RESUMEN
Pancreatic enzyme therapy does not normalize dietary fat absorption in patients with cystic fibrosis and pancreatic insufficiency. Efficacy of LYM-X-SORB (LXS), an easily absorbable lipid matrix that enhances fat absorption, was evaluated in a 12-month randomized, double-blinded, placebo-controlled trial with plasma fatty acids (FA) and coefficient of fat absorption (CFA) outcomes. A total of 110 subjects (age 10.4â±â3.0 years) were randomized. Total FA increased with LXS at 3 and 12 months (+1.58, +1.14 mmol/L) and not with placebo (Pâ=â0.046). With LXS, linoleic acid (LA) increased at 3 and 12 months (+298, +175 nmol/mL, Pâ≤â0.046), with a 6% increase in CFA (Pâ<â0.01). LA increase was significant in LXS versus placebo (445 vs 42 nmol/mL, Pâ=â0.038). Increased FA and LA predicted increased body mass index Z scores. In summary, the LXS treatment improved dietary fat absorption compared with placebo as indicated by plasma FA and LA and was associated with better growth status.
Asunto(s)
Fibrosis Quística/tratamiento farmacológico , Grasas de la Dieta/metabolismo , Insuficiencia Pancreática Exocrina/tratamiento farmacológico , Lípidos/uso terapéutico , Adolescente , Niño , Fenómenos Fisiológicos Nutricionales Infantiles , Fibrosis Quística/complicaciones , Fibrosis Quística/enzimología , Fibrosis Quística/metabolismo , Insuficiencia Pancreática Exocrina/complicaciones , Insuficiencia Pancreática Exocrina/enzimología , Femenino , Humanos , Absorción Intestinal , Ácido Linoleico/uso terapéutico , Masculino , Resultado del TratamientoRESUMEN
Lipid research is challenging owing to the complexity and diversity of the lipidome. Here we review a set of experimental tools developed for the seasoned lipid researcher, as well as, those who are new to the field of lipid research. Novel tools for probing protein-lipid interactions, applications for lipid binding antibodies, enhanced systems for the cellular delivery of lipids, improved visualization of lipid membranes using gold-labeled lipids, and advances in mass spectrometric analysis techniques will be discussed. Because lipid mediators are known to participate in a host of signal transduction and trafficking pathways within the cell, a comprehensive lipid toolbox that aids the science of lipidomics research is essential to better understand the molecular mechanisms of interactions between cellular components. This article is part of a Special Issue entitled Tools to study lipid functions.
Asunto(s)
Lípidos/fisiología , Anticuerpos/fisiología , Membrana Celular/fisiología , Oro/química , Liposomas , Espectrometría de MasasRESUMEN
Accurate determination of lipid concentrations is an obligatory routine in a research laboratory engaged in studies using this class of biomaterials. For phospholipids, this is frequently accomplished using the phosphate assay (Bartlett, G.R. Phosphorus Assay in Column Chromatography. J. Biol. Chem. 234, 466-468, 1959). Given the purity of the currently commercially available synthetic and isolated natural lipids, we have observed that determination of the dry weight of lipid stock solutions provides the fastest, most accurate, and generic method to assay their concentrations. The protocol described here takes advantage of the high resolution and accuracy obtained by modern weighing technology. We assayed by this technique the concentrations of a number of phosphatidylcholine samples, with different degrees of acyl chain saturation and length, and in different organic solvents. The results were compared with those from Bartlett assay, (31)P NMR, and Langmuir compression isotherms. The data obtained show that the gravimetric assay yields lipid concentrations with a resolution similar or better than obtained by the other techniques.
Asunto(s)
Fosfolípidos/análisis , Termogravimetría , Espectroscopía de Resonancia Magnética , Fosfatidilcolinas/química , Tecnicas de Microbalanza del Cristal de Cuarzo , Solventes/químicaRESUMEN
We report the lipidomic response of the murine macrophage RAW cell line to Kdo(2)-lipid A, the active component of an inflammatory lipopolysaccharide functioning as a selective TLR4 agonist and compactin, a statin inhibitor of cholesterol biosynthesis. Analyses of lipid molecular species by dynamic quantitative mass spectrometry and concomitant transcriptomic measurements define the lipidome and demonstrate immediate responses in fatty acid metabolism represented by increases in eicosanoid synthesis and delayed responses characterized by sphingolipid and sterol biosynthesis. Lipid remodeling of glycerolipids, glycerophospholipids, and prenols also take place, indicating that activation of the innate immune system by inflammatory mediators leads to alterations in a majority of mammalian lipid categories, including unanticipated effects of a statin drug. Our studies provide a systems-level view of lipid metabolism and reveal significant connections between lipid and cell signaling and biochemical pathways that contribute to innate immune responses and to pharmacological perturbations.
