/L 0.045 In this example, theophylline dehydrogenase was used with cytochrome c. In the presence of theophylline the re-action produced 1,3 dimethyl uric acid. This product was measured at 292 nm which is the wavelength of maximum ab-sorbance for 1,3 dimethyl uric acid.

., ~ 3 ~3~ 7 ~ ~

The assay mixture contained 50 ~moles/mL potassium phosphate buffer at pH 7.5, 1.8 u/mL of theophylline dehyd-rogenase and 25 nmoles/mL of cytochrome c. To 0.5 mL assay mixture, 25 ~L of a sample containing theophylline at the following concentrations was added in separate cuvettes.
The reaction was carried out in a Gilford spectrophotometer with 10 mm light path cuvette at 30~C. After 20 minutes, the increase in optical density at 292 nm was recorded which was proportionate to the theophylline concentration in the sample.

Theophylline concentrationIncrease in OD at 292 nm 40 mg/L 0.162 30 mg/L 0.125 20 mg/L 0.087 10 mg/L 0.036 In this example, theophylline dehydrogenase was used with potassium ferricyanide as an acceptor which produces potassium ferrocyanide in the presence of theophylline.
The ferrocyanide thus produced is measured chemically by using 4,7 diphenyl-1,10 phenanthroline sulfonate as des-cribed by Avon and Shavit in Anal. Biochem., 6, 549 (1963).

) 7 ~
The assay mixture contained 50 ~moles/mL of potas-sium phosphate buffer at pH 7.5, 1.8 u/mL of theophylline dehydrogenase, and 1.43 ~moles/mL of potassium ferricyan-ide. To 0.35 mL assay mixture, 50 ~L of a sample contain-ing theophylline at the following concentrations was added.The reaction was carried out in a Gilford spectrophotometer with 10 mm light path cuvette at 30~C for 30 minutes. From the above, 50 ~L of assay mixture was mixed with 35 ~L of deionized water and 150 ~L of color producing reagent. The color producing reagent contains 1 M sodium acetate, 0.066 M citric acid, .00055 M ferrichloride in 0.1 M acetic acid, and 83.3 ~g of 4,7 diphenyl-1,10 phenanthroline. After 6 minutes the absorbance was measured at 535 nm wavelength.
The absorbance is proportionate to the concentration of theophylline.

Theophylline concentrationIncrease in OD at 535 nm 40 mg/L 0.319 2030 mg/L 0.241 20 mg/L 0.171 10 mg/L 0.080 5 mg/L 0-045 In this example, theophylline dehydrogenase was used with ferricytochrome c as an acceptor which produces 1,3 dimethyl urie aeid and ferroeytoehrome e in the presenee of theophylline. The formation of ferroeytochrome c is mea-sured by the increase in absorbance at 550 nm wavelength.

The assay mixture contained 50 ~moles/mL of potas-sium phosphate buffer at pH 7.5, 5 u/mL of theophylline dehydrogenase, and 0.25 nmoles/mL horse ferricytochrome c.
To 0.5 mL assay mixture, 25 ~L of sample containing theo-phylline at the following concentrations was added in sepa-rate cuvettes. The reaction was carried out in a Gilford spectrophotometer with 10 mm light path cuvette at 30~C.
After 15 minutes, when the reaction was complete, the in-erease in optical density was measured. As in the other examples, the absorbance has a linear relationship to the concentration of theophylline in the sample.

Theophylline concentrationIncrease in OD at 550 nm 2040 mg/L 0.454 30 mg/L 0.361 20 mg/L 0.264 10 mg/L 0.109 5 mg/L 0 055 Ten by ten mm square filter paper was impregnated with a solution containing theophylline dehydrogenase at 5 various concentrations. For example, 50 u of theophylline dehydrogenase in 0.0 5 M phosphate buffer, pH 7 ~ 5 ~ This solution also contained 1 ~mole/mL of ferricytochrome c.
The paper was dipped in the above solution and air dried.
When 50 ~L of serum containing different concentrations of theophylline, 5-40 mg/L, was added to the filter paper, in-creasingly deeper shades of pink appeared corresponding to the increasing theophylline concentration. The gradation of pink color allowed the estimation of the different theo-phylline concentrations.

