IS THERE A NEED TO REDUCE THE LEVEL OF INDOLE GLUCOSINOLATES
Lloyd D Campbell1, Bogdan A Slominski1 and Soren K Jensen2
1 Depart. of Animal Science, University of Manitoba, Winnipeg, Manitoba, Canada, R3T 2N2
e-mail address: email@example.com
2 Danish Institute of Agricultural Science, Foulum, DK-8880 Tjele, Denmark
Experiments were conducted with weanling rats and broiler chickens to assess the contribution of indole glucosinolates to the overall antinutritive effects of glucosinolates in canola. The potential antinutritive effects of indole glucosinolates was determined in feeding trials with non-canola control diets and diets based on commercial or laboratory-prepared canola meals of varying content and proportions of indole and aliphatic glucosinolates. Studies were also conducted with diets based on relatively pure samples of isolated intact indole glucosinolate (4-hydroxyglucobrassicin) either heat-treated or unheated. The heat- treatment employed for the glucosinolate isolate and in the preparation of the laboratory-produced meals was similar to that involved in the commercial crushing process. In both rat and chicken experiments treatment effects were assessed by measuring feed consumption and growth performance throughout the feeding trials and by determining organ weights, plasma thyroid hormone levels and levels of glutathione and xenobiotic metabolizing enzymes in liver tissue of representative animals sacrificed at the termination of the trials. Biological value of canola protein was also used as an assessment criterion. Feed intake and growth performance effects were noted but were related to aliphatic glucosinolate content of diets rather than indole glucosinolate content. Similarly, treatment effects on thyroid status were influenced by level of aliphatic glucosinolates in the diet. Heat-treatment of 4-hydroxyglucobrassicin did not alter the biological value of the protein in canola. The results indicate minimal involvement of indole glucosinolates as antinutritive factors in canola.
KEYWORDS: Canola meal, glucosinolates, antinutritive, rats, chickens.
The role played by indole glucosinolates in the alleged antinutritive effects of rapeseed glucosinolates is not clear (Duncan, 1992; Fenwick et al., 1989). The situation regarding indole glucosinolates is complicated by the high sensitivity of these compounds to thermal degradation as approximately 70% destruction has been reported during commercial canola seed processing (Campbell and Slominski, 1990). Results have indicated that residual intact glucosinolates, free thiocyanate ion and indole acetonitriles, which were identified as the major indole-derived compounds in meal, had no antinutritive effects when added to the diets of growing chickens, laying hens and rats (Slominski and Campbell, 1991). A similar lack of antinutritive effects of indole glucosinolates was shown by Darroch et al. (1991) in studies with mice although Jensen et al. (1991) reported, in a study with rats, that low dietary levels of heat-treated indole glucosinolate resulted in a reduction in the biological value of casein protein. Indole glucosinolates represent a major component of the total glucosinolate content of current lines of low-glucosinolate canola (Campbell and Slominski, unpublished results) and consequently knowledge of their antinutritive effects is necessary in plant selection programs. This paper will present results of studies conducted to further document the potential antinutritive effects of indole glucosinolates.
Three experiments with male weanling rats (Sprague-Dawley) and one experiment with growing chickens (male broilers, 0-3 weeks-of-age) were conducted to assess the contribution of indole glucosinolates to the overall antinutritive effects of glucosinolates when feeding rapeseed/canola meal as a source of supplementary protein in livestock and poultry diets. Experiment 1 with rats and the experiment with broiler chickens involved the use of 3 different canola meals (produced in a pilot plant) varying in glucosinolate content along with control diets. In the rat experiment a diet containing isolated intact indole glucosinolate (4-hydroxyglucobrassicin) added to the control diet was also used. Control diets included a semipurified (starch/casein) diet and a conventional (wheat/soybean) diet in the rat experiment and two conventional diets (wheat/soybean; wheat/commercial canola meal) in the broiler chicken experiments. Canola meals were added at 30% of the diet in both experiments and the glucosinolate levels varied from a low of 5.3 to a high of 10.1 Fmol/g indole glucosinolates and from a low of 5.2 to a high of 15.9 Fmol/g of aliphatic glucosinolates. The rat diets were formulated to contain 19% protein and 3580 kcal/kg DE and the broiler chicken diets contain 23% protein and 3050 kcal/kg ME (Table 1).
