Weight loss abstract
A method for monitoring the progress of fat loss in a patient during
a weight loss program which comprises, contacting a body fluid sample
from said patient with a solid test strip to provide a color indication
of the presence in said body fluid of .beta.-hydroxybutyrate, optionally
together with acetoacetate and/or acetone.
Weight loss claims
What is claimed is:
1. A solid test strip designed for use in monitoring weight loss
programs and capable of enabling a person to self-monitor weight
loss on a daily basis in a sample of urine, saliva or other bodily
fluid that is noninvasively obtainable, which provides a color signal,
indicative of the .beta.-hydroxybutyrate content of the sample upon
being dipped in the sample, removed, allowed to rest briefly and
read, which solid test strip comprises 1) an inert support layer
and 2) a dried reagent layer comprising a porous material impregnated
with a) .beta.-hydroxybutyrate dehydrogenase enzyme (".beta.-HBD),
which is either (I) obtained from Alcaligenes or another source
such that it is uninhibited by chloride ions or (ii) is obtained
from a source that is inhibited by chloride ions and is present
in a concentration at least 10 to 20 times that used when the .beta.-HBD
is obtained from a source such that it is uninhibited by chloride
ions, b) nicotinamide dinucleotide ("NAD"), c) a tetrazolium
dye precursor d) an electron mediator capable of transferring an
electron to said dye precursor to effect a color change, and e)
a sufficient amount of a buffer having a pH in excess of 8.5 to
maintain the reaction pH above 8.5 when the strip is saturated with
urine or other bodily fluid.
2. A solid test strip according to claim 1 wherein the electron
mediator is a diaphorase enzyme.
3. A solid test strip according to claim 1 wherein the tetrazolium
dye precursor is nitrobluetetrazolium ("NBT") or 2-(indophenyl)-3-(paranitrophenyl)-5-phenyl
tetrazolium chloride ("INT").
4. A solid test strip designed for use in monitoring weight loss
programs and capable of enabling a person to self-monitor weight
loss on a daily basis in a sample of urine, saliva or other non-invasively
obtainable bodily fluid, which provides a color signal indicative
of the combined .beta.-hydroxybutyrate and acetoacetate content
of the sample upon being dipped in the sample, removed, allowed
to rest briefly and read, which solid test strip comprises 1) an
inert support layer and 2) a dried reagent layer comprising a porous
material impregnated with a) .beta.-HBD enzyme, which is either
(i) obtained from Alcaligenes or another source such that it is
uninhibited by chloride ions or (ii) is obtained from a source that
is inhibited by chloride ions and is present in a concentration
at least 10 to 20 times that used when the .beta.-HBD is obtained
from a source such that it is uninhibited by chloride ions, b) NAD
c) a tetrazolium dye precursor d) an electron mediator capable of
transferring an electron to said dye precursor to effect a color
change and e) a sufficient quantity of a buffer having a pH of at
least 7.0 but less than 8.5, to maintain the reaction pH below 8.5
but not less than about 7.0 when the strip is saturated with urine
or other bodily fluid.
5. A solid test strip according to claim 4 wherein the electron
mediator is a diaphorase enzyme.
6. A solid test strip according to claim 4 wherein the tetrazolium
dye precursor is NBT or INT.
7. A solid test strip designed for use in monitoring weight loss
programs and capable of enabling a person to self-monitor weight
loss on a daily basis in a sample of urine, saliva or other non-invasively
obtainable bodily fluid, which provides a color signal indicative
of the combined .beta.-hydroxybutyrate and acetoacetate content
of the sample upon being dipped in the sample, removed, allowed
to rest briefly and read, which solid test strip comprises 1) an
inert support layer and 2) a dried reagent layer comprising a porous
material impregnated with a) .beta.-HBD enzyme, which is either
(i) obtained from Alcaligenes or another source such that it is
uninhibited by chloride ions or (ii) is obtained from a source that
is inhibited by chloride ions and is present in a concentration
at least 10 to 20 times that used when the .beta.-HBD is obtained
from a source such that it is uninhibited by chloride ions, b) NAD,
c) a nitroprusside salt or a diazonuim salt in a quantity sufficient
to react with both endogenous acetoacetate obtained by conversion
thereto of .beta.-hydroxybutyrate in the sample, and d) a sufficient
quantity of a buffer have a pH about 8.5 or higher to maintain the
strip at the same pH when saturated with sample.
