Publications by authors named "Jennifer T Saville"

16 Publications

  • Page 1 of 1

Systemic scAAV9.U1a.hSGSH Delivery Corrects Brain Biochemistry in Mucopolysaccharidosis Type IIIA at Early and Later Stages of Disease.

Hum Gene Ther 2021 Feb 22. Epub 2021 Feb 22.

Genetics and Molecular Pathology, SA Pathology at Women's and Children's Hospital, North Adelaide, South Australia.

Mucopolysaccharidosis type IIIA (MPS IIIA, Sanfilippo A syndrome) is a single gene () childhood onset neurodegenerative disease for which gene therapy is in clinical trial. Theoretically, the transfer of a working gene should enable functional expression of the defective protein and rescue the phenotype when administered before the onset of irreversible disease. Recombinant adeno-associated virus (AAV) is being used as a vehicle for a number of gene therapy applications and the neurotropism of serotype 9 affords utility for monogenetic neurological disorders. To assess the efficacy of restoring the underlying biochemistry in the MPS IIIA brain, tail vein injections of self-complementary AAV9 human -sulfoglucosamine sulfohydrolase (scAAV9.U1A.hSGSH) at 3 × 10 vg/kg were administered to 6- and 16-week-old MPS IIIA mice. Heparan sulfate (HS) and G and G gangliosides were cleared from the cortex, hippocampus and subcortex with residual storage remaining in the brain stem and cerebellum. SGSH activity increased in the brain of the MPS IIIA-treated mice, but remained significantly reduced compared with wild-type. Motor activity as assessed in an open-field arena, and gait length, improved in MPS IIIA mice treated at both 6 and 16 weeks of age. However, functional assessment of cognition in the water cross-maze test, as well as gait width, normalized in mice treated at 6 weeks of age only, with mice treated at 16 weeks performing similar to untreated MPS IIIA mice. Astrogliosis was reduced in mice treated at 6 and 16 weeks of age compared to untreated MPS IIIA mice. These results demonstrate that the gene product is actively clearing primary HS and secondary ganglioside accumulation in MPS IIIA mice, but in older mice, neurocognitive impairments remain. This is likely due to secondary downstream consequences of HS affecting neurological functions that are not reversible upon substrate clearance.
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http://dx.doi.org/10.1089/hum.2020.253DOI Listing
February 2021

Chondroitin sulfate disaccharide is a specific and sensitive biomarker for mucopolysaccharidosis type IVA.

JIMD Rep 2020 Sep 30;55(1):68-74. Epub 2020 Jun 30.

Genetics and Molecular Pathology SA Pathology [at Women's and Children's Hospital] Adelaide South Australia Australia.

Mucopolysaccharidosis type IVA (MPS IVA) is an inborn error of glycosaminoglycan (GAG) catabolism characterized by a deficiency of the lysosomal enzyme, -acetylgalactosamine 6-sulphatase (GALNS). Consequently, partially degraded GAG, chondroitin 6-sulfate (CS) and keratan sulfate (KS), accumulate in the lysosomes of affected cells, primarily in cartilage resulting in skeletal disease. Excessive urinary excretion of these GAG is often used as the initial biochemical parameter to inform a laboratory diagnosis. Here we present the utility of a CS-disaccharide with a non-reducing 6-sulfated -acetylgalactosamine residue (HNAc-UA (1S))-the enzyme's substrate-for the diagnosis and biochemical monitoring of MPS IVA patients. Following implementation of this method into the diagnostic laboratory, we identified one MPS IVA patient over 3 years of MPS urine screening, with no false positive results from 2050 urines tested. Uniquely, urinary concentrations of HNAc-UA (1S) are independent of age meaning that age-related reference ranges are not required. Urinary HNAc-UA (1S) was also able to identify two MPS IVA siblings who remained misdiagnosed with spondyloepiphyseal dysplasia for 5 years because of normal urinary GAG. HNAc-UA (1S) could also be used as a biomarker for monitoring response to enzyme replacement therapy (ERT) as there was a drop in urinary concentration following the administration of ERT in all 12 patients and concentrations correlated with urinary KS ( = 0.92). In conclusion, HNAc-UA (1S) is a reliable, sensitive and specific biomarker for the diagnosis of MPS IVA and can be used to biochemically monitor the response to ERT.
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http://dx.doi.org/10.1002/jmd2.12132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7463049PMC
September 2020

Expanding the clinical utility of glucosylsphingosine for Gaucher disease.

