RESUMO
Precise measurement of sugar phosphates in glycolysis and the pentose phosphate (PP) pathway for 13C-metabolic flux analysis (13C-MFA) is needed to understand cancer-specific metabolism. Although various analytical methods have been proposed, analysis of sugar phosphates is challenging because of the structural similarity of various isomers and low intracellular abundance. In this study, gas chromatography-negative chemical ionization-mass spectrometry (GC-NCI-MS) is applied to sugar phosphate analysis with o-(2,3,4,5,6-pentafluorobenzyl) oxime (PFBO) and trimethylsilyl (TMS) derivatization. Optimization of the GC temperature gradient achieved baseline separation of sugar phosphates in 31â¯min. Mass spectra showed the predominant generation of fragment ions containing all carbon atoms in the sugar phosphate backbone. The limit of detection of pentose 5-phosphates and hexose 6-phosphates was 10â¯nM. The method was applied to 13C-labeling measurement of sugar phosphates for 13C-MFA of the MCF-7 human breast cancer cell line. 13C-labeling of sugar phosphates for 13C-MFA improved the estimation of the net flux and reversible flux of bidirectional reactions in glycolysis and the PP pathway.
Assuntos
Linhagem Celular Tumoral , Análise do Fluxo Metabólico/métodos , Fosfatos Açúcares/análise , Metabolismo Energético , Feminino , Fluorbenzenos , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Isomerismo , Células MCF-7 , Oximas , Espectrometria de Massas por Ionização por Electrospray , Compostos de Trimetilsilil/metabolismoRESUMO
The effect of ractopamine hydrochloride (RH) and zilpaterol hydrochloride (ZH) on slice shear force (SSF) and sensory characteristics of beef from calf-fed Holstein steers was evaluated. All steers were implanted with a progesterone (100 mg) plus estradiol benzoate (10 mg) implant followed by a terminal trenbolone acetate (200 mg) plus estradiol (40 mg) implant. Steers were blocked by weight into pens (n = 32) randomly assigned to 1 of 4 treatments: control, RH fed at 300 mg·steer(-1)·d(-1) (RH 300) or RH fed at 400 mg·steer(-1)·d(-1)(RH 400) for the final 31 d of finishing, or ZH fed at 6.8 g/t for 21 d with a 5-d withdrawal before harvest. Fourteen carcasses were randomly selected from each pen, and two LM samples (1 per side) were excised and aged either 14 or 21 d before SSF testing. For trained panel evaluation, two steaks were collected from each of 60 low Choice strip loins (20 each from control, RH 300, and ZH treatments) and aged either 14 or 21 d. Steers fed RH and ZH produced steaks with SSF values that were 9% to 25% higher than controls. No difference in SSF was detected between the two levels of RH (P > 0.05). Compared to controls, the probability of steaks aged 14 d failing to meet SSF requirements to be certified tender (SSF < 20 kg) was increased 0.15, 0.17, and 0.26 in steers fed RH 300, RH 400, and ZH, respectively. Compared to controls, the probability of steaks aged 21 d having SSF values >20 kg was increased 0.03, 0.08, and 0.16 in steers fed RH 300, RH 400, and ZH, respectively. Steaks from Select carcasses of steers fed ZH aged 21 d postmortem had double the probability (0.39 vs. 0.17) of having SSF values >20 kg compared to steaks from steers fed either level of RH (P < 0.05). This difference tended to be identical in steaks from Select carcasses 14 d postmortem (0.50 vs. 0.33; P = 0.11); however, no difference was found in low Choice samples at 14 or 21 d postmortem. Trained panelists rated steaks aged 14 d from steers fed ZH lower for overall tenderness and flavor compared to controls (P < 0.05); however, no difference was found between controls and those fed RH 300. Steaks from steers fed ZH aged 21 d were rated lower for overall tenderness and juiciness compared to controls and those from steers fed RH 300 (P < 0.05). This study suggests RH and ZH negatively impact sensory attributes of beef from calf-fed Holstein steers.
