Nutrient Metabolism
Dietary Antibiotic Growth Promoters Enhance the Bioavailability of ␣-Tocopheryl Acetate in Broilers by Altering Lipid Absorption1 Ane Knarreborg, Charlotte Lauridsen, Ricarda M. Engberg,2 and Søren K. Jensen Department of Animal Nutrition and Physiology, Danish Institute of Agricultural Sciences, Research Centre Foulum, DK-8830 Tjele, Denmark ABSTRACT The influence of intestinal microbial bile salt deconjugation on absorption of fatty acids and ␣- and ␥-tocopherol was investigated in a trial with Ross 208 broilers. Birds (n ⫽ 1600) were assigned to 4 dietary treatments: no supplementation or supplementation of antibiotics (salinomycin, 40 mg/kg feed and avilamycin, 10 mg/kg feed), and inclusion of either animal fat (10 g/100 g feed) or soybean oil (10 g/100 g feed) in the diet. At d 7, 14, 21, and 35 of age, the intestinal number of the bile salt hydrolase–active bacteria Clostridium perfringens, the concentration of conjugated and unconjugated bile salts, the ileal absorption of fatty acids and tocopherols, and the blood plasma concentrations of tocopherols were measured. All variables were significantly influenced by bird age. C. perfringens counts were lower and bile salt concentrations were greater in birds fed soybean oil. The supplementation of antibiotics reduced the numbers of C. perfringens in the small intestine and reduced the concentration of unconjugated bile salts. The ileal absorption of fatty acids and ␣-tocopherol, as well as the plasma concentration of ␣-tocopherol, was greater in birds fed antibiotics. The absorption and plasma concentration of ␥-tocopherol were not influenced by antibiotics. Unlike ␥-tocopherol, which is present solely as the free alcohol, the major proportion of dietary ␣-tocopherol is present as ␣-tocopheryl acetate, which requires a bile salt– dependent enzymatic hydrolysis before absorption. In conclusion, proper digestion of lipid-soluble compounds is highly dependent on an adequate concentration of bile salts in the small intestine to provide proper lipid emulsification and activation of lipolytic enzymes. J. Nutr. 134: 1487–1492, 2004. KEY WORDS:
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tocopherols
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broiler chickens
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antibiotics
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bile salts
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dietary fat source
activation of CEH (8 –10). Combs (11) proposed that the esterase activity could be the limiting factor in utilizing tocopheryl esters in chicken. In addition, Jensen et al. (12) suggested that the lower absorption of ␣-tocopherol in broilers fed all-rac-␣-tocopheryl succinate compared with all-rac-␣-tocopheryl acetate could be ascribed to the lower ability of CEH to hydrolyze all-rac-␣-tocopheryl succinate. In conjunction with pancreatic lipase and colipase, glycineor taurine-conjugated bile salts play an essential role in the digestion of lipids (13). The majority of gram-positive bacteria inhabiting the small intestine are capable of hydrolyzing the amide bond, liberating the corresponding unconjugated bile salts with markedly different physicochemical properties (14 – 16). Due to a pKa-value around the physiologic pH in the small intestine, unconjugated bile salts easily precipitate (15) and are excreted in the feces (17,18). In addition, the low aqueous solubility of unconjugated bile salts causes less efficient enterohepatic recycling by passive diffusion in the colon (19,20). Clostridium perfringens isolated from the small intestine of broiler chickens was shown to express high levels of bile salt hydrolase activity (16), resulting in a reduced concentration of bile salts available for efficient solubilization of lipid components. The objective of the present study was to investigate, in vivo, the effect of bile salt concentration, lipase, and CEH activity in the small intestine on ileal fatty acid and tocoph-
Because dietary lipids provide a large proportion of the energy in poultry feeds, efficient fat digestion is crucial for chicken growth. Fat digestion in chickens is influenced by numerous factors; of these, fat source and age of the bird are most reported (1– 4). Little information is available, however, on the role of the intestinal microflora in the digestion of fat and fat-soluble components such as tocopherols. The important role of tocopherols in the protection of biological membranes against oxidative damage is well recognized. Vitamin E is usually incorporated into animal feed as dl-␣-tocopheryl acetate (5). Tocopheryl esters must be hydrolyzed after absorption in the small intestine as free alcohols alone or in combination with the emulsified fat products. Carboxyl ester hydrolase (CEH)3 was found to be the principal enzyme hydrolyzing tocopheryl acetate in human duodenal juice (6). The importance of enzymatic hydrolysis was stressed by the results of Chung (7), who found lower plasma concentrations of ␣-tocopherol in weaned piglets fed the acetate ester of ␣-tocopherol compared with the free alcohol. In this context, it was shown in vitro that bile salts are required for 1 Supported by a grant from the Danish Ministry of Food, Agriculture and Fisheries. 2 To whom correspondence should be addressed. E-mail:
[email protected]. 3 Abbreviations used: C, cholate; CDC, chenodeoxycholate; CEH, carboxyl ester hydrolase; TC, taurocholate; TCDC, taurochenodeoxycholate.
