Digoxin-inactivating bacteria: identification in human gut flora.

Digoxin, the most widely used cardiac glycoside, undergoes significant metabolic conversion in many patients to cardioinactive metabolites in which the lactone ring is reduced. This appears to occur within the gastrointestinal tract. An attempt was made to isolate and identify the organisms capable of reducing digoxin from stool cultures obtained from human volunteers. Of hundreds of isolates studied, only Eubacterium lentum, a common anaerobe of the human colonic flora, converted digoxin to reduced derivatives. Such organisms were also isolated in high concentrations from the stools of individuals who did not excrete these metabolites when given digoxin in vivo. When the growth of E. lentum was stimulated by arginine, inactivation of digoxin was inhibited. Neither the presence of these organisms alone nor their concentration within the gut flora appeared to determine whether digoxin would be inactivated by this pathway in vivo.

The mitogenic effect of the lymphocytosis promoting factor from Bordetella pertussis on human lymphocytes.

The purified lymphocytosis promoting factor (LPF) from Bordetella pertussis was found to be a potent mitogen for peripheral blood lymphocytes (PBL) from normal adults as well as for cord blood lymphocytes. Proliferation occurred in autologous plasma or fetal calf serum, regardless of previous exposure to pertussis infection or immunization. Only one adult human serum, from a physician constantly working with B. pertussis, inhibited the mitogenic response to LPF and this serum was shown to contain precipitating antibody against LPF. The proliferative effect of LPF was characteristic of a "nonspecific" mitogen and not of antigen stimulation of sensitized cells.LPF, phytohemagglutinin, and concanavalin A were approximately equal in potency although variation occurred depending upon the cell donor. Experiments with lymphocyte subpopulations obtained by rosetting techniques employing sheep erythrocytes, mouse erythrocytes, and sheep erythrocytes coated with antibody and complement suggested the requirement of a multicellular system for LPF mitogencity.PBL from most patients with chronic lymphatic leukemia and lymphosarcoma cell leukemia were even less responsive to LPF than to phytohemagglutinin, whereas PBL from patients with lymphosarcoma usually responded to both mitogens. It can be inferred from the results of experiments with both normal and leukemic cells that LPF, which is a murine thymus-derived (T)-cell mitogen, is also a T-cell mitogen for human PBL. The exact cell requirement and mode of action, however, are as yet unknown.

Elevation of total homocysteine in the serum of patients with cobalamin or folate deficiency detected by capillary gas chromatography-mass spectrometry.

To determine if levels of serum total homocysteine are elevated in patients with either cobalamin or folate deficiency, we utilized a new capillary gas chromatographic-mass spectrometric technique to measure total homocysteine in the serum of 78 patients with clinically confirmed cobalamin deficiency and 19 patients with clinically confirmed folate deficiency. Values ranged from 11 to 476 mumol/liter in the cobalamin-deficient patients and 77 of the 78 patients had values above the normal range of 7-22 mumol/liter as determined for 50 normal blood donors. In the cobalamin-deficient patients, serum total homocysteine was positively correlated with serum folate, mean corpuscular volume, serum lactate dehydrogenase, serum methylmalonic acid, and the degree of neurologic involvement, and inversely correlated with platelets and hematocrit. In the folate-deficient patients, values for serum total homocysteine ranged from 17 to 185 mumol/liter and 18 of the 19 patients had values above the normal range. Some patients with pernicious anemia who were intermittently treated with cyanocobalamin were found to have elevated serum levels of total homocysteine while they were free of hematologic and neurologic abnormalities. The measurement of serum total homocysteine will help define the incidence of cobalamin deficiency and folate deficiency in various patient populations.

Assay of methylmalonic acid in the serum of patients with cobalamin deficiency using capillary gas chromatography-mass spectrometry.

