METABOLISM AND NUTRITION. Oral Administration of Pravastatin Reduces Egg Cholesterol But Not Plasma Cholesterol in Laying Hens

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1 METABOLISM AND NUTRITION Oral Administration of Pravastatin Reduces Egg Cholesterol But Not Plasma Cholesterol in Laying Hens J. H. Kim,* S. T. Hong, H. S. Lee, and H. J. Kim*,1 *Jinis Biopharmaceuticals Co., , Jang-Dong, Chonju, Chonbuk , Republic of Korea; Institute of Cardiovascular Research and Department of Microbiology, Chonbuk National University Medical School, Chonju, Chonbuk , Republic of Korea; and Institute of Bio-Safety Research and Research Center for Industrial Development of Biofeed Materials, Chonbuk National University, Chonju, Chonbuk , Republic of Korea ABSTRACT The effect of different 3-hydroxy-3-methylglutaryl agreement with previous reports, whereas pravastatin coenzyme A reductase (HMGR)-inhibitors (stat- ins) on plasma and egg cholesterol in laying hens was investigated. Forty-four ISA brown layers were fed lovastatin, simvastatin, or pravastatin at 0.03 or 0.06% for 4 wk. Cholesterol levels in plasma, liver, and egg yolk as well as hen laying performance were studied. Egg weight was significantly lowered with all statin treatments, although egg production was relatively unaffected. Total plasma cholesterol was significantly reduced by 0.06% lovastatin, 0.03% simvastatin, and 0.06% simvastatin, in had no effect. In contrast, liver cholesterol concentrations showed a significant decrease in response to 0.03 and 0.06% pravastatin, implying selective regulation of liver cholesterol synthesis. Furthermore, oral administration with 0.06% pravastatin reduced egg cholesterol levels by almost 20% compared with the control diet. This suggests that pravastatin, unlike other classes of statin, may be a good candidate for commercial production of low cholesterol eggs with limited impact on hen physiology and egg production. (Key words: cholesterol, egg, laying hen, pravastatin) 2004 Poultry Science 83: INTRODUCTION Chicken eggs are an excellent foodstuff from a nutritional standpoint due to their composition of high-quality protein, mono- and polyunsaturated fatty acids, minerals, and vitamins (Cook and Briggs, 1977). However, in addition to these essential dietary components, eggs contain about 200 mg of cholesterol per egg and are considered a major source of dietary cholesterol. Because of recent understanding of the association between total plasma cholesterol levels and the incidence of heart disease, it is surmised that an increased amount of dietary cholesterol may increase the risk of coronary heart disease (Weggemans et al., 2001). Growing numbers of health-conscious consumers exclude eggs from their diets in an effort to limit daily cholesterol consumption to 300 mg/day as recommended by the American Heart Association (National Institutes of Health Consensus Development Panel, 1985). Hence, there has been a steady decrease in per capita egg consumption in developed countries. In the US, increasing public concern over dietary cholesterol is 2004 Poultry Science Association, Inc. Received for publication November 19, Accepted for publication April 26, To whom correspondence should be addressed: hyeonjin_kim@ yahoo.com. reflected in annual per capita egg consumption, which has declined from 303 to 256 during the past 35 years (USDA, 2002). Eggs with reduced cholesterol content may be an attractive, highly nutritious food for health-conscious consumers and a lucrative product for egg producers. To meet the demands of changing consumer dietary preferences, many efforts have been made to lower the cholesterol content of eggs (Naber, 1983; Hargis, 1988; Stadelman and Pratt, 1989; Elkin et al., 1999, 2003). Genetic selection programs have resulted in only modest changes in egg cholesterol levels, with a reported maximum reduction of 7% (Hargis, 1988), and substantial reduction of egg cholesterol levels by dietary means has been even less successful. However, recent studies reported that oral administration of pharmacological agents developed for hypercholesterolemic patients, inhibitors of 3-hydroxy- 3-methylglutaryl coenzyme A reductase (HMGR), was effective in reducing egg cholesterol content in laying hens. Lovastatin, simvastatin, and atorvastatin have been shown to reduce egg cholesterol content as well as liver cholesterol concentration and plasma cholesterol concentration (Elkin et al., 1999; Mori et al., 1999). 3-hydroxy-3- Abbreviation Key: HMGR = 3-hydroxy-3-methylglutaryl coenzyme A reductase. 1539

