Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Reexamination Certificate
2000-09-29
2002-09-10
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
C435S004000, C435S007800, C435S183000, C435S189000
Reexamination Certificate
active
06448028
ABSTRACT:
FIELD OF THE INVENTION
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
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 5&agr; (-androst-16-en-3-one)) or skatole in the fat. Recent results of the EU research program AIR 3-PL94-2482 suggest that skatole contributes more to boar taint than androstenone (Bonneau, M., 1997).
Skatole is produced by bacteria in the hindgut which degrade tryptophan that is available from undigested feed or from the turnover of cells lining the gut of the pig (Jensen and Jensen, 1995). Skatole is absorbed from the gut and metabolised primarily in the liver (Jensen and Jensen, 1995). High levels of skatole can accumulate in the fat, particularly in male pig, and the presence of a recessive gene Ska
1
, which results in decreased metabolism and clearance of skatole has been proposed (Lundström et al., 1994; Friis, 1995). Skatole metabolism has been studied extensively in ruminants (Smith, et al., 1993), where it can be produced in large amounts by ruminal bacteria and results in toxic effects on the lungs (reviewed in Yost, 1989). The metabolic pathways involving skatole have not been well described in pigs. In particular, the reasons why only some intact male pigs have high concentrations of skatole in the fat are not clear. 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. Claus et al. (1994) proposed high fat skatole concentrations are a result of an increased intestinal skatole production due to the action of androgens and glucocorticoids. Lundström et al. (1994) reported a genetic influence on the concentrations of skatole in the fat, which may be due to the genetic control of the enzymatic clearance of skatole. The liver is the primary site of metabolism of skatole and liver enzymatic activities could be the controlling factor of skatole deposition in the fat. Bæk et al. (1995) described several liver metabolites of skatole found in blood and urine with the major being MII and MIII. MII, which is a sulfate conjugate of 6-hydroxyskatole (pro-MII), was only found in high concentrations in plasma of pigs which were able to rapidly clear skatole from the body, whereas high MIII concentrations were related to slow clearance of skatole. Thus the capability of synthesis of MII could be a major step in a rapid metabolic clearance of skatole resulting in low concentrations of skatole in fat and consequently low levels of boar taint.
In view of the foregoing, further work is needed to fully understand the metabolism of skatole in pig liver and to identify the key enzymes involved. Understanding the biochemical events involved in skatole metabolism can lead to novel strategies for treating, reducing or preventing boar taint. In addition, polymorphisms in these candidate genes may be useful as possible markers for low boar taint pigs.
SUMMARY OF THE INVENTION
The present inventors have identified novel metabolites resulting from the phase I metabolism of skatole (3-methylindole, 3MI) by porcine liver microsomes. The metabolites identified 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 hence its susceptibility to boar taint.
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.
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.
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.
The present invention also includes a method for producing pigs that have a lower incidence of boar taint by selecting pigs that have high levels of aldehyde oxidase and/or CYP2A6 and breeding the selected pigs.
Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
REFERENCES:
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patent: 2234349 (1999-10-01), None
Diaz, Gonzalo J et al., Identification of phase I metabolites of 3-methylindole produced by pig liver microsomes.Drug Metabolism and Disposition, v. 27, No. 10, Oct. 1999, pp 1150-1156.
Diaz, Gonzalo J et al., Metabolism of 3-methylindole by porcine liver microsomes: Responsible cytochrome P450 enzymes,Toxicological Sciences, vol. 55, No. 2, Jun. 2000, pp 284-292.
Diaz, Gonzalo J et al., Role of aldehyde oxidase in the hepatic in vitro metabolism of 3-methylindole in pigs,Journal of Agricultural and Food Chemistry, vol. 48, No. 3, Mar. 2000, pp 833-837.
Database WPI AN 1996 107098 Fremgangsmade til at undersoge grise for at bestemme om de er egnet til avl eller formering samt anvendelse af dyr eller saed (Slagteriernes Forskningsinstitut), May 28, 1996.
Thornton-Manning, Janice et al., Metabolism of 3-methylindole by vaccinia-expressed p450 enzymes: Correlation of 3-methyleneindolenine formation and protein-binding,Journal of Pharmacology and Experimental Therapeutics, vol. 276, No. 1, 1996, pp 21-29.
Skordos, Konstantine, W. et al. Evidence supporting the formation of 2,3-epoxy-3-methylindoline: A reactive intermediate of the pneumotoxin 3-methylindole,Chemical Research in Toxicoloy, vol. 11, No. 7, Jul. 7, 1998 pp 741-749.
Potchoiba, M.J. et al., Metabolism and Pneumo Toxicity of 3 Methyl Oxindole Indole 3 Carbinol and 3 methyl indole in goats,American Journal of Veterinary Research, vol. 43, No. 8, 1982, pp 1418-1423.
Diaz Gonzalo J.
Squires E. James
Bereskin & Parr
Prouty Rebecca E.
Rao Manjunath N.
University of Guelph
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