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.

A Novel Technique for Redox Lipidomics Using Mass Spectrometry: Application on Vegetable Oils Used to Fry Potatoes.

Vegetables oils, rich in polyunsaturated fatty acids, are vulnerable to oxidation during manufacturing, processing, and food preparation. Currently, individual oxidation products are not well characterized, and hence, the health impacts of these unique lipid species remain unknown. Here, we introduce an extensive oxidized lipidomics in silico tandem mass spectrometry library and integrate these libraries within a user-friendly software covering a comprehensive redox lipidomics workflow. We apply this workflow to olive, soy, and walnut cooking oil; comparing unheated oil, oil after deep frying potatoes, and oil after oven frying potatoes. We annotated over a thousand oxidized triglycerides across 273 features (many coeluted). This software was validated against traditional chemical assays of oxidation, known oxidized lipids in castor oil, synthesized standards, and an alternate software LPPtiger. Development of these new software programs for redox lipidomics opens the door to characterize health implications of individual oxidation products.

Differences in the Compositions of Vitamin E Tocochromanol (Tocopherol and Tocotrienol) in Rice Bran Oils Produced in Japan and Other Countries.

In this study, we investigated the compositions of vitamin E tocochromanol [tocopherol (Toc) and tocotrienol (T3)] in crude and refined rice bran oil (RBO) produced in Japan and other countries, including Brazil, Thailand, and Vietnam, based on high-performance liquid chromatography analysis. All RBO analyzed contained α-, ß- and γ-Toc and α-, γ- and δ-T3. Japanese crude RBO, although not refined RBO, also contained ß-T3. Furthermore, total Toc contents in both Japanese crude and refined oils were found to be higher than those in the crude and refined RBO from other countries. Total T3 contents in Japanese crude RBO were similar to those in the crude RBO from Brazil and Vietnam. The α-Toc and α-T3 contents in Japanese crude and refined RBO were considerably higher than those in the crude and refined RBO produced in other countries, whereas in contrast, γ-Toc and γ-T3 contents in Japanese crude and refined RBO were lower. Consequently, the ratios of total α-Toc and α-T3 contents to total γ-Toc and γ-T3 contents in Japanese crude and refined RBO (1.75 and 1.91, respectively) were notably higher than those in the crude and refined RBO produced in other countries. Similarly, the ratios of total Toc to total T3 in Japanese crude and refined RBO were higher than those in the crude and refined RBO produced in other countries. These results accordingly indicate that the ratio of total α-Toc and α-T3 contents to γ-Toc and γ-T3 contents could be used as an effective index to discriminate between the RBO produced in Japan and that produced in other countries.

Isolation and structural elucidation of unique γ-oryzanol species in rice bran oil.

Even though γ-oryzanol (OZ) such as 24-methylenecycloartanyl ferulate (24MCAFA) is abundant in purified rice bran oil, we realized that the oil contained the prospect of two additional novels of OZ species. To identify this, we isolated and analyzed their chemical structures by using HPLC-UV-MS, NMR, and IR. We revealed for the first time that the oil had also contained cyclobranyl ferulate (CBFA) and cyclosadyl ferulate (CSFA) which are likely to be exist due to the isomerism of 24MCAFA under acid condition. OZ profile including CBFA and CSFA was roughly similar between commercial rice bran oils and processed foods containing the oils, suggesting that people might have often consumed CBFA and CSFA from rice bran oils and/or processed foods. Since different OZ species are known to have different functionality, this study opens the chance to explore more the contribution of CBFA and CSFA for human health and wellness.

Effects of Sphingolipid Fractions from Golden Oyster Mushroom (Pleurotus citrinopileatus) on Apoptosis Induced by Inflammatory Stress in an Intestinal Tract in vitro Model.

Previously, we reported that the polar lipid fraction from the golden oyster mushroom, Pleurotus citrinopileatus, suppresses colon injuries which result from apoptosis induced by inflammatory stresses in vivo and in vitro (Yamashita et al., J. Oleo Sci., 69, 751-757 (2020)). Here, we investigated the use of lipid classes in mushroom polar lipid fraction in alleviating colon injury using differentiated Caco-2 cells as an intestinal tract model. The mushroom polar lipid fraction was separated into four fractions using silica thin layer chromatography. Each mushroom polar lipid fraction suppressed lipopolysaccharide (LPS)-induced decreases in the viability of intestinal cells, and the effects of sphingolipid fractions were significantly stronger than those of fraction that did not contain sphingolipids. Addition of sphingolipid fractions suppressed the expression of apoptosis-related proteins (e.g., death receptors and caspases) in the LPS-treated cells. Mushroom polar lipids, especially sphingolipids suppress intestinal apoptosis induced by inflammatory stress, and highly polar sphingolipids may exert stronger suppressive effects.

