Comparison of the Catabolic Rates of Linoleic and Oleic Acid Hydroperoxides Using 13CO2 Expired from Mice.

Unsaturated fatty acids, such as oleic and linoleic acids, are easily oxidized by exposure to temperature and light in the presence of air to form unsaturated fatty acid hydroperoxides as primary oxidation products. However, the catabolic rates of unsaturated fatty acid hydroperoxides in the human body remain unknown. In this study, ethyl esters of 13C-labeled linoleic acid (*C18:2-EE) and oleic acid (*C18:1-EE) and their hydroperoxides (*C18:2-EE-OOH and *C18:1-EE-OOH, respectively) prepared by the photo-oxidation of *C18:2-EE and *C18:1-EE, respectively, were administered to mice and their catabolic rates were determined by measuring the expired 13CO2 levels. *C18:2-EE-OOH and *C18:1-EE-OOH were ß-oxidized faster than *C18:2-EE and *C18:1-EE, respectively. Notably, rapid ß-oxidation of *C18:2-EE-OOH and *C18:1-EE-OOH was similar to that of medium-chain fatty acids, such as octanoic acid. Then, degradation products of C18:2-EE-OOH and C18:1-EE-OOH were analyzed under gastric conditions by gas chromatography/mass spectrometry. Major decomposition products of C18:2-EE-OOH and C18:1-EE-OOH were medium-chain compounds, such as octanoic acid ethyl ester, 9-oxo-nonanoic acid ethyl ester, and 10-oxo-8-decenoic acid ethyl esters, indicating that C18:2-EE-OOH and C18:1-EE-OOH isomers formed during photo-oxidation were decomposed under acidic conditions. These findings support previous reports that dietary lipid hydroperoxides are not absorbed into the intestine as lipid hydroperoxides but as degradation products. This is the first study to suggest that dietary lipid hydroperoxides decompose during gastric digestion to form medium-chain compounds that are directly absorbed into the liver via the portal vein and rapidly catabolized via ß-oxidation.

Corrigendum to "Preparation of saturated fatty acid hydroperoxide isomers and determination of their thermal decomposition products - 2-alkanones and lactones" [Food Chem.: X 21 (2024) 101074].

[This corrects the article DOI: 10.1016/j.fochx.2023.101074.].

Inhibition of 7-dehydrocholesterol reductase prevents hepatic ferroptosis under an active state of sterol synthesis.

Recent evidence indicates ferroptosis is implicated in the pathophysiology of various liver diseases; however, the organ-specific regulation mechanism is poorly understood. Here, we demonstrate 7-dehydrocholesterol reductase (DHCR7), the terminal enzyme of cholesterol biosynthesis, as a regulator of ferroptosis in hepatocytes. Genetic and pharmacological inhibition (with AY9944) of DHCR7 suppress ferroptosis in human hepatocellular carcinoma Huh-7 cells. DHCR7 inhibition increases its substrate, 7-dehydrocholesterol (7-DHC). Furthermore, exogenous 7-DHC supplementation using hydroxypropyl ß-cyclodextrin suppresses ferroptosis. A 7-DHC-derived oxysterol metabolite, 3ß,5α-dihydroxycholest-7-en-6-one (DHCEO), is increased by the ferroptosis-inducer RSL-3 in DHCR7-deficient cells, suggesting that the ferroptosis-suppressive effect of DHCR7 inhibition is associated with the oxidation of 7-DHC. Electron spin resonance analysis reveals that 7-DHC functions as a radical trapping agent, thus protecting cells from ferroptosis. We further show that AY9944 inhibits hepatic ischemia-reperfusion injury, and genetic ablation of Dhcr7 prevents acetaminophen-induced acute liver failure in mice. These findings provide new insights into the regulatory mechanism of liver ferroptosis and suggest a potential therapeutic option for ferroptosis-related liver diseases.

A tangible method to assess native ferroptosis suppressor activity.

