Dushmanthi Jayasinghe

Peripheral ethanolamine plasmalogen deficiency: a logical causative factor in Alzheimer's disease and dementia

Although dementia of the Alzheimers type (DAT) is the most common form of dementia, the severity of dementia is only weakly correlated with DAT pathology. In contrast, postmortem measurements of cholinergic function and membrane ethanolamine plasmalogen (PlsEtn) content in the cortex and hippocampus correlate with the severity of dementia in DAT. Currently, the largest risk factor for DAT is age. Because the synthesis of PlsEtn occurs via a single nonredundant peroxisomal pathway that has been shown to decrease with age and PlsEtn is decreased in the DAT brain, we investigated potential relationships between serum PlsEtn levels, dementia severity, and DAT pathology. In total, serum PlsEtn levels were measured in five independent population collections comprising >400 clinically demented and >350 nondemented subjects. Circulating PlsEtn levels were observed to be significantly decreased in serum from clinically and pathologically diagnosed DAT subjects at all stages of dementia, and the severity of this decrease correlated with the severity of dementia. Furthermore, a linear regression model predicted that serum PlsEtn levels decrease years before clinical symptoms. The putative roles that PlsEtn biochemistry play in the etiology of cholinergic degeneration, amyloid accumulation, and dementia are discussed.

Reduced levels of hydroxylated, polyunsaturated ultra long-chain fatty acids in the serum of colorectal cancer patients: implications for early screening and detection

BackgroundThere are currently no accurate serum markers for detecting early risk of colorectal cancer (CRC). We therefore developed a non-targeted metabolomics technology to analyse the serum of pre-treatment CRC patients in order to discover putative metabolic markers associated with CRC. Using tandem-mass spectrometry (MS/MS) high throughput MS technology we evaluated the utility of selected markers and this technology for discriminating between CRC and healthy subjects.MethodsBiomarker discovery was performed using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). Comprehensive metabolic profiles of CRC patients and controls from three independent populations from different continents (USA and Japan; total n = 222) were obtained and the best inter-study biomarkers determined. The structural characterization of these and related markers was performed using liquid chromatography (LC) MS/MS and nuclear magnetic resonance technologies. Clinical utility evaluations were performed using a targeted high-throughput triple-quadrupole multiple reaction monitoring (TQ-MRM) method for three biomarkers in two further independent populations from the USA and Japan (total n = 220).ResultsComprehensive metabolomic analyses revealed significantly reduced levels of 28-36 carbon-containing hydroxylated polyunsaturated ultra long-chain fatty-acids in all three independent cohorts of CRC patient samples relative to controls. Structure elucidation studies on the C28 molecules revealed two families harbouring specifically two or three hydroxyl substitutions and varying degrees of unsaturation. The TQ-MRM method successfully validated the FTICR-MS results in two further independent studies. In total, biomarkers in five independent populations across two continental regions were evaluated (three populations by FTICR-MS and two by TQ-MRM). The resultant receiver-operator characteristic curve AUCs ranged from 0.85 to 0.98 (average = 0.91 ± 0.04).ConclusionsA novel comprehensive metabolomics technology was used to identify a systemic metabolic dysregulation comprising previously unknown hydroxylated polyunsaturated ultra-long chain fatty acid metabolites in CRC patients. These metabolites are easily measurable in serum and a decrease in their concentration appears to be highly sensitive and specific for the presence of CRC, regardless of ethnic or geographic background. The measurement of these metabolites may represent an additional tool for the early detection and screening of CRC.

Metabolic system alterations in pancreatic cancer patient serum: potential for early detection

