- Email: [email protected]
Cholestyramine treatment during pregnancy in the rat results in hypercholesterolemia
Atherosclerosis, 51(1985) Elsevier
139
139-148
ATH 03662
Cholestyramine Treatment during Pregnancy in the Rat Results in Hypercholesterolemia Aslam S. Hassan, Judy J. Hackley
and Laurel L. Johnson
Department of Veterinary Biosciences, Division of Physiolow, University of Illinois. Urbana, IL 61801 (U.S.A.) (Received 14 September, 1984) (Revised, received 25 February, 1985) (Accepted 26 February, 1985)
Summary
Timed pregnant (8 days) Sprague-Dawley rats were fed ground stock diet (CON) or ground stock diet with 4% cholestyramine (CTR) until day 20 of gestation. Animals in both groups gained weight equally well during the study period (CON (n = 7) 308 + 7 g; CTR (n = 6) 315 * 7 g, mean + SEM). At the end of the study period, plasma cholesterol in the CTR group was significantly greater than that in the control group (CON n = 7, 91 + 4 mg/dl; CTR (n = 6) 108 * 5 mg/dl, P < 0.05). The fecal excretion of both neutral steroids and bile acids, studied for 3 days between days 15 and 18 of gestation, was significantly enhanced by CTR treatment. (Neutral steroids: CON, 3.9 * 0.3; CTR, 10.4 f 0.3, P < 0.05. Bile acids: CON, 7.6 f 0.4; CTR, 25.8 + 1.7, P < 0.05, mg/lOO g body wt/day). Bile acid pool size, measured at day 20 of gestation, however, was not significantly different. Consistent
Supported in part by a New Investigator Research Award HL 30934 to Dr. Hassan from National Heart, Lung and Blood Institute, NIH. Part of this work has been published in abstract-form in Physiologist, 27 (1984) 243. Address all correspondence to: Aslam S. Hassan, Ph.D., University of Illinois, Department of Veterinary Biosciences, 2001 South Lincoln Avenue, Urbana, IL 61801, U.S.A. Systematic names of some of the compounds discussed in the text: 7a-hydroxycholesterol = 5cholestene-3P,7a-dial; 7/3-hydroxycholesterol = 5-cholestene-3P,7@-diol; ‘I-ketocholesterol = 3P-hydroxy5-cholesten-7-one; lithocholic acid = 3a-hydroxy-SP-cholan-24-oic-acid; deoxycholic acid = 3a,12adihydroxy-5/?-cholan-24-oic acid; chenodeoxycholic acid = 3a,7a-dihydroxy-SD-cholan-24-oic acid; hyodeoxycholic acid = 3a,6c~-dihydroxy-5/3-cholan-24-oic acid; ursodeoxycholic acid = 3a,7P-dihydroxy-SDcholan-24-oic acid; cholic acid = 3a,7a,l2a-trihydroxy-5~-cholan-24-oic acid; hyocholic acid = 3(~,6(~,12c~-trihydroxy-5/3-cholan-24-oic acid, cr-muricholic acid = 3a,6P.7o-trihydroxy-5/3-cholan-24-oic acid; /3-muricholic acid = 3a,6/3,7/3-trihydroxy-S/3-cholan-24-oic acid.
0021-9150/85/$03.30
0 1985 Elsevier Scientific
Publishers
Ireland,
Ltd.
140
with these results was the finding that hepatic cholesterol 7a-hydroxylase activity (the rate-limiting enzyme of bile acid biosynthesis) measured at day 20 of gestation was significantly enhanced by CTR treatment (CON (n = 4) 14.7 &-1.7; CTR P < 0.05). The atypical finding of hyper(n = 4) 34.8 f. 3.3, pmoles/mg/min, cholesterolemia, despite the CTR-induced enhanced turnover of cholesterol, may be due to changes in the homeostatic mechanisms of cholesterol and bile acid metabolism during pregnancy. Key words: Bile acid pool - Cholesterol 7a-hydroxylase - Fecal neutral sterols and bile acids
Introduction
Cholestyramine, an anion exchange resin binds bile acids in the intestine and promotes their loss [l]. Typically, therefore, cholestyramine feeding results in an enhancement of the activity of hepatic cholesterol 7a-hydroxylase (CH-7A) (EC 1.14.13.17) the rate-limiting enzyme of bile acid biosynthesis from cholesterol [2]. Because of the ‘drain’ of cholesterol into bile acid synthesis, cholestyramine feeding usually also results in a compensatory increase in the activity of hepatic HMGCoAreductase (HMGCoA-R) (EC 1.1.1.34), the rate-limiting enzyme of .cholesterol biosynthesis [3]. Thus, the hypocholesterolemic action of cholestyramine [4] is limited by the extent to which the compensatory increase in biosynthesis of cholesterol can compensate for the cholesterol ‘drain’ induced by CTR. Several studies have shown that cholesterol and bile acid metabolism are altered during pregnancy [5,6], and there is evidence to suggest that estrogens may be involved in the changes [7]. Furthermore, it is also known that a portion of fetal cholesterol is derived from maternal source [8-lo]. Taken together, it is reasonable to assume that the homeostatic mechanisms of maternal cholesterol and bile acid metabolism are altered during pregnancy. Under these conditions there is not a priori reason to believe that cholestyramine would affect cholesterol and bile acid metabolism in a manner similar to that usually seen in a non-pregnant animal. In a recent study, Innis [ll] found that Questran@ (44% cholestyramine resin, 56% sucrose)-fed pregnant rats did not show an increase in CH-7A activity. Yet HMGCoA-R activity was elevated ca. 2.4-fold over that in corresponding controls [ll]. This finding is paradoxical in view of the above-mentioned coordinated regulation of the 2 enzymes. Innis attributed the lack of an increase in CH-7A to cholestasis of pregnancy [5,6]. However, since Questran is 56% sucrose, the animals being fed Questran were receiving a significant amount of sucrose which was not present in the control diet. Whether sucrose has any effect on cholesterol and bile acid metabolism in the pregnant rat is not known. Thus, Innis’ finding of a lack of an increase in CH-‘IA activity with Questran treatment during pregnancy is difficult to interpret. In view of (1) lack of systematic information on the effect of cholestyramine on
141
cholesterol and bile acid metabolism during pregnancy, (2) the potential suggestion of reduced efficacy of cholestyramine during pregnancy [ll], and (3) difficulty in the interpretation of the effect of cholestyramine on CH-7A during pregnancy [ll], we carried out a relatively detailed study on the effect of pure cholestyramine resin on cholesterol and bile acid metabolism in the pregnant rat. Materials and Methods Chemicals
[4-‘4C]Cholesterol was obtained from New England Nuclear (Boston. MA), diluted with unlabeled cholesterol (99 t %, Sigma Chemical Co., St. Louis, MO) and purified by thin-layer chromatography (TLC) on preparative (500 pm thick) thinlayer plates (Analtech, Inc., Newark, DE) developed in benzene/ethyl acetate (2 : 3, v/v). The specific activity of the purified labeled cholesterol was 1.3 x lOI dpm/mole. Nicotinamide and EDTA were obtained from Sigma Chemical Co., and mercaptoethanol was obtained from Eastman Kodak Co.(Rochester, NY). NADPH was obtained from Boehringer-Mannheim (Indianapolis, IN). Hyocholic acid, internal standard for bile acid analysis, was obtained from Calbiochem-Behring (San Diego, CA). All other bile acid standards for gas-liquid chromatography (GLC), 7a-hydoxycholesterol, 7P-hydroxycholesterol and 7-ketocholesterol were obtained from Steraloids (Wilton, NH). All solvents were of reagent grade and used as supplied. Animals
and diet
Timed pregnant (8 days) Sprague-Dawley rats were obtained from Harlan Sprague-Dawley Inc. (Indianapolis, IN). Upon arrival, the rats were randomly assigned to one of two groups and housed individually in cages with wire mesh bottoms. One group (CON) was fed ground stock rat diet while the other group (CTR) was fed ground stock rat diet with 4% cholestyramine (Mead Johnson Co., Evansville, IN). Both diets were prepared in the laboratory from commercially available pelleted stock rat diet (Wayne Rodent Blox, 8604-00, Chicago, IL). Food and water were available ad libitum. Fecal excretion