J Inherit Metab Dis 2020 05 26;43(3):558-563. Epub 2019 Nov 26.

Genetics and Molecular Pathology, SA Pathology [at Women's and Children's Hospital], North Adelaide, South Australia, Australia.

Gaucher disease (GD) is an inherited metabolic disorder characterised by impaired catabolism of the glycosphingolipid, glucosylceramide. The deacetylated derivative, glucosylsphingosine (GluSph, lyso-Gb1) has materialised as a biomarker for GD. Further appraisal of the clinical utility of GluSph is required in terms of its prognostic power to inform disease course and pre-symptomatic testing. In this study, we show that plasma GluSph concentrations are significantly higher in GD patients with neuronopathic disease compared with non-neuronopathic disease, even in the neonatal period. A neonate diagnosed at 1 day of age (homozygous for N370S) due to an affected older sibling, returned GluSph of 70 nmol/L compared with 1070-2620 nmol/L for four neuronopathic patients diagnosed <20 days of age. Given this result shows promise for newborn screening, we developed a rapid, simple, and robust assay for GluSph in dried filter paper blood spots (DBS) and were able to detect 23 GD patients from 220 unaffected individuals. Neuronopathic GD patients also had significantly higher DBS concentrations of GluSph than their non-neuronopathic counterparts. We went on to measure GluSph in tissue extracts prepared from chorionic villus sampling and confirmed concentrations were undetectable in unaffected tissue but elevated in GD tissue demonstrating utility in the prenatal setting. Additionally, GluSph is a pharmacodynamic biomarker, revealing a precipitous drop following initiation of enzyme replacement therapy. In conclusion, GluSph is a reliable and specific biomarker for GD and shows promise for prenatal diagnosis and DBS screening programmes.
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http://dx.doi.org/10.1002/jimd.12192DOI Listing
May 2020

Sphingolipid dyshomeostasis in the brain of the mouse model of mucopolysaccharidosis type IIIA.

Mol Genet Metab 2020 02 29;129(2):111-116. Epub 2019 Aug 29.

Genetics and Molecular Pathology, SA Pathology at Women's and Children's Hospital, 72 King William Road, North Adelaide 5006, Australia; School of Medicine, University of Adelaide, Adelaide 5005, Australia. Electronic address:

Gangliosides are complex glycosphingolipids that are vital for proper brain development and function. Alterations in ganglioside metabolism are evident in neurological disorders including the inherited metabolic disease mucopolysaccharidosis type IIIA (MPS IIIA/Sanfilippo A syndrome). Here we sought to comprehensively analyse alterations in ganglioside metabolism within the brain of a naturally occurring MPS IIIA mouse model at early (one month) and late (six months of age) stages of disease progression, as well as the impact on related sphingolipids in the ganglioside metabolic pathway. The simple gangliosides G and G were elevated in the brain stem, cerebellum and sub-cortex of the MPS IIIA mouse at one month of age, but not in the cortex. By six months accumulation was significant throughout the brain, with G gangliosides also elevated. Elevations in other sphingolipids were limited to the upstream synthetic precursors, ceramide and dihexosylceramide (DHC) species containing 18:0 and 20:0 acyl chains, likely due to the abundance of these fatty acids in the elevated gangliosides. In contrast, sphingomyelin, sulphatide and DHC containing a 24:1 fatty acid, were all decreased in the brain stem of the MPS IIIA mice, suggestive of alterations in myelination. These perturbations in sphingolipid metabolism could provide an avenue for therapeutic intervention by manipulation with specific drugs that target the production of these lipids.
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http://dx.doi.org/10.1016/j.ymgme.2019.08.008DOI Listing
February 2020

Evaluation of biomarkers for Sanfilippo syndrome.

Mol Genet Metab 2019 Sep - Oct;128(1-2):68-74. Epub 2019 May 9.

