The Active-B12 Test (Holotranscobalamin)

Holotranscobalamin represents only 10-30% of the B12 circulating in the blood but is the only form of B12 that is taken up and used by cells of the body, hence it’s other name – active B12.

Active-B12, the next level of B12 testing; Axis-Shield

The active-B12 test (or holoTC / holotranscobalamin test) from Axis-Shield is a novel method for assessing vitamin B12 status. Axis-Shield claims the test offers improved accuracy, sensitivity, and specificity compared to the regular front-line B12 blood test.

Let’s discuss it in detail.

What Is Active B12 (HoloTC)?

Approximately one-quarter of circulating vitamin B-12 binds to transcobalamin and is thereby available for the cells of the body. For this reason, holoTC is also referred to as active vitamin B-12.

Holotranscobalamin, a marker of vitamin B-12 status

Three carrier proteins take part in the transport of B12 within our body. One of them is intrinsic factor (IF), against which the body creates antibodies in cases of pernicious anemia, while the other two are transcobalamin (TC) and haptocorrin (HC).

The selective uptake of cobalamin by humans requires three transporting proteins: intrinsic factor (IF), transcobalamin (TC), and haptocorrin (HC). IF is responsible for intestinal uptake of vitamin B12. TC is the major plasma transporter. HC seems to be a scavenging protein.

Mechanisms of Discrimination between Cobalamins and Their Natural Analogues during Their Binding to the Specific B12-Transporting Proteins

When transcobalamin and haptocorrin bind to the B12, the resulting complexes are known as holotranscobalamin (holoTC) and holohaptocorrin (holoHC).

Of the three carrier proteins, only transcobalamin can carry the B12 which was absorbed into the ileum, and move it further down to the tissues. Once there, the holoTC complex (that is, the “active B12”) is integrated through a specific receptor-mediated uptake, delivering B12 into the cells, where it acts as a co-enzyme for vital cellular functions.

What happens to the remaining B12 circulating in the blood? It is bound to haptocorrin, creating the biologically-inert complex holoHC. We’re not fans of the regular B12 blood test, because it fails to distinguish between holoTC and holoHC. This is a serious concern, because holoTC can make up as little as 10% of your total B12:

As a result, a real B12 deficiency can easily exist within what looks like a normal total-B12 status, and many genuine B12 deficiency cases end up undiagnosed.

From these results it can be concluded that serum concentration of holoTC is a much better predictor of B12 status than total B12.

The usefulness of holotranscobalamin in predicting vitamin B12 status in different clinical settings

Testing For Active B12 vs Total B12

HoloTC has a better diagnostic accuracy than vitamin B(12) and can replace the existing vitamin B(12) assay as a primary screening test in patients suspected of vitamin B(12) deficiency.

Screening for metabolic vitamin B12 deficiency by holotranscobalamin in patients suspected of vitamin B12 deficiency: a multicentre study

HoloTC (“active B12”) is the only B12 complex that the body can utilize, unlike the inactive haptocorrin-bound B12 (holoHC). That’s why an active-B12 test is more reliable than the total-B12 test. Your total-B12 levels are largely irrelevant, because only the transcobalamin-bound B12 can penetrate the cells and tissues. And since holoTC can be as low as 10% of your total-B12 levels, the common B12 blood test can easily give a misleading picture.

Here’s how important holoTC is compared to holoHC:

There’s a genetic condition in which the body has an absence of haptocorrin. However, it’s not life-threatening, and doctors often discover it accidentally. In contrast, the absence of transcobalamin is very serious, and it manifests as the typical symptoms of deficiency in B12. Doctors often diagnose it shortly after birth due to failure to thrive, and it requires immediate B12 therapy, otherwise irreversible neurological damage takes place.

In such cases, and in other situations where transcobalamin is compromised, you may be fooled by the healthy B12 levels that show up in the normal blood test, while an active-B12 test would have discovered the B12 deficiency. Thus, the active-B12 test may one day replace the normal test as the default diagnostic tool. There are good reasons:

Compared to total cobalamin, we observed a better performance of holoTC assay in detecting elevated concentrations of MMA in subjects with normal renal function. The majority of subjects with combined low holoTC and elevated MMA had normal concentrations of total cobalamin. HoloTC can be used as a first line parameter in detecting cobalamin deficiency.

