Digestive Biochemistry

Carbohydrate Digestion and Absorption in the Small Intestine

 Structures of carbohydrates:
- starch: ¬¬glucose joined by alpha 1-4 linkage in a long chain
- amylopectin: glucose chains of alpha 1-4 linkage with alpha 1-6 branching points
- cellulose: glucose joined by beta 1-4 linkages hence undegradable in humans
 Starch digestion:
- Primary locations: preliminary digestion by salivary alpha-amylase in the stomach and then full degraded by pancreatic alpha-amylase in small intestine into maltose, maltotriose and alpha-limit dextrins
- Secondary locations: further breakdown of maltose, maltotriose and alpha-limit dextrins is carried out by the brush borders oligosaccharidases anchored into the lipid bilayer of enterocytes on the intestinal villi producing the final product glucose.
- undigested starch: 40g per day of starch each days are not degraded and is passed to the colon where huge numbers of bacterial ferment it to volatile fatty acids
 Luminal enzymes of starch digestion:
- alpha amylase: salivary and pancreatic enzyme that randomly digest cooked starch such as amylose, amylopectin and glycogen producing a variety of oligosaccharides. Acidic conditions stops salivary amylase.
 Intestinal surface enzymes of starch digestion:
- sucrase: an active 1-2 glucosidase that breaks down sucrose to glucose and fructose but also have 1-4 degradation function and can act non-specifically on maltose/maltotriose substrates.
- Maltase (glucoamylase): digestion of maltose and maltotriose and removal of glucosyl residue from non-reducing end.
- alpha-dextrinase (isomaltase): cleaving the alpha 1-6 branching units of glucose chain or alpha 1-4 linkages
- lactase: hydrolysis of beta 1-4 linkage of disaccharide lactose
 Digestion of alpha limit -dextrin: dextrin are sugar oligosaccharides consisting of 3 -7 units with 1-6 branching linkages.
- Digestion is initiated with enzyme maltase that slowly cleaves glucose units until the branching point is reached
- Alpha-dextrinase digest the alpha 1-6 linkage
- The remaining chain is degraded by sucrase/maltase/alpha-dextrinase
 Sugar absorption: only monosaccharides can be absorbed by enterocytes lining the small intestine and the three levels of intestinal folding optimize this activity. Action is performed by a Na+/glucose symporter.
- Na+/K+ ATPase at the base of the enterocytes actively pumps out Na+ and creates a concentration gradient
- Influx of Na+ into the intestinal cell on the luminal side couples glucose uptake (hence requires energy - utilizes 0.45/3 proportion of Na+ gradient.
- Fructose is absorbed purely by the concentration gradient of the cell through facilitated diffusion
 Fibrous carbohydrate: cellulose with beta 1-4 linkage indigestible by human as the correct pancreatic enzymes are not present. These are passed to colon and fermented by bacteria. Though undegradable, dietary fibres are still important in maintaining health, bulking stools and protecting against number of disease.
 Lactose: the disaccharide of glucose and galactose. When broken down by lactase, both products are absorbed via the same Na+ linked transport protein. Lactase deficiency causes lactose intolerance and lead to diarrhea and dehydration as luminal lactose draws water out paracellularly.
 Galactose-1-phosphate: intermediate of the lactose product and is toxic causing cataract in eye and liver failure

Protein Digestion:

