Pharmacokinetics, Mass Balance, Excretion, and Tissue Distribution of Plasmalogen Precursor PPI-1011.

PPI-1011 is a synthetic plasmalogen precursor in development as a treatment for multiple plasmalogen-deficiency disorders. Previous work has demonstrated the ability of PPI-1011 to augment plasmalogens and its effects in vitro and in vivo, however, the precise uptake and distribution across tissues in vivo has not been investigated. The purpose of this study was to evaluate the pharmacokinetics, mass balance, and excretion of [14C]PPI-1011 following a single oral administration at 100 mg/kg in Sprague-Dawley rats. Further tissue distribution was examined using quantitative whole-body autoradiography after both single and repeat daily doses at 100 mg/kg/day. Non-compartmental analysis showed that following a single dose, PPI-1011 exhibited peak levels between 6 and 12 h but also a long half-life with mean t1/2 of 40 h. Mass balance showed that over 50% of the compound-associated radioactivity was absorbed by the body, while approximately 40% was excreted in the feces, 2.5% in the urine, and 10% in expired air within the first 24 h. Quantitative whole-body autoradiography following a single dose showed uptake to nearly all tissues, with the greatest initial uptake in the intestines, liver, and adipose tissue, which decreased time-dependently throughout 168 h post-dose. Following 15 consecutive daily doses, uptake was significantly higher across the entire body at 24 h compared to single dose and remained high out to 96 h where 75% of the initially-absorbed compound-associated radioactivity was still present. The adipose tissue remained particularly high, suggesting a possible reserve of either plasmalogens or alkyl diacylglycerols that the body can pull from for plasmalogen biosynthesis. Uptake to the brain was also definitively confirmed, proving PPI-1011's ability to cross the blood-brain barrier. In conclusion, our results suggest that oral administration of PPI-1011 results in high uptake across the body, and that repeated dosing over time represents a viable therapeutic strategy for treating plasmalogen deficiencies.

Oral administration of a synthetic vinyl-ether plasmalogen normalizes open field activity in a mouse model of rhizomelic chondrodysplasia punctata.

Rhizomelic chondrodysplasia punctata (RCDP) is a rare genetic disorder caused by mutations in peroxisomal genes essential for plasmalogen biosynthesis. Plasmalogens are a class of membrane glycerophospholipids containing a vinyl-ether-linked fatty alcohol at the sn-1 position that affect functions including vesicular transport, membrane protein function and free radical scavenging. A logical rationale for the treatment of RCDP is therefore the therapeutic augmentation of plasmalogens. The objective of this work was to provide a preliminary characterization of a novel vinyl-ether synthetic plasmalogen, PPI-1040, in support of its potential utility as an oral therapeutic option for RCDP. First, wild-type mice were treated with 13C6-labeled PPI-1040, which showed that the sn-1 vinyl-ether and the sn-3 phosphoethanolamine groups remained intact during digestion and absorption. Next, a 4-week treatment of adult plasmalogen-deficient Pex7hypo/null mice with PPI-1040 showed normalization of plasmalogen levels in plasma, and variable increases in plasmalogen levels in erythrocytes and peripheral tissues (liver, small intestine, skeletal muscle and heart). Augmentation was not observed in brain, lung and kidney. Functionally, PPI-1040 treatment normalized the hyperactive behavior observed in the Pex7hypo/null mice as determined by open field test, with a significant inverse correlation between activity and plasma plasmalogen levels. Parallel treatment with an equal amount of ether plasmalogen precursor, PPI-1011, did not effectively augment plasmalogen levels or reduce hyperactivity. Our findings show, for the first time, that a synthetic vinyl-ether plasmalogen is orally bioavailable and can improve plasmalogen levels in an RCDP mouse model. Further exploration of its clinical utility is warranted.This article has an associated First Person interview with the joint first authors of the paper.

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.

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.

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.

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.

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

BACKGROUND: Circulating 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. METHODS: GTAs 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. RESULTS: Enriched 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-1ß, NOS2 and COX2. CONCLUSIONS: Our 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.

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.

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.

Requirement of N-myristoyltransferase 1 in the development of monocytic lineage.

N-myristoyltransferase (NMT) exists in two isoforms, NMT1 and NMT2, that catalyze myristoylation of various proteins crucial in signal transduction, cellular transformation, and oncogenesis. We have recently demonstrated that NMT1 is essential for the early development of mouse embryo. In this report, we have demonstrated that an invariant consequence of NMT1 knock out is defective myelopoesis. Suppressed macrophage colony forming units were observed in M-CSF-stimulated bone marrow cells from heterozygous (+/-) Nmt1-deficient mice. Homozygous (-/-) Nmt1-deficient mouse embryonic stem cells resulted in drastic reduction of macrophages when stimulated to differentiate by M-CSF. Furthermore, to understand the requirement of NMT1 in the monocytic differentiation we investigated the role of NMT, pp60c-Src (NMT substrate) and heat shock cognate protein 70 (inhibitor of NMT), during PMA-induced differentiation of U937 cells. Src kinase activity and protein expression increased during the differentiation process along with regulation of NMT activity by hsc70. NMT1 knock down in PMA treated U937 cells showed defective monocytic differentiation. We report in this study novel observation that regulated total NMT activity and NMT1 is essential for proper monocytic differentiation of the mouse bone marrow cells.

Identification of the SRC pyrimidine-binding protein (SPy) as hnRNP K: implications in the regulation of SRC1A transcription.

The human SRC gene encodes pp60(c-src), a non-receptor tyrosine kinase involved in numerous signaling pathways. Activation or overexpression of c-Src has also been linked to a number of important human cancers. Transcription of the SRC gene is complex and regulated by two closely linked but highly dissimilar promoters, each associated with its own distinct non-coding exon. In many tissues SRC expression is regulated by the housekeeping-like SRC1A promoter. In addition to other regulatory elements, three substantial polypurine:polypyrimidine (TC) tracts within this promoter are required for full transcriptional activity. Previously, we described an unusual factor called SRC pyrimidine-binding protein (SPy) that could bind to two of these TC tracts in their double-stranded form, but was also capable of interacting with higher affinity to all three pyrimidine tracts in their single-stranded form. Mutations in the TC tracts, which abolished the ability of SPy to interact with its double-stranded DNA target, significantly reduced SRC1A promoter activity, especially in concert with mutations in critical Sp1 binding sites. Here we expand upon our characterization of this interesting factor and describe the purification of SPy from human SW620 colon cancer cells using a DNA affinity-based approach. Subsequent in-gel tryptic digestion of purified SPy followed by MALDI-TOF mass spectrometric analysis identified SPy as heterogeneous nuclear ribonucleoprotein K (hnRNP K), a known nucleic-acid binding protein implicated in various aspects of gene expression including transcription. These data provide new insights into the double- and single-stranded DNA-binding specificity, as well as functional properties of hnRNP K, and suggest that hnRNP K is a critical component of SRC1A transcriptional processes.