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The pathophysiology of mucosal changes observed in infants with chronic protracted diarrhea is poorly understood. We report on two brothers suffering from a special form of sucrase isomaltase (SI) deficiency. The children presented with weight loss and dyspepsia after sucrose exposition. We performed an H respiration test, which showed a pathologic result in the younger brother. Analysis of the brush border enzyme activities showed low expression of lactase and SI. Immunoelectron microscopy of duodenal biopsies showed an isolated SI deficiency in a mosaic pattern [e.g., 42% (14%) crypt enterocytes and 64% (59%) villus enterocytes with decreased amounts of SI on microvilli], whereas lactase and aminopeptidase n (ApN) were present at the apical membrane of all cells in a normal range. The SI mosaic pattern of these patients shows that the enterocytes contain low amounts of SI on the apical membrane but express normal quantities of other disaccharidases. These findings suggest the existence of different clonal expressions or specific (posttranslational) mechanisms of postGolgi transportation for individual brush border enzymes. It remains unresolved whether the mosaic distribution is part of a normal maturation process or caused by a lack of an overall control mechanism in the expression of brush border hydrolases.

Currently 15% of U.S. infants are fed soy formulas that contain up to 14 mg of genistein equivalents/L. Our goal was to investigate the impact of dietary genistein on intestinal development. Piglets (n=8/group) were fed sow milk replacer (MR), MR+1 mg/L of genistein (LG), or MR+14 mg/L of genistein (HG) for 10 d. Formula intake, weight gain, and intestinal length and weight were similar in all groups. Average serum genistein concentration in the HG group was similar to that of soy formula-fed infants. No significant effects of genistein on enterocyte apoptosis, lactase, and sucrase activities or electrophysiologic measures were observed in jejunum or ileum. Jejunal and ileal villus heights were not significantly different, but the percentage of proliferating cell nuclear antigen-positive jejunal crypt cells in the HG was reduced 50% compared with that in MR and LG (p=0.001), indicating decreased proliferation. Enterocyte migration distance in the HG group tended to be 20% less (p=0.1) than LG or MR. Jejunal estrogen receptor beta mRNA expression in HG was half of that in LG (p=0.05), but neither was significantly different from MR. In conclusion, genistein at the level present in soy infant formula is bioactive in the small intestine and results in reduced enterocyte proliferation and migration. The lack of effect of genistein on nutrient transport and enzyme activity suggests that the impact of genistein is greater on proliferating versus differentiated intestinal cells.

Intrauterine growth retardation (IUGR) affects almost 10% of infants born in the United States. It may be responsible for delayed gastrointestinal function and is an important cause of perinatal morbidity and mortality. The New Zealand White rabbit provides an optimal model for the study of naturally occurring IUGR. At term, birth weight is determined by fetal position within the bicornuate uterus. The small intestinal disaccharidase enzymes are indicators of bowel maturity and function. To examine potential differences in disaccharidase development between normal and IUGR fetuses, this rabbit model was investigated. Jejunum was harvested at multiple stages in rabbit development including the third trimester fetus, neonate, and adult. Lactase, maltase, and sucrase enzyme activity, as well as total protein content, was determined. Results were analyzed by the 2-tailed t test and ANOVA. Lactase activity appeared in the mid-third trimester, peaked in the early neonatal period, then declined to adult levels. Maltase activity appeared in the early third trimester and gradually rose to adult levels. Sucrase remained at trace levels until the mid-neonatal period, reaching adult levels by weaning. Both lactase and maltase activity were depressed in IUGR fetuses compared with their normal littermates. This pattern of disaccharidase depression continued into the neonatal period until catch-up growth occurred at 2 wk when levels equalized. This report describes differential small intestinal disaccharidase development between normal and growth-retarded rabbit fetuses in a naturally occurring model of IUGR.

The mechanism(s) by which insulin enhance prematurely the activity of brush border membrane (BBM) hydrolases in rat immature intestine is unknown. Therefore, we have compared the responses of four BBM enzymes [sucrase-isomaltase (SI), maltase, lactase-phloridzine hydrolase (LPH), and aminopeptidase] with exogenous insulin, the analog B-Asp10, IGF-I, and antireceptor MAb [insulin-receptor (IR) MAb] given to preweaning pups. Low doses of insulin caused a precocious induction of SI and of SI mRNA and stimulated maltase activity without effect on LPH nor on aminopeptidase activities. IGF-I given at the same dose as that of insulin had no detectable effect on these enzymes. Administration to sucklings of IR MAb prevented the effect of endogenous insulin by inhibiting the expression of SI and maltase without effect on LPH activity. B-Asp10, an insulin analogue that exhibits in vitro a 3.5-fold increase in receptor affinity with sustained signaling of the receptor tyrosine kinase, caused an overexpression of SI by 3.5-fold and of maltase by 1.5-fold compared with equivalent doses of normal insulin. The premature increases in SI activity, SI mRNA, and maltase activity in response to insulin were dose-dependent and were associated with dose-dependent increases in intracellular spermine and spermidine concentrations. In conclusion, these data suggest that the premature induction of SI by insulin is mediated by a dose-dependent signal initiated by binding of the hormone to its intestinal receptor, which after transduction into the cell indirectly triggers the transcription of the SI gene, possibly by changes in intracellular polyamine concentrations.

During the 3rd wk of postnatal life in the rat, dramatic maturational changes occur in the structure and function of the small intestine, enabling the animal to make the transition from milk to solid food. To investigate the role of GH in the regulation of this complex process, we studied postnatal intestinal maturation in the spontaneous dwarf rat, a strain of Sprague-Dawley rats with an autosomal recessive mutation in the GH gene resulting in complete but isolated GH deficiency. GH-deficient and GH-normal littermates were studied at d 7 and 14 (suckling) and d 23 (postweaned). The body weight of GH-deficient animals was inhibited by 60% at each age. Longitudinal growth of the small intestine was not inhibited, suggesting that longitudinal small bowel growth is independent of GH regulation. Mucosal cell mass was significantly lower in GH deficiency at all ages studied, and digestive hydrolase capacity per cm of intestine was significantly lower in GH-deficient postweaned animals. However, epithelial cell mass increased markedly in association with weaning and the maturation of lactase, sucrase, and aminooligopeptidase proceeded normally in GH deficiency. These data suggest that, although GH is not required for normal postnatal intestinal maturation, the mucosal epithelial hypoplasia found in GH-deficient animals suggests that GH or GH-dependent factors act as an intestinal mucosal growth factor whose function is to promote the homeostatic or steady-state regulation of mucosal epithelial growth.

Despite extensive study in both humans and nonhuman mammals the mechanisms which regulate intestinal lactase activity, particularly during development, are incompletely understood. Our previous studies of human adults are consistent with an important role of lactase-phlorizin hydrolase (LPH) mRNA abundance in determining the lactase persistence/nonpersistence phenotypes. Our intent in the present study was to determine the role of LPH mRNA in the regulation of lactase in children. We therefore studied duodenal mucosal biopsies from 39 children undergoing diagnostic upper endoscopy in whom significant small intestinal and nutritional disease was excluded. We found no relationship between the level of LPH mRNA and lactase enzymatic activity. Our observations suggest the importance of posttranscriptional mechanisms in lactase regulation in human children.

