Cobalamin (Cbl) and folate are essential co-factors for several key metabolic reactions in mammalian metabolism. Deficiency of either vitamin results in interdiction of normal DNA synthesis which leads to megaloblastic anaemia. The underlying biochemical mechanisms are discussed in Chapters 1 and 2. Central to the common haematological outcome of deficiencies of Cbl and folate is the methionine synthase reaction, in which both vitamins participate. It is this reaction that leads to the formation of tetrahydrofolate, the obligate precursor of the form of folate that is required for thymidine production. Deficiency of Cbl or folate can also have other consequences presumed to relate to an interruption of other essential metabolic roles that are restricted to one of the vitamins. Interruption of Cbl or folate-dependent reactions leads not only to a decrease in the products of those reactions, but also to an accumulation of their substrates. From a knowledge of the metabolic roles of Cbl and folate, it has been possible to predict which substrates will accumulate in Cbl or folate deficiencies. In recent years, two metabolites, methylmalonic acid (MMA) and homocysteine (HCYS) have received special attention. Improvements in analytical techniques, in particular those based on gas chromatography-mass spectrometry or high pressure liquid chromatography, have led to assays for the metabolites which have improved precision and are suitable for clinical use. The capability to measure these metabolites in serum and urine has led to several applications which will be reviewed in this chapter. The primary utility of these tests has been for diagnosis of Cbl and folate deficiencies. Recent studies have shown that compared with direct measurement of the vitamins in the blood, metabolite assays provide improved sensitivity and specificity. Metabolite assays can therefore be used in the following settings: (1) for the primary diagnosis of Cbl and folate deficiencies; (2) for further testing in patients found to have subnormal or low normal blood levels of Cbl or folate; (3) to distinguish Cbl from folate deficiencies; (4) for monitoring patients' responses to treatment with Cbl or folate (either to confirm the diagnosis or to assess the adequacy of therapy). In addition, correlation of the patterns of abnormal metabolite accumulation with clinical manifestations of disease may help to elucidate the underlying biochemical mechanisms that are responsible for the clinical manifestations of Cbl and folate deficiencies. Increased levels of the metabolites MMA and HCYS may also be responsible for some of the disease manifestations associated with Cbl or folate deficiency, such as myeloneuropathy. It is also possible that the identification of a particular pattern of metabolites or the finding of some novel abnormal metabolite may shed light on the hitherto unknown metabolic functions of Cbl or folate. Despite these numerous applications of metabolite measurement for the diagnosis of Cbl and folate deficiencies, there are still some caveats and limitations to their use. Apart from vitamin deficiencies, metabolites may also be elevated in a number of other inherited and acquired disorders. Also, because there may be alternative pathways for the conversion or degradation of some metabolites, levels are not invariably raised in deficiencies of Cbl or folate. Even when metabolite levels are raised, the degree of elevation does not always correlate with the extent of vitamin deficiency or with its clinical severity. At this time, MMA and HCYS assays are done in only a few specialized laboratories, and by several different methods. There is some variability in reported normal ranges, which is most evident in the case of HCYS. Perhaps the most significant application of metabolite measurement has been for the detection of early, subtle deficiency states of Cbl and folate including a predisposition to disease that may be associated with early subclinical deficiency of these vitamins. For example, a link has been established between elevated levels of HCYS in the blood and the risk of occlusive vascular disease and there is evidence to implicate deficiency of B-group vitamins, including Cbl and folate, in the production of hyperhomocysteinaemia, even in patients who do not show any of the typical haematological effects of these deficiencies.
