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CIinical Science and Molecular Medicine (1917) 53, 21-33.
Effect of glycylglycine on absorption from human jejunum of
an amino acid mixture simulating casein and a partial
enzymic bydrolysate of casein containing small peptides
P. D. F A I R C L O U G H , D. B. A. S I L K , M. L. C L A R K , D. M. MATTHBWS,")
T. C . MARRS,(I)D. BURSTON")AND K. M. CLEGG(2)
Department of Gastroenterology, St Bartholomew's Hospital, London, (I'Department of Experimental Chemical
Pathology, Vincent Square Laboratories. Westminster Hospital, London. and "'Department of Food Science and
Nutrition, University of Strathclyde, Glasgow, Scotland, U.K.
(Received 25 November 1976; accepted 18 February 1977)
Summary
1. A jejunal perfusion technique has been
used in normal volunteer subjects to study
jejunal absorption of amino acid residues from
a partial enzymic hydrolysate of casein in which
about 50% of the amino acids existed as small
peptides, and also from an equivalent mixture
of free amino acids.
2. The effect of a high concentration of the
dipeptide glycylglycine on the absorption of
amino acid residues from these preparations was
studied to quantify the importance of mucosal
uptake of intact peptides during absorption of
the partial hydrolysate of casein.
3. The results were unexpected. Glycylglycine
significantly inhibited absorption of several
amino acid residues (aspartic acid asparaghe,
serine, glutamic acid + glutamine, proline,
alanine, phenylalanine, threonine and isoleucine) from the free amino acid mixture,
whereas it significantly inhibited the absorption
of only two (serine, glutamic acid fglutamine)
from the peptide-containing partial casein
hydro1ysate.
4. The effect of glycylglycine on absorption
of amino acids from the mixture of free amino
acids was apparently due to inhibition of
amino acid uptake by free glycine liberated
from the dipeptide during perfusion. The
reason for the failure of glycylglycine to cause
extensive inhibition of absorption from the
partial hydrolysate is not clear. It may be due to
glycylglycine being only a weak inhibitor of
+
Correspondence: Dr D. B. A. Silk, Liver Unit,
King's College Hospital, London, S.E.J.
peptide uptake, but the possibility that some
peptides are taken up by a system unavailable
to glycylglycine has to be considered.
Key words: absorption, amino acids, casein,
glycylglycine, peptides.
Introduction
After a protein meal the lumen of the small
intestine contains a complex mixture of free
amino acids and small peptides (Adibi &
Mercer, 1973) but the relative contribution of
mucosal uptake of amino acids and that of
peptides to overall absorption of proteindigestion products is unknown (Matthews,
1975). We have therefore attempted to estimate
the quantitative importance of peptide absorption from a partial enzymic hydrolysate of
casein, in which about 50% of the amino acids
were in the form of small peptides of chain
length two to four amino acids, and about 50%
as free amino acids (Clegg & McMillan, 1974).
We hoped to estimate the contribution of
mucosal uptake of intact peptides to total
amino acid absorption from this mixture by
using another peptide in high concentration,
so as to inhibit absorption of the peptide
fraction of the partial enzymic hydrolysate.
Glycylglycine was chosen as it is known to
inhibit absorption of a number of other diand tri-peptides in vitro (Rubino, Field &
Schwachman, 1971; Addison, Matthews &
Burston, 1974; Addison, Burston, Dalrymple,
Matthews, Payne, Sleisenger & Wilkinson,
27
28
P. D. Fairclough et al.
1975). Available evidence also suggests that it
is mostly absorbed intact and hydrolysed within
the cell cytoplasm (Matthews, Craft, Geddes,
Wise & Hyde, 1968; Adibi, 1971; Hellier,
Holdsworth, McColl & Perrett, 1972; Silk,
1974). It was therefore presumed that it would
cause a minor inhibition of absorption of free
amino acids.
