Acyl Carbon Research Articles

Enantiomeric Separation of Triacylglycerols Consisting of Three Different Fatty Acyls and Their Chiral Chromatographic Elution Behavior.

Chromatographic separation of triacylglycerol (TG) enantiomers is a highly challenging task of analytical chemistry because of the similar physicochemical properties. The analysis of chiral TGs is crucial for improving the knowledge of lipid biochemistry and for understanding the nutritional properties of fats and oils. Thus, this study aimed to systematically investigate the chiral resolution of TGs consisting of three different fatty acyls (FAs). Thirty-three asymmetric TG enantiopairs, including 49 synthesized enantiopure TGs and racemic TGs, were analyzed with a recycling chiral HPLC system. Twenty-six enantiopairs were successfully separated. Overall, having both unsaturated and saturated FAs in the outer positions or a difference in carbon chain length between two saturated FAs at the outer positions favored the separation of enantiomers. The retention time at separation correlated negatively with the sn-3 carbon number of the early eluting enantiomer and positively with the carbon number difference between sn-1 and sn-3. When the samples were studied in separate groups based on unsaturation and regioisomers, both the acyl carbon number and the degree of unsaturation of FAs in all three positions influenced the separation and elution behavior of chiral TGs, indicating an active role of both intermolecular interactions and steric hindrances. This is the first systematic study of the chiral separation of TGs consisting of three different FAs using a large number of enantiopairs. The novel findings on the behavior of TG enantiomers in a chiral environment provide important guidance and reference for a stereospecific study of the chemistry and biochemistry of natural lipids.

A DFT Study of Cenobamate and Its New Isomers as an Effective Medical Treatment for Epilepsy Medications

Many anti-epileptic drugs use cenobamate, a voltage-gated sodium channel blocker, to the fullest extent. This study investigates the electronic, quantum chemical, and photolytic properties of Cenobamate and its novel designed isomers. The results show that the LUMOs levels are higher than the HOMO energies. The HOMO levels are unpaired and of s-character locally around the chlorine atoms. The reactivity and stability are explained by the frontier molecular energy values and different biochemical descriptors. All isomers have smallest energy gap means with a higher reactivity for biological applications. The most frequent reaction sites are found to be the three acyl carbons in the cenobamate molecule, the chloride related to the phenyl ring, the heterocyclic ring, and the carbonyl oxygen and nitrogen in the ester amide group. The novel isomers have high ability to interact with species, and this takes place either in clinical use or under clinical trials. The isomers are soft compounds, and can easily interact with big soft molecules such as DNA.

Bis(acyl)phosphide Complexes of U(III)/U(IV): A Case of a Hidden Redox-Active Ligand.

The recently reported tris(bis(2,4,6-triisopropylbenzoyl)-phosphide)uranium (UIII(trippBAP)3, 2) complex (Inorg. Chem. 2022, 61 (32), 12508-12517) demonstrated a silent 31P NMR spectrum. This complex was described as a U(III) complex with an organic radical ligand fragment. Moreover, the EPR spectrum of 2 was indicative of an organic radical in the ligand framework complexed to uranium, in contrast to that of UIV(mesBAP)4, 1. Herein, with the help of relativistic density functional theory (DFT) calculations, the electronic structures of 1, 2, and U(mesBAP)3 (4) are examined in an effort to understand the unusual 31P NMR spectrum of 2. Results indicate the reduction of the carbonyl bonds and delocalization of the electrons over the ligands, indicative of U → L backbonding. Additionally, the reduced acyl carbons are found to exist as ketyl radicals [O═C• -] that are responsible for the silent 31P NMR spectra of 2. These findings demonstrate the redox noninnocent nature of BAP- in 2 and 4, causing uranium to exist in a formal oxidation state of +4.

Comprehensive lipidomic analysis revealed the effects of fermented Morus alba L. intake on lipid profile in backfat and muscle tissue of Yuxi black pigs.