Oxytocin Ameliorates Impaired Behaviors of High Fat Diet-Induced Obese Mice.

Excessive intake of fat is a major risk factor for lifestyle-related diseases such as heart disease and also affects brain function such as object recognition memory, social recognition, anxiety behavior, and depression-like behavior. Although oxytocin (OXT) has been reported to improve object recognition, social recognition, anxiety behavior, and depression-like behavior in specific conditions, previous studies did not explore the impact of OXT in high-fat diet (HFD)-fed mice. Furthermore, it remains unclear whether intake of HFD affects OXT/oxytocin receptor (OXTR) in the brain. Here, we demonstrated that peripheral OXT administration improves not only social recognition but also object recognition and depressive-like behavior in HFD-fed mice. In contrast, peripheral OXT administration to HFD-fed male mice increased fear and anxiety-related behavior. In addition, we observed that intake of HFD decreased OXTR and c-fos mRNA expression in the hippocampus, specifically. Furthermore, peripheral OXT administration increased OXT mRNA expression in the hypothalamus. Altogether, these findings suggest that OXT has the potential to improve various recognition memory processes via peripheral administration but also has side effects that increase fear-related behavior in males.

Supplementation of Bacillus amyloliquefaciens AS385 culture broth powder containing 1-deoxynojirimycin in a high-fat diet altered the gene expressions related to lipid metabolism and insulin signaling in mice epididymal white adipose tissue.

1-Deoxynojirimycin (DNJ) has been known for its functional properties, such as its anti-hyperglycemic and anti-obesity activities. Previously, we developed a sustainable procedure to produce culture broth powder (CBP) containing DNJ using Bacillus amyloliquefaciens AS385 and demonstrated its regulatory effect on the blood glucose and lipid parameters in C57BL/6J mice. The present study was aimed to determine the molecular mechanism underlying the physiological effects of CBP intake in different concentrations (low, medium and high) towards the development of high-fat diet (HFD)-induced metabolic disorders. Ten-week consumption of CBP-supplemented diets ameliorated HFD-induced adiposity, glucose intolerance, and reduced insulin sensitivity in C57BL/6J mice. To investigate how these physiological events could take place, we analyzed the expression of genes involved in lipid metabolism and insulin signaling in epididymal white adipose tissue and found that CBP had a regulatory effect on the expression of genes related to lipid metabolism (Pparγ, Srebf1c, Acc, Scd, Hsl, Lpl), adiponectin secretion (Foxo1 and Sirt1), and insulin signaling (Irs1 and Akt2). Next, we confirmed that DNJ acted as the main active component in CBP and detected the dose-dependent DNJ uptake in vital metabolic tissues, which may explain the dose-dependent alteration in the metabolic parameters and related gene expressions following the CBP intake in this study. Collectively, our results suggested that DNJ intake in the form of CBP prevented the progression of HFD-induced metabolic disorders through regulation of adipocyte gene expression involved in lipid metabolism and insulin signaling.

Metabolic and pathologic profiles of human LSS deficiency recapitulated in mice.

Skin lesions, cataracts, and congenital anomalies have been frequently associated with inherited deficiencies in enzymes that synthesize cholesterol. Lanosterol synthase (LSS) converts (S)-2,3-epoxysqualene to lanosterol in the cholesterol biosynthesis pathway. Biallelic mutations in LSS have been reported in families with congenital cataracts and, very recently, have been reported in cases of hypotrichosis. However, it remains to be clarified whether these phenotypes are caused by LSS enzymatic deficiencies in each tissue, and disruption of LSS enzymatic activity in vivo has not yet been validated. We identified two patients with novel biallelic LSS mutations who exhibited congenital hypotrichosis and midline anomalies but did not have cataracts. We showed that the blockade of the LSS enzyme reaction occurred in the patients by measuring the (S)-2,3-epoxysqualene/lanosterol ratio in the forehead sebum, which would be a good biomarker for the diagnosis of LSS deficiency. Epidermis-specific Lss knockout mice showed neonatal lethality due to dehydration, indicating that LSS could be involved in skin barrier integrity. Tamoxifen-induced knockout of Lss in the epidermis caused hypotrichosis in adult mice. Lens-specific Lss knockout mice had cataracts. These results confirmed that LSS deficiency causes hypotrichosis and cataracts due to loss-of-function mutations in LSS in each tissue. These mouse models will lead to the elucidation of the pathophysiological mechanisms associated with disrupted LSS and to the development of therapeutic treatments for LSS deficiency.