of neutral steroids and bile acids
In order to examine the effect of CTR treatment on the fecal excretion of neutral steroids and bile acids, quantitative fecal collections (free of food and urine) were made for 3 days between days 15 and 18 of gestation. The fecal samples were pooled and frozen until analyzed. Food intake during the 3 days was also carefully recorded. The weights of the animals taken immediately after the fecal collection period were used to normalize the fecal excretion of neutral steroids and bile acids. Feces were analyzed for neutral steroids and bile acids by the methods of Miettinen et al. [12] and Subbiah [13], respectively. The fecal bile acids were methylated with freshly prepared diazomethane, and the bile acid methyl esters were acetylated using 1 ml of a mixture of acetic anhydride, acetic acid, and perchloric acid (5 : 5 : 0.050, v/v/v) at room temperature for 1 h [14]. The bile acid methyl ester
142
acetates were quantitated by GLC using hyocholic acid as the internal standard. A Perkin Elmer Sigma 2000 (Norwalk, CT) gas-liquid chromatograph equipped with a 6-ft column of 1% OV-225 on Gas Chrom Q (loo/120 mesh) (Supelco, Bellefonte, PA) [15] was used. The operating conditions were: Column -240°C Injector - 250°C and Detector (FID) -260°C Helium was used as the carrier gas at 30 ml/min. Identification of the individual bile acids was based on comparison to relative retention times of authentic standards obtained from Steraloids (Wilton, NH). The major steroid groups in the neutral steroid extract were separated by TLC as described by Miettinen et al. [8]. After elution from the gel, the steroids in each group were quantitated by GLC using Sa-cholestane (Steraloids) as the internal standard. A Hewlett-Packard Model 5840A (Palo Alto, CA) gas-liquid chromatograph equipped with a 6-ft column of 3% OV-17 on Gas Chrom Q (loo/120 mesh) (Applied Science, State College, PA) was used. The operating conditions were: Column - 255°C Injector - 265°C Detector (FID) - 300°C. Nitrogen was used as the carrier gas at a flow rate of 50 ml/mm. [4-i4C]Cholesterol was used to correct for procedural losses of neutral steroids. Fecal excretion of neutral steroids was corrected for variations in fecal flow and steroid losses during intestinal transit by the fecal recovery of dietary /3-sitosterol as described previously [16]. Hepatic CH-7A assay On day 20 of gestation,
the animals were anesthetized with an intramuscular injection of ketamine/xylazine (45 mg/kg body wt and 6 mg/kg body wt, respectively) and exsanguinated by cardiac puncture. Portions of the liver from 4 randomly selected animals in each group were rapidly excised and placed in a chilled solution of 250 mM sucrose, 2.5 mM EDTA, and 50 mM nicotinamide, pH 7.4. Hepatic CH-7A activity was assayed as described below. The remaining liver and the entire gastrointestinal tract (excluding the stomach) were removed and frozen for the analysis of bile acid pool size as described below. Hepatic microsomal CH-7A activity was assayed as described previously [17] based on the methods of Mitropoulos and Balasubramaniam [18] and Shefer et al. [19]. Microsomal protein was measured by the Hartree [20] modification of the Lowry procedure, and CH-7A activity was expressed as picomoles of ;lar-hydroxycholesterol formed/mg microsomal protein/mm.
Analysis of bile acid pool
For the determination of bile acid pool size and composition, the liver and the gastrointestinal tract were frozen in liquid nitrogen and ground into a fine powder using a Waring Blender (Dynamics Corp., New Hartford, CT) with a stainless steel cup. A weighed aliquot of the powder was saponified with 2.5 N NaOH and extracted for bile acids after acidification to pH 3. The extracted bile acids were methylated with freshly prepared diazomethane, and the bile acid methyl esters were purified from fatty acids by TLC as described by Grundy et al. [21]. Purified bile acid methyl esters were quantitated as their acetates as described above for fecal bile acids.
143
Analysis of serum cholesterol Serum obtained from the rats was analyzed for cholesterol according to Morin and Elms [22]. Cholesterol was quantitated by GLC, as described above for fecal neutral steroids, using stigmasterol as an internal standard [22]. Statistical methods Control and experimental values were compared using the Student t-test for unpaired means. A P-value of < 0.5 was considered to be statistically significant. Results
Body weights and serum cholesterol Table 1 shows that animals in both groups gained weight equally well. There was no difference in the mean number of fetuses from animals in either group (CON (n = 7) 10 k 2; CTR (n = 6), 10 k 3, mean f SEM) or in the mean pooled body weights of the fetuses from animals in either group (CON (n = 7), 40.6 + 5.3 g; CTR (n = 6) 38.1 f 2.8 g). Cholestyramine treatment during gestation, however, resulted in a significant elevation of serum cholesterol (CON (n = 7) 91 -t_4; CTR (n = 6) 108 f 5 mg/dl, P < 0.05). This finding was unexpected and the possible mechanism is discussed below. Fecal excretion of neutral steroids and bile acids As shown in Table 2, CTR treatment resulted in the expected enhanced excretion of bile acids (CON (n = 7), 7.6 k 0.4; CTR (n = 6), 25.8 _t 0.7 mg/lOO gm body wt/day, P < 0.05). Fecal excretion of neutral steroids was also significantly enhanced by dietary CTR (CON (n = 7), 3.9 & 0.3; CTR (n = 6), 10.4 + 0.3, P < 0.05) probably, in part, due to the fact that bile acids, which are obligatory for cholesterol
TABLE
1
BODY WEIGHTS AND PLASMA PREGNANT RATS
CHOLESTEROL
IN CONTROL
AND CHOLESTYRAMINE-FED
Timed pregnant rats (8 days) were fed ground stock rat diet (CON) or ground stock rat diet with 4% cholestyramine resin (CTR) until day 20 of gestation. Serum cholesterol was determined at day 20 of gestation. Results are given as the mean & SEM and the numbers in parentheses refer to the number of animals studied. Group
(n)
CON (7) CTR (6)
* Significantly
different
Initial body wt
Final body wt
Serum cholesterol
(8)
(8)
(mg/dB
191*3 192+3
308+7 315f7
91&-4 108+5 *
from control,
P < 0.05.
144
TABLE 2 FECAL EXCRETION OF NEUTRAL STEROIDS CHOLESTEROL 7a-HYDROXYLASE IN CONTROL NANT RATS
AND BILE ACIDS AND HEPATIC AND CHOLESTYRAMINE-FED PREG-
Timed pregnant rats (8 days) were treated as described in Table 1. Quantitative fecal collections were made for 3 days between days 15 and 18 of gestation and analyzed for neutral steroids and bile acids as described under Methods. Hepatic cholesterol 7a-hydroxylase was assayed on day 20 of gestation as described in text. The values are presented as mean + SEM and the numbers in parentheses refer to the number of animals studied. Group
Food eaten (g/100 g body
Fecal steroid excretion (mg/lOO gm body wt/day)
Wday)
Neutral steroids
CON
8.2 k 0.1 (7)
CTR
8.7 + 0.1 (6) *
Bile acids
3.9 f 0.3 (7)
7.6 f 0.4 (7)
10.4 f 0.3 (6) *
25.8 f 0.7 (6) *
Total steroids
Hepatic cholesterol 7cY-hydroxylase (pmoles/mg/min)
11.6+0.5 (7)
14.7 f 1.7 (4)
36.1+ 1.5 (6) *
34.8 k 3.3 (4) *
* Significantly different from control, P < 0.05.
