Effects of Branched and Straight Chain Amines and Azasteroids on Blood and Egg Cholesterol of White Leghorn Chickens

Effects of Branched and Straight Chain Amines and Azasteroids on Blood and Egg Cholesterol of White Leghorn Chickens

Effects of Branched and Straight Chain Amines and Azasteroids on Blood and Egg Cholesterol of White Leghorn Chickens HELENE C. CECIL, JOEL BITMAN, J. ...

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Effects of Branched and Straight Chain Amines and Azasteroids on Blood and Egg Cholesterol of White Leghorn Chickens HELENE C. CECIL, JOEL BITMAN, J. A. SVOBODA, and M. J. THOMPSON US Department of Agriculture, SEA-AR, Avian Physiology Laboratory, Nutrient Utilization Laboratory, and Insect Physiology Laboratory, Beltsville, Maryland 20705 (Received for publication November 21, 1979)

1981 Poultry Science 60:795-804 INTRODUCTION Azasteroids inhibit cholesterol production in insects (Svoboda and Robbins, 1971), in mammals (Thompson et al., 1963), and in birds (Singh et al., 1972; Dam et al, 1979). The azasteroids inhibit the biosynthesis of cholesterol at the reductive step in the conversion of desmosterol to cholesterol. Recently, Robbins et al. (1975) showed that several new secondary and tertiary amines blocked cholesterol synthesis in insects; nonsteroidal amines and the simple azasteroid 25-aza-5o>cholestane blocked cholesterol synthesis in the rat (Svoboda etal., 1977). The possibility that these relatively simple alkyl amines might also exert similar or related effects in birds prompted us to test these

compounds in the laying hen. We wanted to determine whether these nitrogen-containing compounds would act in birds at the reductive step or at a different step in cholesterol biosynthesis. The nature and the time course of the action of the amines on egg and plasma cholesterol were studied during feeding and after withdrawing them from the diet. MATERIALS AND METHODS Test Compounds and Diet Preparation. The straight and branched chain amines were prepared as previously described (Robbins et al., 1975), as were 25-aza-5a-cholestane (azacholestane) and 25-aza-5 |3-cholestane (azacoprostane) (Svoboda et al., 1972). The 20,25-diazacholesterol dihydrochloride (SC-

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ABSTRACT Six branched and straight chain secondary or tertiary amines with chain lengths of 12 to 18 carbons and two azasteroids, 25-aza-5a-cholestane and 25-azacoprostane, were fed to mature White Leghorn hens, and their effectiveness was compared with 20,25-diazacholesterol dihydrochloride (SC-12937), an azasteroid known to lower egg cholesterol. Feed consumption, body weight, egg production, egg and plasma cholesterol and desmosterol, and plasma total lipid were measured. The 6 amines were fed at 200 ppm, and only the C l 2 branched chain amine N,N,3,7,ll-pentamethyldodecanamine reduced plasma and egg cholesterol with a concomitant increase in desmosterol. After 4 weeks, plasma desmosterol was 0, 13, 60, and 75% of total sterol for control, 200 ppm C,j branched chain amine, 5 ppm diazacholesterol, and 5 ppm azacholestane, respectively. Egg production was severely reduced to 6 and 0% by feeding 5 ppm azacholestane for 2 and 4 weeks, respectively, and to 69 and 36% by feeding 5 ppm diazacholesterol. After 4 weeks egg cholesterol was 79 and 36% of the total sterol for the 200 ppm C 12 branched chain amine and 5 ppm diazacholesterol, respectively. Concomitant increases in desmosterol accompanied all reductions in cholesterol. The depletion and repletion rates of egg cholesterol were measured in a subsequent experiment. After 1-Vt weeks of feeding the test substances, egg cholesterol was reduced with concomitant increases in desmosterol. Egg cholesterol was 100, 71, and 50% of the total egg sterol for control, 200 ppm, and 400 ppm C12 branched chain amine, respectively; 58, 13, and 3% for .1, .5, and 1.0 ppm azacholestane; 28, 29, and 18% for 1, 2.5, and 5 ppm azacholesterol; and 23% for 1 ppm azacoprostane. The experimental diets were then withdrawn, and egg cholesterol repletion was studied biweekly. Egg cholesterol was repleted to 100% of the total sterol after withdrawal times of 2 weeks for C 12 branched chain amine, 8 weeks for azacoprostane, 14 to 16 weeks for diazacholesterol, 10 to 16 weeks for the lower levels of azacholestane, and longer than 16 weeks for 1 ppm azacholestane. The increase in desmosterol accompaning the demonstrated reduction in egg cholesterol, particularly with azasteroids, causes one to question the usefulness of this approach to lower cholesterol. (Key words- egg cholesterol, egg desmosterol, egg production, azasterol, azasteroids, amines, A24-sterol reductase)

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CECIL ET AL.

