RESUMO
We used four mutants having albino or pale green phenotypes with disrupted nuclear-encoded chloroplast proteins to analyze the regulatory system of metabolites in chloroplast. We performed an integrated analyses of transcriptomes and metabolomes of the four mutants. Transcriptome analysis was carried out using the Agilent Arabidopsis 2 Oligo Microarray, and metabolome analysis with two mass spectrometers; a direct-infusion Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR/MS) and a gas chromatograph-time of flight mass spectrometer. Among approximately 200 known metabolites detected by the FT-ICR/MS, 71 metabolites showed significant changes in the mutants when compared with controls (Ds donor plants). Significant accumulation of several amino acids (glutamine, glutamate and asparagine) was observed in the albino and pale green mutants. Transcriptome analysis revealed altered expressions of genes in several metabolic pathways. For example, genes involved in the tricarboxylic acid cycle, the oxidative pentose phosphate pathway, and the de novo purine nucleotide biosynthetic pathway were up-regulated. These results suggest that nitrogen assimilation is constitutively promoted in the albino and pale green mutants. The accumulation of ammonium ions in the albino and pale green mutants was consistently higher than in Ds donor lines. Furthermore, genes related to pyridoxin accumulation and the de novo purine nucleotide biosynthetic pathway were up-regulated, which may have occurred as a result of the accumulation of glutamine in the albino and pale green mutants. The difference in metabolic profiles seems to be correlated with the disruption of chloroplast internal membrane structures in the mutants. In albino mutants, the alteration of metabolites accumulation and genes expression is stronger than pale green mutants.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Cloroplastos/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Metaboloma , Transcriptoma , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Cloroplastos/genética , Análise por Conglomerados , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Mutação , Análise de Componente PrincipalRESUMO
In the present study, a high-throughput analytical method for intracellular metabolites using MALDI-MS has been developed. As an analytical tool, the quantitative performance and dynamic range of MALDI-TOF-MS was confirmed to be suitable for characterizing the trends of intracellular metabolism. The technique was tested by investigating the intracellular metabolism of Escherichia coli by analyzing whole cell samples taken consecutively before and after a perturbation of the environmental carbon source. As the result, dramatic changes of metabolite concentrations responding to the perturbation were observed. The whole analysis process (i.e., sample preparation and MALDI-MS analysis for 24 time points in triplicate) was completed within 4 hours. MALDI-FTICR-MS was used to identify the elemental compositions of detected metabolites to support the reliability of the MALDI-MS-based analysis. The MALDI-MS-based analytical method developed herein should be suitable for high-throughput analysis of dynamic intracellular metabolism events.
Assuntos
Citoplasma/metabolismo , Escherichia coli/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Fenômenos BioquímicosRESUMO
We propose a novel strategy for determining the elemental composition of organic compounds using the peak ratio of isotopic fine structure observed by high-magnetic field Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). Using 3'-phosphoadenosine 5'-phosphosulfate and CTU guanamine as standard organic compounds, isotopic peaks derived from (15)N-, (34)S-, and (18)O-substituted forms were separated from (13)C-substituted species. Furthermore, these isotopic peaks were quantitatively detected and closely matched the natural abundance of each element. These data successfully led us to determine the one elemental composition in a standard independent manner. The approach should be particularly amenable to the metabolomics research field.
Assuntos
Análise de Fourier , Íons/química , Espectrometria de Massas/métodos , Compostos Orgânicos/química , Isótopos de Carbono/química , Metabolômica/métodos , Isótopos de Nitrogênio/química , Isótopos de Oxigênio/química , Fosfoadenosina Fosfossulfato/química , Isótopos de Enxofre/químicaRESUMO
Complexes of microcystins (MCs) and metal ions were detected with mass spectrometry. To observe the MCs-metals complexes, CryoSpray ionization ion source was developed and it was equipped on a commercial Fourier transform ion resonance mass spectrometer. Mixtures of two MCs and five metals were applied to Fourier transform ion cyclotron resonance mass spectrometry coupled with CryoSpray. The analyses showed complexes of MC-LR-Fe(II), -Zn, -Cu, -Mg, MC-RR-Fe(II), -Zn, -Cu, and -Mg but MC-LR-Fe(III) and MC-RR-Fe(III) were not observed. Present study suggested that MCs may play roles in metal ion uptake and/or accumulation in the algal cells.