absorption [23], were bound by CTR. While it appears that CTR-treated animals ate a significantly greater amount of food, the increase is more apparent than real. If 4% of the weight of the food consumed is subtracted (because CTR is a non-nutritive, non-absorbable resin), then there is no difference in food consumption between the 2 groups. The dramatic increase in the fecal excretion of bile acids by the CTR-treated animals would be expected to result in an increase in the activity of hepatic CH-7A. As shown in Table 2, CTR-treatment increased CH-7A activity ca. 2.6-fold when compared to that in controls (CON (n = 4) 14.7 f 1.7; CTR (n = 4) 34.8 + 3.3 pmoles/mg/min, P < 0.05). Bile acid pool size and composition
While CTR treatment did not alter the total bile acid pool size (Table 3) there were some differences in the pool sizes of individual bile acids. CTR treatment resulted in an increase in the pool sizes of lithocholic acid and cholic acid, while the pool sizes of hyodeoxy-/ursodeoxy-cholic acids and (Ymuricholic acid were significantly greater in the control animals. When the bile acids were grouped into 2 major pools, ‘cheno’ pool (lithocholic, 3P,A5-cholenoic, hyodeoxy-/ursodeoxy-cholic acids and (Y- and /3-muricholic acids) and ‘cholic’ pool (deoxycholic and cholic acids), CTR-treated animals were found to have a lower ‘cheno’ pool (CON (n = 4), 54.5 * 3.88%; CTR (n = 4), 37.12 f 3.0% P < 0.05) and a higher ‘cholic’ pool (CON (n = 4) 45.5 f 3.88%; CTR (n = 4) 62.88 + 3.0%, P < 0.05) and thus a reduced ‘cheno’ to ‘cholic’ ratio (CON (n = 4), 1.24 + 0.16; CTR (n = 4), 0.6 f 0.08, P < 0.05).
3
POOL
SIZE
AND
COMPOSITION
IN CONTROL
AND
CHOLESTYRAMINE-TREATED
PREGNANT
3.0 +0.7 * (14.26k1.23)”
0.8 f0.2 (4.05 f 2.13)
Lithocholic acid
0.1 io.03 (0.65+0.12)
0.2 fO.l (0.98 f 0.48)
3/3.As-Cholenic acid
2.7 (13.0
i0.4 fO.60)
1.8 +0.2 (8.50k0.72)
Deoxycholic acid
*
1.7 (8.0
+0.3 kO.77)
2.9 kO.4 (13.77 t 1 .O)
Chenodroxycholic acid
* Significantly different frown corresponding control value. P < 0.05. (’ ‘Cheno’ Includes the following hile acid&: Ilthochollc. 3/3.3‘-cholrnoic. ‘Cholic’ includes deoxycholic and cholic acids.
CTR
Control
CiKUp
chenodeoxycholic.
0.1 kO.04 * (0.47+0.17)
0.7 kO.2 (3.50+ 0.89) * *
H yodeoxy-/ ursodeoxycholic
*
RATS
acids.
a-muricholx
0.6 +0.2 (2.8x+0.66)*
2.2 +0.3 (10.x6+1.15)
*
1.5 fO.4 (7.18+ 1.69) 5.3 k1.0 (25.03i4.22)
fl-Muricholic acid
as described in Methods. bile acid pool.
a-Muricholic acid
and ursodeoxy-chollc
9.9 i0.6 (49.89k3.47)
7.6 +0.6 (37.01 t4.31)
Cholic acid
Ilyodeoxy-
acid
Rats were treated as described in Table 1. Pool size and composition was determined in rats killed on day 20 of gestation body wt are given as mea” + SEM for 4 animals in each group. Numbers in parentheses refer to the percentages of total
BILE ACID
TABLE
1.6
”
g
acids.
0.60 + 0.1 *
1.24+0.2
‘Cheno’/ ‘Cholic’
mg/lOO
and &muricholic
20.3 f 2.3
20.9*
Total
The values.
R
146
Discussion
The results of the present study show that cholestyramine fed during gestation can affect cholesterol and bile acid metabolism in the pregnant rat. In the discussion to follow, each of the effects of CTR treatment on cholesterol and bile acid metabolism is discussed separately. CTR treatment and bile acid pool size and composition In a recent study Hassan et al. [24] showed that CTR fed to weanling male rats for 1 week resulted in an expansion of the bile acid pool in addition to a ca. 4-fold increase in the hepatic CH-7A activity. In the present study, feeding CTR to pregnant rats for 12 days did not affect the total bile acid pool size even though CH-7A activity was elevated ca. 2.6 times over that in controls. CTR treatment did, however, result in compositional changes in the bile acid pool. The ‘cheno’/‘cholic’ ratio was significantly lower in the CTR-treated animals. There are at least 2 possible mechanisms which could account for the effect of CTR on the ‘cheno’/‘cholic’ ratio. Cholestyramine has a well-documented greater affinity for dithan for tri-hydroxy bile acids [25]. Thus CTR-induced interruption of the enterohepatic circulation of chenodeoxycholic acid would be greater than that of cholic acid. Since chenodeoxycholic acid also serves as the substrate for the hepatic synthesis of hyodeoxycholic, ursodeoxycholic, and the muricholic acids in the rat [26], a ‘drain’ in the chenodeoxycholic acid pool would be expected to reduce the pool sizes of these bile acids thus resulting in the reduction of the total ‘cheno’ pool. This hypothesis is supported by the finding of a greater increase in the pool of lithocholic acid (3.8-fold) than deoxycholic acid (1.5-fold) (bacterial 7a-dehydroxylation products of chenodeoxycholic acid and cholic acid, respectively) in the CTR-treated animals relative to non-treated controls. Secondly, since there was an actual increase in the cholic acid pool size in the CTR-treated animals, CTR treatment could possibly have increased the activity of hepatic microsomal 12a-hydroxylase and, therefore, increased the synthesis of cholic acid [26]. Both of these mechanisms, alone or in combination, could have resulted in the decreased ‘cheno’/‘cholic’ ratio following CTR treatment in the present study. Effect of
CTR treatment on cholesterol metabolism As mentioned earlier, CTR treatment typically results in a reduction in plasma cholesterol [4]. The degree of CTR-induced hypocholesterolemia is affected by the extent of the compensatory increase in cholesterol biosynthesis. In view of this, it was surprising to find that, in the present study, CTR treatment significantly elevated serum cholesterol in the pregnant rats. The exact mechanism for the hypercholesterolemia is not known. As pointed out earlier, the pregnant animal is faced with maintaining a cholesterol supply both for the developing fetus as well as for itself. CTR treatment during pregnancy imposes an additional ‘load’ on the cholesterol synthetic ability of the mother by dramatically increasing the turnover of cholesterol (Table 2). It is known that CTR treatment enhances the activity of HMGCoA-R both in the liver and intestine [3,27], the 2 major tissues of cholestero-
147
genesis in the rat [28]. Thus, it is possible that maternal total body cholesterogenesis is enhanced in the pregnant CTR-treated rat and overcompensates in order to assure adequate supplies of cholesterol both for fetal and maternal demands thereby resulting in hypercholesterolemia. This finding may have some implications on the use of CTR during pregnancy. Effect of CTR treatment on hepatic CH-7A activity In a recent study, Innis found that pregnant rats treated with Questran did not show an increase in CH-7A activity [ll] while similarly treated non-pregnant rats showed a 3-fold increase. However, since both pregnant and non-pregnant Questran-treated rats showed similar elevation in HMGCoA-R activity relative to non-treated controls [ll], we wished to clarify Innis’ observations on CH-7A activity. To avoid the possible interference by the sucrose content of Questran, we used pure CTR resin. In the present study, CTR treatment during gestation increased CH-7A activity ca. 2.6-fold over controls. This finding is a well-documented effect of CTR treatment on CH-7A activity [1,29] and virtually similar to the finding of Innis [ll] in Questran-treated non-pregnant rats. It is difficult to reconcile the effect of CTR (present study) vs Questran [ll] on CH-7A in the pregnant rat. Since Questran is 56% sucrose, the Questran-containing diet used by Innis [ll] contained a significant amount of sucrose (2.8% or 6.4% depending on whether the diet was 5% in Questran or 5% in resin content), which was not present in the control diet. Whether or not sucrose has any effect on cholesterol and bile acid metabolism in the pregnant rat remains to be determined. In summary, this study has documented the effects of CTR treatment during pregnancy on cholesterol and bile acid metabolism. Pregnant rats respond to CTR treatment with a dramatic increase in the excretion of total fecal steroids and hepatic CH-7A activity. Despite the enhanced turnover of cholesterol, however, plasma cholesterol was significantly greater (ca. 20% increase over controls) in the CTRtreated pregnant rats. References Packard, C.J. and Shepherd, J.. The hepatobiliary axis and lipoprotein metabolism - Effects of bile acid sequestrants and ileal bypass surgery, J. Lipid Res., 23 (1982) 1081. Shefer, S., Hauser, S., Bekersky, I. and Mosbach, E.H., Biochemical site of regulation of bile acid synthesis in the rat, J. Lipid Res., 11 (1970) 404. Goldfarb, S., Regulation of hepatic cholesterogenesis. In: N.B. Javitt (Ed.). International Review of Physiology, Vol. 21 (Liver and Biliary Tract Physiology, I). University Park Press, Baltimore. MD, 1980, p. 317. The Lipid Research Clinics Program, The Lipid Research Clinics Coronary Primary Prevention Trial Results, Part 1 (Reduction in incidence of coronary heart disease) and Part 2 (The relationship of reduction of coronary heart disease to cholesterol lowering), J. Amer. Med. Ass.. 251 (1984) 351. Everson, G.T., McKinley, C., Lawson, M., Johnson, M. and Kern, Jr., F., Gallbladder function in the human female - Effect of ovulatory cycle, proegnancy. and contraceptive steroids, Gastroenterology, 82 (1982) 711. Kern, Jr.. F., Everson, G.T., DeMark, B., McKinley, C., Showalter. R.. Erfling, W.D., Braverman. 2..
