Can LDL cholesterol be too low? Possible risks of extremely low levels.

Following the continuous accumulation of evidence supporting the beneficial role of reducing low-density lipoprotein cholesterol (LDL-C) levels in the treatment and prevention of atherosclerotic cardiovascular disease and its complications, therapeutic possibilities now exist to lower LDL-C to very low levels, similar to or even lower than those seen in newborns and nonhuman species. In addition to the important task of evaluating potential side effects of such treatments, the question arises whether extremely low LDL-C levels per se may provoke adverse effects in humans. In this review, we summarize information from studies of human cellular and organ physiology, phenotypic characterization of rare genetic diseases of lipid metabolism, and experience from clinical trials. Specifically, we emphasize the importance of the robustness of the regulatory systems that maintain balanced fluxes and levels of cholesterol at both cellular and organismal levels. Even at extremely low LDL-C levels, critical capacities of steroid hormone and bile acid production are preserved, and the presence of a cholesterol blood-brain barrier protects cells in the central nervous system. Apparent relationships sometimes reported between less pronounced low LDL-C levels and disease states such as cancer, depression, infectious disease and others can generally be explained as secondary phenomena. Drug-related side effects including an increased propensity for development of type 2 diabetes occur during statin treatment, whilst further evaluation of more potent LDL-lowering treatments such as PCSK9 inhibitors is needed. Experience from the recently reported and ongoing large event-driven trials are of great interest, and further evaluation including careful analysis of cognitive functions will be important.

High levels of 15-oxygenated steroids in circulation of patients with multiple sclerosis: fact or fiction?

15-Oxygenated cholesterol species such as 5α-cholest-8(14)ene-3ß,15α-diol (15HC) and 3ß-hydroxy-5α-cholest-8(14)-en-15-one (15KC) are commercially available synthetic products unlikely to occur in biological systems. Surprisingly, Farez et al. recently reported that these two steroids occur in human circulation at levels considerably higher than those of any other endogenous oxysterol [Farez, M. et al. 2009. Toll-like receptor 2 and poly(ADP-ribose) polymerase 1 promote central nervous system neuroinflammation in progressive EAE. Nat. Immunol. 10: 958-964]. The levels were reported to be increased in patients with multiple sclerosis in a progressive phase and the authors suggested that this could be utilized diagnostically. Based on extensive in vitro experiments exposing cells to the same high levels of 15HC as found in vivo (1000 ng/ml) the authors concluded that 15HC may be an important pathogenetic factor in multiple sclerosis. Using combined gas chromatography-mass spectrometry we fail to detect significant plasma levels of 15HC either in healthy controls or in patients with multiple sclerosis (levels < 2 ng/ml). If 15KC is present in these plasma samples, the concentration of it must be <10 ng/ml. Our failure to detect significant levels of the above steroids could not be due to loss during hydrolysis and work-up because recovery of the added two oxysterols was close to 100%. Autoxidation of lipoprotein-bound cholesterol resulted in extensive conversion of cholesterol into 7-oxygenated but not 15-oxygenated sterols. We conclude that if present there are trace amounts only of the above 15-oxygenated steroids in human circulation and that the role of such oxysterols as pathogenetic factors and biomarkers must be reconsidered.

Enhanced production of 24S-hydroxycholesterol is not sufficient to drive liver X receptor target genes in vivo.

BACKGROUND: Oxysterols such as 24S-hydroxycholesterol (OHC) and 27-OHC are intermediates of cholesterol excretion pathways. In addition, they are putative endogenous agonists of the liver X receptor (LXR) class of nuclear hormone receptors and are thought to be important mediators of cholesterol-dependent gene regulation. 24S-OHC is one of the most efficient endogenous LXR agonists known and is present in the brain and in the circulation at relatively high levels. OBJECTIVES: To explore the regulatory importance of 24S-OHC in vivo. DESIGN: We developed a transgenic mouse model in which human cholesterol 24-hydroxylase, the enzyme responsible for the formation of 24S-OHC, was expressed under the control of a promoter derived from the ß-actin gene. RESULTS: Both male and female transgenic mice had elevated levels of cerebral, plasma, biliary and faecal 24S-OHC. According to the faecal excretion results, production of 24S-OHC was increased four- to sevenfold. Gene expression profiling revealed that the elevated production of 24S-OHC did not result in the anticipated activation of LXR target genes in the brain or liver. CONCLUSION: In spite of the fact that 24S-OHC is a highly effective agonist of LXRs in vitro, it is not a critical activator of target genes to this nuclear receptor in vivo, either in the brain or in the liver.

