347
Atherosclerosis, 32 (1979) 347-357 0 Elsevier/North-Holland Scientific
Publishers,
EFFECT OF MILK CONSTITUENTS GENESIS
AHMED
ASHOUR
Department (U.S.A.)
AHMED
Ltd.
ON HEPATIC
*, R.D. MCCARTHY
of Food Science,
CHOLESTEROL-
and G.A. PORTER
The Pennsylvania State University, University Park, PA 16802
(Received 8 June, 1978) (Revised, received 5 September, (Accepted 6 December, 1978)
1978)
Summary Two preparations active in reducing hepatic cholesterol biosynthesis were isolated from bovine skim milk. One of the inhibitors was in the dialysate and was identified as erotic acid (OA). The other inhibitor, present in the retentate, was not identified. Orotic acid appears to act by inhibiting cholesterol biosynthesis before the formation of mevalonate, whereas the retentate inhibitor exerts its effect beyond the formation of mevalonate in the biosynthetic pathway. Human milk also inhibited the incorporation of both labeled acetate and mevalonate into cholesterol by rat liver. Orotic acid was not detectable in human milk samples employed in this study. Administration of [6-14C]orotate to rats revealed its conversion to uracil in the liver. Subsequent work demonstrated that uracil had inhibitory activity on hepatic cholesterol biosynthesis similar to that of orotate when incubated with rat liver slices. Key words:
Cholesterolgenesis
-Inhibitors
-Liver
-Milk
- Rats
Introduction Several studies document effects of milk on hepatic cholesterolgenesis and blood cholesterol concentrations. Shah [l] reported that the incorporation of labeled mevalonate into digitonin-precipitated sterol was lower in liver homogenates from suckling rats compared to those of weaned rats. He suggested that the activities of one or more of the enzymes catalyzing the conversion of Authorized for publication as paper No. 5359 in the Journal Experiment Station. * Present address: Fatih University, Tripoli. Libya.
Series of The Pennsylvania
Agricultural
348
mevalonate into cholesterol change after weaning. McNamara et al. [2] reported that rat milk contains a thermostable, nondialyzable factor which depresses the activity of hydroxymethylglutaryl-CoA reductase in the liver of adult rats. In 1974, Mann and Spoerry [3] studied Masai Africans. When enough yogurt was consumed to cause weight gain, blood serum cholesterol levels declined. In the same year, Boguslowski and Wrobel [4] established the presence of a compound in bovine and human milk that inhibited hepatic cholesterolgenesis in the rat. The bovine milk factor was heat-stable, dialyzable, and present in the supernatant fraction after the protein had been precipitated with acid. It reduced the incorporation of both acetate and mevalonate into hepatic cholesterol. Malinow and McLaughlin [5] studied the plasma cholesterol levels in rats which were fed, beginning at 2 weeks of age, rat chow mixed with either water or bovine skim milk. The blood plasma cholesterol concentration for those given skim milk was lower in 43- and 64-day-old males and in 64day-old females than in those receiving water. During 1976, Bernstein et al. [6] predicted that the factor in bovine milk described by Boguslowski and Wrobel [4] was erotic acid (OA). Their data, obtained with liver homogenates, indicated that acetate, but not mevalonate, incorporation into cholesterol was reduced by cultured buttermilk, bovine milk, or OA, thus suggesting that the inhibition is at the initial steps in the biosynthetic pathway. Recently, Howard [ 71 and Marks and Howard [8] postulated that the hypochol,esterolaemic factor in milk was calcium, while Mann [9] and Nair and Mann [lo] postulated the effective agent to be /?