Hemostasis Laboratory

Hemostasis Laboratory Volume 7, Number 1, 2014 Table of Contents Triggering Thrombin Generation by Low Dose Dexamethasone Julia Klassen and Thomas Stief

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Modulation of Blood ROS Generation by Low Dose Dexamethasone Ricarda Stumpf and Thomas Stief

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Modulation of Blood ROS Generation by Pre-incubated Low Dose Dexamethasone Ruth Dannhäuser and Thomas Stief

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Triggering Thrombin Generation by High Dose Dexamethasone Christine Dippel and Thomas Stief

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Modulation of Blood ROS Generation by High Dose Dexamethasone Sandra Blumenau and Thomas Stief

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Pre-Incubation Transforms Dexamethasone into a Neutrophil Stimulator Daniela Petri and Thomas Stief

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Triggering Thrombin Generation by Low Dose Hydrocortisone Anna-Lena Neumeier and Thomas Stief

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Modulation of Blood ROS Generation by Low Dose Hydrocortisone Yonca Eroglu and Thomas Stief

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Reactive Oxygen Species (ROS) Generation Modulation by Prolonged Blood Incubation with Low Dose Hydrocortisone Dagmar Heinrich and Thomas Stief

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Paradoxical Increase of Blood ROS Generation by Pre-Incubated Low Dose Hydrocortisone Irina Grischenkov and Thomas Stief

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Triggering Thrombin Generation by High Dose Hydrocortisone Katharina Spengler and Thomas Stief

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Modulation of Blood ROS Generation by High Dose Hydrocortisone Frauke Englert and Thomas Stief

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Modulation of Blood ROS Generation by Pre-Incubated High Dose Hydrocortisone Aybike Süzer and Thomas Stief

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Imipenem/Cilastatin at Lowest Concentrations Triggers Thrombin Generation Anna Köster, Kathrin Zylius, and Thomas Stief

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Imipenem/Cilastatin Inhibits the ROS Generation of Blood Neutrophils Kathrin Zylius, Anna Köster, and Thomas Stief

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Pre-Incubated Imipenem/Cilastatin Stimulates Blood ROS Generation Julia Drajt and Thomas Stief

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Imipenem/Cilastatin: A Pathophysiologic Trigger of Altered Matrix Coagulation Anastasia Busch, Irina Bogun, and Thomas Stief High Dose Imipenem/Cilastatin Suppresses Blood ROS Generation Irina Bogun, Anastasia Busch, and Thomas Stief Urate Added At Ultra-Low Concentrations (0.1µM) to Normal Plasma Triggers Thrombin Generation Dana Stenzel and Thomas Stief

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Urate Triggers Thrombin Generation Laura Schorge and Thomas Stief

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Urate Inhibits Blood ROS Generation Christina Lichtenwald and Thomas Stief

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Urate Stimulates ROS Generation in Pre-Incubated Blood Johanna Grass and Thomas Stief

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Urate Stimulates ROS Generation in Stressed Blood Angela Mühling and Thomas Stief

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Urate Enhances ROS Generation in Pre-Incubated Oxidatively Stressed Blood Thomas Stief

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Blood Reactive Oxygen Species and Urate Thomas Stief, Christina Lichtenwald, Angela Mühling, Laura Schorge, Dana Stenzel, Johanna Grass, and Dagmar Heinrich

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Acetaminophen Suppresses the ROS Generation in Freshest Blood Ricarda Stumpf and Thomas Stief

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Taurine Strongly Enhances the ROS Generation of Blood Neutrophils Anastasia Busch and Thomas Stief

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Caffeine Modulates Blood ROS Generation Angela Mühling and Thomas Stief

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Arginine Suppresses Blood ROS Generation Christina Lichtenwald and Thomas Stief

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Luminescence Kinetic in the Blood ROS Generation Assay (BRGA) Thomas Stief

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Some 2-Macroglobulin/Thrombin Complexes Open by Contact Activation Thomas Stief

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New York

Hemostasis Laboratory Hemostasis (coagulation and fibrinolysis) is of importance in a broad range of medical disciplines. A good hemostasis diagnosis enables a good therapy, which can be rather complicated without laboratory help. “Hemostasis Laboratory” will increase the clinical and academic importance of hemostasis diagnostic. In this Journal, new relevant hemostasis assays are presented in analytical detail. New insights into the biochemistry and physiology of hemostasis are the basis for assay invention and are, therefore, also subjects of Hemostasis Laboratory. Hemostasis Laboratory publishes original articles and reviews on: ● Assay techniques in hemostasis (primary, secondary, tertiary hemostasis, i.e. thrombocytes, plasma, fibrinolysis) that are of clinical relevance; ● Biochemistry / pathobiochemistry of hemostasis that are of clinical relevance; ● Physiology / pathophysiology of hemostasis that are of clinical relevance. EDITOR-IN-CHIEF: Thomas W. Stief, MD Privatdozent in Laboratory Medicine and Hemostasis Department of Clinical Chemistry University Hospital Marburg, D-35043 Germany Hemostasis Laboratory is published quarterly by Nova Science Publishers, Inc. 400 Oser Avenue, Suite 1600 Hauppauge, New York, 11788-3619, U.S.A. Telephone: (631) 231-7269 Fax: (631) 231-8175 E-mail: [email protected] Web: ww.novapublishers.com ISSN: 1941-8493 Subscription Price per Volume (2014) Print: $295

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Hemostasis Laboratory Volume 7, Number 1

ISSN: 1941-8493 © Nova Science Publishers, Inc.

TRIGGERING THROMBIN GENERATION BY LOW DOSE DEXAMETHASONE Julia Klassen and Thomas Stief Institute of Laboratory Medicine and Pathobiochemistry, University Hospital of Marburg, Germany

ABSTRACT Background: The synthetic glucocorticoid dexamethasone is often given in clinical situations of inflammation where the normal blood coagulation is in the pre-phase of pathologic disseminated intravascular coagulation (PIC-0). Since dexamethasone by itself as an organic molecule with one delta-negativelay charged fluor atom and 5 deltanegatively charged oxygen atoms could behave as an important pathophysiologic trigger of altered matrix (AM) – coagulation, here dexamethasone was ultra-finely investigated for its intrinsic power to stimulate AM-mediated thrombin generation, using the recalcified coagulation activity assay (RECA). Material and Methods: 50 µl platelet poor plasma of 4 healthy donors in transparent high quality polystyrene microtiter U-well plates (Brand®781600) were supplemented with 0-0.48 mg/l dexamethasone by immediate repetitive 1+1 dilution on the plate. 5 µl 250 mM CaCl2 were added to start the RECA. After 0, 15, 18, 20 min at 37°C the RECA was stopped by addition of 100 µl 2.5 M arginine, 0.16% Triton X 100®, pH 8.6. After 3 min 25 µl fast chromogenic thrombin substrate HD-CHG-Ala-Arg-pNA in 1.25 M arginine, pH 8.7 were added and the specific increase in absorbance ΔA405 nm was measured by a microtiterplate photometer with a 1 mA resolution (PHOmo). The approximate 200% stimulatory concentrations (approx. SC200) were determined in the important ascending part of the incubation time vs. thrombin generation curve. Results: The approx. SC200 of dexamethasone to trigger AM-thrombin generation in fresh normal plasma supplemented with up to 0.48 mg/l dexamethasone was 0.02±0.01 mg/l dexamethasone (MV±1SD; range: 0.01-0.03 mg/l). Discussion: It is suggested to perform RECA for each individual who needs dexamethasone and to find out if he belongs to a subgroup of patients with increased susceptibility towards intrinsic hemostasis activation by dexamethasone, possibly changing to another glucocorticoid with better approx. SC200 values or combining the therapy with LMWH, aiming to an EXCA-value of about 20% of normal (about 0.5 IU/ml LMWH).

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Correspondence to: PD Dr. med. T. Stief, Central Laboratory, University Hospital, D-35043 Marburg, Germany. E-mail: [email protected]; Tel. : +49-6421-58 64471; FAX: +49-6421-58 65594.

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Julia Klassen and Thomas Stief

INTRODUCTION Dexamethasone is a strong drug against systemic or local inflammation [1]. In these clinical situations the bodie´s coagulation system is in enhanced systemic activation [2]. Normal intravascular coagulation (NIC) with systemic thrombin activity of 100±20% (MV±1SD; 100% = 5.5 mIU thrombin/ ml plasma) can easily change to the pre-phase of systemic intravascular coagulation (PIC-0) or even worse to the typical phase of systemic intravascular coagulation (PIC-1) [3,4]. Lipophilic or (delta-) negatively charged compounds trigger AM-coagulation (= contact phase coagulation = intrinsic coagulation) [5,6]. Dexamethasone, a molecular derivate of the lipophilic cholesterol with inserted one deltanegativelay charged fluor atom and 5 delta-negatively charged oxygen atoms (Figure 1) is an AM-trigger. All xenobiotics that enter the blood stream alter the blood matrix. With the invention of the RECA it became for the first time possible to quantify ultra-finely the discrete F12a/kallikrein/thrombin generation by drugs [7]. There is great inter-individual variety in the response of F12/pre-kallikrein to AM-triggers [8,9]. In a preliminary investigation we could find a blunted approx. SC200 value of approximately 2 mg/l dexamethasone if plasma had been supplemented with up to 100 mg/l dexamethasone [10]. The peculiar behavior of AM-coagulation that the approx. SC200 depends on the maximal concentration of drug added to plasma requires that typical clinical concentrations are chosen prior to plasma dilution [5,6,11]. Here a concentration of only 0.5 mg/l was chosen as the maximal concentration.

Figure 1. Chemical structure of dexamethasone [1]. 9-Fluor-11ß,17,21-trihydroxy-16α-methylpregna-1,4,diene-3,20-dione (C22H29FO5; molecular mass: 392.46 Daltons).

MATERIAL AND METHODS Dexamethasone was from ratiopharm, Ulm, Germany. The 1 ml dosage contained 4.37 mg dexamethasone dihydrogenphosphate disodium salt (= 4 mg dexamethasone dihydrogenphosphate. 50 µl platelet poor plasma of 4 healthy donors that gave written informed consent (4.5 ml venous blood + 0.5 ml 106 mM sodim citrate, pH 7.4 in polypropylene monovettes from Sarstedt (Nümbrecht, Germany) still valid for at least 1 year) in transparent high quality polystyrene microtiter U-well plates (Brand, Wertheim, Germany; article nr. 781600) were supplemented with 0-0.48 mg/l dexamethasone by immediate repetitive 1+1 dilution on the plate. 5 µl 250 mM CaCl2 (Sigma, Deisenhofen, Germany) were added to start the RECA [12,13]. After 0, 15, 18, 20 min at 37°C the RECA was stopped by addition of 100 µl 2.5 M arginine, 0.16% Triton X 100®, pH 8.6. After 3 min 25 µl fast

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chromogenic thrombin substrate HD-CHG-Ala-Arg-pNA (Pentapharm, Basel, Switzerland) in 1.25 M arginine, pH 8.7 were added and the specific increase in absorbance ΔA405 nm was measured by a microtiterplate photometer with a 1 mA resolution (PHOmo; anthos, Krefeld, Germany). The thrombin standard was 40 mIU/ml bovine thrombin (Siemens-DadeBehring, Marburg, Germany) in 5% human albumin (CSL Behring, Marburg, Germany). 50 µl thrombin standard replaced the plasma in 4 wells and resulted in a mean specific ΔA of 1.1 mA/min (37°C). The substrate cleavage was linear up to 40% of the maximal obtainable ΔA which was 1000 mA. The approximate 200% stimulatory concentrations (approx. SC200) were determined in the important ascending part of the incubation time vs. thrombin generation curve, where supra-molar arginine has eliminated the negative effect of antithrombin-1 (nascent uncrosslinked fibrin) [14]. The RECA is the assay to establish even discrete (but clinically relevant) pathologically enhanced thrombin generations (approx. SC200), for decreased thrombin generations (approx. IC50) the INCA (intrinsic coagulation activity assay) or the EXCA (extrinsic coagulation activity assay) are better suited [4].

RESULTS AND DISCUSSION Figures 2-5 demonstrate that the approx. SC200 of dexamethasone to trigger AM-thrombin generation in fresh normal plasma supplemented with up to 0.48 mg/l dexamethasone was 0.02±0.01 mg/l dexamethasone (MV±1SD; range: 0.01-0.03 mg/l).

Figure 2. AM-coagulation activation by dexamethasone in normal plasma 1. The RECA was performed in normal fresh citrated platelet poor plasma 1 that had been supplemented on the microtiter plate with 0-0.48 mg/l dexamethasone by immediate repetitive 1+1 dilution. The approx. SC200 of dexamethasone on intrinsic F2a generation was 0.01 mg/l, seen in the most sensible curve, here RECA-20 (coagulation reaction time (CRT) = 20 min). Some RECA-20 values had to be omitted because they were not in the ascending part of the CRT vs. thrombin generation curve.

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Julia Klassen and Thomas Stief

Figure 3. AM-coagulation activation by dexamethasone in normal plasma 2. The RECA was performed in normal fresh citrated platelet poor plasma 2 that had been supplemented on the microtiter plate with 0-0.48 mg/l dexamethasone by immediate repetitive 1+1 dilution. The approx. SC200 of dexamethasone on intrinsic F2a generation was 0.01 mg/l, seen in the most sensible curve, here RECA-18 (coagulation reaction time (CRT) = 18 min). The RECA-18 y-value for x=0.06 had to be omitted because it was not in the ascending part of the CRT vs. thrombin generation curve.

Figure 4. AM-coagulation activation by dexamethasone in normal plasma 3. The RECA was performed in normal fresh citrated platelet poor plasma 3 that had been supplemented on the microtiter plate with 0-0.48 mg/l dexamethasone by immediate repetitive 1+1 dilution. The approx. SC200 of dexamethasone on intrinsic F2a generation was 0.01 mg/l, seen in the most sensible curve, here RECA-15 (coagulation reaction time (CRT) = 18 min). The RECA-18 and RECA-20 curves had to be omitted because they were not in the ascending part of the CRT vs. thrombin generation curve.

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Figure 5. AM-coagulation activation by dexamethasone in normal plasma 4. The RECA was performed in normal fresh citrated platelet poor plasma 4 that had been supplemented on the microtiter plate with 0-0.48 mg/l dexamethasone by immediate repetitive 1+1 dilution. The approx. SC200 of dexamethasone on intrinsic F2a generation was 0.03 mg/l, seen in the most sensible curve, here RECA-15 (coagulation reaction time (CRT) = 15 min). The y-value of x=0.12 of the RECA-20 curve had to be omitted because it was not in the ascending part of the CRT vs. thrombin generation curve.

It is suggested to perform RECA for each individual that needs dexamethasone and to find out if he belongs to a subgroup of patients with increased susceptibility towards intrinsic hemostasis activation by dexamethasone, possibly changing to another glucocorticoid with better approx. SC200 values or combining the therapy with LMWH (low-molecular-weightheparin), the target would be an EXCA-value of about 20% of normal (about 0.5 IU/ml LMWH) [15,16].

ACKNOWLEDGMENTS The research work of this article has been performed within an intensive hemostasis training course of medical technicians (Marburg University Hospital MTA School) There was neither specific funding nor any conflict of interest.

REFERENCES [1] [2]

www.wikipedia.org Stief TW. Coagulation activation by lipopolysaccharides. Clin Appl Thrombosis/Hemostasis Dec 26, 2007 doi: 10.1177/1076029607309256; 2009; 15: 20919.

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Julia Klassen and Thomas Stief Stief TW. The laboratory diagnosis of the pre-phase of pathologic disseminated intravascular coagulation. Hemostasis Laboratory 2008; 1: 2-20. Stief TW. Thrombin – applied clinical biochemistry of the main factor of coagulation. In: Thrombin: function and pathophysiology. Stief T, ed.; Nova science publishers; New York; 2012; pp. vii-xx. https://www.novapublishers.com/catalog/product_ info.php?products_id=33386 Stief TW. Contact activation of coagulation depends on the maximal lipophilic trigger concentration. Blood Coagulation and Fibrinolysis 2012; 23: 296-8. Stief TW. The maximal plasma concentration of (delta-) negatively charged contact triggers influences plasmatic thrombin generation. In: Thrombin: function and pathophysiology. Stief T, ed.; NOVA science publishers; New York; 2012; pp. 37-46. Stief TW. Drug - induced thrombin generation: the breakthrough. Hemostasis Laboratory 2010; 3: 3-6. Stief TW. Thrombin generation by creatinine. Hemostasis Laboratory 2011; 4: 191-9. Stief TW. Thrombin generation by therapeutic fibrinogen. Hemostasis Laboratory 2011; 4: 467-82. Stief TW. Thrombin generation by corticoids. Hemostasis Laboratory 2010; 3: 71-6. Stief TW, Mohrez M. HMWK increases of decreases F12a generation dependent on the contact trigger concentration in two purified systems. Hemostasis Laboratory 2012; 5: 51-65. Stief TW. Pathological thrombin generation by the synthetic inhibitor argatroban. Hemostasis Laboratory 2009; 2: 83-104. Stief TW. Thrombin triggers its generation in individual platelet poor plasma. Hemostasis Laboratory 2009; 2: 363-78. Mosesson MW. Update on antithrombin I (fibrin). Thromb Haemost. 2007; 98: 105-8. Stief TW. 20% EXCA (0.1 IU/ml thrombin) – the ideal anticoagulant target value. Hemostasis Laboratory 2011; 4: 275-80. Stief TW. LMWH - action-monitoring for all patients. Acta Paediatr. 2012; 101: e314. 10.1111/j.1651-2227.2012.02712.x.



MODULATION OF BLOOD ROS GENERATION BY LOW DOSE DEXAMETHASONE Ricarda Stumpf and Thomas Stief Institute of Laboratory Medicine and Pathobiochemistry, University Hospital of Marburg, Germany

ABSTRACT Background: Dexamethasone (9-Fluor-16α-methylprednisolone) is a synthetic glucocorticoid without mineralocorticoid action that is about 20fold stronger than hydrocortisone. It can be clinically used against systemic inflammation. Dexamethasone induces the synthesis of cytochrome P450 (CYP450). Due to its delta-negative fluor – atom and its 5 oxygen – atoms it might also stimulate the phagocytes. Here we investigated the modulating action of dexamethasone on blood neutrophils, measured by the new blood ROS (reactive oxygen species) generation assay (BRGA). Material and Methods: 40 µl dexamethasone in 0.9% NaCl (final drug conc.: 0-5 mg/l) in black high quality polystyrene microtiter plates (Brand®781608) and 125 µl Hanks´ Balanced Salt Solution (HBSS) were incubated in duplicate with 10 µl fresh citrated blood of 5 healthy donors, 10 µl 5 mM luminol, and 10 µl zymosan A in 0.9% NaCl (final conc.: 0.5 µg/ml or 1.9 µg/ml). After 0-181 min (37°C) the light emissions of the wells were measured by a photons-multiplying microtiter plate luminometer (LUmo) with an integration time of 0.5s/well. Results: The approx. IC50 of dexamethasone on blood ROS generation in BRGA-23 (BRGA with 23 min reaction time) were 0.1 mg/l for samples 1-3. Samples 4 and 5 had approx. IC50 values of 0.2 or 0.5 mg/l, respectively. The approx. IC50 of the mean ROS generation values was 0.1 mg/l. The approx. IC50 of dexamethasone on blood ROS generation in BRGA-48 were 0.1, 0.2, or 0.1 mg/l for 3 samples. 2 samples had no approx. IC50. Instead they had an approx. SC200 of 0.1 mg/l. Discussion: Many patients might be resistant to inhibition of blood ROS generation by dexamethasone, only roughly 60% of blood samples responded to dexamethasone with a favorable IC50 of 0.1-0.2 mg/l at 48 min reaction time. Presumably in many patients dexamethasone with its organically bound fluor and 5 oxygens might be CYP450-metabolized time-dependently into a cell stimulator. Normally, dexamethasone is a drug against hyper-activated phagocytes. There might be many patients that would benefit more from another glucocorticoid (preferably without organic halogen).

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Correspondence to: PD Dr. med. T. Stief, Central Laboratory, University Hospital, D-35043 Marburg, Germany. Email: [email protected]; Tel. : +49-6421-58 64471; FAX: +49-6421-58 65594.

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Ricarda Stumpf and Thomas Stief

INTRODUCTION The long-acting synthetic glucocorticoid dexamethasone is an anti-inflammatory and an immunosuppressant e.g. against rheumatoid arthritis or bronchospasm [1-7]. In autoimmune thrombocytopenia the typical dosage is 40 mg/d for 4 days with 10 days rest [1,8]. In allergic anaphylactic shock high doses of dexamethasone can be useful; many eye drops, devices [9] or nasal sprays contain dexamethasone; in ear drops dexamethasone could be combined with an antibiotic and an antifungal [1]. In bacterial meningitis dexamethasone reduces hyperinflammation by bacterial mediators (Herxheimer reaction) [10]. It is also given against postoperative vomiting [11,12] or in cerebral migraine or edema which e.g. occurs in altitude sickness [13,14]. In multiple myeloma dexamethasone is combined with thalidomide, bortezomib, adriamycin, or vincristine [1,15-20]. The drug is also used in chronic lymphatic leukemia [21] and to induce an iatrogenic osteoporosis to combat better bone metastasis [22]. Dexamethasone promotes the ripening of the fetal lung (synthesis of surfactant factor) [23] and is usually anti-asthmatic [24,25]. The dexamethasone suppression test diagnoses the possible existence of Cushing´s syndrome [1]. The present work is on the modulating action of dexamethasone on blood neutrophils, measured by the new blood ROS generation assay (BRGA) [26].

MATERIAL AND METHODS Dexamethasone was from ratiopharm, Ulm, Germany. The 1 ml dosage contained 4.37 mg dexamethasone dihydrogenphosphate disodium salt (= 4 mg dexamethasone dihydrogenphosphate. 40 µl 0-24.4 mg/l dexamethasone in 0.9% NaCl (final conc.: 0-5 mg/l) in black high quality polystyrene microtiter plates (Brand, Wertheim, Germany; article nr. 781608) and 125 µl Hanks´ Balanced Salt Solution (HBSS; Sigma, Deisenhofen, Germany) were incubated in duplicate with 10 µl fresh citrated blood (venous blood supplemented with 10.6 mM sodium citrate, pH 7.4 in polypropylene tubes from Sarstedt, Nümbrecht, Germany) of 5 healthy donors that gave written informed consent, 10 µl 5 mM luminol (Sigma), and 10 µl zymosan A (ZyA; Sigma) in 0.9% NaCl (final conc.: 0.5 µg/ml or 1.9 µg/ml). After 0-181 min (37°C) the light emissions of the wells were measured by a photons-multiplying microtiter plate luminometer (LUmo; anthos, Krefeld, Germany) with an integration time of 0.5s/well. The measured data were transferred via Autosoft® to Excel data sheets. The approximate 50% inhibitory concentrations (approx. IC50) or the approximate 200% stimulatory concentrations (approx. SC200) of dexamethasone on ROS blood generation were determined.

RESULTS AND DISCUSSION At 48 min incubation time the maximum of 1424-4066 RLU/s was reached in 4 of 5 samples, when stimulated by 1.9 µg/ml zymosan A. One sample reached the maximum of 861 RLU/s not until 100 min reaction time (Figure 1). 0.5 µg/ml ZyA-stimulated blood had a much smaller first maximum of 18 RLU/s, reached at 48 min (Figure 2).

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The approximate 50% inhibitory concentrations (approx. IC50) of dexamethasone on blood ROS generation in BRGA-23 were 0.1 mg/l for samples 1-3. Samples 4 and 5 had approx. IC50 values of 0.2 or 0.5 mg/l, respectively. The approx. IC50 of the mean values was 0.1 mg/l (Figure 3). The approx. IC50 of dexamethasone on blood ROS generation in BRGA-48 were 0.1, 0.2, or 0.1 mg/l for samples 1, 2, 3. Samples 4 and 5 had no approx. IC50. Instead they had an approx. SC200 of 0.1 mg/l (Figure 4a, 4b). The IC50 or SC200 determinations have to be done individually, the mean values of all 5 samples resulted in neither IC50 nor SC200 (Figure 4c). Presumably in many patients dexamethasone with its organically bound fluor and 5 oxygens (Figure 5) might be CYP450-metabolized into a cell stimulator [27-31]. Dexamethasone induces CYP450 and might be hydroxylated into a substance that stimulates phagocytes [32]. Normally, dexamethasone is a drug against hyperactivated phagocytes [33-37].

Figure 1. Kinetic of blood ROS generation upon 1.9 µg/ml ZyA stimulation. The BRGA was performed in 5 different citrated blood samples, final trigger conc. 1.9 µg/ml zymosan A. At 48 min the maximum of 1424-4066 RLU/s was reached in 4 of 5 samples. One sample reached the maximum of 861 RLU/s not until 100 min reaction time. Figure 1a: individual samples, Figure 1b: mean values of the 5 samples.

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Figure 2. Kinetic of blood ROS generation upon 0.5 µg/ml ZyA stimulation. The BRGA was performed in a citrated blood sample, final trigger conc. 0.5 µg/ml zymosan A. At 48 min the first maximum of 18 RLU/s was reached, at > 180 min appeared the second maximum.


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Figure 3. Inhibition of blood ROS generation by dexamethasone. The BRGA values at 23 min reaction time (BRGA-23) were compared for the 5 samples; Figure 3a: x-axis 0-5 mg/l, Figure 3b: x-axis 0-1 mg/l, Figure 3c: mean values of the 5 samples. The approx. IC50 of dexamethasone on blood ROS generation were 0.1 mg/l for samples 1-3. Samples 4 and 5 had approx. IC50 values of 0.2 or 0.5 mg/l, respectively. The approx. IC50 of the mean values was 0.1 mg/l.

The present work demonstrates that there might be many patients that would benefit more from another glucocorticoid (preferably halogen-less). To the known adverse reactions of glucocorticoids (e.g. suppression of cellular fibrinolysis [20,38]) another adverse reaction has to be added: paradoxical stimulation of blood ROS generation. This varies from individual to individual and should be checked prior to administration of the respective drug or device [9].

Figure 4. Continued on next page.

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Figure 4. Inhibition of blood ROS generation by dexamethasone. The BRGA values at 48 min reaction time (BRGA-48) were compared for the 5 samples; Figure 4a: x-axis 0-5 mg/l, Figure 4b: x-axis 0-1 mg/l, Figure 3c: mean values of the 5 samples. The approximate 50% inhibitory concentrations (approx. IC50) of dexamethasone on blood ROS generation were 0.1, 0.2, or 0.1 mg/l for samples 1, 2, 3. Samples 4 and 5 had no approx. IC50. Instead they had an approx. SC200 of 0.1 mg/l.



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ACKNOWLEDGMENTS The research work of this article has been performed within an intensive hemostasis training course of medical technicians (Marburg University Hospital MTA School). There was neither specific funding nor any conflict of interest.

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www.wikipedia.org Gordon PA, Winer JB, Hoogendijk JE, Choy EH. Immunosuppressant and immunomodulatory treatment for dermatomyositis and polymyositis. Cochrane Database Syst Rev. 2012; 8:CD003643. Nagakumar P, Doull I. Current therapy for bronchiolitis. Arch Dis Child. 2012; 97: 82730. De Cassan C, Fiorino G, Danese S. Second-generation corticosteroids for the treatment of Crohn's disease and ulcerative colitis: more effective and less side effects? Dig Dis. 2012; 30: 368-75. Cilliers A, Manyemba J, Adler AJ, Saloojee H. Anti-inflammatory treatment for carditis in acute rheumatic fever. Cochrane Database Syst Rev. 2012; 6: CD003176. Dieleman JM. Corticosteroids for the inflammatory response to cardiopulmonary bypass: an update. Curr Pharm Des. 2013; 19: 3979-91. Hughes RA, Mehndiratta MM. Corticosteroids for chronic inflammatory demyelinating polyradiculoneuropathy. Cochrane Database Syst Rev. 2012; 8:CD002062. Kagoya Y, Hangaishi A, Takahashi T, Imai Y, Kurokawa M. High-dose dexamethasone therapy for severe thrombocytopenia and neutropenia induced by EBV infectious mononucleosis. Int J Hematol. 2010; 91:326-7. de Smet MD. Corticosteroid intravitreal implants. Dev Ophthalmol. 2012; 51:122-33. van de Beek D, Brouwer MC, Thwaites GE, Tunkel AR. Advances in treatment of bacterial meningitis. Lancet. 2012; 380: 1693-702. De Oliveira GS Jr, Castro-Alves LJ, Ahmad S, Kendall MC, McCarthy RJ. Dexamethasone to prevent postoperative nausea and vomiting: an updated metaanalysis of randomized controlled trials. Anesth Analg. 2013; 116: 58-74. Eberhart LH, Graf J, Morin AM, Stief T, Kalder M, Lattermann R, Schricker T. Randomised controlled trial of the effect of oral premedication with dexamethasone on hyperglycaemic response to abdominal hysterectomy. Eur J Anaesthesiol. 2011; 28: 195-201. Giuliano C, Smalligan RD, Mitchon G, Chua M. Role of dexamethasone in the prevention of migraine recurrence in the acute care setting: a review. Postgrad Med. 2012; 124: 110-5. Seupaul RA, Welch JL, Malka ST, Emmett TW. Pharmacologic prophylaxis for acute mountain sickness: a systematic shortcut review. Ann Emerg Med. 2012; 59: 307-317. Wildes TM, Vij R, Petersdorf SH, Medeiros BC, Hurria A. New Treatment Approaches for Older Adults with Multiple Myeloma. J Geriatr Oncol. 2012; 3: 279-90. van de Donk NW, Görgün G, Groen RW, Jakubikova J, Mitsiades CS, Hideshima T, Laubach J, Nijhof IS, Raymakers RA, Lokhorst HM, Richardson PG, Anderson KC.

