Digestion and Absorption of Protein

Digestion and Absorption of Protein

Paul J. Moughan, PhD, DSc, FRSC, FRSNZ and Bruce R. Stevens, PhD

For the body to assimilate protein, it must be first broken down into small peptides and free amino acids. This occurs to a limited extent in the stomach, with most hydrolysis and absorption occurring in the small intestine. The digestion and absorption processes ultimately supply the circulating blood with primarily free amino acids, in addition to very small amounts of physiologically active small peptide fragments. In the absorptive state, amino acids are transported via the portal blood from the small intestine directly to the liver, with subsequent transport to other organs.

The Food and Agriculture Organization, World Health Organization, and United Nations University (FAO/WHO/UNU, 2007) define a “safe” daily intake of dietary protein as 0.83 g high- quality protein per kg body weight, or 58 g/day for the reference 70-kg man and 47 g/day for the reference 57-kg woman. The approximate median intake of protein for adults aged 31 to 50 years in the United States is 100 g/day for men and 65 g/day for women. In addition to food proteins, the body digests 50 to 100 g per day of endogenous protein that is secreted into or sloughed into the lumen of the gastrointestinal tract. These endogenous proteins include proteins in saliva, gastric juice, and other secretions; pancreatic enzymes; mucoproteins; sloughed intestinal cells; and proteins that leak into the intestinal lumen from the blood. Most of this mixture of exogenous and endogenous proteins (115 to 200 g/day) is efficiently digested and taken up by the absorptive enterocytes as free amino acids and dipeptides and tripeptides. Around 85% of the total protein is absorbed anterior to the end of the small intestine (terminal ileum), with around 10 to 20 g of protein entering the colon each day. Daily fecal nitrogen losses amount to the equivalent of about 10 g of protein. The nitrogen excreted in the feces represents primarily endogenous or dietary nitrogen that was not absorbed from the small intestine; this unabsorbed nitrogen was used in the large intestine by the microflora for growth and is therefore mainly present in the feces as part of the bacterial mass.

Digestion of Protein in the Gastrointestinal Tract

An overall concept diagram of the major aspects of protein digestion and absorption is presented in Figure 9-1, and a typical flow of protein in the adult human is shown in Figure 9-2. The normal events of digestion and absorption are grouped into phases corresponding to physiological events. The six major phases covered in this chapter primarily involve the following:

1. Gastric hydrolysis of peptide linkages in the protein

2. Digestion of protein to smaller peptides by action of pancreatic proteases, which are secreted as zymogens and activated in the lumen of the small intestine where they then carry out digestion

3. Hydrolysis of peptide linkages in oligopeptides by apical (brush border) membrane peptidases and transport of amino acids, dipeptides, and tripeptides across the brush border membrane of the absorptive enterocytes

4. Further digestion of dipeptides and tripeptides by cytoplasmic peptidases in the enterocytes

5. Metabolism of some amino acids within the enterocytes

6. Transport of amino acids across the basolateral membrane of the enterocytes into the interstitial fluid from which the amino acids enter the venous capillaries and hence the portal blood

The Gastric Phase: Denaturation and Initial Hydrolysis of Proteins

Protein digestion begins with modest processing by the stomach. Here, gastric hydrochloric acid (HCl) and pepsins partially denature and hydrolyze proteins. The stomach plays a minor role in the overall process of protein digestion and serves primarily to prepare polypeptides for the main events of digestion and absorption that take place within the small intestine. Indeed, complete protein assimilation occurs even after surgical removal of the stomach.

When food is present in the stomach, or if the appropriate vagal cholinergic efferents are activated, the gastric chief cells secrete inactive pepsinogens. Several isozymes of pepsinogen are released, and each is converted to an active pepsin isozyme by cleavage of a peptide from the amino (N) terminus. Activation occurs spontaneously at a pH of less than 5 by an intramolecular process that involves proteolytic cleavage of a highly basic N-terminal precursor segment. After this autoactivation process forms some pepsin, activation of pepsinogen by active pepsin (autocatalysis) also occurs.

