Biomedical subjects
Publications and source records attributed to C J Packard.
The significance of the link between plasma cholesterol concentrations and risk of cerebrovascular disease (CVD) in the elderly is still debated. In a case-control study we found that elderly patients with ischaemic CVD have an atherogenic lipid profile, despite having low or normal total cholesterol concentrations.
We observed the appearance of two intermediate density lipoprotein (IDL) subfractions on gradient gel electrophoresis of lipoproteins in the density range 1.006-1.030 g/ml and estimated their approximate concentrations in plasma in subjects with a wide range of lipid levels, from 0.55 to 28.0 mmol/l plasma triglyceride and 3.75-10.0 mmol/l cholesterol. The larger species, IDL-I (31.7 +/- 0.7 nm, mean +/- SD), showed little variation in size in normal and moderate hyperlipidaemic individuals. The estimated concentration of IDL-I was positively related to plasma triglyceride (r = 0.63, P = 0.0004) and low density lipoprotein (LDL) cholesterol (r = 0.68, P = 0.0003). These findings are consistent with the view that IDL-I is a metabolic intermediate between very low density lipoprotein (VLDL) and LDL. The smaller subfraction, IDL-II (25.7 +/- 2.4 nm) was virtually the only true species observed in subjects with plasma triglyceride < 1.0 mmol/l and its estimated concentration fell as plasma triglyceride increased (r = -0.58, P = 0.0002). IDL-II particle size changed in concert with LDL particle size (r = 0.61, P < 0.0001), suggesting that they were influenced by common metabolic factors. These observations provide further support for the hypothesis outlined by Musliner et al. [1] that IDL-I was part of the delipidation chain from VLDL to LDL, whereas IDL-II arose from a separate source, possibly directly released from the liver. Hence the two subpopulations of IDL differ in their relationship to plasma triglyceride and cholesterol levels.
Lipoprotein (a) (Lp(a)) is recognised as a risk factor for arterial and venous thrombosis, a property which may relate to its structural similarity to plasminogen. Pregnancy is associated with a hypofibrinolytic state. Elevated Lp(a) may influence fibrinolysis and have an unfavourable role in pregnancy outcome. In this study alterations in plasma Lp(a) concentrations during normal pregnancy was examined, in a detailed longitudinal investigation, in ten women together with changes in other lipid parameters. In addition, Lp(a) concentrations were examined in subjects with pre-eclampsia (n=10) relative to matched controls (n=10), since it has recently been reported that a substantial increase in circulating Lp(a) occurs in this disorder. Lp(a) concentration increased steadily in normal pregnancy between 10 and 35 weeks with a doubling of the median value from 14.5 to 27.0 mg/dl (P=0.01). A significant increase in Lp(a) values was observed in all subjects with increasing gestation (median rise 190%, range 117-340%). This increase was intermediate to those seen in plasma triglyceride and cholesterol. No significant difference in Lp(a) values was observed in subjects with pre-eclampsia, compared with matched normal pregnancy controls (median 14 mg/dl [IQR 4.7-69.0] in pre-eclampsia vs 20 mg/dl [9.0-56. 3] in controls; P=0.57), at a median gestation of 32 weeks. In conclusion, there is a 2-fold increase in Lp(a) during normal pregnancy, which may influence fibrinolysis. Circulating Lp(a) is not significantly elevated in women with pre-eclampsia, and thus is unlikely to play a role in the pathophysiology of this disorder.
A specific and robust immunoassay for the lipoprotein-associated phospholipase A(2) (Lp-PLA(2)), platelet-activating factor acetylhydrolase, is described for the first time. The immunoassay was used to evaluate possible links between plasma Lp-PLA(2) levels and atherosclerosis risk amongst susceptible individuals. Such an investigation was important because Lp-PLA(2) participates in the oxidative modification of low density lipoprotein by cleaving oxidised phosphatidylcholines, generating lysophosphatidylcholine and oxidised free fatty acids. The majority of Lp-PLA(2) was found associated with LDL (approximately 80%) and, as expected, enzyme levels were significantly positively correlated to LDL cholesterol. Plasma Lp-PLA(2) levels were significantly elevated in patients with angiographically proven coronary artery disease (CAD) when compared with age-matched controls, even though LDL cholesterol levels did not differ significantly. Indeed, when included in a general linear model with LDL cholesterol and other risk factors, Lp-PLA(2) appeared to be an independent predictor of disease status. We propose, therefore, that plasma Lp-PLA(2) mass should be viewed as a potential novel risk factor for CAD that provides information related to but additional to traditional lipoprotein measurements.
