U.S. patent application number 12/757702 was filed with the patent office on 2010-11-18 for compositions and methods for simultaneous detection of volatile sulfur compounds and polyamines.
This patent application is currently assigned to ALT BIOSCIENCE, LLC.. Invention is credited to Boyd E. Haley, James C. Pendergrass.
Application Number | 20100291699 12/757702 |
Document ID | / |
Family ID | 32092950 |
Filed Date | 2010-11-18 |
United States Patent
Application |
20100291699 |
Kind Code |
A1 |
Pendergrass; James C. ; et
al. |
November 18, 2010 |
COMPOSITIONS AND METHODS FOR SIMULTANEOUS DETECTION OF VOLATILE
SULFUR COMPOUNDS AND POLYAMINES
Abstract
Disclosed are compositions and methods useful for the rapid and
facile simultaneous detection of malodorous bacterial metabolites
in samples of expired breath and other fluids. The invention
enables estimation, by simple visual inspection and comparison
against standards, of the concentration of polyamines and volatile
sulfur compounds in the micromolar to millimolar range.
Inventors: |
Pendergrass; James C.;
(Lexington, KY) ; Haley; Boyd E.; (Lexington,
KY) |
Correspondence
Address: |
VIKSNINS HARRIS & PADYS PLLP
P.O. BOX 111098
ST. PAUL
MN
55111-1098
US
|
Assignee: |
ALT BIOSCIENCE, LLC.
Lexington
KY
|
Family ID: |
32092950 |
Appl. No.: |
12/757702 |
Filed: |
April 9, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10274058 |
Oct 18, 2002 |
7723118 |
|
|
12757702 |
|
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Current U.S.
Class: |
436/111 |
Current CPC
Class: |
Y10T 436/18 20150115;
G01N 31/22 20130101; G01N 33/52 20130101; C12Q 1/04 20130101; Y10T
436/173845 20150115 |
Class at
Publication: |
436/111 |
International
Class: |
G01N 33/00 20060101
G01N033/00 |
Claims
1. A chromogenic composition for intermixture or contacting with a
fluid in which fluid it is desired to determine the concentration
of VSCs and polyamines, said composition comprising a first
reagent, said first reagent capable of reacting with a polyamine to
form a VSC, and a second reagent, said second reagent capable of
reacting with a VSC to form a product, said product possessing an
absorption maximum in the visible portion of the electromagnetic
spectrum.
2-42. (canceled)
Description
FIELD OF THE INVENTION
[0001] The invention relates to, among other fields, the field of
diagnostic tests to detect the presence of pathogenic
microorganisms.
BACKGROUND OF THE INVENTION
[0002] In normal circumstances many microorganisms live on or in
humans without causing harm to these humans. However, an increased
number of certain microorganisms can be associated with a disease
state. Particularly problematic is an increase in the number of
microorganisms that produce significant concentrations of chemicals
that are toxic to humans. Such toxins include: volatile sulfur
compounds (VSCs), such as hydrogen sulfide, methyl mercaptan,
dimethyl disulfide, and dimethyl sulfide; and polyamines, such as
putrescine and cadaverine. Higher concentrations of volatile sulfur
compounds and of polyamines are associated with certain disease
states and with socially stigmatized conditions, such as halitosis,
or bad breath. It would be desirable to enable facile simultaneous
detection of volatile sulfur compounds and polyamines in order to
facilitate detection of the presence of potentially pathogenic
microorganisms and of disease states and conditions with which
these microorganisms are associated.
[0003] The principal VSCs produced by oral bacteria are hydrogen
sulfide (H.sub.2S) and methylmercaptan (CH.sub.3SH). H.sub.2S is
produced by the bacterial enzyme L-cysteine desulthydrase, which
degrades the amino acid L-cysteine to produce H.sub.2S, ammonium
and 2-ketopropanate. The other major VSC, CH.sub.3SH, is produced
by the action of the bacterial enzyme L-methionine gamma lyase,
which breaks down the amino acid L-methionine to produce
CH.sub.3SH, ammonium and 2-ketobutyrate. H.sub.2S levels have been
reported to occur at levels as high as 2 mM in the gingival crevice
of persons with severe periodontal disease.
