1,721,087 research outputs found
Methodology to detect oxidised phospholipids and their relevance in disease
No full textUnsaturated membrane phospholipids are susceptible to oxidation, either by reactive oxygen species or enzymatically, to generate a complex mixture of peroxy and hydroxyl species. They can then spontaneously decompose to truncated oxidised phospholipids composed of aldehyde, carboxyl and hydroxyl species of five to nine carbon atoms chain length, many of which exhibit potent biological activities. In addition, aldehydes can form Schiff's base reactions with protein lysines to form oxidised lipid:protein adducts. While a selection of oxidised phospholipids have been characterised in detail by a range of mass spectrometry techniques, including direct infusion and liquid chromatography mass spectrometry, there are relatively few reports of comprehensive analyses of oxidised phospholipids in disease states. Oxidised phospholipid species are widely thought to be central to the pathology of many diseases, but there is relatively little direct evidence to confirm this in vivo. This review provides an overview of the various analytical methodologies and then summarises their application to examples of chronic and acute disease, cardiovascular disease and acute respiratory distress syndrome, respectively. It highlights the gaps in information and indicates directions for future research.</p
Aspects of hormonal regulation of hepatic carbohydrate and lipid metabolism
No full textThe liver is a major site in the rat for conversion of dietary carbohydrateinto glycogen and triglyceride. Hepatic rates of fatty acid andglycogen synthesis were measured y vivo in response to meal-feeding(2h/day) by the incorporation of from 3H20. This technique has notbeen applied previously to glycogen synthesis and was validated in controland streptozotocin diabetic rats* Hepatic glycogen recycling was low infed adult rats but was apparently greater in foetal rats. The precursorsource for glycogen synthesis in vivo could not be determined from thedistribution pattern of 3H incorporation. Hepatic glycogen synthesis waselevated in control rats for 5h after feeding. During this phase,glycogen could not have been a net precursor for other synthetic pathways®Hepatic fatty acid synthesis in control rats increased 20-fold 2h afterfeeding. This response was impaired and delayed, but not abolished, bystreptozotocin diabetes (55mg/kg). Insulin pretreatment (30 P.Z.I.)restored the low diabetic rate of lipogenesis to normal by 8h afterfeeding. Streptozotocin reduced the hepatic Vmax activities of glucokinase,ATP-citrate lyase and total acetyl CoA carboxylase. None of theseenzyme activities increased when hepatic fatty acid synthesis was stimulatedby feeding in control rats or by feeding and insulin in diabeticrats. Feeding stimulated active acetyl CoA carboxylase in control, butnot diabetic, rats. The regulation of hepatic fatty acid synthesis byboth acetyl CoA carboxylase and increased substrate concentration isdiscussed.In control rats for the first 5h after feeding, hepatic glycogen couldnot have been a net fatty acid precursor. Thus the inhibition of hepaticfatty acid synthesis in this period by glucagon (Img/kg) could not havebeen directly due to depletion of glycogen® The glucagon inhibition oflipogenesis was abolished by adrenalectomy but not potentiated bycorticotropin-treatment, suggesting a permissive role for glucocorticoidhormones. Adrenalectomy also impaired the inhibition of hepatic pyruvatekinase by glucagon but did not abolish the inactivation of pyruvatekinase by 10 )jM-cyclic AMP in vitro« The involvement of L-type pyruvatekinase in the regulation of hepatic fatty acid synthesis is discussed.The integrated regulation of the hepatic pathways of lipogenesis,glycolysis, gluconeogenesis and ketogenesis is considered
Selective changes to phosphatidylcholine and phosphatidylethanolamine molecular species in the developing fetal guinea pig liver and plasma
No full textThe molecular species composition of membrane phospholipids influences the activities of integral proteins and cell signalling pathways. We determined the effect of increasing gestational age on fetal guinea pig liver phosphatidylcholine (PC) and phosphatidylethanolamine (PE), and plasma PC molecular species composition. The livers were collected from fetuses (n = 5/time point) at 5 day intervals between 40 and 65 days of gestation, and at term (68 days). Hepatic PC and PE molecular species composition was determined by electrospray ionisation mass spectrometry. An increasing gestational age was accompanied by selective changes in individual molecular species. The proportion of the sn-1 18:0 species increased relative to the sn-1 16:0 species in liver PC, but not PE, with an increasing gestational age. 1-O-alkyl-2-acyl PC species concentrations decreased significantly between 40 and 45 days of gestation (40%), and 65 and 68 days (54%). Total 1-O-alkenyl-2-acyl PE species concentration increased between days 60 and 65, due to a rise in 1-O-16:0 alkyl/20:4 content, and then decreased until term. Between day 40 and term, PC and PE sn-2 18:2n-6 species concentrations increased 3-fold. PC16:0/18:2 increased gradually throughout gestation, while PC18:0/18:2 content only increased after day 65. The overall increase in PE18:2n-6 content was due to PE18:0/18:2 alone. The composition of plasma PC essentially reflected hepatic PC. Overall, these data suggest differential regulation of hepatic PC and PE molecular species composition during development which is essentially independent of the maternal fatty acid supply
Phosphatidylcholine biosynthesis inside the nucleus: is it involved in regulating cell proliferation?
