Doxorubicin Affects Testicular Lipids with Long-Chain (C18-C22) and Very Long-Chain (C24-C32) Polyunsaturated Fatty Acids

Doxorubicin disrupts spermatogenesis by causing apoptosis of spermatogonia and primary spermatocytes. The aim of this study was to examine the effect of this agent on adult rat testicular lipids and their fatty acids. A single dose (7.5 mg/kg) and a multidose regime (3 mg/kg once a week for 4 weeks) were evaluated. Both treatments resulted in the gradual loss of spermatogenic cells and determined a marked reduction in testicular size and weight 9 weeks after their start. Germ cell loss was accompanied by a decrease in phospholipids, including glycerophospholipids and sphingomyelin. Concomitantly, glycerophospholipids lost selectively their major polyunsaturated fatty acid (PUFA), 22:5n-6, and sphingomyelin lost its major very long-chain PUFA (VLCPUFA), 28:4n-6 and 30:5n-6. The molecular species from which the lost polyenes originated were thus a trait of germ cells. A transient peak of 16:0-ceramide was observed 48 h after the single dose. In both doxorubicin regimes, sphingomyelin and ceramide with reduced amounts of VLCPUFA after about 4 weeks and with no VLCPUFA after 9 weeks resulted. By contrast, triglycerides and especially cholesterol esters (CE) tended to accumulate in the testes undergoing germ cell death, probably in the surviving Sertoli cells, their fatty acid patterns suggesting that initially, these lipids retained part of the PUFA coming from, or no longer used for, the synthesis of germ cell glycerophospholipids. As the latter decreased, CE accumulated massively 9 weeks after starting doxorubicin treatment, 20:4n-6 becoming their major PUFA. Part of these CEs may derive from surviving steroidogenic cells.Fil: Zanetti, Samanta Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Maldonado, Eduardo N.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Aveldaño, Marta Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentin

Long-term biopermanence of ceramides, cholesteryl esters, and ether-linked triglycerides with very-long-chain PUFA in the cadmium-damaged testis

Cadmium is known to harm rat testis by causing the dose-dependent apoptotic or necrotic death of seminiferous epithelium cells. Here we investigated how this affects the lipids with long-chain (C18-C22) and very-long-chain (C24-C32) polyunsaturated fatty acids (VLCPUFA) typical of spermatogenic and Sertoli cells. A severe acute inflammatory reaction resulted from the massive necrotic death of these cells two days after a single high (4 mg/kg) dose of CdCl2. This led to the conversion of most testicular glycerophospholipids to diradylglycerols (DRG) and free fatty acids (FFA) and of most sphingomyelins to ceramides (Cer). By day 30 the testis weight had decreased three fold. The DRG and FFA had been metabolized but, unexpectedly, ceramides persisted. Also slow to disappear were VLCPUFA-containing triacylglycerols from former germ cells and ether-linked triglycerides and cholesteryl esters (CE) from former Sertoli cells. Similar results were observed 30 and 45 days after administering repeated small non proinflammatory CdCl2doses (1 mg/kg). At day 30 after both treatments, an amorphous material replaced the original seminiferous tubules and testicular macrophages populated the interstitium. Species of CE and etherlinked triglycerides containing fatty acids other than VLCPUFA steadily accumulated in the irreversibly damaged testis, a manifestation of the activity of phagocytic cells. The long-term permanence of original VLCPUFA-containing neutral lipids, especially ceramides, indicates that these cells were slow to clear out the acellular material contained in seminiferous tubules, pointing to a form of silent chronic inflammation as an additional outcome of the multifactorial commotion caused in the testis by experimentally administered cadmium.Fil: Zanetti, Samanta Romina. Universidad Nacional del Sur; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET Bahía Blanca. Instituto de Investigaciones Bioquímicas Bahía Blanca (i); ArgentinaFil: Aveldaño, Marta I.. Universidad Nacional del Sur; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET Bahía Blanca. Instituto de Investigaciones Bioquímicas Bahía Blanca (i); Argentin

Unique thermal behavior of sphingomyelin species with nonhydroxy and 2-hydroxy very-long-chain (C28-C32) PUFAs

