Determination of salicin and related compounds in botanical dietary supplements by liquid chromatography with fluorescence detection.

A sensitive and reliable method is described for quantitative determination of salicin (including salicyl alcohol) and salicylic acid in botanical dietary supplements by reversed-phase liquid chromatography (LC) with wavelength-programmed fluorescence detection. One gram sample material was extracted with 20 mL aqueous phosphate buffer (pH 5.0), which was heated in an 80 degrees C water bath for 30 min. After centrifugation and cooling of the extract to room temperature, the supernatant was diluted with additional buffer. A 1 mL portion of diluted extract was mixed with 1 mL beta-glucosidase solution (2 mg/mL) and incubated for 40 min in a 37 degrees C water bath. The extract was passed through a 0.45 micron syringe filter and analyzed by LC. Limits of quantitation for salicin and salicylic acid were 20 and 1 microgram/g, respectively. Recoveries from samples fortified with salicin at 20, 100, and 1000 micrograms/g and with salicylic acid at 5, 20, and 50 micrograms/g ranged from 85 to 110%, with standard deviations less than 7%.

Metabolism of chloral hydrate in mice and rats after single and multiple doses.

Chloral hydrate is a hepatocarcinogen in mice but not rats. To examine this species-related difference, male and female B6C3F1 mice and Fischer (F344) rats were treated by gavage with 1 or 12 doses of chloral hydrate, and concentrations of the drug and its metabolites were determined in plasma at 0.25, 7, 3, 6, and 24 h and 2, 4, 8, and 16 d after the last treatment. Maximum levels of chloral hydrate were observed at the initial sampling time of 0.25 h. By 1 h, levels dropped substantially, and by 3 h, chloral hydrate could not be detected. Trichloroacetic acid was the major metabolite found in the plasma, with peak levels being observed 1-6 h after dosing. The concentrations then slowly decreased such that by 2 d this metabolite could no longer be detected. Trichloroethanol was assayed as both the free alcohol and its glucuronide. Maximum levels of trichoroethanol occurred at 0.25 h, and by 1-3 h approached the limits of detection. A pharmacokinetic model was constructed to describe the metabolic data. The plasma half-life values of chloral hydrate were similar in both species. In mice, the rate of elimination of trichloroacetic acid was significantly increased after multiple doses; this difference was not observed with rats. The half-life of trichloroethanol and its glucuronide was significantly greater in rats as compared to mice. None of the metabolic parameters appears to account for the hepatocarcinogenicity of chloral hydrate seen in mice but not rats.

Reduction of malachite green to leucomalachite green by intestinal bacteria.

Intestinal microfloras from human, rat, mouse, and monkey fecal samples and 14 pure cultures of anaerobic bacteria representative of those found in the human gastrointestinal tract metabolized the triphenylmethane dye malachite green to leucomalachite green. The reduction of malachite green to the leuco derivative suggests that intestinal microflora could play an important role in the metabolic activation of the triphenylmethane dye to a potential carcinogen.

Suppressor cell induction by the anticancer drug spirogermanium.

Spirogermanium is a metal-containing compound reported to have antitumor, antiarthritic, antimalarial and immunoregulatory activity. In this study we have demonstrated that spirogermanium inhibited antibody synthesis to sheep red blood cells in BDF1 mice in vivo. Spleen cells from these treated mice were unable to respond to this antigen in vitro, and suppressed both the antibody response of normal cells to SRBC and the mitogenic response of normal cells to Concanavalin A in co-culture assays. The cells responsible for this suppression did not belong to the T cell lineage since treatment with anti-Thy-1.2 antiserum and complement did not abrogate the suppression. The suppressor cells were found to be radiation resistant and nylon wool adherent. Plastic adherence or passage over Sephadex G10 partially removed the suppression indicating the contribution, at least in part, of a suppressor macrophage. The plastic non-adherent population of cells also contained suppressor cells which were detected following anti-thy-1.2 treatment and selection by panning on anti-IgG coated plates. Fluorescent antibody and flow cytometry technology showed the population of suppressor cells to be 90% immunoglobulin positive, indicative of a B cell lineage.

