Method for determining amounts of nad metabolites from sample and methods and uses related thereto

Abstract

The present invention relates to the fields of life sciences and medicine. Specifically, the invention relates to a method for preparing an extract comprising metabolites such as NAD+, NADP+, NADH and NADPH from a sample of a subject and to a method for determining amounts of NAD+, NADP+, NADH, NADPH, NAD+/NADH ratio, NADPH/NADP+ ratio, NAD pool, and/or NADP pool from a sample obtained from a subject. The present invention also relates to an extract comprising metabolites such as NAD+, NADP+, NADH and NADPH. Also, the present invention relates to a kit comprising an extraction solution, a detection system comprising an electron carrier, chromogen and non-ionic detergent for determining amounts of NAD+, NADP+, NADH, NADPH, NAD+/NADH ratio, NADPH/NADP+ ratio, NAD pool, and/or NADP pool, and optionally GSH, GSSG and/or GSH/GSSG ratio, from a sample of a subject. Still, the present invention relates to use of the kit of the present invention for determining amounts of NAD +, NADP+, NADH, NADPH, NAD+/NADH ratio, NADPH/NADP+ ratio, NAD pool, NADP pool, GSH, GSSG and/or GSH/GSSG ratio or changes thereof in a sample of a subject e.g. for diagnostics purposes or for monitoring changes in health status of a person. Furthermore, the present invention relates to a method and kit for determining disorders of a subject.

1 . The method for determining amounts of NAD+, NADP+, NADH, NADPH, NAD+/NADH ratio, NADPH/NADP+ ratio, NAD pool, and/or NADP pool from a sample obtained from a subject, wherein the method comprises steps A; B or C; and D:
 A) obtaining an extract comprising NAD metabolites NAD+, NADP+, NADH and NADPH of a sample by contacting a sample with pre-heated to at least 40° C. non-buffered alcohol solution, and   B) i) stabilizing NAD+ and NADP+ and eliminating NADH and NADPH by acidic treatment in a first part of the extract, and/or stabilizing NADH and NADPH and eliminating NAD+ and NADP+ by alkali treatment with heating in a second part of the extract for determining amounts of NAD+, NADP+, NADH and NADPH; and carrying out one or more of the following B ii)-v),   ii) contacting NAD+ of the first part of the extract with a first NAD-specific enzyme in the presence of a detection system comprising an electron carrier, chromogen and non-ionic detergent to obtain an enzymatic reaction for determining amount of NAD+,   iii) contacting NADH of the second part of the extract with a first NAD-specific enzyme in the presence of a detection system comprising an electron carrier, chromogen and non-ionic detergent to obtain an enzymatic reaction for determining amount of NADH,   iv) contacting NADP+ of the first part of the extract with a second NADP-specific enzyme in the presence of a detection system comprising an electron carrier, chromogen and non-ionic detergent to obtain an enzymatic reaction for determining amount of NADP+, and/or   v) contacting NADPH of the second part of the extract with a second NADP specific enzyme in the presence of a detection system comprising an electron carrier, chromogen and non-ionic detergent to obtain an enzymatic reaction for determining amount of NADPH; and/or   C) i) contacting NAD+ and NADH together of a third part of the extract with a first NAD-specific enzyme in the presence of a detection system comprising an electron carrier, chromogen and non-ionic detergent to obtain an enzymatic reaction for determining amount of NAD pool, and/or   ii) contacting NADP+ and NADPH together of a fourth part of the extract with a second NADP-specific enzyme in the presence of a detection system comprising an electron carrier, chromogen and non-ionic detergent to obtain an enzymatic reaction for determining amount of NADP pool; and   D) i) determining amounts of NAD+, NADP+, NADH and/or NADPH in the sample by comparing the response of a detection system to added extract with the response of standards with known concentrations to find concentration of NAD metabolite in the extract followed by normalization on protein content, tissue mass or volume of the sample and/or calculation of NAD+/NADH ratio, NADPH/NADP+ ratio and/or NAD pool (NAD+ and NADH together) and/or NADP pool (NADP+ and NADPH together), or   ii) determining amounts of NAD pool and/or NADP pool in the sample by comparing the response of a detection system to added extract with the response of standards with known concentrations to find concentration of NAD metabolite in the extract followed by normalization on protein content or volume of the sample.   
     
