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Effect of a single dose of lactase on symptoms and expired hydrogen after lactose challenge in lactose-intolerant subjects.

The effect of a single dose or oral lactase on symptoms, breath hydrogen concentration, and glucose absorption in lactose-intolerant subjects challenged with lactose was studied. Volunteers underwent a lactose challenge test; those whose breath hydrogen concentrations increased 20 ppm or more and who met other criteria were admitted as subjects. After fasting, the subjects were given three chewable lactase tablets (total lactase dose, 9900 FCC units) or placebo tablets in a randomized, double-blind, crossover manner. The subjects also consumed 8 oz of whole milk in which 37.5 g of lactose powder was dissolved (total lactose content, 50 g). The washout period between lactose challenges was at least one week. Breath hydrogen and plasma glucose concentrations were measured before and at intervals after the challenges, and the subjects completed symptom-evaluation questionnaires every eight hours for four days. Twenty-four subjects completed the study. The maximum mean breath hydrogen concentration was significantly lower after lactase treatment than after placebo treatment. In 21 subjects, the area under the hydrogen concentration-time curve (AUC) was lower after lactase than after placebo; three subjects had hydrogen AUCs more than 300 ppm.hr lower. There were no significant differences in plasma glucose levels. Subjective ratings of the severity of abdominal cramping, belching, flatulence, and diarrhea were lower during the first eight hours after challenge in lactase-treated subjects; ratings for bloating were lower during the next eight hours. Single doses of a chewable lactase tablet reduced the concentration of expired hydrogen and symptoms of lactose intolerance after a lactose challenge.

Abdominal Muscles↗

Intestinal lactase. Shift in intracellular processing to altered, inactive species in the adult rat.

The regulatory mechanism of decline in catalytic activity for intestinal lactase (lactase-phlorizin hydrolase, beta-galactosidase) as mammals mature has not been defined. Solubilized intestinal brush-border membranes from adult male rats (greater than 4 months of age, 200-400 g) were examined by high performance liquid Zorbax GF-450 chromatography, subjected to denaturing acrylamide electrophoresis, blotted to nitrocellulose, and identified by specific polyvalent anti-lactase. Three major species were present within the 235-kDa active lactase peak (225, 130, and 100 kDa). The 100-kDa moiety was also prominent in the approximately 300-kDa region of the GF-450 effluent, suggesting it is a catalytically inactive oligomer. In vivo synthesis and assembly of lactase by intraintestinal pulse [( 35S]methionine, 5 min) and chase (15-120 min) revealed rapid (15 min of chase; maximum, 60 min) intracellular synthesis in the endoplasmic reticulum-Golgi fraction of multiple species (64, 100, 130, 175, and 225 kDa). The 64-kDa species disappeared from the intracellular membrane compartment and was not transferred to the brush-border surface. The 175-kDa moiety appeared to be processed to the 225-kDa unit prior to relocation to the surface membrane. By 120 min, the 100-kDa species became the predominant (approximately 60%) radiolabeled unit in both endoplasmic reticulum-Golgi and brush border. In the adult rat, lactase is assembled in multiple molecular forms that are differentially processed: (a) intracellular degradation (64-kDa unit) or (b) transfer to the brush-border surface as catalytically active (225 and 130 kDa) or inactive (100 kDa) species. Although substantial synthesis of lactase proteins prevails, major changes in processing appear to serve as an important regulatory mechanism producing the maturational decline of catalytic activity. The accompanying article (Castillo, R. O., Reisenauer, A. M., Kwong, L. K., Tsuboi, K. K., Quan, R., and Gray, G. M. (1990) J. Biol. Chem. 265, 15889-15893) extends our studies to synthesis and assembly during the neonatal period of maturation.

Animals↗

Dietary control of lactase expression in the weaning rat.

The decline in lactase activity during weaning has been well established. However, its molecular mechanism remains to be explored. We studied changes in the expression of lactase in terms of the transcription and translation processes in rat microvillus membrane by Northern blot and Western blot analysis, respectively. To examine the effect of dietary change from a milk to a non-milk diet on the developmental pattern of lactase expression, weaning was prevented by keeping the rats under suckling conditions for 27 days after birth. This treatment only suppressed the extent of decline: while the weanlings showed 17 percent activity compared to that of 4-day-old rats, the prolonged suckling rats showed only 42 percent. The changes in the expression of lactase mRNA and protein were parallel with the change of lactase activity. In other words, the fundamental pattern of significant depression of lactase expression occurred relatively independent of dietary modification. This observation indicates that the regulation of lactase expression is firmly determined at the transcriptional level, and that dietary factor such as the termination of lactose ingestion has only a relatively minor effect.

