High-density Lipoprotein Subfractions Research Articles

A simple and precise method for measuring HDL-cholesterol subfractions by a single precipitation followed by homogenous HDL-cholesterol assay

HDL consists of two major subfractions, HDL2 and HDL3. This paper describes a simple method for assaying HDL subspecies by combining a single precipitation with a direct high density lipoprotein-cholesterol (HDL-C) assay. A precipitation reagent (0.06 ml) containing 1,071 U/ml heparin, 500 mmol/l MnCl2) and 12 mg/ml dextran sulfate was added to a serum (0.3 ml). The sample was incubated and centrifuged at 10,000 rpm for 10 min. HDL3-C was measured by a homogenous HDL-C assay in the supernatant, and HDL2-C was estimated by subtracting the HDL3-C from the direct HDL-C. The HDL3-C and HDL2-C values determined by the precipitation method were identical to those determined by ultracentrifugation, and there were excellent correlations between the methods in the measurements of HDL3-C and HDL2-C (r = 0.933 and 0.978, respectively; n = 102). The two methods also proved to be highly correlated in the measurement of apolipoprotein A-I and A-II in HDL subfractions. The HDL-C subfractions determined by ultracentrifugation were more closely associated with the homogenous HDL-C assay than with the total cholesterol assay, especially in the hypertriglyceridemic samples. Our method is far simpler and more precise than the classical dual precipitation method for HDL-C subfractions, and it can be easily performed in a routine chemical laboratory.

Lipid Profiles in Patients with Rheumatoid Arthritis: Mechanisms and the Impact of Treatment

To describe the impact of rheumatoid arthritis (RA), and its treatment, on lipoprotein levels with potential implications for atherosclerosis. A PubMed literature search was undertaken for studies published between 1990 and May 2007, using the search terms "rheumatoid arthritis" AND "lipid" OR "lipoprotein," and including all relevant drug treatment terms for glucocorticoids, disease-modifying antirheumatic drugs, and biologics. Patients with RA face an increased risk of developing premature cardiovascular disease and limited ability to modify risk factors, eg, through exercise. RA is associated with an abnormal lipoprotein pattern, principally low levels of high density lipoprotein (HDL) cholesterol. Most treatments for RA tend to improve the atherogenic index (total/HDL cholesterol ratio), with more evidence for biologics in this regard. The improvement in the lipoprotein profile in RA appears to be associated with suppression of inflammation. Lipid levels should be monitored and managed in patients with RA to minimize the long-term risk of cardiovascular disease. More research is needed to quantify the relationship between systemic inflammation and lipoprotein levels and to determine the impact of specific lipoprotein particles, eg, small dense low-density lipoprotein and subfractions of HDL on long-term risk. Control of inflammation may have an effect on modifying cardiovascular risk.

The lipoprotein subfraction profile: heritability and identification of quantitative trait loci

The HDL and LDL subclass profile is an emerging cardiovascular risk factor. Yet, the biological and genetic mechanisms controlling the lipoprotein subclass distribution are unclear. Therefore, we aimed 1) to determine the heritability of the entire spectrum of LDL and HDL subclass features and 2) to identify gene loci influencing the lipoprotein subfraction pattern. Using NMR spectroscopy, we analyzed the lipoprotein subclass distribution in 1,275 coronary artery disease patients derived from the Regensburg Myocardial Infarction Family Study. We calculated heritabilities, performed a microsatellite genome scan, and calculated linkage. HDL and LDL subclass profiles showed heritabilities ranging from 23% to 67% (all P < 10(-3)) of traits using univariate calculation. After multivariate adjustment, we found heritabilities of 27-48% (all P < 0.05) for HDL and 21-44% for LDL traits. The linkage analysis revealed a significant logarithm of the odds (LOD) score (3.3) for HDL particle concentration on chromosome 18 and a highly suggestive signal for HDL particle size on chromosome 12 (2.9). After multivariate adjustment, we found a significant maximum LOD score of 3.7 for HDL size. Our study is the first to analyze heritability and linkage for the entire spectrum of LDL and HDL subclass features. Our findings may lead to the identification of genes controlling the lipoprotein subclass distribution.

