Total Mucosal Protein Research Articles

Method development for quantification of DMET-proteins in the paediatric intestinal tract via LC-MS/MS using a QconCAT technique

Physiologically based pharmacokinetic (PBPK) modelling is a powerful alternative for paediatric clinical trials. Paediatric PBPK models require data on intestinal drug metabolising enzymes and transporter (DMET)-protein abundances, however only limited data is available. This is the first study to report paediatric duodenal DMET protein quantification using a QconCAT approach. Thirty-six paediatric intestinal biopsies have been obtained from which the total mucosal protein was extracted. Proteins were digested to peptides using FASP and peptide levels were quantified using LC-MS/MS. Preliminary results provide some insight on the effect of age on duodenal protein abundance.

Associations of mucosal disaccharidase kinetics and expression in the jejunum of steers with divergent average daily gain.

The objective of this study was to quantify the differences in the activity of jejunal maltase and isomaltase between two groups of steers with average dry matter intake (DMI) and differing average daily gain (ADG). DMI and ADG were measured in crossbred steers (n = 69; initial body weight = 456 ± 5.0 kg) consuming a finishing diet containing 67.8% dry-rolled corn, 20.0% wet distillers grains with solubles, 8.0% alfalfa hay, and 4.2% vitamin/mineral supplement on a dry matter basis for 84 d. Jejunal mucosal samples were collected from eight steers with the greatest (high) or least (low) ADG and average DMI (± 0.55 standard deviation). Homogenates of jejunal mucosa were incubated with increasing amounts of maltose and isomaltose to determine the disaccharidase kinetics. Total mucosal protein concentration (mg protein/g tissue; P = 0.45) of the mucosa and small intestinal weights (P = 0.69) did not differ between the groups. Neither the Michaelis-Menten constant (Km) of isomaltase (P = 0.15) nor maltase (P = 0.21) differed between groups. The isomaltase maximum velocity (Vmax) expressed per gram of protein tended to differ (P = 0.10) between groups of steers but did not differ (P = 0.13) when expressed on a tissue basis. Similarly, neither the maltase Vmax expressed per gram of protein (P = 0.31) nor tissue (P = 0.32) differed between groups. While previous studies have indicated that disaccharidase expression is associated with differences in ADG, data presented here indicate that differences in enzyme activity at the end of the finishing period are minimal.

Quantitative Proteomics of Clinically Relevant Drug-Metabolizing Enzymes and Drug Transporters and Their Intercorrelations in the Human Small Intestine.

The levels of drug-metabolizing enzymes (DMEs) and transporter proteins in the human intestine are pertinent to determine oral drug bioavailability. Despite the paucity of reports on such measurements, it is well recognized that these values are essential for translating in vitro data on drug metabolism and transport to predict drug disposition in gut wall. In the current study, clinically relevant DMEs [cytochrome P450 (P450) and uridine 5′-diphospho-glucuronosyltransferase (UGT)] and drug transporters were quantified in total mucosal protein preparations from the human jejunum (n = 4) and ileum (n = 12) using quantification concatemer–based targeted proteomics. In contrast to previous reports, UGT2B15 and organic anion-transporting polypeptide 1 (OATP1A2) were quantifiable in all our samples. Overall, no significant disparities in protein expression were observed between jejunum and ileum. Relative mRNA expression for drug transporters did not correlate with the abundance of their cognate protein, except for P-glycoprotein 1 (P-gp) and organic solute transporter subunit alpha (OST-α), highlighting the limitations of RNA as a surrogate for protein expression in dynamic tissues with high turnover. Intercorrelations were found within P450 [2C9-2C19 (P = 0.002, R2 = 0.63), 2C9–2J2 (P = 0.004, R2 = 0.40), 2D6-2J2 (P = 0.002, R2 = 0.50)] and UGT [1A1-2B7 (P = 0.02, R2 = 0.87)] family of enzymes. There were also correlations between P-gp and several other proteins [OST-α (P < 0.0001, R2 = 0.77), UGT1A6 (P = 0.009, R2 = 0.38), and CYP3A4 (P = 0.007, R2 = 0.30)]. Incorporating such correlations into building virtual populations is crucial for obtaining plausible characteristics of simulated individuals.SIGNIFICANCE STATEMENTA number of drug transporters were quantified for the first time in this study. Several intercorrelations of protein abundance were reported. mRNA expression levels proved to be a poor reflection of differences between individuals regarding the level of protein expression in gut. The reported abundance of drug-metabolizing enzymes and transporters and their intercorrelations will contribute to better predictions of oral drug bioavailability and drug-drug interactions by linking in vitro observations to potential outcomes through physiologically based pharmacokinetic models.

