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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.

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.

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 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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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.

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., (2010 Elsevier Inc. All rights reserved.)

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., (2010 John Wiley & Sons, Ltd.)

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 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., (Copyright 2009 John Wiley & Sons, Ltd.)

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., (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.

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.

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.

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.

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.

Electrospray and matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS/MS) experiments were used to investigate an unusual fragmentation in collision-induced dissociation (CID) of sodiated and potassiated perbenzyl ether intermediates obtained in the total synthesis of gallate ester constituents of green tea. Prominent fragments correspond to multiple sequential losses of neutral C14H14 that were not observed in the protonated and ammoniated species, that instead present fragment ion series in which members are separated by C7H6. High-resolution MALDI quadrupole time-of-flight (Q-TOF) and electrospray-Fourier transform mass spectrometry (FTMS) were used to confirm elemental compositions of these and related ions., (Copyright (c) 2005 John Wiley & Sons, Ltd.)

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.

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 gamma-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 individual's 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.

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.

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 alpha-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 15N label denying the intermediacy of the alpha-keto acids, and that alloisoleucine arises as an unavoidable consequence of isoleucine transamination.

Trimethylaminuria (TMAU) results from an accumulation of an excessive amount of unoxidized trimethylamine that is excreted in urine and body secretions. Mutations of the flavin-containing monooxygenase 3 (FMO3) gene (a hepatic phase I drug-metabolizing enzyme) account for the severe recessively encoded form of this condition. We have previously described a number of FMO3 polymorphisms which in vitro exhibit reduced substrate affinity for several FMO substrates. Here we show that three prevalent polymorphisms (E158K, V257M, and E308G) inherited in particular combinations confer a slight decrease in TMA oxidation under normal physiological conditions, which may be clinically "silent." With the use of substrate loading or with the interaction of other known modulators of FMO3 activity such as hormonal influences, these genotypes may predispose to mild TMAU., (Copyright 2001 Academic Press.)

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 33-35;C. 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 alpha-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., (Copyright 2001 Harcourt Publishers Ltd.)

An electrospray mass spectrometric method for the quantification of the depolarizing neuromuscular blocking agent succinylcholine (SUX) is described. An extraction method compatible with direct infusion inlet was developed and leads to an analysis cycle time of 7--8 min instead of 25 min that would be required for HPLC inlet. SUX was extracted from human plasma on C1 solid-phase cartridges and was analyzed using positive ion electrospray tandem mass spectrometry (ESI-MS/MS). SUX plasma concentrations were determined by a stable isotope dilution assay using hexadeuterosuccinylcholine diiodide (SUXd6) as the internal standard. The calibration curve was prepared using the ratio of intensities of the major product ions in the collision-induced dissociation spectrum for known concentration ratios of SUX and SUXd6 in plasma. Calibration curves for the quantification were linear from 25 to 4000 ng/ml. For intraday precision, CV were < or =6% and accuracy ranged from 98 to 103%. For the interday precision, CV were < or =10% and accuracy ranged from 90 to 102%. This method is specific, sensitive, reproducible, and practical in a clinical setting., (Copyright 2001 Academic Press.)

Objective: The authors found considerably lower plasma total homocysteine (tHcy) concentrations in patients with end-stage renal disease (ESRD) on maintenance hemodialysis, who routinely received high-dose parenteral vitamin B12, than in comparable patients receiving much higher doses of folic acid but only replacement-dose oral vitamin B12. They therefore sought prospective evidence that high-dose parenterally administered vitamin B12 may partially ameliorate renal failure-associated hyperhomocysteinemia., Design: Open phase 2 clinical trial., Setting: Outpatient hemodialysis unit., Patients: Fourteen clinically stable patients on maintenance hemodialysis with normal baseline serum vitamin B12 concentrations., Intervention: Three parenteral injections of 1 mg vitamin B12 given at 4-week intervals., Outcome Measures: Plasma tHcy and serum vitamin B12 concentrations were measured before, during and 7 months after the termination of vitamin B12 therapy., Results: The mean (and standard error) baseline plasma tHcy was 26.5 (1.8) micromol/L. The plasma tHcy value fell successively after each vitamin injection to reach a value of 23.6 (1.6) micromol/L 1 month after the final injection (p < 0.05), while the serum vitamin B12 concentration increased from 471 (42) pmol/L to 890 (74) pmol/L (p < 0.05). Seven months after the final injection, the serum B12 concentration had fallen and tHcy had risen to near their original values., Conclusions: Three monthly vitamin B12 injections modestly but distinctly reduced tHcy concentrations in hemodialysis patients whose prior vitamin B12 status was normal. Randomized placebo-controlled clinical trials of longer duration and using larger or more frequent parenteral doses are indicated to determine whether administration of this safe and inexpensive vitamin can improve hyperhomocysteinemia in ESRD.

