Plasma metabolome analysis of patients with major depressive disorder.

AIM: This study sought to characterize the plasma metabolite profiling of patients with major depressive disorder (MDD). METHODS: Psychiatric assessments were made with the Structured Clinical Interview for DSM-IV Axis I Disorders. In the exploratory cohort, plasma metabolite profiles of 34 MDD patients and 31 mentally healthy controls were compared using capillary electrophoresis-mass spectrometry. Among the candidate metabolites, we focused on a metabolite showing the largest difference. The absolute concentrations were measured in two cohorts from a psychiatric primary care clinic to characterize the accuracy of the metabolite biomarker. RESULTS: Among 23 metabolites significantly lower in the MDD group than in healthy controls, we focused on phosphoethanolamine (PEA) as a candidate. The reduction of PEA levels in MDD was checked in independent clinical sample sets. An ion-chromatography-fluorescence detection method was developed to measure plasma PEA levels. In the preliminary cohort, we examined 34 MDD and 43 non-MDD subjects. The area under the receiver-operator curve (AUC) was 0.92, with sensitivity/specificity greater than 88%, at a cut-off of 1.46 µM. In the checking cohort, with 10 MDD and 13 non-MDD subjects, AUC was 0.89, with sensitivity/specificity of 86% and 100%, respectively, at a cut-off of 1.48 µM. Plasma PEA inversely correlated with MDD severity, depressed mood, loss of interest, and psychomotor retardation. CONCLUSION: These results suggest that plasma PEA level could be a candidate biomarker of MDD in the clinical setting. Further studies comparing MDD and mentally healthy controls are needed to confirm the utility of PEA as a biomarker for depression.

15N Tracing Studies on In Vitro Reactions of Ferredoxin-Dependent Nitrite Reductase and Glutamate Synthase Using Reconstituted Electron Donation Systems.

It is known that plants contain ferredoxin (Fd)-dependent nitrite reductase (NiR) and glutamate synthase (GOGAT). The Fd-NiR reaction produces ammonia from nitrite, and the activity is usually measured by nitrite disappearance. The Fd-GOGAT reaction forms two glutamates of different origin, from glutamine and 2-oxoglutarate, and the activity is measured by the oxidation of reductant (NADPH) or by formation of total glutamate. Here, a quantitative probe of the products and efficiency of the process was conducted using (15)N tracing techniques on these reactions in vitro. We quantified the reduction of (15)N-labeled [Formula: see text] to [Formula: see text] and the formation of [(15)N]glutamate and [(14)N]glutamate from [5-(15)N-amide]glutamine plus 2-oxoglutarate by NiR and GOGAT, respectively, with the reductant-Fd-NADP(+) oxidoreductase (FNR)-Fd system as the sequential electron donors. The supply of dithionite or NADPH to recombinant cyanobacterial NiR led to electron donation system-dependent formation of [(15)N]ammonium from [(15)N]nitrite. Addition of 20 mM NaCl and 20 mM Na-ascorbate accelerated nitrite reduction under high concentrations of NADPH. A sufficient supply of NADPH to recombinant Zea mays Fd-GOGAT generated complete GOGAT activity (transferring the [5-(15)N]amide of glutamine to 2-oxoglutarate to form [(15)N]glutamate), whereas a shortage of NADPH resulted in glutaminase activity only, which removed the amide from glutamine and released ammonia and [(14)N]glutamate. We conclude that although the recombinant Fd-GOGAT enzyme has two forms of glutamate synthesis, the first by glutaminase (ammonia release by glutamine amidotransferase) and the second by glutamate synthase (coupling of the ammonia and exogenously applied 2-oxoglutarate), the first works without NADPH, while the second is strictly dependent on NADPH availability.

Concentrations of metals and potential metal-binding compounds and speciation of Cd, Zn and Cu in phloem and xylem saps from castor bean plants (Ricinus communis) treated with four levels of cadmium.

