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.].

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.

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.

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.

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.

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.

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.

Elucidation of decomposition pathways of linoleic acid hydroperoxide isomers by GC-MS and LC-MS/MS.

Food lipid oxidation provides various volatile compounds involved in food flavor via the decomposition of lipid hydroperoxide (LOOH). This study predicted the pathways which can coherently explain LOOH decomposition focusing on hydroperoxy octadecadienoic acid (HpODE) isomers (9-EZ-HpODE, 9-EE-HpODE, 10-HpODE, 12-HpODE, 13-ZE-HpODE, and 13-EE-HpODE) which are the major LOOH contained in edible oils. Each standard was first prepared and thermally decomposed. Generated volatile and non-volatile compounds were analyzed by GC-MS and LC-MS/MS. The results showed that all HpODE decomposition was based on the factors such as favorable scission, radical delocalization, and cyclization. Interestingly, the formation of 8-HpODE and 14-HpODE were demonstrated during HpODE decomposition. The insights obtained in this study would explain the generation pathways of flavor involved in food quality.

Cellular Uptake of Pyrroloquinoline Quinone in Its Intact and Derivatized Forms from the Cell Culture Medium of 3T3-L1.

Following a growing interest in the physiological effects of pyrroloquinoline quinone (PQQ), more cell culture experiments have begun to elucidate its mechanism of action. However, to our knowledge, no reports have used instrumental analysis, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), to study cellular uptake of PQQ. In addition, despite the propensity of PQQ to react with amino acids and other compounds, only a handful of cell culture experiments have been conducted on PQQ derivatives. In the present study, we prepared PQQ derivatives by reacting PQQ with various amino acids and used them as reference standards for optimizing the LC-MS/MS analysis conditions to detect PQQ and its derivatives. Using this method, we evaluated the uptake of PQQ into mouse 3T3-L1 cells and found that most PQQ added to the medium was taken up by the cells in its unchanged form, while some PQQ reacted with amino acids in the medium and was taken up by the cells as PQQ derivatives. These results suggest that PQQ derivatives may contribute to the physiological effects of PQQ. To further elucidate the function of PQQ, it is necessary for future studies to clarify the activity of PQQ derivatives and to evaluate the types of PQQ present in food, animal, and cell samples in more detail.

Possible Glutathione Peroxidase 4-Independent Reduction of Phosphatidylcholine Hydroperoxide: Its Relevance to Ferroptosis.

Ferroptosis is mainly caused by iron-mediated peroxidation of phospholipids and has recently attracted attention due to its involvement in various diseases. At the center of it is supposedly the inability of glutathione peroxidase 4 (GPX4) to reduce excess peroxidized phospholipids (e.g., phosphatidylcholine hydroperoxide (PCOOH)) that trigger ferroptosis. However, the involvement of enzymes other than GPX4 in ferroptosis is scarcely known. To elucidate this matter, we evaluated the uptake of PCOOH in a GPX4 knockout (KO) human hepatoma cell line HepG2 generated using CRISPR-Cas9. After confirming that GPX4 expression in the KO cells was below the detection limit, we cultured both wild-type (WT) and GPX4 KO HepG2 cells in a medium containing 50 µM PCOOH for 1-8 hours. By analyzing the level of PCOOH and its reduction product (phosphatidylcholine hydroxide, PCOH) in cells using liquid chromatography-tandem mass spectrometry, we detected the cellular uptake of PCOOH. On top of this, we detected a large amount of PCOH not only in WT HepG2 but also in GPX4 KO HepG2, thus indicating the notable involvement of enzymes other than GPX4 (e.g., other GPX family, glutathione S-transferase, thioredoxin, or peroxiredoxin) in reducing PCOOH. Further corroboration of these findings hopefully leads to the development of novel methods to prevent ferroptosis-related diseases by targeting enzymes other than GPX4.

Dietary triacylglycerol hydroperoxide is not absorbed, yet it induces the formation of other triacylglycerol hydroperoxides in the gastrointestinal tract.

