1. Protein Digestion and Absorption
Christine Waasdorp Hurtado, MD, MSCS, FAAP
Gregg Kobak, MD
University of Colorado School of Medicine
Children’s Hospital Colorado
Reviewed by:
Michael Narkewicz, MD
Jeremiah Levine, MD

2. NASPGHAN Physiology Education Series
Series Editors:
Christine Waasdorp Hurtado, MD, MSCS, FAAP
Daniel Kamin, MD
This is a lecture series created by faculty from Children’s Hospital Colorado, Boston’s Children’s, Morgan Stanley Children’s Hospital of New York, and UCLA Children’s and supported by NASPGHAN.
To maximize the effectiveness of the physiology series, the text document should be reviewed by lecture attendees before presentation of the PowerPoint. We encourage faculty and fellows to modify the content to meet their educational goals.
Thanks to NASPGHAN for supporting this educational tool.
Christine Waasdorp Hurtado, MD and Daniel Kamin, MD

3. Protein Digestion Objectives
Understand the structure of protein
Understand protein digestion and absorption
Discuss clinical implications

4. What is Protein? Proteins are sequences of amino acids
There are 20 amino acids
9 are essential amino acids
phenylalanine, valine, threonine, tryptophan, isoleucine, methionine, histidine, leucine, lysine
11 are non-essential
4 amino acids are considered conditionally essential
arginine, tyrosine, glutamine, and cysteine (glycine, proline and serine sometimes considered conditionally essential)
Require 0.75g/kg body weight
Increased requirements in infants and illness
Proteins are sequences of amino acids (AA) linked by peptide bonds. There are twenty amino acids of which nine are essential and eleven are non-essential. Essential AA can’t be synthesized by body and must be present in the diet or a deficiency will develop. Four amino acids are considered conditionally essential including arginine, tyrosine, glutamine, and cysteine. Conditionally essential AA are typically present, but in certain conditions a deficiency may develop. An example is found in the disease phenylketonuria (PKU). Individuals living with PKU must keep their intake of phenylalanine extremely low to prevent mental retardation and other metabolic complications. However, they can’t synthesize tyrosine from phenylalanine, so tyrosine becomes essential in the diet of PKU patients.
Essential AA Pneumonic– PVT TIM HaLL (phenylalanine, valine, threonine, tryptophan, isoleucine, methionine, histidine, leucine, lysine)
Clinical correlation – Essential AA are found in animal sources, but vegetable sources have limited AA. Vegans must mix their nutrition sources to meet the needs of essential AA

5. Amino Acids: Structure
Consist of a central carbon atom bonded to: a hydrogen, a carboxylic acid, an amino group, and an additional side group that is unique to each amino acid
The side group creates unique characteristics for each amino acid so they differ in: shape, size, composition, electrical charge, and pH H N C R OH O
Amino Group
Side Group
Carboxyl
Group

6. Amino Acids: Structure
Amino acids (AA) are linked to form proteins
Amino acids are joined by PEPTIDE bonds
Dipeptide – 2 amino acids
Tripeptide – 3 amino acids
Oligopeptides – 4-10 amino acids
Polypeptide – more than 10 amino acids
Most proteins in the body and diet are long polypeptides (100s of AA)

7. Overview of Protein Digestion
A brief overview of protein digestion and absorption
Image from Carolyn Holocroft

8. Protein Digestion Whole proteins are not absorbed
Too large to pass through intact cell membranes
They must be digested into di- and tri-peptides or individual AAs prior to absorption
Additional digestion occurs in the cytosol
Structures of protein are more diverse than carbohydrates
Require broad spectrum peptidases and transporters
protein digestion is more complex than carbohydrate digestion requiring more enzymes and more transporters

