JEJUNAL AND ILEAL ABSORPTION OF DIBASIC AMINO ACIDS AND AN ARGININE-CONTAINING DIPEPTIDE IN CYSTINURIA

GASTROENTEROLOGY 68:1426-1432, 1975 Copyright 1975 by The Williams & Wilkins Co. Vol. 68, No.6 Printed in U.S.A JEJUNAL AND ILEAL ABSORPTION OF DIBASIC AMINO ACIDS AND AN ARGININE-CONTAINING DIPEPTIDE IN CYSTINURIA D. B. A. SILK, M.D., M.R.C.P., D. PERRETT, B.Sc., AND M. L. CLARK, M.D., M.R.C.P Medical Unit, Department of Gastroenterology, St. Bartholomew's Hospital, and St. Leonard's Hospital, London, England Ileal transport of dibasic amino acids has not previosly been stdied in the intestine of healthy volnteers or cystinric patients. Experiments have therefore been designed to compare ileal and jejnal absorption of lysine and arginine both in normal sbjects and cystinric patients. In addition, jejnal perfsion experiments have been carried ot to investigate absorption of the dipeptide L-arginyl-L-lecine. The reslts indicate that, at the concentrations stdied (4.2 mm lysine, 1 mm arginine), severe transport defects exist throghot the whole small intestine for both amino acids in cystinria. Despite the transport defect for free arginine, cystinric patients absorbed the dipeptide L-arginyl-L-lecine normally. Becase of the transport defect for free arginine, it has been possible to show that dring absorption of L-arginyl-L-lecine in cystinria approximately 3% of dipeptide-bond arginine can be recovered from the gt lmen in the free form. These findings indicate that the prime fnction of specific amino acid transport systems dring the absorption of protein digestion prodcts may be as a "recaptre mechanism" for amino acids liberated as a reslt of mcosal cell peptide hydrolysis. Cystinria is de to an atosomal recessive hereditary defect which cases impaired transport of cystine and the dibasic amino acids lysine, arginine, and ornithine by the proximal tblar cells of the kidney. 1 ' 3 Defects in small gt transfer of these dibasic amino acids were demonstrated by oral tolerance techniqes,. 5 indicating that the sitation in cystinria is analogos to that of Hartnp disease, 6 Received Jly 23, 1974. Accepted December 4, 1974. The athors are gratefl to the North East Thames Regional Health Athority and the Medical College of St. Bartholomew's Hospital for financial spport. The athors wold like to thank Professor Sir Eric Scowen for allowing them to stdy patients nder his care, for the se of his laboratory facilities, and for his helpfl discssion dring the corse of the stdy. Dr. A. D. Stephens gave mch help in arranging the stdies, and the athors wold like to thank Dr. A. M. Dawson for his contined encoragement and for the se of his laboratory facilities. 1426 i.e., a genetically determined transport defect affecting both the gt and kidney. In vitro stdies with intestinal mcosa obtained by peroral biopsy from cystinric patients frther spported this concept. 7 8 Recently, however, oral tolerance crves sggest that, althogh a transport defect exists for free arginine, lysine absorption is naffected; 9 1 perfsion stdies carried ot in the jejnm of cystinric patients, however, showed severe impairment of lysine absorption, 11 and it has been sggested that alternative ileal transport mechanisms are sed for lysine absorption in cystinria. 9 In the present stdy we have therefore investigated the possibility of ileal absorption of lysine by sing a perfsion techniqe. Ileal as well as jejnal perfsions with the dibasic amino acids lysine and arginine have been carried ot. Patients with cystinria provide an opportnity to evalate the role of the specific dibasic amino acid transport system

Jne 1974 AMINO ACID AND DIPEPTIDE ABSORPTION 1427 dring normal absorption of dipeptides which contain a dibasic amino acid reside. This is becase dring perfsion of sch a dipeptide any free dibasic amino acids that diffse back into the gt lmen will accmlate rather than be absorbed. Accordingly, jejnal perfsion stdies with an arginine-containing dipeptide, L-arginyl-L-lecine, have been performed. Methods Sbjects. Six adlt homoygos cystinric patients gave their informed consent to jejnal perfsion stdies. Three of these patients also gave their informed consent to ileal perfsion stdies. Normal control sbjects were healthy yong adlt males with no gastrointestinal symptoms. Three of these sbjects also gave their informed consent to ileal perfsion stdies (controls were matched for sex and, as near as possible, for age). Each of the cystinric patients had previosly developed renal calcli, and an excess of cystine and dibasic amino acids was demonstrated in the rine of each of the cystinric patients dring the crrent hospital admission when the intestinal absorption stdy was carried ot. Each was receiving inpatient d-penicillamine therapy which was stopped 24 hr before the stdy. No d-penicillamine or any of its metabolic prodcts was detected in the gt contents aspirated dring the perfsion procedre. Experimental procedre. Each cystinric patient and control sbject was intbated with a doble-lmen perfsion tbe incorporating a proximal occlsive balloon as previosly described. 