The Effect of Carbohydrate Restriction on Lipids, Lipoproteins, and Nuclear Magnetic Resonance-Based Metabolites: CALIBER, a Randomised Parallel Trial.

Low-carbohydrate high-fat (LCHF) diets can be just as effective as high-carbohydrate, lower-fat (HCLF) diets for improving cardiovascular disease risk markers. Few studies have compared the effects of the UK HCLF dietary guidelines with an LCHF diet on lipids and lipoprotein metabolism using high-throughput NMR spectroscopy. This study aimed to explore the effect of an ad libitum 8-week LCHF diet compared to an HCLF diet on lipids and lipoprotein metabolism and CVD risk factors. For 8 weeks, n = 16 adults were randomly assigned to follow either an LCHF (n = 8, <50 g CHO p/day) or an HCLF diet (n = 8). Fasted blood samples at weeks 0, 4, and 8 were collected and analysed for lipids, lipoprotein subclasses, and energy-related metabolism markers via NMR spectroscopy. The LCHF diet increased (p < 0.05) very small VLDL, IDL, and large HDL cholesterol levels, whereas the HCLF diet increased (p < 0.05) IDL and large LDL cholesterol levels. Following the LCHF diet alone, triglycerides in VLDL and HDL lipoproteins significantly (p < 0.05) decreased, and HDL phospholipids significantly (p < 0.05) increased. Furthermore, the LCHF diet significantly (p < 0.05) increased the large and small HDL particle concentrations compared to the HCLF diet. In conclusion, the LCHF diet may reduce CVD risk factors by reducing triglyceride-rich lipoproteins and improving HDL functionality.

Genetically Determined Serum 25-Hydroxyvitamin D Is Associated with Total, Trunk, and Arm Fat-Free Mass: A Mendelian Randomization Study.

PURPOSE: Low serum vitamin D status has been associated with reduced muscle mass in observational studies although the relationship is controversial and a causal association cannot be determined from such observations. Two-sample Mendelian randomization (MR) was applied to assess the association between serum vitamin D (25(OH)D) and total, trunk, arm and leg fat-free mass (FFM). METHODS: MR was implemented using summary-level data from the largest genome-wide association studies (GWAS) on vitamin D (n=73,699) and total, trunk, arm and leg FFM. Inverse variance weighted method (IVW) was used to estimate the causal estimates. Weighted median (WM)-based method, and MR-Egger, leave-one-out were applied as sensitivity analysis. RESULTS: Genetically higher serum 25(OH)D levels had a positive effect on total (IVW = Beta: 0.042, p = 0.038), trunk (IVW = Beta: 0.045, p = 0.023) and arm (right arm IVW = Beta: 0.044, p = 0.002; left arm IVW = Beta: 0.05, p = 0.005) FFM. However, the association with leg FFM was not significant (right leg IVW = Beta: 0.03, p = 0.238; left leg IVW = Beta: 0.039, p = 0.100). The likelihood of heterogeneity and pleiotropy was determined to be low (statistically non-significant), and the observed associations were not driven by single SNPs. Furthermore, MR pleiotropy residual sum and outlier test did not highlight any outliers. CONCLUSIONS: Our results illustrate the potentially causal, positive effect of serum 25(OH)D concentration on total, trunk and upper body appendicular fat-free mass.

Lack of effect of cold water prawns on plasma cholesterol and lipoproteins in normo-lipidaemic men.

OBJECTIVE: Dietary guidelines for the prevention of coronary heart disease (CHD) have restricted the intake of foods rich in dietary cholesterol, on the grounds that the dietary cholesterol will increase blood cholesterol. In the case of shellfish, this recommendation may limit the intake of a valuable dietary source of long chain n-3 polyunsaturated fatty acids (LC n-3 PUFA). The objective of this study was to undertake a dietary intervention to determine the effects of cold water prawns on plasma lipids and lipoproteins. METHODS: 23 healthy male subjects were randomised to receive either 225 g of cold water prawns or an equivalent weight of fish ('crab') sticks as a control for 12 weeks in a cross-over design. Blood samples were taken at the beginning and end of each intervention for the determination of plasma lipids and lipoproteins by routine enzymatic assays and iodixanol density gradient centrifugation respectively. RESULTS: The diets were well matched for the intake of total energy and macronutrients, and body weight remained stable throughout the study. The prawn intervention increased the intake of dietary cholesterol to 750 mg/d against 200 mg/d on the control. The intake of LC n-3 PUFA from prawns was estimated to be between 0.5-0.7 g/d. The consumption of prawns produced no significant effects on the concentration of plasma total or LDL cholesterol, triacylglycerol, HDL cholesterol or apolipoproteins A-I and B relative to the control, or within each intervention group over time. There was also no significant effect on LDL density (particle size) relative to the control, or any difference between and within treatments in total plasma lipoprotein profiles by density gradient centrifugation. CONCLUSION: These findings provide evidence to suggest that the consumption of cold water prawns, at least in healthy, male subjects, should not be restricted on the grounds of this seafood producing an adverse effect on plasma LDL cholesterol.

Separation of plasma lipoproteins in self-generated gradients of iodixanol.