-methylglutarate. The. above divergent observations led to the present effort to purify and define the factor(s) in bovine milk that affect hepatic cholesterol biosynthesis. Materials and Methods Isolation of cholesterol inhibitors from milk One hundred ml of pasteurized bovine skim milk was dialyzed for 48 h at 4°C against two changes of 500 ml distilled water. The dialyzable inhibitor (OA) was isolated by a combination of anion exchange and silicic acid column chromatography and quantitated by GLC [ 111. The retentate inhibitor fraction was obtained by the following technique. Casein was removed from the retentate of skim milk by reducing the pH to 4.6 with concentrated HCl and centrifuging at 17,600 X g for 20 min. The supernatant was freeze-dried and the resulting product dissolved in 10 ml of 0.02 M Tris buffer (pH 7.5) containing 0.2 mM sodium azide. A column (90 cm X 2.5 cm) of Sephadex G-100 (Pharmacia Fine Chemicals, Inc.) was equilibrated with distilled water. A 2.5 ml portion of the sample was placed on the column and eluted with Tris buffer at pH 7.5 with a flow rate of 2 to 2.5 ml/min. Twenty ml portions were collected with absorbance being monitored at 280 nm. Seven pooled fractions (Fig. 2) were dialyzed separately against tap water for 48 h and against distilled water for another 48 h, freezedried, and tested for their effect on cholesterol biosynthesis. Tissue incubation studies Male Wistar rats, 6-8 weeks old, were fed standard
rat chow and water ad
349
libitum. Excised livers were quickly rinsed in cold 0.9% NaCl and liver slices 0.5 mm thick were prepared with a Stadie Riggs tissue slicer. The slices were weighed and immediately transferred to incubation vials containing 3 ml of either Krebs-Ringer bicarbonate buffer or the buffer of Lakshmanan et al. [ 121. Either [l-14C]Na acetate (55.4 mCi/mM) or [2-‘4C]mevalonic acid (8.05 mCi/mM) was added to each flask. Incubations were conducted in a Dubnoff metabolic shaker incubator at 37°C for 3 h in an atmosphere of 95% O2 and 5% COZ, or when CO* was collected, in an atmosphere of laboratory air. Incubations were terminated by the addition of 4 ml of 20% KOH in methanol. For the determination of radioactivity associated with cholesterol, 4 mg of nonradioactive cholesterol was mixed with the sample before it was saponified for 3 h at 65°C. The non-saponifiable fraction was extracted 3 times with 15 ml portions of hexane which were combined and evaporated to dryness under nitrogen. Subsequently, the non-saponifiable residue was dissolved in 5 ml of 1% digitonin in 50% ethanol and permitted to stand for 12 h at room temperature. The digitonide precipitates recovered by centrifugation for 10 min in a clinical centrifuge were washed once each in acetone:ethyl ether (1 : 2, v/v), ethyl ether, and petroleum ether and then dissolved in 3 ml of hot methanol and added to 10 ml of Scintillar (Mallinckrodt, St. Louis, MO) for radioactivity determination (TriCarb Liquid Scintillation Spectrometer Model 3330). External standardization was used for quench correction. The quantitation of cholesterol in liver was performed on triplicate slices obtained from the same rat liver used in the incubation. Following saponification and extraction with hexane, the material was dried and dissolved in a suitable volume of petroleum ether. Cholesterol was analyzed quantitatively by GLC employing a glass column (183 cm X 2 mm) and a silicone coated packing (Sp2100), 3% on 80/100 mesh supelcoport (Supelco, Inc., Bellefonte, PA). Nitrogen at 45 ml/mm served as the carrier gas. The analyses were conducted isothermally,with a column temperature of 255°C detector temperature of 290°C and injection port temperature of 270°C (Hewlett Packard, Model 5750). For each set of. samples, a standard curve was obtained by injecting known quantities of cholesterol (Supelco, Inc., Bellefonte, PA) in a range from 0.18 to 1.4 mg. Radioactive CO2 was determined as described by Baruch and Chaikoff [ 131. The method of Shapiro et al. [14] was applied for the isolation and determination of mevalonic acid. In the studies where the amounts of radioactivity in neutral lipids, free fatty acids, and phospholipids were determined, the incubations were terminated by immersing the sample vials in dry ice-acetone for 30 min. After thawing at room temperature, the total lipids were extracted by a modified Roese
Atherosclerosis, 32 (1979) 347-357 0 Elsevier/North-Holland Scientific
Publishers,
EFFECT OF MILK CONSTITUENTS GENESIS
AHMED
ASHOUR
Department (U.S.A.)
