Biochemical quantification of crypt hyperplastic villous atrophy by aldolase activity assay

Aldolase activity with the two substrates fructose-1-phosphate and fructose-1,6-diphosphate was measured in the homogenate of small intestinal biopsy specimens from children with different malabsorptive diseases (celiac disease, cow's milk protein intolerance, infectious diarrhea, giardiasis, and Crohn's disease) and controls. It is demonstrated that the ratio of fructose-1,6-diphosphate/fructose-1-phosphate activity, which reflects the relative amounts of the crypt enzyme aldolase A (EC 4.1.2.13) and the villous enzyme aldolase B (EC 4.1.2.7), correlates very well with both the ratio of crypt to villous height (correlation factor r = 0.92) and the mitotic index (r = 0.80).     

Iron absorption and iron deficiency in infants and children with gastrointestinal diseases.

Iron status, iron absorption, and intestinal blood loss were studied in 199 children undergoing diagnostic evaluation for suspected malabsorption. Evaluation of iron status included hematological indices, serum ferritin, and transferrin saturation. Iron absorption was assessed by the increment of serum iron after an oral iron load. Iron deficiency was common among patients affected by malabsorptive states, such as celiac disease (84%), cow's milk intolerance (76%), Crohn's disease (72%), and giardiasis (64%), whereas it was less common among patients with postinfectious enteritis (41%) and chronic nonspecific diarrhea (11%). Intestinal blood loss was seen only in patients with Crohn's disease and cow's milk intolerance, irrespective of iron nutritional status. On the other hand, iron malabsorption was very common, affecting 85-95% of the iron-deficient patients in all diagnostic groups, except in chronic nonspecific diarrhea. Iron malabsorption was less common among patients with adequate iron nutritional status than in those with iron deficiency. Iron malabsorption appears to play a major role in the pathogenesis of iron deficiency in patients with malabsorption. The iron absorption test shows greater sensitivity as a screening test for upper intestinal malabsorption than the D-xylose absorption test.     

Follow up study of cow's milk protein intolerant infants

Over a period of 4 years, 88 infants with cow's milk protein intolerance (CMPI) were followed prospectively in order to evaluate the persistence of CMPI and its relationship between either serum IgE levels or RAST results for cow's milk. After exclusion of lactose intolerance, two positive cow's milk elimination challenge tests were considered diagnostic for CMPI. At the age of 1, 2, 3 and 4 years respectively, 85%, 78%, 49% and 33% of the children still were cow's milk intolerant. Initial serum values of IgE 10 kU/l indicated a late development of tolerance to cow' milk proteins. At the age of 4 years, 90% of infants with initial IgE levels <10 k U/l had become tolerant to cow's milk while this was the case for only 47% of infants with initial IgE levels 10 k U/l. Initial RAST results for cow's milk bore no obvious relationship to outcome.     

Selenium content of human milk,cow's milk and cow's milk infant formulas

The selenium content of human milk, cow's milk and cow's milk infant formula were estimated by instrumental neutron activation analysis. The highest values were found in 3 samples of human colostrum (524–865×10^-9 g/g dry weight). There was a significant decrease with increasing time post partum. Mature human milk exhibited a selenium content of 230±79×10^-9 g/g dry weight.The selenium content of 45 samples of cow's milk from the north-western area of Germany was 200±39×10^-9 g/g dry weight. While there was no significant difference between the values of mature human milk and of cow's milk, cow's milk infant formula exhibited significantly (P<0.01) lower values than human milk. The average selenium content of 107 samples of 10 different commercially available fluid and powdered cow's milk infant formulas (range: 18–171×10^-9 g/g dry weight) amounted to about only one third of that in mature human milk.With support of the Deutsche Forschungsgemeinschaft     

Cow's milk protein-sensitive enteropathy. Clinical and histological results of the cow's milk provocation test

Nineteen infants suspected of having cow's milk protein-sensitive enteropathy were studied. They all showed failure to thrive, diarrhoea and/or vomiting when fed a diet of cow's milk, and improved when their diet was changed to casein hydrolysate. Jejunal biopsy was done before and 18--23 hours after a milk challenge. Of the 19 infants, 12 presented histological evidence of cow's milk protein intolerance. Eight suffered from vomiting and diarrhoea within 9 days of the milk challenge, but in 4 cases the histological abnormalities were not accompanied by clinical symptoms. In one case a chicken meat intolerance was documented. The histological appearance of the intestinal mucosa after chicken challenge was identical to that observed after milk challenge. In our opinion, repeated intestinal biopsies before and after an acute challenge is the best method to establish the diagnosis not only of cow's milk protein intolerance but also of intolerance to other alimentary proteins.     

