Etiology and diagnostic evaluation of macrocytosis.

BACKGROUND: Elevation of mean cell volume (MCV) is a common clinical problem, but the etiologic spectrum and optimal diagnostic evaluation of macrocytosis are not well defined. METHODS: We studied 300 consecutive hospitalized adult patients with MCV values > or = 100 fL. Assessment included complete blood counts, morphologic review, liver function tests, and levels of serum cobalamin (Cbl), methylmalonic acid, and total homocysteine. RESULTS: The most common cause of macrocytosis was drug therapy, followed by alcohol, liver disease, and reticulocytosis. Megaloblastic hematopoiesis accounted for less than 10% of cases. MCV values > 120 fL were usually caused by Cbl deficiency. Anisocytosis, macro-ovalocytosis, and teardrop erythrocytes were most prominent in megaloblastic hematopoiesis. Elevated levels of serum methylmalonic acid and total homocysteine were useful in the diagnosis of Cbl deficiency. CONCLUSIONS: Drugs and alcohol are the most common causes of macrocytosis in hospitalized patients in a New York City teaching hospital. We have formulated tentative guidelines for the evaluation of high MCV values in this setting.

Pancytopenia in Zimbabwe.

BACKGROUND: There has been little systematic study of the clinical spectrum of pancytopenia, and the optimal diagnostic approach to pancytopenia remains undefined. METHODS: The authors studied 134 hospitalized pancytopenic patients in Zimbabwe in both consecutive and nonconsecutive fashion. RESULTS: The most common cause of pancytopenia was megaloblastic anemia, followed by aplastic anemia, acute leukemia, acquired immunodeficiency syndrome (AIDS), and hypersplenism. Severe pancytopenia was usually due to aplastic anemia. Patients with aplastic anemia and acute leukemia were usually children, whereas those with megaloblastic anemia were adults. Moderate to severe anemia was noted throughout the series, but was most striking in patients with megaloblastic anemia, aplastic anemia, and acute leukemia. The mean corpuscular volume (MCV) was elevated in most patients with megaloblastic hematopoiesis, aplastic anemia, and acute nonlymphocytic leukemia. Normal or low MCV values were noted in almost one third of patients with megaloblastic anemia. Anisocytosis, poikilocytosis, macroovalocytosis, microcytosis, fragmentation, and teardrop erythrocytes were more prominent on the blood films of patients with megaloblastic anemia. CONCLUSIONS: Megaloblastic anemia, aplastic anemia, and AIDS are the most common causes of pancytopenia in Zimbabwe. Aplasia is the most frequent cause of severe pancytopenia. The authors have formulated tentative guidelines for the evaluation of pancytopenic patients in this setting.

Enhanced levels of biochemical markers for cobalamin deficiency in totally gastrectomized rats: uncoupling of the enhancement from the severity of spongy vacuolation in spinal cord.

The totally gastrectomized (TGX) rat is a new experimental model for studying the pathogenesis of cobalamin (Cbl)-deficient myelopathy, i.e., subacute combined degeneration, total gastrectomy (TG) serving as a surgical paradigm of human pernicious anemia. We determined the serum levels of some biochemical indicators of Cbl deficiency in TGX rats at 2 to 10 months after TG. Methylmalonic acid (MMA) rose within 2 months and progressively increased thereafter until the end of the investigation period. 2-Methylcitric acid (MCA) rose significantly by 6 months and showed a further increment 4 months later. Homocysteine was only clearly elevated much later than the serum MMA, i.e., 10 months after the operation. The concentrations of MMA, MCA, and cystathionine were increased in kidney, liver, and spinal cord (SC) of TGX rats at 10 months. Chronic treatment of TGX rats with Cbl greatly decreased the serum levels of all the metabolic indicators of Cbl deficiency. Chronic peroral administration of the antibiotic lincomycin to TGX rats in an attempt to suppress the enteric flora markedly decreased serum MMA levels. Only Cbl, however, given either for the first 2 months after TG or for the third and fourth postoperative months (i.e., after SC abnormalities had already appeared) significantly decreased the severity of spongy vacuolation in SC white matter, although not completely preventing or repairing the neuropathological damage. Therefore, neither the early impairment in TGX rats of the Cbl-dependent methylmalonyl-coenzyme A mutase reaction nor the more delayed impairment of the Cbl-dependent methionine synthase step, as reflected by changes in serum metabolite levels, seems to be causally related to the TG-induced spongy vacuolation in SC white matter.