0022-3166/04 $8.00 © 2004 American Society for Nutritional Sciences. Manuscript received 30 September 2003. Initial review completed 21 November 2003. Revision accepted 31 March 2004. 1487
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erol absorption and on plasma status of tocopherols. Due to a high genetic homogeneity, commercially raised chickens, which were offered different dietary treatments with varying subtherapeutic levels of antibiotics and different dietary fat sources, were used as experimental animals in this study. MATERIALS AND METHODS Birds, diets, and sampling. A total of 1600 1-d-old male broiler chickens (Ross 208) were obtained from a commercial hatchery (DanHatch A/S) and were housed in 32 floor pens (50 broilers per pen). According to a block design, the pens were randomly assigned to 1 of 4 dietary treatments in which the random allocation of the treatments was balanced in relation to the position in the broiler house (eight replicate blocks). Each pen provided a floor area of 1.7 m2, covered with wood shavings. During the experimental period (d 1–35), the chickens were fed a wheat-based mash diet4 supplemented with either a mixture of lard and tallow (Animal fat, 1.5:1, 10 g/100g), or soybean oil (Soybean oil, 10 g/100 g). Diets were not (⫺) or were (⫹) supplemented with antibiotics, which were a combination of salinomycin (40 mg/kg feed) and avilamycin (10 mg/kg feed). Both substances are approved as feed additives by the European Union for use in broiler production in combination as coccidiostat and antibiotic growth promoter, respectively. Salinomycin and avilamycin were premixed in calcium carbonate before addition to the experimental diets. Chromic oxide (Cr2O3) was added to the diets as a marker substance at a concentration of 0.4%. The birds had free access to feed and water, and a continuous lightning regimen was followed. At 7, 14, 21, and 35 d, 16, 10, 8, and 6 chickens, respectively, were randomly selected from each pen, and killed by cervical dislocation. The number of birds removed at each sampling time was calculated to ensure adequate amounts of intestinal material for analysis, and to avoid major changes in stocking density (kg/m2) during the experiment. The small intestine was ligatured at the proximal and distal end and at the Meckel’s diverticulum, and the 2 segments of the small intestine were rapidly excised. Contents from the proximal and distal part were collected separately and pooled from all chickens within each pen at each sampling time. Samples from the proximal part were divided into aliquots for enumeration of bile salt hydrolase–active bacteria, using C. perfringens as the indicator bacteria (16), and for determination of bile salt concentration, lipase, and CEH-activity. Ileal samples were stored at ⫺20°C until analysis of chromic oxide, fatty acids, and tocopherols. In addition, blood samples were collected and pooled from the vena cutanea ulnaris of 2 chickens/pen into 5 mL sodium-heparinized vacutainer tubes (Becton Dickinson). After centrifugation at 3000 ⫻ g for 10 min, the plasma samples were stored at ⫺20°C until tocopherol analyses. The experiment complied with the guidelines of the Danish Ministry of Justice with respect to animal experimentation and care of animals under study. Enumeration of C. perfringens. Each intestinal sample (10 g) was rapidly transferred under a flow of CO2 into a sterile serum bottle containing 90 mL of a prereduced broth (21). This suspension was poured into a CO2-flushed plastic bag and homogenized in a stomacher laboratory blender (Seward Medical) for 2 min. Subsequently, 10-fold dilutions were made according to Miller and Wolin (22). Counts of C. perfringens were determined by pouring known amounts of sample into Tryptose sulfite agar (MERCK 1972) supplemented with cycloserine (OXOID SR088E). After 24 h of anaerobic incubation at 37°C, black colonies were counted. Determination of carboxylester hydrolase and lipase activities. For the determination of lipase and CEH activity, 1-g samples of
4 Diet composition (g/kg): wheat, 501; soybean meal, toasted, 275.9; peas, 40; fishmeal, 40; fat, 100; sodium chloride, 2; dicalcium phosphate, 19.5; calcium carbonate, 7; sodium bicarbonate, 1.5; DL-methionine (40%), 5; choline chloride, 0.4; chromic oxide, 4; vitamin and mineral mix, 3.5: retinyl acetate, 5.8 mg; cholecalciferol, 0.09 mg; dl-␣-tocopheryl acetate, 42 mg; menadione, 3.5 mg; thiamin, 1.4 mg; riboflavin, 11.2 mg; pyridoxine, 4.2 mg; D-pantothenic acid, 14 mg; niacin, 56 mg; betaine anhydrate, 473 mg; folic acid, 2.1 mg; biotin, 140 g; cyanocobalamin, 28 g; BHT, 140 mg; FeSO4 䡠 7H2O, 112 mg; ZnO, 112 mg; MnO, 140 mg; CuSO4 䡠 5H2O, 21 mg; KI, 840 g; Na2SeO3, 420 g.