4beta-hydroxycholesterol as a marker of CYP3A4 inhibition in vivo - effects of itraconazole in man.

OBJECTIVE: Itraconazole, a triazole antifungal agent, has been demonstrated to act as an inhibitor of the ligand induced pregnane X receptor-mediated transcriptional regulation of the CYP3A4 gene. Here, we study the potential endogenous serum marker of CYP3A4 activity, 4beta-hydroxycholesterol, during therapy with itraconazole. PATIENTS AND METHODS: 8 male patients with onychomycosis received two 1-week cycles of treatment with 400 mg itraconazole once daily in an open, prospective exploratory trial. Fasting serum samples were taken at the beginning and at the end of each cycle. The levels of cholesterol were measured using gas chromatography-flame ionization detection, while cholesterol and bile acid precursors were quantified by gas chromatography-mass spectrometry. RESULTS: Total cholesterol decreased by 10% (p < 0.0005) during the itraconazole treatment. Concentrations of the cholesterol precursor lanosterol and 24, 25-dihydrolanosterol increased 10- and 240-fold, respectively (p < 0.001 for both). Interestingly, the ratio of serum lathosterol to cholesterol, an indicator of endogenous cholesterol synthesis downstream from lanosterol, remained unchanged. Absolute and cholesterol-corrected concentrations of 4beta-hydroxycholesterol, formed by CYP3A4-mediated oxidation, decreased significantly during both cycles, on average by 29.1% (p = 0.0006) and 20.8% (p = 0.0062), respectively. The brain-specific cholesterol metabolite 24S-hydroxycholesterol as well as its ratio to cholesterol increased by 19.7% (p = 0.0422) and 34.9% (p = 0.0013), respectively, while the concentrations of the other bile acid precursors, 7alpha-hydroxycholesterol and 27-hydroxycholesterol, remained unchanged. CONCLUSIONS: In conclusion, 4beta-hydroxycholesterol appears to be a sensitive endogenous surrogate marker in human serum for inhibition of CYP3A4 by itraconazole.

Hepatic uptake of bile acids in man. Fasting and postprandial concentrations of individual bile acids in portal venous and systemic blood serum.

This investigation was undertaken in order to (a) characterize the postprandial inflow of individual bile acids to the liver and (b) determine if peripheral venous bile acid levels always adequately reflect the portal venous concentration, or if saturation of hepatic bile acid uptake can occur under physiological conditions. In five patients with uncomplicated cholesterol gallstone disease, the umbilical cord was cannulated during cholecystectomy, and a catheter was left in the left portal branch for 5 to 7 d. The serum concentrations of cholic acid, chenodeoxycholic acid, and deoxycholic acid in portal venous and systemic circulation were then determined at intervals of 15 to 30 min before and after a standardized meal. A highly accurate and specific gas chromatographic/mass spectrometric technique was used. The sum of the fasting concentrations of the three bile acids averaged 14.04+/-4.13 mumol/liter in portal venous serum, and 2.44+/-0.31 mumol/liter in peripheral venous serum. The estimated hepatic fractional uptake of cholic acid was approximately 90%, and those of chenodeoxycholic acid and deoxycholic acid were 70-80%. This resulted in an enrichment of systemic bile acids in the dihydroxy bile acid species. In response to a standardized meal, portal venous bile acid concentrations increased two- to sixfold, with a peak seen 15-60 min after the meal. The maximum postprandial portal venous bile acid concentration averaged 43.04+/-6.12 mumol/liter, and the corresponding concentration in peripheral serum was 5.22+/-0.74 mumol/liter. The estimated fractional uptakes of the individual bile acids were not affected by the increased inflow to the liver. The peripheral venous concentrations of individual as well as total bile acids were well correlated with those in portal venous serum. The results (a) give a quantitation of postprandial bile acid inflow to the liver and (b) indicate that the hepatic uptake system for bile acids in healthy man cannot be saturated during maximal inflow of endogenous bile acids. Measurement of peripheral serum bile acids can thus give important information on the status of the enterohepatic circulation.

