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D W Jacobsen

Publications and source records attributed to D W Jacobsen.

At least 19 recordsLinked to original sources
PubMed1995

The dynamics of cobalamin utilization in L-1210 mouse leukemia cells: a model of cellular cobalamin metabolism.

The uptake and metabolism of cobalamin (Cbl) has been studied in L-1210 murine leukemia cells propagating in vitro. Extracellular Cbl (protein bound and free) and intracellular Cbl (protein bound and free) were determined after culturing L-1210 cells in the presence of [57Co]cyanocobalamin (CN-Cbl) bound to transcobalamin II (transcobalamin, TC). The intracellular pool of free [57Co]Cbl increased during the first 24 h of culture and a substantial fraction of this free pool was effluxed from the cell to the medium. Upon depletion of extracellular TC-[57Co]CN-Cbl, the intracellular concentration of free Cbl decreased as did the efflux of Cbl to the medium. Internalized [57Co]CN-Cbl was converted to hydroxocobalamin (OH-Cbl), methylcobalamin (Me-Cbl) and 5'-deoxyadenosylcobalamin. These Cbl forms were found in both soluble (cytoplasmic) and insoluble (membrane) fractions. Intracellular protein-bound [57Co]Cbl fractionated with methionine synthase (MS) and methylmalonyl-CoA mutase (MU) activity. The major form of Cbl associated with the two enzymes was OH-Cbl. Cells propagated in medium containing N5-methyltetrahydrofolate and homocysteine showed a substantial increase in MS activity which paralleled the increase in the intracellular concentration of Me-Cbl and the Cbl bound to the enzyme.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1995

Characterization of angiotensin II receptor subtypes in pancreatic acinar AR42J cells.

The AR42J acinar cell line was characterized as a potential cellular model to assess the functional aspects of an exocrine pancreatic angiotensin system. Binding studies revealed that the AR42J cells express high affinity angiotensin II binding sites (Kd = 0.73 +/- 0.06 nM; Bmax = 292 +/- 15 fmol/mg protein, n = 3). Competition studies established that these cells, similar to the intact pancreas, express predominantly the AT2 receptor subtype. The AT2-selective antagonists CGP 42112A, PD 123177, and PD 123319 competed for the majority of angiotensin II binding. However, 10-15% of the angiotensin II binding sites were competed for by the AT1-selective antagonist DuP 753 (Losartan). Affinity labeling of these binding sites with [125I]angiotensin II followed by SDS gel electrophoresis under reducing conditions revealed a single band comprising a molecular mass of 108,000 Da. Competition with unlabeled angiotensin II or the AT2 antagonist, but not the AT1 antagonist, abolished the 108,000-Da band. In intact cells, angiotensin II caused a rapid increase in intracellular calcium (Ca2+) using Fura-2 as a Ca2+ indicator. Pretreatment of the cells with the AT1 antagonist DuP 753 completely inhibited the angiotensin II-induced rise in Ca2+; however, the AT2 antagonists CGP 42112A and PD 123177 were ineffective in blocking the Ca2+ increase. These results demonstrate that this pancreatic acinar cell line expresses both AT2 and AT1 angiotensin II receptor subtypes. The AT1 receptor is coupled to the mobilization of Ca(2+)--a characteristic shared by AT1 receptors in other tissues.

Animals↗
PubMed1995

The frequently low cobalamin levels in dementia usually signify treatable metabolic, neurologic and electrophysiologic abnormalities.

Cobalamin levels are frequently low in patients with dementia, but it is unclear if they represent definable deficiency and what the mechanisms are. Therefore, patients being evaluated for dementia who had low cobalamin levels but no obvious evidence of deficiency were studied hematologically, neurologically and with metabolic tests and were re-evaluated after cobalamin treatment. Abnormalities suggestive of or diagnostic for deficiency were documented in most of the 16 demented and nondemented patients. Metabolic results: 50% of patients tested had abnormal deoxyuridine suppression and 44% had increased serum methylmalonic acid and/or homocysteine levels; these test results correlated with each other. Neurologic results: 73% of patients had clinical abnormalities, primarily mild neuropathies, not attributable to other causes, 75% had electroencephalographic abnormalities, 77% had abnormal visual evoked potentials and 33% had abnormal somatosensory potentials. Metabolic and neurologic dysfunction were present together or absent together in all but 2 cases. Cobalamin therapy improved 50-100% of the various types of abnormalities, although it did not improve cognitive function in the 13 demented patients. Food-cobalamin malabsorption was found in 60% of the patients. Despite the absence of megaloblastic anemia and rarity of traditional malabsorption of free cobalamin, low cobalamin levels in demented patients frequently represent mild cobalamin deficiency and are often associated with food-cobalamin malabsorption. Perhaps most importantly, this is accompanied not only by metabolic changes but by evidence of mild neurologic dysfunction. Their frequent reversibility by cobalamin confirms that these defects indeed arise from cobalamin deficiency. Although the long-standing dementia does not improve, treating such patients with cobalamin has other concrete benefits.