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[30]

[31]

Ricarda Stumpf and Thomas Stief Lenalidomide for the treatment of relapsed and refractory multiple myeloma. Cancer Manag Res. 2012; 4: 253-68. Eshaghian S, Berenson JR. Multiple myeloma: improved outcomes with new therapeutic approaches. Curr Opin Support Palliat Care. 2012; 6: 330-6. Dimopoulos MA, Terpos E, Goldschmidt H, Alegre A, Mark T, Niesvizky R. Treatment with lenalidomide and dexamethasone in patients with multiple myeloma and renal impairment. Cancer Treat Rev. 2012; 38: 1012-9. Palumbo A, Bladé J, Boccadoro M, Palladino C, Davies F, Dimopoulos M, Dmoszynska A, Einsele H, Moreau P, Sezer O, Spencer A, Sonneveld P, San Miguel J. How to manage neutropenia in multiple myeloma. Clin Lymphoma Myeloma Leuk. 2012; 12: 5-11. Cesarman-Maus G, Braggio E, Fonseca R. Thrombosis in multiple myeloma (MM). Hematology. 2012; 17 Suppl 1: S177-80. Smolej L. The role of high-dose corticosteroids in the treatment of chronic lymphocytic leukemia. Expert Opin Investig Drugs. 2012; 21: 1009-17. Toulis KA, Goulis DG, Msaouel P, Koutsilieris M. Dexamethasone plus somatostatinanalog manipulation as bone metastasis microenvironment-targeting therapy for the treatment of castration-resistant prostate cancer: a meta-analysis of uncontrolled studies. Anticancer Res. 2012; 32: 3283-9. Shah SS, Ohlsson A, Halliday HL, Shah VS. Inhaled versus systemic corticosteroids for preventing chronic lung disease in ventilated very low birth weight preterm neonates. Cochrane Database Syst Rev. 2012; 5:CD002058. Cross KP, Paul RI, Goldman RD. Single-dose dexamethasone for mild-to-moderate asthma exacerbations: effective, easy, and acceptable. Can Fam Physician. 2011; 57: 1134-6. Reber LL, Daubeuf F, Plantinga M, De Cauwer L, Gerlo S, Waelput W, Van Calenbergh S, Tavernier J, Haegeman G, Lambrecht BN, Frossard N, De Bosscher K. A dissociated glucocorticoid receptor modulator reduces airway hyperresponsiveness and inflammation in a mouse model of asthma. J Immunol. 2012; 188: 3478-87. Stief T. The routine blood ROS generation assay (BRGA) triggered by typical septic concentrations of zymosan A. Hemostasis Laboratory 2013; 6: 89-98. Iwanaga K, Honjo T, Miyazaki M, Kakemi M. Time-dependent changes in hepatic and intestinal induction ofcytochrome P450 3A after administration of dexamethasone to rats. Xenobiotica. 2013 Jan 23. Sekimoto M, Sano S, Hosaka T, Nemoto K, Degawa M. Establishment of a stable human cell line, HPL-A3, for use in reporter gene assays of cytochrome P450 3A inducers. Biol Pharm Bull. 2012; 35: 677-85. Fisher MB, Henne KR, Boer J. The complexities inherent in attempts to decrease drug clearance by blocking sites of CYP-mediated metabolism. Curr Opin Drug Discov Devel. 2006; 9:101-9. Yoshimoto FK, Desilets MC, Auchus RJ. Synthesis of halogenated pregnanes, mechanistic probes of steroid hydroxylases CYP17A1 and CYP21A2. J Steroid Biochem Mol Biol. 2012; 128: 38-50. Roma LP, Oliveira CA, Carneiro EM, Albuquerque GG, Boschero AC, Souza KL. Nacetylcysteine protects pancreatic islet against glucocorticoid toxicity. Redox Rep. 2011; 16: 173-80.


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[32] Kraaij MD, van der Kooij SW, Reinders ME, Koekkoek K, Rabelink TJ, van Kooten C, Gelderman KA. Dexamethasone increases ROS production and T cell suppressive capacity by anti-inflammatory macrophages. Mol Immunol. 2011; 49: 549-57. [33] Vago JP, Nogueira CR, Tavares LP, Soriani FM, Lopes F, Russo RC, Pinho V, Teixeira MM, Sousa LP. Annexin A1 modulates natural and glucocorticoid-induced resolution of inflammation by enhancing neutrophil apoptosis. J Leukoc Biol. 2012; 92: 249-58. [34] Hirsch G, Lavoie-Lamoureux A, Beauchamp G, Lavoie JP. Neutrophils are not less sensitive than other blood leukocytes to the genomic effects of glucocorticoids. PLoS One. 2012; 7: e44606. [35] Brooks AC, Rickards KJ, Cunningham FM. Modulation of equine neutrophil adherence and migration by the annexin-1 derived N-terminal peptide, Ac2-26. Vet Immunol Immunopathol. 2012 145: 214-22. [36] Huo Y, Rangarajan P, Ling EA, Dheen ST. Dexamethasone inhibits the Nox-dependent ROS production via suppression of MKP-1-dependent MAPK pathways in activated microglia. BMC Neurosci. 2011; 12: 49. [37] Zhou Y, Ling EA, Dheen ST. Dexamethasone suppresses monocyte chemoattractant protein-1 production via mitogen activated protein kinase phosphatase-1 dependent inhibition of Jun N-terminal kinase and p38 mitogen-activated protein kinase in activated rat microglia. J Neurochem. 2007; 102: 667-78. [38] Stief T. Micro-thrombi stimulate blood ROS generation. Hemostasis Laboratory 2013; 6 (issue 2-3)



MODULATION OF BLOOD ROS GENERATION BY PRE-INCUBATED LOW DOSE DEXAMETHASONE Ruth Dannhäuser and Thomas Stief Institute of Laboratory Medicine and Pathobiochemistry, University Hospital of Marburg, Germany

ABSTRACT Background: The synthetic glucocorticoid dexamethasone can be used as antiinflammatory drug. Here the modulating action on blood ROS (reactive oxygen species) generation of low dose dexamethasone that has been pre-incubated with fresh human blood is analyzed. Material and Methods: 40 µl dexamethasone in 0.9% NaCl (final drug conc.: 0-5 mg/l) in black high quality polystyrene microtiter plates (Brand®781608) and 125 µl Hanks´ Balanced Salt Solution (HBSS) were incubated in duplicate with 10 µl fresh citrated blood of 6 healthy donors for 120 min (37°C). 10 µl 5 mM luminol and 10 µl zymosan A in 0.9% NaCl (final conc.: 1.9 µg/ml) were added. After 0-73 min (37°C) the light emissions of the wells were measured by a photons-multiplying microtiter plate luminometer (LUmo) with an integration time of 0.5s/well. Results and Discussion: At 57 min the 6 samples had a maximum ranging from about 1500 to 16000 RLU/s. At 57 min the mean values maximum was reached with 6998 RLU/s. In BRGA-120-43 (120 min pre-incubation and 43 min main incubation) the approx. IC50 values were 0.2±0.2 mg/l dexamethasone for 4 of 6. Two samples did not have IC50 values. Instead, they had an approx. SC200 of 1 or 0.1 mg/l dexamethasone. There is inter-individual difference in the sensibility to pre-incubated low dose dexamethasone. Normally it is expected that a glucocorticoid behaves as a suppressor of blood ROS generation. However, pre-incubated dexamethasone, a molecule with 1 fluor and 5 oxygens, might be metabolized by the cytochrome P450 hydroxylation system to a redox cycler, eventually thereby uncoupling cytochrome P450. The resulting singlet oxygen generation could activate the blood neutrophils.

INTRODUCTION Dexamethasone (9-Fluor-16α-methylprednisolone) is a synthetic glucocorticoid nearly without mineralocorticoid action that is at least 10fold stronger than the physiologic adrenal steroid hydrocortisone [1]. It is a cytochrome P 450 substrate [2,3] and can be clinically used 

Correspondence to: PD Dr. med. T. Stief, Central Laboratory, University Hospital, D-35043 Marburg, Germany. email: [email protected]; Tel. : +49-6421-58 64471; FAX: +49-6421-58 65594.

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against local or systemic inflammation, neoplasmas, or brain edema (altitude sickness) [4-11]. Here we further analyzed the modulating action of dexamethasone on blood neutrophils [12], measured by the blood ROS (reactive oxygen species) generation assay (BRGA) [13].


MATERIAL AND METHODS Dexamethasone was from ratiopharm, Ulm, Germany. The 1 ml dosage contained 4.37 mg dexamethasone dihydrogenphosphate disodium salt (= 4 mg dexamethasone dihydrogenphosphate. 40 µl 0-24.4 mg/l dexamethasone in 0.9% NaCl (final conc.: 0-5 mg/l) in black high quality polystyrene microtiter plates (Brand, Wertheim, Germany; article nr. 781608) and 125 µl Hanks´ Balanced Salt Solution (HBSS; Sigma, Deisenhofen, Germany) were incubated in duplicate with 10 µl fresh citrated blood (venous blood supplemented with 10.6 mM sodium citrate, pH 7.4 in polypropylene tubes from Sarstedt, Nümbrecht, Germany) of 6 healthy donors that gave written informed consent, for 120 min (37°C). Then 10 µl 5 mM luminol (Sigma) (0.26 mM final conc.), and 10 µl 36 µg/ml zymosan A (ZyA; Sigma) in 0.9% NaCl (1.9 µg/ml final conc.) were added. After 0-73 min (37°C) the light emissions of the wells were measured by a photons-multiplying microtiter plate luminometer (LUmo; anthos, Krefeld, Germany) with an integration time of 0.5s/well. The measured data were transferred via Autosoft® to Excel. The approximate 50% inhibitory concentrations (approx. IC50) or the approximate 200% stimulatory concentrations (approx. SC200) of dexamethasone on blood ROS generation were determined.

RESULTS AND DISCUSSION At 57 min the 6 samples had a maximum ranging from about 1500 to 16000 RLU/s (Figure 2). At 57 min the mean values maximum was reached with 6998 RLU/s (Figure 3). In BRGA-120-43 (120 min pre-incubation and 43 min main incubation) the approx. IC50 values were 0.2±0.2 mg/l dexamethasone for 4 of 6. Two samples did not have IC50 values. Instead, two samples had an approx. SC200 of 1 or 0.1 mg/l dexamethasone (Figure 4). The mean BRGA-120-43 values of figure 4 increased slightly by about 2 mg/l dexamethasone when compared with unsupplemented samples (Figure 5).

Low Dose Hydrocortisone in BRGA-120-

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Figure 2. Kinetic of blood ROS generation upon 1.9 µg/ml ZyA stimulation after 120 min preincubation. The BRGA-120- was performed in 6 different normal fresh citrated blood samples, final trigger conc. 1.9 µg/ml zymosan A. At 57 min the 6 samples had a maximum ranging from about 1500 to 16000 RLU/s.

Figure 3. Mean values of figure 2. BRGA-120- mean values of figure 2. At 57 min the mean values maximum was reached with 6998 RLU/s.

There is inter-individual difference in the sensibility to pre-incubated low dose dexamethasone. Normally it is expected that a glucocorticoid behaves as a suppressor of blood ROS generation. However, pre-incubated dexamethasone, a molecule with 1 fluor and 5 oxygens, might be metabolized by the cytochrome P450 hydroxylation system to a redox cycler, eventually thereby uncoupling cytochrome P450. The following singlet oxygen generation could activate the blood neutrophils [14-29].

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Figure 4. Inhibition of blood ROS generation by pre-incubated dexamethasone. The results for BRGA with 120 min pre-incubation at 43 min reaction time (BRGA-120-43) were compared for the 6 samples. The approx. IC50 values were 0.2, -, 0.6, -, 0.1, and 0.05 mg/l dexamethasone, respectively. Two samples (2 and 4) did not have IC50 values. Instead, samples 2 and 4 had approx. SC200 of 1 and 0.1 mg/l dexamethasone. (Figure 4a: x-axis 0-5 mg/l; Figure 4b: x-axis 0-1 mg/l).


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Figure 5. Mean values of figure 4. The mean values of figure 4 demonstrate a tendency for increased blood ROS generation at about 2 mg/l dexamethasone.

ACKNOWLEDGMENTS This research has been performed within an intensive hemostasis training course of medical technicians (Marburg University Hospital MTA School). There was neither specific funding nor any conflict of interest.

REFERENCES [1] [2]

[3]

[4]

[5] [6]

www.wikipedia.org Varis T, Kivistö KT, Backman JT, Neuvonen PJ. The cytochrome P450 3A4 inhibitor itraconazole markedly increases the plasma concentrations of dexamethasone and enhances its adrenal-suppressant effect. Clin Pharmacol Ther. 2000; 68: 487-94. Iwanaga K, Honjo T, Miyazaki M, Kakemi M. Time-dependent changes in hepatic and intestinal induction of cytochrome P450 3A after administration of dexamethasone to rats. Xenobiotica. 2013 Jan 23. Gordon PA, Winer JB, Hoogendijk JE, Choy EH. Immunosuppressant and immunomodulatory treatment for dermatomyositis and polymyositis. Cochrane Database Syst Rev. 2012; 8:CD003643. Nagakumar P, Doull I. Current therapy for bronchiolitis. Arch Dis Child. 2012; 97: 82730. De Cassan C, Fiorino G, Danese S. Second-generation corticosteroids for the treatment of Crohn's disease and ulcerative colitis: more effective and less side effects ? Dig Dis. 2012; 30: 368-75.

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[15]

[16]

[17]

[18]

[19]

[20] [21] [22]

[23] [24] [25]

Ruth Dannhäuser and Thomas Stief Cilliers A, Manyemba J, Adler AJ, Saloojee H. Anti-inflammatory treatment for carditis in acute rheumatic fever. Cochrane Database Syst Rev. 2012; 6: CD003176. Dieleman JM. Corticosteroids for the inflammatory response to cardiopulmonary bypass: an update. Curr Pharm Des. 2013; 19: 3979-91. Hughes RA, Mehndiratta MM. Corticosteroids for chronic inflammatory demyelinating polyradiculoneuropathy. Cochrane Database Syst Rev. 2012; 8:CD002062. Cesarman-Maus G, Braggio E, Fonseca R. Thrombosis in multiple myeloma (MM). Hematology. 2012; 17 Suppl 1: S177-80. Smolej L. The role of high-dose corticosteroids in the treatment of chronic lymphocytic leukemia. Expert Opin Investig Drugs. 2012; 21: 1009-17. Stief TW. Neutrophil granulocytes in hemostasis. Hemostasis Laboratory 2008; 1: 26989. Stief T. The routine blood ROS generation assay (BRGA) triggered by typical septic concentrations of zymosan A. Hemostasis Laboratory 2013; 6: 89-98. Fisher MB, Henne KR, Boer J. The complexities inherent in attempts to decrease drug clearance by blocking sites of CYP-mediated metabolism. Curr Opin Drug Discov Devel. 2006; 9:101-9. Yoshimoto FK, Desilets MC, Auchus RJ. Synthesis of halogenated pregnanes, mechanistic probes of steroid hydroxylases CYP17A1 and CYP21A2. J Steroid Biochem Mol Biol. 2012; 128: 38-50. Roma LP, Oliveira CA, Carneiro EM, Albuquerque GG, Boschero AC, Souza KL. Nacetylcysteine protects pancreatic islet against glucocorticoid toxicity. Redox Rep. 2011; 16: 173-80. Kraaij MD, van der Kooij SW, Reinders ME, Koekkoek K, Rabelink TJ, van Kooten C, Gelderman KA. Dexamethasone increases ROS production and T cell suppressive capacity by anti-inflammatory macrophages. Mol Immunol. 2011; 49: 549-57. Dilger K, Lopez-Lazaro L, Marx C, Bussmann C, Straumann A. Active Eosinophilic Esophagitis Is Associated with Impaired Elimination of Budesonide by Cytochrome P450 3A Enzymes. Digestion. 2013; 87: 110-117. Heo GY, Liao WL, Turko IV, Pikuleva IA. Features of the retinal environment which affect the activities and product profile of cholesterol-metabolizing cytochromes P450 CYP27A1 and CYP11A1. Arch Biochem Biophys. 2012; 518: 119-26. Lewis DF. P450 structures and oxidative metabolism of xenobiotics. Pharmacogenomics. 2003; 4: 387-95. Isin EM, Guengerich FP. Substrate binding to cytochromes P450. Anal Bioanal Chem. 2008; 392: 1019-30. Green RM, Hodges NJ, Chipman JK, O'Donovan MR, Graham M. Reactive oxygen species from the uncoupling of human cytochrome P450 1B1 may contribute to the carcinogenicity of dioxin-like polychlorinated biphenyls. Mutagenesis. 2008; 23: 457-63. Guengerich FP. Mechanisms of cytochrome P450 substrate oxidation: MiniReview. J Biochem Mol Toxicol. 2007; 21:163-8. Poulos TL. Intermediates in P450 catalysis. Philos Transact A Math Phys Eng Sci. 2005; 363: 793-806. Kappus H, Sies H. Toxic drug effects associated with oxygen metabolism: redox cycling and lipid peroxidation. Experientia. 1981; 37: 1233-41.


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[26] Yasui H, Hayashi S, Sakurai H. Possible involvement of singlet oxygen species as multiple oxidants in p450 catalytic reactions. Drug Metab Pharmacokinet. 2005; 20: 1-13. [27] Stief TW. Hemostasis tolerable singlet oxygen - a perspective in AIDS therapy. Hemostasis Laboratory 2008; 1: 21-40. [28] Seike K, Murata M, Oikawa S, Hiraku Y, Hirakawa K, Kawanishi S. Oxidative DNA damage induced by benz[a]anthracene metabolites via redox cycles of quinone and unique non-quinone. Chem Res Toxicol. 2003; 16: 1470-6. [29] De Matteis F, Ballou DP, Coon MJ, Estabrook RW, Haines DC. Peroxidase-like activity of uncoupled cytochrome P450: studies with bilirubin and toxicological implications of uncoupling. Biochem Pharmacol. 2012; 84: 374-82.



TRIGGERING THROMBIN GENERATION BY HIGH DOSE DEXAMETHASONE Christine Dippel and Thomas Stief Institute of Laboratory Medicine and Pathobiochemistry, University Hospital of Marburg, Germany

ABSTRACT Background: Dexamethasone is a synthetic glucocorticoid that is often given in inflammatory diseases. Here the normal blood coagulation can switch to the pre-phase of pathologic disseminated intravascular coagulation (NICPIC-0). Since dexamethasone by itself as an organic molecule with one delta-negativelay charged fluor atom and 5 delta-negatively charged oxygen atoms could behave as an important pathophysiologic trigger of altered matrix (AM) – coagulation, here dexamethasone was ultra-finely investigated for its intrinsic power to stimulate AM-mediated thrombin generation, using the recalcified coagulation activity assay (RECA). Material and Methods: 50 µl platelet poor plasma of 4 healthy donors in transparent high quality polystyrene microtiter U-well plates (Brand®781600) were supplemented with 0-9.6 mg/l dexamethasone by immediate repetitive 1+1 dilution on the plate. 5 µl 250 mM CaCl2 were added to start the RECA. After 0, 15, 18, 20 min at 37°C the RECA was stopped by addition of 100 µl 2.5 M arginine, 0.16% Triton X 100®, pH 8.6. After 3 min 25 µl fast chromogenic thrombin substrate HD-CHG-Ala-Arg-pNA in 1.25 M arginine, pH 8.7 were added and the specific increase in absorbance ΔA405 nm was measured by a microtiterplate photometer with a 1 mA resolution (PHOmo). The approximate 200% stimulatory concentrations (approx. SC200) were determined in the important ascending part of the incubation time vs. thrombin generation curve. Results: The approx. SC200 of dexamethasone to trigger AM-thrombin generation in 4 fresh normal plasmas supplemented with up to 9.6 mg/l dexamethasone was 0.5±0.4 mg/l dexamethasone (MV±1SD; range: 0.2-1 mg/l). Discussion: RECA analysis should be performed for each individual who needs chronic administration of dexamethasone. This would answer prior to drug infusion/ingestion if the patient belongs to a subgroup of patients with increased susceptibility towards intrinsic hemostasis activation by dexamethasone. Then another glucocorticoid with better approx. SC200 values could be chosen or dexamethasone could be combined with LMWH (low-molecular-weight-heparin), a target EXCA-value of about 20% of normal (about 0.5 IU/ml LMWH) could be ideal. Furthermore, new




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Christine Dippel and Thomas Stief drugs should be analyzed by FDA, EMA, PEI for their capacity to trigger intrinsic coagulation prior to their release to the medical market.

INTRODUCTION The glucocorticoid dexamethasone is at least 12fold stronger than the natural cortisol against systemic or local inflammation [1]. Dexamethasone, in contrast to cortisol, does not possess mineralocorticoid action. In systemic inflammatory diseases the human coagulation system is pathologically triggered [2]. Normal intravascular coagulation (NIC) with systemic thrombin activity of 100±20% (MV±1SD; 100% = 5.5 mIU thrombin/ ml plasma) often changes to the pre-phase of systemic intravascular coagulation (PIC-0) or even worse to the typical phase of systemic intravascular coagulation (PIC-1) [3, 4]. Lipophilic or (delta-) negatively charged compounds trigger AM-coagulation (= contact phase coagulation = intrinsic coagulation) [5,6]. Dexamethasone, a molecular derivate of the lipophilic cholesterol with inserted one delta-negativelay charged fluor atom and 5 delta-negatively charged oxygen atoms (Figure 1) is an AM-trigger. Xenobiotics entering the blood stream alter the blood matrix. With the invention of the recalcified coagulation activity assay (RECA) it became for the first time possible to quantify ultra-finely the discrete F12a/kallikrein/thrombin generation by drugs [7]. There is great interindividual variety in the response of F12/pre-kallikrein to AM-triggers [8, 9]. There appeared an approx. SC200 value of approximately 2 mg/l dexamethasone if plasma had been supplemented with up to 100 mg/l dexamethasone [10]. The behavior of AM-coagulation that the approx. SC200 depends on the maximal concentration of drug added to plasma requires that typical clinical concentrations are chosen prior to plasma dilution [5, 6, 11]. Here a concentration of 9.6 mg/l was chosen as the maximal concentration.


MATERIAL AND METHODS Dexamethasone was from ratiopharm, Ulm, Germany. The 1 ml dosage contained 4.37 mg dexamethasone dihydrogenphosphate disodium salt (= 4 mg dexamethasone dihydrogenphosphate. 50 µl platelet poor plasma of 4 healthy donors that gave written informed consent (4.5 ml venous blood + 0.5 ml 106 mM sodim citrate, pH 7.4 in polypropylene monovettes from Sarstedt (Nümbrecht, Germany) still valid for at least 1 year) in transparent high quality polystyrene microtiter U-well plates (Brand, Wertheim, Germany;

High Dose Dexamethasone in RECA

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article nr. 781600) were supplemented with 0-9.6 mg/l dexamethasone by immediate repetitive 1+1 dilution on the plate. 5 µl 250 mM CaCl2 (Sigma, Deisenhofen, Germany) were added to start the RECA [12,13]. After 0, 15, 18, 20 min at 37°C the RECA was stopped by addition of 100 µl 2.5 M arginine, 0.16% Triton X 100®, pH 8.6. After 3 min 25 µl fast chromogenic thrombin substrate HD-CHG-Ala-Arg-pNA (Pentapharm, Basel, Switzerland) in 1.25 M arginine, pH 8.7 were added and the specific increase in absorbance ΔA405 nm was measured by a microtiterplate photometer with a 1 mA resolution (PHOmo; anthos, Krefeld, Germany). The thrombin standard was 40 mIU/ml bovine thrombin (Siemens-DadeBehring, Marburg, Germany) in 5% human albumin (CSL Behring, Marburg, Germany). 50 µl standard replaced the plasma in 4 wells and resulted in a mean specific ΔA of 1.1 mA/min (37°C). The substrate cleavage was linear up to 40% of the maximal obtainable ΔA which was 1000 mA. The approximate 200% stimulatory concentrations (approx. SC200) were determined in the important ascending part of the incubation time vs. thrombin generation curve, where supra-molar arginine has eliminated the negative effect of antithrombin-1 (nascent uncrosslinked fibrin) [14]. The RECA is the assay to establish even discrete (but clinically relevant) pathologically enhanced thrombin generations (approx. SC200), for decreased thrombin generations (approx. IC50) the INCA (intrinsic coagulation activity assay) or the EXCA (extrinsic coagulation activity assay) are better indicated [4].

RESULTS AND DISCUSSION Figures 2-5 demonstrate that the approx. SC200 of dexamethasone to trigger AM-thrombin generation in fresh normal plasma supplemented with up to 9.6 mg/l dexamethasone was 0.5±0.4 mg/l dexamethasone (MV±1SD; range: 0.2-1 mg/l).

Figure 2. AM-coagulation activation by dexamethasone in normal plasma 1. The RECA was performed in normal fresh citrated platelet poor plasma 1 that had been supplemented on the microtiter plate with 0-9.6 mg/l dexamethasone by immediate repetitive 1+1 dilution. The approx. SC200 of dexamethasone on intrinsic F2a generation was 1 mg/l, seen here in RECA-15 (coagulation reaction time (CRT) = 15 min). Many RECA-20 values and some RECA-18 values had to be omitted because they were not in the ascending part of the CRT vs. thrombin generation curve.

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Figure 3. AM-coagulation activation by dexamethasone in normal plasma 2. The RECA was performed in normal fresh citrated platelet poor plasma 2 that had been supplemented on the microtiter plate with 0-9.6 mg/l dexamethasone by immediate repetitive 1+1 dilution. The approx. SC200 of dexamethasone on intrinsic F2a generation was 0.2 mg/l. No increase in basal plasma turbidity due to antithrombin-1 [14].

Figure 4. AM-coagulation activation by dexamethasone in normal plasma 3. The RECA was performed in normal fresh citrated platelet poor plasma 3 that had been supplemented on the microtiter plate with 0-9.6 mg/l dexamethasone by immediate repetitive 1+1 dilution. The approx. SC200 of dexamethasone on intrinsic F2a generation was 0.6 mg/l. No increase in basal plasma turbidity due to antithrombin-1 [14].

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Figure 5. AM-coagulation activation by dexamethasone in normal plasma 4. The RECA was performed in normal fresh citrated platelet poor plasma 4 that had been supplemented on the microtiter plate with 0-9.6 mg/l dexamethasone by immediate repetitive 1+1 dilution. The approx. SC200 of dexamethasone on intrinsic F2a generation was 0.2 mg/l, seen here in RECA-18 (coagulation reaction time (CRT) = 18 min). Two RECA-20 values had to be omitted because they were not in the ascending part of the CRT vs. thrombin generation curve.

RECA analysis should be performed for each individual who needs chronic administration of dexamethasone. This would answer prior to drug infusion/ingestion if the patient belongs to a subgroup of patients with increased susceptibility towards intrinsic hemostasis activation by dexamethasone. Then another glucocorticoid with better approx. SC200 values could be chosen or dexamethasone could be combined with LMWH (lowmolecular-weight-heparin), a target EXCA-value of about 20% of normal (about 0.5 IU/ml LMWH) could be ideal [15,16]. Furthermore, new drugs should be checked by FDA, EMA, PEI for their capacity to trigger intrinsic coagulation prior to their release to the medical market.

ACKNOWLEDGMENTS The research work of this article has been performed within an intensive hemostasis training course of medical technicians (Marburg University Hospital MTA School). There was neither specific funding nor any conflict of interest.

REFERENCES [1] [2]

www.wikipedia.org Stief TW. Coagulation activation by lipopolysaccharides. Clin Appl Thrombosis/Hemostasis Dec 26, 2007 doi: 10.1177/1076029607309256; 2009; 15: 20919.

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[5] [6]

[7] [8] [9] [10] [11]

[12] [13] [14] [15] [16]

Christine Dippel and Thomas Stief Stief TW. The laboratory diagnosis of the pre-phase of pathologic disseminated intravascular coagulation. Hemostasis Laboratory 2008; 1: 2-20. Stief TW. Thrombin – applied clinical biochemistry of the main factor of coagulation. In: Thrombin: function and pathophysiology. Stief T, ed.; Nova science publishers; New York; 2012; pp. vii-xx. https://www.novapublishers.com/catalog/product_ info.php?products_id=33386 Stief TW. Contact activation of coagulation depends on the maximal lipophilic trigger concentration. Blood Coagulation and Fibrinolysis 2012; 23: 296-8. Stief TW. The maximal plasma concentration of (delta-)negatively charged contact triggers influences plasmatic thrombin generation. In: Thrombin: function and pathophysiology. Stief T, ed.; NOVA science publishers; New York; 2012; pp. 37-46. Stief TW. Drug - induced thrombin generation: the breakthrough. Hemostasis Laboratory 2010; 3: 3-6. Stief TW. Thrombin generation by creatinine. Hemostasis Laboratory 2011; 4: 191-9. Stief TW. Thrombin generation by therapeutic fibrinogen. Hemostasis Laboratory 2011; 4: 467-82. Stief TW. Thrombin generation by corticoids. Hemostasis Laboratory 2010; 3: 71-6. Stief TW, Mohrez M. HMWK increases of decreases F12a generation dependent on the contact trigger concentration in two purified systems. Hemostasis Laboratory 2012; 5: 51-65. Stief TW. Pathological thrombin generation by the synthetic inhibitor argatroban. Hemostasis Laboratory 2009; 2: 83-104. Stief TW. Thrombin triggers its generation in individual platelet poor plasma. Hemostasis Laboratory 2009; 2: 363-78. Mosesson MW. Update on antithrombin I (fibrin). Thromb Haemost. 2007; 98: 105-8. Stief TW. 20% EXCA (0.1 IU/ml thrombin) – the ideal anticoagulant target value. Hemostasis Laboratory 2011; 4: 275-80. Stief TW. LMWH - action-monitoring for all patients. Acta Paediatr. 2012; 101: e314. 10.1111/j.1651-2227.2012.02712.x.



