Is the oxidation of high-density lipoprotein lipids different than the oxidation of low-density lipoprotein lipids?

This article gives detailed insight into the kinetics of high-density lipoprotein (HDL) oxidation catalyzed by azobis(2-amidinopropane).dihydrochloride (ABAP) or by copper. ABAP initialized oxidation of human HDL 3-4 times faster than non-human primate HDL with a similar composition. The oxidizability of non-human primate HDL was 1000 times lower than the oxidizability calculated from rate constants derived from liposome oxidation, suggesting that there is a slow step in HDL oxidation not present in liposomes. Saturable binding of copper to HDL was a significant feature of copper-catalyzed oxidation. Binding constants (K(m)) for non-human primate HDL were 2-3-fold lower than those for human HDL. Copper-catalyzed oxidation of non-human primate HDL was slower than that of human HDL, but human HDL(2) and HDL(3) oxidized at about the same rate. Overall, the kinetics describing the oxidation of HDL were mechanistically similar to those reported for LDL, suggesting that HDL lipids were as easily oxidized as LDL lipids and that HDL will be easily oxidized in vivo when exposed to agents that oxidize LDL.     

What is the origin of the lipids of potato tuber plasmalemma?

Plasmalemma isolated from potato tuber cells was prepared according to a flotation method in a sucrose density gradient. When incubated in vitro with various radioactive lipid precursors, the plasmalemma fragments were unable to synthesize any lipid. When labelled lipid precursors ([¹⁴C]acetate of [³²P]phosphate) were furnished to potato slices, radioactive fatty acids or phospholipids were integrated into plasmalemma lipids. When incubated in a suspension of [³²P]liposomes, plasmalemma fragments accepted labelled lipids from the suspension; this exchange process was markedly stimulated by the phospholipid exchange protein prepared from the potato cytoplasmic supernatant. These results suggest that potato tuber plasmalemma is dependent on other cellular fractions for lipid synthesis and exchange.     

Incorporation of lipids into cellular membranes—A spin-label assay

A spin-label method is described for the quantitative assay of lipid incorporation into biological membranes, using computer difference spectroscopy. The incorporation of spin-labeled sphingomyelin into synaptic plasma membranes from calf brain has been studied as a function of sonication time. The spin-label ESR spectra are able to distinguish labeled sphingomyelin which is integrated into the membrane, from the unincorporated label, even if the latter cosediments with the membranes. Spectral subtraction has been used to quantitate the degree of incorporation. The percentage of incorporation increases with increasing sonication time and also with incubation after sonication. The extent of degradation of tritium-labeled sphingomyelin by the neutral sphingomyelinase present in the membrane closely correlates with the dependence of the incorporation of the spin-labeled sphingomyelin on sonication time. This illustrates the utility of the method in the study of membrane-bound, lipid-metabolizing enzymes.     

Effects of membrane lipids on the activity and processivity of purified γ-secretase

The 19-transmembrane multisubunit γ-secretase complex generates the amyloid β-peptide (Aβ) of Alzheimer's disease (AD) by intramembrane proteolysis of the β-amyloid precursor protein (APP). Despite substantial advances in elucidating how this protein complex functions, the effect of the local membrane lipid microenvironment on γ-secretase cleavage of substrates is still poorly understood. Using detergent-free proteoliposomes to reconstitute purified human γ-secretase, we examined the effects of fatty acyl (FA) chain length, saturation and double-bond isomerization, and membrane lipid polar headgroups on γ-secretase function. We analyzed γ-secretase activity and processivity [i.e., sequential cleavages in the APP transmembrane domain that convert longer Aβ species (e.g., Aβ(46)) into shorter ones (e.g., Aβ(40))] by quantifying the APP intracellular domain (AICD) and various Aβ peptides, including via a bicine/urea gel system that detects multiple Aβ lengths. These assays revealed several trends. (1) Switching from a cis to a trans isomer of a monounsaturated FA chain in phosphatidylcholine (PC) increased γ-activity, did not affect Aβ(42):Aβ(40) ratios, but decreased the ratio of long (≥42) versus short (≤41) Aβ peptides. (2) Increasing the FA carbon chain length (14, 16, 18, and 20) increased γ-activity, reduced longer Aβ species, and reduced the Aβ(42):Aβ(40) ratio. (3) Shifting the position of the double bond in 18:1(Δ9-cis) PC to the Δ6 position substantially reduced activity. (4) Gangliosides increased γ-activity but decreased processivity, thus elevating the Aβ(42):Aβ(40) ratio. (5) Phosphatidylserine decreased γ-activity but increased processivity. (6) Phosphatidylinositol strongly inhibited γ-activity. Overall, our results show that subtle changes in membrane lipid composition can greatly influence γ-secretase activity and processivity, suggesting that relatively small changes in lipid membrane composition may affect the risk of AD at least as much as presenilin or APP mutations do.     

