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Relationship between oxidation and conjugation metabolism of skatole in pig liver and concentrations of skatole in fat.

High concentrations of skatole in fat of some intact male pigs are a major cause of boar taint. In this study, we investigated the effect of oxidative and conjugative metabolism of skatole in liver on the concentrations of skatole in the fat of intact male pigs. In Trial 1, 18 Yorkshire intact males were equally divided into two treatments with high (mean, .42; SD, .26 ppm) and low (mean, .06; SD, .02 ppm) fat skatole levels. There was an increased rate of skatole metabolism, an increased glucuronidation activity, and a decreased sulfation activity toward 2-naphthol in liver from pigs with high skatole levels (P < .05). In Trial 2, Swedish Yorkshire x F4 European Wild Pig intact males were used. Among skatole metabolites that were produced in incubations with liver microsomes, pro-MII was conjugated with glucuronic acid and sulfate, and metabolite F-1 was conjugated with glucuronic acid. The rates of formation of various skatole metabolites and conjugation of pro-MII were evaluated for 22 pigs with different levels of cytochrome P4502E1 in the liver. The formation of F-1 and sulfation of pro-MII were negatively correlated with fat skatole levels (r = -.59, and r = -.56, respectively) and were decreased in pigs with high fat skatole levels and low P4502E1 levels (P < .01). The results indicate that oxidation and conjugation reactions of skatole in pig liver have a dramatic effect on skatole levels in the fat. In particular, the formation of F-1 and formation and subsequent sulfation of pro-MII are related to low levels of skatole in the fat, presumably due to rapid metabolic clearance of skatole.

Adipose Tissue↗

Relationship between cytochrome P450IIE1 in liver and levels of skatole and its metabolites in intact male pigs.

The relationships between levels of skatole and androstenone in fat, plasma levels of skatole metabolites, plasma testosterone and estrone sulfate, and levels of cytochrome P450IIE1 in liver were investigated in Swedish Yorkshire pigs and F4 Wild Pig crosses. Levels of skatole, the skatole metabolite MIII, and androstenone were higher in males from the Wild Pig crosses than in the Yorkshire pigs. Plasma levels of the skatole metabolite MII and the ratio between MII and MIII were higher in female Yorkshire pigs than in male pigs. Skatole was correlated with androstenone, cytochrome P450IIE1, and estrone sulfate in the Wild Pig crosses but not in Yorkshire pigs. However, plasma levels of skatole metabolites were not correlated to levels of cytochrome P450IIE1 in liver in either group on animals. Pigs with high levels of cytochrome P450IIE1 in liver had low levels of skatole in fat, and pigs with low levels of cytochrome P450IIE1 in liver had low and high skatole levels. Using stepwise regression analysis, only levels of cytochrome P450IIE1 in liver significantly explained the variation in skatole levels in the Wild Pig crosses. Our results support the hypothesis that low levels of cytochrome P450IIE1 in liver may result in high levels of skatole in backfat of uncastrated male pigs due to decreased metabolism and clearance of skatole. However, these pigs cannot be identified based on plasma levels of skatole metabolites MII and MIII.

Androgens↗

Hepatic metabolism of skatole in pigs by cytochrome P4502E1.

High concentrations of skatole in fat are a major cause of boar taint in intact male pigs. Skatole is metabolized in the liver, and this metabolism could affect concentrations of skatole in fat. In this study, we evaluated the involvement of cytochrome P450, in particular cytochrome P4502E1, in skatole metabolism in pig liver. Liver microsomes from F4 European Wild Pig x Swedish Yorkshire intact male pigs were incubated in a buffer containing NADPH, NADH, and skatole. Several skatole metabolites were detected by HPLC, including 6-hydroxyskatole (pro-MII), 3-hydroxy-3-methyloxyindole (MIII), and five others not identified in this study. Inhibitors of P450 were added to microsomal incubations, and their effect on the formation of skatole metabolites and skatole disappearance was evaluated. The general cytochrome P450 inhibitors SKF 525A, at a concentration of .2 mM and metyrapone, at a concentration of .1 mM decreased the formation of pro-MII (P = .001) to 38.2 and 11.6%, respectively, of that of controls. The SKF 525A also reduced the synthesis of MIII and three other metabolites, whereas metyrapone only reduced the disappearance of skatole and synthesis of pro-MII. Inhibitors specific for cytochrome P4502E1 were more effective in reducing the formation of skatole metabolites than SKF 525A and metyrapone. Chlorzoxazone and diallyl sulfide reduced (P = .001) the synthesis of pro-MII to 9.7 and 30.9% of the control rate. The formation of most of the other skatole metabolites and disappearance of skatole were also reduced with these inhibitors. These results indicate that skatole is metabolized in pig liver to pro-MII and other metabolites by cytochrome P4502E1.

