Brain ethanolamine phospholipids, neuropathology and cognition: A comparative post-mortem analysis of structurally specific plasmalogen and phosphatidyl species.

Reduced cognition in the elderly is associated with low levels of plasmalogens and high levels of lipid rafts, amyloid plaques, and neurofibrillary tangles in the temporal cortex. A systematic integrative analysis of key indices of these pathologies to determine their collective and independent contributions to cognition was performed. Levels of four phosphatidylethanolamines (PE) and four ethanolamine plasmalogens (PL) of identical sn-1 carbon length and desaturation (stearic, 18:0) and identical sn-2 fatty acid compositions of varying side chain lengths and degrees of unsaturation (oleic, 18:1; linoleic, 18:2; arachidonic, 20:4; docosahexaenoic, 22:6), flotillin-1 expression and amyloid plaque and neurofibrillary tangle densities were measured in inferior temporal cortex tissue from 100 elderly subjects (Rush University Memory and Aging Project, 88.5 ± 5.8 years old). Subjects were evenly distributed with respect to gender (52/48, F/M) and cognitive status (38/24/38, no cognitive impairment/mild cognitive impairment/Alzheimer's dementia) proximate to death. Multivariate logistic regression analyses were used to determine the relative and collective associations of the neuropathological indices with cognition. Higher levels of tangles, amyloid, or flotillin and lower levels of PL 18:0/22:6 were significantly associated with lower cognition in the base model (adjusted for age, sex, education). Multivariate analysis revealed that only PL 18:0/22:6 (ß = 0.506; p < 0.00001), tangles (-0.307; p < 0.01), and flotillin (-0.2027; p < 0.05) were independently associated with reduced cognition. PL 18:0/22:6 and PE 18:0/22:6 levels were independently associated with cognition in the presence of tangles, amyloid, and flotillin, but only PL 18:0/22:6 retained its association with cognition when both PL and PE 18:0/22:6 were included in the model indicating that PE 18:0/22:6 levels were associated with PL 18:0/22:6, not cognition. Only high brain levels of PL 18:0/22:6 (>mean+1SD) was predictive of normal cognition (coef = 1.67, p < 0.05) and non-demented state (coef = -2.73, p < 0.001), whereas low levels of PL 18:0/22:6 and high levels of tangles or flotillin were predictive of dementia. The association of high brain polyunsaturated (PUFA)-PL levels with better cognition was independent of amyloid plaque, neurofibrillary tangle, PE, and flotillin-1 expression. Maintenance or augmentation of brain docosahexaenoic (DHA)-PL levels warrants further investigation as a target for preventing cognitive decline or improving cognition in the elderly, respectively.

Targeted Plasmalogen Supplementation: Effects on Blood Plasmalogens, Oxidative Stress Biomarkers, Cognition, and Mobility in Cognitively Impaired Persons.

Plasmalogens are a specific type of glycerophospholipid found in especially high levels in neuronal membranes. Decreased blood and brain levels of docosahexaenoic acid (DHA) containing plasmalogens are associated with decreased cognition and neuromuscular function in humans. Administration of 1-O-alkyl-2-acylglycerol (AAG) plasmalogen precursors containing DHA at the sn-2 position dose-dependently increase blood DHA plasmalogens and are neuroprotective in animal models of neurodegeneration at doses between 10 and 50 mg/kg. We conducted an investigational clinical trial in 22 cognitively impaired persons to evaluate the effects of an escalating oral dosing regimen of DHA-AAG from 900 to 3,600 mg/day over a 4-month period on blood serum plasmalogen and non-plasmalogen phospholipids and oxidative stress biomarkers. Safety, tolerability and therapeutic effects on cognition and mobility were also evaluated. DHA plasmalogen levels increased with increasing dose and remained significantly elevated at all treatment doses and durations. DHA plasmalogen levels were positively associated with catalase activity and negatively associated with malondialdehyde (MDA) levels. DHA-AAG supplementation normalized catalase activity in persons with low baseline catalase activity, normalized MDA levels in persons with high baseline MDA levels, and normalized superoxide dismutase activity in persons with high baseline SOD activity. Cognition improved in nine participants, was unchanged in nine, and declined in four. Mobility improved in twelve, was unchanged in five and declined in four participants. Changes in cognition and mobility were statistically significant versus a random outcome. Baseline DHA-plasmalogen levels were not predictive of clinical response. DHA-AAG was well tolerated at all dosages and no adverse reactions were observed.