2 1540 HAK ET AL. methylglutaryl coenzyme A reductase is the rate-limiting enzyme in cholesterol biosynthesis and HMGR inhibitors (also called statins) are known to lower plasma cholesterol by inhibition of cholesterol biosynthesis in the human liver. However, cholesterol metabolism in laying fowl is not completely understood and may differ importantly from normolipidemic humans. To identify optimal drugs for use in commercial low cholesterol egg production, comprehensive studies are required to compare the effects of different statins in laying hens. To our knowledge, pravastatin, an effective lipid-lowering drug in humans, has not been tested in laying hens. Therefore, the objective of the present work was to evaluate pravastatin in parallel with lovastatin and simvastatin, to compare efficacy of egg cholesterol reduction and effects on hen physiology and laying performance important considerations for commercial producers. Cholesterol levels were measured in the liver, plasma, and eggs, where cholesterol is biosynthesized, transported, and deposited, respectively. The relationship between plasma cholesterol, liver cholesterol, and egg cholesterol levels was also investigated. MATERIALS AND METHODS Birds, Diets, and Management Forty-four 40-wk-old healthy ISA brown hens were assigned randomly to 1 of 7 dietary groups (control group, n = 8 birds; statin-fed groups, n = 6 birds each). Each bird was placed in an individual cage in an environmentally controlled room (25 C, 50% RH). The hens were allowed to adapt to feed (with no additives) and housing for 2 wk. The average numbers of eggs laid during the 14 d before initiation of the experiment were 12.1, 12.0, 11.9, 12.2, 12.1, 12.0, and 12.1 for the 7 dietary groups. Control birds were fed a commercial diet based on corn and soybean meal (Table 1), and birds in experimental groups were fed diets supplemented with 0.03 or 0.06% of lovastatin, simvastatin, or pravastatin, 2 for 4 wk. Feed and water were provided ad libitum throughout the experiment, and a photoperiod of 16L:8D was used. Feed consumption, egg production, and egg weight were recorded daily and feed consumption weekly for 4 wk. The average values for each parameter were calculated for each replicate group. Plasma Cholesterol and Triglyceride Analysis After 4 wk of statin administration, all birds were fasted for 24 h and individual blood samples were obtained 2 Kobiotech, Kozan-Dong, Namdong-Gu, Inchon, Korea. 3 Kit number AM202-K, ASAN Pharm. Co. Ltd., Korea. 4 Kit number AM157S-K, ASAN Pharm. Co. Ltd., Korea. 5 Model Ultraspec 2000, Amersham Pharmacia Biotech Inc., Piscataway, NJ. 6 Sigma C-8003, Sigma Chemical Co., St. Louis, MO. 7 Model GC-2010, Shimadzu, Kyoto, Japan. 8 Model VB-1, Valco Instruments Co., Inc., Houston, TX. TABLE 1. Composition of control hen diet Ingredients and composition % Corn Soybean meal Corn gluten meal 3.88 Rapeseed meal 0.50 Wheat 1.00 Wheat bran 6.02 Tallow 2.00 Limestone 8.44 Tricalcium phosphate 1.58 Salt 0.39 DL-methionine 0.14 Vitamin premix Mineral premix Calculated chemical composition ME, kcal/kg 2,600 Crude protein, % Methionine and cysteine, % 0.50 Calcium, % 3.50 Available P, % 0.40 Choline, mg/kg Vitamin premix provided per kilogram of diet: vitamin A, 5,500 IU; vitamin D 3, 1,100 IU; vitamin E, 11 IU; vitamin B 12, 6.6 µg; riboflavin, 4.4 mg; niacin, 44 mg; pantothenate, 11 mg; choline chloride, 220 mg; menadione, 1.1 mg; folic acid, 0.55 mg; pyridoxine, 2.2 mg; biotin, 0.11 mg; thiamin, 2.2 mg; ethoxyquin, 125 mg. 2 Mineral premix provided per kilogram of diet: Mn, 60 mg; Zn, 100 mg; Fe, 60 mg; Cu, 10 mg; I, 0.46 mg. from the brachial vein using heparinized tubes. Plasma was isolated by centrifuging the blood for 10 min at 1,500 g. Plasma samples were analyzed for cholesterol 3 and triglyceride 4 using an enzymatic calorimetric kit, following the manufacturer s directions. Concentrations of plasma triglyceride and total cholesterol were determined by measuring the color change using a spectrophotometer. 