Nonmyeloablative allogeneic hematopoietic stem cell transplantation for congenital sideroblastic anemia.

Congenital sideroblastic anemia (CSA) is a dyserythropoietic disorder that leads to transfusion dependency and subsequent iron overload. Nonmyeloablative allogeneic hematopoietic stem cell transplantation (NST) was performed for a patient with CSA, who had contraindications to conventional allografting. Conditioning was fludarabine, low-dose total body irradiation and antithymocyte globulin, followed by peripheral blood stem cell transplant. Cyclosporine and mycophenolate mofetil were used for graft-versus-host disease prophylaxis. Complete donor chimerism was observed day +131. Early after transplant, the patient became transfusion independent, allowing a regular phlebotomy program. On day +190, refractory lactic acidosis followed by fatal cardiovascular collapse developed, without evidence of infection. Data from this case demonstrates that NST may correct the erythropoietic defect of CSA.

Folate sufficient subjects do not accumulate additional folates during supplementation.

In a double-blinded placebo-controlled trial of folic acid supplementation in 82 alcoholic subjects, it was found that whole blood folate levels, determined by a mass spectrometric method, do not increase in subjects whose baseline folate levels are above the third quartile (folate sufficiency). Since a state of folate sufficiency can now be identified, a recommended daily allowance (RDA) for folate can be determined using objective means.

Glutathione oxidation in real time by thermospray liquid chromatography-mass spectrometry.

The oxidation and reduction of glutathione and oxidized glutathione were studied in real time by liquid chromatography-mass spectrometry during exposure to hydrogen peroxide and mercaptoethanol. By mass spectrometry mixed disulfides and both reversible and irreversible oxidations of sulfur to higher states (sulfinic and sulfonic acids) were directly observed during exposure to hydrogen peroxide. The irreversible oxidation of glutathione to glutathione sulfonic acid could be detected after 30 min exposure of glutathione to 40 mM H2O2 at 20 degrees C. A peak consistent with glutathione-sulfinic acid was transiently present, suggesting this compound behaved as an oxygen consuming antioxidant. Liquid chromatography-mass spectrometry appears to be an excellent method to study oxidation and reductions of sulfur containing peptides and amino acids.

Quantitation of red blood cell folates by stable isotope dilution gas chromatography-mass spectrometry utilizing a folate internal standard.

We report a new gas chromatography-mass spectrometry (GC-MS) method of measurement of red blood cell folates utilizing a stable isotope-labeled bacterial synthesized folate internal standard. The GC-MS method exploits the fact that the common feature of all folate molecules is a p-aminobenzoic acid moiety sandwiched between a pteridine ring and a polyglutamate chain of varying length. In this method, red blood cell folates together with a folate internal standard are specifically purified using bovine folate binding protein and the folates are subsequently chemically cleaved to p-aminobenzoic acid, pteridines, and glutamic acids. Since all six carbon atoms of the benzene ring in the p-aminobenzoic acid moiety of the folate internal standard are labeled with [13C], it is possible to use selected ion monitoring and stable isotope dilution GC-MS to quantitate folates. The method appears to be sensitive, specific, and accurate. The method has been applied to generate a reference range of red blood cell folates based on assay of 25 normal individuals.

Serum xylose analysis by gas chromatography/mass spectrometry.

A gas chromatography/mass spectrometric (GC/MS) isotope dilution assay for xylose was developed using tertbutyldimethylsilyl-derivatized xylose and [13C]1xylose, and applied to human serum samples. A calibration curve in serum using this assay showed < 3% variation (< 10 mg/L) for any given point. The correlation coefficient for xylose measurements made on 27 sera between a colorimetric method performed by a national commercial reference laboratory and the GC/MS method developed here was .952. However, xylose determinations of 10 of 27 samples differed by > 10% (up to 150 mg/L) when colorimetric values were compared to GC/MS. Two of these samples had borderline-low xylose values by GC/MS, but were well within the normal range by colorimetric analysis. gas chromatography/mass spectrometric isotope dilution assay appears to be an accurate method to measure xylose in serum. These data also suggest that further prospective studies comparing GC/MS to colorimetric methods are indicated for subjects undergoing oral xylose testing.