We examined the concentrations of metals (Cd, Zn, Cu, Fe and Mn) and potential metal-binding compounds [nicotianamine (NA), thiol compounds and citrate] in xylem and phloem saps from 4-week-old castor bean plants (Ricinus communis) treated with 0 (control), 0.1, 1.0, and 10 µM Cd for 3 weeks. Treatment with 0.1 and 1 µM Cd produced no visible damage, while 10 µM Cd retarded growth. Cadmium concentrations in both saps were higher than those in the culture solution at 0.1 µM, similar at 1.0 µM and lower at 10 µM. Cd at 10 µM reduced Cu and Fe concentrations in both saps. NA concentrations measured by capillary electrophoresis-mass spectrometry (MS) in xylem sap (20 µM) were higher than the Cu concentrations, and those in phloem sap (150 µM) were higher than those of Zn, Fe and Cu combined. Reduced glutathione concentrations differed in xylem and phloem saps (1-2 and 30-150 µM, respectively), but oxidized glutathione concentrations were similar. Phloem sap phytochelatin 2 concentration increased from 0.8 µM in controls to 8 µM in 10 µM Cd. Free citrate was 2-4 µM in xylem sap and 70-100 µM in phloem sap. Total bound forms of Cd in phloem and xylem saps from 1 µM Cd-treated plants were 54 and 8%, respectively. Treatment of phloem sap with proteinaseK reduced high-molecular compounds while increasing fractions of low-molecular Cd-thiol complexes. Zinc-NA, Fe-NA and Cu-NA were identified in the phloem sap fraction of control plants by electrospray ionization time-of-flight MS, and the xylem sap contained Cu-NA.

Statistical hypothesis testing of factor loading in principal component analysis and its application to metabolite set enrichment analysis.

BACKGROUND: Principal component analysis (PCA) has been widely used to visualize high-dimensional metabolomic data in a two- or three-dimensional subspace. In metabolomics, some metabolites (e.g., the top 10 metabolites) have been subjectively selected when using factor loading in PCA, and biological inferences are made for these metabolites. However, this approach may lead to biased biological inferences because these metabolites are not objectively selected with statistical criteria. RESULTS: We propose a statistical procedure that selects metabolites with statistical hypothesis testing of the factor loading in PCA and makes biological inferences about these significant metabolites with a metabolite set enrichment analysis (MSEA). This procedure depends on the fact that the eigenvector in PCA for autoscaled data is proportional to the correlation coefficient between the PC score and each metabolite level. We applied this approach to two sets of metabolomic data from mouse liver samples: 136 of 282 metabolites in the first case study and 66 of 275 metabolites in the second case study were statistically significant. This result suggests that to set the number of metabolites before the analysis is inappropriate because the number of significant metabolites differs in each study when factor loading is used in PCA. Moreover, when an MSEA of these significant metabolites was performed, significant metabolic pathways were detected, which were acceptable in terms of previous biological knowledge. CONCLUSIONS: It is essential to select metabolites statistically to make unbiased biological inferences from metabolomic data when using factor loading in PCA. We propose a statistical procedure to select metabolites with statistical hypothesis testing of the factor loading in PCA, and to draw biological inferences about these significant metabolites with MSEA. We have developed an R package "mseapca" to facilitate this approach. The "mseapca" package is publicly available at the CRAN website.

Metabolomic profiling of lung and prostate tumor tissues by capillary electrophoresis time-of-flight mass spectrometry.

Metabolic microenvironment of tumor cells is influenced by oncogenic signaling and tissue-specific metabolic demands, blood supply, and enzyme expression. To elucidate tumor-specific metabolism, we compared the metabolomics of normal and tumor tissues surgically resected pairwise from nine lung and seven prostate cancer patients, using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). Phosphorylation levels of enzymes involved in central carbon metabolism were also quantified. Metabolomic profiles of lung and prostate tissues comprised 114 and 86 metabolites, respectively, and the profiles not only well distinguished tumor from normal tissues, but also squamous cell carcinoma from the other tumor types in lung cancer and poorly differentiated tumors from moderately differentiated tumors in prostate cancer. Concentrations of most amino acids, especially branched-chain amino acids, were significantly higher in tumor tissues, independent of organ type, but of essential amino acids were particularly higher in poorly differentiated than moderately differentiated prostate cancers. Organ-dependent differences were prominent at the levels of glycolytic and tricarboxylic acid cycle intermediates and associated energy status. Significantly high lactate concentrations and elevated activating phosphorylation levels of phosphofructokinase and pyruvate kinase in lung tumors confirmed hyperactive glycolysis. We highlighted the potential of CE-TOFMS-based metabolomics combined with phosphorylated enzyme analysis for understanding tissue-specific tumor microenvironments, which may lead to the development of more effective and specific anticancer therapeutics.

Characterisation of volatile and non-volatile metabolites in etiolated leaves of tea (Camellia sinensis) plants in the dark.

Aroma is an essential factor affecting the quality of tea (Camellia sinensis) products. While changes of volatile compounds during tea manufacturing have been intensively studied, the effect of environmental factors on volatile contents of fresh tea leaves has received less attention. We found that C. sinensis var. Yabukita kept in darkness by shading treatment for 3 weeks developed etiolated leaves with significantly increased levels of volatiles, especially volatile phenylpropanoids/benzenoids (VPBs). Upstream metabolites of VPBs, in particular shikimic acid, prephenic acid, and phenylpyruvic acid, showed lower levels in dark treated than in control leaves, whereas the contents of most amino acids including l-phenylalanine, a key precursor of VPBs, were significantly enhanced. In addition, analysis by ultra performance liquid chromatography-time of flight mass spectrometry, capillary electrophoresis-time of flight mass spectrometry, high performance liquid chromatography, and gas chromatography-mass spectrometry indicated that volatile and non-volatile metabolite profiles differed significantly between dark treated and untreated leaves.

