U.S. patent application number 10/797813 was filed with the patent office on 2005-09-15 for methods of assessing the need for and the effectiveness of therapy with antioxidants.
Invention is credited to Crum, Albert.
Application Number | 20050202521 10/797813 |
Document ID | / |
Family ID | 34920134 |
Filed Date | 2005-09-15 |
United States Patent
Application |
20050202521 |
Kind Code |
A1 |
Crum, Albert |
September 15, 2005 |
Methods of assessing the need for and the effectiveness of therapy
with antioxidants
Abstract
The invention relates to diagnostic methods for assessing the
need of a subject for treatment with an anti-oxidant, or
alternatively, for determining the utilization efficiency and
ultimate effectiveness of anti-oxidant therapy in subjects having
been treated with antioxidants. More specifically, the methods of
the present invention are particularly useful in prophylactic
assessment of individuals at risk for developing diseases or
conditions in which oxidative stress plays a role, such that an
appropriate therapeutic regimen can be prescribed for that
individual, thus leading to alternative therapies and/or life style
changes. The invention further relates to methods for assessing the
need for, the utilization efficiency and the effectiveness of
therapy in subjects having received therapy with specific
antioxidant and immune enhancing formulations. Kits are also
provided for measuring the levels of markers of oxidative stress
and immune cell numbers.
Inventors: |
Crum, Albert; (Brooklyn
Heights, NY) |
Correspondence
Address: |
KLAUBER & JACKSON
411 HACKENSACK AVENUE
HACKENSACK
NJ
07601
|
Family ID: |
34920134 |
Appl. No.: |
10/797813 |
Filed: |
March 10, 2004 |
Current U.S.
Class: |
435/26 ; 436/86;
514/15.1; 514/21.9; 514/3.9 |
Current CPC
Class: |
G01N 33/6815 20130101;
G01N 33/6806 20130101; G01N 33/92 20130101; A61P 43/00 20180101;
G01N 33/493 20130101; G01N 33/6812 20130101; G01N 2800/52 20130101;
G01N 2800/7009 20130101 |
Class at
Publication: |
435/026 ;
436/086; 514/018 |
International
Class: |
A61K 038/05; C12Q
001/32; G01N 033/00 |
Claims
What is claimed is:
1. A method for assessing the need for treatment of a subject with
an anti-oxidant comprising the steps of: a) collecting a sample of
body fluid from a subject suspected of needing such treatment; b)
measuring the amount of lipid peroxide and pyroglutamic acid levels
in said sample; c) measuring the level of blood plasma glutathione;
d) comparing the amount of lipid peroxide and pyroglutamic acid in
said sample with that of a normal standard; and e) comparing the
level of blood plasma glutathione with that of a normal standard;
and wherein the presence of lipid peroxide and pyroglutamic acid in
said sample and the blood plasma levels of glutathione are present
in amounts that lie outside the normal range are indicative of a
need for anti-oxidant treatment.
2. The method of claim 1, wherein said subject in need of treatment
with an anti-oxidant also experiences a reduction in immune cell
number and/or function.
3. The method of claim 2, wherein said immune cell is selected from
the group consisting of a T cell, a B cell or a natural killer
cell.
4. The method of claim 3, wherein said T cell is selected from the
group consisting of a CD4+ T cell or a CD8+ T cell.
5. The method of claim 1, wherein said anti-oxidant comprises a
formulation consisting of a glutathione precursor, wherein said
glutathione precursor is IMMUNE FORMULATION 100.TM. or IMMUNE
FORMULATION 200.TM..
6. The method of claim 1, wherein the sample of body fluid is
urine.
7. A method for measuring the effectiveness of therapy with an
anti-oxidant in a subject receiving treatment with an anti-oxidant
comprising the steps of: a) collecting a sample of body fluid from
a subject being treated with an anti-oxidant; b) measuring the
amount of lipid peroxide and pyroglutamic acid in said sample; c)
measuring the level of blood plasma glutathione; d) comparing the
amount of lipid peroxide and pyroglutamic acid in said sample with
that of a normal standard; e) comparing the level of blood plasma
glutathione with that of a normal standard; and wherein the
presence of normal levels of lipid peroxide and pyroglutamic acid
in said sample and the presence of normal levels of blood plasma
glutathione are an indication of effectiveness of the anti-oxidant
therapy.
8. The method of claim 7, wherein said subject receiving treatment
with an anti-oxidant also experienced a reduction in immune cell
number and/or function prior to the start of therapy with the
anti-oxidant.
9. The method of claim 8, further comprising determining whether
immune cell number and/or function is normalized in said subject,
wherein said normalization is indicative of the effectiveness of
therapy with said anti-oxidant.
10. The method of claim 9, wherein said immune cell is selected
from the group consisting of a T cell, a B cell or a natural killer
cell.
11. The method of claim 10, wherein said T cell is selected from
the group consisting of a CD4+ T cell or a CD8+ T cell.
12. The method of claim 7, wherein said anti-oxidant comprises a
formulation consisting of a glutathione precursor, wherein said
glutathione precursor is IMMUNE FORMULATION 100.TM. or IMMUNE
FORMULATION 200.TM..
13. The method of claim 7, wherein the sample of body fluid is
urine.
14. A method for measuring the utilization efficiency of an
anti-oxidant in a subject receiving therapy with an anti-oxidant
comprising the steps of: a) collecting a sample of body fluid from
a subject being treated with an anti-oxidant; b) measuring the
amount of lipid peroxide and pyroglutamic acid in said sample; c)
measuring the level of blood plasma glutathione; d) comparing the
amount of lipid peroxide and pyroglutamic acid in said sample with
that of a normal standard; e) comparing the level of blood plasma
glutathione with that of a normal standard; and wherein the
presence of normal levels of lipid peroxide and pyroglutamic acid
in said sample and the presence of normal levels of blood plasma
glutathione are an indication of efficiency of utilization of the
anti-oxidant.
15. The method of claim 14, wherein said subject receiving therapy
with an anti-oxidant also experienced a reduction in immune cell
number and/or function prior to the start of therapy with the
anti-oxidant.
16. The method of claim 15, further comprising determining whether
immune cell number and/or function is normalized in said subject,
wherein said normalization is indicative of the utilization
efficiency of said anti-oxidant.
17. The method of claim 16, wherein said immune cell is selected
from the group consisting of a T cell, a B cell or a natural killer
cell.
18. The method of claim 17, wherein said T cell is selected from
the group consisting of a CD4+ T cell or a CD8+ T cell.
19. The method of claim 14, wherein said anti-oxidant comprises a
formulation consisting of a glutathione precursor, wherein said
glutathione precursor is IMMUNE FORMULATION 100.TM. or IMMUNE
FORMULATION 200.TM..
20. The method of claim 14, wherein the sample of body fluid is
urine.
21. A method for determining the amount of IMMUNE FORMULATION
100.TM. or IMMUNE FORMULATION 200.TM. that is necessary to increase
glutathione synthesis or re-synthesis in a patient in need of such
therapy, comprising the steps of: a) collecting a series of body
fluid samples from a patient suspected of being in need of such
treatment, wherein said body fluid samples are collected prior to
the start of treatment, and daily after the start of treatment for
about 14 days b) measuring the amount of lipid peroxide and
pyroglutamic acid in said body fluid samples; c) comparing the
amount of lipid peroxide and pyroglutamic acid in said body fluid
samples with that of normal standards; d) measuring the amount of
glutathione increase in blood samples; e) comparing the amount of
glutathione in said blood samples with that of normal standards;
and wherein the normalization of lipid peroxide and pyroglutamic
acid levels in said body fluid samples correlates with the
synthesis or re-synthesis of glutathione in the patients receiving
IMMUNE FORMULATION 100.TM. or IMMUNE FORMULATION 200.TM..
22. A method for determining the amount of IMMUNE FORMULATION
100.TM. or IMMUNE FORMULATION 200.TM. that is necessary to reduce
urine pyroglutamic acid in a patient in need of such therapy,
comprising the steps of: a) collecting a series of urine samples
from a patient suspected of being in need of such treatment,
wherein said samples are collected prior to the start of treatment,
and daily after the start of treatment for about 14 days; b)
measuring the amount of pyroglutamic acid in said samples; c)
comparing the amount of pyroglutamic acid in said samples with that
of a normal standard; and wherein the reduction of pyroglutamic
acid to normal levels in said samples correlates with the amount of
IMMUNE FORMULATION 100.TM. or IMMUNE FORMULATION 200.TM. sufficient
to achieve a beneficial effect.
23. A method for determining the amount of IMMUNE FORMULATION
100.TM. or IMMUNE FORMULATION 200.TM. that is necessary to reduce
urine lipid peroxide in a patient in need of such therapy,
comprising the steps of: a) collecting a series of urine samples
from a patient suspected of being in need of such treatment,
wherein said samples are collected prior to the start of treatment,
and daily after the start of treatment for about 14 days; b)
measuring the amount of lipid peroxide in said samples; c)
comparing the amount of lipid peroxide in said samples with that of
a normal standard; and wherein the reduction of lipid peroxide to
normal levels in said samples correlates with the amount of IMMUNE
FORMULATION 100.TM. or IMMUNE FORMULATION 200.TM. sufficient to
achieve a beneficial effect.
24. A method for determining an orally anti-oxidative effective
amount of IMMUNE FORMULATION 100.TM. or IMMUNE FORMULATION 200.TM.
sufficient to diminish urine lipid peroxide and pyroglutamic acid
levels and concurrently increase blood plasma glutathione levels,
comprising the steps of: a) collecting blood plasma and urine
samples prior to administration of IMMUNE FORMULATION 100.TM. or
IMMUNE FORMULATION 200.TM. and daily after the start of
administration for about 14 days; b) measuring urine levels of
lipid peroxide and pyroglutamic acid; c) measuring blood plasma
glutathione levels; d) determining whether a decrease in lipid
peroxide and pyroglutamic acid levels correlates with an increase
in glutathione levels; and wherein said correlation establishes an
orally anti-oxidative effective amount of IMMUNE FORMULATION
100.TM. or IMMUNE FORMULATION 200.TM..
25. A method for establishing the interdependence of lipid
peroxides, pyroglutamic acid, glutathione, and immune cell number
and/or function in a subject suffering from oxidative stress,
comprising the steps of: a) collecting a urine sample from a
subject suspected of being under oxidative stress; b) assaying the
urine for the presence of lipid peroxides and pyroglutamic acid; c)
collecting a sample of whole blood; d) separating the cellular
components from the liquid portion of whole blood; e) measuring
glutathione in the liquid portion of whole blood obtained in step
d); f) measuring the number of CD4+ and CD8+ T cells in the
cellular component of whole blood from step d); and g) measuring
the natural killer cell activity from the cellular component of
whole blood obtained from step d); wherein a finding of decreased
plasma glutathione levels, an increase in urinary lipid peroxides
and pyroglutamic acid, and a decrease in the number of CD4+ and
CD8+ T cells and natural killer cell activity provides support for
the interdependence of the level of oxidative stress in said
subject and immune cell number and/or function.
26. A method for determining an immune enhancing effective amount
of IMMUNE FORMULATION 100.TM. or IMMUNE FORMULATION 200.TM.
sufficient to normalize CD4+, CD8+ T cell numbers and natural
killer cell activity in a subject suspected of experiencing
oxidative stress, comprising the steps of: a) collecting whole
blood samples prior to administration of IMMUNE FORMULATION 100.TM.
or IMMUNE FORMULATION 200.TM. and daily after the start of
administration for about 14 days; b) separating the cellular
component of the whole blood from the liquid component; and c)
measuring the number of CD4+ and CD8+ T cells and natural killer
cell activity using the cellular component from step b); wherein a
correlation between the dose of IMMUNE FORMULATION 100.TM. or
IMMUNE FORMULATION 200.TM. that is sufficient to normalize CD4+,
CD8+ T cell numbers and natural killer cell activity establishes an
immune enhancing effective amount of IMMUNE FORMULATION 100.TM. or
IMMUNE FORMULATION 200.TM..
27. A method for determining an orally anti-oxidative effective
amount and an immune enhancing effective amount of IMMUNE
FORMULATION 100.TM. or IMMUNE FORMULATION 200.TM. sufficient to
normalize lipid peroxides, pyroglutamic acid and glutathione levels
in a subject suspected of experiencing oxidative stress, wherein
said normalization of lipid peroxides, pyroglutamic acid and
glutathione levels results in immune enhancement, comprising the
steps of: a) collecting whole blood and urine samples prior to
administration of IMMUNE FORMULATION 100.TM. or IMMUNE FORMULATION
200.TM. and daily after the start of administration for about 14
days; b) measuring urine levels of lipid peroxide and pyroglutamic
acid; c) separating the cellular component of the whole blood from
the liquid component; d) measuring blood plasma glutathione levels
using the liquid component from step c); e) measuring the number of
CD4+ and CD8+ T cells and natural killer cell activity using the
cellular component from step c); f) determining whether a decrease
in urinary lipid peroxide and pyroglutamic acid levels correlates
with an increase in glutathione levels, and whether the
normalization of the levels of all three of these products relates
to a normalization of CD4+ and CD8+ T cell numbers and
normalization of natural killer cell activity; and wherein said
correlation establishes an orally anti-oxidative effective amount
and an immune enhancing effective amount of IMMUNE FORMULATION
100.TM. or IMMUNE FORMULATION 200.TM..
28. The method of claim 27, wherein said correlation further
establishes the interrelationship of lipid peroxides, pyroglutamic
acid, glutathione and immune functions as compared to the level of
oxidative stress in said subject which may result in depressed
immune functions.
29. A kit for measuring oxidative stress in a subject comprising:
a) a solid substrate containing immobilized binding partners
specific for at least three markers for oxidative stress; b)
either: i) an enzyme conjugated second binding partner to the
oxidative stress markers; or ii) a biotinylated second binding
partner to the oxidative stress markers; c) either: i) the enzyme
substrate and the developing reagents specific for the enzyme
conjugated second binding partner from step b) i); or ii) a
streptavidin conjugated third binding partner specific for the
second binding partner of step b) ii); d) buffers for washing and
sample dilution; e) standards for each of the at least three
markers of oxidative stress; and f) instructions for use of said
kit.
30. The kit of claim 29, further comprising additional binding
partners specific for cell surface markers for CD4+ T cells, CD8+ T
cells and natural killer cells.
31. The kit of claim 29, wherein said markers of oxidative stress
are selected from the group consisting of lipid peroxide,
pyroglutamic acid and glutathione.
32. The kit of claim 29, wherein said binding partner is an
antibody selected from the group consisting of a monoclonal
antibody, a polyclonal antibody, a chimeric antibody, and any
combination thereof.
33. A method for providing a course of therapy for an individual
suspected or known to be suffering from oxidative stress comprising
a) determining the identity and level of at least three markers of
oxidative stress in a sample of body fluid from said individual in
accordance with claim 29, said markers being indicative of the
extent of oxidative stress; and b) selecting the appropriate course
of therapy for said individual suffering from oxidative stress and
the sequelae thereof.
34. The method of claim 33, which further includes administering
said appropriate course of therapy to said individual.
35. A method for providing a course of therapy for an individual
suspected or known to be suffering from oxidative stress and
monitoring the success of said therapy comprising: a) determining
the identity and level of at least three markers of oxidative
stress in a sample of body fluid from said individual in accordance
with claim 29, said marker indicative of the extent of oxidative
stress; b) selecting the appropriate course of therapy for said
individual suffering from said oxidative stress; c) administering
said appropriate course of therapy to said individual; and
monitoring the success of said therapy by measuring a normalization
in levels of said markers of oxidative stress.
36. The method of any of claims 33-35, wherein said course of
therapy comprises administering IMMUNE FORMULATION 100.TM. or
IMMUNE FORMULATION 200.TM. to said individual.
Description
FIELD OF THE INVENTION
[0001] The invention relates to diagnostic methods for assessing
the need of a subject for treatment with an anti-oxidant, or
alternatively, for determining the effectiveness of anti-oxidant
therapy in subjects having been treated with antioxidants. More
specifically, it relates to methods for assessing the need for, or
effectiveness of therapy in subjects having received therapy with,
specific antioxidant and immune enhancing formulations.
BACKGROUND OF THE INVENTION
[0002] It is generally recognized that many disease processes are
attributed to the presence of elevated levels of free radicals and
reactive oxygen species (ROS) and reactive nitrogen species (RNS),
such as superoxide, hydrogen peroxide, singlet oxygen,
peroxynitrite, hydroxyl radicals, hypochlorous acid (and other
hypohalous acids) and nitric oxide.
[0003] In the eye, cataract, macular degeneration and degenerative
retinal damage are attributed to ROS. Among other organs and their
ROS-related diseases include: lung cancer induced by tobacco
combustion products and asbestos; accelerated aging and its
manifestations, including skin damage and scleroderma;
atherosclerosis; ischemia and reperfusion injury, diseases of the
nervous system such as Parkinson disease, Alzheimer disease,
muscular dystrophy, multiple sclerosis; lung diseases including
emphysema and bronchopulmonary dysphasia; iron overload diseases
such as hemochromatosis and thalassemia; pancreatitis; diabetes;
renal diseases including autoimmune nephrotic syndrome and heavy
metal-induced nephrotoxicity; and radiation injuries. Diseases of
aging and chronic emotional stress also appear to be associated
with a drop in glutathione levels, which allows ROS to remain
active.
[0004] Certain anti-neoplastic drugs such as adriamycin and
bleomycin induce severe oxidative damage, especially to the heart,
limiting the patient's exposure to the drug.
[0005] Redox-active metals such as iron induce oxidative damage to
tissues; industrial chemicals and ethanol, by exposure and
consumption, induce an array of oxidative damage-related injuries,
such as cardiomyopathy and liver damage. Airborne industrial and
petrochemical-based pollutants, such as ozone, nitric oxide,
radioactive particulates, and halogenated hydrocarbons, induce
oxidative damage to the lungs, gastrointestinal tract, and other
organs. Radiation poisoning from industrial sources, including
leaks from nuclear reactors and exposure to nuclear weapons, are
other sources of radiation and radical damage. Other routes of
exposure may occur from living or working in proximity to sources
of electromagnetic radiation, such as electric power plants and
high-voltage power lines, x-ray machines, particle accelerators,
radar antennas, radio antennas, and the like, as well as using
electronic products and gadgets which emit electromagnetic
radiation such as cellular telephones, and television and computer
monitors.
[0006] Mammalian cells have numerous mechanisms to eliminate these
damaging free radicals and reactive species. One such mechanism
includes the glutathione system, which plays a major role in direct
destruction of reactive oxygen compounds.
[0007] Perhaps one of the most important contributions of
glutathione to mammalian health is its participation in the proper
functioning of the immune system to respond to infection or other
types of trauma. It is known that weakening of the immune system
caused by infection or other traumas occurs concurrently with
depletion of glutathione in body tissues. It is known, also, that
such weakening can be reversed by replenishing the body's level of
glutathione by intracellular synthesis. It is believed that
glutathione accomplishes its salutary effects by protecting immune
cells against the ravages of oxidizing agents and free
radicals.
