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.

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.

Investigation of Fertility Preservation Education Videos for Pediatric Patients Based on International and Historical Survey.

Purpose: Recently, direct communication with children about cancer seems to have shifted, but little is known about communication regarding discussions of future infertility risk due to cancer therapy. This study conducted cross-cultural comparisons between Japan and the United States to clarify communication patterns about cancer notification and develop appropriate information about fertility issues. Methods: An online survey was distributed to members of the Japanese Society of Pediatric Hematology/Oncology in July 2019 and the American Society of Pediatric Hematology/Oncology in July 2020. Based on the results from the survey, we developed three types of educational videos: a prepubertal version A, B, and a pubertal version. Next, we conducted a survey to assess whether these were appropriate for clinical practice. Results: We analyzed 325 physicians in Japan and 46 in the United States. In Japan, 80.5%, 91.7%, and 92.1% of the physicians notified patients aged 7-9, 10-14, and 15-17 years of their cancer diagnosis directly, respectively, compared within the United States, where the rate was 100%, regardless of age. Further, 9% and 45% of physicians in Japan and the United States, respectively, discuss fertility issues directly with patients aged 7-9 years. In the survey to assess the educational videos, 85% of the physicians preferred to use the educational videos in clinical practice. Conclusion: This is the first step in bringing concordance to communication patters for emerging cancer care around the globe and that this study and its intervention arm provide guidance in ways that ensure global equity in care.

Acute and Postacute Clinical Characteristics of Coronavirus Disease 2019 in Children in Japan.

BACKGROUND: The clinical features of coronavirus disease 2019 (COVID-19) in children have been changing because of the emergence and rapid spread of variants of concern (VOC). The increase in cases infected with VOC has brought concern with persistent symptoms after COVID-19 in children. This survey aimed to analyze the clinical manifestations and persistent symptoms of pediatric COVID-19 cases in Japan. METHODS: We analyzed the clinical manifestations of pediatric COVID-19 cases reported between February 2020 and April 2022 in Japan, using a dedicated database updated voluntarily by the members of the Japan Pediatric Society. Using the same database, we also analyzed persistent symptoms after COVID-19 in children who were diagnosed between February 2020 and November 2021. RESULTS: A total of 5411 and 1697 pediatric COVID-19 cases were included for analyzing clinical manifestations and persistent symptoms, respectively. During the Omicron variant predominant period, the percentage of patients with seizures increased to 13.4% and 7.4% in patient groups 1-4 and 5-11 years of age, respectively, compared with the pre-Delta (1.3%, 0.4%) or Delta period (3.1%, 0.0%). Persistent and present symptoms after 28 days of COVID-19 onset were reported in 55 (3.2%). CONCLUSIONS: Our survey showed that the rate of symptomatic pediatric COVID-19 cases increased gradually, especially during the Omicron variant predominant period, and a certain percentage of pediatric cases had persistent symptoms. Certain percentages of pediatric COVID-19 patients had severe complications or prolonged symptoms. Further studies are needed to follow such patients.

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.

A Case of Acute Encephalopathy Associated with Acute Focal Bacterial Nephritis.

Acute encephalopathy is a syndrome characterized by an acute onset of disturbance of consciousness. Many acute encephalopathies are caused by viral infections; however, they can also be a result of bacterial infections. Acute focal bacterial nephritis (AFBN) can cause neurological symptoms, such as irritation, unconsciousness, and seizures. In some cases, AFBN-associated acute encephalopathy has also been reported. This report describes the first case of acute encephalopathy with AFBN without significant findings on brain MRI. The patient was a 3-year-old male, who had two episodes of febrile seizures at the ages of 1 and 2 years. He developed disturbance of consciousness, irritability, excitability, and neck stiffness on the day after admission. There were no abnormal findings on brain MRI; however, a generalized high-voltage slow wave was noted on electroencephalography (EEG). His urinary sediment count was elevated, and Morganella morganii and Enterococcus faecalis were detected in the urinary culture. A diagnosis of acute encephalopathy with urinary tract infection (UTI) was made. Intravenous (IV) antibiotics were administered to treat the UTI, while methylprednisolone pulse therapy and IV immunoglobulin were administered to treat acute encephalopathy. Additionally, AFBN was detected in both kidneys on contrast-enhanced CT. The patient received a second course of methylprednisolone pulse therapy due to the persistent high voltage slow wave noted on the EEG on day 8. Furthermore, contrast-enhanced CT revealed AFBN in both kidneys. The final diagnosis was acute encephalopathy with AFBN; however, we had initially diagnosed febrile seizures associated with UTI. It should be noted that acute encephalopathy is associated with AFBN.

The associations between suicide-related behaviors, prefrontal dysfunction in emotional cognition, and personality traits in mood disorders.