12 13 The tbe was allowed to pass ntil the 3-cm perfsion segment was positioned in the pper jejnm. The final position of the tbe was checked floroscopically so that the infsion orifice was sitated in the 1-cm segment of pper jejnm beyond the dodenojejnal flexre. A proximal occlsive balloon was incorporated in the perfsion tbe to prevent both reflx of the infsed soltions from the perfsion segment and contamination of the infsed soltion within the perfsed segment by proximal secretions, in particlar by proteolytic pancreatic enymes. The soltions were infsed at a rate of 15 ml per min throgh the infsion orifice with a peristaltic pmp (H. R. Flow Indcer, Watson-Marlow, Ltd., Marlow, England) from bottles maintained at 37 C in a water bath. The first 3 min of each perfsion period was allowed for the attainment of a steady state of absorption rates. Preliminary stdies indicated that this interval was sfficient for this prpose. 14 Three 1-min samples were then collected by simple siphonage into plastic bottles containing sfficient slfosalicylic acid crystals to prodce a final ph of approximately 2.. Initial experiments indicated that this procedre inactivates any peptidase activity present. 14 After collection the aspirated samples were filtered throgh Whatman no. 1 filter paper, and the filtrates were stored at - 2 C ntil analysis. After the jejnal perfsions had been completed, the sbjects ndergoing ileal perfsion were permitted oral flids and a light diet ntil midnight. Dring this period the perfsion tbe was allowed to pass distally throgh the small intestine. Ileal perfsion experiments were carried ot the following morning, at which time the mercry bag was sitated 19 to 21 em from the moth of each sbject. The final position of the tbe was checked radiologically to confirm that the infsion orifice appeared to lie in the distal small intestine. Experimental design. Two types of experiments were carried ot. In the first, amino acid absorption from the jejnm and ilem of the cystinric patients was assessed and the reslts were compared with those obtained in normal sbjects. For these stdies soltions containing either glycine ( 4.2 mm) + lysine ( 4.2 mm) or arginine (1 mm) + lecine (1 rm) were perfsed. In the second experiment jejnal absorption of the dipeptide L-arginyl-L-lecine was investigated by perfsing a soltion containing L-arginyl-L-lecine (1 mm) in the jejnm of the cystinric sbjects and normal controls. - Soltions. The amino acids, glycine, L-lysine, L-arginine, and L-lecine, were obtained from Sigma (London) Chemical Company, London, England. The dipeptide L-arginyl-L-lecine was manfactred by Bachem Feinchemikalien AG and spplied by Micro-Bio Laboratories, Ltd., London. All soltions were made isotonic by adding sodim chloride and contained the nonabsorbable marker polyethylene glycol at a concentration of 2.5 g per liter. The ph of all soltions was adjsted to ph 7. by titrating with either molar sodim hydroxide soltion or molar hydrochloric acid. Chemical analysis. The polyethylene glycol content of the perfsion soltions and the intestinal aspirates were determined by the method of Hyden, 15 trbidity being measred by sing a Pye Unicam colorimeter spectrophotometer 13. The amino acid content of the perfsion soltions and intestinal aspirates were qantified with a Technicon NC-1 amino acid analyer sing the bffer system of Prdie et al. 16 Using this system, the dipeptide L-arginyl-L-le-

1428 SILK ETAL. Vol. 68, No.6 cine was fond to be retained on the colmn. A rapid short-colmn system was developed to qantitate the dipeptide content of the samples. A Pyrex colmn (22 by.63 em) maintained at 6 ±.5 C was packed with Technicon Chromobeads type B (bed height, 18 em). The colmn was elted with.25 M sodim citrate bffer (ph 5.25) at a flow rate of 4 ml per min. Under these experimental conditions the dipeptide was elted in 6 min. Calclation of reslts. The absorption rates of the amino acids from the perfsion soltions containing free amino acids and the net amino acid absorption rates from the dipeptide soltion were calclated from formlas previosly described. 17 18 The statistical significances of differences in absorption rates were evalated by the npaired t-test. 19 Reslts Amino Acid Absorption The rates of amino acid absorption from the amino acid soltions perfsed in the jejnm and ilem are shown in figres 1 to4. Jejnm. The two netral amino acids glycine and L-lecine were absorbed normally by the cystinric patients compared with the controls (figs. 1 and 2), whereas there was negligible absorption of both the dibasic amino acids L-lysine and L-arginine (P <.1) dring jejnal perfsion of each cystinric patient with the two amino acid soltions (figs. 1 and 2). Ilem. As in the jejnm, the netral amino acids glycine and L-lecine were absorbed normally by the cystinric patients, whereas there was severe impairment of absorption of both the dibasic amino acids L-lysine and L-arginine dring perfsion of each of the cystinric patients (figs. 3 and 4). Absorption of Amino Acid Resides of L-Arginyl- -lecine The rates of amino acid absorption dring jejnal perfsion of the dipeptide L-arginyl-L-lecine are shown in figre 5, and the concentrations of free amino acids 25 2 "E' l 15 1 ;;; g :;; -5 CYST I NUR IC PATIENTS FIG. 2. Jejnal absorption of arginine and lecine dring the perfsions with the soltion containing arginine (1 mm) + lecine (1 mm). Reslts of stdies carried ot in 6 normal sbjects and 6 cystinric patients. 