Determination of cyclic volatile methylsiloxanes in personal care products by gas chromatography.

OBJECTIVE: Organosiloxanes are prevalent in personal care products (PCPs) due to the desired properties they impart in the usage and application of such products. However, the European Chemical Agency (ECHA) has recently published restriction proposals on the amount of two cyclic siloxanes, octamethylcyclotetrasiloxane (D4) and decamethylcyclotetrasiloxane (D5), allowed in wash off products such as shampoos and conditioners which are discharged down the drain during consumer use. This legislation will require that reliable analytical methods are available for manufacturers and government agencies to use in documenting compliance with the restrictions. This article proposes a simple analytical method to enable accurate measurement of these compounds down to the circa 0.1 weight per cent level in PCPs. METHODS: Although gas chromatography methods are reported in the literature for quantitation of D4 and D5 in several matrices including PCPs, the potential for generation of false positives due to contamination, co-elution and in situ generation of cyclic volatile methylsiloxanes (cVMS) is always present and needs to be controlled. This report demonstrates the applicability of using a combination of emulsion break, liquid-liquid extraction and silylation sample preparation followed by GC-FID analysis as a suitable means of analysing PCPs for specific cVMS. RESULTS: The reliability and limitations of such methodology were demonstrated through several round-robin studies conducted in the laboratories of a consortium of silicone manufacturers. In addition, this report presents examples of false positives encountered during development of the method and presents a comparative analysis between this method and a published QuEChERS sample preparation procedure to illustrate the potential for generation of false positives when an inappropriate approach is applied to determination of cVMS in personal care products. CONCLUSION: This report demonstrates that an approach to determine cVMS levels in personal care products is to perform an emulsion break on the sample, isolate the non-polar phase from the emulsion break and treat with a silylation reagent to abate potential in situ formation of cyclics during the course of GC-FID analysis. Round-robin studies conducted in laboratories representing multiple siloxane manufacturers demonstrated the reliability of the GC-FID method when measuring cVMS in PCPs down to circa 0.1%.

Neuroinflammation in Alzheimer's disease; A source of heterogeneity and target for personalized therapy.

Neuroinflammation has long been known as an accompanying pathology of Alzheimer's disease. Microglia surrounding amyloid plaques in the brain of Auguste D were described in the original publication of Alois Alzheimer. It is only quite recently, however, that we have a more complete appreciation for the diverse roles of neuroinflammation in neurodegenerative disorders such as Alzheimer's. While gaps in our knowledge remain, and conflicting data are abound in the field, our understanding of the complexities and heterogeneous functions of the inflammatory response in Alzheimer's is vastly improved. This review article will discuss some of the roles of neuroinflammation in Alzheimer's disease, in particular, how understanding heterogeneity in the individual inflammatory response can be used in therapeutic development and as a mechanism of personalizing our treatment of the disease.

Metalloprotein complexes for the study of electron-transfer reactions. Characterization of diprotein complexes obtained by covalent cross-linking of cytochrome c and plastocyanin with a carbodiimide.

Cytochrome c (cyt) and zinc cytochrome c (Zncyt) are separately cross-linked to plastocyanin (pc) by the carbodiimide EDC according to a published method. The changes in the protein reduction potentials indicate the presence of approximately two amide cross-links. Chromatography of the diprotein complexes cyt/pc and Zncyt/pc on CM-52 resin yields multiple fractions, whose numbers depend on the eluent. UV-vis, EPR, CD, MCD, resonance Raman, and surface-enhanced resonance Raman spectra show that cross-linking does not significantly perturb the heme and blue copper active sites. Degrees of heme exposure show that plastocyanin covers most of the accessible heme edge in cytochrome c. Impossibility of cross-linking cytochrome c to a plastocyanin derivative whose acidic patch had been blocked by chemical modification shows that it is the acidic patch that abuts the heme edge in the covalent complex. The chromatographic fractions of the covalent diprotein complex are structurally similar to one another and to the electrostatic diprotein complex. Isoelectric points show that the fractions differ from one another in the number and distribution of N-acylurea groups, byproducts of the reaction with the carbodiimide. Cytochrome c and plastocyanin are also tethered to each other via lysine residues by N-hydroxysuccinimide diesters. Tethers, unlike direct amide bonds, allow mobility of the cross-linked molecules. Laser-flash-photolysis experiments show that, nonetheless, the intracomplex electron-transfer reaction cyt(II)/pc(II)----cyt(III)/pc(I) is undetectable in complexes of either type. Only the electrostatic diprotein complex, in which protein rearrangement from the docking configuration to the reactive configuration is unrestricted, undergoes this intracomplex reaction at a measurable rate.

