In Vivo Cellular Phosphatidylcholine Kinetics of CD15+ Leucocytes and CD3+ T-Lymphocytes in Adults with Acute Respiratory Distress Syndrome.

Mammalian cell membranes composed of a mixture of glycerophospholipids, the relative composition of individual phospholipids and the dynamic flux vary between cells. In addition to their structural role, membrane phospholipids are involved in cellular signalling and immunomodulatory functions. In this study, we investigate the molecular membrane composition and dynamic flux of phosphatidylcholines in CD15+ leucocytes and CD3+ lymphocytes extracted from patients with acute respiratory distress syndrome (ARDS). We identified compositional variations between these cell types, where CD15+ cells had relatively higher quantities of alkyl-acyl PC species and CD3+ cells contained more arachidonoyl-PC species. There was a significant loss of arachidonoyl-PC in CD3+ cells in ARDS patients. Moreover, there were significant changes in PC composition and the methyl-D9 enrichment of individual molecular species in CD15+ cells from ARDS patients. This is the first study to perform an in vivo assessment of membrane composition and dynamic changes in immunological cells from ARDS patients.

Exhaled breath particles as a novel tool to study lipid composition of epithelial lining fluid from the distal lung.

BACKGROUND: Surfactant phospholipid (PL) composition plays an important role in lung diseases. We compared the PL composition of non-invasively collected exhaled breath particles (PEx) with bronchoalveolar lavage (BAL) and induced sputum (ISP) at baseline and following endotoxin (LPS) challenges. METHODS: PEx and BAL were collected from ten healthy nonsmoking participants before and after segmental LPS challenge. Four weeks later, PEx and ISP were sampled in the week before and after a whole lung LPS inhalation challenge. PL composition was analysed using mass spectrometry. RESULTS: The overall PL composition of BAL, ISP and PEx was similar, with PC(32:0) and PC(34:1) representing the largest fractions in all three sample types (baseline PC(32:0) geometric mean mol%: 52.1, 56.9, and 51.7, PC(34:1) mol%: 11.7, 11.9 and 11.4, respectively). Despite this similarity, PEx PL composition was more closely related to BAL than to ISP. For most lipids comparable inter-individual differences in BAL, ISP, and PEx were found. PL composition of PEx was repeatable. The most pronounced increase following segmental LPS challenge was detected for SM(d34:1) in BAL (0.24 to 0.52 mol%) and following inhalation LPS challenge in ISP (0.45 to 0.68 mol%). An increase of SM(d34:1) following segmental LPS challenge was also detectable in PEx (0.099 to 0.103 mol%). The inhalation challenge did not change PL composition of PEx. CONCLUSION: Our data supports the peripheral origin of PEx. The lack of PL changes in PEx after inhalation challenge might to be due to the overall weaker response of inhaled LPS which primarily affects the larger airways. Compared with BAL, which always contains lining fluid from both peripheral lung and central airways, PEx analysis might add value as a selective and non-invasive method to investigate peripheral airway PL composition. TRIAL REGISTRATION: NCT03044327, first posted 07/02/2017.

Stratification of asthma by lipidomic profiling of induced sputum supernatant.

BACKGROUND: Asthma is a chronic respiratory disease with significant heterogeneity in its clinical presentation and pathobiology. There is need for improved understanding of respiratory lipid metabolism in asthma patients and its relation to observable clinical features. OBJECTIVE: We performed a comprehensive, prospective, cross-sectional analysis of the lipid composition of induced sputum supernatant obtained from asthma patients with a range of disease severities, as well as from healthy controls. METHODS: Induced sputum supernatant was collected from 211 adults with asthma and 41 healthy individuals enrolled onto the U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes) study. Sputum lipidomes were characterized by semiquantitative shotgun mass spectrometry and clustered using topologic data analysis to identify lipid phenotypes. RESULTS: Shotgun lipidomics of induced sputum supernatant revealed a spectrum of 9 molecular phenotypes, highlighting not just significant differences between the sputum lipidomes of asthma patients and healthy controls, but also within the asthma patient population. Matching clinical, pathobiologic, proteomic, and transcriptomic data helped inform the underlying disease processes. Sputum lipid phenotypes with higher levels of nonendogenous, cell-derived lipids were associated with significantly worse asthma severity, worse lung function, and elevated granulocyte counts. CONCLUSION: We propose a novel mechanism of increased lipid loading in the epithelial lining fluid of asthma patients resulting from the secretion of extracellular vesicles by granulocytic inflammatory cells, which could reduce the ability of pulmonary surfactant to lower surface tension in asthmatic small airways, as well as compromise its role as an immune regulator.