Animals↗

Absorption of nutrients in lactase deficiency.

The influence of malabsorption of lactose, as a result of primary lactase deficiency, on the absorption of the nutrients in milk was tested in four healthy controls and four subjects with lactase deficiency. An ileal perfusion technique was used to quantify arrival in the ileum of nutrients and a nonabsorbable marker (polyethylene glycol, PEG 4000) ingested as a test meal of milk. The meal was 250 ml of whole milk or milk in which the lactose had been hydrolyzed to glucose and galactose. In the fasting state, ileal flow of volume, protein, carbohydrate, and electrolytes was small and not different in controls and lactase-deficient subjects. Ileal flow increased in all subjects after the test meal of milk; more fluid and nutrient was recovered from the ileum in lactase-deficient subjects after whole milk than in control subjects or in lactase deficiency after hydrolyzed milk. Two deficient subjects showed marked malabsorption of lactose (35 and 50%); two did not. Protein, calcium, magnesium, and phosphorus were also recovered from the ileum in greater quantities in lactase deficients after whole milk. However, apart from decreased absorption of lactose, the nutritional consequences of malabsorption in association with primary lactase deficiency in adults are probably minimal.

Adult↗

Cellular origin of lactase decline in postweaned rats.

Intestinal lactase activity falls at weaning reaching low levels in the adult rat. Present work measures cellular migration rates and lactase activity along villi of intestines taken from rats of different ages to determine the cellular basis for this developmentally regulated fall in enzyme expression. An early fall in lactase activity taking place 15 to 23 days after birth was found to be associated with a shortening of the time available for lactase expression in brush-border membranes. A later fall in lactase activity occurring 23 to 46 days after birth was caused by an additional reduction in the rate at which lactase activity appeared in the brush-border membrane. These results are discussed in relation to previous work describing lactase biosynthesis in post-weaned rats.

Age Factors↗

Time- and dose-dependency of intestinal lactase activity in adult rat on starch intake.

Although it is generally accepted that lactase (beta-D-galactosidase, EC 3.2.1.23) activity is not influenced by intake of saccharides containing alpha-linkages, an effect of these carbohydrates on lactase activity was never thoroughly investigated. Activity of lactase and sucrose alpha-D-glucohydrolase, EC 3.2.1 48) was determined in proximal, middle and distal thirds of the jejunoileum of female, 12-week-old rats, fed for 2 weeks a low-starch (5 cal%), high-fat (73%) diet, and in rats, that after this introduction period were fed for 1, 2 and 3 days, an isocaloric middle-starch (40%), middle-fat (36%) diet or an isocaloric high-starch (70%), low-fat (7%) diet. During the entire experimental period, the body weight changes, food intake and the amount of protein per segment were practically the same in all three dietary groups. In all intestinal segments, increased intake of starch was followed by an increase of lactase and sucroase activity (both expressed as per tissue protein or per intestinal segment ) within the first day. The increase continued during the second day and leveled off during the third day. A highly significant linear correlation was found between the search content of the diets and the lactase activity in all three segments. A highly significant correlation was also established in all three segments between sucrase and lactase activities. These studies thus demonstrated a dose- and time-dependency between the intake of starch (a carbohydrate containing only alpha-linkages) and the activity of lactase, a neutral beta-galactosidase in adult rats.

Animals↗

Lactase deficiency among Malaysian children with recurrent abdominal pain.

OBJECTIVES: To determine the prevalence of lactase deficiency among Malaysian children with recurrent abdominal pain and to describe their clinical characteristics. METHODOLOGY: Twenty-four children referred consecutively to the University of Malaya Medical Centre who fulfilled Apley's criteria (at least three episodes of abdominal pain severe enough to affect normal activity over a period longer than 3 months) were tested for lactase deficiency using a pocket breath test analyser (BreatH2 meter; Europa Scientific, Cheshire, England). Lactulose was used to check for hydrogen-producing capacity. RESULTS: There were 14 males and 10 females in the study, consisting of five Malays, 14 Chinese and five Indians. Mean age was 9.9 years. Seventeen of the 24 children (70.8%) with recurrent abdominal pain who underwent the breath hydrogen test had a positive result. In those with a negative result, subsequent lactulose administration resulted in a positive rise in breath hydrogen. None of the 24 children developed abdominal pain during the test. All the Indian subjects, 71.4% of the Chinese subjects and 40% of the Malay subjects with recurrent abdominal pain had lactase deficiency. The proportion of boys and girls with lactase deficiency was similar (71.4 vs 70.0%, respectively). There was no significant difference between lactase sufficient and deficient children with recurrent abdominal pain with regard to sex, age, ethnic group and clinical features. Following a lactose-free diet, none of the children in the breath hydrogen positive and negative groups reported any appreciable difference in pain symptoms. CONCLUSIONS: The prevalence of lactase deficiency among this group of Malaysian children with recurrent abdominal pain was high, but lactase deficiency did not appear to play an important role in causing the symptoms.

