enzymes 3

Question 1

Aspartate Aminotransferase (AST)

Answer 1

Reaction catalyzed:
AST
L−aspartate + α−ketoglutarate → oxaloacetate + L−Glutamate

Nomenclature:
Transferase (class)
Aspartate Aminotransferase
- or Aspartate Transaminase (practical name)
AST (abbreviation)

Coenzyme: Pyridoxal-5’-phosphate (P-5’-P or PLP)
-needed as a prosthetic group → cofactor tightly bound to apoenzyme

Since both apoenzyme (inactive) and holoenzyme (active) exist in serum, methods of analysis incorporate PLP so all AST is measured

Question 2

Aspartate Aminotransferase (AST) Analysis

Answer 2

Karmen Method, 2 steps:
AST
L−Aspartate + α−ketoglutarate → oxaloacetate + L−glutamate

                                          Malate Dehydrogenase Oxaloacetate   +   NADH   +   H+   →       L−Malate   +   NAD+

Decrease in absorbance read at 340 nm per minute (kinetic)

optimum pH for this reaction is 7.3-7.8

Question 3

Isoenzymes of Aspartate Aminotransferase (AST)

Answer 3

• 2 fractions (cytoplasmic and mitochondrial)
• Cytoplasmic isoenzyme most predominant form in serum
• Mitochondrial form increased in disorders that produce cellular necrosis
• Isoenzyme analysis for AST not routinely performed

Question 4

Aspartate aminotransferase (AST) - Clinical Significance

Answer 4

• Evaluation of liver disorders and skeletal muscle involvement

• AST increased in:◦ Viral hepatitis (100X normal)
◦ Other liver disease (4 X)
◦ Skeletal muscle disorders (4-8X)
    - i.e. muscular dystrophy
◦ Acute Myocardial Infarction
    - Rise in 6 - 8 hours
     -  Peak at ~24 hours
     - Normal in  ~5 days

AST is not useful in the diagnosis of AMI due to its distribution in many different tissues.

Question 5

Aspartate Aminotransferase (AST) - Sources of Error

Answer 5

• Avoid hemolysis◦ RBCs contain AST and will cause a false increase (up to 10X normal)
• Stable for 3-4 days at 4oC

Question 6

Alanine Aminotransferase (ALT)

Answer 6

Reaction catalyzed:
ALT
Alanine + α−ketoglutarate → Pyruvate + Glutamate

Nomenclature:
Transferase (class)
Alanine Aminotransferase (practical name)
ALT (abbreviation)
`
Coenzyme: Pyridoxal-5’-phosphate (P-5’-P or PLP) -needed as a prosthetic group → cofactor tightly bound to apoenzyme

Question 7

Alanine Aminotransferase (ALT) Analysis

Answer 7

Coupled enzymatic reaction:
ALT
Alanine + α−ketoglutarate → Pyruvate + Glutamate

                                           LD Pyruvate   +   NADH   +   H+   → Lactate   +   NAD+

Decrease in absorbance at 340 nm
measured continuously and is ~ ALT
activity

optimum pH for this reaction is 7.3-7.8

Question 8

Alanine Aminotransferase (ALT)- Clinical Significance

Answer 8

• Evaluation of hepatic disorders (liver)

• Increased ALT in:
◦ Hepatocellular disorders (largest increase)
-damage to, or inflammation of the hepatocytes
◦ Extrahepatic and intrahepatic obstructive disorders

• Small amount of ALT in cardiac tissue◦ ALT levels remain normal in AMI unless there is also liver involvement
• ALT is more liver specific than AST

Question 9

Alanine Aminotransferase - Sources of Error

Answer 9

• Relatively unaffected by hemolysis

* Stable for 3-4 days at 4oC

Question 10

Gamma-Glutamyltransferase (GGT or g-GT)

Answer 10

Reaction catalyzed:
GGT
[𝑝𝑒𝑝𝑡𝑖𝑑𝑒 𝐴+𝐴𝐴]+𝑝𝑒𝑝𝑡𝑖𝑑𝑒 𝐵 → 𝑃𝑒𝑝𝑡𝑖𝑑𝑒 𝐴+[𝑝𝑒𝑝𝑡𝑖𝑑𝑒 𝐵+𝐴𝐴]
OR
GGT
[𝑝𝑒𝑝𝑡𝑖𝑑𝑒 𝐴+𝐴𝐴]+𝐴𝐴 → 𝑃𝑒𝑝𝑡𝑖𝑑𝑒 𝐴+[𝐴𝐴+𝐴𝐴]

Note: The peptide or amino acid must have glutamate as a terminal residue. Glutamate is transferred from one peptide to another peptide or amino acid.

Nomenclature:
Transferase (class)
Gamma-Glutamyltransferase (practical name)
GGT or g-GT (abbreviation)

Question 11

Gamma-Glutamyltransferase (GGT) Analysis

Answer 11

Most methods use:
GGT
(substrate) 𝛾−𝑔𝑙𝑢𝑡𝑎𝑚𝑦𝑙−𝑝−𝑛𝑖𝑡𝑟𝑜𝑎𝑛𝑖𝑙𝑖𝑑𝑒+𝐺𝑙𝑦𝑐𝑦𝑙𝑔𝑙𝑦𝑐𝑖𝑛𝑒 → 𝛾−𝑔𝑙𝑢𝑡𝑎𝑚𝑦𝑙𝑔𝑙𝑦𝑐𝑜𝑔𝑙𝑦𝑐𝑖𝑛𝑒 + 𝑝−𝑛𝑖𝑡𝑟𝑜𝑎𝑛𝑖𝑙𝑖𝑛𝑒 ( yellow)