5 Liver and Egg Cholesterol Analysis For liver analysis, each hen was killed by decapitation at the end of the experimental period and the liver was removed immediately, weighed, and stored at 70 C until use. For egg cholesterol analysis, eggs from each bird were collected on d 7, 14, 21, and 28. Egg yolk was separated, weighed, and sampled for analysis. Lipids from liver tissue or eggs were extracted by the method of Folch et al. (1957). Briefly, 1 g of sample was saponified with 3 ml of 33% KOH and 30 ml of 95% methanol in a 65 C waterbath for 90 min. After saponification, 10 ml of hexane and 3 ml of water were added, followed by vigorous shaking for 10 min. An internal standard (5αcholestan) 6 was used. Cholesterol content was determined by gas chromatography 7 using a column (30 m 0.25 mm 0.25 µm), 8 with a split ratio of 100:1 and nitrogen as carrier gas for a column flow rate of 0.54 ml/min. Injector, column, and detector temperature were 275, 290, and 340 C, respectively. Statistics All data were analyzed by ANOVA using the GLM procedure of SAS (Steel and Torrie, 1980). Individual

3 REDUCTION OF EGG CHOLESTEROL BY PRAVASTATIN 1541 TABLE 2. Performance of laying hens fed control or HMGR inhibitor-supplemented diets 1 Egg weight Hen-day egg Feed consumption Feed Treatment (g) production 2 (%) (g/d) conversion Control 65.3 ± 0.51 a 74.5 ± ± ± ab 0.03% Lovastatin 63.4 ± 0.45 b 75.6 ± ± ± ab 0.06% Lovastatin 62.5 ± 0.47 b 68.5 ± ± ± a 0.03% Simvastatin 63.8 ± 0.46 b 82.2 ± ± ± b 0.06% Simvastatin 59.2 ± 0.37 d 74.4 ± ± ± ab 0.03% Pravastatin 60.7 ± 0.30 c 71.4 ± ± ± ab 0.06% Pravastatin 61.0 ± 0.72 c 72.0 ± ± ± ab a d Means ± SEM within columns with no common superscript differ significantly (P < 0.01) by Duncan test. 1 Values are means ± SEM of 8 (Control) or 6 (HMGR inhibitor treatment) hens. 2 Hen-day egg production was calculated as (100 number of eggs laid)/(number of hens d). treatment differences were tested by Duncan s multiple range test (Steel and Torrie, 1980). Statements of statistical significance were based on P < RESULTS Hen Laying Performance The effects of 0.03 or 0.06% lovastatin, simvastatin, and pravastatin on the laying performance of 40-wk-old ISA brown hens are presented in Table 2. Egg weight was maintained at more than 60 g after 4 wk of oral administration of lovastatin and pravastatin at both doses and 0.03% simvastatin. Feeding of 0.06% simvastatin caused an approximate 10% reduction in egg weight. Egg production, feed consumption, and feed conversion, however, were not significantly changed by supplementation at either dose of any statin. Plasma Cholesterol and Triglyceride Analysis The effects of different statins on plasma total cholesterol and triglyceride in laying hens are shown in Table 3. Feeding 0.06% lovastatin resulted in a reduction of plasma total cholesterol concentration, a 28% decrease from control (P < 0.05). Administration of simvastatin at 0.03 and 0.06% reduced total cholesterol concentration to 68 and 53 mg/dl respectively, a 36 and 50% decrease, respectively, compared with control. Interestingly, pra- TABLE 3. Hen plasma cholesterol and triglyceride concentrations from hens fed control or HMGR inhibitor-supplemented diets 1 vastatin failed to decrease plasma cholesterol at either dose tested. All statin treatments, with the exception of 0.03% lovastatin, showed a greater than 50% reduction in the average value of plasma triglyceride concentration. However, these values were not significantly different from the control due to large deviations of triglyceride concentrations among samples. Liver Weight and Cholesterol Analysis The effects of different statins on liver cholesterol content are shown in Table 4. Liver weights were significantly decreased by oral administration of 0.03% lovastatin (16.6%), and 0.03% pravastatin (15.1%), but not simvastatin. Liver cholesterol concentration was unchanged by 0.03 and 0.06% lovastatin. Administration of 0.03 and 0.06% simvastatin, however, resulted in slight reduction of liver cholesterol concentration by 11.7 and 8.8%, respectively. Reduction of liver cholesterol concentration was achieved in 0.03 and 0.06% pravastatin groups, a 14.7 and 20.6% decrease, respectively, compared with the control (P < 0.05). Egg Weight and Yolk Cholesterol Analysis Table 5 shows the effects of different statins on egg cholesterol. The average control yolk weight was 16.2 g. Yolk weight showed a slight decrease with statin administration compared with the control. Administration of Total cholesterol Triglyceride Percentage Percentage Treatments (mg/dl) change (mg/dl) change Control ± 7.49 a ± a 0.03% Lovastatin ± 7.88 a ± a % Lovastatin 76.0 ± 8.60 b ± 68.5 b % Simvastatin 67.8 ± b ± b % Simvastatin 53.1 ± 3.39 b ± 53.0 b % Pravastatin ± a ± ab % Pravastatin ± a ± ab 41.5 a,b Means ± SEM within columns with no common superscript differ significantly (P < 0.05) by Duncan test.