Do pre- and postchallenge small intestinal biopsies help to diagnose cow's milk protein intolerance?

Twelve infants suspected of cow's milk protein intolerance were challenged with cow's milk after at least one month of cow's milk-free diet. The challenge was clinically positive in seven. Small intestinal biopsies were taken with a multipurpose capsule both pre- and postchallenge (at 24 h) in eight, prechallenge in three, and postchallenge in one. Two or three biopsy specimens were taken at the same time in 15 of the 20 biopsy occasions. The morphology of the mucosa could vary from normal to slight damage at the same biopsy occasion. No difference was found in morphology judged by light microscopy between pre- and postchallenged biopsies. Light microscopy of small intestinal biopsies taken before and at 24 h after milk challenge seems of doubtful value as a routine diagnostic means in cow's milk protein intolerance.     

Serum IgA and IgG gliadin antibodies and small intestinal mucosal damage in children

Serum immunoglobulin (Ig) A and IgG gliadin antibodies were determined with a simple, rapid, and inexpensive method--diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA)--and the results were related to small intestinal mucosal morphology in 234 children suspected of having malabsorption. Fifty-six of 58 children with flat intestinal mucosa had increased IgA and/or IgG gliadin antibody levels (sensitivity 97%). Fifty-four of the 58 children had celiac disease (CD) (n = 25) or probable CD (n = 29). Four children with flat mucosa had cow's milk protein and/or soy protein intolerance and three of these had increased gliadin antibody levels. Seventeen percent of 132 children with normal intestinal mucosa had increased IgA and/or IgG gliadin antibody levels. IgA and IgG gliadin antibody levels decreased significantly in the celiac children on a gluten-free diet and increased significantly after gluten challenge. Determination of serum IgA and IgG gliadin antibodies by means of DIG-ELISA is a sensitive test for small intestinal mucosal damage in children. When malabsorption is suspected, we suggest that this assay be used to select children for a small intestinal biopsy. It is also very useful for the follow-up of adherence to a gluten-free diet and to determine the effect of gluten challenge in celiac children.     

Jejunal mucosa lymphoid cell subsets and the expression of major histocompatibility complex antigens in children

Using monoclonal antibodies with the immunoperoxidase technique the distribution pattern of class I and class II antigens of the major histocompatibility complex (MHC), and of the lymphocyte subsets have been studied in intestinal biopsies from children without mucosal lesions, from children with coeliac disease (CD) and from infants with cow's milk protein intolerance (CMPI). The staining of the intestinal mucosa for class I antigens is unaltered irrespective of the histological picture or the clinical diagnosis. Class II antigens are only partially or not expressed at all by epithelial cells in untreated cocliac disease and in some cases of cow's milk protein intolerance. The number and the composition of the lamina propria lymphocytes in both CD and CMPI are different from the normal situation. An increase of all lamina propria lymphocyte subsets is observed in untreated CD. A decrease of OKT4⁺ lymphocytes is observed in the lamina propria of CMPI patients. These changes may be involved in the pathogenesis of these diseases.Abbreviations MHC major histocompatibility complex - CD coeliac disease - CMPI cow's milk protein intolerance - IEL intra-epithelial lymphocytes     

Small intestinal mucosa in coeliac disease and cow's milk protein intolerance: Morphometric and immunofluorescent studies