                                                GGT SHORT FORM:  𝐺𝐺𝑃𝑁𝑑  +  𝐺𝐺   →  𝐺𝐺𝐺𝐺  +  𝑃𝑁𝑛(yellow)

GGT transfers glutamate to Glycylglycine (GG)
P-nitroaniline is produced - yellow with absorbance at 405 - 420 nm - amount directly proportional to enzyme activity

Question 12

Gamma-Glutamyltransferase (GGT) - Clinical Significance

Answer 12

• Evaluation of liver and biliary system disorders/disease

• GGT increased in◦ Biliary tract obstruction and liver cancer (30X normal)
◦ Patients receiving enzyme-inducing drugs (4X normal)
- warfarin, phenobarbital, and phenytoin
- these drugs enhance enzyme activity
◦ Chronic alcoholism or heavy drinkers (2-3X normal)
- GGT is used to monitor abstinence in alcohol treatment centers
◦ Small increase in prostate cancer, drug intoxication and fatty liver

• GGT levels will be normal is skeletal disorders and pregnancy
- therefore, can help differentiate the source of elevated ALP

Question 13

Gamma-Glutamyltransferase (GGT) - Sources of Error

Answer 13

• Unaffected by hemolysis
◦ No GGT in RBCs

• Stable for 1 week at 4oC

Question 14

Lactate Dehydrogenase (LD or LDH)

Answer 14

Reaction catalyzed:
LD
𝐿”−” 𝐿𝑎𝑐𝑡𝑎𝑡𝑒+𝑁𝐴𝐷+ ↔𝑃𝑦𝑟𝑢𝑣𝑎𝑡𝑒+𝑁𝐴𝐷𝐻+𝐻+
absorbance measured at 340nm

Reaction is reversible and strongly favors Pyruvate → Lactate direction

Nomenclature:
Oxidoreductase (class)
Lactate Dehydrogenase (practical name)
LD or LDH (abbreviations)

Optimum pH:
Pyruvate → Lactate pH 7.1 - 7.4
Lactate → Pyruvate pH 8.3 - 8.9

Coenzyme: NAD+

Inhibitors: oxalate, EDTA- use heparin or SST for collection

Question 15

Common Measuring Reaction using NAD+ & NADH

Answer 15

NAD+ (nicotinamide adenine dinucleotide) can be reduced to NADH

OR
NADH can be oxidized to NAD+
NAD+ ↔ NADH

If NADH is the endproduct, an ↑ in absorbance @ 340 nm is measured.

If NAD+ is the endproduct, a ↓ in absorbance @ 340 nm is measured.

This is one of the most common indicator reactions used in clinical chemistry.

• Used with: Amylase, AST, ALT, GGT, and LD
• glucose (Hexokinase method)
• triglycerides, cholesterol
• uric acid, urea (coupled enzymatic methods)

Question 16

Isoenzymes of Lactate Dehydrogenase (LD)

Answer 16

• 5 main isoenzymes
• Each has 4 subunits (peptide H & M chains)
- H stands for heart, M stands for muscle
• Classified electrophoretically LD-1 → LD-5
• LD-1 fastest moving to the anode in alkaline medium

Question 17

Isoenzyme Chains of LD

Answer 17

(fastest)
LD-1 LD-2 LD-3 LD-4 LD-5(slowest)
H H H H M
H H H M M
H H M M M
H M M M M
Anode (+) Cathode(-)

Question 18

Isoenzymes of Lactate Dehydrogenase (LD)

Answer 18

• Serum of healthy people – major isoenzyme is LD-2, then LD-1, 3,4,5
• AMI – get a flipped pattern LD-1 > LD-2
• LD-1 & LD-2 - heart, kidney and RBC’s
• LD-4 & LD-5 - liver and skeletal muscle
• LD-6 has been ID’d in patients with arteriosclerotic cardiovascular failure – grave prognosis

Question 19

Methods to differentiate Lactate Dehydrogenase Isoenzymes

Answer 19

• Heat denaturation
◦ Liver (LD-5) destroyed at 65oC; Heart (LD-1) remains active

• Chemical inactivation
◦ 2.0M urea solution inhibits heart
◦ Strong lactate solution inhibits liver

• Immunoprecipitation◦ Antisera to M developed; only LD-1 can be measured
• Electrophoresis – mainly used

Question 20

Lactate Dehydrogenase - Clinical Significance

Answer 20

• LD is increased in:

◦ AMI

• rises 12 - 24 hrs
• peaks48 - 72 hrs
• normal 10 days (LD stays elevated the longest of all enzymes)
• LD-1 > LD-2 in AMI; Flipped pattern (normal is LD-2 > LD-1)

◦ Pernicious Anemia/Megaloblastic Anemias (30X normal)

◦ Liver Diseases
- increase in LD-4, LD-5

◦ Muscular Dystrophy
- increase in LD-5; normal values late in disease

◦ Acute Lymphoblastic Leukemia (ALL)

Question 21

Lactate Dehydrogenase (LD) - Sources of Error

Answer 21

• Any degree of hemolysis is unacceptable
- RBCs have an LD concentration 100-150X that found in serum

• LD is unstable
• Should be stored at 25oC and analyzed within 48 hours
IF doing electrphoresis run within 24 hrs bc LD-5 is the most labile isoenzyme- loses activity quickly and even more so at 4degrees(refrigerated temp)

Question 22

Summary of Enzymes in Disease States

Answer 22

Liver disease/injury:
↑ALP
↑ AST
↑ ALT
↑ GGT
↑ LD

Viral Hepatitis:
↑ AST (100X)
↑ ALP (< 3X)

Chronic Drinkers:
↑ GGT

Heart Attack (AMI):
↑ AST
↑ LD
LD-1 > LD-2

Pancreatitis:
↑ Amylase
↑ Lipase
↑ GGT