Increased Alveolar Heparan Sulphate and Reduced Pulmonary Surfactant Amount and Function in the Mucopolysaccharidosis IIIA Mouse.

Mucopolysaccharidosis IIIA (MPS IIIA) is a lysosomal storage disease with significant neurological and skeletal pathologies. Respiratory dysfunction is a secondary pathology contributing to mortality in MPS IIIA patients. Pulmonary surfactant is crucial to optimal lung function and has not been investigated in MPS IIIA. We measured heparan sulphate (HS), lipids and surfactant proteins (SP) in pulmonary tissue and bronchoalveolar lavage fluid (BALF), and surfactant activity in healthy and diseased mice (20 weeks of age). Heparan sulphate, ganglioside GM3 and bis(monoacylglycero)phosphate (BMP) were increased in MPS IIIA lung tissue. There was an increase in HS and a decrease in BMP and cholesteryl esters (CE) in MPS IIIA BALF. Phospholipid composition remained unchanged, but BALF total phospholipids were reduced (49.70%) in MPS IIIA. There was a reduction in SP-A, -C and -D mRNA, SP-D protein in tissue and SP-A, -C and -D protein in BALF of MPS IIIA mice. Captive bubble surfactometry showed an increase in minimum and maximum surface tension and percent surface area compression, as well as a higher compressibility and hysteresis in MPS IIIA surfactant upon dynamic cycling. Collectively these biochemical and biophysical changes in alveolar surfactant are likely to be detrimental to lung function in MPS IIIA.

Mass spectrometry imaging of phosphatidylcholine metabolism in lungs administered with therapeutic surfactants and isotopic tracers.

Mass spectrometry imaging (MSI) visualizes molecular distributions throughout tissues but is blind to dynamic metabolic processes. Here, MSI with high mass resolution together with multiple stable isotope labeling provided spatial analyses of phosphatidylcholine (PC) metabolism in mouse lungs. Dysregulated surfactant metabolism is central to many respiratory diseases. Metabolism and turnover of therapeutic pulmonary surfactants were imaged from distributions of intact and metabolic products of an added tracer, universally 13C-labeled dipalmitoyl PC (U13C-DPPC). The parenchymal distributions of newly synthesized PC species were also imaged from incorporations of methyl-D9-choline. This dual labeling strategy demonstrated both lack of inhibition of endogenous PC synthesis by exogenous surfactant and location of acyl chain remodeling processes acting on the U13C-DPPC-labeled surfactant, leading to formation of polyunsaturated PC lipids. This ability to visualize discrete metabolic events will greatly enhance our understanding of lipid metabolism in diverse tissues and has potential application to both clinical and experimental studies.

Postnatal adaptations of phosphatidylcholine metabolism in extremely preterm infants: implications for choline and PUFA metabolism.

BACKGROUND: Lipid metabolism in pregnancy delivers PUFAs from maternal liver to the developing fetus. The transition at birth to diets less enriched in PUFA is especially challenging for immature, extremely preterm infants who are typically supported by total parenteral nutrition. OBJECTIVE: The aim was to characterize phosphatidylcholine (PC) and choline metabolism in preterm infants and demonstrate the molecular specificity of PC synthesis by the immature preterm liver in vivo. METHODS: This MS-based lipidomic study quantified the postnatal adaptations to plasma PC molecular composition in 31 preterm infants <28 weeks' gestational age. Activities of the cytidine diphosphocholine (CDP-choline) and phosphatidylethanolamine-N-methyltransferase (PEMT) pathways for PC synthesis were assessed from incorporations of deuterated methyl-D9-choline chloride. RESULTS: The concentration of plasma PC in these infants increased postnatally from median values of 481 (IQR: 387-798) µM at enrollment to 1046 (IQR: 616-1220) µM 5 d later (P < 0.001). Direct incorporation of methyl-D9-choline demonstrated that this transition was driven by an active CDP-choline pathway that synthesized PC enriched in species containing oleic and linoleic acids. A second infusion of methyl-D9-choline chloride at day 5 clearly indicated continued activity of this pathway. Oxidation of D9-choline through D9-betaine resulted in the transfer of 1 deuterated methyl group to S-adenosylmethionine. A very low subsequent transfer of this labeled methyl group to D3-PC indicated that liver PEMT activity was essentially inactive in these infants. CONCLUSIONS: This study demonstrated that the preterm infant liver soon after birth, and by extension the fetal liver, was metabolically active in lipoprotein metabolism. The low PEMT activity, which is the only pathway for endogenous choline synthesis and is responsible for hormonally regulated export of PUFAs from adult liver, strongly supports increased supplementation of preterm parenteral nutrition with both choline and PUFAs.

