Safety evaluation of phytosterols in laying hens: Effects on laying performance, clinical blood parameters, and organ development S. R. Shi,* Y. R. Shen,* L. L. Chang,* C. J. Zhou,† Z. Bo,* Z. Y. Wang,† H. B. Tong,*1 and J. M. Zou*1 *Poultry Institute, Chinese Academy of Agricultural Sciences, Yangzhou, Jiangsu Province, 225125, China; and †College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu Province, 225009, China supplemented with 0, 20, 80, 400, and 800 mg/kg of phytosterols for 12 wk. Throughout the study, clinical observations and laying performance were measured. At the end of the study, birds were subjected to a full postmortem examination: blood samples were taken for clinical pathology, selected organs were weighed, and specified tissues were taken for subsequent histological examination. No treatment-related changes that were considered to be of toxicological significance were observed. Therefore, a nominal phytosterol concentration of 800 mg/kg was considered to be the no-observedadverse-effect level.
Key words: phytosterol, laying hen, safety evaluation, clinical blood parameter, organ development 2014 Poultry Science 93:545–549 http://dx.doi.org/10.3382/ps.2013-03562
INTRODUCTION
intestinal uptake of both dietary and endogenously produced (biliary) cholesterol (Ostlund, 2007). The physiologic effects of phytosterols have been studied extensively in recent years, although public health recommendations regarding phytosterols intake remain controversial (Elkin and Lorenz, 2009). Even so, phytosterols have become popular as animal dietary supplements to lower plasma or product (eggs, muscle) cholesterol (Liu et al., 2010). However, a few conventional subchronic and chronic safety studies have been published for phytosterols in livestock (EFSA, 2012). Therefore, as part of an extensive program of safety evaluation studies, we conducted an 84-d feeding study in laying hens to examine if the high-dose phytosterols could affect the safety of hens, including laying performance, clinical blood parameters, and organ development. The high dosage was chosen to provide significant exposure to the test material and the other dosages were set to provide data adequate for risk assessment purposes.
Phytosterols are natural components of human and animal diets that are structurally similar and functionally analogous to cholesterol (Ostlund, 2002). They are minor constituents of edible vegetable oils present in the unsaponifiable fraction and the most important dietary sources are vegetable oils and products based on vegetable oils, such as margarines. The most common phytosterols in nature are the 4-desmethylsterols, namely β-sitosterol, campesterol, and stigmasterol, which occur in the free form but also esterified to free fatty acids, sugar moieties, or phenolic acids. Phytosterol intake from normal food sources has been estimated to be about 200 mg/d in humans (Morton et al., 1995). Because they are a component of vegetable oil, intake among vegetarians is slightly higher. Phytosterols have been used as blood cholesterollowering agents for more than 50 yr (Kritchevsky and Chen, 2005). They are thought to act primarily in the intestinal lumen, where they compete with cholesterol for incorporation into absorptive micelles and thereby decrease the availability of cholesterol and inhibit the
MATERIALS AND METHODS Study Design
©2014 Poultry Science Association Inc. Received August 15, 2013. Accepted November 29, 2013. 1 Corresponding author: [email protected] or [email protected]
Three hundred sixty 21-wk-old Hy-Line Brown hens were randomly assigned to 5 groups with 6 replicates 545
Downloaded from http://ps.oxfordjournals.org/ at National Chung Hsing University Library on April 9, 2014
ABSTRACT Phytosterols are intended for use as a novel food ingredient with plasma cholesterol-lowering activity. Although phytosterols are naturally present in the normal diet, daily consumption is insufficient to ensure plasma cholesterol-lowering levels. Therefore, phytosterols may be added to the diets to achieve the desired cholesterol-lowering activity. A subchronic laying hen safety study was conducted to examine if high-dose phytosterols could affect the safety of hens. Three hundred sixty 21-wk-old Hy-Line Brown laying hens were randomly assigned to 5 groups with 6 replicates of 12 birds each; after 3 wk, birds were fed diets
546
Shi et al.
of 12 birds each (72 laying hens per group). All birds were acclimated to the basal diet for 3 wk. At 24 wk, the birds were fed diets supplemented with 0 (control), 20 (the minimum recommended available dose), 80 [the maximum recommended available dose (MRAD)], 400 (5-fold of MRAD), or 800 mg/kg (10-fold of MRAD) of phytosterols for 12 wk in the diets. Clinical observations, BW, and feed consumption were measured, and at the end of the scheduled period the animals were killed and subjected to a full postmortem examination. Cardiac blood samples were taken for clinical pathology, selected organs were weighed, and specified tissues were taken for subsequent histological examination.
This trial was carried out at the Poultry Institute, Chinese Academy of Agricultural Sciences. Phytosterols were derived from soybeans (Jiangsu Chunzhigu Biological Products Co. Ltd., Jiangsu, China) with 95% purity, containing 51.74% β-sitosterol, 20.69% campestanol, 16.52% stigmasterol, 2.30% sitostanol, 1.21% campesterol, and 2.54% other sterols. Two birds were housed in individual cages at dimensions of 50 × 40 cm, providing 1,000 cm2 per bird. Table 1 presents the composition of the experimental diets. Water and feed were provided ad libitum during the study. The photoperiod was set at 16L:8D throughout the study. Housing temperature and relative humidity were 20 ± 3°C and 65 to 75%, respectively. All animals handing protocols were approved by Animal Care and Use Committee of the Poultry Institute.
