Chemistry and Sources

There are two forms of vitamin D, vitamin  and vitamin . Vitamin  (cholecalciferol) occurs in some foods but is primarily produced in the skin on exposure to sunlight. Vitamin  (ergocalciferol) does not occur naturally but is manufactured by the ultraviolet irridation of ergosterol, which occurs in molds, yeasts, and plants. Vitamin  differs from vitamin  in that  has a double bond between carbon 22 and carbon 23 and a methyl group on carbon 24 (Figure 1).

Only a few foods (cod liver oil, fatty fish, and egg yolks) contain substantial amounts of naturally occurring vitamin . Unless one has a diet rich in these foods, the amount of vitamin D obtained from food sources and skin synthesis often is insufficient. For this reason food is supplemented with vitamin D in most developed countries. In North America, food is supplemented with both vitamin  and vitamin , with milk being the principal fortified dietary component.

Transport and Metabolism

Once either vitamin  or  enters the circulation, whether ingested or made cutaneously, it is bound to vitamin D-binding protein and transported to the liver where it is hydroxylated on carbon position 25 to produce 25-hydroxyvitamin D (25(OH) vitamin D). 25(OH) vitamin D is released into the circulation once again before reaching the kidney. In the kidney, 25(OH) vitamin D undergoes a second hydroxylation at the 1  position to produce 1, 25-dihydroxyvitamin D (1, 25  vitamin D) (Figure 2). (1)

Figure 1: Structure of vitamin (cholecalciferol) and vitamin (ergocalciferol) and their precursors. 7-Cholecalciferol is produced in the skin from 7-dehydrocholesterol on exposure to sunlight. Ergocalciferol is produced commercially by irradiation of ergosterol. (Adapted from Endres, D.B. and Rude, R.K.: Mineral and Bone Metabolism. In: Tietz. Textbook of Clinical Chemistry and Molecular Diagnotics. 4th ed. Elsevier Saunders. St. Louis, Missouri. 2005: p. 1920. reproduced with permission of the publisher.)

Figure 2: Metabolism of Vitamin D.Vitamin and Vitamin  are enzymatically hydroxylated to 25-hydroxyvitamin D in the liver and 1,25 di-hydroxyvitamin D by the kidneys. 1,25 di-hydroxyvitamin  and 1,25 di-hydroxyvitamin  are the biologically active forms of vitamin D. (Adapted from Endres, D.B. and Rude, R.K.: Mineral and Bone Metabolism. In: Tietz. Textbook of Clinical Chemistry and Molecular Diagnotics. 4th ed. Elsevier Saunders. St. Louis, Missouri. 2005: p. 1921. reproduced with permission of the publisher.)

Biological Actions

25(OH) vitamin D is the major circulating form of vitamin D, but, at physiological concentrations, it is biologically inactive. Although biologically inactive, 25(OH) vitamin D has a longer half-life and is the preferred analyte to measure a patient’s vitamin D status. (2)

The active form of vitamin D is 1, 25 (OH) 2 vitamin D. (3) It is a hormone that responds to decreased serum calcium levels by stimulating the absorption of calcium and phosphate in the intestine and by enhancing parathyroid hormone-induced bone resorption. Elevations in serum calcium produce the opposite effect, that is, decreased intestinal calcium and phosphate absorption and decreased PTH-induced bone resorption.

Clinical Significance

A large number of disorders of bone and mineral metabolism have been associated with abnormal levels of vitamin D and its metabolites (Tables 1 and 2). (4) Although there are many disorders associated with abnormal plasma concentrations of 25 (OH) vitamin D and 1, 25  vitamin D, the most common disorder is vitamin D deficiency.

Table 1: Abnormal Circulating Levels of 25(OH) Vitamin D

Table 2: Abnormal Circulating Levels of 1, 25  Vitamin D

Prolonged vitamin D deficiency leads to osteomalacia, a condition characterized by insufficient mineralization of the newly formed bone. Defective mineralization in childhood leads to rickets, and if untreated, produces severe deformities of bone. Osteomalacia in adults presents more subtly. Classic symptoms of osteomalacia in adults include bone pain, muscle weakness, with skeletal tenderness, and radiographic evidence of generalized thinning of cortical and trabecular bone (demineralization) with poorly healed stress fractures. Biochemically, patients have characteristic increases in PTH, low urinary calcium excretion, and increased indices of bone turnover including alkaline phosphatase and collagen crosslinks, and a deficiency of 25 (OH) vitamin D. (5,6)

Vitamin D deficiency is now recognized as common in elderly people, and the incidence may vary from 5-25% among independent elderly to 60-80% among institutionalized elderly depending on latitude, nutrition, sun exposure, supplementation, use of sunscreens, skin pigmentation, and clinical definition. (7) Opinions vary widely on the definition of vitamin D deficiency and adequate vitamin D status. (7,8,9,10,11,12,13)

A further discussion of the definition of vitamin D status is given below in the reference interval section.