Ferroptosis, a regulated cell death hallmarked by unrestrained lipid peroxidation, plays a pivotal role in the pathophysiology of various diseases, making it a promising therapeutic target. Glutathione peroxidase 4 (GPX4) prevents ferroptosis by reducing (phospho)lipid hydroperoxides, yet evaluation of its actual activity has remained arduous. Here, we present a tangible method using affinity-purified GPX4 to capture a snapshot of its native activity. Next to measuring GPX4 activity, this improved method allows for the investigation of mutational GPX4 activity, exemplified by the GPX4U46C mutant lacking selenocysteine at its active site, as well as the evaluation of GPX4 inhibitors, such as RSL3, as a showcase. Furthermore, we apply this method to the second ferroptosis guardian, ferroptosis suppressor protein 1, to validate the newly identified ferroptosis inhibitor WIN62577. Together, these methods open up opportunities for evaluating alternative ferroptosis suppression mechanisms.

LC-MS/MS analysis of milk triacylglycerol hydroperoxide isomers which are generated corresponding to the photo- and thermal-oxidation.

Milk is a rich source of essential nutrients such as lipids. However, lipid oxidation can be considered a crucial factor in determining the initial stage of milk deterioration. Therefore, it is essential to identify the mechanisms of lipid oxidation, such as photo-oxidation or thermal oxidation, to efficiently prevent it by selecting proper antioxidants. In this study, the oxidation mechanisms of long-life (LL) milk were investigated, and triacylglycerol hydroperoxide isomers generated corresponding to the oxidation mechanisms were analyzed by LC-MS/MS. This study first prepared the standard of TG 4:0_16:0_18:1;OOH isomers, which are the appropriate target for evaluating LL milk's oxidation mechanism. The authentic standards provided the robust analysis of TG 4:0_16:0_18:1;OOH isomers and suggested that LL milk was susceptible to photo-oxidation rather than thermal-oxidation. Furthermore, it was discovered that radicals play a role in the oxidation of LL milk during photo-oxidation. This information could be valuable in effectively preventing photo-oxidation in LL milk. It is important to note that milk is contained in a variety of food products. Hence, these findings would be applicable not only to milk but also to various milk-containing food products.

Identification of Genes Associated with Resistance to Persulcatusin, a Tick Defensin from Ixodes persulcatus.

Antimicrobial peptides (AMPs) are present in a wide range of plants, animals, and microorganisms. Since AMPs are characterized by their effectiveness against emergent antibiotic-resistant bacteria, they are attracting attention as next-generation antimicrobial compounds that could solve the problem of drug-resistant bacteria. Persulcatusin (IP), an antibacterial peptide derived from the hard tick Ixodes persulcatus, shows high antibacterial activity against various Gram- positive bacteria as well as multidrug-resistant bacteria. However, reports on the antibacterial action and resistance mechanisms of IP are scarce. In this study, we spontaneously generated mutants showing increased a minimum inhibitory concentration (MIC) of IP and analyzed their cross-resistance to other AMPs and antibiotics. We also used fluorescent probes to investigate the target of IP activity by evaluating IP-induced damage to the bacterial cytoplasmic membrane. Our findings suggest that the antimicrobial activity of IP on bacterial cytoplasmic membranes occurs via a mechanism of action different from that of known AMPs. Furthermore, we screened for mutants with high susceptibility to IP using a transposon mutant library and identified 16 genes involved in IP resistance. Our results indicate that IP, like other AMPs, depolarizes the bacterial cytoplasmic membrane, but it may also alter membrane structure and inhibit cell-wall synthesis.

Higher Cholesterol Absorption Marker at Baseline Predicts Fewer Cardiovascular Events in Elderly Patients Receiving Hypercholesterolemia Treatment: The KEEP Study.