Luminal Threonine Concentration Acutely Affects Intestinal Mucosal Protein and Mucin Synthesis in Piglets 3

Intestinal mucin synthesis is sensitive to dietary threonine supply, which suggests that the gut’s requirement for threonine may comprise a significant proportion of the whole body requirement. We used a continuously perfused gut loop model and intraluminal flooding dose technique in 6 young pigs to study the acute effects of varying luminal availability of threonine on intestinal protein and mucin syntheses. A complete amino acid mixture containing 0, 21, or 56 mg threonine/g total amino acids (whole body requirement ratio is ∼28 mg/g) was continuously perfused in isolated loops for 120 min, including a 30-min 3H-phenylalanine flooding dose. We measured fractional synthesis rates of total mucosal protein and mucin by analyzing 3H-phenylalanine incorporation. Fractional rates of total mucosal protein synthesis were higher in loops perfused with solutions containing threonine at 56 mg/g (66 ± 4%/d) compared with 0 mg/g (42 ± 9%/d) and 21 mg/g (53 ± 6%/d) (P < 0.05). For mucin, fractional rates of synthesis differed between 0 mg/g (323 ± 72%/d), 21 mg/g (347 ± 49%/d), and 56 mg/g (414 ± 31%/d) (P < 0.05). In addition, total proline and threonine concentrations in the protein hydrolysates increased with luminal threonine concentration (P < 0.05), indicating an increase in threonine- and proline-rich proteins. De novo synthesis of mucosal and mucin proteins is acutely sensitive to luminal threonine concentration, which demonstrates the importance of dietary amino acid supply to gut protein metabolism.

Intestinal protein and LPH synthesis in parenterally fed piglets receiving partial enteral nutrition and enteral insulinlike growth factor 1.

Providing partial enteral nutrition (PEN) supplemented with insulinlike growth factor-1 (IGF-1) to parenterally fed piglets increases lactase-phlorizin hydrolase (LPH) activity, but not LPH mRNA. The current aim was to investigate potential mechanisms by which IGF-1 up-regulates LPH activity. Newborn piglets (n = 15) received 100% parenteral nutrition (TPN), 80% parenteral nutrition + 20% parenteral nutrition (PEN), or PEN + IGF-1 (1.0 mg. kg-1. d-1) for 7 days. On day 7, [2H3]-leucine was intravenously administered to measure mucosal protein and brush border LPH (BB LPH) synthesis. Weight gain, nutrient intake, and jejunal weight and length were similar among the treatment groups. Partial enteral nutrition alone increased mucosal weight, villus width and cross-sectional area, LPH activity, mRNA expression, and high mannose LPH precursor (proLPHh) abundance compared with TPN (P<0.05). Insulinlike growth factor-1 further increased mucosal weight, LPH activity, and LPH activity per unit BB LPH approximately twofold over PEN alone (P < 0.05) but did not affect LPH mRNA or the abundance of proLPHh (one of the LPH isoforms) or mature LPH. Isotopic enrichment of [2H3]-leucine in plasma, mucosal protein, and LPH precursors, and the fractional and absolute synthesis rates of mucosal protein and LPH were similar among the treatment groups. Insulinlike growth factor-1 treatment increased total mucosal protein synthesis (60%, P < 0.05) but not LPH synthesis compared with the other two groups. Because IGF-1 did not affect the fractional synthesis rate of either mucosal protein or LPH, the authors suggest that enteral IGF-1 increases mucosal protein mass and LPH activity by suppressing mucosal proteolytic degradation.

