Testing Vitamin D Metabolites

Inflammatory Markers Of Chronic Disease

Information For Patients and Medical Practitioners

Inflammatory Markers and Immune Function

Testing both of the active D metabolites, 25-Hydroxy Vitamin D, and 1,25 Dihydroxy, are approved for investigation of kidney function and risk of osteoporosis. Anyone who has been on Prednisone or certain other immunomodulatory drugs is at risk of osteoporosis.

By testing both metabolites together, it is possible to identify Vitamin D toxicity, the presence of inflammatory disease, prevention of harmful supplementation, bone density and calcification risk.

see also Clinical Utility of Measurement of Vitamin D-Binding Protein and Calculation of Bioavailable Vitamin D in Assessment of Vitamin D Status 1)

Metabolism and Immunostimulation

Competent Vitamin D receptors regulate a healthy immune system and metabolism. A chronic pathogenic microbiotaThe bacterial community which causes chronic diseases - one which almost certainly includes multiple species and bacterial forms. affects the levels of the D metabolites observed in chronic diseases in several ways. When the immune system is challenged by pathogens, the body activates CYP27B1, causing more 25-DThe vitamin D metabolite widely (and erroneously) considered best indicator of vitamin D "deficiency." Inactivates the Vitamin D Nuclear Receptor. Produced by hydroxylation of vitamin D3 in the liver. to be converted to 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., which, of course, increases activity of the VDRThe Vitamin D Receptor. A nuclear receptor located throughout the body that plays a key role in the innate immune response..

A full understanding of all these mechanisms supports the conclusion that elevated 1,25-D and depressed 25-D are a result rather than a cause of the inflammatory disease process. 1

A vitamin or a steroid hormone?

Vitamins are substances which cannot be made by the body itself. The body synthesizes vitamin D from 7-dehydro-cholesterol. Vitamin D is not a vitamin, it is a Gene-Transcriptional-Activator, a paracrine steroid.

Ingested Vitamin D suppresses the immune system, as do other steroids, and, in the short term, patients often feel better as a result of taking it. 3

In the long term, the ingestion of this steroid allows the human microbiotaThe bacterial community in the human body. Many species in the microbiota contribute to the development of chronic disease. to proliferate unhindered, ensuring disease severity will progress, year by year.

Olmesartan, a VDR agonistA substance such as olmesartan (Benicar) or 1,25-D which activates the Vitamin D Receptor and transcribes the genes necessary for a proper innate immune response., restores innate immune activity, allowing (slow) recovery from advanced disease. 4

The D metabolites test indicate Hypervitaminosis D, Osteoporosis, Osteopenia, Sarcoidosis, Unspecified disorders of Calcium metabolism, Kidney Function, Hypercalcemia, Fatigue, and diseases related to previous use of long term steroids. 5

Therapeutic ranges for Immunostimulation, and to determine if active Immunosuppression is present

It is not necessary to fast for these blood tests. The D metabolites test can be done whether or not the patient has been avoiding ingested vitamin D or sun/lights. This information is taken into consideration when interpreting the test results.

The therapeutic target range for 25-D is 12ng/ml below which the immune system is fully functional. A 25-D above 25ng/ml will not be able to kill pathogenic bacteria. For 1,25-D the target is <20 pg/ml and is a measure of inflammation. 7

Mucosal response and calcium/phosphorus absorption is dependent on a competent vitamin D receptor (VDR), and elevated 1,25(OH)2D reduces VDR competence. Thus, calcium and phosphorus absorption may be inhibited if VDR function is impaired by elevated 1,25(OH)2D. Measuring calcium is primarily affected by PTH status, not VDR competence. 9

Special Handling of the 1,25D Blood sample

The following guidelines for doctors ensures correct handling of the sample for the Vitamin D tests: 1,25-D and 25-D.

Please ensure that collection staff arrange for centrifuging and freezing of the 1,25D sample. The 25-D sample does not require freezing.

The 1,25- D 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 until it reaches the testing lab.

