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1. ISymposium
The use and Interpretation of Assays
for Vitamin D and its Metabolites1«2
MICHAEL F. HOLICK
Boston University School of Medicine, Vitamin D, Skin,
and Bone Research Laboratory, Boston, MA 02118
to form the major circulating form of vitamin D, 25-
ABSTRACT Vitamin D is essential for the mainte hydroxyvitamin D (25-OH-D) (1-3) (Fig. 1). The bio
nance of calcium and bone metabolism in humans. The logically inert 25-OH-D is then hydroxylated in the
recommended daily allowance (RDA) for vitamin D in kidney on carbon 1 to form its biologically active form,
the United States of 200 lu (5.0 /ig) ¡seasonable for
r commonly known as l«,25-dihydroxyvitamin D
adults who receive some exposure to sunlight; however, (1,25(OH)2D) (1-3) (Fig. 1). Circulating 1,25(OH)2D
in the absence of any exposure to sunlight, this rec
ommendation may be 2 to 3 times lower than that ac is taken up by the small intestine, where it enhances
tually required to satisfy the body's needs. Vitamin D the efficiency of intestinal calcium absorption, and by
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was first measured by bioassays. However, bioassays the bone, where it triggers differentiation of stem cells
became obsolete in light of the revelation that vitamin to osteoclasts, which in turn enhance the mobilization
D must be activated first in the liver to 25-hydroxyvi-
tamin D (25-OH-D) and then in the kidney to 1,25- of calcium (Fig. 1) (1-3). 1,25 dihydroxycholecalciferol
dihydroxyvitamin D [1,25(OH)2D] before becoming also stimulates osteoblasts to make osteocalcin and
biologically functional. Current assays measure circu enhances alkaline phosphatase activity in bone. Be
lating concentrations of vitamin D, 25-OH-D or sides the intestine and bone, nuclear receptors for
1,25(OH)2D. The serum vitamin D concentration is of 1,25(OH)2D have also been found in a variety of other
value for determining the role of sunlight in producing
vitamin D in skin and as a provocative test to determine tissues and cells that are not involved in calcium me
the absorption of vitamin D in patients with malab- tabolism, including the stomach, brain, pituitary
sorption syndromes. The serum concentration of 25- gland, gonads, parathyroid glands, epidermis, dermis,
OH-D is most valuable for determining the overall vi monocytes, and activated T and B lymphocytes (1-6).
tamin D status of an individual, since it is an average Although the exact physiological action of 1,25(OH)2D
of dietary and sunlight-induced vitamin D. The mea
surement of the serum 1,25(OH) ,Dconcentrations has in these tissues is unknown, 1,25(OH)2D has been
been most useful in evaluating disorders in calcium shown to inhibit the proliferation and to induce the
and bone metabolism related to acquired and inborn differentiation of many cell types that possess its re-
errors in the conversion of 25-OH-D to 1,25(OH)2D.
J. Hutr. 120:1464-1469, 1990.
1 Presented as part of a conference, quot;Nutrition Monitoring and
INDEXING KEY WORDS: Nutrition Status Assessment,quot; at the first fall meeting of the Amer
ican Institute of Nutrition, Charleston, South Carolina, December
•
vitamin D • vitamin D metabolites •
assays 8-10, 1989. The conference was supported in part by cooperative
•
sunlight •
25-hydroxyvitamin D agreement with HPU880004-02-1 the DHHS Office of Disease Pre
vention and Health Promotion, the USDA Human Nutrition In
formation Service, the DHHS National Center for Health Statistics,
and the International Life Sciences Institute-Nutrition Foundation.
1 The Planning Committee for the meeting consisted of Drs. He
The intensive research activities in the field of vi len A. Guthrie, Roy J. Martin, Linda D. Meyers, James A. Olson,
tamin D during the past two decades have made us Catherine E. Woteki, and Richard G. Allison (ex-officio). The sym
aware that the quot;sunshine vitaminquot; is more than just posium papers were edited by a committee consisting of Dr. James
a fat-soluble vitamin that regulates calcium metabo Allen Olson (coordinator), Dept. of Biochemistry & Biophysics, Iowa
State University, Ames, IA; Dr. Cathy C. Campbell, Division of
lism. Once vitamin D [the term vitamin D represents
vitamin D2 (ergocalciferol) and/or vitamin D3 (chole- Nutritional Sciences, Cornell University, Ithaca, NY; Dr. Roy J.
