SOY PROTEIN-ANOTHER CAUSE OF THE FLAT INTESTINAL LESION

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1 GASTROENTEROLOGY Copyright 1972 by The Williams & Wilkins Co. Vol. 62, No.2 Printed in U. S. A. SOY PROTEIN-ANOTHER CAUSE OF THE FLAT INTESTINAL LESION MARVIN E. AMENT, M.D., AND CYRUS E. RUBIN, M.D. Department of Medicine, University of Washington, Seattle, Washington This is a prospective study of the pathogenesis of a violent gastrointestinal reaction to soy protein in an infant. Within 24 hr of changing this 6-week-old infant's formula to soy milk, he developed sequentially: fever, leukocytosis, cyanosis, vomiting, massive blood-tinged mucoid diarrhea, dehydration, and metabolic acidosis. All symptoms disappeared after discontinuing soy milk. At 6 and 10 months the patient was given a single test feeding of soy milk formula and soy protein isolate respectively. All symptoms recurred promptly. The previously normal jejunal mucosa became acutely inflamed and flat (villi disappeared). The patient recovered completely within 48 hr and villi regenerated within 4 days. Soy lecithin, gluten, and cow's milk neither produced symptoms nor altered intestinal structure. Total hemolytic complement did not change after soy milk exposure nor did circulating eosinophils increase, despite leukocytosis with shift to the left. This is the first documentation of a reversible flat jejunal lesion caused by feeding soy protein to a susceptible infant. Both acute and chronic gastrointestinal reactions of infants to whole cow's milk or to its various proteins are now well established. I - 5 Acutely these patients may exhibit pallor, diarrhea, vomiting, and at times shock. 1, 3 The more chronic reactions may also involve anemia and/or hypoproteinemia 2, 6 resulting from enteric loss of iron2 and protein; 6 steatorrhea has been observed. 4, 5 Attempts to establish the pathogenesis of this illness have not been Received May 14, Accepted September 14, Address reprint requests to: Dr. Cyrus E. Rubin, Department of Medicine, University of Washington, Seattle, Washington This work was supported by Research Grant 20-9R81 from the Children's Orthopedic Hospital and Medical Center; Research Grant CA from the National Cancer Institute, National Institutes of Health, United States Public Health Service; the Clinical Research Center of the University of Washington (National Institutes of Health Grant FR-37) ; Gastroenterology Training Grant 5Tl A,5099 (Dr. Ament); and Career Award CA from the National Institutes of Health, United States Public Health Service (Dr. Rubin). 227 singularly successful; the usually accepted theory of allergic causation has been supported mostly by evidence of circulating antibodies 7 or coproantibodies 8 to whole cow's milk or its derivatives; findings which are notoriously nonspecific. 9, 10, 11 Some fragmentary and contradictory data have accumulated 3, 12 regarding intestinal mucosal structure; but prospective studies with adequate intestinal sampling are lacking, The usual treatment of these patients is to substitute a soy milk formula for cow's milk, Recently even the soy formula has been reported to cause similar acute symptoms, 13 This paper describes several prospective studies in one infant whose gastrointestinal tract reacted violently to a soy milk formula, We determined which part of the soy milk formula caused symptoms and then tested the effect of this fraction on the jejunal mucosa. Prior Course R. H. was born on October 15, 1969 following a 34-week gestation. The mother was a 32-

2 228 AMENT AND RUBIN Vol. 62, No.2 year-old, gravida 5, para 4 white woman. The pregnancy was complicated by prepartal hemorrhage and spontaneous premature labor. Birth weight was 2530 g. The patient's father had lifelong seasonal asthma. The initial feeding was Enfamil [Mead Johnson (skimmed cow's milk, lactose, oleo, corn and coconut oils, soy leci thin, electrolytes, and vitamins)]. During this 1st month of life the patient was asymptomatic but he then developed increasingly frequent loose stools. Because milk intolerance was suspected, he was switched at age 6 weeks to a soy milk formula, Isomil [Ross Laboratories (soy protein isolate, soy lecithin, corn and coconut oil, corn starch, corn syrup solids, sucrose, electrolytes, and vitamins)]. His stools immediately became green, watery, and more voluminous. The following morning he was febrile and vomited all ingested fluids. On hospital admission