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J C Klock

Publications and source records attributed to J C Klock.

At least 19 recordsLinked to original sources
PubMed1999

Direct measurement of unfractionated heparin using a biochemical assay.

A number of investigations have noted that functional biological assays for heparin are not always reliable and may not reflect the actual biochemical level of heparin in patients receiving anticoagulant therapy. This creates the possibility that patients receiving anticoagulant treatment may have an excess or deficiency of circulating levels of heparin. To address this problem, we have developed a direct biochemical measurement of heparin. The heparin assay uses fluorophore-assisted carbohydrate electrophoresis (FACE) to directly measure the predominate disaccharide of unfractionated heparin. In this study, unfractionated heparin was measured in vitro throughout a wide range of heparin concentrations in plasma. Seven in vivo pharmacokinetic studies in five normal subjects given 3,000 USP units of unfractionated heparin intravenously showed a three-phase elimination process with higher peak plasma levels and shorter elimination times than predicted from previous studies. At these doses, heparin is largely eliminated intact through urinary excretion. Body weight has a significant effect on heparin kinetics. When we compared the direct biochemical assay with two biological clotting assays, we found the latter can overestimate biochemical heparin concentrations. The FACE assay, due to its sensitivity, is also able to measure circulating levels of endogenous heparin in plasma and urine. Direct heparin measurement using the FACE technique is practical and useful for studies of the correlation of biochemical and biological activities.

Animals↗
PubMed1996

Fluorophore-assisted carbohydrate electrophoresis in the separation, analysis, and sequencing of carbohydrates.

Carbohydrate analysis has traditionally been viewed as a specialty science, performed only in a few well-established laboratories using conventional carbohydrate analysis technology (e.g. NMR, gas chromatography-mass spectroscopy, high-performance liquid chromatography, capillary electrophoresis) combined with the specialized technical training that has been essential for accurate interpretation of the data. This tradition of specialized laboratories is changing, due primarily to an increase in the number of scientists performing routine carbohydrate analysis. As a result, many scientists who are not trained in traditional carbohydrate analytical techniques now need to be able to perform accurate carbohydrate analysis in their own laboratories. This has created a need for technically simple and inexpensive methods of carbohydrate analysis. In this review, we present application vignettes of a technically simple, yet analytically powerful method called fluorophore-assisted carbohydrate electrophoresis (FACE). FACE can be used for performing routine oligosaccharide profiling, monosaccharide analysis, and sequencing of a variety of carbohydrates.

Carbohydrate Sequence↗
PubMed1986

Structures of O-linked oligosaccharides isolated from normal granulocytes, chronic myelogenous leukemia cells, and acute myelogenous leukemia cells.

O-Linked oligosaccharides were isolated from normal granulocytes, chronic myelogenous leukemia cells, and acute myelogenous leukemia cells by alkaline borohydride treatment. Oligosaccharides were fractionated by Sephadex G-50 gel filtration and QAE-Sephadex column chromatography, and their structures were elucidated by fast atom bombardment-mass spectrometry after permethylation and methylation analysis before and after specific exoglycosidase treatments. Results show that normal granulocytes and chronic myelogenous leukemia cells contain a series of O-linked oligosaccharides with the following structure, (formula: see text) where, in normal granulocytes n = 0 is major and n = 1 or 2, and thus polylactosaminyl oligosaccharides are present as minor components. However, these polylactosaminyl oligosaccharides were barely detectable in chronic myelogenous leukemia cells. On the other hand, acute myelogenous leukemia cells, which represent poorly differentiated myeloid cells, mainly contain short O-linked oligosaccharides with 2----6-linked sialic acid as follows. (formula: see text) These results suggest that structures of O-linked oligosaccharides vary in the different maturation stages along the same cell lineage.

Carbohydrate Conformation↗
PubMed1986

Structure of a novel sialylated fucosyl lacto-N-norhexaosylceramide isolated from chronic myelogenous leukemia cells.

A novel sialylated fucosyl glycolipid, which is present at an elevated level in chronic myelogenous leukemia cells, was isolated. The structure of this fucoganglioside was elucidated by methylation analysis, fast atom bombardment-mass spectrometry, and enzymatic degradation, followed by reaction with anti-Lex, Gal beta 1----4 (Fuc alpha 1----3) GlcNAc beta 1----, monoclonal antibody. The structure of this ganglioside was found to be: (Formula: see text). This structure is unique in that a fucose is attached to the internal N-acetylglucosamine but not to the subterminal N-acetylglucosamine. Since this glycolipid is apparently absent in normal granulocytes or acute myelogenous leukemia cells, it can be a specific marker for chronic myelogenous leukemia cells. Based on the structures of this fucoganglioside and normal granulocyte glycolipids, a biosynthetic pathway of extension, sialylation, followed by fucosylation is proposed.