To study the relative expression of lactase, sucrase-isomaltase, dipeptidyl peptidase IV, and the Na + -dependent glucose transporter mRNA transcripts in small samples of human tissue, we have developed and validated a very simple semiquantitative RNA polymerase chain reaction method that can be used on as little as 5–10 mg of tissue. Here we report the use of this method to study the expression of these genes at different stages of development, in different tissues and in different parts of the intestine, in comparison with another intestinal marker, the colon-specific transcript of carbonic anhydrase 1. Lactase, sucrase-isomaltase, and the Na+-dependent glucose transporter mRNA are expressed predominantly in the small intestine, although lactase mRNA is expressed at a very low level in fetuses. Dipeptidyl peptidase IV mRNA shows a much wider tissue distribution. Sucrase-isomaltase and dipeptidyl peptidase IV mRNA are present at high levels in fetal colon and also at surprisingly high levels in adult colon. Lactase mRNA, on the other hand, is present at very low levels in fetal colon and is not detectable at all in adult colon. The Na + -dependent glucose transporter mRNA in contrast is expressed at higher levels in the adult colon than in the fetal colon. This is also the case for the carbonic anhydrase 1 transcript, although this transcript is not expressed in the small intestine. Thus, each of these genes shows different developmental and cell-specific regulation.

The synergistic effects of dexamethasone (DEX) and thyroxine (T4) on the postnatal maturation of the 13-d-old rodent small intestine has been studied. Previous studies have shown that hydrocortisone and T4 produced a synergistic response in enzyme maturation. However, T4 elevates corticosteroid-binding globulin, which reduces the clearance of hydrocortisone. Thus, the apparent synergy between T4 and hydrocortisone may have been due to increased glucocorticoid availability. DEX, which does not bind to corticosteroid-binding globulin, was given (d 8–12) at 25 pmol (i.e. 0.01 μg)/g body wt/d as established by a dose-response study in which this dose of DEX induced one third the maximum response in sucrase activity. In this way, synergy with T4 (130 pmol/g body wt/d, i.e. 0.1 μg/g body wt/d, d 5–12) could still be observed. Glucoamylase, lactase, acid β-galactosidase, alkaline phosphatase, and sucrase activities were determined in two regions of the small intestine. Overall, the results for the two hormones administered alone showed intestinal maturation to be not significantly affected in the T4 group and partially stimulated in the DEX group. When combined, DEX + T4 synergistically increased jejunal sucrase, ileal glucoamylase, and duodenal alkaline phosphatase, and lowered ileal acid β-galactosidase. The striking exceptions to the general pattern were two brush border enzymes that normally decline during intestinal maturation, namely ileal alkaline phosphatase and jejunal and ileal lactase. For these enzymes, DEX alone did not elicit precocious maturation, and there was no evidence for a synergistic interaction of these two hormones. Serum corticosterone concentrations also were measured. When corticosterone concentrations were compared with enzyme activity, no correlation was found. Thus, DEX + T4 act synergistically in the maturation of four enzymes in the rodent small intestine. Lactase and ileal alkaline phosphatase are regulated differently. Further studies are required to determine the mechanism through which T4 enhances the DEX effect.

To further document the effect of insulin on intestinal maturation, suckling rats were treated either with exogenous insulin (12.5 mU.g body wt, intraperitoneally, twice daily) or with saline from d 8 to 12 postpartum. Sucrase activity in brush border membrane extracts was precociously induced by insulin, whereas the activities of other brush border membrane enzymes (maltase, aminopeptidase, and neutral lactase) were enhanced (+ 30 to + 131%, p less than 0.01 versus controls). The lysosomal enzyme, N-acetyl-beta-glucosaminidase, which normally declines at weaning was significantly (p less than 0.025) decreased in both villus (-51%) and crypt cells (-57%) isolated from the jejunum of insulin-treated rats. The microsomal enzyme, sulfatase C, and the cytosolic enzyme, lactate dehydrogenase, were also sensitive to insulin with decreases in activity ranging from -37 to -63% (p less than 0.05) compared to saline-treated control rats. Insulin at doses of 0.5 or 12.5 mU did not influence plasma total corticosterone levels, which were about 9-fold lower in suckling than in 25-d-old weaned rats. In weaned rats (from d 25 to 32) insulin treatment (12.5 mU) failed to influence the activity of brush border membrane hydrolases or of lysosomal, microsomal, and cytosolic enzymes. The synthesis rate of mature sucrase-isomaltase, measured in weaned rats (32 d) by the incorporation of 14C-leucine into the enzyme precursor protein, was equivalent in both groups. These data demonstrate that the immature enterocyte of the suckling rat is responsive to insulin, whereas the mature enterocyte of the weaned rat is unresponsive.(ABSTRACT TRUNCATED AT 250 WORDS)

Maturation of the fetal gastrointestinal tract (GIT) is influenced by both luminal stimuli (e.g. swallowed fluid) and hormonal factors (e.g. endogenous cortisol release). The aims of the present study were 1) to investigate GIT growth and maturation during the last 20% of gestation in pigs (term = 114 +/- 2 d), and 2) to investigate the effect of esophageal ligation, to prevent fetal swallowing, at 80% to 91% gestation. In normal fetuses, marked increases occurred during late gestation in body weight (+95%), relative intestinal weight (+79%, g kg(-1) body weight), activity of some digestive enzymes (1.5- to 10-fold), and absorption of glucose and intact proteins (3- to 6-fold). Fetuses with ligated esophagi had lowered body weight (-20%), reduced intestinal weight (-43%), aminopeptidase A activity (-24%), and glucose absorption (-27%), while lactase, sucrase, and dipeptidylpeptidase IV activities were increased (+40-50%), compared with sham-operated fetuses (all p < 0.05). Other parameters of GIT function remained unchanged by esophageal obstruction (absorption of amino acids and immunoglobulin, activity of chymosin, amylase, trypsin, chymotrypsin, maltase, aminopeptidase N -- all expressed per gram GIT tissue). Ligated fetuses had elevated cortisol levels, which is known to stimulate fetal GIT maturation. We conclude that the rapid development of GIT function in late gestation is diminished by esophageal obstruction, mainly due to slower GIT growth and not inhibition of normal functional development of enterocytes.

Although mitogen-activating protein (MAP) kinases are crucial signal transduction molecules regulating cellular proliferation, differentiation, and morphology, their ontogenic changes in the small intestine have not been analyzed. Also, it remains unknown which pathway of activated MAP kinases regulates the expression of brush border membrane hydrolases during growth. Therefore, we have analyzed the mucosal distribution, ontogeny, and responses to insulin and to inhibitors of p44, p42, and p38 MAP kinases in immature and mature enterocytes using Western blot analysis and autoradiography after immunoprecipitation, immunohistochemistry, and in vitro phosphorylation assays. Between d 10 and 40 postpartum, diphosphorylated active p44/p42 extracellular regulated protein kinases (ERKs) increased in abundance compared with total immunoprecipitated ERKs, and were highly responsive to exogenous insulin. In concordance, ERK total activity increased by 4-fold during the same period of growth and was further enhanced 2-fold by exogenous insulin. In weaning rats, ERKs were mainly located in membranes of villus cells and with less intensity in crypt cells. By contrast, p38 MAP kinase was unresponsive to insulin and was confined to nuclei. Administration to sucklings of PD 098059, a specific inhibitor of ERKs, not only inhibited the premature stimulation of sucrase, lactase, and maltase total activities in response to exogenous insulin, but also depressed the natural expression of these brush border membrane enzymes in the absence of insulin stimulation. In concordance, administration of SB 203580, a specific inhibitor of p38 MAP kinase, failed to inhibit both the response of brush border membrane hydrolases to insulin and their natural expression in the absence of insulin stimulation. We conclude that the ontogenic expression of brush border membrane hydrolases and their premature stimulation by insulin are regulated at least in part by the activation of p44/p42 ERKs but not by p38 MAP kinase.