that afternoon, he was pale, irritable, listless, and febrile (39.8 C). He was mildly dehydrated. His bowel sounds were hyperactive and his abdomen was flat. Although his cry was strong, his grasp was weak and he demonstrated no desire to suckle. His laboratory values were: hemoglobin 14.4 g per 100 ml, hematocrit 46%, white blood count 42,900 (23%, polymorphonuclear leukocytes, 35% stabs, 25% lymphocytes, 3% monocytes, 10% metamyelocytes, 4% promyelocytes, and 0% eosinophils), serum sodium meq per liter, potassium 5.5 meq per liter, CO 2 5 meq per liter, chloride 118 meq per liter, and blood urea nitrogen 27 mg per 100 ml. A lumbar tap yielded spinal fluid with normal cytology, glucose, and protein. The patient's dehydration and acidosis were corrected with intravenous fluids and electrolytes. Because of his leukemoid hematologic picture a lo-day course of kanamycin 15 mg per kg per. day and ampicillin 200 mg per kg per day was given for suspected sepsis although no enteric pathogens were isolated from stool cultures and no organisms were grown in blood cultures. Following rehydration the hemoglobin dropped to 12 g per 100 ml and the hematocrit to 37%. At that time serum proteins were normal (6.4 g per 100 ml to 4.0 g per 100 ml of albumin, 2.4 g per 100 ml of globulin). The patient recovered after 3 days of intravenous feedings and was then placed on Probana (Mead Johnson) formula (banana powder, partially digested cow's milk protein, casein hydrolysate, medium-chain triglycerides, glucose, lactose, and vitamins). He did well on this formula. Twice in the following 3 weeks he was inadvertently fed a soy milk formula instead of his prescribed formula. On each occasion the volume fed did not exceed 1 ounce of soy milk containing 1.6 g of soy protein isolate. In both instances he became ashen in color and listless within 15 min, vomited within 2 hr, and passed large, pink, guaiac-positive stools by 12 hr. Dehydration required intravenous infusions on both occasions. Over the following 48 hr the patient recovered completely and his stools became guaiac negative and semiformed. Stool cultures did not grow pathogenic bacteria. Sigmoidoscopy was performed 48 hr following the second inadvertent formula feeding. The mucosa was spontaneously friable for 5.0 cm. Rectal suction biopsies showed a few early crypt abscesses and moderate increase in the numbers of eosinophiles within the lamina propria. Upon recognition of the probable association between symptoms and the feeding of a soy milk formula, it was decided to study the patient prospectively. Special Experimental Studies It was unclear whether the infant's initial diarrhea in the 1st month of life was related to cow's milk intolerance or to the soy lecithin which was also present in his initial formula (Enfamil). It was also uncertain which component or components in the soy milk formula (lsomil) caused the violent reaction when the formula was substituted for the cow's milk formula (Enfamil). The presence of lactose in his initial cow's milk formula and of sucrose in his soy milk formula suggested that lactose and sucrose tolerance tests should be performed before challenge with soy milk. At age 10 weeks, on separate days, lactose and sucrose tolerance tests were performed. In each test a dose of 2 g per kg was fed as a 20% solution by gavage tube. Capillary blood true glucose was determined at 1/2 -hr intervals for 2 hr. A normal test was considered a rise in blood glucose of 20 mg per 100 ml or more above base line value. These tests were normal. Base line rectal biopsies were taken with the multipurpose suction biopsy tube 14 1 to 3 cm proximal to the anal mucocutaneous junction using 5 to 7 mm Hg of negative pressure. All biopsies were oriented, embedded, and sectioned by previously

