biochemistry lesson v3

© 2008 HORIBA ABX, All rights reserved.

BIOCHEMISTRY


HORIBA ABXTraining Center

2008


Biochemistry = Chemistry of life

Introduction

Biochemistry is a scientific discipline which explore, in human being, chemical reactions allowing the maintenance of the living status.


WaterGlucidsLipidsProteinsMineral saltOthers

Substrates

Specific Proteins

Enzymes

Ions

Medecine

DrugsTDM

DAT/DAU

CLINICAL

BIOCHEMISTRY

Introduction


Substrate :

Enzyme : Base of Biochemistry

Biochemistry principle

Introduction


Substrate : Molecule or substance which undergo or take action in chemical reaction. After reaction this substrate give a product.

Enzyme : Protein substance,which catalyse chemical reaction. The action of enzyme is substrate specific and action specific (... ase)

.

Specific Protein : Protein with immunogenic properties (characteristics). They can be selectively isolated by

Immunoturbidimetry (antibody)

DAT / TDM : Drug of Abuse Testing. &Therapeutic Drug Monitoring(for medicine or drug tests).

Ions : Electrolytes, mineral compound in biological liquid

Introduction


FOOD

Waste• Liquid Puncture

Organism

• Blood

• Urine

Biological liquids


BLOOD : StructureLiquid element 60%Cell part 40% (Ht)

Biological liquids


Blood : liquid element With or without anticoagulant

Serum

Plasma

Clot (Fibrin + thrombin + coagulation factors)

Ht

Biological liquids

30 min


Urinequalitative

Urine in 24 h (1day) quantitative & qualitativeadditives

Biological liquids


Liquid puncture

CSF CerebroSpinal FluidGlucose status

Others punctures : Knee puncture (synovial)

Uric acid …

Biological liquids


Five type of testsSubstrates

Enzymes

Specific Proteins

Ions

Drug testing

Different techniques

Techniques


Potentiometry (measurement of voltage)

Colorimetry (measurement of absorbance)

Turbidimetry (measurement of level of opacity, cloudy)

Techniques


Techniques

Potentiometry

+

Voltagemeasurement

ElectrodeReference Selective electrode

(for the ion measured) Selective Membrane Electrolyte

(known concentration)

+

Voltagemeasurement




+

+

++

+

++

+

+ +

++

Voltagemeasurement




++

++

++++

++

++++

++

++ ++


Voltage measurement between a selective electrode and a reference electrode.

The intensity is proportional to the selected ion (by the selective electrode : Na, K, Cl,...).

Potentiometry

Techniques


Potentiometry

Techniques


Techniques

Potentiometry


Colorimetry (with spectrophotometer)

Cuvette with reactional mix

Io (initial intensity)

It (intensity after cuvette)

L

Detector

Techniques


T = It / I0 (Transmittance)A = OD = log(1/T) = log(I0/It) (Absorbance)

Each molecule have a specific coefficient of molecular absorption for one wavelength

Beer-Lambert law : OD = .C.L L = length or path of light

C = compound concentration

Colorimetry (with spectrophotometer)

Techniques


Turbidimetry (immunoturbidimetry)Specific protein

recognisedProteins

Specific antibody

Techniques


Antibody coated toLatex beads

Antibody/Antigene complex

(network)

Turbidimetry Latex (immunoturbidimetry)

Techniques


photometry of cloudy solution (Rayleigh law) : I0 incident = I absorb + I transmit + I diffuse

(proportional to the amount of specific protein recognised)

Turbidimetry (immunoturbidimetry)

Techniques


Substrates

Enzymes

Specific Proteins

Ions

Medicine Test

TurbidimetryColorimetryPotentiometry

Techniques


Sample(Substrate, enzyme,…)

Technical Result (OD, …)Calculation mode

Technique

Calculation mode


Method with final point = endpointex : Cs, TG, uric acid

ODODffinal

ODiinitial Time

OD in end of reaction Substrate

concentration

OD measured = OD final- OD initial

Calculation mode


OD

Time

OD

T

Kinetic with fixed time = kinetic ΔOD/min measured is proportional concentration (substrate) or enzyme activity (enzyme)

ex : urea, creat., glu GDH

Calculation mode


OD

Time

OD

T

Kinetic with research of linear zone = kinsearchMeasure of slope (ΔOD/min) of linear zone (at leats 5 points in the same straight),

proportionality with concentration (substrate), or enzymatic activity (enzyme)

Calculation mode


Final Result(concentration, enzymatic activity)

Calibration


Technical Result (OD, …)Calculation Mode

Technique

Calibration


Aim : give a relation between the signal/result measured (Absorbance) and the concentration or the activity measured

Calibration curve : OD = f(Conc)

Calibrator = standard serum or solution with known concentration substance

Calibrator mono & multiparametric

Calibrator alone (for linear calibration)Multi Calibrator (non linear Calibration, algorithm)

CalibrationCalibration


Different types of calibration :

LinearNon linear

Enzyme Case (for Mira)

Calibration


OD

Concentration

Linear

F = Conc(known)/ OD Measured

Conc(known)

OD Measured

Calibration


Linear1- Slope average mode (Slop Avg)ODmeasured using the calibrator allow to calculate the calibration factor :

F = Conc(known)/OD Measured

After for each Sample, you have :Conc = F X OD

2- Linear regression mode (lin reg)Conc = F X OD + Ro (Ro = offset)

Calibration


Non linear

The measurement is not proportional with the concentration or the activity

Calibration curve is given by different types (concentration) of calibrators

Ex. : LIN INTER ; LOGIT/LOG 4 ; LOGIT/LOG 5 ;

EXPONENT 5

N L

conc

sign

alCalibration


Enzymatic activity (Mira case) :The activity of each enzyme is known

you don’t need calibration, you know directly theFACTOR

Remark : F = Vol Total/(Vol Sample * d * )d = optical path ( 0,6 cm) = coefficient of molecular absorptionThis factor is known for each method of analysis ( SFBC, DGKC, IFCC)

Enz Activity = OD/Time x F

Calibration


Final Result(concentration, enzymatic activity )

Calibration


Technical Result (OD, …)Calculation Mode

Technique

Control(checking : reagent, machine, …)

Control


Aim :ensure the validity of calibration curve and reagent

Used Material :Serum control mono or multiparametric1 or 2 level of control (Normal + Pathologic)

Realisation :Control tittered as a SampleTarget values with a confidence range

Obtained results :Measuring value = theoretical value : Cal OK. Run the patients

Measuring value theoretical value : Cal OUT Pb reagent Calibrator Pb Bad control Instrument failure

Control


Hepatic panelTotal Bilirubin, Direct Bilirubin, Transaminase ASAT & ALAT, ALP & GGT

Glucidic panelGlucose, HbA1c, Fructosamine, Micro Albumin

Lipidic panelCholesterol, HDL Chol, LDL Chol, Phospholipid, Trigly, Apo A1, Apo B.

Clinical biochemistry panel


Bone Calcium, Phosphorus, ALP

CardiacCK, ASAT, LDH, Myoglobin

Renal Urea, Creatinine, Uric Acid

Clinical biochemistry panel


Thank you

biochemistry lesson v3

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