intestinal contents were homogenized in 3 mL of 9 g/L NaCl using an Ultra Turrax (T25 basic IKA) equipped with a S25KG-18 g probe (Janke and Kunkel) and centrifuged at 14750 ⫻ g for 20 min at 4°C. The supernatant fractions were assayed for lipase activity according to the titrimetric method of Erlanson-Albertsson et al. (23) and the activity of CEH as described by Jensen et al. (24). One unit of CEH or lipase activity is defined as the hydrolysis of 1 mol substrate in 1 min. Determination of bile salts. Individual bile salts were quantified by reversed-phase HPLC with pulsed amperometric detection (25). The calibration standards were glycine- and taurine-conjugated bile salts and free bile salts of chenodeoxycholate, cholate, deoxycholate, and lithocholate (Sigma). Intestinal samples (1 g) for bile salt determination were diluted 50-fold in a mixture containing 20% acetonitrile, 10% NaOH, and 70% H2O. Ursodeoxycholate (Sigma) was added to the mixture as an internal standard at a final concentration of 40 mol/L. Subsequently, the samples were mixed in a IKAVIBRAX-VXR (IKA-Werke) for 30 s and centrifuged at 5000 ⫻ g for 10 min. The supernatant (1 mL) was passed through a 0.20-m nylon syringe filter membrane (Cameo 17N-DDR02T17NB) before injection onto the HPLC. The chromatographic conditions were described by Knarreborg (26). Determination of chromic oxide, fatty acids, and tocopherols. Diets and ileal samples were freeze-dried and ground before analysis. The concentration of Cr2O3 was determined spectrophotometrically using the method of Schu¨ rch et al. (27). Samples for determination of fatty acids were extracted in a mixture of chloroform and methanol according to Bligh and Dyer (28) after acidification with 3 mol/L HCl to liberate calcium soaps and membrane-bound lipids. The lipid extracts were esterified using NaOH/methanol (30 mol/L). The resulting FAME were separated and quantified by GLC (29). The concentrations of tocopherols in the diets, in the ileal samples, and in the plasma samples were analyzed by HPLC after saponification with KOH and extraction into heptane as described by Jensen et al. (12). Calculations and statistics. Ileal absorption of tocopherols was defined as the ratio of tocopherols to Cr2O3 in the entire ileal contents (from Meckel’s diverticulum to the ileocecal junction) to that in the diet, and was calculated according to the following equation: Tocopherol ileal absorption coefficient ⫽1⫺
冉
关Tocopherols兴ileum 关Cr2O3兴diet ⫻ 关Tocopherols兴diet 关Cr2O3兴ileum
冊
The ileal absorption coefficients of fatty acids were calculated accordingly. The experiment was designed as a split-plot experiment in which dietary treatment was the whole-plot treatment and pens were the whole-plot units. The experimental day (age of birds) was the splitplot factor. Hence, the statistical analysis of data was accomplished using the Mixed procedure of SAS (SAS Institute) and was based on the following model: Yfadbp ⫽ ⫹ ␣f ⫹ a ⫹ ␥d ⫹ b ⫹ 共␣兲fa ⫹ 共␣␥兲fd ⫹ 共␥兲ad ⫹ Ufap ⫹ ⑀fadbp 关M1兴 where Yfadbp is the dependent variable, is the overall mean, ␣f is the systematic effect of fat source (Animal fat, Soybean oil), a is the systematic effect of antibiotic inclusion (⫺, ⫹), and ␥d and b are the systematic effect of experimental day (d 7, 14, 21, 35) and block (1– 8). The interactions between fat and antibiotic inclusion is designated (␣)fa; (␣␥)fd is the interaction between fat source and day, and (␥)ad is the interaction between antibiotic and day. The random effect of pens is designated Ufap and ⑀fadbp denotes the error term. Note that the random pen effect was imposed to account for repeated measurements being made on the same experimental unit (pen). The model was verified by plotting the residuals against the predicted values and by use of quantile plots of the residuals. The 3-factor interaction (␣␥)fad was omitted from the model because it was nonsignificant (P ⬎ 0.15). Statistical analysis with covariates was also performed as model [M2] and included the ileal fatty acid absorption as covariate in the above-mentioned model [M1]; [M3] included the
BIOAVAILABILITY OF TOCOPHEROLS
total bile salt concentration as in [M1], and [M4] included both covariates in [M1]. Results are given as least-square means (LSMeans) with a pooled SEM. Differences were considered significant at P ⱕ 0.05.
