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Recently, the use of Vitamin D in high doses for treatment of several conditions, mainly autoimmune in nature, has been advocated with dubious results. Hypercalcemia is an important side effect of this intervention. Here we describe our findings in samples that presented 25(OH)D in excess of 150 ng/mL (375 nmol/L) and had 1,25(OH)2D also measured. Material and Methods: we used serum samples from our diagnostic routine, received for measurement of 25(OH)D and 1,25(OH)2D according to medical requisition. A first group (group A) included 213 samples collected up to November 2018, used Diasorin’s chemiluminescent assays for 25OHD and 1,25(OH)2D, and a second group (group B), comprising 88 samples, used the same 25(OH)D assay and LC-MS/MS method for 1,25(OH)2D. 1,25(OH)2D measurement in the group A used a chemiluminescent competitive assay (Liason XL, Diasorin). The 1,25(OH)2D LC-MS/MS assay includes a previous sample prep, extraction, derivatization and chromatrography. APCI+ is followed by SRM (Selected Reaction Monitoring) and CAD (Collision Activated Dissociation) fragmentation. Precision studies showed, between run CVs of 6.8% to 7.4% and within run of 2.9% to 5.5%. In vitro investigations testing standards and spiked samples with 25(OH)D3, 25(OH)D2, 3-epimer-25(OH)D3 and 24R,25(OH)2D3 were also used to verify possible analytical interferences in the 1,25(OH)2D LC-MS/MS. Results: in group A, 25(OH)D median was 371 ng/mL (928 nmol/L), range 154 ng/mL to 856 ng/mL; 1,25(OH)2D median of 350 pg/mL (875 pmol/L), range 41 pg/mL to 1280 pg/mL. Correlation (Spearman) between 25(OH)D and 1,25(OH)2 was r= 0.8649 (P<0.001). In group B, 25(OH)D showed a median of 349 ng/mL (872 nmol/L), range 171 to 756 ng/mL; 1,25(OH)2D median of 54 pg/mL (135 pmol/L), range 24 pg/mL to 108 pg/mL. Correlation between 25(OH)D and 1,25(OH)2 was r= 0.185 (P= 0.08). In group A 189 samples had calcium measurement (median 9.7 mg/dL, range of 8.7 to 13.6 mg/dL), 182 creatinine (median of 0.8 mg/dL range of 0.3 to 1.8 mg/dL) and 179 PTH (median 19 pg/mL, range 5 to 68 pg/mL). In group B 75 cases had measurements of calcium (median 9.7, range 8.6 to 16.6 mg/dL), 75 of creatinine (median 0.8, range 0.3 to 2.5 mg/dL) and 75 of PTH (median 20, range 9 to 49 pg/mL). The in vitro tests showed a slight interference from 25(OH)D3, 3-epimer-25(OH)D3 and 24R,25(OH)2D3 molecules in the LC-MS/MS method. Conclusion: our results confirm data already published showing interference of high levels of 25(OH)D in 1,25(OH)2D measured by immunoassay and, in a milder way, by LC-MS/MS (1). V. Care should be taken in the interpretation of 1,25(OH)2D values in samples with high 25(OH)D values. 1. Hawkes CP, Schnellbacher S, Singh RJ, Levine MA. 25-Hydroxyvitamin D can interfere with a common assay for 1,25-dihydroxyvitamin D in vitamin D intoxication. J Clin Endocrinol Metab. 2015; 100:2883-2889.

MON-LB74 The Problem of Measuring 1,25(OH)2 Vitamin D in Patients With High Levels of 25(OH) Vitamin D