An interaction between dietary tryptophan and stress in exacerbating gastric disease

Physiology &Behavior,Vol. 26, pp. 197-200. Pergamon Press and Brain Research Publ., 1981. Printed in the U.S.A.

An Interaction Between Dietary Tryptophan and Stress in Exacerbating Gastric Disease I BENJAMIN

H. NATELSON, 2 LAURA JANOCKO

AND JACOB H. JACOBY 3

Division of Research, Veterans Administration Medical Center and Departments o f Neuroscience and Pharmacology, College of Medicine and Dentistry of New Jersey New Jersey Medical School, East Orange, N J 07018 R e c e i v e d 17 M a r c h 1980 NATELSON, B. H., L. JANOCKO AND J. H. JACOBY. An interaction between dietary tryptophan and stress in exacerbating gastric disease. PHYSIOL. BEHAV. 26(2) 197-200, 1981.--Rats fed a tryptophan deficient diet composed of corn developed significantly more gastric erosive disease when subjected to prolonged immobilization than similarly stressed rats fed a tryptophan enriched diet. No gastric disease developed in control groups which were not immobilized. No significant correlations were found between treatment groups and levels of indoleamines in brain and gut. However, serum tryptophan was significantly lowered only in the group of stressed rats fed a tryptophan deficient diet. This report raises the possibility that people eating preponderantly a corn-based diet, deficient in tryptophan, may be at increased risk of having stress ulcer disease of the gut. Brain

Gut

Nutrition

Rats

Tryptophan

L A R G E doses of reserpine produce ulcer disease in rats [ 11] and cats [7]. The superimposition of stress can dramatically lower the dose of reserpine required to induce disease. F o r example, 0.1 mg/kg of reserpine produces relatively little gut disease in rats or cats but an appreciable amount of disease when reserpinized rats are subjected to immobilization [10] or when reserpinized cats are exposed to electric shock [7]. Importantly, the disease p r o d u c e d in cats by this pharmacologic and behavioral combination appeared in the same areas o f the gut as are affected in human disease--distal stomach or duodenum [15]. To analyze further the biochemical basis of this phenomenon, Doteuchi did a histochemical evaluation of gut monoamines. He noted that low doses (0.1 mg/kg) of reserpine, causing almost complete depletion of gut norepinephrine 24 hour after injection, had little effect on gut serotonin. Electric shock stress, alone, caused no marked changes in either gut norepinephrine or gut serotonin. However, following a low dose of reserpine with a course of shock produced a striking depletion in both gut monoamines [5]. Because syrosingopine, a derivative of reserpine, whose monoamine depleting activity is limited to the periphery, did not produce perforating ulcer in the cat, Doteuchi [6,7] inferred that the ulcers had developed because of reserpineinduced depletion of brain monoamines in the face of concurrent depletion of serotonin in the gut wall.

Serotonin

Stress

5-HIAA

The more recent observation that gut serotonin can also be depleted by feeding rats a diet low in tryptophan [1] suggested to us that a non-pharmacologic--i.e, nutritional-- approach could be employed in combination with stress to study the genesis of peptic ulcer disease. Our plan was to feed rats for several days a corn diet which contains very little tryptophan [8] and then to stress them by immobilization in the hopes of producing a model of spontaneous peptic ulcer disease resembling that seen in man; this does not currently exist [14]. METHOD

Animals and Diet Twenty-eight female Sprague-Dawley rats (Taconic Farms, Germantown, NY), weighing 200-225 g, were used in these experiments. The rats were acclimated for 1 week to our laboratory conditions (singly housed in mesh bottom cages with ad lib Purina rat chow and water; lights on 12 hr a day). After this time, external food holders were removed and feeding jars (provided graciously by Dr. I. Faust) were attached inside the cages. These jars were 7.5 cm high and had a screw-on top with a 4.5 cm opening punched in its center. The jars were packed 2/3 full with one of two diets: a 15% by weight corn/corn oil diet composed of white Masa

~This research was supported by VA medical research funds and a Career Development Award to BHN. ~Address reprint requests to Benjamin H. Natelson, MD, Department of Neurosciences, New Jersey Medical School, 88 Ross Street, East Orange, NJ 07018. aThe authors acknowledge the statistical advice of Jean Chou and the secretarial aid of L. Fox.

