233
J. Physiol. (1975), 253, pp. 233-256 With 8 text-figure. Printed in Great Britain
ABSORPTION OF AMINO ACIDS AND PEPTIDES FROM A COMPLEX MIXTURE IN THE ISOLATED SMALL INTESTINE OF THE RAT
BY M. L. G. GARDNER From the Department of Biochemistry, the University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG
(Received 13 May 1975) SUMMARY
1. Amino acid and peptide absorption from a pancreatic digest of casein at low concentration by an isolated preparation of perfused rat small intestine has been measured. 2. The rate of absorption of each amino acid (free or peptide-bound) is closely proportional to its concentration in the perfusate; this implies a constant Vmax/Km ratio for all amino acids in the mixture. 3. There is a high correlation between the compositions of luminal perfusate and secretion into the tissue fluid (apart from the content of glutamic and aspartic acids and alanine). 4. The concentrations of each free amino acid are, on average, 9 times as great in secretion as in lumen; the total peptide-N concentration in secretion is approximately 4 times that in the lumen. 5. The rate of absorption of each free amino acid is highly negatively dependent on the rate of absorption of that amino acid in peptide-bound form, in addition to being positively dependent on the perfusate concentration of free amino acid. 6. While peptide-bound proline appears to be well absorbed, free proline liberated by hydrolysis appears to pass back into the lumen as well as into the tissue fluid. Substantial back flux of hydrolysis products may occur for all amino acids. 7. About one-third of the amino acids appearing in the secretion on to the serosal surface are peptide-bound. 8. The rate of absorption of peptides appears to determine the rate of their hydrolysis which probably occurs mainly after entry into the mucosal cells.
234
M. L. G. GARDNER INTRODUCTION
Many studies have been made on the absorption of single amino acids from intestine (e.g. Gibson & Wiseman, 1951; Agar, Hird & Sidhu, 1953; Newey & Smyth, 1964; reviews by Wiseman, 1968, 1974). Recently it has become clear that a large proportion of those small peptides which have been investigated can also be absorbed, and that under certain circumstances at least some amino acid residues may be absorbed more rapidly from tri- and di-peptides than from the free amino acid (e.g. Newey & Smyth, 1959; Asatoor, Cheng, Edwards, Lant, Matthews, Milne, Navab & Richards, 1970; Matthews, Craft, Geddes, Wise & Hyde, 1968; Crampton, Gangolli, Simson & Matthews, 1971). However, virtually nothing is known of the relative physiological importance of peptide and amino acid absorption, especially since few investigators have attempted to measure the absorption of a specific amino acid or peptide from complex mixtures such as a natural pancreatic digest of protein. Such studies as have been made have led to the conclusion that the characteristics of absorption of amino acid mixtures are not representative of those of absorption of protein digestion products (e.g. Silk, Marrs, Addison, Burston, Clark & Matthews, 1973; Silk, Clark, Marrs, Addison, Burston, Matthews & Clegg, 1975). The fate of a balanced mixture of amino acids and peptides, as in a natural digest of a first class protein or its equivalent, may be quite different from that of single amino acids or peptides. This paper describes a detailed investigation into the absorption of sixteen amino acids, both free and peptide-bound, from a pancreatic partial digest of casein. The preparation of isolated perfused small intestine from the rat recently developed by Fisher & Gardner (1974) lends itself well to studies of the form in which absorbed solutes are added to the intestinal tissue fluid and therefore has been used throughout the present investigation. It permits serial measurement of absorption rates from the intestinal lumen during single-pass slow perfusion with a definable protein digest. Additionally, the fluid and solutes which are transported across the mucosa drip off as 'secretion' from the serosal surface of the organ, and can be collected for analysis without dilution or contamination by a bloodstream. The steady-state relation between 'absorptive' and secretaryy' activity of the mucosa can therefore be examined directly. The compositions of the casein digest added to the luminal perfusate, of the effluent issuing from the intestinal lumen, and of the transported fluid ('secretion') were studied in detail, before and after total hydrolysis by acid, by means of an automatic ion-exchange amino acid analyser, so that estimates of free and peptide-bound amino acids were available. Hence rates of absorption from the intestinal lumen and also of secretion
