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Digestion and Absorption of Fat - Detailed Biochemistry Notes

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1. Overview of Dietary Lipids

• >90% is triacylglycerol (TAG/triglyceride) - three fatty acids esterified to a glycerol backbone
• Remainder: cholesterol, cholesteryl esters, phospholipids, and free fatty acids (FFA)

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2. Step 1 - Emulsification

Mechanical Emulsification

• Begins during food preparation (grinding, cooking, marinating)
• Continued by chewing and gastric churning (antral peristalsis against a closed pylorus)
• Gastric contents squirt intermittently into the duodenum
• Intestinal peristalsis mixes contents with pancreatic and biliary secretions

Chemical Emulsification (in the duodenum)

• Bile salts (made in liver, stored in gallbladder) are amphipathic derivatives of cholesterol. Conjugated bile salts consist of a hydroxylated sterol ring with a side chain covalently attached to glycine or taurine by an amide bond.
• Bile salts interact with lipid droplets and the aqueous environment, stabilizing droplets and preventing coalescence.
• Phosphatidylcholine from bile also acts as an emulsifier.
• Products of early digestion (fatty acids, monoglycerides) themselves act as additional emulsifiers.
• The emulsion droplet surface is coated with membrane lipids, denatured proteins, and biliary phospholipids/cholesterol. The polar heads project into the water; the charges prevent coalescence.

Medical Physiology (Boron & Boulpaep), p. 1377

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3. Step 2 - Enzymatic Digestion

A. Gastric Phase (Limited)

• Gastric lipase is a 42-kDa glycoprotein stimulated by gastrin
• Both are acid lipases - important in neonates (pancreatic lipase not yet established) and pancreatic insufficiency
• Gastric lipase can hydrolyze ~20-33% of dietary fat in pancreatic insufficiency
• Products (particularly short/medium-chain FAs) signal more distal GI tract to release CCK

Lippincott Illustrated Reviews: Biochemistry 8e, p. 494; Ganong's Physiology 26e

B. Duodenal / Pancreatic Phase (Major)

1. Pancreatic Lipase (Triacylglycerol Lipase)

• Most important enzyme for fat digestion
• Constitutes 2-3% of total pancreatic protein - extremely abundant
• Preferentially cleaves FA at positions 1 and 3 of TAG
• Primary products: 2-monoacylglycerol (2-MAG) + 2 free fatty acids
• The sn-2 bond is hydrolyzed slowly - 2-MAG remains largely intact
• Requires the oil-water interface (acts on emulsified fat)
• Inhibited by bile salts (which coat the droplet surface) unless colipase is present

2. Colipase

• Co-secreted by the pancreas as the zymogen procolipase
• Activated in the intestinal lumen by trypsin
• Binds lipase in a 1:1 ratio, anchoring it at the lipid-aqueous interface
• Overcomes inhibition by bile salts - allows lipase to remain associated with lipid droplets even in the presence of bile acids

3. Cholesterol Ester Hydrolase (Cholesterol Esterase)

• ~4% of total protein in pancreatic juice
• Hydrolyzes cholesterol esters → cholesterol + FFA
• Also cleaves esters of fat-soluble vitamins (A, D, E, K) and phospholipids, and TAG
• Activity greatly enhanced in the presence of bile salts
• A very similar enzyme is found in human milk

4. Phospholipase A2 (PLA2)

• Secreted as the proenzyme (zymogen), activated by trypsin in the intestinal lumen
• Requires bile salts for optimal activity
• Removes FA from carbon 2 of phospholipids → lysophospholipid + FFA
• Phosphatidylcholine → lysophosphatidylcholine
• Remaining FA at carbon 1 can be removed by lysophospholipase → glycerylphosphoryl base (e.g., glycerylphosphorylcholine)

Lippincott Illustrated Reviews: Biochemistry 8e, p. 497

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4. Step 3 - Micelle Formation

How Micelles Form

1. Lipolytic products build up in the surface of the emulsion droplet
2. A multilamellar liquid crystalline layer forms and buds off as multilamellar vesicles (MLVs)
3. Bile-salt micelles transform MLVs into unilamellar vesicles
4. Further transformation into mixed micelles