Martin, Dept. of Foods 6k.Nutrition, University of Georgia, Athens,
calciferol)] is synthesized in the skin or obtained from GA¡ nd Dr. Catherine E. Woteki, Food & Nutrition Board, National
a
the diet, it is hydroxylated on carbon 25 in the liver Academy of Sciences, Washington, DC.
0022-3166/90 $3.00 ©1990 American Institute of Nutrition. Received 11 February 1990. Accepted 11 July 1990.
1464

2. VITAMIN D ASSAYS 1465
VITAMIN D cutaneous production of previtamin D3 include sea
sonal changes, time of day, skin pigmentation, aging,
and sunscreen use (10-14).
The other major source of vitamin D is from the
Liver
VITAMIN D-25- diet. In the United States and Canada, the ingestion
hydroxylase
of fortified milk, which contains 400 IU (10 ¿ig) of
either ergocalciferol or cholecalciferol per quart, pro
vides twice the United States recommended daily al
PO.ond
lowance (USRDA) of 200 IU for this essential fat-sol
OTHER FACTORS uble substance. The only other major dietary source
of vitamin D is from fatty fish and fish liver oils.
Vitamin D concentrations were first measured by
rat and chick bioassays (15-17). The rat bioassay,
1,25-(OH),-D commonly known as the line test, was widely used to
determine the concentration of vitamin D in fortified
foods (15). The development of specific assays for vi
tamin D and its biologically important metabolites
made these bioassays obsolete.
The half-life of circulating vitamin D is only 24 h
(18). Thus, the serum concentration at any time is de
pendent on times of the most recent ingestion of vi
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tamin D as well as of the last exposure to sunlight (18,
19). The quot;normalquot; range of serum vitamin D is 0-310
nmol/L (0-120 ng/mL) (18-20). Consequently serum
ergocalciferol or cholecalciferol is of little value in de
termining the vitamin D status of a patient. Nonethe
less, serum vitamin D assays are useful for determining
the capacity of human skin to produce vitamin D in
response to exposure to solar or simulated solar ultra
violet radiation (18). Furthermore, the measurement
FIGURE I Schematic representation of the hormonal of circulating concentrations of vitamin D 12-24 h
control loop for vitamin D metabolism and function. A re after an oral dose of 50,000 IU (1.25 mg) of ergocal
duction in the serum calcium below ~8.8 mg/mL prompts
a proportional increase in the secretion of parathyroid hor
ciferol provides valuable clinical information for de
mone, which enhances the mobilization of calcium stores termining whether a patient with a malabsorption
from bone. Parathyroid hormone also promotes the synthesis syndrome can absorb vitamin D (20) (Fig. 3).
of 1,25(OH)2D in the kidney, which, in turn, stimulates the Plasma and serum concentrations of vitamin D are
mobilization of calcium from the bone and its absorption determined by: 1) extracting the lipid-soluble vitamin
from the intestine. Reproduced with permission (38). D from 1 to 2 mL of serum or plasma, 2) separating
vitamin D from its metabolites and lipid contaminants
in the extract by rapid reverse-phase cartridge chro-
ceptor (1-3, 7). These insights have been of great value matography, 3) further resolving the vitamin D frac
for the pharmacologie use of 1,25(OH)2D3 in the hy- tion by straight phase high-performance liquid chro-
perproliferative skin disorder psoriasis (7-9). matography (HPLC), and 4) quantitating the amount
of vitamin D by its ultraviolet absorbance in the HPLC
eluate or by a competitive protein binding assay (18,
21-23). The intra-assay and interassay variations are
PHOTOSYNTHESIS, ABSORPTION, ~10 and 12%, respectively.
