D-Light: Vitamin D and good health

There is no clinical laboratory in the past five years that has not experienced a
sudden surge in vitamin D testing. A positive combination of clinical trials, epidemiological studies, and educational journalism has to lead to the discovery that an alarming number of people are vitamin D insufficient or deficient. Major causes of this pandemic are the lack of exposure to the good effects of sunshine and an inadequate supply of vitamin D from natural foods or fortified ones. Other contributing factors towards this trend are obesity, rising aging population, and improved skin care products that block formation of vitamin D3.

Vitamin D, which was previously understood to be solely related to bone mineralization and calcium economy in the body, now enjoys an elevated status having established numerous non-classical functions. More than 36 cell types and 10 extra renal organs have been found to possess the vitamin D receptor, or VDR, as well as the ability to produce the multipotent hormone 1,25-dihydroxyvitamin D [1,25(OH)2D].^1,2 Insufficient vitamin D is increasingly being related to reduced immunological conditions,^3,4 cancers of the breast, colon,^5-7 pancreas,⁸ and prostate as well as heart diseases,⁹, ¹⁰ type 1 diabetes,¹¹ rheumatoid arthritis,¹² cognitive impairment,¹³ and all-cause mortality.¹⁴ This impressive collection of medical conditions accounts for more than 60% of deaths in the Western world.

A fat soluble pro-hormone, vitamin D is a seco-steroid which exists in two forms namely vitamin D2 and D3. While vitamin D2 is obtained from yeast and plant material, vitamin D3 is produced endogenously in the skin by the photochemical conversion of 7-dehydrocholesterol. Vitamin D circulates in the body bound to the vitamin D binding protein,
or VDBP. Both vitamin D2 and D3 are converted to the important biomarker 25-hydoxyvitamin D [25(OH)D] in the liver and undergo further hydroxylation in the kidneys to the bio-active form of the hormone 1,25(OH)2D.

Determining adequacy of vitamin D based on physiology

The cutaneous synthesis of vitamin D, based on the amount of UVB radiation reaching the skin, depends on several factors. Latitude, skin color, age, time of the exposure to sun, body mass index, cloud cover, use of sunscreen and month of the year are significant of mention.

Skin color:
Presence of the skin pigment melanin greatly reduces the synthesis of vitamin D in the body. Darker- skinned people have been found to have higher incidences of rickets, cardiovascular
diseases, and cancers,15,16 Middle East, tropical climates, and regions with religious and social customs that prevent skin exposure have been proved to be lacking in vitamin D.17

Seasonal variation:
It is reasonable to say that 25(OH)D exhibits a seasonal variation, peaking in late summer and showing a trend for a dip in concentration in late winter. Such variations are explained
by greater outdoor activity in summer months. Two cases of such seasonal variation have been reported in studies in south Florida18 and Utah.19 In the Florida case, 212 subjects were tested for 25(OH)D in winter. Hypovitaminosis (defined as 25(OH)D these subjects were re tested in summer. 25(OH)D concentrations were found to increase by 14% in men and 13% in women. A similar seasonal variation was observed when 140 subjects were tested in summer and winter months in the state of Utah. There was an 11-ng/mL difference in mean values noted between summer and winter months. While 39% of these subjects were found to be deficient in summer, the percentage increased drastically to 78% in winter.

Latitude:
Sunlight converts 7-dehydocholesterol into previtamin D3, a precursor of vitamin D3 on the skin. A model developed to study effect of sun exposure to winter sunlight in Boston (42.2^0N) and Edmonton (52^0N) showed that between the months of November through February in Boston and October through March in Edmonton, there was no initiation of cutaneous production of previtamin D3.20 People living at low latitudes are found to be
more prone towards vitamin D insufficiency due to decreased cutaneous production of vitamin D.21 While this is explainable by reduced UVB exposure, the study conducted in South Florida (a region that enjoys year-round sunny weather) and where serum 25(OH)D levels were measured in summer and winter, showed considerable cases of hypovitaminosis in both seasons.18 This has been explained by use of sun screen or preference for an indoor life style due to increased awareness of skin cancer.

