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Parallel measurements of indole and skatole (3-methylindole) in feces and blood plasma of pigs by HPLC
Livestock Production Science, 34 ( 1993 ) 115-126
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Elsevier Science Publishers B.V., Amsterdam
Parallel measurements of indole and skatole ( 3methylindole) in feces and blood plasma of pigs by HPLC R. Clausa, M. Dehnharda, A. Herzoga, H. B e r n a l - B a r r a g a n a a n d T. G i m 6 n e z b aUniversity of Hohenheim, Fachgebiet Tierhaltung, Stuttgart, Germany bClemson University, Animal Science Department, Clemson, SC, USA (Accepted 3 August 1992)
ABSTRACT Claus, R., Dehnhard, M., Herzog, A., Bernal-Barragan, H. and Gim6nez, T., 1993. Parallel measurements of indole and skatole (3-methylindole) in feces and blood plasma of pigs by HPLC. Livest. Prod. Sci., 34:115-126. Skatole and indole (partly) result from microbial degradation of tryptophan in the intestine and may cause an unpleasant fecal odour of pork. A high-performance liquid chromatography method was developed which allows serial measurements (sensitivity, 0.15 ng/ml plasma; coefficient of variation, 10%). Parallel measurements were carried out in blood plasma, drawn through indwelling catheters in the jugular vein and the vena cava, from five ovariectomized sows. Additional determinations were carried out in feces. Concentrations in feces (skatole, ~= 2.3/~g/g; indole, X= 10/tg/g) were significantly correlated (P< 0.001 ) with concentrations in jugular vein plasma. Skatole concentrations in plasma of vena cava (~=0.54 ng/ml) were higher (P<0.001) than in peripheral plasma (0.32 ng/ml). In plasma of the portal vein, concentrations of skatole were 67 ng/ml and those of indole 120 ng/ml. It is concluded that changes in fecal concentration indicate similar changes in blood concentrations. Indole formation is higher than skatole formation and occurs mainly in the proximal part of the large intestine, so that the vena cava does not contribute to peripheral concentrations. Skatole formation also occurs in the distal part, and the vena cava plasma reflects considerable resorption, whereas indole concentrations do not differ significantly in both vessels.
Keywords: pig; skatole; indole; blood plasma.
INTRODUCTION
Skatole (3-methylindole) and indole are formed by microbial degradation of tryptophan in the intestinal tract of ruminant and monogastric species. Ruminal synthesis has been described for the cow and goat (Yokoyama and Carlson, 1979; Yoshihara, 1979; Honeyfield and Carlson, 1990) and it was Correspondence to." R. Claus, University Hohenheim, Fachgebiet Tierhaltung (470), POB 70 05 62, 7000 Stuttgart 70, Germany.
0301-6226/93/$06.00 © 1993 Elsevier Science Publishers B.V. All rights reserved.
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shown that skatole is rapidly absorbed after infusion into the rumen (Hammond et al., 1984), explaining low fecal concentrations in these species (Dehnhard et al., 1991 ). In the pig, skatole and indole formation occur mainly in the large intestine (Yoshihara, 1979; Wilkins, 1990) and the high concentrations of these two substances contribute to the species-specific adverse odor of feces (Burnett, 1969; Yasuhara et al., 1984). Peripheral metabolites of skatole but not indole have been shown to exert a cytotoxic effect in ruminants and rodents on epithelia of the respiratory tract and other organs such as the spleen (Breeze and Carlson, 1982; Updyke et al., 1991 ). In the pig such toxic effects have not been investigated so far. Skatole, however, accumulates in adipose tissue, and may lead to a severe reduction of the organoleptic quality of pork (LundstriSm et al., 1980). The contribution ofindole to the unpleasant taste is lower (Lundstrrm et al., 1984) even if concentrations in fat may occasionally exceed those of skatole (Gibis et al., 1991 ). Measurements in tissue samples from slaughtered pigs revealed a high variability between individuals and genders (Hansson et al., 1980; Porter et al., 1989). Serial measurements in feces were taken to characterize influences on its intestinal formation. Such studies indicate that mechanisms reducing the availability of energy in the hindgut favour the degradation of tryptophan to indole and skatole (Claus et al., 1991 ). Whereas changes of fecal concentrations are likely to reflect the level of skatole formation in the gut, they do not necessarily allow the conclusion that a proportional amount is resorbed and leads to similar changes in blood plasma and adipose tissue. Skatole has been measured hitherto in pig blood plasma after its addition in pharmacological amounts, to clarify the stability of the substance (Lin et al., 1991 ). The sensitivity of the high performance liquid chromatography (HPLC) method was 20 ng/ml and thus not sufficient to determine physiological concentrations. Skatole has also been measured in blood of ruminants after infusion. The lower limits of sensitivity of gas chromatography methods were 10 and 50 ng/ml respectively (Bradley and Carlson, 1974; Williams et al., 1979). Recently an enzyme immunoassay was described which is highly sensitive. The specificity, however, is insufficient because the antibodies cross-react with a variety of other indoles (Weiler et al., 1981; Singh et al., 1988; Singh and Sinha, 1990). The aim of our study was to develop a specific and sensitive HPLC method for the simultaneous measurement of skatole and indole and to determine whether changes in fecal concentrations are paralleled by corresponding changes in blood plasma.
INDOLE AND SKATOLE IN FECES AND BLOOD
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MATERIALS AND METHODS
Animals and blood sampling Five mature German Landrace sows with indwelling catheters into the jugular vein and the vena cava were used for the investigation. The two vessels were chosen to compare peripheral levels (vena jugularis externa) with those reflecting resorption, mainly from the distal part of the colon (Vena cava). In addition, samples were obtained from one Duroc gilt through a portal vein (vena portae) catheter to monitor resorption along the small intestine and the main part of the colon. The sows (average weight 204 kg) were fed twice daily 1.5 kg of a standardized commercial diet (9.1 MJ ME/kg; 16.4% crude protein ). To avoid influences of the stage of cycle the sows were ovariectomized before the experiment. During ovariectomy a catheter was inserted into a main uterine vein branch and gently pushed forward so that the catheter tip was located in the vena cava. The catheter was exteriorized laterally as described earlier (Claus et al., 1990). Additionally all animals were fitted with jugular vein catheters. Ovariectomy and cannulations had been carded out 1 week prior to the start of sampling. Samples were drawn into heparinized vials for a period of 13 days at 4 h intervals both through the jugular vein and the vena cava catheter. Some samples, however, could not be obtained due to transient catheter failure. The blood plasma was stored at - 2 5 °C until assayed. In addition, feces were collected from each individual immediately after defecation (on average four samples per day and animal) and stored deep-frozen. The gilt (90 kg) was sampled twice daily for 9 days through the hepatic vein catheter. These samples were also stored at - 2 5 °C. For comparison, individual blood samples were additionally collected by jugular venipuncture from female mature ruminants (cow, n = 9; sheep, n = 10; goat, n = 7 ) .
Method for skatole and indole determination in feces A rapid and accurate HPLC method for the determination of skatole in feces was available (Dehnhard et al., 1991 ). This method uses internal standardization by 2-methylindole (2-MI) to correct for procedural losses. Samples were extracted with methanol and further purified on Amberlite XAD-8 columns prior to analysis on HPLC by UV detection at 280 nm. The detection limit was 2.5 ng per injection (50 #1) corresponding to 0.2 #g/g feces. The mean coefficients of variation were 7.0% and 11.8% for the intra- and interassay variation respectively (Dehnhard et al., 1991 ). This method allows also the simultaneous determination of indole. The sensitivity and the precision were found to be identical to the skatole measurement.
