American Association for Clinical Chemistry
Better health through laboratory medicine
December 2007 Clinical Laboratory News: The New Vitamin D

December 2007: Volume 33, Number 12

The New Vitamin D  
What’s Putting Fresh Emphasis on Lab Measurements?
By Gina Rollins

Vitamin D is increasingly gaining recognition as a versatile agent that not only supports healthy bone formation and neuromuscular function but also plays a role in the development and progression of certain cancers, autoimmune disorders, and infectious diseases. Recent research has linked vitamin D to type 1 diabetes, multiple sclerosis, tuberculosis, and other conditions such as Alzheimer’s disease and psoriasis. As evidence mounts on the therapeutic potential of vitamin D, clinicians will likely pay closer attention to patients’ serum levels. Yet, questions remain about proper dietary amounts—both for the general population and disease treatment—as well as appropriate target levels of serum vitamin D, placing critical emphasis on the lab’s role in providing accurate test results.

Indeed, the familiar vitamin D has earned a lot of attention lately, say those who study it. “When I entered the field we didn’t know much about how vitamin D worked,” said Sylvia Christakos, PhD, professor of biochemistry and molecular biology at the University of Medicine and Dentistry of New Jersey in Newark. “Now we’re at the point where it is being used therapeutically to affect disease states. For me, the next wave will be when we translate the new discoveries into curing disease.”

The renewed interest in vitamin D among researchers like Christakos originates from many different areas. For example, in the AIPC Study of Calcitriol Enhancing Taxotere Trial (ASCENT), treatment with the high-dose vitamin D metabolite calcitriol in combination with the chemotherapy drug docetaxel improved survival by 8 months in patients with progressive metastatic prostate cancer. Even more far-reaching are the results of a recent meta-analysis that found a correlation between vitamin D supplementation and a 7 percent reduction in all-cause mortality.

A Tightly Regulated System

While researchers continue to investigate the role of vitamin D in cancer and disease, much is already known about the important role of vitamin D in maintaining organ systems. “It’s a very elegant system,” said Hector DeLuca, PhD, Harry Steenbock research professor at the University of Wisconsin-Madison at a 2007 AACC Annual Meeting Symposium, “Vitamin D Reinvented.” DeLuca’s laboratory has been devoted to the understanding of metabolism and mechanism of action of vitamins A and D since 1960.

About 80 percent of vitamin D used in the body is produced photochemically when ultraviolet radiation from sunlight reacts with 7-dehydrocholesterol, a precursor sterol in the skin, producing vitamin D3 or cholecalciferol. This is the opening action of the multi-sequenced vitamin D endocrine system (See Figure). The liver then metabolizes vitamin D3 into 25-hydroxycholecaciferol (25[OH]D3) or calcidiol, the main form of vitamin D circulating in the blood. The kidney, functioning as an endocrine gland, converts this precursor into two principal dihydroxylated metabolites or calcitriol, 1α,25[OH]2D3—the primary active form of vitamin D—and 24R,25(OH)2 D3. After binding to the vitamin D-binding protein, 1α,25[OH]2D3 is transported to various target organs, including bone, the intestines, and kidneys, where it binds to receptors and carries out further biochemical actions.

The primary and best understood action of vitamin D is to maintain plasma calcium and phosphorus levels at concentrations that support normal neuromuscular function and skeletal mineralization. Vitamin D deficiency results from inadequate intake coupled with inadequate sunlight exposure. Shortages of the prohormone leads to rickets in children and osteomalacia in adults (See Sidebar, below). Both develop when, with no other available source, the body robs calcium from the bones. This also leads to secondary hyperparathyroidism from the parathyroid gland’s continual attempts to open the calcium floodgates.



In Target Cells

What has researchers excited is the possibility of preventing or at least slowing the progression of many diseases like cancer via vitamin D’s actions in target cells. A nuclear receptor protein, termed VDR, with ligand- and DNA-binding domains has been found in more than 30 different cell types. Researchers have discovered at least 50 genes that are regulated by this complex; however, there appears to be a threshold amount needed to initiate activity, according to DeLuca. “A certain level of receptor is required before it functions,” he noted. “Why is it expressed in some tissues and not others? I don’t know the answer, nor do I know anyone in the field who knows the answer.”