8. A solid test strip according to claim 7 wherein the electron
mediator is a diaphorase enzyme.
9. A solid test strip according to claim 7 wherein the tetrazolium
dye precursor is NBT or INT.
10. A test strip according to claim 7 wherein ingredient (c) is
sodium nitroprusside.
11. A test strip according to claim 7 wherein ingredient (c) is
a diazonium salt.
12. A test strip according to claim 11 wherein ingredient (c) is
4-nitrobenzene-diazonium fluoborate.
13. A solid test strip designed for use in monitoring weight loss
programs and capable of enabling a person to self-monitor weight
loss on a daily basis in a sample of urine, saliva or other bodily
fluid that is noninvasively obtainable, which provides a color signal,
indicative of the total ketone bodies content of the sample upon
being dipped in the sample, removed, allowed to rest briefly and
read, which solid test strip comprises 1) an inert support layer
and 2) a dried reagent layer comprising a porous material impregnated
with a) .beta.-HBD b) NAD c) a nitroprusside salt or a diazonium
salt in sufficient quantity to (i) immediately react with the acetone
present in the sample and stabilize it against volatilization and
(ii) also react with the endogenous acetoacetate in the sample and
with acetoacetate obtained by the conversion thereto of .beta.-hydroxybutyrate
in the sample and d) an electron mediator e) a sufficient quantity
of a buffer having a pH of about 8.5 or higher to maintain the reaction
pH at the same level when the strip is saturated with sample.
14. A solid test strip according to claim 13 wherein the electron
mediator is a diaphorase enzyme.
15. A solid test strip according to claim 13 wherein the tetrazolium
dye precursor is NBT or INT.
16. A test strip according to claim 13 wherein ingredient (c) is
sodium nitroprusside.
17. A test strip according to claim 13 wherein ingredient (c) is
a diazonium salt.
18. A test strip according to claim 16 wherein ingredient (c) is
4-nitrobenzene-diazonium fluoborate.
19. A method for monitoring the level of .beta.-hydroxybutyrate
present in a sample of urine or another human bodily fluid that
can be noninvasively obtained, which comprises contacting said sample
with a mixture comprising the following ingredients: a) .beta.-HBD
which either (i) has been obtained from Alcaligenes or another source
such that it is not inhibited by chloride ions, or else (ii) has
been obtained from a source such that it is inhibited by chloride
ions and is present in an excess amount from 10 to 20 times the
concentration utilized when the (.beta.-HBD is not inhibited by
chloride ions, b) NAD c) a tetrazolium dye precursor, d) an electron
mediator and e) a buffer having a pH above 8.5
and measuring by electrochemical, spectrophotometric or fluoro
metric means, or by comparison of the develop color to a preestablish
color intensity standard, the amount of .beta.-hydroxyrate in the
sample.
20. A method according to claim 19 wherein the tetrazolium dye
precursor is NBT or INT.
21. A method according to claim 19 wherein the electron mediator
is a diaphoruse enzyme.
22. A method for monitoring the level of combined acetoacetate
and .beta.-hydroxybutyrate in a sample of human bodily fluid which
comprises contacting the sample with a mixture of the following
ingredients: a) .beta.-HBD b) NAD c) a tetrazolium dye precursor,
d) an electron mediator, and e) a buffer having a pH that is over
7.0 but less than 8.5,
and measuring by electrochemical, spectrophotometric or fluorometric
means, or by comparison of the color developed to a preestablished
color intensity standard, the combined amount of .beta.-hydroxybutyrate
and acetoacetate present in the sample.
