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1.
FASEB J ; 15(14): 2623-30, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11726538

ABSTRACT

Apolipoprotein E (apo E) deficiency (or its abnormalities in humans) is associated with a series of pathological conditions including dyslipidemia, atherosclerosis, Alzheimer's disease, and shorter life span. The purpose of this study was to characterize these conditions in apo E-deficient C57BL/6J mice and relate them to human disorders. Deletion of apo E gene in mice is associated with changes in lipoprotein metabolism [plasma total cholesterol (TC) (>+400%), HDL cholesterol (-80%), HDL/TC, and HDL/LDL ratios (-93% and -96%, respectively), esterification rate in apo B-depleted plasma (+100%), plasma triglyceride (+200%), hepatic HMG-CoA reductase activity (-50%), hepatic cholesterol content (+30%)], decreased plasma homocyst(e)ine and glucose levels, and severe atherosclerosis and cutaneous xanthomatosis. Hepatic and lipoprotein lipase activities, hepatic LDL receptor function, and organ antioxidant capacity remain unchanged. Several histological/immunohistological stainings failed to detect potential markers for neurodegenerative disease in the brain of 37-wk-old male apo E-KO mice. Apo E-KO mice may have normal growth and development, but advanced atherosclerosis and xanthomatosis may indirectly reduce their life span. Apo E plays a crucial role in regulation of lipid metabolism and atherogenesis without affecting lipase activities, endogenous antioxidant capacity, or appearance of neurodegenerative markers in 37-wk-old male mice.


Subject(s)
Apolipoproteins E/deficiency , Animals , Antioxidants/metabolism , Apolipoproteins E/genetics , Blood Glucose/metabolism , Body Weight/physiology , Brain/metabolism , Cholesterol/metabolism , Esterification , Genotype , Glial Fibrillary Acidic Protein/analysis , Homocysteine/blood , Humans , Hydroxymethylglutaryl CoA Reductases/metabolism , Hyperlipidemias/blood , Hyperlipidemias/physiopathology , Immunohistochemistry , Kidney/metabolism , Lipase/blood , Lipids/blood , Lipoproteins, HDL/metabolism , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Neurofilament Proteins/analysis , Receptors, LDL/physiology , Survival Analysis , Time Factors , Tissue Distribution
2.
Int J Pharm ; 229(1-2): 75-86, 2001 Oct 23.
Article in English | MEDLINE | ID: mdl-11604260

ABSTRACT

Poloxamer-407 (P-407) is a nonionic surfactant that induces atheroma formation in the aortas of C57BL/6 mice with long-term (14 weeks) administration. The objectives of the present study were to determine the mechanism(s) responsible for the induction of hypercholesterolemia as well as to determine whether this animal model may be of potential use in rank ordering the efficacy (lipid lowering) of various statin drugs. The effect of long-term (16 weeks) administration of P-407 on the catalytic activities of rate-limiting enzymes of cholesterol biosynthesis [HMG-CoA reductase (HMGR)] and catabolism [microsomal cholesterol 7alpha-hydroxylase (C7alphaH) and mitochondrial sterol 27 hydroxylase (S27H)] was assessed in C57BL/6 mice. Effects of P-407 on these enzymes were compared in mice fed an atheroma-inducing diet (high-cholesterol, supplemented with cholic acid) and animals maintained on a basal diet and injected with saline (controls) after 16 weeks. The mean value for the activities of C7alphaH in P-407-injected mice was 24.3+/-3.8 pmol min(-1) mg(-1) and was significantly (P<0.05) less than the mean value determined for sham-injected control animals (37.0+/-14.3 pmol min(-1) mg(-1)). In contrast, the mean values for the catalytic activities of S27H and HMGR did not change with P-407 administration. Neither C7alphaH nor S27H activity in mice fed the high-cholesterol diet differed from values for control animals, whereas the mean HMGR activity was drastically reduced (-94%, P<0.05). The hypercholesterolemic effect of P-407 is not due to altered cholesterol biosynthesis, but is mediated by reduced cholesterol catabolism due to decreased activity of the rate limiting enzyme (C7alphaH) in the classic bile acid synthetic pathway. Plasma triglyceride lowering resulting from the oral administration of equal doses of various statin drugs appeared, in general, to be positively correlated with their relative aqueous solubility and paralleled the efficacy of these agents to lower low-density-lipoprotein-associated cholesterol (LDL-C) in humans. The plasma triglyceride lowering effect of the five statin drugs tested produced the following rank order; pravastatin sodium (-44%)>atorvastatin calcium (-36%)>simvastatin (-33%)>lovastatin (-25%)>fluvastatin sodium (-19%). While reductions in plasma total cholesterol following administration of the statin drugs was not as profound as that observed with triglycerides, the relative rank order or trend was preserved. The percent reduction in plasma triglycerides in the present model appears to be a useful parameter with which to predict the relative reduction in plasma LDL-C expected for these agents in humans.


Subject(s)
Anticholesteremic Agents/therapeutic use , Arteriosclerosis/drug therapy , Hyperlipidemias/drug therapy , Animals , Anticholesteremic Agents/administration & dosage , Arteriosclerosis/blood , Cholesterol/blood , Female , Hydroxymethylglutaryl CoA Reductases/metabolism , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Hyperlipidemias/blood , Liver/drug effects , Liver/enzymology , Mice , Mice, Inbred C57BL , Triglycerides/blood
3.
Metabolism ; 50(10): 1224-9, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11586498