BackgroundThe prognosis of pancreatic cancer (PC) is one of the poorest among all cancers, due largely to the lack of methods for screening and early detection. New biomarkers for identifying high-risk or early-stage subjects could significantly impact PC mortality. The goal of this study was to find metabolic biomarkers associated with PC by using a comprehensive metabolomics technology to compare serum profiles of PC patients to healthy control subjects.MethodsA non-targeted metabolomics approach based on high-resolution, flow-injection Fourier transform ion cyclotron resonance mass spectrometry (FI-FTICR-MS) was used to generate comprehensive metabolomic profiles containing 2478 accurate mass measurements from the serum of Japanese PC patients (n=40) and disease-free subjects (n=50). Targeted flow-injection tandem mass spectrometry (FI-MS/MS) assays for specific metabolic systems were developed and used to validate the FI-FTICR-MS results. A FI-MS/MS assay for the most discriminating metabolite discovered by FI-FTICR-MS (PC-594) was further validated in two USA Caucasian populations; one comprised 14 PCs, six intraductal papillary mucinous neoplasims (IPMN) and 40 controls, and a second comprised 1000 reference subjects aged 30 to 80, which was used to create a distribution of PC-594 levels among the general population.ResultsFI-FTICR-MS metabolomic analysis showed significant reductions in the serum levels of metabolites belonging to five systems in PC patients compared to controls (all p<0.000025). The metabolic systems included 36-carbon ultra long-chain fatty acids, multiple choline-related systems including phosphatidylcholines, lysophosphatidylcholines and sphingomyelins, as well as vinyl ether-containing plasmalogen ethanolamines. ROC-AUCs based on FI-MS/MS of selected markers from each system ranged between 0.93 ±0.03 and 0.97 ±0.02. No significant correlations between any of the systems and disease-stage, gender, or treatment were observed. Biomarker PC-594 (an ultra long-chain fatty acid), was further validated using an independently-collected US Caucasian population (blinded analysis, n=60, p=9.9E-14, AUC=0.97 ±0.02). PC-594 levels across 1000 reference subjects showed an inverse correlation with age, resulting in a drop in the AUC from 0.99 ±0.01 to 0.90 ±0.02 for subjects aged 30 to 80, respectively. A PC-594 test positivity rate of 5.0% in low-risk reference subjects resulted in a PC sensitivity of 87% and a significant improvement in net clinical benefit based on decision curve analysis.ConclusionsThe serum metabolome of PC patients is significantly altered. The utility of serum metabolite biomarkers, particularly PC-594, for identifying subjects with elevated risk of PC should be further investigated.

Membrane plasmalogen composition and cellular cholesterol regulation: a structure activity study

BackgroundDisrupted cholesterol regulation leading to increased circulating and membrane cholesterol levels is implicated in many age-related chronic diseases such as cardiovascular disease (CVD), Alzheimers disease (AD), and cancer. In vitro and ex vivo cellular plasmalogen deficiency models have been shown to exhibit impaired intra- and extra-cellular processing of cholesterol. Furthermore, depleted brain plasmalogens have been implicated in AD and serum plasmalogen deficiencies have been linked to AD, CVD, and cancer.ResultsUsing plasmalogen deficient (NRel-4) and plasmalogen sufficient (HEK293) cells we investigated the effect of species-dependent plasmalogen restoration/augmentation on membrane cholesterol processing. The results of these studies indicate that the esterification of cholesterol is dependent upon the amount of polyunsaturated fatty acid (PUFA)-containing ethanolamine plasmalogen (PlsEtn) present in the membrane. We further elucidate that the concentration-dependent increase in esterified cholesterol observed with PUFA-PlsEtn was due to a concentration-dependent increase in sterol-O-acyltransferase-1 (SOAT1) levels, an observation not reproduced by 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibition.ConclusionThe present study describes a novel mechanism of cholesterol regulation that is consistent with clinical and epidemiological studies of cholesterol, aging and disease. Specifically, the present study describes how selective membrane PUFA-PlsEtn enhancement can be achieved using 1-alkyl-2-PUFA glycerols and through this action reduce levels of total and free cholesterol in cells.