RESULTS Dietary concentrations of fatty acids and tocopherols. The concentrations of dietary total fatty acids were 98 and 115 g/kg dry matter when supplemented with animal fat and soybean oil, respectively. Diets containing animal fat and soybean oil provided on average (g/kg of dry matter): 26.2 and 13.4 palmitic acid, 16.9 and 4.1 stearic acid, 32.8 and 21.2 oleic acid, 13.6 and 63.2 linoleic acid, 1.5 and 10.2 linolenic acid, respectively. The total concentrations of analyzed ␣-tocopherol (mg/kg) were 56.3 and 58.4 for diets containing animal fat and soybean oil, respectively. The diets were supplemented with 42 mg/kg ␣-tocopheryl acetate corresponding to 38 mg analyzed ␣-tocopherol. We assumed that the remaining 18 –20 mg dietary ␣-tocopherol (⬃32–34%) was of natural origin present as the free alcohol. The total concentrations of ␥-tocopherol (mg/kg) in the diets with animal fat were 18.2 and 85.3 in the diet with soybean oil. Because ␥-tocopherol was not added in the vitamin mixture, all of the ␥-tocopherol was present in the free alcohol form. Intestinal numbers of C. perfringens, enzyme activities of CEH and lipase, and concentration of bile acids. The numbers of C. perfringens (Table 1) increased with the age of the birds (P ⬍ 0.001) and were reduced (P ⬍ 0.001) by dietary supplementation with antibiotics. Furthermore, birds fed the diets with animal fat had higher intestinal numbers of C. perfringens than those fed soybean oil (P ⫽ 0.008). The CEH activity in the proximal part of the small intestine was not influenced by dietary antibiotics (P ⫽ 0.95) or fat source (P ⫽ 0.90), but was age dependent (P ⬍ 0.001), showing a decline in the proximal part of the small intestine at 14 d but increasing thereafter (Table 1). Lipase activity was greater in birds supplemented with antibiotics (P ⫽ 0.002). However, a significant interaction between age and dietary supplementation with antibiotics was observed (P ⫽ 0.04) in which no increase in lipase activity occurred in 14-d-old birds. The dominant bile salts present in the intestinal contents were chenodeoxycholate (CDC) and cholate (C), which were conjugated with taurine (TCDC and TC, respectively). The total concentrations of conjugated bile salts (Table 1) in the proximal part of the small intestine were affected by significant interactions between age and supplementation with antibiotics (P ⫽ 0.007), and between fat and supplementation with antibiotics (P ⫽ 0.014). The lowest concentration of total conjugated bile salts was in 14-d old birds, and the concentration of conjugated