     
         2 . The method of  claim 1 , wherein the method comprises normalization of determined NAD+, NADH, NADP+ and/or NADPH levels on total protein amount or sample weight or whole blood volume, and/or calculation of NAD+/NADH and/or NADPH/NADP+ ratios. 
     
     
         3 . The method of any preceding claim, wherein the first NAD-specific enzyme is selected from the group consisting of alcohol dehydrogenase, malate dehydrogenase, lactate dehydrogenase, NAD specific isocitrate dehydrogenase, glyceralde-hyde-3-phosphate dehydrogenase, and any combination thereof; and/or the second NADP-specific enzyme is selected from the group consisting of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, malic enzyme, NADP specific isocitrate dehydrogenase, and any combination thereof. 
     
     
         4 . The method of any preceding claim, wherein the electron carrier is phenazine ethosulfate (PES) and/or the chromogen is 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) for determining NAD+, NADP+, NADH, NADPH. 
     
     
         5 . The method of any preceding claim, wherein the non-ionic detergent is utilized for preventing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) aggregation upon reduction in the course of enzymatic reaction(s). 
     
     
         6 . The method of any preceding claim, wherein the non-ionic detergent comprises a polyethylene oxide unit and/or is selected from the group comprising Tween-20, Triton X-100, and nonyl phenoxypolyethoxylethanol (NP-40). 
     
     
         7 . The method of any preceding claim, wherein the method further comprises determining amount of GSH, GSSG, and/or GSH/GSSG ratio; or the method comprises simultaneous measurement of NAD+/NADH, NADPH/NADP+ and GSH/GSSG ratios or NADPH/NADP+ and GSH/GSSG ratios from the same sample for determining or measuring the redox status of the cell or body. 
     
     
         8 . The method of  claim 7 , wherein the method comprises
 i) removing GSH from a fifth part of the extract of A) by a chemical modification and contacting GSSG of the fifth part of the extract with a a GSH-specific enzyme, such as glutathione reductase in the presence of added NADPH and a reporter for determining GSSG, and/or   ii) contacting GSH and GSSG of a sixth part of the extract of A) with a GSH-specific enzyme, such as glutathione reductase in the presence of added NADPH and a reporter for determining GSH and GSSG.   
     
     
         9 . The method of  claim 8 , wherein the reporter is 5,5′-dithiobis(2-nitrobenzoic acid) named Ellman's reagent. 
     
     
         10 . The method of  claim 8  or  9 , wherein removal of GSH from the fifth part of the extract is achieved by addition of masking reagent 1-methyl-4-vinyl-pyridinium trifluoromethane sulfonate (M4VP) and/or 1-methyl-2-vinyl-pyridinium trifluoromethane sulfonate (M2VP). 
     
     
         11 . The method of any preceding claim, wherein the enzymatic reaction(s) is(are) stopped by allowing a detergent, optionally selected from sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB) and a combination thereof, to contact with the enzymatic reaction(s). 
     
     
         12 . The method of any preceding claim, wherein a first reagent combination comprising a first and/or second part of the extract, an electron carrier, chromogen and non-ionic detergent, and the first NAD-specific enzyme are used for enzymatic reactions for determining amounts of NAD+ and/or NADH. 
     
     
         13 . The method of any preceding claim, wherein a second reagent combination comprising a first and/or second part of the extract, an electron carrier, chromogen and non-ionic detergent, and the second NADP-specific enzyme are used for enzymatic reactions for determining amounts of NADP+ and/or NADPH. 
     
     
         14 . The method of any preceding claim, wherein a third reagent combination comprising a fifth and/or sixth part of the extract, added NADPH and a reporter, and a GSH-specific enzyme, such as glutathione reductase are used for enzymatic reactions for determining amounts of GSH and/or GSSG. 
     
     
         15 . The method of any preceding claim, wherein NAD+ and NADH are determined separately, NADP+ and NADPH are determined separately, GSH and GSSG are determined simultaneously, and/or GSSG is determined separately. 
     
     
         16 . The method of any preceding claim, wherein the method comprises measuring NAD+, NADH, NADP+, NADPH, NAD pool, NADP pool, GSH and/or GSSG, or any combination thereof, by a colorimetric method. 
     
     
         17 . The method of any preceding claim, wherein NAD+, NADP+, NADH, NADPH, NAD pool, and/or NADP pool are determined after an enzymatic reaction or reactions by measuring an absorbance at a wavelength of 560-580 nm or 570-573 nm. 
     
