Functional cobalamin (vitamin B12) deficiency: role of advanced age and disorders associated with increased oxidative stress.

BACKGROUND/OBJECTIVE: Functional cobalamin (Cbl; vitamin B12) deficiency (that is, high levels of the Cbl-dependent metabolites, methylmalonic acid (MMA) and homocysteine (HCys), despite normal serum Cbl values) is common in the elderly and is associated with neurocognitive abnormalities, but its cause is unknown. As only reduced Cbls are metabolically active, the possibility that functional Cbl deficiency is associated with disorders having biomarkers indicative of increased oxidative stress (oxidant risks) was considered. SUBJECTS/METHODS: A retrospective record review of community-dwelling adults evaluated over a 12-year period for Cbl deficiency in a primary care setting who had serum Cbl values ⩾400 pg/ml (n=170). RESULTS: When no oxidant risks were present, older subjects (⩾70 years) had higher metabolite values than younger individuals (<70 years). MMA values were even higher in the elderly when one oxidant risk was present and in younger subjects when two or more oxidant risks were present. Even at Cbl levels ⩾800 pg/ml, MMA values were increased in 73% of elderly subjects with at least one oxidant risk. HCys values were also higher in both age groups when at least two oxidant risks were present. Cyanocobalamin therapy decreased MMA and HCys values in 86 and 76% of subjects, respectively, with nonresponders more likely to have two or more oxidant risks. CONCLUSION: Functional Cbl deficiency is associated with disorders marked by increased oxidative stress particularly in the elderly; it occurs even when Cbl levels are high and is not consistently corrected with high-dose cyanocobalamin therapy. Thus, current approaches to recognizing and managing this disorder may be inadequate.

Proteinuria as a predictor of renal functional outcome after revascularization in atherosclerotic renovascular disease (ARVD).

BACKGROUND: Renal revascularization is performed in 16% of newly diagnosed patients with atherosclerotic renovascular disease (ARVD). Although there may be some improvement in hypertension control as a result of intervention, renal functional outcomes are known to vary. Pre-existing renal parenchymal injury, as manifested by proteinuria, is associated with poor functional outcome in conservatively managed ARVD patients, but this association has not been investigated in patients undergoing revascularization. METHODS: Retrospective case note review of 83 ARVD patients who underwent renal revascularization in four centres within a renal network between 1998 and 2003 was undertaken. Amongst other parameters, baseline proteinuria was correlated with renal functional outcome post revascularization. Renal functional outcome was determined over a mean follow up of 22 months by rate of change of estimated glomerular filtration rate (eGFR) over time. RESULTS: Univariate analysis showed that proteinuria >0.6 g/day was the only significant predictor of poor outcome after revascularization. The relationship persisted with multivariate analysis, and linear regression showed a correlation between baseline proteinuria and decline in eGFR with time (r(2) = 0.058, P = 0.039). CONCLUSION: This study confirms that prior renal parenchymal injury, here reflected by proteinuria at baseline, is a major arbiter of renal functional outcome after renal revascularization in ARVD.

Oral pharmacologic doses of cobalamin may not be as effective as parenteral cobalamin therapy in reversing hyperhomocystinemia and methylmalonic acidemia in apparently normal subjects.

A postmenopausal female evaluated for thrombophilia because of bone infarcts had mild hyperhomocysteinemia, which increased when hormone replacement was discontinued. Serum folate, cobalamin and methylmalonic acid were normal. Compound heterozygosity for C677T/A1298C methylenetetrahydrofolate reductase polymorphisms was present but oral folic acid failed to lower homocysteine and actually increased methylmalonic acid. Oral cobalamin therapy increased serum cobalamin and partially decreased methylmalonic acid but had no effect on homocysteine. Homocysteine remained unchanged after 11 months of oral cobalamin, folic acid and pyridoxine therapy. However, intramuscular cobalamin promptly decreased both metabolites to normal. Thus, parenteral cobalamin therapy may have greater metabolic effects than oral vitamin therapy even in apparently normal subjects.

Urinary C5b-9 excretion and clinical course in idiopathic human membranous nephropathy.

