Problems in the measurement of zinc using heparin as an anticoagulant

Although several reports imply that anticoagulants and preservatives contain zinc, the quantity of zinc in heparin, if any, has not been documented. Zinc concentration was determined by flame atomic absorption spectroscopy in varying dilutions of multiple commercially obtained samples of purified sodium heparin N = 15 (microgram Zn/1000 Units heparin). Rubber stoppers of sterile heparin vials and of blood evacuation tubes were incubated in pre-analyzed water or saline on a mechanical shaker with fluid aliquots obtained up to 27 hours and analyzed for zinc content (microgram Zn/0.1 ml). Heparin, with contact or without contact with rubber stoppers, recorded identical zinc concentrations. Zinc concentrations varied from 0.222 +/- 0.01 (mean +/- SE) to 3.49 +/- 0.005 microgram Zn/1000 Units heparin. Leaching of zinc from rubber stoppers of vacutainer tubes (N = 9) was noted only with those containing known chelators of zinc. These results indicate that zinc is present in certain lots of sodium heparin and that caution must be exercised when reporting zinc concentrations of blood samples that contain sodium heparin as the anticoagulant.     

A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

Laboratory tests can be done on the cellular or fluid portions of the blood. The use of different blood collection tubes determines the portion of the blood that can be analyzed (whole blood, plasma or serum). Laboratories involved in studying the genetic basis of human disorders rely on anticoagulated whole blood collected in EDTA-containing vacutainer as the source of DNA for genetic / genomic analysis. Because most clinical laboratories perform biochemical, serologic and viral testing as a first step in phenotypic outcome investigation, anticoagulated blood is also collected in heparin-containing tube (plasma tube). Therefore when DNA and plasma are needed for simultaneous and parallel analyses of both genomic and proteomic data, it is customary to collect blood in both EDTA and heparin tubes. If blood could be collected in a single tube and serve as a source for both plasma and DNA, that method would be considered an advancement to existing methods. The use of the compacted blood after plasma extraction represents an alternative source for genomic DNA, thus minimizing the amount of blood samples processed and reducing the number of samples required from each patient. This would ultimately save time and resources.The BD P100 blood collection system for plasma protein preservation were created as an improved method over previous plasma or serum collection tubes¹, to stabilize the protein content of blood, enabling better protein biomarker discovery and proteomics experimentation from human blood. The BD P100 tubes contain 15.8 ml of spray-dried K2EDTA and a lyophilized proprietary broad spectrum cocktail of protease inhibitors to prevent coagulation and stabilize the plasma proteins. They also include a mechanical separator, which provides a physical barrier between plasma and cell pellets after centrifugation. Few methods have been devised to extract DNA from clotted blood samples collected in old plasma tubes^2-4. Challenges from these methods were mainly associated with the type of separator inside the tubes (gel separator) and included difficulty in recovering the clotted blood, the inconvenience of fragmenting or dispersing the clot, and obstruction of the clot extraction by the separation gel.We present the first method that extracts and purifies genomic DNA from blood drawn in the new BD P100 tubes. We compare the quality of the DNA sample from P100 tubes to that from EDTA tubes. Our approach is simple and efficient. It involves four major steps as follows: 1) the use of a plasma BD P100 (BD Diagnostics, Sparks, MD, USA) tube with mechanical separator for blood collection, 2) the removal of the mechanical separator using a combination of sucrose and a sterile paperclip metallic hook, 3) the separation of the buffy coat layer containing the white cells and 4) the isolation of the genomic DNA from the buffy coat using a regular commercial DNA extraction kit or a similar standard protocol.     