4 1542 HAK ET AL. TABLE 4. Weights and total cholesterol contents of liver from hens fed control or HMGR inhibitor-supplemented diets 1 Total cholesterol Liver weight Percentage Percentage Treatments (g) mg/g change mg/liver change Control 33.1 ± 1.82 a 3.4 ± 0.10 a ± 6.2 a 0.03% Lovastatin 27.6 ± 1.47 b 3.4 ± 0.13 a ± 5.0 ab % Lovastatin 31.4 ± 3.72 ab 3.4 ± 0.27 a ± 12.7 a % Simvastatin 36.4 ± 1.05 a 3.0 ± 0.02 ab ± 3.2 a % Simvastatin 34.6 ± 1.30 a 3.1 ± 0.06 ab ± 4.0 a % Pravastatin 28.1 ± 0.74 b 2.9 ± 0.08 ab ± 2.2 b % Pravastatin 36.3 ± 2.52 a 2.7 ± 0.18 b ± 6.8 ab 13.0 a,b Means ± SEM within columns with no common superscript differ significantly (P < 0.05) by Duncan test. 0.06% pravastatin caused the biggest reduction in yolk weight, an 11.7% decrease compared with the control (P < 0.05). Lovastatin did not result in any significant change in cholesterol concentration per gram of yolk. However, due to decreased yolk weight, 0.06% lovastatin treatment reduced total cholesterol per yolk by 13%, compared with the control (P < 0.05). In the 0.03 and 0.06% simvastatin groups, yolk cholesterol concentration was reduced by 8.4 and 9.7%, respectively, compared with the control (P < 0.05). When expressed as total yolk cholesterol content, percentage changes compared with that of control were 18.7 and 17.2% at 0.03 and 0.06% simvastatin, respectively. In the 0.03 and 0.06% pravastatin groups, yolk cholesterol concentration was significantly reduced by 8.8 and 8.1%, respectively, compared with the control (P < 0.05). When expressed as total yolk cholesterol content per yolk, percentage changes compared with the control were 11.1 and 19.6% at 0.03 and 0.06% pravastatin, respectively. It should be noted that maximal reduction in total cholesterol content per egg was achieved by 0.06% pravastatin rather than simvastatin, one of the most potent pharmaceutical drugs for hypercholesterolemic human patients. DISCUSSION Efforts to modify egg cholesterol contents have demonstrated that it is extremely difficult to significantly reduce egg cholesterol levels by ordinary dietary means. Thus, much attention has been focused on avian cholesterol metabolism in an attempt to effectively reduce egg cholesterol without affecting other parameters important for commercial application. Laying hens usually meet their needs for cholesterol by de novo synthesis, synthesizing 300 mg/day in the liver and ovary. However, ovariansynthesized cholesterol is rarely transferred to developing oocytes, contributing minimally to egg cholesterol. In laying hens, egg cholesterol is synthesized in the liver and secreted into the blood as very low-density lipoprotein particles, the main yolk cholesterol carrying macromolecules (Andrews et al., 1968). Plasma very low-density lipoprotein particles are then internalized by the oocyte vitellogenesis receptor in the rapidly growing follicles, and deposited to yolk (Hall and McKay, 1993). Therefore, it has been suggested that selective inhibition of liver cholesterol biosynthesis would result in reduction of egg cholesterol content. Several studies have successfully demonstrated the cholesterol-lowering effect of HMGR inhibitors on plasma and egg cholesterol (Elkin and Rogler, 1990; Elkin et al., 1999, 2003; Mori et al., 1999). Lovastatin, simvastatin, and atorvastatin have been shown to inhibit cholesterol biosynthesis in the liver of laying hens, lowering the plasma cholesterol level (Elkin and Rogler, 1990; Elkin et al., 1999). The plasma cholesterol-lowering effect of lovastatin and simvastatin in our study is in agreement with these studies. Egg cholesterol content was reduced TABLE 5. Egg yolk cholesterol content of hens fed control