1 Trade names were used solely for the purpose of providing specific information and do not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture.

equivalent to the amount consumed the previous day by their respective pair-group. Experiment 3. Groups of 10 hens each were fed ad lib diets containing 0, 200, and 400 ppm N,N,—3,7,11-pentamethyldodecanamine (C12 branched chain amine); . 1 , .5, and 1 ppm azacholestane; 1, 2.5, and 5 ppm diazacholesterol; and 1 ppm azacoprostane. After 17 days the treatment diets were withdrawn and replaced with the basal diet; the basal diet was fed an additional 16 weeks. Eggs were saved for sterol analyses at Day 0, 6, and 13 of treatment, Day 5 posttreatment, and biweekly thereafter. Chemical Analyses. The entire contents of individual eggs were freeze-dried in a Virtis RePP Model 100-6 sublimator with a shelf temperature of 21 C and a vacuum of 5 /i Hg. A .5 g sample of the freeze-dried egg was extracted with chloroform-methanol (Washburn and Nix, 1974) and then saponified (Abell et al, 1952). Sterols were quantitated by gas liquid chromatography (GLC) of trimethylsilyl (TMS) derivatives. After evaporation of the petroleum ether phase containing the sterols, 1.0 ml of a 20% solution of TRI-SIL (mixture of hexamethyldisilazane and trimethylchlorosilane, Pierce Chemical Company, Rockford, IL) in pyridine, containing .5 mg cholestane as an internal standard, was added to each vial. The GLC vial was capped and heated at 75 C for 30 min to form the TMS derivatives. The GLC analyses of cholesterol and desmosterol were made on 1.8 m X 2 mm id glass columns containing 3% OV-17 coated on Gas Chrom Q (100 to 120 mesh) with a Model 7620 Hewlett Packard gas chromatograph (column temperature, 250 C; He flow rate, 30 ml/min). Cholesterol and desmosterol were the only sterols present in measurable amounts (>.8 mg/g dried egg). Plasma total lipids were determined by a colorimetric method based on the sulfophosphovanillin reaction (Frings and Dunn, 1970; Postma and Stroes, 1968). Plasma sterols were extracted with acetone:ethanol (1:1), saponified, and determined by GLC as described above. Statistics. Data for body weight and egg production were compared statistically according to the General Linear Models Procedure (Barr et al., 1976) using a one-way analysis of variance comparing the control group with each treatment group within an experiment. Linear regression analysis was performed on the data for cholesterol depletion in Experiment 3.

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12937; diazacholesterol) was a gift of the G. D. Searle Company, Chicago, IL. 1 The structures and the concentrations of the test compounds are reported in Table 1. All test compounds except the water soluble diazacholesterol were dissolved in acetone, and a premix was prepared with the basal diet. The solvent was removed by air drying. Aliquots of the premix were mixed with the basal diet to prepare treatment diets of the appropriate concentrations. The basal diet, a laying mash containing 16% protein and 2.5% calcium, was fed as the control diet. Birds. Mature White Leghorn laying hens were housed in laying batteries and exposed to a 14-hr light regimen. The hens were randomly distributed into individual cages and 10 hens assigned to each treatment group. Each treatment group consisted of 10 cages that had a common feeder and waterer. Feed consumption of each group and body weight of each bird were measured. Daily egg production for each bird was recorded for a 2-week pretreatment period and during the treatment and posttreatment periods. The experimental design is outlined in Table 1, and details of the experiments are given here. Experiment 1. Fifteen groups (10 hens per group) were fed ad lib diets containing 0, 20, and 200 ppm of the six amines and 5 ppm of diazacholesterol and azacholestane. After 4 weeks of feeding, 5 birds in each group were exsanguinated and the ovaries were examined. At this time, the experimental diets of the other birds were replaced with the basal ration. Blood was collected from the brachial vein of four hens in each group on 0, 7, 14, 21, and 28 days of treatment and 7, 14, 21, and 28 days posttreatment. At 7-day intervals, the content of one egg from all hens was saved for sterol analyses. Experiment 2. Experiment 2 was designed as a paired-feeding experiment. Five groups of hens (10 hens per group) were fed ad lib diets containing 0, 1, and 5 ppm diazacholesterol or azacholestane for 4 weeks. Feed consumption was measured daily for each group. An additional 4 groups of control hens were paired by body weight with the 4 treatment groups and fed daily a weighed amount of basal diet

