C18 Reversed-phase High-performance Liquid Chromatography Research Articles

Chemical Identification of a Tumor-Derived Angiogenic Factor

Neoplasms produce substances that induce blood vessel formation (angiogenesis). Fractions from ethanol extracts of the Walker 256 carcinoma were isolated by silica column chromatography and C18 reversed-phase high-performance liquid chromatography. Two of the isolated fractions induced neovascularization when tested in the rabbit corneal micropocket assay. One of the fractions was identified as nicotinamide by desorption-electron impact mass spectrometry, nuclear magnetic resonance spectroscopy, and gas chromatography-mass spectrometry. The second active fraction contained nicotinamide as part of a more complex, as yet unidentified, molecular arrangement. Microgram quantities of commercial nicotinamide induced neovascularization in the corneal micropocket assay and in the chick chorioallantoic membrane assay.

Separation and purification of lymphocyte chemotactic factor (LCF) and interleukin 2 produced by human peripheral blood mononuclear cells

Two T-cell chemotactic factors, lymphocyte chemotactic factor (LCF) and interleukin 2 (IL-2), were separated and characterized from culture supernatants of concanavalin A-stimulated human peripheral blood mononuclear cells. LCF was purified approximately 7800-fold to homogeneity from culture supernatant using gel filtration and high-performance liquid chromatography (HPLC). LCF was found to he distinct from both IL-2 and interleukin-1. Sephadex G-100 gel filtration of crude supernatants from concanavalin A-stimulated mononuclear cells showed two molecular weight regions of T lymphocyte chemotactic activity. A 10,000- to 25,000-Da region contained both IL-2 and LCF and a 45,000- to 75,000-Da region contained only a high molecular weight form of LCF. Both high and low molecular weight species of LCF eluted with 40–44% acetonitrile from a reversed-phase C18 HPLC column. IL-2 present only in the low molecular weight region eluted from the C18 column with 65–75% acetonitrile. The migration of T lymphocytes to IL-2 was totally inhibited by anti-interleukin 2 receptor antibody while the response of T cells to LCF was unaffected. LCF eluting off the C18 column was purified to homogeneity by two subsequent cycles of gel filtration HPLC. The resultant protein showed a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular weight of 10,500. The data presented here demonstrate that IL-2 and LCF are distinct lymphocyte chemotactic factors and although they are not readily separable from crude supernatants by molecular sieve chromatography, they can easily be distinguished by reversed-phase HPLC.

Heterosis and the metabolism of [3H]gibberellin A1 in maize

Seedling growth and the metabolism of high specific activity (1.1 TBq mmol−1) [3H]gibberellin A1 were studied in two Canadian maize (Zea mays L.) inbreds, CM7 and CM49, and in their single cross F1 hybrid. As previously reported, the hybrid grew more rapidly than either parental inbred. Metabolism of the [3H]GA1 was qualitatively similar in all three genotypes and [3H]-labelled metabolites were tentatively identified through sequential, step-elution silicic acid partition chromatography followed by reversed-phase C18 high-performance liquid chromatography, relative to the retention times of authentic GAs. Although the expected 2β-hydroxylation product, [3H]GA8 was not detected, a metabolite was observed at the retention time of GA8-O(2)-glucoside. Additionally, another metabolite(s) eluted at the retention time of glucosyl conjugates of GA1, and enzymic cleavage with cellulase yielded a [3H]-labelled compound which subsequently eluted at the retention time of free GA1. While the ratio of the precursor [3H]GA1 to total [3H]GA conjugate-like metabolites was similar in the three genotypes, the hybrid contained higher levels of the [3H]GA8-glucosidelike metabolite, whereas the inbreds contained higher levels of the [3H]GA1 conjugatelike metabolite(s). Thus, there are differential rates of GA1 metabolism in a maize hybrid relative to its slower-growing inbred parents.

Fractionation of fecal neutral steroids by high performance liquid chromatography.

Fecal neutral steroids were fractionated by high performance liquid chromatography (HPLC) into three major fractions: 5 beta-H, 3-keto steroids; 5 beta-H, 3 beta-hydroxy steroids; and 5 alpha-H and delta 5-3 beta-hydroxy steroids. This separation was achieved in about 10 minutes, with greater than 97% recovery of standards in each fraction. Gas-liquid chromatographic quantitation of fecal steroids fractionated by either HPLC or thin-layer chromatography gave nearly identical results. A method using both C18 reverse phase and silica HPLC to purify radiolabeled sterols is also described.