RESUMO
Functional ecology has long considered the support function as important, but its biomechanical complexity is only just being elucidated. We show here that it can be described on the basis of four biomechanical traits, two safety traits against winds and self-buckling, and two motricity traits involved in sustaining an upright position, tropic motion velocity (MV) and posture control (PC). All these traits are integrated at the tree scale, combining tree size and shape together with wood properties. The assumption of trait constancy has been used to derive allometric scaling laws, but it was more recently found that observing their variations among environments and functional groups, or during ontogeny, provides more insights into adaptive syndromes of tree shape and wood properties. However, oversimplified expressions have often been used, possibly concealing key adaptive drivers. An extreme case of oversimplification is the use of wood basic density as a proxy for safety. Actually, as wood density is involved in stiffness, loads, and construction costs, the impact of its variations on safety is non-trivial. Moreover, other wood features, especially the microfibril angle (MFA), are also involved. Furthermore, wood is not only stiff and strong, but it also acts as a motor for MV and PC. The relevant wood trait for this is maturation strain asymmetry. Maturation strains vary with cell-wall characteristics such as MFA, rather than with wood density. Finally, the need for further studies about the ecological relevance of branching patterns, motricity traits, and growth responses to mechanical loads is discussed.
Assuntos
Gravitropismo/fisiologia , Árvores/fisiologia , Madeira/fisiologia , Fenômenos Biomecânicos , Força Compressiva , Simulação por Computador , Ecologia , Modelos Biológicos , Fenótipo , Caules de Planta/anatomia & histologia , Caules de Planta/crescimento & desenvolvimento , Caules de Planta/fisiologia , Árvores/anatomia & histologia , Árvores/crescimento & desenvolvimento , Vento , Madeira/anatomia & histologia , Madeira/crescimento & desenvolvimentoRESUMO
Gold metallodrugs form a class of promising antiproliferative agents showing a high propensity to react with proteins. We exploit here X-ray absorption spectroscopy (XAS) methods [both X-ray absorption near-edge spectroscopy (XANES) and extended X-ray absorption fine structure (EXAFS)] to gain insight into the nature of the adducts formed between three representative gold(I, III) metallodrugs (i.e., auranofin, [Au(2,2'-bipyridine)(OH)(2)](PF(6)), Aubipy, and dinuclear [Au(2)(6,6'-dimethyl-2,2'-bipyridine)(2)(µ-O)(2)](PF(6))(2), Auoxo6) and two major plasma proteins, namely, bovine serum albumin (BSA) and human serum apotransferrin (apoTf). The following metallodrug-protein systems were investigated in depth: auranofin/apoTf, Aubipy/BSA, and Auoxo6/apoTf. XANES spectra revealed that auranofin, upon protein binding, conserves its gold(I) oxidation state. Protein binding most probably takes place through release of the thiosugar ligand and its subsequent replacement by a thiol (or a thioether) from the protein. This hypothesis is independently supported by EXAFS results. In contrast, the reactions of Aubipy with serum albumin and of Auoxo6 with serum apoTf invariantly result in gold(III) to gold(I) reduction. Gold(III) reduction, clearly documented by XANES, is accompanied, in both cases, by release of the bipyridyl ligands; for Auoxo6 cleavage of the gold-gold dioxo bridge is also observed. Gold(III) reduction leads to formation of protein-bound gold(I) species, with deeply modified metal coordination environments, as evidenced by EXAFS. In these adducts, the gold(I) centers are probably anchored to the protein through nitrogen donors. In general, these two XAS methods, i.e., XANES and EXAFS, used here jointly, allowed us to gain independent structural information on metallodrug/protein systems; detailed insight into the gold oxidation state and the local environment of protein-bound metal atoms was achieved in the various cases.
