Suppression of cholesterol biosynthesis by constituents of Barley Kernel

Suppression of cholesterol biosynthesis by constituents of Barley Kernel

Atherosclerosrs. 51 (1984) Elsevier Scientific 75 75-87 Publishers Ireland, Ltd. ATH 03457 Suppression of Cholesterol Biosynthesis Constituen...

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Atherosclerosrs.

51 (1984)

Elsevier Scientific

75

75-87

Publishers

Ireland,

Ltd.

ATH 03457

Suppression of Cholesterol Biosynthesis Constituents of Barley Kernel * Warren ’USDA’ Department

C. Burger t, Asaf A. Qureshi ‘.*, Zafeer Z. Din * ** and Charles E. Elson * Naji Abuirmeileh

ARS,

Barley and Malt Laboratory,

of Agronomy

and

’ Department

501 N. Walnut

by

I,

St., Madison,

WI 53705

Unioersity

of Wisconsin,

of Nutritional Sciences,

and Madison,

WI 53706 (U.S.A.)

(Received 29 October, 1982) (Revised, received 10 October, 1983) (Accepted 10 October, 1983)

Summary Hepatic P-hydroxy-P-methylglutaryl CoA (HMG-CoA) reductase, cholesterol 7cz-hydroxylase (7a-hyd), and fatty acid synthetase (FAS) activities and cholesterol levels were determined in chicks fed isonitrogenous corn- and high-protein barley flour (HPBF) based diets. HMG-CoA reductase (-27%) 7cY-hyd (-30%), and serum cholesterol ( - 13%) were reduced, whereas FAS increased (28%) in comparison to a corn-based (control) diet. Fractions obtained by serial extractions of HPBF with solvents of increasing polarity were fed at levels equivalent to 20% HPBF in a corn-based diet to female White Leghorn (WHL) chickens for 3 weeks. A petroleum ether-soluble fraction of HPBF produced 3 effects: an increase in body weight (IS%), a strong suppression of HMG-CoA reductase ( - 36%) and FAS ( - 40%) accompanied by decreases in serum triglyceride ( - 9%) and cholesterol levels ( - 23%). The methanol-soluble fraction produced a significant suppression of HMG-CoA reductase (- 49%) and serum cholesterol level ( - 29%), and an increase in FAS activity (95%). This investigation was supported in part by Grant No. 8000597, USDA-SEA/AR Competitive Research Grants Office Human Nutrition Program, Research Grant HL-25591 from the National Heart, Lung Institute, National Institutes of Health, United States Public Health Service and Hatch Fund No. 1718, of the Research Division, College of Agricultural and Life Sciences, University of Wisconsin-Madison. * To whom reprint requests should be sent. ** Cooperative investigation between the Agricultural Research Service, U.S. Department of Agriculture, and the College of Agricultural and Life Sciences, University of Wisconsin-Madison. *** Present address: Director, Department of Biological Sciences, Yarmuk University, Irbid, Jordan.

0021-9150/84/$03.00

0 1984 Elsevier Scientific

Publishers

Ireland,

Ltd.

76

These effects were duplicated in 7-week-old broiler chickens which also showed a significant decrease in chol-LDL (low density lipoprotein) levels by these fractions. The factor(s) lowering serum cholesterol concentration was about equally divided between the polar and nonpolar fractions, and each was significantly more effective than the 20% HPBF in the corn-based diet. The observed effects on lipogenesis and cholesterogenesis might be attibuted to a number of chemical constituents of HPBF. but cannot be attributed to the water-insoluble plant fibers. Key words:

Chickens - Cholesterol biosynthesis - High-protein burlqv flour (HPBF) ~ Lipogenesis - Liver ~ Nonpolar und polar fractions of HPBF - Serum cholesterol - Serum triglycerides