[0008] Until recently, the lack of specific and dependable methods
for evaluating oxidant stress in vivo made it very difficult to
establish a cause and effect relationship between free
radical-generating agents or conditions and disease pathology.
Furthermore, the various treatment strategies with anti-oxidants
have been difficult to monitor due to the lack of techniques
sufficiently sensitive to reliably provide an index of oxidative
damage in vivo.
[0009] For example, there is currently substantial evidence that
oxidation of LDL occurs in vivo, and results of animal studies
suggest that this may lead to the formation and build up of
atherosclerotic plaques. Although epidemiological data support a
role for antioxidants in the prevention of clinical events,
intervention trials thus far have given mixed results (Steinberg D,
Witztum J L. Lipoproteins, lipoprotein oxidation, and
atherogenesis. In Chien KR, ed. Molecular Basis of Cardiovascular
Disease. Philadelphia, Pa.: W.B. Saunders Co., 1998:458-475). This
may be due, in part, to the fact that until now techniques to
adequately provide an index of in vivo lipid peroxidation have not
been available, which could be used to design and monitor effective
antioxidant intervention trials to adequately test the oxidation
hypothesis.
[0010] Furthermore, there are no set measures to identify high-risk
groups that would theoretically benefit most from antioxidant
therapies or interventions. Additionally, there are no reliable
means to measure or determine the effectiveness of such
interventions in vivo. In the absence of such methodology, current
(and future) clinical trials testing natural (or synthetic)
antioxidants, which utilize clinical endpoints, may give incorrect
conclusions regarding the role of antioxidants in specific disease
states. This is a possibility because of the inclusion of
populations that would not be expected to benefit from antioxidant
supplementation, and/or because the dose or agent yielded
insufficient antioxidant protection.
[0011] It is with respect to the development of more sensitive and
accurate assays for assessing the need for intervention with
anti-oxidant therapy and for monitoring the effectiveness and
utilization efficiency of novel anti-oxidants that the current
invention is directed.
SUMMARY OF THE INVENTION
[0012] In its broadest aspect, the present invention relates to
methods for assessing the need of a subject for treatment with an
antioxidant, or alternatively, if a subject is currently being
treated with an anti-oxidant, the invention provides for measuring
the utilization efficiency of the anti-oxidant and the subsequent
effectiveness of therapy with the anti-oxidant. It is also an
object of the present invention to provide a means for determining
the anti-oxidative effective amounts of specific anti-oxidant
formulations for delivery to a subject in need of such therapy.
More particularly, the invention provides for methods for
determining the amount of anti-oxidants necessary to increase
glutathione synthesis or re-synthesis in a patient in need of such
therapy.
[0013] Accordingly, a first aspect of the invention provides for a
method for assessing the need for treatment with an anti-oxidant
comprising the steps of:
[0014] a) collecting a sample of body fluid from a subject
suspected of needing such treatment;
[0015] b) measuring the amount of lipid peroxide and pyroglutamic
acid (PGA) levels in said sample;
[0016] c) measuring the level of blood plasma glutathione;
[0017] d) comparing the amount of lipid peroxide and pyroglutamic
acid in said sample with that of a normal standard; and
[0018] e) comparing the level of blood plasma glutathione with that
of a normal standard; and
[0019] wherein the presence of lipid peroxide and pyroglutamic acid
in said sample and the blood plasma levels of glutathione are
present in amounts that lie outside the normal range are indicative
of a need for anti-oxidant treatment.
[0020] In a particular embodiment, the patient or subject is
preferably an animal, including but not limited to animals such as
monkeys, cows, pigs, horses, chickens, cats, dogs, etc., and is
preferably a mammal, and most preferably human. In one embodiment,
a non-human mammal is the subject. In another embodiment, a human
mammal is the subject. In yet another particular embodiment, the
subject in need of treatment with an anti-oxidant also experiences
a reduction in immune cell number and/or function. In another
particular embodiment, the immune cell is selected from the group
consisting of a T cell, a B cell or a natural killer cell. In yet
another particular embodiment, the T cell is selected from the
group consisting of a CD4+ or a CD8+ T cell.
[0021] In another particular embodiment, the sample of body fluid
is urine. In another particular embodiment, the sample of body
fluid is whole blood. In a yet further particular embodiment, the
sample of body fluid is plasma or serum.
[0022] In another particular embodiment, the anti-oxidant is
selected from the group consisting of glutathione precursors,
IMMUNE FORMULATION 100.TM. and IMMUNE FORMULATION 200.TM..
[0023] A second aspect of the invention provides a method for
measuring the effectiveness of therapy with an anti-oxidant in a
subject comprising the steps of:
[0024] a) collecting a sample of body fluid from a subject being
treated with an anti-oxidant;
[0025] b) measuring the amount of lipid peroxide and pyroglutamic
acid in said sample;
[0026] c) measuring the level of blood plasma glutathione;
[0027] d) comparing the amount of lipid peroxide and pyroglutamic
acid in said sample with that of a normal standard;
[0028] e) comparing the level of blood plasma glutathione with that
of a normal standard; and
[0029] wherein the presence of normal levels of lipid peroxide and
pyroglutamic acid in said sample and the presence of normal levels
of blood plasma glutathione are an indication of effectiveness of
the anti-oxidant therapy.
[0030] In a particular embodiment, the method may further comprise
determining whether immune cell number and/or function is
normalized in the subject, wherein the normalization is indicative
of the effectiveness of therapy with the anti-oxidant. In another
particular embodiment, the immune cell is selected from the group
consisting of a T cell, a B cell or a natural killer cell. The
method of claim 9, wherein said T cell is selected from the group
consisting of a CD4+ T cell or a CD8+ T cell. In another particular
embodiment, The method of claim 7, wherein said anti-oxidant is
selected from the group consisting of glutathione precursors,
IMMUNE FORMULATION 100.TM. or IMMUNE FORMULATION 200.TM.. In yet
another particular embodiment, the sample of body fluid is urine.
In another particular embodiment, the sample of body fluid is whole
blood. In a yet further particular embodiment, the sample of body
fluid is plasma or serum.
[0031] In another particular embodiment, the anti-oxidant is
selected from the group consisting of glutathione precursors,
IMMUNE FORMULATION 100.TM. or IMMUNE FORMULATION 200.TM..
[0032] In yet another preferred embodiment, the method further
comprises measurement of a secondary endpoint for monitoring the
effectiveness of therapy. For example, wherein the patient under
treatment with the anti-oxidant therapy is suffering from a disease
which is, in part, caused by high levels of oxidant stress, or
where a particular drug treatment itself results in oxidative
damage to a particular tissue or organ, such as with chemotherapy,
it is beneficial to measure the effectiveness of therapy with the
anti-oxidant using the steps described above. However, in diseases
such as atherosclerosis or cardiovascular disease, whereby oxidized
low density lipoprotein (LDL) has been implicated in the initiation
and/or exacerbation of the disease process, it would be beneficial
to monitor the effects of the antioxidant therapy not only on lipid
peroxide, PGA and glutathione levels, but also on for example,
cardiac function to determine whether the antioxidant therapy has
effects on the sequelae of high oxidative stress levels, such as
cardiovascular disease or atherosclerosis. The secondary endpoint
may include lowering of triglycerides, LDLs or increasing of high
density lipoproteins (HDLs), or measurement of cardiac function
using standard testing known to one skilled in the art.
[0033] A third aspect of the invention provides a method for
measuring the utilization efficiency of an anti-oxidant comprising
the steps of:
[0034] a) collecting samples of body fluid from a subject being
treated with an anti-oxidant each day after initiation of therapy
and up to 14 days after therapy has been initiated;
[0035] b) measuring the amount of lipid peroxide and pyroglutamic
acid in said samples;
[0036] c) measuring the level of blood plasma glutathione;
[0037] d) comparing the amount of lipid peroxide and pyroglutamic
acid in said samples with that of a normal standard;
[0038] e) comparing the level of blood plasma glutathione with that
of a normal standard; and
[0039] wherein the presence of normal levels of lipid peroxide and
pyroglutamic acid in said samples and the presence of normal levels
of blood plasma glutathione are an indication of efficiency of
utilization of the anti-oxidant.
[0040] In a particular embodiment, the method further comprises
determining whether immune cell number and/or function is
normalized in the subject, wherein the normalization is indicative
of the utilization efficiency of the anti-oxidant. In another
particular embodiment, the immune cell is selected from the group
consisting of a T cell, a B cell or a natural killer cell. In
another particular embodiment the T cell is selected from the group
consisting of a CD4+ or a CD8+ T cell. In yet another particular
embodiment, the sample of body fluid is urine. In another
particular embodiment, the sample of body fluid is whole blood. In
a yet further particular embodiment, the sample of body fluid is
plasma or serum.
[0041] In another particular embodiment the anti-oxidant is
selected from the group consisting of glutathione precursors,
IMMUNE FORMULATION 100.TM. or IMMUNE FORMULATION 200.TM..
[0042] In another preferred embodiment, if the levels of lipid
peroxides, pyroglutamic acid, and glutathione are not normalized,
the levels of antioxidants are increased in dosage until such
normalization occurs.
[0043] A fourth aspect of the invention provides a method for
determining the amount of IMMUNE FORMULATION 100.TM. or IMMUNE
FORMULATION 200.TM. that is necessary to increase glutathione
synthesis or re-synthesis in a subject in need of such therapy,
comprising the steps of:
[0044] a) collecting a series of body fluid samples from a subject
suspected of being in need of such treatment, wherein said body
fluid samples are collected prior to the start of treatment, and
daily after the start of treatment for about 14 days
[0045] b) measuring the amount of lipid peroxide and pyroglutamic
acid in said body fluid samples;
[0046] c) comparing the amount of lipid peroxide and pyroglutamic
acid in said body fluid samples with that of normal standards;
[0047] d) measuring the amount of glutathione increase in blood
samples;
[0048] e) comparing the amount of glutathione in said blood samples
with that of normal standards; and
[0049] wherein the normalization of lipid peroxide and pyroglutamic
acid levels in said body fluid samples correlates with the
synthesis or re-synthesis of glutathione in the patients receiving
IMMUNE FORMULATION 100.TM. or IMMUNE FORMULATION 200.TM..
[0050] In a particular embodiment, the sample of body fluid is
urine. In another particular embodiment, the sample of body fluid
is whole blood. In a yet further particular embodiment, the sample
of body fluid is plasma or serum.
[0051] In another preferred embodiment, if the levels of lipid
peroxides, pyroglutamic acid, and glutathione are not normalized,
the levels of IMMUNE FORMULATION 100.TM. or IMMUNE FORMULATION
200.TM. are increased in dosage and the methods described above are
repeated until such normalization occurs.
[0052] A fifth aspect of the invention provides a method for
determining the amount of IMMUNE FORMULATION 100.TM. or IMMUNE
FORMULATION 200.TM. that is necessary to reduce urine pyroglutamic
acid in a subject in need of such therapy, comprising the steps
of:
[0053] a) collecting a series of urine samples from a subject
suspected of being in need of such treatment, wherein said samples
are collected prior to the start of treatment, and daily after the
start of treatment for about 14 days;
[0054] b) measuring the amount of pyroglutamic acid in said
samples;
[0055] c) comparing the amount of pyroglutamic acid in said samples
with that of a normal standard; and
[0056] wherein the reduction of pyroglutamic acid to normal levels
in said samples correlates with the amount of IMMUNE FORMULATION
100.TM. or IMMUNE FORMULATION 200.TM. sufficient to achieve a
beneficial effect.
[0057] In a preferred embodiment, if the levels of urine
pyroglutamic acid are not normalized, the levels of IMMUNE
FORMULATION 100.TM. or IMMUNE FORMULATION 200.TM. are increased in
dosage until such normalization occurs, and the methods described
above are repeated until such time when normalization is
achieved.
[0058] A sixth aspect of the invention provides a method for
determining the amount of IMMUNE FORMULATION 100.TM. or IMMUNE
FORMULATION 200.TM. that is necessary to reduce urine lipid
peroxide in a subject in need of such therapy, comprising the steps
of:
[0059] a) collecting a series of urine samples from a subject
suspected of being in need of such treatment, wherein said samples
are collected prior to the start of treatment, and daily after the
start of treatment for about 14 days;
[0060] b) measuring the amount of lipid peroxide in said
samples;
[0061] c) comparing the amount of lipid peroxide in said samples
with that of a normal standard; and
[0062] wherein the reduction of lipid peroxide to normal levels in
said samples correlates with the amount of IMMUNE FORMULATION
100.TM. or IMMUNE FORMULATION 200.TM. sufficient to achieve a
beneficial effect.
[0063] In a preferred embodiment, if the levels of urine lipid
peroxide are not normalized, the levels of IMMUNE FORMULATION
100.TM. or IMMUNE FORMULATION 200.TM. are increased in dosage until
such normalization occurs, and the methods described above are
repeated until such time when normalization is achieved.
[0064] A seventh aspect of the invention provides a method for
determining an orally anti-oxidative effective amount of IMMUNE
FORMULATION 100.TM. or IMMUNE FORMULATION 200.TM. sufficient to
diminish urine lipid peroxide and pyroglutamic acid levels and
concurrently increase blood plasma glutathione levels, comprising
the steps of:
[0065] a) collecting blood plasma and urine samples prior to
administration of IMMUNE FORMULATION 100.TM. or IMMUNE FORMULATION
200.TM. and daily after the start of administration for about 14
days;
[0066] b) measuring urine levels of lipid peroxide and pyroglutamic
acid;
[0067] c) measuring blood plasma glutathione levels;
[0068] d) determining whether a decrease in lipid peroxide and
pyroglutamic acid levels correlates with an increase in glutathione
levels; and
[0069] wherein said correlation establishes an orally
anti-oxidative effective amount of IMMUNE FORMULATION 100.TM. or
IMMUNE FORMULATION 200.TM..
[0070] In a preferred embodiment, the levels of lipid peroxide,
pyroglutamic acid and glutathione are measured concurrently. In a
further preferred embodiment, if the levels of lipid peroxides,
pyroglutamic acid, and glutathione are not normalized when first
tested, the levels of IMMUNE FORMULATION 100.TM. or IMMNE
FORMULATION 200.TM. are increased in dosage and the methods
described above are repeated until such normalization occurs.
[0071] An eighth aspect of the invention provides for establishing
the interdependence of lipid peroxides, pyroglutamic acid,
glutathione, and immune cell number and/or function in a subject
suffering from oxidative stress, comprising the steps of:
[0072] a) collecting a urine sample from a subject suspected of
being under oxidative stress;
[0073] b) assaying the urine for the presence of lipid peroxides
and pyroglutamic acid;
[0074] c) collecting a sample of whole blood;
[0075] d) separating the cellular components from the liquid
portion of whole blood;
[0076] e) measuring glutathione in the liquid portion of whole
blood obtained in step d);
[0077] f) measuring the number of CD4+ T cells and CD8+ T cells in
the cellular component of whole blood from step d); and
[0078] g) measuring the natural killer cell activity from the
cellular component of whole blood obtained from step d);
[0079] wherein a finding of decreased plasma glutathione levels, an
increase in urinary lipid peroxides and pyroglutamic acid, and a
decrease in the number of CD4+ and CD8+ T cells and natural killer
cell activity provides support for the interdependence of the level
of oxidative stress in said subject and immune cell number and/or
function.
[0080] A ninth aspect of the invention provides for a method for
determining an immune enhancing effective amount of IMMUNE
FORMULATION 100.TM. or IMMUNE FORMULATION 200.TM. sufficient to
normalize CD4+, CD8+ T cell numbers and natural killer cell
activity in a subject suspected of experiencing oxidative stress,
comprising the steps of:
[0081] a) collecting whole blood samples prior to administration of
IMMUNE FORMULATION 100.TM. or IMMUNE FORMULATION 200.TM. and daily
after the start of administration for about 14 days;
[0082] b) separating the cellular component of the whole blood from
the liquid component; and
[0083] c) measuring the number of CD4+ and CD8+ T cells and natural
killer cell activity using the cellular component from step b);
[0084] wherein a correlation between the dose of IMMUNE FORMULATION
100.TM. and IMMUNE FORMULATION 200.TM. that is sufficient to
normalize CD4+, CD8+ T cell numbers and natural killer cell
activity establishes an immune enhancing effective amount of IMMUNE
FORMULATION 100.TM. or IMMUNE FORMULATION 200.TM..
[0085] A tenth aspect of the invention provides for a method for
determining an orally anti-oxidative effective amount and an immune
enhancing effective amount of IMMUNE FORMULATION 100.TM. or IMMUNE
FORMULATION 200.TM. sufficient to normalize lipid peroxides,
pyroglutamic acid and glutathione levels in a subject suspected of
experiencing oxidative stress, wherein said normalization of lipid
peroxides, pyroglutamic acid and glutathione levels results in
immune enhancement, comprising the steps of:
[0086] a) collecting whole blood and urine samples prior to
administration of IMMUNE FORMULATION 100.TM. or IMMUNE FORMULATION
200.TM. and daily after the start of administration for about 14
days;
[0087] b) measuring urine levels of lipid peroxide and pyroglutamic
acid;
[0088] c) separating the cellular component of the whole blood from
the liquid component;
[0089] d) measuring blood plasma glutathione levels using the
liquid component from step c);
[0090] e) measuring the number of CD4+ and CD8+ T cells and natural
killer cell activity using the cellular component from step c);
[0091] f) determining whether a decrease in urinary lipid peroxide
and pyroglutamic acid levels correlates with an increase in
glutathione levels, and whether the normalization of the levels of
all three of these products relates to a normalization of CD4+ and
CD8+ T cell numbers and normalization of natural killer cell
activity; and
[0092] wherein said correlation establishes an orally
anti-oxidative effective amount and an immune enhancing effective
amount of IMMUNE FORMULATION 100.TM. or IMMUNE FORMULATION
200.TM..
[0093] In a particular embodiment, said correlation further
establishes the interrelationship of lipid peroxides, pyroglutamic
acid, glutathione and immune functions and the level of oxidative
stress in said subject that results in depressed immune
functions.
[0094] An eleventh aspect of the invention provides kits for
measuring oxidative stress in an individual suspected of suffering
from oxidative stress. Such a kit may contain all of the reagents
necessary to measure at least three markers of oxidative stress at
the same time. The assay kit may be formatted for use as a
competitive or non-competitive ELISA assay. Alternatively, the kit
may be structured in much the same way as a take home pregnancy
kit, for example, using a test strip format. The kit may also
contain binding partners, for example, antibodies specific for
certain immune cells, such as CD4+ T cells, CD8+ T cells and
natural killer cells. Thus, the kits of the present invention may
be capable of monitoring both oxidative stress and immune cell
number.