High-Throughput Urinary Neopterin-to-Creatinine Ratio Monitoring of Systemic Inflammation.

BACKGROUND: Systemic inflammation is a marker of ill health and has prognostic implications in multiple health settings. Urinary neopterin is an excellent candidate as a nonspecific marker of systemic inflammation. Expression as urinary neopterin-to-creatinine ratio (UNCR) normalizes for urinary hydration status. Major attractions include (a) urine vs blood sampling, (b) integration of inflammation over a longer period compared with serum sampling, and (c) high stability of neopterin and creatinine. METHODS: A high-throughput ultraperformance LC-MS method was developed to measure neopterin and creatinine together from the same urine sample. The assay was applied in several clinical scenarios: healthy controls, symptomatic infections, and multiple sclerosis. Area under the curve was compared between weekly and monthly sampling scenarios. Analysis of a single pooled sample was compared with averaging results from analysis of individual samples. RESULTS: The assay has excellent intraassay and interassay precision, linearity of dilution, and spike and recovery. Higher UNCR was demonstrated in female vs male individuals, older age, inflammatory disease (multiple sclerosis), and symptomatic infections. In healthy controls, fluctuations in inflammatory state also occurred in the absence of symptomatic infection or other inflammatory triggers. Analysis of a single pooled sample, made up from weekly urine samples, integrates inflammatory activity over time. CONCLUSIONS: UNCR is a useful biomarker of systemic inflammation. The method presented offers simplicity, speed, robustness, reproducibility, efficiency, and proven utility in clinical scenarios. UNCR fluctuations underline the importance of longitudinal monitoring, vs a single time point, to capture a more representative estimate of an individual's inflammatory state over time.

Insight into erythrocyte phospholipid molecular flux in healthy humans and in patients with acute respiratory distress syndrome.

Although the distribution of cellular membrane phospholipid composition is well characterised in human erythrocytes, in-vivo turnover and dynamic flux of phospholipids between plasma and erythrocytes in physiological and in particular during disease states are mostly unknown. Erythrocyte mass primarily consisted of lipids and phosphatidylcholine (PC) contributes to the significant proportion of phospholipid membrane composition. Esterified membrane PC can be utilised during pathological processes to generate pro and anti-inflammatory lipid mediators, which can contribute to the pathogenesis of acute respiratory distress syndrome (ARDS). In this study, utilising isotope labelling of choline and analytical methods with electrospray mass spectrometry (ESI-MS/MS), we characterised individual molecular composition and dynamic exchange of PC, sphingomyelins (SM) and lysophosphatidylcholines (LPC) between plasma and erythrocytes. In ARDS patients, there were significant alterations in PC molecular composition, coupled with a continuous loss of arachidonoyl-PC species over time. Infusion of methyl-D9-choline chloride resulted in enrichment of labelled choline into plasma PC and LPC via CDP-choline pathway with subsequent incorporation into erythrocyte PC. As expected, erythrocyte methyl-D9 PC enrichment is much slower than plasma. Patients had much faster and higher fractional enrichment of all PC and LPC molecules suggesting increased flux between plasma and erythrocytes. There was a particular pattern of incorporation, where the arachidonoyl-PC species achieved equilibrium with plasma rapidly and retained highest concentrations of enrichment compared to the other PC species. Increased enrichment of arachidonoyl-PC coupled with virtually no increase or depletion of its concentrations suggests the possibility of substrate donation for other cell types for the participation of eicosanoid biosynthesis during inflammatory conditions like ARDS. In summary, this study revealed an alerted pattern erythrocyte molecular phospholipid composition and flux in patients with acute respiratory distress syndrome and the pathological consequences of these changes needs further exploration.

Speciation of reactive sulfur species and their reactions with alkylating agents: do we have any clue about what is present inside the cell?

