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The standard vitamin D assessment measures 25(OH)D. When it’s low, as we often see in chronic diseases, it may be due to a rapid, and uncontrolled, conversion to the active metabolite. This vitamin D ‘deficiency’ justifies measuring 1,25(OH)2D which will provide a more complete picture of vitamin D status. If 1,25(OH)2D is elevated (with normal calcium, creatinine and parathyroid hormone), it indicates uncontrolled extra-renal production (stimulated by intracellular bacteria). This diagnosis of dysregulated vitamin D metabolism points to the need for Inflammation Therapy.

These are the recommended vitamin D tests:


25-hydroxyvitamin-D (also known as 25(OH)D or calcidiol)

This is the inactive form of vitamin D.

CPT code: 82306

This is the form that is commonly measured to assess vitamin D deficiency.

It is measured in ng/mL.


1,25-dihydroxyvitamin-D (also known as 1,25(OH)2D3 or calcitriol)

This is the bio-active form of vitamin D.

Measurement is considered medically necessary for conditions that may be associated with defects in vitamin D metabolism or deficiency.

CPT code: 82652

The sample should be frozen, to avoid degradation due to agitation, if it will be sent to another lab.

It may be expensive but it is usually covered by insurance if coded correctly.

ICD-10-CM diagnostic codes:

E55.9 Vitamin D deficiency, unspecified

M81.0 Age-related osteoporosis without current pathological fracture

M89.9 Disorder of bone, unspecified

D86.9 Sarcoidosis, unspecified

E83.50 Unspecified disorder of calcium metabolism

E83.52 Hypercalcemia

E67.3 Hypervitaminosis D

It is measured in pg/mL.



Both tests should be ordered, not just the commonly done 25-D.

When you get the results back, you may send them to CIR via email for a professional analysis (with scientific references) which you can share with your doctor. Sometimes abnormal test results appear to be acceptable because lab ranges have been skewed high.

Correct handling of the blood sample

An increasing number of labs are insisting the blood sample for 1,25-D does not need to be frozen. Quest has changed their testing procedure and now states that freezing for transport is no longer necessary, no matter which code is used. But they will accept samples that are frozen so freezing is still an option.

In our experience results from a nonfrozen sample have often been significantly lower than results from a frozen sample (this may depend on the testing procedure used by a particular lab). Therefore, to increase the odds of an accurate result, we highly recommend that physicians and patients insist that the technician at the drawing (satellite) lab freeze the sample with dry ice (after centrifuging) for shipping.

Keeping the sample frozen during transit will likely ensure an accurate result by preventing degradation due to agitation. If the sample arrives at the testing site frozen and thaws out before testing that is less likely to cause a false-low reading. Quest has done tests of non-transported samples, (room temperature, frozen and refrigerated) and determined that results under these circumstance are similar.

The test for 25-D is done routinely at many local labs and the sample does not require special handling. When the 1,25-D sample is sent out to a testing lab, the same blood sample is used to test both forms of vitamin D. Freezing preserves the sample and will not harm it, even though freezing isn't necessary for 25-D.

Directions for correct handling of the 1,25-D specimen
To facilitate correct handling of the blood sample for 1,25-D and to avoid having to tell the drawing lab how to do their job, the physician could staple the following instructions to the lab order:
  • Please perform the following Vitamin D tests: 1,25(OH)2D and 25(OH)D.
  • Please ensure that collection staff arrange for centrifuging and freezing of the 1,25(OH)2D sample (only).
  • The sample should be allowed to settle and clot at room temperature for at least 30 minutes (but no more than two hours) and then centrifuged. Do not hold on dry ice prior to centrifuging.
  • After centrifuging, freeze at between -2 and -10 C.
  • The sample must be transported on dry ice in order to remain frozen and prevent agitation, until it reaches the testing lab.


Preparation for testing
It’s not necessary to fast for these blood tests but they can be done fasting. The D-metabolites tests can be done whether or not the patient has been avoiding ingested Vitamin D or sun/lights. This information is taken into consideration when assessing the test results. One very small (7 patients), 1982 study demonstrated that the serum concentration of 1,25-D fluctuated with the menstrual cycle. Levels of 1,25-D were shown to be dramatically higher near ovulation in women not on the pill. The higher the level is, the more persuasive is the evidence for treatment. Therefore, it may be advantageous to assess 1,25-D close to mid-cycle (day 15).


The 1,25-D lab report

For informational purposes, many labs provide a total 1,25(OH)2D and also break down the total into two subcomponents; Vitamin D3 and Vitamin D2.

Vitamin D,1,25(OH)2, Total  ___pg/mL
Vitamin D3, 1,25(OH)2
Vitamin D2, 1,25(OH)2

The subcomponent D3 is derived from human or animal sources (both photosynthesis and ingestion of meat/fish or D3 supplements). This is usually the same number as the total because it is the most common source of vitamin D.

The subcomponent D2 is derived from ingestion of plant of fungal sources (the most common source is vitamin D2 supplementation). This is often listed as less than 4 ng/mL because this is not a common source of vitamin D.