Are neuropsychiatric manifestations of folate, cobalamin and pyridoxine deficiency mediated through imbalances in excitatory sulfur amino acids?

Folate, cobalamin and pyridoxine deficiency are associated with psychiatric or neurological symptomatology. Disturbances in sulfur amino acid metabolism leading to accumulation of homocysteine occurs in all three conditions as the metabolism of homocysteine depends on enzymes requiring these vitamins as cofactors. Oxidation products of homocysteine (homocysteine sulfinic acid and homocysteic acid) and cysteine (cysteine sulfinic acid and cysteic acid) are excitatory sulfur amino acids and may act as excitatory neurotransmitters, whereas taurine and hypotaurine (decarboxylation products of cysteic acid and cysteine sulfinic acid) may act as inhibitory transmitters. Homocysteic acid and cysteine sulfinic acid have been considered as endogenous ligands for the N-methyl-D-aspartate (NMDA) type of glutamate receptors. The profile of these sulfur amino acid neurotransmitters could be altered in a similar fashion in states of decreased availability of folate, cobalamin or pyridoxine. It is proposed that the mechanism of neuropsychiatric manifestations in all three conditions result from a combination of two insults to homocysteine catabolism in the brain.

Measurement of excitatory sulfur amino acids, cysteine sulfinic acid, cysteic acid, homocysteine sulfinic acid, and homocysteic acid in serum by stable isotope dilution gas chromatography-mass spectrometry and selected ion monitoring.

Oxidized sulfur-containing amino acids are recognized as agonists of excitatory amino acid receptors in the mammalian nervous system. Homologues of glutamic acid (homocysteine sulfinic acid and homocysteic acid) and aspartic acid (cysteine sulfinic acid and cysteic acid) have been shown to be agonistic to N-methyl-D-aspartate receptors in animal brain and have been demonstrated in brain tissue. Considerable evidence exists for the role of homocysteic acid and cysteine sulfinic acid as endogenous ligands for excitatory amino acid receptors. We report, for the first time, the quantitation of these compounds in normal human serum, by a newly developed gas chromatography-mass spectrometry method that employs stable isotope-dilution selected ion monitoring using internal standards prepared in our laboratory. We also report new methods of synthesis of stable isotope-labeled internal standards used in measuring cysteine sulfinic acid, cysteic acid, homocysteine sulfinic acid, and homocysteic acid.

New anti-lung-cancer antibody cluster 12 reacts with human folate receptors present on adenocarcinoma.

Human folate receptor (hFR, folate-binding protein) is a single-chain glycoprotein with high specific affinity for folic acid and methotrexate. We have created 4 monoclonal antibodies (MAbs) to hFR, all of which react specifically with purified hFR in Western blots. Flow cytometry indicated that the antibodies all had patterns of reactivity against epithelial cell lines similar to that of antibody MW207 (workshop antibody 12), labeling 2 breast-tumor cell lines and 2 of 5 SCLC without labeling the one non-small-cell carcinoma tested. We used the antibodies to trace the in situ distribution of hFR in histologically normal tissues and in lung tumors by a sensitive alkaline-phosphatase-anti-alkaline-phosphatase immunohistochemical technique. In frozen sections of normal lung, hFR was diffusely distributed on cell membranes of type-1 and type-2 pneumocytes and mucociliary and basilar respiratory epithelial cells of distal bronchi. The receptor was focally expressed by mucociliary cells of proximal respiratory mucosa and by macrophages, but was not detected in stromal smooth muscle, fibroblasts or lymphoid cells. In tumors, hFR was heavily and diffusely expressed on cell membranes of 9 of 10 pulmonary adenocarcinomas, 5 of 5 bronchioloalveolar carcinomas and 2 of 2 carcinoid tumors. It was focally expressed in 3 of 5 large-cell lung carcinomas and was absent from 4 of 5 small-cell carcinomas, 18 of 22 invasive squamous carcinomas, 2 of 2 in situ squamous carcinomas, and 13 of 13 squamous dysplasias of bronchial mucosa. We conclude that hFR is heavily expressed in situ by normal alveolar and bronchial epithelium and by adenocarcinoma of lung. It is usually absent from small-cell carcinoma and squamous tumors at levels detectable by immunohistochemistry.