BACKGROUND AND PURPOSE: Posttranslational modifications of cysteine residues represent a major aspect of redox biology, and their reliable detection is key in providing mechanistic insights. The metastable character of these modifications and cell lysis-induced artifactual oxidation render current state-of-the-art protocols to rely on alkylation-based stabilization of labile cysteine derivatives before cell/tissue rupture. An untested assumption in these procedures is that for all cysteine derivatives, alkylation rates are faster than their dynamic interchange. However, when the interconversion of cysteine derivatives is not rate limiting, electrophilic labelling is under Curtin-Hammett control; hence, the final alkylated mixture may not represent the speciation that prevailed before alkylation. EXPERIMENTAL APPROACH: Buffered aqueous solutions of inorganic, organic, cysteine, GSH and GAPDH polysulfide species were used. Additional experiments in human plasma and serum revealed that monobromobimane can extract sulfide from the endogenous sulfur pool by shifting speciation equilibria, suggesting caution should be exercised when interpreting experimental results using this tool. KEY RESULTS: In the majority of cases, the speciation of alkylated polysulfide/thiol derivatives depended on the experimental conditions. Alkylation perturbed sulfur speciation in both a concentration- and time-dependent manner and strong alkylating agents cleaved polysulfur chains. Moreover, the labelling of sulfenic acids with dimedone also affected cysteine speciation, suggesting that part of the endogenous pool of products previously believed to represent sulfenic acid species may represent polysulfides. CONCLUSIONS AND IMPLICATIONS: We highlight methodological caveats potentially arising from these pitfalls and conclude that current derivatization strategies often fail to adequately capture physiological speciation of sulfur species. LINKED ARTICLES: This article is part of a themed section on Chemical Biology of Reactive Sulfur Species. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.4/issuetoc.

Metabolism of a synthetic compared with a natural therapeutic pulmonary surfactant in adult mice.

Secreted pulmonary surfactant phosphatidylcholine (PC) has a complex intra-alveolar metabolism that involves uptake and recycling by alveolar type II epithelial cells, catabolism by alveolar macrophages, and loss up the bronchial tree. We compared the in vivo metabolism of animal-derived poractant alfa (Curosurf) and a synthetic surfactant (CHF5633) in adult male C57BL/6 mice. The mice were dosed intranasally with either surfactant (80 mg/kg body weight) containing universally 13C-labeled dipalmitoyl PC (DPPC) as a tracer. The loss of [U13C]DPPC from bronchoalveolar lavage and lung parenchyma, together with the incorporation of 13C-hydrolysis fragments into new PC molecular species, was monitored by electrospray ionization tandem mass spectrometry. The catabolism of CHF5633 was considerably delayed compared with poractant alfa, the hydrolysis products of which were cleared more rapidly. There was no selective resynthesis of DPPC and, strikingly, acyl remodeling resulted in preferential synthesis of polyunsaturated PC species. In conclusion, both surfactants were metabolized by similar pathways, but the slower catabolism of CHF5633 resulted in longer residence time in the airways and enhanced recycling of its hydrolysis products into new PC species.

Lipidomics of Thalassiosira pseudonana under Phosphorus Stress Reveal Underlying Phospholipid Substitution Dynamics and Novel Diglycosylceramide Substitutes.

