Sulfotransferase 1C2 Increases Mitochondrial Respiration by Converting Mitochondrial Membrane Cholesterol to Cholesterol Sulfate.

HYPOTHESIS: In this communication, we test the hypothesis that sulfotransferase 1C2 (SULT1C2, UniProt accession no. Q9WUW8) can modulate mitochondrial respiration by increasing state-III respiration. METHODS AND RESULTS: Using freshly isolated mitochondria, the addition of SULT1C2 and 3-phosphoadenosine 5 phosphosulfate (PAPS) results in an increased maximal respiratory capacity in response to the addition of succinate, ADP, and rotenone. Lipidomics and thin-layer chromatography of mitochondria treated with SULT1C2 and PAPS showed an increase in the level of cholesterol sulfate. Notably, adding cholesterol sulfate at nanomolar concentration to freshly isolated mitochondria also increases maximal respiratory capacity. In vivo studies utilizing gene delivery of SULT1C2 expression plasmids to kidneys result in increased mitochondrial membrane potential and confer resistance to ischemia/reperfusion injury. Mitochondria isolated from gene-transduced kidneys have elevated state-III respiration as compared with controls, thereby recapitulating results obtained with mitochondrial fractions treated with SULT1C2 and PAPS. CONCLUSION: SULT1C2 increases mitochondrial respiratory capacity by modifying cholesterol, resulting in increased membrane potential and maximal respiratory capacity. This finding uncovers a unique role of SULT1C2 in cellular physiology and extends the role of sulfotransferases in modulating cellular metabolism.

Quantitative determination of plasma cholesteryl ester levels in Japanese preadolescents from the Hokkaido study using liquid chromatography/tandem mass spectrometry.

Cholesteryl esters (CE) are sterols comprising various fatty acyl chains attached to a cholesterol hydroxyl moiety. CEs are often considered plasma biomarkers of liver function; however, their absolute concentrations in the plasma of Japanese preadolescents have not been well explored. This study aimed to determine the plasma CE levels in Japanese preadolescents of different sexes, ages, and body weights living in Hokkaido, Japan using targeted liquid chromatography/tandem mass spectrometry. The analysis was performed on the non-fasting plasma of preadolescents aged 9-12 years (n = 339 healthy volunteers; 178 boys and 161 girls) from Sapporo, Hokkaido, Japan. The analysis results showed that the total CE levels in boys and girls were 871 ± 153 and 862 ± 96 pmol/µL, respectively. CE 18:2 (41 ± 2.9 %) was found to be the most abundant species followed by CE 18:1 (16 ± 1.5 %) and CE 16:0 (13 ± 1.1 %). The ω-3 fatty acid-containing CEs such as CE 18:3 and CE 20:5 were significantly lower in girls than in boys. Despite the different ages, CEs were tightly regulated in the plasma of children's, and the total CEs ranged between 844 and 906 pmol/µL in boys and 824 and 875 pmol/µL in girls. The participants were further classified into three groups based on their body mass index underweight (n = 237), normal weight (n = 94), and overweight (n = 8). Most of the quantified CEs were accumulated in the overweight group. Interestingly, CE 18:3 was significantly upregulated in the overweight group compared to that in the normal range, and the area under the receiver operating characteristic curve was 0.73, suggesting that it could be a possible marker for obesity. This study marks the initial investigation of absolute CE levels in the plasma of children and can help elucidate the relationship between CEs and childhood obesity.

Aberrant lipid accumulation and retinal pigment epithelium dysfunction in PRCD-deficient mice.