C o p y r i g h t © 1981 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/81/020197-04502.00/0

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NATELSON, JANOCKO AND JACOBY

Harina (courtesy of the Quaker Oats Co., Barrington, IL; information about composition of the diet can be obtained from Dr. H. Lockhart of Quaker Oats) and the identical diet which had been enriched by the addition of 0.25 g ltryptophan per 100 g of diet. The corn oil was used to convert the powdered corn diet into a thick cake which could be tightly packed into each jar, thus minimizing spillage. Animals were randomly assigned to either the corn (tryp-poor; n=14) or corn+tryp ( t r y p - s u p p l ; n=14) group and were allowed ad lib access to diet and water. Animals were weighed and food jars replenished on a daily basis.

Procedure On the sixth day, after the rats were weighed, they were tested in an open field. The apparatus used was a square, 113.5 cm across with vertical sides, measuring 32.5 cm. The box was painted black and 100 smaller squares were painted on its bottom with white paint; the inside diameter of each square was 11 cm. Each rat was placed into a corner of the test box, facing the corner. We then timed the latency required for the rat to leave the starting square as well as the number of rears made, the number of squares traversed and the number of fecal boli dropped over a 3 min test period. The rat was then returned to its home cage. On the next day, after each rat was weighed, it was briefly anesthetized with ether and then placed either into a novel cage (an empty, opaque plastic pan with a wire top) or into restraint achieved by snaring each paw with a leather loop and by a 12.5 cm leather flap over the body. Animals in both groups were then placed overnight in a cool room (19-20°C). The next morning, rats were sacrificed by decapitation, and trunk blood was collected and allowed to coagulate over ice. Blood was centrifuged, and serum was then frozen for subsequent analysis for glucose [17] and tryptophan [4]. The brain was dissected, sectioned into two hemispheres and quickly frozen over dry ice. Alternate halves were subsequently used for spectrofluorometric determination of brain tryptophan [4] and of brain serotonin/5 hydroxyindoleacetic acid (5-HIAA) [2]. Stomach and duodenum was removed and a piece of duodenum was frozen for later analysis for gut serotonin [2]. The stomach was then opened along the greater curvature and pinned under Formalin. Subsequently, lesion frequency and lesion length were quantified using a dissection microscope. Data are summarized as means - SEM. Tests for significance of open field data were done using Student's t-test. Because of heterogeneity of variance in the gastric pathologic data, t-tests for differences in pathology were done following log transformation of data+ 1; our a priori hypothesis allowed this test to be one-tailed. Tests for significance of visceral data were done by an analysis of variance; post hoc tests were done by Newman Keuls tests with a significance level of 0.05.

• (61)

36 20

•(45)

n

I m

iNI

IO 0 Tryp-Free Tryp-Suppl NUMBER OF LESIONS

Tryp-Free Tryp-Suppl LENGTH OF LESIONS (mm)

FIG. 1. Left hand panel--vertical axis depicts the number of gastric lesions found following overnight restraint in rats previously fed either a tryp-free or a trypsupplemented diet. One rat in the supplemented group died during the anesthesia given prior to restraint. Right hand panel--vertical axis depicts the length of the gastric lesions (in mm) found from these 2 groups of rats.

Gastric pathology. The data are displayed in Fig. 1. Restrained rats fed a tryptophan-poor diet had significantly more and significantly longer lesions in the gastric corpus than those fed a tryptophan-supplemented (diet tH>l.85, p<0.05). Rats placed in a novel cage overnight were essentially lesion free whether on a tryptophan poor or enriched diet. No obvious duodenal pathology was noted in any group. Other visceral parameters. On the morning prior to stress, after 6 days' access to the 2 diets, rats on the tryptophan-poor diet had lost more weight (26.9 -+ 3.2 g) than those on the enriched diet (16.5 _+ 3.9; t26=2.08, p<0.05) but gross weights for the 2 groups were the same (189.9 - 2.8 and 198.5 _+ 4.3 g, respectively, t2n=l.67). One-way analysis of variance revealed that the concentrations of serum and brain tryptophan and serum glucose varied significantly, F(3,23)=5.53, 3.45, and 5.27, respectively; p<0.05; serum tryptophan was lowest in the restrained group previously fed a tryptophan-poor diet as compared to the other 3 groups (see Table 1). Conversely, serum glucose was significantly elevated only in the group of rats that was restrained after having eaten a tryptophansupplemented diet (Table 1). Although neither gut, nor brain serotonin and 5-HIAA was changed by any of the four specific treatments, an effect of restraint, regardless of prior diet, was found for brain 5-HIAA, F(1,22)=4.17, p=0.054, and for gut 5-HIAA, F(1,22)=6.81, p =0.016: restrained rats had higher brain 5-HIAA (559 _+ 32 ng/g) and lower gut 5-HIAA (2.6 -+ 0.4 /xg/g) than rats placed overnight in a novel cage (brain=490 _+ 14 ng/g; gut=4.1 -+ 0.4/zg/g). DISCUSSION