Phytoplankton replace phosphorus-containing lipids (P-lipids) with non-P analogues, boosting growth in P-limited oceans. In the model diatom Thalassiosira pseudonana, the substitution dynamics of lipid headgroups are well described, but those of the individual lipids, differing in fatty acid composition, are unknown. Moreover, the behavior of lipids outside the common headgroup classes and the relationship between lipid substitution and cellular particulate organic P (POP) have yet to be reported. We investigated these through the mass spectrometric lipidomics of P-replete (P+) and P-depleted (P-) T. pseudonana cultures. Nonlipidic POP was depleted rapidly by the initiation of P stress, followed by the cessation of P-lipid biosynthesis and per-cell reductions in the P-lipid levels of successive generations. Minor P-lipid degradative breakdown was observed, releasing P for other processes, but most P-lipids remained intact. This may confer an advantage on efficient heterotrophic lipid consumers in P-limited oceans. Glycerophosphatidylcholine (PC), the predominant P-lipid, was similar in composition to its betaine substitute lipid. During substitution, PC was less abundant per cell and was more highly unsaturated in composition. This may reflect underlying biosynthetic processes or the regulation of membrane biophysical properties subject to lipid substitution. Finally, levels of several diglycosylceramide lipids increased as much as 10-fold under P stress. These represent novel substitute lipids and potential biomarkers for the study of P limitation in situ, contributing to growing evidence highlighting the importance of sphingolipids in phycology. These findings contribute much to our understanding of P-lipid substitution, a powerful and widespread adaptation to P limitation in the oligotrophic ocean.IMPORTANCE Unicellular organisms replace phosphorus (P)-containing membrane lipids with non-P substitutes when P is scarce, allowing greater growth of populations. Previous research with the model diatom species Thalassiosira pseudonana grouped lipids by polar headgroups in their chemical structures. The significance of the research reported here is threefold. (i) We described the individual lipids within the headgroups during P-lipid substitution, revealing the relationships between lipid headgroups and hinting at the underlying biochemical processes. (ii) We measured total cellular P, placing P-lipid substitution in the context of the broader response to P stress and yielding insight into the implications of substitution in the marine environment. (iii) We identified lipids previously unknown in this system, revealing a new type of non-P substitute lipid, which is potentially useful as a biomarker for the investigation of P limitation in the ocean.

Harmonizing lipidomics: NIST interlaboratory comparison exercise for lipidomics using SRM 1950-Metabolites in Frozen Human Plasma.

As the lipidomics field continues to advance, self-evaluation within the community is critical. Here, we performed an interlaboratory comparison exercise for lipidomics using Standard Reference Material (SRM) 1950-Metabolites in Frozen Human Plasma, a commercially available reference material. The interlaboratory study comprised 31 diverse laboratories, with each laboratory using a different lipidomics workflow. A total of 1,527 unique lipids were measured across all laboratories and consensus location estimates and associated uncertainties were determined for 339 of these lipids measured at the sum composition level by five or more participating laboratories. These evaluated lipids detected in SRM 1950 serve as community-wide benchmarks for intra- and interlaboratory quality control and method validation. These analyses were performed using nonstandardized laboratory-independent workflows. The consensus locations were also compared with a previous examination of SRM 1950 by the LIPID MAPS consortium. While the central theme of the interlaboratory study was to provide values to help harmonize lipids, lipid mediators, and precursor measurements across the community, it was also initiated to stimulate a discussion regarding areas in need of improvement.

Stable isotope analysis of dynamic lipidomics.

Metabolic pathway flux is a fundamental element of biological activity, which can be quantified using a variety of mass spectrometric techniques to monitor incorporation of stable isotope-labelled substrates into metabolic products. This article contrasts developments in electrospray ionisation mass spectrometry (ESI-MS) for the measurement of lipid metabolism with more established gas chromatography mass spectrometry and isotope ratio mass spectrometry methodologies. ESI-MS combined with diagnostic tandem MS/MS scans permits the sensitive and specific analysis of stable isotope-labelled substrates into intact lipid molecular species without the requirement for lipid hydrolysis and derivatisation. Such dynamic lipidomic methodologies using non-toxic stable isotopes can be readily applied to quantify lipid metabolic fluxes in clinical and metabolic studies in vivo. However, a significant current limitation is the absence of appropriate software to generate kinetic models of substrate incorporation into multiple products in the time domain. Finally, we discuss the future potential of stable isotope-mass spectrometry imaging to quantify the location as well as the extent of lipid synthesis. This article is part of a Special Issue entitled: BBALIP_Lipidomics Opinion Articles edited by Sepp Kohlwein.

Membrane cholesterol is essential for triterpenoid saponin augmentation of a saporin-based immunotoxin directed against CD19 on human lymphoma cells.

