Frank Kannenberg

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.Sponsorship: This work was supported by the Central Marketing Agency of the German Agricultural Industry (CMA), the German Union for the Promotion of Oil- and Protein Plants (UFOP), the Austrian Science Foundation, project F00709 (to P.M.A.) and the Brökelmann Ölmühle Company, Hamm, Germany.

Dietary α-Linolenic Acid, EPA, and DHA Have Differential Effects on LDL Fatty Acid Composition but Similar Effects on Serum Lipid Profiles in Normolipidemic Humans

Our aim was to study the effects of increased dietary intake of alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), or docosahexaenoic acid (DHA) on serum lipids and LDL fatty acid compositions. To this end, a controlled parallel study was conducted in 74 healthy normolipidemic men and women aged 19-43 y. Participants were randomly assigned to 1 of 3 interventions and consumed a total intake of 4.4 g/d ALA (ALA group), 2.2 g/d EPA (EPA group), and 2.3 g/d DHA (DHA group) for 6 wk. Fatty acid ethyl esters were incorporated into margarines, which replaced the participants normal spread. The ALA, EPA, or DHA intake led to a significant enrichment of the LDL with the respective (n-3) fatty acid. In addition, LDL EPA contents in the ALA group increased by 36% (P < 0.05) with no changes in LDL DHA. The EPA intervention led to an additional enrichment with DHA (24%; P < 0.001), whereas the DHA intervention further increased the amount of EPA (249%; P < 0.001). ALA, EPA, or DHA intake did not affect fasting serum concentrations of total and LDL cholesterol, but fasting serum triacylglycerol concentrations significantly decreased in the EPA (-0.14 mmol/L) and DHA (-0.30 mmol/L) interventions and also in the ALA intervention (-0.17 mmol/L). DHA intake significantly increased serum HDL cholesterol, whereas no changes were found with ALA or EPA intake. In conclusion, the present data support the hypothesis that isolated dietary ALA, EPA, and DHA intakes lead to differential enrichment in LDL due to interconversion. Moderate amounts of ALA, EPA, and DHA are effective in improving lipid profiles of normolipidemic humans.

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 β-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 β-oxidation: the long known 41-kDa β-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 β-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 β-ketothiolase is involved in peroxisomal β-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 β-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 3α,7α,12α-trihydroxy-27-nor-5β-cholestane-24-one (which is a known bile alcohol derivative of the cholic acid synthetic intermediate 3α,7α,12α-trihydroxy-24-keto-cholestanoyl-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 α- rather than β-oxidation. Our results demonstrate that the SCPx thiolase is critical for β-oxidation of the steroid side chain in conversion of cholesterol into bile acids.

Effects of ezetimibe, simvastatin, atorvastatin, and ezetimibe-statin therapies on non-cholesterol sterols in patients with primary hypercholesterolemia.

ABSTRACT Background: Levels of cholesterol are regulated by its synthesis, absorption, and elimination. Plasma levels of phytosterols (e.g., sitosterol, campesterol) and ratios of these sterols to total cholesterol (TC) are reported to correlate with efficiency of intestinal cholesterol absorption, whereas levels of certain cholesterol precursor sterols (e.g., desmosterol, lathosterol) and their ratios to TC correlate with cholesterol biosynthesis. However, there is a paucity of published data concerning the effects of combined treatment using HMG-CoA reductase inhibitors (statins) and a cholesterol absorption inhibitor (ezetimibe) on these parameters. Objectives: To characterize the effects of ezetimibe co-administered with statins, compared with each treatment alone, on cholesterol precursor sterols and plasma phytosterol levels. Methods: A post-hoc analysis was performed to determine the effects of treatment with ezetimibe 10 mg, simvastatin (10–80 mg), and atorvastatin (10–80 mg), alone or in combination, on these non-cholesterol sterols using plasma samples from two randomized controlled trials involving patients with primary hypercholesterolemia (low-density lipoprotein [LDL-C] = 145–250 mg/dL; triglycerides ≤350 mg/dL; N = 975) but without a recent (≤6-month) history of coronary heart disease (CHD) or either uncontrolled or newly diagnosed diabetes mellitus. Results: Ezetimibe monotherapy significantly reduced plasma sitosterol and campesterol concentrations from baseline compared with placebo (both p < 0.001), whereas statins significantly lowered desmosterol and lathosterol levels (p < 0.001 vs. placebo). Co-administration of ezetimibe and statins significantly decreased plasma levels of all of these sterols (p < 0.001). Conclusions: The observed effects of co-administration of ezetimibe and statins on non-cholesterol sterols are consistent with net inhibition of sterol absorption (driven by ezetimibe) in conjunction with net inhibition of cholesterol synthesis (driven by statins). The potential influence of treatment-induced changes in phytosterols on cardiovascular risk warrants further investigation in long-term, prospective, randomized controlled trials. This post-hoc study was by nature exploratory, and, because data from such analyses are not customarily adjusted for multiple comparisons, some associations may have emerged as statistically significant by chance. Future prospective randomized controlled studies may help to confirm our findings and address other research issues, such as the generalizability of our findings to patients with CHD or diabetes mellitus and possible dose:response relationships between escalating statin (or ezetimibe–statin) doses and circulating non-cholesterol levels.