MODULATION OF BLOOD ROS GENERATION BY HIGH DOSE DEXAMETHASONE Sandra Blumenau and Thomas Stief Institute of Laboratory Medicine and Pathobiochemistry, University Hospital of Marburg, Germany

ABSTRACT Background: Dexamethasone (9-Fluor-16α-methylprednisolone) is a synthetic glucocorticoid without mineralocorticoid action that is 10-20fold stronger than the physiologic hydrocortisone. It is clinically used against systemic inflammation, brain edema, or neoplasma. Dexamethasone is a substrate of cytochrome P450 (CYP450). Due to its delta-negative fluor – atom and its 5 oxygen – atoms it might also stimulate the phagocytes. Here we investigated the modulating action of dexamethasone on blood neutrophils, measured by the new blood ROS (reactive oxygen species) generation assay (BRGA). Material and Methods: 40 µl dexamethasone in 0.9% NaCl (final drug conc.: 0-100 mg/l) in black high quality polystyrene microtiter plates (Brand®781608) and 125 µl Hanks´ Balanced Salt Solution (HBSS) were incubated in duplicate with 10 µl fresh citrated blood of 5 healthy donors, 10 µl 5 mM luminol, and 10 µl zymosan A in 0.9% NaCl (final conc.: 0.5 µg/ml or 1.9 µg/ml). After 0-181 min (37°C) the light emissions of the wells were measured by a photons-multiplying microtiter plate luminometer (LUmo) with an integration time of 0.5s/well. Results: At about 55 min the maximum of about 600-1700 RLU/s (1.9 µg/ml ZyA) was reached; at 194 min 3 of 5 samples had a second increase in blood ROS generation, presumably due to micro-thrombi mediated activation of cellular fibrinolysis. In BRGA-52 (BRGA with 52 min reaction time) the approx. IC50 values of dexamethasone on blood ROS generation were 5, 200 mg/l. The mean values had approx. SC150 values of 4 and 8 mg/l (sensible to middle sensible). Discussion: Imipenem/cilastatin might dangerously increase blood ROS generation in susceptible patients. Whereas the direct action of the drug on blood ROS generation is inhibitory, the action of the drug on blood neutrophils after 60 min pre-incubation is stimulatory. The antibiotic could have been cytochrome P450 transformed into a redoxcycler / uncoupler, generators of H2O2, the mother substance of singlet oxygen, an important cell activator. If cilastatin modulates blood ROS generation, it is suggested to 


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Julia Drajt and Thomas Stief omit cilastatin. Cilastatin might worsen an ongoing acute or chronic inflammation. In conclusion, in the initial phase of drug infusion the patient might be at risk of thrombosis, in the second phase the patient could be at risk of hyper-inflammation.

INTRODUCTION Imipenem is a broad spectrum carbapenem antibiotic that is resistant to typical ßlactamases (penicillinases and cephalosporinases) (Figure1) [1,2]. Often imipenem is combined with the dipeptidase inhibitor cilastatin to increase the plasma concentration of imipenem and to prolong its blood half-time. All antibiotics may modulate the oxidative metabolism of human phagocytes [3]. It is of great clinical interest to know if a given antibiotic would weaken the main cells against invasive bacteria or fungi. Therefore the blood ROS generation assay (BRGA) was performed to study the oxidative power of blood neutrophils in presence of imipenem/cilastatin [4,5].

MATERIAL AND METHODS Imipenem/cilastatin (500 mg imipenem = 530 mg imipenem monohydrate, 500 mg cilastatin = 530 mg cilastatin sodium salt, 20 mg NaHCO3) was from Actavis, Hafnarfjördur, Island. 40 µl imipenem/cilastatin in 0.9% NaCl (final drug conc.: 0-256 mg/l) in black high quality polystyrene microtiter plates (Brand, Wertheim, Germany; article nr. 781608) and 125 µl Hanks´ Balanced Salt Solution (HBSS; without phenol red; Sigma-SAFC Biosciences, Deisenhofen, Germany; article nr. 55037C-1000ML) were incubated in duplicate with 10 µl fresh citrated blood of 6 healthy donors, 1d old blood of 6 healthy donors, 10 µl 5 mM luminol sodium salt (Sigma), and 10 µl zymosan A (Sigma) in 0.9% NaCl (final conc.: 1.9 µg/ml).

Figure 1. Chemical structure of the excellent antibiotic imipenem (above) and the dubious partner cilastatin (below) [1]. Imipenem (N-formimidoyl-thienamycin), C12H17N3O4S (MW: 299.35), is rapidly degraded by renal dehydropeptidase 1 when administered alone, and is therefore combined with cilastatin, C16H26N2O5S (MW: 358.45) to avoid this inactivation.

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During the main incubation (37°C) the light emissions of the wells were measured by a photons-multiplying microtiter plate luminometer (LUmo; anthos, Krefeld, Germany) with an integration time of 0.5s/well. The approximative 50% inhibitory (approx. IC50) or the approx. 200% stimulatory (approx. SC200) concentrations of imipenem/cilastatin were determined.

RESULTS AND DISCUSSION The present data demonstrate that if analyzed at half-maximal to maximal normal time there were 5/12 (42%) sensible blood samples with an approx. SC150 of 1±0.6 mg/l (range: 0.5-2 mg/l) imipenem/cilastatin. 2/12 (16%) were middle sensible with approx. SC150 values of 12 and 70 mg/l. 5/12 (42%) were resistant towards blood ROS generation induction by imipenem/cilastatin with approx. SC150 values > 200 mg/l (Figs. 2-5). The mean values had approx. SC150 values of 4 and 8 mg/l (sensible to middle sensible). Thus, imipenem/cilastatin might dangerously increase blood ROS generation in susceptible patients. Whereas the direct action of the drug on blood ROS generation is inhibitory, the action of the drug on blood neutrophils after 60 min pre-incubation is stimulatory. The antibiotic could have been cytochrome P450 transformed into a redoxcycler/ uncoupler, generators of H2O2, the mother substance of singlet oxygen, an important cell activator. Since penicillin and aminopenicillin do not significantly alter blood ROS generation [3], the partner molecule cilastatin might be the actual stimulator of blood ROS generation. Both drugs need to be analyzed separately. If cilastatin modulates the generation of blood ROS, it is suggested to do without cilastatin. Cilastatin might worsen an ongoing acute or chronic inflammation. In conclusion, in the initial phase of drug infusion the patient might be at risk of thrombosis [6-10], in the second phase the patient could be at risk of hyper-inflammation.

Figure 2. BRGA-60- kinetic in normal human blood. The blood ROS generation assay (BRGA-60-) was performed for 3 fresh blood samples (4-6) and for 3 1d old blood samples (1-3) as described under methods. The ROS maximum was 338-1155 RLU/s, reached after 43-104 min. The ROS maximum of the mean values was 678 RLU/s, reached after 78 min of the main incubation.

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Figure 3. Stimulation of blood ROS generation by imipenem/cilastatin. Normal blood was preincubated with 0-256 mg/l imipenem/cilastatin for 60 min (37°C). The BRGA-60- was performed with 43 min reaction time (BRGA-60-43). The ROS generations were measured by a photo-multiplying microtiter plate luminometer. The approx. SC150 values were: 250, 2, 1, -, 70, - mg/l. The mean values had an approx. SC150 of 8 mg/l imipenem/cilastatin.

Figure 4. BRGA-60- kinetic in normal human blood. The blood ROS generation assay (BRGA-60-) was performed for 3 fresh blood samples (4-6) and for 3 1d old blood samples (1-3) as described under methods. The ROS maximum was 231-3743 RLU/s, reached after 78-97 min. The ROS maximum of the mean values was 1945 RLU/s, reached after 78 min of the main incubation.

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Figure 5. Stimulation of blood ROS generation by imipenem/cilastatin. Normal blood was preincubated with 0-256 mg/l imipenem/cilastatin for 60 min (37°C). The BRGA-60- was performed with 59 min reaction time (BRGA-60-59). The ROS generations were measured by a photo-multiplying microtiter plate luminometer. The approx. SC150 values were: 0.5, 12, 250, -, 1, 0.5 mg/l. The mean values had an approx. SC150 of 4 mg/l imipenem/cilastatin.


REFERENCES [1] [2] [3]

www.wikipedia.org Actavis product information. Imipenem/cilastatin (ATC: J01DH51); approval number: 1–29667. Stief T. Flucloxacillin or cefotaxime (but not penicillin G or ampicillin) inhibit blood ROS generation. Hemostasis Laboratory 2013; 6 (issue 2-3))

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Stief T. The routine blood ROS generation assay (BRGA) triggered by typical septic concentrations of zymosan A. Hemostasis Laboratory 2013; 6: 89-98. [5] Stief T. Glucose initially inhibits and later stimulates blood ROS generation. Journal of Diabetes Mellitus 2013, 3: 15-21. doi: 10.4236/jdm.2013.31003. [6] Stief T. Micro-thrombi stimulate blood ROS generation. Hemostasis Laboratory 2013; 6 (issue 2-3) [7] Stief TW. Neutrophil granulocytes in hemostasis. Hemostasis Laboratory 2008; 1: 26989. [8] Stief TW. The laboratory diagnosis of the pre-phase of pathologic disseminated intravascular coagulation. Hemostasis Laboratory 2008; 1: 2-20. [9] van Golen RF, van Gulik TM, Heger M. The sterile immune response during hepatic ischemia/reperfusion. Cytokine Growth Factor Rev. 2012; 23: 69-84. [10] Kubes P, Mehal WZ. Sterile inflammation in the liver. Gastroenterology. 2012; 143: 1158-72.



IMIPENEM/CILASTATIN: A PATHOPHYSIOLOGIC TRIGGER OF ALTERED MATRIX COAGULATION Anastasia Busch, Irina Bogun, and Thomas Stief Institute of Laboratory Medicine and Pathobiochemistry, University Hospital of Marburg, Germany

ABSTRACT Background: Imipenem is a broad spectrum carbapenem antibiotic that is resistant to penicillinases and to cephalosporinases. Usually imipenem is combined with cilastatin to prolong its blood half-time. Cilastatin with its two negatively charged carboxylate-groups should be an important trigger of the contact phase of plasmatic coagulation (= intrinsic coagulation = altered matrix (AM) - coagulation). The ultra-specific, ultra-sensitive thrombin generation assay recalcified coagulation activity assay (RECA) is an ideal tool to assess the pro-thrombotic power of any compound. Material and Methods: 40 µl normal human plasma of 5 healthy donors and pooled normal plasma (3 individual plasmas mixed) in high quality polystyrene U-wells microtiter plates (Brand®781600) were supplemented with 0-610 mg/l imipenem/cilastatin (Actavis) by repetitive 1+1 dilution on the plate. Immediately thereafter the RECA was started by addition of 4 µl 250 mM CaCl2. After 0-25 min the coagulation reaction time (CRT) was stopped by addition of 80 µl 2.5 M arginine, pH 8.6, 0.16% Triton X 100®. After 3 min 20 µl 1 mM chromogenic thrombin substrate HD-CHG-Ala-Arg-pNA in 1.25 M arginine, pH 8.7 were added and the specific increase in absorbance at 405 nm (ΔA) was measured by a mirotiter plate photometer with a 1 mA resolution (PHOmo). The thrombin standard was 40 µl 40 mIU/ml bovine thrombin in 5% human albumin. The approximative 200% stimulatory (approx. SC200) concentrations of imipenem/cilastatin were determined. Results: The approx. SC200 of imipenem/cilastatin on recalcified thrombin generation was 9±3 mg/l (MV±1SD; range: 5-12). Pooled normal had an approx. SC200 of 10 mg/l imipenem/cilastatin. Discussion: The two carboxylate groups and the lipophilic regions of cilastatin are strong AM-coagulation triggers. It is suggested to omit cilastatin in the antibiotic drug. There might be serious systemic and local pro-thrombotic complications up to aseptic fulminant hepatitis (i.e. hepatic micro-thrombi that induce activation of cellular fibrinolysis). Low concentrations of this excellent reserve antibiotic imipenem might be absolutely sufficient against critical resistant germs. It is suggested to combine imipenem

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Anastasia Busch, Irina Bogun, and Thomas Stief with LMWH at prophylactic to therapeutic dosage, the target being an EXCA-value (extrinsic coagulation activity assay) of 20% of normal.

INTRODUCTION The carbapenem antibiotic imipenem (N-formimidoyl-thienamycin) acts against an extremely broad bacterial spectrum at working concentrations of only about 0.1-1 mg/l [1,2]. Imipenem is indicated in peritonitis, urinary tract infections, peripartal infections, severe dermatitis, complicated endocarditis, and severe pneumonia. The dosage is up to 1000 mg/6h, especially patients infected with less sensible germs (such as Pseudomonas aeruginosa) or with severe infections (as occurring e.g. in neutropenia) need the maximal dosage. Imipenem might be combined with another antibiotic in infections with methicillin resistant Staphylococcus aureus (MRSA) (linezolid) or with Pseudomonas aeruginosa (gentamicin). Described side reactions are venous thrombosis (about 3.1%) or (seldom; micro-thrombi induced?) fulminant hepatitis. Imipenem is not compatible with valproate. Usually imipenem at a dosage up to 500 mg is given in 100 ml infusion within 20 min. Higher dosages should be infused in a 500 ml volume within 60 min. Thienamycin is produced by Streptomyces cattleya. Then the N-formimidoyl group is chemically added. Imipenem is bactericidal due to its affinity to D-alanine carboxypeptidase, peptidoglycan transpeptidase, peptidoglycan endopeptidase (the penicillin binding proteins (PBP), the main enzymes of bacterial cell wall synthesis) and consequently inhibits the enzymatic synthesis of the cell wall of the bacterium, as well in gram-positive as in gram-negative bacteria. Bacteria can be resistant against imipenem because they    

synthesize less porins of the outer membrane (in gram-negative bacteria) pump imipenem outside the bacterium generate modified PBP with no affinity to imipenem generate beta-lactamases that hydrolyze carbapenems (seldom, imipenem is resistant against the typical ß-lactamases such as penicillinases and cephalosporinases).

The plasma concentrations of imipenem (Actavis drug in combination with cilastatin) are 21-58 mg/l (MV: 39 mg/l) 20 min after infusion of 500 mg. The half-time is about 1h. Within 6h the plasma concentrations are below 1 mg/l. 20% of imipenem is bound to plasma proteins. About 70% of the drug are renally eliminated in unchanged form in urine concentrations higher than 10 mg/l within 8h after a 500 mg dosage of imipenem/cilastatin Actavis. About 30% of the drug are renally eliminated as inactive drug metabolites. Imipenem is renally eliminated and inactivated by the renal enzyme dehydropeptidase-1. To prolong the blood half-time of imipenem, the antibiotic is combined with cilastatin (in a mass relation of 1:1), a competitive inhibitor of dehydropeptidase-1. The plasma concentration of cilastatin after a 20-min intravenous infusion was 21-55 mg/l (mean value: 42 mg/l). About 40% of cilastatin bind to human plasma proteins. The half-time of cilastatin is about 1h, about 70-80% of cilastatin leave the body unchanged in urine. About 10 % are renally eliminated as still active N-acetyl-metabolite.

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The best assay to test plasma for coagulation activation is the recalcified coagulation activity assay (RECA) [3,4]. Here imipenem/cilastatin is checked in RECA for its thrombin generating capacity in plasma.

MATERIAL AND METHODS 40 µl normal human citrated platelet poor plasma (4.5 ml venous blood + 0.5 ml 106 mM sodium citrate in polypropylene monovettes from Sarstedt, Nümbrecht, Germany; till 1 year to expiry) of 5 healthy donors that gave written informed consent in high quality polystyrene U-wells microtiter plates (Brand, Wertheim, Germany; article nr. 781600) were supplemented with additional 0-610 mg/l imipenem/cilastatin (500 mg imipenem = 530 mg imipenem monohydrate, 500 mg cilastatin = 530 mg cilastatin sodium salt, 20 mg NaHCO3; Actavis, Hafnarfjördur, Island) by repetitive 1+1 dilution on the plate. Imipenem/cilastatin was supplemented at very high concentrations prior to dilution because such highest concentrations might occur at the point where the infusion enters the blood stream. In the begin and in the end of drug administration and in niches of body fluid there could be critical blood concentrations of a drug. Immediately after drug addition to the plasma samples the RECA was started by 4 µl 250 mM CaCl2. After 0-25 min coagulation reaction time (CRT) the reaction was stopped by 80 µl 2.5 M arginine, pH 8.6, 0.16% Triton X 100® (Sigma). After 3 min 20 µl 1 mM chromogenic thrombin substrate HD-CHG-Ala-Arg-pNA (Pentapharm, Basel, Switzerland) in 1.25 M arginine, pH 8.7 were added and the specific increase in absorbance at 405 nm (ΔA) was measured by a mirotiter plate photometer with a 1 mA resolution (PHOmo; anthos, Krefeld, Germany). The thrombin standard was 40 µl 40 mIU/ml bovine thrombin (Siemens Healthcare Diagnostics, Munich, Germany) in 5% human albumin (CSL Behring, Marburg, Germany).

Figure 1. Chemical structure of the excellent antibiotic imipenem (above) and the dubious partner cilastatin (below) [1]. Imipenem (N-formimidoyl-thienamycin), C12H17N3O4S (MW: 299.35), is rapidly degraded by renal dehydropeptidase 1 when administered alone, and is therefore combined with cilastatin, C16H26N2O5S (MW: 358.45) to avoid this inactivation. The two carboxylate groups [5] and the lipophilic 5C-atoms cluster and the lipophilic 6C-atoms region [6] of cilastatin are AM-coagulation triggers.

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The standard had a linear ΔA of about 1 mA/ min (37°C). Thrombin generation = thrombin activity measured minus basal thrombin activity (0 min CRT). The basal thrombin activity in normal citrated plasma was 6-8 mIU/ml. The approximate 200% stimulatory concentrations (approx. SC200) of added drug on recalcified thrombin generation were measured.

RESULTS AND DISCUSSION Figures 2-6 demonstrate that the approx. SC200 of imipenem/cilastatin on recalcified thrombin generation was 9±3 mg/l (MV±1SD; range: 5-12); pooled normal plasma (out of the 3 individual plasmas of figures 1-3) had an approx. SC200 of 10 mg/l imipenem/cilastatin (Figure5). These values are about 20fold higher than the ones found for plasma supplementation up to 30.5 mg/l imipenem/cilastatin. This is typical for AM-coagulation: the higher the initial trigger concentration prior to dilution the higher the resulting approx. SC200 of the drug on recalcified thrombin generation [6,7]. It seems that highest concentrations of any AM-modifier are anticoagulant towards the initial phase of AM-coagulation [8]. However, this anticoagulant action is not relevant if assayed in the INCA (intrinsic coagulation activity assay), a global assay of intrinsic thrombin generation The INCA but not the RECA [9,10] is the assay to judge the clinically relevant anticoagulant action of compounds that alter intrinsic hemostasis. The RECA is the assay to see any procoagulant tendency of plasma.

Figure 2. Imipenem/cilastatin triggers thrombin generation in normal plasma 1. Normal platelet poor plasma was supplemented with 0-610 mg/l imipenem/cilastatin by repetitive 1+1 dilution on the plate. The RECA was performed as described under Material and Methods. 10 mg/l was the approx. SC200, seen in the most sensible curve, here RECA-20 (RECA with 20 min coagulation reaction time).


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Figure 5. Imipenem/cilastatin triggers thrombin generation in pooled normal plasma. Pooled normal plasma was supplemented with 0-610 mg/l imipenem/cilastatin. The RECA was performed as described under Material and Methods. 10 mg/l was the approx. SC200 in RECA-15. No increase in basal plasmatic turbidity.

Figure 6. Imipenem/cilastatin triggers thrombin generation in normal plasma 4. Normal platelet poor plasma was supplemented with 0-610 mg/l imipenem/cilastatin by repetitive 1+1 dilution on the plate. The RECA was performed as described under Material and Methods. 9 mg/l was the approx. SC200 in RECA-15. No increase in basal plasmatic turbidity.


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The two carboxylate groups and the lipophilic regions of cilastatin are strong AM-coagulation triggers [5,6]. The susceptibility towards AM-coagulation activation differs greatly from individual to individual [11,12]. It is suggested to omit cilastatin in the antibiotic drug. There might be serious systemic [13,14] and local pro-thrombotic complications up to aseptic fulminant hepatitis (i.e. hepatic micro-thrombi that induce activation of cellular fibrinolysis) [15-17]. Low concentrations of this excellent reserve antibiotic imipenem might be absolutely sufficient against critical resistant germs. It is suggested to combine imipenem with LMWH at prophylactic to therapeutic dosage, aiming an EXCA-value (extrinsic coagulation activity assay) of 20% of normal [18-21].


REFERENCES [1] [2] [3]

www.wikipedia.org Actavis product information. Imipenem/cilastatin (ATC: J01DH51); approval number: 1–29667. Stief TW. Drug - induced thrombin generation: the breakthrough. Hemostasis Laboratory 2010; 3: 3-6.

134 [4] [5] [6] [7]

[8]

[9] [10] [11] [12] [13]

[14] [15] [16] [17] [18] [19] [20] [21]

Anastasia Busch, Irina Bogun, and Thomas Stief Stief TW. Pathological thrombin generation by the synthetic inhibitor argatroban. Hemostasis Laboratory 2009; 2: 83-104. Stief TW. Citrate triggers thrombin generation. Hemostasis Laboratory 2011; 4: 59-64. Stief TW. Contact activation of coagulation depends on the maximal lipophilic trigger concentration. Blood Coagulation and Fibrinolysis 2012; 23: 296-8. Stief TW. The maximal plasma concentration of (delta-)negatively charged contact triggers influences plasmatic thrombin generation. In: Thrombin: function and pathophysiology. Stief T, ed.; NOVA science publishers; New York; 2012; pp. 37-46. Stief TW, Mohrez M. HMWK increases of decreases F12a generation dependent on the contact trigger concentration in two purified systems. Hemostasis Laboratory 2012; 5: 51-65. Stief TW. Inhibition of intrinsic thrombin generation. Drug Target Insights 2006; 2: 611. Stief TW. Inhibition of thrombin generation in recalcified plasma. Blood Coagulation and Fibrinolysis 2007; 18: 751-60. Stief TW. Thrombin generation by therapeutic fibrinogen. Hemostasis Laboratory 2011; 4: 467-82. Stief TW. Thrombin generation by creatinine. Hemostasis Laboratory 2011; 4: 191-9. Stief TW. Thrombin – applied clinical biochemistry of the main factor of coagulation (Preface). In: Thrombin: function and pathophysiology. Stief T, ed.; Nova science publishers; New York; 2012; pp. vii-xx. Stief T. Micro-thrombi stimulate blood ROS generation. Hemostasis Laboratory 2013; 6 (issue 2/3, in press) Stief TW. The laboratory diagnosis of the pre-phase of pathologic disseminated intravascular coagulation. Hemostasis Laboratory 2008; 1: 2-20. van Golen RF, van Gulik TM, Heger M. The sterile immune response during hepatic ischemia/reperfusion. Cytokine Growth Factor Rev. 2012; 23: 69-84. Kubes P, Mehal WZ. Sterile inflammation in the liver. Gastroenterology. 2012; 143: 1158-72. Stief TW. 20% EXCA (0.1 IU/ml thrombin) – the ideal anticoagulant target value. Hemostasis Laboratory 2011; 4: 275-80. Stief TW. LMWH - action-monitoring for all patients. Acta Paediatr. 2012; 101: e314. 10.1111/j.1651-2227.2012.02712.x. Stief TW. Antibiotics can trigger thrombin generation. Hemostasis Laboratory 2010; 3: 17-36. Stief TW. Coagulation activation by lipopolysaccharides. Clin Appl Thrombosis/Hemostasis Dec 26, 2007 doi: 10.1177/1076029607309256; 2009; 15: 20919.



HIGH DOSE IMIPENEM/CILASTATIN SUPPRESSES BLOOD ROS GENERATION Irina Bogun, Anastasia Busch, and Thomas Stief Institute of Laboratory Medicine and Pathobiochemistry, University Hospital of Marburg, Germany

ABSTRACT Background: The very broad spectrum antibiotic imipenem is resistant to typical ß-lactamases (penicillinases and cephalosporinases). Imipenem is currently combined with the dipeptidase inhibitor cilastatin to increase the plasma concentration of imipenem and to prolong its blood half-time. Since all antibiotics can modulate the oxidative metabolism of human phagocytes, the clinician should know if a certain drug weakens the main cells against invasive bacteria or fungi. Therefore the blood ROS generation assay (BRGA) was performed that analyzes the reactive oxygen species – generating capacity of blood neutrophils in presence of high concentrations of imipenem/cilastatin that can be found at the infusion/blood interface. Material and Methods: 40 µl imipenem/cilastatin in 0.9% NaCl (final drug conc.: 0-2564 mg/l) in black high quality polystyrene microtiter plates (Brand®781608) and 125 µl Hanks´ Balanced Salt Solution (HBSS) were incubated in duplicate with 10 µl fresh citrated blood of 6 healthy donors, 1d old blood of 6 healthy donors, 10 µl 5 mM luminol, and 10 µl zymosan A in 0.9% NaCl (final conc.: 1.9 µg/ml). During the main incubation (37°C) the light emissions of the wells were measured by a photonsmultiplying microtiter plate luminometer (LUmo) with an integration time of 0.5s/well. The approximative 50% inhibitory (approx. IC50) or the approx. 200% stimulatory (approx. SC200) concentrations of imipenem/cilastatin were determined. Results and Discussion: Imipenem/cilastatin inhibits blood ROS generation in the BRGA at approx. IC50 of 60±41 (MV±1SD) RLU/s, measured at the normal main time to 0.5-1fold maximum (usually around 40 min in the BRGA). Thus, imipenem/cilastatin might dangerously decrease blood ROS generation, especially at high concentrations. If cilastatin significantly modulates blood ROS generation, it is suggested to omit cilastatin. Cilastatin might not only inhibit the neutrophils´ action against bacteria and fungi, cilastatin might also diminish cellular fibrinolysis.

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INTRODUCTION ß-Lactamases (penicillinases and cephalosporinases) are a great clinical problem: excellent antibiotics loose efficiency. Fortunately, we dispose of imipenem, a reserve antibiotic of the carbapenem class that is resistant to typical ß-lactamases (Figure 1) [1,2]. Unfortunately, imipenem is currently combined with cilastatin to inhibit the renal dipeptidase dehydropeptidase- 1. The aim for this is to increase the plasma concentration of imipenem and to prolong its blood half-time. Since all antibiotics may modulate the oxidative metabolism of human phagocytes [3], the clinician should know if a given antibiotic would weaken the main cells against invasive bacteria or fungi or if the drug pathologically increases systemic inflammation. Here the blood ROS generation assay (BRGA) was performed that investigates the oxidative capacity of blood neutrophils in presence of high concentrations of imipenem/cilastatin as they can be found at the infusion sites [4,5].

MATERIAL AND METHODS Imipenem/cilastatin (500 mg imipenem = 530 mg imipenem monohydrate, 500 mg cilastatin = 530 mg cilastatin sodium salt, 20 mg NaHCO3) was from Actavis, Hafnarfjördur, Island. 40 µl imipenem/cilastatin in 0.9% NaCl (final drug conc.: 0-2564 mg/l) in black high quality polystyrene microtiter plates (Brand, Wertheim, Germany; article nr. 781608) and 125 µl Hanks´ Balanced Salt Solution (HBSS; without phenol red; Sigma-SAFC Biosciences, Deisenhofen, Germany; article nr. 55037C-1000ML) were incubated in duplicate with 10 µl fresh citrated blood of 6 healthy donors, 1d old blood of 6 healthy donors, 10 µl 5 mM luminol sodium salt (Sigma), and 10 µl zymosan A (Sigma) in 0.9% NaCl (final conc.: 1.9 µg/ml).

Figure 1. Chemical structure of the excellent antibiotic imipenem (above) and the dubious partner cilastatin (below) [1]. Imipenem (N-formimidoyl-thienamycin), C12H17N3O4S (MW: 299.35), is rapidly degraded by renal dehydropeptidase 1 when administered alone, and is therefore combined with cilastatin, C16H26N2O5S (MW: 358.45) to avoid this inactivation.

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During the main incubation (37°C) the light emissions of the wells were measured by a photons-multiplying microtiter plate luminometer (LUmo; anthos, Krefeld, Germany) with an integration time of 0.5s/well. The approximative 50% inhibitory (approx. IC50) or the approx. 200% stimulatory (approx. SC200) concentrations of imipenem/cilastatin were determined.

RESULTS AND DISCUSSION Imipenem/cilastatin inhibits blood ROS generation in the BRGA at approx. IC50 of 60±41 (MV±1SD) RLU/s, measured at the normal main time to 0.5-1fold maximum (usually around 40 min). Thus, imipenem/cilastatin might dangerously decrease blood ROS generation, especially at high concentrations. Since penicillin and aminopenicillin do not suppress blood ROS generation [3], the partner molecule cilastatin might be responsible for blood ROS generation. Both drugs have to be analyzed separately. If cilastatin significantly modulates blood ROS generation, it is suggested to omit cilastatin. Cilastatin might not only inhibit the neutrophils´ action against bacteria and fungi, cilastatin might also diminish cellular fibrinolysis [6]. Consequently, there could be serious systemic [7] and local pro-thrombotic complications as e.g. aseptic fulminant hepatitis (i.e. hepatic micro-thrombi) [8,9].