235 AMINO ACID AND PEPTIDE ABSORPTION on to the serosal surface for each amino acid, free and peptide-bound, could be calculated. The extensive data handling has been made possible by the development of several digital computer programmes which also calculate and display graphically correlations between the compositions of the perfusate and the secretion, and other relevant inter-relations. METHODS
Animals, anaesthesia and chemicals. These were as described by Fisher & Gardner (1974). Experimentl procedure. Isolated whole jejunum plus ileum (from the Ligament of Treitz to the ileo-caecal valve) were perfused with a segmented flow in a single pass through the lumen as described by Fisher & Gardner (1974). Collections of luminal effluent and of secretion were made over six consecutive 15 min periods. For the reasons given below only the collections during the fourth period (45-60 min perfusion) were subjected to the full amino acid analysis. Perfusion medium. The electrolyte composition was as follows: NaCl 118-5 mM, NaHCO3 24588 mM, KCl 4.73 mM, KH2PO4 1-18 mM, MgSO4 030 mm, CaCl2 1*18 mM. An enzymic hyrolysate of casein (Sigma London Chemical Co. Ltd), prepared by pancreatic digestion of casein, was added to give a concentration of 0-5 mg/ml. The estimated amino acid composition is shown in Table 1. The perfusate also TABLE 1. Amino acid composition of the perfusate, /SM (values are means of five experiments + s.E. of mean)
Amino acid (and Km) Aspartic acid Threonine (13000) Serine Glutamic acid Proline (6200) Glycine (10000) Alanine (6300) Valine (3300) Methionine (5300) Isoleucine (1600) Leucine (2200) Tyrosine (4000) Phenylalanine (1400) Histidine (6000) Lysine (700) Arginine (1200)
Free 51+ 8 *81 + 5 *133 + 5 112+4 77 + 10 33+3 87 + 3 144+ 5
73+1 112+ 2 273+6 61+ 4 102+ 5 47+7 193+10 90+ 2
Peptide-bound *198 + 12 t85+5 t98 ± 12 *529+ 12 327 + 32 104+ 5 86+ 4 140+14 21+3 94+ 3 67+ 10 40+ 6 37 + 4 45+7 45+7 17 + 4
Notes. (1) Owing to inability to resolve asparagine and glutamine during the amino acid analysis some of the estimates are in error (see text). Those which are over-estimated are indicated by *. Those which are underestimated are indicated by t. (2) The digest also contained other amino acids (including tryptophan and cysteine) but these were not routinely estimated. (3) The Km values (IM) shown in parentheses after each amino acid were determined by Larsen, Ross & Tapley (1964) for single amino acids in rat intestine.
236
M. L. G. GARDNER
contained glucose (5 mg/ml.), and was equilibrated with 5 % C02 in 02 at 40° C for at least 1 hr/i. before use. The phenol red used by Fisher & Gardner (1974) was not included in the perfusate since it was found that this was strongly adsorbed on to the ion-exchange column. Amino acid analyses. Samples were applied to a Locarte amino acid analyser equipped with an auto-loader. Protein hydrolysate methodology with stepwise elution was followed according to the manufacturer's instructions. Aliquots were also hydrolysed at 105° C in vacuo for 24 hr with 6N-HCI (Aristar grade, B.D.H. Chemicals Ltd) containing 02 % phenol. This hydrolysate was then dried in vacuo, dissolved in the loading buffer, and applied to the analyzer. It was assumed that losses of free amino acids during the hydrolysis were trivial. No estimate of asparagine or glutamine was available. Since these amides were not resolved from threonine and serine, the values of free threonine and serine will be overestimated. During the acid hydrolysis the amides will be converted to aspartic and glutamic acids; therefore the estimates of peptide-bound aspartic and glutamic acids will be too high while the estimates of peptide-bound threonine and serine will be too low. According to the data of Mercier, Grosclaude & Dumas (1972) approximately half the dicarboxylic amino acids in bovine casein are present as amides. These amides amount to about 11 % of the total amino acids. Each peptide-bound amino acid was estimated by the difference between free amino acid before and after the acid hydrolysis. It was assumed that no peptides in the unhydrolysed material were eluted at the same time as any free amino acid; this is probably valid since the estimated amount of each amino acid was greater after the hydrolysis than before it and no distortions of peak symmetry were seen. Also, the agreement between the estimates of total free amino acids by this ionexchange method and the gasometric ninhydrin method was good (see Table 3). TABLE 2. Apparent rates of nitrogen absorption and secretion during the fourth 15 min period of control experiments with N-free perfusion (/zgN. cm-. hr-1). Means of two experiments
