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The effect of maillard reaction products on the absorption of tryptophan
Comp.
Biochem. Physiol..
1977.
Vol. %A, pp. 473 to 476. Pergamon
Press. Printed
in Grerrr Brrroin
THE EFFECT OF MAILLARD REACTION PRODUCTS ON THE ABSORPTION OF TRYPTOPHAN CHONG MIN LEE*, TUNG-CHING LEE AND C. 0. CHICHESTER Department of Food Science & Technology, University of Rhode Island, Kingston, RI 02881, U.S.A. (Receioed 3 August 1976)
Abstract-l. The kinetics of the absorption of tryptophan in the presence of Maillard reaction products formed in the glucose-tryptophan system was studied by both in vitro everted gut sac method and in uiuo catherization of the portal vein. 2. Fructose-L-tryptophan (Amadori compound) appeared to be the major fraction of the reaction products when fractionated using a cellulose column eluted by water-saturated n-butanol. 3. The absorption of L-tryptophan was partially inhibited in vitro and in uiuo by fructose+tryptophan in a competitive manner with an inhibitor constant (Ki) of 1.1 mM. 4. The relative absorption rate of L-tryptophan was significantly lower in the presence of the Maillard reaction products than in the presence of fructose-L-tryptophan indicating the presence of other inhibitory factors in the reaction products. 5. The in uiuo absorption of fructose-L-tryptophan was almost negligible compared to that of tryptophan. 6. The inhibited absorption by Maillard reaction products may have contributed in part to an incomplete recovery in the growth of the rats when fed a supplemented browned synthetic amino acid diet.
INTRODUCTION The non-enzymatic browning of food products is quite prevalent during processing and storage since most foods are susceptible to this type of reaction. The loss of nutritional value as a result of browning is a well known fact. Only a few studies have been made describing the anti-nutritive role of the browning product. The discrepancies between the availability of amino acids and the biological value of the heat-treated protein products (Baliga et al., 1959; Donoso et al., 1962; Frangne & Adrian, 1967) indicate that the utilization of remaining amino acids is hindered somehow by products formed during browning. The reduced biological value of proteins as a result of browning reaction is not recovered completely by supplementation of amino acids (Rao et al., 1963; Sgarbieri et al., 1973a,b) suggesting that effects of the browning reaction are not limited by the loss of amino acid. The presence of Maillard products (browning products) interfered with the protein digestibility as well as with the utilization of elementary nitrogen (Adrian & Frangne, 1973 ; Araujo Neto et al., 1974). It has also been reported that the cells of Candida utilis stored for prolonged periods in a tropical environment showed a negative effect on the utilization of protein in the diet (Araujo Neto et al., 1974). However, in those reports, no attempts were made to show how these anti-nutritive effects would occur in the presence of browning products. The present study was designed to investigate inhibitory effects of browning products on the absorption of amino acids as one of the possible causes of anti-nutritive effects of browning products. Browning of glucose-tryptophan system was chosen for this * Present Address: Department of Nutrition & Food, Drexel University, Philadelphia, PA 19104, U.S.A.
study because of the relative ease in the preparation of an Amadori rearrangement compound (a major intermediate browning reaction product formed at an early stage of the reaction) and the rapid quantitative determination of its browning products.