ABSTRACT

Sitosterolemia is a rare, recessively inherited disease characterized clinically by accelerated atherosclerosis and xanthomas and biochemically by hyperabsorption and retention of sitosterol and other plant sterols in tissues. Decreased cholesterol biosynthesis and inhibition of 3-hydroxy-3-methylgluratyl coenzyme A (HMG-CoA) reductase and other enzymes in the biosynthetic pathway have been associated with enhanced low-density lipoprotein (LDL) receptor function. We examined the effects of cholesterol and sitosterol on sterol concentrations and composition and HMG-CoA reductase activity in monocyte-derived macrophages (MDM) from 12 control and 3 homozygous sitosterolemic subjects. The cells were cultured up to 7 days in media devoid of plant sterols, but containing increasing amounts of serum cholesterol. Before culture, MDM from the homozygous sitosterolemic subjects contained 22% more total sterols than cells from control subjects. Plant sterols and stanols represented 15.6% of MDM total sterols in sitosterolemic cells, but only 3.8% in control cells. After 7 days of culture in 10% delipidated serum (DLS) (20 microg/mL cholesterol, no sitosterol), all plant sterols were eliminated so that cells from both phenotypes contained only cholesterol. When DLS was replaced with fetal bovine serum (FBS) (300 micromL cholesterol), with and without addition of 200 microg/mL LDL, cholesterol levels in MDM from sitosterolemic subjects increased 108% (P <.05) compared with a 65% increase (P <.04) in control MDM cultured similarly. MDM HMG-CoA reductase activity from the 3 sitosterolemic subjects, which was significantly lower than controls at baseline (24 +/- 3 v 60 +/- 10 pmol/mg/min, P <.05), was not downregulated by increased cellular cholesterol levels, as observed in control cells. Control MDM were also cultured in medium that contained 10% DLS and was supplemented with 100 microg/mL cholesterol or sitosterol dissolved in ethanol or the ethanol vehicle alone. In contrast to cellular cholesterol accumulation, which significantly downregulated HMG-CoA reductase activity (-53%, P <.05), the increase in cellular sitosterol up to 25.1% of total sterols did not change MDM HMG-CoA reductase activity. Evidence of a normal HMG-CoA reductase protein in sitosterolemic cells, which was not derepressed upon removal of cellular sitosterol, and the failure of cellular sitosterol to inhibit normal HMG-CoA reductase activity argue against feedback inhibition by sitosterol as a mechanism for low reductase activity in this disease. The larger accumulation of sterols and inadequate downregulation of HMG-CoA reductase in MDM may be mechanisms for foam cell formation and explain, in part, the increased risk of atherosclerosis in sitosterolemia.


Subject(s)
Cholesterol/pharmacology , Hydroxymethylglutaryl CoA Reductases/metabolism , Macrophages/enzymology , Sitosterols/pharmacology , Adult , Arteriosclerosis/blood , Arteriosclerosis/genetics , Cells, Cultured , Culture Media , Enzyme Activation/drug effects , Female , Humans , Male , Middle Aged , Sitosterols/blood
4.
Metabolism ; 50(9): 1106-12, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11555847

ABSTRACT

The aim of this study was to study the inhibitory effect of dietary stanols (campestanol and sitostanol) fatty acid esters (SE) on intestinal cholesterol absorption. New Zealand white rabbits were fed regular chow alone or enriched with 0.2% cholesterol, 0.33% SE + cholesterol, 0.66% SE + cholesterol, 1.2% SE + cholesterol, 2.4% SE + cholesterol, and 1.2% SE alone. After 2 weeks, plasma cholesterol levels increased 3.6 times in the cholesterol group and did not decrease after addition of 0.33% or 0.66% SE to the cholesterol-enriched diets. However, after addition of 1.2% SE to the cholesterol diet, plasma cholesterol concentration decreased 50% (P <.001), but it did not decrease further after doubling of SE to 2.4%. Percent cholesterol absorption measured by the plasma dual-isotope ratio method was 73.0% +/- 8.1 % in the cholesterol group, which was similar to untreated baseline control. The percent absorption of cholesterol did not decrease significantly after addition of 0.33% or 0.66% SE to the cholesterol diet but decreased 43.8% (P <.001) in the 1.2% SE + cholesterol group, a finding similar to those in rabbits fed 1.2% SE alone. Increasing SE to 2.4% in the cholesterol diet did not further decrease absorption. Hepatic hydroxymethyl glutaryl coenzyme A (HMG-CoA) reductase activity reflecting cholesterol synthesis and low-density lipoprotein receptor-mediated binding unexpectedly decreased 67% (P <.01) and 57% (P <.05) in rabbits fed 1.2% SE alone. Increasing dietary SE intake to 1.2% reduced cholesterol absorption and plasma levels. Dietary SE intake below 1.2% was ineffective and above 2.4% did not further decrease percent absorption or plasma cholesterol levels. These results support the hypothesis that dietary SEs competitively displace cholesterol from intestinal micelles to reduce cholesterol absorption and decrease plasma cholesterol levels.


Subject(s)
Cholesterol/metabolism , Hydroxymethylglutaryl CoA Reductases/metabolism , Intestinal Absorption/drug effects , Phytosterols/pharmacology , Sitosterols/pharmacology , Animals , Anticholesteremic Agents/pharmacology , Bile/metabolism , Cholestanetriol 26-Monooxygenase , Cholesterol 7-alpha-Hydroxylase/metabolism , Cholesterol, Dietary/metabolism , Cytochrome P-450 Enzyme System/metabolism , Dietary Supplements , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Liver/drug effects , Liver/enzymology , Male , Rabbits , Receptors, LDL/metabolism , Steroid Hydroxylases/metabolism
5.
J Clin Invest ; 108(6): 905-15, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11560960

ABSTRACT

Smith-Lemli-Opitz/RSH syndrome (SLOS), a relatively common birth-defect mental-retardation syndrome, is caused by mutations in DHCR7, whose product catalyzes an obligate step in cholesterol biosynthesis, the conversion of 7-dehydrocholesterol to cholesterol. A null mutation in the murine Dhcr7 causes an identical biochemical defect to that seen in SLOS, including markedly reduced tissue cholesterol and total sterol levels, and 30- to 40-fold elevated concentrations of 7-dehydrocholesterol. Prenatal lethality was not noted, but newborn homozygotes breathed with difficulty, did not suckle, and died soon after birth with immature lungs, enlarged bladders, and, frequently, cleft palates. Despite reduced sterol concentrations in Dhcr7(-/-) mice, mRNA levels for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-controlling enzyme for sterol biosynthesis, the LDL receptor, and SREBP-2 appeared neither elevated nor repressed. In contrast to mRNA, protein levels and activities of HMG-CoA reductase were markedly reduced. Consistent with this finding, 7-dehydrocholesterol accelerates proteolysis of HMG-CoA reductase while sparing other key proteins. These results demonstrate that in mice without Dhcr7 activity, accumulated 7-dehydrocholesterol suppresses sterol biosynthesis posttranslationally. This effect might exacerbate abnormal development in SLOS by increasing the fetal cholesterol deficiency.


