metodi enzimatici in diagnostica clinica

Metodi enzimatici in diagnostica clinica

Vantaggi-veloce-molto specifico

Metodi di- Misura in continuo (più facili e più utili)- Misura a tempo fisso

Influenze- pH- temperatura-forza ionica

Enzymatic reaction-based assays

Michaelis-Menten enzyme kinetics

Km

A B CEprim

X Y

Spesso non è possibile un singolo enzima-reazione sequenziale

Eind

t ~ 0 [B] << KmB, vind = Vind.[B]/Km

B - 1st order reaction

t > 0 k.[Eprim] = Vind

vind = Vind.[B]/(KmB + [B]) = Vind/(Km

B/[B] + 1)

[B] 100.KmB - error 1%

It is necessary to use 10 to 100-fold excess of Eind over Eprim!!!

N+

- -N N

NNC

O

NH2

CH2 O P P O

O

O

O

O O

O

OH OH

CH2

O

OH HO

NH2

+

N

- -N N

NN

H+

C

O

NH2

CH2 O P P O

O

O

O

O O

O

OH OH

CH2

O

OH HO

NH2

redox+ 2 [H]- 2 [H]

H

HH

NAD(P)H assay - Wartburg optical test

A + NADH + H+ B + NAD+

Tutte le deidrogenasi NAD o NADP dipendenti utilizzano queso metodo

Biochemistry 2/e - Garrett & Grisham

Copyright © 1999 by Harcourt Brace & Company

Test ottico accoppiato

A + B C + D

C (oD) +NADH E + NAD+

E’ possibile accoppiare una terza reazione



Calcolo attività enzimatica

• 1 UI =1 micromole di substrato trasformato/min = c / min

• Attività enziamtica (mUI / ml) =

A V 1000

min v d

A = variazione assorbimento a 3401000 = fattore di conversione alle mUIV = volume totale della miscela di reazione = coeff. Estinzione molare di 1 mole/ml di NADH a 340

6.22 cm2/molev = volume del campione (ml)d = cammino ottico (1cm)

