Polyunsaturated Phosphatidylcholine Species Research Articles

Tafazzin deficiency causes substantial remodeling in the lipidome of a mouse model of Barth Syndrome cardiomyopathy.

Barth Syndrome (BTHS) is a rare X-linked disease, characterized clinically by cardiomyopathy, skeletal myopathy, neutropenia, and growth retardation. BTHS is caused by mutations in the phospholipid acyltransferase tafazzin (Gene: TAFAZZIN, TAZ). Tafazzin catalyzes the final step in the remodeling of cardiolipin (CL), a glycerophospholipid located in the inner mitochondrial membrane. As the phospholipid composition strongly determines membrane properties, correct biosynthesis of CL and other membrane lipids is essential for mitochondrial function. Mitochondria provide 95% of the energy demand in the heart, particularly due to their role in fatty acid oxidation. Alterations in lipid homeostasis in BTHS have an impact on mitochondrial membrane proteins and thereby contribute to cardiomyopathy. We analyzed a transgenic TAFAZZIN-knockdown (TAZ-KD) BTHS mouse model and determined the distribution of 193 individual lipid species in TAZ-KD and WT hearts at 10 and 50weeks of age, using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Our results revealed significant lipid composition differences between the TAZ-KD and WT groups, indicating genotype-dependent alterations in most analyzed lipid species. Significant changes in the myocardial lipidome were identified in both young animals without cardiomyopathy and older animals with heart failure. Notable alterations were found in phosphatidylcholine (PC), phosphatidylethanolamine (PE), lysophosphatidylethanolamine (LPE), lysophosphatidylcholine (LPC) and plasmalogen species. PC species with 2-4 double bonds were significantly increased, while polyunsaturated PC species showed a significant decrease in TAZ-KD mice. Furthermore, Linoleic acid (LA, 18:2) containing PC and PE species, as well as arachidonic acid (AA, 20:4) containing PE 38:4 species are increased in TAZ-KD. We found higher levels of AA containing LPE and PE-based plasmalogens (PE P-). Furthermore, we are the first to show significant changes in sphingomyelin (SM) and ceramide (Cer) lipid species Very long-chained SM species are accumulating in TAZ-KD hearts, whereas long-chained Cer and several hexosyl ceramides (HexCer) species accumulate only in 50-week-old TAZ-KD hearts These findings offer potential avenues for the diagnosis and treatment of BTHS, presenting new possibilities for therapeutic approaches.

Abstract 4900: Dynamic metabolic response of prostate cancer patients treated with ADT and low carb diet

Abstract Background: Prostate cancer (PrCa) is one of the most common cancers among men and managed through surgery, hormonal therapy, chemotherapy, radiation, and cryotherapy. Depending on the stage of PrCa, androgen deprivation therapy (ADT) is commonly utilized as an intervention. ADT has proven effective in intervening in PrCa progression, although there are several side effects. Recently, the use of low carb diets has been shown to alter patients’ metabolic phenotype and as such may reduce the side effects of ADT. Methods: In this study 35 men, who were beginning ADT, were randomized to low carb diet intervention with recommended exercise or control (no lifestyle change) for 6 months. Primary results have been reported showing significant weight loss at both 3-month (17lb, p<0.01) and 6-month (23 lbs, p<0.01) and improved insulin control at 3-month (HOMA=-19 as compared to baseline, p=0.02) but not at 6-month (HOMA=-4 as compared to baseline, p=0.13). For this exploratory analysis, sera collected at baseline, 3, and 6 months was used for metabolomics analysis utilizing GC/MS TOF, QqQ LC-HILIC-MS/MS, and TripleTOF 6600 LC-RP-MS and lipidomics analysis using TripleTOF 5600+ MS/MSALL workflows to quantify the chemical diversity of the metabolome and lipidome. Results: Over 450 metabolites and 1000 lipid species were quantified. Metabolomics analysis of sera from the control patients (ADT alone) demonstrated alterations in steroid biosynthesis, androgen/estrogen metabolism, fatty acid metabolism, and lysine degradation. Lipidomics analysis of the control patients demonstrated changes in selective long chain polyunsaturated phosphatidylcholine species. Analysis of the ADT plus low carb patients revealed a change in pyruvate, glucose-alanine cycle, selenoamino acid, phenylalanine/tyrosine, and taurine metabolism. Further, lipidomic analysis revealed changes in several triglycerides, plasmalenylethanolamine, and phosphatidic acid species. Conclusion: In summary, integration of metabolomic and lipidomic analysis in hormonal and metabolic interventions of PrCa patients revealed dynamic changes providing novel insight for tailoring therapeutic intervention and monitoring. Citation Format: Stephen Freedland, Pao-Hwa Lin, Emily Y. Chen, Vladimir Tolstikov, Jen-Tsan Chi, Rangaprasad Sarangarajan, Niven R. Narain, Michael A. Kiebish. Dynamic metabolic response of prostate cancer patients treated with ADT and low carb diet [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4900.

