Monomeric and oligomeric procyanidins present in cocoa liquors and chocolates were separated and quantified in four different laboratories using a normal-phase high-performance liquid chromatography (HPLC) method with fluorescence detection. Procyanidin standards through decamers were obtained by extraction from cocoa beans, enrichment by Sephadex LH-20 gel permeation chromatography, and final purification by preparative normal-phase HPLC. The purity of each oligomeric fraction was assessed using HPLC coupled to mass spectrometry. A composite standard was then prepared, and calibration curves were generated for each oligomeric class using a quadratic fit of area sum versus concentration. Results obtained by each of the laboratories were in close agreement, which suggests this method is reliable and reproducible for quantification of procyanidins. Furthermore, the procyanidin content of the samples was correlated to the antioxidant capacity measured using the ORAC assay as an indicator for potential biological activity.

Monomeric and oligomeric proanthocyanidins present in a range of plant-derived foods and beverages were separated by degree of polymerization and identified using a modified normal-phase high-performance liquid chromatography (HPLC) method coupled with on-line mass spectrometry (MS) analysis using an atmospheric pressure ionization electrospray chamber. In addition, ultraviolet (UV) and fluorescence detection were used to monitor the separation of proanthocyanidins, with fluorescence detection demonstrating both increased sensitivity and the ability to reduce interfering signals from other components present in the food and beverage matrices as compared to UV detection. This qualitative study demonstrates the ability of this HPLC/MS technique to separate singly and doubly linked procyanidins, prodelphinidins, and copolymer oligomers, including their galloylated derivatives, present in a range of food and beverage samples.

No abstract

No abstract

The molecular mechanisms of action of a HIV protease inhibitor, ritonavir, on hepatic function were explored on a genomic scale using microarrays comprising genes expressed in the liver of Sprague-Dawley rats (Rattus norvegicus). Analyses of hepatic transcriptional fingerprints led to the identification of several key cellular pathways affected by ritonavir treatment. These effects were compared to a compendium of gene expression responses for 52 unrelated compounds and to other protease inhibitors, including atazanavir and two experimental compounds. We identified genes involved in cholesterol and fatty acid biosynthesis, as well as genes involved in fatty acid and cholesterol breakdown, whose expressions were regulated in opposite manners by ritonavir and bezafibrate, a hypolipidemic agonist of the peroxisome proliferator-activated receptor alpha. Ritonavir also upregulated multiple proteasomal subunit transcripts as well as genes involved in ubiquitination, consistent with its known inhibitory effect on proteasomal activity. We also tested three other protease inhibitors in addition to ritonavir. Atazanavir did not impact ubiquitin or proteasomal gene expression, although the two other experimental protease inhibitors impacted both proteasomal gene expression and sterol regulatory element-binding protein-activated genes, similar to ritonavir. Identification of key metabolic pathways that are affected by ritonavir and other protease inhibitors will enable us to understand better the downstream effects of protease inhibitors, thus leading to better drug design and an effective method to mitigate the side effects of this important class of HIV therapeutics.