Sample Collection and Analytical Determination Observations. Cage-side observations, which included recording any changes in clinical condition or behavior, were made at least twice daily throughout the study. Laying Performance. Body weights of laying hens were determined at the beginning and the end of the study. Feed consumption was recorded on a replicate basis at weekly intervals. Daily egg production and egg weight were monitored during the trial. Egg production is expressed as average hen-day production, calculated from total eggs divided by the total number of hendays. Feed conversion was expressed as grams of feed consumed per grams of eggs produced. Clinical Blood Parameters. At the end of the 84-d feeding period, the wing vein blood samples for hematology and clinical chemistry were taken at necropsy from 60 birds (2 birds from each replicate, 12 birds per group); 1.5 mL of blood was collected in EDTA for hematology, whereas 2.5 mL of blood was collected for blood clinical chemistry. The following hematological parameters were measured on samples collected using a Sysmex XE2100 automated hematology analyzer (Sys-
Statistical Analysis All data were analyzed using SPSS (SPSS 16.0 for Windows, SPSS Inc., Chicago, IL). One-way ANOVA Table 1. Ingredients and nutrient levels of experimental diet1 Item (%, unless otherwise noted) Ingredient Corn Soybean meal (44%) Limestone dl-Met Dicalcium phosphate 50% Choline chloride NaCl Vitamin and trace mineral2 premix Nutrient levels (analyzed) ME (Mcal/kg) CP Ca Available P Met Lys 1Values
Content 62.7 26.3 8.5 0.1 1.0 0.1 0.3 1.0 2.65 16.61 3.5 0.35 0.35 0.85
are expressed on an air-dry basis. provided per kilogram of diet: vitamin A (retinyl palmitate), 7,715 IU; vitamin D3 (cholecalciferol), 2,755 international chick units; vitamin E (dl-α-tocopheryl acetate), 8.8 IU; vitamin K (menadione sodium bisulfate complex), 2.2 mg; vitamin B12 (cobalamin), 0.01 mg; menadione (menadione sodium bisulfate complex), 0.18 mg; riboflavin, 4.41 mg; pantothenic acid (d-calcium pantothenate), 5.51 mg; niacin, 19.8 mg; folic acid, 0.28 mg; pyridoxine (pyridoxine hydrochloride), 0.55 mg; manganese (manganese sulfate), 50 mg; iron (ferrous sulfate), 25 mg; copper (copper sulfate), 2.5 mg; zinc (zinc sulfate), 50 mg; iodine (calcium iodate), 1.0 mg; selenium (sodium selenite), 0.15 mg. 2Premix
Downloaded from http://ps.oxfordjournals.org/ at National Chung Hsing University Library on April 9, 2014
Birds, Diet, and Management
mex Corporation, Kobe, Japan): white blood cells, red blood cells, hemoglobin, hematocrit, mean hematocrit, mean hemoglobin, mean hemoglobin concentration, red cell distribution width, and coefficient variation of red cell distribution width. The following clinical chemistry measurements were made on the serum collected from blood samples centrifuged at 3,500 × g for 10 min under 4°C: total bilirubin, total protein, albumin, glutamic oxalo acetic transaminase, alkaline phosphatase, gamma-glutamyl transpeptidase, lactate dehydrogenase, total cholesterol, triglycerides, low-density lipoprotein cholesterin, glucose, and creatinine. All parameters were measured using a biochemical analyzer (UniCel DxC 800 Synchron, Beckman Coulter, Brea, CA). These parameters were included to cover a wide range of possible toxicities, to include possible effects on electrolyte balance, metabolism (carbohydrates, protein, fat, and minerals), and damage to the major organ systems. Where the target organ is unknown it is necessary to obtain the maximum amount of data to aid interpretation. Necropsy. All blood was collected from birds (n = 60) killed by exsanguination and subjected to a full postmortem examination. Descriptions of all macroscopic abnormalities of birds killed and died during the experiment were recorded. At necropsy, the liver, heart, spleen, abdominal fat, ovary, and oviduct were weighed (paired organs were weighed together).
547
PHYTOSTEROLS IN LAYING HENS Table 2. Laying performance from a phytosterol-safety evaluating study in the hens1 Phytosterol (mg/kg of diet) Item Egg production (%) Feed intake (g/hen per day) Egg mass (g/hen per day) Feed conversion (g of feed/g of egg) 1Results
P-value
0
20
80
400
800
SEM
Phytosterol
Linear
Quadratic
93.16 132.86 55.44 2.40
92.44 132.66 55.27 2.40
91.78 132.57 54.90 2.42
91.77 132.34 54.45 2.43
93.02 132.09 55.54 2.38
0.583 0.213 0.348 0.015
0.920 0.837 0.876 0.829
0.831 0.249 0.815 0.916
0.382 0.896 0.432 0.399
are means with n = 6 per treatment, the same as the tables below.
RESULTS Mortality and Observations No treatment-related adverse clinical signs were observed. No mortality occurred during the study.
Necropsy After 12 wk of treatment with phytosterols, no statistically significant changes were observed in organ development in comparison to the concurrent control animals except for liver and spleen index (Table 5). For birds that survived to termination, no macroscopic findings were noted at necropsy that could be attributed to administration of phytosterols. Similarly, no histological findings were observed that could be attributed to treatment with phytosterols.
Laying Performance
DISCUSSION
The mean laying performance (egg production, feed intake, egg mass, and feed conversion) is shown in Table 2. No statistically significant differences were found among all laying performance parameters observed in either the treated group and the control group.
Clinical Blood Parameters No statistically significant changes were observed in hematology (P > 0.05; Table 3). For clinical chemistry, no parameters were significantly affected by dietary phytosterol supplement (P > 0.05; Table 4) except glucose. Serum glucose increased linearly with increasing dietary phytosterol supplement (P = 0.010). However, in the absence of any significant organ changes or macroscopic histopathological findings in these birds, the magnitude of this clinical chemistry change was such that they were considered to be of no toxicological significance.
