RESUMEN
The objective of this study was to examine the extension of supercritical fluid extraction (SFE) to the extraction of polar drugs. The ultimate aim was to extract veterinary residues from food animal products and thereby demonstrate the versatility of SFE. This technique is shown to have many facets that require careful thought and understanding if it is to be successfully used. Our initial studies indicate that polar drugs may be readily solubilized from relatively inert matrices such as sand, with high recoveries and very little discrimination between related compounds while using only moderate extraction conditions and times. SFE of the same drugs from spiked chicken liver and swine muscle is significantly more difficult and requires more drastic conditions. Close to complete recoveries are achieved for some drugs, while considerably less is found in the worst case. For sulphamerazine, sulphamethizole, sulphamethazine, sulphamethoxypyridazine, sulphamethoxazole, and the major metabolite N4-acetyl-sulphamethoxazole, the recoveries are 97, 66, 94, 79, 53, and 65%, respectively, from spiked liver and 95, 27, 86, 91, 96 and 70%, respectively, from spiked swine muscle. Incurred sulphamethazine is recovered from swine muscle in good general agreement with the reference values provided.
Asunto(s)
Cromatografía Liquida/métodos , Residuos de Medicamentos/análisis , Contaminación de Alimentos/análisis , Carne/análisis , Sulfonamidas/análisis , Animales , Pollos , Hígado/química , Músculos/química , Solubilidad , PorcinosRESUMEN
When incubated in normal human serum, rabbit erythrocytes are haemolysed as a result of activation of the alternative pathway of complement (APC), but sheep erythrocytes do not spontaneously activate the human APC under physiological conditions. The mechanism for this difference has been attributed to differences in the relative affinity of membrane-bound C3b for its natural ligands, factor B and factor H, that favour the formation and stability of the APC C3 convertase on rabbit erythrocytes and inhibit convertase activity on sheep erythrocytes. Previous studies have also suggested that factor I inactivated C3b on sheep erythrocytes more effectively than on rabbit erythrocytes. Further, sheep erythrocytes have recently been shown to have a membrane protein that associates non-covalently with cell-bound C3b, but rabbit erythrocytes lack a predominant C3b binding protein. Together, these results suggested the possibility that sheep but not rabbit erythrocytes have a membrane constituent with factor I cofactor activity. To investigate this hypothesis, rabbit and sheep erythrocytes bearing radiolabelled C3b were treated either with factor I or with factor I and factor H, and conversion to iC3b was assessed by autoradiography. Factor I caused a concentration-dependent conversion of C3b to iC3b on sheep erythrocytes; however, only trace amounts of C3b on rabbit erythrocytes were degraded even when high concentrations of factor I (83 micrograms/ml) were used. While C3b on rabbit erythrocytes was converted to iC3b by the combination of factor H and factor I, much less factor H was required for the same degree of conversion of C3b on sheep erythrocytes. Treatment of sheep erythrocytes with neuraminidase had no effect on either factor I-dependent cofactor activity or the capacity of factor H to serve as a factor I cofactor. Sheep erythrocytes did not manifest decay accelerating activity, however, suggesting that the factor I cofactor constituent is a functional analogue of the human membrane cofactor protein.