To determine the incidence of elevated levels of serum methylmalonic acid in patients with cobalamin deficiency, we utilized a new capillary gas chromatographic-mass spectrometric technique to measure methylmalonic acid in the serum of 73 patients with clinically confirmed cobalamin deficiency. Values ranged from 55 to 22,300 ng/ml, and 69 of the 73 patients had values above the normal range of 19-76 ng/ml as determined for 50 normal blood donors. In the cobalamin-deficient patients, serum methylmalonic acid was significantly correlated with the serum folate level and the degree of neurologic involvement. Some patients with pernicious anemia who were intermittently treated with cyanocobalamin were found to have elevated serum levels of methylmalonic acid while free of hematologic and neurologic abnormalities. A cobalamin-deficient patient is described with a normal serum cobalamin and an elevated serum methylmalonic acid. We conclude that the ability to measure methylmalonic acid in human serum will be useful in studies designed to determine the incidence of cobalamin deficiency in various patient populations.

Schizophrenia and impaired homocysteine metabolism: a possible association.

BACKGROUND: An increased risk of both schizophrenia and neural tube defects was observed in a birth cohort exposed to famine during early gestation. Neural tube defects have been related to a folate-sensitive genetic defect in homocysteine metabolism. If this were also true for schizophrenia, then cases with low folate (LF)--and only these cases--should have increased homocysteine levels compared with controls. METHODS: We compared homocysteine levels of schizophrenia cases and normal controls with low folate (LF) and without low folate (non-LF). Low folate was defined by the bottom tertile for controls. RESULTS: In the LF group (6 cases, 8 controls), mean homocysteine was 10.7 microM in cases compared with 7.7 microM in controls (p = .03). In the non-LF group (11 cases, 16 controls) mean homocysteine did not differ for cases and controls. CONCLUSIONS: These pilot data are compatible with the hypothesis that a folate-sensitive defect in homocysteine metabolism contributes to cases of schizophrenia.

Bioavailability of different lots of digoxin tablets from the same manufacturer.

The bioavailability of single doses from 4 randomly selected lots of digoxin from a single manufacturer that were recently marketed in the United States was compared. In a crossover study in 6 normal volunteers, serum digoxin concentrations for 0 to 6 hr and urinary glycoside excretion for 24 hr after 0.5-mg doses were measured by radioimmunoassay. The variation in bioavailability between the 4 lots was no greater than that seen when one of them was taken twice. Intersubject variation in areas under the serum concentration-time curves and in the 24-hr urinary digoxin measurements was more than twice that of intrasubject variation. The variability of the urinary digoxin determinations was somewhat less than that of the areas under the serum concentration-time curves.

Greater bioavailability of digoxin solution in capsules. Studies in the postprandial state.

A liquid concentrate of digoxin administered as a capsule has recently been found to be better absorbed by normal subjects in the fasting state than a standard tablet or even a solution as such. It was not known whether the enchanced bioavailability of the encapsulated digoxin solution could also be demonstrated when given postprandially. In a crossover study single 0.4 mg doses of digoxin solution in a capsule, a solution in 10% ethanol as such, and a standard tablet were given to 12 normal volunteers immediately following a high-fat breakfast. The mean area under the serum concentration-time curve (AUC) and 6-day cumulative urinary glycoside excretion (CUE) were greater after the capsule than that of either the solution or the tablet. The latter two preparations appeared to be bioequivalent. Intersubject variability in the CUE was less than with the AUC, but did not differ when the three drug formulations were compared. The bioavailability of an encapsulated liquid digoxin concentrate was thus also found to be enhanced when administered postprandially.

Superior bioavailability of digoxin solution in capsules.