BACKGROUND: Higher cholesterol absorption has been reported to be related to a higher incidence of cardiovascular events (CVEs). The KEEP (Kyushu Elderly Ezetimibe Phytosterol) study, a substudy of the EWTOPIA 75 (Ezetimibe Lipid-Lowering Trial on Prevention of Atherosclerotic Cardiovascular Disease in 75 or Older) study, investigated the relationships of cholesterol absorption and synthesis markers with CVEs in older old individuals with hypercholesterolemia, particularly in relation to ezetimibe treatment. METHODS AND RESULTS: Eligible patients were those aged ≥75 years who had low-density lipoprotein cholesterol ≥140 mg/dL, no history of coronary artery disease, and no recent use of lipid-lowering drugs. Participants were randomly assigned into a diet-only or diet-plus-ezetimibe group. Baseline and 24-week follow-up blood samples were analyzed for cholesterol absorption (eg, campesterol) and synthesis markers (eg, lathosterol). Of 1287 patients, 1061 patients with baseline measurement were analyzed. Over a median follow-up of 4.0 years, 64 CVEs occurred. Higher campesterol levels at baseline were significantly associated with a lower risk of CVEs. After adjustment for sex, age, and treatment, the hazard ratios for the lowest to highest quartile categories of baseline campesterol were 1.00 (reference), 0.59 (95% CI, 0.30-1.17), 0.44 (95% CI, 0.21-0.94), and 0.44 (95% CI, 0.21-0.93), respectively (trend P=0.01). This association persisted after further adjustment for hypertension, diabetes, and other cardiovascular risk factors. Neither interactions with ezetimibe treatment nor mediating effects of the changes in cholesterol absorption markers were observed. CONCLUSIONS: The KEEP study indicated that higher campesterol levels without lipid-lowering drugs were associated with a lower incidence of CVEs in older old individuals with hypercholesterolemia who were subsequently treated with diet or ezetimibe. REGISTRATION: URL: https://www.umin.ac.jp; unique identifier: UMIN000017769.

Induction of ferroptosis in human keratinocyte HaCaT cells by squalene hydroperoxide: Possible prevention of skin ferroptosis by botanical extracts.

Ever since the proposal of ferroptosis, it has been studied as a nonapoptotic cell death caused by iron ion-dependent phospholipid (PL) peroxidation. We previously showed that treatment of human hepatoma cell line HepG2 with prepared PL hydroperoxide (PLOOH) resulted in ferroptosis. However, in human sebum, the major hydroperoxide is not PLOOH but squalene hydroperoxide (SQOOH), and to our knowledge, it is not established yet whether SQOOH induces ferroptosis in the skin. In this study, we synthesized SQOOH and treated human keratinocyte HaCaT cells with SQOOH. The results showed that SQOOH induces ferroptosis in HaCaT cells in the same way that PLOOH causes ferroptosis in HepG2 cells. Some natural antioxidants (botanical extracts) could inhibit the ferroptosis in both the cell types. Consequently, future research focus would revolve around the involvement of SQOOH-induced ferroptosis in skin pathologies as well as the prevention and treatment of skin diseases through inhibition of ferroptosis by botanical extracts.

Preparation of saturated fatty acid hydroperoxide isomers and determination of their thermal decomposition products - 2-alkanones and lactones.

As known for quite a long time now, even saturated fatty acids can be oxidized at high temperatures to produce unique aroma compounds, such as 2-alkanones and lactones. Hydroperoxide positional isomers with a hydroperoxy group at the 2-, 3-, 4-, or 5-position are hypothesized to be responsible for the formation of these aroma components, but this hypothesis has not been verified. For the first time, this study successfully prepared a series of glyceryl trioctanoate hydroperoxide (C8TG;OOH) isomers. The isomers were thermally decomposed, proving that 2-heptanone was selectively formed from C8TG;3-OOH, and γ- and δ-octalactones were mainly formed from C8TG;4- and 5-OOH, respectively. C8TG;2-OOH was also involved in lactone formation, whereas C8TG;6- and 7-OOH were not. This proves the long-standing hypothesis. The mechanism revealed in this work is expected to be useful to create favorable aromas (i.e., 2-alkanones and lactones) from saturated fatty acids.

Analysis of oxidized glucosylceramide and its effects on altering gene expressions of inflammation induced by LPS in intestinal tract cell models.

Glucosylceramide (GlcCer) belongs to sphingolipids and is found naturally in plant foods and other sources that humans consume daily. Our previous studies demonstrated that GlcCer prevents inflammatory bowel disease both in vitro and in vivo, whose patients are increasing alarmingly. Although some lipids are vulnerable to oxidation which changes their structure and activities, it is unknown whether oxidative modification of GlcCer affects its activity. In this research, we oxidized GlcCer in the presence of a photosensitizer, analyzed the oxide by mass spectrometric techniques, and examined its anti-inflammatory activity in lipopolysaccharide (LPS)-treated differentiated Caco-2 cells as in vitro model of intestinal inflammation. The results showed that GlcCer is indeed oxidized, producing GlcCer hydroperoxide (GlcCerOOH) as a primary oxidation product. We also found that oxidized GlcCer preserves beneficial functions of GlcCer, suppressing inflammatory-related gene expressions. These findings suggested that GlcCerOOH may perform as an LPS recognition antagonist to discourage inflammation rather than induce inflammation.

