Metodi enzimatici in diagnostica clinica
Enzymatic reaction-based assays
Vantaggi
-veloce
-molto specifico
Metodi di
- Misura in continuo (più facili e più utili)
- Misura a tempo fisso
Influenze
- pH
- temperatura
-forza ionica
Michaelis-Menten enzyme kinetics
Km
Spesso non è possibile un singolo enzima-reazione sequenziale
Eprim
A
Eind
B
X
t ~ 0 [B] << KmB, vind = Vind.[B]/KmB
C
Y
-
1st order reaction
t > 0 k.[Eprim] = Vind
vind = Vind.[B]/(KmB + [B]) = Vind/(KmB/[B] + 1)
[B]  100.KmB - error 1%
It is necessary to use 10 to 100-fold excess of Eind over Eprim!!!
NAD(P)H assay - Wartburg optical test
A + NADH + H+
H
B + NAD+
O
C
+
NH2
N
NH2
O
N
CH2
O
P
O
O
N
O
O
CH2
OH
OH
ox
- 2 [H]
red
+ 2 [H]
H O
C
N
O
O
H
N
-
P
O
HO
OH
-
NH2
N
NH2
O
N
CH2
HO
OH
O
P
-
O
O
P
O
N
N
O
O
CH2
N
O
O
+ H
+
OH
OH
Tutte le deidrogenasi NAD o NADP dipendenti utilizzano queso metodo
Biochemistry 2/e - Garrett & Grisham
Test ottico accoppiato
A+B
C+D
C (oD) +NADH
E + NAD+
E’ possibile accoppiare una terza reazione
Biochemistry 2/e - Garrett & Grisham
Calcolo attività enzimatica
•
1 UI =1 micromole di substrato trasformato/min = c / min
DA V 1000
• Attività enziamtica (mUI / ml) =
min e v d
DA = variazione assorbimento a 340
1000 = fattore di conversione alle mUI
V = volume totale della miscela di reazione
e = coeff. Estinzione molare di 1 mmole/ml di NADH a 340
6.22 cm2/mmole
v = volume del campione (ml)
d = cammino ottico (1cm)
esempi
NH4+
COO-
COO-
C O
CH2
+ NADH + NH4+
CH2
GLUTAMATE DEHYDROGENASE
+ NAD+ + H2O
CH2
CH2
COO-
COO-
2-OXOGLUTARATE
CO2
HC NH2
L-GLUTAMATE
COOC~ O P
+ CO2 + OH-
PHOSPHOENOLPYRUVATE
CARBOXYLASE
COO-
PHOSPHOENOLPYRUVATE
OXALACETATE
COOCH2
COOOXALACETATE
C O
CH2
CH2
C O
COO-
MALATE DEHYDROGENASE
+ NADH + H+
COOHC OH
CH2
COOL-MALATE
+ NAD+
NO3NO3- + NADPH + H+
NITRATE REDUCTASE
NO2- + NADP+ + H2O
H2 O2
H2O2 + LEUCODYE
PEROXIDASE
DYE + H2O
SO32SO32- + O2 + H2O
H2O2 + NADH + H+
SULFITE OXIDASE
NADH-PEROXIDASE
SO42- + H2O2
2 H2O + NAD+
Ethanol, Acetaldehyde
ETHANOL + NAD+
ALCOHOL DEHYDROGENASE
ACETALDEHYDE + NADH + H+
ACETALDEHYDE + NAD+ + H2O
ALDEHYDE DEHYDROGENASE
ACETIC ACID + NADH + H+
Acetate
ACETYL-CoA SYNTHASE
ACETATE + ATP + CoA
ACETYL-CoA + P
CH2
ACETYL-CoA + OXALACETATE + H2O
CITRATE SYNTHASE
COOCOO-
HO
CH2
COO-
CITRATE
MALATE DEHYDROGENASE
MALATE + NAD+
P
OXALACETATE + NADH + H+
+ CoA
Ascorbate
1
O
L- ASCORBATE
OH
OH
PMS
O
O
CH2OH
C
OH OH
+ MTT
O
O
H
C
CH2OH
H
O O
+ MTT- FORMAZANE + H+
MTT = 3 -(4,5 -DIMETHYLTHIAZOLYL-2)-2,5- DIPHENYLTETRAZOLIUM BROMIDE
PMS = 5- METHYLPHENAZINIUM BROMIDE
FORMAZANE
N
N