Asunto(s)
Complemento C3b/metabolismo , Vía Alternativa del Complemento , Eritrocitos/inmunología , Serina Endopeptidasas/inmunología , Animales , Convertasas de Complemento C3-C5/metabolismo , Proteínas Inactivadoras del Complemento C3b/inmunología , Factor H de Complemento , Factor I de Complemento , Relación Dosis-Respuesta Inmunológica , Eritrocitos/química , Humanos , Técnicas In Vitro , Neuraminidasa/farmacología , Conejos , Ovinos , Especificidad de la EspecieRESUMEN
When incubated in acidified serum, the erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) are hemolyzed through activation of the alternative pathway of complement (APC), but normal erythrocytes are resistant to this process. PNH cells are deficient in decay-accelerating factor (DAF), a complement regulatory protein that inhibits the activity of both the classical and the alternative pathways. However, deficiency of DAF alone does not account entirely for the aberrant effects of acidified serum on PNH cells. Recently, we have shown that PNH erythrocytes are also deficient in another complement control protein called membrane inhibitor of reactive lysis (MIRL) that restricts complement-mediated lysis by blocking formation of the membrane attack complex (MAC). To determine the effects of the DAF and MIRL on susceptibility to acidified serum lysis, PNH cells were repleted with the purified proteins. DAF partially inhibited acidified serum lysis by blocking the activity of the amplification C3 convertase. MIRL inhibited acidified serum lysis both by blocking the activity of the MAC and by inhibiting the activity the C3 convertase. When DAF function was blocked with antibody, normal erythrocytes became partially susceptible to acidified serum lysis. By blocking MIRL, cells were made completely susceptible to lysis, and control of C3 convertase activity was partially lost. When both DAF and MIRL were blocked, the capacity of normal erythrocytes to control the activity of the APC and the MAC was destroyed, and the cells hemolyzed even in unacidified serum. These studies demonstrate that DAF and MIRL act in concert to control susceptibility to acidified serum lysis; of the two proteins, MIRL is the more important. In addition to its regulatory effects on the MAC, MIRL also influences the activity of the C3 convertase of the APC. Further, in the absence of DAF and MIRL, the plasma regulators (factor H and factor I) lack the capacity to control membrane-associated activation of the APC.
Asunto(s)
Antígenos de Diferenciación/fisiología , Proteínas Sanguíneas/fisiología , Proteínas Inactivadoras de Complemento/fisiología , Vía Alternativa del Complemento , Eritrocitos/fisiología , Hemoglobinuria Paroxística/sangre , Hemólisis , Glicoproteínas de Membrana/fisiología , Proteínas de la Membrana/fisiología , Animales , Antígenos de Diferenciación/deficiencia , Antígenos de Diferenciación/farmacología , Antígenos CD55 , Antígenos CD59 , Cobayas , Hemólisis/efectos de los fármacos , Humanos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/farmacología , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/farmacología , Valores de ReferenciaRESUMEN
To investigate the mechanism by which treatment of normal human erythrocytes with the sulfhydryl reagent 2-aminoethylisothiouronium bromide (AET) induces susceptibility to complement mediated lysis, the effects of AET on the structural and functional integrity of decay accelerating factor (DAF), membrane inhibitor of reactive lysis (MIRL), and complement receptor type 1 (CR1) were examined. Following treatment with AET, erythrocyte MIRL and CR1 were no longer recognized in situ by antibodies, and antibody binding to DAF was diminished by approximately 50%. These studies indicated that the structural integrity of the three complement regulatory proteins was either partially (DAF) or completely (MIRL and CR1) disrupted by AET. Subsequent experiments showed that functional inactivation paralleled the structural disruption. Treatment of normal erythrocytes with AET induced susceptibility to cobra venom factor-initiated hemolysis, indicating that the functional activity of MIRL had been destroyed. The capacity of erythrocyte CR1 to serve as a cofactor for factor I-mediated cleavage of iC3b to C3c and C3dg was lost following treatment with AET. C3 convertase activity increase markedly following treatment of erythrocytes with AET, but convertase activity on AET cells was approximately 50% less than that observed when DAF function on normal cells was completely inhibited by antibody. Susceptibility of AET cells to acidified serum lysis was shown to be due primarily to inactivation of MIRL. Unexpectedly, in acidified serum the activity of the amplification C3 convertase of the APC was found to be controlled by MIRL as well as by DAF. These studies show that AET induces susceptibility to complement-mediated lysis by disrupting the structural and functional integrity of membrane constituents that regulate the activity of both the C3 convertases and the membrane attack complex of complement.