Table 1. Weight gain, feed intake and antinutritive effects in rats fed diets varying in glucosinolate content (Experiment 1).
Parameter Diet1 ______________________________
1 2 3 4 5 6
Aliphatic glu.2, 0 0 0 5.2 11.3 15.9
Indole glu.2, 0 0 10.0 10.1 9.0 5.3
Weight gain, 152 159 159 146 133 129
g ±8.13 ±4.7 ±9.2 ±7.0 ±7.1 ±4.6
Feed intake, 443 433 442 414 386 386
g ±17 ±15 ±12 ±12 ±9 ±8
Liver, 54.4 52.0 51.8 55.8 55.1 57.8
g/kg BW ±1.3 ±1.0 ±1.8 ±1.5 ±1.9 ±2.0
Glutathione, 3.3 3.18 2.84 2.82 3.06 3.03
mg/g liver ±.17 ±.10 ±.26 ±.25 ±.23 ±.16
Thyroid, 4.5 5.1 5.1 9.5 12.4 14.2
mg/100gBW ±.41 ±.61 ±.40 ±.71 ±1.4 ±.70
Plasma T4, 84.9 75.9 79.8 52.8 41.2 24.5
nmol/l ±5.1 ±2.6 ±2.6 ±2.6 ±1.3 ±2.6
Plasma T3, 2.58 2.06 2.11 1.98 1.88 1.85
nmol/l ±.11 ±.11 ±.10 ±.11 ±.12 ±.10
1 Diet 1, starch/casein control; Diet 2, wheat/soybean control; Diet 3, isolated 4-hydroxyglucobrassicin; Diets 4-6, wheat/canola meal. 2 Sum of aliphatic and indole glucosinolates, respectively. 3 Mean ± std. error.
Experiment 2 with rats involved the use of diets prepared from 4 laboratory-produced canola meals and two control diets. The control diets and canola meal diets were formulated similarly to those described for Experiment 1. The level of glucosinolates in the laboratory-produced meals varied from a low of 2.3 to a high of 8.0 Fmol/g for indole glucosinolates and a low of 1.3 to a high of 15.6 Fmol/g for aliphatic glucosinolates (Table 2).
Table 2. Weight gain, feed intake and antinutritive effects in rats fed diets varying in glucosinolate content (Experiment 2).
Parameter Diet1 ______________________________
1 2 7 8 9 10___
Aliphatic glu.2, 0 0 11.6 1.3 2.1 15.6
Indole glu.2, 0 0 3.0 2.3 6.1 8.0
Weight gain, 143 160 134 147 149 126
g ±5.83 ±5.5 ±4.6 ±6.2 ±5.9 ±4.7
Feed intake, 364 365 327 353 352 305
g ±14 ±12 ±10 ±14 ±12 ±9
Liver, 55.3 54.7 60.8 53.6 57.4 60.2
g/kg BW ±0.8 ±1.9 ±1.6 ±1.5 ±1.1 ±1.3
Glutathione, 4.22 3.62 4.35 4.17 4.05 4.66
mg/g liver ±.25 ±.08 ±.18 ±.30 ±.14 ±.32
Thyroid, 6.9 8.4 13.7 9.8 11.3 16.2
mg/100 g BW ±.50 ±.1.4 ±.50 ±.60 ±.70 ±1.3
Plasma T4, 119 103 60.4 98.2 83.5 38.8
nmol/l ±3.0 ±3.4 ±5.2 ±2.4 ±3.0 ±3.3
Plasma T3, 2.07 1.64 1.52 1.84 1.74 1.52
nmol/l ±.10 ±.08 ±.08 ±.09 ±.09 ±.08
1 Diet 1, starch/casein control; Diet 2, wheat/soybean control; Diets 7-10, wheat/laboratory-produced canola meal. 2 Sum of all aliphatic and indole glucosinolates, respectively. 3Mean ± std. error.