Plasmalogen Deficiency and Overactive Fatty Acid Elongation Biomarkers in Serum of Breast Cancer Patients Pre- and Post-Surgery-New Insights on Diagnosis, Risk Assessment, and Disease Mechanisms.

The polyunsaturated fatty acid (PUFA) elongase, ELOVL5, is upregulated in breast cancer (BC) vs. adjacent normal tissue. We performed a comprehensive lipid metabolomic analysis of serum using high-resolution accurate mass spectrometry from two case-control studies that included non-BC, BC subjects pre-surgery, and BC subjects one-month post-surgery to determine if the metabolic signatures of over-active fatty acid elongation and other lipid changes could be detected in BC vs. non-BC subjects: study 1 (n = 48: non-BC, n = 69: pre-surgery BC); study 2 (blinded validation: n = 121: non-BC, n = 62: pre-surgery BC, n = 31: one month post-surgery). The ratio of the ELOVL5 precursor, linoleic acid (18:2) to a non-ELOVL5 precursor, oleic acid (18:1) was evaluated in multiple lipid pools (phosphatidylethanolamine (PtdEtn), phosphatidylcholine (PtdCho), lyso-PtdCho, and free fatty acids). This ratio was lower in pre-surgery BC subjects in all pools in both studies (p < 0.001). At one-month post-surgery, the 18:2/18:1 ratios increased vs. pre-surgery and were no longer different from non-BC subjects (p > 0.05 expect for lyso-PtdCho). In contrast to the elongation biomarkers, docosahexaenoic acid (22:6n-3) containing ethanolamine plasmalogen (EtnPls) species were observed to be further decreased in BC subjects one-month post-surgery vs. pre-surgery levels (p < 0.001). These results are consistent with the hypothesis that ELOVL5 is upregulated in BC tissue, which would result in the selective depletion of 18:2 vs. 18:1 containing lipid species. Surgical removal of the tumor removes the overactive ELOVL5 effect on serum lipids. In contrast, the low EtnPls levels do not appear to be caused by BC tumor activity and may be pre-existent and a possible risk factor for BC. These results indicate that it may be possible to screen for both breast cancer risk and breast cancer activity using a simple blood test.

Circulating ethanolamine plasmalogen indices in Alzheimer's disease: Relation to diagnosis, cognition, and CSF tau.

INTRODUCTION: Altered lipid metabolism is implicated in Alzheimer's disease (AD), but the mechanisms remain obscure. Aging-related declines in circulating plasmalogens containing omega-3 fatty acids may increase AD risk by reducing plasmalogen availability. METHODS: We measured four ethanolamine plasmalogens (PlsEtns) and four closely related phosphatidylethanolamines (PtdEtns) from the Alzheimer's Disease Neuroimaging Initiative (ADNI; n = 1547 serum) and University of Pennsylvania (UPenn; n = 112 plasma) cohorts, and derived indices reflecting PlsEtn and PtdEtn metabolism: PL-PX (PlsEtns), PL/PE (PlsEtn/PtdEtn ratios), and PBV (plasmalogen biosynthesis value; a composite index). We tested associations with baseline diagnosis, cognition, and cerebrospinal fluid (CSF) AD biomarkers. RESULTS: Results revealed statistically significant negative relationships in ADNI between AD versus CN with PL-PX (P = 0.007) and PBV (P = 0.005), late mild cognitive impairment (LMCI) versus cognitively normal (CN) with PL-PX (P = 2.89 × 10-5 ) and PBV (P = 1.99 × 10-4 ), and AD versus LMCI with PL/PE (P = 1.85 × 10-4 ). In the UPenn cohort, AD versus CN diagnosis associated negatively with PL/PE (P = 0.0191) and PBV (P = 0.0296). In ADNI, cognition was negatively associated with plasmalogen indices, including Alzheimer's Disease Assessment Scale 13-item cognitive subscale (ADAS-Cog13; PL-PX: P = 3.24 × 10-6 ; PBV: P = 6.92 × 10-5 ) and Mini-Mental State Examination (MMSE; PL-PX: P = 1.28 × 10-9 ; PBV: P = 6.50 × 10-9 ). In the UPenn cohort, there was a trend toward a similar relationship of MMSE with PL/PE (P = 0.0949). In ADNI, CSF total-tau was negatively associated with PL-PX (P = 5.55 × 10-6 ) and PBV (P = 7.77 × 10-6 ). Additionally, CSF t-tau/Aß1-42 ratio was negatively associated with these same indices (PL-PX, P = 2.73 × 10-6 ; PBV, P = 4.39 × 10-6 ). In the UPenn cohort, PL/PE was negatively associated with CSF total-tau (P = 0.031) and t-tau/Aß1-42 (P = 0.021). CSF Aß1-42 was not significantly associated with any of these indices in either cohort. DISCUSSION: These data extend previous studies by showing an association of decreased plasmalogen indices with AD, mild cognitive impairment (MCI), cognition, and CSF tau. Future studies are needed to better define mechanistic relationships, and to test the effects of interventions designed to replete serum plasmalogens.