Assuntos
Apoproteínas/química , Compostos de Ouro/química , Soroalbumina Bovina/química , Transferrina/química , Espectroscopia por Absorção de Raios X/métodos , Animais , Auranofina/química , Bovinos , HumanosRESUMO
Ferrociphenol (Fc-diOH) is a new molecule belonging to the fast-growing family of organometallic anti-cancer drugs. In a previous study, we showed promising in vivo results obtained after the intratumoural subcutaneous administration of the new drug-carrier system Fc-diOH-LNCs on a 9L-glioma model. To further increase the dose of this lipophilic entity, we have created a series of prodrugs of Fc-diOH. The phenol groups were protected by either an acetyl (Fc-diAc) or by the long fatty-acid chain of a palmitate (Fc-diPal). LNCs loaded with Fc-diOH prodrugs have to be activated in situ by enzymatic hydrolysis. We show here that the protection of diphenol groups with palmitoyl results in the loss of Fc-diOH in vitro activity, probably due to a lack of in situ hydrolysis. On the contrary, protection with an acetate group does not affect the strong, in vitro, antiproliferative effect of ferrocifen-loaded-LNCs neither the reduction of tumour volume observed on an ectopic model, confirming that acetate is easily cleaved by cell hydrolases. Moreover, the cytostatic activity of Fc-diOH-LNCs is confirmed on an orthotopic glioma model since the difference in survival time between the infusion of 0.36 mg/rat Fc-diOH-LNCs and blank LNCs is statistically significant. By using LNCs or Labrafac to carry the drug, a dose-effect ranging from 0.005 to 2.5mg of Fc-diOH per animal can be evidenced.
Assuntos
Compostos Ferrosos/administração & dosagem , Glioma/tratamento farmacológico , Lipídeos/administração & dosagem , Nanocápsulas/administração & dosagem , Animais , Relação Dose-Resposta a Droga , Portadores de Fármacos/administração & dosagem , Feminino , Glioma/metabolismo , Ratos , Ratos Endogâmicos F344 , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Malignant melanoma is one of the most severe forms of skin cancer, and chemotherapeutic agents currently in use are poorly effective in curing the disease. Here we describe the properties of two organometallic ferrocenyl derivatives, ferrocifen (Fc-OH-Tam) and ferrociphenol (Fc-diOH) that show a specific antiproliferative effect on melanoma cells. After a short incubation period, Fc-OH-Tam is highly cytotoxic on melanoma cells but less toxic on melanocytes. Fc-diOH is slightly toxic at a high concentration but no discrepancy is observed between malignant and normal cells. After a long incubation time the latter is highly toxic for malignant cells but not for normal cells while the former was very highly toxic for primary malignant cells and significantly less toxic for normal cells. We also found that oxidative stress is not implicated in the mechanism of cytotoxicity, since both derivatives neither induce reactive oxygen species (ROS) level in melanocytes nor in melanoma cells. Finally, investigation on hair follicle growth revealed that the two organometallic derivatives induced an irreversible ejection of the hair shaft, thus predicting a potential hair loss side effect if used as a chemotherapeutic treatment.
Assuntos
Antineoplásicos/toxicidade , Compostos Ferrosos/toxicidade , Melanócitos/efeitos dos fármacos , Melanoma/tratamento farmacológico , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Avaliação Pré-Clínica de Medicamentos , Folículo Piloso/efeitos dos fármacos , Humanos , Melanócitos/citologia , Melanócitos/patologia , Melanoma/patologia , Melanoma/secundário , Metástase Neoplásica/patologia , Compostos Organometálicos/toxicidade , Estresse OxidativoRESUMO
During the last years, research in the area of immunoanalysis has been oriented towards the marketing of more reliable, faster and low cost kits. In this field, simultaneous multiple immunoanalysis that offer long-time ignored original and specific advantages, has undergone tremendous improvements triggered by striking technological innovations and the advent of lanthanide chelate based markers. Simultaneous multiple immunoassays have turned from semi-quantitative methods restricted to pharmacology to quantitative methods for medical biology. We chose first to describe several commercial systems among the most representative then we stressed on the main emerging systems based on optical or electrochemical detection as well as some immunosensor devices. The array of simultaneous multiple immunoassays currently available appears attractive enough so as to take a significant area within the analytical arsenal at the biologist disposal.