RESULTS

Openfield. Animals eating the tryp-poor diet behaved the same in the open field as those eating a tryp-supplemented diet: latencies were, respectively, 24 -4- 9 and 17 + 3 sec; tryptophan-supplemented rats traversed an average of 92 _+ 12 squares and reared 10 -+ 1 times in the 3 min test period while rats eating a tryp-poor diet traversed 100 _ 14 squares and reared 9 -+ 1 times. These results are similar to those found after serotonin depletion by pCPA administration [12].

Restrained rats, eating a tryptophan-poor diet, had significantly more gastric erosions than similarly restrained rats eating the same diet plus a tryptophan supplement. The interaction of restraint stress superimposed upon the one week ingestion of a low tryptophan diet produced other changes in visceral parameters as well. Thus, both serum and brain tryptophan were lowest in this group. Also, serum glucose was not elevated above values found in the novel cage groups for the tryptophan-poor group but was significantly higher in the restraint group given the diet with the

STRESS AND LOW TRYPTOPHAN

199

TABLE 1 MEAN SERUM, BRAIN, AND GUT CONSTITUENTS(-+ SEM) AFTER 18 HR FAST Restraint Source

Constituent

Serum Tryptophan (/~g/ml) Serum Brain Brain Brain Gut Gut

Glucose (mg/100 ml) Tryptophan (/zg/g) Serotonin (ng/g) 5-HIAA (ng/g) Serotonin (/xg/g) 5-HIAA (/zg/g)

Tryp-Suppl.

Tryp-Poor

Tryp-Suppl.

n=7

n=6

n=7

n=7

6.3 _+ 0.6

11.6 -+ 1.0

12.5 -+ 0.5

12.0 _+ 1.4

94.7 + 3.8

114.8 _+ 9.2

87.2 _+ 2.7

92.8 _+ 3.6

7.5 -+ 0.6

10.1 ± 0.9

9.2 ± 0.6

11.1 _+ 1.1

362.7 ± 18.3

378.5-+ 2 4 . 4

403.0± 8.4

373.9_+ 11.0

534.3 ± 32.2

584.3± 57.3

494.4± 2 0 . 7

484.6_+ 20.9

2.7 ± 0.5

2.7 ± 0.7

3.3 _ 0.4

3.3 _+ 0.4

2.5 ± 0.5

2.7 ± 0.5

4.0 ± 0.7

4.2 _+ 0.6

TABLE 2 MEAN NON-FASTINGSERUM, BRAIN, AND GUT CONSTITUENTS (± SEM) Source

Constituent

Serum

Tryptophan (/xg/ml) Tryptophan (/~g/g) Serotonin (ng/g) Serotonin (/zg/g)

Brain Brain Gut

Tryp-Poor

Tryp-Suppl.

6.4 _+ 0.4*

13.7 _+ 0.6

1.2 ± 0.1"

3.0 -+ 0.3

313.5 + 13.0"

491.0 _+ 21.7

3.2 -

0.1

Novel Cage

Tryp-Poor

3.6 _+ 0.3

*t(7) at least 7.48, p<0.0005.

tryptophan supplement. That the interaction between nutritional and environmental factors was particularly important under our experimental conditions was seen from the fact that rats fed a tryptophan-poor diet in their home cages and then fasted overnight in novel cages had tryptophan, serotonin and 5-HIAA levels that were not significantly different from levels found in similarly treated animals that had previously been fed a tryptophan-supplemented diet (see Table 1). This finding is quite different from earlier reports [8,20] which note striking differences in concentration of these constituents shortly after rats were put on the same tryptophan-poor, corn diet. Because other workers [3,16] showed that a fast increased brain tryptophan and 5-hydroxyindole levels in rats previously fed a normal diet, we reasoned that the discrepancy lay with the 18 hour fast demanded by our experimental design--a manipulation not employed by the earlier workers. When rats were given 3 days access to either the tryptophan-poor (n=6) or tryptophan-supplemented (n=3) diets and then killed with-

