Form Of High-density Lipoprotein Research Articles

Curcumin Improves Cytokines and HDL in Diabetic Wistar Rats

In type 2 diabetes mellitus, lipid profile is often found in the form of high-density lipoprotein (HDL) decrease. TNFa and IL-6 are proinflammatory cytokines that are elevated in the blood in many patients with diabetes mellitus. Turmeric is one of the natural ingredients that has anti-cholesterol and anti-inflammatory effects through the work of its active substance, curcumin. This study aimed to evaluate the effect of turmeric extract combined with metformin on HDL, TNFa, and IL-6 levels in diabetic rats. Twenty-four Wistar rats were injected intraperitoneally with streptozotocin and niacinamide. The diabetic rats then were divided into four groups; control group, metformin group, turmeric group, and combination group. The dose of metformin and turmeric which were given, respectively, 45mg/KgBW/day and 200 mg/KgBW/day. While the combo group was given half dose of each. Blood serum was taken to examine the HDL, IL-6, and TNFa levels. The levels of HDL, IL-6, TNFa on diabetic rats that received the combination of metformin and turmeric extract (p0.05) were the best among of all the groups. This means that the combination of metformin and turmeric is better in increasing HDL, reducing IL-6, and TNFa levels better than metformin or turmeric extract alone.

Effects of the Melatonin and Vitamin E (Alpha-Tocopherol Acetate) as Antioxidants on Biochemical Blood Parameters, Lipid Profile, and Muscle Vitamin E Concentration in Awassi Lambs Fed a High-Energy Diet and Normal Diet.

Health specialists currently suggest low-cholesterol diets, suggesting that cholesterol in the form of high-density lipoprotein (HDL) reduces the risk of chronic atherosclerosis. The large volume of literature describes the biological roles of vitamin E and its application to preventing disease and improving the health and productive performances of farm animals. The present study aimed to evaluate the effects of vitamin E (Alpha-tocopherol acetate) supplementation and melatonin implants on biochemical blood, lipid profile and muscle vitamin E content of Awassi male lambs fed by a high and normal diet in Iraq. The lambs were divided into teen groups as control normal energy diet T1 (NED) T2 (HED) concentrated lamb fattening feed. Two levels of melatonin (18 and 36 mg implant) were applied to T3, T4, T5, and T6 treatment and 2 levels of Vitamin E (Alpha-tocopherol acetate) diet 200 mg/kg, 400 mg/kg to T7. T8. T9 and T10, respectively. Results from the present study indicate that Vitamin E 200, 400 mg/lamb/day and melatonin implantation 18 mg, 36 mg/lamb/day significantly (P<0.05) increased total protein in serum while decreasing globulin level, glucose concentration in serum, melatonin implantation 36 mg/lamb and vitamin E 400 mg/lamb/day recorded significantly (P<0.05). The same effect on decreasing cholesterol concentration in serum 42.6mg\dl, 40.5 mg\dl, respectively, compared to non-treated groups. Vitamin E 200 mg/kg/lamb recorded the lowest AST level in serum, 43.3. Lambs implanted with Melatonin 36 mg/lamb and fed a high-energy diet (T8) resulted in a significant decrease of serum ALT activity (P<0.05) in comparison to other treated groups 12.7 U/L was achieved. Lambs fed a normal energy diet with vitamin E 200 mg/kg/lamb (T4) exceeded other treated groups, decreasing ALT serum levels by 9.35 U/L. Interestingly, muscle vitamin E concentrations for lambs received 200, 400 mg/lamb/day on the 2nd, 7th and 14th days of the storage period, and fed high energy diet (T10) or normal energy diet (T5) were significantly higher compared to control group (T1, T6).

Characterization of type IV antifreeze gene in Nile tilapia (Oreochromis niloticus) and influence of cold and hot weather on its expression and some immune-related genes.

