Vitamin D3 25-Hydroxyvitamin D
Updated: Nov 27, 2024 Author: Ha Cam Thuy Nguyen, MD; Chief Editor: Sridevi Devaraj, PhD,
DABCC, FAACC, FRSC, CCRP
Reference Range
The major circulating form of vitamin D is 25-hydroxyvitamin D (25(OH)D); thus, the total serum
25(OH)D level is currently considered the best indicator of vitamin D supply to the body from
cutaneous synthesis and nutritional intake.
The reference range of the total 25(OH)D level is 25-80 ng/mL. [1]
Interpretation
There are two principal forms of vitamin D: D2 and D3. Many of the currently available assays measure
and report on both vitamin D2 and D3 metabolites. This can be useful in studies evaluating the
contribution of vitamin D2 and D3 to overall vitamin D status. 25-hydroxyvitamin D (25(OH)D) is the
major circulating form of vitamin D; thus, the total serum 25(OH)D level is currently considered the
best indicator of vitamin D supply to the body from cutaneous synthesis and nutritional intake.
One exception is that 25(OH)D levels do not indicate clinical vitamin D status in patients with chronic
renal failure or type 1 vitamin D-dependent rickets or when calcitriol (1,25-dihydroxyvitamin D) is used
as a supplement.
Interpretation of 25(OH)D can be challenging owing to wide variability in patient’s weight, ethnicity,
assays, laboratory procedures and validation of reference ranges. [2, 3, 4]
Vitamin D deciency
Vitamin D deficiency is defined by most experts as a serum 25(OH)D level of less than 20 ng/mL (50
nmol/L).
Vitamin D insufficiency
Vitamin D insufficiency has been defined as a serum 25(OH)D level of 21-29 ng/mL (52-72 nmol/L).
This is based on the observed physiological changes in calcium absorption and parathyroid hormone
levels that occur with changes in vitamin D levels.
In healthy postmenopausal women with serum 25(OH)D levels averaging 34.4 ng/mL (86.5 nmol/L),
intestinal calcium absorption increased from 45% to 65% compared to those with mean 25(OH)D
, levels of 20 ng/mL (50.1 nmol/L). [5]
Chapuy et al reported an inverse correlation between serum intact parathyroid hormone (iPTH) and
serum 25(OH)D. Serum iPTH held a stable plateau level when serum 25(OH)D values exceeded 31
ng/mL (78 nmol/L) but started to rise when the serum 25(OH)D values fell below this level. [6]
Vitamin D sufficiency
Vitamin D sufficiency has been defined as serum 25(OH)D levels of 30 ng/mL (75 nmol/L) and above
based on analysis of observational studies of vitamin D and various health outcomes.
Bischoff-Ferrari et al reviewed evidence from studies that evaluated the thresholds of serum 25(OH)D
levels and their relation to bone mineral density (BMD), lower-extremity function, dental health, and the
risk of falls, fractures, and colorectal cancer. Their conclusion based on all endpoints was that
25(OH)D levels of 30 ng/mL (75 nmol/L) or greater are more effective in achieving positive outcomes
than lower levels. The best outcomes were seen with 25(OH)D levels from 36-40 ng/mL (90-100
nmol/L). [7]
Vitamin D toxicity
Vitamin D toxicity is observed when serum 25(OH)D levels are greater than 150 ng/mL (374 nmol/L).
Assays for serum 25-hydroxyvitamin D
Currently, there are several assays for measuring serum 25(OH)D. Each of them has its strengths and
weaknesses.
In the early 1970s, Belsey et al and Haddad et al developed a competitive binding assay to measure
serum vitamin D levels. This assay used vitamin D–binding protein (DBP) obtained from rat serum as
the binding agent and exploited the capacity of vitamin D in test samples to displace bound [3
H]25(OH)D from DBP. [8, 9]
3 This assay was limited by issues related to the required extraction and
purification processes; as a result, this method has been replaced by others as indicated below.
Antibody-based assays have been available since the early 1980s. [10] Since then, various antibody-
based methods have been in use, including radioimmunoassay (RIA), enzyme-linked immunosorbent
assay (ELISA), and chemiluminescent assay. [11] In general, these assays depend on an antibody that
recognizes 25(OH)D2 and 25(OH)D3 in equimolar proportions and the competitive displacement of
tracer 25(OH)D (25(OH)D that has been tagged with a radioactive isotope such as 125-I or a
chemiluminescent ligand) by acetonitrile extracted samples. The acetonitrile extraction eliminates the
need for chromatographic purification of vitamin D prior to its actual assay. [12, 13] The advantages
include good precision and ease of use.
The first liquid-chromatography (LC)–based method was presented in 1977. [14] It was based on the
distinctive ultraviolet (UV) spectral absorption peak of vitamin D metabolites at 265 nm. Via UV
detection, this assay allowed direct quantization of not only total 25(OH)D but also 25(OH)D2 and
25(OH)D3 separately. Over the years, this LC-based method has developed into the latest LC-
chromatography–tandem mass spectrometry (LC-MS/MS), which is currently considered the criterion
standard of vitamin D assays. In this method, a mass spectrometer serves as the metabolite detector,
instead of UV light. It measures 25(OH)D2 and 25(OH)D3 with much higher sensitivity and specificity
than the traditional LC methods. [15]
Updated: Nov 27, 2024 Author: Ha Cam Thuy Nguyen, MD; Chief Editor: Sridevi Devaraj, PhD,
DABCC, FAACC, FRSC, CCRP
Reference Range
The major circulating form of vitamin D is 25-hydroxyvitamin D (25(OH)D); thus, the total serum
25(OH)D level is currently considered the best indicator of vitamin D supply to the body from
cutaneous synthesis and nutritional intake.