Progressive Rod-Cone Degeneration (PRCD) is an integral membrane protein found in photoreceptor outer segment (OS) disc membranes and its function remains unknown. Mutations in Prcd are implicated in Retinitis pigmentosa (RP) in humans and multiple dog breeds. PRCD-deficient models exhibit decreased levels of cholesterol in the plasma. However, potential changes in the retinal cholesterol remain unexplored. In addition, impaired phagocytosis observed in these animal models points to potential deficits in the retinal pigment epithelium (RPE). Here, using a Prcd-/- murine model we investigated the alterations in the retinal cholesterol levels and impairments in the structural and functional integrity of the RPE. Lipidomic and immunohistochemical analyses show a 5-fold increase in the levels of cholesteryl esters (C.Es) and lipid deposits in the PRCD-deficient retina, respectively, indicating alterations in total retinal cholesterol. Furthermore, the RPE of Prcd-/- mice exhibit a 1.7-fold increase in the expression of lipid transporter gene ATP-binding cassette transporter A1 (Abca1). Longitudinal fundus and spectral domain optical coherence tomography (SD-OCT) examinations showed focal lesions and RPE hyperreflectivity. Strikingly, the RPE of Prcd-/- mice exhibited age-related pathological features such as lipofuscin accumulation, Bruch's membrane (BrM) deposits and drusenoid focal deposits, mirroring an Age-related Macular Degeneration (AMD)-like phenotype. We propose that the extensive lipofuscin accumulation likely impairs lysosomal function, leading to the defective phagocytosis observed in Prcd-/- mice. Our findings support the dysregulation of retinal cholesterol homeostasis in the absence of PRCD. Further, we demonstrate that progressive photoreceptor degeneration in Prcd-/- mice is accompanied by progressive structural and functional deficits in the RPE, which likely exacerbates vision loss over time.

Butyrate Increases Heparin Synthesis and Storage in Human Mast Cells.

Sulphated glycosaminoglycans (GAGs) such as heparin are a major component of mast cell granules and form the matrix within which biogenic mediators are stored. Since GAGs released from mast cells also play an important role in helminth expulsion, understanding GAG storage can offer new insights into mast cell function. Sodium butyrate (NaBu), a short-chain fatty acid, causes ultrastructural changes within the granules of human mast cells (HMC-1) and increases their histamine content. Therefore, we hypothesized that NaBu treatment would also modify the storage of polysaccharides such as GAGs. NaBu (1 mM) significantly increased GAG content and granularity in a time- and concentration-dependent manner without affecting cell viability and metabolic activity. NaBu increased the expression of enzymes associated with heparin biosynthesis (GLCE, NDST1, NDST2, HS6ST1, and GALT1) in a time-dependent manner. A cholesteryl butyrate emulsion (CholButE) increased heparin content after 24 and 48 h and modestly altered the expression of genes involved in heparin biosynthesis. Similar to NaBu, CholButE reduced cell proliferation without significantly altering viability or metabolic activity. These data show that butyrate increases the synthesis and storage of heparin in human mast cells, perhaps by altering their metabolic pathways.

A Facile LC-MS Method for Profiling Cholesterol and Cholesteryl Esters in Mammalian Cells and Tissues.

Cholesterol is central to mammalian lipid metabolism and serves many critical functions in the regulation of diverse physiological processes. Dysregulation in cholesterol metabolism is causally linked to numerous human diseases, and therefore, in vivo, the concentrations and flux of cholesterol and cholesteryl esters (fatty acid esters of cholesterol) are tightly regulated. While mass spectrometry has been an analytical method of choice for detecting cholesterol and cholesteryl esters in biological samples, the hydrophobicity, chemically inert nature, and poor ionization of these neutral lipids have often proved a challenge in developing lipidomics compatible liquid chromatography-mass spectrometry (LC-MS) methods to study them. To overcome this problem, here, we report a reverse-phase LC-MS method that is compatible with existing high-throughput lipidomics strategies and capable of identifying and quantifying cholesterol and cholesteryl esters from mammalian cells and tissues. Using this sensitive yet robust LC-MS method, we profiled different mammalian cell lines and tissues and provide a comprehensive picture of cholesterol and cholesteryl esters content in them. Specifically, among cholesteryl esters, we find that mammalian cells and tissues largely possess monounsaturated and polyunsaturated variants. Taken together, our lipidomics compatible LC-MS method to study this lipid class opens new avenues in understanding systemic and tissue-level cholesterol metabolism under various physiological conditions.

Analysis of Lipoprotein Signaling in Iris Melanocytes.

