Niemann-Pick Type C-2 Disease: Identification by Analysis of Plasma Cholestane-3ß,5α,6ß-Triol and Further Insight into the Clinical Phenotype.

INTRODUCTION: Niemann-Pick type C disease is a rare disorder caused by impaired intracellular lipid transport due to mutations in either the NPC1 or the NPC2 gene. Ninety-five % of NPC patients show mutations in the NPC1 gene. A much smaller number of patients suffer from NPC2 disease and present respiratory failure as one of the most frequent symptoms. Several plasma oxysterols are highly elevated in NPC1 and can be used as a biomarker in the diagnosis of NPC1. METHODS: Plasma cholestane-3ß,5α,6ß-triol was evaluated as biomarker for NPC2 by GC/MS and LC-MS/MS analysis. The diagnosis was confirmed by Sanger sequencing and filipin staining. RESULTS: We report three NPC2 patients with typical respiratory problems and a detailed description of the nature of the lung disease in one of them. All patients had elevated levels of plasma cholestane-3ß,5α,6ß-triol. In two of these patients, the positive oxysterol result led to a rapid diagnosis of NPC2 by genetic analysis. The phenotype of the third patient has been described previously. In this patient a cholestane-3ß,5α,6ß-triol concentration markedly above the reference range was found. CONCLUSIONS: Measurement of plasma cholestane-3ß,5α,6ß-triol enables to discriminate between controls and NPC1 and NPC2 patients, making it a valuable biomarker for the rapid diagnosis not only for NPC1 but also for NPC2 disease.The measurement of oxysterols should be well kept in mind in the differential diagnosis of lysosomal diseases, as the elevation of oxysterols in plasma may speed up the diagnosis of NPC1 and NPC2.

Modification of dietary polyunsaturated fatty acids via complementary food enhances n-3 long-chain polyunsaturated fatty acid synthesis in healthy infants: a double blinded randomised controlled trial.

OBJECTIVE: To study the effect of modified polyunsaturated fatty acid (PUFA) profiles of complementary food on long-chain (LC) PUFA composition in healthy infants. DESIGN: Double blinded, randomised, controlled intervention trial. SETTING: Dortmund, Germany. PATIENTS: Free-living sample of healthy term infants. METHODS: Participants were randomly assigned within the first 2 months of life. During the intervention period from 4 to 10 months, the control group (n = 53) received commercial complementary meals with corn oil (3.4 g/meal) rich in n-6 linoleic acid (LA), the intervention group (n = 49) received the same meals with rapeseed oil (1.6 g/meal) rich in n-3 alpha-linolenic acid (ALA). Fatty acid intake was assessed from dietary records throughout the intervention period. Fatty acid proportions (% of total fatty acid) in total plasma were analysed before and after the intervention. RESULTS: Plasma fatty acid profiles did not differ between the intervention and control groups before the intervention. During the intervention, the only difference in fatty acid intake between the intervention and control groups was a higher intake of ALA in the intervention group, 21% deriving from study food and a lower ratio of LA/ALA (10.7 vs 14.8). At the end of the intervention, the plasma proportions of total n-3 fatty acids and of n-3 LC-PUFA, but not of ALA, were higher and the ratios of n-6/n-3 fatty acids were lower in the intervention group. CONCLUSIONS: Feasible dietary modifications of the precursor fatty acid profile via n-3 PUFA-rich vegetable oil favoured n-3 LC-PUFA synthesis in the complementary feeding period when LC-PUFA intake from breast milk and formula is decreasing.

Similar serum plant sterol responses of human subjects heterozygous for a mutation causing sitosterolemia and controls to diets enriched in plant sterols or stanols.

