Vitamin B12 Deficiency

Testing for vitamin B12 deficiency generally occurs if a patient has symptoms of vitamin B12 deficiency. Diagnosis of vitamin B12 deficiency it is most often done with a simple blood test. The test measures the amount of vitamin B12, or vitamin B12 analogue in serum. The validity of the test in diagnosing vitamin B12 sufficiency has been questioned for nearly 40 years (England and Linell, 1980: Clarke etal, 2007), as the test does not measure what form of vitamin B12 is present, whether it is Cyanocobalamin (following supplementation), hydroxycobalamin (following supplementation), or the two active forms Adenosyl-Co(III)cobalamin, Methyl-Co(III)cobalamin or the biologically inactive Co(II)cobalamin, or even nitrotrosylcolabalmin. The test, whilst relatively active, therefore basically reflects circulating levels of some form of vitamin of B12 and as such does not measure how much, or what type of biologically activity B12 there is, and as such may not correctly identify true biochemical vitamin B12 sufficiency/deficiency (Schrempf etal, 2011: Clarke etal, 2007; Hermann etal, 2003). Most doctors, however, do not generally understand this. The interpretation of the results is also highly variable, and even the definition of deficiency varies from country to country and lab to lab. This confusion appears to be due to the belief by pathology labs that vitamin B12 is only required for production of Red Blood Cells, and so deficiency is determined in relation to vitamin B12 mediated anemia, a very old and out-dated concept. Thus, some labs define vitamin B12 deficiency at levels of less than 150 pg/ml (110 pmol/L) and others at 200 pg/ml (165 pmol/L). Recent guidelines by the British Haematology Society is a cut-off of 200 pg/ml (148 pmol/L). Recently there has been a push to raise the cut-off to 250-300 pg/ml, but this is not generally accepted/adopted by the majority of laboratories. Thus there is a problem even in defining the cut-off level that defines B12 deficiency. Said cut-off varies from country to country. Even in 2009, the definition of deficiency was still being debated such that in the US, many groups were still using a cut-off of 148 pmol/L, thus grossly under-estimating the incidence of the condition (Allen, 2009). In conditions, such as those caused by functional B2 deficiency (Chronic Fatigue, Autism, Diabetes, Obesity, etc) the assessment of B12 sufficiency by serum B12 levels is almost useless, and generally leads to many false negative.

Active B12 Test

In an attempt to clarify this position a very controversial "Active B12 test" has recently been introduced to the lab testing arena. This name of the test is misleading as the test does not at all measure biologically active vitamin B12, instead it measures active transcobalamin, or more correctly the amount of transcobalamin in serum that has any analogue of vitamin B12, active or inactive, bound to it.

In the assay, a specific antibody recognizes the change in shape of transcobalamin when it binds to vitamin B12. This is regardless of which analogue of vitamin B12 it is, cyanocobalamin, hydroxycobalamin, methylcobalamin, adenosylcobalamin, glutathionylcobalamin, cysteinylcobalamin, sulphitylcobalamin, Co(II)cobalamin or Co(I)cobalamin, all of which are bound with very high affinity to transcobalamin, yet only methylcobalamin and adenosylcobalamin are active.

Transcobalamin with no bound vitamin B12.

Transcobalamin with B12 bound in the binding site. Note the red Cobalt atom. The change in colour denotes a change in confirmation.

Studies by Schrempf and co-workers (2011) showed that there was a very poor correlation between the data from the "active" b12 test, or the serum B12 test and identifying persons with vitamin B12 deficiency symptoms (14.7% and 21.0% respectively, and the test was associated with more false-positive test that true-positive tests (Clarke etal, 2007). Even in patients with low serum B12 levels there was poor concordance between serum B12 and MMA (56%) and homocysteine (62%), even though there was high concordance between MMA and Hcy (82.1%) (Remacha etal, 2014). Studies by Golding (2016) found that the holotranscobalamin test (Active B12), was less useful the the standard B12 test in identifying functional B12 deficiency as judged by MMA and Homocysteine levels. Studies comparing HoloTC measurement to total B12 measurement have been highly  varied whilst there was one study with a good correlation, this study was performed by saturating serum with inactive cyanocobalamin, and so technically the assay could not have been measuring so-called "active B12" because cyanocobalamin is totally inactive (Griffioen et al, 2017) This casts serious doubts on whether the test has any utility at all. Currently to our knowledge there is no commercially available test that will discern the identity of vitamin B12 in serum.

