RESUMO
The degradation of articular cartilage by aggrecanases (ADAMTS-4 and ADAMTS-5) plays a significant role in the pathology of osteoarthritis (OA). To monitor aggrecanase activity in OA, we have developed a sensitive, accurate, and versatile assay for detection of two specific cleavage sites on aggrecan. The assay uses an immunoaffinity-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to detect cleavage at the (374)ARGS site and the (1820)AGEG site. The dynamic range of the assay is more than three orders of magnitude, with interassay precision less than 15%. It has been successfully applied to various biological fluids and species, including rat, bovine, dog, and human. The assay has been analytically qualified for use in human urine and synovial fluid (SF). The limits of detection (LODs) for ARGS in urine and SF are 2.5 and 10 pg/ml, respectively, whereas the LOD for AGEG is 20 pg/ml in SF. Analysis of these biomarkers from OA subjects and normal healthy volunteers revealed a significant elevation of both markers in OA. Similarly, in a rat model of cartilage degradation, both ARGS and AGEG were elevated, demonstrating the utility of these biomarkers for translational research. These data suggest that the ARGS and AGEG biomarkers developed have potential as measures of aggrecanase activity in OA and may contribute to our understanding of OA pathology.
Assuntos
Agrecanas/análise , Líquidos Corporais/química , Cartilagem Articular/patologia , Cromatografia Líquida/métodos , Endopeptidases/metabolismo , Espectrometria de Massas em Tandem/métodos , Agrecanas/química , Agrecanas/urina , Sequência de Aminoácidos , Animais , Biomarcadores/análise , Cartilagem Articular/enzimologia , Bovinos , Cromatografia de Afinidade , Cães , Humanos , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/urina , Ratos , Padrões de Referência , Reprodutibilidade dos Testes , Líquido Sinovial/metabolismo , Pesquisa Translacional BiomédicaRESUMO
OBJECTIVE: Progression of joint damage in osteoarthritis (OA) is likely to result from an imbalance between cartilage degradation and synthesis processes. Markers reflecting these two components appear to be promising in predicting the rate of OA progression. Both N- and C-terminal propeptides of type II collagen reflect the rates of collagen type II synthesis. The ability to quantify the procollagen peptides in biological fluids would enable a better understanding of OA disease pathology and provide means for assessing the proof of mechanism of anabolic disease modifying OA drugs (DMOADs). METHODS: A polyclonal antibody that recognizes the sequence GPKGQKGEPGDIKDI in the propeptide region of rat, dog, and human type II collagen was raised in chicken and peptide-affinity purified. The immunoaffinity liquid chromatography mass spectrometry (LC-MS/MS) was used to extensively characterize N-terminal procollagen type II (NPII) peptides found in biological fluids. The novel competition enzyme-linked immunosorbent assay (ELISA) assay was developed to quantitatively measure the NPII peptides. RESULTS: Several peptides ranging from 17 to 41 amino acids with various modifications including hydroxylations on proline and lysine residues, oxidation of lysines to allysines, and attachments of glucose and galactose moieties to hydroxylysines were identified in a simple system such as ex vivo cultures of human articular cartilage (HAC) explants as well as in more complex biological fluids such as human urine and plasma. A competitive ELISA assay has been developed and applied to urine, plasma, and synovial fluid matrices in human, rat and dog samples. CONCLUSION: A novel NPII assay has been developed and applied to OA and normal human subjects to understand the changes in collagen type II synthesis related to the pathology of OA.
Assuntos
Cartilagem Articular/metabolismo , Colágeno Tipo II/biossíntese , Osteoartrite/metabolismo , Pró-Colágeno/biossíntese , Adulto , Idoso , Animais , Biomarcadores/metabolismo , Cromatografia Líquida , Progressão da Doença , Cães , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Fragmentos de Peptídeos , RatosRESUMO
In previous reports, we described the isolation and characterization of an endogenous digitalislike factor (EDLF). In this report, we describe a unique combination of bioassay and large-scale purification methodology that made possible the purification of sufficient quantities of this inhibitor of Na+,K(+)-ATPase for structural analysis. Using an initial XAD-2 extraction and preparative high-performance liquid chromatography followed by a batch enzyme affinity extraction and two subsequent semipreparative chromatographic steps, 300 l of human plasma was processed, yielding 31 micrograms (53 nmol) of pure EDLF and representing purification on a dry weight basis in excess of 0.6 billionfold. Four divergent pieces of evidence, including chromatographic, mass spectrometric, immunoreactive, and binding characteristics, suggested that the EDLF purified in the present study was either ouabain or an isomer of ouabain. This material may represent a plasma-borne, naturally occurring, selective, high-affinity ligand for the digitalis binding site that may play a significant role in the modulation of the sodium pump and thereby cellular electrolyte homeostasis in humans.