Macrophage activation in rat models of inflammation and arthritis. Systemic activation precedes arthritis induction and progression.

The association between the induction and progression of adjuvant-induced arthritis (AA) and the development of synovial and systemic macrophage activation was assessed by studying the temporal development of these parameters in a rat model. Rats with AA developed significant edema of the uninjected hind leg beginning 10 days post-adjuvant injection, with progressive increases in edema continuing through day 17. Several parameters of macrophage activation, including the enhanced ability to secrete interleukin-1 and prostaglandin E2, kill tumor cells, accumulate fluorescent cyanine dyes, emigrate into the peritoneal cavity and synovium, and express Ia antigen, as well as the decreased ability to secrete superoxide anion, were associated temporally with the development of the arthritic lesion. In addition to the temporal association between macrophage activation and development of arthritis, a positive correlation between macrophage activation and arthritis induction was seen with the use of synthetic adjuvants at arthritogenic and nonarthritogenic doses. These data taken together suggest that induction and progression of AA in rats is associated with both systemic (blood, spleen, and peritoneal cavity) and local (synovium) macrophage activation.

Methodological considerations for implementation of lymphocyte subset analysis in a clinical reference laboratory.

As the diagnostic utility of lymphocyte subset analysis has been recognized in the clinical research laboratory, a wide variety of reagents and cell preparation, staining and analysis methods have also been described. Methods that are perfectly suitable for analysis of smaller sample numbers in the biological or clinical research setting are not always appropriate and/or applicable in the setting of a high volume clinical reference laboratory. We describe here some of the specific considerations involved in choosing a method for flow cytometric analysis which minimizes sample preparation and data analysis time while maximizing sample stability, viability, and reproducibility. Monoclonal T- and B-cell reagents from three manufacturers were found to give equivalent results for a reference population of healthy individuals. This was true whether direct or indirect immunofluorescence staining was used and whether cells were prepared by Ficoll-Hypaque fractionation (FH) or by lysis of whole blood. When B cells were enumerated using a polyclonal anti-immunoglobulin reagent, less cytophilic immunoglobulin staining was present after lysis than after FH preparation. However, both preparation methods required additional incubation at 37 degrees C to obtain results concordant with monoclonal B-cell reagents. Standard reagents were chosen on the basis of maximum positive/negative separation and the availability of appropriate negative controls. The effects of collection medium and storage conditions on sample stability and reproducibility of subset analysis were also assessed. Specimens collected in heparin and stored at room temperature in buffered medium gave reproducible results for 3 days after specimen collection, using either FH or lysis as the preparation method. General strategies for instrument optimization, quality control, and biohazard containment are also discussed.

Long-term follow-up of donors cytapheresed more than 50 times.

11 volunteers who had donated white blood cells or platelets more than 50 times over a 5- to 9-year period were studied to determine whether any adverse consequences of many cytaphereses could be detected. Among the donors no significant differences were found in 18 hematological and biochemical parameters when compared to a group of age- and sex-matched nondonor controls. Despite extensive cumulative lymphocyte losses sustained by these donors, the ratio of T, B, helper and suppressor cells has been maintained within the normal range. No detrimental effects of multiple cytapheresis on the donors' health has been demonstrated to date.

Determination of linear fluorescence intensities from flow cytometric data accumulated with logarithmic amplifiers.

Logarithmic amplifiers are useful in accumulating flow cytometric data with a large dynamic range. However, quantitative comparison of fluorescence intensities for different samples or different subpopulations within a sample is simplified by the conversion of data from log space back to linear space. A method is described in which fluorescent polystyrene spheres of differing intensities are used to construct a calibration curve for the logarithmic intensity scale. This allows calculation of relative linear intensity for each channel of the logarithmically accumulated data and determination of linear fluorescence means and coefficients of variation for comparative purposes. Fluorescent spheres of appropriate intensity may also be used as internal standards to monitor instrument and/or stain stability for samples accumulated using logarithmic amplifiers.