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BACKGROUND There remains an unmet need in objective tests for diagnosing asthma in children. The objective of this study was to investigate the potential of metabolomic profiles of exhaled breath condensate (EBC) to discriminate stable asthma in Asian children in the community. METHODS One hundred and sixty-five Asian children (92 stable asthma and 73 non-asthmatic controls) participating in a population-based cohort were enrolled and divided into training and validation sets. Nuclear magnetic resonance-based metabolomic profiles of EBC samples were analyzed by using orthogonal partial least squares discriminant analysis. RESULTS EBC metabolomic signature (lactate, formate, butyrate, and isobutyrate) had an area under the receiver operator characteristic curve (AUC) of 0.826 in discriminating children with and without asthma in the training set, which significantly outperformed FeNO (AUC = 0.574; P

We have previously shown that GSH depletion alters global metabolism of cells. In the present study, we applied a metabolomic approach for studying the early changes in metabolism in hydrogen peroxide- (H2O2-) treated hepatoma cells which were destined to die. Levels of fructose 1,6-bisphosphate and an unusual metabolite, sedoheptulose 1,7-bisphosphate (S-1,7-BP), were elevated in hepatoma Hep G2 cells. Deficiency in G6PD activity significantly reduced S-1,7-BP formation, suggesting that S-1,7-BP is formed in the pentose phosphate pathway as a response to oxidative stress. Additionally, H2O2treatment significantly increased the level of nicotinamide adenine dinucleotide phosphate (NADP+) and reduced the levels of ATP and NAD+. Severe depletion of ATP and NAD+in H2O2-treated Hep G2 cells was associated with cell death. Inhibition of PARP-mediated NAD+depletion partially protected cells from death. Comparison of metabolite profiles of G6PD-deficient cells and their normal counterparts revealed that changes in GSH and GSSG per se do not cause cell death. These findings suggest that the failure of hepatoma cells to maintain energy metabolism in the midst of oxidative stress may cause cell death.

Metabolic alterations have been documented in peripheral tissues in heart failure (HF). Outcomes might be improved by early identification of risk. However, the prognostic information offered is still far from enough. We hypothesized that plasma metabolic profiling potentially provides risk stratification for HF patients. Of 61 patients hospitalized due to acute decompensated HF, 31 developed HF-related events in one year after discharge (Event group), and the other 30 patients did not (Non-event group). The plasma collected during hospital admission was analyzed by an ultra-high performance liquid chromatography time-of-flight mass spectrometry (UPLC-TOFMS)-based metabolomic approach. The orthogonal projection to latent structure discriminant analysis (OPLS-DA) reveals that the metabolomics profile is able to distinguish between events in HF. Levels of 19 metabolites including acylcarnitines, lysophospholipids, dimethylxanthine, dimethyluric acid, tryptophan, phenylacetylglutamine, and hypoxanthine are significantly different between patients with and without event (p &lt, 0.05). Established risk prediction models of event patients by using receiver operating characteristics analysis reveal that the combination of tetradecenoylcarnitine, dimethylxanthine, phenylacetylglutamine, and hypoxanthine has better discrimination than B-type natriuretic peptide (BNP) (AUC 0.871 and 0.602, respectively). These findings suggest that metabolomics-derived metabolic profiling have the potential of identifying patients with high risk of HF-related events and provide insights related to HF outcome.

Early allograft dysfunction (EAD) is associated with graft failure and mortality after living donor liver transplantation (LDLT). In this study, we report biomarkers superior to other conventional clinical markers in the prediction of EAD and all-cause in-hospital mortality in LDLT patient cohort. Blood samples of living donor liver transplant recipients were collected on postoperative day 1 and analyzed by liquid chromatography coupled with mass spectrometry (LC-MS). Significant metabolites associated with the prediction of EAD were identified using orthogonal projection to latent structures-discriminant analysis (OPLS-DA). A few lipids, more specifically, lysoPC (16:0), PC (18:0/20:5), betaine and palmitic acid (C16:0) were found to effectively differentiate EAD from non-EAD on postoperative day 1. A combination of these four metabolites showed an AUC of 0.821, which was further improved to 0.846 by the addition of a clinical parameter, total bilirubin. The panel exhibits a high prognostic accuracy in prediction of all-cause in-hospital mortality and mortality within 7 postoperative days with AUCs of 0.843 and 0.954. These results show the combination of metabolomics-derived biomarkers and clinical parameters demonstrates the power of panels in diagnostic and prognostic evaluation of LDLT.