In rat germ cells and spermatozoa, sphingomyelin (SM) contains molecular species with nonhydroxy (n) and 2-hydroxy (h) very-long-chain polyunsaturated fatty acids (V), the most abundant being SMs with (n- and h-) 28:4n-6, 30:5n-6, and 32:5n-6 as acyl chains. The aim of this study was to gain information about their thermotropic behavior and interactions with other lipids. After isolation from rat testis, multilamellar and giant unilamellar vesicles from these SMs were examined using fluorescent probes. Only n-32:5 SM and h-32:5 SM displayed a gel-liquid transition temperature (Tt ≈ 21?22°C), the rest remaining in the liquid state in the 5°C?45°C range. The degree of order was larger in bilayers of any of the h-V SMs than in those of their chain-matched n-V SMs. Both, but n-V SM relatively more than h-V SM, decreased the Tt of dimyristoylphosphatidylcholine as their proportion increased in binary phosphatidylcholine:SM liposomes. In contrast to the established ability of 16:0 SM to form lateral cholesterol/SM-rich ordered domains in ternary dioleoylphosphatidylcholine:cholesterol:SM bilayers, neither n-V SM nor h-V SM showed a tendency to do so. Thus, these SMs are in the fluid state and are not involved in this type of domains in spermatozoa at physiological temperatures. However, this state could be altered at the very low temperatures at which these gametes are usually preserved.Fil: Peñalva, Daniel Alejandro. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); ArgentinaFil: Furland, Natalia Edith. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); ArgentinaFil: Lopez, Gustavo H.. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Aveldaño, Marta Isabel. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); ArgentinaFil: Antollini, Silvia Susana. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); Argentin

Polyunsaturated fatty acids of marine macroalgae: potential for nutritional and pharmaceutical applications

As mammals are unable to synthesize essential polyunsaturated fatty acids (PUFA), these compounds need to be taken in through diet. Nowadays, obtaining essential PUFA in diet is becoming increasingly difficult; therefore this work investigated the suitability of using macroalgae as novel dietary sources of PUFA. Hence, 17 macroalgal species from three different phyla (Chlorophyta, Phaeophyta and Rhodophyta) were analyzed and their fatty acid methyl esters (FAME) profile was assessed. Each phylum presented a characteristic fatty acid signature as evidenced by clustering of PUFA profiles of algae belonging to the same phylum in a Principal Components Analysis. The major PUFA detected in all phyla were C-18 and C-20, namely linoleic, arachidonic and eicosapentaenoic acids. The obtained data showed that rhodophytes and phaeophytes have higher concentrations of PUFA, particularly from the n-3 series, thereby being a better source of these compounds. Moreover, rhodophytes and phaeophytes presented. healthier. Sigma(n-6/)Sigma(n-3) and PUFA/saturated fatty acid ratios than chlorophytes. Ulva was an exception within the Chlorophyta, as it presented high concentrations of n-3 PUFA, alpha-linolenic acid in particular. In conclusion, macroalgae can be considered as a potential source for large-scale production of essential PUFA with wide applications in the nutraceutical and pharmacological industries.SEABIOMED [PTDC/MAR/103957/2008]; Foundation for Science and Technology (FCT); Portuguese National Budget; FCT [SFRH/BPD/65116/2009, SFRH/BD/81425/2011

Atypical surface behavior of ceramides with nonhydroxy and 2-hydroxy very long-chain (C28-C32) PUFAs

