Citrate Accumulation Research Articles

Reprogramming mitochondrial metabolism in macrophages as an anti-inflammatory signal.

Mitochondria are master regulators of metabolism. Mitochondria generate ATP by oxidative phosphorylation using pyruvate (derived from glucose and glycolysis) and fatty acids (FAs), both of which are oxidized in the Krebs cycle, as fuel sources. Mitochondria are also an important source of reactive oxygen species (ROS), creating oxidative stress in various contexts, including in the response to bacterial infection. Recently, complex changes in mitochondrial metabolism have been characterized in mouse macrophages in response to varying stimuli in vitro. In LPS and IFN-γ-activated macrophages (M1 macrophages), there is decreased respiration and a broken Krebs cycle, leading to accumulation of succinate and citrate, which act as signals to alter immune function. In IL-4-activated macrophages (M2 macrophages), the Krebs cycle and oxidative phosphorylation are intact and fatty acid oxidation (FAO) is also utilized. These metabolic alterations in response to the nature of the stimulus are proving to be determinants of the effector functions of M1 and M2 macrophages. Furthermore, reprogramming of macrophages from M1 to M2 can be achieved by targeting metabolic events. Here, we describe the role that metabolism plays in macrophage function in infection and immunity, and propose that reprogramming with metabolic inhibitors might be a novel therapeutic approach for the treatment of inflammatory diseases.

Implication of citrate, malate and histidine in the accumulation and transport of nickel in Mesembryanthemum crystallinum and Brassica juncea

Citrate, malate and histidine have been involved in many processes including metal tolerance and accumulation in plants. These molecules have been frequently reported to be the potential nickel chelators, which most likely facilitate metal transport through xylem. In this context, we assess here, the relationship between organics acids and histidine content and nickel accumulation in Mesembryanthemum crystallinum and Brassica juncea grown in hydroponic media added with 25, 50 and 100µM NiCl2. Results showed that M. crystallinum is relatively more tolerant to Ni toxicity than B. juncea. For both species, xylem transport rate of Ni increased with increasing Ni supply. A positive correlation was established between nickel and citrate concentrations in the xylem sap. In the shoot of B. juncea, citric and malic acids concentrations were significantly higher than in the shoot of M. crystallinum. Also, the shoots and roots of B. juncea accumulated much more histidine. In contrast, a higher root citrate concentration was observed in M. crystallinum. These findings suggest a specific involvement of malic and citric acid in Ni translocation and accumulation in M. crystallinum and B. juncea. The high citrate and histidine accumulation especially at 100µM NiCl2, in the roots of M. crystallinum might be among the important factors associated with the tolerance of this halophyte to toxic Ni levels.

Effects of Thioctic Acid on the Oxidative Status Tissues in Rheumatoid Arthritis in Rats

Biochemiluminescence, aconitate hydratase activity, and citrate contents were studied in rat serum and muscle in normal animals and in experimental rheumatoid arthritis; the actions of thioctic acid on the background of the development of this pathology were also studied. The development of rheumatoid arthritis in rats was accompanied by increases in marker values and measures of biochemiluminescence, as well as decreases in aconitate hydratase activity, evidently due to the extreme generation of reactive oxygen species associated with the induction of pathology. Suppression of the activity of this enzyme was linked with the accumulation of citrate in the tissues of the experimental rats. Administration of thioctic acid to rats with rheumatoid arthritis was accompanied by changes in values towards the normal, which was evidently linked with the antioxidant action of this compound.

Optimizing citrate dose for regional anticoagulation in continuous renal replacement therapy: measuring citrate concentrations instead of ionized calcium?