AIM To explore the correlation of metabolomics profiles of gastric cancer (GC) with its chromosomal instability (CIN) status. METHODS Nineteen GC patients were classified as CIN and non-CIN type by The Cancer Genome Atlas Research Group system, based on 409 oncogenes and tumor suppressor genes sequenced. The aqueous metabolites of the GC tumor and its surrounding adjacent healthy tissues were identified through liquid chromatography-mass spectrometry. Groups were compared by defining variable importance in projection score of > 1.2, a fold change value or its reciprocal of > 1.2, and a P value of < 0.05 as a significant difference. RESULTS In total, twelve men and seven women were enrolled, with a median age of 66 years (range, 47-87 years). The numbers of gene alterations in the CIN GC group were significantly higher than those in the non-CIN GC (32-218 vs 2-17; P < 0.0005). Compared with the adjacent healthy tissues, GC tumors demonstrated significantly higher aspartic acid, citicoline, glutamic acid, oxidized glutathione, succinyladenosine, and uridine diphosphate-N-acetylglucosamine levels, but significantly lower butyrylcarnitine, glutathione hydroxyhexanoycarnitine, inosinic acid, isovalerylcarnitine, and threonine levels (all P < 0.05). CIN tumors contained significantly higher phosphocholine and uridine 5’-monophosphate levels but significantly lower beta-citryl-L-glutamic acid levels than did non-CIN tumors (all P < 0.05). CIN GC tumors demonstrated additional altered pathways involving alanine, aspartate, and glutamate metabolism, glyoxylate and dicarboxylate metabolism, histidine metabolism, and phenylalanine, tyrosine, and tryptophan biosynthesis. CONCLUSION Metabolomic profiles of GC tumors and the adjacent healthy tissue are distinct, and the CIN status is associated with downstream metabolic alterations in GC.

To identify the association between metabolites and muscle mass in 305 elderly Taiwanese subjects, we conducted a multivariate analysis of 153 plasma samples. Based on appendicular skeletal muscle mass index (ASMI) quartiles, female and male participants were divided into four groups. Quartile 4 (Men: 5.67±0.35, Women: 4.70±0.32 Kg/m2) and quartile 1 (Men: 7.60±0.29, Women: 6.56±0.53 Kg/m2) represented low muscle mass and control groups, respectively. After multivariable adjustment, except for physical function, we found that blood urea nitrogen, creatinine, and age were associated with ASMI in men. However, only triglyceride level was related to ASMI in women. The multiple logistic regression models were used to analyze in each baseline characteristic and metabolite concentration. After the adjustment, we identify amino acid-related metabolites and show that glutamate levels in women and alpha-aminoadipate, Dopa, and citrulline/ornithine levels in men are gender-specific metabolic signatures of muscle mass loss.

Alterations in metabolism following radiotherapy affect therapeutic efficacy, although the mechanism underlying such alterations is unclear. A new imaging technique—named dynamic nuclear polarization (DNP) carbon-13 magnetic resonance imaging (MRI)—probes the glycolytic flux in a real-time, dynamic manner. The [1-13C]pyruvate is transported by the monocarboxylate transporter (MCT) into cells and converted into [1-13C]lactate by lactate dehydrogenase (LDH). To capture the early glycolytic alterations in the irradiated cancer and immune cells, we designed a preliminary DNP 13C-MRI study by using hyperpolarized [1-13C]pyruvate to study human FaDu squamous carcinoma cells, HMC3 microglial cells, and THP-1 monocytes before and after irradiation. The pyruvate-to-lactate conversion rate (kPL [Pyr.]) calculated by kinetic modeling was used to evaluate the metabolic alterations. Western blotting was performed to assess the expressions of LDHA, LDHB, MCT1, and MCT4 proteins. Following irradiation, the pyruvate-to-lactate conversion rates on DNP 13C-MRI were significantly decreased in the FaDu and the HMC3 cells but increased in the THP-1 cells. Western blot analysis confirmed the similar trends in LDHA and LDHB expression levels. In conclusion, DNP 13C-MRI non-invasively captured the different glycolytic alterations among cancer and immune systems in response to irradiation, implying its potential for clinical use in the future.

Enterovirus 71 (EV71) infection is endemic in the Asia-Pacific region. No specific antiviral drug has been available to treat EV71 infection. Melissa officinalis (MO) is a medicinal plant with long history of usage in the European and Middle East. We investigated whether an aqueous solution of concentrated methanolic extract (MOM) possesses antiviral activity. MOM inhibited plaque formation, cytopathic effect, and viral protein synthesis in EV71-infected cells. Using spectral techniques, we identified rosmarinic acid (RA) as a biologically active constituent of MOM. RA reduced viral attachment and entry; cleavage of eukaryotic translation initiation factor 4 G (eIF4G); reactive oxygen species (ROS) generation; and translocation of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) from nucleus to cytoplasm. It alleviated EV71-induced hyperphosphorylation of p38 kinase and EPS15. RA is likely to suppress ROS-mediated p38 kinase activation, and such downstream molecular events as hnRNP A1 translocation and EPS15-regulated membrane trafficking in EV71-infected cells. These findings suggest that MO and its constituent RA possess anti-EV71 activities, and may serve as a candidate drug for therapeutic and prophylactic uses against EV71 infection.

