Protein digestion and absorption in human small intestine

GASTROENTEROLOGY

76-1415-1421,

1979

Protein Digestion and Absorption in Human Small Intestine YOON CHUL CHUNG, YOUNG S. KIM, AL1 SHADCHEHR, ARTHUR GARRIDO, IAN L. MACGREGOR, and MARVIN

H. SLEISENGER

The Gastrointestinal Research Laboratory, Veterans California, and Department of Medicine, University Francisco, California

The extent of digestion and absorption of bovine serum albumin (BSA) of a mixed meal was quantitatively examined in 6 healthy volunteers by means of a 5-lumen intestinal tube with perfused segments in the proximal jejunum and terminal ileum and a nonperfused sampling site in the proximal ileum. At all times throughout the 4hr postprandial test periods, the ratio of protein to the polyethylene gJycoJ (PEG) meal marker in the proximal jejunal samples was approximately 40% of protein to PEG ratio of the meal, indicating digestion and/or absorption of 60% of meal protein at this level. In the terminal ileum, the protein to PEG ratio was less than 1% of the original meal. About 0.5 g of protein passed through the terminal ileum during the test period. A protein band electrophoretically coincident with BSA on SDS-polyacrylamide gels was detected in samples from the proximal jejunum and proximal ileum throughout the study, but was not found in the terminal iJeaJ samples. The ratio of amino acids to oligopeptides was constant throughout the small intestine. About 200 mg of free amino acid or oligopeptide nitrogen passed into the colon during the 4 hr after the meal ingestion. Trypsin concentration was similar in jejunum and proximal ileum; however, when corrected for reduction of intraluminal volume in the terminal ileum, it was 8% of activity in the proximal jejunum. This study inReceived June 15, 1978. Accepted January 4, 1979. Address requests for reprints to: Young S. Kim, M.D. (151MZ), Gastrointestinal Research Laboratory, Veterans Administration Hospital, 4150 Clement Street, San Francisco, California 194121. This work was supported in part by the Hartford Foundation, by Grant AM-17938 from the United States Health Service, and by Veterans Administration Medical Research Service. This work was presented in part at the annual meeting of the American Gastroenteroiogical Association, Toronto, Canada, April. 1977, and was published as an abstract. 0 1979 by the American Gastroenterological Association 0016-5085/79/061415-07$02.00

Administration of California,

Hospital, San Francisco, School of Medicine, San

dicates that quantitative digestion and absorption of a protein, BSA, is complete by the time an ingested meal has traversed the total small intestine, and that for the completion of the the ileum is important process.

There is relative paucity in data on the fate (i.e., digestion and absorption) of ingested protein in human intestine.” Borgstrom et al. investigated it by determining the disappearance of radioactivity from the intestinal lumen after oral intake of a tracer dose of a labeled protein and concluded that absorption of protein was nearly complete in the proximal small intestine.’ Subsequently, Nixon and Mawe?” reached a similar conclusion with a small amount (15 g) of protein. However, recently, Adibi and Mercer experimented with a substantial amount (50 g) of protein through feeding and reported that ingested protein can be recovered both in the jejunum and ileum as late as 4 hr after feeding.” In all these studies, the concentration of fed proteins and/or digestive products in the intestinal samples were measured, but the quantitative assessment of the degree of digestion or absorption in human gastrointestinal tract was not carried out. The present study was performed to determine the extent of proteolysis in the small intestine as well as the disappearance of protein and products of digestion after a test meal containing 59 g of purified bovine serum albumin (BSA) identical to that used by Adibi and Mercer.’ Using BSA which is water soluble and distinguishable from other proteins by its distinct electrophoretic pattern, we were able to examine the concentration of the fed protein in aspirates at different levels of the small intestine after ingestion of a meal. Since the flow rates of intestinal contents were determined in the proximal jejunum

1416

CHUNG

ET AL.