3 FebrlUlry 1972 SOY PROTEIN-INDUCED INTESTINAL LESION 229 described methods. 15 They were normal as was a sigmoidoscopic examination. The infant became listless and pale within 1 hr of drinking 4 ounces of a soy milk formula (Isomil), containing 6.4 g of soy protein isolate. He began vomiting within 2 hr. By 4 hr his temperature rose to 39.8 C; his white blood count rose from 10,500 to 18,500 and he began passing mushy, guaiac-negative stools. Over the following 12 hr the stools became progressively looser and blood tinged. His weight dropped 300 g. At 24 hr he was recovered and a lactose tolerance test was abnormal. Sigmoidoscopy demonstrated minor mucosal friability although rectal biopsies after exposure showed no changes. Six days later he was challenged with a cow's milk formula containing soy lecithin (Enfamil). The lactose tolerance test was normal immediately preceding this challenge. After 4 ounces of Enfamil he developed watery mushy acid diarrhea and lost 150 g. The following day a lactose tolerance test was flat and caused diarrhea to recur transiently. Rectal biopsies taken over a 24-hr period following cow's milk challenge showed no abnormalities. The infant was discharged home on Probana formula, rice cereal, pears, and applesauce. He remained free from gastrointestinal symptoms on this diet. Age 6 months. At age 6 months the patient was in good health and had neither wheezing nor eczema. He was studied again to assess the effect on jejunal structure of a formula containing both soy protein and soy lecithin, ProSobee [Mead Johnson (soy oil, sucrose, dried glucose syrup, electrolytes, and vitamins)]. Before study the fecal fat excretion was normal (3.2 g per day).16 White blood count was 7100 and the differential count was normal. Proximal jejunal (fig. la) and rectal biopsies were normal. Small intestinal biopsies were obtained with a miniaturized biopsy tube for infants (a smaller diameter more flexible version of the multipurpose tube, available from Quinton Instrument Co., Seattle, Wash.) (fig. 2). They were taken under visual control at the duodenojejunal junction using a modified image amplifier which delivered only 0.12 rad per min to the patient. The infant was premedicated with a mixture of meperidene 1.0 mg per kg; promethazine 0.5 mg per kg, and chlorpromazine 0.5 mg per kg. All subsequent intestinal biopsies were performed in this manner. The patient was fed 7 ounces of ProSobee formula. Within 1 hr he became pale, irritable, febrile, and lethargic. During the first 12 hr he vomited several times and passed a voluminous stool containing mucus and undigested formula. At 12 hr the proximal jejunal biopsy was grossly hemorrhagic. Histologically it showed hemorrhage, edema, absent eosinophils, and polymorphonuclear leukocytic infiltration in the lamina propria; villi were absent, and the surface epithelium was abnormal and had a few microulcerations (fig. Ib). At 24 hr the biopsies were no longer edematous and hemorrhagic but an increased number of plasma cells and lymphocytes were evident in the lamina propria; villi were still absent and the surface epithelium was still abnormal although microulcerations were no longer present (fig. Ie). Rectal biopsies taken during the 24 hr after challenge were normal and no mucosal friability was evident at sigmoidoscopy. To assess the possible allergic nature of the reaction, serum hemolytic complement was measured (complement determinations were performed by Dr. Bruce Gilliland) by a modification of the method of Kabat and Mayer 17 before, and 90 min, 4 hr, and 12 hr after the soy milk feeding. A significant change did not occur. He did develop leukocytosis with neutrophilia. On the 2nd day, his previous Probana formula was restarted and his stools became formed and guaiac-negative. At the time of discharge, gluten-containing foods were added to the diet and the patient continued to do well. Age 10 months. In order to determine if he could tolerate homogenized cow's milk it was decided to readmit the patient for further study. At 10 months he weighed 11 kg. He was in obvious good health despite 4 months on a gluten-containing diet. On physical examination, however, he

4 230 AMENT AND RUBIN Vol. 62, No.2 FIG. 1. Jejunal response to soy milk challenge at age 6 months. a, Normal base line biopsy at the duodenojejunal junction, before challenge. Note normal villi; b, Biopsy at the duodenojejunal junction, 12 hr after challenge. Note absence of villi, surface microulceration-m, edema- E, and hemorrhage- H; c, Biopsy at the duodenojejunal junction, 24 hr after challenge. Note that edema, hemorrhage, and microulcerations have disappeared although villi have not yet regenerated (hematoxylin and eosin, x 115). had diffuse inspiratory and expiratory rhonchi with scattered expiratory wheezing which did not disappear after epinephrine administration. He was in no respiratory distress. Fecal fat excretion was normal (2.2 g per day). Proximal jejunal biopsies were normal. Sigmoidoscopy and white blood count were normal. Initially he was fed 4 ounces of homogenized cow's milk." He developed no symptoms.

