Ziel der vorliegenden Arbeit war die Funktionalisierung von Titanoberflächen mit dem Glycosaminoglycan Heparin, um bei Kontakt des Werkstoffs mit Blut die Gerinnungskaskade nicht auszulösen und das Material für Stents (Gefäßstützen) im arteriellen System einsetzbar zu machen. Für die Modifizierungen wurden als Modell der oxidierten Titanoberfläche sowohl oxidierte cp-Titanplättchen als auch TiO2-Pulver verwendet. Heparin kam zum Einsatz, da es sowohl die Hämostase (Blutgerinnung) als auch die Proliferation (Überwucherung) mit glatten Muskelzellen unterdrückt und somit eine Restenose (Wiederverengung) des in die verengte Arterie eingebrachten Stents verhindert. Die kovalente Immobilisierung des Wirkstoffs erfolgte über bifunktionale Spacer (Haftvermittlermoleküle). Spacer waren 3-(Trimethoxysilyl)-propylamin (APMS), N-(2-Aminoethyl)-3-aminopropyltrimethoxysilan (Diamino-APMS) und N1-[3-(Trimethoxysilyl)-propyl]diethylen¬triamin (Triamino-APMS). Der qualitative und quantitative Nachweis der Funktionalisierung von TiO2 mit Haftvermittler bzw. Heparin erfolgte durch schwingungsspektroskopische Methoden, komplexometrische Farbreaktionen sowie der Bestimmung des Zetapotentials im Elektrolytkontakt. Durch die Anbindung von APMS, Di- und Triamino-APMS stieg das Zetapotential von ca. -26 mV auf positive Werte zwischen +41 und +45 mV. Ein Absinken des Zetapotentials belegte die erfolgreiche Anbindung von Heparin (Werte zwischen -39 und -37 mV) an die verschiedenen Haftvermittler, ebenso wie das Vorhandensein der symmetrischen SO3-Valenzschwingung bei 1040 cm-1. Der quantitative Nachweis der immobilisierten Aminogruppen über die Ninhydrinreaktion ergab für die TiO2-Pulver Werte zwischen 17-20 NH2/nm2, wobei die dichteste Funktionalisierung mit APMS und die niedrigste mit Triamino-APMS erzielt werden konnte. Alle Werte lagen im Bereich von Multilayern, da ein Monolayer aus ca. 2 3 NH2/nm2 besteht. Die immobilisierte Menge an Heparin war bei Verwendung von APMS am größten (53.3±3.6 ng/cm2) und bei Triamino-APMS am geringsten (32.1±5.7 ng/cm2). Die biologische Wirksamkeit des gebundenen Heparins wurde über das chromogene Substrat ChromozymTH® bestimmt und verblieb bei Anbindung an den Spacer mit der größten Moleküllänge (Triamino-APMS) mit ca. 70% am wirksamsten. Neben der kovalenten Anbindung des Wirkstoffs an Spacer zielte diese Arbeit auf die Entwicklung von organisch modifizierten, porösen SiO2-Wirkstoffdepots (P-MA-PS; Poly-methacryl¬oxy¬propylpolysilsesquioxane) für Heparin ab, die sowohl als Volumenwerkstoffe als auch zur Modifikation von Titan(dioxid)oberflächen anwendbar wären. Die Matrices wurden ausgehend von MAS (Methacryl¬oxypropyl¬trimethoxysilan) über den Sol-Gel Prozeß anorganisch und anschließend über photochemische Polymerisation zusätzlich organisch vernetzt. Die Quantifizierung des Polymerisationsgrads erfolgte über die Signalintensität der methacrylischen C=C-Doppelbindung bei 1635 cm-1 durch Integration einer Gauß-Funktion. Über den Polymerisationsgrad der organischen Matrix zwischen 0-71% konnte die Freisetzungskinetik von Heparin je nach therapeutischer Anforderung eingestellt werden. Es konnte gezeigt werden, daß hohe Wirkstoff-Beladungen und niedrige Polymerisationsgrade mit einer schnelleren Freisetzung des Heparins korrelierten, die aufgrund der Endlichkeit des Wirkstoffs im Depot einer Kinetik 1. Ordnung unterlag. Die kumulativ freigesetzten Wirkstoffmengen verhielten sich hierbei proportional zur Wurzel aus der Freisetzungszeit, was dem Higuchi-Modell zur Wirkstofffreisetzung aus porösen Matrices mit einem rein Diffusions-kontrollierten Mechanismus entsprach. Die durch Hydrolyse bedingte Degradation der anorganischen Matrix, die UV-VIS-spektroskopisch bei &#955; = 220 nm gemessen wurde, folgte einer Kinetik pseudo-0. Ordnung. Da das freigesetzte Heparin seine biologische Wirksamkeit beibehielt, sind P-MA-PS Matrices interessant für klinische Anwendungen, wie z.B. für die Beschichtung von Gefäßstützen, die im Blutkontakt stehen. / Aim of this work was the functionalization of titanium surfaces with the glycosaminoglycane heparin to improve the surface hemocompatibility for an application in the field of coronary stenting. Surface modification was performed using both TiO2 powder and titanium sheets as substrates imitating the (oxidized) surface of titanium implants. The