Metabolome analysis of photosynthesis and the related primary metabolites in the leaves of transgenic rice plants with increased or decreased Rubisco content.

Because the comprehensive effects on metabolism by genetic manipulation of leaf Rubisco content are unknown, metabolome analysis was carried out on transgenic rice plants with increased or decreased Rubisco content using the capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS) technique. In RBCS-sense plants, an increase in Rubisco content did not improve light-saturated photosynthesis. Glyceraldehyde 3-phosphate and sedoheputulose 7-phosphate levels increased, but ribulose bisphosphate (RuBP), ATP and ADP levels were not affected. It is considered from these results that RuBP regeneration independent of ATP supply became a bottleneck for photosynthesis. In RBCS-antisense plants, a decline in Rubisco content decreased photosynthesis with a substantial accumulation of RuBP. ATP and ADP levels also increased and were associated with increases in the diphosphate and triphosphate compounds of other nucleosides. These results imply that a decline in Rubisco content slowed down the Calvin cycle and that the resultant excess energy of ATP was transferred to other nucleoside diphosphates and triphosphates. The levels of amino acids tended to decline in RBCS-sense plants and increase in RBCS-antisense plants, probably reflecting the demand for Rubisco synthesis. Starch and carbohydrate levels decreased only in RBCS-antisense plants. Thus, genetic manipulation of Rubisco contents widely affected C and N metabolism in rice.

Pleiotropic modulation of carbon and nitrogen metabolism in Arabidopsis plants overexpressing the NAD kinase2 gene.

Nicotinamide nucleotides (NAD and NADP) are important cofactors in many metabolic processes in living organisms. In this study, we analyzed transgenic Arabidopsis (Arabidopsis thaliana) plants that overexpress NAD kinase2 (NADK2), an enzyme that catalyzes the synthesis of NADP from NAD in chloroplasts, to investigate the impacts of altering NADP level on plant metabolism. Metabolite profiling revealed that NADP(H) concentrations were proportional to NADK activity in NADK2 overexpressors and in the nadk2 mutant. Several metabolites associated with the Calvin cycle were also higher in the overexpressors, accompanied by an increase in overall Rubisco activity. Furthermore, enhanced NADP(H) production due to NADK2 overexpression increased nitrogen assimilation. Glutamine and glutamate concentrations, as well as some other amino acids, were higher in the overexpressors. These results indicate that overexpression of NADK2 either directly or indirectly stimulates carbon and nitrogen assimilation in Arabidopsis under restricted conditions. Importantly, since neither up-regulation nor down-regulation of NADK2 activity affected the sum amount of NAD and NADP or the redox state, the absolute level of NADP and/or the NADP/NAD ratio likely plays a key role in regulating plant metabolism.

sll1961 is a novel regulator of phycobilisome degradation during nitrogen starvation in the cyanobacterium Synechocystis sp. PCC 6803.

The sll1961 gene was reported to encode a regulatory factor of photosystem stoichiometry in the cyanobacterium Synechocystis sp. PCC 6803. We here show that the sll1961 gene is also essential for the phycobilisome degradation during nitrogen starvation. The defect in phycobilisome degradation was observed in the sll1961 mutant despite the increased expression of nblA, a gene involved in phycobilisome degradation during nitrogen starvation. Photosystem stoichiometry is not affected by nitrogen starvation in the sll1961 mutant nor in the wild-type. The results indicate the presence of a novel pathway for phycobilisome degradation control independent of nblA expression.

Distinct modulations of the hexokinase1-mediated glucose response and hexokinase1-independent processes by HYS1/CPR5 in Arabidopsis.