Recent reports suggested that the presence of terminal complement complex (C5b-9) in urine from patients with idiopathic membranous nephropathy (IMN) may indicate on-going immunological damage. This report documents the relationship between C5b-9 excretion and clinical outcome in 35 adult patients with biopsy-proven IMN and progressively declining renal function. There were two groups of patients. Group I received one of three treatment regimens: prednisolone alone, prednisolone and chlorambucil, or prednisolone and cyclophosphamide (N = 22). Group II received no immunosuppressive therapy (N = 17). Three of the 18 patients receiving immunosuppressive drugs had more than one treatment regimen as they experienced a clinical relapse during the study period; hence 22 treatments were available for analysis. Urine samples were collected regularly and urinary C5b-9 (uC5b-9) was determined by ELISA. Both groups were similar with respect to age, sex distribution, and the duration of follow-up. An improvement in proteinuria and creatinine clearance was noted in the immunosuppressed group. Thirty-five patients were excreting C5b-9 initially (18 from group I and 17 from group II); 17 patients continued to excrete C5b-9 at the end of the observation period. These 17 patients had a significantly worse clinical outcome when compared to the 18 patients whose C5b-9 excretion became negative, either spontaneously or with treatment (P < 0.005). These results indicate that continuing C5b-9 excretion is correlated with a poor clinical outcome. They also suggest that uC5b-9 is a dynamic marker of ongoing immunological injury, and therefore may be useful in the initial assessment and monitoring of patients with IMN and in identifying patients who may derive benefit from immunosuppressive therapy.

Donor screening for antibody to hepatitis B core antigen and hepatitis B virus infection in transfusion recipients.

BACKGROUND: Testing for antibody to hepatitis B core antigen (anti-HBc) as a surrogate for hepatitis C viremia is no longer needed for blood donor screening. Currently, the important question is how much its use supplements hepatitis B surface antigen (HBsAg) donor screening in preventing transfusion-transmitted hepatitis B virus (HBV) infection. STUDY DESIGN AND METHODS: In a study conducted in the 1970s, 64 blood donors were associated with 15 cases of HBV (1.0%) in 1533 transfusion recipients. Sera from 61 donors at donation and 29 follow-up visits were available for present-day assays for HBsAg, HBV DNA, anti-HBc, and antibody to HBsAg (anti-HBs). RESULTS: HBsAg was found in four previously negative blood donors; HBV DNA was limited to three of these four. Anti-HBc was detected in six HBsAg-negative donors. Two other donors were negative in all assays at donation, but positive for anti-HBc and anti-HBs 2 to 4 months later. The remaining donors were negative for all HBV markers, which left five recipient cases unexplained. No HBV transmission was observed when anti-HBs sample-to-negative control values were > or = 10. CONCLUSION: Some 33 to 50 percent of cases of hepatitis B that could be transmitted by transfusion of blood from HBsAg-negative donors are prevented by anti-HBc screening. Anti-HBc-positive donors unequivocally positive for anti-HBs should be considered noninfectious for HBV and should be allowed to donate. Anti-HBc screening of paid plasmapheresis donors, supplemented by anti-HBs testing, would reduce the amount of HBV to be processed by virus inactivation and increase the content of anti-HBs in plasma pools.

Unique preS sequence in a gibbon-derived hepatitis B virus variant.

A unique hepatitis B virus (HBV) variant has been identified in a gibbon (Hylobates lar) which could be passed to a chimpanzee by experimental inoculation. This HBV variant had been shown to have no reactivity to a monoclonal anti-preS2 antibody (preS2 mAb 116-34) differentiating it from all human HBV specimens tested. This gibbon sera also was not recognized by an anti-preS1 mAb which binds the preS1 hepatocyte receptor region, amino acids 27-35. In this paper, we report the DNA sequence of the gibbon HBV PreS gene. The lack of preS2 mAb (116-34) binding can be explained by a unique nucleotide substitution of A for C in the second codon of the preS2 region leading to the replacement of glutamine with lysine. Two other unique changes were observed at the seventh and 24th amino acid positions in the preS2 gene leading to a substitution of a valine for threonine and alanine, respectively. Unlike all human derived HBV sequences in the preS1 region, the gibbon HBV had a glutamic acid instead of an aspartic acid at amino acid residue 27. Another unique substitution was a leucine for alanine at preS1 position 33. These amino acid changes in the gibbon HBV may explain its unique preS mAb reactivity.