Mulberry leaf is a widely used protein feed and is often used as a strategy to reduce feed costs and improve meat quality in the livestock industry. However, to date, there is a lack of research on the improvement of meat quality using mulberry leaves, and the exact mechanisms are not yet known. The results showed that fermented mulberry leaves significantly reduced backfat content but had no significant effect on intramuscular fat (IMF). Lipidomic analysis showed that 98 and 303 differential lipid molecules (p < 0.05) were identified in adipose and muscle tissues, respectively, including triglycerides (TG), phosphatidylcholine, phosphatidylethanolamine, sphingolipids, and especially TG; therefore, we analysed the acyl carbon atom number of TG. The statistical results of acyl with different carbon atom numbers of TG in adipose tissue showed that the acyl group containing 13 carbon atoms (C13) in TG was significantly upregulated, whereas C15, C16, C17, and C23 were significantly downregulated, whereas in muscle tissue, the C12, C19, C23, C25, and C26 in TG were significantly downregulated. Acyl changes in TG were different for different numbers of carbon atoms in different tissues. We found that the correlations of C (14-18) in adipose tissue were higher, but in muscle tissue, the correlations of C (18-26) were higher. Through pathway enrichment analysis, we identified six and four metabolic pathways with the highest contributions of differential lipid metabolites in adipose and muscle tissues respectively. These findings suggest that fermented mulberry leaves improve meat quality mainly by inhibiting TG deposition by downregulating medium- and short-chain fatty acids in backfat tissue and long-chain fatty acids in muscle tissue.

Fe]-hydrogenase, cofactor biosynthesis and engineering.

[Fe]-hydrogenase catalyzes the heterolytic cleavage of H2 and reversible hydride transfer to methenyl-tetrahydromethanopterin. The iron-guanylylpyridinol (FeGP) cofactor is the prosthetic group of this enzyme, in which mononuclear Fe(II) is ligated with a pyridinol and two CO ligands. The pyridinol ligand fixes the iron by an acyl carbon and a pyridinol nitrogen. Biosynthetic proteins for this cofactor are encoded in the hmd co-occurring (hcg) genes. The function of HcgB, HcgC, HcgD, HcgE, and HcgF was studied by using structure to function analysis, which is based on the crystal structure of the proteins and subsequent enzyme assays. Recently, we reported the catalytic properties of HcgA and HcgG, novel radical S-adenosyl methionine enzymes, by using an in vitro biosynthesis assay. Here, we review the properties of [Fe]-hydrogenase and the FeGP cofactor, and the biosynthesis of the FeGP cofactor. Finally, we discuss the expected engineering of [Fe]-hydrogenase and the FeGP cofactor.

Protocols for sample preparation and compound-specific stable-isotope analyses (δ2H, δ13C) of fatty acids in biological and environmental samples

Compound-specific stable-isotope analysis (CSIA) of fatty acids is a powerful tool to better understand the trophic transfer of fatty acids and their biochemical fate in and across ecosystems, including tracing animal migration and understanding physiological processes. The non-exchangeable nature of CH bonds in acyl chains, hydrogen (δ2H) and carbon (δ13C) stable-isotope values of fatty acids (FA) provide independent information about the origins of fatty acids. Several technical obstacles must be overcome to ensure accurate and reproducible measurements of FA-CSIA can be made. This protocol describes the sample preparation process for successful stable-isotope analyses of fatty acids obtained from environmental and biological samples. Numerous techniques for the preanalytical processing of fatty acid samples are available, and these often have minimal impact on δ values. Here, we provide an in-depth guide detailing our well-established laboratory protocols, ranging from the initial sample preparation, lipid extraction, and transmethylation to the instrumental arrangement, data collection, and analysis.•Protocol from obtaining a sample to standardized fatty acid specific δ2H and δ13C values.•Separate GC analysis procedures for C and H are recommended for optimal performance.