Altered ethanolamine plasmalogen and phosphatidylethanolamine levels in blood plasma of patients with bipolar disorder.

AIM: Ethanolamine-containing phospholipids are synthesized in endoplasmic reticulum (ER) and mitochondria. ER stress and mitochondrial dysfunction have been implicated in bipolar disorder (BP). In this study, we aimed to examine the relationship of ethanolamine plasmalogen (PLE) and phosphatidylethanolamine (PTE) levels in blood plasma with BP. METHODS: Plasma PLE and PTE levels were compared between 34 patients with BP (DSM-IV) and 38 healthy control participants matched for age, sex, and ethnicity (Japanese). Furthermore, the relationships of plasma PLE and PTE levels with clinical variables were explored. RESULTS: Plasma PLE levels were significantly lower in patients with BP than in healthy controls (P = 0.0033). In subgroup analyses, plasma PLE levels were significantly lower in patients with BP type I (BP I) than in healthy controls (P = 0.0047); furthermore, plasma PTE levels were significantly lower in patients with BP I than in controls (P = 0.016) and patients with BP type II (BP II) (P = 0.010). Receiver-operating characteristic curve analysis revealed that the discriminatory power of plasma PTE levels for distinguishing between BP I and II was fair (area under the curve = 0.78; P = 0.0095). There were no significant correlations of plasma PLE or PTE levels with depression or manic symptoms in patients. CONCLUSIONS: Plasma PLE and PTE levels were associated with BP I, but not with BP II. Moreover, plasma PTE levels differed between patients with BP I and II. Our findings highlight the importance of ethanolamine phospholipids in the pathophysiology of BP, especially BP I.

Plasmalogen deficiency and neuropathology in Alzheimer's disease: Causation or coincidence?

Causation of Alzheimer's disease (AD) is not well understood. It is necessary to look beyond neuropathology to identify the underlying causes of AD and many other common neurological diseases. Lipid abnormalities are well documented in the preclinical phases of many neurological diseases including AD. Here, we use AD as an example to examine the role of lipid abnormalities as an underlying cause of neurodegeneration. Role of lipids, particularly phospholipids, in the optimal function of the nervous system, impact of the aberrations of phospholipid metabolism on ß-amyloid deposition and cholinergic neuronal function, epidemiological evidence on the association of phospholipids with AD, and preliminary data on the possible modulation of risk factors of AD by phospholipids are examined. Implications of these findings on diagnosis and prevention are also discussed.

Relation of Serum Plasmalogens and APOE Genotype to Cognition and Dementia in Older Persons in a Cross-Sectional Study.