Genetics and Molecular Pathology, SA Pathology at Women's and Children's Hospital, 72 King William Road, North Adelaide 5006, Australia; School of Medicine, University of Adelaide, Adelaide 5005, Australia. Electronic address:

Sanfilippo syndrome or mucopolysaccharidosis type III (MPS III) is a childhood metabolic disorder marked by neuropathology arising due to impaired heparan sulphate (HS) catabolism. Consequently, partially degraded HS accumulates in the lysosomes of affected cells and is excreted in the urine. The measurement of HS in urine has long been considered a biomarker of Sanfilippo syndrome although it is largely non-specific. Using blood, urine and CSF collected from a cohort of Sanfilippo patients we investigated the utility of primary and secondary biomarkers to inform on disease activity. These included enzyme activity, specific oligosaccharides with non-reducing end residues reflective of the enzyme deficiency, and gangliosides. The diagnostic oligosaccharides - a HS disaccharide and tetrasaccharide - were elevated in the urine, plasma and CSF of all MPS IIIA and IIIB patients, respectively. There was no correlation between the concentrations in any of the matrices suggesting they reflect specific tissues and not overall disease burden. Enzyme activity did not inform on disease severity, with no measurable activity in CSF and activity approaching normal in MPS IIIA plasma. The concentration of gangliosides, G and G, were significantly higher in the CSF of all MPS III subjects when compared to controls and correlated with the age of onset of first symptoms. Given that these gangliosides reflect delayed brain development they may be useful measures of disease burden, within the limitations of the clinical surrogates. Observation of these biochemical measurements in MPS III patients enrolled in clinical trials may determine whether they represent true pharmacodynamics biomarkers.
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http://dx.doi.org/10.1016/j.ymgme.2019.05.005DOI Listing
April 2020

Disease and subtype specific signatures enable precise diagnosis of the mucopolysaccharidoses.

Genet Med 2019 03 31;21(3):753-757. Epub 2018 Jul 31.

Genetics and Molecular Pathology, SA Pathology at Women's and Children's Hospital, North Adelaide, South Australia, Australia.

Purpose: Expanding treatments for the mucopolysaccharidoses-a family of genetic disorders-place unprecedented demands for accurate, timely diagnosis because best outcomes are seen with early initiation of appropriate therapies. Here we sought to improve the diagnostic odyssey by measuring specific glycosaminoglycan fragments with terminal residues complicit with the genetic defect resulting in precise diagnosis rather than the usual first-line, ambiguous total glycosaminoglycan determinations that return poor diagnostic yield.

Methods: A derivatizing reagent was added to urine aliquots (0.5 μmol creatinine) before separation of the glycosaminoglycan fragments by liquid chromatography and quantification with electrospray ionization-tandem mass spectrometry using multiple reaction monitoring for 10 targeted fragments plus the internal standard.

Results: All 93 mucopolysaccharidosis patients were correctly identified as 1 of 10 subtypes from a total of 723 de-identified subjects-blinded to diagnosis-based on the presence of specific "signature" glycosaminoglycan fragments. Employing reference intervals calculated from 630 unaffected urines, with 99% confidence intervals, provided a laboratory test with 100% specificity and sensitivity.

Conclusion: This novel urine assay allows diagnosis of 10 mucopolysaccharidosis subtypes in a single test. The precise quantification of unique glycosaminoglycan fragments also enables longitudinal biochemical monitoring following therapeutic interventions.
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http://dx.doi.org/10.1038/s41436-018-0136-zDOI Listing
March 2019

Mass spectrometry-directed structure elucidation and total synthesis of ultra-long chain (-acyl)-ω-hydroxy fatty acids.

J Lipid Res 2018 08 15;59(8):1510-1518. Epub 2018 Jun 15.

Central Analytical Research Facility, Institute for Future Environments Queensland University of Technology, Brisbane, Queensland, Australia

The (-acyl)-ω-hydroxy FAs (OAHFAs) comprise an unusual lipid subclass present in the skin, vernix caseosa, and meibomian gland secretions. Although they are structurally related to the general class of FA esters of hydroxy FAs (FAHFAs), the ultra-long chain (30-34 carbons) and the putative ω-substitution of the backbone hydroxy FA suggest that OAHFAs have unique biochemistry. Complete structural elucidation of OAHFAs has been challenging because of their low abundance within complex lipid matrices. Furthermore, because these compounds occur as a mixture of closely related isomers, insufficient spectroscopic data have been obtained to guide structure confirmation by total synthesis. Here, we describe the full molecular structure of ultra-long chain OAHFAs extracted from human meibum by exploiting the gas-phase purification of lipids through multi-stage MS and novel multidimensional ion activation methods. The analysis elucidated sites of unsaturation, the stereochemical configuration of carbon-carbon double bonds, and ester linkage regiochemistry. Such isomer-resolved MS guided the first total synthesis of an ultra-long chain OAHFA, which, in turn, confirmed the structure of the most abundant OAHFA found in human meibum, OAHFA 50:2. The availability of a synthetic OAHFA opens new territory for future investigations into the unique biophysical and biochemical properties of these lipids.
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http://dx.doi.org/10.1194/jlr.M086702DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6071768PMC
August 2018

Glycosaminoglycan fragments as a measure of disease burden in the mucopolysaccharidosis type I mouse.