substrates were modified with heparin to prevent side effects like blood coagulation and neointimal proliferation after implantation, which can both lead to restenosis of the acute artery closure. Surfaces can be modified either by covalent bonding of the drug to the metal by a silane spacer or by embedding the active agent into a polymer matrix for the controlled release over a certain period of time. Covalent attachment of heparin to titanium metal and TiO2 powder was carried out using the coupling agents 3-(Trimethoxysilyl)-propylamine (APMS), N-[3-(Trimethoxysilyl)-propyl]-ethylenediamine (Diamino-APMS) and N1-[3-(Trimethoxysilyl)-propyl]-diethylenetriamine (Triamino-APMS). Additionally, the influence of primary accomplished TiO2 films on the density of surface bound spacer was determined. Therefore, polished titanium substrates were covered with thin (< 2 µm) TiO2 layers by means of thermal/anodic oxidation, physical vapor deposition (PVD) or the sol-gel process and were then modified with the different spacer molecules, respectively. Aim was a correlation between the composition/topography of the TiO2 layers and the amount of bound spacer molecules. At this, two tendencies could be observed. Firstly, the density of surface bound spacer was generally highest for APMS (115-212 nmol/cm2) and lowest for Triamino-APMS (102-176 nmol/cm2), which was due to that smaller molecules underlie less intermolecular sterical hindrance onto surfaces. And secondly, on TiO2 surfaces with a higher roughness and therewith a higher specific surface area more spacer molecules could be immobilized (in the sequence: PVD > heat treated > anodized > sol-gel). The amount of surface bound coupling agent and heparin was quantified photometrically by the ninhydrin reaction and the toluidine-blue test. The biological potency of heparin was determined photometrically by the chromogenic substrate Chromozym TH and fibrinogen adsorption on the modified surfaces was investigated using the QCM-D (Quartz Crystal Microbalance with Dissipation Monitoring) technique. Zeta-potential measurements confirmed the successful coupling reaction; the zeta-potential of the unmodified anatase surface (approx. -26 mV) shifted into the positive range (> +40 mV) after silanisation. Binding of heparin resulted in a strongly negatively charged surface with zeta-potentials of approx. 39 mV. The successful heparinization could also be followed using RAMAN-spectroscopy by the occurrence of the peak at 1040 cm-1 (S=O vibration) caused by the drug. The amount of covalently attached primary amino groups on TiO2 powder was the highest for APMS (20 NH2/nm2) and decreased for Di- and Triamino-APMS (19 & 17 NH2/nm2). The hereby accomplished aminosilane layers were all in the range of multilayers since a monolayer is approx. 2 3 NH2/nm2. On the APMS spacer, the greatest amount of 53.3±3.6 ng/cm2 heparin was immobilized compared to Di- and Triamino-APMS (41.2±6.9 & 32.1±5.7 ng/cm2). The retaining biological activity of heparin was found to be the highest (70%) for the covalent attachment with Triamino-APMS as coupling agent due to the long chain of this spacer molecule and therefore the highest mobility of the drug. Moreover, this work aimed to investigate the use of an organically modified porous silica matrix (Poly(methacryloxypropyl)-poly(silsesquioxane); P-MA-PS) as a release system for heparin. The matrices were obtained from the precursor methacryloxypropyltrimethoxysilane (MAS) via the sol-gel process under acidic conditions following photochemical polymerization and cross-linking of the organic matrix. Modulation of the polymerization degree of the organic matrix in the range 0-71% allowed to adjust the release kinetics of heparin according to therapeutic needs. It was demonstrated that higher drug loads and a decreasing polymerization degree resulted in a faster release profile of heparin, which followed a square root of time kinetic according to the Higuchi model. The hydrolytic degradation of the xerogel was found to follow a zero-order kinetic whereas the heparin concentration did not show an influence on the degradation rate of the anorganic matrix. Since the released heparin retained its biological activity, P-MA-PS matrices may be interesting for clinical application, for instance as coating on drug eluting coronary stents.