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 α-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.Sponsorship:This research was financially supported by the German Federal Ministry of Education and Research (BMBF) within the project ‘NAPUS 2000 – healthy foodstuffs from transgenic rapeseed’.

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.

Rapid Diagnosis of 83 Patients with Niemann Pick Type C Disease and Related Cholesterol Transport Disorders by Cholestantriol Screening

Niemann Pick type C (NP-C) is a rare neurodegenerative disorder caused by an impairment of intracellular lipid transport. Due to the heterogeneous clinical phenotype and the lack of a reliable blood test, diagnosis and therapy are often delayed for years. In the cell, accumulating cholesterol leads to increased formation of oxysterols that can be used as a powerful screening parameter for NP-C. In a large scale study, we evaluated the oxysterol cholestane-3β,5α,6β-triol (c-triol) as potential biomarker for a rapid diagnosis of NP-C. Using GC/MS, c-triol has been analyzed in 1902 plasma samples of patients with the suspicion for NP-C. Diagnosis in patients with elevated oxysterols was confirmed by genetic analysis. 71 new NP-C patients (69 NP-C1 and two NP-C2) and 12 Niemann Pick type A/B patients were identified. 24 new mutations in NPC1, one new mutation in NPC2 and three new mutations in the SMPD1 gene were found. Cholestane-3β,5α,6β-triol was elevated in Niemann Pick type C1, type C2, type A/B and in CESD disease. No other study has ever identified so many NP-C patients, proving that c-triol is a rapid and reliable biomarker to detect patients with NP-C disease and related cholesterol transport disorders. It should replace the filipin test as the first-line diagnostic assay.

Relationship between phytosterol levels and components of the metabolic syndrome in the PROCAM study.

Background Components of the metabolic syndrome such as hypertriglyceridemia, low high-density lipoprotein (HDL) cholesterol and obesity have been shown to be associated with increased cholesterol synthesis and reduced cholesterol absorption. In the present study, we measured the lathosterol/cholesterol ratio as an index of cholesterol synthesis and the ratios of cholestanol, campesterol and sitosterol to cholesterol as indices of cholesterol absorption, as well as components of the metabolic syndrome, in 324 men and 168 women from the PROCAM study, an epidemiological study in which raised sitosterol was previously associated with increased coronary risk. Our aim was to determine if the indices of cholesterol synthesis and absorption show a graded relationship with severity of metabolic syndrome. Results No differences were seen between men and women with regard to the relationship of either the lathosterol/cholesterol or the sitosterol/cholesterol ratios and severity of metabolic syndrome. On multiple regression analysis in men and women together, body mass index showed a positive relationship with the lathosterol/cholesterol ratio (r=0.257, P<0.001) and a negative relationship with the sitosterol/cholesterol ratio (r=-0.221, P<0.001). HDL-cholesterol showed a negative relationship with the lathosterol/cholesterol ratio (r=-0.166, P=0.001). Triglycerides showed a negative relationship with the sitosterol/cholesterol ratio (r=0.141, P=0.005). Overall, these relationships were graded across quintiles of HDL cholesterol, body mass index and triglyceride and across an index of metabolic syndrome severity (number of components present). Only the cholestanol/cholesterol ratio showed a graded relationship with estimated overall coronary risk. Conclusions The metabolic syndrome is associated with increased cholesterol synthesis and reduced cholesterol absorption in a relationship that is graded across severity of the individual components of the syndrome and across an index of the severity of the metabolic syndrome as a whole.

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) μmol/l, +23.0%) and controls (+4.9 (9.9) μmol/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) μmol/l, −54.2%) and controls (−12.2 (9.2) μmol/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.Sponsorship:Supported by a grant of the Stifterverband für die Deutsche Wissenschaft (project number TS022/12372/2002) to MK.