Genotyping of the lactase-phlorizin hydrolase -13910 polymorphism by LightCycler PCR and implications for the diagnosis of lactose intolerance.

BACKGROUND: Hypolactasia and lactose intolerance are common conditions worldwide. Hypolactasia seems to be strongly correlated with genotype C/C of the genetic variant C-->T(-13910) upstream of the lactase phlorizin hydrolase (LPH) gene. We developed a rapid genotyping assay for LPH C-->T(-13910) and investigated the relationship of positive lactose breath hydrogen test (LBHT) results suggesting lactose intolerance with LPH C-->T(-13910) genotype. METHODS: Using automated DNA purification on the MagNA Pure LC and real-time PCR on the LightCycler, we examined samples from 220 individuals to estimate genotype frequencies; we then determined LPH C-->T(-13910) genotype in samples from 54 Caucasian patients with a positive LBHT result and symptoms of lactose intolerance. RESULTS: Genotyping of 220 individuals revealed frequencies of 21.4%, 41.8%, and 36.8% for genotypes C/C, C/T, and T/T. Of the patients with positive LBHT results, only 50% had the C/C genotype suggestive of primary adult hypolactasia in our study population. The other patients had various degrees of secondary hypolactasia or symptoms of lactose intolerance. Patients with C/C genotype had a mean (SD) peak H2 increase in the LBHT [108 (58) ppm] that was significantly higher than in patients with the C/T [65 (54) ppm] and T/T [44 (34) ppm] genotypes. CONCLUSIONS: The new real-time PCR assay provides a rapid, labor-saving means for the genotyping of LPH C-->T(-13910). Use of the assay may assist in differentiating patients with primary hypolactasia from those with secondary hypolactasia and lactose intolerance, who may need further clinical examinations to diagnose their underlying primary diseases.

Investigation of three doses of oral insulin-like growth factor-I on jejunal lactase phlorizin hydrolase activity and gene expression and enterocyte proliferation and migration in piglets.

In a previous study, oral IGF-I at 65 nM increased lactase phlorizin hydrolase (LPH) activity and villus height in piglets, however, the mechanisms were unknown. Herein, the response to a range of doses of IGF-I was investigated and we hypothesized that LPH and villus height would respond to oral IGF-I in a dose-dependent manner. Two 14-d experiments were conducted using cesarean-derived piglets. In experiment 1, piglets (n = 28) were fed formula containing 0, 33, 65, or 131 nmol/L (0, 0.25, 0.5, or 1.0 mg/L) recombinant human IGF-I. In experiment 2, 5'-bromodeoxyuridine was administered to piglets fed formula alone (n = 4) or containing 131 nmol/L IGF-I (n = 4). IGF-I did not affect body weight gain or intestinal weight or length. Jejunal villus height and LPH activity were significantly greater in piglets fed 131 nmol IGF-I/L than control piglets. Villus height and lactase activity in piglets fed the 33 and 65 nmol/L IGF-I doses were similar and intermediate between control and 131 nmol IGF-I/L. Jejunal mRNA expression and LPH polypeptide abundance were investigated in piglets receiving 0 or 131 nmol/L IGF-I. Steady state LPH mRNA abundance was significantly higher (p < 0.05) in IGF-I-treated piglets. The relative abundance of proLPH(h) was not significantly increased (p = 0.06) by IGF-I treatment. Mucosal DNA content and DNA synthesis were greater in piglets receiving 131 nmol IGF-I/L than control, however, enterocyte migration and mucosal protein content were unaffected. Thus, oral IGF-I increased jejunal LPH activity and LPH mRNA abundance and stimulated intestinal cell hyperplasia in normal piglets.

Total intestinal lactase and sucrase activities are reduced in aged rats.

Lactase-phlorizin hydrolase (LPH) and sucrase-isomaltase (SI) are intestinal microvillus membrane hydrolases that play important roles in carbohydrate digestion. Although the expression of these enzymes during postnatal development has been characterized, the effect of old age on disaccharidase activity is poorly understood. In the present investigation, we examined the effect of aging on lactase and sucrase activities and their mRNA levels in the small intestines of 3-, 12- and 24- mo-old rats by sampling from nine equidistant segments of small intestine. Total intestinal disaccharidase activity or mRNA abundance was determined from areas under the proximal-to-distal curves. Rats 24 mo of age had total intestinal lactase and sucrase activities that were 12 and 38% lower, respectively, than the 3-mo-old animals (P < 0.05). In contrast, total LPH and SI mRNA abundance did not change significantly. Thus, total intestinal lactase and sucrase activities decrease with age in a manner that likely involves a posttranscriptional process. The age-related decline in disaccharidase activity, if extrapolated to humans, may have important implications for the digestion of carbohydrate contained in the diet of the elderly.