Assuntos
Proteínas Sanguíneas/isolamento & purificação , Digoxina , Saponinas , Anticorpos , Proteínas Sanguíneas/química , Proteínas Sanguíneas/imunologia , Cardenolídeos , Cromatografia Líquida de Alta Pressão , Reações Cruzadas , Humanos , Ouabaína/imunologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Espectrometria de Massas de Bombardeamento Rápido de ÁtomosRESUMO
A sodium pump inhibitor has been isolated from human plasma and extensively purified. This material, endogenous digitalislike factor, was examined by a variety of mass spectrometric techniques. A low-resolution fast atom bombardment mass spectrometric analysis of a sample of purified endogenous digitalislike factor revealed a single unique molecular ion in the mass range 100-2,500. The accurate mass was determined to be 585.295 Da in a second high-resolution fast atom bombardment mass spectrometric experiment. Based on this accurate mass, the elemental composition of endogenous digitalislike factor was determined and found to be identical to the elemental composition of the known cardenolide ouabain. Direct comparison of ouabain and endogenous digitalislike factor by linked scan tandem mass spectrometry, derivatization with acetic anhydride coupled with fast atom bombardment mass spectrometry, and analytical high-performance liquid chromatography failed to reveal any differences. We conclude that the endogenous digitalislike factor isolated from human plasma is ouabain or a closely related isomer.
Assuntos
Proteínas Sanguíneas/isolamento & purificação , Digoxina , Saponinas , Acetilação , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Cardenolídeos , Cromatografia Líquida de Alta Pressão , Humanos , Espectrometria de Massas , Ouabaína/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Espectrometria de Massas de Bombardeamento Rápido de ÁtomosRESUMO
The pathophysiological importance of reactive oxygen species has been extensively documented in the pathogenesis of hepatic ischemia-reperfusion injury. Kupffer cells and neutrophils were identified as the dominant sources of the postischemic oxidant stress. To test the hypothesis that a direct free radical-mediated injury mechanism (lipid peroxidation; LPO) may be involved in the pathogenesis, highly sensitive and specific parameters of LPO, i.e., hydroxy-eicosatetraenoic acids (HETES), and F2-isoprostanes, were determined by gas chromatographic-mass spectrometric analysis in liver tissue and plasma during 45 min of hepatic ischemia and up to 24 h of reperfusion. A significant 60-250% increase of F2-isoprostane levels in plasma was found at all times during reperfusion; the HETE content increased only significantly at 1 h of reperfusion and in severely necrotic liver tissue at 24 h with increases between 90-320%. On the other hand, in a model of LPO-induced liver injury (infusion of 0.8 mumol tert-butylhydroperoxide/min/g liver), the hepatic HETE content increased two to fourfold over baseline values at 45 min, i.e., before liver injury. A further increase to 12- to 30-fold of baseline was observed during moderate liver injury. Based on these quantitative comparisons of LPO and liver injury, it seems highly unlikely that LPO is the primary mechanism of parenchymal cell injury during reperfusion, although it cannot be excluded that LPO may be important as a damaging mechanism in a limited compartment of the liver, e.g., endothelial cells, close to the sources of reactive oxygen, e.g., Kupffer cells and neutrophils.
