Deletion of the gene encoding G0/G 1 switch protein 2 (G0s2) alleviates high-fat-diet-induced weight gain and insulin resistance, and promotes browning of white adipose tissue in mice.

AIMS/HYPOTHESIS: Obesity is a global epidemic resulting from increased energy intake, which alters energy homeostasis and results in an imbalance in fat storage and breakdown. G0/G1 switch gene 2 (G0s2) has been recently characterised in vitro as an inhibitor of adipose triglyceride lipase (ATGL), the rate-limiting step in fat catabolism. In the current study we aim to functionally characterise G0s2 within the physiological context of a mouse model. METHODS: We generated a mouse model in which G0s2 was deleted. The homozygous G0s2 knockout (G0s2 (-/-)) mice were studied over a period of 22 weeks. Metabolic variables were measured including body weight and body composition, food intake, glucose and insulin tolerance tests, energy metabolism and thermogenesis. RESULTS: We report that G0s2 inhibits ATGL and regulates lipolysis and energy metabolism in vivo. G0s2 (-/-) mice are lean, resistant to weight gain induced by a high-fat diet and are glucose tolerant and insulin sensitive. The white adipose tissue of G0s2 (-/-) mice has enhanced lipase activity and adipocytes showed enhanced stimulated lipolysis. Energy metabolism in the G0s2 (-/-) mice is shifted towards enhanced lipid metabolism and increased thermogenesis. G0s2 (-/-) mice showed enhanced cold tolerance and increased expression of thermoregulatory and oxidation genes within white adipose tissue, suggesting enhanced 'browning' of the white adipose tissue. CONCLUSIONS/INTERPRETATION: Our data show that G0s2 is a physiological regulator of adiposity and energy metabolism and is a potential target in the treatment of obesity and insulin resistance.

Loss of the tumor suppressor LKB1 promotes metabolic reprogramming of cancer cells via HIF-1α.

One of the major metabolic changes associated with cellular transformation is enhanced nutrient utilization, which supports tumor progression by fueling both energy production and providing biosynthetic intermediates for growth. The liver kinase B1 (LKB1) is a serine/threonine kinase and tumor suppressor that couples bioenergetics to cell-growth control through regulation of mammalian target of rapamycin (mTOR) activity; however, the influence of LKB1 on tumor metabolism is not well defined. Here, we show that loss of LKB1 induces a progrowth metabolic program in proliferating cells. Cells lacking LKB1 display increased glucose and glutamine uptake and utilization, which support both cellular ATP levels and increased macromolecular biosynthesis. This LKB1-dependent reprogramming of cell metabolism is dependent on the hypoxia-inducible factor-1α (HIF-1α), which accumulates under normoxia in LKB1-deficient cells and is antagonized by inhibition of mTOR complex I signaling. Silencing HIF-1α reverses the metabolic advantages conferred by reduced LKB1 signaling and impairs the growth and survival of LKB1-deficient tumor cells under low-nutrient conditions. Together, our data implicate the tumor suppressor LKB1 as a central regulator of tumor metabolism and growth control through the regulation of HIF-1α-dependent metabolic reprogramming.

G-protein-coupled receptor 91 and succinate are key contributors in neonatal postcerebral hypoxia-ischemia recovery.

OBJECTIVE: Prompt post-hypoxia-ischemia (HI) revascularization has been suggested to improve outcome in adults and newborn subjects. Other than hypoxia-inducible factor, sensors of metabolic demand remain largely unknown. During HI, anaerobic respiration is arrested resulting in accumulation of carbohydrate metabolic intermediates. As such succinate readily increases, exerting its biological effects via a specific receptor, G-protein-coupled receptor (GPR) 91. We postulate that succinate/GPR91 enhances post-HI vascularization and reduces infarct size in a model of newborn HI brain injury. APPROACH AND RESULTS: The Rice-Vannucci model of neonatal HI was used. Succinate was measured by mass spectrometry, and microvascular density was evaluated by quantification of lectin-stained cryosection. Gene expression was evaluated by real-time polymerase chain reaction. Succinate levels rapidly increased in the penumbral region of brain infarcts. GPR91 was foremost localized not only in neurons but also in astrocytes. Microvascular density increased at 96 hours after injury in wild-type animals; it was diminished in GPR91-null mice leading to an increased infarct size. Stimulation with succinate led to an increase in growth factors implicated in angiogenesis only in wild-type mice. To explain the mode of action of succinate/GPR91, we investigated the role of prostaglandin E2-prostaglandin E receptor 4, previously proposed in neural angiogenesis. Succinate-induced vascular endothelial growth factor expression was abrogated by a cyclooxygenase inhibitor and a selective prostaglandin E receptor 4 antagonist. This antagonist also abolished succinate-induced neovascularization. CONCLUSIONS: We uncover a dominant metabolic sensor responsible for post-HI neurovascular adaptation, notably succinate/GPR91, acting via prostaglandin E2-prostaglandin E receptor 4 to govern expression of major angiogenic factors. We propose that pharmacological intervention targeting GPR91 could improve post-HI brain recovery.