Mol Genet Metab 2018 02 13;123(2):112-117. Epub 2017 Dec 13.

Genetics and Molecular Pathology, SA Pathology, Women's and Children's Hospital, 72 King William Road, North Adelaide, South Australia 5006, Australia; School of Medicine, University of Adelaide, Adelaide, South Australia 5005, Australia. Electronic address:

Glycosaminoglycan (GAG) catabolism involves endo-hydrolysis of polysaccharides followed by the sequential removal of the non-reducing end residue from the resulting oligosaccharides by exo-enzymes. In the inherited metabolic disorder, mucopolysaccharidosis type I (MPS I), a deficiency in the exo-enzyme, α-l-iduronidase, prevents removal of α-l-iduronic acid residues from the non-reducing end of the GAGs, heparan sulphate (HS) and dermatan sulphate (DS). The excretion of partially degraded HS and DS in urine of MPS I patients has long been recognized, but the question of whether they do indeed reflect GAG load in a particular tissue has not been addressed - an important issue in the context of biomarkers for assessment of disease burden in MPS I. Therefore, we measured specific low molecular weight HS and DS oligosaccharides with terminal α-l-iduronic acid residues, in the brain, liver, kidney, serum and urine, and correlated these findings with total GAG in the MPS I mouse model. Six oligosaccharides were identified in the urine, ranging from di- to pentasaccharides. Of these, five were observed in the kidney, four in the liver and brain, with the three most abundant in urine also seen in serum. These oligosaccharides accounted for just 0.1-2% of total GAG, with a disaccharide showing the best correlation with total GAG. The oligosaccharides and total GAG were most abundant in the liver, with the least observed in the brain. The concentration of oligosaccharides as a percentage of total GAG in urine was similar to that observed in the kidney, and both revealed a similar ratio of HS:DS, suggesting that the oligosaccharide storage pattern in urine is a reflection of that in the kidney. Serum, liver and brain had a similar ratio of HS:DS, which was lower to that seen in the urine and kidney. The distribution of oligosaccharides when ranked from most to least abundant, was also the same between serum, liver and brain suggesting that serum more closely reflects the oligosaccharides of the brain and liver and may therefore be a more informative measurement of disease burden than urine. The accumulation of HS and DS oligosaccharides was observed in the brain as early as one month of age, with the disaccharide showing a continuous increase with age. This demonstrates the progressive nature of the disease and as such this disaccharide could prove to be a useful biomarker to measure disease burden in MPS I.
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http://dx.doi.org/10.1016/j.ymgme.2017.12.007DOI Listing
February 2018

Reduced cerebral vascularization in experimental neuronopathic Gaucher disease.

J Pathol 2018 01;244(1):120-128

Department of Medicine, University of Cambridge, Cambridge, UK.

The glycosphingolipidosis, Gaucher disease, in which a range of neurological manifestations occur, results from a deficiency of acid β-glucocerebrosidase, with subsequent accumulation of β-glucocerebroside, its upstream substrates, and the non-acylated congener β-glucosylsphingosine. However, the mechanisms by which end-organ dysfunction arise are poorly understood. Here, we report strikingly diminished cerebral microvascular density in a murine model of disease, and provide a detailed analysis of the accompanying cerebral glycosphingolipidome in these animals, with marked elevations of β-glucosylsphingosine. Further in vitro studies confirmed a concentration-dependent impairment of endothelial cytokinesis upon exposure to quasi-pathological concentrations of β-glucosylsphingosine. These findings support a premise for pathogenic disruption of cerebral angiogenesis as an end-organ effect, with potential for therapeutic modulation in neuronopathic Gaucher disease. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/path.4992DOI Listing
January 2018

Subregional brain distribution of simple and complex glycosphingolipids in the mucopolysaccharidosis type I (Hurler syndrome) mouse: impact of diet.

