Determination of the Duplicated CYP2D6 Allele Using Real-Time PCR Signal: An Alternative Approach.

CYP2D6 duplication has important pharmacogenomic implications. Reflex testing with long-range PCR (LR-PCR) can resolve the genotype when a duplication and alleles with differing activity scores are detected. We evaluated whether visual inspection of plots from real-time-PCR-based targeted genotyping with copy number variation (CNV) detection could reliably determine the duplicated CYP2D6 allele. Six reviewers evaluated QuantStudio OpenArray CYP2D6 genotyping results and the TaqMan Genotyper plots for seventy-three well-characterized cases with three copies of CYP2D6 and two different alleles. Reviewers blinded to the final genotype visually assessed the plots to determine the duplicated allele or opt for reflex sequencing. Reviewers achieved 100% accuracy for cases with three CYP2D6 copies that they opted to report. Reviewers did not request reflex sequencing in 49-67 (67-92%) cases (and correctly identified the duplicated allele in each case); all remaining cases (6-24) were marked by at least one reviewer for reflex sequencing. In most cases with three copies of CYP2D6, the duplicated allele can be determined using a combination of targeted genotyping using real-time PCR with CNV detection without need for reflex sequencing. In ambiguous cases and those with >3 copies, LR-PCR and Sanger sequencing may still be necessary for determination of the duplicated allele.

Alanine-specific appetite in slow growing chickens is associated with impaired glucose transport and TCA cycle.

BACKGROUND: The rate of protein accretion and growth affect amino acid requirements in young animals. Differences in amino acid metabolism contribute to individual variations in growth rate. This study aimed at determining how amino acid needs may change with growth rates in broiler chickens. Experiment 1 consisted of testing amino acid choices in two chicken groups with extreme growth rates (the slowest -SG- or fastest -FG- growing birds in a flock). Essential (EAA) (methionine, lysine and threonine) or non-essential (NEAA) (alanine, aspartic acid and asparagine) amino acids were added to a standard control feed (13.2 MJ/kg; 21.6% crude protein). The chickens were offered simultaneous access to the control feed and a feed supplemented with one of the two amino acid mixes added at 73% above standard dietary levels. Experiment 2 consisted of the selection of the bottom 5 SG and top 5 FG chickens from a flock of 580 to study differences in amino acid metabolism using the proventriculus representing gut sensing mechanism. In this experiment, transcriptomic, proteomic, and genomic analyses were used to compare the two groups of chickens. RESULTS: SG preferred NEAA, while they rejected EAA supplemented feeds (P < 0.05). However, FG rejected NEAA (P < 0.05), and they were indifferent to EAA supplemented feed (P > 0.05). Transcriptomic and proteomic analyses identified 909 differentially expressed genes and 146 differentially abundant proteins associated with differences in growth rate (P < 0.05). The integration of gene expression and protein abundance patterns showed the downregulation of sensing and transport of alanine and glucose associated with increased alanine catabolism to pyruvate in SG chickens. CONCLUSION: Dietary preferences for NEAA in the SG group are associated with a potential cytosolic depletion of alanine following an upregulation of the catabolism into TCA cycle intermediates.

Targeted Genotyping in Clinical Pharmacogenomics: What Is Missing?

Clinical pharmacogenomic testing typically uses targeted genotyping, which only detects variants included in the test design and may vary among laboratories. To evaluate the potential patient impact of genotyping compared with sequencing, which can detect common and rare variants, an in silico targeted genotyping panel was developed based on the variants most commonly included in clinical tests and applied to a cohort of 10,030 participants who underwent sequencing for CYP1A2, CYP2C19, CYP2C9, CYP2D6, CYP3A4, CYP3A5, DPYD, SLCO1B1, TPMT, UGT1A1, and VKORC1. The results of in silico targeted genotyping were compared with the clinically reported sequencing results. Of the 10,030 participants, 2780 (28%) had at least one potentially clinically relevant variant/allele identified by sequencing that would not have been detected in a standard targeted genotyping panel. The genes with the largest number of participants with variants only detected by sequencing were SLCO1B1, DPYD, and CYP2D6, which affected 13%, 6.3%, and 3.5% of participants, respectively. DPYD (112 variants) and CYP2D6 (103 variants) had the largest number of unique variants detected only by sequencing. Although targeted genotyping detects most clinically significant pharmacogenomic variants, sequencing-based approaches are necessary to detect rare variants that collectively affect many patients. However, efforts to establish pharmacogenomic variant classification systems and nomenclature to accommodate rare variants will be required to adopt sequencing-based pharmacogenomics.