Human serum-derived hydroxy long-chain fatty acids exhibit anti-inflammatory and anti-proliferative activity

BackgroundCirculating levels of novel long-chain hydroxy fatty acids (called GTAs) were recently discovered in the serum of healthy subjects which were shown to be reduced in subjects with colorectal cancer (CRC), independent of tumor burden or disease stage. The levels of GTAs were subsequently observed to exhibit an inverse association with age in the general population. The current work investigates the biological activity of these fatty acids by evaluating the effects of enriched human serum extracts on cell growth and inflammation.MethodsGTAs were extracted from commercially available bulk human serum and then chromatographically separated into enriched (GTA-positive) and depleted (GTA-negative) fractions. SW620, MCF7 and LPS stimulated RAW264.7 cells were treated with various concentrations of the GTA-positive and GTA-negative extracts, and the effects on cell growth and inflammation determined.ResultsEnriched fractions resulted in poly-ADP ribose polymerase (PARP) cleavage, suppression of NFκB, induction of IκBα, and reduction in NOS2 mRNA transcript levels. In RAW264.7 mouse macrophage cells, incubation with enriched fractions prior to treatment with LPS blocked the induction of several pro-inflammatory markers including nitric oxide, TNFα, IL-1β, NOS2 and COX2.ConclusionsOur results show that human serum extracts enriched with endogenous long-chain hydroxy fatty acids possess anti-inflammatory and anti-proliferative activity. These findings support a hypothesis that the reduction of these metabolites with age may result in a compromised ability to defend against uncontrolled cell growth and inflammation, and could therefore represent a significant risk for the development of CRC.

Pancreatic cancer serum biomarker PC-594: Diagnostic performance and comparison to CA19-9.

AIM To investigate serum PC-594 fatty acid levels as a potential biomarker in North American pancreatic cancer (PaC) patients, and to compare its performance to CA19-9. METHODS Using tandem mass spectrometry, we evaluated serum PC-594 levels from 84 North American patients with confirmed PaC and 99 cancer-free control subjects. We determined CA19-9 levels by ELISA. Significance between PaC patients and controls, and association with clinical variables was determined by analysis of variance and t-tests. Diagnostic performance was evaluated by receiver-operator characteristic (ROC) curve analysis, and PC-594 correlation with age and CA19-9 determined by regression analysis. RESULTS Mean PC-594 levels were 3.7 times lower in PaC patients compared to controls (P < 0.0001). The mean level in PaC patient serum was 0.76 ± 0.07 μmol/L, and the mean level in control subjects was 2.79 ± 0.15 μmol/L. There was no correlation between PC-594 and age, disease stage or gender (P > 0.05). Using 1.25 μmol/L as a PC-594 threshold produced a relative risk (RR) of 9.4 (P < 0.0001, 95%CI: 5.0-17.7). The area under the receiver-operator characteristic curve (ROC-AUC) was 0.93 (95%CI: 0.91-0.95) for PC-594 and 0.85 (95%CI: 0.82-0.88) for CA19-9. Sensitivity at 90% specificity was 87% for PC-594 and 71% for CA19-9. Six PaC patients with CA19-9 above 35 U/mL showed normal PC-594 levels, while 24 PaC patients with normal CA19-9 showed low PC-594 levels. Eighty-five of the 99 control subjects (86%) showed normal levels of both markers. CONCLUSION PC-594 biomarker levels are significantly reduced in North American PaC patients, and showed superior diagnostic performance compared to CA19-9.

Plasmalogen Augmentation Reverses Striatal Dopamine Loss in MPTP Mice

Plasmalogens are a class of glycerophospholipids shown to play critical roles in membrane structure and function. Decreased plasmalogens are reported in the brain and blood of Parkinson’s disease (PD) patients. The present study investigated the hypothesis that augmenting plasmalogens could protect striatal dopamine neurons that degenerate in response to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment in mice, a PD model. First, in a pre-treatment experiment male mice were treated for 10 days with the docosahexaenoic acid (DHA)-plasmalogen precursor PPI-1011 (10, 50 and 200 mg/kg). On day 5 mice received MPTP and were killed on day 11. Next, in a post-treatment study, male mice were treated with MPTP and then received daily for 5 days PPI-1011 (5, 10 and 50 mg/kg). MPTP treatment reduced serum plasmalogen levels, striatal contents of dopamine (DA) and its metabolites, serotonin, DA transporter (DAT) and vesicular monoamine transporter 2 (VMAT2). Pre-treatment with PPI-1011 (10 and 50 mg/kg) prevented all MPTP-induced effects. Positive correlations were measured between striatal DA contents and serum plasmalogen levels as well as striatal DAT and VMAT2 specific binding. Post-treatment with PPI-1011 prevented all MPTP-induced effects at 50 mg/kg but not at lower doses. Positive correlations were measured between striatal DA contents and serum plasmalogen levels as well as striatal DAT and VMAT2 specific binding in the post-treatment experiment. PPI-1011 treatment (10 days at 5, 10 and 50 mg/kg) of intact mice left unchanged striatal biogenic amine contents. These data demonstrate that treatment with a plasmalogen precursor is capable of protecting striatal dopamine markers in an animal model of PD.