Using a community sample of 1205 elderly persons, we investigated the associations and potential interactions between Apolipoprotein E (APOE) genotype and serum phosphatidylethanolamine (PlsEtn) on cognition and dementia. For each person, APOE genotype, PlsEtn Biosynthesis value (PBV, the combination of three key PlsEtn species), cognition (the combination of five specific cognitive domains), and diagnosis of dementia was determined. APOE genotype and PBV were observed to be non-interacting (p > 0.05) and independently associated with cognition: APOE (relative to ε3ε3:ε2ε3 (Coef = 0·14, p = 4.2 × 10-2); ε3ε4/ε4ε4 (Coef = -0.22, p = 6.2 × 10-5); PBV (Coef = 0.12, p = 1.7 × 10-7) and dementia: APOE (relative to ε3ε3:ε2ε3 (Odds Ratio OR = 0.44, p = 3.0 × 10-2); ε3ε4/ε4ε4 (OR = 2.1, p = 2.2 × 10-4)); PBV (OR = 0.61, p = 3.3 × 10-6). Associations are expressed per standard deviation (SD) and adjusted for serum lipids and demographics. Due to the independent and non-interacting nature of the APOE and PBV associations, the prevalence of dementia in APOE ε3ε4/ε4ε4 persons with high PBV values (>1 SD from mean) was observed to be the same as APOE ε3ε3 persons (14.3% versus 14.0%). Similarly, the prevalence of dementia in APOE ε3ε3 persons with high PBV values was only 5.7% versus 6.7% for APOE ε2ε3 persons. The results of these analyses indicate that the net effect of APOE genotype on cognition and the prevalence of dementia is dependent upon the plasmalogen status of the person.

Improved specificity of serum phosphatidylcholine detection based on side-chain losses during negative electrospray ionization tandem mass spectrometry.

Many current tandem mass spectrometry (MS) methods for measuring phosphatidylcholines (PtdChos) rely only on precursor ion scanning of the common 184 m/z phosphocholine fragment with positive electrospray ionization (+ESI), and thus measure pools of PtdChos rather than specific isoforms. In this paper, we developed and compared an isotope dilution, tandem MS method capable of quantifying PtdChos based on specific fatty acid side-chains to the traditional 184 m/z method. The method is based on the detection of PtdCho ammonium formate (AmF) adduct as parent ions and fatty acid fragment daughter ions under negative electrospray ionization (-ESI). Accuracy, imprecision, and recovery were below 15 %, with acceptable linearity (R 2 > 0.99) up to 5 µg/mL. We used the method to analyze the distributions of PtdChos with common side-chain combinations among 60 subjects and showed that it was possible for two individuals to have the same PtdCho pool concentration based on detection of the 184 m/z fragment, but up to a fourfold difference in the levels of specific isoforms comprising the pool based on our method. We then compared the results of both methods across 572 patients with mild cognitive impairment (MCI), Alzheimer's disease (AD), or no impairment (NI), which showed that statistically significant associations between specific PtdCho isoforms and AD were masked with the 184 m/z method. Our findings demonstrate the importance of isoform specificity for quantifying PtdChos, and suggest caution when interpreting analytical data based on pools of biomarkers.

Serum Metabolite Profiling for the Detection of Pancreatic Cancer: Results of a Large Independent Validation Study.

OBJECTIVES: To improve the detection of pancreatic cancer (PC), a robust diagnostic biomarker is essential. We have previously discovered 4 serum metabolites (PC-594, lysophosphatidylcholine, phosphatidylcholine, and sphingomyelin) in distinguishing patients with PC from healthy controls. Here, we report the results of our validation phase by using larger numbers of independent and blinded samples. METHODS: We collected 3 mL of serum from 116 patients with PC and 138 healthy controls. Samples were blinded and expression of the 4 candidate metabolites in each sample was determined by triple quadrupole tandem mass spectrometry. We then used cutoffs established in the discovery phase to predict the disease state of each of the validation samples. RESULTS: All 4 metabolites showed significantly lower expression in patients with PC compared with healthy controls. PC-594 showed 73.3% sensitivity and 92.0% specificity, whereas the other 3 metabolites showed 58.6% and 92.0%, 76.7% and 69.6%, and 58.6% and 81.9% sensitivity and specificity, respectively. Area under the receiver operating characteristic curve for PC-594 alone was 0.92, whereas a combination method using all 4 metabolites showed 86.2% sensitivity and 84.8% specificity. CONCLUSIONS: Our validation results confirmed that a reduction in PC-594, along with 3 other serum-based choline metabolites, is highly associated with PC.

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.

Metabolic dysfunctions in multiple sclerosis: implications as to causation, early detection, and treatment, a case control study.

