It has gradually become clear that total serum vitamin B12 levels may be misleading. Also, in many studies the metabolic markers, tHcy and MMA are increased and/or typical deficiency symptoms present, whereas cobalamin levels are normal. On the other hand concentrations of homocysteine (Hcy) and methylmalonic acid (MMA) may be elevated even in the face of normal levels of vitamin B12 and holotranscobalamin.
A summary of the pros and cons regarding the use of the current biochemical markers is presented in table 1 (adapted from AM Hvas and E Nexo Haematologica, 2006; 91:1508-14)
Table 1 pros and cons of laboratory tests
| Test |
Rationale and advantage |
Disadvantage |
Cobalamins (total vitamin 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) |
| Methylmalonic acid (MMA) |
Increases in vitamin B12 deficiency
High sensitivity
|
Not easily accessible
Expensive
Specificity debatable; false positive with reduced renal function
|
| 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)
False positive in folate deficiency
False positive in vitamin B6 deficiency
False positive with reduced renal function
|
| Holotranscobalamin (HoloTC) |
Decreases in vitamin B12 deficiency
Expected to have high sensitivity
|
Specificity needs to be further clarified. |
Serum or plasma cobalamin measurement.
Total plasma vitamin B12 concentration is the current standard clinical test for vitamin B12 deficiency. Total vitamin B12 concentrations of less than 148pmol/L (200pg/mL) are generally considered indicative of deficiency. However, a proportion of individuals with vitamin B12 levels that would be considered deficient exhibit no clinical or biochemical evidence of deficiency (1). Conversely, neuropsychiatric (2) and metabolic (1) abnormalities can occur with plasma vitamin B12 concentrations well within the normal reference interval.
The measurement of total plasma vitamin B12 suffers from a number of limitations, most particularly, the majority of vitamin B12 that is measured is that bound to haptocorrin. As Active-B12 (holoTC) 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 (holoTC) concentration (3).
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.
There are many publications attesting to a significant indeterminate zone when using the total vitamin B12 assay:
-
Snow suggests B12 assays discriminate poorly at levels between 100-400 pg/mL (75-300 pmol/l) (1)
- Swain suggests that 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 (2).
-
Hvas and colleagues suggest further testing when B12 levels fall between 125-250 pmol/l (3)
-
Schneede suggests follow-up testing when B12 values fall between 150-250 pmol/l (4).
-
Klee suggests follow-up testing when B12 falls between approx 110-220 pmol/l (5).
-
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 (6).
1. Snow CF Arch Intern Med., 1999 ;159 :1289-98.
2. Swain R J Fam Pract., 1995; 42: 595-61.
3. Hvas AM et al., Ugeskr Laeger., 2003; 165: 1971-6.
4. Schneede J Scan J Clin Lab Invest., 2003; 63: 369-376.
5. Klee G, Clin Chem., 2000; 46; 8(B): 1277-1283.
6. Hermmann W et al Curr. Drug Metab. 2005; 6 : 47-53
Total Hcy and MMA
Both tHcy and MMA may provide information on functional intracellular cobalamin homoeostasis, 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.
Elevated tHcy may indicate vitamin B12 deficiency and/or 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 Cbl 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).
Pepsinogen I and gastrin
Once vitamin B12 deficiency is diagnosed the next challenge is to find the cause (unless obviously dietary). There are a number of markers for estimating the function of the gastric mucosa. Two such tests are serum gastrin and serum pepsinogen I.
Low serum pepsinogen I indicates extensive atrophy of the gastric mucosa. However, this marker has low specificity and pepsinogen I levels can rise in superficial gastritis as observed in H. pylori infections.
High serum gastrin levels generally indicate atrophy in the corpus and fundus of the stomach but may also be caused by a gastrin-producing tumour. Like pepsinogen I the specificity of this marker for vitamin B12 deficiency is low.
Schilling test
The classical Schilling test merely diagnoses a (sub) total lack of IF (genuine pernicious anaemia). However, patients with gastritis and vitamin B12 malabsorption due to an inability to break down the protein-B12 complex in the food, (who have a decreased secretion of gastric acid) may have a normal test. The test is therefore seldom indicated.
