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Assessment of Oxidative Stress Assessment of Oxidative Stress

Oxidative stress involves in various diseases Cancer Inflammation Aging Arteriosclerosis Ischemic Reperfusion Mucosa of Oxidative stress involves in various diseases Cancer Inflammation Aging Arteriosclerosis Ischemic Reperfusion Mucosa of the Gut Disease Hepatic Disease Diabetes Cardiac Disease Respiratory Disorder Renal Disease Cranial Nerves Disease Skin Disease Multiple Organ Failure Ocular Disease (Cataract) (cardiac infarct etc. ) Dr. T Yoshikawa

Balance of Oxidative Stress and Anti-oxidative System ・O 2 OH H O 1 O Balance of Oxidative Stress and Anti-oxidative System ・O 2 OH H O 1 O 2 2 2 LOO・ L・ LO・ OXIDATIVE STRESS Cancer SOD Vitamin E GSH-Px Carotenoids Uric Acid Catalase Flavonoids Vitamin C Other Antioxidant Enzymes Other Antioxidants Balance is Important Life Style-Related Diseases Parkinson disease High blood pressure Arterial sclerosis Myocardial infarction Dementia, Cataract etc. Aging Immune Disorder

Oxidative Stress Markers DNA Oxidation 8 -OHd. G Thymidine Glycol Protein Oxidation Dibromo-Tyrosine Dithyrosine Oxidative Stress Markers DNA Oxidation 8 -OHd. G Thymidine Glycol Protein Oxidation Dibromo-Tyrosine Dithyrosine Lipid Oxidation Acrolein Crotonaldehyde Hexanoyl-Lysine 4 -hydroxy-2 -hexenal 4 -hydroxy-2 -nonenal 7 -ketocholesterol Malondialdehyde Methylglyoxal

Formation of 8 -OHd. G Cell Anti-Oxidative System Reactive Oxygen Species Repair System Oxidative Formation of 8 -OHd. G Cell Anti-Oxidative System Reactive Oxygen Species Repair System Oxidative Stress DNA (d. G) Urine Damaged DNA (8 -OHd. G) Normal DNA (d. G) Normal Base Damaged Base (d. G) (8 -OHd. G) Blood

Anti-8 -OHd. G Monoclonal Antibody (N 45. 1) & 8 -OHd. G ELISA kit Anti-8 -OHd. G Monoclonal Antibody (N 45. 1) & 8 -OHd. G ELISA kit Assessment of Oxidative Stress inside the body. a) Highly specific for 8 -OHd. G. b) Applications: urine, serum, tissue, cultured cells, saliva… c) Assay time: 3. 5 hours (New 8 -OHd. G Check) d) Number of samples (N=3; 18 samples / kit) e) Risk management of health and aging. f) Monitoring the effect of chemicals, UV , radiation etc… g) Assessment the effect of anti-oxidants. Number of Person Concentration of Urinary 8 -OHd. G (250 healthy subjects) Patent No. 3091974 ; Patent No. 2850128 Prized ; Grand invention award (1999 Japan) ; Director-general of the science and technology agency premium (2000 Japan) Urinary 8 -OHd. G (ng/mg CRE)