[0095] In a particular embodiment, the kit for measuring oxidative
stress in a subject comprises:
[0096] a) a solid substrate containing immobilized binding partners
specific for at least three markers for oxidative stress;
[0097] b) either:
[0098] i) an enzyme conjugated second binding partner to the
oxidative stress markers; or
[0099] ii) a biotinylated second binding partner to the oxidative
stress markers;
[0100] c) either:
[0101] i) the enzyme substrate and the developing reagents specific
for the enzyme conjugated second binding partner from step b) i);
or
[0102] ii) a streptavidin conjugated third binding partner specific
for the second binding partner of step b) ii);
[0103] d) buffers for washing and sample dilution;
[0104] e) standards for each of the at least three markers of
oxidative stress; and
[0105] f) instructions for use of said kit.
[0106] In another particular embodiment, the binding partner is an
antibody. In another particular embodiment the kit further
comprises additional compartments to which have been attached
antibodies specific for cell surface markers for CD4+ T cells, CD8+
T cells and natural killer cells. In a preferred embodiment, the
markers of oxidative stress are selected from the group consisting
of lipid peroxide, pyroglutamic acid and glutathione. In another
preferred embodiment, the antibody is selected from a monoclonal
antibody, a polyclonal antibody, a chimeric antibody, and any
combination thereof.
[0107] A twelfth aspect of the invention provides a method for
providing a course of therapy for an individual suspected or known
to be suffering from oxidative stress comprising
[0108] a) determining the identity and level of at least three
markers of oxidative stress in a sample of body fluid from said
individual, said markers being indicative of the extent of
oxidative stress; and
[0109] b) selecting the appropriate course of therapy for said
individual suffering from oxidative stress and the sequelae
thereof.
[0110] In a particular embodiment, the method includes
administering said appropriate course of therapy to said
individual. In another particular embodiment, the method provides a
course of therapy for an individual suspected or known to be
suffering from oxidative stress and monitoring the success of said
therapy comprising:
[0111] a) determining the identity and level of at least three
markers of oxidative stress in a sample of body fluid from said
individual, said marker being indicative of the extent of oxidative
stress;
[0112] b) selecting the appropriate course of therapy for said
individual suffering from said oxidative stress;
[0113] c) administering said appropriate course of therapy to said
individual; and
[0114] monitoring the success of said therapy by measuring a
normalization in levels of said markers of oxidative stress.
[0115] In a preferred embodiment, the method provides for said
course of therapy comprising administering IMMUNE FORMULATION
100.TM. or IMMUNE FORMULATION 200.TM. to said individual.
[0116] Other advantages of the present invention will become
apparent from the ensuing detailed description.
DETAILED DESCRIPTION
[0117] Before the present methods and treatment methodology are
described, it is to be understood that this invention is not
limited to particular methods, and experimental conditions
described, as such methods and conditions may vary. It is also to
be understood that the terminology used herein is for purposes of
describing particular embodiments only, and is not intended to be
limiting, since the scope of the present invention will be limited
only in the appended claims.
[0118] As used in this specification and the appended claims, the
singular forms "a", "an", and "the" include plural references
unless the context clearly dictates otherwise. Thus, for example,
references to "the method" includes one or more methods, and/or
steps of the type described herein and/or which will become
apparent to those persons skilled in the art upon reading this
disclosure and so forth.
[0119] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the invention, the
particular methods and materials are now described. All
publications mentioned herein are incorporated herein by
reference.
DEFINITIONS
[0120] The terms used herein have the meanings recognized and known
to those of skill in the art; however, for convenience and
completeness, particular terms and their meanings are set forth
below.
[0121] "Treatment" refers to the administration of an antioxidant
or the performance of procedures with respect to a subject, for
either prophylaxis (prevention) or to cure the infirmity or malady
in the instance where the subject is afflicted.
[0122] As used herein, "assessing the need for treatment" refers to
determining whether a subject would be a candidate for therapy with
an anti-oxidant. The determination would be made based on the
particular disease and symptoms associated with the disease, and
whether or not the cause of the disease or condition can be
attributed, at least in part, to high levels of oxidation of cells,
tissues, proteins or other molecular or chemical entities which are
candidates for damage caused by oxidative stress, as evidenced by
high levels of lipid peroxide and high levels of PGA in the urine
and low levels of glutathione in the blood.
[0123] A "therapeutically effective amount" is an amount sufficient
to decrease or prevent the symptoms associated with the disease
caused by or attributed to oxidative stress.
[0124] By "patient" or "subject" is meant a human or non-human
mammal that may benefit from the therapies described in the present
application, for example, anti-oxidant therapy. The anti-oxidants
may be administered to subjects already having a disease or
condition whose symptoms and sequelae are attributed to oxidation
of proteins, cells or tissues, or particular molecular entities or
chemical compounds, and whose symptoms or sequelae may be
alleviated by anti-oxidant therapy. Alternatively, the subjects may
be predisposed to diseases or conditions caused by high levels of
oxidation, for which therapy with an anti-oxidant may be
beneficial. Accordingly, the subject may be treated
prophylactically with the anti-oxidant therapy. Diseases or
conditions for which such anti-oxidant therapy would be beneficial
may be selected from the group consisting of autoimmune or
degenerative diseases including acetaminophen poisoning, ADD,
Addison's disease, aging, AIDS, alopecia greata, ALS, Alzheimer's
disease, anemia (hemolytic), ankylosing spondylitis,
arteriosclerosis, arthritis (including osteoarthritis and
rheumatoid arthritis), asthma, autism, autoimmune disease, Behcet's
disease, burns, cachexia, cancer, candida infection, cardiomyopathy
(idiopathic), chronic fatigue syndrome, colitis, coronary artery
disease, cystic fibrosis, diabetes, Crohn's disease, eczema,
emphysema, Epstein Barr Viral (EBV) syndrome, fibromyalgia, free
radical overload, Goodpasture syndrome, Grave's disease, hepatic
dysfunction (liver disease), hepatitis B, hepatitis C, HIV or
patients suffering from AIDS, hypercholesteremia (high blood
cholesterol), herpes, infections (viral, bacterial and fungal),
inflammatory bowel disease (IBD), lupus, macular degeneration,
malnutrition, Meniere's disease, multiple sclerosis, myasthenia
gravis, neurodegenerative diseases, nutritional disorers,
Parkinson's disease, Pemphigus vulgaris, Primary Billiary
Cirrhosis, progeria, psoriasis, rheumatic fever, sarcoidosis,
scleroderma, shingles, stroke, surgery, toxic poisoning, trauma,
vasculitis, vitiligo, and Wegener's granulomatosis
(nutritionadvisor.com/immunocalFAQ.html).
[0125] The term "utilization efficiency" as used herein refers to
how well the body uses the anti-oxidants which are administered to
counteract the damage caused by free radicals or other oxidizing
agents which play a role in the damage to cells and tissues. The
efficiency of use may be determined by either a direct measurement
of the oxidized material, for example, the levels of lipid peroxide
and the levels of PGA in the urine or a specific oxidized protein
such as oxidized low density lipoprotein (LDL), associated with
cells or tissues, or found circulating in the bloodstream. The
"utilization efficiency" is considered to be more effective when
the level of oxidized material is decreased after therapy with an
anti-oxidant compared to its level prior to the start of therapy
with an anti-oxidant.
[0126] By "effectiveness of therapy" is meant that upon treating a
subject with an anti-oxidant, one can determine whether the
treatment has resulted in the desired outcome. For example, in the
case of treating a patient having high levels of an oxidized
protein, for example, such as oxidized LDL (low density
lipoprotein), with an anti-oxidant, one may observe a decrease not
only in the amount of oxidized LDL, but also in the sequelae
associated with oxidized LDL, such as a decrease in the amount of
atherosclerotic plaque which ultimately may lead to an increase or
risk of heart failure. In addition, patients suffering from HIV may
also have sequelae that can be monitored after treatment with an
antioxidant. These may include, for example, changes in the number
of CD4+ T cells and CD8+ T cells and their corresponding
ratios.
[0127] "IMMUNE FORMULATION 100.TM." refers to a non-toxic
nutritional composition useful for increasing glutathione
production in a mammal in order to enhance the immune activity of
the mammal. This composition contains the following as essential
active ingredients:
[0128] a: a catalytic quantity of elemental selenium or a water
soluble selenium precursor;
[0129] b: from about 5% to about 95% of a special whey product
containing from about 65% to about 85% protein which is from about
65% to about 100% undenatured; and
[0130] c: from about 5% to about 95% by weight of colostrum;
[0131] the percent by weight of each component based on the total
weight of the composition. This material is further described and
claimed in U.S. Pat. No. 6,667,063.
[0132] "IMMUNE FORMULATION 200.TM." refers to a nutritional or
therapeutic composition useful for treatment of mammals to enhance
immune activity. This composition contains the following as
essential active ingredients: a catalytic quantity of a selenium
source together with glutathione precursors which are a mixture of
glutamic acid, cystine or another related cystine precursor, and
glycine in a molar ratio of about 1:0.5:1, the amount of
glutathione precursors being effective to increase the content of
glutathione in the body tissue of the mammal above that of a
pretreatment level thereby to enhance immune activity. This
material is further described and claimed in U.S. Pat. No.
6,592,908.
[0133] Glutathione is a tripeptide and a major reducing agent in
the mammalian body. Its chemical structure is: 1
[0134] or, more simply
GLU-CYS-GLY
[0135] Its chemical name is glutamyl-cysteinyl-glycine. Like many
other small peptides in the mammalian body, it is not synthesized
by procedures involving DNA, RNA and ribosomes. Rather, it is
synthesized from the amino acids available in the body and selenium
by procedures utilizing enzymes and other body components such as
adenosine triphosphate as an energy source.
[0136] The term "anti-oxidative effective amount" as used herein
refers to an amount of an anti-oxidant, such as for example, IMMUNE
FORMULATION 100.TM. or IMMUNE FORMULATION 200.TM. which, when
delivered to a patient or subject in need of such therapy results
in reduction of the proteins, cellular components, or tissue
components that have been oxidized and subsequently damaged or have
reduced functional capacity as a result of being oxidized. An
"anti-oxidative effective amount" of an anti-oxidant is the amount
of the anti-oxidant needed to restore certain functional capacities
to the proteins, cells or tissues that are damaged by oxidation.
The anti-oxidants are dietary supplements containing nutritional
products. If an excess of any amino acid is used, it will
presumably be of nutritional value or may be metabolized.
[0137] "Pyroglutamic acid" or "PGA" is a keto derivative of proline
that is formed nonenzymatically from glutamate, glutamine, and
gamma-glutamylated peptides. It is also produced by the action of
gamma-glutamylcyclotransferase. It is also referred to as
5-oxoproline, 5-pyrrolidone-2-carboxylic acid, or
pyrrolidone-5-carboxylate. Elevated levels are often associated
with problems of glutamine or glutathione metabolism.
[0138] "Lipid peroxides" are fats that have been damaged by excess
free radical activity. Lipid peroxides are the products of the
chemical damage done by oxygen free radicals to the lipid
components of cell membranes. This oxidative damage, caused by free
radical pathology, is thought to be a basic mechanism underlying
many diverse pathological conditions--atherosclerosis, cancer,
aging, rheumatic diseases, allergic inflammation, cardiac and
cerebral ischemia, respiratory distress syndrome, various liver
disorders, irradiation and thermal injury, and toxicity induced by
certain metals, solvents, pesticides and drugs. Measurement of
lipid peroxide levels plays a significant role in evaluating
cellular damage caused by oxidative stress and determining an
individual's specific need for antioxidant supplementation. The
level of lipid peroxides is an index of cellular membrane damage
caused by the action of free radicals. The organelle membranes,
such as those of the mitochondria, lysosomes, peroxisomes, and DNA
can be damaged as well. This damage is lipid peroxidation,
resulting from an excess of prooxidants over antioxidants. Such
excess, categorized as oxidative stress, can damage membrane
proteins and cholesterol, as well as membrane lipids. The elevation
of lipid peroxides can serve as an early warning of the long-term
effects of oxidative stress. The natural sequel of oxidative stress
is chronic degenerative disease. One example is that peroxidation
of low density lipoproteins contributes to atherosclerosis. Other
associated diseases include coronary artery disease and cancer, the
leading causes of death in the United States.
[0139] The term "free radicals" refers to a chemical species that
possesses an unpaired electron in the outer (valence) shell of the
molecule. This is the reason they are highly reactive and thus have
low chemical specificity i.e., they can react with most molecules
in their vicinity. This includes proteins, lipids, carbohydrates
and DNA. Free radicals attack the nearest stable molecule, thus
"stealing" its electron. When the attacked molecule loses its
electron, it becomes a free radical itself, thus beginning a chain
reaction. It continues until the final result is the disruption of
a living cell. Free radicals are produced continuously in cells
either as by-products of metabolism or deliberately as in
phagocytosis (Cheeseman, K. H. and Slater, T. F., Br Med Bull. 1993
July; 49(3): 481-93).
[0140] "Surrogate biomarker" or "biomarker" or "marker" as used
herein, refers to a highly specific molecule, the existence and
levels of which are causally connected to a complex biological
process, and reliably captures the state of said process.
Furthermore, a surrogate biomarker, to be of practical importance,
must be present in samples that can be obtained from individuals
without endangering their physical integrity or well-being,
preferentially from biological fluids such as blood, plasma, urine,
saliva or tears. The biomarkers of oxidative damage, as used
herein, include increased lipid peroxides and pyroglutamic acid and
decreased glutathione. The levels of these biomarkers should
reflect the degree of oxidative stress in the body and are the
result of certain diseases or conditions and should continue to
accumulate or remain stable in the body until released, excreted or
neutralized. Furthermore, the presence of these biomarkers, in
particular, lipid peroxides and pyroglutamic acid, should reflect
the need for anti-oxidant therapy. The normalization of these
biomarkers as well as normalization of the levels of glutathione
should also reflect the utilization efficiency and effectiveness of
anti-oxidative therapy as provided in the present application.
[0141] A person "suspected of being in need of such treatment" in
terms of the methods of the present invention may refer to an
individual suffering from symptoms suggestive of lowered immunity,
such as frequent infections or colds.
[0142] By the term "sequelae" of oxidative stress is meant the
conditions following as a consequence of the level or duration of
oxidative stress. This may include a predisposition for acquiring
infections, or a predisposition to certain pathological conditions
or diseases. It may include cellular or tissue damage resulting
from the persistence and/or buildup of free radicals in particular
tissues, for example, in kidney tissue following treatment with
certain drugs such as adriamycin, or following an injury to the
brain or spinal cord.
[0143] The term "antibody" as used herein includes intact molecules
as well as fragments thereof, such as Fab and F(ab').sub.2, which
are capable of binding the epitopic determinant. Antibodies that
bind the proteins or the markers of oxidative stress of the present
invention can be prepared using intact polypeptides or fragments
containing small peptides of interest as the immunizing antigen
attached to a carrier molecule. Commonly used carriers that are
chemically coupled to peptides include bovine or chicken serum
albumin, thyroglobulin, and other carriers known to those skilled
in the art. The coupled peptide is then used to immunize the animal
(e.g, a mouse, rat or rabbit). The antibody may be a "chimeric
antibody", which refers to a molecule in which different portions
are derived from different animal species, such as those having a
human immunoglobulin constant region and a variable region derived
from a murine mAb. (See, e.g., Cabilly et al., U.S. Pat. No.
4,816,567; and Boss et al., U.S. Pat. No. 4,816,397.). The antibody
may be a human or a humanized antibody. The antibody may be a
single chain antibody. (See, e.g., Curiel et al., U.S. Pat. No.
5,910,486 and U.S. Pat. No. 6,028,059). The various portions of the
chimerized antibodies can be joined together chemically by
conventional techniques, or can be prepared as a contiguous protein
using genetic engineering techniques. For example, nucleic acids
encoding a chimeric or humanized chain can be expressed to produce
a contiguous protein. See, e.g., Cabilly et al, U.S. Pat. No.
4,816,567; Cabilly et al., European Patent No. 0,125,023 B1; Boss
et al., U.S. Pat. No. 4,816,397; Boss et al., European Patent No.
0,120,694 B1; Neuberger, M. S. et al., WO 86/01533; Neuberger, M.
S. et al., European Patent No. 0,194,276 B1; Winter, U.S. Pat. No.
5,225,539; Winter, European Patent No. 0,239,400 B1; and Queen et
al., U.S. Pat. Nos. 5,585,089, 5,698,761 and 5,698,762. See also,
Newman, R. et al., BioTechnology, 10: 1455-1460 (1992), regarding
primatized antibody, and Ladner et al., U.S. Pat. No. 4,946,778 and
Bird, R. E. et al., Science, 242: 423-426 (1988)) regarding single
chain antibodies. The antibody may be prepared in, but not limited
to, mice, rats, rabbits, goats, sheep, swine, dogs, cats, or
horses.
[0144] General Description
[0145] Finding a means of protecting cells and/or molecules from
the effects of free radicals is of obvious medical importance.
These free radicals have been associated with a large variety of
conditions in which cells die, resulting in severe clinical
consequences.
[0146] Furthermore, the need for protection of cells and/or
molecules from the damage caused by free radicals has been
addressed, in part, by administration of anti-oxidants. However, it
has been difficult to measure the presence of free radicals due to
their very short half-life. Furthermore, the methods that are
available, such as electron spin resonance and spin trapping
methods (Cheeseman, K. H. and Slater, T. F., Br Med Bull. 1993
July; 49(3): 481-93) using exogenous compounds having a high
affinity for free radicals, have poor sensitivity and are not 100%
accurate, that is, they produce only semi-quantitative data.
[0147] Therefore, it is necessary to find alternate strategies to
assess the presence of free radicals and the level of oxidative
stress in an individual patient. A commonly used technique is to
measure a marker of free radicals rather than the radical itself
(Slater, T. F., Methods Enzymol. 1984; 105:283-93; Pryor W. A. and
Godber S. S., Free Radic Biol Med. 1991; 10(3-4):173). Thus,
various assays have been devised to measure these markers of
oxidative stress.
[0148] Likewise, it is difficult to assess whether an individual is
in need of treatment with an anti-oxidant. Furthermore, once it has
been determined that an individual would benefit from such therapy,
it is important to be able to assess whether the therapy as
delivered is being utilized to its full capacity. And finally, it
is necessary to determine how effective that therapy is by
assessing an individual's response and outcome following such
therapy.
[0149] Accordingly, the present invention provides a
multidimensional and comprehensive method for assessing the need
for treatment of a subject with an anti-oxidant. Previous tests for
measuring an individual's level of oxidative stress has relied
primarily on the measurement of one or two markers of oxidative
stress, such as lipid peroxides. The present invention provides for
the quantitation of several markers of oxidative stress, including
lipid peroxides, pyroglutamic acid, and glutathione. In addition,
since it is becoming more apparent that an individual's immune
status may depend in part on the level of glutathione present in
vivo, the present invention provides for concurrent measurement of
immune cell numbers and activity, in particular, the number of CD4+
and CD8+ T cells, and the level of natural killer cell activity.