Protamine immobilization and heparin adsorption on the protamine-bound cellulose fiber membrane

Immobilization of protamine to the inner lumen of cellulose hollow fibers has been shown useful in preventing both heparin- and protamine-induced complications during an extracorporeal blood circulation procedure. The current study examined the effects of variables on the immobilization of protamine to cyanogen bromide (CNBr)-activated cellulose hollow fibers. The degree of protamine immobilization was controlled by three independent parameters: the amount of CNBr used during the activation process, the duration of the coupling process, and the protamine concentration in the coupling solution. By the adjustment of these parameters, cellulose fibers containing desired amounts of immobilized protamine (ranging from 1 to 20 mg of immobilized protamine per gram of dry fibers) were readily prepared.Heparin adsorption to the protamine-bound cellulose fibers was also examined. The adsorption isotherm followed a Langmuir adsorption model. The amount of heparin adsorbed was dependent on both the heparin concentration in the substrate solution and the protamine loading on the fibers. The Langmuir adsorption constant K was estimated to be 0.37 +/- 0.06 mL/mg, whereas the saturation capacity Q(s) of the protamine-bound fibers increased with increasing the protamine loading.     

Enhancement of blood compatibility of poly(urethane) substrates by mussel-inspired adhesive heparin coating

Heparin immobilization on surfaces has drawn a great deal of attention because of its potential application in enhancing blood compatibility of various biomedical devices such as catheters, grafts, and stents. Existing methods for the heparin immobilization are based on covalent linkage formation and electrostatic interaction between substrates and heparin molecules. However, complicated multistep procedures and uncontrolled desorption of heparin are limitations of these methods. In this work, we report a new heparin derivative that exhibits robust adhesion on surfaces. The derivative, called hepamine, was prepared via conjugation of dopamine, a mussel-inspired adhesive moiety, onto a heparin backbone. Immersion of poly(urethane) substrates into an aqueous solution of hepamine resulted in robust heparin coating of the poly(urethane), the most widely used polymeric material for blood-contacting medical devices. The hepamine-coated poly(urethane) substrate showed significant inhibition of blood coagulation and platelet adhesion. The use of hepamine for surface modification is advantageous for several reasons: for example, no chemical pretreatment of the substrates is necessary, and surface functionalization is a simple, one-step procedure. Thus, the heparin immobilization method described herein is an excellent alternative approach for the introduction of heparin molecules onto surfaces.     

Hydrazinolysis of heparin and other glycosaminoglycans.

Heparin, carboxy-group-reduced heparin, several sulphated monosaccharides and disaccharides formed from heparin, and a tetrasaccharide prepared from chondroitin sulphate were treated at 100 degrees C with hydrazine containing 1% hydrazine sulphate for periods sufficient to cause complete N-deacetylation of the N-acetylhexosamine residues. Under these hydrazinolysis conditions both the N-sulphate and the O-sulphate substituents on these compounds were completely stable. However, the uronic acid residues were converted into their hydrazide derivatives at rates that depended on the uronic acid structures. Unsubstituted L-iduronic acid residues reacted much more slowly than did unsubstituted D-glucuronic acid or 2-O-sulphated L-iduronic acid residues. The chemical modification of the carboxy groups resulted in a low rate of C-5 epimerization of the uronic acid residues. The hydrazinolysis reaction also caused a partial depolymerization of heparin but not of carboxy-group-reduced heparin. Treatment of the hydrazinolysis products with HNO2 at either pH 4 or pH 1.5 or with HIO3 converted the uronic acid hydrazides back into uronic acid residues. The use of the hydrazinolysis reaction in studies of the structures of uronic acid-containing polymers and the implications of the uronic acid hydrazide formation are discussed.     

Should we measure serum or plasma lead concentrations?

