enzymes 3 Flashcards
Aspartate Aminotransferase (AST)
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
Aspartate Aminotransferase (AST) Analysis
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
Isoenzymes of Aspartate Aminotransferase (AST)
- 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
Aspartate aminotransferase (AST) - Clinical Significance
• 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.
Aspartate Aminotransferase (AST) - Sources of Error
- Avoid hemolysis◦ RBCs contain AST and will cause a false increase (up to 10X normal)
- Stable for 3-4 days at 4oC
Alanine Aminotransferase (ALT)
Reaction catalyzed:
ALT
Alanine + α−ketoglutarate → Pyruvate + Glutamate
Nomenclature:
Transferase (class)
Alanine Aminotransferase (practical name)
ALT (abbreviation)
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Coenzyme: Pyridoxal-5’-phosphate (P-5’-P or PLP) -needed as a prosthetic group → cofactor tightly bound to apoenzyme
Alanine Aminotransferase (ALT) Analysis
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
Alanine Aminotransferase (ALT)- Clinical Significance
• 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
Alanine Aminotransferase - Sources of Error
- Relatively unaffected by hemolysis
* Stable for 3-4 days at 4oC
Gamma-Glutamyltransferase (GGT or g-GT)
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)
Gamma-Glutamyltransferase (GGT) Analysis
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
Gamma-Glutamyltransferase (GGT) - Clinical Significance
• 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
Gamma-Glutamyltransferase (GGT) - Sources of Error
• Unaffected by hemolysis
◦ No GGT in RBCs
• Stable for 1 week at 4oC
Lactate Dehydrogenase (LD or LDH)
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
Common Measuring Reaction using NAD+ & NADH
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)
Isoenzymes of Lactate Dehydrogenase (LD)
• 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
Isoenzyme Chains of LD
(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(-)
Isoenzymes of Lactate Dehydrogenase (LD)
- 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
Methods to differentiate Lactate Dehydrogenase Isoenzymes
• 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
Lactate Dehydrogenase - Clinical Significance
• 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)
Lactate Dehydrogenase (LD) - Sources of Error
• 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)
Summary of Enzymes in Disease States
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