The present invention will thus provide for the interrelationship
between lipid peroxides, pyroglutamic acid, and glutathione, as
well as the immune functions associated with adequate levels of
antioxidants, and the markers thereof. There have been no known
studies of which the inventor is aware that provides this
comprehensive and quantitative manner of assessment of oxidative
stress and related immune functions. It is a further object of the
present invention to be able to measure the utilization efficiency
of the anti-oxidant therapy once an individual has started therapy
with an anti-oxidant. It is yet a further object of the present
invention to be able to measure the effectiveness of the
anti-oxidant therapy once an individual has started and then
completed the therapy with an anti-oxidant.
[0150] Furthermore, this invention is based, in part, on the use of
novel IMMUNE FORMULATIONs, in particular, IMMUNE FORMULATION
100.TM. or IMMUNE FORMULATION 200.TM., and provides for methods of
determining the amount of these formulations necessary to increase
glutathione synthesis or re-synthesis in a patient in need of such
therapy and to reduce the amounts of lipid peroxides and
pyroglutamic acid levels in these patients. The ability to monitor
the elevation in plasma glutathione levels while concurrently
measuring a decrease in two oxyradical metabolites, urine lipid
peroxide and pyroglutamic acid, provides a more quantitative and
accurate assessment of a patient's level of oxidative stress, and
allows for adjustment in dose and duration of appropriate
anti-oxidant therapies, including those described herein.
[0151] Accordingly, the present invention provides for tracking two
metabolites from their respective metabolic pathways to establish
anti-oxidant need before treatment with specific anti-oxidant
formulations, and after the administration of the anti-oxidants to
demonstrate the utilization efficiency of the therapy and eventual
effectiveness of the therapy on glutathione synthesis. In preferred
embodiments, the anti-oxidant therapies are IMMUNE FORMULATION
100.TM. or IMMUNE FORMULATION 200.TM..
[0152] Thus, it is important to track the oxyradical metabolite
urine lipid peroxide before and after therapy to determine where
the level falls as compared to the normal range. The normal range
as determined by standard clinical testing, such as that provided
by Metametrix Clinical Laboratory, is about 8.9 to 13.3 nM/mg.
[0153] Furthermore, it is also crucial to track the catabolic
breakdown product of glutathione, known as pyroglutamic acid (PGA)
in the urine and to determine where the level falls as compared to
the normal range. The normal range as determined by standard
clinical testing, such as that provided by Metametrix Clinical
Laboratory, is about <80 .mu.g/mg of creatinine.
[0154] Therefore, establishing the patient's baseline level of
these metabolites will help to establish whether there is a need
for anti-oxidant therapy. If the level falls outside of the normal
range for these metabolites, there is a need for initiation of
anti-oxidant therapy.
[0155] Thus, once certain anti-oxidant deficiencies have been
established which may be responsive to therapies with
anti-oxidants, it would be imperative to determine the utilization
efficiency of such therapy once it has been initiated. Therapies
such as IMMUNE FORMULATION 100.TM. or IMMUNE FORMULATION 200.TM.
would in all likelihood be most beneficial, due to the effects of
these formulations on production of glutathione and its
anti-oxidant activity per se and/or its antioxidant activity with
selenium through glutathione peroxidase. Afterwards, the overall
effectiveness of such therapies must be monitored such as by the
methods of the present invention to determine whether normal levels
of lipid peroxides, PGA and glutathione have been established. The
determination as to whether there is a need for continued therapy
would be made by the health care assistant, physician, or by
self-assessment.
[0156] Methods of Quantifying Lipid Peroxides
[0157] When a fatty acid is peroxidized it is broken down into
aldehydes, which are excreted. Aldehydes such as thiobarbituric
acid reacting substances (TBARS) have been widely accepted as a
general marker of free radical production, with the most commonly
measured TBARS being malondialdehyde (Esterbauer, H, Jurgens, G,
Quehenberger, O, & Koller, E, J Lipid Res 1987, 28:495-509;
Buege, J A & Aust, S D, Methods Enzymol 1978, 52:302-310;
Wallin, B & Camejo, G, Scand J Clin Lab Invest 1994,
54:341-346; El-Saadani, M, Esterbauer, H, El-Sayed, M, Goher, M,
Nassar, A Y, & Jurgens, G A, J Lipid Res 1989, 30:627-630;
Esterbauer, H, Gebicki, J, Puhl, H, & Jurgens, G, Free Radic
Biol Med 1992, 13:341-390).
[0158] Methods of measuring oxidized lipids are well known to those
of skill in the art (see, e.g., Vigo-Pelfrey et al. Membrane Lipid
Oxidation, Volume I-II. CRC Press). such methods include, but are
not limited to mass spectrometry, absorption spectrometry (e.g.,
using UV absorbance at 234 nm), liquid chromatography, thin layer
chromatography, and the use of various "oxidation-state" sensitive
reagents, e.g. in various redox reactions.
[0159] Previously known methods for measuring oxidized lipids (e.g.
lipid peroxides), include the Wheeler method, iron thiocyanate
method, thiobarbituric acid method, and others. The Wheeler method
(Wheeler (1932) Oil and Soap, 9: 89-97) is that in which oxidized
lipid is reacted with potassium iodide to isolate iodine, which is
then titrated with a sodium thiosulfate standard solution. In the
iron thiocyanate method (Stine et al. (1954) J. Dairy Sci., 37:
202) oxidized lipid peroxide is mixed with ammonium thiocyanate and
ferrous chloride, and the blue color from the resulting iron
thiocyanate is calorimetrically determined. In the thiobarbituric
acid method (Tappel and Zalkin (1959) Arch. Biochem. Biophys., 80:
326) the lipid peroxide is heated under acidic conditions and the
resulting malondialdehyde is condensed with thiobarbituric acid to
form a red color dye, which is then calorimetrically measured.
[0160] In another approach, it has been demonstrated that
peroxidase decomposes lipid peroxides and that the resulting
reaction system colors intensely with increasing quantities of
lipid peroxide, if an adequate hydrogen donor is present in the
reaction system (see, e.g., U.S. Pat. No. 4,367,285) Thus, in one
embodiment, the assays of this invention may utilize a peroxidase
and a hydrogen donor.
[0161] Many peroxidases are suitable. The peroxidase employed in
the present invention is preferably any of the commercially
available horseradish peroxidases.
[0162] The hydrogen donor employed may be any of the known
oxidizable compounds which, preferably, generate color,
fluorescence or luminescence upon oxidation. The conventional
coloring, fluorescent, luminescent reagents may be utilized. The
known coloring reagents which may be employed include, but are not
limited to guaiacol, 4-aminoantipyrine with phenol,
4-aminoantipyrine with N,N-dimethylaniline,
3-methyl-2-benzothiazolinone with dimethylaniline,
ortho-dianisidine, and the like. Typically useful fluorescent
reagents include, but are not limited to homovanillic acid,
p-hydroxyphenylacetic acid, and the like. Suitable luminescent
reagents include but are not limited to luminol and the like. The
amount of the hydrogen donor employed is preferably at least
equimolar, preferably not less than two moles, per mole of lipid
peroxide contained in test sample. The amount may be varied
depending upon the size of the sample and the content of the lipid
peroxide in the sample.
[0163] Suitable reaction mediums which may be employed include, but
are not limited to dimethylglutarate-sodium hydroxide buffer
solution, phosphate buffer solution and, Tris-hydrochloric acid
buffer solution is normally from about pH 5 to about pH 9.
[0164] Such factors as the pH at the time of reaction, the reaction
period, the measuring wavelength, etc., may be varied depending
upon the reagents employed. Suitable conditions can be selected
according to the circumstances.
[0165] Another class of assays for oxidized lipids is described in
U.S. Pat. No. 4,900,680. In this approach, an oxidized lipid (e.g.
a hydroperoxide) is reacted with a salt or hydroxide of a
transition metal which produces a cation having a valency of 2, a
complex of a transition metal having a valency of 2, a heme, a heme
peptide, a heme protein, or a heme enzyme. The resultant active
oxygen and oxygen radicals react with a luminescent substance, and
light emitted by this reaction is optically measured. Examples of a
catalyst acting on a lipid hydroperoxide to produce active oxygen
species such as active oxygen or oxygen radicals are: a transition
metal salt which produces a cation having a valency of 2 (e.g.,
ferrous chloride, ferrous sulfate, potassium ferricyanide, each of
which produces Fe.sup.2+; manganous chloride or manganous sulfate,
each of which produces Mn.sup.2+; or cobalt chloride or cobalt
sulfate, each of which produces Co.sup.2+); a hydroxide of the
transition metals described above; a complex of a transition metal
having a valency of 2 (e.g., FeII-porphyrin complex); a heme
protein (e.g., cytochrome C, hemoglobin, or myoglobin); a heme
peptide (e.g., a compound obtained by decomposing a heme protein by
a protease such as chymotrypsin or trypsin); and a heme enzyme
(e.g., horseradish peroxidase or prostaglandin peroxidase).
[0166] Particular catalyst compounds include, but are not limited
to, a heme protein, a heme peptide, or a heme enzyme. Most usually,
the heme protein such as cytochrome C is used due to easy handling.
The concentration of the catalyst compound preferably ranges from
about 0.1 .mu.g/ml to about 1,000 .mu.g/ml and usually falls within
the range of about 1 .mu.g/ml to about 200 .mu.g/ml. For example,
best luminous efficiency can be obtained when the concentration is
about 10 .mu.g/ml for cytochrome C, about 120 .mu.g/ml for
cytochrome C heme peptide; and about 10 .mu.g/ml for horseradish
peroxidase.
[0167] The luminescent substance is not limited to a specific one,
provided it reacts with active oxygen or an oxygen radical to emit
light. Examples of such a compound include, but are not limited to
polyhydroxyphenols (e.g., pyrogallol, perprogalline etc.),
phthaladine derivatives (e.g., luminol, isoluminol, etc.), indol
derivatives (e.g., indoleacetic acid, skatole, tryptophan, etc.);
thiazolidine derivatives (e.g., Cypridinacea luciferin, lophine,
etc.), an acrydine derivatives (e.g., lucigenine), oxalic acid
derivatives (e.g., bistrichlorophenyloxalate); and 1,2-
dioxa-4,5-azine derivatives. The concentration of the luminescent
substance varies depending on the compound used. The concentration
is preferably 0.1 .mu.g/ml or more. When luminol is used, its
concentration is most preferably 1 .mu.g/ml.
[0168] Measurements are preferably performed in a weak basic
solution of a luminescent reagent such as a heme protein and
luminol. A particular pH value ranges from about pH 9 to about pH
10. Many buffers are suitable. One particular buffer is a borate
buffer (H.sub.3BO.sub.3--KOH), a carbonate buffer
(Na.sub.2CO.sub.3--NaHCO.sub.3), a glycine buffer
(NH.sub.2CH.sub.2COOH--NaOH), or the like. The borate buffer is
most preferred.
[0169] In order to prevent oxygen dissolved in the luminescent
reagent solution from interfering with the analysis of a very small
amount of oxidized lipid, the luminescent reagent solution is
desirably purged with an inert gas to remove oxygen to obtain a
stable measurement value. Examples of the inert gas are nitrogen
gas and argon gas.
[0170] The concentration of the oxidized lipid in the sample is
calculated based on a calibration curve. The calibration curve can
be formed according to standard methods, e.g., by using a material
selected from methyl linolate hydroperoxide, arachidonic acid
hydroperoxide, phosphatidylcholine hydroperoxide,
phosphatidylethanolamine hydroperoxide, and triacylglycerol
hydroperoxide.
[0171] The methods of this invention may utilize fluorescent
materials whose fluorescence is altered by oxidation state. Such
fluorescent materials are well known to those of skill in the art
and include, but are not limited to 2'7'-dichlorodihydrofluorescine
diacetate, rhodamine cis-parinaric acid, NBD, cis-parinimic acid
cholesteryl esters, diphenylhexatriene proprionic acid, and the
like. The use of such indicators is illustrated in the
examples.
[0172] It will be appreciated that the foregoing methods of
detecting/quantifying oxidized lipids are intended to be
illustrative and not limiting. Numerous other methods of assaying
oxidized lipids are known to those of skill in the art and are
within the purview of this application.
[0173] The TBA test has been challenged because of its lack of
specificity, sensitivity, and reproducibility. The use of liquid
chromatography instead spectrophotometer techniques help reduce
these errors. In addition, the test seems to work best when applied
to membrane systems such as microsomes. Gases such as pentane and
ethane are also created as lipid peroxidation occurs. These gases
are expired and commonly measured during free radical research.
Dillard (Dillard et al. Free Radic Biol Med. 1989; 7(2): 193-6) was
one of the first to determine that expired pentane increased as VO2
max increased. Kanter et al. (Kanter M M, Nolte L A, Holloszy J O,
J Appl Physiol. 1993 February; 74(2):965-9) has reported that serum
MDA levels correlated closely with blood levels of creatine kinase,
an indicator of muscle damage. Lastly, conjugated dienes (CD) are
often measured as indicators of free radical production. Oxidation
of unsaturated fatty acids results in the formation of CD. The CD
formed are measured and provide a marker of the early stages of
lipid peroxidation (Poirier B, Michel O, Bazin R, Bariety J,
Chevalier J, Myara I. Nephrol Dial Transplant. 2001 August;
16(8):1598-606). A newly developed technique for measuring free
radical production shows promise in producing more valid results.
The technique uses monoclonal antibodies and may prove to be the
most accurate measurement of free radicals. However, until further
more reliable techniques are established it is generally accepted
that two or more assays be utilized whenever possible to enhance
validity.
[0174] When glutathione or glutathione peroxidase (which uses
selenium) or ADP for regeneration of oxidized glutathione is
deficient, blood lipid peroxide and subsequently urine lipid
peroxide would increase.
[0175] Oral ingestion of IMMUNE FORMULATION 100.TM. or IMMUNE
FORMULATION 200.TM. can replenish glutathione levels and have a
vital antioxidant effect, thus, reducing blood lipid peroxide and
consequently urine lipid peroxide.
[0176] This metabolite, urine lipid peroxide, can mirror the body's
antioxidant need and mirror the utilization efficiency of IMMUNE
FORMULATION 100.TM. or IMMUNE FORMULATION 200.TM. to remediate that
deficiency. It can also mirror the effectiveness of glutathione
synthesis from IMMUNE FORMULATION 100 or IMMUNE FORMULATION 200.TM.
during that deficiency. However, the amount of IMMUNE FORMULATION
100.TM. or IMMUNE FORMULATION 200.TM. needed to reverse the
deficiency is to be quantified so as to establish a cause and
effect interrelationship.
[0177] The following equation demonstrates that a toxic peroxide
can be detoxified by glutathione to form water and oxidized
G-S--S-G. 2
[0178] The high level of toxic lipid peroxide in the urine would
indicate that sufficient glutathione or selenium is not available
to detoxify that peroxide. This can be tested and proven by
quantifying the collaboration between the oral ingestion of
specified amounts of IMMUNE FORMULATION 100.TM. or IMMUNE
FORMULATION.sup.200.TM. and the reduction of lipid peroxide in the
urine. Concurrent measurement of plasma glutathione levels would be
made to confirm that the therapy resulted in synthesis or
resynthesis of glutathione levels. The normal range for glutathione
in the plasma is about 200-400 mole/L (AmScot.TM., Cincinnati,
Ohio).
[0179] Therefore, oral ingestion of an antioxidant such as IMMUNE
FORMULATION 100.TM. or IMMUNE FORMULATION 200.TM. should result in
reduced urinary excretion of lipid peroxide and increased levels of
glutathione synthesis in blood plasma, thus proving that these
three functions are interrelated and interdependent.
[0180] There are several methods and kits available and known to
those skilled in the art for measuring the presence of lipid
peroxide in bodily fluids, including urine. Using these methods,
the normal range of urine lipid peroxide is about 8.9 to 13.3 nM/mg
creatinine (Metametrix Clinical Laboratory, Norcross, Ga.). Using
this as the normal range, the physician assessing his or her
patient for the level of oxidative stress can use one of the
validated means for measuring urine lipid peroxide and obtain a
value for that patient to determine where he or she is in relation
to this normal value. If the level increases to a level outside of
the normal range, he or she is in need of therapy with an
anti-oxidant. In one particular embodiment, a patient in need of
anti-oxidant therapy has a urine level of lipid peroxide which is
above the normal range. Preferably, the anti-oxidant of choice is
IMMUNE FORMULATION 100.TM. or 200.TM., although other anti-oxidant
therapies are also contemplated for use with the invention.
[0181] Furthermore, upon initiation of therapy with an
anti-oxidant, it is of use to determine the utilization efficiency
of such therapy by monitoring changes in urine lipid peroxide, to
determine if the level is being adjusted to within the normal
range. In addition, these levels would be monitored throughout the
course of therapy to determine the effectiveness of therapy with
the anti-oxidant. The measurement of urine lipid peroxide may be
done prior to, concurrently with, or shortly thereafter, a measure
of urine pyroglutamic acid and plasma glutathione levels in order
to provide a secondary confirmation and assessment of the patient's
oxidative stress level. Furthermore, these tests may be combined
with other test parameters to determine whether the patient has
recovered sufficiently in order for therapy with the anti-oxidant
to be discontinued. For example, in the case of a patient suffering
from diabetes or high cholesterol or high triglyceride levels, the
physician may assess other parameters such as glycated hemoglobin,
glucose levels or request a full battery of blood chemistries to
assess the overall health status of the individual before
discontinuing therapy with the anti-oxidant.
[0182] The methods for measuring lipid peroxidation products and
lipid peroxidation damage in tissues, cells and body fluids have
been described above. The choice of which method is most
appropriate depends, among other things, on whether the measurement
is needed for strictly research purposes, or for a particular
medical situation or condition wherein the patient is under the
care of a qualified physician, or health care worker, or uses a
self-administered urine kit. In the routine clinical research
laboratory, the determination of thiobarbituric acid reactive
substances (TBARS) under strictly standardized conditions is in
most cases the first choice. The specificity of the colorimetric or
fluorimetric assay can be significantly improved if it is combined
with analysis by HPLC methods. If the level of TBARS is increased,
other more sophisticated assays should be performed for
verification or validation of the values obtained. Several such
assays are available including: Phospholipid- and cholesterylester
hydroperoxides, aldehydic lipid peroxidation products including
4-hydroxynonenal, fluorescent protein adducts (e.g. lipofuscin),
conjugated dienes and antioxidants. The measurement of pentane and
ethane in the exhaled air by gas chromatography has been the only
available non-invasive method, although this assay method has its
own drawbacks in that it is time consuming and cumbersome to run
routinely. Several laboratories have also developed immunological
assays such as the enzyme-linked immunosorbent assay (ELISA) or the
radioimmunoassay (RIA) for determining proteins modified by lipid
peroxidation products (e.g. malondialdehyde, 4-hydroxynonenal) or
autoantibodies against oxidatively modified proteins. These assays
provide a much more convenient and quantitative assessment of the
by-products of oxidation and are much less cumbersome and time
consuming to run as compared to the HPLC or other standard chemical
approaches for assay and quantitation.