 Initial step: food protein is first “softened” by pepsin secreted by chief cells as it passes through the stomach forming a semi-liquid paste called chyme. Acid environment also disrupt the tertiary structure of the protein and allow access of proteases
- pepsin in duodenum can cause peptic ulcer as it degrades the enterocytes
 Chymosin: an alternation of pepsin that breaks down milk particles
 Endopeptidases: pancreatic enzymes such as trypsin, chymotrypsin and elastase that digest peptide bonds within a protein. These are secreted as inactive precursor and activated in the intestinal lumen to prevent unwanted degradation of endogenous tissues
- pepsin: hydrolyze internal bonds of aromatic acids
- trpsin: act on basic amino acids
- chymotrypsin: digest aromatic amino acids, leucine, methionine and glutamine
- elastase: cleaves neutral aliphatic amino acids
 Exopeptidases: pancreatic enzymes that act upon the first peptide bond of the protein terminal.
- Carboxypeptidase A: hydrolyze external peptides bonds of carboxyl end of aromatic and neutral aliphatic amino acids
- Carboxypeptidase B: hydrolyze carboxyl peptide bond of basic amino acids
- aminopeptidase: enzymes attached to brush border that cleaves the amino end from the N-terminal
 Activation of peptidases: enterokinase attaches to the enterocytes in the upper small intestine activate trypsin which acts as the key enzyme to initiate a cascade of activation of other peptidases, i.e. by cleaving off the inactivating factors.
- Chymotrypsin activation: N-terminal has a positive charge and form ionic link with aspartic acid and changes structure of the active site hence activating it
 Degradation efficiency: as each peptidases have their own unique role and substrate, the product of one enzyme immediately becomes the substrate for another enzyme. This complementary relationship maximizes efficiency and produces peptides of only 1-3 amino units long.
 Protein absorption: there are at least 3 Na+-link amino acids transporter plus a peptide transporter that transport only di and tripeptides on the enterocyte. These are broken down later within the cell to yield free amino acids. Amino acids exit enterocytes by facilitated diffusion into the blood streams.
- Approximately 40% of ingested amino acids are absorbed as peptides of 1-3 units through luminal digestion while and the remaining 60% are broken down further by membrane bound peptidases.
- Globular protein are absorbed almost completely
 Difficulties of food digestion:
- digestibility of proteins differs and cooking helps pre-denaturation
- hammering your meat (break lysozyme to digest meat)
- skin + hair, tough tertiary and not digest
- some food rich in proteinase inhibitors may impair digestion

Lipid Digestion and Absorption in the Small Intestine

 Emulsifying agents: amphipatic substances which breaks up fatty compounds into smaller sizes and makes them water-soluble
- bile salt: made in liver and stored in gallbladder
- lecithin: phosholipid present in bile and food membranes
- 2-MAG: 2-monoacylglycerol breakdown production of TAG
 Lipid-digesting enzymes:
- Lingual lipase: active in stomach until it’s inactivated by the acid as it has a optimum pH of neutral. Degrade TAG into DAG or MAG with FA. It is more important in neonates as gastric pH is higher and milk lipid is already emulsified
- pancreatic lipase: active in small intestine and digests triglyceride into 1,2-DAG or 2-MAG. It requires a protein colipase from pancreas to activate the precursor
- Pancreatic non-specific esterase: cholesterol esterase which remove fatty acids from cholesterol esters, 2-MAG, and triglycerides containing short and medium chain fatty acids, and fat soluble vitamin esters.