Nine-gene pharmacogenomics profile service: The Mayo Clinic experience.

PURPOSE: The Pharmacogenomics (PGx) Profile Service was a proof-of-concept project to implement PGx in patient care at Mayo Clinic. METHODS: Eighty-two healthy individuals aged 18 and older underwent genotyping of CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5, SLCO1B1, HLA-B*58:01, and VKORC1. A PGx pharmacist was involved in ordering, meeting with patients, interpreting, reviewing, and documenting results. RESULTS: Ninety three percent were CYP1A2 rapid metabolizers, 92% CYP3A4 normal metabolizers, and 88% CYP3A5 poor metabolizers; phenotype frequencies for CYP2C19 and CYP2D6 varied. Seventy-three percent had normal functioning SLCO1B1 transporter, 4% carried the HLA-B*58:01 risk variant, and 35% carried VKORC1 and CYP2C9 variants that increased warfarin sensitivity. CONCLUSION: Pre-emptive PGx testing offered medication improvement opportunity in 56% of participants for commonly used medications. A collaborative approach involving a PGx pharmacist integrated within a clinical practice with regards to utility of PGx results allowed for implementation of the PGx Profile Service. KEY POINTS: The Mayo Clinic PGx (PGx) Profile Service was a proof-of-concept project to utilize PGx testing as another clinical tool to enhance medication selection and decrease serious adverse reactions or medication failures. Over one-half of participants in the pilot using the PGx Profile Service were predicted to benefit from pre-emptive PGx testing to guide pharmacotherapy. PGx pharmacists played a crucial role in the PGx Profile Service by educating participants, identifying medication-gene interactions, and providing evidence-based (CPIC and DPWG) PGx recommendations for past, current, and future medication us.

Interplay between grain digestion and fibre in relation to gastro-small-intestinal passage rate and feed intake in pigs.

PURPOSE: The combined effects of grain digestibility and dietary fibre on digesta passage rate and satiety in humans are poorly understood. Satiety can be increased through gastric distention, reduced gastric emptying rate and when partially digested nutrients reach the terminal ileum to stimulate peptide release through the ileal/colonic brakes to slow the rate of digesta passage. This study determined the effects of grain digestibility and insoluble fibre on mean retention time (MRT) of digesta from mouth-to-ileum, feed intake (FI), starch digestion to the terminal ileum and faecal short chain fatty acids (SCFA) in a pig model. METHOD: Twelve grain-based [milled sorghum (MS), steam-flaked-sorghum, milled wheat, and steam-flaked-wheat (SFW)] diets with different intrinsic rates of starch digestion, assessed by apparent amylase diffusion coefficient (ADC), and fibre from oat hulls (OH) at 0, 5 and 20% of the diet were fed to ileal-cannulated pigs. RESULT: MRT was affected by grain-type/processing (P < 0.05) and fibre amount (P < 0.05). An approximate tenfold increase in ADC showed a limited decline in MRT (P = 0.18). OH at 20% increased MRT (P < 0.05) and reduced FI (P < 0.05). Ileal digestibility of starch increased and faecal SCFA concentration decreased with ADC; values for MS being lower (P < 0.001) and higher (P < 0.05), respectively, than for SFW. CONCLUSIONS: Lower ileal digestibility of starch, higher faecal SCFA concentration and longer MRT of MS than SFW, suggest the ileal/colonic brakes may be operating. FI appeared to decrease with increasing MRT. MRT increased and intake decreased with grain-based foods/feeds that have low starch digestibility and substantial amounts of insoluble fibre.