Holotranscobalamin in laboratory diagnosis of cobalamin deficiency compared to total cobalamin and methylmalonic acid

This case study demonstrated that 5 different Total-B12 assays from 4 manufacturers all gave falsely elevated results in a patient with confirmed pernicious anaemia. The same failing described by Carmel, Agrawal, Yang and Cook is again apparent in this case study and demonstrates why Total-B12 results can be unreliable.

FALSELY ELEVATED COBALAMIN CONCENTRATION IN MULTIPLE ASSAYS IN A PATIENT WITH PERNICIOUS ANAEMIA: A CASE STUDY

After excluding 20 cases of incident dementia, increased tHcy remained associated with poorer performance in episodic memory, execution functions and verbal expression. Higher holoTC levels tended to be related to better performance in executive functions and psychomotor speed, while elevated serum folate concentrations were significantly related to higher scores in global cognition and verbal expression tests.

Serum homocysteine, holotranscobalamin, folate and cognition in the elderly

We confirm a decrease in cobalamins during pregnancy, and report that the active part of cobalamins (holotranscobalamin, holoTC) remains unchanged. The decrease in cobalamins is explained by a decreased holohaptocorrin (holoHC), suggesting that holoTC rather than cobalamins should be used as a marker of vitamin B12 deficiency during pregnancy.

Holotranscobalamin remains unchanged during pregnancy

No association between serum vitamin B₁₂ concentrations and cognitive decline or dementia was found. However, four studies that used newer biomarkers of vitamin B₁₂ status (methylmalonic acid and holotranscobalamin (holoTC)) showed associations between poor vitamin B₁₂ status and the increased risk of cognitive decline or dementia diagnosis.

Vitamin B12 status, cognitive decline and dementia

The active-B12 test uses a novel monoclonal antibody, which is incredibly accurate at detecting holoTC in a direct manner. It cuts out the need for the complex preliminary steps required by most B12 tests, eliminating a potential source of inconsistency.

One other benefit is that compared to holohaptocorrin (“inactive B12”), holotranscobalamin has a shorter circulating half-life. This means that when you’re approaching a negative B12 balance and entering the low range of B12 levels, the earliest change that actually occurs is very likely to be a decrease in the levels of active B12 specifically.

The Active-B12 Test Has a Misleading Name

With all the benefits we discussed, the active-B12 test is far from perfect. It may perform better than the common total-B12 test, but this is not very impressive given how weak the original test is. Anyway, our issue with the test comes from its misleading name:

The active-B12 test also tests for inactive B12.

Confused? Allow us to explain.

Cobalamin (B12) comes in many forms, depending on the ligand on its cobalt atom. While transcobalamin binds to the two active forms adenosylcobalamin and methylcobalamin, it can also bind to co(II)cobalamin, aquocobalamin, cysteinylcobalamin, nitrosylcobalamin, hydroxocobalamin, cyanocobalamin, aquocobalamin, sulfitocobalamin, chlorocobalamin, glutathionylcobalamin, or thiocyantocobalamin, none of which are biologically active.

Three proteins, intrinsic factor (IF), transcobalamin (TC), and haptocorrin (HC), all have an extremely high affinity for the cobalamins but discriminate these physiological ligands from Cbl analogues with different efficiencies decreasing in the following order:  IF > TC > HC.

Mechanisms of Discrimination between Cobalamins and Their Natural Analogues during Their Binding to the Specific B₁₂-Transporting Proteins

As you can see, transcobalamin is more promiscuous than intrinsic factor, although not as promiscuous as haptocorrin. In other words, it will bind to B12 analogues or any structure that looks like B12. You can even bind polymers or nanoparticles to the cobalt atom, and the transcobalamin will bind to them, showing up on the test results as “active B12”.

When transcobalamin binds to any analogue, its structure changes, and this change is what allows the monoclonal antibody in the holoTC test to recognize it as such. However, that change has nothing to do with whether the cobalamin in the complex is active or not.

In other words, the “active-B12” test measures the absolute amount of any B12 form as long as it’s bound to transcobalamin. The test is quantitative, not qualitative, since it can’t distinguish any analogue from methylcobalamin and adenosylcobalamin, the two true active forms, co-factors for the enzymes methionine-synthase and MMA-CoA mutase.