Regular measurement of systemic and post-filter ionized calcium (iCa) concentrations is imperative to correctly handle regional citrate anticoagulation dose during continuous renal replacement therapy (CRRT). Keeping post-filter iCa within a tight range guarantees optimal circuit function and enhances filter life span [1, 2], whereas a decrease in plasma iCa, with subsequent elevation of the total-to-ionized plasma calcium ratio, can predict systemic citrate accumulation [3]. The new findings (published recently in Critical Care) of Schwarzer et al. expose an alarming inaccuracy for measuring post-filter iCa with currently available blood gas analyzers [4]. This precludes adequate control of citrate flow and raises evident functional and safety issues. On the other hand, Schwarzer et al. found good concordance between all evaluated analyzers for measuring systemic iCa levels [4]. However, the total-to-ionized plasma calcium ratio has occasionally been shown to be a relatively weak indirect marker for citrate accumulation or intoxication [1, 2]. Direct measurement of citrate systemic concentrations could overcome these iCa-related shortcomings. In this perspective, compelling evidence was provided by Italian investigators who adapted a commercially available citrate analyzing kit for measuring systemic and also filter citrate concentrations [5]. Preliminary experience in septic shock patients with liver dysfunction undergoing CRRT suggested a potential clinical use but needs confirmation in a larger and more heterogeneous patient population.

Citrate anticoagulation for CRRT: don't always trust the postfilter iCa results!

Citrate has been recommended as the first-line anticoagulant for continuous renal replacement therapy (CRRT) in critically ill patients. Compared with heparin, citrate anticoagulation is safer and more efficacious. Citrate inhibits the coagulation cascade by lowering the ionized calcium (iCa) concentration in the filter. Monitoring of systemic iCa concentrations is inherent to the protocol, and monitoring of postfilter iCa is recommended to adjust citrate flow and optimize anticoagulation. While systemic iCa targets are in the physiological range, postfilter iCa concentrations are targeted between 0.20 and 0.35 mmol/l. In a previous issue of Critical Care, Schwarzer et al. compared systemic and postfilter iCa measurements of patients receiving citrate-based CRRT between six devices. They highlight the unreliability of iCa concentrations in the postfilter range, because the instruments cannot be validated in the low iCa range. The maximum mean difference between two instruments was as high as 0.33 mmol/l (range 0.21–0.50 mmol/l). The authors call for dialysis companies to revise their protocols. However, the first implication of their study is that the accuracy of blood gas analyzers to measure iCa in the low range needs to improve; and, secondly, clinicians using citrate anticoagulation need to be aware that the postfilter iCa result may be falsely high or low. This is particularly relevant when frequent premature filter clotting is observed despite postfilter iCa results in the seemingly target range. In these situations, citrate flow can be safely increased up to 4 mmol/l blood flow under monitoring of signs of citrate accumulation.

High-intensity sprint training inhibits mitochondrial respiration through aconitase inactivation.

Intense exercise training is a powerful stimulus that activates mitochondrial biogenesis pathways and thus increases mitochondrial density and oxidative capacity. Moderate levels of reactive oxygen species (ROS) during exercise are considered vital in the adaptive response, but high ROS production is a serious threat to cellular homeostasis. Although biochemical markers of the transition from adaptive to maladaptive ROS stress are lacking, it is likely mediated by redox sensitive enzymes involved in oxidative metabolism. One potential enzyme mediating such redox sensitivity is the citric acid cycle enzyme aconitase. In this study, we examined biopsy specimens of vastus lateralis and triceps brachii in healthy volunteers, together with primary human myotubes. An intense exercise regimen inactivated aconitase by 55-72%, resulting in inhibition of mitochondrial respiration by 50-65%. In the vastus, the mitochondrial dysfunction was compensated for by a 15-72% increase in mitochondrial proteins, whereas H2O2 emission was unchanged. In parallel with the inactivation of aconitase, the intermediary metabolite citrate accumulated and played an integral part in cellular protection against oxidative stress. In contrast, the triceps failed to increase mitochondrial density, and citrate did not accumulate. Instead, mitochondrial H2O2 emission was decreased to 40% of the pretraining levels, together with a 6-fold increase in protein abundance of catalase. In this study, a novel mitochondrial stress response was highlighted where accumulation of citrate acted to preserve the redox status of the cell during periods of intense exercise.

Impairment of energy metabolism in cardiomyocytes caused by 5-FU catabolites can be compensated by administration of amino acids.