Methane Emissions from Ruminants in Australia: Mitigation Potential and Applicability of Mitigation Strategies.

Anthropomorphic greenhouse gases are raising the temperature of the earth and threatening ecosystems. Since 1950 atmospheric carbon dioxide has increased 28%, while methane has increased 70%. Methane, over the first 20 years after release, has 80-times more warming potential as a greenhouse gas than carbon dioxide. Enteric methane from microbial fermentation of plant material by ruminants contributes 30% of methane released into the atmosphere, which is more than any other single source. Numerous strategies were reviewed to quantify their methane mitigation potential, their impact on animal productivity and their likelihood of adoption. The supplements, 3-nitrooxypropanol and the seaweed, Asparagopsis, reduced methane emissions by 40+% and 90%, respectively, with increases in animal productivity and small effects on animal health or product quality. Manipulation of the rumen microbial population can potentially provide intergenerational reduction in methane emissions, if treated animals remain isolated. Genetic selection, vaccination, grape marc, nitrate or biochar reduced methane emissions by 10% or less. Best management practices and cattle browsing legumes, Desmanthus or Leucaena species, result in small levels of methane mitigation and improved animal productivity. Feeding large amounts daily of ground wheat reduced methane emissions by around 35% in dairy cows but was not sustained over time.

Next-Generation Sequencing of CYP2C19 in Stent Thrombosis: Implications for Clopidogrel Pharmacogenomics.

PURPOSE: Describe CYP2C19 sequencing results in the largest series of clopidogrel-treated cases with stent thrombosis (ST), the closest clinical phenotype to clopidogrel resistance. Evaluate the impact of CYP2C19 genetic variation detected by next-generation sequencing (NGS) with comprehensive annotation and functional studies. METHODS: Seventy ST cases on clopidogrel identified from the PLATO trial (n = 58) and Mayo Clinic biorepository (n = 12) were matched 1:1 with controls for age, race, sex, diabetes mellitus, presentation, and stent type. NGS was performed to cover the entire CYP2C19 gene. Assessment of exonic variants involved measuring in vitro protein expression levels. Intronic variants were evaluated for potential splicing motif variations. RESULTS: Poor metabolizers (n = 4) and rare CYP2C19*8, CYP2C19*15, and CYP2C19*11 alleles were identified only in ST cases. CYP2C19*17 heterozygote carriers were observed more frequently in cases (n = 29) than controls (n = 18). Functional studies of CYP2C19 exonic variants (n = 11) revealed 3 cases and only 1 control carrying a deleterious variant as determined by in vitro protein expression studies. Greater intronic variation unique to ST cases (n = 169) compared with controls (n = 84) was observed with predictions revealing 13 allele candidates that may lead to a potential disruption of splicing and a loss-of-function effect of CYP2C19 in ST cases. CONCLUSION: NGS detected CYP2C19 poor metabolizers and paradoxically greater number of so-called rapid metabolizers in ST cases. Rare deleterious exonic variation occurs in 4%, and potentially disruptive intronic alleles occur in 16% of ST cases. Additional studies are required to evaluate the role of these variants in platelet aggregation and clopidogrel metabolism.

Concordance between predicted HLA type using next generation sequencing data generated for non-HLA purposes and clinical HLA type.