Lipids are compounds involved in many biologic functions including cell structure, metabolism, energy storage and are involved in signaling. A prominent lipid in these functions is cholesterol. Cholesterol also plays a part in the signaling of melanocytes, which contain melanosomes. The maturation of these melanosomes happens during melanocyte growth. The deficit of melanogenesis or melanosome maturation is associated with ocular albinism in the eye. Aberrations of melanosome maturation are also associated with pigment dispersion syndrome. Albinism and pigment dispersion manifestations are systemic. Both melanogenesis and melanocyte maturation are affected by cholesterol metabolism. Cholesterol signaling is a part of many pathways in the body, and evaluating these signals can have implications in systemic disease processes of melanogenesis and melanosome maturation, like ocular albinism and pigment dispersion. Cholesterol is carried by lipoprotein particles. Low-density lipoprotein (LDL) is usually the transport vehicle for cholesterol to reach tissues and organelles. The LDL uptake on cells often sends out a cascade of internal signaling within the cells. We describe here LDL signaling related to lipase activity changes using enzymatic methods with a kit. We describe analyses of cholesterol esters and free cholesterol with liquid chromatography and gas chromatography with or in tandem with mass spectrometry (GC-MS and LC-MS/MS). These analyses will provide insight into melanosome maturation and melanogenesis. The methods described here are applicable to all melanocytes within the body of a model mammalian organism.

Sterol O-acyltransferase (SOAT/ACAT) activity is required to form cholesterol crystals in hepatocyte lipid droplets.

OBJECTIVE: Excess cholesterol storage can induce the formation of cholesterol crystals in hepatocyte lipid droplets. Such crystals distinguish metabolic dysfunction associated steatohepatitis (MASH) from simple steatosis and may underlie its pathogenesis by causing cell damage that triggers liver inflammation. The mechanism linking cholesterol excess to its crystallization in lipid droplets is unclear. As cholesteryl esters localize to and accumulate in lipid droplets more readily than unesterified free cholesterol, we investigated whether cholesterol esterification by sterol O-acyltransferase (SOAT), also known as acyl co-A cholesterol acyltransferase (ACAT), is required for hepatocyte lipid droplet crystal formation. METHOD: Cholesterol crystals were measured in cholesterol loaded Hep3B hepatocytes, RAW264.7 macrophages, and mouse liver using polarizing light microscopy. We examined the effect of blocking SOAT activity on crystal formation and compared these results to features of cholesterol metabolism and the progression to intracellular crystal deposits. RESULTS: Cholesterol loading of Hep3B cells caused robust levels of lipid droplet localized crystal formation in a dose- and time-dependent manner. Co-treatment with SOAT inhibitors and genetic ablation of SOAT1 blocked crystal formation. SOAT inhibitor also blocked crystal formation in low density lipoprotein (LDL) treated Hep3B cells, acetylated LDL treated RAW 264.7 macrophages, and in the liver of mice genetically predisposed to hepatic cholesterol overload and in mice with cholesterol enriched diet-induced MASH. CONCLUSION: SOAT1-mediated esterification may underlie cholesterol crystals associated with MASH by concentrating it in lipid droplets. These findings imply that inhibiting hepatocyte SOAT1 may be able to alleviate cholesterol associated MASH. Moreover, that either a lipid droplet localized cholesteryl ester hydrolase is required for cholesterol crystal formation, or the crystals are composed of cholesteryl ester.

Propionate prevents loss of the PDIM virulence lipid in Mycobacterium tuberculosis.

Phthiocerol dimycocerosate (PDIM) is an essential virulence lipid of Mycobacterium tuberculosis. In vitro culturing rapidly selects for spontaneous PDIM-negative mutants that have attenuated virulence and increased cell wall permeability, thus impacting the relevance of experimental findings. PDIM loss can also reduce the efficacy of the BCG Pasteur vaccine. Here we show that vancomycin susceptibility can rapidly screen for M. tuberculosis PDIM production. We find that metabolic deficiency of methylmalonyl-CoA impedes the growth of PDIM-producing bacilli, selecting for PDIM-negative variants. Supplementation with odd-chain fatty acids, cholesterol or vitamin B12 restores PDIM-positive bacterial growth. Specifically, we show that propionate supplementation enhances PDIM-producing bacterial growth and selects against PDIM-negative mutants, analogous to in vivo conditions. Our study provides a simple approach to screen for and maintain PDIM production, and reveals how discrepancies between the host and in vitro nutrient environments can attenuate bacterial pathogenicity.