Identification of patients with increased risk of 5-fluorouracil (5-FU)-related toxicity is an important challenge for cancer treatment. Research often focus on dihydropyrimidine dehydrogenase (DPYD) deficiency in this context. However, patients with normal DPYD activity may also develop life-threatening 5-FU adverse effects. DPYD initiates the catabolic route of 5-FU generating metabolites such as fluoroacetate (FAC). The catabolite FAC is known to inhibit the TCA cycle enzyme aconitase, which is supposed to impair mitochondrial energy metabolism. Therefore, we aim for a systems understanding of the association of 5-FU-related cardiac side effects with aconitase inhibition caused by FAC. Using a mitochondrial model of cardiomyocytes we found strong depletion of ATP production and citrate accumulation as main effects of aconitase inhibition. Shadow price analysis revealed that the uptakes of valine, arginine, proline and glutamate are most effective in compensating the impairment of energy metabolism. Our findings suggest that 5-FU catabolism contributes to the occurrence of cardiac adverse effects and are the basis for further biomarker identifications and development of side effect treatment.

Gel-filtration analysis to detect Al-binding ligands in Eucalyptus camaldulensis

Aluminum (Al) toxicity is a major factor limiting plant growth on acid soils; inhibited root elongation is a characteristic symptom. To elucidate mechanisms of Al resistance in plants, we developed a simple method to separate Al-binding ligands from plant roots by gel-filtration chromatography. In this method, an eluent consisting of Homopipes buffer flows through a Sephadex G-25 column. Free Al ions do not pass through the column, but Al-ligand complexes can be fractionated according to differences in their molecular sizes. We applied the method to Eucalyptus camaldulensis (Myrtaceae) and successfully separated Al-binding ligands from roots. One peak of Al-ligand complex increased with Al treatment of seedlings, which can be explained by Al-induced accumulation of citrate in roots.

Sustained low-efficiency dialysis with regional citrate anticoagulation in medical intensive care unit patients with liver failure: A prospective study

Patients with liver failure requiring dialysis are at increased risk for citrate accumulation during sustained low-efficiency dialysis (SLED). The aim of this study was to evaluate the feasibilty of citrate SLED in critical ill patients with liver failure and investigate predictive parameters regarding citrate accumulation. This is a prospective study in 24 medical intensive care unit patients with liver failure and a total of 43 SLED runs (maximum of 3 runs per patient) using citrate anticoagulation. Liver function was characterized before SLED using not only laboratory parameters but also determination of the plasma disappearance rate of indocyanine green. In addition, blood gas parameters as well total calcium and citrate in serum were measured at baseline and defined time points during SLED. Accumulation of citrate could be observed in all SLED runs, which were nearly normalized until the end of SLED and 24 hours after SLED, respectively. However, the critical threshold of total calcium/ionized calcium on ratio of greater than 2.5 was exceeded in only 1 patient. Equalization of initial metabolic acidosis was possible without major disturbances of acid base and electrolyte status. Liver function parameters showed poor predicitve capabilities regarding citrate accumulation. Despite substantial accumulation of citrate in serum, SLED is save and feasible in patients with liver failure using a citrate anticoagulation. Careful monitoring of electrolytes and acid base status is mandatory to ensure patient safety.

A Process-Based Model of TCA Cycle Functioning to Analyze Citrate Accumulation in Pre- and Post-Harvest Fruits.

Citrate is one of the most important organic acids in many fruits and its concentration plays a critical role in organoleptic properties. The regulation of citrate accumulation throughout fruit development, and the origins of the phenotypic variability of the citrate concentration within fruit species remain to be clarified. In the present study, we developed a process-based model of citrate accumulation based on a simplified representation of the TCA cycle to predict citrate concentration in fruit pulp during the pre- and post-harvest stages. Banana fruit was taken as a reference because it has the particularity of having post-harvest ripening, during which citrate concentration undergoes substantial changes. The model was calibrated and validated on the two stages, using data sets from three contrasting cultivars in terms of citrate accumulation, and incorporated different fruit load, potassium supply, and harvest dates. The model predicted the pre and post-harvest dynamics of citrate concentration with fairly good accuracy for the three cultivars. The model suggested major differences in TCA cycle functioning among cultivars during post-harvest ripening of banana, and pointed to a potential role for NAD-malic enzyme and mitochondrial malate carriers in the genotypic variability of citrate concentration. The sensitivity of citrate accumulation to growth parameters and temperature differed among cultivars during post-harvest ripening. Finally, the model can be used as a conceptual basis to study citrate accumulation in fleshy fruits and may be a powerful tool to improve our understanding of fruit acidity.