Figure 2. BRGA kinetic in normal human blood. The blood ROS generation assay (BRGA) was performed for 3 fresh blood samples (4-6) and for 3 1d old blood samples (1-3) as described under methods. The ROS maximum was about 1300-2600 RLU/s, reached after 45-90 min. The ROS maximum of the mean values was about 1500 RLU/s, reached after 65 min. The normal main time to 0.5-1fold maximum was 41 min.

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Figure 3. Inhibition of blood ROS generation by imipenem/cilastatin. Normal blood was incubated with 0-256 mg/l imipenem/cilastatin. The BRGA was performed with 42 min reaction time (BRGA-42). The ROS generations were measured by a photo-multiplying microtiter plate luminometer. The approx. IC50 values were: 50, 100, 170, 40, 30, 20 (68±57) mg/l imipenem/cilastatin. imipenem/cilastatin. The mean values had an approx. IC50 of 60 mg/l imipenem/cilastatin.

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Figure 4. BRGA kinetic in normal human blood. The blood ROS generation assay (BRGA) was performed for 3 fresh blood samples (4-6) and for 3 1d old blood samples (1-3) as described under methods. The ROS maximum was 1447-3132 RLU/s, reached after 39-76 min. The ROS maximum of the mean values was about 1800 RLU/s, reached after 40 min. Samples 3 and 5 had a second ROS maximum at 76 min, presumably due to activation of neutrophils by the micro-thrombi on the plate [6]. The normal main time to 0.5-1fold maximum was 39 min.


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Figure 5. Inhibition of blood ROS generation by imipenem/cilastatin. Normal blood was incubated with 0-256 mg/l imipenem/cilastatin. The BRGA was performed with 39 min reaction time (BRGA-39). The ROS generations were measured by a photo-multiplying microtiter plate luminometer. The approx. IC50 values were: 80, 40, 50, 40, 60, 40 (50±16) mg/l imipenem/cilastatin. imipenem/cilastatin. The mean values had an approx. IC50 of 50 mg/l imipenem/cilastatin.


REFERENCES [1] [2] [3] [4] [5] [6] [7]

www.wikipedia.org Actavis product information. Imipenem/cilastatin (ATC: J01DH51); approval number: 1–29667. Stief T. Flucloxacillin or cefotaxime (but not penicillin G or ampicillin) inhibit blood ROS generation. Hemostasis Laboratory 2013; 6 (issue 2, in press) Stief T. The routine blood ROS generation assay (BRGA) triggered by typical septic concentrations of zymosan A. Hemostasis Laboratory 2013; 6 (issue 1, in press) Stief T. Glucose initially inhibits and later stimulates blood ROS generation. Journal of Diabetes Mellitus 2013, 3: 15-21. doi: 10.4236/jdm.2013.31003. Stief TW. Neutrophil granulocytes in hemostasis. Hemostasis Laboratory 2008; 1: 26989. Stief T. Micro-thrombi stimulate blood ROS generation. Hemostasis Laboratory 2013; 6 (issue 2/3, in press)

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Stief TW. The laboratory diagnosis of the pre-phase of pathologic disseminated intravascular coagulation. Hemostasis Laboratory 2008; 1: 2-20. [9] van Golen RF, van Gulik TM, Heger M. The sterile immune response during hepatic ischemia/reperfusion. Cytokine Growth Factor Rev. 2012; 23: 69-84. [10] Kubes P, Mehal WZ. Sterile inflammation in the liver. Gastroenterology. 2012; 143: 1158-72.



URATE ADDED AT ULTRA-LOW CONCENTRATIONS (0.1 µM) TO NORMAL PLASMA TRIGGERS THROMBIN GENERATION Dana Stenzel and Thomas Stief Institute of Laboratory Medicine and Pathobiochemistry, University Hospital of Marburg, Germany

ABSTRACT Background: Uric acid (trioxy-purine ↔ trihydroxy-purine) circulates in blood as the negatively charged urate. Negatively charged or lipophilic molecules trigger the contact phase of plasmatic coagulation (= intrinsic coagulation = altered matrix (AM) coagulation). The ultra-specific, ultra-sensitive recalcified coagulation activity assay (RECA) is the best thrombin generation assay for any compound at lowest concentrations in blood or plasma. Material and Methods: 40 µl normal human plasma of 4 healthy donors in high quality polystyrene U-wells microtiter plates (Brand®781600) were supplemented with 0-0.15 mg/l additional urate by repetitive 1+1 dilution on the plate. Immediately thereafter the RECA was started by addition of 4 µl 250 mM CaCl2. After 0-25 min the coagulation reaction time (CRT) was stopped by addition of 80 µl 2.5 M arginine, pH 8.6, 0.16% Triton X 100®. After 3 min 20 µl 1 mM chromogenic thrombin substrate HD-CHG-Ala-Arg-pNA in 1.25 M arginine, pH 8.7 were added and the specific increase in absorbance at 405 nm (ΔA) was measured by a mirotiter plate photometer with a 1 mA resolution (PHOmo). The thrombin standard was 40 µl 40 mIU/ml bovine thrombin in 5% human albumin. Results: The approx. SC200 of added urate when given up to 0.15 mg/l (0.9 µM) to normal plasma was 0.02±0.02 mg/l (0.12±0.12 µM; range: 0.02-0.2 µM). Discussion: Urate is an extremely efficient pathophysiologic trigger of AM-coagulation. Urate triggers AM-coagulation about 1000fold stronger than glucose. Spurious amounts of uric acid can cause pathological plasmatic thrombin generation. Presumably, most of urate in blood is complexed to hinder it to fold factor 12/prekallikrein into their respective activated forms. Pathologically elevated urate concentrations have to be lowered to avoid a switch from normal to the pre-phase of pathologic disseminated intravascular coagulation.

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INTRODUCTION The tri-oxy-purine↔tri-hydroxy-purine uric acid (at pH 7.4: urate) is the end product of purine metabolism. Urate is generated by oxidation of xanthine or hypoxanthine by the molybdenum containing enzyme xanthine oxidase [1,2]. The normal urate concentration is below 0.5 mM (10 mg/l=59.48 µmol/l). Pathologically increased concentrations of free urate can change normal to pathologic intravascular coagulation or can cause type 2 diabetes mellitus [1,3-9]. The best assay to test plasma for coagulation activation is the recalcified coagulation activity assay (RECA) [10-12]. Here urate is analyzed in RECA for its thrombin generating capacity.

MATERIAL AND METHODS 40 µl normal human citrated platelet poor plasma (4.5 ml venous blood + 0.5 ml 106 mM sodium citrate in polypropylene monovettes from Sarstedt, Nümbrecht, Germany; till 1 year to expiry) of 4 healthy donors that gave written informed consent in high quality polystyrene U-wells microtiter plates (Brand, Wertheim, Germany; article nr. 781600) were supplemented with additional 0-0.15 mg/l urate (sodium urate was from Sigma, Deisenhofen, Germany; stock solution: 61.5 mg/l urate (61.9 mg/l uric acid) in 0.9% NaCl, checked by analysis on Beckman DXC, Krefeld, Germany) by repetitive 1+1 dilution on the plate. Immediately thereafter the RECA was started by addition of 4 µl 250 mM CaCl2. After 0-25 min the coagulation reaction time (CRT) was stopped by addition of 80 µl 2.5 M arginine, pH 8.6, 0.16% Triton X 100® (Sigma). After 3 min 20 µl 1 mM chromogenic thrombin substrate HD-CHG-Ala-Arg-pNA (Pentapharm, Basel, Switzerland) in 1.25 M arginine, pH 8.7 were added and the specific increase in absorbance at 405 nm (ΔA) was measured by a mirotiter plate photometer with a 1 mA resolution (PHOmo; anthos, Krefeld, Germany). The thrombin standard was 40 µl 40 mIU/ml bovine thrombin (Siemens Healthcare Diagnostics, Munich, Germany) in 5% human albumin (CSL Behring, Marburg, Germany). The standard had a ΔA of 1 mA/min (37°C). Thrombin generation = thrombin activity measured minus basal thrombin activity (0 min CRT). The approximate 200% stimulatory concentrations (approx. SC200) of added urate on recalcified thrombin generation were calculated.

RESULTS AND DISCUSSION The approx. SC200 of added urate when given up to 0.15 mg/l (0.9 µM) to normal plasma was 0.02±0.02 mg/l (0.12±0.12 µM; range: 0.02-0.2 µM) (Figs. 1-4). Urate triggers AM-coagulation about 1000fold stronger than glucose [13,14] ! The susceptibility to AM-coagulation activation varies greatly from individual to individual [15,16]. Urate with its 3 (delta)-negatively charged oxygen atoms should be a trigger of intrinsic coagulation, similar to ellagic acid (Figure1) [17]. Such altered matrix (AM) - coagulation activation generates high blood activities of factor 12a/kallikrein that generate thrombin via intrinsic F10ase. The resulting micro-thrombi are destroyed by cellular fibrinolysis [18-21]. Glucose, triglycerides, cholesterol, lactate have been demonstrated to be efficient

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pathophysiologic AM-coagulation triggers [22-24]. These quantifications have only been made possible by the new ultra-specific, ultra-sensitive thrombin generation assay RECA (recalcified coagulation activity assay) [10-12].

Figure 1. Urate triggers thrombin generation in normal plasma 1. Normal platelet poor plasma was supplemented with 0-0.15 mg/l additional urate. The RECA was performed as described under Material and Methods. 0.02 mg/l was the approx. SC200, seen in the most sensible curve, here RECA-20 (RECA with 20 min coagulation reaction time).


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Figure 3. Urate triggers thrombin generation in normal plasma 3. Normal platelet poor plasma was supplemented with 0-0.15 mg/l additional urate. The RECA was performed as described under Material and Methods. 0.04 mg/l was the approx. SC200 in RECA-20 (RECA with 20 min coagulation reaction time).


In conclusion, urate is an extremely efficient pathophysiologic trigger of AM-coagulation. Urate (disodium or monosodium salt) could be considered as a new trigger substance in a future INCA (intrinsic coagulation activity assay) [17]. Spurious amounts of uric acid when entering the circulating blood can elicit a pathological thrombin generation. Presumably, most of urate in blood is complexed to hinder it to fold factor 12a/prekallikrein. Pathologically elevated urate concentrations have to be lowered to avoid a switch from normal to the pre-phase of pathologic disseminated intravascular coagulation (NICPIC-0)

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[25]. The individual hyperuricemic patient could be tested for the thrombin-generating power of his plasma.

Figure 5. Chemical structure of uric acid [1]. 7,9-dihydro-1H-purine-2,6,8(3H)-trione; C5H4N4O3 ; molecular mass: 168.1 Daltons. The molecule in its 2,6,8-trioxy-purine (keto) form to the left and in its 2,6,8- trihydroxy-purine-(enol) form to the right. The urate ion is demonstrated below.

ACKNOWLEDGMENTS Stephanie Esser is thanked helping to standardize the urate concentration. This research has been performed within an intensive hemostasis training course of medical technicians (Marburg University Hospital MTA School). There was neither specific funding nor any conflict of interest.

REFERENCES [1] [2]

[3] [4]

www.wikipedia.org Okamoto K, Kusano T, Nishino T. Chemical Nature and Reaction Mechanisms of the Molybdenum Cofactor of Xanthine Oxidoreductase. Curr Pharm Des. 2013; 19: 260614. Bellomo G. Serum uric acid and pre-eclampsia: an update. Expert Rev Cardiovasc Ther. 2012; 10: 701-5. Crittenden DB, Lehmann RA, Schneck L, Keenan RT, Shah B, Greenberg JD, Cronstein BN, Sedlis SP, Pillinger MH. Colchicine use is associated with decreased prevalence of myocardial infarction in patients with gout. J Rheumatol. 2012; 39: 145864.

148 [5] [6]

[7]

[8] [9] [10] [11] [12]

[13]

[14]

[15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25]

Dana Stenzel and Thomas Stief Garay RP, El-Gewely MR, Labaune JP, Richette P. Therapeutic perspectives on uricases for gout. Joint Bone Spine. 2012; 79: 237-42. Grassi D, Ferri L, Desideri G, Di Giosia P, Cheli P, Del Pinto R, Properzi G, Ferri C. Chronic Hyperuricemia, Uric Acid Deposit and Cardiovascular Risk. Curr Pharm Des. 2013; 19: 2432-8. Lv Q, Meng XF, He FF, Chen S, Su H, Xiong J, Gao P, Tian XJ, Liu JS, Zhu ZH, Huang K, Zhang C. High serum uric Acid and increased risk of type 2 diabetes: a systemic review and meta-analysis of prospective cohort studies. PLoS One. 2013; 8: e56864. Li C, Hsieh MC, Chang SJ. Metabolic syndrome, diabetes, and hyperuricemia. Curr Opin Rheumatol. 2013; 25: 210-6. Doghramji PP, Wortmann RL. Hyperuricemia and gout: new concepts in diagnosis and management. Postgrad Med. 2012; 124: 98-109. Stief TW. Drug - induced thrombin generation: the breakthrough. Hemostasis Laboratory 2010; 3: 3-6. Stief TW. Pathological thrombin generation by the synthetic inhibitor argatroban. Hemostasis Laboratory 2009; 2: 83-104. Stief TW. Thrombin – applied clinical biochemistry of the main factor of coagulation. In: Thrombin: function and pathophysiology. Stief T, ed.; Nova science publishers; New York; 2012; pp. vii-xx. Stief TW. The maximal plasma concentration of (delta-)negatively charged contact triggers influences plasmatic thrombin generation. In: Thrombin: function and pathophysiology. Stief T, ed.; NOVA science publishers; New York; 2012; pp. 37-46. Stief TW, Mohrez M. Glucose provokes pathologic plasmatic thrombin generation. In: Thrombin: function and pathophysiology. Stief T, ed.; NOVA science publishers; New York; 2012; pp. 23-35. Stief TW. Thrombin generation by therapeutic fibrinogen. Hemostasis Laboratory 2011; 4: 467-82. Stief TW. Thrombin generation by creatinine. Hemostasis Laboratory 2011; 4: 191-9. Stief TW. Ellagic acid as a stable INCA-trigger. Hemostasis Laboratory 2009; 2: 23-32. Stief T. Micro-thrombi stimulate blood ROS generation. Hemostasis Laboratory 2013; 6 (issue 2-3) van Golen RF, van Gulik TM, Heger M. The sterile immune response during hepatic ischemia/reperfusion. Cytokine Growth Factor Rev. 2012; 23: 69-84. Kubes P, Mehal WZ. Sterile inflammation in the liver. Gastroenterology. 2012; 143: 1158-72. Stief TW. Glucose triggers thrombin generation. Hemostasis Laboratory 2010; 3: 93103. Stief TW. Quantification of triglyceride-induced thrombin generation. Hemostasis Laboratory 2011; 4: 39-48. Stief TW. Quantification of cholesterol-induced thrombin generation. Hemostasis Laboratory 2011; 4: 31-8. Stief TW. Quantification of lactate-induced thrombin generation. Hemostasis Laboratory 2011; 4: 23-30. Stief TW. The laboratory diagnosis of the pre-phase of pathologic disseminated intravascular coagulation. Hemostasis Laboratory 2008; 1: 2-20.



URATE TRIGGERS THROMBIN GENERATION Laura Schorge and Thomas Stief Institute of Laboratory Medicine and Pathobiochemistry, University Hospital of Marburg, Germany

ABSTRACT Background: The uric acid (in blood circulating as the negatively charged urate ion) concentration in plasma should never exceed 8 mg/dl. High urate concentrations might pathologically trigger the contact phase of plasmatic coagulation (= intrinsic coagulation = altered matrix (AM) - coagulation). The ultra-specific, ultra-sensitive recalcified coagulation activity assay (RECA) is the best thrombin generation assay for any compound in blood or plasma. Material and Methods: 40 µl normal human citrated plasma of 5 healthy donors in high quality polystyrene U-wells microtiter plates (Brand®781600) were supplemented with 0-1.5 mg/l urate by repetitive 1+1 dilution on the plate. Immediately thereafter the RECA was started by addition of 4 µl 250 mM CaCl2. After 0-25 min the coagulation reaction time (CRT) was stopped by addition of 80 µl 2.5 M arginine, pH 8.6, 0.16% Triton X 100®. After 3 min 20 µl 1 mM chromogenic thrombin substrate HD-CHG-Ala-Arg-pNA in 1.25 M arginine, pH 8.7 were added and the specific increase in absorbance at 405 nm (ΔA) was measured by a mirotiter plate photometer with a 1 mA resolution (PHOmo). The thrombin standard was 40 µl 40 mIU/ml bovine thrombin in 5% human albumin. Results and Discussion: The approx. SC200 of added urate on recalcified thrombin generation in the RECA was 0.3±0.4 mg/l (range: 0.04-0.8 mg/l). There is great interindividual variety in AM-coagulation activation. Individual patients should be tested individually for their recalcified thrombin generation.

INTRODUCTION Uric acid (in blood: urate) is the end product of purine metabolism. Urate is generated by oxidation of xanthine or hypoxanthine by the molybdenum containing enzyme xanthine oxidase [1,2]. The normal urate concentration is below 8 mg/dl (1 mg/dl=59.48 µmol/l). Increased urate concentrations change normal to pathologic intravascular coagulation or can cause type 2 diabetes mellitus [1,3-9]. Urate with its 3 (delta)-negatively charged oxygen atoms should be a trigger of intrinsic coagulation, similar to ellagic acid (Structure 1) [10]. 


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Altered matrix (AM) - coagulation activation generates high blood activities of factor 12a/kallikrein that generate thrombin via intrinsic F10ase. The resulting micro-thrombi are attacked by cellular fibrinolysis [11-13]. Glucose, triglycerides, cholesterol, lactate have been demonstrated to be efficient pathophysiologic AM-coagulation triggers [14-17]. These quantifications have been made possible by usage of the new ultra-specific, ultra-sensitive thrombin generation assay RECA (recalcified coagulation activity assay) [18]. Here uric acid is analyzed for its thrombin generating capacity.

Structure 1. Chemical structure of uric acid [1]. 7,9-dihydro-1H-purine-2,6,8(3H)-trione (2,6,8trioxy-purine); C5H4N4O3 ; molecular mass: 168.1 Daltons.

MATERIAL AND METHODS 40 µl normal human citrated platelet poor plasma (4.5 ml venous blood + 0.5 ml 106 mM sodium citrate in polypropylene monovettes from Sarstedt, Nümbrecht, Germany; till 1 year to expiry) of 4 healthy donors that gave written informed consent in high quality polystyrene U-wells microtiter plates (Brand, Wertheim, Germany; article nr. 781600) were supplemented with additional 0-1.5 mg/l urate (sodium urate was from Sigma, Deisenhofen, Germany; stock solution: 61.9 mg/l uric acid in 0.9% NaCl) by repetitive 1+1 dilution on the plate. Immediately thereafter the RECA was started by addition of 4 µl 250 mM CaCl2. After 0-25 min the coagulation reaction time (CRT) was stopped by addition of 80 µl 2.5 M arginine, pH 8.6, 0.16% Triton X 100® (Sigma). After 3 min 20 µl 1 mM chromogenic thrombin substrate HD-CHG-Ala-Arg-pNA (Pentapharm, Basel, Switzerland) in 1.25 M arginine, pH 8.7 were added and the specific increase in absorbance at 405 nm (ΔA) was measured by a mirotiter plate photometer with a 1 mA resolution (PHOmo; anthos, Krefeld, Germany). The thrombin standard was 40 µl 40 mIU/ml bovine thrombin (Siemens Healthcare Diagnostics, Munich, Germany) in 5% human albumin (CSL Behring, Marburg, Germany). The standard had a ΔA of 1 mA/min (37°C). Thrombin generation = thrombin activity measured minus basal thrombin activity (0 min CRT). The approximate 200% stimulatory concentrations (approx. SC200) of urate on recalcified thrombin generation were calculated. The RECA is the ideal assay to test any prothrombotic tendency of blood or plasma.

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RESULTS AND DISCUSSION The approx. SC200 of added urate on recalcified thrombin generation in the RECA was 0.3±0.4 mg/l (range: 0.04-0.8 mg/l) (Figs. 1-4). There is great inter-individual variety in AM-coagulation activation [19,20].



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Urate is a very efficient pathophysiologic trigger of AM-coagulation. Pathologically elevated urate concentrations have to be lowered to avoid a switch from normal to the prephase of pathologic disseminated intravascular coagulation (NICPIC-0) [21]. The individual hyperuricemic patient could be tested for the thrombin-generating power of his plasma.

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REFERENCES [1] [2]

[3] [4]

[5] [6]

[7]

[8] [9] [10] [11] [12] [13] [14] [15] [16]

www.wikipedia.org Okamoto K, Kusano T, Nishino T. Chemical Nature and Reaction Mechanisms of the Molybdenum Cofactor of Xanthine Oxidoreductase. Curr Pharm Des. 2013; 19: 260614. Bellomo G. Serum uric acid and pre-eclampsia: an update. Expert Rev Cardiovasc Ther. 2012; 10: 701-5. Crittenden DB, Lehmann RA, Schneck L, Keenan RT, Shah B, Greenberg JD, Cronstein BN, Sedlis SP, Pillinger MH. Colchicine use is associated with decreased prevalence of myocardial infarction in patients with gout. J Rheumatol. 2012; 39: 145864. Garay RP, El-Gewely MR, Labaune JP, Richette P. Therapeutic perspectives on uricases for gout. Joint Bone Spine. 2012; 79: 237-42. Grassi D, Ferri L, Desideri G, Di Giosia P, Cheli P, Del Pinto R, Properzi G, Ferri C. Chronic Hyperuricemia, Uric Acid Deposit and Cardiovascular Risk. Curr Pharm Des. 2013; 19: 2432-8. Lv Q, Meng XF, He FF, Chen S, Su H, Xiong J, Gao P, Tian XJ, Liu JS, Zhu ZH, Huang K, Zhang C. High serum uric Acid and increased risk of type 2 diabetes: a systemic review and meta-analysis of prospective cohort studies. PLoS One. 2013; 8: e56864. Li C, Hsieh MC, Chang SJ. Metabolic syndrome, diabetes, and hyperuricemia. Curr Opin Rheumatol. 2013; 25: 210-6. Doghramji PP, Wortmann RL. Hyperuricemia and gout: new concepts in diagnosis and management. Postgrad Med. 2012; 124: 98-109. Stief TW. Ellagic acid as a stable INCA-trigger. Hemostasis Laboratory 2009; 2: 23-32. Stief T. Micro-thrombi stimulate blood ROS generation. Hemostasis Laboratory 2013; 6 (issue 2-3) van Golen RF, van Gulik TM, Heger M. The sterile immune response during hepatic ischemia/reperfusion. Cytokine Growth Factor Rev. 2012; 23: 69-84. Kubes P, Mehal WZ. Sterile inflammation in the liver. Gastroenterology. 2012; 143: 1158-72. Stief TW. Glucose triggers thrombin generation. Hemostasis Laboratory 2010; 3: 93103. Stief TW. Quantification of triglyceride-induced thrombin generation. Hemostasis Laboratory 2011; 4: 39-48. Stief TW. Quantification of cholesterol-induced thrombin generation. Hemostasis Laboratory 2011; 4: 31-8.

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[17] Stief TW. Quantification of lactate-induced thrombin generation. Hemostasis Laboratory 2011; 4: 23-30. [18] Stief TW. Pathological thrombin generation by the synthetic inhibitor argatroban. Hemostasis Laboratory 2009; 2: 83-104. [19] Stief TW. Thrombin generation by therapeutic fibrinogen. Hemostasis Laboratory 2011; 4: 467-82. [20] Stief TW. Thrombin generation by creatinine. Hemostasis Laboratory 2011; 4: 191-9. [21] Stief TW. The laboratory diagnosis of the pre-phase of pathologic disseminated intravascular coagulation. Hemostasis Laboratory 2008; 1: 2-20.



URATE INHIBITS BLOOD ROS GENERATION Christina Lichtenwald and Thomas Stief Institute of Laboratory Medicine and Pathobiochemistry, University Hospital of Marburg, Germany

ABSTRACT Background: Urate is the end product of purine metabolism. This 3fold oxidized purine derivate scavenges oxygen radicals and might be pro-oxidative by interaction with cellular cytochrome P450 enzymes. Therefore we became interested in a possible modulating action of urate on fresh normal blood and analyzed the action of urate on blood neutrophils by the blood ROS (reactive oxygen species) generation assay (BRGA). Material and Methods: 40 µl sodium urate in 0.9% NaCl (final urate conc.: 12.6 mg/l) in black high quality polystyrene microtiter plates (Brand®781608) and 125 µl Hanks´ Balanced Salt Solution (HBSS) were incubated in triplicate with 10 µl fresh citrated blood of 3 healthy donors and 10 µl 5 mM luminol, 10 µl zymosan A in 0.9% NaCl (final conc.: 1.9 µg/ml) were added. During the main incubation (37°C) the light emissions of the wells were measured by a photons-multiplying microtiter plate luminometer (LUmo) with an integration time of 0.5s/well. Results and Discussion: In BRGA with 32 min reaction time (BRGA-32) the approximate 50% inhibitory concentrations (approx. IC50) of urate on blood ROS generation were 0.2, 1.7, 0.8 mg/l. 0.2 mg/l urate is only 1 µM. In BRGA-60 the approx. IC50 of urate was 0.2 (again), - , and 6 mg/l. The longer the main reaction the higher the approx. IC50, unless the neutrophils are in oxidative stress. It seems that the inactivator urate might be metabolized into a stimulator. Thus, there is inter-individual difference in the sensibility to urate. This could be due to the actual oxidative state of the individual and on metabolizing enzymes e.g. of the CYP450 type.

INTRODUCTION Urate is the end product of purine metabolism [1]. This 3fold oxidized purine derivate (Figure1) scavenges oxygen radicals such as superoxide (·O2-) , hydrogen peroxide (HO··OH), hydroxyl radical (HO·) [2] but might also be pro-oxidative by interaction with cellular cytochrome P450 enzymes [3-5]. Therefore we became interested in a possible modulating action of urate on fresh normal blood. Fresh individual normal blood neutrophils [6] should

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be analyzed in a matrix that is close to physiologic, well measurable by the blood reactive oxygen species (ROS) generation assay (BRGA) [7,8].

Figure 1. Chemical structure of uric acid [1]. 7,9-dihydro-1H-purine-2,6,8(3H)-trione (2,6,8- trioxypurine); C5H4N4O3 ; molecular mass: 168.1 Daltons. The 3 oxygen atom can be transformed in hydroxyl-groups. The oxygen atom bound at position 2 of the imidazole ring (1,3-diazacyclopenta-2,4diene; heterocyclic 5-ring) is the one that is negatively charged in urate.

MATERIAL AND METHODS Monosodium urate was from Sigma, Deisenhofen, Germany (article nr. U2875-5G). The stem solution contained 61.9 mg/l uric acid in 0.9% NaCl. 40 µl urate in 0.9% NaCl (final added urate conc.: 0-12.6 mg/l) in black high quality polystyrene microtiter plates (Brand, Wertheim, Germany; article nr. 781608) and 125 µl Hanks´ Balanced Salt Solution (HBSS; Sigma, Deisenhofen, Germany) were mixed in triplicate with 10 µl fresh citrated blood (venous blood supplemented with 10.6 mM sodium citrate, pH 7.4 in polypropylene tubes from Sarstedt, Nümbrecht, Germany) of 3 healthy donors that gave written informed consent. Immediately thereafter 10 µl 5 mM luminol (Sigma), and 10 µl zymosan A (ZyA; Sigma) in 0.9% NaCl (final conc.: 1.9 µg/ml) were added. The samples were stimulated again with 10 µl 36 µg/ml zymosan A, at time points 249 min and 336 min. During the main incubation (37°C) the light emissions of the wells were measured by a photons-multiplying microtiter plate luminometer (LUmo; anthos, Krefeld, Germany) with an integration time of 0.5s/well. The measured data were transferred via Autosoft® to Microsoft®Excel. The approximate 50% inhibitory concentrations (approx. IC50) or the approximate 200% stimulatory concentrations (approx. SC200) of urate on ROS blood generation were determined.

RESULTS AND DISCUSSION Figure 2 demonstrates that the samples 1-3 reached at 41 min their ROS maximum of 5624, 1235, 349 RLU/s. The BRGA with 32 min reaction time (BRGA-32) was compared for the 3 samples. The approximate 50% inhibitory concentrations (approx. IC50) of urate on blood ROS generation were 0.2, 1.7, and 0.8 mg/l, respectively. 0.2 mg/l urate is 1 µM (Figure3). In BRGA-60 the approximate 50% inhibitory concentrations (approx. IC50) of urate on blood ROS generation were 0.2 (again), - , and 6 mg/l, respectively (Figure 4). The longer the main reaction the higher the approx. IC50, unless the neutrophils are in oxidative stress. It seems that the inactivator urate might be metabolized into a stimulator.

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Figure 2. Kinetic of blood ROS generation in fresh normal blood. The BRGA was performed in 3 different normal fresh citrated blood samples, final trigger conc. 1.9 µg/ml zymosan A. At 41 min the samples reached their maximum of 5624, 1235, 349 RLU/s (mean values of duplicate determinations).

Figure 3. Inhibition of blood ROS generation by urate. The results for BRGA with 32 min reaction time (BRGA-32) were compared for the 3 samples. The approximate 50% inhibitory concentrations (approx. IC50) of urate on blood ROS generation were 0.2, 1.7, and 0.8 mg/l, respectively. 0.2 mg/l urate is 1 µM.

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Figure 4. Inhibition of blood ROS generation by urate. The results for BRGA with 60 min reaction time (BRGA-60) were compared for the 3 samples. The approximate 50% inhibitory concentrations (approx. IC50) of urate on blood ROS generation were 0.2 (again), - , and 6 mg/l, respectively.