Background: Plasma total homocysteine (tHcy) concentrations> 15 micromol/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 micromol/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 (r2 = 0.39, P < 0.0001)., 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.

Porin of Haemophilus influenzae type b (341 amino acids; M(r) 37782) determines the permeability of the outer membrane to low molecular mass compounds. Purified Hib porin was subjected to chemical modification of lysine residues by succinic anhydride. Electrospray ionization mass spectrometry identified up to 12 modifications per porin molecule. Tryptic digestion of modified Hib porin followed by reverse phase chromatography and matrix assisted laser desorption ionization time-of-flight mass spectrometry mapped the succinylation sites. Most modified lysines are positioned in surface-located loops, numbers 1 and 4 to 7. Succinylated porin was reconstituted into planar lipid bilayers, and biophysical properties were analyzed and compared to Hib porin: there was an increased average single channel conductance compared to Hib porin (1.24 +/- 0.41 vs. 0.85 +/- 0.40 nanosiemens). The voltage-gating activity of succinylated porin differed considerably from that of Hib porin. The threshold voltage for gating was decreased from 75 to 40 mV. At 80 mV, steady-state conductance for succinylated porin was 50-55% of the instantaneous conductance. Hib porin at 80 mV showed a decrease to 89-91% of the instantaneous current levels. We propose that surface-located lysine residues are determinants of voltage gating for porin of Haemophilus influenzae type b.

Phenylketonuria (PKU) (OMIM 261600) is the first Mendelian disease to have an identified chemical cause of impaired cognitive development. The disease is accompanied by hyperphenylalaninemia (HPA) and elevated levels of phenylalanine metabolites (phenylacetate (PAA), phenyllactate (PLA), and phenylpyruvate (PPA)) in body fluids. Here we describe a method to determine the concentrations of PAA, PPA, and PLA in the brain of normal and mutant orthologous mice, the latter being models of human PKU and non-PKU HPA. Stable isotope dilution techniques are employed with the use of [(2)H(5)]-phenylacetic acid and [2,3, 3-(2)H(3)]-3-phenyllactic acid as internal standards. Negative ion chemical ionization (NICI)-GC/MS analyses are performed on the pentafluorobenzyl ester derivatives formed in situ in brain homogenates. Unstable PPA in the homogenate is reduced by NaB(2)H(4) to stable PLA, which is labeled with a single deuterium and discriminated from endogenous PLA in the mass spectrometer on that basis. The method demonstrates that these metabolites are easily measured in normal mouse brain and are elevated moderately in HPA mice and greatly in PKU mice. However, their concentrations are not sufficient in PKU to be "toxic"; phenylalanine itself remains the chemical candidate causing impaired cognitive development., (Copyright 2000 Academic Press.)

We report a method based upon fast atom bombardment mass spectrometry (FAB-MS) and stable isotope dilution techniques for the measurement of urinary trimethylamine (TMA) and trimethylamine N-oxide (TMAOx). TMA is extracted from urine that was spiked with (15)N-labeled TMA. The extracted TMA isotopomers are quaternized with trideuteromethyl iodide and analyzed in FAB-MS with hexaethylene glycol as matrix. TMAOx is measured by evaporation of another sample of the urine spiked with (15)N-labeled TMAOx on the FAB probe and analyzed as for the TMA. The method allows the ready and simple distinguishing of controls and patients with TMAuria, and is useful in monitoring patients with the disorder. We give examples of its use in determining normal control ranges for these metabolites and in evaluating patients., (Copyright 1999 Academic Press.)

Trimethylsilylation of methylmalonic and ethylmalonic acids in the presence of headspace atmospheric oxygen is shown to produce the trimethylsilyl derivatives of pyruvic and 2-oxobutyric acids, along with 2-hydroxy-2-methylmalonic and 2-hydroxy-2-ethylmalonic acids, respectively. This may lead to overestimation of these keto acids, if they were not oximated in the original sample, and the mistaken reporting of the 2-hydroxymalonates.

