Theory Photochem Anna Horszwald (Michalska)
Free radicals Free radicals inflamation mitochondrial disfuntion Lachance P. A. et al. (2001) Antioxidants an intergarative approach, Nutrition, 17,835-838.
HO hydroxyl radical HO 2 hydroperoxyl radical O - 2 superoxide radical anion NO nitric oxide NO 2 nitric dioxide RO alkoxyl radical ROO peroxyl radical H 2 O 2 hydrogen peroxide 1 O 2 singlet oxygen HOCl hypochlorous acid O 3 ozone * Highly reactive species capable of independent existence containing one or more unpaired electrons Halliwell, 1987
Pathogenesis can be avoided by: optimal tissue levels of antioxidants and free radical scavengers improvement of dietary practices avoiding smoking, environmental pollutants and xenobiotics
Antioxidant [Halliwell, 1995] Any substance that, when present at low concentration compared with those of oxidazable substrate, significantly delays or prevents oxidation of that substrate
Determintion of antioxidants 1) Selective determination of single antioxidant (vitamin C, E, carotenoids, uric acis, bilirubin) 2) Antioxidant capacity (or efficiency, power, potential and activity) chemical reactivity under the specific conditions applied in the assay
The measuremet system for the antioxidative capacity consists of: - generator of free radicals - their detector, which allows the visualisation of the generated species and indicates the variation of the measured signal in the presence of antioxidative substances
Systems generating free radicals
In vitro Antioxidant Capacity Assays Assays involving hydrogen atom transfer reactions ROO + AH ROOH + A ROO + LH ROOH + L ORAC (Oxygen Radical Absorbance Capacity) TRAP Crocin bleaching assay Assays by electron-transfer reaction M(n) + e (from AH) AH + M (n-1) TEAC (Trolox Equivalent Antioxidant Capacity) FRAP (Ferric Ion Reducing Antioxidant Parameter) DPPH Total phenols assays by Folin-Ciocalteu reagent Other assays TOSC (Total Oxidant Scavenging Capacity) Chemiluminescence Elektrochemiluminescence Huang D., Ou B., Prior R. The chemistry behind antioxidant capacity assays, Journal of Agricultural and Food Chemistry, 53, 1841-1856
Choosing the method strongly depends on: task the nature of free radicals physico-chemical properties of the test system (polarity, ph value)
Chemiluminescence Physico-chemical process whereby energy from a chemical reaction is released directly as light with limited emission of heat. (A) + (B) ( ) (Products) + LIGHT where, (A) and (B) reactants ( ) excited intermediate
Example (A) + (B) ( ) (Products) + LIGHT Dye + H 2 O 2 3-APA ( ) 3-APA + LIGHT where, dye is luminol 3-APA is 3-aminophthalate 3-APA ( ) is the excited state fluorescing as it decays to a lower energy levels The decay of the excited state ( ) to a lower energy level is RESPONSIBLE FOR THE EMISSION OF LIGHT
The color of light depends upon the dye Colour Sensitizer Blue Green Yellow-green Yellow Orange Red 9,10-diphenylanthracene, 5-amino-2,3-dihydro-1,4-phthalazinedione 9,10-Bis(phenylethynyl)anthracene Tetracene 1-chloro-9,10-Bis(phenylethynyl)anthracene Rubrene Rhodamine B
Excitation of photosensitizer Optical Photosensitised chemiluminescence PCL method
Photosensitised chemiluminescence The combination of the simple and reliable photochemical generation of free radicals with their very sensitive chemiluminometric detection
www.analytik analytik-jena.de 1. First instrument available for examination of the antioxidant capacity of water-soluble and lipidsoluble substances in a single system 2. Measurement of the total antioxidative capacity of substance mixtures
www.analytik analytik-jena.de 3. Sampling, measurement and rinse cycles are performed automatically 4. Instruments standarised ready-touse kits for broad range of applications
Photosensitiser Luminol -C 8 H 7 N 3 O 2 5-amino-2,3-dihydro-1,4-phthalazinedione A versatile chemical that exhibit chemiluminescence, with a strinking blue glow, when mixed with appropriate oxidazing agent.
Luminol + hv1 L* 3 O 2 [L*O 2 ] L -* + O 2 -* hv1 - UV radiation necessary to excite the luminol L* -the luminol in a triplet state [L*O 2 ] the intermediate product of excited luminol and oxygen L -* -luminol radical O -* 2 -superoxide anion radical
O -* O -* 2 O -* 2 2 O 2 -* O -* 2 O -* 2 A A A A
O 2 -* O 2 -*
O 2 -* L -* O 2 -* L -* O 2 -* + L -*
where AP *2- is excited aminophthalate anion AP 2- is aminophthalate anion O 2 -* L -* O 2 -* L -* O 2 -* + L -* N 2 + AP *2- AP 2- + hv2 quantification by luminometric detection reaction
Photosensitiser Luminol -C 8 H 7 N 3 O 2 5-amino-2,3-dihydro-1,4-phthalazinedione Double role of luminol: 1)Photosensitizer 2)Oxygen radical detection agent
Parameters: 1. Sensitivity nanomolarconcentrations of non-enzymatic antioxidative substances 2. Very short measuring times < 3 min 3. Reproducibility, CV < 2% 4. Computer-controled operation 5. Curve evaluation with comfortable software 6. Comparability with conventional methods: a. good with spectrophotometry b. very good with HPLC
How to measure?