Effects of Recombinant Human Lecithin Cholesterol Acyltransferase on Lipoprotein Metabolism in Humans.

BACKGROUND: LCAT (lecithin cholesterol acyl transferase) catalyzes the conversion of unesterified, or free cholesterol, to cholesteryl ester, which moves from the surface of HDL (high-density lipoprotein) into the neutral lipid core. As this iterative process continues, nascent lipid-poor HDL is converted to a series of larger, spherical cholesteryl ester-enriched HDL particles that can be cleared by the liver in a process that has been termed reverse cholesterol transport. METHODS: We conducted a randomized, placebocontrolled, crossover study in 5 volunteers with atherosclerotic cardiovascular disease, to examine the effects of an acute increase of recombinant human (rh) LCAT via intravenous administration (300-mg loading dose followed by 150 mg at 48 hours) on the in vivo metabolism of HDL APO (apolipoprotein)A1 and APOA2, and the APOB100-lipoproteins, very low density, intermediate density, and low-density lipoproteins. RESULTS: As expected, recombinant human LCAT treatment significantly increased HDL-cholesterol (34.9 mg/dL; P≤0.001), and this was mostly due to the increase in cholesteryl ester content (33.0 mg/dL; P=0.014). This change did not affect the fractional clearance or production rates of HDL-APOA1 and HDL-APOA2. There were also no significant changes in the metabolism of APOB100-lipoproteins. CONCLUSIONS: Our results suggest that an acute increase in LCAT activity drives greater flux of cholesteryl ester through the reverse cholesterol transport pathway without significantly altering the clearance and production of the main HDL proteins and without affecting the metabolism of APOB100-lipoproteins. Long-term elevations of LCAT might, therefore, have beneficial effects on total body cholesterol balance and atherogenesis.

Cholesterol sulfate-mediated ion-pairing facilitates the self-nanoassembly of hydrophilic cationic mitoxantrone.

HYPOTHESIS: Hydrophilic cationic drugs such as mitoxantrone hydrochloride (MTO) pose a significant delivery challenge to the development of nanodrug systems. Herein, we report the use of a hydrophobic ion-pairing strategy to enhance the nano-assembly of MTO. EXPERIMENTS: We employed biocompatible sodium cholesteryl sulfate (SCS) as a modification module to form stable ion pairs with MTO, which balanced the intermolecular forces and facilitated nano-assembly. PEGylated MTO-SCS nanoassemblies (pMS NAs) were prepared via nanoprecipitation. We systematically evaluated the effect of the ratio of the drug module (MTO) to the modification module (SCS) on the nanoassemblies. FINDINGS: The increased lipophilicity of MTO-SCS ion pair could significantly improve the encapsulation efficiency (∼97 %) and cellular uptake efficiency of MTO. The pMS NAs showed prolonged blood circulation, maintained the same level of tumor antiproliferative activity, and exhibited reduced toxicity compared with the free MTO solution. It is noteworthy that the stability, cellular uptake, cytotoxicity, and in vivo pharmacokinetic behavior of the pMS NAs increased in proportion to the molar ratio of SCS to MTO. This study presents a self-assembly strategy mediated by ion pairing to overcome the challenges commonly associated with the poor assembly ability of hydrophilic cationic drugs.

VHL mutation drives human clear cell renal cell carcinoma progression through PI3K/AKT-dependent cholesteryl ester accumulation.