Suicide is a serious public health problem, and it is urgent to identify biomarkers associated with suicide to prevent it. We aimed to clarify the association across suicidal behavior, personality traits, and brain activation by emotional stimulation tasks using near-infrared spectroscopy (NIRS) in patients with mood disorders. 11 mood disorder patients with a history of suicide attempt (MDSA), 18 mood disorder patients with no history of suicide attempt (MDNSA), and 17 healthy individuals were studied. The MDSA patients showed significantly high impulsivity and hopeless compared to healthy subjects, great response to the thread word task in the orbitofrontal cortex (OFC) and dorsolateral prefrontal cortex (DLPFC) compared to MDNSA patients, and the significant correlation between the personality traits and brain activation. The MDNSA did not show the trend. The results suggest that the personality traits and the activation of OFC and DLPFC during the negative emotional cognitive stimuli is associated with suicidal behavior, indicating the findings are involved in the pathophysiology of suicidality in mood disorders.

Mutation in the STXBP1 Gene Associated with Early Onset West Syndrome: A Case Report and Literature Review.

Syntaxin-binding protein1 (STXBP1) is a member of the Sec1/Munc18-1 protein family, which comprises important regulators of the secretory and synaptic vesicle fusion machinery underlying hormonal and neuronal transmission, respectively. STXBP1 pathogenic variants are associated with multiple neurological disorders. Herein, we present the case of a Japanese girl with a mutation in the STXBP1 gene, who was born at 40 weeks without neonatal asphyxia. At 15 days old, she developed epilepsy and generalized seizures. Around 88 days old, she presented with a series of nodding spasms, with the seizure frequency gradually increasing. Interictal EEG indicated hypsarrhythmia and she presented with developmental regression. At 1.5 years old, genetic testing was performed and mutational analysis revealed an STXBP1 gene mutation (c.875G > A: p.Arg292His). Accordingly, she was diagnosed with developmental and epileptic encephalopathy, presenting West syndrome's clinical characteristics caused by the STXBP1 gene mutation. Although drug treatment has reduced the frequency of epileptic seizures, her development has remained regressive. The relationship between the location and type of genetic abnormality and the phenotype remains unclear. Future studies should investigate the genotype−phenotype correlation and the underlying pathophysiology to elucidate the causal relationships among the multiple phenotype-determining factors.

Structural Analysis and Anti-Inflammatory Effect of a Digalactosyldiacylglycerol-Monoestolide, a Characteristic Glycolipid in Oats.

Digalactosyldiacylglycerol- (DGDG-) monoestolide is a characteristic glycolipid in oats. DGDG-monoestolides possess a unique structure whereby a fatty acid of DGDG is replaced by a fatty acid ester of hydroxy fatty acid (FAHFA). While the physiological effects of DGDG and FAHFA have been reported previously, the effects of DGDG-monoestolides are unknown. Hence, we isolated a major DGDG-monoestolide molecular species from oats, analyzed its structure, and evaluated its anti-inflammatory effect. Based on GC-MS, MS/MS, and NMR analyses, the isolated compound was identified as a DGDG-monoestolide that contains the linoleic acid ester of 15-hydroxy linoleic acid (LAHLA) and linoleic acid (i.e., DGDG-LAHLA). The isolated DGDG-LAHLA was evaluated for its anti-inflammatory effect on LPS-stimulated RAW264 cells. The production of nitric oxide and cytokines (IL-6, TNF-α, and IL-10) were significantly decreased by DGDG-LAHLA, suggesting the anti-inflammatory effect of DGDG-LAHLA for the first time. In addition, our data showed a pronounced uptake of DGDG-LAHLA by cells. Some compounds corresponding to the predicted DGDG-LAHLA metabolites were also detected, suggesting that both intact DGDG-LAHLA and its metabolites may contribute to the above anti-inflammatory activities. These results are expected to expand the availability of oats as a functional food.

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.

Antenatal Glucocorticoid Administration Promotes Cardiac Structure and Energy Metabolism Maturation in Preterm Fetuses.

Although the rate of preterm birth has increased in recent decades, a number of preterm infants have escaped death due to improvements in perinatal and neonatal care. Antenatal glucocorticoid (GC) therapy has significantly contributed to progression in lung maturation; however, its potential effects on other organs remain controversial. Furthermore, the effects of antenatal GC therapy on the fetal heart show both pros and cons. Translational research in animal models indicates that constant fetal exposure to antenatal GC administration is sufficient for lung maturation. We have established a premature fetal rat model to investigate immature cardiopulmonary functions in the lungs and heart, including the effects of antenatal GC administration. In this review, we explain the mechanisms of antenatal GC actions on the heart in the fetus compared to those in the neonate. Antenatal GCs may contribute to premature heart maturation by accelerating cardiomyocyte proliferation, angiogenesis, energy production, and sarcoplasmic reticulum function. Additionally, this review specifically focuses on fetal heart growth with antenatal GC administration in experimental animal models. Moreover, knowledge regarding antenatal GC administration in experimental animal models can be coupled with that from developmental biology, with the potential for the generation of functional cells and tissues that could be used for regenerative medical purposes in the future.

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.

Changes Adopted in Asian Pediatric Hospitals during the COVID-19 Pandemic: A Report from the Pediatric Acute and Critical Care COVID-19 Registry of Asia.

