Clinically significant Enymes

• Alkaline Phosphatase or ALP, catalyzes the hydrolysis of various phosphoesters at an alkaline pH
• Optimum pH for ALP is 9-10 pH
• ALP needs magnesium as an activator
• ALP has six isoenzymes:

  1. Liver ALP
  2. Bone ALP
  3. Placental ALP
  4. Intestinal ALP
  5. Regan ALP
  6. Nagao ALP
• Liver ALP is the most anodal, the fastest to migrate to the anode
• Regan ALP is associated with lung, breast and gynecological cancers
• Nagao ALP is associated with adenocarcinoma of pancreas, bile duct and pleural cancer
• ALP is EC 3.1.3.1
• Methods of analysis for ALP include:

  1. Kay and Bodansky
  2. King Armstrong Method
  3. Bowers and McComb
• In Kay and Bodansky the analyte measured is Inorganic phosphate
• In King Armstrong Method the anlyte measured is Phenol
• In Bowers and McComb, the pH is required to be 10.2 pH and is measured at 405 nm. Reference Range is 30-90 U/L (30C)
• ALP interferences:

  1. Hemolysis - slight elevation (ALP is 6 times more concentrated in RBC than in serum
  2. 3–10% increase upon standing at room temp or 4C
  3. Diet: 25% higher in high-fat meal
• Problem in using Electrophoresis in ALP identification is that it has overlapping fractions
• Immunochemical Assay in ALP determination is used for Bone ALP determination
• In heat Inactivation, if 20% is lost after heating it is bone ALP. If it is 20% more after heating, it is liver ALP.
• Phenyl alanine inhibits intestinal and placental ALP
• L-leucine inhibits Nagao ALP
• Acid Phosphatase or ACP catalyzes the same reaction made by ALP but in an acidic environment.
• Acid phosphatase is active as pH 5
• Major Tissue location for ACP is prostate
• Substrate for Kay and Bodansky is Beta-glycerophosphate
• Substrate for King Armstrong method is Phenylphosphate
• Substrate of Bowers and McComb is P-nitrophenyl phosphate
• L-tartrate inhibits prostatic ACP
• 2% Formaldehyde and 0.001M cupric sulfate inhibits RBC ACP
• ACP is tested using Thymolphthalein monophosphate as substrate at pH 5.4. Here thymol is measured at 595 nm
• For calculating Prostatic ACP, L-tartrate-inhibited activity is subtracted from the total ACP activity. This method is used for quantitative end-point reaction.
• For continuous monitoring method in ACP measurement, Nitrophenyl phosphate is the substrate at 5.4 pH. And products are then put into a following reaction to produce the Chromogen that is measured.
• Sources of Error for ACP measurement:

  1. Hemolysis
  2. Decreases upon standing
• Reference range for ACP is 0-3.5 ng/mL
• P2-PSA is an isoform of ACP that is a tumor marker for pancreatic cancer
• Aspartate aminotransferase or AST was originally known as Serum Glutamate Oxaloacetate Transaminase or SGOT
• The decrease in NADH concentration in AST determination is measured at 340 nm.
• The decrease in NADH concentration is proportional to the AST/SGOT activity
• The substrate for AST determination are L-Aspartate and alpha-keto glutarate. The Oxaloacetate is then reacted with NADH to produce malate, NAD and water.
• Pyridoxal-5'-phosphate (P.5'.P) and Pyridoxamine.5'.phosphate are the coenzyme in the amino-transfer that AST catalyzes and these coenzyme increases the AST activity.
• Source of Error for AST:

  1. Hemolysis
• Refernce range for AST/SGOT is 5-30 U/L
• AST principles:

  1. Karmen - main
  2. Wroblewski and LaDue
  3. Henry
  4. Amador and Wacker
  5. IFCC
  6. Bergmeyer et.al.