Vitamin B12 Deficiency Test

B12 Test and Diagnosis 

Vitamin B12 Deficiency Test

What types of B12 deficiency test are there? How are they carried out? How to diagnose a vitamin B12 deficiency through use of a B12 deficiency test.

Vitamin B12 Tests


  • How can you test for a vitamin B12 deficiency?
  • Which tests are worthwhile?
  • Are urine tests also possible?
  • How are individual tests evaluated?
  • What do the results mean?

For people who display typical symptoms of a B12 deficiency or have an increased risk of a vitamin B12 deficiency (e.g. vegans, vegetarians, the elderly), it can make sense to undergo a vitamin B12 test. 

Unfortunately, however, this isn’t quite as simple as it sounds. Anyone who decides to get themselves tested for a vitamin B12 deficiency will quickly discover that there is neither a clear definition for a vitamin B12 deficiency, nor a consensus for a general, standardized test. At present, the best and most reliable tests for a vitamin B12 deficiency are the holoTC test, which uses holotranscobalamin, and the MMA urine test, which uses methylmalonic acid. 

So which tests are actually worth doing? And how is a vitamin B12 deficiency actually defined?

Which Tests Can Check For a Vitamin B12 Deficiency?

Thus far, none of the vitamin B12 tests can give clear information as to the current vitamin B12 status in the body. Presently, a number of tests are in use, all of which have their specific advantages and disadvantages. The standard test in general medical practice is the serum test, which is the oldest but also by far the most unreliable test of all. 

Serum testTotal B12 levels in the blood

Low. Severe deficiencies can be recognized, but higher values have no relevance or significance. The test also includes unavailable B12 and B12-like substances (synthetic B12)

Holo-TCBioavailable B12 which binds with transcobalamin in the blood

High. However with a grey area, in which the diagnosis is uncertain

Homocysteine-TestHomocysteine levels in the bloodMedium. Numerous other factors could also play a role
MMA-UrintestMethylmalonic acid levels in the urineHigh. Only a handful of other factors

Vitamin B12 Deficiency Tests Sensitivity

A vitamin B12 deficiency has several phases, and the various tests unfortunately cannot provide reliable results in all of these phases. 

  • Phase 1: undersupply, B12 body store begins to empty, cells initially still powered by B12 store.
  • Phase 2: B12 store levels critically low, B12 deficiency develops within the cells
  • Phase 3: severe, clinical deficiency

Values Given by Various B12 Tests in the Course of a Deficiency


From this table, we can decipher the following key facts:

HoloTC is the earliest indicator of the start of an insufficient vitamin B12 supply. 
MMA in the urine is also very sensitive and the easiest test for illustrating an actual, existing cellular deficiency. 

It is important to note here that the MMA test is also the only test which can be carried out at home. 

Important Information

  • The serum test has little value
  • Holo-TC is the most exact test
  • The MMA test is reliable and the simplest test, as it can be done at home.

In the following section, the tests will be analyzed individually.

Vitamin B12 Blood Tests

There are two different procedures which measure the vitamin B12 levels directly in the blood:

  1. Serum Test
    Also measures inactive vitamin B12
  2. HoloTC Test
    Only measures active vitamin B12

A vitamin B12 deficiency is presently most often measured via a serum test and is defined as being vitamin B12 serum levels of under 200 pg/mL. However, this value does not describe the actual supply of vitamin B12 within the blood cells, since not all vitamin B12 which is dissolved in blood can be used by the body. The sole decisive factor is the actual supply of vitamin B12 within the cells, which cannot be accurately described through a serum test. 

The result of the serum blood tests is of little importance and the serum test is the most unreliable of all tests available. 

Vitamin B12 Deficiency Definition

Despite its unreliability, the serum test is often used to define a vitamin B12 deficiency. The following table illustrates the definition of a vitamin B12 deficiency which is still being used today. 