MATERIALS AND MmHODS Preparation of Maillard reaction products
Browned product was prepared by dissolving 10 g of o-glucose and 2 g of L-tryptophan in 200 ml of methanol and refluxing for 6 hr. The concentrate obtained after rotary evaporation was reconstituted with water and then freeze-dried. For the preparation of fructose+tryptophan, the concentrate was applied to a cellulose column (12 x 600 mm). The column was packed with Whatman CM,, fibrous powder suspended in water-saturated n-butanol and eluted with the same solvent. Each fraction obtained in this manner was separated by thin layer chromatography using silica gel plates (Type Ql, A. H. Thomas, Co., Philadelphia, PA) with a solvent system consisting of n-butanol, acetic acid and water (4: 1: 1 by vol). After the completion of development, the detection was done under a U.V. light. The chromatogram of each spot was compared with that of frucstose+tryptophan prepared by the method of Heyns & Noack (1964). Fractions rich in fructose+tryptophan were pooled, stored in the refrigerator overnight in order to allow crystallization, and purified by repeating recrystallization twice with n-butanol. A final crystallization was done by suspending it in methanol. The purity of every preparation was ensured by chromatographic examination. In vitro absorption study The absorption test was done following the everted gut sac method established by Wilson & Wiseman (1954) with a slight modification. Male rats of Sprague-Dawley (Charles River Breeding Labs, CD strain, Wilmington. MA) weighing 2OG300 g were killed by a blow on the head after being starved overnight. The small intestine was
413
(‘HONG MIN Ltx. TUNG-CHINGLEL
excised commencing at a distance of 15”,, of the total length of the intestine from pylorus to the ileo-caecal junction. washed out with 0.9”,, NaCl and everted. Four everted sacs (4-5 cm in length) were made (2 for the control and 2 for the experiment) from the same intestine and filled with 0.4 ml of serosal fluid (Krebs-Henseleit buffer. pH 7.4) (Krebs & Henseleit. 1932‘). The pH of the buffer solution was adjusted by gassing NaHCO, stock solution with IOO”,, COZ. They were then transferred into 1Oml Erlenmeyer flasks containing 6 ml of mucosal fluid (KrebsHenseleit buffer) which had been previously gassed (O,:COZ, 95:s by vol) for 5 min. The mucosal fluid contained 5 mM L-tryptophan and different concentrations of browning products. Incubation was carried out at 37’ C for 1 hr at a stirring rate of 140 revimin with an Eberbach Rotator (Eberbach Corp.. Ann Arbor, MI). After incubation, the concentration of tryptophan in the serosal fluid was determined using 15 ml of the sample solution by a combination of thin layer chromatography and calorimetric assay (Speis, 1967). The extraction of tryptophan and fructose-L-tryptophan was done by suspending the spots in 0.7 ml of distilled water, followed by centrifugation. From the supematant, 0.5 ml was taken and assayed calorimetrically with a standard curve prepared from the thin layer chromatogram. The same experimental set was also conducted by substituting the browned product for fructose+tryptophan in order to find out whether there is any other physiologically active fractions other than fructose-L-tryptophan in the browned product. The absorption rate across intestinal membrane is expressed as follows: Absorption rate = (C,) (d)/(CJ (100 mg. intestine) (hr), where C, = concentration in the serosal fluid after incubation; Ci = concentration in the mucosal fluid before incubation; d = dilution factor = combined volume of mucosal and serosal fluid/volume of mucosal fluid. A relative absorption rate of the test sample to the control sample was used for the study of the absorption kinetics. The inhibitor constants of the test samples were determined by the graphical method of Dixon (1953). The stability of fructose-L-tryptophan at incubation temperature was tested under the same experimental condition since fructose-L-tryptophan was found to be easily decomposed at an elevated temperature. In vivo uhsorption
study
In order to compare the results from the in aim system with those from the in vitro system, an in vI’~.ostudy was conducted employing the technique established by GalloTorres & Ludorf (1974). This technique appears to be superior to the various known conventional in ciuo techniques since the physiological functions of the rats remain normal during the test in the light of clinical data (GalloTorres, H.E.. 1974, personal communication); variables in the gastric emptying rate can be excluded; and a continuous test is allowed. Female Sprague-Dawley rats weighed approximately 180 g were starved overnight allowing a free access to water until the next morning when the operation was scheduled. Rats were anesthetized by
the inhalation of pentrane (Methoxyflurane; Abbot Labs., North Chicago, IL) with constant care to avoid overdose. After opening the abdomen by middle incision, the intestine was gently exposed for an easy access to the duodenum. In order to exclude the variables in the stomach emptying rate. duodenal catherization was carried out by introducing a catheter (PE 50: Ace Scientific Co., Linden, NJ) which had been bent in such a way that it formed a right angle I cm from one end into the duodenum at a point 5 mm distal to the pvlorous. The catheter was then secured by silk No. 5 and ii,, (Eastman 910 adhesive; Cadillac Plastic and Chemical Co.. Linden, NJ) and brought out through the abdominal wall. The portal vein was carefully exposed prior to a catheterization. The beveled end
AND
C‘.
0.