Effects of acarbose on fecal nutrients, colonic pH, and short-chain fatty acids and rectal proliferative indices.

Acarbose, an alpha-glycosidase inhibitor, treats diabetes mellitus by delaying the digestion and intestinal absorption of dietary carbohydrates. In effective doses, acarbose induces some passage of carbohydrates into the colon. The effect of such chronic carbohydrate transfer on colonic structure and function is unknown. We studied the effects of 1 year of acarbose administration in diabetes mellitus on fecal energy, protein, and fat, including short-chain fatty acids (SCFA) output, fecal pH, and several metabolizing bacterial species. Changes in colonic histology and epithelial cell proliferation were investigated in rectal biopsies. Fecal macronutrient output was unaffected by acarbose, but pH decreased and total SCFA, butyrate, and acetate output were markedly greater. Breath hydrogen output increased after acarbose, but digoxin-metabolizing bacteria and diacylglycerol (DAG) production were unaltered. Compared with the control, acarbose did not induce hyperplasia or change rectal proliferation. However, total fecal SCFA and butyrate output correlated inversely with proliferation in the rectal upper crypt-a biomarker of risk for colonic neoplasia. In conclusion, long-term acarbose administration does not adversely affect colonic function or fecal nutrient output. If increased fecal SCFA and butyrate reduces upper-crypt proliferation, then acarbose may reduce the risk of colonic neoplasia.

The use of homocysteine and other metabolites in the specific diagnosis of vitamin B-12 deficiency.

Vitamin B-12 (cobalamin) is a cofactor for only two enzymes, methionine synthase and L-methylmalonyl-CoA mutase. The serum vitamin B-12 concentration has been shown to have limitations in specificity and sensitivity in diagnosing vitamin B-12 deficiency and predicting response to therapy in subjects with clinical deficiency syndromes. Serum methylmalonic acid and/or total homocysteine concentrations have been shown to be elevated in almost every patient who has a clinical response to vitamin B-12. In elderly populations serum methylmalonic acid concentrations are elevated in the majority (60-66%) of subjects who have elevated total homocysteine concentrations, suggesting that vitamin B-12 deficiency (with or without associated folate deficiency) and/or chronic renal insufficiency may be the primary cause of most of the elevated total homocysteine concentrations in elderly populations. In such subjects vitamin B-12 and folate concentrations are both frequently in the low or low normal range, making differentiation of the clinical syndromes by use of serum vitamin concentrations problematic. Elevations of 2-methylcitric acid and cystathionine also result from vitamin B-12 deficiency. Serum N-methylglycine concentrations are normal in cobalamin deficiency but are increased in 40% of patients deficient in folate. In conclusion, elevations of methylmalonic acid and total homocysteine are very sensitive and specific in diagnosing vitamin B-12 deficiency and can be used to help differentiate vitamin B-12 deficiency from folate deficiency. Elevated total homocysteine concentrations that may have been attributed to folate deficiency in elderly subjects may in many instances be the result of vitamin B-12 deficiency even though serum vitamin B-12 concentrations are within normal limits.

Effects of vitamin B12, folate, and vitamin B6 supplements in elderly people with normal serum vitamin concentrations.

In a prospective, multicentre, double-blind controlled study, the effect of an intramuscular vitamin supplement containing 1 mg vitamin B12, 1.1 mg folate, and 5 mg vitamin B6 on serum concentrations of methylmalonic acid (MMA), homocysteine (HCYS), 2-methylcitric acid (2-MCA), and cystathionine (CYSTA) was compared with that of placebo in 175 elderly subjects living at home and 110 in hospital. Vitamin supplement and placebo were administered eight times over a 3-week period. Vitamin supplement but not placebo significantly reduced all four metabolite concentrations at the end of the study in both study groups. The maximum effects of treatment were usually seen within 5-12 days. Initially elevated metabolite concentrations returned to normal in a higher proportion of the vitamin than of the placebo group: 92% vs 20% for HYCS; 82% vs 20% for MMA; 62% vs 25% for 2-MCA; and 42% vs 25% for CYSTA. The response rate to vitamin supplements supports the notion that metabolic evidence of vitamin deficiency is common in the elderly, even in the presence of normal serum vitamin levels. Metabolite assays permit identification of elderly subjects who may benefit from vitamin supplements.

Serum betaine, N,N-dimethylglycine and N-methylglycine levels in patients with cobalamin and folate deficiency and related inborn errors of metabolism.