The bioavailability of various formulations of digoxin was assessed after single and multiple doses in a series of crossover studies in human volunteers. Digoxin tablets that were 97% dissolved in 1 hr in vitro were not significantly better absorbed than tablets with a dissolution rate of 78%. A solution given in capsule form had greater bioavailability than tablets of 97% dissolution rate; serum and urinary glycoside levels after 0.4 mg doses of the encapsulated solution were similar to those attained after 0.5 mg doses of tablets with dissolution rates of 78% and 97%. The bioavailability of the solution in capsule form exceeded that of equal doses of the same solution given as a liquid or that of a standard elixir. No increase in gastrointestinal or cardiac toxicity was detected. Inter- and intrasubject variation in bioavailability was not decreased. Above a certain level, dissolution rate is no longer the limiting factor in digoxin absorption. The mechanism of the enhanced bioavailability of concentrated liquid digoxin in capsule form remains to be determined. Such a preparation deserves further consideration as a possible replacement for digoxin tablets.

Variability of steady-state digoxin kinetics during administration of tablets or capsules.

An encapsulated solution of digoxin has been repeatedly shown to have greater bioavailability than tablet forms of the drug. It is predicted that such a preparation would show reduced within- and between-patient variability in absorption, as most studies in normal subjects have shown reduced intersubject variation with the capsule. We tested inter- and intrapatient variability during 4-week periods of dosing with digoxin capsules and tablets in 28 subjects with cardiac disease. In the overall group there were no significant differences between the formulations at steady state in between-patient variability in trough serum digoxin concentrations or 24-hour urinary digoxin excretion. Within-patient variability in urinary digoxin excretion was somewhat lower for the capsules. In a subgroup of six patients who excreted significant amounts of cardioinactive bacterial metabolites (digoxin reduction products [DRP]), the mean (+/- SD) percent urinary DRP excretion was less (p less than 0.05) during capsule (20.5% +/- 15.1%) than tablet (34.4% +/- 10.9%) dosing. Within-patient variability in urinary DRP excretion was much greater after tablets than capsules. Certain subgroups of patients should benefit from the enhanced bioavailability of digoxin capsule preparations.

Lack of difference in digoxin urinary excretion with two intravenous infusion rates.

Ten normal subjects were given 0.4 mg digoxin intravenously by bolus injection over 3 to 5 min and by constant-rate infusion for 1 hr. Urinary excretion of digoxin over the next 6 days, as measured by radioimmunoassay, was similar after both the rates of intravenous injection. In one subject, who excreted substantial amounts of digoxin reduction products, no difference was apparent in the amount of reduced metabolites excreted. These results are not in agreement with previous reports of an effect of intravenous infusion rate on urinary digoxin excretion.

Decreased digoxin cardioinactive-reduced metabolites after administration as an encapsulated liquid concentrate.

The generation by intestinal bacteria of large amounts of cardioinactive metabolites of digoxin with a reduced lactone ring (digoxin reduction products, or DRP) may be associated with increased dosage requirements. Since DRP excretion varies inversely with bioavailability, we compared the 6-day urinary excretion (CUE) of digoxin and DRP after 0.4-mg doses of an encapsulated liquid concentrate and a standard tablet in 22 normal subjects known to form substantial amounts of DRP. Mean (+/- SE) CUE of digoxin was greater with the capsules than the tablets (195.9 +/- 8.6 and 137.5 +/- 6.3 micrograms). CUE of DRP was less after the capsules (60.8 +/- 5.5 and 102.7 +/- 9.5 micrograms). Percent DRP was greater after the tablets in every subject (mean for tablets, 41.2 +/- 2.7%; capsules, 23.5 +/- 1.8%). Patterns of DRP excretion differed with the two preparations, probably reflecting differences in the routes whereby digoxin reached the colon. The use of highly bioavailable capsules in subjects with heavy DRP production should minimize metabolic inactivation during digoxin therapy.

Anemia in alcoholics.