The Arabidopsis mutant hypersenescence 1 (hys1), that is allelic to constitutive expresser of pathogenesis-related genes 5 (cpr5), displays phenotypes related to glucose signalling and defence responses. In the present study, it is shown that the hys1 mutation boosts the inhibitory effects of glucose upon the greening of seedlings and reduces the antagonistic activities of ethylene and cytokinin toward this inhibition. Neither the glucose content nor the sensitivities to ethylene, cytokinin, and abscisic acid were found to differ between wild-type and hys1 seedlings. However, disruption of the gene encoding hexokinase1 (HXK1), which acts as a glucose sensor, partially suppressed the glucose hypersensitive phenotype of the hys1 mutant. These results thus suggest that the hys1 mutation promotes a process associated with the HXK1-mediated glucose response during greening. By contrast, additional hys1 phenotypes, including an increase in salicylic acid (SA), production of abnormal trichomes, and early senescence, were not suppressed by the loss of HXK1. Surprisingly, the hxk1 and hys1 mutations acted synergistically towards an increased SA accumulation. Hence, HYS1/CPR5 appears to be a versatile protein that modulates both the HXK1-mediated glucose response and various HXK1-indepndent processes that are involved in growth control. A possible role for HYS1/CPR5 as a component of the networks that regulate growth control is discussed.

The mutant of sll1961, which encodes a putative transcriptional regulator, has a defect in regulation of photosystem stoichiometry in the cyanobacterium Synechocystis sp. PCC 6803.

In acclimation to changing light environments, photosynthetic organisms modulate the ratio of two photosynthetic reaction centers (photosystem I [PSI] and photosystem II). One mutant, which could not modulate photosystem stoichiometry upon the shift to high light, was isolated from mutants created by random transposon mutagenesis. Measurements of chlorophyll fluorescence and analysis of the reaction center subunits of PSI through western blotting in this mutant revealed that the content of PSI could not be suppressed under high-light condition. In the mutant, transposon was inserted to the sll1961 gene encoding a putative transcriptional regulator. DNA microarray analysis revealed that the expression of sll1773 was drastically induced in the sll1961 mutant upon exposure to high light for 3 h. Our results demonstrate that a transcriptional regulator, Sll1961, and its possible target proteins, including Sll1773, may be responsible for the regulation of photosystem stoichiometry in response to high light.

PsaK2 subunit in photosystem I is involved in state transition under high light condition in the cyanobacterium Synechocystis sp. PCC 6803.

To avoid the photodamage, cyanobacteria regulate the distribution of light energy absorbed by phycobilisome antenna either to photosystem II or to photosystem I (PSI) upon high light acclimation by the process so-called state transition. We found that an alternative PSI subunit, PsaK2 (sll0629 gene product), is involved in this process in the cyanobacterium Synechocystis sp. PCC 6803. An examination of the subunit composition of the purified PSI reaction center complexes revealed that PsaK2 subunit was absent in the PSI complexes under low light condition, but was incorporated into the complexes during acclimation to high light. The growth of the psaK2 mutant on solid medium was inhibited under high light condition. We determined the photosynthetic characteristics of the wild type strain and the two mutants, the psaK1 (ssr0390) mutant and the psaK2 mutant, using pulse amplitude modulation fluorometer. Non-photochemical quenching, which reflects the energy transfer from phycobilisome to PSI in cyanobacteria, was higher in high light grown cells than in low light grown cells, both in the wild type and the psaK1 mutant. However, this change of non-photochemical quenching during acclimation to high light was not observed in the psaK2 mutant. Thus, PsaK2 subunit is involved in the energy transfer from phycobilisome to PSI under high light condition. The role of PsaK2 in state transition under high light condition was also confirmed by chlorophyll fluorescence emission spectra determined at 77 K. The results suggest that PsaK2-dependent state transition is essential for the growth of this cyanobacterium under high light condition.

Physiological roles of free D- and L-alanine in the crayfish Procambarus clarkii with special reference to osmotic and anoxic stress responses.

Under hyper-salinity stress from freshwater to 17 and 25 ppt seawater, red swamp crayfish Procambarus clarkii largely accumulated D- and L-alanine together with glycine, L-glutamine, and L-proline in both muscle and hepatopancreas. The increases of D- and L-alanine in muscle were the highest in all amino acids and reached 6.8- and 5.4-fold, respectively, from freshwater to 25 ppt seawater. These results indicate that both D- and L-alanine are the most potent osmolytes for intracellular isosmotic regulation in crayfish as well as other crustaceans thus far examined. Under anoxia stress below 0.1 mg/l dissolved oxygen for 12 h and subsequent recovery in normoxia for 12 h in freshwater, 17 and 25 ppt seawater, muscle ATP decreased dramatically in all salinity levels and almost depleted in seawater. Along with the decrease of muscle glycogen level, the significant increase of L-lactate was found in muscle, hepatopancreas, and hemolymph for each salinity level, suggesting the transport of L-lactate from muscle into hepatopancreas via hemolymph. Under anoxia, D- and L-alanine also largely increased in both muscle and hepatopancreas for each salinity level. The increase was much higher in seawater than in freshwater. Thus, both D- and L-alanine are possible to be anaerobic end products during prolonged anaerobiosis of this species.