Bruce A. Griffin

Role of plasma triglyceride in the regulation of plasma low density lipoprotein (LDL) subfractions: relative contribution of small, dense LDL to coronary heart disease risk

The concentration of plasma LDL subfractions is described in four groups of normocholesterolaemic (total plasma cholesterol < 6.5 mmol/l) male subjects consisting of men with and without coronary artery disease (CAD+/-), as determined by angiography, post-myocardial infarct survivors (PMI) and normal, healthy controls. The CAD(+) and PMI groups were distinguished from the CAD(-) and controls by raised concentrations of plasma triglyceride, very low density lipoprotein (VLDL) cholesterol, small, dense LDL (LDL-III density (d) 1.044-1.060 g/ml) and lower concentrations of high density lipoprotein (HDL) cholesterol and large, buoyant LDL (LDL-I d 1.025-1.034 g/ml). In all groups, a subfraction of intermediate density, LDL-II (d 1.034-1.044 g/ml), was the predominant LDL species but was not related to coronary heart disease risk. Plasma triglyceride showed a positive association with LDL-II (r = 0.51, P < 0.001) below a triglyceride level of 1.5 mmol/l. Above this threshold of 1.5 mmol/l, LDL-II and LDL-I showed significant negative associations with triglyceride (LDL-II r = -0.5, P < 0.001; LDL-I r = -0.45, P < 0.001). Small, dense LDL-III showed a weak positive association with triglyceride that became highly significant above the 1.5 mmol/l threshold (r = 0.54, P < 0.001). While age was positively related to LDL-II within the control subjects (r = 0.3, P < 0.05), there was no difference in the percentage abundance or concentration of LDL-III within control and CAD(-) subjects above and below the age of 40 years. Smoking was associated with a relative deficiency of the LDL-I subfraction (LDL-I to LDL-III ratio in smokers = 0.77, in ex-smokers = 0.95, in non-smokers = 1.89; P < 0.01), as was beta-blocker medication (% LDL-I, users vs. non-users, P < 0.05). Both of these effects could be explained by their primary influence on plasma triglyceride. Analysis of the frequency distributions for the three LDL subfractions revealed the concentration of small, dense LDL-III to be bimodal around a concentration of 100 mg (lipoprotein mass)/100 ml plasma. The calculation of odds ratios based on this figure indicated relative risk estimates of 4.5 (chi 2: P < 0.01) for the presence of coronary artery disease and 6.9 (chi 2: P < 0.001) for myocardial infarction.(ABSTRACT TRUNCATED AT 400 WORDS)

Long-chain conversion of [13C]linoleic acid and α-linolenic acid in response to marked changes in their dietary intake in men

We studied the long-chain conversion of [U-13C]α-linolenic acid (ALA) and linoleic acid (LA) and responses of erythrocyte phospholipid composition to variation in the dietary ratios of 18:3n-3 (ALA) and 18:2n-6 (LA) for 12 weeks in 38 moderately hyperlipidemic men. Diets were enriched with either flaxseed oil (FXO; 17 g/day ALA, n = 21) or sunflower oil (SO; 17 g/day LA, n = 17). The FXO diet induced increases in phospholipid ALA (>3-fold), 20:5n-3 [eicosapentaenoic acid (EPA), >2-fold], and 22:5n-3 [docosapentaenoic acid (DPA), 50%] but no change in 22:6n-3 [docosahexanoic acid (DHA)], LA, or 20:4n-6 [arachidonic acid (AA)]. The increases in EPA and DPA but not DHA were similar to those in subjects given the SO diet enriched with 3 g of EPA plus DHA from fish oil (n = 19). The SO diet induced a small increase in LA but no change in AA. Long-chain conversion of [U-13C]ALA and [U-13C]LA, calculated from peak plasma 13C concentrations after simple modeling for tracer dilution in subsets from the FXO (n = 6) and SO (n = 5) diets, was similar but low for the two tracers (i.e., AA, 0.2%; EPA, 0.3%; and DPA, 0.02%) and varied directly with precursor concentrations and inversely with concentrations of fatty acids of the alternative series. [13C]DHA formation was very low (<0.01%) with no dietary influences.