Age: Recent
findings22 suggest an inverse relationship between vitamin D and chronological age. One quart of milk fortified with 400 IU (1 IU = 25 ng) of vitamin D provides only 15% of the daily requirement for people over 71 years of age as compared to 50% for people between 19 and 50 years of age. Diminishing renal function could be an explanation for poor synthesis of 1,25(OH)2D in an aged population.23 In a study conducted to assess the circulating levels of 25(OH)D in 1,606 older men randomly selected from six U.S. communities who were enrolled in the Osteoporotic Fractures in Men Study,22 it was observed that deficiency was present in 26% and insufficiency ( augmented with age, low vitamin D intake, and the level of
obesity (BMI

Ethnicity, Gender and Body
Mass Index: Pediatrics’ March 2009 issue monitored close to 3,000 samples and concluded that race, level of education, income, and rural or urban living conditions are independent and statistically significant predictors of vitamin D deficiency.24 According to this study non-Hispanic black adolescents were at increased odds of having serum 25(OH)D <20
ng/mL than Hispanic white adolescents (95% confidence interval [CI]: 12.5 to 33.3). Adolescents with low-income status were at three times increased odds compared to high income status (95% CI: 1.85 to 4.85). Girls were more than twice at odds of being
deficient than boys (95% CI: 1.68 to 3.07) and overweight adolescents had 75% increased odds of being deficient (95% CI: 1.28 to 2.38). Because of the fat soluble nature of vitamin D, supplementation regimes in obese adolescents may not work towards fulfilling adequacy of 25(OH)D levels.

With multiple and varied factors like those cited, playing a crucial role in diagnosing vitamin D
deficiency, even without its physical symptoms, physicians should screen those at risk.

What to measure?

It is important to understand that although serum 1,25(OH)2D is the active state of the hormone, it plays no role in diagnosing vitamin D deficiency. In a tightly regulated endocrinological feedback loop, as renal function declines, there is reduced availability of the enzyme 1alpha hydroxylase (which is responsible for conversion of 25(OH)D to the active form) and, therefore, reduced synthesis of 1,25(OH)2D. This sets the stage for secondary
hyperparathyroidism, promoting increased production of 1,25(OH)2D. The levels of 1,25(OH)2D in such cases may be normal or even high, thereby not indicative of vitamin D deficiency. Skeletal weakness, low calcium levels, elevated PTH, and qualifying in the high- risk groups such as children, pregnant women, malnutritioned, elderly, or institutionalized population, are leading conditions for ordering a 25(OH)D test. While hypercalcemia, renal failure, sarcoidosis, lymphoma, abnormalities of 1-alphahydroxylase, hyphophosphatic rickets, or vitamin D receptor defects call for a 1,25(OH)2D testing.

What is the cut-off level for defining vitamin D deficiency?

Adding to the confusion of which vitamin D test to order is the frustration of establishing an
appropriate reference range for defining cutoffs for deficiency, insufficiency, optimal as well as toxicity levels. With several population/environmental factors playing a key role in deciding
levels of vitamin D in the body, reported deficiency levels range from available for defining sufficiency and toxicity levels. A general consensus veers towards vitamin D deficient state, 21 ng/mL to 29 ng/mL as insufficient and 30 ng/mL to 80 ng/mL as an optimal level with above 150 ng/mL posing a case for possible toxicity. Two recent articles — published in Journal of Bio Medical Research and American Journal of Clinical Pathology — provide insight into defining 30 ng/mL as the optimal cutoff level. Iliac crest bone biopsies and circulating 25(OH)D levels in 675 Northern European subjects (known to have high prevalence of vitamin D deficiency) have shown that there were no pathologic bone mineralization defects
in any subject with circulating 25(OH)D above 30 ng/mL.²⁵ Parathyroid hormone, another measure of the skeletal index, has been used in the second publication¹⁹ in establishing
the appropriateness of 30 ng/mL as the lower reference range for cutoff. The median PTH concentrations plotted for groups with different vitamin D concentrations (257 subjects) show a trend in flattening of the line of regression of the dependency of PTH with 25(OH)D at 30 ng/mL indicating that the cutoff for sufficiency may be correct to be set at 30 ng/mL.