20;2

20;2 20;2 20;2 2O;2 20;2

Exp

Test compound had no effect on egg or plasma cholesterol.

Test compound fed in the diet for 2-Vi weeks, then withdrawn and replaced with basal diet for an additional 16 we

Test compound fed in the diet for 4 weeks.

Test compound fed in the diet for 4 weeks, then withdrawn and replaced with basal diet for an additional 4 weeks

25-Aza-5a-cholestane (azacholestane) R = —H

2 5-Aza-5(3-cholestane (azacoprostane) R = —H

20,25-Diazacholesterol dihydrochloride (diazacholesterol)

CH 2 N(CH 3 ) 2

CH 3 (CH 2 ) 1 0 CH 2 N(CH 3 ) 2 CH 3 (CH 2 ) 1 2 CH 2 N(CH 3 ) 2 CH 3 (CH 2 ) 1 4 CH 2 N(CH 3 ) 2 CH3 (CH 2 ) 16 CH2 NH CH2 CH3 CH 3 (CH 2 ) 1 6 CH 2 N(CH 3 ) 2

N,N-Dimethyldodecan amine N,N-Dimethyltetradecan amine N,N-Dimethylhexadecan amine N-Ethyloctadecan amine N,N-Dimethyloctadecanamine

N,N,3,7,ll-Pentamethyldodecanamine (C12 branched chain amine)

Structure

Test compound

TABLE 1. Amines and azasteroids tested for egg cholesterol lowering effects in White

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In addition to the C 12 branched chain amine, other dietary treatments that had no effect on food consumption or body weight were: 1, .5, and .1 ppm azacholestane (Experiments 2 and 3); 5, 2.5, and 1 ppm diazacholesterol (Experiments 2 and 3); and 1 ppm azacoprostane (Experiment 3). These negative data are not presented. Egg Production. In both Experiments 1 and 2, egg production was severely depressed by feeding 5 ppm of either the diazacholesterol or the azacholestane (Table 3). After 4 weeks of feeding 5 ppm diazacholesterol, egg production was 31 and 36% in the two experiments compared to 67 and 60% for the controls, respectively. However, feeding 1 ppm diazacholesterol did not reduce egg production (Experiment 2, Table 3). Feeding 5 ppm

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Comparisons of the activity of a C 12 branched chain tertiary amine, of four straight chain tertiary amines 12, 14, 16, and 18 carbons long, of the diazacholesterol, and of the azacholestane showed that feeding 20 or 200 ppm of the straight chain C 12 to C ^ tertiary amines had no effect upon plasma or egg cholesterol, feed consumption, or body weight. Therefore, the description of results will be limited to the effects of feeding the C 12 branched chain amine and the three azasteroids—azacholestane, azacoprostane, and 20,25-diazacholesterol. Feed Consumption and Body Weight. In Experiments 1 and 2 the total feed consumption was determined for each treatment group. Because the feed consumption was not measured for each bird, no statistical comparisons were made for feed consumption. Feeding the Ci 2 branched chain amine did not affect feed consumption or body weight. However, results were conflicting after feeding 5 ppm azacholestane (Table 2). In Experiment 1, hens fed 5 ppm azacholestane lost an average of 305 g (17% of initial body weight) and their body weight did not increase after the azacholestane was removed from the diet. These results could not be confirmed in Experiment 2 (Table 2). The weight loss of hens fed 5 ppm azacholestane in Experiment 1 appeared to be caused by a decrease in feed consumption. After removal of the 5 ppm azacholestane from the diets, feed consumption increased to approach that of the control. Although hens fed diazacholesterol in Experiment 1 appeared to consume less feed than control hens, their body weight was not reduced.