Insulin resistance and metabolic derangement are present in patients with type 2 diabetes mellitus (T2DM). However, the metabolomic signature of T2DM in cerebrospinal fluid (CSF) has not been investigated thus far. In this prospective metabolomic study, fasting CSF and plasma samples from 40 T2DM patients to 36 control subjects undergoing elective surgery with spinal anesthesia were analyzed by 1H nuclear magnetic resonance (NMR) spectroscopy. NMR spectra of CSF and plasma metabolites were analyzed and correlated with the presence of T2DM and diabetic microangiopathy (retinopathy, nephropathy, and neuropathy) using an area under the curve (AUC) estimation. CSF metabolomic profiles in T2DM patients vs. controls revealed significantly increased levels of alanine, leucine, valine, tyrosine, lactate, pyruvate, and decreased levels of histidine. In addition, a combination of alanine, histidine, leucine, pyruvate, tyrosine, and valine in CSF showed a superior correlation with the presence of T2DM (AUC:0.951), diabetic retinopathy (AUC:0.858), nephropathy (AUC:0.811), and neuropathy (AUC:0.691). Similar correlations also appeared in plasma profiling. These metabolic alterations in CSF suggest decreasing aerobic metabolism and increasing anaerobic glycolysis in cerebral circulation of patients with T2DM. In conclusion, our results provide clues for the metabolic derangements in diabetic central neuropathy among T2DM patients, however, their clinical significance requires further exploration.

Aging is a complex progression of biological processes and is the causal contributor to the development of diabetes mellitus (DM). DM is the most common degenerative disease and is the fifth leading cause of death in Taiwan, where the trend of DM mortality has been steadily increasing. Metabolomics, important branch of systems biology, has been mainly utilized to understand endogenous metabolites in biological systems and their dynamic changes as they relate to endogenous and exogenous factors. The purpose of this study was to elucidate the metabolomic profiles in elderly people and its relation to lipid disorder (LD). We collected 486 elderly individuals aged &ge, 65 years and performed untargeted and targeted metabolite analysis using nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography&mdash, mass spectrometry (LC/MS). Several metabolites, including branched-chain amino acids, alanine, glutamate and alpha-aminoadipic acid were elevated in LD compared to the control group. Based on multivariate analysis, four metabolites were selected in the best model to predict DM progression: phosphatidylcholine acyl-alkyl (PC ae) C34:3, PC ae C44:3, SM C24:1 and PCae C36:3. The combined area under the curve (AUC) of those metabolites (0.82) was better for DM classification than individual values. This study found that targeted metabolic signatures not only distinguish the LD within the control group but also differentiated DM from LD in elderly Taiwanese. These metabolites could indicate the nutritional status and act as potential metabolic biomarkers for the elderly in Taiwan.

Non-alcoholic fatty liver disease (NAFLD) as a global health problem has clinical manifestations ranging from simple non-alcoholic fatty liver (NAFL) to non-alcoholic steatohepatitis (NASH), cirrhosis, and cancer. The role of different types of fatty acids in driving the early progression of NAFL to NASH is not understood. Lipid overload causing lipotoxicity and inflammation has been considered as an essential pathogenic factor. To correlate the lipid profiles with cellular lipotoxicity, we utilized palmitic acid (C16:0)- and especially unprecedented palmitoleic acid (C16:1)-induced lipid overload HepG2 cell models coupled with lipidomic technology involving labeling with stable isotopes. C16:0 induced inflammation and cell death, whereas C16:1 induced significant lipid droplet accumulation. Moreover, inhibition of de novo sphingolipid synthesis by myriocin (Myr) aggravated C16:0 induced lipoapoptosis. Lipid profiles are different in C16:0 and C16:1-treated cells. Stable isotope-labeled lipidomics elucidates the roles of specific fatty acids that affect lipid metabolism and cause lipotoxicity or lipid droplet formation. It indicates that not only saturation or monounsaturation of fatty acids plays a role in hepatic lipotoxicity but also Myr inhibition exasperates lipoapoptosis through ceramide in-direct pathway. Using the techniques presented in this study, we can potentially investigate the mechanism of lipid metabolism and the heterogeneous development of NAFLD.

Glucose-6-phosphate dehydrogenase (G6PD)-deficient cells are highly susceptible to viral infection. This study examined the mechanism underlying this phenomenon by measuring the expression of antiviral genes—tumor necrosis factor alpha (TNF-α) and GTPase myxovirus resistance 1 (MX1)—in G6PD-knockdown cells upon human coronavirus 229E (HCoV-229E) and enterovirus 71 (EV71) infection. Molecular analysis revealed that the promoter activities of TNF-α and MX1 were downregulated in G6PD-knockdown cells, and that the IκB degradation and DNA binding activity of NF-κB were decreased. The HSCARG protein, a nicotinamide adenine dinucleotide phosphate (NADPH) sensor and negative regulator of NF-κB, was upregulated in G6PD-knockdown cells with decreased NADPH/NADP+ ratio. Treatment of G6PD-knockdown cells with siRNA against HSCARG enhanced the DNA binding activity of NF-κB and the expression of TNF-α and MX1, but suppressed the expression of viral genes, however, the overexpression of HSCARG inhibited the antiviral response. Exogenous G6PD or IDH1 expression inhibited the expression of HSCARG, resulting in increased expression of TNF-α and MX1 and reduced viral gene expression upon virus infection. Our findings suggest that the increased susceptibility of the G6PD-knockdown cells to viral infection was due to impaired NF-κB signaling and antiviral response mediated by HSCARG.