C‘HIC’HIXIX
of heparin-tilled cannula (PE 50) was mm)duccd into the portal vein having its opening face to the operator so 11~1 its tip rests about 5 mm below the cntr! of the hplcnlc vein. The tubing was then fixed in position with a minmum amount of glue to avoid a possihle coalescence and a round piece of polyvinyl plastic (from disposable glove\). 2.5 mm in diameter. placed around the portal opening. The cannular was then drawn out through the abdominal cavity. Subsequent to closure of the ahdomcn. animals were put into restraining cages to restrict their movement. The rat. when recovered from anesthesia. was infused intraduodenally with 0.5 ml of a saline suspension (0.9”,, Na(‘1) of 5 mM L-tryptophan (0.51 mg) containing 0.12 /&‘i ol L-tryptophan-3-“C (New England Nuclear. Boston. MAI. In the same manner. 0.5 ml of a saline suspension of 5 containing 0.12 /i(‘i of r.-trbptomM L-tryptophan, phan)-3-“C and fructose-L-tryptophan (0.92 mg. eqmvaent to the amount to bring its final concentration to 5 mM). was introduced into the rat when the radioactivity returned to the base level. The same amount of 5 mM fructose-r-tryptophan suspended in saline containin! 0.25 PCi of fructose-L-tryptophan-3-“C was introduced In the same manner. For the study of the kinetics of ahsorption. samples of blood were collected with hcparinized capillary tubes (0.025 x 75 mm) (A. H. Thomas Co.. Philadelphia. PA) at different intervals and centrifuged in a hematocrit centrifuge (Clay Adams. Parsippany. NJI. The prepared plasma was then pooled to give 0.1 ml for each time interval and the radioactivity was determined hy liquid scintillation counter (Model Mark I. Nuclear-Chicago. Des Plaines, IL) after dissolving 11 in the scintillation sollent (Scintisol-complete. Isolah. Inc.. Elkharr. IL).
RESULTS AND DlSCC:SSIOh
A quantitative thin layer chromatographic assay showed that 847; of L-tryptophan was converted to fructose+tryptophan indicating that fructose-L-tryptophan (Amadori compound) was a major fraction of the Maillard reaction products. Results of the stability test of fructose-L-tryptophan at 37°C are shown in Table I. Any extent of decomposition of fructose-L-tryptophan results in a corresponding increase in the final tryptophan concentration stoichiometrically. The results showed that the fructose+tryptophan remained relatively stable in aqueous medium at incubation temperature. From the in vitro absorption study. the kinetics of absorption showed that fructose-r_-tryptophan inhibited the transport of tryptophan through the intestinal membrane in a competitive manner (Fig. 1). The inhibition constant (Ki) of fructose-L-tryptophan was determined to be 1.1 mM using a Dixon inhibition plot. The relative absorption rate of L-tryptophan in the presence of fructose-L-tryptophan ranged from 0.75 to 0.80 at 5 mM of tryptophan. while in the presence of the whole reaction product it ranged from 0.709 to 0.766 at 5 mM (Table 2). More inhibition (P < 0.05) by the whole reaction product suggests the presence of other inhibitory fractions beside fructoseL-tryptophan. The in uiuo absorption kinetics of I.-tryptophan in the presence of fructose+tryptophan is shown in Fig. 2. When fructose+tryptophan was added, the absorption rate of tryptophan decreased with a delayed absorption peak. It was also noticed that no appreciable amount of fructose-L-tryptophan W;IS absorbed without showing an apparent absorption
475
Maillard reaction products on the absorption of t~ptophan Table 1. Stability of fructose+tryptophan
Group
Fructose +tryptophan mg/mI
Tryptophan
0
1.02 1.02 1.02 1.02 1.02
1 2 3 4
at incubation temperature (37°C) Tryptophan after incubation
0 0.18 0.36 0.55 0.73
peak. It seems probable
that the absorption of tryptophan was interfered with by fructose+tryptophan in such a way that the unabsorkd fructose+tryptophan competed with L-tryptophan for the absorption site. This finding supports the evidence obtained from
our in vitro study and the result of Buraczewski et at. (1967). The latter su~est~ that the a~um~ation of amino acids and undigested peptides in the small intestine of the rats fed a severely heated fish meal might have hindered absorption of amino acids by saturating the absorption sites. Related evidence was also obtained from our previous study (Sgarbieri et al., 1973a,b). The growth rate of rats fed a supple-
1.03 &-0.005 1.01 + 0.010 1.02 + 0.007 1.06 + 0.003 1.05 + 0.007
mented browned synthetic amino acid diet reached only 63% of that of rats fed a control amino acid diet. The fecal nitrogen content of rats fed a supplemented browned diet appeared significantly higher than that of rats fed a control diet, indicating that a substantial proportion of nitrogenous substances in the diet was unabsorbed. From the results of the present study, as well as from previous observations, we may draw the following conclusion: (1) The inhibited absorption by Maillard reaction products, as well as the nutritional unavailability of Amadori compounds, may be the cause of incomplete recovery in the growth rate and higher
I .7
-I Concentrafbn
0
2 of fructose
3
- L-tryptophan,
mM
Fig 1. In vitro absorption of L-tryptophan in the presence of fru~tos~L-~yptophan; 4 mM tryptophan plus fructoset,-tryptophan (A) and 5 mM tryptophan plus fructose-Ltryptophan (0).