Homocysteine and 5-CH3-tetrahydrofolate (5-CH3-THF) are converted to methionine and THF by the CH3-cobalamin (CH3-Cbl)-dependent enzyme methionine synthase. Serum homocysteine levels are elevated in more than 95% of patients with Cbl or folate deficiency and in patients with inborn errors involving the synthesis of 5-CH3-THF or CH3-Cbl. Homocysteine and betaine are converted to methionine and N,N-dimethylglycine by betaine-homocysteine methyltransferase. It requires neither Cbl nor folate, although N,N-dimethylglycine is converted to N-methylglycine and then to glycine in reactions that both involve the formation of 5,10-CH2-THF from THF. Large amounts of betaine are often given orally to patients with inborn errors, even though little is known about its metabolism in normal subjects or these patients. Thus we developed new gas chromatographic-mass spectrometric assays for serum betaine, N,N-dimethylglycine, and N-methylglycine. In 60 blood donors, we found ranges for normal serum of 17.6 to 73.3, 1.42 to 5.27, and 0.60 to 2.67 mumol/L for the three metabolites, respectively, which were normal in the majority of 50 patients with Cbl deficiency, none of whom had increased levels of N-methylglycine. In 25 patients with folate deficiency, serum betaine level was normal in most, but 76% and 60% had elevations of N,N-dimethylglycine and N-methylglycine levels that ranged as high as 343 and 43.2 mumol/L, respectively. All of seven patients on betaine therapy for inborn errors had high values for betaine (167 to 3,900 mumol/L), N,N-dimethylglycine (15.1 to 250 mumol/L), and N-methylglycine (2.93 to 49.3 mumol/L). Serum total homocysteine levels remained very high at 47.2 to 156 mumol/L (normal, 5.4 to 16.2). In patients with cbl C and cbl D mutations, methionine levels remained low or low-normal at 8.3 to 15.6 mumol/L (normal, 13.3 to 42.7) despite betaine treatment. We conclude that (1) betaine levels are maintained in most patients with Cbl and folate deficiency; (2) levels of N,N-dimethylglycine and N-methylglycine are increased in most patients with folate deficiency; and (3) betaine therapy is relatively ineffective in patients with defective synthesis of CH3-Cbl.

Metabolic abnormalities in cobalamin (vitamin B12) and folate deficiency.

Mammalian cells contain two Cbl-dependent enzymes, L-methylmalonyl-CoA mutase and methionine synthase. The former requires adenosyl-Cbl and catalyzes the conversion of L-methylmalonyl-CoA to succinyl-CoA. The latter requires CH3-Cbl and catalyzes the conversion of 5-CH3-tetrahydrofolate and homocysteine to tetrahydrofolate and methionine, respectively. Biochemical abnormalities related to a decrease in the activity of methionine synthase are thought to be responsible for the indistinguishable hematologic abnormalities seen in both Cbl and folate deficiency. The biochemical basis for the neuropsychiatric abnormalities seen in Cbl deficiency, but not in folate deficiency, is not known although hypotheses have been proposed that implicate one or the other of the two Cbl-dependent enzymes. Recent studies have shown that levels of serum methylmalonic acid, 2-methylcitric acids I and II, total homocysteine, and cystathionine are elevated in most patients with Cbl deficiency and that total homocysteine, cystathionine, N,N-dimethylglycine, and N-methylglycine are elevated in most patients with folate deficiency. Analysis of these metabolic abnormalities in various patient groups fails to support hypotheses that either L-methylmalonyl-CoA mutase or methionine synthase alone are responsible for the neuropsychiatric abnormalities. We suggest that they may result from a third, unknown mammalian Cbl-dependent enzyme or from a combined deficiency of both Cbl-dependent enzymes together with an unknown genetic or environmental factor.

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.

Elevation of serum cystathionine levels in patients with cobalamin and folate deficiency.