Mixed Micelle Structure

• Cylindrical aggregates of bile salts + mixed lipids
• Hydrophobic center contains: FFA, 2-MAG, cholesterol, fat-soluble vitamins
• Hydrophilic exterior faces the aqueous phase
• Diameter: 3-10 nm (much smaller than emulsion droplets)

Why Micelles Are Important

• Further solubilize the hydrophobic lipids
• Provide a transport mechanism through the unstirred water layer (UWL) to the brush border of enterocytes
• Micelles move down their concentration gradient through the UWL
• Lipids diffuse out of micelles at the brush border - a saturated aqueous solution of lipids is maintained in contact with the enterocyte surface

Ganong's Review of Medical Physiology 26e, p. 485; Medical Physiology (Boron), p. 1381

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5. Step 4 - Uptake by Enterocytes

Short- and Medium-Chain Fatty Acids (SCFA/MCFA, ≤12 carbons)

• Water-soluble enough at intestinal pH
• Do NOT require incorporation into micelles
• Absorbed directly by simple diffusion into the enterocyte
• Pass directly into the portal blood (not lymph) bound to albumin
• Transported to the liver via the portal vein
• Do NOT require chylomicron formation

Long-Chain Fatty Acids (LCFA, >12 carbons)

• Poorly soluble in water at physiological pH
• Must be incorporated into micelles for transport
• Uptake at the brush border: by a combination of passive diffusion and facilitated transport (via fatty acid transport proteins - FATP, FAT/CD36)
• Once inside the enterocyte, are bound by fatty acid-binding proteins (FABPs)

Cholesterol Absorption

• Free cholesterol diffuses from micelles into enterocytes
• Mediated partly by NPC1L1 (Niemann-Pick C1-Like 1) transporter - the target of ezetimibe
• ~50% of dietary cholesterol is absorbed

Fat-Soluble Vitamins (A, D, E, K)

• Absorbed from micelles along with LCFA and 2-MAG
• Deficiency occurs when bile salts or pancreatic enzymes are absent (e.g., common bile duct obstruction, pancreatic insufficiency)

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6. Step 5 - Intracellular Processing (Re-esterification)

TAG Re-synthesis (Monoglyceride Pathway - predominant in intestine)

FFA + CoA + ATP → Fatty acyl-CoA + AMP + PPi Enzyme: Fatty acyl-CoA synthetase (Thiokinase)

Enzyme: Acyl-CoA:monoacylglycerol acyltransferase (MGAT)

Enzyme: Acyl-CoA:diacylglycerol acyltransferase (DGAT)

Cholesterol Re-esterification

Cholesterol + Fatty acyl-CoA → Cholesteryl ester Enzyme: Acyl-CoA:cholesterol acyltransferase (ACAT)

Phospholipid Re-synthesis

• Lysophospholipids are re-acylated to form phospholipids in the SER

Lippincott Illustrated Reviews: Biochemistry 8e, Fig. 15.6

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7. Step 6 - Chylomicron Assembly and Secretion

Chylomicron Composition

Assembly Process

1. Re-esterified TAG and cholesteryl esters are very hydrophobic and aggregate
2. They are packaged as lipid droplets coated with:
   • Phospholipids (outer monolayer)
   • Free cholesterol
   • Apolipoprotein B-48 (the structural apolipoprotein)
3. Microsomal TG transfer protein (MTP/MTTP) is essential - transfers lipids onto Apo B-48 in the ER
4. The particle moves through the Golgi where it is glycosylated
5. Released by exocytosis from the basolateral surface of the enterocyte

Apo B-48

• Encoded by the same gene as Apo B-100 (the VLDL apolipoprotein in the liver)
• In the intestinal cell, the primary RNA transcript undergoes RNA editing: a stop codon is generated, producing a protein that is 48% of the size of the hepatic Apo B-100
• Hence the names "B-48" and "B-100"

Route of Chylomicrons

• Released into lacteals (lymphatic capillaries in intestinal villi)
• Lymph containing chylomicrons = chyle (milky appearance after a fat-rich meal)
• Travel via lymphatics → thoracic duct → left subclavian vein → systemic blood
• They are too large to pass through capillary endothelial cell junctions directly

Lippincott Illustrated Reviews: Biochemistry 8e, p. 503-504; Basic Medical Biochemistry 6e, p. 1061

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8. Step 7 - Maturation of Chylomicrons in Blood