ProjectSerum samples may not be appropriate to assess lead (Pb) concentrations because they may contain artificially higher Pb concentrations compared with those measured in plasma samples. Here, we compared Pb concentrations in serum versus heparin plasma separated from blood collected with or without vacuum. We have also examined the effects of sample standing time on Pb concentrations measured in serum, heparin plasma, and EDTA plasma.ProcedureWe studied plasma and serum samples from twelve healthy subjects. Blood samples were collected via venous drainage phlebotomy with and without vacuum into trace metal free tubes containing no anticoagulants (serum), or lithium heparin, or EDTA (to obtain plasma). Variable sample standing times (0, 5, and 30 min) prior to centrifugation were allowed. Plasma and serum Pb and iron concentrations were determined by inductively coupled plasma mass spectrometry. Plasma and serum cell-free hemoglobin concentrations were measured.ResultsPb concentrations in serum and in heparin plasma from blood samples collected with or without vacuum were similar and not associated with significant changes in iron or hemoglobin concentrations. The sample standing time (up to 30 min) did not affect Pb concentrations in serum or in heparin plasma, which were approximately 50% lower than those found in EDTA plasma.ConclusionsSerum or heparin plasma separated from blood samples collected via venous phlebotomy with or without vacuum are appropriate medium to assess Pb concentrations, independently of the sample standing time.     

Activation of heparin cofactor II by heparin oligosaccharides

Heparin was partially depolymerized with heparinase or nitrous acid. The resulting oligosaccharides were fractionated by gel filtration chromatography and tested for the ability to stimulate inhibition of thrombin by purified heparin cofactor II or antithrombin. Oligosaccharides containing greater than or equal to 18 monosaccharide units were active with antithrombin, while larger oligosaccharides were required for activity with heparin cofactor II. Intact heparin molecules fractionated on a column of immobilized antithrombin were also tested for activity with both inhibitors. The relative specific activities of the unbound heparin molecules were 0.06 with antithrombin and 0.76 with heparin cofactor II in comparison to unfractionated heparin (specific activity = 1.00). We conclude that heparin molecules much greater than 18 monosaccharide units in length are required for activity with heparin cofactor II and that the high-affinity antithrombin-binding structure of heparin is not required.     

Effects of citrate, heparin and cation supplementation on mortality and egg production of laboratory-reared horn flies

Abstract. The effects of the blood anticoagulants sodium citrate and sodium heparin on horn fly, Haematobia irritans L., egg production were tested. Sodium citrate was added to freshly collected bovine blood to give final concentrations of 5-100mM while sodium heparin was used in concentrations of 10–70 USP units/ml blood. Small cages containing five male and ten female newly emerged laboratory-reared horn flies were maintained for 8–10 days on these blood samples, and mortality and egg production recorded daily. Results showed that as blood citrate concentration was increased, egg production decreased logarithmically. At sodium citrate concentrations of 50 mM and above, severe impacts on egg production and adult horn fly survival occurred. Although no dose-related response of egg production to increasing heparin concentrations was noted, the 25 USP units heparin/ml blood treatments gave the largest egg production, yielding approximately 28% more eggs than any other treatment. Since citrate is a known chelator of divalent metal cations, the effects of supplemental cation additions to citrated blood were tested for their ability to reverse the egg production decrease seen at 50 mM sodium citrate. Blood samples containing 50mM sodium citrate were supplemented with CaCl₂, calcium lactate, CuCl₂, cupric acetate, FeCl₃, ferric citrate, MgCl₂, magnesium acetate, MnCl₂, ZnSO₄, EGTA or EGTA plus calcium lactate, each at 1 mM except EGTA which was used at 2.5 mM. The magnesium acetate supplement and the combination of calcium lactate plus EGTA resulted in a statistically significant increase in egg production ( P < 0.05).     

Surface plasmon resonance sensor for heparin measurements in blood plasma

Surface plasmon resonance (SPR) was used as an affinity biosensor to determine absolute heparin concentrations in human blood plasma samples. Protamine and polyethylene imine (PEI) were evaluated as heparin affinity surfaces. Heparin adsorption onto protamine in blood plasma was specific with a lowest detection limit of 0.2 U/ml and a linear window of 0.2–2 U/ml. Although heparin adsorption onto PEI in buffer solution had indicated superior sensitivity to that on protamine, in blood plasma it was not specific for heparin and adsorbed plasma species to a steady-state equilibrium. By reducing the incubation time and diluting the plasma samples with buffer to 50%, the non-specific adsorption of plasma could be controlled and a PEI pre-treated with blood plasma could be used successfully for heparin determination. Heparin adsorption in 50% plasma was linear between 0.05 and 1 U/ml so that heparin plasma levels of 0.1–2 U/ml could be determined within a relative error of 11% and an accuracy of 0.05 U/ml.     