Further, in conditions of paradoxical B12 deficiency, the levels of both total B12 and TC-bound B12 (the so-called active B12) are very high.

Biochemically, though, there are many metabolic markers of vitamin B12 deficiency, and several of these have been used as alternative means for determining functional B12 deficiency, one is a test of serum homocysteine, which becomes elevated as levels of methyl B12 drop, and the other is the MMA test (methylmalonic acid test), which becomes elevated as levels of adenosyl B12 drop. Other additional markers, useful in confirming deficiency are elevations in the branched chain organic acids methylsuccinate and ethylmalonate, the neurotransmitter metabolites HVA, VMA, KA, QA and HIAA, and elevations in pyroglutamate, thymine and 3-hydroxymethylglutarate.

Highly elevated total and "active B12" are typically found in conditions such as autism, chronic fatigue syndrome, obesity, depression, diabetes, and in those who supplement with high doses of oral B12 or those who use B12 lozenges. The misleading name of the "active B12" test is resulting in many persons who are functionally deficient in vitamin B12 not receiving appropriate treatment. Thus, clinicians who understood the relevance of the test would not order the test, whilst those who do not understand the relevance may order the test, but then misinterpret its findings.

Pernicious anaemia may often be associated with unexplained weight loss of around 10-15 lb (4-7 kg) occurs in about 50% of patients possibly due to anorexia, which is observed in most patients. Low-grade fever occurs in one third of newly diagnosed patients and promptly disappears with treatment.

Tiredness and fatigue is associated with the anaemia (reduced red cell numbers), which is gradually restored during treatment. However, the cardiac output is usually increased with haematocrit values less than 20%, and the heart rate accelerates. This may in turn lead to congestive heart failure and coronary insufficiency particularly in patients with pre-existing heart disease.

Often a little known condition called "Paradoxical B12 Deficiency" can occur. In this condition, serum levels of vitamin B12 can be much higher than 300 pmol/L, but the patient still exhibits symptoms of B12 deficiency, and upon analysis shows elevated levels of MMA, homocysteine and various organic acids in the urine. Seeing the elevated serum B12 levels the physician often will not treat the deficiency, but even if they do treatment will not be successful. The reason for this appears to due to lack of functional B2 as FMN and FAD. In this situation, lack of FMN and FAD reduces the activity of the two "B12 rescue" enzymes, methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR), with the result that the majority of the vitamin B12 is slowly converted to inactive Co(II)B12. Lack of functional B2 activity also means that if cyanocobalamin or hydroxycobalamin is injected or given as a large dose supplement, it will not be converted to Adenosyl or Methyl B12. This can be confusing to the physician who upon measurement can show that serum B12 has risen but yet symptoms of B12 deficiency have not changed, which causes a paradox. At this time the diagnosis that the symptoms observed are due to vitamin B12 deficiency is abandoned with the "Oh it must be something else" outcome resulting. It is in this situation that tests such as the Organic Acids test become very useful as these will show not only the B12 deficiency markers, but also evidence of functional B2 deficiency. In order to correct the B12 deficiency the functional B2 deficiency MUST be addressed before the B12 deficiency can be overcome. It is very important to recognize Paradoxical B12 deficiency particularly in pregnant mothers, as elevated, yet inactive B12, can give the false impression that a mother and hence the child will be replete in vitamin B12. In Paradoxical B12 deficiency highly elevated levels of serum B12 can be found (>500 pmol/L), and this has been shown to correlate with an increased risk of autism (2.5 times), a condition that is now been shown to be due to functional B12 deficiency in the children. Paradoxical or functional B12 deficiency is common in the elderly, particularly those with "Oxidant" risks where "even at Cbl levels >800 pg/ml, MMA values were increased in 73% of elderly subjects with at least one oxidant risk (Solomon, 2015). Paradoxical B12 deficiency should be suspected when associations are found with disease risk and MMA or Homocysteine levels, but not with vitamin B12 levels (Gopinath etal, 2009).