The foregoing is intended as illustrative of the present invention but not limiting. Numerous variations and modifications may be effected without departing from the true spirit and scope of the invention.

Claims (88)

1. A method for determining theophylline in a sample which comprises contacting the sample with a theophylline-utilizing enzyme selected from the group consisting of microbial theophylline enzymes T-040, T-060 and T-090, determining the amount of signal produced and correlating this amount to the concentration of theophylline in the sample.

2. A method according to claim 1 in which said sample is selected from the group consisting of body fluids, food extracts and medicinal compositions.

3. A method according to claim 1 in which the signal is generated as a result of a decrease in the theophylline present in the sample upon contact with the enzyme.

4. A method according to claim 3 in which the signal is measured by observing a change in the optical density of the sample at a wavelength of from about 260 nm to about 280 nm.

5. A method according to claim 1 in which the signal is a result of a change in the enzyme upon contact with theophylline present in the sample.

6. A method according to claim 5 in which the change in the enzyme is indicated by an increase in optical density of the sample at a wavelength in the range of from about 410 nm to about 550 nm.

7. A method according to claim 1 wherein an electron carrier is present.

8. A method according to claim 7 in which the signal is generated as a result of a decrease in the theophylline present in the sample upon contact with the enzyme.

9. A method according to claim 8 in which the signal is measured by observing a change in the optical density of the sample at a wavelength of from about 260 nm to about 280 nm.

10. A method according to claim 7 in which the signal is a result of a change in the enzyme upon contact with theophylline present in the sample.

11. A method according to claim 10 in which the change in the enzyme is indicated by an increase in optical density of the sample at a wavelength in the range of from about 410 nm to about 550 nm.

12. A method according to claim 10 in which the change is measured electrochemically.

13. A method according to claim 7 in which the electron carrier is an electron donor.

14. A method according to claim 13 in which the electron donor is selected from the group consisting of reduced nicotinamide adenine dinucleotide, reduced nicotinamide adenine dinucleotide phosphate and reduced flavine adenine dinucleotide.

15. A method according to claim 7 in which the electron carrier is an electron acceptor.

16. A method according to claim 15 in which the electron acceptor is oxygen.

17. A method according to claim 15 in which the electron acceptor is selected from the group consisting of cytochromes, ferricyanides, dichlorophenolindophenol, nicotinamide adenine dinucleotide, phenazine methosulfate and methylene blue.

18. A method according to claim 1 in which a reaction product caused by the interaction of the theophylline-utilizing enzyme and theophylline present in the sample generates the measurable signal.

19. A method according to claim 18 in which the reaction product is 1,3-dimethyl uric acid.

20. A method according to claim 19 in which the quantity of the 1,3-dimethyl uric acid is determined by measuring an increase in the optical density of the sample at a wavelength in the range of from about 285 nm to about 305 nm.

21. A method according to claim 18 in which hydrogen peroxide is produced as a reaction product and is the measurable signal.

22. A method according to claim 21 in which the quantity of the hydrogen peroxide produced is measured by a system consisting essentially of a chromogenic reagent which is capable of undergoing a color change in the presence of hydrogen peroxide.

23. A method according to claim 7 in which a change in the electron carrier produces the measurable signal.

24. A method according to claim 7 in which the electron carrier is nicotinamide adenine dinucleotide.

25. A method according to claim 7 in which the electron carrier is ferricytochrome c.

26. A method according to claim 7 in which the electron carrier is potassium ferricyanide.

27. A method according to claim 7 in which the electron carrier is flavine adenine nucleotide.

28. A method according to claim 7 in which the electron carrier is 2,6-dichlorophenolindophenol.

29. A method according to claim 18 in which the theophylline-utilizing enzyme is capable of changing theophylline to a reaction product selected from the group consisting of xanthine and xanthine derivatives.