     
         18 . The method of any preceding claim, wherein GSH and/or GSSG are determined after an enzymatic reaction by measuring an absorbance at a wavelength of 400-420 nm or 410-415 nm. 
     
     
         19 . The method of any preceding claim, wherein the method comprises extracting NAD metabolites together with reduced and oxidized forms of glutathione from the sample, to obtain the extract comprising NAD+, NADP+, NADH, NADPH, GSH and GSSG. 
     
     
         20 . The method of any preceding claim, wherein the method comprises preparing the extract comprising NAD+, NADP+, NADH and NADPH from the sample allowing the sample to contact with an alcohol solution at a high temperature to obtain a mixture of the sample and the alcohol solution, cooling down the obtained mixture, and removing precipitated polypeptides from the mixture to obtain an extract comprising NAD+, NADP+, NADH and NADPH. 
     
     
         21 . The method of  claim 20 , wherein the alcohol solution comprises ethanol, methanol and/or isopropanol; or the alcohol solution is an ethanol, methanol or isopropanol solution. 
     
     
         22 . The method of  claim 20  or  21 , wherein the alcohol concentration of the alcohol solution is about 30-80%, 40-70%, 40-60%, 40-50%, 50-70%, 50-60%, 50% or 60%. 
     
     
         23 . The method of any of  claims 20 - 22  wherein the high temperature is about 40 −90° C., 40-60° C., 45-80° C., 50-80° C., 60-80° C., 70-80° C., 60° C. or 75° C. 
     
     
         24 . The method of any of  claims 20 - 23 , wherein the contacting time at a high temperature is selected from the group consisting of about 10 seconds-10 minutes, about 30 seconds—5 minutes and about 1 minute. 
     
     
         25 . The method of any of  claims 20 - 24 , wherein a period for cooling down the obtained mixture is about 30 seconds-30 minutes, 1-10 minutes, 5-10 minutes or 3-7 minutes. 
     
     
         26 . The method of any of  claims 20 - 25 , wherein the precipitated polypeptides are removed by centrifugation. 
     
     
         27 . The method of any of  claims 20 - 26 , wherein the mixture is a homogenate. 
     
     
         28 . A kit comprising an extraction solution, stock solutions of standards, stop solution, detection system comprising an electron carrier, chromogen, non-ionic detergent and NAD- and NADP-specific enzymes for determining NAD+, NADP+, NADH, NADPH, NAD+/NADH ratio, NADPH/NADP+ ratio, NAD pool, and/or NADP pool from a sample of a subject, and optionally comprising a chromogen and GSH-specific enzyme for determining GSH, GSSG and/or GSH/GSSG ratio from the sample of a subject. 
     
     
         29 . The kit of  claim 28 , wherein the NAD- and NADP-specific enzymes for determining NAD+, NADP+, NADH, NADPH, NAD+/NADH ratio, NADPH/NADP+ ratio, NAD pool, NADP pool are selected from the group consisting of alcohol dehydrogenase, malate dehydrogenase, lactate dehydrogenase, NAD specific isocitrate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and any combination thereof; and/or from the group consisting of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, malic enzyme, NADP specific isocitrate dehydrogenase, and any combination thereof; and/or
 the GSH-specific enzyme for determining GSH, GSSG and/or GSH/GSSG ratio is glutathione reductase.   
     
     
         30 . The kit of  claim 28  or  29 , wherein the detection system comprises an electron carrier phenasine ethosulfate (PES) and/or chromogen 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). 
     
     
         31 . The kit of any preceding claim, wherein the kit comprises a non-ionic detergent for preventing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) aggregation upon reduction in the course of the enzymatic reaction(s). 
     
     
         32 . The kit of any preceding claim, wherein the non-ionic detergent comprises polyethylene oxide unit and/or is selected from the group comprising Tween-20, Triton X-100, and nonyl pheno-xypolyethoxylethanol (NP-40). 
     
     
         33 . The kit of any preceding claim, wherein the kit comprises a detergent, optionally selected from sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB) and a combination thereof, for stopping an enzymatic reaction(s). 
     
     
         34 . The kit of any preceding claim, wherein the kit comprises an extraction solution, NAD+ and/or NADH standards, stop solution and a first reagent combination comprising a first and/or second part of the extract, an electron carrier, chromogen and non-ionic detergent to be used with a first NAD-specific enzyme for determining amounts of NAD+ and/or NADH. 
     