Using an intestinal perfusion technique in
man, we have therefore examined the effect of
high concentrations of glycylglycine on absorption of amino acids from the partial
enzymic hydrolysate of casein, and also from a
mixture of the same amino acids, all of which
were in the free form. As our analysis of the
partial enzymic hydrolysate showed it to
contain a broad spectrum of peptides, the
first investigation tested the effect of a single
dipeptide on absorption of amino acids from a
heterogeneous mixture of peptides and amino
acids.
Methods
Enzymic hydrolysate of casein and amino acid
mixture
The partial enzymic hydrolysate of casein was
prepared by hydrolysis with papain, followed by
hydrolysis with pig kidney peptidases (CIegg &
McMillan, 1974; Silk, Clark, Marrs, Addison,
Burston, Matthews & Clegg, 1975). The
equivalent amino acid mixture simulating the
pattern and molar concentration of casein was
made up in the same proportions as described
by Silk et al. (1975) to a final concentration
of 24.9 mmol of a-amino nitrogen/] (determined
after acid hydrolysis) (Table 1).
Analysis of casein hydrolysate
Free amino acids were removed from the
original partial enzymic hydrolysate of casein
by ligand exchange chromatography on columns
containing Chelex 100 (Biorad, California) in
complex with copper ions. The theoretical
aspects of this method have been described by
Helfferich(1961), and its application to separation of free amino acids from oligopeptides by
Buist & O'Brien (1967) and Asatoor, Milne &
Walshe (1976). Peptides were eluted from the
column with borate buffer (0.1 mol/l), pH 11,
and collected at 0°C with sufficient hydro-
chloric acid (1 mol/l) to neutralize the buffer.
Copper ions were removed from the column
eluate with a methanolic solution of sodium
diethyl dithiocarbamate (Fazakerley & Best,
1965). The resulting solution containing the
peptide moiety of the hydrolysate was subjected
to complete acid hydrolysis and ion-exchange
chromatography to quantify the peptidebound amino acid content of the original
casein hydrolysate. The column eluate, after
removal of copper ions and before acid hydrolysis, was also examined by ion-exchange chromatography to check that complete removal of
free amino acids had been achieved. Free
amino acids in the native casein hydrolysate
were estimated by ion-exchange chromatography of the whole hydrolysate.
Perfusion procedure
Normal subjects (medical students and
laboratory personnel) gave informed consent to
the study. A double-lumen perfusion tube
incorporating a proximal occlusive balloon
(Silk, Perrett & Clark, 1973), was positioned
under radiological control with the 30 cm
perfusion segment in the upper jejunum.
Perfusion solutions were pumped through the
infusion orifice at 20 ml/min with a peristaltic
pump (H.R. Flow Inducer, Watson-Marlow
Ltd, Marlow, Bucks, U.K.) from bottles
maintained at 37°C in a water bath. A 30 min
equilibration period was allowed for the attainment of steady-state absorptive conditions for
each solution before collection of three consecutive 10 min samples from the distal orifice.
Seven subjects (two female and five male;
aged 21-28 years) were perfused with the
enzymic hydrolysate of casein at a concentration of 24.9 mmol of a-amino nitrogen/]
(determined after acid hydrolysis) alone, and
also in the presence of glycylglycine (100
mmol/l). Six further subjects (four female and
two male, aged 21-26 years) were perfused
with the amino acid mixture alone, and also
in the presence of glycylglycine (100 mmol/l).
Solutions, which were perfused in random
order, were adjusted to pH 7 by titration with
sodium hydroxide (1 mol/l), contained the
non-absorbable marker polyethylene glycol
(2.5 g/l) labelled with 1 pCi of [14C]polyethylene glycol/l, and were made iso-osmotic
(285-295 mmol/kg) by adding sodium chloride.
Absorption of amino acids from human jejunum
Glycylglycine was obtained from the Sigma
(London) Chemical Co. Ltd, the purity being
checked before perfusion with an amino acid
analyser.