Dichloroacetate restores drug sensitivity in paclitaxel-resistant cells by inducing citric acid accumulation.

BackgroundThe Warburg effect describes the increased reliance of tumor cells on glycolysis for ATP generation. Mitochondrial respiratory defect is thought to be an important factor leading to the Warburg effect in some types of tumor cells. Consequently, there is growing interest in developing anti-cancer drugs that target mitochondria. One example is dichloroacetate (DCA) that stimulates mitochondria through inhibition of pyruvate dehydrogenase kinase.MethodsWe investigated the anti-cancer activity of DCA using biochemical and isotopic tracing methods.ResultsWe observed that paclitaxel-resistant cells contained decreased levels of citric acid and sustained mitochondrial respiratory defect. DCA specifically acted on cells with mitochondrial respiratory defect to reverse paclitaxel resistance. DCA could not effectively activate oxidative respiration in drug-resistant cells, but induced higher levels of citrate accumulation, which led to inhibition of glycolysis and inactivation of P-glycoprotein.ConclusionsThe abilityof DCA to target cells with mitochondrial respiratory defect and restore paclitaxel sensitivity by inducing citrate accumulation supports further preclinical development.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0331-3) contains supplementary material, which is available to authorized users.

Citrus PH5-like H(+)-ATPase genes: identification and transcript analysis to investigate their possible relationship with citrate accumulation in fruits.

PH5 is a petunia gene that encodes a plasma membrane H+-ATPase and determines the vacuolar pH. The citrate content of fruit cell vacuoles influences citrus organoleptic qualities. Although citrus could have PH5-like homologs that are involved in citrate accumulation, the details are still unknown. In this study, extensive data-mining with the PH5 sequence and PCR amplification confirmed that there are at least eight PH5-like genes (CsPH1-8) in the citrus genome. CsPHs have a molecular mass of approximately 100 kDa, and they have high similarity to PhPH5, AtAHA10 or AtAHA2 (from 64.6 to 80.9%). They contain 13–21 exons and 12–20 introns and were evenly distributed into four subgroups of the P3A-subfamily (CsPH1, CsPH2, and CsPH3 in Group I, CsPH4 and CsPH5 in Group II, CsPH6 in Group IV, and CsPH7 and CsPH8 in Group III together with PhPH5). A transcript analysis showed that CsPH1, 3, and 4 were predominantly expressed in mature leaves, whereas CsPH2 and 7 were predominantly expressed in roots, CsPH5 and 6 were predominantly expressed in flowers, and CsPH8 was predominantly expressed in fruit juice sacs (JS). Moreover, the CsPH transcript profiles differed between orange and pummelo, as well as between high-acid and low-acid cultivars. The low-acid orange “Honganliu” exhibits low transcript levels of CsPH3, CsPH4, CsPH5, and CsPH8, whereas the acid-free pummelo (AFP) has only a low transcript level of CsPH8. In addition, ABA injection increased the citrate content significantly, which was accompanied by the obvious induction of CsPH2, 6, 7, and 8 transcript levels. Taken together, we suggest that CsPH8 seems likely to regulate citrate accumulation in the citrus fruit vacuole.

Participation of citric acid and isocitric acid in the diurnal cycle of carboxylation and decarboxylation in the common ice plant

In this paper we analyzed changes in the concentration of the three carboxylic acids (malic acid, citric acid and isocitric acid) during the salinity-induced C3–CAM shift in Mesembryanthemum crystallinum. We documented that a nocturnal accumulation of citrate preceded that of malate, and that the CAM-dependent increase in citric acid was about 100–times higher than that of malic acid. The induction of CAM was accompanied by an increased activity of cytosolic NADP-dependent isocitrate dehydrogenase (NADP-ICDH, E.C 1.1.1.42) and by decreased activities of mitochondrial enzymes: fumarase (E.C 4.2.1.2) and NAD-dependent ICDH (E.C 1.1.1.41). Moreover, the activity of citrate synthase (E.C 2.3.3.1) was increased during the nocturnal phase of CAM. Altogether, obtained data suggest that some part of the product of a primary carboxylation (oxaloacetic acid) may pass through the mitochondria at night for reduction and, in form of citrate and isocitrate, may leave the mitochondria to support a daytime decarboxylation.