Biosynthesis of bile acids in man. Hydroxylation of the C27-steroid side chain.

The first step in the degradation of the steroid side chain during biosynthesis of bile acids from cholesterol in man was studied in microsomal and mitochondrial fraction of homogenate of livers from 14 patients. The microsomal fraction was found to catalyze an efficient 25-hydroxylation of 5,8-cholestane-3a,7a,12atriol. A small extent of 23-, 24-, and 26-hydroxylation of the same substrate was observed. 53-Cholestane-3a,7adiol was hydroxylated in the 25-position only to a very small extent. The mitochondrial fraction was found to catalyze 26-hydroxylation of cholesterol, 5-cholestene-3P,7a-diol, 5P-cholestane-3a,7a-diol, 7a-hydroxy-4-cholesten-3-one, and 5,0-cholestane-3a,7a,12a-triol. Addition of Mg++ stimulated the 26-hydroxylation of cholesterol but had no effect or an inhibitory effect on 26-hydroxylation of the other substrates, indicating a heterogeneity of the mitochondrial 26-hydroxylating system. The level of 26-hydroxylase activity towards different substrates varied considerably with different mitochondrial preparations. The roles of the microsomal and mitochondrial 26- hydroxylations as well as the microsomal 25-hydroxylation in biosynthesis of bile acids in man are discussed. The results indicate that microsomal 26-hydroxylation is less important than mitochondrial 26-hydroxylation under normal conditions. The possibility that microsomal 25-hydroxylation is important cannot be ruled out.

Defective peroxisomal cleavage of the C27-steroid side chain in the cerebro-hepato-renal syndrome of Zellweger.

Based on in vitro work with rat liver, we recently suggested that the peroxisomal fraction is most important for the oxidation of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) into cholic acid. The cerebro-hepato-renal syndrome of Zellweger is a fatal recessive autosomal disorder, the most characteristic histological feature of which is a virtual absence of peroxisomes in liver and kidneys. This disease offers a unique opportunity to evaluate the relative importance of peroxisomes in bile acid biosynthesis. A child with Zellweger syndrome was studied in the present work. In accordance with previous work, there was a considerable accumulation of THCA, 3 alpha, 7 alpha, 12 alpha, 24-tetrahydroxy-5 beta-cholestanoic acid (24-OH-THCA), 3 alpha, 7 alpha, 12 alpha-trihydroxy-27-carboxymethyl-5 beta-cholestan-26-oic acid (C29-dicarboxylic acid), and 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid in serum. In addition, a tetrahydroxylated 5 beta-cholestanoic acid with all the hydroxyl groups in the steroid nucleus was found. 3H-Labeled 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol was administered intravenously together with 14C-labeled cholic acid. There was a rapid incorporation of 3H in THCA and a slow incorporation into cholic acid. The specific radioactivity of 3H in THCA was about one magnitude higher than that in cholic acid. The conversion was evaluated by following the increasing ratio between 3H and 14C in biliary cholic acid. The rate of incorporation of 3H in cholic acid was considerably less than previously reported in experiments with healthy subjects, and the maximal conversion of the triol into cholic acid was only 15-20%. About the same rate of conversion was found after oral administration of 3H-THCA. Both in the experiment with 3H-5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol and with 3H-THCA, there was an efficient incorporation of 3H in the above unidentified tetrahydroxylated 5 beta-cholestanoic acid. There was only slow incorporation of radioactivity into 24-OH-THCA and into the C29-dicarboxylic acid. From the specific activity decay curve of 14C in cholic acid obtained after intravenous injection of 14C-cholic acid, the pool size of cholic acid was calculated to be 24 mg/m2 and the daily production rate to 9 mg/m2 per d. This corresponds to a reduction of approximately 85 and 90%, respectively, when compared with normal infants. It is concluded that liver peroxisomes are essential in the normal conversion of THCA to cholic acid. In the Zellweger syndrome this conversion is defective and as a consequence the accumulated THCA is either excreted as such or transformed into other metabolites by hydroxylation or side chain elongation. The accumulation of THCA, as well as the similar rate of conversion of 5 beta-cholestane-3 alpha,7 alpha.12 alpha-triol and THCA into cholic acid, support the contention that the 26-hydroxylase pathway with intermediate formation of THCA is the most important pathway for formation of cholic acid in man.