Unveiling an unexpected superoxide-mediated photooxidation mechanism of squalene monohydroperoxides to squalene hydroperoxy cyclic peroxides through ESR and LC-MS/MS analyses.

Lipid cyclic peroxides are a rarely reported and documented class of compounds in the human organism. Recently, we reported the formation of squalene (SQ) hydroperoxy cyclic peroxides derived from SQ monohydroperoxide isomers (SQ-OOHs) for the first time. Notably, we successfully detected and quantified cis-2-OOH-3-(1,2-dioxane)-SQ in the human skin. Nevertheless, the underlying mechanism governing the formation of these compounds remained elusive. Therefore, in the current study, we set to determine the reaction's mechanism. To this end, a comprehensive analysis of the precise conditions involved in the onset and propagation of this conversion was carried out by oxidizing total SQ-OOHs under different conditions, including singlet oxygen (1O2), thermal, and photoinduced oxidations monitored by quantifying the generated 2-OOH-3-(1,2-dioxane)-SQ using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Radical intermediates were thoroughly investigated using Electron Spin Resonance (ESR) with the aid of spin traps and radical references. Moreover, calculations of SQ-OOHs' electrostatic charges were performed on Spartan 18 software. We found that the reaction is ideally induced and favored under photooxidation in the presence of 3O2 in hexane, and that superoxide radical (O2•-) is the first key intermediate in this mechanism, whereas peroxyl radicals were the major species observed throughout the oxidation. Chemical calculations provided an explanation for the targeting of tertiary SQ-OOHs by this reaction and gave further evidence on the proposed heterolytic cleavage initiating the reaction. The novel oxidation mechanism suggested herein offers new insights into understanding lipid secondary oxidation and is a promising finding for further studying lipid cyclic peroxides in general.

Sheep-derived cell immortalization through the expression of cell cycle regulators and biological characterization using transcriptomes.

Sheep are important domestic animals for the production of wool and meat. Although numerous cultured cell lines from humans and mice have been established, the number of cell lines derived from sheep is limited. To overcome this issue, the efficient establishment of a sheep-derived cell line and its biological characterization is reported. Mutant cyclin-dependent kinase 4, cyclin D1, and telomerase reverse transcriptase were introduced into sheep muscle-derived cells in an attempt to immortalize primary cells using the K4DT method. Furthermore, the SV40 large T oncogene was introduced into the cells. The successful immortalization of sheep muscle-derived fibroblasts was shown using the K4DT method or SV40 large T antigen. Furthermore, the expression profile of established cells showed close biological characteristics of ear-derived fibroblasts. This study provides a useful cellular resource for veterinary medicine and cell biology.

The difference in the cellular uptake of tocopherol and tocotrienol is influenced by their affinities to albumin.

Vitamin E is classified into tocopherol (Toc) and tocotrienol (T3) based on its side chains. T3 generally has higher cellular uptake than Toc, though the responsible mechanism remains unclear. To elucidate this mechanism, we hypothesized and investigated whether serum albumin is a factor that induces such a difference in the cellular uptake of Toc and T3. Adding bovine serum albumin (BSA) to serum-depleted media increased the cellular uptake of T3 and decreased that of Toc, with varying degrees among α-, ß-, γ-, and δ-analogs. Such enhanced uptake of α-T3 was not observed when cells were incubated under low temperature (the uptake of α-Toc was also reduced), suggesting that Toc and T3 bind to albumin to form a complex that results in differential cellular uptake of vitamin E. Fluorescence quenching study confirmed that vitamin E certainly bound to BSA, and that T3 showed a higher affinity than Toc. Molecular docking further indicated that the differential binding energy of Toc or T3 to BSA is due to the Van der Waals interactions via their side chain. Overall, these results suggested that the affinity of Toc and T3 to albumin differs due to their side chains, causing the difference in their albumin-mediated cellular uptake. Our results give a better mechanistic insight into the physiological action of vitamin E.