Liquid chromatography/mass spectrometry using electrospray ionisation was used to analyse rhamnolipids produced by a Pseudomonas aeruginosa strain with mannitol or naphthalene as carbon source. Identification and quantification of 28 different rhamnolipid congeners was accomplished using a reverse-phase C(18) column and a 30 min chromatographic run. Isomeric rhamnolipids that were not chromatographically resolved could be identified by interpretation of their mass spectra and their relative proportions estimated. The most abundant rhamnolipid produced on mannitol contained two rhamnoses and two 3-hydroxydecanoic acid groups. The most abundant rhamnolipid produced from naphthalene contained two rhamnoses and one 3-hydroxydecanoic acid group.

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., (Copyright 1999 Academic Press.)

The volume of human extracellular water (ECW) may be estimated from the sulfate space (SS). Although it may better approximate ECW volume than the bromide space, a common alternative, SS measurement is limited by the need to administer a radioactive substance, sodium [35S]sulfate. In this paper, we demonstrate the measurement of the SS using the stable isotope, sodium [34S]sulfate. Eight healthy nonobese men ingested 0.50-0.78 mg (3.47-5.42 micromol) Na234SO4/kg body weight and 30 mg NaBr/kg body weight. Sulfate concentrations and 34SO4 enrichments were measured by electrospray tandem mass spectrometry before and during the 5 h after tracer administration. SS was calculated by linear extrapolation of the natural logarithm of serum 34SO4 concentrations obtained at h 2, 3 and 4 compared with h 3, 4 and 5. The SS obtained using values between h 3 and 5 (187 +/- 17 mL/kg) was similar to published determinations using intravenous or oral radiosulfate, and was 80% of the simultaneously measured corrected bromide space (234 +/- 10 mL/kg, P = 0.01). Oral sodium [34S]sulfate administration is a suitable technique for measuring ECW and avoids radiation exposure.

A reproducible and very sensitive method is described for the quantitation of inorganic sulfate in biological fluids by negative electrospray ionization tandem mass spectrometry. After addition to the sample of (34)S-labeled sodium sulfate internal standard and deproteinization with methanol, interfering bicarbonate anions are removed by acidification and chloride and phosphate by means of a single filtration step. The tandem mass spectrometer is used in neutral loss mode to detect HSO(4)(-) ions free of interference from residual isobaric H(2)PO(4)(-) ions. Organic sulfates do not interfere with the measurement. Serum and urinary inorganic sulfate concentrations measured with this technique agree closely with determinations by ion-exchange chromatography with conductivity detection. Unlike the latter method, this technique does not require dedicated equipment. The method is also suitable for measuring the ratio of (34)S-labeled sulfate to unlabeled sulfate in serum and hence represents an attractive alternative for the use of the radioactive (35)S isotope in human studies of body composition and oxidation of sulfur-containing substrates to sulfate., (Copyright 1998 Academic Press.)

Butter lipids are an important traditional source of dietary energy intake in the form of fat. Butter lost a sizable portion of its market share due to controversies associated with its cholesterol content and high percentage of long-chain saturated fatty acids. Accordingly, the use of vegetable oils and their chemically manipulated counterparts such as those produced by partial hydrogenation or interestrification increased proportionally. However, beginning in 1940, researchers developed several procedures such as temperature-controlled crystallization, refractionation of crystallized butter oil solids, and supercritical carbon dioxide extraction to improve the acceptance of butter oil. Others proposed preparation of synthetic substitutes such as sucrose polyesters to reduce intestinal absorption of fatty acids, thus reducing caloric intake with concomitant reduction in serum cholesterol. The present review provides a summary of the efforts of several attempts to improve the acceptability of butter together with the anticipated epidemiological consequences of long-term consumption of altered butter oil to mammalian health.