Homocysteine can be methylated to form methionine by the cobalamin- (Cbl) and folate-dependent enzyme, methionine synthase; serum levels of total homocysteine are elevated in greater than 95% of patients with either Cbl or folate deficiency. Homocysteine can also condense with serine to form cystathionine in a pyridoxal phosphate-dependent reaction catalyzed by cystathionine beta-synthase. Cystathionine is subsequently cleaved to cysteine and alpha-ketobutyrate by the pyridoxal phosphate-dependent enzyme gamma-cystathionase. To assess levels of cystathionine in Cbl and folate deficiency, we developed a new capillary gas chromatographic-mass spectrometric assay and measured cystathionine in the serum of normal subjects and patients with clinically confirmed deficiencies of these vitamins. The normal range for serum cystathionine was 65 to 301 nmol/L (median = 126 nmol/L) for 50 normal blood donors. In 30 patients with clinically confirmed Cbl deficiency, values for cystathionine ranged from 208 nmol/L to 2,920 nmol/L (median = 816 nmol/L) and 26 (87%) had levels above the normal range. In 20 patients with clinically confirmed folate deficiency, cystathionine concentrations ranged from 138 nmol/L to 4,150 nmol/L (median = 1,560 nmol/L) and 19 (95%) had values above the normal range. Five homozygotes for cystathionine beta-synthase deficiency had high values for serum-total homocysteine and low or low-normal values for serum cystathionine that ranged from 30 nmol/L to 114 nmol/L even though they were on treatment with pyridoxine and had partially responded. One patient with a defect in the synthesis of 5-CH3-tetrahydrofolate and five patients with defects in the synthesis of CH3-Cbl had high values for serum-total homocysteine and high values for cystathionine that ranged from 311 nmol/L to 1,500 nmol/L even though they were on treatment with folic acid and Cbl, respectively, and had partially responded. We conclude that levels of cystathionine are evaluated in the serum of most patients with Cbl and folate deficiency and that they are useful in the differential diagnosis of an elevated serum-total homocysteine level.

High prevalence of cobalamin deficiency in elderly outpatients.

OBJECTIVE: To measure the prevalence of cobalamin (vitamin B12) deficiency in geriatric outpatients as documented by both low serum cobalamin levels and elevations of serum methylmalonic acid and homocysteine and to determine the response to cobalamin treatment. DESIGN: Prospective study screening elderly subjects for cobalamin deficiency using radiodilution cobalamin assays as well as stable isotope dilution gas chromatography-mass spectrometry methylmalonic acid and homocysteine assays. In patients with serum cobalamin levels < or = 300 pg/mL, the response to cobalamin treatment in the group with levels of methylmalonic acid and/or homocysteine > 3 standard deviations (SD) above the mean for normals was compared with that of those without such elevations. SETTING: Outpatient geriatric clinics at the VA Medical Center and University Health Sciences Center, Denver, CO. PATIENTS: One-hundred and fifty-two consecutive outpatients, ages 65 to 99, were screened. Twenty-nine subjects with serum cobalamin levels < or = 300 pg/mL were prospectively evaluated and treated with cobalamin. MAIN OUTCOME MEASURES: Cobalamin, methylmalonic acid, homocysteine, complete blood counts, neurologic examination, and neuropsychological testing. RESULTS: The prevalence of cobalamin deficiency as defined by a serum cobalamin level < or = 300 pg/mL and levels of serum methylmalonic acid and/or homocysteine elevated to > 3 SD was 14.5% of the screened outpatients. A similar proportion of patients with low normal serum cobalamin levels (between 201 and 300 pg/mL) demonstrated elevated metabolites > 3 SD (56%) compared with patients with low serum cobalamin levels (< or = 200 pg/mL) (62%). Cobalamin therapy caused a marked fall or complete correction of the elevated methylmalonic acid and homocysteine levels in each patient who was treated prospectively. Results for complete blood count, lactate dehydrogenase, bilirubin, baseline neurologic score, and baseline neuropsychologic scores did not differ in the group of patients with elevated metabolites compared with those with normal metabolites. The mean red cell volume fell significantly in the patients with elevated metabolites after 6 months of cobalamin treatment. One patient with elevated metabolites had marked improvement in his neurologic abnormalities after 6 months of cobalamin treatment. CONCLUSION: There was a high (14.5%) prevalence of cobalamin deficiency as demonstrated by elevations in serum methylmalonic acid and homocysteine in addition to low or low normal serum cobalamin levels in elderly outpatients. The serum cobalamin level was insensitive for screening since similar numbers of patients with low normal serum cobalamin levels of 201-300 pg/mL compared with patients with low cobalamin levels (< or = 200 pg/mL) had markedly elevated metabolites which fell with cobalamin treatment. Additional studies will be required to define the full clinical benefit from treatment with Cbl in elderly subjects.

Association of infection due to Helicobacter pylori with specific upper gastrointestinal pathology.