ApoE Polymorphism and Fish Oil Supplementation in Subjects With an Atherogenic Lipoprotein Phenotype

The study assessed the efficacy of fish oil supplementation in counteracting the classic dyslipidemia of the atherogenic lipoprotein phenotype (ALP). In addition, the impact of the common apolipoprotein E (apoE) polymorphism on the fasting and postprandial lipid profile and on responsiveness to the dietary intervention was established. Fifty-five ALP males (aged 34 to 69 years, body mass index 22 to 35 kg/m(2), triglyceride [TG] levels 1.5 to 4.0 mmol/L, high density lipoprotein cholesterol [HDL-C] <1.1 mmol/l, and percent low density lipoprotein [LDL]-3 >40% total LDL) completed a randomized placebo-controlled crossover trial of fish oil (3.0 g eicosapentaenoic acid/docosahexaenoic acid per day) and placebo (olive oil) capsules with the 6-week treatment arms separated by a 12-week washout period. In addition to fasting blood samples, at the end of each intervention arm, a postprandial assessment of lipid metabolism was carried out. Fish oil supplementation resulted in a reduction in fasting TG level of 35% (P<0.001), in postprandial TG response of 26% (TG area under the curve, P<0.001), and in percent LDL-3 of 26% (P<0.05). However, no change in HDL-C levels was evident (P=0.752). ANCOVA showed that baseline HDL-C levels were significantly lower in apoE4 carriers (P=0.035). The apoE genotype also had a striking impact on lipid responses to fish oil intervention. Individuals with an apoE2 allele displayed a marked reduction in postprandial incremental TG response (TG incremental area under the curve, P=0.023) and a trend toward an increase in lipoprotein lipase activity relative to non-E2 carriers. In apoE4 individuals, a significant increase in total cholesterol and a trend toward a reduction in HDL-C relative to the common homozygous E3/E3 profile was evident. Our data demonstrate the efficacy of fish oil fatty acids in counteracting the proatherogenic lipid profile of the ALP but also that the apoE genotype influences responsiveness to this dietary treatment.

Rapid isolation of low density lipoprotein (LDL) subfractions from plasma by density gradient ultracentrifugation

High resolution density gradient ultracentrifugation (DGUC) and non-denaturing gradient gel electrophoresis (GGE) indicate that low density lipoprotein (LDL) in both normal and hyperlipidaemic subjects is composed of overlapping particle populations. A new centrifugation procedure has been developed which permits the separation of LDL subspecies directly from plasma within 24 h. The profiles obtained were analogous to those seen on gradient gel electrophoresis. LDL was divided into 3 fractions. The plasma concentration of LDL-I seen in young females was twice that in men (85.6 +/- 28.8 vs. 42.3 +/- 25.7 mg/dl, P less than 0.005). LDL-II was not significantly different in any group while LDL-III was specifically elevated in coronary artery disease (CAD) patients (207.1 +/- 92.6 mg/dl in CAD vs. 87.4 +/- 79.6 mg/dl in normal men, P less than 0.05). The presence of small, dense LDL detected either by density gradient centrifugation or gel electrophoresis was associated with raised triglyceride (TG) and low high density lipoprotein (HDL) cholesterol and may be a risk marker for coronary artery disease.

Influence of plasma lipid and LDL-subfraction profile on the interaction between low density lipoprotein with human arterial wall proteoglycans

Low density lipoprotein (LDL) is known to bind to arterial wall proteoglycans (APG), an interaction which may initiate cholesterol deposition in the arterial wall. The objective of this study was to determine whether a predominance of small, dense LDL (LDL-III, d = 1.044-1.063 g/ml) in the circulation in association with an atherogenic lipoprotein phenotype (ALP) (i.e. LDL-III > 100 mg/dl, an elevated plasma triglyceride and a low high density lipoprotein cholesterol) alters LDL reactivity towards APG. Total LDL (d = 1.019-1.063 g/ml) was isolated from 59 patients undergoing coronary angiography (39 males and 20 females) and the LDL subfraction profile determined by non-equilibrium density gradient centrifugation. A binding assay was developed in which total LDL (0.1 mg/ml apo LDL) was mixed with a standard preparation of APG containing 2.5 micrograms/ml chondroitin sulphate and the extent of APG-LDL complex formation followed by absorbance measurement and the amount of precipitated LDL cholesterol. APG-LDL complex formation was positively associated with (a) the percentage of LDL-III within total LDL (r = 0.48, P < 0.0001); (b) the plasma triglyceride level (r = 0.27, P < 0.04); and negatively associated with (a) the percentage of the buoyant LDL-I (d = 1.019-1.033 g/ml)(r = -0.47, P < 0.0001); and (b) the HDL cholesterol concentration (r = -0.37, P < 0.004). There was no association with the percentage of the major LDL species LDL-II. When the patients were divided according to the presence or absence of an ALP i.e. LDL-III greater or less than 100 mg/dl respectively, proteoglycan-LDL complex formation was significantly higher in the former compared to the latter group of patients (P < 0.0001). This study therefore provides evidence that the extent of the interaction of LDL with APG varies considerably between individuals and is enhanced in the presence of ALP. It is postulated that the increased atherogenicity associated with ALP may in part be due to prolonged and enhanced retention of LDL by the arterial wall.