Antibodies, Monoclonal↗
PubMed1985

Structures of sialylated fucosyl polylactosaminoglycans isolated from chronic myelogenous leukemia cells.

Polylactosaminoglycans were isolated from human chronic myelogenous leukemia cells and their structures were elucidated. The lactosaminoglycan saccharides were isolated by hydrazinolysis and fractionated by QAE-Sephadex. The structures of fractionated oligosaccharides were analyzed by fast atom bombardment-mass spectrometry and methylation before and after treatment with specific exoglycosidases, such as alpha 2----3 specific neuraminidase. Based on these experiments, the structures of sialyl polylactosaminoglycans of chronic myelogenous leukemia cells were found to contain the following unique structure which is absent in normal mature granulocytes: (formula; see text) In addition to this, chronic myelogenous leukemia polylactosaminoglycans can be distinguished from normal granulocyte polylactosaminoglycans by the following characteristics. Leukemic polylactosaminoglycans are (a) shorter, (b) more highly sialylated and contain fully sialylated, tetrasialosyl polylactosaminoglycans, (c) are less fucosylated at C-3 of N-acetylglucosamine of polylactosaminyl side chains, and (d) contain a significant amount of sialyl Lex, NeuNAc alpha 2----3Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----3, structure. These results indicate that chronic myelogenous leukemia cells express unique polylactosaminoglycan structures which are distinct from normal mature granulocytes.

Amino Sugars↗
PubMed1985

Structures of glycosphingolipids isolated from human granulocytes. The presence of a series of linear poly-N-acetyllactosaminylceramide and its significance in glycolipids of whole blood cells.

Structures of glycolipids isolated from human granulocytes were elucidated by fast atom bombardment-mass spectrometry, methylation analysis, and exo- and endoglycosidase treatment. All neutral glycolipids, with saccharide residues ranging from 2 to 10, were found to have linear N-acetyllactosaminyl backbones. The majority of neutral glycolipids contain one or two fucosyl residues attached to N-acetylglucosamine residues through the Fuc alpha 1----3 linkage and were reactive with the monoclonal antibody specific to Gal beta 1----4(Fuc alpha 1----3)GlcNAc, the Lex structure. Their general structure can be expressed as follows: (formula; see text) where n = 0-3. Glycolipids containing sialic acid (gangliosides) were also found to have linear N-acetyllactosaminyl backbones with sialic acid joined to this backbone by either alpha 2----3 or alpha 2----6 linkage. The gangliosides have the following general structure: (formula; see text) where n = 0-3. The ceramide was composed of sphingosine with d18:1 as the long-chain base and C16:0 (as a major component) or C24:1 (as a minor component) fatty acid. Analysis of glycolipids isolated from granulocytes, erythrocytes, and whole blood cells revealed that, among the glycolipids prepared from the whole blood cells, dihexaosylceramide, lactoneotetraosylceramide, and the above described linear lactoneo series neutral glycolipids are present in granulocytes but barely present in erythrocytes.

Antibodies, Monoclonal↗
PubMed1984

Structure of sialylated fucosyl lactosaminoglycan isolated from human granulocytes.

Sialylated fucosyl lactosaminoglycan was isolated from human neutrophilic granulocytes and its structure was elucidated. The lactosaminoglycan glycopeptides were digested by endo-beta-galactosidase and "the core portion" and released oligosaccharides were analyzed by permethylation, fast atom bombardment mass spectrometry, and exoglycosidases. In addition, lactosaminoglycan saccharides were obtained by hydrazinolysis and the structures of fractionated sialyl oligosaccharides were analyzed by fast atom bombardment mass spectrometry and permethylation coupled with exoglycosidase treatment. The structure of one of the major components was found to be: (Formula: see text). This structure is unique in that 1) four linear polylactosaminyl side chains are attached to the core portion, 2) the side chain arising from position 4 of 2,4-linked mannose contains one or more alpha 1----3 fucosyl residues, 3) the side chain arising from position 6 of 2,6-linked mannose is terminated with NeuNAc alpha 2----3Gal(Fuc alpha 1----3)GlcNAc, sialyl Lex, and 4) the side chain arising from position 2 of 2,4-linked mannose is terminated with sialic acid through alpha 2----6 linkage.

Amino Sugars↗
PubMed1984

Isolation and characterization of polyfucosylated lactosaminoglycan from human granulocytes.