The aim of this work is to study the effect of the thermal stress of ambient temperature during winter and summer on the expression of type IV antifreeze gene (ANF IV) in different tissues of Nile tilapia (Oreochromis niloticus) as well as some immune-related genes. At first, genomic ANF IV gene was characterized from one fish; 124 amino acids were identified with 92.7% similarity with that on the gene bank. Expression of ANF IV and immune-related genes were done twice, once at the end of December (winter sample, temperature 14°C) and the other at August (summer sample, temperature 36°C). Assessment of ANF IV gene expression in different organs of fish was done; splenic mRNA was used for assessment of immune-related gene transcripts (CXCl2 chemokine, cc-chemokine, INF-3A, and MHC IIβ). Winter expression analysis of AFP IV in O. niloticus revealed significant upregulation of mRNA transcript levels in the intestine, gills, skin, spleen, liver, and brain with 324.03-, 170.06-, 107.63-, 97.61-, 94.35-, and 27.85-folds, respectively. Furthermore, upregulation in the gene was observed in some organs during summer: in the liver, gills, skin, intestine, and brain with lower levels compared with winter. The level of expression of immune-related genes in winter is significantly higher than summer in all assessed genes. Cc-chemokine gene expression was the most affected in both winter and summer. Variable expression profile of ANF IV in different organs and in different seasons together with its amino acid similarity of N-terminal and C-terminal with apolipoprotein (lipid binder) and form of high-density lipoprotein (HDL) suggests a different role for this protein which may be related to lipid metabolism.

Abstract 397: Aldehyde-Modified Hdl Has CD36-dependent Pro-Atherogenic Effects in Macrophages

The role of high density lipoproteins (HDL) in protecting against cardiovascular disease is compromised when HDL undergoes modification during conditions of oxidative stress; however, the mechanisms underlying these changes in HDL function are not well defined. Reactive aldehydes such as acrolein (a major component in cigarette smoke) or major products of lipid peroxidation such as 4-hydroxynonenal (HNE) or malondialdehyde (MDA) are known to oxidize HDL in cardiovascular disease. To test the hypothesis that modification of HDL with aldehydes impairs HDL’s athero-protective functions in macrophages, we first measured the ability of modified HDL to protect against foam cell formation. Cholesterol-loaded peritoneal macrophages isolated from wild-type C57Bl/J mice were incubated with native HDL, acrolein-modified HDL (acro-HDL), HNE-modified HDL (HNE-HDL) or MDA-modified HDL (MDA-HDL) for 24 h. Contrary to native HDL, oxidized forms of HDL were unable to prevent foam cell formation as shown by increased Oil red-O staining. Next, using a Boyden chamber assay, we demonstrated that acro- and MDA-HDL had impaired abilities to promote macrophage migration (64% and 67% of native HDL cell migration, respectively). Finally, using a secreted alkaline phosphatase reporter THP-1 cell-based assay, we determined that acro-HDL promotes activation of the pro-inflammatory NFkappaB pathway. Interestingly, immunoblot and quantitative RT-PCR analyses revealed that incubation of macrophages with acro- and MDA-HDL leads to increased expression of the pro-atherogenic receptor, cluster of differentiation 36 (CD36). Therefore, we repeated the foam cell formation and migration experiments using similar ligands, but this time, in CD36-null peritoneal macrophages. We found that both of these functions were dependent on CD36; however, the extent of the functional changes varied based on the type of oxidative modification present on HDL. In conclusion, modification of HDL with reactive aldehydes generates a particle that has pro-atherogenic effects in macrophages, many of which are dependent on CD36.

Abstract 393: Covalently Connected Apolipoprotein A-I Molecules Provide Support for the Double Belt Model in Reconstituted HDL