Metabolism of a synthetic compared with a natural therapeutic pulmonary surfactant in adult mice

Secreted pulmonary surfactant phosphatidylcholine (PC) has a complex intra-alveolar metabolism that involves uptake and recycling by alveolar type II epithelial cells, catabolism by alveolar macrophages, and loss up the bronchial tree. We compared the in vivo metabolism of animal-derived poractant alfa (Curosurf) and a synthetic surfactant (CHF5633) in adult male C57BL/6 mice. The mice were dosed intranasally with either surfactant (80 mg/kg body weight) containing universally 13C-labeled dipalmitoyl PC (DPPC) as a tracer. The loss of [U13C]DPPC from bronchoalveolar lavage and lung parenchyma, together with the incorporation of 13C-hydrolysis fragments into new PC molecular species, was monitored by electrospray ionization tandem mass spectrometry. The catabolism of CHF5633 was considerably delayed compared with poractant alfa, the hydrolysis products of which were cleared more rapidly. There was no selective resynthesis of DPPC and, strikingly, acyl remodeling resulted in preferential synthesis of polyunsaturated PC species. In conclusion, both surfactants were metabolized by similar pathways, but the slower catabolism of CHF5633 resulted in longer residence time in the airways and enhanced recycling of its hydrolysis products into new PC species.

No up-regulation of the phosphatidylethanolamine N-methyltransferase pathway and choline production by sex hormones in cats.

BackgroundFeline hepatic lipidosis (FHL) is a common cholestatic disease affecting cats of any breed, age and sex. Both choline deficiency and low hepatic phosphatidylethanolamine N-methyltransferase (PEMT) activity are associated with hepatic lipidosis (HL) in humans, mice and rats. The PEMT expression is known to be upregulated by oestrogens, protecting the females in these species from the development of HL when exposed to choline deficient diets. The aim of the present study was to evaluate the influence of sex hormones on choline synthesis via the PEMT pathway in healthy male and female cats before and after spaying/neutering, when fed a diet with recommended dietary choline content.ResultsFrom six female and six male cats PEMT activity was assayed directly in liver biopsies taken before and after spaying/neutering, and assessed indirectly by analyses of PEMT–specific hepatic phosphatidylcholine (PC) species and plasma choline levels. Hepatic PEMT activity did not differ between intact female and male cats and no changes upon spaying/neutering were observed. Likewise, no significant differences in liver PC content and PEMT-specific polyunsaturated PC species were found between the sexes and before or after spaying/neutering.ConclusionThese results suggest that choline synthesis in cats differs from what is observed in humans, mice and rats. The lack of evident influence of sex hormones on the PEMT pathway makes it unlikely that spaying/neutering predisposes cats for HL by causing PC deficiency as suggested in other species.