9. Protein Digestion - Mouth
No digestion occurs in the oral cavity or esophagus

10. Protein Digestion - Stomach
Gastric phase
Gastrin released from G cells
Stretch receptors stimulate release
AA in stomach stimulate release
Parietal cells secrete HCl
Gastrin stimulates Parietal cells to secrete HCl
HCl denatures proteins - 40, 30, and 20 structures
Converts pepsinogen to pepsin
Pepsin auto-activated at acid pH. Cleaves proteins at neutral AA with large side groups. Inactivated >pH 4.5.
Pepsin breaks proteins down into peptides of various lengths and some AA
Clinical Correlation
Implications for using PPI – pH of stomach will always be above 4.5
Gastric digestion accounts for small portion of protein digestion. Limited effect from PPI and gastric bypass.
See Gastric Secretions module for full details of gastric secretion
Image from: Sweety Mehta

11. Protein Digestion - Stomach
Chief cells secrete Pepsinogen
Stimulated by cephalic vagal input
Secretion enhanced
Acetylcholine, CCK and gastrin
Pepsinogen is auto-activated at pH <4 to Pepsin
Cleavage of an N-terminal peptide
Pepsin can break down collagen
Pepsin cleaves proteins at large aliphatic or aromatic side groups
Completes ~ 10-20% of digestion
Pepsin is inactivated at pH >4.5
Protects intestinal tissues
Protein leaves stomach as mix of insoluble protein, soluble protein, peptides and amino acids

12. Protein Digestion - Intestines
Intestinal phase Overview
Majority of proteolysis occurs in the intestines
70% of proteins are converted to oligopeptides
Still require terminal action at enterocyte membrane
Bulk of proteolysis occurs in the small intestine because of pancreatic proteases.
70% of proteins turned into oligopeptides. Rest are single amino acids.
Mucosal phase of digestion and absorption is also vital
Image from: Sweety Mehta

13. Protein Digestion – Small Intestine
Majority of protein digestion occurs within the intestine due to the action of pancreatic proteases.
Proteases break down polypeptides into smaller peptides and AA
Two main forms of pancreatic enzymes
Endopeptidase – cleaves internal bonds
Ectopeptidase – cleaves AA at C-terminus
Proteins are broken down to Tripeptides, Dipeptides, Free amino acids through the mechanisms of proteases
Enzymes stored in inactive form to avoid auto digestion of the pancreas

14. Protein Digestion - Intestine
Endopeptidase: hydrolyze internal peptide bonds:
Trypsin (Pancreas)
Chymotrypsin (Pancreas)
Elastase (Pancreas)
Pepsin (Gastric)
Ectopeptidase: hydrolyzes external peptide bonds:
Carboxypeptidase A (Pancreas)
Carboxypeptidase B (Pancreas)
Aminopeptidase (Pancreas, Brush Border, Cytoplasm)

15. Pancreatic Proteases
Pancreatic enzymes stored in acinar cells as pro-enzymes (zymogens)
Trypsinogen Trypsin
Chymotrypsinogen Chymotrypsin
Procarboxypeptidase A and B Carboxypeptidase A and B
Proelastase Elastase
Zymogens protect the pancreas from auto digestion

16. Protein Digestion - Pancreas
Zymogens are converted to active form in the intestine
Trypsinogen Trypsin
Endopeptidase
Cleaves on carbonyl side of Lysine & Arginine
Chymotrypsinogen Chymotrypsin
Cleaves carboxy terminal tyrosine, phenylalanine, tryptophan, methionine, and leucine
Procarboxypeptidase A/B Carboxypeptidase
Ectopeptidase
Removes carboxy terminal residues
Enterokinase/ Trypsin
Trypsin
Chyme in the duodenum stimulates the release of enterokinase (= enteropeptidase) which converts trypsinogen into trypsin (active form). Enterokinase is secreted from the cells lining duodenum. Enterokinase cleaves N-terminal hexapeptide of trypsinogen yielding the active enzyme trypsin. Trypsin then converts the other proenzymes to active enzymes. Trypsin has an optimal pH of allowing function in small intestine. Luminal protein digestion produces single amino acids and small peptides (dipeptides, tripeptides and tetrapeptides)
Inhibition of trypsin will slow activation of other proteases
Secretory granules have a trypsin inhibitor to avoid activation and autodigestion
Trypsin