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Method for skatole and indole determination in blood plasma For the determination of skatole and indole in blood plasma a new method was developed.
Extraction of blood plasma. Portions of 0.5 ml blood plasma were extracted with 2 ml diethylether (Merck p.a., redistilled) after the addition of the internal standard (1 ng 2-MI in 25/tl methanol) by vortexing for 30 s. After centrifugation (20 min at 1200 g) the samples were deep-frozen ( - 2 0 ° C ) . The ether phase was decanted into tubes containing 500/tl of the HPLC mobile phase, to avoid losses of the volatile indoles during the following evaporation of the ether phase. Evaporation of ether was carried out in a water bath at 47°C. The mobile phase consisted of water:acetonitrile:2-propanol (60:25:15 v / v / v ; HPLC grade solvents, Merck, Darmstadt). The samples for the calibration curves were prepared by adding amounts between 0.125 and 2.0 ng of both skatole and indole and a constant amount of 1 ng of 2-MI to 0.5 ml portions of 0.05 M Tris buffer (pH 8.3). They were extracted identically to the biological samples. In case of determinations in hepatic vein plasma the calibration curve was extended up to 200 ng/ml. This concentration was still in the linear range. By the addition of the internal standard to both the biological samples and the calibration standards, procedural losses were automatically compensated.
HPLC analysis. The equipment was the same as described for the determination in feces (Dehnhard et al., 1991 ) with the exception that a fluorescence detector (Shimadzu RF 551 ) was used. The extracts were transfered into autosampler vials which had been precoated with silicone. Portions of 100 #1 were injected. The column (LiChrospher RP 18, particle size 5 #m, 125 X 4.6 m m internal diameter) was operated at 40°C; the flow rate of the mobile phase was 1.0 ml/min. An identically packed precolumn ( 10 X 4.6 mm) was used to protect the analytical column. The detection was performed at an excitation wavelength of 275 nm and an emission wavelength of 352 nm. The ratio of the peak areas of the internal standard and the indoles was used for quantification.
Determination of the reliability criteria. The operation conditions of the column were chosen to ensure a clear separation of skatole, indole and 2-MI. The sensitivity was defined as that peak area of the relevant indole which exceeded the maximal peak area of the reagent blank at least two-fold. The reagent blank was determined by measuring extracts of 0.5 ml portions of Tris buffer. For determination of the precision (recovery of added amounts), endogenous indoles were removed from blood plasma by charcoal treatment. Skatole
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and indole were added (0.5, 1.0, 2.0 ng/ml) to this plasma and the resulting concentrations were compared with the known content. The same samples were used to determine the coefficients of the intra- and interassay variation from repeated measurements of the samples on the same day (n = 10) and on consecutive days (n = 10).
Statistical analysis Data are given as mean + s.d. Concentrations of skatole and indole in all compartments were evaluated by an analysis of variance (GLM, SAS ) for the influence of the individual, day of the experiment and time of sampling. Additionally, correlations between all parameters were calculated within animals and between animals (GLM, SAS ). Differences between concentrations in the vena cava and jugular vein were tested for significance by the paired ttest. For the calculation of the coefficients of correlation, concentrations in blood plasma were related to concentrations in feces 8 h later to compensate for the time lag between resorption and defecation (Bergher and Ketz, 1969). RESULTS
Quality criteria of determinations in blood plasma The retention times of the indoles were clearly different from each other and did not interfere with other substances in the blood plasma extracts. The lower limit of detection was 15 pg per injection, corresponding to 0.15 ng/ml blood plasma for both skatole and indole. The precision is given in Table 1. The mean precision (recovery) for skarole was 113% and for indole 95%. The mean intra-assay coefficients of variations were 14% ( s k a t o l e ) a n d 8.6% (indole). These coefficients increased when concentrations decreased. Similarly, the mean interassay variation was 9.2% for skatole and 10.1% for indole. TABLE 1 Precision of skatole and indole determination in blood plasma Substance
Added (ng/ml)
Measured (ng/ml)
Coefficient of variation (%) Intra-assay
Interassay
Skatole
0.5 1.0 2.0
0.66+0.12 1.12+0.16 2.28 + 0.22
18.6 14.7 9.6
8.6 12.3 6.7
Indole
0.5 1.0 2.0
0.47 + 0.06 0.96+0.10 1.94_+0.06
12.4 10.5 2.8
15.9 8.5 5.9
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Concentrations in blood plasma of ruminants Concentrations of skatole and indole in peripheral blood plasma of ruminants are shown in Table 2. The mean skatole concentrations were similar in sheep and goats. In contrast, cow plasma revealed clearly lower concentrations. As shown by the standard deviation and the range, the variation between individuals was high in this species, so that maximal concentrations in cows are not far from mean concentrations in goats. The indole concentrations revealed similar means, standard deviations and ranges to skatole. Concentrations in portal vein plasma of a gilt A total of 17 samples of portal vein plasma was available from one individual gilt. During the 9-day sampling period, the mean concentration of skatole was 67 ng/ml and that of indole nearly two-fold higher (119.6 ng/ml). The concentrations increased along the sampling period so that the minimal concentration of skatole at the beginning of sampling ( 18 ng/ml) increased to a maximum of 141 ng/ml. Similarly, indole increased from 34 ng/ml to 270 ng/ml. The correlation between the two substances was 0.77 ( P < 0.001 ). Concentrations in jugular vein plasma and plasma of vena cava in comparison to concentrations in feces Systematic measurements of the two indoles were carried out in five sows for prolonged periods so that 262 measurements were available from feces, 293 from vena cava plasma and 334 from jugular vein plasma. The mean concentrations and the range for both substances in the three compartments are given in Table 3. The high standard deviation and the broad range of absolute concentrations is partly explained by differences between individuals. For example, mean skatole concentrations in feces over the sampling period varied beTABLE 2 Concentrations of skatole and indole in peripheral blood plasma (ng/ml) of female ruminants Species (n)
Skatole mean+s.d, (range)
Indole mean+s.d. (range)
Sheep (10)
0.68+0.35 (0.22-1.28)
0.51 +0.34 (0.19-1.13)
Goat (7)
0.52+0.29 (n.d.-0.85)
0.49+0.26 (0.23-0.90)
Cow (9)
0.07+0.15 (n.d.-0.43)
0.18+0.12 (n.d.-0.33)
n.d. below detection limit ( < 0.15 ng/ml ).