Introduction

Western diets are generally accepted as producing higher blood cholesterol levels. This has been variously attributed to their higher contents of cholesterol, saturated fatty acids, animal protein, or simple sugars as well as to lower fiber content [l-3]. On the other hand diets rich in cereal grains, principally oats in humans [4] and barley in chickens [5] are hypocholesterolemic. Partial (5-20%) replacement of corn in the diets of 4-6-week old chickens with high-protein barley flour (HPBF) resulted in significant reductions in hepatic /?-hydroxy-P-methylglutaryl coenzyme A (HMG-CoA) reductase activity and lower plasma cholesterol levels, and significant increases in body weight [5]. These findings prompted us to fractionate HPBF in order to obtain an indication of the chemical nature of the active agents and to initiate purification for the eventual identification of the materials. This communication describes the effects of feeding the dried solubles obtained from serial extractions of HPBF with solvents of increasing polarity at levels equivalent to 20% HPBF in a control corn-based diet. The parameters measured consisted of HMG-CoA reductase, cholesterol 7a-hydroxylase (the rate-limiting enzymes in the synthesis and degradation of cholesterol biosynthesis, respectively), and fatty acid synthetase. The activities of these enzymes were measured at their maxima, which was achieved by fasting (48 h) and refeeding (72 h). resulting in an increase in the activities of these enzymes [6]. The effects of the soluble components of HPBF on the levels of total cholesterol, cholesterol in high density lipoproteins (chol-HDL), low density lipoproteins (chol-LDL) and triglycerides in the serum are also described. Materials

and Methods

Experimental materials were purchased from the following sources malonyl-CoA, RS-mevalonic acid, glucose-6-phosphate, dithiothreitol, *

Mention of a trademark or proprietary product product by the U.S. Department of Agriculture other products that may also be suitable.

does not constitute a guarantee and does not imply its approval

*: acetyl-CoA, DL-isocitrate, or warranty of the to the exclusion of

77

NADP+, NADPH, NADH, ATP, glucose-6-phosphate dehydrogenase, cysteamine, Tween-80, triethanolamine hydrochloride, sodium malate, coenzyme A, malate dehynicotinamide, DL-3-hydroxy-3-methylglutaryl-CoA and 6-phosphodrogenase, gluconate (Sigma Chemical Co., St. Louis, MO); cholesterol (Aldrich Chemical Co., Milwaukee, WI) was recrystallized twice in glacial acetic acid, 7a-hydroxycholesterol (5cholesten-3/3-7a-diol) and 7a-ketocholesterol (5-cholesten-3/?-ol-7-one) (Steraloids, Inc., Wilton, NH); EDTA (Fisher Scientific Co., Itasca, IL); bovine serum albumin Cleveland, OH), and DL-3-hydroxy-3(Nutritional Biochemicals Corporation, methyl-[3-‘4C]glutaryl-CoA (specific activity, 26.3 mCi/mmole, and Aquasol (scintillation solution) (New England Nuclear, Boston, MA). HPBF was contributed by the Minnesota Grain Pearling Company, East Grand Forks, MN. TABLE

1

EFFECT OF HPBF ON BODY WEIGHT, CHOLESTEROL IN PAIR-FED S-WEEK-OLD Parameters

HEPATIC FEMALE

ENZYME CHICKENS

Corn (control) 65.5% _

Corn-based diet * High-protein barley flour (HPBF) Soybean meal (44% protein)

26.0%

Initial body weight (g; 5-week-old) Final body weight (g; S-week-old) Percentage gain h Liver weight (g)

ACTIVITIES

AND

SERUM

HPBF _ 79.5% 12.0%

297 _+I8 506 +19 209 * 12.2+ 0.4 * (100) h

303 +12 512 +15 209 * 12.4* 0.5 * (102) h

550.0+39.0 * (100) 2.4_+ 0.5 * (100)

400.0 + 37.0 ** (73) 1.7* 0.2 ** (70)

Serum cholesterol (mg/dl) Serum triglycerides (mg/dl)

237 138

207 143

Fatty acid synthetase e Malic enzyme ’ Citrate-cleavage enzyme g

35.8 + 4.0 * (100) 229 f 12.0 * (100) 10.7 f 0.7 * (100)

P-Hydroxy-b-methylglutaryl-CoA Cholesterol 7a-hydroxylase d

a

reductase



+ll.O * (100) + 7.0*(100)

* 9.0 ** (87) + 6.0 * (104)

45.7zt 4.0 ** (128) 315 + 14.0 ** (138) 14.7+ 1.5 ** (137)