AND ASSAYS FOR VITAMIN D
Sunlight, which contains wavelengths between 290
and 315 nm, photolyzes provitamin D3 (7-dehydro- DETERMINATION AND CLINICAL UTILITY
cholesterol) in the skin to previtamin D3 (Fig. 2). Pre OF THE CIRCULATING CONCENTRATION
vitamin D3, a thermally labile compound, undergoes OF 25-HYDROXYVITAMIN D
an internal isomerization of its double bonds to form
the thermodynamically stable vitamin D3 (2), which The half-life of circulating 25-OH-D is ~3 wk (2).
is then translocated from the epidermis into the dermal Therefore, its steady-state concentration summates the
capillary bed (Fig. 2). Factors that can influence the concentrations of vitamin D derived both from the

3. 1466 HOLICK
SUN
SUN
7- DEHYDROCHOLESTEROL
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BLOOD
DBP-D3 DBP
FIGURE 2 Schematic representation of the formation of previtamin D3 in the skin during exposure to the sun, the thermal
isomerization of previtamin D3 to vitamin D3/ and the specific translocation of vitamin D3 by the vitamin D-binding protein
(DBP) into the circulation. During continual exposure to the sun, previtamin D3 also photoisomerizes reversibly to Iumisterol3
and tachysterol3, which are biologically inert photoproducts (i.e., they do not stimulate intestinal calcium absorption). Because
the DBP has no affinity for Iumisterol3 and has minimal affinity for tachysterol3/ the translocation of these photoisomers into
the circulation is negligible. Subsequently, these photoproducts are sloughed off during the natural turnover of the skin.
When previtamin D3 stores are depleted (due to its thermal isomerization to D3), however, lumisterol and tachysterol, upon
exposure to UV radiation, will photoisomerize to preD3. Reproduced with permission of the American Association for the
Advancement of Science (39).
diet and from photo-formation over a several weeks is considered to be normal. Vitamin D intoxication is
to several months. The separate measurement of 25- usually associated with 25-OH-D concentrations
OH-cholecalciferol and 25-OH-ergocalciferol was above 375 nmol/L (150 ng/mL), with attendant hy-
originally thought to provide information about sun percalcemia and hyperphosphatemia (2).
light-induced vs. dietary sources of vitamin D (24). Serum concentrations of 25-OH-D are measured in
However, since milk and multivitamin preparations several ways (25-30). A competitive protein-binding
are now fortified with both forms of the vitamin, the assay is often used. The vitamin D-binding protein,
separate measurement of these metabolites is of little which has a very high affinity for 25-OH-D (26-28),
value. binds the ligand in a lipid extract of serum or plasma
The 25-OH-D assay is most valuable for determin (0.1 mL). However, other vitamin D metabolites can
ing the vitamin D status of an individual. For its assay, interfere with this assay, even though they usually
commercial diagnostic laboratory services and assay represent <10% of the total binding activity (29). To
kits are available. The normal circulating concentra increase the specificity, 25-OH-D can first be separated
tion of 25-OH-D is usually reported to be between 20 from vitamin D and its metabolites by a rapid straight-
nmol/L (8 ng/mL) and 150 nmol/L (60 ng/mL). Serum phase silica cartridge chromatography (30), followed
values below 25 nmol/L (10 ng/mL) are generally con by the competitive protein-binding assay.
sidered to indicate impending or frank vitamin D de Circulating concentrations of 25-OH-ergocalciferol
ficiency. Although most diagnostic laboratories report and 25-OH-cholecalciferol can be accurately measured
the upper limit of the normal range for 25-OH-D to by first chromatographing a lipid extract from 1 mL
be 150 nmol/L (60 ng/mL), circulating concentrations of serum or plasma on a rapid straight-phase or reverse-
of 250 nmol/L (100 ng/mL) in lifeguards after a full phase cartridge followed by straight-phase HPLC. The
summer of exposure to sunlight is not uncommon and amounts of 25-OH-ergocalciferol and 25-OH-chole-

4. VITAMIN D ASSAYS 1467
80 n 1,25(OH)2D receptor recognizes l,25(OH)2cholecal-
ciferol better than l,25(OH)2ergocalciferol (2, 29). As
a result, this assay underestimated the total circulating
concentration of 1,25(OH)2D. This assay has been im
proved by using the bovine thymus 1,25(OH)2D re
ceptor (29, 33), which reacts equally well with
l,25(OH)2ergocalciferol and l,25(OH)2cholecalciferol.
In addition, the HPLC step has now been replaced by
a rapid silica cartridge Chromatographie step (34, 35).