Intestinal Protein and LPH Synthesis in Parenterally Fed Piglets Receiving Partial Enteral Nutrition and Enteral Insulinlike Growth Factor 1

ABSTRACTBackgroundProviding partial enteral nutrition (PEN) supplemented with insulinlike growth factor‐1 (IGF‐1) to parenterally fed piglets increases lactase–phlorizin hydrolase (LPH) activity, but not LPH mRNA. The current aim was to investigate potential mechanisms by which IGF‐1 up‐regulates LPH activity.MethodsNewborn piglets (n = 15) received 100% parenteral nutrition (TPN), 80% parenteral nutrition + 20% parenteral nutrition (PEN), or PEN + IGF‐1 (1.0 mg · kg−1 · d−1) for 7 days. On day 7, [2H3]‐leucine was intravenously administered to measure mucosal protein and brush border LPH (BB LPH) synthesis.ResultsWeight gain, nutrient intake, and jejunal weight and length were similar among the treatment groups. Partial enteral nutrition alone increased mucosal weight, villus width and cross‐sectional area, LPH activity, mRNA expression, and high mannose LPH precursor (proLPHh) abundance compared with TPN (P &lt;0.05). Insulinlike growth factor‐1 further increased mucosal weight, LPH activity, and LPH activity per unit BB LPH approximately twofold over PEN alone (P &lt; 0.05) but did not affect LPH mRNA or the abundance of proLPHh (one of the LPH isoforms) or mature LPH. Isotopic enrichment of [2H3]‐leucine in plasma, mucosal protein, and LPH precursors, and the fractional and absolute synthesis rates of mucosal protein and LPH were similar among the treatment groups. Insulinlike growth factor‐1 treatment increased total mucosal protein synthesis (60%, P &lt; 0.05) but not LPH synthesis compared with the other two groups.ConclusionsBecause IGF‐1 did not affect the fractional synthesis rate of either mucosal protein or LPH, the authors suggest that enteral IGF‐1 increases mucosal protein mass and LPH activity by suppressing mucosal proteolytic degradation.

Purification and Partial Characterization of Cellular Retinol-Binding Protein, Type Two, from Human Small Intestine

Two forms of human cellular retinol-binding protein, type II [hCRBP(II)A and hCRBP(II)B], were purified from the small intestine in a three-step purification procedure. The N-terminal sequence of CRBP(II)A was determined to 36 residues and found to have only a single residue difference when compared with the N-terminal sequence in rat; the amino acid change maintained charge. hCRBP(II)B was blocked at the N-terminus, presumably because of N-acetylation of the initial threonine residue as previously established for CRBP(II)B from rat. This blockage did not affect the binding properties of the protein. The ability of pure hCRBP(II) to bind all-trans-retinol, retinal and retinoic acid was examined by competitive binding assay and compared with the binding specificity of pure human cellular retinol-binding protein (hCRBP). Retinoic acid did not compete with retinol for binding to either protein. Retinal competed with retinol for binding to hCRBP(II) but not to hCRBP, consistent with what was observed for the homologous proteins of rats. Polyclonal antiserum against hCRBP(II)B was produced that recognized both forms of hCRBP(II) in Western blotting and RIA but did not react with hCRBP. Distribution of hCRBP(II) was determined in the jejunum. Higher concentrations of hCRBP(II) were observed in the proximal portion compared with the distal portion. Although concentrations varied in individual intestinal samples, concentrations up to 1% of total mucosal protein were observed in some samples. These results provide tools for further investigation of the role of hCRBP(II) and suggest that previous results from rats, in this important aspect, are relevant to human metabolism of vitamin A.