These general practice values are considered very unreliable today amongst the scientific community. Other tests with different methods have shown that a vitamin B12 deficiency can exist even with very high B12 serum levels.1-4

Table: Definition of Vitamin B12 Deficiency

Severe deficiency < 150 pg/ml< 110 pmol/l
Deficiency < 200 pg/ml< 150 pmol/l
Potential undersupply 200-300 pg/ml150-220 pmol/l
Normal status 300-900 pg/ml220-665 pmol/l

Some researchers, such as vitamin B12 experts Mitsuyama and Kogoh,Tiggelenand David Brownstein,are convinced that a vitamin B12 deficiency within the cells is possible even with a value of 600 pg/mL, and that the number of deficiency sufferers is thus significantly higher than it is officially given. Numerous studies agree with this statement that even in B12 serum levels far higher than the perceived norm, deficiencies can manifest. 

B12 Serum Test (Total Vitamin B12)

The serum test is the oldest of all tests and is yet still used to provide the current definition of a vitamin B12 deficiency. Most doctors are unaware that much better methods of measurement exist. The serum test is not overly worthwhile, since a large majority (70 – 90%) of the vitamin B12 measured is bonded to proteins like haptocorrin; meaning it cannot be used for metabolism. 

Additionally, the test also measures completely inactive vitamin B12 analogues. These are substances, which are chemically very similar to vitamin B12, but have absolutely no positive, vitamin effect in the body. 

The only vitamin B12 in the blood that the body can use is the cobalamin (B12) bonded to transcobalamin II, which equates to around 20% of all vitamin B12 levels in serum. This demonstrates that the overall levels of vitamin B12 in the blood bear little relevance to the amount of vitamin B12 that the body can actually use. A deficiency can exist even in very high B12 serum levels. 

Usefulness: low

HoloTC Test

HoloTC will accurately determine the levels of biologically available vitamin B12 bonded to transcobalamin II, even in blood serum. A lower level of HoloTC in serum is considered the earliest indicator of a vitamin B12 deficiency, giving an early warning that the body isn’t receiving enough usable vitamin B12 and that the body’s B12 store is beginning to empty. 

The results of a HoloTC test are thus much more useful than those of the serum test, even though it cannot make certain that there is a vitamin B12 deficiency present, as there is a large grey area in which it is unclear whether or not a deficiency exists. Additional factors in the complete blood count can bring more clarity here. 

Usefulness: high

Serum Test and HoloTC Compared

Total Vitamin B12

Holo-TC Interpretation

> 400 ng/l

Low chance of vitamin B12 deficiency

200-400 ng/l

Grey area

Holo-TC > 50 pmol/l

Low chance of vitamin B12 deficiency

Holo-TC 35-50 pmol/l

Further testing needed

Holo-TC < 35 pmol/l

High chance of vitamin B12 deficiency

< 200 ng/l

Vitamin B12 deficiency

Typical results of serum and HoloTC testing. The values are controversial and considered too low by many researchers. Doctor David Brownstein is of the opinion that a total level of 600 ng/l can lead to a deficiency.

Indirect Tests for Vitamin B12 Deficiency

Since both tests fail to give complete clarity regarding a deficiency, testing has been developed to measure a large variety of different metabolic products which are connected to vitamin B12, instead of measuring vitamin B12 directly. These can give a far clearer indication as to whether a deficiency is present, but they give no quantitive information on vitamin B12 levels as such. 

The following indirect tests for vitamin B12 deficiency currently exist:

  1. Homocysteine in the blood
  2. Methylmalonic acid in the urine
  3. Methylmalonic acid in the blood

Homocysteine Test

When a vitamin B12 deficiency is present, homocysteine levels rise significantly. Higher homocysteine levels can be considered a surefire indicator that a vitamin B12 deficiency exists. However, certain diseases can distort this result. A combination of a direct test, such as the HoloTC test, and the homocysteine test can give a very reliable result regarding vitamin B12 deficiency. The homocysteine test is also commonly used to test the success of vitamin B12 therapy. 

The homocysteine level should ideally be under 9 μmol/l. 