Abdominal Pain↗

Effects of diets containing dried whey on the lactase activity of the small intestinal mucosa and the contents of the small intestine and cecum of the pig.

Two studies were conducted to establish the effects of dietary lactose supplied from dried whey on the lactase activity in the contents of the small intestine and cecum as well as the mucosa of the small intestine. In the first study, feeding 0, 10, 25, or 40% dried whey for 120 days did not alter the mucosal lactase specific activity (mumoles galactose released/mg protein) in the duodenum, upper jejunum, or lower ileum. In the second study, total lactase activity in the small intestine was estimated from the total activity found in 10-cm segments of each meter of small intestine. Feeding a diet containing 40% whey continuously from weaning at 5 weeks of age or from 12 weeks of age did not alter the total lactase activity of pigs killed at 21 weeks of age. Dietary lactose tended to increase the lactase activity in the contents of the cecum and small intestine, but the increases were not always statistically significant. In the second study, Chester White pigs had approximately threefold greater mucosal lactase activities than that of Hampshires. These two breeds may be suitable models for detailed studies into the relationships between lactase deficiency and lactose intolerance.

Animals↗

Lactase decline in weaning rats is regulated at the transcriptional level and not caused by termination of milk ingestion.

Lactase activity declines during postnatal development in rats, but little is known about the underlying molecular mechanism of this phenomenon. We attempted to clarify whether the regulation was at the transcriptional or post-transcriptional level and to examine the effects of dietary factors on that regulation. Newborn rats were divided into two groups, prolonged nursing and weaning, at d 21. The prolonged nursing rats were nursed for a further 6 d, whereas weaning rats were separated from their dams and fed nonpurified diet for the same period. The patterns of declining lactase protein and mRNA concentrations during weaning were determined by Western blot and Northern blot analyses, respectively, and compared with lactase activity. There were significant (P < 0.001) correlations between them: r = 0.97 for specific activity vs. protein, r = 0.99 for specific activity vs. mRNA and r = 0.96 for protein vs. mRNA. The lactase activity per milligram DNA showed a pattern similar to that of the specific activity. This result argues against the decline in lactase activity being due to the dilution caused by newly synthesized materials during the weaning period and suggests transcriptional regulation. Furthermore, the prolonged nursing rats showed the same results as weanlings for lactase protein, mRNA, specific activity and activity per milligram DNA. These observations indicate that the regulation of lactase expression is at the transcriptional level and that it is not affected by the termination of milk ingestion.

Actins↗

Effect of lactation on the decline of brush border lactase activity in neonatal pigs.

It has been shown that during the early phase of lactation porcine milk contains high concentrations of hormones and growth factors. The aim of the present investigation was to examine the hypothesis that the temporal coordination of intestinal maturation in piglets can be extrinsically regulated through changes in the composition of milk during the suckling period. Gut morphology and the ontogeny of brush border lactase activity were investigated in piglets reared on two suckling regimens designed to expose the animals to compositionally distinct milk. The first group of animals were cross-fostered onto postcolostrum sows and thereafter suckled normally for up to eight weeks. These normally suckled (N) animals consequently received both early and late lactation products. The second group of piglets were cross-fostered each week, for up to eight weeks, onto newly farrowed sows which were postcolostrum. As a result of this repeated cross-fostering (CF) these animals received only early lactation products. Animals were sacrificed at one, three, five, seven, and eight weeks postpartum. Biochemically active lactase decreased significantly (p less than 0.001) in both groups over eight weeks, but the rate of loss of activity was greater in the CF animals than in the N pigs by approximately 50% at week 3 and 25% at week 8. Quantitative histochemical analysis of lactase activity corroborated the biochemical data. At three weeks maximal enzyme activity was observed approximately 400 microns from the villus/crypt junction. Histochemically detected lactase decreased throughout the suckling period, but the intensity of reaction product was consistently weaker over the entire villus surface in the CF animals. Immunocytochemically detectable lactase was identified at the same sites as the histochemical reaction products. In addition, immunofluorescence microscopy showed the presence of histochemically undetectable enzyme on the basolateral and brush border membranes of both villus and crypt cells. Villus/crypt ratios were significantly lower (p<0.001) in the CF animals than in the N pigs between weeks 3 and 5. The results of this study suggest that lactation products can accelerate the loss of brush border lactase activity. The observed decline in biochemically and histochemically detected lactase was considered to be a consequence of reduced enterocyte lifespan, decreased synthesis of enzyme protein, or altered post-translational modification of enzyme protein, or a combination of there.