One hundred and twenty-five small intestinal biopsies from children with coeliac disease (CD), cow's milk protein intolerance (CMPI) and controls were compared by morphometric analysis, by counting intraepithelial lymphocytes (IEL) and by quantitative evaluation of immunoglobulin-containing cells of lamina propria. The double-stain immunofluorescent technique of Brandtzaeg and Baklien [2] was used, based on defined mucosal tissue units. A patchy enteropathy was found in 60% of CMPI, and in 14% of nonspecific changes, but never in CD. There was a significant difference in crypt depth between CD (360±80 m) and CMPI (217±92 m), even when lesions of equal grade were compared (P<0.015). IEL counts per 1000 epithelial cells showed even better discrimination between the groups (CD: 793±173, CMPI: 320±143, P<0.001). In CD, there was a relatively greater increase of IgM-cells (x4.9) and of IgG-cells (x4.2) than of IgA-cells (x2.6). Ig-cell changes outlasted the morphological lesion in CD on a gluten-free diet. In CMPI, IgM-cells (x2.3), IgG-cells (x2.5), and IgA-cells (x1.7) were proportionately increased, compared to controls. A special increase of IgE-cells in CMPI could not be substantiated.By computerized stepwise discriminant analysis based upon crypt depth, villus/crypt-ratio, IEL-count, and counts of IgM-, IgG-, IgA-containing cells of lamina propria, accurate classification of patient groups was accomplished, even when only the initial biopsy data were analysed.Supported by Deutsche Forschungsgemeinschaft Gr 278/6 and Ste 305/1     

The importance of cow's milk protein intolerance in chronic diarrhoea of children

A survey is given of the cases diagnosed as cow's milk protein intolerance in the last 5 years. The diagnosis was established on the basis of the regression of clinical symptoms after elimination from the diet of cow's milk and their recurrence after milk challenge. In more than half of the cases intestinal biopsy was carried out; three patients were rebiopsied after milk challenge. Intestinal biopsy is indicated solely in cases when the exclusion of coeliac disease is necessary for the correct diagnosis.     

Colonic stricture complicating formula protein intolerance enterocolitis

Cases of formula protein intolerance (FPI), in which many of the features of neonatal necrotizing enterocolitis (NEC) have been exhibited (vomiting, diarrhea, hematochezia, abdominal distention, and pneumatosis intestinalis), have been reported (1-6). This case of combined cow's milk and soy protein intolerance illustrates further the difficulty in distinguishing clinically between NEC and FPI. Moreover, it demonstrates that intestinal stricture is a potential long-term complication of FPI, as it is also of NEC, and suggests that significant morbidity may result when the diagnosis of FPI is not considered as a cause of the NEC syndrome.     

Effect of cow's milk on the gastrointestinal tract: a persistent dilemma for the pediatrician

The confusing area of cow's milk intolerance is explored in an attempt to define the various mechanisms whereby milk affects gastrointestinal function, resulting in clinical symptoms (diarrhea, vomiting, gastrointestinal bleeding, etc.). The adverse reaction of infants to cow's milk ingestion may relate to lactose intolerance (enzymatic), a direct toxic reaction to the mucosal surface resulting in epithelial damage, or it may be immunologically mediated. Factors such as increased intestinal permeability to milk proteins during the newborn period may also contribute to susceptibility of young infants to milk sensitivity. The relative roles of systemic (milk agglutinins) and local immunity (SIgA antibodies) in milk intolerance are discussed and differential immunologic responses (IgE versus IgA/IgM) considered in the pathogenesis. It was concluded that new techniques such as organ culture of intestinal biopsy specimens are needed to establish the diagnosis of hypersensitivity and to begin to provide ways of adequately treating the condition.     

The role of cow''s milk protein intolerance in steroid-resistant nephrotic syndrome

The role of cow's milk protein intolerance in steroid-resistant nephrotic syndrome was evaluated in 17 children. Cow's milk was excluded from the diet for at least 14 days without changing previously ineffective prednisone dosage. Six patients with minimal change or mesangial proliferation went into remission 3 to 8 days after elimination of cow's milk. After a period of 2-3 weeks of remission, cow's milk challenge was positive in three patients. After one year on a cow's milk-free diet, two of six patients became milk tolerant and are in remission of NS, one of six became steroid-dependent, two of six are still unable to tolerate cow's milk and are in remission on a cow's milk-free diet and one of six children was lost from observation. The role of cellular mechanisms in steroid-resistant nephrotic syndrome is suggested.     

THE INTESTINAL MICROVILLUS Ultrastructural Variability in Coeliac Disease and Cow's Milk Intolerance

Abstract. In 8 patients with coeliac disease, 2 patients with cow's milk intolerance and 10 patients with other gastrointestinal disturbances, intestinal biopsies were examined in order to compare the extent of alteration of surface vs. crypt microvilli (MV). In those diseases associated with the ingestion of a noxious agent, namely coeliac disease and cow's milk intolerance, a marked and abrupt difference between the severely damaged surface epithelium and its MV, and the preserved crypt epithelium was noted. The results of the examinations give reason to suppose that these morphological variations are caused by the higher gliadin or milk protein concentration over the avillous surface epithelium as opposed to the lower one in the elongated crypts.     