Symptoms of B12 deficiency

In extreme B12 deficiency, approximately 50% of patients have a smooth tongue with loss of papillae, particularly along the edges of the tongue. The tongue may be painful and beefy red.  Many patients experience burning or soreness, most particularly on the anterior third of the tongue, and may lead to changes in taste and loss of appetite.

Altered bowel movements are common and include constipation or having several semisolid bowel movements daily. These symptoms have been attributed to megaloblastic changes of the cells of the intestinal mucosa, however, it is much more likely that increased production of serotonin, and lack of production of melatonin are causal, particularly since melatonin has been shown to aid in the maturation of villi in the intestine as well as stimulating production of intestinal enzymes such as lactase and histamine-N-methyl transferase. These changes can also be associated with altered uptake of essential nutrients such as B group vitamins. In children with autism this seems to be associated with the development of multiple food intolerances, characteristic of the condition, but also to an intolerance to histamines. Non-specific gastrointestinal (GI) symptoms include anorexia, heartburn, nausea, vomiting, heart burn, flatulence, and a sense of fullness.

Neurological symptoms typical of prolonged vitamin B12 deficiency can be elicited in patients with pernicious anemia. The most common of these are parasthesiae (feeling of pins and needles), weakness, clumsiness, and an unsteady gait. Impaired proprioception is worse in the dark as patients are unable to rely upon vision for compensation. These neurologic symptoms are due to myelin degeneration and loss of nerve fibres in the dorsal and lateral columns of the spinal cord and cerebral cortex.

Symptoms of long term VB12 deficiency can be seen in older patients such as senile dementia, peripheral neuropathy, and psychosis Alzheimer disease; memory loss, irritability, and personality changes delusions, hallucinations, outbursts, and paranoid schizophrenic ideation.

MMA

Serum vitamin B12 concentrations are not always predictive of actual vitamin B12 status and it is often necessary to determine status by another marker, as the predictive value of serum vitamin B12 has been reported to be as low as 22%. One metabolic marker of vitamin B12 status is the level of serum or urinary methylmalonic acid (MMA). Adenosyl-cobalamin is a cofactor for the enzyme methylmalonyl CoA mutase, and in adenosyl B12 deficiency, levels of methylmalonic acid (MMA) increase inversely to levels of functional B12. Comparing MMA as a marker, evidence of vitamin B12 deficiency, as judged by an increase in MMA occurs at around 250 pmol/L vitamin B12. When levels drop to 200 pmol/L urinary MMA levels have increased to around 2.0 umol MMA/mmol creatine, and 0.4 umol/L plasma MMA. Study after study has established that metabolic vitamin B12 deficiency occurs at levels of vitamin B12 that are below 250 pmol/L, yet despite this pathology labs throughout the world stubbornly stick to the definition of vitamin B12 deficiency not occurring until 120, 150, or 180 pmol/L, much to the detriment of the very people who are paying for their services, and who are clinically or sub-clinically vitamin B12 deficient but are not being treated.

Homocysteine

Dietary Methionine (an essential amino acid) performs two critical functions in the body, as a building block in the synthesis of proteins and as an essential amino acid in the methylation cycle, a five step process, which terminates in the production of homocysteine. Homocysteine then represents a branch point at which stage the homocysteine can be converted to cystathionine or can be remethylated to form methionine, at which stage the cycle can repeat itself. The remethylation of homocysteine to form methionine requires the Methyl B12-dependent enzyme methionine synthase. A drop in the level of methyl B12 in the body can thus be measured by an increase in the amount of homocysteine. Elevated homocysteine has been associated with many conditions including, increased risk of stroke, cognitive impairment, dementia, atherosclerosis, myocardial infarction, Parkinson's disease, Multiple Sclerosis, Neural Tube defects, epilepsy,  and peripheral neuropathy amongst others..