Asunto(s)
Inmunidad Innata , Mediadores de Inflamación/metabolismo , Metabolismo de los Lípidos , Lipopolisacáridos/farmacología , Macrófagos/metabolismo , Animales , Línea Celular , Inmunidad Innata/efectos de los fármacos , Inmunidad Innata/fisiología , Mediadores de Inflamación/inmunología , Metabolismo de los Lípidos/efectos de los fármacos , Metabolismo de los Lípidos/fisiología , Macrófagos/inmunología , Ratones , Receptor Toll-Like 4/agonistas , Receptor Toll-Like 4/inmunología , Receptor Toll-Like 4/metabolismoRESUMEN
While profiling the lipidome of the mouse brain by mass spectrometry, we discovered a novel family of N-acylphosphatidylserine (N-acyl-PS) molecules. These N-acyl-PS species were enriched by DEAE-cellulose column chromatography, and they were then characterized by accurate mass measurements, tandem mass spectrometry, liquid chromatography/mass spectrometry, and comparison to an authentic standard. Mouse brain N-acyl-PS molecules are heterogeneous and constitute about 0.1% of the total lipid. In addition to various ester-linked fatty acyl chains on their glycerol backbones, the complexity of the N-acyl-PS series is further increased by the presence of diverse amide-linked N-acyl chains, which include saturated, monounsaturated, and polyunsaturated species. N-Acyl-PS molecular species were also detected in the lipids of pig brain, mouse RAW264.7 macrophage tumor cells, and yeast, but not Escherichia coli. N-Acyl-PSs may be biosynthetic precursors of N-acylserine molecules, such as the recently reported signaling lipid N-arachidonoylserine from bovine brain. We suggest that a phospholipase D might cleave N-acyl-PS to generate N-acylserine, in analogy to the biosynthesis of the endocannabinoid N-arachidonoylethanolamine (anadamide) from N-arachidonoylphosphatidylethanolamine.
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Células Eucariotas/metabolismo , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Animales , Encéfalo/metabolismo , Cromatografía por Intercambio Iónico , Cromatografía Liquida , Ácidos Grasos/análisis , Lípidos/química , Lípidos/aislamiento & purificación , Espectrometría de Masas , Ratones , Estructura Molecular , Fosfolipasa D/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Porcinos , Espectrometría de Masas en TándemRESUMEN
Qualification, preparation, and use of lipid compounds as analytical reference standards are daunting endeavors. The sheer vastness of the number of lipid compounds present in biological samples make it impossible to directly standardize each entity. Available lipid compounds chosen for preparation as standards are difficult to maintain as pure entities of stable concentration due to their physical and chemical interactions. The lipid chemist must understand these constraints for each chosen molecule to construct a standard material, which provides accurate measurement for a practical length of time. We provide methods and guidelines to aid the chemist in these endeavors. These aids include analytical methods for preparation and handling techniques, qualification of candidate materials, packaging, storage, and, finally, stability testing of working standard materials. All information will be provided under the purview of standardization of lipid analysis by mass spectrometry.
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Lípidos/análisis , Lípidos/normas , Espectrometría de Masas/métodos , Control de Calidad , Estándares de ReferenciaRESUMEN
PURPOSE: Fenretinide [N-(4-hydroxyphenyl)retinamide (4-HPR)] is a cytotoxic retinoid that suffers from a wide interpatient variation in bioavailability when delivered orally in a corn oil capsule. The poor bioavailability of the capsule formulation may have limited responses in clinical trials, and the large capsules are not suitable for young children. To support the hypothesis that a novel organized lipid matrix, LYM-X-SORB, can increase the oral bioavailability of fenretinide, fenretinide in LYM-X-SORB matrix and in a powderized LYM-X-SORB formulation was delivered to mice. EXPERIMENTAL DESIGN: Fenretinide was delivered orally to mice as the contents of the corn oil capsule, in LYM-X-SORB matrix (4-HPR/LYM-X-SORB matrix) or in a LYM-X-SORB matrix powderized with sugar and flour (4-HPR/LYM-X-SORB oral powder). Levels of 4-HPR, and its principal metabolite, N-(4-methoxyphenyl)retinamide, were assayed in plasma and tissues. RESULTS: In a dose-responsive manner, from 120 to 360 mg/kg/d, delivery to mice of 4-HPR in LYM-X-SORB matrix, or as 4-HPR/LYM-X-SORB oral powder, increased 4-HPR plasma levels up to 4-fold (P<0.01) and increased tissue levels up to 7-fold (P<0.01) compared with similar doses of 4-HPR delivered using capsule contents. Metabolite [N-(4-methoxyphenyl)retinamide] levels mirrored 4-HPR levels. Two human neuroblastoma murine xenograft models showed increased survival (P<0.03), when treated with 4-HPR/LYM-X-SORB oral powder, confirming the bioactivity of the formulation. CONCLUSIONS: 4-HPR/LYM-X-SORB oral powder is a novel, oral drug delivery formulation, suitable for pediatric use, which warrants further development for the delivery of fenretinide in the treatment of cancer. A phase I clinical trial in pediatric neuroblastoma is in progress.