BACKGROUND: Cholesteryl ester (CE) accumulation in intracellular lipid droplets (LDs) is an essential signature of clear cell renal cell carcinoma (ccRCC), but its molecular mechanism and pathological significance remain elusive. METHODS: Enabled by the label-free Raman spectromicroscopy, which integrated stimulated Raman scattering microscopy with confocal Raman spectroscopy on the same platform, we quantitatively analyzed LD distribution and composition at the single cell level in intact ccRCC cell and tissue specimens in situ without any processing or exogenous labeling. Since we found that commonly used ccRCC cell lines actually did not show the CE-rich signature, primary cancer cells were isolated from human tissues to retain the lipid signature of ccRCC with CE level as high as the original tissue, which offers a preferable cell model for the study of cholesterol metabolism in ccRCC. Moreover, we established a patient-derived xenograft (PDX) mouse model that retained the CE-rich phenotype of human ccRCC. FINDINGS: Surprisingly, our results revealed that CE accumulation was induced by tumor suppressor VHL mutation, the most common mutation of ccRCC. Moreover, VHL mutation was found to promote CE accumulation by upregulating HIFα and subsequent PI3K/AKT/mTOR/SREBPs pathway. Inspiringly, inhibition of cholesterol esterification remarkably suppressed ccRCC aggressiveness in vitro and in vivo with negligible toxicity, through the reduced membrane cholesterol-mediated downregulations of integrin and MAPK signaling pathways. INTERPRETATION: Collectively, our study improves current understanding of the role of CE accumulation in ccRCC and opens up new opportunities for treatment. FUNDING: This work was supported by National Natural Science Foundation of China (No. U23B2046 and No. 62027824), National Key R&D Program of China (No. 2023YFC2415500), Fundamental Research Funds for the Central Universities (No. YWF-22-L-547), PKU-Baidu Fund (No. 2020BD033), Peking University First Hospital Scientific and Technological Achievement Transformation Incubation Guidance Fund (No. 2022CX02), and Beijing Municipal Health Commission (No. 2020-2Z-40713).

Membrane lipid remodeling eradicates Helicobacter pylori by manipulating the cholesteryl 6'-acylglucoside biosynthesis.

BACKGROUND: Helicobacter pylori, the main cause of various gastric diseases, infects approximately half of the human population. This pathogen is auxotrophic for cholesterol which it converts to various cholesteryl α-glucoside derivatives, including cholesteryl 6'-acyl α-glucoside (CAG). Since the related biosynthetic enzymes can be translocated to the host cells, the acyl chain of CAG likely comes from its precursor phosphatidylethanolamine (PE) in the host membranes. This work aims at examining how the acyl chain of CAG and PE inhibits the membrane functions, especially bacterial adhesion. METHODS: Eleven CAGs that differ in acyl chains were used to study the membrane properties of human gastric adenocarcinoma cells (AGS cells), including lipid rafts clustering (monitored by immunofluorescence with confocal microscopy) and lateral membrane fluidity (by the fluorescence recovery after photobleaching). Cell-based and mouse models were employed to study the degree of bacterial adhesion, the analyses of which were conducted by using flow cytometry and immunofluorescence staining, respectively. The lipidomes of H. pylori, AGS cells and H. pylori-AGS co-cultures were analyzed by Ultraperformance Liquid Chromatography-Tandem Mass Spectroscopy (UPLC-MS/MS) to examine the effect of PE(10:0)2, PE(18:0)2, PE(18:3)2, or PE(22:6)2 treatments. RESULTS: CAG10:0, CAG18:3 and CAG22:6 were found to cause the most adverse effect on the bacterial adhesion. Further LC-MS analysis indicated that the treatment of PE(10:0)2 resulted in dual effects to inhibit the bacterial adhesion, including the generation of CAG10:0 and significant changes in the membrane compositions. The initial (1 h) lipidome changes involved in the incorporation of 10:0 acyl chains into dihydro- and phytosphingosine derivatives and ceramides. In contrast, after 16 h, glycerophospholipids displayed obvious increase in their very long chain fatty acids, monounsaturated and polyunsaturated fatty acids that are considered to enhance membrane fluidity. CONCLUSIONS: The PE(10:0)2 treatment significantly reduced bacterial adhesion in both AGS cells and mouse models. Our approach of membrane remodeling has thus shown great promise as a new anti-H. pylori therapy.

Another Brick in the Wall of Tear Film Insights Added Through the Total Synthesis and Biophysical Profiling of anteiso-Branched Wax and Cholesteryl Esters.

The tear film lipid layer (TFLL) plays a vital part in maintenance of ocular health and represents a unique biological barrier comprising unusual and specialized lipid classes and species. The wax and cholesteryl esters (WEs and CEs) constitute roughly 80-90% of the TFLL. The majority of species in these lipid classes are branched and it is therefore surprising that the synthesis and properties of the second largest category of species, i.e., the anteiso-branched species, remain poorly characterized. In this study, we have developed a total synthesis route and completed a detailed NMR spectroscopic characterization of two common anteiso-branched species, namely: (22S)-22-methyltetracosanyl oleate and cholesteryl (22'S)-22'-methyltetracosanoate. In addition, we have studied their structural properties in the bulk state by wide-angle and small-angle X-ray scattering and their behavior at the aqueous interface using Langmuir monolayer techniques. A comparison to the properties displayed by iso-branched and straight-chain analogues indicate that branching patterns lead to distinct properties in the CE and WE lipid classes. Overall, this study complements the previous work in the field and adds another important brick in the tear film insights wall.