Each diet also contains meat scrap-50% protein (5.0%). alfalfa meal-17% protein (1.0%). dicalcium phosphate (1%) calcium carbonate (0.5%). vitamin and mineral mixture (1%). grit (5%) was incorporated at the expense of each diet. Vitamin and mineral mixture contains/kg: vitamin A 3000 I.U., vitamin D, 500 I.U., vitamin E 10 I.U.; vitamin K, 5 mg; choline 1.3 g; thiamine 1.8 mg: niacin 27 mg; riboflavin 2.5 mg; pyridoxine 3 mg: calcium pantothenate 3.0 mg; vitamin B,, 5 mg; lysine-HCI 1 g; methionine 0.72 g; sodium chloride (NaCl) 2 mg; zinc sulphate (ZnSO,) 50 mg and manganese dioxide (Mn,O) 50 mg. Analysis of corn. and high-protein barley flour 8.9% and 17.5% protein, respectively. h Feeding period was 3 weeks; time of killing was 08.00 h; data expressed as mean +SD; N = 9 chickens per group. F pmoles of mevalonic acid synthesized/min/mg of microsomal protein. d pmoles of [‘4C]cholesterol into [‘4C]7a-hydroxycholesterol/min/mg of microsomal protein. e nmoles of NADPH oxidized/min/mg of cytosolic protein. I nmoles of NADP+ reduced/min/mg of cytosolic protein. 8 nmoles of product formed/min/mg of cytosolic protein. h Percentages of respective control activity data are in parentheses. *,** Means on a line and without a common superscript letter are significantly different P < 0.01.

78

Experiment 1: Effect of corn and HPBF on hepatic enzyme activities, cholesterol and fatty acid biosynthesis in pair-fed &week-old female chickens Isonitrogenous diets were prepared with corn or HPBF as the major component (Table 1). A group of nine Sweek-old White-Leghorn female chickens (285-315 g) housed 3 per cage with continuous light was fed the HPBF diet ad libitum. A similar group of 9 chickens was fed the corn-based diet (control) but at levels restricted to the intake of the HPBF diet by the test group. After 16 days, the birds were fasted for 48 h and refed the appropriate diet for 72 h. At 8:00 a.m. on the 22nd day the birds were weighed, killed by severing the carotid arteries, blood (10 ml) was collected, and the liver from each bird was collected. The liver was washed, weighed and placed on ice; serum was isolated and stored at -20°C. The same experiment was repeated 3 times. Experiment 2: Effect of corn and high-protein barley flour and its different fractions on hepatic enzyme activities and serum lipids in I2-week-old chickens Forty two g-week-old White-Leghorn female chickens, weighing approximately 600-680 g were divided into 7 groups, 6 per treatment, and were housed 2 per cage. One group was fed the corn-based diet as a control (Table 2) and the other groups TABLE

2

PERCENT COMPOSITION OF CHICKEN LIVER WEIGHTS OF FEMALE CHICKENS Nutritional

Corn Corn Corn Corn Corn Corn Corn

+ + + + + +

state

20% HPBF petr. ether SF ’ ethylacetate SF ’ methanol SF r water SF ’ residue ’

Diets ’

DIETS

AND

EFFECT

Body weight (g)

Corn

HPBF h Initial’

(%)

(8)

61.5 41.5 60.8 61.0 60.7 59.8 45.2

20.0 0.7 0.5 0.8 1.7 16.3

Finai’

OF HPBF

Liver weight’ (g)

ON

BODY

AND

Liver weight/ 100 g body weight

Gain (g)