Analyticalmethods and calculationof results
TO measure absorption of individual free plus
peptide-bound amino acids from the hydrolysate and amino acid mixture, samples of the
perfusion solutions and their respective intestinal aspirates were hydrolysed under identical conditions in sealed glass tubes with
HCl (6 mol/l) at 110°C for 24 h. Amino acids
were estimated by ion-exchange chromatography with a Locarte Automatic Loading
Amino Acid Analyser (mark 4 Floor model,
The Locarte Co., London, W.14). In solutions
subjected to acid hydrolysis the amides of the
dicarboxylic acids (glutamine and asparagine)
were converted into glutamic acid and aspartic
acid and were measured as such.
'*C radioactivity was measured with a
scintillation counter [Corumatic 2000 with
Diehl Combitron S Computer; ICN Pharmaceuticals (UK) Ltd, Tracerlab, Hersham,
Surrey, U.K.] by the procedure described by
29
Wingate, Sandberg & Phillips (1972) and
Silk, Perrett, Webb & Clark (1974). The
osmolality of perfusion solutions was checked
immediately before perfusion with an Advanced
Osmometer (Advanced Instruments Inc.,
U.S.A.). Percentage amino acid absorption is
the amount of each amino acid disappearing
from the perfused intestinal segment in unit time
expressed as a percentage of the perfused load of
that amino acid. The significanceof differences
in percentage absorption of individual amino
acids was assessed by the paired t-test (Snedecor,
1937).
Results
Composition of the partial epzymic hydroijwate
of casein (Table 1)
The mean recovery of amino acids from the
enzymic hydroIysate of casein was 92%, as
estimated from the sum of the free amino acids
determined by chromatography of the native
hydrolysate and analysis of the peptide-bound
amino acids. This method may give a falsely
low result from unavoidable loss of some
peptide-bound amino acids during column
TABLE
1. Composition of perfusion solutions
N = not measured.
Partial enzymic hydrolysate of casein
Histidine (His)
Lysine (Lys)
Arginine (Arg)
Aspartic acid (Asp)
Asparagine (Am)
Threonine (Thr)
Serine (Ser)
Glutarnic acid (Glu)
Glutamine (Gln)
Proline (Pro)
Glycine (GIy)
Alanine (Ma)
Valine (Val)
hfethionine (Met)
Isoleucine (IIe)
Leucine (Leu)
Tyrosine (Tyr)
Phenylalanine @he)
Cystine (Cys)
Tryptophan (Try)
Amino acid mixture
(mmol/l)
Amino acid content
as free amino
acids (%)
Amino acid
in peptide form
0.6
I .4
0.6
69
56
71
31
44
29
E}
2.0
20
80
41
2.5
39
59
61
17
83
28
50
53
65
60
50
47
35
40
43
45
24
17
34
N
z}
08
1.o
1.6
06
1.2
2.0
09
1.0
02
02
51
55
76
83
66
N
N
(%I
N
P. D. Fairclough et al.
30
separation (Asatoor et al., 1976). Consequently,
the proportions of each amino acid present in
the peptidebound and free forms in the casein
hydrolysate (Table 1) are expressed as percentages. Approximately 50% of the total
amino acids present in the hydrolysate were
peptide-bound. The proportion of individual
amino acids in the form of peptides varied:
for example, 83% of the glutamic acid+
glutamine in the hydrolysate was peptidebound, in contrast to only 17% of the tyrosine.
Absorption of amino acid residues from partial
hydrolysate and equivalent amino acid mixture
The perfusion experiments (Table 2) showed
that there was considerable variation in the
extent to which ihdividual amino acids were
absorbed from both solutions; leucine, isoleucine, tyrosine and arginine were well absorbed, but aspartic acid asparagine, threonine, histidine and glycine were relatively poorly
absorbed.