The effect of anticoagulants, enzyme inhibitors and long term storage on the extraction of known concentrations of methionine enkephalin in human plasma

The stability of methionine enkephalin (M-E) during long term storage was investigated using various anticoagulants and enzyme inhibitors, eg EDTA, heparin, trasylol, citric acid. Plasma was stored for different lengths of time up to six weeks. High pressure liquid chromatography (HPLC) was used to separate and quantify M-E. We found that EDTA, heparin or trasylol per se are ineffective in preserving M-E for short term extraction. Blood collected in chilled heparin tubes with citric acid crystals and the plasma further acidified with hydrochloric acid gave the highest recovery. With storage times up to six weeks further degradation was marked in samples taken in plain tubes but did not occur with tubes containing citric acid crystals and hydrochloric acid.     

Interaction of the polyelectrolyte heparin with copper(II) and calcium

The binding of copper(II) ions by heparin was investigated using equilibrium dialysi techniques, and the effects of this binding on solution properties determined. In neus tral Tris buffer solutions, heparin binds a maximum of twenty-three to twenty-four copper ions in two classes of sites, one containing three to four binding sites, the other containing twenty to twenty-one sites. Cooperative binding is associated with the larger class of sites. In more acidic citrate buffer solution, only one class of sites is observed, containing about four to five binding sites. Association constants are calculated for the classes and the possible chemical nature of the sites is discussed. The binding of calcium ions in neutral buffer is also examined, and these ions appear to be bound by a group of twenty to twenty-one binding sites, with a larger association constant than that for the copper ions. Definite effects on the solution properties of heparin, such as intrinsic viscosity, sedimentation coefficients, and partial specific volume, can be observed only in the cooperative binding of copper ions in neutral buffer. The interpretation of these solution properties in terms of molecular size and shape is analyzed, and it is concluded that the metal ion interactions cause no major change in the apparently random coil conformation of heparin in buffered solution, although some minor changes can be associated with the cooperative uptake of copper ions.     

Monoclonal antibodies specific for oligosaccharides prepared by partial nitrous acid deamination of heparin

Monoclonal antibodies were raised against a conjugate between heparin oligosaccharides and human serum albumin. The oligosaccharides were prepared by partial nitrous acid degradation of heparin and were coupled to human serum albumin by reductive amination. Characterization of the antibodies secreted by one of the resulting clones showed that they recognize a determinant present in the oligosaccharide antigen, but not in intact heparin, nor in a variety of related polysaccharides. Degradation of heparin by nitrous acid generates a 2,5-anhydro-D-mannose residue at the reducing end of the resulting oligosaccharides, and it is concluded that this structure is essential for interaction with the antibodies. Reduced oligosaccharides (containing a terminal anhydromannitol residue) are also active. After gel chromatography of partially degraded heparin, the smallest components capable of binding to the antibodies were found in a tetrasaccharide fraction. Affinity chromatography on immobilized monoclonal antibodies separated this tetrasaccharide fraction into distinct populations of binding and nonbinding species. Structural analysis showed that the tetrasaccharide fraction that bound to the monoclonal antibodies contained one single component with the structure IdoA(2-OSO3)-GlcNSO3 (6-OSO3)-IdoA(2-OSO3)-aManR(6-OSO3), whereas the fraction that did not bind to the antibodies contained a mixture of different structures.     

Conversion of a polysaccharide to nitric oxide-releasing form. Dual-mechanism anticoagulant activity of diazeniumdiolated heparin

We describe heparin/diazeniumdiolate conjugates that generate nitric oxide (NO) at physiological pH. Like the heparin from which they were prepared, they inhibit thrombin-induced blood coagulation. Unlike heparin, they can also inhibit and reverse ADP-induced platelet aggregation (as expected for an NO-releasing agent), suggesting potential utility as dual-action antithrombotics.     

