Lab – ENZYMES CK Total 0.

04
CK-MB 0.04
Experiment:
14 – Aspartate Aminotransferase 1. Amount of reagent needed = 1mL
15 – Alanine Aminotransferase 2. Unit of measurement = U/L
16 – Acid Phosphatase 3. Temperature = 25 C, 30 C
17 – Alkaline Phosphatase 4. All EXCEPT ACP, Amylase, CK
18 – Lactate Dehydrogenase Total, and CK-MB prepare
19 – Y-Glutamyl Transferase Working Reagent:
20 – Amylase 2mL Substrate + 1 Bottle Buffer
21 – CK Total
22 – CK-MB 5. ACP:
Prepare Working Reagent:
Absorbance: (Reagent A)Total:
340nm 405nm Substrate + 15mL Buffer
 AST  ACP
 ALT  ALP (Reagent B) Non-prostatic:
 LD  GGT Substrate + 15mL Tartrate
 CK Total  Amylase (with buffer)
 CK-MB
6. Amylase: Not Specified 
7. CK Total:
Blanks Used: Prepare Working Reagent:
Air Blank Water Blank 1 vial Enzyme/Substrate + 3mL
All except Amylase Amylase Buffer/Substrate sol.

8. CK-MB
Buffers Used: Prepare Working Reagent:
Enzyme Buffer 1 vial Enzyme/Antibody + 3mL Buffer
AST TRIS buffer (pH 7.8) *Incubate for 5 mins at RT
ALT TRIS buffer (pH 7.5)
ACP Citrate buffer (pH 5.2) AST
 Serum glutamic-oxaloacetic
ALP Diethanolamine buffer
transaminase
(pH 7.5)
 EC 2.6.1.1
LD TRIS buffer (pH 7.4)
 Found in Heart, Liver, Muscle
GGT TRIS buffer (pH 8.25)
 Substrate: 2-oxoglutarate
Amylase MES buffer (pH 6.0) L-Aspartate
CK Total Imidazole buffer (pH 6.7)
CK-MB Imidazole buffer (pH 6.7) Prepare Working Reagent:
2mL Substrate + 1 bottle Buffer
Amount of Serum Sample:
Enzyme Serum (mL) Karmen Method
AST 0.2  Coupled enzyme kinetic reaction
ALT 0.2  Indicator enzyme: Malate
ACP 0.1 dehydrogenase
ALP 0.02  Measures change in absorbance of
LD 0.02 NADH -> NAD+
GGT 0.1
Amylase 0.02
  Read A1 after 1 minute and again
after 1, 2, and 3 minutes (every 1
minute after)
2-oxoglutarate -AST-> Oxaloacetate
-MD-> Malate + NAD+
Calculation:
C = [((A2-A1)+(A3-A2)+(A4-A3))/3] 952
ALT
 Serum glutamic-pyruvic
transaminase
 EC 2.6.1.2
 Found in Heart, Liver
 MOST Liver specific enzyme
 UNAFFECTED by hemolysis
 Substrate: 2-oxoglutarate
L-Aspartate
Prepare Working Reagent:
2mL Substrate + 1 bottle Buffer
Karmen Method
 Coupled enzyme kinetic reaction
 Indicator enzyme: Lactate
dehydrogenase
 Measures change in absorbance of
NADH -> NAD+
 Read A1 after 1 minute and again
after 1, 2, and 3 minutes (every 1
minute after)
2-oxoglutarate -ALT-> Pyruvate -LD->
Lactate + NAD+
Calculation:
C = [((A2-A1)+(A3-A2)+(A4-A3))/3] 952
ACP
 Useful for diagnosis of Prostatic
carcinoma
 Hydrolysis of Phosphomonoesters
 Found in Prostate, Erythrocyte,
Bone, Liver, Spleen, Kidney,
Platelets
 Inhibitor: Tartrate (Prostatic ACP)
 Substrate: a-1-Naphthyl phosphate
Fast red TR salt
Prepare Working Reagent:
(Reagent A)Total:
Substrate + 15mL Buffer
(Reagent B) Non-prostatic:
Substrate + 15 mL Tartrate solution
Method
a-1-Naphthyl phosphate –ACP-> a-1-
Naphthol + Phosphate
a-1-Naphthol + Fast Red TR salt -> azo
dye
 Measure A1 of Total and Inhibited
after 5 minutes and A2 after
another 5 minutes
»Calculation:
Total and Non-prostatic:
C = (A2 – A1) X 149
Prostatic:
C = Total – Non-prostatic
Notes: Copper inhibits Non-prostatic ACP
Rape Cases – 4 days in washing
ALP
 Hydrolysis of Phosphomonoesters
 Found in Intestine, Placenta, Bone,
Liver
 Activator: Mg2+
 Substrate: p-nitrophenyl phosphate
Prepare Working Reagent:
2mL Substrate + 1 bottle Buffer