Enterovirus 71 (EV71) infection is an endemic disease in Southeast Asia and China. We have previously shown that EV71 virus causes functional changes in mitochondria. It is speculative whether EV71 virus alters the host cell metabolism to its own benefit. Using a metabolomics approach, we demonstrate that EV71-infected Vero cells had significant changes in metabolism. Glutathione and its related metabolites, and several amino acids, such as glutamate and aspartate, changed significantly with the infectious dose of virus. Other pathways, including glycolysis and tricarboxylic acid cycle, were also altered. A change in glutamine/glutamate metabolism is critical to the viral infection. The presence of glutamine in culture medium was associated with an increase in viral replication. Dimethyl &alpha, ketoglutarate treatment partially mimicked the effect of glutamine supplementation. In addition, the immunoblot analysis revealed that the expression of glutamate dehydrogenase (GDH) and trifunctional carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) increased during infection. Knockdown of expression of glutaminase (GLS), GDH and CAD drastically reduced the cytopathic effect (CPE) and viral replication. Furthermore, we found that CAD bound VP1 to promote the de novo pyrimidine synthesis. Our findings suggest that virus may induce metabolic reprogramming of host cells to promote its replication through interactions between viral and host cell proteins.

Schizonepeta tenuifolia (ST) Briq. is a traditional herbal medicine commonly used to treat allergic skin diseases, where the inflammation process is closely related to symptom severity. This study aimed to explore the immunomodulatory effect of ST by using immunoglobulin E- (IgE-) stimulated RBL-2H3 cell cultures, a common cell line for studying mast cell degranulation and inflammatory cytokine release in vitro. After stimulating the RBL-2H3 cells with IgE, ST at concentrations of 10, 50, or 100 μg/mL was added to the cell cultures. Cell viability, inflammatory cytokines (IL-6, IL-13, IL-4, TNF-α, and IFN-γ), anti-inflammatory cytokine IL-10, and degranulation ability were examined 48 and 72 hours after administration of ST. The markers of inflammation and allergic reaction, IFN-γ, TNF-α, IL-4, and IL-6, were suppressed, especially after treatment with 100 μg/mL ST. However, the anti-inflammation marker IL-10 was also suppressed by ST. Trend analysis showed that a higher ST concentration was associated with lower IFN-γ and TNF-α levels. Moreover, degranulation of RBL-2H3 cells was assessed by measuring the release of β-hexosaminidase, which was suppressed by ST at 10 μg/mL. This study showed an immunomodulatory effect of ST at the cellular level and suggests the role of ST in treating allergic diseases.

Schizonepeta tenuifolia (ST) Briq. is a traditional herbal medicine commonly used to treat allergic skin diseases, where the inflammation process is closely related to symptom severity. This study aimed to explore the immunomodulatory effect of ST by using immunoglobulin E- (IgE-) stimulated RBL-2H3 cell cultures, a common cell line for studying mast cell degranulation and inflammatory cytokine release in vitro. After stimulating the RBL-2H3 cells with IgE, ST at concentrations of 10, 50, or 100 μg/mL was added to the cell cultures. Cell viability, inflammatory cytokines (IL-6, IL-13, IL-4, TNF-α, and IFN-γ), anti-inflammatory cytokine IL-10, and degranulation ability were examined 48 and 72 hours after administration of ST. The markers of inflammation and allergic reaction, IFN-γ, TNF-α, IL-4, and IL-6, were suppressed, especially after treatment with 100 μg/mL ST. However, the anti-inflammation marker IL-10 was also suppressed by ST. Trend analysis showed that a higher ST concentration was associated with lower IFN-γ and TNF-α levels. Moreover, degranulation of RBL-2H3 cells was assessed by measuring the release of β-hexosaminidase, which was suppressed by ST at 10 μg/mL. This study showed an immunomodulatory effect of ST at the cellular level and suggests the role of ST in treating allergic diseases.

Lipids play an important role in the pathogenesis of cardiovascular disease. Changes in lipids of erythrocytes are indicative of the outcome of pathophysiological processes. In the present study, we assessed whether the lipid profiles of erythrocytes from heart failure (HF) patients are informative of their disease risk. The lipidomes of erythrocytes from 10 control subjects and 29 patients at different HF stages were analyzed using liquid chromatography time-of-flight mass spectrometry. The lipid composition of erythrocytes obtained from HF patients was significantly different from that of normal controls. The levels of phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), and sphingomyelins decreased in HF erythrocytes as compared with those of control subjects; however, the levels of lysoPCs, lysoPEs, and ceramides increased in HF erythrocytes. Notably, the oxidized cholesterol 7-ketocholesterol (7KCh) accumulated to higher level in HF erythrocytes than in plasma from the same patients. We further validated our findings with a cohort of 115 subjects of control subjects (n=28) and patients (n=87). Mechanistically, 7KCh promoted reactive oxygen species (ROS) formation in cardiomyocytes; and induced their death, probably through an ATF4-dependent pathway. Our findings suggest that erythrocytic 7KCh can be a risk factor for HF, and is probably implicated in its pathophysiology. Keywords: Heart failure, Lipidomics, 7-ketocholesterol, Oxidative stress