Subject(s)
Dehydrocholesterols/metabolism , Hydroxymethylglutaryl CoA Reductases/metabolism , Oxidoreductases Acting on CH-CH Group Donors , Smith-Lemli-Opitz Syndrome/metabolism , Sterols/biosynthesis , Animals , Animals, Newborn , DNA-Binding Proteins/genetics , Disease Models, Animal , Gene Targeting , Humans , Hydroxymethylglutaryl CoA Reductases/genetics , Mice , Mice, Knockout , Oxidoreductases/chemistry , Oxidoreductases/deficiency , Oxidoreductases/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, LDL/genetics , Smith-Lemli-Opitz Syndrome/genetics , Sterol Regulatory Element Binding Protein 2 , Transcription Factors/genetics
6.
Vestn Ross Akad Med Nauk ; (5): 34-8, 2001.
Article in Russian | MEDLINE | ID: mdl-11510148

ABSTRACT

The paper provides evidence for the pathogenetic approach to treating acute lung lesion (ALL) and adult respiratory distress syndrome (ARDS). An algorithm of the use of Russian lung surfactant preparations: CT-HL and CT-BL has been developed. In involves earlier (the first days following the onset of respiratory failure) use of surfactant, its combined bolus intratracheal or intrabronchial administration in doses of 200-400 mg/m2, followed by continuous (5-day) aerosol inhalation in doses of 20-30 mg/h for children and 30-75 mg/h for adults until pronounced clinical and X-ray effects are shown. Fifty three patients were found to develop ALL and ARDS in the presence of severe pneumonia, postperfusion lung disorders, reperfusion syndrome, pulmonary embolism, long-term artificial ventilation, combined car accident injury and gunshot wounds of the chest, heroine intoxication, septic shock, sepsis, postoperative sequels in cancer patients, and after hepatic transplantation or massive aspiration of gastric contents. Fifty patients were overcome their critical status, 44 survived. The duration of artificial ventilation (AV) ranged from 1 to 6 days. Earlier use of the drugs made it possible to transfer patients to safe AV regimens and to eliminate ALL and ARDS rapidly and to significantly reduce mortality due to critical states.


Subject(s)
Critical Care , Pulmonary Surfactants/therapeutic use , Respiratory Distress Syndrome/therapy , Acute Disease , Adolescent , Adult , Age Factors , Algorithms , Child , Child, Preschool , Humans , Infant , Lung Diseases/drug therapy , Lung Diseases/therapy , Pulmonary Surfactants/administration & dosage , Respiration Disorders/drug therapy , Respiration Disorders/therapy , Respiration, Artificial , Respiratory Distress Syndrome/drug therapy , Respiratory Distress Syndrome/etiology , Respiratory Therapy , Time Factors
7.
J Lipid Res ; 42(8): 1250-6, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11483626

ABSTRACT

We examined the effect of hyodeoxycholic acid (HDCA) on plasma cholesterol levels and atherosclerosis in mice. In wild-type C57BL/6 mice, feeding increasing amounts of HDCA resulted in i) progressive decrease in dietary cholesterol absorption, ii) increased concentrations of HDCA in the gallbladder bile, iii) decreased liver cholesterol content, iv) increased liver cholesterol synthesis, and v) increased plasma concentrations of HDCA. In C57BL/6 LDL-receptor knockouts (LDLR-KO) the addition of HDCA to chow and a 0.5% cholesterol diet decreased their total plasma cholesterol levels by 21% and 62%, respectively, because of a decrease in VLDL and LDL cholesterol. Turnover studies showed that HDCA has no effect on VLDL removal from plasma. Furthermore, the addition of HDCA to chow- and 0.5% cholesterol-fed LDLR-KO mice decreased the aortic root atherosclerosis lesion area by 50% and 80%, respectively. Finally, we tested the effect of HDCA on intestinal tumor formation. Feeding C57BL/6 ApcMin mice with HDCA did not affect the number of tumors but decreased the tumor volume in these animals. These results suggest that HDCA might have beneficial effects in the treatment of increased plasma cholesterol levels and atherosclerosis.


Subject(s)
Arteriosclerosis/prevention & control , Cholesterol/blood , Deoxycholic Acid/therapeutic use , Absorption , Animals , Bile/metabolism , Cholesterol/metabolism , Cholesterol, Dietary/pharmacokinetics , Cholesterol, LDL/blood , Cholesterol, VLDL/blood , Deoxycholic Acid/administration & dosage , Deoxycholic Acid/pharmacokinetics , Hydroxymethylglutaryl CoA Reductases/metabolism , Intestinal Neoplasms/pathology , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, LDL/deficiency , Receptors, LDL/physiology
8.
J Lipid Res ; 42(9): 1438-43, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11518763

ABSTRACT

To study the effect of cholecystectomy on the regulation of classic and alternative bile acid syntheses, gallbladder-intact (n = 20) and cholecystectomized (n = 20) New Zealand White rabbits were fed either chow or chow with 2% cholesterol (3 g/day). After 10 days, bile fistulas were constructed in half of each rabbit group to recover and measure the bile acid pool and biliary bile acid flux. After cholesterol feeding, the bile acid pool size increased from 268 +/- 55 to 444 +/- 77 mg (P < 0.01) with a 2-fold rise in the biliary bile acid flux in intact rabbits but did not expand the bile acid pool (270 +/- 77 vs. 276 +/- 62 mg), nor did the biliary bile acid flux increase in cholecystectomized rabbits. Ileal apical sodium-dependent bile acid transporter protein increased 46% from 93 +/- 6 to 136 +/- 23 units/mg (P < 0.01) in the intact rabbits but did not change in cholecystectomized rabbits (104 +/- 14 vs. 99 +/- 19 units/mg) after cholesterol feeding. Cholesterol 7alpha-hydroxylase activity was inhibited 59% (P < 0.001) while cholesterol 27-hydroxylase activity rose 83% (P < 0.05) after cholesterol feeding in the intact rabbits but neither enzyme activity changed significantly in cholesterol-fed cholecystectomized rabbits. Fecal bile acid outputs reflecting bile acid synthesis increased significantly in the intact but not in the cholecystectomized rabbits fed cholesterol. Removal of the gallbladder prevented expansion of the bile acid pool after cholesterol feeding as seen in intact rabbits because ileal bile acid transport did not increase. As a result, cholesterol 7alpha-hydroxylase was not inhibited.