Allyl Compounds↗

Relationship between metabolism of androstenone and skatole in intact male pigs.

The relationship between the metabolism of androsterone and skatole, the major compounds responsible for boar taint, was investigated in F4 Swedish Yorkshire x European Wild Pig intact males. The metabolism of androstenone and skatole were studied in liver microsomes, and the testicular steroid production was measured in testes microsomes. Including androstenone in the assays of skatole metabolism reduced the formation of 6-hydroxyskatole (pro-MII), and three other skatole metabolites (P<.05). The formation of three additional metabolites was not affected. Liver microsomal incubations of androstenone produced two metabolites, I and II. The rate of the formation of metabolite I and the rate of androstenone metabolism were correlated with the rate of skatole metabolism. Liver metabolism of androstenone was not related to levels of androstenone in fat. Testicular synthesis of 16-androstene steroids was correlated with combined synthesis of estrogens and androgens, plasma levels of androstenone, levels of skatole in fat, and skatole metabolism in the liver (P<.05). Plasma levels of estrone sulfate were correlated with levels of skatole in fat and with androstenone levels in fat and plasma and were negatively correlated with synthesis of skatole metabolite F-1 and pro-MII sulfation. These results indicate that the liver metabolism of androstenone and skatole are related. However, it is likely that the relationship between levels of androstenone and skatole in fat is due more to a link between the testicular synthesis of androstenone rather than to the metabolism of androstenone and skatole in the liver. Sex steroids may affect this relationship because of their biosynthesis along with androstenone and possible inhibition of skatole metabolism in the liver.

Adipose Tissue↗

Cytochrome P450IIE1 (CYP2E1) is induced by skatole and this induction is blocked by androstenone in isolated pig hepatocytes.

Skatole, a derivative of tryptophan, is produced in the hind-gut of pigs and is metabolised via hepatic cytochrome P4502E1 (CYP2E1). Excessive accumulation of skatole together with androstenone, a metabolite of testosterone, in adipose tissue in some pigs is a major cause of 'boar taint' and is associated with defective expression of CYP2E1. This phenomenon is not understood because factors regulating CYP2E1 expression in pig liver have not yet been characterised. Therefore effects of skatole and androstenone on CYP2E1 expression were studied using isolated pig hepatocytes as a model system. Skatole induced CYP2E1 protein expression to the same degree as did acetone, a known CYP2E1 inducer. Induction by skatole was maximum between 20 and 28 h and a half-maximum effect was obtained at a skatole concentration of 0.2 mM. Induction of CYP2E1 by skatole was protein-synthesis dependent. Androstenone antagonised the effect of skatole on CYP2E1 expression but did not affect the CYP2E1 protein level when added alone. These results suggest that defective expression of CYP2E1 in some pigs is due to excessive concentrations of androstenone which prevent CYP2E1 induction by its substrate skatole. As a result, skatole metabolism is reduced and skatole is accumulated in adipose tissue.

Adipose Tissue↗

Effects of butyrate on apoptosis in the pig colon and its consequences for skatole formation and tissue accumulation.