Absorption Total N Free amino-acid N Peptide N
Secretion
-19.5
34-2
-1-24 - 3 04
9.73 4-79
Total free amino acids were estimated by the gasometric ninhydrin technique of Van Slyke, Dillon, MacFadyen & Hamilton (1941) adapted for use with an AutoAnalyzer system similar to that described by Reynolds & Foley (1968). Total-N estimation. Ammonia was estimated on an AutoAnalyzer by a modification of the Berthelot phenol-hypochlorite reaction (Kaplan, 1965) following microKjeldahl ashing with a mercury catalyst. Control experiment. In order to make corrections for loss of endogenous amino acids and peptides from the intestine, control experiments were made in which the casein hydrolysate was omitted from the perfusate. These losses are summarized in Table 2. The assumption is made that these estimates of endogenous nitrogen loss can be used to correct the absorption and secretion rates observed during perfusion with the casein hydrolysate. This may not be valid. In the control experiments the rate of total-N secretion was much greater than the rate of free amino acid-N plus peptide-N secretion (Table 2) indicating the loss of endogenous nitrogen other than that detected by the amino acid analyzer. (If the loss of this material were less when
AMINO ACID AND PEPTIDE ABSORPTION
237
the intestine is perfused with the balanced mixture of amino acids and peptides then this might account for the apparently low rate of total-N secretion given below in Table 5.) Computation of re8ult8. Raw experimental data were used as input to the IBM 370/158 computer at the Edinburgh Regional Computing Centre. Programmes written in IMP (which is an advanced language evolved in Edinburgh from ALGOL and Atlas Autocode) calculated full analytical information, absorption and secretion rates and the differences between them for each total, free, and peptide-bound amino acid, with detailed tabular and graphical outputs. Outputs were recorded both on the line-printer and, for further use, on magnetic tape. Selected parameters were also directed to a card-punch output so that they could be used subsequently as input for the Edinburgh Regional Computing Centre programme package MULTREG which was run in conversational mode from a console attached to the Edinburgh Multiple Access System on the ICL 4-75 computer. This programme performs highly versatile multiple linear regression analyses, and permits the calculation of any desired correlation or partial correlation coefficients and the progressive automatic elimination from a regression of insignificant variables at a variety of levels of significance. Additional programmes calculated, and expressed graphically, mean values of parameters and any desired correlations and partial correlation coefficients calculated for any desired sets of experiments appropriately corrected for the respective control data obtained from the N-free perfusion experiments. Number of experiments. Full analytical data were obtained for five experiments with the casein hydrolysate plus two controls with N-free perfusion. RESULTS
Achievement of steady-state conditions The concentrations of total-N (by the Kjeldahl method), and of free amino acid-N (by the gasometric ninhydrin method) in each sample of luminal effluent and of secretion over the 90 min of perfusion, are shown in Fig. 1 A and 1 B respectively. The marked changes in total-N concentration shown in Fig. 1 B are due to the wash-out of protein and other endogenous total-N from the intestinal tissue fluid (Text-figs. 7 and 9 of Fisher & Gardner, 1974). Preliminary experiments and the data of Fisher & Gardner suggested that steady-state conditions in the secretion would have been achieved by 45-60 min at a normal rate of water absorption. Therefore the complete amino acid analyses were performed always on the fourth 15 min collections. However, as Fig. 1 B shows, the steady state had almost, but not quite, been reached by this fourth collection.
