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Robust scientific evidence shows the sun angle controls water vapour content of the atmosphere, the main component of back radiation, as it cycles annually. Water vapour content measured as the ratio of the number of water molecules to CO2 molecules varies from 1:1 near the Poles to 97:1 in the Tropics. The effect of back radiation on Earth’s atmosphere is up to 200 times larger than that of CO2 and works in the opposite direction. Thus, if CO2 has any effect on atmospheric temperature and climate change we show it is negligible. Consequently, current government policies to control atmospheric temperature by limiting consumption of fossil fuels will have negligible effect. Measured data reported in IPCC report Climate Change 2013: The Physical Science Basis ( AR5) indicate increased water vapour content of the atmosphere is the cause of the 0.5℃ temperature increase from the mid-1970s to 2011.

Does a math problem in IPCC report climate change The physical science basis AR compromise the science

The laws of physics namely the gas laws were applied to the gases in the atmosphere that act as ideal gases The results show that as air temperature increases from winter to summer CO is a cooling gas and from summer to winter it is a warming gas regardless of its concentration in the atmosphere This is contrary to the commonly held belief that CO always warms the atmosphere Back radiation is the sum of the radiation of all of the greenhouse gases back to the Earth It is a measured value and increases with temperature and vice versa Back radiation acts opposite to that of CO methane and the trace gases On average the latter account for of back radiation and water vapor accounts for The effect of CO methane and the trace gases on atmospheric temperature and climate change is so small as to be negligible

The Beer-Lambert law does not apply strictly to the relationship between radiative forcing (RF) of CO2 and concentration in the atmosphere, i.e., ΔRF = 5.35ln(C/Co). It is an approximation because water vapour competes unevenly with CO2 over the IR absorption wavelength range. We propose a quadratic model as an improved approximation. It links concentration to RF thereby allowing RF calculation at any concentration, not just ΔRF. For example, at 378 ppmv of CO2, the level in 2005, it calculates RF = 8.67 W m−2, or approximately 2.7% of the total RF of all the greenhouse gases. A second and independent method based on worldwide hourly measurements of atmospheric temperature and relative humidity confirms this percentage. Each method shows that, on average, water vapour contributes approximately 96% of current greenhouse gas warming. Thus, the factors controlling the amount of water vapour in the air also control the earth's temperature.

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. 15N4-Agmatine was synthesized from 15N4-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. Copyright © 2010 John Wiley & Sons, Ltd.

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.

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 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[2H5]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. Copyright © 2009 John Wiley & Sons, Ltd.

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.

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.

Hepatic encephalopathy (HE) is a neuropsychiatric complication of both acute and chronic liver failure characterized by progressive neuronal inhibition. Some neurosteroids are potent positive allosteric modulators of the gamma-aminobutyric acid (GABA)-A receptor complex, and 'increased GABAergic tone' has been proposed to explain the neuroinhibition characteristics of HE. Brain levels of the neurosteroids pregnenolone, allopregnanolone and tetrahydrodesoxycorticosterone (THDOC) and the functional status of the GABA-A receptor complex were assessed in rats following portacaval anastomosis (PCA). Effects of indomethacin, an inhibitor of the 3alpha-hydroxysteroid dehydrogenase enzyme involved in neurosteroid synthesis, on PCA rat locomotor activity and brain neurosteroid levels were also assessed. Significant increases of the neurosteroid pregnenolone (2.6-fold), allopregnanolone (1.7-fold) and THDOC (4.7-fold) were observed in brains of PCA rats. Brain levels of these neurosteroids were in the nanomolar range, sufficient to exert positive allosteric modulatory effects at the GABA-A receptor. Indomethacin (0.1-5 mg kg(-1)) ameliorated dose-dependently the locomotor deficit of PCA rats and concomitantly normalized brain levels of allopregnanolone and THDOC. Increased brain levels of neurosteroids with positive allosteric modulatory actions at the neuronal GABA-A receptor offer a cogent explanation for the notion of 'increased GABAergic tone' in HE. Pharmacological approaches using agents that either reduce neurosteroid synthesis or modulate the neurosteroid site on GABA-A receptor could offer new therapeutic tools for the management and treatment of HE.

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.

A new metabolite, 2,4-dihydroxyquinoline (DHQ), was identified in cultures of the bacteria Pseudomonas aeruginosa and Burkholderia thailandensis. We found that the biosynthesis of DHQ correlates with the presence of a functional PqsA, which is a product of the pqsABCDE operon responsible for the synthesis of 4-hydroxy-2-alkylquinolines (HAQs) in P. aeruginosa. However, DHQ is not a degradation product or precursor of HAQs. This finding sheds some light on the poorly understood biosynthesis pathway of HAQs, which includes important communication signals regulating the expression of virulence factors.