Animals↗

Temporary depression of lactase activity by thyroxine in suckling rats.

The present study was performed in order to clarify controversies concerning the effect of thyroxine on lactase activity in young mammals. Lactase activity was determined in suckling rats with two reference systems (protein and DNA) after one and three thyroxine injections (0.2 mg/100 g body weight). In comparison with control animals lactase activity was decreased after thyroxine administration. The thyroxine effect was dosage-dependent: three injections caused a more profound and prolonged depression of lactase activity. In both thyroxine-treated groups lactase activity returned to age-specific normal levels before the low lactase activity of adult rats was attained. The transient thyroxine-induced changes of lactase activity were similar to the reference parameters protein and DNA.

Animals↗

Effect of jaundice phototherapy on intestinal mucosal bilirubin concentration and lactase activity in the congenitally jaundiced Gunn Rat.

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.

Animals↗

Rat milk maintains intestinal lactase activity in rat pups whereas artificial formulas do not.

Intestinal lactase activity is maintained at high levels in suckling rats during the first 2 wk after birth. When 12-day-old rat pups were either mother fed (MF) or artificially reared (AR) with natural rat milk or several artificial formulas, the small intestines had gained similar weight in all animal groups by 16 days except in AR rats fed a chemically defined formula. In the ileum, villus length was similar in MF and AR rats, but crypt depth was significantly higher in all groups of AR rats. Ileal absorptive cells in both MF and AR rats showed immature characteristics, including supranuclear vacuoles, apical tubular systems, and pinocytotic vesicles. Jejunal lactase specific activity and total intestinal lactase activity were significantly higher in AR rats fed rat milk than MF rats at 16 days. Ileal lactase specific activity was similar in these two animal groups. In contrast, AR rats fed artificial formulas supplemented with either glucose or lactose as the sole carbohydrate source exhibited significantly lower ileal lactase specific activity and total intestinal lactase activity than MF rats. Intestinal sucrase activity was prematurely elevated in all AR rats, even when fed natural rat milk. Addition of prolactin (3.3 micrograms/ml) to an artificial formula did not prevent the premature decrease in intestinal lactase specific and total activities in AR rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

[Diagnosis of lactase deficiency with the expired hydrogen (H2) test].

To determine the sensitivity and specificity of breath hydrogen (H2) in detecting lactase deficiency, breath H2 collected by end-expiratory sampling and capillary blood glucose were measured after ingestion of 50 g of lactose in 36 patients with biopsy-proved isolated lactase deficiency, 42 with normal lactase activity and 6 with lactase deficiency secondary to mucosal lesions. All patients had digestive symptoms clinically compatible with lactose malabsorption. The maximum increase in breath H2 concentration was more than 1.1 mumol/l (25 ppm) in all patients with isolated lactase deficiency, and less than 0.88 mumol/l (20 ppm) in 88% of patients with normal lactase activity; there were 5 false-positive results, attributed in one case to small bowel colonization and in another case to rapid transit after gastric surgery. Secondary lactase deficiency was accurately detected by neither breath H2 nor blood glucose.

Adult↗

Substrate specificity of small-intestinal lactase. Assessment of the role of the substrate hydroxyl groups.

Lactase-phlorizin hydrolase is a disaccharidase present in the small intestine of mammals. This enzyme has two active sites, one being responsible for the hydrolysis of lactose. Lactase activity is thought to be selective towards glycosides with a hydrophilic aglycon. In this work, we report a systematic study on the importance of each hydroxyl group in the substrate molecule for lactase activity. For this purpose, all of the monodeoxy derivatives of methyl beta-lactoside and other lactose analogues are studied as lactase substrates. With respect to the galactose moiety, it is shown here that HO-3' and HO-2' are necessary for hydrolysis of the substrates by lactase. Using these chemically modified substrates, it has been confirmed that lactase does not behave as a typical beta-galactosidase, since it does not show an absolute selectivity with respect to substitution and stereochemistry at C4' in the galactose moiety of the substrate. However, the glucose moiety, in particular the HO-6, appears to be important for substrate hydrolysis, although none of the hydroxyl groups seemed to be essential. In order to differentiate both activities of the enzyme, a new assay for the phlorizin-hydrolase activity has also been developed.