Ferroptosis driven by radical oxidation of n-6 polyunsaturated fatty acids mediates acetaminophen-induced acute liver failure.

Acetaminophen (APAP) overdose is a common cause of drug-induced acute liver failure. Although hepatocyte cell death is considered to be the critical event in APAP-induced hepatotoxicity, the underlying mechanism remains unclear. Ferroptosis is a newly discovered type of cell death that is caused by a loss of cellular redox homeostasis. As glutathione (GSH) depletion triggers APAP-induced hepatotoxicity, we investigated the role of ferroptosis in a murine model of APAP-induced acute liver failure. APAP-induced hepatotoxicity (evaluated in terms of ALT, AST, and the histopathological score), lipid peroxidation (4-HNE and MDA), and upregulation of the ferroptosis maker PTGS2 mRNA were markedly prevented by the ferroptosis-specific inhibitor ferrostatin-1 (Fer-1). Fer-1 treatment also completely prevented mortality induced by high-dose APAP. Similarly, APAP-induced hepatotoxicity and lipid peroxidation were prevented by the iron chelator deferoxamine. Using mass spectrometry, we found that lipid peroxides derived from n-6 fatty acids, mainly arachidonic acid, were elevated by APAP, and that auto-oxidation is the predominant mechanism of APAP-derived lipid oxidation. APAP-induced hepatotoxicity was also prevented by genetic inhibition of acyl-CoA synthetase long-chain family member 4 or α-tocopherol supplementation. We found that ferroptosis is responsible for APAP-induced hepatocyte cell death. Our findings provide new insights into the mechanism of APAP-induced hepatotoxicity and suggest that ferroptosis is a potential therapeutic target for APAP-induced acute liver failure.

Kinetic Study of Singlet-Oxygen Quenching and Aroxyl-Radical Scavenging Activities of Vitamin E Homologs and Fatty Acids Present in Vegetable Oils.

Recently, singlet-oxygen (1O2) quenching and aroxyl-radical (ArO·) scavenging rates (kQ and kS, respectively) of eight vegetable oils were measured in the ethanol/chloroform/D2O solution. Furthermore, the kQ and kS values and concentrations of four tocopherols and four tocotrienols contained in the vegetable oils were measured. In this study, the concentrations of nine fatty acids (including stearic, oleic, linoleic, and linolenic acids) comprising the above-mentioned eight vegetable oils were determined by gas chromatography. The kQ and kS values for ethyl stearate, ethyl oleate, ethyl linoleate, methyl linolenate, and glyceryl trioleate in the ethanol/chloroform/D2O solution were measured by UV-vis spectrophotometry. Based on the results obtained for the above-mentioned fatty acid esters, the kQ and kS values were estimated for nine fatty acids. Furthermore, comparisons of kQ values observed for the vegetable oils with the sum of the product {∑kQAO-i [AO-i]} of the kQAO-i values obtained for each antioxidant-i (AO-i) and the concentrations ([AO-i]) of AO-i (i.e., four tocopherols (& four tocotrienols) and nine fatty acids) contained in vegetable oils were performed. Based on the results, a detailed comparison of the contributions of the tocopherols (and tocotrienols) and the fatty acids to the 1O2-quenching rate constants (kQ) was performed. This indicated that both the tocopherols (and tocotrienols) and the fatty acids contribute to the 1O2- quenching. A similar comparison was conducted for the ArO· -scavenging rate constants (kS). The results suggested that only the tocopherols (and tocotrienols) contained in the oils contributed to the ArO· -scavenging, with negligible contribution from the fatty acids.

Significance of Squalene in Rice Bran Oil and Perspectives on Squalene Oxidation.

As an intermediate metabolite during the biosynthesis of sterols, squalene is found ubiquitously in plants and animals. In rice, squalene is contained in rice bran, and consequently, squalene in rice bran oil has gained attention. Studies have shown that the intake of squalene from food sources demonstrate various physiological benefits such as the prevention of cancer and cardiovascular disease. Squalene is also known as an effective antioxidant in edible oils. However, due to its chemical structure, squalene is susceptible to oxidation, which may cause a decline in the nutraceutical and antioxidative effects of squalene in edible oils. Oxidative degradation of squalene also results in the formation of scission products (i.e., aldehydes and ketones) which may lead to off-flavor. Since the rate of squalene oxidation depends on the factors that induce its oxidation (i.e., light or heat), emphasis on oxidation mechanisms is necessary. It has been demonstrated in previous studies that the oxidation products formed by the singlet oxygen oxidation and free radical oxidation of squalene are different, and more recently, we demonstrated that different squalene monohydroperoxide isomers are formed by each oxidation mechanism. We herein discuss the significance of squalene in rice bran oil as well as the oxidative degradation of squalene in edible oils with focus on oxidation mechanisms.

