24-25-dihydroxyvitamin-d-3 and Leukemia

A new, highly sensitive HPLC assay method using an electrochemical detector (ECD) for multiple assay of vitamin D metabolites is reported. The assay involves extracting lipids from plasma with methylene chloride and methanol, purification on Zorbax SIL column with 5.5% (v/v) iso-propanol in hexane and quantification by HPLC-ECD. A coulometric system, composed of the dual electrode analytical cell and a guard cell, was used for ECD of the eluting compounds. The potentials applied to detectors 1 and 2 in a dual electrode analytical cell were adjusted to +0.20 V and +0.60 V, respectively. This method is sensitive to 20 pg of 25-hydroxyvitamin D3 [25(OH)D3] and of 24R,25-dihydroxyvitamin D3 [24,25(OH)2D3]. Calibration curves gave linearity from 20-1000 pg for 25(OH)D3 and 24,25(OH)2D3. The detection limit was approximately 50 pg ml-1 for 25(OH)D3 and 24,25(OH)2D3 in plasma. This sensitivity combined with an overall recovery of 25(OH)D3 (81.5 +/- 2.6%, mean +/- S.E.) allows the measurement of trace amount of 25(OH)D3 with only 20 microliters of plasma. Intra- and interassay RSD values were 5.3 and 9.7% for 25(OH)D3 and 6.3 and 9.7% for 24,25(OH)2D3, respectively. Plasma levels of 25(OH)D3 and 24,25(OH)2D3 in normal adults were 15.9 +/- 2.8 ng ml-1 (n = 10) and 1.4 +/- 0.5 ng ml-1 (n = 10), respectively. This method allows the determination of 25(OH)D2 and 25(OH)D3 for evaluating their nutritional and clinical status. From these results, it is concluded that the proposed HPLC-ECD assay system is useful for the determination of vitamin D metabolites in biological fluids as a highly sensitive physicochemical method.

Topics: 24,25-Dihydroxyvitamin D 3; Adult; Case-Control Studies; Chromatography, High Pressure Liquid; Electrochemistry; Humans; Leukemia; Liver Cirrhosis; Osteoporosis; Reference Values; Reproducibility of Results; Sensitivity and Specificity; Vitamin D

Cultured human macrophages from normal donors were examined for their capability to metabolize 25-hydroxyvitamin D3 (25-(OH)D3). Upon exposure to recombinant human interferon-gamma (IFN-gamma) both bone marrow-derived macrophages (BMM) and pulmonary alveolar macrophages (PAM) produced a polar 25-(OH)D3 metabolite which was purified from conditioned media and unequivocally identified as 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) by UV-absorbance spectrophotometry and mass spectrometry. The BMM and PAM also synthesized a second 25-(OH)D3 metabolite which was structurally identified as 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3). The time course of 25-(OH)D3 metabolism by macrophages suggested that the production of 24,25-(OH)2D3 was stimulated by high intracellular levels of 1,25-(OH)2D3 and not by IFN-gamma. The 1,25-(OH)2D3 obtained from BMM and PAM promoted macrophage-like differentiation of promyelocytic HL-60 leukemia cells and inhibited IFN-gamma production by normal human lymphocytes. Our data suggest that locally high levels of 1,25-(OH)2D3 in the microenvironment of IFN-gamma-stimulated BMM and PAM may modulate the function of hormone-responsive cells.

Topics: 24,25-Dihydroxyvitamin D 3; Bone Marrow Cells; Calcitriol; Cell Differentiation; Cell Line; Dihydroxycholecalciferols; Humans; Interferon-gamma; Leukemia; Macrophages; Mass Spectrometry; Pulmonary Alveoli; Recombinant Proteins; Spectrophotometry, Ultraviolet