Assuntos
Agonistas Adrenérgicos beta/metabolismo , Composição Corporal/efeitos dos fármacos , Bovinos/fisiologia , Carne/análise , Músculos Paraespinais/efeitos dos fármacos , Fenetilaminas/metabolismo , Compostos de Trimetilsilil/metabolismo , Agonistas Adrenérgicos beta/administração & dosagem , Ração Animal/análise , Animais , Dieta/veterinária , Suplementos Nutricionais/análise , Implantes de Medicamento/administração & dosagem , Implantes de Medicamento/farmacologia , Estradiol/administração & dosagem , Estradiol/análogos & derivados , Estradiol/farmacologia , Masculino , Músculos Paraespinais/fisiologia , Fenetilaminas/administração & dosagem , Progesterona/administração & dosagem , Progesterona/farmacologia , Resistência ao Cisalhamento , Acetato de Trembolona/administração & dosagem , Acetato de Trembolona/farmacologia , Compostos de Trimetilsilil/administração & dosagemRESUMO
Effects of ractopamine hydrochloride (RH) and zilpaterol hydrochloride (ZH) on saleable yield of carcass sides from calf-fed Holstein steers were evaluated using steers implanted with a progesterone (100 mg) plus estradiol benzoate (10 mg) implant followed by a terminal trenbolone acetate (200 mg) plus estradiol (40 mg) implant. Steers were blocked by weight into pens (n = 32) randomly assigned to one of four treatments: control, RH fed at 300 mgâ¢steer(-1)/d(-1) (RH 300) or RH fed at 400 mgâ¢steer(-1)/d(-1) (RH 400) the final 31 d of finishing, and ZH fed at 60 to 90 mgâ¢steer(-1)/d(-1) (7.56 g/ton on a 100% DM basis) for 21 d with a 5 d withdrawal before harvest. Eight to nine carcass sides were randomly selected from each pen; carcass sides with excessive hide pulls, fat pulls or bruises were avoided. Cutout data were collected within a commercial facility using plant personnel to fabricate sides at a rate of one every 3 to 4 min into items typically merchandised by the facility. All lean, fat and bone were weighed and summed back to total chilled side weight with a sensitivity of ± 2% to be included in the data set. Compared to controls, ß-agonists increased saleable yield of whole-muscle cuts by 0.61%, 0.86% and 1.95% for RH 300, RH 400 and ZH, respectively (P < 0.05). Percent fat was less in carcasses from the ZH treatment compared to controls (P < 0.05); however, this difference was not observed between RH treatments and controls (P > 0.05). Percent bone was less in the ZH treatment due to increased muscle (P < 0.05). The percent of chilled side weight comprised of trimmings was unchanged between treatments, but on a 100% lean basis, RH 400 and ZH increased trim yields (P < 0.05). Analysis of saleable yield by primal showed a fundamental shift in growth and development. Beta-agonists caused a shift in proportion of saleable yield within individual primals, with a greater portion produced from the hindquarter relative to the forequarter, specifically in those muscles of the round (P < 0.05). Beta-agonists increased saleable yield, but these effects were not constant between all major primals. The cutout value gained by packers as a result of ß-agonist use may be influenced more by reduced fatness and increased absolute weight if musculature is primarily increased in the lower priced cuts of the carcass.