bile acids was lower in chickens fed animal fat than in those fed soybean oil, and in chickens not supplemented with antibiotics compared with supplemented chickens. The total concentration of unconjugates was affected by an interaction between fat and age (P ⫽ 0.048) in which the concentration of unconjugates increased to a higher extent in chickens fed soybean oil than in birds fed animal fat. In addition, chickens supplemented with antibiotics had a lower concentration of unconjugates compared with unsupplemented chickens (P ⫽ 0.01). The proportion of unconjugates in chickens supplemented with antibiotics was 3% (21 d) and 8% (35 d) of the total bile salts concentration compared with 7% (21 d) and 11% (35 d) in the antibiotic-free diets. Ileal absorption of fatty acids. The ileal absorption coefficients of total and individual fatty acids (Table 2) were
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TABLE 1 The effect of dietary fat source and antibiotic supplementation on the count of C. perfringens, CEH, and lipase activities, and concentrations of conjugated and unconjugated bile salts in the contents of the proximal part of the small intestine in chickens at various ages1 Fat source Age, d
Animal fat
Antibiotics ⫺
Soybean oil
⫹
C. perfringens, log10 cfu/g digesta 7 3.57a 14 4.17ab 21 4.68b 35 6.70c SEM ⫽ 0.24, P-values: Antibiotic
3.20a 3.55b 4.35c 5.91c ⬍ 0.001, Age ⬍
3.96a 4.77b 5.64c 7.14d 0.001, Fat ⫽
2.82a 2.95a 3.39a 5.48b 0.008
CEH activity, units/g digesta 7 0.24b 14 0.11a 21 0.42c 35 0.41c SEM ⫽ 0.03, P-values: Antibiotic
0.26b 0.12a 0.37c 0.44c ⫽ 0.95, Age ⬍
0.23b 0.28b 0.13a 0.11a 0.42c 0.37c 0.41c 0.44c 0.001, Fat ⫽ 0.90
Lipase activity, units/g digesta 7 6.6a 6.2a 14 5.1a 6.1a 21 11.6c 10.5b 35 6.0b 6.4a SEM ⫽ 0.65, P-values: Antibiotic ⫽ 0.002, Age ⬍ Antibiotic ⫻ Age ⫽ 0.040
5.9a 6.9a 5.7a 5.4a 9.9b 12.2b 4.5a 7.9a 0.001, Fat ⫽ 0.99,
Total conjugates,2 mol/g digesta 7 8.57b 10.71b 9.22b 10.07b 14 6.48a 8.13a 7.12a 7.49a 21 10.01c 12.18c 10.51c 11.68b 35 9.85c 10.74b 8.88b 11.71b SEM ⫽ 0.36, P-values: Antibiotic ⬍ 0.001, Age ⱕ 0.001, Fat ⬍ 0.001, Antibiotic ⫻ Age ⫽ 0.007, Antibiotic ⫻ Fat ⫽ 0.014 Total unconjugates,3 mol/g digesta 7 0.04a 14 0.09ab 21 0.34b 35 0.85c SEM ⫽ 0.09, P-values: Antibiotic Fat ⫻ Age ⫽ 0.048
0.14a 0.20a 0.83b 1.30c ⫽ 0.01, Age ⬍
0.11a 0.07a 0.20a 0.09a 0.81b 0.36b 1.13c 1.02c 0.001, Fat ⬍ 0.001,
1 Values are LSMeans, n ⫽ 8. Means for a variable in a column without a common letter differ, P ⱕ 0.05. 2 Total conjugates ⫽ sum of TCDC and TC. 3 Total unconjugates ⫽ sum of CDC and C.