A third experiment was conducted with weanling rats in which selected canola meal samples used in the first two rat experiments were tested for protein quality according to the rat bioassy method employed at The National Institute of Agricultural Science, Foulum, DK (Jensen, personal communication). A canola meal sample (2.3 and 1.3 Fmol/g, respectively of indole and aliphatic glucosinolates) with added isolated 4-hydroxyglucobrassicin was tested with and without heat-treatment. The amount of added indole glucosinolate amounted to 7.7 Fmol/g meal for a total indole glucosinolate level in the meal of 10.0 Fmol/g in this latter sample.
Glucosinolate analyses were conducted using the methods outlined by Slominski and Campbell (1989). The insolation of 4-hydroxyglucobrassicin from canola seed was done by extraction in methanol and the extracted glucosinolate was purified on DEAE Sephadex and Amberlite IR-120 columns (final purification 88%). To prepare the laboratory-produced meals canola seed was extracted in a soxhlet apparatus and oil-free meal was exposed to heat-treatment by autoclaving the sample at 108EC ±1 for 20 minutes. To obtain the samples of heated and non-heated glucosinolate isolate the autoclave treatment was applied before and after the addition of the glucosinolate isolate to the meal sample.
In both rat and chicken experiments, treatment effects were assessed by measuring feed consumption and growth performance throughout the feeding trials. Antinutritive effects were assessed by determining organ weights, plasma thyroid hormone levels and levels of glutathione (Baker et al., 1990) and xenobiotic metabolizing enzymes (Cytochrome P450; Guengerich et al., 1994 and glutathione-S-transferase; Habig et al;, 1974) in liver tissue of representative animals sacrificed at the termination of the trials.
Responses were noted in weight gain and feed consumption among rats fed canola meal diets varying in glucosinolate amount and type (aliphatic vs indole) and the effects were associated, to a large degree, with the content of aliphatic glucosinolates (Tables 1 and 2). In this regard, rats tended to consume less feed and gain less weight when fed diets containing canola meal with a high proportion of aliphatic glucosinolates. Furthermore, the addition of a relatively pure indole glucosinolate (4-hydroxyglucobrassicin) at a high level (10.0 Fmol/g meal) had no influence on weight gain or feed consumption of rats fed a wheat/soybean control diet. Antinutritive effects as assessed by thyroid and liver weights, liver glutathione levels and plasma T3 and T4 levels also tended to be associated with diet aliphatic glucosinolate level rather than the level of indole glucosinolates. Data for xenobiotic metabolism enzymes in liver tissue corroborated the other antinutritive measures (data not shown). The response of broiler chickens to canola meal diets of varying glucosinolate amount and type were less marked than that for rats with little or no evidence of antinutritive effects (data not shown).
Biological value data measured in rats for the various canola meal samples studied in Experiments 1 and 2, is shown in Table 3. The influence of heat-treatment of indole glucosinolate as a component of a canola meal sample is also indicated.
Table 3. Biological value of canola meal samples used in Experiments 1 and 2 and of meal with added indole glucosinolate with/without heat-treatment.
Diet reference Parameter____________________________
to canola Glucosinolate True Biological Net protein
meal Aliphatic Indole digestibility value utilization
(Fmol/g) (%) (%) (%)______
Diet 4, Exp. 1 5.2 10.1 86.6bc 93.0a 80.5a
Diet 5, Exp. 1 11.3 9.0 91.6a 91.2ab 83.5a
Diet 6, Exp. 1 15.9 5.3 85.5c 91.5ab 78.2b
Diet 10, Exp. 2 15.6 8.0 87.7bc 87.6b 76.8b
Diet 9, Exp. 2 2.1 6.1 86.1bc 93.5a 80.5a
Diet 8, Exp. 2 1.3 2.3 88.7b 92.2ab 81.8a
Diet 8, Exp. 2 1.3 10.01 86.4bc 90.0b 77.8b
Diet 8, Exp. 2 1.3 10.02 87.5bc 89.8b 78.6b
1 Isolated indole glucosinolate (7.7 Fmoles 4-hydroxyglucobrassicin/g) added to canola meal. The isolate was not heat-treated in the preparation of the meal.
