108 CHAPTER 11 Digestion and Absorption of Nutrients to diagnose disaccharidase deficiency if this is a suspected
cause. After an overnight fast, adult patients are fed 50 g of lactose in a 10% aqueous solution (children are usually fed 2 g/kg of body weight). Blood samples are taken for glucose analysis before and at 5, 10, 15, 30, 45, and 60 minutes after lactose administration. An increase in blood glucose of at least 25 mg/dL over fasted levels indicates normal hydroly- sis of lactose and normal absorption of the glucose prod- uct. A flat lactose tolerance curve or failure to observe a rise in blood glucose to more than 25 mg/dL after lactose ingestion indicates low lactase activity. A value between 20 and 25 mg/dL is questionable. In this case the test may be repeated or, if laboratory facilities permit, intestinal biopsy specimens can be collected and examined for enzyme activity. In dealing with biopsy specimens, enzyme activ- ity usually is expressed as units per gram of tissue protein.
Tolerance tests for disaccharides other than lactose are
seldom performed, but a similar procedure would suffice.
Glucose or galactose tolerance tests should be performed to exclude monosaccharide malabsorption as the cause of a flat lactose tolerance curve.
PROTEIN ASSIMILATION
109 CHAPTER 11 Digestion and Absorption of Nutrients
Pancreatic proteases are secreted into the duodenum as inactive precursors. Trypsinogen, which lacks pro- teolytic activity, is activated by enterokinase, an enzyme located on the brush border of duodenal enterocytes.
The exact chemical composition of enterokinase is not known; however, the fact that the molecule is 41% carbo- hydrate probably prevents its rapid digestion by proteo- lytic enzymes. The activity of enterokinase is stimulated by trypsinogen, and it is released from the brush border membrane by bile salts. Enterokinase activates trypsino- gen by releasing a hexapeptide from the N-terminal end of the precursor molecule (Fig. 11.8). Active trypsin, once formed, acts autocatalytically in the manner of enterokinase to activate the bulk of trypsinogen. Trypsin also activates other peptidase precursors from the pan- creas (see Fig. 11.8). Chymotrypsinogen is activated by cleavage of the peptide bond between arginine and isoleucine, which are, respectively, the fifteenth and six- teenth amino acid residues at the N-terminus. Although structural rearrangement occurs, no peptide fragment is released because of a disulfide bond between the cyste- ine residues at positions 1 and 122 in the protein chain.
This configuration is the active form of chymotrypsino- gen. Cleavage at other points in the chymotrypsinogen molecule produces other molecular species of chymo- trypsin having relatively little physiologic importance.
The exact mechanism for activation of proelastase is not
known, and activation of procarboxypeptidases A and B is relatively complicated, involving proteolysis of at least two proenzymes.
Once in the small intestine, pancreatic enzymes undergo rapid inactivation because of autodigestion. Trypsin is the enzyme primarily responsible for inactivation.
Development of Pancreatitis
In developed countries, 80% of the cases of acute pancre- atitis are the result of either biliary stone disease or ethanol abuse. Alcohol consumption is the major cause of chronic pancreatitis.
As pointed out previously, trypsin plays a central role in the activation of other pancreatic zymogens. Normally trypsin remains as inactive trypsinogen until it is secreted into the duodenal lumen. The mechanisms responsible for the intracellular activation of trypsin are not totally clear.
One hypothesis states that it is catalyzed by lysosomal hydrolases after the colocalization of these hydrolases with digestive zymogens within membrane-bound organelles during the early stages of pancreatitis.
Premature activation of trypsinogen within the pancreas initiates the entire activation cascade of other proteases, results in autodigestion of pancreatic cells, and causes acute pancreatitis. The body has several mechanisms to prevent premature trypsin activation. These include synthesis of pancreatic trypsin inhibitors, isolation of zymogens from lysosomes, and a site on trypsin that is susceptible to diges- tion by trypsin itself. Thus when trypsin comes into contact with other trypsin molecules, it is able to cleave them and irreversibly inactivate the enzyme.
This site for autodigestion is an arginine residue in posi- tion 117. Research has located a single guanine-to-adenine gene mutation that results in the substitution of histidine for arginine (R117H). This substitution prevents tryp- sin from being inactivated and was discovered in several families of patients with hereditary pancreatitis. Thus this mutation eliminates one of the failsafe mechanisms pre- venting the premature activation of trypsinogen and is the basis for at least one form of hereditary pancreatitis.
