Current Analytical Chemistry - Volume 3, Issue 2, 2007

Most current methods for determining protein carbonyl content are based on the reaction of carbonyl groups with 2,4-dinitrophenylhydrazine (DNPH) to form 2,4-dinitrophenylhydrazone derivatives, which can then be detected and quantitated spectrophotometrically or immunochemically. In this article, we evaluate different quantitative and semi-quantitative techniques for measuring protein carbonylation. We assessed the sensitivity and accuracy of these techniques using both test samples and tissue extracts. We found that absorbance based quantitation of protein phenylhydrazone content is prone to experimental variation, often generating unreliable results. A previously described microplate ELISA method enabled very sensitive and accurate measurement of the carbonyl content of mixtures of reduced and oxidized BSA but proved to be quite insensitive when complex biological samples were assayed. Immunochemical detection and quantitation of DNPH derivatized samples spotted onto nitrocellulose membrane or processed using unifilter devices often produced inconsistent results. The difficulties observed with each of these methods appear to be associated with the presence of unreacted DNPH and nonprotein carbonyl. We found that short SDS-PAGE of derivatized sample efficiently removes these confounding substances and enables sensitive and reliable immunochemical detection of protein carbonyl after electrophoretic transfer of the proteins onto nitrocellulose membrane.

The advent of the “omics” revolution has reawakened an interest in the mass spectrometric analysis of bile acids, particularly in the related fields of lipidomics and metabolomics. This is due to the presence of bile acids in body fluids and their potential to act as biomarkers. Bile acids and bile alcohols are formed from cholesterol in the liver. Bile acids are excreted from the liver into the small intestine via the bile duct conjugated with glycine or taurine at the side-chain carboxyl group. After assisting in the lipolysis and absorption of fats in the intestinal lumen, bile acids are returned to the liver. Bile acids and bile alcohols undergo further metabolism by bacterial and hepatic enzymes during the enterohepatic circulation, including hydrolysis of conjugates, oxidation and reduction, isomerisation, dehydroxylation and hydroxylation. The hydroxyl groups may also be conjugated with sulphuric acid, glucuronic acid, glucose or N-acetylglucosamine in the liver and in extrachepatic organs including intestine and kidney. Thus, the mixture of metabolic products of cholesterol in biological fluids can be very complex. Here we describe modern mass spectrometric methods used to characterise this diverse range of molecules found in biological fluids.

The objective of this paper is to contribute to the methodology available for extracting and analyzing signal content from protein mass spectrometry data. Data from Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) or Surface-Enhanced Laser Desorption and Ionization Time-Of-Flight (SELDI-TOF) spectra require considerable signal pre-processing such as noise removal and baseline level error correction. After removing the noise by an invariant wavelet transform, we develop a background correction method based on penalized spline quantile regression and apply it to MALDI-TOF spectra. The results show that the wavelet transform technique combined with nonparametric quantile regression can handle all kinds of background and low signal-to-background ratio spectra; it requires no prior knowledge about the spectra composition, no selection of suitable background correction points, and no mathematical assumption of the background distribution. We further present a multi-scale based novel spectra alignment methodology useful in a functional analysis of variance method for identifying proteins that are differentially expressed between different type tissues. Our approaches are compared with several existing approaches in the literature and are tested on simulated and real data. The results indicate that the proposed schemes enable accurate diagnosis based on the over-expression of a small number of identified proteins with high sensitivity.

Virgin olive oil, obtained from Olea europaea L. (Oleaceae) fruits, is an important ingredient in the Mediterranean diet. There is now overwhelming scientific evidence that it has health benefits; that include reduction of risk factors for coronary heart disease and the prevention of several pathologies, including some types of cancer. Olive oil appears to be an example of a functional food with a high content of volatiles that contribute to its palatability. However, because of its high added value, it is often adulterated with less valuable oils. Many analytical techniques have been developed to validate the authenticity of the oil. Among them, SPME seems to be one of the more promising approaches, as demonstrated by the many published studies. Being a solvent-free sample preparation technique, its implementation is fast and simple and couples well with GC-MS and HPLC systems. These validation techniques are very important considering the strict regulations imposed by the USA and the EU. Finally, it can be helpful in geographical certification, giving consumers the assurance that the goods come from an area where a given quality, reputation, or other characteristics of the goods is essentially attributable to their geographical origin.

Calixarenes, following cyclodextrines and crown ethers, are the third generation of supramolecules used in HPLC as stationary phases. They consist of phenol units linked via methylene bridges and can also form inclusion complexes like the other host supramolecules. The resulting interactions influence the retention factors and improve the selectivity of the solutes. Additionally, modification of the calixarenes, for instance by varying the ring size, substitutents, conformations and pKa values, enable a more enhanced interaction spectrum and can improve the specificity for guest molecules. The application of calixarenes in chromatography also includes medical and environmental applications, preparative chemistry as well as the rapidly developing area of supramolecular chemistry. Taking the possibilities and the growing interests of calixarenes into account, the aim of the review is to summarise the application possibilities and interactions of calixarenes as stationary phase in HPLC.