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Genetic markers for skatole metabolism

A skatole, genetic material technology, applied in the field of identifying new enzymes related to skatole metabolism

Inactive Publication Date: 2009-04-15
UNIVERSITY OF GUELPH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Environmental and dietary factors are important (Kjeldsen, 1993; Hansen et al., 1995), but insufficient to explain changes in skatole concentrations in pig body fat

Method used

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  • Genetic markers for skatole metabolism
  • Genetic markers for skatole metabolism
  • Genetic markers for skatole metabolism

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Example 1: Identification of skatole metabolites

[0094] Materials and Methods

[0095] chemical. 3-Methylindole (3MI), indole-3-carbinol (I3C), indole-3-acetaldehyde, indole-3-carboxylic acid, 2-aminoacetophenone, and H-2 from Helix pomatia Type sulfatase was purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada). Oxindole, 3-methyloxindole (3MOI) and 3-hydroxy-3-methyloxindole (HMOI) were synthesized by the methods of Kende and Hodges (1982) and Skiles et al. (1989), respectively. Authentic 5-hydroxy-3-methylindole and 6-hydroxy-3-methylindole (the form of 6-sulfatoxyskatole) are Jens Hansen-Moller (Danish Meat Research Institute, Roskilde, Denmark). To obtain 6-hydroxy-3-methylindole from 6-hydroxyskatosulfate, the compound was hydrolyzed in a total volume of 0.5 ml acetate buffer, pH 5.0, containing 90 units / ml of H-2 type sulfatase. At 40°C, hydrolyze in a shaker water bath for 4 hours, add 0.5ml of ice-cold acetonitrile to terminate the reaction...

Embodiment 2

[0116] Example 2: Aldehyde Oxidase

[0117] Materials and Methods

[0118] chemical. Menadione, quinacrine and allopurinol were purchased from Sigma-Aldrich Canada (Oakville, ON, Canada). Authenticated HMOI was kindly provided by Dr. G.S. Yost, School of Pharmacology and Toxicology, University of Utah. HMI was produced from porcine liver microsomes, isolated and purified by preparative HPLC as previously described (Diaz et al., 1999). Freeze-dry the isolated HMI and store in a desiccator at -20 °C until use.

[0119] Preparation of porcine liver cytoplasm. Liver samples were obtained from 30 virgin boars obtained by backcrossing European wild boar x Swedish Yorkshire F3 boars with Swedish Yorkshire sows (Squires and Lundstrom, 1997). Liver samples were frozen with liquid nitrogen and stored at -80 °C. For the preparation of cytosolic fractions, partially thawed liver samples were minced and washed with 4 volumes of 0.05M Tris-HCl buffer pH 7.4 (containing 0.15M KCl) ...

Embodiment 3

[0134] Example 3: The role of CYP2A6 in the metabolism of 3-methylindole in porcine liver microsomes

[0135] The effect of different cytochrome P450 enzymes on the metabolism of 3-methylindole (3MI) was studied using selective chemical inhibitors. Screening of 8 chemical inhibitors of P450 enzymes for inhibition specificity of 3MI metabolism in porcine microsomes: α-naphthalenesulfone (CYP1A2), 8-methoxypsoralen (CYP2A6), peppermint Menthofuran (CYP2A6), sulphaphenazole (CYP2C9), quinidine (CYP2D6), 4-methylpyrazole (CYP2E1), diethyldithio Diethyldithiocarbamate (CYP2E1, CYP2A6) and troleandomycin (CYP3A4). In microsomal cultures, the production of different 3MI metabolites was only affected by the presence of CYP2E1 and CYP2A6 inhibitors. In a second experiment, a panel of porcine microsomes (n=30) was screened for CYP2A6 content, as well as 7-hydroxylation activity (CYP2A6 activity) by Western blot analysis. Protein content and enzymatic activity correlated with fat co...

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Abstract

Novel metabolites and enzymes involved in skatole metabolism are disclosed. The novel metabolites are 3-OH-3-methylindolenine (HMI); 3-methyloxindole (3MOI); indole-3-carbinol (I-3C); and 2-aminoacetophenone (2-AM). Measuring levels of these metabolites in a pig may be useful in identifying the pig's ability to metabolize skatole and its susceptibility to boar taint. The novel enzymes involved in skatole metabolism are aldehyde oxidase and CYP2A6. Enhancing the activity of these enzymes may be useful in enhancing skatole metabolism and reducing boar taint. The identification of the enzyme also allows the development of screening assays for substances that interact with these enzymes and skatole metabolism. Pigs having high levels of these enzymes may be selected and bred to produce pigs that have a lower incidence of boar taint.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to U.S. Patent Application 10 / 206,118, filed July 29, 2002, which is a continuation of U.S. Patent Application 09 / 672,039, filed September 29, 2000 (now Patent No. 6,448,028) divisional application, and US Patent Application 09 / 672,039 is a continuation of US Provisional Application 60 / 156,935, filed September 30, 1999. All of these applications are hereby incorporated by reference into this application. field of invention [0003] The present invention relates to new metabolites of skatole and to the identification of new enzymes involved in the metabolism of skatole. The present invention is used to develop methods for identifying and reducing boar taint. Background of the invention [0004] Boars raised for meat production are usually castrated shortly after birth to prevent foul odors (boar taint) in the carcass after slaughter. Boar taint is mainly due to high levels of 16-andr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/53C12N9/02C12N
CPCC12Q2600/156C12Q1/6881C12Q1/6888
Inventor 詹姆斯·E·斯夸尔斯贡萨洛·J·迪亚兹
Owner UNIVERSITY OF GUELPH
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