J Neurochem 2017 04 13;141(2):287-295. Epub 2017 Mar 13.

Genetics and Molecular Pathology, SA Pathology at Women's and Children's Hospital, North Adelaide, South Australia, Australia.

Gangliosides are the most complex oligosaccharide-containing glycosphingolipids defined by the presence of sialic acid and although present in all tissues, predominate in the brain. Considering their importance in neural development, it is unsurprising that ganglioside metabolism is altered in neurodegenerative diseases. The severe form of mucopolysaccharidosis type I, Hurler syndrome (HS), is characterised by progressive loss of neuronal function through largely undefined mechanisms. Here, we sought to interrogate brain gangliosides in a murine model of HS and further, assessed whether dietary modulation of lipid metabolism effected correction of the metabolic abnormalities. The simple gangliosides, G , G , G and G were elevated in the five subregions examined - brain stem, cerebellum, cortex, hippocampus, subcortex - in HS mice as early as 2 months of age compared with their wild type counterparts. Their elevation persisted at 6 months of age, imparting protracted neurological development as these simple gangliosides have usually subsided by this stage of brain development. Their immediate synthetic precursor, lactosylceramide, was also elevated, suggesting that their increase arises at this metabolic intermediary, as dihydroceramide, ceramide and monohexosylceramide were unaffected. Dietary linoleic acid supplementation significantly reduced G and G , and furthermore, improved exploratory behaviour as assessed by the open field test, highlighting the possibility of further exploring dietary intervention as a therapeutic consideration.
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http://dx.doi.org/10.1111/jnc.13976DOI Listing
April 2017

Quantification of plasma sulfatides by mass spectrometry: Utility for metachromatic leukodystrophy.

Anal Chim Acta 2017 Feb 19;955:79-85. Epub 2016 Dec 19.

Genetics and Molecular Pathology, SA Pathology, at Women's and Children's Hospital, 72 King William Road, North Adelaide, South Australia, 5006, Australia; School of Medicine, University of Adelaide, Adelaide, South Australia, 5005, Australia. Electronic address:

Impaired sulfatide catabolism is the primary biochemical insult in patients with the inherited neurodegenerative disease, metachromatic leukodystrophy (MLD), and sulfatide elevation in body fluids is useful in the diagnostic setting. Here we used mass spectrometry to quantify fourteen species of sulfatide, in addition to the deacetylated derivative, lyso-sulfatide, using high pressure liquid chromatography-electrospray ionisation-tandem mass spectrometry in both positive and negative ion mode. A single phase extraction of 0.01 mL of MLD plasma identified all 14 sulfatide species in the positive ion mode but none in the negative ion mode. Interrogation of seven major and seven hydroxylated molecular species, as well as lyso-sulfatide, identified the C18 isoform as the most informative for MLD. The C18 produced a linear response and was below the limit of quantification (<10 pmol mL) in control plasma with concentrations in MLD plasma ranging from 12 to 196 pmol mL. Serial plasma samples from an MLD patient post-therapeutic bone marrow transplant proved similar to non-disease controls with C18 sulfatide concentrations below the limit of quantification, as did samples from three individuals with an arylsulfatase A pseudodeficiency - a population variant which appears deficient upon enzymatic assay, without manifestation of disease. These findings emphasise the utility of the C18 sulfatide species for the diagnosis of MLD and biochemical monitoring of MLD patients. Extension of this approach to a newborn screening card correctly identified an MLD patient at birth with elevated C18 sulfatide at levels almost double that present in the newborn card from his unaffected sibling, suggesting the methodology may have applicability for newborn screening.
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http://dx.doi.org/10.1016/j.aca.2016.12.002DOI Listing
February 2017

Determination of ester position in isomeric (O-acyl)-hydroxy fatty acids by ion trap mass spectrometry.

Rapid Commun Mass Spectrom 2016 Nov;30(21):2351-2359

Central Analytical Research Facility, Queensland University of Technology, Brisbane, QLD, 4001, Australia.

Rationale: (O-acyl)-hydroxy fatty acids (OAHFAs) are a recently discovered class of endogenous lipids, generating significant interest for their correlation with enhanced glucose tolerance. Structural variants that differ in the position of the ester linkage have been described, including the ω-OAHFA sub-class, that plays a key role in stabilizing the human tear film. Developing analytical tools for rapid and unambiguous structural elucidation of OAHFAs is essential to understanding their diverse physiological functions.