AHMED
Ltd.
ON HEPATIC
*, R.D. MCCARTHY
of Food Science,
CHOLESTEROL-
and G.A. PORTER
The Pennsylvania State University, University Park, PA 16802
(Received 8 June, 1978) (Revised, received 5 September, (Accepted 6 December, 1978)
1978)
Summary Two preparations active in reducing hepatic cholesterol biosynthesis were isolated from bovine skim milk. One of the inhibitors was in the dialysate and was identified as erotic acid (OA). The other inhibitor, present in the retentate, was not identified. Orotic acid appears to act by inhibiting cholesterol biosynthesis before the formation of mevalonate, whereas the retentate inhibitor exerts its effect beyond the formation of mevalonate in the biosynthetic pathway. Human milk also inhibited the incorporation of both labeled acetate and mevalonate into cholesterol by rat liver. Orotic acid was not detectable in human milk samples employed in this study. Administration of [6-14C]orotate to rats revealed its conversion to uracil in the liver. Subsequent work demonstrated that uracil had inhibitory activity on hepatic cholesterol biosynthesis similar to that of orotate when incubated with rat liver slices. Key words:
Cholesterolgenesis
-Inhibitors
-Liver
-Milk
- Rats
Introduction Several studies document effects of milk on hepatic cholesterolgenesis and blood cholesterol concentrations. Shah [l] reported that the incorporation of labeled mevalonate into digitonin-precipitated sterol was lower in liver homogenates from suckling rats compared to those of weaned rats. He suggested that the activities of one or more of the enzymes catalyzing the conversion of Authorized for publication as paper No. 5359 in the Journal Experiment Station. * Present address: Fatih University, Tripoli. Libya.
Series of The Pennsylvania
Agricultural
348
mevalonate into cholesterol change after weaning. McNamara et al. [2] reported that rat milk contains a thermostable, nondialyzable factor which depresses the activity of hydroxymethylglutaryl-CoA reductase in the liver of adult rats. In 1974, Mann and Spoerry [3] studied Masai Africans. When enough yogurt was consumed to cause weight gain, blood serum cholesterol levels declined. In the same year, Boguslowski and Wrobel [4] established the presence of a compound in bovine and human milk that inhibited hepatic cholesterolgenesis in the rat. The bovine milk factor was heat-stable, dialyzable, and present in the supernatant fraction after the protein had been precipitated with acid. It reduced the incorporation of both acetate and mevalonate into hepatic cholesterol. Malinow and McLaughlin [5] studied the plasma cholesterol levels in rats which were fed, beginning at 2 weeks of age, rat chow mixed with either water or bovine skim milk. The blood plasma cholesterol concentration for those given skim milk was lower in 43- and 64-day-old males and in 64day-old females than in those receiving water. During 1976, Bernstein et al. [6] predicted that the factor in bovine milk described by Boguslowski and Wrobel [4] was erotic acid (OA). Their data, obtained with liver homogenates, indicated that acetate, but not mevalonate, incorporation into cholesterol was reduced by cultured buttermilk, bovine milk, or OA, thus suggesting that the inhibition is at the initial steps in the biosynthetic pathway. Recently, Howard [ 71 and Marks and Howard [8] postulated that the hypochol,esterolaemic factor in milk was calcium, while Mann [9] and Nair and Mann [lo] postulated the effective agent to be /?