Previous work has shown that red wines, grape juices, and other grape products cause endothelium-dependent relaxation (EDR) of blood vessels in vitro by increasing nitric oxide production. In this paper we describe the isolation and characterization of some of the compounds responsible for EDR activity. Concord grape seeds were extracted with methanol and the compounds were separated by Toyopearl TSK HW-40S chromatography. Resulting fractions (primarily phenolic acids, catechins, and proanthocyanidins) were further separated semipreparatively by reversed-phase HPLC, and peaks were collected and bioassayed for EDR activity using the rat aorta preparation. EDR-active compounds were subsequently characterized by HPLC retention times and electrospray-ion-trap mass spectrometry. The compounds exhibiting the most EDR activity were proanthocyanidin trimers, tetramers, pentamers, and polymers and their gallates, as well as a dimer gallate (EC50 values in the range of 0.6-2.5 microg catechin equivalents/mL). These compounds should be useful for in vitro and in vivo studies, particularly as they relate to improvement of cardiovascular function.

We have shown in previous work that extracts of grape seeds (GSE) and skins, grape juice, and many red wines exhibit endothelium-dependent relaxing (EDR) activity in vitro. This EDR activity involves endothelial nitric oxide (NO) release and subsequent increase in cyclic GMP levels in the vascular smooth muscle cells. The NO/cyclic GMP pathway is known to be involved in many cardiovascular-protective roles. The current study focuses on the isolation and identification of EDR-active compounds (procyanidins) from GSE. Crushed Concord grape seeds were extracted with methanol and the extract was separated into seven fractions (A-G) on a Toyopearl TSK-HW-40 column. EDR-active fractions (D-G) were further separated into 25 individual compound peaks by HPLC, 16 of which were EDR active (threshold for relaxation ranged between less than 0.5 microg/mL and greater than 4 microg/mL). Procyanidin identification was accomplished by electrospray-ion trap mass spectrometry (ES-ITMS), MS/MS, and by tannase treatment and acid thiolysis, followed by HPLC and ES-ITMS of the products. Activity of isolated procyanidins tended to increase with degree of polymerization, epicatechin content, and with galloylation. These EDR-active compounds (many of which also possess antioxidant activity), individually or in the form of wines, juices, or nutritional supplements, may be useful in preventing or treating cardiovascular diseases.

Imidazole, 1-methylimidazole, 2-methylimidazole, 4-methylimidazole, and benzimidazole were subjected to electrochemical and chemical oxidation. Chemical oxidation yields oligomers with higher molecular weight (DP > 12) than anodic oxidation (DP >6) as detected by laser desorption mass spectrometry (LDMS).The oligomers have a relatively simple structure in which imidazole moieties are linked by N-N bonds.1-Methylimidazole does not polymerize, and benzimidazole seems to react through the benzene carbon atoms as well as through the nitrogen atoms. Solubility of these products varies with the substituent group on the imidazole ring. We propose a variation of Diaz's mechanism of pyrrole polymerization that we base on cyclic voltammetry, spectral analysis, pH effects, laser desorption mass spectrometry, and computer calculations. The mechanism involves stepwise removal of an electron from the N-3 atom to produce a radical cation that then couples. LDMS data suggest that chemical polymerization occurs by rapid dimerization of imidazole followed by slower polymerization of the dimers. LDMS of electrochemical polymerization products suggests that polymerization occurs by addition of one imidazole unit at a time. None of these products exhibits electrical conductivity.

Al', Cr', Fe+ and Cu+ gas-phase ions have been found to attach efficiently to two polymers, polystyrene and polyethylene glycol (PEG) in laser-desorption time-of-flight experiments, and are shown to be viable alternatives to the commonly used Na', K+ and Ag' ions. At high laser powers, in the range 8.0 X lo7 to 2.0 X lo8 Wlcm', ions can be attached by desorption from a support made of the appropriate metal. An alternative method is to add the metal ion in the form of a salt containing the metal in the +,l oxidation state, when such salts are available. This method requires lower laser powers, in the range 2.0 X 10' to 8.0 X lo7 W/cm2. The two desorption/attachment methods are characterized by different optimal time-delay values for recording of spectra. Polystyrene has been found to have a higher affinity for attachment to Al', Cr+, and Cu+ than to K+, while PEG has been found to have a higher affinity for K' than for Al+, Cr' and Cu'. These preferential attachments are discussed in terms of the hard and soft acids and bases principle.The determination of molecular-weight distributions of synthetic polymers is an important analytical task because the properties of a particular polymer, and therefore its suitability for a particular application, are directly linked to its molecular-weight distribution. The most commonly used methods for determining molecular weights of polymers provide some sort of average molecular weight: either a number-average molecular weight, M,, (osmometry, cryoscopy, ebulliometry, endgroup titration) or a weight-average molecular weight, M , (light scattering) where M , and M , are defined by Eqns (1) and (2)

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