Hypervitaminosis

While the human body has the means to limit the synthesis of vitamin D after 10,000 IU to 20,000 IU of it has been produced via photo chemical conversion of 7 dehydrocholesterol, thereby preventing toxicity,²⁶ vitamin D toxicity in a highly supplemented state is not unheard
of. When there is a sustained intake of vitamin D supplements of 40 IU/d to 50,000 IU/d,²⁷,²⁸ the levels of 25(OH)D in such cases can rise above 220 nmol/L where a total of
100 IU (2.5 ug) of vitamin D raises the blood level of 25(OH)D by 1 ng/mL. These cases of intoxication can be more aggressive in children within a few days of administration.²⁹
Consistent application of analogues of 1,25(OH)2D (calcitriol, 22-oxacalcitriol) to psoriasis patients have also shown to increase hypercalcemia ^{30, 31} due to easy absorption of these derivatives through skin lesions. Vitamin D toxicity can cause constipation, arrhythmia, kidney stones, fatigue, anorexia, muscle weakness, and nausea.

Improved laboratory determination of vitamin D

Measuring vitamin D is no longer a cumbersome and time-consuming procedure compared to the competitive binding protein assay, or CPBA, method that was introduced over 30 years ago.³² Losses due to chromatography and extraction had to be estimated for individual samples and the method was not suitable for a high throughput clinical atmosphere. CPBA has been replaced by radioimmunoassay (RIA), high-performance liquid chromatography (HPLC), and tandem mass spectrometry (MS/MS).

RIA: RIA is a standard non-chromatographic method for quantification of 25OH vitamin D. Developed in the 1980s, this assay measures total vitamin D. Minimum sample prep steps and easy automation are the strong points of this assay.^33,34

Competitive chemiluminescence technology:
This is a random-access automated instrumentation method that uses an antibody as a primary binding agent and measures total vitamin D. After the fully automated chemiluminiscence LIAISON, the most recent addition to the automated platform for measuring 25(OH)D is called vitamin D3 from Roche Diagnostics. While LIAISON measures both D2 and D3 as total vitamin D, the Roche test measures only 25(OH)D3, making it less practical for countries like the U.S., in which both 25(OH)D2 and 25(OH)D3 are required to be quantified.

Direct detection:
HPLC and MS/MS methods separate and measure 25(OH)D2 and 25(OH)D3 independently. HPLC followed by UV is also considered a gold standard in vitamin D estimation, although it
is also associated with large sample volume and low throughput. MS/MS quantifies 25(OH)D2 and 25(OH)D3 individually and is associated with high accuracy, specificity, and sensitivity.
Mass spectrometers, however, are expensive, require costly radioactive internal standards, and cannot be automated. As a detection method, liquid chromatography mass spectrometry (LC-MS or HPLC-MS) compares well with immunoassay techniques.³⁵ While MS/MS affords high accuracy and selectivity, it is often times unable to differentiate between a stereo isomer of 25(OH)D, namely C3-epimer of 25(OH)D found predominantly in infants,³⁶ leading to incorrect estimations.

General frequency of vitamin D insufficiency

The March 2010 issue of the Journal of Clinical Endocrinology & Metabolism points towards an overwhelming 59% of people that are vitamin D insufficient.³⁷ In a cross-sectional study designed to establish a relationship between serum 25(OH)D and the degree of fat penetration in muscle, it was observed that among 90 post-pubertal females aged 16 to 22 years in the state of California, 59% were found to be 25(OH)D insufficient, 24% were deficient, and 41% were sufficient (>=30 ng/mL). Such percentage of vitamin D insufficiency is also reflected in a larger sample study that was made at ARUP laboratories. This model reflects a general sample set from all over the United States collected over a period of five years with no adjustments made to season of collection. The study demonstrated a tenfold increase in sample volume between 2005 to 2009.

Increase in samples which have serum 25(OH)D followed by a decrease between 2008 and 2009. Insufficiency (20 ng/mL to 29 ng/mL) increased. Sufficiency levels remained constant over the period of five years. steadily decreased over the period, indicating possible supplementation.