[0004] In addition to VSCs, oral bacteria have also been shown to
produce high levels of polyamines; chief among these are
1,4-diaminobutane (putrescine) and 1,5-diaminopentane (cadaverine).
Putrescine is produced by the action of the bacterial enzyme
ornithine decarboxylase, which degrades the amino acid L-ornithine
to produce putrescine and carbon dioxide. Cadaverine is produced by
the bacterial enzyme lysine decarboxylase, which breaks down the
amino acid L-lysine to cadaverine and carbon dioxide.
[0005] Millimolar levels of these malodorous bacterial waste
products have been reported in the gingival crevicular fluid (GCF)
of persons with severe periodontal disease. Their presence imparts
a foul odor to the breath of persons with the disease. In addition
to being malodorous, VSCs have also been shown to be toxic and
adversely affect a number of mammalian enzymes. Production of these
waste products by bacteria residing on the dorsum of the tongue
imparts a foul odor to expired breath, resulting in halitosis.
Together these two classes of bacterial waste products combine to
impart a foul odor to the breath of persons with halitosis and
periodontal disease. The levels of these waste products in GCF
correlate directly with the level of periodontal disease activity.
The levels of these waste products in expired air correlate
directly with the level of bad breath or halitosis.
SUMMARY OF THE INVENTION
[0006] Accordingly, the invention provides compositions and methods
for simultaneous detection of volatile sulfur compounds and
polyamines.
[0007] The invention provides inter alia a chromogenic
(colorimetric) biochemical assay to detect volatile sulfur
compounds (VSCs) and polyamines produced by oral bacteria residing
on the dorsum of the tongue and in the gingival crevice.
[0008] The invention provides inter alia an assay to detect VSCs
and polyamines in saliva and expired breath.
[0009] In an aspect of the invention, the absorbance of a
chromogenic composition according to the invention correlates
directly with the concentration of VSCs and polyamines in a fluid
with which the composition is intermixed.
DETAILED DESCRIPTION OF THE INVENTION
[0010] At the outset of the description, it is helpful to note the
meaning of each of several terms recurring throughout the
specification and claims.
[0011] When used in connection with the invention, a "volatile
sulfur compound" or "VSC" refers to a composition having a thiol or
--SH functional group or to a composition having a thiolate or
sulfide anion. Examples of VSCs include hydrogen sulfide and methyl
mercaptan (methanethiol).
[0012] When used in connection with the invention, "polyamine" has
its usual meaning in the biochemical arts and includes a
composition of the general formula NH.sub.2RNH.sub.2. Examples of
polyamines include putrescine and cadaverine.
[0013] When used in connection with the invention, "DTNB" refers to
5,5'-Dithiobis(2-nitrobenzoic acid). DTNB is also known as Ellman's
reagent and has CAS number 69-78-3.
[0014] When used in connection with the invention, "2-IT" refers to
2-Iminothiolane. 2-Iminothiolane hydrochloride is known as Traut's
reagent and has CAS number 4781-83-3.
[0015] The invention provides inter alia a chromogenic
(colorimetric) biochemical assay to detect volatile sulfur
compounds (VSCs) and polyamines produced by oral bacteria residing
on the dorsum of the tongue and in the gingival crevice.
[0016] The invention provides inter alia an assay to detect VSCs
and polyamines in saliva and expired breath.
[0017] In an aspect of the invention, the absorbance of a
chromogenic composition according to the invention correlates
directly with the concentration of VSCs and polyamines in a fluid
with which the composition is intermixed.
[0018] In an embodiment, the invention provides a chemically
stabilized, buffered composition comprising
5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB) to detect VSCs and
2-Iminothiolane (2-IT) to detect polyamines. In this regard, DTNB
and other so-called sulfhydryl reagents are examples of
VSC-reactive means, and 2-IT and other thiolating reagents which
modify primary amino groups are examples of polyamine-reactive
means.