Urinary organic acid measurement is an ideal non-invasive method to monitor metabolic abnormalities, including those that result from deficiency of Adenosyl and Methyl B12. Adenosyl B12 is involved in the metabolism of odd chain fatty acids and also in the metabolism of branched chain amino acids. Lack of metabolism of these molecules leads to an elevation of the by-products, such as MMA, ethylmalonic acid and methylcitrate in urine. Methyl B12 is also involved in the production of CoQ10 and lack of methyl B12 can be measured by increased levels of hydroxymethylglutarate, a precursor in CoQ10 synthesis. Further, methylation is the final step in production of adrenalin (from nor-adrenalin, from dopamine) and melatonin (from serotonin) and recently it has been shown that lack of methyl B12 leads to increased excretion of the organic acids, HVA, VMA, QA, KA and 5-hydroxyindole acetic acid. A further description of this can be found at http://wipeoutautism.org/ .

England JM, Linnell JC. Problems with the serum vitamin B12 assay. Lancet.1980 Nov 15;2(8203):1072-4. PubMed PMID: 6107691.

Herrmann et al Functional vitamin B12 deficiency and determination of holotranscobalamin in populations at risk. Clin Chem Lab Med. 2003 Nov;41(11):1478-88. PubMed PMID: 14656029.

Clarke et al. Detection of vitamin B12 deficiency in older people by measuring vitamin B12 or the active fraction of vitamin B12, holotranscobalamin. Clin Chem. 2007 May;53(5):963-70. Epub 2007 Mar 15. PubMed PMID: 17363419

Schrempf et al Utility of measuring vitamin B12 and its active fraction, holotranscobalamin, in neurological vitamin B12 deficiency syndromes. J Neurol. 2011 Mar;258(3):393-401. doi: 10.1007/s00415-010-5764-4. Epub 2010 Oct 2. PubMed PMID: 20890610.

Remacha etal Role of serum holotranscobalamin (holoTC) in the diagnosis of patients with low serum cobalamin. Comparison with methylmalonic acid and homocysteine. Ann Hematol. 2014 Apr;93(4):565-9. doi: 10.1007/s00277-013-1905-z. Epub 2013 Sep 21.PubMed PMID: 24057896.

Golding PH. Experimental vitamin B12 deficiency in a human subject: a longitudinal investigation of the performance of the holotranscobalamin (HoloTC,  Active-B12) immunoassay. Springerplus. 2016 Feb 25;5:184. doi: 10.1186/s40064-016-1740-5. eCollection 2016. PubMed PMID: 27026880; PubMed Central PMCID: PMC4767712.

Griffioen et al. Measurement of total Transcobalamin employing a commercially available assay for Active B12. Clin Biochem. 2017 Dec;50(18):1030-1033. doi: 10.1016/j.clinbiochem.2017.08.009. Epub 2017 Aug 16. PubMed PMID: 28823760

Allen, LH, 2009 How common is vitamin B12 deficiency. Am J Clin Nutr. 2009 Feb;89(2):693S-6S. doi: 10.3945/ajcn.2008.26947A. Epub 2008 Dec 30

Aparicio-Ugarriza et al. A review of the cut-off points for the diagnosis of vitamin B12 deficiency in the general population. Clin Chem Lab Med. 2015 Jul;53(8):1149-59. doi: 10.1515/cclm-2014-0784.

Ansari et al, 2014 Hyperhomocysteinemia and neurological disorders: a review. J Clin Neurol, 10, 281-288.

Hill et al, 2013 A vitamin B12 supplement of 500 ug/d for eight weeks does not normalize urinary Methylmalonic acid or other biomarkers of vitamin B12 status in elderly people with moderately poor vitamin B12 status. J. Nutrition 143, 142-147

Raghaven, et al. 2018 Maternal multivitamin intake, plasma folate and vitamin B12 levels and Autism Spectrum Disorder risk in offspring. Ped Per Epid 32, 100-111

Solomon LR.Functional cobalamin (vitamin B12) deficiency: role of advanced age and disorders associated with increased oxidative stress. Eur J Clin Nutr. 2015 Jun;69(6):687-92. doi: 10.1038/ejcn.2014.272. Epub 2015 Jan 7.PMID: 25563739

Gopinath etal. The associations between blood levels of homocysteine, folate, vitamin B12, and retinal vascular caliber.Am J Ophthalmol. 2009 Dec;148(6):902-9. doi: 10.1016/j.ajo.2009.07.018. Epub 2009 Oct 1.