Evidence exists that butyrate inhibits apoptosis of colon crypt cells in vivo so that less tryptophan from cell debris is available for skatole formation by microbes in the pig colon. In this study, potato starch containing a high proportion of resistant starch was fed to test the hypothesis that increased butyrate formation will occur in the colon and contribute to reduced epithelial cell apoptosis, thus leading to reduced skatole formation and absorption. Two groups of six barrows were provided with catheters in the jugular vein and fed either a ration with pregelatinized starch (high ileal digestibility; controls) or potato starch (low ileal digestibility; PS) as the main carbohydrate. All pigs were fed 31 MJ of metabolizable energy and 381 g of crude protein per day. The controls were fed for 19 d. The PS group received the same control ration for 10 d, and then changed to the PS ration. The total feeding period of PS consisted of a 5 d adaptation period followed by another 19 d. In the continously sampled feces, pH, short chain fatty acids, and skatole were determined. Skatole was additionally measured in blood plasma that was sampled daily. After killing barrows at the end of the feeding period, fat tissue for skatole measurement and colon tissue for histological quantification of mitosis and apoptosis were obtained. Feeding potato starch led to a rapid 2.2 fold increase of fecal butyrate when compared both with the control period of the PS group and the control group (P < 0.001). PS feeding resulted in a decrease in pH from 7.3 to 5.3 (P < 0.001) and apoptosis from 2.06 cells/crypt to 0.90 cells (P < 0.01), whereas there was no change in mitosis. Consequently, skatole decreased both in feces (controls vs PS group: 120.0 vs 1.9 microg/g; P < 0.001) and in blood plasma (1.6 vs 0.2 ng/mL; P < 0.001). The mean concentration of skatole in fat tissue was 167 ng/g tissue in controls, and below the detection limit (0.8 ng/g) in the PS group (P < 0.001). It is concluded that butyrate-dependent inhibition of apoptosis in the colon due to potato starch feeding efficiently inhibits skatole production in barrows. Because of the depressed skatole levels, improved sensory quality of pork is possible.

Animal Feed↗

Characterizing developmental changes in plasma and tissue skatole concentrations in the prepubescent intact male pig.

The accumulation of skatole in boars to concentrations resulting in carcass taint has been associated with elevated concentrations of steroid hormones in plasma. Studying boar taint in vivo has been challenging because steroid hormones are highly variable between individual boars. However, a peak in steroid hormones occurs between 2 and 4 wk postpartum; therefore, skatole production was investigated in the prepubescent pig. Plasma concentrations of estrone sulphate, dehydroepiandrosterone sulphate, and testosterone peaked between 2 and 4 wk postpartum in intact male pigs, whereas plasma concentrations of these steroid hormones remained low or undetectable in gilts and barrows. However, plasma skatole concentration peaked in all 3 groups of animals between 2 and 3 wk postweaning. The effects of weaning time, intestinal cell turnover, and diet on tissue skatole concentrations were then investigated. Intact male piglets were weaned at 14, 21, 28, or 35 d of age. Plasma skatole concentrations were measured weekly for a period of 63 d and peaked at 17 +/- 1, 14 +/- 1, 13 +/- 1, and 10 +/- 2 d postweaning, respectively. Intestinal cell turnover, as evaluated by villous height:crypt depth ratio, was not correlated with skatole concentrations in cecal contents, suggesting that cellular debris did not constitute a gross source of tryptophan for hindgut fermentation. The inclusion of 10% chicory inulin to piglet diets suppressed the postweaning increase in plasma skatole. Cecal skatole concentrations were also 3.3-fold lower in inulin-supplemented piglets compared with controls. The rise in plasma skatole in the prepubescent intact male pig was not associated with increased steroidogenesis but is likely due to the postweaning adaptation of the intestinal flora to an abrupt dietary change.

Aging↗

Functional polymorphism in porcine CYP2E1 gene: Its association with skatole levels.

Raising intact male pigs would have a significant economic impact on the pork industry. However, the presence of skatole (a major cause of boar taint) in meat from intact male pigs could be highly objectionable to consumer. The excessive accumulation of skatole in fat is a major cause of boar taint, and is associated with defective expression of cytochrome P4502E1 (CYP2E1). In pigs, it has been found that CYP2E1 is negatively correlated with accumulation of skatole. The searching for polymorphism of CYP2E1 and the relevant functional analysis would help develop a genetic marker for the selection of pigs with low skatole levels in fat. The aim of this study was to measure the expression pattern of CYP2E1 mRNA in various tissues of the pig, to identify genetic polymorphisms, and to evaluate the functional relevance of polymorphic sites with respect to the skatole level in fat. We show herein that a substitution of G --> A at base 1423 of the CYP2E1 gene in the liver causes a significant decrease in the expressed CYP2E1 level. Our data suggest that the G --> A substitute might be at least partially responsible for a high level of skatole in pigs. We believe that this is an important step toward the selection of genetic markers for boar taint by lowering fat levels of skatole in fat.