Because of its critical role in high density lipoprotein (HDL) metabolism, significant work has been devoted to studying apolipoprotein (apo)A-I structure in its lipid-bound state, particularly reconstituted forms of HDL (rHDL). Unfortunately, high-resolution structural studies have not been successful because of HDL heterogeneity and compositional dynamics. There is general agreement that apoA-I adopts an antiparallel arrangement on rHDL with the majority of the molecule conforming to the “double belt” model. However, less is understood about the locations of the N- and C-termini in these particles. The classic double belt predicts that, in a particle containing two molecules of apoA-I, all four termini are localized to one area of the particle. However, other models predict that they are far apart. To address this issue, we generated a series of covalently connected apoA-I dimers and tested their ability to generate rHDL particles. The dimeric (d)-apoA-I C-N mutant, locked the C-terminus of one apoA-I molecule to the N-terminus of a second molecule with three intervening Ala residues. Linked by strategically introduced cysteine residues, the d-apoA-I C-C , and d-apoA-I N-N mutants lock the C-termini and the N-termini of two apoA-I molecules together, respectively. When all three of these mutants were reconstituted into rHDL particles using sodium cholate and synthetic phospholipids, the resulting particles closely resembled those generated with wild-type (WT) apoA-I. This indicates a ring-like structure predicted by the double belt. However, when testing the ability of these mutants to spontaneously clear lipid, we found that both the N- and C- termini required the ability to separate and move independently to form normal particles. Native gel analysis showed the dimerized lipid-free mutants present as a tight homogenous band compared to wild-type which runs as a mixture of oligomers. We are currently evaluating if the homogeneity of these mutants can allow for more facile crystallization of full-length apoA-I. Further studies with these stabilized mutants should help us understand the structural basis underlying HDL’s diverse functions, which is essential for developing HDL-based therapies for cardiovascular disease.

Abstract 450: Modification of HDL by Reactive Aldehydes Impairs HDL's Athero-protective Functions

High density lipoproteins (HDL) are athero-protective particles that promote the removal of excess cholesterol from lipid-loaded macrophages and stimulate their migration in order to protect against foam cell formation, a precursor to atherosclerotic plaque build-up. Recently, studies have shown that oxidative modification of HDL prevents HDL from protecting against atherosclerosis; however, the exact mechanisms by which this occurs are not well defined. We hypothesize that oxidative modification of HDL by reactive aldehydes such as acrolein (a major component of cigarette smoke) and 4-hydroxynonenal (HNE; a product of lipid peroxidation) impairs HDL’s athero-protective effects in macrophages. We tested our hypothesis using three different assays. First, we determined that modified forms of HDL upregulate mRNA levels of pro-atherogenic scavenger receptors such as cluster of differentiation 36 (CD36), a known oxidized LDL receptor. Incubation of macrophages with native HDL did not exert similar effects. Second, we tested the ability of oxidized HDL to prevent foam cell formation. Peritoneal macrophages isolated from WT C57Bl/J mice were cholesterol-loaded and incubated with native HDL, acrolein-modified HDL (acro-HDL), or HNE-modified HDL (HNE-HDL). Oil Red-O staining demonstrated that 24% of macrophages had foam cell formation upon incubation with native HDL, whereas 61% and 49% foam cell formation was observed for acro- and HNE-HDL, respectively. Preliminary data suggests this may be CD36-dependent. Finally, using a Boyden chamber assay, we demonstrated that both acro- and HNE-HDL, but not native HDL, had an impaired ability to promote macrophage migration (43% and 72% of HDL cell migration levels, respectively). We determined that the inability of acro- and/or HNE-HDL to stimulate macrophage migration may be due to an impaired ability of these modified lipoproteins to activate the PI3K pathway, as shown by decreased levels of phosphorylated protein kinase B (Akt). In conclusion, we have identified three independent mechanisms by which modification of HDL with acrolein or HNE impairs HDL’s cardio-protective effects and, instead, generates a particle that promotes pathways that lead to atherosclerosis.