68Ok57 643 + 48 603k42 676565 634?58 608&51 653+56

1010~92 983k63 1008&49 979k61 924?76 92Ok94 970*51

330 340 405* 303 290 312 312

27.8 f 26.9 k 29.5*3 26.8 + 27.6 * 28.2 5 26.0 f

3 2 3 4 2 4

2.75 2.73 2.93 * 2.74 2.99 * 3.07 * 2.68

a Each diet also contains soybean meal-44% protein (30.0%); meat-scrap-50% protein (5.0%); alfalfa meal-17% protein (1.0%); dicalcium phosphate (1%); calcium carbonate (0.5%); vitamin and mineral mixture (1%); vitamin and mineral mixture contains/kg: vitamin A 2000 IU. vitamin D, 200 ICU. vitamin E 10 IU, vitamin K, 5 mg, choline 1.3 g. thiamin 1.8 mg, niacin 27 mg. riboflavin 3.6 mg. pyridoxine 3 mg, calcium-pantothenate 10.0 mg, vitamin B,, and sodium chloride (NaCI) 2 mg. zinc sulphate (ZnSO,) 50 mg and manganese dioxide (MnO,) 50 mg: grit (5%) was incorporated at the expense of each diet. h HPBF= High-protein barley flour. After extraction of 250 g of HPBF, the following was obtained: 8.7 g petroleum ether SF; 6.3 g ethyl acetate SF; 10.5 g methyl alcohol SF: 21.5 g water SF; and 203 g residue. ’ Nine-week-old female chickens. ’ Twelve-week-old female chickens. ’ Data expressed as mean k SD; N = 6 chickens per group. ’ SF = Soluble fraction equivalent to 20% HPBF in corn-based diet. * Significantly different from control (P < 0.01).

19

were fed modified forms of the control diet; one modification was the partial replacement (20%) of corn with HPBF. The other modifications involved the addition to the control diet, at a level equivalent to 20% HPBF, the solids obtained by evaporating each solvent extract. These chickens were exposed to a 24-h light period and water was provided ad libitum. At the end of 16 days of feeding, the birds were fasted for 48 h and refed the appropriate diet for 12 h. At 8:00 a.m. on the 22nd day the birds were weighed, killed by severing the carotid arteries, brood was collected, and samples of the liver were taken for analysis to study the lipid metabolism. This experiment was repeated twice. Procedure for the extraction of different fractions of HPBF The HPBF (250 g) was stirred at room temperature with light petroleum ether (500 ml) for 2 h. After standing for 1 h, the solvent was decanted. The procedure was repeated twice. The last extraction was completed by filtering through a sintered glass funnel under vacuum. The 3 extracts were combined and then concentrated to dryness under vacuum at 60°C. The residual HPBF was extracted successively as described above with ethyl acetate, methyl alcohol and water. The final residue was dried overnight in an oven at 60°C. The fractionation yielded 8.7 g petroleum ether-solubles, 6.3 g ethyl acetate-solubles, 10.5 g methyl alcohol-solubles, 21.5 g water-solubles and 203 g of residue. The required amount of each fraction for each diet (equivalent to 20% HPBF) was taken up in a minimal volume of the respective solvent (500 ml for 10 kg feed) and mixed thoroughly with the corn-based diet (Table 2) and the solvent was allowed to evaporate overnight in a pan (air-dried under the hood). These diets were fed to different groups of chickens. Experiment 3: Effect of HPBF, its nonpolar and polar fractions on body, liver weights, and hepatic enzyme activities in 7-week-old female broiler chickens Twenty four 3-week-old female broiler chickens, weighing 485-530 g were divided into 4 groups of 6 birds each. These groups were fed isonitrogenous corn, or corn + 20% HPBF, corn + HPBF petroleum ether (nonpolar)-soluble fraction, or corn + HPBF methanol (polar)-soluble fraction based diets as described in Experiment 2 (Table 2). After 23 days the birds were fasted 48 h, refed 72 h and samples were collected as described in Experiment 1. Preparation of liver homogenates The liver homogenates were prepared in 0.1 M potassium phosphate buffer, pH 7.4, containing 0.004 M MgCl,, 0.001 M EDTA, and 0.002 M dithiothreitol. The tissue was chopped and suspended in the buffer (1: 2, wt : vol) and homogenized at 0-4°C with a Polytron homogenizer for 15 s at setting 8. The homogenate was centrifuged for 10 min at 5000 x g. The supernatant was passed through cheese cloth and the volume of the supernatant was recorded. An aliquot of this supernatant (10 ml) was then centrifuged for 10 and 60 min at 20000 x g and 100000 X g, respectively. The supernatant after each centrifugation was passed through cheese cloth. The 100000 x g supernate (cytosol) and precipitate (microsomes) were treated as