We describe a sensitive, simple and convenient stable isotope dilution assay developed to study endogenous metabolism of administered stable isotope-labeled phenylalanine (Phe) in phenylketonuric (PKU) mice treated experimentally with phenylalanine ammonia lyase (PAL). Mouse urine and plasma containing endogenous and administered labeled Phe together with internal standard Phe bearing a different pattern of labeling are converted by in situ diazotization to 2-chloro-3-phenylpropionic acid (CPP). A single solvent extraction is then used to isolate the isotopomers of CPP along with the trans-cinnamic acid (TCA) produced from Phe by PAL, as well as the TCA metabolites benzoic and hippuric acids. This procedure eliminates the need for a separate ion-exchange isolation step for Phe on a second sample aliquot and separate GC-MS analysis. Extracted CPP and the Phe metabolites are then measured by conversion to the pentafluorobenzyl esters and a single analysis by electron capture negative ion GC-MS. The estimated lower limit of quantitation is 0.1 microM.

We previously reported that human Niemann-Pick Disease type B (NPD-B) is associated with low HDL. In this study, we investigated the pathophysiology of this HDL deficiency by examining both HDL samples from NPD-B patients and nascent high density lipoprotein (LpA-I) generated by incubation of lipid-free apolipoprotein A-I (apoA-I) with NPD-B fibroblasts. Interestingly, both LpA-I and HDL isolated from patient plasma had a significant increase in sphingomyelin (SM) mass ( approximately 50-100%). Analysis of LCAT kinetics parameters (V(max) and K(m)) revealed that either LpA-I or plasma HDL from NPD-B, as well as reconstituted HDL enriched with SM, exhibited severely decreased LCAT-mediated cholesterol esterification. Importantly, we documented that SM enrichment of NPD-B LpA-I was not attributable to increased cellular mass transfer of SM or unesterified cholesterol to lipid-free apoA-I. Finally, we obtained evidence that the conditioned medium from HUVEC, THP-1, and normal fibroblasts, but not NPD-B fibroblasts, contained active secretory sphingomyelinase (S-SMase) that mediated the hydrolysis of [(3)H]SM-labeled LpA-I and HDL(3). Furthermore, expression of mutant SMase (DeltaR608) in CHO cells revealed that DeltaR608 was synthesized normally but had defective secretion and activity. Our data suggest that defective S-SMase in NPD leads to SM enrichment of HDL that impairs LCAT-mediated nascent HDL maturation and contributes to HDL deficiency. Thus, S-SMase and LCAT may act in concert and play a crucial role in the biogenesis and maturation of nascent HDL particles.

HDLs have been proposed to have antiatherogenic properties because of their role in reverse cholesterol transport as lipid acceptors. To elucidate the phospholipid profile of these particles, we used electrospray ionization mass spectrometry to examine the phosphatidylcholine (PC) and sphingomyelin (SM) composition of HDLs purified from plasma and nascently generated in vitro from fibroblasts. We also quantitatively compared the phospholipids present in these lipoproteins between normal and Niemann-Pick disease type B (NPD-B) subjects characterized by sphingomyelinase (SMase) deficiency. We demonstrated that plasma HDLs from NPD-B were significantly enriched in SM by an average of 28%, particularly the palmitoyl SM (with an increase of 95%), which accounted for approximately 25-44% of total SM molecular species. Similarly, we observed an increase of approximately 63% in total SM levels in nascent HDLs prepared from NPD-B fibroblasts. Although PC levels in nascent HDLs were comparable between control and NPD-B cells, there was a 95% increase in total PC levels similar to that of SM in plasma HDLs extracted from NPD-B subjects. These data provide insight into the structure of HDLs and identify potential new roles for SMase in lipoprotein metabolism.