generally greater in birds fed the antibiotic-supplemented diets compared with those fed the unsupplemented diets (P ⬍ 0.05). A significant interaction between age and fat source occurred for the ileal absorption of total (P ⬍ 0.001) and individual fatty acids (except stearic acid), with the lowest absorption coefficients present in 14-d-old birds fed animal fat. In general, the ileal fatty acid absorption in chickens fed soybean oil was higher than in chickens fed animal fat. Ileal absorption of tocopherols. Regardless of treatment, the ileal absorption of ␣-tocopherol (Table 3) was higher in 21- and 35-d old birds than in younger chickens (P ⫽ 0.019). Age did not affect the ileal absorption of ␥-tocopherol (P
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TABLE 2
TABLE 3
Effect of dietary fat source and antibiotic supplementation on ileal absorption coefficients of selected fatty acids in broiler chickens at various ages1
The effect of dietary fat source and antibiotic supplementation on the ileal absorption coefficients of tocopherols in chickens at various ages1
Fat source Age, d
Animal fat
Antibiotics ⫺
Soybean oil
Fat source ⫹
Age, d
0.81b 0.69a 0.83b 0.77ab ⫽ 0.043, Age ⬍
0.65bc 0.56a 0.73c 0.63b 0.001, Fat
0.72b 0.53a 0.81c 0.76b
7 14 21 35 SEM ⫽ 0.010, ⫽ 0.072
0.76b 0.59a 0.83b 0.76b ⫽ 0.011, Age ⬍
⫺
⫹
0.72a 0.78 0.74a 0.78 0.79b 0.80 0.78b 0.77 P-values:2 Antibiotic ⫽ 0.023, Age
0.74a 0.73a 0.79b 0.76ab ⫽ 0.019, Fat
0.76 0.79 0.80 0.80
0.83a 0.92b 0.88ab 0.90b ⬍ 0.126, Fat
0.89 0.90 0.88 0.88
␥-tocopherol
Stearic acid (18:0) 7 0.32b 14 0.18a 21 0.45c 35 0.35b SEM ⫽ 0.04, P-values: Antibiotic
Soybean oil
␣-tocopherol
Palmitic acid (16:0) 7 0.56b 14 0.40a 21 0.71c 35 0.62b SEM ⫽ 0.03, P-values: Antibiotic ⬍ 0.001, Fat ⫻ Age ⫽ 0.03
Animal fat
Antibiotics
0.49b 0.37a 0.59c 0.51bc 0.001, Fat ⬍
0.59b 0.40a 0.68b 0.60b 0.001
0.75b 0.66a 0.87c 0.78b 0.001, Fat
0.81b 0.66a 0.92c 0.89c
7 14 21 35 SEM ⫽ 0.013, ⬍ 0.001
0.77a 0.95 0.85b 0.97 0.80ab 0.96 0.82ab 0.96 P-values:3 Antibiotic ⫽ 0.752, Age
Oleic acid (18:1) 7 0.71b 14 0.55a 21 0.87c 35 0.83c SEM ⫽ 0.03, P-values: Antibiotic ⬍ 0.001, Fat ⫻ Age ⬍ 0.001
0.85b 0.77a 0.92b 0.84ab ⫽ 0.033, Age ⬍
Linoleic acid (18:2) 7 0.69b 14 0.54a 21 0.81c 35 0.74b SEM ⫽ 0.03, P-values: Antibiotic ⬍ 0.001, Fat ⫻ Age ⬍ 0.001
0.94b 0.87a 0.96b 0.91b ⫽ 0.001, Age ⬍
0.80b 0.70a 0.86c 0.79b 0.001, Fat
0.84b 0.72a 0.90c 0.86b
Linolenic acid (18:3) 7 0.74b 0.96b 0.84b 14 0.62a 0.91a 0.76a 21 0.84c 0.97b 0.89b 35 0.77b 0.90a 0.79a SEM ⫽ 0.02, P-values: Antibiotic ⫽ 0.002, Age ⬍ 0.001, Fat ⬍ 0.001, Fat ⫻ Age ⫽ 0.001, Antibiotic ⫻ Age ⫽ 0.07
0.87b 0.77a 0.92b 0.88b
1 Values are LSMeans, n ⫽ 8. Means for a variable in a column without a common letter differ, P ⱕ 0.05. 2 After inclusion of covariates (fatty acid absorption coefficient and concentration of bile salts): Antibiotic ⫽ 0.018, Age ⫽ 0.030, Fat ⫽ 0.416. 3 After inclusion of covariates (fatty acid absorption coefficient and concentration of bile salts): Antibiotic ⫽ 0.380, Age ⫽ 0.235, Fat ⬍ 0.001.
tions of ␣-tocopherol only in blood plasma (P ⫽ 0.01) during the 35-d growth period (Fig. 1). The concentration of ␣-tocopherol was age dependent (P ⬍ 0.001), and the lowest values were measured in 14-d-old birds. The blood plasma concentration of ␣-tocopherol was higher in birds fed the antibiotic-supplemented diets (P ⫽ 0.012). The ␥-tocopherol concentration in blood plasma (Fig. 1) decreased during the
⌺ fatty acids (14–20) 7 0.61b 14 0.45a 21 0.75d 35 0.68c SEM ⫽ 0.02, P-values: Antibiotic ⬍ 0.001, Fat ⫻ Age ⫽ 0.001