Absorption of Digestion Products
Membrane digestion and absorption are closely related phenomena in protein assimilation, and physiologists have been occupied by two fundamental questions concerning them: (1) In what form do products of proteolysis cross the brush border membrane of the epithelial cell? (2) In what form do these products leave the cell to enter the blood?
l-Isomers of some amino acids are absorbed by carrier-mediated mechanisms in much the same man- ner as glucose is absorbed. As with glucose, the cell entry process requires Na+ as part of a ternary complex (see Trypsin
Trypsinogen
Trypsinogen Chymotrypsinogen Proelastase
Procarboxypeptidase A Procarboxypeptidase B
Trypsin Chymotrypsin Elastase
Carboxypeptidase A Carboxypeptidase B Enterokinase
229
Isoleucine 7
1 6
Valine
Precursors Active proteases
1 6
7
Valine Isoleucine
229
Fig. 11.8 Activation of pancreatic proteolytic enzymes. The process begins in the lumen when enterokinase cleaves a hexapeptide from trypsinogen and converts it to the active en- zyme trypsin.
110 CHAPTER 11 Digestion and Absorption of Nutrients
Fig. 11.6), and uphill transfer occurs by secondary active transport. Other amino acids and some of those absorbed by active transport can also be absorbed by facilitated dif- fusion processes that do not require Na+ (Table 11.2). A new class of electrogenic proton-dependent amino acid transporters—PAT proteins—has been cloned. Present on the apical membrane, PAT proteins transport short- chain amino acids (glycine, alanine, serine, proline) into the cell, along with a hydrogen ion (H+). Thus the entry of the amino acid is coupled with the movement of a proton down its electrochemical gradient.
Certain l-amino acids compete with one another for uptake by intestinal cells. Studies of competition have led to the recognition of several different carrier systems for amino acid absorption (see Table 11.2). There is lit- tle doubt that the absorption of free amino acids by gut mucosa is physiologically important. However, amino acids appear in portal blood faster and reach a higher level when peptides from an acid hydrolysate of protein contact the gut mucosa than when there is an equimolar solution of free amino acids (Fig. 11.9). Moreover, greater amounts of total nitrogen are absorbed from a solution of trypsin hydrolysate of proteins than from an equiva- lent solution of amino acids in free form. Competition for transport between two chemically related amino acids is not observed when the same two acids are absorbed after ingestion of their dipeptides and tripeptides. In addition, the site in the intestine for the maximum absorption of
TABLE 11.2 Carrier Systems for the Transport of Amino Acids
Transport System Substrate
Dependence on Sodium Gradient
Brush Border Membrane
Bº Neutral l-amino acids Yes
Bº,t Neutral l- and cationic l-amino acids Yes
bº,t Neutral l- and cationic amino acids, cystine No
IMINO Imino acids Yes
B Taurine, β-alanine Yes
X–AG Anionic amino acids Yes
ASC Neutral l-amino acids Yes
N Glutamine, asparagine histidine Yes
PAT Small neutral amino acids No
Basolateral Membrane
A Neutral l-amino acids Yes
GLY Glycine Yes
Y+ Cationic amino acids No
l Neutral l-amino acids No
Y+l Neutral l-amino acids, cationic amino acids Yes/no
ASC Small l- and d-amino acids No
100 1300
Amino acid absorption (M/min/30 cm) 1100
900
700
500
300
100
10 20 30 40 50 60 70 80 90 Concentration in test solution
(mM) Free
Dipeptide
Leucine
Glycine Leucine
Glycine
Fig. 11.9 Jejunal rates of glycine and leucine absorption (mean ± SEM, five subjects) from perfusion of test solutions containing either l-glycyl-l-leucine or an equimolar mixture of free l-glycine and free l-leucine. (From Adibi SA: Intestinal transport of dipeptides in man: relative importance of hydro- lysis and intact absorption. J Clin Invest 50:2266–2275, 1971.)