Lactosaminoglycan was isolated from human granulocytes and the structure of neutral lactosaminoglycan was elucidated. The lactosaminoglycan glycopeptides and lactosaminoglycan saccharides obtained by hydrazinolysis were analyzed by permethylation. In addition, the lactosaminoglycan was digested by endo-beta-galactosidase and the "core" portion and released oligosaccharides were analyzed by specific glycosidases, permethylation, and fast atom bombardment mass spectrometry. The structure of the major component in the neutral lactosaminoglycan was found to be: sequence in text where m + n + o + p greater than 6, and the mean value of fucose content = 4.5. This structure is unique in that 1) four linear polylactosaminyl chains are attached to the core portions, 2) N-acetylglucosamine residues in the polylactosaminyl side chains are substituted with fucose through an alpha 1----3 linkage and at least 1 mol of Gal beta 1----4(Fuc alpha 1----3)GlcNAc terminal structure is present, and 3) the tetraantennary core is a major component. Since this polyfucosylated lactosaminoglycan is abundantly present in human granulocytes, we propose that this lactosaminoglycan is a major carrier for the granulocyte-specific antigen, Gal beta 1----4(Fuc alpha 1----3)GlcNAc, which is recognized by the My-1 monoclonal antibody (Huang, L. C., Civin, C. I., Magnani, J. L., Shaper, J. H., and Ginsburg, V. (1983) Blood 61, 1020-1023).

Adult↗
PubMed1982

Isolation and characterization of glycosphingolipids from human leukocytes. A unique glycosphingolipid pattern in a case of acute myelomonoblastic leukemia.

Neutral glycosphingolipids and gangliosides were isolated from the malignant cells of a patient with acute myelomonoblastic leukemia. Structural analyses were performed by gas-liquid chromatography and by high-performance liquid chromatography combined with enzymatic hydrolysis of glycosphingolipids using glycosidases. We found that, in contrast to normal leukocytes and chronic leukemia cells which have only a single tetraosylceramide species, these acute myelomonoblastic leukemia cells have approximately equal amounts of both globo- and neolactotetraosylceramide. This is the first population of human leukocytes in which we found two families of neutral glycosphingolipids to be present. The ganglioside fraction was composed of appreciable quantities of both NeuAc alpha 2 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1Cer (GM3, hematoside) and NeuAc alpha 2 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1Cer (sialoparagloboside). These cells did not have the 'leukocyte-specific' N-acetylneuraminosyllactotriaosylceramide found in normal human lymphocytes and neutrophils. These results are discussed in relation to normal leukocyte differentiation and acute leukemia. The present study also illustrates the usefulness of combining enzymatic degradation with high-performance liquid chromatography for glycosphingolipid structural determination.

Carbohydrates↗
PubMed1982

Uptake and metabolism of daunorubicin by human leukemia cells.

Radiolabeled daunorubicin was used to study in vitro uptake of daunorubicin (DNR) by the human promyelocytic leukemia cell line HL-60 and by leukemic cells from five previously untreated patients with acute nonlymphocytic leukemia (ANLL). Uptake of the metabolite daunorubicinol (DOL) and the metabolism of DNR were examined using high-performance liquid chromatography (HPLC). Uptake of DNR and DOL by HL-60 and ANLL cells exhibited a similar kinetic pattern. The uptake of DOL was 35%-50% of the uptake of DNR at the same test concentration in both HL-60 and ANLL cells. Approximately 5%-10% of intracellular DNR was metabolized to DOL by HL-60 and ANLL cells after 24 h of drug exposure. Measurements of DNR or DOL derived from liquid scintillation spectrometry and HPLC permit a sensitive and accurate assessment of the pharmacokinetics of these drugs in human leukemia cells. In addition, the HL-60 cell line can be used as a model for studying in vitro pharmacokinetics of the anthracyclines.

Acute Disease↗
PubMed1982

Cellular and plasma kinetics of daunorubicin given by two methods of administration in a patient with acute leukemia.

Leukemic cell and plasma concentrations of daunorubicin were examined serially in a patient with acute lymphoblastic leukemia following administration of the drug by two different regimens: regimen A-an iv bolus dose of 50 mg/m2, and regimen B-a loading dose of 15 mg/m2 followed by 35 mg/m2 infused over 4 hours. Peak plasma levels were four times higher after regimen A the B, whereas leukemic cell concentrations were not significantly different. No difference in acute toxicity or therapeutic efficacy was seen after either method of administration. However, we have demonstrated that peak plasma levels of daunorubicin can be reduced by continuous infusion without significantly altering target tissue concentrations.