- Phospholipase: removes fatty acids from 2 position of phospholipids such as lecithin to lysolecithin with co-factors such as bile salts and Ca2+. It is activated by trypsin.
- Human milk lipase: bile salt dependent enzyme that degrades fat in milk. The enzyme is stable during passage through the acidic stomach and becomes active in the small intestine
 Bile salts: steroid derivative of cholesterol produced in the liver and stored in the gallbladder between meals. The cholesterol main body is non-polar while the hydroxide groups on carbon 3, 7, and 12 and SO32- end is polar. These have four important functions in food digestion:
- surfactant that emulsify fat droplets
- removal of products from lipid digestion forming mixed micelles
- cofactors required for lipase activity
- increases rate of delivery of products of fat digestion across mucosal membrane of the enterocytes (in the form of mixed michelle)
 Micelles: particles of products of fatty digestion in the gut coated by bile salt to aid absorption.
- lecithin: lecithin with two fatty acid chains is predominantly non-polar, but with the cleaving of one FAC by phospholipase A2, the charge of the molecule is balanced making it amphipathic. The lysolecithin can then act to further stabilize the hydrophobic core in a hydrophilic environment.
 Bile acid reabsorption: bile acid is absorbed in the terminal ileum of the small intestine.
- unabsorbed bile salt is passed into the colon and lost in faeces
- bile duct obstruction can impair fat absorption, steatorrhea and upsets of bacteria in colon by fat.
 Luminal digestion of lipids:
- acid from the chyme are neutralized in the duodenum by bicarbonate ion secreted from the pancreas
- bile salt released from gall bladder emulsifies the fatty compounds, stabilizing its structure in aqueous solution and allowing soluble pancreatic lipases to act
- pancreatic lipases degrades the fatty lipids into their corresponding products and accumulation of the product on the outer rim of the oil phase forms an isotropic phase. A water channel exists to permit entrance of lipase and release of fat. If the layer is too thick, digestion is temporarily stopped.
- the bile salts then pinches off the fatty products from droplet surface as mixed micelles and these are absorbed across the enterocytes
 Milk lipids: milk is already emulsified in the stomach as the lipid is not soluble so degradation occurs in early stage.
 Lipid absorption: there is no need of transporter for lipid as the fatty products can easily traverse the hydrophobic core of the enterocyte bilayer.
 TAG and phospholipid resynthesis in enterocytes: fatty acid bind proteins in the enterocytes transport the fatty acids and monoglyceride to the smooth endoplasmic reticulum where they are reformed into TAG and phospholipid
- preparation of fatty CoA: fatty acid + ATP + HSCoA => Fatty acylSCoA + AMP + PPi
- reesterification: 2-MAG + Fatty acylSCoA => 1,2-DAG + HSCoA
 Lecithin resynthesis: 1,2-DAG formed from the phophotase reaction of phosphatidic acid reacts with a CDP-choline in which a phosphor-choline group is transferred to the 1,2-DAG and leucithin is produced.
 Export of fat:
- Larger fatty materials such as TGA are packaged in the enterocytes along with cholesterol, fatty vitamins and apoproteins into chylomicrons and passes to the lymph and then blood
- Shorter chain fatty acids are carried in the blood by albumin (a nonspecific binding protein). This is especially important in neonates
 Circulation of fat: some hydrolysis of TAG occurs in circulation by interaction with lipase on cell surface of fat cells or muscles cells. Lipoprotein lipase degrade chylomicron and remnants is absorbed by liver.
- LDL complex interact with receptors on liver and absorbed by endocytosis