Tangier Disease

Tangier disease is one of the most severe forms of familial high-density lipoprotein (HDL) deficiency. Since its discovery it has been diagnosed in about 100 patients and is characterized by severe plasma deficiency or absence of HDL, apolipoprotein A-I (apoA-I, the major HDL apolipoprotein) and by accumulation of cholesteryl esters in many tissues throughout the body. The biochemical signs of this condition are plasma HDL concentrations less than 5 mg/dL, low total plasma cholesterol (below 150 mg/dL), and normal or high plasma triglycerides. Tangier disease is caused by mutations in the 'ATP-Binding Cassette transporter A1' (ABCA1) gene, which encodes the membrane transporter ABCA1. This transporter plays a key role in the first step of reverse cholesterol transport, through which the efflux of free cholesterol from peripheral cells is transferred to lipid-poor apoA-I. The Tangier disease clinical phenotype is inherited as an autosomal recessive trait, the biochemical phenotype is inherited as an autosomal co-dominant trait. Nearly all the children affected by Tangier disease were identified on the basis of large, yellow-orange tonsils, while half of the adult patients affected by Tangier disease came to medical attention because of symptoms of neuropathy. Diagnosis in the remaining subjects was related to the clinical features of hepatomegaly, splenomegaly, premature myocardial infarction (about 30% of Tangier disease cases) or stroke, thrombocytopenia, anemia, gastrointestinal disorders, corneal opacities, hypocholesterolemia, low HDL cholesterol, or following a familial screening of Tangier patients. To date there is no specific treatment for Tangier disease. Old and recently designed drugs, known to increase HDL levels, have been shown to be ineffective in Tangier patients. The possible and more realistic therapeutic strategy should be designed to obtain a selective increase of mature HDL concentration to restore cholesterol efflux. Recently designed drugs like the cholesteryl ester transfer protein (CETP) inhibitors dalcetrapib and anacetrapib and reconstituted forms of HDL could be considered until the development of gene therapy.

Antacid co-encapsulated polyester nanoparticles for peroral delivery of insulin: Development, pharmacokinetics, biodistribution and pharmacodynamics

The in vitro/in vivo characterization of antacid-insulin co-encapsulated poly(lactide-co-glycolide) (PLGA) nanoparticles is presented here. The optimized nanoparticle composition has 1% surfactant (didodecyl dimethylammonium bromide) and 2% antacid (magnesium hydroxide or zinc carbonate) in the size range ~136-143nm with ~81-85% entrapment of insulin at a 4% (w/w) initial load to that of polymer. Molecular characterization using circular dichroism, fluorescence and Fourier transform infrared spectroscopy showed that the structural integrity of insulin was maintained during formulation. Furthermore, the encapsulated insulin was well protected under in vitro simulated gastric and intestinal fluids. Nanoparticle insulin results in six fold increase in oral bioavailability to that of plain insulin in healthy rats. In diabetic rats, a 120 IU/kg oral dose of insulin nanoparticles achieved an equivalent blood glucose lowering effect to a 20 IU/kg subcutaneous (sc) dose of insulin solution, the nadir in blood glucose concentration occurring 24h and 1h post-administration, respectively. Both sc insulin and oral nanoparticle insulin partially attenuated hyperglycemia-induced inflammation caused by tumor necrosis factor α, but not by interleukin-6 or C-reactive protein; on the other hand, subcutaneous insulin was found to be more effective on lipid profile measured in the form of high density lipoprotein, cholesterol and triglyceride. Successful oral insulin could be beneficial in type II complications.

ETC-216 for coronary artery disease

Introduction: Increasing attention has focused on the role of high-density lipoprotein function as a target for cardiprotection. Apolipoprotein A-IMilano (AIM) involves a single amino-acid mutation of the major wild-type protein carried on high-density lipoprotein (HDL) particles. Early evidence of beneficial activities of AIM has stimulated support in its development as a potential therapy to reduce cardiovascular risk.Areas covered: The importance of HDL as a target and early data supporting the beneficial effects of AIM are reviewed. All clinical studies of AIM found in PubMed are reviewed.Expert opinion: ETC-216 represents a lipid-deplete form of HDL containing recombinant AIM. While early evidence suggests that administration of ETC-216 promotes rapid regression of coronary atherosclerosis, bringing this compound to clinical practice will require further trials that evaluate its impact on cardiovascular events.

