Vitamin D Levels: Essentials Assessment Implications Siemens Healthcare Diagnostics Inc. All rights reserved.

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1 Vitamin D Levels: Essentials Assessment Implications All rights reserved. 1

2 Vitamin D Levels: Essentials All rights reserved. 2

3 US Population 23% Sufficiency 77% Insufficiency Source: AA Ginde et al, Trends in Vitamin D Insufficiency in the US Population, , Arch. Intern Med, (2009) 169: Page 3 So how many of us are vitamin D insufficient? Is it one in three? Or, is it two in three? No, it s more prominent than that. In the United States, more than 3 out of 4 adolescents and adults are Vitamin D insufficient. This data comes from the NHANES III study that collected data from over 13,000 people in 2001 to This study was careful not to collect data in the winter months in the Northern latitudes of the US so the percentage is probably higher. This large sampling of the nation s health also tells us that Vitamin D insufficiency is higher in Mexican Americans, with 90% being insufficiency and in Non-Hispanic blacks, with 97% having Vitamin D insufficiency. 3

4 Changing Vitamin D Deficiency Deficiency & Insufficiency Sunlight & Diet Page 4 What are the reasons for the increase occurrence of Vitamin D deficiency? As our society is increasingly sedentary we don t need exercise enough and don t get outside enough. This decreased of outdoor activity has reduced the time that our nation spends in the sun. The decrease exposure to sunlight is the most important reason for the marked increase in Vitamin D deficiency. We are using sun screen to reduce our exposure to harmful UV-B wavelengths and this has reduced the incidence of skin cancer. However, blocking these harmful UV-B rays with a sun block will reduce the Vitamin D production by about 95%. Also UV-B radiation does not penetrate glass, so exposure to sunshine indoors through a window does not produce vitamin D. We don t get enough Vitamin D in our diets. Milk is fortified with Vitamin D; however, the amount of milk that we drink has decreased since the Other foods that are high in Vitamin D are fish, especially Salmon, Mackerel and Tuna. Mushrooms also have high Vitamin D content. However, dietary sources are not high enough to offset the lack of direct sunlight. 4

5 High Risk Groups F. R. Bringhurst, et.al., Bone and Mineral Metabolism in Health and Disease ; Chapter 23 of Harrison s Endocrinology Holick M, N Engl J Med (2007); 357: Page 5 Who is at higher risk of Vitamin D deficiency? Breastfed Infants normally human milk has only 25 IU/L and often the mother is Vitamin D deficient herself. Therefore, most infants are prescribed 400 IU of Vitamin D daily. Unfortunately, often our poor can not afford medical care for their infants, so these babies are at a very high risk. Older Adults -- have a reduce level of 7-dehydr-cholesterol, so they can t synthesize Vitamin as well. Also their kidneys are less able to produce the active hormone, 1,25 dihydro Vitamin D. Dark skinned people melanin in the darker skin reduces the ability to produce Vitamin D from sunlight exposure, because it absorbs the sunlight. Limited Sun Exposure this eliminates one of the two sources of Vitamin D. This often happens to the institutionalized people. Obese people because Vitamin D is fat soluble, it is sequestered in the fat; not allowing it to circulate. Also those who have had a gastric bypass don t have a portion of their small intestine and can not absorb Vitamin D as readily. Typical Milk Volume: From birth to 24 hours, colostrum averages about 37 ml From 24 to 96 hours, there is a slow rise in volume Day 5: approximately 500 ml/day 3 to 5 months: 750 ml/day 6 months: 800 ml/d Source: F. R. Bringhurst, et.al., Bone and Mineral Metabolism in Health and Disease ; Chapter 23 of Harrison s Endocrinology, J. Larry Jameson, editor, McGraw-Hill Medical Publishing Division, copyright Reduced skin synthesis Sunscreens, pigmentation, aging, season / latitude, skin grafts for burns Decreased bioavailability Fat malabsorption due to cystic fibrosis, celiac disease, Crohn s disease, medications etc Increased catabolism Anticonvulsants, glucocortoids, AIDS treatments, antirejection medications Breast-feeding Poor vitamin D content in human milk Decreased synthesis of 25 (OH) Vit. D Liver failure Increased urinary loss of 25 (OH) Vit. D or decreased synthesis of 25 (OH) Vit. D Nephrotic syndrome or chronic kidney disease Various heritable or acquired disorders Vit D-dependent rickets type 1, 2, 3, hypophosphatemic rickets, tumor secretion of FGF23, hyperthyroidism and hyperparathyroidism, granulomatous disorders, sarcoidosis, tuberculosis 5

6 Vitamin D Reference Values: Health Based Reference Values Deficient: 20 ng/ml Insufficient: >21 29 ng/ml Sufficient: 30 ng/ml Hollick M. N Engl J Med 2007;357: Total Vitamin D Measurement (ng/ml) Page 6 A simple blood test can be used to measure the amount of vitamin D specifically 25-hydroxyvitamin D. Vitamin D deficiency is any serum 25-hydroxyvitamin D result less than 20 ng/ml. An insufficient level is between 20 and 30 ng/ml, and sufficient levels are above 30 ng/ml. Vitamin D toxicity is rare, but has been observed when 25-Hydroxyvitamin D concentrations are > 200 ng/ml. Although there is no consensus on 25-hydroxyvitamin D Reference ranges, many leading authorities believe that health based Reference Values are preferable. These researchers agree that physiological insufficiency is defined by the 25-OH Vitamin D concentrations below which parathyroid hormone (PTH) levels increase in a population. Because even a slight increases in circulating PTH levels can enhance bone turnover and accelerate bone loss. When 25-Hydroxyvitamin D concentrations are above 30 ng/ml, PTH concentration levels off at its nadir and bone remodeling occurs at a normal rate. If 25-Hydroxyvitamin D concentrations are reduced to between 20 to 29 ng/ml, parathyroid hormone (PTH) concentration increases and signal the increase in intestinal calcium transport. When 25-Hydroxyvitamin D concentrations drop to below 20 ng/ml is when classic sign of various rickets and osteomalicia are observed. 6