Background Identification of novel biomarkers is needed to improve the diagnosis and prognosis of heart failure (HF). Metabolic disturbance is remarkable in patients with HF. Objectives This study sought to assess the diagnostic and prognostic values of metabolomics in HF. Methods Mass spectrometry–based profiling of plasma metabolites was performed in 515 participants; the discovery phase study enrolled 51 normal control subjects and 183 HF patients, and the validation study enrolled 63 control subjects and 218 patients with stage C HF. Another independent group of 32 patients with stage C HF who recovered to New York Heart Association functional class I at 6 and 12 months was profiled as the “recovery” group. Results A panel of metabolites, including histidine, phenylalanine, spermidine, and phosphatidylcholine C34:4, has a diagnostic value similar to B-type natriuretic peptide (BNP). In the recovery group, the values of this panel significantly improved at 6 and 12 months. To evaluate the prognostic values, events were defined as the combined endpoints of death or HF-related re-hospitalization. A metabolite panel, which consisted of the asymmetric methylarginine/arginine ratio, butyrylcarnitine, spermidine, and the total amount of essential amino acids, provided significant prognostic values (p Conclusions Metabolomics demonstrate powerful diagnostic value in estimating HF-related metabolic disturbance. The profile of metabolites provides better prognostic value versus conventional biomarkers.

Aims: Glucose 6-phosphate dehydrogenase (G6PD) is essential for maintenance of nicotinamide dinucleotide hydrogen phosphate (NADPH) levels and redox homeostasis. A number of drugs, such as antimalarial drugs, act to induce reactive oxygen species and hemolytic crisis in G6PD-deficient patients. We used diamide (DIA) to mimic drug-induced oxidative stress and studied how these drugs affect cellular metabolism using a metabolomic approach. Results: There are a few differences in metabolome between red blood cells (RBCs) from normal and G6PD-deficient individuals. DIA causes modest changes in normal RBC metabolism. In contrast, there are significant changes in various biochemical pathways, namely glutathione (GSH) metabolism, purine metabolism, and glycolysis, in G6PD-deficient cells. GSH depletion is concomitant with a shift in energy metabolism. Adenosine monophosphate (AMP) and adenosine diphosphate (ADP) accumulation activates AMP protein kinase (AMPK) and increases entry of glucose into glycolysis. However, inhibition of pyruvate kinase (PK) reduces the efficacy of energy production. Metabolic changes and protein oxidation occurs to a greater extent in G6PD-deficient RBCs than in normal cells, leading to severe irreversible loss of deformability of the former. Innovation and Conclusion: Normal and G6PD-deficient RBCs differ in their responses to oxidants. Normal cells have adequate NADPH regeneration for maintenance of GSH pool. In contrast, G6PD-deficient cells are unable to regenerate enough NADPH under a stressful situation, and switch to biosynthetic pathway for GSH supply. Rapid GSH exhaustion causes energy crisis and futile AMPK activation. Our findings suggest that drug-induced oxidative stress differentially affects metabolism and metabolite signaling in normal and G6PD-deficient cells. It also provides an insight into the pathophysiology of acute hemolytic anemia in G6PD-deficient patients. Antioxid. Redox Signal. 22, 744–759.

No effective drug is currently available for treatment of enterovirus 71 (EV71) infection. Schizonepeta tenuifolia Briq. (ST) has been used as a herbal constituent of traditional Chinese medicine. We studied whether the aqueous extract of Schizonepeta tenuifolia Briq (STE) has antiviral activity. STE inhibited replication of EV71, as evident by its ability to diminish plaque formation and cytopathic effect induced by EV71, and to inhibit the synthesis of viral RNA and protein. Moreover, daily single-dose STE treatment significantly improved the survival of EV71-infected mice, and ameliorated the symptoms. Mechanistically, STE exerts multiple effects on enteroviral infection. Treatment with STE reduced viral attachment and entry; the cleavage of eukaryotic translation initiation factor 4 G (eIF4G) by EV71 protease, 2Apro; virus-induced reactive oxygen species (ROS) formation; and relocation of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) from the nucleus to the cytoplasm. It was accompanied by a decline in EV71-associated hyperphosphorylation of p38 kinase and EPS15. It is plausible that STE may inhibit ROS-induced p38 kinase activation, and subsequent hnRNP A1 relocation and EPS15-mediated membrane trafficking in infected cells. These findings suggest that STE possesses anti-EV71 activities, and may serve as health food or candidate antiviral drug for protection against EV71.

Background Guillain-Barre syndrome (GBS) is an acute inflammatory polyneuropathy resulting in demyelination in peripheral nervous system. Myelin enriched in lipids is easily oxidized by reactive oxygen species during inflammation. Oxidative stress and lipophilic anti-oxidative capacities in GBS patients have not been fully explored. To evaluate the redox status of GBS patients, we measured malondialdehyde (MDA), myeloperoxidase (MPO), lipophilic antioxidants, and tocopherols concentrations in plasma from GBS patients and age-matched healthy controls. Results Concentrations of γ-tocopherol and δ-tocopherol decreased significantly, and α-carotene significantly increased in GBS patients compared to healthy controls. However, no significant changes in MDA and MPO concentrations were detected. In GBS patients, the γ-tocopherol concentration correlated positively with concentrations of δ-tocopherol, α-tocopherol, lutein, Q10, and γ-CEHC, respectively. Similarly, the δ-tocopherol concentration correlated positively with γ-tocopherol, α-tocopherol, lutein, Q10, δ-CEHC, and γ-CEHC concentrations, respectively. The receiver operating characteristics curve analysis showed that γ-tocopherol may serve as a good predictor for GBS. Conclusions Diminished lipophilic antioxidant defense, mainly γ-tocopherol and δ-tocopherol, in GBS patients accounting for their lowered resistance to reactive oxygen species is probably associated with pathogenesis of GBS, and potentially useful for the development of therapeutic strategies.