5 February 1972 SOY PROTEIN-INDUCED INTESTINAL LESION 231 FIG. 2. Infant miniaturized multipurpose biopsy tube below, compared with four-hole adult tube above. Note smaller diameter and greater flexibility of infant tube. Proximal jejunal and rectal biopsies as well as sigmoidoscopy remained normal 24 hr after challenge. Total hemolytic complement and white blood count were normal and unchanged. Over the next 5 days he was fed cow's milk and did not become symptomatic. He was discharged on homogenized cow's milk and a mixed diet free of soy bean products. Age 10 1/ 2 months. He was readmitted 1 week later to determine if soy protein isolate was the injurious substance in soy milk. The infant was well and happy. Proximal jejunal (fig. 3a) and rectal biopsies as well as sigmoidoscopy were normal. He was then fed 4 ounces of water containing 6.5 g of soy protein isolate. (Edi Pro-purified isolated soy proteins in powder form (95% protein-dry weight, 0.18% fat, and 4% moisture and electrolytes). Obtained by Mead Johnson for manufacturing ProSobee from Ralston Purina Co.). During the next 24 hr he developed the same symptoms that he had when challenged with a complete soy milk formula. Rhonchi and wheezes which had been heard before the feeding were no longer present. Proximal jejunal biopsies were taken 24, 48, 72, and 96 hr after soy protein challenge. Two small intestinal biopsies at 24 hr were devoid of villi (fig. 3b) and otherwise identical to those 24 hr following the previous test feeding of soy milk (Pro Sobee). Although the white blood count did not rise following feeding soy protein isolate, there was a significant shift to the left which returned to normal by 48 hr. By 96 hr the proximal jejunal mucosa had become normal morphologically (fig. 3c). No changes were seen on sigmoidoscopy or rectal biopsy at 24 hr after the test feeding. The serum complement levels remained normal throughout this study. Serum precipitins to whole soy protein were analyzed (these assays were performed in the laboratory of Dr. Warren Bierman) using Ouchterlony's technique. 18 Precipitins to soy protein isolate were absent from the patient's serum on the day before the test feeding and were present in 1: 4 dilution 19 days later. No serum precipitins to soy protein isolate were found in an atopic infant of the same age on ProSobee formula nor in a normal adult on a normal mixed diet. The patient was protected from dehydration during the various challenges by continuous intravenous fluid and electrolyte replacement. This project was reviewed and approved by our Biomedical Sciences Review Committee which authorizes all experiments involving human beings. Informed consent was obtained from the parents before each test. All studies from age 6 to 10 1/ 2 months were done in the Clinical Research Center of the University of Washington. Discussion Our evidence indicates that feeding soy proteins to a susceptible infant caused a violent systemic and jejunal reaction. Gluten, soy lecithin, lactose, and sucrose caused no such reaction.. The response to cow's milk was more complicated because this infant first developed loose stools at 4 weeks of age while on a cow's milk formula. At 10 weeks, only moderate diarrhea and transient lactose intolerance developed in response to the same milk formula. This is not surprising because it has been demonstrated that diarrhea or steatorrhea and

6 232 AMENT AND RUBIN Vol. 62, No.2 FIG. 3. Jejunal response to soy proteins at age lov, months. a, Normal base line biopsy at the duodenojejunal junction, before challenge. Note normal villi; b, Biopsy at the duodenojejunal junction, 24 hr after challenge. Note absence of villi and similarity in appearance to biopsy illustrated in figure Ie; e, Biopsy at the duodenojejunal junction, 96 hr after challenge. Note return of villi (hematoxylin and eosin, x 115).

7 FebTlllJry 1972 SOY PROTEIN"INDUCED INTESTINAL LESION 233 transient lactose intolerance may follow exposure to milk proteins in susceptible infants.5 When retested at age 10 months with homogenized cow's milk, our patient developed neither symptoms nor intestinal mucosal changes. Others 3. 6 have observed that intolerance to cow's milk often disappears between age 6 months and 2 years. At age loy2 months our patient still had a violent reaction to soy protein. It will be important to follow this child to determine whether the injurious character of soy proteins will disappear or whether it will persist throughout life. Gluten-containing foods did not cause intestinal injury or symptoms in our patient but one still wonders whether the pathogenesis of our patient's response to soy protein might not be somewhat similar to the injurious effect of gluten in celiac sprue, i.e., the result of a permanent, inherited abnormality. 