Diets containing phytosterols were tolerated well and did not produce any general organ or systemic toxicity when fed to laying hens at doses as high as 800 mg/ kg of the diet for a period of 84 d. No macroscopic observations were noted at necropsy and no histological changes considered to be related to treatment. In addition, these doses did not produce any effects on performance, hematology, or cause any treatment-related clinical signs. Some minor changes were observed in clinical chemistry parameters, but these changes were small and considered to be of no toxicological significance. No significant changes were found in plasma cholesterol levels, as one may have expected from the consumption of phytosterols, which was consistent with the results of Liu et al. (2010) in laying hens. The current data set lies in the use of kinetic measures of cholesterol metabolism (Jones et al., 2000; Wang et al., 2004) to ascertain the physiological mechanisms leading to altered lipid metabolism. However, the poten-
Table 3. Hematology from a phytosterol-safety evaluating study in the hens Phytosterol (mg/kg of diet) Item White blood cells (109/L) Red blood cells (109/L)
Hemoglobin (g/L) Hematocrit (%) Mean hematocrit (fL) Mean hemoglobin (pg) Mean hemoglobin concentration (g/L) Red cell distribution width (%) Coefficient variation of red cell distribution width (%)
P-value
0
20
80
400
800
SEM
Phytosterol
Linear
Quadratic
345.21 2.56 88.58 31.13 119.60 34.63 284.33 39.28 8.93
358.44 2.58 86.83 31.08 120.78 33.73 279.25 39.11 8.99
339.00 2.51 86.08 29.98 122.03 34.28 280.83 39.30 8.95
359.70 2.64 90.50 31.92 120.94 34.32 283.67 40.14 9.38
354.55 2.60 88.33 31.14 119.89 34.01 283.67 38.44 8.89
5.490 0.027 0.821 0.263 0.504 0.202 0.749 0.254 0.059
0.740 0.644 0.507 0.280 0.615 0.715 0.134 0.389 0.019
0.625 0.470 0.594 0.188 0.457 0.655 0.542 0.277 0.011
0.944 0.730 0.538 0.111 0.882 0.699 0.064 0.250 0.010
Downloaded from http://ps.oxfordjournals.org/ at National Chung Hsing University Library on April 9, 2014
followed by a Duncan’s multiple comparison test was used to separate different means among treatments. Data were assumed to be statistically significant when P < 0.05.
548
Shi et al.
Table 4. Blood clinical chemistry from a phytosterol-safety evaluating study in the hens Phytosterol (mg/kg of diet) Item
20
80
400
800
SEM
Phytosterol
Linear
Quadratic
1.43 54.43 26.48 188.08
1.29 57.67 24.96 184.17
1.47 56.59 24.93 199.00
1.39 53.53 26.10 212.17
1.39 52.23 26.90 199.92
0.035 1.206 0.341 3.580
0.583 0.635 0.242 0.085
0.939 0.339 0.402 0.268
0.940 0.294 0.045 0.175
215.50 26.00
240.50 38.50
206.92 30.67
231.42 37.80
241.40 36.17
12.545 1.648
0.896 0.060
0.827 0.098
0.665 0.071
1,640.58 3.05 12.33 0.11
1,708.30 2.89 10.96 0.11
1,629.92 2.66 10.21 0.09
1,726.50 2.38 8.61 0.08
1,512.08 2.55 9.50 0.08
44.055 0.108 0.543 0.008
0.592 0.328 0.229 0.553
0.523 0.810 0.782 0.920
0.550 0.784 0.785 0.800
8.03 12.03
7.87 13.10
7.51 13.35
9.31 13.09
9.02 15.54
0.207 0.562
0.012 0.398
0.010 0.089
0.200 0.637
tial cholesterol-lowering action of plant sterols in avian species has not been thoroughly described. Cholesterol balance in egg-laying hens differs greatly from that in omnivorous mammals. Under current commercial conditions in China, diets of laying hens contain only minor amounts of products of animal origin; therefore, exogenous cholesterol supply is limited. Hens usually meet their body needs for cholesterol entirely by de novo synthesis (Naber, 1983). Phytosterols are poorly absorbed from the intestine in both human and experimental animals, and absorption typically varies between 4 and 5% for sitosterol and stigmasterol and between 9 and 10% for campesterol (Heinemann et al., 1993). These studies have generally been conducted at low levels of intake, but the absorption of sitosterol may be considerably less at higher dietary doses (2–7 g/d; Salen et al., 1970). An exception to the typically low level of absorption of phytosterols is seen in the very rare human disease known as phytosterolaemia (a metabolic disease that causes a defect in cholesterol metabolism), which is characterized by an increased absorption of phytosterols (in addition to cholesterol) and a decreased excretion into the bile by the liver (Lütjohann and von Bergmann, 1997). The major objective of the current study was to examine the clinical and safety parameters during 84 d of consumption of phytosterols in healthy laying hens. Consumption of phytosterols appeared to have no ad-
verse side effects, defined as reported adverse events or undesirable changes in clinical chemical parameters, hematological parameters, and urinalysis. The absence of side effects is in agreement with the observations in earlier clinical and safety studies (Miettinen et al., 1995; Baker et al., 1999; Hallikainen et al., 1999, 2000a,b; Hepburn et al., 1999; Waalkens-Berendsen et al., 1999; Weststrate et al., 1999; Kim et al., 2002; Wolfreys and Hepburn 2002). In conclusion, our study findings revealed that longer-term use of high-dose phytosterols could not induce any treatment-related changes that were considered to be of toxicological significance. Therefore, a nominal phytosterol concentration of 800 mg/kg was considered to be the no-observed-adverse-effect level following daily administration to laying hens for 84 d.
ACKNOWLEDGMENTS This work was financially supported by the Scientific and Technical Support Program of the ‘‘Twelfth Five-Year Plan,’’ Ministry of Science and Technology, Beijing, PR China (2012BAD39B04); Special Fund for Agro-scientific Research in the Public Interest of Ministry of Agriculture, Beijing, PR China (201003011); Feed Quality and Safety Supervision Project of Ministry of Agriculture (2012), Beijing, PR China.