[0019] In a particular embodiment, a composition according to the
invention contains 0.5 millimolar (0.5 mM) DTNB and 0.5 mM 2-IT in
20 mM imidazole or 20 mM sodium phosphate, pH 7.0 buffer containing
0.1 mM manganese chloride (MnCl.sub.2). Such composition is
referred to infra as "reagent mixture." Without intending to be
bound by theory, it is observed that, in such an embodiment, such
concentration of MnCl.sub.2 and such buffer and such pH are
associated with particularly favorable stability, such that the
shelf-life of the reagent mixture is 12 months or longer when
stored at room temperature. A similar embodiment, in which a
composition according to the invention contains 0.1 mM cobalt
chloride or 0.1 mM magnesium chloride instead of the aforementioned
0.1 mM manganese chloride, also possesses particularly favorable
stability. In this regard, imidazole buffer around pH 7, phosphate
buffer around pH 7, manganese chloride, magnesium chloride, and
cobalt chloride are examples of shelf-life-enhancing means. Theory
notwithstanding, it is noted that magnesium is an alkaline earth
metal and that managese and cobalt are transition metals.
[0020] Without intending to be bound by theory, it is known in the
art that DTNB reacts with the sulfide or thiolate anion formed from
the --SH functional group present in H.sub.2S or CH.sub.3SH. In
such reaction, one of the disulfide sulfurs of DTNB is attacked by
the HS.sup.- or CH.sub.3S.sup.- anion, whereupon the
5-thio-2-nitrobenzoate anion (TNB) is released. TNB exists in
equilibrium with the tautomeric thioquinone. The thioquinone has an
absorption (absorbance) maximum at 412 nm with a molar extinction
coefficient of 13,600 M.sup.-1 cm.sup.-1. This allows for the
reaction to be quantified spectrophotometrically or visually by the
increase in the yellow color of the mixture.
[0021] While in the art the colorimetric reaction of DTNB may be
regarded as optimally performed at pH 8.0, DTNB is unstable when
stored for several days at room temperature under such conditions.
It was unexpectedly found that the inclusion of metal salt and the
adjustment of pH around neutrality impart the favorable property of
a markedly extended shelf-life. By way of example and not of
limitation, it was found that a DTNB mixture was maintained stable
for more than one year at room temperature when the mixture
included phosphate or imidazole buffer, pH 7.0, and 0.1 mM
MnCl.sub.2. Hence the invention provides inter alia a chromogenic
composition the absorbance of which correlates directly with the
concentration of VSCs in a fluid with which the composition is
intermixed and which composition has a shelf-life of at least one
year.
[0022] It is known in the art that 2-IT thiolates primary amines
under appropriate conditions. A composition according to the
invention is useful for the detection of primary amines, including
polyamines, such as the malodorous diamines putrescine and
cadaverine. When a composition according to the invention is
intermixed or otherwise contacted with a sample that contains
diamines, 2-IT of the composition reacts with primary amino
(--NH.sub.2) groups of the sample's diamines to yield a reaction
product with at least one free sulfhydryl group. Such a free
sulfhydryl group, or its thiolate anion, reacts with DTNB of the
reagent mixture, releasing the TNB anion and causing an increase in
absorbance at 412 nm.
[0023] Compositions and methods according to the invention are
useful for detecting the presence of malodorous toxins produced by
bacteria residing on the dorsum of the tongue and in GCF obtained
from the gingival sulcus.
[0024] For example, in an aspect of a method according to the
invention, tongue scrapings are obtained by gently rubbing the
cotton tip of a sterile, cotton-tipped applicator against the
dorsum of the tongue to form a scraping-laden applicator tip. The
scraping-laden applicator tip is then contacted for 2 minutes at
room temperature with a volume of 0.1 ml of reagent mixture
contained within a sterile, screw-capped 1.5 ml microcentrifuge
tube, forming a chromogen-developed applicator tip. The color of
the chromogen-developed applicator tip is compared to standards on
a color-coded chart. The chart is scaled in shades of yellow from
LOW (light yellow) corresponding to <0.2 mM H.sub.2S to HIGH
(bright yellow) corresponding to >2 mM H.sub.2S.
