Note: Descriptions are shown in the official language in which they were submitted.
CA 02331953 2001-01-19
RAPID ASSAY FOR TESTING OVERALL OXIDATIVE STRESS
Field of the Invention
The invention relates to an assay for testing oxidative stress of a subject
by measurement of oxidants in biological fluids.
Background of the Invention
Oxidative stress has been implicated as a factor in various diseases and
injury states. However, the extent to which oxidative stress could be
predictive of
clinical outcome was unknown, norwas a satisfactory method for rapidly
assaying total
oxidative stress in a subject available.
Various methods of measuring particular oxidants within biological fluids
are known. However, these methods generally rely on the measurement of only a
single oxidized compound, or a narrow class of compounds, as indicators of
oxidative
activity. Thus, they may not be reliable predictors of overall oxidative
stress. For
example, Draper et al. (Lipids 19:836-843, 1984) disclose the use of urinary
malondialdehyde as an indicator of lipid peroxidation. These approaches may be
limited to the measurement of lipid peroxide breakdown products, and may not
adequately reflect oxidation by hydrogen peroxide and organic peroxides.
Attempts have been made to measure oxidative stress through the
measurement of hydrogen peroxide in urine (Long etal., BBRC, 262,605-609
(1999)).
However, these approaches are limited to the measurement of hydrogen-peroxide
derived oxidative species, and may not provide an accurate assessment of
overall
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oxidative stress.
A method of assaying hydrogen peroxide in fluids using 2-deoxyribose
was developed by Halliwell, et al. (Analytical Biochemistry, 165,215-219
(1987)).
Hydroxyl radicals were generated by the reaction of an Fe3+ EDTA complex with
hydrogen peroxide in the presence of ascorbic acid. These hydroxyl radicals
then
attacked the deoxyribose to form products which, upon heating with
thiobarbituric acid
("TBA") at low pH yield a pink chromogen. The chromogen could then be measured
to provide an estimate of the original hydrogen peroxide concentration.
However,
Halliwell's method is adapted for the measurement of hydroxyl radical, and no
consideration was given to the measurement of overall oxidative stress from a
range
of reactive species. Moreover, the use of ascorbic acid in Halliwell's method
renders
the reagent mixture relatively unstable, and may render it unsuitable for use
in routine
clinical testing, and other applications where stability is important. A
further
disadvantage of Halliwell's method is that the reaction is slow, and of
limited accuracy,
which may make it unsuitable for use in situations where rapid or precise
results are
needed.
Thus, it is an object of the present invention to provide a rapid assay for
the measurement of overall oxidative stress.
Summary of the Invention
In an embodiment of the invention there is provided a method of
determining oxidative stress in a mammalian subject. The method comprises:
obtaining a sample of a biological fluid from the subject; mixing the
biological fluid with
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a solution containing a ferrous reagent and 2-deoxyglucose to form a mixture;
incubating the mixture; and determining the extent of formation of a coloured
reaction product, thereby determining the level of oxidative stress in the
subject.
In an embodiment of the invention there is provided a method of identifying a
mammalian subject in need of medical treatment. The method comprises:
obtaining
a sample of a biological fluid and assaying overall oxidant level in the
biological
fluid using the above mentioned method.
In an embodiment of the invention there is provided a solution suitable for
use in assaying oxidative stress. The solution comprises 2-deoxyglucose, and a
ferrous reagent.
In an embodiment of the invention there is provided a kit suitable for use in
assaying oxidative stress from a biological fluid. The kit comprises a
solution
comprising 2-deoxyglucose and a ferrous reagent. A reference standard is also
included in the kit.
Brief Description of the Drawings
These and other advantages of the invention will become apparent upon
reading the following detailed description and upon referring to the drawings
in
which:
Figure 1 is a graphical depiction of the results of Example 1.
Figure 2 is a graphical depiction of the results of Example 2.
Figures 3a and 3b are graphical depictions of the results of Example 3.
Figure 4 is a colour photograph of the results of Example 4.
While the invention will be described in conjunction with illustrated
embodiments, it will be understood that it is not intended to limit the
invention to
such
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embodiments. On the contrary, it is intended to cover all alternatives,
modifications
and equivalents as may be included within the spirit and scope of the
invention as
defined by the appended claims.
Detail Description of the Preferred Embodiments
The present invention is directed to a rapid assay for the measurement
of overall oxidative stress in biological fluids, and to the use of the method
in predicting
clinical outcome, and identifying subjects warranting further medical
examination.
Oxidative stress has been suspected to be linked to the severity of injury
or disease in numerous disorders. However, it was not clear whether
measurements
of oxidative stress could be useful as a predictor of clinical outcome or
could be used
to guide therapeutic intervention. In particular, previous work had focused on
measurements of particular oxidative species, and had failed to provide a
satisfactory
measure of overall oxidative stress, which could be useful in predicting
clinical
outcome and guiding therapeutic intervention.
