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Methylmalonic Acid

2001; American Medical Association; Volume: 125; Issue: 6 Linguagem: Inglês

10.5858/2001-125-0824-ma

1543-2165

Ronald J. Elin, William E. Winter,

Pharmacological Effects and Toxicity Studies

In 1962, Cox and White1 showed that the excretion of methylmalonic acid (MMA) was a sensitive index of vitamin B12 deficiency. However, it was not until the late 1980s that the biochemical interaction between vitamin B12 and MMA was understood.2 Unlike the serum vitamin B12 assay and the Schilling test, elevated serum MMA levels and urinary excretion of MMA are direct measures of tissue vitamin B12 activity.Untreated vitamin B12 deficiency is a prevalent and serious medical condition most often affecting elderly patients.3 Cobalamin deficiency can have protean morbid effects on health. The classic affected patient who presents with neuropathy and megaloblastic anemia likely represents the minority of subjects with cobalamin deficiency. In community-dwelling older women, metabolically significant vitamin B12 deficiency was associated with a twofold increased risk of severe depression.4 Cobalamin deficiency can also be manifested as psychosis without anemia.5 There are epidemiologic data that show that lower cobalamin concentrations are associated with breast cancer.6 Older data demonstrate an association of gastric carcinoma and carcinoid tumors with pernicious anemia.7 In a study of Scottish patients referred to a geriatric medical unit, 13% of the patients had serum vitamin B12 levels less than 175 pmol/L. Of these patients, only approximately 1 in 4 displayed macrocytosis with a mean corpuscular volume of 100 fl or more. When 34 of the vitamin B12–deficient subjects were treated with vitamin B12, they demonstrated increases in hemoglobin level and decreases in mean corpuscular volume regardless of their initial hemoglobin level.8 Cobalamin deficiency and its effects on health are not limited to elderly patients. Adolescents with cobalamin deficiency with no hematologic signs had a significant impairment in cognitive performance.9The most common cause of vitamin B12 deficiency is “pernicious anemia.” Pernicious anemia results from failure to absorb vitamin B12 due to intrinsic factor (IF) deficiency. Autoimmune destruction of the IF-producing gastric parietal cells leads to IF deficiency and vitamin B12 malabsorption. Normally, the liver has more than a 5-year supply of vitamin B12.Vitamin B12 deficiency affects both white and red blood cell synthesis, producing a macrocytic anemia and hypersegmented neutrophils. Leukopenia and thrombocytopenia may also be recognized. Rarely, platelet counts are low enough to produce purpura. Other rapidly proliferating cells are also affected, and gastrointestinal symptoms can include a sore, smooth, and beefy tongue, anorexia, and diarrhea. The most serious manifestations are neurologic: demyelination followed by axonal degeneration. Because of the possibility of eventual neuronal death, the later neurologic consequences of pernicious anemia can be irreversible. Affected neuronal tissues include the peripheral nerves, posterior and lateral columns of the spinal cord, and cerebrum. Clinical neurologic findings can include numbness and paresthesias, weakness and ataxia, sphincter disturbances, possible diminished position and vibration senses, positive Romberg and Babinski signs, irritability, forgetfulness, and even psychosis or dementia.Vitamin B12 is usually used as a generic term representing various cobalt-containing tetrapyrrole rings with attached nucleotide side chains that are chemically classified as cobalamins or corrinoids. A corrin is the cobalamin tetrapyrrole ring that excludes cobalt and other side chains. Cobalt plus the tetrapyrrole ring without other side chains is termed cobamide. A variety of cobalamins exist in nature that differ according to the side groups bound to the cobalt atom within cobalamin:Dietary vitamin B12 (eg, hydroxocobalamin) is bound by IF secreted by the gastric parietal cells. The cobalt in hydroxocobalamin is in the 3+ state and cobalamin is abbreviated CblIII. The vitamin B12-IF complex is absorbed in the terminal ileum. Absorbed vitamin B12 is then transported in the circulation to the tissues bound predominantly to transcobalamin II (TCII). Cells use pinocytosis