We tested whether expansion of the plasma leucine pool distorts leucine or valine tracer kinetics, causing errors in the derived values of whole body proteolysis. Seven normal adults received a 10-h primed-continuous tracer infusion of L-[5,5,5-2H3]leucine, L-[(1-13)C]valine, and L-[(1-13)C]threonine, during the final 7 h of which L-leucine was infused at a rate that more than tripled the plasma leucine concentration. Leucine, valine, and threonine rates of appearance were converted to a common value of whole body proteolysis on the basis of their concentrations in body proteins. The conversion of labeled leucine and valine to their corresponding branched-chain alpha-keto and alpha-hydroxy acids was also monitored. Before the unlabeled leucine infusion, postabsorptive whole body proteolysis was estimated similarly by the three tracers (approximately 180 mg protein.kg-1.h-1. The leucine infusion reduced proteolysis by an average of 21% (P < 0.006), as estimated by use of valine or threonine kinetics, and by 10% by use of leucine kinetics (P < 0.02). No delay in the conversion of valine to alpha-ketoisovalerate occurred during the leucine infusion. Thus all three tracers indicated similar postabsorptive rates of whole body proteolysis and a reduction of proteolysis during leucine administration, although the magnitude of the effect was underestimated with use of the leucine tracer.

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 reactions of chloroaromatic radical anions with oxygen were studied with a triple quadrupole mass spectrometer. Two chlorobenzenes and eight polychlorinated biphenyls were analyzed by gas chromatography-tandem mass spectrometry under negative ion chemical ionization. The molecular radical anions were selected with the first quadrupole and reacted with oxygen in the collision cell. Under these conditions, [M+O - Cl] ions were obtained with intensities similar to those of the transmitted precursor ions. This dechlorination reaction was not affected by a detectable chlorine isotope effect. The intensities of the [M+O - Cl] ions vary with the nature of the chloroaromatic compounds and with the oxygen pressure and collision energy. Charge transfer reactions are also observed, and the relative amount of O 2 (-.) produced is controlled by the relative electron affinity of the organochlorine. At high collision energies, collision-induced fragmentation of the molecular ion competes for the production of Cl(-).

The title of this article is taken from an interesting Letter to the Editor entitled 'Artificial liver support-Pipe dream or reality' by Cattral and Levy of the Toronto Hospital, Canada, published in the New England Journal of Medicine 1994, in which the authors persuasively propose possibilities of artificial liver support and suggest its advantages. We find that their suggestions agree with the core of our thoughts on this subject. The present article deals with the concept of implanting livers taken from humans, primates or non-primates (e.g. hog) into patients in parallel with their own metabolically fatigued or cirrhotic livers, with minimal surgical manipulation, as a prelude to total artificial liver support via a liver dialysis device. While the possibility exists that the host liver may recover function, a donor liver, whether implanted into the patient's abdomen or connected in vitro to the patient's circulatory system extracorporeally, may provide the host liver respite and a period for recovery and proliferation, if possible. Once recovery is under way, the donor liver may be removed and the patient will not experience the usual risks of rejection and the necessary side-effects of immunosuppression associated with conventional full hepatectomy and donor transplantation. The viability of a liver implantation model in rats is correlated in this article with hepatic acute phase response.

The bioorganic pathway(s) of hepatic acute-phase response in rat to single and compounded traumata triggered either by chemical or physical injury has been re-evaluated for the purpose of advancing a better understanding of mechanisms of hepatic regeneration. These insights would be useful in cases of liver cirrhosis and end-stage liver diseases and may allow avenues of surgical management other than liver transplantation. Mechanisms of acute-phase response in rat to a single inflammatory stimulus, e.g. intoxication with phalloidin, alpha-amanitin, subcutaneous administration of carageenan, subcutaneous implantation of Yoshida sarcoma or i.p. administration of Zajdela ascites are discussed and compared with (a) acute-phase response to intoxication by various factors leading to the development of liver cirrhosis, and (b) acute-phase response of nascent hepatocytes where hepatic regenerative activities were induced by chemical intoxication or surgical partial hepatectomy. Interestingly, hepatic acute-phase response was not limited only to these injuries outlined above but also to psychological conditions.

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.

The electron capture mass spectra of 28 (35)Cl-labeled polychlorinated biphenyls (PCBs) and 4 (37)Cl-labeled 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) metabolites were obtained by using a 20% oxygen in methane mixture as the reagent gas. The degree of regioselectivity of the PCB oxygen addition-induced dechlorination reaction was determined by measurement of the residual amount of label in the M-19 ions produced by addition of O2 and subsequent loss of OCl from the molecule. Chlorine was lost in a random manner from the PCBs, contrary to the dechlorination reaction observed when methane alone was used. For the DDT metabolites, many dechlorination reactions were observed in addition to the one that generated the M-19 ions. Loss of Cl, loss of Cl2, and addition of O2 with the loss of one or two HCl molecules also were seen. These various dechlorination reactions involved only the aliphatic chlorines. Addition of O2 followed by loss of Cl at the beta position of 2,2-bis(4-(37)Cl-chlorophenyl)-1-chloroethylene and 2,2-bis(4-(37)Cl-chlorophenyl)-1,1-dichloroethylene may be due to the ability of the diphenyl methane moiety to stabilize the intermediates. Formation of an ion that corresponds to 4,4'-dichlorobenzophenone also was observed for three of these labeled DDT metabolites.