Morphologic, biochemical, and cytogenetic studies of bone marrow and circulating blood cells in painters exposed to ethylene glycol ethers.

In a previous cross-sectional survey, up to 15% of shipyard painters were found to have mild anemia or granulocytopenia, mostly acquired since employment. Environmental studies had suggested a possible etiologic role for ethylene glycol ethers, solvents to which the men were heavily exposed and which have established myelotoxic potential. To exclude alternative hypotheses, examine possible common patterns of injury, and identify potential risk factors and markers for such an effect, the affected painters were further studied. The painters were matched with two groups of controls: exposed painters without evidence of hematologic abnormality on the previous survey and unexposed controls. Altogether 25 subjects were studied by histopathologic examination of bone marrow, cytogenetic studies of marrow cells, and peripheral lymphocytes and peripheral red cell studies of membrane and metabolic function. Except for an unexpected finding of a race-associated effect on marrow histology, insignificant differences were seen among the groups in terms of marrow morphology and cellularity, stem cell growth kinetics, and marrow or peripheral cytogenetics. Two metabolic abnormalities of peripheral red cells related to exposure or clinical status of the subjects were found. Pyruvate kinase, an established marker of acquired myelodysplasia, was significantly depressed in the subjects with previously abnormal counts. Although reduced glutathione levels and holoenzyme activities of glutathione reductase (GSHR) did not differ among groups, exposed subjects had decreased saturation of GSHR with flavin adenine dinucleotide which could be restored in vitro, suggesting riboflavin deficiency or impaired riboflavin metabolism. Thus, although a unique pattern of bone marrow injury by histologic or genetic assay attributable to ethylene glycol ethers was not defined, biochemical effects of possible mechanistic importance were identified. The relevance of these findings as subclinical disease markers remains to be established.

Enzymatic syntheses of DNA-silicas using DNA polymerase.

Oligothymidylic acids couple to an activated ester silica (N-hydroxysuccinimidyl-silica) only when they contain an added aminoalkyl group. Heteropolymeric oligomers containing other nucleotide bases were shown to also couple by way of the nucleotide base (adenine, cytosine, or guanine); however, when a heteropolymeric oligonucleotide also contains a 5'-aminoalkyl moiety, coupling by way of the latter is the favored reaction. When duplex hybrids of oligonucleotides are formed, the nucleotide bases are protected from chemical coupling. Coupling by way of nucleotide bases would be detrimental to some chromatography experiments. On the basis of these observations, two different procedures were developed to produce DNA-silicas in which a single strand of the DNA is coupled by only its 5'-terminus. In the first of these, the polymerase chain reaction was used with a 5'-aminoalkyl primer to make a duplex DNA with one strand containing the 5'-aminoalkyl group and the duplex DNA is then coupled to the activated ester silica. This yielded a silica containing about 0.17 nmol of a 242-mer per gram silica which bound only probes specific for the coupled strand. In the other procedure, a template DNA strand was poly(A) tailed and hybridized to (dT)18-silica. DNA polymerase I (Klenow large fragments) was then used to copy the template-specified sequence directly onto the 3'-terminus of the (dT)18. This procedure yielded about 1.2 to 2.7 nmol DNA copied/g of silica of a specific 21-mer sequence. The DNA-silica produced selectively hybridized only with complementary sequences and not with DNA lacking that sequence. Either of these procedures thus produces DNA-silicas from heteropolymeric DNA sequences with a predetermined, specific 5'-terminal site of attachment.

Effect of xamoterol in a patient with idiopathic autonomic failure.

We report the effect of xamoterol on autonomic, humoral and haemodynamic function in a patient with idiopathic autonomic neuropathy. Xamoterol produced marked symptomatic improvement, slightly increased heart rates in the sitting, supine and upright tilt positions and higher blood pressures while seated and on recovery from upright tilt. There were, however, no differences in blood pressure responses following a meal or during head-up tilt. We suggest that before ascribing symptomatic benefit to xamoterol in this condition, objective haemodynamic improvement should be recorded. Further studies of treatment in idiopathic autonomic failure require to be placebo controlled.

delta-Aminolevulinic acid dehydratase in rat liver: studies on the effects of ethanol, acetaldehyde, and B6 vitamers.