Table 2. Relative absorption
L-tryptophan 5 5 5 5 5
Time otter dwdanal Infusion.
Fig. 2. In viuo absorption kinetics of tryptophan in the presence of fructose-L-tryptophan; L-tryptophan (o), L-tryptophan plus fructose-L-tryptophan (A) and fructoseL-tryptophan (m).
rates of L-tryptophan in the presence of fructose-L-tryptophan the reaction product
Mucosal fluid Fructose-L-tr~tophan (or reaction product) mM in 6 ml 1’ 2 3 4 5
min
and of
Relative absorption rates/t,-tryptophan Fructose-L-tryptophan Reaction product 0.800 + 0.02 0.780 & 0.014 0.754 & 0.004 0.750 + 0.013
0.766 & 0.01 lb 0.752 _+0.005 0.746 f 0.014 0.724 + OXXX@ 0.709 f 0.W3b
Mean values were obtained from duplicate run. R1 mM of fructose-L-tryptophan and reaction product in 6 ml are equivalent to 2.2 mg and 7.34 mg, respectively. b Significantly different from the fructose+tryptophan group (P e 0.05).
CHONC; MIX’ Ltt:. TIJNG-C‘HINGLEE ANI) C. 0.
416
fecal nitrogenous content of the rats fed a supplemented browned synthetic amino acid diet. (2) After browning, the nutritional availability of the remaining unbrowned portion could be partially diminished by the presence of the browned portion. (3) The impaired protein quality as a result of browning may not be completely recovered by supplementation. Acknowledgement-Supported by HEW/PHS Grant No. 2-ROl-FD-00433-TOX. Rhode Island Agricultural Experiment Station
Contribution
No. 1626.
REFERENCES ADRIAN J. & FRANGNER. (1973) Maillard
reaction 8. Role of premelanoidins on nitrogen digestibility in uivo and on proteolysis in vitro. Ann. Nutr. Aliment. 27, 11 l-123. ADRIAN J., FKANGNE R., PETIT L.. GODON B. & BARBIER J. (1966) Nutritional repercussion of the soluble products formed during Maillard reaction. Ann. Nutr. Aliment. 20, 251-217. ARAUIO NETO J. S., MADI K. & PANEI( A. D. (1974) Longterm storage of cells of Candida utilis: A note on their nutritional evaluation. J. Food Sci. 39. 613-4. BALIGA B. P.. BAYLISS M. E. & LYMAN C. M. (1959) Determination of free lysine e-amino groups in cottonseed meals and preliminary studies in relation to protein quality. Arch. Biochem. Bioph!*s. 94, l--6. BURACZEWSKI S.. BURACZEWSKI L. 8~ FORD J. E. (1967) The influence of heating fish proteins on the course of their digestion. Acta Biochem. Pd.. 14. 121-131. DIXON M. (1953) The determination of enzyme inhibitor constant. Biochem. J. 55. 17&171.
C‘HICHESTEH
DONOXI G., LEWIS 0. A. M., MILL~H D. S. 6t PAYM P. R. (1962) Effect of heat treatment on the nutritive value of proteins: Chemical and balance studies. J. Sri. Food 4yr. 13. 192.-196. FRANGNE R. & AIIRIAN J. (1967) Enzymatic proteolysis of heated food and significances of results. ilnn. Nutr. \vI. I63m 174. GALLO-TORRES H. E. & LUIXW J. (1974) Techniques for the in r>it)ocatheterization of the portal vein in the rat. Proc. Sot. esp. Biol. Med. 145, 249 254. HEYNS K. & NOACK H. (1964) Die umsetzung von L-tryptophan und I.-histidin mit hexoscn. Chr. Ber. 97, 415 418. KR~HS H. A. & HENS~LEITK. (1932) tintersuchungen iiber die harnstoffbilding im tierk8rper. Hoppe-Seder’s Z. Physiol. Chem. 210. 3346. RAO M. N.. SREENIVASH., SWAMINATHANM., CARPENTER K. J. & MORGAN L. B. (1963) The nutritional available
lysine and methionine
of heated
casein-glucose
mixtures.
