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.

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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.

A polarity test mix consisting of acetophenone, N,N-dimethylaniline, naphthalene, decanoic acid, 2-naphthol, and n-tetracosane was spiked onto sand, and extracted with supercritical carbon dioxide, to evaluate the collection efficiency of various solvents and solvent mixtures. Nine single collection solvent systems and four mixed collection solvent systems were studied. When one-component collection solvents were employed, quantitative (above 90 Yo) recovery of all analytes was not possible. With mixed collection solvents, recoveries of 90 % or better with all analytes studied were possible.

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