Assuntos
Ciclotrons , Espectrometria de Massas/métodos , Metais/análise , Microcistinas/análise , Análise de Fourier , Espectrometria de Massas/instrumentação , Metais/química , Microcistinas/química , Propriedades de SuperfícieRESUMO
Mannose is an essential hexose that is required for glycoprotein synthesis. Although circulating mannose levels are known to be influenced by metabolic disorders, how physiological levels of mannose fluctuate in normal and diabetic subjects is largely unknown. We describe a new accurate and sensitive assay for determining circulating mannose levels, which we used to measure plasma mannose levels in 273 normal and diabetic (DM) subjects. Our results revealed a clear correlation (r = 0.754) between fasting plasma mannose (FPM) and fasting plasma glucose (FPG) levels. Our mannose assay showed sensitivity and specificity comparable to that seen for hemoglobin A(1c) (HbA(1c)) assay in subjects with impaired glucose tolerance (IGT) or DM whose FPG levels were normal. Mannose levels were found to increase less than glucose levels in response to an oral glucose tolerance test (OGTT). Furthermore, plasma mannose levels did not significantly change following a meal and more closely correlated with the coefficient of variation (CV) of daily glucose levels than did glucose itself. In conclusion, the close correlation between FPM and FPG levels taken together with the small fluctuations seen in plasma mannose in response to glucose suggests that the measurement of mannose using our assay could potentially play a supplementary role in the diagnosis and screening of patients with mild DM.
Assuntos
Diabetes Mellitus/sangue , Intolerância à Glucose/sangue , Manose/sangue , Adulto , Idoso , Glicemia/metabolismo , Colorimetria , Corantes , Diabetes Mellitus Tipo 2/sangue , Ingestão de Alimentos/fisiologia , Jejum/metabolismo , Feminino , Frutosamina/metabolismo , Teste de Tolerância a Glucose , Hemoglobinas Glicadas/metabolismo , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
In a study population, we compared the level of malondialdehyde-modified LDL (MDA-LDL) with the concentrations of lipid parameters in serum and found a strong correlation between MDA-LDL and apolipoprotein B (apo B) concentrations. Their interrelations had a turning point at an apo B concentration of 1,150 mg/l. In diabetic patients, the ratio of MDA-LDL/apo B increased at apo B concentrations above 1,150 mg/l. This ratio represents the extent of modification of apo B by MDA. In the control subjects, this ratio remained stable. When we divided the patients into medication groups (statins and insulin), we found that the 1,150 mg/l threshold disappeared. At apo B concentrations above 1,150 mg/l, the ratio of MDA-LDL/apo B in the statin group was as high as that in the non-drug group. In the insulin group, the means of MDA-LDL/apo B in all ranges of apo B levels decreased to an extent statistically indistinguishable from those of the control group. In conclusion, insulin therapy represses LDL oxidation even at apo B concentrations > 1,150 mg/l and should be noted for its anti-oxidation properties.
Assuntos
LDL-Colesterol/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Hipoglicemiantes/administração & dosagem , Insulina/administração & dosagem , Adulto , Idoso , Apolipoproteínas B/metabolismo , HDL-Colesterol/metabolismo , Eletroforese , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Hipolipemiantes/administração & dosagem , Lipoproteínas LDL/metabolismo , Masculino , Malondialdeído/metabolismo , Pessoa de Meia-Idade , Triglicerídeos/metabolismo , Receptor fas/metabolismoRESUMO
Several reports have suggested that HDL has anti-oxidative actions. We investigated the relationship between HDL-cholesterol (HDL-C) and malondialdehyde-modified LDL (MDA-LDL) concentrations using enzyme linked immunosolvent assay. We divided our study subjects into four groups on the basis of concentrations of triglyceride (TG) and HDL-C by the following lipid profiles: serum TG < or = 1.69 mmol/L and HDL-C > or = 1.16 mmol/L (control group, n = 26); TG >1.69 and HDL-C < or = 1.16 (high TG group, n = 22); TG >1.69 and HDL-C < or = 0.91 (high TG & low HDL group, n = 67); TG < or = 1.69 and HDL-C < or = 0.91 (low HDL group, n = 21). MDA-LDL concentrations, MDA-LDL/apolipoprotein B (apo B) ratio, and LDL size were different between subjects in high TG & low HDL and control groups. MDA-LDL concentrations in both high TG and low HDL groups did not differ significantly from those in the control. However, MDA-LDL/apo B ratio in low HDL group was significantly higher than that in the control (P < 0.05). The MDA-LDL/apo B ratio reflects the extent of MDA modification of apo B in LDL. Therefore, our data suggest that as HDL-C concentrations fall, the extent of MDA modification per one LDL particle increases. Moreover, accompanied by high TG concentration, LDL size in subjects with lower HDL-C concentrations became smaller.