Aged↗
PubMed1995

Determination of total serum sulfite by HPLC with fluorescence detection.

An estimated 500,000 individuals in the US, mostly steroid-dependent asthmatics, suffer severe adverse reactions to sulfites in foods, beverages, and pharmaceutical products. In an attempt to understand the pathogenesis of sulfite hypersensitivity, we have developed an assay for the determination of total serum sulfite by utilizing: (a) reductive release of serum protein-bound sulfite; (b) derivatization of free sulfite with monobromobimane; (c) separation of sulfite-bimane from thiol-bimanes by reversed-phase HPLC; and (d) quantitation of sulfite-bimane by fluorescence detection. The detection limit of this assay was 0.44 mumol/L serum sulfite. The intra- and interassay CVs for total serum sulfite at 5.4 mumol/L were 8.1% and 22.0%, respectively. The standard addition method was used to determine total serum sulfite in normal subjects. More than 70 samples were prepared in 2-3 h, followed by automated overnight analysis. The mean concentrations (+/- SD) of total serum sulfite in female (n = 41) and male (n = 35) donors were 4.63 +/- 2.33 and 5.16 +/- 2.68 mumol/L, respectively (not statistically significant: P = 0.368). The combined mean concentration of total sulfite in both sexes was 4.87 +/- 2.49 mumol/L. There was no correlation between total serum sulfite and total serum cysteine, cysteinylglycine, homocysteine, subject age, serum cobalamin, or serum folic acid. The reference range (mean +/- 2 SD) for total serum sulfite in normal subjects is 0-9.85 mumol/L.

Bridged Bicyclo Compounds↗
PubMed1994

Serum cobalamin deficiency is uncommon in multiple sclerosis.

OBJECTIVES: To determine the frequency of serum cobalamin (Cbl) deficiency and to clarify the biologic importance of low screening Cbl levels in patients with multiple sclerosis (MS) and idiopathic myelopathy (MYL). BACKGROUND: A significant association between Cbl metabolism and MS has been postulated based on the observations that patients with MS have lower serum Cbl levels, higher unsaturated Cbl binding capacities, and a higher prevalence of macrocytosis than do normal controls. Whether such observations have biologic importance as documented by abnormal accumulation of metabolites that would result from Cbl deficiency has not yet been determined. METHODS: Serum Cbl and folate levels were determined in 208 consecutively evaluated patients seen in an outpatient MS clinic setting during a 7-month period. Necessary blood samples were obtained for 165 of these patients. One hundred twenty-five patients had clinically definite MS, 31 had clinically probable MS, and nine had MYL. Serum methylmalonic acid (MMA) and homocysteine (HCY) concentrations, which rise in biologically severe Cbl deficiency, were subsequently determined in all patients whose Cbl levels were lower than 301 pg/mL. RESULTS: A Cbl level lower than 301 was found in 32 of 156 patients with either clinically definite MS or clinically probable MS but in none of the patients with MYL. Elevated MMA or HCY levels were found in seven of 32 patients with either clinically definite MS or clinically probable MS, six of whom had an elevated HCY level and one of whom had elevated HCY and MMA levels. CONCLUSIONS: Despite the observation that 32 (19.4%) of 165 of these patients with MS and MYL had screening Cbl levels less than 301 pg/mL, only seven (4.2%) of 165 had elevated MMA or HCY levels. The frequency of biologically severe Cbl deficiency in these patients with MS and MYL was very low.

Adult↗
PubMed1994

Rapid HPLC determination of total homocysteine and other thiols in serum and plasma: sex differences and correlation with cobalamin and folate concentrations in healthy subjects.