We explored the feasibility of obtaining accurate HLA type using pre-existing NGS data not generated for HLA purposes. 83 exomes and 500 targeted NGS pharmacogenomic panels were analyzed using Omixon HLA Explore, OptiType, and/or HLA-Genotyper software. Results were compared against clinical HLA genotyping. 765 (94.2%) Omixon and 769 (94.7%) HLA-Genotyper of 812 germline allele calls across class I/II loci and 402 (99.5%) of 404 OptiType class I calls were concordant to the second field (i.e. HLA-A*02:01). An additional 19 (2.3%) Omixon, 39 (4.8%) HLA-Genotyper, and 2 (0.5%) OptiType allele calls were first field concordant (i.e. HLA-A*02). Using Omixon, four alleles (0.4%) were discordant and 24 (3.0%) failed to call, while 4 alleles (0.4%) were discordant using HLA-Genotyper. Tumor exomes were also evaluated and were 85.4%, 91.6%, and 100% concordant (Omixon and HLA-Genotyper with 96 alleles tested, and Optitype with 48 class I alleles, respectively). The 15 exomes and 500 pharmacogenomic panels were 100% concordant for each pharmacogenomic allele tested. This work has broad implications spanning future clinical care (pharmacogenomics, tumor response to immunotherapy, autoimmunity, etc.) and research applications.

PharmVar GeneFocus: CYP2D6.

The Pharmacogene Variation Consortium (PharmVar) provides nomenclature for the highly polymorphic human CYP2D6 gene locus. CYP2D6 genetic variation impacts the metabolism of numerous drugs and, thus, can impact drug efficacy and safety. This GeneFocus provides a comprehensive overview and summary of CYP2D6 genetic variation and describes how the information provided by PharmVar is utilized by the Pharmacogenomics Knowledgebase (PharmGKB) and the Clinical Pharmacogenetics Implementation Consortium (CPIC).

Wheat bran and oat hulls have dose-dependent effects on ad-libitum feed intake in pigs related to digesta hydration and colonic fermentation.

Undigested nutrients and fermentable fibre in the distal ileum and colon stimulate intestinal brakes, which reduce gastric-emptying and digesta-passage-rate, and subsequently limit feed/food-intake. Fibre can also stimulate passage rate potentially increasing feed intake (FI). In order to experimentally determine the relationships between these two hypothesised actions of fibre, five levels of wheat-bran (WB) or oat-hulls (OH) were added to a highly digestible starch-based diet fed to pigs ad-libitum for three weeks. Average-daily-feed-intake (ADFI), faecal short-chain-fatty-acids (SCFA) and related parameters were determined at 7, 14 and 21d. A linear mixed model was fitted to FI and fermentation parameters. Overall, WB diets showed 8-11% lower ADFI (7-14d: p < 0.05; 7-21 & 0-21d: p = 0.053) than OH diets. WB diets produced over 20% more (21d: p < 0.01) SCFA than OH or Control diets. WB at 25% produced 22% more (7d: p < 0.05) SCFA than any other diet. Diets with WB at 25 and 35%, showed higher hydration capacity than any other diet (p < 0.001). OH at 10% had an unusually low FI and a markedly higher hydration capacity. With increasing levels of OH, intake of base diet was 7% more than control at 5% OH, but 8% less than control at 20% OH. With increasing WB content, intake of base diet decreased. From these results, we propose that three mechanisms control the effects of fibre on FI: initial increase in passage rate and feed intake at low concentrations of non-swelling fibres; a depression in FI from high fibre bulk; and reduced feed intake from stimulation of ileal and colonic brakes.

Effect of catechol-O-methyltransferase (rs4680) single-nucleotide polymorphism on opioid-induced hyperalgesia in adults with chronic pain.

