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The assay in serum of non-caeruloplasmin copper, as exchangeable copper after complexation with EDTA (ExCu) and total copper has been evaluated and compared in patients with varying c-reactive protein(CRP). Measurement of ExCu and total copper, range 0.2–47.2 µmol/L, was developed using ICP-MS. The chelating agents EDTA and TEPA were compared over 0.0–10 g/L after incubation with serum for 60 mins followed by ultrafiltration with Amicon 10 kDa filter. The assay for ExCu was optimised with EDTA 3 g/L (8.1 mmol/L) maintained at pH 7.0–8.0 before ultrafiltration. TEPA was not as selective in chelation of copper. Patients n = 82 were studied in relation to changes in inflammatory marker CRP and a group of patients n = 37 with normal CRP. The ExCu assay gave excellent recoveries (94–102 % but poor recovery for free uncomplexed copper), good repeatability, limit of quantitation 0.19 µmol/l with a provisional reference range 0.48 to 1.63 µmol/L (n = 37 patients). The range for relative exchangeable copper (exchangeable copper divided by total serum copper) was 2.49 to 9.96 %. ExCu was elevated in conditions with increased CRP greater than 100 mg/L suggesting an effect of inflammation on the free copper fraction. A reliable and reproducible assay for ExCu and total copper has been developed. The upregulated inflammatory state increases the ExCu suggesting excess free copper. [ABSTRACT FROM AUTHOR]

Metal ecotoxicity testing with microalgae is subject to interferences such as change in bioavailable metal fraction by excreted metabolites. Moreover, high metal loads are usually necessary for obtaining 50% effect concentration (EC50) values based on cell number or chlorophyll a. Thus, sensitive parameters, which promptly respond to low dissolved metal variations at environmentally relevant concentrations, are much needed. The genus Scenedesmus, a coenobium-forming freshwater microalga, is cosmopolitan and inhabits pristine and contaminated waters. The present investigation addressed cellular phenotypic alterations in Scenedesmus acuminatus (Largerheim) Chodat 1902 caused by the presence of copper. Copper toxicity to the alga was investigated under laboratory-controlled conditions using a concentration range of 10-8 mol 1-1 to 10-3 mol 1-1 total dissolved Cu2+, thus including environmentally relevant concentrations. Free Cu2+ concentrations were determined using an ion-selective electrode calibrated with a metal ion buffer. The phenotypic plasticity of S. acuminatus was measured as the number of cells per coenobia and found to be readily affected at low copper concentrations. Under healthy conditions (10-8 mol 1-1 total dissolved Cu2+, 10-11 mol 1-1 free Cu2+) S. acuminatus formed eight-celled coenobia, whereas at 10-7 mol 1-1 total dissolved Cu2+ (3 × 10-10 mol 1-1 free Cu2+) these were absent and at 10-5 mol 1-1 total dissolved Cu2+ (5 × 10-8 mol 1-1 free Cu2+) free cells were predominant. Chlorophyll a synthesis was affected by 5 × 10-8 mol 1-1 free Cu-2+ or higher. EC50 (3.3 × 10-5 mol 1-1 free Cu2+) was obtained using chlorophyll a values after 96-h exposure to the toxicant. The number of cells per coenobia was a more sensitive parameter than chlorophyll a to assess Cu toxicity, and at concentrations that may be encountered in the environment. [ABSTRACT FROM AUTHOR]