Randomized controlled trial of a water-soluble formulation of lutein in humans.

Lutein is poorly absorbed owing to their high hydrophobicity and crystallinity. This double-blind crossover trial involved eight healthy males who were administrated capsules containing either a lutein water-soluble formulation or a lutein oil suspension for 8 days. In the formulation group, plasma and erythrocytes lutein concentrations and baseline-corrected AUC were two-fold higher than those in the oil suspension group.

Evaluation of squalene oxidation mechanisms in human skin surface lipids and shark liver oil supplements.

Squalene is a terpenoid found in human skin surface lipids (SSLs) and foods that possesses beneficial properties. However, since oxidation of squalene causes various complications, it is necessary to identify the mechanisms by which squalene is oxidized. In this study, we aimed to determine the oxidation mechanisms of squalene in SSLs and shark liver oil (SLO) supplements by the analysis of squalene monohydroperoxide (SQOOH) isomers, on the basis of our previous finding that different oxidation mechanisms yield different SQOOH isomers. Liquid chromatography-tandem mass spectrometry analysis of SQOOH isomers revealed that squalene in human SSLs was oxidized by singlet oxygen oxidation, whereas squalene in SLO was oxidized mainly by free radicals. As a result, we have presented the first evidence suggesting that the analysis of SQOOH isomers enables estimation of oxidation mechanisms. Estimating oxidation mechanisms by analyzing SQOOH isomers may provide a foundation for the prevention of skin diseases and food deterioration via regulation of squalene oxidation.

Comparison of Blood Profiles of γ-Oryzanol and Ferulic Acid in Rats after Oral Intake of γ-Oryzanol.

γ-Oryzanol (OZ), a bioactive phytochemical abundant in cereals such as rice, has been reported to be mainly hydrolyzed to ferulic acid (FA) in the body. Meanwhile, in our previous study, we revealed that a part of OZ is absorbed into the body and exists in its intact form. However, the comprehensive absorption profile of OZ and its metabolites (e.g., FA) after OZ intake has not been fully elucidated yet. Therefore, in this study, we measured the concentrations of OZ, FA, and FA conjugates (i.e., FA sulfate and glucuronide) in the blood of rats with the use of HPLC-MS/MS after a single oral administration of 300 µmol/kg body weight of rice bran OZ (RBOZ). As a result, intact OZ along with FA and FA conjugates existed in the blood, which implied that these constituents may all contribute to the physiological effects under OZ intake. Additionally, when an equimolar amount of FA (300 µmol/kg body weight) was administered, it was found that the absorption profile of FA was significantly different from that when RBOZ was administered.

The differential cellular uptake of curcuminoids in vitro depends dominantly on albumin interaction.

BACKGROUND: Curcuminoids, mainly present in the plant rhizomes of turmeric (Curcuma longa), consist of mainly three forms (curcumin (CUR), bisdemethoxycurcumin (BDMC) and demethoxycurcumin (DMC)). It has been reported that different forms of curcuminoids possess different biological activities. However, the mechanisms associated with these differences are not well-understood. Recently, our laboratory found differences in the cellular uptake of these curcuminoids. Therefore, it has been inferred that these differences contribute to the different biological activities. PURPOSE: In this study, we investigated the mechanisms of differential cellular uptake of these curcuminoids. METHOD: Based on our previous study, we hypothesized the differential cellular uptake is caused by (I) polarity, (II) transporters, (III) metabolism rate of curcuminoids and (IV) medium components. These four hypotheses were each investigated by (I) neutralizing the polarities of curcuminoids by encapsulation into poly(lactic-co-glycolic) acid nanoparticles (PLGA-NPs), (II) inhibition of polyphenol-related absorption transporters, (III) analysis of the cellular curcuminoids and their metabolites by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and (IV) use of different mediums in cell study. RESULTS: The differential cellular uptake was not affected by (I-III). However, when investigating (IV), not only CUR but also BDMC and DMC were incorporated into cells when serum free media was used. Furthermore, when we used the serum free medium containing bovine serum albumin (BSA), only CUR was taken up but BDMC and DMC were not. Therefore, we identified that the differential cellular uptake of curcuminoids is caused by the medium components, especially BSA. Also, the fluorescence quenching study suggested that differential cellular uptake is due to the different interaction between BSA and each curcuminoid. CONCLUSION: The differential cellular uptake of curcuminoids was caused by the different interaction between curcuminoids and BSA. The results from this study might give clues on the mechanisms by which curcuminoids exhibit different physiological activities.

