enzymatic standard training
TRANSCRIPT
R-Biopharm AGAn der neuen Bergstraße 1764297 Darmstadt, Germany
Enzymatic Standard Training
Introduction to Enzymatic testing
Dr. Gilbert Garrido
Introduction to enzymatic testing
1. History
2. Basic principles
3. Tests realization4. Tests kits and applications (2 examples)
• Citric acid
• Glucose/ Fructose
5. Trouble shooting6. Photometer linearity
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1. History
3
Definition
The enzymatic analysis is the determination of intermediate catabolic products via enzymes , in other words the application of enzymes as reagents
Analysis of :– Sugars (Carbohydrate)
– Acids
– Alcohols– Others
Ready-to-use test kits
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History (1)
1954 Enzymatic analysis started with routine application in clinical-chemistry 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
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Enzymatic methods in laws and procedures
A Codex Alimentarius Austriacus
B Moniteur Belge-Belgisch Staatsblad
CH Schweizer. LebensmittelbuchD Lebensmittel-Gesetz
E Boletin Oficial del Estado
EU Commission Regulation I Gazzetta Ufficiale della Repubblica Italiana
NL Warenwet
S Statens LivsmedelsverkSF Methods Register of the State Technical Centre in Finland
USA AOAC approval
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Standardized reference methods
B Norme Belge – Belgische Norm NBN
D Deutsches Institut für Normung DIN
F Norme Française NFNL Nederlandse Norm NEN
GB British Pharmacopoeia BP
GUS Russian Standard GOSTEU European standards EN
ISO International Standards Organization
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Instructions from international Organisations
A.I.J.N. Association of the Industry of Juices and Nectars from Fruits and Vegetables of the European Economic Community
ASBC American Society of Brewing Chemists
EBC European Brewery ConventionICUMSA International Commission for Uniform Methods of Sugar
AnalysisIDF/FIL International Dairy Federation
IFU International Federation of Fruit Juice Producers
IUPAC International Union of Pure and Applied ChemistryMEBAK Mitteleuropäische Brautechnische Analysen- Kommission
NMKL Nordisk Metodikkommittée
OICCC Office International du Cacao, du Chocolat et de la ConfiserieOIV Office International de la Vigne et du Vin
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Summary
The Roche (Boehringer Mannheim) methods are the offi cial 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
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2. Basic principles
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Functions of enzymes in the body
the most important proteins in humans
the catalysts in the living cell (108 – 1011 times faster)
catalyse a lot of reaction cycles, without consumption
very specific
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Principle of enzymatic tests
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 a mount of substrate in the sample
Dehydrogenase
A
Substrate
+ B
Coenzyme+
C
Product
+ D
Coenzyme
L-Lactic + NAD+ Pyruvate + NADH + H+L-LDH
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Enzyme and Coenzyme
Substrate (A) Coenzyme (B)
H+ (with 2 e-)
Product (C) Coenzyme (D)
L-Lactic acid (A) + NAD+ (B) Pyruvat (C) + NADH (D) + H+
L-LDH
+ +
H+ (with 2 e-)
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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)
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The Coenzyme NAD or NADP
N
NH2
O
N
NN
NO O
O
CH2
O P
O
O
OH
P O
OH
CH2
O
OO
O
Ribose Ribose
Nic
otin
amid
Adenin
Nicotinamid-adenin-dinucleotid (phosphate)Uptake of H+ after dehydrogenation of the substrate gives thereduced form NAD(P)H
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Abs
orpt
ion
(A)
Wavelength [nm]340 nm
NADH
NAD+
260 nm
Absorption of NADH and NAD+
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Measurement principle
R1 = Buffer
(ggfs R1b / R1c / Wasser )
Time
R2 = Enzyme= Start reagent
Absorption (A)
Sample
Measurement A1
Measurement A2
Time 2Time 1
∆ A = A2 – A1
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Reaction curve NAD →→→→ NADH
A1 = 0,102
A2 = 1,075
A2 – A1 = 0,973
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Reaction curve NADH →→→→ NAD
A1-A2 = 0,87
A1 = 1,72
A2 = 0,85
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General pipeting scheme Procedure
Pipette intocuvettes
Blank Standard Sample
Buffer, saltsSampleStandardWater
1,000 ml--
2,000 ml
1,000 ml-
0,100 ml1,900 ml
1,000 ml0,100 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
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Reagent blank (blank)
Absorption (A)
T =0 (Enzyme)Time
A1 sample
A2 sample
Reagent Blank(water sample)
Sample
A2 Blank
A1 Blank
∆A sample
∆A Blank
∆∆∆∆A = (A 2 – A1)sample – (A2 – A1)Blank∆∆∆∆A = (A 2 – A1)sample – (A2 – A1)Blank
Procedure
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V x MW
ε x d x v x 1000C = 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!
