J. Phyeiol. (1978), 284, pp. 83-104 With 5 text-Mfr Prined in Great Britain
83
AMINO ACID AND PEPTIDE ABSORPTION FROM PARTIAL DIGESTS OF PROTEINS IN ISOLATED RAT SMALL INTESTINE
BY MICHAEL L. G. GARDNER From the Department of Biochemistry, University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG
(Received 8 March 1978) SUMMARY
1. Absorption of each of sixteen amino acids, free and peptide-bound, has been measured in isolated rat small intestine perfused with five partial digests of proteins. 2. At low concentrations net absorption of each amino acid was proportional to its luminal concentration and independent of the nature of the amino acid. 3. A series of first-order multiple regressions was found to describe well the characteristics of absorption. 4. Rate constants for disappearance of free and peptide-bound amino acids from the lumen were closely similar. However, substantial back-flux occurred of amino acids derived from peptide hydrolysis. Hence 60-70 % of the amino-N entering the serosal tissue fluid probably had left the lumen as free amino acids. 5. Intact peptides crossed the mucosa during absorption from a soy bean hydrolysate and in substantial quantities during absorption from one casein digest but not from another. With other hydrolysates there was no evidence for passage of peptides to the serosa. 6. In several cases there was a serious discrepancy between the amount of amino-N absorbed from the lumen and the amount accounted for as peptide or free amino acid in the serosal secretion. 7. The characteristics of absorption were similar (apart from the exceptions in 5 above) for all the digests studied except for soy bean hydrolysate. INTRODUCTION
Although many studies on absorption of protein digests have used single amino acids or simple mixtures it is now established that absorption of small peptides can be quantitatively important (e.g. Matthews, 1975a,b; Matthews & Adibi, 1976; Silk, 1974). However the relative importance of peptide and amino acid absorption in nutrition and the fate of absorbed peptides remain unsolved (e.g. Matthews & Burston, 1977). Also, the fate of amino acids and peptides presented individually to the intestine may well differ from that observed when a balanced mixture is presented (Gardner, 1975). However, it is not known whether the findings of Gardner (1975) refer only to absorption of casein which, although of substantial nutritional significance, is not necessarily typical, since inter alia casein is a phosphoprotein and its phosphopeptides are quite resistant to enzymic hydrolysis (Mellander, 1977, p. 294; Mellander & F6lsch, 1972).
M. L. G. GARDNER The present experiments study absorption from a variety of other partial protein digests. A preliminary abstract has described part of this work (Gardner, 1978a). 84
METHODS
Animal8 Female albino rats (170-220 g) of a local Wistar strain (Centre for Laboratory Animals, Easter Bush, Penicuik, Midlothian) were used. They had free access to water and Oxoid diet 86 (Oxoid Ltd., Basingstoke, Hants.) right up until experiment. Animals were kept in conditions of controlled day-length for at least a week before use.
Experimental procedure Isolated jejunum plus ileum (ca. 90-100 cm from Ligament of Treitz to ileo-caecal valve) were perfused by a segmented flow in a single pass through the lumen as described by Fisher & Gardner (1974). The animal was maintained under ether anaesthesia until after perfusion in
8itu had been established: an experiment was abandoned if the animal died during the setting-up procedure as this produces profound deterioration of viability (Gardner, 1978b). Collections of luminal effluent and of secretion on to the serosal surface were made over four consecutive 15 min periods. Only the collections during the fourth period (45-60 min of perfusion) were fully analysed, by which time the secretion would have reached a steady state (Gardner, 1975). In several experiments with casein and soy bean hydrolysates secretion samples
only were analysed. Perfu8ion media The electrolyte composition was as follows: NaCl 118-5 mm; NaHCO, 24-88 mm; KCl 4-73 mM; KH2PO4 1-18 mM; MgSO4 0-30 mm; CaCl2 1-18 mm. The perfusate also contained either (a) casein hydrolysate (type C/ESX, Serva Feinbiochemica, c/o Uniscience Ltd., Sulivan Road, London), (b) lactalbumin hydrolysate (Sigma London Chemical Co. Ltd.), (c) soy bean hydrolysate (type S/ELB, Serva Feinbiochemica) or (d) bacteriological 'peptone' prepared from muscle (Evans Medical Ltd., Speke, Liverpool) at 0-5 mg/ml. except in control experiments. It always contained glucose (5 mg/ml.; 28 mM), and was equilibrated with 5 % CO 2 in 0 2 at 40 TC for at least 1 h before use. The phenol red used by Fisher & Gardner (1974) was not included in the
perfusate.
Amino acid and peptide analysis Minor refinements were made to the methods used by Gardner (1975, 1976). Amino acids were estimated on a Locarte amino acid analyser before and after total acid hydrolysis. Samples of intestinal secretion were deproteinized with solid sulphosalicyclic acid and all samples were preserved by addition of pentachlorophenol solution in ethanol. Most analyses were performed at least in duplicate, output being directed to a System AA computing integrator (Autolab, Spectra Physics, Ltd., St Albans, Herts.) in addition to the chart recorder. Acid hydrolyses were performed by heating 0-5 ml. perfusate or luminal effluent, or 0-2 ml. secretion, with an equal volume of Aristar hydrochloric acid (B.D.H. Chemicals, Ltd.) containing 0-02 M AnalaR phenol to minimize loss of tyrosine (Blackburn, 1968) for 24 hr at 110 'C in acid-cleaned Pyrex test-tubes evacuated to 0-1-0-5 mmHg. Hydrolysates were taken to dryness in vacuo over NaOH and P205 and were reconstituted with Na citrate buffer at pH 2-2 (1 0 ml. for perfusate and luminal effluents, and 0-4 ml. for secretions): aliquots of 0-5 or 0-05 ml. respectively were applied to the amino acid analyser. If amino acid analysis was not immediate, samples were stored at -20 'C (see Perry & Hansen, 1969). Peptide-bound amino acids were estimated from the increase in free amino acid determined after hydrolysis. The assumption made and partly validated by Gardner (1975) that no peptides in the unhydrolysed samples were eluted at the same time as amino acids was generally true. However, exceptions made resolution of part of the chromatograms of unhydrolysed Evans peptone difficult, especially in the vicinity of methionine and phenylalanine.
ABSORPTION FROM PROTEIN DIGESTS
85
Control exprement2 Data from control experiments with N-free perfusion were taken from Gardner (1975, 1976) and pooled with those from a further four making a total of eight complete experiments. In addition, secretion samples only from a further three experiments were analysed. Intestinal secretions were analysed directly (0-2 ml. applied to the amino acid analyzer) but samples of luminal effluents were concentrated tenfold by lyophilization and reconstitution before application of 0-5 ml. to the amino acid analyzer. Aliquots were also subjected to acid hydrolysis and analysed. The losses of amino acids and peptides from control intestines perfused with the N-free medium are summarized in Table 1 (see below).
Compukaion of remdUr Data were analysed by computer as before (Gardner, 1975). Multiple regression analyses were performed using the Edinburgh program package MULTREG which was run on the 4-75 computer (Gardner, 1975). The goodness of fit of each regression was ascertained by plotting the values of each dependent variable predicted using the regression against the observed values for each amino acid.
Terminology The term absorption denotes net disappearance from the lumen as determined from the analyses of inflowing perfusate and luminal effluent. The term secretion denotes appearance at the serosal surface of the intestine (Fisher & Gardner, 1974). TABLE 1. Apparent rates of absorption (from the intestinal lumen) and secretion (on to the serosal surface) during the fourth 15 min period of control experiments with N-free perfusion. ,ugN. cm-'. hr-1. Values are means of n experiments + s.E. of mean Free amino acid N Free amino acid + peptide N Peptide N (by difference)
Absorption (n = 8) -2-80+ 1-07 - 6-83 2-00 -4-04 + 1-07
Secretion (n = 11) 7-68 + 0-43 11-5 i1-04 3-78 + 0-81
RESULTS
Control experiments Control experiments were made with N-free perfusion in order to correct for loss of endogenous amino acids and peptides. These losses are summarized in Table 1. Amino acid composition of the perfusate The composition of the Sigma casein hydrolysate was given by Gardner (1975). Amino acid analyses for the other hydrolysates are shown in Tables 2 and 3.
Composition of luminal effluents and secretions The over-all distribution of N found in the perfusates, luminal effluents and secretions is shown in Table 3. The free amino acid fraction of the hydrolysate accounts for from 12 % (soy bean) to 68 % (lactalbumin) of the total amino-N in the perfusate. In several cases the free amino acid concentration in the luminal effluent exceeds that in the perfusate, while the peptide-N concentration invariably is lower in the effluent than in the perfusate.
M. L. G. GARDNER
86
_I Xcc oo
¢
+l+l+l -H +l H +l+l+l+l+l+l+l+l+l+l t- 0
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42
7
O
* t t-
.
"-
= O 4
0
cq N,
0 t- = = NOD N
'o _
~~~~~~
~~~~~~1 ~
~
q
+
0
1+
*
0
1+
1+
1+ +
1+
1+
~0
+l 9
+l +l +l +l +l +l +l +l +l +l +l +l +l +l +l +l~~~~~~~
o
|~~~~~~~~~~~~~~~~~~1 X _O e
-4 6
6,
4N
r X O 4o.z~~~~~~~~~~~a
o
-
=
N-
w 0
,
O~
v
II
Oo wIlO _ Ut- N t 4 _* ~ .4 co lOco ~~~~~~~~~
+l +1+l +1+l ++l +l++1+1
t g
c~~
ID
C)0
q
+l +l
-co
C
qC
oo
-
+l +l +l +l +l m
q
_
CIO cq
00
9
+l+H1+1 +l +1+H+1 +l +1 +l -H +l +l +l +I +l + +l +I +l ++l +I +l +I+l +I+l ++l +I +
, ;
0
;
.
_
.
* . .* 1*f._
14 0;~ N _~ ~ -n C@ - ~ 0-O __l N~ ; CX C! O - ""-I0N
|| ~
,=
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t
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~
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_
o -O0oo -
l-
-qco
+
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ws°-° Cvojm~00C-Nw
cl
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+I + +I +I +I +I +I +I +I +I +I +I +I +I +I co + ~4 l N- lO 4f -I m co °
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+
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O O _ N
ABSORPTION FROM PROTEIN DIGESTS
87
Rates of absorption from the lumen and rates of secretion The rates of absorption of N from the intestinal lumen and of N secretion on to the serosal surface are shown in Table 4. These rates have been corrected for losses of endogenous amino acids and peptides by subtraction of the values obtained in the control experiments with N-free perfusion. TABLE 3. Over-all distribution of N in perfusate, luminal effluent and secretion (ug N/ml.) during the fourth 15 min collection period during perfusion with various partial digests of proteins at 0.5 mg/ml. Values are means of n experiments s.E. of mean (actual values, not corrected for loss of N in control experiments) Protein hydrolysate in perfusate Casein* (Sigma), n = 5 Free amino acid N Peptide-bound amino acid N Casein (Serva), n = 7 Free amino acid N Peptide-bound amino acid N Soy bean (Serva), n = 8 Free amino acid N Peptide-bound amino acid N Lactalbumin (Sigma), n = 7 Free amino acid N Peptide-bound amino acid N Peptone (Evans), n = 10 Free amino acid N Peptide-bound amino acid N N-free controls (n = 8) Free amino acid N Peptide-bound amino acid N *
Perfusate
Luminal effluent
31-2+0 57 29*7+ 1*12
20-2+0068 19*6+ 1*14
206+ 15-3 157+48*4
18-0+1-1 36-6 + 2-5
13*0+2*0 25-4+ 3-3
241±+ 115 (n = 10) 6*8 + 14-8 (n = 10)
4*39+0*16
7 49+0-77 24-1+1159
122.9±11*3 (n = 11)
31-2+2-43
22*7 + 2'08 10*9+ 3*76
12*9+ 1*45 10*6+ 3*43
217 + 10*5 - 21*8 + 15*6
11*2+0055 40'9+ 3*01
15*6+ 1*55 22*4 + 4'27
210±7*1 -5*9+ 8*4
0 0
1P35+045 2*02+0*53
Secretion
38-0+121 (n = 11)
51-1+ 2-7 (n = 11) 24*5+ 49(n = 11)
Data of Gardner (1975).
Fig. 1 A and B show the relationships between the net rate of absorption (i.e. disappearance from the lumen) of each amino acid, free and peptide-bound, and its concentration in the luminal perfusate for the experiments with lactalbumin and casein (Serva) respectively. Fig. 3 of Gardner (1975) reported a comparable relationship for experiments with Sigma casein. Note that all the points, for both free and peptide-bound amino acids, conform to a generally similar relationship, which (at the low concentrations studied) is approximately linear between absorption rate and perfusate concentration. Similar relationships were observed for the free amino mixture (Gardner, 1976) for the Evans peptone and (except for the points corresponding to peptide- or amide-bound glutamic and aspartic acids which are present in particularly high concentrations) for the Serva soy bean hydrolysate (Fig. 1 C).
