RESUMO
OBJECTIVE: Mercaptopurine (MP) and pro-drug azathioprine are 'first-line' oral therapies for maintaining remission in IBD. It is believed that their pharmacodynamic action is due to a slow cumulative decrease in activated lymphocytes homing to inflamed gut. We examined the role of host metabolism, lymphocytes and microbiome for the amelioration of colitis by the related thioguanine (TG). DESIGN: C57Bl/6 mice with or without specific genes altered to elucidate mechanisms responsible for TG's actions were treated daily with oral or intrarectal TG, MP or water. Disease activity was scored daily. At sacrifice, colonic histology, cytokine message, caecal luminal and mucosal microbiomes were analysed. RESULTS: Oral and intrarectal TG but not MP rapidly ameliorated spontaneous chronic colitis in Winnie mice (point mutation in Muc2 secretory mucin). TG ameliorated dextran sodium sulfate-induced chronic colitis in wild-type (WT) mice and in mice lacking T and B lymphocytes. Remarkably, colitis improved without immunosuppressive effects in the absence of host hypoxanthine (guanine) phosphoribosyltransferase (Hprt)-mediated conversion of TG to active drug, the thioguanine nucleotides (TGN). Colonic bacteria converted TG and less so MP to TGN, consistent with intestinal bacterial conversion of TG to so reduce inflammation in the mice lacking host Hprt. TG rapidly induced autophagic flux in epithelial, macrophage and WT but not Hprt-/- fibroblast cell lines and augmented epithelial intracellular bacterial killing. CONCLUSIONS: Treatment by TG is not necessarily dependent on the adaptive immune system. TG is a more efficacious treatment than MP in Winnie spontaneous colitis. Rapid local bacterial conversion of TG correlated with decreased intestinal inflammation and immune activation.
Assuntos
Colite/tratamento farmacológico , Microbioma Gastrointestinal/fisiologia , Imunossupressores/uso terapêutico , Mucosa Intestinal/microbiologia , Mercaptopurina/metabolismo , Mercaptopurina/uso terapêutico , Tioguanina/metabolismo , Tioguanina/uso terapêutico , Administração Oral , Administração Retal , Animais , Autofagia/efeitos dos fármacos , Bacteroides thetaiotaomicron/metabolismo , Células Cultivadas , Colite/induzido quimicamente , Colite/genética , Colite/patologia , Colo/microbiologia , Citocinas/genética , Sulfato de Dextrana , Enterococcus faecalis/metabolismo , Células Epiteliais , Escherichia coli/metabolismo , Feminino , Fibroblastos , Interações Hospedeiro-Patógeno , Hipoxantina Fosforribosiltransferase/genética , Imunossupressores/administração & dosagem , Imunossupressores/metabolismo , Macrófagos , Masculino , Mercaptopurina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucina-2/genética , RNA Mensageiro/metabolismo , Linfócitos T/imunologia , Tioguanina/farmacologiaRESUMO
The rates of synthesis, turnover, and half-lives were determined for brain microsomal ether phospholipids in the awake adult unanesthetized rat. A multicompartmental kinetic model of phospholipid metabolism, based on known pathways of synthesis, was applied to data generated by a 5 min intravenous infusion of [1,1-(3)H]hexadecanol. At 2 h post-infusion, 29%, 33%, and 31% of the total labeled brain phospholipid was found in the 1-O-alkyl-2-acyl-sn-glycero-3-phosphate, ethanolamine, and choline ether phospholipid fractions, respectively. Autoradiography and membrane fractionation showed that 3% of the net incorporated radiotracer was in myelin at 2 h, compared to 97% in gray matter microsomal and synaptosomal fractions. Based on evidence that ether phospholipid synthesis occurs in the microsomal membrane fraction, we calculated the synthesis rates of plasmanylcholine, plasmanylethanolamine, plasmenylethanolamine, and plasmenylcholine equal to 1.2, 9.3, 27.6, and 21.5 nmol. g(-1). min(-1), respectively. Therefore, 8% of the total brain ether phospholipids have half-lives of about 36.5, 26.7, 23.1, and 15.1 min, respectively. Furthermore, we clearly demonstrate that there are at least two pools of ether phospholipids in the adult rat brain. One is the static myelin pool with a slow rate of tracer incorporation and the other is a dynamic pool found in gray matter. The short half-lives of microsomal ether phospholipids and the rapid transfer to synaptosomes are consistent with evidence of the marked involvement of these lipids in brain signal transduction and synaptic function.