23. A method according to claim 22 wherein the sample is urine
or another fluid that can be noninvasively obtained and the .beta.-HBD
is either (I) obtained from Alcaligenes or another source such that
it is not inhibited by chloride ions, (ii) or else has been obtained
from a source such that it is inhibited by chloride ions and is
present in an excess amount from about 10 to 20 times the amount
utilized when the .beta.-HBD is not inhibited by chloride ions.
24. A method according to claim 22 wherein the tetrazolium dye
precursor is NBT or INT.
25. A method according to claim 22 wherein the electron mediator
is a diaphorase enzyme.
26. A method for monitoring the level of combined acetoacetate
and hydroxybutyrate in a sample of human bodily fluid which comprises
contacting said sample with a mixture comprising the following ingredients:
a) .beta.-HBD, b) NAD, c) a nitroprusside salt or a diazonium salt
in a quantity sufficient to react with endogenous acetoacetate in
the sample and acetoacetate obtained by conversion thereto of .beta.-hydroxybutyrate
in the sample, and d) a buffer having a pH of about 8.5 or higher
and measuring by electrochemical, spectrophotometric or fluorometric
means, or by comparison of the color developed to a preestablished
color intensity standard, the amount of combined acetoacetate and
.beta.-hydroxybutyrate in the sample.
27. A method according to claim 26 wherein the sample is urine
or another fluid that can be noninvasively obtained and the .beta.-HBD
is either (i) obtained from Alcaligenes or another source such that
it is not inhibited by chloride ions, or else (ii) has been obtained
from a source such that it is inhibited by chloride ions and is
present in an excess amount from about 10 to 20 times the amount
utilized when the .beta.-HBD is not inhibited by chloride ions.
28. A method according to claim 26 wherein the tetrazolium dye
precursor is NBT or INT.
29. A method according to claim 26 wherein the electron mediator
is a diaphorase enzyme.
30. A method according to claim 26 wherein ingredient (c) is a
nitroprusside salt.
31. A method according to claim 26 wherein ingredient (c) is a
diazonium salt.
32. A method according to claim 31 wherein ingredient (c) is 4-nitrobenzene
diazonium fluoborate.
33. A method for monitoring the level of total ketone bodies in
a sample of human bodily fluid which comprises contacting said sample
with a mixture comprising the following ingredients: a) .beta.-HBD,
b) NAD, c) a nitroprusside or diazonium salt in an amount sufficient
to (i) react instantaneously with and stabilize acetone in the sample,
(ii) also react with endogenous acetoacetate in the sample and (iii)
also react with acetoacetate formed by conversion thereto of .beta.-hydroxybutyrate
in the sample, and d) a buffer having a pH of about 8.5 or higher,
and measuring by electrochemical, spectrophotometric or fluorometric
means, or by comparison of the color developed to a preestablished
color intensity standard the amount of total ketone bodies in the
sample.
34. A method according to claim 33 wherein the sample is urine
or another fluid that can be noninvasively obtained and the .beta.-HBD
is either (i) obtained from Alcaligenes or another source such that
it is not inhibited by chloride ions, or else (ii) has been obtained
from a source such that it is inhibited by chloride ions and is
present in an excess amount from about 10 to 20 times the amount
utilized when the .beta.-HBD is not inhibited by chloride ions.
35. A method according to claim 33 wherein the tetrazolium dye
precursor is NBT or INT.
36. A method according to claim 33 wherein the electron mediator
is a diaphorase enzyme.
37. A method according to claim 33 wherein ingredient (c) is a
nitroprusside salt.
38. A method according to claim 33 wherein ingredient (c) is a
diazonuim salt.
39. A method according to claim 38 wherein ingredient (c) is 4-nitrobenzene
diazonium fluoborate.