80

described in an earlier report [7] and stored at -20°C until they were assayed for enzymatic activities. Assays for /3-hydroxy-&methylglutaryl-CoA (HMG-CoA) reductase and cholesterol 7ol-hydroxylase have been described [7,8]. The activities of malic enzyme, citrate-cleavage enzyme, and fatty acid synthetase in the cytosol were assayed spectrophotometrically at 25’C [8]. Protein concentrations were estimated by a modification of the biuret method using bovine serum albumin as a standard [9]. Estimation of serum cholesterol and triglyceride cpncentrations Cholesterol and triglyceride concentrations in serum samples were estimated using the Worthington ‘Cholesterol Reagent’ ‘.and ‘Triglyceride Reagent’ set, obtained from Worthington Diagnostics Division of Millipore Corporation, Freehold, NJ. Low density (LDL) and very low density lipoprotiens (VLDL) were isolated from the serum (100 ~1) by precipitation with a mixture of phosphotungstic acid 9.7 mM (10 ~1) + 0.4 M MgCl, (10 ~1). After standing for 5 min at room temperature, the mixtures were centrifuged at 2000 x g for 10 min, the supernatant was removed and was used to determine the level of cholesterol in HDL. The precipitate was dissolved in 0.1 M sodium citrate buffer (100 ~1) and the level of cholesterol (LDL + VLDL) was estimated using the above method. The estimations of chol-LDL were also calculated from (total-chol) - (chol-HDL + triglycerides/5) according to the reported method [lO,ll], which gave slightly lower values, as expected, from the values of chol-LDL + chol-VLDL. Expression of data and statistical methods Enzyme data are presented as specific activities (units/mg cytosolic or microsomal protein/min). Statistical comparison of results were performed by a one-way analysis of variance. When the F test indicated a significant effect, the differences between the means were analyzed by a protected LSD test [12]. Results The weight gain of 5-week-old chicks fed the HPBF-based diet for 3 weeks ad libitum was equal to that of chicks pair-fed the isonitrogenous corn-based diet (Table 1). In earlier experiments we noted that l-day-old and 2-week-old chicks fed the HPBF-based diet ad libitum grew at rates 70 and 79% respectively, that of chicks fed an isonitrogenous corn-soy diet [5]. These differences were attributed to an inability of chicks of less than 4 weeks of age to effectively utilize high levels of barley or HPBF in the diet. Under the conditions of Experiment 1, the HPBF-diet produced lower hepatic HMG-CoA reductase (73% of control, P < 0.01) and cholesterol 7a-hydroxylase (70% of control, P < 0.01) activities and a lower serum cholesterol level (87% of control, P < 0.01). Similar effects on these parameters were noted in the earlier study with l-day-old and 2-week-old chicks. These changes accompanied increased lipogenie activity estimated by monitoring citrate-cleavage enzyme (137% of control,

3

state

OF HPBF

A-E

I

e

d

c

b

a

ON HEPATIC

6.7f0.2A 3.7 * 0.2 5.0 * 0.4 5.5 * 0.4 3.4 * 0.5 4.1+ 0.4 4.6 + 0.5

(lOO)e B (55) c (75) c (82) B.D (51) D.E (61) C‘.E(69)



ACTIVITIES

Cholesterol 7a-hydroxylase

ENZYME

LIPIDS

62* 8 A (100)’ 72+ 7A(116) 37+ 3’(60) 113~16c(182) 121*14’(195) 151 f 12 D (244) 141+ 11 C.D (227)

Fatty acid synthetase d

AND SERUM

208& sA(lc@)r 178? 9B(86) 164* 7 B (79) 2045 10 A (98) 159+12a(76) 202 * 5 A (97) 201+ 6 * (97)

Cholesterol

Serum (mg/lOO

IN 12-WEEK-OLD ml)

a

242k 308+ 219k 231 f 314k 262 f 247k

when observed.