Methods: Commercially available and synthesized OAHFA standards were dissolved in chloroform and subsequently diluted into methanol with 1.5 mM ammonium acetate. Negative ion collision-induced dissociation (CID) MS spectra were acquired using chip-based nano-electrospray ionization (Advion TriVersa NanoMate) coupled to an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific).

Results: Major product ions observed during CID of [OAHFA - H] ions readily identify the constituent fatty acid and hydroxy fatty acid; however, isomers are not easily distinguished. Interrogation of the hydroxy fatty acid and dehydrated hydroxy fatty acid product ions by MS and ion-molecule reactions yielded diagnostic ions that readily pinpoint hydroxylation position and, thus, the OAHFA ester location. Conversely, these ions are characteristically absent in the MS spectra of ω-OAHFAs. Unimolecular dissociation mechanisms are proposed, which are shown to be consistent with prior isotopic labelling experiments.

Conclusions: A mechanistic rationale is provided to explain the unimolecular dissociation of [OAHFA - H] ions in an ion trap mass spectrometer, thus enabling near-complete de novo structural elucidation of OAHFAs in shotgun lipidomics workflows, even if synthetic standards are unavailable for comparison. Copyright © 2016 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/rcm.7715DOI Listing
November 2016

Selective normalisation of regional brain bis(monoacylglycero)phosphate in the mucopolysaccharidosis 1 (Hurler) mouse.

Exp Neurol 2016 Mar 19;277:68-75. Epub 2015 Dec 19.

Genetics and Molecular Pathology, SA Pathology at Women's and Children's Hospital, 72 King William Road, North Adelaide, South Australia 5006, Australia,; Department of Paediatrics, University of Adelaide, Adelaide, South Australia 5005, Australia. Electronic address:

Bis(monoacylglycero)phosphate (BMP) is a glycerophospholipid highly enriched in the lysosomal network and elevated in lysosomal diseases. To correct this elevation, BMP synthesis was manipulated by dietary fatty acid supplementation and the impact on subregional brain BMP and pathology assessed in the mouse model of mucopolysaccharidosis 1 (Hurler syndrome (HS)). There was widespread elevation of BMP in HS mice across all six sub-regions - brain stem, cortex, cerebellum, hippocampus, olfactory bulb and the sub-cortex - with 22:6/22:6 the most abundant species. Linoleic acid normalised total BMP in all regions except the cortex and cerebellum, although there were differences in fatty acid species; the major finding a decrease in 22:6- and a concomitant increase in 22:5-containing species. A battery of behaviour assessments showed that in the water cross maze both HS and wild type mice performed less well on the linoleic acid diet, and that both HS and wild type mice on the linoleic acid diet performed similarly and better in the exploratory open field test. This may be a consequence of differential subregional BMP composition in the brain. The effects of high fat and docosahexaenoic/eicosapentaenoic acid enriched diets were generally unremarkable. Although major pathologies were not completely abrogated, much of the neurobehavioural testing was confounded by skeletal pathology that did not resolve. This is the first detailed characterisation of subregional brain BMP species informing on the ability to manipulate this phospholipid in the brain, and as such, may hold promise as an adjunct therapy not only for HS but also for other lysosomal diseases.
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http://dx.doi.org/10.1016/j.expneurol.2015.12.012DOI Listing
March 2016

Ceramide accumulation in L6 skeletal muscle cells due to increased activity of ceramide synthase isoforms has opposing effects on insulin action to those caused by palmitate treatment.

Diabetologia 2013 Dec 29;56(12):2697-701. Epub 2013 Aug 29.

Diabetes and Obesity Program, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, NSW, 2010, Australia.

Aims/hypothesis: An accumulation of ceramides has been implicated in the generation of insulin resistance in skeletal muscle upon an oversupply of fatty acid. Different ceramide species are generated through the actions of ceramide synthases (CerSs), which incorporate specific acyl side chains. We tested whether particular CerS isoforms promoted insulin resistance through the generation of more inhibitory ceramide species, thus representing potential targets for intervention.