Regional Differences in the Small Intestinal Proteome of Control Mice and of Mice Lacking Lysosomal Acid Lipase.

The metabolic contribution of the small intestine (SI) is still unclear despite recent studies investigating the involvement of single cells in regional differences. Using untargeted proteomics, we identified regional characteristics of the three intestinal tracts of C57BL/6J mice and found that proteins abundant in the mouse ileum correlated with the high ileal expression of the corresponding genes in humans. In the SI of C57BL/6J mice, we also detected an increasing abundance of lysosomal acid lipase (LAL), which is responsible for degrading triacylglycerols and cholesteryl esters within the lysosome. LAL deficiency in patients and mice leads to lipid accumulation, gastrointestinal disturbances, and malabsorption. We previously demonstrated that macrophages massively infiltrated the SI of Lal-deficient (KO) mice, especially in the duodenum. Using untargeted proteomics (ProteomeXchange repository, data identifier PXD048378), we revealed a general inflammatory response and a common lipid-associated macrophage phenotype in all three intestinal segments of Lal KO mice, accompanied by a higher expression of GPNMB and concentrations of circulating sTREM2. However, only duodenal macrophages activated a metabolic switch from lipids to other pathways, which were downregulated in the jejunum and ileum of Lal KO mice. Our results provide new insights into the process of absorption in control mice and possible novel markers of LAL-D and/or systemic inflammation in LAL-D.

Regulation of lipid droplets and cholesterol metabolism in adrenal cortical cells.

The adrenal gland is composed of two distinctly different endocrine moieties. The interior medulla consists of neuroendocrine chromaffin cells that secrete catecholamines like adrenaline and noradrenaline, while the exterior cortex consists of steroidogenic cortical cells that produce steroid hormones, such as mineralocorticoids (aldosterone), glucocorticoids (cortisone and cortisol) and androgens. Synthesis of steroid hormones in cortical cells requires substantial amounts of cholesterol, which is the common precursor for steroidogenesis. Cortical cells may acquire cholesterol from de novo synthesis and uptake from circulating low- and high-density lipoprotein particles (LDL and HDL). As cholesterol is part of the plasma membrane in all mammalian cells and an important regulator of membrane fluidity, cellular levels of free cholesterol are tightly regulated. To ensure a robust supply of cholesterol for steroidogenesis and to avoid cholesterol toxicity, cortical cells store large amounts of cholesterol as cholesteryl esters in intracellular lipid droplets. Cortical steroidogenesis relies on both mobilization of cholesterol from lipid droplets and constant uptake of circulating cholesterol to replenish lipid droplet stores. This chapter will describe mechanisms involved in cholesterol uptake, cholesteryl ester synthesis, lipid droplet formation, hydrolysis of stored cholesteryl esters, as well as their impact on steroidogenesis. Additionally, animal models and human diseases characterized by altered cortical cholesteryl ester storage, with or without abnormal steroidogenesis, will be discussed.

Association between human blood metabolome and the risk of pre-eclampsia.