Method
p-Nitrophenyl phosphate –ALP->
Phosphate + p-Nitrophenol
 Read A1 after 1 minute and again
after 1, 2, and 3 minutes (every 1
minute after)
Calculation:
C=
[((A2-A1)+(A3-A2)+(A4-A3))/3] 2757
Notes: More specific in Bones than ACP
Phenylalanine: Inhibit intestine and
placenta
Urea: Inhibit bone and liver ALP
Carcinoplacental: Regan & Nagao
Heat Stability:  P, I, B, L 
LD
 Oxidoreductase
 Highest in pernicious anemia and
hemolytic disorders
 Found in Heart, Liver, Muscle,
Kidney, Erythrocytes
 Substrate: Pyruvate
Prepare Working Reagent:
2mL Substrate + 1 bottle Buffer
Method
Pyruvate + NADH –LD-> Lactate + NAD+
 Measures change in absorbance of
NADH -> NAD+
 Read A1 after 1 minute and again
after 1, 2, and 3 minutes (every 1
minute after)
Calculation:
C=
[((A2-A1)+(A3-A2)+(A4-A3))/3] 8095
Notes: LD2 > LD1
AMI =flipped pattern (LD1 > LD2)
LD1-5: normal
LD 6: Alcohol dehydrogenase, bad
prognosis, arterisclerotic
cardio vascular failure
Unstable
GGT
 Liver
 First to rise upon alcohol intake
 Substrate:
L-Y-glutamyl-3-carboxy-4-
nitroanilide
Glycylglycine
Prepare Working Reagent:
2mL Substrate + 1 bottle Buffer
Method
L-Y-Glutamyl-3-carboxy-4-nitroanilide
-GGT-> L-Y-Glutamylglycylglycine
 GGT transfers Y-Glutamyl to
glycylglycine
 Read A1 after 1 minute and again
after 1, 2, and 3 minutes (every 1
minute after)
Calculation:
C=
[((A2-A1)+(A3-A2)+(A4-A3))/3] 1158
Notes: Deficiency = Drug-induced
hemolytic anemia
Increase = Megaloblastic anemia
AMYLASE
 Smallest enzyme
 Hydrolase (Starch and Glycogen)
 Digestion of Starch to produce
Dextrin, Maltose, and Glucose
 Found in Pancreas and Salivary
glands
 Dx of Acute pancreatitis and
Salivary gland lesion
 Does not require ancillary enzymes
 Activators: Ca2+, Cl-
 Substrate:2-chloro-4-nitrophenyl-
maltotrioside
Calculation:
C=
[((A2-A1)+(A3-A2)+(A4-A3))/3] 9864
Notes: Macroamylasemia = Amylase +
Immunoglobulin
Isoenzymes: P (urine) and S
(salivary)
 S-type inhibited by Wheat germ  Enzyme/Substrate:
lectin ADP
Creatine phosphate
CK NADP
 Important in the regeneration of HK
ATP G-6-PD
 ADP phosphorylated to ATP Antibody for M-subunit
 Found in Muscle, Heart, and Brain (goat)
 CK-BB is normally absent in the
blood because of blood brain Prepare Working Reagent:
barrier 1 vial Enzyme/Antibody + 3mL Buffer
*Incubate for 5 mins at RT
CK Total
 Buffer/Substrate: Method:
Glucose Creatine phosphate +ADP –CK-> Creatine
Buffer + ATP
 Enzyme/Substrate:
ADP ATP + Glucose –HK-> G-6-P +ADP
Creatine phosphate
NADP G-6-P + NAD+ –G-6-PD-> 6-
HK phosphoglucolactone + NADPH
G-6-PD
 Antibody binds to M-subunit
Prepare Working Reagent: therefore inhibiting it.
1 vial Enzyme/Substrate + 3mL  CK-BB is not present in the blood
Buffer/Substrate sol. so only CK-MB is measured
 Enzyme activity is multiplied by a
Method: factor of 2 to compensate for the
Creatine phosphate +ADP –CK-> Creatine 50% of CK-MB inhibited
+ ATP  Read A1 after 3 minute and again
after 1, 2, and 3 minutes (every 1
ATP + Glucose –HK-> G-6-P +ADP minute after)

G-6-P + NAD+ –G-6-PD-> 6- »Calculation:

phosphoglucolactone + NADPH
C = [(A2-A1)/5] 8254
 Read A1 after 3 minute and again
after 1, 2, and 3 minutes (every 1
minute after)

Calculation:

C=
[((A2-A1)+(A3-A2)+(A4-A3))/3] 4127

CK-MB
 First to rise and fall during AMI
 Buffer/Substrate:
Glucose
Buffer