[0025] Also by way of example, in an aspect of a method according
to the invention, for detecting the presence of VSCs and polyamines
in GCF, a GCF sample is obtained by inserting a sterile paper
absorbant point into the gingival sulcus and waiting 1 minute for
the GCF sample to absorb, thereby forming a GCF-laden paper point.
The GCF-laden paper point is then contacted for 5 minutes at room
temperature with a volume of 0.1 ml of reagent mixture contained
within a sterile, screw-capped 1.5 ml microcentrifuge tube, forming
a chromogen-developed paper point. The color of the
chromogen-developed paper point is compared to a standard color
chart calibrated linearly from 0 (none detectable) to 5 (extreme),
corresponding to 2 mM hydrogen sulfide.
[0026] Also by way of example, in an aspect of a method according
to the invention, VSCs and polyamines are detected and quantified
spectrophotometrically through use of the reagent mixture. In a
microcentrifuge tube, a GCF-laden paper point is submersed in 450
microliters of 20 mM Tris-HCl, pH 7.75 buffer. When it is desired
to perform a spectrophotometric assay according to the invention, a
volume of 50 microliters of a 10.times. concentrated reagent
mixture in 50% DMSO, 50 mM imidazole, pH 6.4 buffer containing 1.0
mM MnCl.sub.2 is added to and intermixed with the contents of the
microcentrifuge tube, and the resultant mixture is incubated at
room temperature for 8 minutes. Afterwards, the incubated resultant
mixture is transferred to a semi-micro 1.5 ml disposable plastic
cuvet, for example by means of a disposable transfer pipette. The
concentration of VSC and polyamines is determined by measuring the
absorbance of the incubated resultant mixture through use of a
spectrophotometer or colorimeter and by comparison of the measured
absorbance to a reference standard. By measuring the absorbance of
a negative control or a blank, background absorbance readings are
obtained for use in comparison to the measured absorbance of the
incubated resultant mixture.
[0027] A method of detection of VSCs and polyamines in GCF
according to the invention, such as described in either of the two
immediately preceding paragraphs, is observed to possess a lower
limit of detection of no more than about 0.02 mM H.sub.2S in a
sample, and an upper limit of responsive range near about 2 mM
H.sub.2S. If the sample contains polyamines but essentially no
VSCs, this 0.02 mM lower limit corresponds to a concentration of no
more than about 0.02 mM polyamines in the sample. If the sample
contains VSCs but essentially no polyamines, this lower limit
corresponds to a concentration of no more than about 0.02 mM VSCs
in the sample. For detection of polyamines and VSCs according to
the invention, a mode of carrying out the invention whereby a
composition according to the invention is prepared using, as
solvent, reagent grade nanopure water, wherein concentrations of
adventitious metals are extremely low, is in general to be
preferred.
[0028] The invention also provides methods for detection of VSCs
and polyamines in saliva and expired breath.
[0029] For example, in an aspect of a method according to the
invention, for saliva testing, a filter paper disk is impregnated
with the reagent mixture by contacting the disk with the reagent
mixture, and allowing excess solvent to evaporate, to form an
impregnated disk. The impregnated disk is affixed to the bottom
interior of a sterile, graduated specimen cup. A volume of saliva
is then spit by a subject onto the impregnated disk in the specimen
cup. Reaction is allowed to proceed in and on the impregnated disk.
After a fixed period of time, the concentration of VSCs and
polyamines is determined by comparison of the color developed on
the surface of the impregnated disk against a standard such as a
scaled color chart.