The complete targeted profile of the organic acid intermediates of the citric acid cycle using a single stable isotope dilution analysis, sodium borodeuteride reduction and selected ion monitoring GC/MS.

The quantitative profiling of the organic acid intermediates of the citric acid cycle (CAC) presents a challenge due to the lack of commercially available internal standards for all of the organic acid intermediates. We developed an analytical method that enables the quantitation of all the organic acids in the CAC in a single stable isotope dilution GC/MS analysis with deuterium-labeled analogs used as internal standards. The unstable α-keto acids are rapidly reduced with sodium borodeuteride to the corresponding stable α-deutero-α-hydroxy acids and these, along with their unlabeled analogs and other CAC organic acid intermediates, are converted to their tert-butyldimethylsilyl derivatives. Selected ion monitoring is employed with electron ionization. We validated this method by treating an untransformed mouse mammary epithelial cell line with well-known mitochondrial toxins affecting the electron transport chain and ATP synthase, which resulted in profound perturbations of the concentration of CAC intermediates.

A liver-specific defect of Acyl-CoA degradation produces hyperammonemia, hypoglycemia and a distinct hepatic Acyl-CoA pattern.

Most conditions detected by expanded newborn screening result from deficiency of one of the enzymes that degrade acyl-coenzyme A (CoA) esters in mitochondria. The role of acyl-CoAs in the pathophysiology of these disorders is poorly understood, in part because CoA esters are intracellular and samples are not generally available from human patients. We created a mouse model of one such condition, deficiency of 3-hydroxy-3-methylglutaryl-CoA lyase (HL), in liver (HLLKO mice). HL catalyses a reaction of ketone body synthesis and of leucine degradation. Chronic HL deficiency and acute crises each produced distinct abnormal liver acyl-CoA patterns, which would not be predictable from levels of urine organic acids and plasma acylcarnitines. In HLLKO hepatocytes, ketogenesis was undetectable. Carboxylation of [2-(14)C] pyruvate diminished following incubation of HLLKO hepatocytes with the leucine metabolite 2-ketoisocaproate (KIC). HLLKO mice also had suppression of the normal hyperglycemic response to a systemic pyruvate load, a measure of gluconeogenesis. Hyperammonemia and hypoglycemia, cardinal features of many inborn errors of acyl-CoA metabolism, occurred spontaneously in some HLLKO mice and were inducible by administering KIC. KIC loading also increased levels of several leucine-related acyl-CoAs and reduced acetyl-CoA levels. Ultrastructurally, hepatocyte mitochondria of KIC-treated HLLKO mice show marked swelling. KIC-induced hyperammonemia improved following administration of carglumate (N-carbamyl-L-glutamic acid), which substitutes for the product of an acetyl-CoA-dependent reaction essential for urea cycle function, demonstrating an acyl-CoA-related mechanism for this complication.

AMPK is a negative regulator of the Warburg effect and suppresses tumor growth in vivo.

AMPK is a metabolic sensor that helps maintain cellular energy homeostasis. Despite evidence linking AMPK with tumor suppressor functions, the role of AMPK in tumorigenesis and tumor metabolism is unknown. Here we show that AMPK negatively regulates aerobic glycolysis (the Warburg effect) in cancer cells and suppresses tumor growth in vivo. Genetic ablation of the α1 catalytic subunit of AMPK accelerates Myc-induced lymphomagenesis. Inactivation of AMPKα in both transformed and nontransformed cells promotes a metabolic shift to aerobic glycolysis, increased allocation of glucose carbon into lipids, and biomass accumulation. These metabolic effects require normoxic stabilization of the hypoxia-inducible factor-1α (HIF-1α), as silencing HIF-1α reverses the shift to aerobic glycolysis and the biosynthetic and proliferative advantages conferred by reduced AMPKα signaling. Together our findings suggest that AMPK activity opposes tumor development and that its loss fosters tumor progression in part by regulating cellular metabolic pathways that support cell growth and proliferation.

