Enzymes

Why intracellular enzymes appear in blood

Intracellular enzymes enter blood when there is:

• cell damage
• cell proliferation (release of overproduced enzymes)
• increased cell permeability (hypoxia)
• enzyme induction

Clinical importance

1. diagnosis of disease
2. prognosis of disease (course/progression)

Measuring enzymes

Protein level

• ELISA

Enzymatic activity

• colorimetric assay

ELISA (enzyme-linked immunosorbent assay)

Sandwich ELISA (workflow)

• capture antibody captures enzyme/protein of interest on the bottom of wells

• antigen: things that trigger generation of antibodies

• add sample (may or may not contain protein of interest)

• wash with buffers at least 3 times

• secondary antibody binds to the protein of interest (added after wash)

• binds a different epitope on the same molecule

• secondary antibody has a domain for detection antibody that is bound to detection enzyme
• add detection antibody with detection enzyme
• add substrate for the detection enzyme

Notes:

• more than one enzyme can be tested
• example comparison: AST vs ALT

Isoenzymes and serial testing

Isoenzymes

• enzymes with slightly different properties but similar function

Serial enzyme estimation

• measure multiple times

Confounding factors

• pre-existing illness

Examples:

• AST affected by exercise
• CK-MM affected by exercise
• CK-MM elevated after intramuscular injection
• medication

• physiological factors:

• age: AP (alkaline phosphatase) is very high in children

• gender: alcohol dehydrogenase is higher in men

Pancreatic enzymes and pancreas function

• pancreas regions: head, body, tail

• functions:

• endocrine: control blood sugar (hormones to blood vessels)

• exocrine: digestive enzymes to the lumen of the GI tract

Exocrine pancreas (digestive enzymes)

Acinar cells and duct flow

• acinar cells produce digestive enzymes
• enzymes travel via pancreatic ducts in vesicles
• ducts go to the pancreatic head, merge with the bile duct, then to the small intestine (duodenum)

Major enzymes

• trypsin (protein), secreted as trypsinogen
• lipase (lipids): removes fatty acids from glycerol backbone
• amylase (carbohydrates): breaks \(\alpha(1\to4)\) glycosidic bonds

Zymogens and activation

• enzymes are made as zymogens (inactive form)

• masking sequence blocks the active site

• after arriving at duodenum:

• membrane-bound enteropeptidase releases enzymes and removes masking sequence

• activated enzymes can activate other zymogens

Pancreatitis

Causes

• premature trypsin enzyme activity in acinar cells

• may be caused by:

• gallstones (acute)

• alcoholism (chronic; may be caused by calcified plugs)
• blockage of the pancreatic duct

Character

• significant inflammatory event

Diagnosis

• severe upper right abdominal pain that may radiate to the back and worsen

• elevated blood enzymes:

• amylase and lipase: 5×–10× increase

• CT scan findings:

• fluid accumulation near pancreas and/or calcified plugs

Endocrine pancreas

• islets of Langerhans:

• beta cells make insulin

• alpha cells make glucagon

Liver enzymes and function

Enzymes

• alanine transaminase (ALT)

• aspartate transaminase (AST)

• regulate nitrogen and alanine levels (amino groups)

• gamma-glutamyl transferase (GGT)

• adds gamma-glutamyl moiety to alanine to build glutathione (removes ROS)

Other liver roles:

• detoxify ROS
• regulate glucose (store)

Hepatitis, fibrosis, cirrhosis

• hepatitis: liver inflammation
• fibrosis: cell death and connective tissue
• cirrhosis: irreversible scarring
• may finally lead to cancer

Diagnosis

• fatigue, yellowing, flu-like symptoms, abdominal pain
• ALT is typically a better measure (10×–50×)
• AST is also found in heart and muscle
• GGT is also found in biliary tract

De Ritis ratio

Interpretation:

• DRR \(< 1\): liver inflammation from virus
• DRR \(> 1.5\): alcohol-related liver damage and cancer

Imaging/pathology notes:

• circular collagen around blood vessel lumen
• liver under CT is bumpy

Bone enzyme: alkaline phosphatase (AP)

• alkaline phosphatase cleaves phosphate groups
• osteoblasts (bone formation) may use it to release phosphate groups to calcify bones

Rickets (osteomalacia)

• inadequate mineralization for bone hardening
• deficiency in vitamin D, calcium, and/or phosphate

Bone cancer: osteosarcoma

• AP level skyrockets

Diagnosis

Rickets:

• tenderness and softness of bone
• abnormally shaped spine, bow-shaped bones, dental deformity
• treatment may include dietary changes

Bone cancer:

• pain

Muscle and cardiac markers

Creatine kinase (CK)

• CK is a dimer with B and M subunits
• adds/removes phosphate group to creatine
• creatine is an energy reservoir for quick energy

Isoenzymes:

• CK-BB (#1, most in brain)
• CK-MB (#2)
• CK-MM (#3 most in muscle)

Clinical notes:

• CK-MB used to check for heart attacks (blockage of blood to heart)
• CK-BB is related to intestinal cancer (it can’t cross BBB, so brain CK-BB cannot be found in blood)

CK-MB timing in heart attack

• CK-MB leakage: \(t+3\)–\(6\,\text{h}\) to \(t+2\)–\(3\,\text{d}\)
• peak: \(t+24\,\text{h}\)

Other markers:

• AST: \(t+12\,\text{h}\) to \(t+3\)–\(5\,\text{d}\), peak at \(t+1\)–\(2\,\text{d}\)

• LDH: \(t+18\,\text{h}\) to \(t+1\,\text{w}\), peak at \(t+2\)–\(3\,\text{d}\)

• flipped pattern: LDH1 > LDH2

Reasons for different kinetics:

• leakage rates differ
• protein size differs (CK-MB is the smallest)
• clearance time differs

CK-MB index

Interpretation:

• CK-MB index \(> 2.5\): likely heart damage
• CK-MB index \(< 2.5\): muscle damage

Troponin (not an enzyme)

• within 1 hour of heart attack, troponin T and troponin I are specific to myocardium
• peaks at ~12 hours
• clears in ~7 days

Lactate dehydrogenase (LDH)

• ubiquitous enzyme
• converts lactate to/from pyruvate
• tetramer with H and M subunits

Isoenzymes:

• LDH1: H\(_4\) (heart, RBC)
• LDH2: H\(_3\)M (RBC, WBC)
• LDH3: H\(_2\)M\(_2\) (lungs)
• LDH4: HM\(_3\) (kidney, placenta, pancreas)
• LDH5: M\(_4\) (liver, skeletal muscle)