enzymes 2 Flashcards
why measure enzymes
• Enzymes are produced within cells and found in all body tissues.
• Those that are found in the blood stream are either:◦ Plasma specific (have a function in the bloodstream)
E.g. Coagulation enzymes (fibrinolytic enzymes)
◦ Secreted
◦ Cellular
• An increase in secreted or cellular enzymes indicates disease or damage of
the organ.
◦ Disease (e.g. cancer) can cause ↑’d production from the tissue or organ
◦ Damage - causes leakage of enzymes into bloodstream
isoenzymes
•Different forms of an enzyme; differ in amino acid sequence but catalyze the same reaction.
•Have the same active site → can catalyze the same reaction but have different
structures
◦ e.g. ALP (from different organs) always catalyzes the breakage of organic phosphate esters
•Come from different organ/tissues
◦ e.g. ALP isoenzymes come from intestine, liver, bone, spleen, placenta, & kidney
•Why do we ID isoenzymes?
◦ To tell us which organ is involved when there is more than one source
methods to ID isoenzymes
Zone Electrophoresis (AKA protein electrophoresis) ◦ Enzymes have different protein structures ◦ After electrophoresis we get visible bands after exposure to specific chemicals
Selective Inactivation
◦ Increase temperature
◦ Chemical Inactivation
◦ Immunochemical - uses antisera to ID specific isoenzymes
*Temp & chemicals can denature or inhibit certain isoenzymes while
leaving others intact
clinically significant isoenzymes
Alkaline Phosphatase (ALP) Acid Phosphatase (ACP) Amylase (AMS)
Aspartate Transaminase (AST) Alanine Aminotransferase (ALT) Gamma Glutamyltransferase (GGT) Lactate Dehydrogenase (LD)
Creatine Kinase (CK)
Alkaline Phosphatase (ALP)
reaction catalyzed :
ALP
organic phosphate ester + H2O ——> alcohol + PO4
*** group specificity: ALP acts on phosphate ester structure
Nomenclature:
◦ Hydrolase (class)
◦ Alkaline Phosphatase (practical name)
◦ ALP (abbreviation)
***Optimum pH: 9-10
Activators: Mg2+
Inhibitors: oxalate, phosphate, borate, cyanide
◦ Important for blood collection protocol
◦ e.g. do not collect in grey top tube - contains oxalate
Alkaline Phosphatase (ALP) Analysis
Bessy Lowry Brock (BLB) method is most popular. (AKA Bowers McComb)
ALP
4−nitrophenylphosphate+H2O→p−nitrophenol+inorganic phosphate
mg2+
***4−nitrophenylphosphate (substrate)
Substrate is colorless
p-nitrophenol is yellow at alkaline pH (optimal pH: 9-10)
Read at 405 nm → increase in absorbance ~ ALP activity
Automated and manual methods
Isoenzymes of Alkaline Phosphatase (ALP)
• Four major isoenzymes:◦ Bone, Liver, Intestine, and Placenta
• Others: Regan, Nagao, Pa, fast liver → associated with neoplasms
◦ Fast liver found in metastatic liver cancer
• To differentiate between isoenzymes:
◦ Use electrophoresis:
Anode Liver Bone/Placental Intestine Cathode
+ -
◦ Use heat → will determine if bone or placental (placental is the most heat stabile)
◦ Use selective chemical inhibition - urea will inactivate the isoenzymes (bone 1st)
◦ Immunochemical methods - use antibodies; have been developed for placental and intestine and most recently
bone
Alkaline Phosphatase (ALP) - Clinical Significance
• Evaluation of liver and bone disorders/disease
• Increase in ALP seen in:◦ Extrahepatic obstruction (3-10X normal)