A facile synthesis of 2-ethynylpyrroles by ButOK-assisted room temperature deprotection of 2-(acylethynyl)pyrroles

Available N-substituted 2-(acylethynyl)pyrroles undergo room temperature deprotection in the ButOK/THF system to give 2-ethynylpyrroles in 82–92% yields. Quantum-chemical calculations (B2PLYP/6-311G**//B3LYP/6-311G**+C-PCM/THF) show that the cleavage of ethynyl–acyl bond via the proton transfer from ButOK with formation of potassium acylate and 2-methylpropene is thermo-dynamically much more preferred compared to alternative nucleophilic attack of tert-butoxide anion at the acyl carbon (ΔG = –35.1 vs. –2.7 kcal mol-1).

Delivery mechanism of doxorubicin by PEG-DPPE micelles on membrane invasion by dynamic simulations.

Exploiting micelles of polyethylene glycol-dipalmitoylglycerophosphoethanolamine (PEG-DPPE) as a drug delivery approach is of great promise for improving therapeutic targeting and the half-lives of drugs. To optimize the micelle carriers, pending issues concerning the kinetics underlying the carrier-membrane interplay and the specific contributions of the micelle hydrophobic/hydrophilic components remain to be addressed. Relying on MARTINI coarse-grain (CG) molecular dynamics simulations, we explored the carrier-membrane fusion dynamics of PEG-DPPE micelles with different PEG repetitions in delivering doxorubicin (DOX). A bilayer model composed of 20% phosphatidylglycerol (POPG) and 80% phosphatidylcholine (POPC) was constructed to mimic anionic cancer cell membranes. The CG model of DOX was pioneeringly constructed herein, and it was found to distribute at the hydrophilic/hydrophobic interface of the PEGylated micelles, in agreement with experimental results. The free DOXs cause insignificant disorder of the membrane organization, whereas the PEG-DPPE micelles encapsulating DOX lead to a remarkable membrane invasion supported by the order parameter of the lipid acyl carbon tails and the membrane permeation free energy of DOX. The carrier-bilayer interaction shows a stepwise form attributed to the rearrangement of the zwitterionic/anionic lipids upon the absorption of the DOX-micelle complex on a membrane locality, which initiates the rapid release of DOX to the bilayer interior. Benefiting from the enhanced micelle-membrane interplay, the PEG1250-DPPE micelles result in severe bilayer breakage and deeper membrane insertion of DOX compared to the PEG2000-DPPE micelles. This study provides new theoretical insights into the mechanism of PEG-DPPE micelles in delivering drugs through membranes, which is of benefit for further optimization of PEGylated delivery systems.

The human liver lipidome is significantly related to the lipid composition and aggregation susceptibility of low-density lipoprotein (LDL) particles

Background and aimsThe susceptibility of low-density lipoprotein (LDL) to aggregation predicts atherosclerotic cardiovascular disease. However, causes of interindividual variation in LDL lipid composition and aggregation susceptibility remain unclear. We examined whether the lipid composition and aggregation susceptibility of LDL reflect the lipid composition of the human liver. MethodsLiver biopsies and blood samples for isolation of LDL particles were obtained from 40 obese subjects (BMI 45.9 ± 6.1 kg/m2, age 43 ± 8 years). LDL was isolated using sequential ultracentrifugation and lipidomic analyses of liver and LDL samples were determined using ultra-high performance liquid chromatography–mass spectrometry. LDL aggregation susceptibility ex vivo was analyzed by inducing aggregation by human recombinant secretory sphingomyelinase and following aggregate formation. ResultsThe composition (acyl carbon number and double bond count) of hepatic triglycerides, phosphatidylcholines, and sphingomyelins (SMs) was closely associated with that of LDL particles. Hepatic dihydroceramides and ceramides were positively correlated with concentrations of the corresponding SM species in LDL as well with LDL aggregation. These relationships remained statistically significant after adjustment for age, sex, and body mass index. ConclusionsLipid composition of LDL reflects that of the human liver in obese patients. Changes in hepatic sphingolipid metabolism may contribute to interindividual variation of LDL lipid composition and susceptibility to aggregation.