Cryptosporidium parvum produces a prolonged watery diarrhea unresponsive to conventional antimicrobials. Because of reported efficacy of antibody-based immunotherapy, we studied the effect of inexpensive, commercially available oral bovine serum concentrate (BSC) in experimental cryptosporidiosis. Twenty-four calves were treated with 57 g/d BSC (n = 12) or soy protein (n = 12) added to their standard whey protein-based milk replacer (227 g/2 L twice daily). Of the 24, 9 were also treated with L-glutamine (GLN), 8 g/L (50 mM) in the milk (5 calves in the BSC group and 4 in the soy group). Animals were inoculated with 10(8) cryptosporidium oocysts per os on d 8 of life and received oral rehydration on d 12-14. Eight uninfected controls were treated with BSC or soy protein. Fecal and urine volume and urinary Cr-EDTA excretion were measured. Animals were killed on d 18 of life. Cryptosporidiosis induced severe watery diarrhea lasting9 d and produced a 25% increase in intestinal permeability, a 33% decrease in villous surface area, and a 40% reduction in mucosal lactase specific activity. Glutamine treatment had no effect on the diarrhea or any of the intestinal tests; and therefore pooled data were used to compare the 12 calves treated with BSC with the 12 treated with soy. In animals receiving BSC, peak diarrheal volume and intestinal permeability were reduced 33%, fewer oocysts were shed, intestinal crypts were significantly deeper, and villous surface area returned to normal by 9 d after infection (all por= 0.05). BSC should be studied as a treatment for human cryptosporidiosis.

The effect of orally administered IGF-I on intestinal development was assessed in piglets. Cesarean-derived, colostrum-deprived piglets received formula alone or formula containing 65 nM (500 microg/L) of recombinant human IGF-I. IGF-I intake averaged 200 microg/kg/d. On d 7 and 14 postpartum, piglets were killed, organs were removed and weighed, and tissue and blood samples were collected. The small intestine was divided into 13 segments that were weighed and measured. A sample of each segment was fixed in formalin, and the mucosa was scraped for enzyme analyses. Food intake, body and organ weights, intestinal weight, length, protein, DNA and RNA content did not differ between the treatment groups. Serum IGF-I, IGF-II, and IGF-binding protein profiles and tissue IGF-binding protein mRNA expression were also comparable between the treatment groups. In contrast, intestinal enzymes and villus height were increased by oral IGF-I. Lactase was approximately 2-fold higher (p < or = 0.05) in the jejunum and proximal ileum, and sucrase was approximately 50% higher (p < or = 0.05) in the jejunum of IGF-I-treated animals than in controls. Villus height in the terminal ileum was approximately 50% greater in IGF-I-treated animals than in controls (p = 0.03). In conclusion, orally administered IGF-I at 200 microg/kg did not affect whole body or organ growth or serum IGF-I concentrations; however, intestinal disaccharidase activity and ileal villus growth were responsive to orally administered IGF-I, supporting a potential role for milk-borne IGF-I in neonatal intestinal development.

Growth failure is a major complication of chronic hypoxemia, as seen in infants and children with cyanotic congenital heart disease. To determine whether chronic hypoxemia during infancy affects the gastrointestinal tract, we examined small intestinal growth and digestive enzyme activities in chronically hypoxemic newborn lambs and in age-matched controls. Chronic hypoxemia was produced by placing an inflatable occluder around the main pulmonary artery and performing a balloon atrial septostomy. Aortic oxygen saturation was reduced to 60− 74% for 2 wk, after which the small intestine was removed for analysis. During chronic hypoxemia, somatic growth rate was decreased to 60% of control (hypoxemic, 135 $$ 20 versus control, 216 $$ 26 g/d5Jp < 0.02). No differences in caloric intake were found (hypoxemic, 129 $$ 4 versus control, 128 $$ 4 kcal/kg/d). Chronic hypoxemia did not alter small intestinal growth, as measured by jejuno-ileal weight, jejuno-ileal length, mucosal weight, or mucosal protein or DNA contents. However, sp act of lactase, the principal disaccharidase of the infant lamb intestine, were significantly decreased (hypoxemic, 0.08 ± 0.01 versus control, 0.146 ± 0.03 units of enzyme activity/mg DNA, p < 0.05), as were the total small intestinal contents of lactase (hypoxemic, 61.7 ± 7.0 versus control, 120.6 ± 21.7 units of enzyme activity, p < 0.01). There also were decreases in specific and total activities of other digestive enzymes such as maltase, amino-oligopeptidase, and alkaline phosphatase in hypoxemic intestine that did not achieve statistical significance. In conclusion, chronic hypoxemia in infancy is associated with decreases in activities of intestinal lactase, which may result in diminished capacity for absorption of dietary carbohydrate. Alterations in intestinal function may contribute to growth failure associated with chronic hypoxemia.

The hypothesis that dietary supplementation with LC-PUFAs may improve clinical recovery of malnourished children with damaged small intestinal mucosa secondary to chronic diarrhea, was tested. Patients 15d), with dehydration

Lactose intolerance (LI), a consequence of decreased or absent intestinalβ-galactosidase activity is common in the pediatric population. Treatment generally includes dietary lactose restriction, use of exogenous lactase or a combination of the two. To evaluate the effectiveness of these therapies we interviewed 100 patients (58 male, 42 female, aged 4-19 y, mean=11.0 y) 6-36 months after diagnosed as being LI by a positive hydrogen breath test, using a standardized questionnaire. The interview included questions about initial symptoms, present diet, use of exogenous lactase and present symptoms. Participants also graded their symptom severity. Of the 100 subjects interviewed, 60% were on a lactose-free diet, 67% used lactase tablets, and 42% used both. Data analysis showed a significant (p

Premature infants may be intolerant to lactose-containing formulas due to inadequate production of lactase that persists until about 36 weeks gestational age. We hypothesized that a low lactose (< 5% lactose) premature infant formula would ameliorate feeding intolerance in premature infants. We randomized 306 admissions to the NICU who were < 1800 gms and< 36 weeks to receive Enfamil Premature Formula (EPF) 24 kcal/oz or a specially prepared low lactose study formula (LL EPF) which was comparable to EPF 24 except for the replacement of lactose with maltose. All infants were on the same protocol for advancement of feeds. Caretakers were blind to study assignment. We have complete data for 252 study infants, 69 of whom received some human milk. We report data for the 183 infants who received EPF (n=93) or LL EPF (n=90) only. The mean duration of study enrollment was 1 month (31.3 days). Groups were comparable for birth weight (1180 vs. 1150 g), gestational age (30.3 vs. 30.0 wk), and day of first feed (7.0 vs. 7.1). Data are mean(S.D.).

BACKGROUND: Experimental data both supporting and refuting efficient lactose hydrolysis in enterally fed preterm babies is generally derived from fetal studies or using indirect assessments. Measurement of disaccharidase activity in the duodenal fluid of surviving preterm infants is a more direct approach. SUBJECTS: 63 infants, median (range) gestation 30 weeks (24 - 40) and birthweight 1300g (600 -2480).METHODS: Duodenal aspirates were obtained over the first 2 months of life. Disaccharidase activity was determined colorimetrically .RESULTS: 71/92 (77%) of lactase, 52/94 (55%) of sucrase and 59/93(63%) of maltase activities fell below the lower published reference range for older children. There was no increase in enzyme activities with increasing post-conceptual or post-natal age. No infant showed clinical evidence of malabsorption. CONCLUSIONS: Disaccharidase activities are frequently low in the preterm enterally fed newborn compared with older children. However, the absence of clinical evidence of lactose malabsorption suggests that lactose is assimilated in the colon. Preterm milk formulae need not contain less lactose than breast milk.