Subject(s)
Bile Acids and Salts/biosynthesis , Cholecystectomy , Cholesterol 7-alpha-Hydroxylase/antagonists & inhibitors , Cholesterol, Dietary/administration & dosage , Gallbladder/physiology , Hydroxysteroid Dehydrogenases , Membrane Glycoproteins , Membrane Transport Proteins , Animals , Bile Acids and Salts/metabolism , Blotting, Western , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cholestanetriol 26-Monooxygenase , Cholesterol/blood , Cholesterol/metabolism , Cholesterol 7-alpha-Hydroxylase/metabolism , Cytochrome P-450 Enzyme System/metabolism , Feces/chemistry , Hydroxymethylglutaryl CoA Reductases/metabolism , Ileum/metabolism , Liver/metabolism , Male , Organic Anion Transporters, Sodium-Dependent , RNA, Messenger/analysis , Rabbits , Steroid Hydroxylases/metabolism , Symporters
9.
Am J Physiol Gastrointest Liver Physiol ; 281(2): G393-404, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11447020

ABSTRACT

Human obesity is associated with elevated plasma leptin levels. Obesity is also an important risk factor for cholesterol gallstones, which form as a result of cholesterol hypersecretion into bile. Because leptin levels are correlated with gallstone prevalence, we explored the effects of acute leptin administration on biliary cholesterol secretion using lean (FA/-) and obese (fa/fa) Zucker rats. Zucker (fa/fa) rats become obese and hyperleptinemic due to homozygosity for a missense mutation in the leptin receptor, which diminishes but does not completely eliminate responsiveness to leptin. Rats were infused intravenously for 12 h with saline or pharmacological doses of recombinant murine leptin (5 microg x kg(-1) x min(-1)) sufficient to elevate plasma leptin concentrations to 500 ng/ml compared with basal levels of 3 and 70 ng/ml in lean and obese rats, respectively. Obesity was associated with a marked impairment in biliary cholesterol secretion. In biles of obese compared with lean rats, bile salt hydrophobicity was decreased whereas phosphatidylcholine hydrophobicity was increased. High-dose leptin partially normalized cholesterol secretion in obese rats without altering lipid compositions, implying that both chronic effects of obesity and relative resistance to leptin contributed to impaired biliary cholesterol elimination. In lean rats, acute leptin administration increased biliary cholesterol secretion rates. Without affecting hepatic cholesterol contents, leptin downregulated hepatic activity of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, upregulated activities of both sterol 27-hydroxylase and cholesterol 7alpha-hydroxylase, and lowered plasma very low-density lipoprotein cholesterol concentrations. Increased biliary cholesterol secretion in the setting of decreased cholesterol biosynthesis and increased catabolism to bile salts suggests that leptin promotes elimination of plasma cholesterol.


Subject(s)
Biliary Tract/metabolism , Cholesterol/metabolism , Leptin/pharmacology , Liver/metabolism , Obesity/metabolism , Phospholipids/metabolism , Animals , Bile Acids and Salts/chemistry , Cholesterol, VLDL/blood , Male , Models, Biological , Phosphatidylcholines/metabolism , Rats , Rats, Zucker
10.
J Biol Chem ; 276(37): 34579-85, 2001 Sep 14.
Article in English | MEDLINE | ID: mdl-11454857

ABSTRACT

The accumulation of various 25-hydroxylated C(27)-bile alcohols in blood and their excretion in urine are characteristic features of cerebrotendinous xanthomatosis (CTX) a recessively inherited inborn error of bile acid synthesis caused by mutations in the mitochondrial sterol 27-hydroxylase (CYP27) gene. These bile alcohols may be intermediates in the alternative cholic acid side chain cleavage pathway. The present study was undertaken to identify enzymes and reactions responsible for the formation of these bile alcohols and to explain why Cyp27(-/-) mice do not show CTX-related abnormalities. Microsomal activities of 5beta-cholestane-3alpha,7alpha,12alpha-triol 25- and 26-hydroxylases, 5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol 23R-, 24S-, and 27-hydroxylases and testosterone 6beta-hydroxylase, a marker enzyme for CYP3A, in Cyp27(-/-) mice livers were markedly up-regulated (5.5-, 3.5-, 6.5-, 7.5-, 2.9-, and 5.4-fold, respectively). In contrast, these enzyme activities were not increased in CTX. The activities of 5beta-cholestane-3alpha,7alpha,12alpha-triol 25- and 26-hydroxylases and 5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol 23R-, 24R-, 24S-, and 27-hydroxylases were strongly correlated with the activities of testosterone 6beta-hydroxylase in control human liver microsomes from eight unrelated donors. Troleandomycin, a specific inhibitor of CYP3A, markedly suppressed these microsomal side chain hydroxylations in both mouse and human livers in a dose-dependent manner. In addition, experiments using recombinant overexpressed human CYP3A4 confirmed that these microsomal side chain hydroxylations were catalyzed by a single enzyme, CYP3A4. The results demonstrate that microsomal 25- and 26-hydroxylations of 5beta-cholestane-3alpha,7alpha,12alpha-triol and microsomal 23R-, 24R-, 24S-, and 27-hydroxylations of 5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol are mainly catalyzed by CYP3A in both mice and humans. Unlike Cyp27(-/-) mice, CYP3A activity was not up-regulated despite marked accumulation of 5beta-cholestane-3alpha,7alpha,12alpha-triol in CTX.


Subject(s)
Bile Acids and Salts/biosynthesis , Cytochrome P-450 Enzyme System/physiology , Mixed Function Oxygenases/physiology , Steroid Hydroxylases/physiology , Xanthomatosis, Cerebrotendinous/metabolism , Animals , Catalysis , Cholestanetriol 26-Monooxygenase , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/genetics , Ethanolamines , Humans , Hydroxylation , Male , Mice , Mice, Knockout , Microsomes, Liver/metabolism , Middle Aged , Mutation , Nitrates , Steroid Hydroxylases/genetics , Troleandomycin/pharmacology , Up-Regulation
11.
Metabolism ; 50(6): 708-14, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11398149