High-resolution capillary gas chromatography in combination with mass spectrometry for quantification of three major polyamines in postmortem brain cortex.

There is considerable evidence supporting a role of the polyamine system in the etiology and pathology of mental disorders. Changes in the expression and activity of polyamine anabolic/catabolic enzymes, as well as in the levels of individual polyamines, have been found in many psychiatric conditions, including schizophrenia, mood disorders, anxiety, and suicidal behavior. Recent microarray studies have found that spermidine/spermine-N¹-acetyltransferase (SAT1, SSAT), the key enzyme in charge of the polyamine catabolic pathway, is downregulated in brain tissue of individuals who were depressed and died by suicide. To provide further insight into the downstream effects of altered SAT1 expression, we developed a quantitative gas chromatography-mass spectrometry method for measurement of polyamine concentrations in postmortem human brain tissues. This protocol employs a conventional electron ionization method with total ion and selected ion monitoring. This method can accurately measure the levels of the polyamines putrescine, spermidine, and spermine from very small quantities (1-50 mg) of postmortem brain tissues, with quantitation limits down to 10 ng/g of wet tissue for putrescine and 100 ng/g for spermidine and spermine.

Measurement of urinary trimethylamine and trimethylamime oxide by direct infusion electrospray quadrupole time-of-flight mass spectrometry.

Urinary trimethylamine (TMA) and its oxide (TMAOx) are measured separately and as a mixture using (15)N-labeled internal standards and direct infusion electrospray with a quadrupole time-of-flight (Q-ToF) instrument. TMA is quaternized with trideuteromethyl iodide to avoid inclusion of endogenous tetramethylammonium ion in the TMA measurement, whereas TMAOx is measured as the protonated molecule. Measurements reported as percentage TMA made with separate and combined samples agree within 6% of the measured values and demonstrate that both TMA and TMAOx can be measured simultaneously in a single analysis. Moreover, the analysis is simpler and less tedious and time-consuming than some earlier methods.

A novel liquid-liquid extraction and stable isotope dilution NCI-GC-MS method for quantitation of agmatine in postmortem brain cortex.

The group of biologically important amines includes putrescine, spermidine and spermine, as well as agmatine, which is a guanidino-amine. There is considerable evidence supporting a role of these amines in the etiology and pathology of mental disorders. We have previously developed a quantitative GC-MS method for simultaneous measurement of three major polyamines to support our studies linking polyamines to mental disorders. However, a unique GC-MS method is required for agmatine. To efficiently extract agmatine from postmortem brain tissues, we developed an isopropanol based liquid-liquid extraction protocol using potassium carbonate as a salting-out agent which showed a much greater recovery than n-butanol used in earlier methods. The GC-MS analysis employed hexafluoroacetylacetone as derivatization reagent and was carried out using negative chemical ionization with total ion and selected ion monitoring. (15)N(4)-agmatine was synthesized from (15)N(4)-L-arginine and used as internal standard in a conventional stable isotope dilution assay. This method accurately measures the level of agmatine from very small quantities (10-20 mg) of postmortem brain tissue, with a quantitation limit down to 1 ng/g of wet tissue. The limit of detection is 0.01 ng/g of wet tissue.

Evidence of altered polyamine concentrations in cerebral cortex of suicide completers.

Recent studies have implicated alterations in the expression of polyamine-related genes in the brains of suicide completers including widespread downregulation of spermidine/spermine N1-acetyltransferase, the key enzyme in polyamine catabolism, suggesting compensatory mechanisms attempting to increase brain levels of polyamines. Given the complexity of the polyamine system, quantification of the levels of the polyamines is an essential step in understanding the downstream effects of dysregulated gene expression. We developed a method using high-resolution capillary gas chromatography (GC) in combination with mass spectrometry (MS) for quantitation of polyamines from post-mortem brain tissue, which allowed us to accurately measure spermidine and putrescine concentrations in post-mortem brain tissues. Using this method, we analyzed putrescine and spermidine levels in a total of 126 samples from Brodmann areas 4, 8/9, and 11, from 42 subjects, comprising 16 suicide completers with major depression, 13 non-depressed suicide completers, and 13 control subjects. Both putrescine and spermidine levels fell within the expected nanomolar ranges and were significantly elevated in the brain of suicide completers with a history of major depression as compared with controls. These results were not accounted by possible confounders. This is the first GC-MS study to analyze the expression of putrescine and spermidine from post-mortem brain tissue and confirms the hypothesis raised by previous studies indicating alterations in putrescine and spermidine levels in suicide/major depression.