J. Sci. Food Agr. 14, 544-550. SGARBIERIV. C., AMAYA J., TANAKA M. & CHICHE~TEK
C. 0. (1973a) Response of rats to amino acid supplementation of brown egg albumin. J. Nutr. 103, 1731-1738. SGARBIERIV. C., AMAYA J., TANAKA M. & CHICHE~TER C. 0. (I 973b) Physiological consequences of feeding to rat a browned synthetic amino acid-sugar mixture. Arch. Latin.
Nutr.
23, 363-378.
SPEIS J. R. (1967) Determination And. Chem. 39. 1412~1415.
of tryptophan
WILSON T. H. & WISEMAN G. (1954) The
in proteins.
use of sacs of everted small intestine for the study of the transference of substances from the mucosal to the serosal surface. .1. Physiol. 123. I16 I25
Biochem. Physiol..
1977.
Vol. %A, pp. 473 to 476. Pergamon
Press. Printed
in Grerrr Brrroin
THE EFFECT OF MAILLARD REACTION PRODUCTS ON THE ABSORPTION OF TRYPTOPHAN CHONG MIN LEE*, TUNG-CHING LEE AND C. 0. CHICHESTER Department of Food Science & Technology, University of Rhode Island, Kingston, RI 02881, U.S.A. (Receioed 3 August 1976)
Abstract-l. The kinetics of the absorption of tryptophan in the presence of Maillard reaction products formed in the glucose-tryptophan system was studied by both in vitro everted gut sac method and in uiuo catherization of the portal vein. 2. Fructose-L-tryptophan (Amadori compound) appeared to be the major fraction of the reaction products when fractionated using a cellulose column eluted by water-saturated n-butanol. 3. The absorption of L-tryptophan was partially inhibited in vitro and in uiuo by fructose+tryptophan in a competitive manner with an inhibitor constant (Ki) of 1.1 mM. 4. The relative absorption rate of L-tryptophan was significantly lower in the presence of the Maillard reaction products than in the presence of fructose-L-tryptophan indicating the presence of other inhibitory factors in the reaction products. 5. The in uiuo absorption of fructose-L-tryptophan was almost negligible compared to that of tryptophan. 6. The inhibited absorption by Maillard reaction products may have contributed in part to an incomplete recovery in the growth of the rats when fed a supplemented browned synthetic amino acid diet.
INTRODUCTION The non-enzymatic browning of food products is quite prevalent during processing and storage since most foods are susceptible to this type of reaction. The loss of nutritional value as a result of browning is a well known fact. Only a few studies have been made describing the anti-nutritive role of the browning product. The discrepancies between the availability of amino acids and the biological value of the heat-treated protein products (Baliga et al., 1959; Donoso et al., 1962; Frangne & Adrian, 1967) indicate that the utilization of remaining amino acids is hindered somehow by products formed during browning. The reduced biological value of proteins as a result of browning reaction is not recovered completely by supplementation of amino acids (Rao et al., 1963; Sgarbieri et al., 1973a,b) suggesting that effects of the browning reaction are not limited by the loss of amino acid. The presence of Maillard products (browning products) interfered with the protein digestibility as well as with the utilization of elementary nitrogen (Adrian & Frangne, 1973 ; Araujo Neto et al., 1974). It has also been reported that the cells of Candida utilis stored for prolonged periods in a tropical environment showed a negative effect on the utilization of protein in the diet (Araujo Neto et al., 1974). However, in those reports, no attempts were made to show how these anti-nutritive effects would occur in the presence of browning products. The present study was designed to investigate inhibitory effects of browning products on the absorption of amino acids as one of the possible causes of anti-nutritive effects of browning products. Browning of glucose-tryptophan system was chosen for this * Present Address: Department of Nutrition & Food, Drexel University, Philadelphia, PA 19104, U.S.A.
study because of the relative ease in the preparation of an Amadori rearrangement compound (a major intermediate browning reaction product formed at an early stage of the reaction) and the rapid quantitative determination of its browning products.
