to a reduction in the activity of methylmalonyl CoA mutase, the other vitamin B12-dependent enzyme in man (Figure 4). It appears that it is not possible to be functionally deficient in vitamin B12 without a concomitant elevation in MMA, and so a false negative result is not really an issue. However, MMA like plasma homocysteine is also elevated during renal impairment, and while this can to some extent be corrected for by a raised creatinine, it cannot be assumed that elevation of MMA is due to vitamin B12 deficiency. While the estimation of plasma homocysteine is widely available the estimation of MMA requires gas chromatography mass spectroscopy (GC-MS) and has very limited availability in practice. Newer methods to measure vitamin B12 on its transport protein TC II are under development.

For the reasons given above, it is essential that vitamin B12 deficiency is not confused with folate deficiency. As mentioned previously, both conditions present with a morphologically indistinguishable megaloblastic anemia. The inappropriate treatment of vitamin B12 deficiency with folic acid is to be avoided at all costs (see below). Apart from using biochemical assays to measure circulatory levels of the two vitamins and looking for an elevation of the biomarkers plasma homocysteine and MMA, further tests can also implicate vitamin B12 malabsorption, the most common type of severe vitamin B12 deficiency. These include the Schilling test and the detection of antibodies against either intrinsic factor or the parietal cells that manufacture it.

In practice, if vitamin B12 deficiency cannot be ruled out, many clinicians will treat patients with vitamin B12 if uncertain about the diagnosis. If this is followed by a reticulocyte response and complete disappearance of the anemia, it confirms a diagnosis of vitamin B12 deficiency. The appropriate treatment regimen can then be implemented (see later). If treatment with vitamin B12 does not result in improvement of the anemia then the patient is treated for folic acid deficiency, but only after vitamin B12 deficiency has been excluded by all means at the clinician's disposal.

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