Because ethanol ingestion lowers delta-aminolevulinic acid dehydratase (ALAD) activity in liver and red cells, effects of ethanol and acetaldehyde on ALAD in rat liver cytosol were studied. When added to the assay mix, as little as 0.5 mmol/L acetaldehyde competitively inhibited ALAD even in the presence of dithiothreitol, a sulfhydryl reagent. ALAD activity also fell when undiluted cytosol was incubated at 37 degrees with as little as 0.25 mmol/L acetaldehyde for 8 hours before enzyme assay. Inactivation of ALAD by acetaldehyde was prevented by the metabolic inhibitor NaF but not by the aldehyde dehydrogenase inhibitor cyanamide. Incubation of undiluted cytosol with 20 mmol/L ethanol also decreased ALAD activity, but addition of ethanol to the assay mix had no effect. Ethanol-mediated inactivation of ALAD was reduced by inhibition of alcohol dehydrogenase with 4-methylpyrazole, but ALAD activity was not decreased by incubation of undiluted cytosol with acetate or sorbitol or by addition of acetate to the assay mix. The aldehydic B6 vitamers, pyridoxal and pyridoxal phosphate, also inhibited ALAD activity when added to the assay mix. However, these vitamers increased ALAD activity and decreased acetaldehyde-mediated inactivation of ALAD when incubated for 8 hours with undiluted cytosol. We conclude that (1) acetaldehyde decreases ALAD activity both by competitive inhibition with substrate and by inactivation of enzyme protein and that (2) inactivation of ALAD by acetaldehyde may require nonoxidative metabolism of acetaldehyde. The net pharmacologic effect of B6 vitamers on ALAD activity and on inactivation of ALAD by acetaldehyde remains to be determined.

Prospective controlled study of androgen effects on red cell oxygen transport and work capacity in chronic hemodialysis patients.

Effects of androgens on red blood cell (RBC) oxygen transport, RBC metabolism and work capacity in chronic hemodialysis patients were evaluated in a prospective controlled study. Compared to control subjects, patients given nandrolone decanoate had a sustained fall in RBC ATP and a transient rise in RBC DPG but P50 values were unchanged. Despite increases in RBC mass of 16-44% on androgen therapy, exercise on the treadmill improved in only 1 patient and actually declined in 3 others. Thus, these preliminary observations suggest that androgens may not improve hemoglobin-oxygen transport and that androgen-induced increases in red cell mass may only balance increased tissue oxygen requirements produced by these anabolic hormones.

Hypernatraemia in the elderly patient.

The elderly are at risk of developing hypernatraemia because of age-related changes in renal function and body composition. The pathophysiology and aetiology of hypernatraemia are reviewed with emphasis on iatrogenic factors. A rational approach to management is discussed and clear guidelines for treatment described.

Studies on the mechanism of acetaldehyde-mediated inhibition of aspartate aminotransferase (GOT) in human erythrocytes.

The mechanism of acetaldehyde-mediated GOT inhibition was studied in human red cells. In the GOT assay mix, acetaldehyde competitively inhibits activation of apoGOT by pyridoxal phosphate and pyridoxamine phosphate. However, Ki values are 100-1000 times greater than Km values for these B6 vitamers. Moreover, incubation of undiluted lysates with acetaldehyde at 37 degrees inhibits GOT activity without increasing apoGOT levels and without altering affinity of apoGOT for either B6 coenzyme. In undiluted lysates, inhibition is not prevented by disulfiram. However, incubation at 4 degrees prevents both acetaldehyde metabolism and GOT inhibition while preincubation with NaF prevents GOT inhibition without affecting acetaldehyde disappearance. The effect of NaF is completely reversed by pyruvate but only partially reversed by NADH. Glyceraldehyde 3-phosphate, the only glycolytic intermediate which directly inhibits GOT, does not reverse the NaF effect. Thus, inhibition of GOT by acetaldehyde (a) requires nonoxidative metabolism of acetaldehyde and (b) is not mediated either by glycolytic substrates or by impaired binding of B6 vitamers to the GOT apoenzyme. Since NaF had no effect on a lysate deficient in glucose 6-phosphate dehydrogenase, the hexose monophosphate shunt may play a role in acetaldehyde-mediated GOT inhibition.

Bleomycin-iron can degrade DNA in the presence of excess ethylenediaminetetraacetic acid in vitro.