111 CHAPTER 11 Digestion and Absorption of Nutrients
amino acids in small peptide form is different from that for the absorption of free amino acids. The absorptive capacity for dipeptides and tripeptides is greater in the proximal intestine, whereas the capacity for absorbing single amino acids is greater in the distal intestine. The current explanation of these findings is that a separate carrier system for small peptides is involved in absorp- tion. For example, free glycine absorption requires an amino acid carrier system. If saturation of the system occurs under physiologic conditions, the maximum rate of uptake becomes limiting. If, however, a second car- rier for dipeptides or tripeptides of glycine is present, the amino acids can enter the cell in small peptide form. Thus two separate systems for glycine entry exist and work in a parallel manner.
The prevailing concepts regarding protein assimilation are illustrated in Fig. 11.10. Luminal digestion of a protein meal produces approximately 20% free amino acids and 80% small peptides. As with the free acids, dipeptides and tripeptides resulting from digestion can be absorbed intact by a carrier-mediated process. However, tetrapeptides, pentapeptides, and hexapeptides are poorly absorbed;
instead they are hydrolyzed by brush border peptidases to free amino acids or smaller absorbable peptides. Small peptides are absorbed by a carrier with broad specificity.
This transport process is independent of the Na+ gradient and is stimulated by an inwardly directed H+ gradient. The
acidic microclimate pH that normally exists on the luminal surface of the brush border membrane provides the driving force for the peptide transport system. This is referred to as the H+/peptide cotransporter (PEPT1), and it is function- ally coupled to the Na+/H+ exchanger, NHE3, in the same membrane.
Peptides that enter enterocytes are hydrolyzed by cyto- plasmic peptidases to amino acids. These, in turn, diffuse or are moved by carrier-mediated processes from the intra- cellular compartment, across the basolateral membrane, into the blood. As is the case with the apical membrane, several different carriers exist in the basolateral membrane (see Table 11.2). Some of these also are Na+ dependent.
A few peptides enter the blood intact, and this may explain why certain biologically active peptides exert their effects when they are given orally.
Abnormalities in Protein Assimilation
Pancreatic insufficiency caused by various diseases, includ- ing cystic fibrosis and hereditary pancreatitis, may be asso- ciated with a decrease or absence of trypsin and may lead to poor digestion of protein. Cases of primary proteinase deficiency caused by congenital trypsinogen deficiency have been reported. In those patients, chymotrypsin and carboxypeptidase activities are lacking also, because tryp- sin cannot be formed to activate the precursors of these pancreatic proteases.
Small amounts Dipeptides and tripeptides
Cytoplasmic peptidases Amino acids
Amino acids Protein
Di- and tri- peptides
Carrier Peptidases
Large peptides
Carriers Free amino acids Pepsin
Pancreatic proteases
Fig. 11.10 Summary of the digestion and absorption of protein. Luminal digestion yields 20% free amino acids and 80% peptides consisting primarily of two to six amino acid residues.
112 CHAPTER 11 Digestion and Absorption of Nutrients
Intestinal malabsorption and the failure to reabsorb certain amino acids in the proximal renal tubule occurs in some hereditary diseases, thus providing evidence that the carriers are identical in the two tissues. Cystinuria is char- acterized by defective transport of the cationic amino acids (arginine, lysine, and ornithine) and cystine in the kidney and the small bowel (Fig. 11.11). The intestinal malabsorp- tive condition is of little or no consequence in the disability produced by cystinuria, given that the affected amino acids are absorbed as small peptides. However, the disease is severe because cystine has low solubility in water, and as the urine is concentrated in the distal nephron, cystine precip- itates and forms stones. Hartnup’s disease is a hereditary condition in which the active transport of neutral amino acids is deficient in both the renal tubule and the small intestine. An interesting finding is that, although neutral amino acids are not absorbed, they readily appear in the blood after ingestion of their dipeptides. This is compelling evidence that absorption of the dipeptides of certain amino acids is the result of a completely separate process from the one involved in the transport of free amino acids. These patients lose significant amounts of amino acids in their urine, and the condition may become significant in those whose dietary protein intake is low. Because tryptophan is a neutral amino acid, patients with Hartnup’s disease suffer from niacin deficiency (pellagra); approximately
50% of the vitamin is synthesized from this amino acid.
Tryptophan is also the precursor for the neurotransmitter serotonin, and its decreased synthesis may contribute to the neurologic symptoms associated with this disease.