Usefulness: medium

MMA Test – the Important Urine Test

Presently, the best method of determining if a vitamin B12 deficiency exists is the methylmalonic acid (MMA) test. During a deficiency of biologically active B12 in the cells, MMA is increasingly formed, leading to an increased level of MMA in both blood and urine. An advantage of this method is that it can determine very early on whether there is enough active B12 in the cells. A further advantage is the fact that this can be determined with a simple urine sample, making the need for a blood test irrelevant. A deficiency can be unveiled very early and in a reliable and comfortable manner. 

MMA level is tested relative to the creatine level in the body, in order to remove the water content of the urine as a factor. Other than a few, very rare diseases, there are hardly any factors which can distort the results of the MMA test, making it the very best quick test available today. 

The ideal test would be a combination of the HoloTC, homocysteine and MMA tests, but all three would result in a significant financial outlay, which is why the MMA test is most commonly recommended, as many companies offer MMA tests which can be carried out at home. 

Usefulness: high


Serum MMA

Urine MMA

Normal Value

Different measurements:
0.04 – 0.27 μmol/l
0 – 271 nmol/l
0 – 32 µg/l

up to 2 mg MMA/g creatine, aka.
up to 3.6 μmol MMA/mmol creatine

Critical Value

> 0.37 μmol/l
> 271 nmol/l
> 32 µg/l

Higher Value

Vitamin B12 Breath Test

Researchers at the university of Florida have developed a breath test which works by determining the level of vitamin B12 against the CO2 content in the breath. Vitamin B12 plays a decisive role in the metabolization of sodium propionate to CO2. Through administering sodium propionate and measuring the subsequent levels of CO2 expelled from the lungs, it is possible to check the body’s vitamin B12 status. For this, the researchers labelled the propionate with a stable isotope of carbon, and then only measured the resulting CO2 which was labelled. The test is currently still in the research phase, but has already proven itself to be reliable and could be a comfortable alternative to the existing tests in the near future. 

Who can Perform a Vitamin B12 Test?

Serum tests, HoloTC tests and homocysteine tests can presently be carried out by a house doctor. They simply require a blood sample on an empty stomach. MMA urine tests can be ordered from various firms online and must be completed and returned, before results are sent out either by email or mail. 

Readers who think they may suffer a vitamin B12 deficiency should initially take the cheapest and most comfortable variant, the MMA test. This gives a clear indication as to whether a deficiency is present. If the test is positive, a follow-up appointment with a doctor should be made in order to find out the cause of the deficiency and to discuss treatment. 

Those who would rather not visit the doctor, or who believe that their potential deficiency could be caused by an insufficient intake of vitamin B12, for example vegans, can simply start to take regular doses of suitable vitamin B12 supplements and then complete a second MMA test after a few weeks. If this second test returns positive for a deficiency, then it is highly likely that the cause of the problem is absorption difficulties. At this point in time, it is highly recommended to visit a doctor. 

Testing for Causes of a Vitamin B12 Deficiency

Upon discovery of likely malabsorption, testing is carried out as to the cause of this. Among the most common are the following causes, which can be detected in each case by specific tests:

Deficiency in production of the intrinsic factorSchilling test
Irritation or inflammation in stomach and intestinestool examination, blood tests, gastroscopy or colonoscopy
Gastritis (chronic inflammation of the stomach)Gastroscopy, blood test, urease test, 13C-urea breath test
Crohn’s disease (chronic inflammation of the intestine)Blood test, ultrasound, colonoscopy
Interaction with medicineConsultation with a doctor
DiseaseGenetic test

Schilling Test

Of the tests listed above, the Schilling test plays a particularly big role in relation to vitamin B12. The Schilling test can determine whether the body can successfully absorb vitamin B12 via the intrinsic factor, a specialized transport protein. 

The patient is administered radioactive labelled vitamin B12 and the absorption is controlled. The first time, just B12 is administered, the second time a combination of B12 and the intrinsic factor. If the patient is unable to absorb the B12 alone, but succeeds with the intrinsic factor, then the cause of the absorption difficulty can be circled.

If absorption fails in both cases, another cause is responsible. 

Vitamin B12 Deficiency Tests Diagram

The following graphic illustrates systematically how testing for vitamin B12 deficiency is done. Further testing for individual causes is not included in the interest of clarity. 

B12 Deficiency Spectrum


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