No full textDynamic lipidomics with stable isotope labeling
No full textIncorporation of stable isotope labelled precursors enables estimation of the kinetics of lipid synthesis and turnover (dynamic lipidomics) in the clinical as well the experimental setting. Recent advances in tandem mass spectrometry extend the analytical possibilities from measurements of isotope enrichments to determinations of intact substrates. Incorporations of deuteriated choline, ethanolamine and inositol can be determined by precursor and neutral loss scans of phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, respectively. This experimental approach provides information on the kinetics of individual phospholipid molecular species and has considerable potential to probe diseases of lipid metabolism in vivo
Mass spectrometry determination of endonuclear phospholipid composition and dynamics
No full textMammalian cell lipid analyses using tandem electrospray ionization mass spectrometry, in conjunction with stable isotope labeling, permit unparalleled access to membrane phospholipid molecular species compositions and turnover. Lipidomic data from isolable compartments of lipid second messenger generation, such as membrane-free nuclei, can provide dynamic insights into the topology of phospholipid turnover. For example, ESI-MS/MS precursor scans of characteristic phosphocholine m/z 184+ fragments reveal a highly saturated endonuclear phosphatidylcholine pool with homeostatic maintenance properties. A spatially distinct CDPcholine pathway yields, within minutes of choline-d9 labeling, unsaturated endonuclear phosphatidylcholines progressively remodeled to more saturated species evidenced by tracking the deuteriated headgroup through precursor scans of phosphocholine-d9 (m/z 193+ fragment). Among the other endonuclear phospholipids, diacyl phosphatidylethanolamines (neutral loss of m/z 141+) are also highly saturated compared with those of whole cell whereas, phophatidylinositols (precursor scans of m/z 241? fragment) are essentially identical in nuclei and whole cells. Moreover, the pattern of myo-inositol-d6 acquisition into endonuclear phosphatidylinositol (precursor scans of m/z 247? fragment) is inconsistent with compartment-specific synthesis. Endonuclear sphingomyelins (seen in precursor scans of m/z 184+ and confirmed from precursor scans of m/z 168? fragments) are enriched but similar in composition to whole cell species whereas endonuclear phosphatidylserines (neutral loss of m/z 87?) are more saturated than their whole cell counterparts. The focus of described methodologies emphasize their value in probing the compositions and dynamics of endonuclear phospholipids, but in principle may be extended to exploration of other isolable compartments including ER or plasma membranes
Dynamic lipidomic insights into phosphatidylcholine synthesis from organelle to organism
No full textRecent technical improvement and technological innovation in small molecule mass spectrometry have provided powerful tools for the intensive metabolomic biochemical investigations that will necessarily characterise the "post-genomic" era of biomedical research. For membrane phospholipids, use of tandem electrospray ionisation mass spectrometry (ESI-MS/MS) exploiting precursor scanning of class-specific diagnostic fragments, can provide detailed quantitative profiles at the level of individual molecular species for many hundreds of unique lipids. Such "snapshot" measurements provide little information concerning metabolic flux. However, recent use of metabolic labelling with stable isotope derivatives of phospholipid headgroups combined with precursor scans of unlabelled and labelled fragments have yielded considerable insight into phosphatidylcholine metabolism in vivo. Here, we briefly review some of the recent work on pathways of phosphatidylcholine metabolism ranging from studies at subcellular organelle level through to whole organism. The sensitivity, specificity and suitability of this powerful methodological approach to numerous questions of phospholipid metabolism place ESI-MS/MS at the very heart of dynamic lipidomics in the foreseeable future
Analysis of the regulation of surfactant phosphatidylcholine metabolism using stable isotopes
No full textThe pathways and mechanisms that regulate pulmonary surfactant synthesis, processing, secretion and catabolism have been extensively characterised using classical biochemical and analytical approaches. These have constructed a model, largely in experimental animals, for surfactant phospholipid metabolism in the alveolar epithelial cell whereby phospholipid synthesised on the endoplasmic reticulum is selectively transported to lamellar body storage vesicles, where it is subsequently processed before secretion into the alveolus. Surfactant phospholipid is a complex mixture of individual molecular species defined by the combination of esterified fatty acid groups and a comprehensive description of surfactant phospholipid metabolism requires consideration of the interactions between such molecular species. However, until recently, lipid analytical techniques have not kept pace with the considerable advances in understanding of the enzymology and molecular biology of surfactant metabolism. Refinements in electrospray ionisation mass spectrometry (ESI-MS) can now provide very sensitive platforms for the rapid characterisation of surfactant phospholipid composition in molecular detail. The combination of ESI-MS and administration of phospholipid substrates labelled with stable isotopes extends this analytical approach to the quantification of synthesis and turnover of individual molecular species of surfactant phospholipid. As this methodology does not involve radioactivity, it is ideally suited to application in clinical studies. This review will provide an overview of the metabolic processes that regulate the molecular specificity of surfactant phosphatidylcholine together with examples of how the application of stable isotope technologies in vivo has, for the first time, begun to explore regulation of the molecular specificity of surfactant synthesis in human subjects
Exogenous surfactant therapy in acute lung injury/acute respiratory distress syndrome: The need for a revised paradigm approach
No full textRegulation of lung surfactant phospholipid synthesis and metabolism
No full textThe alveolar type II epithelial (ATII) cell is highly specialised for the synthesis and storage, in intracellular lamellar bodies, of phospholipid destined for secretion as pulmonary surfactant into the alveolus. Regulation of the enzymology of surfactant phospholipid synthesis and metabolism has been extensively characterised at both molecular and functional levels, but understanding of surfactant phospholipid metabolism in vivo in either healthy or, especially, diseased lungs is still relatively poorly understood. This review will integrate recent advances in the enzymology of surfactant phospholipid metabolism with metabolic studies in vivo in both experimental animals and human subjects. It will highlight developments in the application of stable isotope-labelled precursor substrates and mass spectrometry to probe lung phospholipid metabolism in terms of individual molecular lipid species and identify areas where a more comprehensive metabolic model would have considerable potential for direct application to disease states
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