to take up TCII hydroxocobalamin. The pinocytotic vesicle fuses with a lysosome that degrades TCII and releases hydroxocobalamin to the cytosol (eg, lysosomal efflux of cobalamin). In the cytosol, hydroxocobalamin can be methylated to methylcobalamin to convert homocysteine to methionine. In this reaction, the methyl group is supplied by methyltetrahydrofolate and the reaction is catalyzed by N5-methyltetrahydrofolate : homocysteine methyltransferase. Folate deficiency can raise homocysteine levels but does not affect the concentration of MMA. On the other hand, vitamin B12 deficiency can elevate both MMA and homocysteine levels. Hydroxocobalamin can also be taken up by the mitochondrion. In the lumen of the mitochondrion, cobalt in the 3+ state is reduced through sequential steps to cobalt in the 2+ state producing CblII and then cobalt in the 1+ state producing CblI. Transfer of an adenosyl group to CblI via the action of adenosyl-transferase produces adenosyl cobalamin. This form of vitamin B12 is essential for the conversion of l-methylmalonyl coenzyme A (CoA) to succinyl CoA (see below).The process starts with the metabolism of various compounds in the mitochondrion to form propionyl CoA (Figure 1). Intramitochondrial propionyl CoA is derived from many sources, including the essential amino acids isoleucine, valine, methionine, and threonine; cholesterol; and odd-chain fatty acids. Propionyl CoA is converted to d-methylmalonyl CoA through the action of propionyl-CoA-carboxylase that requires adenosine 5′-triphosphate, magnesium, and biotin. d-methylmalonyl CoA is isomerized to l-methylmalonyl CoA via methylmalonyl-CoA racemase. Finally, adenosyl cobalamin is essential for the conversion of l-methylmalonyl CoA to succinyl CoA.If vitamin B12 deficiency is present, there is decreased conversion of l-methylmalonyl CoA to succinyl CoA (Figure 2). Elevated l-methylmalonyl CoA raises d-methylmalonyl CoA concentrations and a hydrolase subsequently converts d-methylmalonyl CoA to MMA. Thus, MMA concentrations are elevated with vitamin B12 deficiency. In healthy individuals, 70% of MMA is metabolized to unknown products and only approximately 30% of MMA is excreted in the urine.10The diagnosis of cobalamin (vitamin B12) deficiency is problematic. The development of cobalamin deficiency is usually a slow process involving subtle neurologic changes.11 However, the irreversible neurologic damage that occurs with prolonged cobalamin deficiency necessitates that the diagnosis be made as early as possible because simple and effective treatment is readily available. Furthermore, the physician cannot depend on the presence of macrocytic anemia to make the diagnosis of cobalamin deficiency because neurologic disease can occur in patients with normal hemoglobin concentrations and normal red blood cell indices. Elderly subjects (n = 809) from Ohio were screened for cobalamin deficiency by measuring urinary MMA levels, and 4.4% of them had an elevated urinary MMA concentration as evidence of cobalamin deficiency.12 Although approximately half of these subjects with elevated urinary MMA concentrations had a low serum total cobalamin level (eg, less than 180 pg/mL at hospital 1 or less than 200 pg/mL at hospital 2), 1 in 3 subjects had only a low normal cobalamin level (180 or 200 pg/mL to 350 pg/mL) and 1 in 7 subjects had a normal cobalamin level. These data suggest that using a cobalamin cutoff of 100 pg/mL or less as the sole definition of cobalamin deficiency will fail to detect most cobalamin-deficient subjects.In outpatient geriatric clinics in Denver, 152 consecutive subjects underwent measurements of cobalamin, MMA, and homocysteine.13 Twenty-nine subjects had cobalamin levels of 300 pg/mL or less. A similar fraction of patients with low normal serum cobalamin levels (between 201 and 300 pg/mL) displayed increased metabolites of more than 3 SDs (56%) compared with patients with decreased serum cobalamin levels (≤200 pg/mL) (62%). This study again demonstrates that a definition of cobalamin deficiency based on a serum cobalamin level of 100 pg/mL or less or even 200 pg/mL or less will not detect most subjects with biochemical evidence of cobalamin deficiency based on raised metabolite concentrations and response to cobalamin replacement.In another group of 100 consecutive elderly subjects who visited an outpatient