Pepsins are chemically categorized as endopeptidases because they attack peptide bonds within the polypeptide chain. Their catalytic mechanism involves two carboxylic acid groups at the active site of the enzyme, so pepsins are classified as carboxyl proteases. Most digestive enzymes are relatively permissive in the range of substrates they will accept, and the pepsins partially hydrolyze a broad variety of proteins to large peptide fragments and some free amino acids. Pepsins show a preference for hydrolysis of internal peptide bonds that involve the carboxyl groups of tyrosine, phenylalanine, or tryptophan residues and that do not involve a linkage to the imino group of proline.

Small Intestinal Luminal Phase: Activation and Action of Pancreatic Proteolytic Enzymes

Following partial hydrolysis of protein in the stomach, the polypeptides and amino acids enter the lumen of the proximal small intestine where they stimulate the mucosal enterocytes to release the hormone cholecystokinin (CCK) into the circulation. CCK subsequently reaches the pancreas, where it binds to the acinar cells and stimulates the secretion of a variety of enzymes and zymogens by the exocrine pancreas. These exocrine secretions are delivered to the small intestinal lumen by the pancreatic duct, which joins the common bile duct that drains into the duodenum. In addition to the effects of CCK, stomach distention or the sight and smell of food invoke parasympathetic cholinergic vagal nerve efferents that in turn also stimulate the exocrine pancreatic acinar cells to release enzymes and zymogens. Zymogen is the general term for the inactive proenzyme form of an enzyme. The structure of the zymogen must be modified for it to be enzymatically active. Like the pepsinogens released by the gastric glands in the stomach, all of the pancreatic proteases are released in zymogen form.

Based on work that originated in the Russian laboratory of Ivan Pavlov in the late 1890s, research has established that protein digestion involves a multistep conversion of inactive zymogens to their active states within the lumen of the small intestine. The current understanding of the activation cascade for these pancreatic zymogens is summarized in Figure 9-3.

Pancreatic Zymogens and their Activation Cascade

The major pancreatic zymogens are trypsinogen-1, trypsinogen-2, proelastase, chymotrypsinogen, procarboxypeptidase A, and procarboxypeptidase B. The initial step of the activation cascade is catalyzed by enteropeptidase (also called enterokinase). It is bound to the apical membrane of


Trypsin Inhibitors

Low-molecular-weight proteins or polypeptides that act as protease inhibitors are naturally produced by cells in both animals and plants. In particular, legumes (peas, beans, and lentils) and cereals (wheat, buckwheat, and rice bran) contain trypsin inhibitors that can lower the nutritional quality of their proteins. These trypsin inhibitors can be inactivated to a large extent by wet heating or removed by processing techniques used during protein concentration and isolation (e.g., soy protein). Soybean trypsin inhibitors have been widely studied. Although these are inactivated by heating, animals sometimes ingest large amounts of these inhibitors by consuming raw soybeans.

The pancreas, intestinal cells, liver, and other tissues also synthesize a certain amount of trypsin inhibitors. For example, human pancreatic secretory trypsin inhibitor is secreted from pancreatic acinar cells into the pancreatic duct along with the zymogen precursors of the proteolytic digestive enzymes. The possibility that some symptoms observed in diseases such as acute pancreatitis or gastric ulcer result from an absence of normal synthesis/secretion of these inhibitors is under active investigation (Keim, 2008). The therapeutic use of trypsin inhibitors to treat pancreatitis and other inflammatory conditions is also being tested in animal models.

the enterocytes of the proximal small intestine (duodenum/upper jejunum). Human enteropeptidase is a heavily glycosylated protein with an N-terminal transmembrane domain and a C-terminal extracellular serine protease domain. Enteropeptidase is classified as a serine protease (or serine endopeptidase) because it has a serine residue at its active site as part of a histidine/serine/aspartate catalytic triad.