Verification of the lactase site of rat lactase-phlorizin hydrolase by site-directed mutagenesis.

BACKGROUND & AIMS: Lactase-phlorizin hydrolase (LPH) is an intestinal microvillus membrane glycoprotein that hydrolyzes lactose and phlorizin. These enzymatic activities have been assigned to glutamic acid (E) residues 1271 and 1747 in rabbit LPH. The aim of this study was to determine directly if this assignment was correct and if these two amino acids are the only nucleophiles required for LPH enzyme activity. METHODS: Site-directed mutagenesis of a full-length rat LPH complementary DNA was used to convert the rat homologues E1274 and E1750 to aspartic acid or glycine. Mutants were analyzed by enzyme activity assays. RESULTS: All tested activities of E1274D and E1274G were virtually unaffected. In contrast, mutations E1750D and E1750G resulted in total loss of lactase and cellobiose activities, leaving only low ONP-glc and ONP-gal hydrolase activities detectable. A double mutant containing both E1274G and E1750G had no activity. CONCLUSIONS: These studies directly confirm that the two previously identified glutamic acids are essential to the enzymatic activity of rat LPH. Rat lactase activity is not associated with the E1274 site. This study provides the first evidence that rat LPH has its major catalytic site at E1750, representing all of the lactase and the majority of the phlorizin hydrolase activity.

Immunoelectron microscopic localization of lactase-phlorizin hydrolase in rat small intestine.

To provide insight into the intracellular translocation of lactase-phlorizin hydrolase, an immunoelectron microscopy was performed on rapidly embedded Lowicryl K4M sections of rat jejunum. Lactase-phlorizin hydrolase immunoreactivity was detected not only in the microvillous membranes and in the smooth apical vesicles, but also in the lateral membranes, suggesting an alternative route for intracellular transport of lactase-phlorizin hydrolase via the lateral membranes to the microvilli.

Immunocytochemical heterogeneity of lactase-phlorizin hydrolase in adult lactase deficiency.

BACKGROUND: Adult lactase deficiency may result either from diminished synthesis of precursor lactase-phlorizin hydrolase (phenotype I deficiency) or from altered posttranslational processing of the precursor protein (phenotype II). The aim of this study was to compare the location of lactase-phlorizin hydrolase along the crypt-villus axis in control with adult lactase-deficient subjects. METHODS: The immunocytochemical distribution of lactase-phlorizin hydrolase was studied in subjects with adult hypolactasia and in controls with persistent high lactase expression. RESULTS: Duodenal biopsy samples from controls showed increasing intensity of brush border staining from upper crypt to midvillus. Subjects with phenotype I deficiency showed reduced and more patchy reaction product over brush borders. In some, maximal staining was localized over the upper half of the villi. The subject with phenotype II deficiency showed patchy staining but also intracellular accumulations of immunoreactivity within the apex of enterocytes mainly on the upper half of villi. Subsequent immunoelectronmicroscopy showed nearly a fourfold increase in label over the endoplasmic reticulum. CONCLUSIONS: These findings support prior observations of diminished enzyme synthesis in phenotype I lactase deficiency and suggest that the alterations in posttranslational processing in phenotype II deficiency involve a partial block in transport from endoplasmic reticulum to Golgi apparatus.

Administration of 3,5,3'-triiodothyronine induces a rapid increase in enterocyte lactase-phlorizin hydrolase activity of young pigs on a low energy intake.