7 Types of Vitamin D Ergocalciferol (D2) Cholecalciferol (D3) Page 7 There are two major types of Vitamin D: Vitamin D2 (ergocalciferol) synthesized by plants and is not produced by the human body. Vitamin D3 (Cholecalciferol) is made in large quantities when sunlight strikes your bare skin. It can also be ingested from animal sources. Season, geographic latitude, time of day, cloud cover, smog, skin melanin content, and sunscreen are among the factors that affect UV-B radiation exposure and vitamin D synthesis. The sunlight from November through February above 42 degrees north latitude is insufficient for vitamin D synthesis. 42 degrees north is a line approximately between the northern border of California and Boston. In supplements and fortified foods, vitamin D can be either D2 or D3. The two forms have traditionally been regarded as equivalent based on their ability to cure rickets, but evidence has been offered that they are metabolized differently. Vitamin D3 could be more than three times as effective as vitamin D2 in raising serum 25(OH)D concentrations. This is because of its higher affinity for vitamin D-binding proteins, causing D3 to exist longer in the blood stream. 7

8 Structures of Vitamin D 2 and D 3 Vitamin D 2 (Ergocalciferol) Vitamin D 3 (Cholecalciferol) Page 8 The structures of vitamin D2 and vitamin D3 differ in the area indicated by the circle. 8

9 Vitamin D Synthesis 25(OH)D 7-dehydrocholesterol 1,25(OH) 2 D Source: Adapted from: F. R. Bringhurst, et.al., Bone and Mineral Metabolism in Health and Disease ; Chapter 23 of Harrison s Endocrinology. Page 9 Ultraviolet (UV) B radiation with a wavelength of nanometers penetrates unprotected skin and converts cutaneous 7-dehydrocholesterol to pre-vitamin D3, which then isomerizes to vitamin D3. The other source of Vitamin D is our diets. When we ingest Vitamin D, it is absorbed by the intestines. Once in the blood stream, Vitamin D is bound to Vitamin D Binding Protein or Albumin and carried to the liver. There Vitamin D is hydroxylated to 25 OH-Vitamin D.Then in the kidney, another hydroxylation step takes place to produce 1,25 dihydroxy-vitamin D. 1,25 dihydroxy-vitamin D is the major steroid Hormone involve in mineral homeostasis. Adapted from: F. R. Bringhurst, et.al., Bone and Mineral Metabolism in Health and Disease ; Chapter 23 of Harrison s Endocrinology. 9

10 Vitamin D Hormonal Control Vitamin D Vitamin D-25 hydroxylase Source: Adapted from: F. R. Bringhurst, et.al., Bone and Mineral Metabolism in Health and Disease ; Chapter 23 of Harrison s Endocrinology. - 25(OH)D 3 Phosphorous and Other Factors 1,25(OH) 2 D 3 25(OH)D-1α-hydroxylase PTH PTH - 1,25(OH) 2 D 3 Ca 2+ HPO 4 2- Calcification Blood Calcium Ca 2+ HPO 4 2- Page 10 1,25 dihydroxy-vitamin D is one of the main actors, in this complex hormonal feedback loop.when serum calcium dips to below 8.8 mg/dl prompts a proportional increase in the secretion of parathyroid hormone (PTH). PTH signals to the kidneys to increase the production of 1,25 (OH)2 Vitamin D by increasing the production of 25(OH)Vitamin D-1α-hydroxylase.The increase in 1,25 (OH)2 Vitamin D then stimulates the increase absorption of calcium in the intestines and up-regulates genes in the bone, to stimulate bone remodeling. When the majority of 1,25 (OH)2 Vitamin D is bound to Vitamin D receptor (VDR) -- it acts to meditate bone remodeling. 10

11 Causes of Impaired Vitamin D Action Intestinal Malabsorption Accelerated loss Impaired Production Source: F. R. Bringhurst, et.al., Bone and Mineral Metabolism in Health and Disease ; Chapter 23 of Harrison s Endocrinology. Page 12 We have already discussed the more prominent reasons that Vitamin D deficiency are increasing. Another are several other conditions that can cause this deficiency. Intestinal malabsorption of dietary fats can also lead to Vitamin D deficiency, since Vitamin D is fat soluble. Vitamin D can be metabolized very quickly because the patient is using several drugs, for example barbiturates. And its metabolism can also be increased if the enterohepatic circulation is impaired. Common diseases that impaired the production of 1,25 (OH)2 Vitamin D are liver diseases and kidney failure. Any liver or kidney condition that limits the production of 25 OH Vitamin D or 1,25 dihydroxy Vitamin D will limit the production of this vital hormone. Examples for Accelerated Loss: Phenobarbital (is used to control epilepsy (seizures) and as a sedative to relieve anxiety) appears to interfere with the normal absorption or metabolism of vitamin D.10,11 In turn, this can impair calcium absorption.12 Making sure to get enough vitamin D (and calcium) should help prevent any problems from developing. Isoniazid (is used alone or with other drugs to treat tuberculosis) may interfere with the body's ability to use vitamin D.7,8,9 Although it is not clear whether this actually causes symptoms of vitamin D deficiency,10 it still might be a good idea to take vitamin D supplements at standard U.S. Adequate Intake (AI) dosages. Corticosteroid: One of the most serious side effects of long-term corticosteroid use is accelerated osteoporosis. Although we don't fully understand how this works, corticosteroid interference with calcium and vitamin D is known to play a major role. Calcium and vitamin D supplements are definitely beneficial for fighting ordinary osteoporosis; in addition, there is good evidence that they also protect against osteoporosis brought on by corticosteroids.1 A review of 5 trials enrolling a total of 274 participants found that calcium and vitamin D supplementation significantly prevented bone loss at the lumbar spine and forearm in corticosteroid-treated individuals.2 For example, in a 2-year double-blind placebo-controlled study of 130 individuals, supplementation with 1,000 mg of calcium and 500 IU of vitamin D daily actually reversed steroidinduced bone loss, causing a net bone gain.3 12