Background Hyperhomocysteinemia is a frequent finding after cardiac transplantation, but increased folate intake induces a decrease in total homocysteine concentrations. In 1998, food in Canada was fortified nationwide with folic acid. We assessed the impact of routine folate fortification on homocysteine concentrations in our cardiac transplant population. Methods In 18 subjects, we measured total homocysteine (tHcy), serum folate, and cobalamin concentrations in 1997 (before folate fortification) and in 1998 (after fortification). We repeated the analysis after specific multivitamin supplementation for 10 weeks. Results We found a significant decrease in baseline tHcy concentrations and in folate concentrations between 1997 and 1998. However, we also found a decrease in serum cobalamin concentrations. We found a correlation between decreased cobalamin concentrations and the methionine synthase A2756G genotype, but not with other common polymorphisms associated with homocysteine metabolism. After multivitamin supplementation, we observed a trend toward further decrease in tHcy concentrations and a significant increase in serum folate and cobalamin concentrations. Finally, we measured serum methylmalonic acid concentrations, an index of tissue cobalamin status. We did not find a correlation between increased methylmalonic acid concentrations and decreased serum cobalamin, perhaps related to the confounding effect of altered renal status on methylmalonic acid excretion. Conclusions National folate fortification was associated with decreased tHcy and increased folate concentrations in our cardiac transplant population. Additional administration of vitamin supplements induced a further decrease in tHcy and an increase in folate. Finally, folate fortification unveiled cobalamin deficiency in some patients, associated with the methionine synthase A2756G mutation.

Summary: Purpose: We previously demonstrated that the anticholinesterase eserine provokes interictal-like discharges in the CA3 area of hippocampal slices from rats in which generalized seizures had been induced by pentylenetetrazol (PTZ) when immature. In this study, we investigated several factors as the possible mechanism for this effect, including age at convulsions. Methods: Rats were injected with PTZ on postnatal day (P) 18–20 or >P60, and neuronal activity was recorded intra- and extracellularly from CA3 5–10 or >40 days later. In additional experiments, convulsions were triggered by kainate or were blocked by pentobarbital. Hippocampal (a) acetylcholine (ACh) innervation density was measured by immunocytochemistry, and ACh and γ-aminobutyric acid (GABA) contents were determined by high-performance liquid chromatography (HPLC)-electrospray ionization. Results: The excitatory effect of eserine was the most consistent in slices from rats PTZ-treated when immature and after the long interval, whereas the reverse was true in rats treated as adults. This effect was dependent on the occurrence of a seizure and was less prevalent when the seizure had been provoked by kainate. Adult animals PTZ-treated at P20 did not differ from control in (a) poly- or monosynaptic GABAA and GABAB CA3 inhibitory postsynaptic potentials (IPSPs); (b) density of ACh innervation; or (c) tissue content of ACh and GABA. Conclusions: A PTZ-induced generalized seizure in immature rat provokes endogenous ACh-induced interictal-like discharges in adult hippocampal CA3. This effect is only transiently observed if the seizure was induced in adult. It does not appear to be related to a change in GABAergic inhibition, in density of ACh innervation, or in ACh or GABA content.

Trimethylaminuria is a rare metabolic disorder that is associated with abnormal amounts of the dietary-derived trimethylamine. Excess unmetabolized trimethylamine in the urine, sweat and other body secretions confers a strong, foul body odor that can affect the individuals ability to work or engage in social activities. This review summarizes the biochemical aspects of the condition and the classification of the disorder into: 1) primary genetic form, 2) acquired form, 3) childhood forms, 4) transient form associated with menstruation, 5) precursor overload and 6) disease states. The genetic variability of the flavin-containing monooxygenase (form 3) that is responsible for detoxication and deodoration of trimethylamine is discussed and put in context with other variant forms of the flavin-containing monooxygenase (forms 1-5). The temporal-selective expression of flavin-containing monooxygenase forms 1 and 3 is discussed in terms of an explanation for childhood trimethylaminuria. Information as to whether variants of the flavin-containing monooxygenase form 3 contributes to hypertension and / or other diseases are presented. Discussion is provided outlining recent bioanalytical approaches to quantify urinary trimethylamine and trimethylamine N-oxide and plasma choline as well as data on self-reporting individuals tested for trimethylaminuria. Finally, trimethylaminuria treatment strategies and nutritional support are described including dietary sources of trimethylamine, vitamin supplementation and drug treatment and issues related to trimethylaminuria in pregnancy and lactation are discussed. The remarkable progress in the biochemical, genetic, clinical basis for understanding the trimethylaminuria condition is summarized and points to needs in the treatment of individuals suffering from trimethylaminuria.

A novel minor metabolite 1 with an unprecedented skeleton was isolated from the needles of Taxus canadensis. A biogenesis from taxinine an abundant taxane is proposed. This is the first example of a taxane with a 6/6/8/6-membered ring skeleton.