Adult↗
PubMed1981

Neutral glycosphingolipids in hairy cell leukemia.

The neutral glycosphingolipids of hairy cells from a patient with hairy cell leukemia were chemically analyzed by thin-layer and gas-liquid chromatography, mass spectrometry, combined gas chromatography-mass spectrometry, and glycosidase treatment. These cells were found to have compounds containing one to four sugars with the following structures: Glc1 leads to 1Cer Gal beta 1 leads to 4Glc1 leads to 1Cer Gal alpha 1 leads to 4 Gal beta 1 leads to 4Glc1 leads to 1Cer GalNAc beta 1 leads to 3Gal alpha 1 leads to 4Gal beta 1 leads to 4Glc1 leads to 1Cer These compounds belong to the globo series of neutral glycosphingolipids and are similar to those found in human lymphocytes and chronic lymphocytic leukemia cells. They differ from the neutral glycosphingolipids found in human neutrophils and chronic myelogenous leukemia cells which are of the lactoneo and gala type. Neutral glycosphingolipids may be useful in classifying leukemias of uncertain origin.

Carbohydrates↗
PubMed1981

Isolation and structural characterization of human lymphocyte neutral glycosphingolipids.

The neutral glycosphingolipids of human peripheral blood lymphocytes and of the lymphoid cells from a patient with B cell chronic lymphocytic leukemia were chemically analyzed. Four neutral glycosphingolipids were chemically analyzed. Four neutral glycosphingolipids were isolated from each of these two sources and studied by gas chromatography, methylation analysis, and electron impact--desorption mass spectrometry. The results of these studies indicate that the compounds have the following structures: Glc1 leads to 1Cer Gal1 leads to 4Glc1 leads to 1Cer Gal1 leads to 4Gal1 leads to 4Glc1 leads to 1Cer GalNAc1 leads to 3Gal1 leads to 4Gal1 leads to 4Glc1 leads to 1Cer These compounds, belonging to the globo series, were the only neutral glycosphingolipids found in the lymphoid cells. The ceramide (Cer) moiety of all these compounds contained 4-sphingenine with C16:0, C24:0, and C24:1 as the major fatty acid species. There were no structural differences in the neutral glycosphingolipids of peripheral blood lymphocytes compared to those of chronic lymphocytic leukemia cells. Peripheral blood lymphocytes contained more di- than monohexosylceramide whereas the reverse was true of the chronic lymphocytic leukemia cells. The proportion of tri- and tetrahexosylceramide was less than 10% for both types of cells. The results of our analyses did not support the existence of any differences in the major neutral glycosphingolipids among T, B, and chronic lymphocytic leukemia cells.

Carbohydrate Sequence↗
PubMed1981

Isolation and structural characterization of human lymphocyte and neutrophil gangliosides.

Gangliosides were isolated from purified preparations of human peripheral blood lymphocytes and neutrophils. Structural analyses and comparisons were performed by direct probe mass spectrometry and by degradation studies with the following enzymes: Escherichia freundii endo-beta-galactosidase; Clostridium perfringens and Arthrobacter ureafaciens neuraminidase; and jack bean beta-N-acetylhexosaminidase and beta-galactosidase. This combination of techniques allowed us to obtain carbohydrate composition and sequence information without the aid of methylation or carbohydrate compositional analyses using only 1-2 mg of purified gangliosides. On the basis of these studies we propose that human lymphocytes and neutrophils have gangliosides with the following structures. NeuAc alpha 2 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1Cer Structure A NeuAc alpha 2 leads to ? GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1Cer Structure B NeuAc alpha 2 leads to ? Gal beta 1 leads to 3,4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1Cer Structure C All three compounds were isolated from both cell types with structure A being the major lymphocyte ganglioside and structure C the major neutrophil ganglioside. Structure B is a novel ganglioside and may represent a leukocyte-specific glycosphingolipid. Neuraminidase degradation studies demonstrated that only one ganglioside species of each cell type contains an internally linked sialic acid residue, and on the basis of thin layer chromatographic analysis this component is the same as the major brain ganglioside, GM1 (II3-N-acetylneuraminosyl-gangliotetraosylceramide). In addition, large gangliosides with the general structure NeuAc alpha 2 leads to ?(Gal beta 1 leads to 3,4GlcNAc beta 1 leads to 3)n Gal beta 1 leads to 4Glc beta 1 leads to 1Cer were isolated. These results are discussed as they relate to blood group antigens and specific cell surface markers in human leukocytes.

Carbohydrate Conformation↗