In cells where the vitamin-D receptor is active, including lymphocytes, promyelocytes, and keratinocytes, it forms a complex with the retinoid-X receptor, which binds to DNA—often to activate transcription, but in some instances to suppress it. In most if not all the target tissues, vitamin D also induces the enzyme CYP-24 hydroxylase that starts the pathway to break down vitamin D itself into water-soluble calcitroic acid, which is then excreted into urine. “By initiating its own destruction, there’s a system to prevent overactivity. It’s an auto-regulation,” explained David Feldman, professor of medicine at Stanford University (Palo Alto, Calif.).

The mechanism of how vitamin D plays a role in the prevention or progression of various diseases has not been established definitively. In the case of cancer, 1α,25[OH]2D3 apparently inhibits cell proliferation and stimulates cell differentiation. In certain malignancies, like prostate cancer, it also appears to act as an anti-inflammatory agent by downregulating the activity of cyclo-oxygenase II (COX-II), the critical enzyme for synthesis of inflammatory prostaglandins, while at the same time stimulating prostaglandin dehydrogenase, which destroys prostaglandins. An analogous process may be at work in multiple sclerosis.

“Right now we believe that 1 alpha, 25-dihydroxy vitamin D3 downregulates pro-inflammatory cytokines including interleukin 12, interleukin 2, and interferon gamma, and that it enhances cytokines that inhibit inflammation, including interleukin 4 and interleukin 10,” explained Christakos.

Vitamin D also has been proposed as a mediator of immune response to bacterial antigens like tuberculosis bacillus. Research indicates that 1α,25[OH]2D3 stimulates immune response in antigen-presenting cells like macrophages, while also curtailing excessive response in immune system cells like lymphocytes. Low levels of calcidiol have been found in Alzheimer’s patients, and its neuroprotective role may work by modulating calcium homeostasis and the production of neurotrophins. In addition, a role for vitamin D has been suggested in treating osteoporosis, in increasing muscle function and thereby preventing falls in the elderly, in preventing the development of preeclampsia in pregnancy, in decreasing the risk of hypertension, and even in lowering the chance of postmenopausal women developing overactive bladder.

Spotlight on Epidemiology

Aside from biochemical discoveries, a host of epidemiologic studies also have pointed to a connection between sunlight exposure, a surrogate for vitamin D levels, and various diseases, including multiple sclerosis, type 1 diabetes, and various cancers, most notably breast, colon, and prostate. In general, people who live closer to the equator, where UV rays are stronger, have a lower incidence of these conditions. As latitude increases, however, so does the incidence of these diseases. For instance, a child in Finland is 35 times more likely to develop type 1 diabetes than one in South China or other countries near the equator, according to Cedric Garland, DPH, cancer prevention specialist at Moores Cancer Center at the University of California, San Diego, and a speaker at the AACC symposium on vitamin D.

As epidemiologic evidence has accumulated, researchers have been able to establish a dose–response relationship between sunlight and serum vitamin D levels. “Each degree of latitude corresponds to 1 ng/mL of 25-hydroxy vitamin D3. If you’re at latitude zero degrees at the equator, your 25-hydroxy vitamin D3 is going to be about 60 ng/mL. If you’re at 60 degrees latitude in southern Alaska or Scandinavia, it will be near the range of detection, somewhere between zero and 10 ng/mL in the winter,” Garland explained. He estimates that the naturally occurring level of vitamin D in people with adequate sun exposure is about 65 ng/mL, which can be quite beneficial. His research indicates that serum calcidiol levels of 55 ng/mL are optimal for cancer prevention.

Using data from GLOBOCAN, a new World Health Organization database of cancer incidence, mortality, and prevalence in 177 countries, Garland estimates that up to 250,000 cases of colorectal cancer and 350,000 cases of breast cancer could be prevented worldwide by boosting vitamin D intake to 55 ng/mL.

A Brief History of Vitamin D

First identified in the 1920s, vitamin D is not a vitamin in the classic sense of being a nutrient the body needs in small amounts but can’t make on its own. In reality, it is a prohormone that is biologically inactive until it is metabolized into a secosteroid, similar to classic steroid hormones like testosterone, progesterone, and cortisol. Vitamin D is available both from sunlight and dietary sources such as cod liver oil, fatty fish like salmon, and vitamin D-fortified milk. Individuals who receive enough sunlight on a regular basis do not need vitamin D from their diet.

