Test: 25-hydroxyvitamin D (25-D)

Also known as 25-hydroxyvitamin D, 25-D is the inactive form of vitamin D. Like 1,25-DPrimary biologically active vitamin D hormone. Activates the vitamin D nuclear receptor. Produced by hydroxylation of 25-D. Also known as 1,25-dihydroxycholecalciferol, 1,25-hydroxyvitamin D and calcitirol., 25-D has an affinity for the Vitamin D ReceptorA nuclear receptor located throughout the body that plays a key role in the innate immune response. (VDRThe Vitamin D Receptor. A nuclear receptor located throughout the body that plays a key role in the innate immune response.), but unlike 1,25-D, it inactivates the Receptor.

When researchers are journalists talk about vitamin D deficiency, they are invariably talking about low levels of 25-D as opposed to 1,25-D. According to the Marshall PathogenesisA description for how chronic inflammatory diseases originate and develop., the body purposefully downregulates levels of 25-D so as to upregulate activity of the VDR.

As the vitamin D metabolite calculator states, any 25-D of 20 ng/ml or higher is immunosuppressive and should be countered by restricting consumption of vitamin D.

If a patient's 25-D is low enough, they can enjoy an infrequent splurge of food containing vitamin D.

Patients should ask their physicians to order a baseline 25-D test prior to beginning olmesartan (Benicar)Medication taken regularly by patients on the Marshall Protocol for its ability to activate the Vitamin D Receptor.. Marshall ProtocolA curative medical treatment for chronic inflammatory disease. Based on the Marshall Pathogenesis. patients whose 25-D is above 20 ng/ml should continue to be tested every three months. This allows the doctor and the patient to anticipate a possible increase in immunopathologyA temporary increase in disease symptoms experienced by Marshall Protocol patients that results from the release of cytokines and endotoxins as disease-causing bacteria are killed. (IP), which corresponds to a 25-D level of 20 ng/ml.

Intermittent testing (once every six months or longer) can continue thereafter to verify a patient is continuing to successfully avoid food containing vitamin D.

MP patients getting their 25-D levels tested should ensure the relevant instructions are closely followed including the freezing of blood samples.

Contextual interpretation of a patient's 25-D and 1,25-D results are available using the vitamin D metabolite calculator.

A patient may get a test result, breaking down their 25-D into two different kinds:

• 25 D₃ – synthesized on human skin when exposed to light; enters the body when a person consumes animal products that contain vitamin D
• 25 D₂ – enters the body when a person consumes plants or fungi that contain vitamin D

On the Marshall Protocol study site, 25-D levels are typically discussed using ng/ml units rather than pmol/L. The ratio between the two units is: 2.4 ng/ml = 1 nmol/L. To convert nmol/L into ng/ml, multiply by 0.40, as you see in this example:

60 nmol/L * 0.40 = 24 ng/ml

This is also done automatically with the vitamin D metabolite calculator.

Lower than normal levels of 25-D have been independently associated both with all-cause mortality¹ and dozens of chronic inflammatory diseases ranging from alcoholism ² to allergies³ to prostate cancer.⁴

For this reason, low levels of 25-D can be used (in countries that supplement) as a proxy for chronic disease.

The question of what should be the appropriate reference range for vitamin D involves several issues.

Observational studies show that populations which avoid vitamin D consumption have naturally low levels of 25-D and remain healthy with such levels.

• A study which tested the level of 25-D in 90 “healthy, ambulatory Chilean women” showed that 27% of the premenopausal and 60% of the postmenopausal women had 25-D levels under 20 ng/ml.⁵
• A study on healthy Bangladeshi women found that approximately 80% of the women had a level of 25-D under 16 ng/ml.⁶
• In a 1992 study, healthy full-term infants from China had serum concentrations of 25-D ranging from an average of 5 ng/ml to 14 ng/ml.⁷

There are several molecular pathways activate in chronic inflammatory disease, which cause levels of 25-D to fall to “deficient” levels. It is in the in the interest of such patients to have low levels of 25-D, as low levels increase the activity of the VDR – a receptor which, when activated, plays a key role in innate immune function.