End-stage renal failure (ESRF) is associated with dyslipidemia and accelerated atherosclerosis. Triglyceride-rich lipoproteins accumulate and qualitative changes take place in low-density lipoprotein (LDL), with a predominance of the small dense LDL phenotype. Increased small dense LDL (LDLIII) is a known risk factor for cardiovascular disease. To assess the extent of LDLIII formation in ESRF and identify factors contributing to LDLIII production, we analyzed LDL subfractions by density-gradient ultracentrifugation, very low-density lipoprotein subfractions, and lipase activity in 75 patients with ESRF (25 hemodialysis [HD], 25 peritoneal dialysis [PD], and 25 predialysis patients) and 40 age- and sex-matched controls. The percentage of LDLIII was increased in all three patient groups compared with controls (PD, 33% +/- 29% [mean +/- SD]; P < 0.005; HD, 30% +/- 22%; P < 0.01; predialysis, 26% +/- 26%; P < 0.01; all versus controls, 14% +/- 10%). Plasma LDLIII concentration was increased only in PD patients (median, 84 mg/dL; interquartile range [IQR], 29 to 160 mg/dL versus controls; median, 31 mg/dL; IQR, 26 to 54 mg/dL). In other patient groups, total LDL level was less, with heterogeneity in LDLIII concentrations. Forty percent of PD patients and 28% of HD and predialysis patients had LDLIII concentrations greater than 100 mg/dL compared with 2.5% of controls (P = 0.002). Plasma triglyceride levels (r(2) = 38.4%; P < 0.001) and hepatic lipase activity (r(2) = 6.7%; P < 0.03) were independent predictors of LDLIII concentration. The strong association between LDLIII concentration and triglyceride level was present in all three patient groups (HD, r(2) = 47.9%; PD, r(2) = 45. 2%; predialysis, r(2) = 25.8%); plasma triglyceride levels greater than 177 mg/dL (2.0 mmol/L) had an 86% specificity and 79% sensitivity for predicting an LDLIII concentration greater than 100 mg/dL. We conclude that the atherogenic lipoprotein phenotype predominates in ESRF, with excess LDLIII particularly prominent in PD patients. Atherogenic levels of LDLIII are found in patients with triglyceride levels greater than 177 mg/dL. This is likely to represent a further cardiovascular risk factor in this population.
BACKGROUND: Impaired very low-density lipoprotein (VLDL) clearance contributes to dyslipidemia in nephrotic-range proteinuria. VLDL can be subdivided into large light VLDL1 (Sf 60 to 400) and smaller, denser VLDL2 (Sf 20 to 60). In nephrotic-range proteinuria, the clearance of VLDL1 is delayed. VLDL1 lipolysis is influenced by apolipoprotein CII (apoCII) and apoCIII, whereas apoE regulates receptor-mediated clearance. METHODS: To ascertain whether impaired VLDL1 clearance was related to a deficiency in apolipoproteins on VLDL1, we measured VLDL subfraction concentrations and VLDL1 apolipoprotein and lipid compositions in 27 patients with glomerular disease and urinary albumin> 2 g/24 h along with 27 age- and sex-matched controls. RESULTS: Proteinuric patients had increased plasma VLDL1, VLDL2, apoCII, apoCIII (all P < 0.001), and apoE concentration (P < 0.002). Patients appeared to have smaller VLDL1 particles, as assessed by triglyceride per particle (median + interquartile range, moles per VLDL1 particle): patients, 4.9 (3.0 to 7.9) x103; controls, 7.0 (4.6 to 15.7) x103, P < 0.05, with reduced apoCII, 4.2 (3.1 to 8.2) versus 9.9 (7.4 to 23.2), P < 0.0004; apoCIII, 16.6 (9.1 to 27.2) versus 29.3 (18.5 to 69.4), P < 0.02; and apoE content, 0.17 (0.08 to 0.44) versus 0.48 (0.31 to 1. 31), P < 0.006. The VLDL1 surface free cholesterol to phospholipid results were increased in proteinuric patients (0.55 +/- 0.17 vs. 0. 