MODULATION OF OXIDIZED LDL MEDIATED CELLULAR RESPONSES BY DIFFERENT FORMS OF HDL: PP.2.69

Objectives: Initial phase of atherosclerosis involves influx of low density lipoprotein cholesterol (LDL- C) accumulation, generation of reactive oxygen species and oxidative modulation of LDL-C. The effect of different form of high density lipoprotein (HDL) was studied on oxidized-LDL induced cellular responses in differentiated human monocytic leukemic cell line(THP-1 cells) and monocytes of patients with normal LDL and high LDL. Expression of pro-apoptotic and anti-apoptotic molecules were evaluated at mRNA and protein level. Designs and Methods: Different forms of lipoproteins were separated by ultracentrifugation from normal human plasma. Apolipoprotein A-1 (ApoA-1) was purified and used for preparation of normal high density lipoprotein (nHDL). Differentiated cells (THP-1 cells/cells from hypercholesteremic patients and controls differentiated with 100 nm PMA) were either cultured in presence of oxidized-LDL (Ox-LDL) or preincubated with normal HDL(nHDL) or reconstituted HDL (rHDL-prepared by method of Jonas) or oxidized HDL (Ox-HDL) followed by coincubation with Ox-LDL. Expression of pro-apoptotic (Bax and caspase-3) and anti-apoptotic molecules (Bcl-2 and Bcl-Xl) were assessed. Results: DNA fragmentation analysis revealed that Ox-LDL induced apoptosis in differentiated monocytes. The nHDL/rHDL inhibited Ox-LDL induced apoptosis in differentiated monocytes while Ox-LDL did not show this phenomenon. Studies from differentiated monocytes from hyperLDL patients revealed a higher percentage of apoptotic cells. OxLDL could result in detachment of differentiated THP-1 cells from surface of plate. This phenomenon was reduced by culturing with nHDL/rHDL. An elevated expression of Bax was found in differentiated cells cultured in presence of Ox-LDL while expression of Bcl-2 and Bcl-Xl was downregulated. Expression of Bcl-2 and Bcl-Xl was upregulated when preincubation with nHDL/rHDL was done. Expression of scavenger receptor LOX-1 was decreased in this group. Conclusions: Ox-LDL induced apoptosis in differentiated monocytes was downregulated in presence of nHDL/rHDL but not with OxHDL.Ox-LDL mediates apoptosis through scavenger receptor in differentiated THP- cells and monocytes from patients with high LDL.

Comparison of Synthetic High Density Lipoprotein (HDL) Contrast Agents for MR Imaging of Atherosclerosis

Determining arterial macrophage expression is an important goal in the molecular imaging of atherosclerosis. Here, we compare the efficacy of two synthetic, high density lipoprotein (HDL) based contrast agents for magnetic resonance imaging (MRI) of macrophage burden. Each form of HDL was labeled with gadolinium and rhodamine to allow MRI and fluorescence microscopy. Either the 37 or 18 amino acid peptide replaced the apolipoprotein A-I in these agents, which were termed 37pA-Gd or 18A-Gd. The diameters of 37pA-Gd and 18A-Gd are 7.6 and 8.0 nm, respectively, while the longitudinal relaxivities are 9.8 and 10.0 (mM s)(-1). 37pA has better lipid binding properties. In vitro tests with J774A.1 macrophages proved the particles possessed the functionality of HDL by eliciting cholesterol efflux and were taken up in a receptor-like fashion by the cells. Both agents produced enhancements in atherosclerotic plaques of apolipoprotein E knockout mice of approximately 90% (n = 7 per agent) and are macrophage specific as evidenced by confocal microscopy on aortic sections. The half-lives of 37pA-Gd and 18A-Gd are 2.6 and 2.1 h, respectively. Despite the more favorable lipid interactions of 37pA, both agents gave similar, excellent contrast for the detection of atherosclerotic macrophages using MRI.

Remodeling of Human Plasma Lipoproteins by Detergent Perturbation

Detergent perturbation, the treatment of total human plasma lipoproteins (TLP) with sodium cholate and its subsequent removal, has been used to study lipoprotein dynamics and stability. At physiological TLP concentrations, detergent perturbation converts low-density lipoproteins (LDL) and high-density lipoproteins (HDL) to higher-particle weight species with the concomitant release of apo A-I but not apo A-II as a lipid-poor species. Detergent perturbation of isolated HDL also releases lipid-poor apo A-I and forms larger HDL species, whereas detergent perturbation of an isolated LDL has no effect on its size. A model is presented in which detergent perturbation induces transfer of PC from metastable HDL and LDL to mixed micelles with sodium cholate. The remaining LDL and HDL are unstable because of the loss of their surface components, phospholipid and/or apo A-I, and fuse to give larger LDL and HDL particles. These effects on HDL, i.e., PC transfer, apo A-I dissociation, and particle fusion, emulate the activity of human plasma phospholipid transfer protein. Thus, detergent perturbation is a new and potentially powerful method for determining lipoprotein stability, studying the mechanisms for remodeling of plasma lipoproteins, and preparing new forms of HDL and LDL with unique interactions with lipoprotein transporters and receptors.