Of course, the holoTC test is still better than the total-B12 blood test, because it doesn’t measure forms bound to haptocorrin, which are truly biologically-inert. With holoTC, inactive forms at least have a chance to be converted to an active form downstream:

The transport of the essential micronutrient Cbl (cobalamin; vitamin B12) from food to cells relies on three successive proteins in mammals, HC (haptocorrin), gastric IF (intrinsic factor) and TC (transcobalamin). Cbl is required by cells as the basis for two enzyme cofactors, methyl-Cbl for methionine synthase and 5′-deoxyadenosyl-Cbl (Ado-Cbl) for methyl-malonyl-CoA mutase. Upon initial uptake of Cbl from food, Cbl becomes bound in the stomach to salivary HC. After proteolysis of HC in the duodenum, Cbl is passed on to IF. Mucosal cells in the ileum absorb the IF–Cbl complex via endocytosis mediated by a specific receptor. In the enterocyte, the IF–Cbl complex is degraded and Cbl is transferred to TC which delivers Cbl to cells via the blood. Only the fraction of Cbl bound to TC is taken up via endocytosis by a specific receptor on most cell types. HC that is also present in plasma cannot facilitate cellular uptake of Cbl except in hepatocytes. TC–Cbl is degraded in lysosomes to release Cbl for further conversion into the important cofactors.

Structural study on ligand specificity of human vitamin B12 transporters

Anyhow, nobody should have named it an “active B12 test”. It’s a holoTC test.

Also, transcobalamin inherits the selectivity of intrinsic factor – because IF filters at the intestine, before TC gets the chance to bind cobalamins. Take a look at the illustration we made of B12 absorption cycle:

As you can see in the diagram, IF (which has the highest specificity against analogues) filters cobalamins at the intestine. At step 5 in the blood, HC then captures spillover (it’s the least selective, so it sequesters remaining analogues into possibly a long-term storage), making for a total of the 80% of cobalamins in the blood. TC then gets the “clean” B12, 20% of the blood cobalamins. Remember, this step only comes after IF had already filtered the ingested cobalamins. Hopefully, TC gets predominantly active B12 (adenosylcobalamin & methylcobalamin) for immediate cellular delivery. Hopefully, because some forms (like cyanocobalamin) bind to IF, TC, and HC identically in terms of affinity and kinetic behavior. So holoTC will contain a mix of whatever B12 forms are absorbed through IF, which could include cyanocobalamin from supplements, dietary adenosylcobalamin, dietary methylcobalamin, and any analogues that escape the IF and HC filtration system. once inside cells, the inactive cyanocobalamin needs enzymatic conversion to become metabolically active, whereas adenosylcobalamin and methylcobalamin are already in active coenzyme forms. This conversion happens after TC delivers them to cells—not during transport

HoloTC should be predominantly active B12, because HC sponges up most of the dietary B12 plus any analogues that escape. HC dominates plasma B12 binding, because it has barely any discrimination at all – it binds true cobalamins and analogues with almost equal affinity. HoloHC appears to be a holding pattern for long-term storage.

In-vitro, IF shows the highest specificity for true cobalamins, followed by TC, while HC shows relatively non-selective binding to various corrinoids. However, cyanocobalamin and adenosylcobalamin behaved identically in all binding experiments. HC showed no discrimination between cobalamins and analogues. Essnetially 100% of HC will bind to any corrinoid equally, whether true cobalamin or analogue. TC shows moderate discrimination, and IF shows highest discrimination. In-vivo, however, IF filters the cobalamins before TC gets the chance to bind, so in practice TC inherits the selectivity of IF.

The study linked above reveals an important concern: B12 analogues (inactive compounds structurally similar to B12) can easily bind to HC. The protective role of HC appears to be sequestering these analogues before they can saturate IF or TC, preventing their absorption and cellular uptake. In physiological conditions, calculations show that intestinal IF (50 nM) would be depleted of analogues like cobinamide and pseudo-B12 (at 5 nM concentration) within 100-200 seconds if 10 nM HC is present, demonstrating HC’s protective scavenging function.

While IF has much higher specificity for true cobalamins (200-300-fold discrimination against analogues like pseudo-B12 and adenosyl-factor A, and 100,000-fold against cobinamide), TC shows only 5-fold discrimination against these same analogues. This means TC would still bind contaminating cobalamin analogues more readily than IF. The study above showed that intestinal IF (50 nM) would become depleted of cobinamide and pseudo-B12 analogues (at 5 nM concentration) within 100-200 seconds if 10 nM HC is present.