Binding of platelet factor 4 to heparin oligosaccharides.

Heparin fractions of differing Mr (7800-18 800) prepared from commercial heparin by gel filtration and affinity chromatography on immobilized anti-thrombin III had specific activities when determined by anti-Factor Xa and anti-thrombin assays that ranged from 228 to 448 units/mg. The anti-Factor Xa activity of these fractions could be readily and totally neutralized by increasing concentrations of platelet factor 4 (PF4). That these fractions bound to immobilized PF4 was indicated by the complete binding under near physiological conditions of 3H-labelled unfractionated commercial heparin. An anti-thrombin III-binding oligosaccharide preparation (containing predominantly eight to ten saccharide units), prepared by degradation of heparin with HNO2 had high (800 units/mg) anti-Factor Xa, but negligible anti-thrombin, specific activity. The anti-Factor Xa activity of this material could not be readily neutralized by PF4, and the 3H-labelled oligosaccharides did not completely bind to immobilized PF4. A heterogeneous anti-thrombin III-binding preparation containing upwards of 16 saccharides had anti-thrombin specific activity of just less than one-half the anti-Factor Xa specific activity. This material was completely bound to immobilized PF4 and was eluted with similar concentrations of NaCl to those that were required to elute unfractionated heparins from these columns. Furthermore, increasing concentrations of PF4 neutralized the anti-Factor Xa activity of this material in a manner similar to that of unfractionated heparin. It is concluded that heparin oligosaccharides require saccharide units in addition to the anti-thrombin III-binding sequence in order to fully interact with PF4.     

A selective protein sensor for heparin detection

No clinical assays for the direct detection of heparin in blood exist. To create a heparin sensor, the hyaluronan (HA)-binding domain (HABD) of a protein that binds heparin and HA was engineered. GST fusion proteins containing one to three HABD modules were cloned, expressed, and purified. The affinities of each construct for heparin and for HA were determined by a competitive enzyme-linked immunosorbent assay using immobilized HA or heparin. Each of the constructs showed modest affinity for immobilized HA. However, heparin was 100-fold more potent than HA as a competing ligand. With immobilized heparin, affinity increased as the HABD copy number increased. The three-copy construct, GST-HB3, detected unfractionated free heparin (UFH) as low as 39ng/ml (equivalent to approximately 0.1U/ml) with a signal-to-noise ratio of 5.6. GST-HB3 also showed 100-fold selectivity for heparin in preference to other glycosaminoglycans. The plot of logKd vs log [Na+] showed 2.5 ionic interactions per heparin-HB3 interaction. GST-HB3 showed a linear detection of both UFH (15kDa) and low-molecular-weight heparin (LMWH; 6kDa) added to human plasma. For UFH, the range examined was 78 to over 2000ng/ml (equivalent to 0.2 to 5.0U/ml). For LMWH, the useful range was 312 to over 2000ng/ml. The coefficient of variance for the assay was < 9% for six serial heparin dilutions and <12% for three plasma samples. In clinical use, GST-HB3 could accurately measure therapeutic heparin levels in plasma (0.2 to 2U/ml).     

A lipophilic heparin derivative protects elastin against degradation by leucocyte elastase.

An oleolylated derivative (I) of partially N-desulphated heparin was prepared containing an average number of three oleoyl residues for one molecule of heparin. The inhibitory capacity of I (IC50 = 0.55 microM) for leucocyte elastase resembles that of heparin (IC50 = 0.2 microM). In contrast to heparin, I is also an inhibitor of porcine pancreatic elastase (IC50 = 0.68 microM) and it also has the capacity to protect elastin fibres against the degradation by leucocyte elastase. When insoluble elastin is pretreated with I its degradation by leucocyte elastase is inhibited by almost 90% while pretreatment of elastin with heparin exhibited only a moderate effect on elastolysis (10% inhibition).     