A Thesis Submitted to the Faculty of Medicine University of the Witwatersrand, Johannesburg in fulfillment of the requirements for the Degree of Doctor of Science in Medicine Boston, U.S.A. 1983 / Vascular smooth muscle proliferation follows upon endothelial injury, and is thought to be an early component in the pathogenesis of atherosclerosis, and a possible noxious consequence of vascular surgery. We have shown that heparin suppresses vascular smooth muscle proliferation iri vivo and in vitro. The inhibitory effect is specific for heparin, and not other anions, and is not related to the antithrombin III binding activity of heparin. It is dependent on the size of the molecule, (hexasaccharidees or smaller being ineffective), and O-sulfation, but not N-sulfation / IT2018

Heparin is a mixture of sulfonated polysaccharides which is negatively charged. Heparin is a substance which is important in organism and fundamentally affects its physiology. Main attribute of heparin is anticoagulation - it prevents the complete blood coagulation. This anticoagulant effect balances the hemocoagulation by influencing the coagulation pathway. In some cases a pharmacological application of heparin is needed so the heparin is administrated as a injection of physiological solution of sodium or calcium heparine salt. Monitoring of level of the heparin in blood is problematic - methods used today are based on the measurement of a time required for blood clot formation. The result evaluation is done by comparing a sample with reference solution. These methods are relatively imprecise, can not be used in "on-line" setting and are highly influenced by general health condition of patient. In this work some principles of affinity capillary electrophoresis were adapted from another work - heparin was determined indirectly by monitoring of decrease of the peak area of protamine. Protamine is medically used antidote of heparin because they create a stable complex together. In this work protamine was replaced by well defined tetraarginine because the most frequent amino acid in protamine is...

Class of 2006 Abstract / Objectives: To develop and evaluate a data collection form that could be used to assess the influence of heparin administration on the septic severity index score in patients with Candida blood stream infections. Methods: A data collection form was developed to evaluate the influence of heparin administration on the septic severity index score in patients with Candida blood stream infections. This form was assessed using rating scales for ease of use, applicability of data collection items and availability of data in charts. Results: Data from 10 patient charts was used to assess the instrument. Patient demographics were similar. The strengths of the form included applicability of 9 out of 10 items in the instrument. Four out of 10 items were rated low for ease of use. In addition, 7 out of 10 pertinent data items were not documented in the charts. Conclusion: An extensive reworking of the data collection form as described in the discussion section was required. The form is ready to be implemented if the proposed study should be performed.

SUMMARY Mast cell activation in allergic and inflammatory disease causes increased vascular permeability and edema. This thesis identifies a paracrine mechanism, by which heparin released from intracellular granules, is involved in mast cell-evoked alteration of endothelial barrier function in vivo. Negatively charged heparin initiated factor XII-driven contact activation. Activated factor XII triggered the formation of the inflammatory mediator bradykinin in plasma. Congenital deficiency and pharmacological targeting of factor XII and kinin B2 receptor provided protection from mast cell-heparin-induced leukocyte-endothelial adhesion and hypotension in rats and mice. Intravital laser scanning microscopy and tracer measurements showed that heparin increased leakage with fluid extravasation in skin microvessels in mice. Deficiency in factor XII or kinin B2 receptor conferred resistance to heparin-induced skin edema and largely protected mice from endothelial barrier dysfunction, caused by allergen-induced mast cell activation and anaphylactic reactions. In contrast, heparin and mast cell activation caused excessive edema formation in mice, deficient in