No abstract

Commonly used techniques for trace-element analysis in human biological material are flame atomic absorption spectrometry (FAAS), graphite furnace atomic absorption spectrometry (GFAAS), inductively coupled plasma atomic emission spectrometry (ICP-AES) and inductively coupled plasma mass spectrometry (ICP-MS). Elements that form volatile hydrides, first of all mercury, are analysed by hydride generation techniques. In the absorption techniques the samples are vaporized into free, neutral atoms and illuminated by a light source that emits the atomic spectrum of the element under analysis. The absorbance gives a quantitative measure of the concentration of the element. ICP-AES and ICP-MS are multi-element techniques. In ICP-AES the atoms of the sample are excited by, for example, argon plasma at very high temperatures. The emitted light is directed to a detector, and the optical signals are processed to values for the concentrations of the elements. In ICP-MS a mass spectrometer separates and detects ions produced by the ICP, according to their mass-to-charge ratio. Dilution of biological fluids is commonly needed to reduce the effect of the matrix. Digestion using acids and microwave energy in closed vessels at elevated pressure is often used. Matrix and spectral interferences may cause problems. Precautions should be taken against trace-element contamination during collection, storage and processing of samples. For clinical problems requiring the analysis of only one or a few elements, the use of FAAS may be sufficient, unless the higher sensitivity of GFAAS is required. For screening of multiple elements, however, the ICP techniques are preferable.

Mobilization of iron from ferritin by xanthine oxidase was studied under aerobic and anaerobic conditions. Aerobic iron release amounted to approx. 3.7 nmol/ml in 10 min. This amount was decreased by approx. 30% under anaerobic conditions. Aerobic iron mobilization involved two mechanisms. About 70% was released by O2.- generated by xanthine oxidase. The rest was released by O2(.-)-independent mechanisms, which also accounted for the total iron release when O2 was absent. A possible transfer of reducing equivalents directly from xanthine oxidase to ferritin is discussed. The results imply that, in pathological conditions with increased formation of O2.-, iron may be released from ferritin. Furthermore, in hypoxic tissues xanthine oxidase can release iron from ferritin by an O2(.-)-independent process. Free iron is liable to catalyse the formation of the extremely reactive and damaging OH. radical.

Background Chicken ovomucoid (OM, Gal d 1) has an important role in the pathogenesis of IgE‐mediated allergic reactions to hen's egg white. Objectives The purpose of this study was to clarify the mechanisms of T cell recognition of ovomucoid using intact OM and chemically modified, characterized and homogeneous solid phase synthetic peptides covering the whole molecule. Methods Eighteen overlapping peptides were prepared by solid phase F‐moc polyamide peptide synthesis (SPPS), characterized and high‐pressure liquid chromatography (HPLC) purified. The peptides, together with intact, denatured and oxidized OM, were used to stimulate patient‐derived cell cultures for mapping T cell epitopes. Proliferation responses, T cell phenotype and cytokine secretion using peripheral blood mononuclear cells (PBMC) from eight individuals and T cell lines (TCL) derived from six hen's egg‐allergic patients, were examined. In addition, intact, denatured, oxidized and deglycosylated OM, as well as the peptides solely or with their keyhole limpet haemocyanin (KLH) complexes, were tested. For locating IgE and IgG B cell epitopes, seven egg‐allergic patient sera and three OM‐polyclonal sera were used. Healthy non‐allergic individuals were included as controls. Results Seven peptides were recognized by specific IgE, while OM‐specific TCL recognized 10 peptides. Six of the OM peptides were commonly recognized both by patient S‐IgE and blood‐derived TCL. Among those, one novel epitope, peptide OM 61–74, had the ability to bind IgE. Another peptide, OM 101–114, was recognized by IgE and IgG sera, but not by any of the TCLs. In contrast, the peptides OM 41–56, OM 71–84, OM 131–144 and OM 171–186 were exclusively T cell epitopes with no affinity to specific antibodies. Abundant TCL secretion of IFN‐γ, IL‐6, IL‐4, IL‐13, IL‐10 and TNF‐α in response to OM stimulation indicates the contribution of Th2 as well as Th1/Th0 CD4+ cell subsets. For allergic patients moderate amounts of IFN‐γ, IL‐13, and high amounts of IL‐6, were secreted in response to TCL stimulation by OM peptides. High amounts of IL‐6 were secreted in response to all molecular forms of OM (intact‐, modified‐OM and the peptides 71–84 and 51–64) when TCLs from two non‐allergic donors were used. Conclusions One novel B cell epitope (OM 61–74) and 10 T cell epitopes have been identified. The most reactive epitopes of the OM molecule comprise the motifs 1–14 to 71–84, the overlapping peptide‐pairs OM 121–134 and OM 131–144 and peptides OM 161–174 and 171–186. Peptides OM 1–14 and 171–186 are the only ones capable of inducing IL‐4 secretion. Only one peptide (OM 11–24) induces IL‐10 secretion. Those peptides recognized as both T and B cell epitopes or only T cell epitopes, have the potential to induce T cell secretion of moderate to high amounts of IL‐13, IFN‐γ and particularly IL‐6.