MAbs against enzymes of the brush border provide a new approach to evaluate intestinal lesions in severe PD. The 10 patients with PD in whom a small bowel biopsy was performed during 1992 were evaluated by means of MAbs for lactese (L), sucrase (S), maltsse (M) and aminopeptidase (A); results were compared with those of light mi croscopy, histochemistry lor lactase, disacchandase activities (Dalqvist) and clinical history. Diagnosis was DP and severe malnutrition (MN) in 4 cases, congenital sucrose intolerance (CSI) 1, cow's milk protein intolerance (CMI) 2, celiac disease (CD) 1, sIgA deficiency 1 and cryptosporidias is (Crypt) 1. The latter was the only child with a positive ethiologic study. MAP ofr sucrase detected more enzyme than Dalqvist, which may be interpreted as tht MAP binds non-functional molecules. In patient with CSI, MAP for sucase detedted less enzyme than Dalqvist, which is in agreement with the production of a defective molecule.

Enhanced digestion of yogurt by lactose intolerant individuals is due to inherent beta-galactosidase (lactase) in the culture organisms that aids in the hydrolysis of ingested lactose. We studied yogurt intolerance in 80 children with lactose intolerance, aged 0.5-13 years (x=4.25). Lactose tolerance was evaluated using relevant clinical and laboratory parameters. Small intestinal mucosa was morphologically examined in children with chronic diarrhea. Of 80 patients, 60 (80.00%) had secondary type lactose intolerance, followed by damaged mucosa of small intestine, associated with Rotavirus (23) and Salmonella (1) gastroenteritis, postinfective persistent diarrhea (16), infestation with Giardia lamblia (3) and Ascaris lumbricoides (1) and gluten sensitive enteropathy (20). In 16 (20.00%) it was the case of ontogenic or later onset lactose deficiency, clinically manifested by secondary irritable colon syndrome. In all cases diet treatment involved about 500 ml of jogurt daily, divided in 2-3 portions. None of the cases showed elements of intolerance to the fermented milk preparation. Our results makes jogurt as a well-tolerated source of milk for 1actose-deficient children, confirming that it can be successfully applied in children aged over 6 months.

CREDES is a center for diagnosis and treatment of children with secondary malnutrition. Between January 1990 and June 1991, 21 infants (9.3% of all admissions) suffered from persistent diarrhea and of these, 19 did not respond to the initial dietary management. A locally made comminuted chicken formula (CCF) was assayed as an alternative to the expensive commercial formulae available for nutritional support. It consisted of 10% skinless chicken meat, 20-30% pureed carrots, 3-5% dextrinmaltose, 1-5% glucose and 3-5% vegetable oil. Of the children studied, 35% had birthweight under 2500g, 88% had been breastfed for a month or less and 50% had been hospitalized two or more times. On admission, patients had suffered as an average 13 morbid episodes. Mean W/A, H/A and W/H were 61.7%, 88.3% and 89.5% of the WHO/NCHS standards, respectively. Mean duration of hospitalization was 145.3 days (range 53-283). They were fed CCF for a mean of 145.3 days (59% of their hospitalization period) and they were regraded to a commercial formula before discharge. Restriction of sucrose intake associated to lactose withholding resulted in improvement of clinical symptomatology and of nutritional parameters. None of the patients required parenteral nutrition. The following diagnoses were made: celiac disease (4), cow's milk protein intolerance (2), agammaglobulinemia (1), secondary disaccharidase deficiencies (12, of which 5 were lactase and 10 sucrase deficiencies). On discharge W/A, H/A and W/H were 78.5%, 89.5% and 100,7%, respectively. CCF is a good alternative for the clinical management of these patients bacause of its acceptability, tolerance and low cost.

Eight different monoclonal antibodies which recognize at least three distinct epitopes unique to the lactase protein of human enterocyte have been used to investigate lactase protein expression in hypolactasia of Neapolitan adults by light and electron microscopy with the immunogold technique. All the antibodies gave the same results, namely: strong brush border staining in all the 7 lactase persistent adults tested; no staining at all in 9 of the hypolactasia subjects and a mosaic pattern of staining of enterocytes in other 12 adults with hypolactasia. The percentage of enterocytes showing intense staining for lactase protein varied between 4 and 20% of the total villus cells (mean value±SD:9.86±5.84). Intracellular staining was not apparent in any of the samples examined. Sucrase-isomaltase protein in contrast showed no mosaicism. The mosaic pattern of expression of lactase protein in some hypolactasic subjects suggests that columnar cells display a non homogeneous differentiation along the villus. If the two patterns, mosaicism or absence of detectable lactase protein, are present along the entire small intestine of the individuals tested, the results would suggest that two phenotypes of adult hypolactasia exist in the population we studied.

The development of rat intestinal L-Ph specific activity displays a well-known post weaning decline. In contrast, total lactase activity increases to reach maximal levels around weaning, and remains high subsequently. In order to elucidate the molecular basis for these patterns, a rat lactase cDNA was isolated and characterized, and used in the quantification of lactase mRNA during development. This lactase cDNA uniquely hybridized to a 6.8 kb mRNA in the small intestine. To assess the amount of lactase mRNA encoding for lactase enzyme activity in the small intestine, total intestinal RNA was isolated and analyzed by Northern and dot blot hybridization. The pattern of total lactase mRNA during development followed that of total lactase activity, suggesting that over this time span the level of lactase activity is primarily controlled at the transcriptional level. However, the magnitude of increase of total lactase activity during lactation compared to that of total lactase mRNA suggests that additional mechanisms are involved in regulating lactase levels. Analysis of the regional distribution of lactase mRNA along the small intestine at 14 days revealed that mRNA was high in the proximal three regions, but was dramatically lower in the distal regions. Total lactase activity, in contrast, displayed maximum activity in the mid intestine with decreased levels both proximally and distally. Thus, lactase activity in the intestine appears to be regulated during development predominantly by transcriptional mechanisms, while alterations during lactation, and along the proximal to distal gradient, are the result of other control mechanisms.

In suckling pups treated with exogenous insulin (IN:12.5 mU.g.b.wt), twice daily, from day 8 to 12, sucrase-isomaltase (SI) measured in BBM was precociously induced while maltase, aminopeptidase and neutral lactase were markedly enhanced (+30 to +131%, p < 0.01 vs controls). N-acetyl-β-glucosaminidase (lysosomes), sulfatase C (microsomes) and lactate dehydrogenase (cytosol) were also sensitive to IN with decreases in villus and crypt cells ranging from -37 to -63% (p < 0.05 vs controls). Whatever the dose of IN given, the production of plasma corticosterone remained similar to saline controls. In weaned rats (d. 23 to 32), IN (12.5 mU) failed to influence the activity of BBM, lysosomal, microsomal and cytosolic enzymes. The synthesis rate of mature S-I, measured by the incorporation of 14C-leucine into S-I precursor was equivalent in IN-treated rats (0.039 ± 0.006) and controls (0.034 ± 0.005 ratio cpm 14C-SI/cpm14C BBM prot.). In conclusion : (1) the immature enterocyte of the rat is responsive to IN throughout the nursing period while at weaning, the mature enterocyte lost IN responsiveness (2) the effect of IN on the enterocyte is not mediated by endogenous corticosterone release.