Relationship between 8 -OHd. G and diseases (1) 1) Diabetes (8 -OHd. G in Relationship between 8 -OHd. G and diseases (1) 1) Diabetes (8 -OHd. G in urine) Diabetic patients(macro albuminuria): 19. 2 +/- 16. 8 ug/24 h (24 h urine) Normal controls : 8. 1 +/- 1. 8 ug/24 h [Reference] : Study of urinary 8 -hydroxydeoxyguanosine as a biomarker of oxidative DNA damage in diabetic nephropathy patients. J Pharm Biomed Anal. 2004 Sep 21; 36(1): 101 -4. Xu GW, et. al. 2) Cancer (8 -OHd. G in urine) Normal controls : 1. 11 +/- 0. 62 umol/mol CRE Cancer patients (before treatment): 1. 84 +/- 1. 12 umol/mol CRE After initiation of therapy 2. 18 +/- 1. 44 umol/mol CRE. Irradiation and chemical treatment result in higher 8 -OHd. G formation. [Reference] : Determination of urinary 8 -hydroxydeoxyguanosine by automated coupled-column high performance liquid chromatography: a powerful technique for assaying in vivo oxidative DNA damage in cancer patients. Eur J Cancer. 1995 Jun; 31 A(6): 934 -40. Tagesson C, et. al. 3) Dialysis (8 -OHd. G in serum) Serum 8 -OHd. G concentration from dialysis patient is higher than that from normal controls. [Reference] : Blood 8 -hydroxy-2'-deoxyguanosine is associated with erythropoietin resistance in haemodialysis patients. Nephrol Dial Transplant. 2003 May; 18(5): 931 -6. Kato A, Odamaki M, Hishida A.

Relationship between 8 -OHd. G and diseases (2) 4) Cystic fibrosis (8 -OHd. G Relationship between 8 -OHd. G and diseases (2) 4) Cystic fibrosis (8 -OHd. G in urine) Cystic fibrosis patients : 2. 78 +/- 1. 21 nmol/mol CRE Normal controls : 1. 51 +/- 0. 38 nmol/mol CRE [Reference]: Oxidative damage to DNA in patients with cystic fibrosis. Free Radic Biol Med. 1995 Apr; 18(4): 801 -6. Brown RK, et. al. 5) Alzheimer's disease (8 -OHd. G in tissue and cerebrospinal fluid) 1. 8 -OHd. G levels in the intact DNA from cerebrospinal fluid with AD (9 patients) is higher than normal controls. [Reference]: Increased DNA oxidation and decreased levels of repair products in Alzheimer's disease ventricular CSF. J Neurochem. 1999 Feb; 72(2): 771 -6.  Lovell MA, Gabbita SP, Markesbery WR.   2. 8 -OHd. G levels in brain tissue with AD became high. [Reference]: Oxidative alterations in Alzheimer's disease. Brain Pathol. 1999 Jan; 9(1): 133 -46. Markesbery WR, Carney JM. 6) Administration AZT to HIV Patients (8 -OHd. G in urine) HIV patients (Administration AZT) : 355 +/- 100 pmol/kg/day HIV patients (Avoid AZT) : 182 +/- 29 pmol/kg/day [Reference]: AZT treatment induces molecular and ultrastructural oxidative damage to muscle mitochondria. Prevention by antioxidant vitamins. J Clin Invest. 1998 Jul 1; 102(1): 4 -9. de la Asuncion JG, et. al.

Relationship between 8 -OHd. G and diseases (3) 7) Childhood Bacterial Meningitis (8 -OHd. Relationship between 8 -OHd. G and diseases (3) 7) Childhood Bacterial Meningitis (8 -OHd. G in cerebrospinal fluid) Bacterial meningitis patients’ cerebrospinal fluid 8 -OHd. G levels became high significantly. [Reference]: Oxidative stress in childhood meningitis: measurement of 8 -hydroxy-2'-deoxyguanosine concentration in cerebrospinal fluid. Redox Rep. 2000; 5(5): 295 -8. Tsukahara H, et. al. 8) Periodontitis (8 -OHd. G in Saliva) Untreated patients with periodontitis (78 patients) : 4. 28 +/- 0. 1 ng/m. L Normal controls (17 patients) : 1. 56 +/- 0. 1 ng/m. L [Reference]: New biomarker evidence of oxidative DNA damage in whole saliva from clinically healthy and periodontally diseased individuals. J Periodontol. 2002 May; 73(5): 551 -4. M Takane, et. al.

