constituent amino acids in man'

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1 Gut, 197, 11, Intestinal absorption of arnosine and its onstituent amino aids in man' A. M. ASATOOR, J. K. BANDOH2, A. F. LANT, M. D. MILN, AND F. NAVAB From the Medial Unit of the Westminster Hospital, London S U M MARY Serum onentrations of,b-alanine and L-histidine are ompared in five normal adults after ingestion of the dipeptide arnosine (/-alanyl-l-histidine) and after equivalent amounts of the onstituent free amino aids. The results indiate that absorption is signifiantly more rapid after the ingestion ofthe amino aids than after the dipeptide. The use of the test in a ase of Hartnup disease suggests that arnosine is taken up by intestinal ells as the dipeptide, but subsequent hydrolysis and delivery of the onstituent amino aids to the portal blood is a slower proess than transport of the free amino aids themselves. Current views of intestinal amino aid absorption suggest that at least two mehanisms are involved. Free amino aids are atively transported against a onentration gradient from the intestinal lumen to the portal apillaries (Wiseman, 1951) and small peptides are taken up intat from the intestinal lumen, but are hydrolysed by the peptidases of the intestinal muosal ells and appear in the portal blood or in serosal fluid as the orresponding free amino aids (Wiggans and Johnston, 1959; Newey and Smyth, 196 and 1962). videne supporting intestinal uptake of intat peptides in man has been reported for the di- and tripeptides of glyine (Craft, Geddes, Hyde, Wise, and Matthews, 1968), plasma glyine levels being onsiderably higher 3 minutes after the ingestion of eah peptide than after a orresponding amount of free glyine. This indiated a more rapid ellular uptake of peptide moleules than of the larger number of moleules of free glyine. Beause of the marked differenes in speifi aminopeptidase ativities within the mirovillous membrane of the intestinal epithelium (Rhodes, iholz and Crane, 1967), onlusions drawn from studies with glyyl-glyine may not be generally appliable to the ellular mehanism of absorption of other peptides. Quite apart 'Address for reprints: Medial Unit, Westminster Hospital, Page Street Wing, London, SWI. 'Present address: Military Hospital, Ara, Ghana. from the question of speies differenes in absorptive behaviour, the problem of oligopeptide absorption is ompliated by the fat that about 4 different dipeptides an be formed from the amino aids of proteins, and the figure beomes muh higher if tri- or higher peptides are onsidered. Investigation of peptide absorption by tolerane tests in man involves diffiulties in the supply of oligopeptides of suffiient purity, as these ompounds are in most ases diffiult to prepare in bulk. The dipeptide arnosine (f3-alanyl-lhistidine) is readily available, and is neessarily ingested in onsiderable amounts by all nonvegetarians, as mammalian musle ontains up to -6% of arnosine (Davey, 1957). This paper ompares serum levels of the onstituent amino aids, fl-alanine and L-histidine, after ingestion of the peptide and orresponding quantities of the two free amino aids. Methods Five normal adults were investigated, eah individual ingesting arnosine and equivalent amounts of a mixture of,b-alanine and L- histidine after an overnight fast. The two tolerane tests in eah subjet were arried out at Gut: first published as /gut on 1 Marh 197. Downloaded from on 23 November 218 by guest. Proteted by opyright.