Ceramide Acyl Chain Length and Its Relevance to Intracellular Lipid Regulation.

Ceramides are a class of sphingolipids which are implicated in skin disorders, obesity, and other metabolic diseases. As a class with pleiotropic effects, recent efforts have centred on discerning specific ceramide species and their effects on atopic dermatitis, obesity, type 2 diabetes, and cardiovascular diseases. This delineation has allowed the identification of disease biomarkers, with long acyl chain ceramides such as C16- and C18-ceramides linked to metabolic dysfunction and cardiac function decline, while ultra-long acyl chain ceramides (>25 carbon acyl chain) were reported to be essential for maintaining a functional skin barrier. Given the intricate link between free fatty acids with ceramides, especially the de novo synthetic pathway, intracellular lipid droplet formation is increasingly viewed as an important mechanism for preventing accumulation of toxic ceramide species. Here, we review recent reports of various ceramide species involved in skin abnormalities and metabolic diseases, and we propose that promotion of lipid droplet biogenesis can be seen as a potential protective mechanism against deleterious ceramides.

A Comparative Study of Milk Fat Extracted from the Milk of Different Goat Breeds in China: Fatty Acids, Triacylglycerols and Thermal and Spectroscopic Characterization.

Goat milk (GM) is an excellent alternative to cow milk and has recently been used in commercial infant formula preparation due to its superior fat composition. Here, the fatty acid (FA) composition, triacylglycerol (TAG) molecular species, thermal behavior and infrared spectra of extracted milk fat from the milk of the two main breeds of dairy goat bred in China (Guanzhong GM (GZG) and Xinong Saanen GM (XSG)) are investigated. Gas chromatography, Fourier-transform infrared spectroscopy, differential scanning calorimetry and ultra-performance convergence chromatography with quadrupole time-of-flight mass spectrometry are applied. The obtained results evidence significant fat compositional differences based on the breed that produced the considered GM. The major FAs in both GM fats were capric (C10:0), myristic (C14:0), palmitic (C16:0), stearic (C18:0) and oleic (C18:1 n-9c). GZG presented a higher content of medium-chain saturated FAs, while XSG had higher unsaturated FAs with higher ratios of L/Ln and n-6/n-3. A total of 339 and 359 TAGs were detected and quantified in GZG and XSG, and the major TAGs were those of m/z 740.6712 (14.10 ± 0.27%) and m/z 684.6094 (10.94 ± 0.02%), respectively. Milk TAGs of GZG and XSG showed 24–54 and 26–54 total acyl carbon numbers with a 0–4 and 0–5 double bond number at 68 and 72 various retention times, respectively. Thermal analysis showed that all GM fat samples melted below normal body temperature. Infrared spectra revealed higher absorption values of GZG milk fat. This study provides valuable information to the dairy industry sector about GM fat produced in China, assessing the appropriateness of Chinese GM fat to be applied in Chinese infant formula.

Synthesis and Evaluation of Acylated Derivatives of Hederagenin as Inhibitors of HIV-1 and HCV NS3/4A Proteases

Viral infection imposes a major threat to human health. To develop new potent antiviral agents, Hederagenin (HE), a known inhibitor of HIV-1 and HCV NS3/4A proteases, was used as a starting material to synthesize 4 types of HE derivatives, HE-3,23-diacyl, HES-3,23-diacyl, HES-3-acyl, and HES-3-oxo-23-acyl. We evaluated the in vitro inhibitory activities of the derivatives against HIV-1 and HCV NS3/4A proteases. (3 β,23)-Di- O-diglutaryl-hederagenin (1b) and (3 β,23)-di- O-(3′,3′-dimethylsuccinyl-hederagenin ethyl ester (2b) exhibited potent inhibitory activities against the HIV-1 and/or HCV NS3/4A proteases with IC50 values &lt; 10 μM, but did not appreciably inhibit general human proteases renin and trypsin. The SARs showed that dicarboxylic acid hemiesters of HE significantly enhance the antiviral activities when C3 or C23 are linked with 6 carbon acyl chains.