Assuntos
Agonistas Adrenérgicos beta/metabolismo , Composição Corporal/efeitos dos fármacos , Bovinos/fisiologia , Carne/análise , Músculo Esquelético/efeitos dos fármacos , Fenetilaminas/metabolismo , Compostos de Trimetilsilil/metabolismo , Agonistas Adrenérgicos beta/administração & dosagem , Ração Animal/análise , Animais , Dieta/veterinária , Suplementos Nutricionais/análise , Implantes de Medicamento/administração & dosagem , Implantes de Medicamento/farmacologia , Estradiol/administração & dosagem , Estradiol/análogos & derivados , Estradiol/farmacologia , Masculino , Fenetilaminas/administração & dosagem , Progesterona/administração & dosagem , Progesterona/farmacologia , Acetato de Trembolona/administração & dosagem , Acetato de Trembolona/farmacologia , Compostos de Trimetilsilil/administração & dosagemRESUMO
The Alzheimer's Abeta40 peptide forms soluble oligomers that are extremely potent neurotoxins and strongly impede synapses function. In this study the formation and structure of the large, soluble, neurotoxic Abeta40 oligomer called "beta-ball" were characterized by two-dimensional NMR, circular dichroism, fluorescence spectroscopy, hydrogen exchange, and equilibrium sedimentation. In acidic aqueous solution, half the Abeta40 molecules are in the beta-ball state; the remainder are monomeric. The equilibrium between the two states is slow as judged by NMR linewidths and is stable for months. The kinetics of beta-ball formation from monomer are biphasic with tau1 = 7 min and tau2 = 80 min with no transient helix formation. Monomeric Abeta40 is essentially devoid of stable secondary structure, although the central, Leu17-Ala21, and C-terminal, Gly29-Val40, hydrophobic regions show propensity toward adopting extended structure, and residues 22-25 tended to form a turn. We found that sodium 4,4-dimethyl-4-silapentane-1-sulfonate (DSS) binds to the central hydrophobic region of monomeric Abeta40. DSS binds beta-balls more strongly and caused them to double in size. Plausible micelle-like models for the beta-ball structure with and without bound DSS are presented.
Assuntos
Ácidos Alcanossulfônicos/metabolismo , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/toxicidade , Neurônios/efeitos dos fármacos , Compostos de Organossilício/metabolismo , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidade , Compostos de Trimetilsilil/metabolismo , Sequência de Aminoácidos , Peptídeos beta-Amiloides/química , Animais , Dicroísmo Circular , Humanos , Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Neurotoxinas/química , Neurotoxinas/metabolismo , Neurotoxinas/toxicidade , Ressonância Magnética Nuclear Biomolecular , Células PC12 , Fragmentos de Peptídeos/química , Estrutura Secundária de Proteína , RatosRESUMO
An evaluation of derivatization procedures for the detection of beta(2)-agonists is presented. The study was performed with the beta(2)-agonists bambuterol, clenbuterol, fenoterol, formoterol, salbutamol, salmeterol and terbutaline. Different derivatizating agents were employed, aiming to obtain derivatives with high selectivity to be used in the gas chromatographic/mass spectrometric analysis of beta(2)-agonists in biological samples. Trimethylsilylation was compared with different agents and the role of some catalysts was evaluated. Acylation, combined trimethylsilylation and acylation, and the formation of cyclic methylboronates were also studied. Sterical hindrance caused by different substituents at the nitrogen atom of the beta-ethanolamine lateral chain of beta(2)-agonist molecules is mainly responsible for differences in the abundances of the derivatives obtained. The use of catalysts produces an increase in the derivatization yield, especially for compounds with low steric hindrance (substituents with primary and secondary carbon atoms). The formation of trimethylsilyl (TMS) ethers is not influenced by structural molecular differences when only hydroxy groups are involved in derivatization. Combined trimethylsilylation and acylation showed that compounds with a secondary carbon atom linked to the nitrogen atom form mainly N-TFA-O-TMS derivatives, with a small amount of N-TMS-O-TMS derivatives. Compounds with tert-butyl substituents at the amino group (bambuterol, salbutamol and terbutaline) formed O-TMS derivatives as the main products, although a limited amount of trifluoroacylation at the nitrogen atom also occurred. Cyclic methylboronates were formed with bambuterol, clenbuterol, formoterol, salbutamol and salmeterol. Owing to hydroxy substituents in unsuitable positions for ring formation, this procedure was not effective for fenoterol and terbutaline. Mass spectra of different derivatives and tentative fragmentation profiles are also shown. For screening purpose (e.g. sports drug testing), derivatization with MSTFA or BSTFA alone is recommended as a comprehensive derivatization technique for beta(2)-agonists owing to minimal by-product formation; formation of cyclic methylboronates can be useful for confirmation purposes. Detection limits were obtained for the TMS and cyclic methylboronate derivatives using the derivatizing reagents MSTFA and trimethylboroxine, respectively. For most of the compounds, lower detection limits were found for the TMS derivatives.