The rapid increase in plasma concentration of 3,5,3'-triiodothyronine (T3) which occurs after feeding may invoke changes in lactase-phlorizin hydrolase (LPH) activity of the small intestine. This hypothesis has been tested in 6-week-old pigs living at thermal neutrality (26 degrees C) on a low level of energy intake. Littermate pairs were infused with either saline or T3 at 30 min intervals over a 6 h period, 18-24 h after the last meal. The activity of LPH in mucosal homogenates increased significantly in test compared with control animals (P < 0.05; T3 37% > saline). This was a specific effect on LPH since there was no effect of T3 on the activity of sucrase-isomaltase. Further, it could not be attributed to changes in intestinal morphology since there were no differences in crypt depth, villus height or villus area between the two groups. Enzyme-cytochemical analysis indicated that administration of T3 increases LPH activity at all points along the villus axis, whereas there is no effect on alpha-glucosidase (combined sucrase-isomaltase and maltase) activities. These results indicate that there is unlikely to be a simple causal relation between the immediate increase in plasma T3 after feeding and the initial decline in LPH activity observed previously in young pigs living in a cold environment. By contrast, the subsequent increase in LPH activity could be under the direct control of the food-induced increase in plasma T3 concentration, and the present results suggest a potential role for T3 as an important short-term homeostatic regulator of LPH in the small intestine.

Developmental changes in lactase-phlorizin hydrolase precursor isoforms in the rat.

The purpose of this study was to determine whether the developmental decline in lactase specific activity (mumol/min/g protein) in the rat was associated with (a) changes in the relative quantities of immunoisolated precursor and mature forms of the enzyme purified by SDS-PAGE and/or (b) immunohistologic changes in the jejunal mucosa. We studied 10- and 16-day-old suckling rat pups, 22-day-old weaned rat pups, and adult female rats (nongravid, pregnant, and lactating). Lactase activity was three- to fourfold higher in 10-day-old pups than in adult rats. Lactase activity was 27% greater in lactating compared with nongravid or pregnant rats. Three molecular forms of the enzyme that migrated identically in all animals were observed on SDS-polyacrylamide gels stained with Coomassie blue: 140-kDa (mature brush border form), 200-kDa, and 220-kDa (apparent precursor forms). There was a striking difference in the proportions of the three polypeptides at different ages that was unrelated to animal status, i.e., pregnant or lactating. As the animals aged, the relative amount of the 140-kDa band declined from 86 +/- 1.1% of the total immunoprecipitated lactase in 10-day old suckling pups to 68 +/- 0.7% in adults. Simultaneously, the relative concentration of the 200-kDa band rose from 1.7 +/- 0.4% in the 10-day-old to 19 +/- 0.6% in adults. The relative concentration of the 220-kDa polypeptide did not change as a function of age.(ABSTRACT TRUNCATED AT 250 WORDS)

Biogenesis of intestinal lactase-phlorizin hydrolase in adults with lactose intolerance. Evidence for reduced biosynthesis and slowed-down maturation in enterocytes.

Enzymatic activity, biosynthesis, and maturation of lactasephlorizin hydrolase (LPH) were investigated in adult volunteers with suspected lactose intolerance. Mean LPH activity in jejunal biopsy homogenates of these individuals was 31% compared to LPH-persistent individuals, and was accompanied by a reduced level of LPH-protein. Mean sucrase activity in individuals with low LPH was increased to 162% and was accompanied by an increase in sucrase-isomaltase (SI)-protein. Biosynthesis of LPH, SI, and aminopeptidase N (APN) was studied in organ culture of small intestinal biopsy specimens. In individuals with LPH restriction, the rate of synthesis of LPH was drastically decreased, reaching just 6% of the LPH-persistent group after 20 h of culture, while the rate of synthesis of SI appeared to be increased. In addition, maturation of pro-LPH to mature LPH occurred at a slower rate in LPH-restricted tissue. Immunoelectron microscopy revealed an accumulation of immunoreactive LPH in the Golgi region of enterocytes from LPH-restricted individuals and reduced labeling of microvillus membranes. Therefore, lactose intolerance in adults is mainly due to a decreased biosynthesis of LPH, either at the transcriptional or translational level. In addition, intracellular transport and maturation is retarded in some of the LPH-restricted individuals, and this leads to an accumulation of newly synthesized LPH in the Golgi and a failure of LPH to reach the microvillus membrane.

Glycosylation of lactase-phlorizin hydrolase in rat small intestine during development.