8A(100)r 6 B (127) 4’(91) 7 A.c (95) 6 B (130) 6 A (108) 7 A (102)

Triglycerides

CHICKENS

Feeding period was 3 weeks: time of killing was 08:OO a.m.; Data expressed as mean + SD: N = 6 chickens per group. fi-Hydroxy-/3-methylglutaryl-CoA reductase; pmoles of mevalonic acid synthesized/min/mg of microsomal protein. pmoles of [‘4C]cholesterol into [ “C]7a-hydroxycholesterol/min/mg of microsomal protein. qmoles of NADPH oxidized/min/mg of cytosolic protein. Percentage of respective control activity data are in parentheses. SF = Soluble fraction equivalent to 20% HPBF in corn-based diet. Means within a column and without a common superscript letter are significantly different P < 0.05. Higher levels of significance, reported in the text.

690+44A (100)’ 460+ 42 A,~ (67) 44Ok37 B (64) 460 f 38 A.B (67) 350+37C(51) 370 + 32 ’ (52) 630 of 43 A (92)

HMG-CoA reductase b

FRACTIONS

Corn + 20% HPBF Corn + petr. ether SF ’ Corn + ethyl acetate SF ’ Corn + methanol SF ’ Corn + water SF f Corn + residue ’

corn

Nutritional

EFFECT

TABLE

are

m

A-D

g

I

e

d

c

b

a

ml)

*

5A(lOO)

82 f 9A(100) 233 k 14 A (100) 10.9* l.OA(lOO)

140

+ 7A (104)

126 f 7’(154) 404 k 18 ‘(173) 12.4+ 0.7 A (114)

146

2.82 (92) 1.24 (81)

141 f 13 A (90) 50.0 + 6 A (98) 62.0+ 5 ’ (79)

157 & 11 A (100) 51.0+ 7 A (100) 78.0+ 6 A (100)

3.08 (100) 1.53 (100)

500 + 42 ’ (72) 6.2+ 0.3 ’ (46)

690 +47 A (100) 13.6* 0.6 A (100)

HEPATIC

ENZYME

f

4B(79)

31 f 3’(38) 198 f 12 ’ (85) 8.7 f 0.2 B (80)

111

2.60 (84) 1.14 (75)

121 * 9 B(77) 46.5* 6A(91) 53.0 + 6 ’ (68)

470 f 43 it (68) 4.1 * 0.2 c (30)

1270 k25 ‘(121)s 41.s* 3 A (110)

346 f 34 c (50) 3.4 + 0.2 D (25)

1056 +30A(101)s 39.4+ 3 A (104)

+ 6’(124) 151 f 12 D (184) 380 + 16 B (163) 20.5 + 2.1 c (188)

174

2.44 (79) 0.7 (46)

117 *11 a(75) 48.0 f 7 A (94) 34.0 f 5 D (44)

LIPIDS

Soluble Fraction

AND SERUM

Corn + Methanol

ACTIVITIES

per group.

Corn + Petr. Ether Soluble Fraction

WEIGHTS,

Feeding period was 4 weeks; Time of killing was 08:OO a.m.; Data expressed as mean f SD; N = 6 chickens pmoles of mevalonic acid synthesized/min/mg of microsomal fraction. pmoles of [‘4C]cholesterol into [‘4C]7a-hydroxycholesterol/min/mg of microsomal fraction. nmoles of NADPH oxidized/min/mg of cytosolic fraction. nmoles of NADP+ reduced/min/mg of cytosolic fraction. nmoles of product formed/min/mg of cytosic fraction. Percentages of respective control activity data are in parentheses. Means on a line and without a common superscript letter are significantly different P -c 0.01.



(mg/lOO

Fatty acid synthetase d Malic enzyme e Citrate-cleavage enzyme

Serum triglycerides

Total ChoI/ChoI-HDL Chol-LDL/ChoI-HDL

Serum cholesterol (mg/lOO ml) Cholesterol-HDL (mg/lOO ml) Cholesterol-LDL (mg/lOO ml)

CoA reductase b Cholesterol ‘la-hydroxylase



1216 f20B(116)s 41.5 + 3 A (109)

1050 f30A(100)9 38.0 * 4 A (100)

Body weight (g) Liver weight (g)