Mean rates of absorption of all amino acid
residues from the free amino acid mixture were
less when glycylglycine (100 mmol/l) was
included in the perfusion solutions (Table 2).
By the paired t-test (five degrees of freedom)
the differences were only significant for aspartic
acid asparagine, threonine, serine, glutamic
acid, proline, alanine, isoleucine and phenyl-
+
+
+
alanine. However, serine and glutamic acid
glutamine were the only amino acids whose
absorption from the partial enzymic hydrolysate
was significantly inhibited when glycylglycine
(100 mmol/l) was included in these perfusion
solutions (Table 2).
Hydrolysis of perfused glycylglycine
The concentrations of glycylglycine and free
glycine found in the intestinal aspirates during
perfusion of the amino acid mixture and
hydrolysate in the presence of glycylglycine
(100 mmol/l) are shown in Table 3. The same
concentrations of glycylglycine and free glycine
were found in the intestinal aspirates when
glycylglycine was perfused with the amino acid
mixture and with the hydrolysate.
Discussion
The analysis of the partial enzymic hydrolysate
of casein suggests that a proportion of all the
amino acids present was in the peptide-bound
form. The experiments thus test the effect of
glycylglycine on the absorption of amino acids
from a diverse mixture of small peptides and
free amino acids. This is a marked difference in
experimental design from the studies in vitro
of Addison et al. (1974, 1975) and Das &
Radhakrishnan (1975), who demonstrated the
TABLE
2. Percentage absorption of amino acid residues from an amino acid mixture and a partial enzymic
hydrolysate of casein in the absence and presence of glycylglycine (100 mmolll)
Mean values f s e ~
are shown.NS = not significant.
~
Amino acid mixture (n = 6)
Amino
acid
His
LYs
-b
ASP
Thr
Ser
Glu
Pro
G~Y
Ala
Val
Ileu
Leu
Tyr
Phe
Alone
P
40.1k 7.0
46.22 5.0
65.3 2 7.9
42.2+43
40.12 3.1
48-3+29
43.6k4.2
53.8k7.2
162+ 8 7
51.1k4.8
58-4+ 4 4
725k7.4
72.8-1-7.8
6 4 4 2 8-0
59.0+ 6.2
NS
NS
NS
< 0.01
< 0.025
< 002
< 0.025
< 0.001
-
< 0.02
NS
< 005
NS
NS
< 0.05
~
~~~~
Partial enzymic hydrolysate (n = 7)
With
glycylglycine
17.8f7.1
32.1 f8.0
41.92 11.0
18-628.5
6.8+ 11.3
25-4+ 5.6
19.3k7.4
21.1k4.8
-
31.6k4.5
36.2f 144
4 0 5 k 13.1
42.6+ 13.6
4 0 6 2 109
38.8 7.9
+
Alone
42-lt8.8
42.1 f 6-0
49.6f 9.1
47.1 f 8.1
37.35 7.1
47226.1
34.3+ 6.5
49.1 2 8.0
3 4 8 2I04
53.1 1 6 . 8
54.6+ 8.8
65.41 7.7
63.5+ 6.6
63-8k7.6
524+9-2
P
With
glycylglycine
NS
31-2f8.0
37.9+3-7
56.2f 3.8
342+ 4 2
24.9 4-1
33*5+ 3.9
17.1k5.2
38.1 f 6.3
NS
NS
NS
NS
NS
NS
51.1 k 4.3
58.0+ 2.8
63-7+ 3.4
63.5k 2.9
55.4+ 4.8
546+ 2 6
NS
NS
NS
NS
NS
< 0.01
< 0.01
-
-
Absorption of amino acids from himan jejunum
31
TABLE
3. Concentrations offree glycine and glycylglycine in intestinal aspirates during perfusion
of glycylglycine (100 mrnolll) with amino acid mixture or hybolysate
Concn. in aspirates (mmol/l)
Free glycine
G1ycylglycine
Glycylglycine
Glycylglycine
Glycylglycine
+amino acid mixture
12-8
11.7
23.8
21.1
7.7
14.8
18-2
15.2
16.1
15.7
17.8
15*7+0-7
54.3
18.2
55.7
16.1
78.7
+amino acid mixture +hydrolysate
Mean 16.8k2.8
effects of a number of individual peptides on
absorption of single model peptides.