Biochemical study of the adepidermal granules of Urodela and Anura—Especially on lipids

Summary The lipids of the adepidermal granules of Hynobius tokyoensis and Rana japonica were analysed qualitatively. To extract the lipids from the adepidermal granules, the epidermis of the larvae of both animals were removed from the body. Then the granules were extracted from the surface of the basal lamina by means of wiping with the cotton balls containing the solvent. It was ascertained with the electron microscope whether the adepidermal granules were extracted by the cotton balls or not.By the analysis which was made with thin layer chromatography, the same kind of phospholipids or neutral lipids were detected from the extracts of both R. Japonica and H. tokyoensis. Moreover, four spots which seemed to contain some sialic acids were detected from the both extracts.     

Bacterial species selective toxicity of two isomeric α/β-peptides: Role of membrane lipids

We have studied how membrane interactions of two synthetic cationic antimicrobial peptides with alternating α- and β-amino acid residues (“α/β-peptides”) impact toxicity to different prokaryotes. Electron microscopic examination of thin sections of Escherichia coli and of Bacillus subtilis exposed to these two α/β-peptides reveals different structural changes in the membranes of these bacteria. These two peptides also have very different effects on the morphology of liposomes composed of phosphatidylethanolamine and phosphatidylglycerol in a 2:1 molar ratio. Freeze fracture electron microscopy indicates that with this lipid mixture, α/β-peptide I induces the formation of a sponge phase. ³¹P NMR and X-ray diffraction are consistent with this conclusion. In contrast, with α/β-peptide II and this same lipid mixture, a lamellar phase is maintained, but with a drastically reduced d-spacing. α/β-Peptide II is more lytic to liposomes composed of these lipids than is I. These findings are consistent with the greater toxicity of α/β-peptide II, relative to α/β-peptide I, to E. coli, a bacterium having a high content of phosphatidylethanolamine. In contrast, both α/β-peptides display similar toxicity toward B. subtilis, in accord with the greater anionic lipid composition in its membrane. This work shows that variations in the selectivity of these peptidic antimicrobial peptides toward different strains of bacteria can be partly determined by the lipid composition of the bacterial cell membrane.     

Interaction of methionine–enkephalins with raft-forming lipids: monolayers and BAM experiments

Enkephalins (Tyr-Gly-Gly-Phe-Met/Leu) are opioid peptides with proven antinociceptive action in organism. They interact with opioid receptors belonging to G-protein coupled receptor superfamily. It is known that these receptors are located preferably in membrane rafts composed mainly of sphingomyelin (Sm), cholesterol (Cho), and phosphatidylcholine. In the present work, using Langmuir’s monolayer technique in combination with Wilhelmy’s method for measuring the surface pressure, the interaction of synthetic methionine–enkephalin and its amidated derivative with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), Sm, and Cho, as well as with their double and triple mixtures, was studied. From the pressure/area isotherms measured, the compressional moduli of the lipids and lipid–peptide monolayers were determined. Our results showed that the addition of the synthetic enkephalins to the monolayers studied led to change in the lipid monolayers characteristics, which was more evident in enkephalinamide case. In addition, using Brewster angle microscopy (BAM), the surface morphology of the lipid monolayers, before and after the injection of both enkephalins, was determined. The BAM images showed an increase in surface density of the mixed surface lipids/enkephalins films, especially with double and triple component lipid mixtures. This effect was more pronounced for the enkephalinamide as well. These observations showed that there was an interaction between the peptides and the raft-forming lipids, which was stronger for the amidated peptide, suggesting a difference in folding of both enkephalins. Our research demonstrates the potential of lipid monolayers for elegant and simple membrane models to study lipid–peptide interactions at the plane of biomembranes.     