OBJECTIVE: We investigated the serum phytosterol responses of heterozygous relatives of sitosterolemia patients to diets enriched in phytosterols or stanols. DESIGN: Randomized double-blind crossover design. SETTING: Muenster, Germany. SUBJECTS: Eight heterozygous and 13 control subjects were recruited. One heterozygote and three controls dropped out. INTERVENTIONS: Seven heterozygotes and 10 controls received daily portions of margarine containing 2 g of plant sterols, 2 g of stanols or a control margarine for 6 weeks each in a randomized order. These phases were intercepted by wash-out periods of 6 weeks each. RESULTS: Compared to the control period, serum phytosterol concentrations increased overall by more than 20% when subjects consumed the plant sterol margarine (F((1,15))=8.719, P=0.01), with no significant difference between heterozygotes (mean +14.5 (s.d. 17.2) micromol/l, +23.0%) and controls (+4.9 (9.9) micromol/l, +20.5%; F((1,15))=2.168, P=0.162), but decreased when subjects consumed the stanol-enriched margarine (F((1,15))=12.124, P=0.003), again to a similar extent in heterozygotes (-34.2 (41.2) micromol/l, -54.2%) and controls (-12.2 (9.2) micromol/l, -50.6%; F((1,15))=2.729, P=0.119). The lowest total serum concentrations of cholesterol and phytosterols were seen after the diet enriched in stanols. Serum stanol concentrations increased on this diet, but on a very low level and never exceeded 0.05% of serum cholesterol levels in any subject. CONCLUSIONS: Serum phytosterol concentrations increased only moderately in heterozygotes consuming a diet enriched in phytosterols, indicating that they retained considerable capacity to excrete phytosterols even at higher intakes.

Influence of three rapeseed oil-rich diets, fortified with alpha-linolenic acid, eicosapentaenoic acid or docosahexaenoic acid on the composition and oxidizability of low-density lipoproteins: results of a controlled study in healthy volunteers.

OBJECTIVE: To compare the individual effects of dietary alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) on low-density lipoprotein (LDL) fatty acid composition, ex vivo LDL oxidizability and tocopherol requirement. DESIGN, SETTING AND SUBJECTS: A randomized strictly controlled dietary study with three dietary groups and a parallel design, consisting of two consecutive periods. Sixty-one healthy young volunteers, students at a nearby college, were included. Forty-eight subjects (13 males, 35 females) completed the study. INTERVENTIONS: Subjects received a 2-week wash-in diet rich in monounsaturated fatty acids (21% energy) followed by experimental diets enriched with about 1% of energy of ALA, EPA or DHA for 3 weeks. The omega-3 (n-3) fatty acids were provided with special rapeseed oils and margarines. The wash-in diet and the experimental diets were identical, apart from the n-3 fatty acid composition and the tocopherol content, which was adjusted to the content of dienoic acid equivalents. RESULTS: Ex vivo oxidative susceptibility of LDL was highest after the DHA diet, indicated by a decrease in lag time (-16%, P<0.001) and an increase in the maximum amount of conjugated dienes (+7%, P<0.001). The EPA diet decreased the lag time (-16%, P<0.001) and the propagation rate (-12%, P<0.01). Tocopherol concentrations in LDL decreased in the ALA group (-13.5%, P<0.05) and DHA group (-7.3%, P<0.05). Plasma contents of tocopherol equivalents significantly decreased in all three experimental groups (ALA group: -5.0%, EPA group: -5.7%, DHA group: -12.8%). The content of the three n-3 polyunsaturated fatty acid differently increased in the LDL: on the ALA diet, the ALA content increased by 89% (P<0.001), on the EPA diet the EPA content increased by 809% (P<0.001) and on the DHA diet, the DHA content increased by 200% (P<0.001). In addition, the EPA content also enhanced (without dietary intake) in the ALA group (+35%, P<0.01) and in the DHA group (+284%, P<0.001). CONCLUSIONS: Dietary intake of ALA, EPA or DHA led to a significant enrichment of the respective fatty acid in the LDL particles, with dietary EPA preferentially incorporated. In the context of a monounsaturated fatty acid-rich diet, ALA enrichment did not enhance LDL oxidizability, whereas the effects of EPA and DHA on ex vivo LDL oxidation were inconsistent, possibly in part due to further changes in LDL fatty acid composition.