The mechanism(s) of liver damage initiated by ingestion of toxic components of thermally oxidized lipids was compared in a rat model with the documented mechanisms of hepatic failure and necrosis initiated by acetaminophen. Acetaminophen (50 mg/kg body weight) or oxidized lipids (0.15 ml oxidized trilinolein or 1.05 ml oxidized butter oil per rat) were intubated at 12-h intervals to rats. Treated rats were allowed free access to food and water containing 3% ethanol. Changes in relative concentration of acute-phase plasma proteins, determined by two-dimensional (2D) immunoelectrophoresis, were taken as a marker of liver damage. In contrast to simple inflammation, acute-phase plasma proteins in this study disproportionately increased or decreased as histological damage of the liver due to intubation oxidized lipids or acetaminophen. Histological examination of liver of rats intoxicated with oxidized lipids revealed severe liver cirrhosis at the end of the trial, where the remaining viable hepatocytes were separated in a matrix of collagen. [3H1]Thymidine incorporation in hepatic DNA of acetaminophen or oxidized lipid intoxication increased in the early stages of intoxication, indicative of regenerative activity of the liver. Further progression of the cirrhosis inhibited continued liver regeneration and [3H1]thymidine incorporation into hepatic DNA. The cirrhotic liver at this stage failed to regenerate to the original mass upon 75% partial hepatectomy. Therefore, it may be concluded that hepatic necrosis produced by oxidized lipids or by acetaminophen may have similar mechanisms.

Several acylating reagents are synthesized and used to introduce quatemary phosphonium or ammonium or ternary sulfonium functions into a simple model of a peptido leukotriene (PLT). One of these reagents was selected for further study with LTE4, LTD4, and LTC4. We demonstrate that acylation of the free amine function of PLTs to produce the 5-triphenylphosphoniumvaleryl-amide (TPPV) derivatives enhances chemical stabilities and significantly increases responses in fast-atom bombardment and continuous-flow liquid secondary ion mass spectrometry (CF-LSIMS) relative to the native PLTs. With high-performance liquid chromatography inlet to CF-LSIMS, we demonstrate the facile detection in selected ion monitoring of the TPPV derivative of 3 pg of LTD4.

Plasma concentrations of the branched-chain amino acids leucine, isoleucine and valine, and those of leucine's and isoleucine's transamination products alpha-ketoisocaproic acid (KICA) and alpha-keto-beta-methylvaleric acid (KMVA), respectively, are known to increase after a protein meal or during extended fasting, but little or no increase in the concentration of valine's transamination product, alpha-ketoisovaleric acid (KIVA), has been observed under these conditions. To determine whether this could be explained by the conversion of KIVA to its alpha-hydroxy analogue, we measured the plasma concentrations of KICA, KMVA and KIVA, as well as their alpha-hydroxy analogues [alpha-hydroxyisocaproic acid (HICA), alpha-hydroxy-beta-methylvaleric acid (HMVA) and alpha-hydroxyisovaleric acid (HIVA)], in normal volunteers immediately after a protein meal or during a 60-h fast. We also determined the oxidoreduction equilibrium constants for HIVA/KIVA and HICA/KICA and their extent of plasma protein binding. In subjects in the postabsorptive state, the plasma concentrations of KICA and KMVA were 100 times those of HICA and HMVA, whereas that of KIVA was only twice that of HIVA. Shortly after a protein meal, KICA and KMVA concentrations increased significantly by 30 and 60%, respectively, whereas that of KIVA decreased by 25% (P < 0.05). HICA, HMVA and HIVA concentrations did not change. During prolonged fasting the plasma concentrations of all six metabolites increased gradually. The high plasma keto/hydroxy acid ratios were not related to their K(eq), which favored alpha-hydroxy analogue formation. The reduction of the branched-chain alpha-keto acids to their alpha-hydroxy analogues seems to take place too slowly to attain thermodynamic equilibrium.(ABSTRACT TRUNCATED AT 250 WORDS)