Amino Acid Sequence↗

Effects of fructooligosaccharide on conversion of L-tryptophan to skatole and indole by mixed populations of pig fecal bacteria.

An in vitro study was conducted to examine the effects of fructooligosaccharide (FOS) at levels of 0.5, 1.0, and 1.5% on conversion of L-tryptophan to skatole and indole by a mixed bacterial population from the large intestines of pigs. Microbial suspensions were anaerobically incubated at 38 degrees C for 24 h. Samples were periodically removed for determination of pH and indole compounds. After 24 h incubation, microbial populations in each culture media were analyzed. Addition of 0.5, 1.0, and 1.5% FOS to the slurries with L-tryptophan significantly decreased the skatole concentration, the peak value of indole-3-acetic acid and the medium pH. The viable counts of Bifidobacterium were significantly higher as compared with the control. Addition of 1.0 and 1.5% FOS significantly decreased the rate of tryptophan degradation and the relative rate of skatole production. The relative rate of indole production was significantly increased. The viable counts of Clostridium and Escherichia coli were significantly reduced. The total viable counts of anaerobes were significantly increased. These results suggest that the reduced concentration of skatole observed in the presence of FOS may be caused by the decreased tryptophan degradation due to the increased need for amino acids in the synthesis of bacterial cellular protein, and by shifting microbial metabolism of tryptophan toward indole production at the expense of skatole, which might result from the changed microbial ecosystem and pH. Our observations open the possibility of inhibiting microbial production of skatole and decreasing the skatole concentration in backfat by feeding pigs diets containing FOS, but it remains to be demonstrated in vivo.

Anaerobiosis↗

[The efficiency of photometric determination of skatole in slaughtered swine].

In a group of 264 boars, including both cryptorchides and hermaphrodites, skatole was measured using a photometric method developed in Denmark. It resulted in a concentration of skatole equivalents (SE) in the fat of 0.14 ppm in belly tissue and 0.18 ppm in flomen tissue on average, respectively. 10.6% of belly and 16.3% of flomen tissue exceeded the limiting value of 0.25 ppm skatole for boar taint. The photometric determination of skatole is based on a relatively unspecific reaction according to Ehrlich. Therefore, other derivatives of indole concerning the decomposition of tryptophan are recorded, and the results must be declared as SE. In comparison with HPLC, the photometric method yields values that are too high, indole is always found with skatole in a considerable amount, and the other skatole equivalents seem to play a subordinate part.

Adipose Tissue↗

Age-related variation of plasma concentrations of skatole, androstenone, testosterone, oestradiol-17 beta, oestrone sulphate, dehydroepiandrosterone sulphate, triiodothyronine and IGF-1 in six entire male pigs.

This study describes the age-related variation in boar taint compounds, skatole and androstenone, and testosterone, oestradiol-17 beta (E17 beta), oestrone sulphate (ES), dehydroepiandrosterone sulphate (DHEAS), triiodothyronine (T(3)) and insulin-like growth factor-1 (IGF-1) in six boars. Three pairs of littermates of crossbred entire male pigs (from three Yorkshire x Duroc dams and one Hampshire sire) were included. Blood samples were taken at the age of 9-15 weeks and thereafter at weekly intervals from the age of 20-32 weeks. Plasma concentrations of skatole, androstenone, testosterone, E17 beta, ES, DHEAS, T(3) and IGF-1 were measured. We found that skatole levels in boars increased at the age around puberty after an increase in the levels of testicular steroids. Levels of skatole were not associated with the levels of sex steroids, T(3) and IGF-1. However, the increased level of testicular steroids is probably the underlying factor needed for high skatole levels to occur although the specific mechanism leading to increased skatole levels remains unknown.

Aging↗

Free oestrone in adipose tissue and its relation to androstenone and skatole in entire male pigs.