A bioassay using cultured rat calvarÃ-a an detect pi-
c
cogram quantities of 1,25(OH)2D in the circulation
(36). However, this assay is very time consuming and
requires a tissue culture facility. It is most useful in
verifying the results from a competitive receptor
binding assay.
The half-life of circulating 1,25(OH)2D has been es
24 48 72
timated to be between 4 and 6 h (37). The normal range
HOURS of serum values is between 38 and 144 pmol/L (16-
FIGURE 3 Serum vitamin D concentrations in seven pa 60 pg/mL). As vitamin D deficiency develops, the body
tients with diarrheal syndromes after a single oral dose of responds by increasing the production and secretion
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50,000 IU (1.25 mg) of ergocalciferol. For comparison, the of parathyroid hormone (Fig. 1). Parathyroid hormone
means and standard errors of vitamin D concentrations in turn enhances the 1-hydroxylation of 25-OH-D (1-
measured in seven normal control subjects after a similar 3) (Fig. 1). Thus, secondary hyperparathyroidism ac
dose are indicated by the closed circles and dotted lines celerates the conversion of 25-OH-D to 1,25(OH)2D
(— •€”). Note that two patients, one with Crohn's ileo-
â
colitis (patient F) and one with ulcerative colitis (patient G),
(1-3). Since the circulating concentration of 25-OH-
had essentially normal absorption curves. Five patients, D is about three orders of magnitude higher than
however, showed a dramatic lack of response, with no values 1,25(OH)2D, even very low levels of 25-OH-D can
above 10 ng/mL. Reproduced with permission (20). provide enough substrate for the formation of some
1,25(OH)2D. In vitamin D deficiency, vitamin D is
also efficiently converted to 25-OH-D (18). As a result,
calciferol in the eluate are quantitatively determined a hospital patient with vitamin D deficiency who has
by their UV absorption at either 254 or 265 nm (21, previously obtained a very small quantity of vitamin
25, 29, 30). D from food or exposure to sun can have low or un-
The assay for serum 25-OH-D has clinical utility detectable circulating concentrations of 25-OH-D
in determining the vitamin D status of patients with while having low, normal, or even high circulating
intestinal malabsorption syndromes or with severe concentrations of 1,25(OH)2D (1, 2, 18, 38). Thus
hepatic failure, as well as of the very young and the serum 1,25(OH)2D concentrations are of little value
elderly (2), who may be at risk of vitamin D deficiency. in the evaluation of vitamin D deficiency. Needless to
say, in an absolute vitamin D deficiency state, circu
lating concentrations of 1,25(OH)2D are undetectable.
DETERMINATION AND CLINICAL UTILITY The measurement of circulating concentrations of
OF THE CIRCULATING CONCENTRATION 1,25(OH)2D have been of great value to clinicians for
OF 1,25-DIHYDROXYVITAMIN D the evaluation of patients with acquired and inherited
disorders of 1,25(OH)2D metabolism (1-3, 29, 38).
Specific assays for 1,25(OH)2D (29, 31-33) in serum Serum 1,25(OH)2D levels are routinely assayed by
and plasma are based on the separation of minute commercial diagnostic laboratories and can be mea
quantities of 1,25(OH)2D from lipid contaminants and sured as well by assay kits. Patients with chronic
other vitamin D metabolites. The concentration of renal failure, hyperphosphatemia, hypoparathyroid-
l,25(OH)2cholecalciferol is then determined by a ism, pseudohypoparathyroidism, tumor-induced os
competitive receptor binding assay using a nuclear/ teomalacia, hypercalcemia of malignancy (in most
cytosolic receptor for 1,25(OH)2D (31-33). Initially cases), or vitamin D-dependent rickets type I [an in
1,25(OH)2D in a lipid extract of 2 to 3 mL of serum born error that markedly reduces the conversion of
or plasma was separated from other vitamin D metab 25-OH-D to 1,25(OH)2D] often have low circulating
olites by straight phase HPLC. The 1,25(OH)2D frac concentrations of 1,25(OH)2D (2, 29, 38). Serum con
tion was then subjected to a competitive protein-bind centrations of 1,25(OH)2D are elevated above the nor
ing assay that used the chick cytosolic 1,25(OH)2D mal range in patients with primary hyperparathyroid
receptor (31, 32). However, the chick intestinal ism, vitamin D-dependent rickets type II; [an inborn

5. 1468 HOLICK
error in which the recognition of 1,25(OH)2D by target 11. WEBB, A. R., KLINE, L. & HOLICK, M. F. (1988) Influence of
tissue receptors is defective]; chronic granulomatous season and latitude on the cutaneous synthesis of vitamin D3 :
exposure to winter sunlight in Boston and Edmonton will not
disorders such as sarcoidosis, tuberculosis, and sili promote vitamin D3 synthesis in human skin. /. Clin. Endocrino!.
cosis; and lymphoma (in some patients) (2, 29, 38). Metab. 67: 373-378.