In order to develop a diagnostic approach to the common problem of anemia associated with alcoholism, 121 chronic alcoholics admitted to a general medical service with a low hematocrit were evaluated. Multiple contributing causes of anemia were present in most patients. Megaloblastic marrow change was found in 33.9% of patients, sideroblastic change in 23.1%, absent iron stores in 13.2%, aggregated macrophage iron in 81.0%, and acute blood loss in 24.8%. The MCV was of little value in predicting the presence of megaloblastic change unless markedly elevated (greater than 110 fl). In 15 of 41 patients with megaloblastic marrow morphology (36.6%) the MCV was normal or low. Among 40 patients with MCV values between 100 and 110 fl, megaloblastic change was not present in the bone marrow smears of 24 (60.0%). Neutrophil hypersegmentation was 95% specific but only 78% sensitive for megaloblastic change; in contrast, the presence of macroovalocytosis was 90% sensitive but only 68% specific. Serum lactic dehydrogenase, plasma folate, and erythrocyte folate levels had such low sensitivities and specificities for megaloblastic change as to be of little predictive value. Hematologic responses to folic acid were often inadequate in patients with megaloblastic morphologic changes, apparently because of associated acute and chronic illness. Our findings are consistent with the hypothesis that 2 mechanisms account for the development of megaloblastic hematopoiesis in alcoholics: induction of folate deficiency and a direct toxic effect of alcohol on erythroid precursors independent of folate depletion, as reflected by the presence of normal plasma and erythrocyte folate levels in several patients with megaloblastic change. In no patient was sideroblastic change the sole apparent cause of anemia. Megaloblastic hematopoiesis and aggregated macrophage iron frequently accompanied sideroblastic change. Examination of the blood smear revealed siderocytes in one-third of patients with sideroblastic marrows and dimorphic erythrocyte morphology in the majority. Dimorphic blood smears, however, were neither sensitive nor specific for sideroblastic change. Serum iron concentrations were usually not elevated in the group with sideroblastic abnormalities. In predicting marrow iron stores, serum iron and iron-binding capacity concentrations were often non-diagnostic or misleading. Serum ferritin levels less than 100 ng/ml, however, showed 100% sensitivity and 95% specificity for absent marrow iron stores despite the frequent presence of abnormal liver function. On the basis of our findings, practical guidelines have been formulated for the evaluation and therapy of anemia in alcohol

Neurologic aspects of cobalamin deficiency.

We reviewed 153 episodes of cobalamin deficiency involving the nervous system that occurred in 143 patients seen over a recent 17-year period at 2 New York City hospitals. Pernicious anemia was the most common underlying cause of the deficiency. Neurologic complaints, most commonly paresthesias or ataxia, were the first symptoms of Cbl deficiency in most episodes. The median duration of symptoms before diagnosis and treatment with vitamin B12 was 4 months, although long delays in diagnosis occurred in some patients. Diminished vibratory sensation and proprioception in the lower extremities were the most common objective findings. A wide variety of neurologic symptoms and signs were encountered, however, including ataxia, loss of cutaneous sensation, muscle weakness, diminished or hyperactive reflexes, spasticity, urinary or fecal incontinence, orthostatic hypotension, loss of vision, dementia, psychoses, and disturbances of mood. Multiple neurologic syndromes were often seen in a single patient. In 42 (27.4%) of the 153 episodes, the hematocrit was normal, and in 31 (23.0%), the mean corpuscular volume was normal. Neutropenia and thrombocytopenia were unusual even in anemic patients. In nonanemic patients in whom diagnosis was delayed, neurologic progression frequently occurred although the hematocrit remained normal. In 27 episodes, the serum cobalamin concentration was only moderately decreased (in the range of 100-200 pg/ml) and in 2 the serum level was normal. Neurologic impairment, as assessed by a quantitative severity score, was judged to be mild in 99 episodes, moderate in 39 and severe in 15. Severity of neurologic dysfunction before treatment was clearly related to the duration of symptoms prior to diagnosis. In addition, the hematocrit correlated significantly with severity, independent of the longer duration of symptoms in nonanemic patients. Four patients experienced transient neurologic exacerbations soon after beginning treatment with cyanocobalamin, with subsequent recovery. Followup evaluation was adequate to assess the neurologic response to vitamin B12 therapy in 121 episodes. All patients responded, and in 57 (47.1%), recovery was complete, with no remaining symptoms or findings on examination. The severity score was reduced by 50% or greater after treatment in 91% of the episodes. Residual long-term moderate or severe neurologic disability was noted following only 7 (6.3%) episodes. The extent of neurologic involvement after treatment was strongly related to that before therapy as well as to the duration of symptoms. The percent improvement over baseline neurologic status after treatment was inversely related to duration of symptoms and hematocrit. Some evidence of response was always seen during the first 3 months of treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

Nutritional anemia in alcoholism.