Ascorbate and dehydroascorbate measurements in aqueous solutions and plasma determined by gas chromatography-mass spectrometry.

The tert-butyldlmethylsllyl derivatives of ascorbic acid and dehydroascorbic acid were characterized by gas chromatography-mass spectrometry, and an isotope dilution assay for ascorbate and dehydroascorbate was developed using [13C6]ascorbic acid and [13C6]- and [6,6-2H2]dehydroascorbate. This assay was used to monitor ascorbic acid loss and the resulting rise of dehydroascorbic acid in aqueous solutions and plasma. Ascorbic acid was shown to rapidly decompose in aqueous solutions containing transition metal ions or when exposed to oxygen. Ethylenedlaminetetraacetic acid chelation did not prevent ascorbic acid degradation in aqueous solution, and ascorbate in ethylenedlaminetetraacetic acid chelated plasma was converted to dehydroascorbate on freezing. Gas chromatography-mass spectrometry appears to be a satisfactory method for determining the ascorbate and dehydroascorbate content of solutions including human blood plasma, whether or not there is ongoing oxidation of ascorbate in those solutions.

Mechanism of conversion of human apo- to holomethionine synthase by various forms of cobalamin.

Methionine synthase catalyzes the conversion of N5-methyltetrahydrofolate and homocysteine to tetrahydrofolate and methionine. Methylcobalamin (Me-Cbl) is tightly bound to methionine synthase and is required for enzymatic activity. When added to crude tissue homogenates, Me-Cbl stimulates methionine synthase but similar stimulation is observed with hydroxocobalamin, cyanocobalamin (CN-Cbl), and adenosyl-Cbl, although the mechanisms involved are unknown. We prepared human apomethionine synthase and studied its activation in the presence of [14C]CN-Cbl and [14CH3]Me-Cbl with concentrations of 2-mercaptoethanol ranging from 0.15 to 100 mM. We observed that the removal of the labeled upper axial ligands from CN-Cbl and Me-Cbl both paralleled the activation of human apomethionine synthase. Spectral studies employing CN-Cbl and Me-Cbl showed that both forms of Cbl must be converted to Cob(II)alamin before they can bind to human apomethionine synthase and convert it to its activated holoenzyme form. Studies with 14 different Cbl analogues with alterations in various portions of the corrin ring and the nucleotide showed that all of the analogues were able to fully activate human methionine synthase when they were reduced with 2-mercaptoethanol. Full activation occurred at lower concentrations of many of the Cbl analogues than occurred with Cbl itself. We conclude that Me-Cbl and other forms of Cob(III)alamin do not bind to human apomethionine synthase and that all must first be reduced to Cob(II)alamin before such binding can occur. The fact that human methionine synthase shows little absolute specificity for alterations in various portions of the Cbl molecule suggests that the potent inhibition of mammalian methionine synthase activity observed in vivo with various Cbl analogues is due to inhibition of intracellular Cbl transport or to inhibition of the enzymatic formation of Cob(II)alamin rather than to direct inhibition of mammalian methionine synthase itself.

The conversion of the human membrane-associated folate binding protein (folate receptor) to the soluble folate binding protein by a membrane-associated metalloprotease.