40 +/- 0.18, P < 0.002, all mean +/- SD). For all patients, VLDL1 apoCII, apoCIII, and apoE contents per particle were related to particle size (apoCII, r2 = 61.5%, P < 0.001; apoCIII, r2 = 75.8%, P < 0.001; apoE, r2 = 58.2%, P < 0.001) and inversely to the free cholesterol to phospholipid ratio (apoCII, r2 = 41.6%, P < 0.001; apoCIII, r2 = 38.8%, P < 0.001; apoE, r2 = 11.7%, P < 0.05). Multivariate analysis suggested that the relative lack of apoCII and apoCIII on patients VLDL1 was related to smaller particle size and increased free cholesterol:phospholipid (FC:PL) ratio. Particle size but not free cholesterol determined the apoE content of VLDL1. CONCLUSIONS: We postulate that impaired VLDL1 clearance in nephrotic-range proteinuria results from the appearance of particles deficient in apoCII, apoCIII, and apoE. VLDL1 apoC deficiency is associated with the formation of smaller particles with a high FC:PL ratio, and is likely to cause inefficient lipolysis. VLDL1 apoE deficiency is associated with smaller VLDL1 particles but not altered VLDL1 surface lipid content, and may reduce receptor-mediated clearance of this lipoprotein.
Statin therapy has been conclusively shown to offer patients clinical benefit, virtually irrespective of their baseline risk status. However, the absolute risk reductions observed in different clinical trials, which have recruited patients across a spectrum of lipid levels and vascular disease states, show that baseline global risk determines the absolute benefit gained and in turn will specify the number of patients needed to be treated in order to realize this benefit. Global risk assessment is therefore central to the clinically meaningful use of statin therapy, and a strong case is now argued in the literature for a high-risk primary prevention strategy that goes hand in hand with standard secondary prevention. The routine use of Framingham-based risk assessment tools is advocated because these are the most widely evaluated and have been repeatedly shown to predict the risk of coronary heart disease accurately in western populations. The risk threshold in primary prevention that should determine pharmacological intervention is the subject of controversy. The currently used annual risk figure of 3% would clearly capture all very high-risk individuals but would also deny treatment to many individuals who will subsequently die from their first coronary event. Although a 1.5% annual risk threshold is economically untenable in the present UK health system, a level of 2% is, we believe, both achievable and affordable.
Apolipoprotein B (apoB) metabolism was investigated in 20 men with plasma triglyceride 0.66-2.40 mmol/l and plasma cholesterol 3.95-6. 95 mmol/l. Kinetics of VLDL(1) (S(f) 60-400), VLDL(2) (S(f) 20-60), IDL (S(f) 12-20), and LDL (S(f) 0;-12) apoB were analyzed using a trideuterated leucine tracer and a multicompartmental model which allowed input into each fraction. VLDL(1) apoB production varied widely (from 5.4 to 26.6 mg/kg/d) as did VLDL(2) apoB production (from 0.18 to 8.4 mg/kg/d) but the two were not correlated. IDL plus LDL apoB direct production accounted for up to half of total apoB production and was inversely related to plasma triglyceride (r = -0.54, P = 0.009). Percent of direct apoB production into the IDL/LDL density range (r = 0.50, P < 0.02) was positively related to the LDL apoB fractional catabolic rate (FCR). Plasma triglyceride in these subjects was determined principally by VLDL(1) and VLDL(2) apoB fractional transfer rates (FTR), i.e., lipolysis. IDL apoB concentration was regulated mainly by the IDL to LDL FTR (r = -0.71, P < 0.0001). LDL apoB concentration correlated with VLDL(2) apoB production (r = 0.48, P = 0.018) and the LDL FCR (r = -0.77, P < 0. 001) but not with VLDL(1), IDL, or LDL apoB production. Subjects with predominantly small, dense LDL (pattern B) had lower VLDL(1) and VLDL(2) apoB FTRs, higher VLDL(2) apoB production, and a lower LDL apoB FCR than those with large LDL (pattern A). Thus, the metabolic conditions that favored appearance of small, dense LDL were diminished lipolysis of VLDL, resulting in a raised plasma triglyceride above the putative threshold of 1.5 mmol/l, and a prolonged residence time for LDL. This latter condition presumably permitted sufficient time for the processes of lipid exchange and lipolysis to generate small LDL particles.