NH4+

COO-

C O

CH2

CH2

COO-

COO-

CH NH2

CH2

CH2

COO-

+ NADH + NH4+ + NAD+ + H2O

2-OXOGLUTARATE

GLUTAMATE DEHYDROGENASE

L-GLUTAMATE

CO2 COO-

C O

CH2

P

COO-

C O

CH2

COO-

COO-

C O

CH2

COO-

COO-

CH OH

CH2

COO-

+ CO2 + OH-

PHOSPHOENOLPYRUVATE OXALACETATE

~

PHOSPHOENOLPYRUVATECARBOXYLASE

OXALACETATE

+ NADH + H+

L-MALATE

MALATE DEHYDROGENASE+ NAD+

esempi

NO3- + NADPH + H+ NO2

- + NADP+ + H2O

H2O2 + LEUCODYE DYE + H2O

SO32- + O2 + H2O SO4

2- + H2O2

H2O2 + NADH + H+ 2 H2O + NAD+

NITRATE REDUCTASE

PEROXIDASE

SULFITE OXIDASE

NADH-PEROXIDASE

NO3-

H2 O2

SO32-

Ethanol, Acetaldehyde

ETHANOL + NAD+ACETALDEHYDE + NADH + H+

ACETALDEHYDE + NAD+ + H2O ACETIC ACID + NADH + H+ALDEHYDE DEHYDROGENASE

ALCOHOL DEHYDROGENASE

Acetate

CH2 COO-

COO-

COO-

HO

CH2

ACETATE + ATP + CoA ACETYL-CoA +

ACETYL-CoA + OXALACETATE + H2O

P P

+ CoA

MALATE + NAD+ OXALACETATE + NADH + H+

ACETYL-CoA SYNTHASE

CITRATE

MALATE DEHYDROGENASE

CITRATE SYNTHASE

N N

N

R3

R1 NH

R2

N N

NN

R3

R1 R2

X-

+

+ 2 H+

-2 H+

OO

OH OH

OO

O O

L- ASCORBATE

+ MTT

PMS

+ MTT- FORMAZANE + H+

MTT = 3 -(4,5 -DIMETHYLTHIAZOLYL-2)-2,5- DIPHENYLTETRAZOLIUM BROMIDE

PMS = 5- METHYLPHENAZINIUM BROMIDE

FORMAZANE

BLANK

L-ASCORBATE + O2 ASCORBATE OXIDASE

DEHYDROASCORBATE

+ HX

RED

+ H2O2

C

OH

H

CH2OH

O

O

O2

1

C

OH

H

CH2OH

Ascorbate

578 nm

CITRATE OXALACETATE + ACETATE

PYRUVATE + CO2

OXALACETATE + NADH + H+ L-MALATE + NAD+

PYRUVATE + NADH + H L-LACTATE + NAD+

CITRATE LYASE


L-LACTATE DEHYDROGENASE

L-MALATE + NAD+ OXALACETATE + NADH + H+

OXALACETATE + L-GLUTAMATE L-ASPARTATE + 2-OXOGLUTARATE


ASPARTATE AMINOTRANSFERASE

CH COO-

CH-OOCH2O

FUMARASEL-MALATE+

Citrate

Malate

Fumarate

OXALATEOXALATE DECARBOXYLASE

FORMIATE + CO2

FORMIATE + NAD+ +H2OFORMIATE DEHYDROGENASE

HCO32- + NADH + H+

Oxalate, Formiate

OXALATEOXALATE DECARBOXYLASE

FORMIATE + CO2

FORMIATE + NAD+ +H2OFORMIATE DEHYDROGENASE

HCO32- + NADH + H+

Succinate

Lactate

L-LACTATE + NAD+ PYRUVATE + NADH + H+

PYRUVATE + L-GLUTAMATE L-ALANINE + 2-OXOGLUTARATE

LACTATE DEHYDROGENASE

ALANINE AMINOTRANSFERASE

Pyruvate

PYRUVATE + NADH + H+ L-LACTATE + NAD+L-LACTATE DEHYDROGENASE

2-Oxoglutarate

L-Glutamate

L-Glutamine

2-OXOGLUTARATE + NADH + NH4+

GLUTAMATE DEHYDROGENASE L-GLUTAMATE + NAD+ + H2O

L-GLUTAMINE + H2O GLUTAMINASE

L-GLUTAMATE + NH3

L-GLUTAMATE + NAD+ +H2O 2-OXOGLUTARATE + NADH + NH4+

NADH + IODONITROTETRAZOLIUM CHLORIDE + H+DIAPHORASE

NAD+ + FORMAZAN DYE

GLUTAMATE DEHYDROGENASE

CH2 OH

O

CH2 O-P

O

CH2 O-P

O

O

+ ATP HEXOKINASE+ ADP

-D-GLUCOSE -D-GLUCOSE-6-PHOSPHATE

+ NADP+

GLUCONOLACTON-6-PHOSPHATE

+ NADPH + H+-D-GLUCOSE-6-PHOSPHATE

-D-GLUCOSE-6-PHOSPHATEDEHYDROGENASE

Glucose

SUCROSE + H2OSACCHARASE

-D-GLUCOSE + -D-FRUCTOSE

-D-FRUCTOSE + ATPHEXOKINASE

-D-FRUCTOSE-6-PHOSPHATE + ADP

-D-FRUCTOSE-6-PHOSPHATEPHOSPHOGLUCO ISOMERASE

-D-GLUCOSE-6-PHOSPHATE

Fructose

Sucrose

+ H2O + O2 GLUCOSE OXIDASE

GLUCONOLACTON + H2O2-D-GLUCOSE

CH2

CH

CH2 OH

OH

OHGLYCEROL KINASE

L-GLYCEROL-3-PHOSPHATE + ADP

ADP + PHOSPHOENOLPYRUVATEPYRUVATE KINASE

PYRUVATE + ATP

PYRUVATE + NADH + H+L-LACTATE DEHYDROGENASE

L-LACTATE + NAD+

+ ATP

Glycerol

TRIACYLGLYCEROLS + 3 H2OLIPASE, ESTERASE