Hyperhomocysteinemia adversely affects fundamental aspects of fetal development, adulthood, and aging, but the role of elevated homocysteine levels in these birth defects and adult diseases remains unclear. Mouse models are valuable for investigating the causes and consequences of hyperhomocysteinemia. We used a phenotype-based approach to identify mouse mutants for studying the relation between single gene mutations, homocysteine levels as a measure of the status of homocysteine metabolism, and gene expression profiles as a way to assess the impact of protein deficiency in mutant mice on steady-state transcription levels of genes in the folate-homocysteine pathways. These mutants were selected based on their propensity to produce phenotypes that are reminiscent of those associated with anomalies in folate-homocysteine metabolism in humans. We report identification of new, single-gene mouse models of homocysteinemia and characterization of their molecular and physiological impact on folate-homocysteine metabolism. Mutations in several genes involved in the hedgehog and WNT signal transduction pathways, as well as a gene involved in lipid metabolism, resulted in elevated homocysteine levels and altered expression profiles of folate-homocysteine metabolism genes. These results begin to unravel the complex relations between elevation of a single amino acid in the blood and the diverse birth defects and adult diseases associated with hyperhomocysteinemia.

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. 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. 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. 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.

Reanalysis of the biotransformation of 4(20),11(12)-taxadiene derivatives by Absidia coerulea ATCC 10738a unravelled additional unprecedented rearrangement reactions. 5α,7β,9α,10β,13α-pentaacetoxy-4(20),11(12)-taxadiene (1) was converted to 10 new minor taxane derivatives in addition to the previously reported C-1, C-14-hydroxylations and C-15-hydroxylated abeo-taxane. In addition, 2-deacetoxytaxinine J (2) was successfully metabolized with the same fungi.Key words: natural products, terpenes and terpenoids, microorganism, oxygenation, rearrangement.

5alpha, 7beta, 9alpha, 10beta, 13alpha-Pentahydroxy-4(20),11(12)-taxadiene derivative 1 was converted to two unprecedented 1(15--11)abeo-taxanes and a taxane derivative with a C10-C11 double bond by Absidia coerula ATCC 10738a. A similar compound was obtained from treatment with zinc of a triacetoxy-4(20),11(12)-taxadiene derivative.

The initial catabolic steps of isoleucine by mammals has been misunderstood and misapprehended in the scientific literature for many years. The suggestion that the interconversion of isoleucine and alloisoleucine occurs through the keto–enol racemization of their respective transaminated α-keto acids was first tentatively advanced by Alton Meister in the early 1950s, and accepted without hard confirming evidence by many authors. It will be shown in this brief review that isoleucine is converted to alloisoleucine with conservation of a 15 N label denying the intermediacy of the α-keto acids, and that alloisoleucine arises as an unavoidable consequence of isoleucine transamination.

Intoxication and liver damage induced by carbon tetrachloride (CCl 4 ), aflatoxin B1, diabetes, and subtotal partial hepatectomy (PH 90 ) in rats in which approximately 90% of the total hepatic tissue mass is surgically removed produces an acute-phase response (APR) whose initial stage prior to regression closely mimics the APRs associated with the life-threatening hepatic failure seen in the homeless. Rats treated by PH 90 were either healthy, CCl 4 -intoxicated, diabetic, or alflatoxin B1 (AFB1) intoxicated to the point of 75% liver insufficiency. It is well documented that high rates of mortality following PH 90 in aseptic rats could be minimized by supplementing drinking water with 20% glucose, organic components of L-15 medium and housing animals in cages maintained at 33–35C. Aseptic rats showed a mild 20–30% decrease in APR proteins during the first 4–5 days following PH 90 , while a maximal APR was noted 9–12 days post PH 90 and lasted for ~30 days when it returned to values close to those of healthy controls. This delay in hepatic APR of the remnant caudate lobe favoured replacement of lost basophilic clumps and ribosomes. The newly synthesized ribosomes of the nascent hepatocytes quantitatively maintained the APR signals of the injured caudate hepatocytes, and biosynthesized and released a typical spectrum of APR proteins. We suggest that massively injured liver has decoded an already stored and irreversible DNA-biochemical sequence of events in which priority is given to recovery of lost tissues by delaying an APR response to injury. In PH 90 of diabetic and CCl 4 -intoxicated rats, the hepatic dual functions of regeneration and APR processes associated with intoxication-initiated catabolic signals, created a heavy metabolic burden on the remnant caudate lobe leading to higher rates of mortality. APR of healthy rats to AFB1 parallels that of α-amanitin-induced intoxication. Similarly, within shorter time scale proportional to the severity of surgery, livers undergoing 75% partially hepatectomy (PH 75 ) delayed both the onset and regression of APR. We are therefore led to believe that approaches other than liver transplantation should be considered as viable alternatives in the treatment of various acute and chronic liver diseases to avoid rejection and retransplantation. Scarcity of cadaveric liver has forced the medical community to investigate xenotransplantation with its unknown risks. Concomitantly, it is suggested that in view of the incalculable risks of indifference, the homeless must receive much improved medical care as we have found that two-dimensional immunoelectrophoretic assay of their serum is indicative of acute and chronic liver injury. The scientific and moral interrelationships of related matters are illuminated.