This means:

1. IF filters at the intestine – High specificity prevents most analogues from entering the IF-bound pool. It has 200-300-fold discrimination against base-off analogues and 100,000-fold against cobinamide, so most dietary analogues never get absorbed as IF-B12 complexes
2. HC scavenges remaining analogues – The non-selective HC protein captures dietary analogues that escape IF, preventing them from reaching TC. Even analogues that escape IF are captured by HC: “intestinal IF (50 nM) will be depleted of Cbi and pB (5 nM) in 100-200 s if 10 nM HC is present.
3. TC inherits mostly true B12 – By the time B12 reaches circulation, most analogues have been sequestered by HC. Therefore the B12 that ends up bound to TC should predominantly be at least true cobalamins, even if they’re not all active yet.

So while TC doesn’t inherit IF’s molecular selectivity, the overall body system ensures TC only encounters true B12 through the upstream IF and HC filtration system. This is an elegant protective hierarchy rather than TC’s own selectivity. Some forms though, like cyanocobalamin, are still easily picked up by IF, and then by TC. Hopefully they get converted to one of the active forms. Inactive cyanocobalamin needs enzymatic conversion to become metabolically active.

Any Expert Consensus?

An international expert group of key opinion leaders met in March 2012 to review the current practices of testing for vitamin B12 deficiency. The experts agreed that active-B12 offers a more reliable assessment of vitamin B12 status and as an output from the discussion a consensus statement was produced: “Emerging evidence indicates that active-B12 is a more reliable marker of B12 status than serum vitamin B12”.

Active-B12, the next level of B12 testing; Axis-Shield

In March 2012, a panel of nine internationally recognized experts (Drs. Wolfgang Herrmann, Rima Obeid, Ralph Green, Donald Jacobsen, Dominic Harrington, Per Magne Ueland, Jan Lindemans, Ebba Nexø, and Anne Molloy) issued a consensus statement on the improved utility of the active-B12 assay over total B12. They have suggested the holotranscobalamin test be treated as the optimal marker for true B12 status. Other professionals agree:

Active-B12 has fulfilled our expectations in being a robust assay that improves the assessment of vitamin B12 status. It provides reassurance to us that we have contributed to improved patient care. The routine availability of this assay differentiates our laboratory as a progressive provider of quality pathology.

Dr .Ken Sikaris, Melbourne Pathology, Australia

Serum holoTC was the best predictor, with area under the ROC curve (95% CI) 0.90 (0.86-0.93), and this was significantly better than the next best predictors; serum cobalamin, 0.80 (0.75-0.85), and MMA, 0.78 (0.72-0.83). This study supports the use of holoTC as the first-line diagnostic procedure for vitamin B₁₂ status.

Diagnostic Accuracy of Holotranscobalamin, Methylmalonic Acid, Serum Cobalamin, and Other Indicators of Tissue Vitamin B12 Status in the Elderly

HoloTC levels were more sensitive than the serum vitamin B12 levels for indicating vitamin B12 status. If the serum vitamin B12 level is 151-300 pmol/L, the levels of holoTC alone or in combination with serum vitamin B12 levels are likely to be more useful markers than serum vitamin B12 levels alone.

Relationship between the Levels of Holotranscobalamin and Vitamin B12

Testing for vitamin B12 deficiency should start with holotranscobalamin measurement. Holotranscobalamin between 23 and 75 pM should be followed by MMA testing that can filter substantial number of deficient cases in the grey range in individuals with normal renal function. This diagnostic strategy may significantly improve assessing vitamin B12 deficiency.

UTILITY AND LIMITATIONS OF BIOCHEMICAL MARKERS OF VITAMIN B12 DEFICIENCY

Total serum cobalamin is neither sensitive nor it is specific for cobalamin deficiency. This might explain why many deficient subjects would be overlooked by utilizing total cobalamin as status marker. Concentration of holoTC in serum is an earlier marker that becomes decreased before total serum cobalamin.

Cobalamin deficiency

However, there is no consensus between all experts. For instance, a report by NHANES (National Health and Nutrition Survey) highlighted the need for further studies before using the holoTC test as the primary test. They do see potential in the test though:

Given each biomarker’s sensitivity and specificity problems, the results of a single biomarker measurement would be difficult to interpret. But if NHANES can measure only one biomarker, some experts felt that this measure should reflect the broad range of vitamin B-12 status in the US population. Vitamin B-12 and holoTC increase continuously with increasing intakes, at least until vitamin B-12 saturates the transport proteins, and therefore reflect a broad range of intakes and status. Other roundtable experts felt that the sensitivity of MMA and tHcy to early inadequacies in vitamin B-12 status and marginal vitamin B-12 intakes could support the use of either one as the sole NHANES biomarker. Mean corpuscular volume to detect megaloblastic anemia would not be useful because <10% of patients with vitamin B-12 deficiency have megaloblastic anemia, and high mean corpuscular volume is more likely to reflect alcohol abuse than vitamin B-12 deficiency.