Unique species of ceramide (Cer) with very-long-chain polyunsaturated fatty acid (VLCPUFA), mainly 28?32 carbon atoms, 4?5 double bonds, in nonhydroxy and 2-hydroxy forms (n-V Cer and h-V Cer, respectively), are generated in rat spermatozoa from the corresponding sphingomyelins during the acrosomal reaction. The aim of this study was to determine the properties of these sperm-distinctive ceramides in Langmuir monolayers. Individual Cer species were isolated by HPLC and subjected to analysis of surface pressure, surface potential, and Brewster angle microscopy (BAM) as a function of molecular packing. In comparison with known species of Cer, n-V Cer and h-V Cer species showed much larger mean molecular areas and increased molecular dipole moments in liquid expanded phases, which suggest bending and partial hydration of the double bonded portion of the VLCPUFA. The presence of the 2-hydoxyl group induced a closer molecular packing in h-V Cer than in their chain-matched n-V Cer. In addition, all these Cer species showed liquid-expanded to liquid-condensed transitions at room temperature. Existence of domain segregation was confirmed by BAM. Additionally, thermodynamic analysis suggests a phase transition close to the physiological temperature for VLCPUFA-Cers if organized as bulk dispersions.Fil: Peñalva, Daniel Alejandro. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); ArgentinaFil: Oresti, Gerardo Martin. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); ArgentinaFil: Dupuy, Fernando Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones En Química Biológica de Córdoba (p); ArgentinaFil: Antollini, Silvia Susana. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); ArgentinaFil: Maggio, Bruno. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones En Química Biológica de Córdoba (p); ArgentinaFil: Aveldaño, Marta Isabel. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); ArgentinaFil: Fanani, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones En Química Biológica de Córdoba (p); Argentin

Sphingolipids as emerging mediators in retina degeneration

The sphingolipids ceramide (Cer), sphingosine-1-phosphate (S1P), sphingosine (Sph), and ceramide-1-phosphate (C1P) are key signaling molecules that regulate major cellular functions. Their roles in the retina have gained increasing attention during the last decade since they emerge as mediators of proliferation, survival, migration, neovascularization, inflammation and death in retina cells. As exacerbation of these processes is central to retina degenerative diseases, they appear as crucial players in their progression. This review analyzes the functions of these sphingolipids in retina cell types and their possible pathological roles. Cer appears as a key arbitrator in diverse retinal pathologies; it promotes inflammation in endothelial and retina pigment epithelium (RPE) cells and its increase is a common feature in photoreceptor death in vitro and in animal models of retina degeneration; noteworthy, inhibiting Cer synthesis preserves photoreceptor viability and functionality. In turn, S1P acts as a double edge sword in the retina. It is essential for retina development, promoting the survival of photoreceptors and ganglion cells and regulating proliferation and differentiation of photoreceptor progenitors. However, S1P has also deleterious effects, stimulating migration of Müller glial cells, angiogenesis and fibrosis, contributing to the inflammatory scenario of proliferative retinopathies and age related macular degeneration (AMD). C1P, as S1P, promotes photoreceptor survival and differentiation. Collectively, the expanding role for these sphingolipids in the regulation of critical processes in retina cell types and in their dysregulation in retina degenerations makes them attractive targets for treating these diseases.Fil: Simon, Maria Victoria. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Prado Spalm, Facundo Heber. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Vera, Marcela Sonia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Rotstein, Nora Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentin

Molecular Analysis of Genes Involved in the Biosynthesis of Very Long Chain Polyunsaturated Fatty Acids (VLCPUFAs)

The effective acyl flux between phospholipids and neutral lipids is critical for a high level of the biosynthesis of very long chain polyunsaturated fatty acids (VLCPUFAs) such as arachidonic acid (ARA,20:4-5,8,11,14), eicosapentaenoic acid (EPA, 20:5-5,8,11,14,17) and docosahexaenoic acid (DHA, 22:6-4,7,10,13,16,19) which are essential for human health and wellbeing. Three membrane-bound enzymes, phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT), cholinephosphotransferase (CPT) and ethanolaminephosphotransferase (EPT) from VLCPUFA-producing fungi were selected as candidates for my thesis research based on the hypothesis that these enzymes play important roles in acyl trafficking between phosphatidylcholine (PCs) and diacylglycerols (DAGs) during the biosynthesis of VLCPUFAs. Two putative PDCT cDNAs (PiPDCT1 and PiPDCT2) were cloned from Phytophthora infestans which encode polypeptides with two conserved domains and about 15% of amino acid identity to an Arabidopsis PDCT. However, in vitro assays in yeast Saccharomyces cerevisiae showed they did not have any PDCT activity. Four putative CPT and EPT cDNAs (PiCPT1, PiCPT2, PiEPT and TaCPT) were cloned from P. infestans and Thraustochytrium aureum which encode proteins with a conserved CDP-alcohol phosphotransferase motif and 22% to 26% of amino acid identity to the yeast CPT. In vitro assays indicated PiCPT1 and TaCPT had CPT activity, PiEPT had EPT activity and PiCPT2 did not have either activity. Substrate specificity assays showed that all the three functional CPT and EPT preferred VLCPUFA-containing DAGs as substrates with PiCPT1 being the most specific towards ARA-DAG and DHA-DAG. Real-time qPCR analysis revealed that the expression of PiCPT1 was up-regulated in P. infestans fed with exogenous VLCPUFAs. These results lead us to conclude that PiCPT1 is a VLCPUFAs-specific CPT which may play an important role in shuffling VLCPUFAs from phospholipids to storage neutral lipids, would thus have potential use in metabolic engineering of VLCPUFAs in heterologous systems including oilseed crops