Figure 5. Repeated zymosan A triggering of blood neutrophils. The BRGA was performed in 3 different normal fresh citrated blood samples as indicated in figure 1, triggering them 3 times with 10 µl zymosan A (1.9 µg/ml ZyA final conc.) at time point 0 min, 249 min, and 336 min (37°C). Blood samples 2 and 3 reached very high maxima of about 5000 and 4000 RLU/s at 309 and 332 min. Blood sample 1 with a first maximum of 5624 RLU/s at 41 min hardly had any reserve to respond to subsequent ZyA stimuli.

If the samples were stimulated again with 10 µl 36 µg/ml zymosan A, at time points 249 min and 336 min, the normal blood samples 2 and 3 reached very high maxima of about

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5000 and 4000 RLU/s at 309 and 332 min. Blood sample 1 with a first maximum of 5624 RLU/s at 41 min hardly had any reserve to respond to subsequent zymosan A stimuli (Figure5). Thus, there is inter-individual difference in the sensibility to urate. This could be due to the actual oxidative state of the individual and on metabolizing enzymes e.g. of the CYP450 type [2-5, 9, 10].


REFERENCES [1] [2]

www.wikipedia.org Wojcik M, Burzynska-Pedziwiatr I, Wozniak LA. A review of natural and synthetic antioxidants important for health and longevity. Curr Med Chem. 2010; 17: 3262-88. [3] Hsu CH, Patel SR, Young EW, Vanholder R. Effects of purine derivatives on calcitriol metabolism in rats. Am J Physiol. 1991; 260: F596-601. [4] Ghosh MK, Mukhopadhyay M, Chatterjee IB. NADPH-initiated cytochrome P450dependent free iron-independent microsomal lipid peroxidation: specific prevention by ascorbic acid. Mol Cell Biochem. 1997; 166: 35-44. [5] Yang KH, Lee JH, Lee MG. Effects of CYP inducers and inhibitors on the pharmacokinetics of intravenous theophylline in rats: involvement of CYP1A1/2 in the formation of 1,3-DMU. J Pharm Pharmacol. 2008; 60: 45-53. [6] Stief TW. Neutrophil granulocytes in hemostasis. Hemostasis Laboratory 2008; 1: 26989. [7] Stief T. The routine blood ROS generation assay (BRGA) triggered by typical septic concentrations of zymosan A. Hemostasis Laboratory 2013; 6: 89-98. [8] Stief T. Glucose initially inhibits and later stimulates blood ROS generation. Journal of Diabetes Mellitus 2013, 3: 15-21. doi: 10.4236/jdm.2013.31003. [9] Fisher MB, Henne KR, Boer J. The complexities inherent in attempts to decrease drug clearance by blocking sites of CYP-mediated metabolism. Curr Opin Drug Discov Devel. 2006; 9:101-9. [10] Yoshimoto FK, Desilets MC, Auchus RJ. Synthesis of halogenated pregnanes, mechanistic probes of steroid hydroxylases CYP17A1 and CYP21A2. J Steroid Biochem Mol Biol. 2012; 128: 38-50.



URATE STIMULATES ROS GENERATION IN PRE-INCUBATED BLOOD Johanna Grass and Thomas Stief Institute of Laboratory Medicine and Pathobiochemistry, University Hospital of Marburg, Germany

ABSTRACT Background: Urate, the 3fold oxidized end product of purine metabolism, is a pathophysiologic trigger of altered matrix (AM) - coagulation. In fresh blood urate might scavenge oxygen radicals and might thereby down-regulate the assembly of the NADPH-oxidase of the neutrophils. In stressed blood, i.e. blood pre-activated by AM-triggers the pro-oxidative cell machinery might be up-regulated. Here fresh normal blood is stressed by one or two pre-incubations and analyzed in the BRGA-60- (blood ROS (reactive oxygen species) generation assay with 60 min preincubation. Material and Methods: 40 µl 0-12.6 mg/l urate (final added conc.) in black high quality polystyrene microtiter plates (Brand®781608) and 125 µl Hanks´ Balanced Salt Solution (HBSS) were incubated in duplicate with 10 µl fresh citrated blood of 3 healthy donors for 60 min and 10 µl 5 mM luminol, 10 µl zymosan A (ZyA) in 0.9% NaCl (final conc.: 0.5 or 1 µg/ml) were added. In BRGA-60-147- the blood samples were triggered again, this time with 10 µl 36 µg/ml ZyA. During the main incubation (37°C) the light emissions of the wells were measured by a photons-multiplying microtiter plate luminometer (LUmo) with an integration time of 0.5s/well. Results and Discussion: In BRGA-60-167 the approx. SC200 of urate on blood ROS generation were 0.6 and 2.5 mg/l.. Blood ROS generation could increased about 5-10fold at 3-6 mg/l urate. Urate concentrations > 6 mg/l decreased blood ROS generation. BRGA-60-143- at 50 min the maximum was reached with about 1900 RLU/s. At 24-60 min main incubation the approx. SC200 was 1 mg/l. Blood ROS generation increased about 5fold at about 6 mg/l urate. Thus, there is inter-individual variation in the sensibility to urate. This could be due to the actual oxidative stress state of the individual blood that influence metabolizing enzymes e.g. of the CYP450 type.

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Johanna Grass and Thomas Stief

INTRODUCTION Urate in its tri-oxidized form is the end product of purine metabolism [1]. Urate in the 1 µM range scavenges oxygen radicals such as superoxide (·O2-), hydrogen peroxide (HO··OH), hydroxyl radical (HO·) [2]. However, urate might also be pro-oxidative by metabolization through cellular cytochrome P450 enzymes [3-5]. Fresh individual normal blood neutrophils [6] should be analyzed in a matrix that is as close to physiologic as possible, a condition that is fulfilled in the blood reactive oxygen species (ROS) generation assay (BRGA) [7,8].

MATERIAL AND METHODS Monosodium urate was from Sigma, Deisenhofen, Germany (article nr. U2875-5G). The stem solution contained 61.9 mg/l uric acid. 40 µl urate in 0.9% NaCl (final added conc.: 0-12.6 mg/l) in black high quality polystyrene microtiter plates (Brand, Wertheim, Germany; article nr. 781608) and 125 µl Hanks´ Balanced Salt Solution (HBSS; Sigma, Deisenhofen, Germany) were mixed in duplicate with 10 µl fresh citrated blood (venous blood supplemented with 10.6 mM sodium citrate, pH 7.4 in polypropylene tubes from Sarstedt, Nümbrecht, Germany) of 3 healthy donors that gave written informed consent. After 60 min at 37°C 10 µl 5 mM luminol (Sigma), and 10 µl zymosan A (ZyA; Sigma) in 0.9% NaCl (final conc.: 0.5 or 1 µg/ml) were added. Furthermore, in one blood the BRGA-60- was performed as described above without addition of ZyA. After 143 min 10 µl 36 µg/ml zymosan A were added. The results for this BRGA-60-143- (BRGA with pre-incubation of 60 min, first incubation of 143 min, followed by the main incubation) were analyzed prior to the maximum. During the main incubation (37°C) the light emissions of the wells were measured by a photons-multiplying microtiter plate luminometer (LUmo; anthos, Krefeld, Germany) with an integration time of 0.5s/well. The measured data were transferred via Autosoft® to Microsoft®Excel. The approximate 50% inhibitory concentrations (approx. IC50) or the approximate 200% stimulatory concentrations (approx. SC200) of urate on ROS blood generation were determined.

RESULTS AND DISCUSSION Figure 1 demonstrates the BRGA-60- in 2 different normal fresh citrated blood samples, final trigger conc. 1.9 µg/ml zymosan A. At 249 min the samples reached their maximum of about 2800-3200 RLU/s (mean values of duplicate determinations). In BRGA-60-167 the approx. SC200 of urate on blood ROS generation were 0.6 and 2.5 mg/l, respectively. Blood ROS generation could increase about 5-10fold at 3-6 mg/l urate. Urate concentrations > 6 mg/l decreased blood ROS generation (Figure 2). The BRGA-60-143- kinetic is seen in figure 3 (BRGA with a first pre-incubation time of 60 min and a second pre-incubation time of 143 min, followed by 1.9 µg/ml ZyA addition and the main incubation time). At 50 min the maximum was reached with about 1900 RLU/s.

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At 24-60 min main incubation the approx. SC200 was 1 mg/l. Blood ROS generation increased about 5fold at about 6 mg/l urate. Thus, there is inter-individual variation in the sensibility to urate (Figure 5). This could be due to the actual oxidative stress state of the individual blood that influence metabolizing enzymes e.g. of the CYP450 type [2-5,9,10].

Figure 1. Kinetic of blood ROS generation in BRGA-60- in fresh normal blood. The BRGA-60was performed in 2 different normal fresh citrated blood samples, final trigger conc. 1.9 µg/ml zymosan A. At 249 min the samples reached their maximum of about 2800-3200 RLU/s (mean values of duplicate determinations).

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Figure 2. Stimulation of blood ROS generation by urate. The results for BRGA-60-167 (BRGA with pre-incubation of 60 min and main incubation of 167 min) were compared for 2 samples. The approximate 200% stimulatory concentrations (approx. SC200) of urate on blood ROS generation were 0.6 and 2.5 mg/l, respectively. Blood ROS generation could increase about 5-10fold at 3-6 mg/l urate. Urate concentrations > 6 mg/l decreased blood ROS generation.

Figure 3. Stimulation of blood ROS generation by urate. The BRGA-60-143- (BRGA with a first pre-incubation time of 60 min and a second pre-incubation time of 143 min, followed by 1.9 µg/ml ZyA addition and the main incubation time) was performed for fresh normal blood. At 50 min the maximum was reached with about 1900 RLU/s.

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Figure 4. Stimulation of ROS generation by urate in stressed blood. The reaction was performed as indicated in figure 3. The BRGA-60-143-24, the BRGA-60-143-43, and the BRGA-60-143-60 RLU/s results are demonstrated in dependence of the urate concentration added. The approximate 200% stimulatory concentration (approx. SC200) of urate on blood ROS generation was 1 mg/l. Blood ROS generation increased about 5fold at about 6 mg/l urate.



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URATE STIMULATES ROS GENERATION IN STRESSED BLOOD Angela Mühling and Thomas Stief Institute of Laboratory Medicine and Pathobiochemistry, University Hospital of Marburg, Germany

ABSTRACT Background: Urate, the 3fold oxidized end product of purine metabolism, is a pathophysiologic trigger of altered matrix (AM) - coagulation. In fresh blood urate might scavenge oxygen radicals and might thereby down-regulate the assembly of the NADPH-oxidase of the neutrophils. In stressed blood, i.e. blood pre-activated by AM-triggers the pro-oxidative cell machinery might be up-regulated. Here fresh normal blood is stressed by one or two pre-incubations and analyzed in the BRGA-60- (blood ROS (reactive oxygen species) generation assay with 60 min preincubation. Material and Methods: 40 µl urate (final urate conc.: 12.6 mg/l) in black high quality polystyrene microtiter plates (Brand®781608) and 125 µl Hanks´ Balanced Salt Solution (HBSS) were incubated in duplicate with 10 µl fresh citrated blood of 3 healthy donors for 60 min and 10 µl 5 mM luminol, 10 µl zymosan A (ZyA) in 0.9% NaCl (final conc.: 0.5 or 1 µg/ml) were added. In BRGA-60-147- the blood samples were triggered again, this time with 10 µl 36 µg/ml ZyA. During the main incubation (37°C) the light emissions of the wells were measured by a photons-multiplying microtiter plate luminometer (LUmo) with an integration time of 0.5s/well. Results: In BRGA-60-67 (BRGA with pre-incubation of 60 min and main incubation of 67 min) the approximate 200% stimulatory concentrations (approx. SC200) of urate on blood ROS generation were 0.7±0.4 mg/l (MV±1SD). Urate concentrations > 6 mg/l decreased blood ROS generation in BRGA-60-. The approx. SC200 of urate on blood ROS generation in BRGA-60-149-43 (BRGA with pre-incubation of 60 min, first incubation of 149 min, and main incubation of 43 min, i.e. at about half-maximal RLU/s) were 0.3±0.1 mg/l. Blood ROS generation increased about 10fold at 1-3 mg/l urate. Discussion: There is inter-individual variation in the sensibility to urate. This could be due to the actual oxidative stress state of the individual blood that influence metabolizing enzymes e.g. of the CYP450 type. The oxidative state in stressed blood is prone to be enhanced by urate.

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Angela Mühling and Thomas Stief

INTRODUCTION Urate in its tri-oxidized form is the end product of purine metabolism (Figure 1) [1]. Urate in the 1 µM range scavenges oxygen radicals such as superoxide (·O2-), hydrogen peroxide (HO··OH), hydroxyl radical (HO·) [2]. However, urate might also be pro-oxidative by metabolization through cellular cytochrome P450 enzymes [3-5]. Fresh individual normal blood neutrophils [6] should be analyzed in a matrix that is as close to physiologic as possible, a condition that is fulfilled in the blood reactive oxygen species (ROS) generation assay (BRGA) [7,8].


MATERIAL AND METHODS Monosodium urate was from Sigma, Deisenhofen, Germany (article nr. U2875-5G). The stem solution contained 61.9 mg/l uric acid. 40 µl urate in 0.9% NaCl (final added urate conc.: 0-12.6 mg/l) in black high quality polystyrene microtiter plates (Brand, Wertheim, Germany; article nr. 781608) and 125 µl Hanks´ Balanced Salt Solution (HBSS; Sigma, Deisenhofen, Germany) were mixed in triplicate with 10 µl fresh citrated blood (venous blood supplemented with 10.6 mM sodium citrate, pH 7.4 in polypropylene tubes from Sarstedt, Nümbrecht, Germany) of 3 healthy donors that gave written informed consent. After 60 min at 37°C 10 µl 5 mM luminol (Sigma), and 10 µl zymosan A (ZyA; Sigma) in 0.9% NaCl (final conc.: 0.5 or 1 µg/ml) were added. Furthermore, this BRGA-60- was performed as described above with 0.5 µg/ml or 1 µg/ml zymosan A. After 149 min 10 µl 36 µg/ml zymosan A were added. The results for this BRGA-60-149-43 (BRGA with pre-incubation of 60 min, first incubation of 149 min, and main incubation of 43 min, i.e. at about half-maximal RLU/s) were compared for the 3 samples. During the main incubation (37°C) the light emissions of the wells were measured by a photons-multiplying microtiter plate luminometer (LUmo; anthos, Krefeld, Germany) with an integration time of 0.5s/well.

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The measured data were transferred via Autosoft® to Microsoft®Excel. The approximate 50% inhibitory concentrations (approx. IC50) or the approximate 200% stimulatory concentrations (approx. SC200) of urate on ROS blood generation were determined.

RESULTS AND DISCUSSION Figure 2 demonstrates that the 1 µg/ml ZyA triggered samples 1-3 reached at 95 min their ROS maximum of 139, 199, 258 RLU/s. In BRGA-60-67 (BRGA with pre-incubation of 60 min and main incubation of 67 min) the approximate 200% stimulatory concentrations (approx. SC200) of urate on blood ROS generation were 0.8, 0.3, and 1 mg/l, respectively. Blood ROS generation could increase about 5-10fold at 1-3 mg/l urate. Urate concentrations > 6 mg/l decreased the generation of blood ROS (Figure 3). BRGA-60- was performed as described above with 0.5 µg/ml or 1 µg/ml zymosan A. After 149 min 10 µl 36 µg/ml zymosan A were added. The results for BRGA-60-149-43 (BRGA with pre-incubation of 60 min, first incubation of 149 min, and main incubation of 43 min, i.e. at about half-maximal RLU/s) were compared for the 3 samples. The approx. SC200 of urate on blood ROS generation were 0.4, 0.2, and 0.3 mg/l, respectively (Figure 4). Blood ROS generation increased about 10fold at 1-3 mg/l urate. Urate concentrations > 2 mg/l or > 6 mg/l decreased blood ROS generation in samples stimulated earlier with 0.5 µg/ml or 1 µg/ml, respectively. Thus, there is inter-individual variation in the sensibility to urate. This could be due to the actual oxidative stress state of the individual blood that influence metabolizing enzymes e.g. of the CYP450 type [2-5,9,10].

Figure 2. Kinetic of blood ROS generation in BRGA-60- in fresh normal blood. The BRGA-60was performed in 3 different normal fresh citrated blood samples, final trigger conc. 1 µg/ml zymosan A. At 95 min the samples reached their maximum of 139, 199, 258 RLU/s (mean values of duplicate determinations).

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Figure 3. Stimulation of blood ROS generation by urate. The results for BRGA-60-67 (BRGA with pre-incubation of 60 min and main incubation of 67 min) were compared for the 3 samples. The approximate 200% stimulatory concentrations (approx. SC200) of urate on blood ROS generation were 0.8, 0.3, and 1 mg/l, respectively. Blood ROS generation could increase about 5-10fold at 1-3 mg/l urate. Urate concentrations > 6 mg/l decreased blood ROS generation.

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Figure 4. Strong stimulation of ROS generation by urate in stressed blood. BRGA-60- was performed as described above with 0.5 µg/ml or 1 µg/ml zymosan A. After 149 min 10 µl 36 µg/ml zymosan A were added. The results for BRGA-60-149-43 (BRGA with pre-incubation of 60 min, first incubation of 149 min, and main incubation of 43 min, i.e. at about half-maximal RLU/s) were compared for the 3 samples. The approximate 200% stimulatory concentrations (approx. SC200) of urate on blood ROS generation were 0.4, 0.2, and 0.3 mg/l, respectively. Blood ROS generation increased about 10fold at 1-3 mg/l urate. Urate concentrations > 2 mg/l or > 6 mg/l decreased blood ROS generation in samples stimulated earlier with 0.5 µg/ml (lower curves) or 1 µg/ml (upper curves), respectively.

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URATE ENHANCES ROS GENERATION IN PRE-INCUBATED OXIDATIVELY STRESSED BLOOD Thomas Stief Institute of Laboratory Medicine and Pathobiochemistry, University Hospital of Marburg, Germany

ABSTRACT Background: Uric acid (tri-oxy-purine, in blood circulating as urate) scavenges radicals and might be oxidized by cellular cytochrome P450 enzymes (CP450) in prooxidative compounds or urate might uncouple CP450, generating singlet oxygen. The blood ROS generation assay (BRGA) quantifies the primary reactive oxygen species (ROS) hydrogen peroxide (H2O2) or the secondary one singlet oxygen (1O2*). The BRGA is used to screen urate for a modulating action on blood ROS generation. Material and Methods: 40 µl 0-61.5 mg/l urate in 0.9% NaCl in black high quality polystyrene F-wells (Brand®781608) were incubated with 125 µl Hanks´ Balanced Salt Solution (HBSS) and 10 µl citrated blood of 11 healthy donors (stored for 1-2 days at 23°C). After 0 min (BRGA) or 60 min at 37°C (BRGA-60-) 10 µl 5 mM luminol in 0.9% NaCl (0.26 mM final) and 10 µl 36 µg/ml zymosan A in 0.9% NaCl (1.9 µg/ml final) were added the light emissions of the wells were counted by a photons-multiplying microtiter plate photometer. Results and Discussion: Urate at about 0.2 mg/l (1 µM) within the first initial minutes after addition to blood suppressed neutrophils in oxidative storage stress. This might be due to the scavenging action of urate on oxygen radicals, especially hydroxyl radicals (•OH), depending on the reaction conditions another important secondary ROS. Urate at higher concentrations stimulated blood ROS generation, 10 mg/l urate doubled the BRGA- activity in stressed blood. The BRGA-60- demonstrates that metabolized urate (within 60 min pre-incubation) stimulates neutrophils. Depending on the reaction time in BRGA-60- between 0.8 or 10 mg/l added urate was the approx. SC200. Thus, the stimulatory power of pre-incubated urate was gradually lost during the main incubation. There might be inter-individual variety in the response to urate that deserves further investigation. Urate when incubating for a long time period with blood might be complexed by binding proteins such as albumin. Complexed urate is relatively inert in its biological activity when compared with free urate that directly interacts with factor 12, prekallikrein, or the CYP450 chain, possibly producing micro-thrombi or cancer, or activating neutrophils. Oxidatively stressed blood might increase CYP450 activity.

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Thomas Stief

INTRODUCTION Uric acid (in blood: urate) is the end product of purine metabolism. Urate is generated by oxidation of xanthine or hypoxanthine by the molybdenum containing enzyme xanthine oxidase [1,2]. The normal urate concentration is below 8 mg/dl (1 mg/dl=59.48 µmol/l). Increased urate concentrations change normal to pathologic intravascular coagulation or can cause type 2 diabetes mellitus [1,3-9]. Urate with its 3 (delta)-negatively charged oxygen atoms should be a trigger of intrinsic coagulation, similar to ellagic acid (Figure 1) [10]. Altered matrix (AM) - coagulation activation generates high blood activities of factor 12a/kallikrein that generate thrombin via intrinsic F10ase. Kallikrein might be an activator of neutrophils. Besides, urate cavenges radicals and might be oxidized by cellular cytochrome P450 enzymes (CP450) in pro-oxidative compounds or urate might uncouple CP450, generating singlet oxygen. The blood ROS generation assay (BRGA) quantifies the primary reactive oxygen species (ROS) hydrogen peroxide (H2O2) or the secondary one singlet oxygen (1O2*). The BRGA is used to screen urate for a modulating action on blood ROS generation [11].

MATERIAL AND METHODS 40 µl 0-61.5 mg/l urate (0-61.9 mg/ml uric acid; 0-12.6 mg/l urate final conc.; sodium urate was from Sigma, Deisehofen, Germany; article nr. U2875-5G) in 0.9% NaCl pipetted by an Eppendorf-multipette® with rinsed tips in black high quality polystyrene F-wells (Brand, Wertheim, Germany; article nr. 781608) were incubated in duplicate with 125 µl Hanks´ Balanced Salt Solution (HBSS; Sigma) and 10 µl 10.6 mM sodium citrate supplemented venous blood (drawn in polypropylene monovettes from Sarstedt, Nümbrecht, Germany) of 11 healthy donors that gave written informed consent. After 0 min (BRGA) or 60 min at 37°C (BRGA-60-) 10 µl 5 mM sodium luminol (Sigma) in 0.9% NaCl (0.26 mM final) and 10 µl 36 µg/ml zymosan A (Sigma) in 0.9% NaCl (1.9 µg/ml final) were added the light emissions of the wells were counted upon increasing incubation at 37°C by a photons-multiplying microtiter plate photometer (LUmo; anthos, Krefeld, Germany). The approximate 200% stimulatory concentrations (approx. SC200) or approximate 50% inhibitory concentrations (approx. IC50) of urate on BRGA or BRGA-60- were calculated.

RESULTS AND DISCUSSION Figure 1 demonstrates the kinetic of blood ROS generation in absence and in presence of added urate. Up to half-maximal ROS generation the urate-supplemented samples had higher ROS generations than the control samples. After the maximum at about 70 min with about 5000 RLU/s the control samples had higher ROS generations than the urate-supplemented ones. In oxidatively stressed blood (due to storage) at BRGA-25 0.2 mg/l added urate decreased blood ROS generation by about 25% (approx. IC75). 10 mg/l urate doubled the initial blood ROS generation (approx. SC200) (Figure 2).

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Figure 1. BRGA kinetic in blood with additional 0 mg/l or 13 mg/l urate. The BRGA was performed for 11 normal blood samples, either unsupplemented or with additional 13 mg/l Na – urate. Up to half-maximal ROS generation the urate-supplemented samples had higher ROS generations than the control samples. After the maximum at about 70 min with about 5000 RLU/s the control samples had higher ROS generations than the urate-supplemented ones. Mean values of 11 samples.

At BRGA-34 (BRGA with 34 min reaction time) 0.4 mg/l added urate decreased blood ROS generation by about 25% (approx. IC75). 10 mg/l urate was the approx. SC150 (Figure 3). Figure 4 demonstrates the kinetic of blood ROS generation after 60 min urate preincubation (BRGA-60-) in absence and in presence of added urate. Always the uratesupplemented samples had higher ROS generations than the control samples, especially pronounced for 6.5 mg/l supplemented samples that had a ROS generation maximum of about 12000 RLU/s when compared with about 9000 RLU/s for the controls.

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At BRGA-60-13 (BRGA 60 min pre-incubation and 13 min main incubation) 0.8 mg/l added urate was the approx. SC200 (Figure 5).

Figure 2. Modulation of blood ROS generation by urate. At BRGA-25 0.2 mg/l added urate decreased blood ROS generation by about 25% (approx. IC75). 10 mg/l urate doubled the initial blood ROS generation (approx. SC200).

Figure 3. Modulation of blood ROS generation by urate. At BRGA-34 (BRGA with 34 min reaction time) 0.4 mg/l added urate decreased blood ROS generation by about 25% (approx. IC75). 10 mg/l urate was the approx. SC150.

At BRGA-60-31 (BRGA 60 min pre-incubation and 31 min main incubation) 10 mg/l added urate was the approx. SC200 (Figure 6). In conclusion, urate at about 0.2 mg/l within the first initial minutes after addition to blood suppressed neutrophils. This might be due to the scavenging action of urate (Figure 7) on oxygen radicals, especially hydroxyl radicals (•OH), depending on the reaction conditions


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another important secondary ROS. Urate at higher concentrations stimulated blood ROS generation, 10 mg/l urate doubled the BRGA- activity.

Figure 4. BRGA kinetic in blood with additional 0 mg/l, 6.5 mg/l, or 13 mg/l urate. The BRGA was performed for 11 normal blood samples, either unsupplemented or with additional 6.5 mg/l or 13 mg/l Na – urate. Always the urate-supplemented samples had higher ROS generations than the control samples, especially pronounced for 6.5 mg/l supplemented samples that had a ROS generation maximum of about 12000 RLU/s when compared with about 9000 RLU/s for the controls. Mean values of 11 samples.

The BRGA-60- demonstrates that metabolized urate (within 60 min pre-incubation) stimulates neutrophils. Depending on the reaction time in BRGA-60- between 0.8 or 10 mg/l added urate was the approx. SC200. Thus, the stimulatory power of pre-incubated urate was gradually lost during the main incubation.

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There might be inter-individual variety in the response to urate that deserves further investigation. Urate when incubating for a long time period with blood might be complexed by binding proteins such as albumin. Complexed urate is relatively inert in its biological activity when compared with free urate that directly interacts with factor 12, prekallikrein, or the CYP450 chain, possibly producing anaphylaxis, micro-thrombi or cancer [12-15], or activating neutrophils [15-22]. Oxidatively stressed blood might increase CYP450 activity and tissue inflammation by phagocytes [23] resulting in gout attacks. Macrophages (with their tissue factor that is activity-enhanced by phospholipids [24]) and fibroblasts drive the inflamed tissue towards fibrosis [25].

Figure 5. Modulation of blood ROS generation by urate. At BRGA-60-13 (BRGA 60 min preincubation and 13 min main incubation) 0.8 mg/l added urate was the approx. SC200.


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Figure 6. Modulation of blood ROS generation by urate. At BRGA-60-31 (BRGA 60 min preincubation and 31 min main incubation) 10 mg/l added urate was the approx. SC200.



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www.wikipedia.org Okamoto K, Kusano T, Nishino T. Chemical Nature and Reaction Mechanisms of the Molybdenum Cofactor of Xanthine Oxidoreductase. Curr Pharm Des. 2013; 19: 260614. Bellomo G. Serum uric acid and pre-eclampsia: an update. Expert Rev Cardiovasc Ther. 2012; 10: 701-5. Crittenden DB, Lehmann RA, Schneck L, Keenan RT, Shah B, Greenberg JD, Cronstein BN, Sedlis SP, Pillinger MH. Colchicine use is associated with decreased prevalence of myocardial infarction in patients with gout. J Rheumatol. 2012; 39: 145864. Garay RP, El-Gewely MR, Labaune JP, Richette P. Therapeutic perspectives on uricases for gout. Joint Bone Spine. 2012; 79: 237-42. Grassi D, Ferri L, Desideri G, Di Giosia P, Cheli P, Del Pinto R, Properzi G, Ferri C. Chronic Hyperuricemia, Uric Acid Deposit and Cardiovascular Risk. Curr Pharm Des. 2013; 19: 2432-8. Lv Q, Meng XF, He FF, Chen S, Su H, Xiong J, Gao P, Tian XJ, Liu JS, Zhu ZH, Huang K, Zhang C. High serum uric Acid and increased risk of type 2 diabetes: a systemic review and meta-analysis of prospective cohort studies. PLoS One. 2013; 8: e56864.