The association of infection with Helicobacter pylori and antral (type B) gastritis now is clear, and the development of sensitive and specific serologic assays for IgA and IgG allows for diagnosis of this infection by noninvasive means. With use of these assays, we studied the association of infection with H. pylori and four other upper gastrointestinal inflammatory conditions: Barrett's esophagus, pernicious anemia (which accompanies type A gastritis), and duodenal and gastric ulcers. H. pylori was present in only 39% of 41 patients with Barrett's esophagus whose gastric biopsy specimens were examined histologically. Each serologic assay correctly categorized 39 (95.1%) of the 41 patients. For both assays the frequency of seropositivity noted for 58 patients with Barrett's esophagus was not different from that noted for age- and sex-matched healthy controls. Among 40 patients with pernicious anemia, the results of assays for IgA and IgG were positive for 17.5% and 0%, respectively; these prevalences were significantly less than the 50% (IgA) and 40% (IgG) positivities noted for matched controls (P less than .01 for each; McNemar's test). Among 57 patients with documented duodenal or gastric ulcers, the results of assays for IgG and IgA were positive for 100% and 98.2%, respectively; these prevalences were significantly higher than the rate noted for matched controls (P less than .001 for duodenal ulcers and P = .02 for gastric ulcers for IgA assay). These data suggest that infection with H. pylori is strongly associated with duodenal and gastric ulcers, negatively associated with pernicious anemia, and independent of Barrett's esophagus.

Failure to detect beta-leucine in human blood or leucine 2,3-aminomutase in rat liver using capillary gas chromatography-mass spectrometry.

It has been reported (Poston, J. (1976) J. Biol. Chem. 251, 1859-1863; (1982) 255, 10067-10072; (1984) 259, 2059-2061) that mammalian tissues contain an adenosylcobalamin-dependent enzyme, leucine 2,3-aminomutase, which catalyzes the interconversion of beta-leucine and leucine. It was also reported that beta-leucine is detectable in normal human serum (mean = 4.8 mumol/liter, n = 37) and is elevated in serum from patients with cobalamin deficiency (mean = 24.7 mumol/liter, n = 17). Serum levels of leucine were claimed to be decreased in the cobalamin deficient patients (mean = 52 mumol/liter) as compared with the normal subjects (mean = 81 mumol/liter). It was also reported that rat liver supernatant catalyzed the formation of beta-leucine, leucine, or both amino acids from iso-fatty acids, and that the generation of leucine from iso-fatty acids was stimulated by adenosylcobalamin and inhibited by unsaturated cobalamin-binding protein. We have synthesized t-butyldimethylsilyl derivatives of beta-leucine and leucine and have used capillary gas chromatography-mass spectrometry for their analysis. Using forms of beta-leucine and leucine that contain several deuterium atoms in place of several hydrogen atoms as internal standards, techniques have been developed which make it possible to detect and quantitate as little as 0.1 mumol/liter of beta-leucine or leucine in human serum and in incubations containing rat liver supernatant. beta-Leucine was not detectable, i.e. less than 0.1 mumol/liter, in any sera from 50 normal human subjects or in any sera from 50 cobalamin-deficient patients. The mean level of leucine in the 50 cobalamin-deficient sera was 219 mumol/liter, which was not decreased with respect to that in the 50 control sera (167 mumol/liter). Experiments in which beta-leucine, leucine, isostearic acid, or isocaproic acid were incubated with rat liver supernatant in the presence or absence of adenosylcobalamin or cobalamin-binding protein failed to demonstrate the formation of leucine or beta-leucine or their interconversion under any of the conditions studied. We conclude that beta-leucine is not present in human blood and that the existence of leucine 2,3-aminomutase in mammalian tissues remains to be established.

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.

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

The adenovirus DNA binding protein and adenovirus DNA polymerase interact to catalyze elongation of primed DNA templates.