Table 5. Organs development from a phytosterol-safety evaluating study in the hens Phytosterol (mg/kg of diet) Item (%) Liver index Heart index Spleen index Abdominal fat index Oviduct index Ovary index
Probability
0
20
80
400
800
SEM
Phytosterol
Linear
Quadratic
1.46 0.38 0.11 3.10 3.51 2.35
1.50 0.41 0.11 2.87 3.42 2.69
1.56 0.36 0.11 2.90 3.50 2.31
1.74 0.38 0.10 2.91 3.54 2.30
1.57 0.37 0.13 2.95 3.48 2.36
0.031 0.006 0.003 0.116 0.046 0.069
0.032 0.083 0.036 0.978 0.961 0.363
0.027 0.101 0.036 0.949 0.900 0.308
0.205 0.051 0.286 0.956 0.751 0.177
Downloaded from http://ps.oxfordjournals.org/ at National Chung Hsing University Library on April 9, 2014
Total bilirubin (μmol/L) Total protein (g/L) Albumin (g/L) Glutamic oxalo acetic transaminase (U/L) Alkaline phosphatase (U/L) Gamma-glutamyl transpeptidase (U/L) Lactate dehydrogenase (U/L) Total cholesterol (mmol/L) Triglycerides (mmol/L) Low-density lipoprotein cholesterin (mmol/L) Glucose (mmol/L) Creatinine (μmol/L)
0
P-value
PHYTOSTEROLS IN LAYING HENS
REFERENCES
Liu, X., H. L. Zhao, S. Thiessen, J. D. House, and P. J. H. Jones. 2010. Effect of plant sterol-enriched diets on plasma and egg yolk cholesterol concentrations and cholesterol metabolism in laying hens. Poult. Sci. 89:270–275. Lütjohann, D., and K. von Bergmann. 1997. Phytosterolaemia: Diagnosis, characterisation and therapeutical approaches. Ann. Med. 29:181–184. Miettinen, T. A., P. Puska, H. Gylling, H. Vanhanen, and E. Vartiainen. 1995. Reduction of serum cholesterol with sitostanolester margarine in a mildly hypercholesterolemic population. N. Engl. J. Med. 333:1308–1312. Morton, G. M., S. M. Lee, D. H. Buss, and P. Lawrance. 1995. Intakes and major dietary sources of cholesterol and phytosterols in the British diet. J. Hum. Nutr. Diet. 8:429–440. Naber, E. C. 1983. Nutrient and drug effects on cholesterol metabolism in the laying hen. Fed. Proc. 42:2486–2493. Ostlund, R. E. 2002. Phytosterols in human nutrition. Annu. Rev. Nutr. 22:533–549. Ostlund, R. E. 2007. Phytosterols, cholesterol absorption and healthy diets. Lipids 42:41–45. Salen, G., E. H. Jr. Ahrens, and S. M. Grundy. 1970. Metabolism of beta-sitosterol in man. J. Clin. Invest. 49:952–967. Waalkens-Berendsen, D. H., A. P. M. Wolterbeek, M. V. W. Wijnands, M. Richold, and P. A. Hepburn. 1999. Safety evaluation of phytosterolesters, part 3: Two-generation reproduction study in rats with phytosterol-esters-a novel functional food. Food Chem. Toxicol. 37:683–696. Wang, Y., C. A. Vanstone, W. D. Parsons, and P. J. H. Jones. 2004. Validation of a single-isotope-labeled cholesterol tracer approach for measuring human cholesterol absorption. Lipids 39:87–91. Weststrate, J. A., R. Ayesh, C. Bauer-Plank, and P. N. Drewitt. 1999. Safety evaluation of phytosterol-esters, part 4. Faecal concentrations of bile acids and neutral sterols in healthy normolpidaemic volunteers consuming a controlled diet either with or without a phytosterol ester-enriched margarine. Food Chem. Toxicol. 37:1063–1071. Wolfreys, A. M., and P. A. Hepburn. 2002. Safety evaluation of phytosterol esters, part 7. Assessment of mutagenic activity of phytosterols, phytosterol-esters and the cholesterol derivative, 4-cholesten-3-one. Food Chem. Toxicol. 40:461–470.
Downloaded from http://ps.oxfordjournals.org/ at National Chung Hsing University Library on April 9, 2014
Baker, V. A., P. A. Hepburn, S. J. Kennedy, P. A. Jones, L. J. Lea, J. P. Sumpter, and J. Ashby. 1999. Safety evaluation of phytosterol-esters. Part 1. Assessment of oestrogenicity using a combination of in vivo and in vitro assays. Food Chem. Toxicol. 37:13–22. EFSA (European Food Safety Authority). 2012. Scientific opinion on the safety of stigmasterol-rich plant sterols as food additive. EFSA Journal 10:2659. 10.2903/j.efsa.2012.2659. Elkin, R. G., and E. S. Lorenz. 2009. Feeding laying hens a bioavailable soy sterol mixture fails to enrich their eggs with phytosterols or elicit egg yolk compositional changes. Poult. Sci. 88:152–158. Hallikainen, M. A., E. S. Sarkkinen, H. Gylling, A. T. Erkkila, and M. I. Uusitupa. 2000b. Comparison of the effects of plant sterol ester and plant stanol ester-enriched margarines in lowering serum cholesterol concentrations in hypercholesterolaemic subjects on a low-fat diet. Eur. J. Clin. Nutr. 54:715–725. Hallikainen, M. A., E. S. Sarkkinen, and M. I. Uusitupa. 1999. Effects of low fat stanol ester enriched margarines on concentrations of serum carotenoids in subjects with elevated serum cholesterol concentrations. Eur. J. Clin. Nutr. 53:966–969. Hallikainen, M. A., E. S. Sarkkinen, and M. I. Uusitupa. 2000a. Plant stanol esters affect serum cholesterol concentrations of hypercholesterolemic men and women in a dose-dependent manner. J. Nutr. 130:767–776. Heinemann, T., G. Axtmann, and K. von Bergmann. 1993. Comparison of intestinal absorption of cholesterol with different plant sterols in man. Eur. J. Clin. Invest. 23:827–831. Hepburn, P. A., S. A. Horner, and M. Smith. 1999. Safety evaluation of phytosterol-esters. Part 2. Subchronic 90-day oral toxicity study on phytosterol-esters-a novel functional food. Food Chem. Toxicol. 37:521–532. Jones, P. J., M. Raeini-Sarjaz, F. Y. Ntanios, C. A. Vanstone, J. Y. Feng, and W. E. Parsons. 2000. Modulation of plasma lipid levels and cholesterol kinetics by phytosterol versus phytostanol esters. J. Lipid Res. 41:697–705. Kim, J. C., B. H. Kang, C. C. Shin, Y. B. Kim, H. S. Lee, C. Y. Kim, J. Han, K. S. Kim, D. W. Chung, and M. K. Chung. 2002. Subchronic toxicity of plant sterol esters administered by gavage to Sprague-Dawley rats. Food Chem. Toxicol. 40:1569–1580. Kritchevsky, D., and S. C. Chen. 2005. Phytosterols—Health benefits and potential concerns: A review. Nutr. Res. 25:413–428.