12 Treatments for Vitamin D deficiency Normal Insufficiency 400 IU Vitamin D/day Post Menopausal Women 800 IU Vitamin D/day + Calcium Intestinal Malabsorption Up to 40,000 IU Vitamin D/day Impaired α-hydroxylation 1,25(OH)2 Vitamin D or 1α-hydroxy Vitamin D Page 13 Patients with insufficient Vitamin D level at least 400 IU Vitamin D/daily. Patients with risk of hip fractures post menopausal Women at least 800 IU Vitamin D/daily plus Calcium supplement. In cases of Intestinal Malabsorption, doses up to 40,000 IU per day. When the patient has impaired α-hydroxylation treatment uses a Vitamin D analogue that does not require this metabolic step; such as 1,25(OH)2 Vitamin D and 1α-hydroxy Vitamin D. 13

13 Vitamin D Levels: Assessment All rights reserved. 14

14 Vitamin D Levels: Assessment The most widely used indicator of vitamin D status is the measurement of 25-hydroxyvitamin D [25(OH)D] in either serum or plasma. Because circulating 25(OH)D can arise from hydroxylation of either vitamin D 2 or vitamin D 3, measurement of total 25(OH)D [both 25(OH)D 2 and 25(OH)D 3 ] is essential for accurate assessment of vitamin D status. Phinney KW. Vol. 88, No. 2, 511S-512S, August 2008 Page 15 In this quote, Phinney et al. accurately state that total Vitamin D must include both the contribution of D2 and D3 to get the best result to assess the patient s condition. 15

15 Vitamin D Consensus Panel Recommendations Who should be tested? What is the recommended serum range for 25(0H)D? Who should be supplemented? When should testing be performed? What are the requirements for a test? Sourberbielle JC et al., Autoimmunity Reviews 9 (2010) Page 16 A expert consensus panel met to determine important questions related to vitamin D testing. The investigated the following: Who should be tested? What is the recommended serum range for 25 (0H)D? Who should be supplemented? When should testing be performed? What are the requirements for a test? 16

16 Vitamin D Consensus Panel Recommendations: Who Should have Levels Measured? Classical clinical applications Individuals with or at risk for osteoporosis Elderly subjects with a recent fall Pregnant women Patients with chronic kidney disease Transplants patients Patients with conditions or treatments that can lead to bone loss Obese individuals Non-classical clinical applications Cardiology All individuals with hypertension Autoimmunity Patients with autoimmune disease Subjects at high risk for autoimmune disease Patients starting or already on corticosteroids Oncology All cancer patients undergoing treatment Patients with diabetes Hospitalized patients Patients with bone / muscle pain or aches Souberbielle JC, Autoimmunity Reviews (2010); 9: Page 17 The experts have divided the clinical applications of vitamin D testing into 2 groups: 1-The classical applications for which the level of evidence is very high with numerous Randomized Controlled Trials (RCTs). 2- The non-classical applications for which long-term RCTs are still needed but the amount of evidence is increasing consistently. In both groups, vitamin D testing is recommended in patients at risk of developing the disease or already with the disease! Emphasis on: Premenopausal women with early breast cancer receiving adjuvant chemotherapy or gonadotropin-releasing hormone analogs Breast cancer patients under anti-aromatase therapy Prostate cancer patients under hormone ablative treatment 17

17 Vitamin D Consensus Panel Recommendations: Who, When, What For optimal health benefit patients with or at risk for musculoskeletal, cardiovascular, autoimmune diseases or cancer levels should be >30 ng/ml For individuals with little sun-exposure or dark-skinned individuals supplement first, then test The interval between starting supplementation and measuring/monitoring should be at least 3 months Upper safety limit 100 ng/ml Maintenance dose 800 IU/day Sourberbielle JC et al., Autoimmunity Reviews 9 (2010) Page 18 The consensus panel has recommendations about the who, when, and what for vitamin D testing. For optimal health benefit patients with or at risk for musculoskeletal, cardiovascular, autoimmune diseases and cancer levels should be >30 ng/ml. For individuals with little sun-exposure or dark-skinned individuals supplement first, then test. The interval between starting vitamin D supplementation and measuring/monitoring should be at least 3 months. The upper safety limit is 100 ng/ml. Maintenance doses of 800 IU/day. 18

18 Vitamin D Consensus Panel Recommendations: Assay Management Measure both vitamin D 2 and D 3 If your assay measures vitamin D2 and D3 separately, provide the sum Participate in an external quality scheme Internal quality control is a must Express units in ng/ml Use recommended range not population based reference values Serum is the sample of choice Sourberbielle JC et al., Autoimmunity Reviews 9 (2010) Page 19 In terms of assay management: A test that measures both vitamin D2 and D3. If your assay measures vitamin D2 and D3 separately, provide the sum, Participate in an external quality scheme. Internal quality control is a must, Express units in ng/ml, Use recommended range not population based reference values. Serum is the sample of choice 19

19 Vitamin D Analytical Issues: Imprecision DEQAS 141 participants in October participants in July 2009 (35 countries) Carter GD, et al. J Steroid Biochem Molecular Biology, 2010;121: Page 20 DEQAS is the only international external quality scheme for vitamin D. The number of participants has grown since From 141 in 2004 to 670 participants in 35 countries in There are many analytical issues related to vitamin D testing that is measuring (25-OH D). Chief among these are imprecision and the differences between methods. 20

20 Vitamin D Analytical Issues: Imprecision 1994 to 2009 Imprecision: 32% to 16% Page 21 Inter-laboratory imprecision ranged from 32% to 16%. 21

21 Vitamin D Analytical Issues: Considerable Method Variation There is considerable method variation Positive bias Negative bias Carter GD, et al.; J Steroid Biochem Molecular Biology, 2010;121: Page 22 Among methods there is considerable variation as well. This study shows the differences between methods and the ALTM. The ALTM is calculated as follows: all returned results are ranked, the top and bottom 5% of results removed and the remaining results used to calculate an All-Laboratory Trimmed Mean (ALTM). The accuracy of each result is judged by calculating its % bias from the ALTM (represented by the horizontal axis), although it is recognized that the ALTM is only a surrogate for the true value. Available methods demonstrated both positive and negative bias from the ALTM. There was considerable variation among methods and within the same method overtime. 22

