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Clinical Science( 1986) 71,65-69 65 Effect of peptide ch in length on mino acid and nitrogen absorption from two lactalbumin hydrolysates in the normal human jejunum G. K. GRIMBLE, P. P. KEOHANE, B. E. HIGGINS, M. V. KAMINSKI, JR* AND D. B. A. SILK Department of Gastroenterologyand Nutrition, Central Middlesex Hospital, London, and * The Chicago Medical School, Chicago, Illinois, U.S.A. (Received 6 September 1985/9January 1986; accepted 18 February 1986) Sumrnary 1. A double lumen jejunal perfusion technique has been used in man to study the effect of peptide chain length on absorption of amino acid nitrogen from two partial enzymic hydrolysates of lactalbumin. 2. Copper-chelation chromatography showed that one lactalbumin hydrolysate (LH2) contained 98% peptides with a chain length > 4, whilst the other (LH1) contained a more even spread of chain lengths with 55% < 4. 3. Absorption of total nitrogen and of 14 amino acid residues occurred to a significantly greater extent from the low molecular weight LH1 than from the higher molecular weight LH2. 4. The results suggest that the pattern of nitrogen and amino acid absorption from partial enzymic hydrolysates of whole protein is markedly influenced by peptide chain length and that brush border peptide hydrolysis has an important rate limiting effect on absorption rates. mammalian small intestine [ 1-31. Similar experiments have shown differences in the kinetics of free amino acid and peptide transport [4] and many diand tri-peptide bound residues have been shown to be absorbed faster than when presented in the free form [l-31. Similar observations were also made from human intestinal perfusion studies with partial enzymic hydrolysates of whole protein (consisting of heterogenous mixtures of small peptides [5-7]), but there were considerable variations in amino acid absorption patterns with different hydrolysates [8].The nature of the starter protein and the type of hydrolysis used to produce the peptide mixture have both been shown to influence absorption properties [9]. However, the effect of peptide chain length on absorption was not studied. The aim of the present study was therefore to investigate the influence that peptide chain length has on intestinal absorption of partial enzymic hydrolysates of lactalbumin in man. Methods Key words: amino acid nitrogen absorption, intestinal perfusion, lactalbumin hydrolysate, peptide absorption, peptide chain length. Introduction Recent work has established the existence of at least one transport system which mediates absorption of unhydrolysed model di- and tri-peptides from Correspondence: Dr D. B. A. Silk, Department of Gastroenterology and Nutrition, Central Middlesex Hospital, Acton Lane, London NWlO 7NS. Enzymic hydrolysates and amino acid mixtures Two partial enzymic hydrolysates of lactose-free lactalbumin (approx. 92% protein) were prepared from a single protein source. Papain and trypsin were used to hydrolyse the protein in vitro and the rate and extent of hydrolysis was measured titrimetrically until the reaction was stopped at the desired end-point by heating to 100°C. The remaining enzyme and undigested protein ‘cores’were precipitated by sedimentation and the resulting supernatant was decolorized, using activated charcoal, passed through a 0.45 p m prefilter and ultrafiltered 66 G. K . Grimble et al. using a Millipore Pellicon cassette system (type PTCG with a molecular weight cut-off of 10000) before freeze-drying. On two separate occasions, the reaction time was adjusted to produce two hydrolysates of similar amino acid composition but of different chain length profile. Full details of the method of preparation of hydrolysates are the subject of a US. Patent Application (reference no. 27083-00). TABLE1. Chain length distribution of lactalbumin