Bright luminescence of Vibrio fischeri aconitase mutants reveals a connection between citrate and the Gac/Csr regulatory system.

The Gac/Csr regulatory system is conserved throughout the γ-proteobacteria and controls key pathways in central carbon metabolism, quorum sensing, biofilm formation and virulence in important plant and animal pathogens. Here we show that elevated intracellular citrate levels in a Vibrio fischeri aconitase mutant correlate with activation of the Gac/Csr cascade and induction of bright luminescence. Spontaneous or directed mutations in the gene that encodes citrate synthase reversed the bright luminescence of aconitase mutants, eliminated their citrate accumulation and reversed their elevated expression of CsrB. Our data elucidate a correlative link between central metabolic and regulatory pathways, and they suggest that the Gac system senses a blockage at the aconitase step of the tricarboxylic acid cycle, either through elevated citrate levels or a secondary metabolic effect of citrate accumulation, and responds by modulating carbon flow and various functions associated with host colonization, including bioluminescence.

Novel Nanomolar Potency Mitochondrial Complex I Inhibitor Iacs-1131 Selectively Kills Oxphos-Dependent AML Cells

Recent studies indicate that acute myeloid leukemia (AML) cells, including leukemia-initiating cells, are highly dependent on oxidative phosphorylation (OXPHOS) for survival, while normal hematopoietic stem cells predominantly utilize glycolysis for energy homeostasis. We have reported development of a series of novel, highly potent mitochondrial complex I inhibitors, which in vitro inhibit complex I with IC50 values <10 nM (Marszalek et al., AACR 2014 Abstract #949). These inhibitors offer excellent therapeutic potential in the OXPHOS-dependent cancer models. IACS-1131 was selected as a preclinical tool compound from the series of more than 800 compounds across distinct structural classes. Here, we report the in vitro and in vivo efficacy of IACS-1131 in AML models.Analysis of a panel of AML cell lines showed that a subset of leukemias are markedly dependent on OXPHOS for growth and survival; in this subset, IACS-1131 treatment caused steep decreases in viable cell number via induction of apoptosis. In sensitive cell lines (HL-60, OCI-AML3, KG-1, MV4;11, Kasumi-1), IACS-1131 induced pronounced apoptosis with EC50 between 10 and 100nM, consistent with the IC50 required to inhibit OXPHOS. MOLM13 and OCI-AML2 cells were less sensitive (EC50 250nM and 120nM, and a failure to induce cell death). In primary AML samples from patients with newly diagnosed or relapsed/refractory AML (n=12), the average EC50 for IACS-1131 was 14 ± 8nM in 9 samples, and exceeded 100nM in 3 samples. Consistent with the findings in AML cell lines, 10nM IACS-1131 resulted in partial responses, and 100-250nM resulted in profound loss of viability due to apoptosis induction. In contrast, this treatment caused only a moderate decrease in CD34+ cell numbers and <10% increase in apoptosis in 6 normal bone marrow samples.The effects of IACS-1131 on the two major energy-generating pathways, mitochondrial OXPHOS and glycolysis, were investigated using the Seahorse Bioscience XF96 Analyzer. Treatment for 16 hrs caused a striking dose-dependent decrease in basal oxygen consumption rates (OCR), indicating OXPHOS inhibition; reduced ATP production; and decreased maximal respiratory capacity in OCI-AML3 cells and in primary AML blasts (n=9). We confirmed inhibition of complex I in AML cells using Seahorse mitochondrial electron flow assay. These changes preceded changes in viability or apoptotic markers; as such, loss of the mitochondrial membrane potential, annexin V positivity, and induction of mitochondrial reactive oxygen species were seen only at 72 hrs of exposure. Further time-course analysis demonstrated that 2 hrs of IACS-1131 exposure caused significant inhibition of OCR in both sensitive OCI-AML3 and resistant MOLM13 cells, but in MOLM13 cells there was a greater increase in extracellular acidification rates, suggesting compensation by glycolysis. In turn, inhibition of glycolysis with 2-DG, or blockade of pyruvate dehydrogenase kinase with dicholoroacetate (which forces entry into the TCA cycle) sensitized resistant cells to IACS-1131. The intracellular metabolome (polar fraction) of OCI-AML3 cells was characterized following 2, 4, 12 and 24 hrs of treatment with 100nm IACS-1131 using high-resolution magnetic resonance spectroscopy and high mass accuracy Orbitrap mass spectrometry. IACS-1131 modulated levels of the TCA intermediates, producing increased accumulation of citrate and fumarate and decreased succinate and malate, and increased glutathione, possibly because of the oxidative stress. Furthermore, the metabolic analysis indicated a strong effect on amino acid metabolism, whereby IACS-1131 reduced (between 25% and 62% of control) multiple anaplerotic amino acids (including arginine, leucine, isoleucine, valine, phenylalanine, asparagine, histidine, and glutamine, but not aspartate).Finally, IACS-1131 at 60 mg/kg QD po demonstrated robust anti-leukemia activity in an orthotopic OCI-AML3 model. At this dose, IACS-1131 was well tolerated for >50 days and increased median survival duration by more than 5 times (Fig. 1). Studies exploring the anti-AML efficacy of single-agent IACS-1131 in primary AML xenografts are ongoing.Taken together, these data strongly indicate that OXPHOS inhibition constitutes a novel therapeutic approach that targets a unique metabolic vulnerability of AML cells and indicate that further preclinical evaluation of OXPHOS inhibitors is warranted. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Intoxicación por fluoroacetato de sodio