Assuntos
Isquemia/metabolismo , Peroxidação de Lipídeos , Fígado/irrigação sanguínea , Reperfusão , Alanina Transaminase/sangue , Análise de Variância , Animais , Dinoprosta/sangue , Cromatografia Gasosa-Espectrometria de Massas , Glutationa/análogos & derivados , Glutationa/sangue , Dissulfeto de Glutationa , Isquemia/sangue , Masculino , Peróxidos/farmacologia , Ratos , Ratos Endogâmicos F344 , Espécies Reativas de Oxigênio/farmacologia , Fatores de Tempo , terc-Butil HidroperóxidoRESUMO
The potential role of nitric oxide (NO) was investigated in the pathophysiology of liver injury after priming with 20 min hepatic ischemia-reperfusion and administration of .5 mg/kg Salmonella enteritidis endotoxin. Liver injury during the early reperfusion phase of 4 h was characterized by severe vascular oxidant stress, lipid peroxidation (LPO), neutrophil infiltration, and a 33% reduction of the microvascular blood flow in the liver. Inhibition of NO synthesis with N omega-nitro-L-arginine methyl ester hydrochloride (L-NAME) aggravated liver injury by 90%, reduced LPO, and did not affect liver neutrophils but further impaired microvascular blood flow. Treatment with the NO-donor spermine-NONOate or L-arginine did not affect these parameters in postischemic animals, however, treatment did restore all values of L-NAME-treated animals back to disease control levels. These data suggest that endogenous NO formation is sufficient to limit ischemic liver injury during reperfusion but inhibition of NO synthesis will result in additional ischemic damage. NO may also be involved in scavenging of superoxide in the vasculature and in inducing LPO.
Assuntos
Isquemia/complicações , Fígado/irrigação sanguínea , Óxido Nítrico/biossíntese , Traumatismo por Reperfusão/etiologia , Toxemia/complicações , Alanina Transaminase/sangue , Animais , Arginina/análogos & derivados , Arginina/farmacologia , Endotoxinas/toxicidade , Inibidores Enzimáticos/farmacologia , Sequestradores de Radicais Livres/metabolismo , Glutationa/sangue , Isquemia/fisiopatologia , Peroxidação de Lipídeos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , NG-Nitroarginina Metil Éster , Neutrófilos/efeitos dos fármacos , Neutrófilos/patologia , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/fisiologia , Óxido Nítrico Sintase/antagonistas & inibidores , Ratos , Ratos Endogâmicos F344 , Traumatismo por Reperfusão/fisiopatologia , Toxemia/fisiopatologia , Vasoconstrição/efeitos dos fármacos , Vasoconstrição/fisiologiaRESUMO
We have investigated endothelial function in a two-hit model of multiple organ failure. Male Fischer rats were subjected to 20 min of partial hepatic ischemia followed by reperfusion and administration of .5 mg/kg Salmonella enteritidis endotoxin at 30 min of reperfusion. After either 4 or 24 h of reperfusion, rings of aorta were prepared and suspended in bioassay baths, contracted with phenylephrine, and examined for endothelium-dependent relaxation in response to acetylcholine and endothelium-independent relaxation using nitroglycerin. Endothelium-dependent relaxation to acetylcholine was impaired in rings from animals exposed to endotoxin-enhanced reperfusion injury at both 4 h and 24 h. At 24 h of reperfusion the EC50 for acetylcholine relaxation was significantly increased from 45 +/- 8 nM to 258 +/- 105 nM. Endothelium-independent relaxation to nitroglycerin was not affected. The 21-aminosteroid Tirilazad mesylate (U-74006F) prevented endothelial dysfunction; at 24 h of reperfusion the EC50 for acetylcholine relaxation in U-74006F-treated animals was 55 +/- 8 nM. Thus, endothelial function is impaired in this model of multiple organ failure and this impairment is prevented by Tirilazad mesylate.