or HMGR inhibitor-supplemented diets 1 Egg yolk cholesterol content 2 Yolk weight Percentage Percentage Treatment (g) (mg/g) change (mg/yolk) change Control 16.2 ± 0.29 a ± 0.16 a ± 2.86 a 0.03% Lovastatin 16.1 ± 0.49 a ± 0.16 ab ± 3.74 a % Lovastatin 14.4 ± 0.30 b ± 0.24 a ± 3.32 bc % Simvastatin 14.5 ± 0.27 b ± 0.20 b ± 3.62 cd % Simvastatin 15.2 ± 0.18 ab ± 0.23 b ± 5.58 cd % Pravastatin 15.9 ± 0.52 a ± 0.11 b ± 3.22 b % Pravastatin 14.3 ± 0.37 b ± 0.20 b ± 2.63 d 19.6 a d Means ± SEM within columns with no common superscript differ significantly (P < 0.01) by Duncan test. 2 Egg cholesterol content was expressed as milligrams per gram of yolk (yolk cholesterol concentration) or total milligrams in a yolk (total cholesterol content).

5 REDUCTION OF EGG CHOLESTEROL BY PRAVASTATIN 1543 by 46% in hens fed 0.06% atorvastatin (Elkin et al., 1999). Unfortunately, atorvastatin affected egg production significantly, compromising its potential value for commercial production of low cholesterol eggs. It was therefore important to examine other classes of statins, which may demonstrate significant hypocholesterolemic effects without the negative impact on hen physiology or laying performance. In this study, the effects of pravastatin were compared with lovastatin and simvastatin in laying hens. Pravastatin is a potent HMGR-inhibiting drug, known to cause liver-specific inhibition of cholesterol synthesis (Koga et al., 1990; Hamelin and Turgeon, 1998). However, its effectiveness in reducing egg cholesterol had not been studied in laying hens. We showed that pravastatin reduced yolk weight and yolk cholesterol concentration thereby significantly reducing egg cholesterol content. Administering pravastatin at 0.06% for 4 wk resulted in a 20% decrease in egg cholesterol content, to 154 mg/egg. Importantly, this reduction in egg cholesterol content was not accompanied by lower egg production, in contrast to the atorvastatin study. In agreement with avian data in this study, oral administration of pravastatin did not affect plasma total cholesterol levels in Wister rats at 20 mg/kg per d for 5 wk (Fontaine et al., 2002). Maintenance of normal plasma cholesterol levels in laying hens is very important because low levels affect avian physiology, lowering fertility and synthesis of steroid hormones. Due to the hydrophilic nature of pravastatin, levels in tissues and organs are very low (Hamelin and Turgeon, 1998), reducing the possibility of adverse drug effects during long-term treatment. These data suggest that the physiology of laying hens might not be severely affected by the administration of pravastatin throughout the entire egg-laying period. In conclusion, the present study reports the first assessment of efficacy of pravastatin for the production of low cholesterol eggs. Our data indicate that pravastatin, unlike other types of HMGR inhibitors, is an excellent candidate for further study as a dietary supplement for laying hens. This drug has potential commercial applications for production of low cholesterol eggs without negative impact on egg production or hen physiology. ACKNOWLEDGMENTS We wish to thank Ann Barlow for critical reading of the manuscript. This research was supported by the Program for Cultivating Graduate Students in Regional Strategic Industry in Korea. REFERENCES Andrews, J. W., R. K. Wagstaff, and H. M. Edwards Cholesterol metabolism in the laying fowl. Am. J. Physiol. 214: Cook, F., and G. M. Briggs Nutritive value of eggs. Pages in Egg Science and Technology, 2nd ed. W. J. Stadelman and O. J. Cotterill, ed. AVI Publishing Co., Westport, CT. Elkin, R. G., E. J. Furumoto, and C. R. 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