Pre-eclampsia is a complex multi-system pregnancy disorder with limited treatment options. Therefore, we aimed to screen for metabolites that have causal associations with preeclampsia and to predict target-mediated side effects based on Mendelian randomization (MR) analysis. A two-sample MR analysis was firstly conducted to systematically assess causal associations of blood metabolites with pre-eclampsia, by using metabolites related large-scale genome-wide association studies (GWASs) involving 147,827 European participants, as well as GWASs summary data about pre-eclampsia from the FinnGen consortium R8 release data that included 182,035 Finnish adult female subjects (5922 cases and 176,113 controls). Subsequently, a phenome-wide MR (Phe-MR) analysis was applied to assess the potential on-target side effects associated with hypothetical interventions that reduced the burden of pre-eclampsia by targeting identified metabolites. Four metabolites were identified as potential causal mediators for pre-eclampsia by using the inverse-variance weighted method, including cholesterol in large HDL (L-HDL-C) [odds ratio (OR): 0.88; 95% confidence interval (95% CI): 0.83-0.93; P = 2.14 × 10-5), cholesteryl esters in large HDL (L-HDL-CE) (OR: 0.88; 95% CI: 0.83-0.94; P = 5.93 × 10-5), free cholesterol in very large HDL (XL-HDL-FC) (OR: 0.88; 95% CI: 0.82-0.94; P = 1.10 × 10-4) and free cholesterol in large HDL (L-HDL-FC) (OR: 0.89; 95% CI: 0.84-0.95; P = 1.45 × 10-4). Phe-MR analysis showed that targeting L-HDL-CE had beneficial effects on the risk of 24 diseases from seven disease chapters. Based on this systematic MR analysis, L-HDL-C, L-HDL-CE, XL-HDL-FC, and L-HDL-FC were inversely associated with the risk of pre-eclampsia. Interestingly, L-HDL-CE may be a promising drug target for preventing pre-eclampsia with no predicted detrimental side effects. The study consists of a two-stage design that conducts MR at both stages. First, we assessed the causality for the associations between 194 blood metabolites and the risk of pre-eclampsia. Second, we investigated a broad spectrum of side effects associated with the targeting identified metabolites in 693 non-preeclampsia diseases. Our results suggested that Cholesteryl esters in large HDL may serve as a promising drug target for the prevention or treatment of pre-eclampsia with no predicted detrimental side effects.

CLN3 deficiency leads to neurological and metabolic perturbations during early development.

Juvenile neuronal ceroid lipofuscinosis (or Batten disease) is an autosomal recessive, rare neurodegenerative disorder that affects mainly children above the age of 5 yr and is most commonly caused by mutations in the highly conserved CLN3 gene. Here, we generated cln3 morphants and stable mutant lines in zebrafish. Although neither morphant nor mutant cln3 larvae showed any obvious developmental or morphological defects, behavioral phenotyping of the mutant larvae revealed hyposensitivity to abrupt light changes and hypersensitivity to pro-convulsive drugs. Importantly, in-depth metabolomics and lipidomics analyses revealed significant accumulation of several glycerophosphodiesters (GPDs) and cholesteryl esters, and a global decrease in bis(monoacylglycero)phosphate species, two of which (GPDs and bis(monoacylglycero)phosphates) were previously proposed as potential biomarkers for CLN3 disease based on independent studies in other organisms. We could also demonstrate GPD accumulation in human-induced pluripotent stem cell-derived cerebral organoids carrying a pathogenic variant for CLN3 Our models revealed that GPDs accumulate at very early stages of life in the absence of functional CLN3 and highlight glycerophosphoinositol and BMP as promising biomarker candidates for pre-symptomatic CLN3 disease.

Shotgun lipidomics of human subretinal fluids under rod-dominant retina reveals cone-dominated lipids.

Subretinal fluid (SRF) accumulates between photoreceptor outer segments and retinal pigment epithelium during rhegmatogenous retinal detachment. Biomolecular components such as lipids originate from cells surrounding the SRF. Knowledge of the composition of these molecules in SRF potentially provides mechanistic insight into the physiologic transfer of lipids between retinal tissue compartments. Using mass spectrometry and tandem mass spectrometry analysis on an electrospray ionization quadrupole-time-of-flight mass spectrometer, we identified a total of 115 lipid molecular species of 11 subclasses and 9 classes in two samples from two patients with rhegmatogenous retinal detachment. These included 47 glycerophosphocholines, 6 glycerophosphoethanolamines, 1 glycerophosphoinositol, 18 sphingomyelins, 9 cholesteryl esters, free cholesterol, 3 ceramides, 22 triacylglycerols and 8 free fatty acids. Glycerophosphocholines were of the highest intensity. By minimizing the formation of different adduct forms or clustering ions of different adducts, we determined the relative intensity of lipid molecular species within the same subclasses. The profiles were compared with those of retinal cells available in the published literature. The glycerophosphocholine profile of SRF was similar to that of cone outer segments, suggesting that outer segment degradation products are constitutively released into the interphotoreceptor matrix, appearing in SRF during detachment. This hypothesis was supported by the retinal distributions of corresponding lipid synthases' mRNA expression obtained from an online resource based on publicly available single-cell sequencing data. In contrast, based on lipid profiles and relevant gene expression in this study, the sources of free cholesterol and cholesteryl esters in SRF appeared more ambiguous, possibly reflecting that outer retina takes up plasma lipoproteins. Further studies to identify and quantify lipids in SRF will help better understand etiology of diseases relevant to outer retina.