ABSTRACT

We compared hepatic cholesterol metabolism in apolipoprotein (apo) E-knockout (KO) mice with their wild-type counterparts. We also investigated the effects of treatment with phytosterols or probucol on the activity of hepatic 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase (cholesterol synthesis), cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase (bile acid synthesis), and low-density lipoprotein (LDL) receptor function in this animal model of atherogenesis. These findings were then related to treatment-induced changes in plasma, hepatic, and fecal sterol concentrations. Mouse liver membranes have binding sites similar to LDL receptors; the receptor-mediated binding represents 80% of total binding and is LDL concentration-dependent. These binding sites have higher affinity for apo E-containing particles than apo B only-containing particles. Deletion of apo E gene was associated with several-fold increases in plasma cholesterol levels, 1.5-fold increase in hepatic cholesterol concentrations, 50% decrease in HMG-CoA reductase activity, 30% increase in cholesterol 7 alpha-hydroxylase and 25% decrease in LDL receptor function. Treatment of apo E-KO mice with either probucol or phytosterols significantly reduced plasma cholesterol levels. Phytosterols significantly increased the activity of hepatic HMG-CoA reductase, and probucol significantly increased cholesterol 7 alpha-hydroxylase activity. Neither treatment significantly altered hepatic LDL receptor function. Phytosterols, but not probucol, significantly increased fecal sterol excretion and decreased hepatic cholesterol concentrations. Plasma cholesterol lowering effects of phytosterols and probucol are due to different mechanisms: stimulation of cholesterol catabolism via increased bile acid synthesis by probucol and decreased cholesterol absorption by phytosterols. In the absence of apo E, hepatic LDL receptors could not be upregulated and did not contribute to the cholesterol lowering effects of either agent.


Subject(s)
Anticholesteremic Agents/pharmacology , Apolipoproteins E/deficiency , Bile Acids and Salts/biosynthesis , Cholesterol/biosynthesis , Liver/metabolism , Phytosterols/pharmacology , Probucol/pharmacology , Receptors, LDL/metabolism , Animals , Bile Acids and Salts/blood , Cell Membrane/metabolism , Cholesterol/blood , Feces/chemistry , Iodine Radioisotopes , Lipoproteins, LDL/metabolism , Liver/enzymology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Protein Binding , Receptors, LDL/genetics
12.
Nat Genet ; 27(4): 375-82, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11279518

ABSTRACT

Maturity-onset diabetes of the young type 3 (MODY3) is caused by haploinsufficiency of hepatocyte nuclear factor-1alpha (encoded by TCF1). Tcf1-/- mice have type 2 diabetes, dwarfism, renal Fanconi syndrome, hepatic dysfunction and hypercholestrolemia. Here we explore the molecular basis for the hypercholesterolemia using oligonucleotide microchip expression analysis. We demonstrate that Tcf1-/- mice have a defect in bile acid transport, increased bile acid and liver cholesterol synthesis, and impaired HDL metabolism. Tcf1-/- liver has decreased expression of the basolateral membrane bile acid transporters Slc10a1, Slc21a3 and Slc21a5, leading to impaired portal bile acid uptake and elevated plasma bile acid concentrations. In intestine and kidneys, Tcf1-/- mice lack expression of the ileal bile acid transporter (Slc10a2), resulting in increased fecal and urinary bile acid excretion. The Tcf1 protein (also known as HNF-1alpha) also regulates transcription of the gene (Nr1h4) encoding the farnesoid X receptor-1 (Fxr-1), thereby leading to reduced expression of small heterodimer partner-1 (Shp-1) and repression of Cyp7a1, the rate-limiting enzyme in the classic bile acid biosynthesis pathway. In addition, hepatocyte bile acid storage protein is absent from Tcf1-/- mice. Increased plasma cholesterol of Tcf1-/- mice resides predominantly in large, buoyant, high-density lipoprotein (HDL) particles. This is most likely due to reduced activity of the HDL-catabolic enzyme hepatic lipase (Lipc) and increased expression of HDL-cholesterol esterifying enzyme lecithin:cholesterol acyl transferase (Lcat). Our studies demonstrate that Tcf1, in addition to being an important regulator of insulin secretion, is an essential transcriptional regulator of bile acid and HDL-cholesterol metabolism.


Subject(s)
Bile Acids and Salts/metabolism , Cholesterol/blood , DNA-Binding Proteins/physiology , Nuclear Proteins/physiology , Animals , Base Sequence , Bile Acids and Salts/biosynthesis , DNA Primers , Hypoxia-Inducible Factor 1 , Hypoxia-Inducible Factor 1, alpha Subunit , Ileum/metabolism , Kidney/metabolism , Mice , Mice, Knockout , Transcription Factors/genetics , Transcription Factors/physiology
13.
J Lipid Res ; 42(2): 195-200, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11181748

ABSTRACT

The effects of feeding cholesterol, sitosterol, and lovastatin on cholesterol absorption, biosynthesis, esterification, and LDL receptor function were examined in the rat jejunal mucosa. Cholesterol absorption was measured by the dual-isotope plasma ratio method; the rate-limiting enzyme of cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, was measured as total and expressed enzyme activities (in the absence and presence of a phosphatase inhibitor, NaF, respectively); mucosal total and esterified cholesterol concentrations were determined by gas-liquid chromatography; LDL receptor function was assayed as receptor-mediated binding of (125)I-labeled LDL to mucosal membranes. Feeding 2% sitosterol or 0.04% lovastatin for 1 week significantly (P < 0.01) decreased the amounts of cholesterol absorbed per day (-85% and -63%, respectively). In contrast, feeding 2% cholesterol for 1 week increased the amounts of absorbed cholesterol 27-fold, even though the percent absorption significantly decreased. With all three treatments, there was a coordinate regulation of total HMG-CoA reductase activity and receptor-mediated LDL binding. Cholesterol feeding downregulated both total jejunal HMG-CoA reductase activity (P < 0.05) and receptor-mediated LDL binding (P < 0.01), whereas lovastatin- and sitosterol-supplemented diets significantly upregulated both of these parameters. In the control, cholesterol-fed, and sitosterol-fed animals, about half of the total jejunal HMG-CoA reductase activity was expressed (in functional dephosphorylated form). However, in the lovastatin-treated rats with 4-fold stimulation of HMG-CoA reductase, only 23% of the total enzyme activity was expressed. Changes in total HMG-CoA reductase activity and receptor-mediated LDL binding in all tested groups occurred with no change in total concentrations of mucosal cholesterol, and only cholesterol-fed animals had increased mucosal esterified cholesterol concentrations. Thus, in response to various fluxes of dietary or newly formed cholesterol, HMG-CoA reductase and receptor-mediated LDL binding are coordinately regulated to maintain constant cellular cholesterol concentrations in the jejunum.