[0030] For example, in an aspect of a method according to the
invention, for breath testing, a volume of 0.1 ml of reagent
mixture is contacted with a filter paper disk, and excess solvent
is allowed to evaporate, to form an impregnated disk. The
impregnated disk is then affixed to the distal aperture of a
cylindrical tube, said tube having an aperture at each end, the
proximal aperture being of such length and width, for example, that
a subject might conveniently expire breath through the proximal
aperture, and the distal aperture being of such length and width,
for example, that the impregnated disk covers the entire distal
aperture. A subject then expires breath into the tube through the
proximal aperture of the tube and toward the distal aperture of the
tube, at least a portion of the expired breath contacting the
impregnated disk affixed to the distal aperture of the tube.
Reaction is allowed to proceed in and on the impregnated disk.
After a fixed period of time, the concentration of VSCs and
polyamines is determined by comparison of the color developed on
the surface of the impregnated disk against a standard such as a
scaled color chart.
[0031] While filter paper is an exemplary matrix according to the
invention, cotton is also a suitable matrix according to the
invention, as is any of a number of other inert matrices, such as
those derived from polysaccharides which are low in primary amino
groups.
[0032] When DTNB and 2-IT are included in a MnCl.sub.2-stabilized
buffered mixture according to the invention, the levels of two
principal classes of anaerobic bacterial waste products are
conveniently detected and quantified either visually with a yellow
color scale or spectrophotometrically with a colorimeter or
spectrophotometer at 412 nm. The simplicity of the use of
compositions and methods according to the invention facilitates
in-office testing by health care professionals and home testing by
patients for halitosis and periodontal disease. Compositions and
methods according to the invention thus are useful for the
detection of VSCs in samples comprising air, water, oil, or fluids
in contact with foodstuffs or foods, and hence are also useful for
the detection of products of microbial metabolism, pathogenic
microorganisms, halitosis, VSC pollution, or food spoilage or
contamination.
[0033] The invention provides a diagnostic composition comprising
2-IT, DTNB, a solvent, a buffer, and a metal salt.
[0034] The invention provides a neutral diagnostic solution
comprising from about 0.1 to about 2.5 millimoles of 2-IT per liter
of the solution, from about 0.1 to about 2.5 millimoles of DTNB per
liter of the solution, from about 0.02 to about 0.5 millimoles of
MnCl.sub.2 or MgCl.sub.2 or CoCl.sub.2 per liter of the solution,
and (a) from about 4 to about 100 millimoles of imidazole per liter
of the solution or (b) from about 4 to about 100 millimoles of
sodium phosphate per liter of the solution.
[0035] The invention provides a method of detecting the presence of
a VSC or a primary amine in a sample of a physiological fluid, said
method comprising the step of contacting the sample with a
composition comprising 2-IT, DTNB, a solvent, a buffer, and a metal
salt.
[0036] The invention provides a method of detecting the presence of
a VSC or a primary amine in a sample of a physiological fluid, said
method comprising the step of contacting the sample with a neutral
diagnostic solution comprising from about 0.1 to about 2.5
millimoles of 2-IT per liter of the solution, from about 0.1 to
about 2.5 millimoles of DTNB per liter of the solution, from about
0.02 to about 0.5 millimoles of MnCl.sub.2 or MgCl.sub.2 or
CoCl.sub.2 per liter of the solution, and (a) from about 4 to about
100 millimoles of imidazole per liter of the solution or (b) from
about 4 to about 100 millimoles of sodium phosphate per liter of
the solution.
[0037] The invention provides a chromogen, the color of which
chromogen develops more rapidly after the chromogen is contacted
with a first mixture comprising at least 20 micromolar polyamine or
at least 20 micromolar VSC than the color develops in a second
mixture identical in composition to the first mixture except that
the second mixture comprises no more than 1 micromolar polyamine
and no more than 1 micromolar VSC.
[0038] The invention provides a chromogen, the color of which
chromogen develops more rapidly after the chromogen is contacted
with a first mixture comprising at least 20 micromolar polyamine or
at least 20 micromolar VSC than the color develops in a second
mixture identical in composition to the first mixture except that
the second mixture comprises no more than 1 micromolar polyamine
and no more than 1 micromolar VSC, wherein a neutral solution of
the chromogen has a shelf-life of at least one year.