Weight loss description
BACKGROUND OF THE INVENTION
In Weight-Loss Programs, whether initiated as a diet and/or exercise
regiment due to obesity or as a treatment in certain diseases such
as diabetes, cardiovascular disorders or epilepsy, excess fat in
the body is metabolized into smaller chemical units, called ketone
bodies, comprised of three components: .beta.-hydroxybutyrate, acetoacetate
and acetone.
This invention relates to methods for the measurement of a biochemical
marker, ketone bodies i.e. (a) total ketone bodies consisting of
all three components i.e. .beta.-hydroxybutyrate, acetoacetate,
and acetone. (b) .beta.-hydroxybutyrate in conjunction with acetoacetate
or (c) .beta.-hydroxybutyrate alone. The invention particularly
relates to its convenient use at home to measure loss of fat during
weight-loss programs. The invention includes a disposable, convenient
test strip configuration as a solid-phase or dry-chemistry test,
which measures Total Ketone bodies, all three components--.beta.-hydroxybutyrate,
acetoacetate and acetone. Additionally, the invention relates to
a disposable strip which measures .beta.-hydroxybutyrate and acetoacetate
in one step, and a strip that measures .beta.-hydroxybutyrate alone
which when dipped in urine produces a positive signal, such as a
color indicative of fat loss during weight-loss program. The intensity
of the color on the strip is an indicator of relative concentration
of the analyte(s) present in the sample, thus relates to the relative
loss of fat. Such a non-invasive tool can prove to be very useful
as a psychological stimulator for a person who needs to lose weight.
There are millions of people who are obese and go on some of type
of diet, for example Weight-watcher.RTM., Jenny-Craig , NutriSystem,
Atkins diet, The New Beverly Hill Diet, Liquid diet, The Pritlin
Principle diet, in order to lose weight. However, the majority of
people who go on diets gain back all lost weight within a short
period of time. In the U.S. alone, it is estimated that more than
60% of the population is obese. Obesity is the leading cause of
many serious diseases such as diabetes, hypercholesterolimea that
eventually leads to kidney and liver failure.
Just mere weight loss measurement on a weighing scale during dieting
is not sufficient as overweight people must lose fat during dieting,
not protein. Therefore, it will be of great social and medical benefit
if a biochemical marker is invented which when used on a daily basis
can indicate to a person whether indeed fat loss is taking place
or not while one is dieting. Such a psychological tool can considerably
enhance the efforts of a person in losing weight as well as maintaining
his or her ideal weight.
It has been known that when body fat, i.e. fatty acid, is degraded,
which is the principal component of body fat, it breakdowns ultimately
into small molecules in the form of ketone bodies. Ketone bodies
consist of a group of three chemicals: .beta.-hydroxybutyrate, acetoacetate,
and acetone. .beta.-hydroxybutyrate is a major ketone body comprised
of about 75-80% of total ketone bodies, acetoacetate comprised of
about 20-25% of total ketone bodies and acetone which is present
only in trace quantities less than 2%). Because of acetone's low
concentration and its instability, it is seldom measured by itself.
Instead, acetoacetate and acetone are measured by a nitroprusside
reaction in alkaline conditions. Several patents describe methods
and devices for measurement of acetoacetate using nitroprusside
reaction, such as U.S. Pat. No. 3,212,855 to Mast; U.S. Pat. No.
4,147,514 to Magers and Tabb; U.S. Pat. No. 4,440,724 to Tabb and
Burrows. Acetoacetate test strips have been commercially available
for many years (Bayer diagnostics, Roche diagnostics), which measure
acetoacetate and acetone in urine. They produce purple color and
the intensity of the color approximates the concentration of acetoacetate
present in urine or serum. These strips are generally used by a
patient who has diabetes (especially Type I diabetic patients).
These strips are erroneously referred as to "Test for Ketone
or Ketone body", even though they only measure the minor component
of ketone bodies i.e. acetoacetate and acetate which comprise less
than 20-25% of total ketone bodies and these strips do not measure
the major component, that is, .beta.-hydroxybutyrate at all. Even
though, these strips are insensitive to .beta.-hydroxybutyrate,
such strips have been successfully used in some diet programs such
as the Atkins' diet that is comprised of high fat and very low carbohydrates.
