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Vitamin D Testing

Analytical & Clinical Conundrums

Published: January 2009

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Dr. Singh discusses the various forms of vitamin D, the different tests available for assessing vitamin D stores, and the appropriate use of vitamin D testing based on the clinical picture.

Presenter: Ravinder J. Singh, PhD

Assistant Professor of Laboratory Medicine and Pathology in the Division of Clinical Biochemistry & Immunology at Mayo Clinic

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Welcome to Mayo Medical Laboratories' Hot Topics. These presentations provide short discussion of current topics and may be helpful to you in your practice.

Our presenter for this program is Dr. Ravinder Singh, Assistant Professor of Laboratory Medicine and Pathology in the Division of Clinical Biochemistry & Immunology at Mayo Clinic. Dr. Singh will be discussing the various forms of vitamin D, the different tests available for assessing vitamin D stores, and the appropriate use of vitamin D testing based on the clinical picture.


The objectives for the talk today: I will go through the brief history and physiology of vitamin D deficiency. We will also discuss various forms of vitamin D and related vitamin D testing. We will also discuss a case which will present different clinical conditions.


Clinicians have known disease of rickets for almost a century. When a child presented with a crooked leg, it was probably due to deficiency of calcium or vitamin D. Clinicians treated rickets with calcium and vitamin D supplementation and rickets was cured.

UVB Light

When vitamin D supplementation was not available, children were also treated with exposure to UVB light.

Vitamin D Deficiency

Recently vitamin D deficiency has been reported in adults, especially in the aging population and the population living in nursing homes. Sunlight is the best source for vitamin D production. UVB from sunlight helps in synthesizing vitamin D in the skin from its precursor. Vitamin D is then converted to 25-hydroxy vitamin D by the liver. 25-hydroxy vitamin D is converted to 1,25-dihydroxy vitamin D by the kidney, which helps in resorbing the bone from intestine and thus maintaining calcium in circulation and preserving calcium in bones.

Our aging population cannot get the benefit of UVB from the sun if they apply too much sunscreen, if they have high melanin in their skin, and they live in high latitude and of course, the UVB penetration in winter is also very low.

The aging population can be affected with various conditions like malabsorption. A few examples like Crohn’s, Whipple, cystic fibrosis, celiac. Population affective to the organ failure can also absorb calcium or absorb vitamin D if they have liver disease, renal failure, nephrotic syndrome.

Certain drug also seems to inhibit vitamin D synthesis and vitamin D metabolites are unable to absorb calcium. As a result of vitamin D deficiencies, various diseases have been associated, in addition to bone and muscles: osteoporosis, osteomalacia, osteoarthritis, rickets, muscle aches and weakness.

Coronary heart disease and cardiovascular disease and cancers have also been added to the list, which have been added as a vitamin D deficiency.

Interaction Between Vitamin D and Calcium

The interaction between vitamin D and calcium is very complex. When calcium gets low in circulation, calcium sends a receptor that will result in elevation of PTH. PTH will help resorbing calcium from the bone. 25D will be converted to 1,25-hydroxy vitamin D. 1,25-dihydroxy vitamin D will help in the absorption of calcium and phosphorus from the intestine. Calcium, thus, will be deposited back into the bones.

Vitamin D3

Vitamin D3 can be ingested in the form of diet by consuming fish and meat. Vitamin D is also synthesized by exposure to the sunlight. Once vitamin D enters the circulation it is rapidly converted to 25-hydroxy vitamin D3 by liver. 25-hydroxy vitamin D3 will also be converted to 1,25-dihydroxy vitamin D3, which is in minute amounts in circulation but is the bioactive hormone helping in calcium absorption from the intestine.

Vitamin D2

Similarly, vitamin D2 is also commonly used clinically in the form of supplements. Vitamin D2 goes similarly conversion to 25-hydroxy vitamin D2 and 1,25-dihydroxy vitamin D2 and is considered biologically equally to D3. Thus, all the assays used in the clinical laboratories should mirror equally D2 and D3.

Various Vitamin D Tests

So, we have three pools of vitamin D in circulation: Vitamin D, 25-hydroxy vitamin D, 1,25-dihydroxy vitamin D. Vitamin D test is commonly not used in clinical laboratories. Most of the time, vitamin D status is determined by measuring 25-hydroxy vitamin D in the clinical labs and is the recommended test and is a test of the choice for determining vitamin D status. 1,25-dihydroxy vitamin D should be used only in rare situations.

History of 25-OH Vitamin D Testing

The endocrine lab at Mayo Clinic has a history of vitamin D testing since 1981. We were performing 25-hydroxy vitamin D testing by HPLC. Our volume in 1981 was 50 samples/day. IN 2000, we switched to RIA in 2000, and we were able to perform 150 samples/day. In 2004, we implemented LC-MS/MS into the endocrine lab and we were able to perform 400 samples/day. With this technology today, we are able to perform 3,000-3,500 samples a day to determine the level of 25-hydroxy vitamin D in the blood.

Accreditation Agencies for Precision Among Peers

Our lab also participates in proficiency testing offered by the College of American Pathologists. We also participate in proficiency testing offered by DEQAS, an agency based in England.