GASTROENTEROLOGY

and distal ileum, it was also possible to measure quantitatively the rate of meal passage and trypsin activity. Using new separation methods,8.9 we were also able to quantitate the digestive products of meal protein in the intestinal fluids as amino acids, either free or incorporated in small peptides.

Materials

and Methods

Composition

of Test Meal

The test meal was prepared by dissolving 50 g bovine serum albumin (Sigma Chemical Co., St. Louis, MO.), 120 g cornstarch, 40 g olive oil, 5 g lemon juice, 4.5 g sodium chloride, and 3 g polyethylene glycol 20,000 (PEG) into a final volume of 500 ml of distilled water. The composition of the test meal was identical to that used by Adibi and Mercer.’ The estimated calories of the test meal were 1040.

Experimental

Techniques

All the following aspects of the experimental protocol were approved by the Committee of Human Experimentation, University of California, San Francisco, California, and written informed consent was obtained from each subject before participation in the study. Three caucasian and 1 oriental male and 2 Caucasian female volunteers (body wt 58-65 kg; age 21-27 yr) were studied. Each subject was studied only once. Each subject swallowed a 5-lm polyethylene tube attached to a double latex bag containing 2 ml mercury. The mercury bag was separated from the distal sampling site by a tube 10 cm in length. This design permitted the passage of the bag into the cecum which forms a fixed and radiologically identifiable anatomic landmark. With the bag in the cecum, the distal sampling site was definitely in the terminal ileum. There was an orifice for perfusion 25 cm proximal to the distal sampling site. A similar 25-cm segment was located just

Vol. 76, No. 6

distal to the ligament of Treitz with the jejunal sampling site about 150 cm from the mouth. A third sampling site, unassociated, however, with a perfused segment, was situated 200 cm from the mouth in the proximal ileum. A schematic diagram of the positions of tubes in the small intestine is shown in Figure 1. Twenty-four to thirty-six hours were required for the tube to progress to the desired location. The position of the tube was checked radiologically on the morning of the feeding study. After confirmation of the tube position, perfusion segments was commenced. The nonabsorable markers were Phenol red 50 mg/lOO ml in the proximal segment and Y-PEG 5 $Zi/liter in the terminal ileal segment. Both were perfused at a rate of 0.93 ml/min. After 60 min for equilibration of marker perfusion, intestinal contents were aspirated through the three collection sites with the subjects fasting. After the fasting sample collection, the test meal was consumed over a period of 5-10 min. Intestinal contents were obtained by gentle aspiration continuously and were pooled at intervals of 30 min, 1, 2, 3, and 4 hr after the ingestion of test meal. Total volume of 0.5-1 ml was aspirated at each time period. Immediately after aspiration, intestinal contents were placed into plastic vials positioned in a mixture of dry ice and alcohol, since such immediate freezing prevents in vitro hydrolysis of proteins and peptides by both pancreatic proteolytic enzymes and intraluminal peptidases.‘” At the end of each feeding experiment, the intestinal samples were thawed, then centrifuged at 800 g for 10 min at 4’C. The supernatant was divided into several aliquots. The first aliquot of each sample was immediately assayed for trypsin activity. The second aliquot was immediately heated to 95°C to inactivate pancreatic proteases and intraluminal peptidases and was subsequently assayed for proteins” and the nonabsorbable markers, Phenol red” and PEG.13 The third aliquot was treated with 10% sulfosalicylic acid to precipitate protein followed by centrifugation at 800 g for 10 min. The supernatant was assayed for alpha-amino nitrogen before and after acid hydrolysis. Neither PEG nor Phenol red interfered with protein, trypsin, PEG, or alphaamino nitrogen assays.

Protein

Analysis

Protein in the supernatant and the precipitant fraction of several aliquots from the intestinal aspirate was qualitatively analyzed by electrophoresis on SDS-polyacrylamide gel by the method previously described by DavisI with an analytic polyacrylamide vertical gel apparatus. After electrophoresis, gels were stained for protein with Coomassie blue and then destained to remove excess dye.