Plasma reduced homocysteine concentrations are increased in end-stage renal disease. Background Plasma total homocysteine (tHcy) concentrations> 15 μmol/L are associated with an increased risk of cardiovascular disease. This is especially the case in end-stage renal disease (ESRD), in which tHcy concentrations commonly range between 20 and 30 μmol/L. Adverse vascular or prothrombotic effects associated with hyperhomocysteinemia are assumed to be mediated by the free sulfhydryl (reduced) form of the molecule (rHcy), but data based on fluorescence high-pressure liquid chromatography (HPLC) indicate that rHcy concentrations are not increased in ESRD despite two- to threefold elevations in tHcy. Methods We developed a sensitive method for measuring plasma rHcy concentrations in which freshly drawn blood is incubated with sodium iodoacetate, and the resulting S-carboxymethylhomocysteine is analyzed by gas chromatography mass spectrometry. Results Unlike with the earlier methodology, we found plasma rHcy concentrations two to four times higher than normal in ESRD. These concentrations were lowered by hemodialysis and were proportional to plasma tHcy over the range of tHcy concentrations that has been associated with increased cardiovascular risk ( r 2 = 0.39, P Conclusions These results support the hypothesis that homocysteine could directly mediate vascular disease through mechanisms related to the reactivity of its free sulfhydryl group. It remains to be determined how much of the variability between plasma tHcy and rHcy is due to analytical variation and how much is due to biologic factors that separately influence concentrations of the disease marker, tHcy, and its presumed mediator, rHcy.

An efficient conversion of Taxus canadensis abundant taxane, 9-dihydro-13-acetylbaccatin III to baccatin III is described. Since the synthesis of paclitaxel from baccatin III has been reported, this work can be used for additional supply of this powerful anticancer drug. In addition, new taxanes derived from skeletal rearrangements originating from oxidation–reduction reactions of the Canadian yew major taxane, are reported.

Apolipoprotein (apo) C-III and apoE play a central role in controlling the plasma metabolism of triglyceride-rich lipoproteins (TRL). We have investigated the plasma kinetics of total, very low density lipoprotein (VLDL) and high density lipoprotein (HDL) apoC-III and apoE in normolipidemic (NL) (n = 5), hypertriglyceridemic (HTG, n = 5), and Type III hyperlipoproteinemic (n = 2) individuals. Apolipoprotein kinetics were investigated using a primed constant (12 h) infusion of deuterium-labeled leucine. HTG and Type III patients had reduced rates of VLDL apoB-100 catabolism and no evidence of VLDL apoB-100 overproduction. Elevated (3- to 12-fold) total plasma and VLDL apoC-III levels in HTG and Type III patients, although associated with reduced apoC-III catabolism (i.e., increased residence times (RTs)), were mainly due to increased apoC-III production (plasma apoC-III transport rates (TRs, mean ± SEM): (NL) 2.05 ± 0.22 (HTG) 4.90 ± 0.81 (P < 0.01), and (Type III) 8.78 mg·kg−1·d−1; VLDL apoC-III TRs: (NL) 1.35 ± 0.23 (HTG) 5.35 ± 0.85 (P < 0.01), and (Type III) 7.40 mg·kg−1·d−1). Elevated total plasma and VLDL apoE levels in HTG (2- and 6-fold, respectively) and in Type III (9- and 43-fold) patients were associated with increased VLDL apoE RTs (0.21 ± 0.02, 0.46 ± 0.05 (P < 0.01), and 1.21 days, NL vs. HTG vs. Type III, respectively), as well as significantly increased apoE TRs (plasma: (NL) 2.94 ± 0.78 (HTG) 5.80 ± 0.59 (P < 0.01) and (Type III) 11.80 mg·kg−1·d−1; VLDL: (NL) 1.59 ± 0.18 (HTG) 4.52 ± 0.61 (P < 0.01) and (Type III) 11.95 mg·kg−1·d−1).These results demonstrate that hypertriglyceridemic patients, having reduced VLDL apoB-100 catabolism (including patients with type III hyperlipoproteinemia) are characterized by overproduction of plasma and VLDL apoC-III and apoE.—Batal, R., M. Tremblay, P. H. R. Barrett, H. Jacques, A. Fredenrich, O. Mamer, J. Davignon, and J. S. Cohn. Plasma kinetics of apoC-III and apoE in normolipidemic and hypertriglyceridemic subjects. J. Lipid Res. 2000. 41: 706–718.