Sodium fluoroacetate is known as compound 1080; it was discovered by the German chemicals during World War II. It is a compound soluble in water, odorless and tasteless. The lethal dose 50 in humans is 2 to 4 mg/kg. Because of its lethality, it has been recalled in several countries including Colombia. This compound causes biochemical and physiological effects as citrate accumulation, altered mitochondrial Citrate transporter, the accumulation of lactic acid, impaired glucose regulation and hidroelectrolyte disorders (mainly calcium disturbances). The clinical manifestations of poisoning by this substance are non-specific, which males their identificacion and treatment difficult, making it to increase its lethality. Ethanol increases the level of acetat, thus providing an alternative substrate Krebs cycle and may offer benefits in the acute treatment of these patients; however, management support is the mainstay of management of these cases. Here it is reported a case of intentional ingestion in an adolescent, with delayed diagnosis a successful outcome.

The regulatory network of cluster‐root function and development in phosphate‐deficient white lupin (Lupinus albus) identified by transcriptome sequencing

Lupinus albus serves as model plant for root-induced mobilization of sparingly soluble soil phosphates via the formation of cluster-roots (CRs) that mediate secretion of protons, citrate, phenolics and acid phosphatases (APases). This study employed next-generation sequencing to investigate the molecular mechanisms behind these complex adaptive responses at the transcriptome level. We compared different stages of CR development, including pre-emergent (PE), juvenile (JU) and the mature (MA) stages. The results confirmed that the primary metabolism underwent significant modifications during CR maturation, promoting the biosynthesis of organic acids, as had been deduced from physiological studies. Citrate catabolism was downregulated, associated with citrate accumulation in MA clusters. Upregulation of the phenylpropanoid pathway reflected the accumulation of phenolics. Specific transcript expression of ALMT and MATE transporter genes correlated with the exudation of citrate and flavonoids. The expression of transcripts related to nucleotide degradation and APases in MA clusters coincided with the re-mobilization and hydrolysis of organic phosphate resources. Most interestingly, hormone-related gene expression suggested a central role of ethylene during CR maturation. This was associated with the upregulation of the iron (Fe)-deficiency regulated network that mediates ethylene-induced expression of Fe-deficiency responses in other species. Finally, transcripts related to abscisic acid and jasmonic acid were upregulated in MA clusters, while auxin- and brassinosteroid-related genes and cytokinin receptors were most strongly expressed during CR initiation. Key regulations proposed by the RNA-seq data were confirmed by quantitative real-time polymerase chain reaction (RT-qPCR) and some physiological analyses. A model for the gene network regulating CR development and function is presented.