22 Vitamin D Analytical Issues Standardization Specificity Sensitivity Method comparability Page 23 In addition to imprecision, other issues of concern for measuring vitamin D include the standardization, specificity, functional sensitivity and method comparability. 23

23 Vitamin D Analytical Issues: Standardization SRM 972 has 4 levels Use SRM 972 level 1 (normal serum with 25(OH)D 2 and 25(OH)D 3 ) Wallace AM et al; Steroids 2010;75, Page 24 The newly released reference standard has proved to be disappointing for immunoassays. Only level one (endogenous vitamin D in normal serum) has proven to be useful, the other levels of the reference standard exhibit problems attributed to matrix effects (horse serum) and addition of exogenous vitamin D. Background notes This material (SRM 972) has recently been made available and consists of four standards. Level 1 of SRM 972 was prepared from normal human serum and has not been altered. Level 2 was prepared by diluting Level 1 with horse serum to achieve a lower 25OHD concentration. Level 3 contains normal human serum that has been fortified with 25OHD2, and Level 4 contains normal human serum that has been fortified with 3-epi-25-hydroxyvitamin D3 ( 24

24 Vitamin D Analytical Issues: Specificity and Functional Sensitivity Specificity How much (25OH)D 2 is being detected? Cross-reactivity with other metabolites? Functional Sensitivity / LoQ <10 ng/ml (<25 nmol/l) Wallace AM et al; Steroids 2010;75, Page 25 In terms of specificity, some assays detect metabolites of vitamin D other than (25OH)D. These metabolites can account for up to 10% of circulating levels. Another major challenge is the ability of the assay to detect (25OH)D 2 that is vitamin D 2. Some assays detect only a percentage of vitamin D; this ranges from 20% to 100%.Functional sensitivity/ limit of quantitation (LoQ) should be less than 10 ng/ml to facilitate detection of very severe vitamin D deficiency. 25

25 Vitamin D Analytical Issues: Matrix Interferences Sources are Calibrators Patient sample Release from binding protein Results in Spuriously high values High levels of imprecision Wallace AM et al; Steroids 2010;75, Page 26 Another major issue is matrix interference. This can be from calibrators and/or the patient sample. Lipids are problematic because vitamin D is lipohilic. Vitamin D exists bound to a binding protein, and to measure it, it must be displaced from this protein. Some approaches are more successful than others, in fact there is some evidence that immunoassays have less of a problem with displacing the protein. 26

26 Vitamin D Method Comparison: Additional Issues Manual Immunoassays Staff Waste Matrix effects Sample volume Turnaround time D 2 recovery Automated Immunoassays Staff Waste Matrix effects Sample volume Turnaround time D 2 recovery Direct Detection Staff Waste Matrix effects Sample volume Turnaround time D 2 recovery Wallace AM, et al.steroids 2010;75, Page 27 There are three main types of assays: manual immunoassays, automated immunoassays and direct detection methods. The direct detection methods are high performance liquid chromatography and liquid chromatography-mass spectrometry( HPLC and LC-MS). Several issues should be considered when making your assay choice. They include staff, waste, matrix effects, sample volume, turnaround time and D 2 recovery. Staff time and expertise are issues for manual immunoassay and direct detection methods. Waste is an issue for RIA assays. Matrix effects are a challenge for any immunoassay and less of a problem in direct detection methods. Direct detection method require larger samples, this can be a challenge if you are testing an infant or need to retest. Turnaround time is longer for manual immunoassays and direct detection methods. And for any method, you need to know how much vitamin D 2 it recovers. 27

27 Risk of Vitamin Toxicity Kidney stones Raised calcium levels Nausea, weight loss, vomiting, constipation, loss of appetite, weakness Page 28 The risk of Vitamin D toxicity is very low. However, when a person is taking high concentration of Vitamin D supplement, there serum levels can be increase to above 200 ng/ml, which is potentially toxic. With this increase in Vitamin D, Calcium concentration will rise, causing nausea, weight loss and constipation. If the increased levels of Vitamin D and Calcium persist then it is like that the patient will develop kidney stones. 28

28 Vitamin D Tests: What Do They Measure? Vitamin D 2 Vitamin D Vitamin D 2 Vitamin D Vitamin D 2 Vitamin D Page 29 There are several Immunoassays that do not have the same reactivity to Vitamin D 2 and D 3. As you can see, some of these assays only detect D3 or partial detect D2. Assays that have the same reactivity to Vitamin D 2 and D 3, will give you the most accurate results for all of your patient s samples. 29

29 Disadvantages of Not Measuring 100% of Vitamin D 2 Misclassification of patients Patients on D 2 supplement at risk of toxicity Page 30 By using assays that do not weigh the two forms of Vitamin D equally, the laboratory result may lead one to misclassify the patient s status and potentially miss patients that have toxic level of Vitamin D. 30

30 Example 1: Is this Patient Deficient, Insufficient or Sufficient? 40 Total Vitamin D gives a True Assessment 30 D 2 D 2 D D 3 D 3 D 3 D 3 True Value Test #1 Test #2 Test #3 Page 31 Let s talk about possible sample concentrations that will come in for analysis.first we have the case that the true concentration is just into the sufficient range. If the assay does not detect D2, it is likely that result will be reported as in the insufficient range. And this also can happen if the assay detects only a fraction of the D2 that is present. To get a true reading of the patient s Vitamin D level, one has to use an assay that detects both Vitamin D2 and D3 equally. 31

31 Example 2: Missing Toxicity 300 Total Vitamin D: Only Test to Measure Toxicity D 2 D 2 D 2 D 3 D 3 D 3 D 3 True Value Test #1 Test #2 Test #3 Page 32 When we look at the patient that are being treated for malabsorption with high doseof Vitamin D, the different reactivity for D2 and D3 also can cause the patientstatus to be mis-identified. If the supplement is D2, it is likely to be the largestvitamin D concentration in these patients. So by not having D2 detected orpartial detected, it may result in the under reporting of the total Vitamin D concentration which may result in missing a patient that has levels that aretoxic. 32

