Physiology of Digestion and Absorption 2 Professor John Peters

After this lecture students, should be able to:  Name the main molecular constituents of foodstuffs which can be digested in humans and those which cannot be digested  State how the small intestine is well adapted for absorption  Explain how carbohydrate is digested to the monosaccharides, glucose, galactose and fructose  Appreciate how monosaccharides are transported into and out of enterocytes  Provide an account of protein digestion noting the role of endo- and exo-peptidases  Explain in outline how amino acids, dipeptides and tripeptides are transported into and out of enterocytes  State the problems posed by the digestion of fats and how these are overcome with bile salts etc.  State the events in the formation of small fat droplets and micelles  Describe how free fatty acids and monoglycerides are absorbed in the small intestine  Indicate how the absorption of free fatty acids and monoglycerides differs from that of cholesterol  Explain how chylomicrons are formed, transported and processed  Explain how the absorption of Ca 2+ and iron are regulated processes  Outline the mechanisms that underlie the absorption of water- and fat- soluble vitamins  Explain why the absorption of vitamin B 12 is a special case that requires a complex series of events Learning Objectives

Main Constituents of Food  Carbohydrates – approx. 400 g per day -  Starch (amylose and amylopectin – greater than 50% total carbohydrate ingested)  Cellulose (indigestible in humans - roughage)  Glycogen  Disaccharides (sucrose, lactose)  Lipids – approx g per day -  Triacylglycerols (approximately 90% of total lipid ingested as fats and oils)  Phospholipids  Cholesterol & cholesterol esters  Free fatty acids  Lipid vitamins  Proteins – approx g per day ingested, plus g from endogenous sources  e.g. digestive enzymes and dead cells from GI tract

The Small Intestine is Well Adapted for Absorption  Compared to a simple cylinder of identical dimensions surface area is increased by:  Circular folds  Villi  Microvilli (the brush border) 3-fold 30-fold 600-fold

Carbohydrate Digestion (1)  Mouth  Commences with salivary  -amylase  Stomach  Continues with salivary  -amylase  Small intestine (duodenum)  Pancreatic amylase (enzyme secreted and free in lumen)  Oligosaccharidases (associated with the brush border membrane of enterocytes). Includes isomaltase and  -glucosidase  Disaccharidases (associated with the brush border membrane of enterocytes). Includes sucrase, lactase and maltase

Carbohydrate Digestion (2) Starch, glycogen Amylase - attacks internal  -1,4 glycosidic linkages (not  -1,4 as in cellulose) Maltose MaltotrioseIsomaltose Maltase (Cleaves  - 1,4 bond) Glucose Isomaltase (Cleaves  -1,6 bond) Glucose Enterocyte

Carbohydrate Digestion (3) Sucrose Sucrase (Cleaves  - 1,2 bond) Glucose Enterocyte Lactose Fructose Lactase (Cleaves  - 1,4 bond) Glucose Enterocyte Galactose Nb. Deficiency in lactase causes the common condition lactose intolerance.

Absorption of the Final Products of Carbohydrate Digestion: Glucose, Galactose and Fructose  Glucose and galactose are absorbed by secondary active transport; fructose by facilitated diffusion (occurs in duodenum and jejunum) 2K + 3 Na + 2 Na + 1 Glucose (or galactose) H2OH2O Na + /K + ATPase SGLT1 GLUT2 GLUT5 Glucose (or galactose) Fructose

Digestion of Proteins  Stomach  HCl begins to denature proteins  Pepsin cleaves proteins into peptides  Duodenum  Pancreatic enzymes (trypsin, chymotrypsin) split peptide bonds between different amino acids  Brush border enzymes (aminopeptidase, carboxypeptidase, or dipeptidase) cleave amino acid at ends of molecule, or hydrolyse dipeptide  Amino acids  Dipeptides  Tripeptides  Oligopeptides  (Some intact proteins – very few)  Final products

Protein Absorption  Passive diffusion  Hydrophobic amino acids (e.g. tryptophan)  (Mostly by) active transport – against concentration gradient and also by facilitated transport in small intestine via:  Brush border – at least 7 different mechanisms o 5 are Na + -dependent co-transporters (secondary active transport) o 2 are Na + independent  Basolateral border – at least 3 different mechanisms o Na + independent (facilitated transport) Amino acids Di- and tri-peptides  via H + -dependent mechanism at brush border (co-transport)  Further hydrolysed to amino acids within the enterocyte  Na + -independent systems at the basolateral border (facilitated transport)