     
         35 . The kit of any preceding claim, wherein the kit comprises an extraction solution, NADP+ and/or NADPH standards, stop solution and a second reagent combination comprising a first and/or second part of the extract, an electron carrier, chromogen and non-ionic detergent to be used with a second NADP-specific enzyme for determining amounts of NADP+ and/or NADPH. 
     
     
         36 . The kit of any preceding claim, wherein the kit comprises an extraction solution, GSH and GSSG standards, stop solution and a third reagent combination comprising a fifth and/or sixth part of the extract, added NADPH and a reporter to be used with a GSH-specific enzyme, such as glutathione reductase for determining amounts of GSH and/or GSSG. 
     
     
         37 . The kit of any preceding claim, wherein the kit comprises an alcohol solution for preparing an extract of the sample for determining amounts of NAD+, NADP+, NADH, NADPH, NAD+/NADH ratio, NADPH/NADP+ ratio, NAD pool, and/or NADP pool. 
     
     
         38 . The kit of  claim 37 , wherein the alcohol solution comprises ethanol, methanol and/or isopropanol; or the alcohol solution is an ethanol, methanol or isopropanol solution. 
     
     
         39 . The kit of  claim 37  or  38 , wherein the alcohol concentration of the alcohol solution is about 30-80%, 40-70%, 40-60%, 40-50%, 50-70%, 50-60%, 50% or 60%. 
     
     
         40 . The method or kit of any preceding claim, wherein the first reagent combination comprises a buffer (such as Bicine-NaOH) and a first master mix comprising e.g. EDTA, MTT, PES, non-ionic detergent for preventing MTT aggregation, H 2 O and a substrate for the first NAD-specific enzyme. 
     
     
         41 . The method or kit of any preceding claim, wherein the second reagent combination comprises a buffer (such as Bicine-NaOH) and a second master mix comprising e.g. EDTA, MTT, PES, non-ionic detergent for preventing MTT aggregation, H 2 O and a substrate for the second NADP-specific enzyme. 
     
     
         42 . The method or kit of any preceding claim, wherein the third reagent combination comprises a buffer (such as PBS) and a third master mix comprising e.g. EDTA, DTNB, H 2 O and a substrate NADPH for a GSH-specific enzyme, such as glutathione reductase. 
     
     
         43 . The kit of any preceding claim, wherein the kit comprises two or more multi-well plates for determining amounts of NAD+ and NADH separately, and/or two or more multi-well plates for determining amounts of NADP+ and NADPH separately. 
     
     
         44 . The kit of any preceding claim, wherein the kit comprises reagents for extracting pyridine nucleotides, GSH and GSSG from a sample. 
     
     
         45 . The method or kit of any preceding claim, wherein the sample is selected from the group consisting of cells, cultured cells, blood cells, whole blood, tissue, and adipose tissue. 
     
     
         46 . The kit of any preceding claim, wherein the kit comprises instructions for carrying out a method for determining one or more from the group consisting of NAD+, NADH, NADP+, NADPH, NAD+/NADH ratio, NADPH/NADP+ ratio, NAD pool, NADP pool, GSH, GSSG and GSH/GSSG ratio, of a sample. 
     
     
         47 . The kit of any preceding claim, wherein said kit is for the method of any of the preceding claims. 
     
     
         48 . Use of the kit of any of  claims 28 - 47  for determining amounts of NAD+, NADP+, NADH, NADPH, NAD+/NADH ratio, NADPH/NADP+ ratio, NAD pool, NADP pool, GSH, GSSG and/or GSH/GSSG ratio or changes thereof in a sample of a subject. 
     
     
         49 . A method for preparing an extract comprising metabolites such as NAD+, NADP+, NADH and NADPH from a sample of a subject, wherein the method comprises allowing a sample of a subject to contact with an alcohol solution at a high temperature to obtain a mixture of the sample and the alcohol solution, cooling down the obtained mixture, and removing precipitated polypeptides from the mixture to obtain an extract comprising metabolites such as NAD+, NADP+, NADH and NADPH. 
     
     
         50 . The method of  claim 49 , wherein the alcohol solution comprises ethanol, methanol and/or isopropanol; or the alcohol solution is an ethanol, methanol or isopropanol solution. 
     