The results of the perfusion experiments
were unexpected, so that further studies are
needed to estimate the quantitative importance
of peptide uptake by this type of experiment.
Glycylglycine had a marked inhibitory effect on
absorption from the free amino acid mixture
(Table 2). This was an unexpected result, since
separate transport sytems are thought to be
involved in the mucosal uptake of peptidebound and free amino acids (Rubino et al.,
1971; Addison et al., 1974, 1975; Das &
Radhakrishnan, 1975; Matthews, 1975). Table
3 shows, however, that in the present perfusion
experiments in uivo appreciable amounts of the
dipeptide glycylglycine are absorbed in the
free form, as 16.8 and 15.7 mmol/l of free
glycine was detected in the luminal fluid when
glycylglycine (100 mmol/l) was included in the
perfusion solutions containing the amino acid
mixture and hydrolysate respectively. As the
concentration of individual free amino acids
in the amino acid mixture ranged from only
0.2 to 2.8 mmol/l (Table l), inhibition of
neutral amino acids by the observed concentrations of free glycine is to be expected.
Likewise, the observed inhibition of glutamic
acid and aspartic acid absorption is not altogether unexpected, as absorption of these amino
acids may be inhibited by neutral amino acids
(Matthews, 1975) and both have been shown to
share the transport system used for another
neutral acid (Nathans, Tapley & ROSS,1960).
Unfortunately, since neither the brush-border
nor cytoplasmic peptidase activities of the
perfused mucosa could be measured, we could
607+4-8
Glycylglycine
+hydrolysate
52.2
41.6
37.5
59.8
68.6
60.3
53.1 k 4-1
not determine the source of the free glycine
liberated during perfusion of glycylglycine.
Quite unexpectedly, however, absorption of
only two amino acids (glutamic acid fglutamine and serine) from the partial enzymic
hydrolysate of casein was significantly inhibited
when glycylglycine (100 mmol/l) was included in
the perfusion solutions. Recent studies indicate
that peptides may be either absorbed intact or
hydrolysed at the brush-border membrane of
the cell, with subsequent absorption of the
free amino acids by the normal amino acidtransport mechanisms (Cheng, Navab, Lis,
Miller & Matthews, 1971;Matthews, Crampton
& Lis, 1971;Silk et al., 1974;Silk, Nicholson &
Kim, 1976). One explanation for the lack of
effect of glycylglycine on absorption of amino
acid residues from the hydrolysate could
therefore be that at the concentrationstudied the
peptides of the hydrolysate were all absorbed
as free amino acids. However, one would then
expect inhibition of absorption of the same
amino acids from both the mixture of free
amino acids and the hydrolysate, which did not
OCCW.
Our study was designed on the assumption
that there is only a single peptide-transport
system in mammalian small intestine, as
suggested by Das 8c Radhakrishnan (1975).
Another explanation for our findings may be
that this assumption is not valid for human
small intestine in vivo. The observations that
absorption of aspartic acid, threonine, proline,
alanine, isoleucine and phenylalanine from the
hydrolysate was unaffected by either the free
glycine in the lumen (which as discussed above,
probably inhibited absorption of these amino
32
P . D . Fairclough et al.
acids when presented in the free form), or by
glycylglycine, which has been shown to inhibit
absorption of several di- and tri-peptides
in vitro (Rubino et al., 1971; Addison et al.,
1974; Das & Radhakrishnan, 1975), might be
explained if these amino acid residues were
absorbed by an alternative peptide transport
system which was not shared with glycylglycine.