549
Key words: phytosterol, laying hen, safety evaluation, clinical blood parameter, organ development 2014 Poultry Science 93:545–549 http://dx.doi.org/10.3382/ps.2013-03562
INTRODUCTION
intestinal uptake of both dietary and endogenously produced (biliary) cholesterol (Ostlund, 2007). The physiologic effects of phytosterols have been studied extensively in recent years, although public health recommendations regarding phytosterols intake remain controversial (Elkin and Lorenz, 2009). Even so, phytosterols have become popular as animal dietary supplements to lower plasma or product (eggs, muscle) cholesterol (Liu et al., 2010). However, a few conventional subchronic and chronic safety studies have been published for phytosterols in livestock (EFSA, 2012). Therefore, as part of an extensive program of safety evaluation studies, we conducted an 84-d feeding study in laying hens to examine if the high-dose phytosterols could affect the safety of hens, including laying performance, clinical blood parameters, and organ development. The high dosage was chosen to provide significant exposure to the test material and the other dosages were set to provide data adequate for risk assessment purposes.
Phytosterols are natural components of human and animal diets that are structurally similar and functionally analogous to cholesterol (Ostlund, 2002). They are minor constituents of edible vegetable oils present in the unsaponifiable fraction and the most important dietary sources are vegetable oils and products based on vegetable oils, such as margarines. The most common phytosterols in nature are the 4-desmethylsterols, namely β-sitosterol, campesterol, and stigmasterol, which occur in the free form but also esterified to free fatty acids, sugar moieties, or phenolic acids. Phytosterol intake from normal food sources has been estimated to be about 200 mg/d in humans (Morton et al., 1995). Because they are a component of vegetable oil, intake among vegetarians is slightly higher. Phytosterols have been used as blood cholesterollowering agents for more than 50 yr (Kritchevsky and Chen, 2005). They are thought to act primarily in the intestinal lumen, where they compete with cholesterol for incorporation into absorptive micelles and thereby decrease the availability of cholesterol and inhibit the
MATERIALS AND METHODS Study Design
©2014 Poultry Science Association Inc. Received August 15, 2013. Accepted November 29, 2013. 1 Corresponding author: [email protected] or [email protected]
Three hundred sixty 21-wk-old Hy-Line Brown hens were randomly assigned to 5 groups with 6 replicates 545
Downloaded from http://ps.oxfordjournals.org/ at National Chung Hsing University Library on April 9, 2014
ABSTRACT Phytosterols are intended for use as a novel food ingredient with plasma cholesterol-lowering activity. Although phytosterols are naturally present in the normal diet, daily consumption is insufficient to ensure plasma cholesterol-lowering levels. Therefore, phytosterols may be added to the diets to achieve the desired cholesterol-lowering activity. A subchronic laying hen safety study was conducted to examine if high-dose phytosterols could affect the safety of hens. Three hundred sixty 21-wk-old Hy-Line Brown laying hens were randomly assigned to 5 groups with 6 replicates of 12 birds each; after 3 wk, birds were fed diets
546
Shi et al.
of 12 birds each (72 laying hens per group). All birds were acclimated to the basal diet for 3 wk. At 24 wk, the birds were fed diets supplemented with 0 (control), 20 (the minimum recommended available dose), 80 [the maximum recommended available dose (MRAD)], 400 (5-fold of MRAD), or 800 mg/kg (10-fold of MRAD) of phytosterols for 12 wk in the diets. Clinical observations, BW, and feed consumption were measured, and at the end of the scheduled period the animals were killed and subjected to a full postmortem examination. Cardiac blood samples were taken for clinical pathology, selected organs were weighed, and specified tissues were taken for subsequent histological examination.
This trial was carried out at the Poultry Institute, Chinese Academy of Agricultural Sciences. Phytosterols were derived from soybeans (Jiangsu Chunzhigu Biological Products Co. Ltd., Jiangsu, China) with 95% purity, containing 51.74% β-sitosterol, 20.69% campestanol, 16.52% stigmasterol, 2.30% sitostanol, 1.21% campesterol, and 2.54% other sterols. Two birds were housed in individual cages at dimensions of 50 × 40 cm, providing 1,000 cm2 per bird. Table 1 presents the composition of the experimental diets. Water and feed were provided ad libitum during the study. The photoperiod was set at 16L:8D throughout the study. Housing temperature and relative humidity were 20 ± 3°C and 65 to 75%, respectively. All animals handing protocols were approved by Animal Care and Use Committee of the Poultry Institute.