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EGG CHOLESTEROL DEPLETION BY AZASTEROIDS

TABLE 3. Effect ofazasteroids on egg production of laying bens (Experiments 1 and 2) Treatment*

Egg p r o d u c t i o n (% ± SE) at week

Experiment

Diet

(ppm)

0

1 2 1 2 2 1 2 2

Control Control Azacholestane Azacholestane Azacholestane Diazacholesterol Diazacholesterol Diazacholesterol

0 0 5 5 1 5 5 1

66 70 78 91 83 71 81 70

1 +6 ±4 ± 3 ± 2b ±4b + 5 ± 5b ± 4

56 67 56 81 81 61 80 80

±6 ± 7 ±4 ± 3 ± 3b ± 7 ± 5 ± 3

2

3

4

60 ± 8 67 ± 8 5 + 3b 21±6b 43 ± 5 b 46 + 7 70 + 7 74 ± 3

51+7 78 ± 5 0b 6+ 3b 20 ± 7 b 30 ± 9 b 43 ± l l b 64 ± 3

67± 8 60 ± 7 0b 3± 3b 9± 7b 31 ± l l b 36 ± 9 b 63 ± 6

All diets were fed for 4 weeks. P < . 0 5 , t r e a t e d vs. c o n t r o l for t h e e:xperiment a n d week, after p e r c e n t egg p r o d u c t i o n was c o n v e r t e d t o

azacholestane dramatically reduced egg production and within 9 days all hens in Experiment 1, and 5 of 10 hens in Experiment 2, had stopped laying. When the birds were killed after 4 weeks of treatment, the ovaries had regressed to an immature state. Feeding 1 ppm azacholestane for 4 weeks also severely reduced egg production, although to a somewhat lesser extent than 5 ppm reduced it (Table 3). Although we did not have a pair-fed control group in Experiment 1, clearly the reduced feed consumption did not cause the reduced egg production, because egg production was reduced by the same magnitude in Experiment 2 but with only a slight reduction in feed consumption. Plasma Sterols and Lipids. Plasma total lipids and individual sterols were measured in Experiment 1. Total plasma sterols were not significantly different after 4 weeks of feeding the control, 200 ppm Ci2 branched chain amine, 5 ppm azacholestane, and 5 ppm diazacholesterol and were 169, 218, 182, and 223 mg%, respectively. Azacholestane and diazacholesterol reduced plasma cholesterol markedly, with a concomitant accumulation of desmosterol (Table 4). At 4 weeks, cholesterol accounted for 100, 87, 4 1 , and 25% of the total sterol for the control, Ci 2 branched chain amine, diazacholesterol, and azacholestane, respectively. Within 1 week after withdrawal of the C 12 branched chain amine (Week 5), desmosterol was not detectable in the plasma and cholesterol was 100% of the total sterol. However, 4 weeks after withdrawal of the azasteroid diets (Week 8, Table 4), the synthesis

of cholesterol remained inhibited; plasma cholesterol was 67 and 39% of the total sterol in the diazacholesterol and azacholestane groups, respectively. Plasma total lipids were 1914, 1351, 512, and 1989 mg% after 4 weeks of feeding the control, 200 ppm C 12 branched chain amine, 5 ppm azacholestane, and 5 ppm diazacholesterol, respectively. The cessation of egg production, rather than the azacholestane per se, may account for the plasma lipid reduction in hens fed azacholestane. Egg Sterols. The three nitrogen compounds that increased plasma desmosterol also increased egg desmosterol and reduced egg cholesterol (Table 4). Egg cholesterol was reduced most after feeding 5 ppm azacholestane, and within 2 weeks after the start of feeding, egg cholesterol was reduced to 20% of the total sterol. The reduction in cholesterol was accompanied by an accumulation of desmosterol so that the total sterol in the egg was unchanged. Feeding 5 ppm of diazacholesterol for 2 weeks reduced egg cholesterol to 45% of the total sterol; after 4 weeks, egg cholesterol was reduced to 36% of the total sterol, with a concomitant increase in desmosterol. Feeding 200 ppm Ci 2 branched chain amine reduced egg cholesterol much less than the azasteroids, and egg cholesterol was 80% and 79% of the total sterol after feeding the branched chain amine for 2 and 4 weeks, respectively. Within the limited scope of Experiment 1, apparently the faster the depletion time of egg cholesterol, the slower the repletion time after withdrawal of the treatment diet (Table 4).