30. A method according to claim 18 in which the interaction of the theophylline-utilizing enzyme and theophylline present in the sample to generate a measurable signal is determined in the presence of xanthine oxidase.

31. A method according to claim 18 in which the interaction of the theophylline-utilizing enzyme and theophylline present in the sample to generate a measurable signal is determined in the presence of xanthine dehydrogenase.

32. A method according to claim 18 in which formaldehyde is produced as a reaction product and is the measurable signal.

33. A test composition for the determination of theophylline in a sample comprising a theophylline-utilizing enzyme selected from the group consisting of microbial theophylline enzymes T-040, T-060 and T-090, and a reagent for generating a signal whereby the amount of theophylline in the sample can be determined.

34. A test composition according to claim 33 in which the signal is generated as a result of a decrease in theophylline present in the sample upon contact with the enzyme.

35. A test composition according to claim 34 in which the signal is measured by observing a change in the optical density of the sample at a wavelength of from about 260 nm to about 280 nm.

36. A test composition according to claim 33 in which the signal is a result of a change in the enzyme upon contact with theophylline present in the sample.

37. A test composition according to claim 35 in which the signal is an increase in optical density of the sample at a wavelength in the range of from about 410 nm to about 550 nm.

38. A test composition according to claim 33 in which an electron carrier is present.

39. A test composition according to claim 38 in which the signal is generated as a result of a decrease in the theophylline present in the sample upon contact with the enzyme.

40. A test composition according to claim 39 in which the signal is measured by observing a change in the optical density of the sample at a wavelength in the range of from about 260 nm to about 280 nm.

41. A test composition according to claim 38 in which the signal is a result of a change in the enzyme upon contact with theophylline present in the sample.

42. A test composition according to claim 41 in which the signal is an increase in the optical density of the sample at a wavelength in the range of from about 410 nm to about 550 nm.

43. A test composition according to claim 41 in which the change is measured electrochemically.

44. A test composition according to claim 38 in which the electron carrier is an electron donor.

45. A test composition according to claim 44 in which the electron donor is selected from the group consisting of reduced nicotinamide adenine dinucleotide, reduced nicotinamide adenine dinucleotide phosphate and reduced flavine adenine dinucleotide.

46. A test composition according to claim 38 in which the electron carrier is an electron acceptor.

47. A test composition according to claim 46 in which the electron acceptor is oxygen.

48. A test composition according to claim 46 in which the electron acceptor is selected from the group consisting of cytochromes, ferricyanides, dichlorophenolindophenol, nicotinamide adenine dinucleotide, phenazine methosulfate and methylene blue.

49. A test composition according to claim 33 in which the reagent is responsive to the presence of a reaction product of the theophylline-utilizing enzyme and theophylline.

50. A test composition according to claim 49 in which the reaction product is hydrogen peroxide.

51. A test composition according to claim 49 in which the reagent is responsive to a reaction product selected from the group consisting of xanthine and xanthine derivatives.

52. A test composition according to claim 51 in which the reagent comprises xanthine oxidase.

53. A test composition according to claim 50 in which the reagent comprises peroxidase and an oxidation-reduction indicator.

54. A test composition according to claim 49 in which the reagent is responsive to formaldehyde.

55. A test composition according to claim 49 in which the reagent is responsive to 1,3-dimethyl uric acid.

56. A test composition according to claim 33 in which the signal produced is a signal which can be measured by a measurement selected from the group consisting of spectrophometric measurement, electrochemical measurement, fluorescent measurement, reflectance measurement and polarization measurement.

57. A test device for the determination of theophylline in a sample comprising a solid matrix incorporated with a composition consisting essentially of a theophylline-utilizing enzyme selected from the group consisting of microbial theophylline enzymes T-040, T-060 and T-090 and an indicator reagent.

58. A test device according to claim 57 in which the composition additionally comprises an electron carrier.

59. A test device according to claim 58 in which the electron carrier is an electron donor.

60. A test device according to claim 59 in which the electron donor is selected from the group consisting of reduced nicotinamide adenine dinucleotide, reduced nicotinamide adenine dinucleotide phosphate and reduced flavine adenine dinucleotide.