BACKGROUND: Biochemical changes associated with multiple sclerosis (MS), and its various clinical forms have not been characterized well. Therefore, we investigated the biochemistry of MS in relation to its natural history using targeted lipidomics platforms. METHODS: Cross-sectional serum samples from 24 secondary progressive (SPMS), 100 relapsing remitting (RRMS), 19 primary progressive MS (PPMS), and 55 age-matched control subjects were analyzed by flow injection tandem mass spectrometry for very long chain fatty acid (VLCFA) containing phosphatidyl ethanolamines (PtdEtn), plasmalogen ethanolamines (PlsEtn) and for novel anti-inflammatory gastrointestinal tract acids (GTAs). Changes in analyte levels relative to healthy controls were correlated with the disease stage and disease duration. RESULTS: RRMS subjects having <13 years disease duration had elevated levels (p < 0.05) of anti-inflammatory metabolites (GTAs) and normal levels (p > 0.05) of mitochondrial stress biomarkers (VLCFA-PtdEtn), compared to controls. SPMS subjects had statistically similar levels of anti-inflammatory metabolites (GTAs), elevated mitochondrial stress metabolites (VLCFA-PtdEtn) and elevated peroxisomal metabolites (PlsEtn) compared to controls (p < 0.05). RRMS subjects with > = 13 years disease duration exhibited metabolic profiles intermediate between short-duration RRMS and SPMS, based on statistical significance. Therefore, RRMS cohort appear to comprise of two metabolically distinct subpopulations. The key clinical discriminator of these two groups was disease duration. PPMS patients exhibited metabolic profiles distinct from RRMS and SPMS. CONCLUSIONS: These data indicate that inflammation and mitochondrial stress are intricately involved in the etiology of MS and that progression in MS can potentially be monitored using serum metabolic biomarkers.

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 precursor analog treatment reduces levodopa-induced dyskinesias in parkinsonian monkeys.

L-DOPA-induced dyskinesias (LID) remain a serious obstacle in the treatment of Parkinson's disease (PD). The objective of this study was to test a new target for treatment of dyskinesias, ethanolamine plasmalogens (PlsEtn). PlsEtn play critical roles in membrane structure mediated functions and as a storage depot of polyunsaturated fatty acids such as docosahexaenoic acid (DHA, omega-3) known to reduce dyskinesias. The motor effect of a daily treatment for 12 days of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) Macaca fascicularis monkeys with DHA (100mg/kg) was compared to the DHA-PlsEtn precursor PPI-1011 (50mg/kg). PPI-1011 and DHA reduced LID while maintaining the antiparkinsonian activity of l-DOPA, however the PPI-1011 effect was observed at the first behavioral time point analyzed following drug administration (day 2) whereas the effect of DHA was not observed until after 10 days of administration. DHA treatment increased plasma DHA levels 2-3× whereas PPI-1011 had no effect. DHA and PPI-1011 increased DHA-PlsEtn levels by 1.5-2× while DHA-phosphatidylethanolamine (PtdEtn) levels remained unaffected. DHA treatment also elevated very long chain fatty acid containing PtdEtn and reduced non-DHA containing PtdEtn and PlsEtn levels. PPI-1011 had no effect on these systems. LID scores were inversely correlated with serum DHA-PlsEtn/total PlsEtn ratios levels in DHA and PPI-1011 treated monkeys. Hence, the antidyskinetic activity of DHA and PPI-1011 in MPTP monkeys appears to be associated with the increase of serum DHA-PlsEtn concentrations. This is the first study reporting an antidyskinetic response to augmentation of DHA-PlsEtn using a plasmalogen precursor thus providing a novel drug target for dyskinesias.

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

BACKGROUND: The 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. METHODS: A 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 neoplasms (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. RESULTS: FI-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. CONCLUSIONS: The 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.

Coupling deep transcriptome analysis with untargeted metabolic profiling in Ophiorrhiza pumila to further the understanding of the biosynthesis of the anti-cancer alkaloid camptothecin and anthraquinones.