R1
N
R2
N N R3
O
+
X-
O
2
+ 2 H+
-2 H+
R1
N
N
H
N
R2
R3
+ HX
RED
578 nm
BLANK
L-ASCORBATE + O2
ASCORBATE OXIDASE
DEHYDROASCORBATE
+ H2O2
PYRUVATE + CO2
Citrate
CITRATE LYASE
CITRATE
OXALACETATE + NADH + H+
PYRUVATE + NADH + H
OXALACETATE + ACETATE
MALATE DEHYDROGENASE
L-LACTATE DEHYDROGENASE
L-MALATE + NAD+
L-LACTATE + NAD+
Malate
MALATE DEHYDROGENASE
OXALACETATE + NADH + H+
L-MALATE + NAD+
OXALACETATE + L-GLUTAMATE
Fumarate
ASPARTATE AMINOTRANSFERASE
CH
-OOC
CH
COO+ H2O
L-ASPARTATE + 2-OXOGLUTARATE
FUMARASE
L-MALATE
Oxalate, Formiate
OXALATE DECARBOXYLASE
OXALATE
FORMIATE + NAD+ +H2O
FORMIATE + CO2
FORMIATE DEHYDROGENASE
HCO32- + NADH + H+
O
Succinate
H
N
N
N
N
CH2
COO+
CH2
P
CH2
O
P
P
COO-
SUCCINATE
SUCCINYL-CoA
SYNTHASE
SUCCINYL-CoA + IDP + Pi
+ CoA
ITP
OH OH
OXALATE DECARBOXYLASE
OXALATE
FORMIATE + NAD+ +H2O
FORMIATE + CO2
FORMIATE DEHYDROGENASECOOH
HCO32- + NADH + H+
IDP + PHOSPHOENOLPYRUVATE
PYRUVATE KINASE
O
C
+ ITP
CH3
PYRUVATE
L-LACTATE DEHYDROGENASE
PYRUVATE + NADH + H+
COOHO
H
CH3
L-LACTATE
+ NAD+
Lactate
LACTATE DEHYDROGENASE
PYRUVATE + NADH + H+
L-LACTATE + NAD+
PYRUVATE + L-GLUTAMATE
ALANINE AMINOTRANSFERASE
L-ALANINE + 2-OXOGLUTARATE
Pyruvate
PYRUVATE + NADH + H+
L-LACTATE DEHYDROGENASE
L-LACTATE + NAD+
2-Oxoglutarate
2-OXOGLUTARATE + NADH + NH4+
GLUTAMATE DEHYDROGENASE
L-GLUTAMATE + NAD+ + H2O
L-Glutamate
GLUTAMATE DEHYDROGENASE
L-GLUTAMATE +
NAD+
2-OXOGLUTARATE + NADH + NH4+
+H2O
NADH + IODONITROTETRAZOLIUM CHLORIDE +
H+
DIAPHORASE
NAD+ + FORMAZAN DYE
L-Glutamine
L-GLUTAMINE + H2O
GLUTAMINASE
L-GLUTAMATE + NH3
Glucose
-D-GLUCOSE
H2C
GLUCOSE OXIDASE
+ H2O + O2
OH
GLUCONOLACTON + H2O2
H2C O-P
O
+
O
HEXOKINASE
ATP
-D-GLUCOSE
+
ADP
-D-GLUCOSE-6-PHOSPHATE
-D-GLUCOSE-6-PHOSPHATE
DEHYDROGENASE
H2C O-P
-D-GLUCOSE-6-PHOSPHATE + NADP+
O
+
O + NADPH + H
GLUCONOLACTON-6-PHOSPHATE
Sucrose
SUCROSE + H2O
SACCHARASE
-D-GLUCOSE + -D-FRUCTOSE
Fructose
-D-FRUCTOSE + ATP
HEXOKINASE
-D-FRUCTOSE-6-PHOSPHATE
-D-FRUCTOSE-6-PHOSPHATE + ADP
PHOSPHOGLUCO ISOMERASE
-D-GLUCOSE-6-PHOSPHATE
Glycerol
H2C OH
HC OH + ATP
GLYCEROL KINASE
L-GLYCEROL-3-PHOSPHATE + ADP
H2C OH
PYRUVATE KINASE
PYRUVATE + ATP
ADP + PHOSPHOENOLPYRUVATE
PYRUVATE + NADH + H+
L-LACTATE DEHYDROGENASE
L-LACTATE + NAD+
Triacylglycerols
LIPASE, ESTERASE
GLYCEROL + 3x FATTY ACID
TRIACYLGLYCEROLS + 3 H2O
Free fatty acids
ACYL-CoA SYNTHASE
FATTY ACID + CoA + ATP
ACYL-CoA + O2
ACYL-CoA OXIDASE
AMP + ACYLCoA + P-P
ENOYL-CoA + H2O2
H2O2 + 4-AMINOANTIPYRINE +
2,4,6-TRIBROMO-3-HYDROXYBENZOIC ACID
PEROXIDASE
RED DYE + 2 H2O + HBr
546 nm
Cholesterol
HO
CHOLESTEROL + O2