M. L. G. GARDNER
88
Relationships between the rates of secretion and rates of absorption Table 4 shows that the rate of total amino acid N absorption substantially exceeds the rate of total amino acid N secretion in all instances except for the Sigma casein hydrolysate and the free amino acid mixture. The discrepancy is particularly large for Evans peptone. Fig. 2A and B show the relationship between the rates of secretion and of absorption for each amino acid from Evans peptone and Sigma lactTABiF 4. Over-all rates of N absorption from the intestinal lumen and rates of N secretion on to the serosal surface during the fourth 15 min period of luminal perfusion with various protein hydrolysates. Values (,ug N. cm-'. hr-1) are means of n experiments + s.E. of mean and are corrected for losses during eight or eleven control experiments with N-free perfusion Protein hydrolysate in perfusate Casein* (Sigma), n = 5 Free amino acid N Peptide-bound amino acid N Total amino acid N Casein (Serva), n = 7 Free amino acid N Peptide-bound amino acid N Total amino acid N Soy bean (Serva), n = 8 Free amino acid N Peptide-bound amino acid N Total amino acidN Lactalbumin (Sigma), n = 7 Free amino acid N Peptide-bound amino acid N Total amino acid N Peptone (Evans), n = 10 Free amino acid N Peptide-bound amino acid N Total amino acid N Free amino acid mixturet (n = 4) Free amino acid N Peptide bound amino acid N Total amino acid N
Absorptiont
26-2+1-8 25-6+ 2-6 51-8+4-0
17-3+5-1 35-5 + 8-6
Secretion § 31-4+1-6 27-0+ 6-9 58-4+ 7-7
52-7 + 7-9
29-6+ 1-4 (n = 10) - 2-2 + 2-2 (n = 10) 27-4 + 2-4 (n = 10)
-3-8+ 2-2 25-3+6-2 21-5+6-0
11-7 + 1-5 (n = 11) 2-9+2-3 (n = 11) 14-6+2-1 (n = 11)
23-1+4-2 5-9+ 5-7 29-1 + 6-8
28-0+2-1 -7-8+ 2-3
-3-8+3-6 45-2+ 6-9 41-4 + 7-7
-4-9+ 1-5
25-3+6-2 -6-9+6-7 18-4+ 12-7
20-2 + 1-8
22-2+2-1
17-3 ± 2-2 17-3+6-6 -2-7+3-4 14-6+9-7
* From Gardner (1975).
t From Gardner (1976).
The values for Sigma casein and the free amino acid mixture differ slightly from those originally reported since these data have been recalculated taking into account all eight or eleven control experiments with N-free perfusion now available. I With eight control experiments. § With eleven control experiments.
albumin hydrolysate. In both cases the discrepancy between secretion and absorption applies almost equally to all amino acids in the mixture (see regression analysis below) except for glutamic and aspartic acids. No discrepancy was seen for Sigma casein hydrolysate (Fig. 4 of Gardner, 1975). However, there was no relationship
89 ABSORPTION FROM PROTEIN DIGESTS between the measured rates of secretion and absorption when the perfusate contained soy bean hydrolysate (Fig. 2 C).
Appearance of intact peptides in serosal secretion Table 3 showed that there were bound amino acids, assumed to be peptide-bound, in the steady-state secretion at the serosal surface of the intestine during absorption from the two casein hydrolysates (especially the Sigma hydrolysate) and the soy bean hydrolysate, but not during absorption from the Evans peptone or the lactalbumin hydrolysate. These were not all of endogenous origin, since positive values are shown in Table 4 for the rates of secretion from Sigma casein and Serva soy bean hydrolysates after subtraction of the rates observed in control experiments with N-free perfusion. Gardner (1975) reported corresponding data for the Sigma casein hydrolysate, and concluded that substantial transport of intact peptides had occurred. Rates of secretion of each amino acid, free and peptide-bound, on to the serosal surface from the Serva soy bean and Sigma casein hydrolysates are shown in Fig. 3. These rates are net rates corrected by subtraction of secretion rates in the control experiments. All the amino acids except seine, tyrosine, histidine and arginine from the soy bean hydrolysate appear in the bound-fraction of the secretion. The appearance of bound amino acids is not restricted to the dicarboxylic amino acids which may be amide-bound.
Multiple regression analyses of rates of amino acid absorption First-order multiple regressions were computed by MULTREG with the total rate of absorption of each amino acid (i.e. disappearance from luminal perfusate) as the dependent variable. The perfusate concentrations of free and peptide-bound amino acid were the two independent variables. The results are shown in Table 5. Analysis of variance showed that in all cases, except for soy bean hydrolysate, the regressions fitted very well to the data. An example of the goodness of fit is seen in Fig. 4 for the lactalbumin data, which shows the total absorption rate for each amino acid predicted from the regressions in Table 5 plotted against the observed rate. Clearly there is little scatter about the line of identity: thus the regression does describe well the observed absorption rates. It might be argued that this statistical treatment is not strictly valid since estimation of absorption rates relies on knowledge of the amino acid concentrations in the perfusate. Also, estimation of the perfusate concentrations of peptide-bound amino acids depends on knowledge of the concentrations of free amino acids. Hence the independent variables are not strictly independent, and the regression could be biased. The statistical validity can be tested by using the rates of secretion on to the serosal surface (estimated independently from the perfusate concentrations) instead of absorption rates from the lumen as the measures of transport. The coefficients in these multiple regressions are shown in square brackets in Table 5 and are clearly very similar to the others in Table 5. Note that the coefficients (b2) for concentration of peptide-bound amino acids are notably lower than those (b1) for the concentration of free amino acids for each hydrolysate except Evans peptone. Since Gardner (1975, 1976) had noted substantial backflux of amino acids during
M. L. G. GARDNER 90 absorption of peptides multiple regressions with the net rate of free amino acid absorption as the dependent variable were computed. The independent variables were the perfusate concentration of free amino acid and the rate of peptide-bound acid absorption. Results are shown in Table 6. Again, analysis of variance showed that the fit was good in all cases except for soy -
_
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A Lactalbumin hydrolysate
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0.1 Amino acid concentration (mM)
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Fig. I A and B. For legend
see
facing
page.
I
05
ABSORPTION FROM PROTEIN DIGESTS 91 bean. The excellence of the fit was confirmed by plotting the predicted rates against the observed rates (data not shown). Fig. 1 showed that the rate of disappearance of peptide-bound amino acids from the lumen was proportional to their concentration in the perfusate. Note that disappearance includes both absorption and hydrolysis. Table 7 shows 'rate constants' for this relationship, i.e. first-order coefficients in the regression of rate of disappearance of peptide-bound amino acids against their perfusate concentration. C Soy bean hydrolysate
g1x E 015 us
E'
o.4 0) E
-0
.
0
*
0-10
E
Cu
0o
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0
00
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0-1
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03
Amino acid concentration (mM)
Fig. 1. A, B and C, relationship between the net rate of absorption from the lumen of each amino acid, free (0O) and peptide-bound (@*), and its concentration in the luininal perfusate. The perfusates contained either Sigma lactalbuxnin hydrolysate (A), Serva casein hydrolysate (B), or Serva soy bean hydrolysate (C). All absorption rates have been corrected for pasage of endogenous ammno acids and peptides into the luminal effluents during N-free perfusion. Each point represents a different amino acid: for clarity only extreme points have been identified.
Again, the data for soy bean hydrolysate were the only ones for which a first-order regression was not an excellent fit. The 'rate constants' are closely similar for the Evans peptone, lactalbumin and Serva casein hydrolysates although the value was lower for Sigma casein hydrolysate. They are also very similar to the 'rate constants' for absorption of free amino acids (b1 in Tables 5 and 6). Multiple regression analysis of rates of amino adid secretion Table 5 showed the coefficients in the regression of total rate of amino acid secretion against the perfusate concentrations of free and peptide-bound amino acids. Also the rate of total amino acid N absorption substantially exceeds the rate of total amino acid N secretion during absorption from all perfusates except those containing Sigma casein hydrolysate and the free amino acid mixture (Table 4).
M. L. G. GARDNER Therefore multiple regressions were computed of total rate of amino acid (free + peptide-bound) secretion against the rates of absorption of free amino acid and of peptide-bound amino acid (Table 8); it is clear that the regressions are good fits to the observed data except in the case of the soy bean hydrolysate. The fit to the 92
A Evans peptone
0
0 c ~07 0-10
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-%' E
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0-10 0 15 0-20 Absorption rate from lumen of total (free+peptide-bound) amino acids (,mole . cm-'. hr-1)
B Lactalbumin hydrolysate)
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0-20 0-10 Absorption rate from lumen of total (free + peptide-bound) amino acids (pmole. cm-' . hr-1)
Fig. 2 A and B. For legend see facing page.
93
ABSORPTION FROM PROTEIN DIGESTS C Soy bean hydrolysate
0-10
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0-05 Absorption rate from lumen of total (free+peptide-bound) amino acids (jmole . cm-' hr-')
0-15
Fig. 2. A, B and C, relationships between the rates of absorption from the lumen and secretion on to the serosal surface of each total (i.e. free+ peptide-bound) amino acid. Each point represents a different amino acid. The perfusate contained Evans peptone (A), Sigma lactalbumin hydrolysate (B), or soy bean hydrolysate (C).
TAiBL 5. Multiple regression analyses for total rates of amino acid absorption from various protein hydrolysates. Values shown are computed parameters + S.D. in the equation: free amino acid Total rate of amino [peptide-bound = b,. concentration in acid absorption + b 2 . amino acid + C. from lumen concn. in perfusate perfusate The percentage of the variance in the dependent variable which has been accounted for by the regression is shown in parentheses. The coefficients in braces are those in the corresponding regression with total rate of amino acid secretion on to serosal surface as dependent variable with data for aspartic and glutamic acids and alanine excluded. C b2 b, Perfusate N source (ml. cm-. hr-L) (ml. cm . hr-1) (psmole. cm-'.hr- ) Casein (Sigma) 1-18+ 011 0-44+ 0-05 0-007 + 0-01 (93.2 %) {0-61 + 0-12) (1.41 + 0.15) Casein (Serva) 1-12+0-36 0-90+ 0-11 -0-005 + 0-03 (83-6%)
{1.28 + 0-16} Peptone (Evans) Lactalbumin (Sigma)*
{0-51 + 0.08)
0-65 + 0-26
0-70 + 0-12
{0.42 + 0.16)
{0.35 + 0 10) 0-67 + 0-07 {0.49 + 0.13)
1 12 + 0-09
(1.06 + 0.07} Soy bean (Serva) Soy bean - but excluding glu
0-72 + 1-22 1-13 + 1-01
and asp All data ex. Soy bean pooled
{1-22 + 0.56} 1-01 + 0-091
0-22 + 0-14 0-74 + 0-25 (0.27 + 0.14) 0-75 + 0-043
(130 + 0.087)
{0.61 + 0.065}
*
See Fig. 4.
0-01+ 0-02 (72X5 %) -0-01+ 0-01 (93-3 %)
0-04+0-03 (17-2 %) -0-01+ 0-03 (46.5%) - 0-007 + 0-01 (82-0 %)
M. L. G. GARDNER
94 25
20
- 15
)10
.
5
01
8
A F RSoy bean
6 4 2 ot
:~[H ~.r6hFr0 I
I
ASP
0
TH R
SE R
G LU
PRO
Im
0
m
GLY ALA VAL MET ILE LEU TYR PHE -HIS LYS ARG
Fig. 3. Rates of secretion on to the serosal surface of each amino acid, free (open bars) and bound (hatched bars), during luminal perfusion with (A) soy bean hydrolysate and (B) Sigma casein hydrolysate. Lactalbumin hydrolysate
0
0
0
0-2
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Observed total rate of amino acid absorption from lumen (pmole. cm-'. hr-1)
Fig. 4. Goodness of fit of data for absorption from lactalbumin hydrolysate to multiple regressions shown in Table 5. Each point represents a different amino acid.
ABSORPTION FROM PROTEIN DIGESTS 95 computed regressions was particularly excellent for the Sigma casein and lactalbumin hydrolysates. Peptide hydrolysi by the intestine Table 4 showed that the rates of free amino acid secretion on to the intestine's serosal surface always exceeded the rates of free amino acid absorption during perfusion with mixtures containing peptides. Conversely, the rate of peptide-N TABLE 6. Multiple regression analysis for net rates of free amino acid absorption from various protein hydrolysates. Values shown are the computed parameters + S.D. in the equation: net rate of of amino free amino acid _ b free acid conen. + C. + b8. peptidenbound disappearance from lumen aot The percentage of the variance in the dependent variable which has been accounted for by the regression is shown in parentheses. c b, Perfusate N source (ml. cm-'.hr-1) Casein (Sigma) 1-22 + 0-07 -0-39+004 -0008±+001 (96.6%) Casein (Serva) 1*60+0 15 -0 14+_ 004 -0'03 +0*01 (93.9%) Peptone (Evans) 1.00+ 0*09 -033_+003 - 0*003 + 0-008 (96.6%) Lactalbumin (Sigma) 1-19+0-06 -032±0O05 -0.01 +0-01 (98.1 %) 0*66 + 0-51 Soy bean (Serva) -0-19 + 009 -0-009 + 0.01 (31.5%) All data ex. soy bean pooled 1 19+ 0-056 -0-23 + 0*025 -0-016 + 0*0059 (90.6%) 1.
