Part 2: Enzymes Study Guide

What is the reference range for Lactate Dehydrogenase (LDH)?

• 100 - 225 U/L

Describe Alanine aminotransferase (ALT, SGPT).

• most hepatospecific transaminase

• levels remain normal in acute MI

What does elevation of Alanine aminotransferase (ALT, SGPT) indicate?

• marked elevation → hepatocellular disorders

  • acute viral hepatitis

• mild elevation → obstructive disease

What results would indicate Acute Hepatitis?

• ALT > AST and ALT stays elevated longer (longer half life)

What is the reaction for Alanine aminotransferase (ALT, SGPT)?

• alanine + a-ketoglutarate → pyruvate + glutamate

What is the coupled measurement for Alanine aminotransferase (ALT, SGPT)?

• NADH + pyruvate → NAD+ + lactate

• measures ↓ absorbance at 340

What sample is used for Alanine aminotransferase (ALT, SGPT)?

• non hemolyzed serum

• lithium heparin plasma

What is the reference range for Alanine aminotransferase (ALT, SGPT)?

• 6 - 37 U/L

Describe Aspartate aminotransferase (AST, SGOT).

• highest concentration in liver, then heart and skeletal muscle

  • also kidney, pancreas, and RBC

an elevated Aspartate aminotransferase (AST, SGOT) can indicate what?

• MI - peak 24 hr after event

• pulmonary embolism

• hepatocellular damage - viral hepatitis100x normal

• cirrhosis - 4x normal

• muscular dusophy - 4x normal

What is the forward reaction for Aspartate aminotransferase (AST, SGOT)?

• L-aspartate + a-ketoglutarate → oxaloacetate + L-glutamate

What is the coupled measurement for Aspartate aminotransferase (AST, SGOT)?

• oxaloacetate + NADH → malate + NAD+

  • via malate dehydrogenase, MDH

• measures ↓ absorbance at 340 nm

Describe the DeRitis ratio.

• AST / ALT

• normally < 1

• > 1 = alcoholic liver disease / cirrhosis

What sample is used for Aspartate aminotransferase (AST, SGOT)

• nonhemolyzed serum

• diurnal variation (highest in the afternoon)

What is the reference range for Aspartate aminotransferase (AST, SGOT)?

• 15 - 30 U/L

Describe Alkaline Phosphatase (ALP).

• most clinically significant in hepatobiliary disease

  • especially biliary obstruction and bone disease

What are the major isoenzymes of Alkaline Phosphatase (ALP)?

• liver, bone (osteoblasts), intestine, placenta

  • distinguished by heat stability at 56 C for 10 min

• intestine / placenta: most stabile (>75% remaining)

• liver : 22-55%

• bone: least stable (20%)

What Alkaline Phosphatase (ALP) results indicate bone disease?

• ↑ ALP + normal GGT

What Alkaline Phosphatase (ALP) results indicate liver disease?

• ↑ ALP + ↑ GGT

What sample is used for What Alkaline Phosphatase (ALP)?

• Cofactor: Mg2+

• ONLY HEPARIN is acceptable anticoagulant

  • EDTA, citrate oxalate cheat Mg2+ and inactivate the enzyme

What is the reaction for Alkaline Phosphatase (ALP)?

• p-nitrophenyl-phosphate → p-nitrophenol + phosphate ion

• measures ↑ absorbance at 405 nm

What are sources of error for Alkaline Phosphatase (ALP)?

• high fat meals (intestinal ALP)

• sample left > 8 h at room or refrigerator temp

• freezing / thawing INCREASES activity

What is the reference range for Alkaline Phosphatase (ALP)?

• 30 - 90 U/L

Describe Acid Phosphatase (ACP).

• major cellular origin: prostate

  • also seen in osteoclasts, RBCs, PLTs

• used to detect metastatic prostatic carcinoma

• ↑ in benign prostatic hyperplasia and after prostate surgery

What sample is used for Acid Phosphatase (ACP)?

• tartrate resistant APC is non-hemolyzed serum and is primarily synthesized by osteoclasts

  • useful marker of bone reabsorption

• measured by p-nitrophenol reaction at pH 5

What sources of error can occur for Acid Phosphatase (ACP)?

• hemolysis

• sample left at room temp

  • loses CO2

  • ↑ pH

  • destroys activity

Describe Gamma-glutamyl transferase (GGT)?

• most sensitive marker for chronic alcoholic liver disease

  • alocolo induces GGT synthesis

• detects VERY EARLY changes in liver cell damage

What is the reaction for Gamma-glutamyl transferase (GGT)?

• peptide-substrate analog → p-nitroaniline

• measure absorbance at 405 nm

What is the reference range for Gamma-glutamyl transferase (GGT)?

• at 30 C

  • males: 8 - 37 IU/L

  • females: 5 - 24 IU/L

What do elevated Gamma-glutamyl transferase (GGT) indicate?

• pancreatitis

• diabetes

• MI

• drugs

  • warfarin

  • barbiturates

  • phenytoin

  • valproate

  • methotrexate

What do ALP and GGT results mean together?

• ↑ ALP AND GGT = liver source

• ↑ ALP AND normal GGT = bone source

Describe Amylase.