Relationship between free oestrone and boar taint compounds in adipose tissue were studied in two groups of entire male pigs of different breeds. Group A consisted of 33 entire crossbred male pigs (dam Yorkshire and sire backcross Yorkshire x Wild Boar, generation seven). Group B consisted of 194 entire male pigs of crossbreeds between Swedish Hampshire (H) and Finnish Landrace (L), LH x H, H x LH, LH x LH (dam x sire). The measurements of free oestrone in adipose tissue were performed with a new method developed and validated in our laboratories. The standard curve was linear for concentrations of free oestrone ranging from 0.13 to 5.10 ng/g. The method exhibited parallelism of results between serial dilutions and a mean recovery of 97 +/- 13.7%. Intra-assay variations for samples with concentrations of free oestrone from 0.67 to 2.08 ng/g were from 9.23 to 11.94%. Inter-assay variations for the samples with concentrations of free oestrone from 0.89 to 2.96 ng/g were from 3.78 to 10.11%. The levels of free oestrone in fat from group A were well correlated with fat levels of androstenone (r = 0.66; p < 0.001) and levels of oestrone sulphate in peripheral plasma collected at the same time as the fat (r = 0.74, p < 0.001). The levels of free oestrone in fat from group B were significantly correlated to fat levels of androstenone (r = 0.68, p < 0.001) and skatole (r = 0.29, p < 0.001). In group B, age-related differences in fat levels of free oestrone, androstenone and skatole were studied. Free oestrone and skatole levels increased simultaneously at the age of 22 week (p < 0.05 for both), and androstenone levels increased at the age of 26 week (p < 0.05). It was suggested that the levels of free oestrone in adipose tissue might be used for the evaluation of hormonal status of male pigs as an alternative to plasma levels of testicular hormones. The levels of free oestrone might be involved in the regulation of skatole levels in fat as indicated by both the simultaneous increases in skatole and free oestrone levels in fat and positive correlation between skatole and free oestrone.

Adipose Tissue↗

High-performance liquid chromatographic method for the determination of 3-methylindole (skatole) and indole in adipose tissue of pigs.

A rapid method for the determination of skatole (3-methylindole) and indole in adipose tissue of pigs by reversed-phase high-performance liquid chromatography has been developed. Tissue samples were melted in a microwave oven, and 100 microliters of the liquid fat were dissolved in 1 ml of n-hexane and extracted with acetonitrile-water (75:25, v/v). Portions of 100 microliters of the solution were used for chromatographic analysis. Elution was performed on a reversed-phase column with a mobile phase composed of acetic acid and isopropanol (70:30, v/v). A fluorescence detector was used for quantification. The detection limit was 4 ng/g fat. The mean recoveries of added amounts of skatole and indole were 98.9 and 93.8%, respectively. The mean coefficients of variation were: inter-assay, 6.6% (skatole) and 8.8% (indole); intra-assay, 4.2% (skatole) and 2.9% (indole). Mean skatole concentrations in fat samples from boars (40 ng/g; n = 349) were not significantly higher than those from barrows (24 ng/g; n = 98).

Adipose Tissue↗

Isolation and characterization of anaerobic indole- and skatole-degrading bacteria from composting animal wastes.

Four species of indole-degrading Clostridium and 3 species of skatole-degrading Clostridium were isolated from piggery or chicken manure composting processes. Since type strains of respective isolates did not degrade these compounds, the degradability of the compounds was a novel characteristic. All isolates were mesophilic. The maximum growth allowance concentrations of these isolates were 300 to 800 mg/l in indole and 100 to 300 mg/l in skatole. All isolates showed better growth and utilization of indolic compounds in nutrient-rich medium than in minimal medium. Skatole-degrading isolates degraded some substituted indoles tested, 3-indoleacetic acid, indole and oxindole, but did not degrade 1-methylindole, 2-methylindole, isatin or anthranilic acid. On the other hand, indole-degrading isolates degraded only oxindole. The growth of Clostridium malenominatum A-3 was inhibited by a low concentration (0.005%) of indole or skatole, even when 200-fold excess glucose was present in the medium. When 0.03% indole or skatole was added to the medium, C. malenominatum A-3 showed a lag phase for about 10 and 70 h, respectively. When 0.01% of these compounds was added to the medium, the uptake of glucose was inhibited. C. malenominatum A-3 degraded these compounds under nutrient-rich and minimal conditions.