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Thomas Stief Li C, Hsieh MC, Chang SJ. Metabolic syndrome, diabetes, and hyperuricemia. Curr Opin Rheumatol. 2013; 25: 210-6. Doghramji PP, Wortmann RL. Hyperuricemia and gout: new concepts in diagnosis and management. Postgrad Med. 2012; 124: 98-109. Stief TW. Ellagic acid as a stable INCA-trigger. Hemostasis Laboratory 2009; 2: 23-32. Stief T. The routine blood ROS generation assay (BRGA) triggered by typical septic concentrations of zymosan A. Hemostasis Laboratory 2013; 6: 89-98. Nishino M, Mori N, Yoshimura T, Nakamura D, Lee Y, Tanike M, Makino N, Kato H, Egami Y, Shutta R, Tanouchi J, Yamada Y. Higher serum uric acid and lipoprotein(a) are correlated with coronary spasm. Heart Vessels. 2013; Apr 4. Stief T. Micro-thrombi stimulate blood ROS generation. Hemostasis Laboratory 2013; 6 (issue 2-3) van Golen RF, van Gulik TM, Heger M. The sterile immune response during hepatic ischemia/reperfusion. Cytokine Growth Factor Rev. 2012; 23: 69-84. Kubes P, Mehal WZ. Sterile inflammation in the liver. Gastroenterology. 2012; 143: 1158-72. Mitroulis I, Kambas K, Ritis K. Neutrophils, IL-1β, and gout: is there a link ? Semin Immunopathol. 2013 Jan 24. Steiger S, Harper JL. Neutrophil cannibalism triggers transforming growth factor β1 production and self regulation of neutrophil inflammatory function in monosodium urate monohydrate crystal-induced inflammation in mice. Arthritis Rheum. 2013; 65: 815-23. Ryckman C, Gilbert C, de Médicis R, Lussier A, Vandal K, Tessier PA. Monosodium urate monohydrate crystals induce the release of the proinflammatory protein S100A8/A9 from neutrophils. J Leukoc Biol. 2004; 76: 433-40. Sugioka N, Takai M, Yoshida K, Yasuda K, Fukushima K, Kokuhu T, Okamoto M, Yoshimura N, Takada K. Effect of plasma uric acid on pharmacokinetics of cyclosporine A in living-related renal transplant recipients and pharmacokinetic study in rats with experimental hyperuricaemia. J Clin Pharm Ther. 2010 ; 35: 323-32. Stief T. Singlet oxygen (1O2*) primes blood neutrophils to generate ROS. Hemostasis Laboratory 2013; 6 (issue 4) Dennehy KM, Brown GD. The role of the beta-glucan receptor Dectin-1 in control of fungal infection. J Leukoc Biol. 2007; 82: 253-8. de Oliveira-Junior EB, Bustamante J, Newburger PE, Condino-Neto A. The human NADPH oxidase: primary and secondary defects imparing the respiratory burst function and the microbicidal ability of phagocytes. Scand J Immunol 2011; 73: 420-7. Kruth HS. Sequestration of aggregated low-density lipoproteins by macrophages. Curr Opin Lipidol. 2002; 13: 483-8.

[24] Breimo ES, Østerud B. Generation of tissue factor-rich microparticles in an ex vivo whole blood model. Blood Coagul Fibrinolysis. 2005; 16: 399-405. [25] Østerud B. The role of platelets in decrypting monocyte tissue factor. Dis Mon. 2003; 49: 7-13.



BLOOD REACTIVE OXYGEN SPECIES AND URATE Thomas Stief, Christina Lichtenwald, Angela Mühling, Laura Schorge, Dana Stenzel, Johanna Grass, and Dagmar Heinrich Institute of Laboratory Medicine and Pathobiochemistry, University Hospital of Marburg, Germany

ABSTRACT Background: Urate is the tri-oxidized end product of purine metabolism. Urate scavenges oxygen radicals (especially the primary product of NADPH-oxidase HO··OH or its reaction intermediates) and a cytochrome P450 chain (CYP450) metabolite of urate might act as redoxcycler or CYP450 uncoupler, generating H2O2 or 1O2*. The pathophysiologic action of urate on blood neutrophils is of major clinical importance. So we employed the blood ROS generation assay (BRGA) in presence of different urate concentrations. Material and Methods: 40 µl sodium urate in 0.9% NaCl (final added urate conc.: 0-12.6 mg/l) in black high quality polystyrene microtiter plates (Brand®781608) and 125 µl Hanks´ Balanced Salt Solution (HBSS) were mixed in triplicate with 10 µl fresh citrated blood of 5 healthy donors. Immediately (BRGA) and after 60 min (37°C) (BRGA-60-) 10 µl luminol (0.26 mM final), 10 µl zymosan A in 0.9% NaCl (1.9 µg/ml or 0.1 µg/ml final) were added. During the main incubation (37°C) the light emissions of the wells were measured by a photons-multiplying microtiter plate luminometer (LUmo) with an integration time of 0.5s/well. Results: The approx. IC50 of urate on blood ROS generation in BRGA at approximately half-maximal blood ROS generation was 0.2±0.2 mg/l. In the BRGA-60version of the assay (BRGA with 60 min pre-incubation prior to the main incubation, triggered by 0.1 µg/ml ZyA) instead of an IC50 an SC200 appeared that was about 0.2±0.2 mg/l. Discussion: It depends if urate acts directly on normal blood or if there is a prior preincubation of urate with blood. The direct action of urate is inhibitory, the retarded action of urate on blood neutrophils is stimulatory. There is inter-individual difference in the sensibility to urate. This could be due to the actual oxidative stress state of the individual blood and on metabolizing enzymes e.g. of the CYP450 type. Urate when given to blood primarily is a free molecule that can interact with oxygen radicals, CYP450, or factor 12/prekallikrein. Later-on urate will be complexed by plasma proteins, especially albumin that protects the body from potentially hazardous actions of urate. 


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Thomas Stief, Christina Lichtenwald, Angela Mühling et al.

INTRODUCTION Urate is the end product of purine metabolism [1]. This 3fold oxidized purine derivate (Figure 1) scavenges oxygen radicals such as superoxide (·O2-) , hydrogen peroxide (HO··OH), hydroxyl radical (HO·) [2] but might also be pro-oxidative (H2O2 or 1 O2* ) by cytochrome P450 enzymes metabolization/uncoupling [3-5]. Therefore we became interested in the modulating action of urate on normal blood. Individual normal blood neutrophils [6] should be analyzed in a matrix that is as close to physiologic as possible, a condition fulfilled by the blood reactive oxygen species (ROS) generation assay (BRGA) [7,8].

Figure 1. Chemical structure of uric acid [1]. 7,9-dihydro-1H-purine-2,6,8(3H)-trione (2,6,8- trioxypurine); C5H4N4O3 ; molecular mass: 168.1 Daltons. The 3 oxygen atom can be transformed in hydroxyl-groups. The oxygen atom bound at position 2 of the imidazole ring (1,3-diazacyclopenta-2,4diene; heterocyclic 5-ring to the left) is the one that is negatively charged in urate.

MATERIAL AND METHODS Monosodium urate was from Sigma, Deisenhofen, Germany (article nr. U2875-5G). The stem solution contained 61.5 mg/l urate (61.9 mg/l uric acid) in 0.9% NaCl. 40 µl 0-61.5 mg/l urate in 0.9% NaCl (final added urate conc.: 0-12.6 mg/l) in black high quality polystyrene microtiter plates (Brand, Wertheim, Germany; article nr. 781608) and 125 µl Hanks´ Balanced Salt Solution (HBSS; Sigma, Deisenhofen, Germany) were mixed in triplicate with 10 µl fresh citrated blood (venous blood supplemented with 10.6 mM sodium citrate, pH 7.4 in polypropylene tubes from Sarstedt, Nümbrecht, Germany) of 5 healthy donors that gave written informed consent (2 freshest blood samples, about 0.5h old, and 3 blood samples, about 1d old). Immediately (BRGA) or after 60 min at 37°C (BRGA-60-) 10 µl luminol sodium salt (Sigma) (final conc.: 0.26 mM), and 10 µl zymosan A (Sigma) both in 0.9% NaCl (final conc.: 1.9 µg/ml) were added. During the main incubation (37°C) the light emissions of the wells were measured by a photons-multiplying microtiter plate luminometer (LUmo; anthos, Krefeld, Germany) with an integration time of 0.5s/well. The measured data were transferred via Autosoft® to Microsoft®Excel. The approximate 50% inhibitory concentrations (approx. IC50) or the approximate 200% stimulatory concentrations (approx. SC200) of urate on ROS blood generation were determined.

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RESULTS AND DISCUSSION Figure 2 demonstrates that the 5 samples reached at 49 min their ROS maximum of about 600-3400 RLU/s. The mean values reached their maximum of 1786 RLU/s at 49 min, too (Figure 3).

Figure 2. Kinetic of blood ROS generation in normal blood. The BRGA was performed in 5 normal citrated blood samples, final trigger conc. 1.9 µg/ml zymosan A. Samples 1-3 were 1d (23°C) old, samples 4 and 5 were freshest (0.5h). At 49 min the samples reached their maximum of about 600-3400 RLU/s (mean values of triplicate determinations).

Figure 3. Mean blood ROS generation in normal blood. The mean values of figure 2 were calculated. At 49 min the mean values reached their maximum of 1786 RLU/s.

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Figure 4. Inhibition of blood ROS generation by urate. The results for BRGA with 31 min reaction time (BRGA-31) i.e. at approximately half-maximal blood ROS generation were compared for the 5 samples. The approximate 50% inhibitory concentrations (approx. IC50) of urate on blood ROS generation were 0.1, 0.2, 0.6, 0.14, 0.1 mg/l = 0.2±0.2 mg/l (MV±1SD). 0.2 mg/l urate is 1.2 µM.

The results for BRGA with 31 min reaction time (BRGA-31) i.e. at approximately halfmaximal blood ROS generation were compared for the 5 samples in figure 4. The approximate 50% inhibitory concentrations (approx. IC50) of urate on blood ROS generation were 0.2±0.2 mg/l (MV±1SD). 0.2 mg/l urate is 1.2 µM. Figure 5 reflects the BRGA in unsupplemented controls of further stored blood samples. At about 50-100 min the samples reached their maximum of about 200-1200 RLU/s. At BRGA-40, i.e. at approximately half-maximal blood ROS generation, the approx. IC50 values of added urate on blood ROS generation were 0.4, 0.2, 0.4, 0.1, 0.1 mg/l (0.2±0.2 mg/l). Sample 3 had an additional 200% stimulatory concentration (approx. SC200) of 2 mg/l urate (Figure 6).

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Figure 5. Kinetic of blood ROS generation in normal blood. The BRGA was performed in the 5 normal citrated blood samples of figure 4, stored for two additional hours at 23°C prior to analysis (final trigger conc. 1.9 µg/ml zymosan A). At about 50-100 min the samples reached their maximum of about 200-1200 RLU/s (mean values of triplicate determinations).

In the BRGA-60- (BRGA with 60 min pre-incubation prior to the main incubation) the samples reached their maximum (0.1 µg/ml ZyA stimulation) of about 30-80 RLU/s at 89 min (Figure 7). At BRGA-60-49 (BRGA-60- with 49 min reaction time i.e. at approximately halfmaximal blood ROS generation) the approx. SC200 of added urate on blood ROS generation were 0.2±0.2 mg/l (Figure 8). The approx. SC200 of added urate on mean blood ROS generation at BRGA-60-49 was 0.2 mg/l.


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Figure 6. Inhibition of blood ROS generation by urate. The results for BRGA with 40 min reaction time (BRGA-40) i.e. at approximately half-maximal blood ROS generation were compared for the 5 samples of figure 5. The approximate 50% inhibitory concentrations (approx. IC50) of urate on blood ROS generation were 0.2, 0.2, 0.6, 0.1, 0.1 mg/l (0.2±0.2 mg/l). Sample 3 had an additional 200% stimulatory concentration (approx. SC200) of 2 mg/l urate.

Figure 7. Kinetic of blood ROS generation in normal blood. The BRGA-60- (BRGA with 60 min pre-incubation prior to the main incubation) was performed in 5 different normal fresh citrated blood samples with 0.1 µg/ml ZyA. Samples 1-3 were 1d (23°C) old, samples 4 and 5 were freshest (0.5h). At 89 min the samples reached their maximum of about 30-80 RLU/s (mean values of triplicate determinations).

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Figure 8. Stimulation of blood ROS generation by urate. The results for BRGA-60- with 49 min reaction time (BRGA-60-49) i.e. at approximately half-maximal blood ROS generation were compared for the 5 samples. The approximate 200% stimulatory concentrations (approx. SC200) of added urate on blood ROS generation were 0.04, 0.04, 0.1, 0.2, 0.4 mg/l (0.2±0.2 mg/l).

Figure 9. Stimulation of blood ROS generation by urate. The mean values of the 5 samples of figure 8 were calculated. The approx. SC200 of added urate on mean blood ROS generation was 0.2 mg/l.

In conclusion, it depends if urate acts directly on normal blood or if there is a prior preincubation of urate with blood. The direct action of urate is inhibitory, the retarded action of urate on blood neutrophils is stimulatory. There is inter-individual difference in the sensibility to urate. This could be due to the actual oxidative stress state of the individual blood and on metabolizing enzymes e.g. of the CYP450 type [2-5,9-20]. Urate when given to blood primarily is a free molecule that can interact with oxygen radicals, CYP450, or factor 12/prekallikrein. Later-on urate will be complexed by plasma proteins, especially albumin that protects the body from potentially hazardous actions of urate.

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ACKNOWLEDGMENTS Stephanie Esser and Heidrun Schudarek are thanked for their help standardizing the urate concentration. This research has been performed within the intensive hemostasis training course of medical technicians (Marburg University Hospital MTA School). No specific funding, no conflict of interest.

REFERENCES [1] [2] [3] [4]

[5]

[6] [7] [8] [9]

[10]

[11] [12] [13]

[14] [15] [16]

www.wikipedia.org Wojcik M, Burzynska-Pedziwiatr I, Wozniak LA. A review of natural and synthetic antioxidants important for health and longevity. Curr Med Chem. 2010; 17: 3262-88. Hsu CH, Patel SR, Young EW, Vanholder R. Effects of purine derivatives on calcitriol metabolism in rats. Am J Physiol. 1991; 260: F596-601. Ghosh MK, Mukhopadhyay M, Chatterjee IB. NADPH-initiated cytochrome P450dependent free iron-independent microsomal lipid peroxidation: specific prevention by ascorbic acid. Mol Cell Biochem. 1997; 166: 35-44. Yang KH, Lee JH, Lee MG. Effects of CYP inducers and inhibitors on the pharmacokinetics of intravenous theophylline in rats: involvement of CYP1A1/2 in the formation of 1,3-DMU. J Pharm Pharmacol. 2008; 60: 45-53. Stief TW. Neutrophil granulocytes in hemostasis. Hemostasis Laboratory 2008; 1: 26989. Stief T. The routine blood ROS generation assay (BRGA) triggered by typical septic concentrations of zymosan A. Hemostasis Laboratory 2013; 6: 89-98. Stief T. Glucose initially inhibits and later stimulates blood ROS generation. Journal of Diabetes Mellitus 2013, 3: 15-21. doi: 10.4236/jdm.2013.31003. Fisher MB, Henne KR, Boer J. The complexities inherent in attempts to decrease drug clearance by blocking sites of CYP-mediated metabolism. Curr Opin Drug Discov Devel. 2006; 9:101-9. Yoshimoto FK, Desilets MC, Auchus RJ. Synthesis of halogenated pregnanes, mechanistic probes of steroid hydroxylases CYP17A1 and CYP21A2. J Steroid Biochem Mol Biol. 2012; 128: 38-50. Stief T. Singlet oxygen (1O2*) primes blood neutrophils to generate ROS. Hemostasis Laboratory 2013; 6: (issue 4) Stief T. Light quants of low wave length (405 nm violet photons) prime blood neutrophils for ROS generation. Hemostasis Laboratory 2013; 6 (issue 4) Stief T. Blood neutrophils see UV light: 340 nm ultraviolet A stimulates blood ROS generation nearly half as strong as 405 nm violet photons. Hemostasis Laboratory 2013; 6: (issue 4) Stief T. Blood neutrophils alert each other by photons. Hemostasis Laboratory 2013; 6: (issue 4) Stief TW, Fareed J. The antithrombotic factor singlet oxygen/light (1O2/h). Clin Appl Thrombosis/Hemostasis 2000; 6: 22-30. Stief TW, Fu K, Doss MO, Fareed J. The anti-thrombotic factor singlet oxygen (1O2) induces selective thrombolysis in vivo by massive phagocyte infiltration into the

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[17] [18] [19] [20]

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thrombus. XVII Congress of the International Society on Thrombosis and Haemostasis; Washington; August 14-21, 1999. Stief TW. The blood fibrinolysis / deep-sea analogy: a hypothesis on the cell signals singlet oxygen/photons as natural antithrombotics. Thromb Res 2000; 99: 1-20. Stief TW. Regulation of hemostasis by singlet oxygen (1∆O2). Curr Vasc Pharmacol 2004; 2: 357-62. Stief TW. The physiology and pharmacology of singlet oxygen. Med Hypoth. 2003; 60: 567-572. Stief TW. Hemostasis tolerable singlet oxygen - a perspective in AIDS therapy. Hemost Lab. 2008; 1: 21-40.



ACETAMINOPHEN SUPPRESSES THE ROS GENERATION IN FRESHEST BLOOD Ricarda Stumpf and Thomas Stief Institute of Laboratory Medicine and Pathobiochemistry, University Hospital of Marburg, Germany

ABSTRACT Background: Acetaminophen (AAP) has recently been shown to inhibit the oxidative metabolism of blood neutrophils. The purpose of the present investigation was to analyze the approximate 50% inhibitory concentration (approx. IC50) in freshest blood using the blood ROS generation assay (BRGA). Material and Methods: 40 µl 0-10 mg/ml AAP in 0.9% NaCl in black high quality polystyrene microtiter plates (Brand®781608) and 200 µl Hanks´ Balanced Salt Solution (HBSS) were incubated in duplicate with 10 µl freshest citrated blood of 3 healthy donors. The experiment was also performed with 3 different blood samples incubated with 125 µl HBSS. Immediately 10 µl 5 mM luminol (final conc.: 0.18 mM or 0.26 mM), and 10 µl zymosan A in 0.9% NaCl (final conc.: 1.3 µg/ml or 1.9 µg/ml) were added. During the main incubation (37°C) the light emissions of the wells were measured by a photons-multiplying microtiter plate luminometer (LUmo) with an integration time of 0.5s/well. The approximate 50% inhibitory (approx. IC50) concentrations were determined. Results and Discussion: The approx. IC50 values were 5, 3, 6 mg/l AAP for 3 blood samples assayed with 200 µl HBSS at BRGA-68. For 3 different blood samples assayed with 125 µl HBSS at BRGA-56 the approx. IC50 values were 4, 1, 3 mg/l AAP. Thus, the previously reported very low AAP concentrations necessary for inhibition of blood neutrophils could be confirmed. Freshest blood had even lower values. AAP is a very interesting cheap drug against hyper-activated phagocytes. Its action should be quantitatively monitored by the BRGA. The target level should not be higher than 150% of normal, measured at t-0.5maxn to t-maxn.

INTRODUCTION Acetaminophen (AAP = paracetamol) has recently been shown to inhibit the oxidative metabolism of blood neutrophils. The approx. IC50 went down to only 3 mg/l AAP [1]. The purpose of the present investigation was to check the approximate 50% inhibitory 

Correspondence to: PD Dr. med. T. Stief, Central Laboratory, University Hospital, D-35043 Marburg, Germany. Email: [email protected]; Tel.: +49-6421-58 64471; FAX: +49-6421-58 65594.

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concentration (approx. IC50) in freshest blood (less than 0.5h old) using the blood ROS generation assay (BRGA) [2, 3].

MATERIAL AND METHODS Acetaminophen (Perfalgan®) (C8H9NO2; MW: 151.2 Daltons) was from Bristol-Myers Squibb, Munich, Germany; 100 ml stem solution contained 10 mg/ml. 40 µl 0-10 mg/ml AAP in 0.9% NaCl in black high quality polystyrene microtiter plates (Brand, Wertheim, Germany; article nr. 781608) and 200 µl Hanks´ Balanced Salt Solution (HBSS modified without phenol red; Sigma-SAFC Biosciences, Deisenhofen, Germany; article nr. 55037C-1000ML) were incubated in duplicate with 10 µl freshest citrated blood (venous blood in polypropylene monovettes from Sarstedt, Nümbrecht, Germany, supplemented with 11 mM sodium citrate) of 3 healthy donors. The experiment was also performed with 3 different blood samples incubated with 125 µl HBSS. Immediately 10 µl 5 mM luminol (Sigma; final conc.: 0.18 mM or 0.26 mM), and 10 µl zymosan A (Sigma) in 0.9% NaCl (final conc.: 1.3 µg/ml or 1.9 µg/ml) were added. During the main incubation (37°C) the light emissions of the wells were measured by a photons-multiplying microtiter plate luminometer (LUmo; anthos, Krefeld, Germany) with an integration time of 0.5s/well. The approximate 50% inhibitory (approx. IC50) concentrations were determined.

RESULTS AND DISCUSSION Figure 1 demonstrates the normal blood ROS generation kinetic of freshest citrated blood, using 200 µl HBSS. The unsupplemented blood ROS maxima were about 1900, 600, 1500 RLU/s, reached after 60-70 min (t-maxn). At 136 min there started a second ROS peak, the begin of cellular fibrinolysis, the neutrophils attack the by recalcification generated microthrombi [4-7]. The unsupplemented blood ROS maxima with 125 µl HBSS in 3 different samples were about 3600, 1300, 2200 RLU/s, reached after 70-90 min (t-maxn) (Figure 2). At about 140 min reaction time in samples 1 and 3 blood ROS generation peaked again in cellular fibrinolysis. The approx. IC50 values were 5, 3, 6 mg/l AAP for 3 blood samples assayed with 200 µl HBSS at BRGA-68 (Figure 3). For 3 different blood samples assayed with 125 µl HBSS at BRGA-56 the approx. IC50 values were 4, 1, 3 mg/l AAP (Figure 4). From the mean values it could be deduced that an incubation with 125 µl HBSS renders AAP about twice as effective than one with 200 µl HBSS. In conclusion, the previously reported very low AAP concentrations necessary for inhibition of blood neutrophils could be confirmed. Freshest blood had even lower values. AAP is a very interesting cheap drug against hyper-activated phagocytes. Its action should be quantitatively monitored by the BRGA. The target level should not be higher than 150% of normal, measured at t-0.5maxn to t-maxn.

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Figure 1. BRGA kinetic in normal human citrated blood. The blood ROS generation assay (BRGA) was performed for freshest citrated blood samples, incubating 10 µl of them with 200 µl HBSS. The unsupplemented blood ROS maxima were about 1900, 600, 1500 RLU/s, reached after 60-70 min (t-maxn).

Figure 2. BRGA kinetic in normal human citrated blood. The blood ROS generation assay (BRGA) was performed for 3 different citrated blood samples, this time with 125 µl HBSS. The unsupplemented blood ROS maxima were about 3600, 1300, 2200 RLU/s, reached after 70-90 min (t-maxn). The approximate time to 0.5fold the maximum (t-0.5maxn) of sample 1 was 56 min.

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Figure 3. Inhibition of blood ROS generation by AAP. The blood ROS generation assay at 68 min main incubation (BRGA-68) was performed for freshest citrated blood samples supplemented with acetaminophen. The approx. IC50 values were 5, 3, 6 mg/l AAP.

Figure 4. Inhibition of blood ROS generation by AAP. The blood ROS generation assay at 56 min main incubation (BRGA-56) was performed for freshest citrated blood samples supplemented with acetaminophen. The approx. IC50 values were 4, 1, 3 mg/l AAP.

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REFERENCES [1] [2] [3] [4] [5] [6] [7]

Stief T. Acetaminophen (AAP=paracetamol) suppresses blood ROS generation at an IC50 of only 1-3 mg/l. Hemostasis Laboratory 2013; 6 (issue 2-3) Stief T. The routine blood ROS generation assay (BRGA) triggered by typical septic concentrations of zymosan A. Hemostasis Laboratory 2013; 6: 89-98. Stief T. Glucose initially inhibits and later stimulates blood ROS generation. Journal of Diabetes Mellitus 2013, 3: 15-21. doi: 10.4236/jdm.2013.31003. Stief T. Micro-thrombi stimulate blood ROS generation. Hemostasis Laboratory 2013; 6 (issue 2-3) Stief TW. Neutrophil granulocytes in hemostasis. Hemostasis Laboratory 2008; 1: 26989. Stief TW. The laboratory diagnosis of the pre-phase of pathologic disseminated intravascular coagulation. Hemostasis Laboratory 2008; 1: 2-20. Stief TW, Fareed J. The antithrombotic factor singlet oxygen/light (1O2/h). Clin Appl Thrombosis/Hemostasis 2000; 6: 22-30.



TAURINE STRONGLY ENHANCES THE ROS GENERATION OF BLOOD NEUTROPHILS Anastasia Busch and Thomas Stief Institute of Laboratory Medicine and Pathobiochemistry, University Hospital of Marburg, Germany

ABSTRACT Background: Taurine is a 125-Daltons amino sulfonic acid, related to the normal human amino acids; without a carboxylate group taurine does not participate in the normal protein synthesis. Taurine is semi-essential, our food should contain at least 0.1% taurine. 0.1 % is the approximate percentage of taurine in the human body. Since taurine is the main stabilizing molecule of natural chloramines, important generators of singlet oxygen, here the action of taurine on the generation of reactive oxygen species (ROS) of blood neutrophils was investigated in the blood ROS generation assay (BRGA). Material and Methods: 40 µl taurine in 0.9% NaCl (final drug conc.: 0-26 mM) in black high quality polystyrene microtiter plates (Brand®781608) and 125 µl Hanks´ Balanced Salt Solution (HBSS) were incubated in duplicate with 10 µl freshest citrated blood of a healthy donor or with 1d old blood of 2 healthy donors. Immediately (BRGA) or after 60 min (BRGA-60-), 10 µl 5 mM luminol (final conc.: 0.26 mM), and 10 µl zymosan A in 0.9% NaCl (final conc.: 1.9 µg/ml). During the main incubation (37°C) the light emissions of the wells were measured by a photons-multiplying microtiter plate luminometer (LUmo) with an integration time of 0.5s/well. The approximate 50% inhibitory (approx. IC50) or the approx. 200% stimulatory (approx. SC200) concentrations of taurine were determined. Results: Taurine increased blood ROS generation in fresh healthy citrated blood with an approx. SC200 of 7-8 mM in BRGA-60- and BRGA. An approx. IC50 of 0.2-0.5 mM taurine appeared in BRGA and BRGA-60-, suggesting radicals-catching efficiency of taurine. Prolonged incubation decreased the approx. SC200 to 1-2.5 mM in BRGA-60- and BRGA. 3.3 mM taurine supplemented blood had in the BRGA about 1.5-3fold higher ROS generations especially within the peri-maximal and terminal part of the blood ROS generation kinetic. Blood samples that been stored for 1d at 23°C had about 1.5fold higher SC200 values. Discussion: Since cells can accumulate taurine via specific transporters, an ingestion of just about 500 mg daily could aggravate any systemic inflammatory disorder or complicate a pregnancy. Taurine seems to be a ROS modifier, decreasing the amount of radicalic ROS and favouring the generation of the non-radicalic singlet oxygen (via 


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Anastasia Busch and Thomas Stief N-chloro-taurine). Taurine seems to favour cellular fibrinolysis. This might be beneficial in states of insufficient cellular fibrinolysis but it could also be dangerous in states of hyper-fibrinolysis (e.g. re-bleeding after subarachnoidal bleeding, inflammatory focus in the CNS, systemic inflammation, placental bleeding).

INTRODUCTION Taurine is not a usual amino acid, it is an amino sulfonic acid (Figure 1) [1-6]. Taurine does not contain the for amino acids typical carboxyl group. There are oligopeptides that contain taurine but up to now there has been found no aminoacyl tRNA synthetase specific for translating a genetic code into taurine. The name taurine stems from its first discovery in bull bile (tauros = greek word for bull) by Gmelin and Tiedemann (1827). Taurine in the bile occurs as taurocholic acid. Taurine is dissolvable up to approx. 100 g/l (0.8 M) in water.

Figure 1. Chemical structure of taurine [1]. 2-amino-ethane-sulfonic acid; 125.15 Daltons. Taurine is the most abundant sole amino acid in the human body. Its plasma concentration is about 0.04 mM. The maximum plasma taurine concentration was measured at 1.5 ± 0.6 h after oral administration of g taurine “red bull” as 86±19 mg/l ( 0.69±0.15 mmol/l). The plasma elimination half-life was 1.0±0.3h [7]. The adequate taurine concentration is of vital importance: dogs with low plasma taurine were older, less active, and sicker [8].

About 75% of the bodie´s taurine is in the muscle cells, the rest mainly in brain, heart and blood. A healthy human body contains about 0.1 % taurine. Taurine consumption varies between 40 and 400 mg/day, depending on the meat content of the meal. Taurine is used as an ingredient in energy drinks, containing about 1000 (sometimes 2000 mg) per serving. Absorption of taurine from beverages may be more rapid than from foods. Taurine is semi-essential: any food product approved by the Association of American Feed Control Officials (AAFCO) should have a minimum of 0.1% taurine in dry food and 0.2% in wet food. Taurine is a decarboxylated tri-oxidized (by cysteine dioxygenase) derivative of cysteine (homotaurine is the respective derivative of homocysteine or methionine). Taurine stimulates the membrane fluxes of Ca2+, Na+, and K+. It stabilizes the cell membrane and is anti-arrhythmic. Taurine is important for normal cardiovascular function, and development and function of skeletal muscle, the retina and the central nervous system. Taurine crosses the blood–brain barrier. Human neutrophils chlorinate taurine to N-chloro-taurine (= taurine-chloramine) [4,5,9]. Chloramines are physiologic generators of singlet oxygen, an activator of cellular fibrinolysis [4]. In pathologically increased thrombin generation [10] cellular fibrinolysis increases for compensation: 137 µM is the plasma concentration of taurine in stroke patients, which is about 3fold elevated when compared to healthy individuals [11]. So, taurine prevents diabetes-associated microangiopathy and reduced fatty liver deposits and cirrhosis in rats. Too much of taurine (2000 mg intake per day) activated not only physiologic but also pathologic inflammation e.g. in psoriasis [1].

