Calcifediol, also known as calcidiol, 25-hydroxycholecalciferol, or 25-hydroxyvitamin D3 (abbreviated 25(OH)D3), is a form of vitamin D produced in the liver by hydroxylation of vitamin D3 (cholecalciferol) by the enzyme vitamin D 25-hydroxylase. Calcifediol can be further hydroxylated by the enzyme 25(OH)D-1α-hydroxylase, primarily in the kidney, to form calcitriol (1,25-(OH)2D3), which is the active hormonal form of vitamin D.Calcifediol is strongly bound in blood by the vitamin D-binding protein. Measurement of serum calcifediol is the usual test performed to determine a persons vitamin D status, to show vitamin D deficiency or sufficiency. Calcifediol is available as an oral medication in some countries to supplement vitamin D status. Biology
Calcifediol is the precursor for calcitriol, the active form of vitamin D.  It is synthesized in the liver, by hydroxylation of cholecalciferol (vitamin D3) at the 25-position. This enzymatic 25-hydroxylase reaction is mostly due to the actions of CYP2R1, present in microsomes, although other enzymes such as mitochondrial CYP27A1 can contribute. Variations in the expression and activity of CYP2R1, such as low levels in obesity, affect circulating calcifediol. Similarly, vitamin D2, ergocalciferol, can also be 25-hydroxylated to form 25-hydroxyergocalciferol, (ercalcidiol, 25(OH)D2); both forms are measured together in blood as 25(OH)D.At a typical intake of cholecalciferol (up to 2000 IU/day), conversion to calcifediol is rapid. When large doses are given (100,000 IU), it takes 7 days to reach peak calcifediol concentrations. Calcifediol binds in the blood to vitamin D-binding protein (also known as gc-globulin) and is the main circulating vitamin D metabolite.
A 2021 study showed that mild genotypes associated with LOCAH have a low concordance rate with those phenotypes, probably due to the complex characteristics of 21-hydroxylase genotyping and the limitation of using massive parallel sequencing alone without combining with other comprehensive methods.The following three mutations to the CYP21A2 gene have been found to result in a moderate reduction in enzyme activity associated with that allele (20–60% residual activity), and are associated with LOCAH:
p.V281L (rs6471, c.844G>C, CYP21A2*15);
p.P453S (rs6445, c.1360C>T, CYP21A2*19);
p.P30L (rs9378251, c.92C>T, CYP21A2*8).A point mutation in exon 7 of CYP21A2, (p.V281L), is commonly found in LOCAH-associated alleles. Carriers for this mutation retain 20%–50% of 21-hydroxylase activity, but are at higher risk of symptoms of androgen excess than carriers of the severe mutations and had higher adrenocorticotropic hormone (ACTH) stimulated 17α-hydroxyprogesterone, suggesting heterozygous mutations on CYP21A2 play an important role in disease.The particularly mild clinical symptoms of LOCAH such as hyperandrogenism, hirsutism and acne or infertility overlap with other diseases such as polycystic ovary syndrome. Biochemical parameters like 17α-hydroxyprogesterone may not be elevated in very mild cases of LOCAH, and may vary between labs that makes interpretation difficult. It may not be possible to perform ACTH stimulation tests in all institutions, depending on the availability of the injectable adrenocorticotropic hormone medication. This is why a comprehensive CYP21A2 genotyping (rather than variant-specific assays alone) is a good way to exclude/confirm 21-hydroxylase deficiency and heterozygosity (carrier) status.