hydrolysates Determined by Cu(I1)-Sephadex chromatography as described in the text. Mean chain length Distribution (“hby wt.) Chainlength ... > 4 LH1 3.85 LH2 7.7 40 98 4 3 2 1 23 - 22 - 10 - 5 2 Determination of peptide chain length Peptide chain length was determined by ligand exchange chromatography using Cu(I1)-Sephadex as described by Rothenbuhler et al. [lo]. A column (1.6 cm X 40 cm) was packed with Cu(I1)-Sephadex, equilibrated with 50 mmolA disodium tetraborate (pH 11.0) and the column eluent was monitored at 254 nm to detect Cu(I1)-peptide complexes. This column was calibrated with synthetic di- and tri-peptides (dipeptides: Lys -Asp, Ala - Gly, Pro - Val, Ala - Tyr, Asp - Ala, Arg Tyr, Glu -Val, Ala - His, Gly - Leu, Arg - Arg, Glu - Glu, Leu - Gly, Met - Ser, Leu - Phe, His Leu; tripeptides: Leu - Ala - Pro, Glu - Thr - Tyr, Phe - Gly - Gly, Gly - Ala - Gly, Glu - Cys - Gly), eluted using the same buffer. Approximate retention times were thus obtained for each oligopeptide chain length. For analysis of an unknown oligopeptide mixture, 3 mg of peptide was dissolved in 1 ml of buffer and applied to the column. The mixture was then fractionated into different class sizes according to the retention times predetermined above. The precise mean chain length of each fraction was analysed by determination of a-amino nitrogen, before and after total acid hydrolysis [ll, 121, a method similar to that previously used to determine the mean chain length of hydrolysis products of protein in the rat jejunum [13]. The mean chain length obtained for each oligopeptide class varied somewhat with each separation but was always within the limits: ‘pentapeptides’ 5 f 0.3, ‘tetrapeptides’ 4 k 0.3, ‘tripeptides’ 3 f 0.3, ‘dipeptides’2 f 0.3. The oligopeptide distribution (by weight) was thus obtained by integration of the total quantity in each size-fraction (Table l ) , whilst the percentage of free amino acids was calculated by the difference. The amino acid composition of each preparation was analysed after acid hydrolysis, in 4.0 molA methanesulphonic acid at 110°Cfor 24 h in vacuo, on a Locarte automatic amino acid analyser with an Apple IIe integration system (Locarte Company, London) (Table 2).Two free amino acid mixtures simulating the amino acid composition, of complete acid hydrolysates, of LH1 and LH2 [5-81 were also prepared, taking into account losses of threonine and serine on hydroly- TABLE 2. Amino acid composition of lactalbumin hydrolysates Amino acid Amino acid composition (residued100) Low mol. wt. lactalbumin hydrolysate Higher mol. wt. lactalbumin hydrolysate (LH2) Asx Thr Ser Glx Pro GlY Ala Val CYS Met Ile Leu TYr Phe His TrP LYS ‘4% 9.73 5.75 3.47 19.47 5.75 5.09 8.99 5.91 1.10 2.38 4.36 12.19 1.43 2.57 2.39 1.60 7.53 0.30 10.52 5.95 5.76 16.78 4.86 3.67 7.67 5.99 1.06 2.07 4.63 12.16 2.86 2.90 2.00 n.d. 9.27 1.84 sis. Since the proportion of asparagine and glutamine could not be determined by this method, the amino acid mixture contained aspartate and glutamate. The differences in amino acid composition between hydrolysates are likely to have been caused by removal of some residues by the precipitation and decolorization steps. Perfusion technique Six normal healthy volunteers were intubated with a double lumen perfusion tube incorporating a proximal occlusive balloon [13]. The study was approved by the Ethical Committee of the Central Middlesex Hospital. Full details of the perfusion technique and methods used for collecting intestinal aspirates have been described in detail [5-7, 141. Peptide chain length and jejunal absorption Each subject was perfused in random order with four test solutions, each containing one of the lactalbumin hydrolysates or their free amino acid mixtures. All test solutions contained 100 mmolA total amino acid. The test solutions contained 1 pCi (37 kBq) of ''C-PEGA [15] and the tonicity and pH of the solutions were adjusted to 290-300 mosmol/lcg and 7.0 by addition of NaCl and NaOH respectively. The estimated irradiation to the gut from 14C-PEGwas 20 mrad. Analytical methods and calculation of results The amino acid content of test solutions and intestinal aspirates was determined by automated amino acid analysis (Locarte Company, London) after complete acid hydrolysis with 4.0 moVl methanesulphonic acid in vacua. Nitrogen was measured by an automated chemiluminescence technique [Antek Model 703 nitrogen analyser, Edect (Scientific) Ltd, Northants., U.K.]. The 14CPEG content of samples was measured by liquid scintillation counting with quench correction by the H-number technique (Beckman LD7S00, Beckman-Riic, High Wycombe, Bucks., U.K.). Sodium content of perfusates and aspirates was measured by flame photometry. Amino acid absorption was calculated using previously described formulae [ 161. Luminal disappearance of amino acid residues was taken to be equivalent to absorption. The significance of differences was tested by the t-test. 67 Results Ten amino acid residues (Asx, Thre, Ser, Glx, Ala, Gly, Val, Leu, Phe and His) were absorbed significantly faster ( P< 0.05 or less) from the low molecular weight lactalbumin hydrolysate (LH1)than from its equivalent amino acid mixture (Table 3). In contrast, four amino acid residues (Gly, Ala, Ileu and Leu) were absorbed sigmflcantly slower from the higher molecular weight hydrolysate than from its equivalent free amino acid mixture. Comparison of absorption rates showed that 14 residues (Asx, Thr, Ser, Glx, Gly, Ala, Val, Met, Ile, Leu, Tyr, Phe, His and Lys) were absorbed significantlyfaster from the low molecular weight lactalbumin hydrolysate, LH1, than from the higher molecular weight preparation, LH2 ( P < 0.05 or less) (Table 3). Total nitrogen absorption from the solutions showed a similar pattern. Whilst there was no difference in percentage absorption between the two amino acid solutions equivalent to LH1 and LH2, nitrogen was absorbed from the short-chain hydrolysate LH1 to a markedly greater extent than from LH2 (Table 4). There was significantly slower nitrogen absorption from the higher molecular weight protein hydrolysate than from its equivalent amino acid mixture. The higher nitrogen absorption rate from the short-chain hydrolysate (compared with the longer chain hydrolysate) was also mirrored by water, but not sodium, absorption rates (Table 4). In contrast, TABLE 3. Percentage absorption of amino acid residues during jejunal perfusion of the two lactalbumin hydrolysates and their equivalent amino acid mixtures Results are expressed as percentage absorption of infused load (means k SEM, n = 6). * P < 0.05, **P< 0.01, hydrolysate compared with free amino acid mixture; t P < 0.05, tt P < 0.01, hydrolysate compared with hydrolysate. Low lactalbumin (LH1) Hydrolysate Asx Thr Ser Glx Pro GlY Ala Val CYS Met Ile Leu TYr Phe His LYS Arg 40 f 6**tt 46 f 6*tt 50 -I 5**tt 30 f 3**t 55+2(n=3) 40 f 4*t 57 f 5**tt 65 f 6*tt 27f5 69 f7tt 67 f 7tt 70 f 6*tt 48 f loft 64 f 5**tt 46 f3*tt 44 f5t 28f9 Free amino acid mixture 1457 21 f 2 26f2 14f2 60+9 29f6 39f4 40f4 30f9 62f8 63f5 57f5 3355 32f2 19f8 39f3 28 f14 Higher lactalbumin (LH2) Hydrolysate 14f2 18f3 17f3 14fl 26+9(n=3) 15+2* 17 f 2* 21f5 17f6 19f5 17 f 4* 18-+2* 19f2 25f9 25f5 17f4 33 f13 Free amino acid mixture 20f3 26f4 29f3 14f4 53f16 40f3 46f3 44f7 11f3 51 f 16 64fll 64f4 31 f 15 37f7 21 f 10 39f8 30f9 G. K . Grimble et al. 68 TABLE 4.Absorption of nitrogen, sodium and water f.om two lactalbumin hydrolysales and their equivalent amino acid mixtures All values are expressed as means ~ S E M ,n = 6. * P < 0.05,**P< 0.02, ***P< 0.01, hydrolysate compared with free amino acid mixture; t P < 0.05, t t P < 0.01, hydrolysate compared with hydrolysate. ~~ Solution Rate of