Lin perfisateJ
TABLE 7. First-order 'rate constants' for disappearance (i.e. absorption + hydrolysis) of peptidebound amino acids from the lumen during perfusion with various protein hydrolysates. Values shown are the computed regression parameters in the equation: rate of peptide-bound
b. acid conen. in + C. erfusate The percentage of the variance which has been accounted for by the regression is shown in parentheses. b C Perfusate N source (ml. cm-'.hr-l) (Creole . cm-L . hr-IL) Casein (Sigma) 0*73 0'06 0-02 + 0-01 (91.0 %) Casein (Serva) -0-01 +0-02 (86.5%) 1o10+ 012 Peptone (Evans) 1-13 + 0 16 -0*009+0-02 (78-1 %) Lactalbumin (Sigma) 1.00+ 0.09 -0-016 + 0-008 (89-3 %) Soy bean (Serva) 0 37 0-14 -0 046+ 0019 (32-4%) All data ex. soy bean pooled 1.00+ 0 044 - 0-007 0006 (86-7 %) amino acid
disappearance
=
+
secretion was less than the rate of peptide-N absorption. Also the net rate of free amino acid absorption from the lumen was negative in some cases. The obvious explanation is that a large proportion of the peptides disappearing from the luminal perfusate are hydrolysed to free amino acids by hydrolases located in the brush border and cytoplasm of the mucosal cells. Therefore, the over-all disappearance of peptide-bound acids should be accompanied by a corresponding increase in free amino acids.
Table 9 summarizes the regression coefficients relating the over-all rate of dis-
M. L. G. GARDNER appearance of peptide-bound amino acids from the preparation to the overall rate of appearance of free amino acids. In most instances there is good correspondence between rates of peptide disappearance and of free amino acid appearance. Corresponding data for the Sigma casein hydrolysate were reported by Gardner (1975, Fig. 7). The generally good correspondence seen supports the accuracy of the techniques used. 96
TABLE 8. Multiple regression analyses for total rates of amino acid secretion on to the serosal surface as a function of the respective absorption rates of free and peptide-bound amino acids from the intestinal lumen. Values shown are the computed parameters + S.D. in the equation: total rate of amino acid rate of free rate of peptide-) secretion on to the = bLl. amino acid + b 2. bound amino acid +C. serosal surface absorption absorption The percentage of the variance which has been accounted for by the regression is shown in parentheses. Data for aspartic and glutamic acids and alanine have been excluded C Perfusate N source b2 (#emole . cm-' . hr-1) 1-08 + 0-096 1-14+ 0-09 Casein (Sigma) 0-009 + 0-015 (95.2 %) Casein (Serva) 0-89 + 0-16 0-48 + 0-098 -0-009 + 0-02 (76-8%) Peptone (Evans) 0-53 + 0-13 0-44+ 0-08 - 0-0004 + 0-01 (72.4%) Lactalbumin (Sigma) 0-88 + 0-06 0-63 + 0-09 -0-009 + 0-007 (96-3 %) 0-28 + 0-14 0-29 + 0-36 Soy bean (Serva) 0-032 + 0-012 (29-0 %) All data ex. soy bean pooled 1-05 + 0-08 0-70 + 0-06 - 0-015 + 0-010 (75.1 %) TABLE 9. Relationship between the overall rate of disappearance of peptide-bound amino acids with the over-all rate of disappearance of free amino acid from the perfused intestine. Negative rates of disappearance of free amino acids signify an overall increase in free amino acids, apparently from hydrolysis of peptides. Values shown are the computed parameters+ S.D. in the equation: over-all rate of (over-all rate of disappearance disappearance of = b.o peptide-bound amino acid + C. free amino acid The percentage of the variance which has been accounted for by the regression is shown in parentheses. Data for aspartic and glutamic acids and alpine have been excluded Perfusate N source Casein (Sigma) Casein (Serva) Peptone (Evans) Lactalbumin (Sigma) Soy bean (Serva) All data ex. soy bean pooled
c moleol. 0-921 + 0-085 -0-029+0-007 - 0-547 + 0-072 0-017 + 0-014 0-714+ 0-069 0-028 + 0-012 - 0-786 + 0-076 0-013 + 0-006 - 0-386 + 0-128 -0-024 + 0-011 -0-61 +0-04 0-007+0-005 b
-
-
cm1.
br-1)
(91-5%) (83-8 %) (90-7 %)
(90-6%) (45-2 %)
(80-6%)
Gardner (1975) showed a strong positive correlation between the overall rate of disappearance of peptide-bound amino acids and the rate of their absorption from the lumen for the Sigma casein hydrolysate (Fig. 8 of Gardner, 1975). Similar relationships were also seen in the present investigation (Table 10). Again, the data fit remarkably well to straight lines; the slopes (Table 10) are close to unity except in the case of Sigma casein hydrolysate for which hydrolysis of absorbed peptides has already been found to be incomplete.
ABSORPTION FROM PROTEIN DIGESTS
97
TABLE 10. First-order regression coefficients relating the over-all rate of disappearance of peptidebound amino acid (presumed to be the rate of hydrolysis) to the rate of absorption of peptidebound amino acid from the intestinal lumen. Values shown are the computed parameters + S.D. in the equation: over-all rate of disappearance _ b (rate of absorption of C+ + of peptide-bound amino acid - -peptide-bound amino acid+ The percentage of the variance accounted for is shown in parentheses. Data for aspartic and glutamic acids and alanine have been excluded
b Perfusate N source (ismole. cm". hr-') Casein (Sigma) 0-900 + 0-146 -0-073 + 0-017 (77-5 %) 0-02 + 0-01 (96-1 %) Casein (Serva) 0-99 + 0-06 Peptone (Evans) 1-09+0-04 0-009+ 0-006 (98-4%) Lactalbumin 1-08+0-11 0-03 + 0-006 (90-1%) (Sigma) -0-01+ 0-01 (89-6%) Soy bean (Serva) 0-99 + 0-10 All data pooled 0-94 + 0-048 -0-0004 + 0-007 (83-2 %) DISCUSSION
Relative rates of absorption of different amino acids Gardner (1975, 1976) observed that the rate of absorption of each amino acid in a casein hydrolysate and a synthetic mixture of amino acids depended in a linear fashion on the luminal concentration of that amino acid and apparently not on the nature of the amino acid (see also Bronk & Leese (1974) and Matthews (1975b, pp. 116-117)). Such linear relationships imply a constant ratio of Vmax. to Km for all amino acids (Gardner, 1975). Similar relationships were observed in the present work with each protein digest (Fig. 1A, B and C). Thus in complex mixtures at low concentrations each amino acid appears to be absorbed at a rate which is independent of its chemical structure. This is a contrast to the behaviour of simple pairs of amino acids where competition studies have led to the view that there are separate mechanisms for the transport of acidic, neutral and basic amino acids.
Relative rates of absorption of free and peptide-bound amino acids Since Fig. 1 shows that the free and peptide-bound amino acids conform to the same relationship between absorption rate and perfusate concentration it might appear that free and peptide-bound amino acids are equally rapidly absorbed from a given concentration. However, these absorption rates are net rates measured from the disappearance of amino acids between the perfusate and luminal effluent. If peptides disappear from the lumen and give rise to free amino acids after hydrolysis (superficial or intracellular) then any backflux of amino acids into the lumen will reduce the measured net rate of free amino acid absorption. Hence the net rates of free amino acid absorption shown in Fig. 1 underestimate the true rates of amino acid absorption from the free amino acids in the perfusate. The regression analyses in Table 5 overcome this problem of interpretation. It is 4
PHY
284
M. L. G. GARDNER 98 clear from the analyses of variance and from the specimen plot in Fig. 4 that the equation with first-order terms in free amino acid concentration and in peptidebound amino acid concentration does describe very well the absorption data except for the soy bean hydrolysate. The relative magnitude of the b. and b2 coefficients imply that net absorption occurs substantially faster from free amino acids than from peptides, except in the instance of the Evans peptone (muscle digest) for which the 'rate constants' b1 and b2 are almost equal (Table 5). The same conclusion is drawn when the rate of amino acid secretion on to the serosal surface is the dependent variable (data in square brackets in Table 5).
Backflux offree amino acids into lumen Gardner (1975, 1976) noted substantial backflux of free amino acids into the lumen during absorption of a casein digest. These arose from hydrolysis of peptides, either at the brush border or in the cytosol of the mucosal cells, both of which contain peptide hydrolase activity (e.g. Kim, Birtwhistle & Kim, 1972). Similar backfluxes into the lumen during peptide absorption were reported by others (e.g. Newey & Smyth, 1962; Asatoor, Cheng, Edwards, Lant, Matthews, Milne, Navab & Richards, 1970; Matthews, Lis, Cheng, & Crampton, 1969; Heading, Schedl, Stegink & Miller, 1977). The equations in Table 6 imply that the rate of backflux of amino acids is proportional to the rate of peptide-bound amino acid absorption: on average, 23+ 2-5 % (b2 in Table 6) of absorbed peptide-bound amino acids pass back into the lumen following hydrolysis at the brush-border and/or the cytoplasm (see below and Wiseman, 1977). Total disappearance of peptides from perfusate The 'rate constants', or first-order coefficients, relating the rate of disappearance of peptides from the lumen to the concentration of peptide-bound amino acids were shown in Table 7. Note that disappearance includes both absorption and hydrolysis. These coefficients (b) are very similar to the coefficients (bl) for absorption (or secretion) from free amino acids in Table 5. Thus, the rate of disappearance of a particular amino acid from the lumen is, for a given concentration, independent of whether that amino acid is free or peptide-bound. If however it is peptide-bound a substantial fraction (23 % on average; Table 6) will pass back to the lumen as free amino acid. N balance of perfused intestine Table 4 showed that, except for experiments with Sigma casein and with the synthetic amino acid mixture, the rate of total amino acid absorption from the lumen exceeded the rate of total amino acid secretion on to the serosal surface. The intestine appears to be in positive N balance. Either absorbed amino acids are continuing to accumulate in the tissue or they are appearing in the intestinal secretion in a form which fails to be estimated as free amino acids after the acid hydrolysis. Losses of amino acids could occur if (a) they were transformed so as not to be ninhydrin-positive or so that their elution from the ion-exchange resin was greatly altered, (b) if amino acids entered the secretion in peptides which were stable under the hydrolysis conditions used, or (c) if amino acids entered the secretion in protein
99 ABSORPTION FROM PROTEIN DIGESTS form, either synthesized into proteins or bound to pre-existing proteins, which would be lost when the secretion samples were deproteinized prior to analysis. There is a serious discrepancy between the rate of appearance of nitrogen on the serosal side of the intestine and its rate of absorption from the lumen, and it must be stressed that in no study, in vitro or in vivo, has the nature of all absorbed amino N been accounted for quantitatively. The best estimate appears to be that of Dawson & Porter (1962) who could only account for about 41 % of absorbed amino nitrogen appearing as free amino acids in portal plasma, and this was likely to be an overestimate (Fisher, 1967a). Therefore, Fisher's (1967b) conclusion that '...the form in which the products of protein digestion enter the body is still not established. No one has been able to show that more than a minority of it enters the bloodstream as amino acids...' is still correct. Matthews & Burston (1977) agreed that the possibility of peptides entering the portal blood on a significant scale has not been satisfactorily excluded. Techniques in vitro such as that of Fisher & Gardner (1974) are suited to studies of this kind since the secretion is not diluted or contaminated by a bloodstream. Also one has access to the solutes which would enter the lymphatics as well as the bloodstream and this may be important since the amino-N concentration in the cisterna chyli rises during absorption of a protein meal (Jacobs, Largis & Hanson, 1967; Jacobs & Largis, 1969a, b; Bolton & Wright, 1937). The productprecursor relationship seen by Morris (1956) in intestinal lymph during protein digestion indicated that a significant fraction of the lymph protein was probably synthesized in the intestine (see Fisher, 1967b). The regression coefficients in Table 8 suggest that the discrepancy between secretion and absorption is uniformly greater for the peptide-bound amino acids. The values of b, and b2 for the pooled data (Table 8) suggest that 105 + 8 % of the absorbed free amino acids have appeared in the secretion on the serosal surface while only 70 + 6 % of the absorbed peptides have been recovered in any form. This raises the possibility that some peptides which enter the serosal secretion are either protein-bound (and lost during deproteinization) or are resistant to acid hydrolysis (and escape detection). Alternatively, peptides may be destroyed faster during the acid hydrolysis than free amino acids or peptide-N is continuing to be accumulated within the cells.