Trypsin Assay 0 FOR SAMPLE 0

COLLECTION

FOR PERFUSION

Figure 1. A schematic

diagram

Open circles represent closed circles represent

of intestinal segments studied. sites for marker perfusion and sites for sample aspiration.

The supernatant from the first aliquot of each intestinal sample was used for analysis of trypsin activity.‘” One unit of trypsin activity (U) is equal to the hydrolysis of 1 micromol of p-toluene-L-arginine methyl-ester/min at 25’C and pH 8.0.

PROTEIN

June 1979

Analysis of Free Amino Acid and PeptideBound Alpha-Amino Nitrogen Each supernatant was treated with 10% sulfosalicylic acid to precipitate proteins and then was centrifuged at 800 g. The supernatant from this centrifugation was divided into two parts. One part was directly analyzed for alpha-amino nitrogenI and designated as alpha-amino nitrogen bound to free amino acids. The other part was hydrolyzed using 6 N HCl for 21 hr at 110°C to hydrolyze oligopeptides into free amino acids. The hydrolysates were analyzed for alpha-amino nitrogen, then designated as total alpha-amino nitrogen. The difference between total and alpha-amino nitrogen bound to free amino acid was designated as peptide-bound alpha-amino nitrogen.

Analysis of Free Amino Acid and That Peptide-Bound Amino Acids

of

Samples from the proximal collecting site at z hr after ingestion of the meal were passed through the copper Sephadex G-25 column after sulfosalicylic protein precipitation in order to separate free amino acids and oligopeptides as previously described.8,9 After acid hydrolysis of oligopeptides, amino acid contents were analyzed using a Beckman amino acid analyzer (12OC, Beckman Instruments, Inc., Palo Alto, Calif.). With this method, we were able to achieve satisfactory separation of small peptides from amino acids from a standard mixture containing L-,alanine, glycine, L-leucine, L-alanylglycine, glycylglytine, tri- and tetraglycine and L-alanyltriglycine.

Calculation

of Results

Flow rate of intestinal contents. The flow rates of intestinal contents at the proximal and distal sampling sites were calculated from the formula:

where FR is the flow rate (ml/min) of intestinal contents; Mi, the concentration (mg/ml) for Phenol red or cpm for ‘Y-PEG in the perfusing solutions; MO, the concentration of perfused markers in the samples of intestinal aspirates; and PR, the rate of marker perfusion (ml/min). Rate of meal transit. With knowledge of the flow rate and concentration of PEG in the intestinal aspirates, the taltal amount of PEG passing through the sampling site during given test periods could be calculated. The percentage of total meal PEG passing through the sampling sites per unit time was determined from the formula: x

FR (ml/min) 3000

AND

1417

ABSORPTION

of PEG and protein in the gastrointestinal tract are the same. The actual amount of protein passing during time periods was calculated from the formula: Protein (mg/ml) X FR (ml/min) ______ I’p (‘X,)= -___50,000

x Time (min) x loo

mg

where Pp is the percent of meal protein detected at a particular time and site. As a result the following equation is derived: Percent protein disappeared

= Mp (%) - Pp (%).

The amount of samples collected at each site over a 4-hr period was 3-5 ml. Therefore, a total of 6-10 ml of intestinal contents were removed at both the proximal and middle collection sites. Since the volume of the meal was 500 ml and the additional volume of fluid perfused at proximal site during a 4-hr period was 216 ml, the amount of protein or PEG removed in the proximal sampling sites were minimal and may account for l-2% error in the calculation of PEG and protein in the samples collected at the distal site.

Results Flow rates

of Intestinal

Contents

The flow rate at the proximal jejunal site reached a peak at 30 min and remained above basal level for the 4-hr test period (Figure 2). The flow rate of intestinal content at the terminal ileum did not vary during the test period, and the estimated volume passing into the colon during the 4-hr test period in the terminal ileum was 1.25 &ml, while that of PEG was 4-5 mg/ml, the presence of “C-PEG did not significantly affect the PEG value.