absorption from test segment Nitrogen ("4 LH 1 Amino acid mixture LH2 Amino acid mixture 37.7 f2.89tt 31.3 f3.56 12.1 k 3.12***tt 31.1 f4.24*** sodium uptake from both hydrolysates was greater than from their equivalent amino acid solution, whilst water absorption from LH2 was less than from its equivalent amino acid solution. Discussion In earlier intestinal perfusion experiments, differences were found in the handling of four different protein hydrolysates [S, 6, 81. Although it seemed, at the time, that there could be a number of explanations for the differences (varying amino acid composition of the starter proteins, different peptide chain lengths and hydrolysis method used), direct comparisons of the data could not be made as the experimental conditions differed in some respects [8]. More recently in a controlled study, the native protein and hydrolysis method were both found to influence absorptive profiles of protein hydrolysates [9]. In the present study we have shown that the chain length of the constituent peptides also has an important influence on the absorptive properties of partial enzymic hydrolysates in man. In this case, 14 out of 17 amino acid residues measured were absorbed significantly faster from the low, as compared with the higher, molecular weight lactalbumin hydrolysate and a similar result was obtained for total nitrogen absorption. There was also better absorption of most of the amino acid residues from the low molecular weight lactalbumin hydrolysate (LH1) than from its free amino acid mixture. This would suggest that a significant quantity of amino acid nitrogen in LH1 was absorbed in the form of unhydrolysed peptides [ 171. Recent evidence suggests that uptake of unhydrolysed peptides is restricted to those containing two, three and possibly four amino acid residues [l-3,181and it would appear likely that the component of LH2 with a chain length greater than four required hydrolysis at the luminal surface of the Sodium Water (mmol h" 25 an-') (mlh-' 25 cm-') 18 f lo** 6 f5** 16 f 13*** 6 f4*** 232 f 16t 196f 16 107 f 14*+ 208 f 10* jejunal mucosa by brush-border peptide hydrolases, or absorbed pancreatic proteases, before absorption [19, 201. In contrast to the results of the perfusions with LH1, 13 residues were absorbed at similar rates from LH2 and its free amino acid mixture and the remaining four were actually absorbed faster from the amino acid mixture. The most likely reason for these differences is that in the absence of luminal hydrolysis it is the kinetics of brush border hydrolysis, rather than the kinetics of free amino acid or peptide transport, that controls the overall rate of uptake of amino acid nitrogen from partial enzymic hydrolysates of whole protein. These findings have implications where maximal nitrogen assimilation is required in the rare clinical situations where both luminal hydrolysis and functional absorptive surface area are substantially reduced [21].Thus consideration should be given to administering protein hydrolysates with shorter, rather than longer, peptide chain lengths, thereby avoiding the need for brush border peptide hydrolysis. It should be appreciated, however, that higher loads of amino acid nitrogen were administered during the present perfusion studies (approx. 1400 pmol/min) than are likely to be given during continuous 24 h enteral feeding (approx. 400 pmoV min) [22].Further studies are therefore required before firm recommendations can be made about what constitutes the most 'physiologically based' peptide nitrogen source for use in the so-called 'chemically defined' pre-digested diets. References 1. Matthews, D.M. & Payne, J.W. (1980) Transmemb r a e transport of small peptides. Current Topics in Membranes and Transport, 14, 331-425. 2. Matthews, D.M. & Adibi, S.A. (1976) Peptide absorption. Gastroenterology,71, 151-161. 