Lambert-Beer law
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Linear calculation
3,020 x 180,16
εεεε x 1,00 x 0,100 x 1000C = x ∆ Α
5,441
εεεεC = x ∆ Α
Example Glucose
C = 0,863 x ∆A (at 340 nm)
Linearity up to ∆ A = 1,0
∆ A
Concentration
1
0.1
∆ A
C = F x ∆ A= Linear curve with factor F
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Diluting or increasing the sample volume
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
Procedure
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Following Test-kits use chromogen reagents instead o f 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 t he enzymaticreaction must be accomplished in the dark
Chromogen tests
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3. Test realization
�Reagents�Equipment
�Sample preparation
�Procedure
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Kit content
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.
Reagents
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Quality control
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 O2)
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 w ith the deviation of the control (e.g. + 14 %) !
Procedure
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Equipment
Enzymatic test-kits
Photometer
Cuvettes (disposable) and cuvette holderSpatulas for reagent mixing
Pipettes and pipette tips
Distilled waterDispenser for 1 – 2 ml
Important: check regularly the quality of your equipm ent
Equipment
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Methods of sample preparation
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!
Samples
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Restart and internal control
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
Procedure
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Internal control with half-volume
Recovery =2 x ∆ E Sample+ Standard – ∆ E Sample
X 100 (%)∆ E Standard
The calculation is also valid with the results in g/l
Pipette intocuvettes
Blank Standard SampleSample
+ IC
Buffer, saltsSampleStandardWater
1,000 ml--
2,000 ml
1,000 ml-
0,100 ml1,900 ml
1,000 ml0,100 ml
-1,900 ml
1,000 ml0,050 ml0,050 ml1,900 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
Procedure
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4. Test kits and applications
�Citric acid
�Glucose/Fructose
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Pipette intocuvettes
Blank Control Sample Sample(sensitive)
Solution 1 (buffer) 1.000 ml 1.000 ml 1.000 ml 1.000 ml
Sample - - 0.200 ml 2.000 ml
Control - 0.200 ml - -
Water 2.000 ml 1.800 ml 1.800 ml -
Mix, after 5 min. read absorbancies (A1). Start reaction by addition of:
Solution 2 (enzyme) 0.020 ml 0.020 ml 0.020 ml 0.020 ml
Mix, after 5 min. read absorbencies of the solutions (A2).
Pipeting scheme Citric acid
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Measuring range (points 4. and 5. from the insert)
Sample volume(examples)
Lower detection limit (Sensitivity) Measuring range (Linearity)
∆A = 0.005 (x1) ∆A = 0.010 (x2) ∆A = 0.020 (x4)∆A = 0.100
(x1)∆A = 1.000
(x10)
200 µl 2.3 mg/l 4.6 mg/l 9.2 mg/l 46 mg/l460 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
SensitivityLowest detection limit
measuring rangeUpper Detection limit
c = (V x MW x ∆ A) / (ε x d x v x 1000) in g/lc = 0.460 x ∆ A (g/l) at 340 nm
Citric acid
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Dilution table
Measuring range from von 0,1 (E) to 1,0 (E) = factor 10,so the dilution steps are with factor 10
Citric acid
Estimated amount of citric acid
Dilution with water
Dilution factor F
< 0, 4 g/l0,4 – 4,0 g/l4,0 – 40 g/l
> 40 g/l
-1 + 91 + 991 + 999
110
1001000
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Pipeting scheme for Glucose-Fructose Gluc.-Fruc.