[0039] The invention provides a chromogen, the color of which
chromogen develops more rapidly after the chromogen is contacted
with a first mixture comprising at least 20 micromolar polyamine or
at least 20 micromolar VSC than the color develops in a second
mixture identical in composition to the first mixture except that
the second mixture comprises no more than 1 micromolar polyamine
and no more than 1 micromolar VSC, wherein the color of the
developed chromogen is yellow.
[0040] The invention provides a chromogen, the color of which
chromogen develops more rapidly after the chromogen is contacted
with a first mixture comprising at least 20 micromolar polyamine or
at least 20 micromolar VSC than the color develops in a second
mixture identical in composition to the first mixture except that
the second mixture comprises no more than 1 micromolar polyamine
and no more than 1 micromolar VSC, wherein the chromogen comprises
2-IT and DTNB.
[0041] The invention provides a test kit comprising a chromogen,
the color of which chromogen develops more rapidly after the
chromogen is contacted with a first mixture comprising at least 20
micromolar polyamine or at least 20 micromolar VSC than the color
develops in a second mixture identical in composition to the first
mixture except that the second mixture comprises no more than 1
micromolar polyamine and no more than 1 micromolar VSC.
[0042] The invention provides a test kit comprising a chromogen,
the color of which chromogen develops more rapidly after the
chromogen is contacted with a first mixture comprising at least 20
micromolar polyamine or at least 20 micromolar VSC than the color
develops in a second mixture identical in composition to the first
mixture except that the second mixture comprises no more than 1
micromolar polyamine and no more than 1 micromolar VSC, wherein the
test kit has a shelf-life of at least one year.
[0043] The invention provides a method of detecting the presence of
a microorganism in a sample of a physiological fluid, said method
comprising contacting the sample with a chromogen, the color of
which chromogen develops more rapidly after the chromogen is
contacted with a first mixture comprising at least 20 micromolar
polyamine or at least 20 micromolar VSC than the color develops in
a second mixture identical in composition to the first mixture
except that the second mixture comprises no more than 1 micromolar
polyamine and no more than 1 micromolar VSC.
[0044] The invention provides a method of detecting the presence of
a microorganism in a sample of a physiological fluid, said method
comprising contacting the sample with a chromogen, the color of
which chromogen develops more rapidly after the chromogen is
contacted with a first mixture comprising at least 20 micromolar
polyamine or at least 20 micromolar VSC than the color develops in
a second mixture identical in composition to the first mixture that
the second mixture comprises no more than 1 micromolar polyamine
and no more than 1 micromolar VSC, wherein the microorganism is an
anaerobe or a facultative aerobe.
[0045] The invention provides a method of detecting the presence of
a microorganism in a sample of a physiological fluid, said method
comprising contacting the sample with a chromogen, the color of
which chromogen develops more rapidly after the chromogen is
contacted with a first mixture comprising at least 20 micromolar
polyamine or at least 20 micromolar VSC than the color develops in
a second mixture identical in composition to the first mixture
except that the second mixture comprises no more than 1 micromolar
polyamine and no more than 1 micromolar VSC, wherein the
physiological fluid comprises GCF, saliva, tongue scrapings, or
expired breath.
[0046] It will therefore be readily understood by those persons
skilled in the art that the invention is susceptible of a broad
utility and application. Many embodiments and adaptations of the
invention other than those herein described, as well as many
variations, modifications and equivalent arrangements, will be
apparent from or reasonably suggested by the present invention and
the foregoing description thereof, without departing from the
substance or scope of the present invention. Accordingly, while the
invention has been described herein in detail in relation to its
preferred embodiment, it is to be understood that this disclosure
is only illustrative and exemplary of the invention and is made
merely for purposes of providing a full and enabling disclosure of
the invention. The foregoing disclosure is not intended or to be
construed to limit the invention or otherwise to exclude any such
other embodiments, adaptations, variations, modifications or
equivalent arrangements, the invention being limited only by the
claims appended hereto and the equivalents thereof.
* * * * *