Adverse outcomes of vitamin D insufficiency

Presence of 1,25(OH)2D and vitamin D receptors in a wide variety of tissues ranging from pancreas, colon, brain, liver, muscle, skin, and lung^38-41 speaks of its broad involvement in the functioning of bodily systems. Published literature over several years indicates that the non-bone mineralization effects of vitamin D are autocrine, not endocrine.⁴² Thus, implying that these functions are not based or derived from the amount of circulating 1,25(OH)2D in the body but, rather, due to the intracellular synthesis of 1,25(OH)2D by these tissues. Studies also indicate that the levels of 1,25(OH)2D that are required for these non-calcemic
functions are higher than the levels of normal serum 1,25(OH)2D.⁴³ A good explanation towards the implication of cholecalciferol levels in relation to these non-calcemic functions is provided by Robert P. Heaney in a letter to the editor regarding the effects of vitamin D on cardiovascular system.⁴⁴

Epidemiological evidences have linked the deprived vitamin D condition to osteoporosis, osteoarthritis, obesity, multiple sclerosis, hypertension, type 1 diabetes, and several cancers. Vitamin D is also effective in maintaining low susceptibility to infections including pulmonary diseases.
Table 1 gives a compilation of the disease conditions that are found to be influenced by vitamin D inadequacy.

Vitamin D supplementation

Table 2 shows supplementation regimes presented by the Institute of Medicine, Food and Nutritional Board, Dietary Reference Intakes: Calcium, Phosphorus, Magnesium, Vitamin D and Fluoride that was available in 1997.⁴⁵

Table 2. Adequate Intakes (AIs) for Vitamin D[45].

While this regime may still hold true, a number of studies conducted towards achieving desired levels of serum 25(OH)D, in the recent years have made it amply clear that increase in vitamin D concentration by supplementation are found to be related to the baseline vitamin D levels in the body. While evaluating vitamin D2 against D3, a set of nursing home patients with serum 25(OH)D levels below 20 ng/mL, were treated with 200 IU, 400 IU, and 600 IU vitamin D2 for five months and this regime was found to increase their 25(OH)D levels to 24 ng/mL. Only when the dose was increased to 800 IU did the levels rise to 30 ng/mL. On the other hand, the subjects who had baseline  levels above 25 ng/mL did not see a rise in 25(OH)D levels even after five months of being treated with 200 IU, 400 IU, 600 IU, or 800 IU.⁴⁶ Therefore, it may be important to know the baseline level of 25(OH)D before beginning supplementation. Table 3 shows another relationship of baseline 25(OH)D levels with daily oral dose.⁴⁷

In addition to this information, it is also imperative to realize that while some studies have proved that vitamin D2 is 30% to 50% less effective that vitamin D3 due to its less binding potential with VDBP, ^48,49 others⁵⁰; show that vitamin D2 is as effective as vitamin D3 in
maintaining and raising serum 25(OH)D concentrations. 50,000 IU vitamin D2 administered once a week for eight weeks is used for treating vitamin deficiency and vitamin D sufficiency (30 ng/mL) can be achieved by having 50,000 IU vitamin D2 once every two weeks, according to Holick.⁴⁶

Animal knockout studies of vitamin D receptor and 1-alpha
hydroxylase

Calcitriol receptor or the Vitamin D receptor is a steroid/retinoid nuclear receptor that controls mineral homeostasis while presenting latent tumor suppressive functions.⁵¹ Several VDR ablated transgenic animal models have been studied in recent times bringing to light the influence of VDR on rickets, hypocalcaemia, osteomalacia, hyperparathyroidism, autoimmune diseases, and alopecia.

The mammary gland is capable of independently synthesizing 1-alpha hydroxylase, and it has been shown that both VDR and the enzyme 1-alpha hydroxylase are important for vitamin D signaling in the normal mammary gland. Studies performed on VDR knock out mice have indicated that lack of vitamin D accelerates mammary tumor development and optimal generation of 1,25(OH)2D is required for inhibiting mammary cell proliferation.⁵²

Comparisons of structural and compositional characters of the femur between VDR knock-out mice and wild-type mice for estimating the use of vitamin D in maintaining bone geometry and strength during gestation and lactation reveal that VDR affects the quantity of mineralized bone tissue.⁵³

With the abundancy of VDRs in prostatic epithelial and stromal cells, it has also been shown that there is increased apoptosis of periprostatic adipose tissue in VDR knock-out mice⁵⁴ indicating a possible role of VDR in the signaling pathways of prostate cancer inhibition.