32 Vitamin D Levels: Implications All rights reserved. 33

33 Impact of Vitamin D Deficiency Bone Health Rickets + Skeletal Deformities Cardiovascular Disease Cancer Diabetes Glucose Intolerance Multiple Sclerosis Autoimmune Source: NIH Vitamin D Fact Sheet Page 34 For years we have monitored Vitamin D to help determine our bone health. In the 1930, bone health was improved dramatically by fortifying milk products with Vitamin D. This all but rid our country of Rickets. Fortified Milk is a major source of dietary Vitamin D in our nation. The use of Vitamin D is very important in treating other bone disease, most prominently in osteoporosis in our elderly females today. This is an example that earlier monitoring of calcium and vitamin D may reduce the number of women that suffer with osteoporosis. There have been numerous studies showing a statistical link between Vitamin D deficiency and several other diseases, these include cardiovascular disease, various cancers, diabetes, glucose intolerance, multiple sclerosis and autoimmune diseases. These statistical relationships are the reason that many physicians believe that Vitamin D is a good General Health marker. In non-skeletal disorders, the benefit of Vit D as a treatment is still being explored; the level of evidence varies between the patient populations being studied. Vitamin D deficiency is common in children and adults: In utero and during childhood, it can cause rickets (growth retardation and skeletal deformities) In adults, it can exacerbate osteopenia and osteoporosis, cause osteomalacia (the equivalent of rickets), muscle weakness, and increase the risk of fracture. Increasing evidence is also linking Vitamin D deficiency to many chronic illnesses, including common cancers, autoimmune diseases, and cardiovascular and infectious diseases. 34

34 Vitamin D Levels Focus on Musculoskeletal disease All rights reserved. 35

35 Vitamin D Levels and Fractures Bischoff-Ferrari HA et al. JAMA. 2005;293(18): Page 36 Five RCTs for hip fracture (n=9294) and 7 RCTs for nonvertebral fracture risk (n=9820) met the inclusion criteria. All trials used cholecalciferol. 36

36 Vitamin D Levels and Hip and Nonvertebral Fractures Meta-Analysis of Randomized Controlled Trials Vitamin D Dose 400 IU/d 800 IU/d 2.0 Hip Fracture 2.0 Nonvertebral Fracture Relative Risk 1.0 Relative Risk Achieved 25 (OH) Vit. D Level, nmol/l Achieved 25 (OH) Vit. D Level, nmol/l Adapted from: Bischoff-Ferrari HA et al. JAMA. 2005;293(18): Page 37 Results from studies on vitamin D supplementation and fractures have been heterogeneous. A meta-analysis of 7 RCTs revealed that optimal prevention of both nonvertebral and hip fracture occured ONLY in trials providing IU of vitamin D per day in patients whose baseline concentration of 25 (OH) vit. D was less than 17 ng/ml (42 nmol/l) and whose post-treatment concentration rose to approx. 40 ng/ml)! Importantly, vitamin D deficiency also causes muscle weakness; here too, the meta-analysis concluded that 800 IU per day (plus calcium) were necessary to significantly reduce the risk of falls in nursing home residents. 37

37 Vitamin D Levels Focus on Autoimmune, Cardiovascular, and Oncologic Diseases All rights reserved. 38

38 Vitamin D in Autoimmune Diseases All rights reserved. Vitamin D levels have been linked to autoimmune disease. 39

39 Vitamin D and Autoimmunity: Immune Regulator? Associated with immune system down regulation Dendritic cell maturation (inhibits) Release of cytokines from macrophages (inhibits) IL-2 and IFN-γ secretion by CD4 T cells (inhibits) Th1 cell proliferation and cytokine production (inhibits) B cell antibody production and secretion (inhibits) Adapted from Holick M, N Engl J Med (2007); 357: and Cutolo M et al., Autoimmunity Reviews (2007); 7:59-64 Page 41 The observed effects of the active metabolite of vitamin D (1,25(OH)D) on the immune system is consistent with observation that inadequate levels are associated with various autoimmune conditions. In general vitamin D effects are associated with immune system down regulation, both of cellular immunity and hormonal immunity. The level of evidence in non-musculoskeletal conditions such as autoimmune diseases is growing. This evidence is not as strong as the link between vitamin D levels and musculoskeletal pathologies (i.e. rickets, osteomalacia). 41

40 Vitamin D and Multiple Sclerosis (MS) Among white men and women from the US military personnel, the risk of MS significantly decreased with levels of 25(OH)D above 39.6 ng/ml (99.2 nmol/l). Munger KL et al., JAMA (2006); 296: Page 42 In this retrospective study on stored samples (148 MS cases in white individuals and 296 controls), the association between vitamin D deficiency and MS was most pronounced with samples from individuals less than 20 years of age. Such association was not found in blacks or Hispanic individuals. 42

41 Vitamin D and Multiple Sclerosis In France, this is also correlated with latitude (i.e. sunshine) Vitamin D (nmol/l) 100 Southwest South Southeast Paris Rhone-Alpes Center Nothwest Northeast North Prevalence p Adapted from Pierrot-Deseilligny C et al., J Neurol (2009); 256: Page 43 This graph from a French study confirms the association between low levels of vit.d and prevalence of MS; this association seems also related to the geographical location of the patients with the northern locations having the lowest levels of vit.d and the highest prevalence of the disease. 43