The in vivo presence of triacylglycerol hydroperoxide (TGOOH), a primary oxidation product of triacylglycerol (TG), has been speculated to be involved in various diseases. Thus, considerable attention has been paid to whether dietary TGOOH is absorbed from the intestine. In this study, we performed the lymph duct-cannulation study in rats and analyzed the level of TGOOH in lymph following administration of a TG emulsion containing TGOOH. As we successfully detected TGOOH from the lymph, we hypothesized that this might be originated from the intestinal absorption of dietary TGOOH [hypothesis I] and/or the in situ formation of TGOOH [hypothesis II]. To determine the validity of these hypotheses, we then performed another cannulation study using a TG emulsion containing a deuterium-labeled TGOOH (D2-TGOOH) that is traceable in vivo. After administration of this emulsion to rats, we clearly detected unlabeled TGOOH instead of D2-TGOOH from the lymph, indicating that TGOOH is not absorbed from the intestine but is more likely to be produced in situ. By discriminating the isomeric structures of TGOOH present in lymph, we predicted the mechanism by which the intake of dietary TGOOH triggers oxidative stress (e.g., via generation of singlet oxygen) and induces in situ formation of TGOOH. The results of this study hereby provide a foothold to better understand the physiological significance of TGOOH on human health.

Elucidation of Olive Oil Oxidation Mechanisms by Analysis of Triacylglycerol Hydroperoxide Isomers Using LC-MS/MS.

Despite the importance of the insight about the oxidation mechanisms (i.e., radical and singlet oxygen (1O2) oxidation) in extra virgin olive oil (EVOO), the elucidation has been difficult due to its various triacylglycerol molecular species and complex matrix. This study tried to evaluate the mechanisms responsible for EVOO oxidation in our daily use by quantitative determination of triacylglycerol hydroperoxide (TGOOH) isomers using LC-MS/MS. The standards of dioleoyl-(hydroperoxy octadecadienoyl)-triacylglycerol and dioleoyl-(hydroperoxy octadecamonoenoyl)-triacylglycerol, which are the predominant TGOOHs contained in EVOO, were prepared. Subsequently, fresh, thermal-, and photo-oxidized EVOO were analyzed. The obtained results mostly agreed with the previously reported characteristics of the radical and 1O2 oxidation of linoleic acid and oleic acid. This suggests that the methods described in this paper should be valuable in understanding how different factors that determine the quality of EVOO (e.g., olive species, cultivation area, cultivation timing, and extraction methods) contribute to its oxidative stability.

Carboxylic acids derived from triacylglycerols that contribute to the increase in acid value during the thermal oxidation of oils.

Acid value (AV), is a widely used indicator of oil degradation that, by definition, measures the free fatty acids formed via the hydrolysis of triacyclglycerols. However, based on observations made in previous studies, we hypothesized that the oxidation of triacylglycerols leads to the formation of carboxylic acids with a glycerol backbone which are also calculated as AV. In this study, we aimed to identify such carboxylic acids and prove the above hypothesis. Heating a canola oil at 180 °C for 6 h without the addition of water resulted in an increase in AV from 0.054 to 0.241. However, the contribution of free fatty acids to this increase in AV was minimal; free fatty acid-derived AV before and after heating was 0.020 and 0.023, respectively. Then, via mass spectrometric analyses, we identified two 8-carboxy-octanoyl (azelaoyl) -triacylglycerols (i.e., dioleoyl-azelaoyl-glycerol and oleoyl-linoleoyl-azelaoyl-glycerol) in the heated oil. Azelaoyl-triacylglycerols-derived AV before and after heating the oil was 0.008 and 0.109, respectively, demonstrating that azelaoyl-triacylglycerols contribute to AV. Such an increase in AV by azelaoyl-triacylglycerols was also observed in an oil used to deep-fry potatoes (i.e., an oil with a relatively high water content). These results suggest that AV is also an indicator of the thermal oxidation of triacylglycerols.

Determination of acrolein generation pathways from linoleic acid and linolenic acid: increment by photo irradiation.