METHANOL + H2O2
CHOLESTEROL OXIDASE
CATALASE
4-CHOLESTENON + H2O2
FORMALDEHYDE + 2 H2O
FORMALDEHYDE + NH4+ + 2 ACETYLACETONE
3,5-DIACETYL-1,4-DIHYDROLUTIDINE + 3 H2O
405 nm
Bile acids
3--HYDROXY-BILE ACID + NAD+
3--HYDROXYSTEROID
DEHYDROGENASE
NADH + H+ + NITROBLUE TETRAZOLIUM CHLORIDE
3-OXO-BILE ACID + NADH + H+
DIAPHORASE
NAD+ + FORMAZAN
576 nm
Urea
UREA + H2O
UREASE
2-OXOGLUTARATE + NADH + NH4+
2 NH3 + CO2
GLUTAMATE DEHYDROGENASE
L-GLUTAMATE + NAD+ + H2O
AMP/ADP
AMP + ATP
MYOKINASE
2ADP
ADP + PHOSPHOENOLPYRUVATE
PYRUVATE + NADH + H+
PYRUVATE KINASE
L-LACTATE DEHYDROGENASE
ATP + PYRUVATE
L-LACTATE + NAD+
ATP
GLYCEROL-3-PHOSPHATE + ATP
3-PHOSPHOGLYCERATE
KINASE
GLYCERATE-1,3-DIPHOSPHATE + NADH + H+
GLYCERALDEHYDE-3-PHOSPHATE
GLYCERATE-1,3-DIPHOSPHATE + ADP
GLYCERALDEHYDE-3-PHOSPHATE
DEHYDROGENASE
TRIOSE PHOSPHATE
ISOMERASE
DIHYDROXYACETONE-3-PHOSPHATE + NADH + H+
GLYCERALDEHYDE-3-PHOSPHATE
+ NAD+ + Pi
DIHYDROXYACETONE-3-PHOSPHATE
GLYCEROL-3-PHOSPHATE
DEHYDROGENASE
GLYCEROL-3-PHOSPHATE + NAD+
NAD(P)H - bioluminescence
NAD(P)H + H+ + FMN
FMNH2 + RCHO + O2
FMN-REDUCTASE
LUCIFERASE
BACTERIAL
NAD(P)+ + FMNH2
FMN + RCOOH +

h.
(493 nm)
Metodi in luminescenza
ATP - bioluminescence
ATP + D-LUCIFERIN + O2
LUCIFERASE
FIREFLY
OXYLUCIFERIN + 2 Pi + AMP + CO2 + 
h.
(562 nm)
Biochemistry 2/e - Garrett & Grisham
Diagnostica nell’ischemia
cardiaca
Transaminasi glutamico ossalacetica (GOT)
MALATE DEHYDROGENASE
OXALACETATE + NADH + H+
L-MALATE + NAD+
OXALACETATE + L-GLUTAMATE
ASPARTATE AMINOTRANSFERASE
L-ASPARTATE + 2-OXOGLUTARATE
GOT aumenta dopo 6 ore l’infarto
max a 36 ore
livelli normali 5gg
Biochemistry 2/e - Garrett & Grisham
Creatina fosfato
Fosfocreatina + ADP
H2C
creatina + ATP
OH
• Creatina chinasi
O
+
HEXOKINASE
ATP
-D-GLUCOSE
H2C O-P
O
+
ADP
-D-GLUCOSE-6-PHOSPHATE
-D-GLUCOSE-6-PHOSPHATE
DEHYDROGENASE
-D-GLUCOSE-6-PHOSPHATE + NADP+
H2C O-P
O
+
O + NADPH + H
GLUCONOLACTON-6-PHOSPHATE
Aumenta dopo 3 ore dall’infarto
max 24-30 h
livelli normali 3gg
Biochemistry 2/e - Garrett & Grisham
Lattico deidrogenasi
LACTATE DEHYDROGENASE
PYRUVATE + NADH + H+
L-LACTATE + NAD+
PYRUVATE + L-GLUTAMATE
ALANINE AMINOTRANSFERASE
Aumento entro 2 ore
L-ALANINE + 2-OXOGLUTARATE
max 60 ore
ritorno alla norma lento
Troponine cardiache:
molto specifiche per il cuore
Biochemistry 2/e - Garrett & Grisham
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+
•
•
•


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
DA = elDC
DA = 0,622 – 0,322 = 0.3
DC = DA/el = 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)
•
lattato + NAD+