Validity of multiple regression analyses With the exception of the data for absorption of soy bean hydrolysate, the multiple linear regressions in Tables 5-10 describe remarkably well the observations. The goodness of fit can be seen from the analyses of variance, and particularly from plots of the predicted values of the dependent variables against the observed values (Fig. 4 and unpublished observations). While the validity of the equations in describing the observed processes numerically is therefore proved, the physiological interpretation requires caution. Rate-limiting steps: hydrolysis or transport? Loss of peptides and gain of free amino acids. Table 9 shows that the overall loss of peptide-bound amino acids from the preparation was accompanied by a corresponding gain of free amino acids. Although 4-2
M. L. 0. GARDNER the regression coefficients in Table 9 imply that only 55-92 % (average 61 +4 %) of the disappearing peptide-bound N was accounted for by appearance of free amino acids, the close fit of each amino acid to the relation supports the validity of the measurements of absorption and secretion. Table 10 (also Fig. 8 of Gardner, 1975) showed that the over-all rate of disappearance of peptide-bound amino acids was proportional to the rate of their absorption. Since the rate of peptide absorption was closely proportional to the luminal concentration it appeared that the absorption step was rate-limiting for hydrolysis (Gardner, 1975). However this argument is not valid since a similar relationship could be observed if superficial hydrolysis occurred with no transport of intact peptides. 100
Transport of intact peptides across intestine. Although certain peptides are resistant to mucosal hydrolysis (e.g. glycylglycine, Newey & Smyth, 1959; Peters & MacMahon, 1970; Adibi, 1971; carnosine, Perry, Hansen, Tischler, Bunting & Berry, 1967, and hydroxyproline peptides arising from gelatin digestion, Prockop & Sjoerdsma, 1961; Bronstein, Haeffner & Kowlessar, 1966) and can cross the mucosa in substantial quantities, the conventional view is that most peptides are hydrolysed to free amino acids within or on the mucosal cells (e.g. Matthews, 1975b, p. 72; Bell, Emslie-Smith & Paterson, 1976; Dent & Schilling, 1949). Thus, only free amino acids should enter the circulation in vivo. Fisher (1967a,b) stressed that the evidence for this was poor. Gardner (1975) suggested that about 33 % of the N absorbed from the Sigma casein hydrolysate entered the tissue fluid in peptide form, although the possibility that it was proteinor amide-bound must also be considered. The present experiments show that during absorption from lactalbumin hydrolysate and Evans peptone no significant peptide-bound N was detected in the steadystate secretion on to the serosal surface (Tables 3 and 4). However, small quantities of bound amino acids were detected during absorption from Serva casein and soy bean hydrolysates. Fig. 3 showed that almost all sixteen amino acids were present in bound form in the secretion during absorption from the Sigma casein and the soy bean hydrolysates. A much smaller fraction of the secreted amino-N was peptidebound during perfusion with the Serva casein hydrolysate than with the Sigma casein hydrolysate used by Gardner (1975). Further experiments with the latter casein hydrolysate support the original analyses reported by Gardner (1975), although the size of the transported peptides is still not known. Sleisenger, Pelling, Burston & Matthews (1977) estimated amino acids in portal venous plasma during absorption from a casein hydrolysate and it appeared that a substantial proportion of some amino acids entered the portal blood in peptidebound form. It should be noted that casein contains phosphopeptides, and these are known to be resistant to enzymic hydrolysis (Mellander & F6lsch, 1972; Mellander, 1977, p. 294). Many actively absorbed solutes accumulate at high intracellular concentrations in most preparations of intestine in vitro, so that unphysiological concentrations of peptides may be presented to the peptidases: this may favour hydrolysis of absorbed peptides in vitro. This potential artifact may be reduced by vascular perfusion
ABSORPTION FROM PROTEIN DIGESTS
101
Boyd, 1977). Also, reduction of intracellular accumulation may reduce the backflux of free amino acids into the intestinal lumen if the principal site of peptide hydrolysis is intracellular. While the fate of any intact peptides which enter the portal circulation in vivo is not known, Krzysik, Peterson & Adibi (1975) showed that liver, muscle and kidney also contain substantial dipeptide hydrolase activities. Also, intravenously administered dipeptides were rapidly cleared from plasma (Adibi, 1977; Adibi & Krzysik, 1977; Adibi, Krzysik & Drash, 1977). Assimilation of peptides in i.v. nutrition media is well known in man (e.g. Lidstr6m & Wretlind, 1952; Christensen, Wilber, Coyne & Fisher, 1955).
Similarity of absorption from different proteins Absorption from digests of several different proteins, including animal and plant proteins, milk and meat proteins, and similar proteins from different sources have now been compared. With a few exceptions, the characteristics are very similar, and this justifies extended studies on any one of these digests. The chief exceptions are (i) intact peptide transport into the secretion was only seen from the casein and soy bean digests, (ii) the two different sources of casein gave different amounts of intact peptide secretion, (iii) the soy bean digest data were not well fitted by the multiple regression analyses unlike those for the other digests. In connexion with the anomalous behaviour of soy bean hydrolysate (which had a particularly low free amino acid content, Table 3) it may be relevant that the biological value of raw soy bean is less than expected. One reason appears to be the presence of at least one powerful trypsin inhibitor (Laskowski & Laskowski, 1954). There is indirect evidence that some trypsin inhibitors may also act on intestinal mucosal enzymes (Laskowski, Haessler, Miech, Peanasky & Laskowski, 1958). Concentration ofprotein digests It must be stressed that only a single concentration of the digests, namely 0 5 mg/ ml., has been studied. The conclusions are not necessarily applicable at higher concentrations, and further investigations are needed.
Validity of studies in vitro Accumulation of peptides and free amino acids to high levels in the mucosal cells may have increased the rate of peptide hydrolysis and the rate of backflux of amino acids into the lumen, as discussed above (see also Boyd, 1977). Furthermore, preparations of intestine in vitro lose cytoplasmic peptidases into the luminal or mucosal fluids (Silk & Kim, 1976; Lindberg, Noren & Sj6strom, 1975, pp. 229-230). Thus, unphysiological peptide hydrolysis may proceed within the luminal fluid: also, the cytoplasmic peptidase activity may be low during in vitro studies. However we have evidence (M. L. G. Gardner & J. A. Plumb unpublished observations) that leakage of peptidases is less from the Fisher & Gardner (1974) preparation than from other preparations in vitro. Furthermore, in single-pass perfusion the perfusate peptides can only come in contact with 'leaked' enzymes for a relatively short time and the enzyme concentration in the luminal fluid must be much lower than in incubation or multiple-pass perfusion experiments.
M. L. G. GARDNER
102
Over-all scheme of amino acid and peptide absorption The principal quantitative conclusions can be summarized with the aid of Fig. 5. The rate constants for disappearance of free amino acids (route A) and peptides (route B) from the lumen are similar at around 1 ml. cm-'.hr-I (b, from Tables 5 and 6 and b from Table 7). Substantial backflux of free amino acids arising from peptide hydrolysis occurs by routes C and/or D. The extent of this backflux, 100 w (C+D/B,) is on average 23 % (b2 from Table 6). Hence, 60-70 % of the free amino acids arriving at the serosal surface of the intestine (routes E, F and G) appear to originate from free amino acids in the perfusate by route E: this is reflected by the b, coefficients in Table 5 which are greater than the b2 coefficients. Brush border
Lumen
Mucosal cell
Serosal tissue fluid
A
C
-40=
D
-111
4.
__
a
4 _ _
-=moo,.
lE.
be
F
-HH
B
-I
G
Fig. 5. Schematic representation of the absorption of amino acids and peptides across the intestinal mucosa. Broken lines (---) indicate transport of free amino acids; solid lines ( ) indicate transport of peptides; the symbol *A*+ indicates hydrolysis of peptides. See text.
Superficial membrane hydrolysis (site X in Fig. 5) and intracellular hydrolysis (site Y) cannot be distinguished from these data. Studies on hydrolysis in the presence of dinitrophenol suggest that membrane digestion is important (Gardner, M. L. G. unpublished observations). The amount of intact peptide crossing the mucosa (route H) is negligible during absorption of some digests (Evans peptone and lactalbumin hydrolysate), small during absorption of others (Serva casein and soy bean hydrolysates), but can be substantial during absorption from some digests (Sigma casein hydrolysate) such that (1OOxH)/(E+F+G+H) = 46%. However, in these experiments (except those with Sigma casein hydrolysate) not all the amino-N absorbed (routes A + B - C(- D) has been accounted for by secretion of amino acid or peptide (routes E +F +G+ H). The main discrepancy appears to be in recovery of absorbed peptides (route B) since the b2 coefficients in Table 8 are less than unity. There is no satisfactory evidence whether any absorbed amino-N is synthesized into, or bound to, protein (route J) and this possibility should not be discounted.
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I am indebted to the Medical Research Council for financial support, and to Professor R. B. Fisher and Professor D. M. Matthews for valuable discussions and encouragement. I also thank Miss E. M. Middleton and Mr R. Taylor and the Staff of the workshops and animal house in the Department of Biochemistry for technical assistance. REFERENCES ADiBI, S. A. (1971). Intestinal transport of dipeptides in man: relative importance of hydrolysis and intact absorption. J. dlin. Inve8t. 50, 2266-2275. ADrBI, S. A. (1977). Clearance of dipeptides from plasma: role of kidney and intestine. In Peptide Transport and Hydrolysi8 (Ciba Fdn Symp.), ed. ELuIOTT, K. & O'CoNNOR, M., pp. 265-280. Amsterdam: Associated Scientific Publishers. ADrBi, S. A. & KRZYSIx, B. A. (1977). Effect of nephrectomy and enterectomy on plasma clearance of intravenously administered dipeptides in rats. Clin. Sci. 52, 205-213. ADrBI, S. A., KmzYsix, B. A. & DRAsir, A. L. (1977). Metabolism of intravenously administered dipeptides in rats: effects on amino acid pools, glucose concentration, and insulin and glucagon secretion. Clin. Sci. 52, 193-204. ASATOOR, A. M., CENG, B., EDWARDS, K. D. G., LANT, A. F., MATTrEws, D. M., MILNE, M. D., NAvAB, F. & RIcHARDs, A. J. (1970). Intestinal absorption of two dipeptides in Hartnup disease. Gut 11, 380-387. BELL, G. H., EMsLIE-SMITH, D. & PATERSON, C. R. (1976). Textbook of Physiology and Biochemi8try, 9th edn., p. 128. Edinburgh: Churchill Livingstone. BLAcKBJuRN, S. (1968). Amino Acid Determination: Method8 and Technique8. London: Arnold. BOLTON, C. & WRIGHT, G. P. (1937). The absorption of amino acids and their distribution in the body fluids. J. Physiol. 89, 269-286. BOYD, C. A. R. (1977). Vascular flow and the compartmental distribution of transported solutes within the small intestinal wall. In Intestinal Permeation (Proceedings of the Fourth Workshop Conference Hoechst), ed. KRAMER, M. & LAUTERBACH, F., pp. 41-47. Amsterdam: Excerpta Medica. BRONK, J. R. & LEESE, H. J. (1974). The absorption of a mixture of amino acids by rat small intestine. J. Phy&iol. 241, 271-286. BRONSTEIN, H. D., HAEFNmER, L. J. & KOwLEssAR, 0. D. (1966). The significance of gelatin tolerance in malabsorptive states. Gaetroenterology 50, 621-630. CHRISTENSEN, H. N., WILBER, P. B., COYNE, B. A. & FISHER, J. H. (1955). Effects of simultaneous or prior infusion of sugars on the fate of infused protein hydrolysates. J. din. Inve8t. 34, 86-94. DAWSON, R. & PORTER, J. W. G. (1962). An investigation into protein digestion with 14Clabelled protein. 2. The transport of 14C-labelled nitrogenous compounds in the rat and cat. Br. J. Nutr. 16, 27-38. DENT, C. E. & ScmLwnJa, J. A. (1949). Studies on the absorption of proteins: the amino-acid pattern in the portal blood. Biochem. J. 44, 318-335. Fin m , R. B. (1967a). Absorption of proteins. Br. med. Bull. 23, 241-246. FISHER, R. B. (1967b). Absorption of proteins. Proc. Nutr. Soc. 26, 23-27. FISHER, R. B. & GARDNER, M. L. G. (1974). A kinetic approach to the study of absorption of solutes by isolated perfused small intestine. J. Physiol. 241, 211-234. GARDNER, M. L. G. (1975). Absorption of amino acids and peptides from a complex mixture in the isolated small intestine of the rat. J. Phy8iol. 253, 233-256. GARDNER, M. L. G. (1976). Absorption from a mixture of seventeen free amino acids by the isolated small intestine of the rat. J. Phy8iol. 255, 563-574. GARDNER, M. L. G. (1978a). Absorption of amino acids and peptides from complex hydrolysates of proteins. Gastroent. clin. et biol. 2, 341. GARDNER, M. L. G. (1978b). The absorptive viability of isolated intestine prepared from dead animals. Q. JZ exp. Phy8iol. 63, 93-95. HEADING, R. C., SCHEDL, H. P., STEGINK, L. D. & M aEB, D. L. (1977). Intestinal absorption of glycine and glycyl-L-proline in the rat. Clin. Sci. 52, 607-614. JACOBS, F. A. & LARGIS, E. E. (1969a). Transport of amino acids via the mesenteric lymph duct in rats. Proc. Soc. exp. Biol. Med. 130, 692-696.