Quantitative Lipidomic Analysis of Serum Phospholipids Reveals Dissociable Markers of Alzheimer's Disease and Subcortical Cerebrovascular Disease.

BACKGROUND: Circulating phospholipid species have been shown to predict Alzheimer's disease (AD) prognosis but the link between phospholipid disturbances and subcortical small vessel cerebrovascular disease (CeVD) common in AD patients is not known. OBJECTIVE: Mass-spectrometry lipidomics was applied to quantify serum diacyl, alkenyl (ether), alkyl, and lyso phospholipid species in individuals with extensive CeVD (n = 29), AD with minimal CeVD (n = 16), and AD with extensive CeVD (n = 14), and compared them to age-matched controls (n = 27). Memory was assessed using the California Verbal Learning Test. 3.0T MRI was used to assess hippocampal volume, atrophy, and white matter hyperintensity (WMH) volumes as manifestations of CeVD. RESULTS: AD was associated with significantly higher concentrations of choline plasmalogen 18:0_18:1 and alkyl-phosphocholine 18:1. CeVD was associated with significantly lower lysophospholipids containing 16:0. Phospholipids containing arachidonic acid (AA) were associated with poorer memory in controls, whereas docosahexaenoic acid (DHA)-containing phospholipids were associated with better memory in individuals with AD+CeVD. In controls, DHA-containing phospholipids were associated with more atrophy, and phospholipids containing linoleic acid and AA were associated with less atrophy. Lysophospholipids containing 16:0, 18:0, and 18:1 were correlated with less atrophy in controls, and of these, alkyl-phosphocholine 18:1 was correlated with smaller WMH volumes. Conversely, 16:0_18:1 choline plasmalogen was correlated with greater WMH volumes in controls. CONCLUSION: This study demonstrates discernable differences in circulating phospholipids in individuals with AD and CeVD, as well as new associations between phospholipid species with memory and brain structure that were specific to contexts of commonly comorbid vascular and neurodegenerative pathologies.

Effects of Particle Size of Curcumin Solid Dispersions on Bioavailability and Anti-Inflammatory Activities.

The delivery of curcumin (CUR) using the solid dispersion system (CUR solid dispersions; C-SDs) has been shown to improve CUR bioavailability. However, it is unclear how different particle sizes of C-SDs affect the bioavailability and biological activities of CUR. Hence, we prepared C-SDs in different sizes using food-grade excipients and evaluated their bioavailability and biological activities. By pulverizing large particle sizes of C-SDs using zirconia beads, we successfully prepared C-SDs I-IV (particle size: (I) 120, (II) 447, (III) 987, (IV) 1910 nm). When administrated orally in rats, the bioavailability of CUR was increased with decreasing C-SDs size, most likely by improving its solubility in micelles. When administrated intravenously in rats, blood concentrations of CUR were increased with increasing particle size, suggesting that larger C-SDs presumably control the metabolic conversion of CUR. In RAW264 cells, more CUR was taken up by cells as their sizes reduced, and the more potent their anti-inflammatory activities were, suggesting that smaller C-SDs were taken up through a number of cellular uptake pathways. Altogether, the present study showed an evident effect of C-SDs size on their bioavailability and anti-inflammatory activities-information that serves as a basis for improving the functionality of CUR.

Immortalization of primary cells derived from the endangered Ryukyu long-furred rat.

The Ryukyu long-furred rat is an endangered species confined to the southernmost three small islands of Japan (Amami-Oshima, Tokunoshima, and Okinawa). Its population is rapidly decreasing because of roadkill, deforestation, and feral animals. To date, its genomic and biological information are poorly understood. In this study, we successfully immortalized Ryukyu long-furred rat cells by expressing a combination of cell cycle regulators, mutant cyclin-dependent kinase 4 (CDK4R24C) and cyclin D1, together with telomerase reverse transcriptase or an oncogenic protein, the Simian Virus large T antigen. The cell cycle distribution, telomerase enzymatic activity, and karyotype of these two immortalized cell lines were analyzed. The karyotype of the former cell line immortalized with cell cycle regulators and telomerase reverse transcriptase retained the nature of the primary cells, while that of the latter cell line immortalized with the Simian Virus large T antigen had many aberrant chromosomes. These immortalized cells would be valuable for studying the genomics and biology of Ryukyu long-furred rats.