Assuntos
HDL-Colesterol/metabolismo , LDL-Colesterol/metabolismo , Malondialdeído/metabolismo , Estresse Oxidativo/fisiologia , Adulto , Idoso , Apolipoproteínas B/sangue , Apolipoproteínas B/metabolismo , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Feminino , Humanos , Masculino , Malondialdeído/sangue , Pessoa de Meia-Idade , Triglicerídeos/sangue , Triglicerídeos/metabolismoRESUMO
BACKGROUND: Drug therapy is considered essential to the clinical prevention of atherosclerotic lesions in patients with diabetes mellitus (DM). METHODS: To confirm the effects of fibrate therapy, we determined low-density lipoprotein (LDL) size by gradient gel electrophoresis and malondialdehyde-modified LDL (MDA-LDL) concentrations by enzyme-linked immunosolvent assay (ELISA) and clarified the association between apolipoprotein B (apo B) and MDA-LDL during the fibrate therapy. RESULTS: Mean MDA-LDL concentrations were higher in healthy men than in healthy women. There were no significant differences in mean MDA-LDL concentrations between age groups for males or females. According to the regression equation (y = 0.063x + 10.9) obtained for apo B and MDA-LDL concentrations with fibrate treatment, the apo B concentration in those may need to be decreased to 1260 mg/l to restore the MDA-LDL concentration to the control concentration (65 +/- 25 units/l). This slope of the apoB/MDA-LDL regression line was approximately half of that with no-drug treatment (y = 0.109x - 10.8). CONCLUSIONS: Fibrate therapy had an effect on reducing serum MDA-LDL concentration in diabetic patients.
Assuntos
Bezafibrato/farmacologia , Diabetes Mellitus/sangue , Diabetes Mellitus/metabolismo , Lipoproteínas LDL/sangue , Lipoproteínas LDL/metabolismo , Malondialdeído/química , Adulto , Envelhecimento , Apolipoproteínas B/sangue , Arteriosclerose/sangue , Arteriosclerose/complicações , Arteriosclerose/metabolismo , Arteriosclerose/prevenção & controle , Complicações do Diabetes , Diabetes Mellitus/terapia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Lipoproteínas LDL/química , Lipoproteínas LDL/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Caracteres SexuaisRESUMO
Microcystins (MCs) were purified by DEAE and C(18) cartridge chromatography. Addition of EtOH to the eluents (20%) in DEAE chromatography gave higher resolution than no addition of EtOH. The chromatogram showed three peaks: MC-LR; MC-LY and MC-LF; MC-LW. MC-LR and MC-LW were obtained by one step chromatography with purity of 96 and 88%, respectively. The separation of MC-LF and MC-LW with DEAE chromatography was better than that with reversed-phase chromatography. MC-LY and MC-LF were separated with C(18) cartridge. On the chromatogram, there were three peaks consisting of MC-LY (81% purity), MC-LF (86%), and an unknown compound which was considered as a MC variant judging from the results in HPLC/PDA, FAB-MS, and 1H NMR analyses, but the structure could not be determined. It is concluded that the combination of DEAE and C(18) cartridge chromatography would be a practical approach for the purification of various MCs.