Quality of the 25-OH Vitamin D Testing

This slide shows the data from 2007. The first column shows various methods available for 25-hydroxyvd testing. The next column shows the various number of labs participating in this survey and the number of labs using a particular technology. For example, there are 20 labs using LC-MS method. The mean value obtained by using LC method was 50.8, the standard deviation among labs using LC method was 9.3, leading to the coefficient variation of 18.2.

25-OH Vitamin D Volume

As you all know there is increasing interest in the testing of vitamin D, thus correspondingly the volume of testing for 25-hydroxy vitamin D has been increasing exponentially. A similar trend was observed in our lab. As you can see since we implemented LC-MS/MS in 2004, we are almost performing 50,000 samples a month or more.

Reference Ranges for 25-OH Vitamin D

This slide shows the reference ranges for 25-hydroxy vitamin D. Less than 10 ng/mL describes severe deficiency, which could be associated with osteomalacia or rickets. 10-25 ng/mL will correspond to mild to moderate 25-hydroxy vitamin D deficiency, which may be associated with an increased risk of osteoporosis or secondary hyperparathyroidism. In our review of the literature, 25-80 ng/mL was considered optimum 25-hydroxy vitamin D levels. 80 ng/mL levels or above are considered toxic. 80 ng/mL is the lowest reported level associated with toxicity in patients without primary hyperparathyroidism and with normal renal function.

Various Vitamin D Tests

We already discussed that there are three different forms of vitamin D in circulation, so we can have three different forms of vitamin D tests. Vitamin D itself has not been commonly used; 25-hydroxy vitamin D is the most commonly used test for determining vitamin D deficiency. The question arises, what is the use of 1,25-dihyrdox vitamin D in pt care?

Case Study - Rickets in a Premature Infant

We will address that question by discussing a case which is related to rickets in a premature infant.

Birth History

A brief history of the premature infant is that the infant was born via C-section performed on the mother in whom the ultrasound showed abnormal umbilical blood flow and the intrauterine growth was retarded.

The premature infant had Apgar scores of 6 and 7. The infant was intubated and given Survanta, which is a lung surfactant, to help with breathing. The infant was also quickly transferred to a neonatal intensive care unit for medical management.

Ionized Calcium

This graph shows calcium levels in this premature infant. As you can see, the calcium levels were low and it was very difficult to manage calcium levels in this premature infant, even with the calcium supplementation until this infant was supplemented with vitamin D. Why was this infant supplemented with vitamin D?

Fractures Identified on Routine Radiographic Surveillance for Line Placement

Fractures were identified on routine radiographic surveillance for line placement and fractures were observed in this premature infant. The infant was started on vitamin D supplementation.

Vitamin D Supplementation

Vitamin D supplementation included 2,000 IU of Ergocalciferol per day, and the infant also got Similac Special Care, which provided a lot of calc and vitamin D.

Lab Values After Vit-D Supplementation

This graph shows the increase in vitamin D levels in this premature infant after supplementation. The green line describes an increase in vitamin D levels which increase up to 150 ng/mL at which the clinicians decided to stop the vitamin D supplementation. The blue line describes the vitamin D2 levels, which were determined using the LC-MS/MS method. The pink line describes vitamin D3 levels. The red dot, which is 1,25-dihydroxy vitamin D, was elevated when the vitamin D level and the calcium was low in this premature infant.

When the vitamin D levels reached normal levels, 1,25-dihydroxy vitamin D normalized. PTH and BAP levels were also normalized in this infant.

Mother's History

You would be curious why this child got rickets. Let’s look at the mother’s history. The mother is 25 years-old, gravida 4 and para 3. The mother had a history of chronic hypertension due to polycystic kidney disease. The mother had a history of preeclampsia, she was smoking during pregnancy, she had a history of depression and she was a carrier of cystic fibrosis.

Infant's X-ray

This is the MRI of the mom. As you can see, mom was not doing well. The top red arrows show a lot of cysts in the liver, there were a lot of cysts also in the kidney. The right and left kidney were enlarged; the right measuring 22cm, the left measuring 24cm.

Mother's Creatinine

The creatinine levels also were really elevated when the mother was pregnant. The creatinine levels were up to 1.5 or 2.0.

The Connection Between the Mother and the Infant

What is the connection between the mother and the premature infant who had rickets?

This slide will highlight because of the polycystic kidney in the mother, the mother was unable to convert 25-hydroxy vitamin D to 1,25-dihydroxy vitamin D. Lack of 1,25-hydroxy vitamin D prevented mom from absorbing calcium and phosphorus from the diet, even if mom was taking a diet full of calcium.

Low calcium levels during fetus development led to rickets. If the mom was diagnosed with polycystic kidney disease at the appropriate time, the mother should have been treated with 1,25-dihydroxy vitamin D, not the vitamin D that is used for the normal population because in this mom, vitamin D would not be converted to 1,25-dihydroxy vitamin D as the mother lacks one of the hydroxylase to convert 1,25-hydroxy vitamin D. 1,25-hydroxy vitamin D can be obtained in the form of the drug called Calcitriol, which helps in absorbing the calcium and should only be used in rare situations.


In summary, three different forms of vitamin D exist in human circulation, but 25-hydroxy vitamin D is the recommended marker for determining vitamin D deficiency. Various formats of 25-hydroxy vitamin D assays are available. A lot of literature has been published describing the strengths and weaknesses of those assays. 1,25-dihydroxy vitamin D should be ordered only in rare situations like renal failure or sarcoidosis and should be interpreted only with the help of specialized doctors.