/3-Hydroxy-/3-methylglutaryl-

Corn + 20% HPBF

LIVER

Corn

AND

Parameters

EFFECT OF HIGH-PROTEIN BARLEY FLOUR ON BODY ‘I-WEEK-OLD FEMALE BROILER CHICKENS a

TABLE 4 IN

w

83

P < O.Ol), malic enzyme (138% of control, P < 0.01) and FAS (128% of control, P < 0.01) activities (Table 1). Similar responses in cholesterol and lipid metabolism were observed earlier in younger birds [5] and appear to be affected little by the differences in the growth response of the birds. These observations prompted us to use a sequential extraction with solvents of increasing polarity as a preliminary step in the purification of the active agents in HPBF. The results of feeding the solids thus obtained to 9-week-old chickens for 3 weeks at levels equivalent to 20% substitution of HPBF for corn in the control diet are summarized in Tables 2 and 3. Distributed among the solvent fractions are components which influenced weight gain (Table 2) HMG-CoA reductase. cholesterol 7a-hydroxylase and fatty acid synthetase activities and serum concentrations of cholesterol and triglycerides (Table 3). Components extracted from HPBF with petroleum ether significantly increased weight gain (123% of control, Table 2) and concomitantly decreased fatty acid synthetase activity (60% of control, P < 0.01, Table 3) whereas components extracted from HPBF by more polar solvents tended to decrease weight gain (Table 2) and significantly increase fatty acid synthetase activity (P < 0.01, Table 3). Significantly higher and lower concentrations of triglycerides were recorded for the serum of birds fed the methanol and petroleum ether extracts, respectively, of HPBF (Table 3). Feeding either 20% HPBF or solvent fractions of HPBF significantly lowered hepatic HMG-CoA reductase activity (Table 3); serum cholesterol concentrations were lower in groups fed 20% HPBF (P < O.M), the petroleum ether extract of HPBF (P -c0.01). extract of HPBF (P < 0.05)and the methanol Cholesterol 7a-hydroxylase activity in each of the test groups was lower than the control activity (all P < 0.01, Table 3). Feeding 20% HPBF to 9-week-old chicks failed to elicit the growth response (Table 2) previously found when the diet was fed to l-day-old and 2-week-old chicks [5]. The growth response was demonstrated however, when 3-week-old chicks were fed the diet (Table 4). The effects of feeding the petroleum ether- and methanol-solubles of HPBF on weight gain, fatty acid synthetase activity and serum triglycerides in these chicks were consistent with the effects noted on Tables 2 and 3 for 9-week-old chicks. The changes in other lipogenic enzymes, namely citrate-cleavage enzyme and malic enzyme, mediated by feeding these solvent extracts of HPBF were consistent with the changes noted in fatty acid synthetase activity (Table 4). Significant changes in hepatic HMG-CoA reductase and cholesterol 7a-hydroxylase activities and in serum cholesterol concentration attending the feeding of these solvent extracts (Table 4), are also consistent with those recorded for the older birds (Table 3). The remarkable feature of this significant lowering of the serum cholesterol conoentration mediated by feeding the solvent extracts is that only the LDL cholesterol fraction was lowered to a significant extent (P < 0.01, Table 4). Consequently, the ratio, chol-LDL/chol-HDL is lower than the control in the chicks fed the solvent extracts of HPBF.

121

101 *

Petroleum ethersolubles

Methanolsolubles

Percentages of respective control 3-week-old chicks fed 4 weeks. 9-week-old chicks fed 3 weeks. Nonsignificant from control.

116

20% HPBF

a h ’ *

100

Corn

3b

activity.