Procoagulant activities of plasma factor VIIc and factor Xc are positively and independently associated with concentrations of the high-density lipoprotein apolipoprotein, apo A-II

The pro- and antiatherogenic roles of apolipoproteins B and A-I, respectively, are well-established although the importance of apolipoprotein A-II remains unclear. There is extensive evidence for the involvement of plasma lipoproteins in haemostatic function. However, in-vivo studies of relationships between haemostatic variables and apolipoprotein concentrations are very limited. Plasma fibrinogen, factors VIIc and Xc (FVIIc and FXc, respectively), apolipoproteins (apo) A-I, A-II and B, triglycerides, total, low-density and high-density lipoprotein (HDL) cholesterol, and cholesterol in HDL subfractions 2 and 3 were measured in 186 apparently healthy Caucasian men (aged 26-78 years; body mass index 19.9-37.8 kg/m(2)). Associations between haemostatic, apolipoprotein, lipid and lipoprotein variables were explored in uni- and multivariable analyses. Fibrinogen did not correlate with any of the lipid-related variables. FVIIc and FXc were significant positive univariate correlates of total cholesterol (correlation coefficients 0.26, p<0.001 and 0.19, p<0.05, respectively) triglycerides (0.37, p<0.001 and 0.36, p<0.001), and apoB (0.21, p<0.01 and 0.17, p<0.05) and apoA-II (0.19, p<0.05 and 0.29, p<0.001). HDL(2) subfraction cholesterol correlated negatively with FVIIc and FXc (-0.20, p<0.01 and -0.22, p<0.01, respectively). In multivariable analysis, only the associations of FVIIc and FXc with total cholesterol, triglycerides and apoA-II remained statistically significant. In conclusion, total cholesterol and triglycerides were the major independent lipid correlates of FVIIc and FXc. The independent and positive associations of apoA-II with FVIIc and FXc suggest a prothrombotic involvement for this apolipoprotein.

PROCAM Study: risk prediction for myocardial infarction using microfluidic high-density lipoprotein (HDL) subfractionation is independent of HDL cholesterol

High-density lipoprotein (HDL) subfractions are among the new emerging risk factors for atherosclerosis. In particular, HDL 2b has been shown to be linked to cardiovascular risk. This study uses a novel microfluidics-based method to establish HDL 2b clinical utility using samples from the Prospective Cardiovascular Muenster (PROCAM) Study. Method performance was established by measuring accuracy, precision, linearity and inter-site precision. Serum samples from 503 individuals collected in the context of the PROCAM study were analyzed by electrophoresis on a microfluidics system. Of these, 251 were male survivors of myocardial infarction (cases), while 252 individuals were matched healthy controls. HDL cholesterol, HDL 2b concentration and HDL 2b percentage were analyzed. This novel method showed satisfactory assay performance with an inter-site coefficient of variance of <10% for HDL 2b percentage. Parallel patient testing on 52 samples between two sites resulted in a correlation coefficient of r=0.95. Significant differences were observed in the HDL 2b subfraction between cases and controls independent of other risk factors. Including HDL 2b percentage in logistic regression reduced the number of false positives from 64 to 39 and the number of false negative cases from 48 to 45, in the context of this study. The novel method showed satisfactory assay performance in addition to drastically reduced analysis times and improved ease of use as compared to other methods. Clinical utility of HDL 2b was demonstrated supporting the findings of previous studies.

Changes in lipid metabolism in postmenopausal Japanese women using hormone therapy evaluated by capillary isotachophoresis

The aim of this study was to clarify the changes in lipid metabolism in postmenopausal Japanese women during continuous combined hormone therapy (HT) in detail by using capillary isotachophoresis (cITP). Twenty-three postmenopausal Japanese women with climacteric symptoms were recruited. Blood samples were collected from all participants before HT and after 3 and 6 months of HT, and changes in total cholesterol, triglycerides, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, and apolipoprotein (apo) A-I, apoB, and lipoprotein(a) were assessed. The same blood samples were analyzed for charge-based lipoprotein subfractions using cITP. Total cholesterol (-8.5%), LDL-cholesterol (-16.2%), and lipoprotein(a) (-25.5%) decreased and apoA-I (9.5%) increased significantly from the baseline level at 6 months after starting continuous combined HT. HDL-cholesterol increased nonsignificantly (+2.4%). cITP revealed that the fast HDL subfraction (+24.7%), which contains apoA-I and may be antiatherogenic, significantly increased. Conversely, the fast LDL subfraction (+14.4%), which may possess atherogenic potential, increased during HT. The changes in lipid metabolism in postmenopausal Japanese women receiving HT are inconclusive regarding atherogenicity, ie, it increased fast HDL and simultaneously increased fast LDL. cITP is useful for clarifying the changes in lipid metabolism during HT.