148
7 8 9 10 11
Szczepanik-Van Leeuwen, P. and Klein, P. D., Biliary lipids, bile acids, and gallbladder function in the human female, J. Clin. Invest., 68 (1981) 1229. Kern, Jr., F., Eriksson, H., Curstedt, T. and Sjiivall, J., Effect of ethynylestradiol on biliary excretion of bile acids, phosphatidylcholines, and cholesterol in the bile fistula rat, J. Lipid Res., 18 (1977) 623. Connor, W.E. and Lin, D.S., Placental transfer of cholesterol-4-‘4C into rabbit and guinea pig fetus, J. Lipid Res., 8 (1967) 558. Pitkin, R.M., Connor. W.E. and Lin, D.S., Cholesterol metabolism and placental transfer in the pregnant rhesus monkey. J. Clin. Invest., 51 (1972) 2584. Lin, D.S., Pitkin, R.M. and Connor, W.E., Placental transfer of cholesterol into the human fetus, Amer. J. Obstet. Gynecol., 128 (1977) 735. Innis, S.M., Effect of cholestyramine administration during pregnancy in the rat, Amer. J. Obstet.
Gynecol., 146 (1983) 13. 12 Miettinen, T.A., Ahrens, Jr.. E.H. and Grundy, S.M., Quantitative isolation and gas-chromatographic analysis of total dietary and fecal neutral steroids, J. Lipid Res., 6 (1965) 411. 13 Subbiah, M.T.R., Tyler, N.E., Buscaglia, M.D. and Marai, L., Estimation of bile acid excretion in man - Comparison of isotopic turnover and fecal excretion methods, J. Lipid Res., 17 (1976) 78. 14 Parmentier, G.G., Janssen, G.A., Eggermont, E.A. and Eyssen, H.J., Cz, bile acids in infants with coprostanic acidemia and occurrence of a 3a,7a,l2a-trihydroxy-5P_C29 dicarboxylic bile acid as a major component in their serum, Europ. J. Biochem., 102 (1979) 173. 15 Yousef, I.M., Fisher, M.M., Myher, J.J. and Kuksis, A., Superior gas-liquid chromatography of methyl cholanoate acetates on cyanopropylphenylsiloxane liquid phases, Anal. B&hem., 75 (1976) 538. 16 Grundy, SM., Ahrens, Jr., E.H. and Salen, G., Dietary p-sitosterol as an internal standard to correct for cholesterol losses in sterol balance studies, J. Lipid Res., 9 (1968) 374. 17 Hassan, AS., Gallon, L.S., Zimmer, L.A., Balistreri, W.F. and Subbiah, M.T.R., Persistent enhancement of bile acid synthesis in guinea pigs following stimulation of cholesterol catabolism in neonatal life, Steroids, 38 (1981) 477. 18 Mitropoulos, K.A. and Balasubramaniam, S., Cholesterol 7e-hydroxylase in rat liver microsomal preparations, Biochem. J., 128 (1972) 1. 19 Shefer, S., Hauser, S. and Mosbach, E.H., 7a-Hydroxylation of cholesterol by rat liver microsomes, J. Lipid Res., 9 (1968) 328. 20 Hartree, E.F., Determination of protein - A modification of the Lowry method that gives a linear photometric response, Anal. B&hem., 48 (1972) 422. 21 Grundy, S.M., Ahrens, Jr., E.H. and Miettinen, T.A., Quantitative isolation and gas-liquid chromatographic analysis of total fecal bile acids, J. Lipid Res., 6 (1965) 397. 22 Morin, R.J. and Elms, N.J., Rapid microanalysis of cholesterol in bile and serum by gas chromatography, Ann. Clin. Lab. Sci., 5 (1975) 52. 23 Siperstein, M.D., Chaikoff, I.L. and Reinhardt, W.O., “C-Cholesterol, Part 5. (Obligatory function of bile in the intestinal absorption of cholesterol), J. Biol. Chem., 198 (1952) 111. 24 Hassan, A.S., Srivastava, L.S., Yunker, R.L. and Subbiah, M.T.R., Cholestyramine feeding in weaned rat - Increase in plasma corticosterone levels and bile acid synthesis following adrenalectomy, Atherosclerosis, 51 (1984) 327. 25 Johns, W.H. and Bates, T.R., Quantification of the binding tendencies of cholestyramine, Part 1 (Effect of structure and added electrolytes on the binding of unconjugated and conjugated bile salt anions), J. Pharmacol. Sci., 38 (1969) 179. 26 Beher, W.T., Biosynthesis of primary bile acids. In: D. Kritchevsky, O.J. Pollak and H.S. Simms (Eds.) Monographs on Atherosclerosis, Vol. 6 (Bile Acids - Chemistry and Physiology of Bile Acids and their Influence on Atherosclerosis), S. Karger, New York, 1976, pp. 17-33. 27 Panini, S.R., Lehrer, G., Rogers, D.H. and Rudney, H., Distribution of 3-hydroxy-3-methylglutaryl coenzyme A reductase and alkaline phosphatase activities in isolated epithelial cells of fed, fasted, cholestyramine-fed, and 4-aminopyrazolo-[3,4-dlpyrimidine-treated rats, J. Lipid Res., 30 (1979) 879. 28 Spady, D.K. and Dietschy, J.M., Sterol synthesis in vivo in 18 tissues of the squirrel monkey, guinea pig, rabbit, hamster, and rat, J. Lipid Res., 24 (1983) 303. 29 Myant, N.B. and Mitropoulos, K.A., Cholesterol 7cY-hydroxylase, J. Lipid Res., 18 (1977) 135.