However, the roundtable strongly urged that, given the sensitivity and specificity problems of all vitamin B-12–related biomarkers, future NHANES should concurrently measure at least one biomarker of circulating concentrations of vitamin B-12 (vitamin B-12 or holoTC) and one biomarker of functional vitamin B-12 status (MMA or tHcy). If a choice must be made between serum vitamin B-12 and holoTC, holoTC has the advantage of not being affected by “false-positive” low vitamin B-12 concentrations such as those caused by genetically determined haptocorrin deficiencies. These conditions result in low circulating vitamin B-12 concentrations but have no adverse effect on vitamin B-12 status because they do not affect holoTC, which is the universal transport protein for vitamin B-12 uptake in all cells. However, the roundtable also recognized that holoTC measurement is new and would benefit from additional performance studies to engender full confidence in the use of holoTC for population-based surveys. Therefore, at this time, the roundtable agreed that serum vitamin B-12 is preferable over holoTC as the measure of circulating concentrations of vitamin B-12. MMA is preferable to tHcy if a choice must be made between these biomarkers because it increases with vitamin B-12 inadequacy, but not with folate inadequacy, whereas both of these nutrient deficiencies affect tHcy.

The roundtable generally agreed that, if NHANES reinstates 2 vitamin B-12–related biomarkers, serum vitamin B-12 and plasma MMA would provide continuity with past surveys and provide circulating and functional indicators of vitamin B-12 status. These variables have shown associations with anemia and cognitive decline in previous NHANES and in other studies. Reliable measurement procedures are available, and the NIST is developing reference materials. The roundtable also suggested that NHANES periodically measure vitamin B-12, holoTC, MMA, and tHcy concurrently to provide valuable information on their relations in the general population and on how prevalence estimates vary by biomarker or biomarker combinations.

Biomarkers of vitamin B-12 status in NHANES: a roundtable summary

Also, the latest Medical Policy review still considers the diagnosis of B12 deficiency through the measurement of holoTC investigational:

There is not enough research to know if or how well serum holoTC testing works as an alternative to total serum cobalamin, levels of methylmalonic acid (MMA), or homocysteine to improve diagnosis and management of vitamin B12 deficiency. No clinical guidelines based on research recommend holoTC testing. Therefore, holoTC testing is considered investigational for all indications.

Others completely oppose it. We recently talked to Dr. Gregory Russell-Jones (PhD):

I have been working with vitamin B12, vitamin B12 analogues and the use of vitamin B12 as a targeting agent for some 40 years now. Part of that work involved cloning of Intrinsic Factor (IF), determining the active site of IF for vitamin B12 using photoactivatable cross-linkers, and developing VB12-linked targeting agents for cancer chemotherapeutics.

More recently I have been looking at serum vitamin B12 levels and correlating them with functional vitamin B12 deficiency. This has also involved the use of many analogues of vitamin B12. In addition I have used experiments with radioactive derivatives to look at binding to and displacement of vitamin B12 analogues from intrinsic factor, transcobalamin and haptocorrin. I have also defined a condition, known now in the literature as Paradoxical B12 Deficiency. In this deficiency vitamin B12 levels in serum can be normal (well, according to the pathology labs definition of normal which varies from country to country and lab to lab, so it is nowhere near standard), or elevated.

Through these studies I have come to realize that the only true measurement of vitamin B12 activity is to look at functional markers of deficiency of which there are many, including the standard MMA and homocysteine, but there are plenty more including HVA/VMA/QA/5HIAA, pyroglutamic acid, HMG, citrate, SAM:SAH, GSH:GSSG, creatinine and choline, to name a few. These studies have also shown that the so-called active B12 test is a complete and utter waste of time and money and should NEVER have been approved for use.

The fact that transcobalamin can bind to any corrinoid does not mean the corrinoid is active. In fact in many instances, and particularly in cases when circulating B12 is high, the majority of the B12 is Co(II)B12. This is such basic chemistry. The best tests are either MMA, Hcy or the Organic Acids Test where you can measure functional B12 sufficiency.