A series of discoveries dating from the 1960s established that vitamin D is involved in a tightly regulated endocrine system involving the liver and kidneys, as well as multiple target organs. However, for public health and nutritional reasons it continues to be classified officially as a vitamin. Vitamin D is an umbrella term for vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol), along with metabolites and analogues of these substances.

How Much Intake?

As strong as the role of vitamin D in preventing various diseases appears to be, the medical community hasn’t made sweeping recommendations that the entire population become either sun worshippers or habitual cod liver oil drinkers, for several reasons. First, the sunlight-vitamin D dose-response relationship is multifactorial. Research indicates that the lightest skinned people who sunburn easily and rarely tan need a scant 2 percent to 10 percent the amount of sun exposure to produce a unit of vitamin D as those with the darkest skins.

Latitude, season, cloud cover, pollution, and shade play roles as well. In North America at latitudes above San Francisco and Philadelphia, ultraviolet rays are not powerful enough to generate substantial amounts of vitamin D during the winter. Complete cloud cover halves the energy of ultraviolet rays; shade cuts it by nearly two-thirds. Sunscreens with a sun protection factor of 8 or higher also block UV rays, effectively cutting vitamin D production by 95 percent.

Obesity and age play roles as well. Since vitamin D is fat soluble, it is deposited in fatty tissue, and people who are overweight can’t access it as readily as others. By age 65, changes in the skin may reduce vitamin D production by as much as 60 percent. People with conditions that involve fat malabsorption like Crohn’s disease, pancreatic enzyme deficiency, and celiac disease also absorb less vitamin D.

But experts disagree about the proper levels of vitamin D required to promote health or prevent vitamin D toxicity. When the Food and Nutrition Board of the Institute of Medicine (IOM) set dietary reference intakes for vitamin D in 1997, the body of evidence for optimal or safe levels of vitamin D was not substantial. Lacking sufficient data to set a recommended daily allowance for all healthy individuals, the IOM group instead issued adequate intake levels for various age and sex groups, based on maintaining serum calcidiol levels at 37.5 nmol/L. The adequate intakes are 200 IU (5 mg) per day for all individuals from newborns to age 50, 400 IU (10 mg) for people between the ages of 51 and 70, and 600 IU (15 mg) per day for men and women older than 70.

Recent research suggests that vitamin D insufficiency is widespread, particularly among members of minority groups. For instance, data from a National Health and Nutrition Examination Survey found that 42 percent of African American women had hypovitaminosis, compared with just 4 percent of Caucasians.

But what is the optimal level? “My feeling is that no person should have less than 80 nmol [32 ng/mL]. That’s based on cumulative data,” said Bruce Hollis, PhD, professor of pediatric biochemistry and molecular biology and director of pediatric nutrition sciences at the Medical University of South Carolina in Charleston. He believes the standard for “normal” was set incorrectly decades ago when the first assays for vitamin D were developed. “They sampled asymptomatic populations and measured circulating serum 25-hydroxy vitamin D3 [calcidiol], but if the community was deficient, it’s not a good standard,” he explained. His recent research suggests that optimal vitamin D status may be when there are equimolar concentrations of calcidiol and the parent compound, vitamin D3.

At least one person who sat on the IOM committee that set the original standards believes the recommended intake amounts eventually will be raised to 1,000 IU per day. But, cautions Michael Holick, PhD, MD, professor of medicine, physiology, and biophysics at the Boston University School of Medicine, “It’s not going to happen in the near future.” Christine Stencel, a spokesperson for the IOM, agrees that updates to vitamin D intakes are probably a ways off. “There’s been a lot of discussion on the question of the criteria for when to review the science, and we stand ready to conduct scientific evaluations,” she noted. But without a mandate from a federal agency to conduct a review, the IOM cannot conduct an evaluation.

Another issue up for debate is the amount of vitamin D one can take before inducing toxicity in the form of kidney stones, bone loss, and even calcification of certain organs. As with the intake amounts, the Food and Nutrition Board had little scientific evidence to go by in 1997, so it set what is now considered a very conservative tolerable upper intake level of 2,000 IU (50 mg) per day for children older than age one and all adults.