Assuntos
Agonistas Adrenérgicos beta/análise , Agonistas Adrenérgicos beta/química , Cromatografia Gasosa-Espectrometria de Massas/métodos , Acetamidas , Acilação , Agonistas Adrenérgicos beta/metabolismo , Agonistas Adrenérgicos beta/urina , Compostos de Boro/análise , Compostos de Boro/química , Compostos de Boro/metabolismo , Avaliação Pré-Clínica de Medicamentos , Fluoracetatos , Estrutura Molecular , Sensibilidade e Especificidade , Ácido Trifluoracético/química , Ácido Trifluoracético/metabolismo , Compostos de Trimetilsilil/química , Compostos de Trimetilsilil/metabolismoRESUMO
Peroxynitrite (ONOO-) can react with a wide range of biomolecules resulting in peroxidation, oxidation, and/or nitration and as a consequence cause their inactivation. In this study mass spectrometry (MS) combined with both liquid (LC) and gas chromatography (GC) has been employed to identify the products formed following ONOO- treatment of three peptides at physiological pH: leucine-enkephalin (YGGFL), V3 loop (GPGRAF), and LVV-hemorphin7 (LVVYPWTQRF). LC-MS analysis of leucine-enkephalin following ONOO treatment indicated the formation of products corresponding in mass to mono- and dinitrated forms of the starting material. LC-MS-MS and GC-MS analyses revealed no evidence for the formation of nitrophenylalanine; however, both 3-nitrotyrosine and 3,5-dinitrotyrosine were observed and characterized. GC-MS analysis of hydrolyzed peptides following ONOO- treatment confirmed the presence of nitrated and dinitrated tyrosine. However, when a 20-fold molar excess of ONOO- was reacted with leucine-enkephalin, only about half of the tyrosine originally present in the peptide could be accounted for in the acid hydrolysate. The main product was 3-nitrotyrosine which represented ca. 50% of the original tyrosine; traces of 3,5-dinitrotyrosine (ca. 3% of the original tyrosine) were also present.
Assuntos
Encefalina Leucina/metabolismo , Hemoglobinas/metabolismo , Nitratos/metabolismo , Fragmentos de Peptídeos/metabolismo , Cromatografia Líquida , Encefalina Leucina/química , Cromatografia Gasosa-Espectrometria de Massas , Proteína gp120 do Envelope de HIV/química , Espectrometria de Massas , Fragmentos de Peptídeos/química , Compostos de Trimetilsilil/análise , Compostos de Trimetilsilil/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
The authors developed a technique by which cocaine metabolites as well as cocaethylene can be identified and quantitated using gas chromatography/mass spectrometry (GC/MS), and deuterated standards of benzoylecgonine, cocaine, ecgonine methyl ester, and cocaethylene as internal standards. The authors used a solid phase extraction (Clean Screen, Worldwide Monitoring Corporation, (Horsham, PA)) technique, and rapid derivatization of metabolites using microwave irradiation before GC/MS analysis. The t-butyldimethylsilyl derivatives (prepared by microwave irradiation in 2.5 minutes, 640 Watts [W]) offered better baseline separation and chromatography than conventional trimethylsilyl derivatives. The assay was linear between 0.87-17.3 mumol/L of benzoylecgonine (250-5,000 ng/mL) concentration, 0.83-16.6 mumol/L of cocaine (250-5,000 ng/mL), 0.79-15.8 mumol/L of cocaethylene (250-5,000 ng/mL) and 1.25-25.1 mumol/L of ecgonine methyl ester (250-5,000 ng/mL). The yields are comparable between the microwave technique and conventional heating.