     
         51 . The method of  claim 49  or  50 , wherein the alcohol concentration of the alcohol solution is about 30-80%, 40-70%, 40-60%, 40-50%, 50-70%, 50-60%, 50% or 60%. 
     
     
         52 . The method of any of  claims 49 - 51  wherein the high temperature is at least 40° C., about 40-90° C., 40-60° C., 45-80° C., 50-80° C., 60-80° C., 70-80° C., 60° C. or 75° C. 
     
     
         53 . The method of any of  claims 49 - 52 , wherein the contacting time at a high temperature is selected from the group consisting of about 10 seconds-10 minutes, about 30 seconds-5 minutes and about 1 minute. 
     
     
         54 . The method of any of  claims 49 - 53 , wherein a period for cooling down the obtained mixture is about 30 seconds-30 minutes, 1-10 minutes, 5-10 minutes or 3-7 minutes. 
     
     
         55 . The method of any of  claims 49 - 54 , wherein the precipitated polypeptides are removed by centrifugation. 
     
     
         56 . The method of any of  claims 49 - 55 , wherein the mixture is a homogenate. 
     
     
         57 . The method of any of  claims 49 - 56 , wherein the sample is selected from the group consisting of cells, cultured cells, blood cells, whole blood, tissue, and adipose tissue. 
     
     
         58 . The method of any of  claims 49 - 57 , wherein the method further comprises determining amounts of any metabolite; or amounts of NAD+, NADP+, NADH, NADPH, NAD+/NADH ratio, NADPH/NADP+ ratio, NAD pool, and/or NADP pool from the extract; or the method further comprises simultaneous measurement of NAD+/NADH, NADPH/NADP+ and GSH/GSSG ratios or NADPH/NADP+ and GSH/GSSG ratios from the same sample for determining or measuring the redox status of the cell or body. 
     
     
         59 . An extract comprising metabolites such as NAD+, NADP+, NADH and NADPH, wherein the extract has been prepared with the method of any of  claims 49 - 58 . 
     
     
         60 . A method for determining amounts of NAD+, NADP+, NADH, NADPH, NAD+/NADH ratio, NADPH/NADP+ ratio, NAD pool, and/or NADP pool from a sample of a subject, wherein the method comprises preparing an extract comprising NAD+, NADP+, NADH and NADPH from a sample of a subject according to any of  claims 49 - 58  and determining NAD+, NADP+, NADH, NADPH, NAD+/NADH ratio, NADPH/NADP+ ratio, NAD pool, and/or NADP pool according to any of  claims 1 - 45 . 
     
     
         61 . A method for determining abnormal NAD amounts or ratios in a disorder selected from the group comprising diabetes type I or II, muscle disease, fatty liver disease (non-alcoholic and acquired alcoholic), obesity, Parkinson's disease, Alzheimer's disease, other neurodegenerative diseases such as multiple sclerosis; lung disease, kidney disease, liver disease, thyroid disease, cardiac or cerebral stroke, chronic fatigue syndrome, ataxia disease, ocular disease, mitochondrial disorder, metabolic disorder including inherited and non-inherited, anorexia, cachexia, viral or bacterial infection or related secondary disease such as immune reaction, endocrine disorders, nutrient deficiency, and cancer (such as a breast, colon, stomach, brain, pancreas, prostate, ovary, liver, lung or skin cancer, or leukemia), of children, teenagers or adults, wherein the method comprises determining NAD+, NADP+, NADH, NADPH, NAD+/NADH ratio, NADPH/NADP+ ratio, NAD pool, and/or NADP pool from a sample of a subject. 
     
     
         62 . The method of  claim 61 , wherein an increased or decreased level of NAD+, NADP+, NADH, NADPH, NAD+/NADH ratio, NADPH/NADP+ ratio, NAD pool, and/or NADP pool in the sample of the subject indicates manifestation or progression or need of treatment of diabetes type I or II, muscle disease, fatty liver disease (non-alcoholic and acquired alcoholic), obesity, Parkinson's disease, Alzheimer's disease, other neurodegenerative diseases such as multiple sclerosis; lung disease, kidney disease, liver disease, thyroid disease, cardiac or cerebral stroke, chronic fatigue syndrome, ataxia disease, ocular disease, mitochondrial disorder, metabolic disorder including inherited and non-inherited, anorexia, cachexia, viral or bacterial infection or related secondary disease such as immune reaction, endocrine disorders, nutrient deficiency, and cancer (such as a breast, colon, stomach, brain, pancreas, prostate, ovary, liver, lung or skin cancer, or leukemia), of children, teenagers or adults. 
     