Megaloblastic anemia due to folate deficiency, the result of dietary lack and a weak antifolate action of ethanol, is the most common cause of a low hematocrit in hospitalized alcoholics. Alcoholism in the absence of significant folate depletion is more commonly responsible for macrocytosis, however. Neutrophil hypersegmentation, which typically persists or worsens during the first 1 to 2 weeks of folic acid therapy, is a useful sign of folate depletion. Serum folate concentrations, however, are often misleading. During conversion of the megaloblastic marrow following hospitalization, giant bands and metamyelocytes often persist after erythroid cells become normal. Reversible ineffective erythropoiesis due to sideroblastic anemia, often but not invariably in association with folate deficiency, is also common. In about half the patients, siderocytes in the peripheral blood smear, which may transiently increase in number during recovery, provide a useful diagnostic clue. Despite the presence of hypochromic microcytes, the erythrocyte mean corpuscular volume is typically normal or elevated. The chronic administration of alcohol along with a marginal diet has produced ringed sideroblasts in human volunteers. Inhibition of heme synthesis by ethanol and an unidentified nutritional factor probably play major roles in pathogenesis. Current evidence does not clearly implicate vitamin B6 depletion in sideroblastic anemia in alcoholics.

Oral contraceptive hormones, folate metabolism, and the cervical epithelium.

The currently available evidence concerning disorders of folate metabolism in women taking oral contraceptives has been reviewed. A disturbance in folate balance serious enough to cause symptoms (i.e., megaloblastic anemia) occurs very rarely. In some series, but not in others, serum and/or red cell folate concentrations have been reduced in oral contraceptive users. It is doubtful whether sex steroids affect polyglutamate folate absorption. About 20 percent of women taking contraceptive hormones manifest mild megaloblastic changes on Papanicolaou smears of the cervicovaginal epithelium which disappear after folic acid therapy. The current evidence, however, would not indicate that any significant benefit would ensue from routine folate supplementation in women on oral contraceptives.

Vitamin B-12 deficiency in the elderly: current dilemmas.

Vitamin B-12 deficiency is present in up to 15% of the elderly population as documented by elevated methylmalonic acid with or without elevated total homocysteine concentrations in combination with low or low-normal vitamin B-12 concentrations. Clinical signs and symptoms of vitamin B-12 deficiency are insensitive in elderly subjects and comorbidity in these subjects makes responses to therapy difficult to interpret. Many elderly subjects with hyperhomocysteinemia have undiagnosed vitamin B-12 deficiency with elevated serum methylmalonic acid concentrations. Therefore, such elderly subjects should not receive folic acid supplementation before their vitamin B-12 status is diagnosed. Oral vitamin B-12 supplementation may be effective in lowering serum methylmalonic acid values in the elderly. However, the dose of vitamin B-12 in most common multivitamin preparations is too low for this purpose. Research efforts should be directed toward determining practical methods for diagnosing and treating vitamin B-12 deficiency in the millions of elderly subjects with undiagnosed deficiency.

Prevalence of cobalamin deficiency in the Framingham elderly population.