Pipette into cuvettes Blank Samplesolution 1 1.000 ml 1.000 mlsample solution* - 0.100 mlredist. water 2.000 ml 1.900 mlMix, and read absorbances of the solutions (A1) after approx. 3 minand start reaction by addition of:suspension 2 0.020 ml 0.020 ml Mix, wait for the end of the reaction (approx. 10-15 min), andread the absorbances of the solutions (A2).If the reaction has not stopped after 15 min, continue to readthe absorbances at 2 min intervals until the absorbancesincrease constantly over 2 min. Addsuspension 3 0.020 ml 0.020 ml Mix, read absorbances of the solutions after 10-15 min (A3).
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Reaction curve Glucose/ Fructose Gluc.-Fruc.
E1 E 2 E 3
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5. Trouble shooting
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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
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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
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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
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Checking optical densities when NAD + ⇒⇒⇒⇒ NADH
A1 ≈≈≈≈ 0,3 (A2 ⇑⇑⇑⇑ )
A1 ∆ A A2
Normal sample
< 0,3 > 0,1
< 1,0
OK (< 2,0)
Colouredsample
> 0,3< 1,0
< 2,0
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Checking optical densities when NADH ⇒⇒⇒⇒ NAD+
A1 ≈≈≈≈ 1,6 ( A2 ⇓⇓⇓⇓ )
A1 ≈ 1,6 because NADH is at a constant concentration whatever the kit and the lot
A1 ∆ A A2
Normal sample
≈ 1,6 > 0,1
< 1,0
> 0,6 < 1,6
Colouredsample
> 1,6< 2,2
< 2,0
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Recovery of control solutions = 100 +/- 5 %
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)
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4. The Photometer
�Checking the photometer linearity
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Linearity of the photometers
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 test-reagents
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
Photometer
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Abs
orpt
ion
Concentration
The absorbance increases with the NADH concentration
Some photometer show no linearity in the upper range
Photometer linear range E < 2,000
Photometer linearity Photometer
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Testing procedure
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°10139076035)
– 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
Photometer
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Calculation
SchrittNADH
Volumen (ml)
Gesamt-Volumen
NADHVerdünn. E1
0 0 2.0 0,0000 0.2231 + 0.1 = 0.1 2.1 0,0476 0.4522 +0.1 = 0.2 2.2 0,0909 0.6593 +0.1 = 0.3 2.3 0,1304 0.8534 +0.1 = 0.4 2.4 0,1667 1.0285 +0.1 = 0.5 2.5 0,2000 1.1906 +0.1 = 0.6 2.6 0,2308 1.3367 +0.1 = 0.7 2.7 0,2593 1.4708 +0.1 = 0.8 2.8 0,2857 1.5909 +0.1 = 0.9 2.9 0,3103 1.70310 + 0.1 = 1.0 3.0 0,3333 1.80311 +0.1 = 1.1 3.1 0,3548 1.90212 +0.1 = 1.2 3.2 0,3750 1.99613 +0.1 = 1.3 3.3 0,3939 2.08614 +0.1 = 1.4 3.4 0,4118 2.16215 +0.1 = 1.5 3.5 0,4286 2.238
Photometer
• The NADH concentration is the x-axis
• The absoption A1 is the y-axis
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Spectral bandwidth
The spectral bandwidth is the wavelentgh interval where the measured intensity is 50% of the maximum intensityIf the bandwidth is smaller, the light intensity will also be smaller so that measurement can become less precise
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Example of results Photometer
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Linearity with bandwidth 2 nm Photometer
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Linearity with bandwidth 5 nm Photometer
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Questions? Please!
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