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A fluorometric assay to determine labile copper(II) ions in serum.

ABSTRACT: Labile copper(II) ions (Cu²⁺) in serum are considered to be readily available for cellular uptake and to constitute the biologically active Cu²⁺ species in the blood. It might also be suitable to reflect copper dyshomeostasis during diseases such as Wilson's disease (WD) or neurological disorders. So far, no direct quantification method has been described to determine this small Cu²⁺ subset. This study introduces a fluorometric high throughput assay using the novel Cu²⁺ binding fluoresceine-peptide sensor FP4 (Kd of the Cu²⁺-FP4-complex 0.38 pM) to determine labile Cu²⁺ in human and rat serum. Using 96 human serum samples, labile Cu²⁺was measured to be 0.14 ± 0.05 pM, showing no correlation with age or other serum trace elements. No sex-specific differences in labile Cu²⁺ concentrations were noted, in contrast to the total copper levels in serum. Analysis of the effect of drug therapy on labile Cu²⁺ in the sera of 19 patients with WD showed a significant decrease in labile Cu²⁺ following copper chelation therapy, suggesting that labile Cu²⁺ may be a specific marker of disease status and that the assay could be suitable for monitoring treatment progress.

SUBMITTER: Maares M

PROVIDER: S-EPMC10406877 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature

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A fluorometric assay to determine labile copper(II) ions in serum.

Maares Maria M Haupt Alessia A Schüßler Christoph C Kulike-Koczula Marcel M Hackler Julian J Keil Claudia C Mohr Isabelle I Schomburg Lutz L Süssmuth Roderich D RD Zischka Hans H Merle Uta U Haase Hajo H

Scientific reports 20230807 1

Labile copper(II) ions (Cu2+) in serum are considered to be readily available for cellular uptake and to constitute the biologically active Cu2+ species in the blood. It might also be suitable to reflect copper dyshomeostasis during diseases such as Wilson's disease (WD) or neurological disorders. So far, no direct quantification method has been described to determine this small Cu2+ subset. This study introduces a fluorometric high throughput assay using the nov ...[more]

PMID: 37550465

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Project description:BackgroundTrehalose is a non-reducing disaccharide that is used as an osmolyte, transport sugar, carbon reserve and stress protectant in a wide range of organisms. In plants, trehalose 6-phosphate (Tre6P), the intermediate of trehalose biosynthesis, is thought to be a signal of sucrose status. Trehalose itself may play a role in pathogenic and symbiotic plant-microbe interactions, in responses to abiotic stress and in developmental signalling, but its precise functions are unknown. A major obstacle to investigating its function is the technical difficulty of measuring the very low levels of trehalose usually found in plant tissues, as most of the established trehalose assays lack sufficient specificity and/or sensitivity.ResultsA kinetic assay for trehalose was established using recombinant Escherichia coli cytoplasmic trehalase (treF), which was shown to be highly specific for trehalose. Hydrolysis of trehalose to glucose is monitored fluorometrically and the trehalose content of the tissue extract is determined from an internal calibration curve. The assay is linear for 0.2-40 pmol trehalose, and recoveries of trehalose were ≥88%. A. thaliana Col-0 rosettes contain about 20-30 nmol g-1FW of trehalose, increasing to about 50-60 nmol g-1FW in plants grown at 8°C. Trehalose is not correlated with sucrose content, whereas a strong correlation between Tre6P and sucrose was confirmed. The trehalose contents of ear inflorescence primordia from the maize ramosa3 mutant and wild type plants were 6.6±2.6 nmol g-1FW and 19.0±12.7 nmol g-1FW, respectively. The trehalose:Tre6P ratios in the ramosa3 and wild-type primordia were 2.43±0.85 and 6.16±3.45, respectively.ConclusionThe fluorometric assay is highly specific for trehalose and sensitive enough to measure the trehalose content of very small amounts of plant tissue. Chilling induced a 2-fold accumulation of trehalose in A. thaliana rosettes, but the levels were too low to make a substantial quantitative contribution to osmoregulation. Trehalose is unlikely to function as a signal of sucrose status. The abnormal inflorescence branching phenotype of the maize ramosa3 mutant might be linked to a decrease in trehalose levels in the inflorescence primordia or a downward shift in the trehalose:Tre6P ratio.

Dataset Information

A fluorometric assay to determine labile copper(II) ions in serum.

Publications

A fluorometric assay to determine labile copper(II) ions in serum.

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