There are 400 possible dipeptides, so that our
results cannot with certainty be extrapolated
to the normal biological state. Moreover, if
there is more than one peptide-transport
system, the quantitative importance of peptide
transport during absorption of protein-digestion
products cannot be determined by studies such
as this which investigate the inhibitory effect of
a single peptide.
One reason for the lack of inhibitory effect of
glyclylglycine could be that this dipeptide is a
relatively weak inhibitor of peptide transport.
Indeed, Das & Radhakrishnan (1975) have
suggested that glycylglycine does have an
unusually low affinity for the transport system
used by glycyl-leucine. Nonetheless, glycylglycine significantly inhibits absorption of
several dipeptides in uitro (Rubino et al.,
1971;Addison et af., 1974, 1975). Furthermore,
in the present study the mean total concentration of peptide derived from the partial enzymic
hydrolysate in the intestinal lumen was not
more than 5 mmol/l, whereas the mean luminal
concentration of glycylglycine was about 75
mmolll. It thus seems unlikely that the lack of
inhibition observed was solely due to insufficient saturation of the peptide-transport
system by the inhibitor peptide.
Finally, the above studies in which glycylglycine was shown to inhibit absorption of other
di- and tri-peptides (Rubino et al., 1971;
Addison et al., 1974; Das & Radhakrishnan,
1975) were all performed in vitro. This inhibitory effect may be due to competition for
an energy source which does not occur in uivo,
again emphasizing the difficulty of comparing
experimental data on peptide absorption
in uiuo and in uitro (Silk & Kim, 1976).
Acknowledgments
We are grateful for financial support to the
Medical College and Joint Research Board of
St Bartholomew’s Hospital, the North-East
Thames Regional Health Authority and to the
Medical Research Council (for a grant to
D.M.M.). We also thank Dr A. M. Dawson for
constructive criticism and for the use of his
laboratory facilities.
References
ADDISON,J.M., BURSTON,D., DALRYMPLE,
J.A.,
MATTHEWS,
D.M., PAYNE,J.W., SLEISENGER,
M.H.
& WILKINSON,
S. (1975) Competition between the
tripeptide glycylsarcosylsarcosine and other di- and
tri-peptides for uptake by hamster jejunum in vitro.
Clinical Science and Molecular Medicine, 49, 3 13-322.
ADDISON,J.M., MATTHEWS,D.M. & BURSTON,D.
(1974) Competition between carnosine and other
peptides for transport by hamster jejunum in vitro.
Clinical Science and Molecular Medicine, 46,707-714.
ADIFJI,S.A. (1971) Intestinal transport of dipeptides in
man; relative importance of hydrolysis and intact
absorption. Journal of Clinical Investigation, 50,
2266-2275.
ADIBI,S.A. & MERCER,
D.W. (1973) Protein digestion
in human intestine as reflected in luminal, mucosal
and plasma amino acid concentrations after meals.
Journal of Clinical Investigation, 52, 1586-1594.
ADIFJI,S.A. & SOLEIMANPOUR,
M.R. (1974) Functional
characterisation of dipeptide transport system in
human jejunum. Journal of Clinical Inuestigation, 53,
1368-1374.
ASATOOR,
A.M., MILNE,M.D. & WALSHE,
J.M. (1976)
Urinary excretion of peptides and of hydroxyproline
in Wilson’s disease. Clinical Science and Molecular
Medicine, 51, 369-378.
BUNT,N.R.M. & O’BRIEN,D. (1967) The separation of
peptides from amino acids in urine by ligand exchange chromatography. Journal of Chromatography,
29, 398-402.
CHENG,B., NAVAB,F., LIS, M.T., MILLER,T.N. &
MATTHEWS,
D.M. (1971) Mechanisms of dipeptide
uptake by rat small intestine in uitro. Clinical Science,
40,247-257.
CLEGG,K.M. & MCMILLAN,A.D. (1974) Dietary
enzymic hydrolysates of protein with reduced bitterness. Journal of Food Technology, 9,21-29.