17. Protein Digestion - Intestine
Large peptides from gastric proteolysis are sequentially cleaved in the small intestine
Endopeptidases – cleave in the middle of peptide chains
Trypsin
Chymotrypsin
Elastase
Yield shorter chains with neutral or basic AA at C-terminus
Carboxypeptidase A and Carboxypeptidase B then act
Carboxypeptidase A act on neutral AA
Carboxypeptidase B acts on basic AA
Trypsin and Chymotrypsin responsible for bulk of protease activity

18. Protein Digestion - Trypsin Inhibitors
Small proteins or peptides
Present in plants, organs, and fluids
Soybeans, peas, beans, wheat
Pancreas, colostrum
Decrease activity of trypsin
Decrease all other proteases activated by trypsin
Inactivated by heat
Trypsin inhibitors also present in the secretory vesicles to prevent activation

19. Protein Digestion - Mucosal
Intraluminal degradation of proteins by gastric and pancreatic enzymes is incomplete
Brush border hydrolysis necessary
Diversity of substrates requires diverse hydrolyases
Membrane bound on villi
Not present in crypt cells
Produce free AA and small peptides
Need brush border hydrolyases, Endopeptidases and ectopeptidases, because the luminal material still not suited for intracellular transport.
Digested proteins are transported into cell as small peptides and amino acids via Na co-transporters or endocytosis
Additional digestion occurs in the enterocytes by peptidases breaking peptides down into amino acids
Brush border peptidases are integral membrane proteins that produce single amino acids and smaller peptides from tetrapeptides and larger peptides. Intracellular cytoplasmic peptidases break down dipeptides and tripeptides into single amino acids.

20. Protein Digestion - Mucosal
Small intestine
(brush border)
Aminopeptidases
Cleave at N-terminal AA
Dipeptidases
Cleave dipeptides
Carboxypeptidases
Cleaves at the carboxy-terminal
The peptidases within the microvilli split the remaining polypeptides into tripeptides, dipeptides, and some amino acids for absorption into the enterocyte
Image from John Yaw-Jong Tsai

21. Protein Digestion - Cytosol
The vast majority of di- and tri-peptides are digested into amino acids by cytoplasmic peptidases.
Peptidases
Cleave N-terminal AAs

22. Protein Absorption Overview
Most protein absorption takes place in the duodenum and jejunum
Tripeptides, Dipeptides and AA are absorbed
There is minimal absorption of peptides longer than three amino acids
Most AA are absorbed into the bloodstream, but some remain in the enterocytes and are used to support the cells
>99% of protein enters the bloodstream as amino acids
Intracellular cytoplasmic peptidases digest everything into single amino acids which are absorbed into the bloodstream or used by the cell

23. Protein Absorption - Peptides
Essentially no absorption of peptides longer than three amino acids
Di- and tri-peptides are more rapidly absorbed than free amino acids due to PEPT1
Active transporter – PEPT1
Coupled to sodium-hydrogen exchanger (NHE3)
Accommodates various sizes and charges
Di- and tri-peptides are digested into amino acids by cytoplasmic peptidases
PEPT1
Some amino acids share the same transport system, so if a large amount of one particular amino acid is consumed, the absorption of other AA that share the same transporter may be inhibited
Clinical correlation – Shortly after birth neonates can absorb intact proteins. These proteins pass through the soluble (lumen) and membrane-bound proteases remaining intact. Once mature the enterocytes do not have transporters to transfer intact proteins across the plasma membrane. They can’t permeate tight junctions either. Examples of the intact proteins are immunoglobulins from colostrum which help support immunity. The intestine lose this ability to traffic full proteins.
NHE3
Groff & Gropper, 2000