Mediterranean diet has no effect on markers of inflammation and metabolic risk factors in patients with coronary artery disease.

OBJECTIVE: Mediterranean diet is associated with decreased levels of inflammatory markers and metabolic risk factors in epidemiologic studies and recent trials on patients with metabolic syndrome. Given the recent improvements in medical treatments, it is unclear if such beneficial effects are also present in patients with coronary artery disease (CAD). We therefore investigated the effect of Mediterranean diet on markers of inflammation and metabolic risk factors in patients with treated CAD. DESIGN: Randomized, controlled trial. SUBJECTS: A total of 101 patients (59.4+/-8.6 years, 23% female) with established and treated CAD (80% statins). INTERVENTIONS: Participants were assigned to a Mediterranean diet group (MG; n=48) with a 1-year program of 100 h of education, or to a written advice-only group (AG; n=53). Before and after intervention, we measured serum high-sensitivity C-reactive protein (hs-CRP), fibrinogen, fasting insulin, homocysteine, serum lipids and plasma fatty acids. RESULTS: The Mediterranean diet program increased the intakes of fish, fruits/vegetables and moderately of canola/olive oil and increased plasma concentrations of long-chain n-3 polyunsaturated fatty acids in the MG. Median hs-CRP and mean fibrinogen, homocysteine, fasting insulin, triglycerides and serum cholesterols remained unchanged in both groups. CONCLUSIONS: Adoption of a Mediterranean diet by patients with medically treated CAD has no effect on markers of inflammation and metabolic risk factors. SPONSORSHIP: Alfried Krupp Foundation, Essen, Germany.

Effects of dietary fatty acids on the composition and oxidizability of low-density lipoprotein.

OBJECTIVE: The objective of this study was to compare the effects of dietary monounsaturated fatty acids (MUFA), n-6 and n-3 polyunsaturated fatty acids (PUFA) on LDL composition and oxidizability. DESIGN, SETTING AND SUBJECTS: Sixty-nine healthy young volunteers, students at a nearby college, were included. Six subjects withdrew because of intercurrent illness and five withdrew because they were unable to comply with the dietary regimen. INTERVENTIONS: The participants received a 2-week wash-in diet rich in saturated fatty acids (SFA) followed by diets rich in refined olive oil, rapeseed oil or sunflower oil for 4 weeks. Intakes of vitamin E and other antioxidants did not differ significantly between the diets. RESULTS: At the end of the study, LDL oxidizability was lowest in the olive oil group (lag time: 72.6 min), intermediate in the rapeseed oil group (68.2 min) and highest in the sunflower oil group (60.4 min, P<0.05 for comparison of all three groups). Despite wide variations in SFA intake, the SFA content of LDL was not statistically different between the four diets (25.8-28.5% of LDL fatty acids). By contrast, the PUFA (43.5%-60.5% of LDL fatty acids) and MUFA content of LDL (13.7-29.1% of LDL fatty acids) showed a wider variability dependent on diet. CONCLUSIONS: Enrichment of LDL with MUFA reduces LDL susceptibility to oxidation. As seen on the rapeseed oil diet this effect is independent of a displacement of higher unsaturated fatty acids from LDL. Evidence from this diet also suggests that highly unsaturated n-3 fatty acids in moderate amounts do not increase LDL oxidizability when provided in the context of a diet rich in MUFA.

Disruption of the sterol carrier protein 2 gene in mice impairs biliary lipid and hepatic cholesterol metabolism.