The rapid assay for overall oxidative stress disclosed herein permits
measurement of hydrogen peroxide as well as organic peroxides such as lipid
peroxides in biological fluids, thereby providing a measure of overall
oxidative stress.
The method relies on the conversion of peroxides to strong oxidants using the
ferrous
(Fe2+) ion. These strong oxidants react with 2-deoxyglucose to form further
reactive
products. Additionally, other oxidants such as lipid peroxides, lipid
endoperoxides and
aldehydes, (including a-oxo-aldehydes, and malondialdehyde aldehydes) are
measured by the method of the invention.
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The reaction reagent of the present invention overcomes disadvantages
of the Halliwell method and is better suited for medical and veterinary
applications.
In particular, in a preferred embodiment of the invention, the reaction
reagent is a
ferrous reaction reagent. The use of Fe 2+ in the ferrous reaction reagent
instead of
Fe 3+ renders it unnecessary to use ascorbic acid. Ascorbic acid is relatively
unstable,
and causes Halliwell's reagent to be unstable. In contrast, the ferrous
reaction
reagent of the present invention, comprising Fe2+, has improved stability.
Surprisingly, it was also found that the use of 2-deoxyglucose and Fe 2+
instead of 2-deoxyribose and Fe3+ as used by Halliwell, permits the faster and
more
accurate measurement of oxidant levels.
While it is not intended to limit the invention to any particular theory of
action, it is believed that many strong oxidants can interact both directly
and also
indirectly with thiobarbituric acid ("TBA") by the formation of strong
oxidants resulting
from the reaction of the biological oxidants with 2-deoxyglucose, mediated by
ferrous
(Fe 2+) ions.
The present invention permits oxidative stress to be determined using only
basic laboratory equipment and without requiring invasive procedures such as
intubation. As used herein, the term "minimal method" refers to a method for
determining oxidative stress using only basic laboratory equipment (such as a
single
beam spectrophotometer and graduated cylinders) and capable of being performed
without the introduction of instruments or foreign materials into the body of
the subject.
The present invention also provides a method for assaying oxidative
stress in biological fluids using a solid matrix-associated assay system, such
as a
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"dipstick" method. Such an assay, and a kit for use in such an assay, are
suitable for
use in homes and veterinary clinic settings, and other locations where
sophisticated
laboratory equipment is not readily available.
In one embodiment of the invention there is provided a method for
determining overall oxidative stress comprising the steps of mixing a
biological fluid
of interest with a reaction reagent, and incubating the mixture for an
appropriate time,
following which time the formation of a coloured reaction product is
determined. In
one embodiment of the invention, light absorption at a wavelength of 532 nM is
measured to determine the extent of coloured product formation. Standard
curves
based on known hydrogen peroxide concentrations can be used to determine the
peroxidant equivalent oxidant concentration in biological fluids and such
standard
curves may be prepared by techniques known in the art.
The ferrous reaction reagent preferably comprises a solution of 2-
deoxyglucose, TBA, EDTA, and ferrous sulphate in a suitable solvent, such as
distilled water or a suitable buffer such as a physiological potassium
phosphate buffer.
The ferrous reaction reagent preferably comprises 2-deoxyglucose in a
concentration
of between about 30 and 400 mM, more preferably between about 50 and 200 mM,
even more preferably between about 75 and 150 mM and most preferably of about
100 mM. The ferrous reaction reagent preferably comprises TBA in a
concentration
of between about 10 and 200 mM, more preferably about 20 and 100 mM, even more
preferably between about 40 and 75 mM, most preferably about 50 mM. The
ferrous
reaction reagent preferably comprises EDTA at a concentration of between about
0.5
and 3 mM, more preferably between about 0.7 and 2 mM, even more preferably
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between about 1.0 and 1.6 mM, most preferably of about 1.4 mM. The ferrous
reaction reagent preferably comprises a concentration of ferrous sulphate of
between
about 0.5 and 2 mM, more preferably of between 0.75 and 1.5 mM, and most
preferably of about 1 mM. In an embodiment of the invention, there is an
excess of
Fee+, thus there is no need for an electron donor, thereby enhancing the
chemical
stability of the reaction reagent.
The biological fluid is preferably urine or plasma, although other suitable
biological fluids such as bioreactor medium and respiratory aspirates may be
employed. A suitable number of parts of the biological fluid is preferably
combined
with a suitable number of parts of the reaction reagent.
