Abstract
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(3) (1,25-(OH)(2)D(3)). Catabolism of 25-hydroxyvitamin D(3) (25-OH-D(3)) 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(3) (24,25-(OH)(2)D(3)) is an important factor in optimal bone fracture healing acting via an effector molecule FAM57B2 to produce lactosylceramide. Administration of 24,25-(OH)(2)D(3) was found to restore normal fracture repair in Cyp24a1(-/-) mice devoid of 24,25-(OH)(2)D(3). We set out to study the multi-step catabolism of D(3) metabolites in vivo using LC-MS/MS methods in vehicle or 24,25-(OH)(2)D(3)-treated mice. Vehicle-treated Cyp24a1(+/-) mice possessed normal levels of serum 24,25-(OH)(2)D(3) (7 ng/mL) and 25-OH-D(3)-26,23-lactone (4 ng/mL). We also detected 24-oxo-25-OH-D(3) (3 ng/mL) and 24-oxo-23,25-(OH)(2)D(3) (0.4 ng/mL); which were not detectable in vehicle-treated Cyp24a1(-/-) mice. In 24,25-(OH)(2)D(3)-treated Cyp24a1(+/-) mice, serum 24,25-(OH)(2)D(3) rose to 200 ng/mL while 25-OH-D(3)-26,23-lactone remained unchanged in comparison to vehicle-treated Cyp24a1(+/-) mice Concentration of serum 24-oxo-25-OH-D(3) and 24-oxo-23,25-(OH)(2)D(3) rose by 10-fold, when Cyp24a1(+/-) mice were treated with 24,25-(OH)(2)D(3) Calcioic acid was increased to 0.030 ng/mL for 24,25-(OH)(2)D(3)-treated Cyp24a1(+/-) mice. In 24,25-(OH)(2)D(3)-treated Cyp24a1(-/-) mice, serum 24,25-(OH)(2)D(3) rose further to a striking 830 ng/mL due to lack of catabolism of the 24,25-(OH)(2)D(3) dose. Serum 1,25-(OH)(2)D(3) levels were suppressed in 24,25-(OH)(2)D(3)-treated Cyp24a1(+/-) and Cyp24a1(-/-) mice. Circulating 1,24,25-(OH)(3)D(3) rose from 73 pg/mL to 106 pg/mL when Cyp24a1(+/-) mice were treated with 24,25-(OH)(2)D(3). While undetectable in vehicle-treated Cyp24a1(-/-) mice, 1,24,25-(OH)(3)D(3) rose unexpectedly to 153 pg/mL in 24,25-(OH)(2)D(3)-treated nulls suggesting conversion of 24,25-(OH)(2)D(3) to 1,24,25-(OH)(3)D(3) via 1-hydroxylation. Taken together, amplification of 24,25-(OH)(2)D(3) 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.