Assuntos
Encéfalo/metabolismo , Álcoois Graxos/metabolismo , Microssomos/metabolismo , Éteres Fosfolipídicos/metabolismo , Sinaptossomos/metabolismo , Animais , Autorradiografia/métodos , Transporte Biológico , Colina/biossíntese , Etanolamina/metabolismo , Meia-Vida , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Until recently, brain phospholipid metabolism was thought to consume only 2% of the ATP consumed by the mammalian brain as a whole. In this paper, however, we calculate that 1.4% of total brain ATP consumption is consumed for the de novo synthesis of ether phospholipids and that another 5% is allocated to the phosphatidylinositide cycle. When added to previous estimates that fatty acid recycling within brain phospholipids and maintenance of membrane lipid asymmetries of acidic phospholipids consume, respectively, 5% and 8% of net brain ATP consumption, it appears that phospholipid metabolism can consume up to 20% of net brain ATP consumption. This new estimate is consistent with recent evidence that phospholipids actively participate in brain signaling and membrane remodeling, among other processes.
Assuntos
Encéfalo/metabolismo , Metabolismo Energético , Fosfolipídeos/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Humanos , Fosforilação , RatosRESUMO
Concentration and distribution of long-chain acylcarnitines in control microwaved and ischemic (decapitated) rat brain were measured by electrospray ionization tandem mass spectrometry. The total acylcarnitine concentration from control rat brains equaled 7-8 nmol/g wet weight brain, about one-fourth the total concentration of long-chain acyl-CoA, indicating a small role in buffering the total acyl-CoA pool concentration. Furthermore, acylcarnitine did not differ between ischemic and control rat brain with regard to total concentration or concentrations of molecular species of acylcarnitine. Therefore, the size of the acylcarnitine pool in brain is not affected by the dramatic increase in unesterified fatty acids (approximately 4x) that occurs in ischemia.
Assuntos
Isquemia Encefálica/metabolismo , Encéfalo/metabolismo , Carnitina/análogos & derivados , Animais , Carnitina/química , Carnitina/metabolismo , Masculino , Ratos , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Using a method and model developed in our laboratory to quantitatively study brain phospholipid metabolism, in vivo rates of incorporation and turnover of docosahexaenoic acid in brain phospholipids were measured in awake rats. The results suggest that docosahexaenoate incorporation and turnover in brain phospholipids are more rapid than previously assumed and that this rapid turnover dilutes tracer specific activity in brain docoshexaenoyl-CoA pool due to release and recycling of unlabeled fatty acid from phospholipid metabolism. Fractional turnover rates for docosahexaenoate within phosphatidylinositol, choline glycerophospholipids, ethanolamine glycerophospholipids and phosphatidylserine were 17.7, 3.1, 1.2, and 0.2 %.h(-1), respectively. Chronic lithium treatment, at a brain level considered to be therapeutic in humans (0.6 micromol.g(-1)), had no effect on turnover of docosahexaenoic acid in individual brain phospholipids. Consistent with previous studies from our laboratory that chronic lithium decreased the turnover of arachidonic acid within brain phospholipids by up to 80% and attenuated brain phospholipase A2 activity, the lack of effect of lithium on docosahexaenoate recycling and turnover suggests that a target for lithium's action is an arachidonic acid-selective phospholipase A2.