Triterpenoid saponins from Saponinum Album (SA) exert potent lytic effects on eukaryotic cell plasma membranes and, when used at sub-lytic concentrations, significantly augment the cytotoxicity of saporin-based immunotoxins (IT). To help elucidate the mechanism(s) behind these two phenomena we investigated the role of cholesterol to both. Human Daudi lymphoma cells were lipid deprived using a combination of three different approaches. Following treatment, the total cellular lipid content was analyzed by electrospray ionization mass spectrometry (ESI-MS) and plasma membrane (PM) cholesterol content measured using the lipophilic fluorescent probe NR12S. Maximal lipid deprivation of cells resulted in a complete loss of sensitivity to lysis by SA. Similarly augmentation of the anti-CD19 immunotoxin (IT) BU12-SAPORIN by SA was lost but without a concomitant loss of intrinsic IT cytotoxicity. The lytic activity of SA was restored following incubation of lipid deprived Daudi cells with Synthecol or LDL. The augmentative effect of SA on IT cytotoxicity for Daudi cells was restored following repletion of PM cholesterol levels with LDL. NR12S fluorescence and ESI-MS analysis of cellular lipids demonstrated that restoration of SA lytic activity by Synthecol was entirely due to increased PM cholesterol levels. Restoration of cellular and PM cholesterol levels by LDL also restored the augmentative effect of SA for IT, an effect associated with repletion of PM cholesterol with minor changes in some phospholipid species. These results indicate that the lytic and IT augmentative properties of SA are cholesterol-dependent in contrast to intrinsic IT cytotoxicity that is at least partially cholesterol independent.

Lipid remodelling is a widespread strategy in marine heterotrophic bacteria upon phosphorus deficiency.

Upon phosphorus (P) deficiency, marine phytoplankton reduce their requirements for P by replacing membrane phospholipids with alternative non-phosphorus lipids. It was very recently demonstrated that a SAR11 isolate also shares this capability when phosphate starved in culture. Yet, the extent to which this process occurs in other marine heterotrophic bacteria and in the natural environment is unknown. Here, we demonstrate that the substitution of membrane phospholipids for a variety of non-phosphorus lipids is a conserved response to P deficiency among phylogenetically diverse marine heterotrophic bacteria, including members of the Alphaproteobacteria and Flavobacteria. By deletion mutagenesis and complementation in the model marine bacterium Phaeobacter sp. MED193 and heterologous expression in recombinant Escherichia coli, we confirm the roles of a phospholipase C (PlcP) and a glycosyltransferase in lipid remodelling. Analyses of the Global Ocean Sampling and Tara Oceans metagenome data sets demonstrate that PlcP is particularly abundant in areas characterized by low phosphate concentrations. Furthermore, we show that lipid remodelling occurs seasonally and responds to changing nutrient conditions in natural microbial communities from the Mediterranean Sea. Together, our results point to the key role of lipid substitution as an adaptive strategy enabling heterotrophic bacteria to thrive in the vast P-depleted areas of the ocean.

Antioxidant Role for Lipid Droplets in a Stem Cell Niche of Drosophila.

Stem cells reside in specialized microenvironments known as niches. During Drosophila development, glial cells provide a niche that sustains the proliferation of neural stem cells (neuroblasts) during starvation. We now find that the glial cell niche also preserves neuroblast proliferation under conditions of hypoxia and oxidative stress. Lipid droplets that form in niche glia during oxidative stress limit the levels of reactive oxygen species (ROS) and inhibit the oxidation of polyunsaturated fatty acids (PUFAs). These droplets protect glia and also neuroblasts from peroxidation chain reactions that can damage many types of macromolecules. The underlying antioxidant mechanism involves diverting PUFAs, including diet-derived linoleic acid, away from membranes to the core of lipid droplets, where they are less vulnerable to peroxidation. This study reveals an antioxidant role for lipid droplets that could be relevant in many different biological contexts.

Ready-to-use therapeutic food with elevated n-3 polyunsaturated fatty acid content, with or without fish oil, to treat severe acute malnutrition: a randomized controlled trial.