[0183] Under standard laboratory practice, and for assessment of a
patient's clinical condition, an increased concentration of end
products of lipid peroxidation is the evidence most frequently
cited for the involvement of free radicals in human disease.
However, while it is recognized that the generation of free
radicals and the subsequent oxidative damage resulting from their
presence occurs in most diseases, it is also true that oxidative
damage plays a significant pathological role in only some of these
diseases. This is true, for example, in atherosclerosis and in
exacerbation of the initial tissue injury caused by ischemic or
traumatic brain injury or spinal cord injury, where peroxidation of
the surrounding cells and tissues appears to be extremely important
in the pathology of these conditions. Moreover, it is also becoming
more apparent that stress plays a role in reduction in immune
responsiveness to known pathogens and that this may be due in part
to reduced levels of glutathione. (Leonore A. Herzenberg et al,
Proc. Natl. Acad. Sci. USA Vol. 94, pp. 1967-1972, March 1997)
Oxidative stress can damage many biological molecules including
proteins, DNA and lipids. Many assays are available to measure
lipid peroxidation, but no single assay is an accurate measure of
the whole process. Application of simple diene-conjugate and
thiobarbituric acid (TBA) assays to human tissues and body fluids
can produce artifacts. Thus, it is important to utilize other
methods for assessment of a more accurate clinical profile.
[0184] Some of the methods used to monitor oxidative products
include the following:
[0185] Determination of Urinary Thiobarbituric-Acid-Reacting
Substances (TBARS) This procedure involves the incubation of a
sample of urine with 5% butylated hydroxytoluene (in glacial acetic
acid) and 0.5% aqueous thiobarbituris acid (TBA) solution (Buege J
A, Aust S D: Microsomal lipid peroxidation. Methods Enzymol (1978),
52:302-310; Valenzuela A: The biological significance of
malondialdehyde determination in the assessment of tissue oxidative
stress. Life Sci 1991, 48:301-309). After mixing and incubating the
mixture for about 30 minutes, the absorbance is measured at 532 nm
using a spectrophotometer. The quantity of TBARS is proportional to
the amount of malondialdehyde (MDA), a lipid peroxidation product
generated by the oxidation of lipids by reactive oxygen spoecies.
MDA reacts with TBA to form a 1:2 MDA:TBA adduct that absorbs at
532 nm. To control for urine concentration, data are normalized to
urine creatinine concentrations as described by Coulthard et al.
(Coulthard M G, Hey E N, Ruddock V: Creatinine and urea clearances
compared to inulin clearance in preterm and mature babies. Early
Hum Dev 1985, 11:11-19).
[0186] Measurement of 8-epi-prostaglandin PGF2a (8-epi-PGF2a) The
appearance of 8-epi-prostaglandin PGF2a (8-epi-PGF2a) in plasma or
urine has been suggested by a number of investigators as a reliable
index of in vivo free radical generation and oxidative lipid
formation. There is very strong evidence from animal studies that
8-epi-PGF2a increase in plasma and urine as a result of oxidative
stress, and in human, this product is elevated in smokers.
Comparison with other measures of lipid peroxidation, 8-epi-PGF2a
is specific product of lipid peroxidation, and is very stable. In
addition, its formation is modulated by antioxidant status, and its
level is not affected by lipid content of the diet. The measurement
of 8-epi-prostaglandin PGF2 can be done using a standard
immunoassay using antibodies specific for this by-product of lipid
oxidation.
[0187] ELISA kits for measurement of 8-hydroxy-2'-deoxyguanosine
(8-OHdG) 8-OhdG is a nucleotide which is excised from DNA.
Endonuclease repair enzymes work quickly therefore the amount
excised in urine directly reflects a person's degree of damage in
the body. These kits, which are available from Genox, measure the
amount of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in urine, serum,
plasma, tissue homogenate and digested lymphocyte DNA samples.
Genox is a distributor of products developed by the Japan Institute
for the Control of Aging. Furthermore, Genox also sells a
monoclonal antibody against 8-OhdG for immunohistochemical studies
on tissue samples. These kits contain the following.
1 8-OHdG coated microtiter plate (split type) Primary antibody
(Anti-8-OHdG monoclonal antibody) Primary antibody solution
Secondary antibody (POD-conjugated anti mouse antibody) Secondary
antibody solution Chromatic solution (3, 3', 5,
5'-tetramethylbenzidine) Substrate solution Washing solution
Reaction terminating solution Standard 8-OHdG solution (0.5, 2, 8,
20, 80, 200 ng/ml) Plate seal
[0188] The general procedure is as follows:
[0189] (1) Primary Antibody Reaction (Competitive Reaction):
37.degree. C. for 1 hour
[0190] (2) Secondary Antibody Reaction: 37.degree. C. for 1
hour
[0191] (3) Development of Color Reaction: Room Temperature for 15
min in the dark
[0192] (4) Absorbance Reading (wavelength at 450 nm) and
Calculation of Results
[0193] Additional methods for measuring 8-hydroxy-2'-deoxyguanosine
(8-OHdG) are essentially as outlined in the following references,
which are incorporated in their entirety. (S. S. Kantha, S. Wada,
H. Tanaka, M. Takeuchi, S. Watabe, and H. Ochi Carnosine sustains
the retention of cell morphology in continuous fibroblast culture
subjected to nutritional insult. Biochemical and Biophysical
Research Communications, 223, 278-282 (1996); S. S. Kantha, S.
Wada, M. Takeuchi, S. Watabe, and H. Ochi, A sensitive method to
screen for hydroxyl radical scavenging activity in natural food
extracts using competitive inhibition ELISA for
8-hydroxydeoxyguanosine; Biotechnology Techniques, 10(12), 933-936
(1996); J. Leinonen, T. Lehtimaki, S. Toyokuni, K. Okada, T.
Tanaka, H. Hiai, H. Ochi, P. Laippala, V. Rantalaiho, O. Wirta, A.
Pasternack, and H. Alho, New biomarker evidence of oxidative DNA
damage in patients with non-insulin-dependent diabetes mellitus;
FEBS Letters, 417, 150-152 (1997); H. Tsuboi, K. Kouda, H.
Takeuchi, M. Takigawa, Y. Masamoto, M. Takeuchi, and H. Ochi,
8-Hydroxydeoxyguanosine in urine as an index of oxidative damage to
DNA in the evaluation of atopic dermatitis, British Journal of
Dermatology, 138, 1033-1035 (1998); Y. Miyake, K. Yamamoto, N.
Tsujihara, and T. Osawa Protective effects of lemon flavonoids on
oxidative stress in diabetic rats. Lipids, 33(7), 689-695 (1998);
M-H. Kang, M. Naito, N. Tsujihara, and T. Osawa Sesamolin inhibits
lipid peroxidation in rat liver and kidney. Journal of Nutrition,
128, 1018-1022 (1998); T. Arimoto, T. Yoshikawa, H. Takano, M.
Kohno Generation of reactive oxygen species and
8-hydroxy-2'-deoxyguanosine formation from diesel exhaust particles
components in L1210 cells. Japanese Journal of Pharmacology, 80,
49-54 (1999); M. D. Evans, M. S. Cooke, I. D. Podmore, Q. Zheng, K.
E. Herbert, and J. Lunec, Discrepancies in the measurement of
UVC-induced 8-oxo-2'-deoxyguanosine: Implications for the analysis
of oxidative DNA damage. Biochemical and Biophysical Research
Communications, 259, 374-378 (1999)).
[0194] AntiOxidant Check by Body Balance is a safe, easy-to-use,
and reliable test that uses a small urine sample to measure free
radical activity by measuring lipid peroxide levels. The kit
provides a sample collection device for urine, which is collected
and forwarded to a laboratory for analysis.
[0195] Oxis Bioxytech LPO586 from Oxis International (Seattle,
Wash.) is a colorimetric assay for evaluating lipid peroxidation.
The results are specific for malondialdehyde and 4-hydroxyalkenals,
which are markers of lipid peroxidation.
[0196] Methods for Measuring Glutathione
[0197] Glutathione is recycled and reutilized in the kidney, after
its three constituent amino acids are broken down in the renal
tubules. Any amino acid that is not considered healthy or optimal
is theoretically excreted via the renal tubules, and glutathione
cannot be resynthesized unless it gains an optimal amount of its
three precursor amino acids, cysteine, glycine and glutamate. When
there is a deficiency of one of these amino acids, especially
cysteine, glutathione cannot optimally be resynthesized. It is thus
broken down and excreted in the urine via its constituent parts as
waste products, one of which is pyroglutamic acid, described below.
PGA is a crucial marker of glutathione breakdown and its deficient
reutilization in the kidney. Therefore, it is crucial to establish
the level of this depletion and to determine whether IMMUNE
FORMULATION 100.TM. or IMMUNE FORMULATION 200.TM. can replenish
glutathione levels and reverse the excessive excretion of urine
lipid peroxide. It would be possible to use urine PGA as a marker
of glutathione need, glutathione utilization, and glutathione
resynthesis and of IMMUNE FORMULATION 100.TM. or IMMUNE FORMULATION
200.TM. efficiency. Thus, high urine PGA is a marker for
glutathione need, diminished glutathione utilization and deficient
glutathione resynthesis.
[0198] Several methods are currently available for measuring
glutathione levels. The normal level of plasma glutathione as
measured by AmScot.TM. in Cincinnati, Ohio is about 200-400 mole/L.
The methods for measurement of glutathione are listed below.
[0199] The Bioxytech GSH-400 kit from Oxis International (Seattle,
Wash.) is a non-enzymatic, colorimetric assay specific for
glutathione.
[0200] Cayman Chemical produces a glutathione assay kit, which
utilizes a carefully optimized enzymatic recycling method, using
glutathione reductase for the quantification of GSH. (Baker, M. A.,
Cerniglia, G. J., and Zaman, A. Microtiter plate assay for the
measurement of glutathione (GSH) and glutathione disulfide (GSSG)
in large numbers of biological samples. Anal. Biochem. 190, 360-365
(1990); Eyer, P. and Podhradsky, D. Evaluation of the micromethod
for determination of glutathione using enzymatic cycling and
Ellman's reagent. Anal. Biochem. 153, 57-66 (1986); Tietze, F.
Enzymic method for quantitative determination of nanogram amounts
of total and oxidized glutathione: Applications to mammalian blood
and other tissues. Anal. Biochem. 27, 502-522 (1969)). Briefly, the
sulfhydryl group of GSH reacts with DTNB
(5,5'-dithio-bis-2-nitrobenzoic acid, Eliman's reagent) and
produces a yellow colored 5-thio-2-nitrobenzoic acid (TNB). The
mixed disulfide, GSTNB (between GSH and TNB) that is concomitantly
produced, is reduced by glutathione reductase to recycle the GSH
and produce more TNB. The rate of TNB production is directly
proportional to this recycling reaction, which in turn is directly
proportional to the concentration of GSH in the sample. Measurement
of the absorbance of TNB at 405 or 412 nm provides an accurate
estimation of GSH in the sample. GSH is easily oxidized to the
disulfide dimer GSSG. Because of the use of glutathione reductase
in the Cayman GSH assay kit, both GSH and GSSG are measured and the
assay reflects total glutathione. The kit can also be used to
measure only GSSG by following an alternative protocol. GSH
measurement can be done in plasma, tissue samples, and cultured
cells using this kit. Nearly all samples require deproteination
before assay.
[0201] Methods for Measuring Pyroglutamic Acid
[0202] The presence of pyroglutamic acid (PGA), also known as
5-oxoproline, in the urine is an indication of a defect in the
.gamma.-glutamyl cycle, a series of enzyme-linked reactions
involved in the synthesis, metabolism, and transcellular transport
of glutathione. Furthermore, it is present in urine in patients
suffering from diseases or conditions which induce high levels of
oxidative stress, such as those conditions outlined in the present
application. In these conditions, the presence of abnormally high
levels of PGA is an indication that glutathione levels are low and
that there is a defect in glutathione re-synthesis. The normal
laboratory range for PGA is <80 .mu.g/mg of creatinine
(MetaMetrix Clinical Laboratory, Norcross, Ga.). The levels of
creatinine are measured using standard clinical laboratory
procedures known to those skilled in the art.
[0203] One object of the present invention is to determine if and
how much IMMUNE FORMULATION 100.TM. or 200.TM. would diminish PGA
excretion in the urine and how much IMMUNE FORMULATION 100.TM. or
200.TM. would be needed to increase glutathione synthesis or
resynthesis during this deficiency. Glutathione levels would be
tested in blood plasma while PGA would be tested concurrently in
the urine. Most preferably, both urine lipid peroxides and PGA
would both be measured concurrently with plasma glutathione to aid
in accuracy of the test results and to establish a more precise
assessment of the need for treatment with an antioxidant, such as
IMMUNE FORMULATION 100.TM. or 200.TM.. Therefore, oral ingestion of
an antioxidant such as IMMUNE FORMULATION 100.TM. or IME
FORMULATION 200.TM. should result in reduced urinary excretion of
PGA and increased levels of glutathione synthesis in blood plasma,
thus proving that these three functions are interrelated and
interdependent.
[0204] Thus, analysis of a urine sample for measurement of PGA from
a patient experiencing oxidative stress can be done using standard
gas chromatography-mass spectrometry techniques and would
demonstrate a markedly increased excretion of 5-oxoproline or PGA
in a patient experiencing oxidative stress and would correlate with
lowered plasma glutathione levels.
[0205] PGA measurements can be done by gas chromatography using a
Hewlett-Packard 5890 series II fitted with 7673A autosampler, HP-1
capillary column (25 m.times.0.2 mm.times.0.33-.mu.m film
thickness; Hewlett-Packard), and HP5971A mass-selective detector.
Helium gas flow rate can be 0.6 mL/min (head pressure, 114 kPa).
Split injections (ratio, 100:1) can be made with a 1-.mu.L sample.
A one-step temperature program may be run from 70 to 290.degree. C.
at 7.degree. C./min after an initial time of 0.5 min. The mass
spectrometer in electron ionization mode, connected directly to the
capillary column outlet, would be operated at 70 eV. Data
aquisition can be carried out in the scan mode from m/z 58 to 550,
with dwell time of 100 ms. The method of extraction and preparation
of urine samples for gas chromatography-mass spectrometry and the
method for qualitatively and quantitatively identifying
5-oxoproline are based on those described by Tanaka et al. (Tanaka
K, West-Dull A, Hine D G, Lynn T B, Lowe T. Gas chromatographic
method of analysis for urinary organic acids. I. Retention indices
of 155 metabolically important compounds. Clin Chem 1980;
26:1839-1846; Tanaka K, West-Dull A, Hine D G, Lynn T B, Lowe T.
Gas chromatographic method of analysis for urinary organic acids.
II. Description of the procedure and its application to diagnosis
of patients with organic acidurias. Clin Chem 1980; 26:1847-1853).
The response factor to the internal standard, isopentanoic acid,
could be used to approximate the 5-oxoproline peak as identified by
comparison with published spectra.
[0206] Amino acid measurements, including measurement of
glutathione in urine, plasma, and whole-blood hemolysate could be
made with a Biotronic LC5001 amino acid analyzer
(Eppendorf-Netheler-Hinz, division of Biotronic) and a Trivector
TRIO computing integrator (Trivector Technical Services). A glass
separation column (3.2.times.385 mm) could be used with BTC2710
10-.mu.m separation exchangeresin (Eppendorf-Netheler-Hinz) and
lithium citrate separation buffer (flow rate, 0.30 mL/min).
Separation temperatures would be set at 32.degree. C. for 44 min,
34.degree. C. for 28 min, and 60.degree. C. for 31 min. For
colorimetric peak detection at 570 and 440 nm, 500 .mu.mol/L
aminoethyl-L-cysteine hydrochloride in 37 mmol/L lithium/76 mmol/L
citrate buffer at pH 2.2 would be used as the internal standard.
Plasma, urine, and whole-blood hemolysate would be pretreated with
crystalline 5-sulfosalicylic acid as a deproteinization step.
[0207] Treatment Groups
[0208] As noted above, it is generally recognized that many disease
processes are attributed to the presence of elevated levels of free
radicals and reactive oxygen species (ROS) and reactive nitrogen
species (RNS), such as superoxide, hydrogen peroxide, singlet
oxygen, peroxynitrite, hydroxyl radicals, hypochlorous acid (and
other hypohalous acids) and nitric oxide. Furthermore, subjects
suffering from any of these conditions may benefit from therapy
with anti-oxidants. It is with respect to these particular diseases
and conditions that the current invention would be beneficial,
particularly with respect to assessing the need of the patient for
treatment with anti-oxidant therapy and for monitoring the
effectiveness of such therapy. Moreover, the novel IMMUNE
FORMULATIONs described in U.S. Pat. Nos. 6,667,063 and 6,592,908
would be beneficial for treatment of these patients and the amount
and duration of therapy with these novel compositions, as well as
other anti-oxidant formulations may be monitored effectively using
the methods of the present invention. A summary of particular
diseases for which anti-oxidant therapy would be beneficial and for
which monitoring the need for and effectiveness of such therapy
using the methods described herein follows.
[0209] The methods of the present invention may be utilized to
assess an individual's need for specific therapy with an
antioxidant, and as such, may be considered as a means to assess
either an individual's current medical condition and needs, or
alternatively, the methods may be used as a prophylactic means to
allow identification of individuals of particularly high risk for
diseases known to be associated with high oxidative stress, such
as, but not limited to, atherosclerosis and cardiovascular disease.
Upon such identification, such subjects can adopt more frequent
testing, dietary adjustments, monitoring and regulation of blood
pressure, and the like. As a diagnostic assay, the methods of this
invention supplement traditional testing methods to identify
subjects known to be at risk who may prove resistant to
conventional therapeutic regimens and alter the prescribed
treatment. Thus, for example, where a subject is diagnosed with
early stages of atherosclerosis, a positive test using the assays
of this invention may indicate additional drug intervention rather
than simply dietary or lifestyle changes.
[0210] Cardiovascular Disease
[0211] Oxidative modification of low density lipoproteins (LDL) is
recognized as one of the major processes involved in atherogenesis
and cardiovascular disease. Thus, a measurement of LDL oxidative
susceptibility could be of clinical significance (Scoccia A E et
al, BMC Clin Pathol. (2001) 1(1):1). Furthermore, more precise
measurements of both lipid oxidation and pyroglutamic acid
measurements in the urine, as well as plasma glutathione
measurements as outlined in the present invention could be even
more beneficial in terms of assessing the patients' need for
treatment with both an anti-oxidant as well as lipid lowering drugs
and would also be beneficial in terms of monitoring the utilization
efficiency of these drugs as well as the effectiveness of
therapy.