All that being said, research does show the active-B12 test to have a much higher diagnostic accuracy than the regular total-B12 test. Using MMA as gold standard to detect a B12 deficiency, holoTC correctly identifies 98.9% of the cases detected by MMA, while the regular total-B12 test only identifies 63%. However, both tests have a 50% chance to correctly identify a non-deficiency as discovered by MMA levels. So in about half of the cases where MMA indicates no vitamin B12 deficiency (normal MMA levels), holoTC and total-B12 may give false positives. In other words, low holoTC can classify someone as deficient even though their MMA levels (considered a definitive metabolic marker for B12 deficiency) are normal. This means holoTC is highly sensitive and good for ruling in deficiency but less reliable for ruling out deficiency on its own. For this reason, clinical practice often uses a combination of markers, including holoTC, total B12, and MMA, to improve diagnostic accuracy.

Low Holo-TC levels indicate vitamin B12 deficiency. Our study results demonstrated that Holo-TC levels were low in all patients (n = 94), except for one patient who had normal Holo-TC levels. High MMA levels indicate vitamin B12 deficiency. Present study results demonstrated that 93 (97.9%) patients had elevated MMA levels, while only two patients showed normal MMA levels. Our study showed a sensitivity of 98.9% for Holo-TC, with a specificity of 50.00%. The PPV was 98.91%, and the NPV was 50%. Conversely, the sensitivity of vitamin B12 was 63%, with a specificity of 50%, a PPV of 98.33%, and an NPV of 2.85%. The diagnostic accuracy of HoloTC was observed to be 97.8%, while that of vitamin B12 was 63.15%. 

Comparing Holotranscobalamin and Total Vitamin B12 in Diagnosing Vitamin B12 Deficiency in Megaloblastic Anemia Patients

Urinary MMA is a particularly interesting test that is now more widely available than in the past, where it was only obtainable through Dr. Norman’s website. Impaired renal function influences it far less than the regular blood MMA test, making it particularly useful in older patients with sub-optimal renal function:

We prospectively studied 127 patients with a plasma B12 between 201 and 350 ng/L. We noticed that uMMA/C was not dependent on renal function, contrary to plasma homocysteine and plasma methylmalonic acid. uMMA/C showed a perspective diagnostic performance and the threshold of 1.45 umol/mmol presented a high degree of specificity. In conclusion, uMMA/C is a promising biomarker to assess vitamin B12 status in doubtful cases, notably during renal impairment.

Diagnostic Performances of Urinary Methylmalonic Acid/Creatinine Ratio in Vitamin B12 Deficiency

Reviews, presentations, commentary and letters, on the use of HoloTC as an earlier and more sensitive indicator of vitamin B₁₂ deficiency than total vitamin B₁₂, have been mixed. Several authors supported the claim that HoloTC was superior (Green 2011; Greibe et al. 2012; Nexo and Hoffmann-Lücke 2011). Others raised concerns about the clinical utility of the test (Carmel 2002, 2011, 2012; Devalia 2006; Devalia et al. 2014; Hvas and Nexo 2006; Oberley and Yang (2013). Herrmann et al. (2003b) and Herrmann and Obeid (2007, 2008) recommended the combined use of methylmalonic acid (MMA) and HoloTC. Questioning the superiority of the HoloTC test, Carmel (2002) stated that “what low holo-TC concentrations really tell us remains elusive.”

There are four problematic aspects of HoloTC, where there are major differences between authors: the value of the half-life of HoloTC; the correlation between HoloTC and total vitamin B₁₂; sensitivity of HoloTC to recent absorption of vitamin B₁₂; the value of HoloTC cut-off to detect negative vitamin B₁₂ balance. The latter two differences are the most important because they raise the questions of what it is that HoloTC concentration actually indicates, and whether or not it is possible to use it to reliably detect the early onset of vitamin B₁₂ deficiency.

As noted by this author (Golding 2016), it is possible to alter the apparent relative sensitivity of any pair of analytes by selectively changing the cut-off value of one or both of them. As demonstrated in that experiment, selecting different cut-off values for total vitamin B₁₂changed the relative sensitivities of HoloTC and total vitamin B₁₂; the same applies to the cut-off value for HoloTC. When the HoloTC cut-off is increased, the sensitivity to vitamin B₁₂deficiency is increased, but the specificity of the test is reduced, increasing the number of false positive results. Conversely, if the HoloTC cut-off is decreased, the specificity of the test is increased, but the sensitivity of the test is reduced, increasing the number of false negative results.