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TABLE 3 Mean values and ranges of skatole and indole in feces and blood plasma of the vena cava and jugular vein of five ovariectomized sows Compartment (n)
Skatole mean_+ s.d. (range)
Indole mean_+ s.d. (range)
Feces (/tg/g) (262)
2.32 _+2.97 (n.d.-12.45)
9.96 _+6.58 (0.4-42.73)
Vena cava ( n g / m l ) (293)
0.54_+0.75 (n.d.-5.56)
14.58_+36.31 (0.89-252.70)
Jugular vein ( n g / m l ) (334)
0.32_+0.36 (n.d.-2.05)
19.97_+ 63.71 (0.34-390.8)
n.d., below detection limit.
tween non-detectable and 6.4/tg/g depending on the sow. In addition, the concentrations along the 2-week sampling period varied considerably irrespective of the standardized feeding regime and the exclusion of cycle effects by ovariectomy. Thus the analysis of variance revealed a highly significant ( P < 0.001 ) influence of the individual on skatole and indole concentrations in all compartments. The time of day revealed no significant influence, whereas the day of the experiment affected indole concentrations in all compartments (P<0.001) and skatole in the vena cava and the jugular vein (P
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TABLE 4 Coefficients of between- and within-animal correlation of skatole and indole in feces and blood plasma from jugular vein and vena cava
Skatole, feces Indole, feces Skatole, cava Indole, cava Skatole, jugular
between within between within between within between within between within
Indole jugular
Skatole jugular
Indole cava
Skatole cava
Indole feces
0.32 0.66 0.80 0.33 - 0.10* 0.46 0.95 0.79 0.43 0.59
0.79 0.59 0.47 0.39 0.56 0.42 0.53 0.62
0.43 0.70 0.78 0.33 - 0.01 .... 0.50
0.18** 0.37 - 0.23 0.21"*
0.45 0.34
n.s. = not significant; * P < 0.05; **P< 0.01. All other correlations are highly significant ( P < 0.001 ).
are either non-significantly correlated (cava) or negatively correlated (feces and jugular vein) to indole concentrations. In contrast, an exceptionally high correlation was obvious between indole in cava and jugular vein plasma, as similarly found for the within-animal comparison. DISCUSSION
The data presented in this study are the first systematic measurements in blood plasma and they provide the first results on the relation between skatole and indole in feces and blood plasma of pigs. The method for the determination in blood plasma was proven to have satisfactory reliability criteria. In only a few of the biological samples the concentrations of skatole were below the detection limit. Nevertheless the sensitivity of the method is sufficient, because such low concentrations are the exception and are unlikely to contribute to the skatole problem. The skatole concentrations in blood plasma of small ruminants were clearly higher than those found in peripheral plasma of the ovariectomized sows. In contrast it was shown earlier that fecal concentrations in the same ruminant species were much lower than in intact pigs (Dehnhard et al., 1991 ). This phenomenon is explained by ruminal formation of indoles and their rapid resorption ( H a m m o n d et al., 1984). Differences in indole concentrations between ruminants and monogastrics seem to be due to differences in the microbial population in either the rumen or the colon. Such differences, however, need further clarification. Fecal skatole concentrations in the ovariectomized sows are about one-third of those in intact sows and boars. This further confirms the effect of gonadal
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steroids on skatole formation (Claus et al., 1991 ). In the present study, high variation due to the oestrous cycle was excluded by ovariectomy. Our data show that in the pig concentrations of skatole and indole in peripheral blood plasma are highly significantly correlated with fecal concentrations both within and between animals, demonstrating that a proportional amount of indoles is resorbed from the intestine. Therefore serial measurements in the feces allow us to conclude that similar changes occur in blood plasma. This does not exclude the possibility that gonadal steroids affect resorption of skatole, which has been assumed to explain a tendency for higher concentrations in adipose tissue of boars than in gilts and barrows (Hansson et al., 1980). Our data on comparative determinations of skatole and indole further suggest that the ratio of the two substances differs in feces and blood plasma. In feces the skatole:indole ratio was 1:4.3 and increased up to 1:62.4 in peripheral plasma, suggesting differences in the absorption mechanisms. As shown by the data in the portal vein of one gilt, very high skatole and indole concentrations were found in this compartment. The portal vein values represent the absorption from the small intestine and the main (proximal) part of the large intestine. The concentrations in the vena cava reflect absorption from the distal part of the colon and the rectum. Absorption from this part of the gut apparently contributes moderately to peripheral concentrations of skatole but not to those of indole. The similarity of concentrations of indole in peripheral and cava plasma and their high correlations further support the assumption that the latter compartment does not contribute to peripheral concentrations. In addition, skatole and indole in the vena cava were not significantly correlated, suggesting that the main sites of skatole and indole formation differ within the colon. Such differences in the origin of the two substances are supported by measurements along the intestinal tract (Bernal et al., unpublished results), which reveal that indole formation starts already in the ileum. Maximal concentrations were found in the caecum, whereas concentrations decreased constantly towards the rectum. In contrast, skatole formation is limited to the large intestine and the concentrations increase continuously up to a maximum in the rectum. Therefore skatole formation concentrates to that part of the gut where resorption is comparatively inefficient. Even if skatole and indole are formed by different microbes a positive within-animal correlation and a negative between-animal correlation for indole concentrations in feces and skatole concentrations in the cava was found. This may be taken as an indication that the location of the different microbes, competing for tryptophan degradation in the colon, may vary due to unknown influences. ACKNOWLEDGEMENTS
Part of this work was financially supported by the Ministry for Science and Arts ( M W K ) , Baden-Wiirttemberg. H. Bernal-Barragan was supported by a
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grant from the Universidad de Nuevo Leon, Mexico. We wish to thank Mrs A.Schell for her skilful technical assistance.
REFERENCES Bergher, H. and Ketz, H-A., 1969. Verdauung, Resorption, Intermedi~irstoffwechsel bei landwirtschaftlichen Nutztieren. VEB Deutscher Landwirtschaftsverlag Berlin, pp. 114-115. Bradley, B.J. and Carlson, J.R., 1974. A gas-liquid chromatographic procedure for the determination of indole and 3-methylindole in bovine plasma. Anal. Biochem., 59:214-219. Breeze, R.G. and Carlson, J.R., 1982. Chemical-induced lung injury in domestic animals. Adv. Vet. Sci. Comp. Med., 26: 201-231. Burnett, W.E., 1969. Air pollution from animal wastes. Determination ofmalodors by gas chromatographic and organoleptic techniques. Environ. Sci. Technol., 3: 744-749. Claus, R., Meyer, H.H.D., Gim6nez, T., Hoang-Vu, C. and Miinster, E., 1990. Effect of seminal oestrogens of the boar on prostaglandin F2a release from the uterus of the sow. Anim. Reprod. Sci., 23: 145-156. Claus, R., Bernal-Barragan, H. and Dehnhard, M., 1991. Effect of gonadal hormones in mature cyclic sows on food intake and skatole concentrations in faeces. J. Anim. Physiol. a. Anim. Nutr., 66: 61-68. Dehnhard, M., Bernal-Barragan, H. and Claus, R., 1991. Rapid and accurate high-performance liquid chromatographic method for the determination of 3-methylindole (skatole) in faeces of various species. J. Chromatogr. Biomed. Appl., 566: 101-107. Gibis, M., Dehnhard, M. and Fischer, A., 1991. Bestimmung von Skatol und Indol in Riickenspeck und Muskelfleisch von Schweinen durch Hochleistungs-FliJssigchromatographie (HPLC) mit fluorimetrischer Detektion. Z. Lebensm. Unters. Forsch., 193: 220-223. Hammond, A.C., Glenn, B.P., Huntington, G.B. and Breeze, R.G., 1984. Site of 3-methylindole and indole absorption in steers after ruminal administration of L-tryptophan. Am. J. Vet. Res., 45: 171-174. Hansson, K-E., Lundstr6m, K., Fjelkner-Modig, S. and Persson, J., 1980. The importance of androstenone and skatole for boar taint. Swed. J. Agric. Res., 10" 167-173. Honeyfield, D.C. and Carlson, J.R., 1990. Assay for the enzymatic conversion of indoleacetic acid to 3-methylindole in a ruminal Lactobacillus species. Appl. Environ. Microbiol., 56: 724-729. Lin, R.S., Orcutt, M.W., Patterson, J.A. and Judge, M.D., 1991. Serum skatole detection using gas chromatography and high performance liquid chromatography. Meat Sci., 30" 33-40. Lundstr/Sm, K., Hansson, K-E., Fjelkner-Modig, S. and Persson, J., 1980. Skatole--another contributor to boar taint. Proceedings of the 26th European Meeting of the Meat Research Workers, Colorado Springs, 31 August-5 September 1980, Vol. 1, pp. 300-303. Lundstr6m, K., Malmfors, B., Malmfors, G., Petersson, H., Stern, S., Mortensen, A.B. and Sorensen, S.E., 1984. Boar taint and bitter taste as affected by androstenone and skatole. Proceedings of the 30th European Meeting of Meat Research Workers, Bristol, 9-14 September 1984, pp. 397-398. Porter, M.G., Hawe, S.M. and Walker, N., 1989. Method for the determination of indole and skatole in pig fat. J. Sci. Food Agric., 49" 203-209. Singh, P., Sinha, A., 1990. Detection of skatole for meat quality. United States Patent 4,906, 563. Singh, P., Sinha, A., Afzal, R. and Brock, T.K., 1988. A practical enzyme-linked immunoassay for quantification of skatole. Proceedings of the 34th International Congress on Meat Science and Technology, Brisbane, pp. 692-694.