◦ Intrahepatic obstruction (<3X normal)
◦ Bone disease/bone cancer (highest levels)
◦ Paget’s Disease (bone resorption)
◦ Healing bone fractures/periods of growth and adults > 50 years old
◦ 3rd Trimester of Pregnancy - increase in placental ALP
◦ Increase in ALP also seen with complications
◦ Returns to normal 3-6 days after delivery
Decrease in ALP seen in:
◦ Hypophosphatasia - inherited disorder with absence of bone isoenzyme
◦ Results in inadequate bone calcification
Alkaline Phosphatase (ALP) - Sources of Error
- Avoid hemolysis (ALP 6X higher in RBCs)
- Analyze ASAP (3-10% increase on standing)
- Group O and B secretors (25% higher after a high-fat meal)
Acid Phosphatase (ACP)
Reaction catalyzed:
ACP
𝑂𝑟𝑔𝑎𝑛𝑖𝑐 𝑝ℎ𝑜𝑠𝑝ℎ𝑎𝑡𝑒 𝑒𝑠𝑡𝑒𝑟+𝐻2𝑂 → 𝐴𝑙𝑐𝑜ℎ𝑜𝑙+𝑃𝑂4
Nomenclature:
Hydrolase (class)
Acid Phosphatase (practical name)
ACP (abbreviation)
***Optimum pH: ~ 5.0
Activators: Mg2+
Inhibitors: oxalate, phosphate, borate, cyanide
- Important for blood collection protocol
- e.g. do not collect in grey top tube - contains oxalate
Acid Phosphatase (ACP) Analysis
Bessy Lowry Brock (BLB) method (AKA Bowers McComb):
ACP
4−nitrophenylphosphate +H2O → p−nitrophenol + inorganic phosphate
Roy, Brower and Hayden method:
ACP
𝑇ℎ𝑦𝑚𝑜𝑙𝑝ℎ𝑡ℎ𝑎𝑙𝑒𝑖𝑛 𝑚𝑜𝑛𝑜𝑝ℎ𝑜𝑠𝑝ℎ𝑎𝑡𝑒+𝐻2𝑂 → 𝑇ℎ𝑦𝑚𝑜𝑙𝑝ℎ𝑡ℎ𝑎𝑙𝑒𝑖𝑛+𝑃𝑂4
Both BLB and Roy et al. produce colorless products at acid pH
Must be alkalinized → stops reaction and produces a color
Used for endpoint measurements only (disadvantage - not good for continuous monitoring)
Acid Phosphatase (ACP) - Continuous Monitoring Method
Hillman method:
ACP
α−napthyl phosphate + H2O → α−napthol + PO4
The “α”-napthol is coupled with Fast Red TR to give a compound that absorbs light at 405 nm (with acid pH)
Most continuous monitoring systems use Hillman → “α−napthyl phosphate “(substrate)
Most manual methods employ Roy et al. → Thymolphthalein monophosphate (substrate)
Isoenzymes of Acid Phosphatase (ACP)
• ACP found in liver, bone, spleen, and kidney but highest in prostate.
• Can differentiate prostatic ACP (PAP) from others by:
◦ Earlier manual methods: tartrate added to inhibit prostatic form.
- Total ACP - ACP after tartrate inhibition = PAP
◦ Newer methods (BLB and Roy et al.) are based on the fact that PAP reacts faster than others (breaks down substrate faster). Timing can be used to measure PAP activity.
Roy et al. → thymolphthalein monophosphate is substrate of choice
Acid Phosphatase - Clinical Significance
- Evaluation of prostate carcinoma and used in forensic investigations
- ACP increased in:
◦ Metastatic prostate carcinoma (50X normal)
- Useful to confirm and stage metastatic prostate cancer - Local cancer will show only slight increase or normal values
◦ Vaginal washings after rape
- Can persist up to 4 days following rape - Useful in forensic investigations
◦ Bone Disease
◦ Platelet Damage
Acid Phosphatase (ACP) - Sources of Error
• Red blood cells and platelets have ACP
◦ Separate serum from cells ASAP to avoid a false increase
• ACP is VERY UNSTABLE at room temperature. If not tested immediately,
freeze or acidify to pH < 6.5
◦ Loss of CO2 = ↑ pH = ↓ ACP
◦ So need to acidify to pH <6.5 to preserve ACP
• Activity decreases within 1-2 hours if left at room temperature