Digestive development of the early-weaned pig

Gastric intubation was adopted as a means of comparing the effect of two feeding levels, continuous nutrient supply (C) and restricted nutrient supply (R), on the digestive development of pigs weaned at 14 d of age, during the first 5 d post-weaning. The absolute weights of the stomach and the pancreas were significantly greater (P less than 0.001) in C compared with R pigs. The effect was not significant for pancreas weight when expressed per kg body-weight but was significant (P less than 0.05) for stomach weight. The weights of the small intestine (SI), SI mucosa and total mucosal protein were significantly higher (P less than 0.001) in C pigs but protein content per g mucosa was similar in the C and R groups. There was no significant effect of treatment on the activity of lactase (beta-glucosidase; EC 3.2.1.23) or sucrase (sucrose-alpha-glucosidase; EC 3.2.1.48) irrespective of the basis of comparison used. The specific activity (mumol/min per g protein) of maltase (alpha-glucosidase; EC 3.2.1.20) and of glucoamylase (glucan-1,4-alpha-glucosidase; EC 3.2.1.3) were similar in C and R groups but activities of maltase (mumol/g mucosa) (P less than 0.05), and maltase and glucoamylase (mol/d) (P less than 0.01) were significantly higher in C pigs. Villous height and crypt depth were significantly greater in C pigs (P less than 0.001 and P less than 0.05 respectively). Enteroglucagon was significantly (P less than 0.05) higher in C compared with R pigs. Xylose absorption and the digestibility of energy were not affected by treatment. Digestibility of dry matter, organic matter, crude protein (nitrogen x 6.25) and carbohydrate were significantly higher (P less than 0.001, P less than 0.01, P less than 0.05 and P less than 0.001 respectively) in R pigs compared with C pigs but the differences were small, ranging from 1.3 to 2.5%. These results demonstrate that (1) nutrient intake in the weaned pig affects the anatomy, morphology and function of the gut, (2) there is considerable 'spare capacity' for digestion of cereal-based diets even in pigs weaned at 14 d of age, (3) measurements in vitro of digestive function are of limited value unless supported by information in vivo on absorption/digestibility.

Interaction of malnutrition and difluoromethylornithine-induced intestinal mucosal damage: degree of severity and subsequent recovery.

The interaction between malnutrition and exposure to a mucosal damaging agent, difluoromethylornithine (DFMO), was examined by monitoring the small-intestinal changes in weanling rats. Malnutrition as induced by the expanded-litter method resulted in severe reduction in body weights in the expanded litters as compared to normal litters. Subsequent treatment of malnourished and well-nourished pups with DFMO for 7 days resulted in decreases in small-intestinal weights and enzyme contents. A 2 factors (well-nourished and malnourished) by 2 factors (DFMO-treated and nontreated) analysis of variance showed no interaction between malnutrition and DFMO treatment in terms of food intake, total mucosal protein, and contents of enterokinase, leucine aminopeptidase and sucrase. Very slight and insignificant interactions (p less than or equal to 0.2) were found for body weights, intestinal weights and total DNA content. Only one parameter studied, the maltase content, showed significant interaction between malnutrition and DFMO treatment (p less than 0.05). Three weeks after the withdrawal of DFMO, essentially all the changes caused by DFMO recovered. But those changes caused by malnutrition did not, such that the malnourished group, whether treated with DFMO or not, still remained significantly less than the control group in their small-intestinal parameters. Analysis of variance showed no interaction between malnutrition and DFMO treatment in the recovery phase. The results suggest that malnutrition is a more important factor in determining the intestinal damage and that malnutrition in the immediate postnatal period does not increase the sensitivity of the small intestine to the damaging effect of DFMO.

Vitamin D deficiency, pancreatic and small intestinal enzyme development in rats.