6 5 'c:. E 4. E =>. - 3 2 " 1 " " -1 GLYCINE I LYSINE GLYCINE FIG. 1. Jejnal absorption of glycine and lysine dring the perfsions with the soltion containing glycine (4.2 mm) + lysine ( 4.2 mm). Reslts of stdies carried ot in 6 normal sbjects and 3 cystinric patients. LYSINE

Jne 1974 AMINO ACID AND DIPEPTIDE ABSORPTION 1429 6. 5 5 "c:.e 4 E =< - 3... c.. "" 2 Q 1 :::< -1 GLYCINE LYSINE GLYCINE LYSINE FIG. 3. Ileal absorption of glycine and lysine dring the perfsions with the soltion containing glycine (4.2 mm) +lysine (4.2 mm). Reslts of stdies carried ot in 3 normal sbjects and 3 cystinric patients. 15 25 1 c.e 15 1 5 FIG. 4. Ileal absorption of arginine and lecine dring the perfsions with the soltion containing arginine (1 mm) + lecine (1 mm). Reslts of stdies carried ot in 3 normal sbjects and 3 cystinric patients. detected in the lminal contents aspirated dring the dipeptide perfsions are shown in figre 6. The rates of ptake of both L-arginine and L-lecine were similar dring perfsion of the dipeptide in the cystinric patients compared with the normal control sbjects (fig. 5). Althogh similar concentrations of free L-lecine were detected in the lminal contents aspirated dring dipeptide perfsion of the cystinric patients and the normal control sbjects, higher concentrations of free L arginine were detected dring dipeptide perfsion of the cystinrics (26.7 ± 5.7; n = 6) compared with the controls (7.6 ± 1 "" or------------------- FIG. 5. Jejnal absorption of amino acid resides of L-arginyl-L-lecine dring the perfsions with the soltion containing L-arginyl-L-lecine (1 mm). Reslts of stdies carried ot in 6 normal sbjects and 6 cystinric patients. 2.4; n = 6; P <.2; the vales are the mean ± 1 SEM expressed as micromoles per 1 ml). Discssion Hellier et al. 1 sed the same perfsion techniqe to stdy jejnal absorption of lysine and arginine at six different concentrations in normal hman sbjects. Using the same flow rate and segment length as we have sed in the present stdies, they fond evidence for a satrable transport system for both lysine and arginine. When the reciprocals of absorption were plotted against the reciprocals of sb-

143 SILK ETAL. Vol. 68, No.6 6 5 4 E =>. - 3 2 1.. FIG. 6. Concentrations of free arginine and lecine detected in the lminal contents aspirated dring jejnal perfsion with the soltion containing L-arginyl-L-lecine (1 mm). Reslts of stdies carried ot in 6 normal sbjects and 6 cystinric patients. strate concentration (initial concentrations of amino acid in the test soltions), according to Lineweaver and Brk, K 1 vales for lysine and arginine were fond to be 4.9 and 3.8 mm, respectively. Affinity constants derived in sch a way dring in vivo perfsions provide at best only sefl approximations for the over-all system becase of the technical pitfalls involved. 2 It is likely thogh, at the concentrations perfsed in the present stdy (lysine, 4.2 mm; arginine, 1 mm), that we have stdied specific carrier-mediated transport systems rather than passive diffsion, which cold become significant at higher concentrations. The present reslts that show severe impairment of lysine and arginine ptake from the ilem as well as the jejnm of the cystinric patients sggest that there is a defect of specific carrier-mediated transport of the dibasic amino acids throghot the whole length of the small intestine of the cystinric patients. Asatoor et al. 9 thoght that the reason why lysine appeared to be absorbed normally when fed to cystinric patients, despite the fact that jejnal perfsion stdies indicated a severe transport defect, was that cystinric patients partly reply on transport in distal lengths of gt. Or reslts indicate that this explanation for the discrepancy is nlikely, for nder the present experimental conditions lysine absorption was impaired in the cystinric ilem as well as in the jejnm. A more likely explanation for the discrepancy is that lysine absorption has been stdied at different concentrations by the oral load and perfsion methods. Higher concentrations of lysine are presented to the mcosa for absorption dring the oral load stdies 9 1 than dring the present perfsion stdies. It is qite possible that, dring the oral load tests, the presentation of mch higher concentrations of lysine to the mcosa reslted in absorption by nonspecific diffsion or other amino acid transport systems with overlapping specifities. 