88

123

103 *

100

9=

AND

50

68

72

100

51

64

67

100

25

30

46

loo

51

75

55

loo

9

3

3

9

Cholesterol ‘Icr-hydroxylase

9-WEEK-OLD

HMG-CoA reductase

enzyme activity a

Hepatic

Diets

Weight gain

OF 3-WEEK-OLD

5

THE MAGNITUDES OF THE RESPONSES AND METHANOL EXTRACTS OF HPBF

TABLE

184

38

154

100

3

Fatty acid synthetase

195

60

116 *

loo

9

CHICKENS

AND

75

71

90

100

3

Cholesterol

71

71

80

100

9

124

79

104 *

100

3

Triglyceride

a

ETHER

130

91 *

127

100

9

TO PETROLEUM

Serum concentration

TO 20% HPBF

85

Discussion Effects consistently observed followed the addition of HPBF to the diets of chicks ranging in age from 1 day to several months include the lowering of serum cholesterol level and hepatic HMG-CoA reductase and cholesterol 7ol-hydroxylase activities, and the elevation of hepatic fatty acid synthetase activity. Less consistent is an effect on weight gain, an effect that appears to be modulated by the age of the bird and the level of dietary HPBF. Very young birds fed high levels of HPBF consume less diet and the gain per unit diet intake is lower; older birds fed this diet consume less diet but the gain per unit diet consumed matches that of controls. Diets containing 20% HPBF on the other hand increase the weight performance of birds, the effect being greater in the younger bird. The microsomal enzymes; HMG-CoA reductase and cholesterol 7ti-hydroxylase are generally considered to be the respective rate-limiting enzymes for hepatic cholesterol [13] and bile acid [14] synthesis. The two synthetic pathways are closely linked with cholesterol, the product of the former serving as the substrate of the latter. The two rate-limiting enzymes respond in unison under some conditions including diurnal variation, fasting and refeeding, and hypophysectomy [15-171. The temporal action of the HPBF factor on cholesterol metabolism, suggests that first it is directed toward the inhibition of HMG-CoA reductase, with a lowering of serum cholesterol level and subsequently a substrate-mediated lowering of cholesterol 7a-hydroxylase activity. In support of this temporal relationship it was noted that cholesterol 7a-hydroxylase activity is not significantly lowered by feeding 20% HPBF to restricted ovulator hens, hens which exhibit high levels of serum cholesterol due to a sex-linked genetically transmitted failure to transfer cholesterol to developing yolks [18]. The serial extraction of HPBF revealed the presence of two effecters of lipogenic activity. One effector, extracted with petroleum ether, suppressed fatty acid synthetase activity; the second extracted with more polar solvents following the removal of the first effector, markedly increased fatty acid synthetase activity. The gain in body weight was inverse to fatty acid synthetase activity which suggests that the barley factors directly influenced the form in which dietary energy was retained. Whereas the solvent extraction process revealed two diverse effecters of fatty acid synthetase, an inhibitor of HMG-CoA reductase (and presumably, temporally of cholesterol 7a-hydroxylase) was present in each of the solvent residues. The magnitudes of the responses generated by feeding 20% HPBF. the equivalent petroleum ether-soluble fraction and the equivalent methanol-soluble fraction to 3-week-old and 9-week-old chicks are summarized in Table 5. The relationship between fatty acid synthetase activity and weight gain and between HMG-CoA reductase, serum cholesterol concentration and cholesterol 7a-hydroxylase are apparent. The prevailing concept is that components of plant materials interfere with the reabsorption of bile acids therein causing a reduction in serum cholesterol levels [19-251. The more recent publications suggest that oat-bran [22] and whole grain [23] components exert hypocholesterolemic effects on the LDL fraction. Our data

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obtained by feeding chickens solvent-extracted solids of HPBF as 0.7% (Table 2) of the diet, on the other hand, point to a direct action of the plant material on cholesterol biosynthesis with a concomitant lowering of LDL cholesterol. These results were obtained with a diet free of (Table 2) or containing only 5% (Table 1) animal products. A water-soluble inhibitor of cholesterol biosynthesis, isolated from Lentinus edodes [26], was found to be 2(R), 3(R)-dihydroxy-4-(9-adenyl)-butyric acid [27] with the R groups representing groups of varying degrees of polarity. A water-soluble fraction of garlic also is reported to specifically lower LDL cholesterol [28]. Our studies [29] indicate that this water-soluble component of garlic, added to the diet at levels of 0.2-1.6s in linear fashion inhibited HMG-CoA reductase activity (up to 40%). and lowered LDL cholesterol level by 38%. The above natural products are components of dormant seeds and bulbs. We have also found that AM0 1618, a quaternary ammonium derivative of carvacrol which plays a role in plant dormancy and gibberellin biosynthesis [30], is a powerful in vivo and in vitro inhibitor of HMG-CoA reductase as well [8]. Work on the identification of the active agents from barley is underway. Acknowledgements The authors wish to thank Dr. Burt Olson for the use of the Isocap/300 Liquid Scintillation counter. We also thank Faye Roed for her excellent assistance.

Nuclear editorial

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