A Mass Spectrometric Determination of the Conformation of Dimeric Apolipoprotein A-I in Discoidal High Density Lipoproteins

Discoidal forms of high density lipoproteins (HDL) are critical intermediates between lipid-poor apolipoprotein A-I (apo A-I), the major protein constituent of HDL, and the mature spherical forms that comprise the bulk of circulating particles. Thus, many studies have focused on understanding apoA-I structure in discs reconstituted in vitro. Recent theoretical and experimental work supports a "belt" model for apoA-I in which repeating amphipathic helical domains run parallel to the plane of the lipid disc. However, disc-associated apoA-I can adopt several tertiary arrangements that are consistent with a belt orientation. To distinguish among these, we cross-linked near-neighbor Lys groups in homogeneous 96 A discs containing exactly two molecules of apoA-I. After delipidation and tryptic digestion, mass spectrometry was used to identify 9 intermolecular and 11 intramolecular cross-links. The cross-linking pattern strongly suggests a "double-belt" molecular arrangement for apoA-I in which two apoA-I molecules wrap around the lipid bilayer disc forming two stacked rings in an antiparallel orientation with helix 5 of each apoA-I in juxtaposition (LL5/5 orientation). The data also suggests the presence of an additional double-belt orientation with a shifted helical registry (LL5/2 orientation). Furthermore, a 78 A particle with two molecules of apoA-I fit a similar double-belt motif with evidence for conformational changes in the N-terminus and the region near helix 5. A comparison of this work to a previous study is suggestive that a third molecule of apoA-I can form a hairpin in larger particles containing three molecules of apoA-I.

Lipoprotein receptors, plasma cholesterol metabolism, and the regulation of cellular free cholesterol concentration.

Classical concepts of the regulation of plasma cholesterol levels involve roles for the "forward" delivery of low density lipoprotein (LDL) cholesterol from the liver to the peripheral tissues, mediated by the LDL receptor, and a "reverse" delivery of cholesterol in the form of high density lipoprotein (HDL) from the peripheral tissues to the liver. Candidate receptors for HDL in peripheral tissues and for chylomicrons in the liver have more recently been described, and a receptor of uncertain function recognizing chemically modified LDL has also been identified. The activities of all the well-characterized lipoprotein receptors, as well of major catalytic factors in plasma that regulate cholesterol esterification and cholesteryl ester transfer between lipoproteins, reflect the need to maintain plasma membrane free cholesterol level, and its direct and indirect effects within the membrane, within well-defined limits.

"A" esterases and their role in regulating the toxicity of organophosphates.

Esterases which can hydrolyse organophosphates without being inhibited by them are termed "A" esterases. Using paraoxon and pirimiphos-methyl oxon as substrates, high "A" esterase activity is found in the liver and plasma or serum of a range of mammalian species. In a study of serum "A" esterases of sheep and humans, over 80% of the activity separated into the high density lipoprotein (HDL) fraction following ultracentrifugation. When HDL fractions from sheep serum were run on Sepharose gel columns, most of the paraoxonase activity separated as a single peak of estimated molecular weight 360,000, which corresponds to that of HDL2 of humans. During the course of purification of "A" esterases by three different column procedures, contrasting esterase elution profiles were obtained with organophosphate and pyrethroid substrates. This was strong evidence for the existence of multiple forms of HDL "A" esterases. Levels of "A" esterase activity in plasma and liver of birds were much lower than those of mammals. This appears to be the main reason why birds are much more susceptible than mammals to organophosphates such as pirimiphos-methyl and diazinon which form active oxons that are good substrates for mammalian "A" esterases. No "A" esterase was detected in strains of rust red flour beetle (Tribolium castaneum) which were resistant to organophosphates. Similar observations have been made with strains of other insects resistant to organophosphates, raising the question to what extent esterases of this type are present in insects.

