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.

We claim: 
     
         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. 
     
     
         6 . The method of  claim 1 , wherein the sample of body fluid is urine. 
     
     
         7 .- 28 . (canceled) 
     
     
         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 .- 36 . (canceled) 
     
     
         37 . The method of  claim 1 , comprising determining the amount of lipid peroxides and pyroglutamic acid in the sample with the Wheeler method, iron thiocyanate method, thiobarbituric acid method or with a combination of a peroxidase and a hydrogen donor. 
     
     
         38 . The method of  claim 5 , wherein the glutathione precursor is a composition comprising glycine, cystine and a source of glutmate, together with a source of selenium. 
     
     
         39 . The kit of  claim 29 , wherein the marker of oxidative stress is a lipid peroxide, pyroglutamic acid or glutathione or a combination thereof. 
     
     
         40 . The kit of  claim 29 , wherein the marker of oxidative stress is 4-hydroxynonal (4HNE) or malondialdehyde or a protein modified by 4-HNE or malondialdehyde. 
     
     
         41 . A method for determining the amount of antioxidant formulation sufficient to
 (1) increase glutathione synthesis or re-synthesis in a patient in need thereof;   (2) reduce urine pyroglutamic acid in a patient in need thereof;   (3) reduce urine lipid peroxide in a patient in need thereof; and/or   (4) diminish urine lipid peroxide and pyroglutamic acid levels and concurrently increase blood plasma glutathione levels in a patient in need thereof;   the method comprising   (a) collecting a series of body fluid samples from the patient, 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/or pyroglutamic acid in said body fluid samples;   (c) comparing the amount of lipid peroxide and/or pyroglutamic acid in said body fluid samples with that of normal standards;   (d) optionally measuring the amount of glutathione increase in blood samples;   (e) optionally comparing the amount of glutathione in said blood samples with that of normal standards; and   (f) correlating a change in the levels of lipid peroxide and/or pyroglutamic acid and optionally glutathione in the body samples of patients receiving the antioxidant formulation.   
     
     
         42 . The method of  claim 41 , wherein the levels of lipid peroxide are measured by mass spectrometry, absorption spectrometry, liquid chromatography, thin layer chromatography or a redox reagent. 
     
     
         43 . The method of  claim 41 , wherein the levels of lipid peroxide are measured by Wheeler method, iron thiocyanate method, thiobarbituric acid method or with a combination of a peroxidase and a hydrogen donor. 
     
     
         44 . The method of  claim 41 , which is a colorimetric assay, a fluorescence assay, a luminometric assay. 
     
     
         45 . The method of  claim 41 , wherein the urine levels of lipid peroxide are measured with an immunological assay. 
     
     
         46 . The method of  claim 41 , wherein the immunological assay is enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA). 
     
     
         47 . The method of  claim 41 , wherein the urine levels of lipid peroxide are measured with one or more antibodies to lipid peroxides. 
     
     
         48 . The method of  claim 41 , wherein the measurement of the levels of lipid peroxide is conducted in a fluid phase format, a high throughput format, a test strip format or a test well format. 
     
     
         49 . The method of  claim 41 , wherein the pyroglutamic acid is measured by gas chromatography, mass spectrometry, or NMR spectroscopy. 
     
     
         50 . The method of  claim 41 , wherein the pyroglutamic acid levels are measured with an antibody to pyroglutamic acid. 
     
     
         51 . The method of  claim 41 , wherein the measurement of the levels of pyroglutamic acid is conducted in a fluid phase format, a high throughput format, a test strip format or a test well format. 
     
     
         52 . The method of  claim 41 , wherein the measurement of levels of pyroglutamic acid is conducted in a test-strip format or a test-well format, wherein the test strip or test well comprises an antibody to pyroglutamic acid. 
     
     
         53 . The method of  claim 41 , wherein the levels of lipid peroxide is measured prior to, concurrently with, or shortly after the measurement of urine pyroglutamic acid. 
     
     
         54 . The method of  claim 41 , further comprising measuring the levels of blood plasma glutathione with an enzymatic or non-enzymatic assay. 
     
     
         55 . The method of  claim 41 , further comprising measuring the levels of blood plasma glutathione with an antibody to glutathione. 
     
     
         56 . The method of  claim 41 , wherein the blood plasma and urine samples are collected from a subject or a patient which is a non-human mammal or a human. 
     
     
         57 . The method of  claim 41 , comprising detecting malondialdehyde (MDA) or 4-hydroxynonenal as a marker of lipid peroxide. 
     
     
         58 . The method of  claim 41 , wherein the body fluid sample is urine, blood or a combination thereof. 
     
     
         59 . A method for determining an orally anti-oxidative amount of a nutritional or therapeutic composition 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 the nutritional or therapeutic composition and daily after the start of administration for about 14 days;   b) measuring the urine levels of lipid peroxide and pyroglutamic acid and blood plasma levels of glutathione both prior to and after the start of administration of the nutritional or therapeutic composition;   c) determining whether there is (1) a decrease in lipid peroxide and pyroglutamic acid levels after the administration of the nutritional or therapeutic composition compared to the levels thereof before the administration of nutritional or therapeutic composition and (2) an increase in blood glutathione levels after the administration of the nutritional or therapeutic composition compared to the levels thereof before the administration of the nutritional or therapeutic composition; and   d) correlating the decrease in lipid peroxide and pyroglutamic acid of (c)(1) with the increase in glutathione levels of (c)(2), wherein said correlation establishes an orally anti-oxidative effective amount of the nutritional or therapeutic composition.   
     
     
         60 . The method of  claim 59 , wherein the measurement of the levels of lipid peroxide and pyroglutamic acid is conducted in a fluid phase format, a high throughput format, a test strip format or a test well format. 
     
     
         61 . The method of  claim 59 , wherein the glutathione precursor is a composition comprising glycine, cystine and a source of glutmate, together with a source of selenium.