Are natural lipids UV-screening agents?

Summary Isolated lipids from Deinococcus radiodurans were reconstituted at final concentrations of 1 mg/ml into dioleoyl phosphatidyl choline (DOPC) vesicles and assayed for the ability to protect cells of Escherichia coli against killing by UV light (254 nm). Values of D₃₇ (UV dose required to reduce the number of surviving cells to 37% of the original number) were calculated from killing curves. E. coli was afforded the greatest protection with an individual lipid, identified as vitamin MK8 (D₃₇=310 J//m², compared to D₃₇=67 J/m² for E. coli irradiated in the presence of DOPC alone). Liposome-mediated protection was dependent on UV₂₅₄ absorbance and not on turbidity-related light-scattering. BOth vitamin MK8 from D. radiodurans and vitamin K₁, which is available commercially, showed a similar degree of UV₂₅₄-protection for E. coli. The UV-protective properties of vitamin K₁ were also investigated on mammalian cells in comparison with other natural lipids and known sunscreens. Survival curves were obtained for mouse fibroblast (L) cells irradiated at UV₂₅₄ in the absence or presence of DOPC liposomes into which were incorporated various natural lipids or standard sunscreen ingredients, all at final concentrations of 1 mg/ml. Experimentally determined values of D₃₇ were as follows: Vitamin K₁, 73 J/m²; \-carotene, 44 J/m₂; -tocopherol, 20 J/m₂; sulisobenzone, 156 J/m²; p-aminobenzoic acid (PABA), 113 J/m²; benzophenone, 80 J/m²; oxybenzone, 61 J/m² and DOPC alone. 23 J/m². Vitamin K₁, the most protective lipid tested, was also compared with PABA and oxybenzone (all at concentrations of 20 mg/ml; applied topicall) for its ability to protec Skh-hairless mice from UV₂₅₄-induced erythema, yielding a UV₂₅₄ protection factor of 3.5. In addition, vitamin K₁ (at 100 mg/ml) was able to provide hairless mice with a small degree of UVB protection, as indicated by an experimentally determined Solar Protection Factor of 1.5–2.0. Although it is concluded that vitamin K is not likely to account for the extraordinarily high degree of UV-resistance of D. radiodurans, vitamin K does show characteristics worthy of its consideration as a UV-screening agent. Offprint requests to: R. Anderson     

Tryptophan interactions of gramicidin A′ channels in lipids: a time-resolved fluorescence study

The evolution of the incorporation of cation transport channels into lysolecithin micelles by gramicidin A was followed by measuring the ns time-resolved fluorescence of the tryptophan residues. In all samples, the tryptophan fluorescence could be resolved into three exponentially decaying components. The three decay times ranged from 6 to 8 ns, 1.8 to 3 ns, and 0.3 to 0.8 ns, depending on the emission wavelength. The fractional fluorescence of each component changed with incubation time. The long lifetime component had a reduced contribution to the total fluorescence while the short decay time component increased. The fluorescence spectra could be resolved into three distinct fluorescent components having maxima at 340 nm, 330 nm and 323 nm after 90 min of incubation, and 335 nm, 325 nm and 320 nm after 24 h of incubation. These maxima were, respectively, associated with the long, medium and short decay components. The fluorescence decay behaviour was interpreted as representing three families of tryptophans, the short lifetime component being due to a stacking interaction between tryptophan residues. The variation with incubation time suggests a two-step process in the channel-lipid organization. The first is associated with the conformational change of the polypeptide as it takes up a left-handed helical head-to-head dimer structure in the lipid. The second step is proposed to involve changes originating from membrane assembly and intermolecular interactions between channels as they form hexameric clusters.     

Does dietary magnesium modulate blood lipids?