[0212] Cancer
[0213] The role of oxidative stress in many cancers has been under
investigation for many years. However, it is recently becoming more
apparent that reactive oxygen or nitrogen species may in fact
affect signaling pathways in many hyperproliferative disorders. For
example, prostate cancer (PC) has become the most frequently
diagnosed neoplasm and the second leading cause of cancer-related
mortality in men. Its incidence rate has continued to increase
rapidly during the past two decades, especially in men over the age
of 50 years as they are living longer. The prostate in aging males
is highly susceptible to benign and malignant proliferative
changes. It is believed that about two/thirds of all cancers could
have been prevented based upon lifestyle choices. How environment,
diet and genetics interact to either induce or prevent prostate
cancer (PC) is not known. Free radicals play a significant but
paradoxical role acting as a "double-edged sword" to regulate
cellular processes. That is, because of their effect on cell
signaling pathways and in particular, apoptosis, it appears that
ROS may prevent apoptosis and can thus maintain proliferation in
certain cancer cells. Thus, there appears in certain instances to
be a paradoxical role for ROS in these situations. Recent in vitro
studies using benign prostate hyperplasia (BPH) and PC cell lines
grown under various oxidative stress conditions confirm this
theory. Key signal transduction mechanisms may be involved in ROS
induced effects on prostate cell growth, cell-cycle checkpoints,
apoptosis and transcription factors. Thus, dietary antioxidants may
have a beneficial effect on these mechanisms (Sikka S C Curr Med
Chem (2003) 10(24):2679-92).
[0214] Furthermore, it is also known that many of the therapies
available for treatment of cancers are associated with oxidative
tissue damage. For example, adriamycin is known to induce cardiac
and hepatic toxicity. Studies have been done with specific agents
to determine their effects on such peroxidative damage induced by
adriamycin (ADR). For example, a study was conducted to determine
the effect of a heparin derivative, low molecular weight heparin
(LMWH) on the biochemical changes, tissue peroxidative damage and
abnormal antioxidant levels in adriamycin (ADR) induced cardiac and
hepatic toxicity (Deepa P R et al, Chem Biol Interact (2003)
146(2):201-10). LMWH administration to ADR-induced rats prevented
the rise in serum and tissue levels of LDH, aminotransferases and
ALP, while these parameters were significantly elevated in the ADR
group in comparison with the control group. Cardiotoxicity
indicated by rise in serum CPK in the ADR group was attenuated by
LMWH treatment in group IV. LMWH decreased the cardiac and hepatic
lipid peroxidation induced by ADR. Histologic examination revealed
that the ADR-induced deleterious changes in the heart and liver
tissues were offset by LMWH treatment. Restoration of cellular
normalcy accredits LMWH with cytoprotective role in
adriamycin-induced cardiac and hepatic toxicity.
[0215] Carboplatin is currently being used as an anticancer drug
against human cancers. However, high dose of carboplatin
chemotherapy result in ototoxicity in cancer patients.
Carboplatin-induced ototoxicity is related to oxidative stress to
the cochlea and inner hair cell loss in animals. It is likely that
initial oxidative injury spreads throughout the neuroaxis of the
auditory system later. A study was done to evaluate
carboplatin-induced hearing loss and oxidative injury to the
central auditory system (inferior colliculus) of the rat (Husain,
K. et al. Int J Toxicol. (2003) 22(5):335-42). Carboplatin
significantly increased nitric oxide and lipid peroxidation,
xanthine oxidase, and manganese superoxide dismutase activities in
the inferior colliculus, but not in the cerebellum, indicating an
enhanced flux of free radicals in the central auditory system.
Carboplatin significantly depressed the reduced to oxidized
glutathione ratio, antioxidant enzyme activities, such as
copper-zinc superoxide dismutase, catalase, glutathione peroxidase,
glutathione reductase, and glutathione S-transferase, and enzyme
protein expressions in the inferior colliculus, but not in the
cerebellum, 4 days after treatment. The data suggest that
carboplatii induced oxidative injury specifically in the inferior
colliculus of the rat leading to hearing loss.
[0216] Neurological Diseases and Conditions
[0217] Oxidative stress, which is now recognized as accountable for
redox dysregulation involving reactive oxygen species (ROS) and
reactive nitrogen species (RNS) plays a pivotal role for the
modulation of critical cellular functions, notably for cells in the
neuronal system (Emerit J, Edeas M, Bricaire F. Biomed
Pharmacother. 2004 January; 58(1):39-46; Smith J V et al, J.
Alzheimer's Dis. (2003) 5(4):287-300; Hayashi T. et al, J Cereb
Blood Flow Metab (2003) 23(10):1117-28; Niu K C et al. Clin. Exp.
Pharmacol. Physiol. (2003) 30(10):745-51), in particular, neurons,
astrocytes and microglia, such as apoptosis program activation, and
ion transport, calcium mobilization, involved in excitotoxicity.
Excitotoxicity and apoptosis are the two main causes of neuronal
death. The role of mitochondria in apoptosis is crucial. Multiple
apoptotic pathways emanate from the mitochondria. The respiratory
chain of mitochondria (oxidative phosphorylation), is the fount of
cellular energy, i.e. ATP synthesis, and is responsible for most of
ROS and notably the first produced, superoxide anion. Mitochondrial
dysfunction (i.e. cell energy impairment, apoptosis and
overproduction of ROS), is a final common pathogenic mechanism in
aging and in neurodegenerative disease such as Alzheimer's disease
(AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis
(ALS). Nitric oxide (NO), an RNS, which can be produced by three
isoforms of NO-synthase in brain, plays a prominent role.
[0218] The etiology of neurodegenerative diseases remains
enigmatic; however, evidence for defects in energy metabolism,
excitotoxicity, and for oxidative damage is increasingly
compelling. There is most likely a complex interplay between these
mechanisms. A defect in energy metabolism may lead to neuronal
depolarization, activation of N-methyl-D-aspartate excitatory amino
acid receptors, and increases in intracellular calcium, which are
buffered by mitochondria. Mitochondria are the major intracellular
source of free radicals, and increased mitochondrial calcium
concentrations enhance free radical generation. Mitochondrial DNA
is particularly susceptible to oxidative stress, and there is
evidence of age-dependent damage and deterioration of respiratory
enzyme activities with normal aging. This may contribute to the
delayed onset and age dependence of neurodegenerative diseases.
There is evidence for increased oxidative damage to macromolecules
in amyotrophic lateral sclerosis, Huntington's disease, Parkinson's
disease, and Alzheimer's disease. Potential therapeutic approaches
include glutamate release inhibitors, excitatory amino acid
antagonists, strategies to improve mitochondrial function, free
radical scavengers, and trophic factors. All of these approaches
appear promising in experimental studies and are now being applied
to human studies. (Beal M F, Ann Neurol. 1995 September;
38(3):357-66).
[0219] The role of amyloid beta-peptide (Abeta) in the free-radical
oxidative-stress model of neurotoxicity in Alzheimer's disease (AD)
has received much attention recently. Studies have been done to
study the effects of Abeta on intracellular free radical levels. A
neuroblastoma cell line, which stably expresses an AD-associated
double mutation, which exhibits both increased secretion and
intracellular accumulation of Abeta when stimulated was utilized in
one study. In addition, a transgenic Caenorhabditis elegans
constitutively expressing human Abeta was also used. A rise in
levels of hydrogen peroxide (H2O2) was observed in both in vitro
and in vivo AD-associated transgenic models expressing the Abeta
peptide compared with the wild type controls. Furthermore, an
age-dependent increase in H2O2-related ROS was observed in wild
type C. elegans, which is accelerated in the AD-associated C.
elegans mutant. These results support the hypothesis of the
involvement of Abeta and ROS in association with AD (Smith, J. V.
and Luo, Y. (2003), J. Alzheimers Dis. 5(4):287-3000).
[0220] The endoplasmic reticulum (ER), which plays important roles
in apoptosis, is susceptible to oxidative stress. Because reactive
oxygen species (ROS) are robustly produced in the ischemic brain,
ER damage by ROS may be implicated in ischemic neuronal cell death.
A study was done whereby global brain ischemia was induced in
wild-type and copper/zinc superoxide dismutase (SOD1) transgenic
rats and ER stress and neuronal damage was compared. Phosphorylated
forms of eukaryotic initiation factor 2 alpha (eIF2 alpha) and
RNA-dependent protein kinase-like ER eIF2 alpha kinase (PERK), both
of which play active roles in apoptosis, were increased in
hippocampal CA1 neurons after ischemia but to a lesser degree in
the transgenic animals. This finding, together with the finding
that the transgenic animals showed decreased neuronal degeneration,
indicates that oxidative ER damage is involved in ischemic neuronal
cell death (Hayashi T. et al. (2003), J. Cereb. Blood Flow Metab.
23(10): 1117-1128).
[0221] Inflammatory Diseases
[0222] The role of oxidative stress in inflammatory diseases has
also been investigated. For example, Ramos et al. determined the
level of cellular oxidative stress blood markers and the enzymatic
system of antioxidant defense in patients suffering from juvenile
rheumatoid arthritis (JRA) (Ramos V A et al., (2000), J Pediatr
(RIOJ), 76(2): 125-32). This study included 64 patients. The
patients were separated in three subtypes based on the pattern of
onset within the first six months of disease: polyarticular,
pauciarticular and systemic. The control group included 60 patients
(38 of female sex) following clinical control to diseases of non
inflammatory nature, in the same hospital. The plasmatic levels of
malondialdehyde (MDA), lipoperoxide (LPO), hydroperoxide (HPX),
carbonile groups (CG) of proteins and gluthathione and the
enzymatic activities of Superoxide dismutase (SOD), gluthathione
peroxidase (GSH-Px) and gluthathione reductase were determined. The
results showed that the group of patients with JRA presented high
concentrations of lipid peroxidation products, evaluated by
determining the plasmatic levels of MDA, LPO, and HPX; oxidative
damage of the circulate protein, determined by CG contents of
plasma proteins; elevation of enzymatic activity of SOD and
GSH-Red; decrease of GSH-Px activity and GSH levels. These results
demonstrated the presence of molecular damage that generated oxygen
free radicals in the JRA patients. The SOD activity and the changes
of gluthathione redox enzymatic cycle confirm the decrease of
capacity of cellular defense system against the induced toxicity of
oxidative stress in these patients.
[0223] Drug Induced Oxidation
[0224] The generation of free radicals in vivo can be attributed to
many things. For example, many known drugs can increase the
production of free radicals in the presence of increased oxygen
tensions. These drugs may include antibiotics that depend on
quinoid groups or bound metals for activity (nitrofurantoin),
antineoplastic agents as bleomycin, anthracyclines (adriamycin, see
above) (Fisher, 1988) and methotrexate, which possess pro-oxidant
activity (Gressier et al. 1994). In addition radicals derived from
penicillamine, phenylbutazone, some fenamic acids and the
aminosalicylate component of sulphasalazine might inactivate
protease and deplete ascorbic acid accelerating lipid peroxidation
(Grisham et al. 1992; Halliwel et al. 1992a; Evans et al.
1994).
[0225] Radiation Therapy Induced
[0226] Radiotherapy may cause tissue injury as a result of free
radical generation.
[0227] Electromagnetic radiation, such as X rays or gamma rays, and
particulate radiation, such as electrons, photons, neutrons, alpha
and beta particles, may generate primary radicals by transferring
their energy to cellular components including water. These primary
radicals can undergo secondary reactions with dissolved oxygen or
with cellular solutes.
[0228] Smoking Induced
[0229] The presence of oxidants in tobacco may play a major role in
injuring the respiratory tract. For example, the oxidants in
tobacco smoke severely deplete intracellular antioxidants in lung
cells in vivo. The mechanism for doing so is related to oxidant
stress. The oxidant materials that are present for a time
sufficient to cause damage to the alveoli include aldehydes,
epoxides, peroxides, and other free radicals. In addition, nitric
oxide, peroxyl radicals and carbon centered radicals are present in
the gas phase, while other radicals are present in the tar phase.
Examples of radicals in the tar phase include the semiquinone
moieties derived from various quinones and hydroquinones.
Micro-haemorrhages are most probably the cause of iron deposition
found in smokers' lung tissue. This form of iron may lead to the
formation of the lethal hydroxyl radical from hydrogen peroxide.
Furthermore, smokers have elevated amounts of neutrophils in the
lower respiratory tract. These may contribute to a further
elevation of the concentration of free radicals.
[0230] Providing a Biological Sample for Use in the Methods of the
Present Invention
[0231] In particular embodiments the assays are performed using a
biological sample from the organism/subject of interest. While the
assays are of great use in humans, they are not so limited. It is
believed similar oxidative damage exists essentially in all mammals
and thus the assays of this invention are contemplated for
veterinary applications as well. Thus, suitable subjects include,
but are not limited to humans, non-human primates, canines,
equines, felines, porcines, ungulates, lagomorphs, and the
like.
[0232] A suitable biological sample includes a sample of a
biological material, which may be selected from a blood sample or
urine. As used herein a blood sample includes a sample of whole
blood or a blood fraction (e.g. serum or plasma). The sample may be
fresh blood or stored blood (e.g. in a blood bank) or blood
fractions. The sample may be a blood sample expressly obtained for
the assays of this invention or a blood sample obtained for another
purpose, which can be subsampled for the assays of this invention.
In a preferred embodiment, the bodily sample is preferably plasma
or urine.
[0233] The sample may be pretreated as necessary by dilution in an
appropriate buffer solution, heparinized, concentrated if desired,
or fractionated by any number of methods including but not limited
to ultracentrifugation, fractionation by fast performance liquid
chromatography (FPLC), or precipitation of apolipoprotein B
containing proteins with dextran sulfate or other methods. Any of a
number of standard aqueous buffer solutions, employing one of a
variety of buffers, such as phosphate, Tris, or the like, at
physiological pH can be used.
[0234] Lipid and Water-Soluble Antioxidants
[0235] As described below, there are many known lipid and water
soluble antioxidants known to have beneficial effects in treatment
of various disease conditions, whereby high levels of oxidative
stress may be responsible in part for progression of the tissue
damage associated with such diseases and conditions. It would be
beneficial to utilize the methods of the present invention to
monitor utilization efficiency and effectiveness of these therapies
should it be determined that a patient would benefit from such
therapies. While these antioxidants are believed to be useful in
the treatment of the diseases and conditions described herein, it
is believed that therapy with glutathione precursors, IMMUNE
FORMULATION 100.TM. or IMMUNE FORMULATION 200.TM. would be the most
preferred embodiments.
[0236] Lipid Soluble Anti-Oxidants
[0237] Lutein: A very active lipid-soluble carotenoid antioxidant
(2.3 times higher than vitamin E), which is readily absorbed into
the serum. Lutein and zeaxanthin are major factors in the
prevention of macular degeneration, which is the leading cause of
blindness in the elderly and represents 10% of all blindness in
humans.
[0238] Zeaxanthin: A very active lipid-soluble carotenoid
antioxidant (2.8 times higher than vitamin E) which is readily
absorbed into the serum. Lutein and zeaxanthin are implicated in
the prevention of macular degeneration, which is the leading cause
of blindness in the elderly and represents 10% of all blindness in
humans.
[0239] Beta-Cryptoxanthin:--Probably the most active of the lipid
soluble antioxidants (3.1 times higher than vitamin E) which is
readily absorbed into the serum.
[0240] Lycopene: One of the most active lipid-soluble antioxidants
(2.8 times higher than vitamin E). Research has indicated that
lycopene may be very important in the prevention of prostate
cancer.
[0241] Alpha-Carotene: A known antioxidant and precursor to vitamin
A. Experimental evidence shows that alpha carotene is a stronger
antioxidant and cellular differentiating agent than beta carotene
and therefore may be better in preventing cancer.
[0242] Beta Carotene: A known antioxidant and precursor to vitamin
A, which has been most widely researched and used extensively as a
diet supplement. It is a strong cellular differentiating agent, and
therefore may prevent cancer.
[0243] Retinoi [Vitamin A]: A known antioxidant and cellular
differentiating agent and Therefore may prevent cancer and many
aspects of aging.
[0244] Retinyl Palmitate: The retinol ester that is most commonly
used in dietary Supplements and foods as a source of vitamin A.
[0245] Carotenoid classes: This grouping of carotenoids contain
many uncharacterized carotenoids that most likely are beneficial to
health. This value provides a good overall value of the amounts of
fruits and vegetables being consumed.
[0246] Alpha-Tocopherol (Vitamin E):--One of the best characterized
and diet supplemented lipid-soluble antioxidants. Apart from its
antioxidant capabilities, it has cellular differentiation
properties which are believed to be good in preventing cancer.
[0247] Delta-Tocopherol (Vitamin E): Not much is known about the
beneficial effects of delta-tocopherol to humans, though it is
normally found at lower amounts in foods and human serum.
[0248] Gamma-Tocopherol (Vitamin E): The major type of vitamin E
found in the heart and therefore may be selected for the body
because of its unique properties either as an antioxidant or as a
differentiation agent.
[0249] Ubiquinol [Coenzyme Q10]: Is normally synthesized in cells
as part of the mitochondrial oxidative phosphorylation system and
is present in lipid biomembranes. COQ10 can also be absorbed
through the diet and can act as a very active antioxidant and
protecting LDL from becoming oxidized.
[0250] Water-Soluble Antioxidants
[0251] Vitamin C [Ascorbate] (in serum and saliva) Ascorbic acid
can directly scavenge oxidative species as well as generate other
oxidized antioxidants such as vitamin E. However, under conditions
where there are free prooxidant metals around, such as iron and
copper, vitamin C's strong reductive capacity will catalyze the
production of oxidative free radicals.
[0252] Thiols (in serum and saliva) are very active antioxidants
and reducing agents. Most serum thiols are found in albumin as
indicated by free cysteine and glutathione. Albumin thiols are
thought to act as sacrificial antioxidants that have little
biological consequences of being damaged. Because of their high
antioxidant reactivity and high concentration, albumin thiols act
as a major defense against free radical damage to cell
membranes.
[0253] Uric acid (in serum and saliva) Uric acid is a
methylxanthine (like caffeine) which stimulates brain activity. It
is also known to directly scavenge oxidative species and chelate
prooxidant metals.
[0254] Direct and Total Bilirubin: This is considered as a waste
product of heme metabolism. Bilirubin is known to be a very active
lipid and aqueous soluble serum antioxidant. Direct (conjugated)
bilirubin is the form of bilirubin that can be absorbed and removed
from the body in the bile.
IMMUNE FORMULATION 100.TM. and IMMUNE FORMULATION 200.TM.