Assuntos
Imunoensaio/métodos , Biologia/métodos , Eletrofisiologia , Microscopia Eletrônica de VarreduraRESUMO
The introduction of heavy atoms into protein crystals is sometimes rendered difficult and tedious because of the poor specificity of the available reagents for particular target residues. On the other hand, transition organometallic chemistry offers an almost untouched field for this purpose. In particular, Fischer-type metallocarbene complexes of the general formula (CO)5W=C(OR1)R2 may be attractive reagents because they contain the heavy element tungsten and specifically target amino groups to form stable, covalent aminocarbene adducts. With a small protein such as hen egg white lysozyme (HEWL) with a limited number of potential binding sites, it was possible to form protein-aminocarbene conjugates that have an average of one aminocarbene moiety per protein molecule. RP-HPLC combined with matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS analysis of the conjugates revealed that they were mixtures of the native protein, monoaminocarbenes and diaminocarbenes. Tryptic proteolysis experiments performed on the protein conjugates combined with MALDI-TOF-MS analysis of the aminocarbenic peptides allowed us to determine that lysines 13, 33, 97 and 116 were involved in the reaction of HEWL with (CO)5W=C(OMe)Me.
Assuntos
Proteínas do Ovo/metabolismo , Metano/análogos & derivados , Metano/metabolismo , Muramidase/metabolismo , Compostos Organometálicos/metabolismo , Animais , Sítios de Ligação , Galinhas , Cromatografia Líquida de Alta Pressão , Proteínas do Ovo/química , Feminino , Hidrocarbonetos , Metano/química , Muramidase/química , Compostos Organometálicos/química , Mapeamento de Peptídeos , Ligação Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tripsina/metabolismoRESUMO
The small, compact, robust, and nonpolar units of [CpM(CO)3] (M = Re, Tc) coupled with biomolecules may be considered as bioorganometallic entities of potential interest in the field of medicinal chemistry. However, the short half-life of useful radionuclides (186Re t1/2 = 3.7 d, 188Re t1/2 = 16.8 h, 99mTc t1/2 = 6 h), the risks inherent in their use, and their cost have led chemists to search for novel synthetic strategies that allow the rapid introduction of the [CpM(CO)3] moiety as a late step in the course of synthesizing the target molecule. The present paper describes different strategies recently reported in the literature to tackle this problem.
Assuntos
Ciclopentanos/química , Estrogênios/síntese química , Compostos Organometálicos/síntese química , Compostos de Organotecnécio/síntese química , Radioisótopos/química , Compostos Radiofarmacêuticos/síntese química , Rênio/química , Tamoxifeno/síntese química , Neoplasias da Mama/tratamento farmacológico , Química Farmacêutica/métodos , Estrogênios/química , Meia-Vida , Estrutura Molecular , Octreotida/química , Compostos Organometálicos/química , Compostos de Organotecnécio/química , Peptídeos/química , Proteínas/química , Compostos Radiofarmacêuticos/química , Moduladores Seletivos de Receptor Estrogênico/química , Relação Estrutura-Atividade , Tamoxifeno/análogos & derivados , Tamoxifeno/química , Tamoxifeno/uso terapêutico , Tecnécio Tc 99m Sestamibi/químicaRESUMO
We compare herein the interfacial reactivity of self-assembled monolayers (SAMs) of 11-mercaptoundecanoic acid (MUA), 1-undecanethiol (UDT) and 11-mercaptoundecanol (MUD) on gold surfaces towards aqueous solutions of poly-(L-lysine) (PL). Liquid-phase labelling of PL with the alkyne dicobalt hexacarbonyl cluster 1 combined with analysis of the substrates by Fourier transform infrared reflection-absorption spectroscopy (FT-IRRAS) and X-ray photoelectron spectroscopy (XPS) revealed that irreversible binding of PL occurred in all cases. However, the mechanism of binding involved differed markedly from one monolayer to the other. The main mode of interaction of PL to MUA SAM was of electrostatic nature between the terminal carboxylate of MUA and the ammonium groups of PL. For a similar number of bound thiolate molecules, the UDT adsorbed layer was found less continuous than the MUA one, allowing a higher fraction of PL to directly bind to the gold surface. As for MUD, very little thiolate molecules were adsorbed, leaving bare gold surface areas for non specific adsorption of PL.
