Testing B12 deficiency

Which tests can I get done?

There are 4 different tests that are used to detect a vitamin B12 deficiency:

A Folic Acid test is also important.

The Serum B12-test measures all of the vitamin B12 in the blood, active as well as non-active. In general 5-20% 1 of all vitamin B12 in the blood is of an active vitamin B12 form that can be used by the human body. This means that 80 to 95% of vitamin B12 in the blood is non-active! The body cannot use this. As the level of active vitamin B12 in the blood is lower, the risk of a vitamin B12 deficiency becomes greater. When performing a serum B12-test the amount of active vitamin B12 is unknown, therefore the test cannot exclude a vitamin B12 deficiency.

The HoloTC-test only measures active vitamin B12 in the blood, making it considerably more reliable.

The homocysteine test measures blood levels of homocysteine. High levels of homocysteine result from a vitamin B12 deficiency (a deficiency in methylcobalamin and possibly folic acid/folate (5-MTHF)). If the test indicates an elevated level of homocysteine, you are advised to supplement vitamin B12 in order to reduce homocysteine levels.

A high methylmalonic acid level also results from a vitamin B12 deficiency (a deficiency in adenosylcobalamin).

Best test combination?

If you're going to get tested, a combination of the HoloTC-test, the homocysteine-test and the methylmalonic acid (MMA) test provides the most reliable results for a vitamin B12 deficiency.

The most important thing, however, is whether or not B12 arrives at a cellular level. Cells have to absorb the vitamin B12. At this time, however, there is no test that can measure vitamin B12 levels at a cellular level. The blood levels may be very high, but if the cells do not absorb it, the body is not using vitamin B12. This is why it is advised to look at the symptoms of vitamin B12 deficiency. If your blood levels are good, but you still recognise certain symptoms, it is advisable to try vitamin B12 supplementation.

If you start vitamin B12 supplementation before getting tested, any result afterwards will be distorted, making it difficult to make the right diagnosis "or to rule out a vitamin B12 deficiency as a cause. It may become impossible to make the right diagnosis and prescribe the correct treatment. So if you have signs and symptoms of a vitamin B12 deficiency, make sure to get tested before you start taking vitamin B12 supplements.
Ask your general practitioner for a B12 serum test and a folic acid test, so you have a baseline measurement of both. Your physician will be able to tell if your symptoms are being caused by a different disease. (You can also have ProHealth do these tests).

Once you've been tested, you can start using vitamin B12 lozenges while waiting for your test results.

If you are already taking a high dose (>1000 mcg) of vitamin B12 and you notice your symptoms are diminishing, keep taking the supplements and inform your physician about the vitamin B12 supplements that you are taking. Your physician may want to adjust your treatment (switching to injections for instance), depending on the results of the test and the symptoms.

If you are already taking multivitamins or other products that contain vitamin B12 and you have symptoms, it is still important to get tested. However, do not start new supplements before you get your blood tests done and inform your physician about the vitamin B12 supplements you are currently using.

Quoted from: Could it be B12? "Sally M. Pacholok & Jeffrey J. Stuart, page 213-214

Serum B12-test

This is a measurement of the level of vitamin B12 in the blood serum.
The current threshold used by physicians in the Netherlands to diagnose a vitamin B12 deficiency is: < 200ng/l
Grey zone: between 200 ng/L and 600ng/L
If your results are within the grey zone, but you recognise the symptoms, you should insist on a test treatment with vitamin B12.

The threshold that is used differs per country. In the US for instance, 240 ng/L is used, and there are countries where 300 ng/L is used. Japan has a lower limit of 540 ng/L. In levels below 745 ng/L, there is a vitamin B12 deficiency demonstrable in the cerebral and spinal fluid. Sally M. Pacholok en Jeffrey M. Stuart., "Could it be B12" pages. 241-242 ISBN 978-90-202-0490-2 3 4

