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

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 D (1,25-(OH)D). Catabolism of 25-hydroxyvitamin D (25-OH-D) 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 D (24,25-(OH)D) is an important factor in optimal bone fracture healing acting via an effector molecule FAM57B2 to produce lactosylceramide. Administration of 24,25-(OH)D was found to restore normal fracture repair in Cyp24a1 mice devoid of 24,25-(OH)D. We set out to study the multi-step catabolism of D metabolites in vivo using LC-MS/MS methods in vehicle or 24,25-(OH)D-treated mice. Vehicle-treated Cyp24a1 mice possessed normal levels of serum 24,25-(OH)D (7 ng/mL) and 25-OH-D-26,23-lactone (4 ng/mL). We also detected 24-oxo-25-OH-D (3 ng/mL) and 24-oxo-23,25-(OH)D (0.4 ng/mL); which were not detectable in vehicle-treated Cyp24a1 mice. In 24,25-(OH)D-treated Cyp24a1 mice, serum 24,25-(OH)D rose to 200 ng/mL while 25-OH-D-26,23-lactone remained unchanged in comparison to vehicle-treated Cyp24a1 mice Concentration of serum 24-oxo-25-OH-D and 24-oxo-23,25-(OH)D rose by 10-fold, when Cyp24a1 mice were treated with 24,25-(OH)D Calcioic acid was increased to 0.030 ng/mL for 24,25-(OH)D-treated Cyp24a1 mice. In 24,25-(OH)D-treated Cyp24a1 mice, serum 24,25-(OH)D rose further to a striking 830 ng/mL due to lack of catabolism of the 24,25-(OH)D dose. Serum 1,25-(OH)D levels were suppressed in 24,25-(OH)D-treated Cyp24a1 and Cyp24a1 mice. Circulating 1,24,25-(OH)D rose from 73 pg/mL to 106 pg/mL when Cyp24a1 mice were treated with 24,25-(OH)D. While undetectable in vehicle-treated Cyp24a1 mice, 1,24,25-(OH)D rose unexpectedly to 153 pg/mL in 24,25-(OH)D-treated nulls suggesting conversion of 24,25-(OH)D to 1,24,25-(OH)D via 1-hydroxylation. Taken together, amplification of 24,25-(OH)D 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.