the major inhibitor of factor XII, C1 esterase inhibitor. Hereditary angioedema patients, lacking C1 esterase inhibitor, suffered from allergeninduced edema. The data indicate that mast cell-heparin-initiated bradykinin formation plays a fundamental role in defective barrier function of pathological mast cell-mediated inflammation, hypotension and edema formation. / ZUSAMMENFASSUNG Aktivierte Mastzellen sind bei Allergien und Entzündungskrankheiten an der Ödembildung beteiligt. Diese Arbeit zeigt, wie von aktivierten Mastzellen freigesetztes Heparin die Gefäßpermeabilität erhöht. Mastzell-Heparin aktiviert im Plasma den Blutgerinnungsfaktor XII. Aktiver Faktor XII startet die Bildung des Entzündungsmediators Bradykinin durch Plasmakallikrein-vermittelte Spaltung von hochmolekularem Kininogen. Bradykinin führt zur Ödembildung und Vasodilatation. Bei Ratten und Mäusen blockieren Faktor XII- oder Kinin B2 Rezeptor-Inhibitoren die Heparin-induzierte und Bradykinin-vermittelte Leukozyten-Endothel Adhäsion und den arteriellen Blutdruckabfall. Um den Heparin-vermittelten Flüssigkeitsaustritt aus Mikrogefäßen in der Haut von Mäusen zu analysieren, wurde eine intravitale konfokale Mikroskopietechnik etabliert. Genetische Inaktivierung oder pharmakologische Blockierung von Faktor XII oder Kinin B2 Rezeptoren hemmen den Heparinvermittelten Flüssigkeitsaustritt. Sowohl in systemischen als auch in cutanen passiven Anaphylaxiemodellen waren Faktor XII- und Kinin B2 Rezeptor-defiziente Mäuse vor Allergen-induzierten Ödemen und anaphylaktischen Reaktionen geschützt. Im Gegensatz dazu führte eine Allergenexposition zu einer überschiessenden Ödembildung in C1 Esterase Inhibitor-defizienten Mäusen, bei denen das Gen für den wichtigsten Inhibitor von Faktor XII inaktiviert wurde. Bei Hereditären Angioödem- Patienten, denen funktionaler C1 Esterase Inhibitor fehlt, lösen allergischen Reaktionen Ödemattacken aus. Zusammenfassend zeigen diese Daten erstmals, dass die durch Heparin gestartet Bradykinin-Bildung, eine bedeutende Rolle bei der fehlerhaften Barrierenfunktion der pathologischen Mastzell-vermittelten Entzündungen, Blutdruckabfall und Ödembildung spielt.

Unfractionated heparin (UFH) therapy is frequently used in tertiary paediatric healthcare facilities despite a lack of paediatric-specific research informing the optimal therapeutic intensity, monitoring recommendations or side-effect-profile in infants and children. As a result, the majority of clinical recommendations regarding UFH management in children have been extrapolated from adult evidence. The process of developmental haemostasis, in association with the variable pathogenesis of thromboembolic disease (TED) in children compared to adults, suggests that extrapolation of adult guidelines for UFH management to children is not ideal. / This study hypothesised that the process of developmental haemostasis would influence both the action and effect of UFH in children of different ages. This hypothesis was tested by addressing the following aims: 1. To determine the pharmacokinetics (PK) of UFH in children of different ages; 2. To compare the different methods of monitoring UFH in children of different ages; 3. To identify the impact of competitive plasma binding of UFH in children of different ages; 4. To determine the impact of UFH upon tissue factor pathway inhibitor (TFPI) release in children. / A prospective cohort study of children receiving a single bolus dose of UFH for primary thromboprophylaxis in the setting of cardiac angiography was conducted. Venous blood samples were collected prior to the UFH, then at 15, 30, 45 and 120 minutes post-UFH bolus. Laboratory assays performed included activated partial thromboplastin time (APTT), anti-Xa assay, anti-IIa assay, thrombin clotting time (TCT), protamine titration and TFPI. Levels of two plasma proteins known to competitively bind UFH (vitronectin and platelet factor 4) were determined and the impact of competitive plasma binding upon UFH activity, as measured by the anti-Xa assay, was quantified. A population approach to pharmacokinetic analysis, based on protamine titration results, was performed using WinNonMix™ Professional 2.0.1 (®1998-2000 Pharsight Corporation, Mountain View, CA, USA). Results were analysed according to the following age-groups: less than one year; one