Conclusion

Vitamin D has been shown to have an extensive area of biological influence due to the discovery of VDR and 1-alphahydroxylase enzyme in various extra renal tissues. While its deficiency continues to be associated with latitude and skin color, there is growing evidence that people living in abundant sunlight areas also suffer from its deficiency. Vitamin D levels need to be accurately monitored to prevent several chronic diseases.

Julie A. Ray, PhD, works at ARUP Institute for Clinical and Experimental Pathology, Salt
Lake City, UT, as does A. Wayne Meikle, MD. Dr. Meikle also works in the Departments of Pathology and of Medicine at the University of Utah-SLC.

Published: May, 2010

References

1. Zineb R, et al. Distinct, tissue-specific regulation of vitamin D receptor in the intestine, kidney, and skin by dietary calcium and vitamin D. Endocrinology. 1998; 139(4):1844-1852.
2. Zehnder D, et al. Extrarenal expression of 25-hydroxyvitamin d(3)-1 alpha-hydroxylase.
   J Clin Endocrinol Metab. 2001;86(2):888-894.
3. Cantorna MT, Mahon BD. Mounting evidence for vitamin D as an environmental factor affecting autoimmune disease prevalence. Exp Biol Med (Maywood). 2004;229(11):1136-1142.
4. Cantorna MT, et al. Vitamin D status, 1,25-dihydroxyvitamin D3, and the immune system.
   Am J Clin Nutr. 2004; 80(6 Suppl):1717S-1720S.
5. Gorham ED, et al. Vitamin D and prevention of colorectal cancer. J Steroid Biochem Mol Biol. 2005;97(1-2):179-194.
6. Holick MF. Vitamin D: its role in cancer prevention and treatment. Prog Biophys Mol Biol. 2006;92(1): 49-59.
7. Garland CF, et al. The role of vitamin D in cancer prevention. Am J Public Health. 2006;96(2):252-261.
8. Chiang KC, Chen TC. Vitamin D for the prevention and treatment of pancreatic cancer.
   World J Gastroenterol. 2009;15(27):3349-3354.
9. Nemerovski CW, et al. Vitamin D and cardiovascular disease. Pharmacotherapy. 2009;29(6):691-708.
10. Gouni-Berthold I, Krone W,Berthold HK. Vitamin D and cardiovascular disease. Curr Vasc Pharmacol. 2009;7(3):414-422.
11. Mathieu C, et al. Vitamin D and diabetes. Diabetologia. 2005;48(7):1247-1257.
12. Cutolo, M, et al. Vitamin D in rheumatoid arthritis. Autoimmun Rev. 2007;7(1):59-64.
13. Wilkins CH, et al. Vitamin D deficiency is associated with worse cognitive performance
    and lower bone density in older African Americans. J Natl Med Assoc. 2009;101(4):349-354.
14. Zittermann A, Gummert JF, Borgermann J. Vitamin D deficiency and mortality.
    Curr Opin Clin Nutr Metab Care. 2009;12(6):634-639.
15. Hall LM, et al. Vitamin D intake needed to maintain target serum 25-hydroxyvitamin D
    concentrations in participants with low sun exposure and dark skin pigmentation is substantially higher than current recommendations. J Nutr. 2010;140(3):542-550.
16. Kremer R, et al., Vitamin D status and its relationship to body fat, final height, and
    peak bone mass in young women. J Clin Endocrinol Metab. 2009;94(1):67-73.
17. Mithal A, et al. Global vitamin D status and determinants of hypovitaminosis D.
    Osteoporos Int. 2009; 20(11):1807-1820.
18. Bandeira F, et al. Vitamin D deficiency: A global perspective. Arq Bras Endocrinol Metabol. 2006;50(4):640-646.
19. Kushnir MM, et al. Rapid analysis of 25OH Vitamins D2 and D3 by LC-MS/MS and association of vitamin D and parathyroid hormone concentrations in healthy adults. AJCP-2009-11-0569 (in press)
20. Webb AR, Kline L, Holick MF. Influence of season and latitude on the cutaneous synthesis
    of vitamin D3: exposure to winter sunlight in Boston and Edmonton will not promote vitamin D3 synthesis in human skin. J Clin Endocrinol Metab.1988;67(2):373-378.