Chemical Industry Workers’ Urinary 8 -OHd. G levels became High. [Reference]: Increased urinary excretion Chemical Industry Workers’ Urinary 8 -OHd. G levels became High. [Reference]: Increased urinary excretion of the oxidative DNA adduct, 8 -hydroxydeoxyguanosine, as a possible early indicator of occupational cancer hazards in the asbestos, rubber, and azodye industries. Pol J Occup Med Environ Health, 1993, 6: 4, 357 -68

Oxidative stress by inhalation of asbestos [Reference] : Levels of 8 -hydroxy-2'-deoxyguanosine in DNA Oxidative stress by inhalation of asbestos [Reference] : Levels of 8 -hydroxy-2'-deoxyguanosine in DNA of white blood cells from workers highly exposed to asbestos in Germany. Mutat Res, 2000 Jul, 468: 2, 195 -202

Oxidative stress caused by benzene Benzene cause oxidative damage to DNA in peripheral blood Oxidative stress caused by benzene Benzene cause oxidative damage to DNA in peripheral blood cells. [Reference] : The study of DNA oxidative damage in benzene-exposed workers. Mutat Res, 1996 Oct, 370: 3 -4, 145 -50

Influence of psychological stress 8 -OHd. G in peripheral blood cells from women under Influence of psychological stress 8 -OHd. G in peripheral blood cells from women under higher psychological stress are higher. [Reference] : Relationships between perceived workload, stress and oxidative DNA damage. Int Arch Occup Environ Health 2001 Mar; 74(2): 153 -7

Sperm DNA from smokers [Reference] : Detection of oxidative DNA damage in human sperm Sperm DNA from smokers [Reference] : Detection of oxidative DNA damage in human sperm and the association with cigarette smoking. Reprod Toxicol, 1997 Sep, 11: 5, 675 -80

Vegetable juice(Green Juice) reduce urinary 8 -OHd. G Vegetable juice(Green Juice) reduce urinary 8 -OHd. G

Effect of lemon flavonoids to diabetic rats Effect of lemon flavonoids to diabetic rats

Oxidative Stress Markers DNA Oxidation 8 -OHd. G Thymidine Glycol Protein Oxidation Dibromo-Tyrosine Dithyrosine Oxidative Stress Markers DNA Oxidation 8 -OHd. G Thymidine Glycol Protein Oxidation Dibromo-Tyrosine Dithyrosine Lipid Oxidation Acrolein Crotonaldehyde Hexanoyl-Lysine 4 -hydroxy-2 -hexenal 4 -hydroxy-2 -nonenal 7 -ketocholesterol Malondialdehyde Methylglyoxal

Anti Hexanoyl-Lysine(HEL) Monoclonal Antibody & HEL ELISA kit Assessment of Oxidative Stress inside the Anti Hexanoyl-Lysine(HEL) Monoclonal Antibody & HEL ELISA kit Assessment of Oxidative Stress inside the body. a) Highly specific for HEL. b) Applications: urine, serum, tissue, cultured cells, saliva… c) Assay time: overnight and 2 hours d) Number of samples (N=3; 18 samples / kit) e) Risk management of health and aging. f) Monitoring the effect of chemicals, UV , radiation etc… g) Assessment the effect of anti-oxidants. What is HEXANOYL-LYSINE ?

Relationship between HEL and diseases 1) Dry eyes (HEL in cornea) [Reference] : Selenoprotein Relationship between HEL and diseases 1) Dry eyes (HEL in cornea) [Reference] : Selenoprotein p controls oxidative stress in cornea. PLo. S One. 2010 Mar 29; 5(3): e 9911, Higuchi et. al. 2) Cerebral Infarction (HEL in serum) [Reference] : Effect of prophylactically administered edaravone during antegrade cerebral perfusion in a canine model of old cerebral infarction. J Thorac Cardiovasc Surg. 2007 Mar; 133(3): 710 -6, Suzuki et. al. 3) Atherosclerosis (HEL in tissue) [Reference] : Immunohistochemical detection of oxidative stress biomarkers, dityrosine and N(epsilon)-(hexanoyl)lysine, and C-reactive protein in rabbit atherosclerotic lesions. J Atheroscler Thromb. 2008 Aug; 15(4): 185 -92, Fukuchi et. al.