The membrane-associated (M-FBP) and soluble (S-FBP) forms of human folate binding proteins (FBP) have been well characterized. Although related in a precursor-product manner, the mechanism of conversion and the basis for differences between M-FBP and S-FBP are not known. The conversion of M-FBP to S-FBP in crude human nasopharyngeal carcinoma (KB) cell preparations is demonstrated based on characteristic gel filtration elution profiles of M-FBP and S-FBP (Ve/V0 = 1.3 and 1.7, respectively) in Triton X-100. M-FBP is stoichiometrically converted to S-FBP in a time- and temperature-dependent reaction by a metalloprotease which is: heat-labile; particulate; contained in human KB cell and placental membranes, and rat kidney homogenates; inhibited by EDTA, 1,10-phenanthroline, and parahydroxymercuribenzoate; requires divalent cations; is maximally active at neutral pH; and is active in the presence or absence of detergent. The purified soluble FBP product appears to be identical to S-FBP. Conversion of purified endogenously [3H]leucine-labeled M-FBP yields a soluble FBP characterized by a 45% decrease in specific activity (moles of 3H/mol folate bound) relative to M-FBP and a non-folate binding fragment which contains 45% of the [3H]leucine from M-FBP, requires detergent and/or urea to remain soluble, and migrates aberrantly on gel filtration in 1% (v/v) Triton X-100 and 8 M urea. Based on changes in the specific activity and the gel filtration elution profiles of purified labeled M-FBP associated with conversion to S-FBP, the endoproteolytic cleavage site is predicted between residues 226 and 229 of the cDNA predicted human FBP amino acid sequence. These results suggest that the cDNA predicted hydrophobic carboxyl terminus (residues 227-257) remains intact on the fully processed, membrane-anchored M-FBP, contains the Triton binding domain, and is involved in the formation of the membrane anchor of M-FBP.

Folate and methotrexate interactions in the rat kidney.

The mechanisms controlling the renal retention and urinary output of methotrexate and folates were studied in rats. 125I-Labeled folic acid administered i.v. was shown to be retained in the kidney through a system that could be inhibited by either folic acid or 5-methyltetrahydrofolate. Methotrexate also inhibited this system but required concentrations 50- to 100-fold greater than that required for folic acid and 5-methyltetrahydrofolate. Extracts from solubilized kidneys were shown to contain a folate binder with the same relative affinities for folates and methotrexate as the in vivo system. Methotrexate was shown to cause an increase in the urinary output of endogenous folates in rats when administered as equivalent doses to those used in treating human disease. Conversely, [3H]methotrexate administered i.v. was shown to be retained in the kidney through an additional system that could be inhibited by unlabeled methotrexate, but not by folates. This system was not demonstrable in solubilized kidney. These data demonstrate two systems for the renal retention of methotrexate.

Changes in plasma methionine and total homocysteine levels in patients receiving methotrexate infusions.

Methotrexate reduces intracellular pools of 5-methyltetrahydrofolate and could result in reduced conversion of homocysteine to methionine by methionine synthetase. This study was designed to investigate the effects of moderate dose to very high dose methotrexate on methionine and total homocysteine as reflections of methotrexate induced intracellular events. Methionine and total homocysteine were measured prior to, during, and following twenty-six 24-h i.v. infusions of 33.6 g/m2 methotrexate (very high dose methotrexate) in 16 children with acute lymphocytic leukemia and seven 4-h i.v. infusions of 8 g/m2 methotrexate (high dose methotrexate) in 5 children with osteogenic sarcoma. Amino acids were measured by gas chromatography/mass spectrophotometry. Mean methionine levels decreased by 70.0 +/- 3.1% (SE) with very high dose methotrexate and 72.6 +/- 5.9% with high dose methotrexate at 24 and 4.5 h, respectively, after beginning methotrexate infusions. Mean total homocysteine levels increased by 61.7 +/- 3.1% with very high dose methotrexate and 55.6 +/- 17.5% with high dose methotrexate at 36 and 24 h, respectively, after beginning methotrexate infusions. No consistent or significant changes were noted in levels of total cysteine, leucine, isoleucine, or valine. Similar changes did not occur in patients receiving prednisone, vincristine, daunomycin, and intrathecal methotrexate as therapy for acute lymphocytic leukemia. These changes in homocysteine and methionine may reflect biological effects of methotrexate that may predict cytotoxicity of methotrexate.

Determination of the carbohydrate composition of mammalian glycoproteins by capillary gas chromatography/mass spectrometry.