Plasma concentrations of lipoprotein (a), total cholesterol, high-density lipoprotein (HDL)-cholesterol, low-density lipoprotein (LDL)-cholesterol and triglyceride were measured in 62 women who had suffered an episode of objectively confirmed venous thromboembolism (VTE) at < or = 50 years of age, and in 98 age-matched female controls. The mean body mass index (BMI) of cases was significantly (P < 0.001) higher than that of controls. Plasma triglyceride was significantly higher, and total cholesterol/LDL- and HDL-cholesterol significantly lower, in cases compared with controls. After adjustment for BMI, the plasma total cholesterol and LDL-cholesterol remained significantly lower in cases. No significant differences in mean plasma lipoprotein (a) levels were identified between cases and controls. Lipoprotein (a) does not appear to be significantly associated with the development of VTE in young women. The increased risk of VTE in obese subjects may be mediated, at least in part, via hypertriglyceridaemia, which has previously been demonstrated to have effects on levels of coagulation factors, natural anticoagulants, and plasminogen activator inhibitor type 1.
The PROspective Study of Pravastatin in the Elderly at Risk (PROSPER) is a randomized, double-blind, placebo-controlled trial designed to test the hypothesis that treatment with pravastatin will diminish risk of subsequent major vascular events in a cohort of men and women (70 to 82 years old) with preexisting vascular disease or significant risk of developing this condition. Five thousand eight hundred four men and women in addition to receiving advice on diet and smoking, have been randomized equally to treatment with 40 mg pravastatin/day or matching placebo in 3 centers (Cork, Ireland, Glasgow, Scotland, and Leiden, The Netherlands). Following an average 3.5-year intervention period, a primary assessment will be made of the influence of this therapy on major vascular events (a combination of coronary heart disease, death, nonfatal myocardial infarction, and fatal and nonfatal stroke). A number of additional analyses will also be conducted on the individual components of the primary end point, on men, on women, and on subjects with and without previous evidence of vascular disease. Finally, an assessment will be made of the effects of treatment on cognitive function, disability, hospitalization or institutionalization, vascular mortality, and all-cause mortality.
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Sialic acid is a negatively charged sugar associated with the protein and lipid portions of lipoproteins. Sialic acid has been hypothesised to play an anti-atherogenic role in lipoprotein metabolism through the electrostatic inhibition of lipoprotein interactions with chondroitin-6-sulphate-rich arterial proteoglycans (APG). We conducted a series of studies using native and modified lipoproteins (VLDL1 Sf 60-400, VLDL2 Sf 20-60, IDL1 Sf 16-20, IDL2 Sf 12-16, LDL(A) Sf 8-12, and LDL(B) Sf0-8) that vary in their sialic acid content to examine the relationship between lipoprotein sialic acid content and its interaction with APG. Lipoprotein sialic acid was greatest in VLDL1 and decreased progressively with particle density until the IDL2 fraction (VLDL1 > VLDL2 > IDL1 > IDL2 = LDL(A) = LDL(B)). The pattern of reactivity of each fraction with APG was different from the pattern observed for lipoprotein sialic acid content (IDL2 > LDL(A) > LDL(B) > IDL1 > VLDL2 > VLDL1). Levels of sialic acid were lower in subjects with CHD as compared to control subjects but the presence of CHD had no effect on lipoprotein-APG complex formation when sex and plasma triglyceride levels were taken into account. There was also no significant relationship between the lipoprotein sialic acid content and the reactivity with APG within each lipoprotein fraction. Treatment of hypertriglyceridaemic subjects with ciprofibrate decreased lipoprotein-APG complex formation in all lipoprotein fractions. This was associated with a decrease in the total sialic acid content of apo B100-containing lipoproteins suggesting that the total sialic acid content of apo B100-containing lipoproteins has no influence on lipoprotein-APG complex formation. We next conducted in vitro experiments to manipulate LDL sialic acid content. Enzymatic removal of sialic acid from LDL with neuraminidase resulted in an increase in LDL-APG complex formation. This was accompanied by an increase in the exposure of free amino groups on LDL possibly due to disruption of interactions between free amino groups and sialic acid-containing components on LDL. Increasing LDL sialic acid content through incubation with ganglioside resulted in a decrease in lipoprotein-APG complex formation without any changes in the exposure of free amino groups on LDL. We conclude that total sialic acid content of lipoproteins is not a major determinant of their binding to APG. However, specific sialic acid-containing components on lipoproteins can affect their interaction with APG.