Assuntos
Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Ácidos Docosa-Hexaenoicos/metabolismo , Lítio/farmacologia , Acil Coenzima A/metabolismo , Animais , Ácido Araquidônico/metabolismo , Ácidos Docosa-Hexaenoicos/sangue , Ácidos Graxos não Esterificados/metabolismo , Metabolismo dos Lipídeos , Lítio/administração & dosagem , Masculino , Fosfolipases A/metabolismo , Fosfolipases A2 , Fosfolipídeos/metabolismo , Ratos , Ratos Endogâmicos F344RESUMO
Recent studies from our laboratory have shown that methyl palmoxirate (MEP), an inhibitor of mitochondrial beta-oxidation of long chain fatty acids, can be used to increase incorporation of radiolabeled palmitic acid into brain lipids and reduce beta-oxidation of the fatty acid. Thus, MEP allows the use of carbon labeled palmitate for studying brain lipid metabolism in animals and humans by quantitative autoradiography or positron emission tomography (PET). As it is essential to pretreat human subjects with an acute dose of MEP prior to intravenous injection of [1-11C]palmitate for PET scanning, this study was undertaken to determine the plasma elimination half-life of MEP in rats and human subjects and to provide insight about the drug's absorption and metabolism. A gas chromatographic method was developed to measure MEP in body fluids. Following oral administration of MEP to rats (2.5 and 10 mg/kg) and to humans, the unmetabolized drug could not be detected in plasma or urine (sensitivity of detection was 1 ng). However, when MEP was injected intravenously (10 mg/kg) in rats, a peak initial concentration could be measured in plasma (7.7 microg/mL), the clearance of the drug from plasma was rapid (t1/2 = 0.6 min), which indicates that MEP readily enters tissue lipid pools or is metabolized like long-chain fatty acids. As no adverse experience occured in the 11 human subjects studied, oral administration of a single dose of MEP was safe under the conditions of this study and may be used to increase the incorporation of positron labeled palmitic acid for studying brain lipid metabolism in vivo by PET.
Assuntos
Compostos de Epóxi/administração & dosagem , Compostos de Epóxi/farmacocinética , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/farmacocinética , Propionatos/administração & dosagem , Propionatos/farmacocinética , Administração Oral , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Compostos de Epóxi/efeitos adversos , Humanos , Hipoglicemiantes/efeitos adversos , Injeções Intravenosas , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Oxirredução/efeitos dos fármacos , Propionatos/efeitos adversos , Ratos , Ratos Endogâmicos F344RESUMO
Transient cerebral ischemia (5 min) releases unesterified fatty acids from membrane phospholipids, increasing brain concentrations of fatty acids for up to 1 h following reperfusion. To understand the reported anti-ischemic effect of Ginkgo biloba extract (EGb 761), we monitored its effect on brain fatty acid reincorporation in a gerbil-stroke model. Both common carotid arteries in awake gerbils were occluded for 5 min, followed by 5 min of reperfusion. Animals were infused intravenously with labeled arachidonic (AA) or palmitic acid (Pam), and rates of incorporation of unlabeled fatty acid from the brian acyl-CoA pool were calculated by the model of Robinson et al. (1992), using quantitative autoradiography and biochemical analysis of brain acyl-CoA. Animals were treated for 14 d with 50 or 150 mg/kg/d EGb 761 or vehicle. Ischemia-reperfusion had no effect on the rate of unlabeled Pam incorporation into brain phospholipids from palmitoyl-CoA; this rate also was unaffected by EGb 761. In contrast, ischemia-reperfusion increased the rate of incorporation of unlabeled AA from brain arachidonoyl-CoA by a factor of 2.3-3.3 compared with the control rate; this factor was further augmented to 3.6-5.0 by pretreatment with EGb 761. There is selective reincorporation of AA compared with Pam into brain phospholipids following ischemia. EGb 761 further accelerates AA reincorporation, potentially reducing neurotoxic effects of prolonged exposure of brain to high concentrations of AA and its metabolites.