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Updyke, L.W., Yoon, H.L., Kiorpes, A.L., Robinson, J.P., Pfeifer, R.W. and Marcus, C.B., 1991. 3-Methylindole-induced splenotoxicity: Biochemical mechanisms of cytotoxicity. Toxicol. Appl. Pharmacol., 109: 375-390. Weiler, E.W., Jourdan, P.S. and Conrad, W., 1981. Levels of indole-3-acetic acid in intact and decapitated coleoptiles as determined by a specific and highly sensitive solid-phase enzyme immunoassay. Planta, 153:561-571. Wilkins, C.K., 1990. Analysis of indole and skatole in porcine gut contents. Int. J. Food Sci. Technol., 25: 313-317. Williams, G.D., Rippon, P., Chen, P-M. and Camp, B.J., 1979. An improved gas-liquid chromatographic procedure for the determination of 3-methylindole in rumen liquor, plasma, and tissue of ruminants. Anal. Biochem., 99: 324-331. Yasuhara, A., Fuwa, K. and Jimbu, M., 1984. Identification of odorous compounds in fresh and rotten swine manure. Agric. Biol. Chem., 48: 3001-3010. Yokoyama, M.T. and Carlson, J.R., 1979. Microbial metabolites oftryptophan in the intestinal tract with special reference to skatole. Am. J. Clin. Nutr., 32:173-178. Yoshihara, I., 1979. Simultaneous gas chromatographic microdetermination of indole, skatole and p-cresol in gastrointestinal contents of domestic animals. Agric. Biol. Chem., 43:19851987.
RESUME Claus, R., Dehnhard, M., Herzog, A., Bernal-Barragan, H. et Gim6nez, T., 1993. Mesures parallbles d'indole et de scatol (3-m6thylindole) par HPLC dans les fbces et le plasma de porcs. Livest. Prod. Sci., 34:115-126. en anglais. Le scatol et une partie de l'indole proviennent de la d6gradation microbienne du tryptophane dans l'intestin et peuvent provoquer une odeur f6cale d6sagr6able dans la viande de porc. Une m6thode de dosage par HPLC permettant des dosages en s6rie a 6t6 d6velopp6e (sensibilit6: 0,15 ng/ml, coefficients de variation: 10%). Des mesures en parall6le ont 6t6 effectu6es sur du plasma sanguin pr61ev6 par des cath6ters implant6s dans la veine jugulaire et dans la veine cave de cinq truies ovariectomis6es. Des d6terminations suppl6mentaires ont 6t6 r6alis6es sur les f6ces. Les concentrations dans les f6ces (scatol: X= 2,3/tg/g, indole: ~ = 10/tg/g) sont significativement corr616es (P< 0,001 ) avec les teneurs dans la veine jugulaire. Les concentrations en scatol du plasma sont plus 61ev6es (P<0,001) dans la veine cave (~=0,54 ng/ml) que clans le sang p6riph6rique (0,32 ng/ml). Les concentrations en scatol et en indole sont respectivement de 67 et de 120 ng/ml dans le plasma de la veine porte. II en conclu qu'une modification des teneurs dans les f6ces permet de supposer que des variations similaires des concentrations interviennent dans le sang. La formation d'indole est plus 61ev6e que celle de scatol et se produit essentiellement dans la pattie proximale de gros intestin, si bien que la veine cave ne contribue pas aux concentrations p6riph6riques. La formation du scatol a 6galement lieu dans la par-tie distale, et le plasma de la veine cave refl~te une r6sorption importante tandis que les teneurs en indole ne diff6rent pas significativement entre les deux vaisseaux.
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KURZFASSUNG Claus, R., Dehnhard, M., Herzog, A., Bernal-Barragan, H. und Gim6nez, T., 1993. Vergleichende Messungen von Indol und Skatol in Kot und Blutplasma durch HPLC. Livest. Prod. Sci., 34:115-126 (auf englisch). Skatol, untergeordnet auch Indol, sind f'dr f~ikalartige Geruchsabweichungen in Schweinefleisch verantwortlich. Sie entstehen durch mikrobiellen Abbau der Aminos~iure Tryptophan im Darm. Serielle Messungen sind bisher auf Kot beschr~inkt. Es wurde eine HPLC-Methode entwickelt, um Skatol und Indol im Blutplasma zu messen (Empfindlichkeit 0.15 ng/ml, Variationskoeffizient: 10%). Parallele Messungen wurden im Kot und im Blutplasma durchgeftihrt, das iiber Dauerkatheter aus der V. jugularis und V. cava von ftinf ovariektomierten Sauen gewonnen wurde. Bei einer Jungsau wurde zus~itzlich Blut aus der V. portae mittels Katheter entnommen. Die durchschnittlichen Konzentrationen im Kot (Skatol: 2.3 #g/g, Indol: 10/zg/g) waren signifikant (P-< 0.001 ) mit denen in der V. jugularis korreliert. Die Skatolkonzentrationen in der V. cava (0.54 ng/ml) waren signifikant h~her als die im peripheren Plasma (0.32 ng/ml). In der V. hepatica betrugen die Konzentrationen ftir Skatol 67 ng/ml und ftir Indol 120 ng/ml. Die Ergebnisse zeigen, dab aus Messungen im Kot auf den Konzentrationsverlauf im Blutplasma geschlossen werden kann. Die Indolbildung ist h6her als die Skatolbildung und findet, wie durch die Messungen in der V. hepatica best~itigt, haupts~ichlich im proximalen Dickdarm statt. Die Skatoibildung findet auch im distalen Teil start, so dab ein erheblicher Teil fiber die V. cava resorbiert wird, w~ihrend sich die Indolkonzentrationen zwischen den beiden Gef~iSen nicht signifikant unterscheiden.