RESUMO
New heavy transition metal carbonyl markers for protein labeling, containing an "Mn(CO)11" (M = Ru, Os, n = 3; M = Ir, n = 4) moiety, were prepared by reaction of "lightly stabilized" clusters with an N-succinimidyl ester functionalized phosphine, namely N-succinimidyl 3-diphenylphosphine-propionate (DPPS). The reaction of Os3(CO)11(DPPS) with the model amino acid beta-alanine was performed and led to the expected amide. From the reaction of Mn(CO)11(DPPS) with bovine serum albumin (BSA) in mixed organic/aqueous medium, conjugates bearing a fairly high number of metal carbonyl fragments were obtained, thus demonstrating the usefulness of this class of reagents for the selective and covalent graft of heavy metal clusters to side chain of proteins.
Assuntos
Metais Pesados/química , Proteínas/química , Succinimidas/química , Acilação , Cristalografia por Raios X , Indicadores e Reagentes , Soroalbumina Bovina/químicaRESUMO
As part of our ongoing development of the CMIA nonisotopic immunoassay method, in which the tracers are metal carbonyl complexes and detection is by Fourier transform infrared spectroscopy, we examined the potential use as tracers of the complexes CpFe(CO)2(5,5-diphenylhydantoin) 2d and CpFe(CO)(PPh3)(5, 5-diphenylhydantoin) 3. The present study involved the synthesis of a series of hydantoin complexes (2a-2d), in particular that of the derivative of 5,5-diphenylhydantoin 2d. The structure of 2d was confirmed by X-ray crystallography. The infrared analysis, establishing the position and intensity of the characteristic metal-carbonyl peaks of complexes 2d and 3 in the 1850-2200 cm-1 region, shows that measurement of the absorbance values of these characteristic peaks will permit quantitative analysis in the picomole range, the norm for routine use in immunoassay and thus suitable for use as CMIA tracers. Cross-reaction rates of these tracers with anti-DPH specific antibodies show that 2d and 3 are both recognized by anti-DPH antibodies (cross-reaction rates 43 and 20%, respectively). In developing a CMIA of DPH with these tracers, it was found that 3, with a single, intense band at 1977 cm-1, had very promising IR characteristics for use in multiassay CMIA, but probably owing to its relatively weak affinity for the antibodies, it was not possible to develop a CMIA for DPH using this tracer. Complex 2d, however, showed better recognition by the antibodies, and using this complex as a tracer, it was possible to develop a particularly sensitive monoassay of DPH by the CMIA method.
Assuntos
Anticonvulsivantes/química , Hidantoínas/química , Imunoensaio/métodos , Compostos de Ferro/química , Compostos Organometálicos/química , Fenitoína/química , Ânions , Afinidade de Anticorpos , Especificidade de Anticorpos , Anticonvulsivantes/análise , Anticonvulsivantes/imunologia , Reações Cruzadas , Cristalografia por Raios X , Ciclopentanos/química , Previsões , Análise de Fourier , Indicadores e Reagentes , Fenitoína/análise , Fenitoína/imunologia , Espectrofotometria InfravermelhoRESUMO
We describe here the development of a new, non-isotopic immunological assay termed CMIA (carbonyl metallo immunoassay) that uses metal carbonyl complexes as tracers and Fourier transform infrared spectroscopy (FT-IR) as the detection method. This assay is based on the particular spectral features of these complexes, which show very strong absorption bands in the 1,800-2,200 cm(-1) spectral range where proteins and organic molecules do not absorb. In Section 1, the optimisation of the quantitative detection of these tracers is detailed. In Section 2, the implementation of mono-CMIA is described, including the CMIA assays of three antiepileptic drugs (carbamazepine, phenobarbital, phenytoin). Finally, extension to the simultaneous double- and triple-CMIA of these drugs is reported.