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MATERIAL AND METHODS Taurine was from Sigma, Deisenhofen, Germany. 40 µl in 0.9% NaCl (final drug conc.: 0-26 mM = 0-3282 mg/l) in black high quality polystyrene microtiter plates (Brand, Wertheim, Germany; article nr. 781608) and 125 µl Hanks´ Balanced Salt Solution (HBSS; without phenol red; Sigma-SAFC Biosciences, Deisenhofen, Germany; article nr. 55037C-1000ML) were incubated in duplicate with 10 µl freshest (less than 0.5h old) citrated blood of a healthy donor that gave written informed consent or with 10 µl 1d (23°C) old citrated blood of two healthy donors that gave written informed consent. Immediately (BRGA) or after 60 min (37°C) (BRGA-60-) 10 µl 5 mM luminol sodium salt (Sigma), and 10 µl 36 µg/ml zymosan A (Sigma) in 0.9% NaCl. During the main incubation (37°C) the light emissions of the wells were measured by a photons-multiplying microtiter plate luminometer (LUmo; anthos, Krefeld, Germany) with an integration time of 0.5s/well [12-14]. The approximate 50% inhibitory (approx. IC50) or the approx. 200% stimulatory (approx. SC200) concentrations of taurine were determined.

RESULTS AND DISCUSSION Figure 2 demonstrates the normal blood ROS generation kinetic of freshest citrated blood. The ROS maximum was 1373 RLU/s, reached after 85 min (t-maxn). t-0.25maxn  56 min, t-0.5maxn  74 min.

Figure 2. BRGA kinetic in normal human citrated blood, further supplemented with 0 mM or 3.3 mM taurine. The blood ROS generation assay (BRGA) was performed for a fresh citrated blood sample further supplemented with 0 mM or 3.3 mM taurine (tau) as described under methods. The unsupplemented blood ROS maximum was 1373 RLU/s, reached after 85 min (t-maxn). t-0.25maxn  56 min (352 RLU/s), t-0.5maxn  74 min (967 RLU/s).

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Figure 3. BRGA kinetic in normal human citrated blood. The blood ROS generation assay with 60 min pre-incubation (BRGA-60-) was performed for freshest citrated blood sample as described under methods. The ROS maximum was 2841 RLU/s, reached after 134 min (t-maxn). t-0.25maxn  48 min (649 RLU/s), t-0.5maxn  77 min (1354 RLU/s). 3.3 mM taurine supplemented blood had up to 3fold higher ROS generations (peri-maximal and post-maximal) (Figure 3a). In the initial phase 3.3 mM taurine inhibits blood ROS generation (Figure 3b).

At 170 min there appeared a second ROS peak (651 RLU/s). From 107 min onward up to 194 min the blood ROS generation was rather stable at a level of about 600 RLU/s, this action and the second ROS peak is due to micro-thrombi induced ROS generation [14]. 3.3 mM taurine supplemented blood had about 1.5-3fold higher ROS generations especially within the peri-maximal and terminal part of the blood ROS generation kinetic. In BRGA-60- the ROS maximum was 2841 RLU/s, reached after 134 min (t-maxn). t-0.25maxn  48 min (649 RLU/s), t-0.5maxn  77 min (1354 RLU/s) (Figure 3). 3.3 mM taurine supplemented blood had up to 3fold higher ROS generations (peri-maximal and post-maximal) (Figure 3a). In the initial phase 3.3 mM taurine inhibits blood ROS


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generation (Figure 3b). This means that in the initial phase oxygen radicals -quenchable by taurine- are of great importance to start NADPH-oxidase assembly. In the peri-maximal or post-maximal phase, however, singlet oxygen (N-chloro-taurine being the pro-drug) is the main ROS.

Figure 4. Stimulation of blood ROS generation by taurine. The blood ROS generation assay at 56 min main incubation (BRGA-56) was performed for a fresh citrated blood sample supplemented with 0-13 mM taurine. The approx. IC50 was 0.2 mM added taurine, the approx. SC200 was 8 mM taurine.

Figure 5. Stimulation of blood ROS generation by taurine. The blood ROS generation assay at 74 min main incubation (BRGA-74) was performed for a fresh citrated blood sample supplemented with 0-13 mM taurine. The approx. SC150 was 4 mM taurine.

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Figure 6. Stimulation of blood ROS generation by taurine. The blood ROS generation assay at 170 min main incubation (BRGA-170) was performed for a fresh citrated blood sample supplemented with 0-13 mM taurine. The approx. SC200 was 2.5 mM taurine.

Figure 7. Modulation of blood ROS generation by pre-incubated taurine. The blood ROS generation assay with 60 min pre-incubation at 48 min main incubation (BRGA-60-48) was performed for a fresh citrated blood sample supplemented with 0-13 mM taurine. The approx. SC200 was 7 mM taurine. There was also an approx. IC50 of 0.5 mM taurine.

Taurine increased blood ROS generation in fresh healthy citrated blood with an approx. SC200 of 8 mM or an approx. SC150 of 4-5 mM (Figs. 4,5). An approx. IC50 appeared of 0.2 mM taurine, suggesting radicals-catching efficiency of taurine (Figure 4). Prolonged incubation decreased the approx. SC200 to 2.5 mM (Figure 6). In the BRGA-60- the approx. SC200 was 7 mM taurine. There was also an approx. IC50 of 0.5 mM taurine (Figures 7,8). In prolonged incubation (BRGA-60-184) the approx. SC200 was 1 mM taurine (Figure 9).


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Figure 8. Modulation of blood ROS generation by pre-incubated taurine. The blood ROS generation assay with 60 min pre-incubation at 77 min main incubation (BRGA-60-77) was performed for a fresh citrated blood sample supplemented with 0-13 mM taurine. The approx. SC200 was 7 mM taurine. There was also an approx. IC50 of 0.5 mM taurine. In BRGA-60-184 the approx. SC200 was 1 mM taurine.

Figure 9. BRGA kinetic in citrated blood (1d old). The blood ROS generation assay (BRGA) was performed for two 1d old citrated blood samples as described under methods. The ROS maximum was 358 RLU/s or 2224 RLU/s, reached after about 60-70 min or 84 min (t-maxn).

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Figure 10. Taurine modulates BRGA. The blood ROS generation assay with 43 min main incubation (BRGA-43) was performed for two 1d old citrated blood samples supplemented with 0-26 mM taurine. The approx. SC200 was 11 mM for sample 2; approx. IC50 = 1-1.5 mM taurine.

Figure 11. Taurine modulates BRGA. The blood ROS generation assay with 56 min main incubation (BRGA-56) was performed for two 1d old citrated blood samples supplemented with 0-26 mM taurine. The approx. SC200 was 13 mM or 14 mM taurine.

Blood samples that been stored for 1d at 23°C had higher approx. SC200 values in the range of about 10 mM taurine (Figs. 10-13). The approx. SC150 values in BRGA-60- were 3 mM or 1 mM taurine (Figures 14,15). Thus, taurine in low milli-molar concentrations strongly increases blood ROS generation. Since cells can accumulate taurine via specific transporters [16-20], an ingestion of just about 500 mg daily could aggravate any systemic inflammatory disorder or complicate a pregnancy.


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Figure 12. Taurine modulates BRGA. The blood ROS generation assay with 71 min main incubation (BRGA-71) was performed for two 1d old citrated blood samples supplemented with 0-26 mM taurine. The approx. SC200 was 9 mM or 20 mM taurine. The approx. IC50 was 1 mM or 1.6 mM taurine.

Figure 13. Taurine modulates BRGA. The blood ROS generation assay with 111 min main incubation (BRGA-111) was performed for two 1d old citrated blood samples supplemented with 0-26 mM taurine. The approx. SC200 was 12 mM or 13 mM taurine.

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Figure 14. BRGA kinetic in normal human citrated blood. The blood ROS generation assay with 60 min pre-incubation (BRGA-60-) was performed for two 1d old citrated blood samples as described under methods. The ROS maximum was 3354 or 7706 RLU/s, reached after 72 min (t-maxn).

Figure 15. Taurine modulates BRGA. The blood ROS generation assay with 60 min pre-incubation and 39 min main incubation (BRGA-60-39) was performed for two 1d old citrated blood samples supplemented with 0-26 mM taurine. The approx. SC150 values were 3 mM or 1 mM taurine. The approx. IC50 values were 23 or 20 mM taurine.

Taurine seems to be a ROS modifier, decreasing the amount of radicalic ROS and favouring the generation of the non-radicalic singlet oxygen (via N-chloro-taurine) [21-23]. Taurine seems to favour cellular fibrinolysis. This might be beneficial in states of insufficient cellular fibrinolysis but it could also be dangerous in states of hyper-fibrinolysis (e.g. rebleeding after subarachnoidal bleeding, inflammatory focus in the CNS, systemic inflammation, placental bleeding).


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ACKNOWLEDGMENTS This research has been performed within an intensive hemostasis training course of medical technicians (Marburg University Hospital MTA School). One participant of the course who had consumed an energy drink had several-fold increased blood ROS generations. There was neither specific funding nor any conflict of interest.

REFERENCES [1] [2] [3] [4] [5] [6] [7]

[8]

[9] [10] [11] [12] [13] [14] [15]

[16]

www.wikipedia.org Ripps H, Shen W. Review: taurine: a "very essential" amino acid. Mol Vis. 2012; 18:2673-86. Murakami S. Taurine and atherosclerosis. Amino Acids. 2012 Dec 8. Marcinkiewicz J, Kontny E. Taurine and inflammatory diseases. Amino Acids. 2012 Jul 19. Weiss SJ, Klein R, Slivka A, Wei M. Chlorination of Taurine by Human Neutrophils. Journal of Clinical Investigation 1982; 70: 598–607. Abebe W, Mozaffari MS. Role of taurine in the vasculature: an overview of experimental and human studies. Am J Cardiovasc Dis. 2011; 1: 293-311. Ghandforoush-Sattari M, Mashayekhi S, Krishna CV, Thompson JP, Routledge PA. Pharmacokinetics of Oral Taurine in Healthy Volunteers. Journal of Amino Acids 2010, doi:10.4061/2010/346237. Backus RC, Ko KS, Fascetti AJ, Kittleson MD, Macdonald KA, Maggs DJ, Berg JR, Rogers QR. Low plasma taurine concentration in Newfoundland dogs is associated with low plasma methionine and cyst(e)ine concentrations and low taurine synthesis. Journal of Nutrition 2006; 136: 2525-33. Stief TW. Neutrophil granulocytes in hemostasis. Hemostasis Laboratory 2008; 1: 26989. Stief TW. The laboratory diagnosis of the pre-phase of pathologic disseminated intravascular coagulation. Hemostasis Laboratory 2008; 1: 2-20. Ghandforoush-Sattari M, Mashayekhi SO, Nemati M, Ayromlou H. Changes in plasma concentration of taurine in stroke. Neurosci Lett. 2011; 496: 172-5. Stief T. The routine blood ROS generation assay (BRGA) triggered by typical septic concentrations of zymosan A. Hemostasis Laboratory 2013; 6: 89-98. Stief T. Glucose initially inhibits and later stimulates blood ROS generation. Journal of Diabetes Mellitus 2013, 3: 15-21. doi: 10.4236/jdm.2013.31003. Stief T. Micro-thrombi stimulate blood ROS generation. Hemostasis Laboratory 2013; 6 (issue 2-3) Warskulat U, Brookmann S, Felsner I, Brenden H, Grether-Beck S, Häussinger D. Ultraviolet A induces transport of compatible organic osmolytes in human dermal fibroblasts. Exp Dermatol. 2008; 17: 1031-6. Roig-Pérez S, Moretó M, Ferrer R. Transepithelial taurine transport in caco-2 cell monolayers. J Membr Biol. 2005; 204: 85-92.

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[17] Bitoun M, Tappaz M. Gene expression of the transporters and biosynthetic enzymes of the osmolytes in astrocyte primary cultures exposed to hyperosmotic conditions. Glia. 2000; 32: 165-76. [18] Leibach JW, Cool DR, Del Monte MA, Ganapathy V, Leibach FH, Miyamoto Y. Properties of taurine transport in a human retinal pigment epithelial cell line. Curr Eye Res. 1993; 12: 29-36. [19] Kimelberg HK, Goderie SK, Higman S, Pang S, Waniewski RA. Swelling-induced release of glutamate, aspartate, and taurine from astrocyte cultures. J Neurosci. 1990; 10: 1583-91. [20] Reynolds R, Herschkowitz N. Selective uptake of neuroactive amino acids by both oligodendrocytes and astrocytes in primary dissociated culture: a possible role for oligodendrocytes in neurotransmitter metabolism. Brain Res. 1986; 371: 253-66. [21] Devamanoharan PS, Ali AH, Varma SD. Oxidative stress to rat lens in vitro: protection by taurine. Free Radic Res. 1998; 29: 189-95. [22] Weiss SJ, Lampert MB, Test ST. Long-lived oxidants generated by human neutrophils: characterization and bioactivity. Science. 1983; 222: 625-8. [23] Stief TW, Fareed J. The antithrombotic factor singlet oxygen/light (1O2/h). Clin Appl Thrombosis/Hemostasis 2000; 6: 22-30.



CAFFEINE MODULATES BLOOD ROS GENERATION Angela Mühling and Thomas Stief Institute of Laboratory Medicine and Pathobiochemistry, University Hospital of Marburg, Germany

ABSTRACT Background: Caffeine at about 10 mg/l is a drug for preterm neonates against apnea. These patients often suffer from respiratory dysfunction, distress, or infection. Any compound that pathologically alters the activity of the neutrophils is of great medical interest. Here the methyl-xanthine derivative caffeine is investigated for possible modulation of blood reactive oxygen species (ROS) generation, using the blood ROS generation assay (BRGA). Material and Methods: 40 µl caffeine in 0.9% NaCl (final drug conc.: 0-61.5 mg/l) in black high quality polystyrene microtiter plates (Brand®781608) and 125 µl Hanks´ Balanced Salt Solution (HBSS) were incubated in duplicate with 10 µl freshest citrated blood of a healthy donor or with 1d old blood of 2 healthy donors. Immediately (BRGA) or after 60 min (BRGA-60-), 10 µl 5 mM luminol (final conc.: 0.26 mM), and 10 µl zymosan A in 0.9% NaCl (final conc.: 1.9 µg/ml) were added. During the main incubation (37°C) the light emissions of the wells were measured by a photonsmultiplying microtiter plate luminometer (LUmo) with an integration time of 0.5s/well. The approximate 50% inhibitory (approx. IC50) or the approx. 200% stimulatory (approx. SC200) concentrations of arginine were determined. Results: The approx. IC50 for freshest blood was 25 mg/l caffeine in BRGA-74. In BRGA-170 there appeared an approx. SC150 of 2 mg/l caffeine. The approx. IC50 was 3 mg/l caffeine in BRGA-60-48 for freshest blood. There was an approx. SC200 of 1 mg/l caffeine in BRGA-60-184. Pre-incubation of freshest blood first generated a neutrophil inhibitor later a neutrophil stimulator. Discussion: In conclusion, the reaction of blood neutrophils towards caffeine varies greatly from individual to individual and from the time interval of blood storage prior to analysis. Cell enzymes, e.g. cytochrome P450 or xanthine oxidase, can transform caffeine into an inhibitor or into a stimulator of neutrophils. It is suggested to analyze freshest blood of the individual patient to answer the question if a given (or to given) dosage of caffeine inhibits or stimulates blood ROS generation. It is suggested to administrate caffeine at lowest dosages to avoid critical changes of neutrophils´ oxidative metabolism.

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INTRODUCTION Caffeine (1,3,7-trimethyl-xanthine) stimulates the central nervous system, increases the contractility of the heart, expands the bronchi, is diuretic, stimulates intestinal peristalsis, and promotes glycogenolysis and lipolysis. Caffeine increases the cerebral blood flow and can cross the blood-brain barrier, stimulating psychic attention. Caffeine facilitates weight loss. In overdose, caffeine causes nervousness and insomnia. About 10 g of caffeine is lethal (approximately 100 cups of coffee) [1]. Caffeine counteracts the action of adenosine, that protects the brain from over-exertion by adenosine-receptors that tranquilize nerve cells [1]. Caffeine has a similar structure such as adenosine and so competes for the same receptor. Caffeine acts like a sympathomimetic and against antihistamines and barbiturates. In humans, about 80% of the caffeine is demethylated by the enzyme cytochrome P450 (CYP450) 1A2 to paraxanthine. CYP450 induction (e.g. by smoking) accelerates caffeine degradation. CYP450 inhibition (e.g. by cimetidine or disulfiram) reduce caffeine degradation. 16% of caffeine are converted in the liver to theobromine or theophylline. In urine many different caffeine metabolites, mainly di- and mono-methylxanthine and mono-, di-, and tri-methyl-urate are detected [1]. Caffeine is usually given at 5-20 mg/l is a drug for preterm neonates against apnea or for adults against migraine [1]. Newborns often have respiratory dysfunction, distress, or fungal or bacterial infection. Hyper-activated phagocytes might participate in respiratory distress, hypo-activated neutrophils can result in severe fungal or bacterial sepsis [2,3]. Here caffeine is analyzed for possible modulation of blood reactive oxygen species (ROS) generation, using the blood ROS generation assay (BRGA) [4-6].

Figure 1. Chemical structure of caffeine (left) and adenosine (right) [1]. 1,3,7-trimethyl-xanthine = 1,3,7-trimethyl-3,7-dihydro-2H-purin-2,6-dione (MW: 194.2 Daltons) C8H10N4O2; loss of the methylgroup at the imidazole ring results in theophylline. Caffeine is a competitive inhibitor of adenosine (adenine bound to ribose), a natural tranquilizer.

MATERIAL AND METHODS 40 µl caffeine in 0.9% NaCl (final drug conc.: 0-61.5 mg/l (0-158 µM); Merck, Darmstadt, Germany) in black high quality polystyrene microtiter plates (Brand, Wertheim, Germany; article nr. 781608) and 125 µl Hanks´ Balanced Salt Solution (HBSS; without phenol red; Sigma-SAFC Biosciences, Deisenhofen, Germany; article nr. 55037C-1000ML) were incubated in duplicate with 10 µl freshest citrated blood of a healthy donor or with 1d old blood of 2 healthy donors. Immediately (BRGA) or after 60 min (BRGA-60-),

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10 µl 5 mM luminol (Sigma) (final conc.: 0.26 mM), and 10 µl zymosan A (Sigma) in 0.9% NaCl (final conc.: 1.9 µg/ml) were added. During the main incubation (37°C) the light emissions of the wells were measured by a photons-multiplying microtiter plate luminometer (LUmo; anthos, Krefeld, Germany) with an integration time of 0.5s/well. The approximate 50% inhibitory (approx. IC50) or the approx. 200% stimulatory (approx. SC200) concentrations of caffeine were determined.

RESULTS AND DISCUSSION Figure 2 demonstrates the normal blood ROS generation kinetic of freshest citrated blood. The ROS maximum was 1373 RLU/s, reached after 85 min (t-maxn). t-0.25maxn  56 min (967 RLU/s), t-0.5maxn  74 min (352 RLU/s). At 170 min there appeared a second ROS peak (651 RLU/s). From 107 min onward up to 194 min the blood ROS generation was rather stable at a level of about 600 RLU/s, this action and the second ROS peak is due to micro-thrombi induced neutrophil activation and ROS generation [6,7]. In BRGA-60- the ROS maximum was 2841 RLU/s, reached after 134 min (t-maxn). t-0.25maxn  48 min (649 RLU/s), t-0.5maxn  77 min (1354 RLU/s) (Figure 3). The approx. IC50 for freshest blood was 25 mg/l caffeine in BRGA-74. In BRGA-170 there appeared an approx. SC150 of 2 mg/l caffeine (Figure 4).

Figure 2. BRGA kinetic in freshest normal human citrated blood. The blood ROS generation assay (BRGA) was performed for a freshest citrated blood as described under methods. The unsupplemented blood ROS maximum was 1373 RLU/s, reached after 85 min (t-maxn). t-0.25maxn  56 min (352 RLU/s), t-0.5maxn  74 min (967 RLU/s).

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Figure 3. BRGA kinetic in normal human citrated blood. The blood ROS generation assay with 60 min pre-incubation (BRGA-60-) was performed for freshest citrated blood sample as described under methods. The ROS maximum was 2841 RLU/s, reached after 134 min (t-maxn). t-0.25maxn  48 min (649 RLU/s), t-0.5maxn  77 min (1354 RLU/s).

The approx. IC50 was 3 mg/l caffeine in BRGA-60-48 for freshest blood. There was an approx. SC200 of 1 mg/l caffeine in BRGA-60-184 (Figure 5). Thus, pre-incubation of freshest blood first generates a neutrophil inhibitor later a neutrophil stimulator. The approx. IC50 in BRGA-71 was 1 mg/l caffeine for both 1d old samples. This means that stressed blood processes caffeine into a neutrophil inhibitor, a reaction presumably mediated by cytochrome P450 (CYP450) or xanthine oxidase (XO) [8,9]: freshest blood had an approx. IC50 of 25 mg/l caffeine (Figure 4).



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Figure 4. Modulation of blood ROS generation by caffeine. The blood ROS generation assay at 74 min or 170 min main incubation (BRGA-74 or BRGA-170) was performed for a fresh citrated blood sample supplemented with 0-61.5 mg/l caffeine. The approx. IC50 was 25 mg/l caffeine in BRGA-74. In BRGA-170 there appeared only an approx. SC150 of 2 mg/l caffeine.

There were also approx. SC200 values of 20 or 45 mg/l caffeine in stressed blood (Figures 6,7). The approx. IC50 in BRGA-60-39 was 6 mg/l caffeine or 13 mg/l caffeine for both 1d old samples. Sample 2 also an approx. SC150 of 7 mg/l caffeine (Figures 8,9).


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Figure 5. Modulation of blood ROS generation by pre-incubated arginine. The blood ROS generation assay with 60 min pre-incubation (BRGA-60-) at 48 min and 184 min main incubation was performed for a fresh citrated blood sample supplemented with 0-61.5 mg/l caffeine. The approx. IC50 was 3 mg/l caffeine in BRGA-60-48. There was an approx. SC200 of 1 mg/l caffeine in BRGA-60-184.



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Figure 7. Modulation of blood ROS generation by caffeine. At BRGA-71 performed for two 1d old citrated blood samples supplemented with 0-61.5 mg/l caffeine. The approx. IC50 was 1 mg/l caffeine for both samples. There were also approx. SC200 values of 20 or 45 mg/l caffeine.


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Figure 9. Inhibition of blood ROS generation by pre-incubated caffeine. The blood ROS generation assay with 60 min pre-incubation at 39 min main incubation (BRGA-60-39) was performed for two 1d old citrated blood samples supplemented with 0-61.5 mg/l caffeine. The approx. IC50 was 6 mg/l caffeine for sample 1 (Figure 9a) or 13 mg/l caffeine for sample 2 (Figure 9b). Sample 2 also an approx. SC150 of 7 mg/l caffeine.

In conclusion, the reaction of blood neutrophils towards caffeine varies greatly from individual to individual and from the time interval of blood storage prior to analysis. Cell enzymes, e.g. CYP450 or XO, can transform caffeine into an inhibitor or into a stimulator of neutrophils. It is suggested to analyze freshest blood of the individual patient to answer the question if a given (or to given) dosage of caffeine inhibits or stimulates blood ROS generation. It is suggested to administrate caffeine at the lowest dosage that is clinically necessary [10] to avoid critical changes of neutrophils´ oxidative metabolism.


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REFERENCES [1] [2]

www.wikipedia.org Weiss SJ, Lampert MB, Test ST. Long-lived oxidants generated by human neutrophils: characterization and bioactivity. Science. 1983; 222: 625-8. [3] Stief TW. Neutrophil granulocytes in hemostasis. Hemostasis Laboratory 2008; 1: 26989. [4] Stief T. The routine blood ROS generation assay (BRGA) triggered by typical septic concentrations of zymosan A. Hemostasis Laboratory 2013; 6: 89-98. [5] Stief T. Glucose initially inhibits and later stimulates blood ROS generation. Journal of Diabetes Mellitus 2013, 3: 15-21. doi: 10.4236/jdm.2013.31003. [6] Stief T. Micro-thrombi stimulate blood ROS generation. Hemostasis Laboratory 2013; 6 (issue 2-3) [7] Stief TW, Fareed J. The antithrombotic factor singlet oxygen/light (1O2/h). Clin Appl Thrombosis/Hemostasis 2000; 6: 22-30. [8] Rasmussen BB, Brøsen K. Determination of urinary metabolites of caffeine for the assessment of cytochrome P4501A2, xanthine oxidase, and N-acetyltransferase activity in humans. Ther Drug Monit. 1996; 18: 254-62. [9] Hakooz NM. Caffeine metabolic ratios for the in vivo evaluation of CYP1A2, Nacetyltransferase 2, xanthine oxidase and CYP2A6 enzymatic activities. Curr Drug Metab. 2009; 10: 329-38. [10] Natarajan G, Botica ML, Thomas R, Aranda JV. Therapeutic Drug Monitoring for Caffeine in Preterm Neonates: An Unnecessary Exercise ? Pediatrics 2007; 119: 936.


ISSN: 1941-8493 © 2013 Nova Science Publishers, Inc.

ARGININE SUPPRESSES BLOOD ROS GENERATION Christina Lichtenwald and Thomas Stief Institute of Laboratory Medicine and Pathobiochemistry, University Hospital of Marburg, Germany

ABSTRACT Background: Arginine stabilizes blood plasma against hemostasis activation (almost all hemostasis enzymes are serine proteases). At supra-1-molar concentrations arginine even prevents changes of hemostasis by freezing/thawing of plasma. Not only the infusion of unaltered human plasma but also that of unactivated neutrophils is a great medical challenge in many diseases that threaten the life of the individual patient, such as severe forms of pathologic intravascular coagulation (PIC) to stabilize hemostasis or neutropenia to combat fungi. The present investigation was performed to check by blood ROS generation assay (BRGA) if arginine could stabilize blood neutrophils to prepare them as a drug. Material and Methods: 40 µl 0-1 mol/l arginine in 0.9% NaCl (final drug conc.: 0-205 mM) in black high quality polystyrene microtiter plates (Brand®781608) and 125 µl Hanks´ Balanced Salt Solution (HBSS) were incubated in duplicate with 10 µl freshest citrated blood of a healthy donor or with 1d old blood of 2 healthy donors. Immediately (BRGA) or after 60 min (BRGA-60-), 10 µl 5 mM luminol (final conc.: 0.26 mM), and 10 µl zymosan A in 0.9% NaCl (final conc.: 1.9 µg/ml) were added. During the main incubation (37°C) the light emissions of the wells were measured by a photons-multiplying microtiter plate luminometer (LUmo) with an integration time of 0.5s/well. The approximate 50% inhibitory (approx. IC50) or the approx. 200% stimulatory (approx. SC200) concentrations of arginine were determined. Results and Discussion: Freshest normal citrated blood´s ROS generation was inhibited with IC50 values around 3 mM arginine. 1d old blood´s ROS generation needed twice this amount of arginine. In the BRGA-60- version of the assay sub-millimolar blood concentrations of arginine stimulate blood ROS generation, probably due to activation of the pro-oxidative NO-synthase. Arginine seems to be a promising candidate to stabilize neutrophil infusions.

INTRODUCTION Arginine is a semi-essential amino acid (plasma conc. about 0.1 mM) [1]. No side effects were observed up to a daily intake of 15 g. Arginine is found mainly in nuts. The minimum 


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daily requirement is 2-5g. Arginine arises in the urea cycle out of ornithine and carbamoyl-phosphate (a high-energy nitrogen phosphate compound) reacting to citrulline etc. (Figure 1). Arginine is oxidized by the arginine oxidase = nitric oxide synthase to nitric oxide (NO·). Interestingly, arginine is the sole precursor of NO·, formerly called endotheliumderived relaxing factor (EDRF), an important vasodilatator predominantly acting on the vascular smooth muscle cells in the arterial media. NO· inhibits platelet aggregation and adhesion, too [1]. The amino acid L-arginine in its HCl derivative is used to treat severe alkalosis. It can be found as 1 molar solution in ampoules. Arginine can be given against endothelial dysfunction which is initial atherothrombosis [2] (L-arginine blocks asymmetric dimethylarginine, homocysteine increase is prevented). Arginine is also indicated in tinnitus, hypertension, and type 2 diabetes mellitus. Arginine stimulates insulin secretion and as a competitive inhibitor of serine proteases such as kallikrein reduces the destruction of the pancreas B cells. L-Arginine increases cortisol and strengthens the physiologic immune system. Arginine regulates normal pituitary function (prolactin metabolism) and the synthesis and release of glucagon. Arginine participates in the biosynthesis of creatine, creatine phosphate and ATP being the two main molecular energy stores of the cell [1]. With its guanidino and its amino group arginine buffers well the pH value between pH 8 and pH 10. Since biochemical reactions mostly do not tolerate a pH value > 9.5, arginine is either used at pH 7.4 or it is used at pH 8.7, where arginine could be a 3fold more efficient competitive inhibitor of serine proteases (His···Asp···Ser catalytic triad with the negatively charged Asp binding to a positively charged guanidine group in Arg) or of the lysine-rich antithrombin-1 than at neutral pH. Arginine stabilizes blood plasma against hemostasis activation (almost all hemostasis enzymes are serine proteases). At supra-1-molar concentrations arginine even prevents changes of hemostasis by freezing/thawing of plasma [3]. Not only the infusion of unaltered human plasma but also that of unactivated neutrophils is a great medical challenge in many diseases that threaten the life of the individual patient, such as severe forms of pathologic intravascular coagulation (PIC) [4] to stabilize hemostasis or neutropenia to combat fungi. The present investigation was performed to check by blood ROS generation assay (BRGA) if arginine could stabilize blood neutrophils to prepare them as a drug.

Figure 1. Chemical structure of arginine [1]. 2-amino-5-guanidinopentanoic acid (C6H14N4O2; 174.2 Daltons) can be synthesized in the human body by argininosuccinate synthetase out of citrulline and aspartate.