Assuntos
Toxinas Bacterianas/isolamento & purificação , Cianobactérias/metabolismo , Peptídeos Cíclicos/isolamento & purificação , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia por Troca Iônica/métodos , Etanol/química , Técnicas In Vitro , MicrocistinasRESUMO
OBJECTIVE: Rebamipide tablets, which are used in the treatment of patients with gastric ulcers or gastritis, can be difficult to administer in subjects with reduced swallowing ability or impaired swallowing. The granule formulation may be more easily administered in these patients. The bioequivalence between rebamipide granules (20%/0.5g) and tablets (100mg) was determined in healthy male adult volunteers, in accordance with the Partially Revised Guidelines for Bioequivalence Studies of Generic Products. STUDY DESIGN: In a randomised, nonblind, crossover design, 28 individuals were allocated into two groups of 14 to receive either rebamipide granules or rebamipide tablets. Each individual, under fasting conditions, was administered a single oral dose of rebamipide 100mg followed by a 7-day washout period. Individuals then received a single oral dose of the other rebamipide formulation. Blood samples were collected at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12 and 24 hours. Plasma rebamipide concentrations were measured by validated high-performance liquid chromatography with tandem mass spectrometry. RESULTS: The plasma concentration-time profiles and pharmacokinetic parameters of rebamipide after administration of the granule formulation were similar to those of the tablet in 27 healthy male volunteers. Following administration of the granule formulation, the area under the plasma concentration-time curve from time 0-24 hours (AUC(24h)) was 912.82 mug/L . h, the maximum plasma concentration (C(max)) was 241.82 mug/L, time to maximum plasma concentration (t(max)) was 2.5 hours, and plasma elimination half-life (t((1/2))) was 1.97 hours. Corresponding values for the tablet formulation were 873.55 microg/L . h, 216.19 mug/L, 2.4 hours, and 1.94 hours. The difference in mean log values was 1.01 for AUC(24h) and 1.09 for C(max) after granule and tablet administration. The 90% confidence interval of this difference in mean log value was 0.93-1.10 for AUC(24h), and 0.97-1.21 for C(max). This satisfies the criteria for bioequivalence in the guidelines [within log (0.8) to log (1.25)]. CONCLUSIONS: Rebamipide granules (20%/0.5g) and tablet (100mg) were bioequivalent. Rebamipide granules may therefore be a more practical treatment option in patients with gastric ulcers or gastritis who have difficulty swallowing tablets.
RESUMO
In mass spectrometry (MS)-based metabolomics studies, reference-free identification of metabolites is still a challenging issue. Previously, we demonstrated that the elemental composition (EC) of metabolites could be unambiguously determined using isotopic fine structure, observed by ultrahigh resolution MS, which provided the relative isotopic abundance (RIA) of (13)C, (15)N, (18)O, and (34)S. Herein, we evaluated the efficacy of the RIA for determining ECs based on the MS peaks of 20,258 known metabolites. The metabolites were simulated with a ≤25% error in the isotopic peak area to investigate how the error size effect affected the rate of unambiguous determination of the ECs. The simulation indicated that, in combination with reported constraint rules, the RIA led to unambiguous determination of the ECs for more than 90% of the tested metabolites. It was noteworthy that, in positive ion mode, the process could distinguish alkali metal-adduct ions ([M+Na](+) and [M+K](+)). However, a significant degradation of the EC determination performance was observed when the method was applied to real metabolomic data (mouse liver extracts analyzed by infusion ESI), because of the influence of noise and bias on the RIA. To achieve ideal performance, as indicated in the simulation, we developed an additional method to compensate for bias on the measured ion intensities. The method improved the performance of the calculation, permitting determination of ECs for 72% of the observed peaks. The proposed method is considered a useful starting point for high-throughput identification of metabolites in metabolomic research.
Assuntos
Espectrometria de Massas/métodos , Metaboloma , Metabolômica/métodos , Animais , Isótopos de Carbono/análise , Simulação por Computador , Análise de Fourier , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Isótopos de Nitrogênio/análise , Isótopos de Oxigênio/análise , Isótopos de Enxofre/análiseRESUMO
Although understanding the high-resolution spatial distribution of bioactive small molecules is indispensable for elucidating their biological or pharmacological effects, there has been no analytical technique that can easily detect the naïve molecular localization in mammalian tissues. We herein present a novel in situ label-free imaging technique for visualizing bioactive small molecules, using a polyphenol. We established a 1,5-diaminonaphthalene (1,5-DAN)-based matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) technique for visualizing epigallocatechin-3-O-gallate (EGCG), the major bioactive green tea polyphenol, within mammalian tissue micro-regions after oral dosing. Furthermore, the combination of this label-free MALDI-MSI method and a standard-independent metabolite identification method, an isotopic fine structure analysis using ultrahigh-resolution mass spectrometer, allows for the visualization of spatially-resolved biotransformation based on simultaneous mapping of EGCG and its phase II metabolites. Although this approach has limitations of the detection sensitivity, it will overcome the drawbacks associated with conventional molecular imaging techniques, and could contribute to biological discovery.