Mechanisms of biodegradation of dibenzoate plasticizers.

Biodegradation mechanisms were elucidated for three dibenzoate plasticizers: diethylene glycol dibenzoate (D(EG)DB), dipropylene glycol dibenzoate (D(PG)DB), both of which are commercially available, and 1,6-hexanediol dibenzoate, a potential green plasticizer. Degradation studies were done using Rhodococcus rhodochrous in the presence of pure alkanes as a co-substrate. As expected, the first degradation step for all of these systems was the hydrolysis of one ester bond with the release of benzoic acid and a monoester. Subsequent biodegradation of the monobenzoates of diethylene glycol (D(EG)MB) and dipropylene glycol (D(PG)MB) was very slow, leading to significant accumulation of these monoesters. In contrast, 1,6-hexanediol monobenzoate was quickly degraded and characterization of the metabolites indicated that the biodegradation proceeded by way of the oxidation of the alcohol group to generate 6-(benzoyloxy) hexanoic acid followed by beta-oxidation steps. This pathway was blocked for D(EG)MB and D(PG)MB by the presence of an ether function. The use of a pure hydrocarbon as a co-substrate resulted in the formation of another class of metabolites; namely the esters of the alcohols formed by the oxidation of the alkanes and the benzoic acid released by hydrolysis of the original diesters. These metabolites were biodegraded without the accumulation of any intermediates.

A quantitative GC-MS method for three major polyamines in postmortem brain cortex.

A quantitative method for putrescine (PUT), spermidine (SPD) and spermine (SPM) in homogenized postmortem human brain tissue is described that employs a novel, simple and rapid extractive derivatization with ethylchloroformate and trifluoroacetylation. These amines are metabolites of ornithine and are metabolically interconvertible in mammals. The method was developed to support an ongoing epidemiological study correlating these amines with the frequency of suicide. The isolation methodology is robust and requires less work and time than many previous methods. Analysis is by conventional electron ionization GC-MS with selected ion monitoring using a stable isotope-labeled analog for PUT and a chemical analog for SPD and SPM as internal standards. The time required for chromatographic analysis, about 20 min, is determined by the wide range of the relative volatilities of the derivatized polyamines. The method allows the quantitation of PUT down to 10 ng/g and SPD and SPM down to 100 and 1000 ng/g, respectively of wet tissue.

Metabolites from the biodegradation of 1,6-hexanediol dibenzoate, a potential green plasticizer, by Rhodococcus rhodochrous.

Metabolites from the biodegradation of a potential plasticizer 1,6-hexanediol dibenzoate in the presence of n-hexadecane as a co-substrate by the common soil organism Rhodococcus rhodochrous were identified using GC/MS and Fourier transform mass spectroscopy (FTMS) techniques. Trimethylsilylation of compounds from the biodegradation broth permitted detection of the following metabolites: 1-hexadecyl benzoate, 6-benzoyloxyhexanoic acid, 4-benzoyloxybutanoic acid, 6-benzoyloxyhexan-1-ol and benzoic acid. The presence of these metabolites was confirmed by repeating the biodegradation with 1,6-hexanediol di[(2)H(5)]benzoate, by measurement of their exact masses in FTMS and by comparison with available authentic materials. The results show that biodegradation of 1,6-hexanediol dibenzoate by R. rhodochrous does not lead to the accumulation of persistent metabolites as has been reported for commercial dibenzoate plasticizers.

Oxalic acid excretion after intravenous ascorbic acid administration.

Ascorbic acid is frequently administered intravenously by alternative health practitioners and, occasionally, by mainstream physicians. Intravenous administration can greatly increase the amount of ascorbic acid that reaches the circulation, potentially increasing the risk of oxalate crystallization in the urinary space. To investigate this possibility, we developed gas chromatography mass spectrometry methodology and sampling and storage procedures for oxalic acid analysis without interference from ascorbic acid and measured urinary oxalic acid excretion in people administered intravenous ascorbic acid in doses ranging from 0.2 to 1.5 g/kg body weight. In vitro oxidation of ascorbic acid to oxalic acid did not occur when urine samples were brought immediately to pH less than 2 and stored at -30 degrees C within 6 hours. Even very high ascorbic acid concentrations did not interfere with the analysis when oxalic acid extraction was carried out at pH 1. As measured during and over the 6 hours after ascorbic acid infusions, urinary oxalic acid excretion increased with increasing doses, reaching approximately 80 mg at a dose of approximately 100 g. We conclude that, when studied using correct procedures for sample handling, storage, and analysis, less than 0.5% of a very large intravenous dose of ascorbic acid is recovered as urinary oxalic acid in people with normal renal function.