Plasmalogen precursor mitigates striatal dopamine loss in MPTP mice

Ethanolamine plasmalogens (PlsEtn) are a class of glycerophospholipids characterized by a vinyl-ether bond at the sn-1 position that play an important role in the structure and function of membranes. Previous reports have suggested a link between reduced blood and brain PlsEtn levels and Parkinsons disease (PD). We recently reported that the DHA containing plasmalogen precursor PPI-1011 protected striatal dopamine (DA) against MPTP toxicity in mice. In this paper, we further investigate the specificity requirements of the lipid side chains by testing the oleic acid-containing plasmalogen precursor PPI-1025. Male mice were treated for 10days with daily oral administration of PPI-1025 (10, 50 or 200mg/kg). On day 5 mice received MPTP and were sacrificed on Day 11. Treatment with PPI-1025 prevented MPTP-induced decrease of DA and serotonin, as well as their metabolites. In addition, PPI-1025 treatment prevented the MPTP-induced decrease of the striatal dopamine transporter (DAT) and vesicular monoamine transporter 2 (VMAT2) specific binding. Significant positive correlations were measured between striatal DA concentrations and DAT or VMAT2 specific binding, as well as with serum plasmalogen concentrations. The neuroprotective effect of PPI-1025 displayed a bell-curve dose-dependency losing effect at the highest dose tested. The similar protective response of oleic and docosahexaenoic acid (DHA)-containing plasmalogen precursors suggests that the neuroprotection observed is not only due to DHA but to the oleic substituent and the plasmalogen backbone.

RELATION OF PLASMALOGEN BIOSYNTHESIS TO COGNITION AND AD IN OLDER PERSONS

It has been estimated that 25% of the population over the age of 65 have some form of dementia1, and that the cumulative incidence of dementia in individuals living to the age of 95 is greater than 80%2,3 which means that only 20% of us are protected and that, therefore, the age of onset for 80% of us will be determined by our personal risk factors. Effective prevention or delay strategies, therefore, also need to be personal. This means that we must develop tools that enable the precise and accurate determination of a person’s causal risk factors for specific targeting. The aging population is comprised of a heterogeneous sample of both risk exacerbating and risk reducing genetic, metabolic and behavioral/lifestyle features. Quantifying the interplay of these factors as they relate to specific chronic diseases is at the heart of personalized prevention and longer healthier living. Decreased brain levels of ethanolamine plasmalogens (PlsEtn) in AD were first reported in 19954 and independently confirmed by Han 5, 6 and our lab7. In 2007 we reported that decreased serum PlsEtn levels correlated with both post-mortem pathology and pre-mortem cognitive status in AD and hypothesized that this decrease was causative in AD8 we subsequently showed that pre-mortem cognitive status was more predictive of decreased brain PlsEtn than increased amyloid and that blood and brain levels taken from the same subject were correlated7. In 1998, the first direct evidence of altered membrane structure caused by decreased PlsEtn in AD was reported9. Our hypotheses that decreased membrane PlsEtn played a causative role in the both the cholesterol and amyloid cascades in AD8 were subsequently supported experimentally7,10. Cholesterol, apolipoprotien e4, and PlsEtn are hypothesized to be mechanistically involved in the etiology of dementia and AD pathology. In addition, diabetes and high triglycerides have also been associated with increased incidence of AD. To better understand the role of PlsEtn and their biosynthesis in the risk of AD, serum biomarkers of peroxisomal function and output were measured and their relationship with traditional risk factors such as total cholesterol, High Density Lipoprotein, Low Density Lipoprotein, triglycerides, glucose, and APOE allele status were determined in two well characterized community samples11,12 and the respective contributions to cognition and incident AD determined.