IntroductionExcess energy storage in the form of triglycerides and the release of fatty acids have long been viewed as major functions of adipose tissue in total lipid and energy homeostasis. However, adipose tissue is also the site of the modulation of active metabolism and energy regulation. Fat accumulation in visceral adipose tissue is correlated with the occurrence of diabetes mellitus, hyperlipidemia, hypertension, and atherosclerotic diseases, and is associated with changes in the endocrine and metabolic functions of adipose tissue. Abstract Background: The hypocholesterolemic and hypoglycemic effects of various natural and semisynthetic dietary fibers have been studied for their potential use in the prevention and improvement of metabolic syndrome. Of these dietary fibers, hydroxypropyl methylcellulose (HPMC) has been shown to lower plasma cholesterol and reduce weight gain. However, the underlying mechanisms are not known. In the present study, we examined associations between plasma adipocytokine levels and both lipid metabolism and insulin sensitivity after HPMC intake in golden Syrian hamsters. In addition, endogenous adiponectin from hamster plasma was purified and characterized. Methods: Hamsters were treated with HPMC (2% and 4% in a high-fat diet) or 2% or 4% microcrystalline cellulose (MCC; control diet) for 8 weeks. Plasma glucose, insulin, lipids, adiponectin, leptin, and hepatic lipid levels were assessed using standard techniques. Results: After 8 weeks of feeding, plasma total cholesterol and triglyceride levels in hamsters fed the 4% HPMC-supplemented diet were significantly lower than in hamsters fed the control diet. Moreover, a significant increase in adiponectin levels and a decrease in leptin levels were observed in hamsters fed the 4% HPMC-supplemented diet. Hamster adiponectin was found to be comprised of 244 amino acid residues with an apparent molecular weight of 30 kDa, consistent with the adiponectin reported in other species. Conclusions: Reductions in plasma cholesterol and triglyceride levels were correlated with a decrease in plasma leptin and an increase in adiponectin. These results suggest that adipocytokines are regulated by HPMC and may play a pivotal role in the hypocholesterolemic effect.

Soybean (Glycine max) is considered a major allergenic food. Gly m 4 is one of several soybean allergens that has been identified to cause an allergic reaction, typically the symptoms are localized effects including the skin, gastrointestinal tract, or respiratory tract. Soybean allergens are considered a complete food allergen in that they are capable of inducing specific IgE as well as eliciting a range of severity from mild rashes up to anaphylaxis. In this study, we have isolated, purified, and characterized an endogenous Gly m 4 protein. The endogenous protein has 88.0% sequence homology with the theoretically predicted Gly m 4 sequence. Following detailed characterization, an assay was developed for quantification of endogenous Gly m 4 using two-dimensional liquid chromatography with ultraviolet and mass spectrometric detection (2DLC-UV/MS). A linear relationship (R(2) > 0.99) was observed over the concentration range of 12.5-531.7 μg/mL. Over the linear range, the assay recoveries (percent relative error, % RE) ranged from -1.5 to 10.8%. The assay precision (percent coefficient of variation, % CV) was measured at three different Gly m 4 levels on each of the 4 days and did not exceed 11.2%. The developed method was successfully applied to quantify Gly m 4 level in 10 commercial soybean lines. To the best of our knowledge, this represents the first quantitative assay for an intact endogenous Gly m 4 protein.

Maize (Zea mays) is not considered a major allergenic food; however, when food induced allergenic and immunologic reactions have been implicated to maize, lipid transfer proteins (LTPs) have been identified as major allergens. LTP is an extremely stable protein that is resistant to both proteolytic attack and food processing, which permits the allergen to reach the gastrointestinal immune system in an immunogenic and allergenic conformation, allowing sensitization and induction of systemic symptoms. They are considered a complete food allergen in that they are capable of inducing specific IgE as well as eliciting severe symptoms. We have purified and characterized an endogenous ~9 kDa LTP from maize kernels. The maize LTP consists of 93 amino acid residues and has a M(r) of 9046.1 Da, determined by electrospray ionization mass spectrometry. Following accurate identification and characterization of maize LTP, a highly specific and quantitative assay using liquid chromatography with ultraviolet and mass spectrometric detection was developed. The present assay enables determination of LTP over a concentration range from 29 to 1030 μg/g in maize kernel samples. Assay recovery (percent relative error, % RE) was measured at 11 different concentrations ranging from 4 to 147 μg/mL and did not exceed 5.1%. The precision (percent coefficient of variation, % CV) was measured at 3 concentrations on each of 4 days and did not exceed 14.4%. The method was applied to evaluate the levels of LTP in 14 different maize lines. To our knowledge, this represents the first quantitative liquid chromatography-ultraviolet/mass spectrometry (LC-UV/MS) assay for the determination of LTP for the assessment of a food allergen.

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Top-down approaches for quantification of proteins based on separation and mass spectrometric assays hold promise due to their high specificity and avoidance of both proteolytic steps and need for generation of monoclonal antibodies. In this study, a 2DLC-UV/MS assay was developed for the simultaneous quantification of two intact soybean allergens, hydrophobic protein from soybean (HPS) and Gly m 4. Both of these allergens were purified from soybean seeds followed by complete characterization. The method validation consisted of evaluating linearity, precision, and recovery. A linear relationship (R(2) > 0.99) between concentrations of the two proteins and their respective peak areas was observed over the concentration ranges from 6.9 to 355.1 μg/mL and from 11.9 to 599.8 μg/mL for Gly m 4 and HPS, respectively. For the 4 day validation study, precision range (%CV) was observed to be from 4.7 to 9.2% for HPS and from 6.3 to 9.4% for Gly m 4. The assay recovery range (%RE) was observed to be from -1.1 to -13.7% for HPS and from -3.5 to 15.2% for Gly m 4. The assay was applied on 10 non-transgenic commercial lines to quantify the relative levels of the two allergens. The HPS and Gly m 4 levels ranged from 64 to 479 μg/g and from 204 to 637 μg/g, respectively. To the best of the authors' knowledge, this represents the first 2DLC-UV/MS assay for the simultaneous quantitation of selected allergens at the intact level.