Composition of tuminal effluent and secretion The distribution of N in the perfusate, luminal effluent, and secretion is given in Table 3. Fig. 1 A and B also show the non-free amino acid-N (A) estimated by difference between the Kjeldahl value for total-N and the gasometric ninhydrin value for free amino acid-N. A includes peptide-N and any other nitrogenous material (e.g. nucleic acids) which may be
238 M. L. G. GARDNER released from the perfused intestine. The similarity between the values of A and free amino acids in the perfusate reflects the fact that in this particular casein hydrolysate approximately half the amino acids are in peptides (31.2 ,sg free amino acid-N/ml.; 29*7 ,tg peptide-N/ml., Table 3). A 70 4
Perfusate total-N
60 0
50
oal-N
00 40 W
VPerfusateA AA-N
Perfusate AA-N 30JU~~~U.-
0
U
20 10 0
1
2
3 1 15
4
5
6
min collections
Fig. 1 A and B. Over-all compositions of luminal effluent and secretion respectively over the 90 min of perfusion. Total-N was determined by the Kjeldahl method (0-0), free amino acid N (AA-N) by the automated gasometric ninhydrin method (LI-L), and the non-free amino acid-N (A) was calculated by the difference (U-U). The perfusate concentrations are also shown on Fig. 1 A. Means for four experiments.
Table 4 shows the mean ratio of concentrations of free amino acids in the secretion to those in the intestinal lumen in the 45-60 min samples. Ratios are shown both for secretion to inflowing perfusate and for secretion to outflowing luminal effluent. Amino acids have been ranked in order of the concentration ratios, and the amino acids which are essential for growth have been denoted by E. The over-all concentration ratio is around 9:1. Note that each ratio is significantly greater than unity. In most cases, except for glycine and proline, the value in column 2 is greater than the value in column 1. This reflects the fact that effluent concentrations are lower than those in the inflowing perfusate, owing to absorption.
239
AMINO ACID AND PEPTIDE ABSORPTION B
1,000 I
800 II I I
zZ 600
.t CE
0
I-
0 C
Total-N
'V
U
%
200 I
n
-
S' %s %b
a -
0
4:
AA-N
a
1
2
3
4
--
- - - -
Ai
p
5
6
15 min collections
Fig. 1B
Rates of absorption from the lumen and rates of secretion The corrected rates of absorption of N from the intestinal lumen and of secretion on to the serosal surface are shown in Table 5. Half the N which is absorbed from the digest comes from peptides; also over a third of the nitrogen which is secreted on to the serosal surface is in the form of peptides. Although the variance of this measurement is high, the rate of peptide secretion in each experiment was greater than in the control experiments. The absorption rate from the lumen of each amino acid, from both free
240
M. L. G. GARDNER
TABLE 3. Distribution of N in perfusate, luminal effluent and secretion during the fourth 15 min period (,eg N/ml.). Values are means of five experiments (unless otherwise stated) + s.E. of mean
Total-N (Kjeldahl) Peptide-N + amino acid-N (ion exchange after hydrolysis) Peptide-N (ion exchange difference) Amino-acid-N (ion exchange before hydrolysis) Amino-acid-N (gasometric ninhydrin)
Luminal effluent 50-1+2-8
Secretion 323+10
39-8 1-7 19-6±1-1
t320 + 54(4) t117±37(4)
312+0-6 l* 20-2 0-7 29-2 + 1.9 J 25-7 + 2-2
206+ 15 1* 210 17
Perfusate 61.1+2.3 1 60-8 + 1-5 29-7+1-1
j
J
j
The bracketed values are not significantly different from each other. t One dubious analysis of secretion peptide has been excluded. *
TABLE 4. Secretion to perfusate concentration ratios of free amino acids at the steady state during absorption from a pancreatic digest of casein (0-5 mg/ml.). (E) indicates an essential amino acid Significance of Secretion: Secretion: difference of inflowing perfusate luminal effluent the ratios Glutamic acid 2-7+0-4 2-2+0-4 n.s. Aspartic acid 2-8+0-3 2-6+0-5 n.s. Arginine 3-9 + 0-4 26-8 + 14-8 n.s. Leucine (E) 5-2 + 0-3 12-2+ 0-6 P < 0-001 Phenylalanine (E) 6-1+0-6 11-6+0-7 P < 0.001 Methionine (E) 6-4+0-4 12-8+0-9 P < 0-001 Lysine (E) 6-6+ 0-6 13-6+ 1-0 P
J. Physiol. (1975), 253, pp. 233-256 With 8 text-figure. Printed in Great Britain
ABSORPTION OF AMINO ACIDS AND PEPTIDES FROM A COMPLEX MIXTURE IN THE ISOLATED SMALL INTESTINE OF THE RAT
BY M. L. G. GARDNER From the Department of Biochemistry, the University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG
(Received 13 May 1975) SUMMARY
1. Amino acid and peptide absorption from a pancreatic digest of casein at low concentration by an isolated preparation of perfused rat small intestine has been measured. 2. The rate of absorption of each amino acid (free or peptide-bound) is closely proportional to its concentration in the perfusate; this implies a constant Vmax/Km ratio for all amino acids in the mixture. 3. There is a high correlation between the compositions of luminal perfusate and secretion into the tissue fluid (apart from the content of glutamic and aspartic acids and alanine). 4. The concentrations of each free amino acid are, on average, 9 times as great in secretion as in lumen; the total peptide-N concentration in secretion is approximately 4 times that in the lumen. 5. The rate of absorption of each free amino acid is highly negatively dependent on the rate of absorption of that amino acid in peptide-bound form, in addition to being positively dependent on the perfusate concentration of free amino acid. 6. While peptide-bound proline appears to be well absorbed, free proline liberated by hydrolysis appears to pass back into the lumen as well as into the tissue fluid. Substantial back flux of hydrolysis products may occur for all amino acids. 7. About one-third of the amino acids appearing in the secretion on to the serosal surface are peptide-bound. 8. The rate of absorption of peptides appears to determine the rate of their hydrolysis which probably occurs mainly after entry into the mucosal cells.
234
M. L. G. GARDNER INTRODUCTION
Many studies have been made on the absorption of single amino acids from intestine (e.g. Gibson & Wiseman, 1951; Agar, Hird & Sidhu, 1953; Newey & Smyth, 1964; reviews by Wiseman, 1968, 1974). Recently it has become clear that a large proportion of those small peptides which have been investigated can also be absorbed, and that under certain circumstances at least some amino acid residues may be absorbed more rapidly from tri- and di-peptides than from the free amino acid (e.g. Newey & Smyth, 1959; Asatoor, Cheng, Edwards, Lant, Matthews, Milne, Navab & Richards, 1970; Matthews, Craft, Geddes, Wise & Hyde, 1968; Crampton, Gangolli, Simson & Matthews, 1971). However, virtually nothing is known of the relative physiological importance of peptide and amino acid absorption, especially since few investigators have attempted to measure the absorption of a specific amino acid or peptide from complex mixtures such as a natural pancreatic digest of protein. Such studies as have been made have led to the conclusion that the characteristics of absorption of amino acid mixtures are not representative of those of absorption of protein digestion products (e.g. Silk, Marrs, Addison, Burston, Clark & Matthews, 1973; Silk, Clark, Marrs, Addison, Burston, Matthews & Clegg, 1975). The fate of a balanced mixture of amino acids and peptides, as in a natural digest of a first class protein or its equivalent, may be quite different from that of single amino acids or peptides. This paper describes a detailed investigation into the absorption of sixteen amino acids, both free and peptide-bound, from a pancreatic partial digest of casein. The preparation of isolated perfused small intestine from the rat recently developed by Fisher & Gardner (1974) lends itself well to studies of the form in which absorbed solutes are added to the intestinal tissue fluid and therefore has been used throughout the present investigation. It permits serial measurement of absorption rates from the intestinal lumen during single-pass slow perfusion with a definable protein digest. Additionally, the fluid and solutes which are transported across the mucosa drip off as 'secretion' from the serosal surface of the organ, and can be collected for analysis without dilution or contamination by a bloodstream. The steady-state relation between 'absorptive' and secretaryy' activity of the mucosa can therefore be examined directly. The compositions of the casein digest added to the luminal perfusate, of the effluent issuing from the intestinal lumen, and of the transported fluid ('secretion') were studied in detail, before and after total hydrolysis by acid, by means of an automatic ion-exchange amino acid analyser, so that estimates of free and peptide-bound amino acids were available. Hence rates of absorption from the intestinal lumen and also of secretion