3. Silk, D.B.A. (1981) Peptide transport. Clinical Science, 60,607-615. Peptide chain length and jejunal absorption 4. Burston, D., Taylor, E. & Matthews, D.M. (1980) Kinetics of uptake of lysine and lysyl-lysine by hamster jejunum in vitro. Clinical Science, 59,285-287. 5. Silk,D.B.A., Marrs, T.C., Addison, J.M., Burston, D., Clark, M.L. & Matthews, D.M. (1973) Absorption of amino acids from an amino acid mixture simulating casein and a tryptic hydrolysate of casein in man. Clinical Science and Molecular Medicine, 45, 715-719. 6. Silk, D.B.A., Clark, M.L., Marrs, T.C., Addison, J.M., Burston, D., Matthews, D.M. & Clegg, K.M. (1975) Jejunal absorption of an amino acid mixture simulating casein and an enzymic hydrolysate of casein prepared for oral administration to normal adults. British Journal of Nutrition, 33, 95-100. 7. Hegarty, J.E., Fairclough, P.D., Moriarty, K.J., Kelly, M.J. & Clark, M.L. (1982) Effects of concentration on in vivo absorption of a peptide-containing protein hydrolysate. Gut, 23, 304-309. 8. Silk, D.B.A., Fairclough, P.D., Clark, M.L., Hegarty, J.E., Marrs, T.C., Addison, J.M., Burston, D., Clegg, K.M. & Matthews, D.M. (1980) Uses of a peptide rather than a free amino acid nitrogen source in chemically defined elemental diets. Journal of Parenteral and Enteral Nutrition, 4,548-553. 9. Keohane, P.P., Grimble, G.K., Brown, B., Spiller, R.C. & Silk, D.B.A. (1985) Influence of protein composition and hydrolysis method on intestinal absorption of protein in man. Gut, 26,907-913. 10. Rothenbuhler, E., Waibel, R. & Solms, J. (1979) An improved method for the separation of peptides and alpha-amino acids on copper Sephadex. Analytical Biochemistry, 91,367-375. 11. Habeeb, A.F.S.A. (1966) Determination of free amino acid groups in proteins by trinitrobenzenesulfonic acid. Analytical Biochemistry, 14, 328-336. 12. Nehring, H., Rustow, B. & Hock, A. (1971) Kritische betrachtungen zur quantitativen bestimmung von aminosauren und niederen peptiden in einem gemisch der beiden stoffgrupen. Pharmazie, 26, 449-455. 69 13. Chen, M.L., Rogers, Q.R. & Harper, A.E. (1962) Observations on protein digestion in vivo. IV. Further observation of the gastrointestinal contents of rats fed different dietary proteins. Journal of Nutrition, 16,235-241. 14 Sladen, G.E. & Dawson, A.M. (1970) Further studies on the perfusion method for measuring intestinal absorption in man. The effects of a proximal occlusive balloon and a mixing segment. Gut, 11, 947-954. 15. Wingate, D.L., Sandberg, R.J. & Phillips, S.F. (1972) A comparison of stable and ''C-labelled polyethylene glycol as volume indicators in the human jejunum. Gut, 13,812-814. 16. Silk, D.B.A., Perrett, D. & Clark, M.L. (1973) Intestinal transport of two dipeptides containing the same two neutral amino acids in man. Clinical Science and Molecular Medicine, 45,291-299. 17. Boyd, C.A.R. & Ward, M.R. (1982) A microelectrode study of oligopeptide absorption by the small intestinal epithelium of Necturus maculosus. Journal of Physiology(London), 324,411-428. 18. Chung, Y.C., Silk, D.B.A. & Kim, Y.S. (1979) Intestinal transport of a tetrapeptide, L-leucyl-glycyl-glycylglycine, in rat small intestine in vivo. Clinical Science, 51, 1-11. 19. Smithson, K.W. & Gray, G.M. (1977) Intestinal assimilation of a tetrapeptide in the rat. Obligate function of brush-border membrane aminopeptidases. Journal of Clinical Investigation, 60,665-674. 20. Adibi, S.A. & Morse, E.L. (1977)The number of glycine residues which limits intact absorption of glycine oligopeptides in human jejeunum. Journal of Clinical Investigation, 60,1008-1016. 21. Koretz, R.L. (1984) What supports nutritional support? Digestive Diseases and Science, 29,577-588. 22. Silk, D.B.A., Grimble, G.K. & Rees, R.G. (1985) Protein digestion and amino acid and peptide absorption. Proceedings of the Nutrition Society, 44, 63-72.