Assuntos
Cocaína/análise , Cocaína/metabolismo , Cocaína/análogos & derivados , Cocaína/efeitos da radiação , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Micro-Ondas , Compostos de Trimetilsilil/metabolismo , Compostos de Trimetilsilil/efeitos da radiaçãoRESUMO
Manganese peroxidase (MnP) oxidized 1-(3,5-dimethoxy-4-hydroxyphenyl)-2-(4-(hydroxymethyl)-2-methoxyphenoxy) -1,3-dihydroxypropane (I) in the presence of MnII and H2O2 to yield 1-(3,5-dimethoxy-4-hydroxyphenyl)- 2-(4-(hydroxymethyl)-2-methoxyphenoxy)-1-oxo-3-hydroxypropane (II), 2,6-dimethoxy-1,4-benzoquinone (III), 2,6-dimethoxy-1,4-dihydroxybenzene (IV), 2-(4-(hydroxymethyl)-2-methoxyphenoxy)-3-hydroxypropanal (V), syringaldehyde (VI), vanillyl alcohol (VII), and vanillin (VIII). MnP oxidized II to yield 2,6-dimethoxy-1,4-benzoquinone (III), 2,6-dimethoxy-1,4-dihydroxybenzene (IV), vanillyl alcohol (VII), vanillin (VIII), syringic acid (IX), and 2-(4-(hydroxymethyl)-2-methoxyphenoxy)-3-hydroxypropanoic acid (X). A chemically prepared MnIII-malonate complex catalyzed the same reactions. Oxidation of I and II in H2(18)O under argon resulted in incorporation of one atom of 18O into the quinone III and into the hydroquinone IV. Incorporation of one atom of oxygen from H2(18)O into syringic acid (IX) and the phenoxypropanoic acid X was also observed in the oxidation of II. These results are explained by mechanisms involving the initial one-electron oxidation of I or II by enzyme-generated MnIII to produce a phenoxy radical. This intermediate is further oxidized by MnIII to a cyclohexadienyl cation. Loss of a proton, followed by rearrangement of the quinone methide intermediate, yields the C alpha-oxo dimer II as the major product from substrate I. Alternatively, cyclohexadienyl cations are attacked by water. Subsequent alkyl-phenyl cleavage yields the hydroquinone IV and the phenoxypropanal V from I, and IV and the phenoxypropanoic acid X from II, respectively. The initial phenoxy radical also can undergo C alpha-C beta bond cleavage, yielding syringaldehyde (VI) and a C6-C2-ether radical from I and syringic acid (IX) and the same C6-C2-ether radical from II. The C6-C2-ether radical is scavenged by O2 or further oxidized by MnIII, subsequently leading to release of vanillyl alcohol (VII). VII and IV are oxidized to vanillin (VIII) and the quinone III, respectively.
Assuntos
Basidiomycota/enzimologia , Lignina/metabolismo , Peroxidases/metabolismo , Compostos de Trimetilsilil/metabolismo , Cromatografia Gasosa , Espectrometria de Massas , Oxirredução , Especificidade por SubstratoRESUMO
Urinary metabolites of cannabidiol (CBD), a non-psychoactive cannabinoid of potential therapeutic interest, were extracted from dog, rat and human urine, concentrated by chromatography on Sephadex LH-20 and examined by gas chromatography-mass spectrometry as trimethylsilyl (TMS), [2H9]TMS, methyl ester-TMS and methyloxime-TMS derivatives. Fragmentation of the metabolites under electron-impact gave structurally informative fragment ions; computer-generated single-ion plots of these diagnostic ions were used extensively to aid metabolite identification. Over fifty metabolites were identified with considerable species variation. CBD was excreted in substantial concentration in human urine, both in the free state and as its glucuronide. In dog, unusual glucoside conjugates of three metabolites (4"- and 5"-hydroxy- and 6-oxo-CBD), not excreted in the unconjugated state, were found as the major metabolites at early times after drug administration. Other metabolites in all three species were mainly acids. Side-chain hydroxylated derivatives of CBD-7-oic acid were particularly abundant in human urine but much less so in dog. In the latter species the major oxidized metabolites were the products of beta-oxidation with further hydroxylation at C-6. A related, but undefined pathway resulted in loss of three carbon atoms from the side-chain of CBD in man with production of 2"-hydroxy-tris,nor-CBD-7-oic acid. Metabolism by the epoxide-diol pathway, resulting in dihydro-diol formation from the delta-8 double bond, gave metabolites in both dog and human urine. It was concluded that CBD could be used as a probe of the mechanism of several types of biotransformation; particularly those related to carboxylic acid metabolism as intermediates of the type not usually seen with endogenous compounds were excreted in substantial concentration.