• Apo E - recognized by liver membrane receptors (LDL receptors / remnant receptors) for endocytosis
• Apo C-II - activator of lipoprotein lipase (LPL)

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9. Step 8 - Chylomicron Metabolism in Peripheral Tissues

Lipoprotein Lipase (LPL)

• Located on the luminal surface of capillary endothelial cells, primarily in:
  • Adipose tissue
  • Skeletal and cardiac muscle
  • Mammary gland (during lactation)
• Activated by Apo C-II
• Hydrolyzes TAG in chylomicrons → glycerol + FFA
• FFA are taken up by surrounding cells for:
  • Energy (beta-oxidation) in muscle
  • Re-esterification to TAG (storage) in adipose tissue
• Heparin releases LPL from endothelial cells (used clinically)
• Orlistat (anti-obesity drug): inhibits gastric and pancreatic lipases → reduces fat absorption

Chylomicron Remnants

• After TAG is removed by LPL, the depleted particle = chylomicron remnant
• Remnant still contains cholesterol, cholesteryl esters, Apo E, and some Apo C-II
• Apo C-II is returned to HDL
• Chylomicron remnants are taken up by the liver via Apo E-mediated endocytosis (binding to LDL receptor-related protein, LRP)

Basic Medical Biochemistry 6e, p. 1062

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10. Absorption Site and Efficiency

• On a moderate fat intake, 95% or more of ingested fat is absorbed
• During fasting, the enterocyte secretes VLDL (instead of chylomicrons) into lymphatics

Ganong's Review of Medical Physiology 26e, p. 487

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11. Short-Chain Fatty Acids (SCFA) in the Colon

• Produced by bacterial fermentation of dietary fiber, resistant starch, and complex carbohydrates
• Average colonic concentration: ~80 mmol/L
• Composition: Acetate (~60%), Propionate (~25%), Butyrate (~15%)
• Absorbed via specific transporters (partly by exchange for H+)
• Provide energy (contribute to caloric intake)
• Butyrate is the preferred fuel for colonocytes
• Exert trophic effects on colonic epithelial cells and help combat inflammation

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12. Lipid Malabsorption (Steatorrhea)

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13. Summary Flow Chart

Dietary TAG / Cholesterol Esters / Phospholipids
           ↓
   STOMACH: Lingual + Gastric Lipase (acid lipases, pH 4-6)
   Products: FFA (especially short/medium chain) + 1,2-DAG
           ↓
   DUODENUM: Emulsification by bile salts + mechanical mixing
           ↓
   PANCREATIC ENZYMES:
   - Lipase + Colipase → 2-MAG + FFA (from TAG)
   - Cholesterol esterase → Cholesterol + FFA
   - PLA2 → Lysophospholipid + FFA
           ↓
   MIXED MICELLE FORMATION (bile salts + lipid products)
           ↓
   DIFFUSION through unstirred water layer to brush border
           ↓
   ENTEROCYTE UPTAKE:
   - SCFA/MCFA: direct absorption → portal blood
   - LCFA: via diffusion/FATP → inside enterocyte
           ↓
   INTRACELLULAR RE-ESTERIFICATION (in smooth ER):
   - FFA + CoA → Acyl-CoA (fatty acyl-CoA synthetase)
   - Acyl-CoA + 2-MAG → TAG (MGAT, DGAT)
   - Cholesterol → Cholesteryl esters (ACAT)
           ↓
   CHYLOMICRON ASSEMBLY (Apo B-48 + MTP, in ER → Golgi)
           ↓
   EXOCYTOSIS from basolateral enterocyte → LACTEALS
           ↓
   CHYLE → THORACIC DUCT → LEFT SUBCLAVIAN VEIN
           ↓
   Nascent chylomicrons acquire Apo E + Apo C-II from HDL
           ↓
   LPL (activated by Apo C-II) on capillary endothelium
   → TAG hydrolyzed → FFA taken up by muscle/adipose
           ↓
   CHYLOMICRON REMNANTS → Liver (via Apo E / LRP receptor)

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Key Biochemistry Points to Remember

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• Lippincott Illustrated Reviews: Biochemistry, 8th ed. (Chapter 15)
• Basic Medical Biochemistry: A Clinical Approach, 6th ed. (Chapter 29)
• Ganong's Review of Medical Physiology, 26th ed. (Chapter 26)
• Medical Physiology (Boron & Boulpaep), Chapter 45