2 251 Intestinal absorption of arnosine and its onstituent amino aids in man intervals of at least two weeks. Dosage of arnosine was.286 m mole/kg body weight, orresponding to 2 m mole per standard 7 kg male. Histidine and,b-alanine were taken together in an amount whih would be produed after hydrolysis of the above dose of arnosine. Both the dipeptide and the amino aid mixture were taken dissolved in 5 ml water. Blood samples were obtained at zero time, and at 15, 3, and 45 minutes after ingestion of the solution. Serum for analysis was obtained with the minimum of delay. In one subjet additional samples were analysed at 6 and 9 minutes after the mixture. MATRIALS AND CHMICAL MTHODS Amino aids were analysed on the Tehnion amino aid analyser using the standard proedure desribed in the Tehnion Handbook (1966). Carnosine, fl-alanine, and L-histidine were obtained from ommerial soures and were found to be hromatographially pure. Results Peripheral paraesthesiae ourred in all subjets from about 15 to 45 minutes after ingestion of arnosine and of the mixture of the two amino aids, but otherwise no abnormal symptoms ourred. Sine similar effets have not been desribed after ingestion of larger quantities of histidine alone, the paraesthesiae were presumably due either to,b-alanine or to one of its metabolites. No P-alanine was deteted in the basal serum speimens, but histidine onentration averaged 11.2,umoles/1 ml (range ). All values for histidine in subsequent serum speimens are reorded as inrements e above the basal onentration. Carnosine was not deteted in any serum sample. Tolerane urves in the subjet in whom Z additional serum samples were analysed are e given in Figures 1 and 2. Peak onentrations of,b-alanine ourred in the sample taken after 3 minutes, and of histidine in the 45-minute speimen. In the period of 15 to 3 minutes after the drink the maximum rate of inrease in serum amino aid onentration was 1-25 times as rapid for,b-alanine as for histidine in both tolerane tests. Similarly, the subsequent rate of deline after the peak onentrations was greater in the ase of,-alanine. Comparing the two tolerane urves, the rate of rise of both serum,b-alanine and of histidine in the 15 to 3-minute period was 1-45 times as great after the free amino aids than after arnosine. The individual results of the tolerane tests are given in Table I, and the means of onentrations from the five subjets in Figures 3 and 4. In 19 out of 2 ases inrements of serum histidine 1- w- -._ I 7O 3 6 Histidine + (3-Alandine Fig. 1 Conentrations of serum histidine after ingestion of arnosine and the onstituent free amino aids in a normal subjet. _ 1.- ) I'L 9 9 Fig. 2 Conentrations of serum fi-alanine after ingestion of arnosine and the onstituent free amino aids in a normal subjet. Gut: first published as /gut on 1 Marh 197. Downloaded from on 23 November 218 by guest. Proteted by opyright.

3 252 Histidine at 1 3 minutes 2 A. M. Asatoor, J. K. Bandoh, A. F. Lant, M. D. Milne, and F. Navab and of fi-alanine in the same subjet at 3 and 45 6 minutes after the drink were greater after ingestion of the free amino aids than after arnosine. In the remaining analysis the result was idential. This ould have ourred by hane 2 His alone at a probability value of one in 4 times. As the same individuals were used in both toler- ane tests, the method of the paired t test (Snedeor, 1946) is statistially valid. Using this o 4 tehnique the inrements of serum amino aids are signifiantly higher after ingestion of the free _ amino aids in the 3- and 45-minute samples for a} histidine and in the 3-minute sample for - /-alanine (Table I). In the 45-minute samples for 3-alanine, the results are lower after amosine ingestion, but the differene is not signifiant t 2 (-1 > P > 5). The peak inrement of /3- alanine ourred at 3 minutes in three of the five subjets, and therefore the 45-minute sample in this ase is not a satisfatory representation of * the absorption rate of the amino aid. It an be.o onluded that absorption of both,b-alanine I and of histidine is signifiantly more rapid after ingestion of the free amino aids than after ingestion of the equivalent amount of arnosine. SubJet Free Amino Carnosine Aids (Xi) (X*) S Histidine at 1 45 minutes Total Mean 3. t = 4.3 S fi-alanine at I 3 minutes Total 26- Mean 41-2 t = alanine at 1 45 minutes I5 Total Mean 35.5 t = Total Mean 36.4 t = (X, - X) *7 x = 8-3 p < Deviation (X,-X,-) i i=99 P < x= 1-2 P < t= < P < _ Table I Inrements in serum histidine and,balanine after ingestion of arnosine andfree onstituent amino aids (,gmole/j ml) Squared Deviations S' = ' = S' = = 1159 Fig. 3 Mean serum onentrations of histidine during a 45-minute period after ingestion of arnosine subjets. a I- o In U) 3) Fig. 4 Mean serum onentrations of fi-alanine during a 45-minute period after ingestion of arnosine subjets. Gut: first published as /gut on 1 Marh 197. Downloaded from on 23 November 218 by guest. Proteted by opyright.