104
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JACOBS, F. A. & LARGIS, E. E. (1969b). Effect of protein inhibitors on protein and amino acids in mesenteric lymph. Proc. Soc. exp. Biol. Med. 130, 697-702. JACOBS, F. A., LARGIS, E. E. & HANSON, E. J. (1967). Dietary amino acid transport via lymph. Fedn Proc. 26, 302. Kim, Y. S., BIRTWHSTLE, W. & KIm, Y. W. (1972). Peptide hydrolases in the brush border and soluble fractions of small intestinal mucosa of rat and man. J. dlin. Invest. 51, 1419-1430. KRzYsix, B. A., PETERSON, J. A. & ADIBI, S. A. (1975). The potential of blood, liver, muscle and kidney for dipeptide hydrolysis. Fedn Proc. 34, 466. LASKOWSKI, M., HAEssLER, H. A., MIEcH, R. P., PEANASKY, R. J. & LASKOWSKI, M. (1958). Effects of trypsin inhibitors on passage of insulin across the intestinal barrier. Science, N.Y. 127, 1115-1116. LAsKOWsKi, M. & LASKOWSKI, M. (1954). Naturally occurring trypsin inhibitors. Adv. Protein Chem. 9, 203-242. LIDsTROM, F. & WRETLIND, K. A. J. (1952). Effect of dialysed casein hydrolysate. The effect of intravenous administration of a dialyzed enzymatic casein hydrolysate (Aminosol) on the serum concentration and on the urinary excretion of amino acids, peptides and nitrogen. Scand. J. clin. lab. Invest. 4, 167-178. LINDBERG, T., NORkN, 0. & SJOSTRdM, H. (1975). Peptidases of the intestinal mucosa. In Peptide Transport in Protein Nutrition, ed. MATTnnMws, D. M. & PAYNE, J. W., pp. 204-242. Amsterdam: North-Holland. MATrr1EWs, D. M. (1975a). Intestinal absorption of peptides. Phy8iol. Rev. 55, 537-608. MArrsWs, D. M. (1975 b). Absorption of peptides by mammalian intestine. InPeptide Transport in Protein Nutrition, ed. MArrlEws, D. M. & PAYNE, J. W., pp. 61-146. Amsterdam: NorthHolland. MATTiHws, D. M. & ADIBI, S. A. (1976). Peptide absorption. Gastroenterology 71, 151-161. MATTEwS, D. M. & BURSTON, D. (1977). Intestinal transport of peptides. In Intestinal Permeation (Proceedings of the Fourth Workshop Conference Hoechst), ed. KRAMER, M. & LAUTERBACH, F., pp. 136-143. Amsterdam: ExcerptaMedica. MATTHEWS, D. M., Lis, M. T., CIrENG, B. & CRAMPTON, R. F. (1969). Observations on the intestinal absorption of some oligopeptides of methionine and glycine in the rat. Clin. Sci. 37, 751-764. MELLANDER, 0. (1977). Discussion In Peptide Transport and Hydrolysis (Ciba Fdn Symp.), ed. ELuorr, K. & O'CONNOR, M., pp. 294-295. Amsterdam: Elsevier. MELLANDER, 0. & FOLSCr, G. (1972). Enzyme resistance and metal binding of phosphorylated peptides. In International Encyclopaedia of Food and Nutrition, vol. 11, ed. BIGWOOD, E. J., pp. 569-579. Oxford: Pergamon. MORRIS, B. (1956). The exchange of protein between the plasma and the liver and intestinal lymph. Q. JI exp. Physiol. 41, 326-340. NEWEY, H. & SMYTH D. H. (1959). The intestinal absorption of some dipeptides. J. Phyaiol. 145, 48-56. NEWEY H. & SMYTrH D. H. (1962). Cellular mechanisms in intestinal transfer of amino acids. J.Phyaiol. 164 527-551. PERRY T. L. & HANSEN S. (1969). Technical pitfalls leading to errors in the quantitation of plasma amino acids.Clin. chim. Acta 25, 53-58. PERRY, T. L.,HANSEN, S., TISCELER, B., BuNTING, R. & BERRY, K. (1967). Carnosinemia. A new metabolic disorder associated with neurologic disease and mental defect. New Engl. J. Med. 277, 1219-1227. PETERS, T. J. & MACMAHON, M. T. (1970). The absorption of glycine and glycine oligopeptides bytherat. Clin. Sci. 39, 811-821. PROCKOP, D. J. & SJOERDSMA, A. (1961). Significance of urinaryhydroxyproline in man. J. din. Invest. 40, 843-849. SILK, D. B. A. (1974). Peptide absorption in man. Gut 15, 494-501. SILK, D. B. A. & KIM, Y.S. (1976). Release of peptide hydrolases during incubation of intact intestinal segments in vitro. J. Physiol. 258, 489-497. SLEISENGER, M. H., PELLING, D., BURSTON, D. & MATTEEWS, D. M. (1977). Amino acid concentrations in portal venous plasma during absorption from the small intestine of the guineapig of an amino acid mixture simulating casein and partial enzymic hydrolysate of casein. Clin. Sci. 52, 259-267. WIsEMAN,G. (1977). Site of intestinal dipeptide hydrolysis. J. Physiol. 273, 731-743. a
83
AMINO ACID AND PEPTIDE ABSORPTION FROM PARTIAL DIGESTS OF PROTEINS IN ISOLATED RAT SMALL INTESTINE
BY MICHAEL L. G. GARDNER From the Department of Biochemistry, University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG
(Received 8 March 1978) SUMMARY
1. Absorption of each of sixteen amino acids, free and peptide-bound, has been measured in isolated rat small intestine perfused with five partial digests of proteins. 2. At low concentrations net absorption of each amino acid was proportional to its luminal concentration and independent of the nature of the amino acid. 3. A series of first-order multiple regressions was found to describe well the characteristics of absorption. 4. Rate constants for disappearance of free and peptide-bound amino acids from the lumen were closely similar. However, substantial back-flux occurred of amino acids derived from peptide hydrolysis. Hence 60-70 % of the amino-N entering the serosal tissue fluid probably had left the lumen as free amino acids. 5. Intact peptides crossed the mucosa during absorption from a soy bean hydrolysate and in substantial quantities during absorption from one casein digest but not from another. With other hydrolysates there was no evidence for passage of peptides to the serosa. 6. In several cases there was a serious discrepancy between the amount of amino-N absorbed from the lumen and the amount accounted for as peptide or free amino acid in the serosal secretion. 7. The characteristics of absorption were similar (apart from the exceptions in 5 above) for all the digests studied except for soy bean hydrolysate. INTRODUCTION
Although many studies on absorption of protein digests have used single amino acids or simple mixtures it is now established that absorption of small peptides can be quantitatively important (e.g. Matthews, 1975a,b; Matthews & Adibi, 1976; Silk, 1974). However the relative importance of peptide and amino acid absorption in nutrition and the fate of absorbed peptides remain unsolved (e.g. Matthews & Burston, 1977). Also, the fate of amino acids and peptides presented individually to the intestine may well differ from that observed when a balanced mixture is presented (Gardner, 1975). However, it is not known whether the findings of Gardner (1975) refer only to absorption of casein which, although of substantial nutritional significance, is not necessarily typical, since inter alia casein is a phosphoprotein and its phosphopeptides are quite resistant to enzymic hydrolysis (Mellander, 1977, p. 294; Mellander & F6lsch, 1972).
M. L. G. GARDNER The present experiments study absorption from a variety of other partial protein digests. A preliminary abstract has described part of this work (Gardner, 1978a). 84
METHODS
Animal8 Female albino rats (170-220 g) of a local Wistar strain (Centre for Laboratory Animals, Easter Bush, Penicuik, Midlothian) were used. They had free access to water and Oxoid diet 86 (Oxoid Ltd., Basingstoke, Hants.) right up until experiment. Animals were kept in conditions of controlled day-length for at least a week before use.
Experimental procedure Isolated jejunum plus ileum (ca. 90-100 cm from Ligament of Treitz to ileo-caecal valve) were perfused by a segmented flow in a single pass through the lumen as described by Fisher & Gardner (1974). The animal was maintained under ether anaesthesia until after perfusion in
8itu had been established: an experiment was abandoned if the animal died during the setting-up procedure as this produces profound deterioration of viability (Gardner, 1978b). Collections of luminal effluent and of secretion on to the serosal surface were made over four consecutive 15 min periods. Only the collections during the fourth period (45-60 min of perfusion) were fully analysed, by which time the secretion would have reached a steady state (Gardner, 1975). In several experiments with casein and soy bean hydrolysates secretion samples
only were analysed. Perfu8ion media The electrolyte composition was as follows: NaCl 118-5 mm; NaHCO, 24-88 mm; KCl 4-73 mM; KH2PO4 1-18 mM; MgSO4 0-30 mm; CaCl2 1-18 mm. The perfusate also contained either (a) casein hydrolysate (type C/ESX, Serva Feinbiochemica, c/o Uniscience Ltd., Sulivan Road, London), (b) lactalbumin hydrolysate (Sigma London Chemical Co. Ltd.), (c) soy bean hydrolysate (type S/ELB, Serva Feinbiochemica) or (d) bacteriological 'peptone' prepared from muscle (Evans Medical Ltd., Speke, Liverpool) at 0-5 mg/ml. except in control experiments. It always contained glucose (5 mg/ml.; 28 mM), and was equilibrated with 5 % CO 2 in 0 2 at 40 TC for at least 1 h before use. The phenol red used by Fisher & Gardner (1974) was not included in the
perfusate.
Amino acid and peptide analysis Minor refinements were made to the methods used by Gardner (1975, 1976). Amino acids were estimated on a Locarte amino acid analyser before and after total acid hydrolysis. Samples of intestinal secretion were deproteinized with solid sulphosalicyclic acid and all samples were preserved by addition of pentachlorophenol solution in ethanol. Most analyses were performed at least in duplicate, output being directed to a System AA computing integrator (Autolab, Spectra Physics, Ltd., St Albans, Herts.) in addition to the chart recorder. Acid hydrolyses were performed by heating 0-5 ml. perfusate or luminal effluent, or 0-2 ml. secretion, with an equal volume of Aristar hydrochloric acid (B.D.H. Chemicals, Ltd.) containing 0-02 M AnalaR phenol to minimize loss of tyrosine (Blackburn, 1968) for 24 hr at 110 'C in acid-cleaned Pyrex test-tubes evacuated to 0-1-0-5 mmHg. Hydrolysates were taken to dryness in vacuo over NaOH and P205 and were reconstituted with Na citrate buffer at pH 2-2 (1 0 ml. for perfusate and luminal effluents, and 0-4 ml. for secretions): aliquots of 0-5 or 0-05 ml. respectively were applied to the amino acid analyser. If amino acid analysis was not immediate, samples were stored at -20 'C (see Perry & Hansen, 1969). Peptide-bound amino acids were estimated from the increase in free amino acid determined after hydrolysis. The assumption made and partly validated by Gardner (1975) that no peptides in the unhydrolysed samples were eluted at the same time as amino acids was generally true. However, exceptions made resolution of part of the chromatograms of unhydrolysed Evans peptone difficult, especially in the vicinity of methionine and phenylalanine.
ABSORPTION FROM PROTEIN DIGESTS
85
Control exprement2 Data from control experiments with N-free perfusion were taken from Gardner (1975, 1976) and pooled with those from a further four making a total of eight complete experiments. In addition, secretion samples only from a further three experiments were analysed. Intestinal secretions were analysed directly (0-2 ml. applied to the amino acid analyzer) but samples of luminal effluents were concentrated tenfold by lyophilization and reconstitution before application of 0-5 ml. to the amino acid analyzer. Aliquots were also subjected to acid hydrolysis and analysed. The losses of amino acids and peptides from control intestines perfused with the N-free medium are summarized in Table 1 (see below).
Compukaion of remdUr Data were analysed by computer as before (Gardner, 1975). Multiple regression analyses were performed using the Edinburgh program package MULTREG which was run on the 4-75 computer (Gardner, 1975). The goodness of fit of each regression was ascertained by plotting the values of each dependent variable predicted using the regression against the observed values for each amino acid.
Terminology The term absorption denotes net disappearance from the lumen as determined from the analyses of inflowing perfusate and luminal effluent. The term secretion denotes appearance at the serosal surface of the intestine (Fisher & Gardner, 1974). TABLE 1. Apparent rates of absorption (from the intestinal lumen) and secretion (on to the serosal surface) during the fourth 15 min period of control experiments with N-free perfusion. ,ugN. cm-'. hr-1. Values are means of n experiments + s.E. of mean Free amino acid N Free amino acid + peptide N Peptide N (by difference)
Absorption (n = 8) -2-80+ 1-07 - 6-83 2-00 -4-04 + 1-07
Secretion (n = 11) 7-68 + 0-43 11-5 i1-04 3-78 + 0-81
RESULTS
Control experiments Control experiments were made with N-free perfusion in order to correct for loss of endogenous amino acids and peptides. These losses are summarized in Table 1. Amino acid composition of the perfusate The composition of the Sigma casein hydrolysate was given by Gardner (1975). Amino acid analyses for the other hydrolysates are shown in Tables 2 and 3.
Composition of luminal effluents and secretions The over-all distribution of N found in the perfusates, luminal effluents and secretions is shown in Table 3. The free amino acid fraction of the hydrolysate accounts for from 12 % (soy bean) to 68 % (lactalbumin) of the total amino-N in the perfusate. In several cases the free amino acid concentration in the luminal effluent exceeds that in the perfusate, while the peptide-N concentration invariably is lower in the effluent than in the perfusate.
M. L. G. GARDNER
86
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+
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o
|~~~~~~~~~~~~~~~~~~1 X _O e
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o
-
=
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,
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+l +1+l +1+l ++l +l++1+1
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ABSORPTION FROM PROTEIN DIGESTS
87
Rates of absorption from the lumen and rates of secretion The rates of absorption of N from the intestinal lumen and of N secretion on to the serosal surface are shown in Table 4. These rates have been corrected for losses of endogenous amino acids and peptides by subtraction of the values obtained in the control experiments with N-free perfusion. TABLE 3. Over-all distribution of N in perfusate, luminal effluent and secretion (ug N/ml.) during the fourth 15 min collection period during perfusion with various partial digests of proteins at 0.5 mg/ml. Values are means of n experiments s.E. of mean (actual values, not corrected for loss of N in control experiments) Protein hydrolysate in perfusate Casein* (Sigma), n = 5 Free amino acid N Peptide-bound amino acid N Casein (Serva), n = 7 Free amino acid N Peptide-bound amino acid N Soy bean (Serva), n = 8 Free amino acid N Peptide-bound amino acid N Lactalbumin (Sigma), n = 7 Free amino acid N Peptide-bound amino acid N Peptone (Evans), n = 10 Free amino acid N Peptide-bound amino acid N N-free controls (n = 8) Free amino acid N Peptide-bound amino acid N *
Perfusate
Luminal effluent
31-2+0 57 29*7+ 1*12
20-2+0068 19*6+ 1*14
206+ 15-3 157+48*4
18-0+1-1 36-6 + 2-5
13*0+2*0 25-4+ 3-3
241±+ 115 (n = 10) 6*8 + 14-8 (n = 10)
4*39+0*16
7 49+0-77 24-1+1159
122.9±11*3 (n = 11)
31-2+2-43
22*7 + 2'08 10*9+ 3*76
12*9+ 1*45 10*6+ 3*43
217 + 10*5 - 21*8 + 15*6
11*2+0055 40'9+ 3*01
15*6+ 1*55 22*4 + 4'27
210±7*1 -5*9+ 8*4
0 0
1P35+045 2*02+0*53
Secretion
38-0+121 (n = 11)
51-1+ 2-7 (n = 11) 24*5+ 49(n = 11)
Data of Gardner (1975).