42 Vitamin D and Type 1 Diabetes Among children in Finland with vitamin D deficiency, the risk of type 1 diabetes was significantly increased, even after multiple adjustments. Suspected Rickets No Yes Adjusted Risk Ratio 1 (reference) 3.0 ( ) Given 2000 IU D 3 per day, these children had a risk for type 1 diabetes tremendously decreased (over a 31 years follow-up). Use of Vitamin D Supplements None Irregularly Regularly Adjusted Risk Ratio 1 (reference) 0.16 ( ) 0.12 ( ) Hyppönen E et al., Lancet (2001); 358: Page 44 This large study (n= 12,000) performed in Northern Finland showed that: 1- Children with vit.d deficiency (suspected rickets in the first year of life) had a 3 times higher risk of being diagnosed with type 1 diabetes in the following 30 years. 2- Supplementation with the recommended (for Finland) dose of vit.d considerably decreased the risk of developing type 1 diabetes in the following 30 years. 44

43 Vitamin D and Type 2 Diabetes in Women In a large cohort of middle-aged women (Nurses Health Study), both vitamin D and calcium intakes (from supplements rather than from diet) were significantly associated with a lower risk of type 2 diabetes. Risk Ratio* of Type 2 Diabetes (20 year f/up) Total calcium intake < 600 mg/day > 1,200 mg/day Total vitamin D intake < 400 IU/day > 800 IU/day 1.00 (reference) 0.75 ( ) 0.81 ( ) 0.67 ( ) * After adjustment for age, BMI, hypertension, family history of diabetes, smoking, physical activity, alcohol, state of residence, type of fat, cereal fiber, glycemic load, magnesium and retinol. Pittas AG et al., Diabetes Care (2006); 29: Page 45 Using samples from the Nurses Health Study, the authors looked at the benefit of vitamin D intake alone and in combination with calcium on the risk of developing type 2 Diabetes during the 20 year follow-up period of the study. Vitamin D and calcium had a positive effect on reducing the risk for type 2 diabetes, especially when used in combination. 45

44 Vitamin D in Cancer All rights reserved. Studies have shown an association between cancer risk and vitamin D levels. 46

45 Vitamin D Levels and Colorectal Cancer Mortality N = 16,818 From the Third National Health and Nutrition Examination Survey (NHANES III) Followed from through 2000 Freedman DM, et al. J Natl Cancer Inst 2007;99: Page 47 This very large study included patients from the Third National Health and Nutrition Examination Survey (NHANES III). They investigated whether total cancer mortality was linked to vitamin D levels (25 (OH)D). Abstract Background: Vitamin D has been hypothesized to reduce cancer mortality through its effects on incidence and/or survival. Epidemiologic studies of the association of 25-hydroxyvitamin D [25(OH)D] and the risk of cancer, however, have been largely limited to incident cancers at a few sites. Methods: A total of participants in the Third National Health and Nutrition Examination Survey who were 17 years or older at enrollment were followed from through Levels of serum 25(OH)D were measured at baseline by radioimmunoassay. Cox proportional hazards regression models were used to examine the relationship between serum 25(OH)D levels and total cancer mortality (in the entire population or according to race/ethnicity, sex, age, and retinol status) and mortality from specific cancers. Because serum was collected in the south in cooler months and the north in warmer months, we examined associations by collection season. All statistical tests were two-sided. Results: We identified 536 cancer deaths in person-years. Total cancer mortality was unrelated to baseline vitamin D status in the entire population, men, women, non-hispanic whites, non-hispanic blacks, Mexican Americans, and in persons younger than 70 or 70 years or older. We found no interaction between vitamin D and season or vitamin D and serum retinol. Colorectal cancer mortality was inversely related to serum 25(OH)D level, with levels 80 nmol/l or higher associated with a 72% risk reduction (95% confidence interval= 32% to 89%) compared with lower than 50 nmol/l, P trend =.02. Conclusions: Our results do not support an association between 25(OH)D and total cancer mortality, although there was an inverse relationship between 25(OH)D levels and colorectal cancer mortality. 47

46 Vitamin D Levels and Colorectal Cancer Mortality Vitamin D levels (ng/ml) Relative Risk < to < % CI 0.20 to to to 0.68 Colorectal cancer mortality was inversely related to serum 25(OH)D level, with levels 80 nmol/l [32 ng/ml] or higher associated with a 72% risk reduction (95% confidence interval= 32% to 89%) compared with lower than 50 nmol/l [20 ng/ml] P trend =.02. Freedman DM, et al. J Natl Cancer Inst 2007;99: Page 48 There was no relation between Vitamin D levels and overall cancer mortality. But there was a relationship between vitamin D levels and colorectal cancer mortality. 48

47 Vitamin D and Colorectal Cancer Survival N = 257 Measured before surgery Examined every 0.5 to 2 months Endpoints either death or relapse Mezawa et al. BMC Cancer 2010, 10:347 Page 49 This study investigated the risk of colorectal cancer death and vitamin D levels. Background notes Abstract Background: Recently, serum 25-hydroxyvitamin D (25OHD) levels were shown to be associated with the survival of patients with colorectal cancer. However, 25OHD levels were measured a median of 6 years before diagnosis or were predicted levels. In this study, we directly measured serum 25OHD levels at surgery and examined the association with survival among patients with colorectal cancer. Methods: We started a prospective cohort study to find prognostic factors in patients with colorectal cancer from 2003 to 2008 and stored serum samples and clinical data. As part of a posthoc analysis, serum 25OHD levels were measured by radioimmunoassay. Association between overall survival and serum 25OHD levels were computed using the Cox proportional hazard model adjusted for month of serum sampling as well as age at diagnosis, gender, cancer stage, residual tumor after surgery, time period of surgery, location of tumor, adjuvant chemotherapy and number of lymph nodes with metastasis at surgery. Unadjusted and adjusted hazard ratios (HR) and 95% confidence intervals (95% CI) were determined. Results: Serum 25OHD levels were measured in 257 patients. Only 3% had sufficient levels (30 ng/ml and greater). Based on month of blood sampling, an annual oscillation of 25OHD levels was seen, with levels being lower in spring and higher in late summer. Higher 25OHD levels were associated with better overall survival under multivariate analysis (HR, 0.91: 95% CI, 0.84 to 0.99, P = 0.027).Conclusions: These results suggest that higher 25OHD levels at surgery may be associated with a better survival rate of patients with colorectal cancer. 49