Similarly, U.S. Pat. No. 5,260,219 to Fritz teaches the use of
test strips in the measurement of acetoacetate in diet programs.
Surprisingly, it has been found by the present invention that these
strips measuring acetoacetate/acetone are mostly insensitive when
dipped in the urine of those individuals who are on 1000-1500 calories/day
of "balanced" diets, unlike the Atkins' diet which is
high in fat and very low in carbohydrates content, and therefore
are not useful as biochemical markers for general or common utilization
in weight loss programs. In contrast, the present invention is based
on the discovery that strips, which measure (a) .beta.-hydroxybutyrate
alone (which is about 3 to 4 time more in concentration than acetoacetate)
or (b) both .beta.-hydroxybutyrate and acetoacetate or (c) total
ketone bodies, that is, all three components are very sensitive
and can measure even very small amount of these chemicals in urine
and other biological fluids. Such sensitive strips can successfully
be used during any weight loss program and color produced is reflective
of the presence of these chemicals in the fluid.
SUMMARY OF THE INVENTION
It is, therefore, an object of the present invention to a provide
convenient and sensitive solid-phase methods for measurement of
(a) total ketone bodies, that is all three components, .beta.-hydroxybutyrate,
acetoacetate and acetone; (b) .beta.-hydroxybutyrate and acetoacetate
in one step; and (c) .beta.-hydroxybutyrate alone in biological
samples, by means of a non-invasive sample, such as urine, which
produces color that can be used for detecting the breakdown of fat
(or fatty acids) in persons who are on weight-loss programs.
It is also an objective of the present invention to provide a convenient,
and sensitive solid-phase or dry chemistry device that can be used
for measurement of (a) Total Ketone bodies that is all three components,
.beta.-hydroxybutyrate, acetoacetate and acetone; (b) .beta.-hydroxybutyrate
and acetoacetate in one step; and (c) .beta.-hydroxybutyrate alone
in biological samples, with non-invasive samples, such as urine
which produce color that can be used for detecting the breakdown
of fat (or fatty acids) in persons who are on weight-loss programs.
It is also an objective of this invention to provide strips for
detection of breakdown of fat (or fatty acid) in persons who are
on Weight-Loss programs and are on low calories diet 1000-1500 or
less per day. These strips show positive color when dipped in morning
urine whereas the commercially available strips for ketone, which
only measures acetoacetate and acetone, are insensitive. Thus, the
invention provides for the first time a biochemical marker as a
psychological stimulator to a person who is on weight loss programs.
A method for monitoring the progress of fat loss in a patient during
a weight loss program which comprises, contacting a body fluid sample
from said patient with a solid test strip to provide a color indication
of the presence in said body fluid of .beta.-hydroxybutyrate, optionally
together with acetoacetate and/or acetone.
A method of assaying for .beta.-hydroxybutyrate and acetoacetate
in a sample which comprises: a) contacting a sample with a composition
comprised of .beta.-hydroxybutyrate dehydrogenase (.beta.-HBD) and
nicotinamide adenine dinucleotide (NAD) at a pH of less than 8.5,
whereby (i) .beta.-hydroxybutyrate (.beta.-HB) reacts with NAD to
produce acetoacetate and reduced-type nicotinamide adenine dinucleotide
(NADH), (ii) a portion of the NADH produced in (i) reacts with acetoacetate
in the presence of .beta.-HBD to produce .beta.-HB, and (iii) a
portion of the NADH produced in (i) is converted into a colored
product,; and
detecting the presence of said colored product.
A test strip for assaying for .beta.-hydroxybutyrate, acetoacetate,
and acetone in a sample comprising: a) a support layer; and b) a
reagent layer on said support layer, said reagent layer comprising:
i) .beta.-hydroxybutyrate dehydrogenase (.beta.-HBD), ii) nicotinamide
adenine dinucleotide (NAD), iii) a tetrazolium dye precursor, and
an electron mediator.
