Superior performance of liposomes over enzymatic amplification in a high-throughput assay for myoglobin in human serum.

Myoglobin is one of several cardiac markers which become elevated in the blood following an acute myocardial infarction and can aid in the diagnosis of a heart attack. Here, a sandwich immunoassay for myoglobin was developed, including a thorough optimization of fluorescent dye-encapsulating liposomes versus enzymatic amplification (alkaline phosphatase and horseradish peroxidase) at each step. The optimized microtiter plate-based assay was capable of detecting as low as 11.3 pg/mL myoglobin and was successfully applied for the quantification of myoglobin in human serum. In comparison to enzymatic approaches, the liposomes demonstrated lower limits of detection, significantly reduced limits of quantification, improved signal discrimination through substantial signal enhancement, and reduced assay time. Liposomes were stable and functional at ambient temperatures for over 400 days. Finally, ease of use was greater due to lack of reliance on additional reagents, non-time-based signal enhancement, and excellent photostability. Optimal conditions identified for enzymatic approaches can also be used for liposome amplification, which makes substitution of these liposomes into existing assays straightforward. Thus, the extensive studies carried out here suggest that liposomes may be incorporated into formats currently utilizing enzymatic enhanced fluorescence with a potential for increased performance on various levels.

Influence of dietary quercetin on glutathione redox status in mice.

Flavonoids such as quercetin have been shown to serve as a protective defense against oxidative damage in vivo. However, the bioavailability of quercetin depends on the food source and type of glycosidic moiety linked to the molecule. In this study, mice were fed 1 mg/day quercetin in the form of quercetin aglycone, rutin, apple, or onion, and reduced glutathione (GSH), oxidized glutathione (GSSG), and protein-GSH mixed disulfides were determined to investigate the influence of dietary quercetin on the GSH redox status in metabolically active tissues, mitochondria, and plasma of mice. All quercetin treatment groups produced increases in the GSH:GSSG ratio and decreases in mixed disulfide levels in hepatic tissue. Cardiac tissue did not change in response to dietary quercetin; however, cardiac mitochondria demonstrated a reduction in the GSH:GSSG ratio and an increase in protein mixed disulfide levels. No significant changes were observed in the plasma GSH:GSSG ratio, but mixed disulfide levels were decreased for all of the diets. The changes in plasma redox status did not parallel the changes in the tissues. Onion fed mice demonstrated the greatest increases in GSH:GSSG ratios and the greatest decreases in protein mixed disulfide levels of all diets compared. For all treatment groups, increases in the GSH:GSSG ratios corresponded with decreases in protein mixed disulfide levels. The results of this study indicate that quercetin influences GSH:GSSG ratios and protein thiolation in a tissue-specific manner and that these effects are dependent on food source and bioavailability.

An exploratory study of the nutritional composition of Tanoak (Lithocarpus densiflorus) acorns after potassium phosphonate treatment.

Native American Pomo communities who live in the Northern Coastal range of California and consume acorns from tanoak trees as part of traditional diets are facing the potential loss of many culturally important trees to sudden oak death. Pomo and other Native American communities are reluctant to use the protective fungicide, potassium phosphonate, on trees used for acorn collection without information on how the treatment affects acorn properties. In this study, select macronutrients and polyphenolics were quantified in tanoak acorns to evaluate the influence of potassium phosphonate treatment on the composition and nutritional value of tanoak acorns. Of the fatty acids tested from C14:0 to C20:1, only C17:0 was significantly lower (p < 0.05) in the nontreated and treated acorns after the first year. There were no differences detected in total phenolic content, gallic acid content, or ellagic acid content. Protein, phosphorus, and potassium levels were not significantly affected by fungicide treatment. Soluble glucose and fructose levels were significantly higher (p < 0.05) in both nontreated and treated groups after the first year; soluble sucrose levels did not change. Total glucose, starch, and total nonstructural carbohydrates increased significantly (p < 0.05) in the nontreated group after the first year but not in the treatment group; however, the treatment group values did not differ significantly from the control group values at baseline. The lack of any negative significant differences between acorns from treated and untreated tanoak trees implies that sodium phosphonate application for the prevention of sudden oak death does not impact the predominant polyphenolics or macronutrient quality of tanoak acorns.

Understanding the native Californian diet: Identification of condensed and hydrolyzable tannins in tanoak acorns (Lithocarpus densiflorus).

The tanoak (Lithocarpus densiflorus) acorn was a staple food in the Native American diet and is still used in traditional dishes. Acorns from the genus Quercus have been shown to contain a large range of hydrolyzable tannins. However, neither hydrolyzable nor condensed tannins have been characterized in tanoak acorns. The aim of this study was to identify the full range of hydrolyzable and condensed tannins in extracts of tanoak acorns using liquid chromatography/electrospray ionization-mass spectrometry/mass spectrometry. Condensed tannins were identified as B type oligomers of (epi)-catechin (procyanidins) with a degree of polymerization up to six. Oligomers up to and including tetramers were identified by UV spectra and MS detection whereas pentamers and hexamers were detected only by MS. The total concentration of condensed tannins was 464 mg/100 g acorn pericarp. The concentration of propocyanidin monomers, dimers, trimers, and tetramers in acorn pericarp (mg/100 g acorn pericarp) were 95 +/- 10.9, 148 +/- 35.0, 90 +/- 17.9, and 131 +/- 1.9, respectively. No procyanidins were found in the acorn cotyledon tissue. A total of 22 hydrolyzable tannins were identified in methanolic extracts of acorn cotyledon tissue. Gallic acid derivatives predominated and included galloylated esters of glucose, hexahydrodiphenoyl esters of glucose, and methylated gallates. Galloylated esters of glucose were present as isomers of galloyl glucose, digalloyl glucose, and trigalloyl glucose. Mass spectral fragmentation patterns indicate the presence of one gallic acid-galloyl glucose isomer and two gallic acid-digalloyl-glucose isomers. No isomers of tetragalloyl glucose and pentagalloyl glucose were identified. Ellagic acid and ellagic acid pentoside were also identified.