Unimolecular and bimolecular oxidoreduction reactions involving diprotein complexes of cytochrome c and plastocyanin. Dependence of electron-transfer reactivity on charge and orientation of the docked metalloproteins.

Cytochrome c and plastocyanin form an electrostatic complex, which can be reinforced by amide bonds in the presence of a carbodiimide. Besides this cross-linking, carbodiimide also converts carboxylate side chains into neutral N-acylurea groups. Four derivatives of the covalent diprotein complex, which differ in the degree of this charge neutralization, are separated by cation-exchange chromatography. Electron-transfer reactions at different ionic strengths involving the electrostatic complex and the four derivatives of the covalent complex are studied by laser flash photolysis with flavin semiquinones as reducing agents. The reactivity of the associated proteins toward external reductants cannot be predicted simply on the basis of this reactivity of the separate proteins. Qualitative analysis of the dependence on ionic strength of the reactions between FMN semiquinone and the covalent derivatives indicates sites at which this reductant interacts with the cross-linked proteins. The surprisingly small steric shielding of the protein redox sites in the covalent complex, as deduced from the reactions at high ionic strength, may indicate that the proteins have multiple reaction domains on their surfaces or that the complex is dynamical or both. The intracomplex (unimolecular) electron-transfer reaction is fast in the electrostatic complex (ket = 1300 +/- 200 s-1) but undetectably slow in each of the four derivatives of the covalent complex (ket less than 0.2 s-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Catalytic activity of the serine proteases alpha-chymotrypsin and alpha-lytic protease tagged at the active site with a (terpyridine)platinum(II) chromophore.

Incubation of alpha-chymotrypsin and alpha-lytic protease with chloro(2,2':6',2''-terpyridine)platinum(II), [Pt(trpy)Cl]+, results in attachment of Pt(trpy)2+ tags at both His 57 and His 40 in the former and His 57 in the latter. The [Pt(trpy)His]2+ chromophores are readily detected and quantitated owing to their characteristic, strong UV absorption. Although the tagging of His 57 modifies the catalytic triad (Ser 195, His 57, and Asp 102) and disrupts the charge relay, the platinated enzymes retain significant esterase and amidase activity for both specific and nonspecific substrates. Unlike suicide inhibitors, which inactivate the enzymes by filling the active site and imitating the tetrahedral intermediate, [Pt(trpy)Cl]+ reacts with a particular amino acid and permits binding of substrates. The kinetic constants for the following are reported: two esters and two amides with alpha-chymotrypsin and an amide with alpha-lytic protease. The kcat values are between 1 and 25% of, and the Km values are a little higher than, the corresponding values for the native enzymes. The catalytic activity is not due to the native enzymes, trypsin, or some zinc-containing protease. Activities of the native and of the platinated alpha-chymotrypsin depend similarly on pH although the pKa of His 57 is raised to 9.7 upon platination. The platinated enzymes undergo autodigestion slower than do the native enzymes. Because the Pt(trpy)2+ tags are noninvasive, stable, and yet easily removable by thiourea, [Pt(trpy)Cl]+ may be used to retard autodigestion of stored proteolytic enzymes.