The importance of enteropeptidase is emphasized by the fact that congenital deficiency of this enzyme leads to life-threatening malabsorption of amino acids. Intestinal enteropeptidase cleaves off an amino terminal octapeptide, Ala-Pro-Phe-Asp-Asp-Asp-Asp-Lys, from trypsinogen-2 and cleaves off the same octapeptide or a pentapeptide, Asp-Asp-Asp-Asp-Lys, from trypsinogen-1. This cleavage yields activated trypsin-1 and trypsin-2 enzymes within the intestinal lumen. The specificity of enteropeptidase for trypsinogen is high; the scissile (to be cleaved) peptide bond in trypsinogen is between the carboxyl group of a lysine residue and the amino group of an isoleucine residue.

Trypsin, which is also a member of the serine protease family but which has a very different specificity than enteropeptidase, then activates the other zymogens required for protein digestion (chymotrypsinogen, proelastase, carboxypeptidases A and B), as well as precursors of proteins required for lipid digestion (procolipase and prophospholipase A2), by cleaving off selected peptide sequences. The net result of this cascade is a pool of activated proteases within the lumen of the small intestine. Proteolysis is facilitated by the secretion of pancreatic bicarbonate into the intestinal lumen; the bicarbonate neutralizes the gastric acid in the chyme to bring the pH of the intestinal contents to 6 to 7, which is optimal for activity of pancreatic proteases.

It was formerly thought that once some trypsin was formed from trypsinogen by enteropeptidase, the active trypsin could act on trypsinogen as substrate in an autocatalytic process. Although both trypsin and enteropeptidase cleave at scissile bonds that involve a basic residue (lysine or arginine) attached to an isoleucine residue, it is now recognized that the aspartate-rich sequence in the activation peptide segment of trypsinogen suppresses the ability of trypsin to accept trypsinogen as a substrate for autoactivation. Thus enteropeptidase in the small intestine is essential for activation of trypsinogen and the subsequent zymogen activation cascade.

A benefit of synthesis of proteolytic enzymes as zymogens, with activation occurring after the proenzymes have been secreted into the intestinal lumen, is the prevention of proteolytic digestion and tissue damage within the pancreas and pancreatic duct. In addition to this protective mechanism, pancreatic juice normally contains a small peptide that inhibits trypsin to prevent any small amount of trypsin prematurely formed within the pancreatic cells or pancreatic ducts from catalyzing proteolysis. The absence of this protective mechanism leads to pancreatitis. Gain-of-function mutations and copy number variability of the trypsinogen gene, as well as loss-of-function variants for the pancreatic secretory trypsin inhibitor, have firmly established that prematurely activated trypsin causes chronic pancreatitis (Chen and Férec, 2009).

Pancreatic Digestive Enzymes

The pancreatic enzymes can be divided into two general types—serine proteases and carboxypeptidases. Trypsin, chymotrypsin, and elastase are all serine endopeptidases. They are categorized as endopeptidases because they hydrolyze internal peptide bonds within the polypeptide. They are classified as serine proteases because of their catalytic mechanisms, which involve a serine residue in the catalytic site. Serine proteases are normally synthesized in inactive zymogen or proenzyme form. Each of these serine proteases catalyzes the hydrolysis of peptide bonds but with different selectivities or preferences for the side chains flanking the scissile peptide bond. The site of hydrolysis in the polypeptide substrate (i.e., the scissile peptide bond) is flanked by approximately four amino acid residues in both directions that can bind to the enzyme and impact the reactivity of the peptide bond hydrolyzed; the hydrolyzable bond is designated P1-P′1 and adjacent amino acids are numbered P2, P3, and P4 toward the N-terminus, and P′2, P′3, and P′4 toward the carboxyl (C) terminus. Trypsin is most likely to cleave peptide bonds with a positively charged residue (arginine or lysine) at the P1 site (contributing the carboxyl group to the peptide bond); chymotrypsin prefers bonds in which large hydrophobic amino acid residues, such as tryptophan, phenylalanine, tyrosine, methionine, or leucine, are at the P1 site; and elastase preferentially cleaves peptide bonds that have a small neutral residue, such as alanine, serine, glycine, or valine, at the P1 site. Proline at the P′1 site inhibits cleavage by all three serine proteases. The rate at which particular bonds are cleaved also varies with the identities of the amino acid residues in the adjacent positions (P2-P4 and P′2-P′4).