A technique to determine the carbohydrate composition of glycoproteins using capillary gas chromatography/mass spectrometry (electron impact) with selected ion monitoring is described. This method entails hydrolysis with methanolic-HCl followed by formation of trimethylsilyl methylglycoside derivatives, extraction of the carbohydrate derivatives into hexane, and GC/MS analysis. For those carbohydrates that are present in animal glycoproteins including fucose, mannose, galactose, glucosamine, galactosamine, and N-acetylneuraminic acid (sialic acid), the sensitivity of this assay was approximately 1-3 pmol and the assay was linear over a 100-fold range. The carbohydrate compositions determined on small quantities (1-10 pmol) of various glycoproteins including human transferrin and alpha-1 acid glycoprotein, fetuin, and ovalbumin were identical to their reported carbohydrate content and compositions. Major advantages of this technique include the time required to complete the sample preparation and analysis (less than 8 h), the sensitivity and specificity of the assay, and the fact that all carbohydrate moieties, including sialic acid, can be quantitated in a single hydrolysate of a glycoprotein.

Influence on immunoreactive folate-binding proteins of extracellular folate concentration in cultured human cells.

The influence of extracellular folate concentration on cellular levels of the folate transport protein and its soluble product was studied directly in cultured human nasopharyngeal carcinoma (KB) cells. As determined by radioimmunoassay, levels of the folate transport protein and the soluble folate-binding protein were 58 +/- 17 (mean +/- SD) and 5 +/- 2 pmol/mg cell protein, respectively, in KB cells maintained in standard medium (containing 2,300 nM folic acid). These levels significantly increased to 182 +/- 34 and 26 +/- 6 pmol/mg cell protein, respectively, in KB cells serially passaged in low folate medium (containing 2-10 nM 5-methyltetrahydrofolate). Increases in folate-binding protein levels occurred more rapidly in KB cells serially passaged in very low folate medium containing less than 2 nM folate and were prevented by the addition of 100 nM 5-methyltetrahydrofolate or 0.1-1 microM 5-formyltetrahydrofolate to this medium. When KB cells which had been passaged in low folate medium were passaged back into either standard medium or low folate medium supplemented with reduced folates, the levels of both folate-binding proteins fell linearly towards the levels in KB cells continuously maintained in standard medium. The folate transport protein was identified in and underwent similar changes in human and mouse mammary tumor cells. These studies indicate that the folate transport system is probably regulated by the extracellular folate concentration through changes in intracellular metabolite levels.

Molecular cloning of L-methylmalonyl-CoA mutase: gene transfer and analysis of mut cell lines.

L-Methylmalonyl-CoA mutase (MCM, EC 5.4.99.2) is a mitochondrial adenosylcobalamin-requiring enzyme that catalyzes the isomerization of L-methylmalonyl-CoA to succinyl-CoA. This enzyme is deficient in methylmalonic acidemia, an often fatal disorder of organic acid metabolism. Antibody against human placental MCM was used to screen human placenta and liver cDNA expression libraries for MCM cDNA clones. One clone expressed epitopes that could affinity-purify antibodies against MCM. A cDNA corresponding in length to the mRNA was obtained and introduced into COS cells by DNA-mediated gene transfer. Cells transformed with this clone expressed increased levels of MCM enzymatic activity. RNA blot analysis of cells genetically deficient in MCM indicates that several deficient cell lines have a specific decrease in the amount of hybridizable mRNA. These data confirm the authenticity of the MCM cDNA clone, establish the feasibility of constituting MCM activity by gene transfer for biochemical analysis and gene therapy, and provide a preliminary picture of the genotypic spectrum underlying MCM deficiency.

The isolation, characterization, and comparison of the membrane-associated and soluble folate-binding proteins from human KB cells.