DAS,M. & RADHAKRISHNAN,
A.N. (1975) Studies on a
wide-spectrum intestinal dipeptide uptake system in
the monkey and in the human. Biochemical Journal,
146,133-139.
FAZAKERLEY,
S. & BEST,D.R. (1965) Separation of
amino acids as copper chelates from amino acid,
protein and peptide mixtures. Analytical Biochemistry,
12,290-295.
HELFFERICH, F. (1961) ‘Ligand exchange’: a novel
separation technique. Nature (London), 189, 10011002.
HELLIER,M.D., HOLDSWORTH,
C.D., MCCOLL,I. &
PERRETT,
D. (1972) Dipeptide absorption in man.
Gut, 13,965-969.
MATTHBWS,D.M. (1975) Intestinal absorption of
peptides. Physiological Reviews, 55, 537-608.
MAITHEWS,D.M., CRAFT,I.L.. GEDDES.D.M., WISE,
I.J. & HYDE,
C.W. (1968) Absorption of glycine and
glycine peptides from the small intestine in the rat.
Clinical Science, 31, 751-164.
MATTHEWS,
D.M., CRAMPTON,
R.F. & LIS,M.T. (1971)
Sites of maximal intestinal absorptive capacity for
amino acids and peptides: evidence for an independent peptide uptake system or systems. Journal of’
Clinical Pathology, 24, 882-883.
Absorption of amino acids from human jejunum
MATTHEWS,
D.M., LIS, M.T., CHENO,B. & CRAMPTON,
R.F. (1969) Observations on the intestinal absorption
of some oligopeptides of methionine and glycine in
the rat. Clinical Science, 37, 751-764.
D.F. & Ross, J.E. (1960)
NATHANS,D., TAPLEY,
Intestinal transport of amino acids; studies in uitro
with ~-[~~~I]monoiodotyrosine.
Biochimica et Biophysics Acza, 41,271-282.
H. (1971)
RUBINO,H., FIELD,M. & SCHWACHMAN,
Intestinal transport of amino acid residues of
dipeptides. Journal of Biological Chemistry, 246,
3542-3548.
SILK,D.B.A. (1974) Absorption of peptides in man.
M.D. thesis, University of London.
SILK, D.B.A., CLARK,M.L., MARRS,T.C., ADDISON,
D.M. & CLEGG,
J.M., BURSTON,D. MATTHEWS,
K.M. (1975) Jejunal absorption of an enzymic
hydrolysate of casein prepared for oral adrninistration to normal adults. British JournaZ oflvutrition, 33,
95-100.
SILK,D.B.A. & KIM, Y.S. (1976) Release of peptide
hydrolases during incubation of intact intestinal
33
segments in vitro. Journal of Physiology (London),
2S8.489-491.
-_
-, .- . ... .
SILK, D.B.A., NICHOLSON,
J.A. 8c KIM, Y.S. (1976)
Relationships between mucosal hydrolysis and
transport of two phenylalanine dipeptides. Gut, 17,
870-876.
SILK, D.B.A., PERKETT,D. & CLARK,M.L. (1973)
Intestinal transport of two dipeptides containing the
same two neutral amino acids in man. Clinical Science
and Molecular Medicine, 45, 291-299.
SILK, D.B.A., PERRETT,D., WEBB,J.P.W. & CLARK,
M.L. (1974) Absorption of two tripeptides by the
human small intestine: a study using a perfusion
technique. Clinical Science and Molecular Medicine,
46,393-402.
SNEDECOR,
G.W. (1937) Statistical Methods. Iowa
State Medical College Press, Ames, Iowa.
WINOATE,D.L., SANDBERG,
R.J. & PHILLIPS,S.F.
(1972) A comparison of stable and 14C-labelled
polyethylene glycol as volume indicators in the
human jejunum. Gut, 13,812-815.