12. CLEMENS, . L., HENDERSON, . L., ADAMS,J. S. &.HOLICK,M. F.
T S
(1982) Increased skin pigment reduces the capacity of skin to
synthesize vitamin D3. Lancet 74-76.
CONCLUSION 13. HOLICK,M. F., MATSUOKA,L. Y. & WORTSMAN,J. (1989) Age,
vitamin D, and solar ultraviolet radiation. Lancet ii: 1104-1105.
14. MATSUOKA,L. Y., IDE, L., WORTSMAN,J., MACL.AUGHLIN, . & J
Vitamin D is essential for the maintenance of cal HOLICK, M. F. (1987) Sunscreens suppress cutaneous vitamin
cium and bone metabolism throughout our lives. The D3 synthesis. /. Clin. Endocrino!. Metab. 64: 1165-1168.
major source of vitamin D is casual exposure to sun 15. STEENBOCK, & BLACK,A. (1924) The reduction of growth-
H.
light. In the absence of exposure to sunlight, the av promoting and calcifying properties in a ration by exposure to
erage daily requirement for vitamin D is probably 2 ultraviolet light. /. Bio!. Chem. 61: 408-422.
16. STEENBOCK, . & KLETZIEN,S. W. F. (1932) The reaction of
H
to 3 times higher than the USRDA of 200 IU (5 fig)
chickens to irradiated ergosterol and irradiated yeast as con
(Paris, P., Bondi, K., Luria, S. and Holick, M. F., un trasted with the natural vitamin D in fish liver oil. /. Bio!. Chem.
published results). Among assays for vitamin D and 97: 249-264.
its metabolites, the assay of serum 25-OH-D levels 17. WADDELL,J. (1934) The provitamin D of cholesterol. I. The
has the most utility for determining the vitamin D antirachitic efficacy of irradiated cholesterol. /. Bio!. Chem. 105:
711-739.
status of an individual. Circulating concentrations of
18. CLEMENS, . L., ADAMS,J. S. & HOLICK,M. F. (1982) Measure
T
vitamin D and of 1,25(OH)2D, however, can be of
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ment of circulating vitamin D in man. Clin. Chim. Acta 121:
value for clinicians who are evaluating malabsorption 301-308.
syndromes and acquired and inherited disorders of 25- 19. ADAMS,J. A., CLEMENS,T. L., PARRISH,J. A. & HOLICK, M. F.
OH-D metabolism, respectively (1-3, 29, 38). (1981) Vitamin D synthesis and metabolism after ultraviolet
radiation of normal and vitamin D deficient subjects. N. Eng!.
/. Med. 306: 722-725.
20. Lo, C. W., PARIS,P. W., CLEMENS, . L., NOLAN, J. &. HOLICK,
T
M. F. (1985) Vitamin D absorption in healthy subjects and in
LITERATURE CITED
patients with intestinal malabsorption syndromes. Am. J. Clin.
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P
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H.
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24. HADDAD, H. G. & HAHN, T. J. (1973) Natural and synthetic
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515-517.
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25. CHEN, T., TURNER, A. & HOLICK,M. F. (1990) Method for de
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6. BHALLA,A. K., CLEMENS,T., AMENTO, E., HOLICK, M. F. &. 26. HADDAD,J. G. & CHUY,K. J. (1971 ) Competitive protein binding
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cells: presence in monocytes and induction in T lymphocytes 27. BELSEY, ., CLARK,M. B. BERNAT,M., GLOWACKI,J., HOLICK,
R
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Exper.Med. 19:246-257. assay for 25-hydroxycalciferol and 24R,25-dihydroxycalciferols
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T
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6. VITAMIN D ASSAYS 1469
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