21 It is necessary to carry ot segmental intestinal perfsion stdies at increasing lysine concentrations in cystinric patients before this qestion can be answered; nfortnately, it was not possible to perfse additional soltions dring the present stdy. The reslts of the dipeptide perfsions confirmed that, despite severe impairment of arginine ptake from the free amino acid soltion, the grop of patients with cystinria exhibited normal ptake of arginine from the dipeptide soltion. Dring the corse of the present stdy, normal absorption of another arginine-containing dipeptide, L-arginy l-l-as partate, has been demonstrated in a single cysti-

Jne 1974 AMINO ACID AND DIPEPTIDE ABSORPTION 1431 nric patient, despite severe malabsorption of free arginine. 22 Normal absorption of arginine-containing dipeptides cold explain why normal serm arginine increments were detected after Casilan (a palatable sorce of casein; Glaxo Laboratories, Ltd., Greenford, Middlesex, England) was administered orally to 3 cystinric patients; whereas lower serm arginine increments were detected after a mixtre of free amino acids simlating the amino acid composition of Casilan had been administered. 9 The ntritional significance of these findings with arginine-containing dipeptides is nclear. Despite the loss of active transport of free arginine, cystinric patients show no obvios ntritional defect. L-Arginyl-L-lecine (1 mm) was avidly absorbed by the cystinric patients dring the present stdy, so that peptide-bond transport of arginine per se may compensate for the loss of active transport of free arginine. Normally, free arginine is rapidly released from polypeptide chains by the combined action of trypsin and carboxypeptidase B. 23 Recently, Adibi and Mercer 24 have shown that high concentrations of free arginine can be detected in the jejnal contents aspirated after oral ingestion of a protein test meal to normal hman sbjects. Althogh the data presented by Adibi and Mercer 24 do not distingish between active and passive absorption of free arginine, if high enogh concentrations of free arginine accmlate dring normal digestion in vivo, it is possible that a certain proportion may be absorbed passively. Ths the loss of active transport of arginine in cystinria may be compensated not only by normal peptidebond arginine absorption bt also by passive diffsion of free arginine. The presence of free amino acids in the lminal contents aspirated dring the perfsion of the normal sbjects with L arginyl-l-lecine indicates that some dipeptide hydrolysis occrs dring absorption. Similar observations have been made dring intestinal perfsion of normal sbjects with other dipeptides. 12-14 18 25 In normal sbjects it has never been possible to assess, in qantitative terms, the proportion of dipeptide-bond amino acids that is finally absorbed by specific amino acid transport systems, becase the liberated amino acids are so efficiently recaptred. Higher concentrations of free arginine were detected at the collection site dring L-arginyl-L-lecine perfsion in the cystinric sbjects (fig. 6). In cystinria, free dibasic amino acids liberated dring absorption of dipeptides containing dibasic amino acid resides accmlate in the gt lmen on accont of the transport defects for dibasic amino acids (figs. 1 and 2), and patients with cystinria provide an opportnity to evalate the role of specific dibasic amino acid transport systems dring absorption of sch dipeptides. Ths, dring perfsion of 1 mm L-arginyl L-lecine in the cystinric sbjects,.8 mm intact dipeptide "disappeared," while.27 mm free arginine was recovered in the lminal contents (vales are the mean, n = 6). If there had not been an absorptive defect for free arginine and complete absorption of free arginine had occrred, it follows that 33.8% of the dipeptidebond arginine wold finally have been absorbed in the free form by specific basic amino acid transport systems. This approximate calclation from the cystinric data indicates that in normal sbjects a significant proportion of dipeptide-bond amino acids may be finally absorbed by specific amino acid transport systems. The conclsions drawn from the present stdy, that specific amino acid transport systems may play an important role dring intestinal transport of peptides, raise the qestion as to whether the prime fnction of specific amino acid transport systems dring absorption of protein digestion prodcts is as a recaptre mechanism for amino acids liberated as a reslt of mcosal cell peptide hydrolysis or whether the main fnction is to absorb amino acids released as a reslt of intralr'linal hydrolysis of dietary protein. This qestion will probably remain nanswered becase it is only in sitations sch as Hartnp disease and cystinria that the importance of peptide hydrolysis dring transport can be readily highlighted.