0.88b 0.82a 0.93b 0.87ab ⫽ 0.030, Age ⬍
0.71b 0.64a 0.81c 0.73b 0.001, Fat
0.77b 0.64a 0.86c 0.82c
1 Values are LSMeans, n ⫽ 8. Means for a variable in a column without a common letter differ, P ⱕ 0.05.
⫽ 0.13). The absorption of tocopherols, in particular, ␥-tocopherol, was greater (P ⬍ 0.001) in birds fed soybean oil compared with those fed animal fat. The absorption of ␣-tocopherol was higher (P ⫽ 0.023) in chickens fed the antibiotic-supplemented diets than in unsupplemented birds. In contrast, the absorption of ␥-tocopherol was unaffected by the dietary antibiotics. The antibiotics influenced the concentra-
FIGURE 1 Effect of dietary supplementation with (A⫹) and without antibiotics (A⫺) on the concentration of ␣-tocopherol (␣-toc) and ␥-tocopherol (␥-toc) in blood plasma from chickens at various ages. Values are LSMeans, n ⫽ 8.
BIOAVAILABILITY OF TOCOPHEROLS
growth period (P ⬍ 0.001). Antibiotics did not affect the ␥-tocopherol concentration in the blood (P ⫽ 0.88). Because the ileal absorption of fatty acids and the total concentration of bile salts were correlated (r ⫽ 0.66, P ⬍ 0.001), these variables were included as covariates in the statistical model to clarify whether the observed effect of antibiotics on the absorption of tocopherol could be ascribed to changes in the bile salt concentration and/or the fatty acid absorption. The statistical analysis indicated that the ␣-tocopherol concentration in blood plasma was independent of dietary antibiotics conditional on the presence of the ileal fatty acid absorption and the total bile salt concentration in the model, i.e., the P-value of the antibiotic effect on the ␣-tocopherol concentration in blood plasma was significant (P ⫽ 0.012) in the original model (M1), increased (P ⫽ 0.063) when fatty acid absorption was included as the sole covariate (M2), but increased even further (P ⫽ 0.117) with the bile salt concentration as the sole covariate (M3). By including both covariates (M4), the effect of antibiotics on the ␣-tocopherol concentration in blood plasma disappeared completely (P ⫽ 0.221). For the ileal absorption of ␣-tocopherol, this response was dependent on the antibiotics despite the presence of fatty acid absorption and/or the total concentration of bile salts as covariates in the model (Table 3). The presence of fatty acid absorption as a covariate in the model was significant (P ⫽ 0.031) for the concentration of ␥-tocopherol in blood plasma, whereas this variable only tended (P ⫽ 0.062) to affect the ileal absorption of ␥-tocopherol. However, there was no correlation between the concentration of ␥-tocopherol in blood plasma and fatty acid absorption. The covariate bile salt concentration did not affect the ileal absorption of ␥-tocopherol or the concentration of ␥-tocopherol in blood. DISCUSSION The concentration and composition of bile salts in small intestinal contents found in this study are in agreement with the work of others (30,31). In addition, several studies have shown that bile salt secretion (32–35) and the activity of pancreatic lipase (36,37) are reduced in young birds in which bile salt secretion is considered to be the principal limitation for lipid utilization during the first weeks after hatching. The higher concentration of unconjugated bile salts coincided with a higher number of C. perfringens in birds fed the unsupplemented diets compared with birds fed the antibiotic-supplemented diets. This indicates a higher microbial deconjugation of bile salts in unsupplemented birds. Moreover, the increased number of C. perfringens with age of the birds was accompanied by an increase in the concentration of unconjugated bile salts. Accordingly, the stimulating effects of antibiotics on intestinal lipase activity and fatty acid absorption were more pronounced in older birds in which the number of bacteria capable of deconjugating bile salts was higher. Results from an in vitro study showed that the activity of lipase is influenced by the ratio of unconjugated to conjugated bile salts (38). The influence of dietary fat source on fatty acid absorption was in accordance with the literature, showing a poor utilization of animal fat compared with that of vegetable origin (2– 4,39), which can be ascribed primarily to differences in the ratio of saturated to unsaturated fatty acids. Compared with SFA, unsaturated fatty acids are more polar solutes and have a lower melting point, making them more likely to be in a liquid state at the high body temperature of chickens. Thus, unsaturated fatty acids exhibit a higher solubility in the micellar phase, which enhances the dispersion in the small intestine and reduces the demand for emulsification (40 – 42). The fact