139
139-148
ATH 03662
Cholestyramine Treatment during Pregnancy in the Rat Results in Hypercholesterolemia Aslam S. Hassan, Judy J. Hackley
and Laurel L. Johnson
Department of Veterinary Biosciences, Division of Physiolow, University of Illinois. Urbana, IL 61801 (U.S.A.) (Received 14 September, 1984) (Revised, received 25 February, 1985) (Accepted 26 February, 1985)
Summary
Timed pregnant (8 days) Sprague-Dawley rats were fed ground stock diet (CON) or ground stock diet with 4% cholestyramine (CTR) until day 20 of gestation. Animals in both groups gained weight equally well during the study period (CON (n = 7) 308 + 7 g; CTR (n = 6) 315 * 7 g, mean + SEM). At the end of the study period, plasma cholesterol in the CTR group was significantly greater than that in the control group (CON n = 7, 91 + 4 mg/dl; CTR (n = 6) 108 * 5 mg/dl, P < 0.05). The fecal excretion of both neutral steroids and bile acids, studied for 3 days between days 15 and 18 of gestation, was significantly enhanced by CTR treatment. (Neutral steroids: CON, 3.9 * 0.3; CTR, 10.4 f 0.3, P < 0.05. Bile acids: CON, 7.6 f 0.4; CTR, 25.8 + 1.7, P < 0.05, mg/lOO g body wt/day). Bile acid pool size, measured at day 20 of gestation, however, was not significantly different. Consistent
Supported in part by a New Investigator Research Award HL 30934 to Dr. Hassan from National Heart, Lung and Blood Institute, NIH. Part of this work has been published in abstract-form in Physiologist, 27 (1984) 243. Address all correspondence to: Aslam S. Hassan, Ph.D., University of Illinois, Department of Veterinary Biosciences, 2001 South Lincoln Avenue, Urbana, IL 61801, U.S.A. Systematic names of some of the compounds discussed in the text: 7a-hydroxycholesterol = 5cholestene-3P,7a-dial; 7/3-hydroxycholesterol = 5-cholestene-3P,7@-diol; ‘I-ketocholesterol = 3P-hydroxy5-cholesten-7-one; lithocholic acid = 3a-hydroxy-SP-cholan-24-oic-acid; deoxycholic acid = 3a,12adihydroxy-5/?-cholan-24-oic acid; chenodeoxycholic acid = 3a,7a-dihydroxy-SD-cholan-24-oic acid; hyodeoxycholic acid = 3a,6c~-dihydroxy-5/3-cholan-24-oic acid; ursodeoxycholic acid = 3a,7P-dihydroxy-SDcholan-24-oic acid; cholic acid = 3a,7a,l2a-trihydroxy-5~-cholan-24-oic acid; hyocholic acid = 3(~,6(~,12c~-trihydroxy-5/3-cholan-24-oic acid, cr-muricholic acid = 3a,6P.7o-trihydroxy-5/3-cholan-24-oic acid; /3-muricholic acid = 3a,6/3,7/3-trihydroxy-S/3-cholan-24-oic acid.
0021-9150/85/$03.30
0 1985 Elsevier Scientific
Publishers
Ireland,
Ltd.
140
with these results was the finding that hepatic cholesterol 7a-hydroxylase activity (the rate-limiting enzyme of bile acid biosynthesis) measured at day 20 of gestation was significantly enhanced by CTR treatment (CON (n = 4) 14.7 &-1.7; CTR P < 0.05). The atypical finding of hyper(n = 4) 34.8 f. 3.3, pmoles/mg/min, cholesterolemia, despite the CTR-induced enhanced turnover of cholesterol, may be due to changes in the homeostatic mechanisms of cholesterol and bile acid metabolism during pregnancy. Key words: Bile acid pool - Cholesterol 7a-hydroxylase - Fecal neutral sterols and bile acids
Introduction
Cholestyramine, an anion exchange resin binds bile acids in the intestine and promotes their loss [l]. Typically, therefore, cholestyramine feeding results in an enhancement of the activity of hepatic cholesterol 7a-hydroxylase (CH-7A) (EC 1.14.13.17) the rate-limiting enzyme of bile acid biosynthesis from cholesterol [2]. Because of the ‘drain’ of cholesterol into bile acid synthesis, cholestyramine feeding usually also results in a compensatory increase in the activity of hepatic HMGCoAreductase (HMGCoA-R) (EC 1.1.1.34), the rate-limiting enzyme of .cholesterol biosynthesis [3]. Thus, the hypocholesterolemic action of cholestyramine [4] is limited by the extent to which the compensatory increase in biosynthesis of cholesterol can compensate for the cholesterol ‘drain’ induced by CTR. Several studies have shown that cholesterol and bile acid metabolism are altered during pregnancy [5,6], and there is evidence to suggest that estrogens may be involved in the changes [7]. Furthermore, it is also known that a portion of fetal cholesterol is derived from maternal source [8-lo]. Taken together, it is reasonable to assume that the homeostatic mechanisms of maternal cholesterol and bile acid metabolism are altered during pregnancy. Under these conditions there is not a priori reason to believe that cholestyramine would affect cholesterol and bile acid metabolism in a manner similar to that usually seen in a non-pregnant animal. In a recent study, Innis [ll] found that Questran@ (44% cholestyramine resin, 56% sucrose)-fed pregnant rats did not show an increase in CH-7A activity. Yet HMGCoA-R activity was elevated ca. 2.4-fold over that in corresponding controls [ll]. This finding is paradoxical in view of the above-mentioned coordinated regulation of the 2 enzymes. Innis attributed the lack of an increase in CH-7A to cholestasis of pregnancy [5,6]. However, since Questran is 56% sucrose, the animals being fed Questran were receiving a significant amount of sucrose which was not present in the control diet. Whether sucrose has any effect on cholesterol and bile acid metabolism in the pregnant rat is not known. Thus, Innis’ finding of a lack of an increase in CH-‘IA activity with Questran treatment during pregnancy is difficult to interpret. In view of (1) lack of systematic information on the effect of cholestyramine on
141
cholesterol and bile acid metabolism during pregnancy, (2) the potential suggestion of reduced efficacy of cholestyramine during pregnancy [ll], and (3) difficulty in the interpretation of the effect of cholestyramine on CH-7A during pregnancy [ll], we carried out a relatively detailed study on the effect of pure cholestyramine resin on cholesterol and bile acid metabolism in the pregnant rat. Materials and Methods Chemicals
[4-‘4C]Cholesterol was obtained from New England Nuclear (Boston. MA), diluted with unlabeled cholesterol (99 t %, Sigma Chemical Co., St. Louis, MO) and purified by thin-layer chromatography (TLC) on preparative (500 pm thick) thinlayer plates (Analtech, Inc., Newark, DE) developed in benzene/ethyl acetate (2 : 3, v/v). The specific activity of the purified labeled cholesterol was 1.3 x lOI dpm/mole. Nicotinamide and EDTA were obtained from Sigma Chemical Co., and mercaptoethanol was obtained from Eastman Kodak Co.(Rochester, NY). NADPH was obtained from Boehringer-Mannheim (Indianapolis, IN). Hyocholic acid, internal standard for bile acid analysis, was obtained from Calbiochem-Behring (San Diego, CA). All other bile acid standards for gas-liquid chromatography (GLC), 7a-hydoxycholesterol, 7P-hydroxycholesterol and 7-ketocholesterol were obtained from Steraloids (Wilton, NH). All solvents were of reagent grade and used as supplied. Animals
and diet
Timed pregnant (8 days) Sprague-Dawley rats were obtained from Harlan Sprague-Dawley Inc. (Indianapolis, IN). Upon arrival, the rats were randomly assigned to one of two groups and housed individually in cages with wire mesh bottoms. One group (CON) was fed ground stock rat diet while the other group (CTR) was fed ground stock rat diet with 4% cholestyramine (Mead Johnson Co., Evansville, IN). Both diets were prepared in the laboratory from commercially available pelleted stock rat diet (Wayne Rodent Blox, 8604-00, Chicago, IL). Food and water were available ad libitum. Fecal excretion