48 Vitamin D and Colorectal Cancer Survival: Deficient, Insufficient, or Sufficient? Most were deficient or insufficient P erc e n t (% ) Deficient (<15) Insufficient (<20) Sufficient (>30) Vitamin D (ng/ml) Mezawa et al. BMC Cancer 2010, 10:347 Page 50 Most the patients in the study, like most people in the general population, where either deficient or insufficient. In fact only 3% were sufficient with levels >30 ng/ml. 50

49 Vitamin D and Colorectal Cancer and Death rate: High Levels, Lower Death rates Higher levels are associated with a 84% reduction in death rate Vitamin D levels (ng/ml) Hazard ratio 95% CI to 0.63 Mezawa et al. BMC Cancer 2010, 10:347 Page 51 This study found that higher levels of vitamin D were associated with a 84% reduction in death rate. Or in other terms lower vitamin D levels are associated with a higher risk of colorectal cancer death. Background notes Multivariate hazard ratios, 95% confidence intervals, and P values were adjusted for age at diagnosis (years), gender, calendar month of blood sampling, cancer stage (I, II, III, and IV), residual tumor after surgery (R0, no residual tumor; R1, microscopic residual tumor; R2, macroscopic residual tumor), time period of surgery (between 2003 and 2005 or between 2006 and 2008), and number of lymph nodes with metastasis. 51

50 Vitamin D and Post-menopausal Breast Cancer Risk Selected from the MARIE study (Mamma Carcinoma Risk factor Investigation) 1394 post-menopausal breast cancer cases 1365 controls, matched for year of birth and time of blood collection Abbas A, et al. Carcinogenesis vol.29 no.1 pp.93 99, 2008 Page 52 This large study of post-menopausal women in Germany evaluated vitamin D levels and risk for postmenopausal breast cancer. Background information Abstract Various studies suggest that vitamin D may reduce breast cancer risk. Most studies assessed the effects of dietary intake only, although endogenous production is an important source of vitamin D [25(OH)D] better indicates overall vitamin D status. To assess the association of 25(OH)D serum concentrations with postmenopausal breast cancer risk, we used a population-based case control study in Germany, which recruited incident breast cancer patients aged between 2002 and Information on sociodemographic and breast cancer risk factors was collected by personal interview. For this analysis, we included 1394 cases and 1365 controls, matched on year of birth and time of blood collection. Conditional logistic regression was used to calculate odds ratios (ORs) for breast cancer adjusted for potential confounders. Serum 25(OH)D concentration was significantly inversely associated with post-menopausal breast cancer risk. Compared with the lowest category (<30 nm), OR [95% confidence intervals (CI)] for the higher categories of 25(OH)D (30 45, 45 60, and 75nM) were 0.57 ( ), 0.49 ( ), 0.43 ( ) and 0.31 ( ), respectively (Ptrend < ). Analysis using fractional polynomials indicated a non-linear association. Theassociation was stronger in women never using menopausal hormone therapy (HT) compared with past and current users (Pinteraction < ). Our findings strongly suggest a protective effect for post-menopausal breast cancer through a better vitamind supply as characterized by serum 25(OH)D measurement, with a stronger inverse association in women with low serum 25(OH)Dconcentrations (<50 nm). 52

51 Vitamin D and Postmenopausal Breast Cancer Risk: High levels, Lower risk Vitamin D levels (ng/ml) <12 Adjusted model OR (95% CI) 1 Levels >30 ng/ml associated with a ~70% reduced risk P trend < ( ) 0.49 ( ) 0.43 ( ) Vitamin D Per 4 ng/ml incremental increase Odds Ratio (95% CI) 0.88 ( ) ( ) Abbas A, et al. Carcinogenesis vol.29 no.1 pp.93 99, 2008 Page 53 Higher levels of vitamin D are associated with lower risk for postmenopausal breast cancer. These differences are statistically significant. Levels >30 ng/ml were associated with a risk reduction of 70%. Each 4 ng/ml increase is associated with a 12% drop in risk. This data is from a conditional logistic regression stratified by time of blood collection and year of birth adjusted for age at menopause, first-degree family history of breast cancer, history of benign breast disease, number of pregnancies (28 th week), age at menarche, breastfeeding history, total number of mammograms, use of HT, BMI, education level, smoking status; due to missing values, 40 observations were not included in the adjusted model. 53

52 Vitamin D and Ovarian Cancer Risk Cases 172 Controls 172 Matched for age, parity, date of blood draw Excluded cases diagnosed with cancer within 3 yrs Toriola AT et al., Eur J Cancer (2010), doi: /j.ejca Page 54 This study evaluated ovarian cancer risk and vitamin D levels. It was a case-control study. Each case was matched to a control based on age, parity and date of blood draw. Background notes Abstract Introduction: Ovarian cancer has very few known modifiable risk factors but dietary studies suggest a role for vitamin D and calcium in the prevention of ovarian cancer. Thus, we investigated the association between pre-diagnostic serum calcium and 25- hydroxyvitamin D (25-OHD) on the risk of later development of ovarian cancer. Methods: We conducted a population-based nested case control study within the Finnish Maternity Cohort (FMC). The cohort subset comprised 172 ovarian cancer cases with 172 matched controls (age ± 1 year, parity and season of blood donation ± 2 weeks). Results: We observed a significant inverse association between calcium and ovarian cancer risk. Relative risk (estimated as odds ratio, OR) comparing the highest quartile to the lowest quartile was significantly decreased; 0.41 [95% confidence interval (CI) , P trend 0.004]. Even though a comparable association between 25-OHD and ovarian cancer did not reach statistical significance (OR 0.57, 95% CI , P-trend 0.07), having sufficient (>75 nmol/l) serum 25-OHD levels compared to insufficient serum 25-OHD was associated with a significantly decreased risk of ovarian cancer (OR 0.32; 95% CI , p-value 0.03). No synergistic protective interaction between high levels of calcium and 25-OHD against ovarian cancer was observed, the joint effect being just multiplicative. Conclusion: Calcium and vitamin D act independently to reduce the risk of ovarian cancer. The decreased risk of ovarian cancer associated with pre-diagnostic serum calcium and vitamin D needs to be evaluated further for possible new insights into ovarian cancer prevention. 54