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that unsaturated fatty acids are less dependent on the presence of bile salts may explain why the feeding of soybean oil improved fatty acid absorption to a greater extent in younger chickens in which the bile salt concentration was limited. In agreement, studies showed that dietary supplementation with bile salts increases fat digestion preferentially in young chickens, particularly when animal fat is fed (32–34). It was surprising that unlike lipase activity, the intestinal activity of CEH was not influenced by the dietary treatments. Gabert et al. (43) showed that unsaturated, long-chain fatty acids (fish oil) increased total CEH activities in porcine pancreatic juice more than rapeseed and coconut oil. In accordance with previous studies in pigs (24), CEH activity was strongly influenced by the age of the birds. The lack of influence of dietary antibiotics on CEH activity was not expected because the activation of this enzyme is bile salt dependent (6,10). The importance of pancreatic enzymes for cellular uptake of ␣-tocopherol was also studied in vitro using a human intestinal cell line (9). In this study, the uptake of ␣-tocopherol in its alcohol form occurred in the presence of bile salts and fatty acids, but was not further potentiated by the addition of bile-activated lipase, and it was concluded that pancreatic enzymes are necessary for lipid hydrolysis, but not specifically to facilitate ␣-tocopherol absorption. However, the hydrolysis of tocopheryl esters is a prerequisite for tocopherol absorption. As shown previously in vitro (10), ␣-tocopheryl acetate was not hydrolyzed in the absence of pancreatic enzymes or bile salts. The importance of hydrolysis for the absorption of tocopherols was further stressed in the present study because dietary supplementation with antibiotics clearly increased ␣-tocopherol absorption and the plasma concentration, whereas ␥-tocopherol was not affected. ␥-Tocopherol was provided through the dietary ingredients in its alcohol form, and hence does not depend on hydrolysis before absorption. The observed effect of antibiotics on plasma ␣-tocopherol concentration seemed to be mediated through changes in bile salt concentration. In addition, the digestion of fatty acids, which also depends on the presence of bile salts, contributed to the observed effect of antibiotics on the plasma concentration of ␣-tocopherol, indicating the involvement of the products of the hydrolytic lipid digestion in micelle formation. Inexplicably, the same role of bile salts and lipolytic products in the antibiotic effect on ileal ␣-tocopherol absorption was not demonstrated. However, there was more variation due to the larger numbers of analyses involved. Nevertheless, the lower absorption and lower blood plasma concentrations of ␣-tocopherol in birds fed the unsupplemented diets clearly coincided with a higher incidence of bile salt hydrolase–active bacteria and hence a lower total bile salt concentration in the small intestine compared with chickens fed the antibioticsupplemented diets. The ␥-tocopherol concentrations in the diets supplemented with soybean oil were much higher than in the diets containing animal fat, which may have influenced ␥-tocopherol absorption. However, the higher concentration of unsaturated fatty acids facilitating emulsification and micelle formation (40 – 42) may similarly have contributed to an improved ␥-tocopherol absorption. In conclusion, the present study provides evidence in vivo that the bioavailability of ␣-tocopheryl acetate is highly dependent on an adequate amount of bile salts to generate enzymatic hydrolysis of ␣-tocopheryl acetate and subsequent absorption of ␣-tocopherol to the blood plasma. Thus, conditions facilitating the growth of bile salt hydrolase–active bacteria in the proximal part of the animal or human gut will have a negative influence on the availability of bile salts and
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thereby on the formation of lipid micelles and activation of hydrolytic enzymes. Consequently, the bioavailability of ␣-tocopheryl acetate is decreased. Such conditions may have an even stronger effect on immature animals and humans suffering from limited bile salt synthesis and pancreatic enzyme secretion (44,45). In addition, the absorption of fat and fatsoluble compounds is greatly influenced by the dietary fat source during the beginning of the growth period when the physiologic capacity of chickens, in terms of adequate bile salts, is limited. In contrast, the effect of antibiotics on fat digestion is more pronounced later in the growth period, when the number of bile salt hydrolase–active bacteria is high. ACKNOWLEDGMENTS The authors express their gratitude to all of the technicians involved in the excellent care of the chickens and collection of data. Further, we address special thanks for skillful technical assistance to Mona Dinesen, Karin Durup, Elsebeth Lyng Pedersen, Trine Poulsen, Thomas Rebsdorf, Anne Stouby, and Mette Wu¨ rtz.
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