of neutral steroids and bile acids
In order to examine the effect of CTR treatment on the fecal excretion of neutral steroids and bile acids, quantitative fecal collections (free of food and urine) were made for 3 days between days 15 and 18 of gestation. The fecal samples were pooled and frozen until analyzed. Food intake during the 3 days was also carefully recorded. The weights of the animals taken immediately after the fecal collection period were used to normalize the fecal excretion of neutral steroids and bile acids. Feces were analyzed for neutral steroids and bile acids by the methods of Miettinen et al. [12] and Subbiah [13], respectively. The fecal bile acids were methylated with freshly prepared diazomethane, and the bile acid methyl esters were acetylated using 1 ml of a mixture of acetic anhydride, acetic acid, and perchloric acid (5 : 5 : 0.050, v/v/v) at room temperature for 1 h [14]. The bile acid methyl ester
142
acetates were quantitated by GLC using hyocholic acid as the internal standard. A Perkin Elmer Sigma 2000 (Norwalk, CT) gas-liquid chromatograph equipped with a 6-ft column of 1% OV-225 on Gas Chrom Q (loo/120 mesh) (Supelco, Bellefonte, PA) [15] was used. The operating conditions were: Column -240°C Injector - 250°C and Detector (FID) -260°C Helium was used as the carrier gas at 30 ml/min. Identification of the individual bile acids was based on comparison to relative retention times of authentic standards obtained from Steraloids (Wilton, NH). The major steroid groups in the neutral steroid extract were separated by TLC as described by Miettinen et al. [8]. After elution from the gel, the steroids in each group were quantitated by GLC using Sa-cholestane (Steraloids) as the internal standard. A Hewlett-Packard Model 5840A (Palo Alto, CA) gas-liquid chromatograph equipped with a 6-ft column of 3% OV-17 on Gas Chrom Q (loo/120 mesh) (Applied Science, State College, PA) was used. The operating conditions were: Column - 255°C Injector - 265°C Detector (FID) - 300°C. Nitrogen was used as the carrier gas at a flow rate of 50 ml/mm. [4-i4C]Cholesterol was used to correct for procedural losses of neutral steroids. Fecal excretion of neutral steroids was corrected for variations in fecal flow and steroid losses during intestinal transit by the fecal recovery of dietary /3-sitosterol as described previously [16]. Hepatic CH-7A assay On day 20 of gestation,
the animals were anesthetized with an intramuscular injection of ketamine/xylazine (45 mg/kg body wt and 6 mg/kg body wt, respectively) and exsanguinated by cardiac puncture. Portions of the liver from 4 randomly selected animals in each group were rapidly excised and placed in a chilled solution of 250 mM sucrose, 2.5 mM EDTA, and 50 mM nicotinamide, pH 7.4. Hepatic CH-7A activity was assayed as described below. The remaining liver and the entire gastrointestinal tract (excluding the stomach) were removed and frozen for the analysis of bile acid pool size as described below. Hepatic microsomal CH-7A activity was assayed as described previously [17] based on the methods of Mitropoulos and Balasubramaniam [18] and Shefer et al. [19]. Microsomal protein was measured by the Hartree [20] modification of the Lowry procedure, and CH-7A activity was expressed as picomoles of ;lar-hydroxycholesterol formed/mg microsomal protein/mm.
Analysis of bile acid pool
For the determination of bile acid pool size and composition, the liver and the gastrointestinal tract were frozen in liquid nitrogen and ground into a fine powder using a Waring Blender (Dynamics Corp., New Hartford, CT) with a stainless steel cup. A weighed aliquot of the powder was saponified with 2.5 N NaOH and extracted for bile acids after acidification to pH 3. The extracted bile acids were methylated with freshly prepared diazomethane, and the bile acid methyl esters were purified from fatty acids by TLC as described by Grundy et al. [21]. Purified bile acid methyl esters were quantitated as their acetates as described above for fecal bile acids.
143
Analysis of serum cholesterol Serum obtained from the rats was analyzed for cholesterol according to Morin and Elms [22]. Cholesterol was quantitated by GLC, as described above for fecal neutral steroids, using stigmasterol as an internal standard [22]. Statistical methods Control and experimental values were compared using the Student t-test for unpaired means. A P-value of < 0.5 was considered to be statistically significant. Results
Body weights and serum cholesterol Table 1 shows that animals in both groups gained weight equally well. There was no difference in the mean number of fetuses from animals in either group (CON (n = 7) 10 k 2; CTR (n = 6), 10 k 3, mean f SEM) or in the mean pooled body weights of the fetuses from animals in either group (CON (n = 7), 40.6 + 5.3 g; CTR (n = 6) 38.1 f 2.8 g). Cholestyramine treatment during gestation, however, resulted in a significant elevation of serum cholesterol (CON (n = 7) 91 -t_4; CTR (n = 6) 108 f 5 mg/dl, P < 0.05). This finding was unexpected and the possible mechanism is discussed below. Fecal excretion of neutral steroids and bile acids As shown in Table 2, CTR treatment resulted in the expected enhanced excretion of bile acids (CON (n = 7), 7.6 k 0.4; CTR (n = 6), 25.8 _t 0.7 mg/lOO gm body wt/day, P < 0.05). Fecal excretion of neutral steroids was also significantly enhanced by dietary CTR (CON (n = 7), 3.9 & 0.3; CTR (n = 6), 10.4 + 0.3, P < 0.05) probably, in part, due to the fact that bile acids, which are obligatory for cholesterol
TABLE
1
BODY WEIGHTS AND PLASMA PREGNANT RATS
CHOLESTEROL
IN CONTROL
AND CHOLESTYRAMINE-FED
Timed pregnant rats (8 days) were fed ground stock rat diet (CON) or ground stock rat diet with 4% cholestyramine resin (CTR) until day 20 of gestation. Serum cholesterol was determined at day 20 of gestation. Results are given as the mean & SEM and the numbers in parentheses refer to the number of animals studied. Group
(n)
CON (7) CTR (6)
* Significantly
different
Initial body wt
Final body wt
Serum cholesterol
(8)
(8)
(mg/dB
191*3 192+3
308+7 315f7
91&-4 108+5 *
from control,
P < 0.05.
144
TABLE 2 FECAL EXCRETION OF NEUTRAL STEROIDS CHOLESTEROL 7a-HYDROXYLASE IN CONTROL NANT RATS
AND BILE ACIDS AND HEPATIC AND CHOLESTYRAMINE-FED PREG-
Timed pregnant rats (8 days) were treated as described in Table 1. Quantitative fecal collections were made for 3 days between days 15 and 18 of gestation and analyzed for neutral steroids and bile acids as described under Methods. Hepatic cholesterol 7a-hydroxylase was assayed on day 20 of gestation as described in text. The values are presented as mean + SEM and the numbers in parentheses refer to the number of animals studied. Group
Food eaten (g/100 g body
Fecal steroid excretion (mg/lOO gm body wt/day)
Wday)
Neutral steroids
CON
8.2 k 0.1 (7)
CTR
8.7 + 0.1 (6) *
Bile acids
3.9 f 0.3 (7)
7.6 f 0.4 (7)
10.4 f 0.3 (6) *
25.8 f 0.7 (6) *
Total steroids
Hepatic cholesterol 7cY-hydroxylase (pmoles/mg/min)
11.6+0.5 (7)
14.7 f 1.7 (4)
36.1+ 1.5 (6) *
34.8 k 3.3 (4) *
* Significantly different from control, P < 0.05.