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Elsevier Science Publishers B.V., Amsterdam
Parallel measurements of indole and skatole ( 3methylindole) in feces and blood plasma of pigs by HPLC R. Clausa, M. Dehnharda, A. Herzoga, H. B e r n a l - B a r r a g a n a a n d T. G i m 6 n e z b aUniversity of Hohenheim, Fachgebiet Tierhaltung, Stuttgart, Germany bClemson University, Animal Science Department, Clemson, SC, USA (Accepted 3 August 1992)
ABSTRACT Claus, R., Dehnhard, M., Herzog, A., Bernal-Barragan, H. and Gim6nez, T., 1993. Parallel measurements of indole and skatole (3-methylindole) in feces and blood plasma of pigs by HPLC. Livest. Prod. Sci., 34:115-126. Skatole and indole (partly) result from microbial degradation of tryptophan in the intestine and may cause an unpleasant fecal odour of pork. A high-performance liquid chromatography method was developed which allows serial measurements (sensitivity, 0.15 ng/ml plasma; coefficient of variation, 10%). Parallel measurements were carried out in blood plasma, drawn through indwelling catheters in the jugular vein and the vena cava, from five ovariectomized sows. Additional determinations were carried out in feces. Concentrations in feces (skatole, ~= 2.3/~g/g; indole, X= 10/tg/g) were significantly correlated (P< 0.001 ) with concentrations in jugular vein plasma. Skatole concentrations in plasma of vena cava (~=0.54 ng/ml) were higher (P<0.001) than in peripheral plasma (0.32 ng/ml). In plasma of the portal vein, concentrations of skatole were 67 ng/ml and those of indole 120 ng/ml. It is concluded that changes in fecal concentration indicate similar changes in blood concentrations. Indole formation is higher than skatole formation and occurs mainly in the proximal part of the large intestine, so that the vena cava does not contribute to peripheral concentrations. Skatole formation also occurs in the distal part, and the vena cava plasma reflects considerable resorption, whereas indole concentrations do not differ significantly in both vessels.
Keywords: pig; skatole; indole; blood plasma.
INTRODUCTION
Skatole (3-methylindole) and indole are formed by microbial degradation of tryptophan in the intestinal tract of ruminant and monogastric species. Ruminal synthesis has been described for the cow and goat (Yokoyama and Carlson, 1979; Yoshihara, 1979; Honeyfield and Carlson, 1990) and it was Correspondence to." R. Claus, University Hohenheim, Fachgebiet Tierhaltung (470), POB 70 05 62, 7000 Stuttgart 70, Germany.
0301-6226/93/$06.00 © 1993 Elsevier Science Publishers B.V. All rights reserved.
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shown that skatole is rapidly absorbed after infusion into the rumen (Hammond et al., 1984), explaining low fecal concentrations in these species (Dehnhard et al., 1991 ). In the pig, skatole and indole formation occur mainly in the large intestine (Yoshihara, 1979; Wilkins, 1990) and the high concentrations of these two substances contribute to the species-specific adverse odor of feces (Burnett, 1969; Yasuhara et al., 1984). Peripheral metabolites of skatole but not indole have been shown to exert a cytotoxic effect in ruminants and rodents on epithelia of the respiratory tract and other organs such as the spleen (Breeze and Carlson, 1982; Updyke et al., 1991 ). In the pig such toxic effects have not been investigated so far. Skatole, however, accumulates in adipose tissue, and may lead to a severe reduction of the organoleptic quality of pork (LundstriSm et al., 1980). The contribution ofindole to the unpleasant taste is lower (Lundstrrm et al., 1984) even if concentrations in fat may occasionally exceed those of skatole (Gibis et al., 1991 ). Measurements in tissue samples from slaughtered pigs revealed a high variability between individuals and genders (Hansson et al., 1980; Porter et al., 1989). Serial measurements in feces were taken to characterize influences on its intestinal formation. Such studies indicate that mechanisms reducing the availability of energy in the hindgut favour the degradation of tryptophan to indole and skatole (Claus et al., 1991 ). Whereas changes of fecal concentrations are likely to reflect the level of skatole formation in the gut, they do not necessarily allow the conclusion that a proportional amount is resorbed and leads to similar changes in blood plasma and adipose tissue. Skatole has been measured hitherto in pig blood plasma after its addition in pharmacological amounts, to clarify the stability of the substance (Lin et al., 1991 ). The sensitivity of the high performance liquid chromatography (HPLC) method was 20 ng/ml and thus not sufficient to determine physiological concentrations. Skatole has also been measured in blood of ruminants after infusion. The lower limits of sensitivity of gas chromatography methods were 10 and 50 ng/ml respectively (Bradley and Carlson, 1974; Williams et al., 1979). Recently an enzyme immunoassay was described which is highly sensitive. The specificity, however, is insufficient because the antibodies cross-react with a variety of other indoles (Weiler et al., 1981; Singh et al., 1988; Singh and Sinha, 1990). The aim of our study was to develop a specific and sensitive HPLC method for the simultaneous measurement of skatole and indole and to determine whether changes in fecal concentrations are paralleled by corresponding changes in blood plasma.
INDOLE AND SKATOLE IN FECES AND BLOOD
1 17
MATERIALS AND METHODS
Animals and blood sampling Five mature German Landrace sows with indwelling catheters into the jugular vein and the vena cava were used for the investigation. The two vessels were chosen to compare peripheral levels (vena jugularis externa) with those reflecting resorption, mainly from the distal part of the colon (Vena cava). In addition, samples were obtained from one Duroc gilt through a portal vein (vena portae) catheter to monitor resorption along the small intestine and the main part of the colon. The sows (average weight 204 kg) were fed twice daily 1.5 kg of a standardized commercial diet (9.1 MJ ME/kg; 16.4% crude protein ). To avoid influences of the stage of cycle the sows were ovariectomized before the experiment. During ovariectomy a catheter was inserted into a main uterine vein branch and gently pushed forward so that the catheter tip was located in the vena cava. The catheter was exteriorized laterally as described earlier (Claus et al., 1990). Additionally all animals were fitted with jugular vein catheters. Ovariectomy and cannulations had been carded out 1 week prior to the start of sampling. Samples were drawn into heparinized vials for a period of 13 days at 4 h intervals both through the jugular vein and the vena cava catheter. Some samples, however, could not be obtained due to transient catheter failure. The blood plasma was stored at - 2 5 °C until assayed. In addition, feces were collected from each individual immediately after defecation (on average four samples per day and animal) and stored deep-frozen. The gilt (90 kg) was sampled twice daily for 9 days through the hepatic vein catheter. These samples were also stored at - 2 5 °C. For comparison, individual blood samples were additionally collected by jugular venipuncture from female mature ruminants (cow, n = 9; sheep, n = 10; goat, n = 7 ) .
Method for skatole and indole determination in feces A rapid and accurate HPLC method for the determination of skatole in feces was available (Dehnhard et al., 1991 ). This method uses internal standardization by 2-methylindole (2-MI) to correct for procedural losses. Samples were extracted with methanol and further purified on Amberlite XAD-8 columns prior to analysis on HPLC by UV detection at 280 nm. The detection limit was 2.5 ng per injection (50 #1) corresponding to 0.2 #g/g feces. The mean coefficients of variation were 7.0% and 11.8% for the intra- and interassay variation respectively (Dehnhard et al., 1991 ). This method allows also the simultaneous determination of indole. The sensitivity and the precision were found to be identical to the skatole measurement.
