Note: Descriptions are shown in the official language in which they were submitted.
1~979~
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
- 11
- 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.
12 1~97~
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).
~ g 7 ~ L~
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 ~
16
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.
~39~'3~
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.
* Trade-mark
C
18
~ 3~ 7~
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.
19
~ 3~7~
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.
~ 3~ 3 ~
~;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
22
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
24
~ 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
7 3 1
29
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
~,
,~
~ P3~79~
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.
,,;
32
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 ~
38
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 ~ ~
39
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 ~
42
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~
46
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.
EXAMPLE 11
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
EXAMPLE 12
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
EXAMPLE 13
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.
EXAMPLE 14
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.~ '~
49
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
EXAMPLE 15
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
EXAMPLE 16
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
EXAMPLE 17
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
EXAMPLE 18
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
EXAMPLE 19
In this example, theophylline dehydrogenase was used
54
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
5 5
EXAMPLE 20
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.