The Rubiaceae species, Ophiorrhiza pumila, accumulates camptothecin, an anti-cancer alkaloid with a potent DNA topoisomerase I inhibitory activity, as well as anthraquinones that are derived from the combination of the isochorismate and hemiterpenoid pathways. The biosynthesis of these secondary products is active in O. pumila hairy roots yet very low in cell suspension culture. Deep transcriptome analysis was conducted in O. pumila hairy roots and cell suspension cultures using the Illumina platform, yielding a total of 2 Gb of sequence for each sample. We generated a hybrid transcriptome assembly of O. pumila using the Illumina-derived short read sequences and conventional Sanger-derived expressed sequence tag clones derived from a full-length cDNA library constructed using RNA from hairy roots. Among 35,608 non-redundant unigenes, 3,649 were preferentially expressed in hairy roots compared with cell suspension culture. Candidate genes involved in the biosynthetic pathway for the monoterpenoid indole alkaloid camptothecin were identified; specifically, genes involved in post-strictosamide biosynthetic events and genes involved in the biosynthesis of anthraquinones and chlorogenic acid. Untargeted metabolomic analysis by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) indicated that most of the proposed intermediates in the camptothecin biosynthetic pathway accumulated in hairy roots in a preferential manner compared with cell suspension culture. In addition, a number of anthraquinones and chlorogenic acid preferentially accumulated in hairy roots compared with cell suspension culture. These results suggest that deep transcriptome and metabolome data sets can facilitate the identification of genes and intermediates involved in the biosynthesis of secondary products including camptothecin in O. pumila.

Low-serum GTA-446 anti-inflammatory fatty acid levels as a new risk factor for colon cancer.

Gastrointestinal tract acid-446 (GTA-446) is a long-chain polyunsaturated fatty acid present in the serum. A reduction of GTA-446 levels in colorectal cancer (CRC) patients has been reported previously. Our study compared GTA-446 levels in subjects diagnosed with CRC at the time of colonoscopy to the general population. Serum samples and pathology data were collected from 4,923 representative subjects undergoing colonoscopy and from 964 subjects from the general population. Serum GTA-446 levels were determined using a triple-quadrupole tandem mass spectrometry method. A low-serum GTA-446 level was based on the bottom tenth percentile of subjects with low risk based on age (40-49 years old) in the general population. Eighty-six percent of newly diagnosed CRC subjects (87% for stages 0-II and 85% for stages III-IV) showed low-serum GTA-446 levels. A significant increase in the CRC incidence rate with age was observed in subjects with low GTA-446 levels (p = 0.019), but not in subjects with normal levels (p = 0.86). The relative risk of CRC given a low GTA-446 level was the highest for subjects under age 50 (10.1, 95% confidence interval [C.I.] = 6.4-16.4 in the reference population, and 7.7, 95% C.I. = 4.4-14.1 in the colonoscopy population, both p < 0.0001), and declined with age thereafter. The CRC incidence rate in subjects undergoing colonoscopy with low GTA-446 levels was over six times higher than for subjects with normal GTA-446 levels and twice that of subjects with gastrointestinal symptoms. The results show that a low-serum GTA-446 level is a significant risk factor for CRC, and a sensitive predictor of early-stage disease.

Oral bioavailability of the ether lipid plasmalogen precursor, PPI-1011, in the rabbit: a new therapeutic strategy for Alzheimer's disease.

