Enzymatic Standard Training

Transcription

1 Enzymatic Standard Training Introduction to Enzymatic testing Dr. Gilbert Garrido R-Biopharm AG An der neuen Bergstraße Darmstadt, Germany

2 Introduction to enzymatic testing 1. History 2. Basic principles 3. Tests realization 4. Tests kits and applications (2 examples) Citric acid Glucose/ Fructose 5. Trouble shooting 6. Photometer linearity 2

3 1. History 3

4 Definition The enzymatic analysis is the determination of intermediate catabolic products via enzymes, in other words the application of enzymes as reagents Ready-to-use test kits Analysis of : Sugars (Carbohydrate) Acids Alcohols Others 4

5 History (1) 1954 Enzymatic analysis started with routine application in clinicalchemistry labs 1970 Enzymatic methods were introduced in the Food analysis 1975 Boehringer Mannheim offered the first Test-kits combinations on the market for food & feed analysis 5

6 Enzymatic methods in laws and procedures A Codex Alimentarius Austriacus B Moniteur Belge-Belgisch Staatsblad CH Schweizer. Lebensmittelbuch D Lebensmittel-Gesetz E Boletin Oficial del Estado EU Commission Regulation I Gazzetta Ufficiale della Repubblica Italiana NL Warenwet S Statens Livsmedelsverk SF Methods Register of the State Technical Centre in Finland USA AOAC approval 6

7 Standardized reference methods B Norme Belge Belgische Norm NBN D Deutsches Institut für Normung DIN F Norme Française NF NL Nederlandse Norm NEN GB British Pharmacopoeia BP GUS Russian Standard GOST EU European standards EN ISO International Standards Organization 7

8 Instructions from international Organisations A.I.J.N. ASBC EBC ICUMSA IDF/FIL IFU IUPAC MEBAK NMKL OICCC OIV Association of the Industry of Juices and Nectars from Fruits and Vegetables of the European Economic Community American Society of Brewing Chemists European Brewery Convention International Commission for Uniform Methods of Sugar Analysis International Dairy Federation International Federation of Fruit Juice Producers International Union of Pure and Applied Chemistry Mitteleuropäische Brautechnische Analysen- Kommission Nordisk Metodikkommittée Office International du Cacao, du Chocolat et de la Confiserie Office International de la Vigne et du Vin 8

9 Summary The Roche (Boehringer Mannheim) methods are the official reference methods today They were tested thoroughly in ring trials Data were published They are recognised as reference methods by national and international organisations 31 kits are available and they cover the whole range of applications 9

10 2. Basic principles 10

11 Functions of enzymes in the body the most important proteins in humans the catalysts in the living cell ( times faster) catalyse a lot of reaction cycles, without consumption very specific 11

12 Principle of enzymatic tests A + B Dehydrogenase C + D Substrate Coenzyme + Product Coenzyme L-Lactic + NAD + L-LDH Pyruvate + NADH + H + The enzyme catalyses a specific reaction of a defined substrate The coenzyme is in general NAD(P), and acts as H + acceptor The sum of the NADH produced is equivalent to the amount of substrate in the sample 12

13 Enzyme and Coenzyme L-LDH L-Lactic acid (A) + NAD + (B) Pyruvat (C) + NADH (D) + H + H+ (with 2 e-) + + H+ (with 2 e-) Substrate (A) Coenzyme (B) Product (C) Coenzyme (D) 13

14 Q10- rule Room temperature + 10 C doubling of the reaction speed Room temperature - 10 C division of speed by factor two The enzymes have an optimal reaction temperature around 37 C (above this level the speed decreases again) 14

15 Nicotinamid The Coenzyme NAD or NADP N O O NH 2 O H O 2 C O P O O P O N CH 2 N O N N Adenin O OH OH O O Ribose Ribose Nicotinamid-adenin-dinucleotid (phosphate) Uptake of H+ after dehydrogenation of the substrate gives the reduced form NAD(P)H 15