Evaluation of γ-oryzanol Accumulation and Lipid Metabolism in the Body of Mice Following Long-Term Administration of γ-oryzanol.

γ-Oryzanol (OZ), abundant in rice bran oil, has gained attention due to its physiological activities (e.g., lipid-lowering effects). However, the absorption and metabolism of orally ingested OZ have not yet been fully elucidated. In this study, diets containing normal or high contents of OZ were fed to obesity model mice for 8 weeks, and OZ concentrations in plasma and organs were analyzed by HPLC-MS/MS. To evaluate the relationship between OZ accumulation and lipid metabolism in vivo, lipid concentrations in the mice plasma and liver were also measured. As a result, the accumulation of intact OZ in plasma and organs was seen in mice fed diets containing OZ, where mice fed diets containing higher OZ contents demonstrated higher levels of OZ accumulation and lower amounts of plasma lipids. These results, in combination with our additional data from a single oral administration test, suggest the possibility that intact OZ, along with its metabolites (e.g., ferulic acid), is biologically-active.

Kinetic Study of the Quenching Reaction of Singlet Oxygen by Eight Vegetable Oils in Solution.

A kinetic study of the reaction of singlet oxygen (1O2) with eight vegetable oils 1-8 containing different concentrations of tocopherols (Tocs) and tocotrienols (Toc-3s) was performed. The second-order rate constants (kQ) for the reaction of 1O2 with vegetable oils 1-8 (rice bran, perilla, rape seed, safflower, grape seed, sesame, extra virgin olive, and olive oils) were measured in ethanol/chloroform/D2O (50:50:1, v/v/v) solution at 35°C using UV-vis spectrophotometry. Furthermore, comparisons of kQ values determined for the above oils 1-8 with the sum of the product {∑kQAO-i [AO-i]/105} of the kQAO-i values obtained for each antioxidant (AO-i) and concentration (in mg/100 g) ([AO-i]/105) of AO-i (Tocs and Toc-3s) contained in the oils 1-8 were performed. The observed kQ values were not reproduced by the kQ values calculated using only the concentrations of the four Tocs and Toc-3s. These results suggest that the contribution of fatty acids contained in the oils 1-8 is also necessary to fully explain the kQ values. Recently, the second-order rate constants (kS) for the reaction of aroxyl radical (ArO・) with the same vegetable oils 1-8 were measured in the same solvent at 25℃ using stopped-flow spectrophotometry (Ref. 23). The kS values obtained could be well explained as the sum of the product {Σ kSAO-i [AO-i]/105} of the kSAO-i and the [AO-i]/105 of AO-i (Tocs and Toc-3s) contained in the vegetable oils.

Determination of pyrroloquinoline quinone by enzymatic and LC-MS/MS methods to clarify its levels in foods.

Pyrroloquinoline quinone (PQQ) is believed to be a new B vitamin-like compound, and PQQ supplementation has received attention as a possible treatment for diseases including dementia and diabetes. However, the distribution of PQQ in foods is unclear, due to the difficulty in analyzing the compound. Therefore, in this study, enzymatic and LC-MS/MS methods were optimized to enable an accurate analysis of PQQ in foods. The optimized methods were applied to the screening of foods, in which PQQ contents were identified in ng/g or ng/mL levels. Furthermore, we newly found that some foods related to acetic acid bacteria contain PQQ at 1.94~5.59 ng/mL higher than beer, which is known to contain relatively high amounts of PQQ. These results suggest that the optimized methods are effective for the screening of foods containing PQQ. Such foods with high PQQ content may be valuable as functional foods effective towards the treatment of certain diseases.

Evaluation of the anti-hyperglycemic effect and safety of microorganism 1-deoxynojirimycin.

1-Deoxynojirimycin (DNJ) is a potent α-glucosidase inhibitor and thus beneficial for prevention of diabetes. While we have succeeded in obtaining the culture supernatant extract (CSE) rich in DNJ from microorganism source, information regarding its anti-hyperglycemic effect and safety were still limited. Therefore, this study was aimed to evaluate the anti-hyperglycemic effect and safety of microorganism DNJ. Oral sucrose tolerance test was performed, and the result showed that CSE was able to significantly suppress the blood glucose elevation and suggested DNJ as the main active compound. To determine its safety, the absorption and excretion of microorganism DNJ were evaluated using 15N labeling method. Our findings investigated the recovery rate of 15N from DNJ reached 80% up to 48 hours after oral administration, suggesting its rapid excretion, suggesting the safety of DNJ. This study verified the functional properties and safety of DNJ from microorganisms, suggesting its potential use for functional purpose.