2 Isolated indole glucosinolate added as per footnote 1 above except that the isolate was subjected to heat-treatment (108EC for 20 min.) in the preparation of the meal.
Although differences were evident among the canola meal samples, particularly in true digestibility, the differences were not related clearly to meal content of glucosinolates. Furthermore, the addition of 4-hydroxyglucobrassicin to canola meal had no influence on the biological value of the meal. Heat-treatment of the meal containing the added indole glucosinolate had no effect on biological value of the protein. The lack of effect of indole glucosinolates as an antinutritive factor in canola meal is in agreement with earlier reports by Slominski and Campbell (1991) and Darroch et al. (1991).
Antinutritive effects of glucosinolates were associated with aliphatic glucosinolates. The results showed minimal involvement of indole glucosinolates as antinutritive factors in canola meal and consequently indicate little or no need to reduce the current level of indole glucosinolates present in rapeseed/canola. The data also documented the high biological value of canola protein and indicated no effect of the heat-treatment of 4-hydroxyglucobrassicin on protein quality.
The canola seed used to prepare the laboratory-produced meals was provided by Agriculture and Agri-Food Canada, Saskatoon, SK, Canada. Monsanto, St. Louis, MO, United States of America, provided some of the canola meal samples used in the study. Financial assistance was provided, in part, by the Natural Science and Engineering Research Council of Canada.
Baker, M.A., G.J. Cerniglia and A. Zaman. 1990.l Microliter plate assay for the measurement of glutathione and glutathione disulfide in large numbers of biological samples. Analytical Biochemistry 190:360-365.
Campbell, L.D. and B.A. Slominski, 1990. Extent of thermal decomposition of indole glucosinolates during the processing of canola seed. J. Am. Oil Chem. Soc. 76:73-75.
Darroch, C.S., J.M. Bell, D.I. McGregor and J.H.L. Mills. 1991. The effects of a linear increase in dietary indole glucosinolates on growth and physiology of mice. Can. J. Anim. Sci. 71:887-896.
Duncan, A.J. 1992. Glucosinolates. In: Toxic Components of Plants. ed. J.P.F. Dimello. Royal Society of Chemistry, Cambridge, pp. 126-147.
Fenwick, G.R., R.K. Heaney and R. Mawson. 1989. Glucosinolates. In: Toxicants of Plant Origin. Vol. II Glycosides. ed. R.R. Cheeke, CRC Press, Boca Raton, FL, pp. 1-40.
Guengerick, F.P., 1994. Analysis and charcterization of enzymes. In: Principles and Methods of Toxicology ed. A.W. Hayes, Raven Press Ltd. New York, pp. 1259-1313.
Habig, W.H., M.J. Pabst and W.B. D. Jakoby. 1974. Glutathione-S-transferase: The first enzymatic step in mercapturic acid formation. J. Biol. Chem. 249: 7130-7139.
Jensen, S.K., S. Michaelsen, P. Kacklicki and H. Sorensen. 1991. 4-hydroxyglucobrassicin and degradation products of glucosinolates in relation to unresolved problems with the quality of double low rape. Proceedings of the 8th Int’l. Rapeseed Congress, Saskatoon, Canada, pp. 1359-1364.
Slominski, B.A. and L.D. Campbell. 1987. Gas chromatographic determination of indole glucosinolates - A re-examination. J. Sci. Food Agric. 40:131-143.
Slominski, B.A. and L.D. Campbell. 1991. Influence of indole glucosinolates on the nutritive quality of canola meal. Proceedings of 8th Int’l. Rapeseed Congress, Saskatoon, Canada, pp. 396-401.