DETAILED DESCRIPTION OF THE INVENTION
For the use in weight-loss programs, sensitive, convenient methods
are presented here, specifically a method which (a) simultaneously
measures total ketone bodies, i.e., all three components--.beta.-hydroxybutyrate,
acetoacetate and acetone; (b) simultaneously measures .beta.-hydroxybutyrate
and acetoacetate, and (c) measures only the major Ketone body, that
is, .beta.-hydroxybutyrate in biological fluids including urine,
blood/serum, saliva. In addition, solid-phase devices such as a
test strip are described in the present invention as a means for
(a) simultaneous measurement of Total Ketone bodies, that is all
three components--.beta.-hydroxybutyrate, acetoacetate and acetone;
(b) measurement of .beta.-hydroxybutyrate and acetoacetate; and
(c) measurement of only .beta.-hydroxybutyrate. These test strips
can be used frequently as a non-invasive tool and a biochemical
marker to ensure that indeed fat is being metabolized during various
diet programs. Each of these strips is extremely sensitive, that
is, when dipped in urine frequently or every morning will show a
positive color, if any amount of fat is being metabolized during
the day, thus serving as a psychological stimulator and tool to
a person involved in any weight-loss program.
There is no dry-chemistry method or solid phase device known to
measure Total Ketone bodies (TKB). The present invention, for the
first time provides a novel, convenient method and an impregnated
test strip in a solid-phase or dry-chemistry format for measurement
of TKB that can be used at the patient's site, especially in a weight-loss
program. The method and device use .beta.-hydroxybutyrate debydrogenase
to convert .beta.-hydroxybutyrate to acetoacetate and simultaneously
acetoacetate produced by said reaction, endogenous acetoacetate
and acetone are measured by known colorimetric methods.
A process for measuring TKB was described for use in an automated
analyzer, without employing an impregnated test strip, in U.S. Pat.
No. 5,801,059 to Smith et al. However, as this method required an
expensive automated analyzer and professional personnel to perform
the test, it is not suitable and cannot be used by a patient at
home.
U.S. Pat. No. 5,618,686 to Kojima et al describes a method for
measurement of .beta.-hydroxybutyrate and acetoacetate, which is
referred mistakenly as Total Ketone bodies (TKB). The method is
a two-step process. In the first step, acetoacetate is converted
in .beta.-hydroxybutyrate in the sample with the aid of .beta.-hydroxybutyrate
dehydrogenase (HBD) and NADH (reduced nicotoniamide adnenine dinucleotide)
and in the second step measures both sources of .beta.-hydroxybutyrate,
originally existed in the sample and .beta.-hydroxybutyrate converted
by the first step from acetoacetate with the aid of .beta.-hydroxybutyrate
dehydrogenase and NAD. However, this method suffers from (a) it
can only be used with an automated analyzer and both steps cannot
be combined due to difference in pH requirements for these steps
so it cannot work as a one-step convenient method and (b) it measures
NADH at 340 nm wavelength and does not produce color.
Similarly, another method to measure .beta.-hydroxybutyrate and
acetoacetate is described in U.S. Pat. No. 5,633,143 to Ueda et
al, which is again a two steps process. In this case also, in the
first step acetoacetate is first converted to .beta.-hydroxybutyrate
and then .beta.-hydroxybutyrate is measured using .beta.-hydroxybutyrate
dehydrogenase and a thiol derivative of NAD (thionicotinamide adenine
dinnucleotide), which can be measured at 400 nm wavelength. Although
the method is sensitive, it suffers from the detriments that (a)
NADH and thio-NAD are not compitable and cannot be mixed together
as one reagent in one-step method or device and (b) thio-NAD is
difficult to function as a coenzyme of .beta.-hydroxybutyrate dehydrogenase
as it is not a natural coenzyme and has a very high Km, an unusually
high concentration of .beta.-hydroxybutyrate dehydrogenase is required
and hence becoming uneconomical, and (c) a yellow color produced
at 400 nm is difficult to recognize visually and prone to interference
from hemolysis and presence of bilirubin.