Under such circumstances, a patient who supplements with vitamin D may see a rise in 25-D. However, the increase in serum levels of 25-D would not be quite as high as it otherwise would be in a healthy person.

In other words, the body's enzymatic regulation of the D metabolites can be forcefully overridden by heavy dietary and supplemental intake of D precursors.

The explanation presented here for why 25-D is low in patients with chronic disease runs counter to the more commonly given but incorrect description, namely that patients “use up” vitamin D as they would a true vitamin.

The high rate of chronic disease and the presence of vitamin D supplementation has led to a misunderstanding about what constitutes a healthy or normal range for vitamin D. Laboratories establish a “normal” range for the D metabolites results based on studies purportedly looking at the average of all the “healthy” persons who are tested. Clinicians have yet to recognize the reason for low levels of 25-D and usually recommend supplementation with vitamin D. Therefore, lab ranges for 25-D may be skewed higher and higher by the increasingly prevalent use of dietary supplementation.

Vitamin D values may take years to come into as normal range. Four patients who poisoned themselves with vitamin D supplements were followed over a period of several years of vitamin D near abstinence. The authors describe the rate of decrease as 10.7 ± 3.0 nmol/L per month. Note the y-axis: even after two, three or more years of abstinence, none of the patients have levels of vitamin D that ceased to be immunosuppressive.

The therapeutic range for Marshall Protocol patients is 11 ng/ml or lower. MP patients often have a 25-D below the detectable limit of 5-7 ng/ml.

The rate at which 25-D declines in Marshall Protocol patients tends to vary. Adams et al. (right) showed that the rate of at which 25-D declined among people who taken high amounts of vitamin D supplements and subsequently abstained from supplements is approximately 10.7 ± 3.0 nmol/L.⁸

Sometimes, a patient will be on the MP for 18 months or more, and their 25-D will still be elevated above the therapeutic range – that is greater than 20 ng/ml. It is sometimes the case that a patient has been mistakenly consuming food or supplements containing vitamin D. This explanation must never be overlooked.

The best way to check if a patient is taking vitamin D is to carefully review the possible sources of vitamin D including supplements. Many foods and supplements contain unlisted amounts of vitamin D. Patients can try varying their diet and re-testing themselves to see if this changes their measurable level of 25-D.

• Yeast may be able to produce a 25-D precursor – Patients who have large yeast overgrowth such as Candida may have elevated levels of 25-D as Candida produces ergosterol, a Vitamin D2 precursor.^{9 10} If this is the case, one would expect a large D2 contribution to total 25-D. This could be assessed if the test is broken down in this fashion, which it not always is.

I have found elevated levels of 25-D in compliant patients. It is usually due to an intestinal Candida overgrowth or biofilmA structured community of microorganisms encapsulated within a self-developed protective matrix and living together.. I have found treating with Nystatin, a non-absorbable antifungal azole effective in reducing Candida load and with resultant marked reduction in 25-D levels, often within 1 to 2 months.

Greg Blaney, M.D.

• Minocycline could be activating other receptors – Another explanation is that the MP itself, through activation of the Pregnane X Nuclear Receptor (PXR) is contributing to higher than normal levels of vitamin D. Marshall Protocol patients take minocyclineBacteriostatic antibiotic used by Marshall Protocol patients., which appears to bind the PXR. A similar action may be generated by olmesartan (Benicar). When active, the PXR transcribes the enzyme CYP3A4, which breaks down 1,25-D. When 1,25-D is broken down, 25-D levels escalate. An activated PXR could explain why some MP patients have stubbornly high levels of 25-D even while being extremely careful to avoid consumption of vitamin D.

Endogenous 25-D production will not start to rise until the bacteria have been largely eliminated, which will be in the final few years of MP immunopathology. A healthy 25-D level varies widely, with 25-D being manufactured as the body needs it, and with the maximum level in a healthy person being about 18ng/ml.

Trevor Marshall, PhD

• Forms and structure of vitamin D