Methods: CerS isoforms CerS1, CerS2, CerS4, CerS5 and CerS6 were overexpressed in L6 myotubes using adenovirus, and cells were treated with palmitate and stimulated with insulin. Alternatively, CerS isoforms were knocked down using siRNAs. Sphingolipids were examined by mass spectrometry and tracer incorporation. Phosphorylation of IRS1 and Akt was measured by immunoblotting, while glucose disposal was assessed by measuring GLUT4 translocation and the incorporation of [(14)C]glucose into glycogen.

Results: Palmitate treatment increased the levels of several ceramides but reduced the levels of sphingomyelins, while insulin had no effect. The fatty acid also inhibited insulin-stimulated Akt phosphorylation and glycogen synthesis. Overexpression of CerS isoforms increased specific ceramides. Unexpectedly, the overexpression of CerS1 and CerS6 promoted insulin action, while no isoform had inhibitory effects. CerS6 knockdown had effects reciprocal to those of CerS6 overexpression.

Conclusions/interpretation: Palmitate may increase intracellular ceramide levels through sphingomyelin hydrolysis as well as de novo synthesis, but no particular species were implicated in the generation of insulin resistance. The modulation of ceramides through an alteration of CerS expression does not affect the action of insulin in the same way as ceramide generation by palmitate treatment. Conversely, certain isoforms promote insulin action, indicating the importance of ceramides in cell function.
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http://dx.doi.org/10.1007/s00125-013-3035-5DOI Listing
December 2013

Identification of phospholipids in human meibum by nano-electrospray ionisation tandem mass spectrometry.

Exp Eye Res 2011 Mar 31;92(3):238-40. Epub 2010 Dec 31.

Meibum is believed to be the major source of tear film lipids, which are vital in the prevention of excess evaporation of the aqueous phase. The complete lipid composition of meibum has yet to be established. While earlier studies reported the presence of phospholipids in human meibum, recent mass spectrometric studies have not detected them. In this study we use electrospray ionisation tandem mass spectrometry to investigate the presence of phospholipids in meibum and provide comparison to the phospholipid profile of tears. Lipids were extracted from human meibum and tear samples using standard biphasic methods and analysed by nano-electrospray ionisation tandem mass spectrometry using targeted ion scans. A total of 35 choline-containing phospholipids were identified in meibum and the profile of these was similar to that observed in tears, suggesting tear lipids are derived from meibum. The results shown here highlight the need for a combination of optimised techniques to enable the identification of the large range of lipid classes in meibum.
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http://dx.doi.org/10.1016/j.exer.2010.12.012DOI Listing
March 2011

Detection and quantification of tear phospholipids and cholesterol in contact lens deposits: the effect of contact lens material and lens care solution.

Invest Ophthalmol Vis Sci 2010 Jun 20;51(6):2843-51. Epub 2010 Jan 20.

School of Chemistry, University of Wollongong, Wollongong, New South Wales, Australia.

Purpose: To examine the deposition of tear phospholipids and cholesterol onto worn contact lenses and the effect of lens material and lens care solution.

Methods: Lipids were extracted from tears and worn contact lenses using 2:1 chloroform:methanol and the extract washed with aqueous ammonium acetate, before analysis by electrospray ionization tandem mass spectrometry (ESI-MS/MS).

Results: Twenty-three molecular lipids from the sphingomyelin (SM) and phosphatidylcholine (PC) classes were detected in tears, with total concentrations of each class determined to be 5 +/- 1 pmol/microL ( approximately 3.8 microg/mL) and 6 +/- 1 pmol/microL ( approximately 4.6 microg/mL), respectively. The profile of individual phospholipids in both of these classes was shown to be similar in contact lens deposits. Deposition of representative polar and nonpolar lipids were shown to be significantly higher on senofilcon A contact lenses, with approximately 59 ng/lens SM, 195 ng/lens PC, and 9.9 microg/lens cholesterol detected, whereas balafilcon A lens extracts contained approximately 19 ng/lens SM, 19 ng/lens PC, and 3.9 microg/lens cholesterol. Extracts from lenses disinfected and cleaned with two lens care solutions showed no significant differences in total PC and SM concentrations; however, a greater proportion of PC than SM was observed, compared with that in tears.

Conclusions: Phospholipid deposits extracted from worn contact lenses show a molecular profile similar to that in tears. The concentration of representative polar and nonpolar lipids deposited onto contact lenses is significantly affected by lens composition. There is a differential efficacy in the removal of PC and SM with lens care solutions.
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http://dx.doi.org/10.1167/iovs.09-4609DOI Listing
June 2010