Vitamin D By Andrea M. Rose, PhD, MBA

Medical Laboratory Observer, May 2013 Vol. 45 No. 5
Excerpts by Connie Cailer

Vitamin D has become one of the most widely discussed and intensely scrutinized supplements in recent history. The renewed interest is due to the startling prevalence of vitamin D deficiency worldwide and linking this to multiple clinical conditions other than bone health. Vitamin D is a naturally occurring, biologically inert hormone precursor that exists in two primary forms: vitamin D2(ergocalciferol) and vitamin D3 (cholecalciferol). Both forms are converted in the liver to the body’s main storage form of vitamin D, known as calcidiol, and then in the kidneys to the physiologically active form, or calcitriol. In this final form, vitamin D acts as a hormone. Its main biologic function is to maintain serum calcium and phosphorous concentrations within the normal range by enhancing the efficiency of the small intestine in absorbing these minerals from the diet. The interaction of calcitriol with vitamin D receptors increases the efficiency of intestinal calcium absorption from only 10% or 15% to 30% or 40% and phosphorous absorption from 60% to approximately 80%. 

When dietary intake is inadequate to satisfy the body’s calcium requirement, vitamin D facilitates increased calcium reabsorption in the kidney and works with parathyroid hormone (PTH) to mobilize calcium stores from the bone, effectively increasing serum levels of calcium. Because of its role in maintaining calcium homeostasis, vitamin D is essential to overall bone health, promoting healthy growth and remodeling. An insufficiency leads to thin, brittle or misshapen bones and can con-tribute to rickets in children and in adults, weak bones and osteomalacia. Along with calcium, vitamin D also helps prevent osteoporosis in her physiological roles of vitamin D include maintaining muscle strength, modulating immune function, regulating cellular differentiation, and reducing inflammation. A growing body of research also suggests that vitamin D might play a role in the prevention and treatment of number of diseases, including type 1 and type 2 diabetes, hypertension, glucose intolerance, multiple sclerosis, and other medical conditions, including cancer.

The major source of vitamin D for humans is the sun. VitaminD3 can be synthesized in the skin upon exposure to ultraviolet-B (UVB) radiation from sunlight, sun exposure accounts for about 80% to 90%of vitamin D for most people. According to Mayo clinic, a single exposure to summer sun in a bathing suit for 20 minutes produces the equivalent of 15000 to 20000 IU of vitamin D3. It can also be obtained from the diet, in the form of either D2 or D3. Very few foods naturally contain vitamin D, fatty fish (such as salmon, tuna and mackerel, cheese and egg yolks). These foods primarily contain vitamin D3. Some mushrooms provide vitamin D2 in variable amounts. Most Americans receive the bulk of dietary vitamin D from supplements or fortified food, such as milk, cereals, orange juice, and yogurt. In these sources vitamin D is available in D2 and D3 form, over the counter supplements are typically fortified with vitamin D3 rather than D2. However, preparations of vitamin D for prescription are still in the form of vitamin D2. 

Because vitamin D obtained from sun exposure, food and supplements is biologically inert, it must undergo two processes in the body for activation. The first occurs in the liver, where it is converted to 25-vitamin D status, it allows the detection and monitoring of vitamin D deficiency. It reflects vitamin D produced by the skin (from sunlight) and obtained from food and supplement for activation. The first occurs in the liver, where it is converted to 25-hydroxyvitamin D [25(OH) D], also known as calcidiol. This pre-hormone is the body’s main storage form of vitamin D, and the amount of calcidiol avail-able to the body is what determines vitamin D status. Guidelines for recommended vita-min D levels are referring to calcidiol levels. Mostly bound to vitamin D binding protein, the 25(OH) D is secreted to blood plasma where because of its relatively long half-life of two to three weeks, it serves as reservoir for further hydroxylation. A second hydroxylation occurs in the kidney, where 25(OH) D is converted to physiologically active form 1,25 dihydroxy vitamin D[1,25(OH 2D], known as calcitriol, a potent steroid hormone. The kidney secretes calcitriol into circulation, again bound to vitamin D binding proteins, where it travels to tissues involved in regulation of calcium and phosphorus supply, namely intestine, bone, para-thyroid glands, and the kidney itself. Once in circulation, the half-life of calcitriol is very short compared to that of calcidiol-only about four to six hours. 