Subject(s)
Anticholesteremic Agents/pharmacology , Cholesterol, Dietary/pharmacology , Cholesterol/metabolism , Homeostasis , Jejunum/drug effects , Lovastatin/pharmacology , Sitosterols/pharmacology , Animals , Cholesterol/blood , Hydroxymethylglutaryl CoA Reductases/metabolism , Jejunum/enzymology , Jejunum/metabolism , Male , Microsomes/enzymology , Rats , Rats, Sprague-Dawley
14.
J Lipid Res ; 42(2): 291-300, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11181760

ABSTRACT

Cerebrotendinous xanthomatosis (CTX) is a rare, recessively inherited lipid storage disease characterized by a markedly reduced production of chenodeoxycholic acid and an increased formation of 25-hydroxylated bile alcohols and cholestanol. Patients with this disease are known to have mutations in the sterol 27-hydroxylase (Cyp27) gene. However, one study showed that mice with a disrupted Cyp27 gene did not have any CTX-related clinical or biochemical abnormalities. To explore the reason, hepatic cholesterol, cholestanol, and 12 intermediates in bile acid biosynthetic pathways were quantified in 10 Cyp27(-/-) and 7 Cyp27(+/+) mice, two CTX patients (untreated and treated with chenodeoxycholic acid), and four human control subjects by high resolution gas chromatography-mass spectrometry. Mitochondrial 27-hydroxycholesterol and 5beta-cholestane-3alpha,7alpha,12alpha,27-tetrol were virtually absent in both Cyp27(-/-) mice and CTX patients. In Cyp27(-/-) mice, microsomal concentrations of intermediates in the early bile acid biosynthetic pathway (7alpha-hydroxycholesterol, 7alpha-hydroxy-4-cholesten-3-one, 7alpha,12alpha-dihydroxy-4-cholesten-3-one, and 5beta-cholestane-3alpha,7alpha,12alpha-triol), 25-hydroxylated bile alcohols (5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol, 5beta-cholestane-3alpha,7alpha,12alpha,23R,25-pentol, and 5beta-cholestane-3alpha,7alpha,12alpha,24R, 25-pentol), and cholestanol were all significantly elevated compared with those in Cyp27(+/+) mice, although the levels were lower than those in untreated CTX patients. The intermediate levels in early bile acid biosynthesis were more elevated in male (16;-86% of CTX) than in female Cyp27(-/-) mice (7-30% of CTX). In contrast, 25-hydroxylated bile alcohol concentrations were not significantly different between male and female Cyp27(-/-) mice and were considerably lower (less than 14%) than those in CTX patients.These results suggest that 1) in Cyp27(-/-) mice, especially in females, classic bile acid biosynthesis via 7alpha-hydroxycholesterol is not stimulated as much as in CTX patients; and 2) formed 25-hydroxylated bile alcohols are more efficiently metabolized in Cyp27(-/-) mice than in CTX patients.


Subject(s)
Bile Acids and Salts/biosynthesis , Cytochrome P-450 Enzyme System/physiology , Liver/metabolism , Steroid Hydroxylases/physiology , Xanthomatosis, Cerebrotendinous/metabolism , Adult , Animals , Cholestanetriol 26-Monooxygenase , Cholesterol/metabolism , Cytochrome P-450 Enzyme System/genetics , Female , Humans , Male , Mice , Mice, Knockout , Microsomes, Liver/enzymology , Mitochondria, Liver/enzymology , Steroid Hydroxylases/genetics , Xanthomatosis, Cerebrotendinous/genetics
15.
J Lipid Res ; 41(11): 1883-9, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11060358

ABSTRACT

We measured the percent absorption, turnover, and distribution of campestanol (24-methyl-5alpha-cholestan-3beta-ol) in a sitosterolemic homozygote, her obligate heterozygous mother, and three healthy human control subjects. For reasons relating to sterol hyperabsorption, the homozygote consumed a diet low in plant sterols that contained campestanol at about 2 mg/day. The heterozygote and three control subjects were fed a diet supplemented with a spread that contained campestanol at 540 mg/day and sitostanol (24-ethyl-5alpha-cholestan-3beta-ol) at 1.9 g/day as fatty acid esters. Plasma campestanol concentrations determined by capillary gas-liquid chromatography were 0.72 +/- 0.03 mg/dl in the homozygote, 0.09 +/- 0.04 mg/dl in the heterozygote, and 0.05 +/- 0.03 mg/dl for the control mean. After simultaneous pulse labeling with [3alpha-(3)H]campestanol intravenously and [23-(14)C]campestanol orally, the maximum percent absorption measured by the plasma dual-isotope ratio method as a single time point was 80% in the homozygote, 14.3% in the heterozygote, and 5.5 +/- 4.3% as the mean for three control subjects. Turnover (pool size) values estimated by mathematical analysis of the specific activity versus time [3alpha-(3)H]campestanol decay curves were as follows: 261 mg in the homozygote, 27.3 mg in the heterozygote, and 12.8 +/- 7.6 mg in the three control subjects (homogygote vs. controls, P < 0.001). The calculated production rate (mg/24 h) equivalent to actual absorption in the presence of dietary sterols and stanols was 0.67 mg/day or 31% of intake in the homozygote, 2.1 mg/day or 0.3% of intake in the heterozygote, and 0.7 +/- 0.3 mg/day or 0.1% of intake in the three control subjects. However, the excretion constant from pool A (K(A)) was prolonged markedly in the homozygote, but was 100 times more rapid in the heterozygote and three control subjects.Thus, campestanol, like other noncholesterol sterols, is hyperabsorbed and retained in sitosterolemic homozygotes. However, campestanol absorption was only slightly increased in the sitosterolemic heterozygote and removal was as rapid as in control subjects.