Age-specific changes in glycosylation of rat intestinal lactase-phlorizin hydrolase were analyzed using enzyme immunoprecipitated from microvillus membranes of suckling, weaning, and adult rats, and carbohydrate moieties were examined by lectin affinity binding, metabolic labeling, and neuraminidase treatment. Lectin binding indicated the presence of N-linked and O-linked oligosaccharide chains containing mannose and galactose throughout development. An age-dependent shift in sialic acid and fucose was seen during the period of weaning; no fucose was detectable in lactase-phlorizin hydrolase until after the rats were 20 days of age, whereas sialic acid was reduced in adult lactase-phlorizin hydrolase. The presence of sialic acid in suckling intestines and fucose in adult was confirmed by metabolic labeling with appropriate radioactive precursors. Sodium dodecyl phosphate-polyacrylamide gel electrophoresis analysis of immunoprecipitated lactase-phlorizin hydrolase from the proximal and mid small intestine showed two bands of approximately 220 and 130 kilodaltons in all age groups. In the distal part of the adult small intestine, lactase-phlorizin hydrolase appeared as two bands of similar size to those found in the proximal and mid portions. In contrast, during the suckling and weaning periods, these distal bands were approximately 225 and 135 kilodaltons. [35S]-methionine labeling and fluorography of neonatal intestines confirmed these observations. The size difference between proximal and distal small intestines was virtually eliminated by neuraminidase treatment. These data indicate that the core structure of microvillus membrane lactase-phlorizin hydrolase, consisting of both N-linked and O-linked oligosaccharides, remains constant during development, although terminal sugars shift from predominantly sialic acid during the suckling period to fucose in adulthood. This alteration in glycosylation of the protein occurs in a different pattern from the postweaning decline in lactase specific activity. Consequently, age-dependent changes in glycosylation cannot account for the decrease in lactase-phlorizin hydrolase-specific activity observed during development.

Complete primary structure of human and rabbit lactase-phlorizin hydrolase: implications for biosynthesis, membrane anchoring and evolution of the enzyme.

We report the primary structures of human and rabbit brush border membrane beta-glycosidase complexes (pre-pro-lactase-phlorizin hydrolase, or pre-pro-LPH, EC 3.2.1.23-62), as deduced from cDNA sequences. The human and rabbit primary translation products contain 1927 and 1926 amino acids respectively. Based on the data, as well as on peptide sequences and further biochemical data, we conclude that the proteins comprise five domains: (i) a cleaved signal sequence of 19 amino acids; (ii) a large 'pro' portion of 847 amino acids (rabbit), none of which appears in mature, membrane-bound LPH; (iii) the mature LPH, which contains both the lactase and phlorizin hydrolase activities in a single polypeptide chain; (iv) a membrane-spanning hydrophobic segment near the carboxy terminus, which serves as membrane anchor; and (v) a short hydrophilic segment at the carboxy terminus, which must be cytosolic (i.e. the protein has an Nout-Cin orientation). The genes have a 4-fold internal homology, suggesting that they evolved by two cycles of partial gene duplication. This repetition also implies that parts of the 'pro' portion are very similar to parts of mature LPH, and hence that the 'pro' portion may be a water-soluble beta-glycosidase with another cellular location than LPH. Our results have implications for the decline of LPH after weaning and for human adult-type alactasia, and for the evolutionary history of LPH.

Immunoelectrophoretic studies on human small intestinal brush border proteins. A quantitative study of brush border enzymes from single small intestinal biopsies.

A method for measuring brush border membrane enzymes from small intestinal biopsies by crossed immunoelectrophoresis is presented. The use of a brush border specific antiserum made isolation of the brush border membrane before analysis unnecessary. This prevented loss of material which, together with inactivation of enzymes, was a limiting factor in previous studies of brush border enzymes from peroral biopsies. In 58 biopsies from patients without gastrointestinal disorders a close correlation between antigenic activity and corresponding enzymatic activity was shown for the following enzymes: sucrase-isomaltase (EC 3.2.1.48-EC 3.2.1.10), lactase-phlorizin hydrolase (EC 3.2.1.23-EC 3.2.1.62), microvillus aminopeptidase (microsomal, EC 3.4.11.2) and dipeptidyl peptidase IV (EC 3.4.14.X). The immunoelectrophoretic patterns of intestinal mucosa near the ligament of Treitz, and in jejunum and ileum were established. The method presented is thought to be of value in further studies of the molecular basis of brush border diseases.