Assuntos
Catequina/análogos & derivados , Imagem Molecular/métodos , Polifenóis/farmacocinética , 2-Naftilamina/análogos & derivados , 2-Naftilamina/química , Animais , Biotransformação , Catequina/administração & dosagem , Catequina/química , Catequina/farmacocinética , Íons , Rim/metabolismo , Fígado/metabolismo , Masculino , Desintoxicação Metabólica Fase II , Camundongos , Camundongos Endogâmicos C57BL , Polifenóis/administração & dosagem , Polifenóis/química , Padrões de Referência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Coloração e Rotulagem , Extratos de TecidosRESUMO
In order to develop a new model of diet research, blood was drawn from 12 adult volunteers for 3 wk on regular diets as controls, and for a subsequent 3 wk supplemented with 18.5 g of freeze-dried tofu (Koya tofu) every day. Triplicate aliquots of 0.06 mL each of whole blood were stimulated ex vivo with phytohemagglutinin (PHA)-P, heat aggregated human IgG (HAG), lipopolysaccharide (LPS), zymosan A, and anti-T cell receptor (TCR) monoclonal antibody to activate specific subsets of leukocytes, then the levels of various inflammatory cytokine mRNA were quantified by real time PCR. Koya tofu significantly (p<0.05) augmented the fold increase of PHA-induced tumor necrosis factor superfamily (TNFSF) 15, IL6, and IL8, HAG-induced TNFSF15 and IL8, LPS-induced IL6 and IL8, zymosan-induced TNFSF15, IL6 and IL8, and TCR-induced TNFSF2 in comparison to the regular diet. Such increase was due to the reduction of baseline mRNA expression, not the enhancement of mRNA induction after specific stimulations. Six (TNFSF15), 4 (IL6), and 3 (IL10) subjects showed significant reduction of baseline mRNA during the Koya tofu diet compared to that of the control diet. Despite large individual-to-individual and day-to-day variation of mRNA, the method employed in this study was sensitive enough to identify statistically significant results as a group as well as on an individual basis, which will be a foundation for tailored diet in the future. The results also indicated that Koya tofu had a power to alter mRNA expression in leukocytes, and TNFSF15, IL6, and IL10 would be biomarkers for soy.
Assuntos
Citocinas/sangue , Glycine max , Mediadores da Inflamação/sangue , Inflamação/sangue , Leucócitos/efeitos dos fármacos , Preparações de Plantas/farmacologia , Alimentos de Soja , Adulto , Anticorpos Monoclonais , Biomarcadores/sangue , Citocinas/genética , Suplementos Nutricionais , Feminino , Humanos , Imunoglobulina G , Inflamação/induzido quimicamente , Leucócitos/metabolismo , Lipopolissacarídeos , Masculino , Pessoa de Meia-Idade , Fito-Hemaglutininas , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Adulto Jovem , ZimosanRESUMO
BACKGROUND: Pre-beta 1-HDL participates in a cyclic process involved in the retrieval of cholesterol from peripheral tissues. Although pre-beta 1-HDL can be measured by two-dimensional electrophoresis or crossed immunoelectrophoresis, these methods are time-consuming and require technical expertise. In this study, we separated plasma lipid-poor apolipoprotein A-I (apo A-I) by high-performance size-exclusion chromatography. METHODS: We measured plasma lipid-poor apo A-I in 20 male patients with coronary artery disease [CAD; mean (SD) age, 64.0 (18) years] and 15 male controls [54.7 (17) years] and in 7 female CAD patients [70.3 (7.7) years] and 9 female controls [65.1 (4.7) years]. RESULTS: Lipid-poor apo A-I was most stable when stored at -80 degrees C in the presence of aprotinin (final concentration, 50 kIU/L). The lipid-poor apo A-I concentration decreased during incubation at 37 degrees C, and this was not prevented by the addition of 2 mmol/L of the lecithin:cholesterol acyltransferase (LCAT) inhibitor 5,5'-dithiobis(2-nitrobenzoic acid). Lipid-poor apo A-I was significantly higher in CAD patients than in controls [38.3 (7.9) mg/L for male CAD patients vs 29.3 (7.3) mg/L for male controls; 43.3 (11) mg/L for female CAD patients vs 27.1 (7.4) mg/L for female controls (P <0.01 for both)]. There were no significant differences in LCAT activity or cholesteryl ester transfer protein (CETP) concentration between patients and controls. Moreover, the plasma lipid-poor apo A-I concentration was not significantly correlated with LCAT or CETP activities. CONCLUSIONS: Although the production of lipid-poor apo A-I in plasma is not fully understood, our results indicate that lipid-poor apo A-I could be used as a marker for arteriosclerosis and demonstrate that it is not identical to the pre-beta1-HDL measured by other methods.