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After withdrawal of the branched chain amine, cholesterol content of the eggs increased and no desmosterol was detectable within 2 weeks (Week 6). When the diazacholesterol was withdrawn, the cholesterol content increased slowly, but had not reached the control level 4 weeks after withdrawal (Week 8). The depletion and repletion of egg cholesterol paralleled the changes in plasma sterols (Table 4). Results of the first two experiments indicated that feeding 5 ppm diazacholesterol and 1 and 5 ppm azacholestane for 4 weeks in the diet interfered with egg production (Table 3). Also egg cholesterol did not return to control levels in the subsequent 4-week withdrawal period (5 ppm diazacholesterol, Table 4). Therefore, Experiment 3 was designed to study the depletion and repletion of egg cholesterol. Several dietary levels of the azasteroids (1, 2.5, and 5 ppm diazacholesterol; . 1 , .5, and 1 ppm azacholestane); the isomer of azacholestane (1 ppm azacoprostane); and the amine (200 and 400 ppm C 12 branched chain amine) were fed for 17 days followed by a 16-week withdrawal period. The effectiveness of the compounds in lowering egg cholesterol is shown in Table 5. Feeding 1 ppm of the azasteroids very effectively reduced egg cholesterol. Feeding 200 and 400 ppm of the amine reduced egg cholesterol to a much lesser extent than feeding 1 ppm of the azasteroids. On this basis, the C 12 branched chain amine had less than 1/400th of the activity of the azasteroids in inhibiting cholesterol synthesis. The rate of cholesterol depletion was determined by linear regression analysis of the cholesterol (expressed as percent of total steroid) in eggs on Days 0, 6, and 13 of treatment. Cholesterol depletion with azasteroid treatment was a straight line from 0 to 13 days as the intercept at the y axis was between 99 to 107, and r 2 ranged between .85 and .98. Cholesterol depletion with 200 ppm Cn branched amine also had a y intercept at 98, but r 2 = .79, indicating an increased randomness of the points from 0 time to withdrawal of the amine. In Experiment 1 (Table 4) 200 ppm of the amine was fed for 4 weeks and the cholesterol level in the egg reached a plateau between the first and second weeks of feeding, indicating maximal depletion of cholesterol within 2 weeks. The values for egg cholesterol at the time of

EGG CHOLESTEROL DEPLETION BY AZASTEROIDS

801

TABLE 5. Effect of amines and azasteroids on egg cholesterol (CHOL) and desmosterol (DOL) (Experiment 3)

CHOL

DOL

n

a

b

r2

0

1.00 .79

100 71 50 28 29 18 58 13 3 23

0 29 50 72 71 82 42 87 97 77

24 6 6 16 16 20 8 17 21 8

100 69 54 35 33 23 35 13 5 11

0 1.76 2.57

1 00

-1.61 -2.96 -4.50 -4.27 -4.81 -3.12 -5.26 -5.71 -4.71

.99 .84 .78

77 61 77

ppm

n

a

b

Control C 12 Branched amine

0 200 400 1.0 2.5 5.0 .1 .5 1.0 1.0

9 13 7 6 12 13 11 9 7 15

100 98 100 103 102 100 111 102 100 103

Azacholestane Azacoprostane

Cholesterol repletion regression

X1

Diet

Diazacholesterol

% Total sterol at 17 days

Cholesterol depletion regression"

Treatment

.96 .88 .85 .94 .96 .98 .96

1.82 .87 .68

83 86

2.83

Two to five eggs were analyzed on Day 0, 6, and 13; n = total number of eggs analyzed. Data were fitted to the general linear regression equation, y = a + bx, where y = cholesterol as percent of total sterol, x = days, 2 r = correlation coefficient. Data only at Day 0 and 16 for 400 ppm C 12 branched chain amine. Values calculated from the cholesterol depletion regression equation. Three to six eggs were analyzed at 4 days after withdrawal of the test compound and at 2 week intervals thereafter. Linear regression.analysis performed as above using data from 4 days after withdrawal until the cholesterol in the egg reached 70% of the total sterol. The r2 was not calculated for treatments in which cholesterol was 70% of the total sterol within 2.5 weeks after withdrawal of the test compound.