4 253 Intestinal absorption of arnosine and its onstituent amino aids in man Serum Histidine Serum,3-analine (,u moles/1 mil/min) (ps moles/io ml/min) Subjet After Free After After Free After Amino Aids Carnosine Amino Aids Carnosine Mean normal 1-66 ± ± ± ± 85 Hartnup disease Table II Maximum rates of inrement of serum histidine and,-alanine after ingestion of arnosine subjets and a ase of Hartnup disease Disussion Comparisons of the absorption of amino aids after ingestion of arnosine and of the equivalent amounts of free /3-alanine and histidine in this investigation are the opposite of those obtained by Craft et al (1968), omparing the rates of absorption of free glyine and of the peptides glyyl-glyine and glyyl-glyyl-glyine. We have onfirmed the latter results using the more exat method of ion-exhange hromatography (Tehnion Handbook, 1966). In addition, urinary exretion of free glyine in the 45-minute period after ingestion of glyyl-glyine is approximately twie as high as that after ingestion of the equivalent amount of free glyine, a result to be expeted when, after ingestion of the peptide, serum levels of the amino aid are onsistently higher during this period. A superfiial and obvious interpretation of the onfliting results would be that there is a fundamental differene between the intestinal absorption of the glyine peptides and that of arnosine. The glyine peptides are presumably taken up intat from the gut lumen and subsequently undergo hydrolysis with delivery of free glyine into the portal apillary blood. The whole proess is more rapid than that involved in the orresponding absorption of twie or three times the number of free glyine moleules. By ontrast, the present results might apparently favour the view that arnosine is hydrolysed in the fluid within the intestinal lumen and the derived amino aids are subsequently absorbed, a proess obviously less rapid than absorption of equivalent amounts of the free amino aids not neessitating prior hydrolysis. A repetition of the two tolerane urves in a ase of Hartnup disease to be reported in detail elsewhere (Navab and Asatoor, 197) has, however, shown that this interpretation is almost ertainly inorret. The urrent view of the disordered physiology of Hartnup disease is one of defetive transport of many mono-amino mono-arboxyli amino aids both in the proximal renal tubular ells and in the jejunal epithelium (Milne, Crawford, Girao, and Loughridge, 196;Jepson, 1966).Although not absolutely proven for every single involved amino aid, all the amino aids exreted at abnormally high learane by the kidney are probably poorly absorbed in the jejunum. Clearanes of histidine in Hartnup disease are at the level of the glomerular filtration rate, and are in fat higher than those of any other of the involved amino aids (vered, 1956). By ontrast, /3-alanine, a member of a separate amino aid transport group (Sriver, Pueshel, and Davies, 1966; de la Noue, Newey, and Smyth, 1969), is not exreted in exess in Hartnup disease. Jejunal absorption of,b-alanine is, therefore, likely to be normal, and that of L-histidine to be defetive in Hartnup disease. Table II gives the maximum rate of serum amino aid inrement, during the absorptive phase in the ase of Hartnup disease and in the normal subjets. 3-Alanine absorption is seen to be within normal limits after both arnosine and free amino aid ingestion, whereas histidine absorption in Hartnup disease is normal after arnosine but grossly defetive after ingestion of the free amino aid. The result may explain the reason why ases of Hartnup disease preserve an almost normal nutritional status despite gross defets in absorption of many essential amino aids. Thus, tolerane urves foj the essential amino aid phenylalanine were almost ompletely flat in this ase of the disease, and absorption of the free amino aid would be ompletely inadequate to sustain life. Probably this and other essential amino aids are absorbed mainly as oligopeptides in Hartnup disease, whereas both free amino aid and oligopeptide are absorbed in normal subjets. A possible explanation of the results of this investigation is that arnosine is absorbed as the entire moleule, but that subsequent intraellular hydrolysis is relatively slow, and is in fat the rate-limiting step in the total absorptive proess. By ontrast, intraellular hydrolysis of the glyine peptides must be more rapid and does not ause a signifiant delay in the transport rate. Full onfirmation of this interpretation obviously depends on more omplete knowledge of the enzyme kinetis of human intestinal peptidases hydrolysing glyine peptides and arnosine. Glyyl-glyine dipeptidase is a very speifi enzyme, and is ativated by Co++ or Mn++ (Smith, 