Glucose-6-phosphate dehydrogenase (G6PD) is critical to the maintenance of NADPH pool and redox homeostasis. Conventionally, G6PD deficiency has been associated with hemolytic disorders. Most biochemical variants were identified and characterized at molecular level. Recently, a number of studies have shone light on the roles of G6PD in aspects of physiology other than erythrocytic pathophysiology. G6PD deficiency alters the redox homeostasis, and affects dysfunctional cell growth and signaling, anomalous embryonic development, and altered susceptibility to infection. The present article gives a brief review of basic science and clinical findings about G6PD, and covers the latest development in the field. Moreover, how G6PD status alters the susceptibility of the affected individuals to certain degenerative diseases is also discussed.

In response to infection, neutrophils employ various strategies to defend against the invading microbes. One of such defense mechanisms is the formation of neutrophil extracellular traps (NETs). Recent studies suggest that reactive oxygen species is a signal critical to NET formation. This prompts us to examine whether neutrophils from individuals with glucose-6-phosphate dehydrogenase (G6PD) Taiwan-Hakka variant, which are prone to oxidative stress generation, have altered ability to form NET. We adopted an image-based method to study the NET formation potential in neutrophils from G6PD-deficient patients. Neutrophils from either normal or G6PD-deficient individuals underwent NETosis in response to phorbol 12-myristate 13-acetate (PMA). The extent of NETosis in the former did not significantly differ from that of the latter. Diphenyleneiodonium sulfate (DPI) and 3-methyladenine (MA) inhibited PMA-stimulated NET formation in these cells, suggesting the involvement of NADPH oxidase and autophagy in the process. Glucose oxidase (GO) and xanthine oxidase/xanthine (XO/X) could induce a similar extent of NET formation in normal and G6PD-deficient neutrophils. GO- or XO-induced NETosis was not inhibitable by MA, implying that reactive oxygen species (ROS) can act as an independent signal for activation of NETosis. Mechanistically, enhanced superoxide production in neutrophils was associated with increases in levels of NAD(+) and NADP(+), as well as activation of NAD(+) kinase. Taken together, these findings suggest that G6PD-deficient neutrophils are as equally efficient as normal cells in NET formation, and their deficiency in G6PD-associated NADPH regeneration capacity is largely compensated for by nicotinamide nucleotide biosynthesis.

Glucose-6-phosphate dehydrogenase (G6PD) is crucial to NADPH generation and redox homeostasis. We have recently shown that G6PD deficiency predisposes cells to oxidant-induced cell death, and it is associated with the impairment of glutathione regeneration. It remains unclear what other metabolic pathways are affected by G6PD deficiency and whether the altered metabolism disturbs cellular redox homeostasis and underlies increased susceptibility to oxidants. In this study, we examined the effects of diamide on global metabolite profiles of SK-Hep1-derived SK-i-Gi and SK-i-Sc cells, which could inducibly express short hairpin RNA (shRNA) against G6PD (Gi) and control shRNA (Sc), respectively. There was no significant difference in their metabolite profiles under uninduced conditions. Doxycycline (Dox) addition resulted in over 70% decrease in G6PD activity in SK-i-Gi cells. This was accompanied by relatively minor changes in the metabolome of SK-i-Gi cells. Upon further diamide treatment, the metabolite profiles of both SK-i-Gi and SK-i-Sc cells changed in a time-dependent manner. A number of metabolic pathways, including those involved in energy metabolism and metabolism of amino acids and glutathione, were affected. However, the changes in the metabolite profile of Dox-treated SK-i-Gi cells were distinct from those of control cells (i.e., Dox-treated SK-i-Sc, SK-i-Gi, and SK-i-Sc cells). Cellular glutathione was depleted, whereas its disulfide form increased significantly in diamide, Dox-treated SK-i-Gi cells. Metabolites related to energy metabolism, such as AMP, ADP, and acetylcarnitine, increased to a greater extent in these cells than in diamide-treated control cells. In contrast, NAD and glutathione dropped to lower levels in SK-i-Gi cells than in control cells. The NAD(+) depletion in SK-i-Gi cells was accompanied by a significant increase in NAD kinase activity. Targeted analyses revealed that NADP(+) and NADPH increased significantly in diamide, Dox-treated SK-i-Gi cells compared with similarly treated control cells. Our results suggest that diamide induces oxidation and depletion of glutathione in SK-i-Gi cells under conditions of G6PD shRNA induction and subsequently induces conversion of NAD(+) to NADP(+) through enhanced NAD kinase activity. This may represent a compensatory mechanism to restore cellular NADPH reserve in G6PD-deficient cells. It is accompanied by alteration in pathways of cellular energy metabolism, such as glycolysis and β-oxidation.