Sample Collection and Analytical Determination Observations. Cage-side observations, which included recording any changes in clinical condition or behavior, were made at least twice daily throughout the study. Laying Performance. Body weights of laying hens were determined at the beginning and the end of the study. Feed consumption was recorded on a replicate basis at weekly intervals. Daily egg production and egg weight were monitored during the trial. Egg production is expressed as average hen-day production, calculated from total eggs divided by the total number of hendays. Feed conversion was expressed as grams of feed consumed per grams of eggs produced. Clinical Blood Parameters. At the end of the 84-d feeding period, the wing vein blood samples for hematology and clinical chemistry were taken at necropsy from 60 birds (2 birds from each replicate, 12 birds per group); 1.5 mL of blood was collected in EDTA for hematology, whereas 2.5 mL of blood was collected for blood clinical chemistry. The following hematological parameters were measured on samples collected using a Sysmex XE2100 automated hematology analyzer (Sys-
Statistical Analysis All data were analyzed using SPSS (SPSS 16.0 for Windows, SPSS Inc., Chicago, IL). One-way ANOVA Table 1. Ingredients and nutrient levels of experimental diet1 Item (%, unless otherwise noted) Ingredient Corn Soybean meal (44%) Limestone dl-Met Dicalcium phosphate 50% Choline chloride NaCl Vitamin and trace mineral2 premix Nutrient levels (analyzed) ME (Mcal/kg) CP Ca Available P Met Lys 1Values
Content 62.7 26.3 8.5 0.1 1.0 0.1 0.3 1.0 2.65 16.61 3.5 0.35 0.35 0.85
are expressed on an air-dry basis. provided per kilogram of diet: vitamin A (retinyl palmitate), 7,715 IU; vitamin D3 (cholecalciferol), 2,755 international chick units; vitamin E (dl-α-tocopheryl acetate), 8.8 IU; vitamin K (menadione sodium bisulfate complex), 2.2 mg; vitamin B12 (cobalamin), 0.01 mg; menadione (menadione sodium bisulfate complex), 0.18 mg; riboflavin, 4.41 mg; pantothenic acid (d-calcium pantothenate), 5.51 mg; niacin, 19.8 mg; folic acid, 0.28 mg; pyridoxine (pyridoxine hydrochloride), 0.55 mg; manganese (manganese sulfate), 50 mg; iron (ferrous sulfate), 25 mg; copper (copper sulfate), 2.5 mg; zinc (zinc sulfate), 50 mg; iodine (calcium iodate), 1.0 mg; selenium (sodium selenite), 0.15 mg. 2Premix
Downloaded from http://ps.oxfordjournals.org/ at National Chung Hsing University Library on April 9, 2014
Birds, Diet, and Management
mex Corporation, Kobe, Japan): white blood cells, red blood cells, hemoglobin, hematocrit, mean hematocrit, mean hemoglobin, mean hemoglobin concentration, red cell distribution width, and coefficient variation of red cell distribution width. The following clinical chemistry measurements were made on the serum collected from blood samples centrifuged at 3,500 × g for 10 min under 4°C: total bilirubin, total protein, albumin, glutamic oxalo acetic transaminase, alkaline phosphatase, gamma-glutamyl transpeptidase, lactate dehydrogenase, total cholesterol, triglycerides, low-density lipoprotein cholesterin, glucose, and creatinine. All parameters were measured using a biochemical analyzer (UniCel DxC 800 Synchron, Beckman Coulter, Brea, CA). These parameters were included to cover a wide range of possible toxicities, to include possible effects on electrolyte balance, metabolism (carbohydrates, protein, fat, and minerals), and damage to the major organ systems. Where the target organ is unknown it is necessary to obtain the maximum amount of data to aid interpretation. Necropsy. All blood was collected from birds (n = 60) killed by exsanguination and subjected to a full postmortem examination. Descriptions of all macroscopic abnormalities of birds killed and died during the experiment were recorded. At necropsy, the liver, heart, spleen, abdominal fat, ovary, and oviduct were weighed (paired organs were weighed together).
547
PHYTOSTEROLS IN LAYING HENS Table 2. Laying performance from a phytosterol-safety evaluating study in the hens1 Phytosterol (mg/kg of diet) Item Egg production (%) Feed intake (g/hen per day) Egg mass (g/hen per day) Feed conversion (g of feed/g of egg) 1Results
P-value
0
20
80
400
800
SEM
Phytosterol
Linear
Quadratic
93.16 132.86 55.44 2.40
92.44 132.66 55.27 2.40
91.78 132.57 54.90 2.42
91.77 132.34 54.45 2.43
93.02 132.09 55.54 2.38
0.583 0.213 0.348 0.015
0.920 0.837 0.876 0.829
0.831 0.249 0.815 0.916
0.382 0.896 0.432 0.399
are means with n = 6 per treatment, the same as the tables below.
RESULTS Mortality and Observations No treatment-related adverse clinical signs were observed. No mortality occurred during the study.
Necropsy After 12 wk of treatment with phytosterols, no statistically significant changes were observed in organ development in comparison to the concurrent control animals except for liver and spleen index (Table 5). For birds that survived to termination, no macroscopic findings were noted at necropsy that could be attributed to administration of phytosterols. Similarly, no histological findings were observed that could be attributed to treatment with phytosterols.
Laying Performance
DISCUSSION
The mean laying performance (egg production, feed intake, egg mass, and feed conversion) is shown in Table 2. No statistically significant differences were found among all laying performance parameters observed in either the treated group and the control group.
Clinical Blood Parameters No statistically significant changes were observed in hematology (P > 0.05; Table 3). For clinical chemistry, no parameters were significantly affected by dietary phytosterol supplement (P > 0.05; Table 4) except glucose. Serum glucose increased linearly with increasing dietary phytosterol supplement (P = 0.010). However, in the absence of any significant organ changes or macroscopic histopathological findings in these birds, the magnitude of this clinical chemistry change was such that they were considered to be of no toxicological significance.
Diets containing phytosterols were tolerated well and did not produce any general organ or systemic toxicity when fed to laying hens at doses as high as 800 mg/ kg of the diet for a period of 84 d. No macroscopic observations were noted at necropsy and no histological changes considered to be related to treatment. In addition, these doses did not produce any effects on performance, hematology, or cause any treatment-related clinical signs. Some minor changes were observed in clinical chemistry parameters, but these changes were small and considered to be of no toxicological significance. No significant changes were found in plasma cholesterol levels, as one may have expected from the consumption of phytosterols, which was consistent with the results of Liu et al. (2010) in laying hens. The current data set lies in the use of kinetic measures of cholesterol metabolism (Jones et al., 2000; Wang et al., 2004) to ascertain the physiological mechanisms leading to altered lipid metabolism. However, the poten-
Table 3. Hematology from a phytosterol-safety evaluating study in the hens Phytosterol (mg/kg of diet) Item White blood cells (109/L) Red blood cells (109/L)
Hemoglobin (g/L) Hematocrit (%) Mean hematocrit (fL) Mean hemoglobin (pg) Mean hemoglobin concentration (g/L) Red cell distribution width (%) Coefficient variation of red cell distribution width (%)
P-value
0
20
80
400
800
SEM
Phytosterol
Linear
Quadratic
345.21 2.56 88.58 31.13 119.60 34.63 284.33 39.28 8.93
358.44 2.58 86.83 31.08 120.78 33.73 279.25 39.11 8.99
339.00 2.51 86.08 29.98 122.03 34.28 280.83 39.30 8.95
359.70 2.64 90.50 31.92 120.94 34.32 283.67 40.14 9.38
354.55 2.60 88.33 31.14 119.89 34.01 283.67 38.44 8.89
5.490 0.027 0.821 0.263 0.504 0.202 0.749 0.254 0.059
0.740 0.644 0.507 0.280 0.615 0.715 0.134 0.389 0.019
0.625 0.470 0.594 0.188 0.457 0.655 0.542 0.277 0.011
0.944 0.730 0.538 0.111 0.882 0.699 0.064 0.250 0.010
Downloaded from http://ps.oxfordjournals.org/ at National Chung Hsing University Library on April 9, 2014
followed by a Duncan’s multiple comparison test was used to separate different means among treatments. Data were assumed to be statistically significant when P < 0.05.