Assuntos
Aorta Torácica/fisiopatologia , Toxinas Bacterianas/toxicidade , Endotélio Vascular/fisiopatologia , Endotoxinas/toxicidade , Isquemia/fisiopatologia , Fígado/irrigação sanguínea , Insuficiência de Múltiplos Órgãos/fisiopatologia , Traumatismo por Reperfusão/fisiopatologia , Vasodilatação/fisiologia , Acetilcolina/farmacologia , Animais , Antioxidantes/farmacologia , Constrição , Modelos Animais de Doenças , Peroxidação de Lipídeos , Masculino , Músculo Liso Vascular/fisiopatologia , Nitroglicerina/farmacologia , Fenilefrina/farmacologia , Pregnatrienos/farmacologia , Ratos , Ratos Endogâmicos F344RESUMO
The hypoosmotic lysis curve of freshly collected human erythrocytes is consistent with a single Gaussian error function with a mean of 46.5 +/- 0.25 mM NaCl and a standard deviation of 5.0 +/- 0.4 mM NaCl. After extended storage of RBCs under standard blood bank conditions the lysis curve conforms to the sum of two error functions instead of a possible shift in the mean and a broadening of a single error function. Thus, two distinct sub-populations with different fragilities are present instead of a single, broadly distributed population. One population is identical to the freshly collected erythrocytes, whereas the other population consists of osmotically fragile cells. The rate of generation of the new, osmotically fragile, population of cells was used to probe the hypothesis that lipid peroxidation is responsible for the induction of membrane fragility. If it is so, then the antioxidant, tirilazad mesylate (U-74,006f), should protect against this degradation of stored erythrocytes. We found that tirilazad mesylate, at 17 microM (1.5 mol% with respect to membrane lecithin), retards significantly the formation of the osmotically fragile RBCs. Concomitantly, the concentration of free hemoglobin which accumulates during storage is markedly reduced by the drug. Since the presence of the drug also decreases the amount of F2-isoprostanes formed during the storage period, an antioxidant mechanism must be operative. These results demonstrate that tirilazad mesylate significantly decreases the number of fragile erythrocytes formed during storage in the blood bank.
Assuntos
Antioxidantes , Preservação de Sangue/métodos , Fragilidade Osmótica/efeitos dos fármacos , Pregnatrienos/farmacologia , Dinoprosta/metabolismo , Membrana Eritrocítica/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Técnicas In Vitro , Fatores de Tempo , Vitamina E/sangueRESUMO
The vascular effects of tirilazad mesylate (U-74006F), a 21-aminosteroid antioxidant under development for the treatment of acute CNS trauma and ischemia, have been investigated using isolated rings of rabbit aorta. Endothelium-dependent relaxation was measured in rings contracted with 0.25 microM phenylephrine. Acetylcholine produced a dose-dependent relaxation that was slightly attenuated by the addition of U-74006F (0.1-10.0 microM). At a concentration of 10.0 microM, U-74006F shifted the EC50 for acetylcholine relaxation from 60 +/- 10 to 150 +/- 20 nM and reduced the maximum response by 20%. U-74006F (0.1-10.0 microM), did not reduce relaxation to the endothelium-independent vasodilator nitroglycerin nor did it affect the tone of either resting or phenylephrine contracted rings. ACh relaxation was abolished by a 40 min treatment with the superoxide generating system xanthine oxidase (XO; 0.1 U/ml) plus xanthine (0.4 mM). Relaxation to nitroglycerin was not impaired by XO, nor were the phenylephrine-induced contractions. U-74006F, at doses of 0.05-10 microM, protected against XO mediated damage to endothelium-dependent relaxation. These results demonstrate that tirilazad mesylate can protect endothelial function from damage by reactive oxygen species. Preservation of endothelial function might represent an important component of the activity of tirilazad mesylate in vivo.
Assuntos
Antioxidantes/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Pregnatrienos/farmacologia , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/enzimologia , Cálcio/metabolismo , Bovinos , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/enzimologia , Endotélio Vascular/fisiologia , Técnicas In Vitro , Óxido Nítrico/farmacologia , Coelhos , Vasodilatadores/antagonistas & inibidores , Vasodilatadores/farmacologia , Xantina Oxidase/antagonistas & inibidores , Xantina Oxidase/metabolismoRESUMO
The in vitro vasorelaxant and in vivo cardiovascular effects of synthetic S-nitrosothiols (RSNOs) were compared to standard nitrovasodilators. S-Nitroso-glutathione (GSNO), S-nitroso-N-acetylcysteine (NACysNO), S-nitroso-galactopyranose (GPSNO), S-nitroso-thioglycerol (TGSNO) and S-nitroso-homocysteine (HCysNO) relaxed phenylephrine (PE) contracted rabbit aorta at 50% effective concentrations (EC50s) of 3-46 nM. While nitroglycerin (GTN) exhibited in vitro tolerance after preincubation, the RSNOs were considerably less cross tolerant to GTN. In conscious dogs, GSNO, NACysNO and GPSNO (1-20 mcg/kg/min i.v.) paralleled nitroprusside (SNP) in reducing mean arterial and central venous pressure (MAP; CVP) with mild tachycardia. GSNO, NACysNO and SNP were more hypotensive and more resistant to isosorbide dinitrate (ISDN) cross tolerance than GTN. NACysNO showed mild self tolerance with low infusion (2.5 mcg/kg/min x 4h x 3 days) and blunted GTN's hypotension. These studies demonstrate that GSNO and NACysNO are SNP-like vasodilators in conscious dogs, which exhibit less cross tolerance to ISDN than GTN. Further, RSNOs relax vascular smooth muscle seemingly independent of nitric oxide (NO) liberation, and nitrate tolerance may involve reduced RSNO formation or NO release rather than desensitized guanylate cyclase (GC).