Meal Passage

FR = Mi x PR MO ’

PEG (mg/ml) Mp (%) = _

DIGESTION

x

Knowing flow rates and concentration of meal PEG, we calculated the amount of PEG which passed the sampling site during the test period as percent of total PEG (Figure 3). More than 80% of the 04

150cm

from

teeth

O-0

300cm

from

teeth

Time (min) x loo

mg

wher’e Mp is the percent of meal PEG passage per successive 30- or 60-min time periods during which mean flow rates were determined. The quantity of this meal marker passage through the gut should be equal to the amount of meal protein passage. This is based on the assumptions that no digestion or absorption occurred before the protein reached the sampling site and that the rates of movement

0-r .5

3

1

4

TIME (hours)

Figure 2. Flow rates

(ml/min) of intestinal contents jejunum (O-@) and terminal ileum (O--O) test period.

at proximal during 4-hr

1418

CHUNG

ET AL.

GASTROENTEROLOGY

0150cm

from

teeth

-3OOcm

from

teeth

to be about 500 mg. SDS-polyacrylamide gel electrophoresis of proteins present in the supernatant from intestinal samples showed a band migrating coincident to BSA in aspirate from the proximal and middle sampling site at all time periods except in the 4-hr sample from the proximal collection site. No such band was seen in terminal ileal samples.

100 80

Trypsin

3

4

TIME (hours) Figure

3. Passage (a) of meal marker (PEG) through proximal jejunum (open hors) and terminal ileum (hatched bars)

during 4-hr test period.

meal PEG passed through the proximal collecting site during the 2 hr after ingestion of the meal, while ileum only 30% of the meal PEG passed the terminal during the same period of time. However, almost all of meal PEG passed the distal collecting site during the 4-hr test period.

Disappearance

of Meal Protein

At all time periods, about 40% of ingested protein passed through the proximal jejunum, relative to the passage of PEG (Figure 4). For example, during the 2 hrs after meal ingestion, 77% or 38.5 g BSA should have passed through the proximal collecting site if no digestion or absorption occurred. In fact, 13 g of protein passed through the proximal jejunum during the 2 postprandial hours indicating that 25.5 g protein had been either digested or absorbed. Throughout the whole test period, 60-65% of ingested protein disappeared proximal to the jejunal sampling site, while the other 35-40~~ of the protein meal passed through the sampling site either undigested or unabsorbed. The total amount of protein passing into the colon without digestion during the 4-hr postprandial period of the study was calculated

15Ocm

C.S.

300cm

Digestive

3

4

.51 TIME (hours)

Products

of Protein

As defined in Materials and Methods, the amount and ratio of alpha-amino nitrogen bound to free amino acid or peptides are shown in Figure 6. Only a negligible amount of free amino acid and peptide was detected in the fasting samples. At the proximal site, alpha-amino nitrogen bound to free amino acids tended to increase with time. However, at the terminal ileum, the absolute amount and the ratio of free amino acid to peptide-bound alphaamino nitrogen did not vary with time. Alpha-amino nitrogen passing through the terminal ileum into the colon was estimated to be about 200 mg during the 4hr test period. The amount and ratio of individual amino acid, free or bound to oligopeptides in the proximal jejunal 2-hr samples, are shown in Table 1. No free proline and very little glycine were found in the samples. All amino acids except arginine were present predominantly in the bound (oligopeptide) form.

Discussion must

Before dietary protein can be assimilated, it be broken down by digestive processes into

C.S.

0 2

Activity

Trypsin concentrations were similar at all sampling sites throughout the test period. Trypsin passage through the proximal jejunum peaked at about 30 min and remained above basal level for the 4-hr test period (Figure 5). The total amount of trypsin passing through the terminal ileum was only 8% of that detected at the proximal jejunal collecting site.