The catechol-O-methyltransferase Val158Met polymorphism has been associated with alterations in pain perception, but the influence of the polymorphism on pain perception in patients with chronic pain receiving daily opioid therapy has not been previously reported. The primary aim of this study was to investigate the effects of the catechol-O-methyltransferase Val158Met polymorphism on heat pain perception in a cohort of adults receiving daily opioid therapy for chronic pain. Adults with chronic pain consecutively admitted to an outpatient pain rehabilitation program who met inclusion criteria and were receiving daily opioid therapy were recruited for study participation (N = 142). Individuals were genotyped for catechol-O-methyltransferase Val158Met (rs4680), and the polymorphism was analyzed using an additive and codominant genotype models. The distribution of the Val158Met genotypes was 25% for Val/Val, 41% for Val/Met and 34% for Met/Met (Hardy-Weinberg, P > 0.05). A main effect of genotype was observed for heat pain perception ( P = 0.028). Under the codominant model of allele effects, exploratory post hoc pairwise comparisons adjusted for morphine equivalent dose and pain catastrophizing demonstrated that individuals with the Val/Met genotype were hyperalgesic compared to individuals with the Val/Val ( P = 0.039) and Met/Met ( P = 0.023) genotypes. No significant association was observed between heat pain perception and genotype under the additive model of allele effects. Among patients with chronic pain who were receiving daily opioids, the Val/Met genotype was associated with hyperalgesia using a measure of heat pain perception that has been previously indicative of opioid-induced hyperalgesia in other heterogeneous samples of adults with chronic pain. This study contributes to the emerging understanding of how catechol-O-methyltransferase activity affects pain perception in the context of daily opioid use, and these findings may be useful in the design of future trials aimed at investigating the potential efficacy of ß-2 adrenergic receptor antagonism for opioid-induced hyperalgesia.

Pharmacogenomic Next-Generation DNA Sequencing: Lessons from the Identification and Functional Characterization of Variants of Unknown Significance in CYP2C9 and CYP2C19.

CYP2C9 and CYP2C19 are highly polymorphic pharmacogenes; however, clinically actionable genetic variability in drug metabolism due to these genes has been limited to a few common alleles. The identification and functional characterization of less-common open reading frame sequence variation might help to individualize therapy with drugs that are substrates for the enzymes encoded by these genes. The present study identified seven uncharacterized variants each in CYP2C9 and CYP2C19 using next-generation sequence data for 1013 subjects, and functionally characterized the encoded proteins. Constructs were created and transiently expressed in COS-1 cells for the assay of protein concentration and enzyme activities using fluorometric substrates and liquid chromatography- tandem mass spectrometry with tolbutamide (CYP2C9) and (S)-mephenytoin (CYP2C19) as prototypic substrates. The results were compared with the SIFT, Polyphen, and Provean functional prediction software programs. Cytochrome P450 oxidoreductase (CPR) activities were also determined. Positive correlations were observed between protein content and fluorometric enzyme activity for variants of CYP2C9 (P < 0.05) and CYP2C19 (P < 0.0005). However, CYP2C9 709G>C and CYP2C19 65A>G activities were much lower than predicted based on protein content. Substrate intrinsic clearance values for CYP2C9 218C>T, 343A>C, and CYP2C19 337G>A, 518C>T, 556C>T, and 557G>A were less than 25% of wild-type allozymes. CPR activity levels were similar for all variants. In summary, sequencing of CYP2C9 and CYP2C19 in 1013 subjects identified low-frequency variants that had not previously been functionally characterized. In silico predictions were not always consistent with functional assay results. These observations emphasize the need for high-throughput methods for pharmacogene variant mutagenesis and functional characterization.

Associations of catechol-O-methyltransferase (rs4680) single nucleotide polymorphisms with opioid use and dose among adults with chronic pain.

Catechol-O-methyltransferase (COMT) regulates extracellular catecholamines. A widely studied COMT single nucleotide polymorphism (rs4680) changes the translated amino acid from valine to methionine (Val158Met); the polymorphism has been shown to influence opioid use. The aims of this study were to investigate the influence of COMT Val158Met on the likelihood and dose of opioid use in adults with chronic pain. Adults with chronic pain consecutively admitted to an outpatient pain rehabilitation program were recruited for study participation (N = 298). Individuals were genotyped for COMT Val158Met (rs4680). The polymorphism was analyzed using an additive and codominant genotype model. The distribution of genotypes was 23% (N = 70) for Val/Val, 49% (N = 146) for Val/Met, and 27% (N = 82) for Met/Met (Hardy-Weinberg, P > 0.90). No significant association was observed between opioid use and genotype under the additive model; however, a significant association was observed under the codominant model (P = 0.027). A post hoc comparison demonstrated that the Met/Met genotype was more likely to use opioids compared with the Val/Met genotype (P = 0.0089). No significant association was observed between morphine equivalent dose and genotype under the additive model; however, a significant association was observed under the codominant model (P = 0.0496). A post hoc comparison demonstrated that the Val/Met (P = 0.019) and Met/Met (P = 0.043) genotypes used greater morphine equivalent dose compared with the Val/Val genotype. This study extends key knowledge about the influence of the Met/Met genotype and Met allele on opioid use in adults with chronic pain.