235 AMINO ACID AND PEPTIDE ABSORPTION on to the serosal surface for each amino acid, free and peptide-bound, could be calculated. The extensive data handling has been made possible by the development of several digital computer programmes which also calculate and display graphically correlations between the compositions of the perfusate and the secretion, and other relevant inter-relations. METHODS
Animals, anaesthesia and chemicals. These were as described by Fisher & Gardner (1974). Experimentl procedure. Isolated whole jejunum plus ileum (from the Ligament of Treitz to the ileo-caecal valve) were perfused with a segmented flow in a single pass through the lumen as described by Fisher & Gardner (1974). Collections of luminal effluent and of secretion were made over six consecutive 15 min periods. For the reasons given below only the collections during the fourth period (45-60 min perfusion) were subjected to the full amino acid analysis. Perfusion medium. The electrolyte composition was as follows: NaCl 118-5 mM, NaHCO3 24588 mM, KCl 4.73 mM, KH2PO4 1-18 mM, MgSO4 030 mm, CaCl2 1*18 mM. An enzymic hyrolysate of casein (Sigma London Chemical Co. Ltd), prepared by pancreatic digestion of casein, was added to give a concentration of 0-5 mg/ml. The estimated amino acid composition is shown in Table 1. The perfusate also TABLE 1. Amino acid composition of the perfusate, /SM (values are means of five experiments + s.E. of mean)
Amino acid (and Km) Aspartic acid Threonine (13000) Serine Glutamic acid Proline (6200) Glycine (10000) Alanine (6300) Valine (3300) Methionine (5300) Isoleucine (1600) Leucine (2200) Tyrosine (4000) Phenylalanine (1400) Histidine (6000) Lysine (700) Arginine (1200)
Free 51+ 8 *81 + 5 *133 + 5 112+4 77 + 10 33+3 87 + 3 144+ 5
73+1 112+ 2 273+6 61+ 4 102+ 5 47+7 193+10 90+ 2
Peptide-bound *198 + 12 t85+5 t98 ± 12 *529+ 12 327 + 32 104+ 5 86+ 4 140+14 21+3 94+ 3 67+ 10 40+ 6 37 + 4 45+7 45+7 17 + 4
Notes. (1) Owing to inability to resolve asparagine and glutamine during the amino acid analysis some of the estimates are in error (see text). Those which are over-estimated are indicated by *. Those which are underestimated are indicated by t. (2) The digest also contained other amino acids (including tryptophan and cysteine) but these were not routinely estimated. (3) The Km values (IM) shown in parentheses after each amino acid were determined by Larsen, Ross & Tapley (1964) for single amino acids in rat intestine.
236
M. L. G. GARDNER
contained glucose (5 mg/ml.), and was equilibrated with 5 % C02 in 02 at 40° C for at least 1 hr/i. before use. The phenol red used by Fisher & Gardner (1974) was not included in the perfusate since it was found that this was strongly adsorbed on to the ion-exchange column. Amino acid analyses. Samples were applied to a Locarte amino acid analyser equipped with an auto-loader. Protein hydrolysate methodology with stepwise elution was followed according to the manufacturer's instructions. Aliquots were also hydrolysed at 105° C in vacuo for 24 hr with 6N-HCI (Aristar grade, B.D.H. Chemicals Ltd) containing 02 % phenol. This hydrolysate was then dried in vacuo, dissolved in the loading buffer, and applied to the analyzer. It was assumed that losses of free amino acids during the hydrolysis were trivial. No estimate of asparagine or glutamine was available. Since these amides were not resolved from threonine and serine, the values of free threonine and serine will be overestimated. During the acid hydrolysis the amides will be converted to aspartic and glutamic acids; therefore the estimates of peptide-bound aspartic and glutamic acids will be too high while the estimates of peptide-bound threonine and serine will be too low. According to the data of Mercier, Grosclaude & Dumas (1972) approximately half the dicarboxylic amino acids in bovine casein are present as amides. These amides amount to about 11 % of the total amino acids. Each peptide-bound amino acid was estimated by the difference between free amino acid before and after the acid hydrolysis. It was assumed that no peptides in the unhydrolysed material were eluted at the same time as any free amino acid; this is probably valid since the estimated amount of each amino acid was greater after the hydrolysis than before it and no distortions of peak symmetry were seen. Also, the agreement between the estimates of total free amino acids by this ionexchange method and the gasometric ninhydrin method was good (see Table 3). TABLE 2. Apparent rates of nitrogen absorption and secretion during the fourth 15 min period of control experiments with N-free perfusion (/zgN. cm-. hr-1). Means of two experiments