INTRODUCTION: Docosahexaenoic acid (DHA) and DHA-containing ethanolamine plasmalogens (PlsEtn) are decreased in the brain, liver and the circulation in Alzheimer's disease. Decreased supply of plasmalogen precursors to the brain by the liver, as a result of peroxisomal deficits is a process that probably starts early in the AD disease process. To overcome this metabolic compromise, we have designed an orally bioavailable DHA-containing ether lipid precursor of plasmalogens. PPI-1011 is an alkyl-diacyl plasmalogen precursor with palmitic acid at sn-1, DHA at sn-2 and lipoic acid at sn-3. This study outlines the oral pharmacokinetics of this precursor and its conversion to PlsEtn and phosphatidylethanolamines (PtdEtn). METHODS: Rabbits were dosed orally with PPI-1011 in hard gelatin capsules for time-course and dose response studies. Incorporation into PlsEtn and PtdEtn was monitored by LC-MS/MS. Metabolism of released lipoic acid was monitored by GC-MS. To monitor the metabolic fate of different components of PPI-1011, we labeled the sn-1 palmitic acid, sn-2 DHA and glycerol backbone with (13)C and monitored their metabolic fates by LC-MS/MS. RESULTS: PPI-1011 was not detected in plasma suggesting rapid release of sn-3 lipoic acid via gut lipases. This conclusion was supported by peak levels of lipoic acid metabolites in the plasma 3 hours after dosing. While PPI-1011 did not gain access to the plasma, it increased circulating levels of DHA-containing PlsEtn and PtdEtn. Labeling experiments demonstrated that the PtdEtn increases resulted from increased availability of DHA released via remodeling at sn-2 of phospholipids derived from PPI-1011. This release of DHA peaked at 6 hrs while increases in phospholipids peaked at 12 hr. Increases in circulating PlsEtn were more complex. Labeling experiments demonstrated that increases in the target PlsEtn, 16:0/22:6, consisted of 2 pools. In one pool, the intact precursor received a sn-3 phosphoethanolamine group and desaturation at sn-1 to generate the target plasmalogen. The second pool, like the PtdEtn, resulted from increased availability of DHA released during remodeling of sn-2. In the case of sn-1 18:0 and 18:1 plasmalogens with [(13)C(3)]DHA at sn-2, labeling was the result of increased availability of [(13)C(3)]DHA from lipid remodeling. Isotope and repeated dosing (2 weeks) experiments also demonstrated that plasmalogens and/or plasmalogen precursors derived from PPI-1011 are able to cross both the blood-retinal and blood-brain barriers. CONCLUSIONS: Our data demonstrate that PPI-1011, an ether lipid precursor of plasmalogens is orally bioavailable in the rabbit, augmenting the circulating levels of unesterified DHA and DHA-containing PlsEtn and PtdEtn. Other ethanolamine plasmalogens were generated from the precursor via lipid remodeling (de-acylation/re-acylation reactions at sn-2) and phosphatidylethanolamines were generated via de-alkylation/re-acylation reactions at sn-1. Repeated oral dosing for 2 weeks with PPI-1011 resulted in dose-dependent increases in circulating DHA and DHA-containing plasmalogens. These products and/or precursors were also able to cross the blood-retinal and blood-brain barriers.

Cellular diamine levels in cancer chemoprevention: modulation by ibuprofen and membrane plasmalogens.

BACKGROUND: To develop effective strategies in cancer chemoprevention, an increased understanding of endogenous biochemical mediators that block metastatic processes is critically needed. Dietary lipids and non-steroidal anti-inflammatory drugs (NSAIDs) have a published track record of providing protection against gastrointestinal malignancies. In this regard, we examined the effects of membrane plasmalogens and ibuprofen on regulation of cellular levels of diamines, polyamine mediators that are augmented in cancer cells. For these studies we utilized Chinese hamster ovary (CHO) cells and NRel-4 cells, a CHO cell line with defective plasmalogen synthesis. RESULTS: NRel-4 cells, which possess cellular plasmalogen levels that are 10% of control CHO cells, demonstrated 2- to 3-fold increases in cellular diamine levels. These diamine levels were normalized by plasmalogen replacement and significantly reduced by ibuprofen. In both cases the mechanism of action appears to mainly involve increased diamine efflux via the diamine exporter. The actions of ibuprofen were not stereospecific, supporting previous studies that cyclooxygenase (COX) inhibition is unlikely to be involved in the ability of NSAIDs to reduce intracellular diamine levels. CONCLUSIONS: Our data demonstrate that ibuprofen, a drug known to reduce the risk of colorectal cancer, reduces cellular diamine levels via augmentation of diamine efflux. Similarly, augmentation of membrane plasmalogens can increase diamine export from control and plasmalogen-deficient cells. These data support the concept that membrane transporter function may be a therapeutic point of intervention for dietary and pharmacological approaches to cancer chemoprevention.

In vitro and in vivo plasmalogen replacement evaluations in rhizomelic chrondrodysplasia punctata and Pelizaeus-Merzbacher disease using PPI-1011, an ether lipid plasmalogen precursor.