21 Unlike the other report of a gastrointestinal reaction to soy milk occurring only after multiple exposures,13 our patient reacted after his first exposure. The systemic symptoms and intestinal injury after exposure to soy protein suggest that some substance or substances within this mixture of proteins22 ' 24 injured the bowel locally by some unknown mechanism. The systemic symptoms may have been caused by abnormal absorption of one of the several soy proteins or one of their derivatives through the damaged intestinal mucosa. The rise in circulating precipitins to whole soy protein, which occurred after feeding of soy proteins, supports this theory. Could the reaction to soy protein in this patient have been allergic? It may be, but there is no evidence to support this possibility. The patient was not atopic; complement was not consumed and circulating eosinophiles did not rise during the reaction to soy protein challenge. Furthermore the patient's wheezing did not respond to epinephrine and wheezing disappeared during the adverse response to feeding of soy protein isolate. This is the 1st reported case in which soy protein was shown to cause a flat jejunal lesion with complete loss of villi. Histologically the lesion was indistinguishable from that seen in untreated celiac sprue and several other rarer diseases. 25 The lesion was shown to be reversible and disappeared completely within 4 days after intestinal exposure to soy protein. The mechanism of the reaction is unknown. One wonders whether rare violent gastrointestinal reactions of infants to introduction of other new foods will be shown to have similar reversible jejunal pathology. REfERENCES 1. Goldman AS, Anderson DW Jr, Sellers WA, et al: Milk allergy. I. Oral challenge with milk and isolated milk proteins in allergic children. Pedi atrics 32: , Wilson JF, Heiner DC, Lahey ME: IV. Milkinduced gastrointestinal bleeding in infants with hypochromic microcytic anemia. JAMA 189: , Freier S, Kletter B, Gery I, et al: Intolerance to milk protein. J Pediatr 75: , Davidson M: Malabsorption defect induced by ingestion of beta lactoglobulin. J Pediatr 66: , Liu HY, Tsao MU, Moore B, et al: Bovine milk protein induced intestinal malabsorption of lac tose and fat in infants. Gastroenterology 54:27-34, Lebenthal E, Laor J, Lewitus A, et al: Gastrointestinal protein loss in allergy to cow's milk betalactoglobulin. Isr J Med Sci 6: , Heiner DC, Sears JW, Kniker WT: Multiple precipitins to cow's milk in chronic respiratory disease. Am J Dis Child 103: , Katz J, Spiro HM, Herskovic T: Milk precipita ting substance in the stool in gastrointestinal milk sensitivity. N Engl J Med 278: , Rothberg RM, Farr RS: Antibovine serum albumin and anti alpha lactalbumin in the serum of children and adults. Pediatrics 35: , Saperstein S, Anderson DW Jr, Goldman AS, et al: Milk allergy. III. Immunological studies with sera from allergic and normal children. Pediatrics 31: , Davis SD, Bierman CW, Pierson WE, et al: Clini cal nonspecificity of milk coproantibodies in diarrheal stools. N Engl J Med 282: , Kuitunen P, Visakorpi JK, Hallman N: Histopathology. of duodenal mucosa in malabsorption

8 234 AMENT AND RUBIN Vol. 62, No.2 syndrome induced by cow's milk. Ann Paediatr Fenn 205: 54-63, Mendoza J, Meyers J, Snyder R: Soybean sensitivity: case report. Pediatrics 46: , Brandborg LL, Rubin CE, Quinton WE: A multipurpose instrument for suction biopsy of the esophagus, stomach, small bowel, and colon. Gastroenterology 37: 1-16, Rubin CE, Brandborg LL, Phelps PC, et al: Studies of celiac disease. I. Gastroenterology 38:28-49, Weijers HA, van der Kamer JH: Coeliac disease I. Criticism of the various methods of investigation. Acta Paediatr Scand 42:24-33, Kabat EA, Mayer MM: Experimental Immunochemistry. Second edition. Springfield, Ill, Charles C Thomas Publisher, 1961, p Ouchterlony 0: Diffusion-in-gel methods for immunological analysis. Progr Allergy 5: 1-78, Rubin CE, Brandborg LL, Flick AL, et al: Studies of celiac sprue. ill. The effect of repeated wheat instillation into the proximal ileum of patients on a gluten-free diet. Gastroenterology 43: , Rubin CE, Brandborg LL, Flick AL, et al: Biopsy studies on the pathogenesis of coeliac sprue, Intestinal Biopsy, Ciba Foundation Study Group no. 14. London, J and A Churchill Ltd, MacDonald WC, Dobbins WO, Rubin CE: Studies of the familial nature of celiac sprue using small intestinal biopsy. N Engl J Med 272: , Catsimpoolas N, Ekenstram C, Meyer EW: Separation of soy bean whey proteins by isoelectric focusing. Cereal Chern 46: , Catsimpoolas N, Ekenstram C: Isolation of alpha, beta, and gamma conglycinins. Arch Biochem Biophys 129: , Wolf WJ: Soybean proteins: Their functional, chemical, and physical properties. J Agric Food Chern 18: , Rubin CE, Eidelman S, Weinstein WM: Sprue by any other name. Gastroenterology 58: , 1970