61. A test device according to claim 58 in which the electron carrier is an electron acceptor.

62. A test device according to claim 61 in which the electron acceptor is oxygen.

63. A test device according to claim 61 in which the electron acceptor is selected from the group consisting of cytochromes, ferricyanides, dichlorophenolindophenol, nicotinamide adenine dinucleotide, phenazine methosulfate and methylene blue.

64. A test device according to claim 57 in which the solid matrix is paper.

65. A test device according to claim 57 in which the solid matrix is a polymer.

66. A test device according to claim 58 in which the solid matrix is paper.

67. A test device according to claim 58 in which the solid matrix is a polymer.

CLAIMS SUPPORTED BY SUPPLEMENTARY DISCLOSURE

68. A method for determining theophylline in a sample which comprises contacting the sample with a microbial theophylline dehydrogenase which provides a measurable signal in the presence of theophylline, determining the amount of signal produced and correlating this amount to the concentration of theophylline in the sample.

69. A method according to claim 68 wherein said microbial theophylline dehydrogenase is theophylline dehydrogenase NRRL B-18699.

70. A test composition for the determination of theophylline in a sample comprising a microbial theophylline dehydrogenase capable of producing a measurable signal in the presence of theophylline and a chemical means for correlating the signal to the quantity of theophylline present in the sample.

71. A test composition according to claim 70 wherein said microbial theophylline dehydrogenase is theophylline dehydrogenase NRRL B-18699.

72. A test device for the determination of theophylline in a sample comprising a solid matrix incorporated with a composition consisting essentially of a microbial theophylline dehydrogenase and a reagent capable of producing a measurable signal in the presence of theophylline.

73. A test device according to claim 72 wherein said microbial theophylline dehydrogenase is theophylline dehydrogenase NRRL B-18699.

74. A method for determining theophylline in a sample which comprises contacting the sample with a microbial theophylline demethylase and a reagent which provides a measurable signal in the presence of theophylline, determining the amount of signal produced and correlating this amount to the concentration of theophylline in the sample.

75. A method according to claim 74 wherein said microbial theophylline demethylase is theophylline demethylase NRRL B-18697.

76. A test composition for the determination of theophylline in a sample comprising a microbial theophylline demethylase capable of producing a measurable signal in the presence of theophylline and a chemical means for correlating the signal to the quantity of theophylline present in the sample.

77. A test composition according to claim 76 wherein said microbial theophylline demethylase is theophylline demethylase NRRL B-18697.

78. A test device for the determination of theophylline in a sample comprising a solid matrix incorporated with a composition consisting essentially of a microbial theophylline demethylase and a reagent capable of producing a measurable signal in the presence of theophylline.

79. A test device according to claim 78 wherein said microbial theophylline demethylase is theophylline demethylase NRRL B-18697.

80. A method for determining theophylline in a sample which comprises contacting the sample with a microbial theophylline oxidase which provides a measurable signal in the presence of theophylline, determining the amount of signal produced and correlating this amount to the concentration of theophylline in the sample.

81. A method according to claim 80 wherein said microbial theophylline oxidase is theophylline oxidase NRRL
B-18698.

82. A test composition for the determination of theophylline in a sample comprising a microbial theophylline oxidase capable of producing a measurable signal in the presence of theophylline and a chemical means for correlating the signal to the quantity of theophylline present in the sample.

83. A test composition according to claim 82 wherein said microbial theophylline oxidase is theophylline oxidase NRRL B-18698.

84. A test device for the determination of theophylline in a sample comprising a solid matrix incorporated with a composition consisting essentially of a microbial theophylline oxidase and a reagent capable of producing a measurable signal in the presence of theophylline.

85. A test device according to claim 84 wherein said theophylline oxidase is theophylline oxidase NRRL B-18698.

86. A method according to claim 15 wherein the electron acceptor is NBT or MTT.

87. A test composition according to claim 46 wherein the electron acceptor is NBT or MTT.

88. A test device according to claim 61 wherein the electron acceptor is NBT or MTT.