BACKGROUND: Childhood peroxisomal disorders and leukodystrophies are devastating diseases characterized by dysfunctional lipid metabolism. Plasmalogens (ether glycerophosphoethanolamine lipids) are decreased in these genetic disorders. The biosynthesis of plasmalogens is initiated in peroxisomes but completed in the endoplasmic reticulum. We therefore undertook a study to evaluate the ability of a 3-substituted, 1-alkyl, 2-acyl glyceryl ether lipid (PPI-1011) to replace plasmalogens in rhizomelic chrondrodysplasia punctata type 1 (RCDP1) and rhizomelic chrondrodysplasia punctata type 2 (RCDP2) lymphocytes which possess peroxisomal mutations culminating in deficient plasmalogen synthesis. We also examined plasmalogen synthesis in Pelizaeus-Merzbacher disease (PMD) lymphocytes which possess a proteolipid protein-1 (PLP1) missense mutation that results in abnormal PLP1 folding and it's accumulation in the endoplasmic reticulum (ER), the cellular site of the last steps in plasmalogen synthesis. In vivo incorporation of plasmalogen precursor into tissue plasmalogens was also evaluated in the Pex7 mouse model of plasmalogen deficiency. RESULTS: In both RCDP1 and RCDP2 lymphocytes, PPI-1011 repleted the target ethanolamine plasmalogen (PlsEtn16:0/22:6) in a concentration dependent manner. In addition, deacylation/reacylation reactions resulted in repletion of PlsEtn 16:0/20:4 in both RCDP1 and RCDP2 lymphocytes, repletion of PlsEtn 16:0/18:1 and PlsEtn 16:0/18:2 in RCDP2 lymphocytes, and partial repletion of PlsEtn 16:0/18:1 and PlsEtn 16:0/18:2 in RCDP1 lymphocytes. In the Pex7 mouse, oral dosing of labeled PPI-1011 demonstrated repletion of tissue levels of the target plasmalogen PlsEtn 16:0/22:6 with phospholipid remodeling also resulting in significant repletion of PlsEtn 16:0/20:4 and PlsEtn 16:0/18:1. Metabolic conversion of PPI-1011 to the target plasmalogen was most active in the liver. CONCLUSIONS: Our data demonstrate that PPI-1011 is activated (removal of 3-substitution) and converted to PlsEtn in vitro in both RCDP1 and RCDP2 lymphocytes and in vivo in the Pex7 mouse model of RCPD1 effectively bypassing the peroxisomal dysfunction present in these disorders. While PPI-1011 was shown to replete PlsEtns 16:0/x, ether lipid precursors of PlsEtn 18:0/x and PlsEtn 18:1/x may also be needed to achieve optimal clinical benefits of plasmalogen replacement in these complex patient populations. In contrast, only limited plasmalogen replacement was observed in PMD lymphocytes suggesting that the effects of protein misfolding and accumulation in the ER negatively affect processing of plasmalogen precursors in this cellular compartment.

Targeted metabolomic analyses of cellular models of pelizaeus-merzbacher disease reveal plasmalogen and myo-inositol solute carrier dysfunction.

BACKGROUND: Leukodystrophies are devastating diseases characterized by dys- and hypo-myelination. While there are a number of histological and imaging studies of these disorders, there are limited biochemical data available. We undertook targeted lipidomic analyses of Pelizaeus-Merzbacher disease (PMD) fibroblasts, PMD lymphocytes, and 158JP oligodendrocytes, a murine model of PMD, to define the lipid changes in these cell models. Further targeted metabolomics analyses were conducted to obtain a preliminary evaluation of the metabolic consequences of lipid changes and gene mutations in these cell models. RESULTS: In both PMD fibroblasts and lymphocytes, and 158JP oligodendrocytes, ethanolamine plasmalogens were significantly decreased. Labeling studies with 158JP oligodendrocytes further demonstrated a decreased rate of lipid remodeling at sn-2. Targeted metabolomics analyses of these cells revealed dramatic increases in cellular levels of myo-inositol. Further uptake studies demonstrated increased rates of myo-inositol uptake by PMD lymphocytes. CONCLUSIONS: Our data demonstrating PlsEtn decrements, support previous studies indicating leukodystrophy cells possess significant peroxisomal deficits. Our data for the first time also demonstrate that decrements in peroxisomal function coupled with the PLP1 gene defects of PMD, result in changes in the function of membrane myo-inositol solute carriers resulting in dramatic increases in cellular myo-inositol levels.