GENETIC STUDY OF HUMAN ADULT-TYPE HYPOLACTASIA BY ANALYSIS OF RESTRICTION FRAGMENT LENCHT POLYMORPHISHS (RFLPs) OF THE LACTASE GENE

In order to elucidate the relation between the morphogenesis of the small intestine and the initiation of lactase gene expression, our previously characterized rat lactase cDNA was used as a probe for lactase mRNA. Detection of lactase mRNA by Northern blotting was correlated with intestinal development as assessed by microscopic examination of samples from the same fetus and by enzyme assay. Both mRNA and enzyme activity were detectable as soon as the stratified epithelium of the early intestine had been transformed into a single layer of columnar epithelium lining villi at about 18 days of gestation. Although PAS staining was positive for the brush border, electron microscopy demonstrated that the epithelial cells were still immature, with sparse microvilli, endoplasmic reticulum, and Golgi apparatus, and a relatively homogeneous cytoplasm. Significant levels of lactase mRNA were present in these cells, thus indicating that lactase expression begins as soon as the definitive intestinal epithelial cell develops. During the period of transformation when the columnar epithelium and villi were forming, no lactase mRNA was detectable in the immature distal third of the fetal intestine. CONCLUSIONS: 1) Initiation of lactase expression occurs concurrently with the morphogenesis of columnar epithelial cells. 2) Lactase expression does not require the formation of mature brush border structure. 3) Lactase enzyme activity and mRNA appear simultaneously during development of rat intestine. Supported by NIH grant DK 32658 and the Whitaker Health Sciences Fund.

Intestinal disaccharidase expression in the developing post-natal animal is under a complex system of regulatory mechanisms involving pre-programmed genetic timing as influenced by circulating humoral factors, paracrine factors, and luminal nutrients. The aim of this study was to examine the relative contribution of circulating glucocorticoids and pre-programmed genetic expression of sucrase and lactase in a suckling mouse between post-natal days 13 and 15. We examined primary jejunal and ileal organ culture explants in serum-free medium at 0-2 hours and 22-24 hours of organ culture, respectively. Animals were studied at days 13, 14, and 15, with an additional group of adrenalectomy animals who underwent adrenalectomy on day 13 with organ culture at day 15. The results of sucrase activity are shown below. In summary, day 14 tissues in organ culture for 24 hours reached the same level of expression in organ culture of scrum-free media as in the intact animal on day 15. In addition, adrenalectomy on day 13 did not abolish the time-dependent expression of sucrase. However, the magnitude of expression in the basal state was diminished. We speculate that normal quantitative expression of intestinal disaccharidase in the 15-day suckling mouse is dependent upon a combination of intrinsic tuning in conjunction with circulating glucocorticoids.

Although lactase activity in adult rats is believed to be low, we have found it to be elevated during lactation. Our study relates lactase activity measured enzymatically and histochemically with immunological and morphological changes in the jejunum of lactating, pregnant, and nongravid adult rats, suckling and weanling pups. Jejunal sections were collected for light and fluorescent microscopy. Villus measurements were made on H&E sections using Jandel Video Analysis. Lactase was demonstrated by histochemistry and immunofluorescence (IF). Fluorescent intensity was graded 0-4+. Lactase specific activity (SA) (μmol/min/g protein) was performed using scraped, homogenized mucosal membranes. Histochemically, lactase was present in all animals. Our results show that during 1) weaning, lactase activity decreases independently of changes in villous morphology, 2) early lactation, both villous length (+57%) and lactase SA (+30%) increase, and 3) late lactation, lactase SA decreases to adult values independent of villus length.

Lactase specific activity(SA) rises immediately after birth in the rat, reaches a peak at 9-12 d of age and declines to adult levels shortly after weaning. These studies were intended to determine the relationship of mature(L) to precursor(ProL) forms of lactase in suckling, weanling and adult Sprague-Dawley rats. Homogenized jejunal mucosa was used for measurement of enzyme SA (μmol/min/g protein) and for immunoisolation and purification of L and ProL. The relative quantities of each polypeptide were determined by densitometry. Samples were also treated with Endo H and Endo F. Enzyme SA and distribution are shown below. All bands were sensitive to Endo F treatment, none to Endo H. These studies demonstrate that: 1)the decrease in lactase SA in adult rats is associated with an increase in the level of a 200 kDa protein, 2) these changes occur at weaning when lactase SA is decreasing and may be partially responsible for lower SA seen in older animals. We speculate that these age-related changes may result from an alteration in a post-translational event.

To determine whether zinc has a specific role on intestinal growth and function, three groups of male weanling Sprague-Dawley rats were fed a semipurified zinc-deficient diet: ad libitum fed group received powdered diet and water containing 25 ppm of zinc; force fed (ZN, ZD) groups were fed identical amounts of diet to the ad libitum fed group by intragastric infusion three times per day. The diets were aqueous suspensions made with either deionized water (ZD) or water containing 25 ppm of zinc (ZN), and additional drinking water with (ZN) or without zinc (ZD) was offered ad libitum. Rats were sacrificed after 8 days of feeding. The ZD group showed growth arrest, perioral and periorbital dermal lesions, and abdominal distention within 8 days of feeding. Mucosal DNA, protein, sucrase, maltase, lactase, leucine aminopeptidase, and alkaline phosphatase were significantly decreased in the ZD group, whereas intestinal length, weight, and mucosal weight were unaltered. These results suggest that short-term isolated zinc deficiency impairs growth, digestion, and absorption in the rat small intestine, even in the absence of associated protein calorie malnutrition.

To elucidate the mechanism of the developmental decline in intestinal lactase activity at weaning, we examined lactase synthesis in suckling and adult rats. Lactase was purified to homogeneity from pooled intestines of newborn rats and used to raise a monospecific antibody. Using this antibody, we developed a quantitative immunoprecipitation assay for lactase. Intestinal microvillus membrane proteins were labeled in 15-day and adult rats by intraluminal pulse-chase with 3H-leucine, and newly synthesized lactase quantified by immunoprecipitation. When lactase synthesis was expressed as the quantity of microvillus membrane lactase synthesized relative to total microvillus membrane protein synthesized, a significantly greater proportion of 3H-leucine incorporation into lactase was demonstrated in the suckling animals. No structural differences between newly synthesized suckling and adult lactase were observed when they were compared by SDS-polyacrylamide gel electrophoresis and fluorography. These data suggest that a change in the rate of lactase synthesis plays a role in the postweaning decline in enzyme activity.

The effects of the aqueous phase of human breast milk on the disaccharidase activity of newborn rabbit small intestinal mucosal explants were studied in vitro culture. These explants continuously synthesized protein and normal morphology was maintained for the duration of the cultures. Addition of the aqueous phase resulted in significant increase of lactase (p less than 0.001) and maltase (p less than 0.01) concentrations in these organ cultures. This effect was dose dependent and was observed whether the organ biopsies were derived from fed or starved newborn rabbits. Further purification of the aqueous phase showed that the active ingredient exerting these effects was lactose. These studies suggest that lactose may have an important function in stabilization of newborn intestinal disaccharidase enzymes.