A novel pathway for biosynthesis of cholestanol with 7 alpha-hydroxylated C27-steroids as intermediates, and its importance for the accumulation of cholestanol in cerebrotendinous xanthomatosis.

A mixture of 7 alpha-3H- and 4-14C-labeled cholesterol was administered intravenously to rats. Cholestanol with 20-30% lower ratio between 3H and 14C than in cholesterol could be isolated from different organs. In a healthy human control, cholestanol isolated from feces had a 3H/14C ratio which was 28% lower than in administered cholesterol. Cholesterol and coprostanol reisolated in these experiments had the same ratio between 3H and 14C as in the precursor. A previously unknown pathway for formation of cholestanol, involving 7 alpha-hydroxylated intermediates, may explain these results. Under normal conditions, this pathway is responsible for at most 30% of the cholestanol synthesized from cholesterol. Intravenous administration of the 7 alpha-3H- and 4-14C-labeled cholesterol to a patient with cerebrotendinous xanthomatosis (CTX) resulted in formation of cholestanol which had 70-75% lower 3H/14C ratio. It is concluded that the novel pathway involving 7 alpha-hydroxylated intermediates is accelerated in patients with CTX. This acceleration may contribute essentially to the accumulation of cholestanol, which is a predominant feature of this disease. 7 alpha-Hydroxycholesterol and 7 alpha-hydroxy-4-cholesten-3-one might be intermediates in the novel pathway to cholestanol. After intravenous administration of 7 beta-3H-labeled 7 alpha-hydroxycholesterol in a patient with CTX, significant amounts of 3H were incorporated into plasma and fecal cholestanol. Only small amounts of 7 alpha-hydroxycholesterol and 7 alpha-hydroxy-4-cholesten-3-one are excreted into the intestine, and we therefore conclude that the 7 alpha-dehydroxylation step mainly occurs in the liver. In CTX, the synthesis of cholestanol may be accelerated because the concentrations of 7 alpha-hydroxylated bile acid intermediates in the liver are increased. A possible mechanism for the conversion of a minor fraction of 7 alpha-hydroxycholesterol into cholestanol is suggested.

Role of the 26-hydroxylase in the biosynthesis of bile acids in the normal state and in cerebrotendinous xanthomatosis. An in vivo study.

On the basis of different in vitro studies, we have previously suggested that the basic metabolic defect in the rare inherited disease cerebrotendinous xanthomatosis (CTX) is a lack of a hepatic mitochondrial C27-steroid 26-hydroxylase, involved in the normal biosynthesis of bile acids (1980. J. Clin. Invest. 65: 1418-1430; 1981. J. Lipid Res. 22: 191-200; 22: 632-640). In the present work, this hypothesis was tested in vivo. One patient with CTX and two control subjects received intravenously a mixture of [4-14C]7 alpha-hydroxy-4-cholesten-3-one and [6 beta-3H]7 alpha,26-dihydroxy-4-cholesten-3-one, steroids believed to be important precursors of chenodeoxycholic acid. The ratio between 14C and 3H in cholic acid and chenodeoxycholic acid isolated from bile of the CTX-patient was approximately 1/40 and 1/60 of those of the control subjects, respectively. Another patient with CTX and one control subject received a mixture of [4-14C]5 beta-cholestane-3 alpha,7 alpha-diol and [1,2-3H]5 beta-cholestane-3 alpha,7 alpha,26-triol, both possible precursors to chenodeoxycholic acid. In this case the 14C/3H ratio in cholic acid and chenodeoxycholic acid from the patient with CTX was 1/10 and 1/15, respectively, compared with that of the control subject. The most likely explanation for these findings is that very little of the 14C-precursors, i.e. without a 26-hydroxyl group, can be converted into cholic acid and chenodeoxycholic acid because of a defect of the 26-hydroxylase step. The results obtained are in accord with our previous findings in vitro. The results further underline the importance of the 26-hydroxylase pathway in the normal biosynthesis of cholic acid and chenodeoxycholic acid in man.