Figure

s1

Vol. 76, No. 6

2

3

4

4. Protein disappearance (grams and %) at proximal jejunum (150 cm collecting site [C.S.]) and terminal ileum (300 cm collecting site [C.S.]). Dark oreo represents protein recovered, and light area represents protein disappeared (see text).

lune 1979

Figure

PROTEIN

5. Trypsin activity (p/min) passing num (0-O) and terminal ileum

through proximal jeju(CL-O) during 4-hr test

period.

subunits sufficiently small to be absorbed. Current knowledge of the digestion of ingested protein by the human gastrointestinal tract was derived initially from the classic studies of Borgstrom et al.,’ who measured the absorption of 1 g of radioiodinated human serum albumin in normal volunteers and reported that 60-80% disappeared at the level of the proximal jejunum. Subsequently, Nixon and Mawer’ measured the protein concentration and aminmo acid composition of gut contents at various levels of the intestine after a 15-g protein meal. They claimed that the major part of the meal protein was digested and absorbed in the duodenum and upper jejunum. More recently, Adibi and MerceF fed human subjects 50 g of bovine serum albumin in a mixed meal and sampled from points 110 and 200 cm from the teeth and identified ingested protein by SDS-polyacrylamide gel electrophoresis. They found that undigested protein could be detected from the jejunum and upper ileum for as long as 4 hr after the meal ingestion, and that exogenous protein was the principal contributor to increases above fasting levels of intraluminal amino acids and peptides in the upper gut during this period. They fur-

Figure

6. Protein digestion products at proximal jujunum (150-cm collecting site [C.S.]) and terminal ileum (300-cm collecting site). Dark and light areas of each column represent alpha-amino nitrogen bound to free amino acids or oligopeptides, respectively. Each column, therefore, represents the total a-amino nitrogen associated with free amino acids (dark) and oligopeptides (light). Because of the variation in the duration of collection time (e.g., 0.5 and 1 hr) the amount of o-amino nitrogen associated with free amino acid or oligopeptides for each time period are expressed as mg/ 10 min.

DIGESTION

AND

ABSORPTION

1419

ther reported that greater amounts of amino acids in the intestinal aspirates were present as small peptides than in the free form. Although each of these studies extended the knowledge available at the time of its execution, only the intraluminal concentrations of fed protein and its digestion products were measured. The amount of protein or protein digestion products passing through the intestinal lumen at the sampling sites was not determined.‘,‘.” A five lumen tube was used in the present study which allowed nonabsorbable marker perfusion and collection sites for 25 cm segments that were situated in the proximal jejunum and the terminal ileum. Thus, quantitation of intraluminal flow rates was permitted through these segments. The fifth tube allowed sampling from the proximal ileum, but flow rates were not quantitated at this site. The quantitation of flow rates and the use of a third nonabsorable marker (PEG) in the meal made possible the calculation of the rate of meal passage through the intestine as well as the amount of protein and protein digestion products passing the respective sampling sites per unit time period. Also measured was the fate of trypsin during its passage through the small intestine. Samples were continuously aspirated from the sampling sites and were pooled at 30- and then at 60minute periods. Such infrequent sampling did not allow the detection of minute by minute fluctuations in flow rates, but gave an average value for the collection period. The flow rate at the proximal site reached a peak 30 min after meal ingestion and remained above basal levels during the 4-hr study period. The PEG meal marker measurement showed that more than 80% of the meal passed the proximal sampling site in 2 hr and by the end of the 4hr test period almost all of the PEG had passed into the more distal gut. The continuing increase of the proximal jejunal flow rate above basal levels, even after m

PEPTIDE

m

FREE

BOUND

AMINO

150cm

5

1

ACID

BOUND

C.S

2

300cm

3

A TIME

5 (hours)

1

C.S.

2

3

A

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Table

1.

ET AL.