Calculating results in different units

The molecular weight of 1,25-D is approximately 416.7, yielding the following conversion factors: 1 pmol/L = 0.42 pg/mL; conversely, 1 pg/mL = 2.496 pmol/L.

Use the following calculations to convert lab results from one unit to another as needed. 

25-D: To convert from the conventional units (U.S.A.) of ng/ml into SI units (International Standard), multiply by 2.496. The result will be in nmol/L.

To convert from the SI units (International Standard) of nmol/L into conventional units (USA), divide by 2.496. The result will be in ng/ml.
See this automatic calculator 

1,25-D: To convert from the conventional units (U.S.A.) of pg/ml into SI units (International Standard), multiply by 2.6. The result will be in pmol/L.

To convert from the SI units (International Standard) of pmol/L into conventional units (U.S.A.), divide by 2.6. The result will be in pg/ml.
See this automatic calculator 

1,25(OH)2D Reference Ranges

Measurement of serum 1,25-dihydroxyvitamin D [1,25(OH)2D] is done by radioimmunoassay (RIA). 1,25(OH)2D lab reference ranges are determined by statistical analysis of the people who have had 1,25(OH)2D measured. The "normal" range is where 95% of the people measured fall.

However, the current standard advises measurement of 1,25(OH)2D only in patients with, or suspected of, serious medical conditions such as hypercalcemia, renal failure, sarcoidosis, lymphoma, abnormalities of 1-alphahydroxylase, hyphophosphatic rickets, primary hyperparathyroidism, hypoparathyroidism, pseudohypoparathyroidism, renal osteodystrophy, vitamin D-resistant rickets or vitamin D receptor defects. This population cannot be considered normal. Thus, lab ranges do not provide an accurate assessment of a normal level of 1,25(OH)2D.

Statistics on 1,25(OH)2D in a normal population are very limited. The best data comes from a 1999 cross-sectional study of the influence of smoking on serum parathyroid hormone (PTH), serum vitamin D metabolites, serum ionized calcium, serum phosphate, and biochemical markers of bone turnover. In this study, serum 1,25(OH)2D was measured, via radioimmunoassay, in a cohort of 510 healthy Danish women aged 45 to 58 years. The average 1,25(OH)2D level of the non-smokers in this group was 29.0 pg/mL (plus or minus 9.5 pg/mL) for a range of 19.5 - 38.5 pg/mL in this healthy population. [1]

Furthermore, when measured coincidentally, the significance of elevated 1,25(OH)2D may be overlooked. For example, a 2011 study of the effect of vitamin D and calcium supplementation on patients with multiple sclerosis, revealed elevated 1,25(OH)2D at baseline and one year later (61 pg/mL ± 22.6 pg/mL and 70.7 pg/mL ± 18 pg/mL respectively). [2] All of these 1,25(OH)2D levels were considered normal. Calcium, phosphorus and parathyroid hormone were not measured in those whose 1,25(OH)2D exceeded the normal range.

The Merck Manual of Diagnosis and Therapy (15 Oct 2006 online) listed the range of serum 1,25(OH)2D in healthy persons as 20-45 pg/mL. The 2013 edition lists the normal range as 25–65 pg/mL. The Mayo Clinic lists 18 - 78 pg/mL as the normal range. It's disappointing to note that on March 16, 2015 Labcorp raised their normal range of 1,25(OH)2D from 10 - 75 pg/mL to 19.9 - 79.3 pg/mL.

As the incidence of chronic illness increases and serum 1,25(OH)2D levels rise, authorities simply increase the range of 1,25(OH)2D that is considered normal. This signifies a failure to recognize elevated 1,25(OH)2D as a sign of dysregulated vitamin D metabolism and a marker of a chronic inflammatory disease process. Studies need to be done to determine the serum level of 1,25(OH))2D in healthy populations. Using reference ranges that truly represent normal will promote accurate diagnosis of vitamin D endocrine dysfunction.


1. Brot C, Jorgensen NR, Sorensen OH The influence of smoking on vitamin D status and calcium metabolism. Eur J Clin Nutr. 1999;53:920-6.

2. Kimball S, Vieth R, Dosch HM, et al. Cholecalciferol plus calcium suppresses abnormal PBMC reactivity in patients with multiple sclerosis. J Clin Endocrinol Metab. Sep 2011(96(9)):2826-34.

Lab ranges 25-D

25-D ranges are skewed high by dietary supplementation and flawed thinking in response to studies showing low levels of 25-D in many chronic diseases. They fail to realize that low 25-D doesn't cause chronic disease; the disease process causes low 25-D.

Other tests before you begin Inflammation Therapy

Assessing certain inflammatory markers is recommeded before initiating therapy to establish a baseline before treatment.

The following lab work is suggested to serve as a baseline before treatment begins:

  • Complete blood count (CBC) with differential
  • Comprehensive metabolic panel (CMP)
  • Thyroid function
  • Liver panel
  • Magnesium
  • PTH if calcium is elevated
  • Any other lab work pertinent to the clinical picture (e.g., ACE, triglycerides, INR, CRP, etc.)

Throughout therapy, it’s important to monitor any test results that were not within normal limits or have caused concern.

See also Vitamin D.