Assuntos
Química Encefálica/efeitos dos fármacos , Ácidos Graxos/metabolismo , Ginkgo biloba/química , Ataque Isquêmico Transitório/tratamento farmacológico , Lipídeos de Membrana/metabolismo , Fármacos Neuroprotetores/farmacologia , Fosfolipídeos/metabolismo , Extratos Vegetais , Plantas Medicinais , Traumatismo por Reperfusão/prevenção & controle , Animais , Ácido Araquidônico/metabolismo , Flavonoides , Gerbillinae , Ataque Isquêmico Transitório/metabolismo , Masculino , Fármacos Neuroprotetores/uso terapêutico , Ácido Palmítico/metabolismo , Traumatismo por Reperfusão/metabolismo , VigíliaRESUMO
Previous estimates have placed the energy requirements of total phospholipid metabolism in mammalian brain at 2% or less of total ATP consumption. This low estimate was consistent with the very long half-lives (up to days) reported for fatty acids esterified within phospholipids. However, using an approach featuring analysis of brain acyl-CoA, which takes into account dilution of the precursor acyl-CoA pool by recycling of fatty acids, we reported that half-lives of fatty acids in phospholipids are some 100 times shorter (min-h) than previously thought. Based on these new estimates of short half-lives, palmitic acid and arachidonic acid were used as prototype fatty acids to calculate energy consumption by fatty acid recycling at the sn-1 and sn-2 positions of brain phospholipids. We calculated that the energy requirements for reacylation of fatty acids into lysophospholipids are 5% of net brain ATP consumption. We also calculated ATP requirements for maintaining asymmetry of the aminophospholipids, phosphatidylserine and phosphatidylethanolamine across brain membrane bilayers. This asymmetry is maintained by a translocase at a stoichiometry of 1 mol of ATP per mol of phospholipid transferred in either direction across the membrane. The energy cost of maintaining membrane bilayer asymmetry of aminophospholipids at steady-state was calculated to be 8% of total ATP consumed. Taken together, deacylation-reacylation and maintenance of membrane asymmetry of phosphatidylserine and phosphatidylethanolamine require about 13% of ATP consumed by brain as a whole. This is a lower limit for energy consumption by processes involving phospholipids, as other processes, including phosphorylation of polyphosphoinositides and de novo phospholipid biosynthesis, were not considered.
Assuntos
Encéfalo/metabolismo , Metabolismo Energético , Mamíferos/metabolismo , Modelos Biológicos , Fosfolipídeos/metabolismo , Acil Coenzima A/metabolismo , Acilação , Trifosfato de Adenosina/metabolismo , Animais , Transporte Biológico , Ácidos Graxos/metabolismo , Lisofosfolipídeos/metabolismo , Glicoproteínas de Membrana/metabolismoRESUMO
Awake gerbils were subjected to 5 min of forebrain ischemia by clamping the carotid arteries for 5 min and then allowing recirculation. Radiolabeled arachidonic or palmitic acid was infused intravenously for 5 min at the start of recirculation, after which the brains were prepared for quantitative autoradiography or chemical analysis. Dilution of specific activity of the acyl-CoA pool was independently determined for these fatty acids in control gerbils and following 5 min of ischemia and 5 min of reperfusion. Using a quantitative method for measuring regional in vivo fatty acid incorporation into and turnover within brain phospholipids and determining unlabeled concentrations of acyl-CoAs following recirculation, it was shown that reperfusion after 5 min of ischemia was accompanied by a threefold increase compared with the control in the rate of reincorporation of unlabeled arachidonate that had been released during ischemia, whereas reincorporation of released palmitate was not different from the control. Selective and accelerated reincorporation of arachidonate into brain phospholipids shortly after ischemia may ameliorate specific deleterious effects of arachidonate and its metabolites on brain membranes.
Assuntos
Ácido Araquidônico/metabolismo , Encéfalo/metabolismo , Ataque Isquêmico Transitório/metabolismo , Lipídeos de Membrana/metabolismo , Fosfolipídeos/metabolismo , Reperfusão , Acil Coenzima A/metabolismo , Animais , Autorradiografia , Ácidos Graxos/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Gerbillinae , Processamento de Imagem Assistida por Computador , Técnicas de Diluição do Indicador , Masculino , Palmitatos/metabolismoRESUMO
To ascertain effects of total ischemia on brain phospholipid metabolism, anesthetized rats were decapitated and unesterified fatty acids and long chain acyl-CoA concentrations were analyzed in brain after 3 or 15 min. Control brain was taken from rats that were microwaved. Fatty acids were quantitated by extraction, thin layer chromatography and gas chromatography. Long-chain acyl-CoAs were quantitated by solubilization, solid phase extraction with an oligonucleotide purification cartridge and HPLC. Unesterified fatty acid concentrations increased significantly after decapitation, most dramatically for arachidonic acid (76 fold at 15 min) followed by docosahexaenoic acid. Of the acyl-CoA molecular species only the concentration of arachidonoyl-CoA was increased at 3 min and 15 min after decapitation, by 3-4 fold compared with microwaved brain. The concentration of docosahexaenoyl-CoA fell whereas concentrations of the other acyl-CoAs were unchanged. The increase in arachidonoyl-CoA after decapitation indicates that reincorporation of arachidonic acid into membrane phospholipids is possible during ischemia, likely at the expense of docosahexaenoic acid.
