METHODS: Thirty-two healthy volunteers were randomly allocated to receive saline (Control) or dexamethasone 2 mg, 4 mg or 8 mg intravenously. Venous blood samples were collected at baseline before administration of treatment, and at 4 h, 24 h and one-week post-treatment. At each time point, measurements included blood glucose and macrophage migration inhibition factor (MMIF), full blood count including lymphocyte subsets, monocytes, neutrophils, eosinophils and basophils by flow cytometry, and plasma SPM using liquid chromatography tandem mass spectrometry. The effect of dexamethasone dose and time on all measures was analysed using linear mixed models.
RESULTS: There was a dose-dependent increase in neutrophil count after dexamethasone that persisted for 24 h. In contrast, there was a dose-dependent reduction in counts of monocytes, lymphocytes, basophils and eosinophils 4 h after dexamethasone, followed by a rebound increase in cell counts at 24 h. Seven days after administration of dexamethasone, all cell counts were similar to baseline levels. MMIF concentration, glucose and natural killer cell counts were not significantly affected by dexamethasone. There was a significant gender effect on plasma SPM such that levels of 17-HDHA, RvD1, 17R-RvD1 and RvE2 in females were on average 14%-50% lower than males. In a linear mixed model that adjusted for neutrophil count, there was a significant interaction between the dose of dexamethasone and time, on plasma 17R-RvD1 such that plasma 17R-RvD1 fell in a dose-dependent manner until 4 h after administration of dexamethasone. There were no significant effects of dexamethasone on the other plasma SPM (18-HEPE, RvE2, 17-HDHA, RvD1, RvD2 and 14-HDHA) measured.
DISCUSSION: This is the first study in healthy volunteers to demonstrate that commonly employed antiemetic doses of dexamethasone affect immune cell populations and plasma levels of 17R-RvD1 an SPM with anti-nociceptive properties. If similar changes occur in surgical patients, then this may have implications for acute infection risk in the post-operative period.
DESIGN AND METHODS: The activity of DPD was measured using 5-[2- (14)C]Fluorouracil (5-[2-(14)C]FUra) followed by separation of substrate and product 5-[2-(14)C]FUraH(2) with a 15 x 4.6 mm I.D., 5 microm particle size (d(p)) porous graphitic carbon (PGC) column (Hypercarb(R)) and HPLC with online detection of the radioactivity. This was standardized using the protein concentration of the cytosol (NanoOrange(R) Protein Quantitation).
RESULTS: Complete baseline separation of 5-[2-(14)C]Fluorouracil (5-[2-(14)C]FUra) and 5-[2-(14)C]Fluoro-5,6-dihydrouracil (5-[2-(14)C]FUraH(2)) was achieved using a porous graphitic carbon (PGC) column. The detection limit for 5-[2-(14)C]FUraH(2) was 0.4 pmol.
CONCLUSIONS: By using linear gradient separation (0.1% Trifluoroacetic acid [TFA] in water to 100% Methanol) protocols in concert with PGC columns (Hypercarb(R)), we have demonstrated that a PGC column has a distinct advantage over C-18 reverse phase columns in terms of column stability (pH 1-14). This method provides an improvement on the specific assay for DPD enzyme activity. It is rapid, reproducible and sensitive and can be used for routine screening for healthy and cancer patients for partial and profound DPD deficiency before treatment with 5- FUra.