Control of artifacts in plasma adenosine determinations

The literature concerning the role of adenosine (ADO) in physiology reveals no agreement about plasma ADO concentrations and suggests two main sources of error in these determinations: rapid ADO uptake by red blood cells or rapid ADO production from ADO nucleotides, which may be released by any cell lysis or platelet aggregation during plasma preparation. We therefore studied ADO concentrations in plasma from normal human forearm venous blood. ADO was determined by a high-performance liquid chromatographic procedure with a sensitivity of 3 nM (original plasma). Observed ADO concentrations ranged from 894 nM to 8.2 nM depending on the conditions of plasma preparation. In plasma prepared in plastic tubes from 4.5 ml of blood drawn into a plastic syringe containing 1.5 ml of an isotonic stopping solution (pH 7.4) containing heparin (60 units ml), dilazep (40 microM), EGTA (40 mM, EDTA (40 mM), erythro-9-(2-hydroxy-3-nonyl) adenine (40 microM), and alpha, beta-methylene adenosine-5'-diphosphate (525 nM), the plasma ADO concentration was 13.3 +/- 1.88 nM (SE) after correction for a simultaneous ADO recovery determination. The mean ADO recovery was 78% +/- 3.39. The mean plasma ADO concentration found by this method of collection and preparation is lower then reported by others. Proper collection methods are required to avoid artifacts when determining plasma ADO concentrations.     

Isolation of dermatan sulfate with high heparin cofactor II-mediated thrombin-inhibitory activity from porcine spleen

We prepared dermatan sulfate specimens from various porcine tissues, and compared their heparin cofactor II-mediated thrombin-inhibitory activities and chemical natures, including disaccharide composition. Electrophoresis of the specimens on cellulose acetate membrane indicated that spleen dermatan sulfate was the most acidic of the dermatan sulfates prepared from the various porcine tissues. Analysis of the disaccharide units of the dermatan sulfate specimens by high-performance liquid chromatography revealed that spleen dermatan sulfate was rich in 4,6-di-O-sulfated N-acetylgalactosamine residues as compared with those of the other tissues. Spleen dermatan sulfate exhibited the highest thrombin-inhibitory activity, which may be related to its high content of the disulfated N-acetylgalactosamine residue.     

Effects of glycosylation on heparin binding and antithrombin activation by heparin

Antithrombin (AT), a serine protease inhibitor, circulates in blood in two major isoforms, α and β, which differ in their amount of glycosylation and affinity for heparin. After binding to this glycosaminoglycan, the native AT conformation, relatively inactive as a protease inhibitor, is converted to an activated form. In this process, β‐AT presents the higher affinity for heparin, being suggested as the major AT glycoform inhibitor in vivo. However, either the molecular basis demonstrating the differences in heparin binding to both AT isoforms or the mechanism of its conformational activation are not fully understood. Thus, the present work evaluated the effects of glycosylation and heparin binding on AT structure, function, and dynamics. Based on the obtained data, besides the native and activated forms of AT, an intermediate state, previously proposed to exist between such conformations, was also spontaneously observed in solution. Additionally, Asn135‐linked oligosaccharide caused a bending in AT‐bounded heparin, moving such polysaccharide away from helix D, which supports its reduced affinity for α‐AT. The obtained data supported the proposal of an atomic‐level, solvent and amino acid residues accounting, putative model for the transmission of the conformational signal from heparin binding exosite to β‐sheet A and the reactive center loop, also supporting the identification of differences in such transmission between the serpin glycoforms involving helix D, where the Asn135‐linked oligosaccharide stands. Such intramolecular rearrangements, together with heparin dynamics over AT surface, may support an atomic‐level explanation for the Asn135‐linked glycan influence over heparin binding and AT activation. Proteins 2011; © 2011 Wiley‐Liss, Inc.