MATERIALS AND MmHODS Preparation of Maillard reaction products
Browned product was prepared by dissolving 10 g of o-glucose and 2 g of L-tryptophan in 200 ml of methanol and refluxing for 6 hr. The concentrate obtained after rotary evaporation was reconstituted with water and then freeze-dried. For the preparation of fructose+tryptophan, the concentrate was applied to a cellulose column (12 x 600 mm). The column was packed with Whatman CM,, fibrous powder suspended in water-saturated n-butanol and eluted with the same solvent. Each fraction obtained in this manner was separated by thin layer chromatography using silica gel plates (Type Ql, A. H. Thomas, Co., Philadelphia, PA) with a solvent system consisting of n-butanol, acetic acid and water (4: 1: 1 by vol). After the completion of development, the detection was done under a U.V. light. The chromatogram of each spot was compared with that of frucstose+tryptophan prepared by the method of Heyns & Noack (1964). Fractions rich in fructose+tryptophan were pooled, stored in the refrigerator overnight in order to allow crystallization, and purified by repeating recrystallization twice with n-butanol. A final crystallization was done by suspending it in methanol. The purity of every preparation was ensured by chromatographic examination. In vitro absorption study The absorption test was done following the everted gut sac method established by Wilson & Wiseman (1954) with a slight modification. Male rats of Sprague-Dawley (Charles River Breeding Labs, CD strain, Wilmington. MA) weighing 2OG300 g were killed by a blow on the head after being starved overnight. The small intestine was
413
(‘HONG MIN Ltx. TUNG-CHINGLEL
excised commencing at a distance of 15”,, of the total length of the intestine from pylorus to the ileo-caecal junction. washed out with 0.9”,, NaCl and everted. Four everted sacs (4-5 cm in length) were made (2 for the control and 2 for the experiment) from the same intestine and filled with 0.4 ml of serosal fluid (Krebs-Henseleit buffer. pH 7.4) (Krebs & Henseleit. 1932‘). The pH of the buffer solution was adjusted by gassing NaHCO, stock solution with IOO”,, COZ. They were then transferred into 1Oml Erlenmeyer flasks containing 6 ml of mucosal fluid (KrebsHenseleit buffer) which had been previously gassed (O,:COZ, 95:s by vol) for 5 min. The mucosal fluid contained 5 mM L-tryptophan and different concentrations of browning products. Incubation was carried out at 37’ C for 1 hr at a stirring rate of 140 revimin with an Eberbach Rotator (Eberbach Corp.. Ann Arbor, MI). After incubation, the concentration of tryptophan in the serosal fluid was determined using 15 ml of the sample solution by a combination of thin layer chromatography and calorimetric assay (Speis, 1967). The extraction of tryptophan and fructose-L-tryptophan was done by suspending the spots in 0.7 ml of distilled water, followed by centrifugation. From the supematant, 0.5 ml was taken and assayed calorimetrically with a standard curve prepared from the thin layer chromatogram. The same experimental set was also conducted by substituting the browned product for fructose+tryptophan in order to find out whether there is any other physiologically active fractions other than fructose-L-tryptophan in the browned product. The absorption rate across intestinal membrane is expressed as follows: Absorption rate = (C,) (d)/(CJ (100 mg. intestine) (hr), where C, = concentration in the serosal fluid after incubation; Ci = concentration in the mucosal fluid before incubation; d = dilution factor = combined volume of mucosal and serosal fluid/volume of mucosal fluid. A relative absorption rate of the test sample to the control sample was used for the study of the absorption kinetics. The inhibitor constants of the test samples were determined by the graphical method of Dixon (1953). The stability of fructose-L-tryptophan at incubation temperature was tested under the same experimental condition since fructose-L-tryptophan was found to be easily decomposed at an elevated temperature. In vivo uhsorption
study
In order to compare the results from the in aim system with those from the in vitro system, an in vI’~.ostudy was conducted employing the technique established by GalloTorres & Ludorf (1974). This technique appears to be superior to the various known conventional in ciuo techniques since the physiological functions of the rats remain normal during the test in the light of clinical data (GalloTorres, H.E.. 1974, personal communication); variables in the gastric emptying rate can be excluded; and a continuous test is allowed. Female Sprague-Dawley rats weighed approximately 180 g were starved overnight allowing a free access to water until the next morning when the operation was scheduled. Rats were anesthetized by
the inhalation of pentrane (Methoxyflurane; Abbot Labs., North Chicago, IL) with constant care to avoid overdose. After opening the abdomen by middle incision, the intestine was gently exposed for an easy access to the duodenum. In order to exclude the variables in the stomach emptying rate. duodenal catherization was carried out by introducing a catheter (PE 50: Ace Scientific Co., Linden, NJ) which had been bent in such a way that it formed a right angle I cm from one end into the duodenum at a point 5 mm distal to the pvlorous. The catheter was then secured by silk No. 5 and ii,, (Eastman 910 adhesive; Cadillac Plastic and Chemical Co.. Linden, NJ) and brought out through the abdominal wall. The portal vein was carefully exposed prior to a catheterization. The beveled end
AND
C‘.