2-Propenal (acrolein) is a toxic aldehyde generated from the thermal degradation of edible oils. While previous studies have suggested that linolenic acid (LnA) is the origin of acrolein formation in edible oils, these studies were performed under thermal conditions where only the fatty acid hydroperoxide (FAOOH) isomers derived from radical oxidation were formed. In this study, we reinvestigated the acrolein generation pathway through another oxidation mechanism involving singlet oxygen (1O2) oxidation (type II photo-oxidation). Standards of the main FAOOH isomers (oleic acid hydroperoxide, linoleic acid hydroperoxide (HpODE), and linolenic acid hydroperoxide (HpOTE)) found in edible oils were prepared, and their decomposition products, including those derived from1O2 oxidation (i.e., 10- and 12-HpODE) were analyzed by GC-EI-MS. We found that 1O2 oxidation products of linoleic acid (LA) and LnA but not OA, are significant sources of acrolein formation. The amount of acrolein formed from edible oils high in LA (e.g., rice bran oil) increased by photo irradiation. Further investigation into the mechanism of acrolein generation demonstrated that the amount of acrolein derived from 1O2 oxidation-specific HpOTE isomers (i.e., 10- and 15-HpOTE) was two times greater than that of other HpOTE isomers (i.e., 9-, 12-, 13-, and 16-HpOTE). The results of the present study provide a new pathway of acrolein formation from type II photo-oxidation. This information can be used to inform on oil storage and processing conditions to reduce exposure and dietary intake of acrolein.

Impact of Second-line Chemotherapy on Prognosis: Response of Advanced Gastric Cancer to Taxanes Plus Ramucirumab.

BACKGROUND/AIM: The impact of clinical response to taxanes plus ramucirumab (RAM) on overall survival (OS) has not been clarified for advanced gastric cancer (AGC), although this type of therapy is already in use as second-line chemotherapy (CTx). This study aimed to investigate the prognostic impact of the clinical response to taxanes plus ramucirumab (RAM) for AGC patients. PATIENTS AND METHODS: This study included AGC patients treated with paclitaxel (PTX) or nab-paclitaxel (nab-PTX) and RAM. A retrospective analysis of response and survival rates in consecutive medical records of patients was performed. RESULTS: Forty-two patients were enrolled. Median progression-free survival and OS were 5.4 months [95% confidence interval (CI)=4.440-6.361] and 11.8 months (95% CI=8.648-15.019), respectively. In Cox-hazard multivariate analysis, peritoneal metastasis [hazard ratio (HR)=2.830; 95% CI=1.320-6.067; p=0.008], and disease control rate (HR=0.310; 95% CI=0.129-0.741; p=0.008) were independent factors. CONCLUSION: The response to taxanes plus RAM CTx had an impact on the survival of patients with AGC.

Lipidomics and Redox Lipidomics Indicate Early Stage Alcohol-Induced Liver Damage.

Alcoholic fatty liver disease (AFLD) is characterized by lipid accumulation and inflammation and can progress to cirrhosis and cancer in the liver. AFLD diagnosis currently relies on histological analysis of liver biopsies. Early detection permits interventions that would prevent progression to cirrhosis or later stages of the disease. Herein, we have conducted the first comprehensive time-course study of lipids using novel state-of-the art lipidomics methods in plasma and liver in the early stages of a mouse model of AFLD, i.e., Lieber-DeCarli diet model. In ethanol-treated mice, changes in liver tissue included up-regulation of triglycerides (TGs) and oxidized TGs and down-regulation of phosphatidylcholine, lysophosphatidylcholine, and 20-22-carbon-containing lipid-mediator precursors. An increase in oxidized TGs preceded histological signs of early AFLD, i.e., steatosis, with these changes observed in both the liver and plasma. The major lipid classes dysregulated by ethanol play important roles in hepatic inflammation, steatosis, and oxidative damage. Conclusion: Alcohol consumption alters the liver lipidome before overt histological markers of early AFLD. This introduces the exciting possibility that specific lipids may serve as earlier biomarkers of AFLD than those currently being used.

TRPV2 Promotes Cell Migration and Invasion in Gastric Cancer via the Transforming Growth Factor-ß Signaling Pathway.