The reversibility of the effects of postnatal malnutrition on the intestinal brush border enzymes and somatic and intestinal weights were examined using either ad libitum or restricted feedings. Malnutrition was induced in the immediate postnatal period by expanding newborn rat litters to 20 pups/dam. At 21 days of age, malnourished pups exhibited significantly decreased body and intestinal weights as compared to those from control litters. Malnourished pups also had significantly elevated lactase specific activities whereas sucrase and maltase activities were not affected in the proximal small intestine. With subsequent nutritional rehabilitation by an ad libitum (food available 24 h/day) or restricted feeding regimen (food available 2 h/day), body and intestinal weights remained significantly depressed by 56 days in malnourished as compared to control animals. Rats on 2–h feedings consumed approximately 35% of the food consumed by their ad libitum-fed counterparts. Comparison of the ratio of weight gained to the amount of food consumed did not demonstrate a greater food efficiency with any particular feeding pattern. With ad libitum or restricted feedings, lactase specific activity in the proximal segment attained control values by 14 days. Restricted feedings resulted in an apparent elevation of specific activity of sucrase and of maltase, when rats were sacrificed at one chosen time point. Multiple time studies in a 24–h cycle showed that maximal elevations in enzyme activities were associated with feeding time. There were no significant differences in mean specific daily enzyme activities between the two feeding regimens. Restricted feedings show no advantage in enzyme efficiency or in promoting the rate of recovery of the intestine after postnatal malnutrition.

To investigate the effect of chronic proteincalorie malnutrition on intestinal repair after an enteric infection, we examined small intestinal structure, enzyme activity, and sodium transport in undernourished piglets during the acute and convalescent phases of a viral enteritis, transmissible gastroenteritis (TGE). Gnotobiotic pigs, nutritionally deprived from the age of 7 days, gained less weight than dietary controls from 14 days of age until the end of the study. Animals from malnourished and control diet groups were innoculated with TGE virus at 22-23 days and studied during the acute (40 h) and convalescent (4, 10, and 15 days) stages of this experimental enteritis along with noninfected dietary controls. After TGE infection, we observed a further decrease in weight gain and an increased mortality only in undernourished pigs. In jejunum and ileum of both dietary groups at 40 h after TGE infection, we observed comparable structural lesions, similar decreased activities of mucosal enzymes (sucrase, lactase, sodium-potassium-dependent ATPase), and increased thymidine kinase activities. Also we noted comparable diminution of glucose-stimulated jejunal sodium absorption in both dietary groups at 40 h. In control diet pigs, transport abnormalities recovered by 4 days after TGE infection and normal mucosal structure and enzyme activity returned over 4-15 days. In undernourished piglets, structural repair and enzyme abnormalities were prolonged when compared with the control diet group; glucose-stimulated sodium transport did not recover until 10 days after infection and never regained the enhanced activity seen in noninfected undernourished controls. We conclude that small intestinal recovery of the undernourished animal after viral enteritis is prolonged and speculate that impaired mucosal repair is important determinant of the delayed recovery of structural and functional abnormalities in this animal model of a major human disease.

Summary: Newborn rats born to iron deficient mothers (IDM) were found to have significantly lower hemoglobin, sucrase, lactase and maltase levels compared to control newborn rats. Rats born to IDM and nursed by IDM, when sacrificed at 21 days of age, had statistically significantly lower hemoglobin, serum iron, sucrase, lactase and maltase levels compared to control rats. Rats born to IDM, but nursed by iron sufficient mothers (ISM) and sacrificed at 21 days of age, had hemoglobin, serum iron and sucrase levels compared to control rats whereas lactase and maltase were not corrected by 21 days of nursing by ISM. Rats born to IDM and nursed by either IDM or ISM for 21 days were given intramuscular iron dextran and placed on iron sufficient diet (ISD) for 7 days. These animals experienced correction of the hemoglobin, serum iron, sucrase and maltase levels compared to control rats, whereas intestinal lactase was not corrected by 7 days of ISD and intramuscular iron. Rats born to ISM, nursed by IDM and sacrificed on day 21 had significantly lower hemoglobin, serum iron and intestinal lactase levels compared to control rats. Rats born to ISM and nursed by IDM were given intramuscular iron dextran on day 21 and placed on an ISD from day 21–28. These animals had a return in hemoglobin, serum iron, sucrase and maltase levels comparable to control rats. Rats born to and nursed by ISM and maintained on an iron deficient diet from day 21–84 had significantly lower hemoglobin, serum iron, sucrase, lactase and maltase levels compared to control rats. Rats born to and nursed by ISM, maintained on iron deficient diet from day 21–84, and then given intramuscular iron dextran on day 84 and maintained on an ISD until day 92, experienced correction of the hemoglobin, serum iron and lactase levels compared to control rats. Intramuscular iron and 7 days of ISD did not correct the sucrase and maltase levels in these rats. Lactose tolerance tests in iron deficient rats showed flat curves compared to controls. After iron treatment, lactose tolerance curves returned to control values. Iron deficiency in rats in utero, during the nursing and postweaning period causes, in addition to anemia, a reduction in jejunal disaccharidase activity because of an alteration in the enzymes of the brush border membrane. Varying degrees of reduction and response of certain disaccharidases to iron treatment are dependent on the time of iron deprivation in relationship to the intra-uterine and postnatal development of the digestive and absorptive functions in the small intestine. Alterations in the levels of disaccharidases demonstrated in this paper represent another aspect of the spectrum of biochemical effects of iron deficiency. Speculation: Iron deficiency has a profound effect on protein synthesis. Alterations in the levels of disaccharidases represent only a fraction of this effect. The temporary reduction in lactase and other disaccharidases in iron deficient-infants might predispose them to malabsorption and diarrhea, which contribute to the lowered serum protein and increased incidence of low weight percentiles observed in iron deficient infants.

We compared the absorption of carbohydrate from solutions of glucose oligomers and glucose in jejunal Thiry-Vella fistulae, a preparation deprived of pancreatic secretions. The studies were performed with two concentrations (90 and 360 mg/dl) of both glucose and the glucose oligomers. Carbohydrate absorption from glucose solutions (33.1 +/- 2.8, 115.9 +/- 8.9 micrograms/cm/min) was significantly greater (P less than 0.025; P less than 0.005) than that from oligomer solutions (26.6 +/- 2.1 and 92.4 +/- 9.0 micrograms/cm/min). Thin-layer analyses of the perfusates demonstrate digestion of oligomers with a chain length up to eleven and suggest digestion of oligomers of even greater chain length. Atrophy of the jejunal mucosa occurred over the course of the study as evidenced by a decrease in the ratio of villous height to crypt depth from 3.8 to 0.3, and by a 80% decrease in the activity of maltase, sucrase, and lactase. Atrophy was accompanied by a significant decline in the absorption of both glucose oligomers (P less than 0.005) and glucose (P less than 0.01) from the more concentrated solutions but the decrement in absorption of both carbohydrates was similar: glucose oligomers, 79.3 +/- 19.4 micrograms/cm/min; and glucose, 69.8 +/- 14 micrograms/cm/min (P greater than 0.20). Water absorption was enhanced by both carbohydrates, but there was no demonstrable difference between solutions of glucose and glucose oligomers. The osmolality of the solutions clearly influenced water absorption (P less than 0.025) but failed to effect the absorption of carbohydrates.

