PubMed · 14207620
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A L ZAIDES, A N MIKHAILOV, O I PUSHENKO. [A MODIFIED METHOD OF DETERMINING HYDROXYPROLINE].. https://pubmed.ncbi.nlm.nih.gov/14207620/
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Residual moisture content and formulation are important parameters when preparing lyophilized reference materials containing labile proteins. The protection of Factor VIII and Factor V activities were monitored in a lyophilized plasma preparation following formulation with either no additional excipient, 40 mM Hepes (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid), 10 mg/mL glycine or a combination of 40 mM Hepes and 10 mg/mL glycine. The preservation of Factor VIII activity during freeze-drying was improved by the addition of either stabiliser and improved most, amongst the options studied, by the addition of both glycine and Hepes. The predicted stability at -20 degrees C and 20 degrees C was estimated using accelerated degradation studies. Although for plasma lyophilized alone there was some benefit from further desiccation over phosphorus pentoxide, resulting in very low moistures, for suitably formulated samples the predicted stability was as good for freeze-dried only samples as for those with further desiccation. This study emphasises the importance of optimum formulation on the stability of lyophilized proteins.
It is suggested that power analysis should be formally incorporated into quantification experiment reports in order to substantiate the conclusions derived from experimental data more effectively. The article addressed the issues of power analysis calculation, sample size estimation and appropriate data reporting in quantitative analytical comparisons. Illustrative examples from the literature are used to show how the described power analysis theory could be applied in practice.
Formation of a colored complex between ferric iron and xylenol orange (XO) has been used for the determination of hydroperoxides (FOX method). Original or modified FOX methods were performed on aqueous or organic solutions consisting of a single phase. However, for lipid peroxides in heterogeneous samples, such as biological materials, much of the lipid is sequestered in a separate phase. Organic solvent extraction of these lipids is often incomplete and may result in additional peroxidation during the extraction procedure. In this study, we applied the FOX assay for measurement of the membrane phosphatidylcholine hydroperoxides (PC-OOH) in separated phases. The presence of membranous egg yolk phosphatidylcholine (EYPC) in 60% MeOH shifted the broad peak at 560 nm of Fe(3+)-XO complex to 610 nm with a sharp peak associated with the increased intensity of the absorbance. The shift of the peak is useful to measure the unknown amounts of Fe(3+) because the uncomplexed XO considerably contributed to the absorbance of the peak at 560 nm but did not affect the absorbance at 610 nm. EYPC was required to form the membranes to shift the peak because the shift occurred in 60% MeOH but did not by the treatment with detergents or in 90% MeOH in which EYPC did not form the membranes. The molar absorption coefficient (epsilon 610) was 32,700 M(-1) cm(-1), which was about twice the molar absorption co efficient (epsilon 560) reported. We applied this method to the assay of PC-OOH prepared from EYPC and obtained the molar absorption coefficients (epsilon 610), which were 79,100 and 115,700 M(-1) cm(-1) in the presence and absence of BHT, respectively. This finding allows the determination of PC-OOH concentration even in chemically complex systems.