The second group of proteolytic enzymes secreted by the pancreas, the carboxypeptidases, are exopeptidases that cleave off one amino acid at a time from the C-terminus of the substrate. These exopeptidases can attack the oligopeptides formed by the endopeptidases to sequentially cleave off free amino acids, leaving a mixture of free amino acids and small peptides of two to eight residues. Carboxypeptidases A and B are metalloenzymes that require Zn2+ at the active site, where the cation functions as a Lewis acid. (See Chapter 37 for a discussion of zinc metalloenzymes.) Carboxypeptidase B preferentially cleaves C-terminal lysine or arginine residues of peptides, and carboxypeptidase A selectively hydrolyzes most C-terminal amino acids, except proline, lysine, and arginine, with a preference for valine, leucine, isoleucine, and alanine. Neither carboxypeptidase A nor carboxypeptidase B readily cleaves C-terminal amino acids that are linked to a proline residue.

These pancreatic enzymes act as a team within the small intestinal lumen to hydrolyze many of the peptide bonds in proteins and to efficiently digest protein to yield small peptides (two to eight residues) and free amino acids. It is also important to realize that the upper digestive tract of humans contains an active microflora, and bacteria undoubtedly have a role in the digestive breakdown of food and especially of some of the endogenous proteins. Although the size of the microflora in the colon is much greater than that in the upper digestive tract, there is considerable evidence for microbial activity in the upper tract (Moughan, 2003), and it seems likely, though more experimental evidence is needed, that bacterial enzymes complement to some extent the mammalian proteases.

Small Intestinal Mucosal Phase: Brush Border and Cytosolic Peptidases

The products of pancreatic hydrolysis are free amino acids, tripeptides and dipeptides, and larger peptide fragments called oligopeptides. The free amino acids, dipeptides, and tripeptides are transported across the absorptive epithelial cell brush border membrane by specific carriers. Most larger oligopeptides are not transported but must be further hydrolyzed by epithelial brush border membrane–bound enzymes.

These brush border membrane peptidases are dimers that extend into the lumen about 15 nm from the membrane surface. One subunit is anchored to the membrane, and the other subunit participates in the hydrolysis of luminal peptides. These peptidases are all exopeptidases and are further classified as aminopeptidases or carboxypeptidases, depending on whether they hydrolyze the cleavage of amino acids one at a time from the N-terminus or C-terminus of the peptide. A variety of membrane-bound aminopeptidases exist, but the apical membrane of enterocytes possesses only a single known carboxypeptidase, which is peptidyl dipeptidase. As for the pancreatic proteases, these enzymes also show specificities or preferences for the amino acid residues and peptide sequences that they hydrolyze.

For the majority of tripeptides and dipeptides that are transported into the enterocyte, additional cytosolic aminopeptidases act within the absorptive epithelial cells to complete the process of hydrolyzing proteins and peptides to free amino acids. Most protein nitrogen exits the basolateral membrane to the portal blood as free amino acids. As explained later, certain dipeptides, such as carnosine, and a small fraction (∼1%) of incompletely digested luminal protein and peptides may enter the portal blood intact.

Absorption of Free Amino Acids and Small Peptides

The products of digestion—free amino acids, dipeptides, and tripeptides—are absorbed from the lumen by a variety of transport mechanisms. In this section, free amino acid absorption mechanisms are presented first and are followed by a discussion of dipeptide and tripeptide absorption.

Amino Acid Transporters in the Apical and Basolateral Membranes

Free amino acids are initially taken up by transporters in the luminal-facing apical membrane of villous absorptive enterocytes and subsequently exit those cells via other basolateral membrane transporters. The amino acids can either pass through the enterocyte unmetabolized, be used for protein synthesis in the enterocyte, be partially (or completely) oxidized for energy, or undergo intermediary metabolic conversion into other amino acids or metabolites that, in turn, are subsequently transported out of the cell across the basolateral membrane. Following basolateral membrane transport to the interstitial fluid, the amino acids move into villus capillaries and on to the liver via the portal circulation, as summarized in Figure 9-1. The intestine is highly efficient in extracting the dietary essential and nonessential amino acids from the lumen as free amino acids. This occurs largely because of the activity of brush border and basolateral membrane transporter systems that handle specific amino acids. Some of the absorbed amino acids are used by the enterocytes themselves, most notably glutamine, which is used as the primary fuel source in enterocytes in place of glucose. Enterocyte basolateral membrane transporters also take up enterocyte-sustaining amino acids from the blood circulation, especially in the postprandial state.

