Doran (nee Udovikova), Olena

McGivan, J. D.

Whittington, F. M.

Wood, J. D.

English

UWE Bristol Research Repository

74The role of P450IIE1 protein and mRNA expression in determining adipose tissueskatole levelE. Doran 1, F. Whittington2,   J.D. Wood2  and  J.D. McGivan11Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD, U.K.2Department of Clinical Veterinary Science,School of Veterinary Science, Langford,University of Bristol,Bristol BS405DU,UKIntroduction High concentrations of skatole in adipose tissue are a major factor in boar taint - the offensive odor andtaste  in cooked pork  from some intact male pigs. Skatole is produced by bacterial fermentation  of  tryptophan in thelarge intestine,  absorbed into the blood and  transported into the  liver, where it can be metabolised via the cytochromeP450 system. One reason for high skatole levels in pig adipose tissues could be low expression of hepatic cytochromeP450IIE1 in liver and hence a  reduced rate of skatole clearance, but no correlation was found between the rate ofskatole metabolism and the P450IIE1 content of isolated liver microsomes. The present study re-investigates therelationship between backfat skatole, liver P450IIE1 expression and the rate of microsomal skatole metabolism in twobreeds of pigs.Materials and Methods  85 intact male pigs of  two breed types provided by the Cotswold Pig Development Companywere used. These were Large White x Landrace (LW) and Meishan x Landrace (M).  The pigs were fed  a standardpelleted  diet and slaughtered at the same age to provide carcasses of 63-74 kg. Liver samples were frozen in solid CO2and subsequently stored at –80°C. Skatole in backfat was measured by high resolution gas chromatography.Microsomes were isolated by differential centrifugation. Mitochondrial skatole metabolism was measured by separatingskatole from its products by thin layer chromatography in hexane/ether. P450IIE1 levels in microsomes were measuredby Western blotting with a commercial antibody. mRNA levels were measured by Northern blotting with a cDNAprobe specific for P450IIE1.                                           Results The majority of the LW pigs had skatole levels in the range from 0 to 0.1 µg/g which is below the levelcharacteristic of boar taint.  Only about 5% of LW pigs had  backfat skatole levels which were higher than this. Incontrast, the M breed had very high backfat skatole levels (from 0.2 to 1 µg/g) most of which were above the boar taintthreshold level (0.2µg/g).  When microsomal metabolism was measured under appropriate conditions the rate ofmetabolism was always proportional to the microsomal P450IIE1 content. confirming that metabolism via P450IIE1was the major route of skatole breakdown in liver. Fig 1 shows the correlation between backfat skatole and microsomalP450IIE1 protein. For the LW there was a good inverse correlation for all the pigs tested. All the M pigs expressed  lowlevels of P450IIE1. Backfat skatole levels in M pigs were always high but varied over a ten-fold range. Fig.2 shows thatthere was an inverse correlation between backfat skatole and P450IIE1 mRNA expression in LW. In the M breedP450IIE1 mRNA levels were always low and  backfat skatole levels were relatively   high but there was no clearcorrelation  between mRNA levels and backfat skatole.Fig 1. Microsomal P450IIE1 in LW (l  ) and M (s ) pigs            Fig 2. mRNA  level in liver of LW ( l ) and M(s )pigsConclusions These results confirm the importanace of hepatic cytochrome P450IIE1 protein and mRNA expression indetermining adipose tissue skatole levels. In the LW pigs high backfat skatole is clearly correlated with abnormally lowcytochrome P450IIE1 expression. In M pigs some additional factor increases the skatole level above that determined bythe low level of cytochrome P450IIE1 expression. An understanding of the molecular basis of boar taint will requireelucidation of the factors controlling the expression of cytochrome P450IIE1 in pig liver.Acknowledgements This work was supported by a grant from the Meat and Livestock Commission and  by the DanishNational Committee for pigs.0501001502 0 02 5 03 0 00 0 . 5 1 1.5B a c k f a t  s k a t o l e ,  u g / g00 . 20 . 40 . 60 . 811.21.41.61.80 0 . 2 0 . 4 0 . 6 0 . 8 1 1.2 1.4B a c k f a t  s k a t o l e ,  u g / g

The role of P450IIE! protein and mRAN expression in determining adipose tissue skatole level

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The role of P450IIE! protein and mRAN expression in determining adipose tissue skatole level

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