Specificity and rate of human and mouse liver and plasma phosphatidylcholine synthesis analyzed in vivo

Phosphatidylcholine (PC) synthesis by the direct cytidine diphosphate choline (CDP-choline) pathway in rat liver generates predominantly mono- and di-unsaturated molecular species, while polyunsaturated PC species are synthesized largely by the phosphatidylethanolamine-N-methyltransferase (PEMT) pathway. Although altered PC synthesis has been suggested to contribute to development of hepatocarcinoma and nonalcoholic steatohepatitis, analysis of the specificity of hepatic PC metabolism in human patients has been limited by the lack of sensitive and safe methodologies. Here we incorporated a deuterated methyl-D(9)-labled choline chloride, to quantify biosynthesis fluxes through both of the PC synthetic pathways in vivo in human volunteers and compared these fluxes with those in mice. Rates and molecular specificities of label incorporated into mouse liver and plasma PC were very similar and strongly suggest that label incorporation into human plasma PC can provide a direct measure of hepatic PC synthesis in human subjects. Importantly, we demonstrate for the first time that the PEMT pathway in human liver is selective for polyunsaturated PC species, especially those containing docosahexaenoic acid. Finally, we present a multiple isotopomer distribution analysis approach, based on transfer of deuterated methyl groups to S-adenosylmethionine and subsequent sequential methylations of PE, to quantify absolute flux rates through the PEMT pathway that are applicable to studies of liver dysfunction in clinical studies.

Mass Spectrometric Identification of Molecular Species of Phosphatidylcholine and Lysophosphatidylcholine Extracted from Shark Liver

The profile and structural characterization of molecular species of phosphatidylcholine (PC) and lysophosphatidylcholine (LysoPC) from shark liver using liquid chromatographic/electrospray ionization mass spectrometry (LC-ESI/MS) and tandem mass spectrometry (MS/MS) are described for the first time in this paper. The presence of (i) a relatively high content of ether PC species, such as 1-O-alkyl- and 1-alk-1'-enyl-2-polyunsaturated PC species (about 20%), and (ii) a high percentage of docosahexaenoic acid (DHA)-containing LysoPC (about 27%) is the characteristic of this marine material. 1-Hexadecanoyl-2-docosahexaenoyl-PC (16:0/22:6; about 24%) and 1-docosahexaenoyl-2-hydroxyl-LysoPC (22:6; about 27%) are the two most abundant species in shark liver. The other polyunsaturated PC species including ether PC are tentatively identified as 1-heptadecanoyl-2-docosahexaenoyl-PC (17:0/22:6), 1-octadecyl-2-docosahexaenoyl-PC (alkyl-18:0/22:6), 1-hexadecyl-2-docosahexaenoyl-PC (alkyl-16:0/22:6), 1-octadecenyl-2-eicosapentaenoyl-PC (alkyl-18:1/20:5), 1-octadecenyl-2-eicosatetraenoyl-PC (alkyl-18:1/20:4), 1-hexadecyl-2-docosapentaenoyl-PC (alkyl-16:0/22:5), 1-(1 Z-hexadecenyl)-2-docosahexaenoyl-PC (alkenyl-16:0/22:6), and 1-octadecenoyl-2-docosahexaenoyl-PC (18:1/22:6). These results establish that high contents of ether DHA-PC and DHA-LysoPC species can be obtained from shark liver.

ApoA-I induces a preferential efflux of monounsaturated phosphatidylcholine and medium chain sphingomyelin species from a cellular pool distinct from HDL3 mediated phospholipid efflux

Electrospray ionization tandem mass spectrometry (ESI-MS/MS) was used for a detailed analysis of cellular phospholipid and cholesterol efflux in free cholesterol (FC) loaded human primary fibroblasts and human monocyte-derived macrophages (HMDM) loaded with enzymatically modified LDL (E-LDL). Although both cell models differed significantly in their cellular lipid composition, a higher apoA-I specific efflux was found for monounsaturated phosphatidylcholine (PC) species together with a decreased contribution of polyunsaturated PC species in both cell types. Moreover, medium chain sphingomyelin (SPM) species SPM 14:0 and SPM 16:1 were translocated preferentially to apoA-I in both cell types. In contrast to fibroblasts, HMDM displayed a considerable proportion of cholesteryl esters (CE) in basal and apoA-I specific efflux media, most likely due to secretion of CE associated to apoE. Analysis of HDL3 mediated lipid efflux from HMDM using D9-choline and 13C3-FC stable isotope labeling revealed significantly different D9-PC and D9-SPM species pattern for apoA-I and HDL3 specific efflux media, which indicates a contribution of distinct cellular phospholipid pools to apoA-I and HDL3 mediated efflux. Together with a partial loading of fibroblasts and HMDM with HDL3-derived CE species, these data add further evidence for retroendocytosis of HDL. In summary, analysis of apoA-I/ABCA1 and HDL3 mediated lipid efflux by ESI-MS/MS demonstrated a preferential efflux of monounsaturated PC and medium chain SPM to apoA-I. Moreover, this is the first study, which provides evidence for distinct cellular phospholipid pools used for lipid transfer to apoA-I and HDL3 from the analysis of phospholipid species pattern in HMDM.