16 Absorption of NADH and NAD+ Absorption (A) NADH NAD nm 340 nm Wavelength [nm] 16

17 Measurement principle Absorption (A) Measurement A2 R1 = Buffer A = A2 A1 (ggfs R1b / R1c / Wasser ) R2 = Enzyme = Start reagent Sampl e Measurement A1 Time 1 Time 2 Time 17

18 Reaction curve NAD NADH A 2 = 1,075 A 2 A 1 = 0,973 A 1 = 0,102 18

19 Reaction curve NADH NAD A1 = 1,72 A 1 -A 2 = 0,87 A2 = 0,85 19

20 General pipeting scheme Procedure Pipette into cuvettes Blank Standard Sample Buffer, salts Sample Standard Water 1,000 ml - - 2,000 ml 1,000 ml - 0,100 ml 1,900 ml 1,000 ml 0,100 ml - 1,900 ml Mix, read absorbances A1, then start the reaction with addition of: Start enzyme 0,020 ml 0,020 ml 0,020 ml After the completion of the reaction, read absorbances A2 20

21 Reagent blank (blank) Procedure Absorption (A) A 2 sample Sample A sample A 1 sample A 2 Blank A Blank A 1 Blank T =0 (Enzyme) Reagent Blank (water sample) Time A = (A 2 A 1 ) sample (A 2 A 1 ) Blank 21

22 Lambert-Beer law C = V x MW ε x d x v x 1000 x A C = concentration [g/l] V = final volume [ml] v = sample volume [ml] MW = molecular weight of the substance to be assayed [g/mol] d = light path [cm] ε = NADH extinction coefficient = 6.3 [l x mmol -1 x cm -1 ] at 340 nm Attention: when using higher sample volume change formula! 22

23 Linear calculation Example Glucose 3,020 x 180,16 C = ε x 1,00 x 0,100 x 1000 x Α C = F x A = Linear curve with factor F A 1 Linearity up to A = 1,0 C = 5,441 ε x Α A C = 0,863 x A (at 340 nm) 0.1 Concentration 23

24 Diluting or increasing the sample volume Procedure The Lambert-Beer law must be fulfilled A < 1,0 (A) The signal must be strong enough, so that the error in the measurement stays low: A > 0,1 (A) A > 1 dilute the sample A < 0,1 increase the sample volume 24

25 Chromogen tests Following Test-kits use chromogen reagents instead of coenzyme NAD(P) L-Ascorbic acid (MTT-Formazan, 578 nm) Cholesterol (Lutidine, 405 nm) L-Glutamic acid (Formazan, 492 nm) D-3-Hydroxybuttyic acid (Formazan, 492 nm) D-Sorbitol/Xylitol (Formazan, 492 nm) Attention: The chromogens are light-sensitive so the enzymatic reaction must be accomplished in the dark 25

26 3. Test realization Reagents Equipment Sample preparation Procedure 26

27 Kit content Reagents Test-kits contain all necessary reagents Enzymes and Coenzymes (highly purified) Buffer solutions Salts (sometimes required as activator) most of the test-kits contain a test control Do not shake when adding water to the reagents! Gently swirling and resting for a few minutes is sufficient. 27

28 Quality control Procedure At least one control must be tested in every run They are present in most of the test kits, except in some cases where they are unstable so they must be bought separately like: Acetaldehyde (extremely volatile) Ascorbic acid and sulfite (oxidation through O 2 ) They are set at a low concentration level (where measurement is critical) The control recovery should be 100 ± 5 % If this result is not achieved, the whole test procedure must be checked It is not allowed to recalculate the sample results with the deviation of the control (e.g %)! 28

29 Equipment Equipment Enzymatic test-kits Photometer Cuvettes (disposable) and cuvette holder Spatulas for reagent mixing Pipettes and pipette tips Distilled water Dispenser for 1 2 ml Important: check regularly the quality of your equipment 29

30 Methods of sample preparation Samples General instructions under points 9. and 10. in the insert: Crushing and Homogenisation Extraction with water and other extraction media Dilution (this can be used in order to escape other steps) Filtration or centrifugation Deproteinisation (e.g. perchloric acid method) Carrez reaction De-fating Neutralisation Decolorization (only by strong color with E1 > 0,5) Many methods are described but : use only the one relevant for the samples tested! The less steps as possible! 30