Assuntos
Anticonvulsivantes/análise , Imunoensaio/métodos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Imunoensaio/tendências , Espectroscopia de Infravermelho com Transformada de Fourier/tendênciasRESUMO
Organo-rhenium and organo-tungsten N-succinimidyl and N-sulfosuccinimidyl esters react at amino groups of proteins to form stable amide bonds between the protein and the heavy transition metal complexes. We show here that factors such as pH of the reaction medium and nature of the reagent (coordinating metal, surrounding ligands, type of ester) had a marked influence on the final number of coupled organometallic groups per protein molecule, defined as the coupling ratio. By operating with stoichiometric quantities of reagent with respect to the number of aqino groups, up to 30 organometallic groups were introduced into the model protein, bovine serum albumin (BSA). BSA conjugates were characterized by gel electrophoresis in non-denaturating conditions. Hen egg-white lysozyme organo-tungsten conjugates were prepared in the same manner and the peptides resulting from their tryptic hydrolysis were analyzed by reverse-phase HPLC. A tentative assignment of the preferential amino sites of conjugation is suggested from the latter results.
Assuntos
Compostos Organometálicos/química , Rênio/química , Soroalbumina Bovina/química , Tungstênio/química , Sequência de Aminoácidos , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Cristalografia por Raios X , Ésteres , Hidrólise , Dados de Sequência Molecular , Estrutura Molecular , Mapeamento de PeptídeosRESUMO
The two first transition metal carbonyl isothiocyanates were prepared in high yield within two steps from photolysis of CpFe(CO)2I and 3- or 4-aminophthalimide in the presence of diisopropylamine followed by reaction with thiophosgene/triethylamine. Their reaction with a model amino acid, i.e. beta-alanine, was performed and led to the expected thioureas. When reacted with bovine serum albumin in aqueous medium, conjugates bearing 6-10 iron-carbonyl fragments were obtained and characterized by Fourier transform infrared spectroscopy, thus demonstrating the usefulness of these reagents for the selective and covalent labeling of proteins.
Assuntos
Compostos de Ferro/química , Isotiocianatos/química , Soroalbumina Bovina/química , Espectrometria de Massas , Fotoquímica , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
The feasibility of a double immunoassay of haptens by the nonisotopic carbonyl metalloimmunoassay (CMIA) method is demonstrated. Three different pairings of antiepileptic medications from the groups carbamazepine, diphenylhydantoin, and phenobarbital (for each of which a mono-CMIA is already available) were assayed by double CMIA. The assay method employs as tracers metal-carbonyl complexes that give very strong signals in the range of 1850-2200 cm-1 in the infrared spectrum, permitting quantitative analysis by Fourier-transform infrared spectroscopy. The fact that the signals are individually assignable and of comparable intensity permits quantitative analysis of mixtures of two tracers. The analysis may proceed in one of two ways: in the simpler case, there is no peak overlap with the two tracers and the quantitative analysis can be performed by simply measuring the absorbance of characteristic peaks of the two tracers. In the second case, in which there is partial or total overlap of peaks, a stepwise calculation provides rapid quantification of the two tracers. These findings allowed us to perform the double CMIA of two antiepileptics in which experimental conditions and time of analysis were strictly identical to those for mono-CMIA. We show here that there is a very good correlation between the results obtained in mono- and double-immunoassay by the CMIA method (correlation coefficient > 0.990).
Assuntos
Anticonvulsivantes/análise , Imunoensaio/métodos , Compostos Organometálicos/análise , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Animais , Anticonvulsivantes/química , Anticonvulsivantes/imunologia , Carbamazepina/análise , Carbamazepina/química , Estudos de Avaliação como Assunto , Haptenos/análise , Humanos , Estrutura Molecular , Compostos Organometálicos/química , Fenobarbital/análise , Fenobarbital/química , Fenitoína/análise , Fenitoína/químicaRESUMO
An improved synthesis of 11-oxoestrone-3-acetate-17-ethyleneketal is reported. Adjustments are proposed for the oxidation of estrone by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone into 9(11)-dehydroestrone. A complete hydroboration-oxidation of the resulting ketal, by means of borane-methylsulfide complex, gives the corresponding 11-hydroxy derivative. This latter compound is then acetylated for successful oxidation with pyridinium chlorochromate on alumina. The overall yield is 30%.