In the past, vitamin D deficiency was identified by a physical rather than a biochemical manifestation, the presence of bone disease, rickets or the adult equivalent, osteomalacia. Clinical symptoms of vitamin D deficiency can include chronic, nonspecific musculoskeletal pain, weakness, and fatigue that is non-specific as to age, mobility, sex, or ethnic group. Today, serum concentrations of 25(OH)D (calcidiol) is the best indicator of vitamin D status, it allows the detection and monitoring of vitamin D deficiency. It reflects vitamin D produced by the skin (from sunlight) and obtained from food and supplements. 

Calcidiol functions as biomarker of exposure, but it is not clear to what extent 25(OH) D levels serve as biomarker of effect (i.e., relating to health status or outcomes). 

The Endocrine Society and the National Kidney Foundation have established guidelines as shown below: 

Organization Endocrine Society; NK Foundation.

• Sufficient: 30-100 ng /ml >30 ng /ml 
• Insufficient: 21-29 ng/ml; 16-30 ng/ml 
• Deficiency: <20ng/ml <15ng/ml

The preferred level for vitamin D now recommended by many experts is ≥30ng/ml. 
The Endocrine Society recommends vitamin D screening for individuals at risk for deficiency but not for general population screening for those who are not at risk. 

Diseases and conditions: 
—Rickets, Antiseizure medications African- American& Hispanic children 
—Osteomalacia, Glucocorticoids and adults 
—Osteoporosis, AIDS medications Pregnant and lactating women
—Chronic kidney disease, Antifungals Older adults with history of non-traumatic Hepatic failure, Cholestyramine fractures 
—Malabsorption Syndromes: Obese children and adults (BMI 30kg/m2)) 
—Granuloma-forming disorders: Some lymphomas 

The number of individuals at risk is significant and growing. About one-third of Americans have vitamin D levels that are less than adequate for bone and overall health in healthy individuals. Since 1994, the number of Americans with 25(OH)D levels under 30ng/ml (the NKF threshold for insufficiency/deficiency) has doubled. The downward trend in vitamin D levels is associated with a decline in consumption of milk that is fortified with vitamin D, decrease sun exposure and increase use of sun-screen, and an increase in body mass index (BMI) worldwide. (A rise in BMI causes more vitamin D to be sequestered in subcutaneous fat and not released into the circulation.) 

As research links vitamin D deficiency to disease states other than bone disorders, the volume of vitamin D testing continues to increase throughout the world. New methodologies have been developed to help labs meet the increased demand for testing, including both immunoassay and protein binding assays, some of which run on automated platforms. There are several factors that a lab should assess in order to evaluate ROI and determine which platform and assay best meet its needs: (1) the cost of the equipment, especially if the testing method requires a dedicated system; (2) the complexity of the testing method in terms of staff time and proficiency requirements; and (3) the efficiency of the testing method and the relative balance of throughput with demand. For assessment of vitamin D status in patients at risk for deficiency, Endocrine Society guidelines recommend serum circulating [25(OH) D] level measured by a reliable assay. Otherwise known as total vitamin D assay, it should be able to recognize both vitaminD2 and D3metabolites. 

While LC_MS and immunoassay are the two most common methods used for vitamin D testing today, considerable variability exists among the assays available and among the laboratories that conduct the analyses, due to lack of standardization. In order to address this issue, the NIH office of Dietary Supplements has established the Vitamin D Standardization Program (VDPS) in collaboration with the Centers for Disease Control and Prevention, the National Institute for Standards and Technology (NIST) and Ghent University. The aim of the program is to standardize the laboratory measurement of vitamin D status in national health surveys worldwide by linking them to the NIST reference measurement procedure (RMP). The American Association for Clinical Chemistry (AACC) and the College of American Pathologist (CAP) are among the collaborative partners in the project. The first step in developing a vitamin D standardization protocol is to develop a reference system in order to establish a metrological chain of traceability from an assay to the reference method procedure. This has already been done with the NIST and Ghent labs using LC-MS/MS. 

In addition, the CDC has developed a certification program that will monitor and certify the accuracy and precision of vitamin D test methods on a yearly basis. Once the test method is standardized to the RMP, participants can submit results from four quarterly challenges, which involve 10 blinded, single donor serum samples per challenge. These four challenges are used to determine whether the method can meet an imprecision goal of a CV of <=10% and bias of< =5%. These criteria are based on data on biological variability for vitamin D. So the laboratory may want to confirm that the test manufacturer is participating in this CDC certification program to verify end user performance for its vitamin D assay. 

Connie Cailer, CLS