to five years; six to ten years; 11-16 years. / Sixty-four children were recruited, ranging in age from six months to fifteen-and-ahalf years. The mean dose/Kg of UFH across the entire cohort was 90.9± 15.5 IU/Kg. / Pharmacokinetic model specifications were systematically assessed, investigating the impact of parameter covariates and different error models upon objective function value and/or curve fitting. A first-order kinetic model best fitted the data. This model used weight 0.75 as the covariate of clearance and total weight as the covariate for volume of distribution. Parameter estimates for clearance and volume of distribution both demonstrated variance from adult and small neonatal PK studies of UFH, however methodological differences in PK analysis techniques limited comparisons. The half-life of UFH reported in this study was consistently and significantly shorter than that previously reported for adults, but longer than that reported for neonates. / All measures of UFH-effect demonstrated a significant and prolonged increase post- UFH bolus. The mean APTT was 261 seconds 102 ± 25 minutes post-UFH, representing a seven-fold increase from the mean baseline APTT (38 seconds). Anti- Xa assay levels were within the therapeutic range for TED management (0.35 to 0.7 IU/mL), or greater, at every post-UFH bolus timepoint. This prolonged UFH-effect was evident to nearly two hours post-UFH bolus, without concurrent UFH infusion. Age-related differences in UFH-response were evident for anti-Xa, anti-IIa and protamine titration results. Furthermore, during periods of high UFH concentration, the ratio of anti-Xa to anti-IIa activity in children less than one year of age significantly favoured UFH-mediated anti-Xa effect over anti-IIa effect (1.9), compared to teenagers (1.3). / This study demonstrated poor correlation between protamine titration and both the anti-Xa assay (r2 = 0.47) and APTT (r2 = 0.56). Use of the anti-Xa assay (0.35 to 0.7 IU/mL) or protamine titration assay (0.2 to 0.4 IU/mL) to establish APTT-based reference ranges for therapeutic management of TED resulted in APTT ranges with upper limits greater than 250 seconds. / No age-related quantitative differences in plasma levels of vitronectin or platelet factor 4 were identified across the childhood years. The addition of dextran sulphate (DS) to ex vivo study samples demonstrated no change in anti-Xa activity in samples collected within 20 minutes of UFH bolus, however a significant increase in anti-Xa activity following the addition of DS was evident at all later timepoints post-UFH bolus. / The measurement of TFPI before and after a single bolus dose of UFH demonstrated children have a similar immediate increase in TFPI activity following intravenous UFH compared to adults. However, the children in this series demonstrated a significantly prolonged level of increased TFPI activity, out to 102 ± 25 minutes post-UFH, compared to that reported in adult patients. / This study has developed the first paediatric-specific PK profile of UFH and has elucidated a number of age-dependent UFH-mechanisms of action that contribute to the previously reported age-dependent response to UFH in children. The results of this study support the hypothesis that developmental haemostasis influences both the action and effect of UFH in children of different ages.

Thesis (M.S.)--Oregon State University, 2007. / Printout. Includes bibliographical references (leaves 56-62). Also available on the World Wide Web.

Several studies have found a positive correlation between hypertension and hyperhomocysteinemia. Increasing evidence implicates oxidative stress as one of the initiating events closely linked to the homocysteines ability to damage endothelium, subsequently causing vascular dysfunction. We previously found that heparin protects cultured endothelial cells from free radical injury and oral heparin at 1 mg/kg/48h prevents venous thrombosis in a rat model in vivo. Our objective was to study the protective effects of oral heparin in a rat model with elevated plasma homocysteine (Hcy) concentrations, and begin to elucidate whether the pathophysiological effects of Hcy are mediated through an oxidative mechanism causing endothelial dysfunction.