The above two-step method can be used in liquid assays and can
be adapted to large clinical analyzers even though it requires two
separate steps, however, it cannot be employed to develop a convenient
solid-phase device or dry-chemistry method or device, which requires
one-step process.
In the present invention, it was very surprisingly found, that
if the reaction 1A which converts .beta.-hydroxybutyrate in presence
of NAD (nictonimaide adenine dinucleotide) to acetoacetate and NADH
(reduced NAD), as described below, is carried out at pH less than
8.5, for example pH 8.0, or 7.75 or 7.5, the reaction 1B, that is
conversion of NADH with tetrazolium salts in presence of diaphorase,
becomes slower than the reaction 1A. As a result, part of NADH,
which was not converted to color by diaphorase and tetrazolium salt,
becomes available to be used in the following reaction (2) to measure
acetoacetate that is also present in the sample as shown here. ##STR1##
By this method, the test not only measures .beta.-hydroxybutyrate
in the sample, it was also measuring acetoacetate simultaneously
present in the sample (i.e. endogenoss in a one step process. Moreover,
in the present reaction scheme, it becomes a cyclic reaction between
reaction 1 and 2 and thus continue to produce color over time thus
increasing sensitivity several fold. ##STR2##
This "cyclic" methodology, unexpectedly, leads to a very
sensitive method to measure both .beta.-hydroxybutyrate and acetoacetate
in the sample as it continually produces color over time. Additionally,
the method produces purple color and shades of purple color, which
reflects the relative concentration of .beta.-hydroxybutyrate and
acetoacetate in a sample, which can easily be visually differentiated.
It is also possible to measure NADH by other known methods, such
as electrochemical sensors, but methods producing visual color are
easily adopted by patients at home.
Although, methods of measuring .beta.-hydroxybutyrate in biological
fluids using the enzyme .beta.-hydroxybutyrate dehydrogenase (HBD)
in the presence of NAD which produces NADH that is measured in the
UV region at 340 nm wavelength, (Ref: Williamson et al, 1962, "Enzymatic
Determination of D(-)-beta-Hydroxybutyric Acid and Acetoacetate
acid in Blood", Biochem. J., 82:90-96) have been known for
a long time, there was no easy calorimetric method known to measure
.beta.-hydroxybutyrate. (Ref: Harano et al.,1984, "Development
of Paper-Strip Test for 3-hydroxybutyrate and its Clinical Application",
Diabetic Care, 7, p. 481-485; Harano et al., 1990, "Development
of Stable Film Test for Rapid Estimation of Blood or Plasma 3-hydroxybutyrate,"
Diabetic care, 13: 522-524; and a KetoSite.RTM. test available from
GDS Technology, Inc, Elkhart, Ind. 46514 dated Dec. 19, 1993 product
insert. (See, Tietz Text book of Clinical Chemistry, 3rd edition,
edited by Burtis and Ashwood,1999, p786-787). In these methods,
.beta.-hydroxybutyrate dehydrogenase and NAD are used to react with
.beta.-hydroxybutyrate and the reaction produces NADH which is measured
by the reaction with tetrazolium salt (such as NBT i.e. nitrobluetetrazolium)
in presence of excess of diaphorase enzyme thus producing color
which is proportionate to its concentration in blood. In these systems,
it is necessary to have pH of the reaction higher than 8.5 to derive
the reaction from left to the right, and to have pH of 7.0 or less
to derive the reaction from right to the left as shown in the following
diagram: ##STR3##
These tests have been used for patients who produce .beta.-hydroxybutyrate
in blood during severe ketoacidosis caused by serious insulin deficiencies
in patients with diabetes. Surprisingly, the present inventor has
discovered that the .beta.-hydroxybutyrate strip or card can also
be used by persons using blood as a sample who are on low calories
diet. The intensity of the color produced is reflective of level
of .beta.-hydroxybutyrate in blood. However, the use of blood, as
an invasive method, is not practical for use by a common person