In vivo and vitro studies on formation of bile acids in patients with Zellweger syndrome. Evidence that peroxisomes are of importance in the normal biosynthesis of both cholic and chenodeoxycholic acid.

The last step in bile acid formation involves conversion of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) into cholic acid and 3 alpha,7 alpha-dihydroxy-5 beta-cholestanoic acid (DHCA) into chenodeoxycholic acid. The peroxisomal fraction of rat and human liver has the highest capacity to catalyze these reactions. Infants with Zellweger syndrome lack liver peroxisomes, and accumulate 5 beta-cholestanoic acids in bile and serum. We recently showed that such an infant had reduced capacity to convert a cholic acid precursor, 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol into cholic acid. 7 alpha-Hydroxy-4-cholesten-3-one is a common precursor for both cholic acid and chenodeoxycholic acid. Intravenous administration of [3H]7 alpha-hydroxy-4-cholesten-3-one to an infant with Zellweger syndrome led to a rapid incorporation of 3H into biliary THCA but only 10% of 3H was incorporated into cholic acid after 48 h. The incorporation of 3H into DHCA was only 25% of that into THCA and the incorporation into chenodeoxycholic acid approximately 50% of that in cholic acid. The conversion of intravenously administered [3H]THCA into cholic acid in another infant with Zellweger syndrome was only 7%. There was a slow conversion of THCA into 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-C29-dicarboxylic acid. The pool size of both cholic- and chenodeoxycholic acid was markedly reduced. Preparations of liver from two patients with Zellweger syndrome had no capacity to catalyze conversion of THCA into cholic acid. There was, however, a small conversion of DHCA into chenodeoxycholic acid and into THCA. It is concluded that liver peroxisomes are important both for the conversion of THCA into cholic acid and DHCA into chenodeoxycholic acid.

Metabolism of prostaglandin F2 alpha in Zellweger syndrome. Peroxisomal beta-oxidation is a major importance for in vivo degradation of prostaglandins in humans.

We have recently shown in vitro that the peroxisomal fraction of a rat liver homogenate has the highest capacity to beta-oxidize prostaglandins. In order to evaluate the relative importance of peroxisomes for this conversion also in vivo, we administered [3H]prostaglandin F2 alpha to an infant suffering from Zellweger syndrome, a congenital disorder characterized by the absence of intact peroxisomes. As a control, labeled compound was administered to two healthy volunteers. Urine was collected, fractionated on a SEP-PAK C18 cartridge, and subjected to reversed-phase high-performance liquid chromatography. The Zellweger patient was found to excrete prostaglandin metabolites considerably less polar than those of the control subjects. The major urinary metabolite in the control subjects was practically absent in the urine from the Zellweger patient. The major urinary prostaglandin F2 alpha metabolite from the Zellweger patient was identified as an omega-oxidized C20-prostaglandin, 9,11-dihydroxy-15-oxoprost-5-ene-1,20-dioic acid. The major urinary prostaglandin F2 alpha metabolite from the control subjects had chromatographic properties of a tetranor (C16) prostaglandin, in accordance with earlier published data. The present results, in combination with our previous in vitro data, indicate that peroxisomal beta-oxidation is of major importance for in vivo chain shortening of prostaglandins.