Bioconcentration of Marine Algae Using Lipase Enzyme

Marine algae rich in n-3 PUFA, being a natural and readily available resource, could be an alternative to fish oil derived n-3 PUFA; therefore, it could be of immense potentiality in nutraceutical and pharmaceutical industries. This highlights the sustainable benefits of algae and the many potential gains in creating algal bio-factories. In recent years, the use of lipase as biocatalysts had drawn considerable attention. Lipase is an enzyme that hydrolyzes lipids, the ester bonds in triglycerides, to form fatty acids and glycerol. Among the lipases assayed, the enzyme from the yeast Candida cylindracea is of special interest, as these are proved to be a nonspecific catalyst for many (commercially) interesting reactions such as the modification of oils and fats, reactions in organic solvents, and resolution of racemic mixtures. Hence, the enrichment of microalgae using biolipase from the source Candida cylindracea is of particular attention. Lipase action of Candida cylindracea is investigated as a function of time. It is observed that the lipases display a significant preference to saturated fatty acids; however, the resistance to release EPA and DHA was less as the hydrolysis reaction progresses

Differentiation-linked changes in the biosynthesis and turnover of sphingomyelins in rat male germ cells: genes involved and effects of testosterone

In rodents, sphingomyelins (SMs) species with very-long-chain polyunsaturated fatty acid (VLCPUFA) are required for normal spermatogenesis. Data on the expression of enzymes with roles in their biosynthesis and turnover during germ cell differentiation and on possible effects on such expression of testosterone (Tes), known to promote this biological process, were lacking. Here we quantified, in isolated pachytene spermatocytes (PtS), round spermatids (RS), and later spermatids (LS), the mRNA levels from genes encoding ceramide (Cer), glucosylceramide (GlcCer), and SM synthases (Cers3, Gcs, Sms1, and Sms2) and sphingomyelinases (aSmase, nSmase) and assessed products of their activity in cells in culture using nitrobenzoxadiazole (NBD)-labeled substrates and [3H]palmitate as precursor. Transcript levels from Cers3 and Gcs were maximal in PtS. While mRNA levels from Sms1 increased with differentiation in the direction PtS→RS→LS, those from Sms2 increased between PtS and RS but decreased in LS. In turn, the nSmase transcript increased in the PtS→RS→LS order. During incubations with NBD-Cer, spermatocytes produced more GlcCer and SM than did spermatids. In total germ cells cultured for up to 25 h with NBD-SM, not only abundant NBD-Cer but also NBD-GlcCer were formed, demonstrating SM→Cer turnover and Cer recycling. After 20 h with [3H]palmitate, PtS produced [3H]SM and RS formed [3H]SM and [3H]Cer, all containing VLCPUFA, and Tes increased their labeling. In total germ cells, Tes augmented in 5 h the expression of genes with roles in VLCPUFA synthesis, decreased the mRNA from Sms2, and increased that from nSmase. Thus, Tes enhanced or accelerated the metabolic changes occurring to VLCPUFA-SM during germ cell differentiation.Fil: Santiago Valtierra, Florencia Ximena. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Aveldaño, Marta Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Oresti, Gerardo Martin. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentin

Biosynthesis and Assembly of Very Long Chain Polyunsaturated Fatty Acids in Thraustochytrium sp. 26185

Thraustochytrium is a marine protist producing a specific profile of nutritionally important fatty acids, including very long chain polyunsaturated fatty acids (VLCPUFAs) docosahexaenoic acid (DHA, 22:6n-3), even-chain saturated fatty acids (SFAs) palmitic acid (16:0), and odd-chain SFAs pentadecanoic acid (15:0). However, the mechanism of how these fatty acids are synthesized and assembled into the storage lipid triacylglycerol (TAG) is unclear. Firstly, we reported sequencing of the whole genome and genomic analysis of genes involved in the biosynthesis and assembly of the fatty acids in this species. Analysis of annotated genes from the genome revealed co-existence of both aerobic pathway and anaerobic pathways for the biosynthesis of VLCPUFAs in this species. However, in the aerobic pathway, a key gene encoding stearoyl 9 desaturase introducing the first double bond to long chain saturated fatty acid 18:0 was missing from the genome. A genomic survey of genes involved in the acyl trafficking among glycerolipids showed that, unlike plants, this protist did not possess phosphatidylcholine: diacylglycerol cholinephosphotransferase (PDCT), an important enzyme in bridging two types of glycerolipids, diacylglycerol (DAG) and phosphatidylcholine (PC). Secondly, a series of radiolabeled precursors were used to trace the biosynthetic process of different fatty acids in vivo and in vitro. When Thraustochytrium was fed with long chain fatty acid intermediates such as 14C-oleic acid, 14C-linoleic acid and 14C-α-linolenic acid, no VLCPUFAs were produced, indicating that the aerobic pathway for the biosynthesis of VLCPUFAs was not functional in Thraustochytrium. When fed with 14C-acetic acid, both SFAs and VLCPUFAs were labeled, and when fed with 14C-propionic acid, mainly SFAs were labeled. However, when fed with 14C-acetic acid in the presence of cerulenin, a type I FAS inhibitor, only VLCPUFAs were labeled, and when fed with 14C-propionic acid in the presence of cerulenin, neither SFAs nor VLCPUFAs were labeled. This result clearly indicates that the type I fatty acid synthase (FAS) in Thraustochytrium could use acetic acid and propionic acid as the primers to synthesize even-chain and odd-chain SFAs, respectively, and VLCPUFAs were synthesized by the PUFA synthase using acetic acid as the primer. The in vitro assay with 14C-malonyl-CoA in the presence of cerulenin showed that the crude protein of Thraustochytrium produced only VLCPUFAs, not SFAs, further confirming the role of the PUFA synthase in the biosynthesis of VLCPUFAs. These results have elucidated the biochemical mechanisms for the biosynthesis of all fatty acids in Thraustochytrium. Thirdly, how freshly-synthesized fatty acids are incorporated into different glycerolipids was investigated. The glycerolipid profile of Thraustochytrium at log and stationary growth stages was analyzed by lipidomic tools, and then 14C-acetate and 14C-glycerol were used to trace the flux of fatty acids and backbone in glycerolipids. Lipidomic analysis showed that VLCPUFAs were mostly allocated to phosphatidylcholine (PC) and TAG. PC possessed a relatively stable profile of VLCPUFAs at the two growth stages, whereas TAG species with one or two VLCPUFAs were significantly increased at the stationary phase. Freshly-synthesized VLCPUFAs labeled by 14C-acetate were predominately incorporated into PC initially, while at the late time point of labeling, these fatty acids were mostly found in TAG. Positional analysis showed that PC had either one VLCPUFA at its sn-2 position (PC1) or two VLCPUFAs at both sn-1 and sn-2 positions (PC2), while TAG incorporated these fatty acids almost exclusively at the sn-2 position with similar stereospecific structure as PC1. Similarly, 14C-glycerol was more efficiently incorporated into PC1 than TAG initially, and at the late time point of labeling, it was mostly found in TAG, and DAG and PC1 shared a similar incorporation pattern. These results indicate that VLCPUFAs in TAG are mainly channeled from PC likely through diacylglycerol as the intermediate