The intestinal microvillus. Ultrastructural variability in coeliac disease and cow's milk intolerance.

In 8 patients with coeliac disease, 2 patients with cow's milk intolerance and 10 patients with other gastrointestinal disturbances, intestinal biopsies were examined in order to compare the extent of alteration of surface vs. crypt microvilli (MV). In those diseases associated with the ingestion of a noxious agent, namely coeliac disease and cow's milk intolerance, a marked and abrupt difference between the severely damaged surface epithelium and its MV, and the preserved crypt epithelium was noted. The results of the examinations give reason to suppose that these morphological variations are caused by the higher gliadin or milk protein concentration over the avillous surface epithelium as opposed to the lower one in the elongated crypts.     

Tyrosine and its metabolites in urine and serum of premature and mature newborns: Increased values during formula versus breast feeding

90% of preterm and 10% of mature neonates are reported to have increased tyrosine levels in blood and urine when fed certain cow's milk formulas which are relatively high in protein compared to human milk. It has been suggested that sustained raised tyrosine levels in early infancy might result in decreased intellectual functions at school age.We determined tyrosine by ion exchange and its parahydroxylated metabolites by gas chromatography as oxime-TMS derivatives. Mature neonates (n=30) on adapted cow's milk excreted significantly more (P<0.001) tyrosine, pHPLA, and pHPPA than control neonates (n=7) on human milk. Tyrosine levels in serum on adapted milk were high (17.5 vs. 6.6 Mol/100 ml) compared to human milk, as were pHPLA (7.6 vs. 3.7), pHPAA (1.8 vs. 0.3), and pHPPA (0.4 vs. nil). In prematures (850 to 2500 g, n=40) fed milk with a higher protein content (2.3 g/100 ml), the excretion of tyrosine and its metabolites was significantly raised (P<0.005) when compared to those on human milk (n=7). Excretion was highest in the lowest weight group (850 to 1500 g, n=10), and decreased with increasing birth weight. The low values reached on human milk were not reached by artificially fed prematures. In 5 small neonates (850 to 1700 g) who were followed continuously over the first 7 weeks of life, there were no significant changes in the excretion of tyrosine and its metabolites with increasing age and weight up to 2500 g.It is concluded that breast milk feeding in mature and premature newborns avoids an increase of tyrosine and its metabolites in serum with overspill into the urine.     

Leucocyte Migration Inhibition in Cow's Milk Protein Intolerance

ABSTRACT. The leucocyte migration inhibition (LMI) was determined in an assay after in vitro challenge with beta-lactoglobulin. The assay was considered positive when migration inhibition index was greater than 20 % (mean +3 SD of healthy infants). Ninety-eight infants with protracted diarrhoea and failure to thrive, 16 healthy, 12 malnourished, and 16 infants suffering from acute gastroenteritis were studied. Of the 98 patients with protracted diarrhoea, 12 fulfilled Goldman's criteria for cow's milk protein intolerance, 63 had lactose malabsorption, and in 15 no associated causative factor was identified. The mean index of migration inhibition in the cow's milk allergic group (58.83 ± 11.98) was higher than in healthy controls (8.25 ± 3.91), the difference being statistically significant ( p < 0.05). The test was positive in all patients with caw's milk protein intolerance. The assay was also positive in four other patients suffering from protracted diarrhoea, two of whom had lactose malabsorption. All the infants with acute gastroenteritis and malnutrition had values within the normal range. The migration inhibition index in five patients with cow's milk intolerance had declined to 24.74 ± 4.87 in assays performed 1-6 weeks after return of clinical tolerance to cow's milk ( p < 0.05) but the test was still within the postive range in three of the five infants. These results suggest that this cell mediated immune assay is a sensitive test for the diagnosis of cow's milk protein intolerance in infants. The specificity needs to be reassessed in the light of more objective criteria for the diagnosis of cow's milk protein intolerance.     