Atherogenic, enlarged, and dysfunctional HDL in human PLTP/apoA-I double transgenic mice

In low density lipoprotein receptor (LDLR)-deficient mice, overexpression of human plasma phospholipid transfer protein (PLTP) results in increased atherosclerosis. PLTP strongly decreases HDL levels and might alter the antiatherogenic properties of HDL particles. To study the potential interaction between human PLTP and apolipoprotein A-I (apoA-I), double transgenic animals (hPLTPtg/hApoAItg) were compared with hApoAItg mice. PLTP activity was increased 4.5-fold. Plasma total cholesterol and phospholipid were decreased. Average HDL size (analyzed by gel filtration) increased strongly, hPLTPtg/hApoAItg mice having very large, LDL-sized, HDL particles. Also, after density gradient ultracentrifugation, a substantial part of the apoA-I-containing lipoproteins in hPLTPtg/hApoAItg mice was found in the LDL density range. In cholesterol efflux studies from macrophages, HDL isolated from hPLTPtg/hApoAItg mice was less efficient than HDL isolated from hApoAItg mice. Furthermore, it was found that the largest subfraction of the HDL particles present in hPLTPtg/hApoAItg mice was markedly inferior as a cholesterol acceptor, as no labeled cholesterol was transferred to this fraction. In an LDLR-deficient background, the human PLTP-expressing mouse line showed a 2.2-fold increased atherosclerotic lesion area. These data demonstrate that the action of human PLTP in the presence of human apoA-I results in the formation of a dysfunctional HDL subfraction, which is less efficient in the uptake of cholesterol from cholesterol-laden macrophages.

Effect of Obesity on High‐density Lipoprotein Metabolism

Reduced levels of high-density lipoproteins (HDL) in non-obese and obese states are associated with increased risk for the development of coronary artery disease. Therefore, it is imperative to determine the mechanisms responsible for reduced HDL in obese states and, conversely, to examine therapies aimed at increasing HDL levels in these individuals. This paper examines the multiple causes for reduced HDL in obese states and the effect of exercise and diet--two non-pharmacologic therapies--on HDL metabolism in humans. In general, the concentration of HDL-cholesterol is adversely altered in obesity, with HDL-cholesterol levels associated with both the degree and distribution of obesity. More specifically, intra-abdominal visceral fat deposition is an important negative correlate of HDL-cholesterol. The specific subfractions of HDL that are altered in obese states include the HDL2, apolipoprotein A-I, and pre-beta1 subfractions. Decreased HDL levels in obesity have been attributed to both an enhancement in the uptake of HDL2 by adipocytes and an increase in the catabolism of apolipoprotein A-I on HDL particles. In addition, there is a decrease in the conversion of the pre-beta1 subfraction, the initial acceptor of cholesterol from peripheral cells, to pre-beta2 particles. Conversely, as a means of reversing the decrease in HDL levels in obesity, sustained weight loss is an effective method. More specifically, weight loss achieved through exercise is more effective at raising HDL levels than dieting. Exercise mediates positive effects on HDL levels at least partly through changes in enzymes of HDL metabolism. Increased lipid transfer to HDL by lipoprotein lipase and reduced HDL clearance by hepatic triglyceride lipase as a result of endurance training are two important mechanisms for increases in HDL observed from exercise.