Subject(s)
Cholesterol/analogs & derivatives , Homozygote , Intestinal Absorption , Lipid Metabolism, Inborn Errors/genetics , Phytosterols/metabolism , Sitosterols/blood , Adolescent , Adult , Carbon Radioisotopes , Cholesterol/blood , Diet , Female , Half-Life , Heterozygote , Humans , Kinetics , Male , Middle Aged , Phytosterols/administration & dosage , Phytosterols/blood , Sitosterols/administration & dosage , Tritium
16.
J Neurosci Res ; 61(5): 549-63, 2000 Sep 01.
Article in English | MEDLINE | ID: mdl-10956425

ABSTRACT

Previous reports have suggested that elevated levels of phenylalanine inhibit cholesterol synthesis. The goals of this study were to investigate if perturbations in cholesterol synthesis exist in the PAH(enu2) genetic mouse model for phenylketonuria (PKU), and if so, initiate studies determining if they might underlie the white matter pathology that exists in PKU forebrain. Gross sections and electron microscopy showed that select tracts were hypomyelinated in adult PKU mouse forebrain but not hindbrain. The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), the rate controlling enzyme in the cholesterol biosynthetic pathway, was examined in isolated microsomes from forebrain, hindbrain, and liver to assess if perturbations in cholesterol biosynthesis were occurring. HMGR activity was normal in unaffected PKU hindbrain and was increased 2-4-fold in PKU liver compared to control. HMGR activity in the forebrain, however, was decreased by 30%. Because normal numbers of MBP-expressing glia (oligodendrocytes) were present, but the number of glia expressing HMGR was reduced by 40% in the hypomyelinated tracts, the decreased HMGR activity seemed to result from a down-regulation of HMGR expression in affected oligodendrocytes. Exposure of an oligodendrocyte-like glioma cell line to physiologically relevant elevated levels of Phe resulted in a 30% decrease in cholesterol synthesis, a 28% decrease in microsomal HMGR activity, and a 28% decrease in HMGR protein levels. Measurement of HMGR activity after addition of exogenous Phe to control brain microsomes revealed that Phe is a noncompetitive inhibitor of HMGR; physiologically relevant elevated levels of exogenous Phe inhibited HMGR activity by 30%. Taken together, these data suggest that HMGR is moderately inhibited in the PKU mouse. Unlike other cell types in the body, a subset of oligodendrocytes in the forebrain seems to be unable to overcome this inhibition. We speculate that this may be the cause of the observed pathology in PKU brain.


Subject(s)
Hydroxymethylglutaryl CoA Reductases/metabolism , Phenylketonurias/enzymology , Phenylketonurias/pathology , Prosencephalon/enzymology , Prosencephalon/pathology , Alkyl and Aryl Transferases/antagonists & inhibitors , Animals , Brain Chemistry , Cell Count , Cell Line , Cholesterol/analysis , Cholesterol/biosynthesis , Cholesterol/blood , Demyelinating Diseases/enzymology , Demyelinating Diseases/pathology , Disease Models, Animal , Farnesyltranstransferase , Hydroxymethylglutaryl CoA Reductases/analysis , Hydroxymethylglutaryl-CoA Reductase Inhibitors/metabolism , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Liver/chemistry , Liver/enzymology , Mice , Mice, Neurologic Mutants , Mice, Transgenic , Microsomes/chemistry , Microsomes/drug effects , Microsomes/enzymology , Myelin Basic Protein/biosynthesis , Oligodendroglia/enzymology , Oligodendroglia/pathology , Phenylalanine/metabolism , Phenylalanine/pharmacology , Phenylalanine Hydroxylase/deficiency , Phenylalanine Hydroxylase/genetics , Prosencephalon/ultrastructure , Rhombencephalon/enzymology , Rhombencephalon/pathology , Rhombencephalon/ultrastructure
17.
J Inherit Metab Dis ; 23(5): 464-74, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10947201

ABSTRACT

The Smith-Lemli-Opitz syndrome (SLOS) is a recessively inherited birth disorder caused by a defect in 7-dehydrocholesterol (3beta-hydroxysteroid) delta7-reductase, the final enzyme in cholesterol biosynthesis. To investigate in vivo regulation of the cholesterol biosynthetic pathway in SLOS, we measured hepatic microsomal sterol concentrations and activities of several key enzymes in the pathway, including HMG-CoA synthase, HMG-CoA reductase, squalene synthase and 7-dehydrocholesterol delta7-reductase in liver specimens from a patient with SLOS and 11 controls. Hepatic microsomal 7-dehydrocholesterol delta7-reductase activity in the patient was less than 1% of the control mean, and decreased cholesterol concentration and markedly increased 7- and 8-dehydrocholesterol concentrations were observed in the patient's microsomes. HMG-CoA synthase and squalene synthase activities in the patient were upregulated to 149% and 532%, respectively, while the activity of HMG-CoA reductase, the rate-limiting enzyme in the pathway, was reduced to 39% of the control mean. Downregulation of HMG-CoA reductase activity in SLOS was supported by measuring plasma levels of mevalonic acid, the immediate product of HMG-CoA reductase. The levels in SLOS patients (n = 9) were significantly low compared with age-matched controls (n = 8) (12+/-2 vs 28 + 6nmol/L, p < 0.05). These results suggest that in most SLOS patients in vivo HMG-CoA reductase is not stimulated in spite of blocked cholesterol biosynthetic pathway and reduced plasma and hepatic cholesterol concentrations.


Subject(s)
Cholesterol/biosynthesis , Oxidoreductases Acting on CH-CH Group Donors , Smith-Lemli-Opitz Syndrome/metabolism , Case-Control Studies , Child , Child, Preschool , Farnesyl-Diphosphate Farnesyltransferase/metabolism , Female , Humans , Hydroxymethylglutaryl CoA Reductases/metabolism , Hydroxymethylglutaryl-CoA Synthase/metabolism , Infant , Male , Microsomes, Liver/metabolism , Oxidoreductases/deficiency , Smith-Lemli-Opitz Syndrome/genetics
18.
Gastroenterology ; 119(1): 188-95, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10889168