Regulation of plasma HDL cholesterol and subfraction distribution by genetic and environmental factors. Associations between the TaqI B RFLP in the CETP gene and smoking and obesity.

This study investigated in a healthy population (n = 220) the association of the TaqI B restriction fragment length polymorphism (RFLP) in the cholesteryl ester transfer protein (CETP) gene with plasma high-density lipoprotein (HDL) cholesterol concentration and subfraction distribution. A raised HDL cholesterol level was found in B2B2 homozygotes (B2 cutting site absent) and was associated specifically with a 45% increase in HDL2 compared with B1B1 homozygotes (B1B1, 77 +/- 39 mg/100 mL, mean +/- SD; B2B2, 112 +/- 59 mg/100 mL; P < 0.01). Total plasma, very-low-density lipoprotein, and HDL triglyceride levels did not differ among the genotype groups, nor did plasma apolipoprotein AI levels (B1B1, 1.45 +/- 0.35 mg/mL, mean +/- SD; B2B2, 1.56 +/- 0.33 mg/mL). Thus, the genetic variation appeared to be independent of metabolic factors that are known to regulate HDL levels. Plasma CETP exchange activity was unlikely to be the cause of the association, since it did not differ between genotype groups and was not correlated with HDL2 concentration. Multivariate analysis demonstrated that the TaqI B polymorphism had an effect on HDL cholesterol and HDL2 that was independent of age, sex, body mass index, oral contraceptive use, exercise, alcohol consumption, and plasma triglycerides. In smokers, the presence of the B2B2 genotype did not result in increased HDL cholesterol or HDL2, whereas in obese subjects, the difference between B1B1 and B2B2 individuals was diminished. We conclude that the TaqI B RFLP is associated with a quantitatively significant effect on plasma HDL2 levels that is independent of plasma triglycerides and interacts with lifestyle factors.

“European Panel on Low Density Lipoprotein (LDL) Subclasses”: A Statement on the Pathophysiology, Atherogenicity and Clinical Significance of LDL Subclasses

Aim of the present Consensus Statement is to provide a comprehensive and up to-date document on the pathophysiology, atherogenicity and clinical significance of low density liproproteins (LDL) subclasses. We sub-divided our statement in 2 sections. section I discusses the pathophysiology, atherogenicity and measurement issues, while section II is focused on the effects of drug and lifestyle modifications. Suggestions for future research in the field are highlighted at the end of section II. Each section includes Conclusions.

LIPOPROTEIN ATHEROGENICITY : AN OVERVIEW OF CURRENT MECHANISMS

Raised serum cholesterol does not adequately explain the increased risk of CHD within populations or the relationship between diet and CHD. Nevertheless, the principal transport vehicle of cholesterol in the circulation, LDL, must still be regarded as the most atherogenic lipoprotein species, but not because of its contribution to serum cholesterol. The atherogenic potential of LDL in the majority of individuals arises from an increase in the number of small dense LDL particles and not from its cholesterol content per se. There is now a wealth of evidence from cross-sectional and prospective studies to show that LDL particle size is significantly associated with CHD and predictive of increased coronary risk. Moreover, there are a number of credible mechanisms to link small dense LDL with the atherogenic process. The rate of influx of serum lipoproteins into the arterial wall is a function of particle size, and will thus be more rapid for small dense LDL. Components of the extracellular tissue matrix in the intima, most notably proteoglycans, selectively bind small dense LDL with high affinity, sequestering this lipoprotein in a pro-oxidative environment. The oxidation of LDL promotes the final deposition of cholesterol in the arterial wall, and numerous studies have shown small dense LDL to be more susceptible to oxidative modification than its larger and lighter counterparts. An increase in the number of small dense LDL particles may originate from a defect in the metabolism of triacylglycerol-rich lipoproteins. One mechanism may involve the overproduction and increased residence time of large triacylglycerol-rich VLDL in the postprandial phase, a situation thought to arise through pathways of insulin resistance.