Assuntos
Acil Coenzima A/metabolismo , Isquemia Encefálica/metabolismo , Encéfalo/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Animais , Encéfalo/efeitos da radiação , Humanos , Masculino , Micro-Ondas , Fosfolipídeos/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Transient global cerebral ischemia affects phospholipid metabolism and features a considerable increase in unesterified fatty acids. Reincorporation of free fatty acids into membrane phospholipids during reperfusion following transient ischemia depends on conversion of fatty acids to acyl-CoAs via acyl-CoA synthetases and incorporation of the acyl group into lysophospholipids. To study the effect of ischemia-reperfusion on brain fatty acid and acyl-CoA pools, the common carotid arteries were tied for 5 min in awake gerbils, after which the ligatures were released for 5 min and the animals were killed by microwave irradiation. Twenty percent of these animals (two of 10) were excluded from the ischemia-reperfusion group when it was demonstrated statistically that brain unesterified arachidonic acid concentration was not elevated beyond the range of the control group. Brain unesterified fatty acid concentration was increased 4.4-fold in the ischemic-reperfused animals, with stearic acid and arachidonic acid increasing the most among the saturated and polyunsaturated fatty acids, respectively. The total acyl-CoA concentration remained unaffected, indicating that reacylation of membrane lysophospholipids is maintained during recovery. However, there was a substantial increase in the stearoyl- and arachidonoyl-CoA and a marked decrease in palmitoyl- and docosahexaenoyl-CoA. These results suggest that unesterified fatty acid reacylation into phospholipids is reprioritized according to the redistribution in concentration of acyl-CoA molecular species, with incorporation of stearic acid and especially arachidonic acid being favored.
Assuntos
Acil Coenzima A/metabolismo , Isquemia Encefálica/metabolismo , Encéfalo/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Ácidos Graxos não Esterificados/metabolismo , Gerbillinae , Masculino , Concentração Osmolar , Valores de Referência , Distribuição TecidualRESUMO
In vivo rates of arachidonic acid incorporation and turnover were determined for molecular species of rat brain phosphatidylcholine (PtdCho) and phosphatidylinositol (PtdIns). [3H]Arachidonic acid was infused intravenously in pentobarbital-anesthetized rats at a programmed rate to maintain constant plasma specific activity for 2-10 min. At the end of infusion, animals were killed by microwave irradiation, and brain phospholipids were isolated, converted to diacylglycerobenzoates, and resolved as molecular species by reversed-phase HPLC. Most [3H] arachidonate (> 87%) was incorporated into PtdCho and PtdIns, with arachidonic acid at the sn-2 position and with oleic acid (18:1), palmitic acid (16:0), or stearic acid (18:0) at the sn-1 position. However, 10-15% of labeled brain PtdCho eluted in a small peak containing two molecular species with arachidonic acid at the sn-2 position and palmitoleic acid (16:1) or linoleic acid (18:2) at the sn-1 position. Analysis demonstrated that tracer was present in both the 16:1-20:4 and 18:2-20:4 PtdCho species at specific activities 10-40 times that of the other phospholipids. Based on the measured mass of arachidonate in each phospholipid molecular species, half-lives were calculated for arachidonate of < 10 min in 16:1-20:4 and 18:2-20:4 PtdCho and 1-3 h in 16:0-20:4, 18:1-20:4 PtdCho and PtdIns. The very short half-lives for arachidonate in the 16:1-20:4 and 18:2-20:4 PtdCho molecular species suggest important roles for these molecules in brain phospholipid metabolism and signal transduction.