<p> Elevated plasma Hcy levels were induced by feeding male Wistar Kyoto rats a diet containing an additional 1.7% methionine for 8 weeks. Groups included rats fed additional methionine, methionine plus oral heparin (1 mg/kg/48h by gastric feeding tube), and age-matched controls fed normal rat chow. At the end of 8 weeks of treatment, rats were anesthetized using 1.5% isoflurane in 100% oxygen. Hemodynamics parameters were assessed by inserting a Millar Mikro Tip pressure transducer into the left ventricular chamber and the thoracic aorta. Fasting plasma total Hcy levels were measured using a Hcy immunoassay kit with an Abbott IMx instrument. Malondialdehyde (MDA) concentrations, a lipid peroxidation product and marker for oxidative stress, was measured by a spectrophotometric method in serum and tissue samples. Glutathione (GSH) concentrations, an important antioxidant for low-level oxidative stress was measured by HPLC in plasma and tissues samples. Lastly, tissue samples from each experimental group were stained with the TUNEL method to assess their respective percentage of apoptotic endothelial cells. Results were expressed as mean ± S.E. Unpaired Students two-tailed t-test was employed to assess the difference between groups with p < 0.05 considered significant.<p> Plasma Hcy was significantly elevated after 8 weeks in the methionine (7.17 ± 0.46 umol/L) and methionine plus heparin treated rats (7.02 ± 0.40 umol/L) compared to control (5.46 ± 0.36 umol/L). All measures of arterial pressure, systolic (SP) and diastolic pressure (DP) and mean arterial pressure (MAP), were significantly elevated in rats fed the methionine diet without heparin (119.9 ± 3.9 mmHg; 90.3 ± 3.5 mmHg; 97.7 ± 2.9 mmHg, respectively) compared to controls (107.8 ± 2.5 mmHg; 79.2 ± 2.1 mmHg; 88.8 ± 2.2 mmHg, respectively) but not compared to heparin (114.7 ± 3.3 mmHg; 83.4 ± 2.4 mmHg; 93.8 ± 2.7 mmHg, respectively). Left ventricular end diastolic pressure (LVEDP) was significantly elevated with the methionine diet without heparin (14.2 ± 2.5 mmHg) but not with heparin treatment (8.4 ± 1.9 mmHg) versus controls (7.1 ± 1.1 mmHg). Also, left ventricular systolic pressure (LVSP) was significantly elevated in the methionine fed rats after 8 weeks (122.6 ± 3.2 mmHg) compared to controls (112.3. ± 2.9 mmHg). Heparin treatment had no effect on LVSP (119.9 ± 3.2 mmHg). <p> Additionally, the results of this study showed that oral heparin treatment significantly decreased liver MDA concentrations (2.42 ± 0.28 nmol/mg protein) compared to the methionine treated group (5.10 ± 0.96 nmol/mg protein) and methionine treatment alone significantly reduced MDA concentrations in kidney tissue (1.59 ± 0.12 nmol/mg protein) compared with controls (3.26 ± 0.66 nmol/mg protein). Methionine diet significantly decreased GSH concentrations in plasma (0.59 ± 0.59 µmol/L) compared with controls (4.24 ± 0.94 µmol/L) and oral heparin treatment significantly attenuated the decrease in GSH concentrations in left ventricle tissue samples (0.0229 ± 0.0023 µmol/mg protein) compared with methionine treatment alone (0.0135 ± 0.0016 µmol/mg protein). <p> Elevated plasma homocysteine levels, induced by methionine diet feeding significantly increased the percent of apoptotic endothelial cells in the aortas (17.04 ± 3.74%) and superior mesenteric arteries (17.99 ± 1.90%) of WKY rats compared with control aortas and mesenteric arteries (6.08 ± 3.24%; 7.43 ±1.62%, respectively) and compared to oral heparin treated mesenteric arteries (7.31 ± 1.18%). <p> The results of this study showed that elevated plasma levels of Hcy correlate with the development of hypertension, defined as significantly increased arterial pressure. Oral heparin treatment prevented the significant increase in arterial pressures and LVEDP, decreased MDA concentrations and therefore the oxidative stress on the liver, attenuated the decrease caused by elevated plasma Hcy in left ventricle GSH concentrations, and significantly reduced the number of apoptotic endothelial cells in the superior mesenteric artery of high methionine fed rats. We conclude that elevated levels of plasma Hcy contributes to the development of hypertension and furthermore towards the onset of heart failure likely through an oxidative mechanism and that oral heparin reduces the overall oxidative stress in specific physiological environments, preventing Hcy mediated endothelial cell apoptosis.