• hydrolyzes starch and glycogen

• two main isoenzymes in serum

  • salivary (S-type)

  • Pancreatic (P-type)

• Classic Marker for Acute Pancreatitis along with lipase

What results for Amylase indicate acute pancreatitus

• rises in 2-24 hrs

• returns to normal in 2-3 days

  • persistence beyond 5 days = complication

What are sources of error for Amylase?

• high triglycerides

• EDTA and citrate anticoagulants

What is the reference range for Amylase?

• 35 - 125 U/L

What can Amylase results indicate?

• 10% of acute pancreatitis cases = normal amylase

• up to 30% of amylase elevations are NON-pancreatic

  • salivary disease or intestinal obstruction

What is activator is needed for Amylase?

• Cl- (chloride)

• amylase is one of the few enzymes that is also analyzed in urine

Describe Lipase.

• hydrolyzes ester bonds of fats to glycerol and fatty acids

• more SPECIFIC for acute pancreatitis than amylase but stays elevated longer

  • less affected by renal function

What can inhibit Lipase?

• hemoglobin

  • hemolyzed samples give FALSELY DECREASED results

What is the reference range for Lipase?

• 10 - 200 U/L

What are the isoenzymes of Creatine Kinase (CK)?

• CK-MM

• CK-MB

• CK-BB

Dimer of M (muscle) and B (brain) subunits

Describe CK-MM

• predominant in skeletal muscles

• ↑ in muscle disease

  • muscular dystrophy

  • rhabdomyolysis

Describe CK-MB

• heart muscle

• ↑ in acute MI

• heart tissue has both MM and MB

Describe CK-BB

• brain, smooth muscle

• rarely elevated

• can occur in some cancers or CNS injury

What is the first enzyme to reach peak elevation after a myocardial infection?

• Creatine Kinase (CK)

What activator is needed for Creatine Kinase (CK)?

• Mg2+ only

What is the best assay for CK-MB?

• monoclonal sandwich immunoassay

What are the two types of Cholinesterase (CHE)?

• Acetylcholinesterase

  • in RBC and nerve tissue

• Pseudocholinesterase

  • serum and LIVER (main source)

What is Cholinesterase (CHE)?

• sensitive but non-specific marker for organophosphate (insecticide) poisoning

What can ↓ Cholinesterase (CHE) indicate?

• liver disease

• starvation

• burns

• insecticide poisoning

What are sources of error for Cholinesterase (CHE)?

• hemolysis

  • RBC contain acetylcholinesterase

What do these results indicate?

CK peak first

AST 24 hrs

LDH with flipped LD-1 > LD-2 ratio

Troponin is gold standard

• Acute MI

What do these results indicate?

• very high ALT and AST ( can be 100x normal)

• ALT > AST

• mild ALP rise

• ↑ LD-4 / LD-5

• Acute viral hepatitis

What do these results indicate?

↑ GGT (most sensitive)

AST/ALT ratio > 1

• Chronic alcoholic liver disease / cirrhosis

What do these results indicate?

↑ ALP and GGT (and 5’ - NTP)

ALT / AST only modestly elevated

best enzyme profile: NTP, ALP, GGt

• Biliary obstruction

What do these results indicate?

↑ ALP with NORMAL GGT

↑ TR - ACP

• Bone disease

  • paget’s mets

  • healing fracture

What do these results indicate?

↑ CK-MM

↑ AST

↑ LD-4/LD-5

• Skeletal Muscle disease

  • muscular dystrophy

What do these results indicate?

• ↑ APC (historically)

• monitored by PSA

• Prostate cancer (metastatic)

What do these results indicate?

• ↑ amylase (rises first)

• ↑ lipase (more specific, lasts longer)

• Acute Pancreatitis

What do these results indicate?

↓ Cholinesterase

• Organophosphate poisoning

What is the Michaelis-Menten formula

v = Vmax x (S) / (Km +S)

• when (S) = kM, v = ½ Vmax

What is the Lineweaver-Burk equation?

• 1/v = (Km/Vmax)(1/S) + 1/Vmax

  • y intercept = 1 / vmax

  • x intercept = -1/Km

  • slope = km/Vmax

What is the zero-order assay condition?

• S > 10 - 100 x km

What is 1 IU?

• 1 umol substrate converted to product per minute under defined conditions

• 1 mU/mL = 1 IU/L

Describe NADH

• absorbs at 340 nm

  • NAD+ does NOT

• ↑ absorbance at 340 nm = NADH produced

• ↓ absorbance at 340 nm = NADH consumed

What does a flipped LDH ratio mean?

• LD-1 > LD-2

  • MI

  • hemolysis

  • renal infarction

What does the DeRitis ratio mean?

• AST/ALT > 1 = alcohol / cirrhosis

• AST / ALT < 1 = ordinary hepatitis

What does ↑ ALP + normal GGT mean?

• bone issue

What does ↑ ALP and ↑ GGT mean?

• liver issue

What activator is needed for amylase?

• Cl -

What activator is used for CK?

• Mg2+

What activator is used for ALP?

• Mg2+

Describe LDH and Lipase and what effects them.

• LDH hates hemolysis

• Lipase hates hemoglobin

gets inhibited → falsely low