Assuntos
Compostos de Alumínio , Canabidiol/urina , Compostos de Lítio , Alumínio , Animais , Cromatografia Gasosa , Deutério , Cães , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Lítio , Masculino , Ratos , Especificidade da Espécie , Compostos de Trimetilsilil/metabolismoRESUMO
17-oxogenic steroids have been analysed by gas chromatography (GC) with capillary column and flame ionization detector. The best separation of steroid products was gained after ethyloxime-trimethylsilyl ether formation. The value of the GC method as a screening procedure for patients with suspected adrenal disease is illustrated in selected cases where the diagnosis was equivocal from data derived by RIA analysis of plasma steroids.
Assuntos
17-Hidroxicorticosteroides/urina , Cromatografia Gasosa/métodos , Adolescente , Doenças das Glândulas Suprarrenais/urina , Adulto , Pré-Escolar , Feminino , Humanos , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Oximas/metabolismo , Radioimunoensaio , Compostos de Trimetilsilil/metabolismoRESUMO
The microbial transformation of DDT, DDD and DDE was studied in Gram-negative strain B-206 and a number of phenolic metabolites were identified as the trimethylsilyl derivatives in the bacterial extracts by gas chromatography/mass spectrometry. The major metabolites of DDT were DDD, DDE, DDMU, 1,1,1-trichloro-2-(2-hydroxy-4-chlorophenyl)-2-(4'-chlorophenyl) ethane, 1,1,1-trichloro-2-(2-hydroxy-4-chlorophenyl)-2-(4'-hydroxyphenyl) ethane, and 1,1,1-trichloro-2,2-bis-(2-hydroxy-4-chlorophenyl) ethane. Conversely, DDD was mainly degraded into DDE, 1,1-dichloro-2-(2-hydroxy-4-chlorophenyl)-2-(4'-chlorophenyl) ethane and 1,1-dichloro-2-(2-hydroxy-4-chlorophenyl)-2-(4'-hydroxyphenyl) ethane. Finally, DDE was transformed into DDMU, 1,1-dichloro-2-(2-hydroxy-4-chlorophenyl)-2-(4'-chlorophenyl) ethylene, 1,1-dichloro-2-(2-hydroxy-4-chlorophenyl)-2-(4'hydroxyphenyl) ethylene and 1-chloro-2-(2-hydroxy-4-chlorophenyl)-2-(4'-chlorophenyl) ethylene. The phenolic metabolites exhibited [M - TMSCl]+., [M - HCl - TMSCl]+. and/or [M - HCl - TMSCl - Me]+ fragment ions which reflect the presence of an ortho hydroxyl group in these molecules. Other mass spectral features used to determine their structure are presented and a metabolic scheme accounting for their formation is proposed.
Assuntos
Bactérias/metabolismo , DDT/metabolismo , Biodegradação Ambiental , Biotransformação , DDT/análogos & derivados , Diclorodifenil Dicloroetileno/análise , Diclorodifenil Dicloroetileno/metabolismo , Diclorodifenildicloroetano/análise , Diclorodifenildicloroetano/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/metabolismo , Compostos de Trimetilsilil/metabolismoRESUMO
A gas chromatographic separation of dihydroxy- from monohydroxycannabinoids by the use of homologous trialkylsilyl derivatives is discussed. Trimethylsilyl derivatives produced a group of peaks containing both sets of compounds, sometimes poorly resolved, whereas by increasing the alkyl chain length to n-butyl complete fractionation into two groups, was achieved. The mass spectra of these derivatives resembled those of the trimethylsilyl derivatives with the addition of a set of ions resulting from estimation of the Si-alkyl chains as olefins.