Absorption Total N Free amino-acid N Peptide N
Secretion
-19.5
34-2
-1-24 - 3 04
9.73 4-79
Total free amino acids were estimated by the gasometric ninhydrin technique of Van Slyke, Dillon, MacFadyen & Hamilton (1941) adapted for use with an AutoAnalyzer system similar to that described by Reynolds & Foley (1968). Total-N estimation. Ammonia was estimated on an AutoAnalyzer by a modification of the Berthelot phenol-hypochlorite reaction (Kaplan, 1965) following microKjeldahl ashing with a mercury catalyst. Control experiment. In order to make corrections for loss of endogenous amino acids and peptides from the intestine, control experiments were made in which the casein hydrolysate was omitted from the perfusate. These losses are summarized in Table 2. The assumption is made that these estimates of endogenous nitrogen loss can be used to correct the absorption and secretion rates observed during perfusion with the casein hydrolysate. This may not be valid. In the control experiments the rate of total-N secretion was much greater than the rate of free amino acid-N plus peptide-N secretion (Table 2) indicating the loss of endogenous nitrogen other than that detected by the amino acid analyzer. (If the loss of this material were less when
AMINO ACID AND PEPTIDE ABSORPTION
237
the intestine is perfused with the balanced mixture of amino acids and peptides then this might account for the apparently low rate of total-N secretion given below in Table 5.) Computation of re8ult8. Raw experimental data were used as input to the IBM 370/158 computer at the Edinburgh Regional Computing Centre. Programmes written in IMP (which is an advanced language evolved in Edinburgh from ALGOL and Atlas Autocode) calculated full analytical information, absorption and secretion rates and the differences between them for each total, free, and peptide-bound amino acid, with detailed tabular and graphical outputs. Outputs were recorded both on the line-printer and, for further use, on magnetic tape. Selected parameters were also directed to a card-punch output so that they could be used subsequently as input for the Edinburgh Regional Computing Centre programme package MULTREG which was run in conversational mode from a console attached to the Edinburgh Multiple Access System on the ICL 4-75 computer. This programme performs highly versatile multiple linear regression analyses, and permits the calculation of any desired correlation or partial correlation coefficients and the progressive automatic elimination from a regression of insignificant variables at a variety of levels of significance. Additional programmes calculated, and expressed graphically, mean values of parameters and any desired correlations and partial correlation coefficients calculated for any desired sets of experiments appropriately corrected for the respective control data obtained from the N-free perfusion experiments. Number of experiments. Full analytical data were obtained for five experiments with the casein hydrolysate plus two controls with N-free perfusion. RESULTS
Achievement of steady-state conditions The concentrations of total-N (by the Kjeldahl method), and of free amino acid-N (by the gasometric ninhydrin method) in each sample of luminal effluent and of secretion over the 90 min of perfusion, are shown in Fig. 1 A and 1 B respectively. The marked changes in total-N concentration shown in Fig. 1 B are due to the wash-out of protein and other endogenous total-N from the intestinal tissue fluid (Text-figs. 7 and 9 of Fisher & Gardner, 1974). Preliminary experiments and the data of Fisher & Gardner suggested that steady-state conditions in the secretion would have been achieved by 45-60 min at a normal rate of water absorption. Therefore the complete amino acid analyses were performed always on the fourth 15 min collections. However, as Fig. 1 B shows, the steady state had almost, but not quite, been reached by this fourth collection.