out imposing any fast, serum and brain tryptophan as well as brain serotonin were significantly reduced in the former group (Table 2). These pilot data suggest that the fasted rat previously fed a tryptophan poor diet is able to maintain brain tryptophan levels while the fed r a t - - o n the identical diet--cannot. The fact that superimposition of the fast obfuscated any differences in brain serotonin makes it impossible for us to attribute the additional gut pathology to changes in brain serotonin. In contrast to Colmenares and Wurtman [1], we were unable to show depletion of the gut's store cf serotonin under our conditions. However, restraint stress significantly lowered gut 5-HIAA and tended to lower gut serotonin when compared to data obtained from rats placed in novel cages. An estimation of gut 5-HT turnover would be useful to determine whether these changes result from a massive discharge of gut serotonin with subsequent deamination, or whether such changes are secondary to a stress-induced decrease of serotonin synthesis. Nonetheless, the superimposition of stress in the tryptophan depleted rat produces more severe gastric disease than that found in the tryptophan replete rat (Fig. 1). These data complement an earlier study showing that feeding rats bananas, a food rich in tryptophan and serotonin, reduces the amount of gastric disease induced by immobilization [19]. The notion that stress plus a corn diet can produce serious gut disease has precedence in the literature. One factor thought responsible for acute gastric hemorrhage in swine relates to ingestion of some form of a corn diet [13]. When heat stress is superimposed on corn diets, the incidence of this swine disease increases [18]. Perhaps the imposition of additional metabolic demands on gut tissue already undergoing alterations in protein synthesis due to a relative deficiency of so critical an anabolic amino acid as tryptophan leads to a further compromise in the viability of gut mucosa and then erosion. It is possible that this relation could be important to impoverished humans in Latin America. In areas of countries such as E1 Salvador and Guate-

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NATELSON, JANOCKO AND JACOBY

mala, a corn diet constitutes o v e r 60% o f total caloric intake [9]. Such people could be at risk for acute gastric disease if subjected to environmental stress. Despite our observation o f increased gastric disease in stressed rats on a tryptophan-free diet, no disease o c c u r r e d in distal stomach and d u o d e n u m , sites w h e r e peptic ulcers o c c u r in humans [15] and where gut disease occurs in stressed, reserpinized cats [7]. Thus, a species difference

exists in susceptibility to peptic ulcer disease. One reason for this may lie in the fact that serotonin containing cells are rarely found in rat d u o d e n u m but o c c u r there c o m m o n l y in the cat [5]. This inability to produce gastroduodenal disease at sites comparable to those seen in man would appear to limit the usefulness of employing a rat model for the study of human peptic ulcer disease.

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12. Kohler, C. and S. A. Lorens. Open field activity and avoidance behavior following serotonin depletion: a comparison of the effects of parachlorophenylalamine and electrolytic midbrain raphe lesions. Pharmac. Biochem. Behav. 8: 223-233, 1978. 13. Kowalczyk, T. Etiologic factors of gastric ulcers in swine. Am. J. Vet. Res. 30: 393-400, 1969. 14. Natelson, B. H. Peptic ulcer disease. In: Spontaneous Animal Models o f Human Disease, Vol. 1, edited by E. J. Andrews, B. C. Ward and N. H. Altman. New York: Academic, 1979, pp. 17-20. 15. Oi, M., T. Toriuma, O. Miho and M. Kijima. Location of experimental ulcer as compared with that of human peptic ulcer. In: Peptic Ulcer, edited by C. F. Pfeiffer. Philadelphia: Lippincott, 1971, pp. 244-259. 16. Perez-Cruet, J., A. Tagliamonte, P. Tagliamonte and G. L. Gessa. Changes in brain serotonin metabolism associated with fasting and satiation in rats. Life Sci. 11: 31-39, 1972. 17. Raabo, E. and T. Terkildsen. On the enzymatic determination of blood glucose. Scand. J. clin. Invest. 12: 402-407, 1960. 18. Riker III, J. T., T. W. Perry, R. A. Pickett, C. J. Heidenreich and T. M. Curtin. Influence of controlled ambient temperatures and diets on the incidence of esophagogastric ulcers in swine. J. Anim. Sci. 26: 736-740, 1967. 19. Sanyal, A. K., C. R. Banerjea and P. K. Das. Studies on peptic ulceration. Part II--Role of banana in restraint and prednisolone induced ulcer in albino rats. Arch. int. Pharmacodyn. 155: 244248, 1965. 20. Zambotti, F., M. Carruba, L. Vincentini and P. Mantegazza. Selective effect of maize diet in reducing serum and brain tryptophan contents and blood and brain serotonin levels. Life Sci. 17: 1663-1670, 1976.