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Biomedical subjects

J F Kolhouse

Publications and source records attributed to J F Kolhouse.

At least 19 recordsLinked to original sources
PubMed2003

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.

Adult↗
PubMed2000

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.

Administration, Oral↗
PubMed1999

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.

Chromatography, Liquid↗
PubMed1997

Unpredictable intra-individual variations in serum homocysteine levels on folic acid supplementation.

OBJECTIVE: To determine if changes in serum homocysteine values during folic acid supplementation can identify objectively healthy subjects with subclinical folate deficiency. DESIGN: Blood drawn and processed in a regimented fashion from fasting subjects. Serum homocysteine values determined by gas chromatography-mass spectrometry twice before and on days 5 and 8 of daily folic acid supplementation. SETTING: Outpatient University Hospital Clinical Research Center, Denver, Colorado. SUBJECTS: Subjectively healthy adults with normal hematologic and biochemical screening tests. INTERVENTION: Folic acid 1 mg daily for eight consecutive days. RESULTS: Homocysteine values of the group fell significantly during folic acid supplementation. Values pre supplementation were 7.8 +/- 1.8 and 7.4 +/- 2.4 mumol/L while values on days 5 and 8 of supplementation were 6.5 +/- 2.2 and 6.3 +/- 2.2 mumol/L. However, the homocysteine values of any given individual varied up to 60% (rises up to 7 mumol/L and falls of 5 mumol/L) during folic acid supplementation despite the controlled circumstances of blood handling, and an assay coefficient of variation of 8%. CONCLUSIONS: Although group values of serum homocysteine fall during folic acid supplementation, intraindividual variation is so great that subjects with subclinical folate deficiency can not be identified using this study design. Furthermore, these data suggest than an individuals homocysteine values vary enough that single values must be interpreted with caution.

Adolescent↗
PubMed1995

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.

4-Aminobenzoic Acid↗
PubMed1994

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.

Aspartic Acid↗
PubMed1994

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.

Adenocarcinoma↗
PubMed1994

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.

Amino Acids, Sulfur↗
PubMed1994

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.

Colorimetry↗
PubMed1993

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.

Ascorbic Acid↗
PubMed1993

Identification and perturbation of mutant human fibroblasts based on measurements of methylmalonic acid and total homocysteine in the culture media.

Human and mammalian cells contain two cobalamin-dependent enzymes. Methylmalonyl-CoA mutase isomerizes L-methylmalonyl-CoA to succinyl-CoA in the propionyl-CoA pathway while methionine synthase catalyzes the transfer of the methyl group of 5-methyltetrahydrofolate to homocysteine to form methionine. Decreased activity of mutase leads to an increased methylmalonic acid in the serum of humans while decreased activity of methionine synthase leads to increased homocysteine in the serum of humans. In current studies of cultured fibroblasts, methylmalonic acid levels were specifically increased in media of normal fibroblasts exposed to propionate or in fibroblasts with mutations involving mutase. Homocysteine levels were specifically increased in media of normal fibroblasts exposed to reduced folate concentrations or in fibroblasts involving mutations of methionine synthase. In addition, exposure of normal cells to inhibitory cobalamin analogues resulted in an increase of both methylmalonic acid and homocysteine in the media. This method of analysis appears to be both specific and sensitive for reduced activity of these two enzymes in tissue culture.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1993

A mechanism of inhibition of short-wavelength ultraviolet light-induced deamination of pyrimidine bases by ascorbic acid.

The relation of ascorbic acid to the ultraviolet light-induced deamination of cytosine (to uracil) and 5-methylcytosine (to thymine) was examined using gas chromatography-mass spectrometry. There was an inverse relation between the uracil content measured in solutions of cytosine after exposure to short wavelength (254 nm) ultraviolet light and the ascorbic acid concentration of the solution indicating inhibition of deamination of cytosine by ascorbic acid. Ascorbic acid also inhibited ultraviolet light-induced deamination of 5-methylcytosine. The ascorbic acid was only partially consumed during the exposure suggesting that protection from deamination was not due entirely to the antioxidant properties of ascorbic acid. Mercaptoethanol did not prevent ultraviolet light-induced deamination of either cytosine or 5-methylcytosine. However, near identical protection from ultraviolet light-induced deamination was provided by 4-aminobenzoic acid and ascorbic acid at equivalent ultraviolet light absorbing concentrations. This observation suggests that ascorbic acid prevents short wavelength ultraviolet light-induced deamination through absorbance of ultraviolet light rather than through antioxidant mechanisms.

5-Methylcytosine↗
PubMed1991

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.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1991

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.

Animals↗
PubMed1990

The glycophospholipid-linked folate receptor internalizes folate without entering the clathrin-coated pit endocytic pathway.

The folate receptor, also known as the membrane folate-binding protein, is maximally expressed on the surface of folate-depleted tissue culture cells and mediates the high affinity accumulation of 5-methyltetrahydrofolic acid in the cytoplasm of these cells. Recent evidence suggests that this receptor recycles during folate internalization and that it is anchored in the membrane by a glycosyl-phosphatidylinositol linkage. Using quantitative immunocytochemistry, we now show that (a) this receptor is highly clustered on the cell surface; (b) these clusters are preferentially associated with uncoated membrane invaginations rather than clathrin-coated pits; and (c) the receptor is not present in endosomes or lysosomes. This receptor appears to physically move in and out of the cell using a novel uncoated pit pathway that does not merge with the clathrin-coated pit endocytic machinery.

Animals↗