Heparin given intravenously enhances lipolysis, although fasting lipids are not markedly altered in long-term administration. In the present study we investigated heparin-induced acute perturbation of VLDL subclass metabolism. Eight men were examined during a control study and during an 8.5 h infusion of heparin. 2H3-leucine was used as tracer and kinetic constants derived using a non-steady-state model. Heparin infusion increased both plasma lipoprotein and hepatic lipase activity and raised plasma FFAs two-fold (P < 0.001). The fractional catabolic rate (FCR) of VLDL1 apo B increased on heparin (25.7 +/- 4.2 and 10.8 +/- 1.7 pools/d, heparin vs. control, P < 0.02). The FCR of VLDL2 apo B increased to 12.6 +/- 1.9 pools/d on heparin vs. 8.8 +/- 1.1 pools/d during the control (NS). Total VLDL apo B production was not significantly changed (824 +/- 45 and 692 +/- 91 mg/d, heparin vs. control, NS). We conclude that during heparin infusion, the catabolism of especially large triglyceride-rich VLDL1 apo B is greatly increased. However, although the FFA levels were high during the heparin study, the production of total VLDL apo B did not rise. These findings are consistent with the known action of heparin on lipoprotein lipase but indicate that acute increase in plasma FFA levels does not lead to a rise in VLDL apo B production.
Further understanding of the causative link between plasma lipids and coronary heart disease will come from a deeper appreciation of the impact of lipoprotein heterogeneity on the processes of atherosclerosis and thrombosis. It is now widely appreciated that remnants of triglyceride-rich lipoproteins, IDL and specific LDL subfractions may have a role in atherogenesis disproportionate to the plasma concentrations of these species. Elucidation of the factors that control the distribution of subfractions within the spectrum of apolipoprotein B-containing lipoproteins is underway but far from complete. Important influences are the rate and nature of lipoproteins secreted from the liver, the extent of remodelling by lipid exchange and lipolysis in the circulation and the affinity of the various particles for cell surface receptors.
Previous studies have shown that in preeclampsia, plasma lipids climb substantially above levels seen in normal pregnancies. Such lipid changes may play a role in the endothelial damage characteristic of preeclampsia. Pregnancies complicated by intrauterine growth restriction (IUGR), without preeclampsia, have similar placental pathology to preeclampsia despite the absence of the maternal systemic manifestations of hypertension and proteinuria. The aim of this study was to perform a cross-sectional study of lipid and lipoprotein concentrations in the third trimester, from normal pregnancies, and those complicated by IUGR without preeclampsia. Our hypothesis was that, in contrast to the exaggerated lipid changes seen in preeclampsia, lipid and lipoprotein concentrations in IUGR would be similar to those of matched healthy pregnant controls. Fasting blood samples for lipids and lipoprotein fractions were taken in the third trimester, from eight women with IUGR; and eight women with uncomplicated pregnancies, matched as a group for age, booking weight, parity, and gestational age at sampling. There were no significant differences (P > 0.05) in the median concentrations of triglyceride, high-density lipoprotein, and very-low-density lipoprotein 1 (VLDL1), between cases and controls. However, women with IUGR pregnancies had significantly lower cholesterol [4.95 mmol/L (3.35-7.10) vs. 7.47 (5.75-8.45); median (range) for IUGR patients and controls, respectively; P < 0.01], low-density lipoprotein (LDL)-cholesterol [2.45 mmol/L (0.95-3.60) vs. 4.25 (3.35-5.60); P < 0.01], VLDL2 mass [59.0 mg/dL (37-87) vs. 103.0 (64-168); P < 0.01], intermediate-density lipoprotein mass [56.0 mg/dL (31-110) vs. 125.6 (91-157); P < 0.01], and total LDL mass [221.0 mg/dL (104-237) vs. 380.3 (267-534); P < 0.01]. In addition, it was noteworthy that, with respect to LDL-cholesterol and total LDL mass, there was little or no overlap in the ranges of concentrations measured between cases and controls. Because VLDL2 and intermediate-density lipoprotein are the synthetic precursors to LDL in the circulation, their significantly lower median concentrations imply a failure of appropriate LDL synthesis in IUGR pregnancies. Whatever the mechanism, if our results are confirmed in larger studies and longitudinal investigations, then LDL-cholesterol measurements (when LDL-cholesterol fails to rise appropriately or is low in the third trimester) may be of use in identifying mothers with, or at risk of, a pregnancy complicated by IUGR.