Several studies have found a positive correlation between hypertension and hyperhomocysteinemia. Increasing evidence implicates oxidative stress as one of the initiating events closely linked to the homocysteines ability to damage endothelium, subsequently causing vascular dysfunction. We previously found that heparin protects cultured endothelial cells from free radical injury and oral heparin at 1 mg/kg/48h prevents venous thrombosis in a rat model in vivo. Our objective was to study the protective effects of oral heparin in a rat model with elevated plasma homocysteine (Hcy) concentrations, and begin to elucidate whether the pathophysiological effects of Hcy are mediated through an oxidative mechanism causing endothelial dysfunction.<p> Elevated plasma Hcy levels were induced by feeding male Wistar Kyoto rats a diet containing an additional 1.7% methionine for 8 weeks. Groups included rats fed additional methionine, methionine plus oral heparin (1 mg/kg/48h by gastric feeding tube), and age-matched controls fed normal rat chow. At the end of 8 weeks of treatment, rats were anesthetized using 1.5% isoflurane in 100% oxygen. Hemodynamics parameters were assessed by inserting a Millar Mikro Tip pressure transducer into the left ventricular chamber and the thoracic aorta. Fasting plasma total Hcy levels were measured using a Hcy immunoassay kit with an Abbott IMx instrument. Malondialdehyde (MDA) concentrations, a lipid peroxidation product and marker for oxidative stress, was measured by a spectrophotometric method in serum and tissue samples. Glutathione (GSH) concentrations, an important antioxidant for low-level oxidative stress was measured by HPLC in plasma and tissues samples. Lastly, tissue samples from each experimental group were stained with the TUNEL method to assess their respective percentage of apoptotic endothelial cells. Results were expressed as mean ± S.E. Unpaired Students two-tailed t-test was employed to assess the difference between groups with p < 0.05 considered significant.<p> Plasma Hcy was significantly elevated after 8 weeks in the methionine (7.17 ± 0.46 umol/L) and methionine plus heparin treated rats (7.02 ± 0.40 umol/L) compared to control (5.46 ± 0.36 umol/L). All measures of arterial pressure, systolic (SP) and diastolic pressure (DP) and mean arterial pressure (MAP), were significantly elevated in rats fed the methionine diet without heparin (119.9 ± 3.9 mmHg; 90.3 ± 3.5 mmHg; 97.7 ± 2.9 mmHg, respectively) compared to controls (107.8 ± 2.5 mmHg; 79.2 ± 2.1 mmHg; 88.8 ± 2.2 mmHg, respectively) but not compared to heparin (114.7 ± 3.3 mmHg; 83.4 ± 2.4 mmHg; 93.8 ± 2.7 mmHg, respectively). Left ventricular end diastolic pressure (LVEDP) was significantly elevated with the methionine diet without heparin (14.2 ± 2.5 mmHg) but not with heparin treatment (8.4 ± 1.9 mmHg) versus controls (7.1 ± 1.1 mmHg). Also, left ventricular systolic pressure (LVSP) was significantly elevated in the methionine fed rats after 8 weeks (122.6 ± 3.2 mmHg) compared to controls (112.3. ± 2.9 mmHg). Heparin treatment had no effect on LVSP (119.9 ± 3.2 mmHg). <p> Additionally, the results of this study showed that oral heparin treatment significantly decreased liver MDA concentrations (2.42 ± 0.28 nmol/mg protein) compared to the methionine treated group (5.10 ± 0.96 nmol/mg protein) and methionine treatment alone significantly reduced MDA concentrations in kidney tissue (1.59 ± 0.12 nmol/mg protein) compared with controls (3.26 ± 0.66 nmol/mg protein). Methionine diet significantly decreased GSH concentrations in plasma (0.59 ± 0.59 µmol/L) compared with controls (4.24 ± 0.94 µmol/L) and oral heparin treatment significantly attenuated the decrease in GSH concentrations in left ventricle tissue samples (0.0229 ± 0.0023 µmol/mg protein) compared with methionine treatment alone (0.0135 ± 0.0016 µmol/mg protein). <p> Elevated plasma homocysteine levels, induced by methionine diet feeding significantly increased the percent of apoptotic endothelial cells in the aortas (17.04 ± 3.74%) and superior mesenteric arteries (17.99 ± 1.90%) of WKY rats compared with control aortas and mesenteric arteries (6.08 ± 3.24%; 7.43 ±1.62%, respectively) and compared to oral heparin treated mesenteric arteries (7.31 ± 1.18%). <p> The results of this study showed that elevated plasma levels of Hcy correlate with the development of hypertension, defined as significantly increased arterial pressure. Oral heparin treatment prevented the significant increase in arterial pressures and LVEDP, decreased MDA concentrations and therefore the oxidative stress on the liver, attenuated the decrease caused by elevated plasma Hcy in left ventricle GSH concentrations, and significantly reduced the number of apoptotic endothelial cells in the superior mesenteric artery of high methionine fed rats. We conclude that elevated levels of plasma Hcy contributes to the development of hypertension and furthermore towards the onset of heart failure likely through an oxidative mechanism and that oral heparin reduces the overall oxidative stress in specific physiological environments, preventing Hcy mediated endothelial cell apoptosis.

Amperometric dectection of heparin and glucosamine in flow injection analysis