0.
C‘HIC’HIXIX
of heparin-tilled cannula (PE 50) was mm)duccd into the portal vein having its opening face to the operator so 11~1 its tip rests about 5 mm below the cntr! of the hplcnlc vein. The tubing was then fixed in position with a minmum amount of glue to avoid a possihle coalescence and a round piece of polyvinyl plastic (from disposable glove\). 2.5 mm in diameter. placed around the portal opening. The cannular was then drawn out through the abdominal cavity. Subsequent to closure of the ahdomcn. animals were put into restraining cages to restrict their movement. The rat. when recovered from anesthesia. was infused intraduodenally with 0.5 ml of a saline suspension (0.9”,, Na(‘1) of 5 mM L-tryptophan (0.51 mg) containing 0.12 /&‘i ol L-tryptophan-3-“C (New England Nuclear. Boston. MAI. In the same manner. 0.5 ml of a saline suspension of 5 containing 0.12 /i(‘i of r.-trbptomM L-tryptophan, phan)-3-“C and fructose-L-tryptophan (0.92 mg. eqmvaent to the amount to bring its final concentration to 5 mM). was introduced into the rat when the radioactivity returned to the base level. The same amount of 5 mM fructose-r-tryptophan suspended in saline containin! 0.25 PCi of fructose-L-tryptophan-3-“C was introduced In the same manner. For the study of the kinetics of ahsorption. samples of blood were collected with hcparinized capillary tubes (0.025 x 75 mm) (A. H. Thomas Co.. Philadelphia. PA) at different intervals and centrifuged in a hematocrit centrifuge (Clay Adams. Parsippany. NJI. The prepared plasma was then pooled to give 0.1 ml for each time interval and the radioactivity was determined hy liquid scintillation counter (Model Mark I. Nuclear-Chicago. Des Plaines, IL) after dissolving 11 in the scintillation sollent (Scintisol-complete. Isolah. Inc.. Elkharr. IL).
RESULTS AND DlSCC:SSIOh
A quantitative thin layer chromatographic assay showed that 847; of L-tryptophan was converted to fructose+tryptophan indicating that fructose-L-tryptophan (Amadori compound) was a major fraction of the Maillard reaction products. Results of the stability test of fructose-L-tryptophan at 37°C are shown in Table I. Any extent of decomposition of fructose-L-tryptophan results in a corresponding increase in the final tryptophan concentration stoichiometrically. The results showed that the fructose+tryptophan remained relatively stable in aqueous medium at incubation temperature. From the in vitro absorption study. the kinetics of absorption showed that fructose-r_-tryptophan inhibited the transport of tryptophan through the intestinal membrane in a competitive manner (Fig. 1). The inhibition constant (Ki) of fructose-L-tryptophan was determined to be 1.1 mM using a Dixon inhibition plot. The relative absorption rate of L-tryptophan in the presence of fructose-L-tryptophan ranged from 0.75 to 0.80 at 5 mM of tryptophan. while in the presence of the whole reaction product it ranged from 0.709 to 0.766 at 5 mM (Table 2). More inhibition (P < 0.05) by the whole reaction product suggests the presence of other inhibitory fractions beside fructoseL-tryptophan. The in uiuo absorption kinetics of I.-tryptophan in the presence of fructose+tryptophan is shown in Fig. 2. When fructose+tryptophan was added, the absorption rate of tryptophan decreased with a delayed absorption peak. It was also noticed that no appreciable amount of fructose-L-tryptophan W;IS absorbed without showing an apparent absorption
475
Maillard reaction products on the absorption of t~ptophan Table 1. Stability of fructose+tryptophan
Group
Fructose +tryptophan mg/mI
Tryptophan
0
1.02 1.02 1.02 1.02 1.02
1 2 3 4
at incubation temperature (37°C) Tryptophan after incubation
0 0.18 0.36 0.55 0.73
peak. It seems probable
that the absorption of tryptophan was interfered with by fructose+tryptophan in such a way that the unabsorkd fructose+tryptophan competed with L-tryptophan for the absorption site. This finding supports the evidence obtained from
our in vitro study and the result of Buraczewski et at. (1967). The latter su~est~ that the a~um~ation of amino acids and undigested peptides in the small intestine of the rats fed a severely heated fish meal might have hindered absorption of amino acids by saturating the absorption sites. Related evidence was also obtained from our previous study (Sgarbieri et al., 1973a,b). The growth rate of rats fed a supple-
1.03 &-0.005 1.01 + 0.010 1.02 + 0.007 1.06 + 0.003 1.05 + 0.007
mented browned synthetic amino acid diet reached only 63% of that of rats fed a control amino acid diet. The fecal nitrogen content of rats fed a supplemented browned diet appeared significantly higher than that of rats fed a control diet, indicating that a substantial proportion of nitrogenous substances in the diet was unabsorbed. From the results of the present study, as well as from previous observations, we may draw the following conclusion: (1) The inhibited absorption by Maillard reaction products, as well as the nutritional unavailability of Amadori compounds, may be the cause of incomplete recovery in the growth rate and higher
I .7
-I Concentrafbn
0
2 of fructose
3
- L-tryptophan,
mM
Fig 1. In vitro absorption of L-tryptophan in the presence of fru~tos~L-~yptophan; 4 mM tryptophan plus fructoset,-tryptophan (A) and 5 mM tryptophan plus fructose-Ltryptophan (0).