Na + K+K+ K+K+ Amino acid Amino acid Simplified Scheme for Amino Acid and Peptide Absorption Secondary active transport Na + /K + ATPase Lumen Interstitium Na + H+H+ H+H+ H+H+ Peptide Facilitated transport Amino acid Hydrolysis Amino acid

Digestion of Lipids  Mouth  Lingual lipase (little effect)  Stomach  Gastric lipase (modest effect)  Small intestine  Emulsification by bile  Pancreatic lipase splits into fatty acids and monoglyceride Ingested Lipids  Fats / Oils – triacylglycerols (TAG) – 90 % of total  Phospholipids  Cholesterol and cholesterol esters  Fatty acids All are insoluble in water causing problems for digestion and absorption – only triacylglycerols are considered here.

Lipid digestion of TAG by lipases  In Stomach  Heat and movements in stomach mix food with gastric lipase which begins digestion and forms an emulsion  In duodenum  Pancreatic lipase - main lipid digestive enzyme  Aided by bile salts from gall bladder  HCO 3 - in pancreatic juice neutralises stomach acid - provides suitable pH for optimal enzyme action o Hydrolysis initially slow due to largely separate aqueous/lipid interface o As hydrolysis proceeds, rate increases due to fatty acids produced acting as surfactants breaking down lipid globules aiding emulsification o Emulsified fats ejected from stomach to duodenum H 2 CO C (CH 2 ) 16 CH 3 O HCO C (CH 2 ) 16 CH 3 O H 2 CO C (CH 2 ) 16 CH 3 O Triglyceride O Gastric lipase + H 2 O H 2 CO C (CH 2 ) 16 CH 3 HCO C (CH 2 ) 16 CH 3 O CH 2 OH Diglyceride CH 3 (CH 2 ) 16 COOH Free Fatty acid (stimulates CCK release from duodenum and secretion of pancreatic lipase) +

Role of Bile Salts (1)  Bile salts secreted in bile from the gall bladder in response to CCK act as detergents to emulsify large lipid droplets to small droplets  Failure to secrete bile salts results in:  Lipid malabsorption - steatorrhoea (fat in faeces)  Secondary vitamin deficiency due to failure to absorb lipid vitamins  Bile salts are amphipathic - Hydrophilic (projects from surface of droplet - Hydrophobic (adsorbs onto droplet) Large fat droplet _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Bile salts Increased surface area for action of lipase

Role of Bile Salts (2)  Bile salts increase surface area for attack by pancreatic lipase, but block access of the enzyme to the lipid with the hydrophobic core of the small droplets  Problem solved by colipase, an amphipathic polypeptide secreted with lipase by the pancreas – binds to bile salts and lipase allowing access by the latter to tri- and di-glycerides Small droplet Bile salt Triglyceride Lipase Colipase

Digestion by Pancreatic Lipase Produces 2- Monoglyceride and Free Fatty Acids H 2 CO C (CH 2 ) 16 CH 3 O HCO C (CH 2 ) 16 CH 3 O H 2 CO C (CH 2 ) 16 CH 3 O Triglyceride Pancreatic lipase + 2H 2 O CH 2 OH HCO C (CH 2 ) 16 CH 3 O CH 2 OH 2-monoglyceride + CH 3 (CH 2 ) 16 COOH Free fatty acid

The Final Products of Lipid Digestion are Stored in, and Released From, Mixed Micelles Hydrophobic core Fatty acid Bile salt Monoglyceride Cholesterol Phospholipid

Lipid Absorption (1)  Transfer between mixed micelles and the apical membrane of enterocytes entering by the cell by passive diffusion Free Fatty acids and monoglycerides Fatty acids Monoglycerides  Short chain (i.e.  6 carbon) and medium (i.e carbon ) fatty acids diffuse through the enterocyte, exit through the basolateral membrane and enter the villus capillaries  Long chain fatty (i.e.  12 carbon) fatty acids and monoglycerides are resynthesized to triglycerides in the endoplasmic reticulum and are subsequently incorporated into chylomicrons