Cerebrotendinous xanthomatosis: a defect in mitochondrial 26-hydroxylation required for normal biosynthesis of cholic acid.

Oxidation of side chain of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol was studied in a patient with cerebrotendinous xanthomatosis (CTX) and in control subjects, using various subcellular fractions of liver homogenate and a method based on isotope dilution-mass spectrometry. In the control, 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol was converted into 5 beta-cholestane-3 alpha,7 alpha,12 alpha,26-tetrol and 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid by the mitochondrial fraction, and into 5 beta-cholestane-3 alpha,7 alpha,12 alpha,-25-tetrol by the microsomal fraction. In the CTX patient, liver mitochondria were completely devoid of 26-hydroxylase activity. The same mitochondrial fraction catalyzed 25-hydroxylation of vitamin D3. The microsomal fraction of liver of the subject with CTX contained more than 50-fold the normal amount of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol. The basic metabolid defect in CTX appears to be a lack of the mitochondrial 26-hydroxylase. The excretion in the bile of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25-tetrol and 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24 alpha,25-pentol observed in CTX patients may be secondary to the accumulation of the major substrate for the 26-hydroxylase, i. e., 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol, and exposure of this substrate to the normally less active microsomal 25-and 24-hydroxylases. It is concluded that the major pathway in the biosynthesis of cholic acid in human liver involves a mitochondrial C27-steroid 26-hydroxylation.

The antioxidant butylated hydroxytoluene prevents early cholesterol-induced microcirculatory changes in rabbits.

Microcirculation was studied during 10 wk in untreated rabbits (n = 13) and in rabbits treated with dietary addition of 1% cholesterol (n = 13), 1% cholesterol + 1% of the antioxidant BHT (butylated hydroxytoluene) (n = 11), or 1% BHT (n = 5). The studies were performed by direct intravital microscopic imaging of the left and right conjunctivae with the use of a stereo microscope and a high resolution television camera. Microvessel diameter, erythrocyte flow velocity, and microhemorheologic conditions were evaluated quantitatively via a computer-assisted digital image processing system. Significant and marked changes occurred in all the above variables as a consequence of cholesterol feeding. After 3 wk of feeding there was a dramatic decrease (approximately 30%) in blood flow velocity in arterioli of the third order (P < 0.0001), accompanied by aggregation of cells in 40-50% of the smaller conjunctival vessels (P < 0.0001). These changes were enhanced further during the following 7 wk of treatment. All the above changes in the microcirculation were markedly reduced by the addition of BHT treatment. The diameter of the above arterioli decreased in the purely cholesterol-fed group (P < 0.005), whereas this did not occur in the group fed both cholesterol and BHT. In rabbits fed BHT in the absence of cholesterol, there was no significant effect on any assessed microcirculatory variable. In conclusion, the results demonstrate that the antioxidant BHT prevented early cholesterol-induced microcirculatory changes. This prevention occurred in the absence of a reduction of blood lipid levels. The results provide strong support for the hypothesis that a considerable part of the effects on microcirculation in hypercholesterolemia may be due to cholesterol-induced oxidations and not to cholesterol itself. The results are discussed in relation to the previously demonstrated antiatherogenic effect of BHT and the possible use of antioxidants in the therapy and prophylaxis of atherosclerosis.

Antioxidant treatment inhibits the development of intimal thickening after balloon injury of the aorta in hypercholesterolemic rabbits.