High-performance liquid chromatography with fluorescence detection has been utilized for the rapid determination of total homocysteine, cysteine, and cysteinylglycine in human serum and plasma. Our earlier procedure (Anal Biochem 1989;178:208), which used monobromobimane to specifically derivatize thiols, has been extensively modified to allow for rapid processing of samples. As a result, > 80 samples a day can be assayed for total homocysteine, cysteine, and cysteinylglycine. The method is sensitive (lower limit of detection < or = 4 pmol in the assay) and precise (intra- and interassay CV for homocysteine, 3.31% and 4.85%, respectively). Mean total homocysteine concentrations in plasma and serum were significantly different, both from healthy male donors (9.26 and 12.30 mumol/L, respectively; P < 0.001) and healthy female donors (7.85 and 10.34 mumol/L, respectively; P < 0.001). The differences in total homocysteine between sexes were also significant (P = 0.002 for both plasma and serum). Similar differences were found for cysteine and cysteinylglycine. We found a significant inverse correlation between serum cobalamin and total homocysteine in men (P = 0.0102) and women (P = 0.0174). Serum folate also inversely correlated with total homocysteine in both sexes.

Adult↗
PubMed1992

Glial cells as a model for the role of cobalamin in the nervous system: impaired synthesis of cobalamin coenzymes in cultured human astrocytes following short-term cobalamin-deprivation.

The conversion of cyanocobalamin to adenosyl- and methylcobalamin is impaired in cobalamin-deficient cultured human glial cells. In contrast cultured human skin fibroblasts retained their ability to synthesize coenzyme forms when grown in cobalamin-deficient medium. Cells were pre-conditioned by growing in cobalamin-deficient media for six weeks and then subcultured in medium containing either free or transcobalamin II-bound 57Co-cyanocobalamin. Although both coenzyme levels were low in cobalamin-deficient glial cells, the decrease in methylcobalamin was more marked than that of adenosylcobalamin. Methionine synthase and Cb1 reductase activities were markedly decreased in cobalamin-deficient glial cells but were unchanged in fibroblasts cultured in cobalamin-deficient medium. Our data suggest that in glial cells, cobalamin coenzyme synthesis and function is exquisitely sensitive to short-term cobalamin deprivation. Glial cells apparently synthesize and secrete transcobalamin II since antibodies directed against the transport protein inhibit the uptake of free cobalamin.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1992

Production of angiotensin-(1-7) by human vascular endothelium.

The heptapeptide angiotensin-(1-7) is a circulating biologically active product of the renin-angiotensin system. In this study, we evaluated the role of the vascular endothelium in the formation of angiotensin-(1-7). Metabolism of 125I-angiotensin I was investigated using confluent cultured bovine and human aortic and umbilical vein endothelial cells. The fetal calf serum-supplemented medium was replaced by serum-free medium containing 0.2% bovine serum albumin. One hour later, this medium was replaced by serum-free medium containing 125I-angiotensin I. After incubation of 125I-angiotensin I for various intervals at 37 degrees C, the medium was collected and analyzed for formed products by high-performance liquid chromatography. Products of angiotensin I metabolism were identified by comparison of their retention times with those of radiolabeled standards. The contribution of proteases released into the medium was evaluated by incubation of 125I-angiotensin I with medium previously incubated for 1 hour with endothelial cells. Incubation of 125I-angiotensin I with bovine and human endothelial cells produced a time-dependent generation of 125I-angiotensin-(1-7) greater than 125I-angiotensin II greater than 125I-angiotensin-(1-4). Generation of angiotensin peptides was not due to the presence of proteases in the medium. When human umbilical endothelial cells were incubated in the presence of the angiotensin converting enzyme inhibitor enalaprilat (1 microM), generation of angiotensin II was undetectable. In contrast, angiotensin-(1-7) production increased by an average of 30%.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin I↗
PubMed1991

Stabilization of thermally labile alkylcobalamins by a haptocorrin from chicken serum.

Binding of neopentylcobalamin and benzylcobalamin to the apoprotein of a haptocorrin from chicken serum has been demonstrated spectrophotometrically. The spectra of the protein-bound cobalamins strongly resemble those of base-on alkylcobalamins and show that when unbound these sterically hindered alkylcobalamins are only approximately 75% (benzyl) and 40% (neopentyl) base-on, at neutral pH and at 5 degrees C. The haptocorrin was found to stabilize the spontaneous thermal decomposition of the neutral species of benzylcobalamin and neopentylcobalamin by 470-fold (3.6 kcal) and 166-fold (3.0 kcal), respectively, relative to the protein-free species. After correction of the activation parameters for the thermal decomposition of the protein-free, neutral alkylcobalamins for the relative proportions of base-on and base-off species, the haptocorrin was found to stabilize the base-on species of both alkylcobalamins by 275- to 1400-fold (approximately 3.3 to 4.3 kcal). From the temperature dependence of the decomposition reactions, the enthalpies of activation are found to be essentially identical for the protein-free and protein-bound species of either cobalamin. Thus, stabilization of the thermal decomposition of these sterically hindered alkylcobalamins by haptocorrin is entirely due to entropic factors.