1432 SILK ETAL. Vol. 68, No.6 REFERENCES 1. Harris H, Mittwoch U, Robson EB, et a!: The pattern of amino acid excretion in cystinria. Ann Hm Genet 19:196-28, 1955 2. Harris H, Mittwoch U, Robson EB, eta!: Phenotypes and genotypes in cystinria. Ann Hm Genet 2:57-91, 1955 3. Dent CE, Rose GA: Amino acid metabolism in cystinria. Q J Med 2:25-219, 1951 4. Milne MD, Asatoor AM, Edwards KDG, et a!: The intestinal absorption defect in cystinria. Gt 2:323-337, 1961 5. Asatoor AM, Lacey BW, London DR, eta!: Amino acid metabolism in cystinria. Clin Sci 23:285-34, 1962 6. Milne MD: Disorders of intestinal amino acid transport. J Clin Pathol (sppl) 5:41-44, 1971 7. McCarthy CF, Borland JL, Lynch HJ, et a!: Defective ptake of basic amino acids and L-cystine by intestinal mcosa of patients with cystinria. J Clin Invest 43:1518-1524, 1964 8. Thier SO, Segal S, Fox M, et a!: Cystinria: Defective intestinal transport of dibasic amino acids and cystine. J Clin Invest 44:442-448, 1965 9. Asatoor AM, Crochman MR, Harrison AR, eta!: Intestinal absorption of oligopeptides in cystinria. Clin Sci 41:23-33, 1971 1. Hellier MD, Holdsworth CD, Perrett D: Dibasic amino acid absorption in man. Gastroenterology 65:613-618, 1973 11. Hellier MD, Holdsworth CD, Perrett D, et a!: Intestinal dipeptide transport in normal and cystinric sbjects. Clin Sci 43:659-668, 1972 12. Silk DBA, Perrett D, Clark ML: Intestinal transport of two dipeptides containing the same two netral amino acids in man. Clin Sci Mol Med 45:291-299, 1973 13. Silk DBA, Webb JPW, Lane AE, et al: Fnctional differentiation of hman jejnm and ilem. Comparison of handling of glcose, peptides and amino acids. Gt 15:444-449, 1974 14. Silk DBA: Absorption of peptides in man. MD thesis. University of London, 1974 15. Hyden S: A trbidimetric method for the determination of higher polyethylene glycols in biological materials. K Lantbrttogsk Annlr 22:139-145, 1955 16. Prdie JW, Gravelle RA, Hanafi DE: Slphr containing amino acids. II. Chromatography of dislphides and trislphides with an atomatic analyser. J Chromatogr 38:346-35, 1968 17. Holdsworth CD, Dawson AM: The absorption of monosaccharides in man. Clin Sci 27:371-379, 1964 18. Adibi SA: Intestinal absorption of dipeptides in man: relative importance of hydrolysis and intact absorption. J Clin Invest 5:2266-2275, 1971 19. Langley R: Practical statistics for non-mathematical people. London, England, Pan Books, Ltd., 1968 2. Rey F, Drillet F, Schmit J, et al: Inflence of tion of glcose and lysine in children. Gastroenterology 66:79-85, 1974 21. Holdsworth CD. Transport across the Intestine. Edited by W Brland, P Samel. Edinbrgh and London, Chrchill-Livingstone, 1972 22. Asatoor AM, Harrison BDW, Milne MD, et al: Intestinal absorption of an arginine-containing peptide in cystinria. Gt 13:95-98, 1972 23. Dixon M, Webb EC (editors). Enymes. Second edition. London, Longmans Green & Company, Ltd., 1964 24. Adibi SA, Mercer DW: Protein digestion in hman intestine as reflected in lminal, mcosal and plasma amino acid concentrations after meals. J Clin Invest 52:1586-1594, 1973 25. Hellier MD, Holdsworth CD, McColl I, et a!: Dipeptide absorption in man. Gt 13:965-969, 1972