21. Basile LA, et al. Neonatal vitamin D status at birth at latitude 32 degrees 72′: evidence of deficiency. J Perinatol. 2007;27(9):568-571.
22. Orwoll E, et al. Vitamin D deficiency in older men. J Clin Endocrinol Metab. 2009;94(4):1214-1222.
23. Vieth R, Ladak Y, Walfish PG. Age-related changes in the 25-hydroxyvitamin D versus
    parathyroid hormone relationship suggest a different reason
    why older adults require more vitamin D. J Clin Endocrinol Metab. 2003;88(1):185-191.
24. Saintonge S, Bang H, Gerber LM. Implications of a new definition of vitamin D deficiency in a multiracial us adolescent population: the National Health and Nutrition Examination Survey III. Pediatrics. 2009; 123(3):797-803.
25. Priemel M, et al. Bone Mineralization Defects and Vitamin D Deficiency: Histomorphometric Analysis of Iliac Crest Bone Biopsies and Circulating 25-Hydroxyvitamin D in 675 Patients.
    J Bone Miner Res. 2009.
26. Holick MF. Environmental factors that influence the cutaneous production of vitamin D.
    Am J Clin Nutr. 1995;61(3 Suppl):638S-645S.
27. Heaney RP, et al. Human serum 25-hydroxycholecalciferol response to extended oral dosing with cholecalciferol.
    Am J Clin Nutr. 2003;77(1):204-210.
28. Vieth R. Vitamin D supplementation, 25-hydroxyvitamin D concentrations, and
    safety. Am J Clin Nutr. 1999; 69(5):842-856.
29. Barrueto F Jr, et al. Acute vitamin D intoxication in a child. Pediatrics. 2005;116(3):e453-456.
30. Sato K. [Drug-induced hypercalcemia]. Clin Calcium. 2006;16(1):67-72.
31. Koizumi H, et al. 1,25-Dihydroxyvitamin D3 and a new analogue, 22-oxacalcitriol, modulate proliferation and interleukin-8 secretion of normal human keratinocytes.
    J Dermatol Sci. 1997;15(3):207-213.
32. Haddad JG, Chyu KJ. Competitive protein-binding radioassay for 25-hydroxycholecalciferol. J Clin Endocrinol Metab. 1971;33(6):992-995.
33. Hollis BW, Napoli JL. Improved radioimmunoassay for vitamin D and its use in assessing
    vitamin D status. Clin Chem. 1985;31(11):1815-1819.
34. Hollis BW, et al. Determination of vitamin D status by radioimmunoassay with an 125I-labeled tracer. Clin Chem. 1993;39(3):529-533.
35. van den Ouweland JM. et al. Measurement of 25-OH-vitamin D in human serum using liquid chromatography tandem-mass spectrometry with comparison to radioimmunoassay and automated immunoassay. J Chromatogr B Analyt Technol Biomed Life Sci. 2010.
36. Singh RJ, et al. C-3 epimers can account for a significant proportion of total circulating
    25-hydroxyvitamin D in infants, complicating accurate measurement and interpretation of vitamin D status. J Clin Endocrinol Metab. 2006;91(8):3055-3061.
37. Gilsanz V, et al. Vitamin D status and its relation to muscle mass and muscle fat in
    young women. J Clin Endocrinol Metab. 2010;95(4):1595-1601.
38. Wada K, et al. Vitamin D receptor expression is associated with colon cancer in ulcerative
    colitis. Oncol Rep. 2009;22(5):1021-1025.
39. Kaiser U, et al. Expression of vitamin D receptor in lung cancer. J Cancer Res Clin Oncol. 1996;122(6):356-359.
40. Eyles DW, et al. Distribution of the vitamin D receptor and 1 alpha-hydroxylase in human
    brain. J Chem Neuroanat. 2005;29(1):21-30.
41. Segura C, et al. Vitamin D receptor ontogenesis in rat liver. Histochem Cell Biol. 1999;112(2):163-167.
42. Holick MF. The vitamin D deficiency pandemic and consequences for nonskeletal health:
    mechanisms of action. Mol Aspects Med. 2008;29(6):361-368.
43. Liu PT, et al. Toll-like receptor triggering of a vitamin D-mediated human antimicrobial
    response. Science. 2006;311(5768):1770-1773.