PO23-762 THE EFFECT OF ATORVASTATIN THERAPY ON HDL SUBFRACTIONS, LECITHIN:CHOLESTEROL ACYLTRANSFERASE, CHOLESTERYL ESTER TRANSFER PROTEIN AND HUMAN PARAOXONASE-1

The distribution and composition of high-density lipoprotein (HDL) subclasses were investigated in 14 women with severe hypothyroidism who were studied before and during treatment. The plasma concentrations of triglycerides, total cholesterol, HDL cholesterol, and of the apoproteins (apo) A-I, B, and E were increased in the hypothyroid state, while the apo A-II levels did not change significantly. After normalization of the thyroid function tests, the lipid and apoprotein levels were similar to those of normal individuals. Isopycnic ultracentrifugation in the density range 1.020 to 1.210 g/mL showed increases of both cholesterol and apo B in very-low-density lipoprotein (VLDL) and in low-density lipoprotein (LDL). The distribution of the HDL subclasses was modified in the hypothyroid subjects; both the less dense HDL fraction (d 1.063 to 1.100 g/mL; HDL2b), and the denser subclass (d 1.150 to 1.210 g/mL; HDL3b+3c) were increased, while the intermediate density subfraction (d 1.100 to 1.150 g/mL; HDL2a+3a) did not vary significantly. This redistribution of the HDL subfractions was associated with increased concentrations of cholesterol, phospholipid, and apo A-I in HDL2b, and of phospholipid and apo A-I in DHL3b+3c. Treatment of hypothyroidism decreased the concentrations of these fractions, and HDL2a+3a became the major HDL subclass in the euthyroid state. The particle sizes within HDL subfractions, measured by polyacrylamide gradient gel electrophoresis, were identical in the untreated and treated patients. The increased mass of protein and lipid within HDL2b and HDL3b+3c could therefore be attributed to an accumulation of identical-sized particles. The overall lipid and protein composition of the HDL lipoproteins was similar before and during treatment. The apoprotein composition was however modified, due to the higher apo A-I to apo A-II ratio observed in the HDL subfractions. Analysis of the lipoprotein distribution of apo E by sepharose column chromatography revealed a marked increase in the apo E content of VLDL, while changes in HDL-associated apo E were less pronounced. The major effect of hypothyroidism on apo E metabolism therefore appears to be an enrichment of VLDL with apo E, resulting in a significant increase of the plasma apo E levels.

Speciation of Human Plasma High-Density Lipoprotein (HDL): HDL Stability and Apolipoprotein A-I Partitioning

The distribution of apolipoprotein (apo) A-I between human high-density lipoproteins (HDL) and water is an important component of reverse cholesterol transport and the atheroprotective effects of HDL. Chaotropic perturbation (CP) with guanidinium chloride (Gdm-Cl) reveals HDL instability by inducing the unfolding and transfer of apo A-I but not apo A-II into the aqueous phase while forming larger apo A-I deficient HDL-like particles and small amounts of cholesteryl ester-rich microemulsions (CERMs). Our kinetic and hydrodynamic studies of the CP of HDL species separated according to size and density show that (1) CP mediated an increase in HDL size, which involves quasi-fusion of surface and core lipids, and release of lipid-free apo A-I (these processes correlate linearly), (2) >94% of the HDL lipids remain with an apo A-I deficient particle, (3) apo A-II remains associated with a very stable HDL-like particle even at high levels of Gdm-Cl, and (4) apo A-I unfolding and transfer from HDL to water vary among HDL subfractions with the larger and more buoyant species exhibiting greater stability. Our data indicate that apo A-I's on small HDL (HDL-S) are highly dynamic and, relative to apo A-I on the larger more mature HDL, partition more readily into the aqueous phase, where they initiate the formation of new HDL species. Our data suggest that the greater instability of HDL-S generates free apo A-I and an apo A-I deficient HDL-S that readily fuses with the more stable HDL-L. Thus, the presence of HDL-L drives the CP remodeling of HDL to an equilibrium with even larger HDL-L and more lipid-free apo A-I than with either HDL-L or HDL-S alone. Moreover, according to dilution studies of HDL in 3 M Gdm-Cl, CP of HDL fits a model of apo A-I partitioning between HDL phospholipids and water that is controlled by the principal of opposing forces. These findings suggest that the size and relative amount of HDL lipid determine the HDL stability and the fraction of apo A-I that partitions into the aqueous phase where it is destined for interaction with ABCA1 transporters, thereby initiating reverse cholesterol transport or, alternatively, renal clearance.