Nineteen taxanes were characterized for the first time in the needles of the Canadian yew. Four of these metabolites are novel: taxinine-11, 12-oxide (1), 7-acetoxytaxuspine C (2), 5-cinnamoyltaxin B (3), and 7, 9-deacetyltaxinine B (4). Compound 1 is the second 11,12-epoxy-taxane known and 7-oxygenated-3, 11-cyclotaxane structures are unique to Taxus canadensis. In addition, taxinine and taxinine E were found to be major taxanes in the Canadian yew. The structures of all the taxanes were rigorously established by spectroscopic methods.

The rearrangement of 9-dihydro-13-acetylbaccatin III, the major taxane from Taxus canadensis, has been studied. Two new cyclization products as well as one novel degradation compound are reported. Their structures were determined by spectroscopic techniques.

Trimethylaminuria (TMAuria) (McKusick 602079) first described in 1970 is an autosomal recessive condition caused by a partial or total incapacity to catalyze the N-oxygenation of the odorous compound trimethylamine (TMA). The result is a severe body odor and associated psychosocial conditions. This inborn error of metabolism, previously thought to be rare, is now being increasingly detected in severe and milder presentations. Mutations of a phase 1 detoxicating gene, flavin-containing monooxygenase 3 (FMO3), have been shown to cause TMAuria. Herein we describe a cohort of individuals ascertained in North America with severe TMAuria, defined by a reduction of TMA oxidation below 50% of normal with genotype-phenotype correlations. We detected four new FMO3 mutations; two were missense (A52T and R387L), one was nonsense (E314X). The fourth allele is apparently composed of two relatively common polymorphisms (K158-G308) found in the general population. On the basis of this study we conclude that one common mutation and an increasing number of private mutations in individuals of different ethnic origins cause TMAuria in this cohort.

The first two oxygenation steps post-trichodiene in the biosyntheses of the trichothecenes 3-acetyldeoxynivalenol and sambucinol were investigated. The plausible intermediates 2-hydroxytrichodiene (2alpha- and 2beta-) and 12,13-epoxytrichodiene and the dioxygenated compounds 12,13-epoxy-9,10-trichoene-2-ol (2alpha- and 2beta-) were prepared specifically labeled with stable isotopes. They were then fed separately and/or together to Fusarium culmorum cultures, and the derived trichothecenes were isolated, purified, and analyzed. The stable isotopes enable easy localization of the labels in the products by 2H NMR, 13C NMR, and mass spectrometry. We found that 2alpha-hydroxytrichodiene is the first oxygenated step in the biosynthesis of both 3-acetyldeoxynivalenol and sambucinol. The stereoisomer 2beta-hydroxytrichodiene and 12,13-epoxytrichodiene are not biosynthetic intermediates and have not been isolated as metabolites. We also demonstrated that the dioxygenated 12, 13-epoxy-9,10-trichoene-2alpha-ol is a biosynthetic precursor to trichothecenes as had been suggested in a preliminary work. Its stereoisomer was not found in the pathway. A further confirmation of our results was the isolation of both oxygenated trichodiene derivatives 2alpha-hydroxytrichodiene and 12,13-epoxy-9, 10-trichoene-2alpha-ol as natural metabolites in F. culmorum cultures.

The Canadian yew distinguishes itself from other yews by the nature of its taxane metabolites. We are now reporting a new canadensene taxane whose stereochemistry is rigorously established. The three-dimensional structures of canadensene, 5-epi-canadensene and three other related bicyclic taxanes isolated from other yews were calculated using distance constraints derived from NMR data. The stereochemistry of the substituents, the polar acetate groups and the double bonds determine the 3D models. In addition, three new taxanes were also characterized and some biosynthetic speculations are presented.

