Vol. 25 • Issue 4 • Page 18
Chemistry
In recent years, vitamin D testing has become increasingly popular. The new trend is fueled by a surge of research associating low levels of this prohormone with a weakened immune system and that high or sufficient levels could reduce the incidence of certain cancers and other diseases. But before the assay is ordered, it’s important to understand the mechanics of the testing methods in order to better understand the results.
“Because there are different forms of vitamin D and different mechanisms to identify them, not to mention whether to separate the components or not, there are dozens of ways they can be quantified and measured,” explained Nicole Riddle, MD, laboratory director at Marshall Medical Centers for Cunningham Pathology and member of the College of American Pathologists (CAP). “And making an edict that one specific test should be used is not only illegal in the U.S., but might not actually be the best option, as each lab has different capabilities and could possibly be forced to perform a test they are less capable of, thereby affecting patient care.”
People who have inadequate sun exposure, limited oral intake or impaired intestinal absorption are good candidates for testing, and that group accounts for a large portion of the population. In fact, some studies estimate that more than 50% of the institutionalized elderly and an equal proportion of women undergoing treatment for osteoporosis have inadequate levels.1
“Vitamin D is necessary to absorb calcium and promote bone growth. Inadequate vitamin D intake can result in soft bones in children, called rickets, and a softening of bones, making them fragile in adults, called osteomalacia, and osteoporosis where bones become brittle and weak,” said Toni McKinnon, RN, director of science information at USANA Health Science, explaining vitamin D’s traditional uses.
Testing Methods
The 25-hydroxy vitamin D test is the appropriate measure for most people. It’s the sum of 25-OH-vitamin D2 and 25-OH-vitamin D3.
“Historically, vitamin D was measured by competitive binding methods, high-performance liquid chromatography (HPLC) and radioimmunoassay (RIA). A commonly used RIA kit, developed by DiaSorin S.p.A in Saluggia, Italy, used by many reference laboratories is considered the gold standard,” said Riddle. As vitamin D testing has exponentially increased in recent years and we’ve learned more about the importance of this steroid hormone in conditions such as diabetes, heart disease and cancers, new automated testing methods have been developed.
As a result, many labs like the clinical lab at Prince of Wales Hospital (PWH) in Sydney, Australia, have made the switch to liquid chromatography, tandem mass spectrometry (LC-MS/MS), which measures each concentration individually and then adds them together. In order to prepare the sample, PWH takes the plate, shakes it up, adds all the components together and then centrifuges it into a collection plate, which is put onto the LC-MS. Manually, this protein precipitation process can take 40 minutes to do about 84 patient samples; however, robotic platforms have made this process even faster.2 The next step after that is to clean up the sample.
An RIA method, on the other hand, measures the total 25-hydroxy vitamin D by first dissociating it from its binding protein and binds it to the specific solid phase antibody, according to CAP. A vitamin D-isoluminol tracer is then added, along with a ðchemiluminescent reagent, washing out unbound material. Finally, the light signal is detected by a photomultiplier as relative light units, which are inversely proportional to the 25OD vitamin D concentration.
Complications
Despite these careful procedures, results can still be skewed depending on a number of factors, such as the age of the patient or presence of any underlying medical conditions.
Infants under the age of 1 fall into this sensitive category due to the presence of 3-epimers in their blood. If all the metabolites aren’t accounted for during LC-MS, the test can cloud and overestimate vitamin D2 and D3 levels. This happened in a 2006 study of more than 150 infants in which researchers identified significantly elevated results in some of the newborns. The study was then modified to allow for the detection of the epimers, and researchers concluded that this modification should be made routinely to ensure accurate measurements in children less than 1 year.3
Modifications also have to be made for those whom have high calcium levels or diseases that produce excess amounts of vitamin D, such as sarcoidosis or some forms of lymphoma, as well as in rare cases where an abnormality is found in the enzyme that ðconverts 25-hydroxyvitamin D to 1,25-dihydroxy vitamin D.4
In 2010, the National Institutes of Health (NIH) office of dietary supplements established the Vitamin D Standardization Program to help standardize the laboratory measurement of vitamin D status. Prior to this effort, there was no commutability between labs.5 In response, the program is committed to providing reference materials for clinical labs and research studies so they can be comparable to each other despite variables like location, procedure and time.
“Laboratories are awarded certificates for successfully completing bias and imprecision testing using specific methods, reagent lots, calibrator lots and instrumentation,” explained Crystal Moore, MD, PhD, FCAP, member of CAP. “Laboratories certified by this program have successfully passed the performance criterion of ±5% mean.”
Those studies can accurately identify individuals or whole populations that may have unsafe levels and inform future decisions on safe reference ranges, determining how much they should increase their intake of vitamin D. The current range sufficient for most people was established to be 25 ng/mL to 35 ng/mL by the Institute of Medicine (IOM).6
“Most people obtain vitamin D by cutaneous production from exposure to the sun,” noted McKinnon. “However, there are many factors that can influence the amount of UVB received from exposure to sunlight, including the time of day, season, latitude, altitude, clothing, sunscreen use, pigmentation and even age.”
To get to a sufficient level, people can then increase foods rich in vitamin D, such as fish, eggs and some dairy products, or increase their time in the sun. Sometimes supplements can be a more reliable source of vitamin D if levels are extremely low and daylight is affected by the changing seasons.
“Historically, supplemental replacement in the U.S. was from 25-OH D2, but in recent years, 25-OH D3 is now being used more often,” noted Riddle. “And there are ultra-high-dose forms available in the active 1,25(diOH) D3 (calcitriol) by prescription.”
Retesting should be done in order to monitor the effectiveness of treatments. In rare cases, vitamin D can reach excessive levels, defined as 80 ng/mL to 100 ng/mL, and research suggests adverse effects to vital organs. Vitamin D testing will likely become more popular as improvements in lab methods and new strides in research continue to be made.
Chelsea Lacey-Mabe is a staff writer. Contact: [email protected]
References
1. Holick MF, et al. Prevalence of Vitamin D inadequacy among postmenopausal North American women receiving osteoporosis therapy. J Clin Endocrinol Metab. 2005 Jun;90(6):3215-24.
2. LabManagerVideos. Clinical impact of enhanced resolution of vitamin D metabolites through chromatographic selectivity 2014. Available at: http://bit.ly/1ouw1kH
3. 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 Aug;91(8):3055-61.
4. American Association for Clinical Chemistry. Vitamin D Tests 2015. Available at: http://bit.ly/1ToU6EJ
5. National Institutes of Health. Vitamin D Standardization Program. Available at: http://1.usa.gov/1Rc7hGN
6. National Institutes of Health. Vitamin D Factsheet for Professionals 2014. Available at: http://1.usa.gov/1M5deq6