withdrawal of the treatment on Day 17 were calculated from the linear regression equations for depletion and repletion. The calculated value for cholesterol at 17 days using the depletion regression equation was very similar to the intercept value "a", which was derived from the repletion regression data (Table 5): 71/69, 200 ppm amine; 28/35, 29/33, and 18/23 for 1.0, 2.5, and 5.0 ppm diazacholesterol, respectively; 58/35, 13/13, and 3/5 for . 1 , .5, and 1.0 ppm azacholestane, respectively; 28/11 for 1.0 ppm azacoprostane. The repletion of egg cholesterol during the 16 weeks after withdrawal of the treatment diets is shown in Figures 1, 2, and 3. The three treatments which had the shortest repletion times are shown in Figure 1. Cholesterol was repleted to 100% of the total sterol within 2-Vi weeks after withdrawal of the C 12 branched chain amine diets. However, cholesterol was not repleted to 100% of the total sterol until 8 weeks after withdrawal of the azacoprostane. Each of the three levels of diazacholesterol inhibited cholesterol biosynthesis to approximately the same extent, and cholesterol was not repleted to 100% of the total sterol until 16

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FIG. 1. Cholesterol repletion in eggs from White Leghorn hens after withdrawal of diets containing 200 and 400 ppm N,N,3,7,ll-pentamethyIdodecanamine (C 12 Amine) or 1 ppm azacoprostane (Azacop). The treatment diets were fed for 2-'/2 weeks before withdrawal and replacement with the control diet (Experiment 3).

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The treatment diets were fed for 17 days then withdrawn and replaced with the control diet for an additional 16 weeks.

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FIG. 3. Cholesterol repletion in eggs from White Leghorn hens after withdrawal of diets containing .1, .5, and 1 ppm 25-aza-5a-cholestane. The treatment diets were fed for 2-Vi weeks before withdrawal and replacement with the control diet (Experiment 3).

drawal of the .1 and .5 ppm azacholestane diets. EGG

DISCUSSION

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60

STEROL

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FIG. 2. Cholesterol repletion in eggs from White Leghorn hens after withdrawal of diets containing 1, 2.5, and 5 ppm 20,25-diazacholesterol. The treatment diets were fed for 2-Vi weeks before withdrawal and replacement with the control diet (Experiment 3).

Azasterols have been recognized as hypocholesterolemic drugs in mammals and birds; they decrease cholesterol synthesis by increasing the accumulation of desmosterol. Azasterols have also been recognized as inhibitors of ovulation in birds. Elder (1964) reported that diazacholesterol reduced egg production and virtually eliminated reproduction in captive pigeons for approximately 6 months. As a result, diazacholesterol was evaluated as a chemosterilant for pigeon control (Wofford and Elder, 1967). Diazacholesterol and azacholesterol have also been reported to reduce egg production in chickens and to increase the desmosterol content in blood and egg at the expense of cholesterol (Singh et al., 1972). Chickens treated with triparanol, another hypocholesterolemic drug, showed an increase in egg desmosterol content followed by a cessation of egg production and by ovarian regression (Burgess et al., 1962). In our study, we identified azacholestane as being more potent than diazacholesterol or azacoprostane

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weeks after withdrawal of the diazacholesterol (Fig. 2). The repletion of cholesterol occurred at a diminishing rate: repletion from 25 to 70% cholesterol took 6 weeks; repletion from 70 to 100% was slower and took an additional 10 weeks. The total 16-week repletion period was six times the length of the period required for the depletion of egg cholesterol. Egg cholesterol levels increased slowly after withdrawal of the azacholestane diets (Fig. 3). Egg cholesterol was repleted rapidly to 80% of total sterol within 4 weeks after withdrawal of the .1 ppm diet. After this, repletion progressed at a slower rate and egg cholesterol was not repleted to 100% of the total sterol until 12 weeks after withdrawal of the azacholestane. A similar repletion pattern was seen after withdrawal of the .5 ppm diet. Egg cholesterol increased to 75% of the total sterol within 10 weeks, after which the repletion rate decreased. Egg cholesterol was repleted to 95% of total sterol 16 weeks after withdrawal of the .5 ppm azacholestane diet. Egg cholesterol was repleted linearly to 85% of total sterol 16 weeks after withdrawal of the 1 ppm azacholestane diet. The experiment was not continued long enough to determine whether a slower repletion rate occurred above 85%, as observed after with-