Journal Article↗

Influences on skatole formation from tryptophan in the pig colon.

Variable amounts of skatole (3-methyl-indole) are formed by microbes out of tryptophan in the colon of pigs. It is resorbed and accumulated in fat, leading to a fecal odor of the meat. We investigated the mechanisms by which differences in diet composition lead to variations in skatole concentrations in blood plasma and fat. The experiments were based on the hypothesis that tryptophan is derived from mucosa cell debris from the small intestine. It was found, that gut cell mitosis is stimulated by the growth factor IGF-I. This factor increases when energy in the diet is high. In addition, the mitotic rate is elevated when high amounts of purines are available in the diet, allowing a more rapid DNA- and RNA-synthesis. Thus, high energy combined with high purines in the diet lead to a remarkable increase of gut cell mitosis which is accompanied by an increase of apoptosis. These apoptotic cells ultimately provide the substrate for skatole formation. In consequence, a dramatically rise of skatole in blood plasma and fat was measurable.

Animals↗

Fecal skatole and indole and breath methane and hydrogen in patients with large bowel polyps or cancer.

The object of this study was to explore the use of fecal skatole and indole and breath methane and hydrogen as metabolic markers of the anaerobic colonic flora in patients with unresected large bowel cancer or polyps. Patients with descending or sigmoid colon cancer were more likely to be breath methane excretors than control subjects, patients with proximal colon cancer, and patients with rectal cancer. Control subjects excreting breath methane excreted less fecal skatole than breath methane excretors in the following groups: patients with adenomatous polyps, all patients with colorectal cancer, patients with proximal colon cancer, patients with descending and sigmoid colon cancer, and patients with rectal cancer. These data suggest that fecal skatole excretion equal to or greater than 100 micrograms/g feces might be useful to discriminate colorectal cancer patients from control subjects. Twenty-nine percent (8 of 28) of the cancer patients had both "high" skatole levels and breath methane excretion compared with only 2% (1 of 41) of the control subjects (P less than 0.01).

Bacteria↗

Rapid and accurate high-performance liquid chromatographic method for the determination of 3-methylindole (skatole) in faeces of various species.

A rapid method for the determination of skatole (3-methylindole) in faeces by reversed-phase high-performance liquid chromatography is described. Samples of 0.5 g were extracted with 2 ml of methanol. The extract was purified on Amberlite XAD-8. The lower limit of detection was 2.5 ng per injection (0.2 microgram/g faeces). The mean recovery of skatole was 95%, and the mean coefficients of variation were 7.0% (intra-assay) and 11.8% (inter-assay). Skatole concentrations were clearly lower in faeces from ruminants (average 2.6 micrograms/g for goat, sheep and cattle) than in those from monogastrics. Mean concentrations in human samples were 15.5 micrograms/g, and 10 micrograms/g in mature domestic pigs. An effect of the anabolic status on skatole concentrations in faeces of pigs is likely.

Animals↗

Rapid high-performance liquid chromatographic method for simultaneous determination of androstenone, skatole and indole in back fat from pigs.

A rapid high-performance reversed-phase liquid chromatographic method for the simultaneous quantitative determination of the main boar taint compounds androstenone, skatole (3-methylindole) and indole, in back fat from pigs has been developed. The compounds are extracted by a simple homogenisation of adipose tissue in methanol; interfering lipids are removed by precipitation after cooling and centrifugation. Androstenone is derivatized pre-column with dansylhydrazine (5 min at ambient temperature) using BF3 as catalyst. The compounds are separated on a 60 x 4.6 mm I.D., 3 microns Hypersil ODS column (Hewlett-Packard) using a step-gradient; total time for the separation is 15 min. Fluorescence is used for selective detection. The limit of quantitation for indole and skatole is 30 ng/g and for androstenone 200 ng/g back fat. The results for skatole obtained by the present method were compared with those of colorimetric method, while androstenone determinations were compared with two GC-MS methods and a RIA method. The correlations observed were in the range of 0.946-0.993. The average contents of androstenone and skatole were 640 +/- 700 and 78 +/- 113 ng/g (n = 1162 male pigs), respectively.

Adipose Tissue↗