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Method for skatole and indole determination in blood plasma For the determination of skatole and indole in blood plasma a new method was developed.
Extraction of blood plasma. Portions of 0.5 ml blood plasma were extracted with 2 ml diethylether (Merck p.a., redistilled) after the addition of the internal standard (1 ng 2-MI in 25/tl methanol) by vortexing for 30 s. After centrifugation (20 min at 1200 g) the samples were deep-frozen ( - 2 0 ° C ) . The ether phase was decanted into tubes containing 500/tl of the HPLC mobile phase, to avoid losses of the volatile indoles during the following evaporation of the ether phase. Evaporation of ether was carried out in a water bath at 47°C. The mobile phase consisted of water:acetonitrile:2-propanol (60:25:15 v / v / v ; HPLC grade solvents, Merck, Darmstadt). The samples for the calibration curves were prepared by adding amounts between 0.125 and 2.0 ng of both skatole and indole and a constant amount of 1 ng of 2-MI to 0.5 ml portions of 0.05 M Tris buffer (pH 8.3). They were extracted identically to the biological samples. In case of determinations in hepatic vein plasma the calibration curve was extended up to 200 ng/ml. This concentration was still in the linear range. By the addition of the internal standard to both the biological samples and the calibration standards, procedural losses were automatically compensated.
HPLC analysis. The equipment was the same as described for the determination in feces (Dehnhard et al., 1991 ) with the exception that a fluorescence detector (Shimadzu RF 551 ) was used. The extracts were transfered into autosampler vials which had been precoated with silicone. Portions of 100 #1 were injected. The column (LiChrospher RP 18, particle size 5 #m, 125 X 4.6 m m internal diameter) was operated at 40°C; the flow rate of the mobile phase was 1.0 ml/min. An identically packed precolumn ( 10 X 4.6 mm) was used to protect the analytical column. The detection was performed at an excitation wavelength of 275 nm and an emission wavelength of 352 nm. The ratio of the peak areas of the internal standard and the indoles was used for quantification.
Determination of the reliability criteria. The operation conditions of the column were chosen to ensure a clear separation of skatole, indole and 2-MI. The sensitivity was defined as that peak area of the relevant indole which exceeded the maximal peak area of the reagent blank at least two-fold. The reagent blank was determined by measuring extracts of 0.5 ml portions of Tris buffer. For determination of the precision (recovery of added amounts), endogenous indoles were removed from blood plasma by charcoal treatment. Skatole
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and indole were added (0.5, 1.0, 2.0 ng/ml) to this plasma and the resulting concentrations were compared with the known content. The same samples were used to determine the coefficients of the intra- and interassay variation from repeated measurements of the samples on the same day (n = 10) and on consecutive days (n = 10).
Statistical analysis Data are given as mean + s.d. Concentrations of skatole and indole in all compartments were evaluated by an analysis of variance (GLM, SAS ) for the influence of the individual, day of the experiment and time of sampling. Additionally, correlations between all parameters were calculated within animals and between animals (GLM, SAS ). Differences between concentrations in the vena cava and jugular vein were tested for significance by the paired ttest. For the calculation of the coefficients of correlation, concentrations in blood plasma were related to concentrations in feces 8 h later to compensate for the time lag between resorption and defecation (Bergher and Ketz, 1969). RESULTS
Quality criteria of determinations in blood plasma The retention times of the indoles were clearly different from each other and did not interfere with other substances in the blood plasma extracts. The lower limit of detection was 15 pg per injection, corresponding to 0.15 ng/ml blood plasma for both skatole and indole. The precision is given in Table 1. The mean precision (recovery) for skarole was 113% and for indole 95%. The mean intra-assay coefficients of variations were 14% ( s k a t o l e ) a n d 8.6% (indole). These coefficients increased when concentrations decreased. Similarly, the mean interassay variation was 9.2% for skatole and 10.1% for indole. TABLE 1 Precision of skatole and indole determination in blood plasma Substance
Added (ng/ml)
Measured (ng/ml)
Coefficient of variation (%) Intra-assay
Interassay
Skatole
0.5 1.0 2.0
0.66+0.12 1.12+0.16 2.28 + 0.22
18.6 14.7 9.6
8.6 12.3 6.7
Indole
0.5 1.0 2.0
0.47 + 0.06 0.96+0.10 1.94_+0.06
12.4 10.5 2.8
15.9 8.5 5.9
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Concentrations in blood plasma of ruminants Concentrations of skatole and indole in peripheral blood plasma of ruminants are shown in Table 2. The mean skatole concentrations were similar in sheep and goats. In contrast, cow plasma revealed clearly lower concentrations. As shown by the standard deviation and the range, the variation between individuals was high in this species, so that maximal concentrations in cows are not far from mean concentrations in goats. The indole concentrations revealed similar means, standard deviations and ranges to skatole. Concentrations in portal vein plasma of a gilt A total of 17 samples of portal vein plasma was available from one individual gilt. During the 9-day sampling period, the mean concentration of skatole was 67 ng/ml and that of indole nearly two-fold higher (119.6 ng/ml). The concentrations increased along the sampling period so that the minimal concentration of skatole at the beginning of sampling ( 18 ng/ml) increased to a maximum of 141 ng/ml. Similarly, indole increased from 34 ng/ml to 270 ng/ml. The correlation between the two substances was 0.77 ( P < 0.001 ). Concentrations in jugular vein plasma and plasma of vena cava in comparison to concentrations in feces Systematic measurements of the two indoles were carried out in five sows for prolonged periods so that 262 measurements were available from feces, 293 from vena cava plasma and 334 from jugular vein plasma. The mean concentrations and the range for both substances in the three compartments are given in Table 3. The high standard deviation and the broad range of absolute concentrations is partly explained by differences between individuals. For example, mean skatole concentrations in feces over the sampling period varied beTABLE 2 Concentrations of skatole and indole in peripheral blood plasma (ng/ml) of female ruminants Species (n)
Skatole mean+s.d, (range)
Indole mean+s.d. (range)
Sheep (10)
0.68+0.35 (0.22-1.28)
0.51 +0.34 (0.19-1.13)
Goat (7)
0.52+0.29 (n.d.-0.85)
0.49+0.26 (0.23-0.90)
Cow (9)
0.07+0.15 (n.d.-0.43)
0.18+0.12 (n.d.-0.33)
n.d. below detection limit ( < 0.15 ng/ml ).
