Genetic markers for skatole metabolism

a skatole and gene technology, applied in the field of gene markers for skatole metabolism, can solve the problems of insufficient explanation of the variation in fat skatole concentration in pigs, and achieve the effects of reducing boar taint, enhancing skatole metabolism, and enhancing aldehyde oxidase activity

Inactive Publication Date: 2007-02-08
UNIVERSITY OF GUELPH
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Benefits of technology

[0007] The present inventors have also determined that one of the metabolites of skatole, HMI is metabolized to 3-hydroxy-3-methyloxindole (HMOI) by aldehyde oxidase. As a result, enhancing the activity of the aldehyde oxidase may be useful in enhancing skatole metabolism and reducing boar taint. Accordingly, the present invention provides a method for enhancing the metabolism of 3-methylindole and thereby reducing boar taint comprising enhancing the activity of aldehyde oxidase in a pig. The activity of aldehyde oxidase can be enhanced by using substances which (a) increase the activity of aldehyde oxidase; or (b) induce or increase the expression of the aldehyde oxidase gene.
[0008] The present inventors have further determined that the cytochrome P450 enzyme, CYP2A6, is also involved in the metabolism of skatole by porcine liver microsomes. As a result, enhancing the activity of the CYP2A6 may be useful in enhancing skatole metabolism and reducing boar taint. Accordingly, the present invention provides a method for enhancing the metabolism of 3-methylindole and thereby reducing boar taint comprising enhancing the activity of CYP2A6 in a pig. The activity of CYP2A6 can be enhanced by using substances which (a) increase the activity of CYP2A6; or (b) induce or increase the expression of the CYP2A6 gene.
[0009] The identification of enzymes involved in the metabolism of skatole allows the development of screening assays for substances that interact with these enzymes in skatole metabolism. The screening assays can be used to identify substances that can be used to reduce or treat boar taint.

Problems solved by technology

Environmental and dietary factors are important (Kjeldsen, 1993; Hansen et al., 1995) but do not sufficiently explain the reasons for the variation in fat skatole concentrations in pigs.

Method used

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

Examples

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example 1

Identification of Skatole Metabolites

Materials and Methods

[0085] Chemicals. 3-Methylindole (3MI), indole-3-carbinol (13C), indole-3-aldehyde, indole-3-carboxylic acid, 2-aminoacetophenone and sulfatase type H-2 from Helix pomatia were purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada). The oxindoles, 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-OH-3-methylindole and 6-OH-3-methylindole (in the form of 6-sulfatoxyskatole) were donated by Jens Hansen-Møller (Danish Meat Research Institute, Roskilde, Denmark). In order to obtain 6-OH-3-methylindole from 6-sulfatoxyskatole, the compound was hydrolyzed in a total volume of 0.5 ml acetate buffer pH 5.0 containing 90 units / ml of type H-2 sulfatase. Hydrolysis was conducted for 4 hours in a shaking water bath at 40° C. and then 0.5 ml of ice-cold acetonitrile were added both to stop the reaction an...

example 2

Aldehyde Oxidase

Materials And Methods

[0104] Chemicals. Menadione, quinacrine and allopurinol were purchased from Sigma-Aldrich Canada (Oakville, ON, Canada). Authentic HMOI was graciously provided by Dr. G. S. Yost, Department of Pharmacology and Toxicology, University of Utah. HMI was produced using porcine liver microsomes and it was isolated and purified using preparative HPLC as described before (Diaz et al., 1999). Isolated HMI was freeze-dried and kept in a dessicator at −20° C. until used.

[0105] Preparation of porcine liver cytosol. Liver samples were taken from 30 intact male pigs obtained by back-crossing F3 European Wild Pig×Swedish Yorkshire boars with Swedish Yorkshire sows (Squires and Lundström, 1997). Liver samples were frozen in liquid nitrogen and stored at −80° C. For the preparation of the cytosolic fraction, partially thawed liver samples were finely minced and homogenized with 4 volumes of 0.05 M Tris-HCl buffer pH 7.4 (containing 0.15 M KCl, 1 mM EDTA, and...

example 3

The Role of CYP2A6 IN 3-Methylindole Metabolism by Porcine Liver Microsomes

[0118] The role of different cytochrome P450 enzymes on the metabolism of 3-methylindole (3MI) was investigated using selective chemical inhibitors. Eight chemical inhibitors of P450 enzymes were screened for their inhibitory specificity towards 3MI metabolism in porcine microsomes: alpha-naphthoflavone (CYP1A2), 8-methoxypsoralen (CYP2A6), menthofuran (CYP2A6), sulphaphenazole (CYP2C9), quinidine (CYP2D6), 4-methylpyrazole (CYP2E1), diethyldithiocarbamate (CYP2E1, CYP2A6), and troleandomycin (CYP3A4). The production of the different 3MI metabolites was only affected by the presence of inhibitors of CYP2E1 and CYP2A6 in the microsomal incubations. In a second experiment, a set of porcine microsomes (n=30) was screened for CYP2A6 content by Western blot analysis and also for their 7-hydroxylation activity (CYP2A6 activity). Protein content and enzymatic activity were found to be correlated with 3MI fat conte...

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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 or for genetic screening to identify pigs on the basis of their 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

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims benefit from U.S. application Ser. No. 10 / 206,118 filed Jul. 29 2002, which is a divisional of U.S. application Ser. No. 09 / 672,039, filed Sep. 29,2000 now U.S. Pat. No. 6,448,028 which is a continuation of U.S. provisional application No. 60 / 156,935, filed Sep. 30, 1999 all of which are incorporated herein by reference.FIELD OF THE INVENTION [0002] The present invention relates to novel metabolites of skatole and the identification of novel enzymes involved in the metabolism of skatole. The invention has utility in developing methods to identify and reduce boar taint. BACKGROUND OF THE INVENTION [0003] Male pigs that are raised for meat production are usually castrated shortly after birth to prevent the development of off-odors and off flavors (boar taint) in the carcass. Boar taint is primarily due to high levels of either the 16-androstene steroids (especially 5α(-androst-16-en-3-one)) or skatole in the fat. R...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C07H21/04C12P21/06C12N9/02C12Q1/26H01J49/04
CPCA01K2217/05A01K2227/108C12N9/0008C12Q1/26H01J49/04G01N2333/795G01N2333/80G01N2333/90245C12Q2600/156
Inventor SQUIRES, E. JAMESDIAZ, GONZALO J.
Owner UNIVERSITY OF GUELPH
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