Assuntos
Apolipoproteína A-I/sangue , Doença da Artéria Coronariana/sangue , Lipoproteínas HDL/sangue , Idoso , Proteínas de Transporte/sangue , Proteínas de Transferência de Ésteres de Colesterol , Cromatografia em Gel , Ácido Ditionitrobenzoico/farmacologia , Feminino , Glicoproteínas/sangue , Lipoproteínas de Alta Densidade Pré-beta , Humanos , Masculino , Pessoa de Meia-Idade , Fosfatidilcolina-Esterol O-Aciltransferase/antagonistas & inibidores , PlasmaRESUMO
We established five monoclonal antibodies that reacted with human LCAT and recognized different epitopes on LCAT. These are mouse anti-human LCAT monoclonal antibodies designated 36487, 36454, 36442, 36405, and 36486, which react with the peptides corresponding to human LCAT amino acid residues R159-E179, M258-S273, S274-S294, D352-S376, and N415-E440, respectively. We also successfully used two of these antibodies to develop an ELISA, which uses a solid phase monoclonal antibody, 36486, that reacts with the C-terminus of LCAT, and a detection monoclonal antibody, 36487, that reacts with an epitope located in the center of the LCAT primary structure. We observed a significant positive correlation between the values of LCAT protein determined with ELISA and LCAT activity determined with liposome substrate (r = 0.871, P < 0.001) or the endogenous self-substrate method (r = 0.864, P < 0.001), and we obtained inter- and intra-assay coefficients of variation less than 6.1%, minimum detection limit of 0.1 microg/ml. Highly specific monoclonal antibodies will be useful in the study of the molecular pathology of LCAT. Therefore, this precise and sensitive LCAT assay will help clarify the role of this enzyme in the metabolism of HDLs, and can be used for diagnostic purposes in investigating liver function. We obtained five monoclonal antibodies that recognized different epitopes on LCAT and developed a sandwich-type ELISA. Highly specific monoclonal antibodies provide a sensitive and specific analytical system for measurements of LCAT protein.
Assuntos
Anticorpos Monoclonais/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Epitopos/imunologia , Fosfatidilcolina-Esterol O-Aciltransferase/sangue , Sequência de Aminoácidos , Sítios de Ligação de Anticorpos/efeitos dos fármacos , Humanos , Lipoproteínas/sangue , Lipoproteínas/química , Dados de Sequência Molecular , Oligopeptídeos/farmacologia , Fosfatidilcolina-Esterol O-Aciltransferase/química , Fosfatidilcolina-Esterol O-Aciltransferase/imunologiaRESUMO
Cholesteryl ester transfer protein (CETP) greatly affects the metabolism of all lipoprotein classes including low-density lipoprotein (LDL) and high-density lipoprotein (HDL), both known to constitute powerful risk factors for coronary artery disease (CAD). We now report the successful first cloning and characterization of single-chain antibody fragments specific for CETP. A recombinant phage display library was generated using spleen mRNA isolated from BALB/c mice that had been immunized with highly purified CETP. Screening of the library yielded two single-chain antibody fragments with high affinity for CETP, termed 1CL8 and 1CL10, displaying respective KD values of 4.36 x 10(-9) M and 4.64 x 10(-9) M as determined by affinity sensor technology. Amino acid sequence comparison indicated the complementarity-determining regions of the respective heavy chains to be responsible for CETP high affinity binding. Fragment 1CL8 was successfully employed in clinical chemical quantification systems that uncovered an association in humans between plasma CETP concentration and total body fat mass (r=0.50, p<0.002). Because of the demonstrated superb CETP capturing capacity, combined with high binding affinity to CETP, ready access and unlimited supply, 1CL8 and 1CL10 are expected to prove powerful tools for studies on the role of CETP in atherogenesis.