Hepatic up-regulation of sterol carrier protein 2 (Scp2) in mice promotes hypersecretion of cholesterol into bile and gallstone formation in response to a lithogenic diet. We hypothesized that Scp2 deficiency may alter biliary lipid secretion and hepatic cholesterol metabolism. Male gallstone-susceptible C57BL/6 and C57BL/6(Scp2(-/-)) knockout mice were fed a standard chow or lithogenic diet. Hepatic biles were collected to determine biliary lipid secretion rates, bile flow, and bile salt pool size. Plasma lipoprotein distribution was investigated, and gene expression of cytosolic lipid-binding proteins, lipoprotein receptors, hepatic regulatory enzymes, and intestinal cholesterol absorption was measured. Compared with chow-fed wild-type animals, C57BL/6(Scp2(-/-)) mice had higher bile flow and lower bile salt secretion rates, decreased hepatic apolipoprotein expression, increased hepatic cholesterol synthesis, and up-regulation of liver fatty acid-binding protein. In addition, the bile salt pool size was reduced and intestinal cholesterol absorption was unaltered in C57BL/6(Scp2(-/-)) mice. When C57BL/6(Scp2(-/-)) mice were challenged with a lithogenic diet, a smaller increase of hepatic free cholesterol failed to suppress cholesterol synthesis and biliary cholesterol secretion increased to a much smaller extent than phospholipid and bile salt secretion. Scp2 deficiency did not prevent gallstone formation and may be compensated in part by hepatic up-regulation of liver fatty acid-binding protein. These results support a role of Scp2 in hepatic cholesterol metabolism, biliary lipid secretion, and intracellular cholesterol distribution.

Aberrant oxidation of the cholesterol side chain in bile acid synthesis of sterol carrier protein-2/sterol carrier protein-x knockout mice.

Peroxisomal beta-oxidation plays an important role in the metabolism of a wide range of substrates, including various fatty acids and the steroid side chain in bile acid synthesis. Two distinct thiolases have been implicated to function in peroxisomal beta-oxidation: the long known 41-kDa beta-ketothiolase identified by Hashimoto and co-workers (Hijikata, M., Ishii, N., Kagamiyama, H., Osumi, T., and Hashimoto, T. (1987) J. Biol. Chem. 262, 8151-8158) and the recently discovered 60-kDa SCPx thiolase, that consists of an N-terminal domain with beta-ketothiolase activity and a C-terminal moiety of sterol carrier protein-2 (SCP2, a lipid carrier or transfer protein). Recently, gene targeting of the SCP2/SCPx gene has shown in mice that the SCPx beta-ketothiolase is involved in peroxisomal beta-oxidation of 2-methyl-branched chain fatty acids like pristanic acid. In our present work we have investigated bile acid synthesis in the SCP2/SCPx knockout mice. Specific inhibition of beta-oxidation at the thiolytic cleavage step in bile acid synthesis is supported by our finding of pronounced accumulation in bile and serum from the knockout mice of 3alpha,7alpha, 12alpha-trihydroxy-27-nor-5beta-cholestane-24-one (which is a known bile alcohol derivative of the cholic acid synthetic intermediate 3alpha,7alpha,12alpha-trihydroxy-24-keto-cholestano yl-coenzyme A). Moreover, these mice have elevated concentrations of bile acids with shortened side chains (i.e. 23-norcholic acid and 23-norchenodeoxycholic acid), which may be produced via alpha- rather than beta-oxidation. Our results demonstrate that the SCPx thiolase is critical for beta-oxidation of the steroid side chain in conversion of cholesterol into bile acids.

Defective peroxisomal catabolism of branched fatty acyl coenzyme A in mice lacking the sterol carrier protein-2/sterol carrier protein-x gene function.

Gene targeting in mice was used to investigate the unknown function of Scp2, encoding sterol carrier protein-2 (SCP2; a peroxisomal lipid carrier) and sterol carrier protein-x (SCPx; a fusion protein between SCP2 and a peroxisomal thiolase). Complete deficiency of SCP2 and SCPx was associated with marked alterations in gene expression, peroxisome proliferation, hypolipidemia, impaired body weight control, and neuropathy. Along with these abnormalities, catabolism of methyl-branched fatty acyl CoAs was impaired. The defect became evident from up to 10-fold accumulation of the tetramethyl-branched fatty acid phytanic acid in Scp2(-/-) mice. Further characterization supported that the gene disruption led to inefficient import of phytanoyl-CoA into peroxisomes and to defective thiolytic cleavage of 3-ketopristanoyl-CoA. These results corresponded to high-affinity binding of phytanoyl-CoA to the recombinant rat SCP2 protein, as well as high 3-ketopristanoyl-CoA thiolase activity of the recombinant rat SCPx protein.