Prospective, controlled, multi-center study on the effect of an amino-acid-based formula in infants with cow's milk allergy/intolerance and atopic dermatitis

Cow's milk allergy/intolerance is treated by complete avoidance of cow's milk proteins. Because cow's milk is an important food for infants, its avoidance may lead to an increased risk of growth impairment. Whilst there is evidence for the beneficial effects of extensively hydrolyzed cow's milk formulae (eHF) in infants with cow's milk allergy/intolerance, little is known about the effects of amino-acid-based formulae (AA) in such infants. We therefore performed a prospective, controlled, multi-center trial to study the efficacy of AA in comparison with eHF, on the growth and clinical symptoms of 73 infants (median age 5.7 months) with cow's milk allergy/intolerance and atopic dermatitis. Cow's milk allergy/intolerance was proven in all infants by double-blind, placebo-controlled food-challenge. We observed a significant improvement in the SCORAD index in both groups, from a mean of 24.6, at entry, to a mean of 10.7 (p < 0.0001) after 6 months. In the AA group there was a significant increase in the length standard deviation score (p < 0.04), whilst there was no difference in the eHF group. The weight-for-length values were stable in both groups. The energy intake during the study was similar in both groups. Both an AA and eHF resulted in a significant clinical improvement in infants with an early onset of symptoms of cow's milk allergy/intolerance. Feeding an AA resulted in improved growth compared with feeding eHF, despite similar dietary intakes, and may therefore be considered as a beneficial alternative in infants with severe cow's milk allergy intolerance.     

Malabsorption syndrome with cow's milk intolerance. Clinical findings and course in 54 cases.

Fifty-four infants with the malabsorption syndrome and cow's milk intolerence seen during 1962-1971 were investigated. All had diarrhoea and failed to thrive. Most had vomiting and about 20% had atopic eczema and recurrent respiratory infections. Laboratory investigations revealed malabsorption, raised serum IgA, and precipitins to cow's milk. Biopsies showed that the jejunal mucosa was damaged, and in about half the cases was flat. The patient did well on human milk but reacted clinically to cow's milk challenge, either in a few hours or gradually during 3-4 weeks. Some patients showed first a quick, but later a slow, reaction. Clinical symptoms of cow's milk intolerance disappeared at the age of about one year. At that time 81% had normal faecal fat, but only 29% had a normal proximal jejunal mucosa. Many of the patients developed intolerances to other food proteins, such as soya and wheat, if these were given during the sensitive period. Forty-two patients have been followed up for 2 years on a normal gluten-containing diet. Of these, 37 have a normal or nearly normal jejunal mucosa and 5 (12%) have subtotal villous atrophy indicative of coeliac disease. It is concluded that the malabsorption syndrome with cow's milk intolerance is a clear-cut clinical entity. However, the symptomatology, results of laboratory tests, and jejunal biopsy findings closely resemble those of other entities where damage to the intestinal mucosa causes a malabsorption snydrome. Follow-up studies showed that the disease is transient, but about 10% of the patients have coeliac disease, regarded in such cases as the primary disorder.     

The diagnostic significance of antibodies to various cow's milk proteins (fluorescent immunosorbent test)

Antibodies of various immunoglobulin classes to different cow's milk proteins were studied with the fluorescent immunosorbent test in 601 newborns, infants, children and adults (A). The antibody levels, expressed as the geometric mean (gm) of four antibody titres to casein, -lactoglobulin, -lactalbumin and bovine serum albumin, showed a clear dependence on age. They were compared with the antibody levels in children with cow's milk protein intolerance (C), other gastrointestinal disorders (B) and coeliac disease (D). The 20 children with cow's milk protein intolerance clearly differed (significance level 2×10^-11) from those of the two control groups (A, B) insofar as the criterion adopted was not the titre against a single protein but the gm of the four antibody titres, and insofar as allowance was made for the age of the patients.All patients with cow's milk protein intolerance also showed elevated gm titres of IgE, IgA and IgM antibodies. However, since a number of children in the control groups also showed higher values, particularly with regard to IgE antibodies, the determination of the IgE, IgA and IgM antibodies adds little to the diagnosis and at best provides a further discriminatory aid.Although antibody titres fall immediately after placing the child on a milk-free diet, it is a matter of months before they become negative (titre <120). After challenge titres rise again. In a longitudinal study of 25 children with acute gastroenteritis (E) it was shown that the antibody titres remained unchanged during and after the attack. This contradicts the often expressed opinion that the cow's milk antibodies frequently observed in healthy infants are induced as a consequence of gastroenteritis. In contrast to the other groups, all 26 children with proven coeliac disease (D) had antibodies to gliadin, irrespective of whether their gm cow's milk antibody titre was high or low.Computer Center of the University of Basle