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MATERIAL AND METHODS Arginine·HCl (1 M) was from Braun, Melsungen, Germany. 40 µl in 0.9% NaCl (final drug conc.: 0-205 mM) in black high quality polystyrene microtiter plates (Brand, Wertheim, Germany; article nr. 781608) and 125 µl Hanks´ Balanced Salt Solution (HBSS; without phenol red; Sigma-SAFC Biosciences, Deisenhofen, Germany; article nr. 55037C-1000ML) were incubated in duplicate with 10 µl freshest (less than 0.5h old) citrated blood of a healthy donor that gave written informed consent or with 10 µl 1d (23°C) old citrated blood of two healthy donors that gave written informed consent. Immediately (BRGA) or after 60 min (37°C) (BRGA-60-) 10 µl 5 mM luminol sodium salt (Sigma), and 10 µl 36 µg/ml zymosan A (Sigma) in 0.9% NaCl. During the main incubation (37°C) the light emissions of the wells were measured by a photons-multiplying microtiter plate luminometer (LUmo; anthos, Krefeld, Germany) with an integration time of 0.5s/well [5,6]. The approximate 50% inhibitory (approx. IC50) or the approx. 200% stimulatory (approx. SC200) concentrations of arginine were determined.

RESULTS AND DISCUSSION Figure 2 demonstrates the normal blood ROS generation kinetic of freshest citrated blood. The ROS maximum was 1373 RLU/s, reached after 85 min (t-maxn). t-0.25maxn  56 min (967 RLU/s), t-0.5maxn  74 min (352 RLU/s). At 170 min there appeared a second ROS peak (651 RLU/s). From 107 min onward up to 194 min the blood ROS generation was rather stable at a level of about 600 RLU/s, this action and the second ROS peak is due to micro-thrombi induced ROS generation [7,8]. In BRGA-60- the ROS maximum was 2841 RLU/s, reached after 134 min (t-maxn). t-0.25maxn  48 min (649 RLU/s), t-0.5maxn  77 min (1354 RLU/s) (Figure 3).

Figure 2. BRGA kinetic in freshest normal human citrated blood. The blood ROS generation assay (BRGA) was performed for a freshest citrated blood as described under methods. The unsupplemented blood ROS maximum was 1373 RLU/s, reached after 85 min (t-maxn). t-0.25maxn  56 min (352 RLU/s), t-0.5maxn  74 min (967 RLU/s).

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Figure 3. BRGA kinetic in normal human citrated blood. The blood ROS generation assay with 60 min pre-incubation (BRGA-60-) was performed for freshest citrated blood sample as described under methods. The ROS maximum was 2841 RLU/s, reached after 134 min (t-maxn). t-0.25maxn  48 min (649 RLU/s), t-0.5maxn  77 min (1354 RLU/s).

The approx. IC50 was 3 mM arginine in BRGA-56 and 4 mM arginine in BRGA-74 and BRGA-170. At about 1 mM arginine the blood ROS generation increased by about 20%, due to activation of NO synthase [9-12] and/or positive chaotropic actions of arginine in low concentrations (Figure 4). The approx. IC50 was 3 mM arginine in BRGA-60-48 and 15 mM arginine in BRGA-60-184. There was also an approx. SC150 of 0.6 mM arginine in BRGA-60-48 and an approx. SC200 of only 0.1 mM arginine in BRGA-60-184, suggestive of an activation of the pro-oxidative NO-synthase (Figure 5). The BRGA-60- seems to be a simple assay procedure to test this extraordinarily important enzyme.



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Figure 4. Inhibition of blood ROS generation by arginine. The blood ROS generation assay at 56 min, 74 min, or 170 min main incubation (BRGA-56, BRGA-74, BRGA-170) was performed for a fresh citrated blood sample supplemented with 0-20.5 mM arginine. The approx. IC50 was 3 mM arginine in BRGA-56 and 4 mM arginine in BRGA-74 and BRGA-170. At about 1 mM arginine the blood ROS generation increased by about 20%.

The approx. IC50 was 6 mM or 8 mM arginine in BRGA-71 for 1d 23°C stored blood (Figures 6,7). In BRGA-60-39 the approx. IC50 was 7 mM or 10 mM arginine (Figures 8,9). In conclusion, freshest normal citrated blood´s ROS generation was inhibited with IC50 values around 3 mM arginine. 1d old blood´s ROS generation needed twice this amount of arginine. In the BRGA-60- version of the assay sub-millimolar blood concentrations of arginine stimulate blood ROS generation, probably due to activation of the pro-oxidative NO-synthase. Arginine seems to be a promising candidate to stabilize neutrophil infusions [8,13-15].

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Figure 5. Modulation of blood ROS generation by pre-incubated arginine. The blood ROS generation assay with 60 min pre-incubation (BRGA-60-) at 48 min and 184 min main incubation was performed for a fresh citrated blood sample supplemented with 0-20.5 mM arginine. The approx. IC50 was 3 mM arginine in BRGA-60-48 and 15 mM arginine in BRGA-60-184. There was also an approx. SC150 of 0.6 mM arginine in BRGA-60-48 and an approx. SC200 of only 0.1 mM arginine in BRGA-60-184.



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Figure 7. Inhibition of blood ROS generation by arginine. The BRGA was performed for two 1d old citrated blood samples supplemented with 0-205 mM arginine. The approx. IC50 was 6 mM or 8 mM arginine in BRGA-71.


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Figure 9. Inhibition of blood ROS generation by pre-incubated arginine. The blood ROS generation assay with 60 min pre-incubation at 39 min main incubation (BRGA-60-39) was performed for two 1d old citrated blood samples supplemented with 0-205 mM arginine. The approx. IC50 was 7 mM arginine for sample 1 (Figure 9a) or 10 mM arginine for sample 2 (Figure 9b).


REFERENCES [1] [2]

www.wikipedia.org Stief TW. Thrombin generation by folic acid. In: Thrombin: function and pathophysiology. Stief T, ed.; NOVA science publishers; New York; 2012; pp. 65-72.

Arginine Suppresses Neutrophils [3]

[4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15]

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Stief TW. Thrombin – applied clinical biochemistry of the main factor of coagulation. In: Thrombin: function and pathophysiology. Stief T, ed.; Nova science publishers; New York; 2012; pp. vii-xx. https://www.novapublishers.com/catalog/product_ info.php?products_id=33386 Stief TW. The laboratory diagnosis of the pre-phase of pathologic disseminated intravascular coagulation. Hemostasis Laboratory 2008; 1: 2-20. Stief T. The routine blood ROS generation assay (BRGA) triggered by typical septic concentrations of zymosan A. Hemostasis Laboratory 2013; 6: 89-98. Stief T. Glucose initially inhibits and later stimulates blood ROS generation. Journal of Diabetes Mellitus 2013, 3: 15-21. doi: 10.4236/jdm.2013.31003. Stief T. Micro-thrombi stimulate blood ROS generation. Hemostasis Laboratory 2013; 6: (issue 2-3) Stief TW, Fareed J. The antithrombotic factor singlet oxygen/light (1O2/h). Clin Appl Thrombosis/Hemostasis 2000; 6: 22-30. Stuehr DJ. Mammalian nitric oxide synthases. Biochim Biophys Acta. 1999; 1411: 21730. Gorren AC, Mayer B. The versatile and complex enzymology of nitric oxide synthase. Biochemistry 1998; 63: 734-43. Tejero J, Stuehr D. Tetrahydrobiopterin in nitric oxide synthase. IUBMB Life. 2013 Feb 26. doi: 10.1002/iub.1136. Stuehr DJ, Santolini J, Wang ZQ, Wei CC, Adak S. Update on mechanism and catalytic regulation in the NO synthases. J Biol Chem. 2004; 279: 36167-70. Weiss SJ, Lampert MB, Test ST. Long-lived oxidants generated by human neutrophils: characterization and bioactivity. Science. 1983; 222: 625-8. Stief TW. Neutrophil granulocytes in hemostasis. Hemostasis Laboratory 2008; 1: 26989. Stief TW. The blood fibrinolysis / deep-sea analogy: a hypothesis on the cell signals singlet oxygen/photons as natural antithrombotics. Thromb Res 2000; 99: 1-20.



LUMINESCENCE KINETIC IN THE BLOOD ROS GENERATION ASSAY (BRGA) Thomas Stief Institute of Laboratory Medicine and Pathobiochemistry, University Hospital of Marburg, Germany

ABSTRACT Background: Citrated blood in the blood reactive oxygen species (ROS) generation assay (BRGA) triggered by low concentrations of the specific trigger zymosan A (ZyA) generates ROS with several maxima, all maxima together being about twofold the ROS generation of the first maximum. The present work verifies this ROS generation kinetic in additional samples. Material and Methods: Citrated blood samples of 11 healthy donors (stored for 1-2 days at 23°C) were analyzed in the BRGA. 10 µl sample added to black high quality polystyrene wells that had been prefilled with 125 µl Hanks´ Balanced Salt Solution were incubated immediately (BRGA), after 60 min (BRGA-60-), or after 120 min (BRGA-120-) with 10 µl 5 mM luminol and 10 µl 9, 18, or 36 µg/ml ZyA (0.6, 1.2, 2.3, or 4.4 µg/ml final) in 0.9% NaCl. The light emission per well was determined by a photons-multiplying microtiter plate luminometer (LUmo) with an integration time of 0.5s/well. Results: All samples when triggered with only 0.6 µg/ml ZyA indeed had several ROS generation maxima. In the BRGA version of the assay the following formula describes the appearance of the initial maxima (in 1d old normal samples): y = -0,00006x2 + 0,0332x; in the BRGA-60- version of the assay the following formula describes the appearance of the initial maxima: y = -0.00004x2 + 0,0251x; in the BRGA120- version of the assay the maxima appearance formula is y = -0.00004x2 + 0,0241x. The formula y = -0.00001x2 + 0,0151x describes the calculation of the ROS maximum number in dependence of the main incubation time of the BRGA in freshest normal citrated blood; in freshest EDTA – blood the formula is y = -0.000007x2 + 0,0143x. The reversal function to calculate the main incubation time out of the ROS generation maximum is y = 8,5789x2 + 60,368x for freshest citrated blood. Slightly ZyA-triggered blood develops several ROS generation maxima. The cells seem to keep about 50% of their ROS generation capacity in reserve for following assemblies of active NADPH-oxidase. The 0.5t-maxn (ROS generation at approximately 0.5fold the time to peak) is very important to compare the ROS generations between

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individuals or between different supplemented concentrations of a drug to the same individual blood sample. Keywords: reactive oxygen species, ROS, neutrophils, BRGA, blood ROS generation assay, zymosan A (ZyA), luminescence maxima, RLU/s, relative light units per second

INTRODUCTION Analysis of normal citrated blood in the blood reactive oxygen species (ROS) generation assay (BRGA) triggered by low concentrations of the specific trigger zymosan A (ZyA) [1,2] showed several maxima of ROS, the RLU/s sum of all maxima together being about twofold the ROS generation of the first maximum [3,4]. The present work verifies this ROS generation kinetic in additional samples.

MATERIAL AND METHODS Citrated blood was drawn from 11 healthy donors (stored for 1-2 days at 23°C) after written informed consent (4.5 ml venous blood added to 0.5 ml 106 mM sodium citrate, pH 7.4, in polypropylene monovettes from Sarstedt, Nümbrecht, Germany). The blood ROS generation assay (BRGA) was performed as follows: 10 µl sample were added to black high quality polystyrene wells (Brand, Wertheim, Germany; article nr. 681608) that had been prefilled with 125 µl Hanks´ Balanced Salt Solution (HBSS without phenol red; Sigma, Deisenhofen, Germany) were incubated immediately (BRGA), after 60 min pre-incubation (BRGA-60-), or after 120 min (BRGA-120-) with 10 µl 5 mM luminol (Sigma; 0.26 mM final) and 10 µl 9, 18, or 36 µg/ml zymosan A (Sigma; 0.6, 1.2, 2.3, or 4.4 µg/ml final) in 0.9% NaCl. The light emission per well was determined by a photons-multiplying microtiter plate luminometer (LUmo; anthos, Krefeld, Germany) with an integration time of 0.5s/well.

RESULTS AND DISCUSSION All samples when triggered with only ≤ 1 µg/ml ZyA indeed had several ROS generation maxima (Figures 1,2,4,5,7). In the BRGA version of the assay the following formula describes the appearance of the initial maxima (in 1d old normal samples): y = -0,00006x2 + 0,0332x; in the BRGA-60- version of the assay (BRGA with 60 min pre-incubation prior to the main incubation) the following formula describes the appearance of the initial maxima: y = -0.00004x2 + 0,0251x ; in the BRGA-120- version of the assay the maxima appearance formula is y = -0.00004x2 + 0,0241x. Recalcified samples of blood containing calcium chelators (citrate or EDTA) triggered with about 2 µg/ml ZyA showed one clear maximum and eventually at much larger incubation times a second much lower maximum (Figures 3,6,8). The first maximum in BRGA or BRGA-60- is caused by zymosan A, the following maxima should be caused by the micro-thrombi generated by recalcification [5]. The values described here were obtained in blood stored for 1-2d at 23°C. Freshest blood had similar characteristics [3]: the formula y = -0.00001x2 + 0,0151x describes the calculation of the ROS

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maximum number in dependence of the main incubation time of the BRGA in citrated blood (Figure 10a); in EDTA – blood the formula is y = -0.000007x2 + 0,0143x (Figure 10b).


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Figure 1. ROS generation kinetic in blood triggered by 0.6 µg/ml zymosan A. 5 citrated blood samples (1d 23°C old) were analyzed in the blood ROS generation assay (BRGA) triggered by 0.6 µg/ml zymosan A. The light emissions were measured by a photons-multiplying microtiter plate luminometer (LUmo) with an integration time of 0.5s/well. Figure 1a: 3 normal citrated blood samples, Figure 1b: mean values of the results of figure 1a with 5 ROS maxima, Figure 1c: the formula y = -0.00006x2 + 0,0332x describes the number of the ROS peak (y) dependent on the incubation time (x); e.g. (x│y) = (160│3.776) means that at 160 min incubation time the reaction is approaching the ROS maximum number 4. Figure 1d: 2 citrated blood samples, also with 5 ROS maxima, the third maximum being the highest.

The reversal function to calculate the main incubation time out of the ROS generation maximum is y = 8,5789x2 + 60,368x for citrated blood (Figure 10c). The 0.5t-maxn (blood ROS generation at 0.5fold the time to peak) is of primary importance, t-0.5maxn (main incubation time interval to reach about 0.5fold the luminescence of the normal maximum) is of secondary importance to compare the ROS generations between individuals or between different supplemented concentrations to the same individual blood sample (see this issue of Hemostasis Laboratory).

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Figure 2. ROS generation kinetic in blood triggered by 0.6 µg/ml zymosan A. 6 citrated blood samples (2d 23°C old) were analyzed in the blood ROS generation assay (BRGA) triggered by 0.6 µg/ml zymosan A. The light emissions were measured by a photons-multiplying microtiter plate luminometer (LUmo) with an integration time of 0.5s/well. Figure 2a: 6 normal citrated blood samples, Figure 2b: mean values of the results of figure 2a with 5 ROS maxima, the second maximum being the highest. Figure 2c: the formula y = -0.000009x2 + 0,0191x describes the number of the ROS peak (y) dependent on the incubation time (x). Figure 1d: 2 citrated blood samples, also with 5 ROS maxima, the third maximum being the highest.

If necessary, 0.1-1fold the luminescence of the normal maximum would be possible as well. If main incubation times of BRGA result in luminescences beyond the maximum, it might be advisable to use the BRGA-60- or BRGA-120-. Slightly ZyA-triggered blood develops several ROS generation maxima. The cells seem to keep about 50% of their ROS generation capacity in reserve for following assemblies of active NADPH-oxidase.

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Figure 3. ROS generation kinetic in blood triggered by 1.9 µg/ml zymosan A. 5 normal citrated blood samples (1d 23°C old; Figure 3a) or 6 normal citrated blood samples (2d 23°C old; Figure 3b; mean values in Figure 3c) were analyzed in the BRGA triggered by 1.9 µg/ml zymosan A. The light emissions were measured by a photons-multiplying microtiter plate luminometer (LUmo) with an integration time of 0.5s/well. Sample 3 in Figure 3a has a pronounced second ROS maximum starting at 126 min reaction time, presumably due to sudden zymosan A contamination of the well during 37°C incubation.

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Figure 4. Blood ROS generation kinetic in blood triggered by 1.9 µg/ml zymosan A. 5 normal citrated blood samples (1d 23°C old) or 6 normal citrated blood samples (2d 23°C old) were analyzed in the blood ROS generation assay with 60 min pre-incubation (BRGA-60-) triggered by 1.9 µg/ml zymosan A in the main incubation (Figure 4a). The light emissions were measured by a photons-multiplying microtiter plate luminometer (LUmo) with an integration time of 0.5s/well. The approximation curve to calculate the number of the ROS maximum out of the incubation time (in 1d old samples) is y = -0.00004x2 + 0,0251x (Figure 4b). Figure 4c: approximation curve in 2d old samples.

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Figure 5. Blood ROS generation kinetic in samples triggered by 1 µg/ml zymosan A. 5 normal citrated blood samples (1d 23°C old; Figure 5a) or 6 normal citrated blood samples (2d 23°C old; Figure 5b) were analyzed in the blood ROS generation assay with 60 min pre-incubation (BRGA-60-) triggered by 1 µg/ml zymosan A in the main incubation. The light emissions were measured by a photons-multiplying microtiter plate luminometer (LUmo) with an integration time of 0.5s/well.

Figure 6. Blood ROS generation (BRGA-60-) kinetic in samples triggered by 4.4 µg/ml zymosan A. 11 normal citrated blood samples (1-2d 23°C old) were analyzed in the blood ROS generation assay with 60 min pre-incubation (BRGA-60-) triggered by 4.4 µg/ml zymosan A in the main incubation. The light emissions were measured by a photons-multiplying microtiter plate luminometer (LUmo) with an integration time of 0.5s/well. Mean values ± 1 standard deviation.

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Figure 7. Blood ROS generation (BRGA-120-) kinetic in samples triggered by 0.6 µg/ml zymosan A. 5 normal citrated blood samples (1d 23°C old) or 6 normal citrated blood samples (2d 23°C old) were analyzed in the blood ROS generation assay with 120 min pre-incubation (BRGA-60-) triggered by 0.6 µg/ml zymosan A in the main incubation. The light emissions were measured by a photonsmultiplying microtiter plate luminometer (LUmo) with an integration time of 0.5s/well (Figure 7a). Approximation curve that describes the relationship between main incubation time and number of ROS maximum y = -0.00004x2 + 0,0241x (Figure 7b).

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Figure 8. Blood ROS generation (BRGA-120-) kinetic in samples triggered by 1.2 µg/ml zymosan A. 5 normal citrated blood samples (1d 23°C old) were analyzed in the blood ROS generation assay with 120 min pre-incubation (BRGA-60-) triggered by 1.2 µg/ml zymosan A in the main incubation. The light emissions were measured by a photons-multiplying microtiter plate luminometer (LUmo) with an integration time of 0.5s/well. MV±1SD.

Figure 9. Blood ROS generation (BRGA-120-) kinetic in samples triggered by 2.3 µg/ml zymosan A. 5 normal citrated blood samples (1d 23°C old) were analyzed in the blood ROS generation assay with 120 min pre-incubation (BRGA-120-) triggered by 2.3 µg/ml zymosan A in the main incubation. The light emissions were measured by a photons-multiplying microtiter plate luminometer (LUmo) with an integration time of 0.5s/well (MV±1SD).

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Figure 10. Calculation of the number of ROS generation maximum in freshest normal citrated blood and in normal EDTA-blood. Normal citrated blood or EDTA-blood (both less than 0.5h old) were analyzed in the blood ROS generation assay (BRGA; 125 µl HBSS in black Brand®781608 wells, 10 µl blood, 10 µl 5 mM luminol, 5 µl 18 µg/ml zymosan A). After 0-323 min (37°C) the light emissions per well were measured by a photons-multiplying microtiter plate photometer (LUmo). (Continued …)

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Thomas Stief The formula y = -0.00001x2 + 0,0151x describes the calculation of the ROS maximum number in dependence of the main incubation time of the BRGA in citrated blood (Figure 10a); in EDTA – blood the formula is y = -0.000007x2 + 0,0143x (Figure 10b). The reversal function to calculate the main incubation time out of the ROS generation maximum is y = 8,5789x2 + 60,368x for citrated blood (Figure 10c) (y = 4,1452x2 + 67,416x for EDTA-blood). The t-0.5maxn is very important to compare the ROS generations between individuals or between different supplemented concentrations to the same individual blood sample [3].

ACKNOWLEDGMENTS The research work of this article has been performed within a delightful intensive hemostasis training course of medical technical assistants (Marburg University Hospital MTA School, course 2011-2013). There was neither specific funding nor any conflict of interest.

REFERENCES [1] [2] [3] [4] [5]

Stief T. The routine blood ROS generation assay (BRGA) triggered by typical septic concentrations of zymosan A. Hemostasis Laboratory 2013; 6: 89-98. Stief T. Glucose initially inhibits and later stimulates blood ROS generation. Journal of Diabetes Mellitus 2013, 3: 15-21. doi: 10.4236/jdm.2013.31003. Stief T. Singlet oxygen (1O2*) primes blood neutrophils to generate ROS. Hemostasis Laboratory 2013; 6 (issue 4). Stief T. Photonic Hemostasis. Physiology of Light Signals in the Neutrophil. Nova Science Publishers, New York, 2013. Stief T. Micro-thrombi stimulate blood ROS generation. Hemostasis Laboratory 2013; 6 (issue 2-3).



SOME 2-MACROGLOBULIN/ THROMBIN COMPLEXES OPEN BY CONTACT ACTIVATION Thomas Stief Institute of Laboratory Medicine and Pathobiochemistry, University Hospital of Marburg, Germany

ABSTRACT Background: Thrombin bound in the α2-macroglobulin cage (a2M-F2a) is the biomarker for systemic activation of coagulation. Kallikrein is a known inactivator of a2M. So, the question arises to what extent these complexes are resistant to contact phase (= intrinsic = altered matrix) hemostasis. Material and Methods: 40 µl lyophilized/reconstituted human citrated plasma (control plasma N® = CPN) in high quality polystyrene U-wells microtiter plates (Brand®781600) were supplemented with 0 or 0.17 IU/ml unfractionated heparin (in a 2 µl vehicle of 0.9% NaCl). 2 µl 500 mM CaCl2 and 2 µl 61.5 mg/l uric acid in 0.9% NaCl were added. After 1-10 min or after 13 min at 37°C 80 µl 2.5 M arginine, 0.16% Triton X100®, pH 8.6 were added. After at least 3 min 20 µl 1 mM HD-CHG-Ala-Arg-pNA in 1.25 M arginine, pH 8.7, were added and the specific increase in absorbance at 405 nm (ΔA) was measured by a mirotiter plate photometer with a 1 mA resolution (PHOmo). The thrombin standard was 20 µl 40 mIU/ml bovine thrombin in 5% human albumin. Results: CPN contained about 17 mIU/ml F2a. About 80% (14 mIU/ml) of basal thrombin in control plasma N® was protected within the a2M cage. Only about 20% (3 mIU/ml) of F2a in CPN was outside the cage and could immediately be inactivated by antithrombin-3/heparin. Up to about 30% (4 mIU/ml) of F2a in a2M was lost upon incubation up to 10 min (37°C) with the contact trigger urate. Further incubation resulted in highly increased total plasmatic thrombin activity, e.g. 13 min (37°C) gave about 200 mIU/ml thrombin. Discussion: Intrinsic activation of hemostasis might destabilize F2a-a2M complexes. This action could be due to kallikrein. Nevertheless, the biomarker F2a-a2M does not loose importance because some F2a-a2M might be liberated and inactivated but in these critical phases of intrinsic coagulation much more thrombin is generated as a net effect.

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Correspondence to: PD Dr. med. T. Stief, Central Laboratory, University Hospital, D-35043 Marburg, Germany. Email: [email protected]; Tel.: +49-6421-58 64471; FAX: +49-6421-58 65594.

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INTRODUCTION Constantly about 10% of all thrombin (F2a) systemically generated ends entrapped in the α2-macroglobulin (a2M) cage [1]. Therefore, a2M-F2a is a new biomarker for systemic activation of coagulation, 100% of normal intravascular coagulation (NIC) being 5.5 mIU/ml thrombin [2]. At > 120% of normal the pathophysiology of the pre-phase of pathologic intravascular coagulation (PIC-0) begins, at > 150% of normal the patient enters the typical phase of PIC (PIC-1), at > 200% of normal the patient is in consumption phase of PIC (PIC2) [3]. In vitro the a2M-F2a complexes are stable if EDTA-blood plasma is stabilized by 1.25 M arginine, pH 8.7 [4]. In vivo these complexes might be attacked by oxidants or proteases, resulting in a liberation of the entrapped protease thrombin followed by interaction of thrombin with fibrinogen or antithrombin-3. This instability of the a2M-F2a complexes could be especially problematic in altered matrix coagulation (contact phase = intrinsic hemostasis) [5].

MATERIAL AND METHODS 40 µl lyophilized/reconstituted human citrated plasma (control plasma N® = CPN; Siemens-DadeBehring; Munich-Marburg, Germany) in high quality polystyrene U-wells microtiter plates (Brand, Wertheim, Germany; article nr. 781600) were supplemented with 0 or 0.17 IU/ml unfractionated heparin (Sarstedt, Nümbrecht, Germany) (in a 2 µl vehicle of 0.9% NaCl). 2 µl 500 mM CaCl2 and 2 µl 61.5 mg/l uric acid (both from Sigma, Deisenhofen, Germany) in 0.9% NaCl were added. After 1-10 min or after 13 min at 37°C 80 µl 2.5 M arginine, 0.16% Triton X100®, pH 8.6 (Sigma) were added. After at least 3 min 20 µl 1 mM fast chromogenic thrombin substrate HD-CHG-Ala-Arg-pNA (Pentapharm, Basel, Switzerland) in 1.25 M arginine, pH 8.7, were added and the specific increase in absorbance at 405 nm (ΔA) was measured by a mirotiter plate photometer with a 1 mA resolution (PHOmo; anthos, Krefeld, Germany). The thrombin standard was 20 µl 40 mIU/ml bovine thrombin in 5% human albumin (CSL Behring, Marburg, Germany) it generated a specific ΔA/78 min of 106 mA.

RESULTS AND DISCUSSION CPN contained about 17 mIU/ml F2a (Figure 1). About 80% (14 mIU/ml) of basal thrombin in control plasma N® was protected within the a2M cage. Only about 20% (3 mIU/ml) of F2a in CPN was outside the cage and could immediately be inactivated by antithrombin-3/heparin (Figure 1). Up to about 30% (4 mIU/ml) of F2a in a2M was lost upon incubation up to 10 min (37°C) with the contact trigger urate [6]. Further incubation resulted in highly increased total plasmatic thrombin activity, e.g. 13 min (37°C) gave about 200 mIU/ml thrombin. The findings here are on pooled normal plasma, lyophilized. Fresh individual normal plasma should demonstrate a higher percentual protection within the a2M cage. In fresh samples there is only very few systemic thrombin attaching to antithrombin-1 (fibrin) [7].

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Figure 1. Some instability of F2a-a2M complexes by contact activation. 40 µl pooled human citrated plasma (control plasma N®) in high quality polystyrene U-wells microtiter plates (Brand®781600) were supplemented with 0 or 0.17 IU/ml unfractionated heparin (in a 2 µl vehicle of 0.9% NaCl). 2 µl 500 mM CaCl2 and 2 µl 61.5 mg/l uric acid in 0.9% NaCl were added. After 1-10 min or after 13 min at 37°C 80 µl 2.5 M arginine, 0.16% Triton X100®, pH 8.6 were added and thrombin activity was chromogenically determined (intra-assay CVs < 5%).

In conclusion, intrinsic activation of hemostasis might destabilize the F2a-a2M complex. This action might be due to kallikrein [8,9]. Nevertheless, the biomarker F2a-a2M does not loose importance because some F2a-a2M might be liberated and inactivated but in these critical clinical states of AM-coagulation much more thrombin is generated as a net effect [10].

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REFERENCES [1]

Stief TW. Arginine conserves the hemostasis activation state of plasma even against freezing/thawing. Advances in Medicine and Biology 2011; 19: 275-88. [2] Stief TW. The laboratory diagnosis of the pre-phase of pathologic disseminated intravascular coagulation. Hemostasis Laboratory 2008; 1: 2-20. [3] Stief TW. Thrombin – applied clinical biochemistry of the main factor of coagulation. In: Thrombin: function and pathophysiology. Stief T, ed.; Nova science publishers; New York; 2012; pp. vii-xx. https://www.novapublishers.com/catalog/ product_ info.php?products_id=33386 [4] Stief TW. Specific determination of plasmatic thrombin activity. Clin Appl Thrombosis/Hemostasis 2006; 12: 324-329. [5] Stief TW. Coagulation activation by lipopolysaccharides. Clin Appl Thrombosis/Hemostasis Dec 26, 2007 doi: 10.1177/1076029607309256; 2009; 15: 20919. [6] Stief T, Lichtenwald C, Mühling A, Schorge L, Stenzel D, Grass J, Schudarek H, Heinrich D. Blood reactive oxygen species and urate. Hemostasis Laboratory 2014; 7 (issue 1) [7] Mosesson MW. Update on antithrombin I (fibrin). Thromb Haemost. 2007; 98: 105-8. [8] Obiezu CV, Michael IP, Levesque MA, Diamandis EP. Human kallikrein 4: enzymatic activity, inhibition, and degradation of extracellular matrix proteins. Biol Chem. 2006 387: 749-59. [9] Segal HC, Hunt BJ, Cottam S, Beard C, Francis JL, Potter D, Tan KC. Changes in the contact system during orthotopic liver transplantation with and without aprotinin. Transplantation. 1995; 59: 366-70. [10] Stief TW, Ulbricht K, Max M. Circulating thrombin activity in sepsis. Hemostasis Laboratory 2009; 2: 293-306.