31 Restart and internal control Procedure Restart After the end of the reaction, the control is added to the rube and the reaction restarts A3 A2 is measured, and the control must be recovered (g/l ± 5%) Internal control The control is tested in the same tube than the sample The recovery of OD s is calculated and checked The two methods are different form a normal quality control: These controls are mixed with a single sample where some doubts on possible interferences exist They allow to detect inhibiting substances in this single sample, unlike the QC which is a control for the run in general 31

32 Internal control with half-volume Procedure Pipette into cuvettes Blank Standard Sample Sample + IC Buffer, salts Sample Standard Water 1,000 ml - - 2,000 ml 1,000 ml - 0,100 ml 1,900 ml 1,000 ml 0,100 ml - 1,900 ml 1,000 ml 0,050 ml 0,050 ml 1,900 ml Mix, read absorbances A1, then start the reaction with addition of: Start enzyme 0,020 ml 0,020 ml 0,020 ml 0,020 ml After the completion of the reaction, read absorbances A2 Recovery = 2 x E Sample+ Standard E Sample X 100 (%) E Standard The calculation is also valid with the results in g/l 32

33 4. Test kits and applications Citric acid Glucose/Fructose 33

34 Pipeting scheme Citric acid Pipette into cuvettes Blank Control Sample Sample (sensitive) Solution 1 (buffer) ml ml ml ml Sample ml ml Control ml - - Water ml ml ml - Mix, after 5 min. read absorbancies (A 1 ). Start reaction by addition of: Solution 2 (enzyme) ml ml ml ml Mix, after 5 min. read absorbencies of the solutions (A 2 ). 34

35 Measuring range (points 4. and 5. from the insert) Citric acid c = (V x MW x A) / (ε x d x v x 1000) in g/l c = x A (g/l) at 340 nm Sample volume (examples) Lower detection limit (Sensitivity) A = (x1) A = (x2) A = (x4) Measuring range (Linearity) A = (x1) A = (x10) 200 µl 2.3 mg/l 4.6 mg/l 9.2 mg/l 46 mg/l 460 mg/l ( 0,4 g/l = 80 µg /test) 1000 µl (Factor 5) 0.46 mg/l 0.92 mg/l 1.84 mg/l 9.2 mg/l 92 mg/l 2000 µl (Factor 10) 0.23 mg/l ( 0.25 mg/l) 0.46 mg/l ( 0.5 mg/l) (= 1 µg/test) 0.92 mg/l 4.6 mg/l 46 mg/l Sensitivity Lowest detection limit measuring range Upper Detection limit 35

36 Dilution table Citric acid Estimated amount of citric acid < 0, 4 g/l 0,4 4,0 g/l 4,0 40 g/l > 40 g/l Dilution with water Dilution factor F Measuring range from von 0,1 (E) to 1,0 (E) = factor 10, so the dilution steps are with factor 10 36

37 Pipeting scheme for Glucose-Fructose Gluc.-Fruc. Pipette into cuvettes Blank Sample solution ml ml sample solution* ml redist. water ml ml Mix, and read absorbances of the solutions (A 1 ) after approx. 3 min and start reaction by addition of: suspension ml ml Mix, wait for the end of the reaction (approx min), and read the absorbances of the solutions (A 2 ). If the reaction has not stopped after 15 min, continue to read the absorbances at 2 min intervals until the absorbances increase constantly over 2 min. Add suspension ml ml Mix, read absorbances of the solutions after min (A 3 ). 37