There is considerable disagreement about what a normal result for this test should be, which is why the test is often combined with the MMA-, Homocysteine test and HoloTC- test. The total quantity of serum B12 after all only consists of 5-20% of biologically active vitamin B12 (transcobalamin II). 1 The other two forms (transcobalamin I and transcobalamin III) are assumed to be inactive, however they are included in the total result of the serum B12 test, leading to a higher score which implies your B12 levels are okay.5

It is also important to measure folic acid levels. When folic acid levels are 43 ng/L or more, the serum B12 test becomes invalid. (Folic acid levels can differ per lab)

The test can have misleading results in the following cases:
Reasons why the serum B12 levels can be elevated even though there is a vitamin B12 deficiency:6

  • Active liver disease (hepatitis, alcoholism)
  • Transcobalamin II deficiency
  • Intestinal bacterial overgrowth
  • Myeloproliferative disorders
  • Polycythaemia vera
  • Chronic myelogenous leukaemia
  • Acute promyelocytic leukaemia
  • Chloral hydrate medication
  • Lymphoma

Low serum B12 levels but no vitamin B12 deficiency:6

  • Folate deficiency
  • Pregnancy (although the book "Could this be B12?" questions the validity of this finding because it is possible that B12 deficiency is under-recognized in pregnancy)
  • Multiple myeloma
  • Excessive vitamin C intake
  • Transcobalamin I deficiency

Homocysteine test

This is a measurement of the homocysteine level in plasma. A slight or moderate elevation of the homocysteine levels can be the result of genetic defects or deficiencies of vitamin B12 (methylcobalamin), B6 or folic acid/folate (5-MTHF).

The higher the homocysteine level, the higher the risk of cardiovascular disease. Ralph Green (vitamin B12 expert) "for every 5 µmol increase of the total level of homocysteine in blood plasma, there is a corresponding increase of 40% in the relative risk of developing coronary heart disease."

It is necessary to fast 12 hours before the test starts.

A normal homocysteine value is < 14 µmol/l. Sometimes, homocysteine levels are normal, but the serum B12 level is in the grey zone or lower. In that case, ask for a test treatment with vitamin B12.

Reasons the homocysteine level may be elevated:6

  • Vitamin B12 (methylcobalamin deficiency)
  • Folate (5-MTHF deficiency)
  • Vitamin B6 deficiency
  • Renal insufficiency
  • Intravascular volume depletion
  • Folate deficiency
  • Chronic conditions:
    • hypothyroidism
    • systemic lupus erythematosus
    • severe psoriasis
    • some cancers
    • renal failure
  • Medication:
    • Dilantin
    • Tegretol
    • Nitrous oxide
    • Methotrexate
    • Lipid-lowering agents (colestipol and niacin in combination with thiazide diuretics)
    • Oestrogen-containing oral contraceptives
  • Inherited errors of methionine metabolism:
    • Cystathionine-beta-synthase deficiency
    • Methionine synthase deficiency
    • Methylenetetrahydrofolate reductase deficiency
  • Inborn errors of B12 metabolism

Methylmalonic acid test (MMA-test)

This is a measurement of the amount of methylmalonic acid (MMA) in the urine and/or serum (blood).
An elevated MMA level indicates a vitamin B12 deficiency (adenosylcobalamin).
Adenosylcobalamin is used in the conversion of methylmalonyl-CoA into succinyl-CoA. In case of a deficiency in adenosylcobalamin, the methylmalonyl-CoA is converted into methylmalonic acid, which causes a raise in these values.

Our preference goes to the urine MMA test. Dr. Eric Norman explains "MMA found in urine is concentrated forty times higher than in serum. That is why temporary fluctuations have a much stronger influence on the serum test."7

A normal urine MMA level is below 3.8 µg MMA/mg creatinine (3.6 µmol/mmol creatinine).
A normal serum MMA value is below 0.27 µmol/l.
Higher levels may indicate a vitamin B12 deficiency.

Sometimes your MMA level is normal, but your serum B12 level is within in the grey zone or lower. If this is the case, ask for a test treatment with B12.