Animals↗
PubMed1990

Expression of transcobalamin II receptors by human leukemia K562 and HL-60 cells.

Plasma membrane receptors for the serum cobalamin-binding protein transcobalamin II (TCII) were identified on human leukemia K562 and HL-60 cells using immunoaffinity-purified human TCII labeled with [57Co]cyanocobalamin. The Bmax values for TCII receptors on proliferating K562 and HL-60 cells were 4,500 and 2,700 per cell, respectively. Corresponding dissociation constants (kd) were 8.0 x 10(-11) mol/L and 9.0 x 10(-11) mol/L. Rabbit TCII also bound to K562 and HL-60 cells but with slightly reduced affinities. Calcium was required for the binding of transcobalamin II to K562 cells. Brief treatment of these cells with trypsin resulted in almost total loss of surface binding activity. After removal of trypsin, surface receptors for TCII slowly reappeared, reaching pretrypsin treatment densities only after 24 hours. Reappearance of receptors was blocked by cycloheximide. TCII receptor densities on K562 and HL-60 cells correlated inversely with the concentration of cobalamin in the culture medium. This suggests that intracellular stores of cobalamin may affect the expression of transcobalamin receptors. Nonproliferating stationary-phase K562 cells had low TCII receptor densities (less than 1,200 receptors/cell). However, the density of TCII receptors increased substantially when cells were subcultured in fresh medium. Up-regulation of receptor expression coincided with increased 3H-thymidine incorporation, which preceded the resumption of cellular proliferation as measured by cell density. In the presence of cytosine arabinoside, which induces erythroid differentiation, K562 cells down-regulated expression of TCII receptors. When HL-60 cells were subcultured in fresh medium containing dimethysulfoxide to induce granulocytic differentiation, the up-regulation of TCII receptors was suppressed. This event occurred well before a diminution of 3H-thymidine incorporation and cessation of proliferation. Thus, changes in the regulation of expression of TCII receptors correlate with both the proliferative and differentiation status of cells.

Cell Division↗
PubMed1990

Glutathionylcobalamin as an intermediate in the formation of cobalamin coenzymes.

To evaluate the possible role of glutathionylcobalamin (GS-Cbl) in the intracellular metabolism of cobalamin, the following reactions were analyzed using extracts of rabbit spleen: (i) decyanation of cyanocobalamin; (ii) utilization of GS-Cbl by cobalamin reductase; (iii) participation of GS-Cbl in methionine biosynthesis; and (iv) conversion of GS-Cbl to adenosylcobalamin. Decyanation of cyanocobalamin required reduced glutathione which appeared to form a complex with the cobalamin. This complex decomposed during the extraction steps to sulfitocobalamin which was identified by high-performance liquid chromatography. Cobalamin reductase in spleen extract was more active with GS-Cbl than with aquocobalamin or cyanocobalamin as substrates (specific activities: 10.4, 2.8 and 0.93 nmol/mg/min, respectively). Methionine synthase utilized GS-Cbl as cofactor more efficiently than aquocobalamin or cyanocobalamin based on initial rates of enzyme activity. This suggests that GS-Cbl is a more direct precursor of the coenzyme required for methionine synthase. Formation of adenosylcobalaminm from GS-Cb1 was four times greater than from aquocobalamin alone. Based on these results, we propose that GS-Cbl or a closely related thiol-cobalamin adduct is a proximal precursor in cobalamin coenzyme biosynthesis.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1989

Determination of plasma homocysteine by high-performance liquid chromatography with fluorescence detection.

Severe homocystinemia is frequently associated with vascular disease while the pathological consequences of moderate or slightly elevated plasma homocysteine are unknown. Cobalamin and folate deficiencies may result in an elevation of plasma homocysteine. A sensitive and reproducible assay for total plasma homocysteine has been developed. The essential steps in the assay include (i) conversion of homocysteine disulfides to free homocysteine with borohydride reduction; (ii) conjugation of homocysteine with monobromobimane; (iii) separation of homocysteine-bimane from other plasma thiol-bimane adducts by reverse-phase high-performance liquid chromatography; and (iv) detection and quantitation of homocysteine-bimane by fluorometry. The method has a sensitivity of 4.4 pmol of homocysteine and is highly reproducible (intra- and interassay coefficients of variation = 4.97 and 4.53%, respectively). The mean concentration of total plasma homocysteine in nonfasting adult males (n = 12) and females (n = 12) was 15.8 (range, 7.0-23.7) and 16.5 nmol/ml (range, 8.6-20.7), respectively. Markedly elevated levels of homocysteine were found in patients with cobalamin and folate deficiency. Total plasma homocysteine represents approximately 4% of borohydride-generated thiol reactivity in the plasma of normal individuals.