Polyunsaturated phosphatidylcholine (PC) incorporation into phospholipids is greater in pancreas than in liver and protects against alcohol-induced oxidative stress in both

Polyenylphosphatidylcholine (PPC), a soybean extract, provides protection against alcohol-induced oxidative stress in the liver of non-human primates, and preliminary results indicate a similar effect in pancreas. To determine whether this is associated with incorporation of PPC components into these tissues, PC species, including 18:2-18:2 PC (DLPC), the main constituent of PPC, were determined by HPLC and GC/MS in rats fed for eight weeks with adequate liquid diets rich in vitamin E (30 IU/1000 cal.) and containing PPC (3g/1000 cal. or equivalent amounts of linoleic acid as triglycerides in safflower oil) with either ethanol (36% of energy) or isocaloric carbohydrates. Compared to safflower oil, PPC significantly increased DLPC content in hepatic (0.204±0.019 vs 0.124_+0.008 lamol/g; p<0.05) and pancreatic (1.12 ± 0.22 vs 0.32 ± 0.09 lamol/g; p<0.001) phosphnlipids. Similar results were obtained when expressed per protein. Alcohol tended to decrease DLPC in pancreas, but when PPC was added to the ethanol, it restored the levels to normal or above. There was a similar effect of PPC on hepatic and pancreatic content of 18:1-18:2 PC, another species of PPC, whereas 18:0-20:4 PC and 16:0-20:4 PC, which are primarily endogenous PC species, were not significantly affected by PPC. They both decreased after alcohol, in liver as well as pancreas. The greater effect of PPC on pancreatic than hepatic phospholipids may reflect a correspondingly more rapid phospholipid turnover. Alcohol also produced a significant oxidative stress: 4-hydroxynonenal (4-HNE) and F2-isoprostanes (F2-IP) measured by GC/MS, increased respectively from 33.8±3.6-ng/g to 52.4±6.4 (p<0.01) and 2.07 ±0.06to 2.62_+0.14 (p<0.01) in the liver, and from 8.6± 1.1 to 17.9 _+ 3.3 (p<0.01) and 1.8 ± 0.1 to 2.7 _+ 0.4 (p<0.01) in the pancreas. In both tissues, PPC feeding provided total protection against this oxidative stress, with restoration of 4-HNE and F2-IP to normal. The mechanism of the protection has not been elucidated but does not involve vitamin E, which remained normal and unchanged in both tissues. In liver, and now also in pancreas (Kessova et al, unpublished) it was shown that ethanol induces the activity of cytochrome P450 2El which generates free radicals. PPC did not affect 2El activity. Therefore, it most likely acts by boosting the cellular defense systems against the oxidative stress. Indeed, DLPC readily traps free radicals and may thereby protect the cellular membranes against alcohol. Conclusions: 1) PC species provided as PPC (such as 18:2-18:2 PC and 18:1-18:2 PC) are actively incorporated into liver phospholipids, and even more in those of pancreas. 2) PPC supplementation restores the level of some PC species depleted by alcohol, and corrects the concomitant oxidative stress in the liver and pancreas. Because of the associated protection against tissue injury and fibrosis, PPC is being tested in patients with alcoholic liver disease. Extension to pancreatic disorders should now also be considered since the effects on pancreas were found to be greater than those in liver.