The effects of orlistat and fenofibrate, alone or in combination, on high‐density lipoprotein subfractions and pre‐beta1‐HDL levels in obese patients with metabolic syndrome

We assessed the effect of orlistat and fenofibrate, alone or in combination, on plasma high-density lipoprotein (HDL) subfractions and plasma pre-beta1-HDL levels in overweight and obese subjects with metabolic syndrome (MetS). Patients (n = 89) were prescribed a low-fat low-calorie diet and were randomly allocated to receive orlistat 120 mg three times daily (O group), micronized fenofibrate 200 mg/day (F group) or both (OF group) for 6 months. HDL subfractions were determined using a polyacrylamide gel tube electrophoresis method and pre-beta1-HDL levels using enzyme-linked immunoabsorbent assay. We observed a significant change of high-density lipoprotein cholesterol (HDL-C) levels only in the F group (+3%, p < 0.05). Large HDL-C levels were significantly increased and small HDL-C levels were significantly reduced with O administration. In F group we observed a significant increase of small HDL-C levels. No significant change of large or small HDL-C levels was observed with combination treatment. We observed a significant increase of pre-beta1-HDL levels in all groups, which was significantly greater in OF group compared with O or F monotherapy. OF combination increased the antiatherogenic pre-beta1-HDL levels in overweight and obese patients with MetS. Furthermore, OF combination counterbalanced the reduction of small HDL-C levels observed with orlistat monotherapy.

High density lipoprotein subfractions: isolation, composition, and their duplicitous role in oxidation

The plasma HDLs represent a major class of cholesterol-transporting lipoprotein that can be divided into two distinct subfractions, HDL(2) and HDL(3), by ultracentrifugation. Existing methods for the subfractionation of HDL requires lengthy ultracentrifugations, making them unappealing for large-scale studies. We describe a method that subfractionates HDL from plasma in only 6 h, representing a substantial decrease in total isolation time. The subfractions so isolated were assessed for a variety of lipid and protein components, in addition to their susceptibility to oxidation, both alone and in combination with VLDL and LDL. We report for the first time a prooxidant role for HDL during VLDL oxidation, in which HDL donates preformed hydroperoxides to VLDL in a cholesteryl ester transfer protein (CETP)-dependent process. Examination of the participation of HDL in LDL oxidation has reinforced its classic role as a potent antioxidant. Furthermore, we have also implicated the second major HDL-associated enzyme, LCAT, in these processes, whereby it acts as a potent prooxidant during VLDL oxidation but as an antioxidant during LDL oxidation. Thus, we have identified a potentially duplicitous role for HDL in the pathogenesis of atherosclerosis, attributable to both CETP and LCAT.

African Trypanosomes: Intracellular Trafficking of Host Defense Molecules1

Trypanosoma brucei brucei is the causative agent of Nagana in cattle and can infect a wide range of mammals but is unable to infect humans because it is susceptible to the innate cytotoxic activity of normal human serum. A minor subfraction of human high-density lipoprotein (HDL), containing apolipoprotein A-I (APOA1), apolipoprotein L-I (APOL1) and haptoglobin-related protein (HPR) provides this innate protection against T. b. brucei infection. Both HPR and APOL1 are cytotoxic to T. b. brucei but their specific activities for killing increase several hundred-fold when assembled in the same HDL. This HDL is called trypanosome lytic factor (TLF) and kills T. b. brucei following receptor binding, endocytosis, and lysosomal localization. Trypanosome lytic factor is activated in the acidic lysosome and facilitates lysosomal membrane disruption. Lysosomal localization is necessary for T. b. brucei killing by TLF. Trypanosoma brucei rhodesiense, which is indistinguishable from T. b. brucei, is resistant to TLF killing and causes human African sleeping sickness. Human infectivity by T. b. rhodesiense correlates with the evolution of a human serum resistance associated protein (SRA) that is able to ablate TLF killing. When T. b. brucei is transfected with the SRA gene it becomes highly resistant to TLF and human serum. In the SRA transfected cells, intracellular trafficking of TLF is altered and TLF mainly localizes to a subset of SRA containing cytoplasmic vesicles but not to the lysosome. These findings indicate that the cellular distribution of TLF is influenced by SRA expression and may directly determine susceptibility.