Lipid Absorption (2) – Chylomicron Formation Phospholipid synthesis Apolipoprotein (ApoB-48) Cholesterol esters Central lacteal Carried in lymph vessels to systemic circulation (subclavian vein) via the thoracic duct Exocytosis Monoglyceride Free fatty acid Triglyceride synthesis Chylomicron Nascent chylomicron Endoplasmic reticulum

Lipid Absorption (3) – Chylomicron Processing  Chylomicron enters systemic circulation into the subclavian vein via the thoracic duct and distributed to tissues  Chylomicron triglyceride metabolised in capillaries (particularly muscle and adipose tissue) by lipoprotein lipase present on endothelial cells  Free fatty acids and glycerol released initially bind to albumen and are subsequently taken up by tissues  Remainder of chylomicron is a chylomicron remnant, enriched in phospholipids and cholesterol  Chylomicron remnant undergoes endocytosis by hepatocytes – cholesterol released to: o be stored o secreted unaltered in bile o oxidised to bile salts

Lipid Absorption (4) – Cholesterol Absorption  Once thought to be passive (similar to free fatty acids and monoglycerides)  Now appreciated to be mainly due to transport by endocytosis in clatherin coated pits by Niemann-Pick C1-like 1 (NPC1L1) protein  Ezetimibe binds to NPC1L1, prevents internalization, and thus cholesterol absorption. Used in conjunction with statins in hypercholesterolaemia

Absorption of Ca 2+  Occurs by passive (i.e. paracellular; whole length of small intestine) and active (i.e. transcellular; mainly duodenum and upper jejunum) transport mechanisms  With [Ca 2+ ] in chyme  5 mM absorption is mainly active  Active Ca 2+ absorption is regulated by 1,25- dihydroxyvitamin D 3 (calcitriol) and parathyroid hormone (increases 1,25- dihydroxyvitamin D 3 synthesis) Ca 2+- ATPase (PMCA1) – expression increased by 1,25-dihydroxyvitamin D3 Sodium/calcium exchanger (NXC1) Ca 2+ channel (TRPV6) – expression increased by 1,25- dihydroxyvitamin D3 Ca 2+ (high lumenal Ca 2+ ) Ca 2+ (low lumenal Ca 2+ ) Ca 2+ - calbindin-D Ca 2+ 3Na + Ca 2+ (high lumenal Ca 2+ )

Absorption of Iron  Iron – important constituent of haemoglobin, myoglobin, many enzymes  mg ingested daily – only 3-10 % absorbed (female more than male) Divalent metal transporter 1 (DMT1) Ferroportin (negatively regulated by the hormone hepcidin released from liver when body iron levels are high) – major control on iron absorption Haem carrier protein 1 Haem Fe 3+  Fe 2+ Fe 2+  Fe 3+ (Vit C) Haem oxidase Fe 2+ Apoferratin + Ferratin (storage form of iron) Fe 2+ + Transferrin Transferrin-Fe 2+ e.g. haemoglobin synthesis

Absorption of Vitamin B 12 (cobalamin)  Present in minute amounts in the diet (5-15  g day – daily requirement approximately 6  g per day, hence efficient and selective absorption required Vitamin B 12 ingested in food Salivary glands secrete haptocorin Stomach acid releases vitamin B 12 from food Haptocorin binds vitamin B 12 released in stomach Stomach parietal cells release intrinsic factor Pancreatic proteases digest haptocorin in small intestine, vitamin B 12 released Vitamin B 12 binds to intrinsic factor in small intestine Vitamin B 12 -intrinsic factor complex absorbed in terminal ileum by endocytosis

Absorption of Vitamins Fat soluble vitamins (i.e. A, D, E and K)  Incorporated into mixed micelles  Usually passively transported into enterocytes  Incorporated into chylomicrons, or VLDLs  Distributed by intestinal lymphatics Water soluble vitamins (i.e. B vitamins (but not B 12 ), C, H o Vitamin C – the Na + -dependent vitamin C transporters (SVCT1 and 2)  Transport processes in the apical membrane are similar to those described for monosaccharides, amino acids and di- and tri-peptides o Vitamin H – the Na + -dependent multivitamin transporter (SMVT) For example: o Vitamin B 9 – the Na + -independent proton-coupled folate transporter 1; FOLT)