Assuntos
Ácido Araquidônico/metabolismo , Encéfalo/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidilinositóis/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Masculino , Estrutura Molecular , Ácido Oleico/metabolismo , Ácido Palmítico/metabolismo , Fosfatidilcolinas/química , Fosfatidilinositóis/química , Fosfolipases A , Técnica de Diluição de Radioisótopos , Ratos , Ratos Sprague-Dawley , Espectrometria de Massa de Íon Secundário , Ácidos Esteáricos/metabolismo , Trítio , Fosfolipases Tipo CRESUMO
In vivo rates of palmitate incorporation into brain phospholipids were measured in awake rats following programmed intravenous infusion of unesterified [9,10-3H]palmitate to maintain constant plasma specific activity. Animals were killed after 2-10 min of infusion by microwave irradiation and analyzed for tracer distribution in brain phospholipid and phospholipid precursor, i.e., brain unesterified palmitate and palmitoyl-CoA, pools. [9,10-3H]Palmitate incorporation into brain phospholipids was linear with time and rapid, with > 50% of brain tracer in choline-containing glycerophospholipids at 2 min of infusion. However, tracer specific activity in brain phospholipid precursor pools was low and averaged only 1.6-1.8% of plasma unesterified palmitate specific activity. Correction for brain palmitoyl-CoA specific activity increased the calculated rate of palmitate incorporation into brain phospholipids (0.52 nmol/s/g) by approximately 60-fold. The results suggest that palmitate incorporation and turnover in brain phospholipids are far more rapid than generally assumed and that this rapid turnover dilutes tracer specific activity in brain palmitoyl-CoA pool owing to release and recycling of unlabeled fatty acid from phospholipid breakdown.
Assuntos
Encéfalo/metabolismo , Palmitatos/metabolismo , Palmitoil Coenzima A/metabolismo , Fosfolipídeos/metabolismo , Animais , Meia-Vida , Metabolismo dos Lipídeos , Masculino , Modelos Neurológicos , Palmitatos/sangue , Ratos , Ratos Sprague-DawleyRESUMO
Brain fatty acid incorporation into phospholipids can be measured in vivo following intravenous injection of fatty acid tracer. However, to calculate a cerebral incorporation rate, knowledge is required of tracer specific activity in the final brain precursor pool. To determine this for one tracer, unesterified [3H]arachidonate was infused intravenously in pentobarbital-anesthetized rats to maintain constant plasma specific activity for 1-10 min. At the end of infusion, animals were killed by microwave irradiation and analyzed for tracer specific activity and concentration in brain phospholipid, neutral lipid, and lipid precursor, i.e., unesterified arachidonate and arachidonoyl-CoA, pools. Tracer specific activity in brain unesterified arachidonate and arachidonoyl-CoA rose quickly (t1/2 < 1 min) to steady-state values that averaged < 5% of plasma specific activity. Incorporation was rapid, as > 85% of brain tracer was present in phospholipids at 1 min of infusion. The results demonstrate that unesterified arachidonate is rapidly taken up and incorporated in brain but that brain phospholipid precursor pools fail to equilibrate with plasma in short experiments. Low brain precursor specific activity may result from (a) dilution of label with unlabeled arachidonate from alternate sources or (b) precursor pool compartmentalization. The results suggest that arachidonate turnover in brain phospholipids is more rapid than previously assumed.
Assuntos
Ácido Araquidônico/metabolismo , Barreira Hematoencefálica , Encéfalo/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Fosfolipídeos/metabolismo , Acil Coenzima A/metabolismo , Animais , Ácido Araquidônico/administração & dosagem , Ácidos Graxos não Esterificados/administração & dosagem , Infusões Intravenosas , Cinética , Masculino , Matemática , Modelos Neurológicos , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , TrítioRESUMO
Long-chain acyl-CoA's are important intermediates in fatty acid oxidation and phospholipid metabolism. For quantitative analysis of brain acyl-CoA's, and to avoid decomposition due to high brain acyl-CoA hydrolase activity, a fast and efficient analytical method was developed for isolation and determination of acyl-CoA's. The analysis includes solid-phase extraction by an oligonucleotide purification cartridge and HPLC measurements using a synthetic internal standard. Estimates of concentration in rat brain are oleoyl-CoA (11.0 nmol/g), palmitoyl-CoA (6.0 nmol/g), stearoyl-CoA (4.0 nmol/g), and linoleoyl- and arachidonoyl-CoA (2.0 nmol/g) for a total concentration of 23 nmol/g brain.