CYP2D6 phenotypes are associated with adverse outcomes related to opioid medications.

BACKGROUND: Variation in the CYP2D6 gene may affect response to opioids in both poor and ultrarapid metabolizers, but data demonstrating such associations have been mixed, and the impact of variants on toxicity-related symptoms (e.g., nausea) is unclear. Therefore, we examined the association between CYP2D6 phenotype and poor pain control or other adverse symptoms related to the use of opioids in a sample of primary care patients. MATERIALS AND METHODS: We identified all patients in the Mayo Clinic RIGHT Protocol who were prescribed an opioid medication between July 01, 2013 and June 30, 2015, and categorized patients into three phenotypes: poor, intermediate to extensive, or ultrarapid CYP2D6 metabolizers. We reviewed the electronic health record of these patients for indications of poor pain control or adverse symptoms related to medication use. Associations between phenotype and outcomes were assessed using Chi-square tests and logistic regression. RESULTS: Overall, 257 (25% of RIGHT Protocol participants) patients received at least one opioid prescription; of these, 40 (15%) were poor metabolizers, 146 (57%) were intermediate to extensive metabolizers, and 71 (28%) were ultrarapid metabolizers. We removed patients that were prescribed a CYP2D6 inhibitor medication (n=38). After adjusting for age and sex, patients with a poor or ultrarapid phenotype were 2.7 times more likely to experience either poor pain control or an adverse symptom related to the prescription compared to patients with an intermediate to extensive phenotype (odds ratio: 2.68; 95% CI: 1.39, 5.17; p=0.003). CONCLUSION: Our results suggest that >30% of patients with a poor or ultrarapid CYP2D6 phenotype may experience an adverse outcome after being prescribed codeine, tramadol, oxycodone, or hydrocodone. These medications are frequently prescribed for pain relief, and ~39% of the US population is expected to carry one of these phenotypes, suggesting that the population-level impact of these gene-drug interactions could be substantial.

Clinical UGT1A1 Genetic Analysis in Pediatric Patients: Experience of a Reference Laboratory.

BACKGROUND: Neonatal hyperbilirubinemia can be severe or prolonged and warrant exploration into the underlying etiology, which may include genetic assessment of UGT1A1 for inherited disorders (i.e. Crigler-Najjar syndrome or Gilbert syndrome). METHODS: In our reference laboratory, we performed UGT1A1 gene sequencing analysis on 346 pediatric patients referred for a clinical indication of hyperbilirubinemia. RESULTS: Males (n = 241) had significantly higher mean total bilirubin concentration compared to females (n = 105) (9.7 and 7.3 mg/dL, respectively, p = 0.042); however, no sex-based difference was observed in frequency of known or suspected reduced function UGT1A1 variants. The presence of two UGT1A1 variants (consistent with Gilbert or Crigler-Najjar syndrome) occurred less frequently in neonates (aged ≤28 days) than older children (aged 1-18 years) (31.3% in neonates vs. 85.1%, p < 0.0001), and among neonates there was no significant difference in mean total bilirubin between those with two UGT1A1 variants and those without (p = 0.47). Three novel variants, including c.337T>G (p.Y113D), c.1037C>A (p.A346E), and c.1469A>C (p.D490A) were identified. Among older children, the most common reason for referral was Gilbert syndrome (83.8%) and UGT1A1 genetic analysis confirmed a diagnosis of Gilbert syndrome in 79.0% of those children. CONCLUSIONS: Among neonates, a population in which hyperbilirubinemia is common and often of multifactorial etiology, UGT1A1 genetic testing served as a useful clinical tool in ruling in or ruling out inherited hyperbilirubinemia. Here we describe our experience as a reference laboratory in clinical UGT1A1 full gene sequencing. Our results highlight the challenges in predicting the contribution of genetic variation in UGT1A1 to hyperbilirubinemia based on clinical parameters alone, particularly in neonates, and the utility of UGT1A1 full gene sequencing in the evaluation of neonatal and pediatric hyperbilirubinemia.