Maternal vitamin D deficiency has been shown to lead to reduced body weights in developing rat pups. To evaluate the effects of vitamin D deficiency alone both in dams and pups during the perinatal age on the ontogeny of gastrointestinal enzymes, female weanling rats (3 weeks of age) were divided into three groups. Groups I and II were fed a control (vitamin-D-replete) diet. Group II were fed a vitamin-D-deficient diet. Six weeks afterward they were mated with normal male rats while continuing on their respective diets until sacrifice. Only rats that delivered their pups on the same day from each group were brought into the study. Litter sizes of groups I and II were adjusted to 10, while group III was adjusted to 13 such that the rate of growth paralleled that of group II. At 19 days after birth, all dams and pups were sacrificed. There were no differences in the calcium and phosphorus contents in breast milk obtained from dams of each group. The serum calcium concentration of pups from group II (vitamin-D-deficient) was lower than the other groups. Body weights of pups from groups II and III were significantly lower than those of group I. The mucosal weight, total mucosal protein, mucosal DNA, sucrase, and maltase activities from groups II and III were similar, but lower than group I. Pancreatic weight, total pancreatic protein, DNA, amylase, and lipase activities from groups II and III were also similar, but lower than group I. Vitamin D deficiency was confirmed in both dams and pups from group II.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbohydrate dependent protein synthesis and enzyme activity in the jejunum

The mechanism by which dietary carbohydrate (CH) regulates jejunal disaccharidase activity has not been elucidated. Studies were undertaken to determine whether adaptive changes in disaccharidase levels were accompanied by variations in protein synthesis; and if so, whether they were related to cell migration and RNA synthesis. 200 g rats were fed a CH-free diet (with isocaloric protein replacement) for 7 days and then fed a diet rich in either sucrose (68% cal) or starch (51% cal). Incorporation of 14C amino acids into total mucosal and brush border (BB) proteins (TCA insoluble) was measured. Both sucrose and starch stimulated an increase of 30% in the labeling of total mucosal proteins and greater than 100% rise in that of BB proteins. Simultaneously, there was 100% rise in sucrose and lactase activity and 60% increase in maltase activity in crude jejunal homogenate and BB. When actinomycin-D was injected IP 6 hrs prior to feeding sucrose, RNA synthesis measured 24 hrs later was reduced 50%, and cell migration was arrested. Although neither basal rates of total mucosal protein synthesis nor the increase stimulated by CH feeding were inhibited, 14C labelling of the BB was reduced. Sucrase, lactase and maltase levels in the crude homogenates rose 75–100%, but these activities in the BB did not increase in actinomycin-D treated animals. The data suggest that dietary CH controls disaccharidase acitivity by changes in protein synthesis independent of RNA synthesis and cell migration, and support the concept that there is an RNA-dependent step required for the transfer of newly formed disaccharidases from sites of synthesis to the BB.

The induction of calcium binding protein biosynthesis in intestine by vitamin D 3

The effect of vitamin D 3 on de novo biosynthesis of the calcium-binding protein of chick intestinal mucosa, and the temporal relationship between such synthesis and the stimulation of intestinal calcium transport by the vitamin was investigated. When doses of 20 IU of vitamin D 3 were administered to either frankly D-deficient (“rachitic”) or partially D-deficient (“marginal”) chicks, the rate of biosynthesis of Ca-binding protein as measured by incorporation of [ 3H]leucine was increased. In rachitic chicks, incorporation into Ca-binding protein was initiated from an undetectable to a substantial rate in 24 h. In marginal chicks, a three to four-fold increase in incorporation rate was seen in 12 h. Incorporation of isotope into total mucosal protein was unaffected at the 20 I.U. dose level in either type of chick, but was stimulated 50 % in 24 h by treatment of rachitic chicks with high doses of 500 I.U. This indicated that large doses of the vitamin may elicit rapid non-specific stimulatory effects on growth and metabolism, in contrast to the apparently specific effects on calcium transport of small doses. Simultaneous measurement of the effect of vitamin D on calcium transport and rate of Ca-binding protein biosynthesis in marginal chicks indicated that the rate of Ca-binding protein biosynthesis was stimulated by the vitamin several hours before any effect on transport could be detected. 9 h after the administration of 20 I.U., [ 3H]leucine incorporation into pure Ca-binding protein was stimulated two to three-fold, with no effect whatsoever on calcium transport. Ca-binding protein content in the mucosal tissue appeared to increase in parallel with the rate of calcium transport. These data are consistent with the hypothesis that vitamin D acts at the genetic level, and also suggests that Ca-binding protein may play a primary role in vitamin D-mediated calcium transport.