This is measured by an automated enzyme-linked immunosorbent assay (ELISA) or competitive-binding luminescence assay (CBLA). Normal serum levels range from 118–148 pmol/L (160–200 ng/L) to ~738 pmol/L (1000 ng/L). In patients with megaloblastic anemia due to cobalamin deficiency, the level is usually <74 pmol/L (100 ng/L). In general, the more severe the deficiency, the lower is the serum cobalamin level. In patients with spinal cord damage due to the deficiency, levels are very low even in the absence of anemia. Values between 74 and 148 pmol/L (100 and 200 ng/L) are regarded as borderline. They may occur, for instance, in pregnancy, in patients with megaloblastic anemia due to folate deficiency. They may also be due to heterozygous, homozygous, or compound heterozygous mutations of the gene TCN1 that codes for haptocorrin (transcobalamin I). There is no clinical or hematologic abnormality. The serum cobalamin level is sufficiently robust, cost-effective, and most convenient to rule out cobalamin deficiency in the vast majority of patients suspected of having this problem. However, problems have arisen with commercial CBLA assays involving intrinsic factor in PA patients with intrinsic antibodies in serum. These antibodies may cause false normal serum vitamin B12 levels in up to 50% of cases tested. Where clinical indications of PA are strong, a normal serum vitamin B12 does not rule out the diagnosis. Serum MMA levels will be elevated in PA (see below).
In patients with cobalamin deficiency sufficient to cause anemia or neuropathy, the serum MMA level is raised. Sensitive methods for measuring MMA and homocysteine in serum have been introduced and recommended for the early diagnosis of cobalamin deficiency, even in the absence of hematologic abnormalities or subnormal levels of serum cobalamin. Serum MMA levels fluctuate, however, in patients with renal failure. Mildly elevated serum MMA and/or homocysteine levels occur in up to 30% of apparently healthy volunteers, with serum cobalamin levels up to 258 pmol/L (350 ng/L) and normal serum folate levels; 15% of elderly subjects, even with cobalamin levels >258 pmol/L (>350 ng/L), have this pattern of raised metabolite levels. These findings bring into question the exact cutoff points for normal MMA and homocysteine levels. It is also unclear at present whether these mildly raised metabolite levels have clinical consequences.
Serum homocysteine is raised in both early cobalamin and folate deficiency but may be raised in other conditions, e.g., chronic renal disease, alcoholism, smoking, pyridoxine deficiency, hypothyroidism, and therapy with steroids, cyclosporine, and other drugs. Levels are also higher in serum than in plasma, in men than in premenopausal women, in women taking hormone replacement therapy or in oral contraceptive users, and in elderly persons and patients with several inborn errors of metabolism affecting enzymes in trans-sulfuration pathways of homocysteine metabolism. Thus, homocysteine levels must be carefully interpreted for diagnosis of cobalamin or folate deficiency.
Only vegans, strict vegetarians, or people living on a totally inadequate diet will become vitamin B12 deficient because of inadequate intake. Studies of cobalamin absorption once were widely used, but difficulty in obtaining radioactive cobalamin and ensuring that IF preparations are free of viruses has made these tests obsolete. Tests to diagnose PA include serum gastrin, which is raised; serum pepsinogen I, which is low in PA (90–92%) but also in other conditions; and gastric endoscopy. Tests for IF and parietal cell antibodies are also used, as well as tests for individual intestinal diseases.
This is also measured by an ELISA technique. In most laboratories, the normal range is from 11 nmol/L (2 μg/L) to ~82 nmol/L (15 μg/L). The serum folate level is low in all folate-deficient patients. It also reflects recent diet. Because of this, serum folate may be low before there is hematologic or biochemical evidence of deficiency. Serum folate rises in severe cobalamin deficiency because of the block in conversion of MTHF to THF inside cells; raised levels have also been reported in the intestinal stagnant loop syndrome due to absorption of bacterially synthesized folate.
TREATMENT Cobalamin and Folate Deficiency
It is usually possible to establish which of the two deficiencies, folate or cobalamin, is the cause of the anemia and to treat only with the appropriate vitamin. In patients who enter the hospital severely ill, however, it may be necessary to treat with both vitamins in large doses once blood samples have been taken for cobalamin and folate assays and a bone marrow biopsy has been performed (if deemed necessary). Transfusion is usually unnecessary and inadvisable. If it is essential, packed red cells should be given slowly, one or two units only, with the usual treatment for heart failure if present. Potassium supplements have been recommended to obviate the danger of the hypokalemia but are not necessary. Occasionally, an excessive rise in platelets occurs after 1–2 weeks of therapy. Antiplatelet therapy, e.g., aspirin, should be considered if the platelet count rises to >800 × 109/L. COBALAMIN DEFICIENCY
It is usually necessary to treat patients who have developed cobalamin deficiency with lifelong regular cobalamin injections. In the UK, the form used is hydroxocobalamin; in the United States, cyanocobalamin. In a few instances, the underlying cause of cobalamin deficiency can be permanently corrected, e.g., fish tapeworm, tropical sprue, or an intestinal stagnant loop that is amenable to surgery. The indications for starting cobalamin therapy are a well-documented megaloblastic anemia or other hematologic abnormalities and neuropathy due to the deficiency. Patients with borderline serum cobalamin levels but no hematologic or other abnormality may be followed to make sure that the cobalamin deficiency does not progress (see below). If malabsorption of cobalamin or rises in serum MMA levels have been demonstrated, however, these patients also should be given regular maintenance cobalamin therapy. Cobalamin should be given routinely to all patients who have had a total gastrectomy or ileal resection. Patients who have undergone gastric reduction for control of obesity or who are receiving long-term treatment with proton pump inhibitors should be screened and, if necessary, given cobalamin replacement.