GASTROENTEROLOGY

Concentrations (pmol/ml) of Amino Jejunal Contents 2 Hours After Meal Bovine swum albumin

Acids

Free amino acids

Peptide amino acids

832.2

8.91 -+ 1.01

14.06 ?I 3.92

230.0

2.12 I!z0.19

4.98 +- 1.12

318.1

1.97 f 0.11

0.66 -t 0.21

(wml/g)”

Lysinc Histidine Arginine Aspartic acid Threoninc Serine Glutamic acid Proline

Acids in

691.0

2.04 + 0.14

15.84 f 4.24

459.8

3.78 + 0.60

11.36 f 4.66

362.4

4.16 f 0.46

5.28 + 3.64

1156.0

4.80 + 0.28

18.10 f 4.20 17.60 f 6.24

378.1

0

Glycine

200.1

0.38 f 0.14

9.72 -c 3.46

Alanine

584.1

5.58 +: 0.54

16.56 + 7.18

Valine

382.1

6.62 f 0.82

13.34 -t 4.04

57.1

0.80 f 0.44

1.38 + 0.62

119.2

2.00 I? 0.26

4.28 + 1.26

Methionine Isoleucine Leucine

761.1

Tyrosine

231.0

5.56 + 0.38

5.66 f 2.04

Phcnylalanine

359.1

5.34 I!Z0.83

7.70 f 2.80

57.26 f 6.03

143.90 f 50.34

Total Values

” Amino mined

14.20 k1.14

16.08 + 5.96

are mean

-+ SEM, pmol/ml. acid composition of the protein by acid hydrolysis method

in the test meal as deter-

the meal had passed the sampling site, was presumably due to continued release of gut hormones by the unabsorbed meal in the more distal bowel. During the first three postprandial hours, during which most of the meal marker had passed the proximal sampling site, about 20 g of 40% of ingested protein passed through the proximal jejunum (150 cm from the teeth), indicating that not all meal proteins are absorbed in the proximal small intestine. It is possible that some of this unabsorbed protein was derived from endogenous sources. The study of NasseP and Nixon and Mawe? had suggested that a considerable proportion of intraluminal protein after a meal was from the endogenous sources. However, the study of Adibi and MerceP failed to demonstrate electrophoretically any protein in the intestinal luminal contents after a nonprotein meal. Munro” estimated endogenous protein secretion from the total length of the gastrointestinal tract to be 20-30 g/day. Thus, there is no consistent data by which the proportion of endogenous protein in our samples can be estimated. Although not shown, SDS-polyacrylamide gel electrophoresis demonstrated a protein band migrating coincident with BSA at the proximal and middle sampling sites, confirming the findings of Adibi and Mercer.’ Such a protein band was not seen in samples from the terminal ileum. The amount of protein passing through the terminal ileum was very small, about 0.5 g during the 4-hr study period.

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These data indicate that almost complete digestion and absorption of protein occurs by the time it reaches the terminal ileum. However, the presence of BSA in the proximal ileal content (200 cm) suggests that the ileum is important for normal protein digestion. This finding differs from those of BorgStrom et al.’ and Nixon and Mawe? who claimed that protein digestion and absorption was largely complete in the jejunum. In the present study, however, a physiologic amount of protein entered the gastrointestinal tract, and our technique provided for quantitative assessment of digestion and disappearance of protein. It is suggested that major products of protein digestion by pancreatic proteases are free amino acids and small peptides containing 2-6 amino acid residues.19 In general, over 50% of the total alpha-amino nitrogen in the intestinal aspirate was associated with the peptide fraction. In the proximal jejunum at 0.5 and 1 hr after ingestion of the meal, nearly 70% of alpha-amino nitrogen was in the form of small peptides. These data are consistent with previous findings of Adibi and Mercer.’ The results were the same whether the alpha-amino nitrogen in the supernatant was measured following sulfosalicylic acid precipitation of proteins and large peptides before and after acid hydrolysis, or by separating free amino acids and oligopeptides by copper Sephadex G-25 column. Only about 0.2 g nitrogen associated with free amino acids or small peptides passed through the terminal ileum during the 4-hr test period, which indicates efficient digestion and absorption of protein in the small intestine, Trypsin concentrations were similar throughout the whole of the small intestine. However, the amount of trypsin passing through the terminal ileum was only 8% of the total passing through the proximal jejunum. There appears to be, therefore, a considerable autodigestion, inactivation, or possible intact absorption of trypsin during its passage through the small intestine, but the concentration of the intestinal contents by absorption of fluid maintain adequate trypsin concentration down to the terminal ileum. Other pancreatic enzymes were not measured, and to what extent the activity of trypsin reflects the fate of other pancreatic enzymes is uncertain. This study, therefore, demonstrates that the digestion and absorption of protein fed in a mixed fat, carbohydrate and protein liquid meal is virtually complete by the time the meal has traversed the total small intestine. Undigested meal protein is detected in the proximal ileum, but not in the terminal ileum. Adequate proteolytic enzyme concentration is maintained for continued digestion throughout the whole small intestine. However, autodigestion or in-