Fig. 1 A and B show the relationships between the net rate of absorption (i.e. disappearance from the lumen) of each amino acid, free and peptide-bound, and its concentration in the luminal perfusate for the experiments with lactalbumin and casein (Serva) respectively. Fig. 3 of Gardner (1975) reported a comparable relationship for experiments with Sigma casein. Note that all the points, for both free and peptide-bound amino acids, conform to a generally similar relationship, which (at the low concentrations studied) is approximately linear between absorption rate and perfusate concentration. Similar relationships were observed for the free amino mixture (Gardner, 1976) for the Evans peptone and (except for the points corresponding to peptide- or amide-bound glutamic and aspartic acids which are present in particularly high concentrations) for the Serva soy bean hydrolysate (Fig. 1 C).
M. L. G. GARDNER
88
Relationships between the rates of secretion and rates of absorption Table 4 shows that the rate of total amino acid N absorption substantially exceeds the rate of total amino acid N secretion in all instances except for the Sigma casein hydrolysate and the free amino acid mixture. The discrepancy is particularly large for Evans peptone. Fig. 2A and B show the relationship between the rates of secretion and of absorption for each amino acid from Evans peptone and Sigma lactTABiF 4. Over-all rates of N absorption from the intestinal lumen and rates of N secretion on to the serosal surface during the fourth 15 min period of luminal perfusion with various protein hydrolysates. Values (,ug N. cm-'. hr-1) are means of n experiments + s.E. of mean and are corrected for losses during eight or eleven control experiments with N-free perfusion Protein hydrolysate in perfusate Casein* (Sigma), n = 5 Free amino acid N Peptide-bound amino acid N Total amino acid N Casein (Serva), n = 7 Free amino acid N Peptide-bound amino acid N Total amino acid N Soy bean (Serva), n = 8 Free amino acid N Peptide-bound amino acid N Total amino acidN Lactalbumin (Sigma), n = 7 Free amino acid N Peptide-bound amino acid N Total amino acid N Peptone (Evans), n = 10 Free amino acid N Peptide-bound amino acid N Total amino acid N Free amino acid mixturet (n = 4) Free amino acid N Peptide bound amino acid N Total amino acid N
Absorptiont
26-2+1-8 25-6+ 2-6 51-8+4-0
17-3+5-1 35-5 + 8-6
Secretion § 31-4+1-6 27-0+ 6-9 58-4+ 7-7
52-7 + 7-9
29-6+ 1-4 (n = 10) - 2-2 + 2-2 (n = 10) 27-4 + 2-4 (n = 10)
-3-8+ 2-2 25-3+6-2 21-5+6-0
11-7 + 1-5 (n = 11) 2-9+2-3 (n = 11) 14-6+2-1 (n = 11)
23-1+4-2 5-9+ 5-7 29-1 + 6-8
28-0+2-1 -7-8+ 2-3
-3-8+3-6 45-2+ 6-9 41-4 + 7-7
-4-9+ 1-5
25-3+6-2 -6-9+6-7 18-4+ 12-7
20-2 + 1-8
22-2+2-1
17-3 ± 2-2 17-3+6-6 -2-7+3-4 14-6+9-7
* From Gardner (1975).
t From Gardner (1976).
The values for Sigma casein and the free amino acid mixture differ slightly from those originally reported since these data have been recalculated taking into account all eight or eleven control experiments with N-free perfusion now available. I With eight control experiments. § With eleven control experiments.
albumin hydrolysate. In both cases the discrepancy between secretion and absorption applies almost equally to all amino acids in the mixture (see regression analysis below) except for glutamic and aspartic acids. No discrepancy was seen for Sigma casein hydrolysate (Fig. 4 of Gardner, 1975). However, there was no relationship
89 ABSORPTION FROM PROTEIN DIGESTS between the measured rates of secretion and absorption when the perfusate contained soy bean hydrolysate (Fig. 2 C).
Appearance of intact peptides in serosal secretion Table 3 showed that there were bound amino acids, assumed to be peptide-bound, in the steady-state secretion at the serosal surface of the intestine during absorption from the two casein hydrolysates (especially the Sigma hydrolysate) and the soy bean hydrolysate, but not during absorption from the Evans peptone or the lactalbumin hydrolysate. These were not all of endogenous origin, since positive values are shown in Table 4 for the rates of secretion from Sigma casein and Serva soy bean hydrolysates after subtraction of the rates observed in control experiments with N-free perfusion. Gardner (1975) reported corresponding data for the Sigma casein hydrolysate, and concluded that substantial transport of intact peptides had occurred. Rates of secretion of each amino acid, free and peptide-bound, on to the serosal surface from the Serva soy bean and Sigma casein hydrolysates are shown in Fig. 3. These rates are net rates corrected by subtraction of secretion rates in the control experiments. All the amino acids except seine, tyrosine, histidine and arginine from the soy bean hydrolysate appear in the bound-fraction of the secretion. The appearance of bound amino acids is not restricted to the dicarboxylic amino acids which may be amide-bound.
Multiple regression analyses of rates of amino acid absorption First-order multiple regressions were computed by MULTREG with the total rate of absorption of each amino acid (i.e. disappearance from luminal perfusate) as the dependent variable. The perfusate concentrations of free and peptide-bound amino acid were the two independent variables. The results are shown in Table 5. Analysis of variance showed that in all cases, except for soy bean hydrolysate, the regressions fitted very well to the data. An example of the goodness of fit is seen in Fig. 4 for the lactalbumin data, which shows the total absorption rate for each amino acid predicted from the regressions in Table 5 plotted against the observed rate. Clearly there is little scatter about the line of identity: thus the regression does describe well the observed absorption rates. It might be argued that this statistical treatment is not strictly valid since estimation of absorption rates relies on knowledge of the amino acid concentrations in the perfusate. Also, estimation of the perfusate concentrations of peptide-bound amino acids depends on knowledge of the concentrations of free amino acids. Hence the independent variables are not strictly independent, and the regression could be biased. The statistical validity can be tested by using the rates of secretion on to the serosal surface (estimated independently from the perfusate concentrations) instead of absorption rates from the lumen as the measures of transport. The coefficients in these multiple regressions are shown in square brackets in Table 5 and are clearly very similar to the others in Table 5. Note that the coefficients (b2) for concentration of peptide-bound amino acids are notably lower than those (b1) for the concentration of free amino acids for each hydrolysate except Evans peptone. Since Gardner (1975, 1976) had noted substantial backflux of amino acids during
M. L. G. GARDNER 90 absorption of peptides multiple regressions with the net rate of free amino acid absorption as the dependent variable were computed. The independent variables were the perfusate concentration of free amino acid and the rate of peptide-bound acid absorption. Results are shown in Table 6. Again, analysis of variance showed that the fit was good in all cases except for soy -
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ABSORPTION FROM PROTEIN DIGESTS 91 bean. The excellence of the fit was confirmed by plotting the predicted rates against the observed rates (data not shown). Fig. 1 showed that the rate of disappearance of peptide-bound amino acids from the lumen was proportional to their concentration in the perfusate. Note that disappearance includes both absorption and hydrolysis. Table 7 shows 'rate constants' for this relationship, i.e. first-order coefficients in the regression of rate of disappearance of peptide-bound amino acids against their perfusate concentration. C Soy bean hydrolysate
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Fig. 1. A, B and C, relationship between the net rate of absorption from the lumen of each amino acid, free (0O) and peptide-bound (@*), and its concentration in the luininal perfusate. The perfusates contained either Sigma lactalbuxnin hydrolysate (A), Serva casein hydrolysate (B), or Serva soy bean hydrolysate (C). All absorption rates have been corrected for pasage of endogenous ammno acids and peptides into the luminal effluents during N-free perfusion. Each point represents a different amino acid: for clarity only extreme points have been identified.
Again, the data for soy bean hydrolysate were the only ones for which a first-order regression was not an excellent fit. The 'rate constants' are closely similar for the Evans peptone, lactalbumin and Serva casein hydrolysates although the value was lower for Sigma casein hydrolysate. They are also very similar to the 'rate constants' for absorption of free amino acids (b1 in Tables 5 and 6). Multiple regression analysis of rates of amino adid secretion Table 5 showed the coefficients in the regression of total rate of amino acid secretion against the perfusate concentrations of free and peptide-bound amino acids. Also the rate of total amino acid N absorption substantially exceeds the rate of total amino acid N secretion during absorption from all perfusates except those containing Sigma casein hydrolysate and the free amino acid mixture (Table 4).
M. L. G. GARDNER Therefore multiple regressions were computed of total rate of amino acid (free + peptide-bound) secretion against the rates of absorption of free amino acid and of peptide-bound amino acid (Table 8); it is clear that the regressions are good fits to the observed data except in the case of the soy bean hydrolysate. The fit to the 92
A Evans peptone
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Fig. 2 A and B. For legend see facing page.
93
ABSORPTION FROM PROTEIN DIGESTS C Soy bean hydrolysate
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,0
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Cc ._
0-04 I-
0
_
up
0
0
0
0
0-02 0
3 0
0 10
I
0-05 Absorption rate from lumen of total (free+peptide-bound) amino acids (jmole . cm-' hr-')
0-15
Fig. 2. A, B and C, relationships between the rates of absorption from the lumen and secretion on to the serosal surface of each total (i.e. free+ peptide-bound) amino acid. Each point represents a different amino acid. The perfusate contained Evans peptone (A), Sigma lactalbumin hydrolysate (B), or soy bean hydrolysate (C).
TAiBL 5. Multiple regression analyses for total rates of amino acid absorption from various protein hydrolysates. Values shown are computed parameters + S.D. in the equation: free amino acid Total rate of amino [peptide-bound = b,. concentration in acid absorption + b 2 . amino acid + C. from lumen concn. in perfusate perfusate The percentage of the variance in the dependent variable which has been accounted for by the regression is shown in parentheses. The coefficients in braces are those in the corresponding regression with total rate of amino acid secretion on to serosal surface as dependent variable with data for aspartic and glutamic acids and alanine excluded. C b2 b, Perfusate N source (ml. cm-. hr-L) (ml. cm . hr-1) (psmole. cm-'.hr- ) Casein (Sigma) 1-18+ 011 0-44+ 0-05 0-007 + 0-01 (93.2 %) {0-61 + 0-12) (1.41 + 0.15) Casein (Serva) 1-12+0-36 0-90+ 0-11 -0-005 + 0-03 (83-6%)
{1.28 + 0-16} Peptone (Evans) Lactalbumin (Sigma)*
{0-51 + 0.08)
0-65 + 0-26
0-70 + 0-12
{0.42 + 0.16)
{0.35 + 0 10) 0-67 + 0-07 {0.49 + 0.13)
1 12 + 0-09
(1.06 + 0.07} Soy bean (Serva) Soy bean - but excluding glu
0-72 + 1-22 1-13 + 1-01
and asp All data ex. Soy bean pooled
{1-22 + 0.56} 1-01 + 0-091
0-22 + 0-14 0-74 + 0-25 (0.27 + 0.14) 0-75 + 0-043
(130 + 0.087)
{0.61 + 0.065}
*
See Fig. 4.
0-01+ 0-02 (72X5 %) -0-01+ 0-01 (93-3 %)
0-04+0-03 (17-2 %) -0-01+ 0-03 (46.5%) - 0-007 + 0-01 (82-0 %)
M. L. G. GARDNER
94 25
20
- 15
)10
.
5
01
8
A F RSoy bean
6 4 2 ot
:~[H ~.r6hFr0 I
I
ASP
0
TH R
SE R
G LU
PRO
Im
0
m
GLY ALA VAL MET ILE LEU TYR PHE -HIS LYS ARG
Fig. 3. Rates of secretion on to the serosal surface of each amino acid, free (open bars) and bound (hatched bars), during luminal perfusion with (A) soy bean hydrolysate and (B) Sigma casein hydrolysate. Lactalbumin hydrolysate
0
0
0
0-2
'a
_ 0
3
E
',
0 0 i ._
0 0
_
0.-
0-
0
0
O0 w
'D0
00 .0
.4:
0
00
-
o
0 0
0
I
1I
0.1
0-2
Observed total rate of amino acid absorption from lumen (pmole. cm-'. hr-1)
Fig. 4. Goodness of fit of data for absorption from lactalbumin hydrolysate to multiple regressions shown in Table 5. Each point represents a different amino acid.
