Laboratory Diagnosis of Vitamin B12 Deficiency

 

It has become clear that the levels of biochemical markers of Vitamin B12 status may be misleading.

In many studies, the functional markers Homocysteine (tHcy) and Methylmalonic Acid (MMA) are increased and/or typical deficiency symptoms are present when cobalamin levels are low. On the other hand, concentrations of tHcy and MMA may be elevated even when levels of vitamin B12 and holotranscobalamin (Active B12) are normal or even high, casting some doubt on the usefulness of these markers.

 

Summaries of the utility of the current biochemical markers used in assessing B12 status are shown in Tables 1 and 2 below.

 

Table 1: Comparison of vitamin B12 biomarkers.

(adapted from R Green AJCN, 2011; 94:666S-72S) 

Click here for a PDF of the comparison table.

 

 

 

Table 2: Pros and cons of laboratory tests used to diagnose Vitamin B12 deficiency.

(adapted from AM Hvas and E Nexo Haematologica, 2006; 91:1508-14)

Test	Rationale and advantage	Disadvantage
Total Serum B12	Decreases in vitamin B12 deficiency Accessible. Cheap.	Variation in reference interval due to different methods. Sensitivity and specificity debatable. False positive if haptocorrin is reduced. False negative if haptocorrin is increased (eg in chronic myeloid leukaemia). Large % of indeterminate results (grey-zone)
Methylmalonic acid (MMA)	Increases in vitamin B12 deficiency   High sensitivity	Not easily accessible. Expensive. Low Specificity; False positive with reduced renal function and increases with age
Total Homocysteine (tHcy)	Increases in vitamin B12 deficiency High sensitivity	Special procedure for sample handling is required. Low Specificity; Influenced by lifestyle factors (smoking, alcohol intake, coffee consumption). Folate deficiency, vitamin B6 deficiency and reduced renal function all cause false positives
Holotranscobalamin (Active-B12)	Decreases in vitamin B12 deficiency Expected to have high sensitivity	Improved specificity over other tests. Changes occur early in Vitamin B12 depletion

 

 

 

Total B12 measurement

 

Total B12 concentration is the current standard clinical test for vitamin B12 deficiency. However, the Total B12 test suffers from a number of important limitations:

 

False positives

Total B12 concentrations of less than 148pmol/L (200pg/mL) are generally considered indicative of deficiency.  However, a proportion of individuals with Total B12 levels below this cut-off show no clinical or biochemical evidence of deficiency (1). Low levels of Total B12 therefore cannot be relied upon to indicate deficiency.

It was demonstrated a few years ago by Carmel that low haptocorrin levels are likely to be the one of the most common causes of low total B12 levels, with a frequency approaching 15% for mild haptocorrin deficiency.

 

False negatives

Conversely, neuropsychiatric (2) and metabolic (1) abnormalities can occur with vitamin B12 concentrations well within the normal reference interval. It is reckoned that approximately half of patients with B12 deficiency are not detected when the Total B12 test is used with a cut-off of 148pmol/L (200pg/ml).

 

Measures all circulating Vitamin B12...

...but not all B12 in the blood is available to be used by the body. The majority of vitamin B12 that is measured by the Total B12 test is that bound to haptocorrin, but B12 attached to haptocorrin cannot be used by the vast majority of the cells of the body. Only B12 bound to transcobalamin, Active-B12, is taken up from the blood into the cells of the body. This means that the Total B12 assay could give a misleading result and still be within the normal range, even though levels of Active-B12  are low. Imagine looking at a fuel gauge indicating that the tank was full. However if the tank was wrongly topped up with diesel, on top of the petrol that it really requires, the engine will come to shuddering halt.

 

Late marker of deficiency

Changes in the Total B12 result may not occur until the patient is already Vitamin B12 deficient. As Active-B12 has a shorter circulating half-life compared to holohaptocorrin, the earliest change that occurs on entering negative vitamin B12 balance is very likely to be a decrease in plasma Active-B12 concentration (3). 

 

Large proportion of indeterminate results

There are many publications attesting to a significant indeterminate zone when using the Total B12 assay:

 

• Snow suggests Total B12 assays discriminate poorly at levels between 100-400 pg/mL (75-300 pmol/l) (4)
• Swain suggests that Total B12 should be used as screening test, high levels rule-out deficiency, between 150-300 pmol/l require confirmation, levels below 150 pmol/l probably do not need confirmation (5).
• Hvas and colleagues suggest further testing when B12 levels fall between 125-250 pmol/l (6)
• Schneede suggests follow-up testing when B12 values fall between 150-250 pmol/l (7)
• Klee suggests follow-up testing when B12 falls between approx 110-220 pmol/l (8)
• Based on Active-B12 and metabolite measurements Herrmann estimates vitamin B12 deficiency can occur up to B12 levels of 300pmol/L and that 45% of vitamin B12 deficient subjects would be over-looked if only relying on vitamin B12 as a screening test (9)

 

1. Green R et al., Baillieres Clin Haematol., 1995; 8: 533-6
2. Lindenbaum J et al., New Eng J Med., 1988; 318: 1720-8
3. Herzlich B and Herbert V, Lab Invest., 1988; 58: 332-7.
4. Snow CF Arch Intern Med., 1999 ;159 :1289-98.
5. Swain R J Fam Pract., 1995; 42: 595-61.
6. Hvas AM et al., Ugeskr Laeger., 2003; 165: 1971-6.
7. Schneede J Scan J Clin Lab Invest., 2003; 63: 369-376.
8. Klee G, Clin Chem., 2000; 46; 8(B): 1277-1283.
9. Hermmann W et al Curr. Drug Metab. 2005; 6 : 47-53

 

Total homocysteine (tHcy) and methylmalonic acid (MMA)

 

Both tHcy and MMA may provide information on functional intracellular cobalamin homeostasis, regardless of the underlying reasons for deficiency. The concentrations of both tHcy and MMA may be elevated  in the face of normal cobalamin and holotranscobalamin levels in serum, the reasons for this are not fully elucidated (see Chanarin I and Metz J, Diagnosis of cobalamin deficiency: the old and the new British J Haematol 1997;97,695-697).

 

Many studies have shown that tHcy and MMA levels increase with age and elevated levels are very common among the elderly, even among supposedly healthy elderly persons. More recent research has suggested that only about 17% of the variation in MMA levels between a middle aged and an elderly group could be accounted for by vitamin B12 and renal (kidney) function. In other words, the determinants of MMA levels in an elderly population are largely unknown (Vogiatzoglou  and others).

Elevated tHcy may indicate vitamin B12 deficiency but may also indicate folate deficiency as well as an enzymatic dysfunction in the methylation cycle. Total Hcy is therefore less specific than MMA. MMA is theoretically more specific however, elevated MMA can arise from renal dysfunction and there is also the possibility that in B12 deficiency, some pathways have greater or lesser priority. For example, elevated serum MMA levels in healthy subjects with Cbl levels at the high end of the normal range might suggest that supplying Cbl to MMA-CoA mutase has a low priority. (see Chanarin I and Metz J, Diagnosis of cobalamin deficiency: the old and the new British J Haematol 1997;97,695-697).

 

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