June 1979

activation of pancreatic enzymes, as reflected by trypsin data, suggests that only a small quantity of this endogenously secreted protein passes intact into the callon.

References 1. Borgstrom B, Dahlqvist A, Lundh G, Sjovall J: Studies of intestinal digestion and absorption in human. J Clin Invest 36: 1521-1536. 1957 and absorption or pro2. Nixon SE, Mawer GE: The digestion tein in man. I. The site of absorption. Br J Nutr 24:227-240, 1970 3. Nixon SE, Mawer GE: The digestion and absorption of protein in man II. The form in which digested protein is absorbed. Br J Nutr 24:241-258, 1970 4. Adibi SA, Gray SJ: Intestinal absorption of essential amino acids in man. Gastroenterology 52837-845, 1967 in human intestine 5. Adibi SAA, Mercer DW: Protein digestion as reflected in luminal, mucosal and plasma amino acid concentrations after meals. J Clin Invest 521586-1594, 1973 DM: Protein absorption. J Clin Path01 24 Suppl 6. Matthews (Roy, Co11 Path) 5:29-40, 1971 Crane CW. Newberger A: The digestion and absorption of proteins by normal man. Biochem J 74:313-323, 1959 Gallo-Torres HE, Ludorf J, Moller ON: An ultramicrotechnique for the detection and separation of small molecular weight peptides from amino acids. Anal Biochem 64:260-267, 19’75 Fa’zakerley S, Best DR: Separation of amino acids, as copper

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DIGESTION

AND

ABSORPTION

1421

chelates from amino acid, protein and peptide mixtures. Anal Biochem 12:290-295, 1965 JA, Kim YS: Hydrolysis of peptides 10. Silk DBA, Nicholson within lumen of small intestine. Am J Physiol 231:1322-1329, 1976 11. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with Folin phenol reagent. J Biol Chem 193:256-275, 1951 12. McLeod GM, French AB, Good CH, Wright FS: Gastrointestinal absorption and biliary excretion of phenolsulfonphthalein (Phenol Red) in man. J Lab Clin Med 71:192-205, 1968 SJ, Powell DW: An improved turbidimetric analysis 13. Malawer of polyethylene glycol utilizing an emulsifier. Gastroenterology 53:250-260, 1967 14. Davis BJ: Disc Electrophoresis. II. Method and application to human serum proteins. Ann NY Acad Sci 121:404-412 in Worthington Enzyme Manual. Freehold, N.J. 15. Trypsin Worthington Biochemical Corporation, 1972, p. 125-127 method for primary amino 16. Kabat EA, Mayer MM: Ninhydrin acids. In: Experimental Immunochemistry. Published by Thomas, Springfield, Ill., Charles C. Inc., 1971, p 561-563 tract in protein metabo17. Nasset E: The role of the digestive lism. Am J Dig Dis 9:175-179, 1964 18. Munro HN: Protein secretion into the gastrointestinal tract. In: Postgraduate Gastroenterology. Edited by TJ Thomson, IE Gillespie. London, Baillier, Tindall and Cassell. 1966, p 58-67 on protein di19. Chen ML, Rogers, QR, Harper AE: Observation gestion in vivo. IV. Further observations on the gastrointestinal contents of rats fed different dietary proteins. J Nutr 76:235-239, 1962