Comparative Analysis of Triglycerides From Different Regions and Mature Lactation Periods in Chinese Human Milk Project (CHMP) Study.

The kinds and proportions of triglycerides of human mature milk play an independent role in the growth of infants. In this study, the human milk samples obtained from eight different Chinese cities (Chengdu, Weihai, Lanzhou, Jinhua, Beijing, Guangzhou, Zhengzhou, and Harbin) and six sequential mature lactation times (30, 60, 90, 120, 150, and 180 days) were detected for the triglycerides. The result demonstrated that total 66 triglycerides were detected in mature human milk, with acyl carbon number (ACN) numbers were locating in the range of 34–54 and double bond (DB) numbers were locating in the range of 0–6. In addition, the percentage of OPO, OPL, and OOO was relatively higher than others, accounted for more than 4% of total triglycerides in all the lactation areas and times, and the percentage of U2S and LLL triglycerides was also richest in mature milk. Furthermore, it was obvious that lactation regions had more significant effect on the triglycerides compared with lactation time and the triacylglycerols (TAGs) of human milk in Guangzhou were clearly different from that in other regions. Therefore, the results of this study will provide data reference for the design of infant formula suitable for Chinese babies.

Enantioselective Enzymatic Synthesis of (R)‐Phenyl Alkyl Esters and Their Analogue Amides using Fatty Acids as Green Acyl Donors.

AbstractAn efficient enantioselective synthesis of a set of (R)‐phenylalkylesters and their analogues amides via enzymatic acylation of 1‐phenylethanol (rac‐1) and 1‐phenylethanamine (rac‐2) using carboxylic acids with different chain‐lengths as green acyl donor is reported. Three lipases are used: Candida antarctica B immobilized on acrylic resin (CAL‐B) and two free lipases: Pseudomonas cepacia (PCL) and Candida rugosa (CRL). The CAL‐B shows an excellent selectivity during the acylation of rac‐1 without restriction due to the acyl carbon chain‐length, the (R)‐ esters (1 a–1 f) were obtained enantiopures (ee up to 99 %). For the first time, the PCL catalyzed O‐acylation allows smoothly to (R)‐(1 d–1 f) with high selectivity (E⋙200) is described. The conversions increase with the length of the carbon‐chain of the acyl donor (28.7 %≤C≤40 %). The CRL shows less selective and provided the (R) (1 b–1 e) with 77 %≤eep≤86.4 %. Due to its high thermostability, the CAL‐B is used for the N‐acylation of rac‐2 and provides access to enantioenriched (R)‐fatty amides (2 d–2 f) (60.7 %≤eep≤74.4 %) and the remained (S)‐2 with ees&gt;91 %.

Effect of pasteurisation, homogenisation and freeze‐drying on bovine and buffalo milk fat triacylglycerols profile

Milk fat triacylglycerols (TAGs) analysis is considered to be a challenging task; considerable numbers of fatty acyl moieties form one of the most complicated natural fats, with potentially thousands of molecular species of TAGs. Herein, TAGs fingerprinting of raw and processed bovine and buffalo milk was carried out using ultra‐performance liquid chromatography‐mass spectrometry. Firstly, milk samples were subjected to pasteurisation, homogenisation and freeze‐drying, and the TAGs profile was then analysed. Palmitic, oleic, myristic and stearic were the predominant fatty acids detected. The results revealed that the TAG profile was not significantly influenced by the applied technological treatments. Bovine milk TAGs exhibited 28–54 total acyl carbons, with 0–5 double bonds at 80 different retention times. However, buffalo milk TAGs possessed 26–54 total acyl carbon number containing 0–4 double bonds at 84 different retention times. The findings of this study could offer valuable knowledge to the field of dairy technology.

