Evaluation of reversed-phase nano liquid chromatography conditions by using reversed-phase thin layer chromatography based on Hansen solubility parameters for the analysis of amphiphilic glycosylsphingolipid transformations.

Pulse chase analysis is often used in investigating dynamics of cellular substances. Fluorescently labeled lactosyl sphingosine molecule is useful in chasing its transformation, however the analysis of such metabolites in attomole level is of extreme difficult due to the presence of large amount of endogenous amphiphilic molecules such as glycosphingolipids, sphingomyerin, and glycerophospholipids. Nano LC suites for analyzing the attomole scale metabolites, therefore removal of endogenous substances prior to nano LC and finding appropriate nano LC conditions are necessary. Thus, we focused on the solubility of fluorescent BODIPY-labeled lactosylsphingosine (Lac-Sph-BODIPY) to identify suitable solvents to remove endogenous compounds. In this study, we evaluated solvents by using C18 thin layer chromatography (RP TLC). The mobility (Rf) of Lac-Sph-BODIPY against several solvent mixtures on RP TLC were plotted against polarity and hydrogen bonding capability followed by Hansen solubility parameters (HSPs). The optimum solvent mixture with Rf = 0.3 ±â€¯0.1 was chosen for elimination of endogenous phospholipids on a ZrO2-SiO2 cartridge column and subsequent separation by nano LC. Efficient removal of endogenous phospholipids was demonstrated, and good resolution in nano LC analysis of Lac-Sph-BODIPY extracted from Chinese hamster ovary (CHO)-K1 cells was achieved. It was also shown that the amount of exogenously added compound was important in the investigation of metabolites using cultured cells.

The monocyte-macrophage system in granulomatous inflammation.

Granulomas may be immunologically induced or non-immunologically induced. In immunologically induced granulomas cells of the monocyte-macrophage series take on the appearance of epitheloid cells. Ultrastructurally epithelioid cells may have a secretory appearance with much rough endoplasmic reticulum or take on highly degenerate vesicular appearance. Other epithelioid cells look like activated macrophages. Secretory epithelioid cells may be found associated with acute local inflammation as in borderline tuberculoid leprosy in reaction, the lepromin reaction, following injection of BCG vaccine and in experimental zirconium granulomas. In these situations there may also be strong histological and biochemical evidence of increased fibroblast activity and collagen synthesis. It is suggested that these cells are actively secreting a fibroblast-activating factor. Epithelioid cells may lose their Fc receptors, undifferentiated macrophages in lepromatous leprosy can lose their C3 receptors. It is suggested that in a number of situations granuloma formation may be associated with complement activation through the alternative pathway as in the case of mycobacterial granulomas. Toxic granulomas produced by metals may be caused by C3 being split by plasmin after conversion from plasminogen by activation of the Hageman factor.

The analytical reliability of the zirconyl phosphate method of plasma carcinoembryonic antigen.

We evaluated the analytical reliability of the carcinoembryonic antigen (CEA) method of Hansen. Our experience is based on performing over 23,000 CEA assays in more than 10,000 clinical samples. The sensitivity of the assay is 0.5 ng/ml. The precision must be defined as a function of concentration. In the range 2.6 to 12 ng/ml the coefficient of variation is 4.96 to 7.39%. Long-range studies of the reproducibility of the standard curve, over a period of several years and including 435 standard curves, have shown an overall mean B/Bo of 31.45% which is close to the theoretical optimal of 33%. The long-term 90 and 50% intercepts are 0.55 +/- 0.15 and 7.11 +/- 1.0, respectively. Interlaboratory surveys show good agreement between the means of the survey group and the target values but rather large individual discrepancies. The CEA method studied here is sensitive and reproducible in intralaboratory studies but less so in interlaboratory comparisons. The reagents have performed uniformly and close to specifications over an extended period of time.