ACL ACW
Reagent 1 2 3 (WS) Standard Sample Blank 1500 1000 25 0 0 Calibration 1500 (x) 1000 25 X 0 Measurement 1500 (y) 1000 25 0 Y all volumes in µl
Antioxidant Capacity for Water Soluble Compounds (ACW) Measuring curves Lag time [A] Lag time [C] Voltage proportional to generated luminescence as a function of measuring time Prolongation of a lag phase of the radical detection reaction by standards [B]-[E] (Ascorbic Acid) relative to the blanks [A] [A] Blanks [B] 0.5 nmol Ascorbic Acid x2 [C] 1.0 nmol Ascorbic Acid x2 [D] 2.0 nmol Ascorbic Acid x2 [E] 3.0 nmol Ascorbic Acid x2 or TROLOX
Antioxidant Capacity for Water Soluble Compounds (ACW) assayed by means of the lag phase [L] in seconds L = Lag time [A] Lag time [C] [A] is blank [C] is sample
Antioxidant Capacity for Water Soluble Compounds (ACW) Calibration curve
Reagent 1 2 3 (WS) Standard Sample Blank 2300 200 25 0 0 Calibration 2300 (x) 200 25 X 0 Measurement 2300 (y) 200 25 0 Y all volumes in µl
Antioxidant Capacity for Lipid Soluble Compounds (ACL) Measuring curves The inhibition (reduction) of luminescence by standard [B]-[E] (TROLOX) relative to the blanks [A] [A] Blanks [B] 0.5 nmol Trolox x2 [C] 1.0 nmol Trolox x2 [D] 2.0 nmol Trolox x2 [E] 3.0 nmol Trolox x2
Antioxidant Capacity for Lipid Soluble Compounds (ACL) assayed by degree of PCL inhibition, calculated as: I = 1 S [A]/S [C] [A] integral under the blank curve [C] integral under the sampl curve
Antioxidant Capacity for Lipid Soluble Compounds (ACL) Calibration curve
Calculations
www.analytik analytikjena.de
Applications Food Medicine Pharmacy Chemistry Environmental medicine Cosmetics Biology Neuropathy Food quality, control of technological processing food production, improvement od the shelf life of products Clinical routine diagnosis, disturbance of antioxidative system Analysis od antioxidant activity of new drugs, antioxidant stabilization of pharmaceuticals Determination of the effects of additives Monitoring the antioxidative balance under the environmental noxea Improvement of antiradical properties of cosmetics and control of their long-term stability Examination of oxidative stress, improvement of the resistant to stress factors Effects of the alternative therapies, phytotherapies, oxygen treatment
Food Spice samples Natural plant extracts Concentrated fruit juice Food additives Potato cyto-juice Algae extracts Dried garlic Animal fats and oils Salami extracts Lipid extracts of pork sceletal muscles, animal tissue and salmon Meal sample from infant food Cheese Flours samples Baker yeast Applications Food quality, control of technological processing food production, improvement od the shelf life of products Wine Beer Coffee Green tea Heating oil Walnut oil sample Rapeseed oil Olive fruit extracts Macadamia nut oil Sunflower oil Vegetable oils treated with ozone
Applications Medicine Clinical routine diagnosis, disturbance of antioxidative system Ascorbic acid from blood plasma Blood plasma Serum Pharmacy Analysis od antioxidant activity of new drugs, antioxidant stabilization of pharmaceuticals Pharmaceutical samples Health pills
Applications Chemistry Determination of the effects of additives Fuel oil from round-robin test Biodiesel Technical oils Cosmetics Improvement of antiradical properties of cosmetics and control of their long-term stability Cosmetics products Cosmetics creams Supplements for cosmetics products Deodorant roll-on samples Perfums
Applications Environmental medicine Monitoring the antioxidative balance under the environmental noxea Biology Examination of oxidative stress, improvement of the resistant to stress factors
Examples
Capacity of water-soluble and lipid-soluble antioxidants in different vegetables
Detection of lipid-soluble antioxidants in different cooking oils
Urate-independent antioxidant capacity of blood plasma in patients with mammary tumors.
References: 1. Halliwell B., 1987, Oxidants and human disease: some new concepts. FASEB J., 1: 358 2. Halliwell B., 1995, Antioxidant characterization methodology and mechanism. Biochemical Pharmacology, Vol. 49 (10), 1341-1348. 3. Popov and Lewin, Photosensitized chemiluminescence, medical and industrial applications in the quantification of antioxidants and the assessment of the degree of oxidative damage. 4. Prior R., Wu X., Schaich K., 2005, Standarized methods for the determination of antioxidant capacity and phenolics in food and dietary supplements. Journal of Agricultural and Food Chemistry, 53 (10), 4290-4302. 5. Lachance P. A. et al.,2001, Antioxidants an intergarative approach, Nutrition, 17,835-838.