Vitamin D toxicity, though less common than vitamin D deficiency, does occur. It can result from excessive ingestion of vitamin D, vitamin D analogues, or excessive use of topical calcipotriene (Dovonex) for psoriasis. Vitamin D intoxication can be recognized by hypercalcemia, hyperphosphatemia, and increased levels of 25 (OH) vitamin D.

An endogenous form of vitamin D intoxication, due to extrarenal conversion of 25 (OH) vitamin D to 1, 25  vitamin D, occurs in sarcoidosis (14), cat-scratch disease (15), and certain lymphomas (16). These patients have increased levels of 1, 25  vitamin D. This endogenous form of vitamin D intoxication is, in many reports, the third most common cause of hypercalcemia, following primary hyperparathyroidism and malignancy.

Reference Values

The reference range for 25 (OH) vitamin D is 10-52 ng/ml. This is based on the 95th percentile of apparently healthy adults in the United States. However, many clinicians have observed subclinical hypovitaminosis D in their patients in the low “normal” range. As a result, several studies have been conducted to determine adequate vitamin D status. In these studies, adequate vitamin D status was determined by comparing parathyroid hormone (PTH) levels before and after a period of vitamin D and calcium supplementation. Those individuals whose PTH levels did not significantly change after vitamin D supplementation were considered to have adequate vitamin D status; while individuals whose PTH levels were initially elevated and had their PTH levels decline after vitamin D supplementation were considered vitamin D insufficient. From these studies, the lower limit for adequate vitamin D status was estimated to be 20 or 30 ng/ml of 25 (OH) D. (7,8,13)

However, these studies, although highly suggestive, were based on physiological evidence, and did not prove a connection with morbidity. The publication in 2003 of a large British intervention study provided a crucial piece of needed evidence. With a placebo-controlled, randomized design, Trivedi at al (17) administered 100,000 IU of vitamin D every four months, for five years, to 2686 healthy British participants 65-85 years of age. Serum 25(OH) Vitamin D concentrations were measured and were 21 ng/ml for the placebo group and 30 ng/ml for the Vitamin D treated group. Among the treated group, the risk of all fractures was reduced by 22% and typical osteoporatic fractures were reduced by 33%.

The Vitamin D Dilemma

Based on the above, many clinicians have begun measuring 25(OH) Vitamin D levels in their patients and have initiated therapy when the 25(OH) Vitamin D levels are less than 30 ng/ml. The dilemma lies in the fact that, using this approach, nearly half of the world’s population would require treatment for hypovitaminosis D. For example, in a study by Gordon et al (18), at a lower limit of 20 ng/ml, 42% of healthy adolescents in Boston were vitamin D deficient or insufficient. Using a lower limit of 30 ng/ml, MacFarlane et al (19), found that 75% of healthy adults in Belgium would be similarly classified, and Rucker et al (20) found that 97% of healthy adults in Calgary, Alberta, would again, be classified as vitamin D deficient or insufficient.

To date, there is no definitive classification of 25(OH) Vitamin D levels and Vitamin D status. Based on the available literature, we suggest the following classification (Table 3).

Table 3: Suggested Classification of Vitamin D Status

Treatment of Vitamin D Deficiency

Vitamin D deficiency is most commonly treated with calciferol (Drisdol®, “vitamin “). As stated above, a dilemma exists on who and when to treat a patient for vitamin D deficiency or insufficiency. The interested reader is referred to the recommendations of the Australian Family Physicians (21) and to the paper by Wilkins and Birge (22), which provides specific recommendations on the prevention of osteoporotic fractures in the elderly.

Analytical Methodology

Of the more than 35 metabolites of vitamin  and vitamin  that have been identified only the measurement of 25 (OH) vitamin D and 1, 25  vitamin D has proven clinical value. For clinical purposes, the 25 (OH) vitamin D and 1, 25  vitamin D assays should measure both 25 (OH) vitamin  and 25 (OH) vitamin  or 1, 25  vitamin  and 1, 25  vitamin . Separate measurements of the vitamin  and vitamin  forms does not provide useful information or an indication of dietary and endogenous vitamin D because food is supplemented with both vitamin  and vitamin .(3,23) Because both vitamin  and vitamin  are metabolized to produce dihydroxylated metabolites of nearly equal biological activity, vitamin D assays should measure metabolites of both vitamin  and vitamin , with equi-molar cross-reactivity.(24,25) Warde Medical Laboratory utilizes a chemiluminesent assay for 25 (OH) vitamin D and a radioimmunoassay for 1, 25 , vitamin D, both with equi-molar cross-reactivity.