Real-Time, label-free monitoring of tumor antigen and serum antibody interactions.

Conventional techniques for the detection of biomolecular interactions can be limited by the need for exogenous labels, time- and labor-intensive protocols, as well as by poor sensitivity levels. A refractometer instrument has been reconfigured to detect biomolecular interactions through changes in surface plasmon resonance (SPR). The binding kinetics and affinity values of anti-NY-ESO-1 monoclonal antibody, ES121, to the cancer-testis antigen NY-ESO-1 were determined according to the surface heterogeneity model and resulted in K(D) values of 1.3x10(-9) and 2.1x10(-10) M. The reconfigured instrument was then used to measure the interaction between tumor antigens and serum antibodies against these antigens in preselected cancer patient sera samples. The tumor antigens assayed included NY-ESO-1, SSX2 and p53, all used as recombinant proteins containing polyhistidine tags. These results demonstrated that the instrument is capable of detecting the binding of serum antibodies from cancer patient sera to immobilized tumor antigens, consistent with those observed previously in ELISA-based experiments. These results demonstrate the potential of SPR technology for the rapid diagnosis and monitoring immune responses.

Varietal differences in phenolic content and antioxidant and antiproliferative activities of onions.

Epidemiological studies have indicated that the consumption of fruits and vegetables is associated with a reduced risk for the development of chronic diseases, such as cardiovascular disease and cancer. Phytochemicals, including phenolics and flavonoids, are suggested to be the major bioactive compounds contributing to the health benefits of fruits and vegetables. Onions are a major source of dietary flavonoids; however, there may exist varietal differences in composition, concentration, and beneficial activities. To characterize these differences, shallots and 10 onion (Allium cepa L.) varieties commonly available in the United States (Western Yellow, Northern Red, New York Bold, Western White, Peruvian Sweet, Empire Sweet, Mexico, Texas 1015, Imperial Valley Sweet, and Vidalia) were evaluated for total phenolic and flavonoid contents and antioxidant and antiproliferative activities. Shallots contained the highest total phenolic content (114.7 +/- 10.0 mg/100 g of sample) among the varieties tested, with a 6-fold difference observed when compared to the variety with the lowest phenolic content (Vidalia, p < 0.05). Western Yellow onion variety exhibited the highest total flavonoid content (69.2 +/- 3.7 mg/100 g of onion) of the varieties tested, with an 11-fold difference when compared to the variety with the lowest flavonoid content (Western White, p < 0.05). Shallots exhibited the highest total antioxidant activity (45.5 +/- 2.1 micromol of vitamin C equiv/g of onion), followed by Western Yellow, New York Bold, Northern Red, Mexico, Empire Sweet, Western White, Peruvian Sweet, Texas 1015, Imperial Valley Sweet, and Vidalia. For all varieties, both total phenolic and flavonoid contents were strongly correlated with total antioxidant activity (R (2) = 0.9668, p < 0.05; and R (2) = 0.7033, p < 0.05, respectively). The proliferation of HepG(2) and Caco-2 cells was significantly inhibited in a dose-dependent fashion after exposure to the Western Yellow, shallots, New York Bold, and Northern Red extracts, with Western Yellow, shallots, and New York Bold exhibiting the highest antiproliferative activity against HepG(2) cells and New York Bold and Western Yellow exhibiting the highest antiproliferative activity against Caco-2 cells. However, the varieties of Western White, Peruvian Sweet, Empire Sweet, Mexico, Texas 1015, Imperial Valley Sweet, and Vidalia demonstrated weak antiproliferative activity against both HepG(2) and Caco-2 cells. These results may influence consumers toward purchasing onion varieties exhibiting greater potential health benefits and may significantly affect future breeding efforts to enhance onion nutritional qualities.

Antioxidant and antiproliferative activities of strawberries.

Strawberries contain high levels of antioxidants, which have been correlated with a decreased risk of chronic disease. To more fully characterize the antioxidant profiles and possible associated health benefits of this fruit, the total free and bound phenolic, total flavonoid, and total anthocyanin contents of eight strawberry cultivars (Earliglow, Annapolis, Evangeline, Allstar, Sable, Sparkle, Jewel, and Mesabi) were measured. Cultivar effects on phenolic contents were compared with antioxidant capacities, as measured by the total oxyradical scavenging capacity (TOSC) assay, and to antiproliferative activities, as measured by inhibition of HepG(2) human liver cancer cell proliferation in vitro. Free phenolic contents differed by 65% between the highest (Earliglow) and the lowest (Allstar) ranked cultivars. The water soluble bound and ethyl acetate soluble bound phenolic contents averaged 5% of the total phenolic content of the cultivars. The total flavonoid content of Annapolis was 2-fold higher than that of Allstar, which had the lowest content. The anthocyanin content of the highest ranked cultivar, Evangeline, was more than double that of the lowest ranked cultivar, Allstar. Overall, free phenolic content was weakly correlated with total antioxidant activity, and flavonoid and anthocyanin content did not correlate with total antioxidant activity. The proliferation of HepG(2) human liver cancer cells was significantly inhibited in a dose-dependent manner after exposure to all strawberry cultivar extracts, with Earliglow exhibiting the highest antiproliferative activity and Annapolis exhibiting the lowest. No relationship was found between antiproliferative activity and antioxidant content.