Multidisciplinary model to implement pharmacogenomics at the point of care.

PURPOSE: Despite potential clinical benefits, implementation of pharmacogenomics (PGx) faces many technical and clinical challenges. These challenges can be overcome with a comprehensive and systematic implementation model. METHODS: The development and implementation of PGx were organized into eight interdependent components addressing resources, governance, clinical practice, education, testing, knowledge translation, clinical decision support (CDS), and maintenance. Several aspects of implementation were assessed, including adherence to the model, production of PGx-CDS interventions, and access to educational resources. RESULTS: Between August 2012 and June 2015, 21 specific drug-gene interactions were reviewed and 18 of them were implemented in the electronic medical record as PGx-CDS interventions. There was complete adherence to the model with variable production time (98-392 days) and delay time (0-148 days). The implementation impacted approximately 1,247 unique providers and 3,788 unique patients. A total of 11 educational resources complementary to the drug-gene interactions and 5 modules specific for pharmacists were developed and implemented. CONCLUSION: A comprehensive operational model can support PGx implementation in routine prescribing. Institutions can use this model as a roadmap to support similar efforts. However, we also identified challenges that will require major multidisciplinary and multi-institutional efforts to make PGx a universal reality.Genet Med 19 4, 421-429.

Acetylsalicylic acid supplementation improves protein utilization efficiency while vitamin E supplementation reduces markers of the inflammatory response in weaned pigs challenged with enterotoxigenic E. coli.

BACKGROUND: This experiment was conducted to test the hypothesis that vitamin E (Vit E) and acetylsalicylic acid (ASA), a cyclooxygenase-2 (COX-2) inhibitor, will additively reduce the production of the immunosuppressive molecule prostaglandin E2 (PGE2) and hence reduce inflammatory responses in weaner pigs experimentally infected with an enterotoxigenic strain of E. coli. METHODS: The experiment was conducted in a research facility with 192 individually-housed male weaner pigs (Landrace × Large White) weighing 6.6 ± 0.04 kg (mean ± SEM). The pigs were experimentally infected with an enterotoxigenic strain of E. coli and were allocated to a 2 × 3 factorial design with the respective factors being without and with 125 ppm ASA and three levels of Vit E supplementation (50, 100 or 200 IU/kg diet, dl-α-tocopheryl acetate). RESULTS: Acetylsalicylic acid supplementation improved average daily gain (P < 0.05) and tended to improve feed:gain ratio (P < 0.10) during the first 14 d after weaning. Acetylsalicylic acid supplementation also improved (P < 0.001) amino acid utilization efficiency (as assessed by plasma urea level) and tended to decrease (P < 0.10) PGE2 production in the liver without affecting small intestinal histology and tight junction protein mRNA expression in the jejunal epithelium. Vitamin E supplementation greater than 100 IU/kg diet sustained both the plasma Vit E concentration (P < 0.001) and plasma haptoglobin content (P < 0.001) after weaning. However, there was no additive effects of the combined supplementation of ASA and Vit E on performance, intestinal barrier function and inflammatory responses of weaned pigs. CONCLUSIONS: Although ASA and vitamin E improved amino acid utilization efficiency and reduced acute inflammatory responses, ASA and vitamin E did not additively reduce production of PGE2 and inflammatory responses in weaner pigs experimentally infected with an enterotoxigenic strain of E. coli.