GLYCEROL + 3x FATTY ACID

Triacylglycerols

Free fatty acids

FATTY ACID + CoA + ATPACYL-CoA SYNTHASE

AMP + ACYLCoA + P-P

ACYL-CoA + O2ACYL-CoA OXIDASE

ENOYL-CoA + H2O2

H2O2 + 4-AMINOANTIPYRINE + 2,4,6-TRIBROMO-3-HYDROXYBENZOIC ACID

RED DYE + 2 H2O + HBrPEROXIDASE

546 nm

Cholesterol

Bile acids

OH

CHOLESTEROL + O2 4-CHOLESTENON + H2O2

METHANOL + H2O2CATALASE FORMALDEHYDE + 2 H2O

FORMALDEHYDE + NH4+ + 2 ACETYLACETONE 3,5-DIACETYL-1,4-DIHYDROLUTIDINE 3 H2O+

CHOLESTEROL OXIDASE

405 nm

Urea

3--HYDROXY-BILE ACID + NAD+

3--HYDROXYSTEROID DEHYDROGENASE

3-OXO-BILE ACID + NADH + H+

NADH + H+ + NITROBLUE TETRAZOLIUM CHLORIDEDIAPHORASE

NAD+ + FORMAZAN

576 nm

UREA + H2OUREASE

2 NH3 + CO2

2-OXOGLUTARATE + NADH + NH4+

GLUTAMATE DEHYDROGENASEL-GLUTAMATE + NAD+ + H2O

AMP + ATPMYOKINASE

2ADP

ADP + PHOSPHOENOLPYRUVATEPYRUVATE KINASE

L-LACTATE + NAD+PYRUVATE + NADH + H+ L-LACTATE DEHYDROGENASE

ATP + PYRUVATE

AMP/ADP

ATP

GLYCEROL-3-PHOSPHATE + ATP

3-PHOSPHOGLYCERATEKINASE

GLYCERATE-1,3-DIPHOSPHATE + ADP

GLYCERATE-1,3-DIPHOSPHATE + NADH + H+

GLYCERALDEHYDE-3-PHOSPHATEDEHYDROGENASE

GLYCERALDEHYDE-3-PHOSPHATE+ NAD+ + Pi

GLYCERALDEHYDE-3-PHOSPHATETRIOSE PHOSPHATE

ISOMERASEDIHYDROXYACETONE-3-PHOSPHATE

DIHYDROXYACETONE-3-PHOSPHATE + NADH + H+ GLYCEROL-3-PHOSPHATE

DEHYDROGENASE GLYCEROL-3-PHOSPHATE + NAD+

NAD(P)H + H+ + FMN FMN-REDUCTASE NAD(P)+ + FMNH2

LUCIFERASEBACTERIAL

FMN + RCOOH +

FMNH2 + RCHO + O2

NAD(P)H - bioluminescence

ATP - bioluminescence

LUCIFERASEFIREFLY OXYLUCIFERIN + 2 Pi + AMP + CO2 + ATP + D-LUCIFERIN + O2

h. (493 nm)

h. (562 nm)

Metodi in luminescenza



Diagnostica nell’ischemia cardiaca

L-MALATE + NAD+ OXALACETATE + NADH + H+

OXALACETATE + L-GLUTAMATE L-ASPARTATE + 2-OXOGLUTARATE


ASPARTATE AMINOTRANSFERASE

Transaminasi glutamico ossalacetica (GOT)

GOT aumenta dopo 6 ore l’infarto

max a 36 ore

livelli normali 5gg



• Creatina chinasiCH2 OH

O

CH2 O-P

O

CH2 O-P

O

O

+ ATP HEXOKINASE+ ADP

-D-GLUCOSE -D-GLUCOSE-6-PHOSPHATE

+ NADP+

GLUCONOLACTON-6-PHOSPHATE

+ NADPH + H+-D-GLUCOSE-6-PHOSPHATE

-D-GLUCOSE-6-PHOSPHATEDEHYDROGENASE

Fosfocreatina + ADP creatina + ATP

Aumenta dopo 3 ore dall’infarto max 24-30 h

livelli normali 3gg

Creatina fosfato



L-LACTATE + NAD+ PYRUVATE + NADH + H+

PYRUVATE + L-GLUTAMATE L-ALANINE + 2-OXOGLUTARATE

LACTATE DEHYDROGENASE

ALANINE AMINOTRANSFERASE

Lattico deidrogenasi

Aumento entro 2 ore max 60 ore

ritorno alla norma lento

Troponine cardiache:molto specifiche per il cuore



Dosaggio del substrato• 10 ml di NADH 20 mM (Ci) vengono messi in 2 ml di soluzione tampone

(CfNADH = 100 µM)• 10 ml di piruvato prelevati dal liquido biologico (siero)• 10 ml di lattico deidrogenasi

• Piruvato + NADH + H+ lattato + NAD+

• Sapendo che l’e molare di NADH è 6,22 mM-1 cm-1• 0,1 mM di NADH avrà un assorbanza di 0,622 (Ai)• Nella lettura Af ottengo: 0,322 A = lC A = 0,622 – 0,322 = 0.3 C = A/l = 0.3 / 6,22 mM-1 cm-1 x cm = 0,48 mM = 48 mM• Le micromoli di piruvato nei 2 ml della cuvetta saranno uguali a• 48 mM x 2 ml / 1000 = 0, 096 mmoli• [piruvato] = 0,096 / 10 ml (volume usato) = 9,6 mM

(concentrazione del piruvato)•

metodi enzimatici in diagnostica clinica

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