The biological fluid is preferably assayed within 2 hours of its release from
the body of the subject, more preferably within 1 hour and most preferably
within 15
minutes of its escape. However, it is also effective to store biological fluid
at a
reduced temperature promptly following its release. For example, urine can be
stored
at -20 C for at least 6 months, and plasma can be stored at 4 C for 10 days, -
20 C for
60 days and -70 C for one year without substantially impairing the usefulness
of the
assay. Longer storage times may affect the accuracy of the results obtained,
and
results from such samples must be examined with reference to oxidant level
changes
in the fluid during storage, which can be determined in light of the
disclosure herein.
When the ferrous reaction reagent comprises 100 mM 2-deoxyglucose,
50 mM TBA, 1.4 mM EDTA and 1 mM FeSO4, the final ratio of ferrous reaction
reagent
to biological fluid is preferably between about 20:1 and 1:10. Thus, examples
of a
suitable number of parts of ferrous reaction reagent and a suitable number of
parts of
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biological fluid are one part biological fluid mixed with between 5 and 15
parts of the
reaction reagent. More suitably, one part biological fluid is mixed with
between 7 and
11 parts of the reaction reagent.
A suitable number of parts of biological fluid to be combined with a
particular number of parts of ferrous reaction reagent can be determined by
one
skilled in the art, in light of the disclosure herein, the dilution of the
biological fluid, the
optical density of the biological fluid, and the concentrations of 2-
deoxyglucose, TBA,
EDTA and ferrous sulphate in the reaction reagent.
The mixture of the biological fluid and the ferrous reaction reagent is
preferably incubated at between about 20 and 45 C, more preferably between 25
and
40 C, more preferably at about 37 C. The incubation time is preferably between
about 0 minutes and 24 hours, more preferably between about 1 minute and 4
hours,
even more preferably between about 5 minutes and 30 minutes, and most
preferably
about 15 minutes.
A solid-support reaction system may be employed by absorbing the
reaction reagent on a solid support, such as polyacrylamide gel beads or other
suitably
inert and porous material, and subsequently exposing the biological fluid or a
suitable
dilution thereof to the reagent absorbed on the solid support. For example,
suitable
solid supports include calcium matrices, silica matrices, and porous foam. In
one
embodiment of the invention, solid support material is immobilized on a non-
porous
frame, such as a plastic dipstick.
The reaction reagent is preferably absorbed to a solid support and the
reagent-support combination sealed in a reduced oxygen environment for use at
a
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later date. In particular, the combination can be sold together with a
reference
standard providing one or more colour references for oxidant levels as a kit.
In one embodiment, the reaction reagent is absorbed onto a solid support
which is dipped in freshly captured urine or held in the urine stream of a
patient.
Similarly, where the biological fluid is respiratory aspirates, the reagent-
support
combination is preferably held in the stream of the subject's respiratory
release,
permitting respiratory aspirants to mix with the reaction reagent.
Alternatively,
respiratory aspirates are readily collected from intubated subjects, and
subjects
wearing masks such as face oxygen masks. The patient would then compare the
colour change observed to one or more reference standards. In particular, a
predominantly light pink colour indicates a normal level of oxidative stress
whereas a
predominantly dark pink, red, or brown colour, indicates significant oxidative
stress
warranting medical examination.
Preferably, the subject compares the reaction dipstick to the reference
standard between about 0 minutes to 60 days after exposing the dipstick to the
biological fluid, more preferably between about 0.5 minutes and 1 day, and
even more
preferably between about 10 minutes and 24 hours after exposing the dipstick
to the
biological fluid.
Preferably, the biological fluid is exposed to the reaction reagent
immediately following release of the biological fluid from the subject's body.
However,
storage of samples at reduced temperatures for later analysis is possible, as
previously discussed.
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Example 1 - Preparation of Standard Curve
A ferrous reaction reagent comprising 100 mM 2-deoxyglucose, 50 mM
thiobarbituric acid, 1.4 mM EDTA, and 1 mM ferrous sulphate in triple
distilled water
was prepared. Standard concentrations of hydrogen peroxide, ranging from 1 pM
to
250 pM were prepared in triple distilled water.
500 pL of each hydrogen peroxide standard were added to 500 pL each
of the reaction reagent, and the mixtures were incubated at 37 C for 15
minutes. The
absorbence of the mixtures at 532 nM was measured after 15 minutes incubation.
The results of this experiment are shown in Figure 1, which shows that
absorbence
increased in a linear manner with increasing hydrogen peroxide concentration.
Example 2 - Analysis of Oxidant Levels in Plasma and Urine
Plasma was obtained from whole human blood by standard techniques.