A transport “system” is defined as a physiological functional unit formed from one or more transporter protein subunits. Each transporter subunit type is encoded by a specific gene. A transport system activity may result from the action of a single transporter protein or the multimeric arrangement of transporter proteins within the membrane. Although it is technically correct to use the term transporter to mean only a single protein, scientists often informally also refer to multimeric functional units as transporters. Membrane amino acid transporter systems composed of a single protein (monomeric transport systems) are listed in Table 9-1, whereas heterodimeric transporter systems for amino acids are listed in Table 9-2.


Monomeric Amino Acid Transport Systems in Human Small Intestine or Colon

SLC1 FAMILY              
XAG EAAT3 SLC1A1 9q24 L-Glutamate, D/L-aspartate, cystine (disulfide) H+, Na+, K+ Small intestine Apical
ASC ASCT1 SLC1A4 2p13-p15 Alanine, serine, threonine, cysteine, glutamine Na+ Small intestine Apical
ASC ASCT2 or ATB0 SLC1A5 19q13.3 Alanine, serine, threonine, cysteine, glutamine, branched neutrals Na+ Small intestine, colon Apical
SLC6 FAMILY              
Creatine CRTR SLC6A8 Xq28 Creatine Na+, Cl Small intestine Apical
GLY GLYT1 SLC6A9 1p33 Glycine Na+, Cl Small intestine Basolateral
B0,+ ATB0,+ SLC6A14 Xq23-q24 Neutrals and dibasics, arginine, D-serine Na+, Cl Colon Apical
B0 (or B) B0AT1 SLC6A19 5p15.33 Neutrals, glutamine Na+ Small intestine Apical
IMINO SIT1 SLC6A20 3p21.6 Proline, sarcosine, pipecolate Na+ Small intestine, colon Apical
SLC7 FAMILY              
y+ CAT-1 SLC7A1 13q12-q14 Arginine, ornithine, lysine, histidine, dibasics None Small intestine, colon Basolateral
SLC15 FAMILY              
Pept1 PEPT1 SLC15A1 13q33-q34 Dipeptides & tripeptides, carnosine, β-lactam antibiotics, angiotensin-converting enzyme inhibitors H+ with NHE3 Small intestine Apical
SLC16 FAMILY              
T TAT1 SLC16A10 6q21-q22 Aromatics, L-DOPA None Small intestine Basolateral
SLC22 FAMILY              
OCTN2VT OCTN2 SLC22A5 5q23.3 L-Carnitine, acetyl-L-carnitine Na+ Small intestine Apical
SLC36 FAMILY              
Iminoacid PAT1 SLC36A1 5q33.1 Proline, glycine, β-alanine, GABA, taurine, D-serine H+ with NHE3 Small intestine, colon Apical
SLC38 FAMILY              
A SNAT2 SLC38A2 12q Alanine, asparagine, cysteine, glutamine, glycine, histidine, methionine, proline, serine Na+ Small intestine Basolateral
A SNAT4 SLC38A4 12q13 Alanine, asparagine, cysteine, glycine, threonine Na+ Small intestine Basolateral
N SNAT5 SLC38A5 Xp11.23 Glutamine, histidine, serine, asparagine, alanine Na+, H+ Small intestine (crypt cells) Apical
SLC43 FAMILY              
LAT4 LAT4 SLC43A2 17p13.3 Branched-chain amino acids, phenylalaninine None Small intestine Basolateral

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Feb 26, 2017 | Posted by in PHARMACY | Comments Off on Digestion and Absorption of Protein

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