The St. Thomas' mixed hyperlipidemic (SMHL) rabbit (previously St. Thomas' Hospital rabbit) is a putative model of familial combined hyperlipidemia (FCH). When fed a low (0.08%) cholesterol diet, it exhibits elevations in both plasma cholesterol and triglyceride compared to New Zealand White (NZW) controls. To determine the mechanism for this hyperlipidemia we studied the secretion of apolipoprotein B (apoB)-containing lipoproteins from perfused livers of both young and mature rabbits. During a 3-h perfusion we measured the total cholesterol and triglyceride content of the medium and the cholesterol, triglyceride, and apoB content of very low density lipoprotein (VLDL)(1) (S(f) 60;-400), VLDL(2) (S(f) 20;-60), intermediate (S(f) 12;-20), and low (S(f) 0;-12) density lipoproteins (IDL, LDL). Lipoprotein concentrations increased linearly throughout the perfusion period. The rate of cholesterol output was 3-fold higher (459 vs. 137 ng/g liver/min, P = 0.003) in SMHL versus NZW rabbits whilst that of triglyceride was similar (841 vs. 662 ng/g liver/min, NS). VLDL(1) cholesterol output was elevated 2-fold (232 vs. 123 ng/g liver/min, P < 0.05) and VLDL(2) + IDL + LDL cholesterol output, 4.5-fold (106 vs. 23 ng/g liver/min, P < 0. 005) in SMHL versus NZW rabbits. ApoB output in VLDL1 was 38 ng/g liver per min in SMHL and 14 ng/g liver per min in NZW (NS). In SMHL VLDL(2) + IDL + LDL apoB was increased 9-fold at 53 versus 6 ng/g liver per min in NZW (P < 0.001). We conclude that the SMHL rabbit overproduces apoB-containing lipoproteins particularly in the VLDL(2) + IDL + LDL fraction, a characteristic consistent with its use as a model of FCH.
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Familial ligand-defective apolipoprotein B (apoB) is a group of disorders caused by mutations in the apoB gene. In this report the R3531C mutation is characterized further using a monoclonal antibody MB19/dynamic laser light scattering technique to measure ratios of Cys(3531) to normal low density lipoprotein (LDL) particles. All six subjects studied showed a preferential accumulation of particles carrying the defective apoB allotype. We determined binding properties of LDL from R3531C heterozygotes by measurement of high-affinity binding to LDL receptors on fibroblasts and its ability promote growth of U937 cells. LDL from R3531C heterozygotes, compared to normal LDL, had 49.3% of the binding affinity and was 74% as effective in a U937 cell proliferation assay. To identify new probands, we screened 2570 subjects for the R3531C mutation. Nine probands were found with 15 affected relatives. Of the seven haplotypes we uncovered, two were novel, while five were identical to one initially reported as associated with Cys3531. Three silent mutations were detected also: T3540T, N3542N and T3552T. Analysis of lipid profiles of R3531C families showed, as with the R3500Q mutation, variable expression of the phenotype, modulated by environmental and other genetic factors. Both mutations tend to produce lower plasma levels of LDL in affected subjects than do defects of the LDL receptor (familial hypercholesterolemia, FH). This study shows that the Cys(3531) LDL particles are not only defective at binding to the LDL receptor, as determined by two separate methods, but that in all cases they accumulate preferentially compared to the normal allotype.-Pullinger, C. R., D. Gaffney, M. M. Gutierrez, M. J. Malloy, V. N. Schumaker, C. J. Packard, and J. P. Kane. Apolipoprotein B R3531C mutation: characteristics of 24 subjects from 9 kindreds. .