1,2‐Aryl Migration Induced by Amide C−N Bond‐Formation: Reaction of Alkyl Aryl Ketones with Primary Amines Towards α,α‐Diaryl β,γ‐Unsaturated γ‐Lactams

AbstractRearrangement reactions incorporated into cascade reactions play an important role in rapidly increasing molecular complexity from readily available starting materials. Reported here is a Cu‐catalyzed cascade reaction of α‐(hetero)aryl‐substituted alkyl (hetero)aryl ketones with primary amines that incorporates an unusual 1,2‐aryl migration induced by amide C−N bond formation to produce a class of structurally novel α,α‐diaryl β,γ‐unsaturated γ‐lactams in generally good‐to‐excellent yields. This cascade reaction has a broad substrate scope with respect to primary amines, allows a wide spectrum of (hetero)aryl groups to smoothly undergo 1,2‐migration, and tolerates electronically diverse α‐substituents on the (hetero)aryl ring of the ketones. Mechanistically, this 1,2‐aryl migration may stem from the intramolecular amide C−N bond formation which induces nucleophilic migration of the aryl group from the acyl carbon center to the electrophilic carbon center that is conjugated with the resulting iminium moiety.

1,2-Aryl Migration Induced by Amide C-N Bond-Formation: Reaction of Alkyl Aryl Ketones with Primary Amines Towards α,α-Diaryl β,γ-Unsaturated γ-Lactams.

Rearrangement reactions incorporated into cascade reactions play an important role in rapidly increasing molecular complexity from readily available starting materials. Reported here is a Cu-catalyzed cascade reaction of α-(hetero)aryl-substituted alkyl (hetero)aryl ketones with primary amines that incorporates an unusual 1,2-aryl migration induced by amide C-N bond formation to produce a class of structurally novel α,α-diaryl β,γ-unsaturated γ-lactams in generally good-to-excellent yields. This cascade reaction has a broad substrate scope with respect to primary amines, allows a wide spectrum of (hetero)aryl groups to smoothly undergo 1,2-migration, and tolerates electronically diverse α-substituents on the (hetero)aryl ring of the ketones. Mechanistically, this 1,2-aryl migration may stem from the intramolecular amide C-N bond formation which induces nucleophilic migration of the aryl group from the acyl carbon center to the electrophilic carbon center that is conjugated with the resulting iminium moiety.

PH-triggered pore-forming peptides with strong composition-dependent membrane selectivity

Peptides that self-assemble into nanometer-sized pores in lipid bilayers could have utility in a variety of biotechnological and clinical applications if we can understand their physical chemical properties and learn to control their membrane selectivity. To empower such control, we have used synthetic molecular evolution to identify the pH-dependent delivery peptides, a family of peptides that assemble into macromolecule-sized pores in membranes at low peptide concentration but only at pH < ∼6. Further advancements will also require better selectivity for specific membranes. Here, we determine the effect of anionic headgroups and bilayer thickness on the mechanism of action of the pH-dependent delivery peptides by measuring binding, secondary structure, and macromolecular poration. The peptide pHD15 partitions and folds equally well into zwitterionic and anionic membranes but is less potent at pore formation in phosphatidylserine-containing membranes. The peptide also binds and folds similarly in membranes of various thicknesses, but its ability to release macromolecules changes dramatically. It causes potent macromolecular poration in vesicles made from phosphatidylcholine with 14 carbon acyl chains, but macromolecular poration decreases sharply with increasing bilayer thickness and does not occur at any peptide concentration in fluid bilayers made from phosphatidylcholine lipids with 20-carbon acyl chains. The effects of headgroup and bilayer thickness on macromolecular poration cannot be accounted for by the amount of peptide bound but instead reflect an inherent selectivity of the peptide for inserting into the membrane-spanning pore state. Molecular dynamics simulations suggest that the effect of thickness is due to hydrophobic match/mismatch between the membrane-spanning peptide and the bilayer hydrocarbon. This remarkable degree of selectivity based on headgroup and especially bilayer thickness is unusual and suggests ways that pore-forming peptides with exquisite selectivity for specific membranes can be designed or evolved.