EGG C H O L E S T E R O L D E P L E T I O N BY A Z A S T E R O I D S

The biological consequences of increased

concentrations of desmosterol in the egg are not good. Naber (1976) reported that when more than half of the total egg sterol was desmosterol, hatchability was poor. We cannot ascertain whether the presence of desmosterol, the reduction in the level of cholesterol, or the accumulation of the treatment compound in the egg was responsible for the poor hatchability. Increased desmosterol in eggs is unfavorable on a nutritional basis and would render these eggs unsatisfactory as human foods. Desmosterol accumulation appears to be associated with induced myotonia in humans (Winer et al, 1965), in goats (Burns et al, 1965; Winer et al, 1965), and in rats (Winer et al, 1966; Peter and Fiehn, 1973; Seiler et al, 1975; Dromgoole et al, 1975). The myotonia observed is similar to that of myotonic or muscular dystrophy of humans and is characterized by an impaired relaxation of skeletal muscles after contraction, although none was noted in the present study. ACKNOWLEDGMENTS

The excellent technical assistance of M. Rucker in sample preparation and D. L. Wood in the GLC analysis is greatly appreciated. REFERENCES Abell, L. L., B. B. Levy, B. B. Brodie, and F. E. Kendall, 1952. A simplified m e t h o d for t h e estimation of total cholesterol in serum and d e m o n s t r a t i o n of its specificity. J. Biol. Chem. 195:357-366. Barr, A. J., J. H. Goodnight, J. P. Sail, and J. T. Helwig, 1976. A users guide to SAS 76. Sparks Press, Raleigh, NC. Burgess, T. L., C. L. Burgess, and J. D. Wilson, 1 9 6 2 . Effect of M E R - 2 9 on egg p r o d u c t i o n in the chicken. Proc. Soc. Exp. Biol.Med. 1 0 9 : 2 1 8 - 2 2 1 . Burns, T. W., H. E. Dale, and P. L. Langley, 1 9 6 5 . Normal and m y o t o n i c goats receiving diazacholesterol. Amer. J. Physiol. 2 0 9 : 1 2 2 7 - 1 2 3 2 . Dam, R., M. E. LaBate, S. W. Tarn, and C. CuervoTorres, 1 9 7 9 . Effects of diazacholesterol, triparanol, and /3-sitosterol on egg cholesterol deposition in C o t u r n i x quail. P o u l t r y Sci. 58:985-987. Dromgoole, S. H., D. S. Campion, and J. B. Peter, 1975. M y o t o n i a induced by single doses of 20,25-diazacholesterol: increased muscle and plasma desmosterol levels, unaltered ( N a + + K + )-ATPase activity of e r y t h r o c y t e ghosts. Biochem. Med. 1 3 : 3 0 7 - 3 1 1 . Dvornik, D., and M. Kraml, 1963. A c c u m u l a t i o n of 24-dehydrocholesterol in rats treated with 22,25-diazacholestanol. Proc. Soc. E x p . Biol. Med. 1 1 2 : 1 0 1 2 - 1 0 1 4 . Elder, W. H., 1964. Chemical inhibitors of ovulation in t h e pigeon. J. Wildl. Manage. 2 8 : 5 5 6 - 5 7 5 . Frings, C. S., and R. T. Dunn, 1970. A colorimetric