Animals↗

Differential effects of lipid and carbohydrate on enterocyte lactase activity in newborn piglets.

The influence of enteral feeding in the neonate on lactase-phlorizin hydrolase activity in the small intestine has been determined in newborn piglets fed a series of modified colostra, with a controlled metabolizable energy intake, during the first 31.5 h of life. Striking differences were observed between lactase specific activity in mucosal homogenates and enterocyte lactase activity along the villus axis; compared with newborns, the former decreased after feeding colostrum, whereas the latter increased significantly. When lipid was present in adequate amount, the increase in enterocyte lactase activity occurred when carbohydrate was present as either lactose or galactose. However, when the lipid content of the diet was low, there was a specific effect of carbohydrate composition which was dependent on position along the villus axis: in the lower villus, colostra high in lactose or glucose stimulated an increase in lactase, but there was no such effect with a high galactose intake. It is concluded that colostrum increases enterocyte lactase activity during the first day of life, and that this is dependent on both the nutrient composition of the diet and the position of the enterocytes along the villus.

Animals↗

A novel intestinal trans-factor (NF-LPH1) interacts with the lactase-phlorizin hydrolase promoter and co-varies with the enzymatic activity.

The promoter of the pig lactase-phlorizin hydrolase was cloned and showed to be functional in the human intestinal cell line Caco2. The proximal promoter was analyzed for binding of nuclear proteins from small intestine and liver. DNase I footprinting and electrophoretic mobility shift assays show, that an intestinal nuclear factor (NF-LPH1) binds to a sequence (-40 to -54) located close to the TATA-box. Enterocytes from newborn pigs with high lactase activity contain high amounts of NF-LPH1, whereas enterocytes from adult pigs with low lactase activity contain low amounts of NF-LPH1. The liver does not contain lactase activity, and NF-LPH1 is not present in liver nuclear extracts in detectable amounts. This indicates that NF-LPH1 is involved in the decline of lactase at weaning and may be of importance for the molecular explanation of hypolactasia in humans. It was demonstrated by transfection of two different promoter-reporter gene constructs into Caco2 cells, that there are additional cis-element(s) in the region -142 to approximately -980, which are important for the transcription of the lactase-phlorizin hydrolase gene.

Animals↗

Quantitative analysis of lactase-phlorizin hydrolase expression in the absorptive enterocytes of newborn rat small intestine.

At birth, the mammalian small intestine displays regional differences in morphology as well as complex proximal-to-distal (horizontal) patterns of protein distribution. Lactase-phlorizin hydrolase (LPH), an enterocyte-specific disaccharidase crucial for the digestion of lactose in milk, reveals a characteristic horizontal pattern of expression at birth. However, it is not certain whether this topographic pattern is due to variations in epithelial structure along the length of the small intestine or to regional differences in the transcription of the LPH gene. In order to understand the mechanisms that regulate the regionalization of LPH at birth, we characterized the epithelial structure along the horizontal axis using stereologic techniques and correlated these data with the patterns of lactase activity and LPH mRNA abundance in the small intestine of unsuckled, newborn rats. Epithelial volume and microvillar surface area per unit of intestinal length decreased three-to fourfold from duodenum to distal ileum. In contrast, lactase activity and LPH mRNA abundance were highest in proximal jejunum and lowest in the most proximal and distal ends of the small intestine. Mean lactase activity per cell in proximal duodenum, proximal jejunum, and distal ileum was estimated at 12.0, 26.7, and 5.6 nU/absorptive enterocyte, respectively, and paralleled the concentration of LPH mRNA in the same segments: 20, 45, and 15 molecules of LPH mRNA/absorptive enterocyte. Our data indicate that horizontal gradients of lactase activity in the newborn rat intestine do not depend on epithelial organization or on enteral factors, since the horizontal gradient is established before suckling. Each absorptive enterocyte along the small intestine expresses lactase activity in a position-dependent manner which is controlled at the level of mRNA abundance.

Absorption↗