Although total plasma antioxidant capacity is decreased from normal levels in septic patients, an increase in some oxidants contributes to an increased total antioxidant capacity in septic shock patients.

A method is described for large-scale purification of glycosylphosphatidylinositol-anchored alkaline phosphatase from intestinal mucosa and chyme to homogeneity. Both enzyme preparations contain approximately 2 mol fatty acidmol subunit and exhibit a very similar fatty acid composition with octadecanoate and hexadecanoate as prevalent components.No significant differences between native glycosylPtdIns-anchored and hydrophilic alkaline phosphatases from both sources were found regarding K,,, V,,,,,, the type of inhibition and inhibition constants of the amino acids L-leucine, L-phenylalanine, and L-tryptophan. The purified enzymes of both sources yield diacylglycerol and phosphatidic acid, after treatment with phosphatidylinositolspecific phospholipase C (PtdIns-PLC) and glycosylphosphatidylinositol phospholipase D (PLD), respectively.Enzyme preparations of both sources appear as heterogeneous mixtures of five fractions separable by octyl-Sepharose chromatography. Fraction I corresponds to the anchorless enzyme, fractions 11-V differ in their susccptibility to phospholipases. Fractions TI and IV are completcly split by PtdIns-PLC or PLD action, almost 50% of fraction I11 is split by PtdIns-PLC, while fraction V is resistant. The susceptibility of these two fractions toward the action of PLD is considerably higher. Fatty acid analysis yields molar ratios of fatty acids/alkaline phosphatase subunit of 1.78, 2.58, 2.24, and 3.37 for fractions 11, 111, IV, and V, respectively.Aggregates of glycosylPtdIns-anchored alkaline phosphatase of all fractions are seen in native PAGE in the presence of Triton X-100. By gel chromatography in the presence of Brij 35, fractions 11-V form stable multiple aggregates of dimers and may bind different amounts of the detergent.These data, together with fatty acid analysis, can be interpreted by the following model. Fractions I1 and IV are tetramers and octamers with two molecules fatty acid/subunit. Fraction 111 is a tetramer, bearing one additional fatty acid molecule, localized on the dimer. Fraction V is an octamer, containing glycosylPtdIns-anchor molecules with three molecules fatty acids/anchor molecule. The additional fatty acid residue is possibly located on inositol and responsible for the reduced susceptibility to PtdIns-PLC.The similarity of all measured parameters of both enzymes suggests that the glycosylPtdInsanchored alkaline phosphatase of the mucosa is released into the chyme without changing the anchor molecule constituents.A favoured line of investigation of proteins which are anchored to glycosylphosphatidylinositol (glycosylPtdIns) concerns research on the mechanism of their release by specific anchor-cleaving activities e.g. phospholipases C and D. This process is interesting regarding not only the effects evoked by the proteins released but also regarding the fact that the anchor molecules liberated can potentially act as sig- nals for a broad spectrum of metabolic pathways [l-61. Considerably different susceptibilities to phospholipases have been found for diffe...

An efficient method is presented to determine precision and accuracy of multichannel liquid-handling systems under conditions near to application. The method consists of gravimetrical determination of accuracy and optical determination of precision based on the dilution of absorbing and fluorescent dye solutions in microplates. Mean delivery volume per well can be determined with precision better than a 0.04% coefficient of variation (CV). Optical signal precision, CV(S), is improved by multiwavelength measurements. Precision of absorbance measurement yields a better resolution than precision of fluorescence measurement (0.3% and 1.5%, respectively), indicating that absorbance measurements should be preferred. From CV(S), an upper bound of the precision of the volumes delivered is derived. Method performance is demonstrated with the dispenser CyBi™-Drop and the pipettor CyBi™-Well using different ejection principles; with commonly used fluids; with 96-, 384-, and 1536-well microplates; and with photometric and fluorometric indicators. Precision of the volumes delivered, as obtained with optimized methods, all plate formats, and both devices, is better than 2% CV with 2 µL set volume and about 1% CV with higher set volumes. (Journal of Biomolecular Screening 2004:726-733)