Note that 1,25D levels are exceedingly minute. 1 pg/ml is one millionth part of one millionth part (pico is 10 raised to the -12 power) and the pathology to measure such levels is extremely sensitive to correct handling.

The Innate Immune response

..is the body's first line of defense against, and non-specific way for responding to bacterial pathogens. Located in the nucleus of a variety of cells, the Vitamin D nuclear receptorA nuclear receptor located throughout the body that plays a key role in the innate immune response. (VDR) plays a crucial, often under-appreciated, role in the innate immune responseThe body's first line of defense against intracellular and other pathogens. According to the Marshall Pathogenesis the innate immune system becomes disabled as patients develop chronic disease..

When functioning properly, the VDR transcribes between hundreds and thousands of genes including those for the proteins known as the antimicrobial peptidesBody’s naturally produced broad-spectrum antibacterials which target pathogens.. 6

Antimicrobial peptides are the body's natural antibiotics, crucial for both prevention and clearance of infection. The VDR also expresses the TLR2A receptor which is expressed on the surface of certain cells and recognizes native or foreign substances and passes on appropriate signals to the cell and/or the nervous system. receptor, which is expressed on the surface of certain cells and recognizes foreign substances. 2

The body controls activity of the VDR through regulation of the Vitamin D Metabolites

Although early stage autoimmune disease often succumbs to antibiotic therapy, antibiotics appear to lose utility as disease progresses. Recent sequencing of microbial DNA has confirmed that some pathogens ensure their persistence by reducing expression of, and transcription by, the VDR nuclear receptorIntracellular receptor proteins that bind to hydrophobic signal molecules (such as steroid and thyroid hormones) or intracellular metabolites and are thus activated to bind to specific DNA sequences which affects transcription., which is at the heart of the human innate immune system. 8, 10

Only in homo sapiens is the VDR responsible for expression of the Cathelicidin Family of antimicrobial peptides found primarily in immune cells and transcribed by the Vitamin D Receptor. antimicrobial peptide, the primary defense of the intra-phagocytic innate immune system.

Humans Are Not Tall Mice Without Tails!

Importantly, the innate immune functions of the VDR are unique to Homo sapiens. In Homo sapiens, and only in Homo sapiens, there is one nuclear receptor, the VDR, which expresses genes for TLR2, as well as the cathelicidin and beta-defensin An antimicrobial peptide found primarily in immune cells and transcribed by the Vitamin D Receptor. anti-microbial peptides, all of these are essential to the intracellular innate immune defences.

You cannot study the vitamin D receptor in mice, how it relates to the immune system, and expect it to translate to Homo sapiens. Whereas in man the VDR transcribes cathelicidin, which is the primary anti-microbial producing peptides that protect the phagocytes themselves and TLR2, the primary toll-like receptor which detects pathogens inside the macrophages, there is no such homology in mice. 9

In mice, there are other receptors, and other genes that do that. Even the higher primates do not emulate the cathelicidin innate immunityThe body's first line of defense against intracellular and other pathogens. According to the Marshall Pathogenesis the innate immune system becomes disabled as patients develop chronic disease. of Homo sapiens.


1. Marshall TG: Vitamin D discovery outpaces FDA decision making. BioEssays. 2008 Feb;30(2):173-82 Online ISSN: 1521-1878 Print ISSN: 0265-9247 Preprint of FullText available from URL https://TrevorMarshall.com/BioEssays-Feb08-Marshall-Preprint.pdf

2. Proal AD, Albert PJ, Marshall TG: Dysregulation of the Vitamin D Nuclear Receptor may contribute to the higher prevalence of some autoimmune diseases in women. Contemporary Challenges in Autoimmunity, Annals of the New York Academy of Sciences. 2009 Sep;1173. Http://AutoimmunityResearch.org/transcripts/NY_Annals_Proal_Preprint.pdf

3. Albert PJ, Proal AD, Marshall TG: Vitamin D: the alternative hypothesis. Autoimmunity Reviews. 2009 Jul;8(8):677-81.Preprint available from https://AutoimmunityResearch.org/transcripts/AR-Albert-VitD.pdf

4. Proal AD, Albert PJ, Marshall TG: Autoimmune disease in the era of the Metagenome. Autoimmunity Reviews. 2009 Jul;8(8):639-44. https://AutoimmunityResearch.org/transcripts/AR-Proal-Metagenome.pdf

5. Proal AD, Albert PJ, Marshall TG: Infection, Autoimmunity, and Vitamin D. Infection and Autoimmunity, 2nd Edition. Yehuda Shoenfeld, Noel Rose, Nancy Agmon-Levin Editors; Elsevier 2014.