who is on a low calorie diet. The strip for .beta.-hydroxybutyrate
will obviously be more useful if it can be used for urine testing,
as opposed to blood, since a urine sample is non-invasive and can
be tested conveniently by anyone at anytime. It is also possible
to use other non-invasive fluids such as saliva or sweat as done
for alcohol measurement. However, it was surprisingly discovered
by the present inventor that such test cards, commercially available
by GDS Technology, Inc, for .beta.-hydroxybutyrate cannot be used
for urine for two reasons (a) the reaction is conducted at pH higher
than 8.5 and other components such as sulfahydryl drugs in urine
(also in blood) which can reduce tetrazolium salt itself thus provide
false positive results and (b) the enzyme .beta.-hydroxybutyrate
dehydrogenase (HBD) generally used in the strip is inhibited by
chloride salt which is abundantly present in urine, and thus the
strip is not able to measure .beta.-hydroxybutyrate in urine, which
will be a sample of choice. In the present invention it has surprisingly
been found that it is possible to overcome these problems and develop
a strip for testing .beta.-hydroxybutyrate in urine by either using
a high amount of .beta.-hydroxybutyrate dehydrogenase (e.g, 10 to
20 the times amount than generally is required) or by using .beta.-hydroxybutyrate
dehydrogenase, which is not inhibited by chloride ions. The interferences
from other substances such as sulfahydryl drugs present in urine
have been able to overcome by running the reaction at pH of 8.5
or less.
The strips developed for measurement of .beta.-hydroxybutyrate,
which is generally present at an amount 34 times greater than acetoacetate,
by this invention can, therefore, be successfully used in testing
urine of persons who are on low calories diet or patients suffering
from diabetes. The test strip of the invention for .beta.-hydroxybutyrate
is, therefore, significantly more sensitive to measure than the
test strip commercially available for ketone measurement which only
measures the minor components of acetoacetate and acetone.
There are alternative colorimetric methods to measure .beta.-hydroxybutyrate
that use Ellman's reagent, .beta.-hydroxybutyrate dehydrogenase,
lipomide dehydrogenase, D,L-Lipomide and NAD incorporated into a
carrier matrix for measurement of .beta.-hydroxybutyrate (U.S. Pat.
No. 5,190,863 to Magers, U.S. Pat. No. 5,326,697 to Magers, U.S.
Pat. No. 5,510,245 to Magers. These methods require a complex system,
not cost-effective for general use and do not demonstrate potential
for use in weight loss programs. The signal produced in these methods
is generally a color. An electrochemical sensor measuring NADH as
change in current is commercially available by Abbott (Medisense),
Abbott Park, Chicago, Ill. for the use in ketoacidosis in diabetes.
An attempt was recently made to use this device in a weight-loss
program using whole blood (Byrne et al, 2000, Diabetes care, 23,
500-503). However, as such devices do not produce color and are
instrument based, they are not convenient for home use, especially
for weight loss program. Additionally as described earlier, an non-invasive
sample will be a preferred sample, and above electrochemical device
did not function when urine was used as sample.
Thus, apart from the present invention, there is no sensitive,
easy to use solid-phase device measuring color known that can be
used as a tool for measurement of fat loss during weight loss program.
Surprisingly, it was found that the strips according to the present
inventor--i.e. a strip measuring TKB, a strip measuring .beta.-hydroxybutyrate
and acetoacetate and a strip measuring .beta.-hydroxybutyrate alone,
were extremely sensitive and showed positive color. They were able
to measure trace or small amount of ketone bodies during fat metabolism
when dipped in everyday urine whereas commercially available strip
measuring acetoacetate were negative when person was 1000-1500 calories
balanced diet. |