Elevated methylmalonic acid and total homocysteine levels show high prevalence of vitamin B12 deficiency after gastric surgery.

Elderly persons [1, 2] and persons who have had gastric surgery [3-11] are at increased risk for developing vitamin B12 (cobalamin) deficiency. The hematologic and neurologic manifestations of vitamin B12 deficiency have been well described; however, this deficiency often remains undetected, and some patients receive a misdiagnosis of Alzheimer disease, spinal cord compression, amyotrophic lateral sclerosis, or diabetic or alcoholic peripheral neuropathy [12]. Although megaloblastic anemia is usually reversible with vitamin B12 treatment, the neurologic injuries are reversible only if they are treated soon after their onset [12, 13]. In addition, as do patients with folate deficiency [14], patients with untreated vitamin B12 deficiency have elevated total homocysteine levels. Substantial biochemical and epidemiologic evidence now suggests that an elevated serum total homocysteine level contributes to the development of carotid artery stenosis, coronary artery disease, and peripheral vascular disease [15-18]. Thus, in theory at least, patients with untreated vitamin B12 deficiency may be at increased risk for developing atherosclerotic vascular disease. In the future, the prevalence of vitamin B12 deficiency in the aging population may be expected to increase. Among persons at major risk are those who had subtotal gastrectomy for ulcer disease between the 1930s [19] and 1974 [5, 7] (in 1974, the first histamine-2 blocker, cimetidine, was released [20]). It is not possible to determine how many Americans had gastric surgery during this period, but representative data from the University of Minnesota Hospital suggest that the number is large. At that hospital alone, 1550 patients had subtotal gastrectomy between 1938 and 1950 [4]. Throughout the United States, therefore, hundreds of thousands of patients probably had this surgery. A new operation, gastric bypass for obesity, is currently creating another cohort at risk for developing vitamin B12 deficiency [11]. As these cohorts age, an unknown number of persons will develop vitamin B12 deficiency, and clinicians caring for such persons currently have no accurate guidelines on which to base screening decisions. Previous prevalence estimates are unreliable because clinical manifestations are insensitive and radiodilution vitamin B12 assays were nonspecific [21, 22]. The Schilling test is unreliable after gastrectomy [8, 9], anemia is often absent in vitamin B12-deficient patients [2, 12, 23], and macrocytosis may be masked by coexisting iron deficiency [7, 24]. See editorial comment on pp 509-511. Recently, however, measurements of the metabolites from two vitamin B12-dependent pathways (Figure 1)serum methylmalonic acid [25] and total homocysteine [14]were shown to be highly sensitive detectors of vitamin B12 deficiency [26]. Two enzymes have a known requirement for vitamin B12: L-methylmalonyl-CoA mutase and methionine synthase [22]. Methionine synthase requires folate in addition to vitamin B12 for normal functioning. If the conversion of L-methylmalonyl-CoA to succinyl-CoA is impaired by a deficiency of the vitamin B12 cofactor adenosylcobalamin, the excess methylmalonyl-CoA is cleaved to methylmalonic acid and methylmalonic acid levels in the serum and urine are elevated [25]. Similarly, if the methylation of homocysteine to methionine is impaired by a deficiency of methylcobalamin or methyltetrahydrofolate, serum total homocysteine levels are elevated [14]. The metabolic pathways in which these two enzymes function are not always equally affected by vitamin B12 deficiency. At the time vitamin B12 deficiency is diagnosed, therefore, levels of methylmalonic acid, total homocysteine, or both may be elevated [22]. Figure 1. The two vitamin B12-dependent enzymes, L-methylmalonyl-CoA mutase (left) and methionine synthase (right). In vitamin B12-deficient patients, elevated levels of both serum methylmalonic acid and total homocysteine decrease promptly with adequate vitamin B12 therapy [22, 26]. However, in folate-deficient patients, total homocysteine levels return to normal only after folate replacement [22]. Therefore, in addition to serum vitamin B12 levels, we used methylmalonic acid, total homocysteine, and folate levels to determine whether the prevalence of vitamin B12 deficiency differed between persons who had had gastric surgery and those who had not. Methods Between September 1991 and March 1993, 65 patients who had had gastric surgery were identified at the Philadelphia Veterans Affairs Medical Center. These patients were identified either by review of gastrointestinal radiographs, surveys of the house-staff assigned to the medicine and surgery inpatient services, or referral of outpatients from physicians in the medical clinic. Four of the 65 patients were excluded: Three were receiving vitamin B12 therapy, and one had a hepatoma. Hepatoma can produce increased levels of vitamin