The adenovirus-encoded 140-kDa DNA polymerase (Ad Pol) and the 59-kDa DNA binding protein (Ad DBP) are both required for the replication of viral DNA in vivo and in vitro. Previous studies demonstrated that, when poly(dT).oligo(dA) was used as a template-primer, both proteins were required for poly(dA) synthesis. In this report, the interaction between the Ad Pol and Ad DBP was further investigated using poly(dT).oligo(dA) as well as a linear duplex molecule containing 3' poly(dT) tails. DNA synthesis with the tailed template required Ad Pol, Ad DBP, and an oligo(dA) primer hydrogen bonded to the poly(dT) tails. Incorporation was stimulated 8-10-fold by ATP; however, no evidence of ATP hydrolysis to ADP was observed. Synthesis was initiated at either end of the tailed molecule and proceeded through the duplex region to the end of the molecule. This ability to translocate through duplex DNA and to synthesize long poly(dA) chains suggests that the Ad Pol.Ad DBP complex can act efficiently in the elongation reactions involved in the replication of Ad DNA (both type I and type II). During the replication reaction, substantial hydrolysis of deoxynucleoside triphosphates to the corresponding deoxynucleoside monophosphates occurred. This reaction required DNA synthesis and most likely reflects an idling reaction similar to that observed with other DNA polymerases containing 3'----5' exonuclease activity in which the polymerase first incorporates and then hydrolyzes a dNMP.

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.

Expression in Escherichia coli of a fusion protein product containing a region of the adenovirus DNA polymerase.

The bulk of an open reading frame extending from map coordinates 23.3 to 14.2 in region E2b of the adenoviral genome has been cloned and expressed from a chimeric plasmid in Escherichia coli. The cloning strategy used created a fusion protein of 124,000 daltons, which contained greater than 98% adenovirus-encoded sequences. Antiserum raised against this protein reacted with the authentic 140,000-dalton adenovirus DNA polymerase. Another serum raised against a synthetic hexapeptide whose sequence corresponded to the predicted carboxyl terminus of adenovirus-encoded DNA polymerase also reacted with the fusion protein and authentic adenovirus DNA polymerase. These results demonstrate that the cloned region of DNA encodes the adenovirus DNA polymerase.

Race, poverty, and survival in multiple myeloma.

To identify possible interracial differences in the behavior of multiple myeloma, the records of 52 black myeloma patients at Harlem Hospital Center (HHC) and 46 black and 46 white patients at Columbia-Presbyterian Medical Center (CPMC) were reviewed. In addition to clinical variables such as tumor burden, azotemia, and hypoalbuminemia, the effect of poverty on prognosis was examined, using socioeconomic indices from the United States census block group data of each patient. The median survival of CPMC black and white patients was comparable (34 and 29 months, respectively) whereas that of the HHC group was 12 months (Breslow test, P less than 0.0001). Overcrowding and hypoalbuminemia were the most significant prognostic factors by multivariate regression analysis on all 144 patients (P = 0.001); for HHC patients, overcrowding was the single significant variable affecting survival (P = 0.004). By all socioeconomic indices, HHC patients were more impoverished than CPMC patients (P less than 0.001); they also presented with more advanced disease. Race is not a significant prognostic factor in myeloma, whereas the effect of socioeconomic status on survival appears to equal that of previously described clinical features.

Protein-primed replication of plasmids containing the terminus of the adenovirus genome. I. Characterization of an in vitro DNA replication system dependent on adenoviral DNA sequences.

An in vitro system which replicates plasmid DNA containing the replication origin of adenovirus DNA has been established. Replication of plasmid pLA1 DNA, which contains the left-hand terminus (0-9.4 map units) of adenovirus serotype 5 DNA but which lacks the 55,000-dalton terminal protein, is initiated by a protein-primed mechanism in a manner similar to that found with adenovirus DNA. Initiation of DNA replication using plasmid pLA1 as a template requires (i) that the cloned adenovirus sequence be present at the terminus of a linearized (form III) DNA molecule ( Tamanoi , F., and Stillman , B. W. (1982) Proc. Natl. Acad. Sci. U. S. A., 79, 2221-2225; van Bergen, B. G. M., van der Ley , P. A., van Driel , W., van Mansfield , A. D. M., and van der Vliet , P. A. (1983) Nucleic Acid Res. 11, 1975-1979), and (ii) the presence of the 80,000-dalton precursor to the 55,000-dalton terminal protein and the adenovirus coded DNA-dependent DNA polymerase. In the presence of the four deoxy-nucleoside triphosphates, the preterminal protein, the adenovirus coded DNA binding protein, and an extract prepared from uninfected HeLa nuclei, the adenovirus DNA polymerase can elongate the preterminal-protein dCMP initiation complex formed on pLA1 DNA to full length (6.6 kilobase) DNA molecules. These results suggest that the 55,000-dalton terminal protein covalently linked to the 5' termini of adenovirus DNA is not essential for the replication of this DNA.