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in inhibiting cholesterol synthesis in chickens; azacholestane also caused ovarian regression and a cessation in egg production. Azacoprostane and a C 12 branched chain amine also inhibited cholesterol synthesis but, at the doses fed, did not interfere with egg production. The depletion of cholesterol and concomitant accumulation of desmosterol in the egg is a direct consequence of the inhibition of the A 24 -sterol reductase enzyme involved in the last step of cholesterol biosynthesis; i.e., the conversion of desmosterol to cholesterol. Thompson et al. (1963) showed that 20,25-diazacholesterol resulted in accumulation of desmosterol in the serum, liver, and carcass of rats. Feeding 22,25-diazacholestanol also resulted in an accumulation of desmosterol in rats (Dvornik and Kraml, 1963) and in 14 hypercholesterolemic patients (Sachs and Wolfman, 1963). Thus, use of these structural analogs of cholesterol containing nitrogen atoms blocks cholesterol synthesis, probably by competitive binding to A 24 -sterol reductase enzyme sites. More recently, the simple amines with structures resembling the side chain of cholesterol have been shown to block cholesterol production and normal development in insects (Robbins et al., 1975) and rats (Svoboda et al., 1977). In our study, we characterized the time course of cholesterol depletion and repletion in the egg. The activities of the compounds in depleting cholesterol varied, and the relationship of the depletion to the repletion time was unique for each of the active compounds. The straight chain amines had no effect on plasma or egg cholesterol and thus showed a species difference in comparative biochemical action in insects (Robbins et al., 1975), mammals (Svoboda et al, 1977), and birds (present study). Of the amines tested in the present study, only the C 12 branched chain had activity in the chicken, but it had much less potency than the azasteroids in depleting egg cholesterol. The more rapid depletion of egg cholesterol during the feeding of azasteroids than during the feeding of Ci 2 branched chain amine and conversely, the much slower repletion of cholesterol after the withdrawal of azasteroids than after the withdrawal of the amine from the diet is probably due to the relative rates at which the amine and azasteroids are metabolized. The repletion of cholesterol after the withdrawal of the azasteroids was much slower than the depletion of cholesterol during the feeding of the azasteroids.

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CECIL ET AL. Svoboda, J. A., and W. E. Robbins, 1971. The inhibitive effects of azasterols on sterol metabolism and growth and development in insects with special reference to the tobacco hornworm. Lipids6:113-119. Svoboda, J. A., M. J. Thompson, and W. E. Robbins, 1972. Azasteroids: Potent inhibitors of insect molting and metamorphosis. Lipids 7:553 — 556. Svoboda, J. A., T. R. Wrenn, M. J. Thompson, J. R. Weyant, D. L. Wood, and J. Bitman, 1977. Reduction of blood and liver cholesterol in the rat by straight and branched chain alkyl amines. Lipids 12:691-697. Thompson, M. J., J. Dupont, and W. E. Robbins, 1963. The sterols of liver and carcass of 20,25diazacholesterol fed rats. Steroids 2:99—104. Washburn, K. W., and D. F. Nix, 1974. A rapid technique for extraction of yolk cholesterol. Poultry Sci. 53:1118-1122. Winer, N„ D. M. Klachko, R. D. Baer, P. L. Langley, and T. W. Burns, 1966. Myotonic response induced by inhibitors of cholesterol biosynthesis. Science 153:312-313. Winer, N., J. M. Martt, J. E. Somers, L. Wolcott, H. E. Dale, and T. W. Burns, 1965. Induced myotonia in man and goat. J. Lab. Clin. Med. 66:758-769. Wofford, J. E., and W. H. Elder, 1967. Field trials of the chemosterilant, SC—12937, in feral pigeon control. J. Wildl. Manage. 31:507-515.

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method for determination of total serum lipids based on the sulfo-phosphovanillin reaction. Amer. J. Clin. Pathol. 53:89-91. Naber, E. C , 1976. The cholesterol problem, the egg, and lipid metabolism in the laying hen. Poultry Sci. 55:14-30. Peter, J. B., and W. Fiehn, 1973. Diazacholesterol myotonia: accumulation of desmosterol and increased adenosine triphosphatase activity of sarcolemma. Science 179:910—912. Postma. T., and J.A.P. Stroes, 1968. Lipid screening in clinical chemistry. Clin. Chem. Acta 22: 569—578. Robbins, W. E., M. J. Thompson, J. A. Svoboda, T. J. Shortino, C. F. Cohen,! S. R. Dutky, and O. J. Duncan III, 1975. Nonsteroidal secondary and tertiary amines: inhibitors of insect development and metamorphosis and A 24 -sterol reductase system of tobacco homworm. Lipids • 10:353-359. Sachs, B. A., and L. Wolfman, 1963. 22,25-diazacholestanol dihydrochloride: a new inhibitor of cholesterol biosynthesis. Metabolism 12:608-617. Seiler, D.. W. Fiehn, and E. Kuhn, 1975. Desmosterol accumulation in rats with experimental myotonia. Z. Klin. Chem. Klin. Biochem. 13:225-229. Singh, R. A., J. F. Weiss, and E. C. Naber, 1972. Effect of azasterols on sterol metabolism in the laying hen. Poultry Sci. 51:449-457.