Assuntos
Estrona/análogos & derivados , Estrona/química , Compostos de Espiro/síntese química , Acetilação , Óxido de Alumínio/química , Cromatos/química , Estrona/síntese química , Espectroscopia de Ressonância Magnética , Oxirredução , Compostos de Piridínio/químicaRESUMO
As part of our ongoing work to extend the range of applications of the non-isotopic carbonyl metalloimmunoassay (CMIA), previously developed in our laboratory, we describe here the first CMIA study of carbamazepine. The CMIA method uses a metal carbonyl complex as a non-isotopic tracer, and in this case we chose to employ the dicobalt hexacarbonyl moiety (Co2(CO)6) attached to an alkyne. Two organometallic tracers, 3 and 7, were synthesized, differentiated by the nature and length of the spacer arm of the Co2(CO)6 moiety. Two different coupling methods were subsequently used to synthesize the immunogens 1 and 2, the first one used a carbodiimide, while the second, employed dimethyl adipimidate as coupling agent. Titer values of the antisera obtained by injection of these immunogens into rabbits, were determined by CMIA, using one of the organometallic complexes, 3 or 7, as tracer. Both antisera had higher titer values with the long-chain tracer, 7, than with the short-chain tracer, 3. However these titer values were very different: low for antiserum 1 and high for antiserum 2. The cross-reactivity of antiserum 2 with other antiepileptic drugs was negligible. For competition curves, there was good sensitivity with the antibody 2/3 pairing, while a broad assay range was obtained with antibody 2/7 pairing. These results demonstrate the viability of CMIA as an immunoassay method for carbamazepine, and open the way to development of a simultaneous multiassay by CMIA of the principal antiepileptic drugs.
Assuntos
Anticonvulsivantes/análise , Carbamazepina/análise , Imunoensaio/métodos , Compostos Organometálicos/síntese química , Espectroscopia de Infravermelho com Transformada de Fourier , Animais , Especificidade de Anticorpos , Ligação Competitiva , Carbamazepina/imunologia , Cobalto , Reações Cruzadas , Dibenzazepinas/química , Dicicloexilcarbodi-Imida , Dimetil Adipimidato , Haptenos , Imunização , Compostos Organometálicos/análise , CoelhosRESUMO
New specific reagents for the conjugation of organo transition metal species to proteins are described. These reagents are pyrylium salts bearing a (eta 5-C5H4)M(CO)3 (M = Mn and Re) at position 4. They couple with simple amines (n-butylamine and tert-butylamine) and to lysine side chains of proteins (bovine serum albumin and lysozyme) with varying yields. In almost all cases, the final conjugated species is a pyridinium salt, with the exception of lysozyme, for which the reaction ends at the divinylogous amide form. Differences in reactivity for bovine serum albumin and lysozyme can be explained in terms of differences of isoelectric point and steric local environment around the reactive lysine residue.
Assuntos
Muramidase/química , Compostos Organometálicos/química , Soroalbumina Bovina/química , Acetonitrilas , Ácidos Bóricos , Soluções Tampão , Butilaminas/química , Indicadores e Reagentes , Ponto Isoelétrico , Lisina/química , Espectrofotometria UltravioletaRESUMO
A non-isotopic heterogeneous competitive immunoassay procedure, carbonylmetalloimmunoassay (CMIA) has been applied to the assay of cortisol. The organometallic tracers employed were two stereoisomers (Z and E) of certain cobalt carbonyl complexes of cortisol which have strong, characteristic v(CO) absorptions in the infrared, detectable by Fourier transform infrared (FT-IR) spectroscopy at the femtomole level. The separation of the free and bound organometallic-labelled fractions was achieved by solvent extraction with isopropyl ether. Complete characterization (i.e., dilution, competition and standard curves) of two different polyclonal anticortisol antibodies was possible using the CMIA method. Identical titre values were obtained for the two different stereoisomers used as tracers. In terms of the standard curves, however, the isomers behaved differently depending on which batch of antibody was used. When the best antibody/tracer pair (30 pmol of E isomer; anticortisol 1 antibody) was employed, we obtained a B/B(o)value at 50% of 42 +/- 2.12 pmol and a coefficient of variation of 5%. Finally, preliminary results of a CMIA analysis of plasma cortisol from a patient indicated that reliable and reproducible assays are possible for amounts as small as 50 microliters of serum.