Porcine follicular fluid protein(s) inhibit rat ovary granulosa cell steroidogenesis

We examined the effect of an isolated fraction of follicular fluid, termed follicle regulatory protein, on the production of estrogen, progesterone, and 20α-dihydroprogesterone by cultured rat ovarian granulosa cells. We found a stepwise decrease in estrogen production with an increasing concentration of follicle regulatory protein (9 to 300 μg/ml) when the cells were stimulated by 6.25 ng/ml of ovine follicle-stimulating hormone, but the addition of a saturating dose of ovine follicle-stimulating hormone (50 ng/ml) prevented the inhibitory effect of follicle regulatory protein on estrogen production. Follicle regulatory protein inhibited progesterone production at both low and saturating levels of ovine follicle-stimulating hormone. We examined whether cholesterol substrate in the form of high-density lipoprotein or low-density lipoprotein could overcome the follicle regulatory protein inhibition of progesterone and 20α-dihydroprogesterone production stimulated by follicle-stimulating hormone. At all concentrations of high-density and low-density lipoprotein, 75 μg/ml of follicle regulatory protein inhibited progesterone production by 80% to 90%. In the case of 20α-dihydroprogesterone, the presence of high-density and low-density lipoprotein accentuated the inhibitory effect of follicle regulatory protein. These results demonstrate that the effects of follicle regulatory protein on ovarian steroidogenesis depend on: (1) the concentration of gonadotropin, (2) the concentration of follicle regulatory protein, and (3) the availability of cholesterol substrate.

Isolation of high density lipoproteins from rat intestinal epithelial cells.

Previous studies have defined forms of high density lipoproteins (HDL) in rat mesenteric lymph, suggesting that they have a secretory origin. This study describes the isolation and characterization of intestinal intracellular HDL. Two preparations were made as follows: (a) Rat enterocytes were isolated and a Golgi organelle fraction was prepared. (b) Cell homogenates were subjected to nitrogen cavitation and a cytoplasmic fraction was prepared. Lipoproteins were isolated from both preparations by sequential ultracentrifugation. When the HDL fraction (1.07-1.21 g/ml) was subjected to isopyknic density gradient ultracentrifugation, a peak of apoproteins A-I and B (apoA-I and apoB, respectively) was found at a density of 1.11-1.14 g/ml. Electron microscopy of the fraction showed spherical particles ranging in size from 6 to 13 nm. Immunoelectrophoresis revealed a precipitin arc in the alpha region against apoA-I which extended into the pre-beta region where a precipitin arc against apoB was also seen. ApoB antisera depleted the pre-beta particles whereas the alpha migrating particles remained. Lipid analysis of the whole HDL fraction revealed phospholipid, cholesteryl ester, and triglyceride as the major lipids. [3H]leucine was then administered into the duodenum and a radiolabeled intracellular HDL fraction was isolated. The newly synthesized apoproteins of the HDL fraction, as determined by gel electrophoresis, were apoB, apoA-I, and apolipoprotein A-IV (ApoA-IV). Immunoprecipitation of the apoB particles revealed apoA-I and apoA-IV in the supernatant. These data demonstrate that there are at least two intracellular intestinal forms of HDL particles, one of which contains apoB. The other particle contains apoA-I and apoA-IV, has alpha mobility, is spherical, and resembles a particle found in the lymph.

Partial purification and properties of sheep serum ‘A’-esterases

Using paraoxon and pirimiphos-methyl as substrates, much of the ‘A’-esterase activity of sheep serum was found to be in the high-density lipoprotein (HDL) fraction. A method was developed for the partial purification of ‘A’-esterases by the preparation of a lipoprotein fraction, followed by preparative polyacrylamide gel electrophoresis. The properties of the partially purified preparations of ‘A’-esterase were studied. Although four different preparations all contained a major protein unit, which resembled the core protein of HDL, there was evidence of differences between preparations with regard to substrate specificity, suggesting the existence of multiple enzyme forms. Gel filtration of serum samples indicated that paraoxonase activity is expressed by proteins with mol. wts > 200,000, strongly suggesting that the ‘A’-esterase activity of the lipoprotein fraction is present in one or more forms of HDL 2. The dependence of ‘A’-esterase activity upon Ca 2+ and the problem of esterase classification are discussed.