The succinate receptor GPR91 in neurons has a major role in retinal angiogenesis.

Vascularization is essential for tissue development and in restoration of tissue integrity after an ischemic injury. In studies of vascularization, the focus has largely been placed on vascular endothelial growth factor (VEGF), yet other factors may also orchestrate this process. Here we show that succinate accumulates in the hypoxic retina of rodents and, via its cognate receptor G protein-coupled receptor-91 (GPR91), is a potent mediator of vessel growth in the settings of both normal retinal development and proliferative ischemic retinopathy. The effects of GPR91 are mediated by retinal ganglion neurons (RGCs), which, in response to increased succinate levels, regulate the production of numerous angiogenic factors including VEGF. Accordingly, succinate did not have proangiogenic effects in RGC-deficient rats. Our observations show a pathway of metabolite signaling where succinate, acting through GPR91, governs retinal angiogenesis and show the propensity of RGCs to act as sensors of ischemic stress. These findings provide a new therapeutic target for modulating revascularization.

Hereditary and acquired diseases of acyl-coenzyme A metabolism.

Coenzyme A (CoA) sequestration, toxicity or redistribution (CASTOR) is predicted to occur in many hereditary and acquired conditions in which the degradation of organic acyl esters of CoA is impaired. The resulting accumulation of CoA esters and reduction of acetyl-CoA and free CoA (CoASH) will then trigger a cascade of reactions leading to clinical disease. Most conditions detected by expanded neonatal screening are CASTOR diseases. We review acyl-CoA metabolism, including CoASH synthesis, transesterification of acyl-CoAs to glycine, glutamate or l-carnitine and hydrolysis of CoA esters. Because acyl-CoAs do not cross biological membranes, their main toxicity is intracellular, primarily within mitochondria. Treatment measures directed towards removal of circulating metabolites do not address this central problem of intracellular acyl-CoA accumulation. Treatments usually involve the restriction of dietary precursors and administration of agents like l-carnitine and glycine, which can accept the transfer of acyl groups from acyl-CoA, liberating CoASH. Many hereditary CASTOR patients are chronically ill, with persistent symptoms and continuously abnormal metabolites in blood and urine despite good compliance with treatment. Conversely, asymptomatic patients are also common in hereditary CASTOR conditions. Future challenges include the understanding of pathophysiologic mechanisms in CASTOR diseases, the discovery of reliable predictors of outcome in individual patients and the establishment of therapeutic trials with sufficient numbers of patients to permit solid therapeutic conclusions.

Structural requirements for activation of the 5-oxo-6E,8Z, 11Z,14Z-eicosatetraenoic acid (5-oxo-ETE) receptor: identification of a mead acid metabolite with potent agonist activity.

The 5-lipoxygenase product 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE) is a potent chemoattractant for neutrophils and eosinophils, and its actions are mediated by the oxoeicosanoid (OXE) receptor, a member of the G protein-coupled receptor family. To define the requirements for activation of the OXE receptor, we have synthesized a series of 5-oxo-6E,8Z-dienoic acids with chain lengths between 12 and 20 carbons, as well as a series of 20-carbon 5-oxo fatty acids, either fully saturated or containing between one and five double bonds. The effects of these compounds on neutrophils (calcium mobilization, CD11b expression, and cell migration) and eosinophils (actin polymerization) were compared with those of 5-oxo-ETE. The C12 and C14 analogs were without appreciable activity, whereas the C16 5-oxo-dienoic acid was a weak partial agonist. In contrast, the corresponding C18 analog (5-oxo-18:2) was nearly as potent as 5-oxo-ETE. Among the C20 analogs, the fully saturated compound had virtually no activity, whereas 5-oxo-6E-eicosenoic acid had only weak agonist activity. In contrast, 5-oxo-6E,8Z,11Z-eicosatrienoic acid (5-oxo-20:3) and its 8-trans isomer were approximately equipotent with 5-oxo-ETE in activating granulocytes. Because of the potent effects of 5-oxo-20:3, we investigated its formation from Mead acid (5Z,8Z,11Z-eicosatrienoic acid), which accumulates in dietary essential fatty acid deficiency, by neutrophils. The main Mead acid metabolite identified was 5-hydroxy-6,8,11-eicosatrienoic acid, followed by 5-oxo-20:3 and two 6-trans isomers of leukotriene B(3). We conclude that optimal activation of the OXE receptor is achieved with 5-oxo-ETE, 5-oxo-18:2, and 5-oxo-20:3, and that the latter compound could potentially be formed under conditions of essential fatty acid deficiency.