Measuring cholesterol in macrophages: comparison of high-performance liquid chromatography and gas-liquid chromatography with enzymatic fluorometry.

Cholesterol and cholesteryl esters in human macrophages were analyzed by three different methods. Values obtained by high-performance liquid chromatography and by gas-liquid chromatography were compared with those obtained using enzymatic fluorometry. We also assessed fractional lipid recovery from these cells using radiolabeled cholesterol and cholesteryl ester. Enzymatic fluorometry substantially underestimated cellular cholesterol content. Two reasons for this were found. First, recovery into a variety of solvents was incomplete, particularly when extracted lipids were dried and redissolved in a second solvent. Second, the cells appeared to contain an intrinsic inhibitor of the enzymatic fluorometric method.

An improved method for quantification of cholesterol and cholesteryl esters in human monocyte-derived macrophages by high performance liquid chromatography with identification of unassigned cholesteryl ester species by means of secondary ion mass spectrometry.

The measurement of cholesteryl esters in human monocyte-derived macrophages using previously described high performance liquid chromatography methods is hampered by the presence in these cells of large amounts of triglycerides. We present a simple reversed phase high performance liquid chromatography protocol for quantification of cholesterol and cholesteryl esters in human monocyte/macrophages or other triglyceride-rich cells. Our method requires only lipid extraction and hydrolysis of triglycerides using a solution of ethanolic potassium hydroxide and is of sufficient sensitivity to allow measurement in 10(5) cells. Use of this protocol led to the isolation of eight previously unassigned cholesteryl ester peaks comprising 16% of the total cholesteryl ester content of human monocyte-derived macrophages. Using time-of-light secondary ion mass spectrometry and synthesized authentic standards, seven of these peaks were found to comprise cholesterol esterified with polyunsaturated n-3 (omega 3) (cholesteryl eicosapentaenoate, docosatrienoate, docosapentaenoate, and docosahexaenoate) and n-6 (omega 6) (cholesteryl docosatetraenoate, eicosadienoate, and eicosatrienoate) fatty acids. The remaining peak was shown to be the cholesteryl ester of n-7 (omega 7) palmitoleic acid by comparison with a commercially available standard. The identification of all the cholesteryl esters in cholesterol-loaded human monocyte-derived macrophages will assist future studies of lipid metabolism in these cells.

Smith-Lemli-Opitz syndrome diagnosed by using time-of-flight secondary-ion mass spectrometry.

We describe a rapid and sensitive method involving time-of-flight secondary-ion mass spectrometry (TOF-SIMS) for specific laboratory diagnosis of the Smith-Lemli-Opitz syndrome, which is characterized by massive (approximately 1000-fold) accumulation of the biosynthetic cholesterol precursor 7-dehydrocholesterol. Minute amounts of blood (1-50 microL) were extracted with n-hexane, and aliquots were analyzed by TOF-SIMS. 7-Dehydrocholesterol and its isomers were detected at 491.3 mass units ([M + 107Ag]+) and cholesterol at 495.3 mass units ([M + 109Ag]+). Quantitation of 7-dehydrocholesterol and cholesterol was achieved after saponification and addition of stigmasterol as internal standard. Whereas 7-dehydrocholesterol and isomeric dehydrocholesterol were not detectable in controls, the patients revealed concentrations ranging between 0.84 and 1.25 mmol/L. Comparison with results obtained by gas chromatography indicated that quantitation by TOF-SIMS yielded the sum of 7-dehydrocholesterol, isomeric dehydrocholesterol II, and sterol III, the latter two also being increased in the patients. Consistent with quantitation by gas chromatography, the cholesterol concentrations in the patients ranged between 1.54 and 2.12 mmol/L (controls: 6.10 +/- 1.37 mmol/L).