Abstract —We have previously described patients with familial high density lipoprotein (HDL) deficiency (FHD) having a marked reduction in the plasma concentration of HDL cholesterol and apolipoprotein (apo) A-I but lacking clinical manifestations of Tangier disease or evidence of other known causes of HDL deficiency. To determine whether FHD in these individuals was associated with impaired HDL production or increased HDL catabolism, we investigated the kinetics of plasma apoA-I and apoA-II in two related FHD patients (plasma apoA-I, 17 and 37 mg/dL) and four control subjects (apoA-I, 126±18 mg/dL, mean±SD) by using a primed constant infusion of deuterated leucine. Kinetic analysis of plasma apolipoprotein enrichment curves demonstrated that mature plasma apoA-I production rates (PRs) were similar in patients and control subjects (7.9 and 9.1 versus 10.5±1.7 mg · kg −1 · d −1 ). Residence times (RTs) of mature apoA-I were, however, significantly less in FHD patients (0.79 and 1.66 days) compared with controls (5.32±1.05 days). Essentially normal levels of plasma proapoA-I (the precursor protein of apoA-I) in FHD patients were associated with normal plasma proapoA-I PRs (7.8 and 10.4 versus 10.9±2.6 mg · kg −1 · d −1 ) and proapoA-I RTs (0.18 and 0.15 versus 0.16±0.03 day). The RTs of apoA-II were, however, less in patients (3.17 and 2.92 days) than control subjects (7.24±0.71 days), whereas the PRs of apoA-II were similar (1.8 and 1.9 versus 1.7±0.2 mg · kg −1 · d −1 ). Increased plasma catabolism of apoA-II in FHD patients was associated with the presence in plasma of abnormal apoA-II–HDL (without apoA-I). These results demonstrate that FHD in our patients is characterized, like Tangier disease, by hypercatabolism of mature apoA-I and apoA-II, but unlike Tangier disease, by essentially normal plasma catabolism and concentration of proapoA-I.

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.

We examined the organic chemistry, kinetics, and mechanisms of release of rat serum proteins by aseptic rat donor livers (DL) pathologically intoxicated by CCl4, alloxan, or cholera toxin implanted into the circulatory systems in parallel with the host livers (HL) of guinea pigs (GP). This implant model was biologically functional as hepatocytes of the DL released rat plasma proteins into the host's extracellular compartment (ECC) as monitored in GP serum samples collected during 4 h of the experiment. Furthermore, rat DL hepatocytes retained an in vivo preimplantation pattern of protein biosynthesis acquired in response to a regulatory mechanism most likely involving leukocytic endogenous mediator (LEM), kinins, or platelet activating factor (PAF), and consequently secreted the entire spectrum of acute-phase reaction (APR) serum proteins into the ECC of the host GP. Therefore, the impetus to synthesize and release APR proteins into GP circulation by intoxicated rat liver originated within and was retained by injured rat hepatocytes.

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.

15N,13C6-L-Isoleucine was given by stomach tube to a pair of rats and the urine excreted over the following 6 h period was collected. The urinary amino acid fraction showed that the majority of the L-alloisoleucine produced from the labeled isoleucine was formed with the 15N label intact. This fails to support the commonly held supposition that L-isoleucine and L-alloisoleucine interconversion occurs through the reversible enolization of the 2-keto-3-methylvaleric acids formed by their transamination. In contrast to 15N label conservation in L-alloisoleucine, the majority of the 15N in the administered L-isoleucine underwent exchange with 14N.

The neurotoxin 1-methyl-4-phenylpyridinium (MPP+) has been shown to increase hydroxyl radical formation in the striatum. The production of hydroxyl radicals correlates with the MPP(+)-driven dopamine release which presumably leads to increased metabolism via monoamine oxidase or increased dopamine autoxidation. Both processes result in enhanced production of hydrogen peroxide, which in the presence of iron(II) ions decomposes to the hydroxyl radical. Monoamine oxidase inhibitors decrease the production of hydroxyl radicals as measured by salicylate and 4-hydroxybenzoate trapping. As both MPP+ and monoamine oxidase inhibitors, such as deprenyl and MDL-72,974A, possess aromatic rings, hydroxyl radical adduct formation was investigated in vitro in defined Fenton systems and also in vivo using intra-striatal microdialysis to infuse MPP+ to rats pretreated systemically with either deprenyl or MDL-72,974A. Electrospray mass spectrometric analysis, using full-scan, fragment ion and constant neutral loss spectra, demonstrated ring hydroxylation of all three compounds in the Fenton systems. Spectral comparison of microdialysis samples with spectra from the Fenton reactions indicated the in vivo hydroxyl radical adduct attachment to MPP+, deprenyl and possibly MDL-72,974A.