548
Shi et al.
Table 4. Blood clinical chemistry from a phytosterol-safety evaluating study in the hens Phytosterol (mg/kg of diet) Item
20
80
400
800
SEM
Phytosterol
Linear
Quadratic
1.43 54.43 26.48 188.08
1.29 57.67 24.96 184.17
1.47 56.59 24.93 199.00
1.39 53.53 26.10 212.17
1.39 52.23 26.90 199.92
0.035 1.206 0.341 3.580
0.583 0.635 0.242 0.085
0.939 0.339 0.402 0.268
0.940 0.294 0.045 0.175
215.50 26.00
240.50 38.50
206.92 30.67
231.42 37.80
241.40 36.17
12.545 1.648
0.896 0.060
0.827 0.098
0.665 0.071
1,640.58 3.05 12.33 0.11
1,708.30 2.89 10.96 0.11
1,629.92 2.66 10.21 0.09
1,726.50 2.38 8.61 0.08
1,512.08 2.55 9.50 0.08
44.055 0.108 0.543 0.008
0.592 0.328 0.229 0.553
0.523 0.810 0.782 0.920
0.550 0.784 0.785 0.800
8.03 12.03
7.87 13.10
7.51 13.35
9.31 13.09
9.02 15.54
0.207 0.562
0.012 0.398
0.010 0.089
0.200 0.637
tial cholesterol-lowering action of plant sterols in avian species has not been thoroughly described. Cholesterol balance in egg-laying hens differs greatly from that in omnivorous mammals. Under current commercial conditions in China, diets of laying hens contain only minor amounts of products of animal origin; therefore, exogenous cholesterol supply is limited. Hens usually meet their body needs for cholesterol entirely by de novo synthesis (Naber, 1983). Phytosterols are poorly absorbed from the intestine in both human and experimental animals, and absorption typically varies between 4 and 5% for sitosterol and stigmasterol and between 9 and 10% for campesterol (Heinemann et al., 1993). These studies have generally been conducted at low levels of intake, but the absorption of sitosterol may be considerably less at higher dietary doses (2–7 g/d; Salen et al., 1970). An exception to the typically low level of absorption of phytosterols is seen in the very rare human disease known as phytosterolaemia (a metabolic disease that causes a defect in cholesterol metabolism), which is characterized by an increased absorption of phytosterols (in addition to cholesterol) and a decreased excretion into the bile by the liver (Lütjohann and von Bergmann, 1997). The major objective of the current study was to examine the clinical and safety parameters during 84 d of consumption of phytosterols in healthy laying hens. Consumption of phytosterols appeared to have no ad-
verse side effects, defined as reported adverse events or undesirable changes in clinical chemical parameters, hematological parameters, and urinalysis. The absence of side effects is in agreement with the observations in earlier clinical and safety studies (Miettinen et al., 1995; Baker et al., 1999; Hallikainen et al., 1999, 2000a,b; Hepburn et al., 1999; Waalkens-Berendsen et al., 1999; Weststrate et al., 1999; Kim et al., 2002; Wolfreys and Hepburn 2002). In conclusion, our study findings revealed that longer-term use of high-dose phytosterols could not induce any treatment-related changes that were considered to be of toxicological significance. Therefore, a nominal phytosterol concentration of 800 mg/kg was considered to be the no-observed-adverse-effect level following daily administration to laying hens for 84 d.
ACKNOWLEDGMENTS This work was financially supported by the Scientific and Technical Support Program of the ‘‘Twelfth Five-Year Plan,’’ Ministry of Science and Technology, Beijing, PR China (2012BAD39B04); Special Fund for Agro-scientific Research in the Public Interest of Ministry of Agriculture, Beijing, PR China (201003011); Feed Quality and Safety Supervision Project of Ministry of Agriculture (2012), Beijing, PR China.