To determine whether the increased prevalence of low serum cobalamin concentrations in elderly people represents true deficiency, serum concentrations of cobalamin and folate and of metabolites that are sensitive indicators of cobalamin deficiency were measured in 548 surviving members of the original Framingham Study cohort. Serum cobalamin concentrations < 258 pmol/L were found in 222 subjects (40.5%) compared with 17.9% of younger control subjects (P < 0.001). Serum methylmalonic acid and total homocysteine concentrations were markedly elevated in association with cobalamin values < 258 pmol/L in 11.3% and 5.7%, respectively, of the cohort. Both metabolites were increased in 3.8% of the cohort, associated with significantly lower erythrocyte counts and higher mean cell volumes. Serum metabolites correlated best with serum cobalamin values, even when subnormal determinations were excluded. The prevalence of cobalamin deficiency was > or = 12% in a large sample of free-living elderly Americans. Many elderly people with "normal" serum vitamin concentrations are metabolically deficient in cobalamin or folate.

Folate nutrition in the elderly.

The consensus of this panel is that average dietary intake of folate in the free-living elderly population is probably adequate in most. Certainly more good data are needed; in addition, safe and reasonable dietary goals for folate intake are required. However, patients who have diseases requiring hospitalization or conditions for which institutionalization are required are obviously at greater risk. In addition, there is some evidence that the elderly poor in the US may be at greater risk of deficiency. Similarly, the evidence for folate deficiency based on blood assay data would seem to focus on the lower socioeconomic (largely Black and Hispanic) populations in addition to the hospitalized and institutionalized elderly. An additional factor in the genesis of folate deficiency among the aged is the factor of alcohol use which probably represents the single most important risk factor in folate deficiency among the elderly as well as among the nonelderly population. Although certain drugs such as anticonvulsants and sulfasalazine, may interfere with folate absorption or utilization, the number of elderly patients who are taking these drugs is relatively small and therefore this factor is not considered to be a major contributor to the problem of folate deficiency in the elderly. The question of folate malabsorption in the elderly has been examined. It is our conclusion that disease in the elderly population including gastric surgery and intestinal malabsorption, etc can certainly interfere with folate absorption but these problems are not widespread among the elderly population. There is only limited evidence that the physiological process of aging influences the intestinal absorption of folate.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic evidence that deficiencies of vitamin B-12 (cobalamin), folate, and vitamin B-6 occur commonly in elderly people.

Measurements of the serum concentrations of the metabolites homocysteine, cystathionine, methylmalonic acid, and 2-methylcitric acid, which accumulates when vitamin B-12-, folate-, and vitamin B-6-dependent enzymatic reactions are impaired, should provide a better indication of intracellular deficiency of these vitamins. We measured the serum concentration of these vitamins and the four metabolites in 99 healthy young people, 64 healthy elderly subjects, and 286 elderly hospitalized patients. A low serum vitamin B-12 concentration was found in 6% and 5%, low folate in 5% and 19%, and low vitamin B-6 in 9% and 51%, and one or more metabolites were elevated in 63% and 83% of healthy elderly subjects and elderly hospitalized patients, respectively. These results strongly suggest that the prevalence of tissue deficiencies of vitamin B-12, folate, and vitamin B-6 as demonstrated by the elevated metabolite concentrations is substantially higher than that estimated by measuring concentrations of the vitamins.

Cerebrospinal fluid methylmalonic acid levels in normal subjects and patients with cobalamin deficiency.

We measured methylmalonic acid, which accumulates in the blood and tissues of patients with cobalamin deficiency, in the CSF of 65 patients using capillary-gas chromatography and mass spectrometry. In 58 control patients, methylmalonic acid concentrations were always higher in CSF than in serum (mean CSF: serum ratio, 2.65; range, 1.17 to 7.78). In contrast, in six patients with elevated serum methylmalonic acid levels due to renal failure, CSF concentrations were normal in five and the CSF: serum ratio was less than one in four. In three patients with neuropsychiatric syndromes due to cobalamin deficiency and one patient with a normal serum cobalamin level who was an abuser of nitrous oxide, CSF concentrations were markedly increased (mean level, 600 times that of controls), out of proportion to those in the serum (mean CSF: serum ratio, 8.38; range, 3.5 to 13.5). The potential usefulness of CSF metabolite levels in the diagnosis of cobalamin deficiency is undetermined.