 Liver functions:
- Rapidly uptake nutrient (digested food, carbohydrate, amino acids) via the hepatic portal vein to prevent entrance into the general circulation.
- Facilitation of digestion and regulation of digested product in circulation
- Liver stores glycogen and release glucose when needed
- Synthesis of proteins such as serum albumin and deaminates excess amino acids into urea
 Role of liver in cholesterol regulation:
- Major production center of cholesterol and homeostasis
 Cholesterol: a lipid molecule with several important functions:
- Structural component of membrane to maintain fluidity
- Stabilizer of the plasma lipoprotein structure
- Precursor of bile salt, an emulsifier involved in lipid digestion
- Component of myelin fat
 Liver homeostasis of cholesterol: pathological process can disrupt the regulation of cholesterol causing usually increased input or decreased output.
- Input: endocytosis of cholesterol compounds such as LDL or new synthesis
- Output: bile acid synthesis, bile salt secretion and cholesterol secretion in bile (excretory products 1100 mg per day balances cholesterol)
- Feedback inhibition: excessive cholesterol inhibits HMG-CoA reductase, synthesis of LDL receptors and promote ACAT to form cholesterol stores in liver
 Cholesterol synthesis: synthesis of endogenous cholesterol drops proportionally with increased uptake of cholesterol.
- Sources: 50% in liver, 15% in gut and rest in skin
- about 900mg made in hepatocytes, and 1100mg goes to VLDL
- 82% is made endogenously while rest is taken orally. Synthesis is stimulated by excessive saturated fat as too much acetyl CoA is made and so cholesterol is produced.
 Biosynthesis of cholesterol: cholesterol synthesis is derived from intermediate of ketone body synthesis. HMG-CoA is reduced by HMG-CoA reductase to for mevalonate which is the rate limiting step. Later through a series of reaction in which isoprenoid structure of 5 carbon is combined to form a 30 carbon squalene and eventually forms cholesterol.
- Statin: an antagonist of HMG-CoA reductase which tricks the system into believing too much cholesterol is made. Excessive amount however can cause muscle death as mevalonate, a precursor to ubiquinone and essential for ATP production, is inhibited. Deceased muscle cell release myoglobin and can jam up kidney and patient dies of kidney failure.
 Transport of cholesterol: cholesterol is not usually made in peripheral tissues.
- Cholesterol is packaged along with TGA and exported as VLDL to peripheral tissues for fat supply
- After absorption of TGA, VLDL becomes LDL, a cholesterol-rich particle whose function is to transport cholesterol to peripheral tissues
- HDL converts cholesterol to cholesterol ester to transport cholesterol from peripheral tissues to liver
 Problems of cholesterol
- Congregation of cholesterol forms gall stone
- Cholesterol in bloodstreams may form plaque and cause arthrosclerosis. Ratio of HDL and to LDL governs this.
 Bile salt synthesis: sequence of reactions that turns water-insoluble cholesterol into water-soluble bile salts. This involves addition of hydroxyl groups, saturation of sterol nucleus and side chain shortening
- Side chain shortening: process of removing C25-27 and oxidation of C24 to COOH
- Neutral pathway: first step is catalyzed by cholesterol-7α-hydroxylase which adds a hydroxyl at position 7 (rate-limiting step). This could be followed by hydroxylation at C12 and then reduction of C3 ketone and side chain shortening.
- Products of neutral pathway: primary bile salt made de novo from cholesterol. Major product is 3, 7, 12-trihydroxylcholic acid, minor is 3, 7-dihydroxylcholic acid (chenodeoxycholic acid)
- Acidic pathway: alternative pathway of bile synthesis usually due to pathological processes where neutral pathway is non-existent. In this pathway the cholesterol is first hydroxylated by enzyme CYP27 to give a COOH at C27 (rate limiting step) before continuing a similar step to neutral pathway.
- Products of acid pathway: Minor product is 3, 7, 12-trihydroxylcholic acid, major is 3, 7-dihydroxylcholic acid (chenodeoxycholic acid)
- Bile salt is produced ultimately by conjugation of the C24 COOH group with either glycine or taurine, 4 bile salts formed are glycocholate, glycochenodeoxycholate, taurocholate and taurochenodeoxycholate.
 Properties of bile salt:
- Glycine is a weak acid and taurine is a strong acid. Cholic acid is conjugated to form taurocholic acid and allow emulsification. The SO32- is the hydrophilic end and body is hydrophobic.
 Control of bile synthesis:
- In neutral pathway, bile acid feedback to inhibit CYP7A
- In acidic pathway, CYP27 is down regulated by hydrophobic bile salts and not hydrophilic ones
- CPY12 controls the ratio of cholic acid to chenodeoxycholic acid
 Bile salt secretion: hepatic secretion of bile salts across the canalicular membrane is the main driving force for bile formation. Bile consists of bile salt, organic anions, bilirubin diglucuronide, phospholipids, cholesterol and ions.
- bile salt has an effect of solubilize cholesterol and phospholipid and enhance secretion. P-glycoprotein transporter mediates primary active transport of lipids
 Secondary bile salt: primary bile salts can undergo some deconjugation and then dehydroxylation at C7 by intestinal bacteria to form secondary bile salts. These are deoxycholic acid derived from cholic acid and lithocholic acid form chenodeoxycholic acid. In liver deoxycholate is reconjugated before secretion
- Lithocolic acid is very toxic and insoluble and not absorbed by terminal ileum.
- Ratio of primary bile salts and ratio of primary to secondary bile salts are indicators for liver diseases and bacterial overgrowth respectively
 Intestinal reuptake of bile salt:
- Passive transport: account for more than 50% of uptake. Deconjugated bile salt is passively uptaken in the jejunum and ileum and dehydroxylation to deoxycholate enhances their hydrophobicity and hence enhance passive uptake.
- Na+ dependent uptake: occurs in the terminal ileum to ensure the recovery of all remaining bile salts.
- Crohn’s disease: causes fat to pass into the colon and provoke fluid secretion and diarrhea. When only 100 cm of terminal ileum is resected, bile salt absorption is impaired but fat is not as most is already absorbed. However if more than 100 cm is resected, steatorrhea occurs.
 Enterohepatic circulation:
- Bile salts return to the liver in portal blood and 70 -90% are extracted by the liver in the first pass by carrier mediated transport and passive diffusion. The rest passes into general circulation and represented later by the hepatic artery. Any deconjugated bile salts are reconjugated before secretion in bile.
- Primary and secondary biles salt are excreted across the canalicular membrane by an ATP-dependent process

