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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

BACKGROUND: There 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. METHODS: Biomarker 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). RESULTS: Comprehensive 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). CONCLUSIONS: A 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.

Circulating plasmalogen levels and Alzheimer Disease Assessment Scale-Cognitive scores in Alzheimer patients.

BACKGROUND: Plasmalogens, which are key structural phospholipids in brain membranes, are decreased in the brain and serum of patients with Alzheimer disease (AD). We performed this pilot study to evaluate the relation between the levels of circulating plasmalogens and Alzheimer Disease Assessment Scale-Cognitive (ADAS-Cog) scores in patients with AD. METHODS: We evaluated participants' ADAS-Cog scores and serum plasmalogen levels. For the 40 included AD patients with an ADAS-Cog score between 20 and 46, were tested their ADAS-Cog score 1 year later. The levels of docosahexaenoic acid plasmalogen were measured by use of liquid chromatography-tandem mass spectrometry. RESULTS: We found that the ADAS-Cog score increased significantly in AD patients with circulating plasmalogen levels that were 75%). LIMITATIONS: This was a pilot study with 40 patients, and the results require validation in a larger population. CONCLUSION: Our study demonstrates that decreased levels of plasmalogen precursors in the central nervous system correlate with functional decline (as measured by ADAS-Cog scores) in AD patients. The use of both ADAS-Cog and serum plasmalogen data may be a more accurate way of predicting cognitive decline in AD patients, and may be used to decrease the risk of including patients with no cognitive decline in the placebo arm of a drug trial.

Reduction of novel circulating long-chain fatty acids in colorectal cancer patients is independent of tumor burden and correlates with age.

BACKGROUND: Serum levels of novel hydroxy polyunsaturated ultra long-chain fatty acids (hPULCFAs) have been previously shown to be reduced in pre-treatment CRC patients compared to disease-free subjects, independent of disease stage. However, whether reduced levels of hPULCFAs result from the presence of cancer is currently unknown, as is the distribution of hPULCFAs in the general population. The following studies were carried out to assess whether conventional therapy would result in restoration of systemic hPULCFAs in CRC patients, and to investigate the relationship between hPULCFA levels and age. METHODS: Tandem mass spectrometry was used to determine serum levels of the 28 carbon-containing hPULCFA C28H46O4 (CRC-446) in the following cohorts: two independent Japanese CRC populations following surgical tumor removal (n = 86), a North American Caucasian CRC cohort (n = 150) following post-surgery combination chemo/radiation therapy, 990 randomly selected anonymized serum samples from subjects ranging between 11 and 99 years of age, as well as longitudinally collected serum samples from healthy normals (n = 8, up to 90 weeks) and stage IV CRC subjects on combination therapy (n = 12, up to 63 weeks). RESULTS: Serum CRC-446 levels in CRC subjects were significantly lower than controls (mean of 0.297 ± 0.07 ug/ml in controls versus 0.092 ± 0.03 in CRCs, p < 0.001), and were unaffected by surgical tumor removal or by chemo/radiation treatment (p > 0.05 between pre vs post surgery). CRC-446 levels showed a strong inverse association with age (p < E-11) across the randomly-selected cohort of 990 subjects, with no correlation observed in the CRC-positive subjects. Longitudinal intra-subject results, however, showed relatively stable CRC-446 levels over the short term of up to 90 weeks in both disease-free subjects and late-stage CRC patients. CONCLUSIONS: Our findings show that CRC-446 levels are not affected by conventional CRC treatment and inversely correlate with age, which suggest that reduced serum CRC-446 levels likely exist prior to the development of CRC. Extrapolation of the results to a simple screening scenario showed that, compared to fecal blood testing, pre-colonoscopy screening using serum CRC-446 levels would require 80% fewer colonoscopies, would identify risk in subjects under the age of 50, and would result in increased numbers of early cases detected. The precise role these serum metabolites play in the aetiology of cancer development remains to be determined.

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

BACKGROUND: Disrupted cholesterol regulation leading to increased circulating and membrane cholesterol levels is implicated in many age-related chronic diseases such as cardiovascular disease (CVD), Alzheimer's 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. RESULTS: Using 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. CONCLUSION: The 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.

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.

Triterpenoids from leaves of Elaeophorbia drupifera.

A survey of the hexane extract of the leaves of Elaeophorbia drupifera led to the isolation of a new triterpenoid named elaeophorbate [methyl 4,4,8,10,13,14-hexamethyl-1,17-di (prop-1-en-2-yl)hexadecahydro-1H-cyclo penta[a]phenanthrene-3-carboxylate, 5] and eight known triterpenoids. Based on spectroscopic methods, coupled with high resolution gas chromatography-mass spectrometry the structures of all the compounds were elucidated.