Using reverse phase high performance liquid chromatography as an alternative to resin fractionation to assess the hydrophobicity of natural organic matter.

Resin fractionation is the most widely used technique to isolate and characterize natural organic matter (NOM) based on its hydrophobicity and hydrophilicity, however, it is also recognized as a time consuming technique. This paper describes the use of reverse phase high performance liquid chromatography (RPHPLC) as a rapid assessment technique to determine the hydrophobicity/hydrophilicity of NOM. The optimum column separation condition was achieved and without the need for concentrating the sample prior to analysis and with good reproducibility of the peak retention time and the peak area. The characterization results were further compared with the traditional resin fractionation technique using DAX-8 and XAD-4 resins. The results demonstrated that the polarities defined by the two methods were different but consistent and also that the fractions absorbed onto XAD-4 were less hydrophobic than those absorbed onto DAX-8. The difference in definition between resin fractionation and RPHPLC were further investigated.

Differential effects of glucose on dehydroascorbic acid transport and intracellular ascorbate accumulation in astrocytes and skeletal myocytes.

Skeletal muscle and brain are major sites of glucose transport and ascorbate (vitamin C) storage. Ascorbate is oxidized to dehydroascorbic acid (DHAA) when used as an enzyme cofactor or free radical scavenger. We evaluated the hypothesis that glucose regulates DHAA uptake and reduction to ascorbate (i.e., recycling) by skeletal muscle cells and cerebral astrocytes. DHAA uptake was inhibited partially by glucose added simultaneously with DHAA. Comparison of wild type L6 skeletal muscle cells with an L6-derived cell line (D23) deficient in facilitative hexose transporter isoform 3 (GLUT3), indicated that both GLUT3 and facilitative hexose transporter isoform 1 (GLUT1) mediate DHAA uptake. Preincubation of muscle cells with glucose inhibited the rates of glucose and DHAA uptake, and decreased the intracellular concentration of ascorbate derived from recycling of DHAA. In contrast, glucose preincubation did not depress GLUT1 protein and activity levels or DHAA recycling in astrocytes. These results establish that glucose downregulates subsequent recycling of DHAA by skeletal muscle cells but not astrocytes.