Assuntos
Sistema Cardiovascular/efeitos dos fármacos , Mercaptoetanol , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Compostos Nitrosos/farmacologia , S-Nitrosotióis , Compostos de Sulfidrila/farmacologia , Vasodilatadores/farmacologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Cães , Tolerância a Medicamentos , Feminino , Frequência Cardíaca/efeitos dos fármacos , Técnicas In Vitro , Masculino , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiologia , Óxido Nítrico/fisiologia , Coelhos , Fatores de TempoAssuntos
Leucotrieno B4/isolamento & purificação , Sarcoma de Mastócitos/análise , SRS-A/isolamento & purificação , Sarcoma Experimental/análise , Animais , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Camundongos , Espectrofotometria UltravioletaAssuntos
Ácidos Araquidônicos/biossíntese , Dietilcarbamazina/farmacologia , Ácidos Hidroxieicosatetraenoicos , Sarcoma de Mastócitos/metabolismo , SRS-A/biossíntese , Sarcoma Experimental/metabolismo , Animais , Ácidos Araquidônicos/metabolismo , Feminino , Leucotrieno B4 , Camundongos , Prostaglandinas/biossínteseRESUMO
Destruction of cartilage by matrix metalloproteinases (MMPs) plays a significant role in the pathology of osteoarthritis (OA). A translatable biomarker of MMP activity would enable development of MMP inhibitors for the treatment of OA and potentially the improved diagnosis of OA. A directed approach to identifying specific MMP cleavage products as potential biomarkers has been undertaken. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify peptides generated by MMP-driven degradation of human articular cartilage (HAC) in vivo. It was shown that a 45-mer peptide fragment of collagen type II with five hydroxyprolines (OH) can be selectively produced by the activity of collagenase, an enzyme purported to be involved in the pathology of OA. This 45-mer is the most abundant neoepitope peptide found in biological fluids such as urine and synovial fluid. An immunoaffinity LC-MS/MS assay has been developed to quantify collagen type II neoepitope peptides as biomarkers of collagenase modulation. The lower limit of quantification for this assay was established to be 0.035 nM. The assay was used to measure the levels of collagen type II peptides in the urine of both clinical (healthy human subjects) and preclinical species. The urinary levels of the most abundant peptides are reported for rat, rabbit, guinea pig, dog, and healthy human adult subjects. The utility of this peptide to monitor collagenase activity in vivo has been demonstrated through its detailed characterization in HAC explants as well as in the urine of human and other preclinical species.
Assuntos
Cartilagem Articular/metabolismo , Colágeno Tipo II/química , Epitopos/análise , Metaloproteinases da Matriz/metabolismo , Idoso , Sequência de Aminoácidos , Biomarcadores/análise , Células Cultivadas , Cromatografia Líquida/métodos , Colágeno Tipo II/metabolismo , Colagenases/metabolismo , Feminino , Humanos , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Fragmentos de Peptídeos/químicaRESUMO
S-nitrosothiols (RSNOs) are potent vasodilators. The smooth muscle relaxing activity of RSNOs has been suggested to be mediated by nitric oxide. In order to explore this premise, we examined the biological activity and chemical stability of several RSNOs. A series of eight RSNOs were synthesized and characterized. All of the RSNOs relaxed both vascular smooth muscle (rabbit aorta and mesenteric artery) and nonvascular smooth muscle (guinea pig trachea). The RSNOs also stimulated human platelet-soluble guanylate cyclase and inhibited collagen-induced human platelet aggregation. The biological activities of the synthetic RSNOs varied considerably as a function of structure in each assay. For example, the EC50s for relaxation of rabbit aorta ranged from 4.0 nM for S-nitroso-galctopyranose to 220 nM for S-nitroso-N-acetylpenicillamine. The biological activities also varied considerably between the assays; the rank order of potency for the eight compounds was different in each case. Thus changes in the structure of the R group can influence both the potency and the tissue selectivity of the RSNOs. Solution stabilities were determined for the RSNOs and found to vary considerably; first-order half-lives ranged from 0.023 hr for S-nitrosocysteine to 283 hr for S-nitrosothioglycerol. The solution stabilities of the RSNOs did not correlate with biological activities in any of the bioassays. These results indicate that decomposition of RSNOs in solution with production of nitric oxide cannot explain the biological activities of these compounds.