Assuntos
Acil Coenzima A/análise , Acil Coenzima A/isolamento & purificação , Química Encefálica , Animais , Cromatografia Líquida de Alta Pressão/métodos , Palmitoil Coenzima A/análise , Palmitoil Coenzima A/isolamento & purificação , Ratos , Ratos Sprague-DawleyRESUMO
This study investigates the incorporation of three intravenously administered radiolabeled fatty acids, [9,10-3H]palmitate (3H-PAM), [1-14C]arachidonate (14C-ACH) and [1-14C]docosahexaenoate (14C-DHA), into lipids of intracerebrally implanted tumor and contralateral brain cortex in awake rats. A suspension of Walker 256 carcinosarcoma cells (1 x 10(6) cells) was implanted into the right cerebral hemisphere of an 8- to 9-week-old Fischer-344 rat. Seven days later, the awake rat was infused intravenously for 5 min with 3H-PAM (6.4 mCi/kg), 14C-ACH (170 microCi/kg) or 14C-DHA (100 microCi/kg). Twenty min after the start of infusion, the rat was killed and intracranial tumor mass and brain cortex were removed for lipids analysis. Each radiolabel was incorporated more into tumor than into brain cortex. Ratios of net incorporation rate coefficients (k*) into tumor as compared with brain were 4.5, 3.4 and 1.7 for 3H-PAM, 14C-ACH and 14C-DHA, respectively. Lipid radioactivity comprised more than 80% of total tumor or brain radioactivity for each probe. Phospholipids contained 58%, 89% and 68% of tumor lipid radioactivity, and 58%, 82% and 74% of brain lipid radioactivity, for 3H-PAM, 14C-ACH and 14C-DHA, respectively. Incorporation coefficients (k*i) for a phospholipid class (i)--choline phosphoglycerides (PC), inositol monophosphoglycerides (PI), ethanolamine phosphoglycerides (PE), serine phosphoglycerides (PS), and sphingomyelin (SM)--were greater in tumor than in brain for each fatty acid probe, except that values for k*PE and k*PS using 14C-DHA were equivalent. Differences in k*i between tumor and brain were largest for SM and PC and the change in k*PC accounted for 65-90% of the increase in the net phospholipid incorporation rate for each probe. Differences in k*PI, k*PE and k*PS were smaller than those in were smaller than those in k*PC and k*SM, and varied with the probe. Differences in k*i were related to differences in tumor and brain phospholipid composition and metabolism. The results indicate that suitably radiolabeled fatty acids may be used to image and characterize metabolism of lipid compartments of a brain tumor in vivo using positron emission tomography.
Assuntos
Neoplasias Encefálicas/metabolismo , Encéfalo/metabolismo , Ácidos Graxos/metabolismo , Fosfolipídeos/metabolismo , Animais , Carcinoma 256 de Walker/metabolismo , Masculino , Ratos , Ratos Endogâmicos F344 , VigíliaRESUMO
Standard methods for determining prorenin-renin concentrations in plasma (PRC) and other tissues require the addition of exogenous renin substrate (angiotensinogen) to improve the kinetics of the renin reaction. We studied the effects of substrate prepared from normal human plasma fraction Cohn IV-4, or from nephrectomized (2NX) sheep plasma, on PRC of normal and 2NX human plasmas before and after prorenin activation by acid, cold, and trypsin, and compared the results with plasma renin activities (PRA, no added substrate). Plasmas from 2NX men exhibited negligible basal PRA, indicating that very little, if any, renin had been formed from the extrarenal prorenin they contained, and suggesting the lack of an endogenous prorenin activating mechanism, or "convertase," of probable renal origin. Prorenin was demonstrable by tryptic activation, more than by acid or cold, at up to about 30% of normal. Addition of Cohn IV-4 substrate to 2NX plasma unexpectedly produced (i) a basal PRC value higher than in normal plasma, (ii) total renin values after activation by acid, cold, and trypsin that were much closer to normal values than reflected by PRA methodology, without a commensurate increase (if anything a decrease) in prorenin as a percentage of total renin estimated by all activation methods, and (iii) substantial equalization of activation effects such that trypsin was no longer more effective than acid and cold (and this was also noted with normal plasma). The skewing effect of adding Cohn IV-4 substrate on the PRC of 2NX plasma was much greater than in normal plasma, even though 2NX plasma already had an above normal level of endogenous substrate and should have been influenced less. Enhancement of PRC was very pronounced even when Cohn IV-4 was added to make up only 9% of total (endogenous + exogenous) substrate in the incubation system, suggesting that it was not the added substrate but a renin-generating contaminant that inflated the PRC. Such inflation could be blocked by adding protease inhibitors, suggesting that the responsible protease(s) acted as a prorenin "convertase" that generated new renin from renal and (or) extrarenal prorenin contributed by the added substrate, as well as by the plasma being assayed. One component of convertase could be kallikrein, which was identified by chromogenic assay, the importance of which relative to total convertase activity is unknown.