The antineoplastic drug bleomycin, when complexed to Fe(II), causes both single- and double-stranded lesions in DNA in vitro. EDTA is commonly used to inhibit the reaction of bleomycin-Fe with DNA, presumably by removing the metal cofactor. In this study, we utilized a simple assay involving the conversion of supercoiled plasmid DNA to the nicked or linear forms to further investigate the ability of bleomycin-Fe to degrade DNA in the presence of EDTA. We found that a 1:1 complex of bleomycin and Fe can degrade plasmid DNA even in the presence of a 10(6) molar excess of EDTA over bleomycin. Furthermore, we found that the half-life for inactivation of bleomycin-Fe by excess EDTA is about 1.5 h. Finally, we demonstrate that excess bleomycin associated with the outer plasma membranes of cells can damage DNA after the cells are lysed in buffers containing EDTA and SDS. These results suggest that EDTA may not be an efficient inhibitor of the reaction of bleomycin-Fe with DNA.

Loss of proliferative potential during terminal differentiation coincides with the decreased abundance of a subset of heterogeneous ribonuclear proteins.

The decrease in abundance of a subset of highly conserved basic nuclear proteins is established to correlate with the loss of proliferative potential in association with the process of terminal differentiation in murine mesenchymal stem cells and human keratinocytes. These proteins, designated P2Ps for proliferation potential proteins, have apparent molecular masses of 30-40 kD, are associated with the 30-40S substructures of nuclear hnRNP complexes, and are recognized by antibodies made against core proteins of hnRNP particles. They also share an epitope in common with heat shock protein-90 (hsp90) and are recognized by two mAbs against hsp90. Two-dimensional electrophoretic Western blots furthermore show that P2Ps make up a subset of hnRNP proteins. Cells that possess these proteins express the potential to proliferate whether or not they are traversing the cell cycle. These include rapidly growing cells, reversibly growth-arrested cells, and nonterminally differentiated cells. In contrast, cells that have irreversibly lost their proliferative potential, such as terminally differentiated cells, show a marked reduction in the abundance of P2Ps as determined by immunodetection on Western blots. A correlation, therefore, exists between the presence of this subset of nuclear proteins and the proliferative potential in two cell types. These results raise the possibility that as a subset of hnRNP proteins, P2Ps may mediate posttranscriptional control of the processing of specific RNAs required for cell proliferation.

Inhibition of rat liver transaminases by low levels of acetaldehyde and the pharmacologic effects of B6 vitamers.

To better define the significance and mechanism of acetaldehyde-mediated transaminase inhibition, acetaldehyde metabolism was studied in rat liver homogenates and cytosols. When either preparation was incubated at 37 degrees with 1.5 mM acetaldehyde for 4 hr, acetaldehyde levels fell rapidly in the first 30 min and little inhibition of aspartate aminotransferase (GOT) or alanine aminotransferase (GPT) resulted. In contrast, incubation with 50 mM ethanol also resulted in a peak acetaldehyde level of 1.0 to 1.5 mM by 2 hr, but this level was then maintained for the next 2 hr and transaminases were inhibited by 20-35%. Sequential addition of low dose (125-250 microM) pulses of acetaldehyde to rat liver preparations resulted in a progressive decrease in the rate of acetaldehyde disappearance. When the pulsing schedule was adjusted accordingly to maintain acetaldehyde levels between 50 and 250 microM for 8 hr, transaminases were again inhibited by 20-40%. Finally, addition of 1-5 mM pyridoxal and pyridoxal 5'-phosphate, aldehydic B6 vitamers, to cytosols 2-4 hr after pulsing with acetaldehyde was begun, almost completely prevented further transaminase inhibition. In contrast, the non-aldehydic B6 vitamers, pyridoxine, pyridoxamine and pyridoxamine 5'-phosphate, did not affect acetaldehyde-mediated transaminase inhibition. These findings suggest that (1) prolonged exposure to low levels of acetaldehyde impairs acetaldehyde metabolism in rat liver homogenates and cytosols; (2) acetaldehyde toxicity may be more dependent on sustained exposure to acetaldehyde than on the peak level of acetaldehyde attained; and (3) aldehydic B6 vitamers can modify on-going acetaldehyde-mediated transaminase inhibition.