BACKGROUND: Ready-to-use therapeutic foods (RUTF) are lipid-based pastes widely used in the treatment of acute malnutrition. Current specifications for RUTF permit a high n-6 polyunsaturated fatty acid (PUFA) content and low n-3 PUFA, with no stipulated requirements for preformed long-chain n-3 PUFA. The objective of this study was to develop an RUTF with elevated short-chain n-3 PUFA and measure its impact, with and without fish oil supplementation, on children's PUFA status during treatment of severe acute malnutrition. METHODS: This randomized controlled trial in children with severe acute malnutrition in rural Kenya included 60 children aged 6 to 50 months who were randomized to receive i) RUTF with standard composition; ii) RUTF with elevated short chain n-3 PUFA; or iii) RUTF with elevated short chain n-3 PUFA plus fish oil capsules. Participants were followed-up for 3 months. The primary outcome was erythrocyte PUFA composition. RESULTS: Erythrocyte docosahexaenoic acid (DHA) content declined from baseline in the two arms not receiving fish oil. Erythrocyte long-chain n-3 PUFA content following treatment was significantly higher for participants in the arm receiving fish oil than for those in the arms receiving RUTF with elevated short chain n-3 PUFA or standard RUTF alone: 3 months after enrollment, DHA content was 6.3% (interquartile range 6.0-7.3), 4.5% (3.9-4.9), and 3.9% (2.4-5.7) of total erythrocyte fatty acids (P <0.001), respectively, while eicosapentaenoic acid (EPA) content was 2.0% (1.5-2.6), 0.7% (0.6-0.8), and 0.4% (0.3-0.5) (P <0.001). RUTF with elevated short chain n-3 PUFA and fish oil capsules were acceptable to participants and carers, and there were no significant differences in safety outcomes. CONCLUSIONS: PUFA requirements of children with SAM are not met by current formulations of RUTF, or by an RUTF with elevated short-chain n-3 PUFA without additional preformed long-chain n-3 PUFA. Clinical and growth implications of revised formulations need to be addressed in large clinical trials. TRIAL REGISTRATION: Clinicaltrials.gov NCT01593969. Registered 4 May 2012.

Muscle phospholipid hydrolysis by Bothrops asper Asp49 and Lys49 phospholipase A2 myotoxins--distinct mechanisms of action.

Bothrops snakes are the major cause of ophidian envenomings in Latin America. Their venom contains myotoxins that cause prominent muscle damage, which may lead to permanent disability. These toxins include myotoxins Mt-I and Mt-II, which share the phospholipase A2 (PLA2) fold, but Mt-II lacks enzymatic activity because the essential active site Asp49 is replaced by Lys. Both myotoxins cause sarcolemma alterations, with Ca²âº entry and loss of ATP and K⁺ from muscle cells, but the molecular lesions at the basis of their cellular action are not known, particularly the role of phospholipid hydrolysis. Here we tested their PLA2 activity in vivo, and evaluated the hypothesis that Ca²âº-activated endogenous PLA2s may be involved in the action of Mt-II. The time course of phospholipid hydrolysis by Mt-I and Mt-II in myotubes in culture and in tibialis anterior mouse muscles was determined. Mt-I rapidly hydrolyzed phosphatidylcholine and phosphatidylethanolamine but not phosphatidylserine, but no phospho-lipids were hydrolyzed in the presence of Mt-II. Whole Bothrops asper venom induced a higher extent of phospholipid hydrolysis than Mt-I alone. These results demonstrate in vivo PLA2 activity of Mt-I for the first time, and indicate that it acts only on the external monolayer of the sarcolemma. They also exclude activation of endogenous PLA2s in the action of Mt-II, implying that plasma membrane disruption by this toxin does not depend on phospholipid hydrolysis. Therefore, both Bothrops myotoxins induce Ca²âº entry and release of ATP and cause myonecrosis, but through different biochemical mechanisms.

Phosphatidylinositol transfer protein, cytoplasmic 1 (PITPNC1) binds and transfers phosphatidic acid.

Phosphatidylinositol transfer proteins (PITPs) are versatile proteins required for signal transduction and membrane traffic. The best characterized mammalian PITPs are the Class I PITPs, PITPα (PITPNA) and PITPß (PITPNB), which are single domain proteins with a hydrophobic cavity that binds a phosphatidylinositol (PI) or phosphatidylcholine molecule. In this study, we report the lipid binding properties of an uncharacterized soluble PITP, phosphatidylinositol transfer protein, cytoplasmic 1 (PITPNC1) (alternative name, RdgBß), of the Class II family. We show that the lipid binding properties of this protein are distinct to Class I PITPs because, besides PI, RdgBß binds and transfers phosphatidic acid (PA) but hardly binds phosphatidylcholine. RdgBß when purified from Escherichia coli is preloaded with PA and phosphatidylglycerol. When RdgBß was incubated with permeabilized HL60 cells, phosphatidylglycerol was released, and PA and PI were now incorporated into RdgBß. After an increase in PA levels following activation of endogenous phospholipase D or after addition of bacterial phospholipase D, binding of PA to RdgBß was greater at the expense of PI binding. We propose that RdgBß, when containing PA, regulates an effector protein or can facilitate lipid transfer between membrane compartments.