Extract: Eighteen peroral intestinal biopsies were obtained from children with celiac disease. A marked impairment of the enzymatic equipment was noted in the mucosa of patients with untreated celiac disease. The surface epithelium is clearly deficient in ATP-ase, 5-nucleotidase, monoamine oxidase, leucine aminopeptidase, glucose-6-phosphatase and lactase activities. The histochemical reactions for acid phosphatase, non specific esterases, α-glucosidases and deshydrogenases are often less affected. Only alkaline phosphatase activity seems unaltered in the surface epithelium. After the start of a gluten-free diet, an improvement in the patient's clinical condition occurs within a few weeks. At the same time, there is an increase in the activity of some enzymes. The histochemical reaction for ATP-ase shows a rapid improvement. This reaction is almost normal in specimens taken 2, 3 and 5 weeks after gluten withdrawal, but it is still negative in a biopsy performed one week after the removal of gluten from the diet. In addition, the histochemical stains for several other enzymes, such as 5-nucleotidase and leucine aminopeptidase also return towards normal in a short time. Contrariwise lactase activity remains very low for several months after the beginning of the gluten-free diet. Sometimes the lactase deficiency may be responsible for clinical disturbances, i. e. ‘a secondary intolerance to lactose’. In such cases, lactose exerts some ‘toxic effect’ on the mucosa and prevents the restoration of the villi. Therefore, it is necessary to withdraw lactose as well as gluten from the diet. This intolerance to lactose is nevertheless transient and disappears after the mucosa is repaired. It should be emphasized that there is a marked discrepancy between the histologic improvement, the evolution of the enzymatic abnormalities and the clinical response after gluten withdrawal. In most cases, the biological and clinical disturbances progressively disappear in three to five weeks. The morphologic changes of the duodeno-jejunal mucosa persist much longer. Before the first two months, there is no evident improvement in the histologic appearance of the mucosa. About three months after the start of dietary treatment, the villi are noticeable again, but are still short and broad (partial villous atrophy). Complete repair may require several months and sometimes more than one year. In striking contrast, some enzymatic activities reappear very quickly. This improvement in some histochemical reactions seems to correlate closely with the rapid clinical response and, therefore, suggests that the histoenzymatic abnormalities play a prominent role in the absorptive failure of celiac disease. When the patients are put again on a normal diet, histological studies constantly show the reappearance of the mucosal lesions (subtotal villous atrophy), whereas the evolution of both clinical disturbances and histoenzymatic abnormalities is variable. Sometimes a clinical and biological relapse occurs and the histochemical reactions are again impaired. But in most cases, although severe histologic changes reappear, the clinical state and the biological tests of intestinal absorption remain satisfactory, with normal growth and no steatorrhoea. It is noteworthy to point out that, in these cases without clinical relapse, the enzymatic activities are only slightly reduced. Speculation: Although studies of more cases are needed in order to confirm these data, it appears that histochemical reactions provide a most valuable tool for the study of the mechanism involved in intestinal malabsorption. It seems likely that these reactions reflect the absorptive capacity of the small bowel mucosa. Furthermore, when some patients are put again on a normal diet, the histochemical changes could provide a practical means for predicting a relapse before the impairment of the patient's clinical condition.

The effect of undernutrition during suckling has been investigated on the brush border enzymes and the intestinal uptake of D-glucose and glycine in rats at weaning. The brush border sucrase and alkaline phosphatase activities were drastically reduced, but lactase and leucine aminopeptidase levels were significantly elevated in the intestine of nutritionally deprived pups compared to controls. The uptake of D-glucose and glycine in undernourished rats was also augmented. The chemical composition of the brush border membrane analyzed in nutritionally deficient animals revealed an enhancement of the membrane protein, sialic acid, cholesterol, and phospholipids compared to the control group. [U-14C]D-Glucose incorporation into lipid constituents of the membrane suggested that the observed enhancement of the membrane lipids is the result of an increased synthesis in response to undernutrition.

Summary: In vivo jejunal transport of amino acids, monosaccharides, sodium, and electrolytes were studied in rats made nephrotic with puromycin aminonucleoside (PAN) and in pair-fed controls. Studies were performed 14 days after a single intravenous dose of PAN when rats were no longer edematous, but were still hypoproteinemic. There was decreased absorption of glucose, 3-0-methyl glucose, glycine, phenylalanine, histidine, water, and sodium in the nephrotic animals but transport of fructose, lysine and potassium was similar in the nephrotic and control animals. Enzyme kinetic studies for glucose transport showed a mixed type of inhibition affecting both V m and K m. The jejunal mucosa of nephrotic and control rats had similar ATP content and enzyme activity for lactase, sucrase, maltase and (Na-K)-ATPase and the ratios of RNA to DNA were similar in the nephrotic and control rats. No abnormality of the jejunum was detected by light or electron microscopy. The data suggest that the impairment of absorption is a result of decreased activity of jejunal membrane carrier mechanisms. The altered transport may be secondary to effects related to the metabolic consequences of nephrotic syndrome and does not appear to be related to acute purine aminonucleoside toxicity, edema or malnutrition.

To identify potential tissue-specific characteristics of intestinal glycoprotein synthesis and processing, rat intestinal lactase-phlorizin hydrolase (L-Ph) was studied after pulse-labeling of colonic explants from 5-d-old suckling rats in organ culture and the data compared to similar studies in rat jejunum. Histologic sections of 5-d-old proximal colon showed villus-like structures lined with columnar epithelial cells. Lactase and phlorizin hydrolase activities showed tissue-specific developmental patterns. Using a MAb to small intestinal L-Ph, we were able to immunoprecipitate from colon at different ages a protein that hydrolyzed lactose and phlorizin, and whose activity was not inhibited by p-chloromercuribenzoate. After pulse-labeling for 60 min and chase for 30 min, immunoprecipitated L-Ph from total homogenates of rat colonic explants appeared on fluorography of SDS-PAGE as one band of approximately 205 kD. With increasing time of chase, it took 240 min before the precursor form was converted to the intermediate form (equivalent to the 180-kD form in jejunum) and the mature form (equivalent to the 130-kD form in jejunum), although these conversions in the jejunum were observed within 60 min of chase, and only 30 min of pulse labeling. When compared on SDS-PAGE to immunoprecipitated jejunal L-Ph, the precursor form in the colon had a slightly higher apparent mol wt than the corresponding precursor form found in the endoplasmic reticulum-Golgi fraction of the jejunum. The intermediate as well as the mature L-Ph forms in the colon were also both somewhat higher in apparent molecular weight than the same bands in the microvillus membrane fraction from jejunal explants. Removal of N-linked oligosaccharides from jejunum and colonic forms of L-Ph produced bands on SDS-PAGE with identical mobility, suggesting that the proteins were the same. The data demonstrate that, in neonatal colon, enzymatically active L-Ph undergoes biosynthetic and processing events similar to those in the jejunum. During early life, colonic L-Ph may function in the salvage of lactose not absorbed in the small intestine.

A relationship between disaccharidase and diamine oxidase (DAO) activities was looked for by measuring these enzyme activities in histologically normal small intestinal biopsies of 18 children. The range for disaccharidase activities expressed in U g-1 wet weight was 0.1-5.7, 7.1-36.7, and 2.3-8.5 for lactase, maltase, and sucrase, respectively. The range for DAO activities expressed in nmol h-1 g-1 wet weight was 202-974. Significant correlations were found between disaccharidase and DAO activities (lactase versus DAO: n = 17, r = 0.80, P less than 0.001; maltase versus DAO: n = 18, r = 0.70, P less than 0.001; sucrase versus DAO: n = 18, r = 0.55, P less than 0.05). Our results further support the hypothesis that DAO is a marker of small intestinal functional integrity in children.