Asunto(s)
Antineoplásicos/administración & dosificación , Ácidos Grasos/química , Fenretinida/administración & dosificación , Lisofosfatidilcolinas/química , Monoglicéridos/química , Neuroblastoma/tratamiento farmacológico , Neoplasias del Sistema Nervioso Periférico/tratamiento farmacológico , Administración Oral , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Disponibilidad Biológica , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos , Fenretinida/química , Fenretinida/farmacocinética , Humanos , Ratones , Polvos , Distribución TisularRESUMEN
A high-performance liquid chromatography (HPLC) method was developed to measure levels of d-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (d-threo-PPMP) in mouse plasma and liver. d-threo-PPMP was measured by HPLC with a Luna Pheny-Hexyl column (5 microm, 250 mm x 4.6 mm) employing UV detection at 210 nm using a mobile phase of potassium phosphate buffer (20mM, pH 3.0)-acetonitrile in a 45:55 (v/v) ratio. d-threo-1-phenyl-2-pentadecanoylamino-3-morpholino-1-propanol (PC15MP) was employed as an internal standard (IS). The lower limit of quantitation (LLOQ) was 0.3 microg/ml. The assay was linear over a concentration range of 0.3-10 microg/ml, with acceptable precision and accuracy. Assayed in plasma, the intra- and inter-day validation for all coefficients of variation (R.S.D.%) were found less than 15%. The method was applied to samples from athymic (nu/nu) mice treated with d-threo-PPMP by intraperitoneal injection. d-threo-PPMP levels of approximately 10-20 microg/ml ( approximately 20-40 microM) in plasma and approximately 45 microg/g in liver were obtained. The present method can be used to quantify d-threo-PPMP in mice for bioavailability and dose-response studies.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Hígado/química , Morfolinas/análisis , Esfingolípidos/análisis , Animales , Femenino , Ratones , Ratones Endogámicos BALB C , Morfolinas/sangre , Estándares de Referencia , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Esfingolípidos/sangreRESUMEN
The LIPID MAPS Consortium (www.lipidmaps.org) is developing comprehensive procedures for identifying all lipids of the macrophage, following activation by endotoxin. The goal is to quantify temporal and spatial changes in lipids that occur with cellular metabolism and to develop bioinformatic approaches that establish dynamic lipid networks. To achieve these aims, an endotoxin of the highest possible analytical specification is crucial. We now report a large-scale preparation of 3-deoxy-D-manno-octulosonic acid (Kdo)(2)-Lipid A, a nearly homogeneous Re lipopolysaccharide (LPS) sub-structure with endotoxin activity equal to LPS. Kdo(2)-Lipid A was extracted from 2 kg cell paste of a heptose-deficient Escherichia coli mutant. It was purified by chromatography on silica, DEAE-cellulose, and C18 reverse-phase resin. Structure and purity were evaluated by electrospray ionization/mass spectrometry, liquid chromatography/mass spectrometry and (1)H-NMR. Its bioactivity was compared with LPS in RAW 264.7 cells and bone marrow macrophages from wild-type and toll-like receptor 4 (TLR-4)-deficient mice. Cytokine and eicosanoid production, in conjunction with gene expression profiling, were employed as readouts. Kdo(2)-Lipid A is comparable to LPS by these criteria. Its activity is reduced by >10(3) in cells from TLR-4-deficient mice. The purity of Kdo(2)-Lipid A should facilitate structural analysis of complexes with receptors like TLR-4/MD2.