6. Proal AD, Lindseth IA, Marshall TG: Microbe-Microbe and Host Microbe Interactions Drive Microbiome Dysbiosis and Inflammatory Processes. Discovery Medicine, Volume 23 Number 124, Pages 51-60, January 2017. https://www.ncbi.nlm.nih.gov/labs/journals/discov-med/

7. Vitamin D metabolites as clinical markers in autoimmune and chronic disease. Blaney GP, Ann N Y Acad Sci1173p384-90(2009 Sep) https://mpkb.org/home/publications/blaney_annals_2009.

8. Proal AD, Autoimmune Disease and the Human Metagenome. In Metagenomics and Human Biology, Springer. 2009. P. 231-275.

9. The Salivary Microbiome. Olmesartan overcomes antibiotic resistance. 'Humans are not tall mice without tails'. Immunopathology. Apoptosis. 'Vitamin' D is not a nutrient. Https://mpkb.org/home/publications/marshall_autoimmunity_2010

10. Immunostimulation in the Era of the Metagenome. Proal AD, Albert PJ, Blaney GP, Lindseth IA, Benediktsson C, Marshall, TG. Cellmol.Immunol. 2011 Jan 31. Vol 8. p. 213-225.

Autoimmunity Research Foundation 3423 Hill Canyon Avenue, Thousand Oaks, CA 91360. USA. Website: www.marshallprotocol.com The Autoimmunity Research Foundation is a California-based non-profit organization formed to educate the public about the nature of, and study a treatment for, chronic inflammatory disease.

© May 2018 Important Consideration : Environmental effects in Autoimmunity

Note that collection of blood samples in a high-RF environment may be sub-optimal.

Studies in mice have shown that environmental electromagnetic waves tend to suppress the murine immune system with a potency similar to NSAIDs, yet the nature of any Electrosmog effects upon humans remains controversial. Previously, we reported how the human Vitamin-D receptor (VDR) and its ligand, 1,25-dihydroxyvitamin-D (1,25-D), are associated with many chronic inflammatory and autoimmune diseases. We have shown how olmesartan, a drug marketed for mild hypertension, acts as a high-affinity partial agonist for the VDR, and that it seems to reverse disease activity resulting from VDR dysfunction. We here report that structural instability of the activated VDR becomes apparent when observing hydrogen bond behavior with molecular dynamics, revealing that the VDR pathway exhibits a susceptibility to Electrosmog.

Further, we note that characteristic modes of instability lie in the microwave frequency range, which is currently populated by cellphone and WiFi communication signals, and that the susceptibility is ligand dependent. A case series of 64 patient-reported outcomes subsequent to use of a silver-threaded cap designed to protect the brain and brain stem from microwave Electrosmog resulted in 90% reporting “definite” or “strong” changes in their disease symptoms. This is much higher than the 3-5% rate reported for electromagnetic hypersensitivity in a healthy population and suggests that effective control of environmental Electrosmog immunomodulation may soon become necessary for successful therapy of autoimmune disease. Trevor G. Marshall : Trudy Rumann Heil : PMID 27412293

in this section

Notes and comments

Kim HJ, Ji M, Song J, Moon HW, Hur M, Yun YM. Clinical Utility of Measurement of Vitamin D-Binding Protein and Calculation of Bioavailable Vitamin D in Assessment of Vitamin D Status. Ann Lab Med. 2017 Jan;37(1):34-38. doi: 10.3343/alm.2017.37.1.34.
[PMID: 27834063] [PMCID: 5107615] [DOI: 10.3343/alm.2017.37.1.34]
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