absorption [23], were bound by CTR. While it appears that CTR-treated animals ate a significantly greater amount of food, the increase is more apparent than real. If 4% of the weight of the food consumed is subtracted (because CTR is a non-nutritive, non-absorbable resin), then there is no difference in food consumption between the 2 groups. The dramatic increase in the fecal excretion of bile acids by the CTR-treated animals would be expected to result in an increase in the activity of hepatic CH-7A. As shown in Table 2, CTR-treatment increased CH-7A activity ca. 2.6-fold when compared to that in controls (CON (n = 4) 14.7 f 1.7; CTR (n = 4) 34.8 + 3.3 pmoles/mg/min, P < 0.05). Bile acid pool size and composition
While CTR treatment did not alter the total bile acid pool size (Table 3) there were some differences in the pool sizes of individual bile acids. CTR treatment resulted in an increase in the pool sizes of lithocholic acid and cholic acid, while the pool sizes of hyodeoxy-/ursodeoxy-cholic acids and (Ymuricholic acid were significantly greater in the control animals. When the bile acids were grouped into 2 major pools, ‘cheno’ pool (lithocholic, 3P,A5-cholenoic, hyodeoxy-/ursodeoxy-cholic acids and (Y- and /3-muricholic acids) and ‘cholic’ pool (deoxycholic and cholic acids), CTR-treated animals were found to have a lower ‘cheno’ pool (CON (n = 4), 54.5 * 3.88%; CTR (n = 4), 37.12 f 3.0% P < 0.05) and a higher ‘cholic’ pool (CON (n = 4) 45.5 f 3.88%; CTR (n = 4) 62.88 + 3.0%, P < 0.05) and thus a reduced ‘cheno’ to ‘cholic’ ratio (CON (n = 4), 1.24 + 0.16; CTR (n = 4), 0.6 f 0.08, P < 0.05).
3
POOL
SIZE
AND
COMPOSITION
IN CONTROL
AND
CHOLESTYRAMINE-TREATED
PREGNANT
3.0 +0.7 * (14.26k1.23)”
0.8 f0.2 (4.05 f 2.13)
Lithocholic acid
0.1 io.03 (0.65+0.12)
0.2 fO.l (0.98 f 0.48)
3/3.As-Cholenic acid
2.7 (13.0
i0.4 fO.60)
1.8 +0.2 (8.50k0.72)
Deoxycholic acid
*
1.7 (8.0
+0.3 kO.77)
2.9 kO.4 (13.77 t 1 .O)
Chenodroxycholic acid
* Significantly different frown corresponding control value. P < 0.05. (’ ‘Cheno’ Includes the following hile acid&: Ilthochollc. 3/3.3‘-cholrnoic. ‘Cholic’ includes deoxycholic and cholic acids.
CTR
Control
CiKUp
chenodeoxycholic.
0.1 kO.04 * (0.47+0.17)
0.7 kO.2 (3.50+ 0.89) * *
H yodeoxy-/ ursodeoxycholic
*
RATS
acids.
a-muricholx
0.6 +0.2 (2.8x+0.66)*
2.2 +0.3 (10.x6+1.15)
*
1.5 fO.4 (7.18+ 1.69) 5.3 k1.0 (25.03i4.22)
fl-Muricholic acid
as described in Methods. bile acid pool.
a-Muricholic acid
and ursodeoxy-chollc
9.9 i0.6 (49.89k3.47)
7.6 +0.6 (37.01 t4.31)
Cholic acid
Ilyodeoxy-
acid
Rats were treated as described in Table 1. Pool size and composition was determined in rats killed on day 20 of gestation body wt are given as mea” + SEM for 4 animals in each group. Numbers in parentheses refer to the percentages of total
BILE ACID
TABLE
1.6
”
g
acids.
0.60 + 0.1 *
1.24+0.2
‘Cheno’/ ‘Cholic’
mg/lOO
and &muricholic
20.3 f 2.3
20.9*
Total
The values.
R
146
Discussion
The results of the present study show that cholestyramine fed during gestation can affect cholesterol and bile acid metabolism in the pregnant rat. In the discussion to follow, each of the effects of CTR treatment on cholesterol and bile acid metabolism is discussed separately. CTR treatment and bile acid pool size and composition In a recent study Hassan et al. [24] showed that CTR fed to weanling male rats for 1 week resulted in an expansion of the bile acid pool in addition to a ca. 4-fold increase in the hepatic CH-7A activity. In the present study, feeding CTR to pregnant rats for 12 days did not affect the total bile acid pool size even though CH-7A activity was elevated ca. 2.6 times over that in controls. CTR treatment did, however, result in compositional changes in the bile acid pool. The ‘cheno’/‘cholic’ ratio was significantly lower in the CTR-treated animals. There are at least 2 possible mechanisms which could account for the effect of CTR on the ‘cheno’/‘cholic’ ratio. Cholestyramine has a well-documented greater affinity for dithan for tri-hydroxy bile acids [25]. Thus CTR-induced interruption of the enterohepatic circulation of chenodeoxycholic acid would be greater than that of cholic acid. Since chenodeoxycholic acid also serves as the substrate for the hepatic synthesis of hyodeoxycholic, ursodeoxycholic, and the muricholic acids in the rat [26], a ‘drain’ in the chenodeoxycholic acid pool would be expected to reduce the pool sizes of these bile acids thus resulting in the reduction of the total ‘cheno’ pool. This hypothesis is supported by the finding of a greater increase in the pool of lithocholic acid (3.8-fold) than deoxycholic acid (1.5-fold) (bacterial 7a-dehydroxylation products of chenodeoxycholic acid and cholic acid, respectively) in the CTR-treated animals relative to non-treated controls. Secondly, since there was an actual increase in the cholic acid pool size in the CTR-treated animals, CTR treatment could possibly have increased the activity of hepatic microsomal 12a-hydroxylase and, therefore, increased the synthesis of cholic acid [26]. Both of these mechanisms, alone or in combination, could have resulted in the decreased ‘cheno’/‘cholic’ ratio following CTR treatment in the present study. Effect of
CTR treatment on cholesterol metabolism As mentioned earlier, CTR treatment typically results in a reduction in plasma cholesterol [4]. The degree of CTR-induced hypocholesterolemia is affected by the extent of the compensatory increase in cholesterol biosynthesis. In view of this, it was surprising to find that, in the present study, CTR treatment significantly elevated serum cholesterol in the pregnant rats. The exact mechanism for the hypercholesterolemia is not known. As pointed out earlier, the pregnant animal is faced with maintaining a cholesterol supply both for the developing fetus as well as for itself. CTR treatment during pregnancy imposes an additional ‘load’ on the cholesterol synthetic ability of the mother by dramatically increasing the turnover of cholesterol (Table 2). It is known that CTR treatment enhances the activity of HMGCoA-R both in the liver and intestine [3,27], the 2 major tissues of cholestero-
147
genesis in the rat [28]. Thus, it is possible that maternal total body cholesterogenesis is enhanced in the pregnant CTR-treated rat and overcompensates in order to assure adequate supplies of cholesterol both for fetal and maternal demands thereby resulting in hypercholesterolemia. This finding may have some implications on the use of CTR during pregnancy. Effect of CTR treatment on hepatic CH-7A activity In a recent study, Innis found that pregnant rats treated with Questran did not show an increase in CH-7A activity [ll] while similarly treated non-pregnant rats showed a 3-fold increase. However, since both pregnant and non-pregnant Questran-treated rats showed similar elevation in HMGCoA-R activity relative to non-treated controls [ll], we wished to clarify Innis’ observations on CH-7A activity. To avoid the possible interference by the sucrose content of Questran, we used pure CTR resin. In the present study, CTR treatment during gestation increased CH-7A activity ca. 2.6-fold over controls. This finding is a well-documented effect of CTR treatment on CH-7A activity [1,29] and virtually similar to the finding of Innis [ll] in Questran-treated non-pregnant rats. It is difficult to reconcile the effect of CTR (present study) vs Questran [ll] on CH-7A in the pregnant rat. Since Questran is 56% sucrose, the Questran-containing diet used by Innis [ll] contained a significant amount of sucrose (2.8% or 6.4% depending on whether the diet was 5% in Questran or 5% in resin content), which was not present in the control diet. Whether or not sucrose has any effect on cholesterol and bile acid metabolism in the pregnant rat remains to be determined. In summary, this study has documented the effects of CTR treatment during pregnancy on cholesterol and bile acid metabolism. Pregnant rats respond to CTR treatment with a dramatic increase in the excretion of total fecal steroids and hepatic CH-7A activity. Despite the enhanced turnover of cholesterol, however, plasma cholesterol was significantly greater (ca. 20% increase over controls) in the CTRtreated pregnant rats. References Packard, C.J. and Shepherd, J.. The hepatobiliary axis and lipoprotein metabolism - Effects of bile acid sequestrants and ileal bypass surgery, J. Lipid Res., 23 (1982) 1081. Shefer, S., Hauser, S., Bekersky, I. and Mosbach, E.H., Biochemical site of regulation of bile acid synthesis in the rat, J. Lipid Res., 11 (1970) 404. Goldfarb, S., Regulation of hepatic cholesterogenesis. In: N.B. Javitt (Ed.). International Review of Physiology, Vol. 21 (Liver and Biliary Tract Physiology, I). University Park Press, Baltimore. MD, 1980, p. 317. The Lipid Research Clinics Program, The Lipid Research Clinics Coronary Primary Prevention Trial Results, Part 1 (Reduction in incidence of coronary heart disease) and Part 2 (The relationship of reduction of coronary heart disease to cholesterol lowering), J. Amer. Med. Ass.. 251 (1984) 351. Everson, G.T., McKinley, C., Lawson, M., Johnson, M. and Kern, Jr., F., Gallbladder function in the human female - Effect of ovulatory cycle, proegnancy. and contraceptive steroids, Gastroenterology, 82 (1982) 711. Kern, Jr.. F., Everson, G.T., DeMark, B., McKinley, C., Showalter. R.. Erfling, W.D., Braverman. 2..