ABSORPTION FROM PROTEIN DIGESTS 95 computed regressions was particularly excellent for the Sigma casein and lactalbumin hydrolysates. Peptide hydrolysi by the intestine Table 4 showed that the rates of free amino acid secretion on to the intestine's serosal surface always exceeded the rates of free amino acid absorption during perfusion with mixtures containing peptides. Conversely, the rate of peptide-N TABLE 6. Multiple regression analysis for net rates of free amino acid absorption from various protein hydrolysates. Values shown are the computed parameters + S.D. in the equation: net rate of of amino free amino acid _ b free acid conen. + C. + b8. peptidenbound disappearance from lumen aot The percentage of the variance in the dependent variable which has been accounted for by the regression is shown in parentheses. c b, Perfusate N source (ml. cm-'.hr-1) Casein (Sigma) 1-22 + 0-07 -0-39+004 -0008±+001 (96.6%) Casein (Serva) 1*60+0 15 -0 14+_ 004 -0'03 +0*01 (93.9%) Peptone (Evans) 1.00+ 0*09 -033_+003 - 0*003 + 0-008 (96.6%) Lactalbumin (Sigma) 1-19+0-06 -032±0O05 -0.01 +0-01 (98.1 %) 0*66 + 0-51 Soy bean (Serva) -0-19 + 009 -0-009 + 0.01 (31.5%) All data ex. soy bean pooled 1 19+ 0-056 -0-23 + 0*025 -0-016 + 0*0059 (90.6%) 1.
Lin perfisateJ
TABLE 7. First-order 'rate constants' for disappearance (i.e. absorption + hydrolysis) of peptidebound amino acids from the lumen during perfusion with various protein hydrolysates. Values shown are the computed regression parameters in the equation: rate of peptide-bound
b. acid conen. in + C. erfusate The percentage of the variance which has been accounted for by the regression is shown in parentheses. b C Perfusate N source (ml. cm-'.hr-l) (Creole . cm-L . hr-IL) Casein (Sigma) 0*73 0'06 0-02 + 0-01 (91.0 %) Casein (Serva) -0-01 +0-02 (86.5%) 1o10+ 012 Peptone (Evans) 1-13 + 0 16 -0*009+0-02 (78-1 %) Lactalbumin (Sigma) 1.00+ 0.09 -0-016 + 0-008 (89-3 %) Soy bean (Serva) 0 37 0-14 -0 046+ 0019 (32-4%) All data ex. soy bean pooled 1.00+ 0 044 - 0-007 0006 (86-7 %) amino acid
disappearance
=
+
secretion was less than the rate of peptide-N absorption. Also the net rate of free amino acid absorption from the lumen was negative in some cases. The obvious explanation is that a large proportion of the peptides disappearing from the luminal perfusate are hydrolysed to free amino acids by hydrolases located in the brush border and cytoplasm of the mucosal cells. Therefore, the over-all disappearance of peptide-bound acids should be accompanied by a corresponding increase in free amino acids.
Table 9 summarizes the regression coefficients relating the over-all rate of dis-
M. L. G. GARDNER appearance of peptide-bound amino acids from the preparation to the overall rate of appearance of free amino acids. In most instances there is good correspondence between rates of peptide disappearance and of free amino acid appearance. Corresponding data for the Sigma casein hydrolysate were reported by Gardner (1975, Fig. 7). The generally good correspondence seen supports the accuracy of the techniques used. 96
TABLE 8. Multiple regression analyses for total rates of amino acid secretion on to the serosal surface as a function of the respective absorption rates of free and peptide-bound amino acids from the intestinal lumen. Values shown are the computed parameters + S.D. in the equation: total rate of amino acid rate of free rate of peptide-) secretion on to the = bLl. amino acid + b 2. bound amino acid +C. serosal surface absorption absorption The percentage of the variance which has been accounted for by the regression is shown in parentheses. Data for aspartic and glutamic acids and alanine have been excluded C Perfusate N source b2 (#emole . cm-' . hr-1) 1-08 + 0-096 1-14+ 0-09 Casein (Sigma) 0-009 + 0-015 (95.2 %) Casein (Serva) 0-89 + 0-16 0-48 + 0-098 -0-009 + 0-02 (76-8%) Peptone (Evans) 0-53 + 0-13 0-44+ 0-08 - 0-0004 + 0-01 (72.4%) Lactalbumin (Sigma) 0-88 + 0-06 0-63 + 0-09 -0-009 + 0-007 (96-3 %) 0-28 + 0-14 0-29 + 0-36 Soy bean (Serva) 0-032 + 0-012 (29-0 %) All data ex. soy bean pooled 1-05 + 0-08 0-70 + 0-06 - 0-015 + 0-010 (75.1 %) TABLE 9. Relationship between the overall rate of disappearance of peptide-bound amino acids with the over-all rate of disappearance of free amino acid from the perfused intestine. Negative rates of disappearance of free amino acids signify an overall increase in free amino acids, apparently from hydrolysis of peptides. Values shown are the computed parameters+ S.D. in the equation: over-all rate of (over-all rate of disappearance disappearance of = b.o peptide-bound amino acid + C. free amino acid The percentage of the variance which has been accounted for by the regression is shown in parentheses. Data for aspartic and glutamic acids and alpine have been excluded Perfusate N source Casein (Sigma) Casein (Serva) Peptone (Evans) Lactalbumin (Sigma) Soy bean (Serva) All data ex. soy bean pooled
c moleol. 0-921 + 0-085 -0-029+0-007 - 0-547 + 0-072 0-017 + 0-014 0-714+ 0-069 0-028 + 0-012 - 0-786 + 0-076 0-013 + 0-006 - 0-386 + 0-128 -0-024 + 0-011 -0-61 +0-04 0-007+0-005 b
-
-
cm1.
br-1)
(91-5%) (83-8 %) (90-7 %)
(90-6%) (45-2 %)
(80-6%)
Gardner (1975) showed a strong positive correlation between the overall rate of disappearance of peptide-bound amino acids and the rate of their absorption from the lumen for the Sigma casein hydrolysate (Fig. 8 of Gardner, 1975). Similar relationships were also seen in the present investigation (Table 10). Again, the data fit remarkably well to straight lines; the slopes (Table 10) are close to unity except in the case of Sigma casein hydrolysate for which hydrolysis of absorbed peptides has already been found to be incomplete.
ABSORPTION FROM PROTEIN DIGESTS
97
TABLE 10. First-order regression coefficients relating the over-all rate of disappearance of peptidebound amino acid (presumed to be the rate of hydrolysis) to the rate of absorption of peptidebound amino acid from the intestinal lumen. Values shown are the computed parameters + S.D. in the equation: over-all rate of disappearance _ b (rate of absorption of C+ + of peptide-bound amino acid - -peptide-bound amino acid+ The percentage of the variance accounted for is shown in parentheses. Data for aspartic and glutamic acids and alanine have been excluded
b Perfusate N source (ismole. cm". hr-') Casein (Sigma) 0-900 + 0-146 -0-073 + 0-017 (77-5 %) 0-02 + 0-01 (96-1 %) Casein (Serva) 0-99 + 0-06 Peptone (Evans) 1-09+0-04 0-009+ 0-006 (98-4%) Lactalbumin 1-08+0-11 0-03 + 0-006 (90-1%) (Sigma) -0-01+ 0-01 (89-6%) Soy bean (Serva) 0-99 + 0-10 All data pooled 0-94 + 0-048 -0-0004 + 0-007 (83-2 %) DISCUSSION
Relative rates of absorption of different amino acids Gardner (1975, 1976) observed that the rate of absorption of each amino acid in a casein hydrolysate and a synthetic mixture of amino acids depended in a linear fashion on the luminal concentration of that amino acid and apparently not on the nature of the amino acid (see also Bronk & Leese (1974) and Matthews (1975b, pp. 116-117)). Such linear relationships imply a constant ratio of Vmax. to Km for all amino acids (Gardner, 1975). Similar relationships were observed in the present work with each protein digest (Fig. 1A, B and C). Thus in complex mixtures at low concentrations each amino acid appears to be absorbed at a rate which is independent of its chemical structure. This is a contrast to the behaviour of simple pairs of amino acids where competition studies have led to the view that there are separate mechanisms for the transport of acidic, neutral and basic amino acids.
Relative rates of absorption of free and peptide-bound amino acids Since Fig. 1 shows that the free and peptide-bound amino acids conform to the same relationship between absorption rate and perfusate concentration it might appear that free and peptide-bound amino acids are equally rapidly absorbed from a given concentration. However, these absorption rates are net rates measured from the disappearance of amino acids between the perfusate and luminal effluent. If peptides disappear from the lumen and give rise to free amino acids after hydrolysis (superficial or intracellular) then any backflux of amino acids into the lumen will reduce the measured net rate of free amino acid absorption. Hence the net rates of free amino acid absorption shown in Fig. 1 underestimate the true rates of amino acid absorption from the free amino acids in the perfusate. The regression analyses in Table 5 overcome this problem of interpretation. It is 4
PHY
284
M. L. G. GARDNER 98 clear from the analyses of variance and from the specimen plot in Fig. 4 that the equation with first-order terms in free amino acid concentration and in peptidebound amino acid concentration does describe very well the absorption data except for the soy bean hydrolysate. The relative magnitude of the b. and b2 coefficients imply that net absorption occurs substantially faster from free amino acids than from peptides, except in the instance of the Evans peptone (muscle digest) for which the 'rate constants' b1 and b2 are almost equal (Table 5). The same conclusion is drawn when the rate of amino acid secretion on to the serosal surface is the dependent variable (data in square brackets in Table 5).
Backflux offree amino acids into lumen Gardner (1975, 1976) noted substantial backflux of free amino acids into the lumen during absorption of a casein digest. These arose from hydrolysis of peptides, either at the brush border or in the cytosol of the mucosal cells, both of which contain peptide hydrolase activity (e.g. Kim, Birtwhistle & Kim, 1972). Similar backfluxes into the lumen during peptide absorption were reported by others (e.g. Newey & Smyth, 1962; Asatoor, Cheng, Edwards, Lant, Matthews, Milne, Navab & Richards, 1970; Matthews, Lis, Cheng, & Crampton, 1969; Heading, Schedl, Stegink & Miller, 1977). The equations in Table 6 imply that the rate of backflux of amino acids is proportional to the rate of peptide-bound amino acid absorption: on average, 23+ 2-5 % (b2 in Table 6) of absorbed peptide-bound amino acids pass back into the lumen following hydrolysis at the brush-border and/or the cytoplasm (see below and Wiseman, 1977). Total disappearance of peptides from perfusate The 'rate constants', or first-order coefficients, relating the rate of disappearance of peptides from the lumen to the concentration of peptide-bound amino acids were shown in Table 7. Note that disappearance includes both absorption and hydrolysis. These coefficients (b) are very similar to the coefficients (bl) for absorption (or secretion) from free amino acids in Table 5. Thus, the rate of disappearance of a particular amino acid from the lumen is, for a given concentration, independent of whether that amino acid is free or peptide-bound. If however it is peptide-bound a substantial fraction (23 % on average; Table 6) will pass back to the lumen as free amino acid. N balance of perfused intestine Table 4 showed that, except for experiments with Sigma casein and with the synthetic amino acid mixture, the rate of total amino acid absorption from the lumen exceeded the rate of total amino acid secretion on to the serosal surface. The intestine appears to be in positive N balance. Either absorbed amino acids are continuing to accumulate in the tissue or they are appearing in the intestinal secretion in a form which fails to be estimated as free amino acids after the acid hydrolysis. Losses of amino acids could occur if (a) they were transformed so as not to be ninhydrin-positive or so that their elution from the ion-exchange resin was greatly altered, (b) if amino acids entered the secretion in peptides which were stable under the hydrolysis conditions used, or (c) if amino acids entered the secretion in protein
99 ABSORPTION FROM PROTEIN DIGESTS form, either synthesized into proteins or bound to pre-existing proteins, which would be lost when the secretion samples were deproteinized prior to analysis. There is a serious discrepancy between the rate of appearance of nitrogen on the serosal side of the intestine and its rate of absorption from the lumen, and it must be stressed that in no study, in vitro or in vivo, has the nature of all absorbed amino N been accounted for quantitatively. The best estimate appears to be that of Dawson & Porter (1962) who could only account for about 41 % of absorbed amino nitrogen appearing as free amino acids in portal plasma, and this was likely to be an overestimate (Fisher, 1967a). Therefore, Fisher's (1967b) conclusion that '...the form in which the products of protein digestion enter the body is still not established. No one has been able to show that more than a minority of it enters the bloodstream as amino acids...' is still correct. Matthews & Burston (1977) agreed that the possibility of peptides entering the portal blood on a significant scale has not been satisfactorily excluded. Techniques in vitro such as that of Fisher & Gardner (1974) are suited to studies of this kind since the secretion is not diluted or contaminated by a bloodstream. Also one has access to the solutes which would enter the lymphatics as well as the bloodstream and this may be important since the amino-N concentration in the cisterna chyli rises during absorption of a protein meal (Jacobs, Largis & Hanson, 1967; Jacobs & Largis, 1969a, b; Bolton & Wright, 1937). The productprecursor relationship seen by Morris (1956) in intestinal lymph during protein digestion indicated that a significant fraction of the lymph protein was probably synthesized in the intestine (see Fisher, 1967b). The regression coefficients in Table 8 suggest that the discrepancy between secretion and absorption is uniformly greater for the peptide-bound amino acids. The values of b, and b2 for the pooled data (Table 8) suggest that 105 + 8 % of the absorbed free amino acids have appeared in the secretion on the serosal surface while only 70 + 6 % of the absorbed peptides have been recovered in any form. This raises the possibility that some peptides which enter the serosal secretion are either protein-bound (and lost during deproteinization) or are resistant to acid hydrolysis (and escape detection). Alternatively, peptides may be destroyed faster during the acid hydrolysis than free amino acids or peptide-N is continuing to be accumulated within the cells.