The major classes of plasma lipoprotein, very low density lipoproteins (VLDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL), are characterized on the basis of differences in density and charge ( Table 1 ). Centrifugation is the 'gold-standard' for the analysis of plasma lipo-protein classes and beta quantitation, and other analytical procedures (1-4). Lipoprotein classes are separated by flotation of plasma or serum in a series of centrifugation steps in which the density of the plasma is increased sequentially by addition of potassium bromide. Alternatively, plasma is layered beneath a discontinuous gradient and centrifuged to separate the lipoprotein classes in a single step. However, it is impractical to use these methods in a routine analytical or clinical laboratory because of the long centrifugation steps required. It is also necessary to remove the high salt concentrations used before further analysis (e.g., agarose gel electrophoresis or determination of the cholesterol and/or triglyceride levels)can be carried out. The current method used for the assay of LDL and HDL levels in the chemical pathology laboratory involves determination of total plasma cholesterol followed by selective precipitation of HDL and determination of the cholesterol remaining in the supernatant (1). From the data obtained, the LDL and HDL cholesterol levels are indirectly calculated. It is generally accepted that this method is limited and that the results are compromised by modest elevation in levels of plasma triglyceride, such as frequently occurs in clinical samples (5). Table 1 Plasma Lipoproteins Lipoprotein Protein % PL % Cho1 % TAG % Mobility % Density % Meanvalues in serum (mg/dL) Chylomicrons 2 4 5 89 Start <1.000 <10 VLDL 10 16 17 57 pre-ß <1.006 50-200 LDL 29 24 47 6 ß 1.019-1.063 200-300 HDL 49 29 19 5 α 1.063-1.210 200-300 The mean % composition of the major classis of lipoproteins is given: PL, phospholipids; chol, cholesterol and cholesterol ester; TAG, triglyceride. The mobility from the origin is pre-ß; ß; a see Fig. 1 for example. The density is the band at which the lipoproteins float from salt gradients.

Fractionation of lipoprotein subclasses in self-generated gradients of iodixanol.

Chapter 5 described the use of self-generated gradients of iodixanol for the fractionation of human plasma lipoproteins into the major classes: high-density, low-density, and very low density (HDL, LDL, and VLDL). During the metabolism of plasma HDL and LDL, the lipid and apoprotein composition of the lipoprotein particles changes in such a manner that a series of subclasses exists, each with a distinctive range of densities (1). Thus, in KBr gradients, the two major subclasses, HDL(2) and HDL(3), have densities of 1.063-1.125 g/mL and 1.125-1.21 g/mL, respectively (1). In some individuals a third subclass (HDL1) is recognized (1.055-1.063 g/mL). Electrophoretic (2) and immunological (3,4) techniques have identified additional subfractions. Likewise, subclasses of LDL have been identified and isolated using shallow KBr gradients (5,6). The major LDL subfractions are LDL(1), LDL(2), and LDL(3), which have densities of 1.025-1.034 g/mL, 1.034-1.044 g/mL, and 1.044-1.060 g/mL, respectively (6), and electrophoretic analysis has identified more subfractions (7). The subfractions of LDL are of particular interest, as the presence of small, dense LDL particles in the plasma appears to be associated with a predisposition to cardiovascular disease, and they are recognized as a major causative factor in atherosclerosis (8). Methods for monitoring the LDL subclass pattern in population studies and in dietary and drug intervention trials are thus of considerable interest. This chapter is concerned primarily with the subfractionation of LDL. Although HDL subfractionation systems using iodixanol self-generated gradients have not yet been validated by direct comparison with other methods (e.g., gradient gel electrophoresis or KBr gradient centrifugation), a protocol is included.

Carotenoid composition and antioxidant potential in subfractions of human low-density lipoprotein.

Carotenoids and vitamin E are transported in human plasma complexed with lipoproteins. The bulk of them are associated with low-density lipoprotein (LDL), in which form they may act as antioxidants and thus delay the onset of atherosclerosis. We used a simple, rapid, ultracentrifugation technique to fractionate plasma lipoproteins in self-generating gradients of iodixanol (Optiprep), a non-ionic iodinated density gradient medium. The carotenoid content and composition of a number of LDL subfractions was determined by reversed-phase high-performance liquid chromatography. Lycopene, beta-carotene and beta-cryptoxanthin were mainly located in the larger, less-dense LDL particles whereas lutein and zeaxanthin were found preferentially in the smaller, more dense LDL particles. When the antioxidant content of these fractions was expressed per milligram of LDL protein, significantly lower concentrations of carotenoid and vitamin E were found to be associated with the smaller, protein-rich fractions of LDL. Strong positive correlations were found between total carotenoid and vitamin E plasma concentrations and the lag-time of Cu(2+)-mediated oxidation of LDL subfractions. The more dense LDL subfractions, which had lower levels of these antioxidants, were more readily oxidized, highlighting their possible role in atherosclerotic events.

Expression of an Antirrhinum dihydroflavonol reductase gene results in changes in condensed tannin structure and accumulation in root cultures of Lotus corniculatus (bird's foot trefoil).

Condensed tannins (proanthocyanidins) are an important factor in the nutritive and dietary quality of many forage crops. We report here experiments aimed at altering the levels and monomer composition of condensed tannins (CTs) in 'hairy root' cultures of Lotus corniculatus (bird's foot trefoil) using genetic manipulation. An Antirrhinum majus dihydroflavonol reductase (DFR) cDNA was expressed in sense in L. corniculatus and CT levels in transgenic root cultures were analysed. Two co-transformed lines were noted with decreased CT content relative to controls and these levels were comparable with antisense-DFR phenotypes. In ADFR10, a co-transformed line with the highest CT levels, CT structure was altered in a manner consistent with the substrate specificity of the introduced gene; that is an increase in pro-pelargonidin monomers noted after hydrolysis of CTs. RT-PCR confirmed the expression of endogenous DFR gene(s) in both putatively co-suppressed lines and also in ADFR10. Analysis of selected root culture lines indicated that the monomer composition of CTs did not change during growth and development but that levels of CTs varied in a regulated manner.