Chromatography, High Pressure Liquid↗
PubMed1988

Kinetics and activation parameters of the reaction of cyanide with free aquocobalamin and aquocobalamin bound to a haptocorrin from chicken serum.

The kinetics of the reaction of cyanide with free aquocobalamin (H2OCbl) and with aquocobalamin bound to a vitamin B12-binding protein (haptocorrin) from chicken serum (HC-H2OCbl) have been investigated as a function of temperature and pH. The mechanism of replacement of H2O from protein-bound and free H2OCbl is apparently the same and involves attack of both CN- (k1) and HCN (k2). The reactions with HC-H2OCbl are somewhat slower than those with free H2OCbl, k1 being 22-fold smaller and k2 7-fold smaller at 25 degrees C. The relatively small effect of the protein on the rate constants supports the view that the metal in HC-H2OCbl is readily accessible to solvent. Activation parameters suggest that the transition states for ligand substitution are stabilized by nucleophilic participation (at least by CN-) and that ligand substitution on the protein-bound cobalamin proceeds through more ordered, concerted transition states. The latter effect suggests that the Co-O bond of H2OCbl is strengthened upon binding to the haptocorrin.

Animals↗
PubMed1988

Heteronuclear NMR studies of cobalamins. 31P NMR observations of cobalamins bound to a haptocorrin from chicken serum.

A vitamin B12-binding protein (haptocorrin) from chicken serum has been purified to homogeneity by photodissociative affinity chromatography and characterized by gel electrophoresis and UV-visible spectrophotometry of its aquocobalamin, hydroxocobalamin, and cyanocobalamin complexes. The haptocorrin is a glycoprotein with a molecular mass of about 70 kDa and a protein moiety of about 40 kDa. 31P NMR resonances of the haptocorrin-cobalamin complexes are relatively broad singlets (with or without proton decoupling) shifted downfield by 0.7-1.0 ppm from the position of the free cobalamin resonances. From the line width data, the relaxation of the phosphorus nucleus is found to be dominated by chemical shift anisotropy with a very minor (13%) component from dipolar interaction with the two nearest neighbor protons. The rotational correlation time of the haptocorrin at 25 degrees C is estimated to be 85 ns and the activation energy for rotational correlation 3.9 +/- 0.3 kcal mol-1. The downfield shift of the 31P resonances of cobalamins upon binding to the haptocorrin cannot be due to hydrogen bonding phosphodiester moiety or displacement of the axial base by a group on the protein. Calculations also show that the downfield shift is very unlikely to be due to dipolar deshielding of the phosphorus nucleus by the ring current of an aromatic residue of the protein. It is concluded that the downfield shift of the 31P resonance must be due to sterically induced changes in phosphodiester conformation which may, or may not, involve steric compression of the axial Co-N bond.

Algorithms↗
PubMed1987

Receptors for transferrin and transcobalamin II display segregated distribution on microvilli of leukemia L1210 cells.

Simultaneous addition of uniform latex particles derivatized with transferrin (0.532 micron) and transcobalamin II (0.345 micron) to leukemia L1210 cells resulted in segregated binding to individual microvilli as demonstrated by scanning electron microscopy. This segregated distribution suggests that individual microvilli are endowed either transferrin or transcobalamin II receptors but not both. Intracellular sorting and segregation of newly synthesized or recycling receptors probably occur prior to expression on the plasmalemma microvilli.

Animals↗
PubMed1987

Sulfite hypersensitivity. A critical review.

Sulfiting agents (sulfur dioxide and the sodium and potassium salts of bisulfite, sulfite, and metabisulfite) are widely used as preservatives in foods, beverages, and pharmaceuticals. Within the past 5 years, there have been numerous reports of adverse reactions to sulfiting agents. This review presents a comprehensive compilation and discussion of reports describing reactions to ingested, inhaled, and parenterally administered sulfite. Sulfite hypersensitivity is usually, but not exclusively, found within the chronic asthmatic population. Although there is some disagreement on its prevalence, a number of studies have indicated that 5 to 10% of all chronic asthmatics are sulfite hypersensitive. This review also describes respiratory sulfur dioxide sensitivity which essentially all asthmatics experience. Possible mechanisms of sulfite hypersensitivity and sulfur dioxide sensitivity are discussed in detail. Sulfite metabolism and the role of sulfite oxidase in the detoxification of exogenous sulfite are reviewed in relationship to the etiology of sulfite hypersensitivity.

Administration, Oral↗