44. Guallar E, et al., Vitamin D supplementation in the age of lost innocence. Ann Intern Med. 2010;152(5): 327-329.
45. Institute of Medicine, Food and Nutrition Board. Dietary Reference Intakes: Calcium,
    Phosphorus, Magnesium, Vitamin D, and Fluoride. Washington, DC: National Academy Press, 1997.
46. Holick MF, Chen TC. Vitamin D deficiency: a worldwide problem with health consequences.
    Am J Clin Nutr. 2008;87(4):1080S-1086S.
47. Heaney RP. The Vitamin D requirement in health and disease. J Steroid Biochem Mol Biol. 2005;97(1-2):13-19.
48. Armas LA, Hollis BW, Heaney RP. Vitamin D2 is much less effective than vitamin D3 in humans. J Clin Endocrinol Metab. 2004;89(11):5387-5391.
49. Trang HM, et al. Evidence that vitamin D3 increases serum 25-hydroxyvitamin D more
    efficiently than does vitamin D2. Am J Clin Nutr. 1998;68(4):854-858.
50. Holick MF, et al. Vitamin D2 is as effective as vitamin D3 in maintaining circulating
    concentrations of 25-hydroxyvitamin D. J Clin Endocrinol Metab. 2008;93(3):677-681.
51. Chung I, et al. Role of vitamin D receptor in the antiproliferative effects of calcitriol in
    tumor-derived endothelial cells and tumor angiogenesis in vivo. Cancer Res. 2009;69(3):967-975.
52. Welsh J. Vitamin D and breast cancer: insights from animal models. Am J Clin Nutr. 2004;80(6 Suppl):1721S-1724S.
53. Korecki CL, et al. Effect of the vitamin D receptor on bone geometry and strength during
    gestation and lactation in mice. Calcif Tissue Int. 2009;85(5):405-411.
54. Guzey M, et al. Increased apoptosis of periprostatic adipose tissue in VDR null mice.
    J Cell Biochem. 2004;93(1):133-141.
55. Panda DK, et al. Targeted ablation of the 25-hydroxyvitamin D 1alpha -hydroxylase
    enzyme: evidence for skeletal, reproductive, and immune dysfunction. Proc Natl Acad Sci USA. 2001;98(13):7498-7503.
56. Takeyama K, Yamamoto Y, Kato S. VDR knockout mice and bone mineralization disorders. Clin Calcium. 2007;17(10):1560-1566.
57. Tishkoff DX, et al. Functional vitamin D receptor (VDR) in the t-tubules of cardiac
    myocytes: VDR knockout cardiomyocyte contractility. Endocrinology. 2008;149(2):558-564.
58. Narvaez CJ, et al. Lean phenotype and resistance to diet-induced obesity in vitamin D receptor knockout mice correlates with induction of uncoupling protein-1 in white adipose tissue. Endocrinology. 2009;150(2):651-661.
59. Simpson RU, Hershey SH, Nibbelink KA. Characterization of heart size and blood pressure in the vitamin D receptor knockout mouse. J Steroid Biochem Mol Biol. 2007;103(3-5):521-524.
60. Xiang W, et al. Cardiac hypertrophy in vitamin D receptor knockout mice: role of the
    systemic and cardiac renin-angiotensin systems. Am J Physiol Endocrinol Metab.
    2005;288(1):E125-E132.
61. Froicu M, Zhu Y, Cantorna MT. Vitamin D receptor is required to control gastrointestinal
    immunity in IL-10 knockout mice. Immunology. 2006;117(3):310-318.
62. Nakagawa K, et al. Metastatic growth of lung cancer cells is extremely reduced in Vitamin
    D receptor knockout mice. J Steroid Biochem Mol Biol. 2004;89-90(1-5):545-547.
63. Zinser GM, McEleney K, Welsh J. Characterization of mammary tumor cell lines from wild type and vitamin D3 receptor knockout mice. Mol Cell Endocrinol. 2003; 200(1-2):67-80.
64. Kallay E, et al. Characterization of a vitamin D receptor knockout mouse as a model of
    colorectal hyperproliferation and DNA damage. Carcinogenesis. 2001;22(9):1429-1435.
65. Rowling MJ, et al. High dietary vitamin D prevents hypocalcemia and osteomalacia in CYP27B1 knockout mice. J Nutr. 2007;137(12):2608-2615.