53 Vitamin D and Ovarian Cancer Risk: High levels, lower risk Risk for ovarian cancer is 3-fold lower for higher levels of vitamin D O d d s ra tio fo r o v a ria n c a n c e r > 30 Vitamin D ng/ml Toriola AT et al., Eur J Cancer (2010), doi: /j.ejca Page 55 Higher levels of vitamin D are associated with a lower risk for cancer. About 3-fold lower. 55

54 Vitamin D in Cancer: Key Points Lower levels associated with higher risk for Breast cancer Ovarian cancer Lower levels associated with higher mortality, lower survival in Colorectal cancer Page 56 56

55 Vitamin D in Cardiovascular Disease All rights reserved. Vitamin D levels are also associated with CVD risk. 57

56 Vitamin D Levels CVD and Mortality Risk Drechsler C, et al. European Heart Journal (2010) 31, Page hydroxyvitamin D [25(OH)D] was measured in 1108 diabetic hemodialysis patients who participated in the German Diabetes and Dialysis Study and were followed up for a median of 4 years. Abstract Aims: Dialysis patients experience an excess mortality, predominantly of sudden cardiac death (SCD). Accumulating evidence suggests a role of vitamin D for myocardial and overall health. This study investigated the impact of vitamin D status on cardiovascular outcomes and fatal infections in haemodialysis patients. Methods and results: 25-hydroxyvitamin D [25(OH)D] was measured in 1108 diabetic haemodialysis patients who participated in the German Diabetes and Dialysis Study and were followed up for a median of 4 years. By Cox regression analyses, we determined hazard ratios (HR) for pre-specified, adjudicated endpoints according to baseline 25(OH)D levels: SCD (n ¼ 146), myocardial infarction (MI, n ¼ 174), stroke (n ¼ 89), cardiovascular events (CVE, n ¼ 414), death due to heart failure (n ¼ 37), fatal infection (n ¼ 111), and all-cause mortality (n ¼ 545). Patients had a mean age of 66+8 years (54% male) and median 25(OH)D of 39 nmol/l (interquartile range: 28 55). Patients with severe vitamin D deficiency [25(OH)D of 25 nmol/l] had a 3-fold higher risk of SCD compared with those with sufficient 25(OH)D levels.75 nmol/l [HR: 2.99, 95% confidence interval (CI): ]. Furthermore, CVE and all-cause mortality were strongly increased (HR: 1.78, 95% CI: , and HR: 1.74, 95% CI: , respectively), all persisting in multivariate models. There were borderline non-significant associations with stroke and fatal infection while MI and deaths due to heart failure were not meaningfully affected. Conclusion: Severe vitamin D deficiency was strongly associated with SCD, CVE, and mortality, and there were borderline associations with stroke and fatal infection. Whether vitamin D supplementation decreases adverse outcomes requires further evaluation. 58

57 Vitamin D Levels and CVD and Mortality Risk Hazard ratio Sudden cardiac death Cardiovascular events All-cause mortality >30 ng/ml <10 ng/ml Drechsler C, et al. European Heart Journal (2010) 31, Page 59 Patients with severe vitamin D deficiency <10 ng/ml (30 nmol/l) had a higher risk of sudden cardiac death, cardiovascular events (MI, stroke, death due to heart failure and combined events) and all-cause mortality compared to persons with levels > 30ng/mL (75 nmol/l). 59

58 Vitamin D and Cardiovascular Disease Risk 1739 Framingham Offspring Study participants without CVD Mean follow up 5.4 years 120 developed a first CVD event Wang TJ, et al. Circulation. 2008;117: Page 60 This study use participants from the Framingham Offspring study. All participants were Caucasian due to the selection of the original cohort. None of these patients had CVD at study onset. Mean follow up was 5.4 years and the cutoff with 15 ng/ml. During the course of the study 120 developed a CVD event. CVD events included myocardial infarction, coronary insufficiency (prolonged chest pain with documented ECG changes), angina, stroke, transient ischemic attack, peripheral claudication, or heart failure. Abstract Background Vitamin D receptors have a broad tissue distribution that includes vascular smooth muscle, endothelium, and cardiomyocytes. A growing body of evidence suggests that vitamin D deficiency may adversely affect the cardiovascular system, but data from longitudinal studies are lacking. Methods and Results We studied 1739 Framingham Offspring Study participants (mean age 59 years; 55% women; all white) without prior cardiovascular disease. Vitamin D status was assessed by measuring 25-dihydroxyvitamin D (25-OH D) levels. Prespecified thresholds were used to characterize varying degrees of 25-OH D deficiency (15 ng/ml, 10 ng/ml). Multivariable Cox regression models were adjusted for conventional risk factors. Overall, 28% of individuals had levels 15 ng/ml, and 9% had levels 10 ng/ml. During a mean follow-up of 5.4 years, 120 individuals developed a first cardiovascular event. Individuals with 25-OH D 15 ng/ml had a multivariableadjusted hazard ratio of 1.62 (95% confidence interval 1.11 to 2.36, P0.01) for incident cardiovascular events compared with those with 25-OH D 15 ng/ml. This effect was evident in participants with hypertension (hazard ratio 2.13, 95% confidence interval 1.30 to 3.48) but not in those without hypertension (hazard ratio 1.04, 95% confidence interval 0.55 to 1.96). There was a graded increase in cardiovascular risk across categories of 25-OH D,with multivariable-adjusted hazard ratios of 1.53 (95% confidence interval 1.00 to 2.36) for levels 10 to 15 ng/ml and 1.80 (95% confidence interval 1.05 to 3.08) for levels 10 ng/ml (P for linear trend 0.01). Further adjustment for C-reactive protein, physical activity, or vitamin use did not affect the findings. Conclusions Vitamin D deficiency is associated with incident cardiovascular disease. Further clinical and experimental studies may be warranted to determine whether correction of vitamin D deficiency could contribute to the preventionof cardiovascular disease. 60