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TABLE 3 Mean values and ranges of skatole and indole in feces and blood plasma of the vena cava and jugular vein of five ovariectomized sows Compartment (n)
Skatole mean_+ s.d. (range)
Indole mean_+ s.d. (range)
Feces (/tg/g) (262)
2.32 _+2.97 (n.d.-12.45)
9.96 _+6.58 (0.4-42.73)
Vena cava ( n g / m l ) (293)
0.54_+0.75 (n.d.-5.56)
14.58_+36.31 (0.89-252.70)
Jugular vein ( n g / m l ) (334)
0.32_+0.36 (n.d.-2.05)
19.97_+ 63.71 (0.34-390.8)
n.d., below detection limit.
tween non-detectable and 6.4/tg/g depending on the sow. In addition, the concentrations along the 2-week sampling period varied considerably irrespective of the standardized feeding regime and the exclusion of cycle effects by ovariectomy. Thus the analysis of variance revealed a highly significant ( P < 0.001 ) influence of the individual on skatole and indole concentrations in all compartments. The time of day revealed no significant influence, whereas the day of the experiment affected indole concentrations in all compartments (P<0.001) and skatole in the vena cava and the jugular vein (P
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TABLE 4 Coefficients of between- and within-animal correlation of skatole and indole in feces and blood plasma from jugular vein and vena cava
Skatole, feces Indole, feces Skatole, cava Indole, cava Skatole, jugular
between within between within between within between within between within
Indole jugular
Skatole jugular
Indole cava
Skatole cava
Indole feces
0.32 0.66 0.80 0.33 - 0.10* 0.46 0.95 0.79 0.43 0.59
0.79 0.59 0.47 0.39 0.56 0.42 0.53 0.62
0.43 0.70 0.78 0.33 - 0.01 .... 0.50
0.18** 0.37 - 0.23 0.21"*
0.45 0.34
n.s. = not significant; * P < 0.05; **P< 0.01. All other correlations are highly significant ( P < 0.001 ).
are either non-significantly correlated (cava) or negatively correlated (feces and jugular vein) to indole concentrations. In contrast, an exceptionally high correlation was obvious between indole in cava and jugular vein plasma, as similarly found for the within-animal comparison. DISCUSSION
The data presented in this study are the first systematic measurements in blood plasma and they provide the first results on the relation between skatole and indole in feces and blood plasma of pigs. The method for the determination in blood plasma was proven to have satisfactory reliability criteria. In only a few of the biological samples the concentrations of skatole were below the detection limit. Nevertheless the sensitivity of the method is sufficient, because such low concentrations are the exception and are unlikely to contribute to the skatole problem. The skatole concentrations in blood plasma of small ruminants were clearly higher than those found in peripheral plasma of the ovariectomized sows. In contrast it was shown earlier that fecal concentrations in the same ruminant species were much lower than in intact pigs (Dehnhard et al., 1991 ). This phenomenon is explained by ruminal formation of indoles and their rapid resorption ( H a m m o n d et al., 1984). Differences in indole concentrations between ruminants and monogastrics seem to be due to differences in the microbial population in either the rumen or the colon. Such differences, however, need further clarification. Fecal skatole concentrations in the ovariectomized sows are about one-third of those in intact sows and boars. This further confirms the effect of gonadal
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steroids on skatole formation (Claus et al., 1991 ). In the present study, high variation due to the oestrous cycle was excluded by ovariectomy. Our data show that in the pig concentrations of skatole and indole in peripheral blood plasma are highly significantly correlated with fecal concentrations both within and between animals, demonstrating that a proportional amount of indoles is resorbed from the intestine. Therefore serial measurements in the feces allow us to conclude that similar changes occur in blood plasma. This does not exclude the possibility that gonadal steroids affect resorption of skatole, which has been assumed to explain a tendency for higher concentrations in adipose tissue of boars than in gilts and barrows (Hansson et al., 1980). Our data on comparative determinations of skatole and indole further suggest that the ratio of the two substances differs in feces and blood plasma. In feces the skatole:indole ratio was 1:4.3 and increased up to 1:62.4 in peripheral plasma, suggesting differences in the absorption mechanisms. As shown by the data in the portal vein of one gilt, very high skatole and indole concentrations were found in this compartment. The portal vein values represent the absorption from the small intestine and the main (proximal) part of the large intestine. The concentrations in the vena cava reflect absorption from the distal part of the colon and the rectum. Absorption from this part of the gut apparently contributes moderately to peripheral concentrations of skatole but not to those of indole. The similarity of concentrations of indole in peripheral and cava plasma and their high correlations further support the assumption that the latter compartment does not contribute to peripheral concentrations. In addition, skatole and indole in the vena cava were not significantly correlated, suggesting that the main sites of skatole and indole formation differ within the colon. Such differences in the origin of the two substances are supported by measurements along the intestinal tract (Bernal et al., unpublished results), which reveal that indole formation starts already in the ileum. Maximal concentrations were found in the caecum, whereas concentrations decreased constantly towards the rectum. In contrast, skatole formation is limited to the large intestine and the concentrations increase continuously up to a maximum in the rectum. Therefore skatole formation concentrates to that part of the gut where resorption is comparatively inefficient. Even if skatole and indole are formed by different microbes a positive within-animal correlation and a negative between-animal correlation for indole concentrations in feces and skatole concentrations in the cava was found. This may be taken as an indication that the location of the different microbes, competing for tryptophan degradation in the colon, may vary due to unknown influences. ACKNOWLEDGEMENTS
Part of this work was financially supported by the Ministry for Science and Arts ( M W K ) , Baden-Wiirttemberg. H. Bernal-Barragan was supported by a
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grant from the Universidad de Nuevo Leon, Mexico. We wish to thank Mrs A.Schell for her skilful technical assistance.
REFERENCES Bergher, H. and Ketz, H-A., 1969. Verdauung, Resorption, Intermedi~irstoffwechsel bei landwirtschaftlichen Nutztieren. VEB Deutscher Landwirtschaftsverlag Berlin, pp. 114-115. Bradley, B.J. and Carlson, J.R., 1974. A gas-liquid chromatographic procedure for the determination of indole and 3-methylindole in bovine plasma. Anal. Biochem., 59:214-219. Breeze, R.G. and Carlson, J.R., 1982. Chemical-induced lung injury in domestic animals. Adv. Vet. Sci. Comp. Med., 26: 201-231. Burnett, W.E., 1969. Air pollution from animal wastes. Determination ofmalodors by gas chromatographic and organoleptic techniques. Environ. Sci. Technol., 3: 744-749. Claus, R., Meyer, H.H.D., Gim6nez, T., Hoang-Vu, C. and Miinster, E., 1990. Effect of seminal oestrogens of the boar on prostaglandin F2a release from the uterus of the sow. Anim. Reprod. Sci., 23: 145-156. Claus, R., Bernal-Barragan, H. and Dehnhard, M., 1991. Effect of gonadal hormones in mature cyclic sows on food intake and skatole concentrations in faeces. J. Anim. Physiol. a. Anim. Nutr., 66: 61-68. Dehnhard, M., Bernal-Barragan, H. and Claus, R., 1991. Rapid and accurate high-performance liquid chromatographic method for the determination of 3-methylindole (skatole) in faeces of various species. J. Chromatogr. Biomed. Appl., 566: 101-107. Gibis, M., Dehnhard, M. and Fischer, A., 1991. Bestimmung von Skatol und Indol in Riickenspeck und Muskelfleisch von Schweinen durch Hochleistungs-FliJssigchromatographie (HPLC) mit fluorimetrischer Detektion. Z. Lebensm. Unters. Forsch., 193: 220-223. Hammond, A.C., Glenn, B.P., Huntington, G.B. and Breeze, R.G., 1984. Site of 3-methylindole and indole absorption in steers after ruminal administration of L-tryptophan. Am. J. Vet. Res., 45: 171-174. Hansson, K-E., Lundstr6m, K., Fjelkner-Modig, S. and Persson, J., 1980. The importance of androstenone and skatole for boar taint. Swed. J. Agric. Res., 10" 167-173. Honeyfield, D.C. and Carlson, J.R., 1990. Assay for the enzymatic conversion of indoleacetic acid to 3-methylindole in a ruminal Lactobacillus species. Appl. Environ. Microbiol., 56: 724-729. Lin, R.S., Orcutt, M.W., Patterson, J.A. and Judge, M.D., 1991. Serum skatole detection using gas chromatography and high performance liquid chromatography. Meat Sci., 30" 33-40. Lundstr/Sm, K., Hansson, K-E., Fjelkner-Modig, S. and Persson, J., 1980. Skatole--another contributor to boar taint. Proceedings of the 26th European Meeting of the Meat Research Workers, Colorado Springs, 31 August-5 September 1980, Vol. 1, pp. 300-303. Lundstr6m, K., Malmfors, B., Malmfors, G., Petersson, H., Stern, S., Mortensen, A.B. and Sorensen, S.E., 1984. Boar taint and bitter taste as affected by androstenone and skatole. Proceedings of the 30th European Meeting of Meat Research Workers, Bristol, 9-14 September 1984, pp. 397-398. Porter, M.G., Hawe, S.M. and Walker, N., 1989. Method for the determination of indole and skatole in pig fat. J. Sci. Food Agric., 49" 203-209. Singh, P., Sinha, A., 1990. Detection of skatole for meat quality. United States Patent 4,906, 563. Singh, P., Sinha, A., Afzal, R. and Brock, T.K., 1988. A practical enzyme-linked immunoassay for quantification of skatole. Proceedings of the 34th International Congress on Meat Science and Technology, Brisbane, pp. 692-694.
