enzymes 3

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

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

• 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

• 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.

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

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

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

• 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

• Relatively unaffected by hemolysis * Stable for 3-4 days at 4oC

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)

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

• 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

• Unaffected by hemolysis ◦ No GGT in RBCs • Stable for 1 week at 4oC

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

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)

• 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

(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(-)

• 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

• 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

• 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)

• 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)

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