Analytical, experimental and clinical aspects of total 25-hydroxyvitamin D measurement by two ligand assays are differentially biased by disease-related factors.

BACKGROUND: Pathological concentrations of plasma proteins may confound the results of binding assays. We compared two routinely used total 25-hydroxyvitamin D [t-25(OH)D] methods: a chemiluminescence-immuno-(CLIA) and an electro-chemiluminescence-protein-binding-(ECLPBA) assay. METHODS: Two sub-studies were performed: 1) In an "in vitro" study, exogenous albumin was added to pools of patients' sera with low albumin levels; and 2) In "ex vivo" studies of Cohort_1: sera of hospitalized patients with low albumin levels, and of healthy controls; and of Cohort_2: outpatients with chronic kidney disease in pre-dialysis stage, or on peritoneal dialysis and hemodialysis were investigated by the routine and LC_MS/MS methods. RESULTS: When increasing albumin concentrations were "in vitro" added, t-25(OH)D levels were overestimated by ECLPBA, and underestimated by CLIA. In patients' sera, positive correlations were detected between t-25(OH)D-vitamin D binding protein (DBP) values by both routine methods, and between t-25(OH)D-albumin values by all three methods. Much higher t-25(OH)D was measured by LC_MS/MS in all subgroups. When altering albumin levels with constant DBP concentration, the "in vitro" experiment revealed a higher sensitivity of ECLPBA. The "ex vivo" measurements demonstrated clinically relevant differences between the routine methods. CONCLUSION: Both routine methods are dependent of the matrix effect in hospitalized patients, which is predicted by the DBP/Albumin ratios. In hemodialysis, ECLPBA is recommended because its outcomes differ less from those of LC_MS/MS. The results of LC_MS/MS are reliable, but not routinely available. A guidance would be valuable on how levels measured by the binding methods differ from those by LC-MS/MS in various clinical conditions.

Reverse Na(+)/Ca(2+)-exchange mediated Ca(2+)-entry and noradrenaline release in Na(+)-loaded peripheral sympathetic nerves.

[(3)H]noradrenaline ([(3)H]NA) released from sympathetic nerves in the isolated main pulmonary artery of the rabbit was measured in response to field stimulation (2Hz, 1ms, 60V for 3min) in the presence of uptake blockers (cocaine, 3 x10(-5)M and corticosterone, 5 x10(-5)M). The [(3)H]NA-release was fully blocked by the combined application of the selective and irreversible 'N-type' voltage-sensitive Ca(2+)-channel (VSCC)-blocker omega-conotoxin (omega-CgTx) GVIA (10(-8)M) and the 'non-selective' VSCC-blocker aminoglycoside antibiotic neomycin (3x10(-3)M). Na(+)-loading (Na(+)-pump inhibition by K(+)-free perfusion) was required to elicit further NA-release after blockade of VSCCs (omega-CgTx GVIA+neomycin). In K(+)-free solution, in the absence of functioning VSCCs (omega-CgTx GVIA+neomycin), the fast Na(+)-channel activator veratridine (10(-5)M) further potentiated the nerve-evoked release of [(3)H]NA. This NA-release was significantly inhibited by KB-R7943, and fully blocked by Ca(o)(2+)-removal. However, Li(+)-substitution was surprisingly ineffective. The non-selective K(+)-channel blocker 4-aminopyridine (4-AP, 10(-4)M) also further potentiated the nerve-evoked release of NA in K(+)-free solution. This potentiated release was concentration-dependently inhibited by KB-R7943, significantly inhibited by Li(+)-substitution and abolished by Ca(o)(2+)-removal. It is concluded that in Na(+)-loaded sympathetic nerves, in which the VSCCs are blocked, the reverse Na(+)/Ca(2+)-exchange-mediated Ca(2+)-entry is responsible for transmitter release on nerve-stimulation. Theoretically we suppose that the fast Na(+)-channel and the exchanger proteins are close to the vesicle docking sites.