In Vitro Generation of Human High-Density-Lipoprotein-Resistant Trypanosoma brucei brucei

The host range of African trypanosomes is influenced by innate protective molecules in the blood of primates. A subfraction of human high-density lipoprotein (HDL) containing apolipoprotein A-I, apolipoprotein L-I, and haptoglobin-related protein is toxic to Trypanosoma brucei brucei but not the human sleeping sickness parasite Trypanosoma brucei rhodesiense. It is thought that T. b. rhodesiense evolved from a T. b. brucei-like ancestor and expresses a defense protein that ablates the antitrypanosomal activity of human HDL. To directly investigate this possibility, we developed an in vitro selection to generate human HDL-resistant T. b. brucei. Here we show that conversion of T. b. brucei from human HDL sensitive to resistant correlates with changes in the expression of the variant surface glycoprotein (VSG) and abolished uptake of the cytotoxic human HDLs. Complete transcriptome analysis of the HDL-susceptible and -resistant trypanosomes confirmed that VSG switching had occurred but failed to reveal the expression of other genes specifically associated with human HDL resistance, including the serum resistance-associated gene (SRA) of T. b. rhodesiense. In addition, we found that while the original active expression site was still utilized, expression of three expression site-associated genes (ESAG) was altered in the HDL-resistant trypanosomes. These findings demonstrate that resistance to human HDLs can be acquired by T. b. brucei.

Measurement and meaning of apolipoprotein AI and apolipoprotein B plasma levels

Apolipoproteins AI and B are structural components of lipoprotein particles, and also determinants of the metabolic fate of the encapsulated lipid, cholesterol and triglyceride. Development of accurate assays for these apolipoproteins has opened the way for their use as predictors of coronary heart disease risk. Interpretation of AI and apo B levels is best undertaken with background knowledge of the metabolic status of an individual, especially the lipolytic capacity as reflected in the triglyceride concentration. Those with raised triglyceride, in general, not only have an elevated apo B/apo AI ratio, but also apo B-containing lipoproteins with a prolonged residence time and hence ample opportunity for modification and damage. Assessment of apolipoprotein levels is an aid to risk prediction and can be useful in tailoring treatment.

A Randomized Study Comparing the Effects of a Low‐Carbohydrate Diet and a Conventional Diet on Lipoprotein Subfractions and C‐reactive Protein Levels in Patients With Severe Obesity

To compare the effects of a low-carbohydrate diet and a conventional (fat- and calorie-restricted) diet on lipoprotein subfractions and inflammation in severely obese subjects. We compared changes in lipoprotein subfractions and C-reactive protein levels in 78 severely obese subjects, including 86% with either diabetes or metabolic syndrome, who were randomly assigned to either a low-carbohydrate or conventional diet for 6 months. Subjects on a low-carbohydrate diet experienced a greater decrease in large very low-density lipoprotein (VLDL) levels (difference =-0.26 mg/dL, p = .03) but more frequently developed detectable chylomicrons (44% vs 22%, p = .04). Both diet groups experienced similar decreases in the number of low-density lipoprotein (LDL) particles (difference = -30 nmol/L, p = .74) and increases in large high-density lipoprotein (HDL) concentrations (difference = 0.70 mg/dL, p = .63). Overall, C-reactive protein levels decreased modestly in both diet groups. However, patients with a high-risk baseline level (>3 mg/dL, n = 48) experienced a greater decrease in C-reactive protein levels on a low-carbohydrate diet (adjusted difference = -2 mg/dL, p = .005), independent of weight loss. In this 6-month study involving severely obese subjects, we found an overall favorable effect of a low-carbohydrate diet on lipoprotein subfractions and on inflammation in high-risk subjects. Both diets had similar effects on LDL and HDL subfractions. ( Am J Med. 2004;117:398-405.).

High Dose Alpha Tocopherol therapy does not affect subfractions of High Density Lipoproteins in patients with Coronary Artery Disease on Statin therapy