Dehydroepiandrosterone (DHEA)-induced growth arrest of hepatoma cells is associated with metabolic disturbance. Our previous study has suggested that DHEA may cause cellular energy drain. It is possible that mitochondrial dysfunction may be mechanistically implicated in DHEA-induced changes in cellular phenotype. Treatment of SK-Hep-1 cells with DHEA caused significant reduction in proliferation, colony formation, and growth in semi-solid medium. Such changes in cellular phenotype were associated with mitochondrial depolarization, increase in mitochondrial mass, and decrease in respiratory activity. Level of reactive oxygen species (ROS) increased in DHEA-treated cells. To explore the mechanistic aspect of DHEA-induced mitochondrial dysfunction, we employed SILAC approach to study the changes in the mitoproteome of SK-Hep-1 cells after DHEA treatment. Respiratory chain complex proteins such as NDUFB8 and SDHB were differentially expressed. Of mitochondrial proteins with altered expression, FAST kinase domain-containing protein 2 (FASTKD2) showed significantly reduced expression. Exogenous expression of FASTKD2 in SK-Hep-1 cells increased their resistance to growth-inhibitory effect of DHEA, though it alone did not affect cell growth. FASTKD2 expression partially reversed the effect of DHEA on mitochondria, and reduced DHEA-induced ROS generation. Our results suggest that DHEA induces changes in mitochondrial proteins and respiratory activity, and contributes to growth arrest. FASTKD2 may be an important regulator of mitochondrial physiology, and represent a downstream target for DHEA.

Glucose-6-phosphate dehydrogenase (G6PD) plays a key role in the regeneration of NADPH and maintenance of cellular redox balance. In the present study, we investigate the effect of G6PD deficiency on H(2)O(2)-elicited signaling in HepG2 cells. H(2)O(2) was found to inhibit cellular protein tyrosine phosphatase (PTP) activity, resulting in activation of MAPKs. MKP-1 expression increased in the late phase of H(2)O(2) signaling. Using RNAi technology, we found that G6PD knockdown enhanced the inhibitory effect of H(2)O(2) on PTPs and led to sustained MAPK activation. This was accompanied by delayed expression and inhibition of MKP-1. Using a pharmacological inhibitor and siRNA, we demonstrate that MKP-1 acts as a regulator of MAPK activation in H(2)O(2) signaling. The prolonged MAPK activation in G6PD-knockdown cells was associated with an increased susceptibility to H(2)O(2)-induced apoptosis and growth retardation. Treatment with p38 and JNK inhibitors or N-acetylcysteine ameliorated such cellular effect, while triptolide and MKP-1-siRNA did the opposite. Glucose oxidase treatment had similar effects as addition of H(2)O(2). Taken together, these findings suggest that G6PD knockdown enhances the magnitude and duration of H(2)O(2)-induced MAPK signaling through inhibition of cellular PTPs, and the resultant anomalous signaling may lead to cell demise.

Glutathione peroxidase 1 (GPX-1) has an important role in antioxidant defense and has been suggested to have a protective role against the pathogenesis of atherosclerosis. In the present study, erythrocytic GPX-1 activity from 42 patients with acute myocardial infarction and 285 healthy control subjects of Taiwanese origin were determined. Our data showed that mean GPX-1 activity decreased in patients with acute myocardial infarction (40.7 +/- 9.2 vs 47.3 +/- 16.6 U/g hemoglobin; p0.001). There was a significant inverse correlation between GPX-1 activity and acute myocardial infarction incidence (p = 0.032). In addition, plasma vitamin E inversely correlated with acute myocardial infarction incidence (p = 0.017). The incidence of acute myocardial infarction increased only in patients with low plasma vitamin E and low erythrocytic GPX-1 activity, but not in those with high vitamin E and low GPX-1 activity, low vitamin E and high GPX-1 activity, or high vitamin E and high GPX-1 activity. In conclusion, acute myocardial infarction incidence significantly increased in patients with a concomitant decrease in plasma vitamin E and erythrocytic GPX-1 activity, suggesting that both GPX-1 and vitamin E may be valuable markers for monitoring cardiovascular events, particularly when used in combination.

The host cellular environment is a key determinant of pathogen infectivity. Viral gene expression and viral particle production of glucose-6-phosphate dehydrogenase (G6PD)–deficient and G6PD-knockdown cells were much higher than their counterparts when human coronavirus (HCoV) 229E was applied at 0.1 multiplicity of infection. These phenomena were correlated with increased oxidant production. Accordingly, ectopic expression of G6PD in G6PD-deficient cells or addition of antioxidant (such as α-lipoic acid) to G6PD-knockdown cells attenuated the increased susceptibility to HCoV 229E infection. All experimental data indicated that oxidative stress in host cells is an important factor in HCoV 229E infectivity

Homocysteine (Hcy) has recently been considered as a risk factor for cardiovascular disease. Coronary artery disease (CAD) is the third most common cause of mortality in Taiwan. The objective of our present study is to delineate the relationship between the plasma Hcy level and CAD in Taiwanese using an improved enzymatic method. Blood samples were collected from 86 CAD patients, which include 38 patients with acute coronary syndrome (ACS) and 48 patients with stable CAD, and 89 controls. Plasma Hcy levels were measured by HPLC and enzymatic methods. Plasma Hcy levels were 8.27 +/- 1.74, 8.50 +/-1.88 and 11.76 +/- 4.58 muM in normal controls, patients with stable CAD and those with ACS, respectively. Plasma Hcy levels were elevated in the cohort of ACS patients, as compared with those of normal controls or patients with stable CAD. Within the CAD group, patients with the highest quartile Hcy level (10.18-23.73 muM) had a significantly higher odd ratio for ACS, as compared with those with the lowest quartile Hcy level after multivariate adjustment. Plasma Hcy concentration can be used as an independent risk factor of ACS, particularly for those with a history of CAD. Moreover, our improved enzymatic assay can be automated for large-scale screening of high-risk cohorts.