148
7 8 9 10 11
Szczepanik-Van Leeuwen, P. and Klein, P. D., Biliary lipids, bile acids, and gallbladder function in the human female, J. Clin. Invest., 68 (1981) 1229. Kern, Jr., F., Eriksson, H., Curstedt, T. and Sjiivall, J., Effect of ethynylestradiol on biliary excretion of bile acids, phosphatidylcholines, and cholesterol in the bile fistula rat, J. Lipid Res., 18 (1977) 623. Connor, W.E. and Lin, D.S., Placental transfer of cholesterol-4-‘4C into rabbit and guinea pig fetus, J. Lipid Res., 8 (1967) 558. Pitkin, R.M., Connor. W.E. and Lin, D.S., Cholesterol metabolism and placental transfer in the pregnant rhesus monkey. J. Clin. Invest., 51 (1972) 2584. Lin, D.S., Pitkin, R.M. and Connor, W.E., Placental transfer of cholesterol into the human fetus, Amer. J. Obstet. Gynecol., 128 (1977) 735. Innis, S.M., Effect of cholestyramine administration during pregnancy in the rat, Amer. J. Obstet.
Gynecol., 146 (1983) 13. 12 Miettinen, T.A., Ahrens, Jr.. E.H. and Grundy, S.M., Quantitative isolation and gas-chromatographic analysis of total dietary and fecal neutral steroids, J. Lipid Res., 6 (1965) 411. 13 Subbiah, M.T.R., Tyler, N.E., Buscaglia, M.D. and Marai, L., Estimation of bile acid excretion in man - Comparison of isotopic turnover and fecal excretion methods, J. Lipid Res., 17 (1976) 78. 14 Parmentier, G.G., Janssen, G.A., Eggermont, E.A. and Eyssen, H.J., Cz, bile acids in infants with coprostanic acidemia and occurrence of a 3a,7a,l2a-trihydroxy-5P_C29 dicarboxylic bile acid as a major component in their serum, Europ. J. Biochem., 102 (1979) 173. 15 Yousef, I.M., Fisher, M.M., Myher, J.J. and Kuksis, A., Superior gas-liquid chromatography of methyl cholanoate acetates on cyanopropylphenylsiloxane liquid phases, Anal. B&hem., 75 (1976) 538. 16 Grundy, SM., Ahrens, Jr., E.H. and Salen, G., Dietary p-sitosterol as an internal standard to correct for cholesterol losses in sterol balance studies, J. Lipid Res., 9 (1968) 374. 17 Hassan, AS., Gallon, L.S., Zimmer, L.A., Balistreri, W.F. and Subbiah, M.T.R., Persistent enhancement of bile acid synthesis in guinea pigs following stimulation of cholesterol catabolism in neonatal life, Steroids, 38 (1981) 477. 18 Mitropoulos, K.A. and Balasubramaniam, S., Cholesterol 7e-hydroxylase in rat liver microsomal preparations, Biochem. J., 128 (1972) 1. 19 Shefer, S., Hauser, S. and Mosbach, E.H., 7a-Hydroxylation of cholesterol by rat liver microsomes, J. Lipid Res., 9 (1968) 328. 20 Hartree, E.F., Determination of protein - A modification of the Lowry method that gives a linear photometric response, Anal. B&hem., 48 (1972) 422. 21 Grundy, S.M., Ahrens, Jr., E.H. and Miettinen, T.A., Quantitative isolation and gas-liquid chromatographic analysis of total fecal bile acids, J. Lipid Res., 6 (1965) 397. 22 Morin, R.J. and Elms, N.J., Rapid microanalysis of cholesterol in bile and serum by gas chromatography, Ann. Clin. Lab. Sci., 5 (1975) 52. 23 Siperstein, M.D., Chaikoff, I.L. and Reinhardt, W.O., “C-Cholesterol, Part 5. (Obligatory function of bile in the intestinal absorption of cholesterol), J. Biol. Chem., 198 (1952) 111. 24 Hassan, A.S., Srivastava, L.S., Yunker, R.L. and Subbiah, M.T.R., Cholestyramine feeding in weaned rat - Increase in plasma corticosterone levels and bile acid synthesis following adrenalectomy, Atherosclerosis, 51 (1984) 327. 25 Johns, W.H. and Bates, T.R., Quantification of the binding tendencies of cholestyramine, Part 1 (Effect of structure and added electrolytes on the binding of unconjugated and conjugated bile salt anions), J. Pharmacol. Sci., 38 (1969) 179. 26 Beher, W.T., Biosynthesis of primary bile acids. In: D. Kritchevsky, O.J. Pollak and H.S. Simms (Eds.) Monographs on Atherosclerosis, Vol. 6 (Bile Acids - Chemistry and Physiology of Bile Acids and their Influence on Atherosclerosis), S. Karger, New York, 1976, pp. 17-33. 27 Panini, S.R., Lehrer, G., Rogers, D.H. and Rudney, H., Distribution of 3-hydroxy-3-methylglutaryl coenzyme A reductase and alkaline phosphatase activities in isolated epithelial cells of fed, fasted, cholestyramine-fed, and 4-aminopyrazolo-[3,4-dlpyrimidine-treated rats, J. Lipid Res., 30 (1979) 879. 28 Spady, D.K. and Dietschy, J.M., Sterol synthesis in vivo in 18 tissues of the squirrel monkey, guinea pig, rabbit, hamster, and rat, J. Lipid Res., 24 (1983) 303. 29 Myant, N.B. and Mitropoulos, K.A., Cholesterol 7cY-hydroxylase, J. Lipid Res., 18 (1977) 135.