Heat stress elicits remodeling in the anther lipidome of peanut

Understanding the changes in peanut (Arachis hypogaea L.) anther lipidome under heat stress (HT) will aid in understanding the mechanisms of heat tolerance. We profiled the anther lipidome of seven genotypes exposed to ambient temperature (AT) or HT during flowering. Under AT and HT, the lipidome was dominated by phosphatidylcholine (PC), phosphatidylethanolamine (PE), and triacylglycerol (TAG) species (> 50% of total lipids). Of 89 lipid analytes specified by total acyl carbons:total carbon–carbon double bonds, 36:6, 36:5, and 34:3 PC and 34:3 PE (all contain 18:3 fatty acid and decreased under HT) were the most important lipids that differentiated HT from AT. Heat stress caused decreases in unsaturation indices of membrane lipids, primarily due to decreases in highly-unsaturated lipid species that contained 18:3 fatty acids. In parallel, the expression of Fatty Acid Desaturase 3-2 (FAD3-2; converts 18:2 fatty acids to 18:3) decreased under HT for the heat-tolerant genotype SPT 06-07 but not for the susceptible genotype Bailey. Our results suggested that decreasing lipid unsaturation levels by lowering 18:3 fatty-acid amount through reducing FAD3 expression is likely an acclimation mechanism to heat stress in peanut. Thus, genotypes that are more efficient in doing so will be relatively more tolerant to HT.

Abstract 16607: Age-induced Endoplasmic Reticulum Stress in the Heart: Role of Increased Very Long-chain Ceramides

Introduction: Mitochondrial function is impaired in aged hearts. Increased endoplasm reticulum (ER) stress contributes to the mitochondrial dysfunction observed during aging. Ceramides (CRMD) are sphingolipid metabolites that contribute key roles in cell signaling. Increased CRMD can lead to ER stress. Ceramide synthase enzymes (CerS) generate chain length specific CRMD with the CerS isoform 2 (Cers2) forming very long chain CRMD of ≥ 20 carbon acyl chain lengths. Hypothesis: An increase in CRMD content during aging contributes to age-related ER stress. Methods: Male mice (3, 18, 24 mo.) from the NIA colony were studied. Cardiac mitochondria (MITO), mitochondrial associated membranes (MAM), and ER were isolated from mouse hearts. CRMD content was measured using LC-MS. The contents of CerS enzymes were measured by immunoblotting in myocardial homogenates. Results: ER stress increased progressively during aging with increased contents of cleaved ATF6 and CHOP, indicators of increased ER stress, evident at 18 and 24 mo. (Panel A) (all data mean±SEM; *p&lt;0.05 vs. 3 mo., † p&lt;0.05 vs. 18 mo.). Aging increased very long-chain CRMD (≥C20) in ER (Panel B) at 18 and 24 mo. Similar CRMD trends were observed MAM (Panel C), shared membrane domains where ER and MITO interact. The content of CerS2 was increased at 24 mo. compared to 3 mo. (Panel D, n=4 each age). In contrast, the contents of CerS isoforms 4 and 5, that generate shorter chain CRMD (&lt;C20) were unchanged (not shown). CRMD contents in MITO were unaltered with age (not shown). Thus, increased generation of very long chain CRMD in the ER is the likely mechanism of increased ER stress in the aged heart. Conclusion: Aging increased ER CRMD content by enhancing the formation of very long chain CRMD in ER by an increase in CerS2 content, concomitant with the onset of ER stress. The increase in age-induced ER stress, in turn, leads to mitochondrial dysfunction in the aged heart.