Background This study was designed to investigate the effect of glucose 6-phosphate dehydrogenase (G6PD) deficiency on pro-inflammatory cytokine secretion using a palmitate-induced inflammation HepG2 in vitro model. The modulation of cellular pro-inflammatory cytokine expression under G6PD deficiency during chronic hepatic inflammation has never been investigated before. Methods The culture medium of untreated and palmitate-treated G6PD-scramble (Sc) and G6PD-knockdown (Gi) HepG2 cells were subjected to cytokine array analysis, followed by validation with ELISA and qRT-PCR of the target cytokine. The mechanism of altered cytokine secretion in palmitate-treated Sc and Gi HepG2 cells was examined in the presence of anti-oxidative enzyme (glutathione peroxidase, GPX), anti-inflammatory agent (curcumin), NF-κB inhibitor (BAY11-7085) and specific SiRNA against NF-κB subunit p65. Results Cytokine array analysis indicated that IL-8 is most significantly increased in G6PD-knockdown HepG2 cells. The up-regulation of IL-8 caused by G6PD deficiency in HepG2 cells was confirmed in other G6PD-deficient cells by qRT-PCR. The partial reduction of G6PD deficiency-derived IL-8 due to GPX and NF-κB blockers indicated that G6PD deficiency up-regulates pro-inflammatory cytokine IL-8 through oxidative stress and NF-κB pathway. Conclusions G6PD deficiency predisposes cells to enhanced production of pro-inflammatory cytokine IL-8. Mechanistically, G6PD deficiency up-regulates IL-8 through oxidative stress and NF-κB pathway. The palmitate-induced inflammation in G6PD-deficient HepG2 cells could serve as an in vitro model to study the role of altered redox homeostasis in chronic hepatic inflammation. Electronic supplementary material The online version of this article (doi:10.1186/s12950-015-0078-z) contains supplementary material, which is available to authorized users.

Identification of novel biomarkers is needed to improve the diagnosis and prognosis of heart failure (HF). Metabolic disturbance is remarkable in patients with HF.This study sought to assess the diagnostic and prognostic values of metabolomics in HF.Mass spectrometry-based profiling of plasma metabolites was performed in 515 participants; the discovery phase study enrolled 51 normal control subjects and 183 HF patients, and the validation study enrolled 63 control subjects and 218 patients with stage C HF. Another independent group of 32 patients with stage C HF who recovered to New York Heart Association functional class I at 6 and 12 months was profiled as the "recovery" group.A panel of metabolites, including histidine, phenylalanine, spermidine, and phosphatidylcholine C34:4, has a diagnostic value similar to B-type natriuretic peptide (BNP). In the recovery group, the values of this panel significantly improved at 6 and 12 months. To evaluate the prognostic values, events were defined as the combined endpoints of death or HF-related re-hospitalization. A metabolite panel, which consisted of the asymmetric methylarginine/arginine ratio, butyrylcarnitine, spermidine, and the total amount of essential amino acids, provided significant prognostic values (p0.0001) independent of BNP and traditional risk factors. The prognostic value of the metabolite panel was better than that of BNP (area under the curve of 0.85 vs. 0.74 for BNP) and Kaplan-Meier curves (log rank: 17.5 vs. 9.95). These findings were corroborated in the validation study.Metabolomics demonstrate powerful diagnostic value in estimating HF-related metabolic disturbance. The profile of metabolites provides better prognostic value versus conventional biomarkers.

Humic acid (HA) has been implicated as an etiological factor of Blackfoot disease endemic in the southwest coast of Taiwan. Dysfunction of endothelial cells and vasculopathy have been proposed to explain the onset of ulcerous changes at extremities. However, little is known about the effect of HA on activities of cells in these nonhealing wounds. In the present study, we demonstrate that HA adversely affects the growth properties of fibroblasts, one of the key players in wound repair. HA treatment caused growth arrest and apoptosis in human foreskin fibroblasts (HFF). This was accompanied by a significant increase in the level of 8-hydroxy-2′-deoxyguanosine (8-OHdG) in cellular DNA. The increased fluorescence in dichlorofluorescin (H2DCF)-stained and HA-treated cells suggests the involvement of reactive oxygen species (ROS) in HA-induced biological effects. Conversely, vitamin E pretreatment, which significantly reduced the 8-OHdG formation in HA-treated cells, alleviated the growth-inhibitory and apoptosis-inducing effects of HA. These results indicate that HA initiates oxidative damages to fibroblasts, and leads to their dwindling growth potential and survival. The present study suggests that HA-induced growth retardation and apoptosis of fibroblasts may play a role in the pathogenesis of Blackfoot disease.