Validity of multiple regression analyses With the exception of the data for absorption of soy bean hydrolysate, the multiple linear regressions in Tables 5-10 describe remarkably well the observations. The goodness of fit can be seen from the analyses of variance, and particularly from plots of the predicted values of the dependent variables against the observed values (Fig. 4 and unpublished observations). While the validity of the equations in describing the observed processes numerically is therefore proved, the physiological interpretation requires caution. Rate-limiting steps: hydrolysis or transport? Loss of peptides and gain of free amino acids. Table 9 shows that the overall loss of peptide-bound amino acids from the preparation was accompanied by a corresponding gain of free amino acids. Although 4-2
M. L. 0. GARDNER the regression coefficients in Table 9 imply that only 55-92 % (average 61 +4 %) of the disappearing peptide-bound N was accounted for by appearance of free amino acids, the close fit of each amino acid to the relation supports the validity of the measurements of absorption and secretion. Table 10 (also Fig. 8 of Gardner, 1975) showed that the over-all rate of disappearance of peptide-bound amino acids was proportional to the rate of their absorption. Since the rate of peptide absorption was closely proportional to the luminal concentration it appeared that the absorption step was rate-limiting for hydrolysis (Gardner, 1975). However this argument is not valid since a similar relationship could be observed if superficial hydrolysis occurred with no transport of intact peptides. 100
Transport of intact peptides across intestine. Although certain peptides are resistant to mucosal hydrolysis (e.g. glycylglycine, Newey & Smyth, 1959; Peters & MacMahon, 1970; Adibi, 1971; carnosine, Perry, Hansen, Tischler, Bunting & Berry, 1967, and hydroxyproline peptides arising from gelatin digestion, Prockop & Sjoerdsma, 1961; Bronstein, Haeffner & Kowlessar, 1966) and can cross the mucosa in substantial quantities, the conventional view is that most peptides are hydrolysed to free amino acids within or on the mucosal cells (e.g. Matthews, 1975b, p. 72; Bell, Emslie-Smith & Paterson, 1976; Dent & Schilling, 1949). Thus, only free amino acids should enter the circulation in vivo. Fisher (1967a,b) stressed that the evidence for this was poor. Gardner (1975) suggested that about 33 % of the N absorbed from the Sigma casein hydrolysate entered the tissue fluid in peptide form, although the possibility that it was proteinor amide-bound must also be considered. The present experiments show that during absorption from lactalbumin hydrolysate and Evans peptone no significant peptide-bound N was detected in the steadystate secretion on to the serosal surface (Tables 3 and 4). However, small quantities of bound amino acids were detected during absorption from Serva casein and soy bean hydrolysates. Fig. 3 showed that almost all sixteen amino acids were present in bound form in the secretion during absorption from the Sigma casein and the soy bean hydrolysates. A much smaller fraction of the secreted amino-N was peptidebound during perfusion with the Serva casein hydrolysate than with the Sigma casein hydrolysate used by Gardner (1975). Further experiments with the latter casein hydrolysate support the original analyses reported by Gardner (1975), although the size of the transported peptides is still not known. Sleisenger, Pelling, Burston & Matthews (1977) estimated amino acids in portal venous plasma during absorption from a casein hydrolysate and it appeared that a substantial proportion of some amino acids entered the portal blood in peptidebound form. It should be noted that casein contains phosphopeptides, and these are known to be resistant to enzymic hydrolysis (Mellander & F6lsch, 1972; Mellander, 1977, p. 294). Many actively absorbed solutes accumulate at high intracellular concentrations in most preparations of intestine in vitro, so that unphysiological concentrations of peptides may be presented to the peptidases: this may favour hydrolysis of absorbed peptides in vitro. This potential artifact may be reduced by vascular perfusion
ABSORPTION FROM PROTEIN DIGESTS
101
Boyd, 1977). Also, reduction of intracellular accumulation may reduce the backflux of free amino acids into the intestinal lumen if the principal site of peptide hydrolysis is intracellular. While the fate of any intact peptides which enter the portal circulation in vivo is not known, Krzysik, Peterson & Adibi (1975) showed that liver, muscle and kidney also contain substantial dipeptide hydrolase activities. Also, intravenously administered dipeptides were rapidly cleared from plasma (Adibi, 1977; Adibi & Krzysik, 1977; Adibi, Krzysik & Drash, 1977). Assimilation of peptides in i.v. nutrition media is well known in man (e.g. Lidstr6m & Wretlind, 1952; Christensen, Wilber, Coyne & Fisher, 1955).
Similarity of absorption from different proteins Absorption from digests of several different proteins, including animal and plant proteins, milk and meat proteins, and similar proteins from different sources have now been compared. With a few exceptions, the characteristics are very similar, and this justifies extended studies on any one of these digests. The chief exceptions are (i) intact peptide transport into the secretion was only seen from the casein and soy bean digests, (ii) the two different sources of casein gave different amounts of intact peptide secretion, (iii) the soy bean digest data were not well fitted by the multiple regression analyses unlike those for the other digests. In connexion with the anomalous behaviour of soy bean hydrolysate (which had a particularly low free amino acid content, Table 3) it may be relevant that the biological value of raw soy bean is less than expected. One reason appears to be the presence of at least one powerful trypsin inhibitor (Laskowski & Laskowski, 1954). There is indirect evidence that some trypsin inhibitors may also act on intestinal mucosal enzymes (Laskowski, Haessler, Miech, Peanasky & Laskowski, 1958). Concentration ofprotein digests It must be stressed that only a single concentration of the digests, namely 0 5 mg/ ml., has been studied. The conclusions are not necessarily applicable at higher concentrations, and further investigations are needed.
Validity of studies in vitro Accumulation of peptides and free amino acids to high levels in the mucosal cells may have increased the rate of peptide hydrolysis and the rate of backflux of amino acids into the lumen, as discussed above (see also Boyd, 1977). Furthermore, preparations of intestine in vitro lose cytoplasmic peptidases into the luminal or mucosal fluids (Silk & Kim, 1976; Lindberg, Noren & Sj6strom, 1975, pp. 229-230). Thus, unphysiological peptide hydrolysis may proceed within the luminal fluid: also, the cytoplasmic peptidase activity may be low during in vitro studies. However we have evidence (M. L. G. Gardner & J. A. Plumb unpublished observations) that leakage of peptidases is less from the Fisher & Gardner (1974) preparation than from other preparations in vitro. Furthermore, in single-pass perfusion the perfusate peptides can only come in contact with 'leaked' enzymes for a relatively short time and the enzyme concentration in the luminal fluid must be much lower than in incubation or multiple-pass perfusion experiments.
M. L. G. GARDNER
102
Over-all scheme of amino acid and peptide absorption The principal quantitative conclusions can be summarized with the aid of Fig. 5. The rate constants for disappearance of free amino acids (route A) and peptides (route B) from the lumen are similar at around 1 ml. cm-'.hr-I (b, from Tables 5 and 6 and b from Table 7). Substantial backflux of free amino acids arising from peptide hydrolysis occurs by routes C and/or D. The extent of this backflux, 100 w (C+D/B,) is on average 23 % (b2 from Table 6). Hence, 60-70 % of the free amino acids arriving at the serosal surface of the intestine (routes E, F and G) appear to originate from free amino acids in the perfusate by route E: this is reflected by the b, coefficients in Table 5 which are greater than the b2 coefficients. Brush border
Lumen
Mucosal cell
Serosal tissue fluid
A
C
-40=
D
-111
4.
__
a
4 _ _
-=moo,.
lE.
be
F
-HH
B
-I
G
Fig. 5. Schematic representation of the absorption of amino acids and peptides across the intestinal mucosa. Broken lines (---) indicate transport of free amino acids; solid lines ( ) indicate transport of peptides; the symbol *A*+ indicates hydrolysis of peptides. See text.
Superficial membrane hydrolysis (site X in Fig. 5) and intracellular hydrolysis (site Y) cannot be distinguished from these data. Studies on hydrolysis in the presence of dinitrophenol suggest that membrane digestion is important (Gardner, M. L. G. unpublished observations). The amount of intact peptide crossing the mucosa (route H) is negligible during absorption of some digests (Evans peptone and lactalbumin hydrolysate), small during absorption of others (Serva casein and soy bean hydrolysates), but can be substantial during absorption from some digests (Sigma casein hydrolysate) such that (1OOxH)/(E+F+G+H) = 46%. However, in these experiments (except those with Sigma casein hydrolysate) not all the amino-N absorbed (routes A + B - C(- D) has been accounted for by secretion of amino acid or peptide (routes E +F +G+ H). The main discrepancy appears to be in recovery of absorbed peptides (route B) since the b2 coefficients in Table 8 are less than unity. There is no satisfactory evidence whether any absorbed amino-N is synthesized into, or bound to, protein (route J) and this possibility should not be discounted.
ABSORPTION FROM PROTEIN DIGESTS
103
I am indebted to the Medical Research Council for financial support, and to Professor R. B. Fisher and Professor D. M. Matthews for valuable discussions and encouragement. I also thank Miss E. M. Middleton and Mr R. Taylor and the Staff of the workshops and animal house in the Department of Biochemistry for technical assistance. REFERENCES ADiBI, S. A. (1971). Intestinal transport of dipeptides in man: relative importance of hydrolysis and intact absorption. J. dlin. Inve8t. 50, 2266-2275. ADrBI, S. A. (1977). Clearance of dipeptides from plasma: role of kidney and intestine. In Peptide Transport and Hydrolysi8 (Ciba Fdn Symp.), ed. ELuIOTT, K. & O'CoNNOR, M., pp. 265-280. Amsterdam: Associated Scientific Publishers. ADrBi, S. A. & KRZYSIx, B. A. (1977). Effect of nephrectomy and enterectomy on plasma clearance of intravenously administered dipeptides in rats. Clin. Sci. 52, 205-213. ADrBI, S. A., KmzYsix, B. A. & DRAsir, A. L. (1977). Metabolism of intravenously administered dipeptides in rats: effects on amino acid pools, glucose concentration, and insulin and glucagon secretion. Clin. Sci. 52, 193-204. ASATOOR, A. M., CENG, B., EDWARDS, K. D. G., LANT, A. F., MATTrEws, D. M., MILNE, M. D., NAvAB, F. & RIcHARDs, A. J. (1970). Intestinal absorption of two dipeptides in Hartnup disease. Gut 11, 380-387. BELL, G. H., EMsLIE-SMITH, D. & PATERSON, C. R. (1976). Textbook of Physiology and Biochemi8try, 9th edn., p. 128. Edinburgh: Churchill Livingstone. BLAcKBJuRN, S. (1968). Amino Acid Determination: Method8 and Technique8. London: Arnold. BOLTON, C. & WRIGHT, G. P. (1937). The absorption of amino acids and their distribution in the body fluids. J. Physiol. 89, 269-286. BOYD, C. A. R. (1977). Vascular flow and the compartmental distribution of transported solutes within the small intestinal wall. In Intestinal Permeation (Proceedings of the Fourth Workshop Conference Hoechst), ed. KRAMER, M. & LAUTERBACH, F., pp. 41-47. Amsterdam: Excerpta Medica. BRONK, J. R. & LEESE, H. J. (1974). The absorption of a mixture of amino acids by rat small intestine. J. Phy&iol. 241, 271-286. BRONSTEIN, H. D., HAEFNmER, L. J. & KOwLEssAR, 0. D. (1966). The significance of gelatin tolerance in malabsorptive states. Gaetroenterology 50, 621-630. CHRISTENSEN, H. N., WILBER, P. B., COYNE, B. A. & FISHER, J. H. (1955). Effects of simultaneous or prior infusion of sugars on the fate of infused protein hydrolysates. J. din. Inve8t. 34, 86-94. DAWSON, R. & PORTER, J. W. G. (1962). An investigation into protein digestion with 14Clabelled protein. 2. The transport of 14C-labelled nitrogenous compounds in the rat and cat. Br. J. Nutr. 16, 27-38. DENT, C. E. & ScmLwnJa, J. A. (1949). Studies on the absorption of proteins: the amino-acid pattern in the portal blood. Biochem. J. 44, 318-335. Fin m , R. B. (1967a). Absorption of proteins. Br. med. Bull. 23, 241-246. FISHER, R. B. (1967b). Absorption of proteins. Proc. Nutr. Soc. 26, 23-27. FISHER, R. B. & GARDNER, M. L. G. (1974). A kinetic approach to the study of absorption of solutes by isolated perfused small intestine. J. Physiol. 241, 211-234. GARDNER, M. L. G. (1975). Absorption of amino acids and peptides from a complex mixture in the isolated small intestine of the rat. J. Phy8iol. 253, 233-256. GARDNER, M. L. G. (1976). Absorption from a mixture of seventeen free amino acids by the isolated small intestine of the rat. J. Phy8iol. 255, 563-574. GARDNER, M. L. G. (1978a). Absorption of amino acids and peptides from complex hydrolysates of proteins. Gastroent. clin. et biol. 2, 341. GARDNER, M. L. G. (1978b). The absorptive viability of isolated intestine prepared from dead animals. Q. JZ exp. Phy8iol. 63, 93-95. HEADING, R. C., SCHEDL, H. P., STEGINK, L. D. & M aEB, D. L. (1977). Intestinal absorption of glycine and glycyl-L-proline in the rat. Clin. Sci. 52, 607-614. JACOBS, F. A. & LARGIS, E. E. (1969a). Transport of amino acids via the mesenteric lymph duct in rats. Proc. Soc. exp. Biol. Med. 130, 692-696.
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