Small molecules, like some antibiotics and anticancer agents that bind DNA with high specificity, can represent a relevant alternative as ligands in affinity processes for plasmid DNA (pDNA) purification. In the current study, pDNA binding affinities of berberine, berenil, kanamycin, and neomycin were evaluated by a competitive displacement assay with ethidium bromide using a fluorimetric titration technique. The binding between pDNA and ethidium bromide was tested in different buffer conditions, varying the type and the salt concentration, and was performed in both the absence and presence of the studied compounds. The results showed that the minor groove binder berenil has the higher pDNA binding constant. Chromatographic experiments using a derivatized column with berenil as ligand showed a total retention of pDNA using 1.3M ammonium sulfate in eluent buffer. A selective separation of supercoiled and open circular isoforms was achieved by further decreasing the salt concentration to 0.6M and then to 0M. These results suggest a promising application of berenil as ligand for specific purification of pDNA supercoiled isoform by pseudo-affinity chromatography.

A set of constituents of the erythrocyte membrane lipidome has been proposed to serve as biomarkers for liver disease and acute coronary syndrome. In erythrocytes, sphingomyelin hydrolysis provides ceramide, a signaling lipid necessary for phosphatidylserine exposure and eryptosis. Phosphatidylserine exposure further amplifies hepatic inflammation and fibrosis during non-alcoholic fatty liver disease (NAFLD). In this study, we developed and applied a quantitative TLC for erythrocyte membrane sphingomyelin of NAFLD patients. We also compared 10 extraction methods for the isolation of sphingomyelin from erythrocytes. For quantitative TLC, lipids were separated in Silica gel 60 F254 using a mixture of chloroform/methanol/acetic acid/water (60/50/1/4) (v/v/v/v). The separated lipids were stained in a chamber containing iodine, and the intensity of each of the primary colors (red, green, blue) and the sum of the Red plus Green colors (R+G) was analyzed. The method was linear over a wide range of concentrations, presented acceptable precision (inter-day CV(%) 0.34, 0.006 and 0.44 for 2.5, 5.0 and 10 μg, respectively), good accuracy (recovery range 85.2-97.1%), and excellent limit of detection (0.137 μg/spot) and limit of quantification (0.41 μg/spot). Using this quantitation method, we compared various lipid extraction methods and found that lipid extraction with methanol led to higher yield of erythrocyte sphingomyelin (135.35±1.04% recovery, compared to the Folch method).Application of these methods showed that erythrocytes from NAFLD patients (9 men, 15 women, 57.95±11.11 years old) contained statistically significantly less sphingomyelin (829.82±511.60 vs 1892.08±606.25 μg/ml of packed erythrocytes) compared to healthy controls (4 men, 6 women, 39.3±15.55 years old).

BACKGROUND: Carbohydrates as starch are a staple part of the Mediterranean diet. Starch is digested in the small intestine and the resulting glucose is absorbed into the blood, eliciting an insulin response. The digestion and absorption kinetics (rapid or slow) depends on starch structure. OBJECTIVE: To study the relationship between the in vivo glycemic and insulinemic index and the in vitro digestibility characteristics of six bakery products, made from non-conventional wholemeal/wholegrain flours. METHODS: We analyzed in vitro the rapidly- and slowly- available glucose (RAG and SAG), the rapidly- and slowly- digestible starch (RDS and SDS), and the resistant starch (RS) fraction of the six wholemeal/wholegrain products and one white type of bread. The glycemic and the insulinemic index (GI and II respectively) were estimated by in vivo testing in a group of eleven healthy individuals. RESULTS: The GI of the wholemeal/wholegrain flour biscuits and breads were low, (range 28 +/- 3.2 to 41 +/- 3.9, Mean+SEM) correlating with the II. RAG positively correlated with both GI and II, with fiber having a marginal correlation. CONCLUSIONS: Our findings indicate that both conventional and non-conventional wholemeal/wholegrain bakery products have low GI and moderate II, correlating to in vitro starch digestibility and the type of processing.

The erythrocyte membrane lipidome has been proposed as a set of biomarkers for liver disease and acute coronary syndrome. In erythrocytes, sphingomyelin hydrolysis provides ceramide, a signaling lipid necessary for phosphatidylserine exposure and eryptosis. Phosphatidylserine exposure further amplifies hepatic inflammation and fibrosis during non-alcoholic fatty liver disease (NAFLD). In this study, we developed and applied a quantitative TLC for erythrocyte membrane sphingomyelin of NAFLD patients. We also compared 10 extraction methods for the isolation of sphingomyelin from erythrocytes. For quantitative TLC, lipids were separated in Silica gel 60 F254 using a mixture of chloroform/methanol/acetic acid/water (60/50/1/4) (v/v/v/v). The separated lipids were stained in a chamber containing iodine, and the intensity of each of the primary colors (red, green, blue) and the sum of the Red plus Green colors (R+G) was analyzed. The method was linear over a wide range of concentrations, presented acceptable precision (inter-day CV(%) 0.34, 0.006 and 0.44 for 2.5, 5.0 and 10 ug, respectively), good accuracy (recovery range 85.2-97.1%), and excellent limit of detection (0.137 ug/spot) and limit of quantification (0.41 ug/spot). Using this quantitation method, we compared various lipid extraction methods and found that lipid extraction with methanol led to higher yield of erythrocyte sphingomyelin (135.35 +- 1.04% recovery, compared to the Folch method). Application of these methods showed that erythrocytes from NAFLD patients (9 men, 15 women, 57.95 +- 11.11 years old) contained statistically significantly less sphingomyelin (829.82 +- 511.60 vs 1892.08 +- 606.25 ug/ml of packed erythrocytes) compared to healthy controls (4 men, 6 women, 39.3 +- 15.55 years old).