Plasma was diluted to a fraction of its initial concentration (defined as I")
in 0.01M
potassium phosphate buffer (pH7.4). The peroxide-equivalent overall oxidant
level
was determined, by mixing 100 pL of each plasma dilution with 900 pL of the
ferrous
reaction reagent prepared as in Example 1, incubating the mixtures at 37 C for
15
minutes, and measuring absorbence at 532 nM after 15 minutes. The results are
depicted in Figure 2 (Numbers on the x-axis refer to the dilution of the
plasma. "0"
refers to no plasma present, "0.1" indicates a 10-time dilution and "0.25"
indicates a
4-time dilution. The y-axis depicts the oxidant "units" present in samples,
wherein 1
unit = 5 pM hydrogen peroxide equivalent.) The results indicated that the
assay of the
present invention is an accurate measure of overall oxidant levels in plasma.
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Example 3 - Use of Assay in Predicting Clinical Outcome
The oxidant concentration in urine of patients admitted to hospital for
various conditions was assessed using the method of the present invention, and
was
compared to the oxidant concentration in urine obtained from members of the
general
population.
Hospital patients had urine oxidant concentrations ranging from about 0
units to 1060 units (1 unit = 5 pM hydrogen peroxide equivalent), with a
median of 102
units. The values for the general population ranged from 3 units to 163 units,
with a
median of 62 units. The hospital patients had significantly higher levels of
oxidants
in their urine than did the general population (2-tailed Mann-Whitney test,
p=0.0022).
Figure 3a depicts individual patient oxidant levels, in comparison to oxidant
levels from
individual samples from the general population. Figure 3b depicts oxidant
levels in
patients, with reference to illness or condition.
It is apparent from Figures 3a and 3b that subjects having a urine
peroxide equivalent level in excess of 180 units will frequently be in need of
medical
attention. Furthermore, it is apparent that the assay of the present invention
is useful
in identifying individuals warranting medical examination. In particular,
subjects having
urine peroxide equivalents in excess of 100 units warrant medical examination
as
there is a substantial likelihood of illness or injury. Subjects having urine
peroxide
equivalents in excess of 200 units warrant extensive medical examination, as
there is
a strong possibility of illness or injury.
Thus, the assay of the present invention is useful in identifying subjects
having an underlying medical condition, and in identifying subjects warranting
further
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medical examination.
Example 4 - Solid-Support Assay for Overall Oxidative Stress
Dry polyacrylamide gel (BIOGEL, trade-mark) was reconstituted in a
ferrous reaction reagent comprising 100 mM 2-deoxyglucose, 50 mM TBA, 1.4 mM
EDTA and 1 mM ferrous sulfate in potassium phosphate buffer (pH7.4). One
tablespoon of BIOGELTM was placed in 100 ml of the ferrous reaction reagent
and
allowed to soak at 4 C overnight. The reconstituted BIOGELTM (polyacrylamide)
gel
was added to a disposable polypropylene column. 0.5 ml of urine was added to
the
column and allowed to diffuse, the column was incubated at 37 C for 10 minutes
and
the colour change in the column was compared to the visually apparent colour
change
in columns to which solutions containing known amounts of hydrogen peroxide
were
added. The colour in the column was stable for more than one month.
The results indicate that it is possible to measure overall oxidative stress
in biological fluids using an immobilized reaction reagent according to the
method of
the present invention.
Figure 4 depicts the results for 3 urine samples analyzed according to the
method of Example 4. Tubes 1 and 3 depict the colour reaction of urine from
the
general population, whereas tube 2 depicts the visibly greater colour reaction
of a
patient experiencing extreme oxidative stress and suffering from severe
pancreatitis.
Thus, this method permits the ready identification of subjects experiencing
significant
oxidative stress and subjects warranting medical examination based on visual
analysis
of the colour reaction. The solid support method is suitable for identifying
subjects
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having peroxide-equivalent oxidant levels in the biological fluid assayed of
at least 100
units. Thus, this method is useful in identifying subjects warranting further
medical
attention.
The method of the present invention allows the accurate detection of
overall oxidative stress using biological fluids in cases where the oxidant
levels in the
biological fluid are as low as 5 units. Additionally, it is possible to detect
and roughly
quantify oxidant levels in biological fluids visually using ferrous reaction
reagent
absorbed or otherwise maintained on a solid matrix such as a polyacrylamide
gel
column, indicating that a "dipstick" approach is feasible, as is a home test
kit.
The ferrous reaction reagent of the present invention is relatively stable,
and may be manufactured and sold in appropriate packaging for use in remote
laboratories and veterinary clinics. Additionally, the ferrous reaction
reagent may be
immobilized on a suitable solid support, packaged in a substantially oxidant-
free
package, and sold for home use.
Thus, it is apparent that there has been provided a rapid assay for the
measurement of overall oxidative stress.
While the invention has been described in conjunction with illustrated
embodiments thereof, it is evident that many alternatives, modifications and
variations
will be apparent to those skilled in the art in light of the foregoing
description.
Accordingly, it is intended to embrace all such alternatives, modifications
and
variations as fall within the spirit and broad scope of the invention.