ABSTRACT

BACKGROUND & AIMS: The mechanism for abnormal hepatic bile acid transport was investigated in an 18-month-old Amish boy who presented with pruritus, poor growth, and severe bleeding episodes. Serum bilirubin, gamma-glutamyltranspeptidase, and cholesterol levels were normal, but prothrombin time and partial thromboplastin time were prolonged and bone alkaline phosphatase level was elevated. METHODS AND RESULTS: Cholic acid plus chenodeoxycholic acid levels measured by capillary gas-chromatography were 32 times higher than control in serum (34.7 vs. 1.1+/-0.4 microg/dL) but were not detected in liver and were reduced in gallbladder bile. Treatment with ursodiol, a more hydrophilic bile acid, improved pruritus, produced 37% weight gain, and after 2 years reduced serum primary bile acid concentrations about 85%, while accounting for 71% of serum and 24% of biliary bile acid conjugates. On ursodiol therapy, hepatic bile acid synthesis was enhanced 2-fold compared with controls, and microscopy revealed chronic hepatitis without cholestasis. Three younger sisters with elevated serum bile acids responded positively to ursodiol. Microsatellite markers for the FIC1 (gene for Byler's disease) region in these 4 children were inconsistent with linkage to FIC1. CONCLUSIONS: Conjugated cholic acid and chenodeoxycholic acid were synthesized in the liver and secreted into bile but could not reenter the liver from portal blood and accumulated in serum. In contrast, unconjugated ursodiol entered the liver and was conjugated and secreted into bile. Thus, the enterohepatic circulation of all conjugated bile acids was interrupted at the hepatic sinusoidal basolateral membrane. Unconjugated ursodiol bypassed the hepatic uptake block to enlarge the biliary and intestinal bile acid pools. A mutation in FIC1 recognized among the Amish and linkage of the disorder to FIC1 were excluded.


Subject(s)
Adenosine Triphosphatases/genetics , Bile Acids and Salts/metabolism , Cholagogues and Choleretics/therapeutic use , Ethnicity/genetics , Genetic Linkage/genetics , Liver/metabolism , Ursodeoxycholic Acid/therapeutic use , Bile Acids and Salts/biosynthesis , Biological Transport/drug effects , Chenodeoxycholic Acid/blood , Chenodeoxycholic Acid/urine , Cholagogues and Choleretics/metabolism , Cholic Acid/blood , Cholic Acid/urine , Female , Humans , Infant , Male , Pedigree , Pennsylvania/ethnology , Ursodeoxycholic Acid/metabolism
19.
J Lab Clin Med ; 135(2): 174-9, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10695663

ABSTRACT

Sitosterolemia is an inherited recessive disease characterized by abnormally increased plasma and tissue plant sterol concentrations. Patients hyperabsorb sitosterol. In addition, hepatic, ileal, and mononuclear leukocyte 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-controlling enzyme in the cholesterol biosynthetic pathway, is markedly suppressed in this disease. It is still controversial whether the down-regulation is due to accumulated sitosterol, but the effect of sitosterol on HMG-CoA reductase activity has not been studied in sitosterolemic tissues. To investigate whether sitosterol inhibits HMG-CoA reductase activity in sitosterolemia, we measured the enzyme activities in liver and cultured skin flbroblasts from patients. Hepatic HMG-CoA reductase activities in patients were decreased 76% (P < .05) as compared with results in control subjects. In contrast, HMG-CoA reductase activities in sitosterolemic fibroblasts were not decreased as compared with results in control fibroblasts, and the activities in all cells were up-regulated similarly when they were exposed to delipidated medium. Because the cultured sitosterolemic fibroblasts contained only trace amounts of plant sterols, we added 20 microg/mL sitosterol directly to the cell medium. Raising the intracellular sitosterol concentration to 7% of cellular cholesterol level increased HMG-CoA reductase activity 23% (P < .05), while the addition of the same amount of cholesterol to the cells reduced the activity 46% (P < .05). Thus, when sitosterolemic skin fibroblasts were used, it was possible to distinguish between the effects of cholesterol and those of sitosterol on the activity of HMG-CoA reductase. These results suggest that reduced HMG-CoA reductase activity in this disease is caused by secondary effects of unknown regulator(s) other than sitosterol.


Subject(s)
Cholesterol/pharmacology , Hydroxymethylglutaryl CoA Reductases/metabolism , Lipid Metabolism, Inborn Errors/enzymology , Sitosterols/blood , Sitosterols/pharmacology , Adolescent , Adult , Aorta, Thoracic , Arteriosclerosis/enzymology , Cells, Cultured , Female , Fibroblasts/enzymology , Humans , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Lipid Metabolism, Inborn Errors/blood , Male , Reference Values
20.
J Lipid Res ; 41(3): 442-51, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10706592

ABSTRACT

In classic cholic acid biosynthesis, a series of ring modifications of cholesterol precede side chain cleavage and yield 5beta-cholestane-3alpha, 7alpha, 12alpha-triol. Side chain reactions of the triol then proceed either by the mitochondrial 27-hydroxylation pathway or by the microsomal 25-hydroxylation pathway. We have developed specific and precise assay methods to measure the activities of key enzymes in both pathways, 5beta-cholestane-3alpha, 7alpha, 12alpha-triol 25- and 27-hydroxylases and 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol 23R-, 24R-, 24S- and 27-hydroxylases. The extracts from either the mitochondrial or microsomal incubation mixtures were purified by means of a disposable silica cartridge column, derivatized into trimethylsilyl ethers, and quantified by gas chromatography;-mass spectrometry with selected-ion monitoring in a high resolution mode. Compared with the addition of substrates in acetone, those in 2-hydroxypropyl-beta-cyclodextrin increased mitochondrial triol 27-hydroxylase activity 132% but decreased activities of the enzymes in microsomal 25-hydroxylation pathway (triol 25-hydroxylase and 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol 23R-, 24R-, 24S- and 27-hydroxylases) 13;-60% in human liver. The enzyme activities in both pathways were generally 2- to 4-times higher in mouse and rabbit livers compared with human liver. In all species, microsomal triol 25-hydroxylase activities were 4- to 11-times larger than mitochondrial triol 27-hydroxylase activities but the activities of tetrol 24S-hydroxylase were similar to triol 27-hydroxylase activities in our assay conditions. The regulation of both pathways in rabbit liver was studied after bile acid synthesis was perturbed. Cholesterol feeding up-regulated enzyme activities involved in both 25- (64;-142%) and 27- (77%) hydroxylation pathways, while bile drainage up-regulated only the enzymes in the 25-hydroxylation pathway (178;-371%). Using these new assays, we demonstrated that the 25- and 27-hydroxylation pathways for cholic acid biosynthesis are more active in mouse and rabbit than human livers and are separately regulated in rabbit liver.


Subject(s)
Cholic Acid/biosynthesis , Gas Chromatography-Mass Spectrometry/methods , Steroid Hydroxylases/metabolism , Animals , Cholesterol/metabolism , Humans , Hydrolysis , Hydroxylation , Mice , Microsomes, Liver/enzymology , Microsomes, Liver/metabolism , Mitochondria, Liver/enzymology , Mitochondria, Liver/metabolism , Rabbits
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