The effect of the antioxidant butylated hydroxytoluene (BHT) on the accumulation of intimal smooth muscle cells (SMC) and development of intimal thickening after balloon catheter injury of the aorta were studied in rabbits with dietary-induced hyperlipidemia. Two sets of New Zealand White rabbits (eight rabbits in each group) were fed either 0.25% cholesterol or 0.25% cholesterol/1% BHT for a total of 6 wk. Serum lipid levels did not differ between the two groups. 3 wk after the start of the study, a balloon injury of the aorta was performed, after which the rabbits were kept on their respective diets for another 3 wk. After this period of time, the rabbits were killed and their aortas were investigated. The BHT-treated rabbits had only one fourth of the intimal thickness (P < 0.0001) and half the number of SMC/mm intima (P < 0.001), as compared to the rabbits fed only cholesterol. There was also a lower number of macrophages in the BHT-treated group. T lymphocytes were present in the intima of cholesterol-fed rabbits, whereas no such cells could be identified in the BHT-fed animals. There were significantly lower levels of autooxidation products of cholesterol (7-oxocholesterol, cholesterol-5,6-epoxide, and 7 beta-hydroxycholesterol) in the aortas of BHT-treated rabbits, P < 0.001. In conclusion, the antioxidant BHT effectively inhibited the accumulation of intimal SMC and the development of intimal thickening of the aorta in hypercholesterolemic rabbits after a balloon catheter-induced injury. These results indicate that antioxidants may modify intimal response to injury.

Demonstration of 26-hydroxylation of C27-steroids in human skin fibroblasts, and a deficiency of this activity in cerebrotendinous xanthomatosis.

26-Hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol and other C27-steroids was demonstrated in cultured skin fibroblasts from healthy individuals. Activities in skin fibroblasts were approximately 5-10% of those previously found in human liver homogenates, and were inhibited by CO. The apparent Km was lowest for 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol (1.3 mumol/liter) and highest for 5-cholestene-3 beta, 7 alpha-diol (12 mumol/liter). The rate of 26-hydroxylation was highest with 7 alpha-hydroxy-4-cholesten-3-one. These characteristics are similar to those of hepatic mitochondrial C27-steroid 26-hydroxylase. In skin fibroblasts from three patients with cerebrotendinous xanthomatosis (CTX), 26-hydroxylation of C27-steroids proceeded at a rate of only 0.2-2.5% of healthy controls. No accumulation of endogenous 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol could be demonstrated in these cells, and the lowered formation of radioactive, 26-hydroxylated products could not be explained by dilution of the labeled exogenous substrate. The present results add strong evidence to the concept that the primary metabolic defect in CTX is a deficiency of C27-steroid 26-hydroxylase.

Lack of 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase/isomerase in fibroblasts from a child with urinary excretion of 3 beta-hydroxy-delta 5-bile acids. A new inborn error of metabolism.

Cultured fibroblasts were shown to be capable of catalyzing the conversion of 7 alpha-hydroxy-cholesterol to 7 alpha-hydroxy-4-cholesten-3-one, an important reaction in bile acid synthesis. The apparent Km was approximately 7 mumol/liter and Vmax varied between 3 and 9 nmol/mg protein per h under the assay conditions used. The assay was used to investigate fibroblasts from a patient who presented with a familial giant cell hepatitis and who was found to excrete the monosulfates of 3 beta, 7 alpha-dihydroxy-5-cholenoic acid and 3 beta, 7 alpha, 12 alpha-trihydroxy-5-cholenoic acid in urine (Clayton, P. T., J. V. Leonard, A. M. Lawson, K. D. R. Setchell, S. Andersson, B. Egestad, and J. Sjövall. 1987. J. Clin. Invest. 79:1031-1038). In addition 7 alpha-hydroxy-cholesterol was found to accumulate in the circulation. Cultured fibroblasts from this boy were completely devoid of 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase/isomerase activity. Fibroblasts from his parents had reduced activity, compatible with a heterozygous genotype. The results provide strong evidence for the suggestion that this patient's liver disease was caused by a primary defect in the 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase/isomerase involved in bile acid biosynthesis.

The bile acid synthetic gene 3beta-hydroxy-Delta(5)-C(27)-steroid oxidoreductase is mutated in progressive intrahepatic cholestasis.