-methylglutarate. The. above divergent observations led to the present effort to purify and define the factor(s) in bovine milk that affect hepatic cholesterol biosynthesis. Materials and Methods Isolation of cholesterol inhibitors from milk One hundred ml of pasteurized bovine skim milk was dialyzed for 48 h at 4°C against two changes of 500 ml distilled water. The dialyzable inhibitor (OA) was isolated by a combination of anion exchange and silicic acid column chromatography and quantitated by GLC [ 111. The retentate inhibitor fraction was obtained by the following technique. Casein was removed from the retentate of skim milk by reducing the pH to 4.6 with concentrated HCl and centrifuging at 17,600 X g for 20 min. The supernatant was freeze-dried and the resulting product dissolved in 10 ml of 0.02 M Tris buffer (pH 7.5) containing 0.2 mM sodium azide. A column (90 cm X 2.5 cm) of Sephadex G-100 (Pharmacia Fine Chemicals, Inc.) was equilibrated with distilled water. A 2.5 ml portion of the sample was placed on the column and eluted with Tris buffer at pH 7.5 with a flow rate of 2 to 2.5 ml/min. Twenty ml portions were collected with absorbance being monitored at 280 nm. Seven pooled fractions (Fig. 2) were dialyzed separately against tap water for 48 h and against distilled water for another 48 h, freezedried, and tested for their effect on cholesterol biosynthesis. Tissue incubation studies Male Wistar rats, 6-8 weeks old, were fed standard
rat chow and water ad
349
libitum. Excised livers were quickly rinsed in cold 0.9% NaCl and liver slices 0.5 mm thick were prepared with a Stadie Riggs tissue slicer. The slices were weighed and immediately transferred to incubation vials containing 3 ml of either Krebs-Ringer bicarbonate buffer or the buffer of Lakshmanan et al. [ 121. Either [l-14C]Na acetate (55.4 mCi/mM) or [2-‘4C]mevalonic acid (8.05 mCi/mM) was added to each flask. Incubations were conducted in a Dubnoff metabolic shaker incubator at 37°C for 3 h in an atmosphere of 95% O2 and 5% COZ, or when CO* was collected, in an atmosphere of laboratory air. Incubations were terminated by the addition of 4 ml of 20% KOH in methanol. For the determination of radioactivity associated with cholesterol, 4 mg of nonradioactive cholesterol was mixed with the sample before it was saponified for 3 h at 65°C. The non-saponifiable fraction was extracted 3 times with 15 ml portions of hexane which were combined and evaporated to dryness under nitrogen. Subsequently, the non-saponifiable residue was dissolved in 5 ml of 1% digitonin in 50% ethanol and permitted to stand for 12 h at room temperature. The digitonide precipitates recovered by centrifugation for 10 min in a clinical centrifuge were washed once each in acetone:ethyl ether (1 : 2, v/v), ethyl ether, and petroleum ether and then dissolved in 3 ml of hot methanol and added to 10 ml of Scintillar (Mallinckrodt, St. Louis, MO) for radioactivity determination (TriCarb Liquid Scintillation Spectrometer Model 3330). External standardization was used for quench correction. The quantitation of cholesterol in liver was performed on triplicate slices obtained from the same rat liver used in the incubation. Following saponification and extraction with hexane, the material was dried and dissolved in a suitable volume of petroleum ether. Cholesterol was analyzed quantitatively by GLC employing a glass column (183 cm X 2 mm) and a silicone coated packing (Sp2100), 3% on 80/100 mesh supelcoport (Supelco, Inc., Bellefonte, PA). Nitrogen at 45 ml/mm served as the carrier gas. The analyses were conducted isothermally,with a column temperature of 255°C detector temperature of 290°C and injection port temperature of 270°C (Hewlett Packard, Model 5750). For each set of. samples, a standard curve was obtained by injecting known quantities of cholesterol (Supelco, Inc., Bellefonte, PA) in a range from 0.18 to 1.4 mg. Radioactive CO2 was determined as described by Baruch and Chaikoff [ 131. The method of Shapiro et al. [14] was applied for the isolation and determination of mevalonic acid. In the studies where the amounts of radioactivity in neutral lipids, free fatty acids, and phospholipids were determined, the incubations were terminated by immersing the sample vials in dry ice-acetone for 30 min. After thawing at room temperature, the total lipids were extracted by a modified Roese