There is wide variation in the overall clinical impact of novel coronavirus disease 2019 (COVID-19) across countries worldwide. Changes adopted pertaining to the management of pediatric patients, in particular, the provision of respiratory support during the COVID-19 pandemic is poorly described in Asia. We performed a multicenter survey of 20 Asian pediatric hospitals to determine workflow changes adopted during the pandemic. Data from centers of high-income (HIC), upper middle income (UMIC), and lower middle income (LMIC) countries were compared. All 20 sites over nine countries (HIC: Japan [4] and Singapore [2]; UMIC: China [3], Malaysia [3] and Thailand [2]; and LMIC: India [1], Indonesia [2], Pakistan [1], and Philippines [2]) responded to this survey. This survey demonstrated substantial outbreak adaptability. The major differences between the three income categories were that HICs were (1) more able/willing to minimize use of noninvasive ventilation or high-flow nasal cannula therapy in favor of early intubation, and (2) had greater availability of negative-pressure rooms and powered air-purifying respirators. Further research into the best practices for respiratory support are warranted. In particular, innovation on cost-effective measures in infection control and respiratory support in the LMIC setting should be considered in preparation for future waves of COVID-19 infection.

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.

The inhibition of interaction with serum albumin enhances the physiological activity of curcumin by increasing its cellular uptake.

Based on the free drug hypothesis, we hypothesized that food compounds that bind stronger to BSA than CUR inhibit the binding between BSA and CUR, and that this results in an increase of the cellular uptake and physiological activities of CUR. To verify this hypothesis, food compounds that bind stronger to BSA than CUR were identified. When THP-1 monocytes were co-treated with the identified compounds (e.g., piperine) and CUR, cell viability significantly decreased, suggesting that the physiological activity of CUR was enhanced. Also, when THP-1 macrophages were co-treated with CUR and the identified compounds following LPS + IFNγ treatment, the decrement of TNF-α was higher compared to treatment with CUR only. Furthermore, the cellular uptake of CUR was increased during this co-treatment. Such results verify our hypothesis, and provide insights into the development of ways to enhance the physiological activities of various food compounds via focusing on their interaction with albumin.

The clinical characteristics of pediatric coronavirus disease 2019 in 2020 in Japan.

BACKGROUND: The COVID-19 pandemic has affected the lives of people of all ages. Most reports on pediatric cases suggest that children experience fewer and milder symptoms than do adults. This is the first nationwide study in Japan focusing on pediatric cases reported by pediatricians, including cases with no or mild symptoms. METHODS: We analyzed the epidemiological and clinical characteristics and transmission patterns of 840 pediatric (<16 years old) COVID-19 cases reported between February and December 2020 in Japan, using a dedicated database which was maintained voluntarily by members of the Japan Pediatric Society. RESULTS: Almost half of the patients (47.7%) were asymptomatic, while most of the others presented mild symptoms. At the time of admission or first outpatient clinic visit, 84.0% of the cases were afebrile (<37.5°C). In total, 609 cases (72.5%) were exposed to COVID-19-positive household members. We analyzed the influence of nationwide school closures that were introduced in March 2020 on COVID-19 transmission routes among children in Japan. Transmission within households occurred most frequently, with no significant difference between the periods before and after declaring nationwide school closures (70.9% and 74.5%, respectively). CONCLUSIONS: COVID-19 symptoms in children are less severe than those in adults. School closure appeared to have a limited effect on transmission. Controlling household transmission from adult family members is the most important measure for prevention of COVID-19 among children.

Structural Analysis of Lipid Hydroperoxides Using Mass Spectrometry with Alkali Metals.

Lipid oxidation is involved in various biological phenomena (e.g., oxylipin generation and oxidative stress). Of oxidized lipid structures, the hydroperoxyl group position of lipid hydroperoxides (LOOHs) is a critical factor in determining their biological roles. Despite such interest, current methods to determine hydroperoxyl group positions possess some drawbacks such as selectivity. While we previously reported mass spectrometric methods using Na+ for the highly selective determination of hydroperoxyl group positions, nothing was known except for the fact that sodiated LOOHs (mainly linoleate) provide specific fragment ions. Thus, this study was aimed to investigate the effects of different alkali metals on the fragmentation of LOOHs, assuming its further application to analysis of other complex LOOHs. From the analysis of PC 16:0/18:2;OOH (phosphatidylcholine) and FA 18:2;OOH (fatty acid), we found that fragmentation pathways and ion intensities largely depend on the binding position and type of alkali metals (i.e., Li+, Hock fragmentation; Na+ and K+, α-cleavage (Na+ > K+); Rb+ and Cs+, no fragmentation). Furthermore, we proved that this method can be applied to determine the hydroperoxyl group position of esterified lipids (e.g., phospholipids and cholesterol esters) as well as polyunsaturated fatty acids (PUFAs) including n-3, n-6, and n-9 FA. We anticipate that the insights described in this study provide additional unique insights to conventional lipid oxidation research.