Effect of changing the amount and type of fat and carbohydrate on insulin sensitivity and cardiovascular risk: the RISCK (Reading, Imperial, Surrey, Cambridge, and Kings) trial

BACKGROUND Insulin sensitivity (Si) is improved by weight loss and exercise, but the effects of the replacement of saturated fatty acids (SFAs) with monounsaturated fatty acids (MUFAs) or carbohydrates of high glycemic index (HGI) or low glycemic index (LGI) are uncertain. OBJECTIVE We conducted a dietary intervention trial to study these effects in participants at risk of developing metabolic syndrome. DESIGN We conducted a 5-center, parallel design, randomized controlled trial [RISCK (Reading, Imperial, Surrey, Cambridge, and Kings)]. The primary and secondary outcomes were changes in Si (measured by using an intravenous glucose tolerance test) and cardiovascular risk factors. Measurements were made after 4 wk of a high-SFA and HGI (HS/HGI) diet and after a 24-wk intervention with HS/HGI (reference), high-MUFA and HGI (HM/HGI), HM and LGI (HM/LGI), low-fat and HGI (LF/HGI), and LF and LGI (LF/LGI) diets. RESULTS We analyzed data for 548 of 720 participants who were randomly assigned to treatment. The median Si was 2.7 × 10(-4) mL · μU(-1) · min(-1) (interquartile range: 2.0, 4.2 × 10(-4) mL · μU(-1) · min(-1)), and unadjusted mean percentage changes (95% CIs) after 24 wk treatment (P = 0.13) were as follows: for the HS/HGI group, -4% (-12.7%, 5.3%); for the HM/HGI group, 2.1% (-5.8%, 10.7%); for the HM/LGI group, -3.5% (-10.6%, 4.3%); for the LF/HGI group, -8.6% (-15.4%, -1.1%); and for the LF/LGI group, 9.9% (2.4%, 18.0%). Total cholesterol (TC), LDL cholesterol, and apolipoprotein B concentrations decreased with SFA reduction. Decreases in TC and LDL-cholesterol concentrations were greater with LGI. Fat reduction lowered HDL cholesterol and apolipoprotein A1 and B concentrations. CONCLUSIONS This study did not support the hypothesis that isoenergetic replacement of SFAs with MUFAs or carbohydrates has a favorable effect on Si. Lowering GI enhanced reductions in TC and LDL-cholesterol concentrations in subjects, with tentative evidence of improvements in Si in the LF-treatment group. This trial was registered at clinicaltrials.gov as ISRCTN29111298.

The type and quantity of dietary fat and carbohydrate alter faecal microbiome and short-chain fatty acid excretion in a metabolic syndrome ‘at-risk’ population

INTRODUCTION AND OBJECTIVES:An obese-type human microbiota with an increased Firmicutes:Bacteroidetes ratio has been described that may link the gut microbiome with obesity and metabolic syndrome (MetS) development. Dietary fat and carbohydrate are modifiable risk factors that may impact on MetS by altering the human microbiome composition. We determined the effect of the amount and type of dietary fat and carbohydrate on faecal bacteria and short chain fatty acid (SCFA) concentrations in people ‘at risk’ of MetS.DESIGN:A total of 88 subjects at increased MetS risk were fed a high saturated fat diet (HS) for 4 weeks (baseline), then randomised onto one of the five experimental diets for 24 weeks: HS; high monounsaturated fat (MUFA)/high glycemic index (GI) (HM/HGI); high MUFA/low GI (HM/LGI); high carbohydrate (CHO)/high GI (HC/HGI); and high CHO/low GI (HC/LGI). Dietary intakes, MetS biomarkers, faecal bacteriology and SCFA concentrations were monitored.RESULTS:High MUFA diets did not affect individual bacterial population numbers but reduced total bacteria and plasma total and LDL-cholesterol. The low fat, HC diets increased faecal Bifidobacterium (P=0.005, for HC/HGI; P=0.052, for HC/LGI) and reduced fasting glucose and cholesterol compared to baseline. HC/HGI also increased faecal Bacteroides (P=0.038), whereas HC/LGI and HS increased Faecalibacterium prausnitzii (P=0.022 for HC/HGI and P=0.018, for HS). Importantly, changes in faecal Bacteroides numbers correlated inversely with body weight (r=−0.64). A total bacteria reduction was observed for high fat diets HM/HGI and HM/LGI (P=0.023 and P=0.005, respectively) and HS increased faecal SCFA concentrations (P<0.01).CONCLUSION:This study provides new evidence from a large-scale dietary intervention study that HC diets, irrespective of GI, can modulate human faecal saccharolytic bacteria, including bacteroides and bifidobacteria. Conversely, high fat diets reduced bacterial numbers, and in the HS diet, increased excretion of SCFA, which may suggest a compensatory mechanism to eliminate excess dietary energy.