1951). Carnosinase hydrolyses glyyl-l-histidine, L-alanyl-, L-histidine, P-L-aspartyl- L-histidine, and anserine (f-alanyl-t-methyl-histidine) in addition to amosine, and requires Zn++ or Mn++ for ativity (Hanson and Smith, 1949; Davis, 1956). Similar investigations using these alternative peptides would be both more diffiult and more expensive beause of lak of availability of the hemials in pure and bulk supply. The two peptidases are -widely distributed in animal tissues, and glyyl-glyine dipeptidase is present in high onentration in intestinal muosa. Camosinase has been deteted in onsiderable amount in the intestinal wall of Gut: first published as /gut on 1 Marh 197. Downloaded from on 23 November 218 by guest. 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5 254 A. M. Asatoor, J. K. Bandoh, A. F. Lant, M. D. Milne, and F. Navab the rat (Wood, 1957). Carnosine in the dog is absorbed only after hydrolysis either in the intestinal lumen or in the jejunal ells, but re-synthesis of arnosine ours in the liver, and arnosine is detetable in the plasma of this speies (lwyn, Parikh, and Shoemaker, 1968). Results obtained in the experimental animal may not neessarily be appliable to man. Probably the main importane of this paper is to draw attention to the unertainties whih fae any interpretation of tolerane tests as indies of intestinal absorption in man. If it an be shown, as in the ase of glyine peptides, that plasma levels are higher after ingestion of the peptide than after the free amino aid, intestinal absorption of the intat peptide is at least probable. By ontrast, the onverse result, as in the present investigation, may equally well be interpreted as indiating prior luminal hydrolysis of the peptide, or of absorption of the intat peptide with subsequent slow intraellular hydrolysis. Suh results in isolation are, therefore, purely fatual and any theoretial interpretation is ompletely speulative. Referenes Craft, I. L., Geddes, D., Hyde, C. W., Wise, 1. J., and Matthews, D. M. (1968). Absorption and malabsorption of glyine and glyine peptides in man. Gut, 9, Davey, C. L. (1957). An ion exhange method of determining amosine, anserine and their preursors in animal tissue. Nature (Lond.), 179, Davis, N. C. (1956). Ation of proteolyti enzymes on some peptides and derivatives ontaining histidine. J. biol. Chem., 223, De la Noue, J., Newey, H., and Smyth, D. H. (1969). Transport of alanine isomers by rat small intestine in vitro. J. Physiol. (Lond.), 22, 1-lOIP. lwyn, D. H., Parikh, H. C., and Shoemaker, W. C. (1968). Amino aid movements between gut, liver, and periphery in unanesthetized dogs. Amer. J. Physiol., 215, vered, D. F. (1956). The exretion of amino aids by the human. A quantitative study with ion-exhange hromatography. Biohem. J., 62, Hanson, H. T., and Smith,. L. (1949). Carnosinase: an enzyme of swine kidney. J. biol. Chem., 179, Jepson, J. B. (1966). Hartnup Disease. In The Metaboli Basis of Inherited Disease, 2nd ed., edited by J. B. Stanbury, J. B. Wyngaarden, and D. S. Fredrikson, pp MGraw Hill, New York. Milne, M. D., Crawford, M. A., Girao, C. B., and Loughridge, L. W. (196). The metaboli disorder in Hartnup disease. Quart. J. Med., 29, Navab, F., and Asatoor, A. M. (197). Studies on intestinal absorption of amino aids and a dipeptide in a ase of Hartnup disease. Gut (in press). Newey, H. and Smyth, D. H. (196). Intraellular hydrolysis of dipeptides during intestinal absorption. J. Physiol. (Lond.), 152, Newey, H., and Smyth, D. H. (1962). Cellular mehanisms in intestinal transfer of amino aids. J. Physiol. (Lond.), 164, Rhodes, J. B., ihholz, A., and Crane, R. K. (1967). Studies on the organization of the brush border in intestinal epithelial ells. IV. Aminopeptidase ativity in mirovillus membranes of hamster intestinal brush borders. Biohim. biophys. Ata (Amst.), 135, Sriver, C. R., Pueshel, S., and Davies,. (1966). Hyper- -alaninemia assoiated with,-aminoaiduria and y-aminobutyriaiduria, somnolene and seizures. New ngl. J. Med., 274, Smith,. L. (1951). The speifiity ofertain peptidases. Advan. nzymol., 12, Snedeor, G. W. (1946). Statistial Methods, 4th ed. Iowa State College Press, Ames, Iowa. Tehnion Handbook (1966). Tehniques in Amino Aid Analysis. pp. 14; 114. Tehnion Instruments Co. Ltd., Chertsey, ngland. Wiggans, D. S., and Johnston, J. M. (1959). The absorption of peptides. Biohim. biophys. Ata (Amst.), 32, Wiseman, G. (1951). Ative stereohemially seletive absorption of amino-aids from rat small intestine. J. Physiol. (Lond.), 114, 7-8P. Wood, T. (1957). Carnosine and arnosinase in rat tissue. Nature (Lond.), 18, Gut: first published as /gut on 1 Marh 197. Downloaded from on 23 November 218 by guest. Proteted by opyright.