Table 2. Relative absorption
L-tryptophan 5 5 5 5 5
Time otter dwdanal Infusion.
Fig. 2. In viuo absorption kinetics of tryptophan in the presence of fructose-L-tryptophan; L-tryptophan (o), L-tryptophan plus fructose-L-tryptophan (A) and fructoseL-tryptophan (m).
rates of L-tryptophan in the presence of fructose-L-tryptophan the reaction product
Mucosal fluid Fructose-L-tr~tophan (or reaction product) mM in 6 ml 1’ 2 3 4 5
min
and of
Relative absorption rates/t,-tryptophan Fructose-L-tryptophan Reaction product 0.800 + 0.02 0.780 & 0.014 0.754 & 0.004 0.750 + 0.013
0.766 & 0.01 lb 0.752 _+0.005 0.746 f 0.014 0.724 + OXXX@ 0.709 f 0.W3b
Mean values were obtained from duplicate run. R1 mM of fructose-L-tryptophan and reaction product in 6 ml are equivalent to 2.2 mg and 7.34 mg, respectively. b Significantly different from the fructose+tryptophan group (P e 0.05).
CHONC; MIX’ Ltt:. TIJNG-C‘HINGLEE ANI) C. 0.
416
fecal nitrogenous content of the rats fed a supplemented browned synthetic amino acid diet. (2) After browning, the nutritional availability of the remaining unbrowned portion could be partially diminished by the presence of the browned portion. (3) The impaired protein quality as a result of browning may not be completely recovered by supplementation. Acknowledgement-Supported by HEW/PHS Grant No. 2-ROl-FD-00433-TOX. Rhode Island Agricultural Experiment Station
Contribution
No. 1626.
REFERENCES ADRIAN J. & FRANGNER. (1973) Maillard
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C‘HICHESTEH
DONOXI G., LEWIS 0. A. M., MILL~H D. S. 6t PAYM P. R. (1962) Effect of heat treatment on the nutritive value of proteins: Chemical and balance studies. J. Sri. Food 4yr. 13. 192.-196. FRANGNE R. & AIIRIAN J. (1967) Enzymatic proteolysis of heated food and significances of results. ilnn. Nutr. \vI. I63m 174. GALLO-TORRES H. E. & LUIXW J. (1974) Techniques for the in r>it)ocatheterization of the portal vein in the rat. Proc. Sot. esp. Biol. Med. 145, 249 254. HEYNS K. & NOACK H. (1964) Die umsetzung von L-tryptophan und I.-histidin mit hexoscn. Chr. Ber. 97, 415 418. KR~HS H. A. & HENS~LEITK. (1932) tintersuchungen iiber die harnstoffbilding im tierk8rper. Hoppe-Seder’s Z. Physiol. Chem. 210. 3346. RAO M. N.. SREENIVASH., SWAMINATHANM., CARPENTER K. J. & MORGAN L. B. (1963) The nutritional available
lysine and methionine
of heated
casein-glucose
mixtures.
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C. 0. (1973a) Response of rats to amino acid supplementation of brown egg albumin. J. Nutr. 103, 1731-1738. SGARBIERIV. C., AMAYA J., TANAKA M. & CHICHE~TER C. 0. (I 973b) Physiological consequences of feeding to rat a browned synthetic amino acid-sugar mixture. Arch. Latin.
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of tryptophan
WILSON T. H. & WISEMAN G. (1954) The
in proteins.
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