Table 5. Organs development from a phytosterol-safety evaluating study in the hens Phytosterol (mg/kg of diet) Item (%) Liver index Heart index Spleen index Abdominal fat index Oviduct index Ovary index
Probability
0
20
80
400
800
SEM
Phytosterol
Linear
Quadratic
1.46 0.38 0.11 3.10 3.51 2.35
1.50 0.41 0.11 2.87 3.42 2.69
1.56 0.36 0.11 2.90 3.50 2.31
1.74 0.38 0.10 2.91 3.54 2.30
1.57 0.37 0.13 2.95 3.48 2.36
0.031 0.006 0.003 0.116 0.046 0.069
0.032 0.083 0.036 0.978 0.961 0.363
0.027 0.101 0.036 0.949 0.900 0.308
0.205 0.051 0.286 0.956 0.751 0.177
Downloaded from http://ps.oxfordjournals.org/ at National Chung Hsing University Library on April 9, 2014
Total bilirubin (μmol/L) Total protein (g/L) Albumin (g/L) Glutamic oxalo acetic transaminase (U/L) Alkaline phosphatase (U/L) Gamma-glutamyl transpeptidase (U/L) Lactate dehydrogenase (U/L) Total cholesterol (mmol/L) Triglycerides (mmol/L) Low-density lipoprotein cholesterin (mmol/L) Glucose (mmol/L) Creatinine (μmol/L)
0
P-value
PHYTOSTEROLS IN LAYING HENS
REFERENCES
Liu, X., H. L. Zhao, S. Thiessen, J. D. House, and P. J. H. Jones. 2010. Effect of plant sterol-enriched diets on plasma and egg yolk cholesterol concentrations and cholesterol metabolism in laying hens. Poult. Sci. 89:270–275. Lütjohann, D., and K. von Bergmann. 1997. Phytosterolaemia: Diagnosis, characterisation and therapeutical approaches. Ann. Med. 29:181–184. Miettinen, T. A., P. Puska, H. Gylling, H. Vanhanen, and E. Vartiainen. 1995. Reduction of serum cholesterol with sitostanolester margarine in a mildly hypercholesterolemic population. N. Engl. J. Med. 333:1308–1312. Morton, G. M., S. M. Lee, D. H. Buss, and P. Lawrance. 1995. Intakes and major dietary sources of cholesterol and phytosterols in the British diet. J. Hum. Nutr. Diet. 8:429–440. Naber, E. C. 1983. Nutrient and drug effects on cholesterol metabolism in the laying hen. Fed. Proc. 42:2486–2493. Ostlund, R. E. 2002. Phytosterols in human nutrition. Annu. Rev. Nutr. 22:533–549. Ostlund, R. E. 2007. Phytosterols, cholesterol absorption and healthy diets. Lipids 42:41–45. Salen, G., E. H. Jr. Ahrens, and S. M. Grundy. 1970. Metabolism of beta-sitosterol in man. J. Clin. Invest. 49:952–967. Waalkens-Berendsen, D. H., A. P. M. Wolterbeek, M. V. W. Wijnands, M. Richold, and P. A. Hepburn. 1999. Safety evaluation of phytosterolesters, part 3: Two-generation reproduction study in rats with phytosterol-esters-a novel functional food. Food Chem. Toxicol. 37:683–696. Wang, Y., C. A. Vanstone, W. D. Parsons, and P. J. H. Jones. 2004. Validation of a single-isotope-labeled cholesterol tracer approach for measuring human cholesterol absorption. Lipids 39:87–91. Weststrate, J. A., R. Ayesh, C. Bauer-Plank, and P. N. Drewitt. 1999. Safety evaluation of phytosterol-esters, part 4. Faecal concentrations of bile acids and neutral sterols in healthy normolpidaemic volunteers consuming a controlled diet either with or without a phytosterol ester-enriched margarine. Food Chem. Toxicol. 37:1063–1071. Wolfreys, A. M., and P. A. Hepburn. 2002. Safety evaluation of phytosterol esters, part 7. Assessment of mutagenic activity of phytosterols, phytosterol-esters and the cholesterol derivative, 4-cholesten-3-one. Food Chem. Toxicol. 40:461–470.
Downloaded from http://ps.oxfordjournals.org/ at National Chung Hsing University Library on April 9, 2014
Baker, V. A., P. A. Hepburn, S. J. Kennedy, P. A. Jones, L. J. Lea, J. P. Sumpter, and J. Ashby. 1999. Safety evaluation of phytosterol-esters. Part 1. Assessment of oestrogenicity using a combination of in vivo and in vitro assays. Food Chem. Toxicol. 37:13–22. EFSA (European Food Safety Authority). 2012. Scientific opinion on the safety of stigmasterol-rich plant sterols as food additive. EFSA Journal 10:2659. 10.2903/j.efsa.2012.2659. Elkin, R. G., and E. S. Lorenz. 2009. Feeding laying hens a bioavailable soy sterol mixture fails to enrich their eggs with phytosterols or elicit egg yolk compositional changes. Poult. Sci. 88:152–158. Hallikainen, M. A., E. S. Sarkkinen, H. Gylling, A. T. Erkkila, and M. I. Uusitupa. 2000b. Comparison of the effects of plant sterol ester and plant stanol ester-enriched margarines in lowering serum cholesterol concentrations in hypercholesterolaemic subjects on a low-fat diet. Eur. J. Clin. Nutr. 54:715–725. Hallikainen, M. A., E. S. Sarkkinen, and M. I. Uusitupa. 1999. Effects of low fat stanol ester enriched margarines on concentrations of serum carotenoids in subjects with elevated serum cholesterol concentrations. Eur. J. Clin. Nutr. 53:966–969. Hallikainen, M. A., E. S. Sarkkinen, and M. I. Uusitupa. 2000a. Plant stanol esters affect serum cholesterol concentrations of hypercholesterolemic men and women in a dose-dependent manner. J. Nutr. 130:767–776. Heinemann, T., G. Axtmann, and K. von Bergmann. 1993. Comparison of intestinal absorption of cholesterol with different plant sterols in man. Eur. J. Clin. Invest. 23:827–831. Hepburn, P. A., S. A. Horner, and M. Smith. 1999. Safety evaluation of phytosterol-esters. Part 2. Subchronic 90-day oral toxicity study on phytosterol-esters-a novel functional food. Food Chem. Toxicol. 37:521–532. Jones, P. J., M. Raeini-Sarjaz, F. Y. Ntanios, C. A. Vanstone, J. Y. Feng, and W. E. Parsons. 2000. Modulation of plasma lipid levels and cholesterol kinetics by phytosterol versus phytostanol esters. J. Lipid Res. 41:697–705. Kim, J. C., B. H. Kang, C. C. Shin, Y. B. Kim, H. S. Lee, C. Y. Kim, J. Han, K. S. Kim, D. W. Chung, and M. K. Chung. 2002. Subchronic toxicity of plant sterol esters administered by gavage to Sprague-Dawley rats. Food Chem. Toxicol. 40:1569–1580. Kritchevsky, D., and S. C. Chen. 2005. Phytosterols—Health benefits and potential concerns: A review. Nutr. Res. 25:413–428.
549