Liver 2:

 Function of fat regulation:
- Synthesizes/oxidize fatty acids and produce TAG, phospholipids
- Production of VLDL to distribute TGA and uptake of chylomicron remnants
- Production of HDL to retrieve from cholesterol from extra-hepatic circulation
- Makes ketone bodies from fatty acids during starvation
 Chylomicrons: largest lipoprotein made in the enterocytes and carries TGA from gut to liver and body. Has cholesterol on the surface and cholesterol ester in the center.
- Starve overnight the plasma goes yellow. Forms milky colour after a fat diet due to precipitation (visible as size is roughly the wavelength)
 Chylomicron degradation:
- apoprotein CII on the surface of the chylomicron is recognized by lipoprotein lipase of peripheral tissue (adipose or muscle) to break down TGA into free fatty acid. Only 60% of TGA is degraded while the 40% is resistant as when TGA becomes the minor component of a chylomicron, enzymes can not penetrate.
- Hepatic lipase on the surface of hepatocytes also degrades the chylomicron and remnants are taken up by endocytosis due to recognition of Apo-E by remnant receptor
 VLDL: very low density lipoprotein is synthesized from TAG (to be exported), cholesterol coating and cholesterol esters.
 Degradation of VLDL: Apo C-II having a large extension of the carboxyl terminal allows extrahepatic tissues with lipoprotein lipase to easily recognize the VLDL and degrade the TAG, taking up released fatty acids. Remnants forms IDL and then LDL.
 LDL uptake: LDL can be endocytosed liver or extra-hepatic tissues by LDL receptors that recognize the B-100 component and degraded within the cell as a secondary lysosome. The cholesterol is transferred and stored.
 HDL function: activates LCAT which esterify cholesterol by transferring an ester group from lecithin to cholesterol. Loss of LCAT can lead to high rate artheroschlerosis and cornea is filled with cholesterol.
 Reverse cholesterol pathway: to prevent toxic overload of cholesterol in peripheral cells, HDL delivers the cholesterol to the liver. ATP-binding cassette transporter A1 is formed on the surface of the cells and facilitate cholesterol exit while Apo A-I stimulate LCAT to convert received cholesterol to cholesterol-ester.
- CETP: a negative molecule in the plasma that transfer cholesterol ester to LDL and VLDL particle and is inversely correlated to HDL levels. Suggested to be used as a therapeutic method by inhibiting its expression.
 Degradation of HDL: HRHL, heparin-releasable hepatic lipase hydrolyses the phospholipids on the surface of HDL2 releasing cholesterol for uptake to the liver and forming smaller and more dense HDL3 (no cholesterol). HDL3 then proceed in circulation to pick up more cholesterol
 Density of lipoprotein: lipoprotein with the lowest TGA content has the highest density thus as chylomicron/VLDL is degraded into remnants and LDL etc, the protein to TGA ratio increase and thus increased density.
 Apoproteins:
- mature mRNA of B-100 is twice the length of B-48
 Roles of liver in xenobiotic metabolism: xenobiotics are foreign chemicals including drugs, chemical carcinogens and insecticides etc. metabolism of haem and secretion in bile as bilirubin diglucuronide is a good example.
- Phase I: hydroxylation of enzyme by cytochrome P450
- Phase II: conjugation with various polar metabolites such as glucoronic acid to make them more soluble and capable of excretion urine or bile
- Activities of cytochrome: enzyme is located in the endoplasmic reticulum of liver cells and are affected by factors such as age, sex and other drug effects

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