In a randomized, single-blind, controlled study (400 patients aged 25-63 yr; 374 males, 26 females), 206 subjects were administered a magnesium-rich diet, and 194 subjects their usual diet, for 6 wk. Age, sex, body weight, hypertension, hyperlipidemia, smoking, obesity, diuretic therapy, and diabetes were comparable between the two groups, as were laboratory data at entry to the study. Intervention-group A received a significantly higher amount of dietary magnesium and potassium compared to group B, which received its usual diet. After 6 wk, there was a significant fall in total serum cholesterol (228.5 +/- 46.2 mg/dL), LDL cholesterol 146.5 +/- 75.5 mg/dL), and triglyceride (143.8 +/- 40.5 mg/dL) in group A compared to serum cholesterol (242.5 +/- 58.2 mg/dL), LDL cholesterol (157.0 +/- 78.4 mg/dL), and triglyceride (156.5 +/- 60.0 mg/dL) at entry to study, but no such changes in group-B subjects. HDL cholesterol showed a marginal mean decrease of 0.8 mg/dL in group B and a 2.5 mg/dL increase in group A. The changes in blood lipids were consistent with an increased intake of magnesium and with a rise in serum levels. Although a general blood-lipid-reducing effect of such a diet cannot be excluded, it is possible that dietary magnesium may have contributed to the reduction of total serum cholesterol, LDL cholesterol, and triglyceride, and the marginal rise in HDL cholesterol. More studies with longer follow-up periods are needed to confirm this observation.     

Separation and identification of lipids and fatty acids of the marine algaFucus vesiculosus by TLC and GC—MS

Ten fractions of polar and nonpolar lipids (phospholipids, glyeolipids and neutral lipids) were isolated from the brown marine algaFucus vesiculosus by TLC. In each fraction the quality and quantity of fatty acids were determined by GC—MS.F. vesiculosus is another type of marine alga suitable as a source of polyene fatty acids.     

Role of membrane lipids in the mechanism of bacterial species selective toxicity by two α/β-antimicrobial peptides

We have previously shown that two synthetic antimicrobial peptides with alternating α- and β-amino acid residues, designated simply as α/β-peptide I and α/β-peptide II, had toxicity toward bacteria and affected the morphology of bacterial membranes in a manner that correlated with their effects on liposomes with lipid composition similar to those of the bacteria. In the present study we account for the weak effects of α/β-peptide I on liposomes or bacteria whose membranes are enriched in phosphatidylethanolamine (PE) and why such membranes are particularly susceptible to damage by α/β-peptide II. The α/β-peptide II has marked effects on unilamellar vesicles enriched in PE causing vesicle aggregation and loss of their internal aqueous contents. The molecular basis of these effects is the ability of α/β-peptide II to induce phase segregation of anionic and zwitterionic lipids as shown by fluorescence and differential scanning calorimetry. This phase separation could result in the formation of defects through which polar materials could pass across the membrane as well as form a PE-rich membrane domain that would not be a stable bilayer. α/β-Peptide II is more effective in this regard because, unlike α/β-peptide I, it has a string of two or three adjacent cationic residues that can interact with anionic lipids. Although α/β-peptide I can destroy membrane barriers by converting lamellar to non-lamellar structures, it does so only weakly with unilamellar vesicles or with bacteria because it is not as efficient in the aggregation of these membranes leading to the bilayer-bilayer contacts required for this phase conversion. This study provides further understanding of why α/β-peptide II is more toxic to micro-organisms with a high PE content in their membrane as well as for the lack of toxicity of α/β-peptide I with these cells, emphasizing the potential importance of the lipid composition of the cell surface in determining selective toxicity of anti-microbial agents.     

The beneficial effects of α-cyclodextrin on blood lipids and weight loss in healthy humans

α‐Cyclodextrin (α‐CD) is a soluble fiber derived from corn. It has previously been reported that early intervention with Mirafit fbcx, a trademarked name for α‐CD, has beneficial effects on weight management in obese individuals with type 2 diabetes, and that it preferentially reduces blood levels of saturated and trans fats in the LDL receptor knockout mice. The current investigation involves overweight but not obese nondiabetic individuals and was intended to confirm the effects of α‐CD on both weight management and improving blood lipid levels. Forty‐one healthy adults (age: 41.4 ± 13.6 years) participated in this 2‐month, double‐blinded, crossover study. In 28 compliant participants (8 males and 20 females), when the active phase was compared to the control phase, there were significant decreases in body weight (?0.4 ± 0.2 kg, P < 0.05), serum total cholesterol (mean ± s.e.m., ?0.295 ± 0.10 mmol/l, 5.3%, P < 0.02) and low‐density lipoprotein (LDL) cholesterol (?0.23 ± 0.11 mmol/l, ?6.7%, P < 0.05). Apolipoprotein B (Apo B) (?0.0404 ± 0.02 g/l, ?5.6%, P = 0.06) and insulin levels also decreased by 9.5% (?0.16 ± 0.08 pmol/l, P = 0.06) while blood glucose and leptin levels did not change. These results suggest that α‐CD exerts its beneficial health effects on body weight and blood lipid profile in healthy nonobese individuals, as previously reported in obese individuals with type 2 diabetes.     