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Angiotensinogênio/sangue , Precursores Enzimáticos/sangue , Rim/enzimologia , Renina/sangue , Adulto , Animais , Temperatura Baixa , Ativação Enzimática , Humanos , Calicreínas/metabolismo , Masculino , Pessoa de Meia-Idade , Nefrectomia , Inibidores de Proteases/farmacologia , Ovinos , Tripsina/metabolismoRESUMO
The release of arachidonic acid from thrombin-stimulated platelets can be attributed to the action of phospholipase A2 on membrane phospholipid. Previously, analysis of individual subclasses of phospholipid demonstrated that 1-acyl-2-[3H]arachidonoyl-sn-glycerophosphocholine and to a lesser degree 1-acyl-2-[3H]arachidonoyl-sn-glycerophosphoethanolamine were the main source of [3H]arachidonic acid in thrombin-stimulated cells. In the present work, 1,2-diacyl phospholipid subclasses were analyzed as 1,2-diacylglycerobenzoates by high-pressure liquid chromatography in order to analyze arachidonate release as mass changes in individual molecular species of phospholipid. Following thrombin stimulation (5 U/ml, 5 min, 37 degrees C) all arachidonoyl-containing molecular species of 1,2-diacyl-sn-glycerophosphocholine decreased in mass and [3H]arachidonate content by almost 50%, while those of 1,2-diacyl-sn-glycerophosphoethanolamine decreased by 20%. The mass change was substantial and indicated that these phospholipids are a major source of arachidonate in stimulated cells. No variation was seen in the other non-arachidonate-containing molecular species of either subclass. Thus, deacylation of membrane 1,2-diacylglycerophosphocholine and 1,2-diacylglycerophosphoethanolamine by phospholipase A2 is selective for those molecular species of phospholipid containing arachidonic acid, suggesting that a certain proportion of arachidonoyl-containing molecular species of phospholipid are compartmentalized with the platelet membrane proximal to the site of action of this enzyme. These studies demonstrate that the human platelet is a cell poised and specialized to release rapidly substantial amounts of arachidonic acid upon stimulation.
Assuntos
Ácidos Araquidônicos/metabolismo , Plaquetas/metabolismo , Fosfolipases A/metabolismo , Fosfolipases/metabolismo , Trombina/metabolismo , Ácido Araquidônico , Cromatografia Líquida de Alta Pressão , Humanos , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfolipases A2RESUMO
Resolution of individual molecular species of human platelet 1,2-diradyl-sn-glycero-3-phosphocholines and 1,2-diradyl-sn-glycero-3-phosphoethanolamines by reverse phase high pressure liquid chromatography (HPLC) allowed a thorough analysis of those phospholipids labeled with [3H]arachidonic acid. Approximately 54% and 16% of the total incorporated radiolabel was found in choline glycerophospholipids and ethanolamine glycerophospholipids, respectively, with ca. 90% of this being found in the 1,2-diacyl molecular species. Eighty percent of [3H]-arachidonic acid incorporated into 1-acyl-2-arachidonoyl-sn-glycero-3-phosphocholine in resting platelets was equally distributed between 1-palmitoyl-2-arachidonoyl and 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, while 70% of the radiolabel in 1-acyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine was found in 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine. Thrombin stimulation (5 U/ml for 5 min) resulted in deacylation of all 1-acyl-2-[3H]arachidonoyl molecular species of 1-acyl-2-arachidonoyl-sn-glycero-3-phosphocholine and 1-acyl-2-arachidonoyl-sn-glycero-3-ethanolamine. There was also a slight increase in 1-O-alkyl-2-[3H]arachidonoyl-sn-glycero-3-phosphocholine and a significant increase in 1-O-alk-1'-enyl-2-[3H]arachidonoyl-sn-glycero-3-phosphoethanolamine molecular species of over 300%. Thus, HPLC methodology indicates that arachidonoyl-containing molecular species of phosphatidylcholine and phosphatidylethanolamine are the major source of arachidonic acid in thrombin-stimulated human platelets, while certain ether phospholipid molecular species become enriched in arachidonate.