Clinical Evaluation Based on a New Approach to Improve the Accuracy of 4ß-Hydroxycholesterol Measurement as a Biomarker of CYP3A4 Activity.

This study examines 4ß-Hydroxycholesterol (4ß-HC), which is considered to be a potential marker for the CYP3A4 induction of new chemical entities (NCEs) in drug development. To ensure the use of 4ß-HC as a practical biomarker, it is necessary to accurately measure 4ß-HC and demonstrate that CYP3A4 induction can be appropriately assessed, even for weak inducers. In clinical trials of NCEs, plasma is often collected with various anticoagulants, in some cases, the plasma is acidified, then stored for an extended period. In this study, we examined the effects of these manipulations on the measurement of 4ß-HC, and based on the results, we optimized the plasma collection and storage protocols. We also found that a cholesterol oxidation product is formed when plasma is stored, and by monitoring the compound, we were able to identify when plasma was stored inappropriately. After evaluating the above, clinical drug-drug interaction (DDI) studies were conducted using two NCEs (novel retinoid-related orphan receptor γ antagonists). The weak CYP3A4 induction by the NCEs (which were determined based on a slight decline in the systemic exposure of a probe substrate (midazolam)), was detected by the significant increase in 4ß-HC levels (more specifically, 4ß-HC/total cholesterol ratios). Our new approach, based on monitoring a cholesterol oxidation product to identify plasma that is stored inappropriately, allowed for the accurate measurement of 4ß-HC, and thus, it enabled the evaluation of weak CYP3A4 inducers in clinical studies without using a probe substrate.

Analysis of docosahexaenoic acid hydroperoxide isomers in mackerel using liquid chromatography-mass spectrometry.

Docosahexaenoic acid (DHA) is mostly esterified in food and is easily oxidized by exposure to heat or light. Hydroperoxide positions of DHA mono-hydroperoxide (DHA;OOH) provide information on oxidation mechanisms (e.g., radical- or singlet oxygen oxidation), yet direct identification of esterified DHA;OOH isomers has not been achieved. We previously accomplished the direct analysis of free DHA;OOH isomers with liquid chromatography-mass spectrometry (LC-MS/MS). In this study, we developed an LC-MS/MS method for direct analysis of esterified DHA;OOH based on our previous study. The developed method was capable of distinguishing esterified DHA;OOH isomers in raw- and oxidized mackerel. The result suggested that radical oxidation of esterified DHA can progress even in refrigeration. Different transitions were observed depending on the oxidation mechanism and lipid class. The analytical method and insights obtained in this study would be valuable to further understand and effectively prevent DHA oxidation in food products.

A comprehensive review on the production, pharmacokinetics and health benefits of mulberry leaf iminosugars: Main focus on 1-deoxynojirimycin, d-fagomine, and 2-O-ɑ-d-galactopyranosyl-DNJ.

Mulberry leaves are rich in biologically active compounds, including phenolics, polysaccharides, and alkaloids. Mulberry leaf iminosugars (MLIs; a type of polyhydroxylated alkaloids), in particular, have been gaining increasing attention due to their health-promoting effects, including anti-diabetic, anti-obesity, anti-hyperglycemic, anti-hypercholesterolemic, anti-inflammatory, and gut microbiota-modulatory activities. Knowledge regarding the in vivo bioavailability and bioactivity of MLIs are crucial to understand their role and function and human health. Therefore, this review is aimed to comprehensively summarize the existing studies on the oral pharmacokinetics and the physiological significance of selected MLIs (i.e.,1-deoxynojirimycin, d-fagomine, and 2-O-ɑ-d-galactopyranosyl-DNJ). Evidence have suggested that MLIs possess relatively good uptake and safety profiles, which support their prospective use for oral intake; the therapeutic potential of these compounds against metabolic and chronic disorders and the underlying mechanisms behind these effects have also been studied in in vitro and in vivo models. Also discussed are the biosynthetic pathways of MLIs in plants, as well as the agronomic and processing factors that affect their concentration in mulberry leaves-derived products.