38 Reaction curve Glucose/ Fructose Gluc.-Fruc. E1 E 2 E 3 38

39 5. Trouble shooting 39

40 Trouble shooting (1) : general procedure 1 = Reagent production R-Biopharm is distributing the Roche Enzymatik since 10 years, and during this period of time it was never necessary to withdraw a lot from the market It can happen that a bottle is broken (which is easy to see), or very rarely that one vial is half empty or in bad state (oxydated reagent, etc.. ) 2 = Reagent transport, storage or reconstitution The kits can travel up tp 3 weeks by Room temperature (stress testing is done for every lot) They must be stored between 2 8 C They must be reconstituted with freshly bi-distilled water and stored again between 2 and 8 C 3 = Running Quality controls Recovery of controls musts be within ± 5%, if not it is necessary to check: Instrumentation (pipettes and photometer linearity) Test procedure (steps, volumes, incubation times) 4 = Sample type, sample preparation and general application 40

41 Trouble shooting (2): sample preparation The test procedure is for most of the kits easy, but on the opposite the sample preparation can be very tricky and difficult: The sample is not easy to handle: dried or hygroscopic, volatile, oxidated, etc.. The method chosen (e.g. Carrez method, perchloric acid), must be compatible with the chosen test The method can be very complicated (Starch) The methods for sample preparation are specific for industrial sector (milk, wine, fruit juices, meat, eggs, etc ). Recognized methods can be gathered by the colleagues and by industrial organizations 41

42 Trouble shooting (3) : test procedure Store kit at 2 8 C Water quality is important Bring test kit to room temperature before use Sample dilution according to the dilution table (refer to point 1 in test kit insert) Respect order when pipette ( from above to below ) Use assay control ( standards ) Respect recommended incubation times If possible, make a restart 42

43 Checking optical densities when NAD + NADH A1 0,3 (A2 ) Normal sample Coloured sample A 1 A A 2 < 0,3 > 0,1 > 0,3 < 1,0 < 1,0 OK (< 2,0) < 2,0 43

44 Checking optical densities when NADH NAD + A 1 1,6 ( A 2 ) A 1 1,6 because NADH is at a constant concentration whatever the kit and the lot Normal sample Coloured sample A 1 A A 2 1,6 > 0,1 > 1,6 < 2,2 < 1,0 > 0,6 < 1,6 < 2,0 44

45 Frequent errors Operator manipulation (e.g. dilution errors, quality of water, storage of reagents) Control solution (in case of self prepared solutions: purity, quality, ph) Calculation (used extinction coefficient, A calculated) Spectrophotometer (check wavelength, lamp, calibrate with NADH) Photometer only linear until 2 absorption units (A<2.0) Pipettes (check pipettes for accuracy) Recovery of control solutions = 100 +/- 5 % 45

46 4. The Photometer Checking the photometer linearity 46

47 Linearity of the photometers Photometer It is mandatory to work under the Lambert-Beer conditions, which means it is necessary to be in the linear range of the photometer and of the testreagents For all the reactions which depend on NAD+ or NADH, the absorbance by 340 nm must be propotional to the NADH concentration, which means that the photometer must be linear 47

48 Photometer linearity Photometer Photometer linear range E < 2,000 Absorption The absorbance increases with the NADH concentration Some photometer show no linearity in the upper range Concentration 48

49 Testing procedure Photometer Testing of the linearity takes place with 15 dilutions steps Reagents: content of bottle 1 from the test-kit Citric acid must be reconstituted with water according to package insert (N ) Pipete 2.0 ml bidest. water into a cuvette Measure absorbance A0, then add 0,100 ml NADH solution, mix and Measure absorbance A1, then add 0,100 ml NADH solution, mix and etc Measure absorbance A13, then add 0,100 ml NADH solution, mix and Measure absorbance A14, then add 0,100 ml NADH solution, mix and Measure absorbance A15 49

50 Calculation Photometer Schritt NADH Volumen (ml) Gesamt- Volumen NADH Verdünn. E , = , = , = , = , = , = , = , = , = , = , = , = , = , = , = , The NADH concentration is the x-axis The absoption A1 is the y-axis 50

51 Spectral bandwidth The spectral bandwidth is the wavelentgh interval where the measured intensity is 50% of the maximum intensity If the bandwidth is smaller, the light intensity will also be smaller so that measurement can become less precise 51

52 Example of results Photometer 52

53 Linearity with bandwidth 2 nm Photometer 53

54 Linearity with bandwidth 5 nm Photometer 54

55 Questions? Please! 55