The many faces (and phases) of ceramide and sphingomyelin I – single lipids

Ceramides, the simplest kind of two-chained sphingolipids, contain a single hydroxyl group in position 1 of the sphingoid base. Sphingomyelins further contain a phosphocholine group at the OH of position 1 of ceramide. Ceramides and sphingomyelins show a variety of species depending on the fatty acyl chain length, hydroxylation, and unsaturation. Because of the relatively high transition temperature of sphingomyelin compared to lecithin and, particularly, of ceramides with 16:0–18:0 saturated chains, a widespread idea on their functional importance refers to formation of rather solid domains enriched in sphingomyelin and ceramide. Frequently, and especially in the cell biology field, these are generally (and erroneously) assumed to occur irrespective on the type of N-acyl chain in these lipids. This is because most studies indicating such condensed ordered domains employed sphingolipids with acyl chains with 16 carbons while scarce attention has been focused on the influence of the N-acyl chain on their surface properties. However, abundant evidence has shown that variations of the N-acyl chain length in ceramides and sphingomyelins markedly affect their phase state, interfacial elasticity, surface topography, electrostatics and miscibility and that, even the usually conceived “condensed” sphingolipids and many of their mixtures, may exhibit liquid-like expanded states. This review is a summarized overview of our work and of related others on some facts regarding membranes composed of single molecular species of ceramide and sphingomyelin. A second part is dedicated to discuss the miscibility properties between species of sphingolipids that differ in N-acyl and oligosaccharide chains.     

On the role of peroxisomes in the metabolism of lipids — evidence from studies on mammalian tissues in vivo

Summary Recent investigations into the role of peroxisomes in mammalian lipid metabolism have employed double isotope methodologies to examine the influence of peroxisomal agents on lipid turnover in the liver and extra hepatic tissues of the living animal.The action of these agents, all of which caused extensive changes in the flux of lipid metabolism in the treated animals, may best be viewed in relation to their effects on the common pathway of fatty acid oxidation in peroxisomes.Clofibrate, for example, acts through induction of peroxisomal oxidases and catalase; glycolate and ethanol through activation of this pathway; and aminotriazole and allylisopropylacetamide through inhibition of the catalase step in the sequence.The data from these studies provide support for the concept of an important contributory and regulatory role of peroxisomes in relation to the overall balance of lipid metabolism, and emphasize that these organelles play a significant role in the oxidation of common fatty acids, as well as a potential for the elimination of fatty acids that are poorly oxidized by mitochondria.Additionally, the data raise intriguing questions on the extension of peroxisomal influence to include phospholipid metabolism and the substantial degree of inter-tissue communication which is involved in the balance of lipid metabolism in the whole animal.     

The influence of branched-chain and ω-alicyclic fatty acids on the transition temperature of bacillus subtilis lipids

The influence of branched-chain and ω-alicyclic fatty acids on the transition temperature of Bacillus subtilis lipids was studied by measuring the fluorescence depolarisation of the probe 1,6-diphenyl-1,3,5-hexatriene incorporated into lipid bilayers. Only anteiso-C₁₅ and C₁₇ fatty acid-enriched lipids showed no transition in the observed temperature range. Compared to the transition of normal lipids iso-fatty acid-enriched lipids have a slightly higher transition temperature. The incorporation of ω-alicyclic fatty acids with increasing size of the alicycle leads to a decrease in the transition temperature. A possible role of ω-cyclohexane fatty acids in Bacillus acidocaldarius is proposed.