Composition of tuminal effluent and secretion The distribution of N in the perfusate, luminal effluent, and secretion is given in Table 3. Fig. 1 A and B also show the non-free amino acid-N (A) estimated by difference between the Kjeldahl value for total-N and the gasometric ninhydrin value for free amino acid-N. A includes peptide-N and any other nitrogenous material (e.g. nucleic acids) which may be
238 M. L. G. GARDNER released from the perfused intestine. The similarity between the values of A and free amino acids in the perfusate reflects the fact that in this particular casein hydrolysate approximately half the amino acids are in peptides (31.2 ,sg free amino acid-N/ml.; 29*7 ,tg peptide-N/ml., Table 3). A 70 4
Perfusate total-N
60 0
50
oal-N
00 40 W
VPerfusateA AA-N
Perfusate AA-N 30JU~~~U.-
0
U
20 10 0
1
2
3 1 15
4
5
6
min collections
Fig. 1 A and B. Over-all compositions of luminal effluent and secretion respectively over the 90 min of perfusion. Total-N was determined by the Kjeldahl method (0-0), free amino acid N (AA-N) by the automated gasometric ninhydrin method (LI-L), and the non-free amino acid-N (A) was calculated by the difference (U-U). The perfusate concentrations are also shown on Fig. 1 A. Means for four experiments.
Table 4 shows the mean ratio of concentrations of free amino acids in the secretion to those in the intestinal lumen in the 45-60 min samples. Ratios are shown both for secretion to inflowing perfusate and for secretion to outflowing luminal effluent. Amino acids have been ranked in order of the concentration ratios, and the amino acids which are essential for growth have been denoted by E. The over-all concentration ratio is around 9:1. Note that each ratio is significantly greater than unity. In most cases, except for glycine and proline, the value in column 2 is greater than the value in column 1. This reflects the fact that effluent concentrations are lower than those in the inflowing perfusate, owing to absorption.
239
AMINO ACID AND PEPTIDE ABSORPTION B
1,000 I
800 II I I
zZ 600
.t CE
0
I-
0 C
Total-N
'V
U
%
200 I
n
-
S' %s %b
a -
0
4:
AA-N
a
1
2
3
4
--
- - - -
Ai
p
5
6
15 min collections
Fig. 1B
Rates of absorption from the lumen and rates of secretion The corrected rates of absorption of N from the intestinal lumen and of secretion on to the serosal surface are shown in Table 5. Half the N which is absorbed from the digest comes from peptides; also over a third of the nitrogen which is secreted on to the serosal surface is in the form of peptides. Although the variance of this measurement is high, the rate of peptide secretion in each experiment was greater than in the control experiments. The absorption rate from the lumen of each amino acid, from both free
240
M. L. G. GARDNER
TABLE 3. Distribution of N in perfusate, luminal effluent and secretion during the fourth 15 min period (,eg N/ml.). Values are means of five experiments (unless otherwise stated) + s.E. of mean
Total-N (Kjeldahl) Peptide-N + amino acid-N (ion exchange after hydrolysis) Peptide-N (ion exchange difference) Amino-acid-N (ion exchange before hydrolysis) Amino-acid-N (gasometric ninhydrin)
Luminal effluent 50-1+2-8
Secretion 323+10
39-8 1-7 19-6±1-1
t320 + 54(4) t117±37(4)
312+0-6 l* 20-2 0-7 29-2 + 1.9 J 25-7 + 2-2
206+ 15 1* 210 17
Perfusate 61.1+2.3 1 60-8 + 1-5 29-7+1-1
j
J
j
The bracketed values are not significantly different from each other. t One dubious analysis of secretion peptide has been excluded. *
TABLE 4. Secretion to perfusate concentration ratios of free amino acids at the steady state during absorption from a pancreatic digest of casein (0-5 mg/ml.). (E) indicates an essential amino acid Significance of Secretion: Secretion: difference of inflowing perfusate luminal effluent the ratios Glutamic acid 2-7+0-4 2-2+0-4 n.s. Aspartic acid 2-8+0-3 2-6+0-5 n.s. Arginine 3-9 + 0-4 26-8 + 14-8 n.s. Leucine (E) 5-2 + 0-3 12-2+ 0-6 P < 0-001 Phenylalanine (E) 6-1+0-6 11-6+0-7 P < 0.001 Methionine (E) 6-4+0-4 12-8+0-9 P < 0-001 Lysine (E) 6-6+ 0-6 13-6+ 1-0 P