     
         63 . The method of  claim 61  or  62 , wherein one or more levels of NAD+, NADP+, NADH, NADPH, NAD+/NADH ratio, NADPH/NADP+ ratio, NAD pool, and/or NADP pool of the sample are compared to the corresponding levels of a control sample; or one or more levels of NAD+, NADP+, NADH, NADPH, NAD+/NADH ratio, NADPH/NADP+ ratio, NAD pool, and/or NADP pool of the sample are compared to the corresponding normal levels determined from a set of controls. 
     
     
         64 . The method of any of  claims 61 - 63 , wherein the method comprises preparing an extract comprising NAD+, NADP+, NADH and NADPH from the sample of a subject according to any of  claims 49 - 57  for determining one or more levels of NAD+, NADP+, NADH, NADPH, NAD+/NADH ratio, NADPH/NADP+ ratio, NAD pool, and/or NADP pool. 
     
     
         65 . The method of any of  claims 61 - 64 , wherein NAD+, NADP+, NADH, NADPH, NAD+/NADH ratio, NADPH/NADP+ ratio, NAD pool, and/or NADP pool are determined according to any of  claims 1 - 45  or  60 . 
     
     
         66 . A kit for determining abnormal NAD-amounts or ratios in a disorder selected from the group comprising diabetes type I or II, muscle disease, fatty liver disease (non-alcoholic and acquired alcoholic), obesity, Parkinson's disease, Alzheimer's disease, other neurodegenerative diseases such as multiple sclerosis; lung disease, kidney disease, liver disease, thyroid disease, cardiac or cerebral stroke, chronic fatigue syndrome, ataxia disease, ocular disease, mitochondrial disorder, metabolic disorder including inherited and non-inherited, anorexia, cachexia, viral or bacterial infection or related secondary disease such as immune reaction, endocrine disorders, nutrient deficiency, and cancer (such as a breast, colon, stomach, brain, pancreas, prostate, ovary, liver, lung or skin cancer, or leukemia), of children, teenagers or adults, wherein the kit comprises a detection system comprising an electron carrier, chromogen and non-ionic detergent for determining amounts of NAD+, NADP+, NADH, NADPH, NAD+/NADH ratio, NADPH/NADP+ ratio, NAD pool, and/or NADP pool from a sample of a subject, according to any of  claims 28 - 47 . 
     
     
         67 . Use of the kit of  claim 66  for determining NAD metabolites amounts or ratios to determine manifestation, status, progression or treatment response in a disease selected from the group comprising diabetes type I or II, muscle disease, fatty liver disease (non-alcoholic and acquired alcoholic), obesity, Parkinson's disease, Alzheimer's disease, other neurodegenerative diseases such as multiple sclerosis; lung disease, kidney disease, liver disease, thyroid disease, cardiac or cerebral stroke, chronic fatigue syndrome, ataxia disease, ocular disease, mitochondrial disorder, metabolic disorder including inherited and non-inherited, anorexia, cachexia, viral or bacterial infection or related secondary disease such as immune reaction, endocrine disorders, nutrient deficiency, and cancer (such as a breast, colon, stomach, brain, pancreas, prostate, ovary, liver, lung or skin cancer, or leukemia), of children, teenagers or adults. 
     
     
         68 . Use of NAD+, NADP+, NADH, NADPH, NAD+/NADH ratio, NADPH/NADP+ ratio, NAD pool, and/or NADP pool for determining manifestation, status, progression or treatment response in a disorder selected from the group comprising diabetes type I or II, muscle disease, fatty liver disease (non-alcoholic and acquired alcoholic), obesity, Parkinson's disease, Alzheimer's disease, other neurodegenerative diseases such as multiple sclerosis; lung disease, kidney disease, liver disease, thyroid disease, cardiac or cerebral stroke, chronic fatigue syndrome, ataxia disease, ocular disease, mitochondrial disorder, metabolic disorder including inherited and non-inherited, anorexia, cachexia, viral or bacterial infection or related secondary disease such as immune reaction, endocrine disorders, nutrient deficiency, and cancer (such as a breast, colon, stomach, brain, pancreas, prostate, ovary, liver, lung or skin cancer, or leukemia), of children, teenagers or adults.