The reference range of the total 25(OH)D level is 25-80 ng/mL. [1]
Interpretation
There are two principal forms of vitamin D: D2 and D3. Many of the currently available assays measure
and report on both vitamin D2 and D3 metabolites. This can be useful in studies evaluating the
contribution of vitamin D2 and D3 to overall vitamin D status. 25-hydroxyvitamin D (25(OH)D) is the
major circulating form of vitamin D; thus, the total serum 25(OH)D level is currently considered the
best indicator of vitamin D supply to the body from cutaneous synthesis and nutritional intake.
One exception is that 25(OH)D levels do not indicate clinical vitamin D status in patients with chronic
renal failure or type 1 vitamin D-dependent rickets or when calcitriol (1,25-dihydroxyvitamin D) is used
as a supplement.
Interpretation of 25(OH)D can be challenging owing to wide variability in patient’s weight, ethnicity,
assays, laboratory procedures and validation of reference ranges. [2, 3, 4]
Vitamin D deciency
Vitamin D deficiency is defined by most experts as a serum 25(OH)D level of less than 20 ng/mL (50
nmol/L).
Vitamin D insufficiency
Vitamin D insufficiency has been defined as a serum 25(OH)D level of 21-29 ng/mL (52-72 nmol/L).
This is based on the observed physiological changes in calcium absorption and parathyroid hormone
levels that occur with changes in vitamin D levels.
In healthy postmenopausal women with serum 25(OH)D levels averaging 34.4 ng/mL (86.5 nmol/L),
intestinal calcium absorption increased from 45% to 65% compared to those with mean 25(OH)D
, levels of 20 ng/mL (50.1 nmol/L). [5]
Chapuy et al reported an inverse correlation between serum intact parathyroid hormone (iPTH) and
serum 25(OH)D. Serum iPTH held a stable plateau level when serum 25(OH)D values exceeded 31
ng/mL (78 nmol/L) but started to rise when the serum 25(OH)D values fell below this level. [6]
Vitamin D sufficiency
Vitamin D sufficiency has been defined as serum 25(OH)D levels of 30 ng/mL (75 nmol/L) and above
based on analysis of observational studies of vitamin D and various health outcomes.
Bischoff-Ferrari et al reviewed evidence from studies that evaluated the thresholds of serum 25(OH)D
levels and their relation to bone mineral density (BMD), lower-extremity function, dental health, and the
risk of falls, fractures, and colorectal cancer. Their conclusion based on all endpoints was that
25(OH)D levels of 30 ng/mL (75 nmol/L) or greater are more effective in achieving positive outcomes
than lower levels. The best outcomes were seen with 25(OH)D levels from 36-40 ng/mL (90-100
nmol/L). [7]
Vitamin D toxicity
Vitamin D toxicity is observed when serum 25(OH)D levels are greater than 150 ng/mL (374 nmol/L).
Assays for serum 25-hydroxyvitamin D
Currently, there are several assays for measuring serum 25(OH)D. Each of them has its strengths and
weaknesses.
In the early 1970s, Belsey et al and Haddad et al developed a competitive binding assay to measure
serum vitamin D levels. This assay used vitamin D–binding protein (DBP) obtained from rat serum as
the binding agent and exploited the capacity of vitamin D in test samples to displace bound [3
H]25(OH)D from DBP. [8, 9]
3 This assay was limited by issues related to the required extraction and
purification processes; as a result, this method has been replaced by others as indicated below.
Antibody-based assays have been available since the early 1980s. [10] Since then, various antibody-
based methods have been in use, including radioimmunoassay (RIA), enzyme-linked immunosorbent
assay (ELISA), and chemiluminescent assay. [11] In general, these assays depend on an antibody that
recognizes 25(OH)D2 and 25(OH)D3 in equimolar proportions and the competitive displacement of
tracer 25(OH)D (25(OH)D that has been tagged with a radioactive isotope such as 125-I or a
chemiluminescent ligand) by acetonitrile extracted samples. The acetonitrile extraction eliminates the
need for chromatographic purification of vitamin D prior to its actual assay. [12, 13] The advantages
include good precision and ease of use.
The first liquid-chromatography (LC)–based method was presented in 1977. [14] It was based on the
distinctive ultraviolet (UV) spectral absorption peak of vitamin D metabolites at 265 nm. Via UV
detection, this assay allowed direct quantization of not only total 25(OH)D but also 25(OH)D2 and
25(OH)D3 separately. Over the years, this LC-based method has developed into the latest LC-
chromatography–tandem mass spectrometry (LC-MS/MS), which is currently considered the criterion
standard of vitamin D assays. In this method, a mass spectrometer serves as the metabolite detector,
instead of UV light. It measures 25(OH)D2 and 25(OH)D3 with much higher sensitivity and specificity
than the traditional LC methods. [15]