Human neutrophils convert the sebum-derived polyunsaturated fatty acid Sebaleic acid to a potent granulocyte chemoattractant.

Sebaleic acid (5,8-octadecadienoic acid) is the major polyunsaturated fatty acid in human sebum and skin surface lipids. The objective of the present study was to investigate the metabolism of this fatty acid by human neutrophils and to determine whether its metabolites are biologically active. Neutrophils converted sebaleic acid to four major products, which were identified by their chromatographic properties, UV absorbance, and mass spectra as 5-hydroxy-(6E,8Z)-octadecadienoic acid (5-HODE), 5-oxo-(6E,8Z)-octadecadienoic acid (5-oxo-ODE), 5S,18-dihydroxy-(6E,8Z)-octadecadienoic acid, and 5-oxo-18-hydroxy-(6E,8Z)-octadecadienoic acid. The identities of these metabolites were confirmed by comparison of their properties with those of authentic chemically synthesized standards. Both neutrophils and human keratinocytes converted 5-HODE to 5-oxo-ODE. This reaction was stimulated in neutrophils by phorbol myristate acetate and in keratinocytes by oxidative stress (t-butyl-hydroperoxide). Both treatments dramatically elevated intracellular levels of NADP(+), the cofactor required by 5-hydroxyeicosanoid dehydrogenase. In keratinocytes, this was accompanied by a rapid increase in intracellular GSSG levels, consistent with the involvement of glutathione peroxidase. 5-Oxo-ODE stimulated calcium mobilization in human neutrophils and induced desensitization to 5-oxo-6,8,11,14-eicosatetraenoic acid but not leukotriene B(4), indicating that this effect was mediated by the OXE receptor. 5-Oxo-ODE and its 8-trans isomer were equipotent with 5-oxo-6,8,11,14-eicosatetraenoic acid in stimulating actin polymerization and chemotaxis in human neutrophils, whereas 5-HODE, 5-oxo-18-hydroxy-(6E,8Z)-octadecadienoic acid, and 5S,18-dihydroxy-(6E,8Z)-octadecadienoic acid were much less active. We conclude that neutrophil 5-lipoxygenase converts sebaleic acid to 5-HODE, which can be further metabolized to 5-oxo-ODE by 5-hydroxyeicosanoid dehydrogenase in neutrophils and keratinocytes. Because of its chemoattractant properties, sebum-derived 5-oxo-ODE could be involved in neutrophil infiltration in inflammatory skin diseases.

Room temperature stability of injectable succinylcholine dichloride.

The purpose of this study was to determine the room temperature stability over a period of several months of commercially available intravenous succinylcholine dichloride (Quelicin, 20 mg/mL) in vials. A previously validated electro-spray tandem mass spectrometry method developed for the determination of succinylcholine dichloride in plasma was used. This method was based upon a stable isotope dilution assay using hexadeuterosuccinylcholine diiodide as the internal standard and was shown to be specific, sensitive, and reproducible. Calibration curves were plots of the ratios of intensities of the major product ions in the collision-induced dissociation spectrum for known concentration ratios of succinylcholine dichloride and hexadeuterosuccinylcholine diiodide in solutions. The concentration of succinylcholine dichloride was shown to decline linearly. After 1, 3, and 6 months at room temperature, the vial contents retained approximately 98%, 95%, and 90% of their inital concentration, respectively. We suggest, therefore, that succinylcholine dichloride can be stored safely at room temperature under normal daylight for 6 months.

On the mechanism of demethylation of 5-methylcytosine in DNA.

DNA methylation is an important biological process that programmes gene expression in vertebrates. The methylation pattern is generated by a combination of methylation and demethylation reactions catalyzed by DNA methyltransferases and putative demethylases. MBD2 binds methylated DNA and possesses DNA demethylase activity. We use here direct analysis of the reaction mixture by GC-MS using a water-tolerant gas chromatographic column to avoid the loss of potential volatile products and identify the leaving residue of the demethylation reaction. We show that the DNA demethylase reaction catalyzed by a recombinant human MBD2 purified from SF9 insect cells releases dideuteroformaldehyde from [Me-(2)H(3)]-5-methylcytosine in DNA. A mechanism of the DNA demethylation reaction is proposed based on this observation.