We used expression cloning to isolate cDNAs encoding a microsomal 3beta-hydroxy-Delta(5)-C(27)-steroid oxidoreductase (C(27) 3beta-HSD) that is expressed predominantly in the liver. The predicted product shares 34% sequence identity with the C(19) and C(21) 3beta-HSD enzymes, which participate in steroid hormone metabolism. When transfected into cultured cells, the cloned C(27) 3beta-HSD cDNA encodes an enzyme that is active against four 7alpha-hydroxylated sterols, indicating that a single C(27) 3beta-HSD enzyme can participate in all known pathways of bile acid synthesis. The expressed enzyme did not metabolize several different C(19/21) steroids as substrates. The levels of hepatic C(27) 3beta-HSD mRNA in the mouse are not sexually dimorphic and do not change in response to dietary cholesterol or to changes in bile acid pool size. The corresponding human gene on chromosome 16p11.2-12 contains six exons and spans 3 kb of DNA, and we identified a 2-bp deletion in the C27 3beta-HSD gene of a patient with neonatal progressive intrahepatic cholestasis. This mutation eliminates the activity of the enzyme in transfected cells. These findings establish the central role of C(27) 3beta-HSD in the biosynthesis of bile acids and provide molecular tools for the diagnosis of a third type of neonatal progressive intrahepatic cholestasis associated with impaired bile acid synthesis.

Oxysterols present in atherosclerotic tissue decrease the expression of lipoprotein lipase messenger RNA in human monocyte-derived macrophages.

The presence of oxysterols in macrophages isolated from atherosclerotic tissue and the effect of oxysterols on the regulation of lipoprotein lipase (LPL) mRNA were studied. Both rabbit and human macrophages, freshly isolated from atherosclerotic aorta, show about the same distribution of oxysterols, analyzed by isotope dilution mass spectrometry, except that all three preparations of human arterial-derived macrophages contained high levels of 27-hydroxycholesterol, which was not found in rabbit macrophages. To determine if oxysterols regulate LPL expression, human monocyte-derived macrophages were incubated with different oxysterols. Incubation with 7 beta-hydroxycholesterol and 25-hydroxycholesterol resulted in a 70-75% reduction of LPL mRNA, analyzed by quantitative RT-PCR. Cholesterol and other tested oxysterols showed no effect on macrophage LPL mRNA expression compared with control. LPL activity in the medium was also reduced after exposure of the macrophages to 7 beta-hydroxycholesterol and 25-hydroxycholesterol. In conclusion, we have demonstrated accumulation of oxysterols in macrophage-derived foam cells isolated from atherosclerotic aorta. There was suppression of LPL mRNA in human monocyte-derived macrophages after incubation with 7 beta-hydroxycholesterol and 25-hydroxycholesterol. It is tempting to suggest that an exposure to oxysterols may explain our earlier observation of a low level of LPL mRNA in arterial foam cells.

Hepatic cholesterol metabolism and resistance to dietary cholesterol in LXRbeta-deficient mice.

The nuclear oxysterol-receptor paralogues LXRalpha and LXRbeta share a high degree of amino acid identity and bind endogenous oxysterol ligands with similar affinities. While LXRalpha has been established as an important regulator of cholesterol catabolism in cholesterol-fed mice, little is known about the function of LXRbeta in vivo. We have generated mouse lines with targeted disruptions of each of these LXR receptors and have compared their responses to dietary cholesterol. Serum and hepatic cholesterol levels and lipoprotein profiles of cholesterol-fed animals revealed no significant differences between LXRbeta(-/-) and wild-type mice. Steady-state mRNA levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase, farnesyl diphosphate synthase, and squalene synthase were increased in LXRbeta(-/-) mice compared with LXRbeta(+/+) mice, when fed standard chow. The mRNA levels for cholesterol 7alpha-hydroxylase, oxysterol 7alpha-hydroxylase, sterol 12alpha-hydroxylase, and sterol 27-hydroxylase, respectively, were comparable in these strains, both on standard and 2% cholesterol chow. Our results indicate that LXRbeta(-/-) mice - in contrast to LXRalpha(-/-) mice - maintain their resistance to dietary cholesterol, despite subtle effects on the expression of genes coding for enzymes involved in lipid metabolism. Thus, our data indicate that LXRbeta has no complete overlapping function compared with LXRalpha in the liver.