Plasma lipidomic profiling reveals six candidate biomarkers for the prediction of incident stroke in patients with hypertension.

INTRODUCTION: The burden of stroke in patients with hypertension is very high, and its prediction is critical. OBJECTIVES: We aimed to use plasma lipidomics profiling to identify lipid biomarkers for predicting incident stroke in patients with hypertension. METHODS: This was a nested case-control study. Baseline plasma samples were collected from 30 hypertensive patients with newly developed stroke, 30 matched patients with hypertension, 30 matched patients at high risk of stroke, and 30 matched healthy controls. Lipidomics analysis was performed by ultrahigh-performance liquid chromatography-tandem mass spectrometry, and differential lipid metabolites were screened using multivariate and univariate statistical methods. Machine learning methods (least absolute shrinkage and selection operator, random forest) were used to identify candidate biomarkers for predicting stroke in patients with hypertension. RESULTS: Co-expression network analysis revealed that the key molecular alterations of the lipid network in stroke implicate glycerophospholipid metabolism and choline metabolism. Six lipid metabolites were identified as candidate biomarkers by multivariate statistical and machine learning methods, namely phosphatidyl choline(40:3p)(rep), cholesteryl ester(20:5), monoglyceride(29:5), triglyceride(18:0p/18:1/18:1), triglyceride(18:1/18:2/21:0) and coenzyme(q9). The combination of these six lipid biomarkers exhibited good diagnostic and predictive ability, as it could indicate a risk of stroke at an early stage in patients with hypertension (area under the curve = 0.870; 95% confidence interval: 0.783-0.957). CONCLUSIONS: We determined lipidomic signatures associated with future stroke development and identified new lipid biomarkers for predicting stroke in patients with hypertension. The biomarkers have translational potential and thus may serve as blood-based biomarkers for predicting hypertensive stroke.

The impact of fish oil and/or probiotics on serum fatty acids and the interaction with low-grade inflammation in pregnant women with overweight and obesity: secondary analysis of a randomised controlled trial.

N-3 long-chain PUFA (LC-PUFA) and probiotics are generally considered to induce health benefits. The objective was to investigate (1) the impact of fish oil and/or probiotics on serum fatty acids (sFA), (2) the interaction of sFA with low-grade inflammation and (3) the relation of sFA to the onset of gestational diabetes mellitus (GDM). Pregnant women with overweight/obesity were allocated into intervention groups with fish oil + placebo, probiotics + placebo, fish oil + probiotics or placebo + placebo in early pregnancy (fish oil: 1·9 g DHA and 0·22 g EPA, probiotics: Lacticaseibacillus rhamnosus HN001 and Bifidobacterium animalis ssp. lactis 420, 1010 CFU, each daily). Blood samples were collected in early (n 431) and late pregnancy (n 361) for analysis of fatty acids in serum phosphatidylcholine (PC), cholesteryl esters (CE), TAG and NEFA with GC and high-sensitivity C-reactive protein and GlycA by immunoassay and NMR spectroscopy, respectively. GDM was diagnosed according to 2 h 75 g oral glucose tolerance test. EPA in PC, CE and TAG and DHA in PC, CE, TAG and NEFA were higher in fish oil and fish oil + probiotics groups compared with placebo. EPA in serum NEFA was lower in women receiving probiotics compared with women not receiving. Low-grade inflammation was inversely associated with n-3 LC-PUFA, which were related to an increased risk of GDM. Fish oil and fish oil + probiotics consumption increase serum n-3 LC-PUFA in pregnant women with overweight/obesity. Although these fatty acids were inversely related to inflammatory markers, n-3 LC-PUFA were linked with an increased risk for GDM.