B12-binding protein, which may complicate interpretation of serum vitamin B12 levels. Patients who had not had gastric surgery (controls) were drawn from 127 consecutive patients attending one author's Philadelphia Veterans Affairs Medical Center clinic between November 1992 and March 1993. One hundred seven controls participated, and 20 either declined to participate or did not complete the required blood tests. We determined the type of gastric surgery that had been done either from patient reporting or by reviewing radiologic, endoscopic, or surgical records. In most patients (51 of 61), we determined the year surgery had been done from patient report or chart review. For patients who could not provide the year of surgery but could specify the decade, we used the mid-decade year. For example, if the patient said that the surgery had been done in the 1950s, we recorded the year as 1955. Serum vitamin B12 and folate levels were determined at the Philadelphia Veterans Affairs Medical Center using a commercially available radioligand kit (Bio-Rad, Diagnostics Group, Hercules, California). In the hospital's laboratory, normal values for vitamin B12 and folate levels were 171 to 840 pmol/L and 5 to 39 nmol/L, respectively. The remaining serum samples were frozen at 20 C and were shipped to Denver so that serum methylmalonic acid and total homocysteine levels could be analyzed by the stable isotope dilution gas chromatography-mass spectrometry method [27-30]. The normal range for serum methylmalonic acid levels (determined in 50 normal blood donors 18 to 65 years of age) is 73 to 271 nmol/L, and the normal range for serum total homocysteine levels is 5.4 to 16.2 mol/L [22]. Vitamin B12 deficiency was defined as one of the following: 1) a serum vitamin B12 level less than 221 pmol/L and an elevated methylmalonic acid level; 2) a serum vitamin B12 level less than 221 pmol/L and a total homocysteine level that decreased after vitamin B12 therapy; or 3) in patients unavailable for treatment, a serum vitamin B12 level less than 221 pmol/L, a folate level greater than 9 nmol/L, and an elevated total homocysteine level. Hemoglobin level, hematocrit, and mean corpuscular volume were measured by automatic devices. Macrocytosis was defined as a mean corpuscular volume of 95 fL or less. The peripheral smears of 71% of patients (43 of 61) and 88% of controls (94 of 107) were reviewed by one hematologist who was blinded to each participant's vitamin B12 level, hemoglobin level, hematocrit, and gastric surgery status. Hypersegmentation was defined as five neutrophils with five or more lobes or one neutrophil with six lobes per 100 cells counted. Treatment Vitamin B12 treatment generally consisted of daily intramuscular injections of 1000 g of vitamin B12 for 5 days, followed by monthly injections. Folic acid was given orally, 1 mg/d. Serum vitamin B12, folate, methylmalonic acid, and total homocysteine levels were measured 1 to 6 weeks after treatment. Statistical Analysis Data were examined to determine whether the variables were suitable for parametric analyses. Although relatively modest, the skew for the numeric variables necessitated that several variables be transformed to logs for entry into two-way analysis of variance or be subjected to nonparametric analyses. The comparison between patients and controls for levels of vitamin B12, folate, methylmalonic acid, and total homocysteine was done by two-factor analysis of variance on log-transformed variables. In each analysis of variance, race was included as a factor (along with study group) to control for possible race-by-group interactions. We used unpaired t-tests or Mann-Whitney U tests to do comparisons of other numeric variables, such as hemoglobin and mean corpuscular volume; comparisons between other groups, such as patients with a positive and patients with a negative peripheral blood smear; and comparisons between deficient and nondeficient patients. We used chi-square tests to compare groups on dichotomous variables (such as white patients compared with black patients). Spearman correlations were used to assess the association between the time since surgery and other variables. The Human Studies Subcommittee and the Research and Development Committee of the Philadelphia Veterans Affairs Medical Center approved the study. Results Clinical Characteristics The 61 patients (who had had gastric surgery) and 107 controls (who had not) were similar in the ratio of men to women (60:1 compared with 104:3), age, and race (Table 1). The indications for surgery included peptic ulcer disease (56 patients), obesity (3 patients), gastric cancer (3 patients [2 of whom had previously had surgery for peptic ulcer disease]), and gastric lymphoma (1 patient). The type of gastric surgery could be determined in 36 of 61 patients (59%). The types of surgery were Billroth II (23 patients), repair of perforated ulcer (6 patients), vagotomy and pyloroplasty (2 patients), gastric bypass or gastric banding for obesity (3 patients), Billroth I (1 patient),