clinic, 16% had serum cobalamin levels of 200 pg/mL or less and an additional 21% had levels between 201 and 299 pg/mL (about 1 in 3 subjects overall having cobalamin levels <300 pg/mL).14 Among the 21 subjects with levels of 201 to 299 pg/mL, approximately 50% exhibited clinical disease: 2 patients had peripheral neuropathy and 9 patients had type A gastritis. Megaloblastic changes did not identify any of these affected subjects because none of the subjects with cobalamin levels between 201 and 299 pg/mL displayed a macrocytic anemia. One third of subjects with cobalamin levels from 201 to 299 pg/mL did exhibit elevated MMA and total homocysteine levels as biochemical evidence of cobalamin deficiency. This study suggests that in subjects with cobalamin levels of less than 300 pg/mL, MMA and total homocysteine concentrations should be measured to assess their vitamin status.At present, measurement of the serum cobalamin concentration is ordered by the treating physician when cobalamin deficiency is suspected. However, because cobalamin is highly protein bound, similar to thyroxine, measuring serum (total) cobalamin is problematic and does not reveal the free cobalamin concentration. Furthermore, although there is a test for unbound (free) thyroxine, there is no test for free (bioactive) cobalamin. Thus, the total cobalamin is a relatively poor indicator of bioavailable cobalamin.15Recognizing that MMA concentrations are elevated in states of cobalamin deficiency, measurement of MMA may complement or even replace measurements of serum cobalamin to more accurately diagnose cobalamin deficiency. Several studies have documented the advantages of measuring MMA for the diagnosis of cobalamin deficiency.16–18 In a recent study by Holleland et al,18 a cost-benefit analysis indicated MMA measurement was of value when the serum cobalamin concentration was greater than 60 to 90 pmol/L and less than 200 to 220 pmol/L. In this large study evaluating a total of 76 840 cobalamin analyses, more than 9% of the values fell within this interval, and MMA measurement should be performed for diagnostic accuracy.Knowing that the link between elevated MMA levels and cobalamin deficiency has been recognized for almost 40 years, the following question can be asked: “Why don't physicians order measurements of MMA?” There are 2 possible answers to this question: (1) physicians may not be aware of the limitations of serum cobalamin measurements or the value of MMA measurements and (2) MMA measurements are not readily available. Levels of MMA can be measured using gas chromatography–mass spectroscopy (GC-MS). With GC-MS, a typical serum reference range of 0.08 to 0.56 μmol/L has been reported,19 with a coefficient of variation of less than 8%. The lower limit of detection of MMA in serum can be as low as 0.026 μmol/L.Because MMA measurements require GC-MS or high-performance liquid chromatography,20 the methods are costly and cumbersome and commercial assays are not readily available. However, it is reasonable to expect that clinical chemists and manufacturers should develop generally available, inexpensive methods to measure MMA. A recent article by Magera et al21 describes a liquid chromatography-tandem mass spectroscopy assay with electrospray ionization that is able to accommodate 100 or more assays per day.Once there is sufficient demand for MMA proficiency testing, it is anticipated that the College of American Pathologists will provide proficiency testing for this important analyte. Recognizing the huge volume of cobalamin measurements performed yearly in the United States, MMA measurements as a supplement to cobalamin or in place of cobalamin measurements should be considered as an important diagnostic tool by both laboratorians and clinicians in the diagnosis of cobalamin deficiency. Our proposed approach to cobalamin testing would be to define cobalamin deficiency as any cobalamin level less than 100 pg/mL. For patients with cobalamin levels between 100 and 299 pg/mL, MMA should be measured.22 If MMA levels are elevated in this equivocal group, then cobalamin replacement should be initiated. Although macrocytosis and hypersegmentation are useful findings when present, the absence of hematologic findings does not exclude cobalamin deficiency.We thank Bonnie Deppert for her clerical help with the preparation of the manuscript.