To evaluate the response of the small intestinal mucosa to Saccharomyces boulardii (S.b.), a yeast widely used in some countries as an adjuvant drug with oral antimicrobial therapy, seven healthy adult volunteers were treated with high doses of lyophilized S.b. (250 mg four times per day) for 2 wk. A peroral jejunal suction biopsy was performed on days 0 and 15 of the study. Compared to the initial biopsy, histological examination of the posttrial biopsy revealed no morphological alteration nor change in villus height or crypt depth. After treatment, the specific activity (per U protein) of sucrase, lactase, and maltase was, respectively, increased by 82% (p less than 0.05) 77% (p less than 0.05), and 75% (p less than 0.05) over the basal activity of the enzymes measured on day 0, whereas mucosal protein content remained unchanged. Similar findings were found in the jejunum of adult rats treated for 5 days with either viable or killed S.b. cells. The changes in total enzyme activity (per jejunal segment) paralleled the changes in specific enzyme activity. In vitro assays on freshly prepared suspensions of S.b. (6.0 X 10(8) viable cells/ml) evidenced a high activity for sucrase (mean +/- SE: 8 364 +/- 1280 U X g X protein-1) but no maltase, neutral lactase, acid beta-galactosidase, or aminopeptidase activity. To determine whether treatment with S.b. could influence the incorporation rate of neutral lactase into the brush border membrane, 14-day-old sucklings treated either with saline or with S.b. were given intraperitoneally a dose of 20 microCi D-[1(14)C] glucosamine 3 hours before sacrifice.(ABSTRACT TRUNCATED AT 250 WORDS)

Summary: To evaluate the impact of malnutrition on the developing gut, we studied small bowel structure, epithelial renewal, and enzymes in suckling rats deprived of adequate nutrient from birth. Rat pups were suckled by foster dams fed ad libitum one of three isocaloric, semipurified diets containing 6,9, or 25% (control) protein throughout gestation and lactation. An additional control group consisted of pups raised with their natural, chow-fed mothers. Although survival of the pups, 98% in the chow-fed and 25% protein groups, decreased to 83% in the 9% groups and 53% in the 6% protein groups, body and gut weights were remarkably uniform within each study group. Mean body weight, gut weight, villus height, and crypt depth were markedly and significantly less in the 6 and 9% pups when compared with those in the chow and 25% control groups, the 6% group being significantly more affected was than the 9% group (P < 0.001). Pups raised by chow-fed mothers also weighed significantly less (P < 0.001) than did those raised by dams fed the 25% protein diet. Total small intestinal protein and DNA content of mucosal scrapings were less in 6 than 9% rat pups (P < 0.001), which in turn were less than those in the 25% group (P < 0.05). The protein/DNA ratio in the small intestine of the 6% animals only was reduced when compared with the 25% group (P < 0.05). Epithelial cell migration assessed by autoradiography with [3H]thymidine was significantly slower in both proximal and distal intestinal segments of the 6% animals when compared with those from the 25% group (P < 0.001). Incorporation of the 3H, seen by autoradiography after 1 hr, was also signficantly decreased in the 6% group (P < 0.001). Calculated for the total small intestine, lactase, sucrase, alkaline phosphatase, and thymidine kinase activities were significantly diminished (P ≤ 0.001) in the 6% animals compared with 25% controls. Calculated in relation to mucosal protein content and compared with 25% controls, sucrase and alkaline phosphatase activities were significantly decreased (P < 0.001) in both proximal and distal small intestinal segments of the 6% animals, but thymidine kinase activity was decreased only in the distal segment (P < 0.001). However, lactase specific activity was increased in both proximal and distal segments from the 6% group (P < 0.001). Further studies of this phenomenon demonstrated a significant increase in intracellular acid β-galac-tosidase (P < 0.001), particularly in the distal intestinal segment, but also a marked increase in brush border β-galactosidase. Our data demonstrate that in the small intestinal mucosa of the suckling rat, chronic malnutrition causes a decreased number of cells and impaired epithelial proliferation. These abnormalities reflect a profound direct impact of malnutrition on the gut of the young animal and demonstrate a delay in the normal pattern of postnatal maturation of the small intestinal epithelium.

The effect of phototherapy on intestinal mucosal bilirubin concentration and lactase activity in the Gunn rat was studied. Ten groups of six or seven animals each were studied. Heterozygous (Jj) and homozygous (jj) animals were given 24, 48, or 72 hr of continuous phototherapy, and Jj and jj animals were given 24- and 48-hr sham control treatments. Phototherapy reduced serum bilirubin concentration by 53% at 24 hr, 59% at 48 hr and 68% at 72 hr. The mucosal concentration of bilirubin appeared to parallel the declining serum concentration and was lower in treated than in control jj animals. The jejunal and ileal lactase activity, expressed per mg protein, was not depressed by phototherapy. The lactase activity of jejunum and of ileum of treated jj as compared to treated Jj and control jj at 24 and 48 hr and as compared to treated Jj at 72 hr was never reduced; i.e., jejunal lactase activity in jj treated for 72 hr was 15.1 +/- 4.2 (-x +/- S.D.) units/g protein as compared to 16.2 +/- 4.2 for Jj treated for 72 hr and 12.0 +/- 4.2 for jj 48-hr control animals. The ratio of lactase to sucrase activity demonstrated a significant increase in lactase activity relative to sucrase in all animals treated for 48 or 72 hr. This latter effect is potentially due to alteration in the circadian rhythm while under constant irradiance. These data convincingly demonstrate that the Gunn rat does not develop lactase deficiency consequent to phototherapy.

Summary periods of feeding at lower disaccharide concentrations, which do not induce symp~oms of intolerance. After the high lactose conDisaccharide feedings to post-weaning rats alters their jejunal centration feedings, there was evidence of morphologic damage to barrier to macromolecular absorption. Penetration of horseradish the epithelium of the jejunal mucosa. ~h~ disaccharide-induced peroxidase (HRP) across the jejunum was enhanced after short- increase in HRp absorption may be mediated by transport from term high concentration gavage (30 g*kg-'*da~-l) Of lactose that endocytotic vesicles across the epithelium (10, 12, 43). produces weight loss, osmotic diarrhea, and jejunal mucosal damage. HRP absorption was also increased by-longer term feedings of lower levels of disaccharide that did not produce body weight MATERIALS AND METHODS alterations, diarrhea, or cell damage. Rats without diarihea and gavaged with 7.5 g-kg-' .day-' of either lactose or maltose showed Animals and diet. Postweaning male rats (~rl: (WI) BR, char1e.s an increase in lumen to blood HRP absorption after 21 days of River Breeding Labs, WilmingtOn* MA) weighing g were feedings. Also, lactose or maltose in the solid food diet at 30 g. used. At this stage of development the rats have low lactase levels kg-'.day.' for 21 days did not lead to diarrhea but produced an (36). rats were fed a powdered Purina Rodent Chow (#5001, increase in jejunal lumen to blood HRP absorption. Rats having Ralston Purina Co., St. Louis, MO) and water ad libitum throughno diarrhea and receiving l5 g.kg-~ of lactose or maltose in out the study. The Purina chow diet contained 50% unrefined the drinking water for 21 days, showed an increase in jejunal HRP complex carbohydrate, 23.0% protein. 6.5% fats, and 6.8% minabsorption. When rats were fed either lactose or maltose for 21 erals. The balance corresponded to fiber, vitamins, and moisture. days glucose absorption became dependent upon the sugar In addition to this diet, disaccharides were experimentally adminconcentration in the perfusion medium. The kinetics of this glucose istered the rats. absorption are compatible with a decrease the 66unstirredv layer. Disaccharide administration by gavage with production of diarDisaccharide-induced HRP absorption may be mediated by trans- rhea. In one set of experiments lactose, sucrose or maltose was port from endocytotic vesicles across the jejunal epithelium. administered to the rats by gavage at 30 g. kg-'. day-' for 1 and 7 days. This level of disaccharides was given as a 60% w/v slurry at 5 m1/100 g body weight, carefully resuspended before administraAbbreviations tion. Controls received 0.9% isotonic saline. The lactose-gavaged