Human nasopharyngeal epidermoid carcinoma (KB) cells contain a membrane-associated particulate folate-binding protein which is important in the cellular accumulation of physiologic folates (Antony, A. C., Kane, M. A., Portillo, R. M., Elwood, P. C., and Kolhouse, J. F. (1985) J. Biol. Chem. 260, 14911-14917) and in the binding of methotrexate (Kane, M. A., Portillo, R. M., Elwood, P. C., Antony, A. C., and Kolhouse, J. F. (1986) J. Biol. Chem. 261, 44-49). A soluble folate-binding protein appears in media exposed to proliferating KB cells. We have purified to homogeneity both the membrane-associated and the soluble folate-binding proteins from the KB cell tissue culture system. The purified membrane-associated and soluble folate-binding proteins give single bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent Mr values of 50,000 and 40,000, respectively. The membrane-associated folate-binding protein contains 45,000 g of amino acids and the soluble folate-binding protein contains 24,000 g of amino acids per mole of folate bound. Each of the purified proteins has a single folate-binding site, and the carbohydrate content is approximately 25% for each species of protein. The affinity constants for 5-methyltetrahydrofolate of the membrane-associated and soluble folate-binding proteins are 0.3 and 2.5 X 10(9) liters/mol, respectively. The affinities of various polyglutamated forms of methotrexate are similar for each protein, increase as the chain length of the polyglutamate increases (from approximately 0.004 X 10(9) liters/mol for methotrexate to 0.3 X 10(9) liters/mol for methotrexate heptaglutamate), are equal to the affinity for 5-methyltetrahydrofolate, and exceed the reported increase in affinity of methotrexate polyglutamates for dihydrofolate reductase.

The influence of extracellular folate concentration on methotrexate uptake by human KB cells. Partial characterization of a membrane-associated methotrexate binding protein.

Methotrexate accumulation, subcellular distribution, metabolism, and cytotoxicity were studied in human epidermoid carcinoma (KB) cells that were exposed to a low extracellular concentration of methotrexate (25 nM) following culture in widely differing concentrations of folic acid. KB cells cultured in standard medium with a high folic acid concentration (2.3 microM) had high levels of cellular folate (21.4 pmol/10(6) cells). Five passages through low folate (2.7 nM) medium reduced the level of cellular folate to near physiologic levels (0.4-1.0 pmol/10(6) cells). In contrast to KB cells cultured in standard medium, in KB cells cultured in low folate medium, 1) methotrexate inhibited growth; 2) methotrexate uptake was markedly increased; 3) methotrexate polyglutamation was almost complete; 4) methotrexate binding to dihydrofolate reductase was markedly enhanced; and 5) significant methotrexate binding to a previously undescribed membrane-associated protein occurred. The amount of methotrexate bound to the membrane-associated protein from KB cells cultured in low folate medium equaled the quantities bound by dihydrofolate reductase. Further characterization of this membrane-associated protein indicated that it was soluble in solutions containing Triton X-100, was capable of binding folic acid as well as methotrexate, had an apparent Mr of 160,000 by gel filtration in the presence of Triton X-100, and was precipitated by antiserum to human placental folate receptor. This membrane-associated protein may play an important role in the uptake and metabolism of methotrexate under physiologic conditions.

Studies of the role of a particulate folate-binding protein in the uptake of 5-methyltetrahydrofolate by cultured human KB cells.

The characteristics of the uptake by human epidermoid carcinoma (KB) cells of 5-methyltetrahydrofolate at extracellular concentrations in the physiologic range and the possible role of a membrane-associated folate binder in folate uptake by KB cells have been investigated. Uptake of 5-methyltetrahydrofolate was specific, saturable, and time-, temperature-, and concentration-dependent. Trypsin treatment released 50% of the 5-methyltetrahydrofolate accumulated by KB cells at 4 degrees C, but only 12% at 37 degrees C, indicating that most of the accumulated ligand was intracellular at 37 degrees C, thus demonstrating transport. Accumulated 5-methyltetrahydrofolate was bound to a membrane-associated protein which required detergent for its solubilization, and a significant amount of which was oriented to the cell exterior as demonstrated by its release by trypsin treatment of intact KB cells. The membrane-associated folate binder was immunoprecipitated by antiserum to purified human placental folate receptor, and this antiserum inhibited 5-methyltetrahydrofolate uptake by intact KB cells in a concentration-dependent manner. These data support the hypothesis that the membrane-associated folate-binding protein of human cells participates in the transport of folates under physiologic conditions.