Assuntos
Hidrocortisona/sangue , Imunoensaio/métodos , Compostos Organometálicos , Animais , Anticorpos , Especificidade de Anticorpos , Reações Cruzadas , Estudos de Viabilidade , Humanos , Hidrocortisona/análogos & derivados , Hidrocortisona/síntese química , Compostos Organometálicos/síntese química , Coelhos , Radioimunoensaio , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Solventes , Espectroscopia de Infravermelho com Transformada de Fourier , EstereoisomerismoRESUMO
As part of our ongoing work to develop the new non-isotopic assay method carbonyl metalloimmunoassay (CMIA), whose efficacy has already been proven in the laboratory for phenobarbital and cortisol, we here present the steps involved in establishing CMIA of 5,5 diphenylhydantoin (DPH), one of the most commonly used antiepileptic medications. First, anti-DPH antibodies were obtained by injection of the immunogen DPH-3-valerate-BSA into rabbits. The titer value and specificity of these antibodies were examined by RIA using [14C]-DPH as tracer, and an antibody batch selected for its high titer value and good specificity for metabolites of DPH and other antiepileptic drugs. Next the organometallic complex Cr(CO)3-DPH, chosen as the CMIA tracer, was synthesized and shown to conserve a high recognition value for anti-DPH antibodies (CR = 200%). Isopropyl ether was selected as the best organic solvent for use in separating the free and bound fractions of the tracer. Employing the Cr(CO)3-DPH complex as tracer and FT-IR spectroscopy as the detection method, we were able to obtain a titration curve by CMIA using an amount of tracer identical to that used in RIA. The titer value obtained in CMIA is approximately twice that obtained by RIA. These results demonstrate the feasibility of DPH assay by the CMIA method.
Assuntos
Imunoensaio/métodos , Fenitoína/análise , Animais , Formação de Anticorpos , Reações Cruzadas , Compostos Organometálicos/síntese química , Compostos Organometálicos/imunologia , Fenitoína/análogos & derivados , Fenitoína/síntese química , Fenitoína/imunologia , Coelhos , Radioimunoensaio , Soroalbumina Bovina/síntese química , Soroalbumina Bovina/imunologia , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
We have synthesized organometallic complexes of steroids (cortisol, testosterone, dihydrotestosterone) for potential use as tracers in nonisotopic carbonyl-metal immunoassays (CMIA). An ethynyl/CO2(CO)6 fragment at the end of a five-atom spacer was coupled to position 3 of the steroid skeleton. In the case of cortisol, we exploited the difference in reactivity of the ketone and enone functions toward amines in order to form an enamine which was then made to react with carboxymethylamine to yield 3-[(carboxymethyl)oxime] steroid. Activation of the carboxylic acid function with N,N'-dicyclohexylcarbodiimide in the presence of propargylamine introduced an acetylenic function at the end of the spacer. The triple bond was then complexed by CO2(CO)8 to form complexes 5a-c. Complexes for use in CMIA should be stable in biologic media and effectively recognize specific antibodies. Complexes 5a-c were stable in the buffers we use in biochemical tests. Their cross reactivities for anti-cortisol and anti-testosterone antibodies ranged from 50 to 110% according to batch, indicating, first, that the addition of an organometallic complex in position 3 of the steroid skeleton does not hinder recognition between the organometallic steroid and antibody and, second, that their individual behavior differs substantially according to antibody batch. Although all of these complexes could be used as tracers in CMIA, it is necessary, in each case, to establish which tracer-antibody duo gives rise to the most sensitive immunoassay.