Irreversible liver cirrhosis was induced in rats by supplementing their diet with 0.02% azathioprine and intubating them twice a week with carbon tetrachloride in corn oil. Over period of 3 mo, intoxicated rats showed an atypical acute-phase reaction (APR). The relative concentrations of haptoglobin, beta-lipoprotein, alpha-1-antitrypsin, an unknown peak "X, " and transferrin increased exponentially following a mild initial drop, while albumin, C3c + C3, alpha-1-acid glycoprotein, alpha-1-lipoprotein, and macroglobulin declined continually during the experiment. The accumulated peritoneal fluid was found to contain a similar spectrum of APR proteins. On the other hand, histological examination revealed gradual liver damage manifested as a gradual increase in the areas of collagen separating liver cells, and at the end of the experiment, severe liver damage was evident with isolated hepatocytes in a matrix of collagen. The available data point to the disparity that exists between the physical status of hepatocytes and their biochemical function, which suggests that the remaining metabolically fatigued hepatocytes of the cirrhotic liver continue to biosynthesize and release elevated concentrations of some secretable APR proteins and less of others. Changes in the spectrum of APR plasma components during the progression of inflammatory or physical lesion remain a valid biochemical measure of the pathological function of the acutely intoxicated liver. Partial hepatectomy (PH) of cirrhotic liver displayed a mute APR and no regenerative activity of the remnant hepatic tissue, most likely due to the substantial depletion of hepatic DNA and possible chemical damage to DNA of the remaining viable hepatocytes. A possible cause for the depressed APR to the surgical insult of PH is that the initial azathioprine-CCl4 intoxication had maximally affected APR gene expression and a second injury would then elicit minimal further changes in mRNA levels. Thus, in a compounded pathological condition, the initial inflammatory stimulus on various pre-rRNAs, rRNAs, and mRNAs is rate-limiting to the hepatic biosynthesis and secretion of APR proteins and may not respond linearly, if at all, to a second stimulus.

A new interface has been developed for direct analysis of volatile organic compounds (VOCs) from aqueous solutions without prior sample treatment. The device consists of a nozzle directed vertically downward in a spray chamber, which is in turn coupled through a helium jet separator to an ion trap mass spectrometer. The nozzle reduces the liquid to an aerosol with the help of a helium carrier gas and rapid sample injection. The large surface area of the aerosol permits a rapid equilibration of the VOCs between the gas and liquid phases. The aerosol droplets impinge by their inertia on the floor of the spray chamber, where they coalesce for subsequent removal from the interface. The VOCs are carried from the interface by the helium flowing countercurrent to the spray. The combined VOC-helium mixture passes through a jet separator, which removes most of the carrier gas, and the concentrated VOCs are then analyzed by an ion trap mass spectrometer. This inertial spray extraction mass spectrometry method is very sensitive and has detection limits of

DDT and several of its metabolites were synthesized with their aromatic chlorines enriched with 37Cl. The mass spectra of these compounds were recorded under electron impact and under methane positive-ion and electroncapture negative-ion chemical ionization. The regioselectivity of the dechlorination reactions was determined by measurement of the relative proportions of the unlabeled aliphatic versus the labeled aromatic chlorines in the various fragment ions observed. For all the α,β-unsaturated compounds studied under electron impact ionization, the regioslectivity is small, as both the aromatic and the aliphatic chlorines are lost. Under positive-ion chemical ionization, the dechlorination is highly regioselective, the aliphatic chlorines being almost exclusively lost. Under electron-capture negative-ion chemical ionization, the regioselectivity is also very high and the reactivity of the various chlorines is often controlled by the position of the aromatic chlorines.

SummaryAMPK 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.

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. Electronic supplementary material The online version of this article (doi:10.1007/s11306-013-0521-1) contains supplementary material, which is available to authorized users.

This article will present a personal view of metabolic profiling by mass spectrometry, predominantly gas chromatography/mass spectrometry (GC/MS)—its strengths and its weaknesses, its needs and its wants, and how it might best be used and, perhaps, how it should not. This will be my own perspective on this subject—if it seems narrow of scope it will be so because it has become difficult to be a successful ‘Jack-of-all-trades’ in this business.

γ-Irradiation of DDT and its metabolites DDD and DDE was performed in 2-propanol, cyclohexane, and cyclohexene. The degradation yields of these compounds were quantitated and the dechlorinated products identified by gas chromatography/mass spectrometry. Compounds corresponding to addition products between the solvent and these organochlorines were isolated and identified by mass spectrometry and NMR. Oxidation products of these organochlorines were also observed