Assuntos
Mercaptoetanol , Óxido Nítrico/farmacologia , Compostos Nitrosos/farmacologia , S-Nitrosotióis , Vasodilatadores/farmacologia , Animais , Aorta Torácica/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Plaquetas/enzimologia , Química Farmacêutica , Sinergismo Farmacológico , Gases , Guanilato Ciclase/efeitos dos fármacos , Guanilato Ciclase/metabolismo , Cobaias , Humanos , Técnicas In Vitro , Artérias Mesentéricas/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Óxido Nítrico/fisiologia , Coelhos , Soluções , Relação Estrutura-Atividade , Traqueia/efeitos dos fármacosRESUMO
Lipid peroxidation is a component of various pathologies associated with nitric oxide (NO). It has been suggested that in vivo production of peroxynitrite (ONOO-) is responsible for the detrimental effects of pathologies associated with NO. To investigate the role of NO and ONOO- in the formation of specific lipid peroxidation products, liposomes of phosphatidylcholine were incubated at 37 degrees C for 1 h with various NO sources [NO, diethylamine NONOate (DEA/NO)] or ONOO-sources [3-morpholinosydnonimine-HCl (Sin-1), ONOO-]. Gas chromatography-mass spectrometry was used to quantify the formation of hydroperoxy- and hydroxyeicosatetraenoic acids (HETEs) and F2-isoprostanes. Peroxynitrite (0.5 mm) caused significant oxidation of phosphatidylcholine liposomes as measured by the increased formation of HETEs and F2-isoprostanes. NO, either added directly to the liposomes as a bolus or delivered by slow diffusion or via the donor compound DEA/NO, caused no lipid peroxidation itself and significantly inhibited both iron- and ONOO--induced lipid peroxidation. Sin-l (1 mM), which releases both NO and superoxide, resulted in minor increases in peroxidation and did not inhibit iron-induced HETE formation. We conclude that peroxynitrite but not nitric oxide can directly cause lipid peroxidation and that NO can act as an antioxidant by terminating lipid radical chain propagation reactions.
Assuntos
Dinoprosta/análogos & derivados , Dinoprosta/química , Ácidos Hidroxieicosatetraenoicos/química , Peroxidação de Lipídeos , Lipossomos , Nitratos , Óxido Nítrico , Fosfatidilcolinas , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico , Animais , Bovinos , Cromatografia Gasosa-Espectrometria de Massas , Ácidos Hidroxieicosatetraenoicos/análise , Fígado , OxirreduçãoRESUMO
The analysis of inositol phosphates by anion-exchange HPLC is described. The method employs a citrate buffer gradient to resolve several inositol phosphates including inositol 1-phosphate, inositol 1,4-bisphosphate (IP2), and inositol 1,4,5-trisphosphate (IP3), as well as some of the isomers of these compounds. Since the buffer system does not contain any phosphate, we can use a phosphate assay to examine the chromatographic behavior of phosphate-containing compounds. The method shows good resolution and recovery (greater than 95% for IP2 and IP3). Total analysis time, including reequilibration, is about 90 min. In addition, an isocratic system that can rapidly (less than 10 min) measure IP3 is described. The HPLC system was used to characterize inositol phosphate turnover in thrombin-stimulated platelets and formylmethionyl-leucyl-phenylalanine-stimulated HL-60 cells.