Replenishment of body stores should be complete with six 1000-μg IM injections of hydroxocobalamin given at 3- to 7-day intervals. More frequent doses are usually used in patients with cobalamin neuropathy, but there is no evidence that they produce a better response. Allergic reactions are rare and may require desensitization or antihistamine or glucocorticoid cover. For maintenance therapy, 1000 μg hydroxocobalamin IM once every 3 months is satisfactory. Because of the poorer retention of cyanocobalamin, protocols generally use higher and more frequent doses, e.g., 1000 μg IM, monthly, for maintenance treatment.
Because a small fraction of cobalamin can be absorbed passively through mucous membranes even when there is complete failure of physiologic IF-dependent absorption, large daily oral doses (1000–2000 μg) of cyanocobalamin have been used in PA for replacement and maintenance of normal cobalamin status in, e.g., food malabsorption of cobalamin. Sublingual therapy has also been proposed for those in whom injections are difficult because of a bleeding tendency and who may not tolerate oral therapy. If oral therapy is used, it is important to monitor compliance, particularly with elderly, forgetful patients. This author prefers parenteral therapy for initial treatment, particularly in severe anemia or if a neuropathy is present, and for maintenance.
For treatment of patients with subnormal serum vitamin B12 (B12) levels with a normal MCV and no hypersegmentation of neutrophils, a negative IF antibody test in the absence of tests of B12 absorption is problematic. Some (perhaps 15%) cases may be due to TC I (HC) deficiency. Homocysteine and/or MMA measurements may help, but in the absence of these tests and with otherwise normal gastrointestinal function, repeat serum B12 assay after 6–12 months may help one decide whether to start cobalamin therapy.
Vitamin B12 injections are used in a wide variety of diseases, often neurologic, despite normal serum B12 and folate levels and a normal blood count and in the absence of randomized, double-blind, controlled trials. These conditions include multiple sclerosis and chronic fatigue syndrome/myalgic encephalomyelitis (ME). It seems probable that any benefit is due to the placebo effect of a usually painless, pink injection. In ME, oral B12 therapy, despite providing equally large amounts of B12, has not been beneficial, supporting the view of the effect of the injections being placebo only. FOLATE DEFICIENCY
Oral doses of 5–15 mg folic acid daily are satisfactory, as sufficient folate is absorbed from these extremely large doses even in patients with severe malabsorption. The length of time therapy must be continued depends on the underlying disease. It is customary to continue therapy for about 4 months, when all folate-deficient red cells will have been eliminated and replaced by new folate-replete populations.
Before large doses of folic acid are given, cobalamin deficiency must be excluded and, if present, corrected; otherwise cobalamin neuropathy may develop despite a response of the anemia of cobalamin deficiency to folate therapy. Studies in the United States, however, suggest that there is no increase in the proportion of individuals with low serum cobalamin levels and no anemia since food fortification with folic acid, but it is unknown if there has been a change in incidence of cobalamin neuropathy.
Long-term folic acid therapy is required when the underlying cause of the deficiency cannot be corrected and the deficiency is likely to recur, e.g., in chronic dialysis or hemolytic anemias. It may also be necessary in gluten-induced enteropathy that does not respond to a gluten-free diet. Where mild but chronic folate deficiency occurs, it is preferable to encourage improvement in the diet after correcting the deficiency with a short course of folic acid. In any patient receiving long-term folic acid therapy, it is important to measure the serum cobalamin level at regular (e.g., once-yearly) intervals to exclude the coincidental development of cobalamin deficiency. Folinic Acid (5-Formyl-THF)
This is a stable form of fully reduced folate. It is given orally or parenterally to overcome the toxic effects of methotrexate or other DHF reductase inhibitors, e.g., trimethoprim or cotrimoxazole. PROPHYLACTIC FOLIC ACID
Prophylactic folic acid is used in chronic dialysis patients and in parenteral feeds. Prophylactic folic acid has been used to reduce homocysteine levels to prevent cardiovascular disease and for cognitive function in the elderly, but there are no firm data to show any benefit. Pregnancy
In over 70 countries (but none in Europe), food is fortified with folic acid (in grain or flour) to reduce the risk of NTDs. Nevertheless, folic acid, 400 μg daily, should be given as a supplement before and throughout pregnancy to prevent megaloblastic anemia and reduce the incidence of NTDs, even in countries with fortification of the diet. The levels of fortification provide up to 400 μg daily on average in Chile, but in most countries, it is nearer to 200 μg, so periconceptual folic acid is still needed. Studies in early pregnancy show significant lack of compliance with the folic acid supplements, emphasizing the benefit of food fortification. Supplemental folic acid reduces the incidence of birth defects in babies born to diabetic mothers. In women who have had a previous fetus with an NTD, 5 mg daily is recommended when pregnancy is contemplated and throughout the subsequent pregnancy. Infancy and Childhood
The incidence of folate deficiency is so high in the smallest premature babies during the first 6 weeks of life that folic acid (e.g., 1 mg daily) should be given routinely to those weighing <1500 g at birth and to larger premature babies who require exchange transfusions or develop feeding difficulties, infections, or vomiting and diarrhea.
The World Health Organization currently recommends routine supplementation with iron and folic acid in children in countries where iron deficiency is common and child mortality, largely due to infectious diseases, is high. However, some studies suggest that in areas where malaria rates are high, this approach may increase the incidence of severe illness and death. Even where malaria is rare, there appears to be no survival benefit.