Reasons why urine MMA levels can be elevated:6

  • Vitamin B12 (adenosylcobalamin deficiency)
  • Certain inborn errors of B12 metabolism
  • Unreliable in case of renal insufficiency
  • Unreliable in case of intravascular volume depletion

Reasons why serum MMA levels can be elevated:6

  • Vitamin B12 (adenosylcobalamin deficiency)
  • Certain inborn errors of B12 metabolism
  • Unreliable in case of renal insufficiency
  • Unreliable in case of intravascular volume depletion

Holotranscobalamin test (HoloTc-test)

The HoloTC test only measures the amount of active vitamin B12 in the blood. This is the amount of vitamin B12 in the blood that is attached to HoloTranscobalamin.

This test is not offered in many places in the Netherlands. Hospitals that do take this test are:

It is possible to have your blood sent to one of these hospitals for the HoloTC test.

The normal values for this test are between 27 and 162 ng/L. However, various medical centres advise a threshold of 47 ng/L, and various Germans studies consider levels between 47 and 67 ng/L as a grey zone.

Because the test can only measure the active serum B12 level in the blood, defects in the B12 metabolism can become apparent that a serum B12 test does not show.

Folic acid test

It is important to have a folic acid test done when determining a vitamin B12 deficiency. This is to make sure professionals do not solely look at the blood count.

A vitamin B12 deficiency causes enlarged red blood cells (megaloblastic anaemia). The enlarged red blood cells are seen as a marker for a vitamin B12 deficiency by professionals. A high level of folic acid can normalise red blood cells that otherwise would have been enlarged because of insufficient B12 levels. This means your blood count can look normal, even though you are suffering from a vitamin B12 deficiency. 8
This can occur when the folic acid value is above 31.

List of symptoms

The most comprehensive list of symptoms caused by a vitamin B12 deficiency!!

Overview of medication known to interfere with the absorption of vitamin B12.


List of therapists

map therapeuten vitamine b12 nederland


You can get these tests done at ProHealth or at your local lab.

Test Name of the test at ProHealth
B12-serum Vitamin B12
HoloTC Vitamin B12-active
MMA Methylmalonacid in morning urine
Homocysteine Homocysteine
Folic acid Folic acid serum


  1. Nexo E, Christensen AL, Hvas AM, Petersen TE, Fedosov SN. Quantification of holo-transcobalamin, a marker of vitamin B12 deficiency. Clin Chem 2002;48:561-2 http://www.ncbi.nlm.nih.gov/pubmed/11861448 [Free Full Text]
  2. van Tiggelen, C.J.M., et al., Assessment of vitamin-B12 status in CSF. American Journal of Psychiatry 141, 1:136-7
  3. Mitsuyama, Y., Kogoh, H., Serum and cerebrospinal fluid vitamin B12 levels in demented patients with CH3 - B12 treatment - preliminary study. Japanese Journal of Psychiatry and Neurology 42,1: 65-71
  4. van Tiggelen, C.J.M., Peperkamp, J.P.C., Tertoolen J.F.W., Vitamin-B12 levels of cerebrospinal fluid in patients with organic mental disorder. Journal of Orthomolecular Psychiatry 12, 305-311
  5. Nexo E, Hoffmann-Lücke, E. Holotranscobalamin, a marker of vitamin B-12 status: analytical aspects and clinical utility. Am J Clin Nutr. 2011 Jul; 94(1): 359S-365S http://ncbi.nlm.nih.gov/pmc/articles/PMC3127504 [PDF]
  6. Sally M. Pacholok en Jeffrey M. Stuart., "Is het Misschien B12-tekort?" Blz. 241-242 ISBN 978-90-202-0490-2
  7. Norman, E.J., Morrison, J.A., Screening elderly populations for cobalamin (vitamine B12) deficiency using the urinary methylmalonic acid assay by gas chromatography mass spectrometry. The American Journal of Medicine 94± 589/594. http://www.amjmed.com/article/0002-9343%2893%2990209-8/abstract
  8. Sally M. Pacholok en Jeffrey M. Stuart., "Is het Misschien B12-tekort?" Blz. 35 ISBN 978-90-202-0490-2

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Experience expert Contact: Lavinia Bijl