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Updyke, L.W., Yoon, H.L., Kiorpes, A.L., Robinson, J.P., Pfeifer, R.W. and Marcus, C.B., 1991. 3-Methylindole-induced splenotoxicity: Biochemical mechanisms of cytotoxicity. Toxicol. Appl. Pharmacol., 109: 375-390. Weiler, E.W., Jourdan, P.S. and Conrad, W., 1981. Levels of indole-3-acetic acid in intact and decapitated coleoptiles as determined by a specific and highly sensitive solid-phase enzyme immunoassay. Planta, 153:561-571. Wilkins, C.K., 1990. Analysis of indole and skatole in porcine gut contents. Int. J. Food Sci. Technol., 25: 313-317. Williams, G.D., Rippon, P., Chen, P-M. and Camp, B.J., 1979. An improved gas-liquid chromatographic procedure for the determination of 3-methylindole in rumen liquor, plasma, and tissue of ruminants. Anal. Biochem., 99: 324-331. Yasuhara, A., Fuwa, K. and Jimbu, M., 1984. Identification of odorous compounds in fresh and rotten swine manure. Agric. Biol. Chem., 48: 3001-3010. Yokoyama, M.T. and Carlson, J.R., 1979. Microbial metabolites oftryptophan in the intestinal tract with special reference to skatole. Am. J. Clin. Nutr., 32:173-178. Yoshihara, I., 1979. Simultaneous gas chromatographic microdetermination of indole, skatole and p-cresol in gastrointestinal contents of domestic animals. Agric. Biol. Chem., 43:19851987.
RESUME Claus, R., Dehnhard, M., Herzog, A., Bernal-Barragan, H. et Gim6nez, T., 1993. Mesures parallbles d'indole et de scatol (3-m6thylindole) par HPLC dans les fbces et le plasma de porcs. Livest. Prod. Sci., 34:115-126. en anglais. Le scatol et une partie de l'indole proviennent de la d6gradation microbienne du tryptophane dans l'intestin et peuvent provoquer une odeur f6cale d6sagr6able dans la viande de porc. Une m6thode de dosage par HPLC permettant des dosages en s6rie a 6t6 d6velopp6e (sensibilit6: 0,15 ng/ml, coefficients de variation: 10%). Des mesures en parall6le ont 6t6 effectu6es sur du plasma sanguin pr61ev6 par des cath6ters implant6s dans la veine jugulaire et dans la veine cave de cinq truies ovariectomis6es. Des d6terminations suppl6mentaires ont 6t6 r6alis6es sur les f6ces. Les concentrations dans les f6ces (scatol: X= 2,3/tg/g, indole: ~ = 10/tg/g) sont significativement corr616es (P< 0,001 ) avec les teneurs dans la veine jugulaire. Les concentrations en scatol du plasma sont plus 61ev6es (P<0,001) dans la veine cave (~=0,54 ng/ml) que clans le sang p6riph6rique (0,32 ng/ml). Les concentrations en scatol et en indole sont respectivement de 67 et de 120 ng/ml dans le plasma de la veine porte. II en conclu qu'une modification des teneurs dans les f6ces permet de supposer que des variations similaires des concentrations interviennent dans le sang. La formation d'indole est plus 61ev6e que celle de scatol et se produit essentiellement dans la pattie proximale de gros intestin, si bien que la veine cave ne contribue pas aux concentrations p6riph6riques. La formation du scatol a 6galement lieu dans la par-tie distale, et le plasma de la veine cave refl~te une r6sorption importante tandis que les teneurs en indole ne diff6rent pas significativement entre les deux vaisseaux.
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KURZFASSUNG Claus, R., Dehnhard, M., Herzog, A., Bernal-Barragan, H. und Gim6nez, T., 1993. Vergleichende Messungen von Indol und Skatol in Kot und Blutplasma durch HPLC. Livest. Prod. Sci., 34:115-126 (auf englisch). Skatol, untergeordnet auch Indol, sind f'dr f~ikalartige Geruchsabweichungen in Schweinefleisch verantwortlich. Sie entstehen durch mikrobiellen Abbau der Aminos~iure Tryptophan im Darm. Serielle Messungen sind bisher auf Kot beschr~inkt. Es wurde eine HPLC-Methode entwickelt, um Skatol und Indol im Blutplasma zu messen (Empfindlichkeit 0.15 ng/ml, Variationskoeffizient: 10%). Parallele Messungen wurden im Kot und im Blutplasma durchgeftihrt, das iiber Dauerkatheter aus der V. jugularis und V. cava von ftinf ovariektomierten Sauen gewonnen wurde. Bei einer Jungsau wurde zus~itzlich Blut aus der V. portae mittels Katheter entnommen. Die durchschnittlichen Konzentrationen im Kot (Skatol: 2.3 #g/g, Indol: 10/zg/g) waren signifikant (P-< 0.001 ) mit denen in der V. jugularis korreliert. Die Skatolkonzentrationen in der V. cava (0.54 ng/ml) waren signifikant h~her als die im peripheren Plasma (0.32 ng/ml). In der V. hepatica betrugen die Konzentrationen ftir Skatol 67 ng/ml und ftir Indol 120 ng/ml. Die Ergebnisse zeigen, dab aus Messungen im Kot auf den Konzentrationsverlauf im Blutplasma geschlossen werden kann. Die Indolbildung ist h6her als die Skatolbildung und findet, wie durch die Messungen in der V. hepatica best~itigt, haupts~ichlich im proximalen Dickdarm statt. Die Skatoibildung findet auch im distalen Teil start, so dab ein erheblicher Teil fiber die V. cava resorbiert wird, w~ihrend sich die Indolkonzentrationen zwischen den beiden Gef~iSen nicht signifikant unterscheiden.