Accumulation of low-molecular-weight organic acids in roots and leaf segments of Zea mays plants treated with cadmium and copper

Maize seedlings grown in a nutrient solution were treated with Cd (50, 100 µM) or Cu (50, 100 µM). Roots and separated leaf sections (from the youngest––basal, through the middle––mature, to the oldest––apical) were analysed. Shoot and root fresh weight, and root net growth rates were reduced significantly after Cu application in comparison to Cd. Copper (50, 100 µM) and cadmium (100 µM) decreased the sum of chlorophyll and total carotenoid pools mostly in mature and old leaf sections. The concentration of Cu and Cd increased in the old and mature leaf sections. Analysis of organic acids showed that both metals differently influenced the low-molecular-weight organic acids (LMWOAs) content in maize leaf sections. In mature sections, the excess Cu increased the succinate and tartrate contents, whereas in the young ones mainly the tartrate level was elevated. Cadmium elevated citrate accumulation in mature and old sections, compared to the control plants. Malate, the main LMWOA in maize leaves, increased only after addition of 100 µM of Cd (mature and old sections) or 50 µM of Cu (old sections). Analysis of LMWOAs in roots showed that the excess of Cd or Cu induced higher accumulation of tartrate and malate and, additionally, copper increased the citrate content.

Combined D2‐/L2‐hydroxyglutaric aciduria (SLC25A1 deficiency): clinical course and effects of citrate treatment

Combined D,L-2-hydroxyglutaric aciduria (DL-2HGA; OMIM #615182) is a rare neurometabolic disorder clinically characterized by muscular hypotonia, severe neurodevelopmental dysfunction, and intractable seizures associated with respiratory distress. Biochemically, DL-2HGA patients excrete increased amounts of D- and L-2-hydroxyglutarate (D2HG and L2HG, respectively), with predominance of D2HG, and α-ketoglutarate, and show a decrease in urinary citrate. Impaired function of the mitochondrial citrate carrier (CIC) due to pathogenic mutations within the SLC25A1 gene has been identified as the underlying molecular cause of the disease. CIC mediates efflux of the mitochondrial tricarboxylic acid (TCA) cycle intermediates citrate and isocitrate in exchange for cytosolic malate. Thus, depletion of cytosolic citrate as well as accumulation of citrate inside mitochondria have been considered to play a role in the pathophysiology of DL-2HGA. Here, we report for the first time on a patient with a genetically confirmed diagnosis of DL-2HGA and treatment with either malate or citrate. During malate treatment, urinary malate concentration increased, but beyond that, neither biochemical nor clinical alterations were observed. In contrast, treatment with citrate led to an increased urinary excretion of TCA cycle intermediates malate and succinate, and by trend to an increased concentration of urinary citrate. Furthermore, excretion of D2HG and L2HG was reduced during citrate treatment. Clinically, the patient showed stabilization with regard to frequency and severity of seizures. Treating DL-2HGA with citrate should be considered in other DL-2HGA patients, and its effects should be studied systematically.

Investigation of fatty acid accumulation in the engineered Saccharomyces cerevisiae under nitrogen limited culture condition

In this study, the Saccharomyces cerevisiae wild type strain and engineered strain with an overexpressed heterologous ATP-citrate lyase (acl) were cultured in medium with different carbon and nitrogen concentrations, and their fatty acid production levels were investigated. The results showed that when the S. cerevisiae engineered strain was cultivated under nitrogen limited culture condition, the yield of mono-unsaturated fatty acids showed higher than that under non-nitrogen limited condition; with the carbon concentration increased, the accumulation become more apparent, whereas in the wild type strain, no such correlation was found. Besides, the citrate level in the S. cerevisiae under nitrogen limited condition was found to be much higher than that under non-nitrogen limited condition, which indicated a relationship between the diminution of nitrogen and accumulation of citrate in the S. cerevisiae. The accumulated citrate could be further cleaved by acl to provide substrate for fatty acid synthesis.