From their ROC charts, Heil et al. (2012) selected MMA > 0.45 µmol/L to define vitamin B₁₂ deficiency. With a HoloTC cut-off value of <21 pmol/L, the specificity was impressively high at 88 % but the sensitivity was only 64 % (Heil et al. 2012). By selecting the best trade-off between sensitivity and specificity for HoloTC, with a HoloTC cut-off value of <32 pmol/L, they obtained an impressive HoloTC sensitivity of 83 % but a specificity of only 60 %. To overcome this problem, as did Herrmann and Obeid (2007, 2008), the authors suggest using MMA as a secondary test to confirm vitamin B₁₂ deficiency in cases where HoloTC is <32 pmol/L. This approach would have failed to detect any vitamin B₁₂ deficiency in the subject of this author’s experiment (Golding 2016) because, despite overt metabolic disturbance evident from raised MMA concentration, and the onset of severe symptoms including peripheral neuropathy, HoloTC concentration never fell below 33 pmol/L.

Holotranscobalamin (HoloTC, Active-B12) and Herbert’s model for the development of vitamin B₁₂deficiency: a review and alternative hypothesis

Victor Herbert & Holotranscobalamin

Dr. Herbert was perhaps most famous for his self-experiment, where he induced folate deficiency in himself by eating a diet of boiled vegetables. He almost killed himself in the process, waking up unable to walk. He did all that, in order to document the stages and progress of folate deficiency up until its end point of megaloblastic anaemia.

About forty years later, on November 19, 2002, Dr. Herbert died at his home in NY. At a forum held in Philadelphia less than a month later, Dr. Ralph Green read a tribute:

On Christmas Day 1961, he reached the end of one such study. Unable to move from severe weakness in both lower limbs induced by potassium deficiency caused by ingestion of an experimental diet, and having lost 26 pounds, Dr. Herbert terminated what came to be regarded as a classic self experiment. He had induced in himself nutritional folate deficiency by eating a diet of foods re-cooked to destroy all traces of folic acid. He had, as a consequence, developed megaloblastic anemia. Through this experiment, Dr. Herbert conclusively proved the tie between folate deficiency and megaloblastic anemia. He was also able to carefully document the sequence of changes in the development of folate deficiency. It was this spirit of scientific curiosity and indomitable grit and determination that characterized Victor Herbert’s life and career.

Obituary, Victor Herbert, M.D., J.D.

If you’re interested, here’s another article in memory of Victor Herbert, written by Dr. Charles Halstedand, and published in the American Journal of Clinical Nutrition.

An adaptation of Dr. Herbert’s proposed model is shown in the diagram below, describing the biochemical, metabolic, and clinical changes that take place as vitamin B12 repletion progresses to depletion, and finally to clinical B12 deficiency:

For his contribution to the field of vitamin B12 and to our understanding of its metabolism, we would like to thank Dr. Herbert from the bottom of our heart. As you can see from his model, the first step of B12 deficiency is active-B12 levels decreasing. The argument is that having the active-B12 blood test (holoTC test!) as the first line of defense in diagnosing B12 deficiency may help catch cases while they’re still recoverable and easy to treat. However, some doubt the model’s robustness today:

The problems with Herbert’s model

The absence of an agreed single value for the HoloTC cut-off, together with uncertainty about whether HoloTC represents recent absorption of vitamin B12 or long-term status or both, must raise questions about Herbert’s hypothesis. Some of the most recent original research challenges Herbert’s model for the “Sequential stages in the development of vitamin B12 deficiency”. In reporting their original research results, Remacha et al. (2014) commented that “These data do not support holoTC as the earliest marker of Cbl deficiency and challenge the classification in stages of Cbl deficiency”.

In Herbert’s revised 1994 model (Herbert 1994), the change from Normal to Early Negative Vitamin B 12 Balance is marked by a fall in HoloTC concentration from >37 to <30 pmol/L. Even if it is assumed that such a highly precise and accurate routine clinical assay is possible, there remain two major unresolved fundamental problems with Herbert’s hypothesis. Firstly, as raised by several authors, there is uncertainty about what HoloTC actually represents. Does HoloTC concentration indicate recent absorption of vitamin B12, long-term body store or both? As stated by Chen et al. (2005): “The concept that a test can be used to diagnose both deficiency and malabsorption is problematic because the 2 defects are not identical. If holo-TC II truly reflects both processes, holo-TC II changes would perforce lose all diagnostic specificity for either process”. For the HoloTC immunoassay to have any clinical value in detecting the earliest onset of vitamin B12 deficiency, as Herbert intended, it would need to be either sensitive only to long-term status or be performed after a well-defined period of fasting.