Oxidative Stress Markers DNA Oxidation 8 -OHd. G Thymidine Glycol Protein Oxidation Dibromo-Tyrosine Dithyrosine Oxidative Stress Markers DNA Oxidation 8 -OHd. G Thymidine Glycol Protein Oxidation Dibromo-Tyrosine Dithyrosine Lipid Oxidation Acrolein Crotonaldehyde Hexanoyl-Lysine 4 -hydroxy-2 -hexenal 4 -hydroxy-2 -nonenal 7 -ketocholesterol Malondialdehyde Methylglyoxal

Anti 4 -hydroxy-2 -hexenal (4 -HNE) Monoclonal Antibody (HNEJ-2) Assessment of Oxidative Stress inside Anti 4 -hydroxy-2 -hexenal (4 -HNE) Monoclonal Antibody (HNEJ-2) Assessment of Oxidative Stress inside the body. Specifications a) Highly specific for 4 -HNE. b) Clone #: HNEJ-2 c) Immunogen: 4 -HNE-modified Keyhole Limpet Hemocyanin d) Subclass: Mouse Ig. G 1(kappa) e) Form: Lyophilized Powder (containing 20 ug or 100 ug of Ig. G) c) Applications: Immunohistochemistry and western blotting. Structure of 4 -hydroxy-2 -nonenal

Relationship between 4 -HNE and diseases 1) Sjögren's syndrome (4 -HNE in salivary gland) Relationship between 4 -HNE and diseases 1) Sjögren's syndrome (4 -HNE in salivary gland) [Reference]: Thioredoxin may exert a protective effect against tissue damage caused by oxidative stress in salivary glands of patients with Sjogren's syndrome, J Rheumatol. 2007 Oct; 34(10): 2035 -43, Kurimoto et. al. 2) Active liver disease (4 -HNE in liver tissue) [Reference]: M-30 and 4 HNE are sequestered in different aggresomes in the same hepatocytes. , Exp Mol Pathol. 2007 Dec; 83(3): 296 -300. , Amidi et. al. 3) Chronic obstructive pulmonary disease (COPD)(4 -HNE in lung tissue) [Reference]: Activation of nuclear factor-kappa. B in airway epithelial cells in patients with chronic obstructive pulmonary disease. , Respiration. 2006; 73(5): 610 -6, Yagi et. al.

Antioxidant Assay Antioxidant assay by Test kit for antioxidant (PAO) a) Applications: Human and Antioxidant Assay Antioxidant assay by Test kit for antioxidant (PAO) a) Applications: Human and animal serum samples, foods and beverage samples. b) Assay time: 3 minutes c) Number of samples (N=3; 26 samples / kit) d) Monitoring the effect of chemicals, UV , radiation etc… Principle of this assay:

Relationship between antioxidants and diseases 1) Alzheimer disease (Antioxidants in plasma) [Reference]: Oxidative imbalance Relationship between antioxidants and diseases 1) Alzheimer disease (Antioxidants in plasma) [Reference]: Oxidative imbalance and cathepsin D changes as peripheral blood biomarkers of Alzheimer disease: a pilot study. , FEBS Lett. 2005 May 23; 579(13): 2759 -66, Straface et. al. 2) Coronary artery disease (Antioxidants in plasma) [Reference]: Oxidative stress and its association with coronary artery disease and different atherogenic risk factors. , J Intern Med. 2004 Oct; 256(4): 308 -15, Vassalle et. al.

Conclusion 1) Oxidative stress markers involves in various diseases. 2) 8 -OHd. G, HEL, Conclusion 1) Oxidative stress markers involves in various diseases. 2) 8 -OHd. G, HEL, 4 -HNE are detected in various biological samples such as urine, serum, tissue, cerebrospinal fluid, saliva and cultured cells. 3) For antioxidant assay, PAO is useful. PAO is applicable for serum, foods and beverage samples. 4) Oxidative stress markers and antioxidant assay may be useful for pathological analysis, development of medicines, anti-aging research, or evaluation of functional foods.

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