BACKGROUND: Transient receptor potential vanilloid 2 (TRPV2) is a highly Ca2+-permeable ion channel that is involved in a number of cellular processes. It is expressed in various human cancers; however, the role of TRPV2 in gastric cancer (GC) remains poorly understood. METHODS: TRPV2 gene expression was knocked down in GC cell lines by small-interfering RNA (siRNA), and the biological roles of TRPV2 in the proliferation, migration, and invasion of GC cells were then investigated. The gene expression profile of GC was elucidated using a microarray analysis. TRPV2 expression in tumor tissue sections was analyzed by immunohistochemistry. RESULTS: The migration and invasion abilities of GC cells were inhibited by the knockdown of TRPV2. Moreover, the microarray assay revealed that TRPV2 was associated with the transforming growth factor (TGF)-ß signaling pathway. Immunohistochemical staining showed that the strong expression of TRPV2 correlated with lymphatic invasion, venous invasion, pathological T (pT), pathological N (pN), and a poor prognosis in GC patients. CONCLUSIONS: TRPV2 appeared to promote tumor migration and invasion via the TGF-ß signaling pathway, and the strong expression of TRPV2 was associated with a worse prognosis in GC patients.

Novel Photoinduced Squalene Cyclic Peroxide Identified, Detected, and Quantified in Human Skin Surface Lipids.

Skin surface lipids (SSLs) form the first barrier that protects the human organism from external stressors, disruption of the homeostasis of SSLs can result in severe skin abnormalities. One of the main causes of this disruption is oxidative stress that is primarily due to SSLs oxidation. Squalene (SQ), the most abundant lipid among SSLs, was shown to first undergo singlet molecular oxygen (1O2) oxidation to yield 6 SQ-monohydroperoxide (SQ-OOH) isomers as the primary oxidation products. However, due to the instability and lability of hydroperoxides, we found that when total SQ-OOH isomers are further photooxidized, they form a unique higher molecular weight secondary oxidation product. To generate the compound, we photooxidized total SQ-OOH isomers in the presence of ground state molecular oxygen (3O2), after its isolation and purification, we studied its structure using MS/MS, NMR, derivatization reactions, and chemical calculations. The compound was identified as 2-OOH-3-(1,2-dioxane)-SQ. Photooxidation of individual SQ-OOH isomers revealed that 6-OOH-SQ is the precursor of 2-OOH-3-(1,2-dioxane)-SQ and indicated the possibility of the formation of similar cyclic peroxides from each isomer following the same photoinduced chain reaction mechanism. An HPLC-MS/MS method was developed for the analysis of 2-OOH-3-(1,2-dioxane)-SQ and its presence on the skin was confirmed in SSLs of six healthy individuals. Its quantity on the skin correlated directly to that of SQ and was not inversely proportional to its precursor, indicating the possibility of its accumulation on the skin surface and the constant regeneration of 6-OOH-SQ from SQ's oxidation. In general, research on lipid cyclic peroxides in the human organism is very limited, and especially on the skin. This study shows for the first time the identification and presence of a novel SQ cyclic peroxide "2-OOH-3-(1,2-dioxane)-SQ" in SSLs, shedding light on the importance of further studying its effect and role on the skin.

Investigation of Lipoproteins Oxidation Mechanisms by the Analysis of Lipid Hydroperoxide Isomers.

The continuous formation and accumulation of oxidized lipids (e.g., lipid hydroperoxides (LOOH)) which are present even in plasma lipoproteins of healthy subjects, are ultimately considered to be linked to various diseases. Because lipid peroxidation mechanisms (i.e., radical, singlet oxygen, and enzymatic oxidation) can be suppressed by certain proper antioxidants (e.g., radical oxidation is efficiently suppressed by tocopherol), in order to suppress lipid peroxidation successfully, the determination of the peroxidation mechanism involved in the formation of LOOH is deemed crucial. In this study, to determine the peroxidation mechanisms of plasma lipoproteins of healthy subjects, we develop novel analytical methods using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine hydroperoxide (PC 16:0/18:2;OOH) and cholesteryl linoleate hydroperoxide (CE 18:2;OOH) isomers. Using the newly developed methods, these PC 16:0/18:2;OOH and CE 18:2;OOH isomers in the low-density lipoprotein (LDL) and high-density lipoprotein (HDL) of healthy subjects are analyzed. Consequently, it is found that predominant PC 16:0/18:2;OOH and CE 18:2;OOH isomers in LDL and HDL are PC 16:0/18:2;9OOH, PC 16:0/18:2;13OOH, CE 18:2;9OOH, and CE 18:2;13OOH, which means that PC and CE in LDL and HDL are mainly oxidized by radical and/or enzymatic oxidation. In conclusion, the insights about the oxidation mechanisms shown in this study would be useful for a more effective suppression of oxidative stress in the human organism.