24. Amino Acid Absorption/Transport - Enterocyte
Transporter
Substrate
Ion dependence
Disease
SLC1A1
Anionic amino acids
Na+, K+, H+
Dicarboxylic aciduria
SLC1A5
Ala,Ser,Cys,Gln,Asn
Na+
SLC6A6
Taurine
Na+, Cl-
SLC6A14
Neutral and cationic amino acids
SLC6A19
Neutral amino acids
Hartnup
SLC6A20
Imino acids
SLC7A9/SLC3A1#
Neutral and cationic amino acids, cysteine
None
Cystinuria
SLC36A1
Small neutral amino acids H+
# - heterodimer
Barrett, Gastrointestinal Physiology, 2013

25. Protein Absorption - Amino Acids
The Na+ dependent transporters mechanism
Transporter binds amino acids only after binding sodium
Conformational change occurs - dumping Na+ and the amino acid into the cytoplasm
The transporter then re-orients back to its original form allow transport of the next amino acid
Absorption of amino acids is dependent on the electrochemical gradient of Na+ across the epithelium

26. Protein Absorption - Amino Acids
There are 7 transporters of free amino acids in the brush border
Mechanism varies
Na+-independent carriers (2)
Proton co-transport
Facilitated diffusion
Na+-dependent (5)
Carriers are coupled to Na+ concentration gradient

27. Protein Transport - Enterocyte
The basolateral membrane of the enterocyte contains transporters which export amino acids from the cell into the blood
Diffusion
Both Na+ dependent and independent carriers
Only free amino acids absorbed into blood
Groff & Gropper, 2000

28. Protein Absorption - Intact Proteins
Absorption of intact proteins occurs rarely
Shortly after birth, neonates can absorb intact proteins
Very few proteins can get through the gauntlet of soluble (lumen) and membrane-bound proteases intact
“Normal” enterocytes do not have the transporters needed to carry proteins across the plasma membrane and they can’t permeate tight junctions
Most of these intact proteins are immunoglobulins which can be absorbed from the very first milk (colostrum) and this imparts early neonatal passive immunity.
“Closure” is when the small intestine loses the capacity to absorb intact proteins.

29. Clinical Correlation Hartnup Disease
Abnormal transport of neutral AA (ie – tryptophan)
Mutation in SCL6A19 transporter
Clinical variability
No symptoms to rash or neurologic symptoms
Due to lack of nicotinamide (tryptophan metabolite)
Diagnosis
High levels of neutral AA in the urine
Treatment – supplement with nicotinamide

30. Clinical Correlation Cystinuria
Decreased intestinal absorption of dibasic AA (lysine, arginine, cystine)
Defect in SLC3A1 transporter
Presentation
Kidney stones – accounts for 10%
Diagnosis
Elevated urine cystine
Treatment
Hydration
Limit dietary sodium and methionine
Other transporter defects can present similarly and not all patients with this transport defect present clinically. This is due to large overlap in specificities and function in transporters and absorption process.

31. Protein Digestion - Summary
Proteins are digested into amino acids and di and tri peptides in the stomach and small intestine
The vast majority of digestion is due to pancreatic enzymes
Enterokinase plays a key role in pancreatic enzyme activation
A small percentage of digestion occurs following absorption into the cytosol
Absorption is complex
Several different transporters
Several different mechanisms

32. Review Questions There are 9 essential amino acids. Please list them –
Answer: phenylalanine, valine, threonine, tryptophan, isoleucine, methionine, leucine, lysine, and histidine

33. Review Question
The intestinal phase is responsible for the bulk of protein digestion. What enzymes are active in the intestines?
HCL and Pepsinogen
Endopeptidase and Ectopeptidase
Pepsin and Trypsin
Trypsin, Chymotrypsin and Proelastase
Answer:
B are the pro-enzymes
D are the active enzymes

34. Questions?

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