[0255] The methods described herein contemplate the use of several
anti-oxidant therapies effective at treating various diseases or
conditions in which oxidative stress plays a role. However, a
preferred embodiment provides for the use of IMMUNE FORMULATION
100.TM. or IMMUNE FORMULATION 200.TM. for administration to
subjects experiencing oxidative stress or immune dysfunction. These
formulations were developed for use as a nutritional supplement
that is beneficial if taken on a daily basis. These formulations
have the benefit of supplying the glutathione precursors needed for
resynthesis of glutathione when the levels are depleted due to
disease or metabolic dysfunction. In addition, they are safe and
cost effective for those patients in need of daily consumption.
Furthermore, they can be formulated as chewable tablets or as
nutritive bars or wafers. In another embodiment, it is envisioned
that IMMUNE FORMULATION 100.TM. or IMMUNE FORMULATION 200.TM. can
be used in combination with any of the anti-oxidants listed in
paragraphs [0139] through [0156] of the present invention.
[0256] Immune Formulation 100.TM.
[0257] The essential components in IMMUNE FORMULATION 100.TM. are a
selected whey product, colostrum and a non-toxic catalytic quantity
of elemental selenium or a water soluble precursor of elemental
selenium in an amount sufficient to aid in the production of
glutathione. Selenium precursors are much preferred since they are
easier to handle.
[0258] Selenium is one of numerous trace metals found in many
foods. Selenium may be employed as one of several non-toxic, water
soluble, organic or inorganic selenium compounds capable of being
absorbed by the body. The presently preferred inorganic selenium
compounds are aliphatic metal salts containing selenium in the form
of selenite or selenate anions. However, organic selenium compounds
are more preferred because they are normally less toxic than
inorganic compounds. Other selenium compounds which may be
mentioned by way of examples include selenium cystine, selenium
methionine mono- and di-seleno carboxylic acids with about seven to
eleven carbon atoms in the chain. Seleno amino acid chelates are
also useful. Selenium compounds are utilized in this composition in
amounts to provide selected quantities of elemental selenium.
[0259] A second component of IMMUNE FORMULATION 100.TM. is whey.
Whey is the curd-free portion of milk that remains after the
production of cheese. "Whey" is a term referring to the serum or
watery part of milk after removal of the cheese. Removal of a
substantial portion of the water results in a dry whey. There are
two common types of dry whey. These are dry whey concentrates and
dry whey isolates. The former (WPC) is an off-white to cream
colored product which, depending on the method of manufacture, may
contain from about 15% to 85% protein based on the total weight. It
may additionally contain small amounts of minerals, vitamins and
carbohydrates. Whey protein isolate (WPI) contains more than 85% by
weight of protein. Both types of whey are available from Proliant,
Manhattan, Ill.; Davisco Foods International, Inc. Eden Prairie,
Minn. or Land-O-Lakes, Tulare, Calif. The whey product may be up to
about 35% denatured. The whey product may be completely denatured,
but the cost of wholly denatured whey is such that it is not
feasible to employ wholly undenatured whey in compositions to serve
general human consumption or in animal needs. Accordingly, the
dried whey product utilized in IMMUNE FORMULATION 100.TM. will be a
whey product concentrate or whey product isolate which is up to
about 35% denatured or, conversely about 65% to about 100%
undenatured. Preferably, it will contain from about 65% to about
85% protein. It may comprise from about 5% to about 95% of the
composition based on the total weight of the composition.
[0260] A third component of IMMUNE FORMULATION 100.TM. is
colostrum. Colostrum is a thin milky fluid secreted by the mammary
gland of mammals a few days before or after parturition. It is a
unique combination of beneficial nutrients including protective
antibodies, fat, carbohydrate, vitamins and minerals. The
immunological components of colostrum include IgG, IgM and IgA.
These components confer passive immunity to the neonate and
protection against infection during the initial period after
parturition. After this period, colostrum is no longer absorbed
through the gut and the newborn must depend upon its own developing
immune system for protection. Colostrum is an important factor in
the growth of mammals including humans, bovines, caprines, porcines
and equines. The preferred colostrums for use in the compositions
of this invention are bovine and caprine. Several colostrum
products useful in this formulation are commercially available.
[0261] The daily effective dosage of the products of this invention
will depend upon the size of the individual (human or animal) being
treated, the condition being treated, the age of the individual and
other factors well known to the physician or veterinarian in
attendance. The optimum daily dosage can easily be determined by a
few simple observations. It will generally vary from about 250 mg
to 2000 mg per day for humans and small animals. For large animals
the daily dosage will normally be from about 500 mg to 5000 mg per
day. While these are projected dose ranges, the methods of the
present invention can be utilized to measure utilization efficacy
and ultimate effectiveness of these formulations.
[0262] Immune Formulation 200.TM.
[0263] The essential components of IMMUNE FORMULATION 200.TM. are
precursors of glutathione, namely glutamic acid, cystine or another
cysteine precursor and glycine, together with a catalytic quantity
of a selenium source. The separate components serve as precursors
with selenium for the metabolic formation of glutathione after they
have been transported across the mucous membrane. The glutathione
precursors in this formulation, which are a mixture of glutamic
acid, cystine or another related cystine precursor, and glycine are
in a molar ratio of about 1:0.5:1, the amount of glutathione
precursors being effective to increase the content of glutathione
in the body tissue of the mammal above that of a pretreatment level
thereby to enhance immune activity. This material is further
described and claimed in U.S. Pat. No. 6,592,908. The composition
may be used alone, but normally it will be employed in association
with one or more non-toxic pharmaceutically acceptable carriers
appropriate to the method of administration. If an excess of any
amino acid is used, it will presumably be of nutritional value or
may simply be metabolized.
[0264] IMMUNE FORMULATION 200.TM. will be utilized to increase the
formation of glutathione and thus to enhance the immune activity of
a mammal in need of such treatment. The effect of the treatment is
such that after the treatment, the mammal will be more resistant to
microbial infection or other trauma, diseases, or conditions
adversely affecting immune activity than before such treatment.
[0265] Because of its ability to increase production of
glutathione, IMMUNE FORMULATION 200.TM. is useful to treat a wide
variety of diseases or conditions associated with the presence of
excess free radical or reactive oxygen or nitrogen species. These
include, for example, cancer, Alzheimer's disease,
arteriosclerosis, rheumatoid arthritis and other autoimmune
diseases, cachexia, coronary artery disease, chronic fatigue
syndrome, AIDS and others as described herein.
[0266] The components of this composition are amphoteric and
therefore may be employed as non-toxic metal salts or acid addition
salts. Typically, the salts are alkalic or alkaline earth metal
salts, preferably sodium, potassium or calcium salts. Suitable acid
addition salts include salts of hydrochloric, phosphoric and citric
acid. The amino acids may also be employed in the form of certain
of their derivatives including esters and anhydrides which before
or after transport through the mucous membrane will be modified
into the form in which they will be joined together to form
glutathione. All amino acids employed, except glycine which does
not form optical isomers, are in the natural or L-form. Although
wide variations are possible, it will be apparent that the optimum
ratio of glutamic acid to cystine to glycine in this novel
composition described herein is 1:0.5:1. If an excess of any acid
is used, it will presumably be of nutritional value or may simply
be metabolized.
[0267] It is important for the use of this composition that the
selenium as employed in the composition be capable of transport
through the mucosal membrane of the patient under treatment. For
this reason, water insoluble selenium compounds are not generally
useful.
[0268] For convenience, the term "selenium" is sometimes used
hereinafter to include any of the various water soluble selenium
products which can be transported through the mucosal membrane in
the practice of this invention. It will be understood, however,
that the particular forms of selenium compounds set forth herein
are not to be considered limitative. Other selenium compounds,
which exhibit the desired activity and are compatible with the
other components in the mixture and are non-toxic, can be used in
the practice of the invention. Many of them are available
commercially.
[0269] In fact, the amount of selenium precursor employed in this
novel composition is only enough to provide a catalytic quantity of
the element to activate the glutathione system. The catalytic
quantity of selenium precursor utilized in the compositions of this
invention is such that it will produce either in one dosage unit or
in multiple dosage units sufficient elemental selenium to promote
the production and activation of glutathione.
[0270] Typically, this will be at or near the recommended daily
allowance of selenium for the individual mammal under treatment.
This amount will be well below the toxicity limit for elemental
selenium. By way of non-limiting examples, a representative range
of catalytic quantities of selenium precursors is based on the age
of the individual. The recommended daily allowances for elemental
selenium as reported in The Pharmacological Basis of Therapeutics,
Ninth Edition, page 1540, The McGraw-Hill Companies, 1996. The
recommended daily dosage for humans therefore ranges from 10 to 75
.mu.g per day. For animals the range may depend upon the animal and
its size.
[0271] The tablets or wafers, with fillers will typically weigh
from about 0.5 to 5 grams and will contain a therapeutically
effective amount of the essential ingredients together with the
selected vehicle. Tablets and other forms of the immunoenhancing
compositions can be prepared to provide any quantity of elemental
selenium from less than 1 .mu.g to 7.5 .mu.g. For example, a tablet
containing 10 .mu.g of selenium methionine is capable of delivering
4 .mu.g of elemental selenium, and 7.5 .mu.g of selenium methionine
is capable of delivering 3 .mu.g of selenium. Tablets may be given
several times per day to achieve the desired immune enhancing
effect.
[0272] A one a day tablet weighing two grams may contain 200 mg or
more of the composition. A similar tablet intended to be used every
four hours may contain 50 mg to 100 mg or more of the
therapeutically effective composition.
[0273] Immune Function Analysis
[0274] The methods of the present invention provide for measurement
of specific immune cell numbers and activities, including
quantitation of specific T cell subsets and function of natural
killer cells. In particular, CD4+ T cells and CD8+ T cells provide
immune protection from all forms of pathogens, including bacteria,
viruses, and tumors. These cells act as a source of
cytokines/lymphokines for induction of cytolytic T cells, one of
the primary immune cells that lyse virus infected target cells as
well as tumor targets. These cells also secrete factors that aid in
the induction of specific B cell or antibody producing cell
populations.
[0275] Assays for measuring the numbers of these cell populations
are known to those skilled in the art. For example, as shown below,
the most commonly used procedures are by FACS analysis, whereby the
cell populations are incubated with labeled antibodies specific for
cell surface markers. These antibodies may be labeled with
phycoerythrin or FITC and after a period of time, they are washed
and analyzed in a fluorescent activated cell sorter. Alternatively,
assays can be set up to measure the activity of these cells in a
specific chromium release assay to assess their activity. These
assays are also known to one skilled in the art.
[0276] Natural killer (NK) cells are one of the early defense
mechanisms in the body for protection against a variety of
pathogens. These cells may also be assessed by the use of specific
cell surface markers and FACS analysis. Alternatively, their
activity may be assessed using NK sensitive target cells in a
chromium release assay as described below.
[0277] Assay Formats
[0278] The methods of this invention may use assays which may be
practiced in almost a limitless variety of formats depending on the
particular needs at hand. Such formats include, but are not limited
to traditional "wet chemistry" (e.g. as might be performed in a
research laboratory), high-throughput assay formats (e.g. as might
be performed in a pathology or other clinical laboratory), and
"test strip" formats, (e.g. as might be performed at home or in a
doctor's office).
[0279] Traditional Wet Chemistry
[0280] The assays of this invention can be performed using
traditional "wet chemistry" approaches. Basically this involves
performing the assays as they would be performed in a research
laboratory. Typically the assays are run in a fluid phase (e.g. in
a buffer with appropriate reagents (e.g. lipids, oxidized lipids,
oxidizing agent, etc.) added to the reaction mixture as necessary.
The oxidized lipid concentrations are assayed using standard
procedures and instruments, e.g. as described in the examples.
[0281] High-Throughput Assay Formats
[0282] Where population studies are being performed, and/or in
clinical/commercial laboratories where tens, hundreds or even
thousands of samples are being processed (sometimes in a single
day) it is often preferably to perform the assays using
high-throughput formats. High throughput assay modalities are
highly instrumented assays that minimize human intervention in
sample processing, running of the assay, acquiring assay data, and
(often) analyzing results. In particular embodiments, high
throughput systems are designed as continuous "flow-through"
systems, and/or as highly parallel systems.
[0283] Flow through systems typically provide a continuous fluid
path with various reagents/operations localized at different
locations along the path. Thus, for example a blood sample may be
applied to a sample receiving area where it is mixed with a buffer,
the path may then lead to a cell sorter that removes large
particulate matter (e.g. cells), the resulting fluid may then flow
past various reagents (e.g. where the reagents are added at "input
stations" or are simply affixed to the wall of the channel through
which the fluid flows. Thus, for example, the sample may be
sequentially combined with a lipid (e.g. provided as an LDL), then
an oxidation agent, an agent for detecting oxidation, and a
detector where a signal (e.g. a calorimetric or fluorescent signal)
is read providing a measurement of oxidized lipid.
[0284] In highly parallel high throughput systems samples are
typically processed in microtiter plate formats (e.g. 96 well
plates, 1536 well plates, etc.) with computer-controlled robotics
regulating sample processing reagent handling and data acquisition.
In such assays, the various reagents may all be provided in
solution. Alternatively some or all of the reagents (e.g. oxidized
lipids, indicators, oxidizing agents, etc.) may be provided affixed
to the walls of the microtiter plates.
[0285] In a particular embodiment of the present invention, it is
envisioned that all three products, that is, lipid peroxides,
pyroglutamic acid and glutathione may be measured concurrently. For
example, a 96 well plate may be prepared that contains wells to
which antibodies have been attached for each of the three products.
Thus, one set of thirty two wells would contain an antibody to
lipid peroxides, one set of wells would contain an antibody to
glutathione, and a third set of thirty two wells would contain an
antibody prepared to pyroglutamic acid. Thus, urine samples can be
applied to the wells containing the antibodies to lipid peroxides
and to those wells containing antibodies to pyroglutamic acid.
Whole blood or blood plasma can be added to those wells containing
antibodies to glutathione. After an appropriate incubation time,
for example, 1 hour at 37.degree. C., the plates would be washed,
and secondary antibodies which are conjugated to (labeled with) an
enzyme or fluorophore can be added, incubated in the same manner,
and the amount of secondary label bound can be measured using a
substrate for the enzyme or if the fluorophore method is used, the
amount of label bound is measured using spectrophotometric
techniques at the appropriate wavelength for the fluorophore.
[0286] High throughput screening systems that can be readily
adapted to the assays of this invention are commercially available
(see, e.g., Zymark Corp., Hopkinton, Mass.; Air Technical
Industries, Mentor, Ohio; Beckman Instruments, Inc. Fullerton,
Calif.; Precision Systems, Inc., Natick, Mass., etc.). These
systems typically automate entire procedures including all sample
and reagent pipetting, liquid dispensing, timed incubations, and
final readings of the microplate in detector(s) appropriate for the
assay. These configurable systems provide high throughput and rapid
start up as well as a high degree of flexibility and customization.
The manufacturers of such systems provide detailed protocols. Thus,
for example, Zymark Corp. provides technical bulletins describing
screening systems for detecting the modulation of gene
transcription, ligand binding, and the like.
[0287] "Test Strip" Assay Formats
[0288] The methods of the present invention may also utilize assays
which are provided in "test well" or "test strip" formats. In "test
well" or "test strip" formats, the biological sample is typically
placed in the well or applied to a receiving zone on the strip and
then a fluorescent or calorimetric indicator appears which, in this
case, provides a measure of the protection or repair afforded by
the subject's HDL or components thereof.
[0289] Many patents have been issued which describe the various
physical arrangements for blood testing. These include systems
which involve lateral or horizontal movement of the blood, as well
as plasma testing. For example, U.S. Pat. Nos. 4,876,067,
4,861,712, 4,839,297, and 4,786,603 describe test carriers and
methods for analytical determination of components of bodily
fluids, including separating plasma from blood using glass fibers
and the like. These patents, all teach systems which require some
type of rotation of test pads or a portion of the test pads during
use. U.S. Pat. No. 4,816,224 describes a device for separating
plasma or serum from whole blood and analyzing the serum using a
glass fiber layer having specific dimensions and absorption to
separate out the plasma from the whole blood for subsequent
reaction. Similarly, U.S. Pat. No. 4,857,453 describes a device for
performing an assay using capillary action and a test strip
containing sealed liquid reagents including visible indicators.
U.S. Pat. No. 4,906,439 describes a diagnostic device for
efficiently and accurately analyzing a sample of bodily fluid using
fluid delivery in a lateral movement via flow through channels or
grooves.
[0290] Kits
[0291] The methods of the present invention provide for measuring
the amounts of specific markers of oxidative stress. In a
particular embodiment, at least three markers are quantitated using
standard reagents and kits, which are commercially available to
measure each marker individually. In another particular embodiment,
the methods further comprise the measurement of the number and/or
activity of specific immune cell populations, preferably T cells
and natural killer cells using techniques known to those skilled in
the art. Thus, the present invention provides a more quantitative
and accurate means of assessing a subject's need for antioxidative
therapy by measuring all of these parameters concurrently. To the
inventor's knowledge, no other art currently exists which describes
combining the concurrent non-invasive techniques and measurements
described herein for assessing the need for, and to measure the
effectiveness of, therapy with anti-oxidants.
[0292] However, one of the oxidative markers, pyroglutamic acid, is
measured using non-immunological techniques, in particular, gas
chromatography and mass spectrometry are used. While this procedure
provides the accuracy and sensitivity that is needed for such
measurements in the present invention, the procedure can be time
consuming and requires the use of very specialized equipment.
Accordingly, the present invention also provides for kits
comprising binding partners for the oxidative markers and the
reagents needed for detection of the oxidative stress markers. The
kits of the present invention provide advantages over those
commercially available in that at least three oxidative markers can
be measured concurrently using the same assay format. In another
embodiment, the kits may contain binding partners for the three
oxidative stress markers, that is, for lipid peroxide, pyroglutamic
acid and glutathione, as well as binding partners for cell surface
markers on CD4+ T cells, CD8+ T cells and a cell surface marker for
natural killer cells, such as NK1.1/CD69. Thus, a kit of the
present invention may be useful for monitoring both markers of
oxidative stress as well as markers for immune cells known to be
beneficial against known pathogens.
[0293] Thus, an assay format is preferred in which binding partners
such as antibodies can be obtained or prepared for the analytes
(lipid peroxide, pyroglutamic acid, glutathione, CD4, CD8,
NK1.1/CD69). Biotin-avidin, biotin-streptavidin or other
biotin-binding-reagent reactions can be used to enhance or modulate
the test. However, any such assay can be devised using other
binding partners to the analytes (oxidative stress markers and
immune cell markers), including but not limited to extracellular or
intracellular receptor proteins which recognize the analytes,
binding fragments thereof, hybridization probes for nucleic acids,
lectins for carbohydrates, etc. The particular selection of binding
partners is not limiting, provided that the binding partners permit
the test to operate as described herein. The preselected analytes,
when present, are detectable by binding two binding partners, one
immobilized on the test strip (or whatever format the assay is
provided) and another part of a conjugate. This is taken into
consideration in the selection of the reagents for the assay.
