Calcioic acid: In vivo detection and quantification of the terminal C24-oxidation product of 25-hydroxyvitamin D 3 and related intermediates in serum of mice treated with 24,25-dihydroxyvitamin D 3 .

Calcitroic acid, the excretory form of vitamin D, is the terminal product of a 5-step pathway catalyzed by CYP24A1, commencing with C24-hydroxylation of 1,25-dihydroxyvitamin D3 (1,25-(OH)2 D3 ). Catabolism of 25-hydroxyvitamin D3 (25-OH-D3 ) proceeds via analogous steps culminating in calcioic acid; however this C23-truncated acid has not been reported in the circulation. It has recently been shown that 24,25-dihydroxyvitamin D3 (24,25-(OH)2 D3 ) is an important factor in optimal bone fracture healing acting via an effector molecule FAM57B2 to produce lactosylceramide. Administration of 24,25-(OH)2 D3 was found to restore normal fracture repair in Cyp24a1-/- mice devoid of 24,25-(OH)2 D3 . We set out to study the multi-step catabolism of D3 metabolites in vivo using LC-MS/MS methods in vehicle or 24,25-(OH)2 D3 -treated mice. Vehicle-treated Cyp24a1+/- mice possessed normal levels of serum 24,25-(OH)2 D3 (7 ng/mL) and 25-OH-D3 -26,23-lactone (4 ng/mL). We also detected 24-oxo-25-OH-D3 (3 ng/mL) and 24-oxo-23,25-(OH)2 D3 (0.4 ng/mL); which were not detectable in vehicle-treated Cyp24a1-/- mice. In 24,25-(OH)2 D3 -treated Cyp24a1+/- mice, serum 24,25-(OH)2 D3 rose to 200 ng/mL while 25-OH-D3 -26,23-lactone remained unchanged in comparison to vehicle-treated Cyp24a1+/- mice Concentration of serum 24-oxo-25-OH-D3 and 24-oxo-23,25-(OH)2 D3 rose by 10-fold, when Cyp24a1+/- mice were treated with 24,25-(OH)2 D3 Calcioic acid was increased to 0.030 ng/mL for 24,25-(OH)2 D3 -treated Cyp24a1+/- mice. In 24,25-(OH)2 D3 -treated Cyp24a1-/- mice, serum 24,25-(OH)2 D3 rose further to a striking 830 ng/mL due to lack of catabolism of the 24,25-(OH)2 D3 dose. Serum 1,25-(OH)2 D3 levels were suppressed in 24,25-(OH)2 D3 -treated Cyp24a1+/- and Cyp24a1-/- mice. Circulating 1,24,25-(OH)3 D3 rose from 73 pg/mL to 106 pg/mL when Cyp24a1+/- mice were treated with 24,25-(OH)2 D3 . While undetectable in vehicle-treated Cyp24a1-/- mice, 1,24,25-(OH)3 D3 rose unexpectedly to 153 pg/mL in 24,25-(OH)2 D3 -treated nulls suggesting conversion of 24,25-(OH)2 D3 to 1,24,25-(OH)3 D3 via 1-hydroxylation. Taken together, amplification of 24,25-(OH)2 D3 catabolism by exogenous doses of this metabolite have enabled detection of downstream C24-oxidation pathway products in vivo, including calcioic acid; and provides a platform for studying alternative routes of vitamin D metabolism that may occur in pathological states including hypervitaminosis D and idiopathic infantile hypercalcemia caused by mutations of CYP24A1.