While studies have found no adverse affects from oral doses as high as 10,000 IU, there is not expert consensus about what the upper limit for nontherapeutic consumption should be. In the opinion of Hollis, the present 2,000 IU upper limit is “a disaster. It’s totally wrong. I can’t tell you how many studies have had doses of 10,000 IUs and not observed hypercalcemia,” he proclaimed.

Others sound a note of caution. “I think it should be individualized to some extent,” said Feldman. “That’s the problem with a public health message intended for thousands of healthy people. There are individuals in the population who tend towards making kidney stones, or who may develop vascular calcification and cardiovascular disease, and you have to take them into account in deciding on the recommended dose. I think the dose recommendation definitely should be increased and the range considered vitamin D sufficient should be raised, but I’m leery of a huge dose for the general population until more data proving the benefits in disease prevention are developed. However, in people who already have cancer or other diseases, or who are at increased risk of developing such diseases, higher doses may already be indicated. For example, in such individuals, the risk-benefit ratio is different and they can be given higher amounts.” Many vitamin D-related studies are using high-dose analogues, which have less calcemic activity, often in combination with chemotherapy or anti-inflammatory agents, he noted.

In the absence of official updates, some professional groups are beginning to take the lead in recommending higher intakes. In Canada, for example, Health Canada-endorsed adequate dietary amounts mirror those in the U.S., but the Canadian Paediatric Society issued updated guidelines in September 2007 calling for administration of 2,000 IU per day to pregnant and lactating women. For full-term infants, the guidelines recommend 400 IU per day, with 800 IU per day in winter months at higher latitudes.

Accurate Assays a Must

While scientists and professional groups deliberate on vitamin D intake amounts, laboratorians have a role in the here-and-now of patient care. The quality of commercial tests has improved significantly over the past two decades, according to Graham Carter, MSc, organizer of the Vitamin D External Quality Assessment Scheme

(DEQAS), a London-based program to ensure the analytical reliability of calcidiol and calcitriol assays, in which more than 70 U.S. labs participate. Several of the most commonly used commercial tests are capable of producing consistently accurate results, but there is still a “considerable degree” of variation between laboratories, he says. There also have been problems with certain assays, particularly in having sufficient reactivity to 25-hydroxyvitamin D2. For that reason, Carter urges laboratorians to participate in quality assessment programs and be familiar with the limitations of the methods they are using. “If your assay underestimates 25-hydroxyvitamin D2, then clinicians should be told about it,” he recommended.

In addition to DEQAS, the College of American Pathologists offers quality assessment for vitamin D assays. The National Institute of Standards and Technology (NIST) expects to make standard reference materials available in early 2008, according to Karen Phinney, PhD, a research chemist at NIST.

Laboratorians also can help spread the word about the necessity of vitamin D testing, says Feldman. “They can help put it on the radar screen. Most physicians don’t measure it unless they’re treating patients with osteoporosis, for example. But it needs to be part of the world of good medical practice, what we think of in terms of management of good health.”

Gina Rollins is a freelance writer based in Silver Spring, Md.

Recommended Reading

For more on the link between vitamin D and reduction in all-cause mortality, see:
Autier P and Gandini S. Vitamin D supplementation and total mortality: a meta-analysis of randomized controlled trials. Arch Intern Med 2007;167:1730–1737.

For more on outcomes from the ASCENT trial, see:
Beer TM, Ryan CW, et. al. Double-blinded randomized study of high-dose calcitriol plus docetaxel compared with placebo plus docetaxel in androgen-independent prostate cancer: a report from the ASCENT Investigators. J Clin Oncol 2007;25(6):669–74.

For more on vitamin D epidemiology, see:
Garland CF, Grant WB, et. al. What is the dose-response relationship between vitamin D and cancer risk? Nutr Rev 2007;65(8 Pt 2):S91-5.

For more on vitamin D testing, see:
Hollis BW and Horst RL. The assessment of circulating 25(OH)D and 1,25(OH)2D: where we are and where we are going. J Steroid Biochem Mol Biol 2007; 103(3-5):473-476.