Secondly, the model demands the reliable detection of a change in HoloTC concentration far smaller than the reported range of cut-off values. As noted by Sobczyńska-Malefora et al. (2014), “there has been little consensus with regard to the assigned cut-off to discriminate between replete and deficient states”. Herbert’s required change in HoloTC concentration from >37 to <30 pmol/L, to detect the earliest onset of vitamin B12 deficiency, is inconsistent with the reported range of cut-off values from 20 to 50 pmol/L. Furthermore, the use of various grey zones, for HoloTC concentration (Table 8), is inconsistent with Herbert’s hypothesis in which Early Negative Vitamin B 12 Balance is detected by a universally agreed, and closely specified, fall in HoloTC concentration.

Questioning of Herbert’s hypothesis that HoloTC is the most sensitive marker of Early Negative Vitamin B ₁₂ Balance, or his model for the sequential stages in the development of vitamin B₁₂ deficiency, is likely to raise strong objections.

Firstly, any model that is based on changes from individual normal analyte concentrations, instead of comparison with a universally accepted population normal, conflicts with the dogma that there must be a gold–standard test for vitamin B₁₂ deficiency; either HoloTC or one a yet to be discovered.

Secondly, any model that requires a comparison of current analyte concentrations with a patient’s own normal levels would require proactive testing of all persons at risk, before the onset of any symptoms, to obtain their individual healthy baseline concentrations. Herbert himself suggested proactive testing, saying “serum holoTCIl should be measured every 5 y starting at age 55 because gradual loss of the ability to absorb vitamin B-12 occurs in everyone in a genetically determined, age-dependent pattern.” (Herbert 1994).

A potential alternative to obtaining a longitudinal record for individuals at risk is the use of an algorithm to combine the results of a one-off test of all four analytes into a single parameter. A mathematical model for this has been devised and described in detail by Fedosov (2010), and further tested and refined (Fedosov 2013; Fedosov et al. 2015; Brito et al. 2016). This author is cautious about this approach because, as with all other current methods, it might apply well to a population but not necessarily to an individual. This would need extensive testing on a wide range of subjects, for various causes of vitamin B₁₂deficiency, before it could be validated.

Axis-Shield and others, in promoting the commercialisation of the HoloTC immunoassay, have relied on Herbert’s erroneous hypothesis that “Ho1oTCII falls below the bottom of its normal range long before total serum vitamin B-12 … falls below the bottom of its normal range”, and his flawed model for the staged development of vitamin B₁₂ deficiency (Herbert 1994).

Herbert’s model is flawed because it assumes that a normal minimum concentration for HoloTC may be universally defined. As with total vitamin B₁₂, evidenced by the failure to find such a single cut-off value for HoloTC and instead the discovery of a wide grey zone for HoloTC, the range of normal for an individual is much smaller than for the population. As stated by Herbert (1994): “What is normal for one is not normal for another”.

Herbert’s hypothesis, that HoloTC will respond early to vitamin B₁₂ depletion, before the onset of a clinical deficiency, is erroneous because it does not take into account how enterohepatic recycling regulates the HoloTC concentration. Where the cause of vitamin B₁₂deficiency does not disrupt the enterohepatic cycle, HoloTC will not be an early responder to a deficiency; there will be a Depletion period, in which total vitamin B₁₂ falls, but HoloTC and the metabolites will only respond after the liver store is exhausted. When the cause of vitamin B₁₂ deficiency does disrupt the enterohepatic cycle, the HoloTC will be an early responder but there will be no Depletion period; the metabolites will respond quickly when HoloTC falls below normal for the individual.

The HoloTC immunoassay cannot be used to measure vitamin B₁₂ status any more reliably than total vitamin B₁₂, or to predict the onset of a metabolic deficiency, because it is based on an erroneous hypothesis and a flawed model for the staged development of vitamin B₁₂deficiency.

Holotranscobalamin (HoloTC, Active-B12) and Herbert’s model for the development of vitamin B₁₂deficiency: a review and alternative hypothesis

In Conclusion

To sum up, the active-B12 blood test represents a major step-up over the regular blood test, because it gives a much better picture of the levels of B12 that has any hopes of being absorbed. The test can make the task of B12 deficiency diagnosis easier for the laboratorian, the doctor, and the patient. However, currently available tests like MMA and homocysteine can discover functional B12 deficiencies almost as well, and sometimes better.

Thanks for reading.