Low levels of High density lipoprotein (HDL) are predictive of coronary heart disease (CHD). However, subfractions of HDL are implicated as better indicators of the disease process. The large HDL particles (HDL2) are inversely correlated to the severity of coronary artery disease (CAD). Alpha tocopherol (AT) is the main lipid soluble antioxidant in plasma and is anti-inflammatory. Previously, a small study (n=44) showed that the combination of an antioxidant cocktail (800 IU/d RRR-AT plus 1g Vitamin C, 25 mg β-carotene and 100 ug Se) with simvatstatin –niacin (S-N) therapy attenuated the protective increase in HDL2 seen with S-N alone. Thus, we tested the effect of high dose RRR-AT (1200 IU/d for 2 yrs) supplementation on HDL subfractions in a prospective randomized placebo-controlled study in a larger population of patients (n=90) with stable CAD on AT / placebo at 5 different time points (baseline, 6 months, 1 year, 1½ and 2 years). Importantly, 94% of the patients were on statin therapy. HDL subfractions were analyzed by NMR spectroscopy and divided into large (8.8 to 13.0nm), medium (8.2 to 8.8 nm), and small (7.3 to 8.2 nm) HDL particles. AT levels significantly increased (p<0.05) following therapy compared to placebo. There were no differences in the plasma total cholesterol, TG or LDL-C. AT therapy over 2 years had no significant effect on total (p=0.5), large (p=0.6), medium (p=0.8) and small (p=0.9) HDL particles compared to baseline or placebo. We conclude that high dose RRR-AT therapy over 2 years, in patients with CAD on statin therapy, does not negatively affect HDL subclasses. Thus the negative interaction previously proposed between antioxidant cocktail and statin therapy cannot be ascribed to AT. NIH RO1

Inactivation of penicillin-induced staphylococcal L-forms by human serum high density lipoprotein.

In penicillin-susceptible bacteria, penicillin causes growth of a small fraction of cells as wall-deficient forms if an appropriate osmoprotection is provided (unstable L-forms). A subfraction of human serum high density lipoprotein (HDL3) was shown to have the ability to inactivate unstable L-forms of Staphylococcus aureus. The active principle was distinguishable from the well-documented trypanosome lytic factor 1 with respect to density, size, and other properties. This L-form cytotoxicity therefore seems to represent a novel antimicrobial entity in human serum.

High-density lipoprotein subfractions in normolipidemic individuals without clinical atherosclerosis lipoprotein subfractions in an adult population

This study evaluated the serum concentrations of lipids, lipoproteins, apolipoproteins, and high-density lipoprotein (HDL) subfractions in Brazilian adults. We analyzed the distribution of lipids in HDL2 and HDL3 in a normolipidemic population without evidence of established cardiovascular disease (CVD). A total of 93 males and 92 females, healthy and normolipidemic, volunteered to be submitted to a clinical examination, a blood collection, and to answer a questionnaire aimed at determining signs and symptoms of atherosclerotic disease. Their fasting plasma lipid, lipoproteins, apolipoproteins, and the cholesterol and triglyceride concentrations in HDL2 and HDL3, isolated by microultracentrifugation, were determined by enzymatic-colorimetric methods. The interpercentile intervals (2.5-97.5) for the population were established as being 5-18 mg/dL in men and 4-28 mg/dL in women for HDL2 cholesterol (HDL2chol) and 1-57 mg/dL in men and 2-61 mg/dL in women for HDL3 cholesterol (HDL3chol). HDL2 triglyceride levels (HDL2Tg) in men were 1-26 mg/dL and in women 2-28 mg/dL; moreover, the HDL3 triglyceride (HDL3Tg) intervals were established as 4-46 mg/dL for both sexes. The determination of reference ranges for lipids in HDL subfractions in populations without clinical atherosclerosis, is an useful tool for metabolic, diagnostic, and therapeutic approaches. We determined the intervals for HDL2chol, HDL3chol, HDL2Tg, and HDL3Tg. There were variations with sex and/or age for HDL2chol, HDL3chol, and HDL2Tg in the studied population.

Apolipoprotein M: Progress in understanding its regulation and metabolic functions

ApoM is a novel apolipoprotein mainly present in high‐density lipoprotein (HDL). It belongs to the lipocalin protein superfamily and may bind a small but so far unknown lipophilic ligand. It is secreted without cleavage of its hydrophobic signal peptide, which probably anchors apoM in the phospholipid moiety of plasma lipoproteins. Recent studies suggest that apoM may affect HDL metabolism and have anti‐atherogenic functions. The subfraction of human HDL that contains apoM therefore protects LDL from oxidation and mediates cholesterol efflux more efficiently then HDL without apoM. In addition to hepatocytes, apoM is highly expressed in kidney proximal tubule cells. Recent data suggest that apoM is secreted into the pre‐urine from the tubule cells but is normally taken up again in a megalin‐dependent fashion. Further studies of mice with genetically modified apoM expression will be essential to unravel the potential roles of apoM in lipoprotein metabolism, atherosclerosis and kidney biology.