[0294] The dry test strip may be set up in any format in which
contact of the sample with the reagents is permitted and the
formation and mobility of the immunocomplexes and other complexes
forming therein are permitted to flow and contact an immobilized
reagent at the capture line. Various formats are available to
achieve this purpose, which may be selected by the skilled
artisan.
[0295] The label portion of the mobile, labeled antibody to the
marker may be a visible label, such as gold or latex, an
ultraviolet absorptive marker, fluorescent marker, radionuclide or
radioisotope-containing marker, an enzymatic marker, or any other
detectable label. A visibly detectable marker or one that can be
easily read in a reflectometer is preferred, for use by eye,
reading or confirmation with a reflectometer. Other labels may be
applicable to other semi-automated or automated
instrumentation.
[0296] The conjugates of the invention may be prepared by
conventional methods, such as by activation of an active moiety,
use of homobifunctional or heterobifunctional cross-linking
reagents, carbodiimides, and others known in the art. Preparation
of, for example, a gold-labeled antibody, a conjugate between an
antibody and an analyte (not an immunocomplex but a covalent
attachment which allows each member to independently exhibit its
binding properties), biotinylation of an antibody, conjugation of
streptavidin with a protein, immobilization of antibodies on
membrane surfaces, etc., are all methods known to one of skill in
the art.
[0297] A kit may have at least one reagent for carrying out an
assay of the invention, such as a kit comprising a conjugate
between a biotin-binding reagent and an antibody to an oxidative
marker. Preferably, the kit comprises all of the reagents needed to
carry out any one of the aforementioned assays, whether it be
homogeneous, heterogeneous, comprise a single conjugate of the
marker conjugated to an antibody to the analyte, or comprise two
reagents which serve this function (such as a biotinylated antibody
to the analyte plus a streptavidin-marker conjugate, or a
biotinylated marker plus a streptavidin conjugated to an antibody
to the analyte conjugate), or whether the assay employs an
immobilized antibody to the analyte and a labeled antibody to a
different site on the analyte. Referring to the first analyte as
analyte and the second analyte as marker, and a second binding
partner as a binding partner which recognizes a different epitope
than the first binding partner mentioned, the following kits are
non-limiting examples of those embraced herein:
[0298] In the foregoing kits, the binding partners are preferably
antibodies or binding portions thereof, and both the binding
partner to the analytes (the three oxidative stress markers, and
the markers for immune cells) and the second binding partner to the
analytes capable of simultaneously binding to the analyte. The
immobilized binding partner may be provided in the form of a
capture line on a test strip, or it may be in the form of a
microplate well surface or plastic bead, by way of non-limiting
examples. The kits may be used in a homogeneous format, wherein all
reagents are added to the sample simultaneously and no washing step
is required for a readout, or the kits may be used in a multi-step
procedure where successive additions or steps are carried out, with
the immobilized reagent added last, with an optional washing
step.
[0299] The antibodies specific for the three oxidative stress
markers may be obtained commercially, or can be produced by
techniques known to those skilled in the art.
EXAMPLES
[0300] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to use the methods described herein, and are not
intended to limit the scope of what the inventors regard as their
invention. Efforts have been made to ensure accuracy with respect
to numbers used (e.g., amounts, temperature, etc.) but some
experimental errors and deviations should be accounted for. Unless
indicated otherwise, parts are parts by weight, molecular weight is
average molecular weight, temperature is in degrees Centigrade, and
pressure is at or near atmospheric.
Example 1
Assessment of the Effects of IMMUNE FORMULATION 100.TM. or 200.TM.
on Adriamycin Induced Oxidative Stress Levels in Rats
[0301] Adriamycin, while being an effective anti-cancer agent in
humans, is known to induce nephropathy in a rat model (Zima, T. et
al (1998), Nephrol. Dial. Transplant, 13: 1975-1979). Its
deleterious effects on the kidney are believed to be due in part on
the generation of reactive oxygen species (ROS) following its
metabolism in vivo. Thus, the administration of adriamycin in a rat
model provides an opportunity to study the effects of this compound
on the generation of reactive oxygen species by measuring the
by-products of such ROS, such as urinary lipid peroxides and
pyroglutamic acid, as well as plasma glutathione. Furthermore, one
can also use this model to look at the effects of this toxic
compound on kidney damage, and on numbers of immune cell
populations and on natural killer cell activity. In addition, this
model provides an opportunity to assess the need for treatment of
an animal with an anti-oxidant, as well as monitoring the
utilization efficiency of such treatment, and ultimately, to
monitor the effectiveness of this treatment. Accordingly, this
model will be used to study the effects of IMMUNE FORMULATION
100.TM. or 200.TM. on the normalization of urinary lipid peroxides,
pyroglutamic acid and on plasma glutathione. Studies will also be
done to measure the effects on CD4+ and CD8+ T cell populations, as
well as on natural killer cell activity.
[0302] Materials and Methods
[0303] Rats
[0304] Ninety female Sprague-Dawley rats (200-225 g) are obtained
from Charles River and acclimatized for 7 days after delivery.
Animals are housed in communal cages, fed a rat maintenance diet
and water ad libitum. During experiments, animals are housed in
individual metabolism cages designed to separate and collect feces
and urine, and given powdered diet and water ad libitum. Lighting
is controlled to give a regular 12 h light-12 h dark cycle; room
temperature is maintained at 21.+-.1.degree. C. Urine samples (24
h) are collected over ice and centrifuged (2000 r.p.m., 10 min,
4.degree. C.) to remove hair and food debris and stored
(-80.degree. C.) in aliquots for later analysis. The general
condition of the animals is monitored daily and rats are weighed
twice a week.
[0305] Study Design
[0306] The rats are randomized into three groups of thirty rats per
group. Group I is designated the Placebo control group, Group II is
designated the Adriamycin only group, and Group III is designated
the Adriamycin plus IMMUNE FORMULATION 100.TM. or 200.TM. treatment
group. Twenty-four hours prior to dosing, 1.0 ml of whole blood is
collected from the tail vein of each rat and placed into a
heparinized microcentrifuge tube. Twenty-four hour urines are also
collected from each rat prior to dosing, and the urines are
centrifuged to remove any food or hair and frozen at -80.degree. C.
until assayed for the metabolites lipid peroxide and pyroglutamic
acid. Plasma is separated from whole blood by centrifugation
(2500.times.g for 25 min at 4.degree. C.) and stored at -80.degree.
C. for glutathione analysis.
[0307] On day 1, the rats in Groups II and III are given Adriamycin
at 5 mg/kg i.v., while the rats in Group I are injected with I.V.
saline as placebo. On day 2, the rats in Group III are given either
IMMUNE FORMULATION 100.TM. or 200.TM.. IMMUNE FORMULATION 100.TM.
will be given in a dose ranging from 5 to 125 grams per day
prepared in powdered rat chow. IMMUNE FORMULATION 200.TM. will be
given in a dose ranging from 25 to 100 mg per day by oral gavage.
The rats in group III are dosed daily with the IMMUNE FORMULATIONs
at the doses described. On day 4 and every three days afterwards,
1.0 ml of whole blood is collected from the tail vein of each rat
in the study, and 24 hour urines are also collected. The urines and
blood are treated as described above, and frozen away at
-80.degree. C. for future analysis of lipid peroxides and
pyroglutamic acid.
[0308] The urine samples and plasma samples are analyzed on day
seven for lipid peroxide, pyroglutamic acid and glutathione, using
the methods described below. If the levels of Group III remain
outside of the range of the placebo group, the rats in Group III
are dose adjusted in an increment of 10 gram doses for IMMUNE
FORMULATION 100.TM. and in increments of 10 mg doses for IMMUNE
FORMULATION 200.TM., and dosing is continued for another week.
After the rats in Group III have been dosed for a total of 2 weeks,
the plasma and urine levels are again assessed for glutathione,
lipid peroxide and pyroglutamic acid using the methods described
below. If the ranges of all three are still outside of the range of
the placebo treated Group I rats, the levels of the IMMUNE
FORMULATIONs are again scaled up as described above and dosing
continues another week. After one month of following this testing
and dosing regimen, a final 24 hour urine sample is collected and a
final bleed is done prior to sacrifice of the rats by CO2
asphyxiation. Spleens are removed and single cell suspensions are
prepared for use in natural killer cell assays and for staining
with markers specific for CD4+ and CD8+ T cells.
[0309] Urinary Lipid Peroxide Measurement by Determination of
Urinary Thiobarbituric-Acid-Reacting Substances (TBARS)
[0310] Lipid peroxide levels in urine samples are measured
calorimetrically by the thiobarbituric acid reaction (TBARS) as
described in the following references: Buege J A, Aust S D, Methods
Enzymol 1978, 52:302-310 and in Valenzuela et al (Valenzuela A:
Life Sci 1991, 48:301-309). The level of lipid peroxides in urine
is expressed as equivalents of malondialdehyde (MDA).
Malondialdehyde standards are freshly prepared from
tetraetoxypropane and treated in the same way as the urine samples.
Briefly, 200 .mu.l of urine is combined with 10 .mu.l of 5%
butylated hydroxytoluene (in glacial acetic acid) and 300 l of a
0.5% aqueous thiobarbituric acid (TBA) solution. The samples are
vortexed and are incubated at 100.degree. C. for 30 minutes, and
the absorbance at 532 nm is measured using a PerkinElmer Lamba 3B
spectrophotometer (PerkinElmer, Wellesley, Mass. USA). The quantity
of TBARS is proportionate to the amount of MDA, a lipid
peroxidation product generated by the oxidation of membrane lipids
by reactive oxygen species. MDA reacts with TBA to form a 1:2
MDA-TBA adduct that absorbs at 532 nm. To control for urine
concentration, data is normalized to urine creatinine
concentrations, as described (Coulthard M G, Hey E N, Ruddock V:
Early Hum Dev 1985, 11:11-19). Creatinine can also be measured by a
Sigma diagnostics kit 555-A.
[0311] Urinary Pyroglutamic Acid Measurement
[0312] Using a range of 1 and 2 dimensional 500 MHz 1H NMR
spectroscopic techniques, solid phase extraction and mass
spectrometry, the metabolite pyroglutamic acid (PGA), also known as
5-oxoproline (5OXP), can be measured in the urine. (Ghauri F Y, et
al. (1993), Biochem Pharmacol., September 1; 46(5):953-7.)
Alternatively, PGA measurements can be done by gas chromatography
using a Hewlett-Packard 5890 series II fitted with 7673A
autosampler, HP-1 capillary column (25 m.times.0.2
mm.times.0.33-.mu.m film thickness; Hewlett-Packard), and HP5971A
mass-selective detector. Helium gas flow rate can be 0.6 mL/min
(head pressure, 114 kPa). Split injections (ratio, 100:1) can be
made with a 1-.mu.L sample. A one-step temperature program may be
run from 70 to 290.degree. C. at 7.degree. C./min after an initial
time of 0.5 min. The mass spectrometer in electron ionization mode,
connected directly to the capillary column outlet, would be
operated at 70 eV. Data aquisition can be carried out in the scan
mode from m/z 58 to 550, with dwell time of 100 ms. The method of
extraction and preparation of urine samples for gas
chromatography-mass spectrometry and the method for qualitatively
and quantitatively identifying 5-oxoproline are based on those
described by Tanaka et al. (Tanaka K, West-Dull A, Hine D G, Lynn T
B, Lowe T. Gas chromatographic method of analysis for urinary
organic acids. I. Retention indices of 155 metabolically important
compounds. Clin Chem 1980; 26:1839-1846; Tanaka K, West-Dull A,
Hine D G, Lynn T B, Lowe T., Gas chromatographic method of analysis
for urinary organic acids. II. Description of the procedure and its
application to diagnosis of patients with organic acidurias. Clin
Chem 1980; 26:1847-1853). The response factor to the internal
standard, isopentanoic acid, could be used to approximate the
5-oxoproline peak as identified by comparison with published
spectra.
[0313] Plasma Glutathione Measurement
[0314] A GSH kit can be procured from Calbiochem. Plasma samples
are defrosted and serial dilutions prepared and analyzed for total
GSH per the manufacturer's instructions (Calbiochem).
[0315] Natural Killer Cell Assay
[0316] NK function (i.e., activity) can be measured by .sup.51Cr
release cytotoxicity assays against a suitable target cell. An
example of a suitable target cell by which to measure NK cell
cytotoxic activity is YAC-1. NK cell activation can also be
measured by determining an upregulation of NK1.1/CD69 on cells in
various organs, including spleen, lymph node, lung and liver, by
flow cytometric analysis.
[0317] Cytotoxicity Assay
[0318] A standard 4-hour .sup.51Cr-release assay is used to
quantitate cytotoxic activity present in freshly isolated spleen
mononuclear cells, using YAC-1 cells as targets. Briefly, effector
cells from spleen are added in decreasing concentrations to
duplicate wells of a Linbro plate, to which was then added
5.times.10.sup.3 target cells that had been previously labeled for
1 hour with .sup.51Cr. The plates are incubated at 37.degree. C.
for 4 hours, then supernatants from each well are harvested and the
amount of radioactive .sup.51Cr present is determined by automated
gamma counter. For spontaneous release, only targets were added and
the well was made up to the equivalent volume with medium, for
maximum release 0.1 ml of 2% SDS was added to wells containing
targets only. The percentage specific lysis is calculated as
((experimental release-spontaneous release)/(maximum
release-spontaneous release)).times.100.
[0319] Flow Cytometry
[0320] Upregulation of the early activation marker, CD69, which is
upregulated on activated T cells, B cells, macrophages and NK
cells, can be used to assess early immune cell activation. Single
cell suspensions are prepared from spleens of rats by NH.sub.4Cl
lysis procedure (Sambrook, supra). Cells are analyzed using a
Becton-Dickinson FACSCalibur flow cytometer (Becton Dickinson,
Mountain View, Calif.), with analysis gates set by first gating on
spleen lymphocytes. Between 10,000 and 30,000 gated events are
analyzed for each cell type. For analysis of cell activation,
3-color flow cytometric analysis may be done, using anti-CD69
phycoerythrin (Pharmingen, San Diego, Calif.) to quantitate the
number of CD69 positive cells. Cells can also be dual-labeled to
evaluate T cells (anti-.alpha..beta.TCR antibody (biotin H57.597;
Pharmingen) plus antibodies to either CD4 (FITC RM4-5; Pharmingen)
or CD8 (FITC 53-6.7; Pharmingen). NK cells can be dual-labeled
using anti NK 1.1 (biotin PK136; Pharmingen) and anti CD3 (FITC
2C11). The percentage of double positive cells expressing CD69 can
be determined for each cell type, and the mean (+/-SD) CD69+cells
plotted.
[0321] Assessment of CD4+ and CD8+ T Cell Numbers
[0322] Monoclonal antibodies (mAbs) to murine CD4, GK1.5 (ATCC TIB
207) and murine CD8, 53-6.72 (ATCC TIB 105) are purified from
hybridoma culture supernatants over a recombinant protein G column
(Pharmacia, Piscataway, N.J.). As a control, purified rat IgG is
purchased from Calbiochem (San Diego, Calif.). Flow cytometry is
performed as described above to assess the effectiveness of the
treatment regimen. Standard flow cytometric techniques are used
using Phycoerythrin labeled anti-rat CD4 and FITC labeled anti-rat
CD8 purchased, for example, from Pharmingen (San Diego,
Calif.).
[0323] Statistical Analyses
[0324] Significant differences between groups can be determined by
the Tukey-Kramer HSD multiple comparisons test using JMP.RTM.
statistical discovery software (SAS Institute Inc., Cary, N.C., or
by an Analysis of Variance (ANOVA) with a Bonferroni P value for
multiple comparisons.
Example 2
IMMUNE FORMULATION 100.TM. Tablet Formulation
[0325]
2 Ingredients: Whey (PROLIANT .TM. 8010 or 8200) 1 gm Colostrum 1
gm Selenium methionine 5 .mu.g
[0326] Blend the ingredients together and pass through a 60 mesh
screen and tumble until the components are thoroughly mixed.
Compress using a {fraction (7/16)} inch standard concave punch.
Example 3
IMMUNE FORMULATION 100.TM. Powder Formulation
[0327]
3 Ingredients: Whey (Proliant .TM. 8010 or 8200) 75 gm Colostrum 25
mg Selenium methionine 15 .mu.g
[0328] Thoroughly mix the ingredients in a blender and pass through
a 80 mesh screen.
[0329] This powder may be used for mixing with animal feeds,
frostings, fruit spreads and beverages to be pasteurized.
Example 4
IMMUNE FORMULATION 100.TM. Chewable Tablet Formulation
[0330]
4 Ingredients: Vitamin A USP (dry, stabilized form) 500 USP units
Vitamin D (dry, stabilized form) 400 USP units Ascorbic Acid USP
60.0 mg Thiamine Hydrochloride USP 1 mg Riboflavin USP 1.5 mg
Pyridoxine Hydrochloride USP 1 mg Cyanocobalamin USP 2 .mu.g
Calcium Pantothenate USP 3 mg Niacinamide USP 10 mg Mannitol USP
(granular) 236.2 mg Corn Starch 16.6 mg Sodium saccharin 1.1 mg
Magnesium stearate 6.6 mg Talc USP 10 mg Whey (Proliant .TM. 8010)
8 g Colostrum 500 mg Selenium methionine 7 .mu.g
[0331] Thoroughly mix the ingredients in a blender and compress
using a {fraction (3/8)} inch bevel-edge punch.
Example 5
IMMUNE FORMULATION 200.TM. (TABLET)
[0332]
5 Ingredients: 89 mg cystine 75 mg glycine 147 mg glutamic acid
22.5 .mu.g polyvinylpyrolidone 61.25 mg lactose 4.5 ml alcohol
SD3A-200 proof 9 mg stearic acid 42.3 mg corn starch 10 .mu.g
selenium methionine
[0333] Blend the cystine, glycine, glutamic acid,
polyvinylpyrrolidone and lactose together and pass through a 40
mesh screen. Add the alcohol slowly and knead well. Screen the wet
mesh through a 4 mesh screen. Dry the granulation at 50 degrees
centigrade for 10 hours. Pass the mixture of stearic acid, corn
starch and selenium compound through a 60 mesh screen and tumble
with the granulation until all the ingredients are well mixed.
Compress using a {fraction (7/16)} inch standard concave punch.
Example 6
IMMUNE FORMULATION 200.TM. (TABLET)
[0334]
6 Ingredients: 178 mg cystine 150 mg glycine 294 mg glutamic acid 5
.mu.g selenium methionine 126 mg lactose 78 mg potato starch 96 mg
ethyl cellulose 54 mg stearic acid
[0335] Thoroughly mix the ingredients in a blender, dry, put
through a 12 mesh screen and compress into tablet using a {fraction
(13/32)} inch concave punch.
* * * * *